A Practical Guide to Pharmacological Biotechnology [1st ed.] 978-981-13-6354-2;978-981-13-6355-9

Table of contents :
Front Matter ....Pages i-xiii
General Aspects of Pharmacology Laboratory (Jayanta Kumar Patra, Swagat Kumar Das, Gitishree Das, Hrudayanath Thatoi)....Pages 1-17
Isolated Tissues and Organs (Jayanta Kumar Patra, Swagat Kumar Das, Gitishree Das, Hrudayanath Thatoi)....Pages 19-28
Screening of Drugs Using Cell Lines/Isolated Tissues/Intact Animals (Jayanta Kumar Patra, Swagat Kumar Das, Gitishree Das, Hrudayanath Thatoi)....Pages 29-53
Genotoxicity and Toxicological Studies (Jayanta Kumar Patra, Swagat Kumar Das, Gitishree Das, Hrudayanath Thatoi)....Pages 55-75
Experimental Animal Studies (Jayanta Kumar Patra, Swagat Kumar Das, Gitishree Das, Hrudayanath Thatoi)....Pages 77-108
Clinical Trials (Jayanta Kumar Patra, Swagat Kumar Das, Gitishree Das, Hrudayanath Thatoi)....Pages 109-126
IPR and Ethics in Animal Studies (Jayanta Kumar Patra, Swagat Kumar Das, Gitishree Das, Hrudayanath Thatoi)....Pages 127-142

Citation preview

Learning Materials in Biosciences

Jayanta Kumar Patra Swagat Kumar Das Gitishree Das Hrudayanath Thatoi

A Practical Guide to Pharmacological Biotechnology

Learning Materials in Biosciences

Learning Materials in Biosciences textbooks compactly and concisely discuss a specific biological, biomedical, biochemical, bioengineering or cell biologic topic. The textbooks in this series are based on lectures for upper-level undergraduates, master’s and graduate students, presented and written by authoritative figures in the field at leading universities around the globe. The titles are organized to guide the reader to a deeper understanding of the concepts covered. Each textbook provides readers with fundamental insights into the subject and prepares them to independently pursue further thinking and research on the topic. Colored figures, step-by-step protocols and take-home messages offer an accessible approach to learning and understanding. In addition to being designed to benefit students, Learning Materials textbooks represent a valuable tool for lecturers and teachers, helping them to prepare their own respective coursework. More information about this series at http://www.springer.com/series/15430

Jayanta Kumar Patra • Swagat Kumar Das Gitishree Das • Hrudayanath Thatoi

A Practical Guide to Pharmacological Biotechnology

Jayanta Kumar Patra Research Institute of Biotechnology & Medical Converged Science Dongguk University Goyang-si, Gyeonggi-do South Korea

Swagat Kumar Das Department of Biotechnology College of Engineering and Technology, Biju Patnaik University of Technology Odisha India

Gitishree Das Dongguk University Goyang-si, Gyeonggi-do South Korea

Hrudayanath Thatoi Department of Biotechnology North Orissa University Odisha India

ISSN 2509-6125     ISSN 2509-6133 (electronic) Learning Materials in Biosciences ISBN 978-981-13-6354-2    ISBN 978-981-13-6355-9 (eBook) https://doi.org/10.1007/978-981-13-6355-9 Library of Congress Control Number: 2019933573 © Springer Nature Singapore Pte Ltd. 2019 This work is subject to copyright. All rights are reserved by the Publisher, whether the whole or part of the material is concerned, specifically the rights of translation, reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publisher, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publisher nor the authors or the editors give a warranty, express or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publisher remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Preface

Pharmacology is one of the important subjects in pharmacy and clinical science including biotechnology. Pharmacology deals with the study of drug action, where a drug can be broadly defined as any synthetic, natural, or endogenous molecule which exerts a biochemical or physiological effect on the cell, tissue, organ, or organism as a whole. More specifically, it is the study of interactions that occur between a living organism and chemicals that affect normal or abnormal biochemical function. The field encompasses drug composition and properties, synthesis and drug design, molecular and cellular mechanisms, organ/systems mechanisms, signal transduction/cellular communication, molecular diagnostics, interactions, toxicology, chemical biology, therapy, and medical applications and antipathogenic capabilities. Two important aspects of pharmacology study are pharmacodynamics and pharmacokinetics. The present book titled A Practical Guide to Pharmacological Biotechnology covers the whole range of experiments related to pharmacology. It also contains basic laboratory safety guidelines followed in a laboratory. Each chapter starts with an introduction or background into the basic approach followed by detailed methods sections with easy-to-­ follow workable protocols and comprehensive troubleshooting, calculation, and possible viva voce questions for examination. One of the important aspects of the book manual is the first part which deals with general guidelines of laboratory safety, rules and regulations, and different symbols used commonly in the laboratory. The second part of the book deals with different experiments on basic and advanced pharmacological studies. The book also features different experiments on in vitro tissues, isolated cells, and cell-free biochemical systems focusing on those that are relevant to pharmacology. This book is an indispensable tool for introducing advanced undergraduates and beginning graduate and master students of Pharmacy, Clinical Science, and Biotechnology field to the techniques of pharmacology. There are few books available on practical pharmacology aspects targeting students of the undergraduate and graduate levels. In this regard, this proposed book will be of great help to the students as the book will cover the major experiments on, as per the curricula, the pharmacology of Pharmacy and Biotechnology stream at both undergraduate and graduate levels. The book will be of great use to researchers and scientists in the relevant field of study. v

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Preface

We are honestly grateful to all the literature and search engines that we have referred to write the chapters of this book. We are also thankful to Dr. Sue Lee, Publishing Editor, Biomedical Sciences, and her team of the Springer Nature Singapore Pte. Ltd., Singapore, for their generous cooperation at every stage of the book publication. Gyeonggi-do, South Korea Odisha, India Gyeonggi-do, South Korea Odisha, India

Jayanta Kumar Patra Swagat Kumar Das Gitishree Das Hrudayanath Thatoi

Contents

1 General Aspects of Pharmacology Laboratory ����������������������������������������������    1 1.1 General Safety Features in a Pharmacology Laboratory����������������������������    1 1.1.1 Clothing and Footwear��������������������������������������������������������������������    1 1.1.2 Gloves ��������������������������������������������������������������������������������������������     2 1.1.3 Drinks, Eatables and Smoking��������������������������������������������������������     3 1.1.4 Personal Safety and Hygiene����������������������������������������������������������     3 1.1.5 Handling Chemicals������������������������������������������������������������������������     4 1.1.6 Laboratory Work Place ������������������������������������������������������������������     4 1.1.7 Instrument ��������������������������������������������������������������������������������������     4 1.1.8 House Keeping��������������������������������������������������������������������������������     5 1.1.9 Radiation ����������������������������������������������������������������������������������������     5 1.1.10 Fire��������������������������������������������������������������������������������������������������     6 1.1.11 Laboratory Safety Signs������������������������������������������������������������������     6 1.2 Uses and Management of the Laboratory Animals in a Laboratory ����������     6 1.3 Animal House Facility, Care����������������������������������������������������������������������    10 1.3.1 Environmental Aspects ������������������������������������������������������������������    10 1.3.2 Physical Facilities ��������������������������������������������������������������������������    11 1.3.3 Animal Care������������������������������������������������������������������������������������    12 1.4 Experimental Design����������������������������������������������������������������������������������    13 1.5 Toxicology��������������������������������������������������������������������������������������������������    15 References������������������������������������������������������������������������������������������������������������    17 2 Isolated Tissues and Organs������������������������������������������������������������������������������   19 2.1 Basic Instruments Used for Isolated Tissue Experiments��������������������������    19 2.2 Organ Baths������������������������������������������������������������������������������������������������    22 2.3 Intestinal Muscle Preparations��������������������������������������������������������������������    24 2.4 Skeletal Muscle Preparations����������������������������������������������������������������������    25 2.5 Cardiac Muscle Preparations����������������������������������������������������������������������    26 Referenecs������������������������������������������������������������������������������������������������������������    28

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3 Screening of Drugs Using Cell Lines/Isolated Tissues/Intact Animals����������   29 3.1 Evaluation of Antidiabetic Agents��������������������������������������������������������������    29 3.1.1 Antidiabetic Evaluation of Drug in Induced Diabetic Mice Model����������������������������������������������������������������������    29 3.1.2 Glucose Uptake Assay��������������������������������������������������������������������    32 3.2 Evaluation of Antiulcer Activity ����������������������������������������������������������������    35 3.3 Evaluation of Hepatoprotective Drugs��������������������������������������������������������    37 3.3.1 In vitro Assessment of Hepatoprotective Effect������������������������������    38 3.3.2 In vivo Evaluation of Hepatoprotective Effect��������������������������������    39 3.4 Evaluation of Anti-inflammatory Agents����������������������������������������������������    41 3.5 Evaluation of Antioxidant Activity ������������������������������������������������������������    43 3.5.1 Evaluation of Antioxidant Activity Using Erythrocyte-Based Method ������������������������������������������������������������    43 3.5.2 Evaluation of Antioxidant Activity Using Cell Based Assay Method����������������������������������������������������������������������    45 3.6 Effect of Drug in Rabbit Eye����������������������������������������������������������������������    47 3.7 Evaluation of Local Anaesthetics����������������������������������������������������������������    49 3.7.1 Evaluation of Local Anaesthetics����������������������������������������������������    49 3.7.2 Evaluation of Local Anaesthetics����������������������������������������������������    51 References������������������������������������������������������������������������������������������������������������    52 4 Genotoxicity and Toxicological Studies������������������������������������������������������������   55 4.1 In Vitro Genotoxicity Assay������������������������������������������������������������������������    55 4.2 Mouse Lymphoma Assay����������������������������������������������������������������������������    60 4.3 Comet Assay ����������������������������������������������������������������������������������������������    62 4.4 In Vitro Teratogenicity Testing��������������������������������������������������������������������    66 4.5 Histopathological Studies of Animal Tissues ��������������������������������������������    69 4.6 Drug Poisoning ������������������������������������������������������������������������������������������    73 References������������������������������������������������������������������������������������������������������������    75 5 Experimental Animal Studies ��������������������������������������������������������������������������   77 5.1 Collecting Blood from Mice ����������������������������������������������������������������������    77 5.2 Studies on Different Parameters of Blood��������������������������������������������������    81 5.2.1 Differential White Blood Cell Count (Differential Leukocyte Count)������������������������������������������������������    82 5.2.2 Blood Grouping������������������������������������������������������������������������������    85 5.2.3 Total Red Blood Cell Count������������������������������������������������������������    88 5.2.4 Estimation of Haemoglobin (Hb)���������������������������������������������������    89 5.2.5 Bleeding Time and Clotting Time��������������������������������������������������    91 5.3 Pyrogen Testing������������������������������������������������������������������������������������������    94 5.3.1 Pyrogen Testing by Animal Model (In Vivo Method) ��������������������    94 5.3.2 Pyrogen Testing by LAL Method (In Vitro Method)����������������������    96

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5.4 Effect of Drug on Central Nervous System������������������������������������������������    97 5.4.1 Hypnotic Drugs������������������������������������������������������������������������������    97 5.4.2 CNS Depressant Activity����������������������������������������������������������������    99 5.4.3 Analgesic Drugs������������������������������������������������������������������������������   100 5.5 Experiment on Cardiovascular System ������������������������������������������������������   103 5.5.1 Effect of Cardiac Stimulants and Depressants��������������������������������   103 5.6 Experiments on GI Tract����������������������������������������������������������������������������   106 5.6.1 Muscle Relaxants and Spasmogens������������������������������������������������   106 References������������������������������������������������������������������������������������������������������������   108 6 Clinical Trials ����������������������������������������������������������������������������������������������������  109 6.1 Clinical Pharmacology and Its Genesis������������������������������������������������������   109 6.2 National and International Agencies and Their Role in Clinical Pharmacology ��������������������������������������������������������������������������������������������   113 6.3 Drug Development and Clinical Trials ������������������������������������������������������   113 6.4 Safety Assessment in Clinical Trials����������������������������������������������������������   118 6.5 Ethics in Clinical Trials������������������������������������������������������������������������������   119 6.6 Ethics in Clinical Research ������������������������������������������������������������������������   121 References������������������������������������������������������������������������������������������������������������   125 7 IPR and Ethics in Animal Studies��������������������������������������������������������������������  127 7.1 Introduction������������������������������������������������������������������������������������������������   127 7.2 Intellectual Property Rights and Its Different Categories��������������������������   127 7.2.1 Patents Which Protect Inventions ��������������������������������������������������   128 7.2.2 Copyrights Which Protect Works of Literature and Art������������������   128 7.2.3 Trade Marks������������������������������������������������������������������������������������   130 7.2.4 Trade Secrets����������������������������������������������������������������������������������   130 7.3 Importance of IPR in Drug Development ��������������������������������������������������   130 7.3.1 A New Drug as a New IP����������������������������������������������������������������   131 7.3.2 Safeguard of IP in Finding a New Medicine of Natural Origin����������������������������������������������������������������������������   131 7.3.3 Various Phases in the Natural Product Based Discovery of Drugs Where ‘Inventive’ Contributions Might Occur����������������   132 7.3.4 Most Important Steps in the Natural Based Drug Development Procedure������������������������������������������������������������������   132 7.3.5 Importance of IPR in Drug Development ��������������������������������������   133 7.4 Patenting Cells, Cell Lines and Animals����������������������������������������������������   133 7.5 Ethics in Laboratory Animal Studies����������������������������������������������������������   135 7.5.1 Ethical Considerations��������������������������������������������������������������������   136 7.5.2 Welfare Considerations: The Animal and the Environment������������   136 7.5.3 Ethics in Animal Experimentation��������������������������������������������������   136

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7.6 Risk Assessment and Management in IPR��������������������������������������������������   137 7.6.1 IP Risks ������������������������������������������������������������������������������������������   137 7.6.2 IP Risk Management����������������������������������������������������������������������   137 7.7 Good Laboratory Practices in IPR��������������������������������������������������������������   138 7.8 Principles of GLP����������������������������������������������������������������������������������������   139 References������������������������������������������������������������������������������������������������������������   141

About the Authors

Jayanta  Kumar  Patra  M.Sc. Ph.D., PDF, is currently working as Assistant Professor at Dongguk University, Republic of Korea. His current research is focused on nanoparticle synthesis by green technology methods and their potential application in agricultural, biomedical, and pharmacological fields. He has about 12  years of research and teaching experience in the field of food, pharmacology, and nano-­biotechnology. Dr. Patra completed his Ph.D. (Life Sciences) from North Orissa University, India, in pharmacological application of mangrove plant bioactive compounds and PDF (Biotechnology) from Yeungnam University, South Korea. Since 2007, he has more than 100 papers published in various national and international peer-reviewed journals from different journals of Elsevier, Springer, Taylor & Francis, Wiley, etc. and around 25 book chapters in different edited books. Dr. Patra has also authored eight books for Studium Press (India); Studium Press LLC, USA; Apple Academic Press, Inc., Canada; CRC Press, a Taylor & Francis group; and Springer Nature publisher. Besides, he is Editorial Board Member of several national and international journals.

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About the Authors

Swagat Kumar Das  B.Pharm., M.Tech., Ph.D., is presently working as a Lecturer in the Department of Biotechnology at the College of Engineering and Technology, an autonomous and constituent college of Biju Patnaik University of Technology (BPUT), Rourkela, Odisha, India. He obtained his B.Pharm degree from BPUT, Rourkela, and M.Tech. degree from Rajiv Gandhi Proudyogiki Vishwavidyalaya, Bhopal, and Ph.D. from Ravenshaw University, Cuttack, India. He is also a Fellow, Eurasian Academy of Environmental Sciences (FEAES). He teaches medical and pharmaceutical biotechnology, upstream and downstream process engineering, bioprocess engineering, and intellectual property rights at B.Tech. and M.Tech. levels. He has more than 8 years of teaching and research experience and published more than 20 research and review papers in national and international journals of repute along with some book chapters. He has coauthored one practical book, Practical Biotechnology: Principles and Protocols, published by IK International Publishing House, New Delhi. His research activities involved phytochemical analysis and drug development from mangrove plants for diabetes. His research area also focused on green synthesis of nanoparticles and evaluation of their pharmacological potentials. Gitishree  Das  M.Sc. Ph.D., PDF, is currently working as Assistant Professor at Research Institute of Biotechnology & Medical Converged Science, Dongguk University, Ilsan-dong, Gyeonggi-do, Republic of Korea. She has 10 years of research experience in the field of rice molecular biology, breeding, and endophytic bacteria and 1 year of teaching experience. Dr. Das has completed her Ph.D. (Life Sciences) from North Orissa University, India, on rice gene pyramiding (Central Rice Research Institute, Cuttack, India) and PDF (Biotechnology) from Yeungnam University, South Korea. She has undergone many professional trainings in the field of biotechnology from various international and national reputed organizations. Her current research is focused on the application of endophytes and their bioactive compounds/nanoparticles in biomedical and agricultural fields. To her credit, she has published around 50 research articles in international and national reputed ­journals and 14 book chapters. Dr. Das has also authored four books for Springer Nature publisher, Apple Academic Press and Lambert Academic Publishing. She is Editorial Board Member of Ambika Prasad Research Foundation.

