Drug Delivery Aspects: Expectations and Realities of Multifunctional Drug Delivery Systems: Volume 4: Expectations and Realities of Multifunctional Drug Delivery Systems [1 ed.] 0128212225, 9780128212226

Table of contents :
Cover
DRUG DELIVERY ASPECTS:
EXPECTATIONS AND REALITIES OF
MULTIFUNCTIONAL DRUG
DELIVERY SYSTEMS
Copyright
Contributors
Preface
1
Versatile hyaluronic acid nanoparticles for improved drug delivery
Introduction
Hyaluronic acid
Chemistry
Sources
Physiological role
Turnover and elimination pathways
Preparation of hyaluronic acid nanoparticles
Conjugate formation
Self-assemblies formation
Ionic nanocomplexes formation
Nanogels formation
Applications of HA nanoparticles
Skin applications
Osteoarthritis
Tissue engineering
Cancer targeting
Atherosclerosis
Ocular drug delivery
Insulin sensitivity and diabetes
Theranostic and imaging
Clinical status
Conclusion
References
2
Preclinical testing-Understanding the basics first
Introduction
Preclinical studies
Regulatory aspects of preclinical studies
Preclinical testing and models used
In vitro assays/cell line assays
Animal models and type of tests
In vivo evaluation
Pharmacodynamics
Pharmacokinetic
Toxicology
Advances in the field
Databases
Conclusion
References
3
Aqueous polymeric coatings: New opportunities in drug delivery systems
Introduction
Coating types and polymers used
Aqueous-based coatings for sustained drug release (cellulosic type)
Ethylcellulose
Aqueous-based coatings for enteric drug release
Cellulose acetate phthalate
Hydroxypropyl methylcellulose acetate
Acrylate-type aqueous-based coatings
Ammonio methacrylate copolymers
Ethyacrylate methylmethacrylate
Methacrylic acid copolymers for enteric drug release
Aqueous-based flexible polymer coatings for sustained drug release
Polyvinyl acetate
Strategies to improve storage stability
Optimization of curing conditions
Plasticizer type and content
Addition of high Tg polymers
Addition of hydrophilic excipients
High solids content
Solidification of self-nanoemulsifying drug delivery systems by fluid bed coating
Conclusion
References
4
Large-scale manufacturing of nanoparticles-An industrial outlook
Introduction
Nanosuspensions
Top-down approach
Bottom-up approach
Considerations on scale-up from lab to industrial scale
Large-scale manufacturing unit operation
Lipid nanoparticles
Nanoparticle characterization and analytical techniques used
Other characterizations
Conclusion
References
5
The role of polymers and excipients in developing amorphous solid dispersions: An industrial perspective
Introduction
ASD versus other solubilization techniques
ASD relevance to different pharmaceutical businesses
Considerations in developing ASDs
Thermodynamic and kinetic considerations
Feasibility considerations
Critical functionality attributes of polymers and excipients with respect to the manufacturing process
Manufacturing process considerations
Spray drying
Hot-melt extrusion
Melt rheology considerations
Applications
Future trends and technology landscape
Regulatory considerations
Marketed ASD products
Conclusion
References
Further reading
6
Biologics: Delivery options and formulation strategies
Introduction
Intravenous infusion of biopharmaceuticals
Subcutaneous injection of biopharmaceuticals
Targeted localized delivery of biologics
Brain targeting
Pulmonary delivery
Transdermal delivery
Ocular delivery
Oral delivery
Formulation strategies, degradation routes, and role of excipients
Surfactants
Buffers
Lyoprotectors (sugar)/bulking agents
Salts
Antimicrobial preservatives
Additional formulation considerations
Adjuvants in vaccine formulation
Stability of biologics, typical shelf life and storage considerations
Toxicity and immunogenicity of biologics
Conclusion
References
7
Ethical issues in research and development of nanoparticles
Introduction
Safety of nanoparticles and production methods
Applications and handling of nanoparticles
Impact of nanotechnology on society, environment, and health
Ethical issues and socioeconomic concerns
Conclusions
Acknowledgments
References
8
Sterilization of pharmaceutical dosage forms
Introduction
Challenges in sterilization
Dosage forms and sterilization methods
Compounding and manufacturing of sterile dosage forms
Challenges in manufacturing
Aspects on compounding area
Validation of aseptic processing
Types of sterilization methods
Dry heat sterilization
Moist heat sterilization
Radiation sterilization
Filtration sterilization
Chemical agents
Other techniques
Lyophilization
Quality by design
Process analytical technology
End storage, handling of drugs and drug products
Conclusion
References
Further reading
9
Vaccine delivery strategies against botulism
Introduction
Vaccines
Toxoid and recombinant protein vaccines
DNA vaccines against BoNT
Viral vector vaccines against BoNT
Inactivated rabies virus vector
Influenza A virus vector
Adenovirus-based vectors
VEE vectors
Lactic acid bacterial (LAB) vectors
Botulism antitoxin
Clinical trials of therapeutic and prophylactic vaccine candidates
Conclusion
References
10
Nanotechnological approaches for delivery of antiinflammatory drugs
Introduction
Potential of nanocarriers for delivery of antiinflammatory drugs
Polymeric nanoparticles
Nanoemulsions
Nanosuspensions
Conclusion
References
Further reading
11
Food to medicine transformation of stilbenoid vesicular and lipid-based nanocarriers: Technological advances
Introduction
Stilbenoids
Delivery challenges
Nanocarriers for delivery
Vesicular nanocarriers
Liposomes
Niosomes
Penetration enhancer vesicles (PEVs)
Ethosomes and transfersomes
Lipid-based carriers
Solid lipid nanoparticles (SLNs) and nanostructured lipid carriers (NLCs)
Nanoemulsions and microemulsions
Nanocapsules
Other nanocarriers
Conclusions
References
Index
A
B
C
D
E
F
G
H
I
K
L
M
N
O
P
Q
R
S
T
U
V
W
Z

Citation preview

DRUG DELIVERY ASPECTS

DRUG DELIVERY ASPECTS EXPECTATIONS AND REALITIES OF MULTIFUNCTIONAL DRUG DELIVERY SYSTEMS VOLUME 4 Edited by

RANJITA SHEGOKAR, PHD Capnomed GmbH, Zimmern, Germany

Elsevier Radarweg 29, PO Box 211, 1000 AE Amsterdam, Netherlands The Boulevard, Langford Lane, Kidlington, Oxford OX5 1GB, United Kingdom 50 Hampshire Street, 5th Floor, Cambridge, MA 02139, United States © 2020 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. Library of Congress Cataloging-in-Publication Data A catalog record for this book is available from the Library of Congress British Library Cataloguing-in-Publication Data A catalogue record for this book is available from the British Library ISBN: 978-0-12-821222-6 For information on all Elsevier publications visit our website at https://www.elsevier.com/books-and-journals

Publisher: Andre Gerhard Wolff Acquisitions Editor: Erin Hill-Parks Editorial Project Manager: Pat Gonzalez Production Project Manager: Kiruthika Govindaraju Cover Designer: Mark Rogers Typeset by SPi Global, India

Contributors

Hend Abd-Allah Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt

Riham I. El-Gogary Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt

Sara M. Abdel Samie Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt

Muhammad Irfan Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, GC University Faisalabad, Faisalabad, Pakistan

Mona M.A. Abdel-Mottaleb Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt; PEPITE EA4267, Univ. Bourgogne Franche-Comt
e, Besanc¸on, France

Nirmal Jayabalan Department of Pharmacy, Birla Institute of Technology and Science, Pilani, Hyderabad, India

Abid Riaz Ahmed Merck Darmstadt, Germany

Healthcare

Anđelka B. Kova cevi
c Department of Pharmaceutical Technology, Faculty of Biological Sciences, Institute of Pharmacy, Friedrich-Schiller University Jena, Jena, Germany

KGaA,

Dharmesh Mehta Business Development, Gangwal Chemicals, Mumbai, India Joana Portugal Mota Lecifarma—Laborato´rio Farmac^eutico, Lda, Va´rzea do Andrade—Cabec¸o de Montachique, Lousa; CBIOS-Research Center for Biosciences and Health Technologies, Luso´fona University, Lisbon, Portugal

Akash Chavrasiya Department of Pharmacy, Birla Institute of Technology and Science, Pilani, Hyderabad, India Yanping Chen Center of Emphasis in Infectious Diseases, Department of Molecular and Translational Medicine, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, TX, United States

Mostafa Nakach France

Joa˜o Dias-Ferreira Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal

Sanofi R&D, Vitry sur Seine,

Maha Nasr Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt

Diana Diaz-Ar
evalo Immunology Functional Group, Foundation Institute of Immunology of Colombia-FIDIC, School of Medicine and Health Sciences, Universidad del Rosario, Bogota, D.C., Colombia

Ridahunlang Nongkhlaw Department of Pharmacy, Birla Institute of Technology and Science, Pilani, Hyderabad, India Parameswar Patra Department of Pharmacy, Birla Institute of Technology and Science, Pilani, Hyderabad, India

Sachin Dubey Formulation, Analytical and Drug Product Development, Glenmark Pharmaceuticals, La Chaux de Fonds, Switzerland

Antonello Santini Department of Pharmacy, University of Napoli “Federico II”, Napoli, Italy

Alessandra Durazzo CREA-Research Centre for Food and Nutrition, Rome, Italy

Ahmad Abdul-Wahhab Shahba Kayyali Chair for Pharmaceutical Industries, Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia

Thomas D€ urig R&D and Innovation, Ashland Pharma and Health & Wellness, Ashland Specialty Ingredients G.P., Wilmington, DE, United States

vii

viii

Contributors

Ranjita Shegokar Capnomed GmbH, Zimmern, Germany Vaibhav Sihorkar Formulations, NCE and Innovation, Sai Life Sciences Limited, ICICI Knowledge Park, Genome Valley, Hyderabad, Telangana, India Sarabjit Singh Formulation Mumbai, India

Research,

CIPLA,

Eliana B. Souto Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Coimbra; CEB—Centre of Biological Engineering, University of Minho, Gualtar Campus, Braga, Portugal Haiyan Wang Key Laboratory of Oral Medicine, Guangzhou Institute of Oral Disease, Stomatology Hospital of Guangzhou Medical University, Guangzhou, Guangdong, People’s Republic of China; Center of Emphasis in Infectious Diseases,

Department of Molecular and Translational Medicine, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, TX, United States Yongyong Yan Key Laboratory of Oral Medicine, Guangzhou Institute of Oral Disease, Stomatology Hospital of Guangzhou Medical University, Guangzhou, Guangdong, People’s Republic of China; Center of Emphasis in Infectious Diseases, Department of Molecular and Translational Medicine, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, TX, United States Mingtao Zeng Center of Emphasis in Infectious Diseases, Department of Molecular and Translational Medicine, Paul L. Foster School of Medicine, Texas Tech University Health Sciences Center El Paso, El Paso, TX, United States

Preface

3. facilitate insight sharing within various areas of expertise; and 4. establish collaborations between academic scientists, and industrial and clinical researchers.

The book series titled Expectations and Realities of Multifunctional Drug Delivery Systems covers several important topics on drug-delivery systems, regulatory requirements, clinical studies, intellectual properties trends, new advances, manufacturing challenges, etc., written by leading industry and academic experts. Overall, the chapters published in this series reflect the broadness of nanopharmaceuticals, microparticles, other drug carriers, and the importance of the respective quality, regulatory, clinical, GMP scale-up, and regulatory registration aspects. This series is destined to fill the knowledge gap through information sharing and with organized research compilation between diverse areas of pharma, medicine, clinical, regulatory practices, and academics. Expectations and Realities of Multifunctional Drug Delivery Systems is divided into four volumes: Volume Volume Volume Volume

1: 2: 3: 4:

Innovative cutting-edge developments in micro–nanotechnology offer new ways of preventing and treating diseases like cancer, malaria, HIV/AIDS, tuberculosis, and many more. The applications of micro–nanoparticles in drug delivery, diagnostics, and imaging are vast. Hence, Volume 4: Drug Delivery Aspects in the book series mainly reviews advances in drug delivery areas via targeted therapy with improved drug efficiency at a lower dose, transportation of the drug across physiological barriers, as well as reduced drug-related toxicity. The focus of this volume is on GMP scale-up, regulatory, functional, and preclinical aspects of drug delivery systems. One of the contributions by Abdel-Mottaleb et al. (Chapter 1) discusses new trends in drug delivery area using hyaluronic acid, as an ingredient as well as an active. The pharma industry is always in search of new and functional ingredients. Hyaluronic represents one such example, and is currently being explored as a drug delivery agent for a wide range of routes like nasal, pulmonary, ophthalmic, topical, and parenteral. The authors review its potential in wound healing, osteoarthritis, tissue engineering, cancer targeting, atherosclerosis, diabetes treatment, theranostic, imaging applications, and so on.

Nanopharmaceuticals Delivery of Drugs Drug Delivery Trends Drug Delivery Aspects

The specific objectives of this book series are to: 1. provide a platform to discuss opportunities and challenges in development of nanomedicine and other drug-delivery systems; 2. discuss current and future market trends;

ix

x

Preface

Chapter 2, by Shegokar, highlights basic understandings on preclinical testing. This topic has recently been seriously picked up by industry and regulatory bodies. However, it is not easy to get a clear view on what types of preclinical strategy one must select for specific drug delivery types. Multiple CROs (contract research organizations) exist to support industry/academics on this, but it is a timeconsuming and costly discussion. Therefore, the focus of this chapter is to give readers a basic understanding of preclinical phase and desired testings, guidelines available, and an overview of regulatory requirements, and upcoming trends in the field. The contribution by Ahmed et al. in Chapter 3 describes the importance of aqueous film coatings. The main aim of this chapter is to provide an overview on the curing (postthermal treatment) and storage stability of aqueous coated dosage forms. In this chapter, a novel approach of aqueous coating—solidification of Self-NanoEmulsifying Drug Delivery Systems (SNEDDS)—is discussed, which can significantly enhance the solubility and stability of poorly soluble drugs. A chapter by Shegokar and Nakach (Chapter 4) reviews industrial scale-up aspects of the most employed technique in nanoparticle production, milling. Very few reports are published in literature; however, they describe the scale-up of one particular drug delivery system. The focus of this chapter is to give readers an indepth understanding of the milling technique for production of nanoparticles, GMP settings, regulations associated with it, and an overview of industrial challenges. The compilation by Sihorkar and D€ urig in Chapter 5 aims at discussing amorphous solid dispersion as a formulation enabling technology to a commercially viable technology with a plethora of marketed products across the global pharmaceutical space. The authors discuss the industrial perspective of ASD technology, which has benefited immensely from newer-

generation excipients, especially polymers and processing techniques like HME. This chapter provides readers an in-depth understanding of key parameters in formulating stable solid amorphous dispersions from an industrial point of view. Chapter 6 by Nongkhlaw et al. highlights opportunities and challenges in formulating biopharmaceuticals. It is another trend spotted in pharmaceutical drug delivery in addition to nanotechnology. The authors describe in detail technological potential, industrial advantages, technologies available, and limitations of the same for local (targeting brain, pulmonary, ocular, oral, etc.) and intravenous drug administration of biopharmaceuticals. At the end of the chapter, an overview of formulation strategies and ingredient choice is provided. Nanotechnology is good, but to what extent? It is slowly posing health hazards not only though automobiles but also though food, medicine, cosmetics, and detergents. The topic presented by Souto et al., in Chapter 7 describes the regulatory and ethical issues in nanoparticles, materials, and particles (NMP) research. The chapter further outlines the present and future of nanotechnology, and its applications along the axis of social and ethical concerns. Chapter 8 by Singh and Mehta reviews sterilization opportunities and challenges for drug delivery systems. The team of authors highlights key points like the role of formulations, physical forms, choice of sterilization technique, and regulatory requirements. An industrial perspective is given on this topic by discussing common mistakes and ways to overcome these. Botulism is a paralytic disease caused by intoxication with neurotoxins produced by Clostridium botulinum. Currently, vaccines and antibodies are the only two primary means of treating botulism. The work by Yan et al. (Chapter 9) highlights the vaccine development strategies for botulism. This chapter provides an overview of new vaccine and immunotherapeutic developments like vaccine

Preface

vector updates, immune sequence optimization, and recombinant antibodies. Chapter 10 by Kovacevic discusses challenges in the delivery of nonsteroidal antiinflammatory drugs (NSAIDs), and highlights the recent advances on the use of polymeric nanoparticles, nanoemulsions, and nanosuspensions/nanocrystals, intended for oral, topical, parenteral, and ocular administration to overcome the associated challenges. These new strategies can increase drug-associated poor aqueous solubility and thereby reduce the dose-associated adverse effects. An overview of various nanotechnological approaches for delivery of antiinflammatory drugs is provided in this chapter. Nowadays, patients and consumers demand safer and more natural products for regular infections. This resulted in the evolution of the nutraceutical market. However, many

xi

nutraceuticals have limitations like high dose, poor bioavailability, and toxicity and stability problems. In maximizing their therapeutic potential to their full extent, nanotechnology plays a key role. The last contribution by Abdel Samie and Nasr (Chapter 11) describes food to medicine transformation of stilbenoid via vesicular and lipid-based nanocarriers. In summary, I am sure this book volume and the complete book series will provide you great insights in areas of micro-nanomedicines, drug delivery sciences, new trends, and regulatory aspects. All the efforts of experts, scientists, and authors are highly acknowledged for sharing their knowledge, ideas, and insights about the topic. Ranjita Shegokar, PhD Editor

C H A P T E R

1

Versatile hyaluronic acid nanoparticles for improved drug delivery Mona M.A. Abdel-Mottaleba,b, Hend Abd-Allaha, Riham I. El-Gogarya, Maha Nasra a

Department of Pharmaceutics and Industrial Pharmacy, Faculty of Pharmacy, Ain Shams University, Cairo, Egypt b PEPITE EA4267, Univ. Bourgogne Franche-Comt
e, Besanc¸ on, France

1 Introduction

several thousand repeating disaccharides molecules in the backbone. The molecular weight of HA molecules differs owing to the variable number of these repeating disaccharide units in each molecule, its molecular weight ranges from 1 to 10,000 kDa [3]. HA has an unusual mechanism of biosynthesis and exceptional physical properties. Sodium hyaluronate is the predominant form of HA at physiological pH. Sodium hyaluronate and HA are collectively referred to as hyaluronan. Due to the fact that HA exists as a polyanion, it can self-associate and can also bind water molecules giving it a stiff, viscous quality with jelly-like consistency that causes it to behave like a lubricant [2]. HA was found to be abundantly distributed in cellular surfaces, in the basic extracellular substances of the connective tissues of vertebrates, in the synovial fluid of joints, in the vitreous humor of the eye, and in the tissue of the umbilical cord; all this attracted significant attention regarding its medical applications

Although more than 80 years have passed since the discovery of hyaluronic acid (HA), it still surprises researchers with its unique physicochemical properties and physiological roles in the human body. HA was first discovered by Karl Meyer and John Palmer in 1954 [1]. They isolated an unknown material from the vitreous bodyof a bovine eye, containing two sugar molecules including “uronic acid.” So, by connecting the substitute name for the vitreous—“hyaloid”—with the name of a component of that polysaccharide—“uronic acid”—the name of HA was adopted for this material. HA was first used commercially as a substitute for egg white in bakery products. Later on, its first medical application for humans was initiated as a vitreous replacement during eye surgery in the late 1950s [2]. HA belongs to a group of substances called mucopolysaccharides belonging to the glycosaminoglycans (GAGs) family [1, 2]. HA includes

Drug Delivery Aspects https://doi.org/10.1016/B978-0-12-821222-6.00001-4

1

# 2020 Elsevier Inc. All rights reserved.

2

1. Versatile hyaluronic acid nanoparticles for improved drug delivery

[4]. Although HA has a very simple structure, almost everything else concerning the molecule is unusual. Sometimes its role is mechanical and structural, such as in synovial fluid, the vitreous humor, or the umbilical cord. In other cases, it can interact in low concentrations with cells to trigger important cellular responses. HA’s characteristics, including its consistency, biocompatibility, and hydrophilicity, have made it an excellent moisturizer in cosmetic dermatology and skin-care products. Moreover, its unique viscoelasticity and limited immunogenicity have led it to be used for viscosupplementation in osteoarthritis treatment, as a surgical aid in ophthalmology, and for surgical wound regeneration in dermatology. In addition, HA has currently been explored as a drug delivery agent for different routes such as nasal, pulmonary, ophthalmic, topical, and parenteral [2]. Hence the use of nanotechnology would combine the outstanding properties of HA being biocompatible, biodegradable, nontoxic, and able to bind specific receptors with the different advantages of nanoparticles such as enhanced therapeutic effects and targetability. In this chapter, the use of HA nanoparticles as a versatile drug delivery system will be discussed by highlighting the different production techniques based on the chemical and biological properties of HA.

2 Hyaluronic acid 2.1 Chemistry The exact chemical structure of HA was determined by Weissman and Meyer in 1954. As already noted, HA belongs to a group of substances called mucopolysaccharides belonging to the GAGs family. It is an unbranched nonsulfated GAG composed of repeating disaccharides [β-1,4-D-glucuronic acid (known as uronic acid) and β-1,3-N-acetyl-D-glucosamide], as shown in Fig. 1.1. Both sugars are spatially related to glucose in the beta configuration, thus allowing

CH2OH

COO– H

H

O O

OH H H H H

OH

Glucoronic acid

O

O H

OH H

H H N HCOCH3

N-acetyl glucosamine

FIG. 1.1 Chemical structure of hyaluronic acid unit.

all its bulky groups (the hydroxyls, the carboxylate moiety and the anomeric carbon on the adjacent sugar) to be in sterically favorable equatorial positions, while all the small hydrogen atoms occupy the less sterically favorable axial positions. Thus, the structure of the disaccharide is energetically very stable [3]. The HA backbone is stiffened in physiological solution via a combination of internal hydrogen bonds, interactions with solvents, and the chemical structure of the disaccharide. HA molecular investigations suggested that the axial hydrogen atoms form a nonpolar face (relatively hydrophobic) and the equatorial side chains form a more polar face (hydrophilic) which leads to a twisted ribbon structure for HA called a coiled structure [4]. Owing to this conformational behavior as well as its high molecular weight, the solutions of HA are very viscous and elastic. At very low concentrations, chains entangle with each other, leading to a mild viscosity (molecular weight dependent). However, HA solutions at higher concentrations have a higher than expected viscosity due to greater HA chain entanglement that is shear-dependent. For instance, a 1% solution of high molecular weight HA can behave like jelly, but when shear stress is applied, it will easily shear thinly and can be administered via a fine needle [2]. HA is therefore a “pseudo-plastic” material. This rheological property (concentration and molecular weight dependent) of HA solutions has made

2 Hyaluronic acid

it ideal as a lubricant in biomedical applications. There is evidence that hyaluronan separates most tissue surfaces that slide along each other. The extremely lubricious properties of hyaluronan, meanwhile, have been shown to reduce postoperative adhesion formation following abdominal and orthopedic surgery. HA has several interesting medical, pharmaceutical, food, and cosmetic uses in its naturally occurring linear form. However, chemical modifications of the HA structure represent a strategy to extend the possible applications of the polymer, obtaining better performing products that can satisfy specific demands and can be characterized by a longer half-life. During the design of novel synthetic derivatives, particular attention is paid to avoid the loss of native HA properties such as biocompatibility, biodegradability, and mucoadhesivity [5]. HA can be chemically modified by crosslinking or conjugation reactions. These chemical modifications mainly involve two functional sites of the biopolymer: the hydroxyl (probably the primary alcoholic function of the N-acetyl-D-glucosamine) and the carboxyl groups [6]. Furthermore, synthetic modifications can be performed after the deacetylation of HAN-acetyl groups [7]. Conjugation reactions usually consist of adding a monofunctional molecule onto one HA chain by a single covalent bond, while crosslinking employs polyfunctional compounds to link together different chains of native or conjugated HA by two or more covalent bonds. Crosslinked hyaluronan can be prepared from native HA (direct crosslinking) or from its conjugates. Crosslinking is normally intended to improve the mechanical, rheological, and swelling properties of HA and to reduce its degradation rate, to develop derivatives with a longer residence time in the site of application and controlled release properties [5]. Conjugation of drugs to HA was reported as early as 1991. This approach aimed to form a prodrug by covalently binding a drug to the HA backbone through a bond that ideally should

3

be stable during the blood circulation and promptly cleaved at a specific target site [8]. Owing to HA solubility, it is possible to perform the reaction in water. However, in the aqueous phase, some reactions are pH-dependent and need to be performed in acidic or alkaline conditions, which have been shown to induce significant HA chain hydrolysis [8, 9].

2.2 Sources HA is a natural polymer biologically synthesized by cells in the body by an enzymatic process. HA production is a unique, highly controlled, and continuous process. Approximately half of our body’s HA is distributed in the cutaneous region. It is produced and secreted by cells including fibroblasts, keratinocytes, or chondrocytes with varying molecular weights between 50 and 3000 kDa. The Golgi network is the production site for most GAGs. In tissues such as skin and cartilage where HA comprises a large portion of the tissue mass, HA is synthesized in large amounts. It is naturally synthesized by hyaluronan synthases (HAS1, HAS2, and HAS3), a class of integral membrane proteins [10]. The three enzymes are located on different chromosomes, producing HA with different molecular weights. HAS1 and HAS2 proteins are responsible for the synthesis of high molecular weight HA (2  106 Da) with the latter more active catalytically than the former, whereas the enzyme HAS3 is the most active but can only synthesize short HA chains from 200,000 to 300,000 Da. The different molecular weights of HA chains can lead to different effects on cell behavior. HA performs its biological actions according to two basic mechanisms: it can act as a passive structural molecule and as a signaling molecule. Both mechanisms of action have been shown to be size-dependent [11]. As mentioned above, HA has an essential functional component of almost all tissues in the vertebrate organism. Thus, various animal

4

1. Versatile hyaluronic acid nanoparticles for improved drug delivery

tissues, for example, in rooster combs, shark skin, and bovine eyes have been used as sources of isolation and production of high molecular weight HA. Since HA in biological materials is usually present in a complex linked to other biopolymers, several separation procedures must be applied to obtain a pure compound, such as protease digestion [10]. HA was initially isolated from bovine vitreous humor and later from rooster combs and human umbilical cords [11]. The mean molecular weight of the commercially available “extractive” HA preparations obtained from animal tissues is mostly in the range from several hundred thousand Da up to approximately 2.5 MDa [12]. However, it has been observed that the HA products obtained from rooster combs caused some allergic responses. Further, the technology has been developed recently toward bacterial fermentation to reduce the production cost and complex purification processes. Such alternative sources include attenuated strains of Streptococcus zooepidemicus and Streptococcusequi for the production of HA. The bacterium secretes the HA into fermentation broth and this behavior is an additional advantage for isolation of the HA directly from broth without the need for homogenizing bacterial cells [13, 14]. However, the risk of mutation of the bacterial strains, and possible co-production of various toxins, pyrogens, and immunogens, decreases the application of fermentative HA in clinical practice. This is also why HA samples originating from rooster combs are still currently preferred for human treatment in cases when the HA material is designated for injection, in the eyes or synovial joints. However, these are also not ideal sources of HA, as all HA products obtained from rooster combs are obligated to carry warnings for those who are allergic to avian products. Thus, alternative sources for production of HA are presently a subject of research [12]. One of the promising potential candidates is a genetically modified bacterial strain, Bacillus subtilis, carrying the A gene from Streptococcus equisimilis encoding the enzyme HA synthase.

Such an engineered strain could produce HA with the molecular weight in the 1 MDa range. The advantage of using B. subtilis is that it is easily cultivated on a large scale and does not produce exotoxins or endotoxins, and many products manufactured by this microorganism have received a GRAS (generally recognized as safe) designation in the early 1960s. At present, microbiologically produced HA has been approved for treatment of superficial wounds as well as for use in the cosmetic industry [12].

2.3 Physiological role HA differs from other synthetic polymers in that it is biologically active. Together with HA’s outstanding viscoelastic nature, its biocompatibility and nonimmunogenicity have led to its use in several clinical applications. HA was described as an ubiquitous carbohydrate polymer that is part of the extracellular matrix [15]. A human body weighing 70 kg contains 15 g of HA. The greatest amount of HA is present in the skin, followed by the synovial fluid, the vitreous body, and the umbilical cord. It can also be found in places where friction occurs: the joints, tendons, sheaths, pleura, and pericardium [16]. In the human body, HA occurs in many diverse forms, circulating freely, decorated with a variety of HA-binding proteins (hyaladherins), tissue-associated, intercolated into the extracellular matrix by electrostatic or covalent binding to other matrix molecules. It comprises a major portion of the intimate glycocalyx that surrounds all cells. HA can be tethered to cell surfaces by any of the membrane-associated receptors. Recent evidence indicates that HA also exists within cells, though little is known of the form or function of such HA [17]. HA is a major component of the synovial fluid, and was found to increase the viscosity of the fluid. Along with lubricin, it is one of the fluid’s main lubricating components. It is

2 Hyaluronic acid

considered an important component of articular cartilage, presenting a coat around individual chondrocytes and providing its resistance to compression. The molecular weight (size) of HA in cartilage decreases with age, but the amount of it increases [18]. HA possesses a number of protective physiochemical functions that may provide some additional chondroprotective effects in vivo, explaining its longer-term effects on articular cartilage. HA decreases the nerve impulses and nerve sensitivity associated with pain. In experimental osteoarthritis, HA has protective effects on cartilage [19]. Exogenous HA enhances its synthesis together with proteoglycan in chondrocyte, reduces the production and activity of proinflammatory mediators and matrix metalloproteinases, and alters the behavior of immune cells. These functions are manifested by the scavenging of reactive oxygen-derived free radicals, the inhibition of immune complex adherence to polymorphonuclear cells, and the inhibition of leukocyte and macrophage migration and aggregation [20]. A lubricating role of hyaluronan in muscular connective tissues to enhance sliding between adjacent tissue layers has also been suggested. A particular type of fibroblasts, embedded in dense fascial tissues, has been proposed as being cells specialized for the biosynthesis of the hyaluronan-rich matrix. Their related activity could be involved in regulating the sliding ability between adjacent muscular connective tissues [18]. HA is also a major component of skin. More than half of the total body’s HA is present in the skin [8], where it plays a structural role that depends on its unique hydrodynamic properties and its interactions with other extracellular matrices molecules (ECM) components. HA excellent consistency and tissue-friendliness and being one of the most hydrophilic molecules in nature has caused it to be described as nature’s moisturizer [4]. It gives the skin its properties of resistance and maintenance of the shape, and supports the preservation of

5

the natural degree of hydration of the skin cells. Its concentration in the body tends to decrease with aging, and a lack of it leads to a skin weakness promoting the formation of wrinkles [7]. While it is abundant in extracellular matrices, HA also contributes to tissue hydrodynamics, movement and proliferation of cells, and participates in a number of cell surface receptor interactions, notably those including its primary receptors, CD44 and RHAMM. Upregulation of CD44 itself is widely accepted as a marker of cell activation in lymphocytes. HA’s contribution to tumor growth may be due to its interaction with CD44. The receptor CD44 participates in cell adhesion interactions required by tumor cells. On the cellular level, HA is highly hygroscopic and this property is believed to be important for modulating tissue hydration and osmotic balance. Because of its hygroscopic properties, hyaluronan significantly influences hydration and the physical properties of the extracellular matrix. Hyaluronan is also capable of interacting with several receptors, resulting in the activation of signaling cascades that influence cell migration, proliferation, and gene expression [21].

2.4 Turnover and elimination pathways The concentration of HA in the human body varies from a high concentration of 4 g/kg in umbilical cord, 2–4 g/L in synovial fluid, 0.2 g/kg in dermis, about 10 mg/L in thoracic lymph, and the lowest of 0.1–0.01 mg/L in normal serum [10]. Depending on the location of HA in the body, most of it is catabolized within days. Studies suggested that the normal half-life of HA varies from 1–3 weeks in inert tissues such as cartilages, to 1–2 days in the epidermis of skin, to 2–5 min in blood circulation. The pathways involved in HA catabolism include turnover (internalization and degradation within tissue) and release from the tissue matrix,

6

1. Versatile hyaluronic acid nanoparticles for improved drug delivery

drainage into the vasculature, and clearance via lymph nodes, liver, and kidneys. In structural tissues like bone or cartilage with no or little lymphatic drainage, HA degradation occurs in situ with other ECM such as collagens and proteoglycans. On the other hand, in skin and joints, a minimal fraction (approximately 20%–30%) of HA degrades in situ. Since HA is restricted to the small intracellular space of skin tissue, its half-life is slightly longer for days and weeks [22].

3 Preparation of hyaluronic acid nanoparticles 3.1 Conjugate formation One of the most commonly used techniques for the preparation of HA nanoparticles is the preparation of HA-drug nano-conjugates by establishing a covalent bond between the drug of interest and HA which could improve solubility, pharmacokinetic profile, and in vivo plasma half-life of the conjugated drugs. In most cases, the conjugation is designed to be cleaved after reaching the target site, and in cases of cancer, these conjugates usually have higher accumulation in the tumor sites due to the enhanced permeation and retention effect (EPR) [23]. Due to the presence of multiple functional groups on the backbone of HA like hydroxyl and carboxylic acid groups, it was possible to get conjugated to various compounds and macromolecules such as paclitaxel [24, 25], sodium butyrate [26, 27], and ovalbumin [28]. The use of HA drug conjugates is kind of converting the drug into a prodrug derivative in which the link between the HA and the drug molecule should achieve certain criteria. The most important is that the bond should be stable extracellularly to give the required in vivo halflife, and should be cleaved easily intracellularly to achieve the desired effect. It is evident that the release of intact drug molecule without affecting

the chemical structure is another required property [29]. The two most common sites for the chemical conjugation of drugs to HA are the hydroxyl groups and the carboxylic acid functionalities while the terminal aldehyde group can only be used to prepare terminally modified HA-ligand conjugates as well as to graft HA oligomers to another polymer carrying amino groups.

3.2 Self-assemblies formation Another type of HA nanoparticles are the ones prepared by HA association with hydrophobic polymers with the resulting conjugates able to self-aggregate in the form of hydrophobic micelles. Examples include the hydrophobic association of HA to poly(lactic-co-glycolic acid) (PLGA) [30], poly(ethylene glycol)-poly (ε-caprolactone) copolymers (PEG-PCL) [31], or tetradecylamine [32]. These amphiphilic self-assembling HA derivatives have been prepared by coupling the carboxylic groups of the hydrophilic HA through carbodiimide chemistry to different hydrophobic moieties such as PLGA [33], tetradecylamine [32], and PCL [31]. This kind of nanoparticle would combine the hydrophobic core needed for the encapsulation of many therapeutic agents, and the hydrophilic shell provides longer circulation times by reducing unwanted protein adsorption [23]. Hydrophobically modified HA (HMH) was prepared by the covalent conjugation to the hydrophobic tetradecylamine (TDA) using 1-ethyl-3 (3-dimethylaminopropyl) carbodiimide (EDC) and hydroxysulfosuccinimide (sulfoNHS). This reaction was able to produce nano self-aggregates upon its dissolution and sonication into aqueous phosphate buffer solution [32]. The self-associated HMH nanoparticles ranged in size between 197 and 285 nm depending on the degree of substitution with minor influence from the polymer concentration used. The higher the degree of substitution, the smaller the particles produced. Amphiphilic HA-5b-cholanic acid

3 Preparation of hyaluronic acid nanoparticles

conjugates (HA-CA conjugates) were synthesized by chemical conjugation of hydrophobic bile acid (5b-cholanic acid) to the hydrophilic HA backbone through amide formation in the presence of EDC and NHS. Varying the molar ratio of CA to the carboxylic acid of HA varied the degree of substitution from 2 to 10. For the production of HA nanoparticles, the produced amphiphilic HA-CA conjugates were dissolved in a phosphate buffered saline and the solution was sonicated using a probe-type sonification system followed by filtration step. The produced particles ranged in size from 237 to 424 nm [34].

3.3 Ionic nanocomplexes formation Since HA is a polyanionic polysaccharide, it can be easily allowed to react with cationic compounds and form successful ionic complexes. This reaction can be induced by direct interaction of HA with positively charged cargo molecules like in the case of DNA and plasmids, or it can happen in the presence of another positively charged polymer like chitosan [35]. Examples include the ionic nanocomplex between HA and TRAIL (Tumor necrosis factor Related Apoptosis Inducing Ligand), which was highly stable and long circulating compared to the native TRAIL [36]. Similarly, biopolymeric amphiphiles were prepared from the EDC mediated coupling reaction between HA and deoxcycholic acid producing self-assembled nanocarriers in the size range of 100–600 nm. The strong interaction between HA and chitosan (CS) even led to the faster release of the entrapped negatively charged cargo molecules, whose release could have been hindered in case of interaction with chitosan alone. CS-HA plasmid nanoparticles were prepared by simple mixing of both solutions under magnetic stirring and DNA was then added to form complexes. The mixture was vortexed for 3–5 s and then left at room temperature for the complexes to form completely [37]. It was found that the size of the nanoparticles increased and the zeta

7

potential decreased upon increasing the ratio of HA to CS. Similarly, HA could ionically interact with positively charged metallic compounds such as the anticancer drug cisplatin. CisplatinHA nanoparticles were prepared by simple mixing of HA and the drug, forming nanoparticles in the size range of 80–160 nm. Being large in amount, the release of the drug from the particle matrix was accompanied by disintegration of the particles [38].

3.4 Nanogels formation HA nanogels can be prepared by either physical or chemical crosslinking of HA to provide colloidal stable particles in the micro or nano range. The physical crosslinking would depend on noncovalent attractive forces between the polymer chains such as hydrophobic interactions, hydrogen bonding and ionic interactions. However, chemical crosslinking would provide particles of higher stability and subsequently longer half-lives. To prepare chemically crosslinked nanogel particles, it is preferable to spatially localize the HA molecules and crosslinkers in very small volumes to achieve the required reaction in the nano range. This could be successfully achieved using the inverse w/o microemulsion technique, which is described in Fig. 1.2 [29]. Hyaluronan microspheres were prepared by the surfactant-aided homogenization of HA aqueous solutions and crosslinker in mineral oil. This step is followed by the initiation of crosslinking by adding EDC [39]. Other examples of nanogel particles formation by amidation crosslinking were described in the literature [40, 41]. However, this microemulsion chemical crosslinking techniques demands high energy mechanical stirring or ultrasonication and the use of organic solvents, which are not considered favorable conditions for the labile molecules such as proteins and nucleic acids. Physical crosslinking is considered to be much milder in terms of affecting the labile structures of drug molecules [29].

8

1. Versatile hyaluronic acid nanoparticles for improved drug delivery

Crosslinker or coupling agent

HA and Aqueous drug phase Surfactant

Organic phase

HA

Nano gel = Crosslinks = Drug

FIG. 1.2 Inverse phase water in oil microemulsion technique for the preparation of chemically crosslinked HA nanogels. Reprinted with permission from Ossipov DA. Nanostructured hyaluronic acid-based materials for active delivery to cancer. Expert Opin Drug Deliv 2010;7:681–703. https://doi.org/10.1517/17425241003730399 and Taylor and Francis Ltd.

4 Applications of HA nanoparticles 4.1 Skin applications Although HA is present in most of the biological fluids and tissues and extracellular matrix of soft connective tissues, skin is considered to be most HA-abundant tissue in the human body. Upon aging, especially after the age of 20, skin content of HA continuously decreases [42]. The unique properties of HA, including its biocompatibility, biodegradability, viscoelasticity, and nonimmunogenicity, have made it an ideal material for cosmetic and biomedical applications. It exerts a hydrating effect on the skin, which may help to enhance the penetration of different drugs through the skin. However, its own penetration is very limited due to the high molecular weight as well as the enzymatic degradation risk. It was reported that crosslinked HA proved to permeate through the skin

deeper layers making it suitable carrier for transdermal applications [42]. HA itself as macromolecule is not able to penetrate the skin beyond the surface layers due to the strong barrier properties [43]. HA nanoparticles prepared by the anionic interaction with the cationic polymer protamine were able to penetrate the skin and deliver the HA to the dermis while free HA penetrated no further the stratum corneum. Therefore, these nanoparticles were considered promising for the effective skin delivery of HA to contribute to barrier recovery following UV irradiation [44]. Similarly, nanoparticles of quaternized cyclodextrin-grafted chitosan associated with HA have been also proven promising for cosmetics and skin hydration applications. Their skin hydrating ability as well as their safety on human skin fibroblasts were demonstrated in vitro [45]. Besides the use of nanoparticles for the drug delivery purposes, some cosmetic applications

9

4 Applications of HA nanoparticles

have been proposed. Preparations of slightly crosslinked HA are used as fillers for augmentation, to fill facial wrinkles and depressed scars. Such HA gels are more effective in maintaining cosmetic corrections than collagen-based products. Unlike collagen-based fillers, HA is extremely elastic, providing the elasticity required by spaces in which it is injected and the hyaluronate preparations are more sustained. Examples of the use of HA NPs for cosmetic applications are described in Table 1.1.

mechanisms and hence helping to treat the underlying disease origin. HA-CS-plasmid nanoparticles were prepared as novel nonviral gene delivery vector for the treatment of osteoarthritis. They utilize the ability of HA to bind to the CD44 to be internalized by the targeted cells by the endocytosis pathway. The transfection efficiency of these NPs was found to be superior to the CS-plasmid NPS, suggesting them as a safe and effective nonviral gene delivery vector to chondrocytes [37].

4.2 Osteoarthritis

4.3 Tissue engineering

The use of intra-articular HA injections as a viscosupplement to restore the normal viscosity of synovial fluid in osteoarthritis patients is a well-established therapeutic strategy [11]. Cationic polymeric nanoparticles linked to hyaluronate proved to be effective in the production of ionically crosslinked hydrogels in situ to increase the retention time of a model drug in the synovial cavity [50]. Gene therapy has been proposed as a treatment modality for targeting specific pathological

HA as one of the main components of body tissues has frequently been investigated for tissue engineering applications. HA-based sheets serve as a matrix for soft tissue, cartilage, bone, and skin growth, and as a substrate for tissue regeneration and remodeling. Threedimensional scaffolds of HA-based materials can facilitate restructuring of tissues and assist in regaining function. These materials are ideal for tissue reconstruction, as there is no host immune response, and are particularly useful

TABLE 1.1 Cosmetic applications of HA and HA NPs. Drug

HA nanoparticles

HA

Polyion complex formation with the cationic polymer Protamine

HA

Particle size (nm)

Application

Reference

100

Dermal HA delivery to restore the skin barrier properties in damaged skin

[44]

Polyion complex formation with quaternized cyclodextrin-grafted chitosan

235

Enhanced antiwrinkle effects and moisturizing properties

[45]

HA

HA/lysine NPs by ionic interaction between HA and lysine

134

Topical dermal filler

[46]

Vitamin E

HA-lipid nanoparticles with ethanol injection technique

200

Wound healing and localized delivery for cellular skin layers regeneration

[47]

Lutin

Chitin nanofibril-hyaluronan nanoparticles by ionic gelation

40–200

Antiaging

[48]

Vitamin E

HA-based nanoemulsion

59

Skin care and enhancement of drug percutaneous absorption

[49]

10

1. Versatile hyaluronic acid nanoparticles for improved drug delivery

for burn and trauma patients. Stem cells require an HA-rich environment for maintaining the undifferentiated state. Vascular endothelial cells can be selected, as well as aortic smooth muscle cells for the construction of heart valves, by seeding onto HA sheets and membranes. However, due to HA’s high solubility and fast elimination, its use for scaffold fabrication and structural stability has been challenging. To overcome these limitations, modification and crosslinking of HA have been proposed. Various examples on the use of crosslinked HA nanoparticles for drug delivery are presented in Table 1.2. Water-soluble carbodiimide crosslinking, polyvalent hyadrazide crosslinking, and other techniques have been introduced for tissue engineering applications of HA. Chemical crosslinking is expected to extend the HA degradation process in vivo and provide long-term stability for the various applications in orthopedics, cardiovascular medicine, and dermatology. Elsewhere, photocrosslinked HA hydrogels have been also introduced for different applications such as cartilage tissue engineering, cardiac repair, molecule delivery, valvular engineering, control of stem cell behavior, and microdevices [10]. HA as a natural polymer has been mixed with poly(lactic-co-glycolic acid) nanoparticles to develop an in situ crosslinkable system with drug delivery potential. Although such a system has shown favorable mechanical properties for tissue engineering, its biocompatibility and toxicity in vivo is still a major concern [64].

