Haschek and Rousseaux's Handbook of Toxicologic Pathology, Volume 2: Safety Assessment Environmental Toxicologic Pathology 0128210478, 9780128210475

Table of contents :
Front Cover
HASCHEK AND ROUSSEAUX’S HANDBOOK OF TOXICOLOGIC PATHOLOGY
HASCHEK AND ROUSSEAUX’S HANDBOOK OF TOXICOLOGIC PATHOLOGY
Copyright
Dedication
Contents
Contributors
About the Editors
EDITORS
ASSOCIATE EDITORS
ILLUSTRATIONS EDITOR
Preface
1 - PRODUCT DISCOVERY AND DEVELOPMENT
1 - Overview of Drug Development
1 INTRODUCTION
2 OVERVIEW
2.1 The Stages of the Product Life Cycle
Product Development Stages
Product Introduction
Growth
Maturity
Decline
2.2 The Scope of Drug Discovery and Development
The Challenge
Cost, Time, and Risks in Developing New Drugs
Time Required to Develop a New Drug
The Life Cycle of a Health Product
3 DRUG DISCOVERY AND DEVELOPMENT
3.1 Patents and Intellectual Property
3.2 Discovery
Target Identification and Validation
Target Validation
Lead Generation and Optimization
High-Throughput Assays and Quantitative Structure-activity Relationships (QSAR)
Mechanistic Studies
Innovative Trends in Drug Development
Biomarkers
Toxicity Assessment
4 DEVELOPMENT
4.1 Drug Substance and Drug Product Development (Quality)
Development Pharmaceutics—The Active Pharmaceutical Ingredient
Development Pharmaceutics—The Drug Product
Manufacture of the Finished Dosage Form
Impurities in the Final Drug Product
Excipients
Packaging Materials
Stability Testing of APIs and DPs
4.2 Nonclinical Safety Studies
Selection of Doses for Nonclinical Studies
Toxicity Tests
GENOTOXICITY
BACTERIAL MUTATION (AMES) ASSAY
CHROMOSOMAL ABERRATION ASSAYS
MICRONUCLEUS TEST
MUTAGENICITY TEST
SINGLE-DOSE TOXICITY
REPEATED-DOSE TOXICITY
REPRODUCTIVE TOXICITY
CARCINOGENIC POTENTIAL
4.3 Animal Efficacy Studies
Short-Term Efficacy Studies in Animals
BIOAVAILABILITY AND BIOEQUIVALENCE STUDIES
DIGESTION AND BALANCE STUDIES
Design Considerations for Nonclinical Efficacy Studies
ENDPOINTS
TIMING OF INTERVENTION
ROUTE OF ADMINISTRATION
DOSING REGIMEN
Risk Mitigation Methods
Dose Escalation
5 OTHER HEALTH PRODUCTS
5.1 Biologics
5.2 Medical Devices
5.3 Natural Health Products
5.4 Vaccines
6 CLINICAL TRIALS
6.1 Phase I Clinical Trials
6.2 Phase II Clinical Trials
6.3 Phase III Clinical Trials
6.4 Phase IV Clinical Trials
6.5 Limitations of Clinical Trials
7 POSTMARKETING SURVEILLANCE
7.1 Adverse Drug Events
7.2 Adverse Drug Reactions
7.3 Current Mechanisms and Tools for Identifying and Quantifying ADRs
8 REGULATORY AUTHORITIES
8.1 Overview
International Council on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use
United States and Canada
Europe and the United Kingdom
Asia
Latin America
8.2 Considerations on Legal Frameworks
9 SUMMARY AND CONCLUSIONS
REFERENCES
Selected Links to Regulatory Authorities and Issues
2 - Overview of the Role of Pathology in Product Discovery and Development
1 INTRODUCTION
2 DISCOVERY TOXICOLOGY
2.1 Small Molecules
2.2 Nucleic Acid–Based Pharmaceuticals
2.3 Biologics
2.4 Cell and Gene Therapy
3 DEVELOPMENT TOXICOLOGY
3.1 Small Molecules
3.2 Nucleic Acid Pharmaceuticals
3.3 Biologics
3.4 Cell and Gene Therapy
3.5 Anticancer Drugs
3.6 Adversity and Reversibility
3.7 Clinical Dose Setting and Clinical Safety Assessments
3.8 Regulatory Filings
4 NONSTANDARD STUDIES AND END POINTS
4.1 Pharmacology (Efficacy) Studies
4.2 Investigative (Mechanistic) Toxicology Studies
4.3 Standard and Alternative Animal Models
4.3 Standard
4.3 Alternative
NONMAMMALIAN MODELS
MAMMALIAN MODELS
4.4 Biomarker Considerations
4.5 Interpretation of Unique Findings
REFERENCES
3 - Discovery Toxicology and Discovery Pathology
1 INTRODUCTION
1.1 Discovery Toxicology
1.2 Discovery Pathology
2 KNOWLEDGE INTEGRATION AND THE SPANNING OF DISCIPLINES
3 PATHOLOGY TOOLBOX
4 IN VITRO/IN VIVO CORRELATIONS
5 TARGET SELECTION
6 TARGET VALIDATION
7 TRANSLATIONAL MEDICINE
8 HYPOTHESIS GENERATION, EXPERIMENTAL DESIGN, AND THE ROLE OF INVESTIGATIVE STUDIES
9 DISCOVERY STRATEGY FOR BIOLOGICS
10 COMMUNICATIONS
11 PERSONALITY AND BEHAVIORAL TRAITS THAT ARE HELPFUL TO SUCCEED IN DISCOVERY PATHOLOGY AND DISCOVERY TOXICOLOGY
12 SUMMARY
REFERENCES
4 - Pathology in Nonclinical Drug Safety Assessment
1 INTRODUCTION
2 DRUG SAFETY AND EFFICACY ARE A CONTINUUM
3 THE PATHOLOGIST'S ROLE IN NONCLINICAL SAFETY ASSESSMENT
3.1 Adverse Effects and Pathology Report
3.2 Reversibility and Delayed Toxicity
3.3 Lexicon and Diagnostic Terminology
3.4 GLP Regulations in Pathology
3.5 Pathology Peer Review
3.6 NOAELs and Study Report
4 PATHOLOGY IN NONCLINICAL SAFETY ASSESSMENT OF SMALL MOLECULES
5 PATHOLOGY IN NONCLINICAL SAFETY ASSESSMENT OF BIOTHERAPEUTICS
5.1 Proteins
5.2 Oligonucleotides
5.3 Gene Therapy
5.4 Cell Therapy
5.5 Stem Cell Therapy
5.6 Vaccines
6 PATHOLOGY IN NONCLINICAL SAFETY ASSESSMENT OF NOVEL FORMULATIONS
6.1 Excipients
6.2 Conjugation
6.3 Nanotechnology
7 DIGITAL PATHOLOGY AND COMPUTATIONAL PATHOLOGY
8 NOVEL INVESTIGATIVE TOOLS IN NONCLINICAL SAFETY ASSESSMENT
9 CONCLUSION
REFERENCES
REFERENCES
5 - Carcinogenicity Assessment
1 THE PAST, PRESENT, AND POTENTIAL FUTURE OF CARCINOGENICITY ASSESSMENT
1.1 Brief History of Carcinogenicity Assessment
1.2 Food, Drugs, and Cosmetics
1.3 Other Chemicals
1.4 Relevance of Rodent Findings in Carcinogenicity Hazard Identification Studies for Human Risk
1.5 Evolution from Lifetime Bioassays in Two Rodent Species to the Current Standards
1.6 Looking Forward: ICH Guideline S1B Modifications
1.7 New Approaches in Predicting Carcinogenicity Hazards
1.7 In vivo Approaches in Cancer Hazard Assessment
2 PURPOSE, PLANNING, PREREQUISITE INFORMATION, AND TIMING OF LIFETIME CARCINOGENICITY STUDIES
2.1 Prerequisite Data to Design a Carcinogenicity Study Protocol
2.2 Special Protocol Assessment for Carcinogenicity Studies
2.3 Carcinogenicity Study Planning Timeline
3 TWO-YEAR RODENT CARCINOGENICITY STUDIES
3.1 Study Design
3.2 Managing High Mortality in 2-Year Carcinogenicity Studies
3.2 Low Survival in Treated Groups
3.2 Low Survival in the Control Groups
3.2 Low Survival Observed before 12Months
3.3 Pathology Interpretations
3.4 Historical Control Data
4 ALTERNATIVE GENETICALLY MODIFIED MOUSE MODELS
4.1 The Range-Finding Study
4.2 Carcinogenicity Study Design Using Alternative Models
4.3 Conduct of Carcinogenicity Studies Using Alternative Models
5 CARCINOGENICITY ASSESSMENT OF BIOTECHNOLOGY-DERIVED THERAPEUTIC PROTEINS
6 CARCINOGENICITY ASSESSMENT OF NUCLEIC ACID THERAPIES
7 CARCINOGENICITY ASSESSMENT OF STEM CELL–DERIVED THERAPIES
7.1 Safety Concerns for Stem Cell–Derived Therapies
7.2 In vivo Assessment of Teratoma Formation
8 CARCINOGENICITY ASSESSMENT OF MEDICAL DEVICES
9 CONCLUSION
REFERENCES
2 - PRODUCT-SPECIFIC PRACTICES FOR SAFETY ASSESSMENT
6 - Protein Therapeutics
1 HISTORY OF PROTEIN THERAPEUTICS
2 TYPES OF PROTEIN THERAPEUTICS: STRUCTURAL AND FUNCTIONAL CONSIDERATIONS
2.1 Key Structural and Functional Elements of Antibodies and Related Products
3 GENERAL NONCLINICAL TOXICITY TESTING STRATEGIES
3.1 General Considerations
3.2 Applicable Regulatory Guidelines
3.3 Target Characterization/Species Selection
4 IMMUNOGENICITY
4.1 Overview
4.2 Effect of Immunogenicity
4.3 Predictivity of Nonclinical Immunogenicity
4.4 Assessing Immunogenicity
4.5 Managing Immunogenicity in Toxicity Studies
5 OFF-TARGET PROFILING
5.1 Overview
5.2 History
5.3 Whether and When to Conduct
5.4 Molecules to Test
5.5 Species to Test
5.6 TCR Strategies Supporting FIH (Only for CDR-Containing Test Articles)
5.7 Impact and Regulatory Considerations
6 GENERAL TOXICITY STUDY DESIGN CONSIDERATIONS
6.1 Overview
6.2 Exploratory (Non-GLP) Studies
6.3 FIH-Enabling Studies
6.4 Post-FIH-Enabling Studies
6.5 Dose Selection
6.6 Dosing Interval
6.7 Route of Administration, Formulation
6.8 Recovery
7 IMMUNOTOXICITY
7.1 Overview
7.2 In Vitro Assays for Assessing Immunotoxicity
7.3 In Vivo Assays for Assessing Immunotoxicity
7.4 Immunosuppression
7.5 Immunostimulation
7.6 Program Strategies and Regulatory Considerations
8 SAFETY PHARMACOLOGY ASSESSMENTS
8.1 Overview
8.2 Strategy and Timing
9 DEVELOPMENTAL AND REPRODUCTIVE TOXICITY STUDIES AND JUVENILE ANIMAL STUDIES
9.1 Overview
9.2 Species to Test and Alternative Models
9.3 Study Design Considerations
9.4 Strategies and Timing
9.5 Nonclinical Pediatric Evaluation
10 GENOTOXICITY
11 CARCINOGENICITY STUDIES
12 ADVANCED CANCER
13 ANTIBODY-DRUG CONJUGATES
13.1 Overview
13.2 Mechanisms of Toxicity
13.3 Nonclinical Safety Assessment
14 MULTISPECIFIC MOLECULES
15 BIOSIMILARS
16 TOXICOLOGIC PATHOLOGY OF PROTEIN THERAPEUTICS—PUTTING IT ALL TOGETHER
16.1 On-Target, Off-Target, and Immunogenicity-Related Effects
16.2 Determining Adversity
GLOSSARY
Acknowledgments
REFERENCES
REFERENCES
7 - Nucleic Acid Pharmaceutical Agents
1 INTRODUCTION
2 NUCLEIC ACID PHARMACEUTICALS
2.1 Types of Nucleic Acid Pharmaceuticals
2.1 Antisense Oligonucleotides
2.1 Small Interfering RNA
2.1 MicroRNA
2.2 Advantages of Nucleic Acid Pharmaceuticals over Traditional Therapeutic Modalities
2.3 Specific Indications for Nucleic Acid Pharmaceuticals
2.4 Clinical Trials of Nucleic Acid Pharmaceuticals
3 NUCLEIC ACID PHARMACEUTICAL DESIGN AND DEVELOPMENT
3.1 Chemistry Considerations
3.1 ASOs
3.1 SiRNAs and miRNAs
3.2 Delivery Considerations
3.2 Common Delivery Systems
3.2 Systemic Delivery
3.2 Local Delivery to the Central Nervous System or Peripheral Tissues
3.2 Exploration of Novel Delivery Systems
3.3 Pharmacology Considerations
3.3 Biodistribution
3.3 Detection Methods for Therapeutic Oligonucleotides
4 SAFETY EVALUATION OF NUCLEIC ACID PHARMACEUTICALS
4.1 Regulatory Perspective
4.1 Species Selection for Safety Studies
4.1 Use of Surrogate Sequences
4.1 Genotoxicity Studies
4.1 Reproductive and Developmental Toxicity Studies
4.1 Safety Pharmacology Studies
4.2 Use of Animal Models for Safety Evaluation
4.2 Delivery Models
4.2 Disease-Modifying Models
4.3 Mechanisms of Common Toxicities
4.3 Hybridization-dependent Toxicity
4.3 Hybridization-independent Toxicity
4.3 Tissue Accumulation
5 CONCLUSIONS
GLOSSARY
REFERENCES
8 - Gene Therapy and Gene Editing
1 INTRODUCTION
2 GENERAL PRINCIPLES OF NONCLINICAL RESEARCH AND DEVELOPMENT FOR GENE THERAPY PRODUCTS
2.1 Pharmacology
2.2 Toxicology
2.3 Biodistribution and Viral Shedding
2.4 Role of Pathologists in the Nonclinical Assessment of Gene Therapy Products
3 IN VIVO GENE THERAPY
3.1 General Concepts of In Vivo Gene Therapy
3.2 AAV as a Model Platform for in vivo Gene Therapy
3.3 Nonclinical Pharmacology and Safety Assessment for in vivo Gene Therapy
3.3 Pharmacology
3.3 Biodistribution
3.3 Toxicology
3.4 Contemporary Toxicities Associated With In Vivo Gene Therapy
3.4 Immunotoxicity Associated With AAV-Based Gene Therapy
INNATE IMMUNE RESPONSES TO AAV-BASED GENE THERAPY PRODUCTS
ADAPTIVE IMMUNE RESPONSES TO AAV-BASED GENE THERAPY PRODUCTS
ASSESSMENT OF IMMUNOTOXICITY IN NONCLINICAL STUDIES
3.4 Acute Liver Injury Associated With High-Dose Gene Therapy
3.4 Dorsal Root Ganglion (DRG) Toxicity Associated With AAV Vectors
3.4 DNA Integration, Hepatocellular Carcinoma, and AAV-Based Gene Therapy
4 EX VIVO GENE THERAPY
4.1 General Concepts of Ex Vivo Gene Therapy
4.1 Autologous Lentiviral Vector–Transduced CD34+ Hematopoietic Stem Cell Ex Vivo Gene Therapy
4.1 Retroviral Vectors Derived from Gamma-Retroviruses and Lentiviruses
4.2 Nonclinical Safety Assessment for Ex Vivo Gene Therapy
4.2 Nonclinical Evaluation Strategies for Ex Vivo Gene Therapy
4.2 Proof-of-Concept Studies for Ex Vivo Gene Therapy
4.2 Biodistribution and Pharmacodynamics for Ex Vivo Gene Therapy
4.2 Toxicity Studies (In Vivo Safety Assessment) for Ex Vivo Gene Therapy
4.2 Experimental Design Features Supporting Ex Vivo Gene Therapy Development
ANIMAL MODEL OPTIONS
ANIMAL NUMBERS PER GROUP
STUDY DURATION
CONTROL GROUPS
TIME POINTS
SOURCE OF CELLS
CELL AND VECTOR CHARACTERIZATION
ROUTE OF ADMINISTRATION AND DOSING REGIMEN
VECTOR COPY NUMBER
4.2 Genotoxicity Studies
INTEGRATION SITE ANALYSIS
IN VITRO IMMORTALIZATION ASSAY AND SURROGATE ASSAY FOR GENOTOXICITY ASSESSMENT
4.3 Toxicologic Pathology Considerations With Ex Vivo Gene Therapy
4.3 Conditioning Agents in Ex Vivo Gene Therapy
CONDITIONING
4.3 Immunogenicity
4.3 Tumorigenicity
5 GENOME EDITING
5.1 Harnessing Cellular DNA Repair
5.1 Zinc-Finger Nucleases
5.1 TALENS
5.1 RNA-Guided Endonucleases (CRISPR/Cas Systems)
5.1 Base Editing
5.2 Genome Editing Strategies
5.2 Loss-of-Function
GENE DELETION
5.2 Gain-of-Function
GENE INSERTION
5.2 Gene Correction
NUCLEASE-MEDIATED CORRECTION
5.3 Nonclinical Safety Assessment for Genome Editing Products
5.3 Exaggerated Pharmacology
5.3 Biodistribution
5.3 Toxicology
EFFECTS FROM NANOPARTICLE DELIVERY OF NUCLEIC ACIDS
GENOTOXICITY
METHODS OF ASSESSING GENOTOXICITY
Off-Target Site Discovery
Off-Target Site Verification
CONSIDERATIONS FOR OFF-TARGET RISK ASSESSMENT
CHROMOSOMAL STRUCTURAL VARIATION
GENOTOXICITY SUMMARY AND FUTURE DIRECTIONS
IMMUNE ACTIVATION
6 CONCLUSION
GLOSSARY
REFERENCES
9 - Vaccines
1 INTRODUCTION
2 VACCINE IMMUNOLOGY
3 GENERAL CONCEPTS IN VACCINE TOXICOLOGY
3.1 Vaccine Nonclinical Safety Package: Regulatory Expectations
3.2 Immunogenicity Assays
4 VACCINE MODALITIES
4.1 Protein-Based Vaccines
4.1 Protein and Recombinant Protein Vaccines
4.1 Glycoconjugate Vaccines
4.1 Virus-like Particles Nanoparticle or Generalized Modules for Membrane Antigen Based Vaccines
4.2 Nucleic Acid–Based Vaccines
4.2 RNA-Based Vaccines
4.2 DNA Vaccines
4.2 Inactivated and Attenuated Virus Vaccines
4.2 Viral Vectored Vaccines
4.2 Anti-idiotype Vaccines
4.2 Cell-Based Vaccines
4.2 Oncolytic Virus–Based Modalities
5 VACCINE ADJUVANTS
5.1 Mineral Salts
5.2 Surfactant/Emulsion Adjuvants
5.3 Nucleic Acid/Nucleotide Adjuvants
5.3 CpG-ODN
5.3 Double- or Single-Stranded RNA or RNA Analogs
5.4 Lipid Adjuvants
5.5 Adjuvant Systems and Liposome-Based Adjuvants
5.5 Lipid Nanoparticles for Delivery of mRNA-Based Vaccines
5.6 Carbohydrate-Containing Adjuvants
5.7 Toxin-Based Adjuvants
5.8 Other Adjuvants
6 VACCINE PHARMACOLOGY AND OTHER STUDIES
6.1 Immunogenicity and Efficacy Studies
6.1 Immunogenicity Studies
6.1 Efficacy Studies
INFECTIOUS DISEASE VACCINES
THERAPEUTIC CANCER VACCINES
6.2 Enhanced Disease and Neurovirulence Assessment
6.2 Enhanced Disease
6.2 Neurovirulence Assessments
6.3 Biodistribution and Persistence
6.4 Absorption, Distribution, Metabolism, and Excretion
7 VACCINE SAFETY STUDIES
7.1 Repeat-Dose Toxicity Studies
7.1 Developmental and Reproductive Toxicity Studies
8 VACCINE STUDIES—DESIGN, TECHNICAL CONSIDERATIONS, AND DATA INTERPRETATION
8.1 Species Selection
8.2 Dose Groups
8.3 Technical Considerations Related to Dosing and Dose Administration
8.3 Route of Administration
8.3 Dose Volume
8.3 Animal Husbandry and Dosing
8.3 Injectable Vaccines
8.3 Restraint, Handling, and Dosing
8.4 In-Life Assessments
8.4 Local Toxicity and Reactogenicity
8.5 Clinical Pathology
8.5 Hematology
8.5 Clinical Chemistry
8.5 Coagulation
8.5 Acute Phase Biomarkers
8.5 Therapeutic Vaccine Considerations
8.6 Anatomic Pathology—Post-life Evaluation
8.6 Macroscopic Observations and Sample Collection
8.6 Microscopic Evaluation
8.6 Injection Site—Microscopic Findings
8.6 Draining Lymph Nodes and Spleen
8.6 Other Tissues
8.6 Combined Safety–Efficacy Studies
9 SPECIAL CONSIDERATIONS FOR THERAPEUTIC MODALITIES
9.1 Therapeutic Vaccines
9.2 Other Therapeutic Vaccine Strategies
9.3 Oncolytic Virus-Based Therapeutic Vaccines
10 NONCLINICAL TOXICITY—DETERMINING ADVERSITY
11 NONCLINICAL TOXICITY AND HUMAN TRANSLATION
11.1 Autoimmunity
11.2 Hypersensitivity
11.3 Thrombotic Thrombocytopenia
12 VACCINES AND THE ANTI-VACCINE MOVEMENT
13 CONCLUSIONS
14 GLOSSARY
REFERENCES
REFERENCES
10 - Stem Cell and Other Cell Therapies
1 INTRODUCTION
2 BIOLOGICAL PRINCIPLES OF CELL THERAPY
2.1 Cell Sources
2.1 Autologous Cells
2.1 Allogeneic Cells
2.1 Xenogeneic Cells
2.2 Classes of Transplantable Cells
2.2 Pluripotent Stem Cells
EMBRYONIC STEM CELLS
SOMATIC CELL–DERIVED STEM CELLS
INDUCED PLURIPOTENT STEM CELLS
OTHER SOMATIC-ORIGIN STEM CELLS
2.3 Primary Preclinical Safety Considerations for Cell Therapies
2.3 Test Article–Related Safety Considerations
BIODISTRIBUTION OF IMPLANTED CELLS
DIFFERENTIATION OF IMPLANTED CELLS
PROLIFERATION OF IMPLANTED CELLS
2.3 Procedure-Related Safety Considerations
ANIMAL MODELS OF DISEASE
IMPLANTATION-RELATED TISSUE INJURY
IMMUNOGENICITY TOWARD OR BY IMPLANTED CELLS
3 PRECLINICAL CONSIDERATIONS FOR CELL THERAPIES
3.1 Regulatory Guidance
3.2 Key Endpoints for Preclinical Assessment of Cell Therapies
3.3 Design of Preclinical Safety Studies
3.3 Species Selection
3.3 Group Constitution
3.3 Study Regimen
CBMPS AS THERAPEUTIC TEST ARTICLES
CBMP AS SUBSTRATES TO DELIVER THERAPEUTIC MOLECULES
3.3 Analytical Design
3.3 Principal Cell Therapy-Related Findings in Tissues
EFFECTS DUE TO IMPLANTED CELLS
EFFECTS RELATED TO THE IMPLANTATION PROCEDURE
3.4 The Pathologist's Contribution
4 STEM CELL–BASED TOXICITY TESTING
5 SUMMARY
GLOSSARY
Acknowledgments
REFERENCES
3 - DATA INTERPRETATION AND COMMUNICATION
11 - Biomedical Materials and Devices
1 INTRODUCTION
2 DEFINITIONS OF BIOMATERIALS AND BIOMEDICAL DEVICES
2.1 Types of Biomaterials
2.2 Characteristics of Biomaterials and Medical Devices
3 REGULATORY GUIDELINES
3.1 Regulatory Classification of Devices
3.2 Regulatory Standards
4 SAFETY AND FUNCTIONAL ASSESSMENTS OF BIOMATERIALS AND DEVICES
4.1 Biocompatibility
4.2 Safety, Function, and Efficacy
4.3 Integration, Degradation, and Fate of Implanted Medical Materials
4.3 Integration and Degradation of Medical Materials
4.3 Determination of Complete Biodegradation
4.4 Interactions of the Body and the Device, and Biological Fates of Medical Devices
4.4 Body–Device Interactions
4.4 Device–Body Interactions
4.5 Biologic Fate of and Adverse Reactions to Medical Devices
4.6 Immunotoxicology and Immunopathology
4.7 Carcinogenicity
4.7 Metals and Carcinogenicity
4.7 Silicon and Synthetic Silicone
4.8 Histopathologic Assessment of Device/Host Tissue Interactions
4.8 Local Tissue Effects
4.8 Regional Tissue Effects
4.8 Systemic Effects
5 ORGAN SYSTEMS COMMONLY TREATED WITH BIOMATERIALS/MEDICAL DEVICES
5.1 Multisystemic Uses of Devices/Biomaterials
5.2 Cardiovascular
5.2 Vessels
5.2 Catheters
5.3 Musculoskeletal
5.3 Definitions Used in the Evaluation of Interactions of Bone Implants and Bone Substitutes
5.4 Ophthalmic Devices
5.5 Soft Tissue Repair and Wound Management
5.6 Neural and Endocrine Devices
5.7 Oral/Dental
5.8 Bioengineered Implants, Combination Devices, and Delivery Systems Combination Devices
5.9 Other Organ Systems and Devices
6 CONCLUSION
REFERENCES
REFERENCES
12 - Safety Assessment of Agricultural and Bulk Chemicals
1 INTRODUCTION
2 DEFINITION AND USE OF AGRICULTURAL CHEMICALS
2.1 Different Classes of Agricultural Chemicals
2.2 Screening Strategies for New Compound Classes
3 DEFINITION AND USE OF BULK CHEMICALS
4 SAFETY EVALUATION STRATEGIES
4.1 Regulatory Requirements for Agricultural Chemicals
4.1 North/South America
4.1 Europe
4.1 Asia-Pacific
4.2 Regulatory Requirements for Bulk Chemicals
4.2 North/South America
4.2 Europe
4.2 Asia-Pacific
5 REGULATORY STUDIES FOR REGISTRATION OF AGRO/BULK CHEMICALS
5.1 Toxicity Studies
5.2 Ecotoxicity Studies
6 TOXICOLOGIC PATHOLOGY FINDINGS AND ASSESSMENT
7 SUMMARY AND CONCLUSIONS
8 GLOSSARY
REFERENCES
13 - Preparation of the Anatomic Pathology Report for Toxicity Studies
1 INTRODUCTION
2 OBJECTIVE AND AUDIENCE FOR THE ANATOMIC PATHOLOGY REPORT
3 DEVELOPING THE DATA FOR THE ANATOMIC PATHOLOGY REPORT
3.1 Study Protocol
3.2 Importance of Assessing Correlative Data Before Beginning Microscopic Review of the Tissues
3.3 Value of Data from More than One Time Point
4 REVERSIBILITY
4.1 Presentation and Interpretation of Organ Weight Data
4.2 Presentation and Interpretation of Gross Observations
4.3 Presentation and Interpretation of Microscopic Observations
5 TEXT TABLES
6 THE ANATOMIC PATHOLOGY REPORT DISCUSSION
7 THE ANATOMIC PATHOLOGY REPORT CONCLUSION
8 SIGNING THE ANATOMIC PATHOLOGY REPORT
9 EXAMPLES TO BE AVOIDED IN INTERPRETING/PRESENTING DATA IN THE ANATOMIC PATHOLOGY REPORT
10 PEER REVIEW AND PATHOLOGY WORKING GROUPS
11 CONCLUSION
REFERENCES
REFERENCES
14 - Interpretation of Clinical Pathology Results in Nonclinical Toxicity Testing
1 INTRODUCTION
2 SAMPLE COLLECTION
3 HEMATOLOGY INTERPRETATION (ALSO SEE HEMATOPOETIC SYSTEM, VOL 5, CHAP 5)
3.1 Erythrocytes
3.2 Decreased Red Cell Mass
3.3 Blood Loss
3.4 Hemolysis
3.5 Diminished Erythropoiesis
3.6 Increased Red Cell Mass
3.7 Leukocytes
3.8 Increased Leukocytes
3.9 Decreased Leukocytes
3.10 Platelets
4 INTERPRETATION OF BONE MARROW MORPHOLOGY
5 COAGULATION INTERPRETATION
5.1 Hemostasis
5.1 Primary Hemostasis—Platelets Adhere and Aggregate
5.1 Secondary Hemostasis—Clotting, Soluble Factors Generate Insoluble Fibrin
5.1 Fibrinolysis—Clot Dissolution
5.1 Hypercoagulable and Prothrombotic States
6 CLINICAL CHEMISTRY INTERPRETATION
6.1 Glucose, Cholesterol, and Triglycerides
6.2 Blood Urea Nitrogen (Urea or BUN) and Creatinine
6.3 Total Protein, Albumin, and Albumin/Globulin Ratio
6.4 Calcium and Phosphorus
6.5 Sodium, Chloride, and Potassium
6.6 Markers of Hepatobiliary Injury or Function
6.7 Muscle Injury Markers
7 URINALYSIS AND URINE CHEMISTRY INTERPRETATION
8 NONSTANDARD BIOMARKERS
8.1 Renal Biomarkers
8.2 Hepatobiliary Biomarkers
8.3 Cardiac Biomarkers (see Cardiovascular System, Vol 5, Chap 1)
8.3 Cardiac Troponin
8.3 Creatine Kinase, Aspartate Aminotransferase, Lactate Dehydrogenase
8.3 Atrial and Brain Natriuretic Peptides
8.3 Fatty Acid–Binding Protein and Myosin Light Chain 3
8.4 Inflammatory Biomarkers
8.4 Acute Phase Proteins
8.4 Cytokine/Chemokine Evaluation
8.4 Complement
8.5 Hormones (see Endocrine System, Vol 4, Chap 7)
9 POTENTIAL EFFECTS UNRELATED TO TEST ARTICLE TREATMENT
9.1 Artifacts
9.2 Analytical Methods
9.3 Age/Sex/Genetics
9.4 Anesthesia
9.5 Blood Collection
9.6 Diet/Fasting
9.7 Medications
9.8 Fear/Pain/Stress
9.9 Environment
9.10 Sample Types, Handling, and Stability
9.11 Pregnancy, Neonatal Period, and Estrous Cycle
9.12 In Extremis/Postmortem
10 OVERALL RESULTS INTERPRETATION AND REPORT INTEGRATION
11 COMPARATOR DATA, HISTORICAL CONTROLS, AND STATISTICS
11.1 Comparator Data
11.2 Historical Controls
11.2 Statistics
12 DESCRIPTORS AND BIOLOGIC RELEVANCE
13 REPORT WRITING AND INTEGRATION
13.1 Adversity Reporting in Clinical Pathology
14 CONCLUSIONS
REFERENCES
15 - Assigning Adversity to Toxicologic Outcomes
1 INTRODUCTION AND THE NEED FOR QUANTIFYING ADVERSITY
1.1 History: Adversity, Organ Function, and the NOAEL
2 REGULATORY ASSESSMENT OF ADVERSITY IN THE EU, THE UNITED STATES, AND JAPAN
3 ADVERSE REACTIONS, ADAPTATION, AND REVERSIBILITY
3.1 Adaptation and Adversity
3.2 Reversibility and Adversity
3.3 Exacerbation of Spontaneous Pathology, Historical Control Data, and Adversity
4 THE RELATIONSHIP BETWEEN DOSE RESPONSE AND POTENCY THRESHOLDS IN DEFINING ADVERSITY
5 THE ROLE OF PATHOLOGY IN DEFINING ADVERSITY
5.1 Anatomic Pathology
5.1 Anatomic Pathology and Communicating Adversity
5.1 Additional Factors in Anatomic Pathology Influencing Adversity Decisions
5.1 Anatomic Pathology and Function
5.1 Anatomic Pathology and Severity Grading
5.1 Anatomic Pathology Peer Review in Ensuring the Relevance of Adversity Decisions
5.2 Clinical Pathology
5.2 Clinical Pathology and Functional Assessment
5.2 Clinical Pathology and Dose Response
5.2 Clinical Pathology and Reference Intervals
5.2 Clinical Pathology and the Importance of Quantification in Adversity Decisions
5.2 Clinical Pathology and the Speed and Reversibility of Responses
5.2 Clinical Pathology in Dead or Moribund Animals
5.2 Conclusions
6 CASE EXAMPLES OF ASSESSING ADVERSITY
6.1 Introduction
6.2 Thymic Involution
6.3 Liver Weight Changes
6.3 Regulatory Opinion on Adversity and Liver Weight Increases
6.3 Conclusions
6.4 Progressive Cardiomyopathy—Rat
6.4 Pathophysiology of Rodent Progressive Cardiomyopathy
6.5 Alveolar Macrophage Aggregates
6.6 Squamous Metaplasia in Larynx
6.7 Chemically Induced Exacerbation in Retinal Degeneration
6.8 Treatment-Associated Exacerbation of Chronic Progressive Nephropathy
6.9 Exacerbation of Renal Tubular Mineralization
7 GUIDELINES FOR ADVERSITY DECISIONS
8 NEW APPROACHES TO CHARACTERIZING ADVERSITY IN THE 21ST CENTURY?
8.1 The Adverse Outcome Pathway and Its Relevance to Assessing Adversity
8.2 Assessing Adversity Using Bespoke Studies and Alternative Assays
9 CONCLUSIONS
REFERENCES
16 - Risk Assessment
1 INTRODUCTION
2 CLASSICAL RISK ASSESSMENT
3 ADVANCES IN CONTEMPORARY RISK ASSESSMENT
REFERENCES
17 - Risk Management and Communication: Building Trust and Credibility With the Public
1 DEFINING RISK
2 MANAGING RISK
2.1 General Principles
2.2 Risk Management for Biopharmaceuticals
2.2 Benefit–Risk Principles
2.2 Risk Management in Clinical Development
DOSE, SAFETY MARGINS, AND EXPOSURE MULTIPLES
DETERMINATION OF HIGHEST RECOMMENDED HUMAN DOSES
SAFETY BIOMARKERS
ADDITIONAL CLINICAL DEVELOPMENT CONSIDERATIONS
2.2 Risk Management of Approved Therapeutics
POSTAPPROVAL SAFETY SURVEILLANCE
ENHANCED RISK MANAGEMENT APPROACHES
2.3 Risk Management for Nonpharmaceuticals
2.3 Principles and Considerations
2.3 Risk Management Across Sectors
ENVIRONMENTAL CHEMICALS
FOOD ADDITIVES AND DIETARY SUPPLEMENTS
COSMETICS AND PERSONAL CARE PRODUCTS
CONSUMER PRODUCTS
MEDICAL DEVICES
3 COMMUNICATING RISK
3.1 General Principles: The Science of Risk Communication
3.1 Challenges and Obstacles to Effective Risk Communication
DATA VERSUS INFORMATION
EXPECTATIONS OF SCIENCE
CONTRADICTORY EXPERT OPINIONS
3.1 The Perception and Acceptance of Risk
LIKELIHOOD (SIZE) OF THE ADVERSE EFFECT
PERMANENT OR REVERSIBLE NATURE OF THE ADVERSE EFFECT
WHO BENEFITS FROM ACCEPTANCE OF A RISK?
3.2 Targeting the Right Audience
3.2 Drug Discovery
3.2 Supporting Clinical Development
3.2 Product Approval and Marketing
3.3 Risk Communication: Pharmaceutical Safety Assessment
3.3 Risk Information for Clinical Trials
3.3 Risk Information for Approved Therapeutic Agents
3.4 Risk Communication: NonPharmaceuticals
3.4 Risk Communication Across Nonpharmaceutical Sectors
4 CONCLUSIONS
REFERENCES
Index
A
B
C
D
E
F
G
H
I
J
K
L
M
N
O
P
Q
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Back Cover

Citation preview

HASCHEK AND ROUSSEAUX’S HANDBOOK OF TOXICOLOGIC PATHOLOGY FOURTH EDITION

Volume II: Toxicologic Pathology in Safety Assessment

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HASCHEK AND ROUSSEAUX’S HANDBOOK OF TOXICOLOGIC PATHOLOGY FOURTH EDITION Volume II: Toxicologic Pathology in Safety Assessment Edited by

WANDA M. HASCHEK COLIN G. ROUSSEAUX MATTHEW A. WALLIG BRAD BOLON Associate Editors

KATHLEEN M. HEINZ-TAHENY DANIEL G. RUDMANN Illustrations Editor

BETH W. MAHLER

Academic Press is an imprint of Elsevier 125 London Wall, London EC2Y 5AS, United Kingdom 525 B Street, Suite 1650, San Diego, CA 92101, United States 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom Copyright © 2023 Elsevier Inc. All rights reserved. No part of this publication may be reproduced or transmitted in any form or by any means, electronic or mechanical, including photocopying, recording, or any information storage and retrieval system, without permission in writing from the publisher. Details on how to seek permission, further information about the Publisher’s permissions policies and our arrangements with organizations such as the Copyright Clearance Center and the Copyright Licensing Agency, can be found at our website: www.elsevier.com/ permissions. This book and the individual contributions contained in it are protected under copyright by the Publisher (other than as may be noted herein). Notices Knowledge and best practice in this field are constantly changing. As new research and experience broaden our understanding, changes in research methods, professional practices, or medical treatment may become necessary. Practitioners and researchers must always rely on their own experience and knowledge in evaluating and using any information, methods, compounds, or experiments described herein. In using such information or methods they should be mindful of their own safety and the safety of others, including parties for whom they have a professional responsibility. To the fullest extent of the law, neither the Publisher nor the authors, contributors, or editors, assume any liability for any injury and/ or damage to persons or property as a matter of products liability, negligence or otherwise, or from any use or operation of any methods, products, instructions, or ideas contained in the material herein. ISBN: 978-0-12-821047-5 For information on all Academic Press publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Stacy Masucci Acquisitions Editor: Kattie Washington Editorial Project Manager: Billie Jean Fernandez Production Project Manager: Sreejith Viswanathan Cover Designer: Matthew Limbert Typeset by TNQ Technologies

Dedication To teach is to learn .

(Japanese proverb) To our families, teachers, colleagues, and friends who have supported us in our journeys through life, encouraged us when needed, mentored us in our learning, challenged us in our teaching, joined us in our passion, followed us in our trailblazing, and inspired us in our scholarly pursuits . We are grateful for the opportunities we have enjoyed to advance pathology and toxicology as distinct and blended disciplines, both for our own betterment and in service to our local and global communities.

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Contents Contributors xv About the Editors Preface xix

8.

Regulatory Authorities 38 8.1. Overview 38 8.2. Considerations on Legal Frameworks 43 9. Summary and Conclusions 44 References 45 Selected Links to Regulatory Authorities and Issues 48

xvii

PART 1

Product Discovery and Development 1. Overview of Drug Development

2. Overview of the Role of Pathology in Product Discovery and Development

COLIN G. ROUSSEAUX, WILLIAM M. BRACKEN AND SILVIA GUIONAUD

1. 2.

3.

4.

5.

6.

7.

Introduction 3 Overview 4 2.1. The Stages of the Product Life Cycle 4 2.2. The Scope of Drug Discovery and Development 6 Drug Discovery and Development 9 3.1. Patents and Intellectual Property 10 3.2. Discovery 11 Development 14 4.1. Drug Substance and Drug Product Development (Quality) 15 4.2. Nonclinical Safety Studies 19 4.3. Animal Efficacy Studies 24 Other Health Products 26 5.1. Biologics 26 5.2. Medical Devices 28 5.3. Natural Health Products 30 5.4. Vaccines 30 Clinical Trials 31 6.1. Phase I Clinical Trials 32 6.2. Phase II Clinical Trials 34 6.3. Phase III Clinical Trials 34 6.4. Phase IV Clinical Trials 34 6.5. Limitations of Clinical Trials 35 Postmarketing Surveillance 35 7.1. Adverse Drug Events 35 7.2. Adverse Drug Reactions 35 7.3. Current Mechanisms and Tools for Identifying and Quantifying ADRs 36

JAMES FIKES, CHRISTOPHER HURST AND ERIC TIEN

1. 2.

Introduction 49 Discovery Toxicology 50 2.1. Small Molecules 50 2.2. Nucleic AcideBased Pharmaceuticals 52 2.3. Biologics 52 2.4. Cell and Gene Therapy 53 3. Development Toxicology 53 3.1. Small Molecules 53 3.2. Nucleic Acid Pharmaceuticals 54 3.3. Biologics 54 3.4. Cell and Gene Therapy 55 3.5. Anticancer Drugs 56 3.6. Adversity and Reversibility 57 3.7. Clinical Dose Setting and Clinical Safety Assessments 58 3.8. Regulatory Filings 58 4. Nonstandard Studies and End points 58 4.1. Pharmacology (Efficacy) Studies 59 4.2. Investigative (Mechanistic) Toxicology Studies 59 4.3. Standard and Alternative Animal Models 60 4.4. Biomarker Considerations 61 4.5. Interpretation of Unique Findings 62 References 63

vii

viii

CONTENTS

3. Discovery Toxicology and Discovery Pathology GLENN H. CANTOR, EVAN B. JANOVITZ AND RENE´ MEISNER

1.

Introduction 65 1.1. Discovery Toxicology 66 1.2. Discovery Pathology 67 2. Knowledge Integration and the Spanning of Disciplines 68 3. Pathology Toolbox 73 4. In Vitro/In Vivo Correlations 79 5. Target Selection 81 6. Target Validation 82 7. Translational Medicine 85 8. Hypothesis Generation, Experimental Design, and the Role of Investigative Studies 86 9. Discovery Strategy for Biologics 87 10. Communications 89 11. Personality and Behavioral Traits that Are Helpful to Succeed in Discovery Pathology and Discovery Toxicology 90 12. Summary 91 References 92

6.1. Excipients 116 6.2. Conjugation 117 6.3. Nanotechnology 118 7. Digital Pathology and Computational Pathology 119 8. Novel Investigative Tools in Nonclinical Safety Assessment 120 9. Conclusion 121 References 121

5. Carcinogenicity Assessment AARON M. SARGEANT, ARUN R. PANDIRI, KATHLEEN FUNK, THOMAS NOLTE AND KEVIN KEANE

1.

4. Pathology in Nonclinical Drug Safety Assessment MAGALI R. GUFFROY AND XIANTANG LI

1. 2. 3.

4. 5.

6.

Introduction 95 Drug Safety and Efficacy Are a Continuum 97 The Pathologist’s Role in Nonclinical Safety Assessment 100 3.1. Adverse Effects and Pathology Report 103 3.2. Reversibility and Delayed Toxicity 104 3.3. Lexicon and Diagnostic Terminology 105 3.4. GLP Regulations in Pathology 105 3.5. Pathology Peer Review 106 3.6. NOAELs and Study Report 107 Pathology in Nonclinical Safety Assessment of Small Molecules 108 Pathology in Nonclinical Safety Assessment of Biotherapeutics 109 5.1. Proteins 110 5.2. Oligonucleotides 111 5.3. Gene Therapy 113 5.4. Cell Therapy 114 5.5. Stem Cell Therapy 114 5.6. Vaccines 115 Pathology in Nonclinical Safety Assessment of Novel Formulations 116

2.

3.

4.

The Past, Present, and Potential Future of Carcinogenicity Assessment 126 1.1. Brief History of Carcinogenicity Assessment 126 1.2. Food, Drugs, and Cosmetics 127 1.3. Other Chemicals 128 1.4. Relevance of Rodent Findings in Carcinogenicity Hazard Identification Studies for Human Risk 129 1.5. Evolution from Lifetime Bioassays in Two Rodent Species to the Current Standards 130 1.6. Looking Forward: ICH Guideline S1B Modifications 131 1.7. New Approaches in Predicting Carcinogenicity Hazards 133 Purpose, Planning, Prerequisite Information, and Timing of Lifetime Carcinogenicity Studies 139 2.1. Prerequisite Data to Design a Carcinogenicity Study Protocol 140 2.2. Special Protocol Assessment for Carcinogenicity Studies 142 2.3. Carcinogenicity Study Planning Timeline 143 Two-Year Rodent Carcinogenicity Studies 144 3.1. Study Design 144 3.2. Managing High Mortality in 2-Year Carcinogenicity Studies 147 3.3. Pathology Interpretations 149 3.4. Historical Control Data 150 Alternative Genetically Modified Mouse Models 151 4.1. The Range-Finding Study 152 4.2. Carcinogenicity Study Design Using Alternative Models 153 4.3. Conduct of Carcinogenicity Studies Using Alternative Models 155

CONTENTS

5.

Carcinogenicity Assessment of BiotechnologyDerived Therapeutic Proteins 156 6. Carcinogenicity Assessment of Nucleic Acid Therapies 161 7. Carcinogenicity Assessment of Stem Cell eDerived Therapies 163 7.1. Safety Concerns for Stem CelleDerived Therapies 163 7.2. In vivo Assessment of Teratoma Formation 163 8. Carcinogenicity Assessment of Medical Devices 164 9. Conclusion 166 References 166

PART 2

Product-Specific Practices for Safety Assessment 6. Protein Therapeutics MICHAEL W. LEACH AND KATHERINE HAMMERMAN

1. 2.

3.

4.

5.

History of Protein Therapeutics 176 Types of Protein Therapeutics: Structural and Functional Considerations 176 2.1. Key Structural and Functional Elements of Antibodies and Related Products 179 General Nonclinical Toxicity Testing Strategies 183 3.1. General Considerations 183 3.2. Applicable Regulatory Guidelines 183 3.3. Target Characterization/Species Selection 185 Immunogenicity 189 4.1. Overview 189 4.2. Effect of Immunogenicity 191 4.3. Predictivity of Nonclinical Immunogenicity 191 4.4. Assessing Immunogenicity 192 4.5. Managing Immunogenicity in Toxicity Studies 194 Off-Target Profiling 195 5.1. Overview 195 5.2. History 196 5.3. Whether and When to Conduct 197 5.4. Molecules to Test 197 5.5. Species to Test 197 5.6. TCR Strategies Supporting FIH (Only for CDR-Containing Test Articles) 198 5.7. Impact and Regulatory Considerations 198

6.

General Toxicity Study Design Considerations 199 6.1. Overview 199 6.2. Exploratory (Non-GLP) Studies 199 6.3. FIH-Enabling Studies 201 6.4. Post-FIH-Enabling Studies 202 6.5. Dose Selection 202 6.6. Dosing Interval 204 6.7. Route of Administration, Formulation 204 6.8. Recovery 205 7. Immunotoxicity 206 7.1. Overview 206 7.2. In Vitro Assays for Assessing Immunotoxicity 207 7.3. In Vivo Assays for Assessing Immunotoxicity 207 7.4. Immunosuppression 208 7.5. Immunostimulation 208 7.6. Program Strategies and Regulatory Considerations 208 8. Safety Pharmacology Assessments 209 8.1. Overview 209 8.2. Strategy and Timing 209 9. Developmental and Reproductive Toxicity Studies and Juvenile Animal Studies 210 9.1. Overview 210 9.2. Species to Test and Alternative Models 211 9.3. Study Design Considerations 211 9.4. Strategies and Timing 211 9.5. Nonclinical Pediatric Evaluation 212 10. Genotoxicity 212 11. Carcinogenicity Studies 212 12. Advanced Cancer 213 13. Antibody-Drug Conjugates 213 13.1. Overview 213 13.2. Mechanisms of Toxicity 214 13.3. Nonclinical Safety Assessment 215 14. Multispecific Molecules 216 15. Biosimilars 217 16. Toxicologic Pathology of Protein TherapeuticsdPutting It All Together 217 16.1. On-Target, Off-Target, and Immunogenicity-Related Effects 217 16.2. Determining Adversity 217 Glossary 220 Acknowledgments 220 References 220

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CONTENTS

7. Nucleic Acid Pharmaceutical Agents REBECCA KOHNKEN, CAROLE HARBISON, STEPHANIE KLEIN AND JEFFERY A. ENGELHARDT

1. 2.

Introduction 231 Nucleic Acid Pharmaceuticals 232 2.1. Types of Nucleic Acid Pharmaceuticals 232 2.2. Advantages of Nucleic Acid Pharmaceuticals over Traditional Therapeutic Modalities 236 2.3. Specific Indications for Nucleic Acid Pharmaceuticals 237 2.4. Clinical Trials of Nucleic Acid Pharmaceuticals 242 3. Nucleic Acid Pharmaceutical Design and Development 242 3.1. Chemistry Considerations 242 3.2. Delivery Considerations 243 3.3. Pharmacology Considerations 249 4. Safety Evaluation of Nucleic Acid Pharmaceuticals 251 4.1. Regulatory Perspective 251 4.2. Use of Animal Models for Safety Evaluation 255 4.3. Mechanisms of Common Toxicities 256 5. Conclusions 262 Glossary 262 References 263

4.

Ex Vivo Gene Therapy 293 4.1. General Concepts of Ex Vivo Gene Therapy 293 4.2. Nonclinical Safety Assessment for Ex Vivo Gene Therapy 300 4.3. Toxicologic Pathology Considerations With Ex Vivo Gene Therapy 310 5. Genome Editing 314 5.1. Harnessing Cellular DNA Repair 314 5.2. Genome Editing Strategies 317 5.3. Nonclinical Safety Assessment for Genome Editing Products 319 6. Conclusion 325 Glossary 325 Acknowledgments 326 References 326

9. Vaccines RANI S. SELLERS AND KEITH NELSON

1. 2. 3.

4.

8. Gene Therapy and Gene Editing BASEL T. ASSAF, CLAUDIA HARPER AND JONATHAN A. PHILLIPS

1. 2.

3.

Introduction 269 General Principles of Nonclinical Research and Development for Gene Therapy Products 271 2.1. Pharmacology 272 2.2. Toxicology 272 2.3. Biodistribution and Viral Shedding 273 2.4. Role of Pathologists in the Nonclinical Assessment of Gene Therapy Products 273 In Vivo Gene Therapy 274 3.1. General Concepts of In Vivo Gene Therapy 274 3.2. AAV as a Model Platform for in vivo Gene Therapy 274 3.3. Nonclinical Pharmacology and Safety Assessment for in vivo Gene Therapy 275 3.4. Contemporary Toxicities Associated With In Vivo Gene Therapy 283

5.

6.

Introduction 336 Vaccine Immunology 337 General Concepts in Vaccine Toxicology 338 3.1. Vaccine Nonclinical Safety Package: Regulatory Expectations 338 3.2. Immunogenicity Assays 341 Vaccine Modalities 341 4.1. Protein-Based Vaccines 341 4.2. Nucleic AcideBased Vaccines 344 Vaccine Adjuvants 348 5.1. Mineral Salts 349 5.2. Surfactant/Emulsion Adjuvants 350 5.3. Nucleic Acid/Nucleotide Adjuvants 351 5.4. Lipid Adjuvants 352 5.5. Adjuvant Systems and Liposome-Based Adjuvants 352 5.6. Carbohydrate-Containing Adjuvants 353 5.7. Toxin-Based Adjuvants 353 5.8. Other Adjuvants 354 Vaccine Pharmacology and Other Studies 354 6.1. Immunogenicity and Efficacy Studies 354 6.2. Enhanced Disease and Neurovirulence Assessment 355 6.3. Biodistribution and Persistence 356 6.4. Absorption, Distribution, Metabolism, and Excretion 356

xi

CONTENTS

7.

Vaccine Safety Studies 357 7.1. Repeat-Dose Toxicity Studies 357 8. Vaccine StudiesdDesign, Technical Considerations, and Data Interpretation 359 8.1. Species Selection 359 8.2. Dose Groups 360 8.3. Technical Considerations Related to Dosing and Dose Administration 361 8.4. In-Life Assessments 366 8.5. Clinical Pathology 368 8.6. Anatomic PathologydPost-life Evaluation 370 9. Special Considerations for Therapeutic Modalities 379 9.1. Therapeutic Vaccines 379 9.2. Other Therapeutic Vaccine Strategies 380 9.3. Oncolytic Virus-Based Therapeutic Vaccines 380 10. Nonclinical ToxicitydDetermining Adversity 381 11. Nonclinical Toxicity and Human Translation 382 11.1. Autoimmunity 382 11.2. Hypersensitivity 383 11.3. Thrombotic Thrombocytopenia 383 12. Vaccines and the Anti-Vaccine Movement 384 13. Conclusions 385 14. Glossary 385 Acknowledgments 386 References 386

10. Stem Cell and Other Cell Therapies ALYS E. BRADLEY AND BRAD BOLON

1. 2.

3.

Introduction 397 Biological Principles of Cell Therapy 2.1. Cell Sources 398 2.2. Classes of Transplantable Cells 400 2.3. Primary Preclinical Safety Considerations for Cell Therapies 402 Preclinical Considerations for Cell Therapies 406 3.1. Regulatory Guidance 406 3.2. Key Endpoints for Preclinical Assessment of Cell Therapies 3.3. Design of Preclinical Safety Studies 409 3.4. The Pathologist’s Contribution 418

398

409

4. Stem CelleBased Toxicity Testing 5. Summary 419 Glossary 420 Acknowledgments 420 References 420

419

11. Biomedical Materials and Devices LYN M. WANCKET, JOANN C.L. SCHUH AND ELODIE DREVON-GAILLOT

1. 2.

Introduction 427 Definitions of Biomaterials and Biomedical Devices 428 2.1. Types of Biomaterials 428 2.2. Characteristics of Biomaterials and Medical Devices 429 3. Regulatory Guidelines 429 3.1. Regulatory Classification of Devices 429 3.2. Regulatory Standards 433 4. Safety and Functional Assessments of Biomaterials and Devices 438 4.1. Biocompatibility 438 4.2. Safety, Function, and Efficacy 438 4.3. Integration, Degradation, and Fate of Implanted Medical Materials 439 4.4. Interactions of the Body and the Device, and Biological Fates of Medical Devices 443 4.5. Biologic Fate of and Adverse Reactions to Medical Devices 445 4.6. Immunotoxicology and Immunopathology 445 4.7. Carcinogenicity 449 4.8. Histopathologic Assessment of Device/Host Tissue Interactions 450 5. Organ Systems Commonly Treated with Biomaterials/Medical Devices 455 5.1. Multisystemic Uses of Devices/ Biomaterials 455 5.2. Cardiovascular 455 5.3. Musculoskeletal 457 5.4. Ophthalmic Devices 457 5.5. Soft Tissue Repair and Wound Management 460 5.6. Neural and Endocrine Devices 461 5.7. Oral/Dental 461 5.8. Bioengineered Implants, Combination Devices, and Delivery Systems Combination Devices 462 5.9. Other Organ Systems and Devices 462 6. Conclusion 463 References 463

xii

CONTENTS

12. Safety Assessment of Agricultural and Bulk Chemicals ¨ TERS SIBYLLE GRO

1. 2.

Introduction 467 Definition and Use of Agricultural Chemicals 468 2.1. Different Classes of Agricultural Chemicals 468 2.2. Screening Strategies for New Compound Classes 469 3. Definition and Use of Bulk Chemicals 469 4. Safety Evaluation Strategies 469 4.1. Regulatory Requirements for Agricultural Chemicals 469 4.2. Regulatory Requirements for Bulk Chemicals 474 5. Regulatory Studies for Registration of Agro/Bulk Chemicals 478 5.1. Toxicity Studies 479 5.2. Ecotoxicity Studies 480 6. Toxicologic Pathology Findings and Assessment 485 7. Summary and Conclusions 487 8. Glossary 488 References 489

6.

The Anatomic Pathology Report Discussion 501 7. The Anatomic Pathology Report Conclusion 503 8. Signing the Anatomic Pathology Report 503 9. Examples to Be Avoided in Interpreting/ Presenting Data in the Anatomic Pathology Report 503 10. Peer Review and Pathology Working Groups 504 11. Conclusion 504 References 504

14. Interpretation of Clinical Pathology Results in Nonclinical Toxicity Testing ADAM D. AULBACH, DANIELA ENNULAT AND A. ERIC SCHULTZE

1. 2. 3.

PART 3

Data Interpretation and Communication 13. Preparation of the Anatomic Pathology Report for Toxicity Studies

4.

KEVIN B. DONNELLY AND MAGALI R. GUFFROY

5.

1. 2. 3.

4.

5.

Introduction 495 Objective and Audience for the Anatomic Pathology Report 496 Developing the Data for the Anatomic Pathology Report 496 3.1. Study Protocol 497 3.2. Importance of Assessing Correlative Data Before Beginning Microscopic Review of the Tissues 497 3.3. Value of Data from More than One Time Point 498 Reversibility 498 4.1. Presentation and Interpretation of Organ Weight Data 498 4.2. Presentation and Interpretation of Gross Observations 499 4.3. Presentation and Interpretation of Microscopic Observations 499 Text Tables 500

6.

7. 8.

Introduction 506 Sample Collection 507 Hematology Interpretation 507 3.1. Erythrocytes 507 3.2. Decreased Red Cell Mass 511 3.3. Blood Loss 511 3.4. Hemolysis 515 3.5. Diminished Erythropoiesis 516 3.6. Increased Red Cell Mass 517 3.7. Leukocytes 517 3.8. Increased Leukocytes 517 3.9. Decreased Leukocytes 519 3.10. Platelets 519 Interpretation of Bone Marrow Morphology 520 Coagulation Interpretation 522 5.1. Hemostasis 522 Clinical Chemistry Interpretation 525 6.1. Glucose, Cholesterol, and Triglycerides 525 6.2. Blood Urea Nitrogen (Urea or BUN) and Creatinine 526 6.3. Total Protein, Albumin, and Albumin/ Globulin Ratio 526 6.4. Calcium and Phosphorus 527 6.5. Sodium, Chloride, and Potassium 528 6.6. Markers of Hepatobiliary Injury or Function 528 6.7. Muscle Injury Markers 530 Urinalysis and Urine Chemistry Interpretation 532 Nonstandard Biomarkers 533 8.1. Renal Biomarkers 534 8.2. Hepatobiliary Biomarkers 536 8.3. Cardiac Biomarkers 537

xiii

CONTENTS

8.4. Inflammatory Biomarkers 538 8.5. Hormones 540 9. Potential Effects Unrelated to Test Article Treatment 542 9.1. Artifacts 542 9.2. Analytical Methods 542 9.3. Age/Sex/Genetics 545 9.4. Anesthesia 547 9.5. Blood Collection 548 9.6. Diet/Fasting 549 9.7. Medications 550 9.8. Fear/Pain/Stress 551 9.9. Environment 552 9.10. Sample Types, Handling, and Stability 552 9.11. Pregnancy, Neonatal Period, and Estrous Cycle 553 9.12. In Extremis/Postmortem 555 10. Overall Results Interpretation and Report Integration 555 11. Comparator Data, Historical Controls, and Statistics 555 11.1. Comparator Data 555 11.2. Historical Controls 556 12. Descriptors and Biologic Relevance 556 13. Report Writing and Integration 557 13.1. Adversity Reporting in Clinical Pathology 557 14. Conclusions 558 References 558

15. Assigning Adversity to Toxicologic Outcomes JOHN REGINALD FOSTER AND JEFFERY A. ENGELHARDT

1.

2. 3.

4. 5.

Introduction and the Need for Quantifying Adversity 567 1.1. History: Adversity, Organ Function, and the NOAEL 568 Regulatory Assessment of Adversity in the EU, the United States, and Japan 571 Adverse Reactions, Adaptation, and Reversibility 571 3.1. Adaptation and Adversity 571 3.2. Reversibility and Adversity 574 3.3. Exacerbation of Spontaneous Pathology, Historical Control Data, and Adversity 574 The Relationship Between Dose Response and Potency Thresholds in Defining Adversity 575 The Role of Pathology in Defining Adversity 577

5.1. Anatomic Pathology 577 5.2. Clinical Pathology 582 6. Case Examples of Assessing Adversity 585 6.1. Introduction 585 6.2. Thymic Involution 585 6.3. Liver Weight Changes 587 6.4. Progressive CardiomyopathydRat 593 6.5. Alveolar Macrophage Aggregates 595 6.6. Squamous Metaplasia in Larynx 597 6.7. Chemically Induced Exacerbation in Retinal Degeneration 599 6.8. Treatment-Associated Exacerbation of Chronic Progressive Nephropathy 600 6.9. Exacerbation of Renal Tubular Mineralization 602 7. Guidelines for Adversity Decisions 603 8. New Approaches to Characterizing Adversity in the 21st Century? 604 8.1. The Adverse Outcome Pathway and Its Relevance to Assessing Adversity 605 8.2. Assessing Adversity Using Bespoke Studies and Alternative Assays 607 9. Conclusions 607 References 608

16. Risk Assessment STEPHEN K. DURHAM, DANIEL G. RUDMANN, KEEGAN C. RUDMANN AND JAMES A. SWENBERG

1. 2. 3.

Introduction 617 Classical Risk Assessment 618 Advances in Contemporary Risk Assessment 624 References 627

17. Risk Management and Communication: Building Trust and Credibility With the Public JOHN L. VAHLE, VIRUNYA BHAT AND CHARLES E. WOOD

1. 2.

Defining Risk 629 Managing Risk 632 2.1. General Principles 2.2. Risk Management for Biopharmaceuticals 2.3. Risk Management for Nonpharmaceuticals

632 633 639

xiv 3.

CONTENTS

Communicating Risk 644 3.1. General Principles: The Science of Risk Communication 644 3.2. Targeting the Right Audience 647 3.3. Risk Communication: Pharmaceutical Safety Assessment 649

3.4.

Risk Communication: NonPharmaceuticals 4. Conclusions 653 References 653

Index

657

651

Contributors Sanofi, Inc., Cambridge, MA, United States

Basel T. Assaf

Evan B. Janovitz Nonclinical Research and Development, Bristol Myers Squibb Co., New Brunswick, NJ, United States Kevin Keane Novo Nordisk Research Park Ma˚løv, Denmark

Virunya Bhat Independent Consultant, San Diego, CA, United States Brad Bolon

Stephanie Klein Antisense Drug Discovery, Ionis Pharmaceuticals, Inc., Carlsbad, CA, United States

GEMpath, Inc., Longmont, CO, United States

William M. Bracken LifeCare Medical Strategies, Mundelein, IL, United States

Rebecca Kohnken Preclinical Safety, AbbVie Inc., North Chicago, IL, United States

Alys E. Bradley Charles River Laboratories, Edinburgh, Scotland, United Kingdom Glenn H. Cantor United States

Biogen, Cambridge, MA, United States

Christopher Hurst

Inotiv, Maryland Heights, MO, United

Adam D. Aulbach States

Michael W. Leach

Pfizer Inc., Cambridge, MA, United States

Xiantang Li Drug Safety Research and Development, Pfizer Inc., Groton, CT, United States

Glenn Cantor Consulting, LLC, Bend, OR,

Kevin B. Donnelly Eli Lilly and Company, Indianapolis, IN, United States

Rene´ Meisner Denali Therapeutics, South San Francisco, CA, United States

Elodie Drevon-Gaillot Charles River Lyon SA, Lyon, St. Germain-Nuelles, France

Keith Nelson States

Stephen K. Durham CO, United States

Thomas Nolte Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany

Indepedent Consultant, Fort Collins,

Arun R. Pandiri Molecular Pathology Group, Comparative and Molecular Pathogenesis Branch, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Durham, NC, United States

Jeffery A. Engelhardt Independent Consultant (formerly with Ionis Pharmaceuticals, Carlsbad, CA), CA, United States Daniela Ennulat States James Fikes

GlaxoSmithKline, Collegeville, PA, United

Biogen, Cambridge, MA, United States

John Reginald Foster ToxPath Sciences Ltd. and Ionis Pharmaceuticals, Carlsbad, CA, United States

Intellia Therapeutics, Cambridge, MA,

Colin G. Rousseaux Canada

University of Ottawa, Ottawa, ON,

Keegan C. Rudmann University of Colorado Anschutz Medical Campus, Colorado School of Public Health, Aurora, CO, United States; Banner Health Ft. Collins Medical Center, Ft. Collins, CO, United States

Sibylle Gro¨ters Pathology, Experimental Toxicology and Ecology, BASF SE, Ludwigshafen, Germany Magali R. Guffroy Preclinical Safety, AbbVie Inc., North Chicago, IL, United States

Aaron M. Sargeant Charles River Laboratories, Spencerville, OH, United States

Silvia Guionaud Guionaud Nonclinical Consulting, Ash, Kent, United Kingdom

JoAnn C.L. Schuh United States

Pfizer Inc., Cambridge, MA, United

A. Eric Schultze United States

Carole Harbison Drug Safety Research and Evaluation, Takeda Pharmaceuticals, Cambridge, MA, United States Claudia Harper States

Jonathan A. Phillips United States

Daniel G. Rudmann Charles River Laboratories, Ashland, OH, United States

Kathleen Funk Experimental Pathology Laboratories, Inc., Sterling, VA, United States

Katherine Hammerman States

Charles River Labs, Mattawan, MI, United

JCL Schuh, PLLC, Bainbridge Island, WA, Eli Lilly and Company, Indianapolis, IN,

Rani S. Sellers University of North Carolina, Chapel Hill, NC, United States

Orna Therapeutics, Cambridge, MA, United

xv

xvi

CONTRIBUTORS

James A. Swenberg University of North Carolina, Chapel Hill, NC, United States Eric Tien

Biogen, Cambridge, MA, United States

John L. Vahle Lilly Research Laboratories, Indianapolis, IN, United States

Lyn M. Wancket States

Charles River, PAI, Durham, NC, United

Charles E. Wood United States

Boehringer Ingelheim, Ridgefield, CT,

About the Editors

EDITORS Wanda M. Haschek-Hock, BVSc, Ph.D., is a Diplomate of the American College of Veterinary Pathologists (DACVP), Fellow of the International Academy of Toxicologic Pathology (FIATP), and Honorary Member of the Latin American Society of Toxicologic Pathology. She is Professor Emerita at the University of Illinois College of Veterinary Medicine, Department of Pathobiology. Wanda has over 40 years of experience in comparative, respiratory, and toxicologic pathology with a research focus on natural toxins and food safety. She is a Former President of the Society of Toxicologic Pathology (STP) and of the Society of Toxicology’s (SOT) Comparative and Veterinary Specialty Section and has served as an Associate Editor for Toxicological Sciences and Toxicologic Pathology, as Councilor of the American College of Veterinary Pathologists (ACVP), and as Member of the American Board of Toxicology (ABT). She served as an Editor for the three editions of the Fundamentals of Toxicologic Pathology and Haschek and Rousseaux’s Handbook of Toxicologic Pathology. She is a recipient of the STP’s Lifetime Achievement Award, the SOT Midwest Regional Chapter’s Kenneth DuBois Award, and the University of Sydney Faculty of Veterinary Science Alumni Award for International Achievement in 2016. Colin G. Rousseaux, BVSc, Ph.D., DABT, FIATP, is also a Fellow of the Royal College of Pathology (FRCPath) and Fellow of the Academy of Toxicological Sciences (FATS). He is a Professor (Adjunct) in the Department of Pathology and Laboratory Medicine, Faculty of Medicine, University of Ottawa, Canada. He has over 35 years of experience in comparative and toxicologic pathology with a research focus on herbal remedies, fetal development and teratology, and environmental pollutants. He has described, investigated, and evaluated numerous toxicologic pathology issues associated with pharmaceuticals, pesticides, and agrochemicals. He has served on the editorial board of Toxicologic Pathology. He is a Former President of the STP. Colin served as an Editor for the three editions of the Fundamentals of Toxicologic Pathology and Haschek and Rousseaux’s Handbook of Toxicologic Pathology.

Matthew A. Wallig, DVM, Ph.D., DACVP, is Professor Emeritus in the Department of Pathobiology, College of Veterinary Medicine, the Department of Food Science and Human Nutrition, as well as the Division of Nutritional Sciences at the University of Illinois. His research has focused on the chemoprotective properties and mechanisms of phytochemicals in the diet, in particular those in cruciferous vegetables, soy, and tomatoes. His current interests have expanded to include defining morphologic parameters for diagnostic quantitative ultrasound in pancreatitis, pancreatic and hepatic neoplasia, metastatic disease, and chronic hepatic diseases such as nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH). Matt has served as an Editor for the last two editions of the Fundamentals of Toxicologic Pathology and Haschek and Rousseaux’s Handbook of Toxicologic Pathology. Brad Bolon, DVM, MS, Ph.D., DAVCP, DABT, FATS, FIATP, FRCPath, has worked [sic] as an experimental and toxicologic pathologist in several settings: academia, a contract research organization, pharmaceutical companies (in both biomolecule and traditional small molecule settings), and private consulting. His main professional interests are the pathology of genetically engineered mice (especially embryos, fetuses, and placentas) and toxicologic neuropathology to assess the efficacy and safety of many therapeutic entities (biomolecules, cell and gene therapies, medical devices, and small molecules). He is a Former President of the STP and a Member of the American College of Toxicology (ACT), British Society of Toxicological Pathology (BSTP), and European Society of Toxicologic Pathology (ESTP). Brad served as an Editor for the third edition of the Fundamentals of Toxicologic Pathology and an Associate Editor for the third edition of Haschek and Rousseaux’s Handbook of Toxicologic Pathology.

ASSOCIATE EDITORS Kathleen M. Heinz-Taheny, DVM, Ph.D., DACVP, DABT, currently serves as a Senior Research Advisor at Lilly Research Laboratories. She has 15 years of experience as a pathologist and toxicologist supporting pharmaceutical safety assessment and drug discovery

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ABOUT THE EDITORS

research. Katie’s interests include renal pathology and rodent renal models, immunohistochemistry including tissue cross-reactivity studies, and digital pathology along with image analysis. She has served multiple offices including President of the Association of Reptile and Amphibian Veterinarians and served as Cochair of the 2018 STP conference “Keeping it Renal,” as well as chair of the STP Annual Symposium Committee. In 2019, Katie codirected the Industrial Toxicology and Pathology Short Course with Wanda Haschek-Hock. Daniel G Rudmann, DVM, Ph.D., DACVP, FIATP, is currently a Scientific Director at Charles River Laboratories, Inc. He has 25 years of experience as a pathologist and toxicologist in pharmaceutical discovery and development across therapeutic modalities and has expertise in digital pathology and machine learning, and reproductive toxicologic pathology. He has served as an Associate Editor for the journal Toxicologic Pathology and as Chair or Cochair for various STP committees.

ILLUSTRATIONS EDITOR Beth W. Mahler is employed by Experimental Pathology Laboratories, Inc., located in Research Triangle Park, NC, and works as a contractor at the U.S. National Institute of Environmental Health Sciences (NIEHS) in the Cellular and Molecular Pathology Branch under the Division of the National Toxicology Program (NTP). She has over 42 years of experience as a certified histologist (HT) in the areas of histology, animal necropsy, embryo collection and sectioning, and digital photomicroscopy. Since 2006, she has served as the Illustrations Editor for the journal Toxicologic Pathology. Past illustrative editorship roles include Associate Editor of Pathology of the Mouse, edited by Dr. Robert R. Maronpot, and Illustrations Editor for previous editions of the Fundamentals of Toxicologic Pathology and Haschek and Rousseaux’s Handbook of Toxicologic Pathology.

Preface

Since its inception 3 decades ago, Haschek and Rousseaux’s Handbook of Toxicologic Pathology has been a comprehensive resource covering fundamental knowledge and skills as well as key technical procedures essential for the proficient practice of toxicologic pathology. The reference has found a home in the libraries of numerous academic, government, and industrial institutions engaged in basic and applied biomedical research around the globe, and in doing so has become an indispensable reference for many toxicologic pathologists, toxicologists, regulators, physicians, biomedical researchers, and students. Indeed, the Handbook has been recognized by many as the most authoritative single source of information in the field due to the breadth and depth of coverage for this field. Prior to publication of the inaugural Handbook edition in 1991, information regarding toxicologic pathology had to be gleaned in a piecemeal manner by reading articles in various toxicology and the few toxicologic pathology journals. The success of the one-volume 1st edition and the expanded roles of toxicologic pathologists over time led in due course to the release of updated Handbook versions in subsequent years: a two-volume 2nd edition in 2002 and a three-volume 3rd edition in 2013. The many scientific advances, ongoing and extensive collaboration among global societies of toxicologic pathology, and new regulatory guidance that have occurred since release of the 3rd edition amply prove that another rendition is necessary to maintain, and indeed enhance, the value of this resource to practitioners of the toxicologic pathology craft. For this reason, the Editors and Associate Editors are pleased to offer this new and expanded Handbook to aid your explorations of the toxicologic pathology field. This 4th edition of Haschek and Rousseaux’s Handbook of Toxicologic Pathology has been extensively updated to continue its comprehensive coverage. This update required extensive expansion, which was initially conceived as a four-volume text. However, the unrelenting explosion of information in this field instead has necessitated that this new edition be rendered as a fivevolume set where the contents of Volume 2 of the 3rd edition now split into Volumes 2 and 3 in this version. For the 4th edition, the new five-volume design distributes important concepts in the following way.

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• Volume 1 (released in 2022) covers “Principles and the Practice of Toxicologic Pathology,” including such key topics as basic concepts in biology, pathology, pharmacology, and toxicology as they intersect in the field of toxicologic pathology (Chapters 2e8); primary methods in toxicologic pathology (Chapters 9e16); overviews of major models employed in toxicologic research (Chapters 17e24); and essential practices in generating and interpreting toxicologic pathology data (Chapters 25e29). New chapters in Volume 1 address absorption, distribution, metabolism, and excretion (ADME) principles for biomolecules; toxicologic pathology considerations in developmental and reproductive toxicity (DART) testing; digital pathology; and various animal models including rodents, rabbits, dogs, nonhuman primates, and alternative models (e.g., in silico, ex vivo). Because of the recent decision to split Volume 2, leading to a five-volume 4th edition, chapter cross-referencing in Volume 1 (by volume and chapter number) may be incorrect when referring to contents of other volumes. • Volume 2 addresses “Toxicologic Pathology in Safety Assessment.” Safety assessment covers the application of toxicologic pathology in developing specific product classes (Chapters 1e12) and principles of data interpretation (Chapters 13e17). New chapters in this section consider fundamental attributes of novel therapeutic classes (nucleic acidand protein-based pharmaceutical agents, gene therapy and gene editing, stem cell and other cell therapies, medical devices, vaccines); agricultural and bulk chemicals; and differentiation of adverse from nonadverse effects. These chapters address both pathology and regulatory issues. Previous chapters on such topics as drug discovery and development, toxicity and carcinogenicity testing, report preparation, and risk assessment and communication have undergone extensive revision that includes in-depth discussion of new developments in the field. • Volume 3 addresses “Environmental Toxicologic Pathology and Selected Toxicant Classes.” This volume covers toxicologic pathology as it relates to food, nutrition, and selected toxicant classes in the

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environment. New chapters include herbal products, and animal- and bacteria-derived toxins; for these chapters, particular attention has been paid to the role of such substances in formulating new therapies (either as the active agent or as contaminants of the manufacturing process). All previous chapters (e.g., endocrine disruptors, heavy metals, nanoparticulates, poisonous plants, radiation) have been extensively revised and updated. • Volumes 4 and 5 provide deep and broad treatment of “Target Organ Toxicity” for major systems, emphasizing the comparative and correlative aspects of normal biology and toxicant-induced dysfunction, principal methods for toxicologic pathology evaluation, and major mechanisms of toxicity. New topics incorporated in Volume 4 include the toxicologic pathology of adipose tissue, tendons, and teeth. New information in the organ system chapters for both volumes will address product development and regulatory issues. The expanded coverage of nonclinical testing principles and practices, the new material on various models of toxicity, and the toxicologic pathology of novel toxicant classes (especially new therapeutic modalities) will be a particular strength of this new Handbook edition and should and will continue to justify its long-standing reputation for excellence. The Editors do accept that

information relevant to toxicity research, hazard identification, and risk assessment and management is ever growing, and they acknowledge that readers may benefit by extending their search for up-to-the-minute information in this area to include other textbooks and journals of toxicology and toxicologic pathology. We would like to thank the dedicated efforts of the Associate EditorsdStacey Fossey and John Vahle (Volume 1), Katie Heinz-Taheny and Dan Rudmann (Volumes 2 and 3), Molly Boyle and Mark Hoenerhoff (Volume 4), and Jeff Everitt and Karen Regan (Volume 5); our incomparable Illustrations Editor, Beth Mahler; and the many authors for their outstanding contributions in bringing this book to fruition. In addition, we wish to specifically acknowledge the significant involvement of many leading toxicologic pathologists as essential subject matter experts in contributing to this and previous editions; many are now retired, and a growing number are deceased including our friends (and mentors) Charles C. Capen, Victor J. Ferrans, Gordon C. Hard, Adalbert Koestner, Robert W. Leader, Daniel Morton, John F. Van Vleet, and Hanspeter R. Witschi. Wanda M. Haschek Colin G. Rousseaux Matthew A. Wallig Brad Bolon

P A R T 1

PRODUCT DISCOVERY AND DEVELOPMENT

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C H A P T E R

1 Overview of Drug Development Colin G. Rousseaux1, William M. Bracken2, Silvia Guionaud3 1

University of Ottawa, Ottawa, ON, Canada, 2LifeCare Medical Strategies, Mundelein, IL, United States, 3Guionaud Nonclinical Consulting, Ash, Kent, United Kingdom O U T L I N E

1. Introduction

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2. Overview 2.1. The Stages of the Product Life Cycle 2.2. The Scope of Drug Discovery and Development

4 4 6

3. Drug Discovery and Development 3.1. Patents and Intellectual Property 3.2. Discovery

9 10 11

4. Development 14 4.1. Drug Substance and Drug Product Development (Quality) 15 4.2. Nonclinical Safety Studies 19 4.3. Animal Efficacy Studies 24 5. Other Health Products 5.1. Biologics 5.2. Medical Devices 5.3. Natural Health Products 5.4. Vaccines

26 26 28 30 30

31 32 34 34 34 35

7. Postmarketing Surveillance 7.1. Adverse Drug Events 7.2. Adverse Drug Reactions 7.3. Current Mechanisms and Tools for Identifying and Quantifying ADRs

35 35 35

8. Regulatory Authorities 8.1. Overview 8.2. Considerations on Legal Frameworks

38 38 43

9. Summary and Conclusions

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References

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As recent advances in pharmaceutical research and development are increasingly sciencedriven, laws and regulatory agency guidance have evolved to accommodate this innovation. While drug development occurs globally, it is driven by research and development mainly in the United States as more compounds are in development in the United States than all other regions of the world combined (Keyhani et al., 2010; OECD, 2017). The following chapter is divided into a number of sections addressing the discovery and

1. INTRODUCTION Discovery and development of new medicines (e.g., biomolecules and small molecules) and other therapeutic products (e.g., cell and gene therapies, medical devices, vaccines) is a long and high-risk process that has become more costly and complex over time, even as technology and scientific understanding of disease targets has improved. For drug candidates that reach late-stage clinical development, the probability of success remains low (Hay et al., 2014).

Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-12-821047-5.00016-6

6. Clinical Trials 6.1. Phase I Clinical Trials 6.2. Phase II Clinical Trials 6.3. Phase III Clinical Trials 6.4. Phase IV Clinical Trials 6.5. Limitations of Clinical Trials

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Copyright Ó 2023 Elsevier Inc. All rights reserved.

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1. OVERVIEW OF DRUG DEVELOPMENT

development process of a drug, the laws and regulations on pharmaceutical development in the United States and Europe, and a selection of other countries, and some tips for pathologists and toxicologists regarding the review of data in some regulatory submissions. This discussion is broad but not totally comprehensive, so the reader is directed to the websites that are given in suggested reading at the end of this chapter. The same concepts will apply to nondrug products, although the laws and regulations for such entities will differ to some or a considerable degree from those used for drug discovery and development. The differences related to developing these other product types are not detailed in this chapter.

2. OVERVIEW Imagine that you have discovered a promising New Chemical Entity (NCE), also sometimes referred to as a New Active Substance (NAS), in your laboratory. You are thinking that now you will be able to retire with loads of money arising from the sales of your newfound drug. Sorry to disappoint you! The route from the bench to the market is very time consuming, risky, and expensive. Drug development used to be viewed as only developing the drug for market. That is, once an approval (New Drug ApprovaldU.S. Food and Drug Administration [FDA]) or marketing authorization (European Marketing Authorisation ApplicationdEuropean Medicines Agency [EMA]) is given, the drug is placed on the market for sale. Drugs are now viewed as products that have their own life cycle. For this reason, the life of a health product has a beginning, middle, and end. Each stage of the life cycle is highly complex, requiring expertise from many professionals. The purpose of taking a life cycle approach to drug development is to maximize the return on investment while maintaining the health and safety of the public (Smith and O’Donnell, 2006). Factors influencing the life cycle of a drug include duration of patent exclusivity, indication, and formulation, among others. Patent exclusivity is for a limited duration, so there is a constant race to get products to market to maximize profit before patents expire. So how does the product life cycle relate to health products? There are two main relationships that face the pharmaceutical manufacturer

regarding the drug life cycle management, one of which can be seen as a cost to the manufacturer and the other a benefit to the patient and return on investment for the manufacturer. Postmarketing surveillance (safety and effectiveness assessment during exposure of the population to the drug) is the primary responsibility of the manufacturer, but healthcare professionals are also involved in reporting Adverse Drug Reactions (ADRs). Manufacturers must provide Periodic Safety Update Reports (PSURs) to regulatory authorities for evaluation. The reporting period commences using six month intervals and then decreases as the experience with the drug on the market increases. Manufacturers may be requested to do further studies concerning safety and effectiveness of their drug under specific conditions. The second manner in which the product life cycle relates to drugs is the discovery of new indications or altered formulations that will give the manufacturer increased return on their investments, and new pharmaceutical modalities for the population (Ellery and Hansen, 2012). The cardiac drug minoxidil and the antidepressant bupropion are examples of this phenomenon. Minoxidil is now available to assist in reduction of male pattern alopecia as Rogaine, and bupropion is now available as a smoking cessation aid as Zyban. For each novel use, a new product life cycle starts. The indication for the drug use and health claims triggers the regulatory need, under the appropriate statute, for scientific evidencebased data showing quality, safety, and efficacy of the health product in relation to its particular intended use and route. This triad must be adequate to receive a marketing authorization.

2.1. The Stages of the Product Life Cycle All products and services have certain life cycles, including biopharmaceuticals. The life cycle refers to the period from the product’s first launch into the market until its final withdrawal, and it is split into phases. During this life cycle, significant changes are made in the way that the product is delivered to the market. Since an increase in profits is the major goal of any company that introduces a product into a market, the product’s life cycle management is very important. The product’s life cycle consists of five major steps or phases: product development, product

I. PRODUCT DISCOVERY AND DEVELOPMENT

2. OVERVIEW

introduction, product growth, product maturity, and finally product decline (Stark, 2019). These phases are applicable to all products or services from a certain make of automobile to a drug made by a multibillion-dollar pharmaceutical company. Product Development Stages The product development phase generally includes discovery, nonclinical development (before the first administration to humans), and clinical development (where the product candidate is introduced into humans and the program is carried through to marketing registration). It begins when a company finds and develops a new product concept, which leads to drug discovery (see Overview of the Role of Pathology in Product Discovery and Safety Assessment, Vol 2, Chap 2, Discovery Toxicology and Discovery Pathology, Vol 2, Chap 3). In addition to toxicologic pathology, this stage

FIGURE 1.1

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involves many disciplines and generates an extraordinarily large amount of data. The registration document incorporates all of this information into a new health product submission. Once discovery has been successful, development occurs where the toxicologic pathologist’s work is core to nonclinical drug development (see Pathology in Nonclinical Drug Safety Assessment, Vol 2, Chap 4). Clinical development typically is broken into stages: Phase I (initial testing in human volunteers to assess kinetics and safety), Phase II (efficacy and safety testing in human patients), Phase III (extensive efficacy and safety testing in human patients), and Phase IV (postmarketing monitoring, often with additional efficacy and safety testing). During product development, sales are zero and revenues are negative: it is the time of spending with absolutely no return despite a fair degree of expense (Figure 1.1).

The life cycle of pharmaceutical products.

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1. OVERVIEW OF DRUG DEVELOPMENT

Product Introduction Once a marketing authorization has been obtained, the introduction phase of a product is called the product launch. Marketing materials are usually prepared in the scientific community through a publication strategy that starts during early (Phase I) clinical development. This period can be described as a money sinkhole compared to the maturity phase of a health product. During this phase, the initial pricing is established based on the cost of development, medical need, impact of the drug on the condition, the competitive environment, and a consideration of the payer network (insurers and/or government). The product introduction is an important investment period for the company that will impact the profits when competitors enter the market; those first to market will retain the most profit for the duration of sale. Growth The growth phase offers the satisfaction of seeing the product take off in the marketplace with appropriate timing to focus company resources on increasing the market share. Managing the growth stage is essential for product success. If the product has entered the market (“first-in-class” drugs) ahead of competitor products, then it is in a position to gain and maintain market share relatively easily. However, a new and growing market alerts the competition’s attention. A frequent modification of the drug, such as new delivery methods, is an effective strategy to discourage competitors from gaining market share by copying or generating similar drugs. Other barriers to competition are licenses and copyrights, product complexity, and low availability of product components. Promotion and advertising continue, but not to the extent that was done during the introduction to the market. At this stage, product outreach is oriented to the task of market leadership and not in raising product awareness. A good practice is the use of external promotional contractors, such as drug representatives, and presentation and discussion with formulary managers. This period is the time to develop efficiencies and improve drug availability and service via good coverage in as many critical markets as possible. Maturity Eventually, the market becomes saturated with variations of the drug, and other competitors are represented in terms of an alternative product (“me-too” drugs). In this maturity phase, market share growth is at the expense of someone else’s

business rather than the growth of the market itself. This is the period of the highest returns from the product, where a biopharmaceutical company that has achieved its market share goal enjoys the most profitable time while a company that falls behind its market share goal must reconsider its marketing positioning. Decline During this period of decreased market share, new brands are introduced even when they compete with the company’s existing product. Hence, this is the time to extend the product’s life by producing generic (for small molecules) or biosimilar (for biomolecules) drugs. Once the product reaches the end of the exponential growth phase, it becomes a “cash cow” reaping maximum return on investment. In the decline phase, the product is still being sold but with lower expectations of profit, particularly due to the advent of generic drugs from other drug companies. This may mean that the commercially named product is still sold but has to compete in price with the generics. The product is not necessarily withdrawn. The decision to withdraw a product is a complex task, and many issues need to be resolved before a decision is made to move it out of the market. The obvious causes of withdrawal from the market are associated mainly with safety issues, and sometimes effectiveness. Sometimes it is difficult for a company to recognize the decline signals of a product, although most biopharmaceutical companies are aware of this issue.

2.2. The Scope of Drug Discovery and Development Three parallel streams of research and development interweave at various points during the product life cycle supporting the assessment of efficacy, safety, and drug product quality. Scientific evidence verifying efficacy, safety, and drug product quality is developed and presented by a sponsor, the biopharmaceutical company, to regulatory agencies, and the full body of evidence is the basis for assuring regulators and patients that the product is efficacious as described in the label, has safety consistent with the scope of its use, and can be consistently manufactured from batch to batch. Laws and regulations have been promulgated across the globe to guide this regulatory process. While national differences exist, there is increasing cooperation among developed countries aimed at supporting

2. OVERVIEW

timely introduction of new products across regions. For drugs and biologics, key engines of global cooperation in this regard include the ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH, 2022a) for small molecule and biomolecule drugs; the corresponding purveyor for medical devices is the ISO (International Organization for Standardization (ISO, 2022) and the Organisation for Economic Cooperation and Development (OECD) test guidelines for chemicals (OECD, 2022). The basic development cycle can be viewed from both the sponsor and regulatory perspectives. Sponsors define a strategy and develop plans for implementation geared toward convincing regulatory agencies of a therapy’s efficacy and safety. Regulatory agencies critically review the output of the development program, providing criticism and guidance regarding the Sponsor’s strategy and data package. Negotiations between the two parties lead to approval and commercialization of a product. Regulators have gone to great lengths, in the last decade or so, to create alternate routes of development that accelerate speed to market and decrease costs for the sponsor. From a sponsor’s perspective, successful negotiations with regulators and management that shave time and cost from the development life cycle of a product lead to increased profitability and the ability to reinvest in research and development (R&D). From the regulatory authority perspective, the goal of product development is to assure that the key requirements for a product are metdefficacy, safety, and qualitydwhile worrying less about increasing the speed of product to consumer (except in circumstances when the regulatory agencies are under pressure to develop solutions to pressing national issues, as in the case of developing vaccines against the SARS-CoV-2 virus [sudden acute respiratory syndrome–coronavirus 2] responsible for the COVID-19 pandemic of 2020–21). Meeting the needs of sponsors and regulatory authorities is a complex process. The Challenge Drug development is a precarious business with high risks. The risks are admittedly large, but the higher the risks the greater the corporate rewards and also the societal benefits. Bio/pharmaceutical (“biopharma”) companies traditionally performed both discovery and development research, but an increasingly common model is for large pharma companies to invest in drug discovery by smaller

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start-up pharma and biotechnology (“biotech”) companies (to control research overhead and diversify their product portfolios) and then license intellectual property into the large pharma’s pipeline for development. Although risky, major pharmaceutical companies typically are involved in the new drug development process as a constant influx of new products is essential for success and survival. Biopharmaceutical companies worldwide invest billions of dollars every year to develop new drugs (Goozner, 2004). The trick for maximizing the value of such enormous investments is to increase the odds of success through knowledge, planning, strategic management, and cunning marketing. Cost, Time, and Risks in Developing New Drugs Drug development takes longer from discovery to marketing approval compared to other products and requires mammoth investment from pharmaceutical companies. The cost of developing a single new drug including commercialization has been estimated to range from US $1 billion (Wouters et al., 2020) to US $2.8 billion (DiMasi et al., 2016). The cost of development is also escalating rapidly (OECD, 2017), with an estimated rise annually by 8.5% more than the general inflation rate. It is quite possible that during any stage of development, a drug under review may not make it to the next stage due to reasons such as product quality, safety, or efficacy, thereby increasing the overall cost of development. For every 1000 compounds that are identified by a company, only one drug enters the market. It may take anywhere between 10 and 15 years to develop this single drug (Tamimi and Ellis, 2009). The time to develop a drug is attributed to the numerous investigative steps a drug undergoes before it is ultimately launched in the market (Figure 1.2). The money invested and lost by the company for molecules that fail at some premarketing stage of development is a cost that cannot be recovered, and that cost varies substantially depending on when in the development process of the drug candidate is abandoned. The contribution of the toxicologic pathologist is paramount in terminating development of unsafe entities as early as possible (i.e., “fail fast”) to avoid unnecessary expense that will siphon money away from development programs for other still-viable drug candidates. Not every drug candidate that is developed is marketed. For every 1000 new entities that are

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1. OVERVIEW OF DRUG DEVELOPMENT

FIGURE 1.2

Discovery, development, and marketing authorization.

identified by a company, only about 30 (3%) show promising results (as defined by acceptable kinetic properties and efficacy when formulated in a manner suitable for administration). For every 30 compounds that show promise, only 3 get past the first round of clinical trials and only 1 drug finally enters the market. Compounds usually do not pass the initial development process due to undesirable properties (e.g., toxicity, tolerance, lack of efficacy, and lack of bioavailability) and/or a lack of exclusivity of the drug in humans. Sometimes drugs do not receive regulatory approval. Thus, to introduce one new drug, a company needs to start with many thousands of compounds in drug discovery (see Discovery Toxicology and Discovery Pathology, Vol 2, Chap 3). What this means is that each successful company aims to have one or more “blockbuster” drugs both in the development pipeline and in the market at the same time (Karamehic et al., 2013); blockbuster drugs are drugs that generate annual sales of at least $1 billion

and obtain a majority of the market share (e.g., Lipitor [atorvastatin]). As the concept of “precision medicines” gains favor, where drug use is targeted to specific patient populations as defined by particular genes and proteins, it is unclear whether refined targeting of drugs to subsets of patients (i.e., only those who will clearly benefit from the treatment) will lead to blockbuster-level sales as occurs with more conventional drug use, where many patients are treated but some do not receive the desired level of benefit). Recently, new models have focused on rare disease indications “orphan” where the development cycle is shorter, helping companies diversify their portfolios. Time Required to Develop a New Drug Drug development takes years, but the length of time needed for each phase varies substantially. An “average” timeline includes about 6.5 years of discovery and nonclinical testing (efficacy and toxicity studies); 1.5 years in Phase I clinical

3. DRUG DISCOVERY AND DEVELOPMENT

trials to assess safety in healthy volunteers; then 2 years in Phase II clinical trials with a few hundred patients to evaluate the drug’s effectiveness and side effects. The development process continues with 3.5 years in Phase III clinical trials involving thousands of patients and scores of research centers to confirm effectiveness and evaluate long-term effects, then 1.5 years of regulatory review, where all data are presented. Even after the drug is approved, it may undergo further Phase IV testing so more human safety and efficacy data can be collected (Figure 1.2). The Life Cycle of a Health Product The first event following discovery of a potential therapeutic molecule is legal, where the discovery is patented along with as many similar molecules as possible to keep the New Chemical Entity (NCE) or New Molecular Entity (NME) protected from the competition. Next, discovery screens (typically computer algorithms [in silico] and cell-based [in vitro or ex vivo] assays) are used to explore safety and efficacy profiles of various NCEs or NMEs to find the one entity (i.e., the “lead candidate”) judged to most effectively combine efficacy, safety, and bioavailability traits necessary for successful treatment of patients (see In Silico, In Vitro, Ex Vivo, and Non-Traditional In Vivo Approaches in Toxicologic Research, Vol 1, Chap 24). In addition, chemistry development and analytical methods are undertaken as is the preparation of milligram to gram quantities of the Active Pharmaceutical Ingredient (API). If the candidate compound passes the initial screen (drug discovery), it will enter the development process (nonclinical and clinical development) to establish safety and efficacy, during which time the chemical development of the Drug Substance (DS, or API), the drug without excipients, and Drug Product (DP, or the final dosage form) occurs in parallel to achieve the necessary quality requirements. Following successful development of a highquality API with suitable safety and efficacy profiles in animals, approval to experiment in humans is sought, which entails the preparation of a document that is commonly referred to as an Investigational New Drug submission (INDd [FDA]), Biologic License Application (BLAd [FDA]), Clinical Trial Application (CTAd[EMA] and Health Canada [HC] application). Simultaneously, development of an enabling API synthesis

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and formulation to manufacture supplies for nonclinical toxicity studies and First-In-Human (FIH or Phase I) studies is undertaken, as is the selection of solid dose form and crystallization process and preparation of up to kilogram quantities for use in the nonclinical development stage. If the candidate compound passes the application process, clinical trials may commence. After all clinical studies are completed, a New Drug Application (NDAd[FDA]), Biologic License Application (BLAd[FDA]), Marketing Authorisation Application (MAAd[EMA]), Japanese New Drug Application [NDA], Ministry of Health, Labour and Welfare [MHLW], and a New Drug Submission (NDSd[(HC]) is submitted for review. During this time the API process development, formulation development, characterization, and safety qualification of impurities are done and technical issues regarding large-scale commercial manufacture are solved. At this stage, large (generally kilogram) quantities are prepared, and processing methods scaled to manufacturing large batch sizes are developed. If the clinical trial data for the drug passes a comprehensive regulatory review, a marketing authorization is given, which allows the manufacturer to sell the drug in the region governed by the regulatory body. Following marketing, the manufacturer is expected to report on ADRs (also called “serious adverse events” [SAEs]) and may be required to do more clinical trials to resolve any lingering safety issues. Evaluation of the drug safety and risk–benefit after it has been marketed (a period commonly known as Phase IV) is termed postmarket surveillance or pharmacovigilance. A pictorial view of the life cycle can be found in Figure 1.3.

3. DRUG DISCOVERY AND DEVELOPMENT This section addresses each step of nonclinical and clinical development. The material given below is by no means exhaustive; as the process varies depending on the NCE or New Molecular Entity (NME), form of the Drug Product (DP); and whether the product is a chemically synthesized small molecule or a biologically derived biomolecule. The key difference between a NCE and NME is that a NCE has no active moiety that has ever been approved by the FDA, whereas

I. PRODUCT DISCOVERY AND DEVELOPMENT

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1. OVERVIEW OF DRUG DEVELOPMENT

FIGURE 1.3

Development of safety, efficacy, and quality of health products.

a NME has an active moiety that has not been approved by the FDA previously https://www. fda.gov/media/94381/download; see also Overview of the Role of Pathology in Product Discovery and Safety Assessment, Vol 2, Chap 2, and Discovery Toxicology and Discovery Pathology, Vol 2, Chap 3).

3.1. Patents and Intellectual Property During the discovery stage, patents are applied for. Without patent protection there is no incentive to risk capital in developing new drugs. Patents usually run for 20 years from registration of the patent; however, extensions are possible (Berger et al., 2016). The new product or process being applied for must be something that has not previously been disclosed anywhere in the world before. Usually, the regulatory patent service operates on a “first come, first service” basis. A significant amount of the development budget

is spent on patenting and legal fees. A patent claim relating to a pharmaceutical product may relate to an active ingredient, formulations, salts, prodrugs, isomers, and other characteristics of the product either separately or jointly. This may also include aspects of the manufacturing process for drug substance or drug product as well as use-related claims (e.g., indications, dose, method of treatment, diagnosis). A patent is not renewable. Once the patent expires, the invention becomes part of the public domain, and anyone anywhere can make, use, or commercialize the invention without permission from the inventor. In the pharmaceutical industry, the average interval between the discovery of a new drug and its final approval by the governing drug regulatory authority for human use is approximately 15 years, which includes the time required to conduct clinical research, complete product development, and

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achieve a regulatory authority’s approval (Temima and Ellis, 2009). Pharmaceutical companies can have exclusivity only after the drug is approved for marketing by the regulatory authority. Once the patent expires, the name of the drug may still have trademark protection, but other companies can manufacture and market a generic version of the drug without obtaining permission from the company and sell it as a generic drug, typically at a lower price since they do not have to pay for the cost of developing the drug. Patenting is an expensive activity. In fact, the cost of obtaining and maintaining worldwide coverage for a single patent has been estimated to be as much as $250,000 (Quinn, 2015). This aspect influences the approach to Intellectual Property (IP) management and in particular patenting strategy, where the lower the value of the asset, the lower is the degree of protection, and patent management. Having a limited number of assets means that the patent is managed in a manner that controls the associated costs.

3.2. Discovery Drug discovery is a multifaceted process that requires teamwork across multiple scientific disciplines (see Discovery Toxicology and Discovery Pathology, Vol 2, Chap 3). For most pharmaceutical companies, the discovery process starts with identification of an unmet medical need or marketing opportunity and is followed by defining a strategy for targeted intervention (Smith and O’Donnell, 2006). From the company’s standpoint, discovery arises more often from an idea of possible marketing success than unmet medical need, unless the market is there. Two general paradigms have been followed. The first is physiology based and relies on development of a basic understanding of the underlying biology of the disease and then selection of a specific target in the disease pathway to focus on for drug intervention. Testing of compounds in a battery of target bioassays versus the engineering of a specific compound to “hit” a specific target is used. From the perspective of an established pharmaceutical company, it is important to balance unmet medical need, scientific knowledge and understanding, and manufacturing capabilities with the market opportunity and growth potential to manage a portfolio and choose the most

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attractive medical conditions as opportunities for the business (Abraham, 2010). For the small pharmaceutical company, a narrower focus on one disease, special target knowledge, or a distinctive molecular entity or delivery platform may drive the early stages, with subsequent reliance partnerships with more established firms to obtain complementary skill sets, resources, and capabilities need to take the product into later phases of development. There is no standard method through which drugs are developed. New drug research starts with an understanding of how the body functions, both normally and abnormally, at its most basic levels, usually at the cellular and molecular levels. The questions resulting from this research help determine a concept of how a drug might be used to prevent, cure, or treat a disease or medical condition, which provides the researcher with a target of interest. Next comes validation of the target and the production of multiple chemical series and synthesis of large numbers of molecules within each series (Holgate et al., 2013). At this stage, large numbers of molecules are run through a series of high-throughput screening (HTS) assays designed to identify candidates that are selective for the target and produce a desired biological effect (i.e., pharmacology) in model cellular systems. In addition, disease-related genotype or phenotype, plasma concentration, target occupancy, target activation, physiological measurements, and any modification to the disease process may be used (see Biomarkers: Discovery, Qualification and Application, Vol 1, Chap 14). Target Identification and Validation Target identification and validation involves study of the mechanism and points of intervention for the disease or condition of interest and verification that a potential target is important in initiating and/or sustaining the disease. Strategies and approaches cover the gamut of technologies available to study disease expression including but not limited to molecular biology, functional assays, image analysis, and in vivo studies related to functional assessment. Understanding the degree to which disease models in animals are similar to diseases in humans is critical to building confidence for the translation of findings in animals to predicting the impact on a human disease. Toxicologic pathologists play a pivotal role in identifying target location (target identification) and

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modulation in disease tissues (target validation) as well as evaluating histological endpoints in animal efficacy studies and more. Target Validation Target validation is confirmation that the molecular and/or cellular target(s) of interest have a role in the disease. Enzyme and expression assays are commonly used techniques as are development of knockout/knockin animal models via use of antisense/siRNA strategies and genomics. Translation between humans and animals is an important feature to build confidence on developing screening assays for identification of lead molecules. Lead Generation and Optimization Lead generation uses key attributes of the target to define chemical properties and attributes related to affinity, potency, and selectivity for screening large libraries of chemicals for desired activity. For small molecules and biomolecules, high-throughput screening, computational methods, and more traditional and intuitive medicinal chemistry approaches are used to identify multiple chemical (or protein) series, with relevant chemical functionality associated with the desired target activity and drug characteristics. Assays are usually designed to optimize sensitivity, specificity, positive accuracy, negative accuracy, capacity, and reproducibility and can identify target distribution to anticipate offtarget organ effects. If potential toxicity issues are identified, spot-checking of individual molecules within chemical series using in vitro cellbased cytotoxicity assays may be performed at this early stage in order to characterize potential toxicity risks within a series. Once a suitable chemical series and possible lead candidates have been identified, the candidate to be moved forward into development must be optimized. During optimization, the potency and the selectivity at the target; efficacy in animal models; Absorption, tissue Distribution, Metabolism, and Excretion (ADME) characteristics; information on transporters; pharmacokinetics; solubility and pharmaceutics properties; ability to formulate; and safety are evaluated. Balancing each feature against a desired target profile establishes the optimum characteristics needed for a lead molecule. Pharmacokinetic (PK) modeling to predict a human dose is critical for later development of perspective on anticipated safety margins as well as

dose frequency (see Principles of Pharmacokinetics and Toxicodynamics, Vol 1, Chap 5). Toxicity studies employ novel and state-of-theart in vitro technologies (see In Silico, In Vitro, Ex Vivo, and Non-Traditional In Vivo Approaches in Toxicologic Research, Vol 1, Chap 24) and in vivo models for evaluating target organ toxicity to identify potential safety and development risks. Balancing each feature against a desired target profile establishes the optimum characteristics needed for a lead molecule. Once compounds are identified that successfully meet nonclinical efficacy, ADME, PK, and safety and toxicity criteria, one or more compounds are nominated as candidates for entry into nonclinical development. High-Throughput Assays and Quantitative Structure-activity Relationships (QSAR) Quantitative Structure–Activity Relationships (QSARs) can be used in silico to predict the potential activity of new molecules and thus reduce the chance of in vivo testing of negative compounds. The use of QSARs early in development, when tests are relatively cheap and fast, can increase the proportion of truly active compounds passing through the system into later, more expensive phases of in vivo testing (see In Silico, In Vitro, Ex Vivo, and NonTraditional In Vivo Approaches in Toxicologic Research, Vol 1, Chap 24). At this time, the role of toxicology in lead candidate generation is typically limited to literature reviews of target effects in tissues and identification of potential substructures (known as toxicophores) suggestive of possible toxicity issues for particular molecular series. Toxicology and pathology can also help in identifying target distribution to anticipate possible off-target organ effects. Spot-checking of individual molecules within chemical series using in vitro cellbased cytotoxicity assays may be performed at this early stage in order to characterize potential toxicity risks within a molecular series. Early in discovery, typically several chemically diverse series are initially assessed by highthroughput screening (HTS) until one or more lead series that have appropriate druglike properties and efficacy on the target are identified. This stage is often referred to as “Hit-To-Lead” stage. Once an appropriate lead series is identified, the chemical series then enters into the discovery phase at lead optimization to identify those molecules with the most druglike properties.

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Mechanistic Studies Technical innovation in the life sciences impacts drug development along its pipeline. A key factor for success in modern drug development programs is the appropriate use of mechanistic data to explain the data or sometimes direct the design of pivotal nonclinical studies and/or clinical trials. Innovative Trends in Drug Development The most notable impact of mechanistic studies can be seen in target discovery and validation, translation from in vitro, ex vivo, and in vivo animal experiments to man, as well as in clinical trial design. The first big wave of technical innovation in drug development was the systems biology approach. Beginning in the early 2000s, multiple “omics” platformsdgenomics, transcriptomics, proteomics, and metabolomics, among othersdevolved as key techniques for obtaining a detailed, system-wide assessment of the ways in which a drug candidate may alter cell and organ function (see Toxicogenomics: A Practical Primer, Vol 1, Chap 15). The initial hope to discover hundreds of new drug targets was not realized, but in-depth analysis of gene expression changes as well as protein and metabolite signatures often helped to identify the molecular mechanisms of disease, including affected signaling pathways, and clinically relevant biomarkers. The analysis of multi “omics” has helped to understand which aspects of animal models can be extrapolated to man and which are so different among species that their value in translational medicine is limited. Identification of genetic changes was also one of the enablers of precision (or “personalized”) medicine as specific genetic variants in a person’s genome, or more specific tissue-based mutations in a given disease (e.g., cancer), can be linked to the onset and/or progression of a disease and provide insight regarding whether treatment targeted to correct or minimize the impact of a specific genetic defect will provide a suitable therapeutic approach. Initial systems biology efforts created an avalanche of data but resulted in few tangible benefits in drug discovery, toxicity testing, and risk assessment. The main reason for this was the lack of computational systems that could analyze this amount of data and identify patterns among the complexity. Since 2015, huge advances in the field of artificial intelligence (AI) have now opened the possibility to do just that (Paul et al., 2021). Artificial intelligence is a large field which

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includes machine learning and deep learning. Machine learning has been used in areas like image analysis for decades; it enables algorithms to continually improve their performance with every data set they analyze. In recent years, this has been complemented by deep learning, which is more similar to human neural networks. With this method, it is possible to let algorithms define their own criteria for pattern recognition. Deep learning algorithms have successfully been used in drug development for target and indications discovery, molecular design, image analysis and diagnostics, and clinical trial design. In terms of pathology, molecular detection techniques like immunohistochemistry and in situ hybridization (see Special Techniques in Toxicologic Pathology, Vol 1, Chap 11) as well as single-cell genome sequencing together with AI analysis open the way for a morphology-based systems biology approach that will help us to understand the interaction of cell networks rather than tissues or organ systems (see Digital Pathology and Tissue Image Analysis, Vol 1, Chap 12). The last trend that is starting to gain momentum, and which should confer significant advantages translating both efficacy and safety signals to man, is the use of fully human in vitro systems or in vivo humanized animal models. Human cells or tissue slices have been used for toxicity screening in vitro for some time; the use of threedimensional (3D) cell cultures and microphysiological systems (“organs on a chip”) now allows reactions of human tissues to be studies after exposure to drugs both acutely and over time (see In Silico, In Vitro, Ex Vivo, and NonTraditional In Vivo Approaches in Toxicologic Research, Vol 1, Chap 24). Rodent (mouse > rat) models with humanized liver or immune system (i.e., where animal cells for a given organ are replaced or supplemented by human cells from the corresponding human organ) or less often nonrodents genetically modified to develop a humanlike disease can be employed to interrogate specific pharmacologic properties of drugs on human molecular pathways in vivo that are impossible to study in unmodified nonhuman species (see Genetically Engineered Animal Models in Toxicologic Research, Vol 1, Chap 23). Biopharmaceutical companies and regulators like the FDA are working on approaches to utilize these models in order to predict adverse events of difficult-to-model organ systems (e.g., the immune system) and prevent serious adverse events (e.g., cytokine release syndrome [“cytokine storm”]). Taken together, these technologies create

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the opportunity to address specific safety and efficacy issues and pave the way to a more focused, science-driven form of drug development. Biomarkers A biological marker, or “biomarker,” is in general a substance, a morphology, or other measurable endpoint that can be used as an indicator of a biological state. Biomarkers are characteristics that can be objectively measured and evaluated as indicators of normal biological processes, pathogenic processes, or pharmacologic responses to a therapeutic intervention (see Biomarkers: Discovery, Qualification and Application, Vol 1, Chap 14). Biomarkers may be specific to the cell systems or animal models used in discovery and nonclinical testing, but, in general, effort is expended to identify biomarkers that will perform similarly in test species in nonclinical studies and in human patients in clinical trials. Toxicity Assessment Development-limiting toxicity is often difficult to define at the discovery stage. This is because it depends on factors such as the safety margin; the nature of the toxicity (i.e., is it direct or indirect); its reversibility; and whether or not it can be monitored. It also depends on the route of administration, duration of treatment and intended therapeutic indication being sought, and the potential for occurrence in the human population at large. The Safety Margin, also known as the Therapeutic Index (TI) when applied to human patients in the clinic, is typically defined as the ratio of the NOAEL (No Observable Adverse Effect Level) in animals divided by either (1) the predicted human efficacious exposure level in the “average” responding patient or (2) the

FIGURE 1.4

predicted exposure at the maximum anticipated human dose. More recently, the Minimum Anticipated Biological Effect Level (MABEL) has been used (Suh et al., 2016) though its use is primarily applied in the biotherapeutic arena where safety margins for some product types (e.g., viral gene therapy vectors) tend to be lower. Because it is difficult to accurately predict what the human efficacious dose level will be prior to human clinical trials, often the exposure at an efficacious dose in the nonclinical disease model will be used for the denominator for estimating the safety margin in human patients.

4. DEVELOPMENT The term “drug” in the context of this chapter means any article(s) intended for use in the diagnosis, cure, mitigation, treatment, or prevention of disease in man or other animals. Historically, drugs were small molecules or chemical mixtures (e.g., plant extracts), but recent advances in molecular therapeutics have broadened the definition to include biomolecules such as nucleic acids (e.g., antisense oligonucleotides), proteins (e.g., recombinant or fusion proteins), and combination agents (e.g., antibody–drug conjugates; see Bacterial Toxins, Vol 3, Chap 9). Advice from toxicologic pathologists aids management to decide whether to proceed with development or “kill” the drug candidate while pivoting to a more promising molecule. The drug product must be of a high quality, safe, and effective. Well-characterized safety improves the probability of success as a molecule moves through the drug development stages (Figure 1.4).

Characteristics of well-defined safety risk.

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4.1. Drug Substance and Drug Product Development (Quality) The process of formulation and development of select production runs to produce the final drug product that is intended for marketing should occur at the same time as the beginning of clinical trials (Pharmaceutical Manufacturing Encyclopedia, 2007). If this is not possible, then a test (“research grade”) drug product can be used in Phases I and II. However, in Phase III the final (“clinical grade”) dosage form to be marketed is required for this final stage of safety assessment (Schlindwein and Gibson, 2018). Clinical trials undertaken without use of the final product are not accepted without an accompanying human bioavailability study demonstrating bioequivalence of the test product to the final drug product. Quality issues include all aspects of the chemical, pharmaceutical, and biological development of a new product (Schlindwein and Gibson, 2018). They are covered in the Chemistry and Manufacturing Controls (CMC) in an NDA in the United States, Part II of the Marketing Authorization Application (MAA) in the United Kingdom, and Chemistry and Manufacturing section of the NDS in Canada. In essence, this documentation covers the following for ingredients and finished products: their composition; method of preparation; control of starting materials; control tests carried out on intermediate products; control tests on the finished product; stability data; and bioavailability and bioequivalence in vitro. While most countries issue guidance documents to address quality criteria for the drug substance (active pharmaceutical ingredient [API]) and drug product (DP, final dosage form), they increasingly align with guidance issued by the ICH (see Overview of the Role of Pathology in Product Discovery and Safety Assessment, Vol 2, Chap 2). The benefit of a harmonized approach is that it ensures global consistency in the manufacture and quality of drugs while at the same time facilitating a more expeditious approval process. Development PharmaceuticsdThe Active Pharmaceutical Ingredient Description of the development of a drug substance, also known as the API, requires numerous studies to define its identity. These include the material’s solubility in a variety of solvents, the presence or absence of molecular

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polymorphism, and its pH and pKa (acid dissociation constant) values. Regulators are always keen to be made aware of any unusual aspects associated with the molecular structure during analytical development. The precision and accuracy of the tests used must be described. Any variation in the results, which is especially likely in substances of biological origin, must be detailed. Information on the NCE, or API, may be supplied by the applicant or, if it is manufactured by a third party, using the Drug Master File (DMF) of the manufacturer (see below). The regulator maintains confidentiality between the owner of the DMF and the applicant. Control of the levels of impurities requires critical justification. Third-party manufacturers are distributed globally (Figure 1.5). Manufacturing methods and formulation of the API by a third party may not be available for proprietary reasons to the applicant requesting a marketing authorization (e.g., in the manufacture of generic drugs or in joint ventures). In this case, the third-party manufacturer must supply a detailed description of the manufacturing method, quality control procedures, and process validation direct to the regulatory authority. This material is presented in the form of the DMF. The restricted part of the DMF must detail the manufacturing method, batch analysis, and specification and routine tests that have been applied, among other points. For an API, information on its stability forms a core part of the stability evaluation of the product. The information gained on stability of the active substance is used in determining the stability of the formulated final product. Development PharmaceuticsdThe Drug Product Part of development pharmaceutics is the process of turning an active molecule (API) into a form and strength suitable for human usedthe drug product (DP). A pharmaceutical (drug) product can take any one of several dosage forms (e.g., liquid, tablets, capsules, ointments, sprays, patches) and dosage strengths (e.g., 50, 100, 250, 500 mg). This final formulation (DP) will include the API, vehicle, and excipients, where the latter are substances other than the API added to the DP. Similar considerations apply in principle to other biomedical products, although adjustments are necessary depending on the nature of the product. For example,

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FIGURE 1.5 Distribution of facilities manufacturing active pharmaceutical ingredients (APIs) for the US pharmaceutical market by location in 2019. https://www.statista.com/statistics/1101403/us-registered-drug-chemicalfacilities-producing-api-by-location/ (Accessed December 23, 2020).

biologics, gene therapy vectors, and modified cells typically are administered by injections, and medical devices generally are implanted surgically and may or may not be coated with active biomolecules. Excipients (i.e., an inactive substance that serves as a carrier for an active drug) are added for many reasons (Osterberg and See, 2003). Some of the more common reasons include diluting the API, improving the taste of an oral product, allowing the API to be compounded into stable tablets, delaying the drug’s absorption into the body, or to prevent bacterial growth in liquid or cream preparations. The impact of each excipient on the human body must be evaluated. The API should be compatible with the excipients. All excipients should play a justified role in producing the desired DP, and unusual

excipients need to be comprehensively described and demonstrated to be safe. Also, formulations and changes in excipient physical characteristics should not result in changes in critical properties of the DP (e.g., in bioavailability). Factors in the composition of the DP that need to be specified, controlled, and monitored may include pH, dissolution (in liquids), aggregation, and homogeneity. Dissolution of a solid dosage in vivo after administration needs to be verified. Manufacture of the Finished Dosage Form All drug products must be manufactured according to Good Manufacturing Practices (GMP) (ICH, 2000; ISPE, 2022). This guidance includes recommendations for information to include in the regulatory submission, such as

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the manufacturing formula, the description of the manufacturing process, the description of the manufacturing chain, the validation of the manufacturing process, and special items. The marketing authorization application to the regulatory agency should contain information specific to the medicinal product under review. The main items to be described include the manufacturing formula and manufacturing process, and the main objectives are to guarantee a medicinal product of expected and known specification while keeping variability within experimentally verified limits. The description of the manufacturing process includes the equipment used as well as in process controls. Major changes to the manufacturing process need to be first agreed with the licensing regulatory authority, and will require a nonclinical study confirming the equivalency of the old and new formulations. Impurities in the Final Drug Product The management of impurities is one of the most common rate-limiting steps in quality assessment. The greater the number of ingredients used in the synthetic processes, the greater the risk of the presence of impurities in the finished product. Impurities can arise from the API synthesis itself; from the use of solvents and other materials during purification processes; and the inevitable presence of degradation products should the active substance be shown to be relatively unstable. Biologicals may contain impurities derived from the organism in which they were produced (e.g., endotoxins released from bacterial bioreactors during recombinant protein production; see Bacterial Toxins, Vol 3, Chap 9). The general characteristics of the pharmaceutical form, particularly “pharmacotechnical” characteristics, are determined by physical tests with limits of acceptance of the qualified product. The applicant for the marketing authorization must provide the test procedures used to detect and identify potential impurities, together with the limits imposed on their presence (usually 0.1%). A summary of the test results on impurities in batch samples is also required. It is vital that the results from routine quality control of the active substance

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should be presented. Ideally, these results should be from samples that are to be, or are being, used in clinical trials and in nonclinical toxicity tests. The results should include the date of manufacture, the batch size as well as batch and lot numbers, the place of manufacture, the results of analytical tests, and the intended disposition of the batches (Blacker and Williams, 2011). In general, impurities are difficult to fully eliminate from a synthetic process. Furthermore, lowering them to negligible levels through multiple repurification steps is costly and timeconsuming while doing little to improve efficacy or safety of the final formulation. Therefore, detailed guidance is provided by ICH quality guidelines, and agreed to by many regional regulatory authorities, on the process of qualifying levels of impurities as safe (ICH, 2006a,b, 2019; 2020c, 2021f). Qualification is the process of acquiring and evaluating data that establishes the biological safety of an individual impurity or a given multi-impurity profile (Jacobson-Kram and McGovern, 2007). ICH quality guidelines (Q3A–Q3E) give guidance regarding impurities for drug substances, drug products, and medical devices, while ICH Q6B provides guidance for impurities in biotechnological products (ICH, 2022b). All impurities and degradation products (identified in stability studies at recommended storage conditions) that are present at a level of 0.1% or more must be identified. Pharmacopeial analytical procedures are often used to detect and quantify inorganic impurities and residual solvents. Limits on the presence of such impurities should be stringent and similarly be based on objective analytical data. The reasons for selecting particular impurities for qualification must be based on safety considerations. When nonclinical safety or clinical studies or both have been carried out, the level of any impurity is considered “qualified.” The threshold limits are based upon the maximum daily dose, defined with respect to the level (less than, equal to, or more than) relative to an intake of 20 g of substance per day. Any change in the manufacturing process may lead to the generation of a completely new (previously absent) impurity or an altered

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multi-impurity profile. Again, any decision about the need to identify and qualify the new impurity must be based upon safety considerations. Throughout development, impurities in the DP need to be controlled. For this reason, the applicant will need to describe those impurities that would be expected from the production process as well as suitable tests to demonstrate their presence and that quantities are below acceptable limits. A batch of a final DP may be used for clinical studies, for nonclinical toxicity studies, and/or for stability testing. A tabulated listing of impurities in specific batches must be presented. The data should include the batch identity and size, the date and site of manufacture, the manufacturing process used, the individual and total impurity content, the intended use of the batches, and the analytical procedure used to detect the impurities. The specification for the new substance should include data on both identified and unidentified impurities. Reasons for the inclusion or exclusion of impurities from the specification should take into consideration the likely effects, harmful, or otherwise, of the impurity. Excipients Data about the excipients (inactive components) in the formulation must be developed in a similar way to that for the API. Full details of the excipients in the formulation must be given, including their common name, the amount in which they are being used, and the standard of the material being used. Mixtures of excipients must be qualitatively and quantitatively described. The reasons for the choice of an individual excipient, and standard of material chosen, should be explained. It is not usually a requirement to carry out identity testing and assays of the excipients in a finished product if all the earlier requirements have been fulfilled. There is a wealth of established excipients available to drug developers; in some legislations, lists of innocuous substances are available (e.g., the FDA database of substances Generally Recognized As Safe (GRAS)). In addition, safety studies have been performed and published on a large number of excipients (e.g., cremophor); in recent years, safety profiles of many excipients have been reviewed systematically in the scientific

literature (Patel et al., 2020). If an applicant wants to use a novel excipient, this must undergo its own safety studies. Packaging Materials Packaging materials that come into direct contact with the DP (e.g., the packaging material of a blister pack that surrounds a tablet) require full description in regulatory submissions. Details must include the method of opening, aspects of the design that make the container a multidose one, and measures taken to ensure that the container is both tamper- and childresistant. Any possible interaction between the container and closure and the formulated product must be investigated and documented. Methods used to assess this possibility usually include the extraction from solution of one or more of the ingredients, a problem that usually occurs with glass and rubber containers and closure materials. Plastic materials are inherently unstable. In general, product development must evaluate extractables and leachables in product formulations destined for shipment before use by patients. Extractables are substances removed from packing materials at extreme temperatures in the presence of various acidic, basic, organic, and aqueous solvents; these substances are assessed as potential contaminants that might enter the DP. Extractables can be mutagenic and may need to be safety qualified or controlled below the threshold of toxicological concern, typically 1.5 mg. Leachables are chemical species that can enter into the DP under normal product use or storage conditions, and thus represent a subset of extractables (Markovic, 2007; Ball et al., 2007). Leaching of ingredients might occur from the finished product into the plastic and from the plastic into the pharmaceutical dosage form. The stability of the finished product in contact with the plastic, particularly if it is not a solid dosage form, must be fully characterized. How to evaluate the safety impact of leachables and extractables has received considerable attention (Markovic, 2007) and recently fostered development of an ICH guidance (ICH, 2020b,c). The method of administration, the stability of the product, and any method of sterilization are

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determined. Reasons for the choice of the container in relation to these issues must be fully given. The plastic used in the packaging material should be fully characterized and include details of the technical characteristics of the plastic and any additives in the finished plastic, as well as the plastic’s manufacturer. Stability Testing of APIs and DPs The primary purpose of the ICH guideline regarding stability testing, is to explain what data are required to demonstrate that the quality of an active substance or a medicinal product is maintained under a variety of environmental stresses (e.g., fluctuations in temperature, light, and humidity). These data will then be used to determine the recommended storage conditions, retest periods, and shelf lives. For active substances, at least three batches must be subjected to accelerated and long-term stability tests of at least 12 months’ duration. Accelerated stability testing is where an exaggerated physical environment is used (e.g., heat, to show potential instability over a shorter period). Long-term stability mimics the environmental conditions in which the product will be stored. Equally important, the quality of the ingredients used in the manufacture of the API must remain the same during initial studies, manufacturing scale-up, and the final manufacturing process. This is to permit extrapolation of the stability test results. For the finished DP, data of the stability from laboratory-scale (relatively small) batches are not acceptable as primary stability information. At this stage, a real-life situation involving the containers and closures to be used in distribution of the product are becoming part of the testing scenario and must be used in the stability tests. For the same reasons, the packaging used in the storage and distribution of the API, or the finished DP should be a mock-up of or the same as that used in the testing process. The expiry date is usually based on stability studies; the date often reflects the length of the stability studies undertaken rather than the experimentally determined point at which API degradation exceeds acceptable limits. Where storage instructions are given, they should be specific (e.g., “store in the dark between 10 and 25
C”) and not open to interpretation (e.g., “store at room temperature”).

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4.2. Nonclinical Safety Studies Nonclinical studies in animals, also referred to as preclinical studies, chiefly address safety issues, although the toxicologic pathologist may be called on to evaluate efficacy endpoints as well in some cases (e.g., in combined efficacy/ safety studies of gene therapy products in spontaneous or engineered animal models of disease) (Gad, 2016). They also identify candidate agents, elucidate bioactive pathways, and evaluate efficacy in addition to toxicity in animal models (van Tongeren et al., 2011; Maronpot, 2013). Effects observed are usually separated into “on-target,” “off-target,” and toxic effects. An ICH guidance (ICH, 2009) provides the internationally harmonized recommendations regarding the type and duration of nonclinical safety studies and their timing to support clinical trial designs and marketing authorization, with other ICH guidance documents providing general principles and recommendations for a range of specific types of studies see https:// www.ich.org/page/ich-guidelines (ICH, 2020a). Each therapeutic class of drug candidates must undergo different types of nonclinical research. Key endpoints may include pharmacokinetics (PK), pharmacodynamics (PD), in silico modeling, in vitro toxicity testing (e.g., genotoxicity in bacteria, cytotoxicity in cultured cells), and in vivo nonclinical animal testing (e.g., acute toxicity, chronic toxicity, reproductive toxicity, and carcinogenicity, among others). The nonclinical safety studies, although sometimes limited at the beginning of clinical development, characterize potential effectsdand their adversity and reversibilitydthat occurred in test animals under the specific conditions of the nonclinical study (Kerlin et al., 2016). In turn, these events in animals are viewed as possible consequences to monitor in human patients enrolled in the clinical trial supported by that nonclinical study (see Assigning Adversity to Toxicologic Outcomes, Vol 2, Chap 15). At many times during the early years of the development process, safety assessment constitutes the rate-limiting step in receiving regulatory approval to begin clinical testing. The successful operation of a nonclinical safety assessment program requires that four different streams of the product-related operation be undertaken simultaneously: discovery, applications for FIH trials, clinical efficacy and safety

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research and registration, and postmarketing support. The toxicologic pathologist is core to this safety assessment where essential discussions come from the integration of toxicology, toxicologic pathology, PK, and PD data across several species (including humans). Safety issues cover all aspects of the nonclinical development of a new product and are important in obtaining the authorization to test in humans (Figures 1.6 and 1.7). Nonclinical studies make up the majority of a clinical trial application (CTA, IND, or equivalent) and constitute the major detailed, and possibly mechanistic, studies of the adverse or toxic effects of the molecule. In the marketing authorization application, the nonclinical studies include pharmacology and toxicology studies both in vitro and in vivo. Details regarding the role of the toxicologic pathologist in nonclinical assessment can be found in Pathology in Nonclinical Drug Safety Assessment (Vol 2, Chap 4). Selection of Doses for Nonclinical Studies In selecting the doses to be used for nonclinical studies, dose–response relationships and PK–PD relationships are typically defined using combined PK and single-dose escalation studies. Dose levels in the animal toxicity studies should be selected to provide information on a dose– response relationship, covering the pharmacological dose range and including, where possible, a toxic dose, a maximum tolerated dose (MTD), and an NOAEL or the Minimum Anticipated Biological Effect Level (MABEL) (Suh et al., 2016). The number of animals per dose group in a study may need to be adjusted to anticipate that some drug-exposed animals will experience limited exposure because of immunogenicity to the drug candidate, particularly in long-term studies of human-derived biomolecules where the occurrence of an antibody response is more likely in common nonclinical species. Toxicity Tests In animal testing, two or more species, usually one rodent (typically rats or mice) and one nonrodent (often dogs or nonhuman primates), are tested because a drug candidate may affect one species differently from another. For example, human-derived biomolecule test articles often possess limited pharmacological activity in nonprimates, and the sensitivity of test species to

small molecule–induced toxicity may vary widely among species. The results of these studies, especially responses in the most sensitive animal species, result in the identification of an estimated initial safe starting dose in humans, potential target organs of toxicity, reversibility of adverse effects, and sometimes the mechanism(s) of action responsible for the toxic effects. It may be possible in certain exceptional cases, or where data from conventional animal studies are known to have limited predictive value, to justify entry into clinical trials with very low (“low-risk”) doses based on in vitro data from human cells and tissues, an understanding of the mechanism of action, negative findings in transgenic and knockout mouse models, and in silico computer modeling of projected human PK and PK–PD relationships. Knockout mice studies have revealed new opportunities for treating human diseases. Several examples of breakthrough targets include cyclooxygenase 2 (COX2), phosphodiesterase type 5 (PDE5), and BCR-ABL. The COX2 target was first commercialized in 1998 (Zamrowicz and Sands, 2003). Toxicologic pathology addresses adverse effects related to dose (see Assigning Adversity to Toxicologic Outcomes, Vol 2, Chap 15). Other tests are more general in nature, ranging from acute singleexposure studies to repeat-dose studies in which animals are administered daily doses of a test substance to calculate the NOAEL or MABEL and determine whether one or more organs or systems is adversely affected following exposures of subacute (1-month), subchronic (3-month), and chronic durations from 6 months to 2 years (i.e., a rodent lifetime carcinogenicity bioassay). GENOTOXICITY

There are several tests available for genotoxicity detection (Turkez et al., 2017). The procedure chosen should be of maximum accuracy and of reasonable cost and be capable of detecting the main types of genetic damage: gene mutations, structural damage to chromosomes, and disruption of the genome, along with accounting for the different organization of genetic material in prokaryotes and eukaryotes used in these assays. It should also be recognized that the ability of different organisms and different test systems to metabolize chemicals and drugs greatly varies. Both in vitro and in vivo tests may need to be used in such instances; it is the

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FIGURE 1.6 Nonclinical risk considerations supporting drug development (A). API, active pharmaceutical ingredient; FIH, first in human.

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FIGURE 1.7 Nonclinical risk considerations supporting drug development. API, active pharmaceutical ingredient; HERG, human ether-a-go-go-related gene; Mab, monoclonal antibody; PK, pharmacokinetics.

characteristics of the drug candidate under investigation that determine which combination of tests should be used. The gold standard for genotoxicity testing and its interpretation is briefly summarized below and are presented in more detail in ICH genetoxicity guidance (ICH, 2022b). ICH M7 guidance also addresses how to incorporate in silico approaches for the prediction of genotoxicity (ICH, 2017). BACTERIAL MUTATION (AMES) ASSAY The most widely used tests for gene mutations are performed in bacteria. These assays are carried out using known and well-characterized bacterial strains (e.g., Salmonella typhimurium). Such tests can detect frame shifts and base change mutations in DNA. The microsomal (S9) fraction isolated from homogenized hepatocytes may be added to provide drug-metabolizing enzymes from a given

test species, thereby providing the opportunity to assess metabolites for potential genotoxic activity. CHROMOSOMAL ABERRATION ASSAYS Tests for chromosomal aberrations in mammalian cells in vitro utilize human lymphocytes or mammalian cell lines. Assessment of damage at mitotic metaphase during DNA replication indicates mutagenic potential. The test for gene mutations in eukaryotic systems can be used in both bacteria and organisms with complex eukaryotic chromosomal structures. Complex eukaryotes such as fungi and insects may even be used.

Confirmation of the results from the above in vitro tests is required using a third test, an in vivo test for genetic damage. The micronucleus test is used to determine if a compound is genotoxic by evaluating MICRONUCLEUS

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TEST

4. DEVELOPMENT

the presence of micronuclei. Micronuclei may contain chromosome fragments produced from DNA breakage (clastogens), or whole chromosomes produced by disruption of the mitotic apparatus (aneugens). MUTAGENICITY TEST A fourth test that may be considered is in vivo mutagenicity testing in engineered rodents. These genetically modified rodents, e.g., knockout mice, provide the toxicologic pathologist the opportunity to assess the mechanism of disease for which the knockout mouse was developed (see Genetically Engineered Animal Models in Toxicologic Research, Vol 1, Chap 23). SINGLE-DOSE TOXICITY

The single-dose acute toxicity assessment is an antiquated test which is now incorporated in ascending dose MTD studies and rarely done as a stand-alone study. Single-dose data help determine toxic effects seen in an acute drug overdose, so that extrapolation of health risk to humans can be made. REPEATED-DOSE TOXICITY

In support of initiating human clinical trials, repeated-dose (or sometimes “repeat-dose”) toxicity studies are required (Greaves, 2012). The length of the toxicity study depends upon the expected length of treatment or period of systemic retention of a drug candidate (i.e., antibody) in humans. As development of a drug candidate is a stepwise process, repeated-dose studies of increasing duration (up to 6 months) are conducted to support human clinical trials of a given scope and duration. The maximum length of repeated-dose toxicity studies is 6 months in rodents and 9 months in nonrodents. REPRODUCTIVE TOXICITY

All drug candidates are tested for potential reproductive toxicity, except for some indications where pregnancy after the disease has been treated is not an option (e.g., cancer chemotherapy). Tests should be carried out using male and female animals during the major stages of reproduction (US FDA, 2011). Key reproductive toxicity studies evaluate fertility in rodents (Segment I study, with exposure from before mating through implantation); embryonic/fetal development (Segment II study, with exposure from implantation to the end of organogenesis);

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and prenatal plus postnatal development (Segment III study, with exposure from early organogenesis through weaning). Details can be seen in guidance document S5(R2) (ICH, 2020d). For detection of potential long-term reproductive problems, extended onegeneration reproductive toxicity studies over one or two generations may be required (preSegment III studies) (OECD, 2018). The type of reproductive toxicity study to be undertaken is determined by the expected use of the drug product, its formulation and route of administration, data already collected on toxicity, pharmacodynamics, pharmacokinetics, and the known effects of related molecules. Regardless of study nomenclature, a range of studies must be undertaken covering all stages from conception to sexual maturity. Observations should also be continued through one complete life cycle (from conception in one generation to conception in the next generation). CARCINOGENIC POTENTIAL

Carcinogenicity studies are usually life-time (2-year) rodent or 6-month transgenic mouse assays that will need to be carried out if the drug candidate is to be administered regularly, either continuously or at frequent intervals over at least 6 months. They are also required for drug candidates that are structurally related to a known carcinogenic molecule or where its biological action, long-term toxicity, or mutagenicity may indicate a potential for carcinogenicity. Detection of carcinogenicity is facilitated (but not guaranteed) by the fact that almost all known human carcinogens are also carcinogenic in one or more species of experimental animals. However, the converse is not always true, and extrapolating carcinogenicity results from animal studies to humans is difficult. Factors that affect extrapolation to humans include the intended use; the dose and route of administration; the test system (e.g., the animal species tested, usually rodent, and whether the animals are wild-type vs. engineered to possess enhanced sensitivity to a certain carcinogen class); and the response observed (e.g., increased incidence of tumors or preneoplastic proliferative lesions in addition to other treatment related pathology). Some drugs used in the latter stages of terminal disease are themselves carcinogenic in the longer term (e.g., many antineoplastic chemotherapies).

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If the patient’s life expectancy is shorter than the period in which carcinogenic effects from the health product might develop, carcinogenicity studies are not required during nonclinical safety testing. However, the quality of life needs to be recorded and analyzed for registration, such as improved quality of life and extension of life by 6 months (ICH, 2021c) (see Biotherapeutic ADME and PK/TK Principles, Vol 1, Chap 4, and Pathology in Nonclinical Drug Safety Assessment, Vol 2, Chap 4).

4.3. Animal Efficacy Studies Efficacy is defined as “the extent to which an intervention does more good than harm under ideal circumstances,” whereas effectiveness “is the extent to which an intervention does more good than harm when provided under the usual circumstances of healthcare practice” (Kim, 2013). Others have referred to efficacy as “can it work?” and effectiveness as “does it work?” Efficacy can be assessed in nonclinical studies, while both efficacy and effectiveness may be assessed during clinical studies. Short-Term Efficacy Studies in Animals BIOAVAILABILITY AND BIOEQUIVALENCE STUDIES

Bioavailability is defined as absorption and transport of the drug candidate and its metabolites to the target tissue(s) where it exerts a typical effect. An understanding of four fundamental PK parameters will give the toxicologic pathologist a strong basis from which to connect tissue concentrations of the test substance with altered morphology in that tissue. These parameters are clearance, volume of distribution, half-life, and bioavailability (Benet and ZiaAmirhosseini, 1995). Bioavailability is evaluated by the corresponding specific endpoints (observable or measurable biological, chemical, or functional events), which vary depending on the nature of the molecule. Although it is common to equate “bioavailability” with circulating (blood) levels of an agent, this is not always the case as molecules that concentrate in their target tissue may have low or transient residence in circulation yet be efficacious. For example, azithromycin has low systemic levels following administration but is concentrated in macrophages at the sites of infection.

Bioequivalence is used to assess the expected in vivo biological equivalence of different drugs. If two products are said to be bioequivalent, they would be expected to produce the same response for all relevant effects (e.g., efficacy and toxicity). DIGESTION AND BALANCE STUDIES

The digestibility of oral drugs may be assessed (i.e., the apparent or true, fecal or ileal, availability of digesta as influenced by the drug). Digestibility can be influenced by absorption, distribution, metabolism, and elimination. For this reason, mass balance studies are preferred because they deliver additional information on quantitative excretion and retention of a nutrient or energy. Design Considerations for Nonclinical Efficacy Studies The indication being sought for the drug drives the nonclinical study design (see Experimental Design and Statistical Analysis for Toxicologic Pathologists, Vol 1, Chap 16). The desired outcomes of the study (i.e., product’s effect) should be determined early and then carefully factored into the study design to ensure that the study meets both scientific and regulatory objectives (Wooding, 1994). Adequate and well-controlled animal efficacy studies are required as a base to be used before human clinical trials to demonstrate a proof-ofconcept that the drug may have potential clinical benefit. Vehicle-controlled, masked (“blinded”) animal studies may be performed, since a placebo implies the potential for psychological benefit that usually is not imputed to animals. Blinded animal studies should be used when human testing is unethical as outlined in the Product Development under the Animal Rule (FDA, 2015). If an approved drug or drug combination (“current standard of care”) for the same indication exists, it may be used as an active “head-to-head” comparator in addition to the investigational drug and vehicle arms. Animals of both sexes should be included in most nonclinical studies, excepting those indications that are sex-specific (e.g., oral contraceptives and Hormone Replacement Therapies (HRTs)), and studies should be designed to mimic the clinical scenario (route of administration, dosing regimen, etc.) and achieve meaningful efficacy

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4. DEVELOPMENT

outcomes comparable to the endpoints desired in humans. In addition to the design characteristics already discussed in this section, the following parameters should also be addressed in study protocols for nonclinical studies. ENDPOINTS

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product to be used for pre- or postexposure prophylaxis should be administered during nonclinical studies within a reasonable window before or after exposure to the injurious agent, but before onset of disease, with a time relationship that is adequately justified with respect to the intended administration of the product to humans.

The product studied in the animal model should demonstrate a beneficial effect analogous to the intended outcome in humans. Primary study endpoints generally are the enhancement of survival or prevention/reduction of major morbidity following administration of the drug. The dose–response for these endpoints should be explored fully. In general, only primary endpoints, e.g., blood pressure, serve as the basis for marketing approval in humans, but secondary endpoints (e.g., off-target beneficial response) can provide useful information about the activity of the product.

The route of administration should reflect the disease indication and the anticipated clinical scenario. For example, if a large number of people were exposed to anthrax, a rapidly administered oral or inhaled drug formulation would be preferred over an injectable formulation for postexposure prophylaxis. It may be important to study efficacy when the drug candidate is given by multiple routes since the impact of treatment often varies depending on which route is selected.

TIMING OF INTERVENTION

DOSING REGIMEN

The time to initiate intervention should support the specific indication sought for a product. If the intent is to develop the product for an ongoing disease, intervention before disease is established in the animal model may overestimate the effect that is likely to be seen in humans, and indeed may show an effect in animals when none would be observable in humans. For example, treatment of rodents with induced arthritis or engrafted tumor cells prior to the initial evidence of disease (e.g., swollen joints, detectable masses) frequently produces a robust reduction in disease severity, while initiating treatment only after clinical disease is detected often yields a muted or no evidence of efficacy. A reasonable understanding of the disease course and a trigger for intervention defined by natural history studies in patients will be needed to design the animal efficacy studies for a treatment indication. Therefore, it is important to establish the relationship of time after exposure to effectiveness. With this information, the timing for intervention can be defined, thus differentiating whether the drug should be administered as a pre- and/ or postexposure prophylaxis (e.g., antibiotic candidate given before or at the time of surgery to prevent or minimize infection) rather than as a treatment to mitigate established disease. A

The determination of the dosing regimen (i.e., how often, and for how long, a drug must be given to achieve effectiveness) should rely on sufficient PK and PD data or other relevant product information in animals or humans. The goals should be to (1) determine a regimen in animal studies that is safe and effective for the disease indication; (2) determine the corresponding exposure parameters (i.e., Area Under the Curve [AUC], Maximum or “peak” Concentration [Cmax], Time to reach the Maximum concentration [Tmax], and Half-Life [t1/2]) in animals that is yielded by that dosing regimen; and (3) calculate a dosing regimen in humans that should give an equivalent exposure to that seen in the animal. Obtaining this information will enable initial extrapolation from a dosing regimen found to be efficacious in the animal model to one expected to produce a similar benefit in humans, assuming similar exposure– response relationships for both species.

ROUTE OF ADMINISTRATION

Risk Mitigation Methods Following receipt of authorization to conduct Phase I trials based on adequate nonclinical data, clinical development begins. In nonhigh risk FIH clinical trials, most are completed without any signs of toxicity. However, highrisk FIH trials need close attention to reduce

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1. OVERVIEW OF DRUG DEVELOPMENT

the risk to those humans first exposed to the drug. Here the risk is twofold: safety for the patient and potential negative return on investment depending on the outcome. To address this question, a risk/benefit evaluation will require nonclinical knowledge to provide data regarding potential health risks in those being treated with the drug (see Risk Assessment, Vol 2, Chap 16; Risk Management and Communication: Building Trust and Credibility with the Public, Vol 2, Chap 17). Dose Escalation Dose escalation is a fairly standard procedure in Phase I (dose range finding) clinical studies, and sometimes nonclinical studies, using arithmetical (linear) or geometrical (nonlinear) increases in doses. Various factors that should be considered when developing the plan for dose escalation include the steepness of the slope of dose–effect relationship, steepness of the dose–toxicity relationship (safety margin), therapeutic range in nonclinical efficacy models, predictability (raw estimate) of the effects of the next dose step, potential pharmacological effects, if any, and potential toxic effects. Phase I (dose range finding) studies represent the first in vivo human exposure to new molecules. These trials consist of one or two arms. The usual single-arm dose escalation phase aims to establish the maximum tolerated dose (MTD) using a standard 3 þ 3 design. The traditional standard dose escalation schedule in the development of cancer therapeutics uses the so-called “3 þ 3 design” to avoid selection of a Phase II clinical trial dose that causes a treatment-limiting toxicity in more than 17% of subjects, a standard considered acceptable as an outpatient therapeutic for patients with limited options and life-threatening diseases. In a “3 þ 3 design,” three patients are initially enrolled into a given dose cohort. If there is no Dose Limiting Toxicity (DLT) observed in any of these subjects, the trial proceeds to enroll additional subjects into the next higher dose cohort. If one subject develops a DLT at a specific dose, an additional three subjects are enrolled into that same dose cohort. Development of DLTs in more than one of six subjects in a specific dose cohort suggests that the MTD has been exceeded, and further dose escalation is not pursued (Chabicovsky and Ryle, 2006).

In current practice, however, Phase I trials often are being prolonged by the inclusion of Dose Expansion Cohorts (DECs) in order to better characterize the toxicity profiles of experimental agents and to study disease-specific cohorts. This second dose expansion phase accrues more patients, often with different eligibility criteria, and additional information (e.g., toxicity, pharmacokinetic, efficacy, or other endpoints). The assumption for the larger twoarm Phase I trial is that, regardless of the design being used for dose escalation during the DEC, the neighborhood of a target dose with an acceptable rate of toxicity will be identified. Thus, an initial estimate of the MTD can be refined through using dose expansion close to the initial estimate of the MTD (Iasonos and O’Quigley, 2016a). Phase I studies usually do not include more than one DECs. It is common to see the DEC built on an initial dose escalation study that may have 20–30 patients or more. The uncertainty in the estimation of the MTD obtained during the first Phase I arm, the dose upon which the whole of the DEC is currently based, sometimes requires a dose escalation study prior to the DEC (second Phase I arm), with the purpose of better adapting it to the needs of DEC. Before beginning the DEC phase, those dose levels that will be used for expansion must be identified (Iasonos and O’Quigley, 2016b).

5. OTHER HEALTH PRODUCTS 5.1. Biologics For the most part, discovery and development processes for biological products parallel those for small molecule (chemically derived) drugs (see Protein Therapeutics, Vol 2, Chap 6, and Nucleic Acid Pharmaceutical Agents, Vol 2, Chap 7). Nonclinical studies for biologics and other novel therapeutic classes, such as cell therapies (see Stem Cells and Other Cell Therapies, Vol 2, Chap 10), gene therapies (see Gene Therapy and Gene Editing, Vol 2, Chap 8), and vaccines (see Vaccines, Vol 2, Chap 9), are used to provide proof-of-concept and safety data. In some instances, data from nonclinical studies for biological and cell-based products are of limited use in predicting potential human responses

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5. OTHER HEALTH PRODUCTS

because of the high specificity of many biologicals for the human target. The high specificity for human molecules, which is too divergent from functionally comparable molecules in nonclinical animal species, generally yields limited or no on-target efficacy or toxicity during conventional studies in animals (with the exception in some instances of nonhuman primates). The nonclinical safety testing of biological products is clearly defined in ICH guidance S6(R1) (ICH, 2021c), which emphasizes the importance of using human-relevant nonclinical models for safety testing. Thus, for each model selected it is important to establish what degree of relevance the in vitro or in vivo animal model has to the human target. In some cases, two pharmacologically relevant species are available for toxicity studies, and both are used. In other cases, only one relevant species, or on occasion none, is available. In these situations, an incompletely relevant species (usually nonhuman primates) often is used for characterization of potential off-target effects. Toxicity study types and durations for biomolecule-based products are similar to those conducted for chemically derived drugs. In many cases, the dosing scheme for nonclinical testing of biologics may be less frequent than daily administration, and study design adjustments (e.g., weekly dosing for proteins or nucleic acids, single administration for cell therapies, or gene therapy vectors) are incorporated to better reflect the expected clinical use. Single-dose and repeated-dose toxicity studies are performed and often include integrated safety pharmacology and/or immunotoxicity endpoints. Developmental And Reproductive Toxicity (DART) studies and/or juvenile toxicity studies are conducted (see The Role of Pathology in Evaluation of Reproductive, Developmental, and Juvenile Toxicity, Vol 1, Chap 7), or an analysis is provided that sufficiently establishes the risk or lack of risk for pediatric populations; this information is used to support development of the product label. Genetic toxicity and carcinogenicity studies are often of little use in evaluating the safety of biologics as the established assays are not relevant for many biologically derived molecules due to their specificity for human targets. Immunogenicity is an expected but “off-target” response of the animal immune system to a foreign antigen and is of particular relevance for many

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protein-based products (e.g., peptides, fusion proteins, and monoclonal antibodies). Thus, the immunological properties of the protein or antibody (e.g., formation of AntiDrug Antibodies [ADAs] or initiation of immune-mediated vascular inflammation) should be monitored during nonclinical studies (Leach et al., 2021). The need for ADME studies is addressed in ICH S6(R1) (ICH, 2021c) and summarized here (see Biotherapeutics ADME and PK/PD Principles, Vol 1, Chap 4). These studies should be conducted where there is appropriate species or mechanistic relevance to be studied. Many biologicals have extremely short or very long residence in circulation (bound to blood cells or in plasma), and as with chemically derived drugs it is important to document exposures to establish human relevance. For many protein-based molecules, ADAs can develop and lead to neutralization or clearance of the test molecule (i.e., limited exposure), and it is important to document this in clinical trials as well as animal toxicity studies. For some molecules, the biologic activity is measurable in blood or tissues, and this is an important endpoint to monitor as the activity of the molecule may continue at the active site long after the protein or antibody is no longer measurable (e.g., circulating levels in the blood, receptor occupancy). Should the product be a combination product like an Antibody–Drug Conjugate (ADC), then it is necessary to monitor for the separate components of the product (which may be released if the parent molecule is metabolized) as well as the impact of the complete product (Figure 1.8). Viral contamination of a biological product is a consideration in biologics production. Such contamination can arise from a characterized cell bank used to generate the product, where latent or persistent viral infection is possible, and from adventitious (unintended) viruses introduced during manufacture. Screening for viral contamination is important for all biotechnology products, but cell-derived products such as those harvested from hybridoma cells grown in vivo to release therapeutic proteins into ascites fluid are of particular concern. A similar situation exists for xenotransplantation of pig organs, where endogenous porcine retroviruses have been posited as a concern for cross-species infections (see Genetically Engineered Animal Models in Toxicologic Research, Vol 1, Chap 23).

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FIGURE 1.8 Typical process for producing biopharmaceuticals.

As described in ICH Q5E (ICH, 2021a), which addresses comparability of biotechnology products subject to changes in their manufacturing process, manufacturers of biological products frequently make changes to manufacturing processes of biomolecules and cells both during development and after approval. When changes are made, the manufacturer generally evaluates the relevant quality attributes of the new product against the original formulation. If adequate comparability is not confirmed, it may be necessary to conduct additional nonclinical or clinical studies before the new product can be approved.

5.2. Medical Devices Medical devices are a broad range of products that have either direct or indirect, often extended or even permanent contact with patient tissues. Definitions vary by nation or region, but devices include instruments, machines, implants, and in vitro reagents, among other categories. Devices can be as simple as tongue depressors or examination gloves with direct contact to

patients, pumps and infusion devices as simple as peristaltic pumps or as complex as heart– lung machines that deliver fluids and medicines to the patient, diagnostic equipment such as radiographic (X-ray) or ultrasound machines, or in vitro diagnostic test kits. Combination products incorporate a therapeutic agent and a device (e.g., an implantable or disposable solid material coated with or containing a treatment, such as autoinjectors for biologics delivery and drugcoated vascular stents) or a therapeutic agent and a diagnostic kit (where only patients who achieve a positive signal using the kit will be treated with the drug). A detailed consideration of biomaterials used in devices and considerations for their safety assessment is presented in Biomedical Materials and Devices, Vol 2, Chap 11. During the medical device development process, the goal is to provide a device that consistently achieves the intended performance while doing so in a safe manner. All aspects are tested in a manner that not only includes qualification of materials and performance but also the ability for patients and technicians to properly operate the device.

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The classification or categorization of these devices relates to the level of risk, which in turn has a relationship to the degree of invasiveness or contact the device has with the patient (ISO, 2021) (see Biomedical Materials and Devices, Vol 2, Chap 11). As the degree of risk increases, especially if contact will be for an extended period or permanent, the development and regulatory processes for devices increase in complexity. While there are some differences in device classification or categorization among the United States, Europe, Canada and other national regions, standard convention classifications are based on risks. A generalized classification scheme includes the following categories: 1. Lowest riskdthe device poses little threat to the patient or user when used incorrectly, such as oxygen masks, handheld surgical tools, tongue depressors, elastic bandages, and examination gloves. 2. Intermediate risk (greater possibility of harm)dif the device fails or is used incorrectly, they could pose a risk to both the patient and the user, including contact lenses, radiographic machines, powered wheelchairs, infusion pumps, surgical drapes, surgical needles and suture material, and acupuncture needles. 3. High risk (likely to cause harm)dthe device often will injure the patients and technicians

if they fail or are used incorrectly as they support or sustain life, are implanted in the body, or have the potential for unreasonable risk of illness or injury. This category includes pacemakers, breast implants, digital mammography, invasive and noninvasive glucose monitors, implantable middle ear devices, and some diagnostic tests (e.g., kits to detect human immunodeficiency virus [HIV]). For the lowest risk devices, the manufacturer provides notification to the drug regulatory authority that the device meets the established requirements, and approval for marketing is fast (Premarket Notification in the United States and “self-declare” in EC). In contrast, high-risk devices undergo extensive nonclinical and clinical testing to demonstrate their effective performance and safety (Table 1.1). Device discovery and concept development are the first steps in the process that can vary in complexity depending on the expected device classification. For low-risk devices, the design is likely to be simple, and the development will focus on compliance with established requirements, good manufacturing standards, and establishing a process to monitor and track performance and adverse events. As device complexity increases, the concept design becomes more detailed and includes development of mandatory performance standards and

TABLE 1.1 Categorization of Medical Devices That Informs the Biological Evaluation Requirements

Category

Contact

AdLimited (£24 h) BdProlonged (>24 h to 30 days) CdLong term (>30 days)

Surface medical device

Intact skin

A, B, or C

Mucosal membrane

A, B, or C

Breached or compromised surface

A, B, or C

Externally communicating medical device

Blood path, indirect

A, B, or C

Tissue/bone/dentin

A, B, or C

Circulating blood

A, B, or C

Implanted medical device

Tissue/bone

A, B, or C

Blood

A, B, or C

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device-specific testing requirements along with preparation of a detailed product label with performance instructions and safety information. Devices in this category typically will undergo clinical studies evaluating performance and safety. Early in the development phase a prototype of the device is developed (and designated as not intended for human use) and tested in controlled laboratory settings. This is an important stage where device design and potential use are refined. The prototype process attempts to help reduce risk to people (ISO, 2021). Small devices may be tested in rodents, but large devices often have to be tested in larger nonrodent species (e.g., pigs, sheep) in order to accommodate the full-scale item intended for use in humans. Biological testing covers a broad range of endpoints and study types. With respect to toxicity, these may include cytotoxicity, sensitization, irritation or intracutaneous reactivity, pyrogenicity, implantation effects, hemocompatibility, conventional studies (e.g., acute, subacute, and chronic toxicity; reproductive and development toxicity; carcinogenicity), and degradation of materials in tissues. Beyond testing the intact device for performance and safety, there is a requirement for materials to be tested for extractables and leachables. The type of testing to be conducted is influenced by the categorization, and the degree and type of tissue contact the device requires. The nonclinical testing is expected to adhere to Good Laboratory Practice guidance (see Pathology and GLPs, Quality Control and Quality Assurance in a Global Environment, Vol 1, Chap 27). For devices that have the potential to cause harm, a premarket scientific and regulatory review process precedes approval. This includes providing nonclinical and clinical scientific evidence that the health benefits of the device outweigh the possible risks to the patient. Many national or competent authorities https://www.camd-europe.eu continue to monitor device performance after the device has been authorized. International standards are commonly adopted by national regulatory authorities to support a globally consistent approach to medical device development. Two organizations that issue international standards are the International Organization for Standardization (ISO) (ISO, 2021) and the International Electrotechnical Commission (IEC) (IEC, 2021). The ISO 10993-1 standards address the biological responses to medical devices with a focus

on the nature and duration of the contact with human tissues. It is intended to be used during device development to guide selection of appropriate materials and performance standards that are suitable for the intended use.