About the Authors

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Hrudayanath  Thatoi  M.Sc., M.Phil., Ph.D., is currently working as Professor and Head, Department of Biotechnology, North Orissa University, Odisha, India. He has around 30 years of teaching and research experience. His research activities are basically based on medicinal plants, bioremediation, biodiversity, ethnopharmacology, mangrove biology, etc. Professor Thatoi obtained his M.Phil. and Ph.D. from Utkal University, Odisha, India, and his research work was based on N2 fixation in legume plants under dual inoculation of Rhizobium and vesicular-arbuscular mycorrhiza (VAM) fungi and contributed significantly toward development of technology for mine waste reclamation. Professor Thatoi has handled many research projects from state government and central government organizations like the Department of Science and Technology (DST), Government of Odisha; UGC-DAE, Government of India; Department of Forest, Government of Odisha; etc. Around 20 students have obtained Ph.D. under his guidance and supervision. Besides, several M.Sc., M.Tech., and B.Tech. students have received his guidance for their dissertation works. Professor Thatoi has published more than 200 research papers in various national and international reputed journals and around 30 book chapters. Professor Thatoi has also authored around ten books/manuals by different notable publishers, like Studium Press LLC, USA, IK International Publishing House, Narosa Publishing House, Biotech Books, Ashish Publishing House, etc. He has also authored a textbook on Microbiology and Immunology, published by India Tech Publication, New Delhi, for M.Sc. and B.Sc. students. Professor Thatoi has contributed immensely in the field of microbiology and biotechnology throughout his research and teaching career.

1

General Aspects of Pharmacology Laboratory

1.1

General Safety Features in a Pharmacology Laboratory

Safety of laboratory and the valuation of risk is one of the most vital parts of any laboratory including pharmacology laboratory. The primary objective of laboratory safety standard is the safety of its workers against any type of possible hazardous material or any infection. Further, it is essential to train the students/researchers about the possible hazardous materials/chemicals and equipment that are associated with their work. The laboratory safety rules form a significant feature of code of conduct in the laboratory for students/ researchers that need to be followed strictly as safety rules while working in laboratories and conducting experiments. The different safety rules that are basically followed in a laboratory are discussed as follows:

1.1.1 Clothing and Footwear Laboratory aprons or lab coats normally made of natural cotton fibre and white in color are must to be worn inside a laboratory while conducting experiments in order to provide protection from any unwanted accidents and chemical spills. However there are also different color labcoats and aprons specified for different purposes in the laboratories. Loose fitting clothes, easily combustible clothes, and long, unrestrained hair are not appropriate in the laboratory and should not be worn. Shoes must be worn during the entire time in the laboratory to protect from splashes of chemicals, broken glass and other hazards that may occur in the lab. The shoes must cover the feet without gaps such as open toes.

© Springer Nature Singapore Pte Ltd. 2019 J. K. Patra et al., A Practical Guide to Pharmacological Biotechnology, Learning Materials in Biosciences, https://doi.org/10.1007/978-981-13-6355-9_1

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Laboratory aprons or lab coats and lab shoes

1.1.2 Gloves Gloves are required to protect the hands from different types of chemical spills and potential hazards while conducting experiments in the laboratory. There are different types and specifications of gloves available in the market for specific purposes and works in the laboratory.

Laboratory gloves

1.1 General Safety Features in a Pharmacology Laboratory

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1.1.3 Drinks, Eatables and Smoking It is a thumb rule, that any type of drinking and eating is strictly prohibited inside the laboratory, in order to avoid any type of contamination and infections. Besides, food and drink are also not divided to keep, or prepare inside the laboratory. However, if required, separate refrigerators are to be designated and must be used to store food and also it should be kept away from the chemical storing area and should be handled separately from other biological or poisonous materials and chemicals. Besides, smoking is strictly banned inside the laboratory area.

1.1.4 Personal Safety and Hygiene For protection of the eye, goggles are essential to be worn inside the laboratory. Basically there are different types of goggles available in the market with different properties to be worn for different purposes inside the laboratory. Long hair are avoided in a lab for avoiding any damage caused due to fire or chemicals. Mobile phones, or any shot of electronic gadgets should be switched off or kept in safe place while conducting experiments in a laboratory. Hands should be washed properly with detergents before and after finishing the experiments. Any bruise, burn, scratch, and injury that happened in the laboratory must be reported immediately to the in charge authority.

Safety goggles

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1.1.5 Handling Chemicals A number of chemicals used in the laboratory are hazardous in nature and hence they require utmost attention and care while handling. Chemicals should not be tasted or smelled by nose in the laboratory. Chemicals must not be pipetted by mouth; rather pipette filler should be used. The inflammable chemicals should be kept out of range from open flames inside a special designed container and special care should be taken while opening the pressurized vapours. Chemicals should be taken out of the laboratory by taking permission from the in charge authority.

Safety and storage cabinets for flammable chemicals. (https://www.justrite.com/safety-and-storagecabinets.html)

1.1.6 Laboratory Work Place Laboratory working area must be kept cleaned and all the required chemicals and glass wares should be kept in its appropriate place in an orderly manner. Unnecessary thing should be evaded in the working area. The sink should be cleaned properly every day. The gas outlets and hoods should be used for designated chemicals only.

1.1.7 Instrument Electrical equipment’s should be checked at regular interval for their malfunctioning and also each time before and after its use. Laboratory set up for any instrument should not be changed without informing the laboratory in charge. Instrument should be turned off before leaving the laboratory. Instruments shouldn’t be handled by students without taking permission from instructor. In case of sparks or fire in the electrical equipment, it should

1.1 General Safety Features in a Pharmacology Laboratory

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be turn off the immediately and informed to the person in charge. A fire extinguisher should be kept inside the laboratory and must be used as needed.

Fire extinguisher

1.1.8 House Keeping The broken glasswares should be discarded in the designated container and must not be dumped elsewhere. Chemicals should never be poured down the drain designated for water, rather it should be kept in the specific container and discarded appropriately. Infectious material should be disposed of in the infectious materials bins for biological waste and chemical waste. The pathogenic materials should be either autoclaved or chemically treated before discarding them out. Glassware must always be handled carefully. Glass tubing can easily be broken and can cause severe damage to hands. Contaminated or broken syringes and syringe needles should be placed in designated containers and discarded appropriately.

1.1.9 Radiation One of the major concerns while working with radioisotopes in a laboratory, is the safety of the working personnel as there is chance of exposure to the radio hazards. Extraordinary precautions must be taken in the laboratory while conducting experiments on radioisotopes. Safe disposal of radioisotopes is one of the primary matter of concern for the laboratory working on radio nucleotides. Laboratory certification, protective equipment, clear guidelines for use and disposal, appropriate training, and well-established international and national regulatory authorities help to circumvent such problems with radioisotopes. Radioactive hazards in the laboratory should be dealt separately.

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Fig. 1.1  Warning symbols used in laboratory

1.1.10 Fire The locations of fire blanket, safety shower and fire extinguisher must be known to everyone working in the laboratory. The emergency exits from the laboratory must be clearly labeled and known to all. Fire extinguisher should be kept in each laboratory and its location should be clearly mentioned.

1.1.11 Laboratory Safety Signs Laboratory safety signs and symbols should be kept in the laboratory for informing the working personnel (Fig. 1.1).

1.2

 ses and Management of the Laboratory Animals U in a Laboratory

Different kinds of animals are used in a pharmacology laboratory for experimentation purpose. The commonly used animals for pharmacology experiments are discussed as below:

1.2 Uses and Management of the Laboratory Animals in a Laboratory

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(a) Rana tigrina Name of the animal: Common English name: Strain used: Pharmacological uses:

Rana tigrina Frog Rana esculenta, Rana pipiens and Rana temporaria Studying isolated tissue like rectus, abdominus muscle, heart, sciatic nerve preparation. Studying effect of drug acting on central nervous system, neuromuscular junction and heart. Screening of certain drugs like anesthetics.

Rana tigrina. (Source: http://natureconservation.in/rana-tigrina-hoplobatrachus-tigerinus-completedetail/)

(b) Mus musculus (common house rat) Name of the animal: Common English name: Strain used: Pharmacological uses:

Mus musculus House mouse Balb-c and Swiss albino. Widely used in different toxicity studies. Study related to genetic and cancer research. Screening of analgesic and anticonvulsants. Toxicological and teratogenic study.

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Common house mouse. (Source: https://en.wikipedia.org/wiki/File:House_mouse.jpg)

(c) Rattus rattus (brown rat) Name of the animal: Common English name: Strain used: Pharmacological uses:

Rattus rattus Rat Albino rats of wistar strain, Sprague-Dawley, Wistar Kyoto, Lewis. Bioassay of different hormones such as insulin, oxytocin, vasopressin etc. Psychopharmacological Studies. Acute and chronic toxicity studies. Studies on isolated tissue preparations like uterus, stomach, vasdeferens, anoccoccygeus muscle, fundus strip, aortic strip, heart rate etc.

Brown rat. (Source: https://www.medianauka.pl/mysz-zaroslowa)

(d) Cavia procellus (Guinea pig) Name of the animal: Common English name: Strain used: Pharmacological uses:

Cavia procellus Guinea pig, cavy or domestic cavy Dunkin-Hartley, Peruvian, Anaphylactic and immunological studies. Evaluation of local anaesthetics. Study of tuberculosis and ascorbic acid metabolism. Study of histamine and anti-histamines.

1.2 Uses and Management of the Laboratory Animals in a Laboratory

Guinea pig

(e) Oryctolagus cuniculus (rabbit) Name of the animal: Common English name: Strain used: Pharmacological uses:

Rabbit

Oryctolagus cuniculus Rabbit New Zealand white, American Dutch, Himalayan Black Pyrogen testing. Irritancy tests. Pharmacokinetics studies. Bioassay for different drugs e.g. antidiabetic, hormonal etc.

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(f) Mesocricetus auratus (hamster) Name of the animal: Common English name: Strain used: Pharmacological uses:

Mesocricetus auratus Hamster Mesocriceius auratus, Cricetulus griseus Useful for cytological investigations, genetics, tissue culture and radiation. Study related to virology, immunology and implantation studies.

Hamster. (Source: https://www.proprofs.com/quiz-school/story.php?title=which-hamster-is-rightone-me)

1.3

Animal House Facility, Care

All laboratory animals irrespective of the type of species need to be handled carefully and used for the experiment in an appropriate manner in order to prevent any unwanted injury to them. The requirement of each animal, their environmental conditions and their ­treatment procedures vary with different animal species, protocol and type of experiment. However, their health condition and normal hygiene need to be maintained properly irrespective of their purpose and experimental conditions. Few important aspects in handling the laboratory animal are discussed as below:

1.3.1 Environmental Aspects 1.3.1.1 Temperature Sudden temperature change in laboratory may harm laboratory animals. Therefore, laboratory buildings should be furnished with facilities to maintain appropriate environmental temperatures that suit best for the laboratory animals and the workers.

1.3 Animal House Facility, Care

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1.3.1.2 Humidity Maintenance of the humidity in an animal house facility is of utmost important. Most of the animal can tolerate humidity ranged between 30% and 70%. However, this may change with respect to different types of the animals and their strains. Extreme variance in humidity may result into ill health of animals; hence, dehumidifiers are used in the animal house in order to maintain the humidity balance. 1.3.1.3 Ventilation Normally, an animal house need to be well ventilated. A total air exchange system is preferred way of ventilation. However, if recirculation of air is required, then it need to be fitted with a proper filtration unit for circulation of safe air followed by checking of the quality of air at regular interval. For providing stable environment inside animal house, heating, ventilation, and air-conditioning systems should be fitted with 12–15 air cycles per hour. 1.3.1.4 Light Animal house are properly lighted up with 807–1345 lux of light intensity and light controller machine for better visibility and a regular diurnal lighting cycle. The animal house facility need to be furnished with emergency power supply to avoid chaos in case of power failure. 1.3.1.5 Noise The animal house facility need to be located in a quiet and silent environment and noise free area. Few animals may expect epileptic seizures in case of loud noise. It is recommended to have less than 85 dB noise for rodents. Concrete walls are preferred to avoid noise pollution inside an animal house.

1.3.2 Physical Facilities 1.3.2.1 Building Materials The animal house facility are generally made up of durable, moisture proof, fire resistant, seamless materials including vermin and pest resistance materials. The corridor should be spacious enough to allow free passage of working personnel. The other utilities like water lines, drain pipes, electrical connections etc. must be in corridors outside the animal house. 1.3.2.2 Door, Windows, Floor Animal house must be provided with a rust, vermin and dust proof doors with an observation window for easy monitor of the laboratory animals without any type of infection. The animal house floors should be provided with smooth, moisture proof, non-absorbent, skid proof, resistant to wear, acid, solvents floors.

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1.3.2.3 Drains, Walls, Ceilings Drains must be provided wherever necessary to prevent high humidity, rapid removal of water and for drying of floor surface. However, inlet and outlets of the drains should be fitted with wire mesh rodent guards. The walls and ceilings of the animal house must be free of cracks, unsealed utility penetrations or imperfect junctions. 1.3.2.4 Storage Area Separate storage area are specifically provided inside an animal facility for storage of animal feed, unused cages, bedding and other essential day today requirements of the animal house. Refrigerated storage facility must are provided to store dead animals and animal tissue wastes, if required. 1.3.2.5 Sanitizing Facility Animal house facility are equipped with selected sanitizing facility with adequate water supply for sanitizing animal cages and other auxiliary equipment. 1.3.2.6 Experimental Area Separate area need to be provided in an animal house facility for carrying out different animal related experiments inside it in order to avoid external contaminations. Normally the experimental area are outside of the animal dwelling area but inside the animal facility house.

1.3.3 Animal Care 1.3.3.1 Animal Housing System The animal housing system or caging in an animal house is a significant part of the animal care unit that enable animal care, meet the research requirements, and minimize the ­experimental variables. The housing facility need to be provided with relaxed environment, accessibility to food, ventilation, air for keeping the good health of animal etc. for taking better care of the laboratory animal. 1.3.3.2 Activity, Food, Bedding, Water Animal house need to be provided with specialized locomotors pattern for animals with to express their patterns when kept for long periods. Animals should feed with palatable, non-contaminated and nutritionally rich food on daily basis until any other experimental requirements. The animals should have easy access to food, while avoiding contamination from their urine and faeces. The animal feed should have adequate amount of moisture, crude fibre, crude protein, essential vitamins, minerals crude fat and carbohydrate for providing appropriate nutrition. The animal cage bedding should have absorbent and free of toxic chemicals or any other materials that could injure caged animals. Facility should be there to keep animals dry between cage changes without coming into contact with water-

1.4 Experimental Design

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ing tubes. Bedding material should be replaced with fresh materials regularly to keep the animals clean and dry. Animals must have continuous access to fresh, potable, uncontaminated drinking water as per their particular requirements.

1.3.3.3 Hygiene and Cleanliness Hygiene of the laboratory animal is an important factor that need to be taken care of inside an animal house. The animal house need to be disinfected and sanitized periodically with specified disinfectants and detergents in order to avoid any type of contamination and disease. 1.3.3.4 Waste Disposal Dead animals, animal tissue excreta, bedding, unused diet and all other animal related laboratory waste etc. need to be properly collected in a leak proof metal or plastic containers and destroyed appropriately as per the laboratory rule. 1.3.3.5 Record Keeping The animal house should maintain the records of the laboratory animals such as their, species name, type, age, and body weight etc. for proper maintenance. These aspects are described as below (a) Animal house plans including the typical floor plan, fixtures etc. (b) Both technical and non-technical staff record of Animal house (c) Health record of staff/animals of animal house (d) Clinical record of sick animals (e) Death record of animals (f) SOPs (standard operating procedures) relevant to the animals (g) Breeding, stock, purchase and sales records of animals (h) Training record of staff involved in animal studies activities (i) Minutes of institute Animals Ethics Committee Meetings (j) Water analysis report

1.4

Experimental Design

Experimental study designs are the primary method for testing the efficiency of new therapies and other interventions. Experimental design can lead to reduction in the number of laboratory animals used for the intended study. Experimental design is an essential components in any experimental work (Fig. 1.2). During any experimental work the different aspects of experimental need to be addressed to save substantial time and resources. The experimental design normally depends on a number of factors, including available techniques and materials. A realistic estimate should be carried on both cost and the time involved. The actual design of an

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Fig. 1.2  Sequence of Experimental designing

Fig. 1.3  Steps of experimental design

experiment should include different factors such as hypothesis, controls, statistical analysis etc. Therefore, it is a standard practice to design experiments using standard laboratory practices generating valid reproducible data of sufficient quality. The different steps of experimental design can be discussed as above (Fig. 1.3).

1.5 Toxicology

1.5

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Toxicology

Toxicology is an extensive field that involves different field of areas including chemistry, pharmacology, physiology, biochemistry, anatomy, and numerous others. Normally it can be classified into four broad areas such as analytical toxicology, clinical toxicology, environmental toxicology and industrial toxicology (Fig. 1.4). Analytical toxicology deals in detecting, identifying and measuring a wide variety of compounds for their toxicity. Clinical toxicology study includes detection, identification and measurement of any drugs or other xenobiotics in biological or related specimen that helps in diagnosis, treatment, prognosis etc. Several factors are accountable for the toxicity of a substance such as dose, species, dosing frequency, route administration etc. (Fig. 1.5).

Fig. 1.4  Different branches of toxicology

Fig. 1.5  Factors affecting toxicology property of a substance

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Fig. 1.6  Different stages of toxicology study

The principal objective of toxicology is to assess the safety of potential drug candidates which is accomplished by using relevant animal models and validated procedures. However, the safety evaluation has to be accorded as per the international guidelines. A typical toxicology profile consists of safety pharmacology, genetic toxicology, acute, chronic toxicology, ADME studies, reproductive and developmental toxicology. Toxins can be evaluated both qualitatively and quantitatively. The qualitative analysis provides information of nature of toxins, but quantitative analysis gives information on the chemistry of the toxins and their concentration. Several instruments such as UV-visible spectrophotometer, infrared spectroscopy, gas chromatography, high pressure liquid chromatography, and immunoassay techniques are used to quantify the toxins. The toxicological analysis involves three steps, such as pre-analytical stage, analytical stage and post-analytical stage (Fig. 1.6). Related Questions 1. What are most commonly used animals in pharmacological research? 2. What are the most commonly used strains of rat used in pharmacological research? 3. What are the most commonly used strains of mice used in pharmacological research? 4. What are the most commonly used strains of hamster used in pharmacological research? 5. What are Wistar and Spargue Dawley rats? 6. Why Ascorbic acid or Vitamin C is externally supplemented to Guinea pig? 7. Why rodents are not preferred for antiemetic study? 8. Why experimental designing is important? 9. What does CPCSEA stands for? 10. What is quarantine? 11. What are the optimum environmental parameters in animal laboratory? 12. Why experimental design is important? 13. What is toxicogenomics?