4.4 Cancer targeting Self-assembled polymeric nanoparticles have been investigated for cancer therapy due to their ability to encapsulate the chemotherapeutic agents and release them on a sustained manner. This is even enhanced by rendering their surface hydrophilic which would enhance their circulation time leading to higher accumulation into the tumor tissue with the known EPR effect of nanoparticles [34, 65]. In addition to this passive

targeting strategy, active targeting can be also achieved by binding these NPs to targeting moieties to recognize and bind to the tumor cells and being internalized by receptor-mediated endocytosis. Since HA is distinguished by its ability to bind to various cancer cells that overexpress CD44, it has been conjugated to various drug loaded nanoparticles as a targeting moiety [66]. CD44 is overexpressed in many cancers of epithelial origin and therefore the use of HA nanocarriers could increase the targetability and the retention into the cancer tissue [66]. However, the use of HA nanoparticles as a targeting moiety and drug carrier is a new trend, and extensive studies are needed to understand the factors affecting the affinity to CD44 and the internalization mechanisms of HA nanoparticles. It was found that the slow CD44 representation (24–48 h) led to limited availability of HA internalization receptors. Therefore, a higher affinity nanoparticle and a higher degree of clustering would lead to a lower number of internalized particles. On the other hand, lower affinity systems might lead to less clustering with more efficient HA-mediated delivery of drug payloads [67]. Amphiphilic HA-CA conjugates nanoparticles, which self-assemble into hydrophobic core nanosized particles, surrounded by a hydrophilic HA shell efficiently accumulate into the tumor site compared to the pure watersoluble HA after systemic administration. The in vivo biodistribution of these NPs in tumorbearing mice was investigated using a noninvasive near-infrared optical imaging technique, which revealed a significant accumulation of the HA NPs in the tumor site. The accumulation was much stronger than in normal tissues and was size dependent, which embraces the role of the EPR passive targeting pathway. However, when animals were preinjected with high doses of HA polymer before the injection of HA NPs, the NPs accumulation in the tumor site was remarkably attenuated, suggesting that the interaction between the HA from the NPs to the CD44 receptors on the cancer cell surface is

11

4 Applications of HA nanoparticles

TABLE 1.2 Various applications of HA NPs in drug delivery.

Drug

HA nanoparticles

Plasmid DNA

Polyion complex of HA and chitosan and plasmid DNA

siRNA

Particle size (nm)

Application

Reference

100–300

Nonviral vector for gene delivery for chondrocytes

[37]

Inverse w/o emulsion (nanogel)

200–500

Selective targeting of HCT-116 cells

[51]

Plasmid DNA

Dihydrazide mediated crosslinking

5–20 μm

Controlled rate DNA delivery

[39]

Doxorubicin

HA-PEG-PLGA polymeric nanoparticles by nanoprecipitation

93–186

Selective tumor targeting

[33]

HA

Self-assemblies of hydrophobic 5β-cholanic acid-HA

350–400

Tumor targeting

[52]

HA

Self-assemblies of hydrophobic 5β-cholanic acid-HA

237–424

Passive and active tumor targeting

[34]

Doxorubicin

Bioreducible core-crosslinked polymeric micelle based on hyaluronic acid

148

Tumor targeting

[53]

Cy5.5 and doxorubicin

Self-assemblies of amphiphilic iodinated hyaluronic acid

200

Theranostic system for cancer

[54]

Paclitaxel

Hyaluronate-cholanic acid micelles

258

Targeting CD44 overexpression in cancer cells

[55]

Cy5.5

(PEG)-conjugated self-assembled HA nanoparticles

217–269

Cancer therapy and diagnosis

[56]

Cy5.5

Self-assemblies of hydrophobic 5β-cholanic acid-HA

237–424

Targeting stabilin-2 and CD44 receptors overexpressed in atherosclerosis

[57]

EDC and sulfo-NHS crosslinked HA and cholanic acid

90

Theranostic application in atherosclerosis

[58]

pEGFP or pβ-gal as model plasmid

Hyaluronic acid-chitosan ionotropic gelation

100–235

Gene transfer and targeting to ocular cells

[59]

Dexamethasone

HA-chitosan nanoparticles

–

Enhanced ocular bioavailability

[60]

Insulin

Reverse-emulsion-freeze-drying

182

Oral insulin delivery

[61]

Perfluoropentane

Oil-in-water (O/W) emulsification

350

Ultra-long-acting, liver-specific, Ultrasound contrast agent

[62]

CuS and Cy5.5

EDC and sulfo-NHS crosslinked HA and cholanic acid

227

Image-guided photothermal therapy of cancer

[63]

Cy7 or

89

Zr

12

1. Versatile hyaluronic acid nanoparticles for improved drug delivery

also responsible for the high accumulation on the tumor site [34, 52]. The use of core crosslinked HA micelles prepared with a simple method of disulfide bond formation loaded with doxorubicin has shown an enhanced therapeutic efficiency and tumor accumulation as well as improved stability in vivo compared to the uncrosslinked micelles and the free drug. The superior activity was correlated to the ability of these carriers to unload the drug inside the tumor cell only with the micellar structure dissociated in response to the glutathione at the intracellular level. 2,3,5-Triiodobenzoic acid (TIBA) was conjugated to an HA oligomer as a computed tomography (CT) imaging modality and a hydrophobic residue and self-woven nano-assemblies were produced for the tumortargeted delivery of doxorubicinm which presented a promising theranostic system for cancer diagnosis and therapy of tumors that express CD44 receptors [54]. Polymeric nanoparticulate micelles of HA-CA paclitaxel were found to be specific and efficient chemotherapeutic treatment for CD44 overexpressing tumors and cancer cells [55]. However, a major drawback of HA-based conjugates or nanoparticles for cancer targeting is their preferential accumulation in the liver after systemic administration. PEGylated HA-NPs formed selfassembled nanoparticles in the size range of 217–269 nm of improved cancer accumulation and targetability compared to HA-NPs when tested in tumor-bearing mice [56].

4.5 Atherosclerosis The use of HA-NPs has been proposed as a potential tool for both diagnostic and therapeutic applications in atherosclerosis. A major observation in the pathogenic process of atherosclerosis is the overexpression of receptors of HA such as stabilin-2 and CD44. Selective stronger accumulation of HA-NPs in atherosclerotic lesions was observed, which was probably explained by an active targeting mechanism

after systemic administration [57]. Amine functionalized oligometric hyaluronan conjugated with cholanic ester and labeled with fluorescent or radioactive nanoparticles was tested for targeting atherosclerosis associated inflammation. The 90 nm particles accumulation was 30% higher in atherosclerotic aortas than wild type controls. The plaques treated with the HA nanoparticles contained 30% fewer macrophages compared to control and free HA treated groups. Therefore, these nanoparticles were proposed for PET imaging of atherosclerosisassociated inflammation due to their favorable targeting potential [58].

4.6 Ocular drug delivery Due to the strong defensive mechanisms of the eye, the transport of drugs via topical instillation in the eyes is limited, causing restricted bioavailability. The use of hyaluronan-coated chitosan nanoparticles was investigated for the enhanced ocular delivery of dexamethasone. The HA-coated nanoparticles have shown 2.14-fold higher AUC0-24h compared to dexamethasone solution, which was explained by the prolonged precorneal retention caused by the highly mucoadhesive HA [60]. HA-chitosan nanoparticles with sizes between 100 and 235 nm were also tested for ocular gene delivery and were able to achieve high transfection efficiency without affecting cell viability [59]. HA is also particularly useful as a space-filling matrix in the eye to maintain the shape of the anterior chamber during surgeries. HA solutions also serve as a viscosity-enhancing component of eye drops and as an adjuvant to eye tissue repair.

4.7 Insulin sensitivity and diabetes Recently, it has been demonstrated that CD44 receptors in pro-inflammatory cells in obese adipose tissues are involved in the development of adipose tissue inflammation and insulin

4 Applications of HA nanoparticles

resistance in type 2 diabetes patients. Therefore, empty HA nanoparticles were used as a therapeutic tool for adipose tissue inflammation and insulin resistance by selectively accumulating and clustering the CD44 receptors in inhibiting the interaction of the low molecular weight HA with these receptors, leading to improved insulin sensitivity and glycemic control. The HA nanoparticles are hence proposed as a therapeutic agent in the treatment of type 2 diabetes patients [68]. HA nanoparticles have also been proposed for the oral delivery of insulin as an alternative for insulin injections for the treatment of diabetes. Insulin-loaded HA nanoparticles were prepared by a reverse emulsion freeze-drying method in sizes of approximately 180 nm. The particles produced had very high entrapment efficiency up to 95%. These pH-sensitive nanoparticles provided the required protection for insulin from the acidic environment of the stomach and at the same time had no effect on the junction integrity of epithelial cells, which is an important parameter to consider in case of chronic use of medicaments, which is the usual case of insulin. The presence of HA enhanced the transcellular efflux of insulin transported through Caco-2 cell monolayers via the transcellular pathways. The results of permeability through a rat small intestine showed that insulin transport through the duodenum and ileum was enhanced, and the therapeutic efficiency of the produced nanoparticles was also proved in a diabetic rats model [61].

4.8 Theranostic and imaging For higher diagnostic procedures’ efficiency for different diseases, there is a great need for instant real-time imaging techniques that could favorably target certain organs or tissue types. One of the most commonly used imaging techniques in medical fields is ultrasound imaging. New echogenic HA nanoparticles have been developed and presented as an ultra-long acting, liver-specific ultrasound contrast agent.

13

The particles were prepared with the oil in a water emulsification method and contained perfluoropentane as an ultrasound gas precursor. The particles were more stable compared to the conventional microbubbles used for ultrasound (US) imaging. Their long circulating properties allowed for several systemic circulations followed by intense accumulation at the liver, which embraced their potential as a targeting imaging system for diagnosis of liver diseases. The targetability to the liver was related to both the small hydrodynamic size of the particles as well as the high affinity of HA to the liver. More interestingly, the prepared particles were able to discriminate between the normal liver tissues and liver cancer in a liver tumor-bearing mice model. The cancerous tissue was found to be more compact than the normal tissue, and hence lower US signals were observed [62]. Iron oxide-based magnetic nanoparticles bearing HA on the surface have been developed to target activated macrophages for imaging and therapeutic applications in inflammatory diseases. The particles were prepared by co-precipitation procedures followed by postsynthetic functionalization with both HA and fluorescein. Particles expressed a significant biocompatibility and stability in serum. Significant uptake by activated macrophages was observed, which was HA dependent. In addition, the magnetic core of the particles was found to be only transiently present in the cells, which indicates lower risks of toxicity. Fluorescein was found to be successfully delivered to the cellular nuclei, which showed the potential of using these nanocarriers also as a targeted drug delivery and molecular imaging [69]. HA nanoparticles were synthesized and labeled with the near infrared dye Cys5.5 for imaging purposes. For using the same particles but for photothermal purposes, an additional step of loading the particles with CuS was performed. The obtained particles had a size range of around 200 nm. The fluorescent signal of Cys5.5 was quenched by the presence of CuS. When the enzyme hyaluronidase was

14

1. Versatile hyaluronic acid nanoparticles for improved drug delivery

added to the particles, the fluorescence signal was restored in a time-dependent manner, which suggested that the particles could be used as a nanoprobe to be activated by the highly overexpressed hyaluronidase in the cancerous tumor areas. Therefore, the CuS-loaded HA nanoparticles were used for photoacoustic imaging, utilizing the strong absorbance of the CuS. After intravenous administration, the particles accumulated in the tumor area over time, and when irradiated with a laser, a good tumor inhibition rate of 89.74% was observed on day 5, indicating the great potential of these nanoparticles for theranostic applications [63].

5 Clinical status As already discussed, HA seems to be a very promising molecule for utilization as a vehicle or as an active ingredient for many drug delivery purposes. The use of HA as a dermal filler and for intra-articular delivery is already well established, and several products have been proved safe and approved by the FDA for clinical use [70]. However, most of the scientific research conducted on the use of HA and HA NPs for clinical applications in other areas is still in the phase of laboratory research and preclinical evaluation. This may be due to the consideration of HA as a new chemical entity every time it is linked or conjugated to a drug or any molecules for changing their properties. Due to the complex processes involved in such reactions and the various factors to be studied, HA and its derivatives are also difficult to industrialize [1]. However, several products have reached phase II and phase III clinical trials. Alchemica oncology in Australia have produced several HA-based systems for the management of cancer such as HA-irinotecan, HA-DOX, and HA-5FU. A phase I clinical trial was conducted on 12 patients, and HA-Irinotecan has proved to be safe and well tolerated while preserving

the anticancer activity of irinotecan [23]. Another phase II trial was done on 41 patients, and showed the advantages of the HA nano formulations in terms of progression-free survival and safety [71]. Unfortunately, phase III clinical trials did not achieve the expected results and the reason for this is not yet clear [1]. Therefore, the industrialization and widespread clinical utilization of HA and HA NPs as drug carriers are still rich areas that require more extensive research and have a long way to go.

6 Conclusion HA is one of the most important natural components of human tissues, making it a biocompatible, biodegradable, and promising carrier for various drug delivery applications. The use of nanotechnology has enhanced to a great extent the benefits achieved from this biomaterial. This could be by using systems that can actively target certain tissues or achieve enhanced penetration or permeation through certain body barriers. Future studies are needed to enhance the fabrication techniques and ensure the required efficacy and safety of the produced nanosystems.

References [1] Huang G, Huang H. Application of hyaluronic acid as carriers in drug delivery. Drug Deliv 2018;25:766–72. https://doi.org/10.1080/10717544.2018.1450910. [2] Necas J, Bartosikova L, Brauner P, Kolar J. Hyaluronic acid (hyaluronan): a review. Vet Med (Praha) 2008;53:397–411. [3] Jin Y-J, Ubonvan T, Kim D-D. Hyaluronic acid in drug delivery systems. J Pharm Investig 2010;40:33–43. https://doi.org/10.4333/KPS.2010.40.S.033. [4] Sze JH, Brownlie JC, Love CA. Biotechnological production of hyaluronic acid: a mini review. 3 Biotech 2016;6: https://doi.org/10.1007/s13205-016-0379-9. [5] Fallacara A, Baldini E, Manfredini S, Vertuani S. Hyaluronic acid in the third millennium. Polymers 2018;10:701. https://doi.org/10.3390/polym10070701. [6] Tripodo G, Trapani A, Torre ML, Giammona G, Trapani G, Mandracchia D. Hyaluronic acid and its

References

[7]

[8]

[9]

[10]

[11] [12]

[13]

[14]

[15]

[16] [17]

[18]

derivatives in drug delivery and imaging: recent advances and challenges. Eur J Pharm Biopharm 2015;97:400–16. https://doi.org/10.1016/j.ejpb.2015. 03.032. Wolf KJ, Kumar S. Hyaluronic acid: incorporating the bio into the material. ACS Biomater Sci Eng 2019. https://doi.org/10.1021/acsbiomaterials.8b01268. Mero A, Campisi M. Hyaluronic acid bioconjugates for the delivery of bioactive molecules. Polymers 2014;6:346–69. https://doi.org/10.3390/polym6020346. Schant
e CE, Zuber G, Herlin C, Vandamme TF. Chemical modifications of hyaluronic acid for the synthesis of derivatives for a broad range of biomedical applications. Carbohydr Polym 2011;85:469–89. https://doi. org/10.1016/j.carbpol.2011.03.019. Fakhari A, Berkland C. Applications and emerging trends of hyaluronic acid in tissue engineering, as a dermal filler and in osteoarthritis treatment. Acta Biomater 2013;9:7081–92. https://doi.org/10.1016/j.actbio. 2013.03.005. Fakhari A. Biomedical application of hyaluronic acid nanoparticles. University of Kansas; 2012. Kogan G, Sˇolt
es L, Stern R, Gemeiner P. Hyaluronic acid: a natural biopolymer with a broad range of biomedical and industrial applications. Biotechnol Lett 2006;29:17–25. https://doi.org/10.1007/s10529-0069219-z. Mendichi R, Giacometti Schieroni A. Fractionation and characterization of ultra-high molar mass hyaluronan. 2. On-line size exclusion chromatography methods. Polymer 2002;43:6115–21. https://doi.org/10.1016/ S0032-3861(02)00586-4. Shiedlin A, Bigelow R, Christopher W, Arbabi S, Yang L, Maier RV, Wainwright N, Childs A, Miller RJ. Evaluation of hyaluronan from different sources: Streptococcuszooepidemicus, rooster comb, bovine vitreous, and human umbilical cord. Biomacromolecules 2004; 5:2122–7. https://doi.org/10.1021/bm0498427. Salwowska NM, Bebenek KA, Z˙a˛dło DA, WcisłoDziadecka DL. Physiochemical properties and application of hyaluronic acid: a systematic review. J Cosmet Dermatol 2016;15:520–6. https://doi.org/10.1111/ jocd.12237. Toole BP. Hyaluronan is not just a goo!. J Clin Invest 2000;106:335–6. https://doi.org/10.1172/JCI10706. Kogan G, Sˇolt
es L, Stern R, Schiller J, Mendichi R. Hyaluronic acid: its function and degradation in in vivo systems. In: Studies in natural products chemistry. Elsevier; 2008. p. 789–882. https://doi.org/10.1016/ S1572-5995(08)80035-X. Holmes MWA, Bayliss MT, Muir H. Hyaluronic acid in human articular cartilage. Age-related changes in content and size. Biochem J 1988;250:435–41. https://doi. org/10.1042/bj2500435.

15

[19] Akmal M, Singh A, Anand A, Kesani A, Aslam N, Goodship A, Bentley G. The effects of hyaluronic acid on articular chondrocytes. J Bone Joint Surg (Br) 2005;87-B:1143–9. https://doi.org/10.1302/0301-620X. 87B8.15083. [20] Iturriaga V, Va´squez B, Manterola C, del Sol M. Role of hyaluronic acid in the homeostasis and therapeutics of temporomandibular joint osteoarthritis. Int J Morphol 2017;35:870–6. https://doi.org/10.4067/S0717-95022 017000300012. [21] Dechert TA, Ducale AE, Ward SI, Yager DR. Hyaluronan in human acute and chronic dermal wounds: hyaluronan in human dermal wounds. Wound Repair Regen 2006;14:252–8. https://doi.org/10.1111/j.17436109.2006.00119.x. [22] Drury JL, Mooney DJ. Hydrogels for tissue engineering: scaffold design variables and applications. Biomaterials 2003;24:4337–51. https://doi.org/10.1016/S0142-9612 (03)00340-5. [23] Choi KY, Saravanakumar G, Park JH, Park K. Hyaluronic acid-based nanocarriers for intracellular targeting: Interfacial interactions with proteins in cancer. Colloids Surf B Biointerfaces 2012;99:82–94. https://doi.org/ 10.1016/j.colsurfb.2011.10.029. [24] Luo Y, Prestwich GD. Synthesis and selective cytotoxicity of a hyaluronic acid-antitumor bioconjugate. Bioconjug Chem 1999;10:755–63. https://doi.org/ 10.1021/bc9900338. [25] Rosato A, Banzato A, De Luca G, Renier D, Bettella F, Pagano C, Esposito G, Zanovello P, Bassi P. HYTAD1p20: a new paclitaxel-hyaluronic acid hydrosoluble bioconjugate for treatment of superficial bladder cancer. Urol Oncol 2006;24:207–15. https://doi.org/10.1016/j. urolonc.2005.08.020 [Proc Annu Meet Soc Urol Oncol May 2005 Part II Mol Approaches Early Detect Progn Determ Prosate Cancer Syst Ther Prostate Cancer Angiogenic Targets Clin. Trial Data]. [26] Coradini D, Pellizzaro C, Miglierini G, Daidone MG, Perbellini A. Hyaluronic acid as drug delivery for sodium butyrate: improvement of the anti-proliferative activity on a breast-cancer cell line. Int J Cancer 1999;81:411–6. [27] Coradini D, Zorzet S, Rossin R, Scarlata I, Pellizzaro C, Turrin C, Bello M, Cantoni S, Speranza A, Sava G. Inhibition of hepatocellular carcinomas in vitro and hepatic metastases in vivo in mice by the histone deacetylase inhibitor HA-But. Clin Cancer Res 2004;10:4822–30. [28] Kim KS, Kim H, Park Y, Kong WH, Lee SW, Kwok SJJ, Hahn SK, Yun SH. Noninvasive transdermal vaccination using hyaluronan nanocarriers and laser adjuvant. Adv Funct Mater 2016;26:2512–22. https://doi.org/ 10.1002/adfm.201504879. [29] Ossipov DA. Nanostructured hyaluronic acid-based materials for active delivery to cancer. Expert Opin

16

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37]

[38]

[39]

1. Versatile hyaluronic acid nanoparticles for improved drug delivery

Drug Deliv 2010;7:681–703. https://doi.org/ 10.1517/17425241003730399. Lee H, Ahn C-H, Park TG. Poly[lactic-co-(glycolic acid)]-grafted hyaluronic acid copolymer micelle nanoparticles for target-specific delivery of doxorubicin. Macromol Biosci 2009;9:336–42. https://doi.org/ 10.1002/mabi.200800229. Yadav AK, Mishra P, Jain S, Mishra P, Mishra AK, Agrawal GP. Preparation and characterization of HA-PEG-PCL intelligent core-corona nanoparticles for delivery of doxorubicin. J Drug Target 2008;16:464–78. https://doi.org/10.1080/10611860802095494. Choi KY, Lee S, Park K, Kim K, Park JH, Kwon IC, Jeong SY. Preparation and characterization of hyaluronic acid-based hydrogel nanoparticles. J Phys Chem Solids 2008;69:1591–5. https://doi.org/10.1016/j. jpcs.2007.10.052. Yadav AK, Mishra P, Mishra AK, Mishra P, Jain S, Agrawal GP. Development and characterization of hyaluronic acid-anchored PLGA nanoparticulate carriers of doxorubicin. Nanomed Nanotechnol Biol Med 2007;3:246–57. https://doi.org/10.1016/j.nano.2007. 09.004. Choi KY, Chung H, Min KH, Yoon HY, Kim K, Park JH, Kwon IC, Jeong SY. Self-assembled hyaluronic acid nanoparticles for active tumor targeting. Biomaterials 2010;31:106–14. https://doi.org/10.1016/j.biomaterials. 2009.09.030. Duceppe N, Tabrizian M. Factors influencing the transfection efficiency of ultra low molecular weight chitosan/ hyaluronic acid nanoparticles. Biomaterials 2009;30:2625–31. https://doi.org/10.1016/j.biomaterials. 2009.01.017. Na SJ, Chae SY, Lee S, Park K, Kim K, Park JH, Kwon IC, Jeong SY, Lee KC. Stability and bioactivity of nanocomplex of TNF-related apoptosis-inducing ligand. Int J Pharm 2008;363:149–54. https://doi.org/10.1016/j. ijpharm.2008.07.013. Lu H-D, Zhao H-Q, Wang K, Lv L-L. Novel hyaluronic acid-chitosan nanoparticles as non-viral gene delivery vectors targeting osteoarthritis. Int J Pharm 2011;420:358–65. https://doi.org/10.1016/j.ijpharm.2011.08.046. Jeong Y-I, Kim S-T, Jin S-G, Ryu H-H, Jin Y-H, Jung T-Y, Kim I-Y, Jung S. Cisplatin-incorporated hyaluronic acid nanoparticles based on ion-complex formation. J Pharm Sci 2008;97:1268–76. https://doi.org/10.1002/jps.21103. Yun YH, Goetz DJ, Yellen P, Chen W. Hyaluronan microspheres for sustained gene delivery and sitespecific targeting. Biomaterials 2004;25:147–57. https://doi.org/10.1016/S0142-9612(03)00467-8.

[40] Kumar A, Sahoo B, Montpetit A, Behera S, Lockey RF, Mohapatra SS. Development of hyaluronic acid-Fe2O3 hybrid magnetic nanoparticles for targeted delivery of peptides. Nanomed Nanotechnol Biol Med 2007;3:132–7. https://doi.org/10.1016/j.nano.2007. 03.001. [41] Pitarresi G, Craparo EF, Palumbo FS, Carlisi B, Giammona G. Composite nanoparticles based on hyaluronic acid chemically cross-linked with α,β-polyaspartylhydrazide. Biomacromolecules 2007;8:1890–8. https://doi.org/10.1021/bm070224a. [42] Berko´ S, Maroda M, Bodna´r M, Ero˝s G, Hartmann P, Szentner K, Szabo´-R
ev
esz P, Kem
eny L, Borb
ely J, Csa´nyi E. Advantages of cross-linked versus linear hyaluronic acid for semisolid skin delivery systems. Eur Polym J 2013;49:2511–7. https://doi.org/10.1016/j. eurpolymj.2013.04.001. [43] Abdel-Mottaleb MMA, Lamprecht A. In vivo skin penetration of macromolecules in irritant contact dermatitis. Int J Pharm 2016;515:384–9. https://doi.org/ 10.1016/j.ijpharm.2016.10.042. [44] Tokudome Y, Komi T, Omata A, Sekita M. A new strategy for the passive skin delivery of nanoparticulate, high molecular weight hyaluronic acid prepared by a polyion complex method. Sci Rep 2018;8:2336. https://doi.org/10.1038/s41598-018-20805-3. [45] Sakulwech S, Lourith N, Ruktanonchai U, Kanlayavattanakul M. Preparation and characterization of nanoparticles from quaternized cyclodextrin-grafted chitosan associated with hyaluronic acid for cosmetics. Spec Issue Pharm Innov 2018;13:498–504. https://doi. org/10.1016/j.ajps.2018.05.006. [46] Carneiro J, D€ oll-Boscardin PM, Fiorin BC, Nadal JM, Farago PV, de Paula JP. Development and characterization of hyaluronic acid-lysine nanoparticles with potential as innovative dermal filling. Braz J Pharm Sci 2016;52:645–51. https://doi.org/10.1590/ s1984-82502016000400008. [47] Pereira GG, Detoni CB, Balducci AG, Rondelli V, Colombo P, Guterres SS, Sonvico F. Hyaluronate nanoparticles included in polymer films for the prolonged release of vitamin E for the management of skin wounds. Eur J Pharm Sci 2016;83:203–11. https://doi. org/10.1016/j.ejps.2016.01.002. [48] Morganti P, Palombo M, Tishchenko G, Yudin V, Guarneri F, Cardillo M, Del Ciotto P, Carezzi F, Morganti G, Fabrizi G. Chitin-hyaluronan nanoparticles: a multifunctional carrier to deliver anti-aging active ingredients through the skin. Cosmetics 2014;1:140–58. https://doi.org/10.3390/cosmetics1030140.

References

[49] Kong M, Chen XG, Kweon DK, Park HJ. Investigations on skin permeation of hyaluronic acid based nanoemulsion as transdermal carrier. Carbohydr Polym 2011;86:837–43. https://doi.org/10.1016/j.carbpol.2011.05.027. [50] Morgen M, Tung D, Boras B, Miller W, Malfait A-M, Tortorella M. Nanoparticles for improved local retention after intra-articular injection into the knee joint. Pharm Res 2013;30:257–68. https://doi.org/10.1007/ s11095-012-0870-x. [51] Lee H, Mok H, Lee S, Oh Y-K, Park TG. Target-specific intracellular delivery of siRNA using degradable hyaluronic acid nanogels. J Control Release 2007;119:245–52. https://doi.org/10.1016/j.jconrel.2007.02.011. [52] Choi KY, Min KH, Na JH, Choi K, Kim K, Park JH, Kwon IC, Jeong SY. Self-assembled hyaluronic acid nanoparticles as a potential drug carrier for cancer therapy: synthesis, characterization, and in vivo biodistribution. J Mater Chem 2009;19:4102. https://doi.org/ 10.1039/b900456d. [53] Han HS, Choi KY, Ko H, Jeon J, Saravanakumar G, Suh YD, Lee DS, Park JH. Bioreducible core-crosslinked hyaluronic acid micelle for targeted cancer therapy. J Control Release 2015;200:158–66. https://doi.org/ 10.1016/j.jconrel.2014.12.032. [54] Lee J-Y, Chung S-J, Cho H-J, Kim D-D. Iodinated hyaluronic acid oligomer-based nanoassemblies for tumor-targeted drug delivery and cancer imaging. Biomaterials 2016;85:218–31. https://doi.org/10.1016/ j.biomaterials.2016.01.060. [55] Thomas RG, Moon M, Lee S, Jeong YY. Paclitaxel loaded hyaluronic acid nanoparticles for targeted cancer therapy: In vitro and in vivo analysis. Int J Biol Macromol 2015;72:510–8. https://doi.org/10.1016/j. ijbiomac.2014.08.054. [56] Choi KY, Min KH, Yoon HY, Kim K, Park JH, Kwon IC, Choi K, Jeong SY. PEGylation of hyaluronic acid nanoparticles improves tumor targetability in vivo. Biomaterials 2011;32:1880–9. https://doi.org/10.1016/j.biomaterials. 2010.11.010. [57] Lee GY, Kim J-H, Choi KY, Yoon HY, Kim K, Kwon IC, Choi K, Lee B-H, Park JH, Kim I-S. Hyaluronic acid nanoparticles for active targeting atherosclerosis. Biomaterials 2015;53:341–8. https://doi.org/10.1016/j. biomaterials.2015.02.089. [58] Beldman TJ, Senders ML, Alaarg A, P
erez-Medina C, Tang J, Zhao Y, Fay F, Deichm€ oller J, Born B, Desclos E, van der Wel NN, Hoebe RA, Kohen F, Kartvelishvily E, Neeman M, Reiner T, Calcagno C, Fayad ZA, de Winther MPJ, Lutgens E, Mulder WJM, Kluza E. Hyaluronan nanoparticles selectively target

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

17 plaque-associated macrophages and improve plaque stability in atherosclerosis. ACS Nano 2017;11:5785–99. https://doi.org/10.1021/acsnano.7b01385. de la Fuente M, Seijo B, Alonso MJ. Novel hyaluronic acid-chitosan nanoparticles for ocular gene therapy. Invest Ophthalmol Vis Sci 2008;49:2016–24. https:// doi.org/10.1167/iovs.07-1077. Kalam MA. The potential application of hyaluronic acid coated chitosan nanoparticles in ocular delivery of dexamethasone. Int J Biol Macromol 2016;89:559–68. https://doi.org/10.1016/j.ijbiomac.2016.05.016. Han L, Zhao Y, Yin L, Li R, Liang Y, Huang H, Pan S, Wu C, Feng M. Insulin-loaded pH-sensitive hyaluronic acid nanoparticles enhance transcellular delivery. AAPS PharmSciTech 2012;13:836–45. https://doi.org/ 10.1208/s12249-012-9807-2. Min HS, Son S, Lee TW, Koo H, Yoon HY, Na JH, Choi Y, Park JH, Lee J, Han MH, Park R-W, Kim I-S, Jeong SY, Rhee K, Kim SH, Kwon IC, Kim K. Liver-specific and echogenic hyaluronic acid nanoparticles facilitating liver cancer discrimination. Adv Funct Mater 2013;23:5518–29. https://doi.org/10.1002/adfm.201301131. Zhang L, Gao S, Zhang F, Yang K, Ma Q, Zhu L. Activatable hyaluronic acid nanoparticle as a theranostic agent for optical/photoacoustic image-guided photothermal therapy. ACS Nano 2014;8:12250–8. https://doi.org/ 10.1021/nn506130t. Yeo Y, Ito T, Bellas E, Highley CB, Marini R, Kohane DS. In situ cross-linkable hyaluronan hydrogels containing polymeric nanoparticles for preventing postsurgical adhesions. Ann Surg 2007;245:819–24. https://doi. org/10.1097/01.sla.0000251519.49405.55. Ramzy L, Nasr M, Metwally AA, Awad GAS. Cancer nanotheranostics: a review of the role of conjugated ligands for overexpressed receptors. Eur J Pharm Sci 2017;104:273–92. https://doi.org/10.1016/j.ejps.2017. 04.005. Platt VM, Szoka FC. Anticancer therapeutics: targeting macromolecules and nanocarriers to hyaluronan or CD44, a hyaluronan receptor. Mol Pharm 2008;5:474–86. https://doi.org/10.1021/mp800024g. Almalik A, Karimi S, Ouasti S, Donno R, Wandrey C, Day PJ, Tirelli N. Hyaluronic acid (HA) presentation as a tool to modulate and control the receptor-mediated uptake of HA-coated nanoparticles. Biomaterials 2013;34:5369–80. https://doi.org/10.1016/j.biomaterials.2013.03.065. Rho JG, Han HS, Han JH, Lee H, Nguyen VQ, Lee WH, Kwon S, Heo S, Yoon J, Shin HH, Lee E, Kang H, Yang S, Lee EK, Park JH, Kim W. Self-assembled hyaluronic acid nanoparticles: implications as a

18

1. Versatile hyaluronic acid nanoparticles for improved drug delivery

nanomedicine for treatment of type 2 diabetes. J Control Release 2018;279:89–98. https://doi.org/ 10.1016/j.jconrel.2018.04.006. [69] Kamat M, El-Boubbou K, Zhu DC, Lansdell T, Lu X, Li W, Huang X. Hyaluronic acid immobilized magnetic nanoparticles for active targeting and imaging of macrophages. Bioconjug Chem 2010;21:2128–35. https:// doi.org/10.1021/bc100354m. ´ ngeles G, Nesˇporova´ K, Ambrozˇova´ G, [70] Huerta-A Kubala L, Velebny´ V. An effective translation: the

development of hyaluronan-based medical products from the physicochemical, and preclinical aspects. Front Bioeng Biotechnol 2018;6:https://doi.org/ 10.3389/fbioe.2018.00062. [71] Wickens JM, Alsaab HO, Kesharwani P, Bhise K, Amin MCIM, Tekade RK, Gupta U, Iyer AK. Recent advances in hyaluronic acid-decorated nanocarriers for targeted cancer therapy. Drug Discov Today 2017;22:665–80. https://doi.org/10.1016/j.drudis.2016.12.009.

C H A P T E R

2

Preclinical testing—Understanding the basics first Ranjita Shegokar Capnomed GmbH, Zimmern, Germany

1 Introduction

over several years. It starts with synthesis of the chemical entity for specific biological product and ends with a “dosage form” (tablet/capsule/ injectable, etc.) for “specific indication” in patients. In API industry, mostly IND candidate testing is extended from synthesis screening until Phase 1 testing, while generic drugs have a quicker route via bioequivalence studies. The synthesis step involves standardized SOPs to GLP R&D practices. However, preclinical and Phase 1 are strictly done as per GLP and GCP guidelines, respectively. At the end, commercial manufacturing is performed as per GMP guidelines. On the other hand, pharma companies may start with either their own IND or IND supplied from an API manufacturer. Multiple in vitro, in vivo, and in silico screening assays and testing are performed during preclinical as per GLP guidelines. Depending upon product strategy, the formulation phase may start early or later during this stage, and sometimes last until Phase II. Phase III always tests a final formulation for one specific indication. The process starts with identification of a “biological target” using bioinformatic and phenotype databases for that particular disease

Preclinical testing or assessment is the interlink between drug discovery (i.e., bench—in vitro, in silico, in vivo—research and development, drug candidate selection) and clinical testing and ultimate availability of the drug product to the patient (i.e., bedside). It takes for a drug at least 12–15years of development cycle to go from lab to clinics. Huge amounts of data about a product’s performance, stability, effectiveness, and safety in animals/humans are generated throughout the complete cycle. It is not only about years, but also about compilation of experts’ knowledge from diverse competences such as chemists, biologists, pharmacists, regulatory scientists, toxicologists, statisticians, veterinarians, patent lawyers, clinicians, informatics, etc. The average cost of bringing a drug from the lab to market is around USD $1–3 billion, depending upon the nature of the drug (small vs large molecules). The high attrition rate in drug development cycles also drives up the costs of medications that reach the market [1]. The typical journey of a chemical entity to pharma product (Fig. 2.1) is lengthy and spread

Drug Delivery Aspects https://doi.org/10.1016/B978-0-12-821222-6.00002-6

19

# 2020 Elsevier Inc. All rights reserved.

20

2. Preclinical testing—Understanding the basics first

FIG. 2.1 Exhaustive journey of drug candidate from lab to clinic (**the numbers are based on literature information, as the reported numbers are very diverse, hence the highest number is presented). The color intensities of bars indicate potency of practices from high (dark color) to low (neutral color).

condition. Once the target is identified, chemical agents (called active pharmaceutical ingredients or APIs) are synthesized or obtained by other suitable high-throughput screening of method. These compounds and their modified versions are then tested for their effectiveness to the selected biological target by using various in vitro, ex vivo, and in vivo models. A further choice of optimal performing API is made, and later this new drug candidate is registered as an investigational new drug (IND) and subsequently tested in various clinical trials in its final dosage form (Phases I–IV, fewer patients to more patients) to evaluate their safety, performance, dose determination, etc. As the IND is initially tested in humans in Phase I studies, generally a small population is selected (10–15 healthy humans). In this phase, mainly pharmacokinetic data on IND are collected by administrating a single subtherapeutic dose specifically to evaluate whether the

performance of the IND is comparable to preclinical data. Once the expected data are obtained, the candidate is tested for safety and tolerance (20–100 healthy humans). Various parameters like maximum tolerated dose/escalated dose, relevant and serious adverse effects (AEs), and pharmacological, pharmacodynamic, and pharmacokinetic parameters are evaluated in Phase I. Depending upon the focus on disease and market need, multiple INDs or a single one enter Phase I for initial screening. In Phase II, the therapeutic efficacy of a chemical entity is evaluated in several hundred patients. The study protocol agreed among experts and regulatory clearly defines the selection of population especially inclusion and exclusion criteria, dose selected, and AEs. Beside the IND’s biological therapeutic efficacy, response to the IND vs. placebo and before/after treatment data are evaluated in Phase II. It is a determining clinical phase to assess the safety of the

1 Introduction

drug in the complete cycle and to obtain preliminary data on the IND’s effectiveness, although it is not comprehensive enough to provide sufficient evidence across a wide population. Phase III studies are mainly performed to confirm further therapeutic benefits and safety of the IND in the indication of choice and involves usually up to 10,000 subjects. The population size of Phase III varies depending upon the indication, e.g., in case of platin resistant ovarian cancer on an average between 100–500 patients are recruited. Depending upon the scope and effect of different dosages, combinations with other therapeutic agents are investigated with the aim of gathering information on indications and contraindications over a longer period of time compared to Phases I and II (Fig. 2.2). Phase IV clinical studies occur after marketing approval, and are generally lengthy. Patient information across different regions/continents is collected and analyzed. The cohort of study focus is broad (10,000 patient population). The main aim of this phase is to gather additional data on safety, efficacy, and uses in other indications. Phase IV studies can bring both positive and negative outcomes, e.g., finding effectiveness of a drug in new indications, thereby opening new market opportunity (positive outcome) finding special AEs in wide population

FIG. 2.2

Scope and duration of clinical trial phases.

21

may restrict use of IND only for specific indication (negative outcome) [2–4]. The budgeting on development costs at each phase is generally based on a bottom-up analysis of historical clinical trial costs. The cost may vary depending upon the type of clinical trial, number of patients, type of archetype, type of study design, number of clinical centers, CRO (high to low rating), location of clinical sites, and procedure implemented. R&D and other costs like basic research through lead optimization, chemistry, manufacturing, and controls (CMC), good manufacturing practice (GMP), manufacturing build-up and scale-up costs, regulatory or registration fees (post-Phase III), and all postmarket commitments (e.g., Phase IV pharmacovigilance studies) are on the top of this. A research paper by Terry et al. has impressively compiled info on clinical cost calculations from various sources and interviews. Tables 2.1 and 2.2 provide details on development cost and probability of success depending on the type of archetype and clinical phase planned [5]. Based on the data, preclinical phase needs fair (1–4 years) amount of time to complete compared to complete cycle (10–18years). Hence the main aim of this chapter is to illustrate basic and regulatory understanding on the fundamentals of the preclinical phase and challenges involved.

22

2. Preclinical testing—Understanding the basics first

TABLE 2.1 Development cost assumptions per phase per archetype designated at preclinical and clinical phases. Cost estimates per phase ($ millions) Preclinical Lower bound, upper bound Simple

Phase 1

Point estimate

Lower bound, upper bound

3.3, 10.0

6.7

Complex

8.3, 24.9

New chemical

Simple

Entity (NCE)

Phase 2

Point estimate

Lower bound, upper bound

Point estimate

Lower bound, upper bound

Point estimate

1.8, 2.7

2.2

7.4, 19.0

13.2

56.6, 165.6

111.1

16.6

1.9, 3.0

2.5

7.8, 20.0

13.9

67.9, 198.7

133.3

2.5, 7.5

5.0

1.8, 2.7

2.2

3.7, 7.9

5.8

11.5, 54.1

32.8

Innovative

5.0, 10.0

7.5

4.4, 5.3

4.8

3.9, 8.3

6.1

12.1, 55.4

34.5

Complex

7.5, 12.5

10.0

6.9, 7.9

7.4

4.1, 87

6.4

12.6, 59.6

36.1

Repurposed drug

Simple

Ν/A

N/A

N/A

N/A

3.7, 7.9

5.8

10.0, 25.2

17.6

Complex

2.5, 7.5

5.0

1.7, 2.7

2.2

3.7, 7.9

5.8

10.0, 25.2

17.6

Biologic

Simple

5.4, 16.2

10.8

1.9, 3.0

2.4

4.5, 10.5

7.5

27.7, 80.5

54.1

Complex

16.2, 27.0

21.6

7.0, 8.3

7.6

5.0, 11.6

8.3

30.5, 88.5

59.5

Archetype Vaccine

Diagnostics

Phase 3

Selection and validation of markers

Development

Regulated trials beyond EUO/CE

Assay development

1.0, 5.0

3.0

1.0, 3.0

2.0

1.0, 6.0

3.5

Simple technical platform development

N/A

N/A

50.0, 150.0

100.0

1.0, 6.0

3.5

Obtained from article (5) under open access permission Terry RF, Yamey G, Miyazaki-Krause R, et al. Funding global health product R&D: the portfolio-toimpact model (P2I), a new tool for modelling the impact of different research portfolios. Gates Open Res 2018;2:24.

2 Preclinical studies Preclinical studies are performed in in vitro, in vivo, ex vivo, and in silico models to obtain basic information about the safety and biological efficacy of a drug candidate before testing it in a final target population, i.e., humans. Preclinical studies or tests are mainly performed in compliance with GLP/GSP guidelines (good laboratory practice and good scientific practices) to ensure reliability and reproducibility of results. The FDA/EMA require supporting

basic preclinical data to IND application especially on toxic effects, safety profile, pharmacokinetics, and pharmacodynamics. The data from preclinical trials must be accurate, reliable, and based on the best suitable and comparable model available to the target population. Typically, this means that the IND or drug product must undergo a series of robust tests and experiments using in vitro, in vivo, ex vivo, and in silico models as per the needs of the focused indication and regulatory guidelines.