5.3. Natural Health Products Traditional medicines and herbal remedies do not come under the ICH guidelines (see Herbal Remedies, Vol 3, Chap 4). Some countries do, however, have a regulatory framework for natural health products (NHPs). For example, NHPs sold in Canada are subject to the Natural Health Products Regulations under the Foods and Drug Act, which came into force on January 1, 2004. In the United States, FDA uses the term “botanicals” to regulate products that include plant materials, algae, macroscopic fungi, or combinations of these products (USA FDA, 2016). A botanical drug intended for use in diagnosing, curing, mitigating, or treating disease would meet the definition of a drug under the U.S. Food, Drug and Cosmetic Act and would be subject to regulation as such. The description and intended use of a botanical product are important to defining specific testing and development requirements, and it is advisable to seek input from FDA before submitting an IND application. Pharmacology and toxicology requirements for IND and NDA submissions for a botanical drug are generally considered to be the same as those for a nonbotanical drug.

5.4. Vaccines Vaccine development is primarily targeted to infectious diseases that have global implications as most recently was experienced during the SARSCov-2 pandemic (see Vaccines, Vol 2, Chap 9). While national regulatory authorities develop regional guidance for vaccine manufactures, the World Health Organization (WHO) has an overarching role in establishing expectations for vaccine development. From a nonclinical safety perspective Annex 1 WHO Guidelines On Nonclinical Evaluation Of Vaccines is a valuable resource (WHO, 2005). In addition to use in pre- and postexposure prophylaxis, vaccine development is also occurring for use against infectious diseases (e.g., human immunodeficiency virus [HIV] and human papillomavirus [HPV]). Advances in biotechnology and basic immunology have resulted in development of a broad range of vaccines that include

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6. CLINICAL TRIALS

nucleic acid–based vaccines, viral vector vaccines, recombinant fusion protein vaccines, and genetically altered attenuated live vaccines that may also be combined with novel adjuvants. Nonclinical testing is required before moving a candidate vaccine to the clinic. These studies are aimed at defining the in vitro and in vivo characteristics of candidate vaccines as they relate to safety and immunogenicity. For vaccine research, biosafety requirements are important considerations and studies should be conducted in accordance with GLPs and/or GxPs. The term GxP encompasses a broad range of compliance-related activities such as Good Laboratory Practices (GLP), Good Clinical Practices (GCP), Good Manufacturing Practices (GMP), and others. Potential safety concerns for vaccines include those due to inherent toxicities of the product, toxicities of impurities and contaminants, and toxicities that result from interactions between vaccines components present in the vaccine formulation. The immune response induced by the vaccine may lead to side effects needing evaluation. Nonclinical safety studies are designed to include immunogenicity and pharmacodynamic studies to establish “proof of concept” information and the immunologic characteristics related to dose, schedules, and routes of administration for use in clinical trials. Study of the immunogenicity response should include assessment of humoral and/or cell-mediated immune responses in vaccinated animals. Where possible, functional immune responses (e.g., neutralizing antibodies, opsonophagocytic activity, etc.) leading to protection should be characterized. The basic nonclinical toxicity assessment is similar to that conducted for small molecules and other biological products. The goal is to provide information to ensure that it is reasonably safe to proceed to clinical trials. Where possible, a species sensitive to the biological effects of the vaccine should be used; rabbits are commonly used for testing due to their size, fecundity, and status as nonrodents (see Animal Models in Toxicologic Research: Rabbit, Vol 1, Chap 18). One relevant species is generally sufficient for use in toxicity studies to support initiation of clinical trials. Toxicity studies should address potential for local inflammatory reactions and possible effects on draining lymph nodes, systemic toxicity, and immune system function.

Developmental toxicity studies are usually not needed for vaccines indicated for immunization during childhood. If the target population includes pregnant women or women of childbearing potential, then developmental toxicity or juvenile toxicity studies should be considered. The primary concern is for effects on the developing embryo, fetus, or newborn. It is important to evaluate maternal antibody transfer to verify exposure of the embryo or fetus to the maternal antibody. Genotoxicity and carcinogenicity studies are normally not needed for the vaccine candidate. Specific considerations may be needed for particular types of vaccines. Development of novel adjuvants or additives require evaluation in a manner similar to that of excipients and small molecule drugs. Compatibility of adjuvants and additives with the vaccines (e.g., lack of immune interference) should be evaluated. For live attenuated vaccines, an assessment of the degree of attenuation and stability of the attenuated phenotype are important attributes to be studied. If use of genetically modified organisms is the basis for the live attenuated vaccine, then an environmental risk assessment may be needed.

6. CLINICAL TRIALS Human clinical trials are conducted to investigate the efficacy and safety of a drug candidate in humans. Testing starts with a relatively low systemic exposure in a small number of volunteer subjects (Phase I). This is followed by clinical trials (Phases II and III) in which exposure to the drug candidate usually increases by duration and/or size of the exposed patient population. However, there is a growing trend to merge phases of clinical development. The global distribution of clinical trials for various pharmaceutical products can be seen in Figure 1.9. Clinical trials may be extended based on the demonstration of adequate safety in previous clinical experience as well as on additional nonclinical safety information that becomes available as clinical development proceeds (Brody, 2012). The details of clinical trial ethics, institutional review boards, data handling, and many other subjects central to clinical trial design and execution have been reviewed elsewhere (Turner, 2012). A summary of the clinical trial phases, including

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1. OVERVIEW OF DRUG DEVELOPMENT

FIGURE 1.9 Number of clinical trials by country or region. Source: World Health Organization 2020: Global Observatory on Health R&D. https://www.who.int/observatories/global-observatory-on-health-research-and-development/ monitoring/number-of-clinical-trials-by-year-country-who-region-and-income-group (Accessed July 13, 2022).

objectives, design, duration, population, sample size, and data focus is provided in Table 1.2. Serious adverse clinical or nonclinical findings ascribable to a drug effect influence the decision regarding whether continuation of clinical trials is appropriate. These findings should be evaluated to determine the appropriateness and design of additional nonclinical or clinical studies.

6.1. Phase I Clinical Trials Phase I studies include the initial introduction of a drug candidate into humans to assess whether the drug product is safe. These studies are usually performed in 15–100 subjects and most often are conducted in healthy volunteer male subjects. In certain cases (e.g., antineoplastic agents, cell and gene therapies), Phase I trials are

undertaken in patients since healthy individuals cannot derive benefit from the treatment and therefore ethically cannot be exposed. The FIH exposure is aimed at determining the highest dose that can be given without adverse reactions to healthy volunteers or patients. The aim of Phase I trials is to establish the initial safety profile of the drug product. They are designed to determine the metabolic and pharmacologic actions of the drug in humans, the side effects associated with increasing doses, and frequency of administration and, if possible, to gain early evidence of efficacy. In Phase I studies, regulators can impose a clinical hold for reasons of safety because of a sponsor’s failure to accurately disclose the risk of study to investigators, or because of the observation of unanticipated and/or severe adverse effects in ongoing nonclinical studies.

I. PRODUCT DISCOVERY AND DEVELOPMENT

TABLE 1.2 Comparison of Clinical Trial Phases Phase 1

Phase 2

Phase 3

Phase 4

Objectives

Determine the metabolic and pharmacological actions and the maximum tolerated dose (MTD)

Evaluate effectiveness, select dose for Phase 3, determine the short-term side effects, and identify common risks for a specific population and disease

Obtain additional information about the effectiveness on clinical outcomes and evaluate the overall riskebenefit ratio in a demographically diverse sample

Monitor ongoing safety in large populations and identify additional uses of the agent that might be approved following another marketing authorization application

Factors

BioavailabilityeBioequivalence edose proportionality Metabolism Pharmacodynamics epharmacokinetics

Bioavailability; drugedisease interactions; drugedrug interactions; efficacy at various doses; pharmacodynamics epharmacokinetics; and patient safety

Drugedisease interactions; drugedrug interactions; dosage intervals; riskebenefit information; and efficacy and safety for subgroups

Epidemiological data Efficacy and safety within large, diverse populations Pharmacoeconomics

Data

Vital signs Plasma and serum levels Adverse events

Doseeresponse and tolerance Biomarkers Adverse events Efficacy

Laboratory data Efficacy Adverse events

Efficacy Pharmacoeconomics Epidemiology Adverse events

Design

Single, ascending dose tiers Uncontrolled

Placebo-controlled comparisons Active controlled comparisons Well-defined entry criteria

Randomized Controlled Two to three treatment arms Broader eligibility criteria

Uncontrolled Observational

Duration of study

Up to 1 month

Several months

Several years

Ongoing (following marketing authorization)

Population

Healthy volunteers or individuals with the target disease (e.g., cancer)

Individuals with target disease

Individuals with target disease

Individuals with target disease as well as new age groups, genders, etc.

Number of test subjects

15e100

25e300

Hundreds to thousands

Thousands

Example

Study of a single dose of Drug X in normal subjects

Double-blind study evaluating safety and efficacy of Drug X versus placebo in patients with a given disease

Study of Drug X versus or in addition to a standard treatment in an antidepressant

Study of economic benefit of newly approved Drug X versus standard treatment for hypertension

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1. OVERVIEW OF DRUG DEVELOPMENT

6.2. Phase II Clinical Trials Phase II clinical trials include the early controlled studies conducted to obtain some preliminary data on the effectiveness of the drug for a particular indication or indications, i.e., does it work? Usually in Phase II clinical trials, everyone gets the same dose. However, some Phase II studies randomly assign people to different treatment groups. These groups may get different doses or get the treatment in different ways to see which provides the best balance of safety and response. Placebos (inactive treatments) are not used in Phase II trials. These trials are performed in 25– 300 patients of both sexes who have the disease or condition. The Phases of clinical trials can be expanded to “subphases.” Phase II studies can be broken down into Phase IIa and Phase IIb, where Phase IIa is a pilot clinical trial to evaluate effectiveness and safety in selected populations of patients and Phase IIb studies are wellcontrolled trials to evaluate efficacy and safety in patients. The aim of these studies is to evaluate whether the drug works as intended (i.e., Phase IIa) and to determine the minimally efficacious dose (i.e., Phase IIb) to be tested in Phase III studies. This phase of testing also helps determine the common short-term side effects and risks associated with the drug. These clinical trials usually represent the most rigorous demonstration of a drug candidate’s efficacy and are considered pivotal trials.

6.3. Phase III Clinical Trials Phase III studies are large (multicenter) and expensive, controlled and uncontrolled clinical trials performed in several hundred to several thousand patients of both sexes. These trials aim to determine whether the new drug candidate is safe and effective in the real patient setting, and whether it constitutes an improvement over medications that may be available on the market. The dosage form is typically the final drug product intended for marketing. These studies are large and expensive, generally involving multiple centers around the world. They are also logistically complicated with many sources of potentially confounding variables. Phase III studies should provide an adequate

basis for extrapolating the results to the general patient population and transmitting that information in the product label. Participants are usually randomly assigned to receive the current standard treatment or the new drug product. Sometimes, when there is an efficacious treatment for the condition, it is considered unethical to use the new drug alone and it needs to be compared on top of the standard treatment, looking for improvement. If possible, the trial is double-blinded: neither the researchers nor the participants know which participant receives which drug product. Double-blinded clinical trials help researchers see the actual benefits and side effects of a treatment without bias or outside influence and help to normalize for the placebo effect. Results from a randomized, double-blinded trial are considered more credible than results from a trial that is not randomized or double-blinded. Similar to Phase II trials, Phase III trials can be split into two arms: Phases IIIa and IIIb. The Phase IIIa trials are conducted after effectiveness of the drug candidate is demonstrated in earlier phases, but prior to regulatory submission of a marketing application or other dossier, and in patient populations for which the medicine is eventually intended. They generate additional data on both safety and efficacy in relatively large numbers of patients in controlled and uncontrolled conditions, often providing much of the information needed for the package insert and labeling of the medicine. Phase IIIb clinical trials are initiated after the marketing authorization application is filed, but before such product obtains regulatory approval, the goal of which trial is to provide additional data for marketing support and the launch of such product.

6.4. Phase IV Clinical Trials Postmarketing trials conducted after a medicine is marketed provide additional details about the drug product’s efficacy or safety profile and are known as Phase IV. These studies are primarily observational or nonexperimental in nature, to distinguish them from wellcontrolled Phase IV clinical trials or marketing studies. Different formulations, dosages, durations of treatment, medicine interactions, and

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7. POSTMARKETING SURVEILLANCE

other product comparisons may be evaluated, as can administration to new age groups, races, and other patients with other diseases. Detection and definition of previously unknown or inadequately quantified adverse reactions and related risk factors are an important aspect of many Phase IV studies. If a marketed medicine is to be evaluated for another new indication, then those clinical trials are considered Phase II clinical trials. In general, additional nonclinical studies are not required to support these new Phase II efforts.

6.5. Limitations of Clinical Trials Most trials assess relatively healthy patients with only one disease and mostly exclude specific demographic groups, often those with particular vulnerabilities such as pregnant women, children, and elderly people. Hence, these exclusions limit the patient population during clinical development to a homogeneous group of patients. Since these studies necessarily limit the number of subjects, they may not allow the observation of rare adverse effects that may be seen once thousands of people in the general population are treated. Furthermore, clinical trials have to limit the length of treatment, and therefore typically preclude the discovery of long-term adverse consequences such as cancer. In addition, there is an inability to predict the outcomes associated with real world, day-to-day use of the drug, concomitant dosing with other substances (including “recreational drugs” and alcohol), polypharmacy, and other situations that cannot be predicted from clinical trials.

7. POSTMARKETING SURVEILLANCE Postmarket surveillance is an essential part of the drug product life cycle, which monitors for adverse drug events (ADEs) and addresses the safety, and sometimes effectiveness of a health product. Reporting of such reactions is harmonized across regions by use of the World Health Organization’s (WHO)’s Adverse Reaction Terminology (ART), which will eventually become a subset of the International Classification of Diseases (WHO, 2015).

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7.1. Adverse Drug Events ADEs can be considered an injury resulting from the use of a drug. Under this definition, the term ADE includes ADRs, overdoses, and effects of dose reductions and discontinuations of drug therapy. ADEs may result from medication errors, but most do not.

7.2. Adverse Drug Reactions An ADR is a “response to a drug which is noxious and unintended, and which occurs at doses normally used in humans for prophylaxis, diagnosis, or therapy of disease or for the modification of physiologic function.” Note that there is a causal link between drug exposure (at normal doses) and an ADR. ADRs may occur within minutes or only years after exposure. One ADR is a Life-Threatening Adverse Drug Reaction (LTADR). Such effects may manifest as inpatient hospitalization or prolongation of existing hospitalization, a persistent or significant disability, or a congenital anomaly/birth defect, all of which are classified as Serious Adverse Events (SAEs). Important medical events that may not result in death, be life-threatening, or require hospitalization may be considered a Serious Adverse Drug Experience (SADE) when, based upon appropriate medical judgment, they may jeopardize the patient and may require medical or surgical intervention to prevent one of the outcomes listed in this definition. Examples of such medical events include allergic bronchospasm requiring intensive treatment in an emergency room or at home, blood dyscrasias or convulsions that do not result in inpatient hospitalization, or the development of drug dependency or drug abuse. Unexpected Adverse Drug Experiences (UADEs) are any events that are not listed in the current labeling for the drug product or have not been previously observed, which includes events that may be symptomatically and pathophysiologically related to an event listed in the label but differ from the event because of greater severity or specificity. For example, under this definition, hepatic necrosis would be unexpected if the label only referred to elevated serum hepatic enzyme activities or hepatitis. Similarly, cerebral thromboembolism and cerebral vasculitis would be unexpected if the labeling only listed cerebral vascular accidents. “Unexpected,” as

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1. OVERVIEW OF DRUG DEVELOPMENT

used in this definition, refers specifically to an adverse drug experience that has not been previously observed, and thus was not included in the labeling, rather than generically to all effects that are not anticipated from the pharmacological properties of the pharmaceutical product. Most ADRs are rare, often idiosyncratic, and generally observed first when the drug enters the market. Most countries record ADRs, and routinely evaluate them for possible signals, but the reporting rate for such events is usually less than 10%.

7.3. Current Mechanisms and Tools for Identifying and Quantifying ADRs Protecting society against the adverse effects of drugs requires early detection, valid verification, TABLE 1.3

and quantification. The frequency and severity of adverse events, dose relations, the time course, and susceptibility factors need to be established insofar as possible (Sahu et al., 2014). Guidance on pharmacovigilance and safety reporting has been issued by the ICH (Individual Case Safety Report (ICSR) [E2B(R3)] and Development Safety Update Report (DSUR) [E2F] ICH, 2021f) (Table 1.3). ADEs and ADRs cannot be detected without astute professional observers, with case reports being among the most important tools for observational research. All people exposed to a new drug comprise the potential population susceptible to ADRs. In a hypothetical scenario, a country with some 100 million inhabitants, a 1% yearly cumulative exposure to a drug would equate to 1,000,000 people using the

Current Mechanisms and Tools for Identifying and Quantifying Adverse Drug Reactions

System/tool

Purpose

Example

Medical literature

Rare signal detection

Case reports

Voluntary Spontaneous Reporting Systems

Surveillance of the entire population

Canada: Vanessa’s Law. https://www. canada.ca/en/health-canada/ services/drugs-health-products/ medeffect-canada/adverse-reactionreporting/mandatory-hospitalreporting.html [accessed December 3, 2020]. USA: Adverse Event Reporting System database; MedWatch

Monitoring centers (national)

“Yellow Card” type reporting scheme for severe adverse events, events in new drugs, or children

UK MHRA Yellow Card Scheme

Monitoring centers (WHO)

EU and Japan participate

Confirmation and quantification in denominator-based systems

Follow general or specific populations (e.g., patients with cystic fibrosis) based on prescription/ exposure; allow comparison to unexposed persons and nested caseecontrol studies

WHO Collaborating Centre for Drug Statistics Methodology system; UK modified Prescription Event Monitoring (via questionnaires filled in by general practitioners; Layton and Shakir, 2011); and Population claims event databases

Good pharmacovigilance practice

Set standards

Followed in Japan and EU

Good postmarketing practice

Set standards

Followed in Japan

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7. POSTMARKETING SURVEILLANCE

drug at any time during that year. A rare ADR with an incidence of 1 in 10,000 might be detected in such a population, particularly when the adverse effect has a low background incidence, making it easily recognized. When an adverse effect is nonspecific and has an appreciable background incidence, detection is more difficult. Therefore, the medical literature is probably the most effective system for initial ADR detection as case reports are detailed, assessed for quality by reviewers, mostly independent from commercial incentives, and open to all interested parties. Voluntary spontaneous reporting systems are, undoubtedly, the most cost-effective approach for postmarket identification of new ADRs despite their limitations. The goal of the evaluation of ‘spontaneously’ reported information is to provide a signal of new problems or of an increased incidence of problems. The main drawback of spontaneous reporting is the TABLE 1.4

37

absence of an adequate control group of unexposed patients and, consequently, the difficulty of knowing the relative risk of treated patients, compared to untreated patients, unless the event is extremely rare in the population (Table 1.4). National ADR monitoring centers and the WHO Collaborating Centre for International Drug Monitoring (CCIDM) are also used. They can be productive if active and well-qualified staff consider the detection of adverse reactions as their primary objective and resources are adequate. Most of them are working with a “Yellow Card System”; for example, the UK Medicines and Healthcare Products Regulatory Agency (MHRA) is using this type of system to collect data for severe adverse events, adverse events connected to new drugs, and adverse events in children. The purpose of the scheme is to provide an early warning that the safety of a medicine or a medical device may require further investigation. It is important for people

Pharmacovigilance Procedures and Tools by Health Authority (Selected Countries Only)

Authority

Tool

Examples

USFDA

Voluntary reporting databases

Adverse Event Reporting System (AERS)

EMA

Mandatory updates by marketing authorization holders (MAHs)

Periodic Safety Update Report (PSUR); Development Safety Update Report (DSUR)

Data sharing mechanisms (voluntary, country based)

Postmarketing surveillance of Mutual Recognition Procedure drugs

Annual reassessment

On the anniversary of marketing authorization; considering riskdbenefit ratio in the light of new safety findings

EudraVigilance

Centralized computer system for the capture of adverse events; input from EU regulatory agencies and pharmaceutical companies

Reexamination and reevaluation (mandatory for MAH)

Safety and efficacy must be reconfirmed for 8 years following marketing authorisation

Mandatory adverse drug reaction reporting (MAH)

Ad hoc electronic reporting to PMDA since 2006; periodic safety update reports

Mandatory adverse drug reaction reporting (MAH)

Periodic Safety Update Reports (PSUR)

Voluntary reporting by health professionals and patients

Included in updates of official Product Monographs

Canadian adverse reaction newsletter

Information distribution on adverse events; by regulatory authority Health Canada

Japan

Canada

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1. OVERVIEW OF DRUG DEVELOPMENT

to report problems experienced with medicines or medical devices as these are used to identify issues which might not have been previously known about. Reports can be made by anyone, in electronic or paper format. Denominator-based reporting systems allow for improved detection, quantification, and comparison to unexposed persons. One example is the United Kingdom Modified Prescription Event Monitoring (PEM) that can link prescription-dispensing information (e.g., drug, dose, duration, and date) to data for procedures or other medical interventions via unique patient identifiers (Layton et al., 2011). Finally, exposurespecific registries attempt to identify and follow patients who have been exposed to a drug but have not yet had an adverse outcome. Limitations of any of the above approaches prevent precise quantification of the incidence of drug-related adverse events. These limitations can be due to moderate response rates (e.g., 55% for the modified PEM), imprecise language describing the events, variable temporal relationship, and many more. Taking these limitations into account, monitoring systems probably provide a better picture together than any single method alone. Care should be taken to use and interpret data from these different sources in the proper perspective. The use of several methods and data sources yielding consistent results provides strong support for the hypothesis that a suspected adverse effect is, indeed, drug related. Possible regulatory actions in response to the detection of negative side effects and safety concerns can vary from continuing observation of health products to canceling the marketing authorization. Intermediate steps may include mandating postmarketing studies; product labeling changes and altered packaging; dissemination of information to healthcare professionals and consumers about the risks (e.g., letters, advisories, publications, specialized internet sites); addition of “warnings” in patient information leaflets; issuing public alerts; or conducting market withdrawals.

8. REGULATORY AUTHORITIES 8.1. Overview The purposes of regulatory agencies are that citizens have access to safe and effective

medicines, that drug labeling accurately reflects available product information for the product, and that drug manufacturing standards assure consistency of the product for public use. Many regulatory authorities exist in countries around the world (Brock and Hastings, 2012). The entities guiding drug development that will be discussed here include global multinational consortia seeking international harmonization of development principles and techniques (ICH); regulatory agencies in the United States, Canada, European Union, and the United Kingdom; and selected regulatory authorities in Asia and South America. While there is a general drive toward standardization and harmonization of procedures and quality parameters, significant differences exist in terms of the scope among these authorities, the preference of a centralistic or more diversified system of regulation, the superior state authority, and the alignment with other countries along the fault lines of trade agreements (Table 1.5). International Council on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use Robust, established paths for drug development and licensing have long been available in Europe, North America, and Japan, and this resulted in 1990 in the foundation of the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) (ICH, 2020a). Today, ICH is unique in bringing together the global regulatory authorities and pharmaceutical industry representatives from Europe, Japan, the United States, Canada, Switzerland, Brazil, Singapore, Korea, China, Turkey, and Taiwan along with 17 observer nations to discuss scientific and technical aspects of drug registration. Since its inception, ICH has evolved through its ICH Global Cooperation Group to respond to the increasingly global face of drug development, so that the benefits of international harmonization for better global health can be realized worldwide. ICH’s mission is to achieve greater harmonization to ensure that safe, effective, and high-quality medicines can be developed and registered worldwide in the most resourceefficient manner. In doing this, ICH has established benchmarks for submission documents around the world.

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8. REGULATORY AUTHORITIES

TABLE 1.5

39

Health and Regulatory Authorities and Legislation-Selected Countries

Country

Regulatory/Reviewing Authorities

US

FDA (CDER, CBER)

Section 21 of the Code of Federal Regulations (CFRs); Federal Food, Drug and Cosmetic Act (1906)

Canada

Health Canada

Food and Drugs Act (1920); Food and Drugs Regulation (1947; last amended 2008)

EU

EMA

EU Regulation 2309/93, Directives 2001/82/EC and 2001/ 83/EC; Directive 2010/84/EU and Regulation (EU) No 1235/2010; Regulation (EU) No 1027/2012, Directive 2012/ 26/EU

UK

MHRA (part of the Department of Health and Social Care)

The Human Medicines Regulations 2012 (SI 2012/1916); various amendments of the Human Medicines Regulation

Japan

PMDA (part of the MHLW)

Pharmaceutical Affairs Law (1943, revised 2002) and Law for Partial Amendment of the Pharmaceutical Affairs Law (2006); Law for Ensuring Quality, Efficacy, and Safety of Drugs and Medical Devices (short Pharmaceuticals and Medical Devices Act (2014, revised 2019) and others

China

NMPA (part of the State Administration for Market Regulation) with Centers for Drug and Medical Device Evaluation

Drug Administration Law of China (2019); SFDA Order No. 28 “Provisions for Drug Registrations” (Wang et al., 2019); Technical Guidelines for the Acceptance of Overseas Clinical Trial Data for Drugs (2019)

India

CDSCO (part of MoHFW)

Drugs and Cosmetics Act, 1940 and rules 1945, in particular Rules 122A, B and D and the Appendices I, Ia, and VI of schedule Y; New Drugs and Clinical Trial Rules (2019)

Brazil

ANVISA (COPEC, COPEA)

Federal Law no. 6360 (1976); Law No. 5991 (1973; amended by Law No. 13,097, 2015); Law no. 6437 (1977); and Law no. 9294 (1996)

Mexico

COFEPRIS (part of Ministry of Health)

General Health Law (1984, revised 2021), Health Supplies Regulation, and Official Mexican Norms

Relevant Legislation

Inherent in the ICH process is support for science-based approaches to regulation. Guidance documents are promulgated that provide advice for the degree of scientific and medical understanding that is needed to demonstrate the safety and efficacy attributes of a drug candidate as well as the technical standards for the quality of the drug product and manufacturing process (see Overview of the Role of Pathology in Product Discovery and Safety Assessment, Vol 2, Chap 2). United States and Canada As of today, the United States is still the most economically important area for new drug

filings, with the highest number of new marketing applications per year worldwide (Keyhani et al., 2010). The FDA is the federal agency in the United States that regulates human and veterinary drugs, biologic products (including proteins, nucleic acids, and cell and gene therapies), medical devices, and other therapeutic products to assure that they are safe and effective. In addition, foods, cosmetics, and products that emit radiation fall under their purview. For these products, FDA is charged with ensuring they are also honestly, accurately, and informatively represented to the public.

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1. OVERVIEW OF DRUG DEVELOPMENT

In the FDA, the Center for Drug Evaluation and Research (CDER) is responsible for regulating drugs, and the Center for Biologics Evaluation and Research (CBER) regulates biological and related products including blood, vaccines, monoclonal antibodies, allergenics, tissues, and cellular and gene therapies. FDA’s role in the development of drugs begins with submission of an Investigational New Drug (IND) application by a sponsor to alert the FDA of its intention to test its diagnostic or therapeutic potential in humans (Table 1.6). At that point, the molecule changes in legal status under the Federal Food, Drug, and Cosmetic Act and becomes a new investigational drug subject to specific requirements of the drug regulatory system. Most regulations pertaining to food and drugs documents are provided in Section 21 of the Code of Federal Regulations (CFR) which contains most regulations pertaining to food and drugs documents. The IND is a request for FDA authorization to administer a drug candidate to humans. Such authorization must be secured prior to interstate shipment and administration of any new drug TABLE 1.6

US Federal Regulations Pertaining to an IND Application

Code of federal Regulations (CFRs)

Regulatory Requirements

21CFR Part 312

Investigational New Drug (IND) application

21CFR Part 314

IND and New Drug Applications (NDAs) for U.S. Food and Drug Administration (FDA) approval to market a new drug (new drug approval)

21CFR Part 316

Orphan drugs

21CFR Part 58

Good Laboratory practice for nonclinical laboratory (animal) studies (GLP)

21CFR Part 50

Protection of human subjects

21CFR 56

Institutional review boards

21CFR Part 201

Drug labeling

21CFR Part 54

Financial disclosure by clinical investigators

that is not the subject of an approved new drug application. The IND contains three broad areas of information: animal pharmacology and toxicology studies, and manufacturing information. The typical scientific disciplines that review an IND include pharmacology (animal pharmacology and toxicology), chemistry (manufacturing and processing procedures related to preparing the investigational drug), clinical (medical review of the clinical study protocol), and microbiology when applicable (evaluating in vitro and in vivo effects on the virus and microorganisms targeted by the drug). Like the FDA, Health Canada is involved in drug regulation throughout the development process. Every drug developer has to ask for a Clinical Trial Application (CTA) before testing any drug on the Canadian public. The drug marketing application review (or New Drug Submission [NDS]) is based on the successful operation of the three principles of Benefit-Risk Assessment and Management; the Precautionary Principle; and Shared Responsibility (which includes provincial and territorial governments, health professionals, researchers, industry, and the public). The precautionary principle enables decision-makers to adopt measures when scientific evidence about a human health hazard (or environmental hazard) is uncertain and the stakes are high. Before a prescription or overthe-counter drug can be sold in Canadian stores, the pharmaceutical company must seek a Notice of Compliance (NOC) from Health Canada. Besides monitoring product quality and ADRs, Health Canada also enforces the observation of advertising limitations. Europe and the United Kingdom The European Union (EU) is a distinct case as it is not one country but a federation of independent countries. Therefore, the initial remit of its regulatory authority, the European Medicines Agency (EMA), was to coordinate the scientific evaluation of the safety, efficacy, and quality of medicinal products (including human and veterinary products) across multiple member states. In particular, EMA’s remit is to: 1. Facilitate development medicines. 2. Evaluate applications authorization.

I. PRODUCT DISCOVERY AND DEVELOPMENT

and for

access

to

marketing

8. REGULATORY AUTHORITIES

3. Monitor the safety of medicines across their life cycle. 4. Provide information to patients and healthcare professionals. In the European system, applications for the authorization of medicinal products can be made via a centralized or a number of decentralized procedures. The centralized procedure is compulsory for certain types of products (e.g., biotechnology derived), is made directly to the EMA, and leads to a marketing authorization by the European Commission (EC) that is binding in all EU member states. Decentralized procedures include marketing authorization applications in a number of member states participating in a process involving a “Reference Member” (since 2004), in the mutual recognition of a product that is already licensed in one member state (Mutual Recognition Procedure, MRP), or in purely national marketing authorizations. The legislative framework of the EMA consists of Regulation 2309/93EC and Directives 2001/ 82/EC and 2001/83/EC. The Directives lay down provisions governing the marketing authorization, manufacture, and distribution of drug products. In 2012, a legal framework for pharmacovigilance was introduced that aimed to simplify and streamline processes for detecting, assessing, and managing adverse drug events and give a greater role to EMA (Directive 2010/84/EU and Regulation (EU) No 1235/2010) in accomplishing this task. This advance resulted in the establishment of the Pharmacovigilance Risk Assessment Committee (PRAC). Since leaving the EU in January 2020, the United Kingdom no longer participates in EU institutions and the Medicines and Healthcare products Regulatory Agency (MHRA) is the sole UK regulatory authority. However, until January 1, 2023, Great Britain will continue to adopt new marketing decisions in the community marketing authorization procedure, with special regulations for Northern Ireland. Mechanisms for an accelerated assessment procedure with rolling data reviews exist in both the EU and United Kingdom and were applied in licensing COVID-19 vaccines in 2020 (EMA, 2022). Asia The PMDA (Pharmaceuticals and Medical Devices Agency) (PMDA, 2021) is the Japanese

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regulatory agency under the umbrella of the Ministry of Health, Labour and Welfare (MHLW). Its remit is to assure the safety, efficacy, and quality of pharmaceuticals and medical devices. PMDA conducts scientific reviews of marketing authorization applications of pharmaceuticals and medical devices, and monitoring of their postmarketing safety. They also provide compensation for individuals who experience adverse drug effects. Modern pharmaceutical legislation originated in Japan with the enactment of the Regulations on Handling and Sales of Medicines in 1889. Various regulations apply to the development, manufacture, import, marketing, and proper use of drugs and medical devices in the form of the Law for Ensuring Quality, Efficacy, and Safety of Drugs and Medical Devices (short: Pharmaceuticas and Medical Devices Act (PMDA; 2014, revised 2019), cabinet orders, MHLW ordinances, etc. Formal approval and licenses must be obtained prior to market launch from the Minister of the MHLW or prefectural governors. Drug approval reviews are normally processed in the order of application, but priority status can be assigned (e.g., for drugs treating orphan diseases). Multiple studies have demonstrated that many drugs are metabolized differently in persons of Asian descent, which can lead to serious side effects (Bancroft, 2020). For this reason, Japanese and other Asian regulators are very cautious about accepting foreign clinical trial data and require an assessment of each drug with regard to characteristics that make it sensitive to ethnic factors. If ethnic factors are likely to have an impact on efficacy or toxicity, Japanese authorities require bridging trials in local populations unless a sufficient number of Japanese patients were included in the submitted clinical trials. The Chinese National Medical Products Administration (NMPA) supervises the safety of food, drugs (including traditional Chinese medicines), medical devices, and cosmetics; regulates their registration; and undertakes postmarketing risk management. New licensing applications submitted to NMPA are reviewed by the Center for Drug Evaluation or the Center for Medical Device Evaluation, as appropriate. During the last decade, the NMPA went through a series of organizational and legal changes with the aim to simplify and accelerate the drug licensing process. In the new system,

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foreign companies are entitled to hold marketing authorizations while using a Chinese manufacturer, whereas in the past the marketing authorization was tied to the manufacturer. A number of fast-track options for regulatory approval exist (e.g., conditional approval or priority review process for certain products). Because of the mentioned differences in drug metabolism for persons of Asian descent, China required clinical data collected in China for all marketing authorizations until 2019 (Bancroft, 2020). Development of a drug in China can be integrated in the global development of the drug by simultaneously running some clinical trials in China. Since 2018, NMPA resides under the umbrella of the newly created State Administration for Market Regulation (SFDA). SFDA Order No. 28 “Provisions for Drug Registrations” regulates the registration of new and generic drugs as well as drug imports. It is underpinned by the Drug Administration Law of China (Wang et al., 2019). NMPA is a regulatory member of ICH. The Central Drugs Standard Control Organisation (CDSCO) as part of the Ministry of Health and Family Welfare (MoHFW) is the national regulatory authority of India. Its head is the Drugs Controller General of India. The remit of CDSCO is to assure the safety, efficacy, and quality of drugs including vaccines and biologicals, medical devices, and cosmetics. While CDSCO is responsible for license approval of specified drug categories (e.g., blood products and vaccines), the regulation of other drugs is primarily the concern of the various Indian state authorities, with CDSCO acting as scientific advisor and ensuring harmonization within the Indian market. CDSCO is an observer of ICH, and India has adopted the ICH Common Technical Document (CTD) format for submissions. The legal framework for the regulation of drugs in India is set out in the Drugs and Cosmetics Act (1940) and Rules (1945), in particular, Rules 122A, B, and D and Appendices I, Ia, and VI of schedule Y, which collectively describe the information required for drug import or approval. The Drugs and Cosmetics Act also specifies the respective responsibilities of the CDSCO and the State authorities, which creates a significant degree of complexity for drug submissions in India due to differences among the various authorities (Nupur et al., 2015).

In the past, India did not accept foreign clinical trial data, necessitating clinical trials in local populations. This changed drastically in 2019 with the issuing of the “New Drugs and Clinical Trial Rules” (Bancroft, 2020). Local trials can now be waived if the drug is already approved in the United States, EU, the United Kingdom, Japan, or Australia; no major adverse effects have been observed; and the drug effect is not considered to be influenced by ethnic factors. Efforts to harmonize the regulation of pharmaceuticals and their registration have intensified worldwide since the 1990s. The Association of Southeast Asian Nations (ASEAN) is an observer of ICH, and numerous ICH guidance documents have been adopted by its member states (ICH, 2021e). ASEAN has also adapted its own version of the CTD document. The climate in ASEAN countries is typically hot and humid, therefore drug stability studies receive special attention. Latin America In Latin America, substantial harmonization efforts have been ongoing since the 1990s, mainly through the initiative of the Pan American Health Organization (PAHO) via the Pan American Network for Drug Regulatory Harmonization (PANDRH). PAHO currently recognizes six reference authorities for medicines in Latin America (Mexico, Brazil, Argentina, Chile, Cuba, and Colombia; https://www.paho.org/en/topics/ medicines-and-health-technologies). In spite of these harmonization efforts, marketing applications currently need to be planned according to local national requirements. Mexico and Brazil are discussed here as regional examples. The Mexican federal commission for sanitary risk (Comisio´n Federal para la Proteccio´n contra Riesgos Sanitarios, COFEPRIS) holds mutual recognition agreements with Health Canada and the FDA for the regulation of drugs and medical devices and is connected with the United States and Canada through the North American Free Trade Agreement (NAFTA). Through its strong performance, COFEPRIS acquired a reputation for early, speedy approvals of medicines. COFEPRIS is an administrative authority of the Ministry of Health, and the regulatory authority for pharmaceuticals in Mexico. The relevant legislative framework is laid out in the General Health Law (Ley General de Salud 1984,

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revised 2021), Health Supplies Regulation, and Official Mexican Norms. The scope of this regulatory framework includes drugs, biological products, homeopathics, medical devices, and traditional medicines. Clinical trials are also regulated by the Health Law Regulations for Health Research (Reglamento de la Ley General de Salud en Materia de Investigacio´n para la Salud, last revised 2014) (RLGSMIS) and the Official Mexican Standards (Normas Oficiales Mexicanas) (NOMs) for Health Research in Human Beings. Currently, local harmonization and mutual recognition agreements are in place with the regulatory and health authorities of Chile, El Salvador, Ecuador, and Colombia (Valverde, 2015). Together with Argentina, Paraguay, Uruguay, and Venezuela, Brazil is a member of the Mercosur trade bloc established in 1991. Since 1995, Mercosur members share common standards on requirements and inspection of pharmaceutical drug production facilities, requirements for pharmaceutical ingredients and raw materials, as well as common guidelines on GMP (Valverde, 2015). The Brazilian health authority ANVISA (Agencia Nacional de Vigilancia Sanitaria), founded in 1999, has a particularly extensive remit. It includes the safety of food, drugs, cosmetics, and medical devices as well as drug licensing and regulation of clinical trials; the authorization of drug manufacturers; and the examination of drug patents. ANVISA is home to the department for Coordination of Clinical Research with Drugs (COPEC) which is dealing with drugs and biologicals, and the department for Coordination of Clinical Research with Devices and Food (COPEA). ANVISA is a regulatory member of ICH. The legal framework is set out in several Brazilian statutes. Federal Law No. 6360 of September 23, 1976, is the main statute for pharmaceutical products. It regulates the production, commercialization, advertising, labeling, inspection, quality control, penalties, importation, and marketing approval of medicines, drugs, active ingredients, and medical devices; Law No. 5991 (1973; amended by Law No. 13,097, 2015) established sanitary controls of drugs; Law no. 6437 (1977) provided penalties and criminal sanctions; and Law no. 9294 (1996) described restrictions on use and advertising of medicines and therapies.

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8.2. Considerations on Legal Frameworks The foundations and framework for the approval process for new drugs in the United States, Europe, Canada, and Japan are provided within each country’s legislation. In general, the main legislation deals with the question of what to regulate (e.g., drugs for human use, but this can extend also to veterinary drugs, homeopathics, ethnic medicines, medical devices, and cosmetics). Additional topics addressed in each nation’s legal system include how to define these items; acceptance criteria for safety and efficacy; and scientific guidance for the regulatory path toward marketing authorization. Early regulations were concerned with ensuring that food and drugs were not adulterated. The 1876 “Act to Impose License Duties on Compounders of Spirits and to amend the Act Respecting Inland Revenue to Prevent the Adulteration of Food, Drink and Drugs” was Canada’s first consolidated effort to regulate the safety of food. In the United States, the Federal Food, Drug and Cosmetic Act (FD&C) was originally enacted in 1906 and subsequently amended to strengthen and broaden the mandate. The underlying requirement is for a sponsor to provide substantial evidence to support claims of effectiveness for new drugs. Inherent in the concept of effectiveness are the three major areas of focus for regulatorsdefficacy, safety, and quality. There are three defining provisions of the FD&C Act that continue to shape the new drug development process. The first is definition of the “drug” and the products to be regulated as drugs: “articles intended for use in diagnosis, cure, mitigation, treatment, or prevention of disease in man .” and “articles (other than food) intended to affect the structure or any function of the body of man ..” Second, the meaning of “new drug” is defined, thereby identifying which products are subject to the requirement of the new drug approval process (excluding animal drugs and animal feed containing a new animal drug). Finally, general criteria are identified that all new drugs must meet in order to gain marketing approvaldeach drug candidate must be the subject of an FDA-approved new drug application (NDA), which must contain adequate data and information on the drug’s safety and “substantial evidence” of the product’s effectiveness.

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The interpretive and discretionary powers granted to the FDA under the FD&C Act provide wide-ranging authority. Within this context, it is the FDA that defines what a sponsor must present as substantial evidence for each new drug. This is done on a case-by-case basis to determine the scientific testing needs and the nonclinical and clinical data required to support new drug registration. The FDA perspective and interpretation on all aspects of drug development are shared in guidance documents. The so-called Redbook (Toxicological Principles for the Safety Assessment of Direct Food Additives and Color Additives Used in Food) is a good place to start https://www.fda. gov/regulatory-information/search-fda-guidance -documents/redbook-2000-i-introduction. FDA supports the ICH process as a mechanism to achieve broad consistency across regions. Concerns about the safety of drugs that have been counterfeited continue to our time. On April 2008, an amendment to the Food and Drugs Act, Canadian Bill C-51 was tabled in the House of Commons. The purposes of this bill were to modernize the regulatory system for foods and therapeutic products, to strengthen the oversight of the benefits and risks of therapeutic products throughout their life cycle, to support effective compliance and enforcement actions, and to enable a greater transparency and openness of the regulatory system. Some of the proposed amendments were to make the sale and importation of products that have knowingly been adulterated illegal, to make the sale of counterfeit therapeutic products illegal, and to clarify in the Food and Drugs Act requirements for therapeutic products to have market authorization, which has been required by Health Canada for many years. Major failures of drug development can prompt momentous legislative changes. In Canada, the thalidomide tragedy of the early 1960s prompted a complete revision of regulations to strengthen the Department of Health’s regulatory abilities. The revision marked the first appearance of the requirement for manufacturers to submit evidence of efficacy in seeking a Notice of Compliance for marketing authorization. In the EU, Directive 2010/84/EU and Regulation (EU) No 1235/2010 were implemented in 2012 in order to simplify and streamline pharmacovigilance processes. Following the withdrawal

of Mediator (benfluorex), legislation was further amended to allow prompt notification and assessment of safety issues (Regulation (EU) No 1027/2012, Directive 2012/26/EU). The collection of information on adverse events (AEs) is now supported by mandatory risk management plans for all new medicines; the requirement for annual PSURs; a central EMA repository for PSURs; the requirement for authorization holders to run a Pharmacovigilance Master File; and an improved legal basis for postauthorization safety studies (with input from PRAC). There is a commercial side to drug development, and this is reflected in the Canadian Proprietary or Patent Medicine Act (1909). It was the first Canadian legislation to register medicines, which was limited to secret-formula, nonpharmacopeial packaged medicines. The Act was also the beginning of the protection of the public against drugs administered without medical supervision. Finally, the Patented Medicine Prices Review Board (PMPRB) was created in Canada in 1987 under the federal Patent Act. This is an independent, quasi-judicial body responsible for ensuring that the prices of all patented medicines, including prescription and over-the-counter drugs, vaccines, biologics, and veterinary drugs, sold in Canada are not excessive. The Board was created as a quid pro quo for the abolition of compulsory licensing of medicines. In the United Kingdom, the National Institute for Health and Care Excellence fulfills a similar function. During the late 1990s and early 2000s, Brazil led a global initiative to ensure affordable access to AIDS medication for patients worldwide that involved a World Trade Organization (WTO) dispute with the United States. In this context, Brazil in 2001 introduced a resolution entitled “Access to Medication in the Context of Pandemics such as HIV/AIDS” to the UN Commission on Human Rights, which was duly ratified. This activism changed global attitudes to patient access and drug pricing worldwide (Nunn et al., 2009).

9. SUMMARY AND CONCLUSIONS As reflected in this chapter, the life cycle of a drug is a multifaceted process that occurs over an extended period, with the most beneficial

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REFERENCES

and enduring drugs being marketed worldwide for years or even decades (e.g., aspirin). A drug’s discovery is rooted in science and a deep understanding of the underlying disease process. With knowledge of the associated biologic pathways, identification of a trigger or inhibitory step leads to an intense effort to identify a chemotype that allows the opportunity to maximize potency and the chemical and pharmaceutical attributes that make a chemical ‘druggable.’ Ensuing pharmacology and animal safety studies start the process of evaluating the drug’s benefit–risk profile. These animal data are extrapolated to design early human clinical trials that lead to the conduct of larger, definitive clinical trials to evaluate the drug’s benefit in the intended patient population. Benefit–risk profiles are not universal but are targeted to the disease condition and patient population. Risk tolerance for adverse effects on a drug for a debilitating or lifethreatening condition like cancer will be higher than for a drug for a disease or condition that has a milder expression, is nonlife threatening, and for which one or more alternate drug therapies provide adequate intervention (e.g., headaches). In parallel with studies evaluating the safety and therapeutic benefit, extensive research is conducted to develop a high-quality manufacture process. Manufacturing must consistently deliver the intended performance of the product from batch to batch. Upon approval the marketplace acceptance of a new drug is dependent on a combination of the inherent efficacy of the drug for the disease or condition as well as comparative benefits with competitor drugs vying for market share. The safety of a drug continues to be monitored postapproval and continues throughout the life cycle of the product. The advertising and promotion of the drug must remain true to the approved use claims. As a drug’s use is expanded to larger patient populations and the number of patient-years of drug exposure increase, new ADRs that were not observed during clinical trials may become evident due to the expanded use. Identification of significant safety signals lead to updates to product labels and, in a worse-case scenario, could lead to a drug product being removed from the market. In the absence of safety concerns, a drug product will remain in the marketplace as long as the therapeutic benefits

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and sales continue to warrant a company’s effort to market the product. The globalization of drug development has expanded the opportunity to extend the benefit of new drug therapies to patients worldwide. This global expansion further complicates the drug life cycle, as regional differences in language, laws, and regulatory requirements need to be addressed to achieve approval around the world. While harmonization of regulatory agency advice and guidance has begun through the efforts of international consortia like the ICH, much work remains to be done to fully achieve this goal. On the horizon, expanded emphasis by government regulators on the scientific integrity of drug development, more in-depth safety reviews in the early postmarketing days that could lead to limitations on product introductions, expansion of personalized medicine and healthcare reforms that influence the sales, marketing, and reimbursement of medicines will further evolve and influence the life cycle of drugs. The challenge of managing all facets of this process will become increasingly complex, and the greater awareness pharmaceutical scientists from all disciplines have of the product life cycle, its critical milestones, and how to influence critical decisions the greater the contributions these scientists will make toward improving the probability of success.

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Zambrowicz BP, Sands AT: Knockouts model the 100 bestselling drugs–will they model the next 100? Nat Rev Drug Discov 2(1):38–51, 2003.

Selected Links to Regulatory Authorities and Issues Australia Therapeutic Goods Administration (TGA) http:// www.tga.gov.au (Accessed December 23, 2020). Code of Federal Regulations. 21CFR.312.23. “Drugs for Human Use.” Investigational Drug Application. IND Content and Format. http://www.accessdata.fda.gov/ scripts/cdrh/cfdocs/cfcfr/cfrsearch.cfm?cfrpart¼312 (Accessed December 23, 2020). European Medicines Agency (EMA): http://www.ema. europa.eu (Accessed December 23, 2020). EMEA: Committee for human medical products, final report on the pilot joint EMEA/FDA VXDS experience on qualification of nephrotoxicity biomarkers, 2008. In http://www.ema.europa.eu/ docs/en_GB/document_library/Regulatory_and_procedural_gui deline/2009/10/WC500004205.pdf. (Accessed 23 December 2020). FDA. Guidance for industry: product development under the animal rule, 2015. https://www.fda.gov/regulatoryinformation/search-fda-guidance-documents/product-de velopment-under-animal-rule. (Accessed June 22, 2021). Health Canada: http://www.hc-sc.gc.ca/index-eng.php (Accessed December 23, 2020). Health Canada link to Access to Therapeutic Products: the regulatory process in Canada: https://www.canada. ca/en/health-canada/services/drugs-health-products.html (Accessed December 23, 2020).

International Conference on Harmonization of Technical Requirements for Registration (ICH) of pharmaceuticals: http://www.ich.org/ (Accessed December 23, 2020). MedEffect – Health Canada: http://www.hc-sc.gc.ca/dhpmps/medeff/index-eng.php (Accessed December 23, 2020). Medicines and Healthcare products Regulatory Agency (MHRA): http://www.mhra.gov.uk (Accessed December 23, 2020). Pharmaceutical and Medical Devices Agency (PMDA), Japan: http://www.pmda.go.jp/english/index.html (Accessed December 23, 2020). US Food and Drug Administration (FDA) link to website: http://www.fda.gov Accessed December 23, 2020. US FDA link to Development and approval process for drugs: http://www.fda.gov/Drugs/DevelopmentApprovalProcess/ default.htm Accessed December 23, 2020. US FDA link to Product application and petition review process: http://www.fda.gov/ForIndustry/FDABasicsforIndustry/u cm234626.htm Accessed December 23, 2020. WHO (World Health Organization): WHO Expert Committee on Biological Standardization, Fifty-fourth Report, 2005. http:// apps.who.int/iris/bitstream/handle/10665/43094/WHO_ TRS_927_eng.pdf;jsessionid=E0741A291EB421CDB8F6886 A6ADF1F07?sequence=1 WHO Technical Report Series, No. 927, 2005 (Accessed April 13, 2021). WHO: (World Health Organization): WHO Pharmacovigilance indictors: a Practical Manual for the Assessment of Pharmacovigilance Systems, 2015. https://apps.who.int/iris/bitstream/ handle/10665/186642/9789241508254_eng.pdf (Accessed December 23, 2020).

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C H A P T E R

2 Overview of the Role of Pathology in Product Discovery and Development James Fikes, Christopher Hurst, Eric Tien Biogen, Cambridge, MA, United States O U T L I N E 1. Introduction

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2. Discovery Toxicology 2.1. Small Molecules 2.2. Nucleic AcideBased Pharmaceuticals 2.3. Biologics 2.4. Cell and Gene Therapy

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3. Development Toxicology 3.1. Small Molecules 3.2. Nucleic Acid Pharmaceuticals 3.3. Biologics 3.4. Cell and Gene Therapy 3.5. Anticancer Drugs

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3.6. Adversity and Reversibility 3.7. Clinical Dose Setting and Clinical Safety Assessments 3.8. Regulatory Filings

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4. Nonstandard Studies and End points 4.1. Pharmacology (Efficacy) Studies 4.2. Investigative (Mechanistic) Toxicology Studies 4.3. Standard and Alternative Animal Models 4.4. Biomarker Considerations 4.5. Interpretation of Unique Findings

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find themselves in discussions regarding the selection and testing of new drug candidates and chemicals, in decisions of whether to pursue or terminate research programs, in characterizing the potential hazards associated with products in development, in defining how those hazards can be monitored in human subjects, and in translating those hazards into human risk assessments and the benefit:risk analyses critical to regulatory approval. The goal of this chapter is to introduce these diverse roles for a pathologist using the biopharmaceutical discovery and development process as a useful framework to put these activities into better context.

1. INTRODUCTION Pharmaceutical or biopharmaceutical, food, and agrochemical discovery and development require a multidisciplinary approach. The benefit of this approach is the synergy of many diverse methods, experience, expertise, research tools, and opinions that culminate in optimally designed hypothesis testing, problem resolution, and product development over the least amount of time. Pathologists play key roles in these activities spanning from assessment of new targets to hazard identification in toxicity studies to the investigative efforts exploring unexpected findings. Pathologists may

Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-12-821047-5.00013-0

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2. DISCOVERY TOXICOLOGY The origination of a drug discovery program starts with a hypothesis that intervention at a particular target in just the right way will lead to a therapeutic benefit for patients suffering from a disease. As a program starts to develop the nonclinical pharmacologic evidence that supports this hypothesis, the toxicology team begins assessment of the potential issues that may arise with this type of intervention (Blomme and Will, 2016). Toxicities associated with drug discovery come in many flavors and can be related to the target, off-target profile, or modality, among other things. Safety is one of the most common reasons for the discontinuation of a molecule or a program (Weaver et al., 2020). One of the earliest safety assessments that can be conducted for a new program is a so-called Target Safety Assessment intended to evaluate the target biology and potential impacts of pharmacologic intervention. Excessive pharmacology in organs other than the desired organ can lead to toxicities that are potentially identifiable as possible liabilities. The pathologist, with the unique combination of scientific acumen and knowledge of animal physiology, can be a major contributor to not only identifying potential toxicities based on biology but also potential methods to screen/detect said toxicities as well as potential translatability of such effects to humans (Figure 2.1). The specifics of the safety finding(s) that lead to the discontinuation of a program can come from nonclinical studies, clinical studies, or both. In discovery stage nonclinical studies, the contributions, and conclusions of the pathologist play a major role as the pathology end points are a significant contributor to the selection of new molecules that could be safely justified for use in clinical trials (see Discovery Toxicology and Discovery Pathology, Vol 2, Chap 3 for more details). In early discovery, standalone toxicology studies may not be a high priority as a project seeks to verify potential therapeutic benefit and identify a promising molecule or entity for further study. However, even in early discovery, a pathologist may be called upon to perform histopathologic assessment of tissues or interpretation of clinical pathology measurements from pharmacology studies for evidence of efficacy and/or early identification of target organ systems for

toxicity (see Pharmacology (Efficacy) Studies, Section 4.1). While sometimes useful to help identify target organ toxicities or derisk certain off-target profiles, these types of studies should be evaluated with caution as they are not intended to assess toxicity and therefore might use suboptimal study designs, animal models of disease that have not had a thorough histomorphological phenotypic assessment, or compounds/molecules that are not well characterized. The identification of a promising lead molecule typically marks the beginning of a formal toxicology program. Depending on the modality, the scope of these studies in the discovery space could cover multiple studies in multiple species or be limited in scope to simple toxicokinetic and tolerability assessments. The following sections outline some of the basic discovery stage approaches for different modalities. It should be noted that a pathologist should have a significant role in designing a customized approach to best suit the needs of the safety assessment plan.

2.1. Small Molecules A classic small molecule drug is a chemical entity typically under 1 kDa in size. While there are exceptions for small molecules isolated from natural biological sources, the majority are chemically synthesized. Compared to other modalities, small molecules require the most comprehensive toxicology evaluation due to factors such as generally lower specificity, highly permeable nature, and biodistribution/exposure to a wide range of tissues and organ systems. The large number of complex studies makes the involvement of the pathologist a critical engagement to ensure these studies are comprehensive enough to provide sufficient information to justify clinical investigation of any small molecule. In the discovery stage, small molecules are tested in a battery of in vitro and in vivo toxicity assessments. In vitro assessments including, but not limited to, off-target activity, genetic toxicity, and cardiovascular safety tend not to require involvement from the pathologist as these are cell-based or biochemically based assays that are intended for rapid screening of compound promiscuity or evaluating well-established mechanisms of toxicity (Thompson et al., 2012).

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FIGURE 2.1 Major product discovery stage (blue) and development stage (gold) milestones with associated activities (boxes) with potential roles for a pathologist. Activities are categorized as in vitro studies (yellow box), in vivo studies (red boxes), and other activities (green boxes).

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In vivo assessments start with simple pharmacokinetic assessments in multiple species, followed by single or very limited multiple dose tolerability studies and generally conclude with repeat-dose toxicity testing. For discovery projects, repeat-dose toxicity studies in rodents and nonrodents can vary in length from a few days to a few weeks in duration depending on individual sponsor’s strategy and proposed clinical plan. The pathologist plays a large role in the design of these studies due to the associated assessment of a broad range of tissues via histology as well as clinical pathology and safety biomarker parameters. Each study should be customized to support potential or known toxicity concerns and based on the biological rationale for the project and/or history with older molecules, the end points should be customized by the pathologist to suit the safety assessment and both standard and nonstandard end points can be considered. If well designed, these discovery stage repeat-dose studies identify the major potential hazards (target organs of toxicity) and establish initial no-observedeffect level (NOEL) and/or no-observedadverse-effect level (NOAEL). These results, combined with the projected efficacious doses in humans, will determine the therapeutic index (TI) or safety margin. This information contributes to an organization’s decision to progress the molecule on to development stage activities and inform the design of subsequent development stage investigational new drug (IND) application-enabling toxicology studies.

2.2. Nucleic Acid–Based Pharmaceuticals Oligonucleotides are unmodified or chemically modified single-stranded DNA molecules which hybridize to the targeted base sequence and results in gene-specific modulation. For example, modulation of targeted genes occurs through several different mechanisms including RNAse H-mediated degradation of target mRNA, splicing modifications, or by targeting miRNAs (Bennett et al., 2017). These molecules are oftentimes chemically synthesized and generally evaluated for safety under the guise of small molecules even though they tend to be larger than the traditional small molecule (Evers et al., 2015) and are discussed in more detail in Nucleic Acid Pharmaceutical Agents,

Vol 2, Chap 7. However, these agents may have some features like biologics that must be considered such as high specificity for a single target and limitations regarding cross-species activity in nonclinical species. Therefore, the pathologists may be involved early in considerations of relevant species and animal models for efficacy and discovery stage in vivo toxicity studies for these molecules.

2.3. Biologics Biologics encompass a broad range of proteinbased therapeutics such as monoclonal antibodies (mAbs), fragment antigen binding (Fab) protein (a truncated form of a mAb), and recombinant/replacement proteins (see Protein Therapeutics, Vol 2, Chap 6). These drugs are usually manufactured using cell culture systems to produce the protein with further steps to isolate and purify said proteins. Some replacement proteins such as insulin can even be isolated (Heller et al., 2007) or generated through “biopharming” (see Genetically Engineered Animal Models in Toxicologic Research, Vol 1, Chap 23) from animals and used in humans. Contrary to small molecules, biologics generally have more targeted activity with less potential for off-target activity, have a more restricted biodistribution pattern in that they cannot easily pass blood–tissue barriers (i.e., brain, retina, testes) after peripheral administration, cannot be administered orally, and cannot penetrate the nucleus of cells. These general characteristics can serve to streamline the toxicity assessment for biologics. The primary concerns with biologics center around either platform-related toxicities such as immunologic responses and immune complex formation (Yin et al., 2015) and on-target toxicities, both of which can be evaluated by the pathologist in the context of toxicology studies. The most common type of biologic is the mAb. From a toxicity evaluation standpoint, mAbs tend to have similar profiles from molecule to molecule. Unlike a small molecule where the off-target and pharmacokinetic profile can vary widely between molecules of similar structure, mAbs often have very similar physiochemical and pharmacokinetic profiles. This allows for a reasonable prediction of the distribution and clearance of mAbs which means that certain

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gene product either to enhance, repress, or correct its function. At present, the most common viruses used for this purpose are either adeno-associated virus (AAV) or lentivirus therapies. Both cell and gene therapy, as with other modalities, rely heavily on pharmacology data in the discovery stage of drug development. As noted earlier, a pathologist may be involved in interpretation of these studies depending on the models being used and the types of evaluations that are to be conducted.

properties could be logically extrapolated from one program and applied to another, even if the intended target is different (Glassman and Balthasar, 2019). This molecule-to-molecule concordance can help with toxicity assessment as not as many studies would need to be conducted. Recombinant proteins are full or partial length proteins that may or may not have been modified to give them more favorable druglike properties. Unlike mAbs, recombinant protein characteristics will be more specific to the individual molecule and likely not easily translatable from program to program. Common modifications include such alterations as PEGylation, Fc fusion, and chimeric proteins. As with mAbs, however, once a sufficient understanding of the pharmacokinetic–pharmacodynamic (PKPD) relationship is obtained, a recombinant protein would likely be suitable for toxicity testing. In the discovery stage, a biologic may only need a sufficient understanding of the general PKPD relationship to proceed to development (see ADME Principles in Small Molecule Drug Discovery and DevelopmentdAn Industrial Perspective, Vol 1, Chap 3, and Biotherapeutic ADME and PK/PD Principles, Vol 1, Chap 4) and the start of Good Laboratory Practice (GLP) studies (see Pathology and GLPs, Quality Control and Quality Assurance in a Global Environment, Vol 1, Chap 27). Therefore, prior to development, the pathologist role is most focused on the following: (1) selection of biologically relevant animal species for in vivo studies, (2) evaluation of target distribution in tissues, and (3) assessing histologic, clinical pathology, and biomarker parameters from efficacy studies.

As described above, early pharmaceutical candidates are evaluated for both safety and efficacy. By contrast, the development stage activities focus primarily on safety. The objectives of these nonclinical safety studies are to understand the dose–response relationship, identify target organs of toxicity, determine whether observed toxicities are potentially reversible, and determine dose levels at which toxicity does not occur. This information is then presented as a safety margin where the NOAEL dose from animal studies is compared to a projected or predicted clinical dose. This safety margin is then used to determine a safe starting dose for Phase I clinical trials. As clinical development continues, longer duration, repeat-dose toxicology studies are conducted, along with studies evaluating the potential for reproductive and developmental toxicity and carcinogenicity. Even after marketing approval investigative or mechanistic studies may be performed in response to data generated in humans.

2.4. Cell and Gene Therapy

3.1. Small Molecules

Cell and gene therapy can be broadly broken down into two classes: cell-based therapies and viral-based therapies (for more details see Gene Therapy and Gene Editing, Vol 2, Chap 8, and Stem Cells and Other Cell Therapies, Vol 2, Chap 10). Like biologics, these are manufactured using cell-based processes. Cell-based therapies involve the removal and ex vivo manipulation of a patient’s own cells to correct a particular defect and the reintroduction of those cells into a patient. Gene therapies are viral based where viral particles are used as a delivery mechanism meant to adjust the expression of a particular

After a successful transition from discovery into development stage, small molecules are tested in a battery of in vitro and in vivo toxicity assessments. The objective of these studies is to build on the nonclinical safety information obtained during the discovery stage and to evaluate safe-use conditions. The design of toxicology studies in discovery is generally conducted with less rigor as the objective is to make a quick decision to terminate a molecule with an unacceptable safety profile. Toxicology studies in development, however, are more robust as they contain more end points, have larger sample

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size, and are conducted under GLP conditions. These studies would include a battery of genetic toxicology studies, a battery of safety pharmacology studies (cardiovascular, respiratory, and central nervous system), and in vivo repeatdose toxicology studies in rodent and nonrodent species (Figure 2.1). If the molecule is deemed to have an acceptable safety profile, longer duration repeat-dose toxicology studies are conducted to understand the progression and potential reversibility of adverse findings. Based on the patient population and length of treatment, reproductive and developmental toxicity and carcinogenicity studies are best approached on a case-by-case basis. The pathologist provides an important contribution to the nonclinical safety understanding of the development candidate by meticulously characterizing target organs of toxicity, evaluating clinical pathology and safety biomarker data and contributing to the determination of an NOAEL.

3.2. Nucleic Acid Pharmaceuticals While many of the principles of traditional small molecule development are employed in designing the appropriate toxicology studies, unique aspects of this therapeutic modality must be considered when assessing nonclinical safety. For example, toxic effects may be mediated by both hybridization-dependent and hybridization-independent mechanisms. Not only must the potential toxicity of modulating the specific gene be assessed, but one must also consider the impact of the antisense oligonucleotide (ASO) binding to off-target genes. This unique modality provides ample opportunity for inclusion of nontraditional end points in toxicology studies for the pathologist to investigate. For example, pro-inflammatory consequences are a known class effect associated with the hybridization-independent ASOs. For this reason, additional immunotoxicity end points are often included in the clinical pathology investigation (Henry et al., 2008). Nucleic acid therapeutics are in development and have been approved for a number of clinical indications. These include, but are not limited to, oncology and diseases of the eye, brain and spinal, and metabolism (Sridharan and Gogtay, 2016). One of the challenges of antisense therapies is that it is difficult to achieve efficient

delivery to target organs other than the liver. Nucleic acids are negatively charged with a high molecular weight resulting in poor transport across membranes. Targeting ocular or neurologic disorders bring about new challenges as these therapeutics do not cross vascular endothelium, dense extracellular matrix, and cell and nuclear membranes and must be delivered directly into the eye or cerebrospinal fluid (CSF), to reach their intended targets. For example, indications where the target is in posterior regions of the eye, intravitreal or subretinal injections are required to achieve sufficient drug concentrations. Similarly, unique delivery routes (intracerebroventricular/intrathecal) to administer test articles to nonclinical species directly into the central nervous system (CNS) require skillful evaluation to determine potential procedure-related lesions from antisense-related lesions. In addition, expanded neuropathology assessments often are included in nonrodents with more complex cortical development (e.g., dogs, nonhuman primates) to provide a more robust evaluation of the CNS. This not only includes H&E evaluation but the inclusion of special neurohistochemical procedures (e.g., immunohistochemistry for cell type–specific biomarkers and histochemical stains to assess for neuronal cell death and myelin integrity) to highlight key neural domains (see Nervous System, Vol 4, Chap 8).

3.3. Biologics As previously mentioned, a significant portion of the nonclinical safety assessment of biologics is undertaken during development stage activities. Unlike small molecules where toxicity may be mediated by both on-target and off-target mechanisms, the toxicity of biopharmaceutical products is often attributed to exaggerated pharmacology due to their more narrowed target specificity. General differences between small molecules and biopharmaceuticals are presented in Table 2.1. Examples of safety concerns linked to specific biologically mediated activity include immunosuppression leading to increased risk for infections and tumors, lymphocyte stimulation leading to “cytokine storm,” and tumor lysis syndrome following the initiation of antineoplastic biologics. This requires the nonclinical safety

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TABLE 2.1

General Differences Between Pharmaceuticals and Biotechnology Products.

Pharmaceuticals

Biotech products

Previous examples

Unique

Historical database

Concurrent controls

Species independent

Species specific

Nonimmunogenic

Immunogenic

Metabolized

Degraded

Short acting

Long acting

Chronic daily dosing

Intermittent dosing

Toxicity

Exaggerated pharmacology

Specific mechanisms

Pleiotropic mechanisms

Linear dose eresponse

Bell-shaped dose eresponse curve

Direct effects

Complex temporal relationships

Complex formulations

Simple formulations

Oral route

Parenteral routes

Bioequivalence

Comparability

Modified from Cavagnaro (2002).

scientist to take a science-based approach to the nonclinical safety plan. Antibody formation to administered biotechnology products may lead to local injection-site reactions or systemic reactions; inhibition of endogenous proteins, changes in the pharmacokinetics and the potential decrease in efficacy due to neutralizing antibodies against the product (Cavagnaro, 2002). Selection of relevant species which includes an understanding of expression, distribution and primary structure of the target, binding and occupancy of the target, as well as functional consequences (including cell signaling) is critical to the nonclinical safety assessment of biologics and a pathologist should be a part of these critical considerations. In some cases, suitable test animal species (rodent/nonrodent) may require use of alternative approaches, such as the use of transgenic models, assessment of an animal surrogate molecule (homologous proteins), and/or animal

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models of human disease. Each of these options carries significant risks in making accurate potential safety claims and the models should be thoroughly evaluated and understood in advance to ensure that results generated from these models are not spurious. As with any animal model used for safety evaluation, it should be well characterized to include extensive review by the pathologist to understand background anatomic and clinical pathology characteristics of the models (see Section 4, Standard and Alternative Animal Models and Models of Toxicity: Genetically Engineered Animals (rodent, non-rodent, nonmammalian), Vol 1, Chap 23). While the safety assessment between biologics and small molecules shares some overlap (i.e., use of rodent and nonrodent species if pharmacologically relevant, general considerations for design and duration of repeat-dose toxicity studies, and immunotoxicity evaluation if relevant), there are notable differences. For example, carcinogenicity studies have generally not been required for therapeutic antibodies and peptides. This is based upon the evidence that these molecules are not able to gain access to the nucleus and interact with DNA. Unique to antibody and antibodylike molecules, tissue cross reactivity (TCR) studies are generally performed for mAbs on a panel of tissue cryosections from human and nonclinical toxicology species (e.g., nonhuman primate) to assess tissue binding profiles of mAbs by evaluating patterns of on-target (CDRmediated) and off-target (non-CDR-mediated) immunolabeling. Immunolabeling similarities between the two species have been used to increase the confidence that hazards identified in the animal species might be relevant to predicting outcomes in human studies as well. The pathologist is critical to this assessment as they are involved not only in optimization of the immunohistochemistry reagents and protocol but also in the interpretation of the study.

3.4. Cell and Gene Therapy Gene therapy treatment modality is based on the introduction of exogenous nucleic acid into cells to alter the course of a medical condition or disease. Gene therapy products mediate their effects by replacing a missing or defective gene, introducing a gene variant, or introducing a novel gene for the purpose of inducing endogenous

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gene editing. This modality poses specific safety issues that need to be addressed before clinical use, to protect subjects to whom they will be administered. Like nonclinical development for biotherapeutics, species selection for gene therapy products is critical. Considerations include susceptibility to infection with the viral vector, transduction tropism, pharmacological response to the transgene, similar anatomy and physiology to humans, immunogenicity, and route of administration (Bolt et al., 2020). The pathologist and toxicologist work closely to evaluate and decide on an appropriate species to enable characterization of safe-use conditions for these products. In-depth review of nonclinical safety considerations can be found in Gene Therapy and Gene Editing, Vol 2, Chap 8, and Stem Cells and Other Cell Therapies, Vol 2, Chap 10, but a key somewhat unique component of the nonclinical toxicology studies is an assessment of the biodistribution of the vector DNA and transgene expression (mRNA or protein) in tissues. Polymerase chain reaction (PCR) can be used to detect vector DNA and transgene expression in blood, major organs, and gonads (Assaf and Whitely, 2018). The general organ biodistribution can be further refined by a pathologist using histomorphological-based techniques such as in situ hybridization (ISH) for vector DNA and ISH and immunohistochemistry (IHC) for transgene expression of mRNA and protein, respectively. This information is critical to assess both safety and efficacy. Like other modalities, cell and gene therapies present their own challenges for the pathologist. There are several potential platform/modalityrelated toxicities that must be monitored and evaluated. For cell-based therapies, oncogenesis is of concern since the cells are being manipulated with the intent of effecting a permanent change before reintroduction into a patient. For gene- and viral-based therapies, oncogenesis is also a concern owing to the potential integration of viral or transgenic DNA into the patient genome particularly with lentivirus-based therapies that are intended to integrate into the genome. Gene-editing technologies that are utilized in both cell and gene therapies also fall within this type of concern. In nonclinical studies, the pathologist evaluation of any

possible signs of tumorigenesis or oncologic activity, coupled with detailed molecular analyses, contributes to the risk assessment. Nonclinical study design elements provide unique opportunities for the pathologist as these studies employ nonstandard routes of administration, have underlying toxicities (Hordeaux et al., 2020; Batty and Lillicrap, 2019) associated with this modality, and need to evaluate potential immune responses in response to the transgene. Furthermore, there may be instances where immunosuppression is needed to mitigate a potential toxicity and the pathologist, toxicologist, and clinical representatives would be discussing an appropriate regimen that is closely aligned with what is being proposed in the clinic (Bolt et al., 2020).

3.5. Anticancer Drugs For anticancer drug candidates, especially ones that might be indicated for serious, lifethreatening, tumors, the nonclinical development may be more accelerated and differ from the traditional approach for small molecule and biological drugs. This is because the clinical population is typically terminal cancer patients and the effective dose may be close to the toxic dose (ICH S9, 2009). However, nonterminal anticancer therapies or drugs delivered to improve the quality of life in cancer patients typically undergo the same nonclinical safety assessment required for most therapeutic candidates (ICH M3, 2009). For anticancer drugs, pathologists are routinely involved during discovery stage activities as the evaluation of anticancer candidates normally includes a combination of rodent models of human tumors for in vivo efficacy assessment. The most used models are immunomodulated rodents with subcutaneous or orthotopically placed xenografts of human cancer cell lines or fresh human patient derived tumors. These models are used for target evaluation, understanding functional modulation, and impact on tumor growth. In their evaluation of the target expression and tumor response to treatment, pathologists leverage routine and molecular techniques (ISH/IHC) as well as image analysis. As an anticancer candidate moves into later stage discovery and on into development, the pathologist’s project involvement is typical for that previously described

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for small molecules and biologics. In addition to the cytotoxic chemotherapeutics initially used for oncology, there has been a tremendous increase in molecularly targeted therapeutics. This has made it possible to develop companion diagnostics to help identify patients most likely to respond and/or least likely to show adverse events in association with a specific treatment. These diagnostics, also known as “personalized medicine,” serve as predictive biomarkers for selecting the right drug for the right patient. Many of these companion diagnostics use IHC or ISH to identify the presence or overexpression of the target in a tumor biopsy and pathologists are involved in the development and validation of these diagnostic tests in parallel to the development of the companion therapeutic. Treatment of cancer is one area that has witnessed notable use of combining multiple molecules together to form a multifunctional pharmaceutical agent such as antibody-drug conjugates or bispecific antibodies. These are, in one sense, no different than any other product in that they require rigorous toxicity testing but they generally will need more customized approaches that borrow principles and standards from the different modalities and molecule types that are involved. A pathologist would be expected to weigh in heavily regarding the potential toxicity of the individual components and whether those toxicities would be present in the combined product as well as if any new, previously unseen toxicities might arise.

3.6. Adversity and Reversibility As described previously in this section, the major objectives of nonclinical safety assessment studies are to determine target organs, the dosage or exposure level where toxicities are observed, and whether toxicities are potentially reversible. This information contributes to considerations for safe starting dose decisions and overall risk assessment for human clinical trials. Inclusion of a treatment-free (nondosing) period following the main dosing-phase of a toxicology study is included in at least one nonclinical safety study when merited to understand whether toxicities observed during or at the end of the dosing period are partially or fully reversible. If a treatment-related finding in a nonclinical study demonstrates reversibility, it

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can be extrapolated that should a similar finding be seen in patients that there may be a reasonable expectation the change is not permanent. Evidence of reversibility, especially if changes can be monitored (preferably in a noninvasive or minimally invasive manner), may allow exploration of doses in clinical studies with smaller safety margins (Rosenfeldt et al., 2010). By contrast, if a toxicity in a nonclinical species is irreversible, it suggests the possibility the injury may be permanent in patients and the safety factor for the first-in-human (FIH) starting dose may be increased (FDA, 2005). As part of a development team, a pathologist is essential during study design discussions of whether to include a recovery group(s) in toxicology studies. Extending this to study execution, pathologists are uniquely qualified because of their training, knowledge of cellular and molecular processes, and experience to assess from recovery group animals the toxicological significant of findings, the potential clinical translation of clinical pathology and histopathologic findings, the likelihood of lesion reversal, and whether non/minimally invasive methods (i.e., imaging, fluid-based, biomarkers, or clinical pathology) are sufficient to monitor recovery without histopathology (Pandher et al., 2012; Perry et al., 2013; Tomlinson et al., 2016). Although a treatment-induced effect may demonstrate partial or full reversal, that is not sufficient grounds to interpret a finding as nonadverse. Treatment-induced effects that do not damage the stromal framework of the extracellular matrix (some kinds of necrosis in a parenchymal organ) are completely reversible and yet may be adverse. Determination of adverse findings and ultimately the NOAEL are critical aspects of nonclinical toxicity studies and are discussed in detail in Assigning Adversity to Toxicologic Outcomes, Vol 2, Chap 15. Publications by the Society of Toxicologic Pathology (STP) and European Society of Toxicologic Pathology (ESTP) have aimed to facilitate a more consistent approach to the interpretations of test article– related effects as adverse, assigning an NOAEL, and clearer communication of these critical results in nonclinical study reports (Kerlin et al., 2016; Palazzi et al., 2016). Fundamentally, adverse is defined as a term indicating “harm” to the test animal, while nonadverse indicates lack of harm. In addition, it is

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important that adverse findings in study reports should be defined in relation to effects on the test species used and within the context of the given study. Through the primary histopathology evaluation and peer-review process, the study pathologist makes the determination of whether an effect is treatment related followed by whether the finding is considered adverse in the context of the study. The ultimate assignment of an NOAEL for a toxicology study requires the integrated consideration of all serious findings; but frequently, treatment-related pathology findings are the basis for the NOAEL. Therefore, decisions regarding adverse findings and the NOAEL in the final study report should combine the expertise of all contributing scientific disciplines to include the pathologist. Ultimately, the pathologist, along with other contributing subject matter experts who interpret data from nonclinical studies, should be active participants in assessing and communicating human risk.

3.7. Clinical Dose Setting and Clinical Safety Assessments Throughout the staged approach to nonclinical drug development, results of the nonclinical safety studies must be synthesized and claims about the potential impact on human health must be conveyed to regulatory authorities. For example, after completion of an IND-enabling package, the nonclinical findings in the toxicology species are examined and assessments are made based on information about the pharmacology, pharmacokinetics, and toxicity of the molecule. The process for recommendation on the starting dose in the clinic is outlined in the FDA guidance on Estimating the Maximum Safe Starting Dose in Initial Clinical Trials for Therapeutics in Adult Healthy Volunteers. Simply, the guidance recommends determination of the NOAEL in the animal species, conversion of the NOAEL to a human equivalent dose (HED), selection of the most sensitive species and then application of a safety factor. In general, the safety factor is usually a factor of 10 or more. Not only has the pathologist analyzed the histopathology data from the individual study reports, but now the totality of the data is synthesized to determine the most sensitive/most relevant species to aid in starting

dose selection. While there are other approaches for determination or modification of starting dose such as the minimal anticipated biological effect level or pharmacologically active dose (EMEA, 2017; Leach et al., 2021), pathology input is equally important for all methods and they play an integral role in determination of the NOAEL as well as FIH dose selection. Another important responsibility of the pathologist is the recommendation for inclusion of additional safety end points or biomarkers in clinical studies based on the results of the nonclinical safety assessments.

3.8. Regulatory Filings Nonclinical safety data from the GLP toxicology studies are an integral part of the submissions to regulatory agencies. These data include a characterization of the toxic effects with respect to target organs, dose dependence, relationship to exposure, and potential reversibility. Results of those studies are used to extrapolate to humans, in terms of setting a safe starting dose for FIH clinical trials (ICH M3, 2009; Seaton, 2014). With the progression of clinical development, additional nonclinical studies are conducted to evaluate the potential effects of chronic dosing, reproductive and developmental toxicity, and carcinogenicity. The nonclinical safety data are integrated and combined with the clinical data and submitted to regulatory bodies for marketing approval. At each phase of clinical development, the pathologist is evaluating, interpreting, and putting the toxicology findings into context to support future development.

4. NONSTANDARD STUDIES AND END POINTS The previous sections provided a modalitybased framework for where pathologists have a role in new product discovery and development. However, in vivo studies evaluating pharmacology (efficacy) and investigating product mechanism of action and early toxicity may have broad application across modalities and are further highlighted in this section. In addition, special, study-related parameters a pathologist may have a role in, such as the choice and

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qualification of animal models and biomarkers and the interpretation of unique findings, are also highlighted in this section.

4.1. Pharmacology (Efficacy) Studies New pharmaceuticals go through a series of pharmacology studies (see Principles of Pharmacodynamics and Toxicodynamics, Vol 1, Chap 5) to investigate the mechanism of action (MOA) of a new compound and demonstrate desired effects (efficacy) against the target (see Discovery Toxicology and Discovery Pathology, Vol 2, Chap 3 for more details). These discovery stage pharmacology studies generally consist of a combination of in vitro assays and/or in vivo disease models. The in vitro assays allow early assessment of a product’s potential for on- and off-target binding, cellular activity such as changes in gene expression and protein profile, release of biochemical mediators, changes in cell cycle, cytotoxicity, and cell proliferation. In vivo disease models are designed to recapitulate some aspect(s) of the targeted disease process and are used to further augment the in vitro efficacy findings. Collectively, these studies provide information to refine a chemical series of analogues to optimize the desired pharmacological response, as well as provide data useful in determining effective concentrations (in vitro) and exposures (in vivo) that can be used to model potentially clinically effective exposures for patients. Pathologists are routinely involved in the design and interpretation of in vivo pharmacology studies where anatomical/clinical pathology, biomarker, and/or imaging end points are used to indicate efficacy. Knockout (KO) and transgenic mice are frequently used in these studies and it is important that these animal models are fully characterized in advance as animal models of human disease. This characterization should include an extensive histopathology evaluation as a component of the phenotyping of the animal model. There are a variety of special quantitative microscopy and imaging techniques available to pathologists to evaluate pharmacology studies. Examples would include morphometry, stereology, computed tomography (CT), magnetic resonance imaging (MRI), and matrix-assisted laser desorption ionizationdmass spectrometry (MALDIMS) (for more details see Digital Pathology and Tissue Image Analysis, Vol 1, Chap 12, and

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In Vivo Small Animal Imaging: A Comparison to Gross and Histopathologic Observations in Animal Models, Vol 1, Chap 13). These techniques can be used to evaluate and quantify parameters such as cell proliferation and apoptosis, tumor volume, neovascularity and blood flow in tissues of interest, trabecular bone volume, and compound biodistribution. Molecular pathology techniques such as ISH and IHC can be used to assess the in situ distribution of molecular targets in tissue from an animal disease model compared to that of the natural disease (see Special Techniques in Toxicologic Pathology, Vol 1, Chap 11 for more details). In addition, IHC is also routinely used to characterize target distribution in a comparative manner in normal tissues from animals and humans. This comparative profile of target distribution helps identify potential target tissues in humans and animals and is taken into consideration for species selection for nonclinical efficacy and safety studies. Although toxicologic changes are not necessarily expected to be seen during pharmacology studies, histopathologic evaluation of known or suspected target organs of toxicity is frequently added to in vivo pharmacology studies in an early attempt to identify potential hazards associated with new compounds. This is especially true if little is known about the novel product and in cases where in vivo models are very long in duration, resulting in significant tissue exposure over time.

4.2. Investigative (Mechanistic) Toxicology Studies As previously described in Section 2 Discovery Toxicology, it is now common to incorporate nonclinical safety assessment evaluation into the discovery process to better understand and potentially derisk compound toxicity in advance of a new product’s full progression to development. This discovery stage effort is generally directed at the assessment of intended or unintended pharmacological activity and may provide very early hazard identification. The identification of potentially important toxicities early in the overall development process enables the initiation of investigative (also called mechanistic) toxicology studies. Related to this, unexpected toxicities can also be observed during full development of a product in later stage in vivo toxicity studies or in clinical trials.

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Investigative toxicology efforts tend to be hypothesis driven and generally directed at elucidating the mechanism of a toxicity instead of broader hazard identification. A better understanding of the pathophysiology of an adverse effect allows it to be put into better perspective regarding exposure, dose–response, translatability, and long-term consequence. Some toxicities may be found to be specific to the species used in the study and have no relevance to humans. This information contributes to the decision of whether a toxicity challenge may be manageable or is too difficult to overcome. During discovery stage activities, this facilitates the rational termination of a compound if appropriate at a point when there has been a relatively small investment. This facilitates a quicker pivot to considerations of whether a backup molecule should be brought forward and/or should chemistry or protein science efforts be redirected or reinitiated. The ultimate goal of investigative efforts is to improve safety margins or contribute to redesigning molecules to eliminiate such liabilities altogether (Kramer et al., 2007). Pathology is generally a critical component of investigative efforts. Often, the toxicity finding that instigates the investigation is identified through pathological assessment or end points conducted on a pharmacology or toxicology study. Investigative in vivo studies will routinely include anatomical/clinical pathology or biomarker end points and pathologists should be involved in the design and interpretation of these studies. Pathologists are well suited to guide the use of molecular pathology techniques such as ISH and IHC to assess the in situ distribution of molecular targets in tissue, evaluate cellular response (e.g., proliferation, apoptosis), and characterize inflammatory cell infiltrates. In addition, there are a variety of special quantitative microscopy and imaging techniques, such as those mentioned in Section 4.1, that are available to pathologists to facilitate the evaluation in an investigative study.

4.3. Standard and Alternative Animal Models Over the past three decades, there has been significant effort devoted to the development of in vitro and ex vivo models for investigating toxicity and screening of new candidate

products (see Alternative Models in Biomedical Research: In Silico, In Vitro, Ex Vivo, and NonTraditional In Vivo Approaches, Vol 1, Chap 24). However, animal models are still essential for safety testing of new products (Prior et al., 2019). Companies constantly strive to improve the efficacy and safety predictability of in vivo studies and the translation of these findings to the consumer or clinical setting. As part of discussions with research and safety scientists, a pathologist can provide important comparative anatomy, physiology, and medical input during the consideration and the selection of a standard or alternative species and/or animal model for evaluation of a new product. In vivo animal models can be divided arbitrarily into “standard” and “alternative” categories. Standard Mammalian species generally considered as standard models for drug metabolism and pharmacokinetics (DMPK) and toxicity testing include rats and mice (see Animal Models in Toxicologic Research: Rodents, Vol 1, Chap 17); rabbits (see Animal Models in Toxicologic Research: Rabbit, Vol 1, Chap 18); dogs (see Animal Models in Toxicologic Research: Dog, Vol 1, Chap 19); pigs (see Animal Models in Toxicologic Research: Pig, Vol 1, Chap 20); and nonhuman primates (see Animal Models in Toxicologic Research: Nonhuman Primate, Vol 1, Chap 21). These standards species are extensively covered in the respective chapters listed. Alternative Alternative species have been found to have distinctive anatomic, physiologic, or genetic attributes or specific advantages that cannot be recapitulated in standard mammalian models, and therefore make them particularly useful in certain applications. NONMAMMALIAN MODELS

A large group of alternative models is made up of nonmammalian species such as birds, reptiles, amphibians, fish, and invertebrates (see Animal Models in Toxicologic Research: Nonmammalian, Vol 1, Chap 22). Nonmammalian species have been found to be particularly suited for screening and investigative research into mechanism of toxicity and DMPK attributes a new product and for environmental toxicology assessment.

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MAMMALIAN MODELS

Alternative mammalian models include less commonly used species such as Guinea pigs and hamsters (see Animal Models in Toxicologic Research: Rodents, Vol 1, Chap 17) and bioengineered disease models (see Genetically Engineered Animal Models in Toxicologic Research, Vol 1, Chap 23). Guinea pigs have been employed in the areas of respiratory, nervous, and immune systems diseases. Gerbils and Guinea pigs have a similar hearing range to humans which makes them useful models for human otologic physiology and disease. Hamsters have been used for research of various cancers, metabolic disorders, cardiovascular disease, and infectious diseases. Genetically modified animals (especially rodents) are now widely used in efficacy and safety evaluation of new products. During the discovery stage, findings from KO rodent models can be viewed as the “worst case scenario” of blocking a biochemical target due to the complete removal of the targeted gene’s function. Unexpected toxicities observed in nonclinical and clinical studies may be investigated using KO models, where targets can be modulated or even replaced, and if a particular target is responsible for the toxicity observed in wild-type animals, the effect should not be seen in the bioengineered model. Genetically engineered mice are also used for evaluating and investigating the adsorption, distribution, metabolism, and excretion (ADME) properties of new products and the potential for drug–drug interactions. Examples of this would include knocking out a gene coding for specific metabolic enzymes, signaling transduction elements, and membrane transporter proteins to investigate the effect on the product’s profile. Both bioengineered rat and mouse models may be used as a second or third tier mutagenicity assessment when positive or equivocal results are observed in the standard in vitro testing genetic toxicology testing battery. Genetically engineered rodent models are also used in the evaluation of the biodistribution, differentiation, growth, toxicity, and carcinogenicity of human cell–based therapies. This is a result of evaluation using immunocompetent animals that would be complicated by an immune response, potentially resulting in spurious results and rejection of the administered

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material. As a final example, genetically engineered mice (GEMs) are routinely used in carcinogenicity testing (see Carcinogenicity Assessment, Vol 2, Chap 5, and Genetically Engineered Animals (Rodent, Non-rodent, Nonmammalian), Vol 1, Chap 23). In recent years, the historical data set for these models has expanded and there has been clear regulatory authority acceptance of the models demonstrated, leading to 75% of all mouse carcinogenicity studies submitted to the FDA using the rasH2 for assessing carcinogenicity hazard identification (Jacobs and Brown, 2015). Regardless of how they are utilized, pathologists play a key role in the optimal development and use of genetically engineered models in these diverse applications. Pathologists have the critical role of providing histopathology and clinical pathology assessments as components of the phenotyping of genetically engineered models. Pathologists, in collaboration with their research and safety colleagues, can provide valuable input on the selection suitability of GEM model(s) for the intended purpose, study design, and ultimately assessment and interpretation of histopathology, clinical pathology, and biomarker parameters when included the study design.

4.4. Biomarker Considerations Toxicologic and investigative pathologists have long employed routine end points as indicators of treatment long before biomarkers came of age. Examples of these would include physiologic parameters (blood pressure), clinical pathology (alanine transaminase (ALT) and aspartate transaminase (AST) activities, platelet count), and histopathology. Biomarkers have been defined as “a defined characteristic that is measured as an indicator of normal biological processes, pathogenic processes, or responses to an exposure or intervention, including therapeutic interventions” (NIH-FDA Working Group on Biomarker Definitions, 2020). Since a biomarker, regardless of type, provides an indication of a response to treatment, it becomes readily apparent why they would have potential widespread application in nonclinical studies to better detect and characterize efficacy and toxicity (covered in detail in Biomarkers: Discovery, Qualification and Application, Vol 1,

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Chap 14). Overall, biomarker data should then enhance the success rate of bringing forward, into and through development, products that are more likely to pass the rigors of late-stage clinical trials and ultimate regulatory approval. During discovery, biomarker parameters can provide insight on a new product’s desired or unexpected target engagement, efficacy, and/or an early indication of target organ toxicity in standard species and animal models of human disease. This contributes to enhanced confidence in decisions made during the lead selection process. Once into development, safety and efficacy biomarkers may also be integrated into general and special nonclinical safety studies. Because of their training and experience, a pathologist is well suited to work closely with discovery and development scientists in the consideration and selection of efficacy and safety biomarkers and ultimately in their interpretation when used in animal studies. As an extension of this, pathologists should also contribute a comparative pathology perspective during discussions with clinical scientists on the translatability to humans of a nonclinical biomarker and findings in animal studies. Biomarkers that can be translated from the nonclinical to clinical setting may better inform the benefit:risk consideration because of the ability to monitor for a hazard identified in nonclinical toxicology studies. This may allow progression of a product into and through clinical trials that might otherwise have to be stopped.

4.5. Interpretation of Unique Findings During toxicity studies, pathologists are routinely involved in the assessment and overall interpretation of unanticipated in-life observations such as unexpected moribundity, unscheduled euthanasia, and deaths. Uniquely among these are observations of tumors and infections. A constellation of factors must be considered in these situations to properly place the observation in the context of treatment with the test product. The process starts with the pathologist ensuring the correct diagnosis and interpretation of gross and/or microscopic findings and then contributing to the appropriate correlation of the pathology findings with any relevant clinical observation(s). Procedure-related factors may be considered initially, and this may lead to

a clear explanation of the cause of the unexpected findings. In-life procedure-related causes of unexpected findings may include improper dosing, sampling, and handling. A pathologist’s evaluation of an affected animal may detect macroscopic and microscopic evidence of dosing mishaps such as an esophageal puncture associated with oral gavage in rodents, misplaced administration of test article or repeated CSF sampling via the cisterna magna, and perivascular changes associated with repeated attempts at intravenous injection. Evidence of animal handling errors during dosing or husbandry activities might be bruising, lacerations, and/or fractures. In addition, routine necropsy procedures and the mishandling of tissues may induce changes in tissue that must subsequently be separated from treatment-related histologic changes. Examples of necropsy-related procedure-induced histopathology changes in just the brain that may have to differentiate from being related to treatment might include extravasated red blood cells present in the meninges as a result of tissue tearing during removal of the brain from the calvarium and myelin vacuolization and dark neurons observed as a result of mishandling the brain prior to fixation (Garman, 2011). In situations where a clear procedural effect cannot be determined, a potential association with the test product must be considered. Although findings may be unexpected in a study, the pathologist can consider if the observations may represent “excessive pharmacology” based on the pharmacologic mechanism of the product or potential off-target effect based on known pathophysiologic mechanisms. These unique observations may have been brought on by using higher dose levels and longer treatment time periods than in previous studies or use of a new animal strain or species with a different DMPK and/or tolerability profile. An example of species differences in response would be the lower tolerance and appearance of lesions in rats versus mice associated with inhalation and intravenous exposure to some nanomaterials (Bahamonde et al., 2018; Dekkers et al., 2018). The appearance of tumors in repeat-dose rodent toxicology studies may come as a surprise to some, but common spontaneous tumors may occur, especially in longer term (13-week and chronic) studies (Lanzoni et al., 2007; Son and

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REFERENCES

Gopinath, 2004). When tumors are observed in general toxicity studies, pathologists can take into consideration what is known regarding the pharmacologic mechanism of the test article and off-target effects, historical control data of the species being used, evidence of potential preneoplastic histologic changes (e.g., hyperplasia, metaplasia, cellular atypia), and any findings from in vitro and in vivo genotoxicity assays. Another potentially challenging situation where pathologists should be involved is in the interpretation of infections observed in toxicity studies. This situation is most commonly associated with studies investigating new immunosuppressive biotherapeutics or when incorporating them into the study design for a specific immunosuppressive effect. During their assessment, pathologists can consider the potential for adventitial and subclinical disease in rodent and especially nonrodent toxicology studies (Simmons, 2010). Immunosuppression has been reported to lead to an increased risk of opportunistic bacterial, fungal, or parasitic infection, chronic viral infection, and viralinduced cancers (Brennan et al., 2010; Carville and Mansfield 2008; Simmons, 2010; Werner et al., 2020). Related to this, stress induced by test products may lead to immunosuppression with activation of subclinical infections (see Issues in Laboratory Animal Science that Impact Toxicologic Pathology, Vol 1, Chap 29). The accurate interpretation of these findings in association with immunosuppression is especially important as this information contributes to the benefit:risk considerations of a new product’s risk of increasing infection and cancer. In summary, the list is endless of potential unique and/or unexpected in-life or pathologyrelated findings that may be encountered in a nonclinical study. Based on their training in veterinary medicine or human medicine and additional training in pathology, pathologists bring critical expertise to the project team facing an unexpected finding. Accurate characterization and interpretation of these types of observations is essential to inform their proper classification as incidental, procedure-, or treatment-related. This enables decision-makers to then have more confidence as they consider continuing with the development of a new candidate or terminate it because of apparent unacceptable safety risk.

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REFERENCES Assaf BT, Whitely LO: Considerations for preclinical safety assessment of adeno-associated virus gene therapy products, Toxicol Pathol 46:1020–1027, 2018. Bahamonde J, Brenseke B, Chan MY, et al.: Gold nanoparticle toxicity in mice and rats: species differences, Toxicol Pathol 46:431–443, 2018. Batty P, Lillicrap D: Advances and challenges for hemophilia gene therapy, Hum Mol Genet 28(R1):R95–R101, 2019. Bennett CF, Baker BF, Pham N, et al.: Pharmacology of antisense drugs, Annu Rev Pharmacol Toxicol 57:81–105, 2017. Blomme E, Will Y: Toxicology strategies for drug discovery: present and future, Chem Res Toxicol 29:473–504, 2016. Bolt MW, Whiteley LO, Lync JL, et al.: Nonclinical studies that support viral vector-delivered gene therapies: an EFPIA gene therapy working group perspective, Mol Ther Methods Clin Dev 19:89–98, 2020. Brennan FR, Morton LD, Spindeldreher S, et al.: Safety and immunotoxicity assessment of immunomodulatory monoclonal antibodies, mAbs 2:233–255, 2010. Carville A, Mansfield KG: Comparative pathobiology of macaque lymphocryptoviruses, Comp Med 58:57–67, 2008. Cavagnaro J: Preclinical safety evaluation of biotechnologyderived pharmaceuticals, Nat Rev Drug Discov 1:469–475, 2002. Dekkers S, Ma-Hock L, Lynch I, et al.: Differences in the toxicity of cerium dioxide nanomaterials after inhalation can be explained by lung deposition, animal species and nanoforms, Inhal Toxicol 30:273–286, 2018. EMEA/CHMP/SWP/28367/07: Guideline on strategies to identify and mitigate risks for first-in-human clinical trials with investigational medicinal products, 2017. https://www.ema. europa.eu/en/documents/scientific-guideline/guidelinestrategies-identify-mitigate-risks-first-human-early-clinicaltrials-investigational_en.pdf. (Accessed 22 March 2021). Evers M, Toonen J, van Roon-Mom W: Antisense oligonucleotides in therapy for neurodegenerative disorders, Adv Drug Deliv Rev 87:90–103, 2015. FDA (Food and Drug Administration): guidance for Industry: Estimating the maximum safe starting dose in initial clinical trials for therapeutics in adult healthy volunteers, 2005. https://www. fda.gov/regulatory-information/search-fda-guidancedocuments/estimating-maximum-safe-starting-dose-initialclinical-trials-therapeutics-adult-healthy-volunteers. (Accessed 29 November 2020). Garman RH: Histology of the central nervous system, Toxicol Pathol 39:22–35, 2011. Glassman P, Balthasar J: Physiologically-based modeling of monoclonal antibody pharmacokinetics in drug discovery and development, Drug Metabol Pharmacokinet 34:3–13, 2019. Heller S, Kozlovski P, Kurtzhals P: Insulin’s 85th Anniversarydan enduring medical miracle, Diabetes Res Clin Pract 78:149–158, 2007. Henry SP, Kim T-W, Kramer-Strickland S, et al.: Toxicological properties of 2’-O-methoxyethyl chimeric antisense

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inhibitors in animals and man. In Crooke ST, editor: Antisense drug technology: principles, strategies, and applications, Boca Raton, 2008, Taylor and Francis, pp 327–363. Hordeaux J, Buza EL, Dyer C, et al.: Adeno-associated virusinduced dorsal root ganglion pathology, Hum Gene Ther 31: 808–818, 2020. ICH (International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use): guideline for industry: M3(R2) Guidance on nonclinical safety studies for the conduct of human clinical trials and marketing authorization for pharmaceuticals, 2009. https:// database.ich.org/sites/default/files/M3_R2__Guideline. pdf. (Accessed 26 January 2021). ICH (international conference on harmonisation of technical requirements for registration of pharmaceuticals for human use): guideline for industry: S9 nonclinical evaluation for anticancer pharmaceuticals, 2009. https://database.ich.org/sites/ default/files/S9_Guideline.pdf. (Accessed 26 January 2021). Jacobs A, Brown P: Regulatory forum opinion piece: transgenic/alternative carcinogenicity assays: a retrospective review of studies submitted to CDER/FDA 1997-2014, Toxicol Pathol 43:605–610, 2015. Kerlin R, Bolon B, Burkhardt J, et al.: Recommended (‘‘best’’) practices for determining, communicating, and using adverse effect data from nonclinical studies, Toxicol Pathol 44:147–162, 2016. Kramer JA, Sagartz JE, Morris DL: The application of discovery toxicology and pathology towards the design of safer pharmaceutical lead candidates, Nat Rev Drug Discov 6:636–649, 2007. Lanzoni A, Piaia A, Everitt J, et al.: Early onset of spontaneous renal preneoplastic and neoplastic lesions in young conventional rats in toxicity studies, Toxicol Pathol 35:589– 593, 2007. NIH-FDA working group on biomarker Definitions: BEST (biomarkers, EndpointS, and other tools) resource, 2020. https://www.ncbi.nlm.nih.gov/books/NBK326791/. (Accessed 12 June 2020). Leach M, Clarke D, Dudal S, et al.: Strategies and recommendations for using a data-driven and risk-based approach in the selection of first-in-human starting dose: an international consortium for innovation and quality in pharmaceutical development (IQ) assessment, Clin Pharmacol Therapeut 109:1395–1415, 2021. Palazzi X, Burkhardt JE, Caplain H, et al.: Characterizing ‘‘adversity’’ of pathology findings in nonclinical toxicity

studies: results from the 4th ESTP international expert workshop, Toxicol Pathol 44:810–824, 2016. Pandher K, Leach MW, Burns-Naas LA: Appropriate use of recovery groups in nonclinical toxicity studies: value in a science-driven case-by-case approach, Vet Pathol 49:357– 361, 2012. Perry R, Farris G, Bienvenu J-G, et al.: Society of toxicologic pathology position paper on best practices on recovery studies: the role of the anatomic pathologist, Toxicol Pathol 41:1159–1169, 2013. Prior H, Monticello T, Boulifard V, et al.: Integration of consortia recommendations for justification of animal use within current and future drug development paradigms, Int J Toxicol 38:319–325, 2019. Rosenfeldt H, Kropp T, Benson K, et al.: Regulatory aspects of oncology drug safety evaluation: past practice, current issues, and the challenge of new drugs, Toxicol Appl Pharmacol 243:125–133, 2010. Seaton M: The study pathologist’s role in GLP studies: a regulator’s perspective, Toxicol Pathol 42:285, 2014. Simmons JH: Herpesvirus infections of laboratory macaques, J Immunot 7:102–113, 2010. Son W-C, Gopinath C: Early occurrence of spontaneous tumors in CD-1 mice and Sprague-Dawley rats, Toxicol Pathol 32:371–374, 2004. Sridharan K, Gogtay NJ: Therapeutic nucleic acids: current clinical status, Br J Clin Pharmacol 82(3):659–672, 2016. Thompson RA, Isin EM, Li Y: In vitro approach to assess the potential risk of idiosyncratic adverse reactions caused by candidate drugs, Chem Res Toxicol 25:1616–1632, 2012. Tomlinson L, Ramaiah L, Tripathi NK, et al.: STP best practices for evaluating clinical pathology in pharmaceutical recovery studies, Toxicol Pathol 44:163–172, 2016. Weaver R, Blomme E, Chadwick A, et al.: Managing the challenge of drug-induced liver injury: a roadmap for the development and deployment of preclinical predictive models, Nat Rev Drug Discov 19:131–148, 2020. Werner JA, Ishida K, Wisler J, et al.: Phosphatidylinositol 3Kinase d inhibitor-Induced immunomodulation and secondary opportunistic infection in the cynomolgus monkey (Macaca fascicularis), Toxicol Pathol 48:949–964, 2020. Yin L, Chen X, Vicini P, et al.: Therapeutic outcomes, assessments, risk factors and mitigation efforts of immunogenicity of therapeutic protein products, Cell Immunol 295: 118–126, 2015.

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3 Discovery Toxicology and Discovery Pathology Glenn H. Cantor1, Evan B. Janovitz2, Rene´ Meisner3 1

Glenn Cantor Consulting, LLC, Bend, OR, United States, 2Nonclinical Research and Development, Bristol Myers Squibb Co., New Brunswick, NJ, United States, 3Denali Therapeutics, South San Francisco, CA, United States O U T L I N E 8. Hypothesis Generation, Experimental Design, and the Role of Investigative Studies

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a comprehensive discipline that integrates biology, pathophysiology, various branches of toxicology, cell and molecular biology, systems biology, bioinformatics, and pathology for the purpose of providing the earliest possible hazard identification and risk assessment of a new pharmacologic target or new pharmaceutical candidate molecules. In a discovery toxicology department or as members of discovery research teams, the discovery pathologists function as well-rounded whole-body biologists. Discovery pathologists understand the pathophysiology of organs and systems and how they interact in the whole animal. They are trained in integrating multiple data sets and, importantly, in how to

1. INTRODUCTION Veterinary anatomic and clinical pathologists play a key role in discovery pathology and discovery toxicology in the pharmaceutical industry and are termed “discovery pathologists” in this chapter. This role in drug discovery is broad and extends well beyond safety concerns. For that reason, in this chapter, we define discovery pathologist in a much broader sense than is conventionally done. In addition to knowledge and technical skills, we also describe behavioral and personality traits that enable this interactive, multidisciplinary role. In the context of this chapter, “discovery toxicology” is defined as

Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-12-821047-5.00020-8

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learn and assimilate new material quickly. Their unique skill sets allow them to provide critical perspectives on a wide variety of issues relevant to the successful selection of new targets, characterization and validation of disease models to assess efficacy, and development of new pharmaceutical candidates. These include identifying, characterizing, and validating animal models of disease and efficacy, phenotyping genetically engineered rodents, determining whether target expression in animals is representative of what is known in humans, and designing experiments to assess whether exaggerated pharmacology results in toxicity.

1.1. Discovery Toxicology The power of the pharmaceutical industry lies in its ability to coordinate the efforts of specialists in many diverse fields toward a common scientific goal. An impressive example of this multidisciplinary effort is the establishment of discovery toxicology groups, in which classical toxicologists work together with discovery pathologists, in vitro biologists, cell and molecular biologists, genotoxicity specialists, cardiologists, electrophysiologists, bioinformaticians and genomicists, reproductive biologists, immunologists, and others (Table 3.1). In addition to contributing to target selection, the multidisciplinary discovery toxicology approach can often identify safety liabilities at an early stage of drug discovery or development and provide insight into how to minimize or manage these liabilities (Figure 3.1). The cost to a pharmaceutical company of a compound failing because of irresolvable safety concerns increases exponentially as drug development progresses. Discontinuation of a drug during Phase II or III clinical trials can result in the loss of tens or hundreds of millions of dollars in direct investment, plus the difficult-to-quantify opportunity costs had companies spent their time and resources on a different project. Obviously, there is a major economic advantage in identifying pharmaceutical candidates that are likely to fail as early as possible to reduce these losses and to enable project teams to select alternative molecules that are more likely to succeed. Simple analysis shows the power of this approach: If attrition can be reduced from 90% to 80%, then the number of successful molecules doubles (10% in the first case; 20% in the second),

and the productivity of a research organization grows impressively. Significantly, the philosophy of the discovery toxicology approach is not to avoid all liabilities and stop drug development, but rather to partner with discovery teams by providing an early assessment of potential liabilities without forestalling progress by being overly cautious. Often, important liabilities such as interactions with off-target receptors can be recognized at an early stage, and appropriately customized screening assays can be designed to help guide discovery of more selective molecules through structure–activity relationships (SARs). Sometimes, comparative biology investigations may enable better understanding of whether a liability in a nonclinical species is likely to translate and be a problem in humans. If a drug is intended for treatment of advanced cancer or other lifethreatening disease, it may be possible to accept the risk of a liability, especially if the liability is well understood and can be monitored. As the discipline of discovery toxicology has evolved and our scientific “tool box” has TABLE 3.1

Discovery Toxicology is a Multidisciplinary Scientific Field, Composed of Many Specialists, All With a Creative and Innovative Mind Set, Who Work Together to Solve Problems

Toxicologists Pathologists In vivo biologists Cell biologists Molecular biologists Genotoxicity specialists Cardiologists Electrophysiologists Safety pharmacologists Bioinformaticians Genomicists, proteomics, metabolomics, epigenetics specialists Systems biologists Reproductive biologists Immunologists

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FIGURE 3.1 Drug discovery process for a small molecule or monoclonal antibody (large molecule). Straight arrows indicate the normal progression from one stage to another. Curved arrows indicate potential paths if problems are encountered. Although discovery toxicology is involved in all activities, the areas in bold indicate particular involvement of discovery pathology and discovery toxicology. LM, large molecule; SM, small molecule.

expanded, it has become apparent that we can actually do too much. Because the potential risk of toxicologic liabilities can be investigated in so many ways, the extra investment of time and resources can be excessively costly and unduly prolong the time needed to develop drugs. Accordingly, principles of optimization must be applied. For example, every highthroughput assay has a certain rate of false positives. Unless thoughtful science is applied throughout the assessment process, one could inadvertently discard high-quality drug candidates or would need to perform elaborate, expensive, and time-consuming follow-up studies. At the early stages of drug discovery and development, it may be cost-effective for a pharmaceutical company to identify and exclude major risks, accept some of the less likely risks, and move drug candidates through early nonclinical development in a more expedient way, while acknowledging upfront that liabilities may be encountered at a later stage of development. An optimized, cost-effective strategy will allow accelerated development of a larger number of drug candidates, with the occasional failure of a few candidates at a later stage than would have been detected otherwise. Another consideration in optimization is that the level of optimal risk in continuing to develop a drug with an identified hazard, but uncertain translatability, varies depending on the target and intended patient population. Certain targets warrant taking more risk, depending on the disease and the

competitive situation. Clearly, there is no one way to reach this optimum (see Risk Assessment and Risk Management and Communication, Vol 2, Chaps 16 and 17, respectively). Pharmaceutical companies have wrestled with this issue and are continuously trying to find the optimal balance between speed and cost. Undoubtedly any solution must be reevaluated periodically in a changing scientific, regulatory, and business environment.

1.2. Discovery Pathology In a discovery environment, discovery pathologists work closely with biologists to identify and validate new targets and to evaluate efficacy of drug candidates in animal models. This process often requires the use of disease models, such as animals with either naturally occurring, genetically engineered, or induced disorders. Understanding the entire systemic biology and pathology of these animal models, beyond just the organ of interest, can be critical to their proper usage. Another important role of discovery pathologists is to phenotype genetically engineered rodents. Genetically engineered mice (GEMs) are often generated to create a disease model that mimics a human disease so that efficacy of pharmaceutical molecules can be evaluated. Knockout mice are used to help validate the anticipated efficacy of a target that is to be inhibited or antagonized pharmacologically.

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Such mice also provide an opportunity to evaluate abnormalities that might not otherwise be suspected as a consequence of the proposed pharmacology. This is useful in predicting ontarget toxicity of pharmacologic antagonists. In addition, they are useful in studies to evaluate target-based toxicity of pharmacologic agonists. If an agonist drug causes toxicity in wild-type mice but the drug does not cause toxicity in knockout mice, this is useful evidence that the toxicity is on-target. Transgenic or humanized mice, in which the human target is expressed in the mouse, are often used as models for human-specific drugs, and key tools for biology and efficacy studies. Further discussion of GEM tools is in Section 6 (Target Validation). Training and experience in mouse genetics, physiology, anatomy, and pathology are important in evaluating the biology and histopathology of GEMs. This is necessary to accurately discriminate between relevant and confounding phenotypic characteristics (Treuting et al., 2017). A discovery pathologist can play a key role in not only examining the tissue sections but also in designing the phenotyping strategy, including selecting or developing appropriate in vivo assessments to evaluate the animals’ behavior and physiology. Discovery pathologists should also participate in characterizing pharmacologic target distribution and functional expression in animal models in a comparative manner. This is particularly relevant for selecting the most appropriate nonclinical species for toxicity studies. Identifying a nonclinical species with a tissue distribution similar to humans is helpful in demonstrating drug activity that is translatable to humans. When target distribution or function in a nonclinical species significantly differs from that in humans, the relevance of using this species for human risk assessment is questionable, and it could be possible to use these differences to provide perspective on certain toxicities observed in nonclinical studies. Target distribution studies are also useful for oncology discovery programs. Performing immunohistochemistry (IHC) on a large number of otherwise morphologically similar neoplasms can contribute to identification of therapeutic targets that are found in high percentages of cancers or help select which type of cancers (indications) to treat. Discovery pathologists can also assist discovery teams by use of specialized

morphologic and analytical techniques to answer specific research questions. It is often the discovery pathologist who can bring to a team expertise in IHC, electron microscopy, laser capture microscopy, morphometry, confocal microscopy, tissue MALDI-mass spectroscopy, digital automated image analysis, spatial transcriptomics or multiomics, or applications of artificial intelligence pathology techniques.

2. KNOWLEDGE INTEGRATION AND THE SPANNING OF DISCIPLINES A discovery pathologist should contributemore value than examining tissues on a glass slide, recognizing or describing morphologic lesions, and providing data reports for others to interpret. It is paramount to have the ability to integrate key data across many scientific disciplines and to provide insightful interpretation along with recommendations to solve problems. Thus, the discovery pathologist plays the important, and often overlooked, role of whole-body biologist, not only diagnosing diseases or toxicities but also realistically assessing their significance. Insightful assessment can not only provide essential perspective but also lead to incisive decision-making or experiments to investigate an issue further (Figure 3.2). Critical thinking skills are absolutely required. It is important to have enough experimental scientific training and experience to design solid, robust experiments with all of the appropriate controls. It is equally important to be able to assess and troubleshoot the experiments of others, without disturbing the collaborative nature of discovery research teams. Critical analysis includes “big-picture” thinking about the broad, long-range goal of the discovery research team, the rationale and objectives of an experiment, and how the experiment fits into the overall investigative program, as well as careful critique of the experimental details. A discovery pathologist needs to be as broadly trained as possible, since a discovery pathologist functions as a discovery toxicologist as well. Most pathologists are well trained in pathophysiology, and this is very important. A wide base in biology, including whole-body, cellular, and molecular biology, provides a solid

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FIGURE 3.2 translation.

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Interrelations of requisite knowledge base, special pathology techniques, animal models, and human

foundation. Additionally, it is absolutely essential to have a working knowledge of pharmacokinetics when interpreting the significance of potential drug-induced pathology. Pathology trainees typically do not have sufficient background in pharmacokinetics to participate effectively in a discovery research team. Therefore, anyone aspiring to become a discovery pathologist in the pharmaceutical industry should fill this knowledge gap as soon as possible (please refer to ADME Principles in Small Molecule Drug Discovery and DevelopmentdAn Industrial Perspective, Vol 1, Chap 3, Biotherapeutic ADME and PK/PD Principles, Vol 1, Chap 4 and Principles of Pharmacodynamics and Toxicodynamics, Vol 1, Chap 5). This knowledge can be accomplished by taking an intensive short course, appropriate mentored reading, or in some other way. Toxicity is frequently driven by drug metabolites of small molecules; hence a working knowledge of drug transformation is essential. This requires a basic understanding of organic and medicinal chemistry, which for most veterinary pathologists require review of long-forgotten course work. Since small molecule discovery research teams always include medicinal

chemists, discovery pathologists should leverage opportunities to work with and learn from them. Experienced medicinal chemists can provide a different perspective on problem-solving. For example, they are often willing to synthesize compounds that are not drug candidates per se but as enantiomers or metabolites of the compound in question provide invaluable tools for testing hypotheses. One of the delights of working in discovery pathology and toxicology is the need for continuous lifetime learning. A discovery pathologist interacts with a wide variety of specialists in a discovery toxicology department. It is helpful to have at least some familiarity with as many of these areas as possible and learn how disciplines, traditionally considered outside classic pathology, can be leveraged to solve problems. These disciplines include systems biology, including “omics” such as epigenetics, genomics, proteomics, and metabolomics; statistics; electrophysiology; genetics and biochemistry, including the biochemistry of DNA damage and repair; receptor–ligand interactions; and cell signaling. These are fast-moving disciplines, so continued, self-motivated learning is essential, rather than relying on what was taught in a training program.

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Core training in toxicology is essential in the day-to-day activities of a discovery pathologist in a discovery toxicology department. This training includes distinctions between pharmacokinetics and toxicokinetics, and between adverse or nonadverse effects (see ADME Principles in Small Molecule Drug Discovery and DevelopmentdAn Industrial Perspective, Vol 1, Chap 3, Biotherapeutic ADME and PK/PD Principles, Vol 1, Chap 4 and Principles of Pharmacodynamics and Toxicodynamics, Vol 1, Chap 5, and Assigning Adversity to Toxicologic Outcomes, Vol 2, Chap 15). The key role of a discovery toxicology department is early recognition, interpretation, and communication of toxicities, including identification of hazards and assessment of dose– response relationships (see Risk Assessment, Vol 2, Chap 16). Toxicities often do not have a morphologic component that results in a recognizable lesion in tissues. Therefore, a comprehensive discovery toxicology department also includes specialists in safety pharmacology, including electrophysiologists as well as in vivo cardiac, respiratory, and neurologic experimentalists; genetic toxicologists; and reproductive toxicologists. A pathologist must be capable of communicating with these specialists, understand their findings, and when necessary be capable of explaining their findings to the discovery research teams and company management (see Overview of Drug Development, Vol 2, Chap 1). In a discovery toxicology setting, the important goal is to provide a rapid, reasonably costeffective, and ideally predictive assessment of potential liabilities, while acknowledging that more thorough studies will be done later if the drug candidate progresses into further development. Predictive safety assessments are useful not only in preventing drug candidates from advancing and consuming corporate resources only to fail later, but also for helping to guide medicinal chemists in selecting the most promising molecules. Thus, relatively rapid, less costly assays, usually performed under non-GLP conditions, enable testing of multiple compounds and selection of the ones with the higher probabilities of success. In allocating resources to a discovery toxicology effort, it is useful to consider the most likely reasons for a drug to fail at later stages of development and to structure the discovery toxicology program to address and prevent these

failures. Early and inexpensive identification of common or obvious “no-go” liabilities enables rapid decision-making and allocation of resources into more fruitful directions. Examples include early identification of invalid or unsafe targets, mutagens, hERG blockers, or subacute hepatotoxins. While the reasons for failure vary among companies, cardiovascular safety issues are one of the most common causes of offtarget small molecule drug failure. Accordingly, some discovery toxicology departments emphasize cardiovascular safety assessments, going beyond the paradigm of rapid in vitro assays and performing early testing of compounds in telemeterized animals. This approach is expensive, of course, and is only practical with selected, otherwise optimized compounds. In vivo telemetry studies allow detailed analysis of cardiac and hemodynamic parameters in addition to changes in locomotion, repiratory rates and body temperature, providing an early indication of safety pharmacology. Studies with telemeterized animals can identify liabilities such as altered blood pressure, heart rate, myocardial contraction or relaxation, cardiac conduction, or cardiac rhythm. These liabilities could impact safety, if they occur at pharmacologically relevant exposures. Serum cardiac troponins can be useful biomarkers of cardiac necrosis, but many key cardiovascular safety endpoints do not involve tissue injury. While few pathologists have the expertise to be competent cardiovascular safety pharmacologists, as veterinarians they should be able to reacquire enough knowledge to understand the science and communicate effectively. The successful discovery pathologist in a discovery toxicology environment must have sufficient understanding of pharmacology to communicate effectively, as well as deep enough knowledge to design or assess experiments. Drugs act by interacting with target molecules, usually receptors or enzymes. Classically, when acting on a receptor, a drug can be an agonist, biased agonist, inverse agonist, or antagonist. Many other approaches to drug discovery are now underway, including molecules that enhance target degradation, modifiers of transcription or translation, and others. Understanding the dynamics of these pharmacologic interactions is at the essence of drug discovery (see Principles of Pharmacodynamics

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and Toxicodynamics, Vol 1, Chap 5). This includes understanding the biological and chemical consequences of the interactions, and the kinetics of the interactions with varying concentration and time. It is quite helpful to have familiarity with terms such as Kd, Ki, on/off rates, and Vmax, and with suitable assays to quantitatively detect these interactions and their downstream events. Experiments are conducted in both in vitro systems and in vivo, either sequentially or in parallel. When conducting in vivo experiments, characterizing a compound’s pharmacokinetic profile, including in some cases determining its concentration in various body compartments at various times, can provide a basis for explaining both desirable and undesirable effects. Linking pharmacokinetics with pharmacodynamics in the same in vivo experiment is particularly powerful, showing the effect of the drug at different concentrations and times and helping to ensure that the drug has reached the target tissue. When an adverse effect occurs only at a dose that far exceeds exposures necessary for the maximum pharmacologic effect or at a dose that fails to achieve pharmacology, an off-target toxicity should be investigated. This is useful information, since off-target effects may be

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identified by rapid in vitro counterscreens or short-term in vivo studies and often can be eliminated in newer molecules. From a toxicology perspective, these quantitative analyses provide an early risk assessment including estimations of safety margins or potential therapeutic indices, i.e., the ratio between unsafe and efficacious exposures. Organizationally, in many pharmaceutical companies, discovery toxicology representatives, including discovery pathologists, work in a crossdisciplinary team formed around a specific pharmacologic target, with the goal of discovering and developing a drug to inhibit or stimulate the target. In the authors’ experience, this is termed a Discovery Working Group (Figure 3.3). The role of the discovery toxicology representative is to interact with all other team members; listen and react to their concerns, questions, and suggestions; and integrate all aspects related to toxicology in communications with the team. Smooth, productive interactions between the discovery pathologist and medicinal chemists contribute greatly to efficient small molecule drug discovery. Medicinal chemists work from a starting molecule, often identified through a high-throughput robotic screen of hundreds of thousands of molecules. Starting molecules

FIGURE 3.3 Discovery Working Groups are highly interactive and collaborative groups, designed to carry out the discovery and early nonclinical development of drugs targeted to specific targets. The group is composed of individual scientists who represent many specialties and can bring in additional input as required. Illustrated here is typical membership of a small molecule Discovery Working Group. The group also may include a scientist with expertise in designing and implementing high-throughput assays. In an antibody Discovery Working Group, membership would include a hybridoma specialist, protein engineer, and others with relevant protein expertise. I. PRODUCT DISCOVERY AND DEVELOPMENT

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that emerge from the screen contain a pharmacophore that interacts with the therapeutic target and may have a desired biological effect at high doses, but generally lack many of the properties that are necessary for a successful drug, such as structural novelty, stability, adequate absorption, and selectivity, i.e., freedom from cross-reactivity with other molecular targets. Once reliable highthroughput in vitro assays are validated and established, medicinal chemists use an iterative process to define the structure-activity relationships (SAR), methodically changing a portion of the molecule and observing the effects. Once the SAR is understood, medicinal chemists can rapidly focus their synthetic efforts on those portions of the molecule that impact its pharmacologic effects. In many cases, structural biochemists and computer-assisted drug design engineers can elucidate a target’s crystal structure and how the molecule interacts with it. This allows medicinal chemists a more rational approach to SAR. As prospective drug candidates are evaluated in vitro and in vivo, rapid feedback from the pathologist and other team members enables this incremental process to move forwards toward success. Organ- or pathway-specific pathology findings from an in-life study can expedite understanding a molecular mechanism of toxicity, which can lead to the use of an assay to counterscreen against the problem. For example, if an adverse effect can be linked to a molecule’s interactions with a particular off-target receptor, then it might be possible to design a high-throughput counterscreen assay in cells transfected with the gene encoding that receptor. In the small molecule realm, the discovery pathologist must collaborate with specialists in biotransformation, since xenobiotic metabolism is an essential pillar of toxicology and pharmacology. Drug metabolites and/or reactive intermediates can be toxic in their own right, and can cause toxicities that are not caused by the parent drug (Shu et al., 2008) (see ADME Principles in Small Molecule Drug Discovery and DevelopmentdAn Industrial Perspective, Vol 1, Chap 3). Recognizing molecular substructures that may be toxic once the parent molecule is metabolized can avoid toxicology issues later in development (Zhang et al., 2008; Limban et al., 2018). This relatively common phenomenon can be investigated by using inhibitors of drug metabolism, including

broad-spectrum inhibitors such as 1aminobenzotriazine (ABT) or more specific inhibitors of particular cytochrome P450s (Cyps) (Mico et al., 1988; Otieno et al., 2017). Providing medicinal chemists with a solid rationale to synthesize metabolites or weak or inactive enantiomers can be useful. The advantage of identifying this type of toxicity in the early stages of drug discovery is that it may be possible to redesign the molecule so that particular metabolic “soft spots” can be altered to avoid metabolism at those sites. A discovery pathologist also interacts closely with pharmaceutical scientists. For small molecules, pharmaceutical scientists are responsible for characterizing physical forms of drug candidates and for developing optimal vehicles, in which the molecule is stable while in storage and, when orally dosed, is accessible to the body at a desirable and reliable rate. Solubility and permeability parameters of the molecule are involved. The rate of absorption of an oral drug can be altered by selection of different salts or vehicles. For protein therapeutics, which are generally delivered by either intravenous or subcutaneous routes, pharmaceutical scientists are responsible for ensuring that the protein has suitable physicochemical characteristics and identifying the optimal vehicles for intravenous or subcutaneous delivery. Vehicle composition is selected to optimize protein stability, and this varies depending on the physicochemical nature of the protein. Importantly, choice of vehicle affects the maximum concentration of a drug in solution. Since the volume that can be dosed to an animal is limited, the maximal concentration, together with the rate of absorption (in the case of oral drugs), dictate the highest drug exposures that can be tested in a toxicology setting. If that maximal exposure is not sufficiently large, relative to the exposure that is known to be efficacious in animal models, then conducting acceptable and informative nonclinical toxicology studies may become impossible. Bioanalytic chemists are crucial partners of this effort to measure drug concentrations in plasma or tissues. Since the molecules under investigation are new inventions, it may not always be possible to use off-the-shelf, preexisting bioanalytic techniques, and bioanalytic chemists must design and optimize new protocols of quantitative analysis. The environment of a molecule, called the matrix, influences the

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assay. For example, an assay to detect a drug molecule is optimized differently depending on whether the molecule is in a sample of plasma, liver homogenate, brain, or urine. Chemically synthesized molecules are detected by a variety of analytical techniques, such as liquid chromatography followed by mass spectroscopy (LC-MS) and variants thereof. Because a small molecule drug is metabolized, it is necessary to identify and quantify the major metabolites as well. Protein therapeutics present a different challenge to the analyst and can be assessed by immunological assays such as ELISAs (enzyme-linked immunosorbent assays) or immunocapture assays. The advent of systems biology, which uses large data sets composed of epigenetic, genomic, transcriptomic, proteomic, lipidomics, or metabolomics information, has expanded the practice of discovery pathology and toxicology. Discovery pathologists must be versatile, or at least conversant, with the use of these so-called “omics” and “multiomics” technologies because they can be used to generate testable hypotheses for otherwise unexplainable toxicities. Actively seeking and reaching out to gene, protein, or metabolomics analysts and with chemometricians or other informatics specialists can be quite fruitful. If there is a gap in in-house expertise, one can collaborate with external scientists, either at contract research organizations or at universities. The collaboration is bidirectional and simultaneous. These specialists seek the assistance of pathologists in anchoring the lists of gene, protein, or metabolite changes to specific, definable biologic processes or lesions. Discovery pathologists should make themselves known to the larger discovery research community. Pathologists can also play a reality check in the “omics” field by recommending systemic nomenclature that is suitable for the results that automated systems biology analyses depend on. A discussion of the skill sets required of the discovery pathologist would not be complete without emphasizing the importance of critical literature reviews. In order for a pathologist to have productive collaboration with different disciplines, a well-rounded up-to-date scientific awareness is required. In addition to a lifetime of routine background scientific reading, a discovery pathologist needs to be aware of any scientific advances that could impact the

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various research project teams on which they participate. It is not enough, though, to read and assimilate factual knowledge from papers; it is essential to read and think critically, distinguishing good science from bad. It is important to be curious and to absorb and synthesize what one reads, incorporating new ideas and approaches. But old ideas are not always wrong. It is humbling to read that Dr. Youyou Tu, the recipient of the 2015 Nobel Prize in Physiology or Medicine for the discovery of the antimalarial drug artemisinin, made the breakthrough improvement in her method of chemical extraction based on techniques described in a paper written by Ge Hong (283–343 CE) during the Jin Dynasty in China (265–420 CE) (Miller & Su, 2011). No matter how hard we try to keep up our reading, most of us will never have working command of 1700 years of scientific literature, but it is nonetheless inspirational.

3. PATHOLOGY TOOLBOX The core toolbox of pathologists, of course, consists of careful, experience-based gross necropsy, microscopic examination of slides, including histopathology and cytology, and assessment of clinical pathology data. A key challenge for discovery pathologists is prioritization and efficiency. Prioritization applies at every level of a company from the individual discovery pathologist all the way to senior management. At the level of an individual discovery pathologist, the decision to work longer on one project means that less time will be available to work on others. At the level of the company, resources must be allocated to projects most likely to give return on investment. The discovery pathologist plays an important role in prioritization at the ground level by customizing study protocols. For example, in an early-stage discovery toxicity study, a full complement can be collected, but the number of tissues to examine microscopically can be limited to tissues that are known to express the pharmacologic target or accumulate high concentrations of drug, vital organs, and tissues that are known to commonly show toxicity. The goal of prioritization is to identify in a cost-conscious manner those changes that are likely to impede the development of a drug,

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while acknowledging that rare lesions might escape detection at this early stage and not be detected until later in development. This is an effort to strike a balance between the risk of missing an opportunity to identify toxicity in an unusual tissue, and the reward of minimizing resource expenditure and accelerating the drug development process. Prioritization permits distribution of scarce pathology resources, thus allowing a company to pursue a larger number of programs. One successful business strategy is to recognize that not every program is the same and to base allocation of resources on the most critical problems to solve, combined with consideration of the importance of the programs to the company portfolio. On the other hand, other companies choose a more uniform approach, for example, by evaluating a complete tissue set for essentially every multiple dose study, based on the assumption that the overall cost of such a study is still much lower than waiting until later in the development process when GLP standards have to be met. In addition, it can be argued from a 3R (Replacement, Reduction, and Refinement) perspective that one should attempt to glean as much information as possible from any study that uses animals for experimentation, in the off-chance that development of a drug candidate with less common liabilities can be avoided. Sometimes, soluble biomarkers can be incorporated into discovery toxicology studies to improve efficiency, especially if an issue is already known to a specific program (see Biomarkers, Vol 1, Chap 14). For example, bone biomarkers such as CTX-1 (carboxy-terminal cross-linked telopeptide of type 1 collagen) or P1NP (procollagen type 1 N-terminal propeptide) may change well before morphologic bone lesions appear (Kuo & Chen, 2017). However, some caution is necessary in using highly sensitive assays without knowing the correlations between the magnitude of change and its biological relevance. Sometimes, small but statistically significant changes in a soluble biomarker do not turn out to be toxicologically relevant. In the discovery stages, it is helpful to have full knowledge of the program and its issues before examining the slides, rather than reading the slides in a blinded way. In that way, the pathologist can refresh oneself beforehand on the

detailed histology of the most relevant tissues, pay attention to possible subtle changes that might otherwise be overlooked, and interpret the changes relative to background changes. After identifying a putative lesion, it is often useful to shuffle the slides, including treated and control, and examine the slides again without looking at the labels (“targeted masked evaluation”) to confirm that changes are a result of the drug administered. Of course, knowledge of the issues of a particular program and awareness of potential lesions must not divert one’s attention away from other unsuspected lesions, but a well-trained pathologist should be capable of examining the whole animal at necropsy or the entire tissue on a slide and identifying unexpected lesions. Clinical pathology is discussed elsewhere in this book series (see Clinical Pathology in Nonclinical Toxicology Testing, Vol 1, Chap 10, and Interpretation of Clinical Pathology Results in Nonclinical Toxicity Testing, Vol 2, Chap 14). It is valuable to have a veterinary clinical pathologist in a discovery toxicology department. It is worth noting that studies during the early stages of nonclinical drug development typically use small numbers of animals. In this setting, it is particularly valuable to examine individual animal data, including, when possible, longitudinal data for individual animals, rather than only mean data. Visualization tools are particularly useful for quickly evaluating interrelations between analytes or assessing multiple parameters at the same time. In discovery pathology, the distribution of a molecular target and the correlation of the target’s expression with disease status are important parameters to understand. If one knew that in humans, a target was expressed in a certain set of tissues, then it would be desirable to select a nonclinical toxicology species with a similar distribution pattern so that the toxicology could be mimicked in the most relevant way. Comparative tissue expression can begin with publicly available gene expression data sets (Brennan, 2017). An emerging technology is single-cell RNASeq, in which gene expression is determined on an individual cell basis, enabling a much more focused understanding of potential safety liabilities. An interesting example is how a single-cell RNASeq data set led to the discovery of CD19 expression on mural cells

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surrounding capillaries in the brain, which may explain CD19 CAR-T cell neurotoxicity (Parker et al., 2020). Most data sets are limited to human and mouse. Rat data are emerging, but dog and cynomolgus monkey data are lagging. Some institutions create their own databases. Gene expression provides a starting point, which can focus protein expression (IHC) evaluation to not only show whole-tissue expression, but cellular expression and distribution. Some companies use IHC on a wide variety of tissues from humans and multiple nonclinical species at an early stage of discovery to characterize the distribution of the pharmacologic target. This can be done either on a slide-by-slide basis, or by tissue microarrays, in which a single slide contains spots, typically 1–2 mm diameter, of many tissues so that the IHC can be done on a minimal number of slides, thus conserving reagents and time. Even with tissue microarrays, the examination of each spot on the array is laborious and, in some circumstances, it may be that automated digital pathology or Western blotting of protein extracts from multiple organs is more efficient. A limitation of examining whole-tissue protein extracts is that it is not possible to identify which specific cells express the target. Another use of large-scale IHC is assessment of tumors for expression of a proposed new target (see Section 7, Translational Medicine). For example, if a newly proposed target is expressed on a large proportion of breast cancer sections from thousands of patients, then it may be economically feasible to initiate a drug discovery program focused on that target. A key role for a discovery pathologist is optimizing and troubleshooting the IHC assays, since false positives and false negatives are common problems. This is discussed in more detail elsewhere in this book (see Special Techniques in Toxicologic Pathology, Vol 1, Chap 11). It is also necessary to validate the identity and quality of the tissues that are used for the IHC assays. Source tissues for IHC analyses are often derived from internal studies or purchased from biosample vendors. When samples are collected from in vivo biology or pharmacology studies, study personnel can be trained to collect samples for pathology. Otherwise highly competent biologists may inadvertently err in tissue collection, handling, storage, or transfer of

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specimens. It is often useful for discovery pathologists to manage, train, or advise technicians. Many institutions create and maintain tissue banks, in which tissues from the various nonclinical species are collected from normal, nondiseased animals, for the purpose of IHC target distribution studies. For human tissues, this is more difficult, but donors should be as nondiseased as possible (Sandusky et al., 2007). Donor demographic data (age, sex, concurrent disease, medications) is important in understanding the relevance of any IHC labeling. Furthermore, the interval from death to tissue collection should be minimized to reduce loss of antigen expression and avoid postmortem artifact. It is advisable that the best tissue samples be cataloged and stored in an organized tissue bank in formalin-fixed paraffin-embedded blocks, individualized or in tissue microarrays, and in the frozen state, along with relevant metadata about the animals or subjects. Whether commercial or internally generated, all antibodies used for IHC should be qualified in terms of target specificity and species cross-reactivity (Janardhan et al., 2018). Alternatively, in situ hybridization can be done to identify DNA or RNA. In situ nucleic acid–based molecular techniques, e.g., in situ hybridization, allow the identification of specific RNA or DNA within the morphologic context of a tissue or cell. Occasionally, assays are performed to identify apoptotic cells in tissues. This can be done with an in situ terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay to detect cells with fragmented DNA. The apoptotic cells can be quantified by various imaging techniques and compared with those in other tissues or other treatment groups. In situ matrix-assisted laser desorption/ionization followed by mass spectroscopy (MALDIMS) can be used to identify small molecules or proteins in particular locations within a tissue. The tissue section on a slide is scanned pixelby-pixel by a laser beam, which ionizes molecules, which are then captured, analyzed, and identified by mass spectroscopy. The mass spectroscopy data are then correlated with the anatomic position, such that molecules of interest can be quantitatively mapped within a histopathologic image of a tissue. This has been used, for example, to confirm that crystals in a tissue are

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composed of precipitated drug or drug metabolite rather than an endogenous compound, or to measure the concentration of the drug or its metabolites within renal tubules or substructures of the eye, such as the lens, retina, or ciliary body (Drexler et al., 2007; Ruepp et al., 2019). Morphometrics is useful in some investigative efforts (see Digital Pathology, Vol 1, Chap 12). For example, certain bone parameters, such as growth plate thickness and trabecular bone volume, can be quantified quite precisely. Morphometrics approaches can be especially powerful when combined with automated imaging techniques which can identify, count, and summarize specific cell types or features in a tissue section. When using these automated imaging techniques, the user first uses algorithms to train the instrument which cells are considered positive and which are negative, so that background signal is minimized. Such optimization procedures are necessary and require a significant time commitment to ensure highquality data. Once an acceptable correlation has been established between the automated and manual approaches to cell identification and characterization, the tissue sections can be efficiently scanned, generating large amounts of quantitative data to test investigative hypotheses. Increasingly, automated multiplexed IHC is used to analyze immune infiltrates in tumors or bone marrow. Multiplexed analyses can be especially powerful and informative when combined with morphometric analyses to identify location of the immune cells relative to the tumor, structures such as tertiary lymphoid structures, or intercellular distances as a surrogate measure of potential cell–cell interactions (Cabrita et al., 2020; Patel et al., 2019). Newer, more powerful multiplex spatial imaging techniques are coming into use, including techniques to simultaneously evaluate large numbers of mRNA transcripts, proteins, and even epigenetic features on a slide, in the context of the histopathology (Pietrobon et al., 2021). A caveat, though, is that while digital techniques can generate an abundance of quantitative data, it is important to think about the study objectives and hypotheses beforehand and decide if and when it is worth the effort. The digital data should not replace qualitative assessment by a trained pathologist, which

should be conducted in conjunction with the quantitative analysis. Digital imaging of slides is useful for sharing slides and seeking others’ opinions in a timely way. As pathology becomes more global, this becomes increasingly valuable (see Digital Pathology and Tissue Image Analysis, Vol 1, Chap 12). Challenges include the quality of resolution, speed of access, including the speed at which a remote pathologist can move among different areas of the image or change the magnification, and cost of the system. Increasingly, these challenges are being overcome. Pathologists can now better view slides remotely and with ease. This will certainly facilitate peer-review and consultation. Specialized microscopic techniques include confocal, laser capture, and electron microscopy. In confocal microscopy, a precisely focused plane of section allows detailed intracellular localization of proteins, which are typically labeled with fluorescent markers. Different proteins can be labeled by antibodies conjugated to a spectrum of fluorophores, and then, by combining the images, colocalization of proteins can be assessed. For example, colocalization may suggest that two or more proteins of interest are located in mitochondria, or may form heteromers, or may colocate in lipid rafts in the cell membrane. Confocal microscopy is particularly useful on live cells, in which the dynamic effects of drug exposure including biological properties such as receptor internalization can be visualized. This principle is also used extensively in high-content imaging procedures, in which cells are grown in multiple-well plates and the wells are imaged and evaluated for multiple parameters concurrently. Alternatives to confocal microscopy are two-photon (or, more recently, multiphoton) excitation microscopy, a fluorescence imaging technique which allows deeper tissue penetration and less background (So et al., 2000; Parodi et al., 2020), or light sheet fluorescence microscopy, which can acquire images faster than confocal microscopy and can be used in living cells (Lemon & McDole, 2020). Superresolution microscopy is a series of techniques in optical microscopy that allow images to have resolutions higher than those imposed by the diffraction limit of light (Schermelleh et al., 2019). The resolution of conventional

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fluorescence microscopy is limited by diffraction to several hundred nanometers; about the size of an organelle. There are several optical microscopy techniques that improve spatial resolution by an order of magnitude beyond the diffraction limit. The properties of the fluorescent probes are used to distinguish emissions from two nearby molecules within a diffraction-limited region. This highly powerful technique allows for visualization of drugs within organelles, e.g., the lysosome, and can aid in identifying subcellular distribution to better understand biology of a target or toxicity of a drug. Laser capture microscopy (LCM) allows a pathologist to remove a specified portion of a tissue section for further analysis (Simic et al., 2013). The slide is examined by light microscopy; the area of interest is identified and isolated, typically enabled by a software application, excised by the laser, and collected into a receptacle. Captured tissues or cells can be used for protein-, RNA-, or DNA-based assays, biochemical tests, or other purposes. This procedure is particularly valuable when molecular comparisons between diseased tissue and adjacent normal or reactive tissue are desired. For example, separating neoplastic cells from their adjacent stroma facilitates genetic comparison of the neoplastic genome with the nonneoplastic genome from the same individual. Nucleic acids can be harvested from formalin-fixed, paraffin-embedded tissues, so this is a powerful tool even for archival slides. Transmission electron microscopy (TEM) is covered in detail in another chapter in this book (see Morphologic Manifestations of Toxic Cell Injury, Vol 1, Chap 6). If properly applied, TEM is a useful and complementary investigative tool, facilitating hypothesis-directed research. When considering the potential use of TEM in a study, it is important to plan ahead and collect suitable specimens, with minimal postmortem interval, small enough sample size for optimal fixation, and optimal fixative. TEM is rarely of value post hoc. While TEM can be performed on routinely collected formalin-fixed, paraffinembedded tissues, it is suboptimal, making interpretation difficult or even erroneous. We recommend including potential TEM evaluation along with details of proper collection and fixation techniques in standard necropsy protocol templates so that it is given consideration

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whenever an in vivo toxicity study is contemplated. The protocol section in the template can be deleted from the final protocol if TEM is determined not to be of value. When TEM evaluation is undertaken, it is advisable that the objective be explicitly stated so that ambiguity can be avoided. For example, at an early stage of an investigation, one might need to determine if hepatocellular cytoplasmic granules seen by light microscopy are peroxisomes or mitochondria, since the pathogenesis of peroxisome proliferation is quite different than mitochondrial expansion. Employing EM to hunt for “drug-related changes” not otherwise specified is a recipe for confusion and indecision. TEM can also be employed as part of in vitro studies, to evaluate cells or organoids and demonstrate that they recapitulate or confirm in vivo toxicity. Another area in which discovery pathologists can be involved is validation or interpretation of microphysiological systems, also referred to as “organs-on-chips.” Sometimes, relevant biology can be mimicked with in vitro systems that include key features of a tissue, either by mixtures of cells that provide essential cell–cell interactions, provision of key matrices to support growth and biological processes, threedimensional structures, or relevant mechanical features such as rhythmic stretching or flow (Huh et al., 2009). Microphysiological systems that capture key biological processes can be quite useful in answering questions that require higher throughput. Input from pathologists can be valuable in ascertaining whether these in vitro systems actually mimic in vivo conditions or only show an artifact of the methods. Most discovery pathologists are stretched thin by the demands of multiple projects, timelines, and tight budgets that are inadequate to pay for everything that one might want to do. Strategic application of standard pathology is necessary to obtain important, decisional information. There is a huge number of “knowledge gaps,” areas of science that are inadequately understood. As we learn more, we realize that there are more knowledge gaps, and it could be argued that our knowledge gaps are infinite. For most pathologists, it is no longer acceptable to study phenomena simply because one has curiosity about a knowledge gap; there must be a rationale to justify the funding and resources. In the setting of a pharmaceutical company, the overall success

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of the enterprise takes precedence over individual interests and pursuits. The most important rationale is to enable decision-making. This includes providing options and key experiments that enable go/no-go decisions with the weight of evidence clearly spelled out. This needs to be the predominant focus of pathologists. In drug development, there are always risks and there is rarely certainty. For example, finding severe drug-induced lesions in rats at a low exposure of a tool compound may reduce the probability of success of a drug discovery project and make it more financially risky, but it does not necessarily mean that humans would be adversely affected by an optimized drug. Risks must be identified, understood, communicated, and prioritized, such that decisions can be made in a way that is most likely to lead to costeffective development of successful drugs to improve the lives of patients who otherwise have limited options with currently available therapies. Since resources must be allocated judiciously, pathologists can sometimes make use of pharmacology and other studies to obtain valuable toxicology information. In the jargon of the pharmaceutical industry, this is called “leveraging,” in which one obtains highly valuable, decision-driving information from relatively small investment. Historically, in drug discovery, biologists performed a number of efficacy studies using animal models before they engaged in a meaningful way with pathologists and toxicologists. Only after efficacious and proprietary molecules were discovered were they subjected to formal toxicology evaluation, in studies exclusively to provide hazard identification and risk assessment and that include doses that are considerably higher than the efficacious doses. Alternatively, a company can use a hybrid, potentially more efficient, approach, in which safety and efficacy issues can be addressed together in the same experiment. Discovery teams that embed a discovery pathologist or toxicologist can identify and address safety liabilities earlier. This can be done in the context of efficacy studies with clinical pathology testing of blood samples from nonterminal studies or necropsy and histopathology of animals given efficacious doses. This form of leveraging can provide valuable early insight

into toxicology issues associated with pharmacological manipulation of the target, as well as issues that may be unique to the specific chemotype under evaluation. It is helpful to spend time training discovery biologist colleagues in how to make important clinical observations, collection of body weight and organ weight data, tissue or blood collection, gross lesions, and even microscopic screening, so that they can contribute to the overall assessment of key questions and leverage the pathologist’s time. Conversely, highly experienced in vivo biologists can teach the pathologist concepts that are not available in textbooks, such as unusual behaviors or atypical observations in new strains of genetically modified mice. Clearly, in the interest of time management, the pathologist must discriminate among the many opportunities available to engage in early efficacy studies. The number of molecules in early efficacy testing far exceeds the capacity of most pathologists. On the other hand, dedicated toxicology studies are expensive. Thoughtful and interactive teams can work out optimal ways of making better use of early studies to obtain decisional information. Another way that pathology can be strategically applied is in customizing endpoints in studies. These may be specific tissues to examine by histopathology, clinical pathology analytes, levels of expression of certain mRNAs, or many other endpoints. The pathologist on a project team should acquire a complete understanding of the target and its potential liabilities, through any means available, including dialogue with subject matter experts, reading, and attending meetings. Input from clinicians is useful to better understand the planned use of the drug in the clinical setting. Based on this knowledge and on carefully honed scientific thinking, specific, nonroutine endpoints can be built into a study during the planning stages. If applied in a cost-disciplined way, these additional endpoints can greatly enhance the value of the results and the ability of the data to guide decision-making. In other situations, there may not be preexisting knowledge to guide selection of nonroutine endpoints, but the pathologist can play a role in selecting a default set of endpoints that are rational, experience-based, and costeffective.

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4. IN VITRO/IN VIVO CORRELATIONS

4. IN VITRO/IN VIVO CORRELATIONS As part of the optimization process for a molecule in discovery, a wide array of in vitro data are generated, addressing its suitability as a drug based on potency, efficacy, absorption, metabolism, formulatability, biophysical characteristics, and safety. In vitro assays can inform translatability of effects by comparing the toxicology species to humans. While some targets and their biology are highly conserved among species, others are poorly conserved or even have no animal analogy. Pathologists can facilitate experiments that determine if any rodent or large animal is a suitable model and encourage development of assays that are species specific when necessary. To fully understand the impact of these in vitro data, it is necessary to correlate the in vitro results and in vivo findings. This not only helps validate the in vitro findings for a particular molecule that is progressing in development, but is also useful for putting the in vitro findings into a better context for assessing the target and other molecules of a similar chemotype. With strong in vitro/in vivo correlations, a program can move forward more rapidly and with more confidence. A pathologist often plays a central role in building this in vitro– in vivo correlation. Standard in vitro safety screening assays of small molecules include assessments of a molecule’s cytotoxicity, binding to, and functional activity on cardiac ion channels, metabolic enzyme inhibition and induction, and target selectivity. Many drugs of various chemotypes can inhibit cardiac ion channels, particularly those for potassium, calcium, and sodium. Cardiac ion channel effects can be assessed in a high-throughput mode in flux assays, in which indicator dyes, which fluoresce with varying intensity depending on the concentration of the ion that is measured, are used to monitor the movement of ions into or out of cells. These assays have the advantage of very high throughput, in multiple-well plates in which the reactions can be performed by robots. Sometimes, there can be false positives, and findings should be confirmed for molecules that are going forward in development by patch-clamp techniques. In patch-clamp assays, genes encoding

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the predominant human cardiac ion channels are cloned and expressed in cells. Under a microscope, ion channel–expressing cells are pierced with a narrow-bore glass pipette, such that the intracellular milieu of the cell is continuous with the interior of the pipette and electrical impulses can be monitored. Then, the cells are stimulated in the presence of various concentrations of drug to determine if there is an inhibitory effect and determine the IC50. Typically, these assays are run in the absence of plasma proteins or with reduced plasma proteins, so if a molecule is known to bind tightly to plasma proteins such as albumin, then there will be a low concentration of free drug available in vivo and the in vitro assay may overpredict toxicity (see below). Solubility also must be taken into account. In vitro assays are often invalid when the molecule being tested is insoluble or poorly soluble in the assay media. If the soluble forms of a molecule are at an insufficient concentration to cause an effect, a molecule could be falsely categorized as not toxic or, conversely, falsely categorized as not efficacious. High-throughput cytotoxicity assays are a standard early screen for new small molecules. A variety of immortalized human cell lines are useful for this purpose, but none are completely accurate predictors of human toxicity. However, they are useful for screening purposes. Because drug-induced liver injury including direct hepatotoxicity is a relatively common cause for failure of drug development or drug withdrawal from the market, many of these in vitro assays use hepatocytes. One reason such assays can produce false-negative results is that in vitro hepatocytes lose much of their in vivo functionality, including their ability to express metabolizing enzymes and transporters. Phase I metabolizing enzymes, particularly the cytochrome P450s (Cyps), may detoxify a drug or enhance its toxicity, so a cell without metabolizing capacity may not provide a true reflection of in vivo toxicity. Similarly, hepatocytes have a variety of transporters which import or export molecules, and expression of these transporters in vitro often differs from the in vivo situation. Cell lines are available that express metabolizing enzymes and transporters to a greater degree, and there has been substantial work on enhancing expression by mimicking the hepatic

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environment with various matrices and threedimensional structures. One can also use transfected cell lines that are engineered to overexpress specific human metabolizing enzymes. Human pluripotent embryonic stem cells can be induced to differentiate into a variety of functional cell types such as hepatocytes, neurons, or cardiac myocytes that can then be exposed in vitro to compounds of interest (Medine et al., 2013; Corbett & Duncan, 2019). A common liability of pharmaceutical molecules is inhibition or induction of metabolizing enzymes, such as cytochrome P450s (Cyps) and others. This can lead to drug–drug interactions (see ADME Principles in Small Molecule Drug Discovery and DevelopmentdAn Industrial Perspective, Vol 1, Chap 3). In real life, patients generally take multiple medicines at the same time, and alteration of metabolizing enzymes can increase or decrease the concentrations of some of these comedications. In some cases, the compound of interest can induce metabolizing enzymes which can alter the concentrations of other medications that a patient may be taking. In other cases, exposure of the compound of interest may be either increased or decreased by the actions of other medications, leading to toxicity or lack of efficacy. Accordingly, there is considerable effort at the early stages of drug discovery to avoid inhibition or induction of metabolizing enzymes. Highthroughput screening assays are used to measure metabolic stability and the effects of pharmaceutical molecules on the common cytochrome P450s. Depending on the history of a particular program and chemotype, more detailed analyses can be performed as needed. High-throughput in vitro screening assays are also used to identify and deselect molecules that interact with a broad panel of off-target receptors and enzymes that may lead to toxicity (Bowes et al., 2012). Notably, the initial panels of screening assays generally identify binding, which does not necessarily mean there will be a functional change, such as agonism or inhibition. Key positive results from binding assays can be followed up with a functional assay. Receptor panel screening assays can provide drug concentrations at which binding occurs. While those concentrations may be much higher than the in vitro potency or the efficacious plasma concentration of a drug and thus may not be deemed relevant, it is possible that those concentrations would be achieved in a toxicity

study, in which high doses are given, or in organ such as liver where a compound or its metabolites are at higher concentration than in plasma. Concentrations within the GI tract may be higher than in plasma, which may affect local transporters or metabolizing enzymes. Thus, it is quite helpful for a pathologist to be aware of the results of the receptor panel assays so that relevant organ systems can be scrutinized carefully. Not all adverse events have morphologic hallmarks that cause gross or microscopic lesions. Depending on the receptor, it may be appropriate to follow up with safety pharmacology tests to evaluate the cardiovascular, respiratory, or central nervous system function, or it might be necessary to design more specific tests to evaluate toxicity. Ideally, if one is aware of potential toxicities from a receptor panel assay, one could anticipate serious liabilities and build informative endpoints into the design of a standard toxicity study, to avoid additional investigative studies afterward. Proper interpretation of in vitro data requires an understanding of their limitations. An important consideration is a compound’s solubility. If a compound is poorly soluble, then an in vitro assay may give a false-negative result. Sometimes, the scientist who performs the assay, or the analytical output itself, informs the end user that the compound has precipitated, but this cannot be assumed. It is important to review all data even in a high-throughput system where large numbers of compounds are assayed simultaneously in multiwell, robot-driven plates. Nephelometry is a useful way to identify solubility issues. This measures light scatter, which is enhanced if there are small, undissolved particles in a solution. However, a poorly soluble compound may not be detected by nephelometry. The user of in vitro data must be aware of this issue when interpreting results. To enhance solubility, many compounds are dissolved in DMSO, which can affect certain assay endpoints. Compound sample impurities, including solvents and catalysts, can also confound data interpretation. At the early stages of drug discovery, newly synthesized compounds are usually tested in high-throughput in vitro assays without purity characterization. When interpreting in vitro data, it is important to consider the possibility that effects may be due to an impurity rather than the compound of interest. Undesired cytotoxicity can also confound interpretation of in vitro cellular assays. For

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example, the Ames assay for mutagenicity is based on the frequency of random mutations that allow otherwise replication-incompetent Salmonella to grow on media that lacks histidine. Bacteria with these mutations grow on the selective media, and the resultant colonies are counted. However, if the compound happens to be bactericidal, then there will be few if any colonies, and the compound would be falsely categorized as nonmutagenic or “clean.” To prevent these errors of interpretation, appropriate controls are essential when designing in vitro assays. Compounds often bind avidly to proteins such as albumin, e.g., via hydrogen bonds, and protein binding can significantly impact interpretation of in vivo results. Most in vitro assays do not simulate plasma protein concentrations and may be performed in protein-free tissue culture media. This can result in a larger percentage of the molecules that are free, rather than protein bound, in the in vitro test solution, as compared with what would occur in vivo. If the concentration of nonprotein-bound drug, i.e., free drug, is what drives pharmacology or toxicity in vivo, then in vitro results can overpredict in vivo relevance. For example, a drug that is 99% protein bound, and therefore only 1% free, may test positive for a liability at 1 mM in vitro, but might require 100 mM to cause the anticipated effect in vivo! The concentrations at which adverse effects are seen in an in vitro assay should be interpreted in light of the efficacious concentrations in pharmacological assays or in vivo studies. Sometimes, in vitro assays show effects at concentrations that are hundreds of fold higher than the efficacious concentrations, so the findings may not be relevant to the in vivo situation. Additionally, some toxicities are more serious than others, and the impact of the predicted effect on the patient should be considered. As an example, if an uncommon metabolizing enzyme is inhibited at a concentration that is much higher than the efficacious concentration, the relevance to patients might be low. However, if a cardiac ion channel is inhibited at a similarly high concentration, this could result in cardiac conduction impairment in a fraction of the patient population that happens to have high drug levels and cause fatal arrhythmias. A consideration is if the toxicity can be easily

monitored in patients, either by biomarkers or early premonitory signs. Importantly the therapeutic indication should be considered. Adverse effects may be more manageable and acceptable if the drug is intended for serious lifethreatening diseases such as cancer, while adverse effects are not tolerated at all for indications such as hair growth, obesity, or erectile dysfunction. However, the indication should not be used as an excuse for accepting compounds with serious safety liabilities. Clearly, cancer patients would prefer drugs with fewer adverse effects, and there is a huge competitive advantage in marketing safer drugs for any indication. With any drug, risk assessment is necessary, and potential adverse effects should be defined and ideally minimized in the early stages of drug discovery.