References

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4. What are vehicle and sham controls? 1 15. What are random and systemic errors? 16. What is acute toxicity study?

References Badyal D (2008) Practical manual of pharmacology. Jaypee Brothers Medical Publishers, New Delhi BM Swamy V, Jayaveera KN, Reddy V (2014) Experimental pharmacology and toxicology. S. Chand & Company, New Delhi Control of the Animal House environment 1976. Laboratory Animals Handbooks CPCSEA Guidelines CPCSEA guidelines for laboratory animal facility (2003) Indian J Pharmacol 35:257–274 Goyal RK (2017) Practical in pharmacology. B. S. Shah Prakashan, Ahmedabad http://www.alexu.edu.eg/index.php/en/research/4401-regulatory-framework-in-ethics-of-animalresearch. Accessed on 19 Nov 2018 https://slideplayer.com/slide/6184688/. Accessed on 19 Nov 2018 https://vdocuments.site/code-sanitary-56290a602456c.html. Accessed of 22 November 2018 https://www.justrite.com/safety-and-storage-cabinets.html. Accessed on 19 November 2018 Johnson PD, Besselsen DG (2002) Practical aspects of experimental design in animal research. IIAR J 43:202–206 Kale SR, Kale RR (2017) Practical pharmacology and toxicology. Nirali Prakashan, Mumbai Medhi B (2017) Practical manual of experimental and clinical pharmacology. Jaypee Brothers Medical Publishers, New Delhi Ritter JM, Lewis LD, Mant TGK, Ferro A (2008) A text book of clinical pharmacology and therapeutics. Hodder Arnold, London Salmon DM (2014) Practical pharmacology for the pharmaceutical sciences. Wiley, Chichester Sierra LM, Gaivao I (eds) (2014) Genotoxicity and DNA repair a practical approach. Springer Protocols. Humana Press, London Singhal KC (1997) Pharmacology laboratory manual. CBS Publishers & Distributor, New Delhi Thatoi HN, Dash S, Das SK (2017) Practical biotechnology, principle and protocols. I.K. International, New Delhi Tripathi KD (2013) Essentials of medical pharmacology. Jaypee Brothers Medical Publishers, New Delhi Turner RA, Hebborn P (eds) (1971) Screening methods in pharmacology. Academic, New York Vogel HG (ed) (2002) Drug discovery and evaluation pharmacological assays. Springer, Berlin Woolley A (2008) A guide to practical toxicology. Informa Healthcare USA, New York

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Isolated Tissues and Organs

2.1

Basic Instruments Used for Isolated Tissue Experiments

Background Over the years, the use of isolated tissue and/or organ preparations has provided biologists a convenient biological model to work without the systemic influences of the intact animal. The isolated organ/tissue preparations are generally carried out in groups of 2, 4, 8 or more and changes can easily be studied as compared to intact animals. Traditionally, the in vitro dose responses of various drugs are carried out in tissue organ baths by using tissue preparations from various species such as frog, mice, guinea pig, rabbit etc. Different tissue preparations including smooth or skeletal muscles, arterial rings or strips, uterine tissue or vas deferens, ileum, colon, atrial or ventricle, diaphragm etc. are used for these studies. Under controlled environment and perfused with an oxygenated physiological solution condition these experiments are performed. Objective To study the equipments used for isolated tissue experiments. Basic Instruments The in vitro isolated preparation represents an isolated organ or a piece of living tissue which has been taken from freshly sacrificed animal. The basic instruments and other requirements used should provide optimum environment to maintain the isolated tissue or organ in living state. Different instruments are used for isolated tissues/organs in experimental pharmacology that include Organ Bath, Thermostat, Aerator cum tissue holder, Recording lever, Sherrington Recording Drum and Drum cylinder etc.

© Springer Nature Singapore Pte Ltd. 2019 J. K. Patra et al., A Practical Guide to Pharmacological Biotechnology, Learning Materials in Biosciences, https://doi.org/10.1007/978-981-13-6355-9_2

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Organ Bath Organ bath is one of the most commonly used pharmacology laboratory experimental set up for investigation of physiological and pharmacological aspects of in vitro tissue preparations. The isolated organ bath assay serves as screening tool for assessment of concentration to response curve of different drugs in contractile tissues. This also serves as a useful experimental tool for elucidating the mode of action of any drug preparation along with optimizing the lead compound. The exterior of the organ bath is double walled through which water can be circulated which helps in regulating the temperature of the interior physiological buffer. The organ bath is also facilitated with inlets to allow the entry and outlets for exit of water to the warming jacket. Further, arrangements are there to allow easy replacement of the physiological buffer by draining and filling. The physiological buffer is stored in a large beaker above the organ bath that may work as reservoir in which a tube is placed. A siphon is established so that when the tap is opened, it allows passage of the buffer to the organ bath. Ideally, the buffer is first passed through a warming coil which is a simple condenser with an outer water jacket and the required temperature is maintained by circulating the water. The supply of air or oxygen is done through a polyethylene capillary tube tied to a tissue holder in which the tissue is suspended and the other end is connected to the lever of the recording device (Fig. 2.1).

Fig. 2.1  Schematic presentation of organ bath

2.1 Basic Instruments Used for Isolated Tissue Experiments

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Reservoir: An ideal reservoir has an arrangement to deliver the physiological solution at a fixed rate and with constant pressure. Glass separators can also be used as reservoirs. Margate bottle: It consists of an aspirator bottle fitted with tight stopper perforated by a glass tube reaching nearly the bottom of the bottle. Levers: Levers are meant for recording and magnifying the responses of isolated tissues to drugs. The levers are attached to the isolated tissues and are used to record various types of contractions in them. Different types of livers are used as discussed below: Frontal Writing levers: This is used for recording of isotonic contraction of the isolated tissues. In this lever the writing end (stylus) can freely rotate around its axis. This minimizes the friction between the stylus and the kymograph. With frontal writing lever, the contraction of the isolated tissues are recorded as straight lines. Simple/Side way writing lever: This is used for recording of isotonic contractions of the isolated tissues. The responses recorded by simple lever are curvilinear. Starling’s heart lever and Broodie’s Universal lever: This is used for recording of isometric contractions of the isolated tissues. This type of lever is used for recording of rapid and multiple contractions in the isolated tissues. Gimble lever: The friction between the writing end and the kymograph is minimum in the Gimble lever because the pressure of stylus on the kymograph depends on gravity. Paton’s Auxotonic lever: it is designed in such a way that the load on the tissue goes on increasing as tissue contracts. Cannula: Cannula is generally made of glass or steel. They are used to infuse the physiological salt solution or drug solution in to an isolated organ (tissues) or for administration of physiological salt solution or drug solution to the experimental animal. Sherrington Recording Drum and Drum Cylinder Sherrington recording drum is used to record the contraction and relaxation of muscle against any particular drug. The instrument consists of different components including heavy base and a vertical shaft. The other components are as follows: • Base hoofs (legs) with adjustable levelling screws to keep drum horizontal if surface of the table is uneven. • Side hoof to turn the drum on its side so that shaft becomes horizontal. • Gear rod arrangement with fast, slow and neutral gears and clutch (starter). The gear rod is attached to a cone wheel which has four pulley grooves. Desirable speed of drum can be obtained by changing gear position and shaft drum pulley connections. • Contact screw on the surface. A wire can be fixed from main plug to convey the current through base. • Contact foil with a contact screw mounted on an insulated material on the superior surface of the base. Second wire can be connected here. Drum cylinder is a brass or iron cylinder around which a paper is wrapped and smoked. Drum with smoked paper is fitted on vertical shaft. At the base of vertical shaft, there are two projecting strikers

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which can be drawn apart to set any desired angle between them. When the striker makes the contact with foil, the make the circuit occurs. These days electrical drum is more commonly used. This is similar to Sherrington recording drum but speed is controlled electrically with the help of gear. Rotating Drum Smoke Drum: The responses are recorded on smoked drum. The smoked drum is prepared as follows: The glazed paper is laid on the table, keeping glazed surface downward. One end of the paper is gummed. The drum cylinder is placed in the middle of the paper. The proximal ungummed end is rolled around the drum and held tightly between the thumbs. The other end is also rolled on other side and the gummed and is pasted on the proximal ungummed end. The cylinder with paper is passed over a road fixed in smoking rack. A shooty flame is obtained by passing the gas through benzene or using a mixture of benzene and kerosene in the ratio of 1:9. The burner is brought nearer to the drum which is rolled uniformly at the maximum possible speed. The outer orange zone of flame should touch the paper. Fixing the graph: The paper is cut after obtaining the recording and then it is dipped in a solution resion (colophony) in methylated spirit. This solution is prepared by dissolving 150 gm of resion in 2 l of spirit. After passing the paper through the solution, it is drained and then allowed to try. Recording of responses on drum cylinder without smoking: The responses with the help of frontal writing levers can be recorded on drum cylinder using unsmoked paper. Simple sketch-pen tip can be tied with the help of cotton thread with very small amount of wool and a drop of ink (or eosin) can be placed before start of recording. This avoided the trouble of smoking as well as varnishing of the graph.

2.2

Organ Baths

Objective To study the equipment of organ bath used for isolated tissue experiments. Principle The organ bath, first used by Rudolf Magnus in 1904 is a traditional experimental tool for screening and assessing the concentration response relationships in contractile tissue. The organ bath assay helps in cardiovascular research by using isolated aortic rings, papillary muscle, left ventricles of heart tissue and arteries. This assay is also useful in studying gastro intestinal effects of ileum and colon preparations along with studying gastric antral muscle and sphincter. The organ bath assays are also used in pre-clinical safety studies. The organ bath should have an optimal capacity so that there will not be unnecessary wastage of chemicals and other solvents. Although the volume of the organ bath is not

2.2 Organ Baths

23

important, however a standard volume of 20  ml is often convenient. However, small volume organ baths (1–5 ml) are also useful to reduce the amount of drug administered. But the volume of the organ bath is important if the tissue is electrically stimulated. Student’s Organ Bath A commonly used organ bath used in the laboratory is known as Student’s Organ Bath. A typical Student’s Organ Bath (Fig. 2.2) has following components: Outer jacket: It is generally made of Perspex or glass and holds tap water warmed thermostatically (at 37  °C) and helps to maintain the environment of isolated tissue at physiological temperature (Fig. 2.3). Organ tube: The isolated tissue is suspended in the organ tube and connected to the reservoir containing physiological salt solution. It has varying size depending upon the tissue to be mounted. Glass coil: It is also called as preheating coil and is about double the capacity of organ tube. It holds the physiological salt solution at 37 °C to avoid fluctuations in the temperature of physiological salt solution during washing of the isolated tissue. Oxygen delivery tube or Aeration tube: Air or oxygen is supplied to the isolated tissue. Through an opening in aeration tube, Carbogen (mixture of 95% oxygen and 5% Carbon dioxide) is supplied to the isolated tissue. The speed of aeration is maintained at one to two bubbles per second. Thermostat: maintains the temperature of water in the outer jacket at 37 °C. Heater: Warms the water in the outer jacket.

Fig. 2.2 Sherrington recording drum

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Fig. 2.3  Schematic diagram of a Student’s organ bath

Stirrer: Circulates the water held in the outer jacket and helps in distribution of the heat generated by thermostat. Aerator: It is a device used for supply of the air or mixture of air and oxygen.

2.3

Intestinal Muscle Preparations

Background Identification and excision of tissue or muscle plays a very important role in in vitro efficient bioassay techniques. The tissue or muscle is first selected on the basis of sensitivity of the drug on it and then the procedure is continued with the selected animal (rat). Objective To prepare the intestinal muscle from the rat. Requirements Adult rat Physiological salt solution (PSS) Organ bath Experimental Procedure . The rats were kept fasted at least 4 h before, however water was supplied ad libitium. 1 2. The rats were sacrificed by cervical dislocation after anaesthetized them.

2.4 Skeletal Muscle Preparations

25

. Then the dead rats were fixed on the dissecting board by tying its legs. 3 4. The abdomen of the rat is opened by incision and the stomach and cecum were located. 5. For selecting the stomach section, 10 cm above ileum section adjacent to cecum was dissected and then required length of ileum was dissected. 6. Then 1.5–2 cm of the ileum was dissected out and washed properly in physiological salt solution (PSS). 7. Then the tissue is tied with a thread and mounted to an Organ bath.

2.4

Skeletal Muscle Preparations

Background Unlike the situation in the intact animal, the isolated muscle model makes it possible to study particular effects of hormones, uncomplicated by side effects of other substances that might occur in vivo. However, this model in vitro does not optimally reflect the situation in vivo. Objective To prepare the mouse skeletal muscle. Requirements Swiss-Webster mice weighing 30–32 g w Krebs-Ringer solution (PSS) Organ bath Experimental Procedure 1. The mice were kept at constant-temperature (22 ± 1° C) room on a 12 h-light/12 h-dark cycle and had free access to water and standard laboratory feed. 2. Briefly, at the time of the experiment the mice were anaesthetized with phentanyl/fluanisone (0.05 ml/g body wt., subcutaneously). 3. The rats were sacrificed by cervical dislocation after anaesthetized them. 4. Then the dead rats were fixed on the dissecting board by tying its legs. 5. From the hindlimbs both the soleus and the extensor digitorum longus muscles were carefully removed, weighed on a Mettler balance and mounted on a stainless-steel V-shaped clip. 6. Each muscle was individually immersed at 0  °C in an oxygenated (O2/CO2, 19:1) Krebs-Ringer bicarbonate buffer, pH 7.4 containing 4% (w/v) BSA. 7. Then they were transferred to plastic vials containing 1.5  ml of Krebs-Ringer bicarbonate. 8. Then the tissue is tied with a thread and mounted to an Organ bath.

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2  Isolated Tissues and Organs

Cardiac Muscle Preparations

Background Oscar Langendorff established the isolated perfused mammalian heat preparation first in 1897. This isolated cardiac muscle heart preparations helps in studying the contractile force, heart rate, coronary resistance and other parameters of the heart in vitro condition under controlled physiological conditions. This kind of study also can be carried out without the interference of any neural and hormonal complications of the animal. It has become a mainstay of pharmacological and physiological research. Objective To prepare cardiac muscle from rat. Principle This is based on the principle of retrograde flow or constant pressure. The entire perfusate enters through coronary arteries via the ostia at the aortic root. The PSS passes through the coronary vessels and then pefusate drains into right atrium via the coronary sinus. The retrograde perfusion creates pressure that closes the aortic valve and forces the solution to coronary circulation via the coronary sinus into the right atrium. The heart continuously contacts in this state so that different cardiac parameters can be measured upon administration of drugs (Fig. 2.4). 95%O2+5%CO2

reservoir perfusion fluid sintered glass gas distributor

perfusion cannula with side arm perfusion pressure transducer

transducer

heart chamber

balloon coronary effluent

Fig. 2.4  Schematic of the working heart model of an isolated perfused heart preparation

2.5 Cardiac Muscle Preparations

27

Requirements Albino rats (1 year old and of 300 g weight) Physiological salt solution (PSS) Pump providing a constant flow system Experimental Procedure 1. The rats were kept at constant-temperature (22 ± 1° C) room on a 12 h-light/12 h-dark cycle and had free access to water and standard laboratory feed. 2. Briefly, at the time of the experiment the mice were anaesthetized with pentobarbital (60 mg/kg IP). 3. Heparin (1000 IU/kg) is administered intravenously in the right femoral vein to prevent coagulation. 4. A cannula is placed in the trachea for ventilation. 5. A longitudinal incision is made on skin and muscle to open the abdomen from the diaphragm to the throat. 6. Then the diaphragm is cut free from the ribs. 7. The thorax is opened following the bone-cartilage border on the left and right sides parallel to the sternum from the diaphragm cranially to the first rib. 8. The complete anterior thoracic wall was turned upwards over the head to expose the heart. 9. The pericardium is removed and the ascending aorta is separated from the connective tissue and the pulmonary artery using blunt dissection. 10. Heart is dissected out and removed rapidly and transferred to cold Kreb’s solution. 11. Then the ascending aorta is separated gently using forceps from pulmonary artery and the heart is dissected with 1 cm intact aorta. 12. The heart is gently squeezed several times to remove blood from the heart and to prevent formation of thrombus. 13. A glass cannula is placed about 0.5 cm into the aorta and firmly kept in place using a thread. 14. Care should to be taken to prevent formation of air emboli inside the system. 15. Langendorff’s preparation system uses a constant head reservoir system used for providing constant pressure to the heart. 16. Alternatively, a peristaltic pump is used to set the rate for about 15–20  ml/min to maintain the perfusion chamber and aerated the Kreb’s solution at 37° C. Precautions 1 . The PSS flow is maintained adequately to prevent formation of edema in heart. 2. Cannula should be inserted firmly to avoid its insertion into aorta. 3. Sufficient hydrostatic pressure should be maintained.