23

3 Regulatory aspects of preclinical studies

TABLE 2.2 Probability of success (attrition rate) and cycle time (length of phase) per phase and archetype Length of phase (years) Archetype

Probability of success (%)

Preclinical

Phase 1

Phase 2

Phase 3

Simple

3.36

1.57

2.23

2.33

41.0

68.4

45.9

70.8

Complex

3.33

1.97

3.71

3.50

41.0

50.0

21.6

63.6

Simple

2.49

1.80

3.38

3.18

65.0

59.7

38.8

69.1

Innovative

2.70

1.81

3.35

3.10

60.0

51.9

28.4

57.8

Complex

2.87

1.93

3.51

2.80

55.0

57.2

19.7

40.3

Repurposed drug

Simple

0.00

0.00

2.14

2.14

100.0

100.0

45.7

68.1

Complex

2.33

1.63

2.14

2.14

75.0

58.5

45.7

68.1

Biologic

Simple

3.29

1.62

2.47

2.10

75.0

66.2

44.3

70.9

Complex

3.24

1.49

4.16

3.38

77.0

69.6

32.2

62.5

Diagnostics

Assay development

1.00

1.25

1.33

0.00

50.0

100.0

100.0

100.0

Diagnostics

Simple technical platform development

0.00

2.50

2.00

0.00

100.0

75.0

100.0

100.0

Vaccine

New chemical Entity (NCE)

Preclinical

Phase 1

Phase 2

Phase 3

Adapted from (5) under open access permission Terry RF, Yamey G, Miyazaki-Krause R, et al. Funding global health product R&D: the portfolio-to-impact model (P2I), a new tool for modelling the impact of different research portfolios. Gates Open Res 2018;2:24.

3 Regulatory aspects of preclinical studies Preclinical studies mainly follow a combination of: • • • •

• • • •

GLP (21CFR, 58) and compliance monitoring; OECD principles; ICH/M3; Common Technical Document/CTD module 4 (article 6 of Regulation (EC) No. 726/2004, and with respect to the Annex I to Directive 2001/83/EC); Pharmacopoeial or codex requirements; 3R principles; Local animal ethical committee guidelines and requirements; and ISO regulations

The “set of tests” changes as per type of products, e.g., category (pharma, food, or veterinary), type (chemical or device), use (human/ non-human), and other many more factors. Table 2.3 lists a few tests reported in the literature for medical devices. As discussed above, the list is exhaustive. The type of tests to be performed must be decided as per intended use of product. For further details, readers are requested to refer to the above regulatory guidelines and full articles on this topic. The literature contains vast information on disease, organ, and bioactivityspecific assays and preclinical tests. A list of regulatory guidelines and supporting articles is provided in Tables 2.3–2.5.

24

2. Preclinical testing—Understanding the basics first

TABLE 2.3 Typical tests for medical device. Functional tests Functional implantation studies (bone, dental, hemostasis, cardiovascular, etc.) Identification and quantification of degradation products (ISO 10993-15) Packaging validation* (ISO 11607) EO residue determination* (ISO 10993-7) Biocompatibility testing Sensitization Irritation Pyrogenicity* Implantation Hemocompatibility Microbiological testing of medical devices Test for sterility Endotoxins Bioburden Test for specified microorganisms/microbial limit test Test for antimicrobial preservation Test for antimicrobial efficacy Evaluation of reusables for the intended reprocessing procedure (cleaning, disinfection, sterilization)

TABLE 2.4 Typical tests for chemical entity and pharma drugs. • Cell viability assays Thymidine incorporation assays Cell Titer-Glo cell viability assays MTS assay ATPlite cell viability assay SRB assay other specific tests • Cell proliferation assays MTT cell proliferation assay cyQuant direct proliferation assay BrdU cell proliferation ELISA assay Label-free and real time proliferation assay using IncuCyte other specific tests • Cell apoptosis assays Caspase-Glo 3/7 activation assay Reaction oxygen species (ROS) assay

TABLE 2.4 Typical tests for chemical entity and pharma drugs—cont’d Mitochondria membrane potential assay Apoptosis analysis by flow cytometry other specific tests • Cell angiogenesis assay Matrigel plug assay Tube formation assay Platelet activation assay Co-culture angiogenesis assay other specific tests • Related assays Cell migration assay Tumor invasion assay Chemotaxis assay Cellular phosphorylation assay Cell cycle assay Endotoxin assay Genetic stability testing Endocrine disruption Drug-drug interaction Drug efficacy test 3D angiogenesis assay 3D invasion assay Inflammation assay Receptor binding assay Enzyme inhibition 20 messenger analysis Plasma stability Plasma protein binding assay other specific tests • In vitro permeability and transporter assays Caco-2 permeability assay MDCK permeability assay Parallel artificial membrane permeability assay (PAMPA) Transporter assays, e.g., P-glycoprotein (P-gp), BCRP, OCT2 other specific tests • Type of toxicity assays 2D-based hepatotoxicity assay 3D-based hepatotoxicity assay Cardiotoxicity Neurotoxicity Nephrotoxicity Genotoxicity Carcinogenicity other specific tests

5 Animal models and type of tests

TABLE 2.4 Typical tests for chemical entity and pharma drugs—cont’d • Animal tests Teratogenicity Pharmacokinetic Local tolerance Pharmacodynamic Fertility studies Single dose toxicity in animal Repeated dose toxicity in animal other specific tests

TABLE 2.5 Regulatory guidelines and related publications.  Sanjay Jain, Michael Edwards, Louise Spencer, Regulatory Rapporteur—Vol 13, No 6, June 2016, European Medicines Agency (2007a). Advances and challenges in the development of drug delivery systems— A European perspective  Guideline on strategies to identify and mitigate risks for first-in human trials with investigational medicinal products. London: European Medicines Agency. Retrieved 18 Oct, 2019, from http://www.ema.europa. eu/docs/en_GB/document_library/Scientific_ guideline/2009/09/WC500002988.pdf  European Medicines Agency (2007b). Guideline on requirements for first-in-man clinical trials for potential high-risk medicinal Retrieved 18 Oct, 2019, from http:// www.ema.europa.eu/docs/en_GB/document_library/ Scientific_guideline/2009/09/WC500002989.pdf  European Medicines Agency (2009). ICH guideline M3(R2) on non-clinical safety studies for the conduct of human clinical trials and marketing authorisation for pharmaceuticals. London: European Medicines Agency. Retrieved 18 Oct, 2019, from http://www.ema.europa. eu/docs/en_GB/document_library/Scientific_ guideline/2009/09/WC500002720.pdf  Innovation in Development, Regulatory Review, and Use of Clinical Advances—A Vital Direction for Health and Health Care Michael Rosenblatt; Christopher P. Austin; Marc Boutin; William W. Chin et al., https://nam.edu/wpcontent/uploads/2016/09/Innovation-in-DevelopmentRegulatory-Review-and-Use-of-Clinical-Advances.pdf  ICH efficacy guidelines https://www.ich.org/page/ efficacy-guidelines  ICH safety guidelines https://www.ich.org/page/ safety-guidelines  M3-nonclinical safety studies/ICH https://www.ich. org/page/multidisciplinary-guidelines

25

4 Preclinical testing and models used 4.1 In vitro assays/cell line assays In vitro assays to evaluate basic effects are simple, fast, and cost-efficient. Dedicated cell lines, tissue, blood cells, organ cultures, or components of the same are used. As these assays are performed under GLP, they allow tight control over experimental parameters and design of experiments. Furthermore, depending upon the intended use of the drug, other tests might be needed like carcinogenicity, genotoxicity, and reproductive toxicity. For example, carcinogenicity is performed in two species (rat and mouse). It involves a number of animals and a specific study design plan as per the advice of the regulatory authorities. The costs of such studies are in excess of $ 2.5 million and they are typically performed as 2-year bioassays. Although the basic information provided by these assays is helpful, one disadvantage is that isolated cells may not mirror the exact performance when they are in vivo, due to missing the microenvironment of that particular organ. Depending upon the target disease, indication, and organ, cell line assays are selected. Mostly cell line assays are used as in vitro model systems. Cell lines provide an indefinite source of biological material if grown under optimum conditions. The major challenge includes selection of a proper cell line, strict control of experimental conditions, and proper design of the experiment to avoid cross-contamination and misleading results (Tables 2.6 and 2.7).

5 Animal models and type of tests 5.1 In vivo evaluation Various animal models are reported in the literature, including rats, mice, guinea pigs, hamster, rabbits, cats, monkey apes, and dogs. One must refer to specific disease physiology,

26

2. Preclinical testing—Understanding the basics first

TABLE 2.6 Key references on in vitro models (as per diseases/target organ). In vivo and in vitro models for scanning drug substances in malaria: Prestudy

Turkiye Parazitol Derg. 2017 Sep;41(3):156–163. https:// doi.org/10.5152/tpd.2017.5365

In vitro and in vivo models of HIV latency

Adv Exp Med Biol. 2018;1075:241–263. https://doi.org/ 10.1007/978-981-13-0484-2_10

In vitro and ex vivo corneal penetration and absorption models

Drug Deliv Transl Res. 2016 Dec;6(6):634–647

In vitro skin models in the optimization of skin formulations

Acta Pharm Hung. 2017;87(1):3–12

In vitro models of Mycobacterium tuberculosis dormancy, and in vivo models of latent tuberculosis infection

Klin Lab Diagn. 2019;64(5):299–307. https://doi.org/10. 18821/0869-2084-2019-64-5-299-307

In vitro three-dimensional (3D) models in cancer research: an update

Mol Carcinog. 2013 Mar;52(3):167–82. https://doi.org/ 10.1002/mc.21844. Epub 2011 Dec 7

Improved in vitro human tumor models for cancer gene therapy

Hum Gene Ther. 2015 May;26(5):249–56. https://doi. org/10.1089/hum.2015.028. Epub 2015 Apr 20

Beyond 3D culture models of cancer

Sci Transl Med. 2015 Apr 15;7(283):283 ps9. https://doi. org/10.1126/scitranslmed.3009367

In vitro models for evaluation of periodontal wound healing/ regeneration

Periodontol 2000. 2015 Jun;68(1):41–54. https://doi. org/10.1111/prd.12079

In vitro cell and tissue models for studying host–microbe interactions: a review

Br J Nutr. 2013 Jan;109 Suppl 2:S27–34. https://doi. org/10.1017/S0007114512004023

In vitro and in vivo methods for studying retinal ganglion cell survival and optic nerve regeneration

Methods Mol Biol. 2018;1695:187–205. https://doi. org/10.1007/978-1-4939-7407-8_16

In vitro models to study human lung development, disease and homeostasis

Physiology (Bethesda). 2017 May;32(3):246–260. https://doi.org/10.1152/physiol.00041.2016

In vitro modeling of the interaction between human epithelial cells and lymphocytes upon influenza infection

Influenza Other Respir Viruses. 2016 Sep;10(5):438–42. https://doi.org/10.1111/irv.12394. Epub 2016 May 17

Towards 3D in vitro models for the study of cardiovascular tissues and disease

Drug Discov Today. 2016 Sep;21(9):1437–1445. https://doi.org/10.1016/j.drudis.2016.04.014. Epub 2016 Apr 23

In vitro models of the blood-brain barrier

Acta Neurobiol Exp (Wars). 2011;71(1):113–28

In vitro models of the blood-brain barrier for the study of drug delivery to the brain

Mol Pharm. 2014 Jul 7;11(7):1949–63. https://doi.org/ 10.1021/mp500046f. Epub 2014 Apr 18

In vitro cardiac tissue models: current status and future prospects

Adv Drug Deliv Rev. 2016 Jan 15;96:203–13. https:// doi.org/10.1016/j.addr.2015.09.011. Epub 2015 Sep 30

In vitro models of the blood-brain barrier

Methods Mol Biol. 2014;1135:415–37. https://doi.org/ 10.1007/978-1-4939-0320-7_34

Nonanimal models of wound healing in cutaneous repair: in silico, in vitro, ex vivo, and in vivo models of wounds and scars in human skin

Wound Repair Regen. 2017 Apr;25(2):164–176. https://doi.org/10.1111/wrr.12513. Epub 2017 Feb 20

In vitro psoriasis models with focus on reconstructed skin models as promising tools in psoriasis research

Exp Biol Med (Maywood). 2017 Jun;242(11):1158–1169. https://doi.org/10.1177/1535370217710637

Further development of an in vitro model for studying the penetration of chemicals through compromised skin

Toxicol In Vitro. 2017 Feb;38:101–107. https://doi.org/ 10.1016/j.tiv.2016.10.004. Epub 2016 Oct 14

5 Animal models and type of tests

27

TABLE 2.6 Key references on in vitro models (as per diseases/target organ)—cont’d In vitro skin models as a tool in optimization of drug formulation

Eur J Pharm Sci. 2015 Jul 30;75:10–24. https://doi. org/10.1016/j.ejps.2015.02.018. Epub 2015 Mar 5

Improved in vitro models for preclinical drug and formulation screening focusing on 2D and 3D skin and cornea constructs

Eur J Pharm Biopharm. 2018 May;126:57–66. https:// doi.org/10.1016/j.ejpb.2017.11.014. Epub 2017 Dec 2

TABLE 2.7 Key references on ex vivo models (as diseases/target organ). Human lung ex vivo infection models

Cell Tissue Res. 2017 Mar;367(3):511–524. https://doi.org/10.1007/ s00441-016-2546-z. Epub 2016 Dec 20

Ex vivo models of chronic granulomatous disease

Methods Mol Biol. 2019;1982:587–622. https://doi.org/10.1007/9781-4939-9424-3_35

Human ex vivo wound healing model

Methods Mol Biol. 2013;1037:255–64. https://doi.org/10.1007/978-162,703-505-7_14

Modeling tuberculosis pathogenesis through ex vivo lung tissue infection

Tuberculosis (Edinb). 2017 Dec;107:126–132. https://doi.org/10.1016/ j.tube.2017.09.002. Epub 2017 Sep 12

Epithelial ovarian cancer experimental models

Oncogene. 2014 Jul 10;33(28):3619–33. https://doi.org/10.1038/onc. 2013.321. Epub 2013 Aug 12

Ex vivo rabbit and human corneas as models for bacterial and fungal keratitis

Graefes Arch Clin Exp Ophthalmol. 2017 Feb;255(2):333–342. https:// doi.org/10.1007/s00417-016-3546-0. Epub 2016 Nov 14

In vivo and in vitro models of type 2 diabetes in pharmaceutical drug discovery

Diabetes Obes Metab. 1999 Mar;1(2):75–86

Human pulmonary 3D models for translational research

Biotechnol J. 2018 Jan;13(1). https://doi.org/10.1002/biot.201700341. Epub 2017 Sep 20

metabolic pathways, target organ, specific regulatory guidelines, and requirements of drug product and financial aspects before selecting an animal model. Animal studies are strictly governed by ethical committees to avoid unnecessary harm to animal species. Appropriate study protocol and related explanations are needed to obtain official ethical approval to perform animal studies. Advances like noninvasive imaging, microsampling, and telemetric monitoring are valuable methods to obtain relevant data. Unlike in vitro cell line assays, in vivo models do not allow too much control on experimental parameters due to variations in animals. The most popular animal models are mice and rats;

however, these models have several limitations on the pharmacokinetic and pharmacodynamic fronts. Despite the limitations, rodent models are still helpful to get basic information and ideas on toxicity at the preliminary stages of development. Advances in rodent models like the one below provides new platform to test drug substances, not to mention this test methodology its also has its own limitations. Some examples include: • • • • •

Immune deficiency mice; Cell line derived xenograft (CDX) models; Patient-derived xenograft (PDX) models; Orthotopic tumor models; and Genetically engineered mice (GEM). etc

28

2. Preclinical testing—Understanding the basics first

5.2 Pharmacodynamics In a pharmacodynamics study, the effects of a drug on the body/biological environment (in this case, animal) are observed based on the concentration of the drug. This is generally referred to as the dose response relationship, categorizing the drug into the category potent, cytotoxic, or safe. Drugs are evaluated for desired pharmacological effects and AEs. These studies indicate the therapeutic index and therapeutic window of a drug.

5.3 Pharmacokinetic Pharmacokinetic and biodistribution studies determine how a body handles a drug, meaning how it distributes and eliminates the drug. These effects are studied by checking the drug distribution profile in an organ or particular tissue (target site), and by plasma profiling. Absorption, distribution, metabolism, and excretion (ADME) of the drug is key information when determining safer dose ranges and drug

administration regime for the next clinical trial, e.g., oral medication is challenged by first pass effect whereas intravenous medication is not, which has an effect on bioavailability. Furthermore, this study can be extended to determine a drug’s affinity for plasma proteins and the drug’s molecular interaction with a tissue or organ’s microenvironment. During metabolism, a drug is altered chemically into pharmacologically active or inert metabolites. It is vital to ensure a therapeutic effect; an adequate and safe steady-state concentration of the drug should be maintained in the body. Multiple factors affect metabolism, such as health condition, age, race, type of dosage form, etc. Lastly, clearance is mainly achieved via the renal and hepatic routes, and the metabolites are excreted from the body at the end. Depending upon the administration route, the requirement for preclinical testing changes, e.g., for a dermal product, biodistribution studies are not required, more imp would be local tissue distribution studies (Tables 2.8 and 2.9).

TABLE 2.8 Key references on animal models (diseases/target organ). Animal models for cutaneous vaccine delivery

Eur J Pharm Sci. 2015 Apr 25;71:112–22. https://doi.org/10.1016/j. ejps.2015.02.005. Epub 2015 Feb 14

Animal models used to study direct peripheral nerve repair: a systematic review

Neural Regen Res. 2020 Mar;15(3):491–502. https://doi.org/10. 4103/1673-5374.266068

Animal model of asthma, various methods and measured parameters: a methodological review

Iran J Allergy Asthma Immunol. 2016 Dec;15(6):445–465

Animal models of spinal cord injury: a systematic review.

Spinal Cord. 2017 Aug;55(8):714–721. https://doi.org/10.1038/ sc.2016.187. Epub 2017 Jan 24

Animal models in cardiovascular research: hypertension and atherosclerosis

Biomed Res Int. 2015;2015:528757. https://doi.org/10. 1155/2015/528757. Epub 2015 May 3

Animal models for percutaneous absorption

J Appl Toxicol. 2015 Jan;35(1):1–10. https://doi.org/10.1002/ jat.3004. Epub 2014 Oct 27

Animal models of osteoarthritis: classification, update, and measurement of outcomes

J Orthop Surg Res. 2016 Feb 2;11:19. https://doi.org/10.1186/ s13018-016-0346-5

Animal models in influenza research

Methods Mol Biol. 2018;1836:401–430. https://doi.org/10. 1007/978-1-4939-8678-1_20

Animal models as a tool in hepatocellular carcinoma research: a review

Tumor Biol. 2017 Mar;39(3):1010428317695923. https://doi. org/10.1177/1010428317695923

29

5 Animal models and type of tests

TABLE 2.8 Key references on animal models (diseases/target organ)—cont’d Animal models for evaluation of oral delivery of biopharmaceuticals

J Control Release. 2017 Dec 28;268:57–71. https://doi.org/10. 1016/j.jconrel.2017.09.025. Epub 2017 Sep 19

Search for an animal model to investigate selective pulmonary vasodilation

Lab Anim. 2017 Aug;51(4):376–387. https://doi.org/10. 1177/0023677216675384. Epub 2016 Nov 25

Animal models in myopia research

Clin Exp Optom. 2015 Nov;98(6):507–17. https://doi.org/10. 1111/cxo.12312

Latest animal models for anti-HIV drug discovery

Expert Opin Drug Discov. 2015 Feb;10(2):111–23. https://doi. org/10.1517/17460441.2015.975201. Epub 2014 Oct 28

Animal models of tuberculosis: lesson learnt

Indian J Med Res. 2018 May;147(5):456–463. https://doi.org/ 10.4103/ijmr.IJMR_554_18

Animal Models of Tuberculosis: an overview

Microbiol Spectr. 2016 Aug;4(4). https://doi.org/10.1128/ microbiolspec.TBTB2-0004-2015

Animal models in tuberculosis research—where is the beef?

Expert Opin Drug Discov. 2015;10(8):871–83. https://doi.org/ 10.1517/17460441.2015.1049529. Epub 2015 Jun 13

Malaria in pregnancy: the relevance of animal models for vaccine development

Lab Anim (NY). 2017 Oct 6;46(10):388–398. https://doi.org/10. 1038/laban.1349

Animal models of efficacy to accelerate drug discovery in malaria

Parasitology. 2014 Jan;141(1):93–103. https://doi.org/10.1017/ S0031182013000991. Epub 2013 Jun 21

The use of animal models in diabetes research

Br J Pharmacol. 2012 Jun;166(3):877–94. https://doi.org/10. 1111/j.1476-5381.2012.01911.x

TABLE 2.9 Key references on in silico models (diseases/target organ). In silico ADME-Tox modeling: progress and prospects

Expert Opin Drug Metab Toxicol. 2017 Nov;13(11):1147–1158. https://doi.org/10.1080/17425255.2017.1389897. Epub 2017 Oct 13

In silico skin model: a multiscale simulation study of drug transport

J Chem Inf Model. 2017 Aug 28;57(8):2027–2034. https:// doi.org/10.1021/acs.jcim.7b00224. Epub 2017 Jul 31

In silico models for hepatotoxicity

Methods Mol Biol. 2016;1425:201–36. https://doi.org/10. 1007/978-1-4939-3609-0_11

In silico models of stem cell and developmental systems

Theor Biol Med Model. 2014 Jan 8;11:1. https://doi.org/ 10.1186/1742-4682-11-1

In vitro and in silico liver models: current trends, challenges and opportunities

ALTEX. 2018;35(3):397–412. https://doi.org/10.14573/altex. 1803221. Epub 2018 May 28

In silico modeling for tumor growth visualization

BMC Syst Biol. 2016 Aug 8;10(1):59. https://doi.org/10.1186/ s12918-016-0318-8

In silico models for acute systemic toxicity

Methods Mol Biol. 2016;1425:177–200. https://doi.org/ 10.1007/978-1-4939-3609-0_10 Continued

30

2. Preclinical testing—Understanding the basics first

TABLE 2.9 Key references on in silico models (diseases/target organ)—cont’d In silico modeling of the immune system: cellular and molecular scale approaches

Biomed Res Int. 2014;2014:371809. https://doi.org/10. 1155/2014/371809. Epub 2014 Apr 6

In silico prediction of genotoxicity

Food Chem Toxicol. 2017 Aug;106(Pt B):595–599. https://doi.org/10.1016/j.fct.2016.12.013. Epub 2016 Dec 12

In silico cancer research towards 3R

BMC Cancer. 2018 Apr 12;18(1):408. https://doi.org/ 10.1186/s12885-018-4302-0

In silico assessment of adverse drug reactions and associated mechanisms

Drug Discov Today. 2016 Jan;21(1):58–71. https://doi. org/10.1016/j.drudis.2015.07.018. Epub 2015 Aug 10

The use of in silico models within a large pharmaceutical company

Methods Mol Biol. 2016;1425:475–510. https://doi.org/ 10.1007/978-1-4939-3609-0_20

In silico modeling of gastrointestinal drug absorption: predictive performance of three physiologically based absorption models.

Mol Pharm. 2016 Jun 6;13(6):1763–78. https://doi.org/ 10.1021/acs.molpharmaceut.5b00861. Epub 2016 May 5

5.4 Toxicology In toxicity studies, the safety of a drug and its dose relation are determined. Additionally, this study is used to establish biomarkers for monitoring AEs in later clinical phases. The toxicity studies are mainly performed for single and multiple dose, i.e., single and repeated dose toxicity over a period of months, to analyze toxic effects of the drug and multiple administrations. Toxicity studies are planned based on the intended drug administration regime and the route of administration. Selecting the appropriate species for a program of work is one of the most critical aspects of drug development to enable first in man application and/or dose administration. Various regulatory guidance including the ICHs and M3R2 gives explicit information on these approaches, and will indicate other applicable regulations to the test drug type, route of administration, and the intended indications. Generally, these follow the use of two species (a rodent and nonrodent) for small molecules. For biotherapeutics, on the other hand, this is mostly akin to pharmacological relevance and/or receptor targeting/occupancy, and may typically result in the use of single species (nonhuman primates or genetically

modified murine species, etc.) programs. Marmosets are also useful nonhuman primate alternatives, but are relatively challenging to handle and are significantly less robust due to their nature and practicalities such as blood sampling limitations and breeding. The dog is arguably the most widely used nonrodent species for regulatory toxicology, followed closely by nonhuman primates and, in more recent times, minipigs. The rodent of choice falls mainly into two categories/species, rats and mice, with the majority of rodent toxicity studies being performed in the rat model/ species. It has also been demonstrated over the years that using two species that are distinct phylogenetically provides a far superior coverage of a chemical’s risk to humans.

5.5 Advances in the field Advances in bioinformatics, virtual reality, and the internet of things over the past decades have made in silico studies a supporting and next-generation model. Computer simulations are used to predict in vivo and in vitro performances of chemical entities. For the in silico model, structure-activity relationship (SAR) and quantitative structure-activity relationship

31

6 Conclusion

(QSAR) are commonly used approaches in the field of testing. These in silico models are very complex and are based on compiled knowledge from statistics, mathematics, bioinformatics, biochemistry, and molecular biology. In addition, from a preclinical perspective for example, the no observed adverse effect level (NOAEL) [6] has typically served as the focal point for selecting the most appropriate dose levels for the conduct of first in human (FIM) studies. The NOAEL continues to be used and followed extensively as prescribed in various regulatory guidance documents, for selecting safe multiples of animal doses for extrapolating to humans within the food, pharmaceutical, and chemical industries. There have been several publicized limitations of this approach in recent times, and a resurgence of a collaborative effort to focus on the advancement of existing/novel alternatives to complement the use of this well-established method in regulatory science. A few of these perceived alternatives are the BenchMark Dose (BMD) models [7] and/or adverse outcome pathways (AOPs) [8], both of which are in silico-based models and are fully implemented by prominent regulatory agencies to support human safety. There are still some reservations for virtually all these concepts, but the coming decades could see their full use and adoption.

5.6 Databases In June 2016, the FDA commissioner proposed building a database that would gather information before clinical trials, meaning from preclinical research (lab animals or cells/cell lines). This proposal of a ClinicalTrials.gov database for preclinical work received mixed reactions from the scientific community. Currently, if a molecule becomes successful, all the drug development data become trade secret; if it does not, the data are stored in archives, and very rarely published despite their high scientific value. The idea of this database supports learning and sharing from unand successful trials. Even though the idea is

interesting, the scientific community is unsure about the future of it, due to several reasons, such as reliability, funding, legal aspects, government and regulatory scrutiny, etc. https://www. preclinicaltrials.eu is trying to promote transparency in preclinical research. The main objective of this database is to increase transparency, avoid duplication of animal studies, reduce bias reporting, increase data sharing on protocols, study designs, and outcomes, and foster research collaborations. The UK’s Catapult database (https://ct.catapult.org.uk/resources/cell-andgene-therapy-catapult-uk-preclinical-researchdatabase) provides one-stop information for ongoing preclinical research activity related to cell, gene, and other advanced therapies. Elsevier’s PharmaPendium database (https:// www.elsevier.com/solutions/pharmapendiumclinical-data) collects data on drug safety, the FDA Adverse Event Reporting System (FAERS), and ADME and drug-drug interactions. This database offers risk-benefit analyses and drug candidate assessments. Researchers can search FDA and EMA regulatory documents, committee meeting minutes, FAERS, data on pharmacokinetics, efficacy, safety, metabolizing enzymes’ and transporters’ potential drug-drug interactions, and guidance on validation of the best animal model. Other databases include http://fprd.ihes.fr (a French preclinical database) and https://dtp.cancer.gov (the NCI’s Development Therapeutics Program, DTP).

6 Conclusion The high costs of drug development (for both successful and unsuccessful candidates) results in exorbitant drug prices, leading to high healthcare expenditure. The only solution to reduce this burden is open communication between all science streams involved during the complete cycle, better alignment on “regulatory requirement” and “right experimental design,” along with transparency in sharing result

32

2. Preclinical testing—Understanding the basics first

and interpretation. This helps to reduce costs associated with drug development at each milestone, including the preclinical stage. It is clear that preclinical is a turning point for any drug, and involves millions of dollars. Selection of optimal experimental design, study protocol, assay, cell lines, and animal model with significant translation value have a tremendous impact on the completion of the preclinical phase with desired results. A significant amount of research is needed in generating sophisticated preclinical models to understand and evaluate the effects of disease-related factors, cellular microenvironments, and therapy-related (drug + device) parameters. Although 3D bioprinting, in silico are showing some promise, but still the question is whether these techniques are reducing— long experiments—to get better results or they are just generating more information in the pool of existing data. Databases can certainly help, but questions about data protection, safety, company policy, and openness to share all information remain unanswered. Importantly, outsourcing preclinical studies to Contract Research Organizations (CROs) can support companies and speed up preclinical development to a significant extent.

Optimal, cost-effective, and timely collaborative research is the way forward to reduce health expenditure.

References [1] Honek J. Preclinical research in drug development. Med Writ 2017;26(4). [2] Mahan VL. Clinical trial phases. Int J Clin Med 2014; 5:1374–83. https://doi.org/10.4236/ijcm.2014.521175. [3] https://www.hqsc.govt.nz/assets/Consumer-Engagement/Publications/Training-guide/019-clinical-trialsresearch-review.pdf. [4] The International Conference on Harmonisation of Technical Requirements for Registration of Pharmaceuticals for Human Use (ICH). www.ich.org. [5] Terry RF, Yamey G, Miyazaki-Krause R, et al. Funding global health product R&D: the portfolio-to-impact model (P2I), a new tool for modelling the impact of different research portfolios. Gates Open Res 2018;2:24. [6] Dorato MA, Engelhardt JA. The no-observed-adverseeffect-level in drug safety evaluations: use, issues, and definition(s). Regul Toxicol Pharmacol 2005; 42:265–74. [7] Haber LT, Dourson ML, Allen BC, Hertzberg RC, Parker A, Vincent MJ, Maier A, Boobis AR. Benchmark dose (BMD) modeling: current practice, issues, and challenges. Crit Rev Toxicol 2018;48(5):387–4159. [8] Edwards, et al. Adverse outcome pathways—organizing toxicological information to improve decision making. J Pharmacol Exp Ther 2016;356(1):170–81.

C H A P T E R

3

Aqueous polymeric coatings: New opportunities in drug delivery systems Abid Riaz Ahmeda, Joana Portugal Motab,c, Ahmad Abdul-Wahhab Shahbad, Muhammad Irfane a

Merck Healthcare KGaA, Darmstadt, Germany Lecifarma—Laborato´rio Farmac^eutico, Lda, Va´rzea do Andrade—Cabec¸ o de Montachique, Lousa, Portugal c CBIOS-Research Center for Biosciences and Health Technologies, Luso´fona University, Lisbon, Portugal d Kayyali Chair for Pharmaceutical Industries, Department of Pharmaceutics, College of Pharmacy, King Saud University, Riyadh, Saudi Arabia e Department of Pharmaceutics, Faculty of Pharmaceutical Sciences, GC University Faisalabad, Faisalabad, Pakistan b

1 Introduction

polymer dispersions can be prepared by emulsion polymerization of a monomer (latex) or by emulsification of a polymer (pseudolatex) [12]; physical properties and method of preparation of pure aqueous dispersion are summarized in Table 3.2. The formation of a thin and transparent film from aqueous latex or pseudolatex dispersion takes place in parallel with evaporation of water. Film formation is illustrated in Fig. 3.1. During spraying of the aqueous dispersion onto a substrate, water evaporates at a constant rate and the particles form a closed and ordered packing. Deformation and fusion of the colloidal particles lead to a film with water-filled cavities. Finally, particle coalescence occurs when the capillarity forces are strong enough [13–15].

Aqueous film coatings have been extensively applied to solid substrates as tablets, pellets, granules, and capsules, for several purposes. They include odor and taste masking, improvement of dosage form appearance, protection of the drug from environmental conditions (light, oxygen, and water vapor), isolation of incompatible materials, easy identification of drug products for both patients and healthcare professionals and to modify drug release (Table 3.1) [5–7]. Aqueous-based coatings are an attractive alternative to organic-based coatings, due to environmental reasons and reduced processing time (high polymer content) [8–11]. The aqueous

Drug Delivery Aspects https://doi.org/10.1016/B978-0-12-821222-6.00003-8

33

# 2020 Elsevier Inc. All rights reserved.

34

3. Aqueous polymeric coatings: New opportunities in drug delivery systems

TABLE 3.1 Examples of aqueous based polymers used for immediate and modified release coatings. Immediate release

Polymers (Trade name)

Ref.

Taste and odor masking

• Hydroxy ethylcellulose (Natrosol), hydroxy proylcellulose (Klucel), hydroxy propylmethylcellulose (Methocel) • Polyvinyl alcohol-polyethylene glycol graft copolymer (Kollicoat IR) • Polyvinyl alcohol-polyethylene glycol graft copolymer and polyvinyl alcohol (Kollicoat Protect) • Aminoalkyl methacrylate copolymer (Eudragit E 100)

[1–3]

Sustained release

• • • •

Ethylcellulose (Aquacoat ECD and Surelease) Ammonio methacrylate (Eudragit RS 30 D, Eudragit RS 30 D and combination of them) Ethylacrylate methylmethacrylate (2:1) (Eudragit NE 30 D and Eudragit NM 30 D) Polyvinyl acetate (Kollicoat SR 30 D)

[2, 4]

Enteric/colonic release

• • • •

Cellulose acetate phthalate (Aquacoat CPD) Hydroxypropyl methylcellulose acetate succinate (AQOAT) Methacrylic acid copolymer (Eudragit L 30 D 55) Polymethylacrylate-co-methyl methacrylate-co-methacrylic acid (Eudragit FS 30 D)

Moisture protective Easy identification Improving ingestion and swallowing Isolation of incompatible materials Improving appearance Modified release

TABLE 3.2 Physical properties and method of preparation of pure aqueous dispersions. Aqueous dispersion

Polymer

Additives (%)

Method of preparation Tg (°C)

MFT (°C)

Ref

Aquacoat ECD

Ethylcellulose

Sodium dodecyl sulfate (0.9–1.7) Cetyl alcohol (1.7–3.3)

Emulsification of 90 polymer (pseudolatex)

81

[30, 31]

Surelease

Ethylcellulose

Oleic acid (1.9) Dibutyl sebacate (3.5)

Inverse emulsification (pseudolatex)

32

[32, 33]

Aquacoat CPD

Cellulose acetate phthalate

Poloxamer (7)

Emulsification of 40 polymer (pseudolatex)

Information not found

[16, 34]

AquaSolve

Hydroxypropyl methylcellulose acetate succinate

Oleic acid (1.9) Dibutyl sebacate (3.5)

Redispersion of a polymeric powder

N/A

[35]

Eudragit RS 30 D

Ammonio methacrylate type B

Sorbic acid (0.25) NaOH (0.1)

emulsification of 55 polymer (pseudolatex)

45

[1, 36]

Eudragit RL 30 D

Ammonio methacrylate type A

—

N/A

40

50

Depends on substitution degree

50

35

1 Introduction

TABLE 3.2 Physical properties and method of preparation of pure aqueous dispersions—cont’d Aqueous dispersion

Polymer

Additives (%)

Method of preparation Tg (°C)

MFT (°C)

Ref

Eudragit NE 30 D

Ethylacrylate methylmethacrylate (2:1)

Nonoxynol 100* (1.5)

Emulsification polymerization (latex)

5

[1, 37]

Eudragit NM 30 D

N/A

PEG stearyl ether (0.7)

N/A

N/A

N/A

[1, 38]

Eudragit L 30 D 55

Methacrylic acid copolymer

Sodium lauryl sulfate (0.7) Polysorbate 80 (2.3)

N/A

>100

25

[1, 39]

Kollicoat SR 30 D

Polyvinyl acetate

Polyvinyl pyrrolidone (2.9) Sodium dodecyl sulfate (0.1)

N/A

46

18

[40, 41]

8

N/A, not applicable.

I

Aqueous dispersion deposited on surface

Water evaporation and particles packing II

Closely packed spheres with water filled voids Water evaporation and polymer deformation III

Apparently continuous polymer film Water diffusion and polymer interdiffusion

FIG. 3.1 Film formation of thin films from polymeric lattices.

In order to obtain a good-quality film (free of cracks), the coating temperature is usually 10–20°C above the minimum film formation temperature (MFT) [5, 16, 17] of the aqueous

polymeric dispersion. The MFT depends on the elastic modulus (resistance to particle deformation). Another important characteristic is the glass transition temperature (Tg) of the polymer [18] (Fig. 3.2). Below the Tg, the polymer exists in a rigid and glassy state with extremely limited polymer segment mobility. Above the Tg, the polymer is in a soft and rubbery state, and the polymer chains present a significant movement. Where the Tg of a polymer is higher than the desired operating coating temperatures, a plasticizer should be added to obtain good film formation. Generally, plasticizers reduce the cohesive forces along with polymer chains, leading to an increase in flexibility, reduction in tensile strength, and reduction of Tg and MFT, facilitating the polymer particle coalescence [19–22]. A plasticizer should be compatible (miscible) with the polymer and show no (or little) tendency to migrate [23]. Plasticizers are high boiling point organic compounds, with low vapor pressures and different water solubility (Table 3.3) [12]. The water-soluble plasticizers dissolve in the polymer aqueous dispersion, whereas the water insoluble plasticizers are emulsified in the

36

3. Aqueous polymeric coatings: New opportunities in drug delivery systems

aqueous phase of the dispersion. The most widely used plasticizers are: triethyl citrate (TEC) and triacetin (TA) as water-soluble plasticizers and acetyltriethyl citrate (ATEC), acetyltributyl citrate (ATBC), dibutyl sebacate (DBS), diethyl phthalate (DEP), and tributyl citrate (TBC) as waterinsoluble plasticizers. The decrease of the MFT and thus the effectiveness of the plasticizer depend on the plasticizer amount and its effectiveness for each aqueous polymeric dispersion. The effects of type and concentration of plasticizers on MFT of aqueous polymeric dispersions are shown in Table 3.4. In addition, the type and amount of plasticizer can also change the diffusivity of the coating and thus the drug release rate [42–45]. In the case of polymers with low Tg or where the plasticizer strongly decrease the Tg and MFT, glidants and antitacking agents are used to avoid tackiness or agglomeration during coating, drying, curing, and storage. Talc, magnesium stearate, kaolin, and glycerol monostearate are frequently used. The content of the antitacking agent depends on its nature—for example, talc is used in the range of 25%–100% (w/w, based on dry polymer), while 5%–20% (w/w, based on dry polymer) of glycerol monostearate is highly effective [5, 46]. Pigments are generally added to polymeric dispersions to enable easy product identification and to improve the elegance of pharmaceutical dosage form. In addition, titanium dioxide has been incorporated into film coating formulations as an opacifying agent to improve the stability of light-sensitive drugs [47, 48]. The concentration of pigments should not exceed critical pigment volume concentration (CPVP) and the CPVP is characteristic of each polymer-filler combination [49]. Surfactants such as sodium lauryl sulfate, poly vinylpyrrolidone, and polysorbates are used to stabilize the aqueous polymeric dispersion [1]. Pore formers can also be incorporated in the coating to modify the drug release. Polyethylene glycol, polyvinylpyrrolidone, hydroxypropyl cellulose, and in particular hydroxypropyl methylcellulose (HPMC) [24, 50] have been used to increase permeability of the coatings. However, destabilization of the aqueous

dispersions may occur [51]. Recently, polyvinyl pyrrolidone, polyvinyl alcohol-polyethylene glycol graft copolymer, and carrageenan have been successfully used to modify drug release from aqueous-based coatings [52–54] as Aquacoat ECD-coated dosage forms without destabilizing the aqueous dispersion. After completion of the coating process, a curing step (short thermal treatment) is regularly performed to improve polymer particle coalescence. This curing step is of utmost importance to promote complete film formation from a polymer aqueous dispersion [55, 56]. The curing can be performed in the coating equipment or in an oven. A curing temperature 10–20°C above the MFT of the aqueous dispersion is recommended to promote the “further gradual coalescence” (microscopic coalescence). The specific temperature, time, and presence of humidity on the curing step is dependent on each formulation as aqueous polymeric dispersion properties, plasticizer type, presence/absence of talc, and coating conditions, to name a few. Tackiness and agglomeration can occur during the curing process, and to circumvent these problems, a thin HPMC overcoat can be used [46, 57] or talc can be blended with coated dosage forms before placing them in the oven [16]. During the curing step, drug migration through the coating can occur, especially if the drug has a low melting point and affinity for the polymeric coating [56, 58]. In this case, a subcoating can avoid drug migration during the curing step [58]. After the curing step, coated dosage forms are stored under high temperature and humidity conditions (the International Committee on Harmonization recommends 25°C/60% RH and 40°C/75% RH) for a defined time. Often, drug release rate decreases after these storage conditions due to the well-known physical aging. Physical aging or enthalpy relaxation is observed with all amorphous polymers. Amorphous materials are not in thermodynamic equilibrium when they are cooled below their Tg [59]. Since this is an energetically unfavorable state, the polymer chains in a glassy film will reorient to achieve the equilibrium over time [60].

37

Specific volume

1 Introduction

Truly glassy state

Aging range

Equilibrium line

Tg

Tβ Temperature FIG. 3.2

Origin of physical aging. Tg is the glass transition temperature of the polymer and Tβ is the temperature of the highest secondary transition. Adapted from Guo J-H. Aging processes in pharmaceutical polymers. Pharm Sci Technol Today 1999;2(12):478–483.

TABLE 3.3 Physical properties of plasticizers commonly added to aqueous polymeric dispersions. Plasticizer

Water solubility (%)

Solubility parameter (J/cm3)1/2

Boiling point (°C)

Vapor pressure (mmHg)

TEC

6.5–6.9

20.4–21.1

294

1 at107°C

9.7

[12, 24–28]

Triacetin

7.0–7.1

18.5

258

1 at 100°C

7.52

[12, 26–28]

ATBC

< 0.01

17.7

326

0.8 at 170°C

ATEC

0.72

18.3

294

–

DBS

0.01

18.8

344–394

10 at 200°C

DEP

0.15

19.1

295

100 at 220°C

TBC

< 0.01

19.5

322

1 at 170°C

Vapor density (air 5 1)

14.1

[12, 26–28]

–

[26–28]

10.8

[12, 24, 28, 29]

7.66

[12, 24, 27, 28]

12.4

[12, 25–28]

TABLE 3.4 Effect of plasticizer type and content on MFT from different aqueous dispersions. Aqueous dispersion

Plasticizer

(%)

MFT (°C)

Ref.

Aquacoat ECD

TEC

25

30

[79]

30

84% CN in solution while Stugeron® showed significant CN precipitation, leaving only 7% CN in solution. On the stability prospect, the moisture-sealed ML-SNEP showed significant enhancement of CN chemical stability compared to liquid SNEDDS and SL-SNEP. In particular, ML-SNEP coated with Kollicoat Smartseal 30D showed robust stability and maintained 95% within all the storage conditions. The observed stability enhancement is attributed to the complete isolation between CN and SNEDDS layer as well as the effective moisture protection provided by Kollicoat Smartseal 30D. Hence the degradation problem could be eradicated. In addition, the incorporation of silicon dioxide, as an anti-adherent, had an important role in the inhibition of pellet agglomeration upon storage. Accordingly, ML-SNEP played a vital role in the stabilization of CN within lipid-based formulations and upon exposure to humidity. This innovative design offers several advantages such as enhancement of drug dissolution by SNEDDS technology, dual protection of unstable drugs against lipid-based excipients and atmospheric moisture, enhancing the product physical stability along with flexibility to

incorporate a wide range of drugs with various solubilization and stabilization requirements.

5 Conclusion The advantages and success of aqueousbased coatings are well documented in the literature. One of the major challenges is the storage stability of the aqueous-based coated dosage forms. Decreased drug release profiles due to continuous film formation are often reported. The continuous film formation or physical aging of aqueous-based coatings is highly dependent on coating formulation, coating conditions, and curing conditions. It is a sequential and interconnected process that must be evaluated carefully. Despite the fact that there are standard and recommended procedures, each application of aqueous polymeric dispersion is somehow unequaled. For instance, after any modification in the coating formulation, the coating and curing conditions need to be adjusted to reduce the aging effects as much as possible. If this adjustment is not performed, misinterpretation of the results can lead to confusing outcomes. In addition, the curing conditions must guarantee completeness of film formation (no further change in dissolution profiles) prior to long-term storage. Another problem faced by the formulator scientist is the long time of the storage stability tests. Consequently, it is crucial to identify possible aging effects and determine the underlying mechanism as early as possible. To understand better these complex mechanisms and possible interactions, coupled techniques are required to investigate the physical changes in the aqueous-based coatings. Besides drug release studies, thermal analysis, mechanical properties, film permeability, and free volume measurements may help to characterize the physical aging further. Recently, several methods have been successfully carried out to prevent aging, and stable

52

3. Aqueous polymeric coatings: New opportunities in drug delivery systems

drug release profiles were achieved during storage at different conditions. Moreover, it is important to consider the correct or appropriate packaging for the coated dosage form during storage. Fluid bed coating was recently introduced as an innovative application of aqueous-based coating to solidify SNEDDS into free-flowing pellets. This technique showed great potential to achieve enhanced solubilization and stabilization of poorly soluble drugs.