5. TARGET SELECTION In many pharmaceutical companies, initial target selection falls into the domain of biologists, bioinformaticians, or from the literature or competitors. Increasingly, though, discovery pathologists and discovery toxicologists play an important role. A useful tool for this endeavor is a Target Safety Assessment (TSA) document, which is a compilation of available data on the intended target and potential safety liabilities, as well as an initial risk assessment for the intended patient population. New targets must be considered holistically by discovery pathologists who should compile a target safety assessment dossier by combining their literature review with their own experiences in a living document. Discovery pathologists should lead or participate in the writing of a TSA, which can serve as a valuable resource throughout the program. Knowing where the target is expressed can often enable an early estimate of potential safety liabilities. It is valuable to know if there are polymorphisms, heterozygosity, or deletions in the human population, as well as in animal models. An important consideration is how the target distribution in the animal models matches the target distribution in humans, including healthy humans and diseased patients (see below, Translational medicine). IHC is one technique in assessing tissue distribution of a proposed target.

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Discovery pathologists can help ensure that the assays are performed in a reliable, properly controlled way, and that results are correctly interpreted. Considerable information is available from published and on-line sources, including databases and competitors’ information. A useful guide to information resources is Brennan (2017), Kuo and Chen (2017).

6. TARGET VALIDATION Validating a target that has been identified and deemed of interest to the company requires demonstrating with a reasonable degree of certainty that pharmacologic modification of the target will impact the disease in a beneficial way. Once a pharmacologic target is identified and endorsed by the company, a drug discovery team is formed and empowered, research activities from many disciplines quickly ramp up, and resource expenditures increase exponentially. Accordingly, selection of good pharmacologic targets, with a reasonably high probability of efficacy and safety, is absolutely critical to the success of a company. Target validation is not a straightforward, “check-box” process. Each target requires a combination of brute force effort and finesse or ingenuity, as new biology is understood and new technologies are applied. Often a target is not completely validated until late-stage clinical trials, but it starts with providing evidence that modulation of the target is likely to have a beneficial effect on humans with a particular disease. Equally important, target validation includes providing evidence that potential adverse effects will be manageable in the context of the disease that the drug is intended to treat. Of course, the ultimate test of a target does not occur until a drug has been tested in human beings, but early efforts toward validating a target can save considerable resources. Animal models of efficacy and safety are usually indispensable and almost always are required by management and eventually by health authorities, e.g., the FDA or EMA. The choice of animal model can have a serious impact on whether a target is accurately characterized or not. As George Box famously said, “all models are wrong, but some are useful“ (Box, 1979). No model perfectly replicates all characteristics

of human beings, especially ones with the targeted disease. The biology of the test species must be considered and details matter. Some considerations are quite obvious to a veterinary pathologist but many others require selflearning. For example, inhibitors of cholesteryl ester transfer protein (CETP) cannot be evaluated in wild-type mice, since wild-type mice lack CETP and are resistant to atherosclerosis. Thus, investigations of lipid biology typically use other species, use mice with specific genetic alterations such as CETP transgenic mice, or at least take the absence of CETP into account when interpreting results. Discovery pathologists need to be fully informed and be able to recognize naturally occurring or so-called “background” lesions in each animal model. For example, a common model of diabetes is the Zucker Diabetic Fatty (ZDF) rat. Although these rats develop spontaneous diabetes, they commonly have hydronephrosis as well. The renal lesions can be quite severe, and this can confound studies of diabetic nephropathy (Marsh et al., 2007). Ob/ob mice have a moderate background incidence of pancreatitis, usually involving peripherally located lobules and associated with steatitis or fat necrosis. Inexperienced pathologists or nonpathologists could be confused by finding pancreatitis in rats being used as animal models of diabetes. Rather than understanding it as a spontaneous background lesion (Matveyenko et al., 2009; Chadwick et al., 2014), they might attribute it to a drug effect, especially because drugs such as GLP1 analogues and DPP4 inhibitors (which enhance the half-life of GLP1) have been associated rarely with pancreatitis in the clinic (DeVries & Rosenstock, 2017). GEMs are often valuable in target validation (see Models of Toxicity: Genetically Engineered Animals, Vol 1, Chap 23). Their proper use requires a basic understanding of genetic manipulation techniques. Use of knockout and transgenic mice by discovery biologists and pathologists to show the biological effects of target modulation is commonplace. Phenotyping of GEMs provides important insight into potential target liabilities (Cantor, 2010). Firstline phenotyping, especially standard clinical chemistry, hematology, gross necropsy, and histopathology, can be performed on unstressed GEMs in a basal or resting state. After

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6. TARGET VALIDATION

characterizing the baseline or “natural state,” especially valuable information can be attained when a discovery pathologist can influence the design of efficacy studies that use these GEMs to include customized clinical or pathologic examination. Often this approach can be reassuring with regard to target safety under relevant physiological stressors or pharmacologic conditions, but sometimes can reveal potential target liabilities that require further investigation. It is beyond the scope of this chapter to discuss GEM models in detail, and only the principles will be discussed here. The pathologist should be aware of the details of the model construction and of the genetic background of the GEM. For transgenic GEMs, this includes whether the transgene is randomly inserted or targeted to a specific locus, and for knockouts, the specific details of what portion of a gene is deleted. The choice of promoter influences the tissues in which the altered gene is expressed. Selection of the genetic background is important to reduce the likelihood that naturally occurring traits or lesions of the background strain will obscure the results or confound the interpretation of pharmacological studies. GEM phenotyping strategy is a balance between the potential usefulness of the results and the cost. The power of using GEMs for target validation is that they can reveal previously unsuspected abnormalities in animals as a whole or in specific body systems. When a target is largely unexplored, the desire to undertake extensive phenotyping is quite understandable. On the other hand, resources are almost always limiting. Comprehensive phenotyping may include growth and development measurements, behavioral assessments, and physical examinations. It is helpful to communicate closely with the mouse breeders and animal caretakers to learn about any unusual observations, and it can be useful for the pathologist to go into the vivarium personally and see the mice when a situation is out of the ordinary. Other assays include physiological and immunological tests, clinical pathology evaluations, in addition to necropsy and histopathology. Results should be compared to appropriate control animals, ideally wild-type littermates, and this requires advanced planning, especially when the breeding strategy is determined.

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GEMs can provide important insights and direct further research, but certain caveats need to be kept in mind. The phenotype in a particular line of GEMs may vary depending on the background strain. Phenotypes depend not only on the gene of interest but also a complex network of modifier and regulatory genes. Thus, it is possible to be misled by findings in a line of GEMs. Spontaneous background lesions or “nonlesions,” i.e., normal anatomic or morphologic variants that are nonpathologic, of the background strain must be considered. This can be especially problematic when spontaneous lesions are uncommon. In phenotyping studies, group size is typically small, and just by chance, if a spontaneous background lesion appears in several animals in the GEM group and not in the wild-type control group, it could be wrongly attributed to the gene of interest and thereby generate unnecessary concern and timewasteful follow-up investigative efforts. On the other hand, dismissing exacerbation of background lesions can also be a mistake. In the experience of one of the authors, there was a study with an increased incidence of atrial thrombosis after drug treatment. Later, it was learned that the drug was increasing the heart rate and blood pressure, which was probably enhancing the background incidence of this finding. Superficially, it might seem that a knockout mouse is an ideal surrogate for predicting the worst-case scenario should a drug completely inhibit or antagonize a target. However, consideration should be given to effects on development that would not likely manifest in an adult population. Conventional knockout mice have an important difference from humans who are being treated with a drug that targets the functional protein, e.g., receptor or enzyme, encoded by the deleted gene. In conventional knockout mice, the gene of interest is deleted from conception, including throughout embryonic development, whereas humans are only treated after they are diagnosed with disease, often as an adult. In the developing knockout mouse embryo, alternative pathways may be activated to adapt or compensate for the absence of the deleted gene of interest. This can result in a misleading model. It may be necessary to generate a temporally conditional knockout, in which gene excision is induced at a specific time. Another use of temporally conditional knockouts is when gene deletion is lethal to embryos.

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Understanding the molecular genetics that underlie construction of a GEM is important and its potential impact on interpretation of the GEM phenotype can be critical (Ku¨hn & Wurst, 2009; Markossian & Flamant, 2016). Even with molecular verification of targeted gene deletion, the discovery pathologist must be on the lookout for other genetic consequences. Knockout mice are typically constructed by deletion of a single key exon, rather than the entire genetic locus. However, alternative splicing is common, and if the particular exon that is deleted is also shared by other mRNAs that encode other proteins, then those proteins also will be missing or lack some aspect of their function. It is possible that the knockout phenotype is due to the absence of one of those proteins, rather than the target of interest. It is also possible that the insertion of genetic material into the genome results in unintended disruption of the genetic locus into which the gene of interest is inserted. Thus, there may be genetic alterations other than those intended. Although mouse and rat research has contributed enormously to our knowledge of biology and led to all sorts of medical advancements, there are unique aspects of human biology that cannot be mimicked by rodent models. A variety of “humanized” models are available to answer specific questions. Mice with altered cytochrome P450s (CYPs), nuclear receptors, and transporters are discussed in Genetically Engineered Animal Models in Toxicologic Research, Vol 1, Chap 23. A more complete attempt to replicate human biology has led to replacement in the mouse of entire tissues by their human counterparts. Several groups have designed mice in which most of the native, mouse hepatocytes are replaced by human hepatocytes (Tateno et al., 2004; Strom et al., 2010) (Genetically Engineered Animal Models in Toxicologic Research, Vol 1, Chap 23). These in vivo models have been useful for investigating differences between mouse and human drug metabolism, including speciesspecific conversion of nontoxic drugs to toxic metabolites. Mice with humanized livers have also been used for efficacy testing of antihepatitis C virus compounds, since hepatitis C virus does not replicate in cells from mammals other than humans or great apes, such as chimpanzees, although with some caveats (Berggren

et al., 2020). Another application of mice with human hepatocytes is the study of aspects of drug distribution or biology that depend on human plasma proteins. Since the humanized liver mice have functional hepatocytes, they express human serum albumin, human acid– alpha1-glycoprotein, other acute phase reactant proteins, certain human complement factors, coagulation factors, and others. For example, humanized liver mice could be used in the study of certain coagulation factor inhibitors. While humanized liver mice have opened up new research opportunities, the current state of the technology comes with important caveats. Replacement of mouse hepatocytes by human hepatocytes is incomplete, and these humanized livers actually consist of a mixture of human and mouse hepatocytes. Nonetheless, the mouse hepatocytes are metabolically active and can produce mouse-specific metabolites that can confound interpretation of experimental results. Also, if cells other than hepatocytes are of investigative interest, it is important to note that these are not humanized. Bile duct epithelial cells, endothelial cells, Kupffer cells, and Ito cells remain of mouse origin and presumably retain mouse-like biology. Also, the human hepatocytes tend to not link to the mouse biliary tract, resulting in an inherently cholestatic model with mixtures of circulating conjugated bile acids that may have different secondary effects on engrafted human hepatocytes (Chow et al., 2017). Another caveat is that the human cells that are transplanted into the mice are generally only from one, or a few, individual human donors. There can be substantial genetic differences among individual humans in expression of metabolizing enzymes, and unless one repeats the experiments with a number of independently transplanted humanized mice, which would be quite expensive, the results could be misleading. Concurrent diseases or drug treatments that the donor received can also alter results. With those caveats, however, when the right questions are asked or when the models are carefully applied, these humanized models are quite powerful tools. Mice with humanized immune systems are also available (Allen et al., 2019) and are discussed in detail in Genetically Engineered Animal Models in Toxicologic Research, Vol 1, Chap 23. Because immune cell interactions are complex,

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7. TRANSLATIONAL MEDICINE

interactions between human immune cells and other murine cells are not easily characterized. For example, interactions between immune cells and endothelial cells are paramount for normal function, and in the humanized models, in which the endothelial cells are of mouse origin, the human immune cells may not bind or signal in a way that recapitulates either the normal mouse or the normal human immune system. Nonetheless, important questions can be addressed with these tools. For example, mice with humanized immune systems have been used to optimize the selection of protein therapeutics based on minimizing immunogenicity. These in vivo assessments with humanized mouse models can be used to complement or in special cases replace in silico evaluation of amino acid sequences and in vitro assessment with functional human peripheral blood mononuclear cells. Of course, these humanized mouse models are expensive and complicated to use effectively, so they are generally more suitable for resolving specific issues than for unfocused screening.

7. TRANSLATIONAL MEDICINE A common source of failure in the pharmaceutical industry has been lack of translation of findings from mouse or other models to humans. Sometimes, animal models underpredict human safety and/or efficacy, and sometimes they overpredict. A compound which brilliantly cures cancer in a mouse model may have no efficacy in human patients if the underlying biology is different between the species. Accordingly, there is now considerable effort to understand whether findings in animal models will translate to human patients, an effort that may be of second nature to many veterinary pathologists. Sometimes, there are specific populations of patients who might benefit or who are at increased risk, leading to “patient stratification.” Translational medicine also can work the other way. Findings in human medicine may stimulate nonclinical researchers to ask different questions or test new hypotheses. Formerly in many companies, translational medicine was a subdiscipline of clinical medicine, reserved for late-stage compounds undergoing clinical testing, after drugs were shown

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to be efficacious and safe in animal models. Now, translational medicine activities have a prominent role even in the earliest stages of drug discovery, and discovery pathologists should embrace the opportunity to collaborate in these endeavors (Seyan, 2019). Success rates can be markedly improved if projects ensure that the underlying biology of an animal model is relevant to human disease and that there are assays in place to measure whether a drug candidate has the intended pharmacodynamic effect once the drug enters clinical trials. These translational efforts include understanding of the target distribution, function, and target binding affinity in humans. Discovery pathologists may partner with clinical teams to identify useful tissue-based markers that can be identified by IHC. Sometimes, this leads to companion diagnostics, assays that are developed and validated to identify individual patients with a higher probability of response to a drug, or to pharmacodynamic assays that can be applied in biopsy specimens to better gauge response to therapy. Biomarkers have become a key expected tool and are almost essential for successful drug development because their appropriate use enhances confidence in clinical outcomes encouraging the company to move forward (see Biomarkers: Discovery, Qualification and Application, Vol 1, Chap 14, and Interpretation of Clinical Pathology Results in Nonclinical Toxicity Testing, Vol 2, Chap 14). Biomarkers can be used to identify patient populations who are more likely to benefit from a drug, to inform pharmacokinetic/pharmacodynamic (PK/PD) relationships and thus dose selection in the clinics, and to monitor toxicity. A key to successful development of a drug is the parallel development of appropriate biomarkers to assess in each patient if a compound is efficacious and potential toxicities are detected before they progress to serious adverse events. When properly validated and applied, biomarkers enable proof-of-confidence in a variety of ways, including the mechanism of action and regulatory approval with surrogate endpoints. Biomarkers also offer opportunities for individualized medicine, identifying patients who can or cannot be successfully treated. With some drugs, adverse effects cannot be avoided because they are integral with the pharmacology of the drug, such as cytotoxic cancer

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drugs. However, appropriately selected safety biomarkers allow physicians to identify patients at risk and discontinue drug therapy before there is irreversible injury. Thus, identification and validation of biomarkers can be crucial for regulatory agencies to approve a drug. Biomarkers are especially useful in selecting patients for treatment with expensive drugs or when patients with serious, life-threatening diseases must choose among different therapeutic options. For example, patients with metastatic colorectal cancer with certain KRAS mutations may have less benefit from treatment with the epidermal growth factor (EGF) receptor inhibitor cetuximab (Erbitux), whereas those with wild-type k-Ras often do respond favorably (Evaluation of Genomic Applications in Practice and Prevention (EGAPP) Working Group, 2013). Based on this observation, KRAS status in colon cancer samples is now a routine biomarker that is tested before cetuximab therapy is initiated. Considerable effort has gone into biomarker development to predict response to immunooncology drugs, including biomarkers such as tumor PD-L1 expression, tumor mutation burden, T cell infiltration into tumors, and more (Butterfield et al., 2018). Biomarkers include many modalities and range from blood tests to complicated imaging studies. In some cases, biomarkers are obvious and have been available for decades, such as the use of alanine aminotransferase (ALT) to monitor liver injury. In other cases, biomarkers must be discovered and validated for each drug development project, based on deep understanding of the biology of the disease and the mechanism of action of the specific drug. The quest for more useful and predictive biomarkers is initiated at the early stages of drug discovery, at the time when discovery pathologists and discovery toxicologists are engaged, and continues through the life cycle of a drug.

8. HYPOTHESIS GENERATION, EXPERIMENTAL DESIGN, AND THE ROLE OF INVESTIGATIVE STUDIES Another valuable role of a discovery pathologist is to define an issue, formulate hypotheses, and test them in an animal model or in vitro

system when appropriate. While some experiments may end with go/no-go outcomes, others are designed to provide a clearer understanding of hazards or more precisely define risk assessment. Even though considerable resources are expended to lay out flowcharts of efficient processes for drug discovery and development, the actual course of drug discovery and development is rarely, if ever, smooth and predictable. It is a scientific endeavor, not a productmanufacturing process. It is fully expected that there will be unexpected findings and issues. Simplistically, a discovery organization could set up rigorous screening tiers in predesigned decision trees and select only those compounds that pass all screening assays for further consideration as drug candidates. On the other hand, very few, if any, compounds are perfect. The only perfect compounds may be the endogenous ligands, so everything that we make, except perhaps recombinant proteins, could be considered as xenobiotic. It can be highly productive and eventually profitable to investigate an issue and resolve it. In some cases, an adverse effect may be specific to a particular species and not relevant to humans. Addressing a serious nonclinical safety concern by designing a mechanistic study that demonstrates its lack of relevance to human safety can convince regulatory authorities of the safety of a new drug and result in successful marketing approval (Tirmenstein et al., 2015). In other cases, the discovery and validation of a biomarker that can be used in humans to identify a reversible change may be the essential step in obtaining regulatory agency approval. Even at the earliest stage of drug discovery, medicinal chemists must make choices to select which types of core chemical structures to pursue, and protein engineers may need to determine which immunoglobulin isotype, target affinity, and mutations are best suited for a particular target. Such choices can be influenced by collaboration with, and feedback from biologists, toxicologists, and pathologists. Creative and scientifically sound hypothesis generation leading to decision-driving experiments can provide crucial guidance to chemists. Discovery pathologists, who are trained to think broadly about all body systems, must be more than classic slide-readers and play a key role in hypothesis generation and experimental design.

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Pathologists with strong research training, interest, and aptitude are especially valuable. Basic principles of scientific thinking should be emphasized, including the importance, whenever possible, of studies that show causation, not just correlation. Discovery pathologists in a research role within drug discovery provide the greatest value when they engage other scientists and leverage their knowledge and experience to ask the most impactful questions, and then formulate ways to answer those questions in a focused manner. These discovery pathologists do not spend the majority of their time performing microscopic examinations on an exhaustive list of tissues from numerous animals on each study. Instead, they often focus on specific tissues in a smaller number of animals to rapidly provide decisionenabling information.

9. DISCOVERY STRATEGY FOR BIOLOGICS As a convenient generalization, drugs are often classified as “small molecules,” which are chemically synthesized, or “biologics,” which are larger molecules, generally proteins, that are genetically engineered and then expressed by living organisms such as Escherichia coli, yeasts, or mammalian cell lines (see Overview of the Role of Pathology in Product Discovery and Development, Vol 2, Chap 2; Protein Therapeutics, Vol 2, Chap 6; Biotherapeutic ADME and PK/PD Principles, Vol 1, Chap 4). These include antibodies, fusion proteins, and recombinant proteins. In addition, there are many newer modalities in development. For example, antisense oligonucleotides are synthesized and are generally 10–20 bases in length (approximately 7000 MW) (see Nucleic acid Pharmaceutical Agents, Vol 2, Chap 7). There are also longer mRNA molecules, such as those used in the SARSCoV2 vaccines (see Vaccines, Vol 2, Chap 9), and altered cells, such as CAR-T or CAR-NK cells, and these are covered in more detail in other chapters. Typically, small molecules are in the 300–700 molecular weight range and have other pharmaceutical attributes that generally allow for oral bioavailability and long-term stability at room

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temperature. These molecules are subject to classic principles of absorption, distribution, metabolism, and elimination (ADME). In contrast, biologicals generally require parenteral administration, either intravenous or subcutaneous, and are often not stable at room temperature. Discovery and development of biologics come with a set of special challenges that differ from those of small molecules (Cavagnaro, 2008). In the discovery stage, often large numbers of hybridoma clones are available, but only a small number (or even just one) must eventually be selected for characterization. At this early stage, it is highly desirable to consider species crossreactivity. If a biologic only binds to the human target and not its orthologue in any nonhuman species, the route of development will be difficult. Sometimes there are affinity differences, such that there is reduced binding and pharmacologic activity in a nonhuman species. To some extent, affinity differences can be overcome by increased dosing, but it might not be possible to fully translate to humans, especially when higher doses do not provide the same degree of pharmacology as anticipated in the clinic. With such a molecule, it would be problematic to perform proof-of-concept testing in animal models, develop human dose projections, and conduct nonclinical toxicology testing. Selection of biologics that cross-react with cynomolgus monkey orthologues is highly desirable. This species is often selected because of its historic use and relatively more similar anatomy and physiology to humans. If possible, it is useful to select biologics that also cross-react with rodent orthologues, as this is typically the pharmacology model. In situations where the human target is unique and cross-reactivity is not possible, then it may be desirable to develop a transgenic mouse model which expresses the human target. Alternatively, some companies pursue discovery and parallel development of a “surrogate biologic,” a biologic that reacts with a nonclinical species, typically a rodent. This is discovered and developed in parallel and is used for investigational purposes even though it will never be used therapeutically in humans. Another reason to develop a surrogate might be because the human protein is so highly immunogenic in nonhuman species that adequate exposures or function in those species

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cannot be achieved and testing is not possible. Another strategy is the use of anti-CD4 antibodies in pharmacology studies, which generally prevents the formation of antidrug antibodies (Sun et al., 2014). If it is necessary to develop a surrogate, this effort should be started as early as possible because of the long timelines involved. Also, it should be recognized that the surrogate will not identically replicate all of the properties of the actual clinical candidate, and that some findings with the surrogate may be irrelevant. The cost and risk of investigating irrelevant findings can greatly complicate the process. A potential problem when therapeutic proteins are administered systemically is the development of immune responses against them, i.e., the formation of antidrug antibodies (ADAs) or “immunogenicity” (Krishna & Nadler, 2016) (see Biotherapeutic ADME and PK/ PD Principles, Vol 1, Chap 4). ADA is often formed in nonclinical species due to the administration of a human (foreign or xenogeneic) protein. Presence or absence of an in vivo immune response in a rodent or nonhuman primate does not usually translate into the same response in humans (Husar et al., 2017). In general, the formation and potential adverse effects of this ADA response, including hypersensitivity reactions, are not translatable to human immunogenicity. Predicting immunogenicity during discovery and preclinical development is an inexact science. Development of ADA in the clinics is especially common when the therapeutic protein differs from proteins normally found in the human body. There are many approaches to reduce immunogenicity, including “humanization” of biologics by replacing large portions of the encoding genes with human genetic material. Generally, the human response to a biologic consisting of a human protein is less than the animal response to the same protein. In silico approaches, as well as cell-based assays using human peripheral blood mononuclear cells, can be used to inform the likelihood of immunogenicity in humans, and may be employed in the discovery phase to select preferred drug candidates. If a biologic is immunogenic, nonclinical animals or patients may produce polyclonal antibodies, which can target different portions of the biologic protein. These include neutralizing

antibodies that could reduce target engagement and therefore the efficacy of the biologic, and opsonizing antibodies that enhance clearance of the biologic and reduce exposure. In some cases, antibodies bind to the biologic but have no effect on its medicinal properties. Thus, not every immune response to a biologic is harmful. Immune responses vary among individuals, however. In some cases, antigen–antibody complexes can form and their deposition can result in immunopathology (Rojko et al., 2014). Another consideration is the effect of immune responses in nonclinical species. This should be considered when designing toxicology studies, since it may reduce the exposure and necessitate higher doses and/or more frequent dosing. Repeated use of individual nonhuman primates such as cynomolgus monkeys for multiple pharmacokinetic studies using small molecules is common because these animals are expensive and have to be trained. However, the same multipurpose approach may not be glibly applied to biologics. The backbone Fc region of many therapeutic antibodies is the same or similar. Prior exposure to a monoclonal antibody from previous studies can lead to a persistent immune response, which has the potential of significantly altering the pharmacokinetics of future monoclonal antibodies if studied in the same monkey. Thus, whenever possible, naive animals which have not been exposed to other biologics of a similar type should be used. Alternatively, nonnaı¨ve monkeys can be screened for ADA, and those monkeys without ADA selected for studies (Han et al., 2015). Typically, the pharmacokinetics of biologics is quite different from small molecules. Monoclonal antibodies generally have half-lives of several weeks. Half-lives of antibody fragments are substantially shorter but can be prolonged by conjugation to polyethylene glycol (PEG) or other time extension moiety. This is an advantage because dosing, which is typically done by injection rather than oral route to avoid digestion, can be done at wider intervals which is more convenient to the patients. However, it is a potential problem if there is an adverse event attributed to pharmacology because it takes a long time for the antibody to be eliminated from the body. Because of long half-lives, a single-dose experiment during the early discovery and development stage can be used to test toxicity

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over a considerable period of time, unlike a small molecule, where single-dose studies provide limited information other than toxicokinetics and acute tolerability. In contrast, short peptides are rapidly excreted through the kidney by glomerular filtration and renal elimination. Extremely short half-lives can be an impediment to toxicity studies and to efficacy. A consideration in the use of PEG as a half-life extender is the potential for PEG itself to cause tissue alterations and subsequent evaluation of whether these changes are adverse (Ivens et al., 2015). Another consideration is bioanalytical support. The total plasma antibody concentration may not reflect the pharmacologically active antibody. For example, the antibody might be in a complex with soluble targets. A bioanalytical approach may have to be developed to distinguish between bound and unbound antibody. Due to the length of time to develop moleculespecific reagents, in the discovery phase generic (often antihuman IgG) ELISA assays are developed to evaluate the pharmacokinetics of biologics in nonclinical species. Often ADA is inferred from the pharmacokinetics profile, i.e., increased clearance with repeat exposure.

10. COMMUNICATIONS A pharmaceutical company is a highly matrixed environment, and a discovery pathologist works with an extraordinarily wide variety of specialists as well as with company management. In contrast to later stages of drug development, where pathologists generate reports in a standardized style and format for inclusion in the overall study report and regulatory filings, a discovery pathologist must be able to communicate effectively with a much wider range of specialists. This requires a different style of communication, which is more flexible and free of jargon, including highly specialized pathology terms that may seem pedantic. Although occasional formal written reports are needed, a pathologist in the discovery environment often communicates less formally, including by PowerPoint slides, presentations at meetings, teleconferences, video conferencing, or e-mails. When communicating to a group of pathologists, it is sufficient to show a photomicrograph of a lesion and assume that the viewers have a good idea of

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what is normal and what is not. The goal of sharing histopathology images to a project team which includes nonpathologists is to provide a visual representation of important findings. The goal is not to show all the findings and all the severity levels. However, a demonstration of how a minimal finding is different from a severe finding is helpful, particularly if a call of adversity is made on the one level of severity and not another. When communicating to nonpathologists, it is helpful to show side-by-side photomicrographs of control tissues and to carefully label the anatomic substructures for orientation. Clear and simple explanatory captions are essential. It is useful to state the pathologic diagnosis and interpretation on the slide, even as the title of the slide, rather than expecting the viewers to draw their own conclusions. Thus, instead of a general title such as “Study xxxx: Histopathology Results,” it is better to use an informative title such as “Compound Y, 5 mg/kg/day: Hepatocellular Necrosis.” In interactions with other specialists, it is important to realize that as scientists, they are trained to be skeptical and question new data critically before accepting it. In presentations, it is common for nonpathologists to intensively query the pathologist’s interpretations. To a beginner, this can be intimidating, and it may seem that the other scientists are challenging one’s results in a belligerent or confrontational way. However, that form of dialogue is the normal and healthy process by which most new scientific data are examined. It is a good idea to be well prepared, anticipate questions, and be fully capable of justifying diagnoses and interpretations. At the same time, it is important to avoid the temptation to seem arrogant or hyper-knowledgeable. When you are not certain of an answer, freely admit it and assure the team you will investigate and report back on or before an agreeable date. The rewards of good communication are building a better, more functional team and being given the opportunity to be taught by experts in other fields, as well as making new friends. In that way, after explanation of the pathology results, discovery team members more readily accept the advice and recommendations of the discovery pathologist. Such advice includes a broad, multifactorial assessment of the impact of the results on the validity of the target and/or developability of the

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compound, and when possible on how to progress the program (see Risk Management and Communication: Building Trust and Credibility with the Public, Vol 2, Chap 17). Timely communications are particularly important in a team environment. A discovery team, which fosters innovation and risk-taking, is much less formal than a development team, and many teams expect ongoing updates during the progress of a study and a prompt preliminary report at the conclusion of a study. Sometimes, it is helpful to discuss results with leaders or key team members before a meeting so that a consensus can be built easier during the meeting. One should avoid springing surprises on “high-level” stakeholders such as management during team meetings. Sometimes, a discovery pathologist needs to communicate critical data quickly, at times even presenting preliminary conclusions to a discovery team on the same day that the slides are received. Input from an experienced discovery pathologist is well suited for helping a discovery team navigate this fast-paced exchange. However, while rapid communication with discovery teams is important, it is also helpful to take the time to communicate with your pathology and toxicology colleagues in development. They can often provide perspectives or share experiences that you haven’t thought of or seen before.

11. PERSONALITY AND BEHAVIORAL TRAITS THAT ARE HELPFUL TO SUCCEED IN DISCOVERY PATHOLOGY AND DISCOVERY TOXICOLOGY The discovery realm is the flexible and fastmoving area of the pharmaceutical industry. In the most productive discovery research teams, ideas are exchanged rapidly in a free-thinking, brainstorming mode; strategies and experiments are designed and executed as quickly as possible; and results are challenged and debated vigorously. Except for inescapable aspects of corporate life such as shifting management priorities, organizational changes, and internal politics, there tends to be much less hierarchy than in later stage development or commercialization. New ideas, alternative hypotheses, and innovative

methods are welcomed, especially if thoughtfully and convincingly presented. Not every pathologist is well suited for this culture. Flexibility and the ability to admit that one is wrong must be part of the discovery pathologist’s daily routine. Quick thinking, consideration of multiple viewpoints, and the ability to synthesize multidisciplinary information into new hypotheses are all important skills. A broad scientific base, a burning sense of curiosity, and the ability and desire to learn rapidly are all necessary. A sense of purpose, of being a “drug hunter,” of yearning to help patients with unmet medical needs is essential. Passion for the truth and for good science must be the motivating force. The ability to work well in a team environment is absolutely essential for a successful and effective discovery pathologist. Discovery pathologists do not spend the majority of their working day with their office door closed, peering through the eyepieces of their microscopes. They must be recognized by their discovery team colleagues as committed, fully engaged members of the team who share the team goals, including dedication to the end-goal of bringing innovative new drugs to humans with unmet medical needs. Although a discovery pathologist sometimes has to report program-halting results, he or she must learn how to communicate these results as a team member and offer realistic plans to resolve the issues that were uncovered, rather than appear as the Grim Reaper. If the team perceives the pathologist as sharing these goals, rather than as an obstructionist who delights in finding flaws with the compounds or the project, it is much easier to communicate effectively. Whenever possible, it is helpful to engage with a team as early as possible, showing up for team meetings even when the subject matter does not involve pathology or toxicology questions, to learn what the issues are and establish a cordial working relationship with colleagues. This attentive engagement builds team cohesiveness and can repay dividends later when difficult decisions must be addressed. The discovery pathologist must be able to take a firm, sometimes unpopular stand when there is strong evidence of an unacceptable toxicity, and it can be difficult to resist the peer pressure from the team. Even in these circumstances, discussing your findings and concerns with the team leaders

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12. SUMMARY

one-on-one or in a small group prior to informing the whole group by email or in a large meeting is usually appreciated and builds respect and trust. At these times, one’s communications and collaborative skills can be put to the test. When there is a disagreement, it is helpful to try to understand others’ points of view, and that can lead to finding common ground. It is important to accept, though, that scientific certainty is rare and more often decisions must be based on finding a balance between risk and potential benefit. Even the finding of severe toxicity in a nonclinical species does not necessarily mean there will be toxicity in humans, although it does mean that the team would face the need to conduct an investigative study to explain the toxicity and live with the risk that after putting resources into the investigative study, the results might not support further development. Pathologists in the discovery environment work on multiple projects at the same time, and time management can be stressful. Prioritization is essential to meet deadlines and help ensure that the most important work gets done. Work that aids in project decisions takes a high priority. At the same time, one must collaborate closely with colleagues, so it is difficult to completely avoid work that is not such a high priority, especially since most discovery team leaders consider their own project to have the highest priority. In those cases, it is important to convey an attitude of concern and cooperation, so that others understand that while the work of interest to them will not get done immediately, it is still in the queue and will receive careful attention in the near future. It is also necessary to recognize that one cannot do everything, nor can the organization provide resources to support every idea, so it is sometimes necessary to communicate directly that one cannot undertake certain projects or activities because of resource limitations. There are many personal skills required to successfully work in a collaborative team environment. It is particularly valuable to seek mentorship from experienced people and give mentorship to those who are less experienced. One must be willing to accept responsibility, including taking on work that is important, doing the work well, and bearing personal responsibility if there are failings that are within one’s own control. A group succeeds if members

recognize each other’s accomplishments. Sometimes, this can be simply a kind word or an acknowledgment. Networking, both within the organization and among professionals elsewhere in the country or world, is important. This can lead to enhanced collaborations and incorporation of new technologies to resolve problems. In conclusion, discovery pathologists have an exciting and highly beneficial role in the drug discovery process. Their skills, knowledge base, and problem-solving abilities can have farreaching positive results and contribute to improving human lives. Discovery pathology and toxicology is a rapidly moving field which quickly incorporates other areas of science and challenges the pathologist to continue to learn and grow.

12. SUMMARY Discovery veterinary anatomic and clinical pathologists play a key role in drug discovery research groups in the pharmaceutical industry because their expertise extends well beyond conventional pathology and toxicology. Discovery pathologists function as wellrounded whole-body pathophysiologists and biologists. Their unique skill sets provide research teams with critical perspectives on a wide variety of issues. These skills enable the impactful decisions necessary for the next stages of developing a successful new pharmaceutical candidate. These include providing input on selection of new targets; identifying, characterizing, and validating animal models of disease and efficacy; phenotyping genetically engineered animals; defining cross-species target expression and function in animals and comparing to what is known in humans; and designing experiments to assess whether exaggerated pharmacology results in toxicity. When toxicities are identified, pathologists are uniquely suited to distinguish between target-based and off-target effects and advise the team on the implications of these findings, including biomarkers for monitorability and reversibility. Within a department of discovery toxicology, discovery pathologists are an integral part of a larger team, in which they work together with specialists in a large number of allied disciplines. In the discovery toxicology context, classical discovery toxicologists work together with discovery pathologists, in vitro

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biologists, cell and molecular biologists, genotoxicologists, cardiologists, electrophysiologists, bioinformaticians and genomicists, reproductive biologists, immunologists, in vivo scientists, and others. This multidisciplinary approach can often identify safety liabilities at an early stage of drug discovery or development and provide insight into how to avoid or manage these liabilities.

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Rebelatto MC, Schenck E, Horvath C: Formation, clearance, deposition, pathogenicity, and identification of biopharmaceutical-related immune complexes: review and case studies, Toxicol Pathol 42:725–764, 2014. Ruepp S, Janovitz E, Brodie T, White R, Santella J, Hynes J, Carmen J, Pan D, Wu Y, Hanumegowda U, Gemzik B, Megill J, DiPiero J, Drexler D, Su C-C, Hageman M: Assessing the risk of drug crystallization in vivo, J Pharmacol Toxicol Methods 96:1–8, 2019. Sandusky GE, Teheny KH, Esterman M, Hanson J, Williams SD: Quality control of human tissuesdexperience from the Indiana University Cancer Center-Lilly Research Labs human tissue bank, Cell Tissue Bank 8:287–295, 2007. Schermelleh L, Ferrand A, Huser T, Eggeling C, Sauer M, Biehlmaier O, Drummen GPC: Super-resolution microscopy demystified, Nat Cell Biol 21:72–84, 2019. Seyan AA: Lost in translation: the valley of death across preclinical and clinical divide: identification of problems and overcoming obstacles, Transl Med Commun 4:18, 2019. https://doi.org/10.1186/s41231-019-0050-7. Shu Y-Z, Johnson BM, Yang TJ: Role of biotransformation studies in minimizing metabolism-related liabilities in drug discovery, AAPS J 10:178–192, 2008. Simic D, Simutis F, Euler C, Thurby C, Peden WM, Bunch RT, Pilcher G, Sanderson T, Van Vleet T: Determination of relative Notch1 and gamma-secretase-related gene expression in puromycin-treated microdissected rat kidneys, Gene Expr 16:39–47, 2013. So PTC, Dong CY, Masters BR, Berland KM: Two-photon excitation fluorescence microscopy, Annu Rev Biomed Eng 2:399–429, 2000. Strom SC, Davila J, Grompe M: Chimeric mice with humanized liver: tools for the study of drug metabolism, excretion, and toxicity, Methods Mol Biol 640:491–509, 2010. Sun B, Banugaria SG, Prater SN, Patel TT, Frederickson K, Ringler DJ, de Fougerolles A, Rosenberg AS, Waldmann H, Kishnani PS: Non-depleting anti-CD4 monoclonal antibody induces immune tolerance to ERT in a murine model of Pompe disease, Mole Genet Metabolism Rep 1:446–450, 2014. Tateno C, Yoshizane Y, Saito N, Kataoka M, Utoh R, Yamasaki C, Tachibana A, Soeno Y, Asahina K, Hino H, Asahara T, Yokoi T, Furukawa T, Yoshizato K: Near completely humanized liver in mice shows human-type metabolic responses to drugs, Am J Pathol 165:901–912, 2004. Tirmenstein M, Horvath J, Graziano M, Mangipudy R, Dorr T, Colman K, Zinker B, Kirby M, Cheng PTW, Patrone L, Kozlosky J, Reilly TP, Wang V, Janovitz E: Utilization of the Zucker Diabetic Fatty (ZDF) rat model for investigating hypoglycemia-related toxicities, Toxicol Pathol 43:825–837, 2015. Treuting PM, Dintzis SM, Montine KS: Comparative anatomy and histology: a mouse, rat, and human atlas, 2nd ed., 2017, Academic Press, 570 pp. Zhang D, Zhu M, Humphreys WG: Drug metabolism in drug design and development: basic concepts and practice, 2008, Wiley-Interscience, 607 pp.

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C H A P T E R

4 Pathology in Nonclinical Drug Safety Assessment Magali R. Guffroy1, Xiantang Li2 1

Preclinical Safety, AbbVie Inc., North Chicago, IL, United States, 2Drug Safety Research and Development, Pfizer Inc., Groton, CT, United States O U T L I N E 1. Introduction

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3. The Pathologist’s Role in Nonclinical Safety Assessment 3.1. Adverse Effects and Pathology Report 3.2. Reversibility and Delayed Toxicity 3.3. Lexicon and Diagnostic Terminology 3.4. GLP Regulations in Pathology 3.5. Pathology Peer Review 3.6. NOAELs and Study Report

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5.3. 5.4. 5.5. 5.6.

Gene Therapy Cell Therapy Stem Cell Therapy Vaccines

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6. Pathology in Nonclinical Safety Assessment of Novel Formulations 6.1. Excipients 6.2. Conjugation 6.3. Nanotechnology

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7. Digital Pathology and Computational Pathology 119

4. Pathology in Nonclinical Safety Assessment of Small Molecules 108

8. Novel Investigative Tools in Nonclinical Safety Assessment 120

5. Pathology in Nonclinical Safety Assessment of Biotherapeutics 109 5.1. Proteins 110 5.2. Oligonucleotides 111

9. Conclusion

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past several decades, pharmaceutical companies have invested in discovery and development of novel biotherapeutic modalities such as proteins, nucleotides, vaccines, and gene- and cell-based therapeutics (Assaf and Whiteley, 2018; Sellers et al., 2020; Yu et al., 2019). Traditional understanding of how chemical and

1. INTRODUCTION Drug research and development (R&D) is a time-consuming, resource-demanding, and highly regulated process (Figure 4.1). Traditionally, drug R&D has been focused on small molecules (Blanco and Gardinier, 2020). During the

Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-12-821047-5.00033-6

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FIGURE 4.1 Drug research and development is a time-consuming, resource-demanding, and highly regulated process. Pathologists play important roles in this process, from initial ideas for therapeutic target selection through the loss of exclusivity of a patented drug, focusing on nonclinical assessment and risk management with regard to both drug efficacy and safety profiles. Figure revised, based on a Pfizer poster, with permission. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 24.1, p. 726, with permission.

physical properties of small molecules mediate their efficacy and safety has been challenged by these diverse biotherapeutic modalities. To add to this complexity, both traditional chemical and biotherapeutic modalities may require innovative formulations and novel targeted delivery methods (Figure 4.2). The innovative formulation and targeted delivery may improve drug efficacy and safety profiles but may also present new safety challenges (Joubert et al., 2020). When drugs are formulated at nanoscale, for example, the unique size-specific properties and biological behaviors may create unique safety concerns (see Nanoparticulates, Vol 3, Chap 13). Novel therapeutic modalities, especially biotherapeutics, may also require innovative and complex processes of manufacturing, transportation, and storage.

The process-related product variations, impurities, residuals, degradation products, and contaminants may present additional safety challenges (Figure 4.2). Pathology studies the nature, pathogenesis, and consequences of diseases, through characterization of morphologic and molecular changes in body tissues and/or fluids. In the pharmaceutical or chemical industry, pathologists characterize toxicologic effects on body tissues and/or fluids after exposures to test articles of diverse modalities under controlled nonclinical test conditions. Pathologists play important roles in the drug discovery and development process, from initial selection of therapeutic target and molecule through the loss of exclusivity of a patented drug (Figure 4.3). In the discovery phase, discovery pathologists not only can help

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specific agencies, such as the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH), Organisation for Economic Co-operation and Development (OECD), US Food and Drug Administration (FDA), Health Canada, European Medicines Agency (EMA), Therapeutic Goods Administration (TGA), Japan Pharmaceuticals and Medical Devices Agency (PMDA), Brazilian Health Regulatory Agency (ANVISA), and China National Medical Products Administration (NMPA). Nonclinical safety studies to support clinical trials must also be conducted in compliance with Good Laboratory Practice (GLP) regulations (see Pathology and GLPs, Quality Control and Quality Assurance, Vol 1, Chap 27) and with the principles of the 3Rs (Replacement, Reduction, and Refinement) for humane animal research. FIGURE 4.2 Novel therapeutic modalities, innovative formulations, complex production, and packaging processes may present unique and unanticipated safety concerns from conventional perspectives. Pathologists must take the whole product life cycle into consideration, based on a deep understanding of the pathophysiological processes involved in any potential toxicity. Each specific modality should be assessed on a case-by-case basis, including its unique target, indication, relevant regulatory guidance, formulation method, and production process. Figure modified from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 24.2, p. 728, with permission.

interpret animal model study data for efficacy endpoints, but also can implement approaches to identify predictable safety issues earlier in the testing paradigm, in conjunction with selection and validation of therapeutic targets and drug molecules (see Discovery Toxicology and Discovery Pathology, Vol 2, Chap 3). In the development phase, toxicologic pathologists are involved in nonclinical safety assessment in compliance with diverse regulatory requirements, and thus they are also known as regulatory pathologists. Toxicologic pathologists identify the hazard and characterize potential human risk associated with diverse therapeutic modalities to support clinical trials. This chapter focuses on toxicologic pathologists’ roles in nonclinical safety assessment. Nonclinical safety assessment is highly regulated by international, regional, and country-

2. DRUG SAFETY AND EFFICACY ARE A CONTINUUM Conceptually, drug R&D consists of three basic steps: selecting the right therapeutic target, selecting the right drug molecule, and finally selecting the right patient population (Figure 4.3). In the initial stage of drug discovery, promising therapeutic targets are identified for a variety of unmet medical needs (see Overview of Drug Development, Vol 2, Chap 1). It is generally believed that once a drug molecule has been selected, the risk is already baked into that selection. Thus, careful selections of both target and molecule and clear understanding of off-target effects are critical for the successful development of drugs. Pathology expertise is important in discerning the safety risk inherent with the proposed target and in contributing to the selection of the right therapeutic modality and molecule (see Risk Assessment, Vol 2, Chap 16). Pathology expertise is also critical in teasing out toxicity related to exaggerated pharmacologic effects and off-target effects, which will facilitate the selection of the very best and most viable target and molecule. Drug efficacy and safety are essentially a function of the continuum of drug exposures and duration. With both small molecules and biotherapeutics, toxicity comes in two main flavors,

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FIGURE 4.3 Drug research and development conceptually consists of three basic steps, i.e., selection of the right therapeutic target, the right molecule, and the right patient population. Once the target and molecule are selected, the candidate drug molecule will be assessed in a series of nonclinical safety studies in compliance with regulatory requirements (e.g., ICH guidelines) and the 3Rs principle of animal uses. BLA, biologics license application; CTA, clinical trial application; FIH/FIP, first in human/first in patient; IND, investigational new drug; MAA, marketing authorisation application; NDA, new drug application.

that associated with binding to the intended target, i.e., exaggerated primary pharmacologic effects, and that associated with unintended interaction with secondary targets, i.e., offtarget effects (Figure 4.4). Regarding exaggerated pharmacologic or on-target effects, excessive target stimulation with sustained high drug exposures may have toxic effects (see Principles of Pharmacodynamics and Toxicodynamics, Vol 1, Chap 5). In this situation, thresholds for efficacy and safety may be arbitrarily drawn and the right dosage and dosing duration may differentiate the remedy from the toxicity. Exaggerated pharmacology effects are often associated with the intended drug mode of action given at high doses for prolonged durations, including carcinogenic effects (Vahle et al., 2010). The excessive and sustained pharmacologic effects could nonspecifically affect downstream pathways or produce secondary effects in animals, which may not occur in humans at therapeutic doses. Some drugs may bind to the same receptor but induce differential effects depending on the dose or duration of exposure. For example, gamma-aminobutyric acid (GABA) binds to inhibitory neurons in the

central nervous system (CNS) and diminishes neuronal excitability at low doses (Ngo and Vo, 2019). Thus, GABA receptor agonists are used to calm anxiety and/or increase thresholds for convulsions. However, at high doses some agonistic drugs may act as antagonists at the same GABA receptor, leading to increased neuronal excitation and seizures. Another classic example of exaggerated pharmacology is the cytokine release syndrome (Fajgenbaum and June, 2020). Cytokines released by targeting lymphocytes or macrophages may increase cytotoxic T cell or NK cell activity against tumors. However, at higher doses such effects may be exaggerated, resulting in a “cytokine storm” and a cascaded release of large amounts of cytokines with the potential for causing great harm, including death. There are many other examples of exaggerated pharmacology effects, such as increased bone mass and bone tumors in rats after long-term dosing with parathyroid hormone intended for the treatment of osteoporosis, and excessive lymphocyte proliferation in monkeys dosed with immunostimulatory biotherapeutics (Figure 4.5). Whereas secondary pharmacology effects or off-target toxicities are traditionally more

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FIGURE 4.4 Safety concerns about therapeutics often come in two forms: exaggerated pharmacology and offtarget toxicity. With small molecules, drugs tend to be distributed in multiple organs or systems (A). Off-target toxicity is a common observation. With biotherapeutics and novel targeted drug delivery system (B), drugs mainly affect the target organ. Exaggerated pharmacology is a more common toxicity finding. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 24.3, p. 729, with permission.

associated with small molecules that may chemically bind to a variety of unexpected targets, large molecules also can induce secondary effects through cross-binding to the target expressed on other cell types or cross-reactivity with closely related targets on the same or different cell types. Many homologous human proteins or nucleotides have been demonstrated with heterogeneity in receptors or ligands within and between species (human and animal model), races, and genders, and may result in unanticipated off-target effects. These off-target effects

may be screened through in vitro and in vivo safety pharmacology specialized assays. In some cases, the off-target toxicity can also be associated with the mechanism driving the efficacy, where the intended mode of action has multiple and sequential actions or effects on different tissues at high doses and/or for prolonged durations. For example, prolonged overdose of clotting factors for hemophilia has resulted in thrombosis, coagulopathy, and mortality in animal models. Another classical example would be the nonsteroidal

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FIGURE 4.6 Kidney of a dog dosed with a nonsteroidal antiinflammatory drug (NSAID). NSAIDs inhibit cyclooxygenase and thus block synthesis of prostaglandins and prostacyclins, which directly reduce renal medullary blood flow and lead to ischemic renal papillary necrosis (N). Note the pelvis (P) and medulla (M). Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 24.5, p. 730, with permission.

FIGURE 4.5 Immunohistochemistry (IHC) stain with anti-CD79a antibody for B cells illustrating immunostimulation by a CD40 agonist monoclonal antibody (mAb) in monkeys. (A) Spleen from a control monkey shows normal germinal/follicular centers (GC/FC) and marginal zones. (B) CD79a IHC of the spleen of a monkey dosed with CD40 agonist mAb shows excessive B cell proliferation in the enlarged germinal centers or lymphoid follicles. Bar ¼ 160 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 24.4, p. 730, with permission.

antiinflammatory drugs (NSAIDs), which inhibit cyclooxygenases and thus block synthesis of prostaglandins (D2, E2, and F2), thromboxane A2, and prostacyclin (Radi and Khan, 2019). Although decreased prostaglandins and thromboxane may have beneficial effects on pain perception and inhibit inflammation through a similar mechanism on different cell types, decreases in prostaglandins and prostacyclins may also directly reduce renal medullary blood flow leading to ischemic renal papillary necrosis (Figure 4.6).

Exaggerated pharmacology is intrinsic to the drug mode of action. On the other hand, offtarget effects can often be eliminated by chemical structure design, biologic sequence alteration, or innovative formulations to increase the ligand/ receptor binding affinity and specificity. Pathologists contribute their expertise to elucidate the mechanism for any given toxicity, which adds value to the drug development. By studying responses and evaluating the tissue changes from animals used in target modulation studies, pathologists provide critical insight into the pathophysiological mechanisms associated with drug efficacy and safety, which will facilitate the risk management associated with toxicities.

3. THE PATHOLOGIST’S ROLE IN NONCLINICAL SAFETY ASSESSMENT Once the target and molecule are selected, the nascent drug molecule will be assessed in a series of stepwise nonclinical safety studies to ensure human safety in clinical trials in compliance with regulatory requirements (Figure 4.3), mainly the ICH Safety (S) guidelines, and the

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3Rs principles of animal use (see Overview of the Role of Pathology in Product Discovery and Development, Vol 2, Chap 2). ICH M3(R2) is an overarching guideline on nonclinical safety studies to support conduct of clinical trials as well as marketing authorization for human pharmaceuticals. Potentially mutagenic drug molecules are weeded out using a series of in vitro and in vivo genotoxicity assays (ICH S2). Primary and secondary pharmacologic effects are screened in a panel of in vitro and in vivo safety pharmacology studies (ICH S7). Target organ toxicity and reversibility, dose responses and exposures, safety margins, and safety biomarkers are evaluated in general toxicology studies in animal species (ICH S3 and S4). Developmental and reproductive toxicity is assessed through evaluation of effects on one complete life cycle from conception to adults of both sexes (ICH S5). Carcinogenicity potential will be assessed on a weight-of-evidence approach (ICH S1). Special nonclinical safety assessment may be necessary on a case-by-case basis for specific concerns such as immunotoxicology (ICH S8) and phototoxicity (ICH S10), specific therapeutic

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modalities such as biologics (ICH S6) and gene therapeutics (ICH S12), and specific therapeutic indications or patient populations such as anticancer therapeutics (ICH S9) and pediatric patients (ICH S11). Toxicologic pathologists play key roles in supporting general toxicology studies and carcinogenicity studies. The majority of toxic effects are identified from anatomic and/or clinical pathology evaluation, which accounts for w80% of adverse study findings that affect the no-observable-adverse-effect level (NOAEL) and lead to drug attrition in Pfizer experience (Palazzi et al., 2022; Ramaiah et al., 2017, Figure 4.7) (see Assigning Adversity to Toxicologic Outcomes, Vol 2, Chap 15). Cardiovascular, hepatobiliary, gastrointestinal, CNS, and renal organs are among the most common target organs of toxicity with various therapeutic indications and drug modalities. General toxicology studies are conducted in animal species and include numerous study endpoints that encompass thorough clinical pathology and anatomic pathology assessments (Figure 4.7). Histopathology remains the

FIGURE 4.7 General toxicology studies are conducted in animal species, with numerous study endpoints. Pathology is one of the key study endpoints. NOAEL is defined based on adversity evaluation of all study endpoints.

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primary and routine tool to characterize test article–related toxic effects in tissues. Protocol development of well-designed animal studies requires pathologists’ input to ensure that pathology endpoints are appropriately included, particularly for nonstandard toxicity studies. In addition to routine histopathology, samples may be collected at necropsy with due regard to the potential for electron microscopy, histochemistry, immunohistochemistry (IHC), or other methods requiring frozen tissues or special fixatives. Additionally, many other structural, functional, and molecular pathology techniques are also available to investigate mechanisms of pathology findings of interest. These techniques or endpoints are often included in dedicated investigative studies but can also be part of routine GLP toxicology studies, although not necessarily in compliance with GLP regulations. Rodent carcinogenicity assessment is a critical part of the safety assessment of investigational drugs, as almost the entire burden of carcinogenic risk assessment for humans lies in results obtained from testing in rodents. The primary goals of the rodent carcinogenicity studies are to determine whether a drug candidate has a carcinogenic potential, i.e., increases the tumor

incidence and/or shortens the tumor latency period, and to provide an assessment regarding human relevance. The assumption is that the same tumor induced in rodents by the drug candidate will arise in humans and the tumor development is accelerated in rodents. However, the predictivity and relevance of rodent carcinogenicity study outcomes to human risk have been debated in past decades. Carcinogenicity testing paradigms have been evolving from standard 2-year carcinogenicity studies in rats and mice to an integrative weight-of-evidence approach to assess human risk (see Carcinogenicity Assessment, Vol 2, Chap 5). Pathology findings in nonclinical safety studies can be generally classified into three categories: test article–related findings, background findings, and artifacts (Figure 4.8). Broadly speaking, findings related to in-life study procedures or experimental manipulations and microorganism infections are considered background findings. Artifacts should be considered for postmortem tissue changes introduced artificially by the necropsy, histology and other laboratory procedures. The toxicologic pathologist is expected to accurately describe or diagnose the test article–related effects, understand their

FIGURE 4.8 A decision tree for assessing and communicating adverse (i.e., harmful) or nonadverse test article– related findings in nonclinical safety studies.

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pathogenesis and biologic significance, and advise on their human relevance and potential risks associated with the intended clinical use. Test article–related findings must be further characterized as adverse or nonadverse with appropriate justification of the determination (Kerlin et al., 2016; Palazzi et al., 2016). Uncertainty or ambiguity should be avoided. The pathologist is the subject matter expert, closest to the study dataset, and therefore in the best position to make the determination. Background changes and artifacts in tissues can mask or mimic a toxicologic tissue response (McInnes and Scudamore, 2014). Tissue artifacts can be produced along the processes of necropsy, tissue sample collection and preparation, histology slide preparation, and many other special procedures. The histology slide quality and tissue representation are vitally critical for accurate pathology diagnoses. Technical artifacts may occur from the onset of animal death (autolysis) through necropsy and histological process. Background findings can be defined as broadly as all tissue findings other than test article–related findings, such as sex- and age-related tissue changes, species-specific spontaneous changes, in-life experimental manipulations (e.g., sutures and catheters), or unique species-specific histology features. Certain background changes may be exacerbated or accelerated by drug candidates, such as chronic progressive nephropathy (CPN) in rats. CPN is a rat-specific disease process and has no counterpart in humans. Exacerbation or acceleration of CPN changes by general systemic debilitation may have no relevance to human kidneys. However, CPN exacerbation or acceleration by a primary nephrotoxic drug candidate may affect human kidney. Clinical monitoring for renal alterations may be performed with greater vigilance if the drug progresses to human trials. Relevant historical control datasets are essential for pathology assessments, especially for pathology findings of low incidences (Keenan et al., 2009). Historical control datasets can be found in literature or other various sources, but the concurrent study control animals are the most relevant controls for determining test article–related effects in a study. Recent study control datasets from the same laboratory or facility will likely be more relevant than those

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from other laboratories or literature sources. A dataset collected internally in recent 3–5 years will be appropriate for general reference use. Published datasets or other laboratory datasets are useful as a weight-of-evidence approach to determine the relevance of a pathology finding.

3.1. Adverse Effects and Pathology Report In regulated general toxicology studies, the pathology report is often handled as a standalone contributing or substudy report appended to the main toxicology study report as with other contributing or substudy reports (Figure 4.7). In toxicologic pathology reports, pathologists characterize pathology changes associated with test articles, including target organs of toxicity, dose responses, exposures and safety margins, and reversibility (Kerlin et al., 2016; Palazzi et al., 2016) (see Preparation of the Anatomic Pathology Report for a Toxicity Study, Vol 2, Chap 13). Pathologists should understand the drug mode of action, target biology and the pathophysiology of on-target and off-target effects, which may help differentiate between adverse and nonadverse test article effects, in addition to background changes and artifacts (see Assigning Adversity to Toxicologic Outcomes, Vol 2, Chap 15). The assessment of the adversity of test article– related findings is a key deliverable by the study pathologist that contributes to the risk evaluation of a drug candidate (Figure 4.8). There have been no universally accepted criteria for identifying adverse effects. In general, adverse effects have been defined as “a test item-related change in the morphology, physiology, growth, development, reproduction or life span of the animal model that likely results in an impairment of functional capacity to maintain homeostasis and/or an impairment of the capacity to respond to an additional challenge” (Palazzi et al., 2016). By this definition, potential adverse effects are determined specifically for the test animal species within the context of a given study and should not take into consideration translatability or human relevance of the findings, although the latter may be brought up in the discussion of the pathology report. Test article–related findings harmful to the test animal species under given test conditions are considered adverse, whereas those that are not harmful are considered nonadverse.

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In the pathology report or subreport, adverse effects should be stated unambiguously and justified accordingly (Figure 4.8). The test article–related findings in a given study should not be extrapolated to or from other studies, even if performed with the same test article. The NOAEL of a toxicology study will be determined in the main study report based on a collective evaluation of all adverse study findings reported in both the main study report and all contributing substudy reports, including the pathology report. However, the adverse effects and NOAEL in toxicology studies are primarily driven by the adverse pathology findings. Determination of adversity of pathology findings requires deep understanding of comparative pathophysiology, medicine, toxicokinetics, and other disciplines that are often best embodied in well-trained and experienced pathologists.

3.2. Reversibility and Delayed Toxicity Reversibility and delayed toxicity are controversial aspects of toxicology study design, with regard to duration, doses, and endpoints to be evaluated, which requires understanding of the pathophysiology of the pathology finding from experience and literature (Perry et al., 2013). Being mindful of resources, the study should be designed in a logical, consistent manner that will give regulatory agencies and clinicians the confidence to test the drug candidate in human with minimal risk of irreversible harm. There is not a “one size fits all” protocol design for reversibility phases. Some pathology findings are well known to be adaptive, and a complete recovery is expected with cessation of dosing. There is no need to prove the recovery in such a study. For pathology findings associated with significant parenchymal cell death in an organ with limited regenerative capacity, such as retina, heart, or brain, these changes are not expected to reverse completely, so there is arguably no point in sacrificing animals to confirm current knowledge and a recovery study may not be productive. For pathology findings that fall between these two extremes, there are scientific justification and regulatory expectation that reversibility be demonstrated in the study and the design of the recovery phase should be discussed with the study pathologist.

The length of the recovery period may involve many factors, including regulatory requirements, drug modality and mode of action, halflife of the drug candidate, therapeutic indication and patient population, and the nature and severity of the target organ toxicity. For example, an extensive exhaustion or depletion of B lymphocytes may take 10–12 months for a full recovery. A rule of thumb would be to ensure that the drug exposure at the recovery sacrifice is negligible or at least below the exposure at the NOAEL from previous shorter-term studies. When recovery data are required, a smaller number of animals (50% of dosing groups) are added to at least the high-dose and control groups. The reversibility of adverse effects can be informed or monitored with tissue morphology and/or, if possible, with appropriate biomarkers. In many cases, a partial reversibility can predict the likelihood of complete reversibility in time, based on sound scientific reasoning. Alternatively, in the case of some resolution, the pathologist can make the case for functional reversibility and the relevance of any remaining changes in the tissue. As an example, renal tubular necrosis may be a very serious change resulting in compromise of renal function, but by 2 weeks without exposure to the drug this may be completely reversed, especially if the infrastructure (tubular basement membranes, renal vasculature, etc.) is unaltered. If the pathologist does not see complete return to quiescence, but the presence of numerous regenerating basophilic tubules with lower cytoplasm/nuclear ratio, it may hint a strong likelihood of complete return to normality with time. Drug indication or patient population is also a factor to consider with respect to reversibility assessment. For example, partial reversibility may not be acceptable for a minor toxic effect in the lung for a drug intended for asthma in juveniles, whereas severe, even life-threatening, and irreversible toxicity with a drug that is efficacious in end-stage solid tumors may be manageable. Testing for delayed toxicity can be more challenging than for reversibility, and the critical or uncertain factor is the length of the postdosing period. For reversibility, the toxicity or lesion is known, and a study design can add a recovery phase accordingly. For delayed toxicity, both

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onset lengths and target organs of toxicity are unknown for the drug candidate, perhaps other than literature information on the drug class. Although for a pure reversibility assessment the pathologist only needs to evaluate tissues or organs with treatment effects in dosing-phase animals, determination of delayed toxicity will require microscopic evaluation of all tissues, including those not previously identified as target organs. This may become a default for test articles with long half-lives (such as antibodies) or with myriad, unpredictable, and extensive effects on many tissues (such as oncology drugs).

3.3. Lexicon and Diagnostic Terminology In order to share pathology findings in a consistent and unambiguous manner, toxicologic pathologists should use appropriate diagnostic terminology and align with best practices agreed upon by international professional societies, such as the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) and Standardized System of Nomenclature and Diagnostic Criteria (SSNDC) guides. These published guides provide standardized nomenclature or terminology to diagnose and classify tissue findings by tissue and species. The publications include definitions, synonyms, and reference illustrations. INHAND and SSNDC guides can facilitate pathologist’s communication of tissue changes consistently worldwide (see Nomenclature and Diagnostic Resources in Anatomic Toxicologic Pathology, Vol 1, Chap 25). The use of INHAND terminology along with the Standard of Exchange for Nonclinical Data (SEND) data format required by FDA for the standardized communication of nonclinical data allows for increased efficiency of data review and for more effective data mining for regulatory submissions (Briggs, 2017; Choudhary et al., 2018). The standardized terminology brings consistency in nonclinical datasets within and across drug development programs and the industry, and provides cross-referenceable datasets worldwide, including for historical control databases. Pathologists should thoughtfully choose diagnostic terminology entries. Specific diagnostic terms may eventually drive the adversity

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determination since some pathology findings are considered inherently adverse (e.g., retinal and neuronal necrosis). Any inconsistency or change in diagnostic terminology among regulated nonclinical studies for the same drug candidate program during development could inevitably raise confusions or concerns and thus requires thorough justifications. In addition to appropriate diagnostic terms for the pathology findings, additional descriptors should be provided to characterize anatomical or cellular locations, severity, distribution, and extent for each pathology finding whenever possible. Collectively, these additional descriptors are also informative for the adversity assessment. Ultimately, a written description of a pathology diagnosis should enable any professional reader to understand the nature of the pathology finding.

3.4. GLP Regulations in Pathology Much of GLP discussion in toxicologic pathology has been focused on the nature of raw data for pathology endpoints. Per GLP regulations, raw data are the first recording of an original experimental observation and are necessary for the reconstruction and evaluation of the study and study report. The interim pathology notes and diagnoses are not considered necessary for reconstructing the study report. Per GLP final rule, only the signed and dated pathology report by the study pathologist represents the pathology raw data (FDA, 2021). Accordingly, pathology specimen retention is required, so that pathology findings can be verified independently, if necessary (see Pathology and GLPs, Quality Control and Quality Assurance, Vol 1, Chap 27). Macroscopic or “gross” observations and descriptions made at the time of necropsy had been considered as gross pathology raw data. The gross observation made at necropsy cannot be revisited since in most cases only parts or samples of the organ or tissue will be saved for subsequent histopathology. The next discussion point is around the definition of “at the time of necropsy.” For example, it could be argued that this rigidly means that every word written down at necropsy becomes raw data immediately. Others consider that it may be at the end of each animal necropsy, at the end of the entire necropsy session, or even at the time of operator

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sign-off, which may be some days after euthanizing the first animal. The gross observations made at the start of the necropsy may be modified with more information about the nature of findings associated with controls as well as treated animals. Recording of gross findings should use descriptive terms as much as possible, including location, shape, size, color, consistency, odor, etc. Since the gross findings will be subsequently evaluated using histopathology in most cases, gross findings should be descriptive, not diagnostic. For example, a red spot on the liver should be called just focal red discoloration, not hemorrhage, since histopathology may show focal congestion, telangiectasia, or hemorrhage. This also avoids potential diagnostic discrepancy between the necropsy table and histopathology table. The necropsy observations and descriptions remain accurate and objectively true whatever the subsequent histopathology finding is. Due to the qualitative and subjective nature of the generation and classification of histopathology diagnoses, the histopathology data are uniquely different from most types of other data under the GLP regulations. Original histopathology observations or diagnoses may be subsequently modified and improved as data tables become more complete. Initially entered diagnoses may be changed accordingly to better represent the fact. For example, if changes that were initially “lumped” under a common background finding are in fact test article–related findings, a subsequent reexamination of that tissue can be performed to “split” out the true article–related finding from the background finding. This lumping versus splitting diagnosis strategy occurs frequently in complicated pathologies such as the previously discussed rat spontaneous chronic progressive nephropathy (CPN). CPN comprises a constellation of progressive age-related renal changes, including renal tubular degeneration and regeneration. When a drug candidate produces renal tubular degeneration and regeneration in rats, the CPN renal changes may be exacerbated. The challenge for pathologists is to determine if an effect on the renal tubules by a drug candidate is really due to an exacerbation of CPN, perhaps due to general debilitation at the toxicologically high dose, or whether the drug has a specific deleterious effect on the renal tubular epithelium.

Each of these scenarios has a different regulatory implication. The final pathology report after signed and dated by the study pathologist will become the pathology raw data (FDA, 2021). Traditionally, necropsy observations, histopathology slides (and tissue paraffin blocks), or histopathology datasets had been considered as the pathology raw data. The process of iterative refinement of pathology diagnoses is different from the way most other raw data are created. Although the first observations are original observations, it may be necessary for the pathologist to reevaluate and refine the diagnosis and report until the pathologist finalizes the database and signs off on the pathology report.

3.5. Pathology Peer Review Pathology peer review has been widely implemented in the industry as an extra step to ensure quality and accuracy of pathology diagnoses, interpretations, and reports (FDA, 2021; Morton et al., 2010). Each pathologist may develop specialized expertise in certain organ systems, animal species, drug classes, or special techniques such as IHC, genomics, or electron microscopy. The peer-review pathologist often has areas of pathology expertise or experience that are complementary to those of the study pathologist. Thus, the peer-review pathologist may provide additional insight to support diagnosis and interpretation of pathology findings. Peer reviews are usually performed by another pathologist from the same testing facility or a pathologist representing the sponsor for an outsourced study. The sponsor peer-review pathologist is usually familiar with the mechanism of action of the test article and understands expected on-target and off-target effects in the animal model, which will contribute to the refinement of the pathology interpretation. The peer-review pathologist ensures that all relevant test article–related changes are identified and appropriately characterized, and that terminology and grading system are utilized consistently across studies. The FDA has recently issued a draft guidance regarding management and conduct of pathology peer reviews performed for GLPcompliant toxicology studies (FDA, 2021). The

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pathology peer review can be conducted prospectively before finalization of the pathology report or retrospectively after the pathology report is finalized and signed off by the study pathologist. The peer-review process should be well documented and transparent and guided by SOPs and protocols to establish the extent of the review and ensure the integrity of the study data. The peer-review pathologist should review appropriate histopathology slides and pathology data to assist the study pathologist in refining pathology diagnoses and interpretations (Morton et al., 2010). The peer-review pathologist should generate a signed and dated peer-review statement for inclusion in the study file and/or final study report. If the peer-review pathologist concurs with the study pathologist’s diagnoses and interpretations, the peer-review statement is worded to indicate and reflect the consensus opinions of both pathologists. Any changes to the interpretations by the study pathologist in a retrospective peer review must be documented in an amendment to the final pathology report. Unresolved differences in interpretation, resolution of any differences, and the process of resolution should be appropriately documented, especially in retrospective peer reviews. If differences between the study and peerreview pathologists cannot be resolved, additional experienced pathologists may be consulted for consensus, including assembling a formal pathology working group (PWG). The PWG should consist of the study pathologist, the peer-review pathologist, other pathologists who may possess relevant knowledge of the target organ toxicity and pathology findings, and/or subject matter experts (Morton et al., 2010). A PWG may review the overall pathology interpretations within a study to resolve pathology diagnoses. If the PWG cannot reach a consensus agreement, discrepancies may be resolved by majority vote. Records of communications and meeting summaries should be retained in the study file. If more than one formal pathology peer review is conducted for a given study, the peer-review memos should be prepared and signed for each peer-review process. If a formal PWG is convened, the peer-review memo should document the issue or issues addressed, the specimens and data examined, the PWG members,

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and the conclusions of the PWG. The peerreview memos should be signed by all PWG members. For additional information (see Pathology Peer Review, Vol 1, Chap 26).

3.6. NOAELs and Study Report The NOAEL denotes the highest dose level that has no observable adverse effects in a given study, i.e., based on a collective assessment of all adverse effects identified in that study (Figures 4.7 and 4.8). The NOAEL should be set in the main study report. Minimally, toxicologists, pathologists, and other study contributing scientists should be actively involved in the NOAEL determination. As with the adversity designation, the NOAEL is also study-specific for the test species under the test conditions of a given study. The NOAEL should not be defined in the pathology report or other substudy reports. As for other substudy reports, the pathology results will be summarized in the main study report, and the pathology report will be appended to the main study report. The NOAEL is the prime endpoint in the nonclinical risk assessment. One NOAEL is identified in each study. However, one study may have two NOAELs, one for males and one for females, if there are sex differences in drug exposures, tissue metabolism, and/or unique target organ toxicities. Multiple nonclinical studies in different animal species and for different dosing durations will generate multiple NOAELs for a test article. The NOAELs should be discussed and communicated in regulatory overview documents such as the nonclinical overview (NCO). Selection of the NOAEL in the most sensitive species and relevant study duration requires analyses of data from all nonclinical studies of the test article, and clinical and literature information on the same or similar drug class. The NOAEL-based approach has been frequently used in the starting dose selection for first-in-human (FIH) clinical trials (FDA, 2005). The minimal-anticipated-biological-effect level (MABEL) or minimum effective dose (MED)-based approach is increasingly used for the FIH starting dose selection for biologics, especially for those with agonistic properties. The MABEL-based approach may be used also

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for therapeutics without relevant animal species to conduct toxicity studies. Data from in vitro studies (e.g., target binding affinity, activity, cytotoxicity, or cytokine release) and in vivo studies (e.g., efficacy studies in relevant animal models) are used for MABEL determination. The selections and executions of in vitro and in vivo studies depend on the therapeutic modality and the intended pharmacology. There are no universal approaches for determining MABEL, and various approaches have been used (Leach et al., 2021). The MABEL-based approach is generally considered a conservative method to maximize human subject safety. A consistent approach to determining, communicating, and utilizing information on adversity and NOAELs should be developed to facilitate risk assessment, including in the nonclinical study reports and regulatory submission documents (Figure 4.8). Mechanistic studies designed to understand the pathogenesis of a nonclinical adverse finding may influence the human risk characterization. Involving knowledgeable pathologists within risk management teams will contribute to successful development of a well-articulated risk management strategy.

4. PATHOLOGY IN NONCLINICAL SAFETY ASSESSMENT OF SMALL MOLECULES Nonclinical safety assessment of small molecules involves the conduct and reporting of a series of nonclinical studies to identify risks in major areas of concern, if any, to support clinical trials (Figure 4.3). Nonclinical safety assessment studies follow relatively standardized study design principles for small molecules, guided by relevant regulations from international and government regulatory agencies (including ICH guidelines) and conducted in compliance with GLP regulations and the 3Rs principle of animal uses. Nonclinical safety studies to support FIH clinical trials include a series of genetic toxicity, safety pharmacology, and general toxicity studies (Avila et al., 2020). Briefly, genetic toxicity studies are designed to evaluate risks for genetic toxicity (ICH S2). Potentially

genotoxic drug molecules are weeded out using a series of in vitro and in vivo genotoxicity studies. Safety pharmacology studies are conducted to evaluate primary and secondary pharmacologic or other off-target effects in vitro in a panel of various well-known pharmacology receptors, enzymes, and ion channels and in vivo in well-understood pharmacology models for cardiovascular, neurological/behavioral, and respiratory functions (ICH S7). General toxicity studies are performed to characterize target organs of toxicity and reversibility, dose responses and exposures, NOAEL and safety margins, and safety biomarkers in animal species (ICH S3 and S4). Pathologists contribute mainly to the conduct and interpretation of general toxicity studies. Some key elements in the general toxicology study design include the selection of relevant animal species, appropriate dose levels, and dosing duration (ICH M3, S4, and S6). For small molecules, animal species selection is based mostly on the similarity of pharmacology and metabolism profiles between humans and animal test species. The rodent species is usually the rat and less frequently the mouse, and the nonrodent species is usually the dog and less commonly the monkey or other species. Dose selection and dosing duration recommendations are provided in ICH M3 guideline. A recovery or reversibility phase may be added if there are known adverse findings characterized in previous studies and the recovery duration depends on the nature of adverse findings (Perry et al., 2013). For general toxicology studies, a standard list of approximately 40–50 tissues or organs will be observed grossly, collected, and evaluated histologically from animals after the in-life study phase is completed. Unless special procedures are required regarding tissue sampling, there is little need for pathologists to provide input to tissue selection and collection for a standard study. Pathologists may provide meaningful advice for special tissue collections based on the therapeutic target and drug molecule information from earlier in vitro or in vivo studies or literature. After successful conduct and reporting of FIH/IND-enabling GLP studies, primary and secondary pharmacologic effects, potential target organs of toxicity, dose–response relationship,

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drug exposure and toxicokinetics, maximum tolerated dose, NOAEL, safety margin, reversibility, and safety biomarkers are largely understood. NOAEL is a critical nonclinical reference for setting safe starting and stopping doses and escalation paradigms in clinical trials of new drug candidates. After initial proof of clinical efficacy and safety in clinical studies, additional nonclinical studies will be conducted to support the full development of the drug molecule, such as long-term general toxicity studies of 3–9 months in duration, developmental and reproductive toxicity studies (ICH S5), and rodent carcinogenicity studies (ICH S1). Carcinogenicity potential will be assessed in rodents on a weight-of-evidence approach.

5. PATHOLOGY IN NONCLINICAL SAFETY ASSESSMENT OF BIOTHERAPEUTICS The advancement of biomedical research and innovative approaches used in biotechnology and pharmaceutical sciences along with the discovery of ever-increasing numbers of bioactive large molecules have opened a new era of TABLE 4.1

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biologics-based therapies. Of the over 20,000 human genes and their encoded protein products, many have been or are being explored as potential drug candidates or therapeutic targets in various therapeutic modalities. Pathologists are increasingly challenged with novel and complex therapeutic modalities with multiple, often interdependent, components and various purposely designed and modified functionalities. Although small molecules and biotherapeutics complement each other in accessing therapeutic target repertoires, there are some fundamental differences in their mechanisms of toxicity (Table 4.1). For example, small molecules can induce carcinogenesis through genetic mechanisms, i.e., direct DNA or gene damage, whereas biotherapeutics induce carcinogenesis mainly through nongenetic mechanisms such as enhanced cell proliferation, decreased apoptosis, hormonal perturbations, and immune suppression. Small molecules can easily access most types of cells or tissues due to small molecular size, and thus may potentially induce a broad spectrum of off-target toxicities in virtually any cells/tissues at high doses (molecular charge/lipophilicity effects notwithstanding) (Figure 4.4). Small molecules can have markedly differing physicochemical

Key Differences Between Small-Molecule and Biotherapeutic Modalities

Characteristics

Small Molecules

Biotherapeutics

Size (molecular weight)

Small (1000 Da)

Source

Chemically synthesized

Produced in living system

Structure

Simple

Complex to highly complex

Well defined

Often difficult to define with current analytical methods

Purity

Homogenous

Heterogeneous

Manufacture processes

Simple, well controlled

Highly complex, processes define products

Reproducible (generics)

Difficult to reproduce (biosimilars)

Therapeutic modality

Simple

Diverse and complex to highly complex

Target intervention

Primarily intracellular, due to small sizes

Primarily extracellular or cell surface due to large sizes

Safety concern

Primarily off-target safety concern

Primarily exaggerated pharmacologic effect

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properties, varying from highly hydrophilic to extremely lipophilic, and in this category can vary from soft and waxy to hard and crystalline. Although this is also the case with biologics, these molecules are usually much larger and can have multiple components that can vary from mostly hydrophilic to mostly lipophilic. In general, though, the biological molecules often mimic the biodistribution properties of the natural ligands for the receptor target. Generally, with biotherapeutics such as monoclonal antibodies, for example, most toxic effects result from exaggerated pharmacology or immunogenicity in animals, due to the usually much higher doses used in nonclinical studies than are required for efficacy in humans. The effects related to exaggerated pharmacology can be translated clinically to humans if their basic biology and mode of action are similar in animals and humans (Dixit et al., 2010). Significantly, effects related to immunogenicity of human proteins in wildtype animals have not been predictive of the same effects in humans and genetically engineered humanized mouse models are not yet considered mature enough for this purpose (Kissner et al., 2021). Typical safety assessment challenges with biotherapeutic modalities include the selection of relevant animal species (Dixit et al., 2010). Since most toxicity is related to exaggerated pharmacology, toxicity or safety studies should generally be conducted in pharmacologically relevant animal species which express the appropriate receptors and reproduce downstream effector processes. Lack of a relevant animal species creates significant issues for nonclinical testing. A complex and often costly approach is to use alternative animal models or surrogate animal molecules (where the human drug does not bind animal receptors) when a pharmacologically relevant animal species is not available. Other challenges to safety assessment of large molecules include their unique and diverse modalities (as creative as the human mind can devise), long half-life, immunogenicity, immunomodulation (activation or inactivation), cytokine release effects, etc. Each modality should be assessed on a case-by-case basis, using scientific rationale (either tested or extrapolated from the literature) and regulatory guidance (Hey et al., 2021).

5.1. Proteins Protein- or peptide-based biologics has been the most exploited modality to date and includes a diverse array of biotherapeutic possibilities, including antibodies, cytokines, hormones, growth factors, enzymes, clotting factors, and other protein or peptide derivatives (see Protein Therapeutics, Vol 2, Chap 6). Many proteinbased therapeutics have been approved, or are emerging for various diseases such as cancer, autoimmunity, infectious, and metabolic diseases. It is interesting to remember that the first recombinant protein therapeutic to be approved by US FDA was human insulin in 1982 and it has been the major therapy for diabetes mellitus ever since. In general, protein-based biotherapeutics affect toxicity primarily through exaggerated pharmacology on tissues or organs that express the target. Other safety challenges of proteinbased biotherapeutics include the selection or availability of relevant animal species, tissue cross-reactivity, inappropriate cytokine release, immunogenicity, immune modulation, and nongenetic carcinogenesis. Tissue cross-reactivity (TCR) studies are generally required by regulatory agencies for therapeutic monoclonal antibodies and related products (Leach et al., 2010), but are usually not needed for drugs developed for oncology indications that fall under ICH S9. TCR screens for unintended antibody binding to related epitopes in human and animal cells or tissues other than the target site. TCR involves an IHC assay performed on a panel of frozen human and animal tissues, using the monoclonal antibody product as the primary antibody. Pathologists play key roles in the design, execution, and interpretation of TCR studies. Currently, challenges in conducting TCR screening include technical feasibility and application inconsistency, which contribute to the suboptimal performance of this assay for assessment of antibody specificity. Indeed, TCR labeling patterns in tissues often do not correlate with observed or expected in vivo effects of monoclonal antibody products. The relevance and value of the TCR screening should continually be assessed as experiences in animals and humans accumulate. In addition, in the last few years, non-IHC array-based platforms have emerged that allow

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for screening close to 80% of the human membrane proteome, indicating a viable alternative and/or addition to the IHC methods (MacLachlan et al., 2021). Initial experience with these protein/cell arrays has been largely positive. Major platforms currently available include HuProt Human Proteome Microarray (Cambridge Protein Arrays, Cambridge, UK), Retrogenix Cell Microarray (Retrogenix, High Peak, UK), and Membrane Proteome Array (Integral Molecular, Philadelphia, PA). Although these array screens lack tissue context with regard to antibody binding, they offer additional information regarding potential antibody crossreactivity, and they will likely become more commonly incorporated into development strategy to assess antibody specificity. Cytokine release by therapeutic agents is a significant safety concern. Certain protein products may bind to cell surface receptors or other molecules and stimulate cytokine release. Excessive cytokine release could induce a lifethreatening cytokine storm as observed in a Phase I clinical trial of TGN1412 in 2006, the TeGenero Incident (Suntharalingam et al., 2006). TGN1412 is a monoclonal antibody that activates CD28 receptors on human T cells. Some of the volunteers that received TGN1412 suffered massive cytokine release in this disastrous clinical trial, resulting in life-threatening systemic effects. There are now commonly used in vitro models available for hazard identification of this risk (Grimaldi et al., 2016), which use unstimulated human cells in plate-bound and/or soluble formats with appropriate positive and negative controls. In addition, pathologists should be aware of the potential for cytokine release in safety assessment of protein products, including signpost tissue changes, alterations in key clinical pathology endpoints, and other biomarkers. Immunogenicity refers to the host immune system response to drug protein as foreign antigen, with production of antibodies against the drug, i.e., antidrug antibodies (ADAs). Many biotherapeutics are antigenic to a variable degree, particularly in nonhumans, and may carry risks of immunogenicity in animals and humans. The ADAs may not only decrease drug exposure but also potentially induce tissue injury secondary to immune-complex deposition (Rojko et al., 2014). The ADAs can bind,

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neutralize, and eliminate the drug, thus reducing drug exposure and pharmacological activities over time, which can make long-term toxicity and efficacy studies difficult in animal models. This effect may become worse with increasing “evolutionary distance” between animals and humans; for example, ADA for human antibodies is usually greater in rodents than in monkeys. Drug–ADA complexes can deposit in various tissues, commonly in the renal glomeruli or in a variety of other vascular beds, and induce immune-complex-associated tissue injury (Figure 4.9). IHC characterization of immune-complex-associated tissue injury should demonstrate granular deposits with colocalization of drug, ADA, and complement components. IHC is usually performed using immunoperoxidase stains on paraffin sections because of improved morphology compared with cryosections. ADA responses in animals may aid in interpretation of toxicity study results, including the dose–response relationship. In general, the immunogenicity and consequent effects in animals (such as glomerulonephritis) are not very predictive of immune-response-related effects in humans. Immune modulation may occur with protein products. As nonnative proteins, they may enhance immune responses, induce hypersensitivity, or suppress immune responses, depending upon the protein structure or modality (Figure 4.5). Like immunogenicity, immune modulation in animals is not very predictive of human effects.

5.2. Oligonucleotides Oligonucleotides are short DNA- or RNAbased synthetic polymers that range from 18 to 30 nucleotides in length. The major classes of oligonucleotides developed for therapeutics include antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs, aptamers, and decoys. These agents can inhibit gene expression or impede protein function by binding to a specific sequence of a target gene or protein. In general, native oligonucleotides without chemical modifications have poor druglike properties. They are rapidly degraded by exonucleases and endonucleases and have poor plasma protein binding affinity, leading to rapid elimination after parenteral

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FIGURE 4.9 Electron micrograph illustrating immunecomplex-mediated glomerulonephropathy in a monkey treated with a protein therapeutic. (A) Glomerulus from a control monkey kidney shows normal thin and homogenous basement membrane between the endothelial cell (arrows) and podocyte processes (P). (B) Glomerulus from a kidney of a monkey dosed with a protein therapeutic shows irregular or nodular (N) thickening of the basement membrane between the endothelial cell (arrows) and podocyte processes (P), with fibrinous or granular material deposited (G) between the basement membrane and podocyte processes. Note the erythrocytes (RBC) in the capillary lumen and the urinary spaces (USs). Images reproduced courtesy of Pfizer Electron Microscopy Lab. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 24.6, p. 736, with permission.

administration. In addition, unmodified oligonucleotides are highly negatively charged polyanionic macromolecules and have poor membrane permeability, which significantly

impairs their intracellular distribution. To achieve clinical utility, various chemical modifications have been required to confer druglike properties onto DNA- or RNA-based therapeutics. Major synthetic optimizations, which have led to improved stability, cellular delivery, biodistribution, and pharmacokinetics, have included changes to the phosphate backbone (incorporation of phosphorothioate [PS] moiety) and sugar moieties (introduction of 20 -Omethoxyethyl [MOE]). Major effects of oligonucleotides are usually independent of the nucleotide sequences and due to nonspecific protein binding (in plasma or tissue) or tissue accumulation (Frazier, 2015; Tessier et al., 2021). For example, plasma protein binding may lead to coagulation inhibition or complement activation while cellular protein binding may have pro-inflammatory effects. Inhibition of the coagulation cascade (intrinsic pathway) has been observed in both rodents and nonrodents and the transient prolongation of APTT is considered related to nonspecific plasma protein binding and interaction with the intrinsic tenase complex (Sheehan and Thao, 2001). This effect is mostly observed with the oligonucleotides modified to enhance plasma protein binding such as those with PS backbone linkages. Diligent evaluation of coagulation times in relation with the pharmacokinetic drug profile is therefore expected in nonclinical toxicology studies. Another common class effect of oligonucleotides is the activation of the alternative pathway of the complement system through reversible binding to complement Factor H (Henry et al., 2016). This effect is primarily observed in nonhuman primates and may lead to secondary hemodynamic alterations and/or inflammatory changes. Oligonucleotides can be distributed in many tissues after systemic or local administration and accumulate commonly in the liver, kidney, and mononuclear phagocytes of various tissues such as lymphoid organs, lung, and bone marrow. Oligonucleotides usually appear histologically as basophilic granules in the cytoplasm or nuclei of renal epithelial cells and hepatocytes, or in the cytoplasm of macrophages, and excessive amounts may be associated with cellular hypertrophy or degenerative changes (Figure 4.10).

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FIGURE 4.10 Antisense oligonucleotide nephropathy in a rat illustrating accumulation of basophilic granular material in the cytoplasm (arrows) and eosinophilic homogenous material in the nuclei (arrowheads) of proximal tubular epithelial cells, with interstitial infiltrates of predominantly mononuclear cells. Note the glomerulus (G) is not affected. H&E stain; Bar ¼ 35 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 24.7, p. 736, with permission.

The knowledge on the class effects of these drugs continues to grow and will provide good foundational information to guide the nonclinical and clinical development of novel oligonucleotide classes. For additional information (see Nucleic Acid Pharmaceutical Agents, Vol 2, Chap 7).

5.3. Gene Therapy Gene therapy holds remarkable potential for treating a wide range of diseases through the therapeutic delivery of genetic material into a patient’s cells in order to restore diseasecausing gene function. Although advanced biotechnology tools can target or alter specific genes very precisely, all techniques in gene therapy carry potential risks that need to be thoroughly characterized to avoid possible disastrous consequences. As an example, zinc-finger nucleases are highly specific nucleases that can delete or swap genes by recognizing and cutting specific DNA sequences in the genome. When early researchers used zinc-finger nucleases to remove the host gene that allows the HIV to enter

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immune cells, the viral vector inserted itself near cancer-causing genes. This off-target insertion resulted in leukemia in humans in early clinical trials. Robust nonclinical safety assessment is vital for gene therapies and often involves early interaction with regulatory agencies and innovative approaches (Assaf and Whiteley, 2018). Gene delivery is usually achieved using viral vectors due to their high delivery efficiency, although there is an interest in safer nonviral vectors such as those based on polymers or cationic lipids. The most commonly used viral vectors are the adeno-associated viruses (AAVs), which combine low immunogenicity and pathogenicity, as they are replication deficient and do not integrate into host genome but remain episomal. Retroviral viruses, on the other hand, integrate permanently into the host genome with long-term transgene expression. Nonclinical safety assessment needs to be conducted in relevant species with characterization of transduction efficiency of viral vector and pharmacological activity of transgene in selected species. If appropriate, studies may also be conducted in diseased animal models that reflect the targeted patient population and enable the characterization of the safety profile in the disease setting. Of note is that nonrodents included in toxicology studies should be screened for preexisting neutralizing antibodies to viral vector capsids as natural virus exposure may occur in some species and confound in vivo gene therapy studies. Specific endpoints will be included in the toxicology studies to address safety concerns related to viral vector insertion, transgene expression, and immunogenicity (Salmon et al., 2014; Assaf and Whiteley, 2018). The assessment will include an evaluation of biodistribution of viral vector and characterization of transgene expression by polymerase chain reaction (PCR), in situ hybridization (ISH), and/or IHC in a defined set of tissues including blood, major organs, and gonads. Unique concerns of vector DNA are related to potential DNA integration into host genome and genotoxic effects (insertional mutagenesis), germline transmission, and environmental shedding into biofluids. Although AAVs usually remain in an episomal form in the nucleus of infected cells, recent reports have shown AAV integration into host genome

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that revived oncogenic safety concerns of the modality. Specifically, in six dogs that were treated with an AAV-based gene therapy for hemophilia up to 10 years earlier, evaluation of liver samples showed integration of therapeutic DNA into host genome, sometimes near genes known to play a role in cell growth (Nguyen et al., 2021). Such data reinforce the need to monitor for host genome integration of therapeutic DNA, even for AAV vectors. Additional safety concerns relate to the off-target or unregulated expression of the transgene and immunogenicity of viral capsid and transgene product. For additional information (see Gene Therapy and Gene Editing, Vol 2, Chap 8).

5.4. Cell Therapy Cell therapy involves the administration of modified human living cells into a patient to drive therapeutic effects. A variety of cell types can be used in cell therapy, including pluripotent cells, multipotent cells, and differentiated or primary cells. Pluripotent and multipotent stem cells are discussed in the following section. Examples of cell therapy that uses primary cells include blood transfusion (transfusion of red blood cells, white blood cells, and platelets) and immune cell therapy, such as chimeric antigen receptor (CAR) T cell therapy and other types of adoptive cell therapies. CAR T cell technologies represent a most promising and innovative advancement in personalized cancer treatment and there are over 100 companies developing CAR T cell therapies, which are generated by genetically engineering donor T cells to express a novel chimeric receptor specific for a tumor antigen. The two main sources of T cells that can be engineered into CAR T cells include those derived from a patient (autologous) and those derived from a healthy donor (allogeneic). There are currently three approved CAR T cell agents, all of which target CD19 for the treatment of advanced B cell lymphomas. Major toxicities associated with these agents and preventing their widespread use include cytokine release syndrome and neurologic toxicity (Brudno and Kochenderfer, 2019). There is no standard approach for the nonclinical safety assessment of CAR T cell therapies and strategies are tailored to the specific programs. The assessment must include (Sharpe, 2018):

characterization of target expression and cellular localization in normal human tissues by IHC, ISH, and/or PCR to determine potential offtumor on-target liabilities; in vitro immunosafety assays; toxicity evaluation in in vitro assays (e.g., cytotoxicity in human primary cells and cell lines) and/or in vivo models (e.g., monkey given autologous CAR T cells; xenograft immunodeficient mouse given human CAR T cells); in vivo biodistribution to evaluate expansion and persistence of T cells.

5.5. Stem Cell Therapy Stem cell–based therapies with targeted delivery of pluripotent cells could potentially treat a wide range of disease conditions and are especially interesting to the pharmaceutical industry in the area of tissue regeneration and repair. Currently, research stem cells are derived from three major sources: embryonic stem cells, adult stem cells, and inducible stem cells. They all are pluripotent cells that can self-renew and differentiate into various endodermal, ectodermal, and mesodermal cells or tissues. Once introduced or transplanted in vivo, stem cells will interact intimately and dynamically with host cells or tissues and may continue their self-renewal, expansion, and differentiation or dedifferentiation. Because of their pluripotent nature, stem cell differentiation, integration, migration, and proliferative potentials provoke great safety concerns (Herberts et al., 2011; Sharpe et al., 2012). Undifferentiated stem cells may have the risk of unregulated growth and have the capacity to differentiate or dedifferentiate into teratomas or other neoplasms. A comprehensive safety assessment of stem cell therapies is therefore vitally important for therapeutic use (Sharpe et al., 2012), including assessing cell sources, production, manipulation, differentiation, and characterization. Stem cell differentiation, stability, and maturity should be screened in vitro and in vivo, and the efficacy and toxicity of stem cells should be assessed in appropriate animal models where cellular viability is maximized and graft rejection is absent, or at least similar to that to be encountered in the human recipients. Common safety issues for stem cell therapeutics include abnormal, unexpected cell

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integration, cell migration and engraftment, ectopic growth and tumorigenicity, immune modulation, allogenicity, and graft rejection. In addition to general safety assessments, in vitro differentiation and in vivo biodistribution of stem cells can be screened by comprehensive histopathology and molecular biotechnology methods. An in vivo teratoma assay can be conducted in immunologically incompetent rodent models (Wesselschmidt, 2011). Histopathology assessment of standard tissues, injection sites, and target organs can be useful for screening anatomic and functional integration, distribution, and differentiation of transplanted stem cells. Biodistribution, biological activity, and toxicity of stem cells can be further screened using IHC, ISH, bioimaging, and other molecular or biotechnology tools with specific probes. For additional information (see Stem Cell and Other Cell Therapies, Vol 2, Chap 10).

5.6. Vaccines Vaccines are therapies designed to induce specific host immune responses. Host lymphocytes and antibodies are the actual effectors. Traditionally, most vaccines are prophylactic vaccines for preventing infections. Limited nonclinical safety assessment has been conducted for prophylactic vaccines, depending on the indication and patient population (Ahmed et al., 2011; Sellers et al., 2020). For most prophylactic vaccines, a well-designed and executed repeat-dose toxicity study will be adequate. For live virus vaccines and vector platform vaccines, additional studies may be necessary to assess the risk of neurovirulence, DNA integration, and biodistribution in animals. Modern vaccine modalities and delivery systems are becoming increasingly complex. The vaccines vary from simple antigens or peptides/proteins, such as inactivated toxins, to complex constructs or conjugates, such as DNA plasmids and engineered messenger RNAs. New vaccine antigens may be poor immunogens and may require novel adjuvants to enhance the host immune response. Increasingly, therapeutic vaccines are being developed against noninfectious diseases, e.g., cancers and central nervous system diseases, and for smoking cessation. There has been no specific regulatory guidance for the development

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of therapeutic vaccines, but safety hurdles are likely to be high. Therapeutic vaccines may require specific animal models to assess their efficacy and safety (Matsumoto et al., 2014). Adjuvants may present safety concerns independently, or in conjunction with other components of the vaccine. New adjuvants may be required to undergo standard safety assessment, including the potential for carcinogenicity. The multiple components of a vaccine may be tested together, with multiple controls making some of these studies large and costly. Vaccines are most often administered parenterally, e.g., via intramuscular, subcutaneous, or intravenous routes. Local tissue reactions or tolerances have been a safety concern for vaccine use. For example, postvaccination inflammation may persist for many months in vaccination or injection sites. Postvaccination sarcomas, composed of neoplastic mesenchymal tissue and vaccine residues, have been observed in vaccination sites in animals (Figure 4.11) (Vascellari et al., 2003). The carcinogenic mechanisms of the sarcomas are unknown but are suspected to arise within a background of persistent inflammation in vaccination or injection sites.

FIGURE 4.11 Postvaccination sarcoma at the local vaccine application site in a cat. Postvaccination sarcomas, composed of spindle cells and vaccine residues, have been observed in vaccination sites in animals, commonly in cats. Note the mitotic figures (arrows). H&E stain; Bar ¼ 35 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 24.8, p. 738, with permission.

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Finally, vaccines, especially those with potent adjuvants, may have the potential to generally stimulate the host immune system and affect host immune homeostasis, e.g., anaphylaxis, auto-immune response, complement activation, cytokine release, etc. Careful assessment of animals in toxicity studies for changes that may indicate some of these more unusual responses requires experienced pathologists and integration of the results of a variety of immunotoxicity assays. For additional information (see Vaccines, Vol 2, Chap 9).

6. PATHOLOGY IN NONCLINICAL SAFETY ASSESSMENT OF NOVEL FORMULATIONS 6.1. Excipients Excipients are substances other than active pharmaceutical ingredients (APIs) in finished pharmaceutical dosage forms. Almost all drug dosage forms include some kind of excipient to guarantee the dosage, stability, and bioavailability. Currently, approximately 1000 excipients of more than 40 functional categories are used in marketed pharmaceutical products. Excipients range from inert and simple to active and complex substances that can be difficult to characterize. Traditionally, excipients were often structurally simple, biologically inert, and of natural origin, such as corn, wheat, sugar, and minerals. Many more novel and increasingly complex excipients have been developed as novel drug formulation and delivery systems emerge and evolve. Many excipients are potential toxicants at high doses in animals, though safe in humans at therapeutic doses, including commonly used excipients such as cyclodextrins, dextrans, and polyethylene glycol (Enright et al., 2010; Thackaberry et al., 2010; Gad et al., 2016). Cyclodextrins (cycloamyloses) are macrocyclic oligosaccharides composed of a (1,4)-linked glucopyranose subunits, with cagelike lipophilic inner cavities and hydrophilic outer surfaces. Cyclodextrins can solubilize and stabilize drugs by forming noncovalent inclusion complexes with various inorganic, organic, organometallic, cationic, anionic, or neutral molecules. Reversible toxicities in animals include increased liver

enzyme activities, decreased erythrocyte parameters, epithelial vacuolation of renal proximal tubules and urinary bladder, accumulation of foamy macrophages in the lung, and vacuolation of Kupffer cells in the liver (Figure 4.12). Increased tumor incidences have been noted in the pancreas and intestines of the rat (Stella and He, 2008). However, cyclodextrins are generally well tolerated in humans at therapeutic doses, although minor diarrhea has been observed clinically. Dextrans are complex straight (a-1,6 linkages) or branched (a-1,3 linkages) polysaccharide polymers made of glucose molecules. Dextrans of low molecular weights have been used clinically as antithrombotic (antiplatelet) blood thinners and blood volume expanders. Dextran has been relatively safe to use clinically, although anaphylaxis, pulmonary and cerebral edema, platelet dysfunction, or acute renal failure have been noted. Dextrans may be absorbed or phagocytized by renal tubular epithelial cells when passing through the kidney. Renal tubular epithelial cell vacuolation has been noted in animals at high doses due to indigestible dextrans accumulating in the lysosomes or cytoplasm, which appear as vacuoles after tissues are processed and stained (Figure 4.13).

FIGURE 4.12 Kidney from a rat treated with cyclodextrins as an excipient. Note the prominent intracytoplasmic vacuolation (V) within the proximal renal tubular epithelial cells. The distal renal tubular epithelial cells (D) were not affected. H&E stain; Bar ¼ 40 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 24.9, p. 739, with permission.

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Polyethylene glycol (PEG) is an oligomer or polymer of ethylene oxide, with the synonym polyethylene oxide or polyoxyethylene based on the molecular weight or mass. PEG may contain potentially toxic impurities such as ethylene oxide and 1,4-dioxane. PEG has been widely used as a laxative in medicine and an excipient in pharmaceutical products. PEG, when conjugated with peptides or proteins, induces cytoplasmic vacuolation in animals in multiple tissues in both epithelial cells and macrophages that is not associated with demonstrable tissue damage or dysfunction and is reversible (Figure 4.14) (Irizarry Rovira et al., 2018). The safety profiles of new excipients need to be evaluated systematically for potential risk in humans. Depending on data available, excipients may have to be assessed experimentally for genotoxicity, general toxicity, carcinogenicity, or other specialized toxicity endpoints. Excipients should also be evaluated for their source, quantity, purity, degradation profiles, and potential interactions with APIs and other components in the dosage form.

FIGURE 4.13 Liver from a rat dosed with a soluble dextran polymer shows acute hepatocyte necrosis (N) predominantly around the central vein (CV), with mononuclear cell infiltration in the periportal regions (PPs). H&E stain; Bar ¼ 65 mm. Image reproduced courtesy of Pfizer. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 24.11, p. 740, with permission.

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FIGURE 4.14 Kidney from a rat dosed with a PEGconjugated monoclonal antibody therapeutic with prominent cytoplasmic vacuolation (arrows) of the proximal renal tubular epithelial cells. Note unaffected glomerulus (G) (H&E stain; Bar ¼ 40 mm). Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, 2013, Fig. 24.10, p. 739, with permission.

6.2. Conjugation Conjugation, in this context, is the process of covalently linking drugs or prodrugs to various natural or synthetic molecule carriers for specific applications, e.g., polymers, polypeptides or proteins, lipids, and carbohydrates. Conjugation has become an important tool for controlled drug release and targeted drug delivery of both small molecules and biotherapeutic modalities, such as polymer–drug conjugation, protein–drug conjugation, antibody–drug conjugation, and T cell dual affinity retargeting conjugation. Conjugation of drugs to carriers can improve drug stability and pharmacokinetic/pharmacodynamic properties and alter toxicity profiles. In polymer conjugations, the drug is usually linked to a water-soluble, biocompatible, polymeric molecule. The polymer can, for example, enhance drug solubility, improve drug pharmacokinetic profiles, protect the drug against enzymatic degradation, prevent efflux by P-glycoprotein, or trigger drug release in target sites, e.g., due to change in pH or presence of local enzymes. In sophisticatedly designed polymer conjugates, a target-seeking group or function (such as antibody light chains or ligands) may be added for targeted drug delivery. Some

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complex polymer conjugates or carriers, such as PEG and dextran polymers, have been shown to be toxic to the liver, kidney, or other target organs in animals at high doses (Figures 4.13 and 4.14). Antibody–drug conjugation (ADC) has become a platform of targeted drug delivery. In the ADC, the drug or payload (usually a cellular toxin) is typically attached to a monoclonal antibody carrier by a chemical linker. The monoclonal antibody provides selective targeting to, for example, tumor cells. Classically, the ADC is designed to be stable in the bloodstream but to release drug at targeted sites by enzymatic cleavage or pH changes. The drug is capable of reaching specific targets in conjugated form and there is reduced drug exposure at nontarget sites. This high selectivity ideally reduces the systemic toxic effects of traditional chemotherapy. Although driven mostly by the effect of the cytotoxic drug, the toxicity of the ADC may differ qualitatively from that of the free cytotoxin, since the antibody is going to drive the cellular distribution of the cytotoxin (Mahalingaiah et al., 2019; Pretto and FitzGerald, 2021). The elegance of this concept, along with the wave of recent approvals, makes this an attractive approach being investigated by most pharmaceutical companies. In T cell dual affinity retargeting (TCDART), two different single-chain antibodies are covalently linked together. One chain recognizes the antigen on T lymphocytes and the other recognizes surface antigen on target cells. TCDART conjugates are designed to encourage cytolytic synapses to form between target cells and T cells, which in turn induce target cell membrane perforation and lysis, or apoptosis of the target cells induced by T cell cytokines or enzymes. In addition to the inherent toxicity of the drugs themselves, pathologists must be aware that conjugating carriers, linkers, and other functional moieties can be toxic in their own right. Conjugation of PEG to oligonucleotides causes hemodilution. Conjugation of PEG to antibody or peptide induces cytoplasmic vacuolation in epithelial cells and/or macrophages in multiple tissues (Figure 4.14). For protein or antibody carriers, nonspecific tissue cross-reactivity and cytokine release could be potential safety concerns. For new carriers or linkers, a systemic safety assessment may be warranted, including rodent carcinogenicity assays.

6.3. Nanotechnology Nanotechnology is the study and manipulation of matter at atomic and molecular levels, with particle sizes of 1–100 nm in at least one dimension. Small particles in these nanometer ranges display size-specific biological activities and behave differently from their larger counterparts (see Nanoparticulates, Vol 3, Chap 13). For example, inhaled carbon particles at nanoscale sizes can translocate to the brain through the olfactory nerve while larger particles of the same material cannot. Hypothetically, nanoparticles are small enough to enter the body, circumvent the body barriers, and interact with cellular machinery in a variety of different ways that larger molecules cannot do (Hoshyar et al., 2016). The implications of nanotechnology are being debated, especially regarding the toxicity and environmental impact of nanomaterials. Nanomaterials are widely present in nature in various shapes and forms, such as micelles, liposomes, dendrimers, nanoshells, quantum dots, or fullerenes. Variable toxicity has been shown with industrial and environmental nanomaterials which are relatively insoluble and biopersistent. Nanomaterials have been shown to accumulate or persist in macrophages or parenchymal cells of multiple organs, such as kidney, liver, brain, lung, and aorta (Kumar et al., 2012; Gustafson et al., 2015). In the pharmaceutical industry, nanotechnology has been explored for the potential to formulate drugs in order to solubilize relatively insoluble molecules, improve target access, and modulate the pharmacokinetic profile (Cmax, AUC, and sustained release) of the active pharmaceutical ingredient. Nanoparticles may be formulated simply by nanomilling to reduce drug particle sizes or, in many creative ways, to target the drug delivery. A variety of relatively nontoxic, water-soluble, and nonbiopersistent materials have been evaluated, with or without drug payloads. A variety of target organ toxicities have been noted in the kidney, liver, lung, etc. (Figures 4.15 and 4.16). Nanotechnology, and certainly nanotoxicology, is a science in its infancy. We are still far from understanding of how particle parameters govern mechanisms of nanoparticle interactions within the nonclinical biological systems that we use, and their relationship to human risk.

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FIGURE 4.16 Photomicrograph of lung from a rat injected intravenously with nanoformulated materials (without drug) shows pulmonary microgranulomas (arrows) in the alveolar septa. The birefringent material (arrowheads) within the microgranulomas might be the nanoformulated materials. H&E stain; Bar ¼ 75 mm. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, Fig. 24.15, p. 743, with permission.

FIGURE 4.15 (A) Photomicrograph of liver from a rat dosed with a nanoformulated vehicle (without drug) showing Kupffer cell hypertrophy and hyperplasia (arrows) in the lining of the sinusoidal spaces. Note the pale and foamy cytoplasm of the affected Kupffer cells around the central vein (CV) and periportal regions (PPs). H&E stain; Bar ¼ 35 mm. (B) Electron micrograph of liver from a rat dosed with a nanoformulated vehicle (without drug) showing Kupffer cell hypertrophy and foamy or vacuolated cytoplasm (V) in the sinusoidal spaces. Note the hepatocytes (HCs) and Ito cell (IC) with cytoplasmic fat droplets (F) adjacent to the Kupffer cell (KC) and erythrocytes (RBC) in the sinusoidal space. Image courtesy of Pfizer Electron Microscopy Lab. Figure reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Handbook of toxicologic pathology, ed 3, Academic Press, Fig. 24.14, p. 742, with permission.

Particle size and surface physicochemical properties have been recognized as key parameters in determining potential biological and

toxicological impacts of nanoscaled material. Pathologists should also consider other particle attributes in safety assessment, such as shape, surface area, crystal structure, potential for agglomeration or aggregation, and solubility at different pH values. Combined effects of the biochemical composition and the physical form of drugs at nanoscale create safety challenges that are probably much more complex than simply an effect of the size range.

7. DIGITAL PATHOLOGY AND COMPUTATIONAL PATHOLOGY The field of digital pathology has made tremendous progress over the last decade and is likely to transform, in the near future, the practice of pathology in the clinical diagnostic and experimental settings, including in toxicologic pathology (Turner et al., 2020). Digital pathology has benefited from the rapid progress of whole-slide imaging (WSI) technology coupled with advances in computational technology and storage. WSI refers to the ability to digitize an entire pathology glass slide and current whole-slide scanners are capable of producing very high-resolution

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images, which accurately replicate glass slides, with a relatively high throughput. The first WSI system to receive clearance by the FDA was the PIPS (Philips IntelliSite Pathology Solution), which was approved in 2017 for review and interpretation of clinical surgical pathology slides. WSI in toxicological pathology is improving diagnostic efficiency by enabling remote primary pathology evaluation, peerreview evaluation, and consultation across distant sites, without any requirement for slide shipment. However, detailed guidelines regarding the nature and extent of validation required for the use of WSI on GLP toxicology studies are still lacking, which has limited the deployment and adoption of digital pathology in toxicological pathology. In addition to enabling viewing of microscope slides on a computer screen, digitized image data can be leveraged to create diagnostic algorithms and build artificial intelligence (AI) solutions. Advances in the development of novel powerful computational tools for image analysis are indeed giving enhanced opportunities for tissue interrogation and extraction of information, leading to a wide range of applications in toxicological pathology, such as computer-assisted abnormality detection, quantitative morphological assessments, and content-based image search and retrieval (Mehrvar et al., 2021). An interesting and currently futuristic application is to develop algorithms to interrogate “digital slides” in order to separate out normal tissues from “abnormal” tissues. This could vastly decrease the workload per pathologist, who would only have to view slides which the computer allocated as abnormal. Although computational pathology is still in its infancy in the field of toxicological pathology, major recent progresses are poised to converge to ultimately improve pathologist diagnostic efficiency and accuracy. For additional information (see Digital Pathology and Tissue Image Analysis, Vol 1, Chap 12).

8. NOVEL INVESTIGATIVE TOOLS IN NONCLINICAL SAFETY ASSESSMENT Since the first compound microscope was invented in 1595, it has taken over 400 years to

achieve the state-of-the-art microscopes that pathologists use today. For more than four centuries, pathologists have been using microscopes as major tools to study and diagnose diseases. Largely through structural changes observed in cells, tissues, and organs that underlie the cause, pathogenesis, and functional consequence of diseases, pathologists provide the foundation for clinical care and therapy and, in the pharmaceutical industry, for safety assessment and risk management of drug development. Traditionally, pathology assessment of drug safety has been mainly by histopathology examination of, and measurements obtained from, cells and tissues, supplemented with electron microscopy, histochemistry, IHC, ISH, and/or clinical pathologic endpoints (see Special Techniques in Toxicologic Pathology, Vol 1, Chap 11, and Clinical Pathology in Nonclinical Toxicity Testing, Vol 1, Chap 10). Although there is no other good direct substitute for histopathology assessment so far, safety assessment of novel modalities and formulations may utilize novel scientific tools, such as genomics, proteomics, metabonomics, immunolomics, cellomics, and bioimaging, to supplement morphology observations. Modern “omics” techniques have provided valuable insights into the dynamic and multiparametric host responses to drug effects (Alexander Dann et al., 2018; Sutherland et al., 2018) (see Toxicogenomics: A Practical Primer, Vol 1, Chap 15). Using DNA microarray technology, gene expression and function can be profiled on a whole- or partial-genome scale. Metabonomics has provided quantitative metabolic profiles in tissues and body fluids, such as urine, saliva, and plasma. Proteomics analyzes cellular, gene, and protein functions by examination of peptide or protein expression. Immunolomics profiles lymphocyte, antibody, and cytokine responses in a comprehensive manner. Cellomics can provide valuable knowledge of cellular phenotype and functions using various cell analysis techniques such as flow cytometry and high-content throughput. Ultimately, these “omics” techniques can provide valuable insights into the mechanisms and biomarkers of drug efficacy and toxicity. Bioimaging has provided a useful tool to assess drug exposure, distribution, target binding, and pharmacologic effects. Bioimaging

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REFERENCES

can track the migration, occupancy, binding, and biodistribution of drugs in vivo in real time. Imaging biomarkers of mechanism, target, efficacy, and safety often have a good nonclinical to clinical translatability. Continued advancements in bioimaging techniques will allow many in vivo drug-related biological and biochemical events to be visualized or monitored in real time with reasonable resolution. With increasingly complex and novel drug modalities and formulations, pathologists need new tools to effectively understand and manage potential risks. The entire spectrum of host responses to drugs may be characterized from initial gene expression to clinical toxicity, including evaluation of unique biological signals (biomarkers), mechanistic pathways, and intermediate and final metabolites, all in relation to drug exposure and dose responses. With novel drug modalities and formulations, target organ toxicity and dose–response relationships may show radical departure from the conventional norm. In some cases, such as unique animal disease models and nanoformulations, existing tools might not be capable of assessing potential and plausible risks. New tools need to be developed to address the unique and unanticipated safety challenges.

9. CONCLUSION This chapter has provided an introduction to the complex science of toxicologic pathology and highlighted the critical contributions of toxicologic pathologists to drug development, through the characterization and investigation of drug-induced toxicities in nonclinical species that drive the assessment of clinical translation. Challenges are multifold and include in particular: demanding regulatory requirements, everincreasing complexities of novel therapeutic modalities, and proliferation of molecular and computational methods providing deeper insights into toxicity mechanisms. The success and impact of toxicologic pathologists will reside in their ability to integrate in matrix organization of multidisciplinary teams and keep up with the scientific advancements of the field.

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a report of the 9th BioSafe European annual general membership meeting, mAbs 13:e1938796, 2021. Kumar V, Kumari A, Guleria P, Yadav SK: Evaluating the toxicity of selected types of nanochemicals, Rev Environ Contam Toxicol 215:39–121, 2012. Leach MW, Clarke DO, Dudal S, et al.: Strategies and recommendations for using a data-driven and risk-based approach in the selection of first-in-human starting dose: an international consortium for innovation and quality in pharmaceutical development (IQ) assessment, Clin Pharmacol Ther 109:1395–1415, 2021. Leach MW, Halpern WG, Johnson CW, Rojko JL, MacLachlan TK, C.han CM, Galbreath EJ, Ndifor AM, Blanset DL, Polack E, Cavagnaro JA: Use of tissue crossreactivity studies in the development of antibody-based biopharmaceuticals: history, experience, methodology, and future directions, Toxicol Pathol 38(7):1138–1166, 2010. https:// doi.org/10.1177/0192623310382559. Epub 2010 Oct 6. MacLachlan TK, Price S, Cavagnaro J, Andrews L, Blanset D, Cosenza ME, Dempster M, Galbreath E, Giusti AM, HeinzTaheny KM, Fleurance R, Sutter E, Leach MW: Classic and evolving approaches to evaluating cross reactivity of mAb and mAb-like moleculesda survey of industry 2008–2019, Regul Toxicol Pharmacol 121:104872, 2021. Mahalingaiah PK, Ciurlionis R, Durbin KR, Yeager RL, Philip BK, Bawa B, Mantena SR, Enright BP, Liguori MJ, Van Vleet TR: Potential mechanisms of target-independent uptake and toxicity of antibody-drug conjugates, Pharmacol Ther 200:110–125, 2019. Matsumoto M, Komatsu S, Tsuchimoto M, Matsui H, Watanabe K, Nakamura K, Amakasu K, Ito K, Fueki O, Sawada J-I, Maki K, Onodera H: Considerations for nonclinical safety studies of therapeutic peptide vaccines, Regul Toxicol Pharmacol 70:254–260, 2014. McInnes EF, Scudamore CL: Review of approaches to the recording of background lesions in toxicologic pathology studies in rats, Toxicol Lett 17:134–143, 2014. Mehrvar S, Himmel LE, Babburi P, Goldberg AL, Guffroy M, Janardhan K, Bawa B: Deep learning approaches and applications in toxicologic histopathologydcurrent status and future perspectives, J Pathol Inf 12:42, 2021. https:// doi.org/10.4103/jpi.jpi_36_21. Morton D, Sellers RS, Barale-Thomas E, Bolon B, George C, Hardisty JF, Irizarry A, McKay JS, Odin M, Teranishi M: Recommendations for pathology peer review, Toxicol Pathol 38:1118–1127, 2010. Ngo DH, Vo TS: An updated review on pharmaceutical properties of gamma-aminobutyric acid, Molecules 24:2678, 2019. Nguyen GN, Everett JK, Kafle S, Roche AO, Raymond HE, Leiby J, Wood C, Assenmacher C-A, Merricks EP, Long CT, Kazazian HH, Nichols TC, Bushman FD, Sabatino DE:

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REFERENCES A long-term study of AAV gene therapy in dogs with hemophilia A identifies clonal expansions of transduced liver cells, Nat Biotechnol 39:47–55, 2021. Palazzi X, Burkhardt JE, Caplain H, Dellarco V, Fant P, Foster JR, Francke S, Germann P, Gro¨ters S, Harada T, Harleman J, Inui K, Kaufmann W, Lenz B, Nagai H, Pohlmeyer-Esch G, Schulte A, Skydsgaard M, Tomlinson L, Wood CE, Yoshida M: Characterizing “adversity” of pathology findings in nonclinical toxicity studies: results from the 4th ESTP international expert workshop, Toxicol Pathol 44:810–824, 2016. Palazzi X, Li X, Khan NK, Burkhardt JE: Introduction to toxicologic pathology. In Sahota PS, Wojcinski Z, Spaet R, Hardisty J, editors: Toxicologic pathology: an atlas, 2022, CRC Press/Taylor & Francis. in progress/print. Perry R, Farris G, Bienvenu J-G, Dean C, Foley G, Mahrt C, Short B: Society of toxicologic pathology position paper on best practices on recovery studies: the role of the anatomic pathologist, Toxicol Pathol, 2013:1159–1169, 2013. Pretto F, FitzGerald RE: In vivo safety testing of Antibody Drug Conjugates, Regul Toxicol Pharmacol 122:104890, 2021. https://doi.org/10.1016/j.yrtph.2021.104890. Radi ZA, Khan KN: Cardio-renal safety of non-steroidal antiinflammatory drugs, J Toxicol Sci 44:373–391, 2019. Ramaiah L, Tomlinson L, Tripathi NK, Cregar LC, Vitsky A, von Beust B, Barlow VG, Reagan WJ, Ennulat D: Principles for assessing adversity in toxicologic clinical pathology, Toxicol Pathol 45:260–266, 2017. Rojko JL, Evans MG, Price SA, Han B, Waine G, DeWitte M, Haynes J, Freimark B, Martin P, Raymond JT, Evering W, Rebelatto MC, Schenck E, Horvath C: Formation, clearance, deposition, pathogenicity, and identification of biopharmaceutical-related immune complexes: review and case studies, Toxicol Pathol 42:725–764, 2014. Salmon F, Grosios K, Petry H: Safety profile of recombinant adeno-associated viral vectors: focus on alipogene tiparvovec (Glybera), Expet Rev Clin Pharmacol 7:53–65, 2014. Sharpe ME: T-cell immunotherapies and the role of nonclinical assessment: the balance between efficacy and pathology, Toxicol Pathol 46:131–146, 2018. Sheehan JP, Thao PM: Phosphorothioate oligonucleotides inhibit the intrinsic tenase complex by an allosteric mechanism, Biochemistry 40:4980–4989, 2001. Sellers RS, Nelson K, Bennet B, Wolf J, Tripathi N, Chamanza R, Perron Lepage M-F, Adkins K, Laurent S, Troth SP: Scientific and regulatory policy committee points to consider: approaches to the conduct and interpretation of vaccine safety studies for clinical and anatomic pathologists, Toxicol Pathol 48:257–276, 2020.

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Sharpe ME, Morton D, Rossi A: Nonclinical safety strategies for stem cell therapies, Toxicol Appl Pharmacol 262:223–231, 2012. Stella VJ, He Q: Cyclodextrins, Toxicol Pathol 36:30–42, 2008. Suntharalingam G, Perry MR, Ward S, Brett SJ, CastelloCortes A, Brunner MD, Panoskaltsis N: Cytokine storm in a phase 1 trial of the anti-CD28 monoclonal antibody TGN1412, N Engl J Med 355:1018–1028, 2006. Sutherland JJ, Webster YW, Willy JA, Searfoss GH, Goldstein KM, Irizarry AR, Hall DG, Stevens JL: Toxicogenomic module associations with pathogenesis: a network-based approach to understanding drug toxicity, Pharmacogenomics J 18:377–390, 2018. Tessier Y, Achanzar W, Mihalcik L, Amuzie C, Andersson P, Parry JD, Moggs J, Whiteley LO: Outcomes of the European federation of pharmaceutical industries and associations oligonucleotide working group survey on nonclinical practices and regulatory expectations for therapeutic oligonucleotide safety assessment, Nucleic Acid Therapeut 3: 7–20, 2021. Thackaberry EA, Kopytek S, Sherratt P, Trouba K, McIntyre B: Comprehensive investigation of hydroxypropyl methylcellulose, propylene glycol, polysorbate 80, and hydroxypropyl-beta-cyclodextrin for use in general toxicology studies, Toxicol Sci 117:485–492, 2010. Turner OC, Aeffner F, Bangari DS, High W, Knight B, Forest T, Cossic B, Himmel LE, Rudmann DG, Bawa B, Muthuswamy A, Aina OH, Edmondson EF, Saravanan C, Brown DL, Sing T, Sebastian MM: Society of toxicologic pathology digital pathology and image analysis special interest group Article: opinion on the application of artificial intelligence and machine learning to digital toxicologic pathology, Toxicol Pathol 48:277–294, 2020. Vascellari M, Melchiotti E, Bozza MA, Mutinelli F: Fibrosarcomas at presumed sites of injection in dogs: characteristics and comparison with non-vaccination site fibrosarcomas and feline post-vaccinal fibrosarcomas, J Vet Med A Physiol Pathol Clin Med 50:286–291, 2003. Vahle JL, Finch GL, Heidel SM, Hovland Jr DN, Ivens I, Parker S, Ponce RA, Sachs C, Steigerwalt R, Short B, Todd MD: Carcinogenicity assessments of biotechnologyderived pharmaceuticals: a review of approved molecules and best practice recommendations, Toxicol Pathol 38:522– 553, 2010. Wesselschmidt RL: The teratoma assay: an in vivo assessment of pluripotency, Methods Mol Biol 767:231–241, 2011. Yu S, Yi M, Qin S, Wu K: Next generation chimeric antigen receptor T cells: safety strategies to overcome toxicity, Mol Cancer 18:125, 2019.

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C H A P T E R

5 Carcinogenicity Assessment Aaron M. Sargeant1, Arun R. Pandiri2, Kathleen Funk3, Thomas Nolte4, Kevin Keane5 1

Charles River Laboratories, Spencerville, OH, United States, 2Molecular Pathology Group, Comparative and Molecular Pathogenesis Branch, Division of the National Toxicology Program, National Institute of Environmental Health Sciences, Durham, NC, United States, 3Experimental Pathology Laboratories, Inc., Sterling, VA, United States, 4Boehringer Ingelheim Pharma GmbH & Co. KG, Biberach an der Riss, Germany, 5Novo Nordisk Research Park Ma˚løv, Denmark O U T L I N E 1. The Past, Present, and Potential Future of Carcinogenicity Assessment 126 1.1. Brief History of Carcinogenicity Assessment 126 1.2. Food, Drugs, and Cosmetics 127 1.3. Other Chemicals 128 1.4. Relevance of Rodent Findings in Carcinogenicity Hazard Identification Studies for Human Risk 129 1.5. Evolution from Lifetime Bioassays in Two Rodent Species to the Current Standards 130 1.6. Looking Forward: ICH Guideline S1B Modifications 131 1.7. New Approaches in Predicting Carcinogenicity Hazards 133 2. Purpose, Planning, Prerequisite Information, and Timing of Lifetime Carcinogenicity Studies 139 2.1. Prerequisite Data to Design a Carcinogenicity Study Protocol 140 2.2. Special Protocol Assessment for Carcinogenicity Studies 142 2.3. Carcinogenicity Study Planning Timeline 143 3. Two-Year Rodent Carcinogenicity Studies 3.1. Study Design 3.2. Managing High Mortality in 2-Year Carcinogenicity Studies

Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-12-821047-5.00006-3

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3.3. Pathology Interpretations 3.4. Historical Control Data 4. Alternative Genetically Modified Mouse Models 4.1. The Range-Finding Study 4.2. Carcinogenicity Study Design Using Alternative Models 4.3. Conduct of Carcinogenicity Studies Using Alternative Models

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7. Carcinogenicity Assessment of Stem CelleDerived Therapies 163 7.1. Safety Concerns for Stem CelleDerived Therapies 163 7.2. In vivo Assessment of Teratoma Formation 163 8. Carcinogenicity Assessment of Medical Devices 164 9. Conclusion

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1. THE PAST, PRESENT, AND POTENTIAL FUTURE OF CARCINOGENICITY ASSESSMENT 1.1. Brief History of Carcinogenicity Assessment Since the 1700 and 1800s, sporadic examples of occupational, pharmaceutical, and environmentally induced cancers have been recognized in human beings. Occupational cancers were first identified in 1713 by B. Ramazzini, considered the “father of occupational medicine,” who reported that nuns have higher rates of breast cancer than do married women. The first recognition of cancer induced by an exogenous agent (and an occupational cancer) was the identification of scrotal cancer in chimney sweeps in 1775 by P. Pott. Lifestyle cancers were first recognized in 1761, when J. Hill identified the oral cancer risk from the regular use of snuff (Huff, 1999). In 1888, J. Hutchinson reported cancer induced by the therapeutic use of arsenical solutions in topical application for various maladies, thus supplying the first example of cancer attributed to a pharmaceutical agent. An occupational cancer related to a particular industrial process, specifically urinary bladder cancer caused by chronic exposure to aniline dyes in factory workers, was first identified by L. Rehn in 1895. Despite all these human cases, it was not until 1918 that Yamagiwa and Ichikawa first reported the results of an animal study investigating cancer induction by exogenous agents, specifically cancer induced by the repeated topical application of coal tar (Jacobs and Hatfield, 2012). The US National Cancer Institute (NCI) was created in 1937, and the Food, Drug, and Cosmetic Act of 1938 (creating the US Food and Drug Administration [FDA]) established the modern regulatory basis for controlling exposures to carcinogens in foodstuffs, drugs, and cosmetics. However, regulatory agency and industrial expectations for cancer risk assessment of new products were not established until the second half of the 20th century. As human life expectancy dramatically and progressively increased throughout the 20th century, a significant related rise in cancer incidence naturally occurred, thereby increasing the public’s expectations for government and industry action

relevant to cancer as early as the 1950s (Jacobs and Hatfield, 2012). Similarly, the vast majority of new knowledge and enhanced understanding in carcinogenesis has occurred in the past 50 years. The modern era of industrial and regulatory practices in cancer hazard identification, risk assessment, and regulation has led to substantive progress in increasing longevity and decreasing ageadjusted cancer rates in the United States. The accumulated learning over this period has enabled identification of the known biological attributes and novel mechanisms of chemical/ pharmaceutical/food agent-related human cancer for the 21st century, specifically hormonal disruption, immunosuppression, genotoxicity, and chronic toxicity (see Carcinogenesis: Mechanisms and Evaluation, Vol 1, Chap 8). A plethora of theories of carcinogenesis exist, such as the “loss of contact inhibition” popular in the 1990s as well as “genetic addiction” (to enhanced oncogene expression and/or decreased tumor suppressor gene availability) and “altered methylation” as an epigenetic mechanism that gained large followings during the past decade. However, actual examples of chemicals/foods/drugs causing cancer exclusively through these mechanisms have yet to be confirmed as independent from the pathways already recognized to cause neoplastic diseases, such as hormonal, immunosuppressive, or genotoxic mechanisms, or nongenotoxic influences like chronic tissue injury and receptormediated effects. The most important current, generally accepted risk factors affecting cancer rates include genetics, lifestyle (e.g., obesity, sedentary habits, ultraviolet light [UV] exposure, smoking, and alcoholism), environment (e.g., aflatoxin exposure), immunosuppression (e.g., acquired immunodeficiency syndrome [AIDS]), chronic inflammation, and age. In the modern era of cancer risk assessment practices, the activities of toxicologists/pathologists in the United States are driven and controlled, in part, by regulations established by Congress and other regulatory bodies (FDA Redbook, OECD Guidelines, EPA Guidelines). Similar rules have been developed by regulatory acts in other developed nations (ECHA Guideline). Some background on the regulating authorities, relevant regulations, and other agencies relevant to cancer hazard identification

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practices and laboratory practices is an important means for providing context for modern cancer risk assessment paradigm. Practices today originated in the 1960s, with continual refinement but few major changes taking place over the subsequent years. Major changes have included the empirical move to conduct bioassays only in rodents in the 1970s and the evidence-based move to utilization of alternative genetically modified mouse models in 1998 in lieu of the lifetime mouse bioassay (for pharmaceuticals) (Tamaoki, 2001). Differences in regulations as well as some practices exist for different classes of products based on the specific agency that has regulatory oversight and responsibility for product approval.

1.2. Food, Drugs, and Cosmetics In 1958 the Delaney Clause, an amendment to the Food, Drug, and Cosmetic Act of 1938, banned the use of food additives that are demonstrated to cause cancer in animals. Animals had already been demonstrated to be potential sentinels for human cancer risk. For instance, in the 1930s, diethylstilbestrol (DES) was shown to be carcinogenic in mice. In the absence of a translational science mindset, DES was approved by the FDA in 1941 for use in pregnant women in the belief that it improved gestational outcomes in certain circumstances, as well as for use in teenage girls to suppress growth in height. In 1971, DES was reported to cause vaginal cancer in the daughters of pregnant women who were treated with DES during pregnancy, after which the FDA listed pregnancy as a contraindication for DES therapy (Huff, 1999). Systematic testing of new foods, cosmetics, and drugs to identify hazards and characterize potential risks did not actually begin until the 1960s. The FDA (via its drug division) first requested cancer studies via a letter to various sponsors in July 1967. Guidelines for carcinogenicity testing were first published in the FDA Papers (the name from 1967 to 1972 of the magazine subsequently published through 2007 as FDA Consumer; Dr. Abigail Jacobs, personal communication). Although animal cancer studies supporting pharmaceutical development became common beginning in the late 1960s, testing procedures were not standardized. As examples from the 1960s, 7-year dog studies

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and 10-year monkey studies were utilized in development of birth control drugs such as levonorgestrel and norelgestromin, while the US Army commissioned a 7-year dog study of irradiated meat to assess safety of this method for preserving food. Other key events in the second half of the 20th century affecting the strategy for carcinogenicity hazard assessment included the discovery and development of the Ames test (a bacterial assay for the detection and classification of mutagens) in 1973 and the implementation of the Good Laboratory Practices (GLP) Act in 1978 (Title 21, Code of Federal Regulations, Part 58) that established rigorous expectations for the administrative quality of the nonclinical cancer testing process (see Pathology and GLPs, Quality Control and Quality Assurance, Vol 1, Chap 27). In 1982, the FDA Red Book, published by the Bureau of Foods, became the first regulatory guidance with detailed carcinogenicity testing requirements (FDA Redbook). Although this guidance document called for 3-year rat carcinogenicity studies, the 2-year study was more commonplace, with study duration limited by obesity-related decreases in longevity occurring as a consequence of selective breeding of rats for improved fecundity. International standards for cancer hazard identification for pharmaceuticals have evolved under the auspices of the International Council for Harmonisation (ICH). The ICH was launched in April 1990 at a meeting of the International Federation of Pharmaceutical Manufacturers and Associations (EFPIA) in Brussels, and was expanded to include regulatory authorities from Europe, Japan, and the United States. The ICH provides guidance on the need for long-term rodent carcinogenicity studies of pharmaceuticals (S1A), testing for carcinogenicity of pharmaceuticals (S1B), and dose selection for carcinogenicity studies of pharmaceuticals (S1C). The S1B guideline is under active review at the time of this writing as discussed in Section 1.6 (Looking Forward: ICH Guideline S1B Modifications). The ICH has greatly enhanced standardization of international pharmaceutical regulatory expectations for the rodent lifetime cancer bioassay. Now, 30 years later, the ICH process is quite active in addressing and modernizing cancer hazard identification and risk assessment practices (see Pathology and GLPs, Quality Control and Quality Assurance, Vol 1, Chap 27).

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1.3. Other Chemicals Although the US National Cancer Institute (NCI) was created in 1937, the NCI bioassay program was not instituted until 1961. The NCI is part of the US National Institutes of Health (NIH), 1 of 11 agencies that are part of the US Department of Health and Human Services (HHS). In the 1950s, concurrent with the beginning of the NCI bioassay program and the passage of the Delaney Clause for foods, Rachel Carson published articles on chemicals in the environment repeatedly in Reader’s Digest. Her seminal work, the book Silent Spring (published in 1962), led the public to recognize the importance of environmental hazards, including cancer, posed by certain pesticides. Carson is considered the founder of the modern-day environmental activist movement, and is credited with leading the effort to eliminate dichlorodiphenyltrichloroethane (DDT) and chlorinated hydrocarbon pesticides from commercial use in the United States. In response to growing public environmental concerns, the US Environmental Protection Agency (EPA) was created in 1970 to control chemicals not regulated by the FDA. The EPA was charged with protecting human health and the well-being of the environment by formulating and enforcing regulations based on laws passed by Congress. One of the chapter’s authors (CLA, third edition) recalls a highschool classmate on a neighboring farm in the late 1950s whose father died from organophosphate (OP) poisoning while unloading OPtreated seed corn in an enclosed workspace. Similar experiences undoubtedly were replicated in many farm communities across the United States. Currently there are over 30 Acts of Congress governing EPA activities and actions concerning the air, land, and water. Two major acts created the foundation for regulatory control of chemicals in our environment. The Federal Insecticide, Fungicide, and Rodenticide Act (FIFRA), first created in 1947 and extensively revised in 1972, charged the EPA with responsibility for pesticide regulation. This revised Act established a registration process to create data supporting the effectiveness and safety of pesticides and established labeling requirements. The Toxic Substances Control Act (TSCA) of 1976 regulates the introduction of new chemicals

in commerce that do not fall under the jurisdiction of the Food, Drug, and Cosmetic Act or FIFRA for the purpose of defining and limiting risk of injury to human health or the environment (see Agricultural and Bulk Chemicals, Vol 2, Chap 12). New chemicals are defined as any agents manufactured domestically or imported subsequent to December 1979 (i.e., chemicals not on the TSCA inventory of existing agents in December 1979). To date, there are about 86,000 chemicals within the TSCA inventory. In the United States, it is estimated that more than 8800 manufactured or imported chemicals are produced at quantities exceeding 10,000 pounds annually. Some 62,000 chemicals in commercial use have never been tested for carcinogenicity risk. The EPA, when necessary, has successfully banned or restricted chemicals in commerce, including polychlorinated biphenyls (PCBs), chlorofluorocarbons (CFCs), dioxins, asbestos, and hexavalent chromium. Several government institutes in the United States have special relevance to cancer hazard identification and risk assessment. The US National Institute of Environmental Health Sciences (NIEHS) was created in 1966 as 1 of 27 institutes and centers of the National Institutes of Health (NIH). The NIEHS supports basic research, clinical research, and training to better understand environmental causes of human diseases. Of specific interest to toxicologists and pathologists, NIEHS scientists have been instrumental in developing genetically altered mice to improve the toxicity and carcinogenicity screening of toxins. The NIEHS also administers the US National Toxicology Program (NTP), an interagency program established in 1978 (as a successor to the NCI bioassay program of 1961) to coordinate, evaluate, and report on the toxic effects and mechanisms of chemicals, foods, and drugs. The NTP played a major role during the late 1970s and early 1980s in shaping the high standards of current rodent carcinogenicity testing. Differences exist between the NTP testing practices and the typical industry testing practices. For example, the NTP traditionally employed the F344 rat (replaced with the Harlan Sprague Dawley rat in the late 2000s) and B6C3F1 mouse, in contrast to the routine use by industrial firms of Sprague Dawley and Wistar rats and CD-1 mouse.

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1.4. Relevance of Rodent Findings in Carcinogenicity Hazard Identification Studies for Human Risk Modern cancer risk assessment practices have served to protect the health of the public in the workplace and in the home. Current industry and regulatory practices can be credited in part with the continuous increase in life expectancy since 1900, as well as the decreasing ageadjusted cancer rates since 1990 in the United States. Today, universally recognized biologic mechanisms of chemical carcinogenesis in humans include genotoxicity, hormonal disruption, immunosuppression, and chronic toxicity (chronic tissue damage and repair) (see Carcinogenesis: Mechanisms and Evaluation, Vol 1, Chap 8). Rodent carcinogenicity studies are reasonable but not consistently reliable predictors of cancer risk for known human carcinogens. For example, human carcinogens associated with genotoxicity, chronic toxicity, and hormonal risk factors are typically positive in rodent cancer bioassays. In contrast, human carcinogens related to immunosuppression are only sporadically tumorigenic in rodent studies (Bugelski et al., 2010). The International Agency for Research in Cancer (IARC) carcinogens classified in Group 1 (confirmed in humans) and 2A (probable in humans) predominantly test positive in rodent cancer studies. Therefore, the modern version of the rodent bioassay has enabled dramatic advances in our biological understanding of carcinogenic mechanisms and improved our capabilities in cancer hazard identification and risk assessment. As one early example, the work on dietary (i.e., caloric) restriction in the rat dates as far back as 1935, thus revealing obesity as a risk factor for carcinogenesis even though general acceptance of this link is relatively recent. The molecular mechanisms of carcinogenicity in rodent bioassays are typically conserved in humans too; however, numerous rodentspecific lesions and cancer mechanisms have been characterized to demonstrate the lack of human relevance (see Carcinogenesis: Mechanisms and Evaluation, Vol 1, Chap 8). These agents incite neoplasia in various rat or mouse tissues, but act typically via nongenotoxic modes of action involving species-specific endocrine feedback loops that does not occur in the corresponding human tissues (Alison et al., 1994).

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The following are some examples of rodentspecific carcinogenicity mechanisms induced by chemicals/foods/drugs. • a2u-globulin nephropathy leads to tubular neoplasia in the male rat kidney (D-limonene, decalin, unleaded gasoline, 2,2,4Trimethylpentane) (Swenberg, 1993). Chemically induced chronic renal tubular degeneration leads to persistently elevated levels of epithelial hyperplasia (increased cell division) as a reparative response. Several petroleum companies transiently added warning labels on gasoline pumps describing the male rat kidney tumor response until the unique vulnerability of the male rat to this condition was confirmed. The food ingredient D-limonene, the predominant natural component of orange oil, is an additive for flavoring and an important component of fragrances. The increase in renal tubular neoplasms in the D-limonene rat bioassay created a challenge for the Bureau of Foods because the Delaney Clause usually would ban the use of a rodent carcinogen as a food additive. Recognition that the rat findings were not relevant to humans based on research in the early 1980s convinced the Agency not to apply the Delaney Clause to oil of the orange and orange-flavored products. US regulators did not require warnings on orange juice cartons (stating “carcinogenic for male rats”), as occurred for refiners of unleaded gasoline, since orange juice is a natural product known to have substantial health benefits. • Urothelial tumorigenesis in the rat urinary bladder (ascorbate, saccharin) has been linked to excessive formation of calcium phosphate precipitates in urine after administration of high doses of sodium salts. The presumed mechanism is urothelial irritation with secondary increases in cell division due to chronic crystalluria (Cohen et al., 1995). • Neoplasms of the liver, pancreas, and testis are induced by peroxisome proliferators (agonists of peroxisome proliferator–activated receptor [PPAR]-a) in rodents, but not humans, because rodent tissues have a higher PPAR-a density compared to the corresponding human tissues (Corton et al., 2018).

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• Testicular Leydig cell tumors in rats (finasteride, flutamide) arise via increased 17b-estradiol levels secondary to induction of aromatase (a key enzyme in estrogen biosynthesis) in the liver. Decreased estrogen leads to decreased conversion of estrogen to testosterone by Leydig cells. In response to decreased testosterone, the pituitary secretes luteinizing hormone (LH) at increased levels, stimulating Leydig cell proliferation leading to neoplasia (Cook et al., 1999). • Thyroid follicular tumors in rats are caused by chemicals that induce cytochromes P450, thereby accelerating the metabolism of thyroid hormones (loratadine, cetirizine, doxylamine, phenobarbital). The decline in serum levels of thyroid hormones results in positive feedback to the pituitary gland, elevation of thyroidstimulating hormone secretion, and eventual increases in follicular cell proliferation in response to persistent stimulus for thyroid hormone production. This chronic upregulation stimulates thyroid tumor development over the rodents’ lifespan. These drugs also induce mouse liver tumors in traditional lifetime bioassays, so they are considered transspecies, multi-organ rodent carcinogens (Hill et al., 1998; Bartsch et al., 2018). • Pancreatic exocrine tumors (e.g., corn oil and uncooked soy protein) result from persistent elevation of plasma cholecystokinin (CCK). This peptide hormone serves as a trophic stimulus for the pancreatic acinar epithelium (Woutersen et al., 1991). • Gastrointestinal endocrine cell neoplasia (carcinoids) caused by treatment with antiacid ATPase inhibitors (omeprazole, lansoprazole) results from chronic repression of gastric acid secretion that stimulates persistent enterochromaffin-like cell (ECL) hyperplasia and increased gastrin secretion in a vain attempt to restore gastric acid secretion to normal levels. Increased ECL proliferation over the lifetime of the rat eventually leads to ECL neoplasia (Havu, 1986; Hirth et al., 1988; Betton et al., 1988). • Mesovarian leiomyomas result from chronic smooth muscle proliferation in the mesovarium stimulated directly in the rat by b2 adrenergic agonists (b2-adrenergic receptor stimulants, e.g., bronchodilators) (Kelly et al., 1993).

• Prolonged hyperprolactinemia produces pituitary and mammary gland tumors (antidopaminergic compounds, betaadrenergic blocking agents) (Alison et al., 1994). • Nongenotoxic induction of forestomach, Zymbal’s gland, and Harderian gland tumors often is considered irrelevant to human risk assessment because these anatomic structures do not occur in humans (Alison et al., 1994). • Pheochromocytoma incidences have been noted in rats that also have concurrent toxic effects in the kidney (with chronic progressive nephropathy), and lung (with space occupying lesions). However, the human relevance of these lesions is currently debatable (Greim et al., 2009). These examples of false-positive and falsenegative results as they relate to human risk identification underscore the idea that the rodent cancer bioassay as currently performed cannot be considered a discriminating test.

1.5. Evolution from Lifetime Bioassays in Two Rodent Species to the Current Standards Now, after thousands of animal cancer bioassays over five decades, more effective and efficient processes are evolving including novel animal models such as rasH2 mice, complex mechanistic approaches using microphysiological systems, as well as high-throughput screening approaches. Comparing animal carcinogenicity tests of pharmaceuticals with human experience has provided insights typically not available for environmental chemicals. A recent review of this comparative database revealed several conclusions (Alden et al., 2011). First, most drugs will elicit a positive carcinogenic response in test animals because the past bias for setting the high dose in rodent cancer studies led to very elevated and usually toxic exposures; indeed, all rodent cancer bioassays would be likely to yield a positive result if the sample size and duration of testing were significantly increased, as chronic toxicity is a risk factor for carcinogenesis (Alden et al., 2011). In like manner, 82% of chemicals tested in the NTP rodent bioassay program are reported to be positive for carcinogenic potential. Since myriad toxicity mechanisms exist, the variability in the

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link between chronic toxicity and cancer response would also be predicted. This variation is problematic because weak carcinogens cannot be identified with standard group sizes of 50–65 rodents treated over a 2-year period or even a longer lifetime. Evidence-based refinements during this past decade have resulted in setting the high dose for pharmaceutical agents in cancer studies at nontoxic levels if sufficient multiples (usually a dose multiple >25 times) above the expected human exposure can be produced in rodents without toxicity. Rodent carcinogenicity bioassays yield poorly reproducible results. Follow-up studies are estimated to generate conflicting carcinogenicity results approximately 50% of the time (Gottmann et al., 2001). Of even greater concern, lifetime rodent cancer bioassays have failed to detect a number of notable human carcinogens, including both environmental pollutants (cigarette smoke) and common pharmaceutical agents (e.g., hormone modulators and immunosuppressive drugs). A false-negative rate of 15% has been reported in this regard (Alden et al., 2011). Indeed, the rodent cancer bioassay as currently performed cannot be considered a discriminating test. A large number of “generally recognized as safe” (GRAS) substances as well as several commonly marketed pharmaceuticals may be carcinogenic in rodents, if rodents are exposed to them under the conditions of a typical rodent cancer bioassay (Alden et al., 2011; Boorman et al., 1994). Examples include over-the-counter pharmaceuticals (loratadine, cetirizine, doxylamine, phenobarbital, acetaminophen, sodium fluoride, benzoyl peroxide, omeprazole, lansoprazole, and minoxidil) as well as prescription products (gemfibrozil, clofibrate, simvastatin, lovastatin, griseofulvin, sertraline, albuterol, cimetidine, and terazosin). Discrepancies between the outcome in test animals and the lack of response in human patients demonstrate some of the limitations of the rodent cancer bioassay and may compromise the lay public’s perceptions regarding the credibility of the test interpretation by industrial toxicologists and pathologists and the subsequent regulatory review. These deficiencies have led to the development of the current ICH S1 guidance, under which regulatory authorities accept that carcinogenicity can be assessed via a 2-year rat bioassay plus a shortterm mouse study using a transgenic model.

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The latter in practice has become the rasH2 mouse (see Section 4). Additional refinements to carcinogenicity testing are also warranted to improve human risk assessment and to assure the public that efforts to guard human health are effective. In order to understand how carcinogenicity hazard identification and risk assessment will develop, it is important to understand the strengths and weaknesses of the current testing paradigm. Several retrospective investigations into the outcome of 2-year rodent bioassays were performed to assess their contribution to regulatory decisions in addition to that published by Alden et al. (2011). An early and comprehensive one was performed by the US FDA, European Medicines Agency (EMA), and Japanese Pharmaceuticals and Medical Device Agency (PMDA) in the 1990s. It revealed that transspecies carcinogens may not only be identified by 2-year bioassays in rats and mice but also by one 2-year bioassay combined with an alternative in vivo assay (Contrera et al., 1997). This outcome was an important basis for the ICH guideline S1B on carcinogenicity testing of pharmaceuticals. About 15 years later, an evaluation of carcinogenicity data of medical products authorized by the European centralized procedure between 1995 and 2009 was performed (Friedrich and Olejniczak, 2011). It revealed that 94 of the 144 compounds evaluated were positive in either carcinogenicity or repeat-dose toxicity studies. The majority of tumor findings were considered not to be of human relevance. The authors concluded that the value of the current testing strategy for carcinogenicity appears questionable (Friedrich and Olejniczak, 2011).

1.6. Looking Forward: ICH Guideline S1B Modifications An evaluation of the concordance of histopathology findings of 6-month and 12-month rat studies and the outcome of 2-year carcinogenicity studies in rats indicated that the absence of histopathology risk factors for neoplasia is a reliable predictor for a negative outcome of a 2-year study on an animal basis, but not on a tissue basis (Reddy et al., 2010). Stimulated by this result, a Pharmaceutical Research and Manufacturers of America (PhRMA) consortium investigated 182 marketed and nonmarketed pharmaceuticals

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and concluded that a 2-year rat carcinogenicity study may not have relevant contribution to carcinogenicity risk assessment, if the agent fulfills the following criteria: lack of genotoxicity, lack of certain histopathologic risk factors for rat neoplasia in chronic toxicology studies, and lack of hormonal perturbation. The authors reasoned that such compounds should be exempted from testing for carcinogenicity in a 2-year carcinogenicity study in rats (Sistare et al., 2011). These and other investigations gave the impetus to revise the existing guidance on carcinogenicity testing for pharmaceuticals. A concept was endorsed by the ICH Steering Committee and published in 2012 (ICH, 2012). The aim was to modify the ICH S1 carcinogenicity testing guidelines by adding the option that for certain compounds a positive or negative outcome of a 2-year rat study could be predicted and, thus, the compound be exempted from a 2year rat study. The underlying hypothesis was validated in a prospective evaluation period in which pharmaceutical companies submitted a prediction of the outcome of 2-year rat studies for development compounds based on all data available. Regulatory authorities made their own prediction based on submission reviews. These predictions were compared to the outcome of the final reports of the 2-year rat study. From the 46 complete submissions received, 21 predicted that the 2-year rat study will be negative or only rat-specific tumors may occur (ICH, 2020). The comparison of the predictions with the final study reports confirmed the working hypothesis. Consequently, an addendum to the guideline on testing for carcinogenicity of pharmaceuticals has been issued to modify the existing testing paradigm (ICH, 2021). The Step 2 document recommends an integrative weight of evidence (WoE) approach in the assessment of the human carcinogenic risk of a small molecule pharmaceutical compound. Possible outcomes of this assessment may be that the compound is likely carcinogenic/noncarcinogenic in humans or that the prediction is uncertain. A 2-year rat study may add value to the carcinogenic risk assessment only in the uncertain cases, while compounds predicted to be carcinogenic or noncarcinogenic may be exempted from a 2year rat study and labeled according to the WoE assessment. The draft guideline provides a comprehensive and thoroughly elaborated

listing of factors that need to be included in the WoE assessment. Readers are encouraged to familiarize themselves with this listing and the examples given in the guideline appendix. Currently, there seems to be no established process among ICH member states/regions for mutual recognition of an exemption for a 2-year rat study. And it is out of the scope of ICH to propagate such an alignment. Thus, sponsors will have to seek alignment with all relevant regulatory authorities before deciding not to conduct a 2-year rat study based on the WoE approach. This new approach for carcinogenicity risk assessment has the potential to substantially impact current approaches for nonclinical testing of small molecules. It will be important for programs to include endpoints that sufficiently inform the WoE assessment (see ICHS1B(R1) guideline). Therefore, end points like an offtarget toxicity receptor screen, reversibility of histopathological findings, toxicity mode of actions, effects on hormones, or toxicogenomics will become an integral part of many nonclinical safety development programs. In addition, it will be imperative for sponsors and regulators to carefully consider target-based cancer risks and address these concerns in the development program. The additional information gathered from these end points will enable a more science-based assessment of the carcinogenic risk, may reduce resources, and in certain cases substantially reduce development timelines all without compromising patient safety. The ICHS1B(R1) Step 2 document also gives guidance on the second species for carcinogenicity testing, which usually is the mouse. A mouse study would still be expected in most cases, either a 2-year bioassay in wild-type animals or a 6-month medium-term in vivo assay in a transgenic model. Even here the guideline offers flexibility in that no mouse carcinogenicity study may be required if that can be based on strong scientific evidence. Such evidence would include limitations in maximum exposure to only subtherapeutic and pharmacologically inactive drug plasma levels in mice. For development programs limited to the EU it may be important to note that in this region no mouse study is expected when the WoE evaluation indicates that the 2-year rat study indicates no value. For most development programs a carcinogenicity study in mice will still be required for submission. This could be a conventional 2-year bioassay or an alternative test. In recent years,

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a 6-month study in the rasH2 transgenic mouse was more frequently used in carcinogenicity testing programs of pharmaceuticals than a 2year bioassay in wild-type mice (Morton et al., 2002; Jacobs and Brown, 2015). The usage of the rasH2 mouse model will likely further increase in the future, as a 2-year mouse study may be on the critical path to submission of a project for which a 2-year rat study would not be required. Results from a 6-month rasH2 mouse study may add to the WoE assessment as to whether or not conduct a 2-year rat study. Furthermore, retrospective investigations revealed that a combination of the 2-year rat bioassay with the rasH2 mouse model yielded a higher correct determination of 85% of human carcinogens than the two species 2-year rodent bioassay, which resulted in a correct determination in 69% (Pritchard et al., 2003). The difference between the two test strategies was due to a higher false-positive rate in the case of the two species 2-year rodent bioassay. Of note, neither strategy missed any human carcinogen. The pharmacokinetic end point for high dose selection for carcinogenicity studies, as outlined in ICH guideline S1C(R2) (ICH, 2008), has not been globally accepted for studies conducted in rasH2 mice so far. The revision of ICH S1B will introduce this end point also for rasH2 mice: drug plasma exposure 50 times higher than human exposure at the maximum recommended therapeutic dose is considered sufficient. This factor is based on a retrospective examination of 53 studies in rasH2 mice which indicated that doses in excess of the factor 50 did not add to cancer risk identification (Hisada et al., 2022). In addition, the revision states that wild-type rasH2 littermates may be used for dose range finding studies and for toxicokinetic monitoring. Transgenic mouse models are more sensitive to genetic drift than wild-type rats or mice employed in the 2-year bioassay (Long et al., 2010). Therefore, positive controls are generally included in studies with rasH2 mice intended for submission to a regulatory agency. Literature reports indicate that the positive controls produced tumors as expected in various studies conducted over a period of 15 years (Bogdanffy et al., 2020; Paranjpe et al., 2013). For laboratories that conduct such studies on a regular basis this may offer future options not to run a positive control in every study but only at certain time intervals.