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Questions 1. Why is it important to maintain a constant time cycle in isolated tissue organ bath experiments? 2. What is the recommended time cycle for organ bath experiments? 3. What is the function of transducer? 4. What are the different components of organ bath? 5. What are the essential features of physiological salt solution? 6. Name the first synthetic solution designed to maintain an isolated organ. 7. What is the composition of carbogen? 8. What is the use of a stimulator? 9. What are the different parameters controlled by stimulator? 10. Define the following terms: tachyphylaxis, selective antagonism and competitive antagonism. 11. What do you mean by quantal and graded bioassays? 12. What are the different types of graded bioassays? 13. How to construct concentration to response curve in static organ bath? 14. Which kind of tissue preparations required electrically stimulation? 15. Why vas deferens, or ductus deferens isolated tissue preparations are not so widely used despite their role in several landmark pharmacological discoveries?

Referenecs Badyal D (2008) Practical manual of pharmacology. Jaypee Brothers Medical Publishers, New Delhi BM Swamy V, Jayaveera KN, Reddy V (2014) Experimental pharmacology and toxicology. S. Chand & Company, New Delhi Goyal RK (2017) Practical in pharmacology. B. S. Shah Prakashan, Ahmedabad Kale SR, Kale RR (2017) Practical pharmacology and toxicology. Nirali Prakashan, Mumbai Katzung BG, Masters SB, Trevor AJ (2012) Basic & clinical pharmacology. McGraw-Hill, New York Medhi B (2017) Practical manual of experimental and clinical pharmacology. Jaypee Brothers Medical Publishers, New Delhi Ritter JM, Lewis LD, Mant TGK, Ferro A (2008) A text book of clinical pharmacology and therapeutics. Hodder Arnold, London Salmon DM (2014) Practical pharmacology for the pharmaceutical sciences. Wiley, Chichester Sierra LM, Gaivao I (eds) (2014) Genotoxicity and DNA repair: a practical approach. Springer protocols. Humana Press, London Singhal KC (1997) Pharmacology laboratory manual. CBS Publishers & Distributor, New Delhi Tripathi KD (2013) Essentials of medical pharmacology. Jaypee Brothers Medical Publishers, New Delhi Turner RA, Hebborn P (eds) (1971) Screening methods in pharmacology. Academic, New York Vogel HG (ed) (2002) Drug discovery and evaluation pharmacological assays. Springer, Berlin Woolley A (2008) A guide to practical toxicology. Informa Healthcare USA, New York

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Screening of Drugs Using Cell Lines/Isolated Tissues/Intact Animals

3.1

Evaluation of Antidiabetic Agents

3.1.1 Antidiabetic Evaluation of Drug in Induced Diabetic Mice Model Background Animal models have been extensively used for pharmaceutical evaluations of antidiabetic drugs and active principles of plant extracts for evaluation of their potency, mechanism of action and possible side effects if any. However, no animal model is good enough to represent entirely the manifestations and characteristic feature of particular type diabetes as the pathogenesis of diabetes may show heterogeneous expressions in man. Hence, several animal models have been used in antidiabetic studies, each displaying a different set of features as per the condition observed in human diabetic conditions. Amongst the different models used, small rodents are the widely used animal models as they are comparatively inexpensive to maintain. Further, they also show a rapid onset of diabetes when experimentally induced which is usually consistent with their shorter lifespan. Objective To evaluate the antidiabetic potential of the drug (Glibenclamide) in diabetic mice model. Principle The alloxan (ALX) or streptozotocin (STZ) induced mice models for are quite useful to study diabetes and its associated complications as they show remarkable similarity in deducing the possible role of various environmental factors in development destructive pancreatic disorders leading to pathogenesis of diabetes. STZ, is an antibiotic derived from Streptomyces achromogenes. It has been reported to cause diabetes by destructing the beta cells in pancrease. STZ have structural similarity with glucosamine derivative of

© Springer Nature Singapore Pte Ltd. 2019 J. K. Patra et al., A Practical Guide to Pharmacological Biotechnology, Learning Materials in Biosciences, https://doi.org/10.1007/978-981-13-6355-9_3

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nitrosourea that generates free radicals leads to destruction of beta cells. Apart from this STZ also causes alkylation and DNA breakage and increase the level of poly-ADP-ribose synthetase causing energy deprivation and death of beta cells. The beta cells destruction in pancrease causes deficiency in insulin production leading to development of diabetes in mice. Requirements Adult Balb/c male mice Streptozotocin (dissolved in 0.05 M citrate buffer, pH 4.5) Glibenclamide (dissolved in 0.05 M citrate buffer, pH 4.5) 0.05 M citrate buffer, pH 4.5 Glucostrips (One Touch Glucometer) Experimental Procedure 1. Healthy, adult male Balb/c mice weighing 28–37 g are maintained in controlled temperature condition of 23 ± 2 °C, humidity and a 12 h light–dark cycle. 2. They are kept in sanitised polypropylene cages haing paddy husk as bedding. They are having free access to standard mice pellet and water ad libitum. The animals are kept for a period 1 week for acclimatization before experimentation. 3. Mice are fasted overnight before diabetes was induced with streptozotocin (STZ). 4. STZ is dissolved in 0.05 M citrate buffer (pH 4.5) and injected intraperitoneally (i.p.) to overnight fasted mice at a single dose of 110 mg/kg body weight. 5. The animals are allowed to drink 5% glucose solution to overcome the drug induced hypoglycemia. 6. Seventy two hours after STZ administration, blood samples are collected from tail and glucose levels are estimated by glucostrips. 7. Mice having fasting blood glucose levels above 200 mg/dl are considered as diabetic and subsequently used for the experimental purpose (Fig. 3.1). 8. A total of 18 mice of 2 months old and average 30 g body weight were used. They were randomly taken as 12 diabetic and 6 normal mice. The above mice are divided into 3 groups of 5 each as follows. Group-1: Normal control mice supplemented with vehicle. Group-2: Diabetic control mice supplemented with vehicle. Group-3: Diabetic mice treated with glibenclamide drug (3 mg/kg b.w.) 9. The glibenclamide dissolved in 0.05  M citrate buffer (pH  4.5) and are given once daily for 30 consecutive days at glibenclamide (3 mg/kg b.w.) to Group-3 animals. 10. All animals were treated for 28 days. On 0, 7, 14 and 28th days, the blood samples are collected from each animal by puncturing the tail veins (Fig. 3.2).

3.1 Evaluation of Antidiabetic Agents

31

Fig. 3.1  Induction of diabetes in mice by injecting with STZ intraperitoneally

Result and Observation The blood glucose levels of all mice are checked by puncturing the tail vein and recording the blood glucose level of each mouse and are tabulated in Observation table. Observation Table Mice Group-­1 Group-­2 Group-­3

Glucose level Glucose level Glucose level (0th day) (7th day) (14th day)

Glucose level (28th day)

% of change in Blood glucose level

Interpretation The percentage in change in blood glucose level in drug treated mice (Group-3) as compared to the diabetic control mice (Group-2) is calculated as follows: % of change in blood glucose level =

( Mean blood glucose level in Group-2 mice- Blood glucose level in Group-2 mouse )

Mean blood glucose level in Group-2 mice

On the basis of the results obtained from the above study, the antidiabetic potential of the drug is calculated.

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Fig. 3.2  Checking of blood glucose level using glucose strip from tail vein of mice

Precaution 1. The blood glucose level of the mice should be observed at same condition throughout the experimental period i.e. in overnight fasted condition. 2. The STZ can be given in one single large dose varying between 110 and 200 mg/kg b.w. in mice or else at multiple dosing of 40 mg/kg b.w. for a period of 5 consecutive days

3.1.2 Glucose Uptake Assay Background Insulin resistance by decreased GLUT-4 translocation to plasma membrane is one of the important factors for NIDDM (non-insulin dependent diabetes mellitus) development. Hence, glucose uptake by cells can be considered as a useful technique to study cell signalling and glucose metabolism associated in diabetic condition. 2-deoxyglucose (2-DG) that bears a structural similarity to glucose is a commonly used method to assay the glucose uptake at cellular level. Both muscle and adipose tissue are regarded as primary insulin responsive tissues and express insulin-sensitive glucose transporter GLUT-4. Then in response to insulin, GLUT-4 translocates from intracellular vesicles to the plasma membrane leading to transport of glucose across the muscle and fat cells.

3.1 Evaluation of Antidiabetic Agents

33

Objective Principle The principle of this method is based upon the accumulation of 2-DG-6-phosphate (2-DG6P), a metabolized product of 2-deoxyglucose (2-DG). Usually, 2-deoxyglucose (2-DG) is taken up by cells using the by glucose transporters and inside the cells it get metabolized into 2-DG6P by hexokinase. However, 2-DG6P couldn’t be further be metabolized leading to its accumulation inside the cells. Therefore, concentration of 2- DG6P can be directly linked to uptake of 2-DG by cells. Since, 2-DG bear structural resemblance with glucose, accumulation of 2-DG6P can be correlated with uptake of glucose by cells. Usually 3 T3-L1 cells behave like primary adipocytes and provide an excellent in vitro model system to study insulin action and signalling (Fig. 3.3). Requirements 3 T3-L1 cell line Glucose assay kit Dulbecco’s Modified Eagle medium (DMEM) Fetal bovine serum (FBS) 10% Insulin Dexamethasone 3-isobutyl-1- methyl xanthine (IBMX) Dimethyl sulfoxide (DMSO) Krebs-Ringer-Phosphate-Hepes (KRPH) buffer BSA Extraction buffer Neutralization buffer 2-Deoxy glucose 2-DG uptake assay buffer Enzyme mix Picoprobe (in DMSO)

Fig. 3.3  Working principle in Glucose uptake assay

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2-DG6P standard (0.1 mM of 2-DG6P standard is prepared by diluting 2-DG6P with deuterated water. Further, 0, 2, 4, 6, 8 and 10 μL of diluted 2-DG6P is further diluted with deuterated water in 96 well plate in duplicate to generate 0, 200, 400, 600, 800 and 1000 pmol/well of 2- DG6P. The volume is made up to 50 μL with assay buffer.) Test compound (different concentrations of test compounds are prepared by dissolving the test compound in DMSO solvent). Experimental Procedure Preparation of Cell 1. 3  T3-L1 cell cultures are grown in 10% FBS (Fetal bovine serum) supplemented DMEM (Dulbecco‘s Modified Eagle medium) with 100 IU/ml penicillin and 100 μg/ ml streptomycin maintained at 37 °C in a humidified atmosphere of 95% air and 5% CO2. 2. The cells are seeded in DMEM with 10% FBS at a density of 4 × 102 cells/ml in 96 well culture plates and allowed to differentiate for 48 h after supplemented with DMEM having different factors such as 10 μg/ml insulin, 1 μM dexamethasone and 0.5 mM 3-isobutyl-1- methyl xanthine (IBMX). 3. Cells are maintained with10% FBS DMEM with 10  μg/ml insulin alone and the medium was changed every alternative day. 4. After 9 days of induction of differentiation, test compounds at different concentrations (100–1000 μg/ml) are added for an additional 3 days. 5. The control cells are treated with DMSO only and the final concentration of DMSO was kept below 0.1% overnight. 6. The cells are seeded at a density of 1500 cells per well in a 96-well plate and allowed to differentiate for another 96 h to get mature adipocytes. 7. The adipocytes are then washed with PBS and starved overnight in 100  μL Krebs-­ Ringer-­Phosphate-Hepes (KRPH) buffer supplemented 2% BSA. Glucose Assay Protocol 1. The adipocytes cells are then incubated for 20 min by adding 10 μL of 10 mM 2 DG to each well. 2. Cells are then gently washed with PBS (3 times) to remove exogenous 2-DG. 3. The cells are then lysed with 90 μL of extraction buffer, freeze thawed once and heated at 85 °C for 40 min. 4. The cell lysate is then cooled on ice for 5  min and neutralized by adding 10  μL of neutralization buffer. 5. 50 μL of test compound and/or standard are added per sample and the final volume is adjusted to 50 μL with assay buffer.

3.2 Evaluation of Antiulcer Activity

35

6. 50 μL of reaction mixture containing 47 μL of assay buffer, 1 μL of Picoprobe and 2 μL of enzyme mix is then added and incubated at 37 °C for 40 min in dark. 7. The Fluorescence was measured at Excited/emmitted at 535/587 nm respectively. 8. The cells were exposed with increasing concentrations of positive control Insulin (1 μM) for 20 min to activate glucose transporter. Result and Observation The accumulation 2-DG6P in cells can be linked with concentration of 2-DG of the test sample as: 2 - DGuptake = Sa / Sv ( pmol / mL )



where, Sa is the amount of 2-DG6P (in pmol) in the samples well calculated from standard curve. Sv is the sample volume (in μL) added in to the sample wells. Interpretation The amount of glucose remaining in the medium with positive control and with test sample are measured, using a glucose assay Kit. Precaution 1 . Proper sterilization condition should be maintained to avoid contamination of cells. 2. The enzyme solution should be kept on ice during working.

3.2

Evaluation of Antiulcer Activity

Background Peptic ulcer can be described as a circumscribed ulceration of mucus membrane penetrating through muscularis mucosa. It mostly occurred in areas frequently bathed by gastric HCl and pepsin. Depending upon the location of ulcer it can be named as peptic ulcer (in stomach) or duodenal ulcer (in duodenum). Gastric ulcer occurred due to abnormality in secretion of gastric acid or abnormal mucosal defence. In laboratory animals like rats or mice, the peptic ulcer is induced by certain drugs like aspirin, indomethacin etc. Several approaches are employed to treat the ulcer and they can be summarized as agents that (i) reduce gastric acid secretion, (ii) neutralize gastric acid, (iii) ulcer protectives and (iv) Anti-H pylori drugs. Objective To evaluate the anti ulcer activity of test drug (Pantoprazole).

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3  Screening of Drugs Using Cell Lines/Isolated Tissues/Intact Animals

Principle Pylorus ligation is one of the most commonly used methods for screening of antiulcer activity which is also known as ‘shay rat’ method. Pantoprazole is a proton pump inhibitor and after absorption, it diffuses into epithelial parietal or oxyntic cells of stomach of and accumulates there. It further undergoes a conversion to a more active form of drug i.e. reactive thiophilic sulphonamide cation which then binds to –SH group of cysteines in the H  +  K  +  -ATPase and irreversibly inactivating the pump thereby decreasing the acid secretion by stomach and providing prolonged suppression of acid secretion. Requirements Drug (Pantoprazole) Albino rats Ether (anesthesia) Saline solution

Experimental Procedure 1. Male albino rats (120–150 g) of Sprague-Dawley strain. Animals are housed in clean acrylic cages and maintained in standard conditions of 12 h light/12 h dark cycle at a controlled temperature at 20–22 °C and 60% humidity. 2. Rats are fed with a standard rat pellet diet having free access to water ad libitum and are kept for 1 week for acclimatization. 3. After 1 week of acclimation, the rats were made to fast overnight before treatment. Five rats are used per dose. 4. Before pylorus ligation, rats were fasted for 36 h under anesthesia. 5. Under ether anaesthesia, a midline abdominal incision is made and the pylorus is ligated carefully without damaging the blood supply or causing traction to the pylorus. 6. The abdominal wall is closed by sutures. 7. The test drug (Pantoprazole) is given intra duodenally. 8. The animals are sacrificed after 6 h followed which abdomen is opened. 9. Then a ligature is placed around the oesophagus close to diaphragm. 10. The stomach is then removed and the contents are drained in a centrifuge tube. 11. The stomach is opened along the greater curvature, washed with normal saline and pinned in cork plate. 12. The numbers of ulcers are noted down and severity is recorded.

Result and Observation The numbers of ulcers are counted and severity of ulcer is recorded with the following scoring.

3.3 Evaluation of Hepatoprotective Drugs

37

0 = No ulcer or normal mucosa 1 = Superficial ulcer 0 = Deep ulcer 0 = Perforation The ulcer index is calculated by Ui = Un + Us + Up* 10 -1 Where Ui is ulcer index; Un is average number of ulcer per animal; Us is average severity and Up is percentage of animals with ulcers. Observation Table Sl. No.

Control Un

Us

Treatment with drug Un Us

Ulcer Index

1 2 3

Interpretation The ulcer index and gastric content of Pantoprazole treated and control rats are recorded. Precaution The stomach should not be grasped with instruments as ulceration may develop at such points.

3.3

Evaluation of Hepatoprotective Drugs

Background The liver is the most significant digestive gland that metabolizes drugs via oxidation, reduction, hydration, hydrolysis, condensation, conjugation, or isomerization. Hepatic drug metabolism undergoes two stages to convert into conjugated water-soluble substances via P450 enzymes, which are excreted via urine or bile. Disruption of these processes can lead to hepatotoxicity. Hepatotoxicity may occur due to hepatocytes disassembling, apoptosis of hepatocytes, bile duct injury, mitochondrial inhibition, and cytolytic T-cell activation. Origin of hepatotoxicity may be linked with loss in weight, hepatitis, dyspepsia, coagulation of blood, oedema, pruritus etc. Liver is highly susceptible to damage from drugs and some other substances. The liver injury may also happen because of alteration of drugs to chemically active toxic metabolites that can react with cellular macromolecules like proteins, lipids, and nucleic acids, leading to dysfunction of proteins, peroxidation of lipids, damage of nucleic acids, oxidative stress etc.