References [1] Skalsky B, Petereit H-U. Chemistry and application properties of polymethacrylate systems. In: McGinity JW, Felton LA, editors. Aqueous polymeric coatings for pharmaceutical dosage forms. 3rd ed. New York: Informa Healthcare; 2008. p. 237–77. [2] Miller DA, McGinity JW. Aqueous polymeric film coating. In: Augsburger LL, Hoag SW, editors. Pharmaceutical dosage forms: tablets. New York: Informa Healthcare; 2008. p. 399–438. [3] Kollicoat® protect technical information. Limburgerhof: BASF; 2010. [4] Gupta VK, Beckert TE, Deusch NJ, Hariharan M, Price JC. Investigation of potential ionic interactions between anionic and cationic polymethacrylates of multiple coatings of novel colonic delivery system. Drug Dev Ind Pharm 2002;28(2):207–15. [5] Petereit H-U, Weisbrod W. Formulation and process considerations affecting the stability of solid dosage forms formulated with methacrylate copolymers. Eur J Pharm Biopharm 1999;47(1):15–25. [6] Lecomte F, Siepmann J, Walther M, MacRae RJ, Bodmeier R. Polymer blends used for the aqueous coating of solid dosage forms: importance of the type of plasticizer. J Control Release 2004;99(1):1–13. [7] Bruce LD, Petereit H-U, Beckert T, McGinity JW. Properties of enteric coated sodium valproate pellets. Int J Pharm 2003;264(1–2):85–96. [8] Wesseling M, Bodmeier R. Drug release from beads coated with an aqueous colloidal ethylcellulose dispersion, Aquacoat®, or an organic ethylcellulose solution. Eur J Pharm Biopharm 1999;47(1):33–8. [9] Liu J, Williams RO. Long-term stability of heathumidity cured cellulose acetate phthalate coated beads. Eur J Pharm Biopharm 2002;53(2):167–73.

[10] Siepmann F, Siepmann J, Walther M, MacRae R, Bodmeier R. Aqueous HPMCAS coatings: effects of formulation and processing parameters on drug release and mass transport mechanisms. Eur J Pharm Biopharm 2006;63(3):262–9. [11] Thoma K, Bechtold K. Influence of aqueous coatings on the stability of enteric coated pellets and tablets. Eur J Pharm Biopharm 1999;47(1):39–50. [12] Carlin B, Li J-X, Felton LA. Pseudolatex dispersions for controlled drug delivery. In: James W, McGinity LAF, editors. Aqueous polymeric coatings for pharmaceutical dosage forms. 3rd ed. New York: Informa Healthcare; 2008. p. 1–46. [13] Lin F, Meier DJ. A study of latex film formation by atomic force microscopy. 1. A comparison of wet and dry conditions. Langmuir 1995;11(7):2726–33. [14] Keddie JL. Film formation of latex. Mater Sci Eng R: Rep 1997;21(3):101–70. [15] Zheng W, Sauer D, McGinity JW. Influence of hydroxyethylcellulose on the drug release properties of theophylline pellets coated with Eudragit® RS 30 D. Eur J Pharm Biopharm 2005;59(1):147–54. [16] Williams Iii RO, Liu J. Influence of processing and curing conditions on beads coated with an aqueous dispersion of cellulose acetate phthalate. Eur J Pharm Biopharm 2000;49(3):243–52. [17] Amighi K, Moes A. Influence of plasticizer concentration and storage conditions on the drug release rate from Eudragit RS 30D film-coated sustained-release theophylline pellets. Eur J Pharm Biopharm 1996;42 (1):29–35. [18] Okhamafe AO, York P. Studies of interaction phenomena in aqueous-based film coatings containing soluble additives using thermal analysis techniques. J Pharm Sci 1988;77(5):438–43. [19] Wu C, McGinity JW. Influence of ibuprofen as a solidstate plasticizer in Eudragit RS 30 D on the physicochemical properties of coated beads. AAPS PharmSciTech 2001;2(4):24. [20] Felton LA, McGinity JW. Influence of plasticizers on the adhesive properties of an acrylic resin copolymer to hydrophilic and hydrophobic tablet compacts. Int J Pharm 1997;154(2):167–78. [21] Gutierrez-rocca JC, McGinity JW. Influence of aging on the physical-mechanical properties of acrylic resin films cast from aqueous dispersions and organic solutions. Drug Dev Ind Pharm 1993;19(3):315–32. [22] Banker GS. Film coating theory and practice. J Pharm Sci 1966;55(1):81–9. [23] Pearnchob N. Evaluation of new film coating processes and materials. Berlin: Freie Universitaet Berlin; 2002.

References

[24] Frohoff-H€ ulsmann MA, Lippold BC, McGinity JW. Aqueous ethyl cellulose dispersion containing plasticizers of different water solubility and hydroxypropyl methyl-cellulose as coating material for diffusion pellets. II. Properties of sprayed films. Eur J Pharm Biopharm 1999;48(1):67–75. [25] Felton LA, Shah NH, Zhang G, Infeld MH, Malick AW, McGinity JW. Physical-mechanical properties of filmcoated soft gelatin capsules. Int J Pharm 1996;127 (2):203–11. [26] Gutierrez-Rocca J, McGinity JW. Influence of water soluble and insoluble plasticizers on the physical and mechanical properties of acrylic resin copolymers. Int J Pharm 1994;103(3):293–301. [27] Wang C-C, Zhang G, Shah NH, Infeld MH, Waseem Malick A, JW MG. Influence of plasticizers on the mechanical properties of pellets containing Eudragit® RS 30 D. Int J Pharm 1997;152(2):153–63. [28] Rowe RC, Sheskey PJ, Quinn ME. Handbook of pharmaceutical excipients. 6th ed. Pharmaceutical Press and American Pharmacists Association; 2009. [29] Mark JE, Zeng W, Du Y, Xue Y, Frisch H. Solubility parameters. In: Physical properties of polymers handbook. Springer New York; 2007. p. 289–303. [30] Aquacoat ECD product brochure. Philadelphia: FMC BioPolymer; 1996. [31] Lippold BC, Lippold BH, Sutter BK, Gunder W. Properties of aqueous, plastic Izer-containing ethyl cellulose dispersions and prepared films in respect to the production of oral extended release formulations. Drug Dev Ind Pharm 1990;16(11):1725–47. [32] Surelease product information. West Point: Colorcon; 2006. [33] Porter SC. Controlled-release film coatings based on ethylcellulose. Drug Dev Ind Pharm 1989;15 (10):1495–521. [34] Aquacoat CPD product brochure. Philadelphia: FMC BioPolymer; 1996. [35] AQOAT product brochure. Shin-Etsu: Tokyo; 2005. [36] Specifications and test methods for EUDRAGIT® RL 30 D and EUDRAGIT® RS 30 D. Darmstadt: EVONIK R€ ohm GmbH; 2007. [37] Specifications and test methods for EUDRAGIT® NE 30 D. Darmstadt: Evonik R€ ohm GmbH; 2007. [38] Specifications and test methods for EUDRAGIT® NM 30 D. Darmstadt: EVONIK R€ ohm GmbH; 2007. [39] Specifications and test methods for EUDRAGIT® L 30 D-55. Darmstadt: EVONIK R€ ohm GmbH; 2007. [40] Kossena G, Charman W, Boyd B, Porter C. A novel cubic phase of medium chain lipid origin for the delivery of poorly water soluble drugs. J Control Release 2004;99 (2):217–29.

53

[41] Wei H, Li-Fang F, Bai X, Chun-Lei L, Qing D, YongZhen C, et al. An investigation into the characteristics of chitosan/Kollicoat SR30D free films for colonic drug delivery. Eur J Pharm Biopharm 2009;72(1):266–74. [42] Crawford RR, Esmerian OK. Effect of plasticizers on some physical properties of cellulose acetate phthalate films. J Pharm Sci 1971;60(2):312–4. [43] Bodmeier R, Paeratakul O. Process and formulation variables affecting the drug release from chlorpheniramine maleate-loaded beads coated with commercial and selfprepared aqueous ethyl cellulose pseudolatexes. Int J Pharm 1991;70(1–2):59–68. [44] Johnson K, Hathaway R, Leung P, Franz R. Effect of triacetin and polyethylene glycol 400 on some physical properties of hydroxypropyl methylcellulose free films. Int J Pharm 1991;73(3):197–208. [45] Rohera B, Parikh N. Influence of plasticizer type and ˆ ® film properties. Pharm Dev coat level on SureleaseA Technol 2002;7(4):407. [46] Maejima T, McGinity J. Influence of film additives on stabilizing drug release rates from pellets coated with acrylic polymers. Pharm Dev Technol 2001;6(2):211. [47] Bechard SR, Quraishi O, Kwong E. Film coating: effect of titanium dioxide concentration and film thickness on the photostability of nifedipine. Int J Pharm 1992;87(1–3):133–9. [48] Rowe RC. Modulus enhancement in pigmented tablet film coating formulations. Int J Pharm 1983;14(2–3):355–9. [49] Felton LA, McGinity JW. Influence of insoluble excipients on film coating systems. Drug Dev Ind Pharm 2002;28(3):225–43. [50] Frohoff-H€ ulsmann MA, Schmitz A, Lippold BC. Aqueous ethyl cellulose dispersions containing plasticizers of different water solubility and hydroxypropyl methylcellulose as coating material for diffusion pellets. I. Drug release rates from coated pellets. Int J Pharm 1999;177(1):69–82. [51] Wong D, Bodmeier R. The impact of the use of flocculated colloidal dispersions on the drug release from drug-containing films and coated tablets. Pharm Res 1994;11:s-185. [52] Siepmann F, Muschert S, Zach S, Leclercq B, Carlin B, Siepmann J. Carrageenan as an efficient drug release modifier for ethylcellulose-coated pharmaceutical dosage forms. Biomacromolecules 2007;8(12):3984–91. [53] Dashevsky A, Ahmed AR, Mota J, Irfan M, Kolter K, Bodmeier RA. Effect of water-soluble polymers on the physical stability of aqueous polymeric dispersions and their implications on the drug release from coated pellets. Drug Dev Ind Pharm 2010;36(2):152–60. [54] Siepmann F, Hoffmann A, Leclercq B, Carlin B, Siepmann J. How to adjust desired drug release patterns

54

[55]

[56]

[57]

[58]

[59] [60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

3. Aqueous polymeric coatings: New opportunities in drug delivery systems

from ethylcellulose-coated dosage forms. J Control Release 2007;119(2):182–9. Gilligan CA, Li Wan Po A. Factors affecting drug release from a pellet system coated with an aqueous colloidal dispersion. Int J Pharm 1991;73(1):51–68. Bodmeier R, Paeratakul O. The effect of curing on drug release and morphological properties of ethylcellulose Pseudolatex-coated beads. Drug Dev Ind Pharm 1994;20(9):1517–33. Harris M, Ghebre-Sellassie I, Nesbitt R. Water based coating process for sustained release. Pharm Technol 1986;10(9):102–7. Dashevsky A, Wagner K, Kolter K, Bodmeier R. Physicochemical and release properties of pellets coated with Kollicoat® SR 30 D, a new aqueous polyvinyl acetate dispersion for extended release. Int J Pharm 2005;290 (1–2):15–23. Guo J-H. Aging processes in pharmaceutical polymers. Pharm Sci Technol Today 1999;2(12):478–83. Kucera SA, Felton LA, McGinity JW. Physical aging of polymers and its effect on the stability of solid oral dosage forms. In: Felton LA, McGinity JW, editors. Aqueous polymeric coatings for pharmaceutical dosage forms. 3rd ed. New York: Informa Healthcare; 2008. p. 445–74. Greiner R, Schwarzl FR. Volume relaxation and physical aging of amorphous polymers. I. Theory of volume relaxation after single temperature jumps. Colloid Polym Sci 1989;267(1):39–47. Priestley RD, Ellison CJ, Broadbelt LJ, Torkelson JM. Structural relaxation of polymer glasses at surfaces, interfaces, and in between. Science 2005;309 (5733):456–9. Guo J-H, Robertson RE, Amidon GL. Influence of physical aging on mechanical properties of polymer free films: the prediction of long-term aging effects on the water permeability and dissolution rate of polymer film-coated tablets. Pharm Res 1991;8(12):1500–4. Bodmeier R, Paeratakul O. Plasticizer uptake by aqueous colloidal polymer dispersions used for the coating of solid dosage forms. Int J Pharm 1997;152(1):17–26. Siepmann J, Paeratakul O, Bodmeier R. Modeling plasticizer uptake in aqueous polymer dispersions. Int J Pharm 1998;165(2):191–200. Wesseling M, Bodmeier R. Influence of plasticization time, curing conditions, storage time, and core properties on the drug release from aquacoat-coated pellets. Pharm Dev Technol 2001;6(3):325–31. Yang QW, Flament MP, Siepmann F, Busignies V, Leclerc B, Herry C, et al. Curing of aqueous polymeric film coatings: importance of the coating level and type of plasticizer. Eur J Pharm Biopharm 2010;74 (2):362–70.

[68] Hutchings D, Kuzmak B, Sakr A. Processing considerations for an EC latex coating system: influence of curing time and temperature. Pharm Res 1994;11 (10):1474–8. [69] Hutchings D, Clarson S, Sakr A. Studies of the mechanical properties of free films prepared using an ethylcellulose pseudolatex coating system. Int J Pharm 1994;104 (3):203–13. [70] Chowhan ZT, Amaro AA, Chi L-H. Comparative evaluations of aqueous film coated tablet formulations by high humidity aging. Drug Dev Ind Pharm 1982;8 (5):713–37. [71] McConnell E, Tutas J, Mohamed M, Banning D, Basit A. Colonic drug delivery using amylose films: the role of aqueous ethylcellulose dispersions in controlling drug release. Cellulose 2007;14(1):25–34. [72] Lorck CA, Grunenberg PC, J€ unger H, Laicher A. Influence of process parameters on sustained-release theophylline pellets coated with aqueous polymer dispersions and organic solvent-based polymer solutions. Eur J Pharm Biopharm 1997;43(2):149–57. [73] Surelease. The influence of post coating thermal treatment on film properties and drug release from ethylcellulose barrier membrane coating systems. Colorcon; 2009. [74] Missaghi S, Fegely KA, Rajabi-Siahboomi AR. Investigation of venlafaxine HCl release from extruded and spheronized beads coated with ethylcellulose using organic or aqueous coating systems. In: Controlled release society annual meeting; 2008. [75] Sheen P-C, Sabol PJ, Alcorn GJ, Feld KM. Aquesons films coating stuied of sustabined release niciotine acid fellets: an in-vitro evaluation. Drug Dev Ind Pharm 1992;18(8):851–60. [76] Rekhi GS, Porter SC, Jambhekar SS. Factors affecting the release of propranolol hydrochloride from beads coated with aqueous polymeric dispersions. Drug Dev Ind Pharm 1995;21(6):709–29. [77] Duerig T, Harcom WW, Kinsey BR, Tewari D, Lewis RK. Fundamental studies on modified release pellets coated with ethylcellulose. Aqualon: Wilmington; 2005. [78] Williams RO, Liu J. The influence of plasticizer on heathumidity curing of cellulose acetate phthalate coated beads. Pharm Dev Technol 2001;6(4):607–19. [79] Obara S, McGinity JW. Influence of processing variables on the properties of free films prepared from aqueous polymeric dispersions by a spray technique. Int J Pharm 1995;126(1–2):1–10. [80] Meier C. Physico chemical properties of anionic EUDRAGIT® polymers and their influences on film properties. In: 4th international GI-targeting workshopEvonik R€ ohm GmbH: Darmstadt; 2008.

References

[81] Wesseling M, Kuppler F, Bodmeier R. Tackiness of acrylic and cellulosic polymer films used in the coating of solid dosage forms. Eur J Pharm Biopharm 1999;47 (1):73–8. [82] Bhattacharjya S, Wurster D. Investigation of the drug release and surface morphological properties of filmcoated pellets, and physical, thermal and mechanical properties of free films as a function of various curing conditions. AAPS PharmSciTech 2008;9(2):449–57. [83] Hamed E, Sakr A. Effect of curing conditions and plasticizer level on the release of highly lipophilic drug from coated multiparticulate drug delivery system. Pharm Dev Technol 2003;8(4):397–407. [84] Noemi CG, Kalidas K, Kenneth RM. Investigation of film curing stages by dielectric analysis and physical characterization. J Pharm Sci 1997;86(3):329–34. [85] Wurster DE, Bhattacharjya S, Flanagan DR. Effect of curing on water diffusivities in acrylate free films as measured via a sorption technique. AAPS PharmSciTech 2007;8(3). [86] Nimkulrat S, Suchiva K, Phinyocheep P, Puttipipatkhachorn S. Influence of selected surfactants on the tackiness of acrylic polymer films. Int J Pharm 2004;287(1–2):27–37. [87] Lin A, Muhammad N, Pope D, Augsburger L. A study of the effects of curing and storage conditions on controlled release diphenhydramine HCl pellets coated with ˆ ® NE30D. Pharm Dev Technol 2003;8(3):277. EudragitA [88] Mota J. Matrix- and reservoir-type oral multiparticulate drug delivery systems. Berlin: Freien Universit€at Berlin; 2010. [89] G€ opferich A, Lee G. The influence of endogenous surfactant on the structure and drug-release properties of Eudragit NE30D-matrices. J Control Release 1992;18 (2):133–44. [90] Lin AY, Muhammad NA, Pope D, Augsburger LL. Study of crystallization of endogenous surfactant in Eudragit NE30D-free films and its influence on drugrelease properties of controlled-release diphenhydramine HCl pellets coated with Eudragit NE30D. AAPS PharmSci 2001;3(2). [91] Bajdik J, Pintye-Ho´di K, Regdon G, Fazekas P, Szabo´Revesz P, Ero˝s I. The effect of storage on the behaviour of Eudragit NE free film. J Therm Anal Calorim 2003;73 (2):607–13. ´ , S€ [92] Zelko´ R, Orba´n A uvegh K. Tracking of the physical ageing of amorphous pharmaceutical polymeric excipients by positron annihilation spectroscopy. J Pharm Biomed Anal 2006;40(2):249–54. [93] Felton L, Baca M. Influence of curing on the adhesive and thermomechanical properties of an applied acrylic polymer. Pharm Dev Technol 2001;6(1):53.

55

[94] Shao Z, Morales L, Diaz S, Muhammad N. Drug release from kollicoat SR 30D-coated nonpareil beads: evaluation of coating level, plasticizer type, and curing condition. AAPS PharmSciTech 2002;3(2):87–96. [95] Irfan M, Ahmed AR, Dashevsky A, Kolter K, Bodmeier R. Formulation parameters affecting the adhesion of Kollicoat® SR 30 D coatings to the drug layer in coated pellets and their implications on curing phenomena. Copenhagen, Denmark: Controlled Release Society; 2009. [96] K€ orber M, Hoffart V, Walther M, Macrae RJ, Bodmeier R. Effect of unconventional curing conditions and storage on pellets coated with Aquacoat ECD. Drug Dev Ind Pharm 2010;36(2):190–9. [97] Wu C, McGinity JW. Influence of relative humidity on the mechanical and drug release properties of theophylline pellets coated with an acrylic polymer containing methylparaben as a non-traditional plasticizer. Eur J Pharm Biopharm 2000;50(2):277–84. [98] Zheng W, McGinity JW. Influence of Eudragit® NE 30 D blended with Eudragit® L 30 D-55 on the release of phenylpropanolamine hydrochloride from coated pellets. Drug Dev Ind Pharm 2003;29(3):357. [99] Wu C, McGinity J. Influence of an enteric polymer on drug release rates of theophylline from pellets coated with Eudragit® RS 30D. Pharm Dev Technol 2003;8 (1):103. [100] Siepmann F, Muschert S, Leclercq B, Carlin B, Siepmann J. How to improve the storage stability of aqueous polymeric film coatings. J Control Release 2008;126(1):26–33. [101] Kranz H, Gutsche S. Evaluation of the drug release patterns and long term stability of aqueous and organic coated pellets by using blends of enteric and gastrointestinal insoluble polymers. Int J Pharm 2009;380 (1–2):112–9. [102] Kucera SA, McGinity JW, Weijia Z, Shah NH, Malick AW, Infeld MH. Use of proteins to minimize the physical aging of EUDRAGIT® sustained release films. Drug Dev Ind Pharm 2007;33(7):717–26. [103] Ensslin S, Moll K, Haefele-Racin T, M€ader K. Safety and robustness of coated pellets: self-healing film properties and storage stability. Pharm Res 2009;26 (6):1534–43. [104] Skultety PF, Sims SM. Evaluation of the loss of propylene glycol during aqueous film coating. Drug Dev Ind Pharm 1987;13(12):2209–19. [105] Muschert S, Siepmann F, Cuppok Y, Leclercq B, Carlin B, Siepmann J. Improved long term stability of aqueous ethylcellulose film coatings: importance of the type of drug and starter core. Int J Pharm 2009;368(1–2):138–45.

56

3. Aqueous polymeric coatings: New opportunities in drug delivery systems

[106] Lei Y, Lu Y, Qi J, Nie S, Hu F, Pan W, et al. Solid selfnanoemulsifying cyclosporin a pellets prepared by fluid-bed coating: preparation, characterization and in vitro redispersibility. Int J Nanomed 2011;6: 795–805. [107] Wang Z, Sun J, Wang Y, Liu X, Liu Y, Fu Q, et al. Solid self-emulsifying nitrendipine pellets: preparation and in vitro/in vivo evaluation. Int J Pharm 2010;383 (1–2):1–6. [108] Shahba AA, Alanazi FK, Mohsin K, Abdel-Hamid M. Stability assessment of cinnarizine in self-emulsifying drug delivery systems. Latin Am J Pharm 2012;31 (4):549–54. [109] Gumaste SG, Dalrymple DM, Serajuddin ATM. Development of solid SEDDS. V. Compaction and drug release properties of tablets prepared by adsorbing lipid-based formulations onto Neusilin® US2. Pharm Res 2013;30(12):3186–99. [110] Bansal T, Mustafa G, Khan ZI, Ahmad FJ, Khar RK, Talegaonkar S. Solid self-nanoemulsifying delivery systems as a platform technology for formulation of poorly soluble drugs. Crit Rev Ther Drug Carrier Syst 2008;25(1):63–116. [111] Gupta S, Kesarla R, Omri A. Formulation strategies to improve the bioavailability of poorly absorbed drugs

[112]

[113]

[114]

[115]

[116]

[117]

with special emphasis on self-emulsifying systems. ISRN Pharm 2013;2013:16. Abdalla A, M€ader K. Preparation and characterization of a self-emulsifying pellet formulation. Eur J Pharm Biopharm 2007;66(2):220–6. Shahba AA, Ahmed AR, Mohsin K, Abdel-Rahman SI, Alanazi FK. Solidification of cinnarizine selfnanoemulsifying drug delivery systems by fluid bed coating: optimization of the process and formulation variables. Pharmazie 2017;72(3):143–51. Shahba AA, Alanazi FK, Abdel-Rahman SI. Stabilization benefits of single and multi-layer self-nanoemulsifying pellets: a poorly-water soluble model drug with hydrolytic susceptibility. PLoS One 2018;13(7). Byrn SR, Xu W, Newman AW. Chemical reactivity in solid-state pharmaceuticals: formulation implications. Adv Drug Deliv Rev 2001;48(1):115–36. Shahba AA-W, Ahmed AR, Alanazi FK, Mohsin K, Abdel-Rahman SI. Multi-layer self-nanoemulsifying pellets: an innovative drug delivery system for the poorly water-soluble drug Cinnarizine. AAPS PharmSciTech 2018; Shahba A, Alanazi F, Ahmed A. Multi-layer selfnanoemulsifying pellets for dual enhancement of drug solubility and stability. Saudi patent no# 6357. Kingdom of Saudi Arabia. 2019.

C H A P T E R

4

Large-scale manufacturing of nanoparticles—An industrial outlook Ranjita Shegokara, Mostafa Nakachb a

Capnomed GmbH, Zimmern, Germany b Sanofi R&D, Vitry sur Seine, France

1 Introduction The literature describes multiple academic level lab-scale processes for batch sizes up to 1 liter, and very limited reports are published on pilotscale and industrial-scale manufacturing of nanoparticles [1]. Pharmaceutical companies need fast implementable, adaptable (formulation type, sterility requirement), and easy to maintain (in terms of cleaning) production lines to produce nanoparticles for clinical trials and commercial use. The transfer of manufacturing process from lab scale to large scale is a complex operation and may lead to inconsistent product quality if not done properly. It is clear that the proper adaptations of manufacturing lines to process scaleable formulation and regular checks on key process parameters play an important role in determining the quality of the end product. Depending on the quality, target nanoproduct specifications, desired physico-chemical parameters, and sterility requirements, existing technology can be employed or adapted. The selection of large-scale nanoparticle production methods in the pharmaceutical industry depend on three factors:

A prerequisite for introduction of any particulate or colloidal nanoparticulate drug delivery system into the pharmaceutical market is the availability of a GMP certified large-scale production/manufacturing process. The process itself needs to be qualified and validated, and to be accepted by the regulatory authorities. In addition to all these factors, manufacturers look for cost-effective, easy, and consistent production and operation setup. Ideally, the manufacturing process should yield a product of a high quality with “zero” to “acceptable” levels on contamination set by regulatory authorities or in-house criteria, e.g., elemental impurities from manufacturing equipment or residual solvent. Fig. 4.1 depicts commonly studied nanoparticulate systems in literature whereas Fig. 4.2 shows currently approved nanoproducts and those under investigation as per type of drug delivery system. For decades, large-scale production methods have been established for microparticles, but there are still major challenges in establishment of largescale manufacturing process for nanoparticles.

Drug Delivery Aspects https://doi.org/10.1016/B978-0-12-821222-6.00004-X

57

# 2020 Elsevier Inc. All rights reserved.

58

4. Large-scale manufacturing of nanoparticles—An industrial outlook

Polymeric nanoparticles Liposomes Solid lipid nanoparticles Nanoemulsions Nanostructured lipid carriers Polymer lipid hybrid Nanosuspension Cubosomes

FIG 4.1 Typically studied drug delivery systems in the pharma literature (PubMed databased accessed on 02.07.2019 July at 10.15 European standard time).

2%

2% 4%

10%

20%

Inorganic (5)

30%

Nanocrystal (2) 3% 3%

Inorganic (2) 56%

Micelles (1) 34%

Proteins (2)

Polymer (11)

15%

Polymer (15) Nanocrystal (15)

Lipsome (33)

3%

Lipsome (10)

18%

Micelles (9) Proteins (1) Dendrimer (2)

FIG. 4.2 Nanoparticles approved by authorities to date for clinical use (left) and for investigational drugs. Figure obtained with permission from article by Lee Ventola C. Progress in nanomedicine: approved and investigational nanodrugs. P&T 2017;42 (12):742–55.

- type of nanoparticles; - type of approach used (bottom-up vs. topdown); and - regulatory requirements. Other determining parameters such as the nature of active pharmaceutical ingredient (API), temperature stability of API, use of solvent,

and selection of excipients used may vary depending upon the above three factors, e.g., conventional liquid systems like micro-multiple emulsion systems (pharma or nutritional), liposomes, etc. can be produced by high shear mixing techniques, high pressure homogenization (HPH), cross-flow methods, or microfluidics. Depending upon the technique selected and API

59

2 Nanosuspensions

temperature stability and solubility, the required key manufacturing process parameters change as well as the excipient selection. According to the biopharmaceutical classification system (BCS), class II drugs have poor solubility but high permeation profile while class IV drugs have poor solubility and permeability, which generally limits their application. The challenge is more intense for APIs having high log P value. Various techniques like salt selection, co-crystals, amorphous solid dispersions, complexation with beta-cyclodextrins, lipid formulation, micronization, and nanoparticles formation can help to overcome the solubility challenge. However, percentage drug loading and total solubility enhancement remain low with many drug delivery systems; in addition, each technique has its own advantages and limitations. The manufacturing process must be chosen based on several factors on a case-by-case basis. However, it is highly likely that the selection of technology will be driven by drug properties and the purpose of drug delivery system (e.g., to reduce toxicity or to sustain release). Among the several approaches used to overcome solubility challenges, nanosuspension is the most common [2]. Nanosuspensions have the following characteristics: - highly practical approach; - easily adaptable and transferrable into solid form; - industrially feasible to scale up; - offer increased dissolution velocity; - enhanced saturation solubility; - improve the bioavailability of the drug; - suitable for a wide range of administration routes; and - can be directly injected into system circulation (if in the desired size range). In addition to nanosuspension, lipid nanoparticles are a widely investigated drug delivery system which offers patient-friendly drug delivery [3]. Lipid nanoparticles like nanosuspension

offer various advantages to deliver drugs therapeutically to the target site without severe toxicity [4, 5]. Keeping in mind that increasing use and popularity of nanosuspension or lipid nanoparticles by scientific pharma community motivated the design of this chapter. The main aim of this chapter is to discuss the large-scale manufacturing aspects and considerations of two widely studied and established drug delivery systems viz. nanosuspensions and lipid nanoparticles, which are increasingly used by the scientific community. Some steps, such as mixing, filtration, solvent removal, lyophilization, conversion into solid product, and sterilization, are excluded from this chapter due to limited scope. However, all other core unit operations and related factors of the manufacturing process are described in detail.

2 Nanosuspensions The in vivo activity of a drug depends on its solubility and permeability profile. Therefore, poorly soluble compounds are difficult to apply. Today there are many strategies to overcome poor solubility, e.g., micelles, self-emulsifying drug delivery systems, microemulsions, and cyclodextrins. However, one of the most successful strategies is the production of drug nanocrystals. Key features of nanocrystals are an increased solubility and dissolution velocity, as well as an increased adhesiveness on biological membranes. These aspects are very well known and have led to more than 10 oral products already being in the pharmaceutical market. These products possess an increased oral bioavailability, a reduced inter-patient bioavailability, and reduced food effect on the bioavailability of the drug. In comparison to the solutions (i.e., drug is dissolved), suspensions lead to a constant release of the drug over time and to a higher dermal bioavailability [6]. For the manufacturing of nanosuspensions, two approaches are mainly used [4, 5, 7, 8]: top-down and bottom-up.

60

4. Large-scale manufacturing of nanoparticles—An industrial outlook

(i) The top-down approach uses larger (macroscopic) starting structures which can be externally controlled in the processing and transformed into nanostructures via application of severe plastic deformation using mechanical, chemical, or other forms of energy. Examples include wet media milling or bead milling (shear forces) and HPH (cavitation force). (ii) The bottom-up approach includes the miniaturization or synthesis of materials components (up to atomic level) with a further self-assembly process to form nanostructures. Examples include sol-gel processing, precipitation, supercritical, aerosol-based, chemical vapor deposition (CVD), plasma or flame spraying synthesis, laser pyrolysis, and atomic or molecular condensation.

TABLE 4.1 Comparison of two commonly used industrially feasible technologies.

Both approaches can be implemented in gas, liquids, supercritical fluids, or solid states, or in a vacuum environment. However, the key attributes to control remain the same: particle size, particle shape, size distribution, particle composition, degree of particle agglomeration, and others as needed.

- brittleness of the API; - percentage of solid content in recipe; - type and percentage of wetting or dispersing agent used; and - processing parameters:

2.1 Top-down approach A limited number of studies are published on large-scale manufacturing of nanosuspensions based on top-down approaches [9]. Typically employed techniques are wet media milling and HPH (Table 4.1). It is well proven that bead milling is a more robust technique than HPH due to the tremendous shear forces applied in HPH; however, HPH gives tighter particle size controls. In addition, each technique has unique features and needs to be selected depending upon the desired final product profile and nature of the API. The ideal indicator to determine the performance and effectiveness of technique is particle size distribution (PSD). Fig. 4.3 indicates key parameters to consider during

Milling

HPH

- High energy operation - Shear forces resulted from collisions of media and API - API properties are key - Both cold and room temperature processing possible

- Cavitation forces - High energy operation - Particle breakage due to force and collision among particles - API properties are key - Cold and room temperature-assisted processing possible

R&D and before finalization of technology for large-scale production. Typical parameters that determine the PSD distribution of the final product are:







batch size; energy supplied; length of milling time; continuous vs. discontinuous mode operation; and
temperature. Unit operations performed at a high amount of energy (more than 200 kJ/kg3) are common in the pharmaceutical industry. Nakach et al. designed a study plan to understand impact of two techniques, bead milling and HPH, by evaluating the effect of the processing parameters on the quality attributes of the final product. A total seven poorly soluble APIs of different properties were selected. Nanosuspensions containing 10%–20% solid content were processed using stabilizers selected: PVP K30, PVP K25, and poloxamer in combination with either SDS or mPEG 2000 DSPE. The processing of nanosuspensions was carried out as described in

61

2 Nanosuspensions

FIG. 4.3

Key parameters to consider for technology selection on a large scale.

Table 4.2. The authors found that the impact of the stabilizer content and type is critical, and proper selection of type of surfactant and concentration was key to reach a stable particle size profile. Different APIs responded differently to the surfactants chosen; this could be due to the difference between APIs in terms of degree of brittleness, surface area available, defect density, and grinding limit. The milling kinetic profile used for each composition was different. Authors reported no specific correlation between milling ability and physico-chemical characteristics of APIs. The total percentage of solid content in formulation impacted the milling outcome as expected. HPH followed a similar set of rules. For bead milling and HPH, a different set of milling or homogenization cycles was required to achieve similar particle size of the final product when percentage of solid in formulation (ultimately the viscosity) is increased above 20% compared

TABLE 4.2 Process setting used for manufacturing of nanosuspensions by Nakach et al. Milling

HPH ®

1. Stirred pin (Labstar ) milling system [volume (555 mL), diameter (95 mm) and length of the milling chamber (117 mm)] 2. Stirred pin (LMZ2®) milling system [volume (1600 mL), diameter (128 mm) and length of the milling chamber (194.5 mm)]

1. Panda homogenizer® with a homogenization capacity of 10 L/h at 1400 bars 2. NS 2006 homogenizer® with a homogenization capacity of 35 L/h at 1400 bars 3. NS3024 homogenizer® with a homogenization capacity of 300 L/h at 1400 bars

Experimental settings:

Experimental settings:

- Cross-linked polystyrene bead and zirconium oxide bead (500 μm) milling media - Temperature (15°C) and rotation speed constant

- Homogenization pressure was 600–1400 bar, keeping the temperature at 15°C

62

4. Large-scale manufacturing of nanoparticles—An industrial outlook

to 10%: HPH has a limited impact on particle size while milling has a higher impact based on percentage of solid content. In both technologies, authors found that the increase of milling energy leads to a faster milling kinetic. They also reported that zirconium oxide beads performed better in terms of milling kinetics compared to polystyrene beads due to their higher density of 6.1 g/mL than that of 1.1 g/mL for polystyrene. Authors reported successful large-scale production of nanosuspensions by both technologies with optimized milling and homogenization parameters from 10 to 300 L/h. In another published report [10], apigenin nanocrystals were scaled up from lab scale (20 g) to pilot scale (3 kg) by a scale-up factor of 150. Apigenin is a known flavonoid with potential application in pharma, cosmetic, and nutritional benefits. Two different techniques were applied for production of nanosuspension based on the top-down approach (Table 4.3). - lab scale batch: 20 g—HPH and - pilot scale batch: 3 kg—smartCrystal combination technology (CT), combining pearl milling and a subsequent HPH (1 cycle, 300 bar) in continuous mode. Although the pilot scale-up batch size was 3 kg, as the mode of operation was continuous,

a 100 kg batch could be produced; it was only due to limitations on cost that a 3 kg batch was produced. As discussed before, in HPH particles pass through a “μm” gap under pressure, causing the particle size reduction by cavitation forces, whereas in smartCrystal technology, a combination of HPH and milling is used, meaning synergy of shear and cavitation forces. In pilot scale-up, an author used the discontinuous mode; this arrangement is transferable to largescale production to generate a concentrated suspension which can then be further diluted to a batch size in “tons.” The author reported that the nanocrystals from both production processes were comparable in size, stability, and crystallinity profile. The smartCrystal technology offered robust processing of viscous suspension (with higher amounts of solid), meaning a smaller concentrated total volume to be processed in large-scale production which can later be diluted to a suitable concentration. The authors suggested that one can reduce particle size further by reducing the size of milling media to, e.g., 0.2–0.4 mm bead size. However, the major challenge in doing so would be to separate these smaller beads from a nanosized product, which will make the separation process more tedious and costly. The authors confirmed comparable particle sizes of lab scale (250 nm)

TABLE 4.3 Production settings used for scale-up of apigenin nanosuspensions. HPH

SmartCrystal technology

Composition: Apigenin 10.0% (w/w) + Plantacare 2000 (1% w/w) + water (q.s. to 100%)

Composition: Apigenin 10.0% (w/w) + Plantacare 2000 (1% w/w) + water (q.s. to 100%)

Batch size: 20 g

First step-milling

Micro LAB 40 (APV Deutschland GmbH,

Batch size: 3 kg (>100 kg concentrate is possible)

Germany)

Discontinuous mode: seven passages

Low pressure premilling Two cycles each at 200, 500 and 1 cycle at 1000 bar

Switzerland) Media: Yttria stabilized zirconia milling pearls of size 0.4–0.6 mm

High pressure milling Actual homogenization was performed by

Second step-low pressure homogenization The milled nanosuspensions were diluted with 1.0% (w/w) aqueous

applying 30 cycles at 1500 bar.

surfactant solution and passed through an Avestin C50 piston-gap

Equipment: agitating pearl mill B€ uhler PML-2 (B€ uhler AG,

homogenizer (Avestin Europe GmbH, Mannheim, Germany) at 300 bars (1 cycle)

63

2 Nanosuspensions

FIG. 4.4 XRD patterns (from bottom to top):

Intensity

a—aqueous apigenin nonpreserved scaled-up nanosuspension, b—nanosuspension after addition of preservative Euxyl PE 9010, e c—apigenin coarse powder, d—nonpreserved d nanosuspension after drying, e—nanosuspension after addition of preservative c Euxyl PE9010 after drying.

b a 2 theta

and scaled-up batch (approx. 200 nm) due to implementation of robust milling technology and stable formulation composition. Fig. 4.4 clearly indicates no changes in crystallinity of apigenin nanosuspensions at lab scale and at pilot scale when processed using two different techniques from the top-down class. The addition of preservative did not affect the crystallinity of the scaled-up product. The degree of crystallinity of the original drug is higher in intensity (graph c) and is significantly reduced after nanonization, although keeping the fingerprint the same, which clearly indicates an absence of amorphous state generation. To get the final pharmaceutical form, nanosuspension can be kept in liquid form if it is physically and chemically stable. In the opposite case, nanosuspension is converted in solid dosage form (lyophilized or spray-dried); depending upon the technique employed, degree of crystallinity may vary according to the use of cryoprotectant or spraying material however the fingerprint of original drug is followed even after drying (graphs d and e)

2.2 Bottom-up approach The typically used bottom-up approach in the pharmaceutical industry for nanoparticle production includes precipitation, antisolvent diffusion, solvent emulsification, etc. [11]. As the main focus of this chapter is on key technologies (like top-down), the authors prefer not to

discuss the bottom-up approach in detail. However, key process attributes of both technologies are listed in Table 4.4. The bottom-up technology is applied for producing a wide variety of nanocrystalline or amorphous suspensions, lipid nanoparticles, emulsions, etc. The process usually involves the use of organic solvents which are later removed in the manufacturing step, by either extraction or evaporation methods (Fig. 4.5). The process starts from the step where drugs are dissolved in an organic solvent and mixed with the aqueous phase, causing precipitation. Sometimes nanoparticle formation is induced by condensation methods by nucleation and crystal growth. In this section, only the precipitation technique is discussed. Readers are invited to refer to specific literature for more details [12, 13]. In addition, stabilizer agents are added to the organic or water phase to protect the nanosuspension against flocculation and Ostwald ripening over time. In desired cases such as where API has degradation in aqueous media, suspensions can be converted into solid form by sprayor freeze-drying [14]. Solvent emulsification techniques can be combined with additional homogenization or a microfluidic step to reduce particle size further. This will result in an extra processing step and a cost increase. Fig. 4.6 shows the distribution of commonly employed techniques in the literature for production of nanoparticles. It can be noted that 99% of

TABLE 4.4 Comparison of top-down and bottom-up manufacturing processes. Top-down

Bottom-up

The process is free from organic solvent

Organic solvent is needed

APIs that are poorly soluble in organic and aqueous media can be processed using the top-down process

APIs must have a good organic solubility

The number of manufacturing steps are easy to adapt and reproducible

Process steps are critical; small error during processing will change final product characteristic

The risk of generating an amorphous state or polymorphic transition is reduced to negligible

Mostly amorphous and polymeric transition is a common challenge

The target particle size of the suspension is more easily obtained and controlled

Target particle size is very much dependent on each step of processing

It is possible to process highly concentrated suspensions (up to 40% w/w)

There is a limitation on percentage of API solubility in organic solvent

The scaling up of the process is feasible and well established

The scale-up process is challenging; small variations can cause severe product-to-product variation

It is a robust, adaptable, reproducible manufacturing process

Sensitive, exhaustive processing steps are only reproducible to some extent

Wear of grinding media is possible mainly when stirred bead mills are used

Removal of organic solvent by extraction or vacuum distillation is required to make the product acceptable with regard to amounts of residual solvents for the safety of the patient (ICH guideline Q3C (R6) on impurities: guideline for residual solvents)

API + Solvent + Polymer + surfactant1

Water + aq. surfactant + other

Organic phase

Water phase

Precipitation

Solvent removal

Lyophilization

FIG. 4.5

Concentrate formation

Flow chart of manufacturing of nanosuspension using the solvent diffusion method.

2 Nanosuspensions

Sonication

65 FIG. 4.6 Technologies employed to produce

HPH

nanoparticles in the literature (PubMed database accessed on 02.07.2019 at 23.43 European time).

Supercritical

Media milling

Microfluidic nanosupension

Solvent evaporation

Precipitation

Microfluidic nanoparticles

published reports are academic and have no possibility to use sophisticated industry standard equipment, hence only easily available techniques like solvent evaporation or sonication are commonly employed. In industry, although most of the data held are confidential and not published, through various reports it is clear that HPH, media milling, and microfluidics are key technologies used in commercial production in the pharmaceutical industry [15].