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1.7. New Approaches in Predicting Carcinogenicity Hazards The value of the rodent cancer bioassays in predicting a carcinogenic hazard is well recognized and continues to be the regulatory mainstay for cancer hazard identification. However, due to the requirement of large number of animals, expense, long duration, uncertainty of the outcomes related to equivocal evidence of carcinogenicity, marginal increases in rare tumors, high incidences of background/spontaneous tumors, and limited translational relevance of some nongenotoxic carcinogens, there is a continuing need to refine these carcinogenicity assays. That said, significant progress has been made in rodent carcinogenicity testing, i.e., there has been a continuous refinement of the rodent carcinogenicity model which has progressed from counting the “lumps and bumps” to understanding the molecular mechanisms and the translational relevance of defined preneoplastic and neoplastic lesions from rodents. However, there is an urgent need to develop high confidence assays with higher throughput to test the vast catalog of chemicals with no information on their carcinogenic hazard potential. These new approaches will utilize predominantly nonanimal testing approaches. In this section, the strategies outlined in the Integrated Approaches to Testing and Assessment (IATA) (OECD, 2020a) for chemical hazard identification will be discussed. None of the approaches described below are currently used by pharmaceutical regulatory agencies and most of them are a work in progress. IATA uses a comprehensive WoE approach to weigh and integrate available information from various in silico, in vitro, and in vivo approaches in the context of adverse outcome pathways (AOPs) to characterize and predict a cancer hazard. In addition, new assay developments to measure qualitatively and quantitatively the key characteristics of carcinogens will also be discussed. Some of the approaches discussed below are also reflected in the recently updated preamble for the IARC’s monographs and these include strengthening of systematic review methods; critical evaluation of the epidemiological data including exposure assessment; have greater emphasis on mechanistic evidence based on key characteristics of carcinogens; harmonize evaluation criteria for the different evidence

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streams; and integrate mechanistic evidence on cancers in humans and experimental animals for reaching overall evaluations (Samet et al., 2020). Adverse outcome pathways (AOPs): AOPs use a mode (and/or mechanism) of action (MOA) framework to organize existing knowledge from various sources to establish causal linkages on biologically plausible molecular events at various levels of biological complexity (cells > tissues > organs > organism) to examine the adverse outcomes of regulatory concern (OECD, 2018a,b). A typical AOP starts with the description of the interaction of the chemical/agent with a protein/cell (molecular initiating event (MIE)) that initiates a cascade of sequential biological causal events that progress through several interrelated key events (KEs) that ultimately lead to an adverse outcome at the level of the whole organism or population (Ankley et al., 2010) (Figure 5.1A). A single MIE can initiate a series of biological events and

based on the potency of the MIE (in terms of dose, duration, and target), there can be numerous adverse outcomes (i.e., toxicity, carcinogenicity, etc.), or a single MIE can cause the same adverse outcome through various pathways. So, the biological effects of a single chemical may be represented in multiple interconnected AOPs. AOPs are modular and composed of reusable elements named KE and key event relationships (KERs). The KEs are essential and causally linked with other KEs (or an adverse outcome), and must be experimentally measurable. If a biological intermediate step is redundant and not essential, then it is not a KE. KERs define the type of association between the KEs, i.e., a positive or negative relationship, and the strength of the association directly influences the weight of evidence (biological plausibility and empirical support) of that relationship. KERs may link two sequential KEs or link KEs that are not adjacent to each

FIGURE 5.1 (A) A graphic representation of a typical linear adverse outcome pathway (AOP). The anchor points are initial molecular initiating event (MIE) and the downstream endpoint of regulatory concern (adverse outcome (AO) in the organism or the population). Each of the essential biological responses (called Key events (KEs)) contribute to the subsequent essential biological effects and the KEs are linked by key event relationships (KERs) that regulates the KEs in a positive or negative manner. In essence, each of the individual KEs function as biological dominoes in a domino effect. (B) A hypothetical example of an AOP network with five AOPs. AOP1 linking MIE1 to AO1. AOP2 linking MIE1 to AO3. AOP3 linking MIE2 to AO3. AOP4 linking MIE3 to AO3. AOP5 linking MIE3 to AO4. An AOP network reflects the pleotrophic effects of KEs in a typical biological process.

1. THE PAST, PRESENT, AND POTENTIAL FUTURE OF CARCINOGENICITY

other (i.e., an unknown KE is in between), this approach allows for increasing the preciseness of the AOP as new information is obtained and also provides an opportunity to identify data gaps and prioritize research to refine existing AOPs. In general, no cancer adverse outcome is a simple linear pathway with one MIE, and a few KEs and KERs, but is almost always composed of a complex network of multiple AOPs (Figure 5.1B). Hence, multiple AOP modules are usually combined into a network to explain a complex biological effect such as cancer. Even though the practical application of each AOP begins with a chemical under consideration, the AOPs by definition are chemical agnostic and generalizable to any adverse outcome, i.e., an AOP developed for a particular chemical may be used for other chemicals that result in a similar adverse outcome. Finally, AOPs are living documents, as new knowledge is generated, they can be updated with greater granular information. An AOP, depending on its “completeness,” can serve a number of uses within the regulatory context, such as (1) prioritization for further testing, (2) hazard identification, (3) classification and labeling, and (4) risk assessment. As we proceed from (1) to (4), the level of uncertainty that can be tolerated decreases and the level of evidence (qualitative and quantitative) to support the biological plausibility of the KEs/ KERs and the AOP increases. So, depending on the extent of development, a partially developed AOP (i.e., not all KEs/KERs are known) may be useful for objectives (1)–(4) to varying degrees. To provide data to support an AOP evaluation, a range of in silico, in vitro, and in vivo information on KEs and KERs, as well as data from highthroughput screening (HTS) assays, high-content screening (HCS), and omics approaches, can be used. In the context of chronic toxicity and cancer, a number of the above modalities address the qualitative and quantitative aspects of KEs/KERs to address the AOPs. In the qualitative AOPs, the KEs can be measured and the KERs are supported by empirical evidence, biological plausibility, or statistical inference to support the WoE of the AOP, while in the quantitative AOPs, the KEs can be measured and KERs provide a quantitative understanding on the magnitude of effects on the downstream KEs due to precise changes in the upstream KEs (Villeneuve et al., 2014). In general, most AOP development cycles go

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through a putative AOP > qualitative AOP > quantitative AOP > further iterative refinement based on new data. In the concept of AOP development for cancer hazard identification, the development and refinement of various assays that reflect KEs/KERs are always a work in progress to increase assay throughput while obtaining biologically complex information. In cancer AOP development, either a single assay can provide a predictive value for multiple AOPs, or a battery of assays that are highly specific for predicting a specific endpoint of regulatory concern may be used. By using the AOPs in a systematic manner for cancer hazard identification, there is an opportunity to obviate the need for 2-year rodent cancer bioassays based on the confidence in measuring the causal MIEs, KEs/KERs, and the adverse outcome. Based on the MOA of the cancer hazard of an agent/chemical, a WoE based on data with lower biological complexity may be sufficient to predict an adverse outcome (Meek et al., 2014). In these cases, more expensive and time-consuming assays may not be needed. For example, a highly genotoxic chemical predicted based on in silico/ in chemico approaches accompanied by some in vitro data is sufficient to predict a cancer hazard. However, a nongenotoxic compound may require data from assays with higher biological complexity to predict a cancer hazard. The next few paragraphs will cover various assay modalities with decreasing throughput and increasing biological complexity used in cancer hazard identification. In order to understand the context of these approaches and the underlying concepts of cancer biology such as genotoxicity and nongenotoxicity, the reader is encouraged to refer to the chapter on Carcinogenesis: Mechanisms and Evaluation, Vol 1, Chap 8. In general, most of the approaches discussed below consider the broad mechanistic classification of carcinogens, i.e., genotoxic and nongenotoxic. In silico approaches for cancer hazard assessment: In silico approaches refer to virtual experiments on a computer (silicon in computer chips) using existing experimental data to predict a biological outcome based on the structural similarities of related or unrelated chemicals. These approaches help to direct future in vitro or in vivo experiments and are used extensively in pharmaceutical development to identify theoretical or existing chemicals with undesirable qualities and refine the drug development process

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(fail-fast and iterate principle). These approaches are also used in environmental toxicology to prioritize testing chemicals in more biologically complex test systems. The goal of these in silico approaches is to predict mutagenicity responses in the Ames test and predict carcinogenicity in rodent models. In general, all these models are more successful in predicting a mutagenic potential than a carcinogenic potential due to the latter’s biological complexity. The three commonly used in silico toxicology methodologies include statistical-based (Quantitative Structure–Activity Relationship (QSAR)) models, expert rule-based, and read-across. A statistical-based methodology or QSAR uses a mathematical model that, for a given chemical structure, will estimate a value for a specific type of biological effect. An expert rule-based system (alerts) uses a reference dataset to assess the predictive performance of any “structural” alert that describes a part of the chemical structure that is associated with a specific toxic effect or mechanism. Read-across is an approach to predict toxicity using experimental data from one or more chemical analogs to predict toxicity for a chemical with no data (target chemical). In most cases, all three methodologies are used to build a WoE on the potential mutagenicity of a compound (Myatt et al., 2017). While the in silico approaches can predict various biological outcomes, for carcinogenicity assessment, the in silico methods are reasonably successful in predicting genotoxicity and to a less extent nongenotoxic MOAs. In general, genotoxic carcinogens are either electrophiles or can be activated to electrophilic reactive intermediates (proelectrophiles). There are a number of reference databases with information on the mutagenicity and carcinogenicity as well as software tools for predicting the unknown chemicals (Myatt et al., 2017; Serafimova et al., 2010). The ICH M7 guideline that deals with the assessment and control of DNA reactive (mutagenic) impurities in pharmaceuticals (to limit potential carcinogenic risk) recognizes the ability of (Q) SAR to relate chemical structure and bacterial mutagenicity (Benigni et al., 2020). In vitro approaches to cancer hazard assessment: The most common in vitro assays to predict a cancer hazard are based on the identifying genotoxicity and other nongenotoxic MIEs (Table 5.1). Please refer to the chapter Carcinogenesis: Mechanisms and Evaluation, Vol 1, Chap 8 as

well as OECD 471 for detailed descriptions on guideline genotoxicity assays (OECD, 2020b). In addition, major federal initiatives such as Tox21 (a collaborative effort from EPA, FDA, and NIH) and EPA’s Toxicity Forecaster (ToxCast) have contributed to the bulk of the in vitro data using high-throughput screening assays. Tox21 used about 70 HTS assays on 8500 chemicals and ToxCast used about 500 HTS assays on 2000 chemicals to generate 100s of millions of data points that can inform on various MIEs and KEs/KERs (Richard et al., 2021; Dix et al., 2007). These initiatives are continuing to refine the in vitro assays using cell lines with and without metabolizing capability to generate translationally relevant toxicity information that can be used to build chemical-specific MOAs and chemicalagnostic AOPs. Also, recent publications on the key characteristics of carcinogens (KCCs) as well as previously published hallmarks of cancer (HMCs) have become focal points for the development of novel assays aimed at predicting KEs/KERs as well as adverse outcomes (Smith et al., 2020; Hanahan and Weinberg, 2000; Hanahan and Weinberg, 2011). A recent publication by Smith and colleagues (2020) contains exhaustive collated information on several assays (in various stages of validation) that measure each of the KCCs with the goal of identifying KEs/KERs before the cancer endpoint. Unlike cancer hallmarks that are characteristics of neoplasia (that already occurred), these measurable KCCs can inform on the precarcinogenic effects and may predict a potential cancer hazard with some certainty. The currently available assays to measure each of these KCCs have several limitations where most are qualitative, and fewer are quantitative and do not provide concrete evidence with respect to potency (effect size and duration) that would result in an adverse outcome. Depending on the assay, they would indicate a perturbation in a defined KCC that may potentially lead to neoplasia and these approaches may replace the rodent carcinogenicity assays for at least genotoxic carcinogens. However, with respect to prediction of nongenotoxic carcinogens, significant progress needs to be made before these approaches can replace the rodent carcinogenicity assays. Currently, these additional data add value to the in vitro and in vivo carcinogenicity assessments but cannot be used in isolation.

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TABLE 5.1

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Data Required to Design Carcinogenicity Studies

1. Test Article Data Test article impurity profile Comparison to impurity profile of marketed product 2. Pharmacokinetics (PK) and absorption, distribution, metabolism, excretion (ADME) data Mouse, rat, and human PK Plasma protein binding in mouse, rat, and human Mouse and rat radiolabeled tissue distribution/metabolite characterization Human radiolabeled metabolite characterization Human clinical dose(s) and corresponding exposure Area under the curve (AUC) Maximal concentration (Cmax) 3. Genetic toxicology data In vitro mutation assay In vitro cytogenetics assay In vivo cytogenetics assay 4. Dose justification data Range-finding study by intended clinical route Ratd3 months or longer for a 2-year study Mousedsame as above for conventional mice; or 4 weeks for a 6-month study in a genetically engineered strain (wild-type littermates can be used for the range-finding study) 5. Dose selection and rationale Maximum tolerated dose (MTD; primary rationale for environmental and occupational assessments) 25
clinical AUC (only applicable to 2-year studies) Limit dose (1500 mg/kg) Saturation of systemic exposure Maximum feasible dose (MFD; 5% of diet if drug given in diet) Pharmacodynamic limit dose Other Reproduced from Haschek’s and Rousseaux’s Handbook of Toxicologic Pathology, Vol 3, Table 27.1, p. 815 with permission.

There are several limitations to some of the KCCs. Not all the KCCs are amenable for in silico or in vitro assay development since KCCs such as chronic inflammation and immunosuppression need in vivo experiments to provide confident data. Certain KCCs such as electrophilicity, genotoxicity, alterations in DNA repair and genome instability can each independently contribute to a cancer outcome. However,

certain KCCs such as epigenetic alterations or oxidative stress do not have a definitive causal role in causing carcinogenicity but influence other KCCs and contribute to carcinogenesis. In addition, perturbation of some KCCs, especially those related to nongenotoxic carcinogenesis MOAs such as epigenetic alterations and modulation of receptor-mediated effects, may or may not lead to carcinogenesis and the thresholds

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that need to be exceeded to cause carcinogenicity must be determined on a case-by-case basis using in vivo assays. Nevertheless, when these approaches are applied as a battery, they provide sufficient information to help in prioritizing chemicals for testing in assays of higher biological complexity and help in read-across for chemical analogs. The KCC approach also helps to identify data gaps in assay development and helps prioritize the use of resources optimally. In vivo Approaches in Cancer Hazard Assessment While the current rodent carcinogenicity assay is more than 50 years old, there are several opportunities to refine it by incorporating methods to identify cancer hazards along with the underlying mechanisms and the translational relevance. As discussed earlier, the rodent carcinogenicity assay is usually preceded by a dose range finding study and a subchronic study. In this section, we’ll discuss the potential opportunities to predict a cancer outcome based on molecular analysis of tissues from subchronic studies. Ideally, before initiating the animal studies, data from a battery of in silico/in vitro assays focused on the KCCs would provide an insight into the potential mode of action of toxicity and/or carcinogenicity. These in vitro data would help build a tentative AOP with MIEs and some KEs and KERs with reasonable confidence and may obviate the need for an in vivo carcinogenicity assay especially if a genotoxic mode of action is confirmed. In addition, this tentative AOP may help to identify data gaps that would need animal studies to address. In this context, the in vivo approaches will add a tremendous amount of mechanistic information especially with regard to nongenotoxic MOAs. In general, mechanistic studies are only considered in the industry as a derisking measure to show a lack of translational relevance of a particular rodent tumor. However, if prospective mechanistic studies are designed for tissues obtained from routine in vivo studies, significant mechanistic and translational value may be derived from the subchronic studies. In general, with repeated dose exposures, there is an initial phase of tissue adaptation followed by adverse toxic effects in various target organs that subsequently may lead to preneoplasia and neoplasia. Preneoplastic lesions are usually the

earliest morphological manifestation of carcinogenicity, and these lesions may appear after a 3– 6 month duration in a repeat-dose exposure, i.e., more realistically most carcinogens seldom exhibit preneoplastic lesions in subchronic studies. However, the molecular alterations underlying carcinogenic processes may already be established in spite of the absence of a morphological manifestation of preneoplasia. Over the past two decades, omics technologies such as microarrays and RNASeq allowed the generation of gene signatures that can provide an insight into the mechanisms underlying toxicity and carcinogenicity using tissues obtained from short-term (5–90 days) animal studies. Gene signatures that can discriminate and predict various genotoxic and nongenotoxic carcinogens with varying degree of sensitivity and specificity have been published. For more details on the promise and limitation of the transcriptomic approaches for predicting a cancer hazard based on short-term in vivo studies, please refer to Toxicogenomics: A Primer for Toxicologic Pathologists, Vol 1, Chap 15. The advent of highly sensitive next-generation sequencing (NGS) technologies provides an opportunity to probe alterations in the DNA, mRNA, noncoding RNA, as well as other epigenetic alterations. It is generally accepted that past exposures and life history leave traces of genomic and epigenomic alterations, whereby systematic cataloging and identification of these signatures provide an opportunity to predict the downstream end points such as cancer. Recent NGS studies on human cancers, in vitro exposures, as well as rodent carcinogenicity studies have demonstrated that each of the exposures leaves signature hallmarks in the form of single base substitution (SBS) mutation signatures that serve to provide an insight into the unifying causal molecular mechanisms (Alexandrov et al., 2020; Kucab et al., 2019; Riva et al., 2020). In addition, each of the cancers also has other types of mutation signatures based on small insertions–deletions, doublet mutations, and also harbor mutations in key cancer driver genes. Examination of mutation signatures helps in capturing the biological signal that is directly related to neoplasia rather than an MIE that may lead to neoplasia. It is highly plausible that tissues with no lesions from subchronic exposures harbor mutation signatures that may provide an insight into potential cancer outcomes (Hu et al., 2020; Helleday et al., 2014; Martincorena et al., 2015).

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2. PURPOSE, PLANNING, PREREQUISITE INFORMATION, AND TIMING

Recent advances in NGS technologies such as error corrected duplex sequencing technologies that barcode both strands of DNA with unique molecular identifiers (UMIs) provide a powerful tool to detect rare cancer driver mutations in nontumor tissues from subchronic exposures (Abascal et al., 2021; Salk et al., 2018). Another exciting development in this area is the examination of the circulating cell-free DNA (ccfDNA) in plasma/serum (“liquid biopsy”) collected from subchronic studies for genetic and epigenetic signatures as well as mutations in cancer driver genes. Since the circulating tumor DNA is shed into the blood, a priori knowledge on the tumor target tissue is not necessary (Zhu et al., 2021). As the costs of these NGS technologies decrease, there is a potential for these approaches to be commonly adapted. Similar to mutation signatures, epigenetic alterations in DNA such as methylation, histone modification, and other chromatin remodeling events, as well as alterations in noncoding RNA such as lncRNA and miRNA, may help in predicting a carcinogenic outcome by examining tissues from short in vivo studies (Baylin and Jones, 2016; Herceg et al., 2013). Aberrant DNA methylation related to various exposures such as tobacco smoke, estrogen modulators, hepatitis virus infections, as well as those due to chronic inflammation and aging has been identified. Similar to aberrant methylation signatures, studies also demonstrated lncRNA and miRNA signatures specific to various cancers (Calin and Croce, 2006; Carlevaro-Fita et al., 2020). Recent data suggest that combination of the genomic and epigenomic alterations can have a greater predictive performance for identifying potential cancer outcomes (Yamashita et al., 2018; Takeshima and Ushijima, 2019). For a brief outline on epigenetic mechanisms in chemical carcinogenesis, please refer to Carcinogenesis: Mechanisms and Evaluation, Vol 1, Chap 8. The future of carcinogenicity testing will likely be less dependent on the 2-year rodent cancer assay and instead will focus on a multipronged WoE approach that utilizes data from various high-throughput in vitro assays with increasing biological complexity to obtain sufficient information for regulatory action. In some cases, chemicals may be prioritized for subchronic studies to generate data from the whole animal and the predictive genomic, epigenomic, and

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transcriptomic approaches will provide actionable data. The 2-year rodent cancer bioassays will likely be reserved for exposures where sufficient actionable information was not obtained from the multimodal approaches described above.

2. PURPOSE, PLANNING, PREREQUISITE INFORMATION, AND TIMING OF LIFETIME CARCINOGENICITY STUDIES The assessment of carcinogenicity potential is an integral part of pharmaceutical development as well as for ingredients used as food additives, and also for chemicals resulting in environmental and occupational exposures such as pesticides, water disinfection by-products, and industrial chemicals. Rodent carcinogenicity testing of pharmaceuticals is needed especially when they are expected to be used continuously for at least 6 months or intermittently for the treatment of chronic or recurrent conditions but not for drugs used infrequently or for short duration of exposure (e.g., anesthetics and radiolabeled imaging agents) (ICH S1A guideline). Carcinogenicity studies are also considered when there is a concern about the carcinogenic potential of a pharmaceutical especially when there is evidence of carcinogenic potential in the product class, structure–activity relationship, positive genotoxicity findings, evidence of preneoplastic lesions in repeat-dose toxicity studies, and long-term tissue retention of the pharmaceutical or its metabolite(s) disrupting tissue homeostasis (ICH S1A guideline). The guidelines for determining the need for rodent carcinogenicity studies for environmental exposures are different since they can potentially affect all life forms including humans, and animals in various ecological niches. For high production volume (HPV) chemicals (>1 million pounds in the United States or in the OECD >1000 tons) and chemicals/agents for which widespread environmental exposures are suspected, the rodent carcinogenicity studies are needed to assess the carcinogenic hazard associated with these exposures. Of course, not every HPV chemical is a carcinogen, so epidemiological studies and exposure data (occupational and lifestyle) usually provide a justification for carcinogenicity testing (Pandiri et al., 2017).

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The modern assessment process will include a variable mix of in vitro and in vivo studies to explore genotoxicity and chronic toxicity, including lifetime (2-year) carcinogenicity studies in rats, 2-year studies in conventional mice or 6-month carcinogenicity studies in genetically modified mice (for pharmaceuticals only), and, in many cases, shorter studies to address potential MOA. There are exceptions to the need for, or the feasibility of, conducting lifetime rodent carcinogenicity studies. Anticancer drugs and drugs for short-term administration in humans may not be tested in carcinogenicity studies in animals because the risk of druginduced neoplasia is very low and therefore the risk is acceptable to treat the medical condition. Carcinogenicity assessment of recombinant therapeutic proteins uses a WoE approach and may not include rodent carcinogenicity studies for a variety of factors (ICH S6(R1)). Although alternative models (rasH2 and p53þ/ mice) are now accepted for human pharmaceutical risk assessment, they are not yet accepted for veterinary drugs, food additives, and environmental chemicals in the absence of a 2-year rodent study. The conduct of carcinogenicity studies in rodents to support the registration of human pharmaceuticals can be broken down into three phases. The first is the design of the protocol, including negotiations to obtain FDA agreement to the study design. The second is the study execution phase. The third phase includes the analysis, interpretation, and reporting of the study results. This section will focus on the planning steps for conducting 2-year lifetime and 6-month alternative carcinogenicity studies in rodents to support the approval of a new chemical entity (NCE, i.e., a nonbiological, small molecule) as a human drug (see below for special issues to consider in carcinogenicity testing as applied to protein-based, nucleic acid–based, and stem cell–based therapeutics, and medical devices). It will include the procedures expected by the FDA’s Center for Drug Evaluation and Research (FDA-CDER). The FDA will review study designs and dose selection for studies based upon appropriate range-finding data and make recommendations on dose selection. If the sponsor and the FDA agree on the study design and dose selection, the FDA will accept the study results even if the maximum tolerated dose (MTD) is exceeded and high mortality occurs in

one or more treatment groups. Many of the concepts and procedures described for small molecules in the United States will apply to other regulatory agencies worldwide and also to other categories of product development. However, some aspects will be specific to prescription drugs and their passage through the US regulatory process.

2.1. Prerequisite Data to Design a Carcinogenicity Study Protocol Table 5.1 lists the various data sets required for study design for new drug entities. These data are useful both in providing sound scientific justification for study design and also in assuring eventual regulatory acceptance of the sponsoring institution’s carcinogenicity assessment plan. Many of these data sets must be completed prior to initiating the carcinogenicity testing phase. Accordingly, pathologists and toxicologists who engage in the design, conduct, analysis, and interpretation of carcinogenicity studies ideally should be given early access to this information. • Test Article Data. The batch(es) of test article to be used in the carcinogenicity assessment should be analyzed. The impurity profile of the test article should be compared to that of the product intended for human use. This allows for the qualification of impurities in the eventual marketed product. Ideally, the impurity profile of the test article used in the carcinogenicity studies will be the same as that of the marketed product, and the impurity exposure in the animal studies should be comparable to the exposure in humans at the highest approved dose. • Pharmacokinetics and Metabolism Data. In vivo pharmacokinetics used to plan carcinogenicity studies should be characterized in mouse, rat, and humans. Specifically, the human maximum concentration (Cmax) and area under the curve (AUC) for the parent drug candidate and any significant metabolites at the highest dose to be marketed will be used to calculate the exposure levels to be sought in the rodent carcinogenicity studies. In vitro plasma protein binding should be measured in mouse, rat, and human blood to facilitate comparisons

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of free plasma exposures to drug and major metabolites, thus enabling estimation of levels of unbound chemicals that will be available to bind with the intended target at each of the rodent dose levels. In some cases, drug plasma protein binding is similar across species. In other cases, there can be significant differences in plasma protein binding across species. This disparity may be critical to the dose justification argument. • Absorption, Distribution, Metabolism, and Excretion (ADME) Data. The tissue distribution and metabolite production should be assessed in rodents that have been given radiolabeled parent compound. These studies will determine if there is any accumulation of label in any site (i.e., potential target organs) and identify the major metabolites produced. In addition, human radiolabeled metabolite characterization should be conducted. The rodent metabolites can then be compared to those identified in humans to determine whether or not all human metabolites are also present in rodents. This information will support the appropriateness of the rodent species/strain selected for assessing the carcinogenic risk of the drug candidate (and its major metabolites) to humans. • Genetic Toxicology Data. The potential for the test article to produce genetic mutations and/or chromosomal aberrations should be evaluated in a battery of in vitro and in vivo tests. The composition of the battery usually will include an in vitro bacterial mutation assay (e.g., the Ames test), an in vitro human cytogenetics assay, and an in vivo cytogenetics assay (often done as an evaluation of bone marrow micronuclei as part of the first GLP rodent toxicity study or as a separate study). • Dose Range–Finding Study Data: The minimum dose range–finding study to support a 2-year rodent carcinogenicity study should be 3 months in duration and utilize the intended clinical route of exposure. If the outcomes of longer studies are available, such as a 6-month study, the resulting data should be included when the dose selection proposal is submitted for review by regulatory authorities. Key endpoints from range-finding studies for consideration in carcinogenicity study design should include mortality, clinical signs, body weight changes, and target organs effects.

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Range-finding studies of 4 weeks duration are generally appropriate for 6-month alternative carcinogenicity studies in engineered mouse models. These range-finding studies can be conducted in the wild-type littermates of the appropriate genetically modified mouse model. Although there is some train of thought (based on greater toxicity sensitivity related to smaller body size) that rangefinding studies for rasH2 carcinogenicity studies should use transgenic rasH2 mice rather than the wild-type B6C3F1 strain (Paranjpe et al., 2017), the vast majority (79%) of range-finding studies use the wild-type mice and most have been found to have a similar toxicity and exposure profile as that observed in the 6-month transgenic studies (Bogdanffy et al., 2020). • Dose Selection and Rationale. The dose selection for the 2-year cancer study is critical in establishing the translational relevance to human health risk and is generally based on the toxicity data from the subchronic studies. If the doses are too low, the animals might be insufficiently challenged leading to a falsenegative response and if the doses are too high, it might overwhelm physiologic homeostasis resulting in a false-positive response. The lowest dose should also receive considerable attention since it should attempt to reflect environmental exposures or clinically relevant exposures and aid in the determination of NOAELs. Due to the inherent limitations of the statistical power of a bioassay that typically employs w50 animals/dose group, it is essential to maximize the sensitivity of the bioassay and make sure that the animals are challenged to the maximum extent without causing overt toxicity that impacts the natural lifespan of the animal on the study. It is also important that the animals be challenged at the MTD to give confidence in case of a negative study. The maximum tolerated dose (MTD) is the most common approach used to justify the upper dose in a rodent carcinogenicity study. Simply, it is the highest dose that is thought to be well tolerated by rodents over the duration of the study. The MTD is defined by evidence of increased mortality, body weight changes, and/ or target organ toxicity. For example, if mortality

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is observed in the high-dose group in the rangefinding study, the high dose recommended in the carcinogenicity bioassay should be one-third to half of the lowest dose associated with treatment-related mortality. The choice of using one-third or half of a mortality-inducing dose is based on the duration of the range-finding study; a 3-month range-finding study typically can support a one-third factor, while a 6-month study can support the half factor. Complicating issues include the time-course for the mortality and whether the cause of death is understood. Body weight (BW) or body weight gain (BWG) can influence the choice of the doses. For example, if there is no mortality in a rangefinding study, any dose that produced a BWG decrease of >10% in rats in a 3-month rangefinder would likely be unsuitable for a high dose in a 2-year carcinogenicity study. A BW decrease (not a BWG decrease) of >10% compared to controls in rats in a 6-month study would also likely be unsuitable. The highest dose that does not exceed these limits would be considered the MTD. In general, the doses in the short-term repeat-dose studies should be spaced in such a way that an NOAEL, the MTD, and a dose–response curve may be generated. The animals should be exposed by the most appropriate route of exposure that is relevant to the human exposure scenario. Effects specific to certain routes of exposures, such as gavage versus dietary or drinking water exposures, should also be taken into consideration as they determine the toxicokinetics/toxicodynamics of the chemical exposure. Target organ toxicity can influence the choice of doses, especially in those cases where there is no mortality or BW change. The key determinant will be whether the target organ changes observed in the dose range–finding study might be incompatible with survival over the course of the 2-year study. Examples might include toxicity to viscera (e.g., kidney, liver, or digestive tract) or any adverse effects due to the route of exposure (e.g., changes in the lung if by inhalation, or skin if topical). The 25
Clinical AUC approach, which sets the high dose as one that produces a rodent AUC that is > 25
the human clinical AUC, is an acceptable dose-setting method for 2-year carcinogenicity studies, whether genetic toxicology results are positive or negative. This approach

is usually utilized only when the clinical dose range is well defined. The 25
clinical AUC approach is not currently acceptable for the 6month alternative carcinogenicity studies in engineered mice (see Section 1.6, Looking Forward: ICH Guideline S1B Modifications). Many drugs cannot use the 25
clinical AUC because excessive pharmacology (rather than toxicity per se) at such an elevated exposure will not be tolerated. Extended periods of supraphysiological activity may be considered to exceed the MTD. The Limit Dose approach may be used for compounds that have very low toxicity and are very well tolerated. This strategy uses a high dose of 1500 mg/kg/day. It is considered appropriate if the human clinical dose is  500 mg/ day, the drug is not genotoxic, and this dose produces a rodent AUC that is  10
the human AUC. The Saturation of Systemic Exposure approach occurs with some drugs and can be used to justify the high dose. If the administration of higher doses has been shown not to increase exposure (perhaps due to rate-limiting absorption), there is little sense in administering higher doses to assess systemic carcinogenic risk. The Maximum Feasible Dose (MFD) approach can be employed in dose-setting if dosage is limited by physical criteria of the test agent such as solubility, suspendability, stability, aerosol generation limit, and also physiological limits of the animal such as 1% incidence) or rare (1% incidence) in the particular strain of rodent. Lin et al. however, noted an overall falsepositive rate of approximately 10% when utilizing these decision rules based on levels of significance for drug effects on individual tumor types (Lin and Rahman, 2018). The Society of Toxicologic Pathology Peto Working Group also found limitations with

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TABLE 5.2

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Example of a Study Outline for a Rat Lifetime (2-Year) Carcinogenicity Bioassay

Sprague Dawley or Wistar Han IGS (CRL:WI [Han]) Age at initial dose: 6e8 weeks old Route: Oral (gavage or in diet) Frequency: Once daily by gavage, unless otherwise justified; ad libitum if administered in diet Groups: 4 (1 vehicle control group þ 3 treated groups [low, mid, and high]) Animals/sex/group at initial dose: • Sprague Dawley, 60e70 in all dose groups (due to higher incidence of early death in this stock) • CRL:WI(Han), 50 in all dose groups In-life observations: • Clinical signs: Once weekly • Morbidity: Twice daily, early and near the end of the day • Palpable masses: Once weekly from month 6 • Ophthalmology: Pretest and at 1 year • Body weights: Predose day 1, weekly for 6 months, then monthly • Food consumption: Weekly for 6 months, then monthly • Toxicokinetics: Near week 26, minimum of 3 time points; 3 sex/dose/time point Postmortem endpoints: • Full gross evaluation • Full histopathological evaluation • Clinical pathology and ophthalmic assessment only if warranted by specific, previously identified findings Statistics: • Consult an experienced biostatistician in advance • A trend test and pairwise comparison tests often are used to compare tumor incidences • The Peto test or an alternative method may be used to adjust for time from initiation of treatment to the first observation of each neoplastic process and early mortality in treatment groups, if present (see Experimental Design and Statistical Analysis for Toxicologic Pathologists, Vol 1, Chap 16) Reproduced from Haschek’s and Rousseaux’s Handbook of Toxicologic Pathology, Vol 3, Table 27.2, p. 821 with permission.

the draft guidance and recommended, for example, that only one test should be routinely required which would usually be the trend test (Morton, 2001). An outline of the design of a typical 2-year rat carcinogenicity study is provided in Table 5.2.

3.2. Managing High Mortality in 2-Year Carcinogenicity Studies Lower than expected survival in 2-year carcinogenicity studies can confound analysis and interpretation of the results (Long, 2004; Roth et al., 2007). Regulatory guidelines have been published addressing appropriate responses to such situations, but there are inconsistencies, gaps,

and ambiguities in available regulatory guidance. When low survival is observed in one or more groups in an ongoing carcinogenicity study supporting pharmaceutical development, the FDA encourages the sponsor to consult with the FDA regarding appropriate action. It generally is not practical to specify in detail how low survival will be handled in study protocols because the specific circumstances, the most appropriate response, and the advice of regulatory authorities will vary on a case-by-case basis. For studies supporting pharmaceutical registration, the primary reviewing division of the US FDA should be contacted when survival approaches 50% in one sex and group to permit adequate time for negotiating an action plan. Although not mentioned in published guidance,

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the FDA has consistently recommended termination when survival in one sex in one treated group drops to 15 animals (from 50 to 70 at the start). Once a course of action has been determined, the study protocol should be amended to incorporate the designated procedures. Based on the previous authors’ experience (Morton et al., third edition), the FDA’s standard responses are generally consistent and are summarized below. These guidelines may be appropriate for carcinogenicity studies involving chemicals other than pharmaceuticals. Statistical considerations related to managing early termination of specific groups for low survival have been reviewed elsewhere (Roth et al., 2007). Low Survival in Treated Groups • If survival in one sex of the high-dose group drops to 20 prior to Week 100, treatment of this dose and sex may be halted to extend survival of the remaining animals. • If survival in one sex in any treated group (not controls) drops to 15 prior to Week 100, the affected sex in the affected dose group should be terminated. This includes high-dose animals after cessation of treatment. The FDA typically has not recommended terminating any of the control animals when an individual group treated with test article is euthanized for low survival prior to week 100. • If survival in one sex in any treated group (not controls) drops to 15 during or after Week 100, terminate the affected sex in all dose groups. Low Survival in the Control Groups Low survival in the control group(s) has serious implications. If it occurs quite early in the study, the validity of the entire study may be questioned. • If survival in one sex of a control group drops to 10 multiples, so that if measured exposures result in multiples that are less than predicted, they are still likely to meet the 10 margin. This often leads to doses that target 15–20 of the highest intended clinical exposure when there is more uncertainty about the modeling, predictions, and/or clinical plan. From the standpoint of humane animal care and use, it should be emphasized that doses/exposures should not exceed any known maximum tolerated dose/ exposure in the test species unless there is a clear justification for such a strategy. Finally, if the high dose is based on a maximum feasible dose, the rationale why higher doses are not possible should be clearly articulated. Figure 6.3 shows an algorithm that can be used in high-dose FIGURE 6.3 Selection of high dose for toxicity studies with protein therapeutics. *Adjustments are usually not necessary if the highest dose and exposure are derived from the maximum pharmacologic effect in the test species, as higher doses or exposures would not be expected to have any greater PD effect. PD, pharmacodynamic; MTD, maximum tolerated dose. From Leach MW: Antibodies (abs) and related products containing complementarity-determining regions (CDRs). Chapter 20. In Cavagnaro J, Cosenza ME, editors: Translational medicine. Optimizing preclinical safety evaluation of biopharmaceuticals, 1st ed., Boca Raton, 2021, CRC Press.

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selection for any given study based on the most current information available from the program. Experience has shown that high doses of protein in the context of toxicity studies can be administered to animals for long periods of time. Therefore, there is currently no rationale to limit the high dose based on concerns about protein overload. One minor finding that may occur at higher mAb doses and exposures is slightly higher total protein and slightly to mildly higher globulins (with subsequent lower albumin:globulin ratio) during standard clinical chemistry evaluations. The amount of change is generally similar to the amount of test article that is present in the serum, although experience suggests an exact match should not be expected. In general, the low dose should be targeted to match around 1–2 of the highest planned clinical exposure, and the intermediate dose is selected to further characterize the range of toxicity between the high and low doses, usually a geometric mean (e.g., 10, 30, and 100 mg/kg). In cases where adverse toxicity may be a concern at the high dose and clinical doses may be based on the intermediate dose, it may be useful to consider whether the intermediate dose will provide a sufficient margin and modify if needed. As with the high dose, adjustments may be needed for differences in affinity/ potency. When using different dose routes, it may be appropriate to target the geometric mean of the exposure (or some modification if necessary based on expected toxicity), not the dose in mg/kg [e.g., 25 SC, 30 IV, and 100 IV in mg/kg might provide a reasonable range of exposures because the SC exposure is likely well below the IV exposure]. Input from PK modeling is essential. Although common historically, the same dose or exposure should usually not be used to test different routes of administration unless there is a clear justification (e.g., avoid having two high-dose groups, with one using the IV route and the other using the SC route; instead only use one route). Involvement of the clinical team is recommended to ensure appropriate dose selection. It is likely that the highest planned clinical dose will change over time, and that possibility should be considered in study designs; it may be appropriate to use lower doses in a chronic toxicity study compared with earlier studies, for example, if the clinical dose/exposure is lower, or raise them if the clinical dose/exposure is higher.

For studies where there is no intermediate dose, as might occur with nontoxic test articles, it is recommended that the high dose be determined as described above, and the low dose be 2–4 the highest planned clinical dose. This provides some exposure multiple if adverse findings are seen in the high-dose group.

6.6. Dosing Interval The dosing interval should usually be the same or more frequent in the nonclinical toxicity studies compared with the clinical trial dosing interval. Because the half-lives of molecules with an Fc that bind the FcRn are typically long (2 weeks or more in humans) due to recycling of the molecule back into the circulation (Ovacik and Lin, 2018), humans are usually dosed once weekly or less frequently with Fc-containing molecules (although it should be recognized that some Fc-containing molecules have shorter half-lives). For these longer half-life molecules, animals are often dosed weekly or once every other week. It should be noted that less frequent dosing of animals (less than once a week) with mAbs and related molecules has sometimes been associated with increased immunogenicity (Leach et al., 2014), and for that reason many study designs with mAbs and related products will often dose weekly even if clinical dosing is less frequent. It is also important to recognize that the dosing interval will impact the exposure and therefore the dose that is needed in mg/kg.

6.7. Route of Administration, Formulation Generally, the route of administration in nonclinical toxicity studies should be based on the planned clinical route(s) that will be tested (e.g., IV, SC, intrathecal, etc.). For FIH-enabling toxicity studies, if only one route will be tested in the clinic, then the toxicity study(ies) should usually use only that route (unless the clinical route does not provide sufficient systemic exposure, and such exposure is deemed necessary). If more than one route will be tested in the FIH-enabling study (e.g., SC and IV), then the low-dose group receiving the test article should receive the drug by the route most likely to be the final commercial route of administration. The high-dose group should receive the drug by the IV route to maximize exposure. The

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intermediate-dose group can be SC or IV. If cardiovascular (CV) safety pharmacology endpoints are included in the intermediate-dose group, then the IV route is preferred. In a single control group, individual animals should receive the vehicle by all the routes being tested whenever possible to minimize animal use (e.g., they might receive both SC and IV doses). Post-FIHenabling studies should also use the planned clinical route(s) that will be tested. When feasible and available, the clinical formulation should be used in nonclinical toxicity studies. However, the clinical formulation is not always available, especially for FIH-enabling toxicity studies. The IV route in particular may result in infusion reactions due to various mechanisms (Mease et al., 2017). Moving from an IV bolus to an IV infusion where the dose is given over a longer time (e.g., 30 min, 1 h, etc.) can often reduce the reactions and may allow dosing to continue (Mease et al., 2017). This does add some logistical challenges to the study, but IV infusion can usually be accomplished technically. There is a belief that SC administration may result in greater induction of immunogenicity compared with the IV route, in part because the test article will be delivered to local lymph nodes, which, coupled with migration of local antigen presenting dendritic cells to local lymph nodes, is thought to potentially enhance an immune reaction (Fathallah et al., 2013; Hamuro et al., 2017; Turner and Balu-Iyer, 2018). In practical experience, some studies show more immunogenicity by the SC route, others show more immunogenicity by the IV route, and still others suggest no difference (reviewed in Hamuro et al., 2017, and Davda et al., 2019). Thus, there is not clear data at the present time to generalize that SC administration is more likely to result in greater immunogenicity compared with the IV route. That said, if data are available on a given test article that suggests greater immunogenicity by a certain route, it should be considered when designing future studies in that specific species.

6.8. Recovery According to ICH S6(R1) (2011), recovery from pharmacological and toxicological effects with potential clinical impact should be understood. Historically recovery phases were also

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conducted to assess delayed toxicity or potential immunogenicity; however, ICH S6(R1) (2011) clearly states these are not appropriate reasons, and the purpose of the recovery phase is to understand reversibility of effects seen during the dosing phase. ICH S6(R1) (2011) also states that reversibility of clinically-relevant effects should usually be demonstrated in at least one study, be evaluated using at least one dose, and demonstration of complete reversibility is not essential. In most cases, recovery groups should only be needed in one study or one study duration (if testing in two species). In some cases, recovery groups may not be necessary on any study (e.g., no toxicity suspected, no significant concerns with the target, reversibility of the finding is well understood). A decision on whether to include recovery groups, and on which study or studies, and at what doses should be determined based on program needs and existing data on a case-by-case basis. The default should not be to include recovery groups in all dose groups, or on all studies. For many biologics, recovery groups have not provided impactful information (Sewell et al., 2014). Recovery phases are not usually added to exploratory studies if these studies are conducted. However, if exploratory studies are conducted, the study design may include a prolonged period after the last dose to assess PK and/or evaluate a prolonged PD effect (especially with long half-life molecules such as mAbs); this may provide some assessment of recovery in these studies. There is an ongoing debate about whether it is better to include recovery phases on FIH-enabling toxicity studies, or on chronic studies, if recovery groups are considered necessary (Pandher et al., 2012; Perry et al., 2013; Sewell et al., 2014). There are pros and cons with both approaches, and ultimately the project team should determine what is most appropriate. From a practical standpoint, one may not know if recovery animals are really needed until the study is underway. Key questions when determining whether recovery is needed on a study include: (1) is the test article predicted to produce an adverse effect at clinically meaningful exposures, (2) has reversibility already been adequately demonstrated in another study, and (3) can an assessment of recovery be made based on knowledge of the finding(s) without using recovery animals

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(Perry et al., 2013). When included, recovery animals are usually in the control and highdose groups, but it is also possible to instead assess recovery in the intermediate-dose group if there is concern that toxicity in the high-dose group may be too great (e.g., if toxicity has not yet been well characterized, which is more likely in FIH-enabling toxicity studies). Because ICH S6(R1) (2011) states that recovery should be understood at clinically-relevant exposures, it is not necessarily a requirement to have recovery in the high-dose group. The length of the recovery phase should allow enough time for an assessment of reversibility and should consider the half-life of the drug and the expected duration of the PD effects, with longer half-life drugs and/or those with longer lasting PD effects requiring longer duration recovery phases. A common mistake with Abs and related products with a long half-life is to have a recovery phase that is too short. The recovery duration should be scientifically based on when the expected test article concentration has gone below the concentration at which pharmacology is expected, and then allowing time for recovery of any effects; this may be many months for potent molecules given at high doses. Assessing recovery in the intermediate- or even lowdose group (vs. the high-dose group) may reduce the duration of the recovery phase needed, provided these groups have exposures that still meet or exceed clinical exposures. In cases where there is an acceptable biomarker available (e.g., B cells for a B cell-depleting drug), the length of the recovery phase could be based on recovery of the biomarker using an adaptive type of study design where the recovery phase might end when the biomarker has demonstrated recovery to certain values identified in the protocol. In cases where there is no biomarker, one may consider empirically targeting approximately five half-lives. Conducting a recovery phase that is too short will not provide an assessment of reversibility, could lead to misinterpreting the likelihood of recovery, and does not represent appropriate animal use. If no toxicity is observed after the dosing phase, it may be possible to reduce data collections or even terminate the recovery phase early (Pandher et al., 2012). However, from a practical standpoint, because dosing phase data are often not available until close to the end of the

recovery phase, and it is “better safe than sorry,” the recovery phase usually continues up through collection of tissue at necropsy so that all information is potentially available. After the recovery phase, it is only necessary to microscopically examine tissues from the target organs identified in the dosing phase in both rodent and nonrodent studies, unless clinical observations, clinical pathology, or necropsy data suggest otherwise. If there are no test article-related microscopic findings at the end of the dosing phase, and no test article-related clinical signs were noted during the recovery phase, then it may not be necessary to microscopically examine any tissues from the recovery phase animals. There are also disussions in industry about whether control monkeys from the recovery phase could be returned to the colony for other uses if there is no need to microscopically evalaute the recovery phase animals and this decision can be made in time (usually more feasible with longer recovery phases).

7. IMMUNOTOXICITY 7.1. Overview Evaluating the intended and unintended effects on the immune system by test articles, including protein therapeutics, is an important aspect of drug development. There is currently limited approved guidance on assessing immunotoxicity of protein therapeutics from a regulatory perspective, as the ICH guidance directly covering immunotoxicity “does not apply to biotechnology-derived pharmaceutical products” and was finalized over 15 years ago (ICH S8, 2005), ICH S6(R1) (2011) mentions immunotoxicity only briefly, and an approved FDA guidance also excludes biologics (FDA, 2002). Immunotoxicity assessments are only briefly mentioned in EMA/EMEA guidances (EMEA, 2000; EMA, 2007). The USFDA has a draft guidance that does cover biologic products (USFDA, 2020), but the final version is not available at present time. Although clear regulatory guidance does not presently exist, a number of publications addressing the immunotoxicity of biologics have been published from industry and academia (Descotes, 2009; Brennan et al., 2010; Lebrec, 2013; Sathish et al., 2013).

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Immunotoxicity with protein therapeutics can be classified in various ways, but at a high level typically includes immunosuppressive or immunostimulatory effects, and those related to immunogenicity (Descotes, 2009; Brennan et al., 2010; Lebrec, 2013); immunogenicity can also be considered a cause of immunostimulation (Evans, 2014). Immunogenicity is covered in Section 4 of this chapter. Immunosuppressive and immunostimulatory effects are often related to the intended pharmacology of the test article. Adverse immunostimulation has also been reported due to impurities in biopharmaceuticals (Reijers et al., 2019). An assessment of potential immunotoxicity should be begin with a thorough understanding of the test article biology, the intended pharmacology, whether an Fc (or part of an Fc) is present, what modifications have been made to alter halflife, and what types of effector functions (if a mAb or related molecule) are likely (Brennan et al., 2010). For example, a mAb with enhanced ADCC to kill tumor cells more effectively could also kill normal immune cells that express the target antigen, resulting in immunotoxicity. In contrast, a test article lacking an Fc that targets the same molecule would have a short half-life and lack effector function, making it less likely to directly kill unintended immune cells and result in immunotoxicity. After the biology of the molecule is determined, decisions can be made regarding what additional assays are needed, if any.

7.2. In Vitro Assays for Assessing Immunotoxicity The immunologic properties of molecules with an Fc should be thoroughly evaluated (ICH S6(R1), 2011). Test articles that contain an Fc should be evaluated for binding to Fcg receptors and complement. The Fc functions in the test article may be wild-type, or via molecular engineering they may be enhanced, diminished, or eliminated (Wang et al., 2018, see also Section 2). If there is binding to Fcg receptors and/or complement, the activity should be further characterized in some manner. These types of analyses are often conducted during the engineering of the molecule in discovery. Additional functional assays for ADCC, ADCP, and/or CDC may be

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conducted. Overall, these assays can provide information about likely effects on the immune system, which can translate into intended pharmacology, exaggerated pharmacology, and potential off-target unintended immune effects. Cytokine release assays (CRAs) should be considered for certain molecules where there is a reasonable concern about cytokine release based on expected pharmacology, but should not be conducted as a default strategy. If the test article binds to immune cells, or is designed to stimulate the immune system, then CRAs may be appropriate. Alternatively, if a test article is designed to bind to a soluble mediator not involved in immune stimulation, CRAs are likely not necessary. CRAs are currently best considered as a tool for hazard identification, and not for risk assessment (Grimaldi et al., 2016). While historically there have been a variety of assays available to assess cytokine release and standardization has been lacking (Grimaldi et al., 2016), efforts are underway across industry to better standardize CRA platforms (Vessillier et al., 2020).

7.3. In Vivo Assays for Assessing Immunotoxicity A wide range of in vivo assays are possible in rodents and large animals to test for immunotoxicity. Clinical signs, clinical pathology assessments (hematology and clinical chemistry), organ weights (thymus, spleen, and possibly lymph nodes [lymph node weights can be particularly variable and potentially misleading]), and microscopic evaluation of lymphoid tissues (thymus, spleen, lymph nodes, bone marrow, gut-associated lymphoid tissue [GALT], etc.) in toxicity studies should be considered as baseline evaluations; similar assessments can also be conducted in efficacy studies (Brennan et al., 2010). Additional assays or studies should only be included if necessary, and many of these can be included in standard toxicity studies. For example, it is possible to collect peripheral blood for assessment of complement activation (e.g., C3a, C4a, C5b-9, Bb) and cytokine concentrations (e.g., IL6, TNF, INFg). Immunophenotyping of peripheral blood, lymphoid tissues, or bone marrow can be used to reveal changes in cell surface markers and cell numbers (Lebrec, 2013). Assessment of the T-dependent antibody response can

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be added to general toxicity studies (Lebrec, 2013). It is important to note that some assays may require addition of more animals to the study groups, as some assays may require samples that cannot be collected from main study animals or may require sampling throughout a study. This may be especially true for rodents due to their smaller size. Adding animals will require additional test article.

7.4. Immunosuppression The primary concerns with immunosuppression are increased risk of infections and tumor development, which can occur in animals or humans (Descotes, 2009; Brennan et al., 2010; Hutto, 2010; Sathish et al., 2013). A variety of pathogens including bacteria, viruses, and fungi can be involved in infections. Cell proliferation and tumors may be virally induced. Evidence of immunosuppression may be seen in in vivo efficacy or toxicity studies, with clinical, clinicopathologic, macroscopic, and/or microscopic evidence of infection, cell proliferation, or tumors. Alternatively, more subtle effects such as decreased cellularity in tissues such as lymph nodes, spleen, thymus, or bone marrow may be observed (Elmore, 2012). It is uncommon that all animals in a given test article-dosed group will be affected in a similar manner. Due to the variability and low incidence of infections in nonclinical toxicity studies, it may not be possible to derisk the potential for functional immune suppression that could lead to opportunistic infections or viral recrudescence in humans. It is often necessary to continue to monitor for these events in the clinic, particularly if the pharmacology of the test article and/or target warrants.

7.5. Immunostimulation Immunostimulation can be the direct result of intended pharmacology (e.g., killing of normal cells that express the target antigen by T cells stimulated via T cell-recruiting bispecific molecules), or it can be secondary to more general immune stimulation (e.g., autoimmune-like conditions associated with blockade of checkpoint inhibitors like anti-PD-1 and anti-CTLA-4), although normal animals may underpredict toxicities observed in humans, especially if given as monotherapies (Keler et al., 2003; Wang et al.,

2014; Selby et al., 2016; Ji et al., 2019). Toxicities related to immunostimulation are particularly common with immuno-oncology molecules, which are designed to stimulate the immune system to respond against cancers (Shimabukuro-Vornhagen et al., 2018; Ji et al., 2019). Mechanistically, a test article can cause immunostimulation from cytokine release (including cytokine release syndrome or cytokine storm), immune cell proliferation and activation, direct complement activation, and effector activity related to Fc receptor binding (i.e., ADCC, ADCP) or complement activation (CDC) (Brennan et al., 2010; Evans, 2014; Ji et al., 2019). One or more mechanisms may be operative at the same time. A wide variety of potential negative effects are possible including infusion reactions, leukocytosis, inflammatory cell infiltrates in tissues (including heart, colon, liver, salivary glands, and endocrine organs), and autoimmune-type syndromes (Descotes, 2009; Evans, 2014; Ji et al., 2019). As discussed in Section 4.4 Assessing Immunogenicity, it is necessary, though potentially difficult, to differentiate test article-related immunostimulatory effects, which have potential relevance to humans, from immunogenicity, which is not predictive for humans. In these cases, additional investigations may be warranted and may include trying to further understand the pharmacology of the test article and target expression, determine the presence of ADA in the study, understand the microscopic appearance and localization of the findings, and/or find correlative findings suggesting primary pharmacology versus ICD. Additional investigative work (EM, IHC) with the affected tissues to evaluate for evidence of IC deposition may also be informative.

7.6. Program Strategies and Regulatory Considerations From a regulatory standpoint, ICH S6(R1) (2011) states that routine tiered testing approaches or standard testing batteries are not recommended for evaluating immunotoxicity. Immunotoxicity can be assessed in standard toxicity studies by evaluating routine assessments including clinical signs, hematology, organ weights, and microscopic examination of tissues. If additional assessments or studies are under

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consideration, scientists should carefully determine whether they will truly add value to the safety assessment, as knowledge of the mechanism of action of the test article often provides such information (e.g., one probably does not need additional studies with anti-TNF molecules to know they will be immunosuppressive in humans), and it may take studies in large numbers of humans to fully understand potential risk, making additional nonclinical work unnecessary. If additional in vitro or in vivo assays are needed, they should be carefully selected. It is easy to run numerous assays but interpreting the data can be difficult due to variability (Lebrec, 2013). When considered necessary, it is recommended that in vitro assays including CRAs be conducted before in vivo toxicity studies are conducted, so that they can inform the in vivo study designs.

8. SAFETY PHARMACOLOGY ASSESSMENTS 8.1. Overview In addition to ICH S6(R1) (2011) and ICH S9 (2009), ICH S7A (2000) provides supplementary guidance on safety pharmacology studies for human biopharmaceuticals. In accordance with regulatory requirements, assessments of CV, central nervous system (CNS), and pulmonary function must be performed prior to FIH; this group of assessments is often termed the core battery. It should be noted that there are currently questions about whether respiratory safety pharmacology studies should remain as part of the core battery for all therapeutic modalities (small molecules, biotherapeutics, etc.) based on a perceived lack of value (Paglialunga et al., 2019). In vitro CV studies, such as hERG assessments, are not considered appropriate as protein therapeutics are generally specific in their targeting, and in addition they are not able to access the inner pore of the hERG channel to exert an effect (Vargas et al., 2008; ICH S6(R1), 2011; Vargas et al., 2013). Safety pharmacology core battery endpoints can usually be added to general toxicity studies (Vargas et al., 2013; ICH S6(R1), 2011; ICH S7A,

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2000), and standalone studies are typically not needed and are not an appropriate use of animals. However, if the target is known or suspected to be associated with safety pharmacology risks, standalone studies may be appropriate if sufficient information cannot be collected in general toxicity studies (Vargas et al., 2013). Rodents should be used for standalone safety pharmacology studies if they are pharmacologically relevant and the necessary assays can be conducted. Beyond the core battery, the gastrointestinal and renal systems are also sometimes evaluated, especially if they are likely targets of the therapeutic effect; these assessments usually require specific, standalone studies. Table 6.8 shows some considerations for safety pharmacology core battery assessments for lower and higher risk test articles in FIHenabling general toxicity studies.

8.2. Strategy and Timing It is strongly recommended that appropriate safety pharmacology strategies based upon the perceived level of risk be in place prior to planning the first in vivo toxicity studies. For programs with a high level of concern, for example, where safety pharmacology findings might terminate the program, relevant safety pharmacology assessments may be conducted prior to non-GLP exploratory studies, or as part of these studies. When there is no perceived risk, assessments can be added to the FIHenabling general toxicity study. If no signals of concern are seen, additional safety pharmacology assessments are not usually needed in chronic studies. If standalone studies are conducted and the duration is longer than 5 days, it is recommended that samples for ADA be collected in case some assessment of immunogenicity is needed. Because of the potential for immunogenicity, and long half-lives with mAbs, crossover and dose escalation designs are usually not used. In addition, animals should not have been previously administered biotherapeutics unless they are tested and found negative for ADA. Finally, due to potential development of ADA, it is recommended that safety pharmacology assessments be conducted not only prior to and at the

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TABLE 6.8 Selected Considerations for in Vivo Safety Pharmacology Assessments With Protein Therapeuticsa in FIHEnabling Toxicity Studies to Meet Regulatory Guidelines

Assessment

Lower risk test articles

Higher risk test articles (conduct studies below in addition to assessments listed for lower risk molecules)

Cardiovascular

Surface lead ECG in restrained large animal (usually monkey) at baseline and end of study. Because of concerns about impact of ADA, data may also be collected on w Day 2, prior to potential development of ADA.

Consider incorporation of implanted telemetry evaluation onto select dose groups as part of an FIH-enabling GLP large animal toxicity study. Standalone single-dose telemetry study may be warranted; conduct in rodent if pharmacologically relevant.

Central Nervous

Body temperature and appropriately timed standard clinical observations in rodent and large animal studies, if pharmacologically relevant.

Consider detailed clinical neurological exam (for monkeys) or functional observational battery/Irwin test (for rodents if pharmacologically relevant).

Respiratoryb

Clinical signs of respiration in large animal.

Consider whole body plethysmography assessment in rodent study if pharmacologically relevant.

a

If a protein therapeutic is conjugated to a small molecule(s), these may necessitate a more traditional small molecule safety pharmacology assessment of linkers and payloads. b The need for respiratory assessments is currently being reconsidered by the pharmaceutical industry and regulatory agencies. ADA, anti-drug antibody; ECG, electrocardiogram; FIH, first-in-human; GLP, good laboratory practice.

end of the study, but also on w Day 2 when exposures are likely high and before potential development of ADA.

9. DEVELOPMENTAL AND REPRODUCTIVE TOXICITY STUDIES AND JUVENILE ANIMAL STUDIES 9.1. Overview ICH M3(R2) (2009), ICH S5(R3) (2020), ICH S6(R1) (2011), and ICH S9 (2009) all refer in some regard to DART studies, and ICH S11 (2020) refers to Juvenile Animal Studies (JAS) (see The Role of Pathology in Evaluation of Reproductive, Developmental, and Juvenile Toxicity, Vol 1, Chap 7). An assessment of whether the test article can likely cross the placenta in the test species should be done. While many proteins are typically too large to diffuse across the placenta, some molecules may have receptors to transport them. Molecules with an Fc can bind to the Fc receptors on the placenta, in particular FcRn, allowing active transport across the placenta by receptor-mediated transcytosis

(Roopenian and Akilah, 2007; DeSesso et al., 2012). This is the same receptor responsible for the long half-life of Fc-containing biotherapeutics, as discussed above. It is known that Fc-containing biotherapeutics can cross the placenta in mice, rats, rabbits, and cynomolgus monkeys, although the timing of exposure (in terms of development) varies between species (Bowman et al., 2013). Differences in exposure based on isotype have also been noted, with greater transfer of IgG1 in cynomolgus monkeys, compared with IgG2 and IgG4 (Bowman et al., 2013). A complete DART safety assessment includes evaluation of effects on development (evaluating the potential for maternal toxicity as well as preand postnatal developmental toxicity, including juvenile toxicity), and effects on fertility (reproduction) (Denny and Faqi, 2017). Developmental toxicity studies typically dose and evaluate animals during pregnancy [an embryo-fetal development (EFD) study] and evaluate offspring in utero and following delivery [a pre- and postnatal development (PPND) study], providing critical information on the potential for drug-induced birth defects. Male and female

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fertility studies typically dose animals before, during, and after mating to determine potential adverse effects on fertility and early embryonic development. While these studies are relatively easy to conduct in rodents and rabbits, in monkeys they are time-consuming, expensive, and subject to variable and sometimes high fetal loss that can make interpretation difficult, which has led to modified assessments in monkeys compared with rodents and rabbits (Weinbauer et al., 2011, 2013; Mecklenburg et al., 2019). If monkeys are used, fertility assessments are conducted by evaluating the reproductive tissues in general toxicity studies of at least 3 months duration using confirmed sexually-mature monkeys (ICH S5(R3), 2020; ICH S6(R1), 2011), which provides a long enough duration to assess any potential toxic effects. Developmental assessments in monkeys are typically done using an enhanced (e)PPND study (Luetjens et al., 2020; Weinbauer et al., 2011; Weinbauer et al., 2013).

9.2. Species to Test and Alternative Models Details on which species to test based on which are pharmacologically relevant are discussed in Section 3.3. Generally, if rodents are pharmacologically relevant, they should be used for DART studies, and efforts should be made to minimize use of large animals (usually monkeys). Rabbits may also be used in EFD studies if they are pharmacologically relevant. Monkeys should only be used for DART studies if they are the only pharmacologically-relevant species and DART studies are considered necessary. Before using monkeys, alternatives such as testing in genetically-modified animals should be considered (Martin et al., 2009; Wright et al., 2012). Studies in genetically-modified animals have been adequate to support drug development.

9.3. Study Design Considerations Test articles with relatively short half-lives may be dosed daily; however, test articles that contain an Fc and have a long half-life will likely have infrequent dosing (likely similar to other in vivo toxicity studies with that test article). The need for dose range finding (DRF) studies should be made on a case-by-case basis. If no

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toxicity is anticipated and reasonable estimates of doses can be made, the DRF studies are likely unnecessary; DRF studies are particularly discouraged when monkeys are being used. Doses should be selected as described for general toxicity studies. It is not necessary to cause maternal toxicity in developmental studies if sufficient (minimum of 10) exposure multiples are achieved. Loading doses, or two doses close to one another early in a study, may be useful to achieve steady-state concentrations quickly. For molecules that are anticipated to be nontoxic, fewer than three test article-dosed groups may be used. Fewer dose groups should also be considered whenever monkeys are being used. Similar to other in vivo studies, immunogenicity may be an issue. This is usually less of a concern with studies that dose for less than 14 days (e.g., rodent EFD studies), although ADA can develop in the 7–10 day range for highly immunogenic molecules. Thus, it is often possible to complete rodent and rabbit developmental toxicity studies without impact from immunogenicity. For monkey ePPND studies, where dosing is usually from GD20 until birth approximately 150 days later, there is a much greater chance for development of immunogenicity. It is recommended that toxicokinetic and ADA samples be collected from dams, and possibly from fetuses or infants.

9.4. Strategies and Timing Scientists should understand the role of the target pathway in development and reproduction, the potential for off-target effects, and the risk tolerance of the patient population. After this assessment, a determination on whether there is sufficient knowledge to inform patient safety about potential risks to reproduction and development should be made. If there is insufficient information, there should be a determination of what toxicity testing is warranted and when it is needed. Alternatively, it has recently been suggested that a weight of evidence approach could be used instead of conducting in vivo developmental toxicity study(ies) if there is sufficient information to inform patient risk (Rocca et al., 2018). This approach is supported by ICH S5(R3) (2020); however, precedence for this approach is currently limited. Therefore,

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engagement of regulatory agencies is recommended prior to using this strategy to ensure regulatory acceptance and prevent program delays. The timing of DART studies will depend on the clinical program (primarily when women of child-bearing potential or pregnant women are enrolled, how many will be enrolled), what species are pharmacologically relevant, and company policy (some companies have guidelines that are more stringent than those of regulatory agencies). For example, if cynomolgus monkeys are the only relevant species and a developmental or ePPND toxicity study in monkeys is deemed necessary, the study is usually conducted during Phase III with careful attention to contraception during clinical trials. In contrast, if rodents or rabbits are pharmacologically relevant and WOCBP are being enrolled in Phase II, then these studies might be conducted in time to support Phase II or III, depending on clinical needs, and the findings might support less stringent contraceptive measures and thus likely aid in clinical study enrollment. When using monkeys for DART assessments, careful planning well in advance of needing reports is necessary to avoid program delays. Inclusion of fertility assessments in general toxicity studies requires using sexually-mature animals, which may be difficult to obtain and typically require advanced planning. All animals should be confirmed to be sexuallymature prior to beginning the study (Leach, 2013; Mecklenburg et al., 2019). Fertility assessments collected in monkeys during the course of general toxicity studies (3 months duration) should include organ weights and microscopic examination of reproductive organs consistent with ICH S5(R3) (2020) guidelines. The pathologist should record microscopic evidence of sexual maturity for each animal examined on the study. If there are specific causes for reproductive concern based on mechanism of the test article, target, or findings in previous toxicity studies, then additional evaluations may be needed. For ePPND studies in monkeys, slots are limited and booked well in advance, and the studies may take over 2 years to complete and obtain a final report once dosing has initiated.

9.5. Nonclinical Pediatric Evaluation If the age of the target pediatric population is younger than the corresponding developmental age that was evaluated in existing general toxicity study package, a JAS may be warranted as described in ICH S11 (2020), which also discusses study design considerations. The design of any studies, if required, should be based on the intended age of the pediatric population, target organs in adult humans and animals (theoretical and identified in clinical and nonclinical studies), and the pharmacological target. JAS should be conducted in rodents if they are pharmacologically relevant, and typically one species is sufficient. The timing of studies is dependent on the clinical pediatric development plan. In general, if a JAS is required it should be conducted prior to initiation of clinical trials in pediatric populations. Use of alternatives such as genetically-modified animals can also be considered.

10. GENOTOXICITY ICH S6(R1) (2011) indicates that genotoxicity studies are usually not needed for biotherapeutics. A review of 53 biologics that were tested for genotoxicity supports the lack of value of these assays for proteins and peptides containing only natural amino acids (Sawant et al., 2014). However, if small molecule linkers or conjugates are associated with the protein test article, they will likely need to be assessed in a manner similar to standard small molecules (ICH S2(R1) (2011); Hinrichs and Dixit, 2015; Lansita et al., 2015).

11. CARCINOGENICITY STUDIES Carcinogenicity testing is not needed for therapeutics that have a limited duration of dosing (2-fold increase over controls). This combination of clinical pathology alterations and anatomic pathology findings is a common cause for NAP attrition at this development stage, as was the case for ALN-AAT (an Nacetylgalactosamine-coupled siRNA targeting alpha-1 antitrypsin mRNA) in Phase I/II (Huang, 2017). Hepatocellular vacuolation (lipid accumulation) may also be observed following repeated NAP administration in rats but not NHPs as a nonadverse finding that is not typically associated with clinical pathology correlates, and progression to degeneration/necrosis has not been observed (Janas et al., 2018a). Hybridization-independent Toxicity Injection site reactions and flu-like symptoms are commonly observed in patients administered

oligonucleotides because of innate immune stimulation due to the nucleic acid–based structure or LNP formulations. The severity of these symptoms may cause some patients to discontinue therapy or require premedication with immunomodulatory drugs to manage the effects. As an example, a liposomal formulation of an miRNA-34 mimic resulted in frequent reports of fever, cytokine release syndrome, hepatic failure, and other immune-mediated toxicity in Phase I trials that necessitated termination of the trial (Hong et al., 2020). The mechanism of toxicity was unclear, and immune activation was not directly attributable to the liposomal carrier (Beg et al., 2017). Importantly, preclinical studies in monkeys did not predict the immune activation profile seen in the clinic (Hong et al., 2020). Improvements in screening for NAP candidates with reduced immunostimulatory properties and more potency have greatly decreased the occurrence of injection site reactions and flu-like symptoms in patients for second-generation NAPs. Pro-inflammatory mechanisms related to interactions with extracellular, intracellular, or cell-surface proteins leading to immune stimulation may also occur with ASOs and siRNAs (Frazier, 2015). Their appearance may be related or unrelated to the sequence used. NAP sequence-specific interactions with Toll-like receptors in rodents (especially in rats) may result in cytokine release and subsequent lymphoid hyperplasia and multi-organ mononuclear cell infiltrates (Senn et al., 2005). In addition, in monkeys, pro-inflammatory ASOs can cause vasculitis by activation of the alternative complement pathway via serum Factor H binding, thereby leading to damage of the vascular endothelium (Frazier et al., 2014; Henry et al., 2002) (Figure 7.4F). Complement activation and subsequent vasculitis is a low risk in human patients as monkeys are substantially more sensitive to Factor H stimulation. Highdose administration in clinical trials has not caused an effect on complement activation (Henry et al., 2008). All these accumulative or pro-inflammatory stimuli are reversible when dosing is stopped, but due to the long tissue half-life can take weeks or months to recover. Ligand-conjugated NAP candidates show substantially reduced pro-inflammatory effects and reduced injection site reactions. This has

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FIGURE 7.4 Typical Histologic Changes Associated with NAPs. (A) Kidney. Basophilic granules (arrows) present in proximal tubular epithelium in an NHP treated once weekly with a 20 -MOE ASO. Basophilic granules are common in NHPs, rats, and mice in this location with ASOs and siRNAs but are more prominent with ASOs in NHPs. The number of granules tends to achieve a steady state that mirrors the tissue kinetics for the NAP being studied and is fully reversible with discontinuation of treatment. 20 objective. (B) Kidney. Vacuolation of proximal tubular epithelium in an NHP treated once weekly with a 20 -MOE ASO. This alteration has been rarely encountered in rodents and has been demonstrated to be an artifact due to extraction of the ASO from lysosomes during tissue processing. 40 objective. (C) Lymph node. Granular/vacuolated macrophages in sinusoids from an NHP treated once weekly with a 20 -MOE ASO. Cytoplasmic appearance is due to ingestion of ASO and possible accumulation of cytokines/chemokines. Accumulation of macrophages in lymph nodes and other tissues reaches a steady state that also tends to mirror the tissue kinetics for the NAP being studied. This alteration is fully reversible with

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been widely beneficial for siRNA using lipid vehicle delivery. Moreover, no injection site reactions or flu-like symptoms have been observed with GalNAc-conjugated 20 -MOE-modified ASOs (Crooke et al., 2018b). Other NAP–protein interactions that stimulate complement activation and increase coagulation times have been demonstrated to be generally sequence-independent and related to chemistry where modifications that increase protein binding to increase plasma t1/2 also increase these unintended effects (Bennett et al., 2017). Injection site reactions, fever, chills, and rigors (shivering) were the most common observed side effects attributed to this issue, but efforts to identify and eliminate these sequences in addition to the use of chemical modifications have successfully mitigated many of these undesirable effects (Bennett et al., 2017; Yamakawa et al., 2019). In particular, the shorter base-pair length used in LNA gapmer ASO designs seems to have reduced their pro-inflammatory characteristics as compared to earlier designs (Frazier, 2015). Tissue Accumulation GalNAc-conjugated or LNP-encapsulated siRNAs rapidly distribute to hepatocytes and renal proximal tubular epithelial cells (PTECs) and may be taken up by cells of the reticuloendothelial system (tissue-resident macrophages) in the liver (Kupffer cells), spleen, lymph nodes, testes, and/or near the subcutaneous injection site. The bulk of the siRNAs concentrate in the endosomal–lysosomal system in cells and are visible on H&E-stained slides as basophilic granules or lightly basophilic vacuoles (Figure 7.4A). These granules are not associated with cell/ tissue damage or apparent cell dysfunction, but given the long tissue half-life of siRNAs these granules may take many weeks to months to

fully regress. Rats more frequently have basophilic granules in the kidneys, whereas granules are more common in Kupffer cells and lymph node macrophages in NHPs (Janas et al., 2018a) (Figure 7.4C). In some cases, hybridization-independent effects may account for hepatotoxicity observed in nonclinical species. The mechanism for this may be related to NAP accumulation in Kupffer cells and subsequent cytokine release, hepatocellular protein interactions, or lysosomal breakdown leading to intracellular enzyme release although the specific mechanism may not always be identified (Frazier, 2015; Henry et al., 2008). Compound accumulation in the proximal renal tubule is also a well-known NAP class effect. With siRNAs, the uptake occurs by an unknown mechanism and accumulation of test article results in basophilic granules on light microscopy, but no functional effects have been demonstrated in nonclinical studies or clinical trials. Unlike siRNAs, ASOs are readily bound to plasma proteins so that most are not filtered by the glomeruli, but a small unbound fraction of ASOs gets filtered through the glomeruli and then reabsorbed by the proximal tubular epithelial cells. Accumulation of ASOs within lysosomes may reduce the absorptive capacity of PTEC and has been shown to lead to proteinuria particularly in rats (Henry et al., 2008). The pathogenesis could also be a result of ASO hybridization with unintended mRNA targets that have either complete or partial sequence. Increased mesangial matrix and increased glomerular cellularity related to local pro-inflammatory activity rarely lead to glomerulonephritis with ASOs, where immune-meditated deposits form along the glomerular basement membranes in preclinical species (Frazier et al., 2014). Crooke et al. analyzed data of approximately 2400

= discontinuation of treatment but is the slowest compartment to recover. 10 objective. (D) Lymph node. Ultrastructure appearance of dilated phagolysosome containing immunogold-labeled granular material (arrows) identifying these as ASO particles. 4000. (E) Hippocampus. Vacuolation of hippocampal neurons is commonly seen with ASOs in NHPs after three or more once-monthly IT injections of a 20 -MOE ASO. Vacuolation in the hippocampus has also been demonstrated to be an artifact due to extraction of the ASO from lysosomes during tissue processing. 10 objective. (F) Heart. Perivascular/vascular inflammation of a coronary artery secondary to repeated activation of the alternative complement pathway in an NHP following multiple once weekly doses of a 20 -MOE ASO. The presence of vascular lesions is considered adverse but likely not clinically relevant due to the species propensity for complement activation relative to humans. 10objective.

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patients and found no clinically relevant impairment of renal function following ASO administration (Crooke et al., 2018a). A single case of acute tubular necrosis was diagnosed after administration of a modified LNA test article in a patient receiving a weekly dose of 5 mg/ kg in a Phase I clinical trial. While this effect was most likely sequence-related, regulatory authorities recommended increased surveillance of PS-modified ASOs as a class. Markers of kidney injury such as the more traditional markers BUN (blood urea nitrogen) and CREA (creatinine) or urine protein:creatine ratios can help in monitoring potential renal damage (Frazier, 2015). Newer serum markers of acute kidney injury like Kim-1 (kidney injury molecule 1) for glomerular damage or NGAL (neutrophil gelatinase–associated lipocalin) for injured tubular epithelial cells also can be added to the preclinical protocols. An increased incidence and severity of early-onset chronic progressive nephropathy (CPN, a species-specific background finding in rats) has been observed following ASO exposure (personal observation, JE) and highlights the renal sensitivity of this species. Artificial vacuolation of the proximal tubule, especially the S1–S2 segment, can be found in preclinical species. This effect is dose dependent and is caused by initial test article accumulation in phagolysosomes followed by osmotic imbalances during tissue processing after formalin fixation that leads to lysosomal dilation (Lenz et al., 2018). Tubular vacuolation can be avoided by using modified Karnovsky’s solution as a fixative or by preparing freshfrozen sections (Engelhardt, 2016). About 10% of ASOs with a PS backbone lead to mildly reduced platelet counts in rats and monkeys, and very rarely in mice. This effect is dose- and sequence-dependent. Usually a steady state is reached, and the platelet numbers recover fully within days/week when dosing is ceased (Henry et al., 2008). However, a doseindependent, severe thrombocytopenia (TCP, indicated by platelet counts 12 weeks). While platelets rebound within days after dosing is stopped, the TCP would return when the affected animals were rechallenged. Several hypotheses (Frazier, 2015) have been tested, but the exact mechanism for TCP has not been

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discovered yet. A rare but notable reduction of platelet count was found in human patients treated with drisapersen, inotersen, and volanosersen ASOs. Dose-related TCP was noted in a small subgroup of patients induced by chronic dosing using the three different ASOs with different indications and nonoverlapping sequences (Levin, 2019). Since implementing platelet monitoring during ASO treatment, no serious sequelae secondary to TCP have been observed (Crooke et al., 2018b). Findings in tissues outside the liver, kidney, and immune cells with LNP-formulated or GalNAc-conjugated siRNAs have rarely been reported. In clinical trials with revusiran for hereditary ATTR amyloidosis, an apparent increase in peripheral neuropathy and patient deaths were reported in treated groups, but these outcomes may have been associated with the underlying disease, and no clear links of neuropathy or cardiotoxicity to revusiran administration have been identified (Judge et al., 2020; Setten et al., 2019; Zanazzi et al., 2019). Additionally, in nonclinical studies with revusiran, minimal necrosis of lymphoid follicles was observed in the spleen, and injection site mixed cell infiltrates (with or without transient clinical observations of edema after dosing) were reported, the former only in acute toxicity studies with daily dosing. These findings were not considered to be adverse (Sutherland et al., 2020). For novel NAPs, toxicities in other organs may appear more often as targeted delivery mechanisms to other tissues (e.g., muscle, gastrointestinal tract, and lungs) are advanced in development. Similarly, intrathecal delivery of siRNAs or ASOs intended for CNS indications may also be associated with unique procedural, class-effect, and sequence-specific toxicities that have yet to be evaluated in detail (Figure 7.4E). In NHP and rats, minimal focal axonal degeneration with gliosis and minimal mononuclear cell (lymphohistiocytic) infiltrates in surrounding meninges are rarely found at the cauda equina close to the injection site. These findings have been observed equally in dosed and vehicle-treated animals and represent a procedure-related lesion (Ionis internal observation). Neuronal macrovacuolation (up to 40 mm) of the hippocampus in NHP can frequently be found with repeat dosing. An indepth analysis of this entity revealed

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accumulation of ASO inside lysosomes that artificially became enlarged during processing. Therefore, these vacuoles do not represent a safety concern (Lamb et al., 2022).

5. CONCLUSIONS Nucleic acid pharmaceuticals represent an exciting and emerging drug class with many potential advantages over traditional targeted therapeutics, as well as some challenges. There have been several therapeutic advances/ approvals in recent years for orphan indications and challenging targets such as Onpattro for hereditary transthyretin-mediated amyloidosis, Exondys 51 for Duchenne muscular dystrophy, and Spinraza for spinal muscle atrophy. Expected therapeutic benefits are also in sight for NAPs in clinical trials for indications such as cancer, Huntington’s disease, and viral diseases. The pharmacology of NAPs allows for direct targeting of the genetic origin for many types of disease, the opportunity to affect multiple cellular pathways, and the ability to limit the development of resistance mechanisms while avoiding many off-target toxicities that plague traditional biologics. In addition, the temporary (albeit long-lasting) effect on mRNA/protein reduction is a significant advantage to lifelong adjustments induced by gene therapy. Major advances in NAPs have come in the form of new chemistries, novel delivery systems allowing tissue-specific targeting by parenteral administration, and direct tissue delivery systems such as intratumoral and intraparenchymal delivery to the CNS. Recent optimization of chemical modifications and delivery conjugate technologies are resulting in improved stability, nuclease resistance, tissue distribution and uptake, improved target binding, and expanded safety margins. Nonclinical assessment of NAPs has also been a rapidly evolving field, in part due to the unique pharmacology driving innovative considerations for safety and efficacy assessments. Improved engineering and screening of target engagement and prediction of hybridization-dependent effects using in silico modeling techniques has reduced the need for animal studies. For the pathologist, NAPs provide a unique field of play, to review variations on “expected”

findings such as lysosomal accumulations but also to evaluate and explore novel toxicities that may result from the unique exaggerated pharmacology, hybridization-dependent, or deliverymediated toxicological effects. As more biotechnology and biopharmaceutical companies as well as academia expand the definition of a “drug” into the fields of nucleic acids, cellbased and engineered therapeutics, it is critical for the pathologist to stay agile, engaged, and ready for the new frontiers of drug discovery and development.

GLOSSARY Antisense oligonucleotide (ASO) A class of engineered linear nucleic acids which act through degradative and nondegradative mechanisms to regulate the activity of target messenger RNAs. Aptamer A class of small nucleic acid ligands that are composed of RNA or single-stranded DNA oligonucleotides and have high specificity and affinity for their targets and exert their pharmacodynamic effect by binding to proteins and modifying their function. Drug class The specific chemistry used in a sequence for a drug platform. Drug delivery conjugate Linkage of an NAP to a ligand specific for a cell membrane receptor on a target cell of interest to facilitate targeting and uptake of the NAP by the desired cell population. Drug platform The general type of nucleic acid pharmaceutical under development, such as antisense oligonucleotide or smallinterfering RNA. Gapmer Single-stranded oligonucleotides containing a central DNA sequence flanked by a locked nucleic acid sequence that induces RNase H1 activation to mediate nuclease digestion of their target RNA. They exhibit cellular entry by gymnosis, without need for any carriers, conjugation, or transfection. MicroRNA (miRNA) Small 20- to 22- nucleotide single-stranded noncoding RNA molecule that influences protein expression by binding target mRNA within the RISC, causing mRNA degradation, silencing, or translational repression. MiRNA mimic Synthetic double-stranded microRNA sequence that replaces the function of a downregulated miRNA in order to downregulate target proteins. Morpholino Synthetic antisense oligonucleotides that are designed to bind and block the translation initiation complex of mRNA sequences. This technology has been used to test the role of specific genes by transient inhibition of protein expression, particularly during embryologic development. Small-interfering RNA (siRNA) Small 20- to 25- nucleotide doublestranded noncoding RNA molecules that influence protein expression by binding target mRNA within the RISC and causing mRNA cleavage or translational repression. TargomiR Synthetic miRNA therapeutic coupled with a nanoparticle delivery system and a targeting moiety that directs the NAP to target-expressing cells.

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8 Gene Therapy and Gene Editing Basel T. Assaf1, Claudia Harper2, Jonathan A. Phillips3 1

Sanofi, Inc., Cambridge, MA, United States, 2Orna Therapeutics, Cambridge, MA, United States, 3Intellia Therapeutics, Cambridge, MA, United States O U T L I N E 1. Introduction 2. General Principles of Nonclinical Research and Development for Gene Therapy Products 2.1. Pharmacology 2.2. Toxicology 2.3. Biodistribution and Viral Shedding 2.4. Role of Pathologists in the Nonclinical Assessment of Gene Therapy Products

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3. In Vivo Gene Therapy 274 3.1. General Concepts of In Vivo Gene Therapy 274 3.2. AAV as a Model Platform for in vivo Gene Therapy 274 3.3. Nonclinical Pharmacology and Safety Assessment for in vivo Gene Therapy 275 3.4. Contemporary Toxicities Associated With In Vivo Gene Therapy 283

1. INTRODUCTION Gene therapy (GTx) is the transfer of exogenous (foreign or “non-self”) genetic material into the patient’s cells to treat a specific genetic disease. Introduction of foreign DNA into another cell via a vector is termed transduction. Depending on the disease, GTx functions by several mechanisms of action. Basic components of GTx products are composed of a vehicle (viral

Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-12-821047-5.00034-8

4. Ex Vivo Gene Therapy 293 4.1. General Concepts of Ex Vivo Gene Therapy 293 4.2. Nonclinical Safety Assessment for Ex Vivo Gene Therapy 300 4.3. Toxicologic Pathology Considerations With Ex Vivo Gene Therapy 310 5. Genome Editing 5.1. Harnessing Cellular DNA Repair 5.2. Genome Editing Strategies 5.3. Nonclinical Safety Assessment for Genome Editing Products

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or nonviral vectors) capable of delivering the genetic payload (a transgene) into the target cells. GTx exerts its therapeutic benefit in one of several ways: by transferring a functional copy of a defective or missing gene, where the copy is capable of producing a functional protein missing from the affected cells (gene replacement); by transferring a gene expressing an RNA interference element (such as short hairpin RNA [shRNA] or microRNA [miR])

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capable of suppressing a gain-of-toxic-function gene in the affected cells (gene suppression); or by transferring genetic material capable of directly editing the host genome to eliminate a genetic mutation at its chromosomal source (such as CRISPR/Cas technology [gene editing]) (Figure 8.1). GTx products can be administered directly into the patient (in vivo GTx) or by engineering of cells extracted from the patient and modified ex vivo prior to reinfusion of the modified cells back into the patient (ex vivo GTx). GTx can be classified based on the requirement of transferred genetic material to integrate into the host genome to exert its therapeutic function (such as lentivirus [LV] or other retrovirus GTx vectors) or function in an extrachromosomal (nonintegrating) fashion (such as recombinant adeno-associated virus [AAV] GTx vectors). Despite the promise of current GTx clinical trials and approved products, the field was initially marred (in the late 1990s) by several serious safety concerns associated with acute fatal systemic inflammatory outcomes secondary to adenovirus-based GTx and several patients developing T cell leukemia due to insertional mutagenesis associated with firstgeneration g-retroviral vectors (Board of the European Society of Gene and Cell Therapy, 2008; Cavazzana-Calvo and Fischer, 2007; PikeOverzet et al., 2007; Raper et al., 2003).

Nonetheless, approaches to mitigating such safety concerns observed in early clinical trials ultimately culminated in approval of several GTx products by the U.S. Food and Drug Administration (US FDA) and the European Medicines Agency (EMA). Luxturna (voretigene neroparvovec-rzyl) is the first directly administered in vivo gene therapy approved by the FDA and uses the AAV GTx platform for the treatment of RPE65 mutation-associated retinal dystrophy. Zolgensma (onasemnogene abeparvovec-xioi), an AAV-based GTx for the treatment of spinal muscular atrophy in patients with mutations in the survival motor neuron 1 (SMN1) gene, followed shortly thereafter. Prior to that, the EMA granted conditional marketing approval to Glybera (alipogene tiparvovec), another AAV GTx therapy for familial lipoprotein lipase deficiency (which has been withdrawn from the market due to lack of commercial value [Senior, 2017]), and most recently approved Zynteglo (betibeglogene autotemcel), an LV-based GTx, for the treatment of beta thalassemia. This chapter will discuss GTx as it relates to the treatment of genetic diseases and not those associated with modulating immunological function as a means of therapy (such as vector-based vaccines, cancer gene therapy, or genetically modified Chimeric Antigen Receptor [CAR] T-cell therapy).

FIGURE 8.1 Gene therapy is the transfer of exogenous genetic material into the patients’ cells to treat a specific genetic disease. The transfer of genetic material can occur by direct administration into the patient (in vivo GTx) or by modification of cells extracted from the patient followed by reinfusion of the modified cells back into the patient (ex vivo GTx). The transferred genetic material (green symbol) can exert its therapeutic function on a defective gene (red symbol) by replacing the defective/ missing gene (gene replacement), by suppressing a gain-of-toxicfunction gene (gene suppression), or by directly editing (scissors symbol) the host genome to eliminate a chromosomal mutation (gene editing). II. PRODUCT-SPECIFIC PRACTICES FOR SAFETY ASSESSMENT

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TABLE 8.1

Biological Features of Commonly Used Gene Therapy Platforms

Feature/Vector Type

Adenovirus

Adeno-Associated Virus

Retrovirus/ Lentivirus

Nonviral

Genome*

dsDNA

ssDNA

ssRNA

DNA

Integration

Rare

Rare

Yes

Rare

Packaging capacity

8e30 kb

5 kb

8 kb

>5 kb

Immunogenicity

Extensive

Limited

Limited

Limited

Main utility

Vaccines

In vivo gene therapy

Ex vivo gene therapy

All applications

* ds, double-stranded; ss, single-stranded.

The choice of GTx platform depends on various factors (Hutt et al., 2022) (Table 8.1). Viral vectors, such as AAV and LV, are widely used as compared with nonviral vectors due to the superior ability of viruses to transfer genes into target cells. However, nonviral vectors, such as nanoparticles and exosomes, are being considered due to their capacity for packaging larger genes and their lower toxicity profiles relative to viral vectors. Recombinant AAV vectors are derived from nonpathogenic viruses and widely employed for in vivo GTx due to their acceptable safety profile and the ability to infect dividing and nondividing cells. However, the majority of the AAV genome forms extrachromosomal episomes that will gradually get diluted in proliferating tissues, such as the livers of infant patients, so such vectors eventually will lose their therapeutic benefit. Therefore, AAV vectors are more suited for targeting longlived, terminally differentiated cells such as neurons, myocytes, or hepatocytes in livers that have matured out of their exponential growth phase. Conversely, LV vectors, which are generally based on human immunodeficiency virus (HIV), are more suited for stem cell transduction because the vector stably integrates into the host genome. Integration permits the introduced gene to persist undiluted by passing to daughter cells upon cell division. Despite the recent traction in GTx, the field is still relatively new, and the platforms and nonclinical and clinical trial designs are still under development. Consequently, the typical approach to assessing the pharmacology and toxicology related to GTx products is focused on scienceand evidence-based, case-by-case evaluations depending on the product attributes (such as

integrating vs. nonintegrating vectors) and indication (e.g., disease of the central nervous system [CNS] in young patients vs. disease of the liver in adults). Despite the case-by-case approach and the continuously evolving health authority regulations of gene therapy, the goals and general principles of nonclinical safety evaluation for other therapeutic modalities are still applied to the safety assessment of GTx products albeit with modifications to some practices (Hutt et al., 2022).

2. GENERAL PRINCIPLES OF NONCLINICAL RESEARCH AND DEVELOPMENT FOR GENE THERAPY PRODUCTS The goals of the nonclinical program for GTx products are to demonstrate a safe and therapeutically beneficial treatment within a defined effective dose range following a specific route of administration in the most relevant animal species that will ultimately support the proposed safe starting dose and dose escalation plan for First-in-Human (FIH) clinical trials. The nonclinical program to support GTx product relies on three fundamental – and familiar – pillars: (1) pharmacology, (2) toxicology, and (3) biodistribution and viral shedding studies. Depending on the explored gene therapy platform and the “Risk Group” classification of viral vector intended for the program, experiments involving recombinant or synthetic gene transfer may require institutional biosafety committee approval before the study is conducted in a biosafety containment facility (National Institute of Health, 2019).

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2.1. Pharmacology Typical gene therapy programs start with a predetermined gene therapy platform (e.g., viral vs. nonviral or AAV vs. LV vectors). Subsequently robust pharmacology studies are designed to define the best vector attributes to demonstrate the intended biological activities and support the proof-of-concept studies in relevant animal models of disease. For example, pharmacology studies of AAV GTx products define the virus capsid serotype (based on preferential tropism of certain serotypes for specific cell populations), transgene DNA sequence, and other regulatory elements such as promoter design in the final transgene expression cassette (Hutt et al., 2022). Additionally, pharmacology studies identify the best route of administration required to achieve the intended therapeutic benefit, and potentially assess any special devices that may be required to support less common routes of administration (e.g., catheters for intracerebroventricular infusion). Once a specific GTx product is identified and the biological activity and therapeutic benefit have been demonstrated at a particular dose level, a dose range–finding study is carried out to identify the effective dose range of that product. The goals of dose range–finding studies are to identify (1) a minimally effective dose below which the therapeutic benefit is lost and (2) a maximum effective dose beyond which no additional therapeutic benefit is observed. However, nonclinical interspecies differences in effective dose range may exist and therefore not translate directly to predicting the likely human response (Collaud et al., 2019; Jiang et al., 2006).

2.2. Toxicology Once a GTx product candidate is defined and an effective dose range is identified for a specific route of administration, nonclinical toxicity studies are carried out to assess the safety of the product. The goals of toxicity studies for GTx test articles are to assess the potential toxicities associated with the product and to understand the relationship between the incidence and severity of toxicities to administration of the test article. Ultimately, toxicity studies define an effective dose level that is not associated with

toxicity (No-Observed-Adverse-Effect Level [NOAEL]) or an optimal biological dose that is effective with manageable levels of risk (e.g., Highest Nonseverely Toxic Dose [HNSTD]). A common GTx study design includes a vehicle control group and 3 (or sometimes 2) test article dose groups. Similar to other therapeutic modalities, toxicity studies for GTx test articles are performed in the most sensitive nonclinical animal species; however, unlike other modalities a single nonclinical species may suffice to support the safety assessment of GTx products (U.S. Food and Drug Administration, 2013). The low and middle GTx doses assessed in toxicity studies typically reflect the minimal and maximum effective doses that were determined in the pharmacology studies. The principle in choosing the high dose in GTx toxicity studies is like other modalities: to determine the safety margin beyond which test article–related toxic effects are observed. However, the current acceptable safety margins for GTx products are generally narrower compared to other modalities, such as small molecules and other biomolecules (Hutt et al., 2022), ranging from 5- to 10-fold above the estimated maximum dose in humans and as low as 2-fold if the maximum feasible dose is limiting (e.g., dose formulation constraints with respect to concentration of the product in a reasonable dose volume). The acceptable safety margin is influenced by the risk–benefit considerations of the patient population as well as the observed toxicity profile. The choice of safety margin is often discussed in advance with the regulatory agency and is defined depending on several risk–benefit considerations. Toxicity studies assess the potential toxic effect at maximum and persistent exposure to components of the GTx test article, including exposure to the delivery system (viral capsid proteins or components of the nanoparticle) and the transgene product (mRNA and/or protein) as well as manufacturing process–related impurities. Unlike therapeutic modalities that rely on pharmacokinetic (PK) and pharmacodynamic (PD) studies to determine the kinetics of exposure and biological effect of the test article, gene therapy relies on biodistribution (BD) studies to assess exposure to components of the gene therapy product (particularly vector nucleic acid and the product of transgene expression)

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and determine the time points required to assess the toxicity of the GTx product at maximum and persistent exposures (Chowdhury et al., 2021; European Medicines Agency, 2008; Kamiya et al., 2003; Parra-Guillen et al., 2010; U.S. Food and Drug Administration, 2013). Assessing toxicity at persistent exposures to GTx test article is of a particular importance because, unlike other therapeutic modalities, the effect of GTx is designed to be durable for the lifetime of the patient, with generally no ability to remove or counter its effect. Endpoints in GTx toxicity studies typically include the routine measurements of clinical signs, body weight, food consumption, clinical pathology analytes, organ weights, and macroscopic and microscopic pathology evaluations. Safety pharmacology endpoints may also be included to address specific concerns. There is no default requirement to assess special classes of toxicity (e.g., immunotoxicity, genotoxicity, reproductive toxicity, and carcinogenicity) for GTx products. These assessments are considered on a case-by-case basis depending on certain risks expected or observed with a particular GTx product (Hutt et al., 2022). For example, integration analysis is an expectation for GTx products that rely on integrating LV vectors because of the increased risk of tumorigenicity due to insertional mutagenesis. Integration analysis is not an expectation for nonintegrating AAV vectors for first-in-human clinical trials (U.S. Food and Drug Administration, 2013, 2020). However, because low frequency of integration may occur with AAV vectors, regulatory authorities may require an integration assessment during later stages of development to fully characterize the risk– benefit profile for a specific patient population (U.S. Food and Drug Administration, 2021).

2.3. Biodistribution and Viral Shedding Biodistribution studies are performed to assess the peak and persistent exposure to the GTx products. They can be performed as standalone studies, but current practices often include preliminary BD endpoints in the pharmacology and/or exploratory toxicity studies and pivotal BD endpoints during the pivotal toxicity studies. Exposure to GTx product is assessed by

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measuring the vector DNA by quantitative polymerase chain reaction (qPCR) and by measuring the transgene mRNA by reverse-transcription PCR (RT-PCR). Additional assessment of BD spatially within tissue sections using in situ hybridization (ISH), immunohistochemistry (IHC), or immunofluorescence (IF) may allow localization of vector DNA and transgene expression within a specific tissue compartment or cell types. Biodistribution includes at a minimum (per recently released guidance (U.S. Food and Drug Administration, 2020)) nine key tissues: the site of administration, blood, brain, gonads, heart, kidneys, liver, lungs, and spleen. However, other tissues may be added on a case-by-case basis. Assessment of vector shedding in urine, feces, saliva, or other body secretions into the environment in nonclinical toxicity studies is occasionally required by certain regulatory agencies. Vector shedding is assessed by the detection of vector DNA in body secretions using the same PCR assay developed for the assessment of BD (albeit in a different matrix) and can be performed as an additional endpoint in the nonclinical toxicity studies.

2.4. Role of Pathologists in the Nonclinical Assessment of Gene Therapy Products Pathologists in their diverse capacities as discovery, comparative, and toxicologic pathologists support the nonclinical development of gene therapy in a wide array of functions throughout the life cycle of the product development. In addition to the routine functions such as the macroscopic, microscopic, and clinical pathology support for toxicity studies, pathologists commonly are involved in assessing vector tropism and BD in tissues utilizing molecular tools such as IHC and ISH. For example, multiplex IHC and/or ISH can aid in differentiating the ability of different AAV capsid serotypes to transduce neurons versus glial cells. Considering that several GTx products remain tissue bound and are not released into the circulation, pathologists utilize qualitative and quantitative digital pathology and image analysis tools to identify tissue exposure to the test article, the level of

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transgene expression, or the level of genemodified cell engraftment (by IHC or ISH) that ultimately aid in defining the effective dose range of the product. Pathologists also support the longitudinal analysis of transgene products secreted in biological fluids by determining any biological activities of the GTx product, such as assessing the transgene expression and biological activity of Factor IX in improving coagulation parameters and decreasing bleeding incidents in hemophilia B animal models of disease. Considering the genetic basis of gene therapy indications, development programs for GTx test articles often rely on transgenic animal models of disease that are not completely characterized. Pathologists are heavily involved in supporting such programs through detailed phenotypic analysis to validate such unique disease models so that they can be employed to support the pharmacology assessment of GTx products (see Genetically Engineered Animal Models in Toxicologic Research, Vol 1, Chap 23). Gene therapy also often targets rare diseases for which biomarkers are scarce or unexplored. Pathologists use their skill set to support the identification and qualification of biomarkers that bridge the nonclinical program with clinical monitoring and data interpretation. Endpoints generated by the pathologist may influence the safety strategy of GTx products and improve on the success rate of product development. For example, while immunotoxicity assessments are not routinely required during the development of GTx products, the identification of tissue inflammation or elevated circulating levels of fibrinogen, C-reactive protein (CRP), or other species-specific acutephase proteins (see Clinical Pathology in Nonclinical Toxicity Testing, Vol 1, Chap 10) by the pathologist may trigger additional derisking studies to address the immunotoxic potential of the GTx product. Conversely, while the identification of vector DNA in gonads by PCR may indicate potential risk of germline transmission and require reproductive toxicity studies, the pathologist may aid in derisking such PCR observations and eliminate the need to conduct reproductive toxicity studies if the presence of vector DNA in gonads can be confirmed by ISH to occur in interstitial cells and not gametes.

3. IN VIVO GENE THERAPY 3.1. General Concepts of In Vivo Gene Therapy In vivo gene therapy involves the local or systemic delivery of the therapeutic gene directly into patients in order to modify the genetic content of their cells. The therapeutic genes are typically delivered via viral or nonviral vectors, with the former being more widely used. Common viral platforms for in vivo GTx include adenovirus, AAV, and LV vectors. The use of adenovirus vectors for the treatment of genetic diseases has decreased due to their strong immunogenic potential, but consequently the strong immune response against adenovirus vectors has widened their application to become a preferred viral vector in vaccine development (Tatsis and Ertl, 2004). Lentivirus vectors are used primarily for ex vivo gene therapy, as described in detail later in this chapter. The utility of LV vectors has been successfully demonstrated with in vivo gene therapy by several groups; nonetheless, they are not widely used in this fashion due to the higher risk associated with widespread vector DNA integration into the host genome. Various AAV vectors have gained tremendous traction for in vivo gene therapy in the past 15 years due to their favorable safety profiles. Wild-type AAV and the recombinant AAV (rAAV) vectors are naturally nonpathogenic; they do not induce robust immune responses that may constrain the efficacy and safety of treatment, and they generally do not integrate into the host genome. The first two in vivo GTx approved in the United States, Luxturna and Zolgensma, are AAVbased GTx products, and the bulk of GTx test articles currently in development for in vivo GTx are based on the AAV vector platform. Therefore, this section will primarily focus on AAV vectors. However, the principles and practices of the safety assessment for AAV test articles are directly applicable to all approaches of in vivo GTx (Hutt et al., 2022).

3.2. AAV as a Model Platform for in vivo Gene Therapy Adeno-associated viruses are naturally occurring, replication-defective, nonpathogenic

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viruses that require help from elements of adenovirus or herpesvirus to complete their replication cycle. They are composed of 4.7 kilobases (Kb) of single-stranded DNA (ssDNA) carrying open reading frames (ORFs) responsible for the generation of capsid (cap) and replication (rep) proteins and flanked by inverted terminal repeats (ITRs) (wtAAV in Figure 8.2). Viral DNA is enclosed within a 20-nM-diameter icosahedral capsid composed of three viral proteins (VP1, VP2, and VP3) arranged in a ratio of 1:1: 10. Capsid proteins are assembled by the aid of the assembly activating protein (AAP), which is a nonstructural protein encoded within the cap gene (Maurer et al., 2018). All capsid proteins are encoded by the same cap gene, and they contain an amino acid sequence that always

FIGURE 8.2 Recombinant adeno-associated viruses (rAAV) are composed of an icosahedral capsid (composed of VP1, VP2, and VP3 subunits) enclosing a DNA construct (single-stranded [ssAAV] or doublestranded, self-complimentary [scAAV; not shown]). In wild-type AAV (wtAAV), the DNA encodes the viral capsid (cap) and replication (rep) genes, while in recombinant vectors the DNA contains promoter, transgene, and polyadenylation (polyA) sequences capable of producing a desired transgene product. Modified from Hutt JA, Assaf BT, Bolon B, et al.: Scientific and Regulatory Policy Committee points to consider: nonclinical research and development of in vivo gene therapy products, emphasizing adeno-associated virus vectors, Toxicol Pathol 50:118–146, 2022, by permission.

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overlaps with that of VP3. However, the Ntermini of VP1 and VP2 contain additional amino acid sequences with biological activity of phospholipase A2 and nuclear localization signal necessary for endosomal escape and nuclear entry (Bleker et al., 2006; DiPrimio et al., 2008; Johnson et al., 2010). Natural differences in the peptide sequence of capsid proteins in various viral serotypes are responsible for the presence of numerous naturally occurring AAVs. Serotypes are represented by numbers (AAV1, AAV2, etc.). To date, there are 12 wildtype AAV serotypes from both human and nonhuman primates (NHPs) and over 100 AAV variants identified across all species (Balakrishnan and Jayandharan, 2014; Wu et al., 2006). Differences in the peptide sequence of capsid proteins and their interactions with cellular receptors determine the cell and tissue tropism profile of each AAV serotype (Van Vliet et al., 2008). AAV vectors for GTx are generated by the removal of viral rep and cap ORFs and replacing them with the therapeutic transgene and supportive regulatory elements such as upstream promoters, enhancers, introns as well as downstream polyadenylation (polyA) termination sequences (single-stranded AAV [ssAAV] vectors in Figure 8.2). The ITRs are the only component of wild-type AAV retained in rAAV vectors, and they are typically derived from wild-type AAV2. Naturally occurring capsid proteins are typically used for AAV vectors. However, novel serotypes may be developed by directed evolution of cap genes, thereby yielding rationally designed modifications to (1) alter vector tropism, (2) evade the vector cross-reactivity against neutralizing antibodies of naturally occurring AAV serotypes, and (3) improve vector product yields during manufacturing.

3.3. Nonclinical Pharmacology and Safety Assessment for in vivo Gene Therapy Pharmacology Gene therapy programs generally target disease entities with well-characterized monogenetic (single gene) mutations affecting previously defined tissue(s). Such knowledge influences the choice of AAV vector serotype

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and the design of the DNA construct to ensure optimal efficacy and safety profiles of the GTx product. For example, Duchenne muscular dystrophy (DMD) is a generalized muscular disorder caused by mutations in the dystrophin (DMD) gene resulting in the translation of a defective cytoskeletal protein followed by myofiber degeneration (Boland et al., 1996; Mendell et al., 2021). Consequently, AAV9 serotype has been widely employed for the treatment of DMD due to its strong tropism to skeletal muscle. The AAV9 serotype is also used for the genetic treatment of cardiomyopathies (e.g., Friedreich’s ataxia) and neurodegenerative diseases (e.g., spinal muscular atrophy [SMA]) due to its ability to transduce cardiac muscle and to cross the blood–brain barrier (BBB) and efficiently transduce neurons, respectively. On the other hand, hemophilias that result from the genetic loss of a coagulation factor (Factor VIII for hemophilia A and Factor IX for hemophilia B) produced by the liver are targeted with AAV serotypes that readily transduce hepatocytes, such as AAV2, AAV6, and AAV8 (Kattenhorn et al., 2016). DNA construct designs bearing the therapeutic transgene of interest are also influenced by the pathogenesis of the disease. Transgene choice may transcribe and translate into a therapeutic protein to compensate for the lost protein function, such as mutated forms of dystrophin in DMD, Factor IX in hemophilia B, and survival motor neuron (SMN) for SMA. Alternatively, the transgene may encode a microRNA to suppress a gene with gain-of-toxic-function in repeat expansion disorders such as amyotrophic lateral sclerosis (ALS) and Huntington’s disease (HD) (Qu et al., 2019). The transgene may produce a secreted/circulating protein (e.g., Factor IX for hemophilia B) or nonsecreted/ membrane-bound/intracellular protein (e.g., frataxin for Friedreich’s ataxia), and the cellular localization will influence the pharmacology and toxicology profiling of such products. For example, the kinetics of circulating transgene products can be assessed longitudinally through repeat blood sampling, while intracellular transgene products will require tissue-based analyses such as IHC and ISH assays on specimens collected by biopsy or at necropsy. The choice of capsid, promoters, and other regulatory elements influences transgene expression and

defines the transcriptional activity in specific cell types. Promoters can be ubiquitous or tissue specific. The choice of promoter is of particular importance following systemic administration of the GTx product to limit the transgene expression in off-target organs as well as fine-tuning the strength of on-target transgene expression to minimize the potential of transgene overexpression-mediated toxicities (Gray et al., 2011; Nieuwenhuis et al., 2021; Powell et al., 2015). A limitation of the ssAAV vectors (Figure 8.2) is their small packaging capacity of 4.7 Kb, which can be further reduced by half in the case of self-complementary AAV (scAAV) vectors. The scAAV vectors are used to promote efficient transduction by bypassing the host cell– dependent rate-limiting step of second-strand DNA synthesis to produce the complementary strand (McCarty et al., 2001). Regardless of DNA construct configuration (ssAAV or scAAV), transgenes larger than the vector’s packaging capacity are carefully modified by deletions to enable the transgene to fit within the DNA construct while maintaining biological function of the translated transgene product. For example, various versions of the w14 Kb DMD gene have been designed to generate microdystrophin constructs where the protein product is reduced in size but remains functional; these microdystrophin variants are currently being evaluated in several AAV gene therapy clinical trials (Duan, 2018; Seto et al., 2012). Any modifications to the transgene are taken into consideration when designing primers for the PCR, probes for ISH, and antibodies for IHC assays. The size of the promoter and other regulatory elements required for expression may further impact the packaging capacity. Once the vector design is completed (for example, capsid and DNA construct for AAV) and preliminarily assessed for integrity and ability to express the transgene product in the appropriate in vitro systems, studies in animals commence with initial screening of the vector’s ability to transduce the intended target cells and tissues as well as its ability to express the intended therapeutic transgene product. Nonclinical in vivo studies of efficacy and safety are designed to recapitulate the intended clinical plan whenever possible, such as dose range, route of administration (ROA), and any delivery

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device used to introduce the in vivo GTx product. The choice of ROA is influenced by the intended target tissues and will influence target cell transduction, vector BD, and local and systemic body exposure to the vector and transgene product (Francis et al., 2021; Igarashi et al., 2013; Pang et al., 2008; Rubin et al., 2019; Santry et al., 2017). Systemic intravenous (IV) injection will typically result in high exposure in the liver (Bostick et al., 2007; Salmon et al., 2014). On the other hand, localized delivery is tailored for targeting specific organs (e.g., the eye or a specific brain region) while minimizing systemic exposure, immunogenicity (when targeting immunologically privileged sites such as eye and brain), and potential off-target toxicities (Barker et al., 2009; Hinderer et al., 2018; Hordeaux et al., 2019; Hordeaux et al., 2018a; Li et al., 2008; Seitz et al., 2017; Willett and Bennett, 2013; Xu et al., 2018). Consequently, the impact of ROA on BD and exposure to the GTx product will affect the range of potential organ toxicity and determine the extent of tissue collection (e.g., additional brain or liver samples, tissuedraining lymph nodes). ROA also influences the choice of nonclinical species due to animal size (e.g., larger animals require larger quantities of test article) and other anatomical constraints that may limit the application of the intended clinical device or ROA in the nonclinical species (e.g., cerebrospinal fluid [CSF] volume and turnover in rodents varies substantially from that of primates, especially humans (Vuillemenot et al., 2016)). This is particularly evident for intraparenchymal administration into the brain or intraocular routes where mouse brain and eye, respectively, neither of which fully recapitulates their human counterparts anatomically and both of which are too small to allow for delivery by the device intended for clinical use. The choice of nonclinical animal species for the assessment of efficacy and safety of GTx products is determined based on factors similar to those used for other therapeutic modalities (Cavagnaro and Silva Lima, 2015). The GTx product must be able to transduce relevant target cells, and the transgene product must be biologically active in the nonclinical species to adequately assess its therapeutic effect and any associated potential toxicities. Proof-of-concept (POC) studies use animal models of disease where the

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GTx candidate has been administered at different dose levels (e.g., low, middle, and high) to identify the dose range at which efficacy is observed (efficacious dose range). Such POC studies may be repeated several times and in different disease models until a minimally efficacious dose (lowest dose at which signs of efficacy are evident) and maximum efficacious dose (the dose beyond which no additional therapeutic benefit is observed) or optimal biological dose (a dose that offers a maximal therapeutic benefit with manageable risk) are identified (U.S. Food and Drug Administration, 2013). POC studies can also serve to simultaneously generate the required safety endpoints in conjunction with efficacy endpoints to reduce the number of animals used for nonclinical studies (European Medicines Agency, 2008; U.S. Food and Drug Administration, 2013). However, the phenotype of the animal model of disease must be thoroughly characterized to ensure the ability to discriminate test article– related findings from those of a background nature associated with progression of the disease. In addition, the fidelity of the animal model relative to the human condition and the inherent variability in disease phenotype and disease progression must be well defined before the model can be used rationally (Cavagnaro, 2002; Morgan et al., 2013, 2017b). This detailed characterization commonly includes selected functional analyses (clinical assessment, neurobehavioral testing, ophthalmic examinations, etc.) and structural evaluation (e.g., routine anatomic and pathology endpoints). As noted earlier, animal species should be able to support the intended clinical ROA as well as the proposed clinical dose, dose volume, and delivery rate (bolus vs. infusion). For example, NHPs are better suited for GTx studies using intrathecal (IT) injection or infusion for product administration considering the relatively small CSF volume and intrathecal spaces in rodents (Vuillemenot et al., 2016). Once confidence in the GTx candidate is achieved in terms of efficacy (POC) and the efficacious dose range, BD and toxicity profiles of the candidate within the efficacious dose range and using the clinically intended ROA commence as the next steps in nonclinical development.

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Biodistribution Biodistribution (BD) studies to characterize the presence, persistence, and clearance of vector recombinant genetic material (DNA or RNA) and its transgene product (mRNA or protein) in target and nontarget tissues and bodily fluids are used to assess the exposure kinetics (PK) and dynamic effect (PD) of the GTx product on the host (Chowdhury et al., 2021; European Medicines Agency, 2008; International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, 2021; Kamiya et al., 2003; ParraGuillen et al., 2010; U.S. Food and Drug Administration, 2013). BD studies employ bioanalytical methods, primarily quantitative PCR assays (qPCR) or droplet digital PCR (ddPCR), to detect the onset, peak, persistence, and clearance of the GTx product (vector nucleic acid) as well as transgene product (by RT-PCR) in tissues and body fluids. PCR assays used for BD profiling need to be sensitive and validated, with a limit of quantitation (LoQ) of
50 vector copies/1 mg genomic DNA according to the US FDA (U.S. Food and Drug Administration, 2020). Understanding the GTx product BD profile can start early during the identification of vector capsids (for viral vectors), particularly for genetically engineered (recombinant) novel capsids. These studies may employ reporter genes (e.g., green fluorescent protein [GFP]) and ubiquitous strong promoters (e.g., the recombinant CAG promoter (formed by linking the human cytomegalovirus immediate early enhancer to the chicken b-actin promoter and the 30 splice sequence of the rabbit beta-globin gene (Doll et al., 1996; Niwa et al., 1991)) to facilitate BD assessment and inform the design of nonclinical toxicity studies, such as the tissue collection list and necropsy time points to evaluate peak and persistent exposures to GTx product. However, IND (Investigational New Drug)-enabling BD studies should assess the intended clinical product using the intended ROA and a dose similar or higher to the maximum anticipated clinical dose. While INDenabling BD studies can be conducted as standalone studies, often BD endpoints are included in Good Laboratory Practice (GLP)-compliant toxicity studies. This approach maximizes the value of IND-enabling GLP toxicity studies by facilitating the correlation of potential toxicity findings with the peak and persistence of vector genome copy numbers and transgene expression levels. Additional ancillary assays, such as

western blotting (WB) and enzyme-linked immunosorbent assay (ELISA) for proteins extracted from tissues or IHC and ISH to assess the total or spatial localization of vector (DNA or RNA) and transgene products (mRNA or protein) within tissues, aid in the understanding of dose–response relationships and the potential correlation of histopathologic findings to spatial localization of the GTx product. Several factors may influence the BD profile of a GTx product. For example, AAV-based gene therapy with double-stranded scAAV bypasses the rate-limiting second-strand DNA synthesis and results in earlier onset of transgene expression, which in turn shifts the transgene onset and peak expression time points (McCarty, 2008). For example, onset and peak GFP transgene expression of scAAV serotype 2 vector is achieved at 1 and 6 weeks postadministration, respectively, while an identical vector with single-strand DNA configuration (ssAAV, which requires second-strand synthesis before transgene expression can begin) is only detected at 2 weeks postadministration and peaks approximately 6 months later (Wang et al., 2003). The choice of nonclinical animal species may also influence the BD profile depending on vector-specific receptor distribution within the host. For example, AAV-PHP.B (a variant of the AAV9 serotype) is able to cross the BBB in C57BL/6J, C57BL/6N, SJL/J, FVB/N, CD1, and DBA/2 mice following IV infusion of the vector. In contrast, AAV-PHP.B does not penetrate the BBB in BALB/c, C3H/HeJ, and CBA/J mice or in monkeys, which results in lower transgene expression in the CNS after IV infusion (Batista et al., 2020; Hordeaux et al., 2018c; Matsuzaki et al., 2019). This differential BD and BBB penetration has been attributed to the expression of Ly6a receptor on BBB to facilitate AAV-PHP.B crossing and transduction of the CNS in permissive mouse strains (Batista et al., 2020; Huang et al., 2019). Interestingly, direct CNS administration of AAVPHP.B into the cisterna magna in rhesus monkeys yields higher transduction efficiency in the CNS compared with animals receiving an equivalent dose of vector by the IV route, highlighting the role of ROA on influencing the BD profile of a GTx product (Liguore et al., 2019). The tissue list for assessing the BD profile of a GTx product varies depending on many factors. Consequently, health authorities require justification of selected tissues for analysis. The

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minimum list of nine tissues (U.S. Food and Drug Administration, 2020)dthe injection/infusion site(s), blood, brain, gonads, heart, kidney, liver, lung, and spleendmay be expanded depending on the need to examine the tissue tropism of novel AAV serotypes, inclusion of other target organs (e.g., ear), tissues related to the ROA (such as draining cervical lymph nodes for intracerebral injections), or potentially preidentified platform-related toxicities. For example, the high incidence of AAV-associated dorsal root ganglion (DRG) findings reported by several groups warrants the collection of multiple DRGs as a potential target tissue even though this organ is not routinely collected in GLP toxicity studies for other test article classes (Hordeaux et al., 2020a; Hutt et al., 2022). Advanced tissue collection practices may be required for BD profiling. For example, exsanguination and potentially intravascular perfusion with phosphate-buffered saline (PBS) may help minimize or eliminate cross-contamination by residual circulating vector components. Collection of tissues anticipated to harbor from the least to most amount of vector genome copy numbers, exchanging or decontaminating surgical instruments, and/or frequently changing gloves between tissues and between animals will further minimize the potential for cross-contamination between collected tissues. The principles of BD assessment apply to all in vivo GTx products. However, modifications to the BD study design may be required under certain circumstances. For example, BD profiling of replication-competent viral vectors may require additional time points to assess second peaks of vector expression. Immune responses against vector capsid or transgene protein products that occur in the animal but that are not translatable to humans (e.g., human-derived transgene protein products are foreign proteins and consequently may be immunogenic in animals but not humans) may, respectively, result in neutralization of vector particles in animals seropositive to the vector capsid and/ or elimination of transduced cells expressing the foreign transgene protein product. Such immune responses consequently preclude adequate BD profiling of the GTx product. In such cases, immunosuppression regimens or using a species-specific ortholog to assess transgene expression and activity in the nonclinical

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animal species may be used to mitigate such limitation. The BD pattern is largely independent of the transgene, so BD profiling may be abbreviated if sufficient data exist from other programs with similar product attributes but with different transgenes (Cearley and Wolfe, 2006; Gonin and Gaillard, 2004; Tiesjema et al., 2010; Zincarelli et al., 2008). In certain circumstances BD profiling may not be feasible, so BD data may be justifiably waived if no biologically relevant animal species is available (Huang et al., 2016; Kiem et al., 2014). Evaluation of GTx product dissemination through body secretions (urine, semen, saliva, sweat, etc.) and excreta (feces) addresses the concern of unintended vector transmission outside the treated patient. PCR-based bioanalytical assays identical to those utilized for BD profiling are typically used to assess vector shedding, and several in-life time points are collected to assess the peak, persistence, and clearance of shed GTx product. Assessing shedding potential is an expectation in all clinical trials and is frequently included in nonclinical toxicity studies. However, not all health authorities require such assessment at the nonclinical stage. Toxicology The goal of toxicity studies is to assess the local and systemic safety profile of the GTx candidate in biologically relevant nonclinical animal species using a dose range encompassing and exceeding the predicted efficacious dose range in humans. The GTx candidate is delivered using the intended clinical ROA and device with the ultimate goal of identifying an efficacious dose level with NOAEL. Information generated from POC and BD studies are essential to design an adequate toxicology package required for an IND application to support the first-in-human (FIH) clinical trial. Two nonclinical species (typically rodent and nonrodent) are required for small molecule drugs and large molecules (biologics). However, this two-species approach is not a regulatory requirement for GTx products, and a single nonclinical species may suffice with scientific justification (European Medicines Agency, 2008; U.S. Food and Drug Administration, 2013). The choice of nonclinical species should be permissive to vector transduction, biological activity of the transgene, and support the intended clinical

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ROA. Consideration should also be given to the immune status of such species for the human transgene protein product to ensure that exposure is adequate and irrelevant immune responses that are unique to the animals (and will not translate to the human) will not confound interpretation. Standalone toxicity studies in healthy wild-type animals are typically performed to support safety evaluation of the GTx product. However, safety endpoints may also be combined with efficacy endpoints in POC studies (i.e., hybrid pharmacology/ toxicity studies) or as standalone studies performed in a relevant animal model of disease. Animal models of disease have several advantages and disadvantages for evaluating the safety of GTx products, but their utility must be thoroughly considered. Unintended modified exposure to the GTx product may occur in an animal model of disease and result in toxicity that may or may not be translatable to the target clinical population. For example, the administration of AAV gene therapy to the acid sphingomyelinase deficiency (ASMD) knockout (KO) mouse model results in cardiovascular and liver toxicity due to rapid breakdown of accumulated sphingomyelin (associated with the disease model) into ceramide that is not observed in wild-type mice, rats, and dogs receiving 3-fold higher doses (Murray et al., 2015). Conversely, severe neurotoxicity uniquely develops in healthy, wild-type cynomolgus monkeys (Macaca fascicularis) but not mouse, cat, and sheep models of GM2 gangliosidosis due to aand b-hexosaminidase transgene overexpression following intracranial administration of AAVrh8, demonstrating the occurrence of toxicity in conventional animals but not disease models (Golebiowski et al., 2017). These examples reinforce the need for detailed phenotypic characterization of animal disease models prior to their use in assessing the safety of GTx products. Toxicity studies are typically performed with two time points (one acute and one chronic) reflecting peak and persistent transgene expression, respectively. Common choices include 4 or 6 weeks for the acute time point, and 13 weeks or occasionally 26 weeks for the chronic time point. Occasionally, longer-term studies may be required to assess the potential for delayed toxic effects and/or to demonstrate reversibility of toxic findings observed at earlier time points.

Kinetics of transgene expression and determination of peak, persistence, and clearance of the GTx product (vector DNA and transgene product) must be thoroughly characterized in the POC and BD studies. BD studies also support the list for tissue collection at necropsy during GLP-compliant, IND-enabling studies, particularly when the vector genome is detected in nonstandard tissues (such as the DRG, a known target tissue for AAV-based GTx products). Dose selection for toxicity studies is informed by the POC efficacy study. Commonly, the low dose in a GTx toxicity study is usually the minimally efficacious dose, the middle dose is usually the maximum efficacious dose or the optimal biological dose, and the high dose is selected to produce a toxic effect or provide an adequate safety margin (e.g., 5- to 10-fold) relative to the estimated maximum dose in humans (U.S. Food and Drug Administration, 2013). Toxicity studies for GTx programs are generally conducted in conjunction with BD studies as the latter provide the equivalent of PK endpoints necessary to correlate potential toxicity findings to exposure to the GTx product. Standard in-life endpoints such as clinical signs, physical examination, intermittent body weight measurements, and food consumption are included during the in-life phase of GTx toxicity studies. Other endpoints, such as safety pharmacology, immunotoxicity, and development and reproductive toxicology, are determined on a caseby-case basis depending on the GTx program’s need and relevance to the planned clinical trial. The anatomic pathology (see Basic Approaches in Anatomic Toxicologic Pathology, Vol 1, Chap 9) and clinical pathology (see Clinical Pathology in Nonclinical Toxicity Testing, Vol 1, Chap 10) analyses in general toxicity studies remain the gold standard for safety assessment of GTx products. Accordingly, primary aims in GTx safety studies are to characterize the incidence and onset of toxicity, address the dose–response relationship to toxicity findings, determine adversity and reversibility, and identify the NOAEL (Assaf and Whiteley, 2018; Hinderer et al., 2018; Hordeaux et al., 2020a; Hordeaux et al., 2018a). Comprehensive tissue list and clinical pathology analytes relevant to GTx products are provided in Tables 8.2 and 8.3, but special modifications may be required depending on several factors such as ROA and BD profile of the gene therapy

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TABLE 8.2

Recommended Clinical Pathology Sampling in Developing rAAV Test Articles for In Vivo Gene Therapya

Hematologic Evaluation

Complete blood count Leukocyte differential (automated) Platelet count Blood smear (cytological evaluation of cell morphology, performed if warranted) Bone marrow smear (cytological evaluation of cell morphology and numbers, performed if warranted)

Serum chemistry analysis (Minimum list of tests)

Albumin Globulin Total protein Albumin/Globulin (A/G) ratio Alkaline phosphatase (ALP) Alanine aminotransferase (ALT) Aspartate aminotransferase (AST) Gamma-glutamyl transferase (nonrodents) (GGT) Blood urea nitrogen (BUN) Creatinine Phosphorus Calcium Glucose Total bilirubin Triglycerides Total cholesterol Sodium Chloride Potassium Tissue-specific biomarkers (as warranted)dcreatine kinase (CK) for skeletal muscle, etc.

Coagulation

Activated partial thromboplastin time (APTT) Prothrombin time (PT) Fibrinogen

Urinalysis

Volume Color/Clarity pH Specific gravity Glucose Protein Ketones Blood Bilirubin

a

Table reproduced from Hutt et al. (2022), by permission of SAGE.

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TABLE 8.3 Recommended Tissue Sampling in Developing Test Articles for rAAV In Vivo Gene Therapy* Major Target Organs

Administration Sitea

(minimal tissue set, for exploratory studies)

Adrenal glandb Bone marrowa (femur, with marrow exposed, or sternum) Braina,b Dorsal root ganglia (cervical, thoracic and lumbar, multiple per segment)c Gonada,b (ovary or testis) Hearta,b Intestine, large e Colon Intestine, small e Jejunum Kidneya,b Livera,b Lunga Lymph node (mesenteric, and [if warranted] draining) Pancreas Skeletal muscle Skin Spleena,b Thymus Gross lesionsa

Ancillary organs (Comprehensive tissue list, for GLPcompliant toxicity studies)

Accessory sex glands, male e prostate gland, seminal vesicle Aorta Bone (vertebrae from cervical, thoracic, and lumbar segments) Connective tissue e brown and white adipose tissue Esophagus Intestine, small e duodenum, ileum (with Peyer’s patch) Intestine, large e cecum, rectum Eye/optic nerve Gall bladder Ganglion (trigeminal) Joint (femorotibial [“knee”]) Mammary gland Nerve (sciatic  tibial) Pituitary gland Reproductive organs, female e cervix, vagina, uterus Reproductive organs, male e epididymis, ductus deferens (Continued)

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TABLE 8.3

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Recommended Tissue Sampling in Developing Test Articles for rAAV In Vivo Gene Therapy*dcont’d

Ancillary organs (Comprehensive tissue list, for GLPcompliant toxicity studies) [cont’d]

Salivary glands e mandibular ( parotid and sublingual) Skeletal muscle e diaphragm, gastrocnemius Stomach Spinal cord (cervical and lumbar intumescence  midthoracic) Thyroid gland ( parathyroid gland) Urinary bladder

* Table reproduced with minor modifications from Hutt et al. (2022), by permission of SAGE. a Minimal list of tissues (italicized) that is recommended by the FDA (U.S. Food and Drug Administration) for evaluating biodistribution (along with blood) of gene therapy test articles (U.S. Food and Drug Administration, 2020). b Minimal list of organs to be weighed. c Dorsal root ganglia (DRG) should be collected per Society of Toxicologic Pathology (STP) recommended best practice for peripheral nervous system (PNS) sampling (Bolon et al., 2018) in nonclinical studies for recombinant adeno-associated virus (rAAV) test articlesdthough histopathologic evaluation may be performed at the Sponsor’s discretiondsince this organ is a sensitive target for the rAAV platform. Sampling several sacral DRGs also is advised considering their large size, ease of access compared with other DRGs, and proximity to the lumbar cistern (a common intrathecal injection site).

product (Hutt et al., 2022). For example, multiple DRGs are routinely collected in nonhuman primate studies considering the high incidence of DRG toxicity associated with AAV vector administration following intra-CNS and IV injections in this species (Hordeaux et al., 2020a).

3.4. Contemporary Toxicities Associated With In Vivo Gene Therapy The field of in vivo GTx provides a novel approach to treating diseases with unmet medical needs. Like other therapeutic modalities, patterns of toxicities associated with in vivo gene therapy are emerging in nonclinical studies as well as clinical trials. While still not fully characterized, such toxicity findings are encountered with reasonable frequency and contribute to common challenges faced during the development of in vivo gene therapy products. In the following section, we present four of the most discussed contemporary toxicology concerns associated with AAV-based in vivo gene therapy and attempt to draw similarities of such findings with other viral vectors (U.S. Food and Drug Administration, 2021). Immunotoxicity Associated With AAV-Based Gene Therapy All in vivo GTx platforms, viral and nonviral vectored, are inherently immunogenic due to their

foreign (“non-self”) nature to the animal or human host, albeit at different levels of immunogenicity. For viral vector–based GTx, viral capsid or envelope proteins can induce both innate and adaptive immune responses. The transferred genetic material (DNA or RNA in both viral and nonviral vectored platforms) is recognized by the innate immune system as pathogenassociated molecular patterns (PAMPs), and the initial innate response ultimately influences the character of the adaptive immune response (Newton and Dixit, 2012; Wright, 2020a,b). Although transgene protein products are intended to express the naturally occurring endogenous human protein and preclude the induction of immunogenicity in human patients, in certain instances transgenic proteins will be immunogenic in patients (e.g., complete loss of endogenous protein and consequently a lack of developmentally acquired tolerance for it). The importance of immunotoxicity for in vivo GTx products cannot be exaggerated as the entire field was negatively impacted following the immune-mediated death of GTx patient Jesse Gelsinger in 1999. This 18-year-old man with partial ornithine transcarbamylase (OTC) deficiency died acutely following administration of an adenovirus type 5 (Ad5)-based GTx candidate expressing human OTC due to systemic inflammatory response syndrome with multiorgan failure (Raper et al., 2003). In the lungs,

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hemorrhage, edema, fibrin, neutrophils, and necrosis were consistent with the final diagnosis of acute respiratory distress syndrome (ARDS). Necrosis was also observed in liver, spleen, and kidneys. In the bone marrow, bone marrow erythroid depletion was marked, and a left shift in granulopoiesis indicated a compensatory rise in myeloid cell proliferation secondary to an explosive increase in neutrophil consumption. There was diffuse cerebral edema and global anoxic encephalopathy. As a result, the GTx field dedicated extensive resources to hunting for a safer vector with lower immunogenic potential. This search ultimately resulted in the identification of AAVs and the development of many AAV serotypes as versatile GTx vectors. More recently, AAV vectors have been shown to be capable of inducing immunotoxicity in animals and human, and potentially resulting in fatality as evidently demonstrated in nonclinical monkey studies (Hinderer et al., 2018; Hordeaux et al., 2018b; Shirley et al., 2020; Zaiss et al., 2002). In fact, immune responses are considered the main driver for most reported AAV-associated toxicities seen in nonclinical studies and clinical trials (Hordeaux et al., 2018a; Samulski and Muzyczka, 2014). It is worth noting that the immune response against GTx products is not only a safety concern but also of concern to efficacy and patient eligibility for treatment. Natural (nonrecombinant or “wild-type”) adenoviruses and AAVs are prevalent in human populations, and the presence of preexisting neutralizing antibodies against the wild-type viral capsids will counteract the vector’s ability to exert its therapeutic effect. This leads to exclusion of patients who are seropositive for the vector serotype used in the viral vector–based GTx product. Additionally, neutralizing antibodies that develop following GTx administration limit the ability of vector readministration, which means that patients will only have a single chance for treatment with such therapeutic products. Cell-mediated immunity against viral vector capsids and transgene products also results in elimination of transduced cells as well as subsequent loss of efficacy and potential tissue injury (Figure 8.3). The efficacy and safety concerns associated with immunogenicity and immunotoxicity of GTx products, respectively, indicate that immune responses to GTx products will remain

a major hurdle to the field for the foreseeable future (Mays and Wilson, 2011; Nidetz et al., 2020; Verdera et al., 2020). INNATE IMMUNE RESPONSES TO AAV-BASED GENE THERAPY PRODUCTS

Many molecular mechanisms of innate and adaptive immune responses against AAV-based GTx products have been identified, and the field continues to evolve to elucidate additional mechanisms and effectively modulate such immune responses (Dauletbekov et al., 2019; Kuranda et al., 2018; Rabinowitz et al., 2019; Shirley et al., 2020). Almost all components of AAVbased GTx product (capsid, DNA construct, and transgene product) have the potential for inducing immunogenicity and associated toxicities. Capsid proteins are recognizable by the cell surface receptor Toll-like receptor 2 (TLR2), resulting in activation of the transcription factor nuclear factor kB (NFkB) and the production of inflammatory cytokines as demonstrated in primary human liver Kupffer cells in vitro (Rabinowitz et al., 2019). Vector DNA, particularly the unmethylated cytosine-phosphate-guanine (CpG) motifs, is recognized by the endosomal TLR9 in plasmacytoid dendritic cells ultimately through the myeloid differentiation primary response 88 (MyD88)-mediated pathway, resulting in the release of type I interferons (IFNs) (Zhu et al., 2009). These cytokines ultimately contribute to the induction of T-cell and B-cell adaptive immune responses (Zhu et al., 2009). Other elements may also induce the innate immune response against AAV-based GTx products. For example, double-stranded RNA (dsRNA) formed following AAV transduction can activate the cytosolic sensor melanoma differentiation–associated gene 5 (MDA5) (Shao et al., 2018). The viral dsRNA serves as a PAMP, and its binding to MDA5 induces expression of IFNa, IFNb, and other proinflammatory cytokines (Brisse and Ly, 2019). rAAV vectors carrying a scAAV induce a higher innate immune response mediated in a TLR9dependent pattern and also a stronger adaptive immune response directed against the vector capsid (Martino et al., 2011). AAV capsid can interact with the complement system (Zaiss et al., 2008), and several clinical trials for the treatment of DMD have been placed on hold due to AAV-related complement activation

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FIGURE 8.3 rAAV vectors can trigger adaptive immune responses follow vector administration. Periportal (A) and intraparenchymal (B) mononuclear cell infiltrates are readily demonstrated by immunoreactive (C) CD8þ T-cells in the liver of cynomolgus macaques 4 weeks post rAAV administration. (D) This is an IgG negative control. Adapted from Bolt MW, Brady JT, Whiteley LO, et al.: Development challenges associated with rAAV-based gene therapies, J Toxicol Sci 46:57–68, 2021 by permission.

with concomitant reductions in platelets and red blood cell counts as well as renal failure. ADAPTIVE IMMUNE RESPONSES TO AAV-BASED GENE THERAPY PRODUCTS

Antigenic epitopes on vector capsid proteins and/or the transgene protein product can induce humoral and cell-mediated adaptive immunity, ultimately generating long-lived memory B-cells and memory T-cells. The adaptive immune response against vector components may neutralize the required persistent transgene expression leading to loss of efficacy, preclude patients from receiving a second treatment due to the generation of neutralizing antibodies (particularly against capsid proteins), and may pose safety risks associated with product-related immunopathology. Infections with one or more wild-type AAV serotypes are prevalent in animals and humans (Boutin et al., 2010; Wang et al., 2018). Seroprevalence and neutralizing antibody titers against capsid proteins vary depending on the targeted AAV serotype and the animal or human population. Preexisting neutralizing antibodies to

a particular AAV serotype will bind and neutralize a GTx product of the same or crossreactive serotypes resulting in failure of treatment; such antibodies are a major limitation in recruiting seropositive animals and patients to nonclinical studies and clinical trials, respectively (Masat et al., 2013). Neutralizing antibodies may develop in seronegative patients following vector administration and will limit the repeat dosing in the patient, underscoring the importance of achieving long-lasting efficacy in the patient by a single GTx dose. Neutralizing antibodies can also develop against the transgene protein if it lacks homology in the nonclinical species or the human patient lacks the usual immune tolerance to such proteins due to inborn homozygous gene defects leading to the absence of the protein during the self-tolerizing period of immune system development. Cell-mediated immune responses carry the highest overall concerns for GTx (Vandamme et al., 2017). Cytotoxic CD8þ T cells directed against AAV2 capsid proteins have been shown to eliminate transgene-expressing hepatocytes in

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a hemophilia B clinical trial, resulting in loss of efficacy. Transgene-specific T-cell responses similarly eliminated dystrophin and a-1-antitrypsin expression in DMD and a-1-antitrypsin (AAT)deficient patients, respectively, following administration of AAV vectors, thereby leading to reduced or lost therapeutic efficacy. Serious adverse events (SAEs) of substantially increased serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities have been reported following administration of AAV9 expressing SMN1 approximately 3 weeks after treatment in two patients receiving 6.3  1013 vg/kg or 2  1014 vg/kg. Both patients demonstrated CD8þ T cell infiltration in liver biopsies, and the increases in serum activities of ALT and AST were attenuated by prednisolone treatment, collectively indicating that the liver damage was mediated in part by an adaptive immune response presumably directed against the GTx vector and/or transgene product (Chand et al., 2021; Feldman et al., 2020). Injection of AAV GTx intramuscularly (IM) or into the liver through the portal vein can generate regulatory T-cells that reduce the adaptive immune response and permit prolonged transgene expression. ASSESSMENT OF IMMUNOTOXICITY IN NONCLINICAL STUDIES

Immune responses following the administration of a GTx product can serve as either a primary mechanism of toxicity driven by anticapsid and/or antitransgene immune responses or as a secondary (indirect) mechanism of toxicity associated with primary tissue degeneration and necrosis driven by other product-related mechanisms (e.g., transgene overexpression leading to disrupted cell homeostasis). Regardless of the mechanism, immune responses to viral GTx typically manifest as mononuclear cell infiltrates (predominantly composed of CD3þ T-cells) in the affected tissues. Over time, the leukocyte infiltration may lead to inflammation (i.e., leukocyte accumulation resulting in tissue damage) and some degree of parenchymal degeneration and necrosis, particularly of transgene-expressing target cells. Depending on the target tissue and the extent of tissue damage, stromal inflammatory and repair reactions such as fibrosis in nonneural tissues, microgliosis and astrogliosis in CNS tissues, and hypertrophy of resident phagocyte populations (e.g., sinusoidal Kupffer cells in liver) can be observed.

Considering the biological nature of GTx and the potential for immunotoxicity, any GTx program must have a strategic immunogenicity assessment plan to evaluate the potential impact of immune responses on the efficacy and safety of the GTx product. Immunogenicity is expected with administration of a human-derived GTx product to animals, and standard immunogenicity testing may not discriminate its impact on efficacy and safety. Immunotoxicity assessment for GTx is generally accomplished by collecting appropriate samples from nonclinical in vivo studies for potential future analysis if evidence of immunotoxicity is observed in the animals; such analyses essentially employ the general principles for assessing biologic products as discussed in ICH S6(R1) “Preclinical Safety Evaluation of Biotechnology-Derived Pharmaceuticals” (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, 2011). The two primary triggers for assessment of immunogenicity and immunotoxicity for the GTx product are 1) low transgene expression and 2) evidence of immunotoxicity histologically (e.g., observation of mononuclear cell infiltration in target tissues, particularly centered around transgeneexpressing cells). While primarily considered an efficacy endpoint, loss of transgene expression may impact the adequate assessment of toxicity due to the diminished exposure to the protein product. Measurement of antitransgene antibodies in serum samples will aid in addressing this possibility. Frozen lymphocytes harvested (when possible) pre- and postadministration of the GTx product, such as peripheral blood mononuclear cells (PBMCs) and lymphocytes isolated from disaggregated tissues (e.g., lymphoid tissues and liver), may be collected and stored for future assessment of capsid- and/or transgene-specific T-cells by immunological methods such as flow cytometry or enzymelinked immunospot (ELISpot). Additional serum samples may also be collected, particularly for investigative toxicity studies, to gain further understanding of the innate immune responses shortly after vector administration such as production of C-reactive protein or other acutephase proteins and cytokines like tumor necrosis factor alpha (TNF-a) and interleukin-6 (IL-6). Such innate responses have been frequently associated with immunotoxicity during GTx programs forced to use high doses of AAV vectors to achieve sufficient cell transduction to provide efficacy. These additional approaches

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are typically performed in nonrodent species where the larger body size permits repeated sampling from the same animal over time. Despite the major advances in understanding the immune response in GTx and its effect on efficacy and safety, several nonclinical studies have failed to predict immunogenicity in humans. For example, a lack of immune responses has been reported in hemophilia B dogs and monkeys failed to mount an immune response to eliminate transgene-expressing cells (Mingozzi and High, 2013; Niemeyer et al., 2009). Therefore, the translatability of nonclinical findings should be critically considered, and additional investigations may be needed to improve the predictive value of nonclinical findings with respect to potential implications in the clinic. Acute Liver Injury Associated With HighDose Gene Therapy In vivo administration of GTx products permits systemic distribution of the test article within the host. rAAV serotypes have widespread tropism to various tissues in vivo (Srivastava, 2016; Zincarelli et al., 2008). Different viral serotypes favor particular organs, a feature that has controlled the choice of rAAV vector to use for GTx against diseases stemming from diverse organs (Pipe et al., 2019). However, the potential for multiorgan tropism increases the chance of off-target effects by transducing and expressing the transgene product in an undesired tissue or cell type. Such off-target expression may increase the potential safety liability without improving the therapeutic benefit of GTx treatment. Certain organs are impacted by GTx products more than others. For example, the liver is considered a key target site of the rAAV platform. Hepatocytes are effectively transduced by rAAV following systemic (e.g., IM, IV) or organ-targeted (e.g., intracisternal [ICM], intracerebroventricular [ICV], or intrathecal [IT] for the CNS; subretinal or intravitreal for the eye) administration. Exposure to high doses of AAV (>1  1014 viral genomes (vg) per kilogram of body weight) particularly following systemic administration may produce toxic levels of transgene expression that induce hepatocellular degeneration (single cell or one or more areas with multiple cells) and ultimately necrosis (Hinderer et al., 2018). Additionally, and as noted in the previous section, immune responses against capsid components of rAAV may result in hepatocellular degeneration.

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Acute hepatotoxicity generally manifests as transient and asymptomatic increases in serum activities of hepatocyte cytosolic enzymes like ALT and AST early after rAAV administration (Markusic and Herzog, 2012; Verdera et al., 2020). However, severe and occasionally fatal hepatotoxicity associated with symptoms of liver failure has been reported following high-dose rAAV administration in both animals and humans (Anonymous, 2020; Hinderer et al., 2018). The first lethal case of acute GTx-related hepatotoxicity in a human patient (in 1999) was associated with a systemic injection of 6  1014 Ad5 particles/kg (total dose ¼ 3.8  1013 particles) expressing the OTC gene into the right hepatic artery (Raper et al., 2003). The patient developed an altered mental status and jaundice 18 h postadministration, indicating hepatotoxicity, and death from multiorgan failure and ARDS occurred at 98 h postdose. Histopathological examination reflected widespread necrosis in the liver. Two decades later (in 2018), the laboratory that reported the fatal human case following Ad5 administration published similar severe toxicities in rhesus macaques (Macaca mulatta) following high-dose systemic (IV) administration of AAVhu68 vectors expressing the SMN1 gene (Hinderer et al., 2018). Severe, acute hepatotoxicity proved to be fatal in one of three monkeys that received a high dose (2  1014 vg/kg) (Figure 8.4). In a second report, acute hepatotoxicity occurred in a single rhesus monkey following IV injection of AAV-PHP.B expressing GFP at a dose of 7.5  1013 vg/kg (Hordeaux et al., 2018c). The hepatotoxicity in this animal was characterized by increased serum activities of aminotransferases on Day 3 that decreased on Day 5, but ensuing profound thrombocytopenia and diffuse bleeding was observed and required euthanasia by Day 5 postadministration. Histologically, there was acute hemorrhage in the diaphragm, heart, and subcutis as well as marked multifocal hepatocellular degeneration and necrosis. An AAV-PHP.B vector expressing SMN1 (AAV-PHP.B-CBh-SMN1) at doses of 2  1013, 5  1013, or 1  1014 vg/kg resulted in acute hepatotoxicity and disrupted coagulation in cynomolgus monkeys 3–4 days following vector administration with one monkey found dead on Day 4 postdose (Palazzi et al., 2021). The hepatotoxicity in these monkeys was likely related to the direct vector effect on the liver as the hepatocyte degeneration and necrosis was not associated with inflammatory cell infiltrates.

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FIGURE 8.4 High-dose rAAV administered intravenously to juvenile rhesus monkeys required euthanasia on study day 4 due to massive acute hepatocellular necrosis (A) with activation of the clotting cascade as shown by sinusoidal fibrin deposition (B, arrowheads) and acute fibrin thrombi (C, arrow) in portal veins. Fibrin deposition is visualized more easily by IHC against fibrinogen (D). Adapted from Hinderer C, Katz N, Buza EL, et al.: Severe toxicity in nonhuman primates and piglets following high-dose intravenous administration of an adeno-associated virus vector expressing human SMN, Hum Gene Ther 29:285–298, 2018 by permission.

The coagulation disorder in these monkeys was characterized by decreased platelets and fibrinogen, increased prothrombin time (PT) and activated partial thromboplastin time (APTT), and detectable D-dimers (a fibrin degradation product produced when thrombi are dissolved), suggesting a consumptive coagulopathy. Similar hepatotoxicity or coagulopathy was not observed in Wistar Han rats receiving the same vector at similar dose levels, suggesting that such hepatoxicity against the tested vector was highly species specific (Palazzi et al., 2021). The clinical manifestation of acute liverrelated findings (increased serum AST and ALT activities) in nonclinical toxicity studies has

generally translated well, predicting the potential for similar findings in human clinical trials that involve the administration of high-dose AAV GTx products. Acute liver injury was reported in pediatric patients receiving onasemnogene abeparvovec (Zolgensma; an AAV9 expressing SMN1) with 90% of patients demonstrating increased serum ALT and/or AST activity(ies) during therapy, and two patients receiving 6.3  1013 vg/kg or 2  1014 vg/kg were reported for developing serious adverse events (SAEs) of substantially increased serum ALT and AST activities (Chand et al., 2021; Feldman et al., 2020; Mendell et al., 2021). The transaminase increases were biphasic, with initial

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increases beginning at 1 week following vector administration and a second peak at the time of the tapering and discontinuation of systemic corticosteroid therapy at 1 month postdose (suggesting the second peak as a result of an adaptive immune response). Although the mechanism of high-dose rAAV toxicity remains to be determined, the acute onset of the toxicity and the lack of a robust cellular immune response suggest that direct hepatocyte injury and/or activation of the innate immune system post vector administration might be the primary mechanisms of acute high-dose rAAV toxicity. Liver targeting and acute liver toxicity with transient and asymptomatic increases in liver aminotransferases are not unique to rAAV products. Dogs administered LV vectors demonstrate slightly increased serum ALT and AST activities within days postinfusion that return to baseline on Day 7 postadministration (Cantore et al., 2015). This finding was attributed to an anaphylactoid reaction to the GTx product as antihistamine and antiinflammatory treatments prevented the occurrence of similar acute hepatotoxicity in an additional dog administered the same vector. Similar self-limiting acute hepatocellular toxicity with transient slight increases in serum ALT and AST activities was observed in pig-tail macaques (Macaca nemestrina) a few days following IV injection of LV vector (Milani et al., 2019). Dorsal Root Ganglion (DRG) Toxicity Associated With AAV Vectors DRG toxicity has recently emerged as a platform concern for rAAV vectors despite their generally safe profile. Such vectors can readily access DRG following IV administration or direct CNS delivery into the cerebral spinal fluid (e.g., ICM, ICV, or IT injection). The DRGs are readily exposed to AAV because they are not included within the BBB. Instead, DRG vasculature is lined by a fenestrated endothelial lining (Bernal et al., 2002; Gray et al., 2013), and DRGs reside within the spinal subarachnoid space allowing exposure via the CSF. Accordingly, DRG sensory neurons and their projecting axons are readily accessed for transduction by AAV delivered into the blood or the CSF (Federici et al., 2012). A comprehensive review of AAV GTx products involving 256 NHPs across 33 nonclinical studies reported a high incidence of DRG pathology, with 83% and 32% of animals affected, respectively, following intra-CSF or IV administration of

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rAAV vector (Hinderer et al., 2018; Hordeaux et al., 2020a; Hordeaux et al., 2018a; Hordeaux et al., 2018b). This analysis shows that DRG pathology is associated with exposure to AAVbased GTx products in general, occurring across 5 different capsids, 5 different promoters, and 20 different transgenes. Therefore, this finding appears to be a modality-related safety concern almost universally present following administration of AAV GTx, at least in NHP with administration directly into the CSF; the comparative sensitivity of DRG in other nonclinical species is uncertain due to the scarcity of reports describing evaluation of this tissue. Milder findings related to AAV-associated DRG pathology are characterized by increased numbers of satellite glial cells (“increased cellularity” in current INHAND [International Harmonization of Nomenclature and Diagnostic Criteria] parlance for neural findings in nonclinical studies) and occasionally infiltration of mononuclear cells (macrophages and/or lymphocytes) around and sometimes impinging on DRG sensory neurons (Figure 8.5). More severe DRG findings include sensory neuron degeneration or necrosis, sometimes with ongoing removal of neuronal debris by phagocytic cells (“neuronophagia” in INHAND), with secondary nerve fiber degeneration of DRG axons projecting through the spinal nerve roots (mainly dorsal ones) and ascending tracts of the spinal cord dorsal funiculi, and to a lesser degree the dendrites passing in peripheral nerves to reach the DRG. In this 256 NHP review, most findings were of minimal severity (i.e., affecting 10% or less of the affected DRG); however, moderate to marked lesions (affecting 50% or more of the involved tissue) were observed in a few DRG. The severity of findings in the spinal cord white matter tracts carrying DRG axons was generally higher compared with DRG themselves, which is consistent with spinal cord serving as the summation of input from many DRG simultaneously. For a given animal, DRG pathology was not uniform across the entire spinal column, indicating that a review of one or a few DRG may not adequately assess DRG toxicity associated with AAV GTx. Accordingly, a more comprehensive sampling of multiple DRG across the cervical, thoracic, lumbar, and sacral regions is warranted to screen the extent of DRG pathology within the animal (Bolon et al., 2018). Sacral DRG also might be examined, especially if the AAV product was delivered locally by IT injection into the lumbar

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FIGURE 8.5 DRG toxicity and secondary axonopathy after rAAV intra–cisterna magna administration. Axonopathy (top left) manifests as clear vacuoles that are either empty or filled with macrophages and cellular debris (arrow). DRG lesions (top right and bottom left): Arrow shows neuronal cell body degeneration, whereas circle indicates mononuclear cell infiltration. Bottom right picture shows immunostaining for the transgene encoded by AAV [green fluorescent protein (GFP) in this case]. Figure and legend are adapted with minor modification from Hordeaux J, Buza EL, Jeffrey B, et al.: MicroRNA-mediated inhibition of transgene expression reduces dorsal root ganglion toxicity by AAV vectors in primates, Sci Transl Med 12, 2020b by permission.

cistern. In like manner, inclusion of trigeminal ganglia (which are also susceptible to AAVassociated pathology) may be considered, especially for vectors given by the ICM or ICV routes. Regardless of the extent of DRG changes, most of the 256 NHPs remained clinically asymptomatic. Only 3 out of 204 animals administered AAV by ICM or lumbar IT injections developed clinical signs of ataxia and/or tremors. Similarly, 2 of 56 animals assessed by electrophysiological (nerve conduction velocity) testing demonstrated a reduction in the amplitude of sensory action potentials in the median nerves that correlated with axonopathy and periaxonal fibrosis in the

median nerve while still remaining clinically asymptomatic. DRG toxicity in NHP is further supported by others reporting similar findings following IT administration of AAV9 (Pardo et al., 2020; Perez et al., 2020; Therapeutics, 2019). While most reports highlight DRG toxicity in NHP, others have reported DRG toxicity in other nonclinical species such as mice, rats, and pigs (Bolt et al., 2021; Hinderer et al., 2018; Palazzi et al., 2021; Van Alstyne et al., 2021). The same group reported an earlier study of more severe pathology and overt toxicity involving progressive proprioceptive deficits and ataxia in 3 piglets (1–4 weeks of age) within 14 days of

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high-dose AAVhu68 injection (2  1014 vg/kg IV). Interestingly, NHPs (14-month-olds) in the same study given an identical AAV exposure (2  1014 vg/kg IV) of the same test article also exhibited the DRG pathology but with no clinically apparent sensory deficits (Hinderer et al., 2018). The small numbers of pigs and the disparity in developmental age at exposure provide no explanation regarding why infant pigs developed symptomatic AAV-induced DRG pathology while juvenile NHPs did not. Concern regarding the potential risk posed by DRG toxicity is further indicated by the potential that humans are as sensitive as NHP to AAVassociated DRG pathology. For instance, a clinical hold was placed on the experimental IT administration of AVXS-101 (now available as onasemnogene abeparvovec [Zolgensma]), an AAV9-based GTx product approved for the IV treatment of SMA. This hold was placed due to the presence of mononuclear cell inflammation and occasional neuronal degeneration in the DRG of NHP following IT but not IV administration. No adverse sensory deficits are reported in human patients who have already received the same product by IT or IV injection (Novartis, 2019). Similarly, in a human patient with familial amyotrophic lateral sclerosis (ALS), IT delivery of AAVrh10-microRNA to knockdown the expression of mutated superoxide dismutase 1 (SOD1) was associated with clinical sensory neuropathy, contrast enhancement of lumbar DRGs and the cauda equina on magnetic resonance imaging, and, upon autopsy, histological evidence of sensory neuron depletion, gliosis, and T-cell infiltration of DRGs and spinal cord (Mueller et al., 2020). This patient also showed skeletal muscle denervation in the gastrocnemius muscle as well as increased serum activities of hepatic aminotransferases related to centrilobular hepatic injury. This single case did not permit differentiation regarding how much of the DRG injury was related to the AAV GTx product versus the chronic neurodegenerative disease itself. Several hypotheses have been proposed for the mechanism of AAV-associated DRG toxicity. Viruses in general may damage cells by a direct effect on cellular processes (e.g., induction of proapoptotic pathways) or incite indirect cytotoxic T-cell responses against virus-infected cells. Transgene overexpression resulting in supraphysiological levels of transgene protein product may induce the unfolded protein response

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(UPR), an intracellular safety checkpoint normally triggered to eliminate misfolded or unfolded proteins to prevent stress on endoplasmic reticulum (ER) function. However, if the UPR fails to restore cellular homeostasis, the cell undergoes programmed cell death. This mechanism of ER stress and subsequent activation of UPR and cell death has been proposed as one of the primary mechanisms of AAVassociated degeneration and cell death for both hepatocytes and DRG neurons (Balakrishnan et al., 2013; Hordeaux et al., 2020a; Hordeaux et al., 2020b; Sen et al., 2014). In the comprehensive NHP analysis mentioned earlier, the severity of DRG pathology was greatly impacted by the choice of transgene. Moreover, knockdown of AAVhu68-mediated transgene overexpression for GFP or alpha-iduronidase (hIDUA, loss of which leads to mucopolysaccharidosis type I) by including microRNA183 (miR183) target sequences in the GTx vector prevents the occurrence of DRG toxicity compared with monkeys that received a GTx product bearing the same transgene but without protective miR183 target sequences. The inclusion of miR183 target sequences within the transgene expression cassette results in targeted degradation or blocking of the translation of the transgene mRNA within DRG neurons (due to high expression of miR183 in DRG neurons) while maintaining high transgene expression in other cell types. In the same analysis, the administration of exogenous steroids to suppress antiGTx immune responses did not mitigate DRG toxicity in the absence of miR183 targeting sequences, supporting transgene overexpression and not immune responses against capsid or transgene proteins as a primary mechanism of DRG toxicity. Neuronal degeneration associated with chronic activation of UPR in the brain of cynomolgus macaques was also reported following b-N-acetylhexosaminidase transgene overexpression mediated by intrathalamic AAVrh8 infusion (Golebiowski et al., 2017). While UPR-mediated cell death may explain toxicities related to protein overproduction, it does not address DRG toxicities associated with transgene mRNA (including microRNA transgene products). DRG toxicity was also reported following AAVrh10-mediated microRNA against SOD1 in ALS patient. These reports indicate that transgene mRNA products may drive cell death and lead to DRG toxicity. Moreover, the presence of mononuclear cells infiltrating the affected DRG

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in some reports suggests a primary or secondary role for immune responses in the initiation or exacerbation of DRG toxicity, respectively. The role of immune responses in DRG toxicity is highlighted by studies that expressed foreign and “non-self” transgene proteins. AAV-mediated expression of GFP results in CD8þ T-cell-mediated inflammation and necrosis in the striatum of cynomolgus macaques with poor hand–eye coordination, impaired balance, and ataxia following intraparenchymal infusion into the putamen (Samaranch et al., 2014). DRG toxicity has also been observed in Wistar rats receiving GFP-expressing AAV6 (Mason et al., 2010). Modification of GFP that renders this protein nonimmunogenic (“stealth” GFP) prevents GFP-mediated inflammation in intercostal nerves of Wistar rats transduced by LV vectors (Hendriks et al., 2007). A robust immunosuppression regimen (e.g., the addition of rituximab and sirolimus to methylprednisolone) in a second ALS patient treated with AAVrh10-microRNA targeting SOD1 for the treatment of ALS mitigated the inflammatory response and prevented the occurrence of sensory dysfunction (Mueller et al., 2020), further suggesting a role of immune responses in AAVassociated DRG toxicity. Moreover, several elements of rAAV vectors can induce the innate immune system, activity of which may ultimately shape the inflammatory response against vector components and lead to DRG toxicity (Hutt et al., 2022). These data suggest that the mechanism of DRG toxicity is likely multifactorial and may differ depending on many factors such as the AAV vector capsid serotype, the nature of the transgene expression and function, immunogenicity, and the dose-dependent transgene expression. DNA Integration, Hepatocellular Carcinoma, and AAV-Based Gene Therapy Administration of GTx products is intended to provide life-long therapeutic benefit to the patient by correcting gene deficits and maintaining persistent gene expression in the modified cells following gene transfer. Genotoxicity with a potential for carcinogenicity are concerns associated with several gene therapy platforms. The premise of such concerns stems from the possibility that the introduced genetic material may insert into the host genome and create a mutagenic effect (insertional mutagenesis) that results in a genotoxic effect and eventual neoplastic transformation.