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3  Screening of Drugs Using Cell Lines/Isolated Tissues/Intact Animals

3.3.1 In vitro Assessment of Hepatoprotective Effect Objective To evaluate the hepatoprotective activity of the test agent by in vitro technique. Principle Different hepatotoxic agents’ viz. carbon tetrachloride (CCl4), paracetamol, D-galactosamine are used to induce hepatotoxicity in laboratory models to estimate the hepatoprotective action of these drugs. The hepatoprotective potential of any drug is evaluated by its ability to prevent or mitigate biochemical, histological changes associated with hepatotoxic agents and normalization of the volume of the liver. The hepatoprotective effect can be assessed by using both in vitro and in vivo models. Requirements Rat liver Test agent (dissolved in DMSO) mol/l CCl4 (dissolved in DMSO) HEPES buffer I, pH 7.4 HEPES (N-2-hydroxyethylpiperazine-N-2-ethanesulphonic acid): 0.01 mol/L NaCl: 0.142 mol/L KCl: 0.0067 mol/L HEPES buffer II, pH 7.6 HEPES: 0.1 mol/L NaCl: 0.0667 mol/L KCl: 0.0067 mol/L Collagenase type IV: 0.5% RPMI-1640 media (supplemented with 10% calf serum, HEPES and 1  μg/mL gentamycin) Experimental Procedure 1. The livers are isolated under aseptic conditions and placed in HEPES buffer I. 2. The livers are cut into small pieces and then incubated in HEPES buffer II for 45 min at 37 °C in an incubator with constant shaking. 3. Then it is filtered and centrifugation is carried out at 200 rpm at 4 °C for 2 min thrice. 4. Following which the hepatocytes are collected and suspended in HEPES buffer I. 5. The collected hepatocytes are assessed for their viability by trypan blue exclusion method. 6. Then the freshly isolated viable hepatocytes are suspended in RPMI-1640 culture medium containing 10% calf serum, HEPES and 1 μg/mL gentamycin.

3.3 Evaluation of Hepatoprotective Drugs

39

7. Then approximately 1–1.2 × 106 per ml cells are then seeded into culture bottles and incubated at 37 °C in a CO2 dioxide incubator for 24 h. 8. Upon incubation for 24 h the hepatocytes formed a monolayer appeared as individual cells. The medium is then decanted and the culture is washed with HEPES buffer-I and followed by suspended in Buffer-I. 9. Triplicate of hepatocyte suspensions (0.1  mL) are distributed into various culture tubes and labelled as control, toxicant, and test. 10. The control group contain 0.1  mL of vehicle (30% DMSO) and toxicant groups received 0.1  mL of CCl4, while the test groups received 0.1  mL of respective test solutions (250, 500 and 1000 μg/mL). 11. Then hepatic cytotoxicity is then induced in isolated rat hepatocytes by mixing 0.1 mol/L CCl4. 12. Then volumes of all culture tubes are made up to 1  mL with HEPES buffer I and incubated in a CO2 incubator at 37 °C for 24 h. 13. Hepatocyte suspensions are then collected after incubation and cell viability is assessed by trypan blue exclusion method. 14. The hepatocyte suspensions are then centrifuged at 200 rpm and leakage of hepatocellular enzymes e.g. ALT and AST are then evaluated from the supernatant by using standard kit. Result and Observation 1. The effect of test agent on the protection of liver is determined by calculating the increase in percentage of viable cells incubated with test agent, as compared to control and toxicant groups. 2. The reversal in the elevated enzyme level is also considered for evaluation of hepatoprotective activity. Interpretation The hepatoprotective effect of the test agent is determined on the basis of higher % of viable hepatocytes and decrease in ALT and AST enzyme levels. Precaution The hepatocytes should be 95–96% viable and confirmed by trypan blue exclusion test.

3.3.2 In vivo Evaluation of Hepatoprotective Effect Objective To evaluate the hepatoprotective activity of the test agent by in vivo technique.

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Principle Carbon tetrachloride (CCl4) induced liver damage is considered to be the most frequently employed method to promote liver damage in experimental animals. Different parameters such as morphological, biochemical and histopathological changes are frequently studied to assess the hepatoprotective effect of any drug or bioactive agent. The morphological study include weight (organ/body weight ratio) and sometimes volume of the liver; the biochemical study include evaluation of different enzymatic and non-enzymatic parameters like AST, ALT, ALP, LDH, bilirubin, biliviridin, triglycerides, total cholesterol etc. Requirements Wistar rats CCl4 CMC (Carboxy methyl cellulose) Liver enzyme diagnostic kit Experimental Procedure 1. About eighteen albino Wistar rats each weighing about 180-220 g is divided into four groups of six rats each. 2. Group I having normal rats served as control group. Group II rats receiving 1% CMC 2 ml/kg b.w. served as CCl4 toxicant control and Group III rats are fed with receiving test agent suspended in 1% CMC at different dose levels (250, 500 and 1000 μg/ml b.w.). 3. All the rats are treated for 1 week and on the 7th day, the toxicant CCl4 (500 μl/kg i.p.) is administered to all groups of rats except Group I. 4. After 24 h of administration, all the animals are anesthetized and blood was collected by sino-orbital puncture. 5. The collected blood is centrifuged for 10 min. at 2000 rpm. The serum is then separated and various biochemical parameters such as AST, ALT, and ALP are estimated by using liver enzyme diagnostic kit. 6. All the rats are then sacrificed livers are perfused and excised followed which the tissues are kept at −70 °C for estimation of lipid peroxidation. 7. The level of lipid peroxidation (LPx) in liver homogenate is quantified by their reactivity with thiobarbituric acid in acidic conditions.

Result and Observation The levels of AST, ALT, ALP and LPX in serum are determined and tabulated.

3.4 Evaluation of Anti-inflammatory Agents

41

Observation Table Mice Group- I Group- II Group- III

AST

ALT

ALP

LPx

Interpretation 1. The different biochemical parameters e.g. AST, ALT, ALP of different group of mice are recorded and the hepatoprotective potential of the test agent is determined. The lowering of enzyme levels is a definite indication of hepatoprotective action of the test agent.

3.4

Evaluation of Anti-inflammatory Agents

Background The process of inflammation is associated with pain and increased vascular permeability, protein denaturation along with increase in membrane alteration. Inflammation is generally considered as a protective response to tissue injury by a stimulus characterized by redness, pain, heat, swelling and loss of function in the affected area. Inflammation may be described as acute or chronic inflammatory response. In response to harmful stimuli, increased movement of plasma and leukocytes occurs from blood to affected area which is known as acute inflammation. On the other hand in chronic inflammation, destruction and healing of the tissue in the affected area goes on side by side simultaneously. Both in vivo and in vitro experimental methods are used for evaluating the ant-inflammatory potency of drugs. Objective To evaluate the anti-inflammatory property of test agent (drug) against carrageenan-­ induced paw edema in rats. Principle Upon injection of several irritants viz. carrageenan, formalin, bradykinin, histamine, mustard or egg white in dorsum of foot, the rats can produce acute paw edema within few minutes. The carrageenan induced paw edema is the most commonly study method of evaluating anti-inflammatory activities of drugs in rat model. The principle behind anti-­ inflammatory agents is based on the capacity to reduce the edema caused by any irritants. The change in the volume of edema is estimated by plethysmograph, which measures the volume of rat paw upon treatment with anti-inflammatory drug.

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Requirements Rats (150–200 g) Plethysmograph (This apparatus is used to measure the paw volume. This apparatus contains mercury which gets displaced when the paw is dipped and the displaced amount is directly read from the scale attached to mercury column.) Carrageenan Test agent (Indomethacin) 20 mg/kg dose. Stock solution is prepared at a concentration of 4 mg/ml. Experimental Procedure 1. The animals are weighed and animals are divided into two groups with six animals in each group. 2. The animals are numbered and the both left and right hind paws are marked beyond tibio-tarsal junction. 3. The initial paw volume of each rat is measured by using Plethysmograph. 4. The Group-1 rat are injected with saline and the Group-2 rat are injected with Indomethacin (0.5 ml/100 g bw of rat) subcutaneously (s.c.). 5. About 0.1 ml of 1% w/v carageenan is injected subcutaneously in the plantar region of the left paw after 30 min of treatment both in control as well as Indomethacin-treated group. The non-inflammed right paw served as reference paw for comparison. 6. The paw volume of both legs of the rat are checked at 15, 30, 60, 120 min after carageenan challenge (Fig. 3.4).

Result and Observation The percentage difference in the right and left paw volume of each rat of Group-1 and Group-2 are calculated. The percentage of protection after treatment is calculated and tabulated in observation table. Observation Table Paw edema (15 min) Paw edema (30 min) Paw edema (60 min) Paw edema (120 min) Sl. No. Right paw Left paw Right paw Left paw Right paw Left paw Right paw Left paw 1 2 3 4

Interpretation On the basis of the capacity of decreasing the volume of the paw, the anti-inflammatory property of the drug is assessed.

3.5 Evaluation of Antioxidant Activity

43

Fig. 3.4  Plethysmograph used for measuring the paw volume. (Source: www.labthai.co.th/ugoinflame.html)

Precaution The rats should be marked properly to avoid mixing of untreated and treated groups. The paw should be cleaned and marked.

3.5

Evaluation of Antioxidant Activity

3.5.1 E  valuation of Antioxidant Activity Using Erythrocyte-Based Method Background Reactive oxygen species (ROS) is regarded as molecules responsible for generating oxidative stress inside the cell and thereby disturbing the balance between oxidant-­antioxidant systems inside the cell. The excessive generation of ROS under physiological condition may destruct different molecules like protein, carbohydrates and lipids inside cells by making them them functionally impaired thereby leading to development of many oxidative stress associated diseases and ageing. Antioxidant can be defined as any substance capable of delaying, preventing or removing oxidative damage of a target molecule. The

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3  Screening of Drugs Using Cell Lines/Isolated Tissues/Intact Animals

antioxidant effect at sub cellular level as well as their involvement in metabolic pathways can be revealed by using several cellular and molecular biology approaches. Cell based assay methods are used to identify compounds with target the antioxidant defence. Furthermore, a number of cell types such as fibroblasts neural cells, osteoblasts can be tested. Erythrocyte-based assays represent a good choice because they are quick, easy to perform, and do not need specialized equipment. Objective To assess the antioxidant potential of the test compound by erythrocyte-based method. Principle The erythrocytes mimic the cellular environment as well as the membrane structure possess both enzymatic and non-enzymatic antioxidant system that can neutralize ROS thereby limiting oxidative stress damage. The erythrocyte-based antioxidant assay method is based on the principle that AAPH (2,2-azobis(2-amidinopropane) dihydrochloride) releases ROS spontaneously causing haemolysis of RBCs which can be avoided by presence of antioxidant compound. So the antioxidant activity of the test compound can be assessed on the basis of capacity to prevent hemolysis of erythrocytes. Requirements Wistar rats AAPH (150 mM) Isotonic saline (PBS), pH 7.4 22.2 mM Na2HPO4 5.6 mM KH2PO4 123.3 82 mM NaCl mM glucose in Distilled water

Experimental Procedure 1 . The blood is collected from retro-orbital plexus of Wistar rats. 2. Red blood cells (RBCs) are separated from the plasma and buffy coat by centrifugation at 1000 g for 10 min and the plasma is removed from the supernatant. 3. Then the erythrocyte layer is washed thrice with isotonic phosphate-buffered saline (PBS). 4. Cells are then suspended at a density of 12.5% in isotonic saline solution. 5. Then 250 μL of 12.5% erythrocyte suspension is incubated at 37 °C for 90 min with constant shaking in the presence of AAPH (150 mM) for achieving complete hemolysis. 6. Concentrations ranging between 15 and 10,000 μg/mL of the test compound dissolved in PBS are added to the erythrocyte suspension containing AAPH.

3.5 Evaluation of Antioxidant Activity

45

7. Erythrocyte controls are kept in all of the experiments for detecting any spontaneous haemolysis of the erythrocytes. 8. After the incubation of 90 min, cells are centrifuged and the absorbance is is determined spectrophotometrically at 540 nm to observe the haemolysis pattern. 9. Then the percentage of haemolysis is calculated by using the following formula.

Result and Observation

The percentage haemolysis = éë( Abscontrol - Abssample ) / Abscontrol ùû ´100

Interpretation The antioxidant potential of the test compound is assessed on the basis of inhibition in percentage of haemolysis.

3.5.2 E  valuation of Antioxidant Activity Using Cell Based Assay Method Objective To assess the antioxidant activity of the test compound using cellular antioxidant activity (CAA) technique. Principle The CAA assay uses cell-permeable 2′,7′-dichlorofluorescin diacetate (DCFH-DA) which is fluorescence probe that can be taken up by the cells. Inside the cells the DCFH-DA is deacetylated to form 2′,7′-dichlorofluorescein (DCFH) in presence of cellular esterase enzymes. The reactive oxygen molecule 2, 2′-azobis (2-amidinopropane) dihydrochloride (AAPH) spontaneously releases peroxyl radical which then oxidize the intracellular DCFH to form a fluorescent compound dichlorofluorescein (DCF). So the level of fluorescence formed within the cells is directly proportional to the level of oxidation. Antioxidant compounds that could quench peroxyl radical generation can inhibit the formation of fluorescent DCF which can be measured as decrease in cellular fluorescence. So the antioxidant potential of any compound can be assessed by observing the decrease in fluorescence of DCF. Requirements PC12 cells Media RPMI-1640 with l-glutamine: 85% Heat-inactivated Horse serum: 10% Fetal bovine serum (FBS): 5%

46

3  Screening of Drugs Using Cell Lines/Isolated Tissues/Intact Animals

Loading medium (99% RPMI-1640 and 1% FBS) 100 U/ml penicillin G sodium 100 mg/ml streptomycin sulphate The cells are maintained in collagen-coated plates at 37 °C with 5% CO2. The culture medium is changed twice in a week and the cells are splitted to 1:8 every week. 6-Carboxy-29,79-dichlorofluorescin diacetate (DCFHDA) 2, 2 ́-azobis (2-amidinopropane) dihydrochloride (AAPH) KRH Buffer (Krebs-Ringer HEPES Buffer) NaCl 116 mM KCl 4 mM 1 mM MgCl2 1.8 mM CaCl2 Glucose 25 mM HEPES acid 10 mM Adjust pH to 7.4.

Test compound Experimental Procedure 1. PC12 cells are splitted and viability is checked by trypan blue exclusion assay. Then, 1 day before the experiment the viable cells are plated in collagen-coated 96-well plates at a concentration of 104/well. 2. On the day of the experiment, the media is removed and plated cells are washed with KRH buffer. 3. The cells are then incubated with 100 mM DCFH-DA in the loading medium in 5% CO2/95% air at 37 °C for 30 min. 4. Then DCFH-DA is removed and the cells are washed and incubated with KRH buffer containing test compound. 5. As positive control 1 mM H2O2 diluted in KRH buffer is incubated with cells. 6. The fluorescence of the cells from each well was measured and recorded. The excitation filter was set at 485 ± 10 nm and the emission filter was set at 530 ± 12.5 nm. 7. The absorbances are taken every 5 min for 30 min.

Result and Observation Percentage increase in fluorescence per well was calculated by the formula éë( Ft 30 - Ft0 ) / Ft0 ùû ´ 100 Where, Ft30 = fluorescence at time 30 min and Ft0 = fluorescence at time 0 min.

3.6 Effect of Drug in Rabbit Eye

47

Interpretation Quantifying cellular oxidative stress by CAA is an efficient method with low variability which can be used to quantify the efficiency of antioxidants against ROS the in various cell lines. Precaution . Biosafety level 1 precautions should be followed when handling cells. 1 2. Any material containing cells should be discarded as bio hazardous waste.

3.6

Effect of Drug in Rabbit Eye

Background A large number of drugs are used for their local action in the eye as eye drops, or eye ointments. The eye is supplied by both the sympathetic and parasympathetic nerves. The superior palpebral muscle and the dilator pupillae of iris have sympathetic supply. The sphincter paplillae of the iris has parasympathetic supply which exercises dominant control. The ciliary muscle is also supplied by the parasympathetic nerve. Contraction of ciliary muscle causes body to move inwards and forwards direction towards the axis of the eye. The Iris is composed of the circular and the radial muscle fibres. The circular muscles are supplied with parasympathetic or cholinergic nerve fibres and the radial muscles are innervated by sympathetic or adrenergic nerve fibres. Together both the sympathetic and parasympathetic nerves stimulate to produce mydriasis and miosis respectively whereas paralyses of these nerves lead to produce the opposite effects. Objective To study the effect of drugs on the rabbit eye. Principle Pupil of the eye is dilated by paralysis of parasympathetic or stimulation of sympathetic system known as Mydriasis. Similarly due to stimulation of parasympathetic system and sympathetic system, the circular muscle fibre of the iris and the radial muscle fibres contract respectively. Miosis is the constriction of pupil occurred due to stimulation of parasympathetic nerve, oculomotor nerve or paralysis of sympathetic nerves. Requirements Rabbit Dropper Drug solution

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3  Screening of Drugs Using Cell Lines/Isolated Tissues/Intact Animals

(Pilocarpine 1%, Adrenaline 0.1%, Atropine 1%, Ephedrine 5%, Cocaine 1%, Phenylephrine 3%) Experimental Procedure 1. The rabbit’s are weighed and marked. 2. Rabbits are put in the holders so that head will be protruding outside. 3. The eyelashes of both the eyes are clip off. 4. One eye (either right or left eye) as control and the other as test. 5. Saline is applied in the control eye and a drug in the test eye. 6. 1–2 drops of saline or drug solutions are instilled using dropper. The lower eyelid is pulled upwards and kept in contact with conjunctiva for 1–2 min. 7. Different parameters like Size of pupil, Light reflex, Touch reflex (Corneal reflex), are observed. 8. The diameter of both the pupils are measured with a pupilometer made up of cardboard or hard paper. 9. Light reflex is checked with a torch. The light is always put from side (back) and brought to the front. The changes in the diameter of pupil are observed when light is put into eye. Any decrease or increase in pupillary diameter is recorded. 10. It is tested with a fine cotton wool wick. The peripheral part of cornea is touched with tip of cotton wick. Blinking represents presence of corneal reflex. 11. All the observation are repeated thrice. 12. Then the response is noted and both control and test eyes are compared (Fig. 3.5).

Result and Observation The pupillary size, light reflex and corneal reflex are recorded after 5 min of drug instillation and observations are tabulated.