2.3 Considerations on scale-up from lab to industrial scale Any nanoparticulate drug delivery system will follow the path sketched in Fig. 4.7. Typically, at R&D level or lab-scale level, initial screening of formulation and technology is done depending upon the characteristics of API and route of administration within required regulatory limitations, e.g., in case of nanosuspensions, API characteristics like crystallinity, degree of brittleness, solubility profile, and morphology are studied. The technology used to improve solubility is then selected, considering the following factors:

- selection of manufacturing technique (topdown or bottom-up); - batch production mode (recycling mode or discrete passages); - desired quality and target product profile based on past experiences; and - availability-adaptation of current production step up. As an example, if HPH is used for production of nanosuspensions, a similar technique can be applied for production of lipid nanoparticles, emulsions, or liposomes just by changing the temperature conditions and other process parameters. During R&D, ideally the technology that allows easy scalability must be selected to speed up the process engineering phase [16]. In general, the technology must be suitable for a wide range of dosage forms, e.g., sterile injectable parenterals to oral tablet or dermal product. In the case of parenteral route administration, the product needs to be preserved from any microbial or foreign particle contamination during processing. The milling can be performed in either batch (discontinuous) or continuous mode (recirculation). Several mill designs are available on

66

4. Large-scale manufacturing of nanoparticles—An industrial outlook

Other • Bead size • Type of beads • Homogenization pressure • Number of homogeinzation cycles

Techniques • Batch mode • Recirculation mode • Passage mode Formulation ingredients • API • Stabilizer • Surfactant/wetting agents • Buffering agents • Thickening agent - Cloud point modifier - Solvents-if any - Cryoprotectants

Techniques • Top down : HPH, milling • Bottom up : Precipitation, emulsification

Formulation Engineering • Screening of excipients Technology selection • Physico-chemical characterization • Technology selection • QbD study design • Selection of Initial • Accelerated stability testing process parameters

FIG. 4.7

Other requirements • Sterilization • Lyophilization • Spray drying • Safety on cytotoxics

Lab scale

R&D workflow for development of nanosuspensions.

the market, and they differ mainly in their chamber capacity and stirring geometry. Typical mills employed in nanocrystals production are discs mills, pin stirred bead mills, and annular mills. Fig. 4.8 depicts industrial requirements on the production line for nanosuspensions. This means an in-process check on product quality attributes is equally important to access: (1) key physico-chemical properties like the PSD, zeta potential, pH, and optionally morphology of the final product particles and

(2) analytical parameters like assay, sterility, level of contaminants, stability of the final product over time. The flow of product from R&D to the production unit and related requirements and operations performed during stages are depicted in Fig. 4.9. The flow clearly shows that an R&D scientist has to keep in mind that the formulation recipe must be so robust that it survives all steps of process engineering with no or limited fine-tuning. Drastic changes in product quality after scale-up

67

2 Nanosuspensions

FIG. 4.8 Industrial requirements on the production line.

Adaptable to sterility and cleaning requirement Clinical and commercial production

Nitrogen/oxy gen line supply

Production line

Allows fine tuning of process variables

Robust and digitalisable

Technology selection • Technology selection • Selection of initial process parameters

Formulation engineering • Screening of excipients • Physico-chemical characterization • QbD study design • Accelerated stability testing

Lab scale

Consistent product output

Support variable batch sizes and formulation type

Process engineering • Selection of reliable and robust technology • Free of contaminants • Low energy consumption • Safe and cost effective • Optimization of process parameters • Production line qualification • IPQC set and monitoring SOP • Stability study

1 or multiple batches as per clinical study protocol

Clinical batch

Scale up

ICH stability Process /batch validation

Commercial batch

Packaging validation

Technical batch ICH stability

20–500 L 1 mL to 5 L

FIG. 4.9

5–300 L

Typical engineering flow of product and related batch sizes.

20–500 L

500 L to several tons

68

4. Large-scale manufacturing of nanoparticles—An industrial outlook

are not at all desired. The performance of product must be stable throughout the chain. The collaboration of process engineers with R&D formulation scientists at an early stage is crucial.

2.4 Large-scale manufacturing unit operation As mentioned earlier, generic product development methodology needs a robust and scalable formulation to ensure continuity of product quality attributes. Fig. 4.10 represents important aspects for large-scale production of nanoparticles. As businesses like to save cost and reduce resources on implementation of multiple production lines, it would be ideal if an available

API dispensing under isolator

Recycling loop

Cooling exchanger

Particle size reduction using bead mill or HPH

production line were adaptable for a wide range and type of nanoformulations, e.g., HPH can be used for emulsions, lipid nanoparticles, and nanosuspensions. In addition, if a drug is cytotoxic, specific safety and production facilities (clean room, isolator) need to be implemented as per safety classification of drug. On the other hand, if it is a drug device combination, a separate set of regulatory constraints (associated with administration rule) needs to be performed. Besides that, nanoparticles depending upon the composition and stability can be sterilized either during operation or at the end of the production cycle. Sterilization can occur during manufacturing by filtration, or the product can be completely processed aseptically. Some Stabilizer

Water

Vessel for stabilizer solution manufacturing

Double filtration 0.22 µm

Vessel for suspension manufacturing

Water

Cryoprotector

Vessel for cryoprotector manufacturing Feeding pump

Clarifying filtration ?? µm

Double filtration 0.22 µm Vessel for final bulk suspension manufacturing Primary container filling

FIG. 4.10 Typical manufacturing unit operation for intravenously injectable nanosuspension using the top-down process.

69

3 Lipid nanoparticles

operations such as autoclaving, filtration, and lyophilization can be done on separate secondary process setup. However, transfer of product from the main production line to the secondary line needs to be validated to minimize contamination and to guarantee all the quality attributes of the original product are maintained. Furthermore, the manufacturing unit must be compliant with pharmaceutical regulations such as current good manufacturing practices (cGMPs) with regard to qualification and validation, 21 CFR part 11, USP, and EP. The production unit needs to be qualified according to all current safety and cleaning regulations. The manufacturing equipment, facilities, and utilities have specific requirements as per class of drug, e.g., cross-contamination, facility temperature, and humidity monitoring are key subparameters to bear in mind. As per new health authority regulations, for highly potent drug products, dedicated manufacturing facilities and safety requirements are mandatory. Depending upon the development stage and batch size, some equipment parts like vessels, filters, and piping can be disposed of or reused. This proposal will depend on the cost of APIs, risk of crosscontamination, and the cost of complete operation and that specific machine part. For sterile nanoformulation production, all equipment that comes in contact with the product needs to be sterilized either by autoclaving or by radiation. Ideally, the equipment used must be robust for processes like the sterilization in place (SIP) and cleaning/drying in place (CIP/DIP) procedure to prevent damage and to save time. The filters (for liquid and gas) can be sterilized using clean steam while isolator must be sterilized using hydrogen peroxide vapors. The sterilization process must be validated using a microbiological growth medium (media fill) and all the data must be recorded. In addition, timely and continuous monitoring on the pressurization procedure, temperature, humidity, sterilization, cleaning, and exchange of machine parts needs to

be monitored and maintained for audits. The production line must be easily connected to other utility areas like weighing rooms, flow meters, connections to WFI (water for injection), purified water, nitrogen, clean steam, and liquid for heating and cooling amenities. All the personnel working in such areas must be trained for the quality and safety aspects of the product in a timely manner. During manufacturing operation, control of the environment is equally important to ensure the product is free from any microbial, pyrogen, and particle contamination. The typical manufacturing of parenteral nanoformulations is performed in C grade rooms (class 10,000: ISO 14644-1 Cleanroom Standards), which are equipped with laminar air flow hoods. A D grade room (class 100,000: ISO 14644-1 Cleanroom Standards) can be used if a closed barrier system is used to avoid any external contamination and to facilitate easy supervision and control of the manufacturing line.

3 Lipid nanoparticles In the literature, various types of lipid-based drug delivery systems like liposomes, niosomes, virosomes, ufasomes, nanoemulsion, cubosomes, solid lipid nanoparticles (SLNs), nanostructured lipid nanoparticles (NLCs), lipospheres, ethosomes, discosomes, cryptosomes, transfersomes, vesosomes, and phytosomes are described. However, the most commonly studied DDS from the lipid nanoparticles category includes liposomes, SLNs, and NLCs [3]. In this session, large-scale production of SLNs is described in detail as a case study; however, the same principle can be applied to other lipid-based nanoparticles by implementing a suitable preproduction step. Briefly, SLNs are nanoparticles made from lipids that are solid at body temperature. SLNs can be used for a wide range of biomedical

70

4. Large-scale manufacturing of nanoparticles—An industrial outlook

application. NLCs are second-generation lipid nanocarriers, which contain an additional lipid phase, which is liquid at room temperature. Both systems can be produced by top-down or bottom-up techniques [17]. This session will focus only on the top-down technique by homogenization. The main advantage of this system is that: -

it is composed of biocompatible materials; it contains already known ingredients; is easy to preparation; is stable; is industrially scalable; has a higher penetration-permeation profile; is suitable for a wide range of activechemical entity to biomolecules/genetic material; - has high encapsulation efficiency; - exhibits low cytotoxicity; and - has low immunogenicity. Several techniques are used in the literature for preparation of SLNs/NLCs, including: -

cold high-pressure homogenization; hot high-pressure homogenization; microfluidics; solvent emulsification; coacervation; microemulsions template; phase inversion temperature (PIT) techniques; - supercritical fluid technology; and - the membrane contactor method. Most of the published reports on lipid nanoparticle production are from academics and at lab scale; few reports are published that discuss scaling up of batch size. This session gives an overview of them. The first report as per our information on medium-scale production of SLNs was published in 2002 by Jenning et al., using the HPH technique. The batch was scaled up from 40 mL to 50 L, and the authors reported that

increased batch size affected the product quality in terms of PSD and physical storage stability. This could be due to homogenizer performance of machine from two different suppliers, although reproducibility and batch-to-batch uniformity was well established and was known. As this was academic research, no automation, facility control, or equipment qualification was performed. In addition, this study reported influence of pressure and temperature during the process and cooling process of the final product as key contributors for product quality. Fast cooling of the final product deteriorated the product quality, as recommended cooling settings by this group was with cold water at 18°C [18]. In 2011, Shegokar et al. [19] published a systematic comparison of lab scale to pilot scale to industrial scale by increasing the batch scale stepwise (e.g., 40 g, 2–10 kg, 50 kg, and up to half a ton and more) for production for HIV drug-loaded SLNs. Throughout the study, machines provided by one supplier were employed to avoid supplier-to-supplier variations on machine configuration in one group. In another group, equipment from a second supplier was evaluated. The homogenizers employed in this study included the following: Supplier 1: • APV Gaulin LAB 40 (lab scale: 20–40 g); • APV Gaulin Micron LAB 60 (pilot scale: 2–10 kg); and • APV Gaulin 5.5 (industrial scale: 50 kg and up to half a ton and more). Supplier 2: • Avestin C50. In addition to the above range of Gaulin homogenizers, Avestin C50 was included in the study from Supplier 2 with different equipment design of the homogenization chamber. All homogenizers followed the piston gap size reduction principle by cavitation (Table 4.5).

71

3 Lipid nanoparticles

TABLE 4.5 Scale-up parameters used for large-scale production of SLNs. Lab-scale production (LAB 40)

Pilot-scale production (LAB 60)

• Hot HPH • One homogenization valve • DIS: 800 bar 5 passes • Homogenizer temp was maintained at 80°C (circulating water bath connection)

Batch size 40 g

Industrial-scale production Supplier 1: Gaulin 5.5

Supplier 2: Avestin C50

• Hot HPH • Capacity 60 L/h • Two homogenization valves • Continuous (CNT) and discontinuous (DIS) mode • 800 bar—5 passes (DIS) • 800 bar—30 min (CNT) • Pipes were maintained at 80°C (water bath)

• Hot HPH • Capacity is 160 L/h • Continuous (CNT) And discontinuous (DIS) mode • 800 bar—5 passes (DIS) • 800 bar—30 min (CNT) • Pipes were maintained at 80°C (water bath)

• Hot HPH • Capacity 55 L/h • Continuous (CNT) And discontinuous (DIS) mode • 800 bar—5 passes (DIS) • 800 bar—30 min (CNT) Pipes were maintained at 80°C (water bath)

Batch size 10 kg

Batch size 20 kg

Batch size 10 kg

CNT mode: 2 min homogenization— 67 nm 30 min homogenization— 115 nm DIS mode: 5 homogenization cycle—63 nm

CNT mode 2 min circulation—61 nm 30 min—107 nm DIS mode: 1 homogenization cycle—48 nm

The final product was cooled to room temperature DIS: 53 nm

CNT mode: 2 min homogenization— 67 nm 30 min homogenization—70 nm DIS mode: 1 homogenization cycle—55 nm

Processing and related factors affecting quality of end product • Temperature of homogenizer • Number of homogenization cycles • Pressure • Cooling of product

• • • • • • • • • •

Product temperature Residence time of product in hot mixing container Mixing settings of preemulsion Transfer temperature Temperature of homogenizer/piping Number of homogenization cycles Pressure cycles Cooling step (time and temp) of product Temperature of cooling pipes Stirring and residence time during cooling cycles

This group successfully scaled up SLNs formulation from 40 g to 20 kg (factor of 500) without changing production parameters (pressure, temperature, passes number). However, depending upon requirements, process parameters can be varied to achieve larger or smaller

particle size of the same recipe. The authors did not assess differences in drug loading as a function of batch size, assuming they must be the same due to unchanged physical properties. Scaled-up formulations showed good long-term stability. The authors reported flexibility of

72

4. Large-scale manufacturing of nanoparticles—An industrial outlook

Solvent : oil or emulsion and organic solvent for suspension or liposomes Phospholipds for liposomes Lipids for emulsion Solvent evaporation only liposomes or suspension

API dispensing under isolator

Phospholipids or lipids

Solvent

Stabilizer

Stabilizer Vessel for organic phase manufacturing

Water

Recycling loop

Double filtration 0.22 µm Cooling exchanger

Water Vessel for suspension/ emulsion/ liposomes manufacturing

Particle size reduction using bead mill or HPH Feeding pump

Solvent evaporation

FIG. 4.11

Double filtration 0.22 µm for emulsion or simple clarifying filtration ?? µm for suspension

Cryoprotector

Vessel for cryoprotector manufacturing

Double filtration 0.22 µm

Vessel for final bulk suspension/ emulsion/ liposomes manufactur ing Primary container filling

Typical manufacturing unit operation for suspensions, liposomes, or emulsions.

manufacturing operation in continuous and discontinuous mode for fine-tuning production parameters of nanoparticles, e.g., the discontinuous mode can be used, just by increasing the capacity of the coarse product supply hopper. Fig. 4.11 highlights the typical industrial flow for manufacturing of lipidic nanoparticles. A similar platform can be used for production of liposomes, emulsions, and precipitated nanocrystalline suspensions. All related factors that need to be considered during industrial production of lipid nanoparticle systems are compiled in Fig. 4.12. In 2015, another research group presented a simple and industrially accessible method for producing liquid crystalline lipid nanoparticles

with various internal structures. They produced bilayer vesicles based on phytantriol, Pluronic F127, and vitamin E acetate, deionized water upon evaporation of solvent-ethanol. The vesicle structure can be adapted to hexagonal lattices (inverted cubic Pn3m), lined or coiled pattern (inverted hexagonal H2), and disordered structure (inverse microemulsion, L2), just by playing around with final recipe and temperature settings. The authors claimed that this method could produce lipid nanoparticles of characteristics similar to those produced via the conventional method. Further advantages of this technique involved simple tooling, adaptable mixing chambers, and setup for ethanol evaporation. At the present stage, the assembly supports production of up to a 10 kg batch of lipid nanoparticles [20].

73

4 Nanoparticle characterization and analytical techniques used

Scalability

Tunable on pressure and evaporation steps

Stable for cold and hot processing

Adaptable to wide range of lipid nanoparticles

Robust formulation (especially lipid + stabilizer)

Allows digitalized control of parameters

Cost effectiveness

Min. batch to batch variation

FIG. 4.12

Reproducibility

Lipid nanoparticle manufacturing

Adaptable to extra filtration-sterilizationand lyophilization operations

Factors to consider during manufacturing of lipid nanoparticles.

In 2016, a report was published on production of large-scale Q10 NLC formulation using a modular production line [21]. The parameters such as emulsification, homogenization pressure, or homogenization cycles can be well controlled or adapted as required. The production line manufactured NLC of 210 nm particle size at a speed of 25 kg/h and a lipid phase flow rate at 0.4 kg/ min. The author reported that the main influencing parameters on the characteristics of the final NLC product were preemulsification temperature, homogenization pressure, and number of homogenization cycles or passes. For that particular Q10 loaded NLC composition, an emulsification temperature of 70°C, homogenization pressure of 600–800 bar, and 3–4 homogenization cycles were optimal to obtain a narrow PSD. The production line used allowed digitalized networking of machine parts and adaptable features for fast and cost-effective nanoparticle production. Both batches (lab and large scale) exhibited excellent stability at room temperature. All these reports are based on academic research; in industry, the setup is much more

sophisticated and modern [3]. However, all these studies provide the confidence that by fine-tuning formulation, processing, and hardware aspects, one can produce lipid nanoparticles (majority types of lipid systems) with reproducible and consistent product quality. Like nanosuspensions, lipid nanoparticles can be spray-dried or lyophilized in a second processing step. The rest of the industry requirements like a clean room, dedicated production area, equipment validation, flooring, safety, and personnel training remain the same for lipid nanoparticle products. As mentioned earlier, depending upon the technology selected and the type of formulation, the scope of the manufacturing process changes.

4 Nanoparticle characterization and analytical techniques used Unlike the conventional formulation, characterization of nanoparticles is more complex. Generally, a set of highly sophisticated

74

4. Large-scale manufacturing of nanoparticles—An industrial outlook

analytical techniques are used for characterization of diverse aspects of nanoparticles. This section describes typically applied techniques at industrial-scale manufacturing operation and during manufacturing as process control. Fig. 4.13 shows commonly used techniques for characterization of nanoparticles. In the case of nanosuspension, along with particle size measurement and crystallinity measurement, it is important to access surface morphology information. This package allows determination of quantitative and qualitative impact of milling or homogenization on API properties. In addition, the crystallinity pattern also assists in detecting the presence of the amorphous phase, if any, and evaluating morphological modifications after milling. SEM

DDS/characterization

PS

ZP

PDI

highlights that both technologies led to nanosuspensions exhibiting the same morphology but differing PSDs (Fig. 4.14). In the case of nanosuspensions, contaminant residues from milling media and milling chamber due to abrasion might get introduced in the suspension. Chemical element contamination is also feasible. Inductively Coupled Plasma-Atomic Emission Spectroscopy (ICP-AES) is used to evaluate the contaminant content in milled suspensions. Types of bead used are the main source of contamination and their selection is critical, e.g., cross-linked polystyrene beads (minimal to below 5 ppm) are far more robust media milling materials than zirconium oxide or stainless steel where the contaminant levels (tungsten and iron) are significantly higher (25–500 ppm).

Osmolarity Morphology Crytallinity (if i.v.)

Nanosuspension

Thermal profiling

Brittleness

Surface area

Elemental compo

Porosity

if dried

SLN NLC Nanoemulsions Micelles Polymeric nanoparticles PLH Liposomes

FIG. 4.13 Matrix of characterization parameters for nanoparticulate drug delivery systems (green ¼ required, orange ¼ optional, gray ¼ not mandatory, but can be selected as required).

FIG. 4.14

Impact of the technique used on API morphology.

4 Nanoparticle characterization and analytical techniques used

4.1 Other characterizations APIs respond differently to different techniques. It is important that a patient receives a product of a consistent quality, e.g., HPH is less impacted by the thermal exposure than bead milling, which could impact the morphology of suspensions and thereby their stability. Depending upon the regulatory requirements of the engineering development phase, a stability study is planned: 1. adapted/customized short-term stability programme at R&D level for initial screening;

75

2. accelerated stability studies; and 3. extensive long-term stability studies as per ICH guidelines. Nanoparticles are typically characterized for mean particle size, PSD, zeta potential, osmolarity, dispersibility, and drug content. In short, any technology can be applied and optimized to obtain the desired target product profile, provided the initial formulation composition used is robust and stable. Table 4.6 lists various techniques used for characterization of varied aspects of nanoparticles (obtained with permission from Mourdikoudis

TABLE 4.6 Nanoparticle characterization techniques with permission under open access right from [22]. Entity characterized

Characterization techniques suitable

Size (structural properties)

TEM, XRD, DLS, ΝΤA, SAXS, HRTEM, SEM, AFM, EXAFS, FMR, DCS, ICP-MS, UV-Vis, MALDI, NMR, TRPS, EPLS, magnetic susceptibility

Shape

TEM, HRTEM, AFM, EPLS, FMR, 3D-tomography

Elemental-chemical composition

XRD, XPS, ICP-MS, ICP-OES, SEM-EDX, NMR, MFM, LEIS

Crystal structure

XRD, EXAFS, HRTEM, electron diffraction, STEM

Size distribution

DCS, DLS, SAXS, ΝΤA, ICP-MS, FMR, superparamagnetic relaxometry, DTA, TRPS, SEM

Chemical state-oxidation state

XAS, EELS, XPS, M€ ossbauer

Growth kinetics

SAXS, NMR, TEM, Cryo-TEM, liquid-TEM

Ligand binding/composition/density/ arrangement/mass, surface composition

XPS, FTIR, NMR, SIMS, FMR, TGA, SANS

Surface area, specific surface area

BET, liquid NMR

Surface charge

Zeta potential, EPM

Concentration

ICP-MS, UV-Vis, RMM-MEMS, ΡΤA, DCS, TRPS

Agglomeration state

Zeta potential, DLS, DCS, UV-Vis, SEM, Cryo-TEM, TEM

Density

DCS, RMM-MEMS

Single particle properties

Sp-ICP-MS, MFM, HRTEM, liquid TEM

3D visualization

3D-tomography, AFM, SEM

Dispersion of NP in matrices/supports

SEM, AFM, TEM

Structural defects

HRTEM, EBSD

Detection of NPs

TEM, SEM, STEM, EBSD, magnetic susceptibility

Optical properties

UV-Vis-NIR, PL, EELS-STEM

Magnetic properties

SQUID, VSM, M€ ossbauer, MFM, FMR, XMCD, magnetic susceptibility

76

4. Large-scale manufacturing of nanoparticles—An industrial outlook

et al., Nanoscale, 2018, 10, 12871, DOI: 10.1039/ C8NR02278J under a Creative Commons Attribution 3.0). Readers are requested to refer to this article for abbreviations and major highlights on each technology. The type of drug delivery system and the final administration route will decide which types of parameters need to be controlled and checked before the product is released. The checklist of tests and type tests may vary from industry to industry, although basic characterization of assay, particle size, crystallinity, and morphology will always be a high priority to determine the physical and analytical properties of a product.

5 Conclusion Nanosuspensions and lipid nanoparticles are very promising drug delivery systems for overcoming drug-related challenges, and to develop better therapeutic systems for patients. Both systems have a wide application in biomedicine as well as in other fields of science. This chapter is a first compilation to our knowledge where only large-scale manufacturing as per industry standard for both drug delivery systems has been discussed. It is critical and important to control formulation and process-related variables to produce nanoformulations of desired quality. Timely communication with regulatory bodies, equipment suppliers, and excipient suppliers is beneficial to understand the impact of changed parameters on the quality of a product. Similarly, it is equally important to break the silos within an organization and to foster collaborations between different experts, especially formulation scientists, engineers, regulatory scientists, analysts, and packaging experts. This will speed up the process of nanoparticles formulation and process development. This chapter guides scientists, engineers, and other experts on the industrial outlook of nanoparticle production, requirements, and challenges.

References [1] Shegokar R, M€ uller RH. Nanocrystals: industrially feasible multifunctional formulation technology for poorly soluble actives. Int J Pharm 2010. https://doi.org/ 10.1016/j.ijpharm.2010.07.044. [2] Shegokar R. Nanosuspensions: a new approach for organ and cellular targeting in infectious diseases. J Pharm Investig 2013. https://doi.org/10.1007/ s40005-013-0051-x. [3] Muller RH, Shegokar R, Keck CM. 20 years of lipid nanoparticles (SLN & NLC): present state of development & industrial applications. Curr Drug Disc Technol 2011. https://doi.org/10.2174/157016311796799062. [4] Shegokar R. Wet media milling: an effective way to solve drug solubility issue. In: Handbook of nanoparticles. 2015. https://doi.org/10.1007/978-3-319-15338-4_20. [5] Shegokar R. Nanoparticles’ promises and risks: characterization, manipulation, and potential hazards to humanity and the environment. Nanotoxicity: must consider aspect of nanoparticle development; 2015. https://doi.org/10.1007/978-3-319-11728-7_6. [6] Shegokar R. What nanocrystals can offer to cosmetic and dermal formulations. In: Nanobiomaterials in galenic formulations and cosmetics: applications of nanobiomaterials. 2016. https://doi.org/10.1016/ B978-0-323-42868-2.00004-8. [7] Nakach M, Authelin JR, Perrin MA, Lakkireddy HR. Comparison of high pressure homogenization and stirred bead milling for the production of nano-crystalline suspensions. Int J Pharm 2018. https://doi.org/ 10.1016/j.ijpharm.2018.05.042. [8] Nakach M, Shegokar R, Authelin J-R, Tadros T. Nanocrystalline suspensions by top down processes: review of manufacturing considerations. Drug Deliv Lett 2018. https://doi.org/10.2174/2210303108666180706144005. [9] Leung DH, Lamberto DJ, Liu L, Kwong E, Nelson T, Rhodes T, Bak A. A new and improved method for the preparation of drug nanosuspension formulations using acoustic mixing technology. Int J Pharm 2014. https://doi.org/10.1016/j.ijpharm.2014.05.003. [10] Al Shaal L, M€ uller RH, Shegokar R. SmartCrystal combination technology—scale up from lab to pilot scale and long term stability. Pharmazie 2010. https://doi. org/10.1691/ph.2010.0181. [11] Du J, Li X, Zhao H, Zhou Y, Wang L, Tian S, Wang Y. Nanosuspensions of poorly water-soluble drugs prepared by bottom-up technologies. Int J Pharm 2015. https://doi.org/10.1016/j.ijpharm.2015.09.021. [12] Fern JCW, Ohsaki S, Watano S, Pfeffer R. Continuous synthesis of nano-drug particles by antisolvent crystallization using a porous hollow-fiber membrane module. Int J Pharm 2018. https://doi.org/10.1016/j.ijpharm. 2018.03.025.

References

[13] Tran TTD, Tran PHL, Nguyen MNU, Tran KTM, Pham MN, Tran PC, Van Vo T. Amorphous isradipine nanosuspension by the sonoprecipitation method. Int J Pharm 2014. https://doi.org/10.1016/j.ijpharm. 2014.08.017. [14] Van Eerdenbrugh B, Van den Mooter G, Augustijns P. Top-down production of drug nanocrystals: nanosuspension stabilization, miniaturization and transformation into solid products. Int J Pharm 2008. https:// doi.org/10.1016/j.ijpharm.2008.07.023. [15] Lestari MLAD, M€ uller RH, M€ oschwitzer JP. The scalability of wet ball milling for the production of nanosuspensions. Pharm Nanotechnol 2019. https://doi.org/ 10.2174/2211738507666190401142530. [16] Verma S, Lan Y, Gokhale R, Burgess DJ. Quality by design approach to understand the process of nanosuspension preparation. Int J Pharm 2009. https://doi.org/ 10.1016/j.ijpharm.2009.05.006. [17] Doktorovova S, Shegokar R, Souto EB. Role of excipients in formulation development and biocompatibility of lipid nanoparticles (SLNs/NLCs). In: Nanostructures for novel therapy: synthesis, characterization and applications. 2017. https://doi.org/10.1016/B978-0-32346142-9.00030-X.

77

[18] Jenning V, Lippacher A, Gohla SH. Medium scale production of solid lipid nanoparticles (SLN) by high pressure homogenization. J Microencapsul 2002. https:// doi.org/10.1080/713817583. [19] Shegokar R, Singh KK, M€ uller RH. Production & stability of stavudine solid lipid nanoparticles—from lab to industrial scale. Int J Pharm 2011. https://doi.org/ 10.1016/j.ijpharm.2010.08.014. [20] Kim DH, Lim S, Shim J, Song JE, Chang JS, Jin KS, Cho EC. A simple evaporation method for large-scale production of liquid crystalline lipid nanoparticles with various internal structures. ACS Appl Mater Interfaces 2015. https://doi.org/10.1021/acsami.5b06413. [21] Hu C, Qian A, Wang Q, Xu F, He Y, Xu J, et al. Industrialization of lipid nanoparticles: from laboratory-scale to large-scale production line. Eur J Pharm Biopharm 2016. https://doi.org/10.1016/j.ejpb.2016.10.018. [22] Mourdikoudis S, Pallares RM, Thanh NTK. Characterization techniques for nanoparticles: comparison and complementarity upon studying nanoparticle properties. Nanoscale 2018. https://doi.org/10.1039/ c8nr02278j.

C H A P T E R

5

The role of polymers and excipients in developing amorphous solid dispersions: An industrial perspective Vaibhav Sihorkara, Thomas D€ urigb a

Formulations, NCE and Innovation, Sai Life Sciences Limited, ICICI Knowledge Park, Genome Valley, Hyderabad, Telangana, India b R&D and Innovation, Ashland Pharma and Health & Wellness, Ashland Specialty Ingredients G.P., Wilmington, DE, United States

1 Introduction

the precedence (of amorphous) in early/late phase development of new drugs. This has paved the way for strengthening fundamental understanding of physicochemical, molecular, and thermodynamic properties, and developing an understanding of the amorphous state to create process and products with a consistent performance. Despite a plethora of published research studies by academic and industrial scientists on amorphous systems, the implementation of these systems in marketed products has been far from impressive. However, the understanding of these systems has been increasing with evolving orthogonal analytical methodologies and better polymeric excipients and processes/technologies. Besides that, a better understanding is evolving on the molecular and thermodynamic aspects of the pharmaceutical amorphous

Amorphous systems are ubiquitous in nature and have emerged as a major material science gain across knowledge-intensive sciences and technologies. They differ from crystalline forms in not being confined to predefined templates for molecular arrangements, and provide a high-energy continuum that enables them to be of tremendous application in the fields of material science, pharmaceuticals, and polymer industries, among others [1]. Additionally, the amorphous systems offer unique physicochemical, bulk, and mechanical properties such as kinetic solubility, viscosity and viscoelastic properties, thermo-plasticity, compressibility, and stability [2]. The realization has grown in last few decades to design and develop amorphous materials as a development strategy (APIformulation connect) in generics, along with

Drug Delivery Aspects https://doi.org/10.1016/B978-0-12-821222-6.00005-1

79

# 2020 Elsevier Inc. All rights reserved.

80

5. The role of polymers and excipients in developing amorphous solid dispersions: An industrial perspective

systems (also termed as “glass”), which are prerequisites for the scientific rationale-based pharmaceutical product development and consistent quality and performance throughout the product’s shelf life. The phenomena like strong or weak glass-forming ability, glass fragility, glass transition temperature (Tg), and relaxation behavior of an active pharmaceutical ingredient (API) in its amorphous form have been understood better with the advent of advanced analytical tools. The opportunities that currently exist are greater than ever in the pharmaceutical field to shed light on developing a finished dosage form with an amorphous API or otherwise stabilizing amorphous API molecularly dispersed in a polymer (solid dispersions, also termed APIpolymer pre-mix) before developing the finished dosage forms [3,4]. This chapter focuses on the evolution of amorphous pharmaceutical technologies in the pharmaceutical industry and explains in detail the role of excipients in amorphous solid dispersion (ASD) product design and process technologies, and their success in the market.

2 ASD versus other solubilization techniques Amorphous forms of an API and ASD in general set their footprint in pharmaceuticals nearly five decades ago, owing to poor solubility and oral bioavailability of pharmaceutical actives [5–7]. To improve the solubility and oral bioavailability of poorly water-soluble drugs, several methods and technologies, such as ASD, nanocrystalline dispersions, complexation, lipidbased systems, particle engineering approaches like micronization and nanosizing, and other chemical modifications like salt and co-crystals, have been widely explored. Among these, ASD is one of the most prevalent and established methods [8, 9]. A key advantage of ASDs is their ability to provide a reasonably higher Δ solubility advantage

compared with other technologies, especially where oral bioavailability is limited by solubility or dissolution. Another benefit is the easy scalability of the powdered ASD with wellestablished manufacturing technologies like spray drying (or solvent evaporation) and hotmelt extrusion (fusion-based methods) to the final drug product in the form of a tablet or capsule, the most common oral dosage forms. Compared with lipid-based systems, ASDs allow formulation with much higher dosage levels. Lipid-based formulations like selfemulsifying and self-micro-emulsifying ones typically contain very high levels of surfactants, co-surfactants, oils, and co-solvents, and are still not able to provide a high drug load (mostly limited up to around 100 mg of API) [10]. On the other hand, ASDs have much more leverage in terms of delivering high doses and drug loadings of as much as around 500mg or more of the active ingredient. Though both lipid-based systems and ASDs are high-energy systems, the lipid-based systems tend to be more complex and sometimes pose issues such as batch-to-batch variability, sensitivity of constituent lipids to oxidation, and less flexibility to convert to solid dosage forms [11]. Cyclodextrin complexation provides another means to solubilize drugs [12]. However, it is mostly limited to low or moderate dose drugs. The stochiometric molar ratio required for inclusion complexation to take place between the drug and cyclodextrins may lead to feasibility and scaling-up issues, due to a very high bulk volume of complexed powder product. This is not practically feasible to manufacture at times to a commercially viable solid oral dosage form. ASDs, however, do pose challenges of physical and chemical stability of drugs during manufacturing and through their shelf life. The growth in marketed products over the last decade in the ASD space suggests several challenges to overcome on both the formulation and manufacturing fronts. Moreover, the advent of modular and continuous manufacturing technologies like hot-melt extrusion has given a

3 ASD relevance to different pharmaceutical businesses

progressive momentum to ASD technology, in providing more flexibility to its commercial viability in years to come [13,14].

3 ASD relevance to different pharmaceutical businesses The reasons to explore amorphous pharmaceuticals have been expanded from their primary focus of increasing solubility and dissolution (and hence oral bioavailability) of poorly soluble new drugs to their use as re-positioning, re-purposing, and differentiating strategies. The historical reasons for breaking the crystal lattice of an API and generating a highly active (reactive) amorphous form were driven by the fact that the new chemical entities (NCEs) had less favorable bio-pharmaceutical properties. The chemical scaffolds (hit, lead, and candidates) profiled for their druggability in early discovery stages originated from high throughput screening and combinatorial chemistry, and were more lipophilic. These lipophilic scaffolds had high affinities for the therapeutic receptor/targets (most of them offer a lipophilic ligand/motif ); however, they lacked desired (bio)-pharmaceutical properties. Although these druggability priorities generated thousands of hits/leads, they had poor developability properties, most specifically poor aqueous solubility. The enormous amount of time and financial investment on these “druggable leads” required more resource and time to be spent in the early drug discovery and development phase. Therefore, the improvement of solubility of these molecules with the aid of high-energy technologies has helped in moving them from the nonclinical to the clinical stages of development. Solidstate modifications in general and amorphous strategy specifically have emerged as a preferred way of enhancing solubility and optimizing delivery of poorly soluble leads/drugs. An innovator company perspective (discovery and development pharmaceutics) of amorphous materials and ASDs has been discussed in detail

81

by Newman [15]. Once an ASD concept is being used at discovery stages to profile a hit/lead compound, it should be assessed for its potential to be developed into a first-in-human product later during clinical development (early or late phase). Some of the druggable descriptors could be preferred indicators to explore amorphous formulation strategies like highly planar/rigid structure, high melting point (>200°C), very low solubility (5 w/w %), PS is partially soluble (1–5 w/w %), I is insoluble (300 biologics have been approved since then, used to treat millions of people, and about 900 biologics are under development. The importance of biologics in today’s world continues to increase, but many challenges still need to be addressed during their development. While ensuring the therapeutic effectiveness of biologics, there is a growing interest from many researchers in identifying ways to overcome these challenges. Biologics are pharmaceutical products manufactured or extracted through a biological process (involving biotechnology methods) from living organisms rather than chemical synthesis. Types of biologics

Drug Delivery Aspects https://doi.org/10.1016/B978-0-12-821222-6.00006-3

include antibodies, interleukins, and protein-, peptide-, and vaccine-based products (Fig. 6.1 classifies different types of biologics). Recently, biologics have rapidly emerged as an important class of pharmaceuticals due to several advantages. Biologics are in clinical use for several life-threatening and rare diseases such as cancer, diabetes, anemia, rheumatoid arthritis, multiple sclerosis, etc. Biologics are considerably more complex and expensive than a small molecule pharmaceutical product. Nearly 30% of all drugs approved by the U.S. Food and Drug Administration (FDA) in 2015–18 were biologics [1]. In the near future, biologics-based products are expected to attain a significant market share among various pharmaceuticals. Biologics have further got great impetus by the advent of biosimilars. A biosimilar is a biological product that is highly similar to and has no clinically meaningful differences from an existing FDA-approved reference product. In simpler terms, biosimilars are medicines that are highly

115

# 2020 Elsevier Inc. All rights reserved.

116

6. Biologics: Delivery options and formulation strategies

Biologics

Vaccines

FIG. 6.1

Blood components

Allergenics

Gene therapy

Human tissue

Proteins and Peptides

Types of biologics.

similar and clinically equivalent to complex biologics medicines approved for use against serious life-threatening diseases in the fields of immunology, gastroenterology, and oncology. In terms of product, biosimilars are divided into recombinant nonglycosylated protein, recombinant glycosylated protein, and recombinant peptides. The biosimilar market is expected to reach >23 billion USD by 2023. >60 biosimilar products are now in the pipeline in therapeutic areas such as oncology, immunology, and diabetes. There are many blockbuster biologic products of pharmaceuticals companies, such as Remicade, Rituxan, and Herceptin, and other patents have expired in the recent past, and many will

expire in the upcoming decade. This creates new opportunities for many pharmaceutical companies to launch their product and enter the biologics market. The various importances of biologics by virtue of sales are summarized in Table 6.1; they are becoming more and more significant. Currently, the majority of biologics are delivered either by intravenous infusion or by subcutaneous injections; alternative delivery options for biologics have been explored to some extent, but are largely elusive in the current situation. The main aim of this chapter is to provide an update on the current status with alternative routes and to highlight the possibilities with these routes.

TABLE 6.1 Revenue from some of the top products in the USA. Product name

Biologics (active)

Manufacturer

Indication/Disease

Sales in 2018

Humira

Adalimumab

AbbVie Inc.

Rheumatoid arthritis, plaque psoriasis, Crohn’s disease

$20.2 b

Revlimid

Lenalidomide

Celgene

Multiple myeloma

$ 9.2 b

Enbrel

Etanercept

Pfizer/Amgen

Rheumatoid arthritis

$ 7.3 b

Eylea

Aflibercept

Bayer

Wet macular degeneration

$ 6.5 b

Avastin

Bevacizumab

Roche

Metastatic cancers

$ 6.4 b

Rituxan

Rituximab

Roche

Non-Hodgkin’s lymphoma

$ 6.4 b

Herceptin

Trastuzumab

Roche

Breast cancer

$ 6.4 b

Remicade

Infliximab

Johnson & Johnson

Rheumatoid arthritis

$ 6.3 b

Keytruda

Pembrolizumab

Merck & Co.

Metastatic melanoma

$ 6.1 b

Xarelto

Rivaroxban

Johnson & Johnson/Bayer

Oral anticoagulant

$ 6.1 b

2 Intravenous infusion of biopharmaceuticals

2 Intravenous infusion of biopharmaceuticals Intravenous administration is one of the common delivery options for biopharmaceuticals— this route of administration allows maximum bioavailability, and for certain indications (e.g., oncology) where patients are in hospital, this is often the most convenient option. Intravenous administration is usually used when rapid onset of action is required (e.g., emergency medicine and anesthesia) or the drug is unable to be administered orally because of some inherent physicochemical properties of the drug molecule. Intravenous administration is advantageous over other routes for biopharmaceutical administration as it bypasses the absorption barriers and other metabolic pathways. By this route, high-concentration biopharmaceuticals can be administered rapidly to the relevant organ, which may also reduce toxic effects. Drugs in the form of suspensions or oily solutions cannot be given intravenously. IV administration bypasses many of the absorption barriers, efflux pumps, and metabolic mechanisms. Drugs administered via the intravenous route reach the brain directly, with onset occurring within 20–40 s. IV infusions may be used to achieve a constant level of drug in the bloodstream. Multiple doses can be administered through an intravenous infusion, with a high degree of flexibility and control. Biologic products such as infliximab, abatacept, rituximab, tocilizumab, vedolizumab, etc. commonly used for various clinical indications like hypertension, rheumatoid arthritis, Crohn’s disease, ulcerative colitis, psoriasis, psoriatic arthritis, etc. are administered via the IV route. Intravenous administration can be achieved by two ways: bolus injections or continuous infusion. With a bolus injection, complete medication is given in a small period of time—for example, in 16 kDa are taken up by lymphatic vessels, while smaller ones (2.5mL are associated with pain in injection, leakage, tissue distortion, and tissue back pressure. Higher SC injection volumes have also been evaluated using permeation enhancers. The volume barrier within the ECM is created by GAG hyaluron together with collagen. Hyaluronidase cleavage of hyaluron enables delivery of biologics through the SC route by increasing the tissue surface area and reducing tissue back pressure. This leads to fast administration of SC injection volumes >2.5mL with a reduced induration and leakage at the injection site. In spite of the known potential of hyaluronidase, ways to purify it from human body tissue need to be established to overcome the limitations of immunogenicity [9] or an alternative supply means are required to be established. Surface charge, molecule shape, solvent viscosity, pH, ionic strength, temperature, and shear rate [8] are some variables that affect the viscosity of a protein solution. The development of high-concentration formulation (due to high dose) of monoclonal antibodies poses great challenges due to the chances of aggregation, viscosity, subvisible and visible particles, as well as the method to concentrate the proteins. The translation of SC animal data to humans has not been clearly established. Relatively very few data report the mechanism of SC absorption of biologics in different species. The role of lymph and blood capillaries in systemic absorption, cross-species differences in hypodermis morphology and physiology, drug formulation, stability of the molecule, the site of injection, the depth of injection, as well as the molecular properties of the biologics themselves are major parameters affecting the absorption

TABLE 6.3 List of biologics for the SC route approved by the FDA. Date of approval

Brand name (drug name)

Indications

Target

Company name

Udenyca (pegfilgrastim)

Reduction in the duration of neutropenia

Leukocyte growth factor

11/02/2018 Coherus (U.S.) Bioscience

Fulphila (pegfilgrastim)

Neutropenia in adult patients

Leukocyte growth factor

06/04/2018 Mylan (U.S.)

Hyrimoz (adalimumab)

Rheumatoid arthritis, juvenile arthritis, Crohn’s disease, ankylosing spondylitis, psoriasis

TNF-α

10/30/2018 Sandoz (U.S.)

Tegsedi (inotersen)

Polyneuropathy of hereditary transthyretin-mediated amyloidosis in adults

Human transthyretin (TTR) protein

10/05/2018 Ionis (U.S.) Pharmaceuticals

Emgality (galcanezumab)

Migraine

Calcitonin gene-related peptide (CGRP)

09/27/2018 Eli Lily (U.S.)

Ajovytm (fremanezumab)

Migraine

Calcitonin gene-related peptide (CGRP

09/14/2018 Celltrion (U.S.)

Takhzyrotm (lanadelumab) Prevent attacks of hereditary angioedema (HAE)

Plasma kallikrein

8/23/2018 (U.S.)

Nivestym (filgrastim)

Acute leukemia, neutropenia

Leukocytes growth factor

07/18/2018 Pfizer (U.S.)

Palynziq (pegvaliase)

Phenylketonuria

Phenylalaninemetabolizing enzyme

05/24/2018 Biomarin (U.S.) Pharmaceuticals

Aimovigtm (erenumab)

Migraine

Calcitonin gene-related peptide receptor

05/17/2018 Amgen (U.S.)

Retacrit (epoetin alfa)

Chronic kidney disease

Progenitor stem cells

05/17/2018 Pfizer (U.S.)

Crysvita (burosumab)

X-linked hypophosphatemia (XLH)

Fibroblast growth factor 23 (FGF23)

04/17/2018 Ultragenyx (U.S.)

Ilumya (tildrakizumab)

Plaque psoriasis

Interleukin-23

03/21/2018 Merck Sharp (U.S.)

Admelog (insulin lispro injection)

Types I and II diabetes mellitus

Glucagon-like peptide 1 (GLP-1) receptor

12/05/2017 Sanofi Aventis (U.S.)