However, viral vectors commonly used for GTx vary in their abilities to integrate into the host genome. Recombinant AAV vectors are considered “nonintegrating” GTx products as the vector genome forms a monomeric or concatemeric (i.e., multiple linked copies), circular, double-stranded episome that allows viral DNA to persist in an extrachromosomal form (Balakrishnan and Jayandharan, 2014). Recombinant AAV vector genomes do not need to integrate into the host genome to be biologically active. In contrast, retroviruses and LV are “integrating” vectors in which the vector genome must be permanently inserted into the host chromatin to exert its intended biological activity. This section discusses safety concerns associated with nonintegrating AAV-based GTx products, while integrating retrovirus and LV GTx products are considered below under ex vivo gene therapy. Wild-type AAV integrates into the host cell DNA, establishes latency in the absence of a helper virus, and integrates into a specific chromosomal locus termed the adeno-associated virus integration site (AAVS). Wild-type AAV integration into oncogenes has been described in human patients with HCC (Balakrishnan and Jayandharan, 2014). Site-specific integration of wild-type AAV has been linked to activities within the native ITR and Rep proteins. The Rep proteins are eliminated from rAAV genomes, which lower the risk of rAAV integration. Nonetheless, rare integration events of the rAAV vector genome have been recognized at a very low frequency (of less than 104 insertional events per diploid genome 1 mg of total DNA) following systemic administration (IV or IM) in NHPs (Nowrouzi et al., 2012). Despite this ultralow frequency of rAAV integration, reports from as early as 2001 have highlighted the genotoxic potential of rAAV as rare inducers of HCC in a murine model of mucopolysaccharidosis type VII (Donsante et al., 2001). A growing number of similar reports have identified several risk factors associated with AAVinduced HCC including dose, strong promoter/ enhancer elements, and transduction of rapidly dividing cells such as hepatocytes in neonatal (still developing) animals, organs undergoing substantial repair (e.g., after partial hepatectomy), or organs with extensive inflammation (e.g., nonalcoholic fatty liver disease [NAFLD]) (Chandler et al., 2015; Dave and Cornetta, 2021; Logan et al., 2017). Integration of rAAV DNA associated with murine HCC is reported mostly in a region within the RNA Imprinted and Accumulated in

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Nucleus (Rian) locus on mouse chromosome 12, a mutation-prone site that is not present in the human genome (Chandler et al., 2015). Hence, the relevance of such mouse findings to human risk assessment remains unclear. In addition to mice, GTx vector integration has been reported in rats, monkeys, and humans, but with no apparent pathological consequences (European Medicines Agency, 2010). However, clonal expansion of hepatocytes in the livers of six out of nine Factor VIII (FVIII)-deficient hemophilia A dogs has been reported, with two animals showing gradual increase of FVIII activity starting 4 years after treatment (Nguyen et al., 2021). However, no HCC was observed in these dogs, and the site of integration differed from those observed in HCC human patients with wild-type AAV integration. Consequently, while the association between rAAV and HCC remains unclear, the potential of rAAV GTx as a rare cause of genotoxicity is gaining traction and cannot be completely ignored in nonclinical discovery and development of GTx products. Accordingly, vector integration analysis may be warranted in some rAAV programs on a case-by-case basis going forward, with the decision being driven primarily by risk–benefit considerations for the patient population. Integration analysis is more likely to be useful for conditions where an acceptable standard of care already exists (e.g., hemophilia) or if evidence of cellular alterations is observed, such as the presence of nodular cellular proliferation, clonal expansion of transgene-expressing cells, or tumor formation.

4. EX VIVO GENE THERAPY 4.1. General Concepts of Ex Vivo Gene Therapy Autologous Lentiviral Vector–Transduced CD34þ Hematopoietic Stem Cell Ex Vivo Gene Therapy For the purpose of this chapter, ex vivo LVbased genetically modified autologous cell therapies (ex vivo GTx) are based on gene addition of human autologous CD34þ hematopoietic stem and progenitor cells (HSPCs) through the transfer of a therapeutic transgene using LV vectors. There are different HSPC subpopulations that express the CD34þ stem cell surface marker including hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). This mixed CD34þ cell population is included in autologous

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ex vivo GTx. HSCs are the most undifferentiated stem cell type and can either self-renew as HSC or asymmetrically differentiate into MPP (Figure 8.6). This section will be referring to autologous ex vivo CD34þ HSC GTx with the understanding that other HSPCs are also included. LV vectors are used to introduce a therapeutic transgene into the DNA of the recipient CD34þ HSC, after which the genetically modified HSCs are infused into the patient to serve as cellular vehicles to deliver therapeutic proteins into the circulation, cross-correcting tissues, and the CNS or to restore function to the genetically modified cells (Ferrari et al., 2021). One of the potential applications for ex vivo LV-transduced CD34þ HSC GTx is to overcome the patient’s missing or mutated gene by adding a functional copy of the gene (therapeutic gene or transgene) into the patient’s HSCs. This approach is well suited for the treatment of many inherited monogenic diseases such as blood-borne and metabolic disorders (lysosomal storage diseases [LSDs]). There are numerous ongoing clinical trials using ex vivo HSC GTx with an LV vector being evaluated for the treatment of metabolic and blood disorders, and several ex vivo GTx products have been approved using an LV or gammaretroviral vector (GRV) for clinical use such as Skysona, Strimvelis, and Zynteglo. Allogeneic hematopoietic stem cell transplantation (HSCT) has also been used as a treatment for blood disorders and considered a clinical standard to correct congenital or acquired defects in blood cell production or immune function as well as restoring hematopoiesis following cytotoxic oncology treatment. The first successful transplantations for the treatment of X-linked severe combined immunodeficiency (SCID-X1) occurred in 1968 using allogeneic HSCT (Ferrari et al., 2021). Since then, significant improvements have been made with regard to donor matching, management of graft-versus-host disease (GvHD), infection, and toxicity as well as conditioning regimens. In the oncology setting, conditioning regimens are administered in allogeneic hematopoietic cell transplantation (HCT) to provide sufficient immunoablation to prevent graft rejection and reduce the tumor burden. These advances allow the use of allogeneic HSCT for the treatment of many genetic diseases. However, the application of allogeneic HSCT is limited by the availability of suitable immunocompatible matched donors and the potential risk of GvHD. In contrast, use of autologous

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FIGURE 8.6 Hematopoietic Stem Cells (HSCs) Hierarchy. Bone marrow–resident hematopoietic stem and progenitor cells express the CD34þ cell surface marker. Bone marrow–resident HSCs and hematopoietic progenitor cells replenish blood and tissues with new mature cells. Both HSCs and hematopoietic progenitor cells express the cell surface marker CD34, which is used to enrich a mixture of hematopoietic stem and progenitor cells for transplantation and gene therapy. HSCs can be classed as long-term hematopoietic stem cells (LT-HSCs) or shortterm hematopoietic stem cells (ST-HSCs). ST-HSCs progressively acquire lineage specifications to differentiate into lineage-committed progenitors and eventually terminally differentiated cells, which are released into the peripheral blood. A simplified scheme of human hematopoiesis is presented here. Alternative models have been postulated on the basis of cell surface marker analyses, in vitro and in vivo functional assays, clonal tracking by insertion analyses in hematopoietic stem and progenitor cell gene therapy studies, and single-cell RNA analyses. Mendelian genetic disorders can affect self-renewal, differentiation, and/or the function of different blood and immune cells. Examples of genetic diseases for which gene therapy is under investigation or approved are represented in white boxes below affected cell types. Wiskott–Aldrich syndrome affects platelets and other lineages. CDP, common dendritic progenitor; CID, combined immunodeficiency; CLP, common lymphoid progenitor; CMP, common myeloid progenitor; GMP, granulomonocytic progenitor; LMMP, lymphoid-myeloid primed progenitor; MEP, megakaryocytic–erythroid progenitor; MPP, multipotent progenitor; NK cell, natural killer cell; preB, pre-B cell; preT, pre-T cell; SCID; severe combined immunodeficiency. Adapted from Ferrari G, Thrasher AJ, Aiuti A: Gene therapy using haematopoietic stem and progenitor cells, Nat Rev Genet 22:216–234, 2021 by permission.

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genetically corrected HSCs avoids these limitations since the source of HSCs originates from the patient, which allows the engrafted “self” cells to avoid immune reactions directed against the “non-self” antigens present on allogeneic HSCs. This section will focus on LV-mediated ex vivo genetically modified autologous CD34þ human HSCs (hHSCs) to express a functional therapeutic transgene product to treat inherited monogenetic metabolic disorders such as lysosomal storage diseases caused by enzyme deficiencies resulting in pathological accumulation of substrate. CD34þ cells can be classified as long-term hematopoietic stem/progenitor cells (LTHSPCs) or short-term hematopoietic stem/ progenitor cells (ST-HSPCs). In the bone marrow, HSCs maintain their numbers while also differentiating to replenish the entire blood system. They undergo asymmetric cell division which is the mechanism that balances HSC self-renewal and differentiate into lineagecommitted progenitor cells producing all types of blood-forming cells (Figure 8.6). HSPC reconstitution transpires in two phases where shortterm reconstitution occurs approximately 3 months posttransplantation as compared to 6–9 months for the long-term HSPCs reconstitution (Biasco et al., 2016; Ferrari et al., 2021; Morgan et al., 2017a; Naldini, 2019). The ST-HSPC is composed of a larger cell population compared to the long-term engrafting cells (LT-HSPCs), which are responsible for achieving hematopoietic steady state (as shown in the Human Hematopoietic Reconstitution Model and described later in this section; Figure 8.7) (Biasco et al., 2016; Biasco et al., 2018). Fundamental differences exist between autologous ex vivo LV gene-corrected HSCs, allogeneic

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HSCT, and in vivo GTx approaches. First, HSCspecific GTx relies on ex vivo gene transfer by transducing the patient’s own HSCs outside the body with a vector (usually an integrating vector) and then reintroducing the modified cells back into the patient by IV infusion. The genetically modified HSCs migrate to the bone marrow where they engraft and differentiate thereby producing a new population of modified cells effectively passing the transgene to daughter blood cells including daughter cells (monocytes) that engraft in the brain as microglia (Ferrari et al., 2021). The drug product is referred to as autologous ex vivo LV-transduced CD34þ hHSCs that have been genetically modified to express the functional protein. Second, allogeneic HSCT collects HSCs from a healthy donor and transplants the unmodified cells that naturally express the desired protein into the patient. These donated HSCs also migrate and subsequently engraft in the bone marrow, where they differentiate and produce daughter cells expressing the naturally expressed protein. In contrast, gene addition is accomplished by ex vivo transduction. Lastly, in vivo GTx products can be administered directly into the patient as described in the previous section. HSCs are well suited as a reservoir for ex vivo LV-transduced GTx as they are long-lived, multipotent, and quiescent. Cellular quiescence is a reversible cell cycle arrest that is poised to reenter the cell cycle in response to a combination of cellintrinsic factors and environmental cues. In HSCs, a coordinated balance between quiescence and differentiating proliferation ensures longevity and protects the cells from both genetic damage and stem cell exhaustion (Yamada et al., 2013). CD34þ HSCs represent much less than FIGURE 8.7 Human Hematopoietic Reconstitution Model. Hematopoietic reconstitution occurs in two distinct clonal waves. Steady-state hematopoiesis after transplant is maintained by both hematopoietic stem cells (HSCs) and multipotent progenitors (MPPs). Adapted from Biasco L, Pellin D, Scala S, et al.: In vivo tracking of human hematopoiesis reveals patterns of clonal dynamics during early and steady-state reconstitution phases, Cell Stem Cell 19:107e119, 2016 by permission.

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FIGURE 8.8 Autologous ex vivo gene therapy using lentiviral vector transduced CD34þ hematopoietic stem and progenitor cells (HSPCs). Autologous HSPCs are collected from the patient through multiple aspirations from the iliac crests or by apheresis or leukapheresis following the administration of growth factor and a chemokine antagonist. The collected material is enriched for CD34þ cells cultured in the presence of cytokines and genetically modified either by gene addition using gammaretroviral or lentiviral vectors or gene editing using zincfinger, CRISPReCas9, or transcription activatorelike effector nuclease programmable endonucleases. Before gene therapy, a conditioning preparatory regimen is usually administered to patients to deplete endogenous HSPCs. The intensity of conditioning ranges from reduced intensity (partial) to myeloablative, depending on the disease and the engraftment level required to reach the therapeutic threshold. The medicinal product is represented by the gene-corrected cells, ready for infusion at the end of the manipulation or after a cryopreservation and thawing step. Quality control tests performed on the drug product may include those on viability, sterility, endotoxin level, mycoplasma, immune phenotype, number of vector copies per diploid genome, transduction efficiency, transgene expression, vector production impurities, and whether the vector is replication competent. In the case of fresh product or rapidly progressive diseases, a two-step strategy is used to allow urgent treatment without completion of all tests. NGS, nextgeneration sequencing. Adapted from Ferrari G, Thrasher AJ, Aiuti A: Gene therapy using haematopoietic stem and progenitor cells, Nat Rev Genet 22:216e234, 2021 by permission.

1% of the normal adult human bone marrow stem cell population. They are collected via bone marrow aspiration or apheresis (a procedure that involves removing whole blood from the patient and separating the blood cells into different components postmobilization). Compared to bone marrow aspiration, when possible, apheresis is the preferred procedure for collecting the CD34þ HSCs because it yields more stem cells, allows faster hematopoietic reconstitution in the bone marrow postinfusion,

and is more convenient, less painful, and presents a lower risk of sample contamination (Figure 8.8) (Ferrari et al., 2021). To increase the number of circulating CD34þ HSCs in the peripheral blood before apheresis, patients can be given HSC-mobilizing agents such as granulocyte colony-stimulating factor (G-CSF) and plerixafor (AMD3100), which is a bicyclam molecule that antagonizes the binding of the chemokine stromal cell–derived factor-1 (SDF-1) to its cognate receptor CXC chemokine receptor

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(CXCR4) (Uy et al., 2008). Following apheresis, the sample is enriched for CD34þ cells. The enriched CD34þ HSCs are then transduced ex vivo with an LV vector containing a ribonucleic acid (RNA) transcript that, after reverse transcription, results in codonoptimized complementary deoxyribonucleic acid (cDNA) that, upon its integration into the human genome, encodes for functional therapeutic protein. Prior to reinfusing the ex vivo LV-transduced CD34þ hHSCs, the patient receives a conditioning agent to create space in the bone marrow to enable engraftment resulting in a chimeric cell population (mixed

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population of transduced and nontransduced cells) in the bone marrow and/or CNS. Given the specific disorders, the transgene introduced into HSCs needs to be expressed in the cells derived from one or more of the hematopoietic lineages such as the erythroid, myeloid, and lymphoid lineages (Figure 8.6) (Morgan et al., 2017a). In the case of LV-based GTx applied to LSDs caused by enzyme deficiencies within lysosomes leading to the accumulation of substrate, the genetically modified stem cells differentiate into macrophages and microglia in the CNS producing functional enzyme in the affected tissue (Figure 8.9).

FIGURE 8.9 Ex vivo lentiviral vector transgene deliver in lysosomal storage diseases. Lysosomal storage diseases are characterized by the accumulation of intracellular substrates caused by deficiencies in lysosomal enzymes. Substrate accumulation can disrupt normal cell function and can affect the function of organs such as the kidney, eye, liver, heart, and bones, and the peripheral nervous system. Gene-corrected cells may release functional enzyme into the circulation and at a local level following migration into the tissue to treat these diseases, as therapeutic enzymes can be taken up by noncorrected cells expressing mannose 6-phosphate receptor and break down accumulated intracellular substrates. Some lysosomal storage diseases affect cells of the central nervous system (CNS), leading to demyelination and cognitive and motor degeneration. To reach the CNS, cells need to cross the blood–brain barrier, which can be facilitated by conditioning and, in some cases, the underlying disorder. Hematopoietic stem and progenitor cells (HSPCs) may engraft in the CNS, expand locally, and differentiate into corrected myeloid and microglia-like cells, which then release the therapeutic enzyme. In addition, corrected myeloid progenitors or monocytes released from the bone marrow may migrate into the CNS, differentiate into macrophages, and produce enzymes locally. The relative contribution of HSPCs and mature cells to correction is still unclear, although mouse models suggest a predominant role of progenitors. HSPC gene therapy may be advantageous over enzyme replacement therapies for targeting the CNS due to the ability of HSPCs to cross the blood–brain barrier. MLD, metachromatic leukodystrophy; MPSI, mucopolysaccharidosis type I; MPSIII, mucopolysaccharidosis type III. Adapted from Ferrari G, Thrasher AJ, Aiuti A: Gene therapy using haematopoietic stem and progenitor cells, Nat Rev Genet 22:216–234, 2021 by permission.

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Retroviral Vectors Derived from GammaRetroviruses and Lentiviruses Retroviruses are enveloped, single-stranded, positive-sense RNA viruses that belong to the family Retroviridae. The family Retroviridae is divided into 2 subfamilies, Orthoretrovirinae and Spumaretrovirinae. The subfamily Orthoretrovirinae is further subdivided into six genera: Alpharetrovirus, Betaretrovirus, Gammaretrovirus, Deltaretrovirus, Epsilonretrovirus, and Lentivirus, whereas the subfamily Spumaretrovirinae includes only a single genus, Spumavirus. All viruses belonging to the Retroviridae family have the ability of incorporating their genome into the genome of the host. Upon entering the cytoplasm of host cells, the retroviral RNA is reverse-transcribed into double-stranded DNA (dsDNA) that subsequently integrates into the host cell DNA. This process is termed reverse transcription. Following DNA integration into the genome, the host cell transcribes the viral genes using normal host cell machinery for gene transcription, thereby producing mRNA for viral proteins. Retroviral reverse transcription and the ability to integrate are essential for the use of retroviridae-based vectors as gene transfer vehicles. Viral vectors provide an efficient means for modification of eukaryotic cells and are used in academia and industry for clinical gene therapy applications (Milone and O’Doherty, 2018). There are several members of the family Retroviridae that have been used as vectors in ex vivo HSC GTx and most gene addition approaches have used viral vectors derived from GRV and LV to integrate a therapeutic transgene into HSC due to their ability to transduce mammalian cells and ability to stably integrate into the host cell genome. This section will compare these vectors as used in ex vivo HSC GTx. The main advantage of both LV and GRV vectors is their ability to integrate into the cell genome. GRV must access the host genome during mitosis, when the nuclear envelope is disassembled in order for transduction to occur. HSCs are quiescent and cycle infrequently during hematopoiesis and are not well suited for GRV transduction (Morgan et al., 2017a). GRV tends to integrate close to transcriptional start sites. The long terminal repeats (LTRs) act as a strong promoter and enhancer. Strong enhancers are capable of recruiting a number of transcription factors that can override innate cellular

transcriptional control of neighboring genes, leading to transactivation (Milone and O’Doherty, 2018; Morgan et al., 2017a). Several patients in clinical trials using murine leukemia virus (MLV)-based GRV vectors have developed leukoproliferative complications (T-cell acute lymphoblastic leukemia [T-ALL]) several years posttreatment due to the LTR-driven GRV vector integration upstream of proto-oncogenes and ectopically activating their expression (Morgan et al., 2017a). Improvements of Retroviridae vector technology have led to the design of self-inactivating (SIN) GTx vectors to address the GRV tendency to potentially promote enhancer-mediated leukemogenesis. SIN vectors have a deletion in the 30 LTR enhancer–promoter sequence, and an internal promoter is added to control transgene expression (Modlich et al., 2009). These modifications can provide better regulation of tissuespecific transgene expression and reduce the risk of cellular gene transactivation. The SIN vector design reduced the likelihood that replication-competent retroviruses would originate during vector production or in vivo and the design also reduces the risk of recombination with wild-type HIV in an infected patient. Therefore, SIN GRV vectors are safer than conventional GRV vectors, and subsequent clinical trials demonstrated that SIN system reduced the likelihood of insertional mutagenesis and potential genotoxicity (Morgan et al., 2017a). LVs integrate in quiescent and nondividing host cells, which make them the preferred vector for the transduction of HSCs. LVs typically integrate inside the transcriptional units and are recognized as having a safer integration site profile. LVs do not exhibit a preference for integrating near known proto-oncogenes, regulatory gene regions, or transcriptional start sites that have all been linked to the development of cellular transformation as seen in the past with GRV such as Moloney murine leukemia virus-based vectors in clinical trial (Braun et al., 2014; Hacein-Bey-Abina et al., 2003; Ott et al., 2006; Stein et al., 2010). LVs are recognized as having a better integration profile reducing the theoretical risk of cellular transformation seen in the past with GRV. LV is the principal vector platform used for ex vivo CD34þ HSC GTx in humans and generally is based on human immunodeficiency virus type 1 (HIV-1) that has been extensively investigated and optimized over the past two decades.

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Third-generation SIN LVs (described below) have been used in multiple clinical trials (Milone and O’Doherty, 2018). The HIV-1 genome contains nine genes (env, gag, pol, vif, vpr, vpu, nef, tat, rev). Gag, pol, and env genes are common to all Retroviridae. The other six genes include two essential regulatory genes (tat and rev) involved in viral replication and four accessory genes (vif, vpr, vpu, and nef) important for in vivo replication and pathogenesis. LV vectors are designed and produced by combining several different plasmids (transfer plasmid, packaging plasmid, and envelope plasmid). These plasmids are cotransfected into a packaging cell line, such as HEK293T (human embryonic kidney cells), to produce the vector. Following incubation of the transfer, packaging, and envelope plasmids in the cell line, the supernatant containing the LV vectors is isolated. Each plasmid serves a different function in producing the LV vector. The transfer plasmid encodes the transgene or therapeutic sequence of interest. It is flanked by LTR sequences facilitating the integration of the transfer plasmid into the CD34þ HSC DNA. Transfer plasmids are designed to be self-inactivating (SIN). The transfer plasmids may also contain a deletion in the viral promoter located in the LV LTR, rendering the vector SIN during integration into the CD34þ HSC (Modlich et al., 2009). Vector design (e.g., the choice of promoter) regulates transgene expression in specific cell types. For example, genetically modified myeloid cells differentiate into monocytes, macrophages, and microglia (Ferrari et al., 2021) in contrast to regulated expression of a transgene directing erythroid-specific expression of b-globin (Morgan et al., 2017a). The packaging plasmid and the envelope plasmids encode components of the viral capsid and envelope, respectively, which are required for the assembly of the viral particles. Separating the viral genes needed for viral infection and replication on separate plasmids during LV vector production increases the safety feature of the vector and avoids the risk of homologous recombination. The envelope plasmid encodes the envelope protein, which is commonly derived from the vesicular stomatitis virus glycoprotein (VSV-G) that is recognized by a ubiquitously expressed receptor and allows the LV vector to transduce a wide range of cells (Amirache et al., 2014). LVs can be designed

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with different viral envelopes (a process called “pseudotyping”) that alter LV tropism for different primary cell types (GutierrezGuerrero et al., 2020). Several generations of LV vectors have been developed and are briefly described below. The third-generation LV vector is recognized as one of the safest and easiest-to-use GTx vectors for ex vivo CD34þ HSCs. A fourth-generation LV vector design has been proposed (Berkhout, 2017; Gouvarchin Ghaleh et al., 2020), but this generation is not commonly used at present and so is not discussed further in this chapter. First-generation Lentiviral Vectors. The firstgeneration conserved eight of the nine genes from the HIV genome (gag, pol, vif, vpr, vpu, nef, tat, rev). Only the env gene coding for the envelope proteins gp120 and gp41 was removed. Gp120 and gp41 are responsible for attachment of the LV to CD4 T cells during fusion. Second-generation Lentiviral Vectors. In the second-generation, the four accessory gene sequences encoding proteins supporting HIV virulence were deleted and the packaging genes were removed (vpr, vif, vpu, nef). The secondgeneration LV is also a three-plasmid system but the packaging genes gag, pol, rev, and tat were all on one plasmid and the envelope gene encoding VSV-G env was on another plasmid. Third-generation Lentiviral Vectors. The third-generation uses a three- or four-plasmid system (1 or 2 packaging plasmids, an envelope plasmid [VSV-G], and a transfer plasmid). The third-generation LV vectors usually encode three of the nine HIV-1 genes (gag, pol, rev) expressed on 2 separate packaging plasmids, thereby separating the gag and pol genes from the rev gene. The use of multiple plasmids further improves safety by reducing the theoretical risk of homologous recombination. In addition, the transcriptional activator gene (tat) can be removed by adding a promoter on the transfer plasmid to drive the production of viral vector transcripts (Milone and O’Doherty, 2018). Third-generation LV vectors represent some of the safest and easiest-to-use vectors available for the ex vivo delivery of genes into mammalian cells. Their genetic composition is well characterized, they effectively deliver genetic material in an ex vivo setting, and they maintain long-term and stable transgene expression in target cells (White et al., 2017).

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4.2. Nonclinical Safety Assessment for Ex Vivo Gene Therapy The nonclinical safety assessment of gene and cell therapy products is generally done on a caseby-case basis. Factors influencing the design of development programs for these products include such variables as the disease indication, patient population, product characteristics, mechanism of action, clinical route of administration, dosing plan, and targeted anatomic sites or tissues, among others. Importantly, there is no standard set of nonclinical tests applicable to all ex vivo HSC products. Nonetheless, the overarching general principles of nonclinical testing as applied to all therapeutic test articles are applicable to gene and cell therapy products. Nonclinical Evaluation Strategies for Ex Vivo Gene Therapy Nonclinical safety and efficacy assessment of autologous ex vivo HSC GTx should include a benefit–risk assessment for the use in humans. The in vivo effects of the transgene, vector backbone, plasmid-derived DNA sequences, and transduced CD34þ HSCs should be evaluated using relevant in vitro assays and in vivo studies. This section focuses on in vivo testing of ex vivo LV-transduced CD34þ HSCs. In vivo assessment of ex vivo HSC GTx products should include characterizing the vector used as the therapeutic transgene delivery tool, characterizing the nontransduced and transduced HSC, the transduction process, conditioning, and the HSCs’ ability to engraft, differentiate, and produce a functional protein from the transgene. The objectives of the nonclinical program are to support the rationale and design of the clinical trials. Nonclinical evaluation typically involves efficacy, safety, and BD assessments. The nonclinical approach should include at least one POC study performed in a relevant animal model of disease (often in mice, rarely in nonrodents) to assess pharmacological activity and BD of the transgene as well as toxicity studies in immunocompromised mice using the ex vivo HSC GTx product. Depending on the program and regulatory agency input, hybrid POC and toxicity study designs using a mouse model of disease may be used to supplement or replace standalone toxicity studies in immunodeficient mice used

to assess the human drug product. The majority of the animal models used in testing may be based on spontaneous or engineered mutations in mice. There are other species that may have suitable disease models, but mice are the standard due to technical limitations associated with xenotransplantation and conditioning regimen and are an accepted model by regulatory agencies. Proof-of-Concept Studies for Ex Vivo Gene Therapy The goal of performing POC studies in mouse models of disease is to demonstrate the in vivo effects of administering genetically modified HSC expressing a functional protein in animals where the protein is either lacking altogether or is functionally abnormal. Mutant mice (usually engineered transgenic or knockout strains) attempt to recapitulate a clinical indication and are commonly used in POC studies to demonstrate the biological responsiveness of the animal to the engrafted LV-transduced HSC carrying the therapeutic transgene and assess the effect of the transgene product in vivo. The POC study can evaluate the integrity of the transgene, protein expression, protein function, therapeutic effect, and distribution of genetically modified cells and their transgene. Depending on feasibility, current practice for POC studies uses syngeneic (genetically similar [i.e., immunologically compatible]) bone marrow cells to demonstrate the ability of LV-transduced mouse HSCs (mHSCs) to restore biological function in a mutant mouse engineered to recapitulate a biological dysfunction in the clinical indication of interest (Figure 8.10). Occasionally, safety endpoints are included in POC studies in mouse models of disease in combined efficacy/toxicity studies. Data from such hybrid study designs can supplement and possibly replace standalone toxicity studies. In many instances, the adequacy of the nonclinical package to support FIH clinical trials for ex vivo HSC GTx products is made on a case-by case basis. The mouse models ideally should be relevant as indicated by their similarity (in terms of disease-related anatomic and functional changes) to the patient population in the proposed clinical indication. Demonstration of this similarity requires that the animal model be wellcharacterized. In terms of pathology endpoints,

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FIGURE 8.10 Illustration of Proof-of-Concept study following intravenous administration of ex vivo lentiviral vector–transduced mouse Lin- hematopoietic and progenitor cells. Reprinted with permission from AVROBIO, Inc.

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conventional anatomic pathology endpoints (see Basic Approaches in Anatomic Toxicologic Pathology, Vol 1, Chap 9) and clinical pathology parameters (see Clinical Pathology in Nonclinical Toxicity Testing, Vol 1, Chap 10) represent a minimum data set for characterizing the model. Special pathology techniques such as IHC and ISH (Special Techniques in Toxicologic Pathology, Vol 1, Chap 11) and noninvasive imaging (In Vivo Small Animal Imaging: A Comparison to Gross and Histopathologic Observations in Animal Models, Vol 1, Chap 13) also may be useful in fully characterizing animal models of disease. Additional means for assessing stem cell–based products may be explored in Stem Cell and Other Cell Therapies (Vol 2, Chap 10). For POC studies evaluating ex vivo HSC GTx, bone marrow cells are collected from an allogeneic donor mouse femur or tibia, and the mHSCs are isolated using immunomagnetic beads. Lineagenegative cells are depleted for differentiated hematopoietic cell lineages including T- and Blymphocytes, erythroid and myeloid elements, platelets, etc. The lineage depletion Lin (LinScaþcKitþ) (LSK) mouse HSCs are separated as this cell population includes both progenitors and long-term HSCs. This cell population is enriched for LSK cells, but cells expressing Sca-1 (“stem cell antigen-1,” a biomarker for HSCs) and c-Kit (a stem cell factor receptor that also is referred to as CD117) can be used to further enrich long-term repopulating stem cells. The enriched mHSCs are then transduced ex vivo with the LV to express the therapeutic transgene of interest. The recipient mice are conditioned and then dosed with a single IV administration of ex vivo genetically modified mouse Lin HSCs. The conditioning protocol consists of either total body irradiation (TBI) or administration of busulfan, an alkylating agent, to generate space within the bone marrow niche and CNS of recipient mice to facilitate Lin HSC engraftment postinjection. The total dose range of busulfan to condition wild-type mice is not standardized and ranges between 100 and 125 mg/kg total dose by intraperitoneal (IP) injection, with a daily maximum dose of 25 mg/kg, for 4–5 days. Ideally, the POC study using a mouse model of disease should demonstrate that the modified mHSCs carrying the therapeutic gene have engrafted in the bone marrow, reconstituted the hematopoietic system following the conditioning

regimen, produced protein arising from transgene expression, and demonstrated efficacy or another biological response to the expressed protein. Since the POC is performed in a mouse model of disease with the intended clinical disease indication, biological effect should be measurable and is important in defining the benefit–risk profile. If safety endpoints are included to support clinical development, common parameters include hematology and clinical chemistry parameters as well as macroscopic and microscopic evaluations of major organs for on-target or off-target effects. Biodistribution and Pharmacodynamics for Ex Vivo Gene Therapy Due to the autologous nature of ex vivo HSC GTx product, the standard battery of ADME (absorption, distribution, metabolism, elimination) and pharmacokinetic (PK) studies are not conducted to support the clinical development program. Biodistribution of the drug product can be evaluated in the efficacy and/or safety studies in mice through evaluation of VCN/dg which measures the transgene (vector copy number [VCN]; the mean number of proviral DNA copies in transduced cells) per diploid genome (dg) of transduced cells by quantitative or digital droplet polymerase chain reaction (qPCR, ddPCR) in blood and tissues. The BD of both the modified HSCs (distribution, durability) and the transgene product (mRNA or protein levels) should be defined in mouse blood and tissues following engraftment of the genetically modified HSCs to confirm the presence of the proviral DNA insert in vivo and transgene protein expression and function when applicable. Depending on the program, significant time and effort may be required to develop the required bioanalytical tools needed to identify and quantify the presence of the proviral DNA insert and transgene product in vivo. Biodistribution and PD of the proviral DNA insert and transgene-expressed protein can be assessed in POC studies by measuring enzyme activity and substrate reduction; performing functional evaluations (locomotion, cardiac, and respiratory); flow cytometry; IHC; and VCN to name a few. These endpoints will vary depending on the disease indication. If possible, combining PD and BD evaluation in the same study generates comprehensive and integrated data.

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Toxicity Studies (In Vivo Safety Assessment) for Ex Vivo Gene Therapy Safety assessments of ex vivo HSC GTx products include performing a nonclinical toxicity study in either a relevant mouse model of disease with ex vivo LV-transduced Lin- mouse HSC cells (as described in the POC section above) or using xenotransplantation of the ex vivo LVtransduced CD34þ human HSCs in immunocompromised mice such as an NSG (“NOD/ SCID/IL2Rg triple-null” strain; formal nomenclature: NOD.Cg-Prkdcscid Il2rgtm1Wjl/SzJ) mouse (see Genetically Engineered Animal Models in Toxicologic Research, Vol 1, Chap 23) to prevent rejection of the human cells (Figure 8.11). In either scenario, the toxicity study aims to identify, characterize, and quantify potential local or systemic toxicities, their time of onset, the latency to and degree of recovery. BD endpoints can be incorporated in the toxicity study design to correlate study findings to the GTx product exposure as measured by vector distribution and if possible, the levels of transgene product. If feasible, the IND-enabling nonclinical toxicity study should meet GLP standards. To date, one of the preferred animal models to assess the safety of ex vivo LV-transduced hHSC GTx products expressing therapeutic proteins to correct metabolic disease, for example, is the immunodeficient NSG mouse model. This engineered mouse lacks T-cells, B-cells, and natural killer (NK) cells and also exhibits impaired cytokine production (see Genetically Engineered Animal Models in Toxicologic Research, Vol 1, Chap 23). The significant decrease in normal immune responses readily permits bone marrow engraftment of human CD34þ HSCs. To facilitate engraftment, NSG mice are conditioned with busulfan at a dose lower than that for wild-type mice: 20–25 mg/kg IP for two consecutive days (40–50 mg/kg total dose) (Chevaleyre et al., 2013). Although TBI can be used to support toxicity studies in mice, busulfan is preferred if feasible since this is the conditioning agent used in clinical protocols. However, regulatory guidance for testing GTx does not require that the same regimen for bone marrow conditioning be employed in animals and humans. The protocols used should effectively produce myeloablation and facilitate HSC engraftment in the bone marrow that is followed by

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multilineage reconstitution. This engraftment is limited in NSG mice; while this mouse strain is one of the best models for evaluating human HSCs in vivo, multilineage reconstitution is limited. Any toxicity associated with the conditioning regimen should be characterized in the nonclinical toxicity studies intended for the development of clinical programs. The toxicity study design for ex vivo HSC GTx products follows the standard practice in nonclinical toxicity studies. Assessments include mortality, clinical observations, body weights, routine clinical pathology endpoints (hematology and serum chemistry at minimum), and macroscopic and microscopic examinations of a full tissue list. Appropriate control groups will be needed to differentiate any background findings in mutant mouse strains from any test article–related effects. For example, in NSG mice there are certain background histological findings that have been associated with the model such as cystic structures in the thymus, absence of follicles in the spleen, and reduced cellularity of lymph nodes. It is important to use animal models that are accurately characterized and use supportive historical control databases from previous studies (Bonapersona et al., 2021; Knoblaugh et al., 2018). Experimental Design Features Supporting Ex Vivo Gene Therapy Development Nonclinical programs supporting development of ex vivo HSC GTx products present scientific, technological, and regulatory challenges. The broad range of questions inherent in such challenges is beyond the scope of this chapter, so interested readers should read relevant review articles to obtain additional information not covered in this section (Garcia-Perez et al., 2020; Morgan et al., 2017a). Additional information regarding study design and pathology evaluation for cell-based products may be investigated in this book by reviewing Stem Cell and Other Cell Therapies, (Vol 2, Chap 10). The nonclinical study design supporting clinical trial development varies on a case-by-case basis depending on the clinical indication and relevant governing and regulatory guidelines. This section will consider some of the typical design features included in nonclinical studies for these products.

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FIGURE 8.11 Illustration of safety toxicity study following intravenous administration of ex vivo lentiviral vector-transduced CD34þ hematopoietic and progenitor cells. Reprinted with permission from AVROBIO, Inc.

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ANIMAL MODEL OPTIONS

The relevance of the animal model and the factors determining its suitability for any intended nonclinical assessment should be evaluated in the context of the clinical indication, patient population, mechanism of action, biological responsiveness, etc. The animal model should allow the determination of the pharmacological activity and BD profile of the proviral DNA insert (distribution, durability) and transgene product (protein levels) in cells, body fluids, and tissues. In some instances, the model may need to be generated de novo or licensed from a third-party organization. Relevant spontaneous and engineered mouse models are commonly used for the nonclinical assessment to demonstrate biological responsiveness and evaluate the efficacy driven by the transgene expression of a functional recombinant protein. These models are essential to characterize potential physiological, morphological, functional, or behavioral changes in response to the infusion of genetically modified Lin- mHSC as well as characterizing phenotypic traits that may be confounding variables associated with genetic backgrounds of mouse strains. For example, mice with a C57BL/6 genetic background have a higher incidence of age-related mortality due to spontaneous lymphoma and hematopoietic neoplasms as well as vascular neoplasms, acidophilic macrophage pneumonia, nephropathy, urinary obstructive syndrome, and cardiac changes (Brayton et al., 2012). There are different approaches that can be used to evaluate the BD of the modified donor mHSC in vivo. For example, CD45 is encoded by the protein tyrosine phosphatase receptor type C (Ptprc) gene and is expressed on the surface of all nucleated cells of hematopoietic origin except for differentiated erythrocytes and platelets. There are different isoforms of CD45 in mice. The allelic variant Ptprca encodes CD45.1 and occurs in the SJL mouse strain. The Ptprcb allele encodes CD45.2, which is expressed in the C57BL/6 strain. The CD45.1 allele has been backcrossed onto the C57BL/6 genetic background and has been widely used in immunological studies to track the contribution of specific genes to hematopoietic cell development (Jafri et al., 2017). CD45 hematopoietic cell markers (CD45.1 and CD45.2) have been established as a marker system to track hematopoietic cells following congenic mouse bone marrow

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transplants to distinguish between donor and recipient hematopoietic cells after transplantation, which permits the BD of the transduced cells to be determined (Biasco et al., 2018; Jafri et al., 2017). In contrast, the toxicity assessments of ex vivo hHSC GTx products are performed in immunodeficient mouse models to prevent rejection of engrafted human cells. Several highly immunodeficient models such as the NSG strain, the NOD.CgPrkdcscid Il2rgtm1Wjl Tg(CMV-IL3,CSF2,KITLG) 1Eav/MloySzJ (typically abbreviated as NSGS or NSG-SGM3) strain, and humanized mice (i.e., an animal into which human genetic material or human cells has been inserted) have a suitably permissive immunological profile but differ in their cytokine profiles, multilineage engraftment potential, conditioning requirements, etc. The proposed animal model used to support safety assessment and/or to demonstrate efficacy must be well understood to ensure that the data are suitable to support regulatory submissions. The NSG mouse model is a highly immunodeficient strain and commonly used for ex vivo LVtransduced CD34þ hHSC toxicity assessment as it readily permits engraftment of human CD34þ HSC, as well as other human-derived cells such as PBMCs, patient-derived xenografts (PDX or “avatars”), or adult stem cells and tissues (Greiner et al., 1998; Ito et al., 2002). They carry two mutations on the NOD/ShiLtJ genetic background: severe combined immune deficiency (scid) and a complete null allele of the IL2 receptor common gamma chain (IL2rgnull). The scid mutation is in the DNA repair complex protein Prkdc leading to B- and T-cell deficiencies. The IL2rgnull mutation prevents cytokine signaling through multiple receptors, leading to functional NK cell, T-cell, and B-cell deficiencies. The severe immunodeficiency allows the mice to readily become engrafted with human cells. In the context of assessing the toxicity of ex vivo LV-transduced CD34þ hHSC GTx products, the NSG model allows effective HSC engraftment and widespread BD. Engraftment of human HSC and differentiation into specific lineages can be evaluated in the hematopoietic organs such as bone marrow, spleen, and thymus, peripheral blood and brain posttransplantation (Ellison et al., 2019). However, a limitation with using the NSG mouse is the inability to evaluate GTx product-specific immune responses (due to the

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profound immunodeficiency of the strain), so test article–related immune reactions can be assessed in a POC study using immunocompetent animal model of disease. NSG mice are not permissive to all multilineage engraftment of all the compartments and not suitable to assess test article–related immune responses. The NSG-SGM3 (or NSGS) mouse strain is an improved model to understand hematopoiesis, HSC engraftment, and potential effects produced by GTx products delivered using HSCs (Wunderlich et al., 2018). The NSG-SGM3 strain expresses three human transgenes: colonystimulating factor 2 (CSF2, also known as granulocyte-macrophage colony-stimulating factor [GM-CSF]); IL-3; and stem cell factor (SCF, also known as KIT ligand [KITLG]). This combination merges the extremely immunodeficient phonotype of the NSG mouse with a cytokine profile that supports the stable engraftment of HSCs, especially myeloid lineages and regulatory T (Treg) cells. ANIMAL NUMBERS PER GROUP

Adequate numbers of animals per sex that are appropriately randomized to each group are required to reduce study bias as much as possible. The number of animals required for each group will vary depending on the safety concerns for the investigational drug product, the species, model, and the number of arms in the study. Group sizes in POC and hybrid efficacy/toxicity studies vary, and there are no universal standards dictating study designs. Group sizes for mice typically range between 5 and 10 per sex per group. However, some studies include a much larger number of animals per group depending on the scientific objective. STUDY DURATION

Although the required durations of POC and toxicity studies for ex vivo GTx products are not specified, study length should be defined based on transgene expression (peak and persistence) and the intended morphological, functional, and behavioral effects to be evaluated. In conditioned mice, it takes approximately 12– 16 weeks for mHSC or hHSC to engraft. Daughter cells from the long-term engrafting HSCs are responsible for producing a sustained level of the transgene product. Therefore, it will require longer than 4 months to fully assess the

effects of the transgene product such as enzyme activity levels, substrate reduction, or morphological changes. The total duration of toxicity studies for ex vivo modified HSCs in mice should be greater than 4 months and typically should include an interim time point (usually at 1–2 months) or a midtime point depending on the duration of the study. Nonclinical studies range from 4 to 12 months depending on the disease indication, animal model, targeted assessments, and the ability to demonstrate a durable therapeutic response. In contrast to small molecules and other biologics, a recovery period is not included in the evaluation of ex vivo modified HSCs. Recovery periods usually determine whether toxicity is reversible in the absence of the drug product. CONTROL GROUPS

Suitable control groups to evaluate ex vivo modified HSCs in nonclinical studies should be considered based on the purpose of the study, knowledge of the vector, character of the cells, and the animal model. The “Guidance for Industry: Preclinical Assessment of Investigational Cellular and Gene Therapy Products” (U.S. Food and Drug Administration, 2013) provides general suggestions on the types of controls for consideration. Justification should be provided for the specific control group(s) selected. Examples of potential animal control groups that may be considered in the nonclinical assessment for ex vivo modified HSC GTx products include: • Nontransduced HSC transplanted controls, • HSC transduced with vector particles without the RNA transcript (empty particles without viral vector genome), • HSC transduced with vector particles containing a mutated RNA transcript resulting in a noncoding proviral DNA being integrated, • HSC transduced with vector resulting in transduced cells producing a fluorescent protein, • Untreated animal model of disease, and • Untreated wild-type mice. Complex study designs where multiple HSC products bearing different GTx products may require arms to control for infusion of each product as well as an arm where animals receive

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both products (Puig-Saus et al., 2019). Conditioning regimen controls can also be evaluated. TIME POINTS

Interim time points help define the fate of the proviral DNA insert and transgene product in both the early and late phases of hematopoietic reconstitution comparing the engraftment of the short-term precursors (ST-HSPC) compared to the long-term (LT-HSPC) engrafting cells responsible for achieving hematopoietic steady state. This study design assesses safety, durability, BD, and PD at the acute and peak stages of engraftment. Later time points give the opportunity to correlate recombinant protein levels as well as morphological, functional, and behavioral changes as warranted. For example, an interim time point could evaluate engraftment (e.g., flow cytometry, vector copy number, hematologic parameters) and confirm hematological reconstitution while histopathological evaluation can be performed at the interim and terminal time points and contribute to the safety assessment. SOURCE OF CELLS

For ex vivo modified HSC GTx in animal studies, the HSCs used for LV transduction are either collected from mouse bone marrow (animals from the same inbred strain) as the Lincell fraction or the CD34þ cells are collected by apheresis from healthy human donors. In both cases, these cells are considered surrogates since the clinical GTx product will be autologous CD34þ cells derived from the patient cells as opposed to a healthy donor or syngeneic Lin– mouse cells transduced with the clinical vector. CELL AND VECTOR CHARACTERIZATION

Characterization and quality control testing is performed for the ex vivo GTx product used in the nonclinical studies for safety assessment. Analytical methods are used to characterize: (1) the LV vector containing transgene, (2) the nontransduced HSC, and (3) the vector-transduced HSC. The characterization demonstrates that the test material used in the nonclinical studies is representative of the ex vivo GTx product intended for use in the clinical trial and also representative of the intended manufacturing processes to produce the vector and accomplish the ex vivo cell transduction. The following are examples of parameters that may be evaluated

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for the transduced and nontransduced hCD34þ cells that will be administered to animals for a toxicity study in NSG mice: cell count, cell viability, sterility, endotoxin concentration (see Bacterial Toxins, Vol 3, Chap 9), transduction efficiency, VCN/dg, vector titer, and potency assay (enzyme activity, for example). The value “multiplicity of infection” (MOI) is used to define the number of transducing units of LV vector used per cell during a transduction of target cells. MOI is often set during vector characterization to target a VCN in the drug product. Different vector batches may result in different MOIs needed for a certain transduction efficiency/VCN to be achieved using the drug product. Titer is a standard biological method for measuring LV vector transducibility and transducing/integrating units are measured by endpoint dilution on permissive cells. Some of this information should be included in the certificate of analysis (COA) for the GTx test article and thus will be included in the regulatory submission package (Garcia-Perez et al., 2020). Modifications that might impact the characteristics of the final GTx product may require additional safety evaluation based on the appropriate regulatory guidelines. ROUTE OF ADMINISTRATION AND DOSING REGIMEN

The route of administration (ROA) used in nonclinical studies should mimic the intended clinical ROA. Ex vivo GTx products are usually administered with a single IV infusion into a peripheral vein following conditioning. In common mouse models, the lateral tail vein is used for this procedure. As noted above (Table 8.3), the infusion site should be collected for histopathologic evaluation. The drug product is often a cryopreserved CD34þ hHSC suspension stored in infusion bag(s), which is thawed prior to use and is given as a one-time IV infusion. In humans, the minimum dose commonly given is approximately 3  106 total cells/kg body weight of modified HSCs. In mice, the number of donor mHSCs given IV to the recipient mice ranges between 5  105 and 1  106 cells per mouse, but in some cases the numbers may be much greater. The safety margin between the cell dose (cell/ kg) administered in mice and the highest anticipated clinical dose varies for HSC GTx products and ranges between 2- to 5-fold which is

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considered acceptable for ex vivo GTx products. However, there are no standard safety factors since the clinical dose from the patient is dependent on how many cells were originally harvested from the patient. VECTOR COPY NUMBER

Vector copy number measures the proviral DNA per diploid genome (dg) of transduced cells (VCN/dg). VCN/dg informs the biodistribution of the ex vivo GTx product and is typically used for BD and safety endpoints (Garcia-Perez et al., 2020). VCN/dg is determined using a quantitative PCR-based assay (or digital droplet PCR [ddPCR]) to measure the number of proviral DNA in transduced cells, and it is expressed as copy number of viral genome per diploid genome. VCN tracks the presence of integrated proviral DNA in vivo to inform BD and possible exposure. A VCN/dg analysis may be performed at multiple time points (Garcia-Perez et al., 2020). Biodistribution includes at a minimum nine key tissues: blood, the site of administration, gonads, brain, liver, kidneys, lungs, heart, and spleen (U.S. Food and Drug Administration, 2020). However, other tissues may be added on a case-by-case basis. Tissues analyzed for VCN must be collected at necropsy in a manner that prevents cross-contamination and blood must be removed from the samples to assure accuracy. Tissue perfusion with cold physiological saline or similar solution (but not fixative) at necropsy should be considered to remove potential circulating transduced cells in the blood that carry the transgene and affecting the VCN values of the homogenized tissue samples. Regulatory authorities such as the EMA and FDA require integration site studies and longterm follow-up (LTFU) on products such as ex vivo LV-based GTx that have a capacity to integrate permanently into the host cells and to persist for a long time in treated individuals. As a result, patients in ex vivo GTx clinical trials may be monitored for an “LTFU” period lasting as long as 15 years. Although there are currently no regulatory requirements for the minimum or maximum vector copy number in humans or in animals, in nonclinical studies, the currently targeted vector copy number is less than 5 copies per genome, however, there are numerous examples where the VCN exceeds 5. This is a theoretical number

that has been used as a benchmark in the industry but may change as the field continues to evolve (European Medicines Agency, 2013; U.S. Food and Drug Administration, 2020; Zhao et al., 2017). It is important to note that ex vivo GTx used in the clinic have demonstrated efficacy where functional protein was expressed by the genetically modified stem cell at lower VCN levels. Genotoxicity Studies New molecular entities (NMEs) and gene therapies are not evaluated in the standard panel of genotoxicity assays traditionally applied to new chemical entities (NCEs) such as smallmolecule pharmaceuticals. Ex vivo GTx use unique genotoxicity assays such as in vitro immortalization assay (IVIM) and the newer molecular surrogate assay for genotoxicity assessment (SAGA), and Integration Site Analysis (ISA) from in vivo samples, which are not generally applicable to other platforms. These assays are used in nonclinical studies to support clinical development of ex vivo GTx programs by assessing the transforming potential of LV vectors in development (Biasco et al., 2018; Bushman, 2020; Schwarzer et al., 2021). INTEGRATION SITE ANALYSIS

Integration Site Analysis (ISA) is an in vivo genotoxicity assay that determines the integration profile and clonal distribution of the transduced cells. ISA aims to evaluate the tendency for aberrant oligoclonality and/or insertion of the vector near “problematic” sites, such as proto-oncogenes or transcription start sites (TSSs), in tissues from transplanted animals. In animals, ISA is used as a read-out for the genotoxicity potential of the viral vectors used, but it is only a tool to pick up the dominant clones or leukemic clones that contain an integrated vector and to assess the general integration preference. In humans, ISA has been used in earlier clinical studies of ex vivo GRV-transduced HSC GTx products for the treatment of Wiskott– Aldrich Syndrome (WAS) to identify dominant clones in several patients with vector integration at the LMO2, MDS1, or MN1 locus leading to leukemogenesis (Braun et al., 2014). LV vectors have a much more favorable safety profile compared to GRV vectors due to their insertion pattern, and the third-generation LV system has an inactive viral promoter element (U3) in

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the LTR in addition to other safety features (see previous section). Review articles describe insertion site identification and mapping used for safety and clonal tracking studies on human and animal samples used as an indicator of vector safety in genetically modified HSC transduced using LV (Biasco et al., 2018). ISA is added in the nonclinical safety assessment to evaluate the overall integration profile of the vector and the presence of any clusters of integrations (Biasco et al., 2018). The molecular basis of insertional oncogenesis is believed to be secondary to insertional deregulation of one or more genes leading to clonal expansion and eventually transformation of HSCs. Polyclonal hematopoietic reconstitution in HSC-engrafted mice indicates a balanced clonal composition where multiple HSC clones are established in bone marrow niches and then differentiate. Hematopoietic repopulation with an oligoclonal distribution may be observed in individual mice engrafted with a lower number of transduced stem cells or that develop very low chimerism with no sign of vector-induced clonal dominance and a typical LV proviral DNA integration pattern (Poletti et al., 2018). IN VITRO IMMORTALIZATION ASSAY AND SURROGATE ASSAY FOR GENOTOXICITY ASSESSMENT

IVIM is an in vitro genotoxicity assay performed in primary murine bone marrow–derived HSC. This method relies on the induction of a survival advantage in transduced cells such as might occur due to insertional activation of cellular proto-oncogenes, which may become apparent when the transduced primary mHSCs are cultured under differentiating cytokine conditions and plated in a limiting dilution fashion. The murine primary HSCs are transduced at an artificially high MOI with the LV vector. Upon culturing and replating, the selective outgrowth advantage of LV-transduced, transformed cells is established by their increased survival and expansion relative to transduced cells that lack genotoxic profile. Although the IVIM assay is useful to help assess the risk of insertional mutagenesis, cells are cultured in a myeloid-inducing differentiation medium favoring the readout of selective myeloid mutants (Evi1 and Prdm16) as opposed to B- or T-cell mutants. The cytokine conditions used in the IVIM support the default

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differentiation route of HSPCs in culture, which is the myeloid lineage. IVIM is a short-duration assay (2 weeks in culture) and demonstrates good sensitivity. A next-generation genotoxicity assessment assay is the Surrogate Assay for Genotoxicity Assessment of Gene Therapy Vectors (SAGA) (Garcia-Perez et al., 2020; Adrian Schwarzer et al., 2016; A. Schwarzer et al., 2021). SAGA is a molecular-based assay that integrates into the IVIM analysis where it increases the reproducibility and sensitivity of the read-out (Adrian Schwarzer et al., 2016). SAGA classifies integrating Retroviridae-based vectors using machine learning to detect this gene expression signature during the course of in vitro immortalization. On a set of benchmark vectors with known genotoxic potential SAGA achieved an accuracy of 90.9%. The assay is performed on mouse stem cells (Lin- HSPCs) to quantify the mutagenic potential of the GTx product in comparison to several benchmark positive control vectors such as GRV with strong spleen focus-forming virus (SFFV) promoter/enhancer elements (LTR.RV.SFFV) known to have a high risk of genotoxicity and shows a high incidence of insertional mutants. SAGA is faster and delivers a better nonclinical risk assessment of ex vivo LV-transduced HSC GTx products compared to conventional IVIM (A. Schwarzer et al., 2021). In summary, the IVIM assay assesses whether vector transduction results in increased immortalized cell clones and the SAGA evaluates gene expression profiles (mRNA) for the presence of upregulation of an oncogenic “signature” to inform on potential mutagenic risk. Both are in vitro assays in which the vectors are tested by transducing mouse stem cells (Lin-HSPCs) and can inform about insertional perturbations of cellular proto-oncogenes. Depending on the protocol, mouse HSPCs are incubated with LV at multiplicity of infection (MOI) which is greater than the MOI for transduction of human patients. For example, mouse HSPCs could be incubated with LV at MOI of 60, which is six times greater than the MOI of 10 selected for transduction of human patients HSCs in order to maximize mutagenic potential by testing a value beyond the anticipating clinical manufacturing MOI. The insertional profile and risk for each test LV is compared with positive controls RSF91 and lv-SF, which are

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gamma-retroviral vectors with documented in vivo and in vitro mutagenic potential, respectively.

4.3. Toxicologic Pathology Considerations With Ex Vivo Gene Therapy Study design considerations for nonclinical studies of ex vivo GTx products vary from product to product. That said, pathology evaluations for such studies are comparable to those used routinely by toxicologic pathologists when assessing other therapeutic test articles. This section considers common endpoints suitable for assessing the BD, efficacy, and safety of ex vivo GTx products as well as key considerations needed to discriminate test article–related effects from procedural effects (e.g., consequences of conditioning or the basic phenotype of the disease model). Conditioning Agents in Ex Vivo Gene Therapy CONDITIONING

In autologous ex vivo GTx, the goal is to attain a stable, mixed chimeric HSC population in the bone marrow. Various conditioning agents create a bone marrow niche to allow establishment of the genetically modified exogenous HSCs alongside the uncorrected endogenous cells. There are many different conditioning strategies, and they vary depending on the disease indication, animal model, and cell origin, for example. Common conditioning agents used in nonclinical studies include alkylating agents and TBI, and recently monoclonal antibodies directed against endogenous HSCs are being evaluated (Bernardo and Aiuti, 2016; Ferrari et al., 2021). TBI has been extensively used in mouse studies where recipient mice are irradiated prior to receiving HSC for ex vivo GTx. TBI targets mitotically active cells, and it is not selective. The TBI-exposed animals are immunosuppressed and susceptible to opportunistic infections such as Pseudomonas sp., Escherichia coli, Clostridium sp., and Klebsiella sp. until their immune system is reconstituted. Some of the risks and complications associated with TBI include weight loss, anemia, infection, gastrointestinal hemorrhage, and secondary neoplasia (Dange et al., 2007; Green and Rubin, 2014).

Changes attributed to TBI exposure in mice depend on the mouse strain, sex, age, and TBI dose level and duration. Monoclonal antibody conditioning approaches are being evaluated in mice and NHPs. The antibodies target the recipient’s HSCs by binding particular hematopoietic cell surface markers (e.g., CD117 [also called c-Kit] and CD45). Antibody binding permits removal of cells expressing the specific marker, which leads in turn to clearance of a discrete bone marrow niche. Another approach targets hematopoietic stem cell–specific CD45 receptor internalizing saporin (SAP) immunotoxin as a means of depleting endogenous HSCs (Ferrari et al., 2021; Palchaudhuri et al., 2016). Alkylating agents used for conditioning, such as busulfan, have progressively been replacing TBI for bone marrow transplantation in mice. In nonclinical studies, busulfan is commonly used in POC or toxicity studies in mice to support clinical development of ex vivo GTx therapies. The dose regimen administered in NSG mice is 20 mg/kg IP administered daily for 2 days (40 mg/kg total dose) and in most mouse models of disease receive 25 mg/kg IP administered daily for 4–5 days (100–125 mg/kg total dose) (Ellison et al., 2019). As a point of comparison, in rodents, busulfan is administered by IP injection and in humans it is administered intravenously. However, it is recommended to perform a tolerability study with new mouse strains or mouse disease models. Busulfan conditioning generates bone marrow mixed chimerism where resident endogenous mHSCs intermingle with genetically modified HSCs (transduced and nontransduced cells). Anticipating and characterizing the clinical and pathological changes attributed to conditioning regimens in mice is important. Busulfan has a limited therapeutic margin and may result in toxicities when high doses are administered; the occurrence of clinical signs, physiological changes, and/or lesions induced by busulfan can confound the interpretation of nonclinical studies for GTx studies. For example, transient supportive care may need to be given to mice during the conditioning regimen as they may develop transient lethargy, reduced appetite, and body weight loss. If the genetically modified HSCs do not engraft, moribundity may be observed within the 2 weeks thereafter due to

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poor bone marrow reconstitution and cell differentiation, which rapidly leads to depletion of red blood cells (i.e., anemia) or white blood cells (often indicated by secondary bacterial infections). Poor engraftment of genetically modified HSCs also may occur due to an inadequate conditioning regimen, poor HSC viability, or low numbers of infused HSCs. Changes associated with busulfan conditioning in mice that may be identified during histopathological evaluation include lenticular degeneration (affecting the eye), atrophy of reproductive organs, lymphoma, and nonproliferative lesions with differing incidences and distributions across strains and mouse models of diseases (Chanut et al., 2021). Busulfan is lipophilic, and unlike other alkylating agents it does not bind to plasma protein in the blood and thus can cross the BBB. Busulfan kinetics have been studied in the rat; the ratio of brain/plasma concentration is 0.74, demonstrating similar distribution to both compartments (Hassan et al., 1988). Busulfan has become the nonclinical conditioning agent of choice to study engraftment of ex vivo LVtransduced HSCs in the brain of mice since monocytes of the myeloid lineage are able to cross the BBB into the CNS and differentiate into microglia-like cells following busulfan conditioning (Bartelink et al., 2016; Capotondo et al., 2012; Ellison et al., 2019; Ferrari et al., 2021; Wilkinson et al., 2013). The unique combination of ex vivo LV-transduced HSCs and busulfan conditioning enables genetically modified HSCs to target the CNS in mice with neurodegenerative metabolic diseases. Nonclinical studies of ex vivo GTx products given by IV injection have demonstrated efficacy in ameliorating brain pathology, cognitive and behavioral abnormalities, and reductions in substrate levels, indicating the feasibility of brain-targeted HSC GTx administered intravenously (Capotondo et al., 2012; Ellison et al., 2019; Gleitz et al., 2018). Several different conditioning protocols have been used in the clinic for autologous ex vivo GTx treatments. Both target concentration intervention (TCI) and therapeutic drug monitoring (TDM) can be considered as approaches to concentration controlled dosing (CCD) of individual patients (Holford et al., 2020). There are different approaches in achieving individualized busulfan dosing but in the context of using

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ex vivo GTX as a treatment modality, TCI is superior to TDM in providing a safe, consistent, and reproducible conditioning regimen. TDM involves measurement of the drug levels in plasma or blood and then individualization of drug dosage to achieve and maintain a drug concentration within a targeted therapeutic range (Choong et al., 2018). TCI brings more clinical benefit because of the precision of the approach and the ability to link TCI to principles of PK and PD to predict the dose required by an individual (Holford et al., 2020). To improve clinical outcomes busulfan conditioning is used as a single agent and targets a cumulative area-under-the-curve [AUC] ranging between 78 and 101 mg x h/L for optimal exposure (Figure 8.12) to attain tolerability and achieve the target concentration to permit the genetically modified CD34þ cells to engraft in the bone marrow (Bartelink et al., 2016). In a clinical setting, the out of range toxicity associated with busulfan can be mitigated with the use of PK monitoring by performing serial evaluations of drug concentrations followed by dose adjustment to define the AUC (Bernardo and Aiuti, 2016; Ferrari et al., 2021) using TCI (Holford, 2018). The proposed conditioning strategy used in nonclinical studies supporting clinical development should be the same when feasible. However, using a different conditioning agent in the nonclinical studies, such as TBI, can be used to support regulatory submissions even when the intended clinical conditioning agent is busulfan. Immunogenicity There are several regulatory guidances that address immunogenicity assessment in nonclinical studies (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, 2011; U.S. Food and Drug Administration, 2014). Immunogenicity is evaluated in cases when there is: 1) evidence of altered PD activity; 2) unexpected changes in exposure in the absence of a PD marker; or 3) evidence of immune-mediated reactions (immune complex disease, vasculitis, anaphylaxis, etc.). While case-by-case evaluation is important, immunogenicity assessment may not be needed for ex vivo GTx programs when there is no evidence of (1) immunogenic concern

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FIGURE 8.12 Pharmacokinetic model for optimal busulfan exposure predicting survival and toxicity after hematopoietic cell transplantation in children and young adults. Retrospective analysis, including 674 patients (41% malignant, 59% nonmalignant) ranging from 0.1 to 30.4 years receiving busulfan-based conditioning regimen. The polynomial Weibull model of the association between busulfan cumulative area-under-the-curve (AUC) and event-free survival (EFS) is able to reproduce the central tendency in the observed EFS data, shown using D 5 mg  h/L AUC groups (dots) in the training (blue solid line) and internal validation dataset (blue dashed line). The busulfan cumulative AUC and EFS model stratified by malignant (red solid line) and nonmalignant (blue dashed line) underlying disease shows that the optimum AUC does not depend on indication. The Weibull model produced an optimal cumulative AUC of 90 mg  h/L (10% event probability optimum ¼ 78–101 mg  h/L). Shaded areas represent 95% confidence intervals. These results demonstrate that improved clinical outcomes may be achieved by targeting the busulfan-AUC to 78–101 mg  h/L using this validated pharmacokinetic model for all indications. Adapted from Bartelink IH, Lalmohamed A, van Reij EM, et al.: Association of busulfan exposure with survival and toxicity after haemopoietic cell transplantation in children and young adults: a multicentre, retrospective cohort analysis, Lancet Haematol 3:e526–e536, 2016 by permission.

with the specific transgene; (2) loss of efficacy; (3) PK issue; or (4) adverse events observed in nonclinical studies. Immunogenicity risk in autologous ex vivo HSC GTx is far less compared to in vivo GTx. For example, autologous cell product composed of CD34þ HSCs that have been genetically modified to express the functional protein to treat LSD metabolic disorders produces a functional enzyme for the lysosomes which is either intracellular or released from the cell thus crosscorrecting affected tissue. In this case, an antidrug antibody (ADA) evaluation for immunogenicity in nonclinical studies could be assessed in immunocompetent mouse models of disease but not in NSG mice due to their immune status. It is well recognized that nonclinical ADA assessment is typically not predictive of the human risk for developing immunogenicity (Bugelski and Treacy, 2004; Leach et al., 2014).

Therefore, with scientific justification, immunogenicity studies may not be required to support the development of ex vivo LV-based GTx programs when there is no evidence of immunogenic concerns with the specific transgene, loss of efficacy, impact on PK, or adverse events observed in the animals. Nonclinical strategies may involve the development of an ADA assay and collection and storage of terminal plasma samples from animals for eventual analysis if needed. Additionally, there are reports that busulfan conditioning regimens in human may induce tolerance of preexisting antibody-based immune responses. For example, results from a Phase I/II clinical trial in Hurler disease demonstrated preexisting antibodies induced by enzyme replacement therapy (ERT) before GTx was initiated. The preexisting antibodies rapidly become undetectable after conditioning and the GTx did not induce new antibodies (Gentner et al., 2019).

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Immune tolerance induction though HSC translation as a general mechanism has also been seen in allogeneic stem cell transplantation (Saif et al., 2012). Tumorigenicity Regulations and guidance on gene therapy products are rapidly evolving as scientific data accumulate for these modalities. The existing EMA and FDA (European Medicines Agency, 2018; U.S. Food and Drug Administration, 2013) gene therapy guideline and guidance, respectively, and the more recent EMA draft guideline (European Medicines Agency, 2019) on quality, nonclinical, and clinical requirements for investigational advanced therapy medicinal products (ATMPs) in clinical trials, all state that tumorigenicity studies should be performed before humans are administered gene therapy, but that standard lifetime rodent carcinogenicity studies are not generally required. This is consistent with ICH Guideline S6 (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use, 2011), which states that standard carcinogenicity bioassays are generally inappropriate for biotechnology-derived pharmaceuticals. Existing FDA guidance on Preclinical Assessment of Investigational Cellular and Gene Therapy Products (U.S. Food and Drug Administration, 2013), however, states that studies conducted in animals to assess tumorigenicity should use the intended clinical product, not analogous animal cells; should be of sufficient duration to demonstrate a cell therapy survives long enough to elicit tumors in vivo; that positive and negative control groups be included, if available; and that adequate numbers of animals be included per group so that the statistical analysis applied to any biological observations is appropriately powered. There is currently no scientific consensus regarding the selection of the most relevant animal models to evaluate tumor forming potential in vivo or the ability of current animal models to predict clinical outcome (U.S. Food and Drug Administration, 2013). The main approach to assess tumorigenicity potential for ex vivo LV-transduced HSCs treatment in animals (in addition to the analyses to evaluate integration risk) is to evaluate tumorigenicity by examining the fate of the cells

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postadministration (engraftment, migration, differentiation, tumorigenicity) in mice following sufficient length of time (typically multiple months) to allow for potential tumor formation. In the context of autologous ex vivo GTx nonclinical studies, tumorigenicity evaluation should be assessed in conjunction with ISA and clonal analysis. As described in the previous section, ISA evaluates the tendency for aberrant oligoclonality and/or insertion of the vector near proto-oncogenes or TSS in tissues and bone marrow. An oligoclonal distribution indicates persistent clonal dominance (i.e., marrow population arising from progeny of one or a few engrafted HSCs) and bone marrow reconstitution occurred from a small number of HSCs possessing a competitive advantage (Nash et al., 1988). Polyclonal distribution usually indicates a balanced clonal composition (marrow population arising from progeny of several engrafted HSCs) and the objective of ISA is to determine if a polyclonal insertion site pattern is observed or whether specific integrations confer a selective advantage due to possible insertional mutagenesis effects. Furthermore, general characteristics, like the proximity to the TSS of genes, the vicinity of integrations to known proto-oncogenes, clonal contribution of individual insertions to the overall pool of integrations, and shared integration sites among recipients should be analyzed. It is important to note that oligoclonality may occur in the absence of tumor formation. An oligoclonal composition with one or a few dominant clones that do not exhibit vector integration in mice with tumors indicates that the tumor formation is a spontaneous event and not a consequence of GTx. Oligoclonal distribution may occur in mice engrafted with a lower number of transduced stem cells, with very low bone marrow chimerism (Poletti et al., 2018). Additionally, studies show that aged control mice have oligoclonal hematopoiesis suggesting that with age, certain hematopoietic clones can predominate, and similar findings have been noted in NHPs and humans (Koeffler and Leong, 2017; Williams et al., 1984). Population-based genetic analyses of blood cells from large cohorts of aged humans have revealed oligoclonal or even clonal hematopoiesis, including evidence for specific clonal expansion of cells derived from progenitors with mutations in genes known to be associated with myelodysplastic

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syndromes (MDSs) or acute myeloid leukemia, such as DNMT3A and TET2 (Yu et al., 2018). Most individuals acquire clonal hematopoiesis during aging but will not develop MDS. In fact, clonal hematopoiesis may be detected with some frequency in healthy individuals with few or no hematologic findings. Therefore, acquisition of somatic mutations that drive clonal expansion in the absence of disease is termed clonal hematopoiesis of indeterminate potential (CHIP) (Steensma et al., 2015). MDS was diagnosed in a patient treated during a clinical trial with autologous ex vivo LV-transduced CD34þ HSCs for the treatment of sickle cell anemia (SCA) (Hsieh et al., 2020). The patient did not have MDS prior to conditioning, and developed MDS post LentiGlobin treatment. Multiple assays demonstrated the absence of vector integration in the CD34þ blasts and excluded LV vector–mediated oncogenesis as the cause of MDS (Hsieh et al., 2020). Potential possibilities that were discussed for these cases included busulfan, insertional mutagenesis, both, or neither (Jones and DeBaun, 2021). An alternative hypothesis is that after gene therapy for SCD, the stress of switching from homeostatic to regenerative hematopoiesis by transplanted cells drove clonal expansion and leukemogenic transformation of preexisting premalignant clones, eventually resulting in AML/MDS (Jones and DeBaun, 2021). Other reports of MDS were investigated in a cerebral adrenoleukodystrophy (CALD) clinical trial, but further investigation concluded that findings were misdiagnosed (Servick, 2021). Nonclinical studies evaluating ex vivo LVtransduced HSCs are generally conducted in mice for a minimum of 4–6 months. Longer term studies have been conducted in mice to assess tumorigenicity, but confounding findings may be observed depending on the conditioning agent, mouse genetic background, and/or ageassociated morbidities. For example, it is well known that mice exposed to TBI may eventually develop secondary solid organ and hematologic malignancies (e.g., thymic lymphomas, leukemias) due to radiation-induced mutations (Boniver et al., 1990; Dange et al., 2007; Hasapis et al., 2021) and that mouse strains on a C57BL/6 genetic backgrounds have a high incidence of age-related mortality due to spontaneous lymphoma and hematopoietic neoplasms

(Brayton et al., 2012). Therefore, it is important to identify experimental variables that may confound research outcomes when attempting to assess the risk of tumor formation.

5. GENOME EDITING 5.1. Harnessing Cellular DNA Repair Gene therapy is the introduction of genetic modifications to cells for the purpose of beneficially altering the cell’s biochemistry. Genome editing is a type of GTx that aims to improve the precision of induced genetic alterations. Targeted disablement of pathogenic genes, addition of healthy genes, or correction of dysfunctional genes can be accomplished with genome editing. Cellular DNA repair mechanisms are central to gene editing. The connection between DNA injury, in particular double-strand breaks (DSBs), and stimulation of DNA repair affords significant control over the genome editing process. Double-strand breaks are most commonly repaired through nonhomologous end joining (NHEJ) or less frequently by homology-directed repair (HDR) (Maeder and Gersbach, 2016; Takata et al., 1998). Other DNA repair pathways can be invoked, but NHEJ and HDR represent the predominant mechanisms in mammalian cells. Broken DNA ends can be repaired by NHEJ through direct reattachment to each other. One aspect of NHEJ is the propensity to incorporate small indels (a contraction of “insertions or deletions”) at the repair site (Lieber et al., 2003). The error-prone nature of NHEJ resulting from the unplanned gain or loss of genetic material can be useful for disrupting genomic regulation, structural features, or coding elements by introduction of frame shifts (Bibikova et al., 2002; Doyon et al., 2008; Geurts et al., 2009; Meng et al., 2008; Santiago et al., 2008). Addition of a repair template can invoke HDR (Szostak et al., 1983). The presence of a template that supplies the correct sequence of the original DNA strand renders HDR a high-fidelity repair process relative to NHEJ. Studies characterizing HDR have revealed that DSBs stimulate homologous recombination more often than NHEJ by orders of magnitude, providing an advantage

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for targeted genome editing (Choulika et al., 1995; Rouet et al., 1994; Smih et al., 1995). Programmable nucleases are used to introduce appropriately positioned DSBs in DNA and trigger a sequence of DNA repair events. After DNA repair is completed, the result is a permanent, location-specific change. The expected outcome of therapeutic genome editing is a permanent alteration in genomic DNA sequence. Two key features are common to programmable nucleases: (1) a targeting mechanism, and (2) a specific catalytic activity toward nucleic acids. The advent of programmable nucleases has made it possible to control the location of DSBs and leverage endogenous DNA repair machinery to engineer a wide variety of genomic alterations in a site-specific manner. Different programmable nucleases utilize different targeting mechanisms and thus have divergent utility in genome editing (Table 8.4) TABLE 8.4

(see also Genetically Engineered Animal Models in Toxicologic Research, Vol 1, Chap 23). Zinc-Finger Nucleases Zinc-finger nucleases (ZFNs) are modular enzymes comprised of two domains: a DNAbinding zinc-finger protein (ZFP) domain and the nuclease domain. Each ZFP recognizes a 3base pair (bp) sequence (Wolfe et al., 2000). Tandem placement of up to 6 ZFPs as an engineered fusion protein enables sequence-specific targeting of up to 18 bp. New targeting domains can be formed by connecting previously characterized ZFPs to form unique structures. The nuclease domain of ZFNs originates from the FokI restriction enzyme (Y. G. Kim et al., 1996). The FokI DNA-binding domain is replaced with ZFPs to create ZFNs. Since the FokI nuclease requires dimerization to cleave DNA, two ZFN monomers are needed to form an

Comparison of Programmable DNA-Editing Enzymes TALEN

RNA-Guided Endonucleases

Targeting mechanism Zinc-finger binding proteins

Transcription activation-like effector proteins

Single-stranded Fusion with nucleic guide RNA (sgRNA) acid targeting protein (e.g., cas)

Catalytic function

Fusion to endonuclease or nickase

Fusion to endonuclease or nickase

DNA nicking, cutting Biochemical conversion of target nucleic acid

Specific examples

Site-specific ZFN string with FokI endonuclease domain

Site-specific TALE with carboxyl end FokI endonuclease domain

CRISPR/Cas: Cas9 þ sgRNA Streptococcus pyogenes (SpyCas9) Neisseria meningitidis (Nme2Cas9)

Adenine deaminase: Adenine base editor (ABE; changes A•T to G•C)

Ease of targeting

Difficult

Difficult

Easy

Easy

Target site restrictions

G-rich

T as first nt, a as last nt

PAM site defined by CRISPR system (e.g., NGG or NAG for SpyCas9)

PAM site defined by the CRISPR system

Target site span

18-36 bp

>1 bp

20 bp þ PAM

18-20 bp þ PAM

Targeting precision (“off-target” effects)

High

Low

Variable

Variable

Platform

ZFN

Base Editors

Abbreviations: ABE, Adenine Base Editor; bp, basepair; Cas, CRISPR-associated protein; Cas, CRISPR-associated endonuclease; CRISPR, clustered regularly interspaced short palindromic repeats; nt, nucleotide; PAM, Protospacer adjacent motif; sgRNA, single guide RNA; TALE, Transcription activator-like effector; TALEN, Transcription activator-like effector nucleases; ZFN, Zinc finger nuclease.

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active nuclease (Bitinaite et al., 1998). Heterodimerization effectively doubles the recognition site length contributing to the ZFN specificity. Off-target effects from homodimerization have been described and can be substantially reduced by modifying ZFN variants to require heterodimerization (Kim and Kim, 2014; Miller et al., 2007; Szczepek et al., 2007). Genome editing with ZFNs faces several challenges. Flexibility for targeting all potential genomic loci is limited by the availability of ZFPs. Since a given ZFP can recognize a specific 3-bp combination, there are 64 DNA triplet permutations possible; currently, some of the triplets among the 64 possible combinations do not have corresponding ZFPs. In addition, a novel ZFN may not efficiently cut its target site. Consequently, each unique ZFN must be empirically tested to confirm activity (Ramirez et al., 2008) and rule out cytotoxicity from off-target effects (Cornu et al., 2008). Finally, generation of new ZFPs is relatively slow due to the complexity of protein synthetic techniques that are needed to ensure the appropriate sequence and folding of each ZFP. TALENS Transcription activator–like effector nucleases (TALENs) are similar in concept to ZFNs. Their structure contains a nuclease domain and a DNA-binding domain. Also similar to ZFNs, the nuclease domain of TALENs is derived from the FokI restriction enzyme. The DNAbinding domain of TALENs is from a class of proteins known as transcription activator–like effectors (TALEs) (Deng et al., 2012; Mak et al., 2012). TALEs are composed of sequences containing 33–35 amino acids. Nucleotide specificity is determined by two key amino acids known as “repeat variable diresidues” (RVDs) (Boch et al., 2009; Moscou and Bogdanove, 2009). Four different RVD modules can be used to recognize all four nucleotides in DNA. To target a given genomic locus, up to 20 RVDs are typically used to generate a site-specific TALEN. The availability to encode specificity at the single-nucleotide level gives TALEN design the capacity to target virtually any genomic location. As with ZFNs, generation of new TALES requires protein synthetic chemistry that slows the pace at which TALEN-based genome editing may be deployed.

RNA-Guided Endonucleases (CRISPR/Cas Systems) RNA-guided endonucleases (RGENs) were first identified as part of an acquired antiviral “immune system” characterized in prokaryotes (Barrangou et al., 2007; Makarova et al., 2006). Prokaryotic immunity to previously invading viral pathogens can be encoded into the host’s genetic “memory” as part of certain RGEN systems. Prior viral encounters are recalled by the retention of fragments of viral nucleic acid sequences within the bacterial genome. These viral DNA fragments, known as “clustered regularly interspaced short palindromic repeats,” or CRISPR sequences, represent the identification method for detecting and resolving subsequent viral invasions. To enable RNA-guided activity, prokaryotic systems utilize two RNAs derived from the captured CRISPR sequences. The transcribed CRISPR RNA (crRNA) gives rise to the targeting crRNA. A target-independent transactivating crRNA (tracrRNA) is also transcribed. Together, the crRNA and tracrRNA are combined with the catalytic protein to form an active ribonucleoprotein (RNP) complex. CRISPR-associated proteins (Cas) are a family of enzymes with nuclease activity. For example, Cas9 is an enzyme with DNA-cleaving endonuclease activity (Jiang and Doudna, 2017). Complexing with the crRNA and tracrRNA triggers a conformational change in Cas9 creating an access region within the RNP for target DNA to enter and bind. The result is an active endonuclease with specificity to the w20-bp targeting sequence within the crRNA. Recombinant engineering using standard nucleic acid biochemistry techniques resulted in development of a single-guide RNA (sgRNA) that reduced the key structural and targeting components of both tracrRNA and crRNA into a single nucleic acid (Jinek et al., 2012). Varying the targeting region of the optimized sgRNA is readily feasible with modern oligonucleotide synthesis methods, which allows tremendous genomic editing flexibility. This genome editing technique is preferred by many investigators since it can be adapted readily to insert one or many genes simultaneously and can be deployed in any species (prokaryotic or eukaryotic). Current applications include genetic engineering to produce new, especially nonrodent

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models of disease; in vivo or ex vivo correction of genetic defects; and production of genetically modified plants and animals as food sources. The CRISPR/Cas system has also been suggested as a means of removing integrated viral sequences from donor tissues or modifying endogenous antigens from animal organs to improve compatibility for xenotransplantation. Not all sgRNA target sequences result in a cleavable genomic locus (Koike-Yusa et al., 2014). Experimental confirmation of RGEN activity to a specific genomic locus is required to identify a tractable CRISPR/Cas9 combination. Base Editing In addition to DNA cleavage, Cas proteins can be modified to remove or reduce their endonuclease activity and recombined with other enzymatic functions to edit nucleic acids. Base editing is a genome editing approach that leverages RNA-programmable Cas proteins to catalyze targeted biochemical conversion of single nucleotides. The attractiveness of this technique is that point mutations may be corrected without producing DSBs or indels. These systems allow single-nucleotide variations without generating double-strand breaks. DNA base editors use CRISPR targeting functionality of Cas proteins combined with enzymatic function of deaminases (Shin et al., 2017; Tsai et al., 2015; Zhang et al., 2015). RNA base editors have also been described using RNA targeting components and deaminases (Rees and Liu, 2018). Base editors introduce point mutations by introducing specific chemical changes to a nitrogenous base that results in conversion to another base via mismatch repair pathways. The chemical conversion essentially switches the nucleotide identity allowing for precise editing without severing the helix. Cytosine base editors (CBEs) convert C•G basepairs into T•A basepairs (Gaudelli et al., 2017; Komor et al., 2016; Komor et al., 2017; Nishida et al., 2016), whereas adenosine base editors (ABEs) convert A•T basepairs to G•C basepairs. These two base editor classes can mediate all four possible basepair transitions. Certain RNA-targeting base editors can convert adenosine into inosine (Gaudelli et al., 2017; Montiel-Gonzalez et al., 2013; Vogel et al., 2018).

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5.2. Genome Editing Strategies Genome editing is generally a two-step process. First, programmable nucleases allow creation of precise DNA changes. Depending on the nature of the DNA alteration, certain endogenous DNA repair mechanisms are recruited to the site of DNA damage. These cellular repair systems then resolve the nucleic acid blemish, resulting in a permanent genetic edit. The type of programmable nuclease activity typically drives the recruitment of specific cellular repair systems. For example, programmable endonucleases cause DSBs, which stimulate NHEJ and to a lesser extent HDR mechanisms. In contrast, programmable deaminases can result in basepair mismatching, which recruits baseexcision repair mechanisms. Overall, the nature of DNA damage, and most importantly its genomic coordinates, can be managed by rational design of programmable nucleases. However, the editing process is completed by endogenous cellular DNA repair mechanisms. One of the first examples of genome editing was described when enhanced homologous recombination rates in mouse stem cells were observed following introduction of targeted DSBs by rare-cutting endonucleases (Kim and Kim, 2014; Rouet et al., 1994). Improved homologous recombination rates were also observed with ZFNs (Bibikova et al., 2003). In addition to homologous recombination, other DNA repair mechanisms are leveraged for genome editing. In the absence of donor DNA template, DSBs are efficiently repaired by relatively error-prone NHEJ, typically resulting in small indels (Bibikova et al., 2002). NHEJ or HDR of site-specific DSBs induced by programmable nucleases can lead to site-specific genetic modifications, including gene disruption, deletion, or insertion. Likewise, altering the catalytic function of programmable nucleases to biochemically change individual nucleotide identity allows for site-specific base editing. In this section, we discuss various strategies to select, design, and apply programmable nucleases to achieve a variety of functional genomic changes. Loss-of-Function The root cause of many monogenic diseases is expression of a pathogenic gene. Conventional

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therapeutic approaches aim to diminish the function of the pathogenic genes (proteins) through small molecule inhibition or function-blocking antibodies. Gene editing provides the opportunity to remove a pathogenic gene at the genomic level rather than quenching the activity of its product. Permanent removal of the disease-causing gene offers the potential for a functional cure. A common gene editing approach to knockout pathogenic genes makes use of NHEJ. Introduction of DSBs stimulates NHEJ repair, which can result in removing (deletion) or adding (insertion) a few nucleotides at the DSB site. These small, precise indels can result in a frameshift of the coding sequence disrupting gene expression. Transcription of frameshifted genes results in rapid degradation of those mRNA transcripts through nonsense-mediated decay (NMD) (Chang et al., 2007). For example, in diseases such as transthyretin amyloidosis (ATTR), misfolding and aggregation of the protein product arising from the pathogenic gene result in the disease phenotype. Targeted DNA DSBs in the TTR gene can result in NHEJ-induced indels, disrupting the reading frame of the gene (Gillmore et al., 2021). Cellular NMD processes reduce TTR mRNA levels and subsequent protein production. As residual protein concentration decreases through natural clearance, ATTR disease progression is expected to halt, or possibly reverse. GENE DELETION

In addition to small indels resulting from NHEJ, it is possible to delete large segments of DNA by introducing two DSBs bracketing the targeted span. This approach has introduced genomic deletions up to several megabases in size (Canver et al., 2014; Lee et al., 2010). This approach is useful for therapeutic strategies that may require the removal of an entire genomic element (e.g., one or several mutant exons). Duchenne muscular dystrophy (DMD) is caused by a frameshift mutation in the dystrophin gene. Deletion of one or more exons can correct the reading frame by “skipping” the frameshifted exon. Exon skipping by targeted deletion can restore expression of a shortened, but partially functional, dystrophin protein (Li et al., 2015; Nelson et al., 2016; Ousterout et al., 2015; Tabebordbar et al., 2016).

Gain-of-Function GENE INSERTION

Conventional GTx most commonly uses naked DNA or viral vectors to insert a transgene of interest into the genome. Spontaneous recombination or insertion of therapeutic DNA into DSBs is a stochastic event that can happen at locations throughout the genome with low predictability. Since the insertion site cannot be controlled when conventional GTx methods are used, the transgene and its regulatory sequences can be incorporated in the host cell genome at unintended sites. Misdirected insertion increases the risk of insertional mutagenesis. As a byproduct of conventional GTx, insertional mutagenesis may inactivate essential genes or activate proto-oncogenes, thereby leading to cell death or transformation (Hacein-Bey-Abina et al., 2003). Programmable nucleases can enhance control of the location where transgene insertion occurs. The efficiency of homologous recombination increases approximately 100-fold when site-specific DSBs are created by programmable nucleases (Li et al., 2011; Lombardo et al., 2011). Controlled creation of DSBs provides preferential openings in the genomic sequence for new genetic material to fit. This strategy allows the use of rationally designed templates coupled with programmable nucleases to direct targeted insertion of therapeutic genes into genomic locations considered to be safe (Lombardo et al., 2011). To further tune genome editing, sequence orientation can be controlled by leveraging HDR. Introducing flanking homology arms to the gene of interest promotes HDR-directed insertion of the construct. Matching homology arms to sequences surrounding the genomic cut site can orient the construct in the chosen direction relative to the cut site. An alternative approach omitting homology arms is used where orienting the transgene sequence is not critical. With this strategy, template DNA is ligated to the genomic target locus using NHEJ repair (Ran et al., 2013). In practice, the editing nuclease is simultaneously administered with an insertion template to achieve targeted genomic insertion (Moehle et al., 2007). Gene Correction The largest class of human pathogenic mutations is single-nucleotide polymorphisms (SNPs),

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or point mutations (Shastry, 2009). Safely correcting pathogenic SNPs is essential to permanently treat monogenic diseases. The greatest challenge to addressing the root cause of SNPs is sorting through the patient’s entire genome to specifically alter an individual nucleotide without unintended changes to nucleotides at other locations. NUCLEASE-MEDIATED CORRECTION

In contrast to the variety of repair structures and mutations resulting from NHEJ, targeted DSBs can induce precise gene editing by stimulating HDR with an exogenously supplied donor template. Predominantly during cell division, HDR repairs DNA using the sister chromatid as a reference DNA template (Ciccia and Elledge, 2010; Heyer et al., 2010). Borrowing from this system, genome editors can control repair using an exogenously supplied DNA template sequence. Simultaneous delivery of programmable nucleases with exogenous templates can initiate DSB formation and HDR to correct pathogenic SNPs with reasonably high fidelity (Rouet et al., 1994; Smih et al., 1995). This type of correction approach is similar to gain-of-function insertions with the exception that the native gene and structure is preserved, altering only a few nucleic acids to restore a healthy genotype. Base editing is a genome editing method with potential to precisely change point mutations without generating DSBs, using exogenous DNA templates, or relying on HDR (Rees and Liu, 2018). In cases where monogenic disease is caused by a defined point mutation, base editing can be employed to chemically convert the pathogenic base into the normal base. For example, alpha-1-antitrypsin disease is caused by a point mutation where a single adenine replaces the wild-type guanine. This point mutation results in misfolded alpha-1-antitrypsin, potentially resulting in pathogenic lung and/or liver phenotypes. Targeted reversion of the mutant adenine into guanine by an ABE strategy could potentially correct the disease.

5.3. Nonclinical Safety Assessment for Genome Editing Products Exaggerated Pharmacology Target-specific considerations include the biological influence of achieving the desired

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gain- or loss-of-function. Separate from the toxicological assessment of test article administration, genome-edited products carry the expectation of permanent pharmacology long after the GTx components have cleared the body. Exaggerated pharmacology studies should aim to test dose ranges achieving maximum pharmacological effects for extended periods of time. Depending on the mechanism and potential biological role(s) of the target gene of interest, exaggerated pharmacology studies in animals typically extend for several months; 6 months is common to demonstrate the chronic influence of an exaggerated pharmacological effect stemming from a single-dose GTx application. An additional consideration related to study duration is the relative fraction of the animal’s lifetime during which a long-acting GTx product is active. In rodents, 6 months can represent approximately one quarter of the animal’s lifespan, whereas in NHPs, 6 months represents approximately 2% of the animal’s natural lifespan (Choi et al., 2016). These aspects can influence claims associated with the relevance of a potential exaggerated pharmacology study. Biodistribution

Biodistribution of genome editing elements depends predominantly on the delivery system. Viral and nonviral delivery systems exhibit tropism that can vary widely depending on the cell or tissue type. Biodistribution studies aim to characterize the product tropism. For genome editing, characterization of delivery system components is useful for describing the relative tropism and potential for editing activity. For viral delivery approaches, PCR assays targeted to specific portions of the viral genome are typically used to estimate relative distribution to tissues. For nonviral approaches, methods measuring one or more representative components of the delivery system can provide information about the tissues where genome editing may occur. A unique consideration for GTx products is the question of whether the product distributes to gonads, which contain germ cells. Currently, human germline genome modification is outlawed in multiple countries. Characterizing the potential for delivery of genome editing

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components to germ cells is an important step in limiting risk of vertical germline transmission. Assessing germline transmission risk can be considered a biodistribution assessment since absence of gonadal distribution indicates a low risk of germline editing. If biodistribution is detected in germ cell–containing matrices, such as semen, or tissues, such as ovary or testis, a functional study can be performed as a definitive assessment. Mouse models are ideal for use in breeding studies to definitively assess the risk of vertical germline transmission. Guidance on nonclinical testing of inadvertent germline transmission, focused on viral GTx, has been published by the EMA (European Medicines Agency, 2007). The guidance document contains a detailed experimental and decision framework outline and is readily adaptable to viral and nonviral delivery approaches. Toxicology Genome editing carries many of the same safety considerations mentioned earlier in this chapter relevant to conventional gene and cell therapies. Most toxic effects of genome editing can be attributed to either the delivery system (e.g., viral capsid of a GTx) or effects related to gain or loss of the target gene function (i.e., exaggerated pharmacology). Many of these effects are well covered in earlier sections of this chapter. In this section, we focus on key toxicity issues specific to genome editing. In particular, significant emphasis is placed on genome safety, considering the mode of action involves direct and ideally permanent manipulation of a patient’s genetic material. Additional considerations for nonviral lipid nanoparticle delivery of nucleic acids will be reviewed briefly. EFFECTS FROM NANOPARTICLE DELIVERY OF NUCLEIC ACIDS

Separate from exaggerated pharmacology, GTx toxicity is most commonly associated with chemical components of the delivery system. Nanoparticle delivery of gene editing technologies represents a breakthrough advance in nucleic acid therapy (see Nanoparticulates, Vol 3, Chap 13). Naked nucleic acids will quickly elicit an innate immune response. In particular, RNAs are highly sensitive to endogenous nucleases and will degrade quickly without protection.

Nanoparticles have been developed to protect nucleic acids from rapid clearance, cloak them from immune surveillance, and in most cases improve tropism to a targeted tissue or cell type. In exchange for advantages associated with nanoparticles, several features have been described as common unintended occurrences in nanoparticle nucleic acid delivery systems. Nanoparticles are often designed with features similar to viral vectors to mimic cellular uptake. Nanoparticles can avoid some of the main drawbacks of viruses that limit exposure and/or safety. For example, preexisting antibodies to environmental viral components can limit exposure to cross-reactive therapeutic viral vectors. Considering nanoparticles resemble synthetic virus–like structures, it is not surprising that some (but not all) safety features appear to be similar between viral and nanoparticle delivery systems. Depending on physical/chemical nanoparticle characteristics, various plasma proteins can adsorb to the particle surface. The concentration of certain adsorbed proteins can bias particle tropism to certain tissues. For example, the adsorption of ApoE to nanoparticle surfaces can confer specificity to hepatocytes, which are rich in the ApoE receptor LDLR. Increased local concentrations of nanoparticles in the liver can drive hepatotoxicity, which is a common consequence of nanoparticle administration. GENOTOXICITY

Potential unintended effects of incorporating new genetic material into a cell’s genome are typically related to the precision of gene manipulation. Off-target mutations from programmable nucleases occur predominantly at other sites in the genome with DNA sequences that are highly homologous to their intended target sites. Measuring off-target effects of programmable nucleases is critical to assess potential unintended biological effects and developing means to reduce such off-target effects is a key effort toward improving the safety of genome editing products. A variety of strategies are available to characterize off-target effects of programmable nucleases. Selecting target sites with unique genomic sequences is a logical first step to limiting METHODS

OF

ASSESSING

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off-target activity. Whole-genome sequencing analyses of ZFNs, TALENs, and RNA-directed nucleases support the notion that target sites with exclusively unique genomic sequences do not induce substantial off-target mutations in cells (Cho et al., 2013). Whole-genome sequencing requires significant sample input to allow for sufficient sensitivity to detect rare events at all nucleotide positions. For this reason, whole-genome sequencing is generally not feasible during nonclinical or clinical studies for adequately characterizing gene editing therapeutics for human use. Despite these limitations, assessment from a genome-wide perspective for off-target editing is possible using a combination of methods. Staging the evaluation to initially survey the genome and identify the most likely off-target locations helps to focus the analysis to highest risk areas. Narrowing the search allows sequencing depth to be focused on the most plausible off-target activity sites. Increasing sequencing depth at the most likely affected genomic loci allows for the most sensitive detection of rarely formed indels. A stepwise approach increases confidence in confirming or ruling out whether off-target activity may occur at the candidate sites. Off-Target Site Discovery Off-target discovery aims to sensitively identify the most likely genomic locations where editing may occur outside the intended editing site. Multiple orthogonal methods (i.e., distinct methods assessing a question, such as IHC and ELISA to confirm transgene expression in a tissue) should be applied to rationally predict and empirically identify potential off-target sites. Computational prediction based on sequence homology and biophysical binding rules is a broadly applied practice to identify potential off-target sites in the genome. For ZFNs and TALENs where editing is targeted by protein binding, off-target prediction can be challenging due to limited understanding of biophysical relationships governing the stringency of protein and nucleic acid interactions. Published computer algorithms are available to predict off-target candidate sites from ZFNs and TALENs (Fine et al., 2014; Kim et al., 2013). For programmable nucleases, computational prediction focuses on nucleic acid sequence homology. Variation of sequence

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mismatches, potential bulges, nucleotide insertions or deletions can be tuned within the prediction algorithm. Off-target site prediction for RNA-guided nucleases leverages biophysical rules related to mismatch base pairing. Several computational approaches to predict potential RNA-guided nuclease off-target sites have also been described (Bae et al., 2014; Heigwer et al., 2014; Hsu et al., 2013). Despite good mechanistic evidence supporting the underlying rules for computational offtarget prediction, not all off-target sites can be reliably identified in silico. To complement in silico prediction, experimental methods to identify potential off-target sites are used to increase confidence in a comprehensive genome-wide assessment. Empirical approaches for identifying possible off-target editing sites make use of the specific activity of the programmable nuclease in a relevant experimental assay system, such as cell-free (e.g., SITE-seq) or cellbased (e.g., GUIDE-seq) methods (Cameron et al., 2017; Tsai et al., 2015). Programmable nucleases with DSB activity can be experimentally evaluated by a variety of methods currently available, with new peer-reviewed methods appearing regularly in the literature (Chaudhari et al., 2020; Naeem et al., 2020). The combined set of in silico and empirically identified off-target discovery sites can then be categorized into three functional classifications: intergenic, intronic, and exonic regions. Intergenic regions are spans of noncoding DNA between genes. Since intergenic DNA has no known function, potential off-target sites located in intergenic regions are considered to be of lowest risk. Intronic regions are noncoding DNA spaces within genes that are removed posttranscriptionally by RNA splicing before translation into protein. Intronic DNA may contain splice sequences or other expression control elements. For these reasons, any potential intronic offtarget sites should be functionally annotated and considered for further risk assessment pending verification of off-target editing. Finally, exonic sites contain protein-coding sequences, and thus carry the greatest risk for altering biological function. Potential off-target sites identified in exonic regions should be carefully evaluated to verify absence or presence of off-target editing since mutations in this region are likely to disrupt protein and therefore biological function.

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Off-Target Site Verification A compiled list of potential off-target sites identified during the discovery phase is generated to advance into the verification phase. This combined set of genomic coordinates can then be subject to deep sequencing for determining the rate, if any, of nucleotide sequence variation within those regions. Currently, next-generation sequencing is the gold standard for confirming the absence or presence of indels formed by off-target genome editing activity. Limiting the scope of sequencing to a focused set of locations allows for rich sampling of those areas, which greatly increases the likelihood of observing low editing rates. Increased sensitivity for detecting low editing rates maximizes confidence in confirming or ruling out rare instances of off-target editing. Narrowing the search to a targeted list of the most likely off-target locations can improve sequencing to an average coverage of 5000-fold per site, as compared to 50-fold coverage with the best wholegenome approaches. Some genomic locations may not be amenable to sequencing due to various technical limitations. From a bioinformatic perspective, collecting quality metrics helps improve confidence that interpretation for each candidate site can be compared in an appropriate context relative to all other sites, genome-wide. Genetic sample material to verify off-target editing should be representative of the therapeutic approach. For cell therapy approaches, samples generated with an engineering process representative of the clinical process should be submitted for off-target verification. For in vivo administered products, human tissue and/or cell types from the intended target organ(s) should be treated with relevant doses of the clinical therapeutic construct, and those samples submitted for off-target verification. Despite potentially highly conserved genetic identity to humans, animal models have limited relevance for assessing off-target editing due to incomplete genomic homology. Subtle genetic variation within the human genome warrants testing of multiple tissue/cell donors to improve confidence in identifying rare offtarget events. Biodistribution data may provide additional information for systemically administered products. CONSIDERATIONS FOR OFF-TARGET RISK ASSESSMENT Risk assessment should prominently

consider the functional genomics of each potential off-target locus. Exonic loci carry the greatest risk given the role protein-coding sequences have in biological function. Off-target activity in exonic regions can potentially disrupt protein expression through frameshifting indels. Intronic regions are generally considered lower risk relative to exonic regions. Special attention should be paid to candidate off-target sites within intronic regions to identify possible splice sites and other features that may influence gene expression or regulation. Intergenic regions are spans of noncoding DNA between genes. Rarely do intergenic regions have genetically functional properties beyond structural spacing and for these reasons are considered the lowest offtarget risk category. Totals in the hundreds to thousands of candidate off-target sites are typically identified during the off-target discovery phase. Statistically, potential off-target editing sites within genes (either intronic or exonic) will be unavoidable. A general review of all potential off-target sites should be performed to identify any genes with known roles in cellular proliferation, tumor-suppressor or oncogenic effects. Several additional considerations should be applied if a site demonstrates validated offtarget activity within a gene. In general, the impact of losing expression of the off-target gene within the target cell type should be projected. For systemically administered products, biodistribution data can also inform the risk relative to on-target activity in unintended tissues, suggesting the potential for off-target editing in that tissue type. Finally, derisking experiments may be warranted to define the true influence of editing to off-target gene expression. CHROMOSOMAL STRUCTURAL VARIATION Any source of DNA DSBs can give rise to chromosomal structural variations (SVs) upon repair. Programmable nucleases focus DSB activity to precise locations in the genome, which allows for high-confidence prediction of potential varied chromosomal structure. Chromosomal SVs are most common at the on-target site. Deletions, duplications, and inversions of up to a few megabasepairs of chromosomal segments have been achieved using ZFNs (Lee et al., 2010; Lee et al., 2012), TALENs (Carlson et al., 2012), or CRISPR/Cas (Cong et al., 2013). Sister chromatid

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exchange without indel formation represents a structural variation with no expected functional consequence. This category of SV is also termed balanced rearrangement (Figure 8.13). When evaluating the on-target site for SVs by next-generation sequencing (NGS), concordant sequencing paired ends are identified. In this scenario, forward (F) and reverse (R) probes are oriented in agreement with the reference genome. When discordant paired ends are identified by NGS, various types of SVs common to on-target editing may be present. Confirmed discordant paired ends are considered true

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chromosomal translocations. Acentric and dicentric interchromosomal rearrangements occur within the target chromosome (Figure 8.13). Loss, gain, or inversion of large DNA spans may also result. The potential for chromosomal SV increases with editing activity and with the number of sites simultaneously edited. Complex editing at multiple on-target sites within the same chromosome represents a possible strategy for intentionally generating a deletion, an intended form of chromosomal SV. Unintended effects of this approach include inversions of the target region,

FIGURE 8.13 Potential chromosomal repair structures resulting from a double-strand DNA break, at a single genomic locus. Predominant repair structures can be assessed by next-generation sequencing (NGS) using locusspecific forward (F) and reverse (R) reagents. Concordant paired end outcomes include perfect repair and balanced repair. Concordant repair structures preserve native chromosomal structure and are considered to be the vast majority of chromosomal repair outcomes. Discordant paired ends are considered the rarest chromosomal structural variants resulting from targeted DSBs. Discordant paired ends most commonly appear as interchromosomal allelic translocations within the same chromosome number. Dicentric and acentric structural variants, which can be detected by NGS, are typical discordant structural variants stemming from a single, allelic DSB. Nonallelic interchromosomal translocations require exchange with two separate chromosomes. Discordant paired end intrachromosomal structural variants involve a single locus and may reflect large insertions and/or deletions. Copyright 2021 by Intellia Therapeutics.

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in addition to potential acentric and dicentric chromosomes. The introduction of off-target activity on different chromosomes increases the likelihood of additional types of structural variants. Interchromosomal translocations can be generated in cases where genome editing activity is occurring at multiple sites simultaneously. The interchromosomal type of structural variation is more commonly associated with neoplastic conditions. Overall, the possibility of unwanted chromosomal structural variations should be considered when programmable nucleases are used for gene or cell therapy. GENOTOXICITY SUMMARY AND FUTURE DIRECTIONS Programmable nuclease technology is

relatively new and will benefit from further characterization leading to improvements. Better definition of relationships between the biophysics and biochemistry of programmable nucleases can lead to improved nuclease design, specificity, potency, and optimization of undesirable effects (Sternberg et al., 2014). Improved methods for empirically identifying off-target editing activity will increase confidence in genome editing precision and likely will influence the pace at which the technology is adopted (Gabriel et al., 2011). Currently, only programmable nuclease activity can be directly controlled. Influencing DNA repair pathways represents an additional opportunity to develop high-precision genome editing approaches. Pharmaceutical (i.e., transient) or genetic (i.e., lasting) modification of DNA repair mechanisms offers the potential for total control of the editing process. IMMUNE ACTIVATION

Given similarities in size, structure, and nucleic acid delivery to viral vectors, activation of immune responses can be an expected feature of nanoparticle administration. These effects can resemble infusion reactions clinically, with signs of elevated body temperature and heart rate, hypertension or hypotension, dyspnea, flushing, and/or erythema. Symptoms can vary from absent to severe and may be lethal depending on individual subject sensitivity. Observation of clinical signs may be present with a single treatment, without requiring sensitization from prior exposure. Mechanistically, nanoparticle-mediated immune reactions tend to involve components of

the innate immune arm including acute complement activation, inflammatory cytokine release, or both. Complement plays an important role in innate immunity to provide defense against pathogens. Activation of plasma complement may occur in response to nucleic acid–delivering nanoparticles. The specific complement pathway and severity of complement activation can vary in response to a combination of factors, including nanoparticle size, surface chemistry, surface charge, and nanoparticle morphology. Acute responses with symptoms matching Type I hypersensitivity reactions, but that are not mediated by IgE antibodies, have been reported to occur in a high percentage (up to 45%) of patients within minutes of infusions with therapeutic antibodies, micellar drug formulations, and nanoparticles (Moghimi et al., 2012). These reactions are often referred to collectively as Complement Activation Related Pseudoallergy (CARPA). Although CARPA has been observed in the clinic with various types of therapeutic products, nanoparticles and especially those with a polyethyleneconjugated (“PEGylated”) formulation are frequent causes of CARPA in humans (Salvador-Morales and Sim, 2013; Szebeni et al., 2007). Activation of plasma complement is initiated via three main pathways: the classical, the alternative, and the lectin pathway. Each of these pathways converges at the complement component 3 (C3) to form a common, terminal pathway. Complete activation of the terminal pathway leads to formation of the membrane attack complex (Sahu and Lambris, 2001). The membrane attack complex forms a pore in cell membranes to disrupt structural integrity and destroy the pathogenic cell. Cleavage of C3 and additional complement components C4 and C5 results in generation of several split products. The larger split products (C3b, C4b, and C5b) bind to and accelerate the clearance of microbial pathogens. Split products that bind to nanoparticles can increase recognition and rapid clearance by macrophages of the reticuloendothelial system that bear complement receptors (e.g., hepatic Kupffer cells, splenic marginal zone and red pulp macrophages, blood monocytes, etc.).

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GLOSSARY

The smaller split products (C3a, C4a, and C5a) are potent anaphylatoxins, which are the main triggers of the symptoms observed in CARPA. Generation of complement-derived anaphylatoxins can lead to release of cytokines and chemokines from a variety of immune cells, which may subsequently initiate anaphylaxis in some individuals.

6. CONCLUSION The turn of the 21st century enabled the field of GTx by providing access to molecular signatures of numerous genetic diseases that would have been otherwise left enigmatic and far from clinical reach. At the same time the scientific revolution of genetic engineering bolstered the field of GTx by providing access to technologies that permitted the manufacturing of vectors at a larger scale to support nonclinical and clinical trials. Despite the tremendous success in proofof-concept nonclinical studies and the fastpaced progress and success into clinical trials, several theoretical and real toxicological risks have recently emerged and occasionally been identified as GTx class-effect toxicities that require closer attention as the field moves forward. The goal of this book chapter is to introduce the reader to basic concepts on GTx development and dive deeper into the key safety concerns and toxicology testing parameters as they pertain to the most common GTx platforms. Tremendous traction and growth are occurring in the field of GTx with thousands of nonclinical programs and clinical trials. Nonetheless, the current state of GTx drug development remains on a case-by-case basis dependent on disease indication and product attributes. Therefore, toxicologists and toxicologic pathologists involved in the nonclinical safety assessment of GTx products need to recognize both the emerging patterns of GTx platform-specific toxicities, become familiar with often innovative parameters in the design and execution of effective nonclinical studies, and continue building the knowledge and path for a standardized approach to the safety assessment of novel gene therapies.

GLOSSARY Allogeneic – involving tissues or cells that are genetically dissimilar (i.e., “non-self”) and hence immunologically incompatible even though derived from individuals of the same species Apheresis – a procedure that withdraws blood from the body, separates the cell and plasma fractions, and then reintroduces the cells to the patient’s body Autologous – involving cells or tissues obtained from the same individual (i.e., “self”) Biodistribution studies – experiments performed to assess the peak, persistent, and clearance of gene therapy products by measuring the vector DNA by quantitative polymerase chain reaction (qPCR) and by measuring the transgene mRNA by reverse-transcription PCR (RT-PCR) Clonal dominance – population of bone marrow by progeny arising from one or a few engrafted hematopoietic stem cells Clonal hematopoiesis of indeterminate potential – acquisition of somatic mutations that drive clonal expansion in the absence of disease Concatemeric – multiple linked copies of the same transgene DNA (linked in tandem and forming a long continuous DNA sequence) inserted at the same site in the genome Conditioning regimen – application of chemotherapy, monoclonal antibody therapy, and/or radiation to the entire body to (1) create space in the patient’s bone marrow for newly transplanted hematopoietic stem cells to colonize and (2) help prevent the patient’s immune system from rejecting the transplanted cells DNA integration – covalently linked (i.e., stable) insertion of foreign genetic material into a host DNA sequence on a chromosome Episome – (for adeno-associated viral vectors) circular, doublestranded extrachromosomal DNA Gene editing – transfer of genetic material (such as CRISPR/Cas technology) capable of directly editing the host genome to eliminate a genetic mutation at its chromosomal source Gene replacement – transferring a functional copy of a defective or missing gene, where the copy is capable of producing a missing functional protein from the affected cells to restore their functional abilities Gene suppression – transferring a gene expressing an RNA interference element (such as short hairpin RNA [shRNA] or microRNA [miR]) that is capable of suppressing a gain-of-toxic-function gene in the affected cells Gene therapy – a technique that seeks to modify the expression of a gene or to alter the biological properties of living cells for therapeutic use to treat or cure disease Highest Nonseverely Toxic Dose (HNSTD) – a dose that is biologically effective with a manageable level of toxicity Homology-directed repair – a precise DNA repair pathway utilizes either an endogenous gene (second copy of the broken gene on the sister chromosome) or exogenous piece of homologous DNA as a template to repair a sequence fo damaged DNA Humanized mouse – a mouse that has been engrafted with human genetic material or human cells/tissues Insertional mutagenesis – usually random insertion of introduced genetic material into the host genome at a site that disrupts a gene and alters its function Leukapheresis – a procedure that separates white blood cells, including hematopoietic stem cells, from the blood and then returns the remaining blood fractions to the body Maximum efficacious dose – the dose beyond which no additional therapeutic benefit is observed Minimally efficacious dose – lowest dose at which signs of efficacy are evident

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Mobilizing agents – drugs that induce transient mobilization of hematopoietic stem cells from the bone marrow into circulation so that they can be collected by leukapheresis Multiplicity of infection (MOI) – the number of transducing units of viral particles per cell used during a transduction of target cells Neutralizing antibody – an antibody that binds to a virus and interferes with its ability to infect a cell Nonhomologous end joining – a set of genome maintenance pathways in which two DNA double-strand break (DSB) ends are (re) joined by apposition, processing, and ligation without the use of extended homology to guide precise repair of the sequence Oligoclonality – clones derived from one or a few cells or molecules Optimal biological dose – a dose that offers a maximal therapeutic benefit with manageable risk Orthogonal methods – distinct methods assessing a single question, such as immunohistochemistry (IHC) and enzyme-linked immunosorbent assay (ELISA), that are used to confirm transgene expression in a tissue Pathogen-associated molecular patterns (PAMP) – molecules with conserved motifs that are associated with pathogen infection and that serve as ligands for host pattern recognition molecules such as Toll-like receptors Progenitor cells – descendants of stem cells that further differentiate to create specialized cell types Pseudotyping – the process of producing viruses or viral vectors in combination with foreign viral envelope proteins. For viral vectors, pseudotyping by acquisition of novel surface proteins derived from other viruses modifies the tropism of the viral vector particles Replication-defective virus – virus defective for one or more functions that are essential for viral genome replication or synthesis and/or assembly of viral particles Self-complementary – vector construction for recombinant adenoassociated viral vectors in which a dimeric inverted repeat DNA molecule can form a double-stranded DNA sequence without the need for the host cell–dependent rate-limiting step of second-strand DNA synthesis needed for efficient transgene transduction Self-inactivating – introduction of a deletion in the U3 region of the 30 long terminal repeat (LTR) region of retroviral vector DNA that eliminates the enhancer–promoter sequence and provides better regulation of tissue-specific transgene expression, thereby reducing the risk of cellular gene transactivation and the likelihood of forming replication-competent (i.e., viable) virus Stem cells – undifferentiated cells that are able to both self-renew and differentiate into various specialized cell types Syngeneic – involving genetically similar or identical cells and tissues that hence are immunologically compatible, thereby allowing transplantation without provoking an immune response Transduction – introduction of foreign DNA into a cell Transgene – a gene artificially transferred into a host cell by any of several gene therapy techniques Tumorigenicity – the tendency for agents to induce cells to undergo neoplastic transformation Viral genomes (vg) – the recombinant genetic material (DNA or RNA) carried by the vector for introduction into the host cells Vector – a system (viral or nonviral) used to deliver foreign DNA (the genetic payload; a transgene) into target cells Viral tropism – the specificity of a virus for a particular host cell or tissue type, determined in part by the interaction of viral surface molecules with receptors present on the surface of the host cell

Acknowledgments The authors thank Drs. N.P. van Til and Lawrence O. Whiteley for their critical review of the book chapter and Ms. Beth Mahler for assistance with optimizing the figures.

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C H A P T E R

9 Vaccines Rani S. Sellers1, Keith Nelson2 1

University of North Carolina, Chapel Hill, NC, United States, 2Charles River Labs, Mattawan, MI, United States O U T L I N E 7.1. Repeat-Dose Toxicity Studies

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1. Introduction

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2. Vaccine Immunology

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3. General Concepts in Vaccine Toxicology 3.1. Vaccine Nonclinical Safety Package: Regulatory Expectations 3.2. Immunogenicity Assays

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4. Vaccine Modalities 4.1. Protein-Based Vaccines 4.2. Nucleic AcideBased Vaccines

341 341 344

8. Vaccine StudiesdDesign, Technical Considerations, and Data Interpretation 8.1. Species Selection 8.2. Dose Groups 8.3. Technical Considerations Related to Dosing and Dose Administration 8.4. In-Life Assessments 8.5. Clinical Pathology 8.6. Anatomic PathologydPost-life Evaluation

5. Vaccine Adjuvants 5.1. Mineral Salts 5.2. Surfactant/Emulsion Adjuvants 5.3. Nucleic Acid/Nucleotide Adjuvants 5.4. Lipid Adjuvants 5.5. Adjuvant Systems and Liposome-Based Adjuvants 5.6. Carbohydrate-Containing Adjuvants 5.7. Toxin-Based Adjuvants 5.8. Other Adjuvants

348 349 350 351 352

9. Special Considerations for Therapeutic Modalities 9.1. Therapeutic Vaccines 9.2. Other Therapeutic Vaccine Strategies 9.3. Oncolytic Virus-Based Therapeutic Vaccines

379 379 380 380

352 353 353 354

10. Nonclinical ToxicitydDetermining Adversity

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6. Vaccine Pharmacology and Other Studies 6.1. Immunogenicity and Efficacy Studies 6.2. Enhanced Disease and Neurovirulence Assessment 6.3. Biodistribution and Persistence 6.4. Absorption, Distribution, Metabolism, and Excretion

354 354

11. Nonclinical Toxicity and Human Translation 11.1. Autoimmunity 11.2. Hypersensitivity 11.3. Thrombotic Thrombocytopenia

382 382 383 383

12. Vaccines and the Anti-Vaccine Movement

384

355 356

13. Conclusions

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14. Glossary

385

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Acknowledgments

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7. Vaccine Safety Studies

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References

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Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-12-821047-5.00029-4

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1. INTRODUCTION Vaccines against infectious diseases, termed prophylactic vaccines, have had a tremendous impact on world health, eliminating or significantly reducing diseases such as polio, smallpox, measles, mumps, rubella, diphtheria, pneumococcus, meningitis, tetanus, and most recently, COVID-19. Vaccines are also being developed as therapeutics, in which the immune system is manipulated to aid in the treatment of diseases, most commonly cancer but also chronic conditions such as Alzheimer’s disease, hypercholesterolemia, and to treat some infectious diseases such as human immunodeficiency virus (HIV). The concept of exposure-based protection for infectious disease has been understood for centuries. Variolation (i.e., deliberate inoculation with Variola virus–contaminated secretions from smallpox victims) had been used as early as the 10th century in China to protect against disease, and the practice spread to Europe and the North American colonies of European powers in the early 1700s (Riedel, 2005). Advances in protection against smallpox using a similar but less deadly orthopox virus, cowpox (Vaccinia virus), came in the late 1700s. Edward Jenner, an English physician, has been hailed as the father of modern vaccination with his publication in 1798 of a paper entitled ’An Inquiry into the Causes and Effects of the Variolæ Vaccinæ; a Disease Discovered in some of the western counties of England, particularly Gloucestershire, And known by the name of The Cow Pox’. The association between cowpox infection and smallpox protection, however, was well understood by many at that time, and inoculation with cowpox for that reason was already in practice. Nevertheless, Jenner was the first to scientifically assess, publish, and promote vaccination with cowpox to cross-protect against deadly smallpox (Riedel, 2005). Vaccines developed today have little resemblance to those developed during the 18th, 19th, and even 20th centuries (Plotkin & Plotkin, 2018). Thanks to advances in molecular and protein technologies, antigen components used in modern vaccines induce a more antigenspecific immune response, and vaccines are safer and more consistently manufactured. Further, novel molecular modalities and advances in chemistry have allowed for very rapid development and deployment of vaccines against

pathogens and patient-specific tumor antigens. Vaccine products are highly varied, and include modified live and inactivated microbes; purified or recombinant proteins; polysaccharide or polysaccharide-conjugated; DNA (recombinant viruses or plasmids); RNA (recombinant viruses or mRNA); synthetic peptides; virus-like particles (VLPs); and cellular modalities (e.g., dendritic cell–based vaccines). Oncolytic viruses may also fall under the therapeutic vaccine umbrella. Vaccine formulations may include adjuvants, which enhance the immune response to the antigen. Additionally, vaccine administration may utilize specialized delivery systems (ranging from nanoparticles to medical devices), which must also be evaluated for safety in nonclinical studies and clinical trials. Because of the diversity of the modalities, the nonclinical testing paradigm for vaccines is designed on a case-by-case basis. The rapid development of vaccines against severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), the virus responsible for the ‘coronavirus disease 2019’ (COVID-19) pandemic, exemplifies the power of modern molecular technologies for vaccine development. The first human clinical trials of a COVID-19 vaccine were initiated within 3 months of the publication of the SARS-CoV-2 sequence (Zhu et al., 2020). This rapid vaccine generation was the result of advances in ribonucleic acid (RNA) technology allowing for the development of RNA-based vaccines. These mRNA vaccines were engineered to encode the SARS-CoV-2 S (“Spike”) protein in its prefusion conformation, and the RNA was encapsulated in a lipid nanoparticle (LNP) to ensure effective cell entry (Corbett et al., 2020; Polack et al., 2020). The role of the toxicologic pathologist in vaccine development cannot be understated as their expertise connects basic science, immunology, physiology, and toxicology. The pathologist is uniquely positioned to integrate immunological data with safety and efficacy data and should be involved in the programs from the earliest stages of vaccine development. This chapter will review the nonclinical development of prophylactic vaccines and vaccine-based therapeutics, including study design and interpretation, as well as review the types of toxicity studies needed to support first-in-human (FIH) clinical studies and vaccine registration.

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2. VACCINE IMMUNOLOGY

2. VACCINE IMMUNOLOGY Vaccine efficacy requires robust antigenspecific immune responses. Long-term vaccine efficacy, particularly for prophylactic vaccines, most often relies on the generation of highaffinity neutralizing antibodies by plasma B cells, antigen-specific T cells, and long-term immune memory cells to ensure rapid protective immune responses upon subsequent pathogen exposure (Pulendran & Ahmed, 2011; Siegrist, 2018). Antibodies may mediate protection through an assortment of mechanisms, including binding to viral proteins and preventing cellular entry, binding bacterial proteins and promoting clearance by macrophages and neutrophils (opsonophagocytosis), by activating complement and promoting macrophage clearance, or by binding to active sites to prevent receptor binding (e.g., toxoids). Although B cells are the source of antibodies, T cells are essential for the generation of highly specific antibodies with high affinity as well as immune memory. Antigen-specific T cell responses are also important in the elimination of intracellular pathogens (e.g., viruses, mycobacteria). Cancer vaccine approaches rely heavily on the T cell response for efficacy. An innate immune response is essential for generating adaptive immune responses, including improved antibody production and improved B and T cell memory, leading to enhanced vaccine potency. Immune responses are initiated at the site of vaccine administration and in the draining lymph node (or, in the case of attenuated live viral pathogens, in multiple secondary lymphoid tissues). Vaccine antigens and/or adjuvants (substances that enhance immune responses) activate pathogen recognition receptors (PRRs) on local antigen-presenting cells (APCs) such as monocytes and dendritic cells (DCs) to initiate an innate immune response (Schaefer, 2014). PRRs recognize certain pathogen-associated molecular patterns (PAMPs) or damage-associated molecular patterns (DAMPs) and include Toll-like receptors (TLRs) which are present both on the surface of the cell as within endosomes, retinoid acid–inducible gene like receptors (RLRs), NODlike receptors (NLRs), C-type lectin receptors, absent in melanoma 2 (AIM2) like receptors, etc. (Schaefer, 2014). Detail of these immune responses are out of scope for this chapter, but there are many excellent review articles on this subject

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(Fernandes-Alnemri et al., 2009; Kawasaki & Kawai, 2014; Platnich & Muruve, 2019; Wen et al., 2013). Upon activation, APCs differentiate and mature, releasing cytokines that promote the extravasation and migration of mononuclear cells, granulocytes, and natural killer (NK) cells to the administration site. Activated APCs from the injection site incorporate antigen and present it in association with MHC class II antigens and migrate to the Tcell zone of regional lymph nodes where they interact with and activate naı¨ve CD4þ T helper cells promoting their differentiation into follicular T helper cells (Tfh). Antigens unassociated with DCs may also drain into the lymph node, where they are phagocytosed by subcapsular macrophages and may drive differentiation of Tfh cells. Differentiating Tfh in the lymph node drive antigen-specific B and T cell responses. The generation of long-lived specific Tfh memory cells is important for an enduring immune response to antigens. Tfh migrate to the B cell region (lymphoid follicle), interacting with antigen-presenting B cells, driving differentiation of both cell types and migration into the lymphoid follicle. Antigen-bearing follicular dendritic cells (FDCs) attract these antigen-specific B and Tfh cells, promoting clonal expansion of one antigen-specific B cell which forms the follicular germinal center. During the rapid proliferation phase of the B cell, there is class switching from IgM to IgG/IgM/IgA and extensive somatic hypermutation in the variable-region segments of the immunoglobulin genes. This hypermutation results in high-affinity immunoglobulins which have a competitive advantage for binding the small quantities of available antigen. These B cells process and present antigen via MHC II to Tfh, increasing Tfh activation and maturation. The combined effect of antigen-specific B cells, antigen-bearing FDCs, and antigen-specific Tfh cells in the germinal centers promotes the generation of antigen-specific B cells and provides signaling needed for B cell differentiation into plasma cells and memory B cells (Siegrist, 2018). Tfh also drive differentiation of antigenspecific T cells into memory and active cytotoxic T cells. CD8þ T cell responses are triggered primarily by the presentation of cytoplasmically expressed microbial epitopes (e.g., viral proteins generated in the cell cytoplasm) on the infected

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host cell’s surface in association with MHC class I. CD8þ T cells may control infection by directly killing infected cells (e.g., release of perforins [pore-forming cytolytic proteins]) or through secondary mechanisms such as the release of pro-inflammatory cytokines (e.g., interferongamma [IFNg] and tumor necrosis factor-alpha [TNFa]) (Akondy et al., 2017; Siegrist, 2018). Memory CD8 T cells rapidly proliferate when they reencounter a pathogen. Ideally upon reexposure to the pathogen, the presence of memory B and T cells to the antigen will generate a rapid and protective immune response. Vaccine administration is typically intramuscular (IM), intradermal (ID), or subcutaneous (SC), although there are some mucosal vaccines administered intranasally or orally. Mucosal vaccines are generally modified live organisms as they can induce strong serum and secretory IgA responses to limit virus at the mucosal surface. As noted, the induction of an antigenspecific immune response requires activation of APCs at the injection site with presentation of antigen in the draining lymph node (typically by DCs). DCs are distributed higher in the dermis and lower in the subcutis (adipose) compared to skeletal muscle, making intradermal vaccination highly efficient and subcutaneous administration less efficient in generating immune responses. With the exception of modified live organisms, nasal and oral vaccines are challenging to develop due to an assortment of issues including physical and chemical (e.g., mucosal enzymes) barriers (Siegrist, 2018). Immune responses vary by species, strain, sex, age, exposure history, antigen processing and presentation, etc., as well as by variations in the microbiome. It is important to note that immunogenicity data from animals cannot be readily extrapolated to humans due to species differences in immunological responses and exposure history. Species differences in epitope recognition, antigen processing, and adjuvant responses all contribute to limitations in translatability of findings in laboratory animals to humans. An assortment of assays is used to confirm antigen-specific immune responses in nonclinical studies, and a demonstrated response is essential for a valid study. In all vaccine studies, the primary concern is immune-mediated toxicity. Immunotoxicity in vaccine studies is primarily assessed as part of nonclinical general toxicity studies.

3. GENERAL CONCEPTS IN VACCINE TOXICOLOGY Vaccines may be prophylactic or therapeutic. Prophylactic vaccines contain foreign antigens (from infectious agents) whereas therapeutic vaccines may contain endogenous antigens (e.g., disease-related proteins like amyloid), neoantigens (e.g., tumor proteins), or foreign antigens (e.g., infectious agents like human immunodeficiency virus [HIV], human papillomavirus [HPV], tuberculosis, viral hepatitis) (Boukhebza et al., 2012). Vaccine effectiveness depends entirely on the immune response generated to the administered antigen(s), and in this way they are quite different from small molecules or biologics where test article activity is dependent on binding to the target molecules. Because prophylactic vaccines are intended to be administered to millions of healthy individuals, there is little tolerance for test article–related toxicity. For therapeutic vaccines intended to treat chronic diseases such as Alzheimer’s disease, diabetes, hypercholesterolemia, and cancer, the risk–benefit is greater and tolerance for test article–related toxicities may be higher (Guo et al., 2013; Matsumoto et al., 2014; Moingeon et al., 2003; Nakagami & Morishita, 2018). The ideal vaccine is one that is effective in preventing or reducing the severity of a specific disease, provides long-term protection against the disease, achieves immunity with a minimum number of doses, and has no undesirable side effects. Generating an immune response, however, even a robust immune response, may not confer protection against disease, or may provide only short-term protection clinically due to virus mutations (e.g., vaccines against coronavirus, influenza). To optimize clinical protection, vaccines may incorporate several different antigens or the same antigen from different pathogen serotypes (termed multivalent vaccines) to broaden protection and improve chances of success.

3.1. Vaccine Nonclinical Safety Package: Regulatory Expectations The standard nonclinical risk assessment paradigm for vaccines is considerably different from other pharmaceuticals. Prophylactic vaccines generate an immune response to foreign antigens

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and are intended (and designed) to have no endogenous targets. Thus, many of the typical genetic toxicity and safety pharmacology studies used for small molecule and biologic pharmaceuticals may not be applicable for vaccines (Table 9.1). For prophylactic and therapeutic vaccines, safety assessment in a single species is often adequate to support FIH studies (Forster, 2012; Plitnick, 2013; Sellers et al., 2020). Exceptions to this rule occur with novel vaccine components including adjuvants, excipients (e.g., stabilizers), or vectors (WHO, 2014) or where toxicity is a concern. If the vaccine requires the use of a specialized medical device for delivery, the device itself will need an adequate nonclinical safety package to support clinical testing and subsequent approval (see Biomedical Materials and Devices, Vol 2, Chap 11). Vaccine nonclinical studies should be performed either with the intended clinical device or with a device that closely matches that intended for use in the clinical setting, considering limitations related to size in smaller nonclinical species. Regulatory guidance around prophylactic vaccines is generally adaptable to various modalities (e.g., protein, nucleic acid, etc.) (Table 9.2). For prophylactic vaccines, most regions of the world follow the guidelines published by the World Health Organization (WHO, 2005, 2014). However, there may be local health authority requirements that are not covered by the harmonized guidelines (Ministry of Health, 2010; Plitnick, 2013). For example, subcutaneous local toleration studies are presently expected for vaccines intended for use in infants in Japan. Additionally, there may be other regulatory guidelines for specific types of vaccines, such as those developed to prevent seasonal influenza, Ebola hemorrhagic fever, AIDS, and COVID-19 (FDA, 2006, 2007a, 2020; WHO, 2016b). The Ebola and SARS-CoV-2 guidance documents may serve as templates for rapid development of vaccines against emerging life-threatening pathogens. Vaccines mostly fall outside the scope of the ICH (International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use) guidelines except for the ICH S5(R3) guideline on reproductive toxicology, where vaccines are in scope. Guidance around development of therapeutic vaccines is often less clear due to the more

TABLE 9.1

Regulatory Expectations for Nonclinical Studies Supporting Vaccine Regulatory Submissions

Study type

Requirement

Single-dose toxicity

Single-dose toxicity study endpoints are assessed within the repeat-dose toxicity study and are not routinely required

Repeat-dose toxicity

• Single species • Immunogenicity assessed N þ 1 (number of doses) • Chronic toxicity studies typically unnecessary

Exploratory toxicity

Generally not performed

Developmental and reproductive toxicity (DART)

• Single species • Female fertility and fetal/ offspring development assessed • Vaccine administered premating and during gestation • Immunogenicity assessed

In vivo safety pharmacology

Generally not performed

In vitro safety pharmacology (hERG)

Generally not performed

Genotoxicity

Generally not performed

Carcinogenicity

Generally not performed

Juvenile toxicity

Generally not performed

Other toxicity (modality dependent)

• Biodistribution and integration (nucleic acidebased vaccines) • Neurovirulence studies (some live viral vaccines) • Delivery modalities (electroporation, nanoparticles, etc.) • Absorption, distribution, metabolism, and excretion (ADME) studies generally not necessary except for novel ingredients or excipients • Small moleculeetype toxicity studies may be needed for novel adjuvants or excipients

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340 TABLE 9.2

9. VACCINES

Regulatory Guidelines Relevant to Vaccine Studies

Vaccine type

Guideline

All vaccines

WHO: Guidelines on the nonclinical evaluation of vaccines (2005)

All adjuvanted vaccines

WHO: Guidelines on the nonclinical evaluation of vaccine adjuvants and adjuvanted vaccines (2014) EMA/CHMP: Guideline on adjuvants in vaccines for human use (2005)

Country-specific

Ministry of Health, Labour and Welfare, Japan: Guideline for nonclinical studies of vaccines for preventing infectious diseases (MHLW, 2010) State Food and Drug Administration, China: Technical guidelines for preclinical research on preventive vaccines (SFDA, 2010)

Vaccines for women of childbearing potential and pregnant women

ICH S5(R3). Step 5. Committee for Medicinal Products for Human Use guideline on reproductive toxicology: Detection of toxicity to reproduction for human pharmaceuticals (2020) US FDA: Guidance for industry: Considerations for developmental toxicity studies for preventive and therapeutic vaccines for infectious disease indications (2006)

RNA vaccines

WHO: Evaluation of the quality, safety, and efficacy of RNA-Based prophylactic vaccines for infectious diseases: Regulatory considerations (draft) (Dec. 2020)

DNA vaccines

US FDA: Guidance for industry: Considerations for plasmid DNA vaccines for infectious disease indications (2007) WHO: Guidelines for assuring the quality and nonclinical safety evaluation of DNA vaccines (2005).

Viral vectored vaccines

EMA: Guideline on quality, nonclinical and clinical aspects of live recombinant viral vectored vaccines (EMA, 2010) FDA: Characterization and qualification of cell substrates and other biological materials used in the production of viral vaccines for infectious disease indications (FDA, 2010)

Recombinant protein/peptide vaccines

US FDA: Points to consider in the production and testing of new drugs and biologicals produced by recombinant DNA technology (FDA, 1985) International Conference on Harmonisation (ICH) S6 and ICH S6 (R1): Preclinical safety evaluation of biotechnology-derived pharmaceuticals. (ICH, 1997, 2011)

Combination vaccines

EMA: Note for guidance on pharmaceutical and biological aspects of combined vaccines (1998) US FDA: Guidance for industry for the evaluation of combination vaccines for preventable diseases production, testing and clinical studies (1997)

Therapeutic cancer vaccines

US FDA: Clinical considerations for therapeutic cancer vaccines (2011) US FDA: Guidance for industry: Preclinical assessment of investigational cellular and gene therapy products (2013) EMA: Guideline on the evaluation of anticancer medicinal products in man (2013) EMA: Guidance for industry: Gene therapy clinical trialsdObserving subjects for delayed adverse events (2006)

EMA, European Medicines Agency; US FDA, United States Food and Drug Administration; WHO, World Health Organization.

novel modalities (e.g., DC-based vaccines, oncolytic viruses, etc.) used for this purpose (Table 9.2). Because of this, several guidance documents may need to be considered

together, along with consultations with regulators to develop an appropriate and acceptable strategy for clinical testing and registration.

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3.2. Immunogenicity Assays A key component of the nonclinical evaluation is the measurement of the immune response to the administered antigen(s). Ideally, immunogenicity assays in nonclinical studies should be comparable to the intended clinical assays (Sellers et al., 2020). Immune assessments vary depending on the vaccine modality and target antigen. Prophylactic vaccine immunogenicity assays used for nonclinical studies frequently assess humoral antibody responses rather than cell-mediated responses, as the cell-mediated response assays tend to be more labor- and reagent-intensive. Assessment of the cell-mediated immune response to a vaccine is often performed in non-GLP pharmacology studies or as part of efficacy studies, but may be a component of GLP toxicity studies for modalities where the efficacy depends on a cell-mediated immune response (e.g., tumor cytotoxicity). A commonly used endpoint to assess the cellmediated immune response is the enzyme-linked immunospot assay (ELISPOT). The ELISPOT assay measures antigen-specific IFNg release from lymphocytes. Flow cytometry may also be utilized if measurements of changes in select immune cell types are of interest. These assays may be more challenging to perform under GLP conditions and may require a GLP exception. Key pharmacodynamic (PD) endpoints should also be included for therapeutic vaccines as appropriate. For toxicity studies, a qualified fit-for-purpose assay performed according to Good Laboratory Practice (GLP) is acceptable to evaluate an antigen-specific immune response (WHO, 2016a). Assays may be species-specific, requiring optimization for cells and proteins of the test species. Assays to measure the humoral immune response may include functional assays (i.e., demonstrate an ability to neutralize the pathogen) and/or antibody titers. Examples of functional assays are opsonophagocytic activity (OPA) assays, toxoid neutralizing assays (TNAs), and neutralizing antibody assays. Assays to measure antibody titer include enzyme-linked immunosorbent assays (ELISAs) or similar multiplex assays (e.g., Luminex).

4. VACCINE MODALITIES Until the 1980s, antigens for vaccines were primarily derived through extraction of bacterial

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proteins (such as the toxoid vaccines), inactivation of whole organisms with chemicals such as formaldehyde, or from live organisms attenuated through propagation in cell culture (e.g., the measles, mumps, rubella [MMR] vaccine). Over the last several decades, however, advances in molecular biology have led to the development of more specific and targeted approaches, such as recombinant protein antigens and molecularly modified viruses as well as nucleic acid– based (RNA, DNA) and viral vector vaccines (Table 9.3) (Francis, 2018; Rauch et al., 2018). Different modalities may be utilized for the same disease; for example, COVID-19 vaccines include RNA vaccines (e.g., Moderna, Pfizer), viral vectored vaccines (e.g., Johnson and Johnson, AstraZeneca), recombinant protein vaccines (e.g., Novavax), and inactivated viral vaccines (e.g., Sinovac). The availability of these modalities have improved the safety, specificity, and efficacy of vaccines, although the reduction of residual viral and cellular components in vaccines has had the effect of slightly lower immunogenicity, requiring the addition of immune-enhancing adjuvants to boost the immune response (Francis, 2018).

4.1. Protein-Based Vaccines Protein and Recombinant Protein Vaccines Protein antigens must be generated such that they are conformationally stable, exposing protective epitopes to the immune system. In general, protein vaccines effectively elicit strong B cell and T cell responses and produce good immunological memory (Saylor et al., 2020). The earliest protein vaccines were derived from chemically inactivated or heat-inactivated bacterial toxins and included the diphtheria and tetanus toxoid vaccines which were widely used in the early part of the 20th century. In recent years, most protein vaccines are recombinant vaccines in which antigens are expressed from DNA constructs. The first genetically engineered vaccine became available in 1986 and was for the Hepatitis B virus (Beardsley, 1986). Improving vaccine efficacy may also require the use of multiple antigens in a single formulation (i.e., multivalent vaccines). Many protein or polysaccharide vaccines are multivalent as different strains or subtypes

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TABLE 9.3 Examples of Vaccine Modalities Vaccine type

Description of antigen

Examples of licensed vaccines

Live or live attenuated pathogen

Attenuated or related replication-competent organism

ACAM 2000 (smallpox) Dengvaxia (dengue fever) FluMist (influenza) Imovax (rabies) MMR II (measles, mumps, rubella) YF-vax (yellow fever) Vaxchora (cholera) Vivotif (typhoid)

Inactivated pathogen

Inactivated nonreplicating organism

BioThrax (anthrax) Fluad quadrivalent (Influenza) IPOLdIPV salk (Polio) Ixiaro (Japanese encephalitis)

Subunit/conjugate/ protein/toxoid

Recombinant or purified protein; polysaccharide alone or conjugated to carrier protein; inactivated toxin

Cervarix (human papillomavirus) DAPTACEL (diphtheria, tetanus toxoids, acellular pertussis) HEPLISAV-B (hepatitis B) Prevnar13 (pneumococcus pneumonia) Shingrix (varicella zoster) Trumenba (meningococcal B meningitis)

DNA

Plasmid DNA or modified viral expression vector

Janssen; AstraZeneca (COVID-19 vaccines) Ervebo (ebola)

RNA

Stabilized or self-amplifying mRNA

COMIRNATY; SPIKEVAX (COVID-19 vaccines (modified RNA))

Anti-idiotype

Antibodies which mimic antigen

Racotumomab (NSCLC glycosylated gangliosides)

Cell-based vaccines

Allogenic, autologous, or dendritic cell based

No marketed products Example: GVAX for pancreatic cancer

NSCLC, non–small cell lung cancer.

may have incomplete cross-immunoprotection. As mentioned previously, the increased purity of protein vaccines as a result of recombinant technology has increased the need for coformulation with adjuvants as residual material such as other membrane proteins and lipids, which have adjuvant properties, are no longer present in engineered molecules to help boost immune responses (Siegrist, 2018). Retention of antigenic three-dimensional (3D) structure is often essential for driving effective antipathogen immune responses, and X-ray crystallography and neutralizing monoclonal antibodies have contributed significantly to structure-based vaccine design (Graham et al., 2019). Antigens that promote nonneutralizing

responses may have reduced or no efficacy and could promote enhanced disease in vaccinated individuals who are subsequently exposed to the pathogen (Kapikian et al., 1969; Scott, 1987). Therefore, accurate protein engineering is essential for generating antigens in which the conformation is both stable and induces a neutralizing immunological response (Graham et al., 2019; Jimenez-Sandoval et al., 2020; Pallesen et al., 2017). Expression system conditions and vaccine formulations may modulate protein conformation and protein aggregation (Saylor et al., 2020). For example, the respiratory syncytial virus (RSV) F protein exists as a trimer on the viral envelope (“prefusion” F protein) and is responsible for fusion with the host cell

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membrane (McLellan, 2015; McLellan et al., 2011). Upon fusion, the F protein changes conformation into a “postfusion” F protein. Antibodies against the prefusion F protein in its trimeric conformation induce a strong neutralizing antibody titer; in contrast, the postfusion F protein does not incite a strong immune response and thus is not as protective (Phung et al., 2019). Therefore, it is critical to maintain the F protein in its immunogenic prefusion conformation as a vaccine antigen. Similar considerations on stabilizing the prefusion conformation may also be important for optimal immune protection with the SARS-CoV-2 S(“spike”) based vaccines. Glycoconjugate Vaccines Conjugation of bacterial capsular polysaccharide antigens to carrier proteins was developed as a mechanism to improve the effectiveness of vaccines against certain strains of bacteria. Unconjugated oligosaccharides (OSs) and polysaccharides (PSs) are generally weak antigens, driving mostly B cell but not T cell responses, thereby producing poor immunological memory (T-independent B cell responses) (Weintraub, 2003). To enhance T cell engagement, the PS or OS is covalently linked to a carrier protein to create a “glycoconjugate.” Extracted and purified bacterial carbohydrate antigens or synthetically generated carbohydrate antigens are chemically conjugated by random linkage to a carrier protein to form a very high molecular weight glycoconjugate antigen. Traditionally the PS was attached somewhat randomly to amino and carboxyl groups on the carrier proteins, but efforts are ongoing to create more consistency in conjugation sites (Francesco Berti & Micoli, 2020; Hu et al., 2016). The most utilized carrier proteins are derived from tetanus toxoid (TT), diphtheria toxoid, and the nontoxic mutant of diphtheria toxin cross-reactive-material (CRM197) (Berti & Adamo, 2018). Conjugate vaccines are often administered in adjuvantcontaining formulations (e.g., aluminum phosphate). Presently there are efforts to improve immune activation of the PS conjugates with further linkage to innate immune activators such as TLR-7 agonists or components of the lipopolysaccharide (LPS) O antigen (TLR-4 agonists) (Francesco Berti & Micoli, 2020).

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Virus-like Particles Nanoparticle or Generalized Modules for Membrane Antigen Based Vaccines Virus-like particles (VLPs), which mimic viruses, are nanoparticles comprised of viral antigens which form icosahedral or helical structures. These multimeric VLPs induce a more potent immune response against viral antigens than monomeric proteins (Schiller & Lowy, 2018) because of the rigid and repetitive nature of the VLP. VLP-based vaccines are designed to generate strong B cell responses through MHC II–based Th-cell activation by APCs. However, the particulate nature of the VLPs results in efficient cross-presentation of VLP-derived peptides on MHC class I molecules to induce CD8þ T cell responses (Makarkov et al., 2019; Win et al., 2011). Several characteristics of the VLP are important in vaccine development, including a size range of 20–200 nm (which facilitates diffusion directly into lymphatic system), the maintenance of native viral protein structure (to optimize B cell activation), and VLP packaging with adjuvants such as TLR activators (e.g., ssRNA, dsRNA, TLR7/8, CpGs) to augment the immune response. Further, chemical and genetic modifications of the proteins may also improve antigenicity. Several VLP-based vaccines are licensed to prevent infection by human papillomavirus (HPV), hepatitis B virus (HBV), and malaria. For example, for the HPV vaccines Gardasil and Cervarix, the VLP is comprised of L1, the major protein capsid of the HPV virus. The L1 proteins, which are produced in yeast or insect cells, self-assemble and form immunogenic VLPs that induce high neutralizing antibody titers (Mohsen et al., 2017). Generalized molecules for membrane antigens (GMMAs)-based vaccines use outer membrane vesicles (OMVs) naturally released from gramnegative bacteria. These bacteria have been genetically engineered to generate more vesicles (exosomes), express a less reactogenic Lipid A, and to express the antigen of interest. The GMMAs resemble the bacterial surface as well as have intrinsic adjuvanticity, which drives a strong immune response to the expressed antigens (Micoli et al., 2018; Raso et al., 2020). A GMMA vaccine against Shigella sonnei 1790GAHB has been evaluated recently in

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clinical trials with good immunogenicity and safety results (Launay et al., 2019).

4.2. Nucleic Acid–Based Vaccines RNA-Based Vaccines RNA-based modalities are utilized increasingly for both prophylactic and therapeutic vaccines and are amenable to rapid development in the face of epidemics or pandemics. The advantages of the RNA modality are in its versatility (antigen sequences can be swapped into the constructs easily), ease of manufacturing (in vitro transcription from a DNA template), general safety, and intrinsic immune activation (“selfadjuvant”) (Pardi et al., 2018). Unlike with DNA vaccines, there is no risk of genomic insertion, and unlike modified or attenuated live viral vaccines, RNA vaccines are not infectious. The RNA, once in the cytoplasm of the cell, uses cellular translational machinery to produce the antigen of interest, which may be released, localize to the cell membrane, or be expressed as part of the MHC I complex to drive an antigen-specific immune response (Pardi et al., 2018). There are presently two RNA platforms that are used for prophylactic or therapeutic vaccines: nonreplicating mRNA and self-amplifying RNA (saRNA) (Brito et al., 2015; Pardi et al., 2018). Nonreplicating mRNA platforms utilize mRNA which has 50 and 30 untranslated regions, a 50 cap, and a polyadenylated (PolyA) tail as well as sequences for the antigen of interest. Modifications that improve stability and translatability include the use of modified nucleosides (e.g., pseudouridine or methylpseudouridine), codon optimization, increased GC nucleotide content, and specific cap types (Pardi et al., 2018; Schaefer, 2014). saRNA utilizes a viral RNA backbone, usually an alphavirus, in which the sequences for replication competence are retained but the regions expressing structural genes are replaced by sequences for the antigen of interest (Brito et al., 2015). The benefit of this modality is the ability to administer a lower RNA dose to get a similar amount of antigen expression. saRNA administration has been reported to be associated with increases in ALT and AST, which are not thought to be of liver origin (Stokes et al., 2020). The increases may have been of skeletal muscle

origin, given the IM administration route and likely transfection of skeletal muscle cells with the saRNA. It is uncertain if the nonstructural proteins in the constructs, which are essential to support self-amplification, will generate immunologic responses that subsequently decrease the effectiveness of the vaccine. While naked RNA has been administered and has demonstrated ability to express the protein of interest, its efficiency of entry into cells is limited, largely because the hydrophobic nature of RNA hinders cell entry (Pardi et al., 2018). Additionally, RNA is inherently unstable and rapidly destroyed by extracellular ribonucleases (Rauch et al., 2018). Therefore, most mRNA vaccines are complexed with cationic lipids or polymers (e.g., lipid nanoparticles [LNPs] or lipoplexes). Lipid delivery approaches are highly variable, and often involve novel lipids. For an excellent review of the types of lipid delivery systems and RNA modalities, please see Pardi et al., (2018). Clinical signs associated with RNA administration in humans and animals are generally due to activation of the innate immune system. These responses are driven by proinflammatory cytokines, particularly type 1 interferons, and are generally more severe with unmodified mRNA. saRNA may stimulate a more rigorous immune response for a number of reasons. First, saRNA has dsRNA intermediates which activate additional PRRs; second, these molecules may not be as amenable to the inclusion of less reactogenic modified nucleosides (such as pseudouridine) found in modRNA vaccines; third, the self-amplifying nature of saRNA results initially in progressive increases in the foreign mRNA in the transfected cells before it begins to be cleared whereas modRNA clearance initiates immediately after transfection. Clinical vaccine reactions correlate to those observed in nonclinical models, including notable injection site reactions and elevations in body temperature (Hassett et al., 2019; Sedic et al., 2018). Nonclinically, the vaccine reactions may manifest as negligible to transient edema, redness, or scabbing at the injection site in rats. Elevations in circulating white blood cells, particularly neutrophils, monocytes, and large unstained cells, are common, as are alterations in acute phase proteins. Administration of RNA complexed with lipids sometimes produces additional findings in

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nonclinical studies. Intramuscular administration of RNA-LNP vaccines is associated with transient minimal to mild portal hepatocyte vacuolation which is interpreted to reflect lipid distribution to the hepatocytes; the observation of this finding may depend on the dose and types of lipids administered. When present, the finding is not associated with evidence of hepatocyte damage or disfunction. Transient decreases in reticulocytes have also been observed in rats after administration of RNA-LNP vaccines and products (e.g., the RNAi product, Onpattro). DNA Vaccines Nonviral DNA vaccines utilize plasmids (pDNA) to express the antigen(s) of interest. The advantages of DNA vaccines mirror those of RNA vaccines: they are highly versatile, safe, and easy to manufacture. The inserted gene of interest is flanked by a 50 eukaryotic promoter and a 30 PolyA signal as well as genetic material to stabilize expression (Ghaffarifar, 2018; Mazid et al., 2013). However, unlike RNA modalities, the materials must be made within a cell culture system, often using Escherichia coli. In order for the plasmid to be transcribed, it must enter into the host cell nucleus. As such, IM or ID administration of plasmid-based vaccines has not produced strong antigenspecific immune responses. Uptake of these vaccines into the cell and thus antigen expression and immune responses are notably improved through the use of special delivery devices. Devices include electroporators and gene guns (Broderick & Humeau, 2017; Ghaffarifar, 2018; Ravi et al., 2015), nanoparticles and liposomes (Ghaffarifar, 2018), and respiratory delivery technology (Mazid et al., 2013; Rajapaksa et al., 2014). pDNA vaccines, while immunogenic, may require coadministration of adjuvants to improve their immunogenicity. Immunogenicity may also be enhanced by coexpression or coadministration of pDNA plasmids expressing natural adjuvants, such as interleukins (IL-12, IL-15) or PAMPs such as TLR-4 agonists (Ghaffarifar, 2018; Saade & Petrovsky, 2012). Because pDNAs may persist in the vaccinated individual, particularly at the injection site, sometimes for years (Armengol et al., 2004), there is a risk of genomic integration, potentially driving oncogenesis. While the risk of genetic

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integration with subsequent mutagenesis or oncogenesis is very low, the FDA recommends genomic integration studies to be included whenever the pDNA persists at copy numbers of greater than 30,000 per mg host DNA in any tissue at the end of the study (FDA, 2007b, 2013; WHO, 2013). Inactivated and Attenuated Virus Vaccines Attenuated live and inactivated viral vaccines can be effective in generating strong and lasting protective immune responses. Inactivated (killed) viral vaccines are generated through radiological, thermal, or chemical inactivation of whole virus (Siegrist, 2018). These vaccines generally do not require the addition of adjuvants as the virus and its associated proteins, carbohydrates, etc., initiate a strong innate immune response. Attenuated viruses for vaccines are generated through in vitro passaging, whereas modified live viruses in vaccines have been genetically engineered to be less pathogenic. Live vaccines tend to result in robust immune responses, including good B cell and T cell memory (Siegrist, 2018). Live attenuated viral vaccines were traditionally generated by propagating the virus in systems that were not “natural” to the virus, such as in cells from a different species or at suboptimal temperatures, forcing virus adaptation (Bull et al., 2018; Minor, 2015). Some of these adaptations resulted in decreased efficiency of viral replication in the natural host and could be used as a vaccine strain. Attenuated viral vaccines generally result in viremia, which is usually shorter in duration with lower viral replication and/or less ability to infect cells than occur with the unattenuated or wild-type virus (Bull et al., 2018). This transient viremia produces immune responses at both the draining lymph node and lymphoid sites distant from the draining lymph nodes, driving a robust antiviral immune response. Transmission from vaccinated to unvaccinated individuals after vaccination with attenuated viral vaccines may occur, though infrequently. The use of genetic modification technology has allowed for alterations in the genome which modulate viral replication and gene translation. These include codon optimization, gene rearrangement, or gene deletions (Bull et al., 2018). One risk of live viral vaccines is reversion after administration to

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a more virulent form either through mutation or recombination with wild-type virus during coinfection in an individual. The risk of these types of events appear to be greater when there have been large deletions within the viral genome to generate the vaccine rather than multiple modifications. Viral Vectored Vaccines Viral vectored vaccines are those which utilize a live attenuated or nonreplicating virus to express a vaccine antigen. Expression of the antigen within the host system allows for accurate protein modifications (e.g., glycosylation), protein folding, and multimerization (e.g., formation of dimers and trimers). Examples of viruses used as vectors are the measles virus, adenoviruses, cytomegalovirus, vesicular stomatitis virus, and vaccinia virus (Humphreys & Sebastian, 2018). This approach results in the generation of antigen-specific cellular immune responses and neutralizing antibodies. The relative ease of antigen modification and general safety of viral vectored vaccines have made them a commonly used modality for vaccines against emerging viral diseases, such as Chikungunya, Zika, Ebola, COVID-19, Lassa Fever, and Nipah virus infection (Frantz et al., 2018; Humphreys & Sebastian, 2018; Rauch et al., 2018). Preexisting immunity to some of a viral vector (e.g., adenovirus) will impact antigen expression (and therefore antigen-specific immune responses) as the underlying immune response against the vector may clear the virus quickly. Therefore, these modalities may require use of rare viral serotypes or strains derived from other species (e.g., chimpanzee adenoviruses) (Guo et al., 2018). One consideration for DNA virus vectors or RNA vectors with a DNA intermediate is the risk of genomic integration resulting in insertional mutagenesis (Rauch et al., 2018; Romano, 2012). Therefore, genomic integration assessments may be requested by regulators. Such studies tend to be complicated and outside the scope of this chapter. Approaches have been described for adeno-associated virus (AAV), a vector commonly used in gene therapy (Bolt et al., 2021). Additionally, these types of vaccines are categorized as genetically modified organisms (GMOs) and typically are assessed using the regulations related to these organisms.