Fig. 3.5  Effect of drug on Rabbit’s eye

3.7 Evaluation of Local Anaesthetics

49

Observation Table Drug Saline Pilocarpine 1%, Atropine 1%, Ephedrine 5% Cocaine 1% Phenylephrine 3%

Sl. no. 1 2 3 4 5 6

Size of pupil Normal Constriction Dilation Dilation Dilation Dilation

Light reflex Present Present Absent Present Present Present

Corneal reflex Present Present Absent Present Absent Present

Interpretation The effect of drugs on rabbit eye are observed and reported. Precaution 1. Do not touch central part of cornea it can cause corneal ulcers/opacities. This can lead to blindness as central part of cornea is the main part of cornea used for visibility. 2. The drug or the solution to be tested should not hurt or cause permanent damage to the animal eye.

3.7

Evaluation of Local Anaesthetics

Background Local anaesthetics (LAs) are the agents when applied topically or injected locally can cause reversible loss of sensory perception in the applied area of the body. When come in contact, these agents can block the stimuli generation and conduction of nerve impulse at any part without causing any kind of structural damage. During upstroke of action potential, these agents can block conduction of nerve impulse by decreasing the entry of Na+ ions. So with increase in the concentration of the LA, the rate of nerve conduction slows down as local depolarization unable to reach the threshold potential leading to blockage of conduction.

3.7.1 Evaluation of Local Anaesthetics Objective To study the effect of Local anaesthetics (LAs) by surface anaesthesia method. Principle The cornea of eye is one of the sensitive parts of eye and responds to even very light touch. Upon applying LAs to eye, the LAs abolish the neuronal activity in cornea. So when any plug of cotton is touched to the cornea, the corneal reflex shown by blinking is not observed.

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3  Screening of Drugs Using Cell Lines/Isolated Tissues/Intact Animals

Requirements Procaine hydrochloride Animal holder Cotton stop watch Rabbit Experimental Procedure . The rabbit’s are weighed and marked. 1 2. Rabbits are put in the holders so that head will be protruding outside. 3. The eyelashes of both the eyes are clip off. 4. One eye (either right or left eye) as control and the other as test. 5. Saline is applied in the control eye and a drug in the test eye. 6. 1–2 drops of saline or drug solutions are instilled using dropper. The lower eyelid is pulled upwards and kept in contact with conjunctiva for 1–2 min. 7. The corneal reflex is checked by touching cotton plug to the cornea from side of the rabbit. 8. The corneal reflex is checked at every one min interval for 5 min.

Result and Observation The corneal reflex for each eye is observed and recorded in the observation table. Sample Saline Drug

Eye Left Right

Corneal reflex (minutes) 1 2 3

4

5

Interpretation On the basis of the observation of corneal reflex, the Las effect of the drug is validated. Precaution 1. Do not touch central part of cornea it can cause corneal ulcers/opacities. This can lead to blindness as central part of cornea is the main part of cornea used for visibility. 2. The drug or the solution to be tested should not hurt or cause permanent damage to the eye of the animal

3.7 Evaluation of Local Anaesthetics

51

3.7.2 Evaluation of Local Anaesthetics Objective To study the effect of Local anaesthetics (LAs) by intradermal method. Principle LAs reversibly block impulse conduction along nerve axons and other excitable membranes that utilises the Na+ Channels as primary means of action potential generation causing loss of sensation against external stimuli. Requirements Lignocaine 2% Animal holder Guinea pig (250–350 g) Experimental Procedure 1 . The animals are weighed and marked. 2. The hair is removed on its flanks on both sides. 3. One side is marked as ‘C’ as the Control side where saline or control jelly is applied. 4. The other side is marked as ‘T’ as the Test side where Lignocaine jelly is applied. 5. The anaesthetic activity is checked by giving a small inch with forcep on the control side and then on treatment side and the n checking the animal response. 6. The responses are taken at 0, 5, 10, 15, 20, 30 min after the induction of drug. Result and Observation The corneal reflex for each eye is observed and recorded in the observation table. Sample Saline Drug

Flank Left (C side) Right (T side)

Squeak response (minutes) 1 2 3

4

5

Interpretation On the basis of the observation of corneal reflex, the Las effect of the drug is validated. Precaution The hair is removed carefully from either side of flanks with blade. The control and treatments are marked properly to avoid confusion.

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3  Screening of Drugs Using Cell Lines/Isolated Tissues/Intact Animals

Questions 1. What are the different classes of antidiabetic drugs? 2. How carbohydrate metabolizing enzyme inhibition drugs act? 3. What is the principle behind α-amylase and α-glucosidase inhibition assay? 4. What is the principle behind glucose uptake assay? 5. How STZ induces diabetes in rodents? 6. What are the different approaches to treat ulcer? 7. What is Pylorus ligation? 8. What is the mode of action of Pantprazole drug? 9. How to calculate ulcer index? 10. What do you mean by hepatoprotective drugs? Give examples. 11. How hepatotoxicity originates? 12. How chemical metabolite causes hepatic injury? 13. What are the different histopathological changes associated with hepatic injury? 14. What is liver function test? 15. What are the enzymes or observed for assessment of liver injury? 16. How inflammation is characterized? 17. Classify inflammation on the basis of response? 18. How anti-inflammatory drugs act? 19. What is plethysmograph? 20. What is ROS? How it is generated? 21. How antioxidants work? 22. What are the different antioxidant enzymes? 23. What are the different methods of antioxidant assays? 24. What is mydriasis and miosis? 25. How local anaesthesia acts?

References Apak R, Esra C, Fereidoon S (eds) (2018) Measurement of antioxidant activity & capacity recent trends and applications. Wiley, Hoboken Badyal D (2008) Practical manual of pharmacology. Jaypee Brothers Medical Publishers, New Delhi BM Swamy V, Jayaveera KN, Reddy V (2014) Experimental pharmacology and toxicology. S. Chand & Company, New Delhi Botta A, Martínez V, Mitjans M, Balboa E, Enma C, Vinardell MP (2014) Erythrocytes and cell line-based assays to evaluate the cytoprotective activity of antioxidant components obtained from natural sources. Toxicol In Vitro 28(1):120–124 Goyal RK (2017) Practical in pharmacology. B. S. Shah Prakashan, Ahmedabad Kale SR, Kale RR (2017) Practical pharmacology and toxicology. Nirali Prakashan, Mumbai Martinez V, Ugartondo V, Vinardell MP, Torres JL, Mitjans M (2012) Grape epicatechin conjugates prevent erythrocyte membrane protein oxidation. J Agric Food Chem 60(16):4090–4095

References

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Medhi B (2017) Practical manual of experimental and clinical pharmacology. Jaypee Brothers Medical Publishers, New Delhi Ogu C, Maxa J  (2000) Drug interactions due to cytochrome P450. Proc Bayl Univ Med Cent 13:421–423 Ritter JM, Lewis LD, Mant TGK, Ferro A (2008) A text book of clinical pharmacology and therapeutics. Hodder Arnold, London Salmon DM (2014) Practical pharmacology for the pharmaceutical sciences. Wiley, Chichester Seglen PO (1976) Preparation of isolated rat liver cells. Methods Cell Biol 13:29–83 Sierra LM, Gaivao I (eds) (2014) Genotoxicity and DNA repair a practical approach. Springer Protocols, Humana Press, London Singhal KC (1997) Pharmacology laboratory manual. CBS Publishers & Distributor, New Delhi Thatoi HN, Dash S, Das SK (2017) Practical biotechnology, principle and protocols. I.K. International, New Delhi Tingstrom A, Obrink B (1989) Distribution and dynamics of cell surface-associated cell CAM 105 in cultured rat hepatocytes. Exp Cell Res 185:132–142 Tripathi KD (2013) Essentials of medical pharmacology. Jaypee Brothers Medical Publishers, New Delhi Turner RA, Hebborn P (eds) (1971) Screening methods in pharmacology. Academic, New York Vogel HG (2002) Drug discovery and evaluation pharmacological assays. Springer, Berlin Woolley A (2008) A guide to practical toxicology. Informa Healthcare USA, New York www.labthai.co.th/ugo-inflame.html Yang C, Shahidi F, Tsao (2017) Biomarkers of oxidative stress and cellular-based assays of indirect antioxidant measurement. In: Apak R, Capanoglu E, Shahidi F (eds) Measurement of antioxidant activity & capacity recent trends and applications. Functional Food science Technology/Wiley, Hoboken, p 179

4

Genotoxicity and Toxicological Studies

Background Genotoxicity can be defined as the toxic changes on the genetic material (DNA, RNA) of the cell leading to destruction of nucleotide strand breakage. Genotoxins are the mutagens and cause genotoxicity by damaging chromosomal material causing mutation. Genotoxins includes both chemical substance and radiation. Genetic material damage in somatic cells may lead to malignancy whereas damage in germ cells may lead to heritable mutations causing various birth defects. Therefore, assessment of genotoxicity is important for evaluation of safety aspects of substances for protection of health and the environment. The results of the genotoxicity tests form the scientific basis for risk assessment and are used for classification and labelling of chemical substances. Genotoxicity Test Methods Genotoxicity basically affect at the level of gene and chromosome. In assessing the Genotoxicity of any new chemical is assessed by several tests to determine their mechanism. Generally, genotoxicity studies are designed in such a manner that it follows a broad pattern of other toxicity tests. Genotoxicity assays are conducted by both in vitro and in vivo methods. Few important genotoxicity methods and their method of evaluation have been tabulated in Table 4.1.

4.1

In Vitro Genotoxicity Assay

Background Genotoxicity test is essential for safety assessment of different substances such as pharmaceuticals, chemicals, pesticides, biocides, food additives, cosmetics, veterinary drugs for protection of human and animal. The in vitro genotoxicity test comprises of bacterial

© Springer Nature Singapore Pte Ltd. 2019 J. K. Patra et al., A Practical Guide to Pharmacological Biotechnology, Learning Materials in Biosciences, https://doi.org/10.1007/978-981-13-6355-9_4

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Table 4.1  Commonly used genotoxicity test methods Sl. no. Test method Bacterial reverse 1 mutation test (Ames test)

2

Type of assay In vitro

3

Mammalian chromosome aberration test Mammalian cell gene mutation test or, mouse lymphoma test

4

Comet assay

5

Micronuclei test or, In chromosome vivo aberration test

In vitro In vitro

In vivo

Working principle Salmonella typhimurium and Escherichia coli strains are used for bacterial reverse mutation test to detect substitution, deletion or addition of one or few of base pairs of DNA. Following identification of mutation it reverts back and restores the functional capability of the mutant cell to synthesize Histidine This test helps in identifying the factors that cause structural mutations in chromosomes. Different chromosomal changes e.g. polyploidy and duplication are identified using this test This test assesses chemical substances that can cause gene mutations in cell lines. Further, it can detect the different end points of mutation such as thymidine kinase (TK) and hypoxanthine-guanine phosphor ribosyltransferase (HPRT), and a transgene of xanthineguanine phosphor ribosyltransferase (XPRT) This test detects DNA damage in a variety of primary DNA lesions which other tests can’t identify. This test is also useful for assessment of hazardous agents which have potential for genotoxicity or mutagenicity The mutagen exposure leads to damage and division of cell and form smaller micronucleus

reverse mutation assay, in vitro mammalian chromosomal aberration test, in vitro mammalian cell gene mutation test and in vitro mammalian cell micronucleus test. Further, in vitro test are validated by several in vivo genotoxicity studies such as mammalian erythrocyte micronucleus test, mammalian bone marrow chromosomal aberration test, transgenic rodent somatic and germ cell gene mutation assay. Further, in vivo comet assay can also evaluate the genotoxicity of any compound (Fig. 4.1). Objective To assess the mutagenicity potential of the test agent by Ames test or bacterial reverse mutation assay. Principle The Ames test is used to evaluate the potential carcinogenic effect of chemicals that are capable of causing genetic damage leading to gene mutation by using the bacterial strain Salmonella typhimurium. In this assay mutated Salmonella strains are used that are incapable of synthesize histidine of their own because of mutations in histidine operon at different gene level. As a result in histidine lacking medium, they can’t grow and form colonies. Therefore, upon treated with mutagenic chemicals, these mutant bacterial cells

4.1  In Vitro Genotoxicity Assay

57

Fig. 4.1  In vitro geneotoxicity tests. (Reproduced with permission from Corvi and Madia (2017))

undergoes mutation enabling them to grow in histidine lacking media. Upon positive in Ames test, the mutagenic chemicals are further tested for carcinogenic effect in animal model. Requirements Colony counter Sterile petri dishes Sterile syringes (5, 10, and 50 mL) Membrane filters (0.22 and 0.45 μm) Salmonella tester strains (TA97, TA98, TA100, TA102, TA104, TA1535, TA1537, and TA1538) Nutrient Broth Glucose Minimal (GM) Agar Plates (This is prepared by taking 15 g Agar and 930 ml of distilled water in a 2 L flask. Then it is autoclaved and cooled to 65 °C. Followed which 20 mL Vogel-Bonner salts (50×) and 50 mL glucose (40%) are added. Then the solution is stirred thoroughly followed which plating was carried out in 100 × 15 mm petri dishes containing approximately 25 mL/plate) Vogel-Bonner Salts (50×) Ingredients per litre: Magnesium sulfate (MgSO4 · 7H20) Citric acid monohydrate (C6H8O7·H2O) Potassium phosphate, dibasic, anhydrous (K2HPO4) Sodium ammonium phosphate (NaHNH4.PO4·4H20) Warm distilled water (about 45 °C)

10 g 100 g 500 g 175 g 670 mL

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4  Genotoxicity and Toxicological Studies

Top Agar Supplemented with Histidine/Biotin (The solution is prepared by taking 6 g of Agar and 5 g sodium chloride to 900 ml of distilled water in a flask. Then it is autoclaved followed by addition of 100 mL of histidine and biotin solution (0.5 mM) in a laminar flow hood) Histidine/Biotin Solution (0.5 mM) Ingredients per 250 mL D-Biotin L-Histidine Distilled water

0.0309 g 0.0244 g 250 mL

Crystal Violet Solution (0.1% w/v) Metabolic Activation System (S 9 Mix) Composition for 50 mL: Phosphate buffer (0.2 M, pH 7.4) NADPa 0.1 M D-Glucose-6-phosphate (1 M) KCl (1.65 M)–MgCl2 (0.4 M) salts Liver S-9 fraction (4%) Distilled water

25 mL 2 mL 0.25 mL 1 mL 2 mL 19.75 mL

Phosphate Buffer (0.2 M, pH 7.4) Ingredients per 500 mL: Sodium dihydrogen phosphate (0.2 M) 60 mL (NaH2PO4 · 4H2O) (13.8 g/500 mL) Disodium hydrogen phosphate (0.2 M) 440 mL (Na2HPO4) (14.2 g/500 mL) Nicotinamide Adenine Dinucleotide Phosphate (NADP) (0.1 M) Ingredients per 10 mL: NADP Sterile distilled water

0.07654 g 10 mL

(It is prepared by dissolving the NADP in sterile distilled water followed by filtering through membrane filter (0.45  μm) and then stored in a sterilized screw cap vial. This solution should be prepared freshly.) Test compound (Different dilutions of the test compounds)

4.1  In Vitro Genotoxicity Assay

59

Experimental Procedure 1. About 0.05 mL of tester strain(s) cultures is inoculated in 50 mL of nutrient broth and incubated at 37 °C for 12–16 h. 2. Then appropriate number of GM agar plates and sterile glass test tubes for each test chemicals are prepared. Each experiment should contain a series of duplicate plates for (a) negative control (solvent); (b) Positive controls and (c) at least five or more concentrations of the test substance. 3. Then molten agar supplemented with 0.05 mM histidine and biotin (2 ml), S-9 mix (0.50 ml), test compound of different dilution or control (0.5 ml) and overnight grown culture of the Salmonella strain (0.1 ml) are taken in separate sterile glass tubes and maintained at 43 °C. 4. Then contents of each test tube are poured carefully to the surface of the corresponding GM plates and swirled gently to distribute the molten agar evenly. 5. Upon solidification of agar, plates are inverted and placed at 37  °C for 48  h in an incubator. 6. After 48 h of incubation the plates are checked for growth of bacterial colony size and if the sizes of the colonies are smaller than anticipated then the plates are incubated for another 12–24 h. 7. Then the bacterial colonies are counted by using electronic colony counter or manually. Result and Observation The test results are expressed as number of revertant colonies per plate. Interpretation 1. Positive: If increase in the number of revertant colonies is observed then the test compound is considered mutagenic. 2. Negative: If increase in the number of revertant colonies is not observed then the test compound is considered a non-mutagenic substance. 3. Inconclusive: If mutagenicity or non-mutagenicity could not be established from the observation, then the compound is called inconclusive. Precaution Metabolic Activation System (S 9 Mix) must be prepared in a laminar flow hood and all components must be kept in ice bath.