Ozempic (semaglutide)

Glycemic control in adults with type 2 diabetes mellitus

Glucagon-like peptide 1 (GLP-1) receptor

12/05/2107 Novo Nordisk (U.S.)

Dyax Corp.

Hemlibra (emicizumab)

Hemophilia A with factor VIII

Factor VIII

11/16/2017 Genetech (U.S.)

Fasenra (benralizumab)

Asthma

Interleukin-5 receptor alpha

11/14/2017 Medimmune/ (U.S.) Astra zenneca

Fiasp (insulin)

Glycemic control

GLP-1

09/29/2017 Novo Nordisk (U.S.)

Cyltezo (adalimumab)

Rheumatoid arthritis, juvenile arthritis, Crohn’s disease, ankylosing spondylitis, psoriasis

TNF-α

08/25/2017 Boehringer (U.S.) Ingelheim Pharmaceuticals

Tremfya (guselkumab)

Psoriasis

Interleukin 23

07/13/2017 Janssen Biotech (U.S.)

Rituxan hycela (rituximab Chronic lymphocytic leukemia and hyaluronidase human)

CD20, Hyaluridase

06/23/2017 Genentech (U.S.)

Haegarda (C1 esterase inhibitor subcutaneous)

Hereditary angioedema

C1 Esterase

06/22/2107 CSL Behring (U.S.)

Kevzara (sarilumab)

Rheumatoid arthritis

Interleukin 6

05/22/2017 Sanofi & (U.S.) Regeneron

Dupixent (dupilumab)

Severe eczema

Interleukin-4 receptor alpha

03/28/2017 Regeneron (U.S.)

Siliq (brodalumab)

Plaque psoriasis

Interleukin 17 receptor

02/15/2017 Valeant Pharma (U.S.)

Basaglar (insulin glargine injection)

Type II diabetes

GLP-1

12/16/2016 Eli Lilly (U.S.)

Xultophy (insulin degludec Diabetes mellitus type II

GLP-1

11/21/2016 Novo Nordisk (U.S.)

Soliqua 100/33 (insulin glargine and lixisenatide injection)

Diabetes mellitus type II

GLP-1

11/21/2016 Sanofi (U.S.)

Amjevita (adalimumab)

Rheumatoid arthritis, juvenile idiopathic arthritis, Crohn’s disease, ulcerative colitis, alkylosing spondylitis, plaque psoriasis

TNF-α

09/23/2016 Amgen (U.S.) Continued

TABLE 6.3 List of biologics for the SC route approved by the FDA—cont’d Date of approval

Brand name (drug name)

Indications

Target

Company name

Cuvitru, immune globulin

Primary immune deficiencies

Immunoglobulin

09/14/2016 Shire (U.S.)

Adlyxin (lixisenatide)

Type II diabetes mellitus

GLP-1

07/28/2016 Sanofi (U.S.)

Zinbryta (daclizumab)

Multiple sclerosis

Interleukin 2 receptor

05/27/2016 Biogen (U.S.)

Taltz (ixekizumab)

Plaque psoriasis

Interleukin 17A

03/22/2016 Eli Lilly (U.S.)

Amjevita (Adalimumab)

Rheumatoid arthritis, juvenile arthritis, Crohn’s disease, ankylosing spondylitis, psoriasis

TNF-α

09/23/2016 Amgen (U.S.)

Erelzi (etanercept)

Rheumatoid arthritis, juvenile arthritis, ankylosing spondylitis, psoriasis

TNF-α

08/30/2016 Sandoz (U.S.)

Basaglar (insulin glargine injection)

Type II diabetes mellitus

GLP-1

12/16/2015 Eli Lilly

Nucala (mepolizumab)

Asthma

Interleukin 5

11/04/2015 GlaxoSmithKline (U.S.)

Strensiq (asfotase alfa)

Perinatal, juvenile and infantile hypophosphatasia

Alkaline phosphatase

10/23/2015 Alexion (U.S.)

Tresiba (insulin degludec injection)

Diabetes mellitus

GLP-1

10/16/2015 Novo Nordisk (U.S.)

Ryzodeg 70/30 (insulin Diabetes mellitus degludec and insulin aspart injection)

GLP-1

10/16/2015 Novo Nordisk (U.S.)

Repatha (evolocumab)

Treatment for low density level cholesterol (LDL)

PCSK9 (proprotein 08/27/2015 Amgen convertase subtilisin kexin (U.S.) type 9)

Praluenttm (alirocumab)

Treatment for low density level cholesterol (LDL)

PCSK9 (proprotein 07/24/2015 Sanofi convertase subtilisin kexin (U.S.) type 9)

Neupogen (filgrastim)

Neutropenia

Leukocyte growth factor

03/06/2015 Novartis (U.S.)

Toujeo (insulin glargine injection)

Diabetes mellitus

Cosentyxtm (secukinumab) Plaque psoriasis

GLP-1

02/25/2015 Sanofi (U.S.)

Interleukin 17

01/23/2015 Novartis (U.S.)

Zarxio (filgrastim-Sndz)

Incidence of infection‚ as manifested by febrile neutropenia‚ in Leukocytes growth factor patients with nonmyeloid malignancies

03/06/2015 Sandoz (U.S.)

Trulicity (dulaglutide)

Diabetes mellitus

09/18/2014 Eli Lilly (U.S.)

GLP-1

Hyqvia [immune globulin Immune globulin with a recombinant human hyaluronidase Immune globulin and infusion 10% (human) with hyaluronidase recombinant]

09/12/2014 Baxter (U.S.)

Plegridy (peginterferon beta-1a)

Multiple sclerosis

Interferon beta

08/15/2014 Biogen (U.S.)

Tanzeum (albiglutide)

Diabetes mellitus type II

GLP-1

04/15/2014 GlaxoSmithKline (U.S.)

Mircera (methoxy Anemia associated with chronic renal failure polyethylene glycol-epoetin beta)

Erythropoiesisstimulating agent (ESA)

11/14/2014 Vifor (U.S.)

Herceptin SC (trastuzumab) Breast cancer

Human epidermal growth 02/10/2014 Genentech factor receptor 2 (HER2) (U.S.)

Kynamro (mipomersen sodium)

Apo-B Lipoprotein

01/17/2013 Genzyme (U.S.) Corporation

Gattex (teduglutide [rDNA Short bowel syndrome origin])

GLP-2

12/21/2013 NPS Pharma (U.S.)

Benlysta (belimumab)

B-lymphocyte stimulator (BLyS)-specific

03/09/2011 Human Genome (U.S.) Sciences

Immune globulin

03/04/2010 CSL Behring (U.S.)

Antilipidemic

Positive systemic lupus erythematosus

Hizentra, immune globulin Primary immunodeficiency Prolia (denosumab)

Postmenopausal with osteoporosis, to increase bone mass in Antireceptor activator of men with osteoporosis at high risk for fracture, nuclear factor kappa-B glucocorticoid-induced osteoporosis in men and women at ligand (RANKL) high risk for fracture

06/01/2010 Amgen Inc. (U.S.)

Continued

TABLE 6.3 List of biologics for the SC route approved by the FDA—cont’d Date of approval

Brand name (drug name)

Indications

Target

Stelara (ustekinumab)

Plaque psoriasis

Interleukin 12 and 23

09/25/2009 Janssen Biotech (U.S.)

Extavia (inteneron beta-l b) Multiple sclerosis

Interferon beta

08/15/2009 Novartis (U.S.)

Ilaris (canakinumab)

Interleukin-1β

06/17/2009 Novartis (U.S.)

Pegasys (peginterferon alfa- Chronic hepatitis C, hairy cell leukemia, chronic 2a) myelogenous leukemia

IFN-α-2a

08/28/2009 Genentech (U.S.)

Simponi (golimumab)

Rheumatoid arthritis, ankylosing spondylitis, psoriasis

TNF-α

04/24/2009 Janssen Biotech (U.S.)

Cimzia (certolizumab pegol)

Refractory Crohn’s disease

TNF-α

04/23/2008 Bayer Schering (U.S.)

Arcalysttm (rilonacept)

Cryopyrin-associated periodic syndromes (CAPS) disorders

Interleukin-1

02/27/2008 Regeneron (U.S.)

Accretropin (somatropin)

Pediatric growth failure

Somatropin

01/24/2008 Cangene (U.S.)

Valtropin

Growth deficiencies

Somatropin

04/19/2008 LG Lifesciences (U.S.)

Omnitrope (somatropin [rDNA origin])

Growth hormone deficiencies

Human growth hormone

05/30/2006 Sandoz/ (U.S.) Novartis

Zostavax (Zoster vaccine live)

Herpes Zoster

–

05/25/2006 Merck (U.S.)

Vivaglobin

Primary immunodeficiency

Immune globulin

01/09/2006 ZLB Behring (U.S.)

Cryopyrin Associated periodic syndrome

Company name

Iplex (mecasermin rinfabate Growth deficiency [rDNA origin] injection)

Insulin like growth factor 1 12/12/2005 Insmed Inc. (U.S.)

Hylenex recombinant (hyaluronidase human injection)

Hyaluronidase

To enhance delivery of local anesthesia

12/05/2005 Halozyme (U.S.) Therapeutics

Hydase (hyaluronidase injection)

To enhance delivery of local anesthesia

Hyaluronidase

Increlex (mecasermin [rDNA origin] injection)

Growth deficiency

Insulin like growth factor 1 08/31/2005 Tercica Inc. (U.S.)

Levemir (insulin detemir [rDNA origin] injection)

Diabetes mellitus

Insulin receptors

06/17/2005 Novo Nordisk (U.S.)

Orencia (abatacept)

Rheumatoid arthritis

CTLA-4/Fc fusion

12/23/2005 Bristol-Myers (U.S.) Squibb

Amphadase (hyaluronidase To enhance delivery of local anesthesia injection)

Hyaluronidase

10/24/2004 Amphastar (U.S.)

Fuzeon (enfuvirtide) for Injection

HIV-1 infection

HIV fusion inhibitor

10/15/2004 Hoffman (U.S.) La-Roche

Luveris (lutropin alfa for injection)

Infertility treatment

Stimulation of follicular development

05/24/2004 Serono Inc. (U.S.)

Vitrase (hyaluronidase injection)

To enhance delivery of local anesthesia

Hyaluronidase

05/04/2004 ISTA Pharm (U.S.)

Apidra (insulin glulisine [rDNA origin] injection)

Diabetes mellitus

GLP-1

04/16/2004 Aventis (U.S.)

Xolair (omalizumab)

Asthma

IgE

06/20/2003 Genetech (U.S.)

Iprivask (desirudin for injection)

Deep vein thrombosis

Thrombin inhibitor

04/03/2003 Aventis (U.S.)

Humira (adalimumab) injection

Rheumatoid Arthritis

TNF-α

12/30/2002 Abbott (U.S.)

Forteo (teriparatide)

Osteoporosis

Parathyroid hormone (PTH)

11/26/2002 Eli Lilly (U.S.)

Rebif (interferon beta-1a)

Multiple sclerosis

–

03/07/2002 Serono (U.S.)

Neulasta (pegfilgrastim) injection

Nonmyeloid malignancies

Leukocyte growth factor

01/31/2002 Amgen (U.S.)

PEGylated IFN-α-2b

10/16/2002 Schering (U.S.) Corporation

Pegasys (peginterferon alfa- Chronic hepatitis C, hepatitis B 2b)

10/25/2005 Prima Pharm (U.S.)

Continued

TABLE 6.3 List of biologics for the SC route approved by the FDA—cont’d Date of approval

Brand name (drug name)

Indications

Target

Company name

PEG-intron (Peginterferon Alfa-2b

Chronic hepatitis C

PEGylated IFN-α-2b

08/07/2001 Schering (U.S.) Corporation

Kineret (anakinra)

Rheumatoid arthritis

Interleukin-1

11/14/2001 Amgen (U.S.)

Lantus (insulin glargine injection)

Glycemic control in adults and children with type 1 diabetes GLP-1 mellitus and in adults with type 2 diabetes mellitus

04/20/2000 Sanofi Aventis (U.S.)

Actimmune (interferon gamma-1b)

Chronic granulomatous disease, osteopetrosis

IFN-γ-1b

25/02/1999 Horizon Pharma (U.S.)

Enbrel (etanercept)

Rheumatoid arthritis, juvenile arthritis, ankylosing spondylitis, psoriasis

TNF-α

11/02/1998 Amgen (U.S.)

Avonex (interferon beta-1a) Multiple sclerosis

IFN-β-1a

27/05/1996 Biogen Idec (U.S.)

Betaseron (interferon beta1b)

Multiple sclerosis

IFN-β-1b

07/23/1993 Bayer Health (U.S.) care pharm

Humulin (insulin human)

Glycemic control in adults and children with type 1 and type 2 GLP-1 diabetes mellitus

10/28/1982 Eli Lily (U.S.)

Notes: Nonexhaustive list, mainly to show examples of different categories. Information have been obtained for different products from www.accessdata.fda.gov (accessed on June 22, 2019).

4 Targeted localized delivery of biologics

process [10–12]. In addition, the anatomy and physiology, and lymphatic absorption, may differ among species. Hence, it is important to control and maintain standard experimental protocols to reduce sources of variability [13]. The injection site and injection rate play an important role in the drug absorption after SC injection due to various factors. Namely, the thickness of hypodermis differs across various sites of the body and from person to person [14]. The lymphatic absorption and lymph node uptake of proteins is also determined by the site of injection. Differences in SC uptake are generally attributed to differences in blood flow to those areas and/or to regional variations in lymph flow [12]. Apart from these pressure gradients, lymph movement, variable lymph flow, and blood flow affect the drug absorption after an SC injection [15]. However, the role of the injection site is mostly unexplored. Another important factor affecting the uptake from the SC site is molecular size. Lymphatic uptake is usually around 10–100 nm [16]. Reports suggest that 100 nm molecules/liposomes have been found to be trapped at the site of injection, resulting in decreased uptake [17] (Fig. 6.2).

4 Targeted localized delivery of biologics The above two delivery routes, IV infusions and SC injections, cover approximately 95% of biological delivery. Nevertheless, several studies have been performed for alternative nonstandard routes of administration from a delivery of biologicals perspective. The majority of these alternative routes focus on localized delivery, rather than targeting systemic delivery. Localized drug delivery is one delivery option wherein a drug is administered specifically to a particular organ to reduce the unwanted systemic exposure, thereby avoiding the side effects and to increase the therapeutic concentration in

127

the particular organ (Table 6.4). There are persistent efforts in this direction, and Table 6.5 summarizes all the ongoing efforts along with their development stages. In the upcoming subsections, organ-specific delivery options will be discussed in detail.

4.1 Brain targeting In the next 20 years there will be an increase in global drug development for senior citizens and patients with central nervous system (CNS) disorders. Unfortunately, the drug development for brain diseases has a poor success rate. The aim of brain drug delivery is to treat brain disorders by passing through the tight junctions called the blood-brain barrier (BBB). The BBB is a dynamic diffusion barrier which occurs between the endothelial lining and the cerebral microvasculature, made up of special tight junctions to protect the brain from unwanted and harmful substances entering via the bloodstream [19, 20]. The areas of BBB researched until now show that it is simply not a barrier that blocks the drugs, but a complex, wellcoordinated, ever-adapting interface which facilitates the communication between the CNS and blood [19]. Biologics like proteins, peptides, and monoclonal antibodies are macromolecules which have greater resistance to reach the target as they do not cross the BBB. Apart from this, it has been identified that the presence of enzymes that could lead to inactivation of the drug pose a major hindrance after passing through the BBB. There is a need to understand these target junctions’ barriers and transporters in order to promote the permeation of drugs through the brain to reach the target [21]. Strategies have been put forward to improve the therapeutic efficacy of the products at the site of action. One of the reports suggests reengineering the therapeutic protein to get through the BBB with the help of a molecular

128

6. Biologics: Delivery options and formulation strategies

Figure legend: Skin architecture and cellular components

= Blood vessels

= Lymphatics

= Collagen fiber = Elastin fiber

= Fibroblast

= Adipocyte

= Macrophage

Administered = MAb

= Langerhan’s cell

=

Activated Langerhan’s cell

Dermal = dendritic cell

Activated = dermal dendritic cell = T-cell

FIG. 6.2

Challenges and opportunities for subcutaneous delivery of biologics [18].

Trojan horse technology. This is a peptide monoclonal antibody molecule which, when fused together with the biologic molecule and introduced into the brain, binds with the endogenous transporter present in the BBB. This mechanism

of binding helps the transport of the drug through these barriers in the brain [21]. Another approach of delivering the therapeutic drug were reported by Klyachko et al. in 2017 using immune cells, macrophages and monocytes, etc., which

TABLE 6.4 List of biologics approved for localized delivery.

Drug name (brand name)

Date of approved by FDA ROA

Indications

Target

Company name

Brineura (cerliponase alfa)

Slows the loss of ambulation in symptomatic pediatric patients 3 years of age and older with late infantile neuronal ceroid lipofuscinosis type 2 (CLN2), also known as tripeptidyl peptidase 1 (TPP1) deficiency

Lysosomal N-terminal tripeptidyl peptidase

Spinraza (nusinersen)

Treatment of spinal muscular atrophy (SMA) in pediatric and adult patients

Motor 12/23/ neuron-2 2016 (SMN2)directed antisense oligonucleotide

Intrathecal

Biogen, Inc.

Affreza (insulin human)

Improve glycemic control in adult patients with diabetes mellitus

Glucagon like 07/27/ peptide-GLP-1 2014

Inhalation

Mankind Corporation

Exubera (insulin human, rDNA)

Types 1 and 2 diabetes

Glucagon like 01/27/ peptide-GLP-1 2006

Oral inhalation Pfizer

Pulmozyme (dornase alfa)

Cystic fibrosis (CF) patients to improve pulmonary function

DNAse

12/30/ 1993

Inhalation solution

Influenza A and B

05/09/ 2011

For intradermal Sanofi Pasteur use only

04/01/ 2014

Sublingual

Brain drug delivery 04/27/ 2017

Intraventricular BioMarin

Pulmonary drug delivery

Genentech

Transdermal drug delivery Fluzone intradermal quadrivalent Prevention of influenza disease caused by (influenza vaccine) influenza A subtype viruses and type B viruses contained in the vaccine Oral drug delivery Orallair (sweet vernal, orchard, perennial rye, timothy, and 7 Kentucky blue grass mixed pollens allergen extract)

Grass pollen-induced allergic rhinitis with or Eye as target without conjunctivitis organ

Greer Labs

Continued

TABLE 6.4 List of biologics approved for localized delivery—cont’d Date of approved by FDA ROA

Drug name (brand name)

Indications

Target

Company name

Live, monovalent, human attenuated rotavirus stain (rotavirus vaccine, live, oral)

Prevention of rotavirus gastroenteritis caused by G1 and non-G1 types (G3, G4, and G9)

Rotavirus

04/03/ 2008

Oral suspension

Oxervate (cenegermin)

Neurotrophic keratitis

High affinity nerve growth factor

08/22/ 2018

Topical Dompe Pharmaceuticals ophthalmic use

Luxturna (voretigene neparvovec)

Vision loss due to confirmed biallelic RPE65-mediated inherited retinal disease,

Biallelic RPE65 12/19/ 2017

Subretinal injection

Spark Therapeutics

Eylea (Aflibercept)

Diabetic retinopathy in patients with diabetic macular edema (DME)

Vascular endothelial growth factor (VEGF)

07/29/ 2014

Intravitreal injection

Regeneron Pharmaceuticals

Jetrea (ocriplasmin)

Symptomatic vitreomacular adhesion

Alpha-2 antiplasmin

10/17/ 2012

Intravitreal injection

Thrombo Genics Inc.

Lucentis (ranibizumab)

Diabetic macular edema

Vascular endothelial growth factor (VEGF)

08/10/ 2012

Intravitreal injection

Genentech

Macugen (pegaptanib)

Macular degeneration

VEGF

12/17/ 2004

Intravitreal injection

Pfizer

GlaxoSmithKline

Ocular drug delivery

Data collected from FDA website and product information leaflets.

TABLE 6.5 List of biologics under clinical trials for localized delivery. Drug name (brand name)

Indications

Target

Status

Delivery approaches

Company name

Brain delivery Glial cell line-derived neurotrophic Parkinson’s disease factor

r-metHuGDNF Phase II

Convection enhanced North Bristol NHS delivery Trust

Pulmonary delivery Human insulin (Dance 501)

Pharmacokinetic and pharmacodynamic profiles of Dance 501 in healthy subjects without diabetes but with mild to moderate asthma or COPD

Beta-cells

Phase I, II

Inhaler

Dance Biopharm

Recombinant modified vaccinia virus Ankara expressing antigen 85A (MVA85A)

Tuberculosis vaccine

Mycobacterium tuberculosis

Phase I

Aerosol

Oxford university

PUR003

Flu vaccine

Not listed

Phase I

Nebulizer

Pulmatrix

Recombinant replication-deficient human adenoviral tuberculosis vaccine containing immunodominant antigen Ag85A (Ad5Ag85A)

Tuberculosis vaccine

Human adenovirus

Phase I

Aerosol

McMaster University

Teriparatide (MicroCor PTH)

Osteoporosis

Human Phase II parathyroid hormone (increase bone growth)

Dissolving microneedles

MicroCor PTH (1–34) (Corium)

Abaloparatide (baloparatide-TD)

Osteoporosis in postmenopausal women

Human Phase II parathyroid hormone (increase bone growth)

Transdermal microneedle patch

Radius Health

Insulin (icronJet600)

Determining pharmacokinetics and pharmacodynamics of insulin

Beta-cells

Transdermal drug delivery

Early Phase Hollow microneedles I

Nanopass Continued

TABLE 6.5 List of biologics under clinical trials for localized delivery—cont’d Drug name (brand name)

Indications

Target

Status

Delivery approaches

Company name

C19-A3 GNP (proinsulin) MicronJet600

Immunotherapy for diabetes

Beta-cells

Phase I

Hollow microneedles

Nanopass

Glucagon (ZP-glucagon)

Hypoglycemic

Glucagon

Phase I

Transdermal patch

Zosano

Gonadotropin releasing hormone

Infertility

Gonadotropin releasing hormone (GnRH)

Phase II

Iontophoretic patch

Ferring Pharmaceuticals

Mixture of peptides from islet autoantigens multipeptide

Induce or restore immunological tolerance to β-cells

Phase I

NanoPass MicronJet technology

Kings College London

Inactivated polio vaccine

Polio vaccine

Phase II

NanoPass MicronJet 600 microneedle device

Nanopass

Phase I

NanoPass MicronJet technology

Astellas

Single multivalent peanut lysosomal associated membrane protein DNA plasmid (ASP0892) Recombinant hepatitis B vaccine (hepatitis B virus vaccine)

Hepatitis B vaccine

Liver virus, hepatitic B virus

Phase II/III

NanoPass MicronJet technology

Octreotide (mycapssa)

Acromegaly

Growth hormone

Phase III

Capsule using the Chiasma proprietary technology platform transient permeability enhancer

Semaglutide (NN9924)

Type 2 diabetes

Glucagon like peptide receptor

>25 trials; Phase III/I

Tablet with Novo Nordisk absorption-enhancing excipients

GnRH

Phase II

Oral drug delivery

Leuprolide (ovarest)

Pharmacokinetic and pharmacodynamic profiles in healthy female volunteers

Peptelligence: improved solubility and absorption of peptides for oral delivery

Enteris Biopharm Inc.

Salmon calcitonin

Postmenopausal osteoporosis in women

Calcitonin receptor

Phase III/II

Tablet

Tarsa therapeutics

Interferon-α

Idiopathic pulmonary fibrosis

Viral RNA

Phase II

Oral lozenge

Texas Tech University Health Sciences Center/ Amarillo Biosciences, Inc.

Anti-CD3 monoclonal antibody

Hepatitis C

CD-3

Phase II

Oral delivery

Inspira medical

AMD

Vascular endothelial growth factor

Phase II/III

Intravitreal injection

Hemera Biosciences

Ocular drug delivery AAVCAGsCD59

Data collected from clinical trial website (https://clinicaltrials.gov).

134

6. Biologics: Delivery options and formulation strategies

have the capability to infiltrate into the brain during inflammation and were shown to be successful in a mouse model to deliver the therapeutic neurotropic factors and other enzymes [22]. Manipulation of transporters, secretory functions, extracellular pathways, and adsorptive transcytosis are examples of promising approaches for drug development [23]. Furthermore, the application of nanocarriers like polymeric nanoparticles, liposomes, Dendrimers, micelles, etc. could be a useful approach in delivering biologicals into the brain [24]. Over the last two decades, the use of viral vectors like lentivirus, herpes simplex virus, and adeno-associated virus has proven to be one of the options in brain delivery. In addition, exosomes, which are extracellular small vesicles produced by the cells, could offer more advantages over nanoparticles because of their nonimmunogenicity in nature. These have been used to deliver proteins and nucleic acids with the aim to cross the barriers in the brain [25]. Delivering drugs through another route like the intranasal route, which could bypass the BBB and reach the brain directly, have also been explored and could be one of the options in directing the delivery of biologics in future [26]. In relation to this, most of the biological drugs have been approved by the FDA (Food and Drug Administration) like natalizumab for the treatment of multiple sclerosis and bevacizumab for brain cancer, but the commercial success of these drugs is low because of poor permeation through the tight junctions (BBB) in the brain. Due to the major challenges in crossing the BBB and various other reasons, very few biologics have been approved by the FDA for treating brain disorders. However, these drugs require direct injection into the cerebrospinal fluid to exert therapeutic efficacy. Intrathecal ziconotide (2.6 kDa) for chronic pain, intrathecal nusinersen (
7 kDa) for spinal muscular atrophy (SMA), and intraventricular cerliponase alfa (59 kDa) are some biologics approved by the FDA for CNS diseases [27]. A few biologics for

multiple sclerosis have emerged as clinical successes in treating the disease. DNAse or deoxyribonucleases is another biologic that has been identified by researchers as playing an important role in the apoptotic process. These biologics are the enzymes which consist of DNAse 1 and DNAse 2. These enzymes cleave the DNA at a molecular level for various applications like RNA isolation, DNA fragmentation, etc. Reports suggest end-stage Alzheimer’s disease could be improved by injecting DNAse 1 into the brain [28].

4.2 Pulmonary delivery Pulmonary drug delivery appears to be the easy route of administration as this allows the delivery of drugs directly into the lungs and hence drug deposition through this route is more efficient due to the large absorptive surface area (between 70 and 100 m2) and the thinness of the alveolar epithelium (between 0.5 and 1.0 μm) [29]. Due to the limited presence of drug metabolizing enzymes in the lungs, delivery of biologics does not pose a hindrance like other routes. Pulmonary drug delivery can be self-administered by patients. Although this route offers many advantages, many challenges associated with the physicochemical properties of biologics and delivery systems like the instability of biologics, size of particles, and shape and delivery mechanism need to be addressed. Lungs have a complex defense mechanism to pass inhaled drug particles out of them and to eliminate these once deposited. All these factors have a greater impact on delivering biologics through this route. Many other approaches have been taken into consideration in developing products that can overcome these challenges. Studies have reported that only particle sizes ranging from 1 to 5 μm are suitable for pulmonary delivery, and the shape of particles should also be taken into consideration. Interestingly,

4 Targeted localized delivery of biologics

nonspherical shapes are mostly preferred to avoid the particles getting captured by macrophages [30]. Maintaining the size of particles for pulmonary drug delivery is key to overcome the challenges. The size has to be optimized properly because the large particles get deposited in the central surface of airways with more drugs per unit surface area, whereas with small particles, the drug gets deposited in the periphery of the airways with less amount of drug per unit surface area. Micronization for controlling particle size is one option to deal with this challenge [31]. Other approaches like developing particulate nanocarrier systems such as microparticles, nanoparticles [32], and liposomes have been widely used and provide benefits in improving delivery of drugs [33]. Many other approaches have been taken into consideration in developing products that can overcome these challenges. The design of the device to deliver the drugs during inhalation also has a greater impact in deciding the bioavailability of the drug. The most available devices for inhalation at present are the pressurized meter dose inhalers (pMDIs), nebulizers, and dry powder inhalers (DPIs). Each of these has its advantages and disadvantages [34]. There is a need to design an ideal device that can be used to deliver drugs in an ideal manner [35]. The pipelines of biologics for pulmonary delivery remain a smaller portion in the market due to the abovementioned challenges. Currently biologics approved by the FDA for pulmonary drug delivery system are dornase alfa, pulmozyme, exubera (insulin—withdrawn 2007), and afrezza [36] (Table 6.4). Among all the biologics, DNAse is one of the first promising proteins that was approved by the FDA in 1993 (Pulmozyme, Genentech, Inc.). It works by hydrolyzing the DNA strand or breaking down the strands of DNA in the airway tract and clears the sputum to improve the complications of diseases. DNAse has played an important role as a protein therapeutic which is applicable clinically at the time of writing for

135

pulmonary drug delivery [37] to treat the most commonly affected diseases like cystic fibrosis (CF). CF is a disease that results in abnormal functioning of the lungs due to respiratory tract infections, which results in high concentrations of DNA from degenerating polymorphonuclear leukocytes in lungs. Due to a high concentration of DNA, the viscosity of the sputum in the airway tract increases and hence results in various respiratory inflammatory responses. Pulmozyme has proved preclinically as well as clinically to have more advantages over traditional inhalants, such as improved lung deposition, decreased rate of administration, and improved stability of the protein. All these advantages could reduce patient compliance, which is usually faced with the traditional inhalants for treating such types of diseases. Various studies have been done in order to investigate the effect of DNAse as a therapeutic protein to treat these types of diseases. Yang et al. investigated inhalable antibiotic delivery using a dry powder co-delivering recombinant deoxyribonuclease and ciprofloxacin for treatment of cystic fibrosis [38]. The study was based on co-delivering ciprofloxacin and DNAse through a single particulate system, and it was suggested that this single particulate system could be one of the strategies to improve the delivery of antibiotics as the DNAse enhanced the penetration activity of antibiotics and thereby cleared off the high concentration of sputum viscosity in cystic fibrosis patients. This study was done to compare the efficiency of antibiotics alone and in combination with DNAse. The study showed that the effectiveness of this particulate system was able to kill the bacteria more efficiently when compared to ciprofloxacin alone, because DNAse improved the penetration of the antibiotics to diffuse into the airway tract [39]. The delivery route is still in the exploratory phase and requires significant development before it can be considered a standard delivery option for biopharmaceuticals.

136

6. Biologics: Delivery options and formulation strategies

4.3 Transdermal delivery One of the potentially ideal routes to deliver therapeutic proteins and peptides is transdermal drug delivery. This route offers several advantages like noninvasiveness and constant delivery of drugs, hence avoiding frequent interventions. The degradation of therapeutic proteins can be avoided as this route has limited proteolytic enzymes which can degrade the proteins compared to other routes, and hence the bioavailability of proteins and peptides can be improved. It is also convenient for patients as it can be self-administered by placing the patch on the skin, which is not painful. Physiologically, it is well known that skin is the largest organ in the body, and it acts as an excellent natural barrier, preventing most drug and foreign particles from entering the body. This mechanism is governed by the presence of the outermost layer of the skin called the stratum corneum, which is about 10–15 um thick and consists of several bricktype structures called corneocytes organized in a bilayer lipid form. Therefore, only the small molecules (approximately 500 kDa) that have these physicochemical properties can pass through this limiting barrier, whereas the large macromolecules cannot diffuse through it [40]. The success of transdermal drug delivery is limited to the properties of the skin barrier, mainly the presence of the stratum corneum, which restricts the hydrophilic macromolecules from entering the skin. Considering the challenges faced by macromolecules, many strategies and approaches have been developed to address the problem by various physical and chemical enhancement techniques. Some commonly used physical techniques are microporation, iontophoresis [41, 42], electroporation, and sonophoresis [43, 44]. Similarly, chemical enhancement techniques involve the use of permeation enhancers like alcohols, surfactants, sulfoxides, etc. in designing a formulation which could be

used in physical methods to deliver across skin barriers [45]. The application of nanocarriers such as liposomes, micelles, nanoparticles, etc. has been widely used across the globe. In addition, the prodrug approach is also an option that could improve the permeation process—for example, the conjugation of acyl derivatives with INF-α improves the permeation rate up to 2.5- to 5-fold [46]. Although skin has a limited number of proteolytic enzymes when compared to other routes, protease inhibitors are being used for peptide formulations so that the delivery process could be enhanced much more [45]. Combinations of multiple transdermal techniques have further advanced the delivery of biologics with promising results in recent years [47–49]. >20 biologics have been reported and investigated for delivery through the skin. Currently >10 biologics are under development in clinical trials (phases II and III), and >15 products are based on the microneedle system. Most biologic products for transdermal drug delivery are proteins, peptides, and vaccines. Some of the products tested are available in hydrogel formulation—for example, single-chain antiTNF-α antibody DLX105 hydrogel, for which phase II trials (NCT01936337) have been completed. This study was investigated to demonstrate the safety, efficacy, and tolerability of DLX105 hydrogel to treat mild to moderate psoriasis vulgaris. Many strategies were tested preclinically using novel peptides for delivering biologics across the skin using various mechanisms. The delivery of biologics like smallinterfering RNA (siRNA), glyceraldehyde-3phosphate dehydrogenase (GAPDH), interleukin-10, and insulin were delivered successfully using pore-forming peptides such as magainin, cell-penetrating peptides, peptides that create transient openings in the skin, skinpenetrating peptides, and peptides with protein transduction domains. Details of the undergoing clinical trials product for transdermal drug delivery are listed in Table 6.5 [50].

4 Targeted localized delivery of biologics

4.4 Ocular delivery In the present scenario, treatments for various ocular diseases have proved successful using biologic products. Many of the biologic products used for ocular treatment are monoclonal antibodies, proteins, and peptides. However, the blood ocular barriers like the blood-aqueous barrier (BAB) and blood-retinal barrier (BRB) of the eye pose a great challenge for the delivery of large molecular weight biologics to treat both anterior and posterior segment eye diseases. To overcome the BRB, direct intravitreal injections of monoclonal antibodies like anti-vascular endothelial growth factor (anti-VEGF) are the standard of care for treating many posterior segment eye diseases like age related macular degeneration (ARMD), diabetic retinopathy (DR), diabetic macular edema (DME), etc. However, the clinical success of these therapeutics is challenged by a higher frequency of injection [51]. In addition, treatment with such types of biologics pose a challenge because of poor permeability across the retinal barriers, poor availability due to their large molecular size, stability, and additionally the dynamic clearance mechanism of the biologics from the eye. There is an unmet medical need for these challenges to improve patient compliance. There has been a growing interest and enormous attention for researchers to overcome these challenges by developing several novel technologies like a sustained drug delivery system using nanotechnology for delivering biologics [52]. Many approaches have been studied on how to overcome the various ocular barriers, and to minimize the clearance mechanism. The nanotechnology at the time of writing is one strategy that could respond to the challenges using both physical and chemical methods. The enhancement of permeation by different nanocarriers like nanoparticles, micelles, and liposomes was shown to have potential by many researchers in delivering drugs to different ocular tissues [51].

137

DNAse is also one of the biological products that has been used in the treatment of dry eye diseases. Dry eye disease is a condition where the eye lacks moisture and lubrication on the ocular surface, which could lead to visual discomfort, irritation, etc. A few studies have reported that severe dry eye disease results from inflammation due to decreases in tear film nucleases (e.g., DNAse). Treatment of dry eyes with DNAse1 eye drops has been studied in clinical trials. The first eye drop of DNAse (Pulmozyme 0.1%) administered four times a day was studied topically by a Tibrewal and group. They determined the reduction of extracellular DNA in dry eye patients by using DNAse eye drops. Results showed that DNAse eye drops could reduce the excessive extracellular DNA from dry eye patients and improved the dry eye symptoms and inflammation as well [53]. Monoclonal antibodies and their subunits (e.g., bevacizumab and ranibizumab) have found a use in the treatment of ophthalmological conditions. Many others have also shown positive effects and are likely to be used in future. Additional care has to be taken with the ophthalmic delivery route mainly in terms of skilled injection procedure as well as particle control.

4.5 Oral delivery The oral route of administration offers advantages in delivering proteins and peptides because of its ease of drug administration for patients of all ages, and so the demand is high due to patient convenience. Hence, oral drug delivery of biologics remains a “holy grail” at the time of writing. Unfortunately, the success of this route for therapeutics is very poor because of acidic environment and enzymatic degradation due to the presence of intestinal enzymes in the intestinal gut; on the other hand, permeability of these agents is low due to high

138

6. Biologics: Delivery options and formulation strategies

molecular weight, which leads to low bioavailability. The high demand for oral delivery of proteins and peptides has led many researchers to put great effort into developing an oral drug delivery system that can overcome these challenges. Different strategies to overcome the challenges of oral drug delivery are: (i) use of mucoadhesive polymers to increase contact time of the drug with the physiological membrane [54]; (ii) compounds that can disrupt the membrane barrier and promote the transient opening of the epithelial tissues [55]; (iii) materials that can inhibit the degrading proteolytic enzymes in the gut [56] and materials that can promote dissociation of protein (e.g., cyclodextrins) [57]. All these nanotechnology-based mediated forms of oral delivery have not yet improved beyond the preclinical level. Although various approaches are claimed and being investigated by many researchers, many unaddressed challenges remain to be considered to make oral drug delivery of biologics a reality. Clinically, it has been reported that several biologics are being investigated for oral delivery across the globe. Commercially, there are two peptides which are available in the market: (1) Linzess (Iron Wood Pharmaceuticals) and Trulance (Synergy Pharmaceuticals). Trulance was approved in January 2017 for treating chronic idiopathic constipation (CIC). Interestingly, in January 2018, Trulance was approved for the treatment of another disease called irritable bowel syndrome with constipation (IBS-C) [58]. A few peptides are undergoing clinical trials (Phase III), e.g., octreotide for acromegaly, semaglutide for diabetes, insulin for diabetes, salmon calcitonin for osteoporosis, and desmopressin for diabetes. In addition, about four vaccines have been approved clinically (against vibrio, cholera, typhoid and rotavirus). The biologics for oral delivery that are undergoing clinical trials are listed in Table 6.5.

5 Formulation strategies, degradation routes, and role of excipients Biologics (peptide, proteins, mAbs, and vaccines) are naturally less stable than small molecules, and formulation development is often challenging due to their complex structure. Complex molecular structure, lack of welldefined analytical tools, and multiple degradation mechanisms create major problems for the formulation development of biologics and vaccines. Because of the limited availability of drug substance for initial screening and development, formulation scientists perform forced degradation studies using limited analytical techniques, which help in developing optimal formulation and provide insight in understanding degradation pathways of active molecules. Formulation development is one of the most important aspects of drug development, as proper formulation ensures appropriate shelf life and safety to patients. For biologics, the formulation development is generally divided into three interconnected stages: preformulation, formulation, and process development. The preformulation stage involves biochemical analysis, amino acid sequencing, and biophysical characterization of large molecules in the presence of pH, ionic strength, along with development of stability indicating assay. This study also helps researchers to understand degradation pathways and therefore provides an approach to select appropriate excipients of the required characteristics. A thorough understanding of macromolecular behavior can be obtained using high-throughput formulation approaches to explore changes in structure (e.g., secondary, tertiary) and function (e.g., activity, potency, binding wherever possible) in the presence of stress conditions (e.g., pH, temperature, freezethaw, drying, sheer). In the case of liquid formulations, if the initial strategy fails to meet the target product profile, it is recommended to

5 Formulation strategies, degradation routes, and role of excipients

develop a lyophilized product using a suitable lyophilization process. Application of quality by design (QbD) in formulation development is also useful to obtain a stable product with the desired target profile. Through extensive stability studies (both real-time and accelerated) and in vivo animal model testing (when applicable), a potential formulation can be selected for preclinical and clinical study. To minimize the cost and complexity of clinical study, it is recommended that process and formulation optimization should occur at an early stage. Peptide, protein, and other biologically derived macromolecules have rapidly emerged as a major class of pharmaceuticals because of their multiple advantages. Excipients are pharmacologically inactive substances formulated along with the active pharmaceutical ingredients (APIs) to impart specific physicochemical properties to pharmaceutical products. Excipients have a defined functional role in pharmaceutical products like maintaining pH, osmolality, solubility and bioavailability enhancement, antioxidant effect, emulsifying action, stabilization, etc. The stability of the finished product depends on the selection of the excipients, their concentrations and the interaction between drug-excipients and excipientsexcipients interaction. It is important during the early stage development phase that the excipients are carefully screened and optimized based on the interactions mentioned above. Owing to their clinical and commercial success, biologics are the fastest growing class and have become dominant over other conventional therapy. Protein-based macromolecules now constitute a major proportion of therapeutic imperative for the treatment of various diseases. But due to the fragile nature and complex structure of proteins, it is very challenging to maintain integrity during processing, administration, and distribution of these products. Two types of degradation process are responsible for the

139

instability of protein molecular structure: physical instability and chemical instability (Table 6.6). Various excipients like buffers, salts, sugars, and surfactants are designed to improve the stability of protein-based biologics. These excipients are used to optimize the environment surrounding the protein to maintain its conformational and colloidal stability, reduce interactions with neighboring proteins, and block interactions with container surfaces. This section will provide information about majorly used excipients along with their role and functionality (Table 6.6). Prevention against these degradation routes requires careful selection of stabilizing excipients. The choice of the excipients is based on the compatibility, functionality, and variability of critical material characteristics within a certain acceptable range for development of a particular drug product. Selected excipients’ concentrations and characteristics can modulate the finished product performance (e.g., bioavailability and stability). Excipients used for the product formulation should preferably be multicompendial [European Pharmacopeia (EP), Japanese Pharmacopeia (JP), United States Pharmacopeia (USP), etc.] so that the product can be easily registered across the globe with the same version of excipient. Currently, approximately 1000 excipients of >40 functional categories are used in marketed pharmaceutical products. Conventional excipients are simple in structure, well known, pharmacologically inert, and of natural origin such as sugar, minerals, and wheat, but in recent years, many more novel and highly complex excipients have been developed and evolved for specific usage, especially for novel drug delivery systems like lipids, polymers, dextrans, cyclodextrins, polyethylene glycol, etc. Excipients play a vital role in the formulation development of both small and macromolecule pharmaceutical products. The type and level of excipients’ usage depends on various factors like type of API, dosage form,

140

6. Biologics: Delivery options and formulation strategies

TABLE 6.6 Common chemical degradation pathway of amino acid sequences. Reaction

Corresponding amino acid involved

Deamidation

Asparagine and glutamine

Peptide bond hydrolysis

Primarily at aspartic

β-Elimination

Cystine

Disulfide bond reshuffling

Cystine

Oxidation

Cystine and methionine

Thiol-disulfide exchange

Cystine and cysteine

Racemization

Aspartic acid and glycine

Hydrolysis

Aspartic acid, serine and threonine

route of administration, indication, target population, etc. As per the growing status of protein, peptide therapeutics, and vaccines development, the stabilization of biologics during processing and storage poses a significant challenge for product development scientists. Biologics and vaccines are highly unstable in nature and heavily prone to degradation by physical and chemical (oxidation, hydrolysis, deamination, photo-oxidation, and thiooxidation) mechanisms. Excipients play a significant role in the development of stable biologics product and thereby the selection and use of appropriate excipients is very important at the product development stage. Excipients in biologics products can be used for different functionality, such as: (a) controlling pH and tonicity; (b) enhancing the solubility of the active molecule; (c) preventing its aggregation and degradation; (d) enhancing the process and stability of the active molecule; (e) maintaining its conformation; and (f ) other functions like antioxidants, preservatives, bulking agents, etc. The understanding of drug-excipient interactions is also critical in the rational design of formulations to stabilize protein-based therapeutic drugs and vaccines. The complexity of formulation development is often more pronounced for the development of high-concentration antibody formulations due to additional constraints of viscosity, analytical characterization at high

concentration, and the desire to formulate an isotonic formulation while maintaining the ratio of excipient to antibody to provide the stability. Commonly used excipients in biologics and vaccine formulation are summarized in Table 6.7. Regulatory acceptance of excipients is also critical for their selection and inclusion in the final formulation. Regulatory agencies across the globe, maintain information about approved excipients which are already in use with various commercial products. USFDA maintains the list of approved excipients in a database (inactive ingredient database; IID) with their dosage form, route of administration and concentration. In Japan, Japanese Pharmaceutical Excipients Dictionary (JPED) provides compilation of all excipients for which there is a precedence of use in drug products, route of administration, and patient exposure. It includes monographs from the JP or Japanese Pharmaceutical Excipients Council (JPEC) as well as all nonmonograph excipients that have been previously used.