Anti-idiotype Vaccines Anti-idiotype “vaccines” are antibodies (antiId) that structurally mimic an antigen. The region of an antibody that recognizes and binds to an antigen is termed an idiotype. Jerne and colleagues hypothesized that the immune system not only produces antigen-specific antibodies, but will also then produce antibodies against the idiotype (the anti-Id) (Jerne, 1974; Kohler et al., 2019). This anti-Id antibody will present as a new idiotype, for which another anti-Id will be created. This new idiotype can then bind to and inactivates the original antigen (Kohler et al., 2019). Essentially, administration of an antigen results in antigen-specific antibodies, termed Ab1. A series of anti-Id antibodies are then generated against Ab1, which are termed Ab2. Immunization with the Ab2 antibodies (with or without an adjuvant) would be expected to generate anti-anti-Id antibodies, termed Ab3, which will recognize the original antigen identified by Ab1. This modality has been used to develop specific antitumor antibodies for use as a cancer therapy. Because most of tumor-associated antigens (TAAs) are self-antigens, breaking immune tolerance is essential for antitumor efficacy. It is thought that the anti-Id antibodies can serve as antigen surrogates and thus will be less impacted by immune tolerance. Clinical studies have demonstrated that some anti-Id cancer vaccines can induce clinically beneficial humoral and cellular immune responses. Unfortunately, there has been little clinical success with these modalities, with the exception of Racotumomab, which induces an immune response against specific glycosylated gangliosides (NeuGcGM3) present in some non–small cell lung cancers (Kohler et al., 2019; Ladjemi, 2012; Ladjemi et al., 2011). Cell-Based Vaccines Cell-based vaccines include allogenic or autologous cell vaccines and DC vaccines. Allogenic/ autologous cell vaccines historically have been composed of killed or modified tumor cells from a patient which are subsequently administered back to the patient (autologous) or to another patient (allogenic) with a similar tumor type in the hopes of generating a strong cellular immune response against the tumor. Because these tumor cells often express an assortment of

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TAA on their surface, they behave like a multivalent tumor vaccine which may increase tumor cell killing (Kelly & Giaccone, 2011). One example of a modified allogenic vaccine is GVAX, which is comprised of irradiated tumor cells transfected with a nonreplicating adenoviral vector expressing GM-CSF (Le et al., 2015; Salgia et al., 2003). Recently, the use of autologous pluripotent stem cells expressing a broad spectrum of tumor antigens to the immune system has shown promise as a therapeutic cancer vaccine approach (Wang et al., 2019). Dendritic cell vaccines are comprised of DCs loaded with allogeneic tumor cell lysates. DCs have the capacity to both prime and activate T cells while also preserving tolerance to selfantigens (Amon et al., 2020; Sabado et al., 2017), making them useful for therapeutic purposes. Treatment of monocytes with GMCSF and IL-4 in vitro will drive differentiation to moDCs. Maturation of these moDCs can be initiated by in vitro treatment with various cytokines, such as IL-1b, IL-6, TNFa, and PGE2. Antigen-specific targeting of these mature DCs is initiated through an assortment of methods, including incubation with the antigen of interest, tumor cell lysates, transfection with TAA expressing DNA, etc. Further genetic manipulation designed to improve immune responses may also be used. These antigen-primed DCs can then be transferred to the patient. Oncolytic Virus–Based Modalities While not classically considered a vaccine, the ability of oncolytic viruses to mediate prolonged antitumor immunity results in its classification by some as a vaccine. Oncolytic viruses (OVs; Table 9.4) are being widely investigated for use in cancer therapy, typically in combination with immune checkpoint inhibitors. The first FDA-approved oncolytic virus therapy was an oncolytic herpes simplex virus (HSV)-containing granulocyte/macrophage colony-stimulating factor (GM-CSF) (talimogene laherparepvec, TVEC) in 2015 to treat melanoma (Appleton et al., 2015; Johnson et al., 2015). OVs are most commonly delivered intratumorally or intravascularly but may also be delivered intranodally. Oncolytic viruses are modified to preferentially infect tumor cells with limited infection of normal host cells (Bommareddy et al., 2018; Kohlhapp & Kaufman, 2016). The majority of

TABLE 9.4 Oncolytic Viruses Used for Cancer Therapy Viruses

Representative Species

DNA VIRUSES

ssDNA

Parvovirus

dsDNA

Adenovirus, herpesvirus, poxvirus

RNA VIRUSES

(+)ssRNA

Alphavirus, picornavirus, retrovirus

(e)ssRNA

Paramyxovirus, rhabdovirus

dsRNA

Reovirus

OVs are replication competent but have been modified to selectively replicate in tumor cells rather than normal host cells, thereby primarily inducing tumor cell death (oncolysis) with limited damage to other cells. For examples, the OVs may be genetically modified to be thymidine kinase defective, promoting viral replication in tumor cells, which generally have high endogenous TK levels compared to normal cells (Twumasi-Boateng et al., 2018a,b). The effect of oncolytic viruses is biphasic. In the first phase, viruses enter the tumor cell, proliferate, and kill the host cell. Cell lysis promotes immune cell entry into the tumor resulting in immune responses to an assortment of tumor-specific neoantigens. The potential for oncolytic viruses to induce cell lysis depends on virus type and tropism, susceptibility of tumor cells to virus-mediated death, and the administered dose. Tumor oncolysis results in the release of PAMPs and DAMPs that activate the innate immune and adaptive immune responses. These responses promote DC infiltration and cross-presentation of TAAs that promote neoantigen-specific cytotoxic T lymphocyte (CTL) responses. In this way, the oncolytic virus acts as a “vaccine” by generating a tumor-specific immune response. Viral infection and subsequent cell lysis activate PAMP and DAMP receptors to induce a strong innate immune response which supports the generation of specific adaptive antitumor immune responses (the “vaccine” effect of oncolytic viruses) (Lawler et al., 2017; Russell & Peng, 2018; Russell & Barber, 2018). In addition

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to channeling the oncolytic efficacy of OVs, the OV may be further modified with the insertion of transgenes which may alter the tumor microenvironment to promote immunogenic tumor cell death (Bommareddy et al., 2018; Ghouse et al., 2020). Inclusion of immune checkpoint inhibitors has demonstrated improved efficacy with oncolytic virus therapies (LaRocca & Warner, 2018). Data suggest that combining immune checkpoint inhibitors with OVs does not increase the clinical safety risk relative to either modality alone. A review of clinical trials of combinations of oncolytic viruses and PD-1/ PD-L1 inhibitors described no tolerability or safety issues, including when the OV expresses cytokines such as IL2 (Chen & Han, 2015; Chesney et al., 2018; Chiu et al., 2020; LaRocca & Warner, 2018; Mahalingam et al., 2020).

5. VACCINE ADJUVANTS As vaccine design and manufacture has become more regulated, many of the “impurities” of historical vaccines (such as bacterial flagella or other bacterial or cell wall proteins/LPS or bacteria/viral DNA and RNA), which activated innate immunity, have been removed from the vaccine formulations (Aposto´lico et al., 2016; Bonam et al., 2017). While vaccines without these components are considered safer, initiating a robust immune response has required the addition of adjuvants into the vaccine formulation. Adjuvants are defined as “. substances or combinations of substances that are used in conjunction with a vaccine antigen to enhance (e.g. increase, accelerate, prolong and/or possibly target) or modulate to a different type (e.g., switch a Th1 immune response to a Th2 response, or a humoral response to a cytotoxic T-cell response) the specific immune response to the vaccine antigen in order to enhance the clinical effectiveness of the vaccine” (WHO, 2014). Different adjuvants have a range of immunological effects, and thus one adjuvant may be better suited to improve protective immune responses for a given pathogen (Table 9.5) (Del Giudice et al., 2018). Adjuvants ideally induce innate responses which promote adaptive responses (Iwasaki & Medzhitov, 2010). Adjuvants may impact the immune response to antigens through several mechanisms, the most typical being (1)

TABLE 9.5 Examples of Vaccine Adjuvants Type of molecule

Representative adjuvant

Mineral salts

Al(OH)3, AlPO4

Surfactants/ emulsions

MF59, LPS, saponins (QS-21), mannide monooleate, GLA-SE, SLA-SE, ISCOMATRIX, Freund’s adjuvant (complete and incomplete)

Nucleic acid/ nucleotide adjuvants

CpG-ODN, poly(I:C), and poly(ICLC)

Inert substances

Gold particles

Lipid-based adjuvants

MPLA, a-GalCer

Liposome-based adjuvants

ALFQ, AS01b, AS02, AS03 (þ vitamin E), AS04

Endogenous human immunomodulators

hIL-12, hGM-CSF, IL-12, IL-15

Combinations

ALFQ (QS-21, MPLA) AS01b, AS02 (MPL, QS-21) AS04 (aluminum, MPLA)

a-GalCer, a-galactosylceramide; Al(OH)3, aluminum hydroxide; AlPO4, aluminum phosphate; AS, adjuvant system (AS01b, AS02, AS03, AS04); ALFQ, army liposome formulation containing QS-21; CpG-ODN, CpG oligodeoxynucleotides; GLA-SE, glucopyranosyl lipid A stable emulsion; hGMCSF, human granulocyte-macrophage colony-stimulating factor; hIL, human interleukin; IL, interleukin; LPS, lipopolysaccharide; MF-59, oil-in-water emulsion of squalene oil; MPL and MPLA, monophosphoryl lipid A; Poly(I:C), polyinosinic: polycytidylic acid; Poly(ICLC), polyinosinic-polycytidylic acid stabilized with poly L-lysine; QS-21, quillaja Saponaria extract 21; SLA-SE, second generation lipid adjuvant stable emulsion.

enhanced antigen delivery to APCs at the injection site or lymph nodes (e.g., aluminum salts, MF59 and AS03, and liposomes), (2) immune stimulation or potentiation, e.g., MPLA, which activates Toll-like receptor 4, QS21, CpG ODN, and cytokines), or (3) a combination of both enhanced antigen delivery and immune stimulation (“adjuvant systems”). Some adjuvants may also bind antigen to stabilize it or to help reduce local and systemic reactogenicity induced by the injected material (Ko & Kang, 2018; Novicki, 2013). Only a limited number of adjuvants have been used in approved vaccines and include MF59, aluminum salts, CpG-ODN, AS04, and AS01B (Table 9.5).

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Adjuvants are included in vaccines only if they have a demonstrated advantage or support use of a decreased antigen dose (Del Giudice et al., 2018). Well-established adjuvants have been used in marketed products or have an extensive safety database. These include aluminum hydroxide, aluminum phosphate, potassium aluminum sulfate, and some proprietary oil-inwater emulsions (e.g., AS02, AS03, MF59) (Del Giudice et al., 2018), and more recently CpG oligodeoxynucleotides (CpG-ODN). The use of different and potentially more robust adjuvants has been slow to progress because of reactogenicity safety concerns as vaccines are given to millions of healthy individuals. In toxicity studies, the use of saline and adjuvant control groups helps differentiate antigen- and adjuvant-related effects as well as in identifying adjuvant-specific effects. Identifying adjuvant effects separately within the report may benefit other programs using the same or similar adjuvant for comparative purposes. Novel adjuvants, depending on their modality, may require additional assessments, including toxicity studies similar to those for small molecules. The WHO guideline (WHO, 2014) indicates that only one species is necessary for assessing a vaccine with a novel adjuvant. While unlikely, other regulatory bodies may request a second species either with the vaccine or with the adjuvant alone. Further, genotoxicity, biodistribution, immunotoxicity, and safety pharmacology studies may be warranted and should be considered in collaboration with regulatory authorities on a case-by-case basis. Species differences in innate responses to adjuvants should be considered when designing nonclinical studies. Adjuvant-associated enhancement of immune responses to antigens will vary by species and cannot be anticipated to always translate to predict the response in humans. Speciesspecific variations in immune responses, where known, will be included within the specific adjuvant sections below.

5.1. Mineral Salts By far, the most common type of adjuvant used in vaccine formulations are the inorganic mineral adjuvants, typically aluminum-based materials (Lindblad & Duroux, 2017; Oleszycka & Lavelle, 2014). The word “alum” is occasionally used to describe all aluminum-containing adjuvants, but this is inaccurate as alum is specific to

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a sulfated salt comprised of aluminum and potassium. The most used aluminum salts are comprised of aluminum phosphate (AlPO4) or aluminum hydroxide (Al(OH)3), with other aluminum salts less commonly used. The mechanism by which aluminum or calcium salts stimulate immune responses is not fully understood. Aluminum salts in vaccines are regarded as safe (i.e., generally recognized as safe [GRAS]) when used within the specified guidelines (0.85 mg/ dose FDA or 1.25 mg/dose EMA). Traditionally, any vaccine formulations with aluminum or calcium salt must either be in liquid formulation or formulated as a lyophilized antigen with a separate aluminum-containing diluent. This requirement was based on the premise that vaccines containing aluminum salts cannot be frozen, and thus cannot be lyophilized. However, methods of creating lyophilized aluminum-containing vaccines have been reported recently (Chisholm et al., 2019). In general, aluminum adjuvants are not good inducers of cellular immunity (Hogenesch, 2012; Oleszycka & Lavelle, 2014) as they are poor inducers of DCs. Therefore, aluminum adjuvants have limited effect on protective TH1 responses. Aluminum has been demonstrated to initiate TH2 responses in mice and mixed TH1 and TH2 responses in humans (Del Giudice et al., 2018; Hogenesch, 2012). Aluminum adjuvants form insoluble aluminum particles which are taken up by macrophages. In vitro, aluminum adjuvants induce the production of IL-1b and IL-18 by mouse macrophages (Eisenbarth et al., 2008). In mice this appears to be dependent on activation of the NLRP3 (NOD-, LRR-, and pyrin-domain-containing protein 3) inflammasome (Eisenbarth et al., 2008; Sharp et al., 2009). Additionally, in vitro, aluminum interacts with lipids on the DC membrane to induce cell activation via the Syk-PI3 kinase pathway (Flach et al., 2011). The aluminum binding with DC membrane lipids appears to be mediated by intercellular adhesion molecule 1 (ICAM-1) and lymphocyte function–associated antigen 1 (LFA1), and this activation may occur in the absence of inflammasome activation (Flach et al., 2011). An important component of the immunepotentiating effects of aluminum in vaccines is, in many cases, likely related to its ability to adsorb antigen. Antigen adsorption to aluminum helps to retain the antigen at the injection site, allowing time for recruitment of

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DCs and other APCs to be exposed to antigen. Further, soluble antigens adsorbed to aluminum may precipitate, improving antigen phagocytosis and DC activation (Ghimire et al., 2012; Hogenesch, 2012).

5.2. Surfactant/Emulsion Adjuvants The terms “surfactant” and “emulsion” are often used interchangeably. Emulsion adjuvants are generally oil-in-water mixtures which create a depot at the site of injection, resulting in slow release of the antigen to promote more prolonged immune stimulation. The oil-in-water emulsions are stabilized by surfactant molecules. These have been beneficial in promoting T cell responses with recombinant protein vaccines (Shah et al., 2015). Surfactant adjuvants may be derived from synthetic or natural sources. Surfactant adjuvants typically have a polar head with at least one fatty chain (Petrovsky & Cooper, 2011). The most common surfactant adjuvant used in licensed vaccines is a saponin (QS-21) extracted from the soapbark (Quillaja saponaria) tree, which is native to South America. It has been hypothesized that QS-21 promotes binding to the cell surface lectins on APC to facilitate antigen uptake as well as serving as a costimulatory molecule by binding to T cell receptors to promote T cell activation (Fleck et al., 2019). QS-21 activates the NLRP3 inflammasome complex (Lacaille-Dubois, 2019). QS-21 is typically combined into an adjuvant system for use in vaccines (see below). ISCOMATRIX adjuvant, which has been evaluated in clinical trials but is not included in any currently licensed vaccines, contains a saponin extract of Q. saponaria combined with cholesterol and phospholipid (Pearse & Drane, 2005; Sun et al., 2009). In liposomal formulations, it has been proposed that cholesterol drives endocytosis of QS-21 by dendritic cells with subsequent lysosomal disruption leading to the production of pro-inflammatory cytokines (Welsby et al., 2016). Data from rats indicate that the use of QS-21 alone in water results in coagulation necrosis of skeletal muscle at the injection site (Garc¸on et al., 2007; Reagan et al., 2020). It was demonstrated that combination of QS-21 in an oil emulsion is not associated with muscle necrosis (Garc¸on et al., 2007). Other emulsion adjuvants are primarily formulated with squalene oil. Squalene is a precursor to cholesterol found naturally in

plants and humans. Squalene oil used in vaccines is most commonly extracted from shark liver (O’Hagan et al., 2012). Examples of such adjuvants include MF59 (squalene oil with Tween 80 and Span 85); Montanide ISA51, Emulsigen (mannide monooleate in a squalene-based oilin-water emulsion); GLA-SE (glucopyranosyl lipid adjuvant [GLA], formulated in a squalene-based oil-in-water emulsion [SE], a synthetic TLR4 agonist); and SLA-SE (synthetic lipid-A [SLA] formulated in SE, another synthetic TLR4 agonist) (Del Giudice et al., 2018). Many of these adjuvants have been studied in clinical trials, but at this time only MF59 is included in licensed vaccines for human use. MF59 is internalized by and activates monocytes and DCs (Dupuis et al., 2001; O’Hagan et al., 2012), which then produce chemokines that recruit inflammatory cells (neutrophils, monocytes, DCs) to the injection site. MF59 immune effects are TLR independent, and MF59 promotes the differentiation of monocytes to DCs, increases antigen uptake, and promotes DC migration to draining lymph nodes (Ko & Kang, 2018; Seubert et al., 2008). It has been suggested that after IM injection, apoptotic macrophages containing MF59 and antigen in the draining lymph node induce “danger” signals (DAMPs) in DCs to enhance immune responses to the antigen. Perhaps the best known emulsion adjuvants are the Complete Freund’s (CFA) and Incomplete Freund’s (IFA) adjuvants. CFA is comprised of heat-killed Mycobacterium tuberculosis in mineral oil and mannide monooleate, whereas IFA contains only the oils without the mycobacterial component. Although not used in human vaccines, these adjuvants are widely used in the research setting (Broderson, 1989). CFA is an excellent immune stimulator, driving TH1 and TH17 responses (Shenderov et al., 2013). Mycobacterium is also known to activate TLR-2 (Brightbill et al., 1999; Quesniaux et al., 2004) and TLR-4 (Raghavendra et al., 2004) as well as have TLR-independent mycobacterial effects mediated in part by nucleotide-binding oligomerization domain (NOD)-like and C-type lectin receptors (Saiga et al., 2011). The use of CFA has been limited by the significant side effects, including notable pain at the injection site (Gould, 2000), dermal necrosis, and the risk of granulomas at sites distant to the injection site

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(Broderson, 1989). Additionally, administration of CFA renders tuberculin skin testing invalid as the test will be positive because of the exposure to mycobacterial proteins in the adjuvant.

5.3. Nucleic Acid/Nucleotide Adjuvants Nucleic acids (DNA and RNA) have been demonstrated to activate innate immune responses through activation of several TLRs (i.e., TLR3, TLR7, TLR8, TLR9) and other RNA sensors such as retinoid acid–inducible gene 1 (RIG-I), melanoma differentiation–associated protein (MDA5), and protein kinase RNAactivated (PKR). In this way, the RNA vaccines and inactivated or attenuated live viral vaccines have intrinsic adjuvant activity. It is important to note that rodents do not respond to the same TLR8 ligands as humans (Sarvestani et al., 2012). Rabbits have limited TLR7 and reduced TLR8 activity compared to humans (Lai et al., 2014), but rodents may have more robust TLR7 responses than humans (Clarke et al., 2009). These differences further highlight that immune responses in test species may not reflect immune responses in humans, and this should be taken into consideration when selecting test species and interpreting data from toxicity studies. CpG-ODN Synthetic oligodeoxynucleotides (ODNs) contain unmethylated CpG motifs (CpG-ODN) which mimic bacterial and viral DNA and potently activate TLR9. There are three major classes (A, B, and C) of CpG-ODN which vary in immunological effects (Bode et al., 2011). CpG-ODN are typically 18–30 mers and contain multiple CpG motifs. They are often synthesized to be nuclease resistant. Smaller CpG molecules are more cost-effective as long as potency is maintained. CpG-ODN drives a TH1-type immune response, inducing the maturation and activation of APCs. The immune activation of CpG-ODN may have a species-specific component (Rankin et al., 2001). For example, rats and mice develop immunological responses to CpG-ODN that simulate responses in humans, but rabbits do not (J. Liu et al., 2012); this difference in response is postulated to be due to differences in protein homology at the receptor binding

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domain (Chuang et al., 2014). CpG-ODN appears to be more potent immune activators in mice than in humans, which may be the result of a wider cellular distribution of the ODN in this species (Chuang et al., 2014). CpG-ODN specifically designed to function in rabbits have been reported (Chuang et al., 2014). The antigen-specific immune enhancement of CpGODN appears to require close association of the antigen with the CpG motif. CpG-ODN and aluminum hydroxide (Al(OH)3) are typically used in combination as both CpG and protein bind well to the Al(OH)3, resulting in enhanced immune responses to vaccine antigens (Bode et al., 2011). When coadministered with Al(OH)3 by IM injection, CpG-ODN induces a more robust immune response than aluminum-containing adjuvants alone. At this time, there is only one licensed vaccine containing a nucleic acid adjuvant, Heplisav (HBV), which contains a CpG-ODN. Double- or Single-Stranded RNA or RNA Analogs Both single-stranded (ss) and double-stranded (ds) RNA can be recognized by PRR in mammalian (Tatematsu et al., 2018). TLR-7 and TLR-8, which are located on the endosome, recognize ssRNA and are present in many cell types. In immune cells, it has been reported that TLR7 is distributed in plasmacytoid dendritic cells (pDCs) and B cells, while TLR8 is distributed to monocytes, macrophages, and myeloid dendritic cells (mDCs) (Tatematsu et al., 2018). These receptors may be activated by imidazoquinolines (synthetic chemical compounds). Positive-strand RNA viruses produce an intermediate dsRNA, which may be detected by endosomally located TLR3. Doublestranded RNA analogs, such as poly(I:C), have been identified as effective immune stimulators of TLR3 as well as other PRRs such as retinoic acid–inducible gene-1 (RIG-1), and melanoma differentiation–associated gene 5 (MDA5), which are cytosolic PRRs (Tatematsu et al., 2018; Yu & Levine, 2011). Although the nucleic acid analogs poly(I:C) and poly(ICLC) and imidazoquinolines have been used extensively in the research setting, there are presently no licensed vaccines containing these adjuvants (Del Giudice et al., 2018). As mentioned previously, species differences in PRRs may impact translatability of nonclinical findings to humans.

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5.4. Lipid Adjuvants Lipid adjuvants typically stimulate TLR4 receptors. The most widely used of these adjuvants is monophosphoryl lipid A (MPL or MPLA) or synthetic lipid A (Casella & Mitchell, 2008). MPLA is a monophosphorylated mixture of chemically modified LPS (endotoxin) derived from Salmonella minnesota R595, first described by Edgar Ribi. MPLA has the immunostimulatory effects of LPS without the adverse effects of the native endotoxin (Sarkar et al., 2019). MPLA is the only lipid-based adjuvant in currently licensed vaccines. Examples of other adjuvants in this class include a-galactosylsphingamides (a-GalCers), and RC529. a-GalCers are synthetic glycolipid(s) that bind to CD1d and are the prototype ligand for activation of invariant NKT cells (Del Giudice et al., 2018; Dubois Cauwelaert et al., 2016). Lipid adjuvants should not be confused with liposome-based adjuvants, but lipid adjuvants are frequently a component of liposome-based adjuvants which contain an assortment of lipids and potentially other adjuvants (e.g., AS01b, AS02, AS04, ALFQdsee below) (Del Giudice et al., 2018; Didierlaurent et al., 2017; Garc¸on et al., 2007; Nathalie Garc¸on et al., 2011).

5.5. Adjuvant Systems and Liposome-Based Adjuvants Adjuvant systems utilize a variety of adjuvants in order to activate different immune pathways, thereby potentiating the immune response to antigens. These typically include aluminum salts, liposomes, and oil-in-water emulsions combined with various immunostimulatory compounds. Examples of such systems include Matrix M, Army liposome formulation with QS-21 (ALFQ), and the GlaxoSmithKline proprietary adjuvant systems AS01b, AS02, AS03, and AS04 (Del Giudice et al., 2018; Garc¸on et al., 2007). Matrix M is comprised of Quillaja saponins formulated with cholesterol and phospholipids into nanoparticles. AS01 is also a liposomebased adjuvant containing QS-21 and MPLA as well as 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) and cholesterol. AS02 contains MPLA

and QS-21 in an oil-in-water emulsion. AS03 contains two lipids, squalene oil and a-tocopherol, as well as Tween 80. Inclusion of a-tocopherol may potentiate the innate and adaptive immune response (Novicki, 2013). AS04 is also an oil-in-water emulsion similar to AS03 but with the addition of aluminum salts and MPL. Liposomes, vesicles lined by a phospholipid bilayer, often include cationic lipids such as 1,2-dioleoyl-3-trimethylammonium-propane (DOTAP) and other phospholipids such as 1,2dioleoyl-sn-glycero-3-phosphocholine (DOPC), 1,2-dimyristoyl-sn-glycero-3-phospho-(10 -rac-gl ycerol) (DMPG), or 1,2-dimyristoyl-sn-glycero-3phosphocholine (DMPC). Liposomes enhance antigen uptake by APCs (Tandrup Schmidt et al., 2016), where the antigen is ultimately processed and presented on the cell surface in association with MHC I and MHC II. Liposomes enhance immune responses to antigens with which they are associated, and range in size from 50 nm up to 2 mm (Schwendener, 2014). Liposome size will impact immunogenicity and inflammation at the injection site; smaller particles have been suggested to have increased Th2 responses while larger liposomes drive Th1 responses. Larger liposomes (>500 nm) appear to be more readily taken up by APC at the injection site and may result in greater inflammation at the injection site, whereas smaller particles may be cleared quickly by lymphatics to the regional lymph nodes and have somewhat less inflammation at the injection site (RSS, personal communication) (Perrie et al., 2016). The physicochemical properties of the liposome will impact uptake and processing by APCs, as well as retention at the injection site and distribution. Lipid Nanoparticles for Delivery of mRNABased Vaccines Encapsulating mRNA vaccines in lipid or polymer-based nanoparticles improves cellular uptake in vivo. Details of nanoparticle delivery systems for mRNA vaccines tend to be lipid- or lipoplex-based, and the efficiency and cell targeting depend on the physiochemical properties of the carrier complexed with the mRNA and are

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reviewed elsewhere (Pardi et al., 2018). LNPs used for delivery of mRNA vaccines or therapeutics (e.g., siRNA modality Onpattro) are comprised of four lipid components. These include structural lipids (cholesterol and a phospholipid), a “stealth” lipid (may be lipid linked to polyethylene glycol [PEG]) and an ionizable amino lipid. The ionizable lipid promotes selfassembly into nanoparticles (usually 50– 100 nm) and allows for the release of the cytoplasmic mRNA after endosomal fusion. PEGylated lipids help to stabilize the LNP and prolong its half-life. The phospholipid component (often a naturally occurring lipid) supports the lipid bilayer, and cholesterol promotes particle stability (Pardi et al., 2018). Changes in the lipid components will impact the physicochemical characteristics of the LNP-mRNA (e.g., particle size and the structure), and may be a useful method for altering reactogenicity and immunogenicity.

5.6. Carbohydrate-Containing Adjuvants A detailed review of these adjuvants may be found in a publication by Pifferi et al. (2021) and Petrovsky and Cooper (2011). Carbohydrate adjuvants may be natural or synthetic derivatives of natural chemicals (Pifferi et al., 2021). Naturally derived adjuvants have limitations, including availability and purity. There are several carbohydrate-containing adjuvants that may enhance immune responses to vaccine antigens. Carbohydrate adjuvants include dextran, zymosan, beta-glucan, muramyl dipeptide (MDP), as well as adjuvants that fall under other classes, such as MPLA a-galactosylceramide (a-GalCer) and QS-21, which activate a wide assortment of PAMPs. The mechanism of action of these adjuvants has not been clearly elucidated but appears to be mediated through direct effects on APCs leading to cytokine and chemokine release (Petrovsky & Cooper, 2011). Carbohydrate adjuvants are recognized by an assortment of PRRs and lead to strong proinflammatory cytokine signals (e.g., IL-1 and TNF-a). Carbohydrate adjuvants may trigger the complement cascade, giving rise to C3a, C3b, C3d, C5a, and C5b. These factors may act as opsonins and chemokines, which may be important in their adjuvant activity (Petrovsky & Cooper, 2011). a-GalCer holds promise as an

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adjuvant, and variants have been covalently linked to protein or polysaccharide antigens to improve their immunogenicity. a-GalCer can be chemically modified to promote TH2 or TH1 skewing of immune responses (Pifferi et al., 2021).

5.7. Toxin-Based Adjuvants Presently there are no licensed vaccines using toxin-based adjuvants. However, polysaccharide vaccines are often conjugated to modified tetanus toxoid (TT) or nontoxic mutants of diphtheria toxoids (CRM197) to make them more immunogenic (Weintraub, 2003). Polysaccharides cannot be presented on MHC because they lack the peptides needed for binding. Unconjugated polysaccharide vaccines result in limited immune responses; however, when conjugated to toxoids will generate robust B cell and T cell responses. Conjugation to TT or CRM197 improves T cell responses to the antigen (de Roux et al., 2008; Rappuoli, 2018; Rino Rappuoli et al., 2019) (e.g., Prevnar13). Mutants of tetanus toxoid have been similarly used as a carrier protein (e.g., Nimenrix). Other examples of toxoid adjuvants not presently in licensed vaccines include the Clostridioides difficile toxins, E. coli heat-labile (LT) and mutant LT toxin (LTK63), shiga toxin, staphylococcal enterotoxins, LPS, and cholera toxin (CT) and derivatives. Cholera toxin (CT) and E. coli heatlabile enterotoxin (LT) are highly homologous (Gilligan et al., 1983). Cholera toxin and LT and their B subunit are known to bind to the GMl ganglioside found in cell membranes (Berenson et al., 2013). It has been postulated that this binding ability confers their mucosal immunogenicity by aiding uptake by M cells or by trapping mucosal lymphocytes or macrophages or both (McKenzie & Halsey, 1984). For this reason, they have been considered for use in mucosal vaccines (oral or intranasal), but toxicity (e.g., diarrhea, Bell’s palsy, impaired olfactory function) is a significant issue (Fukuyama et al., 2015). However, the use of the B subunits (CTB or LTB) alone or as carrier proteins does not induce toxicity but instead may be good mucosal adjuvants (Hagiwara et al., 1999; Jan Holmgren et al., 2005; Holmgren et al., 2013).

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5.8. Other Adjuvants Many other potential vaccine adjuvants have been studied in animals. These include bacterial proteins such as flagellin and profilin, small molecule immune potentiators (SMIPs) such as TLR7/8 activators, TLR4 activators, and cytokines and chemokines such as GM-CSF, Type I IFNs, IL-1, IL-2, IL-6, IL-12, IL-15, IL-18, IL-21, and CCL2. The use of these immune activators presently is more within the therapeutic vaccine space, particularly for cancer vaccines, but also is being explored for therapeutic vaccines against chronic infectious diseases (e.g., AIDS) (Del Giudice et al., 2018; Li & Petrovsky, 2016). Regulators may request toxicity studies independent of the antigen for cytokine and chemokine adjuvants depending on the available nonclinical, clinical, and published data. Of particular concern with cytokine and chemokine adjuvants may be species selection, as homologs, orthologs, and paralogs may function differently between humans and laboratory animal species.

6. VACCINE PHARMACOLOGY AND OTHER STUDIES 6.1. Immunogenicity and Efficacy Studies Immunogenicity and efficacy studies are key components of the regulatory package for vaccine candidates. Typically, efficacy and immunogenicity data are included in the pharmacology section of the NCO. If platform data are being utilized to support entry into Phase One clinical trials without GLP toxicity studies, it may be valuable to add nonclinical safety endpoints onto immunogenicity studies. Basic in-life data, such as body weight, clinical observations, body temperature, and injection site reactogenicity, may be adequate. Collection of other endpoints (e.g., clinical and anatomic pathology) can be included on a case-by-case basis if requested by regulators and will be dependent in part on the species selected for these studies. Immunogenicity Studies Most early vaccine studies assess immunogenicity in various nonclinical species. These studies do not typically have pathology endpoints. Nonetheless, it is important to have

pathologist involvement, even at the very early stages of antigen and formulation selection. Early immunogenicity studies provide the opportunity for the pathologist to evaluate endpoints which would reveal unexpected local effects. For example, a vaccine component may cause acute muscle necrosis after IM administration, which would likely stop further clinical development (e.g., QS-21 in aqueous solution) (Garc¸on et al., 2007; Reagan et al., 2020); to identify such undesirable effects prior to GLP toxicity studies is advantageous for conserving financial reserves, time, and animal resources. In the future, safety testing for platform vaccines in which all components remain the same except for the antigen might be able to be limited to non-GLP immunogenicity studies that include clinical observations, clinical pathology, and if possible histopathology endpoints rather than requiring GLPcompliant toxicity studies (https://cdn.who. int/media/docs/default-source/biologicals/callfor-comments/bs.2021.bs2402_who-regulatoryconsiderations-for-mrna-vaccines_final.pdf?sf vrsn¼c8623b32_5). Efficacy Studies Most nonclinical species have been used as animal models for human pathogens, although many do not fully recapitulate the human disease. However, different models may replicate components of the human disease, and data from various models can be used to help in predicting vaccine efficacy. The mouse is the most used animal model because of its small size and low cost, even if not ideally suited to the pathogen. The NHP is a common model for infectious diseases, particularly if no other natural disease model exists, although there are limitations related to cost, housing, and ethics in using monkeys as efficacy models. The variation in immune responses and background inflammation among mouse strains (Radaelli et al., 2018) and between monkeys of different geographic origins (e.g., Asian vs. Mauritian cynomolgus monkeys) may impact data interpretation (Colman, 2016). The ferret (Mustela putorius furo) is a commonly used species to model respiratory disease, particularly influenza virus, as it is susceptible to human influenza viruses and develops comparable disease (Albrecht et al., 2018; Enkirch &

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von Messling, 2015). The Syrian hamster has been demonstrated to be useful in efficacy studies against SARS-CoV-2 (Chan et al., 2020; Dhakal et al., 2021; Imai et al., 2020). INFECTIOUS DISEASE VACCINES

Several approaches have been used for human pathogens that do not naturally infect or cause disease in laboratory animal species. One method is to utilize species-optimized pathogens or similar species-specific microbes to recapitulate human disease. For example, mice are not naturally infected by the SARS-CoV-2 virus as the viral spike protein binds to the human angiotensin-converting enzyme 2 (ACE-2) but poorly to the mouse ACE-2. Mouse adapted and genetically modified SARS-CoV-2 viruses have been generated in which the spike protein binds to the mouse ACE-2 and causes disease (Dinnon et al., 2020; Zeiss et al., 2021). Additionally, mice genetically modified to express the human ACE-2 have also been developed as models for SARS-CoV-2 pathogenicity (Bao et al., 2020; Lutz et al., 2020; Veenhuis & Zeiss, 2021; Zeiss et al., 2021). Species-specific or species-adapted pathogens have been used to cause disease similar to that in humans. Malaria studies are common in mice, but human malaria poorly infects mice. Therefore, researchers use mouse-adapted malarial strains (e.g., Plasmodium yoelii, Plasmodium berghei) for these studies (Harris et al., 2012; Li et al., 2001; Smith et al., 2020). Smallpox and vaccinia studies utilize mouse-adapted pox viruses (e.g., Western Reserve strain) or rabbit poxvirus to generate animal models of disease, although NHPs appear to be similarly susceptible to the pox viruses that infect humans (e.g., Copenhagen strain of the vaccinia virus) (Chapman et al., 2010; Jacobs et al., 2009). Mouse-adapted influenza viruses (e.g., A/H1Ni/WSN) generate lung inflammation that recapitulates the pulmonary inflammation identified in humans (Aeffner et al., 2015). Rarely, the pathologist may be involved in animal models of pathogen transmission since such studies may be a component of vaccine development. These models may assess transmission between animals of the same species or transmission via vectors. Such studies involve the use of mosquitos, ticks, or other insects that transmit disease, and the pathologist may be

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called upon to assess for evidence of macroscopic or microscopic disease. THERAPEUTIC CANCER VACCINES

Generally, in vivo efficacy models for cancer therapeutic vaccines are performed in mice. For tumor studies, syngeneic mouse models are typically used, although GEM models are also employed (see Genetically Engineered Animal Models in Toxicologic Research, Vol 1, Chap 23). Patient-derived xenograft (PDX) models are not used for vaccine studies, as an intact immune system is required for vaccine efficacy. Humanized mice with PDX are also generally not utilized, as the graft-vs-host response by the humanized mouse to the PDX confounds study endpoints. GEM models are somewhat longer in duration and less controlled as to tumor burden compared to syngeneic tumor models. In either case, the vaccine antigen must be against a tumor-expressed antigen, which may be naturally occurring or expressed through genetic engineering of the tumor cells. If the therapeutic vaccine or coadministered product is or expresses a cytokine or other modulator, the expressed protein should have an appropriate biological effect in the test species in order to fully evaluate efficacy (i.e., may require a species-specific surrogate) (Kaumaya et al., 2020).

6.2. Enhanced Disease and Neurovirulence Assessment There are a few other types of animal models related to vaccine development and safety with which the toxicologic pathologist should be familiar, such as for vaccine-associated enhanced disease or neurovirulence. Neurovirulence studies are generally performed only for modified live viral vaccines in which neurovirulence has been identified as a component of the natural viral infection. Enhanced Disease Pathologists should be engaged in both the design and interpretation of enhanced disease animal model studies. Vaccine-associated enhanced disease is rare; however, under certain circumstances, vaccination could exacerbate rather than mitigate viral infection. The mechanism is usually either through antibody-dependent

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enhanced disease or TH2-mediated enhanced disease. Enhanced disease has occurred clinically, most notoriously with RSV and Dengue vaccines, but is generally quite rare. Dengue vaccines have been associated with antibody-dependent disease enhancement (e.g., DENGEVAX) (Bentsi-Enchill et al., 2013; Thomas & Yoon, 2019). Antibodydependent enhanced disease is due to the induction of nonneutralizing antibodies (i.e., antibodies that do not neutralize virus) which also mediate viral entry into cells via the Fc gamma receptor or compliment receptors. After facilitated entry, the viruses replicate and cause disease which may be more severe than without vaccination. In the case of RSV, enhanced respiratory disease has been attributed to the induction of TH2 type cellular immune responses rather than a protective TH1 response (Acosta et al., 2015; Kapikian et al., 1969). In the 1960s, a vaccine composed of formalin-inactivated, whole virus RSV was administered to infants and toddlers. RSV-naı¨ve toddlers administered the vaccine who were subsequently exposed to RSV developed unexpectedly severe respiratory disease. Since then, despite the significant need for a vaccine against this virus, companies have been very wary of this viral target (Russell et al., 2019). Recently many companies have begun developing RSV vaccines, and regulators expect these will be tested in a model to predict enhanced respiratory disease. Of the animal models of enhanced disease considered relevant to humans, the most utilized nonclinically is the cotton rat (Sigmodon hispidus) model for RSV vaccine-associated enhanced disease (Boukhvalova & Blanco, 2013). The cotton rat model of enhanced respiratory disease, while not ideal, is accepted by regulators. When administered the formalin-inactivated, whole virus vaccine and challenged with RSV, cotton rats develop more severe lung inflammation (neutrophilic infiltrates into alveoli, increased alveolar wall thickness) compared to unvaccinated cotton rats challenged with RSV. While the inflammation in the cotton rat lungs related to the vaccine followed by RSV challenge is generally minimal, it is more severe than in unvaccinated rats challenged with RSV. Neurovirulence Assessments Neurovirulence (NV) studies are used to assess modified live virus vaccines in which the wild-type (WT) virus has been demonstrated to

infect neural cells. NV studies are expected to demonstrate that there is attenuation of NV in the vaccine as well as to identify any potential for reversion of the modified virus to the WT phenotype. These studies have traditionally been performed in monkeys (Rubin & Afzal, 2011). However, rodent models have been developed that appear to be acceptable for assessing NV risk. These include neonatal mouse and rat NV studies (Kim et al., 2014; Rubin et al., 2005; Zhang et al., 2010). Neurovirulence studies often utilize survival as an endpoint, but inclusion of confirmation of brain inflammation at least in a subset of animals to confirm cause of moribundity/mortality may be beneficial.

6.3. Biodistribution and Persistence Biodistribution and persistence studies are used for any modified live microbial or nucleic acid vaccines to understand target organs and can help inform nonclinical study designs. For these studies to be considered valid, the animal model must be able to support replication of the vaccine virus (Planty et al., 2020; Stokes et al., 2020). These studies generally do not have pathology endpoints; however, the pathologist should be involved in the selection of tissues for evaluation. Regulators may expect both nucleic acid and protein distribution data, particularly for new modalities.

6.4. Absorption, Distribution, Metabolism, and Excretion In general, absorption, distribution, metabolism, and excretion (ADME) studies (see Biotherapeutic ADME and PK/PD Principles, Vol 1, Chap 4) are not required for vaccines. However, such studies may be required if associated with nanoparticle delivery methods or with novel adjuvants. These studies generally do not have pathology endpoints but may be valuable if there are unexpected findings in nonclinical studies. For example, in mRNA-LNP vaccines administered IM, transient minimal portal vacuolation of hepatocyte cytoplasm has been identified without evidence of hepatocellular damage. ADME data of the LNP lipids demonstrated hepatic distribution, indicating that the vacuoles

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were likely LNP lipids that distributed to the hepatocytes.

7. VACCINE SAFETY STUDIES Many prophylactic vaccines will require only one repeat-dose toxicity study with a recovery arm for assessment of reversibility and delayed toxicity in a single species. Generally, singledose toxicity and safety pharmacology studies are not required for vaccines or may be addressed using data collected during the repeat-dose toxicity study. For changes in vaccine formulation which may impact local tolerance but do not warrant full toxicity studies, local toleration studies may be performed.

7.1. Repeat-Dose Toxicity Studies Designing an effective vaccine study requires careful consideration of the appropriate animal species or strain, dose volume, frequency of dosing, route of dosing, technical details of test material administration, animal handling limitations, clinicopathologic sampling intervals and evaluation, tissue sampling, analytical endpoints, and a host of other items (Sellers et al., 2020). While prophylactic and therapeutic vaccines have different objectives, the mechanism of action to generate an immune response against a specific antigen or antigens is shared. In both cases, the immune response to the antigen and its impact on tissues is the key endpoint of nonclinical toxicity studies, along with any effect of the vaccine formulation components (including any impurities or contaminants). Any off-target or severe expected toxicity is generally not acceptable in prophylactic vaccine development. The dosing schedule in nonclinical toxicity studies need not reflect that schedule anticipated for use in humans. For example, a vaccine for human use may be anticipated to have a booster dose 6 months after the initial vaccination. Such a dosing paradigm would not be pragmatic for nonclinical studies. Therefore, timing between doses in nonclinical studies may occur at shorter intervals (as short as 1 week) and should be based on a demonstrated antigen-specific immune response during the dosing phase of the study. Frequency of dosing may vary between 1 and 4 weeks to allow for the development of an humoral immune response in the

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subject. Single-dose toxicity may be evaluated as part of the repeat-dose toxicity study, as vaccines are generally administered at 2- to 3week intervals allowing for assessment of toxicity after the first dose administration. Most prophylactic and therapeutic vaccines need at least one GLP-compliant repeat-dose toxicity study with a recovery phase to support entry into clinical trials. Dose range finding studies are typically not included as part of the nonclinical safety package as translatability of the animal immune response to humans is inconsistent and not generally predictable. Dose range finding may be appropriate for novel virus–based modalities, modified live viral vaccines, and novel adjuvanted vaccines. Prior to nonclinical toxicity studies, however, the immune response to the vaccine antigen in the test species should be understood, including the kinetics of immune induction and duration. Chronic toxicity studies are not required to support licensing of prophylactic vaccines. Chronic studies might be required for therapeutic vaccines depending on the condition being treated and the anticipated duration of treatment. The recovery phase of nonclinical vaccine studies serves a slightly different purpose from recovery studies for small molecules. Although the recovery phase is intended to confirm recovery from any test article–related findings, given that the mechanism of action is through induction of an immune response, the recovery phase is also intended to identify any delayed immunemediated toxicities. Because delayed toxicities are a concern in vaccine development, there is an expectation to microscopically evaluate either all tissues or a selected subset of major organs in all animals. Therefore, additional time needs to be built into vaccine repeat-dose toxicity studies for these extended assessments. To the authors’ knowledge, no delayed toxicity has been identified in nonclinical prophylactic vaccine studies. Whenever possible, the vaccine should be administered at least at the highest anticipated clinical dose and dose volume (generally 0.5 mL), and by the clinical route of administration. If there are issues around feasibility, the WHO guidance allows for dose justification based on a milligram per kilogram basis (WHO, 2005). The route of administration for nonclinical studies is typically IM, but SC, ID, intranasal, oral, or intravenous (IV) administration have all been utilized for prophylactic and therapeutic vaccines, as well as intratumoral or intranodal administration for therapeutic vaccines and oncolytic viruses. The

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number of doses should equal the number of anticipated clinical doses provided within a year. The inclusion of one additional dose beyond that anticipated number for clinical use should be considered (known as an “N þ 1” study design) because this approach allows for administration of one additional dose clinically if experience suggests that a further exposure will be prophylactically or therapeutically beneficial to patients. Developmental and Reproductive Toxicity Studies Studies to evaluate developmental and reproductive effects are expected for vaccines and are performed to support Biologic License Application (BLA) submission for vaccines intended for use in women of childbearing potential or prior to clinical studies in pregnant women (maternal vaccines). These studies are combined fertility, embryofetal, and postnatal development studies, and a single study in a single species is usually adequate. However, novel modalities, adjuvants, or excipients may require a study in a second species. Studies are typically performed in rats or rabbits. The updated ICH S5(R3) guidance on developmental and reproductive toxicity (DART) studies includes vaccines but does not specifically give an exemption for vaccines to male fertility or postweaning studies (ICH, 2020). These studies are frequently not

warranted for vaccines, and data from the repeat-dose toxicity studies will help inform the risk relative to male fertility. As with repeat-dose toxicity studies, the DART species should ideally be administered the full human dose. Rats and rabbits are the most common DART species. Maternal transfer of antibodies tends to occur primarily by placental transfer during gestation in the rabbit whereas it occurs primarily through the nursing postpartum in rats. Dosing is initiated prior to mating to allow for adequate vaccine titers at the time of mating. Frequently doses are administered twice before mating, 2–3 weeks apart (e.g., 21 and 7 days prior to mating); this design assesses potential effects on female fertility. Dosing during organogenesis (e.g., rats at gestation day 6 and rabbits at gestation day 10) and again late in gestation (e.g., rats at gestation day 20 and rabbits at gestation day 24) will allow for identification of vaccine effects during embryofetal development (Figure 9.1). Dosing of dams during lactation may heighten confidence in the safety of the vaccine formulation for use in lactating women but is typically not expected by regulators. It is recommended to perform DART studies at a facility with an extensive historical control database to allow for appropriate interpretation of rare findings.

FIGURE 9.1 Overview of DART study design. Regardless of test species, timing of vaccine administration prior to mating should ensure adequate antigen-specific immune responses. This typically requires a prime dose followed by a boost 2–3 weeks later. Additional vaccine doses are given during organogenesis and in late gestation.

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8. VACCINE STUDIESdDESIGN, TECHNICAL CONSIDERATIONS, AND DATA INTERPRETATION 8.1. Species Selection Species selection is based on demonstration of an immune response to the administered antigen (WHO, 2005, 2014). If an adjuvant is included in the vaccine formulation, the nonclinical species ideally should display enhanced immune responsiveness to the antigen plus adjuvant relative to the antigen alone. There is no expectation that the immune response in the nonclinical species extrapolates directly to humans; higher doses of antigen do not necessarily produce increased immunogenicity. The WHO guidelines also suggest that the nonclinical species ‘be sensitive to the pathogenic organism or toxin under consideration’ (WHO, 2005). This specification may be related to vaccines using live viral

TABLE 9.6

vectors, in which biodistribution and virulence may be important to understanding toxicity. While not always the case, many human viral pathogens have limited or no effects in nonclinical species making fulfillment of this recommendation challenging. Demonstrated evidence of viremia or biodistribution studies may help to justify the selected nonclinical species for modified live virus modalities. The traditional species used for prophylactic vaccine studies is the rabbit (see Animal Models in Toxicologic Research: Rabbits, Vol 1, Chap 18), although rats are also commonly used (see Animal Models in Toxicologic Research: Rodents, Vol 1, Chap 17). Considerations around species selection for regulatory toxicity studies are presented in Table 9.6. Nonhuman primates may be used as the nonclinical species (see Models of Toxicity: Nonhuman Primate, Vol 1, Chap 21) for prophylactic vaccines, but for ethical reasons should only be used if there are no alternatives.

Considerations in Species Selection Rabbit

Rat

Mouse

Monkey

Ferret

Pig

Dog

Guinea Pig

Cost housing

M

L

L

H

M

H

H

L

Full human dose (0.5 mL)

Y

N

N

Y

N

Y

Y

N

Harmonized and standard system of nomenclature

Y

Y

Y

N

N

N

N

N

Technical staff comfort

N

Y

Y

Y

N

N

Y

N

Reagents readily available

Y

Y

Y

Y

N

N

N

N

Published resources

L

H

H

H

M

M

H

L

Additional concerns

LHD TLR7-NR TLR8-RR

TLR8-NR

TLR8-NR

LHD

LHD

PS-80 hypersensitivity

LHD Ig hypersensitivity

Y, yes; N, no. Ig, immunoglobulin. L, low; M, medium; H, high. LHD, limited historical data. TLR, toll-like receptor. NR, not responsive. RR, reduced responsiveness.

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Recommended Administration Volumes

Species (mean body weights)

Intradermal

Intramuscular (up to 2 sites/day)

Subcutaneous (multiple sites dependent on species)

Intranasal (upper respiratory tract mucosa)

Dose vol./site (mL) Dose vol./site (mL) mL/kg/site Dose vol./Site (mL) Dose vol./nare (mL)

Mouse (25 g)

50e100

0.025e0.05

5e10

0.12e0.25

35

Rat (250 g)

50e100

0.1e0.25

1e5

0.25e1.25

50

Ferret (1 kg)

50e100

0.25e0.5

2

2

100

Rabbit (3 kg)

50e100

0.5e1

1e2.5

3e7.5

250

Beagle dog (7 kg)

50e100

1.5e3

0.5e1

3.5e7

500

Cynomolgus macaque (2.5e3.0 kg)

50e100

0.5e2

0.5e1

1e3

200

Go¨ttingen minipig (15 kg)

50e100

3e5

1

15

N/A

Values compiled from Hull, 1995; Diehl et al. 2001; Morton et al. 2001; Turner et al., 2011; and Gad et al., 2016.

Larger species have the advantage of allowing for administration of the full human dose as well as having adequate blood volume for intermittent sample collection. Administration of the full human dose to rats will generally require administration of the test article at more than one site. Mice are acceptable for nonclinical toxicity studies, but alternative approaches for dosing may be required, including using the maximum feasible dose, a milligram per kilogram approach, or administering a more concentrated antigen formulation. For details on acceptable dose volumes, see Table 9.7 (Diehl et al., 2001; Turner et al., 2011).

8.2. Dose Groups When designing a vaccine study, at minimum the study should include a control group (generally dosed with saline or formulation buffer) and vaccine group(s). Vaccine dose groups may consist of only a single-dose or multiple-dose levels depending on program needs. The vaccine dose administered should ideally be at the highest anticipated clinical dose in humans whenever

feasible. Inclusion of adjuvant control groups is not a regulatory requirement; in instances where a known adjuvant is being used, the vaccine combined with the adjuvant is considered to be the test article and an adjuvant control is not necessary (WHO, 2014). Inclusion of adjuvant control groups may be beneficial to more clearly delineate adjuvant-specific effects from those caused by the vaccine. Animal numbers for vaccine study dose groups are determined similarly to routine toxicity studies, with specific recommendations available in vaccine-specific WHO guidance documents (WHO, 2005, 2014). In rodents, the recommended number of animals is 15 animals/sex/group, of which 5/sex/group are relegated to the recovery arm. In general, no satellite animal groups are necessary for routine prophylactic vaccine studies. However, for small species such as mice, satellite animals may be necessary for serology, biodistribution, or biomarker analyses due to the low blood volume obtainable from individual rodents. In nonrodents, recommended group sizes are 7–10 animals/sex/group, with 3–5 of those relegated to the recovery arm.

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8.3. Technical Considerations Related to Dosing and Dose Administration Route of Administration The route of administration (IM, SC, ID, IV, mucosal, or intranasal) will affect a wide range of factors in the study, ranging from husbandry to pathology evaluation, as well as the response of the test animals to the vaccine. The volume of the dose should also be considered as different routes and different species may limit the potential volume of dosing, necessitating multiple dose sites or alternate methods of administration. Vaccines have traditionally been administered to patients through IM injection or SC injection. However, they may also be given by ID injection, IV administration, mucosal delivery, or intranasal inoculation. If dosed IM, common sites are the large muscle groups of the hind limb (quadriceps, biceps femoris/gluteus, or gastrocnemius) or the dorso-lumbar epaxial muscles. Larger animals (dogs or minipigs) may be dosed in the muscles of the proximal forelimb or, in the case of pigs, the neck (though this is complicated by abundant subcutaneous and intramuscular fat). If dosed SC, the dorsum is most common, though the flanks or hind limbs may also be used for larger animals with less mobility in the skin over the underlying muscle. Rodents may also be dosed SC at the base of the tail. Intramuscular dosing is one of the primary routes of administration. Injection of vaccines into skeletal muscle optimizes immunogenicity of most vaccines and minimizes adverse reactions (Cook, 2008). The comparatively extensive blood supply and active metabolic nature of the muscle tissue is thought to provide for increased immunogenicity through increased acute exposure of APCs to the injected antigen. Reduction of inflammatory reactions may be due to both the more highly vascularized tissue, which provides more rapid clearance, and the mechanical advantage of myofiber contraction spreading the injected material along fascial planes. Often, inflammatory reactions are noted in the overlying subcutaneous tissues, where the test material may spread in a retrograde fashion along the injection track. Appropriate delivery of the injected vaccine to the muscle is therefore important for correct evaluation of histopathology at the site of delivery, accurate evaluation of

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pharmacokinetics due to variance in tissue absorbance and distribution of injected test material, accurate evaluation of the course and degree of immune response due to optimization of immunogenicity from IM injection compared to injection into connective tissues or adipose tissue, and, if appropriate, accurate evaluation of biodistribution (Cook, 2008). Proper selection of materials (needle length and gauge), appropriate dose volume selection, and proper training for technical administration are important factors in maintaining consistent intramuscular delivery. Subcutaneous injections are the second most widely used route of administration for vaccines. This may be due to the subcutis providing a more easily accessible target for delivery or allowing increased volume when dosing rodents. However, when compared directly to IM injections, there is evidence of increased severity and incidence of inflammatory reactions at SC injection sites. This may be due to decreased removal of antigen and increased chronic exposure of DCs to antigen, resulting in chronic inflammation around the injection site and formation of a granuloma in association with adjuvant materials. Subcutaneous injections may also have decreased immunogenicity due to lower blood perfusion and increased sequestration of the injected material (Cook, 2008). Subcutaneous injections will often spread out within the subcutis, providing a wider field for evaluation, but also confounding histopathological examination of control sections if they are taken close to the delivery site. Intradermal injections are not commonly used as a route of administration but are currently used in humans in Bacille Calmette-Gue´rin (BCG) vaccination, vaccinia (smallpox) vaccination, and some rabies prophylaxis protocols. There has been investigation of the use of this route of administration as an alternative to intramuscular or subcutaneous dosing, in the interest of reducing required vaccine dose and reducing unexpected events, such as accidental needle injuries to providers, inoculation of bacteria deep within tissues, or the like. Technical demands for increased training for providers and unfamiliarity with the intradermal dose route have been factors reducing the adoption of this route of administration. However, with increasing use of transdermal or intradermal

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microneedle dosing via patches or needle-free injector dosing (as with the Bioject system), we may see the ID route becoming more common for vaccination protocols. Benefits include decreased dose requirements, and, in conjunction with cutaneous absorption, jet injection, or microneedle dosing modalities, offers a “needle-free” approach to vaccination (Schnyder et al., 2020, 2021). However, development of appropriate modalities, concern regarding adverse local immune responses, and concern regarding long-term antibody responses have slowed the development of this route of administration. Intranasal vaccine dosing is the primary route by which mucosal vaccine delivery is performed. Intranasal vaccines provide antigenic stimulation at the site of the nasal mucosa and submucosa, which is important as such tissues are one of the primary sites of viral infection for many respiratory viruses. Rather than relying on a purely systemic overall response, antigenic stimulation at the site of possible infection provides increased immunologic surveillance, increased mucosal cell-mediated immunity, and higher local IgA antibody production at that site as well as systemically as its main advantages (Lycke, 2012). It also provides a noninvasive, needle-free delivery system, requiring a lower antigenic dose and contributing to increased patient compliance for humans. Intranasal delivery of vaccines carries with it the potential, in humans, for passage of the vaccine to the olfactory bulb of the brain, so species with nasal anatomy similar to that of humans, such as the cynomolgus macaque, should be preferentially used for these safety evaluations (Chamanza & Wright, 2015; Emami et al., 2017). The intranasal route requires specific intranasal dosing regimens and careful attention to the correct dose volume and methodology. Inaccurate dosing can be an issue, particularly if using larger dose volumes in animals that are only restrained physically. Instead, for this dosing route, animals will often be sedated, at increased time and cost, but with dramatically decreased individual animal stress, potential handling-associated injuries, and loss of test material due to animals expelling it by sneezing. Additionally, dosing via the upper respiratory tract will often result, as dose volumes increase,

in carryover dosing of the lower respiratory tract or even the stomach. In dosing via this route, it may be prudent to include routine sampling of not only the nares, nasal cavity and turbinates, nasopharynx and oropharynx, but also the esophagus, stomach, trachea, larynx, and lungs to capture these area where carryover dosing can occur. Dose Volume The dose volume used may, in part, depend on the model species, and dosing guidelines are available through the AALAC (American Association for Laboratory Animal Care) (Turner et al., 2011). Dose volume is often selected to reflect that given to humans (0.5–1.0 mL). This is one reason why rabbits and other nonrodents may be preferred to rodent species as larger animals can tolerate larger dose volumes. For example, rats are generally limited to maximal IM dose volumes of 200–250 mL and mice limited to 100 mL, per recommended guidelines (Diehl et al., 2001) (personal experience), while rabbits can tolerate up to 0.5 mL IM dose volumes. While dose volume may be limited, rodents are easier to maintain and utilize in larger numbers than nonrodent species, as well as having a generally wider array of commercial reagents available for cytokine and immunological assays. Additionally, many research organizations may have a more comprehensive database of historical control data in mice or rats than in rabbits or other nonrodent species, making evaluation of possible background findings simpler. Other routes of administration have different recommended dosing volumes (Table 9.7). Recommended volumes may be reduced, depending on the degree to which nontarget tissue dosing is tolerated. Only a small number of publications address dose volume restriction, suggesting the potential need for pilot studies to evaluate dose volume, particularly for nonstandard routes (such as intranasal or intradermal dosing). For example, in mice, while publications have suggested that the 100 mL IM dose volume is acceptable (Diehl et al., 2001), an elegant study using contrast media injection and imaging demonstrated that only doses of 50 mL or less remained within the muscle, and larger doses (including 100–200 mL volumes) extended beyond the muscle into adjacent fascial

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and subcutaneous tissues (Gehling et al., 2018). Overly large-volume IM injections may result in inadvertent muscle damage and necrosis due to increased pressure within the muscle. Although this will not directly translate to humans, the volume-associated changes may mask other effects and render evaluation more difficult. Additionally, for routes such as intranasal dosing, lower dose volumes may be readily retained within the nasal cavity while higher volumes will result in either loss of test material through the nares or aspiration or swallowing of test material, with subsequent dosing to the lungs or stomach. This may or may not affect safety evaluations of the test article, and discussion of this potential spill-over effect should be part of study design. Assessment and approval of the use of increased dose volumes in specific species for individual studies may be done by the Institutional Animal Care and Use Committee (IACUC) of the institution, in conjunction with pilot studies to assess appropriate dose volumes, when sufficient experimental work in the literature is not available to set proper dose volumes. Animal Husbandry and Dosing Administration of vaccines should be done by experienced technicians with appropriate attention to detail regarding anatomy, animal handling, dose site selection, materials selection, and marking of dose sites for future assessment. Many of these factors can be outlined in the study design and protocol, but specific features should be emphasized as accurate delivery of the vaccine to the appropriate site and assessment of vaccine effects are equally dependent on these initial technical components. Clearly the need for specific details varies depending on the route of administration, as some routes (e.g., intranasal dosing) would not require marking of the dose site. Injectable Vaccines Selection of the dose site is a critical component of the study design. This is particularly important for IM injections, as muscles vary in size and ease of injection as well as ease of specimen collection for histopathological examination. Additionally, the dose site selection will impact which lymph nodes should be collected

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for evaluation as draining nodes for the injection site. Use of clear language and directions for the anatomic location and dosing procedure will benefit the pathologist and provide a more accurate interpretation of potential toxicity or injection site effects. The need for clear identification and description of the dose site begins at dosing and carries through the necropsy evaluation and tissue collection. In-life techniques for facilitating the accurate administration of the test material include (1) specific anatomic localization of the injection site; (2) consideration of species variations in anatomy; (3) use of specific dosing parameters, including needle length/gauge, injection approach, and restraint methodology; (4) manual or, if necessary, chemical restraint during dosing; and (5) utilization of appropriate marking of the area of the injection. At necropsy, the anatomic localization techniques may be coupled with appropriate marking by in-life technicians and collection of wide margins to ensure the accurate collection of the injection site(s). The use of multiple dose sites, while beneficial for reducing trauma to animals, can present challenges in safety evaluation. If a larger dose is spread among multiple injection sites at each dosing time point, it is advisable to collect each injection site and evaluate them separately. However, diagnoses may be reported as a combined assessment rather than including each site as a unique tissue. If the dose is administered on alternating legs, evaluating each site as a unique tissue may allow for the assessment of temporal changes in the development of the injection site reaction, comparing findings at different postdose recovery time points. In our experience, this may demonstrate some degree of difference in histopathological findings between the most recent and the earliest injection sites, but those sites with more subacute findings are often not readily discernible from one another. When using multiple dose sites or delivering multiple injections to the same muscle group, there should be a clear and consistent way to identify the dose site(s) and track clinical findings at that site as well as appropriately sample the injection site for histopathological examination. Commonly used mechanisms include marking the injection site with a marking pen or tattooing the area of the injection in addition

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to using specific anatomic landmarks for the injection. When using a marking pen, the question of how to mark or identify the site is one that can affect the conduct of the study as the time between doses is often quite prolonged and sufficient for ink or pen markings to fade. Therefore, a procedure needs to be in place to refresh the marking at the site. This commonly results in a drift in the region being marked and thus potentially a drift in dosing as well as a reduced ability for accurate site examination that is gradually compounded over the multiple repetitions of the marking process. The use of tattoos to delineate a dosing area can eliminate some of that drift as it isolates a specific region of skin and underlying tissue for evaluation. If done, it is recommended that the tattoos be limited to dots at the corners of a square or triangular dosing area, with dosing done into the middle of that area; limiting the amount of tattoo ink at the center of the injection prevents exogenous pigment from being confused with vaccinerelated material within macrophages. Tattooing the skin of the animal needs to be done at least 1–2 weeks prior to study initiation, but otherwise produces little to no interference with site evaluation, either clinically or microscopically. Histologically, tattoo ink pigment may be associated with minimal lymphohistiocytic infiltrates and pigment within the dermis (Figure 9.2). Exterior marking has the benefit of easy identification of the injection site for the technical

staff and may have benefit for the necropsy staff. However, the highly mobile nature of the skin of many species, such as the rabbit and rodents, may make skin marking an inconsistent way to delineate the desired IM or SC target. More robust methods of identifying the injection site other than by marking may allow for more consistent and better dosing and collection. The use of anatomic landmarks, particularly bones in the region of the injection site, may provide consistent localization of the site of administration, assisting staff during in-life and postmortem procedures. For example, the femur is a commonly used landmark for injections into the quadriceps femoris or biceps femoris muscles, with the articulation of the coxofemoral joint (“hip”) and the femorotibial joint (stifle or “knee”) forming 2 points of reference and the shaft of the femur forming a third point. By palpating the hip and knee joints, the midfemur may be located, and the vaccine consistently administered into the adjacent defined muscle belly (Figure 9.3). Along the dorsum, use of the vertebral column, ribs, and pelvic bones may provide similar landmarks for SC, ID, or IM injections. This method also improves collection of sites, as necropsy and tissue trimming staff can use the same landmarks to locate the injection site region to be sampled. Clearly, handling and restraint of animals at the time of injection must be consistent to minimize relative movement of the skin and muscles with respect to the associated skeletal landmarks.

FIGURE 9.2 Epidermis and superficial dermis with tattoo pigment and associated small numbers of lymphohistiocytic infiltrates. Ferret. H&E 10.

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FIGURE 9.3 Injection location diagram for biceps femoris and quadriceps femoris of the rabbit hind limb.

The use of consistent anatomic locators to delineate the site(s) of injection may provide the most consistency but also requires increased personnel training and dosing time. When using anatomic locators for dosing sites, there should be an emphasis on training and anatomic knowledge of technical staff involved in dosing and in necropsy activities. Clear directives, with use of normal skeletal anatomy to isolate and identify the targeted injection muscle, skin, or subcutaneous area along with specific handling procedures to correctly orient the tissues are key aspects to ensuring consistency and accuracy in dosing, sample collection and eventual evaluation of findings. Restraint, Handling, and Dosing Handling of animals being used for vaccine studies is, in general, similar to handling for general toxicity studies. The route of administration will dictate handling concerns. Manual restraint is generally sufficient for dosing injectable vaccines, though for consistency in dosing as well as personnel and animal safety during dosing it may be advisable to have two technicians, one to hold the animal and one to perform the injection and any marking of the site. For example, in rabbits the standard hold for injectable dosing may need modification to reduce movement of the skin and muscle and to more closely mimic the position in which they will be necropsied. Such modifications are key because rabbits in a live natural position have their hind limbs drawn up, with the muscles and skeletal structure in a different posture than when they

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are being necropsied. Slight extension of the hind limb at the time of dosing places the muscle and skeletal structure of the hind limb in a similar position to the position it will assume at necropsy, as well as facilitating palpation of skeletal structures and identification of the appropriate muscle groups for injection. Some test facilities have found it advantageous to sedate small species for IM injections to ensure accuracy of administration and reduce local trauma due to animal movement during the injection. The use of sedation has not been associated in the literature with any confounding effects in study parameters. For intranasal administration, it is suggested to use sedation to facilitate ease of dosing and reduce animal stress and discomfort. Handling of the animal to produce consistent approaches to the injection delivery is important. Technical staff should be trained to have consistent orientation, injection site direction, and injection depth. For example, if the area of injection is marked on the skin, the technical staff should pay attention to where the material is ultimately injecteddthe needle might enter the skin in the delineated region but be injected outside the demarcated area depending on the angle at which the injection device is inserted. This is particularly a concern for SC injections but is also important for IM injections since the skin may be misaligned with the underlying muscle in species with loose subcutaneous connective tissues. IM injections should be oriented consistently (preferably with the needle perpendicular to the muscle) to provide greater accuracy and reproducibility, and reduced potential for offtarget effects on regional structures such as the sciatic nerve and knee joint. Depth of injection can be controlled to some degree by the choice of needle length but should be considered when designing the study procedures so as to deliver the test article to the appropriate tissue compartment. Needle gauge and length are critical features to consider both within a study and between studies to reduce nonspecific variation in the injection site histology. This is particularly of concern in IM injections, as ID and SC injections are more easily palpated, and the site of delivery visualized externally as a bleb. Regarding needle length, in performing IM injections, longer needles may run the risk of causing deeper than

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desired injection of material, such as into the stifle joint region, muscle bundles deep to the targeted muscle, and in or around the sciatic nerve. Conversely, shorter needles may result in delivery of a test article into the subcutis rather than the muscle in an IM injection. A needle length of 1 cm is appropriate for IM delivery in the majority of laboratory animals, including rabbits, monkeys, dogs, and ferrets. The use of longer needles (1.5 cm) may be associated with administration of material into underlying muscle beds, even in NHP or canines (Figure 9.4). Conversely, for minipigs, the extensive and thick subcutaneous fat (particularly along the dorsum) means that longer needles even up to three or 4 cm may be needed to inject into certain muscle groups (Figure 9.5). This is particularly pronounced in Yucatan minipigs, though even Go¨ttingen minipigs will need longer needles. Even shorter needles (0.5 cm) may be appropriate in smaller species, such as mice, rats, and hamsters and the use of these shorter needles should be considered on a case-by-case basis. Age of the animal should also be considered, with older animals often requiring a longer needle since they have more abundant subcutaneous fat and thicker dermis,

as well as more developed and larger muscles. Variations in needle length may impact the consistency of the location of where material is injected, even if other factors are consistent. Inappropriate needle length may result in several potential issues, including delivery to an incorrect muscle group, leading to failure to collect appropriate or sufficient tissues to evaluate the injection site at necropsy. Inappropriate needle length may also result in delivery outside of the muscle, into the deep fascia or subcutaneous fat, which can affect immunogenicity, pharmacokinetics, and inflammatory response due to variations in tissue response and uptake of injected vaccine. Finally, with longer needles there may be delivery deep to the muscles, resulting in dosing around or into the sciatic nerve or joints, producing increased trauma, pain, and morbidity in affected animals. Needle gauge and speed of injection are components affecting future evaluation of the injection site. A narrow-gauge needle will result in a more forceful ejection of solution into the tissue compartment, along with accompanying tissue trauma. Additionally, in larger animals, narrow gauge needles may be bent or broken by muscle contraction during injection. This must be balanced against the potential for increased trauma and pain associated with the use of larger gauge needles. Speed of injection is known to increase muscle damage when volumes are large, presumably due to mechanical trauma to the muscle fibers. Viscosity, pH and other aspects of the injected solution may also impact the injection site findings, but the use of appropriate controls is a strong mitigating factor. Use of needle-free options, such as the Biojector system (Figure 9.6), mitigates the need for needles and provides a consistent dosing force, but the compressed air delivery can be overly traumatic in animals with insufficient muscle mass, due to the speed and force of the injection.

8.4. In-Life Assessments FIGURE 9.4 Epaxial muscle injections in feline. The 3/800 needle IM injection site (blue) is at left, with the longer 5/800 needle IM injection (green) at right, using equal injecta volume. The blue dye, injected via the shorter needle, is contained within the muscle, while the green dye spills into the deep margin. Image is of formalin-fixed tissue.

The in-life assessments in vaccine studies are the same as for other repeat dose toxicity studies with inclusion of postvaccine administration-specific endpoints, such as body temperature and injection site evaluations. Specific recommendations are detailed in the WHO recommendations and regulatory guidance documents (WHO, 2005, 2014).

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FIGURE 9.5 Yucatan minipig neck injections. IM injection of dye solution with needles of varying lengths in Yucatan minipig neck. (A) 4 cm needle, with dye injection into deep muscle. (B) 2 cm needle, with dye injection in subcutaneous adipose tissue.

FIGURE 9.6 Biojector used in pilot injection of dye solution into biceps femoris of euthanized rabbit to test depth of injection.

Local Toxicity and Reactogenicity Evaluation of the vaccine administration site is an essential component of the in-life assessment in vaccine studies. Injection site assessment may be done by a specific scoring system or by close observation and accurate description of findings in individual animals. Overall assessment of the animal is generally performed as expected for any safety study, with the addition of regular

body temperature monitoring for systemic fever around the time(s) of injection. In-life assessment of the injection site often utilizes a prospectively defined scoring system. Many endpoints may be scored including swelling (edema) and redness (erythema) (such as a modification of the Draize method (Draize et al., 1944) as well as characterization of other changes such as vesiculation, ulceration, eschar formation and any potential evidence of significant toxicity, including limb impairment. However, use of a specific scoring system is not a requirement if in-life injection site observations over the course of the study are accurately captured and described. In the case of an intranasal administration, the nasal mucosa, nares, and possibly oral mucosa should be evaluated carefully following dosing. Close monitoring of pulmonary parameters also is important in case of inadvertent dosing of the lower respiratory tract. Body temperatures prior to and following vaccine administration are evaluated and may be measured at the same time as the injection site observations. Body temperatures may rise from 1 to 2 C in a rabbit or rodent model, though a febrile response would not necessarily be expected. Regulatory recommendations (WHO, 2014) suggest evaluation of body temperatures at each administration prior to dosing, 3–8 h

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postdose, and again 24 h postdose with ongoing monitoring from there until body temperature returns to baseline. Daily measurements of food consumption and body weight are also recommended for 1 week following each dose, to monitor for morbidity (WHO).

8.5. Clinical Pathology As with small molecule regulatory toxicity studies, clinical pathology for both prophylactic and therapeutic vaccine studies typically include routine hematology, clinical chemistry, coagulation assessments, and urine analysis (see Clinical Pathology in Nonclinical Toxicity Testing, Vol 1, Chap 10). In addition, one or more serum acute phase proteins (APP)dsuch as C-reactive protein (CRP), fibrinogen, haptoglobulin, and serum amyloid A (SAA)dare measured as indicators of a systemic immune response (Cray et al., 2009; Green, 2015). Cytokines may also be measured, but their use is less common and should be carefully considered due to high interand intra-animal variability, short half-lives, and wide physiological range of variation that make the interpretation of cytokine changes more difficult. Blood collection is usually predose, 1–3 days after the first dose, at the dosing phase necropsy (1–3 days after the last vaccine dose), and again at the recovery phase necropsy (Table 9.8). Other time points may be included as necessary, particularly for therapeutic vaccines. Blood volume limitations may both limit additional blood collections and preclude exhaustive testing for

an array of APP or cytokines, particularly for rodents. Blood collections after the first dose primarily identify induction of an acute inflammatory response, including induction of APP, which generally have a short response window (24–72 h) (Reagan et al., 2020). Understanding of the correct window of evaluation for the APP of interest is important, as different APPs have different peak response times. For rat studies in which multiple blood collections for clinical pathology and serology are required, half the rats in a group may be used for collection of one set of assays (e.g., hematology) and the other half for the remaining serology assays (e.g., APP or cytokines, clinical chemistry, and other biomarkers). Some markers, such as fibrinogen, may only be assessed at necropsy in rodents, due to blood volume limitations. Serum collection for assessing the antigen-induced immune response occurs prior to dose initiation and at the end of the dosing and recovery phases, and overlaps blood collection for clinical pathology, although interim serology time points may be collected depending on study needs. Hematology Vaccines induce an acute inflammatory response which may result in increases in white blood cells, particularly in vaccines formulated with adjuvants. Low magnitude and transient decreases in RBC mass or platelets may occur with robust innate immune responses (RSS personal experience). Findings should be compared to concurrent controls, even when baseline data (prior to dose administration) is

TABLE 9.8 Recommended Clinical Pathology Sampling Intervals for Commonly Used Species Hematology

Clinical Chemistry D APPs

Coagulation

Serology

Mouse

Day 4, necropsy

Day 4, necropsy

Necropsy

PID, necropsy

Rat

Day 4, necropsy

Day 4, necropsy

Necropsy

PID, necropsy

Rabbit

PID, day 4, necropsy

PID, day 4, necropsy

PID, day 4, necropsy

PID, day 4, necropsy

Monkey

PID, day 4, necropsy

PID, day 4, necropsy

PID, day 4, necropsy

PID, day 4, necropsy

Dog

PID, day 4, necropsy

PID, day 4, necropsy

PID, day 4, necropsy

PID, day 4, necropsy

Pig

PID, day 4, necropsy

PID, day 4, necropsy

PID, day 4, necropsy

PID, day 4, necropsy

APPs, acute phase proteins. PID, pre-initial dose (w7–10 days prior to study start but after acclimation).

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available, as hematology data can be influenced by study procedures and the inflammatory response may be quite variable depending on the adjuvant and the antigen. Typically, the increased white blood cell count (WBC) is evident within 3 days of the first dose administration and is often higher after the last dose. The elevations in WBC are due to higher numbers of neutrophils (heterophils in rabbits), monocytes, and large unstained cells (LUC, mainly lymphocytes and/ or monocytes) as well as relatively slight increases in eosinophil and basophil counts. Different adjuvants can result in different impact on the severity and character of hematology changes. For example, more severe hematology changes are observed in rats administered aluminumcontaining formulations in combination with CpG than with aluminum-containing formulations alone (personal experience). Effects of procedure-related stress or other procedural manipulations (e.g., anesthesia, repeated handling, injection-associated trauma, etc.) may also result in increased neutrophil, monocyte, and/or lymphocyte counts and should be taken into consideration when evaluating potential vaccine/adjuvant-related hematology changes. Clinical Chemistry The clinical chemistry panel typically includes various endpoints for assessment of general health and to detect evidence of liver, kidney, and gastrointestinal effects with assays for various APP. Creatine kinase (CK) may be included as a marker of skeletal muscle damage at the injection site, and may be used to differentiate acute phase responses associated with the vaccine components from those occurring secondary to physical trauma. However, CK is a nonspecific biomarker, and data suggests that there is not good concordance between local tissue injury (measured by CK) and the systemic inflammatory response (measured by the CRP response) (Green, 2015). Other indicators of muscle damage, including serum lactate dehydrogenase (LDH) or aspartate aminotransferase (AST), are even more nonspecific and in the absence of other confirmatory findings should not be used as the sole determinants for the degree of muscle damage.

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Coagulation The coagulation panel for vaccine studies typically includes routine coagulation times (prothrombin time [PT] and activated partial thromboplastin time [APTT]) and fibrinogen. Fibrinogen is a clotting factor and an APP and is present in higher basal concentration (generally 200–300 mg/dL range) in the blood to maintain normal clotting. Due to its high basal concentration and the upper limit of the assay, the maximal increase in fibrinogen is typically not more than 4- to 5-fold, making it a minor or moderate reactant for most species, including humans. However, fibrinogen is still an effective marker for inflammation/acute phase reaction in most species, and small increases are usually meaningful. Interestingly, very slight prolongation of APTT has been identified in vaccine studies in rabbits, monkeys, and rats. A mechanism for this change is not known, although a relationship to inflammation has been noted as well as CRP-related interference on assay results in rabbits, dogs, and humans (Cheng et al., 2009; Liu et al., 2018; Planty et al., 2020). Acute Phase Biomarkers Acute phase proteins (APPs) increase within 1–3 days of vaccine administration, so timing of blood collection is essential to obtain meaningful data. Other changes consistent with an acute phase response include increased globulins (typically a response that occurs later in the dosing phase), decreased albumin (typically a response that occurs earlier in the dosing phase), and increased fibrinogen. Changes in globulin and albumin are often quite small and may be overlooked or misinterpreted as biological variation. At the end of 3–4 weeks of recovery, albumin will typically have returned to control levels, but globulins may continue to be elevated in vaccinated animals. Because of the lack of sensitivity in the routine clinical chemistry parameters for monitoring an acute phase response, fibrinogen and speciesspecific APPs (see Clinical Pathology in Nonclinical Toxicity Testing, Vol 1, Chap 10) are routinely measured to assess potential vaccine reactogenicity. APP evaluation is often used to confirm an immune response (Reagan et al., 2020).

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Species differences in APPs must be considered when designing a vaccine regulatory toxicity study (Kilicarslan et al., 2013). For example, CRP is a sensitive indicator of inflammation and the most commonly utilized major APP for humans, rabbits, dogs, and NHPs, and is specifically recommended in WHO guidelines (Green, 2015; WHO, 2014). However, CRP is a poor marker of an acute phase response in rodents. Instead, the major APPs for rodents are a1-acid glycoprotein and a2-macroglobulin (Cray et al., 2009). APPs will also vary in timing of the response and duration following dosing, with major APPs (e.g., CRP, SAA, a1-acid glycoprotein, a2-macroglobulin) generally peaking at levels over 10-fold higher relative to the normal range within the first 48 h and then declining rapidly, whereas more moderate APPs (e.g., fibrinogen, haptoglobin) will have a slower onset of increase and decrease in conjunction with a less pronounced response (Sellers et al., 2020). Some APP will even have a negative response, decreasing over time (e.g., albumin, transferrin, transthyretin, retinol-binding protein, etc.). Care must be taken to confirm the sensitivity of the assay as slight vaccinerelated changes might not be distinguishable with some assays and data comparison among different methods or laboratories is not recommended. Changes or lack thereof in APP in vaccine studies is, however, of little significance to the overall study conclusions of the study so long as there is an immune response to the antigen and no unexpected systemic findings. The addition of cytokine panels may be desirable for certain modalities or with new adjuvants. However, the inclusion of cytokine panels in routine vaccine studies typically adds little value due to their short half-lives and high inter- and intra-animal variation. Therapeutic Vaccine Considerations Therapeutic vaccines, which contain other active components, may have changes related to those components or to the vaccine target. For example, immuno-oncology checkpoint inhibitors can elevate the WBC count, particularly lymphocytes. Therapeutic vaccines may also have intended alterations in clinical pathology parameters. For example, vaccines targeting cholesterol biogenesis should have reductions in serum cholesterol concentrations as

a therapeutic endpoint. Assessment of antidrug antibodies in the case of coadministered biological products is an important endpoint to understand exposure changes and nonclinical study findings.

8.6. Anatomic PathologydPost-life Evaluation Macroscopic and microscopic evaluation and assessment of vaccine study animals is similar in many ways to evaluation of general toxicity studies. There are often limited systemic effects, with most findings seen at the injection site(s) and surrounding local tissues (sciatic nerve, joints, etc.), the draining lymph nodes, spleen, and, less often, bone marrow. Macroscopic Observations and Sample Collection At the time of necropsy, visual evaluation of the injection site(s) and careful sample collection are key factors. Like many other tissues, the collection of the injection site should be done when feasible by referring to specific anatomical landmarks as well as any markings, although those markings are often less accurate than using anatomical landmarks. Animals with particularly loose skin, such as rabbits, may have significant slippage of the skin in relation to the underlying muscle, so will need to be carefully handled to ensure accurate evaluation of all tissue compartments related to the injection track. For IM injections, the injection site muscle and overlying skin with subcutaneous compartment should be collected and evaluated. For SC injections, the underlying muscle, overlying skin, and the subcutaneous compartment should be collected and evaluated. For ID administration, it is sufficient to collect the skin, dermis, and subcutis, without ensuring the collection of the underlying skeletal muscle. For intranasal instillation of vaccines, it is important to take multiple cross-sections of the nasal cavity, including mucosa, submucosa, and turbinates as well as nasal bones. Macroscopic findings at the site of the injection are uncommon. More common changes include pale, tan, or red discoloration of the site, while more rarely seen changes consist of newly formed nodular masses, thickening of the muscle or

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subcutis, or even ulceration or abscesses. For prophylactic and therapeutic vaccines, macroscopic observations of draining lymph nodes are limited as well, with enlargement, red discoloration, edema, or similar observations most prevalent if any findings are present. Occasionally, splenic enlargement may be identified due to pronounced lymphoid expansion or increased extramedullary hematopoiesis (rodents) as a part of the immune response. It is not recommended that the prosector extensively section through the injection site at necropsy to evaluate potential macroscopic findings. Instead, serial sectioning and examination should be reserved for the time of trimming, after tissues have been well fixed. This ensures that the muscle, skin and subcutis are fixed in place relative to the surrounding tissues, thus minimizing artifactual mechanical separation and allowing more accurate evaluation of the separate tissue compartments by the pathologist. It is not necessary to keep the skin and underlying muscle apposed but doing so facilitates the evaluation of potential needle tracks and effects of injected vaccines on adjacent tissues. If attempting to keep skin and muscle apposed, it is important to recognize that skin and muscle have different contraction rates when undergoing formalin fixation, which may result in significant displacement between surface markings for injection sites and actual underlying locations of vaccine delivery in the tissue compartment of interest (Figure 9.7). This can be mitigated by tying the skin to the underlying muscle via sutures or staples (Figure 9.8). The additional time taken at necropsy to do this is rewarded by improved processing time at trimming and improved accuracy in evaluation of findings by the pathologist. When sampling injection sites at necropsy, it is important to take broad margins, as the injection process, remarking of injection sites, variability in determination of anatomical landmarks, and variation in injection and sampling on an individual basis may combine to produce substantial variance in location of comparable injection sites among animals across a study. For smaller animals with injections in the hind limb or forelimb musculature, taking the entire limb and fixing it in formalin with the injection sites left in situ may give improved recovery of injection sites. An additional key component of evaluating

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FIGURE 9.7 Formalin fixation-associated skin contraction at hind limb. Skin contracts asymmetrically from the muscle. The blue circle represents the actual IM injection site and the arrow is pointing to the associated dose site marking. If muscle sections were collected from only the marked site at trimming, the actual site of dose delivery would not be included. This shift was w3–4 cm.

FIGURE 9.8 Securing skin to underlying muscle. (A) Staples used to appose cut edge of skin and muscle. (B) Excised epaxial muscle block with two dose sites. Skin affixed to underlying muscle to reduce movement.

injection sites is ensuring that multiple crosssections are sampled and viewed by the pathologist, giving a robust view of the entire injection site area and addressing the potential issue of variability in site identification. Three to five cross-sections of the region for histological evaluation (depending on the animal size) are recommended as there will be variation in the extent of the change depending on whether the

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FIGURE 9.9 Successful identification of injected material (black ink) in only three of five sections cut through the dose site. Note that injecta varies in depth and amount present in each section.

evaluation is at the center of the injection site or along the margins (Figure 9.9). This is true for both SC and IM injections, though less so for ID vaccine administration. In rodents, three cross-sections through the injection site, with each separately processed to slide, is considered adequate due to their smaller muscle mass. In nonrodents, it is suggested that five crosssections of the injection be performed, incorporating the putative center of the injection site as well as two separate samples lateral to the site (distal and proximal if on a limb) (generally at 0.5–1 cm intervals). Inclusion of the deep margins of the muscle at the injection site is often helpful in evaluating the full extent of the local changes, and it is not uncommon for much of the injected material to be delivered to the deep edge of the muscle, particularly if needle length was not considered fully prior to administration (Figure 9.10). Evaluation of adjacent tissues is important in understanding potential effects of vaccine spillover into these nearby off-target tissues, whether it is retrograde spread along a needle track or inadvertent delivery into other tissue compartments. For intranasal vaccines, collection of multiple levels of the nasal cavity at standard intervals allows the pathologist to evaluate effects on both respiratory and olfactory epithelium, as well as potential effects on nasal-associated lymphoid tissue (NALT), submucosa, turbinates, and other structures (Pereira et al., 2011; Randall et al., 1988; Young, 1981). Tonsillar tissue (where present), the nasopharynx/pharynx, larynx, trachea, draining lymph nodes for the respiratory tract (e.g., [trachea]bronchial lymph nodes), lungs, and the brain (particularly the olfactory lobes) are additional tissues that should be

FIGURE 9.10 Injection site with material at the deep margin. In this instance, the test material was delivered predominantly in the fascia and deeper muscle, and deeper sections revealed more extensive microscopic findings. H&E stain. 0.5.

collected and evaluated histopathologically. In rodents (which lack tonsils), NALT located on the ventral aspects of the caudal nasal cavity at the entrance of the nasopharynx is the equivalent of the Waldeyer’s ring in humans and can be assessed on nasal cavity sections. In monkeys, NALT is present throughout the nasal cavity, and tonsillar tissue is present within the nasopharyngeal septum (Chamanza & Wright,

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2015). Dogs have abundant tonsillar tissue that can be sampled separately, but dogs are rarely used in IN vaccine studies since the presence of a transverse lamina and olfactory recess in the nasal cavity in this species limits their utility in predicting brain delivery-associated effects. Skeletal muscle damage and microscopic changes in the local area around injection sites, such as the sciatic nerve or joint in IM hind limb injections, are commonly observed after an IM injection. Such findings may provide an interpretive dilemma if they are more frequently identified in the vaccine groups (Figure 9.11). Not only is collection of these local tissues for evaluation of potential vaccine-associated effects important, but also collection of appropriate control tissues as well. To provide appropriate controls and rule out potential questions regarding systemic effects on the skeletal muscle, nerves or joints, it may be of value to collect examples of these tissues from sites distant to the site of vaccine administration. For example, if both the hind limbs were used for injection, collection of a section of tibial nerve (a major distal branch of the sciatic nerve) and/ or forelimb skeletal muscle, joints, and nerves or, for smaller animals, the entire forelimb, may be beneficial. These additional tissues can be held in reserve for evaluation if needed to investigate findings in the routinely processed tissues or may be processed to slide proactively and evaluated as part of the study.

FIGURE 9.11 Rat, quadriceps femoris. Sciatic nerve surrounded by injecta and inflammation. H&E stain. 20.

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Microscopic Evaluation A typical vaccine study will have only limited microscopic findings outside of the administration site, regional (draining) lymphoid tissues, or systemic lymphoid tissues (e.g., spleen). It is rare for there to be systemic findings, particularly in prophylactic vaccine administration. In therapeutic vaccines, particularly those with other active components in the treatment regimen, systemic findings may be more prevalent. Adjuvants may also produce findings in the lymphoid tissues, particularly draining lymph nodes and spleen. Comparison of microscopic findings of the vaccine formulation to a saline control is important in understanding the overall effects of the clinically administered formulation, but it is also valuable for the researcher and reviewer to understand the degree to which the chosen adjuvant contributes to microscopic findings. When evaluating microscopic findings, it is important to differentiate effects of the expected antiantigen response from effects produced by adjuvants. This may be managed by comparing changes in test article groups to those in an adjuvant control group. If an adjuvant control is not available, historical control data for comparative studies in which the adjuvant was tested in isolation may be used as a yardstick. This is of particular importance in instances where a development team may be able to use different adjuvant formulations to mitigate tissue effects. Injection SitedMicroscopic Findings The injection site is the location of most microscopic findings in vaccine studies. Thus, careful evaluation by the pathologist of the injection site is a critical component of any study in which histopathology is performed. Most vaccine studies have multiple doses administered over the period of two or more weeks. Determining how many injection sites should be evaluated microscopically should be based on the study design. If the dose was administered at multiple sites on each dosing day, then it may be adequate to evaluate a single site (while collecting all sites). However, evaluation of all sites is generally recommended as it provides a more thorough assessment of the range of local responses to the vaccine and it minimizes the likelihood that inaccurate sample collection will prevent a thorough tissue analysis. If a full dose was

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administered in alternating sites, then evaluation of all sites gives a temporal understanding of the injection site findings, as microscopic findings at an individual site may range from acute to chronic in nature. For therapeutic vaccines, different components of the regimen may be administered at different sites (e.g., pDNA at one site, viral vector at another, biologic adjuvant therapy at another, etc.). Injection sites for each unique component should be evaluated independently. Regardless of how many injection sites are evaluated, multiple levels of each injection site should be reviewed to identify the microscopic characteristics and severity (Figures 9.9 and 9.12). The findings from these levels should generally not be separately detailed, but instead combined to form an aggregate description and interpretation of the findings in the overall injection site. If microscopic findings are not identified, particularly with formulations where findings would be expected (e.g., those containing adjuvants), it may be of value to either section deeper into the block (in rodents) or retrim wet tissue to ensure complete categorization of injection site findings.

Most injection sites include some degree of inflammation or inflammatory cell infiltration, muscle degeneration or necrosis, edema, hemorrhage, and/or fibrosis, depending on the timing of necropsy postadministration. When large volumes of material or persistent adjuvants are used, foreign material and even cavities may be seen where the injecta were removed during processing. Even IM administration of saline may result in a small amount of inflammation, often comprised of scattered small aggregates of macrophages or other mononuclear inflammatory cells with or without individual myocyte degeneration or necrosis, edema, hemorrhage and/or fibrosis (Figure 9.13). These modest inflammatory reactions may be exacerbated by large volumes, with increased myocyte degeneration or necrosis, more extensive inflammatory infiltrates, and other changes induced by the acute trauma and pressure of the injection on the surrounding tissue, or by variation in pH or osmolality of the formulation. Trauma from the needle insertion will also create a thin track through the muscle, subcutis, and dermis along which inflammatory cell infiltrates, fibrosis,

FIGURE 9.12 Rat, quadriceps femoris. Multiple adjacent sections presented in order, demonstrating the variable nature of findings within each section. H&E stained. 0.5.

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FIGURE 9.13 Intramuscular (IM) injection site with saline. Hemorrhage, myofiber degeneration and necrosis, and slight inflammation present. H&E stain. 10.

hemorrhage, and myofiber degeneration or necrosis may be seen. Some antigens are highly immunogenic and may cause notable inflammation at the injection site, even when formulated without an adjuvant. Most vaccine antigens present with microscopic changes at the injection site like those identified with saline administration. The implementation and use of consistent diagnostic terminology across studies, programs, and organizations will improve comparisons of findings. When evaluating injection sites, it may be helpful to subcategorize the injection site tissue compartments or, at a minimum, use modifiers to clearly identify which compartments are affected by a given finding. For an IM injection, this would consist of evaluating and compartmentalizing the injection site into skin, subcutis, and muscle. Separate evaluation of each compartment will allow the pathologist and reader to readily identify the affected tissues and survey for off-target effects. One approach to terminology is to use single, simple diagnoses

such as “inflammation, mixed cell”; “inflammation, mononuclear cell”; “inflammation, neutrophil”; “inflammation, granulomatous”; or “infiltrate, mononuclear cell” (or lymphocytic or histiocytic). Careful selection of diagnostic terminology helps to limit the findings recorded in the data capture system to diagnoses most likely to effectively define the safety profile, with limited use of other diagnoses (e.g., fibrosis) to differentiate between test groups. The microscopic details of these findings would then be specified and/or elaborated on in the text of the study report; for example, “inflammation, neutrophil” is characterized by not only neutrophils, but also by muscle degeneration/ necrosis, edema, hemorrhage and rare other inflammatory cells. Another potentially useful approach (especially to external regulatory reviewers) to injection site terminology would include the use of a limited number of potential microscopic findings, including inflammation or infiltration, degeneration or necrosis, hemorrhage, fibrosis, edema, and, if appropriate,

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foreign material. These would be further defined by tissue sublocation and, in the case of inflammation, the type of cell affected (histiocyte, neutrophil, mononuclear cell, etc.). Then, within the text of the report, a consolidated and integrated interpretation of the constellation of findings may be made. No matter which approach is utilized, clear description and characterization of any findings within the text of the report is extremely helpful in ensuring that consistent terminology is used and the interpretation is understood by both the pathologist and the reader. When inflammation includes many macrophages, it is suggested that macrophage or histiocyte be used as modifiers for the finding of inflammation rather than granulomatous (since the latter is evocative of infectious etiologies such as fungal or mycobacterial infection). However, some formulations which contain oil or crystalline material may induce a foreign body response, and in such cases, the pathologist may opt to use the modifier granulomatous (Sellers et al., 2020). The severity grading should take into consideration the amount of tissue affected and the intensity of the finding. For example, if three sections of the injection site were evaluated microscopically and each affected, the severity grade should be based on the average severity across all three sections. The presence of inflammation in multiple sections is an indicator of an overall increased area of tissue affect. In this, as in all grading, consistency in application of criteria for severity within the study on the part of the study pathologist is critical. The criteria for the severity grades are typically described within the pathology text. Additionally, species- or sex-specific anatomic features of the injection site must be taken into account when interpreting findings. For example, in rats the severity of findings is often greater at the injection site in females than in males simply because of their smaller muscle size. In larger animals, a finding may seem quite pronounced but affect only a fraction of the overall muscle tissue. As mentioned previously, specific adjuvants will cause microscopic findings at the injection site regardless of the accompanying antigen. Aluminum-containing formulations are common and consistently result in inflammation at the injection site. Nonaluminum-containing adjuvants will variably incite inflammation at the

FIGURE 9.14 IM injection with inflammation tracking along a fascial plane. H&E stain. 4.

injection site. Inflammation and edema tracking along the muscle fascial planes may occur with some adjuvants, presumably because they are nonviscous and nonparticulate (Figure 9.14). Acute (1–2 weeks postdose) findings at aluminum-dosed IM or SC injection sites are characterized by infiltrates of macrophages (generally without multinucleated giant cells), neutrophils (termed heterophils in rabbits), and eosinophils, with variable degrees of hemorrhage and individual muscle cell degeneration/ necrosis (Figure 9.15). When aggregates of

FIGURE 9.15 Intramuscular (IM) injection site with acute muscle necrosis, neutrophilic and lymphoplasmacytic inflammation and acute hemorrhage. H&E stain. 10.

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granulocytes (neutrophils/heterophils and/or eosinophils) are present within the lesion, they generally resolve over time; their presence should be interpreted as evidence for bacterial contamination of the injection site, although they may represent an antigen effect and should be assessed with that possibility in mind. Basophilic to gray granular material (interpreted to be aluminum and often coded in data entry as “foreign material, basophilic”) is typically present both within infiltrating macrophages and extracellularly at the injection site (Figure 9.16). Macrophages may surround and infiltrate into aggregated areas of extracellular adjuvant material, resulting in tissue displacement, often with small numbers of individual

FIGURE 9.16 Injection track with alum adjuvant. Macrophages and lightly basophilic granular adjuvant material present between muscle fibers. H&E stain. 4.

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degenerating myocytes around the edges where the material has compressed the muscle. In the chronic stage, there will be fewer granulocytic cells and the adjuvant-containing macrophages will often be surrounded by lymphocytes and fewer plasma cells. There may be some slight fibrosis associated with areas of inflammation. These changes are considered indicators of repair. Adjuvant material and the associated macrophages are usually present at recovery, although often slightly less prominent, with aluminum adjuvant material persisting for at least 2 months following injection (Verdier et al., 2005). Administration of oligodeoxynucleotide (ODN) adjuvants, which drive TLR-9 activation, produce histological changes similar to saline. However, when ODN adjuvants are coadministered with aluminum, the microscopic changes are generally more severe than for aluminum alone. Lipid-based adjuvants, such as squalene and other oils, may also produce areas of macrophagic inflammation and pockets of injected material that persist and displace the surrounding muscle (Figure 9.17). With IM administration, it is common to see extension of the injection material outside the region of administration, whether by extension along fascial planes or as a result of inaccurate injection placement. Particularly with rats and mice, inadvertent administration of material into an incorrect location is not uncommon, including unintentional delivery of the vaccine into the wrong muscle bed (e.g., administration into the gastrocnemius rather than the caudal thigh muscles [biceps femoris or semitendinosus]). Because of this, regional structures may FIGURE 9.17 IM injection with lipid-based vehicle. Central area of foreign material surrounded by foreign body macrophages, neutrophils, and lymphoplasmacytic inflammation. Fibrosis and scattered mineralization are also present. H&E stain. 4.

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material, may be seen within the joint capsule. In the pathology report, these findings may be described as an extension of the injection site to differentiate it from an immune-mediated lesion.

FIGURE 9.18 Extension of IM injection site inflammation to periarticular region of the knee joint, rat. Inflammatory cell infiltrates are present adjacent to the femur, just proximal to the knee joint. H&E stain. 5.

be at risk for needle-related damage or vaccineassociated inflammation. The extent of this effect will vary depending on the site of administration, the size of the test species, and the experience of the technicians administering the material. In the hind limb, inflammation around the sciatic nerve is common regardless of the species (Figure 9.11), though this may be mitigated to some degree by selecting needles of an appropriate length and choosing a suitable approach for injection. In smaller species, like the rat and mouse, periosteal or periarticular extracapsular inflammatory infiltrates may be evident around bones of the knee joint (Figure 9.18), and rarely, inflammatory infiltrates, with or without injection FIGURE 9.19 Lymph node with aggregates of macrophages containing basophilic adjuvant. H&E stain. 20.

Draining Lymph Nodes and Spleen Microscopic evaluation of the draining lymph nodes may be challenging simply because of the variability of findings observed in lymph nodes and inconsistencies in organ orientation at tissue embedding and sectioning, particularly for rodents. This variation should be taken into consideration when interpreting the findings as there may be extensive variation in apparent density of lymphocytes and overall size of lymph node and subcompartments due to nothing more than variance in sectioning. The most common finding in draining lymph nodes is increased lymphocyte cellularity of the germinal centers in association with antigens with or without adjuvants, although adjuvants may increase the severity of this finding. With some vaccines, increased sinusoidal plasma cells (mature and immature) may be evident in the draining lymph nodes (personal experience). Increased germinal center cellularity may also be present in the spleen, although this is not as common as in the draining lymph nodes and may vary with the vaccine formulation. In animals administered aluminumcontaining formulations, the draining lymph nodes will have aggregates of macrophages containing basophilic to gray granular material reflecting accumulation of aluminum transferred from the injection site (Figure 9.19). As with other changes, the regions of the lymph node within the histological section can dramatically impact

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the apparent severity of macrophage aggregates; although aggregates may form within the cortex, the sinuses tend to have the greatest accumulation of these aggregates. While germinal center changes indicative of an active immune reaction typically recover within 4 weeks from the last dose, the accumulation of aluminum-containing macrophages in the node is often unchanged from that seen at the end of dosing phase (as at the injection site). Other particulate or lipidbased adjuvants may present with similar lymph node findings, depending on the distribution of the adjuvant to the regional lymph nodes and the antigenicity of the particular adjuvant. Other Tissues Vaccine-related microscopic findings in other tissues are uncommon for prophylactic vaccines. There may be exacerbations of species-specific background findings as a result of study-related stress, as has been seen in rabbits with increased incidence of cardiac findings (Sellers et al., 2017). If there is a systemic response to a prophylactic vaccine, it may present as an overall increase in inflammatory cell infiltrates in tissues, though this is rare. However, therapeutic vaccines (for which the target is typically an endogenous protein) may be associated with a wider distribution of changes consistent with an auto-immune response (i.e., systemically increased inflammatory cell infiltrates into tissues). Most commonly, these findings are present in animals and humans administered immune checkpoint inhibitors, for which there may be underlying antiself-antibodies (Hughes et al., 2015; Iwama et al., 2014; Selby et al., 2016). However, understanding the relationship of treatment to inflammatory infiltrates into tissues may be challenging, as the primary species for testing therapeutic vaccines is the NHP, which have notable inter-animal variation in the background incidence and severity of inflammatory cell infiltrates (particularly with macaques of Chinese origin) (Kozlosky et al., 2015). Additionally, studies using NHP generally have fewer animals per group. In these cases, the historical control database may be of notable value in interpreting the study findings. Combined Safety–Efficacy Studies The microscopic endpoints for nonclinical combined safety and efficacy studies incorporate

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both an evaluation of the injection site and a broad range of tissues as well as a specific evaluation of the targeted tissues for any presumed efficacious response. It is important to include negative control groups given vehicle alone and adjuvant alone that both received and did not receive the infectious agent to permit differentiation of any formulation-related changes (e.g., adjuvant-related immune activation or inflammation), as well as at least one control group given the vaccine that did not receive the infectious agent to allow evaluation of any potential safety concerns for the vaccine in isolation. For prophylactic vaccines, tissue evaluation is similar to that in a normal safety study. However, any tissues of specific interest in assessing the efficacy of the treatment may be more widely sampled or reviewed using a scoring system specific to determining efficacy. For example, in ferrets dosed with an intranasal influenza vaccine with subsequent evaluation of efficacy by inoculation with influenza, the careful evaluation of a wide range of tissues from the upper and lower respiratory tracts is recommended, to include the nasal cavity and turbinates, oropharynx and nasopharynx, multiple sections of trachea, and all lung lobes. Study-specific criteria and scoring may be applied to assess disease progression, or reliance on the standard histopathological scoring scheme may be considered sufficient.

9. SPECIAL CONSIDERATIONS FOR THERAPEUTIC MODALITIES 9.1. Therapeutic Vaccines Therapeutic vaccines use an assortment of modalities, and the considerations of those modalities are like prophylactic vaccines. Effective therapeutic vaccines generally require induction of both humoral and cellular immune responses. Therapeutic vaccines have been developed primarily for the treatment of cancer (Morse et al., 2021). However, such vaccines also have been investigated to treat other conditions including chronic diseases such as hypercholesterolemia (Toth et al., 2020), Alzheimer’s disease (Hull et al., 2017; Mantile & Prisco, 2020), and asthma (Licari et al., 2017); addictions to drugs such as nicotine, cocaine, and opioids (Fraleigh et al., 2021; Heekin et al., 2017; Oliva et al., 2019); or persistent infections like HIV

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(de Jong et al., 2019; Leal et al., 2018) and HPV (Yang et al., 2016). Similar considerations apply to both cancer and noncancer indications. Therapeutic vaccines should ideally have durable responses, such that there is no disease recurrence or immune escape. For vaccines targeting endogenous proteins, the vaccine will require coadministration with immune checkpoint inhibitors to break immune tolerance, such as anti-PD1 (programmed cell death protein 1) or anti-CTLA4 (cytotoxic T-lymphocyte-associated protein 4) antibodies. Inclusion of immune checkpoint inhibitors will increase the risk of nonantigen-related autoimmunity. Cancer vaccines work to eradicate neoplasms through activation of a patient’s immune system against tumor cells. Patients are vaccinated with antigens that induce TAA-specific active (humoral or cell-mediated) immunity to destroy tumor cells. TAAs may be against normal endogenous proteins that are overexpressed in the cancer (e.g., prostate specific antigen [PSA]) or may be a neoantigen that is either common to a particular type of tumor or unique to a patient’s tumor. A multi-antigen approach is likely key to the success of therapeutic vaccines against tumors. To understand potential off-target immune responses, it is essential to also evaluate the antigen as it might be presented in association with MHC. To do this, 8–15 amino acid fragments of the antigen can be assessed for homology to other endogenous proteins (e.g., BLAST [Basic Local Alignment Search Tool] search) to better predict unexpected off target effects. Given that the targets for therapeutic vaccines are often endogenous molecules, there are several considerations required before starting nonclinical safety studies. First, the target antigen should be expressed in the nonclinical species, and it should have high protein homology to the human protein. Second, the tissue distribution and antigen expression should be comparable between the human and nonclinical species. This equivalence is explored by such methods as tissue cross-reactivity studies and molecular quantification in homogenized tissues (see Special Techniques in Toxicologic Pathology, Vol 1, Chap 11). Third, homologies to other closely related proteins should be assessed for potential off-target immune effects. Fourth, antigens must be available for immune targeting.

Fifth, the nonclinical species should be responsive to any coadministered drugs/biologics or have a suitable surrogate. For therapeutic vaccines, NHP are more commonly used as sequence homology to the targeted self-antigen is often greatest and coadministered biological compounds may not have pharmacodynamic effects in other nonclinical species. Nonclinical safety studies are ideally performed in a species that expresses the target antigen, although this may not always be possible. The study should be designed to reflect one clinical cycle, with components ideally administered at the highest anticipated clinical dose.

9.2. Other Therapeutic Vaccine Strategies There is an assortment of vaccine strategies that include oncolytic virus therapies, antiidiotype vaccines, and cell-based vaccines. Nonclinical studies for all of these approaches will require close interactions with regulatory authorities and using one or more relevant guidelines. Nonclinical efficacy and safety studies should be performed in animal species which demonstrate a biological response to the therapeutic similar to that expected in humans. In some cases, the use of nonstandard species, such as genetically modified animals, may be appropriate. For additional details on approaches to cell therapies, please refer to guidance documents, such as Guidance for Industry Preclinical Assessment of Investigational Cellular and Gene Therapy Products (FDA, 2013).

9.3. Oncolytic Virus-Based Therapeutic Vaccines Repeat dose toxicity studies for oncolytic viruses may require studies in syngeneic tumor-bearing animals, most commonly mice. These models are thought to best indicate the potential toxicity of an oncolytic virus as it is anticipated that there will be increased and prolonged exposure to virus from replication in and release from infected tumor cells. The route of administration should reflect that anticipated in the clinic (e.g., intratumoral vs. IV injection). These studies do not need to be performed entirely under GLP regulations, although all

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aspects of the study that can be conducted in compliance with GLP (e.g., clinical and anatomic pathology) should be performed in such fashion. It may not be necessary to perform studies in nontumor-bearing animals, depending on the modality and target. Discussions with regulators are essential to best determine the nonclinical safety package for any oncolytic virus therapies. Another consideration with oncolytic virus therapies is that the susceptibility to viral infection may vary notably among species. In some cases, surrogate viruses may be needed to demonstrate proof of concept. Further, vaccines or viruses that express cytokines or other modulators must have efficacy in the test species and may also require expression of species-specific surrogates (e.g., mouse IL-2 in place of human IL-2). The lack of genetic heterogeneity in the mouse strains used in most studies and the divergence in immune responses between humans and mice may limit predictivity of these mouse models to treat human cancer. However, the use of different models and varied in vivo approaches can give useful data for moving product candidates into human clinical trials. For oncolytic virus therapies or therapies with coadministration of another biological product (e.g., a CTLA4 or PD-1 inhibitor), inclusion of antidrug antibody (ADA) assays may be beneficial when attempting to interpret study data. The development of ADA to any product is anticipated to reduce its efficacy or may result in ADA-related findings (see Biotherapeutic ADME and PK/PD Principles, Vol 1, Chap 4). Further, inter-animal variation in ADA may impact the toxicity profile. This data must be interpreted together with toxicokinetic data for those molecules, as well as PD markers (such as increased circulating or intratumoral lymphocyte numbers/types with immune checkpoint inhibitors). There are safety considerations with this modality which may need to be addressed nonclinical and/or clinically, such as assessing neurovirulence, genomic integration, biodistribution and shedding, and safety in immunosuppressed patients.

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10. NONCLINICAL TOXICITYdDETERMINING ADVERSITY Because prophylactic vaccines are intended for wide use in a healthy population, there is little or no tolerance for vaccine-associated adverse events (McNeely et al., 2014). In prophylactic vaccines, local tolerability is often the primary endpoint. The most common findings in humans relative to tolerance with prophylactic vaccines are discomfort or swelling at the injection site and sometimes fever or malaise. These effects are mostly minimal to mild and resolve quickly (Herve´ et al., 2019). Significant adverse events are rarely identified during the nonclinical stage of assessment, but such effects may become evident during human clinical trials or at the postmarketing stage. In nonclinical studies, the primary findings are at the injection site and/or related to immune activation: inflammation at the injection site (particularly in vaccines containing adjuvants) and increased lymphocyte numbers (usually in germinal centers) in lymphoid organs, particularly the draining lymph node. Accumulations of macrophages containing aluminum may be present at the injection site and draining lymph nodes in aluminum-containing formulations. The injection site inflammation may be robust, particularly when formulated with an immune activator, and may sometimes be associated with focal myofiber degeneration or necrosis. Therapeutic vaccines may have similar findings at the injection sites and in lymphoid tissues, although target- or modality-related findings may also be present in other tissues. When unaccompanied by clinical signs (other than transient elevations in body temperature and redness at the injection site) and with evidence of reversibility (i.e., no evidence of long-term impairment, excessive scarring/ fibrosis, progression), injection site findings are often considered nonadverse. This is aligned with U.S. FDA-CDER (FDA, 2005): “[A]s a general rule, an adverse effect observed in nonclinical toxicology studies used to define a NOAEL for the purpose of dose-setting should be based on an effect that would be unacceptable if produced by

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the initial dose of a therapeutic in a phase one clinical trial .”. Additionally, clinical pathology findings, including acute phase protein concentrations, are generally not considered adverse but instead support determinations of adversity in conjunction with other study findings (Ramaiah et al., 2017). Recent “best practice” recommendations for “Determining, Communicating, and Using Adverse Effect Data from Nonclinical Studies” provides an excellent review of relevant literature on adversity and, along with other recent publications, presents some parameters for defining adversity in nonclinical studies (Kerlin et al., 2016; Pandiri et al., 2016; Ramaiah et al., 2017). Occasionally, however, a vaccine or adjuvant development candidate can be associated with atypical findings that may be considered adverse (e.g., any effect that impairs the animal’s physical or physiological function (Kerlin et al., 2016)). Ulceration of the skin overlying the IM injection site, severe edema or abscessation, extensive necrosis, progression rather than reversibility, evidence of systemic toxicity, unexpected organ findings, etc. may require additional efforts and discussions to determine mechanism and adversity. However, even with atypical findings, defining adversity at the injection site may be challenging. For example, some formulations containing potent immune potentiators cause ambulatory difficulty. In defining adversity, how much difficulty is too much or too long? Ultimately, the decision on clinical tolerability of any vaccine, prophylactic or therapeutic, will be based on a risk:benefit analysis. Since a precise threshold for local tolerability may be difficult to define, it is often helpful to place nonclinical study findings in context by comparison to other marketed products. There is available published literature characterizing local injection site findings for various vaccines and long-acting parenteral depot formulations (Paquette et al., 2014).

11. NONCLINICAL TOXICITY AND HUMAN TRANSLATION Many of the common vaccine-related adverse events in humans are reliably identified in nonclinical species and include redness/swelling/pain at the injection site, swollen lymph nodes, and increased body temperature. General malaise

identified in people may manifest in nonclinical species as decreased food consumption and/or decreased body weight gain. Most of these findings, as in humans, are low grade and transient. While nonclinical study findings often reflect clinical reactogenicity, nonclinical studies may not be predictive of rare immune-mediated adverse events. Such events are very uncommon, and often only become evident after hundreds of thousands or millions of doses have been administered to humans. Nonclinical studies (and also clinical trials) are not powered to identify rare events, and the immune responses in animals do not necessarily translate to humans.

11.1. Autoimmunity Most rare events are considered to be due to induction of autoimmunity, potentially through molecular mimicry; however tissue infection by modified live microbes may also result in immune-mediated tissue damage. The most common reported auto-immune event associated with vaccines in humans is Guillain-Barre´ syndrome (GBS), a rare immune-mediated polyneuropathy caused by antibodies that cross-react with gangliosides in axons and myelin sheaths (Principi & Esposito, 2019). GBS most commonly occurs within 4–6 weeks of microbial infections (Rodrı´guez et al., 2018). Patients who develop GBS may have underlying genetic susceptibility (Rodrı´guez et al., 2018; van den Berg et al., 2014). GBS has widely been reported to be caused by the influenza virus vaccine, but many published studies examining the risk of GBS after influenza vaccination (as well as other vaccines) have found little to no increased risk (Haber et al., 2009). In contrast, the risk of developing GBS after influenza infection is several times greater than the risk after influenza vaccination (Vellozzi et al., 2014). It is for this reason, however, that assessment of nerves is an important component of the safety evaluation for a nonclinical vaccine study. Often the vaccine administration in the hind limb results in inflammation at or around the sciatic nerve through local extension. Therefore, it is wise to include other myelinated nerves in the tissue collection from sites distant to the injection site. Note that collection of forelimb nerves may have background axonal degeneration in rats, presumably due to restraint (Pardo et al., 2020). Less frequently, transverse

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myelitis has been reported with vaccination and has been associated with viral and bacterial infections (less commonly fungal or protozoal infections) (Rodrı´guez et al., 2018; van den Berg et al., 2014). An unusual example of a vaccine-associated adverse event was identified after use of the monovalent H1N1 monovalent influenza vaccine Pandemrix. This vaccine used in the 2009–10 influenza season and was associated with narcolepsy in vaccinated individuals (Weibel et al., 2018). Although an auto-immune cause has been postulated, the mechanism for the increased rate of narcolepsy in vaccinated individuals has not been determined (Sarkanen et al., 2018; Wallenius et al., 2019).

11.2. Hypersensitivity Immediate (Type 1) hypersensitivity reactions are rare after vaccination, estimated to occur approximately in 1.3 of every 1,000,000 individuals vaccinated (McNeil & DeStefano, 2018). Hypersensitivity reactions are generally IgEmediated and infrequently are related to antigenic components of the vaccine. Immediate hypersensitivity reactions caused by IgEmediated mast-cell, eosinophil, and basophil degranulation have been reported to occur as a result of preexisting allergies to excipients or residual components (e.g., preservatives/antimicrobials, albumin, egg proteins, gelatin, latex, and yeast proteins) (https://www.vaccinesafety. edu/do-vaccines-cause-hypersensitivity-reactio ns/; https://www.vaccinesafety.edu/potentialallergens-in-vaccines-per-0-5-ml-dose/). Non-IgE-mediated mechanisms of acute hypersensitivity may also occur and may be referred to as “pseudoallergy.” Clinically, allergy and pseudoallergy are indistinguishable. NonIgE-mediated mast cell degranulation may be triggered by complement components (C3a and C5a) or by direct binding to mast cell receptors by immunoglobulins. Complement activation of pseudoallergy (CARPA) has been described for drugs containing polysorbate-20 or polysorbate-80 (PS-80), phospholipids, and PEGylated (polyethylene glycol [PEG]conjugated) compounds including liposome drug formulations (Stone et al., 2019; Wenande & Garvey, 2016). Dogs may be particularly sensitive to PS-80-mediated hypersensitivity reactions, thus development of vaccines containing

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PS-80 should use a species other than dog for safety testing (Qiu et al., 2013). Induction of CARPA may be due to an assortment of causes, including lipid surface charge, size, and for pegylated (PEG) lipids in LNPs, PEG itself (De´zsi et al., 2014). Underlying PEG IgM antibodies have also been suggested to drive anaphylactoid reactions. CARPA-like reactions are unlikely to be identified in routine nonclinical toxicity studies. If there is a suspicion that CARPA is a risk, specialized studies for complement activation with vaccine components in mice, rats, or pigs should be implemented (De´zsi et al., 2014). Such models evaluate changes in blood pressure and cardiac output, as well as biomarkers of complement activation after vaccine administration. IgG-mediated mechanisms of hypersensitivity have been reported in the guinea pig with vaccination (Al-Laith et al., 1993), but they are not considered reliable models to identify hypersensitivity risk.

11.3. Thrombotic Thrombocytopenia Thrombotic thrombocytopenia (TTP) is a rare and serious syndrome characterized by vascular thrombosis and low platelet count in association with low fibrinogen and increased D-dimers. This syndrome is most commonly reported in pregnancy and with oral contraceptives and hormone replacement therapy. Nonhormonal drug-related causes for TTP are uncommon, with heparin therapy being the most well characterized (Goor et al., 2002). Patients who develop TTP have generated antibodies against the platelet factor 4 (PF4) complexed with heparin. This antibody-heparin-PF4 complex both drives platelet clearance by the spleen (leading to thrombocytopenia) and platelet activation (leading to thrombosis). A form of TTP, termed vaccine-induced thrombotic thrombocytopenia (VITT) has been seen rarely in association with adenoviral vector-based COVID-19 vaccines roughly 7 days after vaccination, typically in younger women (Cines & Bussel, 2021). VITT is different from TTP, in that the thromboses tend to be in the cerebral venous sinus and the splanchnic bed rather than in deep veins and the lungs. In VITT patients, there is evidence of circulating anti-PF4 antibodies, which suggests a similar mechanism to heparin-associated TPP (Arepally & Ortel, 2021; Huynh et al., 2021; McGonagle et al., 2021; Sharifian-Dorche et al.,

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2021). There is no evidence that the anti-PF4 antibodies in these patients are the result of anti-S protein immune responses to the vaccine. An explanation for how the SARS-CoV-2 adenoviral vaccine initiates VITT and what underlies susceptibility is an area of active investigation.

12. VACCINES AND THE ANTIVACCINE MOVEMENT For as long as there have been vaccines, some individuals have feared them and rejected them as unsafe (aka “anti-vaxxers”). Vaccineassociated serious side effects occur, albeit rarely, and include life threatening allergic reactions, febrile seizures, neurologic disorders (such as of Guillain-Barre´ syndrome [GBS]), and myocarditis (smallpox vaccines) (Halsell et al., 2003). Certain vaccines may also cause a minimal disease, with the most notable being the variola virus vaccine to protect against smallpox. Vaccine hesitancy has resulted in disease outbreaks in the last decade by pathogens which had been considered, for the most part, to have been eradicated. These include measles, mumps, and Neisseria meningitis. Without widespread vaccination, however, the ability to reach a community immunity (aka “herd immunity”) is key. Community immunity occurs when a sufficient percent of the community is immune to an infectious disease; this percentage will vary by the infectious disease and are in part driven by how easily the pathogen is spread between individuals (Fontanet & Cauchemez, 2020). Highly infectious diseases require a much higher percentage of community immunitydfor example, community immunity against measles is estimated to be achieved when >90% of the population is immune; for COVID-19, the percentage has been estimated at w 70%, but likely needs to be higher for good community protection (Aschwanden, 2020). In an effort to promote vaccination, the scientific community continues to try to educate people, resulting in slogans such as “vaccines cause adults.” The true cause of vaccine hesitancy is, however, driven by mythology admixed with historical events and the fear of vaccine-related side effects. Perhaps one of the most important historical

causes for vaccine fear in the United States was the “Cutter Incident” (Offit, 2005). In 1955, Cutter Laboratories was producing Salk’s formaldehyde-inactivated polio virus vaccine, which was administered to children by IM injection. It was noticed that children administered this vaccine were developing paralysis starting in the vaccinated arm, rather than the typical polio progression which started in the legs. It was soon discovered that the method of inactivation of the virus was defective, but not before 200,000 people received the vaccine leading to polio in 40,000 people, of which 10 ultimately died and hundreds of others suffered lifetime neurological deficits. Salk’s inactivated virus was replaced by Sabin’s attenuated vaccine, which was administered orally. Additional issues developed with the polio vaccine when it was discovered that the monkey cells used to grow the vaccine were contaminated by the simian virus SV40, which was demonstrated to cause cancer in hamsters. However, no increased risk of cancer was identified in people administered the vaccine (Institute of Medicine Immunization Safety Review, 2002). Mythical causes of vaccine rejection have been propagated through inaccurate associations and bad science. For example, after getting the inactivated influenza virus, people may experience local injection site reactions, slight fever, and achiness, leading them to conclude that the vaccine “gave” them the flu. Another example is the myth that administration of the measles, mumps, and rubella (MMR) vaccine may cause encephalitis. While this does have biological plausibility and numerous studies have looked at the association, there has been no conclusive evidence for a relationship between the MMR vaccine and encephalitis/encephalopathy. Another myth was that sudden infant death syndrome (SIDS) was caused by vaccines. This myth dissipated, in large part, because of the dramatic reduction in SIDS after the “back to sleep” program started (babies put on their back to sleep). Perhaps the greatest and most damaging myth related to vaccines and disease was that propagated by Andrew Wakefield and his colleagues in his 1998 (Wakefield et al., 1998) with a publication in the Lancet “Ileal-lymphoid-nodular hyperplasia, nonspecific colitis, and pervasive developmental disorder in children.” In this publication, which was based on 12 brief case

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reports, suggested that the MMR vaccine may predispose to behavioral regression and pervasive developmental disorder in children (essentially autism). The paper received global attention, despite its uncontrolled design and low number of patients. It was not long after that the number of MMR vaccinations in children plummeted. Even after the paper was debunked and retracted a decade later (2010), and it was revealed that Wakefield had been funded by lawyers to help with legal cases for parents claiming vaccine-associated autism, people still cite the increased risk of autism as a key reason for not vaccinating their children. Another important myth driving the relationship between vaccines and autism was the presence of thimerosal, a mercury-containing constituent of vaccines (Taylor et al., 2014). In the 1990s, there were concerns over the effects of environmental methylmercury, primarily from fish. It was noted that thimerosal, which is an ethyl mercury, in vaccines was higher than the levels allowed for methylmercury. In 2005, an article published in the Rolling Stone (written by the environmental lawyer and notorious perpetuator of vaccine misinformation, Robert F. Kennedy), alleged that the mercury in vaccines could cause autism, and the government was covering up the evidence. However, there is no clinical evidence of any association between thimerosal and autism (MrozekBudzyn et al., 2010; Taylor et al., 2014; Uno et al., 2015). Pertussis vaccines against Bordetella pertussis (causes whooping cough) were also the subject of extensive misinformation and fear of vaccine side effects which resulted in an upsurge in clinical cases. The whole-cell pertussis vaccines, which have been replaced with acellular vaccines, did rarely result in significant adverse events in children, including febrile seizures. Additionally, various physical, neurological, and intellectual disabilities were blamed on the vaccine, which were subsequently refuted, were highlighted in a 1982 TV documentary, “DPT: Vaccine Roulette.” Such misinformation decreased the vaccination rate for pertussis, resulting in a resurgence of this disease. In 1981, the number of reported pertussis cases in the US was 1248; the incidence steadily increased starting in 1982, reaching a high of

48,277 in 2012 and in 2019, had decreased to 18,617 (https://www.cdc.gov/pertussis/survreporting/cases-by-year.html).

13. CONCLUSIONS Vaccines have had a tremendous impact on world health by reducing or eliminating infectious diseases. In addition, vaccination against bacterial pathogens has reduced our dependence on antibiotics, helping to contain development of antibiotic-resistance by bacteria. With the worldwide resurgence of a range of viral, parasitic, and bacterial infectious diseases, fueled by pathogen resistance to other therapeutics, preventative vaccines remain of great importance. In the last few decades, vaccines have moved from disease prevention into the arena of disease therapy, with therapeutic vaccines holding great promise in the treatment of an assortment of maladies, from cancer to HIV. In this chapter, we have reviewed some of the key background information on vaccine immunology, modalities, and adjuvants as well as reviewed considerations for the toxicologic pathologist in nonclinical vaccine study design and interpretation. Involvement of the toxicologic pathologist in all stages of vaccine study design is a critical component of ensuring the accurate evaluation and interpretation of the nonclinical vaccine study and translation into the human or target species studies. Toxicologic pathologist have a broad perspective on science and in nonclinical safety, and as many are veterinarians, they also have an unique understanding of “herd health”. As such, they should be a key partner in vaccine research and development from concept to clinic.

14. GLOSSARY Adjuvant “substances or combinations of substances that are used in conjunction with a vaccine antigen to enhance (for example, increase, accelerate, prolong and/or possibly target) or modulate to a different type (e.g., switch a Th1 immune response to a Th2 response, or a humoral response to a cytotoxic T cell response) the specific immune response to the vaccine antigen in order to enhance the clinical effectiveness of the vaccine” (WHO, 2014) Adjuvanticity the ability to induce an adjuvant response Antigen the active ingredient in a vaccine (or generated by a vaccine) against which a specific immune response is raised (WHO, 2014)

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Attenuated viral vaccine Live virus is altered such that it is less virulent Excipient an inactive substance that is a component of a vaccine formulation Inactivated microbial vaccine Viral or bacterial vaccines in which the infectious agent has been killed through chemical, thermal, or other means Lipid nanoparticle nanoparticles composed of various types of lipids; most commonly consists of both structural and ionizable lipids. Local tolerance/toleration studies studies are intended to identify the potential effects of a vaccine at the site of administration Modified live viral vaccine see Attenuated viral vaccine Monovalent vaccine vaccine has only a single antigenic component Multivalent vaccine vaccine has 2 or more antigenic components; the components may be against strains of the same organism or against different organisms. Nanoparticle particle which is less than 100 nm in diameter, although the term may be used for particles up to 500 nm. Oncolytic virus a virus that preferentially infects and kills cancer cells Opsonophagocytosis phagocytosis by mononuclear cells most commonly mediated by antibody or complement binding to the organism Prophylactic vaccine vaccine intended to prevent disease, typically against infectious diseases Reactogenicity generally refers to the manifestations of the immune response at the site of administration, such as redness, swelling, and pain. Therapeutic vaccine vaccine intended to treat a disease or condition Toxoids a chemically modified microbial toxin, which renders it nontoxic but which retains antigenicity and can be used in vaccines. Variolation obsolete method of immunization against smallpox through infecting an individual with exudate from a pustule of an infected individual Virus-like particle (VLP) are vaccines comprised of particles usually ranging from 40 to 400 nm in diameter which have densely arranged viral surface proteins that are conformationally appropriate to elicit strong T cell and B cell immune responses

Acknowledgments

The authors would like to thank Sean Troth, Bindu Bennet, Niraj Tripathi, Marie-France Perron LaPage, Sebastien Laurent, Karissa Adkins, Jayanthi Wolf, and Ronnie Chamanza for intellectual contributions to this chapter.

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C H A P T E R

10 Stem Cell and Other Cell Therapies Alys E. Bradley1, Brad Bolon2 1

Charles River Laboratories, Edinburgh, Scotland, United Kingdom, 2GEMpath, Inc., Longmont, CO, United States O U T L I N E 3.3. Design of Preclinical Safety Studies 3.4. The Pathologist’s Contribution

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1. INTRODUCTION Cell-based medicinal products (CBMPs) encompass various products in which viable human-derived therapeutic cells (TCs) are administered to patients. The TCs may be of allogeneic origin (i.e., collected from a healthy donor and given to a different individual with an illness) or autologous derivation (i.e., obtained from the patient and then returned to that same person). The TCs may or may not have been genetically modified (e.g., via ex vivo lentiviral gene therapy to produce chimeric antigen receptor–expressed T-lymphocytes [CAR-T cells]) and may or may not be combined with a medical device, mesh, or scaffold (Dong et al., 2021; Evans et al., 2006). The current chapter focuses on CBMPs involving nonmodified or reprogrammed TCs alone. Cell therapies Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-12-821047-5.00011-7

involving ex vivo modification to enhance their function may be explored further in the chapter on Gene Therapy and Gene Editing, (Vol 2, Chap 8), while TCs in combination with medical devices are addressed briefly in the chapter on Biomedical Materials and Devices (Vol 2, Chap 11). A glossary of essential terms and definitions is included in Section 6. In general, CBMPs are administered to rejuvenate, repair, or replace the function of a damaged cell population or a degenerating tissue structure in order to ameliorate or ideally cure a previously untreatable disease or injury. Common disease indications for cell therapies include degenerative, genetic, inflammatory, and neoplastic diseases. The TCs may be implanted directly at the site of degeneration or injury (e.g., stem cells to restore deep brain nuclei or joint cartilage) or introduced by intravenous injection (e.g., hematopoietic

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stem cells). The fates of engrafted TCs depend on their differentiation state and any laboratory modifications imparted to them. For example, some TCs become incorporated into the parenchyma in place of damaged cells. In other cases, the implanted TCs secrete various factors that coordinate the functions and interactions of nearby or distal cells (e.g., TCs designed to secrete hormones). Scientific, clinical, and public expectations are that cell therapies will soon offer significant long-term health benefits and healthcare cost savings by successfully reviving and extending organ function while substantially improving the quality of life for patients who suffer from one or more serious diseases.

2. BIOLOGICAL PRINCIPLES OF CELL THERAPY Appropriate design of preclinical studies for CBMPs requires some understanding of the basic concepts that form the foundation for this therapeutic modality. This section briefly reviews key elements that impact cell selection and whether or not the TCs require modification to achieve optimal efficacy.

2.1. Cell Sources In general, CBMPs are obtained from humans. Cells may be harvested by blood collection to remove circulating stem cells, such as leukapheresis to collect hematopoietic stem and precursor cells (HSPCs), or by tissue biopsy to sample sites with higher population of existing (e.g., adipose tissue, bone marrow) or inducible (e.g., skin) stem cells. In terms of CBMPs currently in development, three main sources should be considered. Autologous Cells Isolation of a patient’s own stem cells is the basis of autologous cell therapy. In vitro expansion of the cells permits their reintroduction into the patient in greater numbers than might be available in damaged tissue. Modification using specific cocktails of cytokines and/or growth factors drives stem cell differentiation toward certain cell lineages that are more effective at filling structural defects left by degeneration of

the original tissue (e.g., defects in hyaline cartilage of joints during osteoarthritis). This approach is utilized commonly for stem cell therapy in regenerative medicine (Figure 10.1). Another option is to use nonviral or viral means of cell transduction to enhance the functional capability of the isolated stem cells, thereby enhancing their activity when returned to the patient. This approach is relevant to anticancer CAR-T immunotherapy (Sterner and Sterner, 2021). Several CAR-T products involving ex vivo modified autologous stem cells have been approved to treat acute B-lymphocytic tumors, including tisagenlecleucel (Kymriah), brexucabtagene autoleucel (Tecartus), and axicabtagene ciloleucel (Yescarta). Additionally, sipuleucel-T (Provenge) is an autologous T-cellbased immunotherapy for the treatment of prostate cancer. Autologous cells have no or extremely low immunogenicity and generally will not invoke a graft-versus-host disease (GvHD) response or be rejected by the recipient. The absence of immunogenicity is a significant advantage in achieving a sustained response following administration of autologous TCs. However, the primary shortcomings of autologous cell therapy are the extended times required for cell expansion, the variable quality of blood or tissue samples acquired from ill patients, the complex coordination and process manufacturing logistics, and the lofty expense of this personalized medicine approach relative to the economies of scale provided by conventional “off-the-shelf” allogeneic population-oriented treatments. Allogeneic Cells Isolation of cells from a healthy donor for introduction into a patient is the basis of allogeneic cell therapy. As for autologous cell therapy, the collected allogeneic stem cells are expanded ex vivo and then reintroduced into a patient. The donor cells and patient cells typically express an identical human leukocyte antigen (HLA) signature to limit GvHD. For this reason, allogeneic donors usually are close relatives of the patient. Allogeneic cells offer some advantages over autologous TCs. Cells from a single donor may be used to treat multiple patients with the same HLA genotype. Moreover, these TCs retain their efficacy when frozen and thus may be biobanked until needed, which allows their use over an

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FIGURE 10.1 Schematic diagram demonstrating a stem cellebased regenerative medicine approach for treating degenerative joint disease. Primary (differentiated) somatic cells from the joint are harvested and then exposed to either lineage-specific factors (which direct a cell to adopt a particular differentiation pathway) or reprogramming factors (which drive reversion of primary somatic cells to a “stem cell” phenotype). The resulting induced pluripotent stem (iPS) cells then differentiate to produce cells with a desired cell type that can be used for basic research (e.g., drug discovery and disease modeling) or cell therapy. Graphic prepared by Mr. Timothy Vojt.

extended period of time rather than only near the time of harvesting. Challenges related to allogeneic TCs include cell heterogeneity related to a patient’s own genetic profile, variable purity, and the propensity for viable engrafted immune cells to incite GvHD. Xenogeneic Cells Isolation of cells from healthy animals for introduction into a human patient is the foundation for xenogeneic cell therapy. This approach is a form of xenotransplantation, which is defined by the U.S. Food and Drug Administration (FDA) as any procedure that involves the introduction into a human recipient of either (a) live cells, tissues, or organs from a nonhuman animal source or (b) human body fluids, cells, tissues, or organs that have had ex vivo contact with live nonhuman animal cells, tissues, or organs (FDA, 2016b). Xenogeneic cell therapy is being explored as a potential means for restoring the function of depleted cell populations for which autologous tissue or HLA-matched allogeneic human tissue is of insufficient quantity and/or quality for ex vivo cell expansion (Huang et al., 2021a). Tissues for which xenogeneic cells have been tested in animal models include brain (neurons

in basal nuclei), liver (hepatocytes), and endocrine pancreas (insulin-producing b cells from islets of Langerhans). Pigs represent a favored animal source for xenogeneic cells based on their similarities with humans in terms of physiology and size (Sykes and Sachs, 2019). Principal advantages of porcine cells for cell therapy include the ability to rigorously characterize the genetic background and pathogen profile of the individual donor (and herd), to harvest both partially and fully differentiated cells from any organ, and to maintain the animals and collect the cells under rigorously controlled conditions. In addition, the consistency, quality, and safety of pig-derived cells may be optimized by growing the animals under specific pathogen-free (SPF) conditions and generating the cells utilizing Good Manufacturing Practice (GMP) principles (Schuurman, 2009; Spizzo et al., 2016). Current pig donors may be screened for the presence of endogenous porcine retroviruses, which are a potential concern in terms of zoonotic transmission (Denner and Schuurman, 2021; Semaan et al., 2013), but in the future genome editing (e.g., gene editing using clustered regularly interspaced short palindromic repeats [CRISPR] technology (see Genetically Engineered Animal

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Models in Toxicologic Research, Vol 1, Chap 23) may be used more widely to remove endogenous viral sequences to further clean xenogeneic cells (Barrangou and Doudna, 2016). Interestingly, pig stem cells have much lower expression of endogenous retroviruses compared to human and mouse embryonic stem cells (Glinsky, 2016; Kru¨ger et al., 2021), suggesting that pigs may serve as stem cell donors for human patients. Obviously, a clear challenge to the use of animal cells for cell therapy in humans is their intrinsic immunogenicity. Porcine cells are encapsulated in biocompatible polymers (Mulroy et al., 2021) or semipermeable but cell-impermeable membranes (Zhu et al., 2015) to protect them from the host’s immune response. Furthermore, the host immune reaction may be advantageous in some settings, such as injecting them into solid tumors to produce a bystander antitumor response as the injected animal cells are attacked (Huang et al., 2021a).

2.2. Classes of Transplantable Cells Many cell types may be isolated for CBMPs. Cells may be collected from human (autologous or allogeneic) or xenogeneic sources. This section briefly introduces the major cell classes that are being evaluated in preclinical studies and/or clinical trials. Pluripotent Stem Cells Stem cells are elements that act as “master” cells in populating tissues and organs. They can be classified according to their regenerative potential as totipotent, pluripotent, multipotent, oligopotent, and unipotent. Totipotent stem cells are present very early in embryonic development and possess the capacity for extensive self-renewal. Properly stimulated, totipotent cells can give rise to any cell kind in the body via their ability to produce pluripotent stem cells. Evolving slightly later in development (see Embryo, Fetus, and Placenta, Vol 5 , Chap 11), pluripotent stem cells may divide to produce either more pluripotent stem cells or partially committed (multipotent) stem cells that can give rise to many kinds of mature (terminally differentiated) cells. The germ layers arising from pluripotent stem cells may reflect one of three basic cell lineages: ectoderm,

endoderm, or mesoderm. Multiple kinds of pluripotent stem cells have been identified. Conceptually, stem cells gradually are converted to terminally differentiated cells through two additional stages. In the first, stem cells (totipotent or pluripotent) evolve into highly proliferative cells that slowly lose the capacity for self-renewal while simultaneously becoming dedicated to a limited (multipotent) range of cell fate decisions. In the second stage, these partially differentiated ancestral cells will gradually adopt a more limited capability of selfrenewal coupled to the ability to differentiate into only a few cell lineages (oligopotent) or a single cell type (unipotent). These two stages often are referred to as precursor cells and progenitor cells, but unfortunately both of these terms are applied to both stages (e.g., compare among (Anonymous, 2019; Martı´nez-Cerden˜o and Noctor, 2018; Tajbakhsh, 2009; Udayangani, 2019)) or even generically used as a synonym for “stem cells.” Therefore, in designing and communicating preclinical and clinical stem cell studies care must be taken to define the intended specific meanings of “precursor cells” and “progenitor cells” to avoid confusion. EMBRYONIC STEM CELLS

Pluripotent stem cells derived from the inner cell mass of a blastocyst (approximately 3- to 5day-old embryo, depending on the species) are termed embryonic stem cells (ESCs, or occasionally ES cells). These cells possess the capacity for indefinite self-renewal and readily differentiate into any of the three germ layers. Moreover, ESCs have a normal karyotype (chromosome number), which permits their use not only in discovery research but also as a potential platform for clinically relevant TCs. To date, ESCs have been derived for multiple species including rodents, pigs, nonhuman primates, and humans (reviewed in Rippon and Bishop, 2004). The key advantage of ESCs is that they may be used to produce TCs arising from any cell lineage. Therefore, they may be utilized to fulfill any tissue repair or replacement need. Three main difficulties have been encountered when developing ESCs as potential TCs. The main biological challenge is that ESCs typically are derived from “generic” embryos, such as those that remain after unused embryos collected for in vitro fertilization are discarded. Therefore,

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therefore are unlikely to invoke an immunemediated immune response that destroys the engrafted cells. That said, large-scale cell therapy programs using somatic cell–derived stem cells are likely to be allogeneic in origin.

FIGURE 10.2 Representative section of a teratoma produced by injecting human embryonic stem cells (hESCs) into the subcutis of a congenic scid/beige mouse (formal designation: CB17.Cg-PrkdcscidLystbg-J/J), where the scid (severe combined immunodeficiency) mutation yields deficits of B- and T-lymphocytes while the beige mutation produces defective natural killer (NK) cells. After 8 weeks, tumor growth at the implantation site produced a mass with cells derived from all three germ layers: endoderm (inner layer), such as hepatocytes (L) and respiratory epithelium (R); mesoderm (middle layer), including bone (B), cartilage (C), and undifferentiated mesenchyme (M); and ectoderm (outer layer), including neuroepithelium (N) and retinal pigmented epithelium (E). Stain: H&E.

ESCs generally are allogeneic with respect to the patient and thus are likely to incite the patient’s immune system to mount a transplant rejection response. A second biological problem is that ESCs can devolve into teratomas, a usually benign but rapidly expanding neoplasm comprised of cells from all three germ layers (Bulic-Jakus et al., 2016; Gutierrez-Aranda et al., 2010; Hentze et al., 2009; Prokhorova et al., 2009) (Figure 10.2). The final issue with respect to the use of human ESCs (hESCs) for cell therapy rests with ethical concerns grounded in the unavoidable destruction of human embryos in harvesting the cells. SOMATIC CELL–DERIVED STEM CELLS

Three major types of pluripotent stem cells have been produced by repurposing somatic cells into stem cells. The advantage of these cells is that they may be obtained from the patient and

INDUCED PLURIPOTENT STEM CELLS Pluripotent stem cells derived from terminally differentiated adult somatic cells are designated as induced pluripotent stem cells (iPSCs or sometimes iPS cells). These cells have been reprogrammed ex vivo through forced expression of specific genes needed to maintain a pluripotent status. Various methods may be employed to return adult cells to pluripotency including epigenetic reprogramming, protein-mediated transduction, and viral-mediated transduction (Hockemeyer et al., 2008; Jensen et al., 2021; Kim et al., 2012; Okahara-Narita et al., 2012; Park et al., 2008; Rajasingh, 2012; Takahashi et al., 2007; Takahashi and Yamanaka, 2006; Ye et al., 2013; Yu et al., 2007). This process has been performed successfully in many species including mice (Takahashi and Yamanaka, 2006), monkeys (Okahara-Narita et al., 2012), and humans (Takahashi et al., 2007). Once reprogrammed, iPSCs have similar biological properties and growth characteristics to ESCs, including persistent self-renewal and the ability to generate other, more mature cell types. Retained epigenetic memory of the parental somatic cell source has been postulated to limit the transdifferentiation efficiency of iPSCs into cell lineages from other tissues (Kim et al., 2010; Noguchi et al., 2018). Over time, iPSCs may revert to their original differentiation state (Heng et al., 2009). In addition, they also have been shown to form teratomas (Bulic-Jakus et al., 2016; Fong et al., 2010; Gutierrez-Aranda et al., 2010). OTHER SOMATIC-ORIGIN STEM CELLS Inducible tissue-specific stem cells (iTSCs) are pluripotent stem cells derived from somatic cells of a particular tissue. These cells differentiate more efficiently into cells of the organ of origin than do ESCs, a trait thought to reside in the parental cell source (Noguchi et al., 2018; Noguchi et al., 2015). Their dedication to forming cells from a single tissue has been suggested to limit their potential to form teratomas (Miyagi-Shiohira et al., 2018).

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Nuclear transfer stem cells (ntSCs) are pluripotent stem cells generated by somatic cell nuclear transfer. In this procedure, a somatic cell nucleus is inserted into an enucleated oocyte. These cells have been proposed as a platform for therapeutic cloning for use in regenerative medicine (Matoba and Zhang, 2018). These cells seem to have less parental cell memory relative to iPSCs. Adult stem cells (ASCs) are a rare, resident population of undifferentiated stem cells present within a differentiated organ (Goodell et al., 2015; Gurusamy et al., 2018). These cells are multipotent elements with limited self-renewal capabilities that function in maintaining tissue homeostasis. In vivo, ASCs occupy a particular niche, which is the combination of the physical location and extrinsic microenvironmental signals that integrate to determine stem cell behavior (Boulais and Frenette, 2015; Ferraro et al., 2010). When activated, ASC proliferation is able to generate new differentiated cells to replace damaged or lost tissue. These cells can only differentiate into cell types within the tissue in which they reside. Specific examples of ASCs include endothelial progenitor cells (EPCs), hematopoietic stem cells (HSCs), and mesenchymal stem cells (MSCs). These cells may be heterogeneous in terms of the donor, the tissue source, and the various stem cell populations in that tissue (Zha et al., 2021).

BIODISTRIBUTION OF IMPLANTED CELLS

Administration of stem cells may be to a specific site (e.g., implantation into a particular brain nucleus) or into a compartment (e.g., intravascular infusion of HSCs) ensuring systemic distribution to a niche located at multiple sites. Accordingly, biodistribution of engrafted TCs must be examined to ensure that they arrive at and are incorporated into the appropriate target tissue (Gu et al., 2012). Conventional histopathologic evaluation has been the primary approach for ex vivo analysis of stem cell engraftment at the implantation site. This assessment begins with the evaluation of routine hematoxylin and eosin (H&E)-stained tissue sections from the target tissue. Detection of small grafts or isolated scattered TCs using H&E alone is challenging, so serial sections often are used to highlight cell type–specific biomarkers to confirm the presence of the TCs at the target location. For example, immunohistochemical (IHC) labeling to detect human-specific markers such as human leukocyte antigen (HLA) (Shultz et al., 2010); human cytoplasmic protein STEM121 (found in brain, pancreas, and liver) (Kawabata et al., 2016); human nuclear antigen (usually HNA or HuNu [Figure 10.3]) (Arpornmaeklong et al., 2010);

2.3. Primary Preclinical Safety Considerations for Cell Therapies The main preclinical safety concerns for cell therapies may be divided into test article–related and procedure-related categories. Toxicologic pathologists are integral to assessing the presence, severity, and implications of both categories of safety concerns. Test Article–Related Safety Considerations Key concerns related to the test article include its biodistribution, inappropriate differentiation, and unsuitable proliferation (Baker and Assaf, 2015; Bradley and Black, 2021; Doi et al., 2020). A corollary efficacy consideration is to ensure that implanted cells have differentiated correctly.

FIGURE 10.3 Representative injection site in the striatum (a basal nucleus in the brain) of an athymic nude rat (formal designation: Crl:NIH-Foxn1rnu) injected with human embryonic stem cells (hESCs) showing dispersion of cells labeled with human nuclear antigen (HNA or HuNu [dark brown elements]). Counterstain: hematoxylin. Image provided courtesy of Dr. Gary Steinberg, Stanford University School of Medicine.