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4  Genotoxicity and Toxicological Studies

4.2

Mouse Lymphoma Assay

Background The Mouse Lymphoma Assay (MLA), also known as in vitro mammalian cell gene mutation test is used to study mutations induced by different chemicals or/and their metabolites. The assay is useful technique in detecting both point and chromosomal mutations. The assay uses Thymidine kinase gene (Tk1) as the target for mutation induction. Objective To evaluate the metagenicity of the test compound by Mouse Lymphoma Assay technique. Principle Tk gene codes for a cytosolic protein which produces a thymidine kinase (TK) phosphotransferase enzyme that plays an important role in metabolism of pyrimidine nucleotide in salvage pathway. The thymidine kinase enzyme phosphorylates deoxythymidine to deoxythymidine 5′-phosphate which is then incorporated into DNA. This study involves exposure of thymidine kinase (TK) proficient cells to the pyrimidine analogue trifluorothymidine (TFT) so that the cellular metabolism will be inhibited and cell division will be halted. However, TK deficient cells will show resistance to the cytotoxicity of TFT and will proliferate to form colonies. So upon treatment with any test compound if the number colonies increased, then it can be correlated with the mutagenic nature of the said compound. The toxicity is measured by calculating the relative cloning efficiency. Requirements 1. Maintenance of cell culture: L5178Y TK+/− mouse lymphoma cells are maintained as suspension culture in RPMI 1640 media incubated at 37 °C and 5% CO2. 2. RPMI 1640 media (The media contains thymidine (T), hypoxanthine (H), methotrexate (M) and glycine (G) and is used to select against newly arising TK−/− mutants) 3. Metabolic Activation System (S 9 Mix) Composition for 50 mL: Phosphate buffer (0.2 M, pH 7.4) NADPa 0.1 M D-Glucose-6-phosphate (1 M) KCl (1.65 M)–MgCl2 (0.4 M) salts Liver S-9 fraction (4%) Distilled water

4. Methylmethanesulphonate (MMS)

25 mL 2 mL 0.25 mL 1 mL 2 mL 19.75 mL

4.2 Mouse Lymphoma Assay

61

5. Cyclophosmphamide 6. Benzo(a)pyrene 7. Test compound Experimental Procedure 1. L5178Y TK+/− mouse lymphoma cells maintained in RPMI 1640 media. The cells are further maintained in the RPMI 1640 media containing THMG for a week to select against newly arising TK−/− mutants. Then it is maintained in the RPMI 1640 media containing THG for 1–3 days prior to mutagenesis study. 2. Different concentration of test agent freshly prepared in distilled water is selected in the mutagenesis assay such that the survival in highest dose should be approx. 10–15% and in the low concentration the survival should be closer to the negative control. 3. Then about 6 × 106 maintained mouse lymphoma cells in 10 ml medium are treated with test compound in presence and absence of S9 metabolic activation system at 37 °C for 3–4  h. Further, methylmethanesulphonate (without S9 mixture) and cyclophosmphamide and benzo(a)pyrene (with S9 mixture) dissolved in DMSO treated cells are used as the positive controls. 4. Then the chemicals are removed by centrifugation and cells are washed twice in non-­ selective RPMI 1640 media and again resuspended in non-selective RPMI 1640 medium at a density of 3 × 105 cells/ml and incubated at 37 °C for 2 days. 5. Then for determination of survival and mutation frequency, about 106 cells are seeded in 96 well culture plates. 6. Further, for viability measurement at each dose, two 96 well culture plates containing 106 cells/well are set up in non selective RPMI-1640 medium. 7. Another two 96 well culture plates with 2000 cells/well in selective medium containing trifluorothymidine (TFT) at 4 μg/ml concentration are used for mutant counting. 8. Then colonies are counted and allowed to grow for 11–14 days at 37 °C. The mutant frequency is then calculated. Result and Observation Mutant Frequency (MF) is determined from the plating efficiencies of mutant colonies (PEM) and plating efficiencies of viable cells (PEV) from the same culture. Mutant frequency ( MF ) =

PEm PEv

PEm and PEv are calculated by using the number of colonies and the total number of cells used for the cloning:



PEm =

Cm Tm

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4  Genotoxicity and Toxicological Studies

PEv =



Cv Tv

where CM is the total number of colonies on the selective plates, TM is the total number of cells used for selection, CV is the total number of colonies on the viability plates, and TV is the total number of cells used for viability. CFE = Number of colonies / Number of cells plated



Where CFE is colony forming efficiency. The colony forming efficiency (CFE) indicated the toxicity of the test agent as the decrease in CFE is correlated to toxicity of the test agent. Increase in mutation frequency indicated the mutagenicity of the test agent. Interpretation • A test agent is considered positive or mutagenic if it induces a statistically significant dose-related increase in the mutant frequency. • A test agent is considered negative or non-mutagenic if it fails to produce a statistically significant dose-related increase in mutant frequency in any one of the tested concentrations.

4.3

Comet Assay

Background The Comet Assay or single cell gel electrophoresis (SCGE) assay was first described by Ostling and Johanson (1984). It is a sensitive and rapid technique used to detect DNA damage at the level of a single cell. The size, shape and DNA amount within the ‘comet’ play decisive role in evaluating the level of damage. Comet assay can measure the transient genetic damage which is not a fixed change to DNA. The comet assay can be performed by both in vitro and in vivo ways. The in vitro method utilizes single cell from immortalized cell lines where as cell suspension dispersed from any tissue are used for in vivo assay (Fig. 4.2). Objective To detect the extent of DNA damage by Comet assay. Principle When current is passed through agarose gel containing DNA, the negatively charged loops/fragments of DNA are drawn through the agarose gel. In comet assay, when the electric field passes through the gel, DNA migrates out of the cell and move in the direction of the anode, appearing like a ‘comet’. The migration of DNA depends upon the extent of

4.3 Comet Assay

63

Fig. 4.2  Model image of comet

damaged DNA present in the cells. The size, shape and distribution of DNA within the comet correlate with the extent damage of DNA within the cell. Requirements Dimethylsulfoxide (DMSO) Disodium EDTA Ethidium Bromide Histopaque Normal Melting Agarose (NMA) Low Melting Point Agarose (LMPA) Phosphate Buffered Saline (PBS) (Ca2+, Mg2+ free) Sodium Chloride (NaCl) Sodium Hydroxide (NaOH) Triton X-100 Trizma Base Lysing Solution: Ingredients per 1000 mL NaCl (2.5 M) EDTA (100 mM) Trizma base (10 mM) NaOH dH2O

146.1 g 37.2 g 1.2 g 8 g Volume make up to 1000 ml

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Electrophoresis Buffer (300 mM NaOH/1 mM EDTA): Prepare of stock solution: NaOH (10 N) EDTA (200 mM) Stored at room temperature

200 g NaOH dissolved in 500 ml of dH2O 14.89 g of EDTA is dissolved in 200 ml dH2O

For preparation of 1× running electrophoresis buffer NaOH (10 N) EDTA (200 mM) dH2O

30 ml 5.0 ml q.s. to 1000 ml

The solution are mixed properly and the pH of the buffer should be maintained at above>13. Neutralization Buffer (pH 7.5) Tris (0.4 M) dH2O

48.5 g q.s. to 1000 ml

The pH is maintained at 7.5 and stored at room temperature. Staining Solution Ethidium Bromide (EtBr) EtBr stock solution (10×) – 20 μg/mL 10 mg of ethidium bromide in 50 mL dH2O Stored at room temperature. EtBr working solution (1×) 1 ml of stock solution and 9 ml dH2O is mixed. Experimental Procedure Preparation of Slides: 1. About 1% and 0.5% LMPA in PBS and 1.0% NMA in Milli Q water is prepared by heating until near boiling to dissolve agarose. 5 mL sample aliquot of LMPA are put into scintillation vials and refrigerate until needed. LMPA vial is placed in water bath at 37 °C to cool and stabilize the temperature. 2. The slides are dipped in methanol and burn them over a blue flame to remove any oil or dust. 3. The conventional slides are dipped up to one third area while NMA agarose is hot and gently remove. 4. The underside of slides are wiped to remove agarose and laid in a tray on a flat surface for drying following which they are stored at room temperature.

4.3 Comet Assay

65

Cell Isolation/Treatment: Whole Blood About 75 μL of LMPA (0.5%; 37 °C) is mixed with 10 μL lymphocytes (About 10,000 nos.) and added to the coated slide. The coverslips are then placed and slides are kept on a slide tray resting on ice packs until the agarose layer hardens. Isolated Lymphocytes: 1. 20 μL whole blood is mixed with 1 mL RPMI 1640 in a microcentrifuge tube followed by addition of 100 μL Ficoll histopaque below the blood/media mixture. 2. Then it is centrifuged for 3 min at 2000×g followed by removal of 100 μL of bottom of the media/top of Ficoll layer. Then, 1 mL media and mix is added and centrifuged for 3 min at 2000×g to pellet lymphocytes. 3. The supernatant is poured off and the pellet is resuspended in 75 μL LMPA and process as above. 4. Then the coverslip is removed and slide is lowered slowly into cold, freshly made lysing solution. 5. The slides are protected from light and refrigerate for 2 h at 4 °C. Electrophoresis of Microgel Slides 1. The slides are removed gently from the Lysing solution after 2 h. Then slides are placed side by side on the horizontal gel box near one end as close together as possible. 2. Then reservoir is filled with freshly prepared electrophoresis buffer so that it completely covers the slides. 3. The slides are then allowed to immerse in the alkaline buffer for 20  min allowing unwinding of DNA. 4. Then the power supply is turned on to 24 V (0.74 V/cm) and the current is adjusted to 300 mA and electrophoresis is carried out for 30 min. 5. The power supply is then turned off and the slides are gently lifted from the buffer and placed on a drain tray. Then neutralization buffer is poured and slides are left as such for at least 5 min. 6. Then the slides are drain and the procedure is repeated twice. 7. Slides are stained with 80 μl 1× EtBr and left for 5 min and then dipped in chilled distilled water to remove excess stain. 8. The cover slip is then placed over it. Result and Observation 1. The EtBr-stained DNA is observed under a microscope (40× objective) to visualize damage of DNA. 2. The length and percentage of migrated DNA are observed to assess the DNA damage.

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Fig. 4.3  Images of comets (from lymphocytes) and represented for visual scoring on the basis of 0–4 score

Interpretation Different parameters like amount of migration per cell, number of cells with increased migration, extent of migration among damaged cells, and viability are compared by analysing the image to assess the quantitative and qualitative extent of DNA damage (Fig. 4.3). Precaution Handle EtBr with adequate precaution as it is known carcinogen.

4.4

In Vitro Teratogenicity Testing

Background The capacity of drug or any other agent to cause foetal abnormalities when administered to the pregnant mother is known as teratogenicity. It has been a common practice to check for teratogenicity effect of any drug since the thalidomide tragedy of 1961. There are four manifestations of prenatal toxicity such as (i) embryo or fetal death, (ii) malformation, (iii) retardation and (iv) functional impairment out of which the first three stages can be detected by any standard teratogenicity assay. However, teratogenicity test in vivo is the only one segment of reproductive toxicity screening. Alternatively, several other approaches have also been proposed in last few decades which can be used for studying developmental toxicity. The four major categories are: (i) established cell line, (ii) primary cell culture, (iii) non-eutherian embryos and (iv) culture mammalian embryos or primordial.

4.4  In Vitro Teratogenicity Testing

67

Objective To test the teratogenicity of the agent by employing micromass cultures. Principle The primary culture of limb bud cells reproduces cartilage histogenesis and exhibited several other developmental processes e.g. proliferation and differentiation of cells along with cell to cell and cell to extracellular matrix interactions. Therefore, interference with these developmental aspects of cell would lead to expression of primordial teratogenic endpoints. The micromass method is based on the principle that a particular chemical that inhibit the formation of foci or the number of cells within foci is positive in nature. Requirements Tissue culture incubator, 37 °C, humidified, 5% CO2 in air. Laminar flow hood Heating block Water bath Binocular dissecting microscope Inverted phase contrast microscope 96 well plate spectrophotometer Haemocytometer Cells: Undifferentiated rat embryo limb bud cells Hanks’ balanced salt solution, with phenol red Ham’s F-12 nutrient mixture Fetal calf serum (FCS) Hanks’ BSS and Ham’s F-12 are prepared according to the supplier’s instructions. Penicillin G/streptomycin 100× liquid in saline The medium is made by adding FCS, 5% v/v in Ham’s F-12 and 10 ml of 100× Penicillin G per lit followed by filtering through 0.22 μm membrane. Trypsin 10× liquid Neutral red Alcian blue 37% Formaldehyde Ca/Mg free phosphate buffered saline (CMF) % trypsin in CMF 0.9% w/v Saline 0.4% neutral red in sterile distilled water 1% w/v alcian blue in 0.1 N HCI. Acid alcohol: 1.0%v/v acetic acid in 50% ethanol. Formol-Calcium: 1 ml 37% formaldehyde plus 10 ml of 0.1 g/ml calcium chloride dihydrate, made up to 100 ml with distilled water Penicillin G (benzylpenicillin)

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Sodium 5-fluorouracil (Sigma F-6627) Test compound Experimental Procedure 1. Different concentration of the test compound is produced by diluting 1000 μg/ml of test chemical to 100 μg/ml, 10 μg/ml, 1 μg/ml, 100 ng/ml, 10 ng/ml, 1 ng/ml, 0.1 ng/ml by tenfold serial dilutions. 2. Embryos are obtained from Wistar rats on day 14 of gestation and the limb buds are isolated and single cell suspension is prepared by trypsinization. 3. Subsequently, cells are spotted into 96 well plates. 4. Then the 96 well plates are placed in the incubator, followed by addition of 300  μl medium with or without test chemical and incubated for 5 days at 37 °C. 5. At the end, the total number of viable cells and of differentiated and number of foci were determined. Measurement of Total Number of Viable Cells 6. The medium from wells are removed and blotted to remove as much medium as possible. 200 μl of 0.005% neutral red in medium at 37 °C is added to each well and incubated at 2–3 h at 37 °C. 7. Then neutral red is removed and rinsed thrice with saline. 8. Then 200 μl formol-calcium is added to each well allowed to stand for about 1 min. and then removed. 9. 200 μl acid alcohol is added to each well and left for 30–60 min on a mixing table, until all the neutral red is extracted from cells. 10. Then the absorbance in read using Spectrophotometer at 540 nm. Measurement of Number of Differentiated Cells and Foci 11. The medium from wells are removed and blotted to remove as much medium as possible. 12. 200 μl of 0.005% neutral red in medium at 37 °C is added to each well and incubated at 2–3 h at 37 °C. 13. Then neutral red is removed and rinsed thrice with saline. 14. Then 200 μl formol-calcium is added to each well allowed to stand for about 1 min. and then removed. 15. 200 μl acid alcohol is added to each well and left for 30–60 min on a mixing table, until all the neutral red is extracted from cells. 16. Then the wells are rinsed three times with saline. 17. 200 μl 1% alcian blue in 0.1 N HCl is added to each well and left overnight to stain and then alcian blue.

4.5 Histopathological Studies of Animal Tissues

69

8. Then the plate was rinsed three times with saline. 1 19. The number of foci can be counted at this point. 20. Or else, 200 μl 6 M guanidine hydrochloride (freshly made) is added to each well and left for at least 2 h to elute stain from cell foci. 21. Then the absorbance was read at 620 nm. Result and Observation In the micromass test two endpoint values are determined: ID50 (50% inhibition of cells differentiation) for cells differentiation and IC50 (IC50: 50% inhibition of cell viability and growth) for cells viability/growth. Interpretation On the basis of the data, the test chemicals can be classified as test chemicals into three toxicity classes of in vitro embryotoxicity: non, weakly and strongly. Precaution Each experiment must include one plate with the full range of positive control concentrations by adding 5-fluorouracil at 1000, 500, 250 & 125, 62.5, 31.25, 15.625 ng/ml along with a column of negative controls containing 500 μg/ml penicillin.

4.5

Histopathological Studies of Animal Tissues

Background Histopathology is an investigative medical tool to study different tissues of human or animal origin under microscope to examine for any pathological condition. Histopathology study involves microscopic preparation of biological tissues, staining, visualization and microscopic examination of biological tissues. This technique helps in recognizing specific microscopic structural changes associated with pathological condition of any disease. This technique basically compares any diseased or altered tissues with respect to normal or control counterparts. Objective To learn technique of histopathology. Principle When the cell undergoes autolysis, water and electrolytes comes out of the cell leading to uncontrolled and of alteration of enzymatic activity leading to complete destruction of tissue architecture. Therefore, tissues are preserved using fixative that permanently cross-­ link its proteins and stabilize it resulting in counteracting further acting autolysis and preserving the tissue architecture. Formaldehyde fixes tissue by cross-linking the proteins, primarily the residues of the basic amino acid lysine. Xylene is used as the clearing agent because the tissues contain alcohol so it has to be removed from the tissue to make it firm

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for the purpose of section cutting. Following this tissues are cut into fine sections and tissues are stained with different stains. After staining the stained tissues are examined under microscope for any structural changes of the tissue. Requirements Tissue section from animal (Liver) Fixative solution (usually commercially available formalin). Phosphate buffer (pH = 6.8). Forceps Containers for histological specimens, cassettes and permanent labels. Disposable plastic cassettes for histology Absolute ethyl alcohol. 90% and 70% ethanol solutions. Paraffin solvent Paraffin wax for histology, melting point 56–57 °C Eosin solution. Clearing agent (xylene) Staining dishes and Coplin jars suitable for staining. Permanent mounting medium Glass cover slips (25 × 60 mm) Tissue embedding station Paraffin wax for histology, melting point 56–57 °C Rotary microtome. Tissue water bath with a thermometer. Experimental Procedure Fixation . A portion of the liver was excised and placed in ice-cold normal saline. The tissue was 1 properly cleaned, freed of connective tissues and clotted blood, pat dried on filter paper. 2. Small pieces of the tissue were fixed in sublimate formol (nine parts of saturated HgCl2 and one part of formaldehyde) for 24 h followed by washing in running water for 24 h to remove excess fixative. 3. (Tissue immersion in formaldehyde or glutaraldehyde is the most frequently used fixation method in biomedical research. Formalin (formaldehyde) is commercially available as 38–40% or 10% neutral phosphatebuffered solutions. It is generally accepted that a volume ratio of tissue to fixative of 1:10 to 1:20 is necessary for optimal fixation. Tissue samples are usually fixed at room temperature after 12–48 h.) 4. The fixative container is placed under a fume hood and tissue samples are plunged in the fixative solution. The fixative container is gently stirred so that the tissue sample does not stick to the container surface and then the cap is replaced over the container after each tissue.