5.1 Surfactants Surfactant are surface active agents, which reduces surface tension of liquids or interfacial tension between a multiple-phase system (usually a two-phase system) by adsorbing on the surface/interface. The role of surfactants in the

5 Formulation strategies, degradation routes, and role of excipients

141

TABLE 6.7 Commonly used excipients and their general range in biologics and vaccine formulations. Category of excipients

General range

Examples

Buffers

5–100 mM

Acetate, succinate, citrate, histidine

Amino acid

–

Arginine, aspartic acid, glutamic acid, lysine

Stabilizer/bulking agents

1%–10%

Lactose, trehalose, dextrose, sucrose, sorbitol

Surfactants

0.01%–0.1% (w/v)

Tween 20, Tween 80, Plurenic F68

Antimicrobial/preservative

–

Benzyl alcohol, m-cresol, phenol, 2-phenoxyethanol

Metals ions/Chelators

–

Ca, Zn, EDTA

Cyclodextrine based

–

Hydrxypropyl β-cyclodextrine

Salts

0–300 M

NaCl

protein-based formulation is to block the protein’s interaction with hydrophobic surfaces (e.g., vial walls or air interfaces), which otherwise can cause denaturation and aggregation of proteins. Some biologics products need surfactants to block the particular subregions of the protein or to block the protein-protein interaction that could lead to denaturation. The surfactants like polysorbate (Polysorbate 80 or Polysorbate 20) and Poloxamer 188 (PO188) are mainly used in protein-based biologics formulation. Even in the presence of these surfactants, protein drug stability is often insufficient, particularly because of agitation-induced aggregation. In recent reports, N-myristoyl phenylalanine Jeffamide (FM1000) has been proven to have better interfaces blocking capacity and stabilizes faster than other conventional surfactants.

5.2 Buffers The role of buffers in formulation is to maintain the pH for optimal solubility and stability of product during manufacturing, storage, and reconstitution. The chemical stability of biologics such as monoclonal antibodies is pH-dependent and slightly acidic conditions are favorable for stability of the formulation. The widely used

buffers in biologics products are acetate, citrate, histidine, succinate, and phosphate buffers. The self-buffering potential of proteins becomes more relevant for high-concentration biologics. Buffer capacity and the possibility of buffer catalysis are crucial characteristics for liquid biologics.

5.3 Lyoprotectors (sugar)/bulking agents The bulking agents are required to provide a matrix to carry the drug which are normally present in low quantities. Generally, mannitol, lactose sucrose, dextran, trehalose, and glycine are used as bulking agents. Bulking agents always play a dual role as filler and cryoprotectors in lyophilized products. These agents have the potential to turn entire formulations into a crystalline (mannitol and glycine) or amorphous state (sucrose). An appropriate selection of lyoprotector results in better product quality (stability and moisture levels and reconstitution time) and facilitates freezedrying and scaling up to commercial batch. The concentration of bulking agents utilized will determine the rationale use (as stabilizer, eutectic temperature modifier or matrixforming agents).

142

6. Biologics: Delivery options and formulation strategies

Aqueous solution-based biologics may pose stability issues because of hydrolytic degradation of active molecules. Lyophilization is a technique used to stabilize pharmaceutical products by removing water from drug aqueous solution. Lyophilization is the most frequently used strategy to achieve the desired stability of a finished product according to its target product profile. It is also proven to be a superior technique for the stabilization of thermolabile biologics, especially vaccines. Lyophilization consists of three phases: freezing followed by primary and secondary drying under vacuum. During the freezing step, protein denaturation can occur either in the freeze-concentrate state or at frozen surface interfaces. To counter the relevant criticality, the formulation can be designed using salts and buffer, which can minimize protein denaturation during the lyophilization process. Lyoprotectant, along with cryoprotectant, is normally needed for stabilization of biologics. Lyoprotectant plays a vital role in protection of the product during the drying phase while cryoprotectant stabilizes the frozen product during the freezing phase. Bulking agents are also added to lyophilized product to prevent product “blowout” especially in the case of low concentration product ( 1% solid). They are also useful in increasing the product collapse temperature and/or to improve product elegance (aesthetic appearance of the cake—maintaining proper shape and lacking irregularities). Sucrose, trehalose, lactose, raffinose, dextran, and hydroxyethyl starch (HES) are commonly used amorphous bulking agents while glycine and mannitol are commonly used crystalline bulking agents. Excipients with high glass transition temperatures (Tg0 ) for amorphous excipients or high eutectic temperatures (Teu) for crystalline excipients allow for faster primary drying. However, the presence of the hydrate form of mannitol and possibility of glass breakage during manufacturing (due to high fill volume, incorrect freezing protocol, and/or high concentration) might

limit the selection of mannitol during excipient screening (Table 6.8).

5.4 Salts The presence of salt in solution is required to maintain the optimal ionic strength, which leads to physical stability of the protein. Salts possess properties to stabilize proteins by inhibiting aggregation. Salts also minimize catalytic activity of phosphate which is present in certain protein/peptide products. Commonly used slats are NaCl, LiCl, Tris HCl, etc.

5.5 Antimicrobial preservatives Antimicrobial preservatives are mainly required for multidose units to ensure the microbiological safety of a solution during use and storage. The addition of preservatives in biologics can trigger compatibility issues between protein and antimicrobial agents that need to be addressed by researchers during product development activities. Some commonly used preservatives are benzyl alcohol, metacresol, phenol, etc.

5.6 Additional formulation considerations 5.6.1 Adjuvants in vaccine formulation Vaccine formulations often contain an important component that may improve the longevity, breadth, magnitude, and intrinsic immunogenicity to antigens with minimal toxicity and lasting immune effects on their own. Strategic formulations of agonists of the innate immune system and carriers that selectively present at the target site of antigen evolve a class of pharmaceutical “adjuvants,” which significantly impact immunity resulting from vaccination. The effects of adjuvants in vaccines could reduce both the amount of antigen and/or number of immunizations required to achieve the efficacy.

TABLE 6.8 Various excipients and their role in biologics formulation depicting typical ranges used for different routes of administrations. Role

Product

Route of admin.

Date Concentration approved

Sodium phosphate

Buffering agent

Reopro

IV bolus

0.01 M

12/22/1994 (U.S.)

Dibasic sodium phosphate anhydrous

Buffering agent

Herceptin

IV bolus

0.335 mg

09/25/1998 (U.S.)

Monobasic sodium phosphate monohydrate

Buffering agent

Orencia

SC

0.114 mg

12/23/2005 (U.S.)

Monobasic sodium phosphate

Buffering agent

Remicade

IV infusion

2.2 mg

08/24/1998 (U.S.)

Dibasic sodium phosphate

Buffering agent

Remicade

IV infusion

6.1 mg

08/24/1998 (U.S.)

Sodium phosphate, monobasic, monohydrate Buffering agent

Tysabri

IV infusion

123 mg

11/23/2004 (U.S.)

Sodium phosphate, dibasic, heptahydrate

Buffering agent

Tysabri

IV infusion

17 mg

11/23/2004 (U.S.)

Zinc acetate

Buffering agent

Nutropin depot

IV infusion

1.2 mg

12/22/1999 (U.S.)

Zinc carbonate

Buffering agent

Nutropin depot

IV infusion

0.8 mg

12/22/1999 (U.S.)

Acetate

Buffering agent

Udenyca (pegfilgrastim)

SC

0.35 mg

11/02/2018 (U.S.)

Acetate

Buffering agent

Nivestym (filgrastim)

IV infusion

0.59 mg

07/20/2018 (U.S.)

Acetate

Buffering agent

Fulphila (pegfilgrastim)

SC

0.7 mg

06/04/2018 (U.S.)

Monobasic sodium phosphate

Buffering agent

Remicade

IV infusion

2.2 mg

08/24/1998 (U.S.)

Dibasic sodium phosphate

Buffering agent

Remicade

IV infusion

6.1 mg

08/24/1998 (U.S.)

Excipients (category) Buffer

Continued

TABLE 6.8 Various excipients and their role in biologics formulation depicting typical ranges used for different routes of administrations—cont’d Product

Route of admin.

Date Concentration approved

Sodium phosphate, monobasic, monohydrate Buffering agent

Tysabri

IV infusion

123 mg

11/23/2004 (U.S.)

Sodium phosphate, dibasic, heptahydrate

Buffering agent

Tysabri

IV infusion

17 mg

11/23/2004 (U.S.)

Sodium phosphate monobasic monohydrate

Buffering agent

Retacrit (epoetin alfa)

IV

1.3 mg

05/15/2018 (U.S.)

Threonine

Buffering agent

Retacrit (epoetin alfa)

IV

0.25 mg

05/15/2018 (U.S.)

Disodium succinate hexahydrate

Buffering agent

Ixifi (infliximab)

IV infusion

12.1 mg

12/13/2017 (U.S.)

Sodium phosphate (monobasic, monohydrate) Buffering agent

Mvasi (bevacizumab)

IV infusion

23.2 mg

09/14/2017 (U.S.)

Sodium phosphate (dibasic, anhydrous)

Buffering agent

Mvasi (bevacizumab)

IV Infusion

4.8 mg

09/14/2017 (U.S.)

Monobasic sodium phosphate monohydrate

Buffering agent

Renflexis (infliximab)

IV infusion

5.55 mg

04/21/201 (U.S.)

Sodium citrate

Buffering agent

Erelzi (etanercept)

SC

13.52 mg

08/30/2016 (U.S.)

Sodium dihydrogen phosphate monohydrate Buffering agent

Inflectra (infliximab)

IV infusion

2.2 mg

04/05/2016 (U.S.)

Potassium dihydrogen phosphate

Buffering agent

Shingrix (zoster vaccine recombinant, adjuvanted)

IM

0.54 mg

10/20/2017 (U.S.)

Sodium dihydrogen phosphate dihydrate

Buffering agent

Shingrix (zoster vaccine recombinant, adjuvanted)

IM

0.160 mg

10/20/2017 (U.S.)

Disodium phosphate anhydrous

Buffering agent

Shingrix (zoster vaccine recombinant, adjuvanted)

IM

0.15 mg

10/20/2017 (U.S.)

Sodium phosphate

Buffering agent

Luxturna (voretigene neparvovecrzyl)

Intra retinal

10 mM

12/19/2017 (U.S.)

Sodium phosphate, dibasic dodecahydrate

Buffering agent

Heplisav-b (hepatitis b vaccine recombinant)

IM

1.75 mg/mL

11/09/2017 (U.S.)

Excipients (category)

Role

Disodium phosphate anhydrous

Buffering agent

Shingrix (zoster vaccine recombinant, adjuvanted)

IM

0.15 mg

10/20/2017 (U.S.)

Sodium phosphate

Buffering agent

Luxturna (voretigene neparvovec)

Intra retinal

10 mM

12/19/2017 (U.S.)

Sodium phosphate, dibasic dodecahydrate

Buffering agent

Heplisav-b (hepatitis b vaccine recombinan)

IM

1.75 mg/mL

11/09/2017 (U.S.)

Sodium phosphate, monobasic dihydrate

Buffering agent

Heplisav-b (hepatitis b vaccine (recombinant)

IM

0.48 mg/mL

11/09/2017

Polylactide-coglycolide (PLG)

Controlled release polymer

Nutropin depot

IV infusion

68.9 mg

12/22/1999 (U.S.)

Sodium chloride

Isotonicity

Reopro

IV

0.15 M

12/22/1994 (U.S.)

Sodium chloride

Isotonicity

Enbrel

SC

120 Mm

11/02/1998 (U.S.)

Sodium chloride

Isotonicity

Truxima (rituximab)

IV infusion

9 mg

11/28/2018 (U.S.)

Sodium

Isotonicity

Udenyca (pegfilgrastim)

SC

0.02 mg

10/30/2018 (U.S.)

Sodium chloride

Isotonicity

Nivestym (filgrastimaafi)

IV infusion

4.93 mg

07/20/2018 (U.S.)

Sodium chloride

Isotonicity

Erelzi (etanercept)

SC

1.5 mg

08/30/2016 (U.S.)

Sodium chloride

Isotonicity

Shingrix (zoster vaccine recombinant, adjuvanted)

IM

4.385 mg

10/20/2017 (U.S.)

Sodium chloride

Isotonicity

Luxturna (voretigene neparvovec)

Intra retinal

180 Mm

12/19/2017 (U.S.)

Sodium chloride

Isotonicity

Heplisav-b (hepatitis b vaccine recombinant)

IM

9.0 mg/mL

11/09/2017 (U.S.)

Sodium chloride

Isotonicity

Synagis (palivizumab)

IM

0.9 mg

06/19/1998 (U.S.)

Potassium chloride

Isotonicity

Lemtrada (alemtuzumab)

IV infusion

0.2 mg

Salts

Continued

TABLE 6.8 Various excipients and their role in biologics formulation depicting typical ranges used for different routes of administrations—cont’d Excipients (category)

Role

Product

Route of admin.

Date Concentration approved 05/07/2001 (U.S.)

Sodium chloride

Isotonicity

Zevalin (ibritumomab tiuxetan)

IV infusion

0.9%

02/19/2002 (U.S.)

Sodium chloride

Isotonicity

Erbituxtm (cetuximab)

IV infusion

8.48 mg/mL

02/12/2004 (U.S.)

Mannitol

Stabilizer

Humira

SC

16.8 mg

12/31/2002 (U.S.)

Sucrose

Stabilizer

Cimzia

SC

100 mg

04/22/2008 (U.S.)

Sorbital

Stabilizer

Simponi

SC

20.5 mg

04/24/2009 (U.S.)

Sucrose

Stabilizer

Enbrel

SC

1%

11/02/1998 (U.S.)

Α,Α-trehalose

Stabilizer

Herceptin

IV bolus

381.8 mg

09/25/1998 (U.S.)

Dihydrate

Stabilizer

Herceptin

IV bolus

381.8 mg

09/25/1998 (U.S.)

Sucrose

Stabilizer

Orencia

SC

68 mg

12/23/2005 (U.S.)

Sucrose

Stabilizer

Remicade

IV infusion

500 mg

08/24/1998 (U.S.)

Α,Α-trehalose

Stabilizer

Herzuma (trastuzumab)

IV infusion

839 mg

12/14/2018 (U.S.)

Mannitol

Stabilizer

Hyrimoz (adalimumab)

SC

9.6 mg

10/30/2018 (U.S.)

Sorbitol

Stabilizer

Nivestym (filgrastimaafi)

IV infusion

50 mg

07/20/2018 (U.S.)

Stabilizer

D-Sorbitol

Stabilizer

Fulphila (pegfilgrastim)

SC

30 mg

06/04/2018 (U.S.)

Sucrose

Stabilizer

Ixifi (infliximab)

IV infusion

250 mg

12/13/2017 (U.S.)

D-Sorbitol

Stabilizer

Ogivri (trastuzumab)

IV fusion

322.6 mg

12/01/2017 (U.S.)

Trehalose dihydrate

Stabilizer

Mvasi (bevacizumab)

IV infusion

240 mg

09/14/2017

Sucrose

Stabilizer

Renflexis (infliximab)

IV infusion

500 mg

04/21/2017 (U.S.)

Sucrose

Stabilizer

Amjevita (adalimumab)

SC

36 mg

09/23/2016 (U.S.)

Sorbitol

Stabilizer

Zarxio (filgrastim)

SC

25 mg

03/06/2015 (U.S.)

Sucrose

Stabilizer

Inflectra (infliximab)

IV infusion

500 mg

04/05/2016 (U.S.)

Sucrose

Stabilizer

Shingrix (zoster vaccine recombinant, adjuvanted)

I M injection 20 mg

10/20/2017 (U.S.)

Sucrose

Stabilizer

Simulect (basiliximab)

IV

10 mg

05/12/1998 (U.S.)

Mannitol

Stabilizer

Simulect (basiliximab)

IV

40 mg

05/12/1998 (U.S.)

Sucrose

Stabilizer

Andexxa [coagulation factor Xa (recombinant)]

IV

2% w/v

05/03/2018 (U.S.)

Mannitol

Stabilizer

Andexxa [coagulation factor Xa (recombinant)]

IV

5% w/v

05/03/2018 (U.S.)

Priming fluid

Mylotarg (gemtuzumab ozogamicin) IV bolus

41.0 mg

05/17/2000 (U.S.)

Polysorbate 80

Solubilizing agent

Reopro

IV bolus

0.001%

12/22/1994 (U.S.)

Polysorbate 80

Solubilizing agent

Humira

SC

0.4 mg

12/31/2002 (U.S.)

Dextran-based excipients Dextran 40 Surfactant

Continued

TABLE 6.8 Various excipients and their role in biologics formulation depicting typical ranges used for different routes of administrations—cont’d Excipients (category)

Role

Product

Route of admin.

Date Concentration approved

Polysorbate

Solubilizing agent

Cimzia

SC

0.1 mg

04/22/2008 (U.S.)

Polysorbate 80

Solubilizing agent

Simponi

SC

0.08 mg

04/24/2009 (U.S.)

Poloxamer 188

Solubilizing agent

Orencia

SC

3.2 mg

12/23/2005 (U.S.)

Polysorbate 20

Solubilizing agent

Herceptin

IV bolus

1.7 mg

09/25/1998 (U.S.)

Polysorbate 80

Solubilizing agent

Cathflo activase

IV bolus

0.2 mg

11/13/1987 (U.S.)

Polysorbate 80

Solubilizing agent

Tysabri

IV infusion

7.24 mg

11/23/2004 (U.S.)

Polysorbate 80

Solubilizing agent

Remicade

IV infusion

0.5 mg

08/24/1998 (U.S.)

Polysorbate 20

Solubilizing agent

Herzuma (trastuzumab)

Solubilizing agent

1.7 mg

12/14/2018 (U.S.)

Polysorbate 80

Solubilizing agent

Truxima (rituximab)

IV infusion

0.7 mg

11/28/2018 (U.S.)

Polysorbate 20

Solubilizing agent

Udenyca (pegfilgrastim)

SC

0.02 mg

11/02/2018 (U.S.)

Polysorbate 80

Solubilizing agent

Nivestym (filgrastim)

IV infusion

0.04 mg

07/20/2018 (U.S.)

Polysorbate 80

Solubilizing agent

Ixifi (infliximab)

IV infusion

0.5 mg

12/13/2017 (U.S.)

Polysorbate 20

Solubilizing agent

Mvasi (bevacizumab)

IV infusion

1.6 mg

09/14/2017 (U.S.)

Sodium acetate trihydrate

Solubilizing agent

Cyltezo (adalimumab)

SC

2.4 mg

08/25/2017 (U.S.)

Polysorbate 80

Solubilizing agent

Amjevita (adalimumab)

SC

0.4 mg

09/23/2016 (U.S.)

Polysorbate 80

Solubilizing agent

Zarxio (filgrastim)

SC

0.02 mg

03/06/2015 (U.S.)

Polysorbate 80

Solubilizing agent

Inflectra (infliximab)

IV infusion

0.5 mg

04/05/2016 (U.S.)

Polysorbate 80

Solubilizing agent

Shingrix (zoster vaccine recombinant, adjuvanted)

IM

0.08 mg

10/20/2017 (U.S.)

Poloxamer 188

Solubilizing agent

Luxturna (voretigene neparvovecrzyl)

Intra retinal

0.001%

12/19/2017 (U.S.)

Polysorbate 80

Solubilizing agent

Heplisav-b [hepatitis b vaccine (recombinant)

IM

0.1 mg/mL

11/09/2017 (U.S.)

Polysorbate 80

Solubilizing agent

Rituxan (rituximab)

IV infusion

0.7 mg

11/26/1997 (U.S.)

Polysorbate 80

Solubilizing agent

Lemtrada (alemtuzumab)

IV infusion

0.1 mg

05/07/2001 (U.S.)

Polysorbate 80

Solubilizing agent

Andexxa (coagulation factor xa (recombinant)

IV

0.01% w/v

05/03/2018 (U.S.)

Polysorbate 80

Solubilizing agent

Kineret (anakinra)

SC

0.70 mg

11/14/2001 (U.S.)

Polysorbate 20

Solubilizing agent

Neulasta (pegfilgrastim)

SC

0.02 mg

01/31/2002

Benzyl alcohol

Preservative

Enbrel

SC

0.9%

11/02/1998 (U.S.)

Glacial acetic acid

Preservative

Amjevita (adalimumab-atto)

SC

0.24 mg

09/23/2016 (U.S.)

Metacresol

Preservative

Humulin (insulin human)

SC

2.5 Mg

10/28/1982 (U.S.)

Antimicrobial/preservative

Continued

TABLE 6.8 Various excipients and their role in biologics formulation depicting typical ranges used for different routes of administrations—cont’d Excipients (category)

Role

Product

Route of admin.

Date Concentration approved

Metacresol

Preservative

Humalog (insulin lispro injection)

SC

3.15 Mg

06/14/1996 (U.S.)

m-Cresol

Preservative

Lantus (insulin glargine injection)

SC

2.7 mg

04/20/2000

Metacresol

Preservative

Novolog (insulin aspart injection)

SC

1.72 mg/mL

06/07/2000 (U.S.)

Phenol

Preservative

Novolog (insulin aspart injection)

SC

1.50 mg/mL

06/07/2000 (U.S.)

Glycerin

Solvent

Humulin (insulin human)

SC

16 mg

10/28/1982 (U.S.)

Glycerin

Solvent

Humalog (insulin lispro injection)

SC

16 mg

06/14/1996 (U.S.)

Retacrit (epoetin alfa-epbx)

IV

0.01 mg

05/15/2018 (U.S.)

Calcium chloride dihydrate Glycine

Solvent

Retacrit (epoetin alfa-epbx)

IV

7.5 mg

05/15/2018 (U.S.)

Glycine

Solvent

Simulect (basilixiroab)

IV

20 mg

05/12/1998 (U.S.)

Glycine

Solvent

Synagis (palivizumab)

IM

0.1 mg

06/19/1998 (U.S.)

Glycerol 85%

Solvent

Lantus (insulin glargine injection)

SC

20 mg

04/20/2000

Glycerin

Solvent

Novolog (insulin aspart injection)

SC

16 mg/mL

06/07/2000

and L-histidine monohydrochloride monohydrate

Antioxidant

Simponi

SC

0.44 Mg

04/24/2009 (U.S.)

L-Arginine

hydrochloride

Antioxidant

Enbrel

SC

25 Mm

11/02/1998 (U.S.)

L-Histidine

Hcl monohydrate

Antioxidant

Herceptin

IV bolus

9.5 Mg

09/25/1998 (U.S.)

Amino acid and small acids L-Histidine

L-Histidine

Antioxidant

Herceptin

IV bolus

6.1 Mg

09/25/1998 (U.S.)

L-Arginine

Antioxidant

Cathflo Activase

IV bolus

77 Mg

11/13/1987 (U.S.)

Antioxidant

Herzuma (trastuzumab)

IV infusion

9.5 Mg

12/14/2018 (U.S.)

Adipic acid

Antioxidant

Hyrimoz (adalimumab)

SC

2.69 Mg

10/30/2018 (U.S.)

Leucine

Antioxidant

Retacrit (epoetin alfa)

IV

1 Mg

05/15/2018 (U.S.)

Antioxidant

Retacrit (epoetin alfa)

IV

0.25 Mg

05/15/2018 (U.S.)

Phenylalanine

Antioxidant

Retacrit (epoetin alfa)

IV

0.5 mg

05/15/2018 (U.S.)

Succinic acid

Antioxidant

Ixifi (infliximab)

IV infusion

0.6 mg

12/13/2017 (U.S.)

L-Histidine

Antioxidant

Ogivri (trastuzumab)

IV infusion

9.4 mg

12/01/2017 (U.S.)

Glacial acetic acid

Antioxidant

Cyltezo (adalimumab)

SC

0.13 mg

08/25/2017 (U.S.)

Phenylalanine

Antioxidant

Retacrit (epoetin alfa)

IV

0.5 mg

05/15/2018 (U.S.)

Succinic acid

Antioxidant

Ixifi (infliximab)

IV infusion

0.6 mg

12/13/2017 (U.S.)

L-Histidine

Antioxidant

Ogivri (trastuzumab)

IV infusion

9.4 mg

12/01/2017 (U.S.)

Citric acid

Antioxidant

Kineret (anakinra)

SC

1.29 mg

11/14/2001 (U.S.)

Lactic acid

Antioxidant

Cimzia

SC

0.9 mg

04/22/2008 (U.S.)

Citric acid

Antioxidant

Erelzi (etanercept)

SC

0.786 mg

08/30/2016 (U.S.)

L-Histidine

L-Glutamic

Hcl

acid

hydrochloride monohydrate

hydrochloride monohydrate

Continued

TABLE 6.8 Various excipients and their role in biologics formulation depicting typical ranges used for different routes of administrations—cont’d Role

Product

Route of admin.

Date Concentration approved

Zinc oxide

–

Humulin (insulin human)

SC

0.017 mg

10/28/1982 (U.S.)

Zinc ion

–

Humalog (insulin lispro injection)

SC

0.046 mg

06/14/1996 (U.S.)

Disodium edetate dihydrate

–

Lemtrada (alemtuzumab)

IV infusion

0.0187 mg

05/07/2001 (U.S.)

Disodium EDTA

–

Kineret (anakinra)

SC

0.12 mg

11/14/2001 (U.S.)

Zinc

–

Lantus (insulin glargine injection)

SC

30 μg

04/20/2000 (U.S.)

Zinc

–

Novolog (insulin aspart injection)

SC

19.6 μg/mL

06/07/2000 (U.S.)

Polyethylene glycol

Vehicle

Ogivri (trastuzumab)

IV infusion

94.1 mg

12/01/2017 (U.S.)

Macrogol 3350

Vehicle

Ogivri (trastuzumab)

IV infusion

NA

12/01/2017 (U.S.)

Cholesterol

Emulsifying agent

Shingrix (zoster vaccine recombinant, adjuvanted)

IM

0.25 mg

10/20/2017 (U.S.)

Excipients (category) Metal ions/chelators/others

153

References

The most commonly used adjuvant formulations include aluminum hydroxide and aluminum phosphate for human use because of their historic safety and efficacy profiles. For the selection of adjuvants, it is important to understand the interaction between antigenadjuvant and their impact on stability and immunogenesity. While alum-based adjuvants are very traditional, quite successful, and very commonly used, newer adjuvant mechanisms are being explored, including delivery mechanisms like liposome/virosome-based/inspired systems. 5.6.2 Stability of biologics, typical shelf life and storage considerations Biologics are fragile in nature especially when compared to small molecules. One of the main challenges is their stability, mainly in liquid form. Because of their complex nature, they are prone to several routes of degradation— most of the time, more than one degradation pathway is observed. Typically, biopharmaceuticals are stored and maintained under refrigerated conditions, most of them at 5  3 °C. The liquid forms typically have a shelf life of 24 months while the lyophilized form has a shelf life of 36–48 months. More and more ready to use options (e.g., prefilled syringes, autoinjectors) are becoming available, which pose additional challenges to secure stability during the shelf life of the product. 5.6.3 Toxicity and immunogenicity of biologics Over the journey of biopharmaceutical development and especially for monoclonal antibodies, the trend over the last 40 years has been to get as close to the human proteins sequence as possible. At first, biopharmaceuticals had sequences coming from animal origins (e.g., mouse), but slowly the sequences have started to become more humanized and now a large

majority are fully human. Newer formats and sequences are being developed as well, but their development does involve consideration on sequences already present/exposed to humans. The main reason for getting the protein sequence close to that of humans is to minimize any risk of immunogenicity, which is the main reason for toxicities of biopharmaceuticals.

6 Conclusion Biologics are an important class of molecules and they continue to gain in significance. Currently the options to deliver them are largely limited to intravenous and subcutaneous injections. While the existing systems have been employed for several years, there is a need to understand the operational mechanistics for these routes, especially for subcutaneous administration. Improving their efficiency while pushing the existing limits will help clinicians on a broad level. On the other hand, there is a need to develop drug delivery options for localized application if not systematic delivery. Clinically meaningful efforts are being attempted which hold promise for the future; multiple studies and different approaches are being developed. Having these delivery options will open new therapeutic possibilities with patient ease and comfort at the center.

References [1] Anselmo AC, Gokarn Y, Mitragotri S. Non-invasive delivery strategies for biologics. Nat Rev Drug Discov 2018;18:19. [2] Jackisch C, et al. Subcutaneous administration of monoclonal antibodies in oncology. Geburtshilfe Frauenheilkd 2014;74(4):343–9. [3] Kling J. Highly concentrated protein formulations. Westborough, MA: BioProcess International; 2014. [4] Leveque D. Subcutaneous administration of anticancer agents. Anticancer Res 2014;34(4):1579–86.

154

6. Biologics: Delivery options and formulation strategies

[5] Supersaxo A, Hein WR, Steffen H. Effect of molecular weight on the lymphatic absorption of water-soluble compounds following subcutaneous administration. Pharm Res 1990;7(2):167–9. [6] Harris RJ, Shire SJ, Winter C. Commercial manufacturing scale formulation and analytical characterization of therapeutic recombinant antibodies. Drug Dev Res 2004;61(3):137–54. [7] Shire SJ. Challenges in the subcutaneous (SC) administration of monoclonal antibodies (mAbs). In: Monoclonal antibodies. Woodhead Publishing; 2015. p. 131–8. [8] Yadav S, Shire SJ, Kalonia DS. Factors affecting the viscosity in high concentration solutions of different monoclonal antibodies. J Pharm Sci 2010;99(12):4812–29. [9] Haller MF. Converting intravenous dosing to subcutaneous dosing with recombinant human hyaluronidase. Pharm Technol 2007;31(10):118–32. [10] McLennan DN, Porter CJ, Charman SA. Subcutaneous drug delivery and the role of the lymphatics. Drug Discov Today Technol 2005;2(1):89–96. [11] Porter C, Charman W. Transport and absorption of drugs via the lymphatic system. Adv Drug Deliv Rev 2001;50(1–2):1–2. [12] Porter CJ, Charman SA. Lymphatic transport of proteins after subcutaneous administration. J Pharm Sci 2000;89(3):297–310. [13] Beshyah SA, et al. The effect of subcutaneous injection site on absorption of human growth hormone: abdomen versus thigh. Clin Endocrinol (Oxf ) 1991;35 (5):409–12. [14] Gibney MA, et al. Skin and subcutaneous adipose layer thickness in adults with diabetes at sites used for insulin injections: implications for needle length recommendations. Curr Med Res Opin 2010;26(6):1519–30. [15] Richter WF, Bhansali SG, Morris ME. Mechanistic determinants of biotherapeutics absorption following SC administration. AAPS J 2012;14(3):559–70. [16] Swartz MA. The physiology of the lymphatic system. Adv Drug Deliv Rev 2001;50(1–2):3–20. [17] Oussoren C, Storm G. Liposomes to target the lymphatics by subcutaneous administration. Adv Drug Deliv Rev 2001;50(1–2):143–56. [18] Turner MR, Balu-Iyer SV. Challenges and opportunities for the subcutaneous delivery of therapeutic proteins. J Pharm Sci 2018;107(5):1247–60. [19] Ramlakhan N, Altman J. Breaching the blood-brain barrier: The brain has gatekeepers that let in essential nutrients but keep out substances in the blood that would interfere with its nerve cells. Studies of this barrier may lead to ways of supplying drugs to the otherwise inac; 1990. [20] Rubin L, Staddon J. The cell biology of the blood-brain barrier. Annu Rev Neurosci 1999;22(1):11–28. [21] Pardridge WM, Boado RJ. Reengineering biopharmaceuticals for targeted delivery across the blood-brain

[22]

[23] [24] [25]

[26]

[27]

[28]

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

[37] [38]

[39]

barrier. In: Methods in enzymology. Elsevier; 2012. p. 269–92. Klyachko NL, et al. Macrophages with cellular backpacks for targeted drug delivery to the brain. Biomaterials 2017;140:79–87. Freskga˚rd P-O, Urich E. Antibody therapies in CNS diseases. Neuropharmacology 2017;120:38–55. Teleanu D, et al. Blood-brain delivery methods using nanotechnology. Pharmaceutics 2018;10(4):269. Ha D, Yang N, Nadithe V. Exosomes as therapeutic drug carriers and delivery vehicles across biological membranes: current perspectives and future challenges. Acta Pharm Sin B 2016;6(4):287–96. Lochhead JJ, Thorne RG. Intranasal delivery of biologics to the central nervous system. Adv Drug Deliv Rev 2012;64(7):614–28. Keren N, Flynn D. Enabling novel treatments for nervous system disorders by improving methods for traversing the blood-brain barrier. In: Proceedings of a workshop; 2018. Tetz V, Tetz G. Effect of deoxyribonuclease I treatment for dementia in end-stage Alzheimer’s disease: a case report. J Med Case Rep 2016;10(1):131. Olsson B, et al. Pulmonary drug metabolism, clearance, and absorption. In: Controlled pulmonary drug delivery. Springer; 2011. p. 21–50. Morales JO, et al. Challenges and future prospects for the delivery of biologics: oral mucosal, pulmonary, and transdermal routes. AAPS J 2017;19(3):652–68. Ruffin RE, et al. The effects of preferential deposition of histamine in the human airway. Am Rev Respir Dis 1978;117(3):485–92. Klingler C, M€ uller BW, Steckel H. Insulin-micro-and nanoparticles for pulmonary delivery. Int J Pharm 2009;377(1–2):173–9. Chono S, et al. Aerosolized liposomes with dipalmitoyl phosphatidylcholine enhance pulmonary insulin delivery. J Control Release 2009;137(2):104–9. Chung SW, Hil-lal TA, Byun Y. Strategies for noninvasive delivery of biologics. J Drug Target 2012; 20(6):481–501. Jitendra P, Bansal S, Banik A. Noninvasive routes of proteins and peptides drug delivery. Indian J Pharm Sci 2011;73(4):367. Clarke JG. Where have all the inhaled biologics gone?; 2017. https://aerosol-soc.com/abstracts/inhaledbiologics-gone/. Accessed 20 January 2020. MacConnachie A. Dornase-Alfa (DNase, Pulmozyme) for cystic fibrosis. Intensive Crit Care Nurs 1998;14(2):101–2. Yang Y, et al. Inhalable antibiotic delivery using a dry powder co-delivering recombinant deoxyribonuclease and ciprofloxacin for treatment of cystic fibrosis. Pharm Res 2010;27(1):151. Yeo Y. Battling with environments: drug delivery to target tissues with particles and functional biomaterials. Ther Deliv 2010;1(6):757–61.

References

[40] Choy YB, Prausnitz MR. The rule of five for non-oral routes of drug delivery: ophthalmic, inhalation and transdermal. Pharm Res 2011;28(5):943–8. [41] Dubey S, Kalia YN. Electrically-assisted delivery of an anionic protein across intact skin: cathodal iontophoresis of biologically active ribonuclease T1. J Control Release 2011;152(3):356–62. [42] Kalaria DR, Dubey S, Kalia YN. Clinical applications of transdermal iontophoresis; In: Benson HAE, Watkinson AC, editors. Topical and transdermal drug delivery: principles and practice. Wiley; 2011. p. 67–83 [43] Banga AK. Transdermal and intradermal delivery of therapeutic agents: application of physical technologies. CRC Press; 2011. [44] Prausnitz MR, Langer R. Transdermal drug delivery. Nat Biotechnol 2008;26(11):1261. [45] Magnusson B, Runn P. Effect of penetration enhancers on the permeation of the thyrotropin releasing hormone analogue pGlu-3-methyl-His-Pro amide through human epidermis. Int J Pharm 1999;178(2):149–59. [46] Foldvari M, et al. Dermal and transdermal delivery of protein pharmaceuticals: lipid-based delivery systems for interferon α. Biotechnol Appl Biochem 1999; 30(2):129–37. [47] Dubey S, Kalia YN. Understanding the poor iontophoretic transport of lysozyme across the skin: when high charge and high electrophoretic mobility are not enough. J Control Release 2014;183:35–42. [48] Dubey S, et al. Noninvasive transdermal iontophoretic delivery of biologically active human basic fibroblast growth factor. Mol Pharm 2011;8(4):1322–31.

155

[49] Yu J, Dubey S, Kalia YN. Needle-free cutaneous delivery of living human cells by Er:YAG fractional laser ablation. Expert Opin Drug Deliv 2018;15(6):559–66. [50] Anselmo AC, Gokarn Y, Mitragotri S. Non-invasive delivery strategies for biologics. Nat Rev Drug Discov 2018. [51] Mandal A, et al. Ocular delivery of proteins and peptides: challenges and novel formulation approaches. Adv Drug Deliv Rev 2018;126:67–95. [52] Joseph M, et al. Recent perspectives on the delivery of biologics to back of the eye. Expert Opin Drug Deliv 2017;14(5):631–45. [53] Tibrewal S, et al. Tear fluid extracellular DNA: diagnostic and therapeutic implications in dry eye disease. Invest Ophthalmol Vis Sci 2013;54(13):8051–61. [54] Leitner VM, Guggi D, Bernkop-Schn€ urch A. Thiomers in noninvasive polypeptide delivery: in vitro and in vivo characterization of a polycarbophil-cysteine/ glutathione gel formulation for human growth hormone. J Pharm Sci 2004;93(7):1682–91. [55] Ahsan F, et al. Enhanced bioavailability of calcitonin formulated with alkylglycosides following nasal and ocular administration in rats. Pharm Res 2001;18(12):1742–6. [56] Bernkop-Schn€ urch A. The use of inhibitory agents to overcome the enzymatic barrier to perorally administered therapeutic peptides and proteins. J Control Release 1998;52(1–2):1–16. [57] Loftsson T, Brewster ME. Pharmaceutical applications of cyclodextrins. 1. Drug solubilization and stabilization. J Pharm Sci 1996;85(10):1017–25. [58] News Provided By Reportlinker. Oral proteins and peptides market. May 08, 3rd ed; 2018.

C H A P T E R

7

Ethical issues in research and development of nanoparticles Eliana B. Soutoa,b, Joa˜o Dias-Ferreiraa, Ranjita Shegokarc, Alessandra Durazzod, Antonello Santinie a

Department of Pharmaceutical Technology, Faculty of Pharmacy, University of Coimbra, Coimbra, Portugal b CEB—Centre of Biological Engineering, University of Minho, Gualtar Campus, Braga, Portugal c Capnomed GmbH, Zimmern, Germany d CREA-Research Centre for Food and Nutrition, Rome, Italy e Department of Pharmacy, University of Napoli “Federico II”, Napoli, Italy

1 Introduction The first time the idea of handling small objects was made public was in the presentation that Nobel laureate Richard Feynman gave in 1959 entitled “There’s Plenty of Room at the Bottom” [1]. In the 1970s, Norio Taniguchi coined the term “nanotechnology” to describe the manipulation of materials at the nanoscale [2]. In 1986, K. Eric Drexler published “Engines of creation: the coming era of nanotechnology,” launching the concept of nanotechnology worldwide [3]. In the 1990s and together with Robert Freitas Jr., Drexler expanded the concept to medicine, giving birth to nanomedicine. For the 20th anniversary, an updated and expanded edition of Drexler’s book was launched in 2006 [4]. Through this, Drexler earned the title of “Father of Nanotechnology.”

Drug Delivery Aspects https://doi.org/10.1016/B978-0-12-821222-6.00007-5

Over the last 20 years, we have witnessed the exponential scientific and technological advances that document the added-value of nanotechnology in several industries (e.g., pharmaceuticals, nutraceuticals, cosmetics, medical devices, and electronics). Along with technological developments, social and ethical issues arise from basic and applied research in fields that stretch the limits of human knowledge. Nanotechnology refers to research and development of materials at the nanoscale, to create nanomaterials at atomic, molecular, or macromolecular levels, with at least one dimension between 1 and 100 nm. Due to their reduced size, nanomaterials exhibit singular properties that differ from their bulk counterparts, and can be exploited for several applications including nanomedicine. This cutting-edge technology is being used in different fields of knowledge by

157

# 2020 Elsevier Inc. All rights reserved.

158

7. Ethical issues in research and development of nanoparticles

exploiting the high surface-area-to-volume ratio. Although several advantages exist in the use of nanomaterials, the transformation of bulk materials into their nanosized range requires serious follow-up because of the associated enhanced reactivity both in vitro and in vivo [5, 6]. Depending on the shape, size, morphology, and concentration, nanoparticles may interact with cellular processes and compromise cell function, resulting in increased risk of cytotoxicity and/or genotoxicity [7–9]. Although most complex living organisms have sophisticated immune systems, the effects of nanoparticles still need to be assessed at a preclinical level [10]. For a nanoformulation to reach clinical trials, its quality, efficacy, and safety must be ensured [11, 12]. According to the journal Nanoethics (Springer), launched in 2007, this topic is a recent discipline that comprises “Ethics for Technologies that Converge at the Nanoscale” [13]. From a search in the Web of Science (WoS) using “ethics and nanoparticles” and “ethics and nanotechnology” as keywords, only 344 publications appeared indexed, of which only 110 works were published in ethics-related editions, which highlights a new forum of debate (Fig. 7.1).

The application of nanotechnology in medicine—nanomedicine—covering therapy and diagnosis, is a growth field in scientific research. According to the Forbes Councils member de Boeck, nanotechnology is one of the most relevant technologies to watch over the coming decade [14]. It aims at developing new drug delivery systems (DDS), i.e., nanoparticles loaded with a drug, to overcome the problems encountered with the treatments currently available. Several advantages can result from the use of nanoparticles as drug delivery systems, namely: (i) to improve the solubility of drugs belonging to class II and class IV of the Biopharmaceutical Classification System (BCS); (ii) to avoid the burst release of drugs from classical dosage forms and modify the release profile in a controlled fashion [15–18]; (iii) to reduce systemic toxicity due to generalized distribution of drugs; (iv) to enhance the permeability and retention effect by passive targeting, accumulating the drugs in solid tumors; (v) to achieve site-specific delivery by their surfacetailoring with targeting moieties to the site of action; and (vi) to increase drug bioavailability by improving absorption in the gut and in other tissues. In addition, the cost of the therapy can

FIG. 7.1 Number of publications indexed in the Web of Science. Keywords: “ethics and nanoparticles” and “ethics and nanotechnology”; search date: 24.01.2020.

2 Safety of nanoparticles and production methods

also be reduced with the reduction of the dose of drug required to achieve the therapeutic effect. Personalized medicine is another potential use of nanomedicines, as they can be tailored to a patient’s specific needs. According to Resnik and Tinkle, who addressed this topic in a 2009 review, ethical issues in nanomedicine are still linked to risk assessment and management, raised over the course of clinical trials [19]. Ten years later, human trials have not been fully replaced by animal studies yet, while other concerns, such as access to improved medicines, costs of the research, and environmental impact, raise social and ethical challenges in the field. This chapter provides an overview of the safety of different types of nanoparticles, their methods of production, and socioeconomic ethical aspects related to their applications.