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Ku80 (another human nuclear antigen) (Jensen et al., 2021); or Clone 113–1 (an antibody against a human mitochondrial protein) (Saludas et al., 2019) may be used to distinguish implanted human TCs from endogenous cells of the preclinical test species (Baker and Assaf, 2015; Bradley and Black, 2021). Biodistribution also may be confirmed through other analytical methods. Homogenized tissue may be screened by polymerase chain reaction (PCR) to detect TC-specific genes. For instance, expression of the male-specific sex-determining region Y (SRY) protein may be used as a marker if the TCs are derived from a male source and implanted in female test animals (Isakova et al., 2006). Fluorescent markers may be used to label stem cells for fluorescence-activated cell sorting (FACS) (Abujarour et al., 2013), and in principle such sorting might be applied to tissue homogenates as well if quantity rather than location is the major point of interest. DIFFERENTIATION OF IMPLANTED CELLS

Proper cell maturation is required if TCs are to function effectively in providing therapeutic efficacy. Histopathologic evaluation is useful for demonstrating appropriate differentiation at the target site. Morphologic characteristics include the correct cytoarchitectural features and proper cell integration into the existing tissue. For instance, implantation of neural TCs into the brain may result in formation of neurons with a large nucleus, single nucleolus, and abundant cytoplasmic aggregates of rough endoplasmic reticulum (i.e., Nissl substance). Similarly, MSCs engrafted in a joint are meant to become incorporated into articular cartilage, assume chrondrocytic features, and proliferate (Li et al., 2016; Yang et al., 2015). Ideally, mature TCs will integrate fully into the implantation site. This incorporation tends to be relatively more complete for TCs designed to fill a structural defect and restore tissue integrity or slow the degeneration of any remaining endogenous tissue (Li et al., 2016). In contrast, differentiated cells may fail to integrate properly in tissues with complex intercellular connections, like the brain (Henriques et al., 2019). While not incorporated directly into existing neural circuits, implanted neural TCs that differentiate into neurons positively boost brain function by increasing neurotrophic factor levels in the

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microenvironment and reducing inflammation (Henriques et al., 2019). PROLIFERATION OF IMPLANTED CELLS

Many TCs are designed to proliferate to some degree when introduced into the proper target site. The presence and degree of TC proliferation may be confirmed by microscopic evaluation of tissue sections processed by conventional methods. For example, mitotic figures may be counted in H&E-stained sections, or IHC-labeled sections may be assessed to detect markers present during the active (G1, S, G2, and M) phases of the cell cycle, such as Ki67 (Sun and Kaufman, 2018). An appropriate degree of TC proliferation typically is indicated by the limited expansion or absence of TC clusters within the target site, which shows that proliferation has not escaped normal growth control pathways. Unregulated cell proliferation is a concern following TC delivery since implanted cells typically cannot be removed once they have been introduced. Several factors impact the capacity for tumor (specifically teratoma) formation of TC test articles. For example, the class of stem cell dictates their tumorigenicity. Highly pluripotent stem cells such as ESCs, iPSCs, and ntSCs have been linked to teratoma development (Fong et al., 2010), and iPSCs have been reported to be more tumorigenic relative to ESCs (Gutierrez-Aranda et al., 2010). In contrast, less pluripotent ASCs usually do not form tumors (Ng et al., 2013) even though endogenous adult stem cells may serve as cancer cells of origin in vivo (White and Lowry, 2015). Tumorigenicity rises as the number of engrafted stem cells is increased or if colonies (i.e., clustered cells) are injected rather than isolated cells (Hentze et al., 2009; Lee et al., 2009). In immunodeficient mice, the propensity for tumor formation varies with the implantation site, with the most reliable development occurring when stem cells are injected beneath the kidney capsule or into the testis (Hentze et al., 2009; Prokhorova et al., 2009). Finally, purity of the TC preparation impacts tumorigenic potential. The presence of residual undifferentiated stem cells among the differentiated somatic TCs may lead to tumor formation, while purification to eliminate residual stem cells greatly reduces tumorigenicity (Chour et al., 2021; Hentze et al., 2009; Polanco and Laslett, 2013) (Figure 10.4). Contamination

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FIGURE 10.4 Schematic diagram showing that contamination of cell preparations with a desired phenotype (e.g., neural cells [gray ectoderm-derived cells]) with pluripotent stem cells (SC, orange cells with brown nuclei) leads over time to aberrant differentiation characterized by production of other cell lineages, including mesenchymal (elongated blue cells) and endodermal (columnar pink cells) elements. Injection of unpurified (stem cell– contaminated) preparations produces teratomas, while purified (stem cell–free) preparations do not. Schematic prepared by Mr. Timothy Vojt based on the graphic abstract from Chour T, Tian L, Lau E, et al.: Method for selective ablation of undifferentiated human pluripotent stem cell populations for cell-based therapies, JCI Insight 6(7):e142000, 2021. https://doi. org/10.1172/jci.insight.142000 under a Creative Commons 4.0 International License.

by undifferentiated stem cells may be assessed in vitro using flow cytometry and/or quantitative reverse transcription PCR (RT-PCR) to detect the expression of markers for differentiated and nondifferentiated cells (Doi et al., 2020). Evaluation for tumorigenicity in vivo by evaluation of tissues at the implantation site and lungs (for metastases) is a common task for toxicologic pathologists engaged in developing CBMPs. Procedure-Related Safety Considerations Key concerns associated with the procedure include underlying anomalies related to the animal disease model, damage induced directly during implantation, and subsequent immunogenicity (of both the host tissue toward the engrafted cells and GvHD). In general, these issues are best dealt with by inclusion of appropriate concurrent control groups in the preclinical study design. Where possible, the evaluation is facilitated when the study pathologist has previous experience analyzing tissues from both the animal disease model and examining cell-based test articles. ANIMAL MODELS OF DISEASE

Many animal efficacy studies and combined efficacy/toxicity studies are performed in mice

that are either immunodeficient (spontaneous or engineered) or humanized (engineered to insert human immune cells to replace the corresponding mouse cells). Details regarding these models may be found in Genetically Engineered Animal Models in Toxicologic Research (Vol 1, Chap 23). Some common considerations for immunodeficient mouse models include altered hematologic values and the greatly modified structure of bone marrow and secondary lymphoid organs, all of which differ considerably from wild-type mice that retain their normal complements of immune cells. Successful engraftment of allogeneic stem cells into wild-type mice for efficacy and sometimes safety testing may require preconditioning to suppress the native immune system before the TC test article may be implanted. For example, busulfan (an anticancer chemotherapy agent (Hayakawa et al., 2009; Uchida et al., 2019)) or total body irradiation (Boieri et al., 2016) may be utilized to ablate the bone marrow, thereby permitting the injected HSCs to colonize the appropriate niche. Side effects of these procedures may include altered cerebellar lamination (if neonatal animals are treated during the first 5 days after birth, when the cerebellum is developing); fibrosis affecting the stroma of organs

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with relatively rapid cell turnover (e.g., bone marrow and intestine); and inflammation. Certain animal models may be induced by chemical treatments and/or surgical interventions (see chapters in the Animal and Alternative Models in Toxicologic Research section, Vol 1, Part 3). These manipulations will result in modest to profound changes in normal tissue morphology. For example, rodent models of osteoarthritis to test MSCs as means for cartilage repair may be incited by intraarticular injection of monoiodoacetic acid (Udo et al., 2016) or surgical induction of cartilage trauma (Tawonsawatruk et al., 2018), removal of meniscus tissue (Ali et al., 2018), or transection of key ligaments (Tawonsawatruk et al., 2018). Special stains (e.g., toluidine blue for cartilage matrix integrity in rodent models of joint disease [Bolon et al., 2011]) often are useful ancillary procedures for fully characterizing anatomic changes produced by these disease-inducing procedures. IMPLANTATION-RELATED TISSUE INJURY

The implantation procedure will involve a degree of tissue injury as cells are introduced into the target organ. The degree is often slight, consisting of a narrow needle track visible as a very thin line of tissue disruption (Figure 10.5). Slightly more tissue damage might be induced if the implantation method requires a large-gauge needle or surgical incision to insert a cell-containing device. In general, the injury produced during implantation is modest and nonadverse. Insertion of cells generally may be accomplished without undue impact on the organism’s function, even if cells are placed in such remote sites as deep brain nuclei (Isakova et al., 2006). IMMUNOGENICITY TOWARD OR BY IMPLANTED CELLS

Immune reactions to engrafted cells are a frequent concern for cell therapies, particularly for allogeneic and xenogeneic cells. The host immune system recognizes these cells as “nonself” based on mismatched donor HLA, major histocompatibility complex (MHC), and ABO blood group antigens and will seek to mount an immune response to reject the implanted cells (Heslop et al., 2015; Worel, 2016). Concurrently, surviving stem cells with immune functions may launch an attack against host organs (GvHD). Either side of these competing immune

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FIGURE 10.5 Representative injection site in the striatum of an athymic nude rat (formal designation: Crl:NIH-Foxn1rnu) injected with human embryonic stem cells (hESCs), showing a thin linear needle track (horizontal line of disrupted tissue in the image center) bordered by a narrow margin of neuropil containing slightly more glial cells (visible as small, dark, round nuclei). Stain: H&E. Image provided courtesy of Dr. Gary Steinberg, Stanford University School of Medicine.

reactions is capable of infringing on adjacent host tissues. Autologous cells are less immunogenic, but iPSCs are capable of invoking a host immune response (Liu et al., 2017b). Undifferentiated and incompletely differentiated iPSCs incite a more robust immune response than their differentiated progeny, and distinct progeny lineages arising from the same stem cell cause divergent levels of immunogenicity (Haworth and Sharpe, 2021; Wood et al., 2016; Zhao et al., 2015). Interestingly, MSCs possess a distinctive ability among ASCs to control the host immune response directed against donor cells (Aggarwal and Pittenger, 2005). Rejection of stem cell allografts is mediated primarily by the adaptive immune response. The primary mechanism of host-versus-graft reactions reflects host T-lymphocyte recognition of foreign MHC antigens on the implanted stem cells. Host helper (CD4þ) T-cells drive the reaction. Mechanisms participating in graft rejection include alloantigen presentation to CD4þ T-cells by antigen-presenting cells (APCs), leading to release of proinflammatory cytokines, recruitment of cytotoxic (CD8þ) T-cells, and later CD4þ T-cell activation of B-lymphocytes to

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produce antibodies against the alloantigen (de Almeida et al., 2013). In histopathologic sections, the rejection response is dominated by mononuclear cell infiltration (chiefly macrophages and small lymphocytes), progresses in severity over time, and may be accompanied by degeneration and necrosis of the encircled stem cells (Daldrup-Link et al., 2017). The major clinical outcome of host-versus-graft disease is loss of the TCs and any functional restoration they were intended to provide. In contrast, GvHD associated with introduction of functional allogeneic or xenogeneic stem cells may lead to systemic inflammation with arteriopathy and/or sclerosis of connective tissue–rich organs. Activated alloreactive T-cells populate secondary lymphoid organs and then migrate to host tissues to drive the GvHD response locally (Boieri et al., 2016). Common target organs include the digestive tract (including liver), lungs, and skin (Greenblatt et al., 2012; Nikolic et al., 2000). The attack involves additive Th1 (cell-mediated) and Th2 (antibody-focused) responses, with each response impacting different target organs. For example, Th1-driven events are more important in liver and skin involvement (Nikolic et al., 2000). Cell infiltrates in tissue sections consist mainly of macrophages and small lymphocytes with scattered plasma cells, and severe inflammatory reactions may be accompanied by single-cell degeneration and necrosis of parenchymal tissue.

3. PRECLINICAL CONSIDERATIONS FOR CELL THERAPIES For many categories of therapeutic candidates, guidance regarding the design and evaluation of animal safety studies is relatively detailed. Such guidance is available from health authorities (e.g., the European Medicines Agency [EMA], FDA, Japanese Ministry of Health, Labour and Welfare [MHLW], and United Kingdom Medicines and Healthcare products Regulatory Agency [MHRA]) as well as global organizations aiming to provide uniform guidelines to foster biomedical product development (e.g., the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human

Use [ICH], International Organization for Standardization [ISO], and Organisation for Economic Co-operation and Development [OECD]). Biomedical products covered by such guidance include conventional drugs (Overview of Drug Development, Vol 2, Chap 1), nucleic acids (Nucleic Acid Pharmaceutical Agents, Vol 2, Chap 7), proteins (Protein Therapeutics, Vol 2, Chap 6), medical devices (Biomedical Materials and Devices, Vol 2, Chap 11), and vaccines (Vaccines, Vol 2, Chap 9) as well as nextgeneration innovations like gene therapies (Gene Therapy and Gene Editing, Vol 2, Chap 8). Guidance for developing CBMPs is scarce at present (Table 10.1), and this paucity represents a principal challenge to preclinical study design. Instead, major considerations in designing preclinical and clinical investigations have been covered more effectively in several scientific publications (Goldring et al., 2011; Polanco and Laslett, 2013; Sharpe et al., 2012). The rapidly evolving principles and practices for investigating the efficacy and safety of cell therapies means that preclinical studies must be designed and conducted on a case-by-case basis. This chapter describes current considerations for this field as they apply to toxicologic pathology analysis. The focus is deliberately flexible to encourage early and regular interactions among the scientific team involved in designing preclinical studies as well as between these teams and health authorities to ensure that development programs for cell therapies provide appropriate data for assessing safety. In particular, toxicologic pathologists are essential to the safety evaluation of cell therapies and thus should play a leading role at initial preclinical study design draft, INitial Targeted Engagement for Regulatory Advice on CBER producTs (INTERACT) meetings, and pre–investigational new drug (preIND) meetings with regulators.

3.1. Regulatory Guidance Guidance documents for cell therapies with relevance to safety studies are presented in Table 10.1. The bulk of these documents has been produced within the last 15 years as the field has become more mature, and efforts are ongoing to provide greater regulatory harmonization among different health authorities (Cozzi et al., 2016). Most are applicable to treating

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TABLE 10.1

Principal Regulatory Guidance on Cell-Based Therapies

Source

Title

Year

Reference

EMA/MHRA

Regulation on advanced therapy medicinal products

2007

Commision (2007)

FDA

Guidance for human somatic cell therapy and gene therapy

1998

FDA (1998)

FDA

Eligibility determination for donors of human cells, tissues, and cellular and tissue-based products

2007

FDA (2007)

FDA

Considerations for allogeneic pancreatic islet cell products

2009

FDA (2009)

FDA

Cellular therapy for cardiac disease

2010

FDA (2010)

FDA

Potency tests for cellular and gene therapy products

2011

FDA (2011a)

FDA

Preparation of IDEs and INDs for products intended to repair or replace knee cartilage

2011

FDA (2011b)

FDA

Preclinical assessment of investigational cellular and gene therapy products

2013

FDA (2013)

FDA

Considerations for the design of early-phase clinical trials of cellular and gene therapy products

2015

FDA (2015)

FDA

Donor eligibility and manufacturing of cellular therapies for animals

2021

FDA (2021)

MHLW

Act on the safety of regenerative medicine

2014

MHLW (2015)

MHLW

Amendment of the enforcement regulations of the act on securing quality, efficacy and safety of pharmaceuticals, medical devices, regenerative and cellular therapy products, gene therapy products, and cosmetics related to reprocessed single-use medical devices (R-SUDs)

2017

PSEHB (2017)

MHLW

Basic principles on utilization of registry for applications

2021

PSEHB (2021)

Abbreviations: EMA, European Medicines Agency; FDA, U.S. Food and Drug Administration; MHLW, Ministry of Health, Labour and Welfare (Japan); MHRA, Medicines and Healthcare products Regulatory Agency (United Kingdom).

human patients, but opportunities for cell therapies in animals are being studied (Gattegno-Ho et al., 2012; Voga et al., 2020) and are the subject of recent draft guidance by the FDA (FDA, 2021). In the European Union, the main guidance for CBMPs is Regulation (EC) No. 1394/2007 on advanced therapy medicinal products (Commision, 2007). This guidance defines cell therapies as medicinal products in which more than minimal manipulation has been undertaken prior to their introduction into patients or where the intended use differs from the normal function of those cells within the body. The guidance acknowledges that no “one size fits all” approach is suitable for all cell products. Information needed for regulatory review will unite Good Manufacturing Practice (GMP) production data (“chemistry,” manufacturing, and control [CMC] for the cells) and Good Laboratory Practice (GLP)-compliant preclinical (efficacy and

safety) data; reports from clinical trials; basic research findings published in peer-reviewed scientific journals; and perhaps cross-references to comparable CBMPs. The primary regulatory body is the EMA, which coordinates closely with national regulatory authorities and the European Commission’s Directorate–General for Health in reviewing candidate cell therapies. The EMA guideline on human cell-based medicinal products (EMEA/CHMP/410869/2006) (EMA, 2008) indicates that tumorigenicity studies should be carried out “if the risk of cellular transformation can be foreseen.” In Japan, CBMPs are regulated according to the Act on the Safety of Regenerative Medicine (ASRM), which was promulgated to address policy set in the Regenerative Medicine Promotion Act (2013) (Tobita et al., 2016). Cell products are divided into three “Regenerative Medicine” classes based on the potential risk

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(MHLW, 2015). Class I is for high-risk test articles such as ESCs and iPSCs, Class II is for ASCs, and Class III is for processing somatic cells. The regulatory body in Japan is the MHLW via its Pharmaceutical and Medical Devices Agency (PMDA) and specifically the Pharmaceutical Safety and Environmental Health Bureau (PSEHB). Guidelines on ensuring the quality and safety of CBMPs have been issued and are available in English translations in the amendment on securing medical products of suitable quality, efficacy, and safety (Notifications PSEHB 0731–7 (2017) (PSEHB, 2017) and in guidance defining the basic principles for completing applications for registering common medical device (PSEHM/MDED) and regenerative medical products (PSEHB/PED) Notification No. 0323–1 (2021) (PSEHB, 2021). The Forum for Innovative Regenerative Medicine (FIRM), the Japanese industry association for regenerative medicine, established the Committee for Non-Clinical Safety Evaluation of Pluripotent Stem Cell-derived ProducT (FIRM-CoNCEPT) in 2016 to provide regulatory science-based globally acceptable consensus for safety evaluation in the development of CBMPs derived from pluripotent human stem cells. In the United Kingdom and European Union, CBMPs are classed as Advanced Therapy Medicinal Products (ATMPs) and assessed using Directive 2001/83/EC (UK, 2001) as amended by the ATMP Regulation 1394/2007 (Commision, 2007), and include combination ATMPs. All regulatory enquiries about regenerative medicines in the United Kingdom go through the MHRA Innovation Office. As noted above for the EMA, preclinical studies to assess tumorigenicity (the ability to produce a neoplasm [“tumor”]) and/or oncogenicity (the capacity of an acellular agent [e.g., contaminating oncogenic viruses] to induce normal cells to become tumor cells) may be performed if potential transformation of engrafted stem cells is a concern. In the United States, CBMPs are regulated by the FDA via the Office of Tissues and Advanced Therapies (OTAT) under the Center for Biologics Evaluation and Research (CBER). The number of guidance documents related to cell therapies has been expanding gradually since the turn of the century (FDA, 2016a), with the bulk released in the last decade (Table 10.1). The most relevant for toxicologic pathologists addresses preclinical

assessment (FDA, 2013). Principal effects to investigate include local reactions (e.g., altered tissue function, inappropriate cellular differentiation and/or tumorigenicity, or inflammatory infiltrates); systemic sequelae (e.g., distant migration leading to ectopic tissue formation and/or tumorigenicity); and delayed reactions (e.g., immunogenicity of the cells or associated device). Both acute and chronic safety should be evaluated in vivo. A single relevant animal species may provide a suitable assessment of efficacy and safety; animal models of disease may be used if available. The FDA’s Center for Veterinary Medicine (CVM) recently issued two draft guidance documents for CBMP to be used as treatments for animals, such as MSC therapies for equine tendon repairs (FDA, 2021). Draft guidance #253, “Good Manufacturing Practices for Animal Cells, Tissues, and Cell- and Tissue-Based Products,” provides manufacturers of animal cells, tissues, and cell- and tissue-based products (ACTPs) with recommendations for meeting requirements for current good manufacturing practices. It addresses the methods, facilities, and controls used for manufacturing ACTPs, including steps in recovery, processing, storage, labeling, packaging, and distribution. Draft guidance #254, “Donor Eligibility for Animal Cells, Tissues, and Cell- and Tissue-Based Products,” if finalized, will assist sponsors, firms, or establishments that participate in the manufacture of ACTPs or perform any aspect of the ACTP donor eligibility determination by providing CVM’s recommendations on screening, testing, and selecting appropriate donors. In discussions with the FDA, sponsors generally should arrange informal, nonbinding exchanges with CBER due to the innovative nature and unique challenges posed by these products. These INTERACT discussions (formerly known as pre-pre-IND meetings) offer sponsors the opportunity for very early consultation on possible issues and solutions for key CMC, efficacy, and safety questions. The dearth of regulatory oversight for this field extends to postmarketing surveillance. Long-suffering patients have turned to stem cell clinics in the hope of mitigating or even reversing chronic damage associated with severe conditions such as dementia and degenerative joint disease. Decisions by some regulatory agencies to extend a grace period to permit

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clinics to demonstrate the value of cell therapies have yielded an enormous proliferation of regenerative medicine clinics but variable progress in providing rigorous evidence to support many stem cell procedures (Perrone, 2021). This suspension of enforcement actions by regulatory agencies is unlikely to continue given the huge unmet medical need and the genuine value offered by CBMPs once protocols to ensure their efficacy and safety have been fully established.

3.2. Key Endpoints for Preclinical Assessment of Cell Therapies Safety considerations for CBMPs differ to a considerable degree from those experienced using conventional biologics (e.g., nucleic acids and proteins) and small molecule drugs but are somewhat similar to those encountered for gene therapies. Once administered, cell and gene therapies typically cannot be withdrawn, and often cannot be given again (though cells within cellimpermeable scaffolds that exclude immune cells sometimes may be injected again). An additional concern, especially for CBMPs, is the potential that rogue stem cells may differentiate inappropriately and form local or even systemic tumors. Guidance by the FDA on preclinical assessment of cell therapies (FDA, 2013) and the World Health Organization (WHO) on tumorigenicity evaluation (WHO, 2010) are useful starting points when designing studies. The extent of the preclinical program for a CBMP will depend on the nature of the cell line and will diverge for unmodified or minimally modified cell lines versus those with engineered genomes. Seven primary questions must be answered to assess the suitability of new CBMPs as therapeutic candidates. Cell characterization studies (in vitro, ex vivo, and sometimes in vivo) are used to confirm (1) what the cells are and (2) what they can do. In vivo discovery studies are performed in animals to determine (3) what is the best system in which to perform preclinical testing; the answer may be a disease model for evaluating efficacy and toxicity or a wild-type animal for a more focused analysis of safety. Subsequently, additional in vivo testing will define (4) where do implanted cells go (i.e., biodistribution); (5) how long do they stay; (6) are they functional (i.e., efficacy [proof-of-concept]);

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and (7) are they safe (i.e., local and systemic toxicity, including tumorigenicity). Taken together, principal issues in translating animal data to humans include difficulty in verifying stem cell containment and integration at the delivery site, inefficient stem cell differentiation, and the potential for tumor formation. The complexity of development programs as well as the lack of boilerplate templates for CBMP development necessitates frequent discussions among discovery researchers, preclinical scientists (including toxicologists and toxicologic pathologists), and regulatory specialists. The program must show that the test article is well characterized, biocompatible, and can be manufactured consistently. The distinctive needs of each CBMP require a case-by-case approach to preclinical development. Accordingly, sponsors should converse with regulatory authorities before animal studies are initiated to ensure that suitable animal models and study designs are selected to answer key safety questions. Development programs typically encompass both non-GLP and GLP-compliant studies. Pilot studies to explore cell biodistribution, confirm the “dose” levels (i.e., number of implanted cells), confirm efficacy, and document persistence are typically performed using non-GLP conditions. Different time points may be examined if cell persistence is problematic and/or if the proper timing of cell injection must be matched with a given disease state in an animal model. Subsequent GLP studies with GMP test article should be conducted using a dosing regimen and route of administration that mimics those to be used in clinical trials, and the cells should be introduced using the device proposed for clinical use. Ultimately, the preclinical studies should define the minimal efficacious dose and noobserved-adverse-effect level (NOAEL), hazard identification, and characterize the spectrum of tissue distribution and responses of/to the injected cells. Animal studies also may be employed to examine any possible interactions of the CBMP with any concomitant therapy.

3.3. Design of Preclinical Safety Studies Considerations for designing experiments to explore the efficacy and safety of CBMP therapies are comparable to those of other candidate therapeutics. A test system (one or more animal

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species) and study regimen (dose, duration, route, and time points) must be selected to assess beneficial and harmful tissue responses. Studies may be focused pharmacology (proof-of-concept [POC]) or toxicity studies or hybrid POC/ toxicity studies. As noted previously, tumorigenicity and/or oncogenicity studies may be performed if potential transformation of engrafted stem cells is a concern. This section reviews important elements to weigh in planning preclinical studies for CBMPs. In general, safety studies to support clinical trials should be GLP-compliant. Species Selection In preclinical testing of CBMPs, regulatory guidance does not specify a default test system (animal species) for safety assessment. Written justification should explain why the chosen species affords the appropriate environment for testing the implanted cells. Because the designs of toxicity studies should mirror those of clinical trials, the chosen test system needs to be both a host in which the engrafted cells can survive as well as a suitable species for evaluating the clinical delivery device and route of administration. Common test systems for CBMP safety testing include wild-type rats (including neonates), mice, minipigs, and nonhuman primates (NHPs); immunocompromised models (typically mice); and induced (genetically engineered or surgically altered) or spontaneous models of disease. A substantial benefit of using animal models of disease is that both efficacy and safety can be tested in a single, well-designed first-inhuman (FIH)- or IND-enabling study. An additional advantage of immunocompromised models is to reduce the likelihood of the host rejecting the implanted cells, which reduces one major confounder during data interpretation. Anatomic and physiological factors are major parameters in species selection, and the use of nonhuman primates is not essential to enable human dosing. The test system must have organs and tissues of sufficient size that they may accommodate the targeted delivery device and accept the administered cell dose and formulation volume at the intended location. For example, cell scaffold implants to hold engrafted retinal pigmented epithelium (RPE) are inherently bulky since they are scaled for placement in human eyes, so the test system needs to have

eyes large enough to accept the scaffold. In contrast, species of all sizes may receive unencapsulated cells in small numbers given in a limited fluid volume using a narrow-gauge needle (e.g., neural stem cells placed into a basal ganglion in the brain). Group Constitution Group assignments are comparable to safety studies for other categories of test articles. The key considerations include designating treatments and group sizes. Animals should be assigned among groups using suitable randomization procedures. Justification should be provided for the relevance of the selected control group(s) and the composition (i.e., numbers, sexes, ages) per group. The number of groups needed for a study ranges from 2 to 15 or more depending on the study objective. Studies for CBMPs may assess one or more test articles. Typical designs include a single TC line tested at several doses and comparison of one or more novel TC lines to an existing TC line (held in a master cell bank, which is a series of multiple cryopreserved vials produced from the original stem cell line as a guard against genetic variation over time). Appropriate control groups are essential, and many options are available. Frequent choices include no manipulation (“untreated [naı¨ve] control”), placement of the delivery device without (“sham control”) or with (“vehicle control”) introduction of the carrier liquid, placement of an empty scaffold (“device control”), injection of undifferentiated or unmodified cells (“negative cell control”) or incompletely modified cells (i.e., having only one of several genetic modifications needed for complete test article efficacy), or insertion of cells to demonstrate the ability to detect key toxicity endpoints (“positive cell control”). In some studies, one or more “tumorigenicity controls” consisting of the differentiated TCs with variable proportions of stem cells may be used to examine the degree of stem cell contamination that might result in tumor formation. Additional controls may be necessary for the disease model and/or genetic background (see Genetically Engineered Animal Models in Toxicologic Research, Vol 1, Chap 23). Adequate animal numbers should be allocated to each group, but numbers differ depending on the primary endpoint. For biodistribution, group

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sizes typically are 5–10/sex/group for rodent studies and 3–5/sex/group for nonrodents. For safety studies, group sizes often are 15–25/sex/ group for rodent studies and 5/sex/group for nonrodents. In some cases, fewer animals or only one sex per group, or inclusion of multiple ages, may be warranted. The rationale for the chosen design should be explained in the study report. Study Regimen The study regimen for safety testing of CBMPs depends on the attributes of the test article. CBMPS AS THERAPEUTIC TEST ARTICLES

For CBMPs destined to regenerate damaged tissue, key aspects of the study regimen to address the influence of the engrafted cells include the duration, doses, route of administration, and time points for analysis. As with other aspects of the study design, choices for these parameters should be justified in the report documentation. The study duration will reflect the anticipated persistence of the cells at the engraftment site(s). A duration of 1–3 weeks is suitable for rapidly cleared products while 6–12 months may be necessary for products with a sustained presence. Longer study durations often are warranted for products given in multiple doses over time or where cell engraftment and integration are expected to be permanent. The duration may be influenced by the nature of the test system. For instance, a 1-month period may be suitable if the question is to assess the impact of implanted cells on early (predebilitation) stages of an animal disease model. The dose levels (as defined by variable numbers of implanted cells) will vary with the scientific question. Common doses range from 1  104 to 1  108 cells per site. Chosen doses usually include one at or near the estimated minimal efficacious dose (to examine potential effects that might be observed in patients) as well as one representing the maximum feasible number of cells that can be injected (to assess tumorigenic potential). For preclinical studies supporting clinical trials, the cells should be the GMP product proposed for clinical use. The route of administration for preclinical studies should be identical to that proposed for human clinical trials. The same delivery device slated for clinical use should be employed to

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introduce cells to the chosen implantation site. Where feasible, cells are engrafted at the orthotopic site in animals that is equivalent to the one proposed for clinical use. Preclinical safety studies of CBMPs often include two or more time points. A common early time is set between 48 h and 7 days postinjection. This timing addresses the potential for acute toxicity related to the implanted cells and the procedure. A frequent intermediate time point occurs between 2 to 4 weeks and 3 months postimplantation. This timing explores the possibility of chronic toxicity and provides a measure of cell integration at the implantation site. Tumorigenicity risk has been primarily linked to CBMP derived from ESCs and iPSCs (Sato et al., 2019). Tumorigenicity is defined as “the capacity of a cell population inoculated into an animal model to produce a tumor by proliferation at the site of inoculation and/or at a distant site by metastasis”; the related term oncogenicity is defined as “the capacity of an acellular agent to induce normal animal cells to become tumor cells” (WHO, 2010). Sufficient time points should be used in tumorigenicity and oncogenicity studies to assess chronic and off-target effects. Longterm time points of 6 months, or less often 9 or 12 months, may be selected for this purpose since longer time points typically are necessary to adequately evaluate the potential for tumor formation related to stem cell implantation or the chronic placement of associated devices (per ISO 10993). Recovery groups are not necessary since CBMPs cannot be removed. CBMP AS SUBSTRATES TO DELIVER THERAPEUTIC MOLECULES

For CBMPs that serve as carriers for other bioactive entities, considerations for assessing stem cell safety concerns are added to those for the intended therapeutic molecules. For example, CAR-T therapies employ ex vivo– modified HSCs as substrates for introducing gene therapies to patients. Continuous cell lines (CCLs) used as substrates for biological vaccines have to be shown to lack tumorigenicity. Immortalized CCLs are the substrates of choice because of the ease with which they can be transfected and engineered as well as their rapid growth to achieve a high density. Primary cell cultures (PCCs) from different tissues have been used

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worldwide in producing live and inactivated viral vaccines, such as Madin Darby Canine Kidney (MDCK) cells used in the manufacture of influenza vaccines. Other mammalian cell lines employed in biomedical research include epithelial and mesenchymal cells from chicken embryos (Intarapat and Stern, 2013), Vero kidney epithelial cells from monkeys, and Chinese hamster ovary (CHO) cells. Insect cell lines derived from mosquitos and Drosophila flies are also used in vaccine production. The aim is to generate a cell line that will not undergo neoplastic transformation once introduced into the host. An advantage of using insect cell lines as stem cell xenografts is because the mammalian tissue environment into which they will be injected is not compatible with further growth of the insect cells. The essential incompatibility of high-temperature vertebrate tissues with the growth requirements of low-temperature insect cells (among other variable conditions) reduces the risk of xenograft implantation. In vivo tests remain the current gold standard for assessing tumorigenicity and oncogenicity until robust in vitro methodologies to assess tumorigenicity have been developed. Conventional rodents cannot be used in tumorigenicity and oncogenicity studies because assays require inoculation of xenogeneic or allogeneic cells, which necessitates that the test system animals be rendered deficient in cytotoxic T lymphocyte (CTL) activity. The test systems most often used for in vivo tumorigenicity studies are the athymic nude (Foxn1nu) mouse or the NOD-SCID (nonobese diabetic with severe combined immunodeficiency disease [Prkdcscid]) mouse. The test systems recommended for oncogenicity studies are juvenile nude mice, hamsters, or rats (WHO, 2010). For novel cell lines, oncogenicity tests are performed in all three species, whereas for cell banks previously shown to be negative only one species (usually the juvenile nude mouse) is used to check the latest cell passage. In tumorigenicity and oncogenicity studies, the cells and cell lysates to be characterized must be from the in vitro cell passage that is going to be used for production of the vaccine. The cells typically must have been propagated to at least three population doublings beyond the limit to be used for the vaccine production run. The objective of such studies is to test if the tumorigenic phenotype or oncogeneic

potential of a banked cell line has significantly changed; any substantial differences relative to prior tests indicate that something affecting the characteristics of that cell bank has occurred (virus infection, mutation and oncogenic activation by a mutagen or stress, etc.) which will render the cell line not suitable for use. Because male athymic mice often display aggressive traits against each other when housed together, some laboratories prefer to use only female mice in tumorigenicity studies. The inoculation site needs to be technically easy and to give reliable results (e.g., subcutaneous) and ideally should be the same as in the clinical application to evaluate tumorigenic potential of the product in the tissue microenvironment similar to that intended in the clinical setting. The number of cells to be administered depends on the cell number to be used in the final GMP product but preferably will be 10-fold to 100-fold higher than the dose that the patients will receive. Positive control cells are used to assure that an individual test is valid by demonstrating that the animal model has the capacity to develop tumors from inoculated cells (i.e., a negative result is unlikely to be due to a problem with the in vivo model). The most common positive control cells used in preclinical studies are HeLa cervical carcinoma cells and Syrian hamster embryo (SHE) sarcoma cells. These cells should be sourced from a WHO-approved master cell bank. Usually, the negative control for such studies is a cell-free injection of the cell culture medium. The standard protocol regimen is that each animal in the celltreated groups should receive 5  107 viable cells, suspended in a volume of 0.1 mL of phosphatebuffered saline (PBS) or Dulbecco’s Modified Eagle Medium (DMEM) (WHO, 2010). A routine study consists of 10 animals dosed with test article cells, 10 animals dosed with positive control cells, and 2 animals dosed with cell-free cell culture medium to serve as negative procedural controls. The reduced number of negative control animal is warranted as the rates of spontaneous neoplastic disease in nude mice are very low (Bradley and Black, 2021), which offers both sufficient scientific justification and is in keeping with current animal welfare 3Rs objectives for replacement, reduction, and refinement of animals in biomedical research. Oncogenicity testing is performed with test article cell lysates and cell DNA. The test system

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used is new-born (i.e., 1000 tons/year, Annex X prescribes assays such as prenatal developmental toxicity study (OECD 414), extended one-generation reproductive toxicity study (OECD 443), or even carcinogenicity study; for ecotoxicological assessment, fate and behavior in the environment has to be determined. The registration dossier is submitted to ECHA, who performs a compliance check and evaluates individual registrations. The EU Member States

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FIGURE 12.4 Requirements of Annexes VI to X of REACH. According to the tonnage band of the chemical, dossiers must fulfill different requirements according to the respective Annex of REACH.

evaluate selected substances to clarify initial concerns for human health or for the environment. Authorities and ECHA’s scientific committees assess whether the risks of substances can be managed. Asia-Pacific To manufacture and/or import a product that includes a new chemical substance in Japan, studies and registration are necessary in accordance with the Chemical Substance Control Law (CSCL) under the auspices of the Ministry of Economy, Trade and Industry (METI). The purpose of CSCL is to prevent environmental pollution caused by chemical substances that pose a risk of impairing human health and interfere with the population and growth of flora and fauna. The scope covers chemical substances, which means compounds created through chemical reaction and industrial chemicals, which are not regulated otherwise (e.g., pesticides). For new chemicals, a notification to and evaluation by the government are required before manufacture or import. For existing chemicals, an annual report of manufacture/import volume and usage is mandatory. The government conducts risk assessment based on this annual notification and may request additional toxicity information from the manufactures/importers, if necessary. The risk assessment is conducted in two phases, both before and after placing the substance on the market. Based on the result of the risk assessment, METI may take measures to control risks

associated with the chemical. New chemicals will be assessed and evaluated by a premarketing notification and evaluation process and are divided into different categories: more than 1 ton/year, Small Volume (less than 1 ton/year), Intermediates, and Polymers of Low Concern (PLC). They will get a confirmation, unless there are no certain levels of risk concern, the volume is less than 10 tons/year (low volume), and there is no bioaccumulative or persistency risk. Depending on the stepwise risk assessment, new chemicals and existing chemicals are classified as General Chemicals, Priority Assessment Chemicals, Class II Specified Chemicals, Monitoring Chemicals, and Class I Specified Chemicals. The evaluation criteria are biodegradation, bioaccumulation, toxicity, and ecotoxicity with their respective OECD test guidelines (METI, 2017). New substance or hazardous substance registration in China is similar to REACH legislation in Europe or registration under TSCA in the United States and has to be done prior to manufacture or importation. China’s chemical inventory of existing chemical substances is IECSC, which stands for the Inventory of Existing Chemical Substances Produced or Imported in China (IECSC). Currently, there are approximately 45,000 substances listed in IECSC. Other countries in the Asia-Pacific region, e.g., Korea, Australia, and New Zealand, have similar regulations as compared to REACH or TSCA, whereas others have not or are still under development (e.g., India).

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5. REGULATORY STUDIES FOR REGISTRATION OF AGRO/BULK CHEMICALS To fulfill the requirements for the different endpoints (e.g., bioaccumulation, biopersistence, ecotoxicity, genotoxicity, mammalian toxicity), which are necessary for (re)registration of an (agro)chemical, several different studies must be performed. What they all have in common is that they must be performed according to existing, internationally accepted testing guidelines. The most relevant internationally accepted test guidelines are the OECD Guidelines for the Testing of Chemicals (OECD_GL, 2021), the Office of Prevention, Pesticides and Toxic Substances (OPPTS) or Office of Chemical Safety and Pollution Prevention (OCSPP) Test Guidelines for Pesticides and Toxic Substances issued by the US EPA (US_EPA, 2021), and the “Guidelines related to the study reports for the registration application of pesticide” issued by the JMAFF (JMAFF, 2000). All guidelines are regularly updated, and their most recent version must be used. Harmonized test guidelines reduce the burden on chemical producers and conserve scientific resources, including the minimal use of laboratory test animals. They also form a basis for work sharing and cooperation among all OECD countries and others. The OECD Guidelines for the Testing of Chemicals are a unique tool for assessing the potential effects of chemicals on human health and the environment. Accepted internationally as standard methods for safety testing, the guidelines are used by professionals in industry, academia, and government involved in the testing and assessment of chemicals (industrial chemicals, pesticides, personal care products, etc.). These guidelines are continuously expanded and updated to ensure they reflect the state-of-theart science and techniques to meet member countries regulatory needs. The guidelines are elaborated with the assistance of experts from regulatory agencies, academia, industry, environmental, and animal welfare organizations. OECD Test Guidelines are covered by the OECD MAD system. Under this system, laboratory test results related to the safety of chemicals that are generated in accordance with OECD Test Guidelines and OECD Principles of GLP are

accepted in all OECD countries and adherent countries for the purpose of safety assessment and other uses relating to the protection of human health and the environment. The OECD test guidelines are split into five sections: 1) Physical Chemical Properties, 2) Effects on Biotic Systems, 3) Environmental Fate and Behavior, 4) Health Effects, and 5) Other Test Guidelines. The US EPA’s test guidelines for pesticides and toxic substances specify EPA-recommended methods to generate data that are submitted to EPA to support registration of a pesticide under the FIFRA, setting of a tolerance or tolerance exemption for pesticide residues under section 408 of the FFDCA, or the decision-making process supporting potential regulation of an industrial chemical under the TSCA act. Prior to April 22, 2010, OCSPP was known as OPPTS. To distinguish these guidelines from guidelines issued by other organizations, the numbering convention adopted in 1994 specifically included OPPTS as part of the guideline’s number. Any test guidelines developed after April 22, 2010, will use the new acronym (OCSPP) in their title. The test guidelines of the EPA are organized by series numbers in 11 chapters, for example, Series 870 for Health Effects Test Guidelines (subdivided into different groups that include Group A for Acute Toxicity, Group B for Subchronic Toxicity, Group C for Chronic Toxicity) and Series 890 for Endocrine Disruptor Screening Program Test Guidelines. The EPA’s test guidelines are harmonized with those established by the OECD and EPA works closely with other government agencies and with other countries through the OECD to facilitate the harmonization of test guidelines. The Japanese test guidelines are very similar to the OECD and US EPA test guidelines and differ only in minor variations. Nevertheless, all guidelines must be read very carefully and especially pathology endpoints (specifically organ weight assessment, organ preservation, histopathology) may vary in some details. Most regulatory authorities of other countries rely on those international accepted test guidelines issued by OECD, US EPA, or JMAFF, whereas some countries, e.g., India (MoA&FW, 2017) or China (National Standard of the People’s Republic of China, Toxicological Test Methods for Pesticides Registration, GB/T

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15670, Ministry of Agriculture of the People’s Republic of China (China, 2017)), issued their own test guidelines, which are very similar to those previously mentioned. During the last few years, beside general toxicity testing especially regarding carcinogenicity and reproductive toxicity, endocrine disruption (ED) assessment has become more and more important, which is reflected in several international initiatives (e.g., Endocrine Disruptor Screening Program by EPA, EDSP), regulations, and guidance documents (OECD_GD_150, 2018), released by regulatory authorities. The EU introduced new scientific criteria for the determination of ED properties (Commission Regulation EU 2018/605) (EU_Commission, 2013), which have been applicable since 2018 to all applications for the approval of active substances, including pending applications. Therefore, all dossiers and assessment reports should include an assessment of the substance’s ED properties. A guidance document on the topic “Guidance for the identification of endocrine disruptors in the context of Regulations (EU) No 528/2012 and (EC) No 1107/2009” (ECHA_EFSA, 2018) has been developed for the identification of endocrine disruptors in accordance with the new criteria, as defined in the respective Commission Regulation. The EPA’s EDSP uses a two-tiered approach to screen pesticides, chemicals, and environmental contaminants for their potential effect on estrogen, androgen, and thyroid hormone systems. Tier 1 screening data are used to identify substances that have the potential to interact with the endocrine system. Chemicals that go through Tier 1 screening and are found to exhibit the potential to interact with the estrogen, androgen, or thyroid hormone systems will proceed to Tier 2 for testing. Tier 2 testing data identify any adverse endocrine-related effects caused by the substance and establish a quantitative relationship between the dose and that adverse effect. The results of Tier 2 testing are combined with other hazard information and exposure assessment on a given chemical resulting in the risk assessment. Regarding assessment of ED properties, the main difference between European Regulatory Authorities as EFSA/ECHA and the US EPA is a hazard-driven assessment with clear cut-off

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criteria for the EU and a risk-based assessment for the United States. This means that any substance with a proven ED property cannot be registered any longer in the EU, whereas the US EPA also performs a classic risk-based assessment for such substances.

5.1. Toxicity Studies For registration of an (agro)chemical, the complete registration dossier must include robust and reliable test results covering different endpoints such as efficacy, (eco)toxicity, mutagenicity, persistence, and bioaccumulation. Toxicity studies to determine hazard to humans and domestic animals have to be performed in different species (e.g., rat, mouse, rabbit, dog) from acute to subchronic to chronic/long-term exposure times with different application routes (e.g., oral, inhalation, dermal) and endpoints. The mandatory endpoints for an (agro)chemical include information for hazard and risk assessment of carcinogenicity, reprotoxicity, teratogenicity, and mutagenicity. This includes studies starting at acute oral toxicity up to combined chronic/carcinogenicity studies in at least two species (rat and mouse), reproductive and teratogenicity studies in at least two species, as well as neurotoxicity studies. For mammalian testing, the most relevant health effects test guidelines are described in section 4 of OECD Test Guidelines (OECD_GL, 2021) and in Series 870 of OPPTS/OCSPP (US_EPA, 2021). Here, in vivo and in vitro test guidelines are summarized from acute to chronic exposure up to carcinogenicity studies as well as reproductive, genotoxicity, and neurotoxicity testing. In general, for active ingredients of plant protection products, testing starts with first dose range finding and palatability studies in one species and with a limited number of animals. These are followed by 28-day (OECD 407: Repeated Dose 28-day Oral Toxicity Study in Rodents; OPPTS 870.3050: Repeated Dose 28-day Oral Toxicity Study in Rodents) and 90-day (OECD 408: Repeated Dose 90-Day Oral Toxicity Study in Rodents; OPPTS 870.3100: 90-Day Oral Toxicity in Rodents) studies for dose setting for the combined chronic/carcinogenicity studies (OECD 453: Combined Chronic Toxicity/Carcinogenicity Studies; OPPTS 870.4300: Combined Chronic Toxicity/Carcinogenicity) in at least two species

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(rat and mouse). The chronic/carcinogenicity toxicity study is an important part of the registration dossier and has relevant impact on the classification of the test substance. Therefore, a thorough and careful gross examination, and histopathologic assessment by an experienced toxicologic pathologist, is mandatory. In addition, a peer review should be performed to assure the results as described in the OECD Guidance Document 116 (OECD_GD_116, 2014). Furthermore, test results from subchronic toxicity studies with nonrodent species are mandatory, which means that at least a 28-day as well as a 90-day oral study (OECD 409: Repeated Dose 90-Day Oral Toxicity Study in NonRodents; OPPTS 870.3150: 90-Day Oral Toxicity in Nonrodents) in dogs must be performed. The scientific and regulatory necessity of the chronic 1-year dog study (OECD 452: Chronic Toxicity Studies; OPPTS 870.4100: Chronic Toxicity) is under discussion for years and its necessity seems doubtful, which means that many regulatory authorities do not require this study type anymore. Therefore, the routine inclusion of a 1-year dog study as a mandated regulatory requirement for the safety assessment of pesticides is no longer justifiable and a globally harmonized approach should be taken to match the latest legislation worldwide (Kobel et al., 2010, 2014; Spielmann, 2019). Endpoints for potential reproductive and/or developmental toxicity are derived from several different study types, including 1- or 2generation reproduction toxicity studies (OECD 416: Two-Generation Reproduction Toxicity, OECD 443: Extended One-Generation Reproductive Toxicity Study; OPPTS 870.3800: Reproduction and Fertility Effects) and prenatal developmental studies (OECD 414: Prenatal Developmental Toxicity Study; OPPTS 870.3700: Prenatal Developmental Toxicity Study). Finally, neurotoxicity testing should clarify potential toxicity on central and peripheral nervous system (see Nervous System, Vol 4, Chap 8). For this endpoint, a neurotoxicity screening battery (OECD 424: Neurotoxicity Study in Rodents; OPPTS 870.6200: Neurotoxicity Screening Battery) is applied, from acute up to chronic testing, depending on study results. In the case of potential developmental neurotoxicity issues, a developmental neurotoxicity (DNT) study (OECD 426: Developmental Neurotoxicity Study; OPPTS 870.6300: Developmental Neurotoxicity Study) must be performed. Alternatively, intended cohorts (Cohort 2A and 2B) of the

Extended One-Generation Reproductive Toxicity Study (OECD 443) assess the potential impact of chemical exposure on the developing nervous system. Those investigations require highly standardized DNT neuropathology evaluation, particularly procurement of highly homologous brain sections and collection of the most reproducible morphometric measurements (Bolon et al., 2013; Garman et al., 2016). Interpreting DNT neuropathology data and their presumptive correlation with neurobehavioral data requires an integrative weight-of-evidence approach including consideration of maternal toxicity, body weight, brain weight, and the pattern of findings across brain regions, doses, sexes, and ages (Garman et al., 2016). In addition to mandatory regulatory studies for (re)registration of agrochemicals it is common practice to perform mechanistic studies to elucidate the mode-of-action of certain histopathological findings, e.g., liver tumors in rodents. This might be important in the discussion of possible human relevance and therefore for the setting of ADIs and derived classification. As already mentioned above, toxicity studies for (re)registration of new chemicals – beside agrochemicals – are also performed according to the mentioned test guidelines. The scope of the studies might depend on the intrinsic properties of the substance, on the tonnage band (e.g., under REACH), on the intended use, and so on. Therefore, a very complex spectrum (e.g., Extended 1-Generation Reproductive Toxicity Study, OECD 443) of toxicity studies must be performed for each substance.

5.2. Ecotoxicity Studies Ecotoxicity studies, which investigate the biotic and environmental effects of (agro)chemicals, are summarized in section 2 of OECD Test Guidelines (Effects on Biotic Systems) (OECD_GL, 2021) and in Series 850 (Ecological Effects test Guidelines) and 890 (Endocrine Disruptor Screening Program test Guidelines) of OPPTS/OCSPP (US_EPA, 2021). They are also part – beside others – of the “Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption” (OECD_GD_150, 2018). Data from those studies should determine hazard to nontarget organisms and possible toxicological effects, including ED effects, on birds, fish, terrestrial, and aquatic invertebrates. These tests include

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short-term acute, subacute, and reproduction testing arranged in a hierarchical or tier system. Under the EDSP of US EPA, the Amphibian Metamorphosis (Frog) Assay (AMA; OPPTS 890.1100; OECD 231) and the Fish Short-Term Reproduction Assay (FSTRA; OPPTS 890.1350; OECD 229) are listed as Tier 1 studies. Tier 2 studies include the Medaka Extended One Generation Reproduction Test (MEOGRT; OPPTS 890.2200; OECD 240) and the Larval Amphibian Growth and Development Assay (LAGDA; OPPTS 890.2300; OECD 241) (see Figures 12.5 and 12.7). In the OECD GD 150, the AMA and FSTRA are listed under Level 3 (in vivo assays providing data about selected endocrine mechanisms/pathways) for nonmammalian toxicology. Level 4 (in vivo assays providing data on adverse effects on endocrinerelevant endpoints) describes Fish Early Life Stage Toxicity Test (FELS; OECD 210; OPPTS 850.1400), LAGDA, and Fish Sexual Development Test (FSDT; OECD 234), whereas MEOGRT and Zebrafish Extended One Generation Reproduction Test (ZEOGRT, draft OECD TG) are listed under Level 5 (in vivo assays providing more comprehensive data on adverse effects on endocrine-relevant endpoints over more extensive parts of the life cycle of the organism) (see Figures 12.6 and 12.7). Although (histo)pathological investigation in these studies is performed on an individual basis, the intention of these studies is to evaluate a possible impact on the population and the ecosystem. The FSTRA (OPPTS 890.1350; OECD

FIGURE 12.5

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229) describes an in vivo screening assay: sexually mature male and spawning female fish are placed together and exposed to a chemical during a limited part of their life cycle (21 days). At termination of the 21-day exposure period, two biomarker endpoints are measured in males and females as indicators of endocrine activity of the test chemical (vitellogenin and secondary sexual characteristics). Gonads are also preserved, and histopathology may be evaluated to assess the reproductive fitness of the test animals and to add to the weight of evidence of other endpoints. Generally, beside sex determination and general histopathological assessment of the gonads, staging of testis and ovary will be performed as described in OECD Guidance Document 123 (OECD_GD_123, 2010) and the maturity index can be determined (Baumann et al., 2013). The FSDT (OECD 234) protocol is in principle an enhancement of FELS (OECD TG 210; OPPTS 850.1400), where fertilized eggs are placed in test chambers and exposed to a range of concentrations of the test chemical. This is continued for a species-specific time period that is necessary for the control fish to reach a juvenile life stage. In FSDT, the exposure is continued until the fish are sexually differentiated, i.e., about 60 days posthatching, to assess early life-stage effects and potential adverse consequences of putative endocrine disrupting chemicals (e.g., estrogens, androgens, and steroidogenesis inhibitors) on sexual development. The combination of

Tests/Assays of the Endocrine Disruptor Screening Program (US EPA).

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FIGURE 12.6 The organisation for economic Co-operation and development (OECD) Conceptual framework for testing and assessment of endocrine disrupting chemicals. Taken from “Revised Guidance Document 150 on Standardised Test Guidelines for Evaluating Chemicals for Endocrine Disruption.” https:// www.oecd-ilibrary.org/environment/guidance-document-on-standardised-test-guidelines-for-evaluating-chemicals-for-endocrine-disruption-2nd-edition_ 9789264304741-en OECD Series on Testing and Assessment, OECD Publishing, Paris.

FIGURE 12.6

Cont’d.

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FIGURE 12.7 Comparison of test/assay of Endocrine Disruptor Screening Program (EDSP) and OECD Guidance Document (GD) 150 for nonmammalian toxicity testing.

the two core endocrine endpoints, vitellogenin (VTG) concentration and phenotypic sex ratio, enables the test to indicate the mode of action of the test chemical. The sex ratios (proportions of sex) are determined via gonad histology, whereas additional histopathology (evaluation and staging of oocytes and spermatogenetic cells) is optional. The MEOGRT (OECD 240; OPPTS 890.2200) and ZEOGRT (draft OECD TG) describe a comprehensive test based on fish exposed over multiple generations to give data relevant to ecological hazard and risk assessment of chemicals, including suspected endocrine disrupting chemicals (EDCs, see New Frontiers in Endocrine Disruptor Research, Vol 3, Chap 12). Exposure continues until hatching (until 2 weeks postfertilization) in the second generation. Several biological endpoints are measured, namely, population relevant parameters including survival, gross development, growth and reproduction as well as vitellogenin mRNA or protein, phenotypic secondary sex characteristics as related to genetic sex, and histopathology. Histopathology includes – beside histological evaluation of gonadal sex – evaluation of kidneys, liver, and gonads with staging of testis/ovary as described in OECD Guidance Document 227 part 1–4 (OECD_GD_227, 2015). The AMA (OPPTS 890.1100; OECD 231) is a screening assay intended to empirically

identify substances which may interfere with the normal function of the hypothalamic–pituitary–thyroid (HPT) axis. Study design calls for exposure of stage 51 Xenopus laevis tadpoles for a minimum of 21 days. The observational endpoints include hind limb length, snout to vent length (SVL), developmental stage, wet weight, and daily observations of mortality as well as thyroid gland histopathology as described in OECD Guidance Document 82 (OECD_GD_82, 2007). The LAGDA (OECD 241; OPPTS 890.2300) describes a toxicity test using an amphibian species that considers growth and development from fertilization through the early juvenile period. It is an assay (typically 16 weeks) that assesses early development, metamorphosis, survival, growth, and partial reproductive maturation. Endpoints evaluated during the course of the exposure include those indicative of generalized toxicity: mortality, abnormal behavior, and growth determinations (length and weight), as well as endpoints designed to characterize specific endocrine toxicity modes of action targeting estrogen, androgen, or thyroid-mediated physiological processes. Histopathology includes evaluation of gonads, reproductive ducts, kidneys, and liver as described in OECD Guidance Document 228 (OECD_GD_228, 2015).

6. TOXICOLOGIC PATHOLOGY FINDINGS AND ASSESSMENT

6. TOXICOLOGIC PATHOLOGY FINDINGS AND ASSESSMENT In mammalian animal studies, necropsy, slide reading, and assessment of pathological findings in regulatory studies for registration of (agro) chemicals do not differ from other regulatory animal studies performed, for example, for pharmaceutical compounds. Standard trimming procedures (Ruehl-Fehlert et al., 2003; Kittel et al., 2004; Morawietz et al., 2004) and nomenclature according to International Harmonization of Nomenclature and Diagnostic Criteria for Lesions in Rats and Mice (INHAND) (Mann et al., 2012) should be used. The developmental target of a new pesticide (e.g., insecticide, herbicide, or fungicide) is, in general, not to treat disease in humans or animals but to protect plants against pests. Therefore, the main developmental goal is to have high efficiency regarding plant protection with the lowest toxicological profile possible, which means test animals are treated at limit dose (1000 mg/ kg bw/d) or maximum tolerable dose (MTD). The main hurdles for (re)registration of an (agro)chemical are carcinogenicity, reproductive toxicity/teratogenicity, mutagenicity, and endocrine disruption properties. Besides regulatory studies, mandatory for any registration worldwide, quite a number of different mechanistic studies – in vitro as well as in vivo – are performed to elucidate possible mode of actions in test species and to determine their possible human relevance and environmental impact. A common finding (see Figure 12.8) in animal studies with (agro)chemicals is liver enzyme induction and its pathological correlates of increased liver weight, hepatocellular hypertrophy, increase in cell proliferation, and potential for hepatocarcinogenesis (Hall et al., 2012) (see Liver and Gall Bladder, Vol 4, Chap 2). This might induce secondary or subsequent findings in other organs, e.g., hypertrophy/hyperplasia of thyroid gland due to increased degradation of T3 and T4 in the liver (Huisinga et al., 2020) (see Endocrine System, Vol 4, Chap 7). As endocrine disruption is a critical endpoint in assessment of (agro) chemicals, the presumed mode of action and possible human relevance has to be demonstrated by additional investigations, outlined in the respective testing guidelines and guidance documents by regulatory authorities (see New Frontiers in Endocrine Disruptor Research, Vol 3, Chap 12). A disturbance of thyroid hormone balance might

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also impact neurodevelopmental outcome, which means that often additional developmental neurotoxicity studies must be performed. Appropriate techniques to investigate primary or secondary neurodevelopmental findings are under discussion (Gilbert et al., 2020). Other findings, which might be seen more often in animal studies with (agro)chemicals, are Leydig cell hyperplasia or tumors in the testes (Rasoulpour et al., 2014), alpha 2u-globulin nephropathy (Hard, 2008), or findings specific for certain compound classes. For example, 4hydroxyphenylpyruvate dioxygenase (HPPD) inhibitor herbicides induce tyrosinemia in animals with typical findings such as macroscopic corneal opacity (Lock et al., 2006). Many chemicals interact with lysosomes resulting in accumulation of endogenous or exogenous material in lysosomes (Lenz et al., 2018) leading to findings such as phospholipidosis. The adversity of each finding has to be assessed and described in the individual study report (Palazzi et al., 2016). If possible, the human relevance of findings should be assessed. As some of the examples mentioned above are clearly only rat-specific findings with no human relevance (e.g., alpha 2u-globulin nephropathy in male rats), those findings are not relevant for deriving MRL, ADI, or ARfD. Pathology is done not only for animal studies performed in mammalian species, but also in regulatory studies with other species such as fish and amphibians, which are relevant for ecotoxicity assessment (see Animal Models in Toxicologic Research: Non-mammalian, Vol 1, Chap 22). As already mentioned above, some of those ecotoxicity studies regularly include histopathology, e.g., FSTRA, FSDT, MEOGRT, AMA, or LAGDA. Histopathology in ecotoxicity studies is a common endpoint in toxicologic bioassays and has become more and more important for detection of possible endocrine disruptive properties and signs of systemic toxicity. Therefore, histopathology is not limited to gonads. Histopathological evaluation and assessment of fish (e.g., fathead minnow, medaka, zebrafish) and amphibia (e.g., frog, Xenopus laevis) used for regulatory ecotoxicity testing require specialized knowledge and extensive experience in this area. Otherwise, assessment and interpretation of data might not be credible (Wolf and Wheeler, 2018). Because of the subjective nature of the histopathology endpoint, and the advanced level of specialized training required for its effective utilization, the reliability of histopathology data

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FIGURE 12.8 Examples of common lesions in toxicity studies for (agro)chemicals. 1 (control) þ 2 (high dose): Liver, Crl:WI(Han) rat, female Centrilobular hepatocellular hypertrophy (arrow), grade 3, HE stain, 100  3 (control) þ 4 (high dose): Kidney, Crl:WI(Han) rat, male Alpha 2u-globulin nephropathy with intratubular eosinophilic droplets (arrows) and basophilic tubules (star), grade 3, HE stain, 100/400  5 (control) þ 6 (high dose): Pancreas, Crl:WI(Han) rat, male Vacuolation, acinar and ductal (phospholipidosis), grade 3, HE stain, 200. II. PRODUCT-SPECIFIC PRACTICES FOR SAFETY ASSESSMENT

7. SUMMARY AND CONCLUSIONS

can be inconsistent (Wolf and Maack, 2017). Differentiating salient histopathologic changes from normal anatomic features or tissue artifacts can be decidedly challenging, especially for the novice fish pathologist. As a consequence, findings of questionable accuracy may be reported inadvertently, and the potential negative impacts of publishing inaccurate histopathologic interpretations are not always fully appreciated (Wolf et al., 2015). A relevant endpoint in fish studies is gonadal development or sex reversal, therefore special emphasis is on gonadal histopathology (Ankley and Johnson, 2004). Besides altered testicular staging grades, common findings in the testis include increased interstitial cells, increased spermatogonia, proteinaceous fluid, testicular germ cell degeneration, testicular oocytes, mineralization, or granulomatous inflammation (OECD_GD_123, 2010). One cause of increased interstitial cells can be decreased circulating androgens due to antiandrogens, steroidogenesis synthesis inhibitors, or possibly exogenous estrogens as occurs with ketoconazole. Increased spermatogonia can be seen after exposure to exogenous estrogens (e.g., 17b-estradiol), in which the overall appearance of testis is similar to immature testis, but the tubules of the testis are filled with mature spermatozoa and the exogenous estrogen causes maturation arrest. Testicular oocytes can be an endocrine-specific finding induced by estrogens or antiandrogens but can also be seen spontaneously in fathead minnow control males. In female fish, decreased postovulatory follicles, decreased yolk formation, changed ovarian stage scores, increased oocyte atresia, or spermatogenesis can be commonly seen. For example, fadrozole, an aromatase inhibitor, causes decreased yolk formation in the ovary due to decreased estrogen status as well as perifollicular cell hyperplasia/ hypertrophy in Japanese medaka. Increased oocyte atresia can be endocrine related but is also seen as spontaneous background lesion due to normal egg resorption (OECD_GD_123, 2010). Histological processing and histopathology is challenging for the MEOGRT/ZEOGRT: beside evaluation of the gonads, at least liver and kidney have to be evaluated with additional optional evaluation of thyroid gland, eye, and pituitary gland (OECD_GD_227, 2015). Therefore, assessment of normal variation versus artifact and treatment-related finding is even more complex. Common findings for the liver are increased/decreased basophilia, increased/ decreased vacuolation, or cystic degeneration.

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In the kidney, increased/decreased tubular eosinophilia as well as an estrogen-induced nephropathy can be seen. The thyroid gland can reveal follicular cell hypertrophy/hyperplasia or hyaline degeneration. It should be also considered that anatomical differences regarding certain organs, e.g., testis, exist between fish species. The fathead minnow has separate right and left testes, and spermatogenesis occurs simultaneously throughout the testis, whereas Japanese medaka have fused right and left testes and spermatogenesis occurs from the periphery to the central efferent duct. The AMA and LAGDA are primarily designed for detection of ED properties and the histopathology endpoint in AMA is thyroid gland histopathology (OECD_GD_82, 2007). In addition, liver, kidneys, gonads, and gonadal ducts are investigated in LAGDA (OECD_GD_228, 2015). Thyroid findings include follicular cell atrophy, follicular cell hypertrophy, or colloid changes. Other findings which can be seen include: increased/decreased vacuolation (liver), changed gonadal/gonadal duct stage scores, proteinaceous fluid (kidney), testicular oocytes (testis), estrogen-induced nephropathy (kidney), or increased oocyte atresia (ovary).

7. SUMMARY AND CONCLUSIONS (Agro)chemicals are widely regulated and a large amount of testing must be performed before (re)registration. The registration process differs in different countries and regions but is overall highly complex, although much harmonization is already done. Besides registration of agrochemicals, the regulation of chemicals has become much more complex over the last few decades and new legislation has been introduced, e.g., REACH in Europe. In addition to the well-known endpoints of carcinogenicity, reproductive toxicity/teratogenicity, and mutagenicity, the hot topic of endocrine disruption has been a dominating issue over the last few years, especially for (re) registration of an (agro)chemical (see New Frontiers in Endocrine Disruptor Research, Vol 3, Chap 12). All scientists involved in developing, testing, and registering an (agro)chemical, together with the regulatory authorities, have the responsibility to only bring products to market which are safe for humans, animals, and the environment.

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Abbreviation ACVM

ARfD CAA CCPR CEFIC CLP CSCL CSS EC ECHA ED EDSP EFSA EU FAMIC FELS FFDCA FIFRA FQPA FSC FSDT FSTRA GAP GHS

Name Good Laboratory Practice

IECSC

Amphibian Metamorphosis (Frog) Assay Agencia Nacional de Vigilancia Sanitaria Australian Pesticides and Veterinary Medicines Authority Acute Reference Dose Consumer Affairs Agency Codex Committee on Pesticide Residues The European Chemical Industry Council Classification, Labelling and Packaging of substances and mixtures Chemical Substance Control Law Chemicals Strategy for Sustainability European Commission European Chemicals Agency Endocrine Disruption Endocrine Disruptor Screening Program European Food Safety Authority European Union Food and Agricultural Materials Inspection Center Fish Early Life Stage Toxicity Test Federal Food, Drug, and Cosmetic Act Federal Insecticide, Fungicide, and Rodenticide Act Food Quality Protection Act

JMAFF LAGDA MAD

Inventory of Existing Chemical Substances Produced or Imported in China Japanese Ministry of Agriculture, Forestry and Fisheries Larval Amphibian Growth and Development Assay Mutual/multilateral Acceptance of Data

MAF MAFF MEOGRT METI MHLW

Ministry of Agriculture and Forestry Ministry of Agriculture, Forestry and Fisheries Medaka Extended One Generation Reproduction Test Ministry of Economy, Trade and Industry Ministry of Health, Labour and Welfare

MoARA MPI MRL OCSPP OECD OPPT OPPTS PCPA PMN PMRA PRIA RAR REACH

Food Safety Commission Fish Sexual Development Test Fish Short-Term Reproduction Assay Good Agricultural Practice Globally Harmonised System of Classification and Labelling of Chemicals

RMS TSCA US EPA ZEOGRT

Ministry of Agriculture and Rural Affairs Ministry for Primary Industries Maximum Residue Limit Office of Chemical Safety and Pollution Prevention Organisation for Economic Co-operation and Development Office of Pollution Prevention and Toxics Office of Prevention, Pesticides and Toxic Substances Pest Control Products Act Premanufacture Notice Health Canada’s Pest Management Regulatory Agency Pesticide Registration Improvement Act Renewal Assessment Report Registration, Evaluation, Authorisation and Restriction of Chemicals Rapporteur Member State Toxic Substances Control Act US Environmental Protection Agency Zebrafish Extended One Generation Reproduction Test

8. GLOSSARY

II. PRODUCT-SPECIFIC PRACTICES FOR SAFETY ASSESSMENT

AMA ANVISA APVMA

Abbreviation GLP

12. SAFETY ASSESSMENT OF AGRICULTURAL AND BULK CHEMICALS

ADI

Name Approvals and Agricultural Compounds and Veterinary Medicines Group Acceptable Daily Intake

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C H A P T E R

13 Preparation of the Anatomic Pathology Report for Toxicity Studies Kevin B. Donnelly1, Magali R. Guffroy2 1

Eli Lilly and Company, Indianapolis, IN, United States, 2Preclinical Safety, AbbVie Inc., North Chicago, IL, United States O U T L I N E

1. Introduction

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3. Developing the Data for the Anatomic Pathology Report 3.1. Study Protocol 3.2. Importance of Assessing Correlative Data Before Beginning Microscopic Review of the Tissues 3.3. Value of Data from More than One Time Point 4. Reversibility 4.1. Presentation and Interpretation of Organ Weight Data 4.2. Presentation and Interpretation of Gross Observations

5. Text Tables

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6. The Anatomic Pathology Report Discussion

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7. The Anatomic Pathology Report Conclusion

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8. Signing the Anatomic Pathology Report

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9. Examples to Be Avoided in Interpreting/ Presenting Data in the Anatomic Pathology Report

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10. Peer Review and Pathology Working Groups

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11. Conclusion

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References

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1. INTRODUCTION This chapter describes the assessment and reporting of the anatomic pathology findings in a toxicity study conducted under Good Laboratory Practices (GLP) or non-GLP conditions for test articles including chemicals, biologics, or devices. Accompanied by descriptions in the narrative of the anatomic pathology report, the findings must be organized such that they can be readily presented in computer generated, summary Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-12-821047-5.00009-9

4.3. Presentation and Interpretation of Microscopic Observations 499

incidence tables and as in-text tables of selected findings that can be incorporated directly into the body of the study report. Tables are important in presenting to the reader the significant findings in a study in a format that facilitates the visualization of the important findings by both dose and sex, and possibly time. The anatomic pathology data must be interpreted in light of the available animal clinical observations, clinical pathology findings, toxicokinetic data, and any other unique parameters collected during the study.

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Copyright Ó 2023 Elsevier Inc. All rights reserved.

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In most toxicity studies, it is the data in the pathology report that identifies the target organ(s) of toxicity and contribute directly to the determination of a no-observed-adverseeffect level (NOAEL). If not factually correct, complete, clearly written, and easily understood, the pathology report can lead to considerable effort on the part of others to ensure that the actual changes and meaningful data are communicated correctly. Failure to do so may potentially lead to a permanent misunderstanding of the results of the study. A poorly written report often occurs when the visualization, presentation, and evaluation of the data are not clear, which can complicate the safety assessment process and delay market authorizations. While quality control procedures and peer review help mitigate this risk, a rigorous and well-written initial report by the study pathologist is critical and must accurately and comprehensively represent the important findings. The key elements of the anatomic pathology report should seamlessly integrate into the Results Section of the body of the report for the entire study. For further information on the pathology report in toxicity studies see Morton et al. (2006).

2. OBJECTIVE AND AUDIENCE FOR THE ANATOMIC PATHOLOGY REPORT The objective of the anatomic pathology report prepared for a toxicity study is to present a clearly written description of the pathophysiologic alterations that are test article–induced in the animal model. This evaluation incorporates the relationship between the findings or lesion(s), the dose level(s) administered, and the associated local or systemic exposures. The lesion descriptions should be accurately recorded using terms, descriptions, and grading systems that are understood by the reader and represent best practice (e.g., INHAND terminology; see Nomenclature and Diagnostic Resources in Anatomic Toxicologic Pathology, Vol 1, Chap 25). They must create an understanding of the lesion, where it is located, how it is distributed, the approximate timecourse in the tissue (acute, subacute, chronic), overall severity, and, whenever possible, its etiopathogenesis. The report should correlate the

microscopic alterations with animal clinical observations, organ weights, gross observations, clinical pathology parameters, and/or other biomarkers utilized in the study. When they occur, the report should also describe and explain findings associated with morbidity leading to unscheduled termination or spontaneous mortality of animals. If a reversibility period is included in the study, the anatomic pathology findings present at the end of the reversibility period should be described and interpreted in light of the observations made at the end of the dosing period based on the duration of the reversibility period and/or potential mechanism of action. There are at least four groups among the audience for the anatomic pathology report. The first group consists of other principal investigators involved in the study including the clinical pathologist, toxicokineticist, and the study director, of whom the latter will incorporate the findings from the anatomic pathology report into the final report for the entire study. The second group includes the sponsors of the study, such as the scientists and managers of the organization for whom the study was conducted, who make decisions about whether to advance or how to advance the test article. The third group are the clinical trial scientists, such as the clinical pharmacologists and physicians, who will design, conduct, monitor, and interpret the test article’s behavior in human volunteers or patients. The fourth group are the regulatory reviewers from global agencies who will assess the completeness, accuracy, and interpretation of the data and reach regulatory conclusions. Ultimately, the anatomic pathology report should contribute to the characterization of the nonclinical safety profile of the test article in the particular animal species with reference to the experimental model used and the duration of the study, and contribute to the determination and justification of an NOAEL in the study.

3. DEVELOPING THE DATA FOR THE ANATOMIC PATHOLOGY REPORT As the pathologist sets out to interpret, describe, and report the anatomic pathology findings in a toxicity study, it is important to

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3. DEVELOPING THE DATA FOR THE ANATOMIC PATHOLOGY REPORT

begin with the end in mind. The anatomic pathologist’s report should include a concise but thorough narrative and also standardized in-text summary tables for data presentation and analysis.

3.1. Study Protocol Developing the data for the anatomic pathology report requires that the pathologist first has an in-depth understanding of the study protocol and its stated objective(s), the general methods used in the study, and which, if not all, dose groups are to be evaluated microscopically. The protocol will provide key information on the number of dose levels and animals per dose group, the route of administration, the nature of the control/vehicle group(s), and the formulation (with or without special excipients) used in the study. It also identifies if there is a reversibility period and how the tissues from the animals in that part of the study are to be evaluated. Being familiar with the protocol will ensure that the pathologist is aware of the tissues to be weighed, collected at necropsy, and processed and evaluated microscopically. The protocol will also describe the statistical methods and the levels of significance to be used to determine if specific data in the dosed animals is to be considered similar or different than the same data in the concurrent controls. The protocol may also reference the historical data for the animal species to be used in the interpretation of study findings. For further information on designs of toxicity and other nonclinical safety assessment studies see Avila et al. (2020).

3.2. Importance of Assessing Correlative Data Before Beginning Microscopic Review of the Tissues Before beginning a microscopic assessment of the tissue sections from a toxicity study, the pathologist should review previous published data including toxicity study reports, if available, and carefully review the animal clinical observations, organ weight data, gross observations, and clinical pathology data from the study. This review may identify previously identified target

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organ(s) and guide the microscopic assessment. Specialized biomarkers may be present based on the mechanism of action of the test article or a previously observed finding. It is important that the pathologist be aware of the nature of the biomarker and how its significance and interpretation are to be used if the suspected change is present in the species and at the doses used in the current study. For further reading on the interpretation of clinical pathology data see Interpretation of Clinical Pathology Results in Nonclinical Toxicity Testing, Vol 2, Chap 14. The toxicokinetic data for small molecules or biotherapeutics, and other assessments of biodistribution (e.g., gene therapy agents or implanted medical devices) should also be made available to and utilized by the pathologist in support of their assessment of the pathology data. Considering the magnitude of the separation of exposures between dose levels as it relates to the occurrence, incidence, or severity of pathology findings between groups helps the pathologist judge dose relatedness of the observed findings. The ability to assign adversity or nonadversity of a test article–related pathology finding will facilitate the eventual determination of a study NOAEL. For detailed information on pharmacokinetics and toxicokinetics in toxicity studies see ADME Principles in Small Molecule Drug Discovery and Development - An Industrial Perspective, Vol 1, Chap 3; Biotherapeutic ADME and PK/PD Principles, Vol 1, Chap 4; Principles of Pharmacodynamics and Toxicodynamics, Vol 1, Chap 5; and Schrag and Regal (2013). Other study data that can also impact the pathologist’s interpretation includes safety pharmacology, reproductive, and/or immunologic assessments (Guidance for Industry, ICH S7A, 2001; Guidance for Industry, ICH S8, 2006). The pathologist may also request tissue sample special analysis including histochemical or immunohistochemical assessments, or morphometric analysis. It is important to note that there are occasions where the pathologist may not have access to some of the data listed above. Hence, evaluation of data impacting pathology endpoints may remain incomplete in the anatomic pathology report and it should be stated clearly what data were and were not available for assessment.

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3.3. Value of Data from More than One Time Point Studies with more than one time point during the dosing/testing phase may help the pathologist with their overall assessment. Examples of these data include interim or serial clinical pathology assessments, surgical biopsy specimens, diagnostic imaging, nontraditional biomarkers, and/ or interim scheduled termination of special cohorts of animals. The data help build an understanding of the temporal nature of a particular change, patterns of severity, or in the case of special biomarkers, provide insight into the pathogenesis of findings in the final anatomic pathology assessment. On the other hand, it can be just as important in the pathologist’s assessment that no anatomic pathology finding(s) were correlated with a transient change in a parameter. An additional set of data from interim time points may be present in a toxicity study in the form of data from unscheduled terminations and/or spontaneous deaths. These data should be evaluated carefully with the study’s full termination data and integrated into the overall study assessment. In most toxicity study reports, the description and interpretation of the findings from unscheduled terminations or spontaneous deaths form a separate section of both the toxicity study report and pathology report. Interpreting data from unscheduled terminations or spontaneous deaths is difficult because of the low number of animals in this category (rarely even on the same date) and the lack of concurrent controls. It is incumbent on the pathologist to utilize all available data from these animals. The constellation of clinical observations and veterinary interventions prior to termination or death may help to determine a pathology basis for the morbidity leading to euthanasia in the case of unscheduled termination, or the cause of death in the case of spontaneous death. The pathologist should use historical control and scheduled termination data in support of their interpretation and conclusions, and avoid speculation.

4. REVERSIBILITY The reversibility period of a toxicity study requires a careful comparison of the recovery

findings to the important test article–related findings from the histopathology assessment from the dosing period. The pathologist also commonly assesses the reversibility of gross observations or organ weights. A reversibility period is often restricted to the control and high dose level, and animal numbers are reduced compared to the dosing period which can make the interpretation of reversibility more difficult. The pathologist’s assessment of all toxicity data when combined with the macroscopic, organ weight, and microscopic changes in target organs or tissues adds to the overall interpretation of the significance of findings in a toxicity study. These data may help the pathologist determine if there are organ-specific functional changes in support of assigning adversity. The reversibility period adds important context to the observations made at the end of the dosing period. The representation and correlation of the pathology and toxicity findings are important to decision-makers among the audience (study sponsors, regulators, and physicians) who need to know if a developing toxicity can be identified and monitored in vivo, if the toxicity is reversible, and how long after cessation of dosing reversibility occurs.

4.1. Presentation and Interpretation of Organ Weight Data Usually, organ weight data are presented as an absolute value and related to either body or brain weight or both. The body weight of an animal influences the relative weight for most organs. Higher body weights may lower the relative weights for an organ even if the absolute weight of that organ is not different from control. Similarly, lower body weights may increase the relative weights of the organ even though the absolute weight of the organ is not different from control. There are published best practices for the reviewing pathologist in assessing organ weights (Sellers et al., 2007). Statistical analysis is commonly applied to animal studies and the pathologist should follow established rules of interpretation of any statistically significant parameters. Nonstatistically significant changes may also be meaningful and may be identified as trends or patterns when a dose relationship exists. A careful comparison of the differences

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4. REVERSIBILITY

in absolute versus relative weights is needed to identify a potentially altered organ weight compared to controls. Differences are to be interpreted in light of the affected organ since not all organs fluctuate with changes in body weight (e.g., brain and testis). The organ weight relative-to-brain weight may be helpful because of the stable weight of the brain compared to body weight, although one should be aware of any statistically significant differences in the absolute weight of the brain between the controls and the treated groups when interpreting this parameter, see Experimental Design and Statistical Analysis for Toxicologic Pathologists, Vol 1, Chap 16. The criteria for identifying a meaningful effect on organ weights should be described by the pathologist in the pathology report allowing for less ambiguity as to how the pathologist has interpreted the data.

4.2. Presentation and Interpretation of Gross Observations The gross observations noted at necropsy must be described and recorded in a systematic manner that facilitates meaningful tabulation of the findings. Tabulated summaries of the gross observations should consistently track the findings by organ, location within the organ, size, color, distribution, and incidence organized by sex and dose. To accomplish this requires procedural controls (SOPs) that establish the sequence to be used in describing grossly evident lesions. The terminology should be standardized into a lexicon accessible within the data entry system used in the necropsy room, see Nomenclature and Diagnostic Resources in Anatomic Toxicologic Pathology, Vol 1, Chap 25. Using a structured method of describing gross changes in any given organ prevents the situation where the same lesion in different animals in the same dose group is tabulated differently because the sequence of terms used in describing the change was not the same. In the anatomic pathology report, a narrative description and interpretation of gross observations should accompany the tabulated summaries and account for the relationship of the observations to concurrent controls, dose level, duration on study, sex, and to correlative data such as clinical pathology findings, organ weights, and microscopic

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observations. Indeed, while certain gross observations serve as stand-alone findings indicative of target organ toxicity, most gross observations serve to point the histopathologist to carefully assess the affected tissue microscopically and characterize potential effects.

4.3. Presentation and Interpretation of Microscopic Observations When describing microscopic changes, the same holds true as for gross observations. Limiting the description of microscopic findings to a lexicon in an electronic data capture system prevents the scenario that similar lesions are tabulated as different lesions within and between groups or even between studies. Microscopic descriptions should include the severity and distribution of the lesion. This information makes it possible to tabulate the severities of a given lesion by dose group to determine if there is a dose-related increase in the severity of a lesion as the dose increases. The severity descriptions or codes for the microscopic data also require that legends be included with the microscopic findings to clearly define for the reader/reviewer each of the descriptive (e.g., minimal, slight, moderate, marked, severe) or numerical (e.g., scale of 1–5) severity codes. If needed by the pathologist to reach an accurate interpretation, the microscopic findings can also be further characterized using special histochemical stains, immunohistochemistry, or any other appropriate diagnostic tools. The objective of these additional investigations is to elaborate on the changes observed in order to provide pathophysiologic context to the findings. For consistency in the practice of microscopy in toxicologic pathology, pathologists are referred to the International Harmonization of Nomenclature and Diagnostic Criteria (INHAND) publications on internationally accepted nomenclature for proliferative and nonproliferative lesions by organ system in laboratory animals (www.toxpath.org/inhand.asp). There are occasions where more than one pathologist may be assigned to interpret portions of the histopathology data from a toxicity study, such as by sex of the animals in the study, or when specific organ systems (e.g., neurologic, reproductive, immune system, etc.) may be

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subcontracted to a specialty pathologist designated as a “primary investigator” (PI) or “consultant pathologist.” In these situations it is important for each pathologist to identify which systems were evaluated, and that ultimately when more than one pathology report is submitted, the study pathologist takes responsibility to assure that the total pathology data are assessed in an integrative manner.

5. TEXT TABLES To facilitate the visualization and understanding of the significant findings in the anatomic pathology data, the inclusion of intext summary tables in the report is essential. In-text tables are an effective way to present significant findings by dose level and sex for organ weights, gross observations, or microscopic findings. Using in-text tables can markedly reduce the verbal descriptions of the important findings in the postmortem data and allow the reader to quickly grasp the information that impacts the interpretation of test article– related and dose-related toxicities. The goal is a presentation of the data that is correct, clear, and concise, known as the “three C’s rule”. In-text summary tables of gross observations, organ weights, and microscopic findings should be presented in a summary table format for males and females, at all dose levels, sacrificed at the end of the dosing period, at the end of the reversibility period, and from animals terminated early during the study, as appropriate. Individual animal findings are usually also placed in the appendices to the pathology report enabling the reader to explore data beyond the summaries provided by the author. As shown below, in-text tables make it possible to visualize on one table the important changes in a parameter. Table 13.1 describes the organ weight test article–related changes in the males including lower weights for the thymus (5.0 and 20.0 mg/kg/day), spleen (20 mg/kg/ day), and prostate (5.0 and 20.0 mg/kg/day). In the females, weights for the thymus (5.0 and 20.0 mg/kg) and pituitary (5.0 and 20.0 mg/ kg/day) were lower than control while spleen weights were higher than control at the 20.0 mg/kg/day dose. Note that the absolute heart weights for the males were significantly

lower than control (5.0 or 20.0 mg/kg/day) but the relative weights changed in the opposite direction and were significantly higher than control at 20.0 mg/kg/day. Therefore, the heart weights were not interpreted to reflect a test article–related change. In-text tables are also effective in presenting by dose and sex the significant gross and microscopic observations. The table of microscopic observations (Table 13.2) presents a suggested format for presenting these types of data. The in-text table of dose-related microscopic findings (Table 13.2) concisely and clearly summarizes for the reviewer the target organs of toxicity, the lesions, and their severity within each target organ, and demonstrates the dose and sex relationships. It also reveals those target organs in which the lesions were reversing but had not completely reversed during the recovery period. The findings in the heart demonstrate that the initial lesion of degeneration and necrosis progressed during the recovery period to fibrosis of minimal severity. Effective in 2016, the US Food and Drug Administration adopted The Standard for Exchange of Nonclinical Data (SEND) as an implementation of the CDISC Standard Data Tabulation Model (SDTM) for nonclinical studies (https://www. cdisc.org/standards/foundational/send). This program specifies a standardized presentation of nonclinical data from animal toxicity studies conducted during test article development to support submissions to FDA. The process originates with collection of data in toxicity studies in a way that results in the generation of specified electronic-format files of the entirety of the study raw data with uniform nomenclature. These data are searchable and manipulatable by a variety of data-handling systems to produce detailed, cross-correlated analyses. The uniform nomenclature used in SEND is called Controlled Terminology, the details of which are maintained and periodically updated on a National Institute of Health web portal (https://www.cdisc.org/standards/terminology /controlled-terminology). Based on the standardized platform of SEND, multiple vendors provide electronic data-handling and presentation programs for manipulation and visualization of the data in nonclinical toxicity studies. These tools are deployed by regulatory reviewers during analysis of test article approval packages

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TABLE 13.1

Test Article–Related Organ Weight Changes – Day 29 [Oral Gavage Toxicity Study in Rats] Dose (mg/kg/day) Males

Females

0

1.0

5.0

20.0

0

1.0

5.0

20.0

(g)

0.39

0.27

0.12b

0.09b

0.40

0.38

0.16b

0.10b

(% bwt)

0.08

0.06

0.03b

0.03b

0.14

0.13

0.06b

0.04b

(g)

0.99

0.81a

0.66b

0.54b

0.64

0.64

0.54

0.48b

(% bwt)

0.21

0.18

0.17

0.17a

0.23

0.22

0.20

0.19

(g)

1.73

2.03

2.10a

1.99

1.38

1.43

1.43

1.73b

(% bwt)

0.37

0.45

0.57b

0.64b

0.50

0.50

0.50

0.66b

1.14

1.04

0.74b

0.48b

-

e

e

e

0.24

0.23

0.20

a

b

e

e

e

e

(g)

3.49

3.56

2.99

2.50b

e

e

e

e

(% bwt)

0.75

0.79

0.81

0.80

e

e

e

e

1.56

1.56

1.36b

1.21b

1.06

1.10

1.09

1.05

b

0.39

0.39

0.42

0.42

THYMUS

SPLEEN

LUNGS

PROSTATE

(g) (% bwt)

0.15

TESTES

HEART

(g) (% bwt)

0.33

0.34

0.36

0.38

(g)

0.01

0.01

0.009b

0.009b

0.015

0.013a

0.011b

0.010b

(% bwt)

0.002

0.002

0.002

0.002

0.005

0.004a

0.004b

0.004b

PITUITARY

a b

P < .05. P < .01.

and pathologists are recommended to familiarize and utilize these resources likewise. For additional information on the industry approach to nonclinical data standardization under SEND, see Kropp et al. (2013).

6. THE ANATOMIC PATHOLOGY REPORT DISCUSSION The discussion section in the anatomic pathology report briefly summarizes the important gross, organ weight, and microscopic findings in the study and correlates them with

dose-related and exposure-related changes observed in clinical observations, body weights, hematology, clinical chemistry, and urinalysis. The discussion may also be used to highlight important lesions or changes that are not test article–related but, instead, are interpreted to represent spontaneous findings for the species and age group or procedural changes. The discussion is also the place to indicate whether the findings in pathology do or do not correlate with specific biomarkers that have been included in the study. If the pathologist is aware of findings in previous studies conducted with the test article or a similar class of

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TABLE 13.2

Test Article–Related Microscopic Findings [28-Day Oral Gavage Toxicity Study in Rats] Dose (mg/kg) Males

Females

0

1

10

50

0

1

10

50

10

10

10

10

10

10

10

10

0

4

8

8

0

0

10

10

0

0

9

8

0

0

7

8

Minimal

0

0

5

7

0

0

5

7

Mild

0

0

3

3

0

0

0

1

Minimal

2

2

3

5

0

0

6

6

Mild

0

0

7

5

0

0

0

0

0

0

3

6

0

0

3

6

Minimal

0

0

1

3

0

0

1

2

End of reversibility (n)

5

5

5

5

5

5

5

5

0

1

2

1

0

0

1

1

0

0

1

2

0

0

2

1

1

3

3

2

0

1

0

0

0

0

4

4

1

0

1

0

End of dosing (n) KIDNEY

Hyalin droplets e tubular Minimal Tubular vacuolation Minimal LUNG

Alveolar macrophage accumulation

HEART

Degeneration/necrosis

THYMUS

Lymphoid atrophy/necrosis Minimal SPLEEN

Lymphoid atrophy/necrosis

KIDNEY

Hyalin droplets Minimal LUNG

Alveolar macrophage accumulation Minimal HEART

Degeneration/necrosis Minimal Fibrosis Minimal

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9. EXAMPLES TO BE AVOIDED IN INTERPRETING/PRESENTING

compound, it is appropriate to discuss the similarity or differences in findings in the current study as compared to other studies conducted previously or in parallel. This discussion requires appropriate reference(s) to those studies by title and report number. Whenever possible, the pathologist should endeavor to identify whether the changes observed are considered direct or indirect effects of the test article. For example, with the observation of test article–related chronic renal injury, is there secondary parathyroid hyperplasia? This type discussion must also include appropriate references to the literature. It is important to note that regulatory reviewers expect the primary pathologist to provide an integrated assessment and interpretation of the pathology findings, and not just a mere catalog of all findings as available from the data summary tables. It is also critical that the pathologist provide input to the study director on the study significance (e.g., adverse, nonadverse, or adaptive) of the findings. Even insignificant or trivial findings may need to be discussed to ensure alignment on the interpretation. While the pathologist contributes to the assessment of the adversity of individual findings, the final determination of the NOAEL for the study rests with the study director. The pathologist should avoid including speculative statements regarding lesion pathogenesis and human translation.

7. THE ANATOMIC PATHOLOGY REPORT CONCLUSION The conclusion for the pathology report should not be a summary of the observations. The conclusion should be the result of the pathologist’s evaluation of the data and directed to the information the audience expects to learn from the pathology report, i.e., target organs and nature of findings, assessment of dose and/or exposure response, and assessment of adversity and reversibility. While the pathologist is not responsible for determining the study of NOAEL, the adversity and importance of anatomic pathology findings must be stated. For further information on establishing adversity of pathology findings in toxicity studies for nonclinical safety assessment, see Assigning Adversity to Toxicologic Outcomes, Vol 2, Chap 15, and Kerlin et al. (2016).

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8. SIGNING THE ANATOMIC PATHOLOGY REPORT Upon completion and review of the pathology report, the pathologist who interpreted the histology slides and prepared the written report must sign and date the report to indicate ownership of its contents. It is important, for the benefit of the audience, to include, after the pathologist’s signature, the degree(s) and board certification(s) achieved by the individual. Although these are not necessarily an indication of the experience of the individual as a nonclinical safety study pathologist, they do suggest the pathologist has the necessary educational background to understand and interpret the data.

9. EXAMPLES TO BE AVOIDED IN INTERPRETING/PRESENTING DATA IN THE ANATOMIC PATHOLOGY REPORT Examples of practices or items to be avoided when preparing the pathology report include: inconsistent microscopic terminology or description of similar lesions within and across studies, long written diagnostic microscopic terminologies that make tabulation impossible, lack of a conclusion as to identification of the primary target organs and secondary organ changes, lack or inconsistent determination and justification or adversity, poorly organized tabular data, use of the present tense in the narrative regarding events and findings from the now-concluded study, and the use of idiom that may not be understood by a wider audience and usually do not translate correctly into other languages. There are statements to be avoided when drafting the pathology report. Statements to be avoided are those that suggest to the reader that the writer is unsure of their conclusions. There is nothing wrong with stating that the test article relationship or significance of a finding is unclear or unknown. However, the following statements, for example; “is considered to increase the likelihood”, “clearly demonstrating an indirect versus a direct effect is problematic,” “cannot be ruled out”, “the possibility exists”, or “played at least some role”, leave the interpretation of a finding up to the reader of the report, and the reader with the notion the author did not understand the data.

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10. PEER REVIEW AND PATHOLOGY WORKING GROUPS When, at the conclusion of the primary interpretation of the pathology data in a toxicity study, there is doubt as to the actual target organs and lesions and their adversity, it may be necessary to request a retrospective peer review by a second pathologist or a pathology working group of the microscopic observations for a given organ or the entire study. The reader is referred to Peer Review and Pathology Working Groups, Vol 1, Chap 26 for specific information regarding this important practice. An additional best practice for the authoring pathologist may include an informal review of the draft pathology report by a pathology colleague to check for clarity and correct use of language.

11. CONCLUSION The purpose of a well-written anatomic pathology report is to accurately present, in an easily visualized and understandable fashion, the results of the pathology data captured in a toxicity study. The “three Cs (correct, clear, and concise)” rule should be followed for writing and for constructing text tables. A wellwritten and presented pathology report that includes peer-reviewed microscopic data adds significant credibility to the conclusions of the final report for the entirestudy conducted to assess the toxicologic potential of a test article. The goal of the pathology report is to describe for the study director, sponsor management, regulatory reviewers, and physicians undertaking clinical trials the target organs, the changes induced in the target organs, their relationship to dose and/or systemic exposure levels, and their significance and adversity.

REFERENCES Avila A, Bebenek I, Bonzo J, Bourcier T, Davis Bruno K, Carlson D, et al.: An FDA/CDER perspective on nonclinical testing strategies: classical toxicology approaches and new approach methodologies (NAMs), Regul Toxicol Pharmacol 114:104662, 2020. Controlled Terminology, SEND/CDISC, National Institute of Health. https://www.cdisc.org/standards/terminology/ controlled-terminology. (Accessed January 2021). Guidance for industry S7A safety pharmacology studies for human pharmaceuticals, 2001, USFDA/ICH, https://www.fda.gov/ regulatory-information/search-fda-guidance-documents/ s7a-safety-pharmacology-studies-human-pharmaceuticals. (Accessed January 2021). Guidance for industry S8 immunotoxicity studies for human pharmaceuticals, 2006, USFDA/ICH, https://www.fda.gov/ regulatory-information/search-fda-guidance-documents/s8immunotoxicity-studies-human-pharmaceuticals. (Accessed January 2021). International Harmonization of Nomenclature and Diagnostic Criteria (INHAND). www.toxpath.org/inhand.asp. (Accessed January 2021). Kerlin R, Bolon B, Burkhardt J, Francke S, Greaves P, Meador V, Popp J: Scientific and regulatory policy committee: recommended (‘‘Best’’) practices for determining, communicating, and using adverse effect data from nonclinical studies, Toxicol Pathol 44(2):147–162, 2016. Kropp T, Rosario L, DeHaven S, Houser W, Kramer L, Nath S, Smyrnios T, Wally J: FDA engages collaborators to address nonclinical data challenges, Ther Innov Regul Sci 47(1):41– 45, 2013. Morton D, Kemp RK, Francke-Carroll S, Jensen K, McCartney J, Monticello TM, Perry R, Pulido O, Roome N, Schafer K, Sellers R, Snyder PW: Best practices for reporting pathology interpretation within GLP toxicity studies, Toxicol Pathol 34:806–809, 2006. Schrag M, Regal K: Pharmacokinetics and toxicokinetics. In Faqi A, editor: A comprehensive guide to Toxicology in preclinical test article development, Waltham, MA, USA, 2013, Elsevier/Academic Press. Sellers R, Mortan D, Michael B, Roome N, Johnson J, Yano B, Perry R, Schafer K: Society of toxicologic pathology position paper: organ weight recommendations for toxicity studies, Toxicol Pathol 35:751, 2007. The Standard for Exchange of Nonclinical Data (SEND)/ CDISC Standard Data Tabulation Model (SDTM). https:// www.cdisc.org/standards/foundational/send. (Accessed January 2021).

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C H A P T E R

14 Interpretation of Clinical Pathology Results in Nonclinical Toxicity Testing Adam D. Aulbach1, Daniela Ennulat2, A. Eric Schultze3 1

Inotiv, Maryland Heights, MO, United States, 2GlaxoSmithKline, Collegeville, PA, United States, 3Eli Lilly and Company, Indianapolis, IN, United States O U T L I N E

1. Introduction

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2. Sample Collection

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3. Hematology Interpretation (also see Hematopoetic System, Vol 5, Chap 5) 3.1. Erythrocytes 3.2. Decreased Red Cell Mass 3.3. Blood Loss 3.4. Hemolysis 3.5. Diminished Erythropoiesis 3.6. Increased Red Cell Mass 3.7. Leukocytes 3.8. Increased Leukocytes 3.9. Decreased Leukocytes 3.10. Platelets

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4. Interpretation of Bone Marrow Morphology

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5. Coagulation Interpretation 5.1. Hemostasis

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6. Clinical Chemistry Interpretation 525 6.1. Glucose, Cholesterol, and Triglycerides 525 6.2. Blood Urea Nitrogen (Urea or BUN) and Creatinine 526 6.3. Total Protein, Albumin, and Albumin/Globulin Ratio 526 6.4. Calcium and Phosphorus 527 6.5. Sodium, Chloride, and Potassium 528 6.6. Markers of Hepatobiliary Injury or Function 528 6.7. Muscle Injury Markers 530 7. Urinalysis and Urine Chemistry Interpretation 532

Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-12-821047-5.00027-0

8. Nonstandard Biomarkers 533 8.1. Renal Biomarkers 534 8.2. Hepatobiliary Biomarkers 536 8.3. Cardiac Biomarkers (see Cardiovascular System, Vol 5, Chap 1) 537 8.4. Inflammatory Biomarkers 538 8.5. Hormones (see Endocrine System, Vol 4, Chap 7) 540 9. Potential Effects Unrelated to Test Article Treatment 9.1. Artifacts 9.2. Analytical Methods 9.3. Age/Sex/Genetics 9.4. Anesthesia 9.5. Blood Collection 9.6. Diet/Fasting 9.7. Medications 9.8. Fear/Pain/Stress 9.9. Environment 9.10. Sample Types, Handling, and Stability 9.11. Pregnancy, Neonatal Period, and Estrous Cycle 9.12. In Extremis/Postmortem

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10. Overall Results Interpretation and Report Integration

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11. Comparator Data, Historical Controls, and Statistics 11.1. Comparator Data 11.2. Historical Controls

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12. Descriptors and Biologic Relevance

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Copyright Ó 2023 Elsevier Inc. All rights reserved.

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13. Report Writing and Integration 13.1. Adversity Reporting in Clinical Pathology

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14. Conclusions

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1. INTRODUCTION Accurate interpretation of clinical pathology and biomarker test results in nonclinical toxicity studies is dependent upon the proper design of a protocol optimized to meet study objectives. The elements required to describe and interpret clinical pathology data effectively, including the identification and characteristics of parameters to be assayed along with the number and timing of specimen collections, should be reviewed indepth during protocol preparation. This chapter focuses on clinical pathology data interpretation and should be read in conjunction with Clinical Pathology in Nonclinical Toxicity Testing, Vol 1, Chap 10, which identifies characteristics of test parameters included in clinical pathology and novel biomarker components of toxicity studies. Identification of test article–related alterations and discussion of the potential adverse effects are important goals of nonclinical toxicity studies (Schultze et al., 2020). Interpretation of clinical pathology findings in a nonclinical study cannot be made in isolation and must consider the design of the study, clinical observations, measurements of food consumption and/or body weight, and histopathology and associated organ weight changes. This is imperative in order to understand whether an effect is directly test article–related or due to physiological factors such as weight loss, emesis, or seizures. The Regulatory Affairs Committee of the American Society for Veterinary Clinical Pathology (ASVCP) has developed general guidelines for interpretation of clinical pathology data (Weingand et al., 1992, 1996). These guidelines state that the relationship of a change in value of a clinical pathology parameter to test article administration and interpretation of its importance are accomplished through comparison of individual animal and group mean data with concurrent controls regardless of species. In studies with dogs, nonhuman primates

References

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(NHPs), or other large animals, because of smaller group sizes, it is necessary to determine the magnitude of change and relationship between posttreatment and pretreatment values. Statistical significance alone or in comparison to historical control data does not indicate that the change in a clinical pathology parameter is necessarily test article–related and does not indicate the biological or toxicological relevance. In dogs and NHPs, statistical analysis of data in standard toxicity studies is generally not appropriate due to the low number of animals per treatment group and interanimal and intraanimal variability. However, the use of statistics can augment but not replace comparison to pretreatment and/or concurrent controls on an individual animal basis. The determination of whether test article– induced clinical pathology findings are adverse is based on the nature and magnitude of changes in all concurrently evaluated study parameters, as well as toxicokinetic data, and pharmacological class effects. In-life observations, body weights, food consumption, organ weights, and gross and histopathology data provide additional indicators of test article–relatedness and adverse effects. Assessment of the toxicokinetic data is helpful for the evaluation of results for individual animals or dose groups with incongruent changes in clinical pathology values and/or clinical observations. Finally, evaluation of all the data from a study allows identification of the number of affected animals per dose group, dose-specific and/or sex-specific responses, and the reversibility of those changes. These data are collated and integrated in the final study report to identify test article–related changes and determine whether these changes are adverse. Combination of these studyspecific interpretations with data from other studies with the test article in the same or alternative nonclinical species provides important information for assessment of the risk profile

III. DATA INTERPRETATION AND COMMUNICATION

3. HEMATOLOGY INTERPRETATION

for humans and is of prime interest to regulatory bodies. A dose–response effect is characterized by an increased magnitude and/or incidence of a change corresponding to increasing doses of the test article. However, absence of a dose response does not exclude the possibility of a test article– related effect. For example, effects on the immune system may not follow a classic dose–response pattern due to antigen or antibody excess at the highest dose group or the lack of response in all individuals in a dose group. The lack of dose– response effects on clinical pathology test results may also occur when systemic exposure to the test article or metabolites does not follow a dose– response as determined by toxicokinetic evaluation. Acronyms and abbreviations used in this chapter can be found in Table 14.1.

2. SAMPLE COLLECTION The quality of blood and urine samples submitted to the clinical pathology laboratory for analysis in toxicity testing is critically important for satisfactory sample analysis. Blood, serum, and/or plasma samples intended for analysis should be of adequate volume to complete the number of tests requested and should be free from lipemia, hemolysis, and icterus. Readers are referred to Clinical Pathology in Nonclinical Toxicity Testing, Vol 1, Chap 10, for discussion of required sample volumes for hematology, hemostasis, and clinical chemistry determinations; appropriate sample matrix; anticoagulants; and methods/technology used for sample analysis. Additional information regarding clinical pathology tests requested, sample matrix, blood sample collection sites, and methods to obtain adequate urine sample volume and sample stability are listed in Table 14.2. Common artifacts encountered in analysis of samples submitted to the clinical pathology laboratory are described in detail later in this chapter. There are numerous anatomic sites from which blood may be obtained in laboratory animals (NC3Rs, 2020; IQ Consortium, 2020). Not all sites provide equal sample volume or ease of phlebotomy. Some phlebotomy procedures require anesthesia whereas others can be performed on conscious animals with minimal

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restraint. For example, blood samples obtained from the jugular vein or periorbital venous sinus in rats are usually of adequate volume for CBC and clinical chemistry panel determinations. Larger blood volumes from vessels of higher flow as suggested for hemostasis testing usually require collection from the abdominal vessels (aorta or vena cava) in anesthetized rats. Blood obtained from the periorbital venous sinus may show platelet clumping and release of tissue thromboplastin from tissue injury during blood collection and is generally not used for hemostasis testing in rodents. Urine from laboratory animals submitted for complete urinalysis with or without quantitative chemistry determinations are usually obtained by cage pan collection or metabolism cages. Other methods of urine collection often provide insufficient volume for complete urinalysis with or without urine chemistry determinations. Ideally, blood and urine samples submitted to the clinical pathology laboratory are analyzed shortly after receipt. If immediate analysis is not possible, some samples may be held at room temperature, chilled in a refrigerator, or frozen until analysis. Please refer to Table 14.2.

3. HEMATOLOGY INTERPRETATION (ALSO SEE HEMATOPOETIC SYSTEM, VOL 5, CHAP 5) 3.1. Erythrocytes The erythroid parameters included on toxicity studies generally include erythrocyte count, hemoglobin concentration, hematocrit, and reticulocyte count, as well as several erythrocyte indices (e.g., MCV, MCH, MCHC, RDW, CHCM, and/or HDW). Circulating red cell mass reflects the balance of erythrocyte production and erythrocyte loss, destruction, and natural turnover (senescence). Because erythrocytes circulate and are suspended in fluid, plasma volume also influences overall red cell mass. The term “red cell mass” generally refers to erythrocyte count, hemoglobin concentration, and hematocrit, and in the absence of substantial effects on erythrocyte size or hemoglobin concentration, and changes in these endpoints generally occur in parallel. Erythropoietin is the principal growth factor driving erythropoiesis and is produced by the

III. DATA INTERPRETATION AND COMMUNICATION

TABLE 14.1 Acronyms and Abbreviations Acronym/ Abbreviation

Meaning

Acronym/ Abbreviation

Meaning

a

Alpha

IFN-a

Interferon-a

a-GST

Alpha glutathione S-reductase

IL

Interleukin

ACTH

Adrenocorticotropic hormone

KIM-1

Kidney injury molecule-1

ADA

Antidrug antibody

L-ALP

Liver alkaline phosphatase isozyme

A/G

Albumin/globulin ratio

LDH

Lactate dehydrogenase

ALP

Alkaline phosphatase (total activity)

LH

Luteinizing hormone

ALT

Alanine aminotransferase

LOS

Letter of support

ANP

Atrial natriuretic peptide

MAC

Membrane attack complex

APP

Acute phase protein

MCH

Mean corpuscular hemoglobin

APTT

Activated partial thromboplastin time

MCHC

Mean corpuscular hemoglobin concentration

AST

Aspartate aminotransferase

MCP-1

Monocyte chemoattractant protein-1

ASVCP

American Society for Veterinary Clinical Pathology

MCSFR

Macrophage colonystimulating factor receptor

B-ALP

Bone alkaline phosphatase isozyme

MCV

Mean corpuscular volume

BNP

Brain natriuretic peptide

M:E

Myeloid to erythroid ratio

BSEP

Bile salt export pump

MIP

Macrophage inflammatory protein

BUN

Blood urea nitrogen

miR-122

microRNA-122

C-ALP

Corticosteroidinducible alkaline phosphatase isozyme

MPC

Mean platelet component

CBC

Complete blood count

MPV

Mean platelet volume

CETP

Cholesterol ester transfer protein

MyHC

Myosin heavy chain

CH50

Total complement

Myl3

Myosin light chain 3 (Continued)

TABLE 14.1 Acronym/ Abbreviation

Acronyms and Abbreviationsdcont’d Meaning

Acronym/ Abbreviation

Meaning

CHCM

Corpuscular hemoglobin concentration mean

NAG

N-acetyl-/ b-glucosaminidase

CIC

Circulating immune complex

NGAL

Neutrophil gelatinase-associated lipocalin

CK

Creatine kinase

NHP

Nonhuman primate

CK-18

Keratin 18

NOAEL

No-observedadverse-effect level

CRO

Contract research organization

NT-proANP

Amino cleavage equivalent of ANP

CRP

C-reactive protein

NT-proBNP

Amino cleavage equivalent of BNP

CSR

Contributing scientist report

OAT1B1

Organic anion transporting polypeptides

cTn

Cardiac troponin

OPN

Osteopontin

cTnC

Cardiac troponin C

P5P

Cofactor pyridoxal 50 phosphate, the active form of vitamin B6

cTnI

Cardiac troponin I

PCR

Polymerase chain reaction

cTnT

Cardiac troponin T

PDW

Platelet distribution width

Cys-C

Cystatin-C

PRL

Prolactin

D-dimer

A specific, crosslinked fibrin degradation product

PT

Prothrombin time

DIC

Disseminated intravascular coagulation

PTH

Parathyroid hormone

DIKI

Drug-induced kidney injury

RBC

Red blood cell (erythrocyte)

DILI

Drug-induced liver injury

RDW

Red cell distribution width

DME

Drug-metabolizing enzyme

ROC

Receiver operating characteristic

ECF

Extracellular fluid

RPA-1

Renal papillary antigen-1

EDTA

Ethylenediaminetetraacetic acid

SAA

Serum amyloid A

(Continued)

TABLE 14.1 Acronyms and Abbreviationsdcont’d Acronym/ Abbreviation

Meaning

Acronym/ Abbreviation

Meaning

eGFR

Estimated glomerular filtration rate

SCF

Stem cell factor

ELISA

Enzyme-linked immunosorbent assay

sCr

Serum creatinine

Fabp3

Fatty acid binding protein 3

SDH

Sorbitol dehydrogenase

FDA

US Food and Drug Administration

SDMA

Symmetrical dimethyl arginine

FDP

Fibrinogen/fibrin degradation product

sTnI

Skeletal troponin I

FSH

Follicle-stimulating hormone

STP

Society of Toxicologic Pathology

G-CSF

Granulocyte colonystimulating factor

T3

Triiodothyronine

GFR

Glomerular filtration rate

T4

Thyroxine

GGT

Gamma-glutamyl transferase

TAT

Thrombin eantithrombin complex

GLDH

Glutamate dehydrogenase

TBIL

Total bilirubin

GLP

Good laboratory practice

Th1

T helper type 1 response

HCD

Historical control data

Th2

T helper type 2 response

Hct

Hematocrit

TNF-a

Tumor necrosis factor-a

HDL

High-density lipoprotein cholesterol

Tnni2

Fast twitch sTnI subunit

HDW

Hemoglobin distribution width

TSH

Thyroid-stimulating hormone

H and E

Hematoxylin and eosin

TMP-SMX

Trimethoprimsulfamethoxazole

HESI

Health and Environmental Sciences Institute

UGT1A1

Uridine diphosphate glucuronosyltransferases

Hgb

Hemoglobin

US

United States

HMGB1

High mobility group box 1

VLDL

Very-low-density lipoprotein

I-ALP

Intestinal alkaline phosphatase isozyme

WBC

White blood cell (leukocyte)

3. HEMATOLOGY INTERPRETATION

renal peritubular interstitial cells in response to tissue hypoxia (Stockmann and Fandrey, 2006). Erythropoiesis occurs throughout the bone marrow in erythroblastic islands that consist of a central iron-containing macrophage surrounded by developing erythroid precursors. Extramedullary hematopoiesis (myeloid and/ or erythroid) can also occur in various extramedullary tissues (e.g., spleen, liver, lymphoid tissues) in response to increased tissue demand. Distinctive cytologic stages of erythropoiesis include rubriblasts, prorubricytes, rubricytes, metarubricytes, reticulocytes, and mature erythrocytes. As erythroid precursors mature, they become progressively smaller, and accumulate increasing concentrations of hemoglobin during the later stages of maturation (Figure 14.1). The nucleus of metarubricytes is ultimately extruded prior to the reticulocyte stage. Reticulocytes typically spend 2–3 days in the bone marrow before being released into circulation (Stockham and Scott, 2008). The number of reticulocytes in circulation during health varies considerably by age and species. Rodents have a higher number of reticulocytes in circulation than NHPs, dogs, and pigs, although the number of reticulocytes in circulation can increase dramatically under periods of erythropoietic stress, as is commonly seen as an adaptive change (reactive thrombocytosis) following repeated phlebotomy during toxicity studies (see Clinical Pathology in Nonclinical Toxicity Testing, Vol 1, Chap 10; Aulbach et al., 2017). Erythrocyte indices (e.g., MCV, MCH, MCHC, CHCM, RDW) are a collection of results generated from measurements of various characteristics (i.e., size, density, hemoglobin content) of RBC populations. Although erythrocyte indices can sometimes be used to provide additional supportive or mechanistic information, they most often are limited in their interpretive value and largely reflect expected changes associated with RBC turnover (regeneration and kinetics). MCV is a measure of RBC size while RDW relates to degree of size variability overall in the RBC population. MCH, MCHC, CHCM, and HDW are indices related to hemoglobin content and the overall degree of variability of hemoglobin content with the RBC population (Stockham and Scott, 2008). Unlike most of the erythrocyte indices including MCHC, CHCM is a direct measure of hemoglobin content within RBCs which can be used in conjunction with other

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markers to help characterize certain hematologic abnormalities.

3.2. Decreased Red Cell Mass Decreases in red cell mass are commonly observed in nonclinical toxicity studies and are frequently multifactorial in nature (e.g., studyrelated blood collection, direct/indirect test article effects). Most often, test article–related decreases in red cell mass occur as a result of reduced erythrocyte production, increased erythrocyte destruction, hemolysis, and/or internal or external blood loss as illustrated in Table 14.3. It is imperative to examine other measures of erythrocyte production (e.g., reticulocyte counts, erythrocyte indices, blood smear evaluation, and/or histopathology) in conjunction with clinical observations and in consideration of study design in order to characterize the underlying cause. Increases in reticulocyte counts indicate stimulation of the bone marrow in response to demand (tissue hypoxia). In most species, increases in reticulocyte counts may be observed in the peripheral blood 3–4 days after a stimulus, with maximal reticulocyte production 7–10 days following the stimulus (Olver, 2010; Stockham and Scott, 2008). The magnitude of reticulocyte count increases should be proportional to the severity of the decreases in red cell mass. Decreases in reticulocyte counts, or a lack of an appropriate increase in response to blood loss, indicate a nonregenerative response and suggest diminished erythropoiesis in the bone marrow.

3.3. Blood Loss Study procedure-related blood loss is the most common cause of decreased red cell mass in toxicity studies, particularly in large animal studies where repeated blood collections for toxicokinetic, pharmacodynamic, and/or biomarker analyses are routinely performed. Comparison to concurrent controls is often a more appropriate comparator for interpreting effects on red cell mass in most studies regardless of species for this reason (Aulbach et al., 2019a, b). Test article–related blood loss great enough to result in discernible effects on red cell mass is uncommon in nonclinical safety studies. Decreased red cell mass secondary to blood loss of any cause is commonly associated with increased reticulocyte counts and decreased

III. DATA INTERPRETATION AND COMMUNICATION

TABLE 14.2 Specimen Collection Information for Standard Clinical Pathology Testing Procedures Tests Selected

Matrix

Sample Collection Sites/Methods

Mice/Rats: Hematology (CBC): EDTAanticoagulated blood Tail vein and Erythrocyte count (RBC), hematocrit saphenous vein: (Hct), hemoglobin lower flow concentration (Hgb), vesselsdblood mean corpuscular volumes may be volume (MCV), mean limited; consider use corpuscular for individual clin hemoglobin (MCH), chem test requests. mean corpuscular Jugular veind hemoglobin excellent site to concentration obtain blood samples (MCHC), reticulocyte for hematology and count, platelet count, clin chem analysis. and leukocyte (WBC, Peri-orbital venous total and differential sinusdrequires leukocyte) counts anesthesia; used primarily in the Citrated plasma Hemostasis: United States; Activated partial acceptable to obtain thromboplastin time blood for CBC and (APTT), prothrombin clin chem analysis; time (PT), fibrinogen not recommended for concentration hemostasis testing due to platelet clumping and release of tissue thromboplastin. Abdominal vena cava/aorta: Collection requires anesthesia, usually followed by Serum, plasma Clinical chemistry: euthanasia; excellent (EDTA or heparin). Concentrations of high flow vessels to Note: Some clin chem creatinine, urea, obtain blood samples parameters cannot be sodium, chloride, for hemostasis, potassium, inorganic measured in EDTA hematology, and clin phosphorus, calcium, plasma due to chem testing. chelation of cations albumin, total Cardiac puncture: (calcium, protein, cholesterol, Collection requires magnesium, some triglycerides, anesthesia; site enzymes) glucose, total acceptable for Check with clinical bilirubin, and hematology and pathology laboratory activities of alanine some clin chem personnel a priori to aminotransferase testing. Not assure appropriate (ALT), alkaline recommended for test/sample matrix phosphatase (ALP), cardiac injury agreement gamma-glutamyl parameters (cTn, CK, transferase (GGT), AST) or hemostasis aspartate testing. aminotransferase Decapitation: Not (AST), and creatine recommended for kinase (CK) routine use; some

Submission Instructions

Sample Stability

Transport EDTAblood samples at room temperature in an approved biologics safety transport container to the clinical pathology laboratory as soon as possible (1 h) for immediate analysis.

Stable for minimum of 5 h at room temperature. If analysis is delayed for more than 5 h, refrigerate EDTA blood sample for up to 24e72 h. Return sample to room temperature before analysis.

Transport citratedblood samples at room temperature in an approved biologics safety transport container to the clinical pathology laboratory as soon as possible (1 h). Spin anticoagulated blood in centrifuge to obtain citrated plasma. Analyze immediately.

Citrated plasma samples are stable at room temperature for 4 h. If analysis is delayed, freeze citrated plasma at 20 C or 80 C for up to 10 days. Thaw plasma and analyze immediately.

Transport blood samples at room temperature in an approved biologics safety transport container to the clinical pathology laboratory as soon as possible (1 h) for immediate analysis. Allow blood to clot. Spin in a centrifuge to produce serum. Analyze immediately.

Many clin chem parameters are stable in serum frozen at 20 to 80 C for 6 months. Consult clinical pathology laboratory personnel a priori to check freeze stability of desired analytes.

(Continued)

TABLE 14.2 Specimen Collection Information for Standard Clinical Pathology Testing Proceduresdcont’d Tests Selected

Matrix

Sample Collection Sites/Methods

Submission Instructions

Sample Stability

institutions require anesthesia; site used more frequently for blood collection for hormone analysis. Dogs: Jugular vein, cephalic vein, saphenous veinduseful phlebotomy sites for hematology, hemostasis, and clin chem testing. NHPs: Femoral vein/arteryduseful phlebotomy sites for hematology, hemostasis, and clin chem testing. Standard urinalysis: Fresh urine (without Volume, color, clarity, preservative) specific gravity; pH, protein, glucose, bilirubin, ketones, blood, urobilinogen; and microscopic examination of the sediment

Cage pan collection: Collect urine from rats, dogs, and NHPs overnight chilled; usually provides adequate volume for complete urinalysis and quantitative urine chemistry determinations.

Deliver fresh urine to the clinical pathology laboratory within 30 min of collection for standard urinalysis. If unable to deliver urine sample immediately, refrigerate urine for up to 24 h.

Urine for standard urinalysis may be analyzed immediately, or chilled in a refrigerator for up to 24 h prior to analysis. Do not freeze. Return to room temperature prior to analysis.

Fresh urine (without preservative). Some methods for urine calcium and phosphorus analysis may suggest acidification of urinedconsult the clinical pathology laboratory for specific collection instructions Fresh urine (without Urine biomarkers: NAG, KIM-1, NGAL, preservative) clusterin, albumin, osteopontin, cystatin C

Metabolism cage: collect urine from rats and dogs overnight chilled; usually provides adequate volume for complete urinalysis and quantitative urine chemistry determinations.

Urine samples intended for quantitative urine chemistry or biomarker determinations should be spun in a centrifuge to remove cells and debris. Best to analyze immediately.

Urine samples intended for quantitative urinalysis should be analyzed immediately or may be frozen for up to 7 days for most analytes.

Quantitative urine chemistry: Sodium, chloride, potassium, phosphorus, calcium, protein, albumin, glucose, and creatinine

Catheterization: Used infrequently for Urine samples may some timed samples be frozen until in dogs in physiology analysis. studies. Urine volume may be variable, and procedure requires skilled technician. Cystocentesis: Often done at necropsy; provides limited urine volume for select tests, especially in rodents.

If possible, analyze immediately. Urine for biomarker determinations is often frozen prior to analysis. Check with clinical pathology laboratory personnel for stability of individual biomarkers.

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FIGURE 14.1 Reproduced with permission from Clinical Hematology Atlas, 4th Edn, by BE Rodak and JH Carr, Elsevier, 2012, Figure 2.1.

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TABLE 14.3

Common Causes of Decreased Red Cell Mass in Laboratory Species Pathology and/or InLife Findings

Root Process

Clinical Pathology Findings

Decreased erythropoiesis

• Y Reticulocyte counts • þ/ Y WBC and/or platelet counts

• Y Bone marrow cellularity • [ M:E ratio • Weakness, pallor

• Myelosuppressive/ chemotherapeutic agents • Chronic inflammation • Reduced food consumption

Hemolysis

• [ Reticulocyte counts • [ MCV and RDW • Y MCHC • Polychromasia/ anisocytosis • RBC shape changes • [ TBIL concentration • [ Platelet counts (reactive thrombocytosis)

• [ Bone marrow cellularity • Y M:E ratio • Erythrophagocytosis and/or iron pigment in the liver, spleen, or kidney • Weakness, pallor

• Oxidative injury • Mechanical trauma (vascular pathology, medical devices) • Traumatic venipuncture • Immune-mediated • Formulation hemo-incompatibility

Blood loss

• [ Reticulocyte counts • [ MCV and RDW • Y MCHC • Polychromasia/ anisocytosis • Y Serum protein concentrations • [ Platelet counts (reactive thrombocytosis)

• Hemorrhage grossly or microscopically • Repeated blood collections • Weakness, pallor

• Coagulopathy • Y Platelet counts (3–4 doses) (Everds and Tarrant, 2013). These reactions are usually nondose-responsive and occur sporadically in test article–treated animals. Erythrophagocytosis and/or iron pigment may be observed microscopically in the spleen, liver, and/or kidneys of affected animals. Oxidative injury to erythrocytes is typically associated with Heinz bodies and/or eccentrocytes, although other shape abnormalities can also occur (Stockham and Scott, 2008). These morphologic abnormalities develop in response to oxidation of hemoglobin and/or red cell membrane components and can be identified on microscopic blood smear evaluation. Intravascular hemolysis is relatively uncommon in toxicity studies, but typically occurs secondary to red cell swelling or direct damage to the red cell membrane following intravenous administration of the test article. Drug-induced intravascular hemolysis can also occur by complement fixation to red blood cells following repeated exposure to some test articles (Everds and Tarrant, 2013). Increased reticulocyte counts and hemoglobin concentration in the serum and/or urine may occur depending on the severity and duration of the hemolysis. In vitro hemolysis testing can be performed to help identify test articles that are incompatible with blood. Mechanical fragmentation of erythrocytes, uncommon in toxicity studies, may cause hemolysis (see Table 14.3). Fragmented erythrocytes, including schistocytes and keratocytes, may be seen during microscopic examination of blood smears and are sometimes associated with lesions of the cardiovascular system (i.e., vasculitis, endothelial, or cardiac injury). Alterations in erythrocyte membrane lipids have also been reported to increase erythrocyte fragility resulting in fragmentation of erythrocytes (Bauer, 1996).

3.5. Diminished Erythropoiesis Diminished erythropoiesis is commonly seen on toxicity studies and is typically indirectly test article–related and secondary to reduced food consumption, renal effects, declining health condition, chronic disease and inflammation, and/or endocrine disease (Moriyama et al., 2008; Stockham and Scott, 2008; Olver, 2010).

Findings include mildly decreased red blood cell mass without evidence of regeneration (i.e., decreased or nonresponsive reticulocyte counts) or any clear underlying cause. Animals may exhibit mild nonspecific clinical signs that reflect general ill-health, including hunched posture, unkempt appearance, decreased activity, reduced food consumption/inappetence, and decreased body weight or body weight gain. Overall, these effects suggest decreased erythropoiesis, and may be attributable to reduced metabolic rate or decreased tissue oxygen demands secondary to inactivity. Reduced food consumption is also a common cause of decreased erythropoiesis and reticulocyte counts (and decreased hematopoiesis in general) on toxicity studies (Hall, 2001, 2013). These changes are typically most pronounced in rodents and may also be associated with decreases in lymphocyte and platelet counts, as well as decreases in serum glucose, protein, and/or phosphorus concentrations while triglyceride and cholesterol concentrations tend to be variable (Moriyama et al., 2008; Zeng et al., 2010). Chronic disease and inflammation can contribute to decreased erythropoiesis in animals on longer term studies (e.g., 1 month) by disruption of normal iron handling (mediated by hepcidin), decreased red cell survival, and inhibition of erythropoiesis by inflammatory cytokines (Stockham and Scott, 2008). Myelosuppressive agents causing direct test article–related effects on hematopoietic tissues are commonly encountered in toxicity studies. The rate and timing of changes in the peripheral blood secondary to administration of a bone marrow toxin are dependent on several factors including compound class, cell line targeted, and the circulating life span of the cell line. Reticulocyte counts are typically the first cell line decreased in the peripheral blood and these changes may be rapid and dramatic. Due to the longer circulating half-life of erythroid cells, decreases in red cell mass may or may not be seen, depending on the length of the study, dosing scheme, and extent of marrow toxicity. The timing of blood collections is critical to assess hematopoietic effects in these cases as decreased cellularity of the bone marrow will be observed if sampled during the period of myelosuppression; however, hypercellularity may be observed during recovery of

III. DATA INTERPRETATION AND COMMUNICATION

3. HEMATOLOGY INTERPRETATION

myelosuppression, and will precede recovery of cell counts in the peripheral blood.

3.6. Increased Red Cell Mass Increased red cell mass in toxicity studies is most commonly indirectly test article–related and secondary to dehydration, hemoconcentration, and reduced plasma volume. Decreased food consumption, gastrointestinal fluid losses (e.g., emesis and diarrhea), and renal effects (e.g., increased urine output) are common causes of dehydration in animal studies (Hall, 2001, 2013). Increased concentrations of urea nitrogen and creatinine, urine specific gravity, serum electrolyte, and protein concentrations can corroborate dehydration depending on the underlying cause. Increased red cell mass may also occur following administration of erythropoiesis stimulating agents, as these compounds directly or indirectly mimic the actions of endogenous erythropoietin. Increases in reticulocyte counts and red cell mass, in conjunction with increased extramedullary hematopoiesis and bone marrow erythroid hyperplasia, may be seen following administration. These changes can be marked and dramatic depending on the dose and duration of administration.

3.7. Leukocytes Standard toxicity studies typically include total leukocyte and WBC with differential (relative and absolute) counts of neutrophils, lymphocytes, monocytes, eosinophils, basophils, and large unstained (or unclassified) cells (large peroxidase-negative cells that cannot be further classified). The relative percentage and leukocyte differential counts among laboratory animals can vary between species; in NHPs and dogs neutrophils are the primary leukocyte type (>75%) whereas lymphocytes comprise the majority of circulating blood leukocytes in rodents (70%– 90%) (Hall, 2001). Interpretation of leukocyte responses should generally be made based on absolute rather than relative percentage counts. Changes in the number of individual leukocyte types in blood reflect the balance of cell production and release to the blood, margination (adhesion to

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endothelial cells), migration to tissues, and destruction/consumption. Leukocyte production primarily occurs in the bone marrow. Granulopoiesis refers to production of granulocytes (neutrophils, eosinophils, and basophils), and is largely governed by the cytokine granulocyte colony-stimulating factor (G-CSF). Stem cell factor (SCF), interleukin-3 (IL-3), interleukin-6 (IL-6), and granulocytemacrophage colony-stimulating factor (GMCSF) are also important for granulopoiesis (Stockham and Scott, 2008). Stages of granulopoiesis include myeloblast, promyelocyte, myelocyte, metamyelocyte, band cell, and mature granulocyte. Primary granules appear at the promyelocyte stage in neutrophil, eosinophils, and basophils. Secondary specific granules of neutrophils, eosinophils, and basophils appear at the myelocyte stage, and allow microscopic determination of the cell lineage. Under basal conditions, approximately 6 days is required for neutrophil production in the marrow; however, this can be shortened to as little as 2–3 days under periods of increased demand (e.g., inflammation).

3.8. Increased Leukocytes Increases in leukocyte counts in the peripheral blood are most often caused by excitement and stress (mediated by catecholamines and corticosteroids), inflammation, or direct test article– related stimulation (Table 14.4). Stress, fear, excitement, or exercise causes an acute release of catecholamines (epinephrine, norepinephrine, etc.) and increased heart rate and blood pressure which can result in mild transient increases in neutrophil, monocyte, and/or lymphocyte counts. This phenomenon is often referred to as a physiological leukocytosis and is largely the results of leukocytes shifting from the marginating to the circulating pools within the vascular space. Effects of catecholamines are most pronounced in animals that have not yet become habituated to new environmental conditions or study procedures. For large animal studies, performing two baseline blood collections several days apart prior to the initiation of dosing typically helps acclimate animals reducing the impact of procedure-related stress responses on prestudy hematology data (Everds et al., 2013; Aulbach et al., 2017). Additionally,

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TABLE 14.4 Factors Affecting Leukocyte Counts in Laboratory Species Pathology and/or In-Life Findings

Root Process

Clinical Pathology Findings

Associated Causes

Stress/excitement

• [ WBC, neutrophil, monocyte • þ/ Y Lymphoid counts tissue cellularity (stress) • þ/ [ PLT counts • Bone marrow • Y Lymphocyte typically unaffected and/or eosinophil counts • Y BW and/or FDC • Stress behaviors

Inflammation

• [ WBC, • þ/ [ Bone marrow • Direct/off-target tissue neutrophil, monocyte counts cellularity toxicity • þ/ [ Lymphocyte and/or • [ M:E ratio • Immune reactions eosinophil counts • Tissue inflammation • Indwelling catheters • [ Platelet counts (reactive • Findings associated with • Surgical procedures thrombocytosis) immune reactions • Wounds/injury • þ/ Y Albumin, [ globulin • [ Fibrinogen

• Handling/restraint • Dosing/phlebotomy • Travel/shipment • New environment, caging/cage mates • Morbidity/ill health

Myeloid suppression • Y WBC, neutrophil, monocyte, • þ/ Y Bone • Direct/off-target tissue and/or eosinophil counts marrow cellularity effect • þ/ Y PLT or reticulocyte • Y M:E ratio • Morbidity/ill health counts • Lymphoid tissues typically • Y FC (rodents) • Lymphocyte count typically unaffected unaffected Lymphoid suppression

• Y Lymphocyte counts • Y Lymphoid • Direct/off-target tissue • þ/ Y WBC counts tissue cellularity effect • Neutrophil, monocyte, eosinophil • Bone marrow and M:E ratio • Morbidity/ill health typically unaffected • Y FDC (rodents) counts typically unaffected

BW, body weight; FC, food consumption; M:E, myeloid:erythroid; PLT, platelet; WBC, white blood cell. Findings may be variable based on timing/severity/duration of stimulus

the blood collection closest to the initiation of dosing (most recent) from habituated animals often provides leukocyte differential results that are less impacted by stress and excitement-related influences. The corticosteroid component of the stress response results in increased counts of neutrophils and/or monocytes with decreases in lymphocyte and eosinophil counts in the peripheral blood. Similar effects are also seen following exogenous administration of corticosteroids. These effects tend to be superimposed with the influences of catecholamines resulting in

increased total WBC and neutrophil counts with decreased lymphocytes and eosinophil counts. Stress-related changes in leukocytes may be pronounced or extreme in moribund animals, or in animals in poor clinical condition. Careful evaluation of clinical pathology data, clinical observations, and histopathology of lymphoid organs (lymphoid depletion of the thymus, spleen, and lymph nodes is commonly seen) are often necessary to make an accurate interpretation of leukogram effects (Everds et al., 2013). Inflammatory responses are common in toxicity studies and can vary from minor local

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injection site reactions, which will often not result in any notable effects on hematology data, to widespread acute to chronic systemic inflammation, which can have profound impacts among a variety of hematology endpoints. Effects on hematology endpoints most often associated with inflammation include increased neutrophil counts [with or without immature band neutrophils (i.e., “left shift”)], neutrophil cytoplasmic changes (e.g., foamy vacuolation, diffuse basophilia, and/or Do¨hle bodies), and/ or mild increases in lymphocyte counts (Stockham and Scott, 2008). Increased lymphocyte counts may also occur as a result of immune stimulation. Alterations in serum and plasma proteins (e.g., increased fibrinogen and globulin with or without concurrent decreases in albumin concentrations) also support the conclusion that leukocyte changes are attributable to inflammation (Table 14.4). Injection or infusion sites, skin wounds, and bacterial infections are common sources of inflammation in toxicity studies (Hall, 2001, 2013). Evaluation of clinical observations and histopathology data are also useful to identify the source of inflammation, although in some instances, inflammatory changes lack a definitive source and may be classified as a primary inflammatory process (i.e., no specific tissue “inflammation” observed). Increases in neutrophil counts may also result from direct stimulation of granulopoiesis following G-CSF analog administration (e.g., pegfilgrastim). These effects are often dramatic with release of immature neutrophils (myelocyte or earlier), neutrophils with pronounced cytoplasmic change, and extreme WBC counts (>200  103 cells/mL) and are related to accelerated granulopoiesis. Increased leukocytes (primarily myeloid or lymphoid cells) may also be seen secondary to hematopoietic neoplasia which may be either spontaneous or test article–related.

3.9. Decreased Leukocytes Decreased leukocyte counts in the peripheral blood are most commonly caused by bone marrow and lymphoid toxicity. Drug-induced direct bone marrow toxicity (and resultant decreased hematopoiesis) may result in decreased leukocytes in the peripheral blood. Decreases in neutrophil counts are often the most pronounced findings in these cases although

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decreases in monocyte, eosinophil, and basophil counts may also be seen (Table 14.4). Increased susceptibility to infections and fever secondary to low neutrophil counts can occur following bone marrow toxicity. Decreases in leukocyte counts secondary to bone marrow toxicity are commonly accompanied by decreases in other cell types produced in the bone marrow including reticulocytes and/or platelets (Hall, 2001, 2013). These changes may correlate with a microscopically hypocellular bone marrow. Although lymphocytes are present in relatively abundant numbers in the bone marrow, peripheral lymphocyte counts generally do not reflect bone marrow lymphocyte density as peripheral lymphocyte counts are influenced by the rate of production, circulation, use, destruction, and tissue distribution (Reagan et al., 2011). A rebound in neutrophil count, sometimes with a left shift, may occur during recovery from bone marrow toxicity, and may result in hypercellular bone marrow. Extramedullary hematopoiesis may also be observed in tissues like the spleen and liver in rodents. Lymphoid toxicity commonly results in decreased lymphocyte counts in the peripheral blood and/or depletion of lymphocytes in multiple peripheral lymphoid tissues (thymus being most sensitive) (Haley, 2013). It is important to differentiate lymphoid toxicity from stress, as blood and tissue effects may appear similar. Consideration of the drug class and relationship of lymphoid and clinical effects will help differentiate these important causes of decreased lymphocyte counts. Flow cytometry can be also used to further characterize subset(s) of lymphocytes with test article–related effects (e.g., B cells, helper T cells, cytotoxic T cells, NK cells).

3.10. Platelets Platelets are small cytoplasmic fragments of megakaryocytes, and are composed of a phospholipid membrane, open canalicular system, a cytoskeleton composed of actin and myosin, a dense tubular system of endoplasmic reticulum, and granules that contain various components involved in hemostasis and platelet activation (Stockham and Scott, 2008). Megakaryocytes reside in hematopoietic tissue of the bone marrow and spleen, and are occasionally seen in the liver or lung, and shed cytoplasmic fragments (platelets) into the blood. Thrombopoietin is the

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primary cytokine regulating the proliferation and differentiation of megakaryocyte progenitor cells and will increase secondary to decreased platelet mass. Platelet numbers in circulation vary considerably by species, and reflect a balance of production, destruction, consumption, and redistribution to tissues like the spleen. Platelet indices (e.g., MPV, PDW, MPC) are calculated results generated from measurements of various characteristics (i.e., size, density, internal granularity) of platelet populations that have gained favor in recent years and can be useful in supporting conclusions regarding the dynamics and activity of platelet populations. Mean platelet volume (MPV) is related to the average size of platelets and is related to thrombopoietic activity. Increased MPV is typically associated with increased thrombopoiesis (platelet production) and is often inversely related to overall platelet concentration (Stockham and Scott, 2008). Platelet distribution width (PDW) is related to the overall degree of size variability in the platelet population and also tends to correlate to increased thrombopoiesis. Mean platelet component (MPC) is a measurement of the refractive index of platelets and is an indicator of internal granularity. Decreases in MPC are seen when platelets become activated and have been associated with certain hypercoagulable states (Macey et al., 1999). Decreased platelet counts may occur from decreased production by megakaryocytes, peripheral consumption, or myelosuppression. Because the circulating life span of platelets is approximately 5–9 days, low platelet counts resulting from decreased production will not be immediately apparent and are typically noted 1–2 weeks following induction of myelosuppression. Platelet activation and accelerated consumption may occur at sites of hemorrhage, vasculitis, or in association with indwelling catheters or other foreign devices in the vascular system. Acute and pronounced decreases in platelet counts may be seen following postdose “pseudoallergic” reactions in primates following repeated large molecule administration (Everds and Tarrant, 2013). Sequestration of platelets in the spleen, liver, or lungs secondary to endotoxemia or severe hypothermia may also result in decreased platelet counts in the peripheral blood. Infusion of large volumes of fluids or platelet-poor blood products may also reduce

platelet counts by a dilutional effect. Low platelet counts may also occur erroneously when a portion of the platelets are excluded from the platelet count (i.e., not counted) by automated methods. This commonly occurs with in vitro platelet clumping secondary to release of tissue factor during phlebotomy which can be exacerbated by traumatic blood collection. Clinical signs resulting from low platelet counts (generally 60–80%) decrease in hepatic functional mass is needed to impact circulating levels of these proteins, therefore decreases in albumin concentration or increases in coagulation times due to hepatic insufficiency are extremely rare in nonclinical studies. Other parameters also associated with liver function such as serum cholesterol or triglyceride concentrations and even urea nitrogen concentration are influenced by extrahepatic processes and, because of this lack of specificity for liver, must be interpreted in the context of other hepatobiliary marker changes and liver histopathology to be accurately attributed to hepatic functional changes. General guidelines for identification of liver injury in nonclinical studies recommend use of at least two of the following markers of hepatocellular injury: ALT, AST, GLDH, or SDH; and at least two of the following for hepatobiliary injury: ALP, GGT, total bilirubin, total bile acids,

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or 50 nucleotidase (Tomlinson et al., 2013). Species differences in marker biology determine which marker is most appropriate as an indicator of hepatobiliary injury for that species. For example, ALT, the predominant hepatocellular injury marker used in nonclinical and human studies, is inappropriate for use in minipig due to low liver expression; GLDH and/or SDH are therefore recommended as hepatocellular injury markers for this species. Differences in circulating half-life influence both marker utility and interpretation. For example, ALT circulates for up to 45 h in dogs, NHP, and humans, and less than 8 h in rodents. In contrast, AST circulates for less than 2 h in the rat and 12 h in the dog (Hoffmann et al., 1999). Both the pattern and time course of changes in these markers can be used to evaluate target organ injuryde.g., ALT is more specific and more highly expressed in liver than in skeletal muscle, while AST is widely expressed with liver and skeletal muscle as the major source of serum AST. Thus, liver injury is associated with larger increases in ALT than AST. Similarly, because of a shorter circulating half-life, serum AST activities are expected to decrease faster than ALT activities and can be used to monitor recovery from liver injury. ALT increases are usually associated with either morphologic evidence of hepatocellular injury such as hepatocellular necrosis/apoptosis or degeneration, or in the absence of morphologic evidence of hepatobiliary pathology, can be associated with biochemical evidence of cholestasis (increased concentrations of total bilirubin and/or bile acids, and increased activity of ALP or GGT) (Ennulat et al., 2010a). Not all liver microscopic changes are associated with changes in clinical chemistry parameters. For example, rats with microscopic evidence of bland lipid accumulation (steatosis) or hepatocellular hypertrophy that is not associated with degenerative hepatocellular changes generally do not manifest changes in serum ALT, triglycerides, or cholesterol based on data from a large meta-analysis of rats given test articles including hepatotoxicants, nonhepatotoxicants, or specialized diets (Ennulat et al., 2010a). Increases in ALT that coincide with hepatocellular hypertrophy are often erroneously attributed to hepatic drug-metabolizing enzyme (DME) induction. This practice is based on

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circumstantial evidence in the older literature where data were often derived from single toxicant studies, many of which evoked complex liver histopathologic findings in addition to hepatocellular hypertrophy, the expected microscopic correlate of hepatic DME induction (Ennulat et al., 2010b; Maronpot et al., 2010). For example, corticosteroids have long been known to stimulate hepatic gluconeogenesis and be inducers of cytochrome P450 3A (CYP3A), and ALT and AST are gluconeogenic enzymes (Ennulat et al., 2010b). In several older studies, increased liver and/or serum ALT levels were interpreted as evidence of hepatic ALT induction, but recent studies of dexamethasone, an extremely potent glucocorticoid, have shown that increases in serum ALT activity exceeded increases in liver ALT activity and were associated with microscopic evidence of hepatocellular single cell necrosis in addition to the expected pharmacologically mediated cytoplasmic glycogen accumulation (Jackson et al., 2008; Ennulat et al., 2010b). Similarly, an analysis of data from rats given prototypic inducer compounds (n ¼ 17 compounds, 254 male Sprague–Dawley rats) which included cytochrome P450 gene expression, liver histopathology and weight, and liver clinical chemistry data demonstrated that, while all of the rats had evidence of phase I DME induction and increased liver weights, and most had hepatocellular hypertrophy, ALT was only increased in the rats given the inducer compounds that caused hepatocellular necrosis and/or cholestasis in addition to hypertrophy (Ennulat et al., 2010b). Thus, in the absence of microscopic evidence of hepatocellular necrosis or degeneration or biochemical evidence of cholestasis, hepatic DME induction is not a cause of increased serum ALT. Hepatic DME induction can, however, be associated with induction of hepatic ALP or GGT. In the dog, prototypic inducer compounds such as phenobarbital and phenytoin are known to increase serum ALP, while in the rat, hepatic DME may be associated with increases in serum GGT. Total serum ALP concentrations include isoenzymes from a variety of tissues, including liver (L-ALP), bone (B-ALP), and intestine (IALP). Dogs given nonsteroidal inducer compounds may have mildly increased serum ALP activity due to induction of L-ALP. Dogs

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also have a unique corticosteroid-inducible ALP (C-ALP) isoenzyme produced in response to exogenous or endogenous corticosteroids. In rats the intestinal isoenzyme is the main circulating form of ALP. Hepatic DME induction is sometimes associated with increases in serum GGT, although this is an uncommon finding with DME induction in rats, potentially because GGT has a very short circulating half-life (minutes) in the rat. In addition to DME induction through phase I biotransformation reactions, hepatobiliary marker changes can also be associated with xenobiotic effects on phase II detoxification pathways, most notably inhibition of uridine diphosphate glucuronosyltransferases (e.g., UGT1A1), organic anion transporting polypeptides (e.g., OAT1B1), and the bile salt export pump (BSEP). UGT1A1 and OAT1B1 inhibition can be associated with increases in conjugated bilirubin, while BSEP inhibition may be associated with increases in serum total bile acid concentrations. Total bilirubin concentration is routinely measured in nonclinical studies, and large increases in total bilirubin concentrations in the absence of other causes of cholestasis or increased bilirubin turnover (e.g., hemolysis) in toxicology studies may be due to UGT1A1 inhibition and can be further characterized by measurement of bilirubin subfractions to determine the proportion of conjugated versus unconjugated hyperbilirubinemia, and also by in vitro profiling. BSEP inhibition is recognized as a risk factor for cholestatic drug-induced liver injury (DILI) in the clinic, and in vitro screens are increasingly being applied in drug development. However, species differences in bile acid composition (e.g., humans have more hydrophobic bile acids than rats and dogs) and metabolism (rats metabolize bile acids to more hydrophilic and less cytotoxic bile acids through a Cyp2 family unique to rodents) impact translation of preclinical data to human risk assessment (Kenna et al., 2018). Total bile acid concentration can be highly variable across species and, beyond BSEP function, is influenced by many factors including inhibition of other transporters (e.g., organic anion transporters or OATPs), fasting state, gut microbiome metabolic activity, and extrahepatic cholestasis. As a result, total bile acids are not routinely measured in nonclinical safety studies or clinical studies.

6.7. Muscle Injury Markers The conventional markers most often used to evaluate skeletal muscle injury include AST, CK, and lactate dehydrogenase (LDH). These markers are not specific for skeletal muscle and therefore must be interpreted in the context of histopathology and changes in other markers (e.g., cardiac troponin-I or hepatobiliary markers) to identify skeletal muscle injury. Aldolase and skeletal troponin-I (sTnI) are also often measured in nonclinical and clinical studies to improve identification of skeletal muscle injury. Although highly expressed in skeletal muscle, AST is also present in liver, heart, and red blood cells; AST activity increases can therefore occur with liver or cardiac injury or hemolysis. CK is generally measured as total CK activity; however, CK circulates as homodimers or heterodimers of M and B subunits encoded by the CKM (muscle) and CKB (brain) genes which can be evaluated to help determine the source of CK increases. Although there are species differences, in general, CK-MM is most highly expressed in skeletal muscle, while cardiac muscle has predominately CK-MB with a lesser amount of CK-MM. Measurement of CK-MM protein has been shown to be more sensitive and specific than total CK activity for skeletal muscle injury in rats (Burch et al., 2016). LDH activity is present in all tissues, particularly skeletal and cardiac muscle, liver, kidney, and red blood cells. LDH circulates as five different tetramers of two different subunits: M and H. Measurement of total LDH activity is of no value for identification of muscle injury because of the lack of tissue specificity, and measurement of the various LDH isoenzymes is not often done because of the availability of newer, more specific biomarkers. Aldolase is sometimes used in clinical laboratory medicine as a marker of muscle injury, and, because of its role in energy metabolism, is also receiving attention as a potential therapeutic target in oncology. Aldolase is a glycolytic enzyme involved in the conversion of fructose 1,6-bisphosphate to glyceraldehyde 3phosphate and dihydroxyacetone phosphate. Aldolase has three different isoenzymes that are derived from three different genes with different tissue expression: aldolase A is present

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in muscle, aldolase B is found in liver and kidney, and aldolase C, within the central nervous system. Although less affected by preanalytic variables such as muscle mass or ethnicity than CK, measurement of aldolase activity in clinical patients has yielded disparate results. For example, increases in plasma aldolase A concentrations preceded increased plasma CK activity and were predictive of development and progression of myopathy in systemic sclerosis patients (Tole´dano et al., 2012). In contrast, serum aldolase activity provided no added value over measurement of CK activity in statin myopathy because aldolase had greater biological variability than CK activity (Wu et al., 2009). Measurement of total serum aldolase activity has been used to a limited extent in nonclinical studies, particularly in rats where the circulating half-life of aldolase is around 20 h (Schapira et al., 1960), compared with minutes for CK (Grindem et al., 2018). Because of potential cardiac or hepatic sources of increased CK or AST activities, measurement of serum aldolase activity can be used to help discriminate between muscle and liver injury in studies in rats and other nonclinical species (Vassallo et al., 2009). However, as with other conventional markers of skeletal muscle injury, aldolase changes must be interpreted in the context of histopathology findings as well as changes in other muscle or hepatobiliary injury markers. Increases in serum or plasma activities of the above conventional markers do not discriminate between skeletal and cardiac muscle injury; however, measurement of troponins, striated muscle-specific structural and regulatory proteins that mediate calcium signaling, and actin–myosin interactions are often used to improve specificity for cardiac or skeletal muscle injury. Cardiac and skeletal muscle troponins (cTn and sTn, respectively) contain three subunits with discrete functions: troponin C (TnC) serves as a calcium sensor, troponin I (TnI) inhibits formation of the tropomyosin complex at low intracellular calcium concentrations, and troponin T (TnT) binds tropomyosin and is involved in regulating actin and myosin interactions during muscle contraction. Cardiac troponin I (cTnI) and T (cTnT) are most often used for detection of cardiomyocyte injury; troponin C is not specific for heart or skeletal

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muscle and is not measured. More information on cardiac biomarkers can be found in sections below. In addition to increasing specificity for the detection of skeletal muscle injury, measurement of sTnI can also help characterize muscle injury. Skeletal myofibers were formerly characterized using physiological and histochemical assays as type I or slow-twitch (oxidative or “red”) muscle, and type II or fast-twitch (“white” or glycolytic) muscle. Skeletal muscle is now known to contain four main myofiber types that differ in myosin heavy chain (MyHC) composition: MyHC 1, 2A, 2B, and 2X. Skeletal muscle fiber type composition varies by muscle and between species. In general, rodent myofibers are highly oxidative and consist predominantly of 2X and 2B fibers, whereas human muscles have fewer oxidative enzymes and contain primarily type 1 and 2A fibers. In rats, the soleus muscle contains predominantly type I fibers, whereas quadriceps femoris, gastrocnemius, and rectus femoris contain primarily type II fibers with abundant MyHC-2B expression. In contrast, human fast-twitch muscles essentially lack MyHC-2B and express primarily 2A. Druginduced skeletal muscle injury is often specific to myofiber type, for example, statin-induced myopathy primarily involves fast-twitch or type II myofibers, whereas peroxisome proliferator–induced skeletal muscle injury predominantly affects slow-twitch or type I myofibers (Vassallo et al., 2009). Markers that can discriminate between injury to slow-twitch and fasttwitch myofibers have not been qualified in nonclinical species. Of the markers currently available, a rat sTnI ELISA which detects the fast-twitch sTnI subunit (Tnni2) has demonstrated specificity for skeletal muscle injury in rats (Burch et al., 2016; Vassallo et al., 2009; Tonomura et al., 2009) and dogs (Vlasakova et al., 2017); however, specificity of this assay for fast twitch or type II skeletal muscle has not been determined in animals. Further, due to species differences in myofiber type distribution, functional (e.g., shortening velocity), and metabolic physiology, use of myofiber type-specific assays in nonclinical studies may be of limited translational relevance. Other protein markers of skeletal and cardiac muscle injury recently evaluated in rat studies include fatty acid–binding protein 3 (Fabp3),

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myosin light chain 3 (Myl3), and CK-MM (Ckm) (Burch et al., 2016; Tonomura et al., 2009; Vassallo et al., 2009). Fabp3, also known as hearttype fatty acid–binding protein (H-Fabp), is involved in intracellular fatty acid transport and is expressed in a variety of tissues including cardiac and skeletal muscle as well as liver. Because of the prominent role of the liver in lipid metabolism, Fabp3 can be highly upregulated in response to metabolic demands, for example, hepatic Fabp3 protein was upregulated 50-fold in rats given peroxisome proliferator–activated receptor-a agonists (Kochansky et al., 2018). Although serum Fabp3 and other skeletal muscle injury markers were not increased in rats given various hepatotoxicants (Tonomura et al., 2009), the use of serum Fabp3 as a marker of skeletal or cardiac muscle injury may be limited by the potential for increases in serum Fabp3 of liver origin. Myl3 is highly expressed in type I skeletal muscle as well as cardiac muscle, and therefore is best interpreted in the context of histopathology and changes in other markers of skeletal and cardiac muscle injury. CK-MM is the main isoform of CK found in skeletal muscle and has clear advantages over CK activity measurement as a specific marker of skeletal muscle injury. Myl3 and CK-MM have demonstrated utility for skeletal muscle injury in rats (Burch et al., 2016; Tonomura et al., 2009) and dogs (Vlasakova et al., 2017). Skeletal TnI, Myl3, CK-MM, and Fabp3 have received a Letter of Support encouraging their use as markers of skeletal muscle degeneration/necrosis in preclinical and clinical studies (Burch et al., 2016). In summary, conventional skeletal muscle injury markers such as CK and AST activity lack specificity for skeletal muscle and must be interpreted in the context of other study findings including histopathology, other biomarker data including traditional biomarkers of liver and cardiac injury, and potentially troponins such as cTnI and sTnI to confirm skeletal muscle injury. Skeletal TnI, Fabp3, Myl3, and CK-MM have proven to be exceptional early markers of skeletal muscle injury in rats and dogs, generally preceding and having a greater dynamic range than AST and CK activities. However, evaluation of muscle injury markers can produce falsenegative results or changes inconsistent with

the severity of muscle injury because of timing of sample collection and differences in biomarker biology. As a result, identification and characterization of skeletal muscle injury may also require further evaluation of muscle function in investigative studies.

7. URINALYSIS AND URINE CHEMISTRY INTERPRETATION Urinalysis is commonly included in nonclinical toxicity study protocols and is used to assess kidney function, urinary tract health, and other metabolic disturbances (see Kidney, Vol 5, Chap 2 and Lower Urinary Tract, Vol 5, Chap 3). While standard urinalysis endpoints are a regulatory requirement, routine urinalysis is often an imprecise tool as assays are largely qualitative or semiquantitative, and routine collection urine practices are prone to preanalytical variability. Components of urinalysis vary slightly among laboratories but usually contain the following: urine volume; physical examination (color, clarity, and specific gravity or osmolality); chemical examination by reagent strip methods (e.g., pH, protein, glucose, ketones, heme [occult blood], bilirubin, urobilinogen, and also potentially nitrite and leukocyte esterase); and/or microscopic examination of unstained or stained sediment to identify erythrocytes, leukocytes, bacteria, casts, crystals, epithelial cells, and other nondissolved materials. Quantitative urinalysis refers to the measurement of specific urine components (e.g., volume, protein, albumin, glucose concentrations) using quantitative methods, generally over a defined urine collection time period which can range from 6 to 16 or more hours depending on species. Additional parameters that are less often included in quantitative urinalysis are electrolyte excretion and specific urinary protein biomarkers that have been evaluated in nonclinical species and qualified in rat and human (Ennulat et al., 2018). These protein markers can help localize the nephron segment involved and include proximal tubule injury markers such as kidney injury molecule-1 (KIM-1) or low molecular weight markers (e.g., urinary cystatin C (uCysC), b2-microglobulin, or a1-

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microglobulin), and general renal injury markers (e.g., lipocalin-2 (NGAL), clusterin, or osteopontin). Additional information is found in the novel renal biomarker sections of this chapter and Clinical Pathology in Nonclinical Toxicity Testing, Vol 1, Chap 10. Urine composition is highly variable and is heavily influenced by preanalytical factors such as the method of collection (cystocentesis vs. free catch vs. timed collection in a metabolism cage), feeding regimen, or experimental design. Urine collection by cystocentesis or free catch methods provides urine biomarker results for a single point in time and therefore can be associated with considerable variability. Timed urine collections are more resource intensive but yield results with less variability that are more representative of the state of renal function or structural injury. Normalization of urinary constituent concentrations can be used to mitigate the inherent variability in urine composition. Although consensus is lacking, common methods of normalization include normalization to urinary creatinine, urine volume, or collection period, each with their own drawbacks. With severe acute nephrotoxicity, use of urine creatinine values for normalization can actually overestimate urine biomarker changes due to differences between the rate of urine biomarker release and the rate of sCr increase because urinary Cr excretion reflects glomerular filtration of sCr, and sCr increases associated with compromised renal function in acute renal injury are known to lag behind renal structural injury (Ennulat and Adler, 2015). Similarly, increases in urine glucose excretion may occur when the tubular maximum for glucose reabsorption is exceeded during hyperglycemia; however, glucosuria in the absence of hyperglycemia (normoglycemic glucosuria) can be an early indicator of proximal tubular functional compromise or injury prior to the development of increases in sCr or urea nitrogen. For timed urine collections, normalization to urine volume or collection period may be preferred in cases of severe acute renal injury. Regardless of the method used, data should be normalized using the same method throughout the study. Understanding the normal variability of the urine analytes is essential when interpreting

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urinalysis data. Urine is continually excreted, is of an inconstant volume, and is more dynamically variable in composition than blood constituents which circulate within a defined vascular space and are generally more tightly regulated. For example, sCr is a parameter that has extremely low variability in control animals; however, it is not uncommon to have more than two-fold differences in urine analytes in control animals, therefore tolerance ranges for urinary parameters must be accordingly sized. Due to the large degree of variability in urine parameters, single “spot” samples normalized to urine creatinine may be less accurate than timed collections regardless of normalization method. However, timed urine collection methods have their own sources of preanalytical variability. For example, rodents fasted overnight will have decreased water consumption which can concentrate urine potentially leading to false-positive results. Conversely, water contamination can dilute urine collected in metabolism cages, leading to falsenegative results. In summary, biological variability in quantitative urinalysis can be considerably greater than biological variability in blood analytes. In addition to consideration of collection method used, changes in urine parameters must also be interpreted in the context of other study findings including clinical observations (e.g., emesis or diarrhea), clinical chemistry, and histopathology.