2 Safety of nanoparticles and production methods With the possibility to reduce the drug dosage required for a therapeutic effect, the loading of drugs into nanoparticles has become an instrumental approach to create nanomedicines with reduced adverse side effects and risk of toxicological events. The greatest challenge encountered in developing nanomedicines is thus to produce biocompatible and biodegradable carriers, able to deliver the drug payload to the site of action in sufficient amounts. In addition, the selected production method should be fast, reproducible, inexpensive, and not pose any toxicological risk. The obtained product should be sterile (or sterilizable), in particular, if intended for parenteral, ocular, or pulmonary administration. Nanoparticles can be produced from materials that are chemically very different, using either bottom-up or top-down technologies. Bottom-up technologies refer to either chemical or biological methods used for the synthesis of nanoparticles; top-down technologies refer to

159

physical methods starting from the bulk material, which undergoes mechanical fractionation into smaller pieces. Both techniques are reliable, with distinct advantages—bottom-up methods are more appropriate for nanostructures generation with high precision, while top-down methods are ideal for industrial processes. The characteristics of the final product are dependent on the method chosen (biological, chemical, or physical) and the starting materials employed [20, 21]. Physical methods require energy from thermal or electrical sources, variances in mechanical pressure, radiation incidence, or even phenomena such as condensation or evaporation. Chemical methods comprise techniques such as vapor deposition, micro-emulsionbased nanosystems, solvent-gel synthesis, or hydrothermal synthesis. The most frequently used methods are based on synthesis of nanoparticles. Among them, and in addition to being expensive, chemical synthesis of nanoparticles may require the use of toxic reagents (e.g., organic solvents), creating not only environmental issues but also the need to ensure that no toxic residues remain in the final product. As an alternative to chemical methods, the use of biologically friendly approaches, such as enzymatic procedures or microorganism application, in the synthesis are more eco-friendly and safer for human use [22]. Metal-based nanoparticles are the most popular type of inorganic nanoparticles, showing a set of interesting biological applications, and can be used as antibacterial, antifungal, antiviral, anticancer, antiangiogenic, and antiinflammatory agents. Among them, silver nanoparticles are being exploited for their antimicrobial activity [23–25], while others, such as gold nanoparticles and iron oxide cores, have been used for cancer treatment and diagnosis [26]. Any potential risk of toxicity posed by these particles depends on the concentration used, their physicochemical properties, and administration route, all of which govern their pharmacokinetics and pharmacodynamics.

160

7. Ethical issues in research and development of nanoparticles

The most commonly used polymers in the production of polymeric nanoparticles are synthetic poly (lactic-co-glycolic) acid (PLGA) or poly (D,L-lactic) acid (PLA) [17, 27–32], and polysaccharides (e.g., chitosan [16, 33–37], alginate [38, 39]). Both produce biodegradable nanoparticles with the capacity to modify the release profile of the loaded drugs using reproducible production methods. Silica nanoparticles are another type of versatile polymeric nanoparticles [40–42]. They show high thermal stability and low toxicity, and can be surface-modified for site-specific targeting. These particles can also achieve a high drug payload by controlling the surface chemistry, and have been exploited for theragnostics (a single system combining therapy and diagnosis). Lipid nanoparticles (e.g., solid lipid nanoparticles, nanostructured lipid carriers) are also versatile drug delivery systems of low toxicity [8–10, 43, 44], with the advantage of being produced from lipids resembling those existing in the human body and in food, which increases biocompatibility and biotolerability [45, 46]. In addition, their lipid matrix enables them to be loaded with lipophilic and poorly water-soluble drugs [47, 48]. As lipids are known to be absorption/penetration enhancers, the bioavailability of such drugs can be improved when loaded in such particles. Nanoparticles can be surface-tailored with different targeting moieties (e.g., transferrin, folic acid, monoclonal antibodies, aptamers) [18, 49] for site-specific drug delivery by interacting with specific receptors. The half-life of particles in the blood can also be extended by tailoring their surface with hydrophilic polyethylene glycol chains (PEGylation) [30, 31, 50, 51], thereby avoiding the reticuloendothelial system (RES) and remaining in systemic circulation for a longer time for drug release over a sustained period. An additional challenge is to ensure that the surface properties (chemical modification, electrical charge) and parameters (size, polydispersity) remain the same during scale-up. As

nanoparticles are more complex systems than their bulk counterparts, both production and storage can be more costly, particularly as batches may be required to feed preclinical and clinical trials.

3 Applications and handling of nanoparticles Nanoparticles are currently used in several sectors of society. The food industry is presently considered to be safer than 50 years ago, due to continuous innovations in significant areas, such as efficient growth of crops, efficiency of product fabrication, and nutritional value optimization. Yet nanoparticles can still be applied in a range of uses in this sector, e.g., in coatings and packaging of food materials, nanoprobes for food assessment, filters to remove objectionable compounds from food products, and even in additives such as vitamins, minerals, antioxidants, and antimicrobials [52]. For instance, nanolaminate shields protect food products from loss of taste and color, or even from exterior contamination [53]. Silver and gold nanoparticulated systems have also been exploited in nutraceutical formulations [54]. Gold and palladium nanoparticles are used to remove toxic substances from water [55]. In nanomedicine, a range of nanoparticles are impacting gene therapy, fluorescence for tumor imaging, enabling the smartest and cheapest obtention of biological samples, and contributing to molecular biology improvements, tissue engineering, and surgery applications [56]. Furthermore, nanotechnology has been implicated in cleaning contaminated water, in nanoprobes for detection of compounds in several matrices, nanosystems for remediation of petrol accidents, and even (nano) security against bioterrorism using nanofilters [57, 58]. Current daily products—such as cosmetics, sports equipment, luggage, car parts, food processing, and security systems— frequently have something derived from

4 Impact of nanotechnology on society, environment, and health

nanotechnology in their composition. Nanosizing makes every material stronger and lighter in comparison to conventional counterparts [59]. Handling materials at the nanometer scale allows us to reveal optical, magnetic, and electrical properties that differ from those of the bulk material.

4 Impact of nanotechnology on society, environment, and health Nanotechnology has allowed science to reformulate the shape and purpose of materials, from both natural and synthetic sources. Long-term and short-term benefits, advantages, and limitations of nanotechnology must also be transmitted to key industrial stakeholders. The impact of nanotechnological developments is expected to grow continuously, bringing added-value to society; e.g., to improve diagnosis and therapeutics and automate surgeries, which will positively impact waiting lists in the health-care services, reducing errors by health-care professionals and the associated costs of treatment and public health support. With improved quality of life, nanotechnology is expected to contribute to the human life span, and thus cuttingedge health-care services will be in demand [60]. Progression in the field of nanotechnology faces several challenges, and also potential risks concerning both health and the environment. The need to establish adequate methodologies and protocols to determine the risks associated with the usage of nanoparticles is critical and should be implemented by regulatory agencies. Four major challenges are raised in the short term: (i) the technological progression; (ii) the technological prospect; (iii) the capacity of the technology to be accredited and understood; and (iv) public policy enunciation [61–63]. The application of nanotechnology in a range of industrial sectors has triggered a wave of renewal of processes and products at a truly global level. This unprecedented development

161

has occurred in the virtual absence of specific legislation for this type of product. The complexity of nanoparticles and their applications cover several disciplines, such as genetics, physiology, chemistry, physics, and engineering, among others, which is considered a reinforcing factor in the demand for legislators to make a judgment about the creation of appropriate policies to regulate such goods [64]. The foremost problem related to this market is the possibility of producing a correct evaluation of the opportunities and uncertainties related to the risk of its implementation. Since all emerging technologies have advantages and limitations, the determination of their associated risks is mandatory. However, the uncertainty associated with these potential risks should be addressed in terms of legislation applied to industrial production processes, such as methodologies and materials used in manufacturing. The risk assessment should reflect these needs [65, 66]. Nanoparticles are a similar size to biomolecules (e.g., proteins, nucleic acids) and they can therefore mimic their effects on cells via rapid absorption. However, not all nanoparticles have the same capacity to be equally internalized by cells; this property is dependent on the surface energy of these particles, shape, electric charge, and solubility. This may even determine the organ targeting of particles to the liver, heart, kidneys, and brain [67, 68]. Due to their small size, nanoparticles are more reactive than the bulk material. The chemical arrangement of particles is another leading factor governing the risk of toxicity. Interaction between nanoparticles and the biological surroundings is also dependent on other factors, such as biodegradability. Nonbiodegradable particles may undergo accumulation in the body and induce adverse reactions and/or toxicological events [69, 70]. Nanoparticles interact with the biological environment through, e.g., inhalation, specific drug delivery, or via other routes such as enteric absorption (from the gut). Depending on their lipophilic character and surface

162

7. Ethical issues in research and development of nanoparticles

properties, nanoparticles can cross barriers such as the blood-brain barrier and reach organs such as the brain. Other compounds are used in nanoparticles (e.g., nickel, cobalt, polystyrene, latex, and titanium dioxide), and these can be toxic in certain concentration ranges [71, 72]. Toxic welfare also depends on the properties of the given materials to generate reactive oxygen species (ROS), damaging the cells. Nanoparticles in cosmetics comprise another concern requiring attention, without much data having been collected so far. Sunscreens and other dermocosmetic formulations are composed of several types of nanoparticles. More in-depth studies are required, as the long-term impact of these products on human health is not yet fully understood. The main environmental concerns are raised in particular with the use of semiconductors and metal oxide nanoparticles. The environmental risk of toxicity is governed by the concentration of the nanoparticles and the risk of accumulation in soil, biological organisms, and water [73, 74]. In water, nanoparticles act as colloids and frequently collide, binding together, and yield a completely new system with different (and frequently worse) properties. As nanoparticles come into closer contact with the soil, they become more attached to the minerals and all other compounds present in the environment, remaining there for a long time or being decomposed, depending on the structural characteristics of the particles [75]. Despite these effects, some nanoparticles have a positive impact on the environment, such as those based on silver. However, the majority act as endocrine disruptors or pollutants in the soil [76, 77]. Many other ethical problems can be linked with nanotechnology, such as the use of extremely toxic starting materials, despite the final nanoparticles being considered environmentally friendly, which is ultimately a paradox [78]. Nanotechnology generates concerns about pollution requiring further processes, such as

remediation and continuous monitoring, as well as expenditure in the purification of water and soil for agricultural purposes. High energy and water consumption is frequently associated with the production of nanoparticles, which may pose environmental concerns thereby requiring proper regulation [79, 80]. Nanomaterials represent, above certain limits, a new type of toxic material. As particle size decreases, in many nanomaterials the production of ROS increases, as does toxicity. Many current products that employ nanoparticles were tested and proved to cause destructive effects on biological systems via destruction of DNA and inhibition or stimulation of pathways, even leading to cell death. An upcoming opinion about the effect of nanomaterials in living organisms argues that these compounds present toxicity, even for common indicators in the environment such as algae and some fish species [69, 81–83].

5 Ethical issues and socioeconomic concerns Ethical issues raised in nanotechnology research and development are not only linked to the risk of dual use or misuse. Overall, the technology is used to contribute to a better world by offering high-quality products such as food, clean water, a safe environment, medical services, well-defined education, social insurance, democracy, and freedom, as well as many others [84]. Attention should be paid to efficiency and economy regarding the circulation of products and facilities generated by nanotechnologies. Nanotechnological applications in military/army needs must be used only for defense purposes and to ensure security [85]. Knowledge about these applications should also be available to the public. Experts in this field should hold a solid background in concepts of ecology and environmental safety and, ultimately, be responsible for damage arising from

5 Ethical issues and socioeconomic concerns

possible self-destructive practices in science. The research performed on nanotechnologies and materials should also be based on reliable sources. The construction of these products must be based on models incorporating sustainable methods, such as economy of resources, green industrial processes, lack of toxic materials, fair earnings for workers, and legal and humane rules in the workplace. Within the frame of responsible research in nanotechnology, four main goals have been set: (i) responsible development; (ii) education of workers and future workforce; (iii) transfer of knowledge; and (iv) continuous efforts in research and development of modern technologies in sustainable infrastructures [86–88]. The National Nanotechnology Initiative (NNI) proposed setting up a committee of experts on questions of ethical, legal, and social affairs connected with nanotechnology purposes to plot proper connections between the prior terms and consumers, researchers, producers, government and nongovernment institutions, and even legislators. To establish their work properly, they should take into consideration the benefits and risks of these new achievements. The NNI and stakeholders, together, may also generate valuable resources as a result of combining intellectual property and ethics in these developments. The cumulative knowledge about nanotechnology must be passed on to future generations [89, 90]. EU law enforcement agencies have developed patent protection for biotechnology purposes; similar solutions should also be implemented for nanotechnology-based products. Biotechnology legislation can in some cases be applied to nanotechnology products, because they are derived from manipulations of living entities or inert matter on a nanoscale. For a given technology to succeed in the market, it must be well accepted, and to achieve this, the claims of society and its values must be considered to avoid moral judgments.

163

A simple but concise definition for socioeconomic analysis (SEA) may be a parallel of impact analysis (IA). The former is applied by public authorities to achieve an equilibrium on the potential benefits and costs. IA is performed to prepare the legislation for a certain purpose, while SEA is a functional terminology when the legislation is implemented; both are considered the same method when they are applied to analysis of cost-benefit (ACB). Started in France during the 19th century, the idea has subsequently been worked on in several countries, such as the United States, and more widely within the European Union (EU), primarily applied in projects for public infrastructures [91]. Because of growing requirements, the ACB concept has been applied to several public aspects, such as public health issues, environmental restrictions regarding pollutants, and many other public statements. The EU has released several regulations to guide the design of projects according to a public safety ideology, but room is reserved for the contribution of each Member State. SEA promotes a complete, well-balanced, and structured perspective of pros and cons on setting fresh projects, products, or technologies in the market/social context. In the context of novel materials production, SEA generates an inventory of existing positive and negative impacts of the materials to avoid failures in evaluation. For SEA, the utility of a product is instrumental to understand its added-value in comparison to alternatives [85, 92]. Several new properties of nanomaterials will add socioeconomic value to developed products. Indeed, nanotechnologies are currently developing very fast, and socioeconomic issues must follow-up these developments. At present, nanotechnologies are making breakthroughs in several industrial fields. Due to their innovative profiles, it is often difficult for SEA to establish comparisons between marketed products and new products sharing equal functions [93].

164

7. Ethical issues in research and development of nanoparticles

6 Conclusions Better governance of technological innovation in the field of nanotechnology with clearer sustainability aims and higher quality environmental risk and life cycle assessment are required. In many ways, nanotechnology is an example of attempted technological-fixing of problems requiring social, economic, and political solutions. Nanotechnologies will underpin a new wave of industrial expansion that will expand existing resources, and are expected to bring benefits to society, as measured by economic growth, improved health and longevity, environmental protection, strengthened security, social vitality, and enhanced human capabilities.

Acknowledgments This work was partly financed through the projects M-ERA-NET/0004/2015-PAIRED and UIDB/04469/2020. It received support from the Portuguese Science and Technology Foundation, Ministry of Science and Education (FCT/ MEC) through national funds, and was also co-financed by FEDER, under the Partnership Agreement PT2020.

References [1] Feynman R. There’s plenty of room at the bottom. Eng Sci 1960;23:22–36. [2] Sun M, Sen Gupta A. Vascular nanomedicine: current status, opportunities, and challenges. Semin Thromb Hemost 2019; https://doi.org/10.1055/s-0039-1692395. [3] Drexler KE. Engines of creation: The coming era of nanotechnology. New York, NY: Anchor Books, Doubleday; 1986. [4] Drexler KE. Engines of creation 2.0—The coming era of nanotechnology. wowio; 2006. e-book. [5] Gwinn MR, Vallyathan V. Nanoparticles: health effects— pros and cons. Environ Health Perspect 2006;114:1818–25. https://doi.org/10.1289/ehp.8871. [6] Doktorovova S, Santos DL, Costa I, Andreani T, Souto EB, Silva AM. Cationic solid lipid nanoparticles interfere with the activity of antioxidant enzymes in hepatocellular carcinoma cells. Int J Pharm 2014;471:18–27. https://doi.org/10.1016/j.ijpharm. 2014.05.011. [7] Doktorovova S, Shegokar R, Rakovsky E, GonzalezMira E, Lopes CM, Silva AM, Martins-Lopes P,

[8]

[9]

[10]

[11]

[12]

[13] [14]

[15]

[16]

[17]

Muller RH, Souto EB. Cationic solid lipid nanoparticles (cSLN): structure, stability and DNA binding capacity correlation studies. Int J Pharm 2011;420:341–9. https://doi.org/10.1016/j.ijpharm. 2011.08.042. Doktorovova S, Silva AM, Gaivao I, Souto EB, Teixeira JP, Martins-Lopes P. Comet assay reveals no genotoxicity risk of cationic solid lipid nanoparticles. J Appl Toxicol 2014;34:395–403. https://doi.org/10. 1002/jat.2961. Doktorovova S, Souto EB, Silva AM. Nanotoxicology applied to solid lipid nanoparticles and nanostructured lipid carriers—a systematic review of in vitro data. Eur J Pharm Biopharm 2014;87:1–18. https://doi.org/ 10.1016/j.ejpb.2014.02.005. Doktorovova S, Kovacevic AB, Garcia ML, Souto EB. Preclinical safety of solid lipid nanoparticles and nanostructured lipid carriers: current evidence from in vitro and in vivo evaluation. Eur J Pharm Biopharm 2016;108:235–52. https://doi.org/10.1016/j.ejpb. 2016.08.001. Souto EB, Silva GF, Dias-Ferreira J, Zielinska A, Ventura F, Durazzo A, Lucarini M, Novellino E, Santini A. Nanopharmaceutics: part I—clinical trials legislation and good manufacturing practices (GMP) of nanotherapeutics in the EU. Pharmaceutics 2020;12: 146. https://doi.org/10.3390/pharmaceutics12020146. Nardecchia S, Sa´nchez-Moreno P, de Vicente J, Marchal JA, Boulaiz H. Clinical trials of thermosensitive nanomaterials: an overview. Nanomaterials (Basel, Switzerland) 2019;9:191. https://link.springer.com/journal/11569 (Accessed 27 January 2020). De Boeck J. Nanotechnology: how small chips are creating a bigger, brighter future. Forbes 2019;(28th February 2019). Araujo J, Garcia ML, Mallandrich M, Souto EB, Calpena AC. Release profile and transscleral permeation of triamcinolone acetonide loaded nanostructured lipid carriers (TA-NLC): in vitro and ex vivo studies. Nanomedicine 2012;8:1034–41. https://doi.org/10.1016/ j.nano.2011.10.015. Jose S, Fangueiro JF, Smitha J, Cinu TA, Chacko AJ, Premaletha K, Souto EB. Predictive modeling of insulin release profile from cross-linked chitosan microspheres. Eur J Med Chem 2013;60:249–53. https://doi.org/ 10.1016/j.ejmech.2012.12.011. Abrego G, Alvarado H, Souto EB, Guevara B, Bellowa LH, Garduno ML, Garcia ML, Calpena AC. Biopharmaceutical profile of hydrogels containing pranoprofen-loaded PLGA nanoparticles for skin administration: in vitro, ex vivo and in vivo characterization. Int J Pharm 2016;501:350–61. https://doi.org/ 10.1016/j.ijpharm.2016.01.071.

References

[18] Jose S, Cinu TA, Sebastian R, Shoja MH, Aleykutty NA, Durazzo A, Lucarini M, Santini A, Souto EB. Transferrin-conjugated docetaxel-PLGA nanoparticles for tumor targeting: influence on MCF-7 cell cycle. Polymers (Basel) 2019;11. https://doi.org/10.3390/ polym11111905. [19] Resnik DB, Tinkle SS. Ethics in nanomedicine. Nanomedicine (London) 2007;2:345–50. https://doi.org/ 10.2217/17435889.2.3.345. [20] Luttge R. Nanotechnology. In: Microfabrication for industrial applications. Boston: William Andrew Publishing; 2011. p. 91–146. https://doi.org/10.1016/ B978-0-8155-1582-1.00004-6 [Chapter 4]. [21] Ramsden JJ. Nanosystems and their design. In: Nanotechnology. Oxford: William Andrew Publishing; 2011. p. 199–211. https://doi.org/10.1016/B978-0-08096447-8.00010-7 [Chapter 10]. [22] Durazzo A, Lucarini M, Novellino E, Souto EB, Daliu P, Santini A. Abelmoschus esculentus (L.): bioactive components’ beneficial properties-focused on antidiabetic role-for sustainable health applications. Molecules 2018;24:38. https://doi.org/10.3390/molecules 24010038. [23] Sa´nchez-Lo´pez E, Gomes D, Esteruelas G, Bonilla L, Lopez-Machado AL, Galindo R, Cano A, Espina M, Ettcheto M, Camins A, et al. Metal-based nanoparticles as antimicrobial agents: an overview. Nanomaterials 2020;10:292. https://doi.org/10.3390/nano10020292. [24] Barbosa GP, Debone HS, Severino P, Souto EB, da Silva CF. Design and characterization of chitosan/ zeolite composite films—effect of zeolite type and zeolite dose on the film properties. Mater Sci Eng C 2016;60:246–54. https://doi.org/10.1016/j.msec. 2015.11.034. [25] Souto EB, Ribeiro AF, Ferreira MI, Teixeira MC, Shimojo AAM, Soriano JL, Naveros BC, Durazzo A, Lucarini M, Souto SB, et al. New nanotechnologies for the treatment and repair of skin burns infections. Int J Mol Sci 2020;21. https://doi.org/10.3390/ijms 21020393. [26] da Silva PB, Machado RTA, Pironi AM, Alves RC, de Araujo PR, Dragalzew AC, Dalberto I, Chorilli M. Recent advances in the use of metallic nanoparticles with antitumoral action—review. Curr Med Chem 2019;26:2108–46. https://doi.org/10.2174/09298673256 66180214102918. [27] Araujo J, Vega E, Lopes C, Egea MA, Garcia ML, Souto EB. Effect of polymer viscosity on physicochemical properties and ocular tolerance of FB-loaded PLGA nanospheres. Colloids Surf B: Biointerfaces 2009;72:48–56. https://doi.org/10.1016/j.colsurfb.2009.03.028. [28] Canadas C, Alvarado H, Calpena AC, Silva AM, Souto EB, Garcia ML, Abrego G. In vitro, ex vivo and in vivo characterization of PLGA nanoparticles

[29]

[30]

[31]

[32]

[33]

[34]

[35]

[36]

165 loading pranoprofen for ocular administration. Int J Pharm 2016;511:719–27. https://doi.org/10.1016/j. ijpharm.2016.07.055. Cano A, Ettcheto M, Chang JH, Barroso E, Espina M, Kuhne BA, Barenys M, Auladell C, Folch J, Souto EB, et al. Dual-drug loaded nanoparticles of epigallocatechin-3-gallate (EGCG)/ascorbic acid enhance therapeutic efficacy of EGCG in a APPswe/ PS1dE9 Alzheimer’s disease mice model. J Control Release 2019;301:62–75. https://doi.org/10.1016/j. jconrel.2019.03.010. Jose S, Sowmya S, Cinu TA, Aleykutty NA, Thomas S, Souto EB. Surface modified PLGA nanoparticles for brain targeting of Bacoside-A. Eur J Pharm Sci 2014;63:29–35. https://doi.org/10.1016/j.ejps. 2014.06.024. Sanchez-Lopez E, Egea MA, Cano A, Espina M, Calpena AC, Ettcheto M, Camins A, Souto EB, Silva AM, Garcia ML. PEGylated PLGA nanospheres optimized by design of experiments for ocular administration of dexibuprofen-in vitro, ex vivo and in vivo characterization. Colloids Surf B: Biointerfaces 2016;145:241–50. https://doi.org/10.1016/ j.colsurfb.2016.04.054. Silva AM, Alvarado HL, Abrego G, Martins-Gomes C, Garduno-Ramirez ML, Garcia ML, Calpena AC, Souto EB. In vitro cytotoxicity of oleanolic/ursolic acids-loaded in PLGA nanoparticles in different cell lines. Pharmaceutics 2019;11. https://doi.org/10.3390/ pharmaceutics11080362. Ataide JA, Gerios EF, Cefali LC, Fernandes AR, Teixeira MDC, Ferreira NR, Tambourgi EB, Jozala AF, Chaud MV, Oliveira-Nascimento L, et al. Effect of polysaccharide sources on the physicochemical properties of bromelain-chitosan nanoparticles. Polymers (Basel) 2019;11:https://doi.org/10.3390/polym11101681. Cefali LC, Ataide JA, Eberlin S, da Silva Goncalves FC, Fernandes AR, Marto J, Ribeiro HM, Foglio MA, Mazzola PG, Souto EB. In vitro SPF and photostability assays of emulsion containing nanoparticles with vegetable extracts rich in flavonoids. AAPS PharmSciTech 2018;20:9. https://doi.org/10.1208/s12249-0181217-7. Jose S, Fangueiro JF, Smitha J, Cinu TA, Chacko AJ, Premaletha K, Souto EB. Cross-linked chitosan microspheres for oral delivery of insulin: Taguchi design and in vivo testing. Colloids Surf B: Biointerfaces 2012;92:175–9. https://doi.org/10.1016/j.colsurfb. 2011.11.040. Jose S, Prema MT, Chacko AJ, Thomas AC, Souto EB. Colon specific chitosan microspheres for chronotherapy of chronic stable angina. Colloids Surf B: Biointerfaces 2011;83:277–83. https://doi.org/10.1016/j.colsurfb.201 0.11.033.

166

7. Ethical issues in research and development of nanoparticles

[37] Severino P, Souto EB, Pinho SC, Santana MH. Hydrophilic coating of mitotane-loaded lipid nanoparticles: preliminary studies for mucosal adhesion. Pharm Dev Technol 2013;18:577–81. https://doi.org/10.3109/1083 7450.2011.614250. [38] Severino P, Chaud MV, Shimojo A, Antonini D, Lancelloti M, Santana MH, Souto EB. Sodium alginate-cross-linked polymyxin B sulphate-loaded solid lipid nanoparticles: antibiotic resistance tests and HaCat and NIH/3T3 cell viability studies. Colloids Surf B: Biointerfaces 2015;129:191–7. https://doi.org/ 10.1016/j.colsurfb.2015.03.049. [39] Severino P, da Silva CF, Andrade LN, de Lima Oliveira D, Campos J, Souto EB. Alginate nanoparticles for drug delivery and targeting. Curr Pharm Des 2019;25:1312–34. https://doi.org/10.2174/13816128256 66190425163424. [40] Andreani T, Fangueiro JF, Severino P, Souza ALR, Martins-Gomes C, Fernandes PMV, Calpena AC, Gremiao MP, Souto EB, Silva AM. The influence of polysaccharide coating on the physicochemical parameters and cytotoxicity of silica nanoparticles for hydrophilic biomolecules delivery. Nanomaterials (Basel) 2019;9. https://doi.org/10.3390/nano9081081. [41] Andreani T, Kiill CP, de Souza AL, Fangueiro JF, Fernandes L, Doktorovova S, Santos DL, Garcia ML, Gremiao MP, Souto EB, et al. Surface engineering of silica nanoparticles for oral insulin delivery: characterization and cell toxicity studies. Colloids Surf B: Biointerfaces 2014;123:916–23. https://doi.org/10.1016/j. colsurfb.2014.10.047. [42] Andreani T, Miziara L, Lorenzon EN, de Souza AL, Kiill CP, Fangueiro JF, Garcia ML, Gremiao PD, Silva AM, Souto EB. Effect of mucoadhesive polymers on the in vitro performance of insulin-loaded silica nanoparticles: interactions with mucin and biomembrane models. Eur J Pharm Biopharm 2015;93:118–26. https://doi.org/10.1016/j.ejpb.2015.03.027. [43] Silva AM, Martins-Gomes C, Coutinho TE, Fangueiro JF, Sanchez-Lopez E, Pashirova TN, Andreani T, Souto EB. Soft cationic nanoparticles for drug delivery: production and cytotoxicity of solid lipid nanoparticles (SLNs). Appl Sci 2019;9:4438. [44] Silva AM, Martins-Gomes C, Fangueiro JF, Andreani T, Souto EB. Comparison of antiproliferative effect of epigallocatechin gallate when loaded into cationic solid lipid nanoparticles against different cell lines. Pharm Dev Technol 2019;24:1243–9. https://doi.org/ 10.1080/10837450.2019.1658774. [45] Souto EB, Doktorovova S. Chapter 6—Solid lipid nanoparticle formulations pharmacokinetic and biopharmaceutical aspects in drug delivery. Methods Enzymol 2009;464:105–29. https://doi.org/10.1016/S0076-6879 (09)64006-4.

[46] Souto EB, Muller RH. Lipid nanoparticles: effect on bioavailability and pharmacokinetic changes. Handb Exp Pharmacol 2010;115–41. https://doi.org/10.1007/9783-642-00477-3_4. [47] Gokce EH, Ozyazici M, Souto EB. Nanoparticulate strategies for effective delivery of poorly soluble therapeutics. Ther Deliv 2010;1:149–67. https://doi.org/ 10.4155/tde.10.4. [48] Teixeira MC, Carbone C, Souto EB. Beyond liposomes: recent advances on lipid based nanostructures for poorly soluble/poorly permeable drug delivery. Prog Lipid Res 2017;68:1–11. https://doi.org/10.1016/j. plipres.2017.07.001. [49] Souto EB, Doktorovova S, Campos JR, Martins-Lopes P, Silva AM. Surface-tailored anti-HER2/neu-solid lipid nanoparticles for site-specific targeting MCF-7 and BT-474 breast cancer cells. Eur J Pharm Sci 2019;128: 27–35. https://doi.org/10.1016/j.ejps.2018.11.022. [50] Sanchez-Lopez E, Egea MA, Davis BM, Guo L, Espina M, Silva AM, Calpena AC, Souto EMB, Ravindran N, Ettcheto M, et al. Memantine-loaded PEGylated biodegradable nanoparticles for the treatment of glaucoma. Small 2018;14. https://doi.org/ 10.1002/smll.201701808. [51] Sanchez-Lopez E, Ettcheto M, Egea MA, Espina M, Cano A, Calpena AC, Camins A, Carmona N, Silva AM, Souto EB, et al. Memantine loaded PLGA PEGylated nanoparticles for Alzheimer’s disease: in vitro and in vivo characterization. J Nanobiotechnol 2018;16:32. https://doi.org/10.1186/s12951-018-0356-z. [52] Patil M, Mehta DS, Guvva S. Future impact of nanotechnology on medicine and dentistry. J Indian Soc Periodontol 2008;12:34–40. https://doi.org/10.4103/0972-124 X.44088. [53] Azadmanjiri J, Berndt CC, Wang J, Kapoor A, Srivastava VK. Nanolaminated composite materials: structure, interface role and applications. RSC Adv 2016;6:109361–85. https://doi.org/10.1039/ C6RA20050H. [54] Martirosyan A, Schneider Y-J. Engineered nanomaterials in food: implications for food safety and consumer health. Int J Environ Res Public Health 2014;11:5720–50. https://doi.org/10.3390/ijerph110605720. [55] Huifeng Q, Pretzer LA, Velazquez JC, Zhun Z, Wong M. Gold nanoparticles for cleaning contaminated water. J Chem Technol Biotechnol 2013;88:735–41. https:// doi.org/10.1002/jctb.4030. [56] Zdrojewicz Z, Waracki M, Bugaj B, Pypno D, Cabala K. Medical applications of nanotechnology. Postepy Hig Med Dosw (Online) 2015;69:1196–204. [57] Kunduru KR, Nazarkovsky M, Farah S, Pawar RP, Basu A, Domb AJ. 2—Nanotechnology for water purification: applications of nanotechnology methods in wastewater treatment A2—Grumezescu, Alexandru

References

[58]

[59]

[60]

[61]

[62]

[63]

[64]

[65]

[66]

[67]

[68]

[69]

[70]

Mihai. In: Water purification. Academic Press; 2017. p. 33–74. https://doi.org/10.1016/B978-0-12-8043004.00002-2. Gehrke I, Geiser A, Somborn-Schulz A. Innovations in nanotechnology for water treatment. Nanotechnol Sci Appl 2015;8:1–17. https://doi.org/10.2147/NSA. S43773. Mihranyan A, Ferraz N, Strømme M. Current status and future prospects of nanotechnology in cosmetics. Prog Mater Sci 2012;57:875–910. https://doi.org/10.1016/j. pmatsci.2011.10.001. Schulte PA, Salamanca-Buentello F. Ethical and scientific issues of nanotechnology in the workplace. Environ Health Perspect 2007;115:5–12. https://doi.org/10.1289/ ehp.9456. Iavicoli I, Leso V, Ricciardi W, Hodson LL, Hoover MD. Opportunities and challenges of nanotechnology in the green economy. Environ Health 2014;13:78. https:// doi.org/10.1186/1476-069X-13-78. Qiu J. Nanotechnology development in China: challenges and opportunities. Natl Sci Rev 2016;3:148–52. https://doi.org/10.1093/nsr/nww007. Cheng HN, Doemeny LJ, Geraci CL, Grob Schmidt D. Nanotechnology overview: opportunities and challenges. In: Nanotechnology: Delivering on the promise volume 1, vol. 1220. American Chemical Society; 2016. p. 1–12. Brianna S. Perspectives on FDA’s regulation of nanotechnology: emerging challenges and potential solutions. Compr Rev Food Sci Food Saf 2009; 8:375–93. https://doi.org/10.1111/j.1541-4337.2009. 00088.x. Maynard AD. Nanotechnology: assessing the risks. Nano Today 2006;1:22–33. https://doi.org/10.1016/ S1748-0132(06)70045-7. Hwang M, Lee EJ, Kweon SY, Park MS, Jeong JY, Um JH, Kim SA, Han BS, Lee KH, Yoon HJ. Risk assessment principle for engineered nanotechnology in food and drug. Toxicol Res 2012;28:73–9. https://doi.org/ 10.5487/TR.2012.28.2.073. Bogunia-Kubik K, Sugisaka M. From molecular biology to nanotechnology and nanomedicine. Biosystems 2002;65:123–38. https://doi.org/10.1016/S0303-2647 (02)00010-2. Salata OV. Applications of nanoparticles in biology and medicine. J Nanobiotechnol 2004;2:3. https://doi.org/ 10.1186/1477-3155-2-3. Bahadar H, Maqbool F, Niaz K, Abdollahi M. Toxicity of nanoparticles and an overview of current experimental models. Iran Biomed J 2016;20:1–11. https://doi.org/ 10.7508/ibj.2016.01.001. Yang Y, Qin Z, Zeng W, Yang T, Cao Y, Mei C, Kuang Y. Toxicity assessment of nanoparticles in various systems and organs. Nanotechnol Rev 2017;6:279.

167

[71] Singh S, Nalwa HS. Nanotechnology and health safety—toxicity and risk assessments of nanostructured materials on human health. J Nanosci Nanotechnol 2007;7:3048–70. [72] The dose makes the poison. Nat Nanotechnol 2011;6:329. https://doi.org/10.1038/nnano.2011.87. [73] Smita S, Gupta SK, Bartonova A, Dusinska M, Gutleb AC, Rahman Q. Nanoparticles in the environment: assessment using the causal diagram approach. Environ Health 2012;11:S13. https://doi.org/10.118 6/1476-069X-11-S1-S13. [74] Taghavi SM, Momenpour M, Azarian M, Ahmadian M, Souri F, Taghavi SA, Sadeghain M, Karchani M. Effects of nanoparticles on the environment and outdoor workplaces. Electron Physician 2013;5:706–12. https://doi. org/10.14661/2013.706-712. [75] Lee W-M, Kim SW, Kwak JI, Nam S-H, Shin Y-J, An Y-J. Research trends of ecotoxicity of nanoparticles in soil environment. Toxicol Res 2010;26:253–9. https://doi. org/10.5487/TR.2010.26.4.253. [76] Jiang Y, Yu L, Sun H, Yin X, Wang C, Mathews S, Wang N. Transport of natural soil nanoparticles in saturated porous media: effects of pH and ionic strength. Chem Speciat Bioavailab 2017;29:186–96. https://doi. org/10.1080/09542299.2017.1403293. [77] Klitzke S, Metreveli G, Peters A, Schaumann GE, Lang F. The fate of silver nanoparticles in soil solution—sorption of solutes and aggregation. Sci Total Environ 2015;535:54–60. https://doi.org/10.1016/j. scitotenv.2014.10.108. [78] Huang Y-W, Cambre M, Lee H-J. The toxicity of nanoparticles depends on multiple molecular and physicochemical mechanisms. Int J Mol Sci 2017;18:2702. https://doi.org/10.3390/ijms18122702. [79] G€ okc¸ay B, Arda B. Nanotechnology, nanomedicine; ethical aspects, Rev Rom Bioet 2015;13. http://www. bioetica.ro/index.php/arhiva-bioetica/article/view/ 829/pdf. [80] Amenta V, Aschberger K, Arena M, Bouwmeester H, Botelho Moniz F, Brandhoff P, Gottardo S, Marvin HJP, Mech A, Quiros Pesudo L, et al. Regulatory aspects of nanotechnology in the agri/feed/food sector in EU and non-EU countries. Regul Toxicol Pharmacol 2015;73:463–76. https://doi.org/10.1016/j. yrtph.2015.06.016. [81] Fu PP, Xia Q, Hwang H-M, Ray PC, Yu H. Mechanisms of nanotoxicity: generation of reactive oxygen species. J Food Drug Anal 2014;22:64–75. https://doi.org/ 10.1016/j.jfda.2014.01.005. [82] Shinde SK, Grampurohit ND, Gaikwad DD, Jadhav SL, Gadhave MV, Shelke PK. Toxicity induced by nanoparticles. Asian Pac J Trop Dis 2012;2:331–4. https://doi.org/10.1016/S2222-1808(12) 60072-3.

168

7. Ethical issues in research and development of nanoparticles

[83] Yang Z, Liu H, Liu D. Spatial regulation of synthetic and biological nanoparticles by DNA nanotechnology. NPG Asia Mater 2015;7:e161. https://doi.org/10.1038/ am.2015.2. [84] Florczyk SJ, Saha S. Ethical issues in nanotechnology. J Long-Term Eff Med Implants 2007;17:271–80. [85] Patenaude J, Legault G-A, Beauvais J, Bernier L,  Genest J, Beland J-P, Boissy P, Chenel V, Daniel C-E, Poirier M-S, et al. Framework for the analysis of nanotechnologies’ impacts and ethical acceptability: basis of an interdisciplinary approach to assessing novel technologies. Sci Eng Ethics 2015;21:293–315. https://doi. org/10.1007/s11948-014-9543-y. [86] Dowling AP. Development of nanotechnologies. Mater Today 2004;7:30–5. https://doi.org/10.1016/S13697021(04)00628-5. [87] Cozzens S, Cortes R, Soumonni O, Woodson T. Nanotechnology and the millennium development goals: water, energy, and agri-food. J Nanopart Res 2013; 15:2001. https://doi.org/10.1007/s11051-013-2001-y. [88] Salamanca-Buentello F, Persad DL, Court EB, Martin DK, Daar AS, Singer PA. Nanotechnology and the developing world. PLoS Med 2005;2:e97. https:// doi.org/10.1371/journal.pmed.0020097.

[89] National Research Council (US) Committee for the Review of the National Nanotechnology Initiative. Preliminary comments, review of the national nanotechnology initiative. Washington (DC): National Academies Press (US); 2001. 1, Description of the national nanotechnology initiative. [90] Guinee JB, Heijungs R, Vijver MG, Peijnenburg WJGM. Setting the stage for debating the roles of risk assessment and life-cycle assessment of engineered nanomaterials, Nat Nanotechnol 2017;12:727. https://doi.org/ 10.1038/nnano.2017.135. https://www.nature.com/ articles/nnano.2017.135#supplementary-information. [91] Dunphy Guzma´n KA, Taylor MR, Banfield JF. Environmental risks of nanotechnology: national nanotechnology initiative funding, 2000–2004. Environ Sci Technol 2006;40:1401–7. https://doi.org/10.1021/ es0515708. [92] Cinelli M, Coles SR, Sadik O, Karn B, Kirwan K. A framework of criteria for the sustainability assessment of nanoproducts. J Clean Prod 2016;126:277–87. [93] Singh AK. The past, present, and the future of nanotechnology. In: Engineered nanoparticles. Boston: Academic Press; 2016. p. 515–25. https://doi.org/10.1016/B978-012-801406-6.00010-8 [Chapter 10].

C H A P T E R

8

Sterilization of pharmaceutical dosage forms Sarabjit Singha, Dharmesh Mehtab a

b

Formulation Research, CIPLA, Mumbai, India Business Development, Gangwal Chemicals, Mumbai, India

1 Introduction Sterile dosage forms are free from microorganisms, dust, and foreign particles, and should be isotonic. Parenteral products including injectables, transfusion fluids, suspensions, solutions, emulsions, and ophthalmic preparations like eye drops, eye lotions, eye gels, contact lens solutions always need to be sterile. For products like injectables, implants, and ophthalmic products, sterility is a key product attribute for the safety of a product [1]. As per WHO guidelines, sterility can be defined as the freedom from the presence of viable microorganisms. Different sterility test methods like culture-based and nonculturebased test methods are used to evaluate the sterility of different finished formulations. For culture-based test methods, various parameters like composition of culture media, growthpromotion test requirements, and incubation conditions (time and temperature) are to be considered. In the case of nonculture-based test methods, composition of test components along with different test parameters and

Drug Delivery Aspects https://doi.org/10.1016/B978-0-12-821222-6.00008-7

controls are used to verify the method’s ability to detect the presence of viable contaminating microorganisms [2].

2 Challenges in sterilization Different challenges are faced with regard to sterility of different dosage forms like solution, suspension, etc. The challenges also differ according to the sterility method being used. A few of the challenges observed include maintaining sterility assurance in aseptic manufacturing through supply of presterilized consumables and raw materials, and transfer of presterilized product containers into filling lines using barrier technology. Another challenge involves compliance with regulatory requirements for sterile manufacturing, which are becoming more stringent. On December 20, 2017, the European Commission issued a long-awaited draft of Annex 1 Manufacture of Sterile Medicinal Products, which aimed at adding clarity, incorporating the principles of quality risk management (QRM) to enable

169

# 2020 Elsevier Inc. All rights reserved.

170

8. Sterilization of pharmaceutical dosage forms

inclusion of new technologies and processes while ensuring that microbial, particulate, and pyrogen contamination associated with microbes is prevented in the final medicinal product [3]. Techniques in the field of sterility include: (1) chemical methods—various chemicals used for sterilization include glutaraldehyde, chlorine dioxide, orthophthaldehyde, superoxidized water; (2) physical methods, such as pulsed light sterilization, ultra-high pressure sterilization, and autoclave; (3) physicochemical methods, such as gas plasma sterilization; and (4) synergistic methods such as Psoralen and UVA, ultrasound, and bactericide. This chapter provides details about different aspects with regard to sterile dosage forms. It gives information about different steps involved in compounding and manufacturing of sterile dosage forms along with different requirements for the same. It also provides details about the different methods of sterilization along with their applications for different dosage forms. It offers insights into different tools like QbD (quality by design) and PAT (process analytical technology), which are used in product development.

Other sterile products include: • • • •

topical ophthalmic medications; topical wound healing medications; solution for irrigation; and sterile devices (e.g., syringes, administration sets, implantable systems).

Injectable formulations mainly involve delivery of drugs by the parenteral route. Delivery of injectable formulations includes subcutaneous, intravenous, intramuscular, intracardiac, intraperitoneal, and intraarticular routes of administration. Examples of injectables include epinephrine autoinjectors given by IM route, EpiPen administered by SC route, and Depo Provera and Haloperidol Decanoate given as a depot injection. Typical steps involved in manufacturing of injectables are depicted in Figs. 8.1 and 8.2.

Warehouse (unclassified)

Dispensing

Formulation (US:ISO 8 or better; EU:ISO 7 or better)

2.1 Dosage forms and sterilization methods

Filtration, filling, and stoppering

In the following section, we discuss different types of sterile dosage forms along with various steps involved in compounding and manufacturing of the same. Sterile dosage forms are basically classified into three broad categories: • conventional small (1–30 mL); • conventional large (100–1000 mL); and • modified release (1–5 mL) [4].

Capping/ sterilization (ISO 5) Loading and unloading freezedryer (ISO 5)

volume

injectables

volume

injectables

Finishing (unclassified)

(depot)

injectables

FIG. 8.1 Steps involved in manufacturing of injectable dosage form.

171

2 Challenges in sterilization

Calculate the maximum possible contribution of each component and categorize risk of maximum Figure 2: Endotoxin levels flow chart potential contribution

High risk

Medium risk

Low risk

(≥100% of specification)

(5%–100% of specification)

(