8. NONSTANDARD BIOMARKERS Nonstandard biomarkers continue to play an increasingly important role in drug and chemical development studies and have become expected if not formally required as an adjunct component of many safety studies. Although there are many “biomarkers” that would be considered traditional in the sense that they have been in use for many years and utilize conventional methodologies (i.e., standard hematology and clinical chemistry assays), most of the biomarkers discussed in this section are newer safety biomarkers used to characterize biologic, physiologic, and/or pathologic signals that have demonstrated usefulness when used in conjunction with standard clinical and anatomic pathology endpoints. These assays typically

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utilize a wide range of relatively novel immunoassay methodologies using commercially available assay kits/reagents that are proprietary and not consistently regulated or standardized. For this reason, more scrutiny is often placed on methodology, assay performance, and proof of biologic relevance for biomarker assays (Bioanalytical Method Validation (CDER), 2018; Khan et al., 2015). Due to their use in pathology characterization, soluble nature, and similarity to like-methodologies, interpretation of biomarker assays is most effective when integrated with clinical and anatomic pathology results using a weight-of-evidence approach (Ramaiah et al., 2017; Aulbach et al., 2019a, b). In the context of interpreting biomarker results it is important to keep in mind the relative quantitative nature of these assays. When compared to standard clinical pathology endpoints which usually yield similar test results regardless of instrument, methodology, or laboratory, biomarker assays can sometimes give dramatically different results/ absolute values from laboratory to laboratory depending on the methods used. Assuming satisfactory assay performance, what becomes more important than the absolute values in these cases are the pattern and magnitude of signals generated in response to a biologic or pathologic stimulus. When used in conjunction with traditional clinical and anatomic pathology assessment, biomarkers can be powerful tools in characterizing pathologic/biologic responses with increased sensitivity and specificity. These features also emphasize the importance of appropriate assay validation practices. A recent study comparing renal biomarkers across different platforms revealed differences in individual biomarkers that ranged up to 15-fold and that these differences were probably due to the use of different antibodies with varying degrees of affinity and specificity (Gautier et al., 2014). This phenomenon is not uncommon and emphasizes the importance in establishing laboratory or method-specific reference intervals. As important as analytical performance and method consistency, biologic validation (or proofof-concept studies) and assessment of samples in species-specific matrix are fundamental components of contemporary biomarker paradigms. Biologic validations involve obtaining positive control samples, either generating them using animal models, tissue homogenates, or purchased

commercial sources, as a component of method validation. However, for relatively novel biomarkers and/or methods that are new to a laboratory, incorporation of a positive control model can be an important component of a method validation. In addition, given the antibody-based nature of many of these methods, incorporating analysis of samples in speciesspecific matrix during the validation is also essential and expected based on current regulatory guidance (Lee et al., 2006; Khan et al., 2015).

8.1. Renal Biomarkers New urinary biomarkers of acute kidney injury have recently been evaluated by consortia of representatives from industry, academia, and regulatory organizations leading to regulatory qualification (FDA, 2020a) or letters of support (FDA, 2020b) for use of these markers in nonclinical and clinical studies in conjunction with traditional clinical chemistry parameters. The second-generation markers qualified for detection of drug-induced kidney injury (DIKI) in rat definitive or good laboratory practice (GLP) studies include the conventional markers urine albumin, total protein, N-acetyl-b-D-glucosaminidase (NAG), and the low molecular weight proteins b2microglobulin and cystatin C (CysC). Additional urine proteins qualified for rat GLP studies include kidney injury molecule-1 (KIM-1), clusterin, and renal papillary antigen-1 (RPA-1, FDA 2020a). Subsequent qualification submissions have yielded letters of support (LOS) which recommend the use of neutrophil gelatinaseassociated protein or lipocalin-2 (NGAL) and osteopontin (OPN) in nonclinical and exploratory clinical studies, and urinary alpha-glutathione-Stransferase (a-GST), clusterin, CysC, KIM-1, NGAL, OPN, albumin, and total protein in early clinical studies (FDA, 2020b). An overview of these and other urinary markers of renal injury can be found in Clinical Pathology in Nonclinical Toxicity Testing, Vol 1, Chap 10 and recent references (Charlton et al., 2014; Ennulat et al., 2018; Harpur et al., 2011; Phillips et al., 2016). Briefly, these markers include constitutive urinary proteins released into the urine such as albumin and total protein, several low molecular weight proteins and NAG, and proteins that are induced within various segments of the nephron in response to renal

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injury including KIM-1, NGAL, clusterin, and OPN. Most of the conventional and next-generation urinary markers reflect proximal tubular injury or functional compromise, often well in advance of increases in sCr or urea nitrogen concentration. The proximal tubule is the most common site of renal injury because of its major role in concentrating the glomerular filtrate, high rate of endogenous and xenobiotic metabolism, and limited antioxidative and antiapoptotic capacity. Proximal tubular injury often precedes glomerular injury in acute kidney injury and progression to chronic renal disease, and the proximal tubules constitute more than 50% of the renal parenchymal mass (Chevalier, 2016). Consequently, characterization of the nature of proximal tubular injury or functional status adds value to both clinical diagnosis and risk assessment; and quantitative urinalysis of conventional and secondgeneration renal injury markers provides added value over measurement of sCr or in the assessment of drug-induced kidney injury. Due to the massive functional and structural reserve capability of the kidney, increases in urinary excretion of analytes such as glucose, electrolytes, and proteins of varying molecular weights often precede increases in sCr or urea nitrogen concentration. Increases in urinary excretion of albumin, total protein, or low molecular weight proteins occur with altered

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glomerular permeability and saturation of proximal tubular reabsorptive capacity or following structural injury of proximal tubules (e.g., degeneration/necrosis). Similarly, KIM-1, NAG, or a-GST is increased in response to structural injury to proximal tubules. There can also be differences between the time of onset of urine protein changes after proximal tubular structural injury, with excretion of constitutive proteins such as albumin preceding that of induced proteins such as KIM-1 in drug-induced proximal tubular injury by hours or even days (Lambert et al., 2010; see Figure 14.2). Although the proximal tubule is the most common site of renal injury, distal nephron segments also can sustain injury as a result of direct or segment-specific nephrotoxicity (Bonventre et al., 2010) or nonspecific collateral damage following proximal tubular injury. Several markers induced in response to general renal injury including NGAL, clusterin, and osteopontin have been evaluated in nonclinical species and humans, as well as RPA-1, a constitutive marker of collecting duct injury unique to the rat. While many of these markers are upregulated within specific segments of the distal nephron, several including NGAL and osteopontin are not kidney-specific and may cross the glomerular filtration barrier and enter the urine from the systemic circulation because of low molecular weight.

FIGURE 14.2 Chronology of Constitutive Versus Inducible Protein Biomarker Changes Following Proximal Tubular Injury in Rats Given Hexachlorobutanedione (HBCD) or Potassium Dichromate (K2Cr2O7). (A) Albumin and (B) KIM-1 were measured over 96-hour in the urine of male albino Wistar rats given a single injection of hexachlorobutadiene i.p. (n ¼ 4), potassium dichromate s.c. (n ¼ 4), or vehicle (n ¼ 2). For both toxicants, increases in albumin excretion preceded excretion of KIM-1 by approximately 24 h, consistent with the more rapid release expected of a constitutive protein relative to the later release of an inducible protein biomarker. Courtesy of A. Chiusolo, K.M. Lynch and P Cristofori.

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As with any quantitative urinalysis data, interpretation of changes in these next-generation urinary biomarkers requires an understanding of their biological variability and dynamic range. Meta-analyses of the diagnostic utility of these markers in preclinical species have largely been based on studies using tool nephrotoxicants, often with severe renal injury, which demonstrated improved sensitivity and specificity for renal injury relative to sCr and urea nitrogen concentration as comparators. Many of the same compounds, most notably cisplatin and gentamicin, were used repeatedly and at doses leading to structural renal injury that far exceeded the kidney injury seen in nonclinical toxicity evaluation, leading to “spectrum bias” and overestimation of diagnostic utility of these next-generation markers. Evaluation of diagnostic performance using metaanalytic methods such as receiver operating characteristic (ROC) analysis or even the Youden Index (value of the biomarker with the greatest sensitivity and specificity for the reference disease state in an ROC analysis) does not inform interpretation of changes in these next-generation renal injury markers and they do not reflect actual changes seen in traditional nonclinical toxicity studies. In practice, the diagnostic performance of conventional urinary biomarkers such as urinary protein, albumin, or glucose has been essentially comparable to that of the novel urinary biomarkers, and these markers are readily applicable across species.

8.2. Hepatobiliary Biomarkers Clinical DILI markers including miR-122, GLDH, keratin 18 (CK-18), high mobility group box 1 (HMGB1), osteopontin, and macrophage colony-stimulating factor receptor (MCSFR1) have recently received LOS from regulatory agencies for exploratory use in clinical studies alone or in association with traditional liver injury markers (Roth et al., 2020). Although there have been no formal regulatory qualification submissions for markers of DILI in nonclinical species, a number of studies conducted by industry and academia have helped characterize the performance of some of these markers. The most common markers evaluated in rodent liver injury models have been miR-122 and GLDH. MicroRNAs (miRs) are small noncoding RNAs that are highly tissue specific and regulate posttranslational gene expression in development,

differentiation, and metabolism. Circulating miRs are promising translational biomarkers because they are highly conserved across species and can be quantified using sensitive assays such as realtime polymerase chain reaction (PCR), and they have better storage stability than protein or enzymatic activity biomarkers, making them amenable to retrospective assessment in archival samples. miR-122 is highly expressed in liver, representing about 70% of the total hepatic miR pool, and is considered liver specific in both humans and rodents (Jopling, 2012). miR-122 is initially held considerable clinical interest as a biomarker of liver injury; however, clinical consortia have since deprioritized miR-122 due to its extensive variability and potential for release under normal physiological conditions. In rodent studies, miR122 changes generally parallel changes in traditional markers such as ALT and GLDH. miR-122 is relatively specific for hepatobiliary injury in the rat, as increased circulating miR-122 levels were noted in rats given a variety of tool hepatotoxicants with hepatobiliary injury, but not with phenobarbital-mediated hypertrophy or extrahepatic (doxorubicin) injury (Starckx et al., 2013). The diagnostic performance of serum miR-122 levels was slightly better than ALT and comparable with GLDH or AST in rats given various development compounds or tool hepatotoxicants (Sharapova et al., 2016). Serum miR-122 increased prior to and to a greater degree than ALT in rats given acetaminophen, suggesting it may be a useful early marker of liver injury (Park et al., 2016). In a rat warm ischemia-reperfusion model, increases in circulating miR-122 paralleled increases in ALT and were not associated with increase in hepatic miR-122 expression (Caster et al., 2015). In rodent nonalcoholic fatty liver disease models, serum miR-122 was increased in the absence of an increases in ALT, consistent with its role in lipid metabolism and suggesting that it might also be a candidate marker of steatosis (Yamada et al., 2015). As in preclinical species, use of GLDH in humans has demonstrated that, while GLDH correlates strongly with ALT activity in liver injury, GLDH has advantages over ALT because it is more liver specific and can be used to help differentiate between ALT increases of liver versus skeletal muscle origin, is not vulnerable to age or sexrelated differences, and has little inter- and intrasubject variability. In rats, GLDH is less widely used than ALT although it has a larger dynamic

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range and longer circulating half-life. Although GLDH activity can become more variable with increasing age in the rat (e.g., studies of 6 months or longer duration), GLDH is nevertheless a useful biomarker in the rat because of this large dynamic range. Both GLDH and miR-122 are useful adjunct markers for addition to nonclinical toxicity studies because they provide greater specificity and sometimes sensitivity for liver injury than traditional hepatobiliary injury markers. As with other new markers, these and other exploratory nonclinical markers should be evaluated in the relevant nonclinical drug development “space.” With increased implementation as exploratory markers of liver injury, the sensitivity and specificity of miR-122, GLDH, and other next-generation DILI markers can and should be evaluated across a broad range of studies in future meta-analyses.

8.3. Cardiac Biomarkers (see Cardiovascular System, Vol 5, Chap 1) Cardiac Troponin Cardiac troponin I and T (cTnI and cTnT) are sensitive and specific biomarkers of cardiac muscle injury. These globular proteins are released from cardiac muscle and may be detected within an hour of injury. There are two pools of cardiac troponin within cardiac myocytes. The cytoplasmic pool is very small and is released quickly with cardiomyocyte injury. The much larger sarcomeric pool of cardiac troponin is released more slowly upon injury. Cardiac troponins have short half-lives and serum values may peak within hours after initial injury (Berridge et al., 2009; Walker, 2006). Effective study design needs to accommodate assessment of cardiac troponins shortly after initial test article administration to catch potential transient elevations. Serial determinations of cTn concentration within a toxicity study provide the greatest probability of success in detection and monitoring subtle cardiac injury and should include measurements early, mid, and at termination of the study at a minimum. Cardiac troponins are highly translatable biomarkers of cardiac injury in laboratory animals and people and use of this biomarker is well accepted by regulatory bodies (O’Brien, 2006, 2008). Most assays in routine use in toxicity safety studies measure cTn values in ng/mL.

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The newer ultrasensitive or high sensitivity assays detect cTn in the low pg/mL range, thus increasing the sensitivity and precision of detection of baseline cTn concentrations in healthy, resting control populations at levels that were previously undetectable using earlier generation commercial assays, and thereby potentially increasing the ability to detect test article–induced myocardial injury (Schultze et al., 2008, 2009; Reagan et al., 2013). An additional benefit of some of the ultrasensitive assays is a smaller sample volume requirement that makes the assays easier to use in rodents (Schultze et al., 2008, 2009). Use of an ultrasensitive cTnI assay has demonstrated variability in baseline measurements among/ between strains, sexes, and surgical alterations (i.e., castration or ovariectomization) as well as different ages in rats (Herman et al., 2011, 2014). Investigators have used the ultrasensitive cTnI assay in longitudinal studies of Sprague–Dawley rats (Schultze et al., 2009) and NHPs (Schultze et al., 2015) to demonstrate the effects of handling, transport, and/or gavage dosing on the biologic variability in cTnI values. Administration of rosiglitazone (Mikaelian et al., 2011), hydralazine (Mikaelian et al., 2009), and isoproterenol (Schultze et al., 2008) has caused increased concentrations of cTnI in blood from rats. While experimentally induced cardiac lesions are associated with increased serum cTn concentration in animals, cTn can increase in the absence of apparent alterations in myocyte morphology. Differences between Sprague–Dawley rats in mortality, histopathologic cardiac injury, and increases in cTnI have been demonstrated following administration of the cardiotoxicant isoproterenol (Schultze et al., 2011). The amount of cTnI released is affected by tissue content. Partial depletion of tissue cTnI such as with inanition, weight loss, and heart failure has been reported in rats. The time of peak cTn response and duration of response depend on the mechanism of cardiac injury, dose, and frequency of test article administration. In many situations, cTn release can occur within minutes of injury. Faster kinetics of clearance occurs in animals compared with humans. Creatine Kinase, Aspartate Aminotransferase, Lactate Dehydrogenase Prior to use of cTn in toxicity studies, degenerating/necrotizing cardiac injury was detected by

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increases in the activities of creatine kinase (CK), aspartate aminotransferase (AST), and/or lactate dehydrogenase (LDH). Of these three, CK is generally the most sensitive, with peak activity reached within about 6–12 h following insult. CK has a relatively short half-life (about 2–4 h), and activity returns rapidly to normal following cessation of myodegeneration or necrosis. Liver and muscle are considered the main sources of AST activity in serum. Maximal AST activity is reached about 24–48 h postinjury. All tissues contain various amounts of LDH activity, which make it nonspecific as a marker. LDH activity peaks about 48– 72 h postinjury. Interpretation of changes in CK, LDH, and AST activities in toxicity studies should be made with caution, as the variability of these parameters can be quite wide in healthy animals, and these enzymes lack tissue specificity. Species differences exist in the predominant tissue source of CK and LDH serum activities (Preus et al., 1989). Without separation of various isozymes of CK and LDH, there is only small value in determination of total enzyme activity in detection of cardiac injury. Due to expense, time required for analysis, and limited availability, measurement of total enzyme activity and isozyme separation of these enzymes is rarely used for detection of cardiac injury in toxicity studies today. Atrial and Brain Natriuretic Peptides In healthy animals the natriuretic peptides control blood volume, blood pressure, and cardiac growth. Atrial natriuretic peptide (ANP) and brain natriuretic peptide (BNP) and their amino cleavage equivalents NT-proANP and NTproBNP, respectively, are translational biomarkers of cardiac function used to detect and monitor increased cardiac stretch due to heart failure, increased plasma volume, and some specific forms of cardiac hypertrophy in rats, dogs, and NHPs. ANP and BNP have short half-lives. The amino cleavage equivalents are measured more often in toxicity studies because of their longer stability and greater assay availability (Engle and Watson, 2016; Dunn et al., 2017). Assays for ANP and NT-proANP that will cross-react with rat are more numerous than assays for BNP and NT-proBNP used in this species. Hemodynamic-induced increases in ANP concentrations usually last longer than increases in BNP concentrations (Dunn et al., 2017; Vinken et al., 2016). In example, daily

administration of a PPARa/g compound to Sprague–Dawley rats caused increased concentration of NT-proANP at 2, 4, and 8 days of test article treatment. After 14 days of daily treatment, concentrations of NT-proANP and NT-proBNP were increased and left ventricular cardiac hypertrophy was detected. At 28 days, concentrations of NT-proANP remained increased but concentrations of NT-proBNP were no longer different than control (Engle et al., 2010). Fatty Acid–Binding Protein and Myosin Light Chain 3 The biomarkers fatty acid–binding protein 3 (Fabp3) and myosin light chain 3 (Myl3) have been used to evaluate cardiac and skeletal muscle injury in investigational and toxicity studies (Schultze et al., 2011; Pritt et al., 2008). Fabp3 concentrations in serum or plasma increase following cardiac damage at times similar to cTn. My13 concentrations increase more slowly after cardiac injury and concentrations remain increased longer than cTn. Neither Fabp3 nor Myl3 is considered a specific marker for cardiac injury.

8.4. Inflammatory Biomarkers Contemporary approaches to assessing inflammation in nonclinical toxicity studies involve combining traditional toxicity assessment modalities (e.g., hematology, clinical chemistry, anatomic pathology) with immune system biomarkers such as APPs, cytokines/chemokines, markers of complement activation, immunoglobins, and/or other mediators of inflammation (e.g., histamine, tryptase). It is important to consider that each of these classes of biomarkers has different kinetics and response patterns yielding different magnitudes of change depending on when samples are collected in relation to dose administration and ultimately the timing of the pathologic response. For example, cytokines and complement tend to respond acutely (24–72 h) (Tarrant, 2010; Cray et al., 2009). Given these factors, a weight-of-evidence and integrated approach is often most appropriate when combining the results from various study endpoints. Utilizing well-designed combinations

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of these markers can be helpful in characterizing a variety of immune and inflammatory conditions including vaccine responses, postdose immune and hypersensitivity reactions, development of autoimmune and immunogenicity responses, and other inflammatory conditions. In the following sections interpretation of specific groups of inflammatory biomarkers is discussed. Acute Phase Proteins APPs are blood proteins primarily synthesized in the liver that are produced as part of the innate immune response and are useful in characterizing pro-inflammatory effects in humans and animals. Increases in these biomarkers can be seen in response to bacterial infection, tissue trauma, and surgery (Cray et al., 2009). APPs can be either positive, they increase with inflammation (e.g., fibrinogen, C-reactive protein (CRP), serum amyloid A (SAA)), or negative (e.g., albumin), they decrease with inflammation. Most APPs will increase within 24–72 h following an inflammatory stimulus which should be considered when choosing phlebotomy or blood collection intervals and during data interpretation. In addition, APPs are generally classified into major (>10-fold increase) and minor (1- to 10-fold increase) categories which have predictable species-specific response patterns (Cray et al., 2009). For example, in human, NHP, and dogs CRP is the major APP, while in rodents, the CRP response will be comparatively minor. In the rat and mouse a1acid glycoprotein and haptoglobin are useful APPs, respectively. Decreases in serum albumin and albumin/globulin ratio continue to be sensitive and highly useful biomarkers for the detection of pro-inflammatory signals in any species (Stockham and Scott, 2008; Cray et al., 2009). Cytokine/Chemokine Evaluation Cytokines are a heterogeneous collection of peptides that serve as signaling molecules between cells and elicit biological responses, including cell activation, proliferation, growth, differentiation, migration, and apoptosis (Tarrant, 2010; Gwaltney-Brant, 2014). The evaluation of cytokines has had widespread use for the last decade for the purposes of hazard identification, mechanistic characterization, as markers of pharmacodynamic activity and/or efficacy, as well as to monitor for adverse drug

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reactions including immune complex disease, with both in vivo and in vitro applications (Tarrant, 2010; Finco et al., 2014). Cytokines are routinely used to monitor and/or characterize potentially adverse immune responses, in both clinical and nonclinical settings, in a variety of therapeutic classes (e.g., biotherapeutics, gene/ cell therapies, CAR-T cell, RNA) (Everds and Tarrant, 2013; Vahle, 2018). Blood cytokine measurements are an attractive option due to the ability to perform serial monitoring using readily accessible samples on a multitude of available assay platforms. However, the use of cytokines can pose some challenges related to their short half-life and release dynamics, lack of specificity, lack of standardized methodologies, and general overall expense. When applying cytokine measurements to nonclinical safety studies, it is important to take samples at intervals that are physiologically relevant to the specific immunomodulation or immunopathology being induced. Cytokines are released quickly and rapidly cleared; hence serial blood sampling time points should occur at several intervals within the first 24 h of dose administration (Everds and Tarrant, 2013). A standard approach is to sample 2–3 times within 24 h of dose administration (e.g., 2–6, 12, and 24 h postdose), although this may need to be customized somewhat based on the known pharmacology of the compound. Ongoing immune/inflammatory stimulation may allow positive signals to be detected at later intervals although this is more the exception than the rule. Increases in cytokine concentrations (2– 24 h) will slightly precede increases in neutrophil count (12–36 h), which precede increases in APPs (24–72 h). Most cytokine responses will completely resolve within 24–36 h following an acute stimulus. Substantial procedure-related increases in cytokines can be seen following various in-life procedures (restraint, slings, intravenous dose administration) and in these cases it is imperative to have a control group and/or a robust enough study to interpret through the procedure-related “noise” so common to cytokine measurements (Sakai et al., 2016; Kuo et al., 2006). Some of the most common cytokines being evaluated include interleukin-6 (IL-6), monocyte chemoattractant protein-1 (MCP-1), tumor necrosis factor (TNF-a), and interferon gamma (IFN-g) with more advanced panels including

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granulocyte-macrophage colony-stimulating factor (GM-CSF), interferon beta (IFN-b), macrophage inflammatory protein (MIP-1a), interleukin-1RA (IL-1RA), interleukin-2 (IL-2), interleukin-4 (IL-4), interleukin-8 (IL-8/KC/ GRO), interleukin-10 (IL-10), interleukin-12 (IL12), interleukin-13 (IL-13), and/or interleukin17a (IL-17a). Most often cytokines are run in panels focused on identifying general inflammation (e.g., IL-6, TNF-a, MCP-1) or more specific immune or mechanistic responses [T helper type 1 response (Th1) (e.g., IFN-g, TNF-a, TNFb) or T helper type 2 response (Th2) (e.g., IL-4, IL-5, IL-10, and IL-13)]. Complement The complement system/cascade is a component of the innate immune system and is comprised of dozens of circulating and cellbound blood proteins that can be activated through three distinct pathways: the classical pathway, the alternative pathway, and the lectin pathway, each with different recognition molecules (Shih and Murali, 2015; Dunkelberger and Song, 2009). Upon proteolytic activation, an enzymatic cascade ensues ultimately resulting in recruitment of inflammatory cells, enhancement of phagocytic ability of mononuclear cells, and formation of the membrane attack complex that promote the lysis of microbes. In the context of nonclinical safety studies, complement assays have been used to characterize postdose immune reactions, hypersensitivity reactions, and other immunemeditated and inflammatory conditions (Engelhardt et al., 2015; Everds & Tarrant, 2013). Some of the more common complement endpoints utilized in nonclinical studies include assessment of complement split products like Bb, C1q, C3a, C4a/d, sC5b-9, and C5a; circulating immune complexes (CICs); and/or total complement (CH50), which is a measurement of functional capacity of the complement system (Kirschfink and Mollnes, 2003). These tests are typically arranged in panels aimed at characterizing complement activation, function, and/or which complement pathway is being activated. For example, classical pathway activation results in increases in complement split products C1q, C4d, and C4bc, while the alternative pathway triggers changes in Bb, C3, C3a, while activation of the terminal arm of the pathway induces increases in C5a and SC5b-9. Upon activation

of the cascade, complement split products (e.g., Bb, C1q, C3a, C5a) will tend to increase as they are enzymatically cleaved and become free in circulation while parent proteins like total C3 or C5 will tend to decrease upon complement activation as they are cleaved into split products resulting in decreased circulating concentrations. Central to the cascade are split products C3a and C5a which are anaphylatoxins and mediate many of the physiologic and vascular (e.g., increased permeability, vasodilation) changes associated with complement activation (Dunkelberger and Song, 2009). Downstream activation of the terminal arm of the complement cascade results in increases in sC5b-9 (soluble form of the membrane attack complex (MAC)) which will often follow increases in C3a and C5a. The total complement activity assay, also referred to as CH50, is a functional assay and will decrease upon strong activation indicating the lack of, or exhaustion of, complement activity (Kirschfink and Mollnes, 2003). Interpretation of these endpoints is most effective when using a panel of pro-inflammatory and compliment endpoints in order to fully characterize the immune response.

8.5. Hormones (see Endocrine System, Vol 4, Chap 7) Hormone concentrations are not routinely measured in nonclinical regulatory studies except where needed to characterize a mechanistic basis for study findings or to identify translatable markers for monitoring potential clinical safety risks. Thyroid hormone measurement to characterize changes in thyroid gland related to hepatic drug-metabolizing enzyme induction is the most common hormone panel evaluated in traditional repeat-dose studies. Hormones are also commonly used as pharmacodynamic markers in some cases. However, because of the considerable variability in serum hormone concentrations due to a variety of factors including circadian or diurnal rhythm, pulsatility, stage of the reproductive cycle, and stress associated with blood collection, customized investigative studies are more appropriate for mechanistic investigation of endocrine changes than traditional toxicity studies. These investigative studies should be designed to address specific questions or hypotheses, and they should be powered with larger numbers of animals than are used in traditional

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toxicity studies to mitigate the large variability inherent in circulating hormone concentrations. For both investigative and repeat-dose studies, hormone data should be evaluated in the context of histopathology findings, organ weight data, changes in other functionally related hormones, and, in some cases, pharmacology of the test article (Stanislaus et al., 2012). In nonclinical safety evaluation, the hormones most commonly assessed include hormones of the male (testosterone, follicle-stimulating hormone [FSH], luteinizing hormone [LH]) and female (estrogen, progesterone) hypothalamic pituitary gonadal axis; hypothalamic–pituitary–thyroid axis hormones including triiodothyronine (T3), thyroxin (T4), and thyroid-stimulating hormone (TSH); hypothalamic–pituitary–adrenal axis hormones such as cortisol or corticosterone, adrenocorticotropic hormone [ACTH], and aldosterone; hormones regulating energy metabolism such as insulin, glucagon, and leptin; and less frequently measured hormones such as prolactin, natriuretic peptides, oxytocin, vasopressin, and growth hormone. General information on the implementation and interpretation of some of these hormones is presented below and in Clinical Pathology in Nonclinical Toxicity Testing, Vol 1, Chap 10, and specific guidance for the interpretation of hormone data is beyond the scope of this chapter but can be found in several excellent reviews (Andersson et al., 2013; Chapin and Creasy, 2012; Cooke et al., 2004; Keane et al., 2015; Rehm et al., 2008; Stanislaus et al., 2012). Briefly, important considerations for protocol design for evaluation of endocrine changes include minimizing variables such as stress related to husbandry practices and method of euthanasia, ensuring consistency in the time of sample collection, diet and feeding status throughout the study, selection of the most relevant endpoints based on species differences in hormone biology, and adequately powering group sizes to detect biologically meaningful changes in hormone concentrations. Interpretation of hormone changes in nonclinical studies requires a weight of evidence approach that must include evaluation of effects on the associated target organs and endocrine axis, in-life data such as changes in body weight or food consumption, sexual maturity, and stage of the reproductive cycle. For example, the age and duration of food restriction initiated prior to sexual maturity in Sprague–Dawley rats can cause lower serum

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testosterone levels with stage-dependent testicular changes (pachytene spermatocyte degeneration in stage VII tubules and spermatid retention in stage XII tubules) in association with decreases in body weight, testis, and seminal vesicle weights (Rehm et al., 2008). However, comparable food restriction typically does not result in similar male reproductive effects in studies of longer duration initiated in rats at later stages of sexual maturation. Similarly, in peripubertal male and female Wistar rats, food restriction leading to >9% body weight loss in males was associated with decreases in thyroid hormones without effects on thyroid histology or reproductive endpoints, whereas no changes in thyroid or reproductive endpoints occurred in the food-restricted females with >19% body weight loss, illustrating the importance of dose selection to minimize effects on food consumption and body weight, and the potential for sex-related differences in sensitivity to endocrine perturbation. Circadian effects on hormone concentrations and pulsatility of hormone secretion are well recognized and vary by hormone system and species. For example, circadian or diurnal fluctuations of glucocorticoids and ACTH parallel activity patterns, where cortisone or corticosterone peaks before the active phase (early morning in humans and early evening in rodents). Prolactin (PRL) secretion is highly pulsatile; however, circadian patterns differ between speciesdfor example, PRL secretion in men and NHPs peaks during the night, while dogs do not have circadian periodicity for PRL, and male rats do not consistently have a circadian pattern of PRL release. These species differences highlight the importance of consistency of sampling time and consideration of species differences in experimental design and execution. Statistically significant changes may not be biologically meaningful due to the large interand intraindividual animal variability inherent in circulating hormone concentrations. For example, circulating testosterone levels are highly variable in rats and other species, and minor statistically significant decreases in serum testosterone may not be biologically meaningful in a study of short duration in the absence of an effect on androgen-dependent accessory sex gland weights, testicular histopathology, or related trophic (LH) hormones. Study design must also address the biologic variability of the specific hormone system and species being evaluateddfor example, using power calculations

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based on the coefficient of variation of a specific hormone in a particular species, the group size in rats needed to detect a 50% change in testosterone concentration with 80% power is estimated to be an n of 30 rats/group. Similarly, in dogs and monkeys, an n of 20 animals/group would be needed if a single sample were to be collected; however, group size in nonrodent species can be reduced by serial sampling and pooling of the samples collected from individual animals (Chapin and Creasy, 2012). In females, the stage of the reproductive cycle can have profound effects on hormone levels, with as much as 10-fold variability in certain hormones in rats and dogs depending on the stage of the estrus cycle (Stanislaus et al., 2012). Staging of the estrus cycle using vaginal cytology is commonly used in rats, dogs, and monkeys to evaluate the synchrony of the estrus cycle within groups and, in some cases, to determine the optimal time of sample collection based on the stage of the cycle and objective of the study. Ultimately, integration of female reproductive tract and mammary gland histopathology findings with endocrine data is essential for interpretation of endocrine effects on female reproductive system. In summary, evaluation of hormone data in nonclinical studies is best performed in dedicated investigative studies that are designed to address specific questions. Interpretation of hormone data in nonclinical studies requires a weight of evidence approach that should involve interaction between the clinical and anatomic pathologist, study director, and, potentially, subject matter experts in endocrinology or reproductive toxicology.

9. POTENTIAL EFFECTS UNRELATED TO TEST ARTICLE TREATMENT 9.1. Artifacts Artifactual results may arise from a variety of causes. Hemolysis, lipemia, and icterus are common causes of analytical interference that have potential to change clinical pathology data results. Hemolysis, also known as erythrolysis, refers to the destruction of erythrocytes (red blood cells) within blood. Hemolysis may occur in vivo (pathologic) or in vitro (artifact). Common causes for in vitro hemolysis include poor phlebotomy technique; traumatic handling, transport, and storage of the blood specimen;

and excessive temperature changes such as freezing of the specimen (Latimer et al., 2003; Stockham and Scott, 2008 [Erythrocytes]). Hemolysis may affect several parameters measured in complete blood counts and numerous analytes in clinical chemistry panels. Lipemia is defined as the translucent to opaque, milklike appearance of plasma or serum that occurs due to increased concentrations of triglycerides (chylomicrons and VLDL) in blood. Lipemia may be physiologic or pathologic in origin. Postprandial lipemia is a common cause of altered clinical pathology test results. Lipemia may interfere with the spectrophotometric measurement of several chemistry parameters, flame photometric electrolyte determinations, proteins measured by refractometry, and in hemoglobin determinations (Latimer et al., 2003; Evans and Duncan, 2003). Icterus is the term for the yellow to orange color of plasma or serum due to the increased amounts of bilirubin pigments. Increased bilirubin concentrations may occur in blood of rodents, dogs, and NHPs due to hemolytic events (indirect or unconjugated bilirubin) or cholestatic disease (direct or conjugated bilirubin). The increased matrix pigment may cause artifactual changes in both the complete blood count results and several analytes in the clinical chemistry panel. Blood specimens should be evaluated for quality upon receipt in the clinical pathology laboratory and any hemolysis, lipemia, and/or icterus in the serum or plasma should be recorded and reported along with results to the clinical pathologist for accurate data interpretation. The amount of change in any specific clinical pathology parameter is dependent upon the magnitude of the hemolysis, lipemia, and/or icterus and the scientific methods used for analyte determination (Stockham and Scott, 2008 [Erythrocytes]; Glick et al., 1986; Cornell University College of Veterinary Medicine EClinPath: Interference indices, 2020). Numerous other, less frequently encountered causes of artifacts and their sequalae in clinical pathology test results are listed in Table 14.5.

9.2. Analytical Methods There are multiple analytical methods available for clinical pathology tests, and each method may produce different results. Some considerations include the reagent vendor, instrument, wavelength for absorbance readings, time point of taking the absorbance reading, reaction temperature or

TABLE 14.5

Artifactual Changes in Hematology And/or Clinical Chemistry Parameters by Cause

Hematology

Clinical Chemistry

Prevention and/or Remediation

Increases in AST, ALT, CK, and/or LDH activity; increased total protein, phosphorus; and in some species, potassium concentrations

Review phlebotomy technique and syringe and needle sizes; avoid excessive shaking or trauma to blood sample during sample mixing and transport to laboratory; avoid freezing whole blood samples

Increased glucose, phosphorus, calcium, and total bilirubin concentrations; decreased concentrations of total protein and albumin [spectrophotometric determinations]; increased potassium and sodium concentrations (flame photometry), increased plasma protein concentration (refractometry)

If possible, fast animals prior to collection of blood for clinical pathology determinations; lipemic serum/plasma samples may be refrigerated to allow separation of matrix and lipids or ultracentrifugation may clear turbidity

IN VITRO HEMOLYSIS

Hematology: Pink hue to plasma, decreased erythrocyte count and hematocrit, increased MCHC and/or MCH

LIPEMIA

Hematology: Increased hemoglobin concentration, MCH, and MCHC

ICTERUS

No change to minimal increase Decreased creatinine and total protein in hemoglobin concentration, concentrations; increased ALT, AST, MCH, and MCHC and ALP activity

None

IMPROPER PHLEBOTOMY SITE AND/OR TECHNIQUE

Platelet clumping on blood smear with false decrease in platelet count Clotting of blood sample

Avoid blood collection in rodents from sites of low blood flow or tissue trauma such as tail veins or periorbital venous sinus, respectively Prolonged slow blood flow; excessive tissue trauma

Practice phlebotomy technique, gently invert blood tube immediately upon collection to cause adequate mixing of anticoagulant and blood cells

BLOOD DRAWN VIA CARDIAC PUNCTURE

Increases in cardiac troponin concentration and CK activity

Collect blood intended for measurement of cardiac biomarkers from site other than heart

DELAY IN SEPARATING ERYTHROCYTES FROM SERUM/PLASMA

Increased mean cell volume, mean platelet volume, and/or hematocrit; decreased MCHC

Decreased glucose concentration and increased AST activity

Separate serum/plasma from cells within 1 h of blood specimen collection

Prolonged period between EDTA blood collection and measurement of complete blood count

Perform complete blood counts within 4 h of receipt of blood by clinical pathology laboratory to prevent erythrocyte swelling interference with measurement of MCV

EVAPORATION OF STORED SERUM/PLASMA SAMPLES

Increased concentrations of sodium, chloride, potassium, total protein, albumin, and globulin

Analyze sample immediately upon receipt in the clinical pathology laboratory or tightly cap any stored samples of serum/plasma until analysis (Continued)

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TABLE 14.5 Artifactual Changes in Hematology And/or Clinical Chemistry Parameters by Causedcont’d Hematology

Clinical Chemistry

Prevention and/or Remediation

DILUTIONAL EFFECT FROM UNDERFILLING CITRATE ANTICOAGULANT TUBES

Prolonged PT and APTT

Maintain 9:1 ratio of blood: Citrate anticoagulant during phlebotomy

ASSAY INTERFERENCE DUE TO CEPHALOSPORIN TREATMENT

Increased creatinine concentration

Select enzymatic creatinine determination versus Jaffe reaction

PROLONGED EXPOSURE OF BLOOD TO EDTA ANTICOAGULANT

Crenated, shrunken erythrocytes; vacuolated neutrophils and macrophages; increased MPV

Collect full draw of blood in vacuum tubes to maintain appropriate EDTA: Blood ratio; analyze whole anticoagulated blood within 4 h of phlebotomy.

Neutrophil morphologic changes consisting of discrete, clear cytoplasmic vacuoles, irregular distribution of cytoplasmic granules, uneven cell membranes and pyknosis that occur in the absence of cytoplasmic basophilia

Make blood smears from fresh blood at time of phlebotomy or deliver EDTA anticoagulated blood to laboratory within 1 h for prompt smear preparation

ALP, alkaline phosphatase; ALT, alanine aminotransferase; APTT, activated partial thromboplastin time; AST, aspartate aminotransferase; CK, creatine kinase; EDTA, ethylenediaminetetraacetic acid; LDH, lactate dehydrogenase; MCH, mean corpuscular hemoglobin; MCHC, mean corpuscular hemoglobin concentration; PT, prothrombin time.

pH, substrate, buffer, or methodology (colorimetric vs. electrophoresis vs. high-pressure liquid chromatography vs. radioimmunoassay vs. ionspecific electrode (Stockham and Scott, 2008). For example, enzymatic creatinine methods are likely to have lower values than a colorimetric Jaffe assay because interfering chromogens will not be measured in the enzymatic assay (Palm and Lundblad, 2005). However, these differences may be minor, depending on the species. Some other common methodology options in a clinical pathology laboratory include choosing the dye binding method for albumin (Cornell University College of Veterinary Medicine EClinPath: Albumin, 2020), inclusion or not of pyridoxal phosphate (P5P) for ALT and AST activity measurements (Mesher et al., 1998), or the use of a glycerol-blanked method or not for triglyceride measurement (needed in pigs) (Weingand, 1988). Inclusion of immunologic (antibody-mediated) chemistry assays into the clinical pathology laboratory has presented unique challenges associated with a lack of universal reagent cross-species

reactivity. For example, several assays for cTn that work well for dogs and NHPs utilize reagent antibodies that fail to cross-react or react very poorly with rats (Apple et al., 2008). Differences in analytical methods are not often factors in data interpretation if an appropriate method has been chosen but may become confusing when comparing studies that are conducted at more than one facility or using different methodologies. Comparison of current study results to those of historical studies at the same institution is also subject to the challenges of comparison of technology and method updates that may confound comparison of clinical pathology data results. Published reference intervals do not often specify details about the analytical methods used or even the reference group, and the use of published reference intervals for decision-making is not advised, especially if there are instrumentation, reagent, or reference group differences (Mahaffey, 2003; Stockham and Scott, 2008).

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9.3. Age/Sex/Genetics There are several age-related changes in clinical pathology data that occur in all species of mammals. Differences associated with immaturity in young animals of most species include higher absolute reticulocyte count, MCV, polychromasia, ALP activity, and inorganic phosphorus concentration compared to values in mature animals. The higher serum ALP values are due to greater activity of the bone isoenzyme in young growing animals that have not reached their mature body size. Higher phosphorus concentrations in young animals also reflect the influence of rapid bone growth. Less consistent differences include higher concentrations of cholesterol, triglycerides, thyroxine, glucose, and/or bilirubin; and higher LDH and GGT activities compared to adult animals (Wolford et al., 1988). Young animals may also have lower erythrocyte count, hematocrit, hemoglobin concentration, total protein, and globulin concentrations, and, less consistently, lower ALT, AST, and/or sCr values compared with those of adult animals. The difference in sCr values is most likely due to the differences in body mass between young and mature animals (Lowseth et al., 1990). The example illustrated in Table 14.6 shows typical age-related changes in male Beagle dogs during a 1-year study. Results were similar in female dogs (data not shown). Age-related decreases occurred during the 1year study in ALP activity, phosphorus, and cholesterol concentrations, but urea nitrogen and creatinine concentrations increased slightly TABLE 14.6

between 16 and 22 weeks of age. Age-related differences in serum cholesterol and triglyceride concentrations are not consistent between Sprague Dawley, Wistar, and Fisher 344 rats, underscoring that strain as well as age may impact clinical pathology data (Story et al., 1976). Higher serum triglyceride and glucose concentrations and ALT and AST activities were seen in C57BL/6J mice at 20 weeks of age compared to 6 weeks of age (Zhou and Hansson, 2004). Aged mice have been shown to have suboptimal immune responses, including defects in B-cell differentiation and mature B-cell function (Landin et al., 2009). Sex-associated differences in clinical pathology values are expected, although they vary from species to species. ALP activity, hematocrit, and total leukocyte and absolute lymphocyte counts tend to be higher in male rats. Higher plasma and blood volumes were found in female Sprague Dawley rats, which corresponded to lower hematocrit in females (Probst et al., 2006). In the example provided in Table 14.7, age- and sex-related differences are seen in these Sprague Dawley rats (Wolford et al., 1987). Between ages 7 and 40 weeks, ALP activity, phosphorus concentration, absolute reticulocyte count, and MCV decrease. Total protein, globulin, urea nitrogen, and cholesterol concentrations and erythrocyte count increase. Males have higher ALP activity, phosphorus concentration, erythrocyte count, and reticulocyte count. They have lower total protein and cholesterol concentrations compared to females at all intervals (with the exception that

Age-Related Changes in Serum Clinical Chemistry in Control Male Beagles During a 1-Year Study (Mean (SD))a

Study Interval

Week 1

Week 6

Week 13

Week 26

Week 39

Week 51

Animal age

16 weeks

22 weeks

29 weeks

42 weeks

55 weeks

67 weeks

ALP (U/L)

133 (29.6)

98 (24.6)

76 (19.6)

44 (9.9)

37 (7.3)

36 (5.7)

Phos (mg/dL)

7.4 (0.56)

6.7 (0.59)

5.8 (0.50)

4.9 (0.13)

4.6 (0.35)

4.4 (0.43)

BUN (mg/dL)

11.0 (1.12)

15.2 (1.41)

13.7 (1.02)

15.6 (1.27)

14.2 (1.25)

14.0 (1.09)

Cr (mg/dL)

0.4 (0.0)

0.6 (0.05)

0.6 (0.10)

0.7 (0.06)

0.7 (0.12)

0.8 (0.06)

Chol (mg/dL)

168 (33.4)

152 (28.1)

141 (15.9)

136 (13.2)

130 (18.8)

134 (16.4)

a

Four per group. Table reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, 3 ed, Academic Press, 2013, Table 29.8, p. 875, with permission.

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TABLE 14.7

Age- and Sex-Related Changes in Sprague Dawley Rats (Mean (Range))

ALP (U/L)

Phos (mg/dL)

T Pro (g/dL)

Glob (g/dL)

BUN (mg/dL)

Chol (mg/dL)

RBC (106/mL)

MCV (fL)

Retic (103/mL)

Wk 7 M

286 (173e405)

10.4 (8.7e13.3)

6.0 (5.4e6.4)

1.8 (1.3e2.2)

13.1 (8.1e17.9)

57 (34e88)

7.8 (6.42e8.06)

61.2 (56.2e69.9)

352.3 (215.3e543.7)

Wk 7 F

193 (113e298)

9.5 (7.6e11.9)

6.2 (5.6e6.9)

1.7 (1.2e2.1)

14.5 (10.4e20.8)

70 (38e101)

7.28 (6.07e8.28)

58.7 (52.4e671).

224.2 (118.3e450.0)

Wk 10 M

198 (112e310)

8.8 (7.1e12.7)

6.4 (5.8e7.0)

2.2 (1.7e2.7)

13.7 (8.8e19.3)

55 (34e84)

8.14 (7.20e9.22)

56.1 (51.0e63.6)

195.7 (100.3e346.8)

Wk 10 F

121 (65e195)

7.9 (6.0e10.6)

6.8 (6.1e7.6)

2.2 (1.6e2.7)

15.6 (10.5e21.8)

67 (42e103)

7.98 (7.04e8.83)

54.8 (50.3e61.5)

155.5 (77.3e287.3)

Wk 14 M

144 (88e233)

8.1 (6.5e9.8)

6.5 (5.8e7.2)

2.3 (1.7e2.8)

14.9 (10.2e20.2)

58 (35e89)

8.51 (7.56e9.36)

52.9 (48.9e59.0)

189.2 (111.9e435.9)

Wk 14 F

87 (46e145)

7.2 (5.1e9.6)

7.1 (6.3e8.0)

2.3 (1.7e2.9)

16.9 (11.4e24.9)

72 (44e107)

8.04 (6.91e8.95)

53.4 (49.4e60.2)

174.7 (91.4e531.2)

Wk 17 M

115 (72e165)

7.5 (6.2e9.3)

6.8 (6.2e7.5)

2.5 (1.9e3.2)

14.4 (11.1e20.0)

58 (38e106)

8.75 (7.65e9.79)

51.3 (48.0e56.0)

187.1 (104.5e360.2)

Wk 17 F

68 (37e112)

6.8 (5.2e8.8)

7.2 (6.5e8.3)

2.5 (2.0e3.0)

15.5 (10.8e22.9)

76 (45e111)

8.17 (7.04e9.12)

52.7 (49.1e59.8)

163.7 (87.1e314.7)

Wk 21 M

91 (59e143)

7.0 (5.4e9.3)

6.9 (5.6e9.3)

2.6 (2.0e3.2)

14.3 (10.7e18.5)

63 (38e101)

9.01 (8.12e9.9)

50.5 (46.7e56.4)

146.4 (83.2e224.6)

Wk 21 F

55 (30e93)

6.3 (4.5e9.2)

7.4 (6.5e8.5)

2.4 (1.9e3.0)

15.8 (11.8e21.0)

76 (43e124)

8.36 (7.44e9.19)

52.3 (48.1e58.4)

127.1 (66.4e189.4)

Wk 40a M

88 (52e154)

6.7 (5.5e8.3)

6.7 (6.3e7.6)

2.7 (2.1e3.2)

14.3 (11.6e18.5)

67 (36e115)

9.05 (8.33e9.87)

50.8 (46.2e57.0)

135.9 (73.1e219.7)

Wk 40a F

50 (27e90)

6.1 (4.5e8.1)

7.5 (6.7e8.5)

2.5 (2.0e3.5)

16.3 (12.7e21.7)

84 (50e138)

8.28 (7.25e9.02)

52.8 (49.2e58.2)

121.4 (56.2e187.2)

Wk 40 ¼ Weeks 22–40. F, females; M, males; Wk, Week. Table reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, 3 ed. Academic Press, 2013, Table 29.9, p. 876, with permission.

a

14. INTERPRETATION OF CLINICAL PATHOLOGY RESULTS

III. DATA INTERPRETATION AND COMMUNICATION

Age Sex

9. POTENTIAL EFFECTS UNRELATED TO TEST ARTICLE TREATMENT

erythrocyte counts were similar for males and females at Week 7). In a study comparing 10 different strains of mice, absolute neutrophil counts; urea nitrogen, sodium, cholesterol, and globulin concentrations; and ALT activity were higher in male mice in most strains. Total leukocyte and absolute lymphocyte counts, hematocrit, ALP activity, albumin concentration, and albumin/ globulin (A/G) ratios were higher in female mice of most tested strains (Serfilippi et al., 2003). However, there is much disparity in the literature on sex differences in mice, and the readers are advised to become familiar with the sex- and age-related differences in the strain and source of the mice used in their facility and not to rely on published reference intervals or data from other laboratories. Sex-associated differences in pathophysiology and disease treatment are an evolving area of human medicine. For example, sex-associated differences in metabolic syndrome and the associated risks for diabetes and cardiovascular disease are recognized in humans. Assessing sex differences in laboratory animals may provide guidance in selecting the best model for a human disease. For example, because of their more atherogenic profile due to higher concentrations of C-peptide, insulin, triglyceride, total cholesterol, HDL-cholesterol, and leptin and insulin resistance, female Go¨ttingen minipigs may be better models for metabolic syndrome than males (Christoffersen et al., 2007). Physiological differences between groups of animals may also be attributed to the breeding facility. Differences in kidney function and the onset of chronic progressive nephrosis characterized by differences in protein excretion have been reported in young Sprague Dawley rats of the same strain, but from different breeders (Palm, 1998). The use of genetically altered mouse models is an important tool in medical research and allows for targeted genetic manipulations to create mice that are homologous to genetically based human diseases. In order to interpret alterations in clinical pathology data, which may be secondary to administration of test material, it is critical to understand the clinical pathology results in the background strain of mice, as well as in the genetically altered model prior to testing.

547

Pharmacologic and toxicologic responses can be influenced by genetic variability. Breeding of rodents has resulted in genetically homogeneous models and strains that have been well characterized with regard to clinical pathology and other parameters. Cynomolgus macaques (Macaca fascicularis) and rhesus macaques (Macaca mulatta) are the most commonly studied nonhuman primates in research and have been used for many years without careful evaluation of their genetic diversity. Cynomolgus monkeys used in research may be derived from several geographical sources in Asia. China does not have an indigenous population of cynomolgus monkeys, and their breeding populations originate from other geographical areas. Genetic studies have shown M. mulatta alleles in the M. fascicularis populations from Indonesia, suggesting hybridization between rhesus and cynomolgus monkeys (Stevison and Kohn, 2009). In general, populations of monkeys from a mainland geographical area have more diversity than those that originated on an island, due to the likelihood of movement and interbreeding of diverse populations in mainland areas. Genetic studies have demonstrated that Chinese and Vietnamese cynomolgus populations (mainland) are more genetically heterozygous than those from Mauritius (island). Genetic variation and admixture were also greater in the Chinese-derived rhesus macaques when compared to the Indian rhesus macaques. Differences in clinical pathology parameters between geographically sourced cynomolgus monkeys have been recognized and include larger erythrocytes (higher MCV) from Chinese and Vietnamese cynomolgus monkeys compared to those from Indonesia, Mauritius, or the Philippines. This new information on genetic diversity suggests that NHPs used for nonclinical toxicity studies likely have variable genetic backgrounds, which may include both cynomolgus and rhesus alleles.

9.4. Anesthesia Anesthesia, or chemical restraint of an animal, may affect clinical pathology data (Deckardt et al., 2007; Gonzalez et al., 2010). Effects due to ketamine anesthesia, used alone or in combination, are reported often. When administered by intramuscular injection, ketamine causes

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muscle irritation, necrosis, and fibrosis with release of muscle enzymes including CK, AST, and LDH. These effects have been reported in rats, rabbits, goats, and NHPs. In addition, metabolism of ketamine affects the liver and can lead to hepatic inflammation and increased hepatic enzyme activity in serum independent of the route of ketamine administration. In rhesus monkeys, decreased erythrocyte counts; hemoglobin concentration; hematocrit; total leukocyte and absolute lymphocyte counts; and concentrations of glucose, total protein, albumin, and electrolytes occurred 15 min after ketamine injection (Bennett et al., 1992). Repeat intramuscular dosing of ketamine for 14 days in cynomolgus monkeys resulted in increased absolute neutrophil counts; activities of ALT, AST, CK, GGT, and LDH; and decreased concentrations of albumin, chloride, cholesterol, glucose, potassium, sodium, and triglyceride. Various anesthetic articles are used in rabbits and may result in numerous clinical pathology effects depending on the type, dosage, and route of administration of anesthetic article and time evaluated. In general, intravenous ketamine with xylazine or diazepam has been associated with higher ALT and AST activities, and concentrations of creatinine, urea nitrogen and triglyceride, and transient changes in electrolytes (Gonzalez et al., 2002, 2003). Thiopentone has been associated with higher serum ALT, AST, and GGT activities in rabbits (Gonzalez et al., 2004, 2005). Serum AST and/or ALT activity increased after intraperitoneal injection of tribromethanol, pentobarbital, or ketamine/xylazine in mice (Thompson et al., 2002). Serum glucose, urea nitrogen, and phosphorus concentrations and CK activity differed between five anesthetic regimes in guinea pigs (including ketamine–xylazine administered subcutaneously, intramuscularly, or intraperitoneally, pentobarbital administered intraperitoneally, and medetomidine administered intramuscularly). Hematology results were comparable among the different regimes. These data illustrate that anesthetic effects on clinical pathology values are variable depending on the type, route, and dose of anesthesia (Dang et al., 2008). Studies have reported that anesthetic agents suppress the number and function of various cells of the immune system. Inhalation or injectable anesthesia may cause increased glucose and

glucagon concentrations due to impairment of insulin secretion and glucose utilization (Penicaud et al., 1987; Brown et al., 2005; Tanaka et al., 2009). The magnitude and duration of effect will vary depending on the anesthetic article, and species and strain of animal. While not an anesthetic, use of carbon dioxide inhalation to euthanize mice, rats, and guinea pigs can cause marked elevations in serum potassium concentration (Walter, 1999). Khokhlova and colleagues (2017) compared carbon dioxide and tiletamine– zolazepam–xylazine anesthesia for collection of terminal blood samples intended for analysis of hematology, clinical chemistry, and coagulation parameters. Mice given tiletamine–zolazepam– xylazine anesthesia by intramuscular injection had shorter APTT and lower fibrinogen concentration, platelet count, erythrocyte count, hemoglobin concentration, and total leukocyte count compared to mice euthanized by carbon dioxide inhalation. Lower concentrations of calcium, cholesterol, triglycerides, total protein, albumin, and globulin and activities of ALT and ALP occurred in mice given the tiletamine–zolazepam–xylazine anesthesia compared to those euthanized with carbon dioxide.

9.5. Blood Collection The anatomic site and animal order of blood sample collection can substantially influence clinical pathology and biomarker results (Dameron et al., 1992; Nemzak et al., 2001; Fernandez et al., 2010; Hamlin et al., 2017). Blood samples for clinical pathology testing during nonclinical toxicity studies should always be collected and analyzed in a random or rotational group order to avoid creation of a block effect (Schultze et al., 2020). Collecting all samples from one dose group or “block”, then all samples from another dose group, etc., introduces bias because of the time differences in sample collection, processing, and analysis which will influence test results. Results may also differ depending on the method of blood collection (vacutainer vs. syringe vs. capillary tube) (Neptun et al., 1985; Smith et al., 1986). Numerous references illustrate differences in clinical pathology results based on the site and method of blood collection, and a detailed summary is beyond the scope of this publication. There are several references that report clinical pathology ranges for various species, without

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9. POTENTIAL EFFECTS UNRELATED TO TEST ARTICLE TREATMENT

citing the site or method of blood collection. Readers are cautioned to use discretion in comparing results from a set of animals in a study to a published reference range/interval without knowing the site and method of blood collection, as well as the numerous other variables discussed in this section (Walter, 1999; Schnell et al., 2002). Serial phlebotomy is a common practice in studies utilizing large laboratory animals, such as dogs and NHPs, in which the same individuals are bled for toxicity endpoints (such as clinical pathology) as well as toxicokinetic/ pharmacokinetic endpoints (Ooms et al., 2004). Recently, this practice has been extended to rats in some situations (Scipioni et al., 1997). The volume of blood collected may affect clinical pathology values, and the time to recovery from the effects of the bleed is proportional to the volume removed (Nahas and Provost, 2002). Because of differences in clinical pathology parameters secondary to blood collection, it is important to collect the same amount of blood from control and test article–treated animals. The effects of intravenous administration of fluid and blood collection are illustrated in Table 14.8. Male cynomolgus monkeys were administered 10% dextrose intravenously for 14 days (96 mL/kg/day on Day 1 followed by 56 mL/kg/day on subsequent days). On Days 1 and 16, 8 mL of blood was collected for toxicokinetic evaluation. Decreases in clinical pathology parameters on Day 16 were primarily due to intravenous fluid administration, with blood loss due to blood collection also contributory to the decreased red cell parameters; total protein, albumin, and, indirectly, calcium concentrations. Clinical pathology results may also be affected by handling of animals during study procedures, including blood collection. Table 14.9 shows TABLE 14.8

nondose-related increased mean AST and ALT activities (with large standard deviations) in a 2-week oral gavage study in B6C3F1 mice. There were no apparent test article–related liver microscopic alterations. The mice were particularly difficult to dose, and the increases in aminotransferase activity were attributed to a procedural effect (firm grasping of the abdominal musculature over the liver region while dosing the resistant mice).

9.6. Diet/Fasting Fasting of animals, increased or decreased feed consumption, and the specific composition of the feed can influence experimental outcomes and impact clinical pathology data. Animals may be fasted to reduce lipemia in serum or plasma samples, reduce the variability in results of analytes which are sensitive to the duration of fasting (glucose and triglycerides), reduce the contents of the intestinal tract prior to necropsy, and provide more uniform liver histology via glycogen depletion. There are numerous references that provide information on fasting of various species, although results vary among studies, most likely due to the length of fasting, and the age, sex, and strain of the animals (Keenan et al., 1995, 2005; Zeng et al., 2010). When interpreting clinical pathology data in toxicity studies, it is important to consider that some groups of animals may have decreased body weight and food consumption because of test article administration in addition to an overnight fast. Fasting-induced changes in clinical pathology results in rats were consistent with hemoconcentration due to decreased water consumption and altered nutrition and metabolic function, with most changes occurring at 16 h, and minimal subsequent change between 16 and 48 h of fasting

Effects of IV Infusion and TK Blood Collection on Control Male Cynomolgus Monkeys (Mean Values)a

RBC (106/mL)

HGB (g/dL)

HCT (%)

BUN (mg/dL)

Chol (mg/dL)

T Pro (g/dL)

Alb (g/dL)

Glob (g/dL)

CaD2 (mg/dL)

Trig (mg/dL)

Phos (mg/dL)

Day 7

5.44

13.1

43.6

20

155

8.2

4.9

3.3

11.4

36

7.5

Day 16

4.80

11.7

37.8

17

114

7.2

4.3

2.8

10.2

24

5.4

a

Three per group. Table reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, 3 ed. Academic Press, 2013, Table 29.10, p. 878, with permission.

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TABLE 14.9 Clinical Chemistry Results in 2 Week Mouse Study (Mean (SD)) 0 mg/kg/day AST (U/L) ALT (U/L)

3 mg/kg/day

10 mg/kg/day

30 mg/kg/day

100 mg/kg/day

Males

68 (14.6)

61 (2.9)

123 (78.1)

198 (175.2)

139 (79.6)

Females

80 (27.4)

126 (97.9)

131 (34.0)

149 (34.8)

202a (53.4)

Males

26 (4.9)

24 (3.9)

31 (10.4)

91 (83.0)

40 (18.3)

Females

26 (1.8)

35 (26.0)

33 (9.2)

31 (7.5)

42 (7.8)

P  05. Table reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, 3 ed. Academic Press, 2013, Table 29.11, p. 879, with permission. a

(Apostolou et al., 1976; Waner and Nyska 1993; Matsuzawa and Sakazume 1994; Kale et al., 2009). Overnight fasting of rats may result in increased erythrocyte count; hemoglobin concentration; Hct; PT; APTT; sCr and bilirubin concentrations; and AST, SDH, and CK activities. Decreases may occur in total leukocyte counts; the concentrations of serum glucose, urea nitrogen, calcium, cholesterol, triglycerides, and carbon dioxide; and ALT and ALP activities. Overnight fasting of mice has been associated with decreased body weight; glucose and triglycerides concentrations; and increased concentrations of urea nitrogen, albumin, total protein, and phosphorus; and ALP, AST, and CK activities. The magnitude of body weight loss and decreased glucose values were considered adverse to the health of the mice fasted overnight (Walter et al., 2013). Mice tend to dehydrate rapidly, and overnight fasting of mice is discouraged (Jensen et al., 2019). Studies of overnight fasting in NHPs are few and results varied moderately (Zeng et al., 2010). The effects of overnight fasting, feeding, or sucrose supplementation prior to necropsy have been investigated in rats. Decreased serum ALP activity has been recognized in rats following fasting or feed restriction and is most likely associated with a decrease in ALP isozyme of intestinal origin, which is the primary form of ALP in blood in rats. ALP activity decreases secondary to decreased food consumption are illustrated in Table 14.10 Providing a simple carbohydrate to rats overnight has been suggested to minimize the effects of withholding food. Blood glucose and urea nitrogen concentrations, ALP and ALT activities, liver weights, and liver and pancreatic histology differed in rats supplemented with sucrose compared to fasted or chow-fed rats, and these changes were considered potentially to impact study

outcomes. Therefore, sucrose supplementation was not recommended (Turner et al., 2001). Results of a 2-week food restriction study in rats showed decreased total leukocyte, lymphocyte, and platelet counts; decreased triglycerides, cholesterol, and total protein concentrations; and ALT and ALP activities. There were increased serum bilirubin concentrations, and electrolyte derangements (Levin et al., 1993). Another 2week feed restriction study in Sprague Dawley rats noted hemoconcentration, decreased glucose concentration, increased ALP, ALT, and AST activities, and hemoglobinuria in the severely restricted group (Moriyama et al., 2008). The effects of feed restriction on serum AST, ALT, and ALP activities in rats are variable according to various published references (Hubert et al., 2000). Ad libitum feeding of laboratory rodents results in obesity and early onset of renal, cardiac, and degenerative diseases, metabolic and endocrine disruption, and diet-related tumors. Moderate dietary restriction results in improved survival and decreased disease incidence and decreased study-to-study variability. However, the ad libitum-overfed Sprague Dawley rat (specifically the Charles River CD rat) may serve as a good model of polygenic adult-onset human and animal diabesity and has higher blood cholesterol and triglyceride concentrations compared to rats with moderate or marked dietary restriction (Keenan et al., 2005).

9.7. Medications Laboratory animal veterinarians are entrusted with maintaining the health of large colonies of mice, rats, dogs, and NHPs that may be used in toxicity studies. It is beyond the scope of this chapter to cover every medication that might be

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TABLE 14.10

Clinical Chemistry Results in Male Rats Administered Gentamicin (Mean (SD))

BUN (mg/dL)

Cr (mg/dL)

Phos (mg/dL)

ALP (U/L)

GGT (U/L)

AST (U/L)

ALB (g/dL)

NaD (meq/dL)

CIL (meq/dL)

Vol (mL)

FE Na

Day 2

14.4 (2.47)

0.3 (0.14)

8.9 (0.43)

257 (70.1)

1.0 (0.0)

95 (11.9)

3.6 (0.15)

148 (1.6)

105 (1.2)

16.5 (12.4)

0.12 (0.008)

Day 8

127.6 (67.16)

3.7 (2.11)

11.8 (3.8)

169 (52.1)

1.7 (0.85)

222 (64.0)

2.9 (0.13)

139 (4.2)

91 (6.3)

7.4 (6.6)

3.72 (0.390)

Day 13

111.6 (41.02)

2.8 (2.48)

14.0 (2.81)

117 (44.2)

1.5 (1.07)

116 (40.8)

3.4 (0.26)

126 (24.7)

76 (15.8)

15.9 (6.6)

262.82 (14.12)

Day 18

38.5 (14.64)

0.6 (0.19)

7.6 (0.95)

141 (58.5)

1.5 (0.55)

100 (11.1)

3.5 (0.23)

144 (1.7)

99 (1.5)

11.7 (3.0)

0.63 (0.023)

Table reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, 3 ed. Academic Press, 2013, Table 29.5, p. 865 with permission.

used to treat illness in laboratory animals and list expected effects on pathology parameters. This section focuses on two drugs used frequently to maintain rodent colony health. Trimethoprim-sulfamethoxazole (TMP-SMX) is administered to immunocompromised mice to prevent Pneumocystis murina pneumonia. Therapeutic doses of SMX may cause decreased total and free thyroxine (T4) levels in dogs and mice, increased TSH concentration in mice, and thyroid hypertrophy and hyperplasia in mice, rats, and dogs. The presence and magnitude of effects are dependent on the daily dose of TMP-SMX (Altholtz et al., 2006). Fenbendazole is an anthelmintic drug used to treat and prevent pinworm outbreaks in laboratory rodents. This treatment is typically incorporated into rodent feed at a target dose of 8– 12 mg/kg/day. Data in nonrodent species, including sheep, dogs, porcupines, tortoises, and columbiform birds, indicate possible effects of fenbendazole on the bone marrow and lymphocyte proliferation and function (Gozalo et al., 2006). Limited and disparate findings about fenbendazole effects have been reported in rodents (Villar et al., 2007; Cray et al., 2008). Differences in the results of studies are most likely due to differences in study designs and endpoints evaluated.

9.8. Fear/Pain/Stress The acute stress response includes an initial rapid increase in mediators of neural origin with a short duration of action, followed by a slower increase in endocrine-derived

mediators with a longer duration of action. Fear, pain, and stress will affect certain clinical pathology analytes, most notably leukocyte differential count and glucose concentration. A transient response to fear, excitement, or muscular exertion is the “fight or flight” response mediated by catecholamines. Epinephrine promotes glycogenolysis in hepatocytes and myocytes, with a resulting increase in blood glucose concentration. The alteration in the leukogram, called a physiologic leukocytosis, occurs with seconds to minutes and may be characterized by an increase in all leukocyte types. However, a physiologic leukocytosis is more often characterized by increased absolute neutrophil count (without release of immature neutrophils) and, in some species, absolute lymphocyte count and is caused by a redistribution of circulating neutrophils and lymphocytes. Monocyte, eosinophil, and basophil counts are seldom changed. The duration of the physiologic leukocytosis is generally 20–30 min (Webb and Latimer, 2011; Everds et al., 2013). Excessive endogenous corticosteroids released in response to pain and other stressful events promote gluconeogenesis, with a resulting increase in blood glucose concentration. Factors such as housing, cage changes, exercise, lactation, weaning, surgery, anesthesia, feed deprivation, emotional strain, handling, noise, temperature, social interactions, and investigative procedures such as blood collection and test article or vehicle administration can lead to increased corticosteroid secretion and the manifestation of a “stress response” (Brown et al., 2000; Pekow, 2005; Everds et al., 2013). In

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addition to changes in corticosteroid release, alterations also occur in levels of catecholamines, thyroxine, prolactin, somatostatin, growth hormone, b-endorphin, adrenocorticotropic hormone, glucagon, insulin, vasopressin, and substance P. A stress leukogram is characterized by increased absolute neutrophil count (and absolute monocyte count in some species), and decreased absolute lymphocyte and absolute eosinophil counts due to increased release of neutrophils from the bone marrow, decreased migration of neutrophils into the tissues, and a shift from the vascular marginal pool to the circulating pool. The decreases in lymphocyte and eosinophil counts associated with a stress leukogram are due to redistribution of circulating lymphocytes and eosinophils (Webb and Latimer, 2011). If corticosteroids are chronically increased, lysis of thymic cortical lymphocytes and uncommitted lymphocytes in the lymph nodes and spleen occurs. Thymic medullary, bone marrow, and effector T and B lymphocytes are resistant to corticosteroid-induced lysis. Assessment of stress or distress in laboratory animals necessitates a weight of evidence approach, which might include behavioral and physiological endpoints, such as body temperature, alertness, arousal, food consumption, heart and respiratory rates, as well as anatomic and clinical pathology findings (Everds et al., 2013). A chronic stress response is more variable than an acute stress response. In chronic stress, catecholamine levels may return to normal while corticosteroid levels may increase, decrease, or return to baseline. Methods used to collect data to evaluate these stresses in animals can have a significant impact on results. Fecal corticoid metabolites have been evaluated to assess stress in rats and mice (Cavigelli et al., 2006). This method of collection avoids the need to handle or anesthetize animals to collect data, but it must be emphasized that measurement of fecal corticoid metabolites is an involved analysis and is not at all routine. Readers are referred to the comprehensive review article of Everds et al. (2013) for further description of the biology and proper assessment of stress responses in toxicity studies.

9.9. Environment Environmental factors can influence study outcomes, including clinical pathology data. Materials used for animal bedding such as pine- or cedar-based products are known to induce liver drug-metabolizing cytochrome P450 enzymes in rats and mice (Buddaraju and Van Dyke, 2003; Davey et al., 2003). Animal housing density and group size can affect physiological parameters in laboratory animals (Chvedoff et al.,1980; Ortiz et al., 1985; O’Malley et al., 2008). Environmental effects in mice vary depending on strain, as well as several other factors such as frequency of bedding changes (Rosenbaum et al., 2009). Differences in hematology parameters in mice may differ with cage size, housing density, strain, and time of day (Peng et al., 1989; Nicholson et al., 2009). Increased housing density has negative effects on body weight gain, corticosterone, behavior, and immune parameters in BALB/c mice, with less consistent effects in C57BL/6 mice (Laber et al., 2008). Group versus individual housing influences glucose and triglyceride concentrations in rats (Perez et al., 1997). Environmental factors can influence the results of animal health and, subsequently, clinical pathology results. Readers are cautioned to consider the large amount of variation in the literature on these topics, and that multiple factors, including strain of animal and study design, contribute to the outcome of environmental effects.

9.10. Sample Types, Handling, and Stability The type of sample may influence test results. Clinical chemistry tests from animals in nonclinical toxicity studies in the United States are usually performed on serum samples. However, in Europe plasma samples are more commonly used. Accordingly, differences are expected for results of protein testing since soluble coagulation factors (many of which are b-globulins) and fibrinogen are consumed in the clotting process and therefore are not present in serum but are present in plasma. Because potassium is released from platelets during the clotting process, serum potassium concentration is

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9. POTENTIAL EFFECTS UNRELATED TO TEST ARTICLE TREATMENT

higher than that of plasma. Results of other clinical chemistry tests will differ between serum and plasma samples, and sample type is an important parameter to consider when comparing results to a reference interval. The type of anticoagulant will affect hematology and coagulation results. Platelet aggregates are more frequent in blood anticoagulated with citrate compared to EDTA. This can result in lower platelet counts and inaccurate results for MPV and mean platelet component concentration (MPC) (Stokol and Erb, 2007). The use of serum or anticoagulants (heparin, EDTA, or citrate) produced significant differences in results of canine C-reactive protein (CRP), serum amyloid A (SAA), and ceruloplasmin. Hemolysis, lipemia, and hyperbilirubinemia may cause interference with some test methodologies, including those for canine CRP and ceruloplasmin, but not SAA (Martinez-Subiela and Ceron, 2005). Results can also be affected by sample storage conditions. Sample stability may differ between serum and plasma samples. Delayed analysis of hematology samples will result in artifactual changes, the earliest of which are increased hematocrit, MCV, and MPV (Medaille et al., 2006; Furtanello et al., 2006; Ameri et al., 2011). In general, refrigeration of hematology samples will improve stability compared to samples held at room temperature. Other than rat lipase, dog and rat serum analytes were stable up to 24 h at 4 C and up to 120 days at 80 C (Boysza et al., 2005). Advanced planning of toxicity studies including study design, sample collection, and proper storage of samples, if necessary, is the responsibility of the investigators. Scientists with questions regarding analyte stability and proper storage of samples prior to analysis or postanalysis should be directed to clinical pathology laboratory personnel who should be prepared to discuss these criteria for each test performed in the laboratory.

9.11. Pregnancy, Neonatal Period, and Estrous Cycle Clinical pathology evaluation is not a standard component of many reproductive toxicity studies. However, for those studies in which clinical pathology evaluations are conducted,

553

knowledge of differences between pregnant and nonpregnant animals is valuable. In Segment II teratology/embryo-fetal development studies, animals are generally dosed from Gestation Day (GD) 6 to GD 16/17 of pregnancy for rats, and GD 6 to GD 18/19 for rabbits. Animals are usually necropsied on GD 20–21 in rats, and GD 29 in rabbits. Clinical pathology testing is most commonly done just after the last dose of test article or at necropsy. Interpretation of hematology and clinical chemistry test results obtained in reproductive toxicity studies requires separate reference or control data for pregnant and nonmated animals. Anticipated findings in pregnant animals include decreased total protein, albumin, and urea nitrogen concentrations due to plasma volume expansion. Pregnancy is a dynamic condition and some clinical pathology parameters will vary during the pregnancy and will depend on the stage of gestation, as well as the species and strain of animal. This accounts for numerous disparities in the literature regarding clinical pathology values during pregnancy. In humans, decreased erythrocyte count, hemoglobin concentration, hematocrit, and albumin, total protein, bilirubin, calcium, creatinine, and urea nitrogen concentrations are commonly reported. Although albumin and total protein concentrations typically decrease, increases occur in a-1, a-2, and b-globulin, fibrinogen, C-reactive protein, ceruloplasmin, a2-macroglobulin, and most soluble coagulation factors. On GD 18/19, pregnant Wistar Hannover and Sprague Dawley rats had lower erythrocyte counts, hemoglobin concentration, and hematocrit, and higher neutrophil, reticulocyte, and platelet counts compared to nonmated controls (LaBorde et al., 1999; Kim et al., 2000; Liberati et al., 2004). Pregnant Wistar Hannover rats also had lower MCH and MCHC values and higher total leukocyte, lymphocyte, and monocyte counts compared to nonmated female rats. Higher cholesterol and triglycerides concentrations, and lower albumin, glucose, and chloride concentrations and ALP activity occurred in both pregnant Sprague Dawley and Wistar Hannover rats compared to nonmated controls. Triglyceride concentration was approximately

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14. INTERPRETATION OF CLINICAL PATHOLOGY RESULTS

four-fold higher than in nonmated controls (LaBorde et al., 1999; deRijk et al., 2002; Liberati et al., 2004). Pregnant Wistar Hannover rats also had lower urea nitrogen concentration and CK activity, and higher total bilirubin, calcium, phosphorus, and globulin concentrations, and ALT activity than nonmated rats. The significantly higher total bilirubin concentration in Wistar Hannover rats was not seen in Sprague Dawley rats or rabbits during pregnancy. In studies that evaluated clinical pathology at multiple intervals in pregnant rats, most clinical pathology parameters fluctuated during gestation and literature references are inconsistent, further illustrating the need for gestationmatched controls. Several reports of clinical pathology in pregnant New Zealand White and Japanese White rabbits include the following changes: decreased erythrocyte counts, hemoglobin concentration, and hematocrit from about GD 10 through the rest of gestation; increased reticulocyte counts and triglyceride concentration through GD 18, then decreased through the remainder of gestation; increased fibrinogen concentration and APTT later in gestation (GD 18 and GD 28); and decreased total protein, albumin, cholesterol, calcium, glucose, and urea nitrogen concentrations; and ALP activity and minimally decreased creatinine concentration at most time points relative to nonmated controls (Bortolotti et al., 1989; Wells et al., 1999; Haneda et al., 2010; Mizoguchi et al., 2010). There are disparities in literature references for alterations in leukocyte and platelet counts and ALT activity TABLE 14.11

in pregnant rabbits. Clinical pathology changes between Gestation Day (GD) 7 and GD 29 are illustrated in the example provided in Table 14.11. Reticulocyte counts and triglyceride concentration peaked on GD 20. Values for total protein, albumin, urea nitrogen, glucose, calcium, cholesterol concentrations, and ALP activity decreased between GD 7 and GD 29. Lower absolute lymphocyte counts; total protein, albumin, urea nitrogen, cholesterol, calcium, phosphorus, sodium, and chloride concentrations; and ALP activity and higher globulin concentration were reported in pregnant rhesus monkeys (M. mulatta) compared to age-matched nonmated controls, although the stage of gestation was not reported and clinical pathology data were evaluated at only one time point (Buchl and Howard, 1997). It is not uncommon to see nucleated erythrocytes in circulation in a pregnant animal, as well as in a neonate. Clinical pathology values in fetal and postnatal animals are different than those in adults. Most serum chemistry values increase after birth; however, ALP activity, calcium and potassium concentrations decrease postnatally compared to fetal values. Neonatal animals generally have lower erythrocyte counts and higher total leukocyte counts, MCV, and MCH compared to adults. Hematologic and serum biochemical values may be affected by the phase of the estrus or menstrual cycle in many species, including humans (Ulutas et al., 2009). High circulating progesterone concentration, characteristic of diestrus, can influence clinical pathology

Clinical Pathology Results in Pregnant New Zealand White Rabbits (Mean (SD))a

Retic (103/mL)

T Pro (g/dL)

AIb (g/dL)

BUN (mg/dL)

ALP (U/L)

Gluc (mg/dL)

Ca2D (mg/dL)

Chol (mg/dL)

Trig (mg/dL)

GD 7

168.4 (81.7)

6.0 (0.31)

4.7 (0.24)

20.1 (3.24)

54 (10.1)

147 (13.2)

14.9 (0.47)

59 (12.2)

74 (26.1)

GD 20

370.2 (47.6)

5.6 (0.50)

4.4 (0.41)

16.4 (4.95)

34 (4.9)

138 (13.2)

14.8 (0.50)

19 (5.8)

143 (42.0)

GD 29

80.7 (39.6)

4.6 (0.34)

3.4 (0.24)

17.3 (2.73)

25 (6.7)

129 (21.2)

11.9 (1.56)

11 (4.5)

60 (17.8)

a

Five per group. GD, gestation day. Table reproduced from Haschek WM, Rousseaux CG, Wallig MA, editors: Haschek and Rousseaux’s handbook of toxicologic pathology, 3 ed. Academic Press, 2013, Table 29.12, p. 882 with permission.

11. COMPARATOR DATA, HISTORICAL CONTROLS, AND STATISTICS

values. The typically long diestrus phase of dogs has been associated with higher eosinophil counts and cholesterol values and lower erythrocytic and AST values (Willson et al., 2012).

9.12. In Extremis/Postmortem Analyzing clinical pathology samples from animals in toxicity studies that are moribund or in extremis is not recommended. The clinical pathology and/or biomarker data most often reflect the moribund state of the animal, and any test article effects are confounded by the pathophysiological state. Collection of samples postmortem from an animal that has died on study is unwarranted, and clinical pathology and biomarker results in this situation cannot be interpreted.

10. OVERALL RESULTS INTERPRETATION AND REPORT INTEGRATION The purpose of a clinical pathology report in a nonclinical toxicity study is to identify, describe, and interpret test article–related effects as they relate to safety evaluation. The report should attempt to make specific mention of the relationship of notable clinical pathology changes to dose administration, dose level, sex, reversibility of effects, and correlations to relevant in-life and anatomic pathology findings. When interpreting effects on clinical pathology endpoints it is advocated to use a weight-of-evidence and integrated approach incorporating information from in-life, anatomic pathology, biomarker, toxicokinetic, antidrug antibody (ADA) formation, and/or any factor that may impact or be impacted by clinical pathology results (Aulbach et al., 2019a, b). Any relevant study information should always be considered during interpretation of clinical pathology data. There is a natural and mutual synergy when interpreting clinical pathology, anatomic pathology, immunology, and biomarker data in combination; it also promotes multidisciplinary collaboration. It is appropriate to include brief, targeted, specific, and accurate correlation phrasing within the clinical pathology report when it is relevant or provides perspective on clinical pathology findings. This is most

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commonly done with correlative in-life (body weight, food consumption, clinical observations, morbidity) and anatomic pathology findings (e.g., liver, kidney, lymphoid, bone marrow), particularly specific (or lack thereof) microscopic calls. Along with using a weight-of-evidence approach, there are several general patterns of change (listed below) that are indicators that a given change among clinical pathology data is more likely to be “real” or related to the test article, and not due to nontest article–related sources of variation (Aulbach et al., 2019a, b). However, ultimately it is important to consider these patterns in combination with all available data and not rely too heavily on a single trend as definitive support for identifying a change as test article related. • Effects are dose-dependent • Effects are consistent between sexes • Effects are observed in multiple related endpoints (e.g., ALT, AST, GLDH) • Effects have direct microscopic or in-life correlates • Effects are consistent with compound class or known effects • Effects are of sufficient magnitude to be distinguished from background • Absence of similar effects in control animals • Effects are consistent over time • Effects are reversible following cessation of dosing

11. COMPARATOR DATA, HISTORICAL CONTROLS, AND STATISTICS 11.1. Comparator Data In the context of interpreting clinical pathology results, comparator groups are the animals, groups, and/or values that are used to reference against those receiving the test article. Using in-study control and/or baseline (e.g., predose/pretest) values typically serve as the best comparator groups for identification of test article–related effects in test article–treated animals. In nonclinical studies, it is generally recommended to report findings compared to concurrent on-study controls in small animal (i.e., rodent) studies, while reporting

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comparisons to baseline/pretest is often preferable in large animal studies (i.e., dog, NHPs, pig) which often contain fewer animals and may lack a concurrent control group (Aulbach et al., 2019a, b; Weingand et al., 1996; James, 1993). When there are multiple baseline values as in large animal studies, it is recommended to use the time point nearest to the study start as a comparator and not to use the average of the baselines. Most endpoints in large animal studies should be compared to baseline/pretest, although there are several scenarios listed below where comparisons to concurrent control may be appropriate: • Endpoints sensitive to procedure-related influencesdactivities such as blood collection, animal handling and restraint, surgical manipulation, and other stressors can have profound influences on certain endpoints (e.g., decreased red cell mass) as described previously (Aulbach et al., 2017), making comparison to concurrent controls more appropriate. • Endpoints influenced by animal agedshould be compared with concurrent controls in longer term studies (i.e., >13 weeks), particularly in dogs as a variety of clinical pathology endpoints will undergo substantial changes over the course of a 6- to 12-month study (e.g., ALP, phosphorus, urea nitrogen, sCr, reticulocytes, red cell mass).

HCD range. Most subject matter experts have advocated that data from concurrent on-study control animals are the most appropriate comparator for treated animal comparison, more so than historical reference ranges (Tomlinson et al., 2013; Aulbach et al., 2019a, b; Siska et al., 2017). However, HCD can be helpful in circumstances lacking concurrent controls (e.g., in extremis, single sick animal, too few animals per group, and/or no control group), to identify aberrant outliers in control groups, or to aid in identification of extreme values that may present a health concern during animal assignment to study. HCD can also be used as a nonspecific quality control measure or to provide perspective on expected analyte variability. Statistics Although statistical analysis of clinical pathology data sets is required by many regulatory agencies it should not be used as a primary tool to identify test article–related effects among clinical pathology data in nonclinical toxicity studies. Mention of statistical results in interpretive reports is not required and should be minimized when possible as extensive discussion of statistical results may lead the reader to believe that statistical significance represents a substantial component of the interpretation. Clinical pathology data should be interpreted based on the pathophysiologic and biologic relevance of measured results in individual animals, not based solely on results of statistical analysis.

11.2. Historical Controls Historical control data (HCD) sets represent a range of endpoint-specific values that are considered expected in “normal” individuals from a given species within a set of stratified categories (e.g., age, sex, collection site, source/ vendor). HCD are usually generated from values obtained from stock colony animals residing in the test facility, or from individual control animals and/or pretest values from actual studies, and hence, the utility of historical control data is limited to the test site from which it was generated. Individual test results are subjected to basic statistical calculations removing outliers, and the outermost 2.5% of the values at each of end of the distribution (retaining central 95% of the range) meaning 1 of every 20 “normal” test results will be outside of

12. DESCRIPTORS AND BIOLOGIC RELEVANCE At face value, identifying statistical differences and/or values outside of historical ranges among clinical pathology data seems at best a mundane computational exercise. However, the true essence of an interpretation is in providing a more holistic, comprehensive perspective on what effects actually mean to the animal, or their biologic relevance. In order to accomplish this in clinical pathology reports both quantitative and qualitative descriptors are used to provide both objective and subjective perspectives on clinical pathology changes, respectively. Inherent to the numeric nature of clinical pathology data, quantitative descriptors are often

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13. REPORT WRITING AND INTEGRATION

used to express the magnitude of a change among endpoints relative to a comparator group (i.e., x-fold or % change). Quantitative descriptors are objectively determined calculations that allow comparisons across multiple studies and drug programs, but largely fail to provide context on the biologic or toxicologic importance of a change, particularly for readers with limited experience in toxicologic clinical pathology data interpretation. Tables of x-fold calculations alone do not provide adequate context as to the biologic or pathologic relevance of any given effect without additional perspective. Qualitative descriptors are also used to describe clinical pathology changes which, much like histopathology, are subjective to the individual interpreting scientist and require specific training and expertise. When assigning qualitative descriptors consideration must be given to expected biologic variation of the endpoint, species, age of animal, time over which the change occurred, concurrent changes in other related endpoints, influence of study-related procedures, and/or analytical characteristics of the endpoint (Aulbach et al., 2019a, b). Due to the unique biologic behavior of each endpoint, assigning a qualitative descriptor requires a thorough understanding of the endpoint, associated physiology, and analytical aspects of the test. Therefore, each endpoint will require an individualized approach which may vary across studies. For example, a 20% increase in sodium concentration would be considered a marked/severe effect while a 20% increase in alkaline phosphatase activity would be considered a minimal effect at best. Qualitative descriptor severity score scales are typically 4- (minimal, mild, moderate, marked) or 5-point (minimal (or slight), mild, moderate, marked, severe) scales although the latter is gaining favor due to the SEND initiative (Mann et al., 2012). Ultimately the use of both qualitative and quantitative descriptors by individuals with specific training in toxicologic clinical pathology is the most effective way to interpret and integrate clinical pathology results.

13. REPORT WRITING AND INTEGRATION Contemporary toxicologic clinical pathology reports often integrate study results among

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a variety of separate “sub” disciplines in order to help form a well-founded mechanistic or pharmacologic explanation effects and/or to identify potential adverse test article–related effects. Correlations/associations among clinical pathology data with other study endpoints, including clinical observations, food consumption/body weights, toxicokinetic data, and anatomic pathology, are particularly useful to explain pathophysiologic relationships, provide context, and integrate study findings, which may help to support conclusions. The scope of conclusions should be limited to the conditions of the study without extrapolation to other conditions (e.g., longer duration) or to humans. For example, a decrease in lymphocyte count should not be attributed to stress in the absence of sufficient corroborative evidence (Everds et al., 2013). It is appropriate not only to describe the effect (e.g., a moderate decrease in phosphorus concentration was seen), but to further identify the effect as direct (due to the test article directly) or indirect (a secondary sequela) if such a distinction is possible (e.g., a moderate decrease in phosphorus concentration was considered indirectly test article–related, and secondary to reductions in food consumption). Because there is no formal regulatory guidance for the format of clinical pathology reports, report structure and its integration with other clinical pathology contributing scientists’ reports varies between organizations. A standalone clinical pathology contributing scientist report (CSR) should be an all-inclusive document which generally includes an overview of the study design, clinical pathology and biomarker testing protocols, data tables and results, and a comprehensive integrated interpretation based on all available study data. The purpose of a standalone report is to provide all information related to clinical pathology assessment and results for a given study. This format is the most common, and the preferred approach for studies conducted under GLP (good laboratory practice) conditions.

13.1. Adversity Reporting in Clinical Pathology Current philosophies on assigning adversity in clinical pathology reports support that an adverse effect is not limited to a specific clinical pathology

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value, or microscopic change, but is an overall effect of a test article on an organ or organ system that is considered to be adverse based on a weight-of-evidence approach that includes consideration of other study findings (Ramaiah et al., 2017; Kerlin et al., 2016, see Assigning Adversity to Toxicologic Outcomes, Vol 2, Chap 15). Therefore, in most cases, individual and isolated changes among single clinical pathology endpoints are rarely adverse in and of themselves (i.e., hyperkalemia can be adverse while increased ALT activity without other associated findings is not). Additionally, classifying each change among individual clinical pathology endpoints as adverse or not in an itemized fashion is highly discouraged. Adversity of clinical pathology findings is best addressed as part of a constellation of findings that are collectively deemed adverse at a given dose. Clearly adverse clinical pathology effects can be identified, addressed, and expanded upon in the clinical pathology report, particularly if it assists in determination of the no-observed-adverseeffect level by the study director.

14. CONCLUSIONS Interpretation of clinical pathology and biomarker findings in nonclinical toxicity studies must include consideration of all available information and study data including clinical observations, changes in food consumption and/or body weight, and any associated histopathology and organ weight findings. It is of upmost importance to understand and consider the influence of study design (e.g., sample collection timing), sample collection practices (e.g., restraint, animal handling, anesthesia), and any other relevant preanalytical influences (e.g., age, species, diet, housing, medications/supportive treatments) during interpretation or when identifying test article–related effects. The determination of whether test article–related findings among clinical pathology endpoints are adverse should be based on a weight-of-evidence approach that considers findings from all aspects of the study including in-life, anatomic pathology, any related disciplines (e.g., immunology), and toxicokinetics, and should be made in conjunction with the study director and any other relevant study scientists. Contemporary approaches to clinical

pathology interpretation utilize a comprehensive, if not holistic, approach in utilizing both subjective and objective information to characterize test article–related clinical pathology and biomarker effects, and to sensibly frame those findings within the context of other study results.

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C H A P T E R

15 Assigning Adversity to Toxicologic Outcomes John Reginald Foster1, Jeffery A. Engelhardt2 1

ToxPath Sciences Ltd. and Ionis Pharmaceuticals, Carlsbad, CA, United States, 2Independent Consultant (formerly with Ionis Pharmaceuticals, Carlsbad, CA), CA, United States O U T L I N E 1. Introduction and the Need for Quantifying Adversity 1.1. History: Adversity, Organ Function, and the NOAEL 2. Regulatory Assessment of Adversity in the EU, the United States, and Japan 3. Adverse Reactions, Adaptation, and Reversibility 3.1. Adaptation and Adversity 3.2. Reversibility and Adversity 3.3. Exacerbation of Spontaneous Pathology, Historical Control Data, and Adversity

Thymic Involution Liver Weight Changes Progressive CardiomyopathydRat Alveolar Macrophage Aggregates Squamous Metaplasia in Larynx Chemically Induced Exacerbation in Retinal Degeneration 6.8. Treatment-Associated Exacerbation of Chronic Progressive Nephropathy 6.9. Exacerbation of Renal Tubular Mineralization

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4. The Relationship Between Dose Response and Potency Thresholds in Defining Adversity 575 5. The Role of Pathology in Defining Adversity 5.1. Anatomic Pathology 5.2. Clinical Pathology

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6. Case Examples of Assessing Adversity 6.1. Introduction

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1. INTRODUCTION AND THE NEED FOR QUANTIFYING ADVERSITY At the highest level, toxicology is a process used to identify, assess, and manage risk (Figure 15.1). Toxicity studies in animals are intended to identify potential hazards of industrial and agricultural xenobiotics and new medicinal substances. Their intent is to determine safe exposure limits

Haschek and Rousseaux’s Handbook of Toxicologic Pathology, Fourth Edition. https://doi.org/10.1016/B978-0-12-821047-5.00002-6

6.2. 6.3. 6.4. 6.5. 6.6. 6.7.

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585 587 593 595 597 599 600 602 603

8. New Approaches to Characterizing Adversity in the 21st Century? 604 8.1. The Adverse Outcome Pathway and Its Relevance to Assessing Adversity 605 8.2. Assessing Adversity Using Bespoke Studies and Alternative Assays 607 9. Conclusions

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for regulatory authority decision-making with respect to permitting clinical studies to proceed, or to permit the safe use of industrial chemicals and pesticides in society (see Risk Assessment, Vol 2, Chap 16). A critical part of this exercise is the assessment of doses/concentrations where adverse responses do and do not occur. The cut-off between adverse and non-adverse outcomes sets the upper boundaries of safe

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Hazard Identification

Hazard Assessment

Risk Management

FIGURE 15.1 Approaching the call of adversity: Identify the lesion/hazard, assess that lesion relative to potential to cause harm, use that assessment to determine risk of exposure and how to mitigate that risk (RUN!).

exposure. Assignation of “adverse” to a particular histopathologic finding or set of findings is many times the hardest part of the evaluation of an animal toxicity study, due principally to a diversity of opinions regarding what constitutes an adverse finding. For nearly a century, regulators have required sponsors to provide some sort of study, most often in animals, to demonstrate the safety and/or potential risks of their product for consumers (Popp and Engelhardt, 2019). Interpretation of the outcome of the studies and endpoints included in them has greatly matured over this time. In the beginning, safety was based on lethality, the ultimate adverse effect. The lethal dose for 50% of the exposed animals (LD50) was one of the earliest measures for adversity and safety (and no longer is used as a stand alone study). In more contemporary settings, a progression of animal studies evaluates target organ effects and defines the adverse and non-adverse effects and the dose/concentrations associated with the finding(s). A lack of an accepted definition for adversity led initially to the inconsistent interpretation of findings between sponsors and regulators. Widely accepted definitions and best practices have now been established that provide a strong foundation for adversity assessment.

1.1. History: Adversity, Organ Function, and the NOAEL In an initial collective step to address adversity best practices, the European Centre for

Ecotoxicology and Toxicology of Chemicals (ECETOC) convened a task force to address adversity in toxicity studies. The report of the initiative (Lewis et al., 2002) presented more specific definitions to assist toxicologists and toxicologic pathologists in their crafts. The two key definitions were: Adverse effect: A biochemical, morphological, or physiological change (response to a stimulus) that either singly or in combination adversely affects the performance of the whole organism or reduces the organism’s ability to respond to additional environmental challenge. Non-adverse effect: Those biological effects that do not cause biochemical, morphological, or physiological changes that affect the general well-being, growth, development, or life span of an animal. The circular argument of using “adversely” in the definition still complicated the interpretation of findings and their use in regulatory decisionmaking. The decision tree presented in the paper, though, was a great beginning into how one can approach the process of evaluating adversity (Lewis et al., 2002). A subsequent paper by Dorato and Engelhardt (2005) focused on the definition of the noobserved-adverse-effect level (NOAEL) rather than specifically on adversity, although they did bring in the concept of “harmful” to the test animal as a feature of adversity. In using harm to mean adverse, they went on to describe the NOAEL and its dependencies on adversity

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FIGURE 15.2 The Margin of Safety (MOS) is the range in dose/concentration from the clinically effective dose or exposure/application concentration to where no unacceptable toxicity occurs (NOAELs) in relationship to where overt toxicity may occur.

and how to determine the margin of safety (MOS; Figure 15.2). When the final decision is made, regardless of the intended use of the toxicity data, the scientist can declare which dose or concentration did not cause adverse effects. Setting this threshold value is likely the most important part of the decision tree for toxicity studies as it allows a safety margin to be determined where safe exposure of humans, be they patients with intentional exposure or workers with incidental/accidental exposure, is possible. This NOAEL value is critical for establishing safe starting doses in human clinical studies. It is important to be aware that minimal toxic and/or pharmacodynamic effects may be present at the NOAEL, but these have not achieved a level of severity to pose a cause for concern during the risk assessment. Having set an NOAEL allows the scientist to define the dose/concentration where no physiological or morphological effects have occurred. This non-adverse exposure level is the noobserved-effect level (NOEL). Both values, NOAEL and NOEL, are necessary in order to establish a thorough nonclinical risk assessment for the molecule being tested. Identification of adverse findings in a toxicity study need not interfere with the continued development of the test item as the adverse finding only contributes to an understanding of the dose-limiting toxicity or maximum tolerated dose (MTD) of the compound. It is the safety margin or margin of exposure that determines whether or not further development will take place.

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The importance of the NOAEL in regulatory decision-making and risk assessment is indicated by the many issues that impact how to define what the NOAEL represents and numerous previous attempts at evolving a workable approach for addressing adversity (Dorato and Engelhardt, 2005). The authors provided several examples of definitions and how available words within a particular language that described events in toxicity studies may cause difficulty in translation, particularly in Japan. Key concepts forwarded by the authors were that “lack of statistical significance alone does not constitute an NOAEL” and that “statistical significance, by itself, does not make an event adverse.” Notably, the NOAEL is not risk-free, as minimally toxic effects or pharmacodynamic responses that are important for human safety assessment also may be observed at this dose. By consistent application of diagnostic criteria for morphologic alterations and utilization of pathology peer review to ensure uniformity, the assignment of the NOAEL should become a more objective, science-based exercise where decisions can be clearly justified. As such, the designation of adverse effects is a necessary prerequisite to confidently defining an appropriate NOAEL for a toxicity study (Dorato and Engelhardt, 2005; Engelhardt and Dorato, 2020). Additional groups followed with attempts at defining adversity and how it should be presented in reports (Keller et al., 2012; Kerlin et al., 2016; Palazzi et al., 2016; Hall et al., 2021). Palazzi et al. (2016). These presented a useful definition of “adverse” in the exercise of naming an NOAEL and appropriately conducting a risk assessment for a test item. In assigning adversity to pathologic alterations, the spectrum of associated changes (e.g., hematology, clinical chemistry) must also be considered in addition to the severity and distribution of the alteration. Their definition of “adverse” represents an expansion of an earlier concept (Lewis et al., 2002) and is quite useful in conjunction with their decision tree (Figure 15.3). Adverse effect: In the context of a nonclinical toxicity study, an adverse effect is a test item-related change in the morphology, physiology, growth, development, reproduction or life span of the animal model that likely results in an impairment of functional capacity to maintain

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FIGURE 15.3 Workflow diagram illustrating the tiered approach for evaluating adversity. From Palazzi X, Burkhardt JE, Caplain H, et al.: Characterizing “adversity” of pathology findings in nonclinical toxicity studies: results from the 4th ESTP international expert workshop. Toxicol Pathol 44:810–824, 2016.

homeostasis and/or an impairment of the capacity to respond to an additional challenge. In distilling all the papers that have attempted to define “adverse,” it comes down to the dose or concentration of a test item that causes harm to the test animal (Palazzi et al., 2016; Kerlin et al., 2016). The expert professional judgment of

assessing adversity and risk itself is a progressive and iterative process done by the pathologist, with a frame of reference to the test item dose or exposure duration or both. When viewed within the context of a particular nonclinical study, adversity is used to guide the design of a human clinical trial or recommendations for acceptable environmental exposure duration. Framing the safety assessment in such a way to be readily understood

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by regulatory authorities around the globe depends on the consistent interpretation of findings as adverse and outlining the rationale used to reach that conclusion (Engelhardt and Dorato, 2020).

2. REGULATORY ASSESSMENT OF ADVERSITY IN THE EU, THE UNITED STATES, AND JAPAN Clearly the application of the concept of an adverse effect, as a risk assessment tool for extrapolation from nonclinical studies to human exposures, is different for unintentional exposures, from xenobiotics such as pesticides or industrial products, versus those involved in the preparation for intentional human exposure to drugs where both the dose and timing of exposure is generally much better defined. While the principles involved in assigning adversity for drugs and industrial chemicals/pesticides are essentially the same, the risk-to-benefit evaluation for drugs is more easily justifiable/understandable than it is for the “nondrugs”, even though there are considerable benefits to society from the use of pesticides and industrial chemicals. The risks for “nondrugs” compel regulatory agencies to adopt a considerably more conservative approach to the application of non-adverse effect levels from nonclinical laboratory animal studies. The U.S. Food and Drug Administration (FDA) defines the NOAEL as “ . the highest dose that does not produce a significant increase in adverse effects (i.e., responses that are statistically significant or that may be clinically significant even if they are not statistically significant)” (US FDA-CDER 2005). This interpretation is where an adverse effect is one “. that would be unacceptable if it occurred in a human clinical trial . .” (Engelhardt and Dorato, 2020). Global approaches to the use of nonclinical laboratory animal studies to assess risk for humans also exist, especially for nondrugs, and most countries adhere to the principles set down in international guidelines designed to harmonize product development processes across geographic regions. Examples of harmonized guidance include those from the World Health Organization (WHO) International Programme on Chemical Safety (IPCS) (WHO, 2009, 2015) and Organisation for Economic Cooperation and Development (OECD) (OECD,

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2018) for chemicals and the International Council for Harmonisation of Technical Requirements for Pharmaceuticals for Human Use (ICH) (ICH, 2020) for various therapeutic agents. For nondrug applications the adverse call from an animal toxicity study is used to set healthbased guidance values for acute and chronic toxicity endpoints (WHO, 2015), whereas for pharmaceutical development the adverse call is used to set safe starting doses for subsequent tolerability studies in human volunteers or patients dependent upon the therapeutic indication and nonclinical safety profile of the potential drug (EMA, 2013; FDA, 2005; ICH, 2009).

3. ADVERSE REACTIONS, ADAPTATION, AND REVERSIBILITY 3.1. Adaptation and Adversity In the past, pathological changes were frequently evaluated as to whether they were “adaptive”, with the assumption that adaptive (i.e., protective) changes were less likely to have a detrimental effect on the organism as a whole or in the affected organ compared to nonadaptive changes. This association still holds true in that adaptation by a tissue generally means that the effects of exposure to a toxic chemical becomes less than in the absence of adaptation; thus, in many cases adaptation would permit the continued existence of the whole organism or of cells in the affected organ/tissue where a lack of adaptation would result in cell or even organism death. Describing a histological change as adaptive should be of less concern than any pathology that becomes progressively worse with increasing duration of exposure. However, adaptive changes in an organ or tissue do not necessarily equate with a non-adverse change since some adaptive changes clearly result in a loss of function in the affected tissue or organ. An example of this is the squamous metaplasia that occurs in the larynx following continued exposure to inhaled irritants such as cigarette smoke. Exposure to the chemical and particulate melange in smoke results in damage to the delicate respiratory epithelium. Under normal circumstances, the cells respond by a combination of increased metabolic capacity (to provide faster detoxification of chemicals) and heightened turnover

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(proliferation to replace lost cells). In extreme instances, the degree of injury leads to cell death of the respiratory epithelium and its replacement by production of squamous epithelial cells, which represent an abnormal but hardier cell population to sustain some degree of function for the laryngeal epithelium, and the regenerative response does not replace functional laryngeal epitheial cells but results in a fibrotic reaction which allows the larynx to respond to the environmental change. While undoubtedly beneficial under the circumstances, it is clearly not desirable in that while it stabilizes the structure of the larynx, the end product is a loss of laryngeal parenchyma resulting in organ dysfunction (Mallat and Lotersztajn, 2013). Other changes, such as the hepatic hypertrophy that occurs in rodents following exposure to peroxisome proliferating activating receptor alpha (PPARa) and constitutive androstane receptor (CAR) ligands, simply reflect the increased metabolic burden placed upon the liver by exposure to the respective xenobiotics and, provided they remain unaccompanied by necrotic or inflammatory cell infiltration, are adaptive and non-adverse (Hall et al., 2012). For many of these liver hypertrophic xenobiotics, however, there is a clear dose response relationship where at low doses hepatocellular hypertrophy alone occurs whereas at higher dose levels, excessive fat accumulation can occur together with hepatocellular necrosis and inflammation (Maronpot et al., 2010). Dose levels where hypertrophy occurs in the absence of any other changes are non-adverse, but dose levels where hypertrophy is accompanied by additional pathological changes (especially necrosis) may be considered as adverse as they imply a loss of hepatocyte functional mass. Most xenobiotics that cause hepatocellular hypertrophy also induce the activities of several drugmetabolizing enzyme systems such as the cytochromes P450 (CYP) enzymes, produce liver growth and increased cell replication, and also affect changes in bile secretion and bile flow (Paumgartner et al., 1971; Okolicsanyi et al., 1986). The dose responses for these endpoints will generally overlap, but the sensitivity for changes in each endpoint can be subtly different so that in many cases increased liver weight is the most sensitive endpoint observed at the lowest affected dose level and therefore defines the NOAEL (Hall et al., 2012).

Adaptation can be considered as an evolutionary strategy to ensure survival in a new environment where the xenobiotic is present continuously. While typically considered protective in relation to the new circumstance, it is still possible that the adaptive changes are adverse. Adaptation can be viewed as a resetting of the normal homeostatic adjustments that occur on a day-to-day basis. The fluctuations involved in adaptation generally lie outside of the daily dynamic range seen in “normal” situations but fall within limits that do not ultimately lead to a compromise in the function of the affected tissue/organ (represented diagrammatically in Figure 15.4). When the fluctuations in homeostasis occur outside of that required to easily return to normal, then the adaptive changes become adverse and may lead to permanent failure in function (represented in Figure 15.5). Adjusting for fluctuations involved in the daily resetting of the homeostatic equilibrium requires a certain amount of energy, which becomes progressively greater as the magnitude of fluctuations increase beyond the normal range. The model implies that pathologic changes arise when the amount of energy required to maintain the normal functional state becomes so great that it is no longer possible to sustain, leading to physiological changes in an attempt to decrease the energy demand. Such changes may involve changes in the histologic structure, enzymology or other cell molecules or mechanisms in the affected tissue/organ (Figure 15.5). An alternative way of distinguishing when an initially adaptive and non-adverse response can evolve into an adverse change is

FIGURE 15.4 Schematic illustrating the normal, everyday fluctuations that occur in the physiology of tissues which can be corrected through the operation of normal homeostatic mechanisms without the development of disease.

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FIGURE 15.5 Schematic illustrating what happens in a quantitative sense when a tissue reacts by adapting to xenobiotic exposure by adapting its biochemistry as occurs during hepatic enzyme induction. The adaptation results in a stable situation but the change increases the energy level required to maintain the enhanced biochemical state, but this is still a non-adverse change. Additional factors, such as increased stress or higher concentrations of exposure to the xenobiotic, can result in an unstable metabolic change, such as the generation of additional, toxic, metabolites, and these can then result in pathology and disease.

FIGURE 15.6 Schematic of the proposed changes that occur on exposure to chemicals that determine the development of non-adverse and adaptive changes which can, on continued exposure, become adverse, and result in toxicity. Adapted from NRC: Toxicity testing in the Twenty-first century: a vision and a strategy. National Research Council, National Academies Press, Washington, DC, 2007. https://www.nap.edu/resource/11970/Toxicity_Testing_final.pdf. Accessed 24th May 2020.

illustrated in Figure 15.6. This flow pattern implies that exposure to a low-dose concentration for a set time period may show an adaptive, and non-adverse, organ or tissue change which at higher doses and/or for longer durations may lead to perturbation in the function of the affected tissue/organ such that the change becomes excessive and eventually adverse.

In the context of a toxicity study, adaptation is the process whereby a cell or organism responds to a xenobiotic so that the cell or organism will survive in an environment that contains the xenobiotic. As has been described above, adaptation frequently results in an enhancement of normal function, as in the case of induction of hepatic drug metabolism, and in these cases the

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changes should be regarded as non-adverse. On the contrary, certain types of adaptation are accompanied by structural changes that lead to subsequent impairment of function or secondary tissue degeneration, thus defining the effect as clearly adverse. The dose level at which the secondary tissue damage occurs is a low observed adverse effect level (LOAEL), rather than an NOAEL.

3.2. Reversibility and Adversity Adaptive or toxic responses to toxicant exposure may be characterized by reversibility, and it is common to design toxicity studies with a dosing phase followed by a period of nondosing, the so-called reversibility or recovery phase. Such studies are designed to describe the effects of dosing during the dosing phase of the study, and to investigate whether the changes that remain present at the end of the dosing phase can reverse (partially or completely) at the end of the defined recovery phase. Such studies are especially valuable in drug development as they can help to set starting dose levels for subsequent clinical studies in humans as well as in estimating the seriousness and potential ability to monitor any adverse reactions, as determined by the reversibility, of any changes that might occur during the clinical trial. Demonstrating reversibility of a histopathologic finding may cause less concern regarding potential adversity decisions than a nonreversible one (Andrade et al., 2016). The demonstration of histopathologic reversibility does not necessarily equate with the original effect being non-adverse. The reversibility of any change is dependent upon a number of factors including the severity of the effect, the innate regenerative capacity or reparative mechanisms of the affected tissue, the length of time of treatment, and the length of the recovery period. An adverse lesion may, or may not, be reversible, but by definition an adaptive change should be reversible when exposure to the chemical inducing the adaptation is removed. Hence, non-adversity for a dose level is not necessarily defined by the reversibility of the effect indeed, several potentially adverse lesions, such as squamous metaplasia that occurs in the larynx with irritant xenobiotics and hepatic foci of cellular alteration, are reversible on cessation of exposure to

the causative agent (Osimitz et al., 2007; Kaufmann et al., 2009).

3.3. Exacerbation of Spontaneous Pathology, Historical Control Data, and Adversity Exacerbation of a histological finding commonly observed in control animals can be observed after dosing with xenobiotics. In practice this can present as an increase in the incidence and/or severity of a particular finding in the treatment groups over that present in the concurrent vehicle control group (Hard et al., 2013; Melnick et al., 2012). There are several considerations when evaluating an apparent increase in the incidence and/or severity of a background histological finding in laboratory animal studies. Probably the most important consideration relates to the fact that many, if not all, spontaneous histopathologic findings can vary considerably among control animals depending on many factors including their species, strain, age, sex, housing conditions, diet, route of dosing, etc. (Haseman et al., 1994; Keenan et al., 1994). In comparing control and xenobiotic dose groups, it is critical that these potential confounders are taken into account, especially for studies where the numbers of animals in a given group may be small (Deschl et al., 2002; Haseman et al., 1984; Haseman, 1995). The reliance on historical control pathology to resolve interpretive dilemmas may also be undependable since pathologists who populate historical control databases may have different thresholds for recording spontaneous findings (McInnes and Scudamore, 2014). Recommendations for the appropriate use of historical control data (Keenan et al., 2009) and a standardized nomenclature for the diagnosis of laboratory animal pathology (INHAND program; goRENI) (Keenan et al., 2015; STP, 2020; goRENI, 2020) should help standardize the application of historical control data and limit the vagaries associated with the use of differing nomenclatures for the same pathologies. However, the consistency of recording background still affects the value of historical histopathology control data for nonneoplastic pathologies. Where questions remain regarding whether or not a particular background finding’s incidence

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or severity is treatment-related or not, the best solution is to generate contemporary incidence and severity grading by reviewing control tissue from the last 10 or so studies of similar duration using a single pathologist, preferably the one reading the study in question. This can also be done by a panel of pathologists in a pathology working group (PWG; see Pathology Peer Review, Vol 1, Chap 26) to ensure consistency in recording and grading. While statistics can help in evaluating a treatment-related effect, the correct statistical approach needs to be applied alongside an intimate knowledge of the variables involved in the appearance or disappearance of any background pathological findings (Elmore and Paddada, 2009) (see Experimental Design and Statistical Analysis for Toxicologic Pathologists, Vol 1, Chap 16). As with many of the decision points in considering if a particular finding is adverse, biological plausibilitydwith an understanding of the probability that an effect is due to the known pharmacological action of the test articledweighs heavily in favor of any effect being related to treatment (ECETOC, 2002).

4. THE RELATIONSHIP BETWEEN DOSE RESPONSE AND POTENCY THRESHOLDS IN DEFINING ADVERSITY Paraphrasing the paradigm attributed to Paracelsus, “it is the dose of a given xenobiotic that determines the poison” (Borzelleca, 2000). Hence all xenobiotics that enter the body are poisonous at and above a threshold dose, below which the given agent is safe. This threshold toxic dose is known as the lowest observed effect level (LOEL). This threshold is intimately related to the NOAEL, and determines where on the dose response curve the NOAEL lies (see Section 1). Xenobiotics that activate a common molecular target or affect a common target structure within cells may show different toxicity-dose relationships depending on several factors. Key influences include their pharmacokinetics, their toxicodynamics, and their potency in affecting the target molecule. Potency is determined through a combination of factors but is exemplified especially well for those xenobiotics that

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interact with hormone receptors (Borgert et al., 2013). Typical extracellular concentrations of functionally active hormones are in the range of 10
11 to 10
9 M whereas those of structurally similar molecules, both endogenous signaling factors and xenobiotics, that bind to the same hormone receptors are typically in the range of 10
5 to 10
3 M (Chedrese and Celuch, 2009; Grannar, 1993). Hence there are large differences in the ability of the respective molecules to bind to, let alone activate, individual hormone receptors. Normal functioning of endocrine cells is exquisitely dependent on high-affinity binding of a hormone or hormone mimetic to the receptor. This relationship explains why the estrogen receptors in human females can be specifically activated by estradiol, with a mean plasma concentration of w5 pg/mL, but not by the chemically similar molecule testosterone, which is present at approximately 40-fold higher concentrations than estradiol (w200 pg/mL) but engages the estrogen receptor with much less affinity (Sower et al., 2001; Scarabin-Carre´ et al., 2014). The inability to distinguish hormones from structurally similar molecules in the body would prevent the endocrine system from functioning. The ability of receptors to differentiate structurally similar molecules is based on highly specific binding of hormones with their respective receptors so that only energetically favored interactions (based on close structural pairings between ligand and receptor molecular groups) will produce biological activation and downstream signaling effects. This pairing is called the affinity between the ligand (whether endogenous hormone or xenobiotic) and receptor; while the system permits structurally similar molecules to bind receptors, much higher local concentrations of low-affinity ligands will be required to displace highaffinity ligands (Chedrese, 2009; Chedrese and Celuch, 2009). The example of estrogen and testosterone clearly illustrates this principle. However, a xenobiotic that can also bind the receptor with low affinity is able to produce a cellular response if a sufficient concentration can be achieved at the receptor site to competitively displace the natural hormone ligand (Figure 15.7). At low concentrations, such xenobiotics would be unable to displace the natural high-affinity endogenous hormones from their specific receptors.

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FIGURE 15.7 Concentrationdependent hormone response when there is a proportional relationship among hormone concentration, receptor occupancy, and biologic response. Data are plotted on a semilog scale and demonstrate that the entire doseeresponse spans at least six orders of magnitude. Adapted and redrawn from Lucier GW, Portier CJ, and Gall MA: Receptor mechanisms and dose-response models for the effects of dioxins. Environ Health Perspect 101:36e44, 1993.

Differences in the ability of analogous chemical series to activate a given molecule can be readily measured in vitro as the concentration required to produce a 50% inhibition in a particular endpoint, or alternatively a 50% activation of the endpoint. These measures of potency are known, respectively, as the half-maximal inhibitory concentration (IC50) and halfmaximal (or median) effective concentration (EC50) (Sebaugh, 2011). The importance of potency in determining the dose at which a toxic endpoint manifests is exemplified in the activation of the aryl hydrocarbon receptor (AhR) by polycyclic aromatic hydrocarbons (PAHs) such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD), polychlorinated biphenyls (PCBs), and polybrominated biphenyls (PBBs). While all of these are ligands for activating the AhR, the EC50 values among the xenobiotics in the series differs by four orders of magnitude between the most and least potent analogs (Van den Berg et al., 1998; Becker et al., 2015). Since activation of the AhR determines most, but not all, of the toxicities observed with these chemicals, it follows that the dose level at which effects become adverse will also show similar degrees of differences (Schmitz et al., 1995) with the most potent, TCDD, having the lowest NOAEL for those toxicities mediated via the AhR. The differences in affinity and hence the range separating the dose levels at which a desirable

response (efficacy) is elicited by structurally similar endogenous molecules imply potency thresholds in the activation of undesirable responses (toxicity) as well (Borgert et al., 2013). Although such biological thresholds would vary for different types of binding sites and with the kinds of toxic outcomes, the degree of binding that induces toxicity and any detectable response resulting from binding will require an appropriate concentration of chemical with sufficient potency. These requirements determine the thresholds for toxicity (Figure 15.8). Target cells will almost always have multiple (a few to hundreds of) molecular target sites. For structurally related xenobiotics, the potency of their responses on binding to a molecular target reflects the affinity of that chemical for the respective molecule (Lucier et al., 1993). Evaluation of dose-response relationships for receptor-mediated toxicities requires information on the quantitative relationships among ligand concentration, receptor occupancy, and biologic response. Figure 15.8 illustrates a situation where there is a proportional relationship between receptor occupancy and biological response. In this situation, occupancy of one receptor would produce a response, although it is highly unlikely that this base-level response would be detected. The biological significance of such a response is likely negligible, and it will almost certainly vary with the endpoint as well as with

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FIGURE 15.8 Proportional relationship among hormone concentration, receptor occupancy, and biologic response using the same data set as in Fig. 15.7 plotted on an arithmetic scale. In this case, linearity of response is clearly seen in the low concentration region, followed by saturation at the higher concentrations. Adapted and redrawn from Lucier GW, Portier CJ, and Gall MA: Receptor mechanisms and doseresponse models for the effects of dioxins. Environ Health Perspect 101:36e44, 1993.

developmental stage and cell type (Lucier et al., 1993). If the same data are plotted on an arithmetic scale, the shape of the dose-response curve readily shows a linear relationship between target receptor occupancy and biological response at lower concentrations with target receptor saturation and a plateaued response at higher concentrations (Figure 15.8). Such a simple proportional relationship does not explain the diversity of biological responses that can be elicited by a single chemical using a single receptor. For example, low concentrations of insulin produce much greater effects on fat cells than on muscle cells. These differences are due to tissue- and cell-specific factors such as the number and density of receptors, and the intensity of the signal transduction response that occurs on binding of hormone to receptor (Dimitriadis et al., 2011; Soda and Tavassoli, 1983; Cuatrecasa et al., 1971; Kaplan, 1984). These factors are responsible for modulating the qualitative relationship between target binding and response. In the case of dioxin, the induction of hepatic CYP1A1 and CYP1A2 does not correlate with hepatocyte proliferation rates and numbers of foci of cellular alteration (preneoplastic lesions), with the latter tissue responses occurring at considerably higher dose levels than do changes in the expression of the CYP genes (Clark et al., 1991; Popp et al., 2006; Becker et al., 2015). These data suggest that our ability to predict doseresponse relationships for the effects of

a relatively simple chemical like dioxin are limited by our substantial lack of understanding of the association between changes in gene expression and subsequent biological effects. Figure 15.9 provides the sequence of events leading to biological changes for dioxin (Lucier et al., 1993). By analogy with hormone ligand-binding models, the term “fractional occupancy” nicely illustrates the fraction of target molecules occupied at a particular concentration of a xenobiotic (Figure 15.10A). The principle states that the fractional occupancy is equal to the number of occupied target binding sites divided by the total number of binding sites, which essentially means that a given chemical will express toxicity at dose levels that permit binding to a given fraction of the target sites within a cell (Figure 15.10A) below which no toxicity (i.e., no adverse effects) will occur (Salahudeen and Nishtala, 2017). For low-affinity xenobiotics, the number of chemical to target bindings would have to be much higher than for high-affinity xenobiotics in order to result in toxicity (Figure 15.10B).

5. THE ROLE OF PATHOLOGY IN DEFINING ADVERSITY 5.1. Anatomic Pathology Histopathology is frequently the most sensitive indicator of toxicity in animal studies, often

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the resulting data cannot be extrapolated to predict responses in other study scenarios or by other species (including human). Decisions based on adversity data from animal studies is the strict domain of risk assessment (see Risk Assessment, Vol 2, Chap 16). The theoretical extrapolation from a short-duration, often highdose study into the consequences of longerduration, typically lower-dose study, with subsequent exacerbation of a low-grade lesion, is fraught with problems associated with the biological responses of adaptation, modification and profound changes occurring in the incidence and severity of a lesion seen on shortduration studies (Kerlin et al., 2016).

FIGURE 15.9 Postulated mode of action representation of the sequence of molecular and biological events involved in dioxin-mediated toxicity. Redrawn from Lucier GW, Portier CJ, and Gall MA: Receptor mechanisms and dose-response models for the effects of dioxins. Environ Health Perspect 101:36–44, 1993.

occurring at lower dose levels than other test article-related changes such as altered clinical biochemistry values, food or water intake, and organ weight changes (Palazzi et al., 2016). There are several broad rules that can be applied when judging if a test article effect in an animal toxicity study is adverse or non-adverse (Pandiri et al., 2017). Whatever the reasons behind the adversity decision, the rationale must be clearly stated, with scientific justification, in the study report (Kerlin et al., 2016). A very important principle when assigning adversity, but one that is frequently forgotten in a toxicity study, is that the application of the decision should be limited to the test species used, and to the dose levels and duration of the study in question;

Anatomic Pathology and Communicating Adversity For drug toxicity studies, a NOAEL for a test article should be stated within the final study report with a clear explanation for which toxic effect(s) the NOAEL is being applied and what other effects may have been dismissed as being non-adverse. For pesticides and other nondrug products, the individual studies will normally be used alone to set health-based guidance values such as the acute reference dose (RfD) and/or acceptable daily intake (ADI) values for human risk assessment based upon the dose level in animal studies where the most sensitive toxic endpoint is not seen. With respect to safety assessment studies on xenobiotics intended for drug use, where the intention is to set safe starting dose levels for the xenobiotic entering human clinical trials, the respective NOAELs obtained from various in vivo and in vitro laboratory animal studies need to be considered in their entirety before recommendations of starting doses for “first in man” studies are made to regulatory authorities (WHO, 2015; U.S. FDA-CDER, 2005). In this regard, the NOAELs for individual safety studies are assessed together to define the NOAEL indicative of the most sensitive effect in the most sensitive test species. Clearly all available data, including histopathology, from animal studies must be evaluated together to define potential toxicities and to set NOAELs. Communication of adverse findings and NOAEL should include direct participation of contributing scientific disciplines (pathologists, toxicologists, etc.) in assessing and

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FIGURE 15.10 (A) This figure illustrates the concept of “fractional occupancy” for a dose response when a xenobiotic interacts at a high affinity with a cell receptor. At low doses there is tight binding to a limited number of receptors and adaptation in the absence of toxicity occurs. In contrast, at high concentrations there is extensive binding to the target molecule resulting in activation of the function of the target receptor and toxicity. (B) This figure illustrates a theoretical situation in a dose–response relationship where the xenobiotic ligand has poor binding affinity to the target receptor. In this situation, irrespective of the dose administered, there is poor association between the xenobiotic and receptor and no toxicity irrespective of dose. Adapted from Salahudeen MS, Nishtala PS: An overview of pharmacodynamic modelling, ligand-binding approach and its application in clinical practice. Saudi Pharmaceut J 25:165–175, 2017.

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communicating human risk (Kerlin et al., 2016; Pandiri et al., 2017) (see Risk Management and Communication, Vol 2, Chap 17). Additional Factors in Anatomic Pathology Influencing Adversity Decisions Many factors should be taken into account when considering an adversity determination for a pathological effect. Key questions highlighting these factors include: • Are there related pathological findings? • Is there a known or biologically plausible underlying mechanism to explain such findings? • What are the severity criteria used by the pathologist, and have these been sufficiently defined in the report to support the decision? • What is the background incidence for the finding (historical control)? A call of adversity for a finding in a toxicity study should not be based on: • Extrapolation across species (including humans) or • Extrapolation across studies of varying durations, doses, and/or delivery routes in the same species. An adverse finding can be: • An exacerbation of a background lesiondeither increased in incidence or severity. • An exaggeration of a presumed suprapharmacological (expected, or “on target”) effect. • Secondary to an effect on hormones, the immune system, food/water consumption, etc. • Transient or reversible. Does the severity decrease on continued exposure? • An adaptive response that occurs when a lesion that begins early in a study is altered in character with prolonged dosing. It is generally recommended that a conclusion of adversity is not simply dependent on a statistical analysis but rather should take into consideration all available evidence from within the study. An effect that only occurs in one sex can be adverse. However, an effect seen in both sexes would potentially be of more concern and engender considerably more discussion in making adversity decisions.

Anatomic Pathology and Function When the study findings do not compromise normal tissue physiology, do not impair functional capacity, and do not increase susceptibility to other influences, then they typically are considered non-adverse. However, the detailed reasoning behind an interpretation of nonadversity still may need to be fully explained and supported by adequate literature references for submission to regulatory authorities. The reasoning behind adversity decisions for given endpoints should be accompanied by a sufficiently detailed description to permit regulatory reviewers to reach their own conclusions. Such details should be present within each study report, but also may be communicated in multiple study reports and collated in a nonclinical overview document integrating several study reports. For example, a compilation of all data from multiple reports may be used in support of setting a starting dose for a first-in-man study. The details on the pathogenesis and mechanism of the adverse responses need to be provided, including the full explanation for the morphological criteria used for making diagnoses and assigning severity grades. The pathology report for each study needs to clearly characterize any biologically relevant changes (background or test article-related) and to fully explain the reasoning behind the decision regarding whether a given change is adverse or non-adverse to the test species for that study. If necessary, there is merit in using outside pathology and toxicology peer review as well as in providing independent expert interpretations that review the spectrum of observed effects. Of vital importance in adding credibility to these professional judgments is that all written reports are supplemented with relevant published literature related to the incidence and severity of various adverse findings and their characterization as related (or unrelated) to test article exposure. Anatomic Pathology and Severity Grading The call of adversity may depend on an increased severity of a spontaneous finding also present in the vehicle-treated control animals (Schafer et al., 2018). In such circumstances, it is essential that the histopathology changes accurately reflect the severity grade definitions and that these are precisely defined in the

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“Materials and Methods” section of the pathology report. In these cases, it is the severity of the pathologic change that defines the conclusion of adversity rather than presence of the finding per se. Anatomic Pathology Peer Review in Ensuring the Relevance of Adversity Decisions Threshold values (e.g., NOAELs) are most frequently determined by the study director based on the histopathological data and often by test article-related increases in the severity of a particular finding. Assigning low-, medium-, and high-grade changes in particular findings can be highly variable even for a single pathologist, and the potential “diagnostic drift” over time becomes more important to control the longer it takes to complete a study (Eighmy, 1996; Schafer et al., 2018). Typically, a lifetime (2year) rodent carcinogenicity study (w400 animals) can take between 12 and 16 weeks to evaluate the thousands of tissue sections, so that measures such as colleague or supervisor review must be put in place to ensure consistency in making diagnoses and assigning severity grades for the various lesions (Boorman et al., 2002, 2010; Morton et al., 2010). The problem of drift is fairly limited for the recording of neoplastic findings (which are typically identified as “present”) but is a challenge for nonneoplastic changes (for which a number of similar diagnostic terms and severity grading systems might exist). Ensuring consistent diagnostic terminology, severity grading, and data recording over a prolonged period has been recognized as a possible problem for many years, and the accuracy and sensitivity of the study findings depends on limiting the variability inherent in the iterative process of histopathologic diagnosis. By its very nature, anatomic pathology to generate microscopic diagnoses and severity grades is a somewhat subjective discipline with its dependency on the particular education, training, and experience of the pathologist carrying out the evaluation (Schafer et al., 2018; Bolon et al., 2010). In recognition that the assessment of tissue specimens is based on the professional judgment of the pathologist, it is a best practice for many test facilities to implement a pathology peer review process whereby a given

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percentage of the slides from a study are assessed by another (or rarely several other) pathologist(s) (Morton et al., 2010) either based within the same facility as the study pathologist or increasingly by a pathologist from outside the laboratory conducting the study (see Pathology Peer Review, Vol 1, Chap 26). The peer review pathologist carries out an independent review with clearly defined objectives that checks the diagnostic accuracy and assures the quality of the data interpretation. While the issue has been the subject of intense debate for some years, there is no requirement in the Good Laboratory Practice (GLP) principles to conduct a peer review (see Pathology and GLPs, Quality Control and Quality Assurance, Vol 1, Chap 27), although many regulatory authorities now expect that some level of peer review will be performed for microscopic data from animal toxicity studies (OECD, 2014). The pathology peer review process (Figure 15.11) can, by its very nature and intent, lead to changes in the microscopic diagnoses and severity grades, and thus potentially to changes in interpretation of the pathology data for a given toxicity study (McInnes and Scudamore, 2014; Morton et al., 2010). The study sponsor may request that the slides are reviewed by a specific peer review pathologist with a particular expertise or who may be experienced with the physiological/pharmacological effects of the test article under investigation. This approach can help to ensure consistency in diagnostic terminology, descriptions, and interpretations across different but related studies of the same test article carried out in different species and/or at different laboratories (FDA, 2019). Pathology peer review expectations within sponsors (usually companies), research institutions, and regulatory agencies worldwide have evolved over the last decade, but the ultimate aim of the endeavor is to improve diagnostic quality and pathology data interpretation by ensuring that treatment-related effects are accurately, and consistently, recorded both in terms of their incidence and severity grade. Most relevant to the current discussion is that pathology peer review can help in confirming the adversity of individual pathology findings either through affecting the incidence, severity, or clarifying the

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FIGURE 15.11 Decision tree for assigning severity grades to histopathology findings in assigning adversity to the change. Adapted from Schafer KA, Eighmy J, Fikes JD, et al.: Use of severity grades to characterize histopathologic changes. Toxicol Pathol 46:256e265, 2018.

biological significance of the key findings that affect dose setting for clinical trials or healthbased guidance values for other test articles.

5.2. Clinical Pathology In comparison to anatomic pathology, guidelines for setting adversity using clinical pathology data are considerably more limited. One important reason for this divergence is that historically clinical pathology changes were considered secondary to anatomic histopathology findings indicative of cell or tissue damage, so the existence of the former, in the absence of histopathology, was frequently dismissed as being of no or limited relevance with respect to assessing the degree of xenobioticrelated toxicity. A European Society of Toxicologic Pathology (ESTP) workshop on the characterization and definition of adversity in the context of toxicity studies concluded that clinical pathology changes should not be considered adverse if they occurred in the absence of associated anatomic pathology or adverse in-life consequences (Palazzi et al., 2016). This principle was further endorsed by the outcome of a combined initiative of the Society of Toxicologic Pathology (STP) and the American Society of Veterinary Clinical Pathology (ASVCP) (Ramaiah et al., 2017).

Clinical Pathology and Functional Assessment In many situations, substantial changes in clinical pathology parameters provide important, and in some cases the only, evidence of test article–related tissue injury. Altered clinical pathology values serve as biomarkers of functional changes in affected organs and tissues. Hence while they may not be considered harmful (adverse) per se, clinical pathology signals may be key to setting non-adverse dose levels where histopathologic findings and/or organ weight changes are accompanied by changes in clinical pathology markers of organ function. For those few organs that have clinical pathology measurements assessed during conventional toxicity studies and clinical trials, they are an indispensable tool in deciding where adverse effects on tissue/organ function and structure begin. Clinical Pathology and Dose Response The dose response for clinical pathology changes is frequently different relative to those associated with similar anatomic pathology changes. Clinical pathology alterations for some organ systems (e.g., the liver and kidney) can be more sensitive than histopathology, developing at lower dose levels compared to those

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where histopathological changes are first observed. High blood urea nitrogen (BUN) levels, or high creatinine concentrations, in the presence of minimal histological changes in the kidneys confirm that the dose level at which these changes occur is an adverse one. In contrast, the same degree of histological changes in the kidney in the absence of these clinical pathology changes would support a conclusion of non-adversity as defined by the absence of functional effects on the kidney (Hall et al., 2012). Similarly, an organ weight effect in the presence of significant clinical pathology changes (e.g., liver enlargement with high serum activities of hepatocyte cytosolic enzymes) may be considered adverse even in the absence of any additional, associated, histopathologic findings (Hall et al., 2012). Hence the assessment of adversity will always be based upon a pattern of changes and their pathophysiologic implications, of which clinical pathology is an important consideration. Currently, certain clinical pathology changes that are considered reliable indicators of altered function may occur in the absence of histopathology and as such should, if the definitions of adversity are strictly adhered to, be considered adverse in the absence of histopathology at the respective dose level. An example of this is an increase in serum concentrations of bile acids or bilirubin which, at least in rodents, can be seen in the absence of significant histopathology but is a considered to be a reliable indicator of cholestasis as an adverse effect where histopathology may be an insensitive measure of liver damage (Carroll et al., 2018; Kwon et al., 2008; Starckx et al., 2013). As with virtually all endpoints measured in toxicity studies, there are broad principles that are applied in assessing if a particular change in a clinical pathology parameter is treatmentrelated or not, before the second stage of considering if the observed change is adverse (WHO, 2015). With respect to clinical pathology analytes, related parameters such as organ weight or histopathology should change in the same predicted direction as the clinical pathology change (increase or decrease); if not then the relationship of the clinical pathology change to treatment, or to the associated anatomic pathology endpoint, will be in doubt. The dose dependency of the clinical pathology change should also be apparent if it is truly a treatmentrelated effect rather than a reflection of

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a sporadic variability that some parameters exhibit due to dynamic changes in systemic physiological processes. Clinical Pathology and Reference Intervals The importance of suitable reference intervals (the range of normal variation in historical control data for a given clinical pathology parameter) is critical in the ultimate interpretation of any changes observed in test article-exposed animals (WHO, 2015; Hall, 1997). There are several factors, including species, strain, sex, age, diet, dosing route, etc. that can affect historical control data, and these need to be carefully controlled to ensure appropriate interpretation (Barnes et al., 2015; Hall, 1997, 2016; Hall et al., 2012; Hall and Everds, 2013; Weingand et al., 1996). In studies in dogs and nonhuman primates where the group sizes are small for ethical reasons, the changes in individual animal values, including those baseline measurements from study animals acquired during the pretreatment period, are often more helpful than group mean values for a given treatment group due to the variability inherent in the individual animal values. Information on hematological, blood and urine chemical, and coagulation parameters of structurally similar xenobiotics or xenobiotics with a similar mode of action is also useful where questions arise over the relationship of a clinical pathology change to treatment (WHO, 2015). Clinical Pathology and the Importance of Quantification in Adversity Decisions The question of when a quantitative effect becomes adverse (i.e., when the degree of change from normal is accompanied by functional defects) is exemplified by the inhibition of acetylcholinesterase (AChE). Low-level inhibition of this enzyme is generally well-tolerated without clinical signs, whereas inhibition of 20% or more is considered to be significant for risk assessment purposes. In this context, recommendations exist that all available data on brain, blood, and any other tissue cholinesterase activity as well as the presence or absence of inlife clinical signs and neuropathologic findings should be evaluated when assessing the safety of cholinesterase-inhibiting xenobiotics such as organophosphorus and carbamate pesticides. Such specialized analyses are carried out on a case-by-case basis, using a weight-of-evidence

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approach, but the inconsistent practice does open the door for conflicting views due to the paucity of historical control data for unusual endpoints. Inconsistent treatment of xenobiotics with common molecular targets will often be resolved by a reversion to the most conservative conclusions with respect to adversity decisions and risk assessment (ECETOC, 1992; Padilla et al., 1994; USEPA, 1998). A subset of organophosphate agents, exemplified by tri-o-cresyl phosphate and leptophos, produce a neurotoxic condition known as organophosphate-induced delayed neuropathy (OPIDN) that develops some weeks or months after acute or repeated exposure. This slowly arising condition is fundamentally different from the acute toxicity experienced by most animals in the treatment group where exposure leads to degeneration and gradual demyelination of long axons in peripheral nerves and the spinal cord. The sensitivity to developing OPIDN differs considerably among animal species with the adult hen being a sensitive animal model of choice. Humans are also considered to be highly susceptible. The observed neural degeneration and clinical ataxia is irreversible, and extensive investigative studies have shown that the initiation of OPIDN is associated with the inhibition and subsequent permanent binding (“aging”) of the chemical to “neuropathy target esterase” (NTE) (Johnson, 1990; Richardson, 1995). These studies have shown that, at least experimentally, a threshold of inhibition requires greater than 70% of the target enzyme to be inhibited before the development of OPIDN can occur. This again exemplifies that the onset of adversity is a quantitative phenomenon, exhibiting clear and definable thresholds, rather than a qualitative “all or nothing” response. Clinical Pathology and the Speed and Reversibility of Responses Additional factors that have been introduced into the evaluation of adversity for clinical pathology endpoints are the rapidity of onset of the biomarker change and the persistence of the particular change with repeated dosing. Both of these factors need to be considered when assessing adversity (Ramaiah et al., 2017). In most standard rodent toxicity studies, clinical pathology changes are only assessed prior to dosing and at the end of dosing such that the rapidity of onset of a particular parameter

cannot be determined. Where they exist, specially designed studies incorporating multiple sampling time points, often including baseline (predosing) values, would answer this question and can help evaluate a potentially dose-limiting change. Acute and severe changes in biomarkers such as serum potassium concentrations, leading to cardiac dysfunction, have been associated with sudden death in some toxicity studies (Vormberge et al., 2006; Ben Salem et al., 2009). Hypokalemia (low potassium levels) has been induced by b2 agonists (Clausen and Flatman, 1977), calcium channel blockers (Tishler and Armon, 1986; Oe et al., 1998), insulin (Burghen et al., 1980), catecholamines (Brown, 1985; Seck et al., 1996) and many other therapeutics (Ben Salem et al., 2009). In contrast to sudden changes in clinical pathology parameters where clear clinical symptoms can be observed, slow onset of clinical pathology changes (even if of similar severity) can result in few if any clinical signs as physiological adaptation can occur if the alterations develop gradually. For example, acute and severe hemorrhage resulting in decreases in hemoglobin, hematocrit, and red blood cell mass will be adverse and have immediate and serious functional consequences (Ettinger and Barrett, 1995; Redondo et al., 1995). In contrast, a gradual loss of blood will normally result in an adaptive response (hematopoiesis) in tissues such as those in the bone marrow, spleen and liver that maintain relatively normal hemoglobin, hematocrit, and red blood cell mass. These compensated changes might still result in an adverse decision for any dose level resulting in the observed changes (Suttie, 2006; Johns and Christopher, 2012; Pechereau et al., 1997). Certain clinical pathology endpoints, such as those relating to red cell and white cell counts, are tightly regulated within the bodies of laboratory animals, and significant perturbations outside the normal ranges may result in death. In these circumstances, lethal disturbances are clearly adverse. If lethality does not occur, reactive and/or associated histopathological changes leading to moderation of the red cell and white cell effects will occur that may moderate the adversity decision. An exception to this example possibly exists for platelet counts where druginduced, severe depletion typically is associated with excess bleeding, but where sufficient information may be available in the literature to set

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a platelet count threshold where an effect will be considered adverse even in the absence of hemorrhage (Narayanan et al., 2019; Mitta et al., 2019). Platelet counts of less than 10,000/ mL in dogs or less than 25,000/mL in mice are known to produce a high risk of hemorrhage (Morowski et al., 2013; Ramaiah et al., 2017). A drug-induced decrease in neutrophil counts is another clinical pathology parameter where extensive experience permits the setting of a threshold for adversity decisions based on cell numbers likely to increase the risk of infection even if an infection is not present (Giguere, 2013). Neutrophil counts of around 50/mL in rats and 500/mL in cynomolgus monkeys are associated with an increased risk of infection. There are clear species differences in susceptibility, with rodents being generally far more resistant and requiring far lower neutrophil numbers to result in increased susceptibility relative to monkeys or dogs (Dinauer and Coates, 2005; Johnson et al., 1985; Yarrington et al., 1991; Kristiana et al., 2013). Clinical Pathology in Dead or Moribund Animals Very pronounced changes in clinical pathology parameters can occur in moribund animals as organs and systems shut down. Therefore, real care needs to be exercised in interpreting such changes as being related to treatment and/or adverse consequences of treatment as opposed to being nonspecific indicators of the terminal medical condition. Moribund animals can exhibit nonspecific changes in clinical pathology parameters related to such factors as poor sample quality (due to difficulty in blood collection related to dehydration, extreme stress, or shock), immune system dysregulation, and acid–base imbalances (Hall, 2013; Everds et al., 2013; Everds, 2015). General guidance in reporting clinical pathology data from moribund (or deceased) animals is the same as that for reporting other data from such individuals: such values should be reported separately from the main body of the results from nonmoribund animals surviving for the intended duration of the study (Ramaiah et al., 2017). Conclusions In conclusion, defining adverse dose levels should seldom be done on the basis of clinical

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pathology endpoints alone. Instead, current recommendations are that clinical pathology values should be evaluated in conjunction with other data obtained from toxicity studies in a weight-of-evidence evaluation to establish NOAELs or NOELs (Kerlin et al., 2016).

6. CASE EXAMPLES OF ASSESSING ADVERSITY 6.1. Introduction There are many excellent examples of histopathological changes in laboratory animals that have traditionally been considered non-adverse (Table 15.1). These include changes that occur during development and maturation, such as thymic involution; retention of embryonal features, such as remnants of the thyroglossal duct in the thyroid gland; changes that are part of the normal aging process, such as bile duct hyperplasia/degeneration in the liver and cardiac degeneration in rats; or a consequence of administering xenobiotics in particular vehicles, such as occurs with the presence of lipidladen macrophages in the draining lymph nodes. While in many cases these findings can be easily dismissed as irrelevant in setting nonadverse dose levels, others can prove to be problematic and lead to many hours of debate regarding their significance.

6.2. Thymic Involution Involution (physiological atrophy) of the thymus is a very common spontaneous change that occurs during the aging process in most laboratory animals, although at differing rates depending on the species and individual (see Immune System, Vol 5, Chap 6). Involution is characterized by reduced thymus volume and weight secondary to thymic lymphocyte depletion (Figure 15.12A). Age-related thymic involution is a gradual, nonreversible, change that is thought to be associated with an imbalance in the circulating levels of sex- and stress-related steroids. Differentiating age-related involution from chemically induced atrophy can be difficult, but age-related involution can be characterized by decreased cortical cellularity with a blurring of the cortico-medullary junction,

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TABLE 15.1 Examples of Pathologies That May Not Be Concluded to Be Adverse Organ

Effect/Pathology

Species Affected

Dose Route

Literature Reference

Thymus

Involution

Rat, dog

Various

Pearce (2006), Kobayashi et al. (1994), Sato et al. (2012)

Liver

Bile duct proliferation

Rat

Various

Hailey et al. (2014)

Liver

Hypertrophy/ increased weight

Rat, mouse

Various

Carmichael et al. (1997), Hall et al. (2012), Maronpot et al. (2010)

Thyroid

Embryonal rests

Rat, mouse

Various

Bra¨ndli-Baiocco et al. (2018)

Liver

Extramedullary hematopoiesis

Rat, mouse

Various

Thoolen et al. (2010).

Liver

Altered hepatic foci

Rat

Various

Mahon (1989)

Larynx

Squamous metaplasia

Rat

Inhalation

Kaufmann et al. (2009)

Kidney

Chronic progressive nephropathy

Rat

Various

Hard et al. (2013)

Heart

Progressive cardiomyopathy

Rat

Various

Chanut et al. (2013)

Eye

Retinal degeneration

Rat

Various

Yamashita et al. (2016)

Lung

Phospholipidosis

Rat

Various

Nonoyama and Fukuda (2008), Sawada et al. (2005)

Lung

Histiocytosis

Rat

Inhalation

Nikula et al. (2014)

Kidney

Tubular mineralization

Rat

Oral

Frazier et al. (2012)

Kidney

Tubular karyomegaly

Oral

Boorman et al. (1992), Frazier et al. (2012)

Lymph node

Pigment laden macrophages in sinuses

Oral gavage/oil feeding studies

Firriolo et al. (1995), Elmore (2006), Smith et al. (1996), WillardMack et al. (2019)

Rat, mouse

adipocyte infiltration of the capsule and parenchyma, increased prominence and/or hyperplasia of medullary epithelial cells, and formation of follicular-like B-cell aggregates in the medulla (Pearce, 2006). As the incidence of thymic involution increases with the age of the animal, it is more difficult to distinguish agerelated involution from a chemically induced

thymic atrophy in longer-duration studies. Nonetheless, the normal rules relating a finding to treatment apply including clear doserelationships for both the incidence and severity of the effect. Additionally, many toxicity protocols also incorporate a recovery phase that can clarify any apparent treatment-related thymic change present on the dosing phase of the study.

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studies (Kobayashi et al., 1994; Sato et al., 2012). The situation is further confounded by the small numbers of animals per group in beagle dog (and cynomolgus monkey) toxicity studies. In such cases, a single instance of “thymic atrophy” in dosed groups can be problematic in determining if the change is treatment-related, let alone whether the thymic change is adverse or not. Stress during toxicity studies is a major factor in producing secondary thymic atrophy. Both chronic elevated endogenous, and therapeutically administered exogenous, glucocorticoids are known to cause thymic atrophy. Having excluded a glucocorticoid agonistic activity of the xenobiotic, thymic atrophy occurring in the high-dose group, in the absence of a doserelationship is highly likely to be a secondary product of stress (i.e., endogenous glucocorticoid production) rather than a primary, chemically induced toxicity (Everds et al., 2013). Recovery of a more normal thymic morphology following a period of abstinence from chemical treatment is good evidence that the atrophy was indeed chemically induced rather than an age-related involution, which would fail to show recovery (Schuurman et al., 1991). FIGURE 15.12 (A) Thymus from a 6-month-old male, control beagle dog showing the normal anatomy of cortex and medulla. (B) Thymus from a 6-month-old control male beagle dog showing marked involution. The triangular organ on the top right hand corner is the spleen from the same dog. Both slides are from hematoxylin and eosin stained sections. 0.3 magnification.

As such, longer-term studies (6 or more months) may become less sensitive in detecting a chemically induced thymic toxicity than do shorterterm studies (e.g., 28- and 90-days). Degrees of change in the thymus outside of those normally associated with age-related involution for the specific age of laboratory animal may be adverse based on the severity and incidence of the change and evidence for immune system dysfunction (i.e., lymphopenia). Another potential confounder is the laboratory animal species. For instance, the incidence of thymic atrophy in young beagle dogs tends to be higher than that present in laboratory rodents of equivalent developmental age and is commonly diagnosed in control dogs in 28-day

6.3. Liver Weight Changes There are three common measures of organ weight used during toxicity studies: the absolute organ weight, the organ weight relative to brain weight, and the organ weight relative to body weight (Nirogi et al., 2014; Bailey et al., 2004). Since the size and weight of the liver is correlated closely with the body weight (Figure 15.13), the inclusion of the relative weights is intended to account for any significant changes in body weight, the most common one being a loss due to changes in food consumption or toxicity of a test article that might inhibit normal feeding or nutrient uptake. Under these circumstances, the liver weight would also decrease in relation to the loss of body weight, typically exhibiting a dosedependent decline with the greatest loss occurring at the highest dose tested. Figure 15.14B shows the histological appearance of the liver from an animal with low liver and body weights where the hepatocytes show a decreased size, with less cytoplasm and a more dense packing

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FIGURE 15.13 Relationship between the absolute liver weight, absolute brain weight, and the body weight in the rat in a study where the animals in the top dose group 4 showed a significant decrease in final body weight when compared to the concurrent controls. In comparison to the liver, the brain weight failed to show any effect of the decreased body weight and was maintained at levels similar to those in the control group.

of cells in comparison to a control rat liver (Figure 15.14A). These images were taken from a feeding study in Wistar rats where the compound proved only partially palatable, and where dosing resulted in a significant loss of body weight. In contrast to a loss of liver weight, an increase in liver weight, independent of an effect on body weight, is a common response to exposure to xenobiotics and is frequently accompanied by a histopathological diagnosis of hepatocellular hypertrophy (Hall et al., 2012; Maronpot et al., 2010). On some occasions, the increase in liver weight occurs in the absence of histopathological findings, but a histological diagnosis of hepatocellular hypertrophy and sometimes hyperplasia is the most common change accompanying an increase in absolute and relative liver weight (Figure 15.15) (see Liver and Gallbladder, Vol 4, Chap 2). A xenobiotic-induced increase in liver weight can be due to several different causes including the accumulation of glycogen, water, fat, and organelles (e.g., smooth endoplasmic reticulum) associated with the induction of drug metabolizing enzymes (Maronpot et al., 2010). Table 15.2 below summarizes the microscopic changes associated with increased liver weight together with xenobiotics that induce the respective changes.

In addition to hepatic enzyme induction inside cells, clinical chemistry changes often accompany the liver weight increase. Common alterations are increases in the plasma activities of hepatocyte-derived enzymes, especially alanine aminotransferase (ALT), alkaline phosphatase (ALP), and gamma-glutamyltransferase (GGT) (Ramaiah et al., 2017). Histopathologically, liver weight increases due to induction of drug-metabolizing enzymes or peroxisomes may be accompanied by distinct tinctorial and structural changes in the cytoplasm. It is common to find references to “ground glass change”, “increased eosinophilia”, and/or “increased granularity” present within the hypertrophic hepatocytes that correspond to the accumulation of smooth endoplasmic reticulum or peroxisomes that occurs within the hepatocyte cytoplasm (Figure 15.16). These changes frequently show a zonal preference, with the centrilobular or zone 3 hepatocytes being most commonly affected (Maronpot et al., 2010). Liver weight increases due to the intracellular accumulation of glycogen will appear as increased clear cytoplasmic vacuolation in hepatocytes (resulting from glycogen extraction during processing and sectioning of the liver) (Figure 15.16). Cell nuclei remain centered in cells (i.e., are surrounded by glycogen) The loss

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FIGURE 15.15 Rat liver from a rat given sodium phenobarbitone for 14 days showing zonal hypertrophy of the hepatocytes. The cells in the bottom left hand corner can be seen to be considerably smaller than those in the top right hand corner which are hypertrophic due to proliferation of smooth endoplasmic reticulum in their cytoplasm and induction of drug metabolizing enzymes. Hematoxylin and eosin staining. 20 microscope magnification.

FIGURE 15.14 (A) Liver from a control rat. (B) Atrophic liver from a rat in the high dose group showing smaller and more densely packed hepatocytes, with reduced cytoplasmic volume. Hematoxylin and eosin– stained sections. Both sections taken at the same magnification as shown by the size bars in the bottom right hand corner. Photographs courtesy of P. Maslej, CRL, Hungary.

of glycogen can be obviated with special fixatives or by sectioning the liver at increased thicknesses of around 10 mm (compared to the usual 4–5 mm), such that the glycogen is prevented from dropping out of the section during suspension/expansion of the sections on the water bath prior to mounting on the glass slide (Smitherman et al., 1972). Stains such as periodic acid– Schiff (PAS) can be used to reveal the glycogen inside cells. Liver weight increases due to accumulation of lipid will appear as clear, unstained, round

vacuoles within the hepatocyte cytoplasm in routinely processed tissue since the lipid will have been extracted during processing (Figure 15.17). Cell nuclei may be displaced peripherally in cells with very large lipid droplets. Since the lipid is unaffected by fixation, confirmation of any vacuolation as being composed of lipid can be achieved through retrieval of fixed liver samples, cryo-sectioning at around 10 mm thickness, and staining with lipophilic histochemical stains such as Oil red O or Sudan Black (Pearse, 1961). Regulatory Opinion on Adversity and Liver Weight Increases There has been an ongoing debate regarding liver weight increases and what criteria determine when this finding becomes adverse and when it is merely adaptive. As the dose level increases, the capacity of the original compliment of hepatocytes to metabolize the xenobiotic is exceeded and the liver adapts by increasing the quantity of metabolizing enzymes in each hepatocyte (hypertrophy) and later increasing the numbers of hepatocytes (hyperplasia). If not excessive, both the hypertrophic and

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TABLE 15.2 List of Xenobiotics Associated With Liver Weight Increases and Their Effect on the Liver. Hepatocyte Change

Chemical

Literature Reference

Hydropic degeneration/fat accumulation

Carbon tetrachloride, bromobenzene

Uemitsu et al. (1986), Uemitsu and Nakayoshi (1984), Dodd et al., (2013)

Glycogen accumulation

Dexamethazone, prednisolone

Roussel et al. (2003), Onyesli (1986), Hall et al. (2012), Maronpot et al. (2010)

Fat accumulation

High fat diet, ethanol

Karac¸or et al. (2014), Meli et al., (2013), Habib-ur-Rehman et al. (2011)

Proliferation of endoplasmic reticulum

Sodium phenobarbitone, pregnenolone 16alphacarbonitrile

Cattley and Popp (1989), Hall et al. (2012), Kourounakis et al. (1976), Maronpot et al. (2010)

Proliferation of peroxisomes

Diethylhexylphthalate, clofibrate, nafenopin

Price et al. (1986), Parmar et al. (1988), Rowdhwal and Chen (2018), Isenberg et al. (2001), Mackerer (1977)

Most of these changes will show dose–response relationships for their severities, with threshold dose levels above which the mere presence of these changes indicates an adverse effect on the liver.

hyperplastic responses can be considered adaptive and not adverse as they will not compromise the function of the liver, but rather optimize it in the face of a persistent challenge (Hall et al., 2012; Maronpot et al., 2010). However, evidence

FIGURE 15.16 Liver from a dog given a high dose of the glucocorticoid steroid, prednisolone, for 6 months. There is severe clear vacuolation of the cytoplasm of the hepatocytes which was confirmed as glycogen by periodic acid-Schiff staining. This change has been described as “steroid induced hepatopathy”, and dogs appear to be especially sensitive to showing the change. Hematoxylin and eosin–stained section. 10 microscope magnification.

with classic inducers of drug metabolism, such as sodium phenobarbitone (phenobarbital), have shown that higher doses result in exuberant hepatocyte hypertrophy and

FIGURE 15.17 Liver from a rat given a high dose of perchloroethylene by oral gavage for 28 days. The liver has accumulated clear, round vacuoles following processing into paraffin wax. The appearance of the droplets is typical of neutral lipid which can be confirmed by cutting thick, frozen sections of formalin-fixed liver and staining with Oil Red O or Sudan Black, without processing into wax, which extracts the lipid during the solvent stage of processing. Hematoxylin and eosin– stained section. 20 microscope magnification.

III. DATA INTERPRETATION AND COMMUNICATION

6. CASE EXAMPLES OF ASSESSING ADVERSITY

hyperplasia, which over time may incite potentially adverse histopathologic changes including cell degeneration and necrosis, fatty change, and an inflammatory response normally consisting of a mixed mononuclear cells but also including a neutrophilic cell migration into sites of necrosis (Maronpot et al., 2010). These phenotypic changes can be seen in studies of 28 days and longer and reflect saturation of normal hepatic clearance mechanisms, the accumulation of the parent xenobiotic or active metabolites to toxic levels within the hepatocytes, or incorporation of harmful metabolic routes (Maronpot et al., 2010); local ischemia due to sinusoidal constriction secondary to hepatocyte hypertrophy serves as an additional contributing factor. The key question with respect to adversity decisions for initially adaptive changes is what to decide when liver weight increases occur in the absence of accompanying histopathology? Is any liver weight increase that is not accompanied by histologic or clinical pathology alterations, irrespective of its degree, nonadverse? The answer depends on the context. For an individual study, adversity decisions should be based on the findings observed for that test species, test article, dose, and treatment duration (Kerlin et al., 2016; Palazzi et al., 2016). However, in the context of an entire development program, adversity decisions may be based on the cumulative results of multiple studies using a weight-of-evidence approach. Evidence suggests that liver weight increases of between 40% and 50% greater than concurrent controls in 28- and 90-days toxicity studies in rodents are predictive of increased incidences of liver cancer in longer-duration rodent studies (Carmichael et al., 1997; Haseman, 1995; Maronpot et al., 2010). In contrast, evidence exists that dose levels of xenobiotics in rodents that lead to liver weight increases of