4.5 Histopathological Studies of Animal Tissues

71

Pre-embedding 5. In this step, tissue samples are infiltrated with paraffin so that water content in tissue is replaced by this wax material. 6. Pre-embedding consists of dehydration of tissues with sequential increased concentrations of alcohol. 7. Following this the alcohol is gradually replaced by paraffin. 8. For this purpose, metal cassettes are used which are clean and free of spilt fluids and wax. 9. Then the tissues are gradually dehydrated by putting the tissues in metal cassettes as follows: 10. The dehydration is carried out in graded series of ethanol as follows: (a) Washed in 70% ethanol for 1 h. (b) followed by washing in 95% ethanol for 1 h (two times). (c) followed by washing in absolute ethanol for 1 h (two times). 11. Then tissues are embedded in paraffin for 1 h (two times). Embedding 12. Once tissue samples are infiltrated by paraffin, they are removed from the cassettes and carefully positioned inside a metal base mold with proper orientation. 13. The specimen is positioned at the centre in the mould ensuring that paraffin entirely surrounds the edge of the tissue. 14. The mould is then filled with melted paraffin and then placed carefully on a cooling surface until the paraffin has hardened. 15. Then the mould is snapped off and the paraffin blocks are brought together. 16. The paraffin blocks are stored at room temperature until sectioning. Sectioning 17. The paraffin block is mounted on the microtome holder. 18. The angle between the blade edge bevel and the block is set at about 2–5°. 19. The blade is locked in place and the microtome hand-wheel is locked properly. 20. The edge of one block is trimmed with a sharp razor blade. 21. The thickness is set at 4–5 μm. 22. Then a series of paraffin sections are cut and a ribbon of serial sections is obtained. 23. The ribbon is separated from the knife edge with a paint brush. 24. The piece of ribbon is transferred to the surface of the water bath set at 45 °C. 25. The floating sections are gently separated on the water bath with pressure from the tips of forceps. 26. The sections are collected on a clean glass slides by sliding vertically in the water bath beneath the floating section and then carefully lifting up the slide to enable the tissue to adhere the glass slide. 27. The slides are allowed to dry horizontally on a warm plate for 10 min so that sections are firmly adhereed to the glass slide.

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Fig. 4.4  Microtome used for tissue sectioning

8. The slides are labelled with a histopen or pencil. 2 29. The slides are then stored in dry boxes at room temperature (Fig. 4.4). Staining and Mounting Then 5 μm sections were routinely stained with haematoxylene and eosin and assessed in a light microscope. For study of histopathology three randomly selected slides were taken per animal and data were scored at ten different focuses per slide. (However, as per need different set of dyes can be used for staining). Result and Observation All alterations from the normal structure were registered. Interpretation Upon completion of microscopic examinations statistical analyses are carried out the results are interpreted based upon the findings. Precaution • Absolute ethanol is highly is inflammable and can cause irritation to the eye and should not be handled closer to naked flame or heat or eye. • Xylene moderately inflammable and a mild irritant to eye and mucous membrane that may cause dermatitis. • In case of immunohistochemistry slides should be stored at 4 °C to minimize antigen loss.

4.6 Drug Poisoning

73

• Histology grade paraffin wax should be used as it has a melting point around 56 or 57 °C, that does not alter the structures and key morphologic characteristics of tissues.

4.6

Drug Poisoning

While poisons can be defined as the substance that cause temporary or permanent damage to the body when swallowed, ingested, inhaled or contacted, drug poisoning can be stated as a condition of taking of prescribed drug in overdoses either accidentally and/or intentionally. Drug overdosing leads to drug poisoning and it may be a source of morbidity, mortality, and health care expenditure. Among adults, the most common exposures of drug poisoning is due to analgesics (11.6%), sedatives and antipsychotics (10.1%), and antidepressants (6.7%). The drug poisoning can be broadly discussed under 3 categories, such as: self poisoning, accidental poisoning and criminal poisoning. Diagnosis of Drug Poisoning A meticulous, rapid but accurate clinical examination is essential because the symptoms and signs may be characteristic of certain poisons. The clinical manifestations of some common poisons are summarized in Table 4.2. Routine investigation of the drug overdose in patient also include blood glucose and biochemical determination of plasma electrolytes, urea, creatinine, oxygen saturation and arterial blood gases. In case of drug poisoning, the affected patients are managed with intensive supportive therapy so that the drug is eliminated naturally by the body. However, different methods are also employed to eliminate the toxin rapidly from the patient. Few methods are being tabulate in Table 4.3. Further, antidotes are also available against a few numbers of poisons (Table 4.4).

Table 4.2  Clinical manifestations of some common drugs causing drug poisoning Common drugs Benzodiazepines and other hypnosedatives, alcohol Opioids Tricyclic antidepressants, phenothiazines; other drugs with anticholinergic properties Amphetamines, MDMA, anticholinergic agents Tricyclic antidepressants, phenothiazines, carbon monoxide, monoamine oxidase inhibitors, mefenamic acid, theophylline, hypoglycaemic agents, lithium, cyanide Salicylates

Symptoms Coma, hypotension, flaccidity Coma, pinpoint pupils, hypoventilation Coma, dilated pupils, hyper-­reflexia, tachycardia Restlessness, hypertonia, hyper-reflexia, pyrexia Convulsions

Tinnitus, overbreathing, Salicylates pyrexia, sweating, flushing, usually alert

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Table 4.3  Methods of poison elimination Drug poisoning Salicylates, phenobarbital Salicylates, methanol, ethylene glycol, lithium, phenobarbital Barbiturates, theophylline, disopyramide Salicylates, theophylline, quinine Most anticonvulsants, digoxin

Method Alkaline diuresis Haemodialysis Charcoal haemoperfusion Gastro-intestinal dialysis Multiple-dose activated charcoal

Table 4.4  Commonly used antidotes for treatment in case of drug overdosing Overdose drug Paracetamol Iron Cyanide

Antidote Acetylcysteine i.v., Methionine p. o. Desferrioxamine Oxygen, dicobalt edentate i.v. or sodium nitrite i.v. followed by sodium thiosulphate i.v Benzodiazepines Flumazenil i.v Beta-blockers Atropine, Glucagon, Isoprenaline Carbon monoxide Oxygen, Hyperbaric oxygen Methanol/ethylene glycol Ethanol, Fomepizo Lead (inorganic) Sodium EDTA i.v. Penicillamine p.o.Dimercaptosuccinic acid (DMSA) i.v. or p.o Mercury Dimercaptopropane sulphonate, (DMPS) Dimercaptosuccinic acid (DMSA), Dimercaprol, Penicillamine Opioids Naloxone Organophosphorus Atropine, pralidoxime insecticides Digoxin Digoxin-specific fab antibody fragments Calcium-channel Calcium chloride or gluconate i.v. blockers

Accidental poisoning with drugs causes between 10 and 15 deaths per annum in children. In adults, accidental poisoning most commonly occurs at work and usually involves inhalation of noxious fumes. Factory and farm workers are at particular risk. Criminal poisoning is one mode of non-accidental injury of children. Questions 1. What is xenobiotic? 2. What are in vitro tests for detecting gene mutations? 3. What are characteristic toxic effects of chemicals on the skin? 4. Define LD50? 5. What is teratogenic study? 6. What are the different animals used for teratogenic studies? 7. What are the different genotoxicity testmethods.

References

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8. What is the principle of Ame’s test? 9. What is the principle of Comest assay? 10. What is reverse mutation assay? 11. What is the role of Thymidine kinase gene (Tk1) in mouse lymphoma assay? 12. What is the principle of mouse lymphoma assay? 13. What is mutant frequency? How to calculate it? 14. How colony forming efficiency (CFE) helps in mutagenicity evaluation? 15. What are the factors affecting the comet? 16. What is teratogenicity? 17. What are the different approaches for studying developmental toxicity? 18. How histology is different from histopathology study? 19. How histopathology study helps? 20. What is microtomy? 21. What are the different stains used in histopathology study? 22. What stain haematoxylene and eosin? 23. What is immunohistochemistry? 24. How to diagnose drug poisoning? 25. What is an antidote? Discuss its role in drug poisoning?

References Badyal D (2008) Practical manual of pharmacology. Jaypee Brothers Medical Publishers, New Delhi BM Swamy V, Jayaveera KN, Reddy V (2014) Experimental pharmacology and toxicology. S. Chand & Company, New Delhi Corvi R, Madia F (2017) In vitro genotoxicity testing – can the performance be enhanced? Food Chem Toxicol 106:600–608 Goyal RK (2017) Practical in pharmacology. B. S. Shah Prakashan, Ahmedabad Kale SR, Kale RR (2017) Practical pharmacology and toxicology. Nirali Prakashan, Mumbai Katzung BG, Masters SB, Trevor AJ (2012) Basic & clinical pharmacology. McGraw-Hill, New York Medhi B (2017) Practical manual of experimental and clinical pharmacology. Jaypee Brothers Medical Publishers, New Delhi Ostling O, Johanson KJ (1984) Microelectrophoretic study of radiation-induced DNA damages in individual mammalian cells. Biochem Biophys Res Commun 123:291–298 Ritter JM, Lewis LD, Mant TGK, Ferro A (2008) A text book of clinical pharmacology and therapeutics. Hodder Arnold, London Salmon DM (2014) Practical pharmacology for the pharmaceutical sciences. Wiley, Chichester Sierra LM, Gaivao I (eds) (2014) Genotoxicity and DNA repair: a practical approach. Springer protocols. Humana Press, London Singhal KC (1997) Pharmacology laboratory manual. CBS Publishers & Distributor, New Delhi Thatoi HN, Dash S, Das SK (2017) Practical biotechnology, principle and protocols. I.K. International, New Delhi Tripathi KD (2013) Essentials of medical pharmacology. Jaypee Brothers Medical Publishers, New Delhi Turner RA, Hebborn P (eds) (1971) Screening methods in pharmacology. Academic, New York Vogel HG (2002) Drug discovery and evaluation pharmacological assays. Springer, Berlin Woolley A (2008) A guide to practical toxicology. Informa Healthcare USA, New York

5

Experimental Animal Studies

5.1

Collecting Blood from Mice

Blood sample from experimental animals are frequently required for study of effect of drugs on biochemical parameters and for the study of pharmacokinetics of drugs in the experimental animals. One important aspect of the blood sample collection is that the experimental animals should feel least stress and pain as the blood collection under stressful condition may affect the outcome of the experiment. The blood sample is generally collected from venous, arterial blood vessels or heart chambers. However, several factors need to be considered for collection of blood from experimental animals. Such as: • • • • • • •

Species type Size of the animal Age and health of the animal Volume of sample required Frequency of sampling Experience of the trained personnel Anesthesia type

Sample volume should always be the minimum volume of blood which satisfies experimental needs. To minimize risk of injury to the animal and personnel working in the laboratory appropriate physical or chemical restraint should be employed. The blood samples can be collected from experimental animals by employing the following techniques.

© Springer Nature Singapore Pte Ltd. 2019 J. K. Patra et al., A Practical Guide to Pharmacological Biotechnology, Learning Materials in Biosciences, https://doi.org/10.1007/978-981-13-6355-9_5

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• Blood collection not requiring anesthesia –– From saphenous vein and dorsal pedal vein of rat, mice. • Blood collection requiring anesthesia –– From tail vein, orbital sinus, jugular vein, temporary cannula of rat and mice –– From tail snip of mice –– From blood vessel cannulation of rat, guinea pig, ferret –– From tarsal vein of guinea pig –– From marginal ear vein/artery of rabbit • Terminal procedure –– By cardiac puncture (e.g. rat, mice, guinea pig, rabbit, ferret) –– From orbital sinus and posterior vena cava of rat, mice. The sampling procedures for blood collections are of two types such as: non-terminal blood collection and terminal blood collection. Non-terminal Blood Collection  When blood is collected from the conscious or unconscious animals through a single or multiple withdrawals it is known as non-terminal blood collection. Animal are not sacrificed after blood collection in this procedure. It can be done by different ways: (i) Collection of blood from lateral tail vein or ventral/dorsal artery: This procedure is applied in both rats and mice. It is done by cannulating the blood vessel or by nicking it superficially at a perpendicular to the tail. • Obtainable volume: Mouse – small to medium [50–100 μl] Rat – medium [0.2–0.4 ml] • This procedure is carried out in the conscious mice or rat. Prior to the collection of blood, the tail is dipped in warm water (approx. 50–60 °C) or else xylol is applied to the tail to increase the circulation through tail vein. Then, the needle (25–27 gauge, 0.5–1 length) is inserted in the distal portion of tail vein. Then, the blood is slowly aspirated avoiding the collapse of vein. Advantages and Disadvantages • Sample quality decreases with prolonged bleeding times and tail stroking. • Repeated collection of blood is possible. • This procedure is relatively non-traumatic. • This procedure is routinely done without anesthesia, although effective restraint is required. • In most cases warming the tail with the aid of a heat lamp or warm compresses becomes useful as it will increase blood volume. • Arterial sampling produces comparatively faster and involves uptake of larger volumes of blood but it requires special care to ensure adequate hemostasis. • Piercing the tail vein with a needle is usually done when it is required to collect a very small quantity of blood sample (Fig. 5.1).

5.1 Collecting Blood from Mice

79

Fig. 5.1  Blood lateral collection from lateral vein

Fig. 5.2  Collection of blood from mandibular vein

(ii) Collection of blood from Mandibular Vein/Artery: This procedure can be used in both rats and mice by piercing the mandibular vein or artery with a needle (20G) or stylet (Fig. 5.2). • Obtainable volume: medium to large (100–200 μl, mouse; 0.4–0.5 ml, rat). • This procedure is customarily performed on an unanesthetized animal, but effective restraint is required during blood collection. • Arterial sampling produces are done for large volumes and are performed very rapidly. • Venous sampling produces are done for medium volumes and are performed more slowly. • Usually gentle pressure is applied for approximately 30 s post-collection to ensure hemostasis.

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(iii) Collection of blood from saphenous/lateral tarsal: • This procedure can be used in both rats and mice by piercing the saphenous vein with a needle (23–25G: mouse, 21–23G: rat) (Fig. 5.3). • The obtainable blood volumes are small to medium (mouse: 100 μl; rat: 0.4 ml). • By this method repetition of sampling is possible. • This procedure is customarily done on an unanesthetized animal; however, effective restraint is required. • This procedure is more time-consuming than that of other methods. • Care must be taken to ensure adequate hemostasis following the blood sampling. (iv) Collection of blood from retro-orbital: • This procedure can be used in mice by penetrating the retro-orbital sinus with a glass capillary tube of 0.5 mm in diameter or via the retro-orbital plexus in rats with a capillary tube (Fig. 5.4). • It must be performed by a skilled operator. • Follow up observations are required for 24–48 h after blood collection. If complications such as squinting or bulging of the eye are noted, then it must be reported. • Obtainable volume: medium to large. • Collection is limited to once per eye. Precaution The retro-orbital sampling possesses a greater in the hands of an unskilled operator than other blood collection routes due to the following complications:

Fig. 5.3  Collection of blood from saphenous vein

5.2 Studies on Different Parameters of Blood

81

Fig. 5.4  Retro-orbital collection of blood

• Hematoma and excessive pressure on the eye result in retro-orbital hemorrhage. • Excessive pressure in eye may result into persistent bleeding causing hematoma, corneal ulceration, keratitis, rupture of the eyeball or micro-ophthalmia etc. • It may leads to damage of optic nerve and other intra-orbital structures resulting into vision deficits or blindness. • It may result in fracture of the bones of the orbit and neural damage along with loss of vitreous humour due to penetration of the eyeball. Terminal Blood Collection In this type of blood collection, a large volume of blood is collected in single or multiple withdrawals from the anaesthtized experimental animals. Animal is generally sacrificed during or after such blood collection by cardiac puncture or axillary cut down which are considered as terminal procedures. This procedure is done after ensuring that the animal is under surgical anesthesia. The post-mortem performed immediately after euthanasia.

5.2

Studies on Different Parameters of Blood

Background Analysis of blood has become a standard routine procedure in medical diagnosis. Blood is a complex fluid consisting of different blood cells suspended in straw-coloured (yellowish) liquid known as plasma. The blood cells comprise a mixture of red cells (erythrocytes), white cells (leucocytes) and platelets (thrombocytes). The blood plasma which makes up about 55% of total blood volume contain different types of proteins, chemical substances, clotting factrs and other metabolic substances. The concentration of various substances and number of other factors help the physician to diagnose and monitor treatment for a large number of disease condition. Analysis of blood is often carried out for research

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purposes, as a number of diseases and physiological processes alter its characteristic values and composition.

5.2.1 D  ifferential White Blood Cell Count (Differential Leukocyte Count) Background The white blood cells are classified as either granular or agranular, depending on their cytoplasmic granules that can be visualized by staining. Granular leukocytes include neutrophils, eosinophils and basophils. The agranular cells include lymphocytes and monocytes. The numbers of WBCs are calculated by a differential count, which reports the percentage of presence of different types of WBCs i.e. neutrophils, lymphocytes, monocytes, eosinophils and basophils in blood. The differential count of blood helps in detecting abnormal or immature cells and can be used to diagnose an infection, inflammation, leukemia, or an immune system disorder. The blood smear is also helps in examination of the morphology of WBCs and identification of pathological cells in the blood (Table 5.1 and Fig. 5.5). Objective To determine the relative number of each type of white blood cell presents in the blood by performing differential cell counts on normal blood smears.

Table 5.1  Type of white blood cells (WBCs), and their clinical significance % of cells in normal Type of cells physiological condition Neutrophils 60–70

Eosinophils

1–5

Basophils

0–1

Lymphocytes 20–30 Monocytes

2–6

High count may indicate Bacterial infection, burns, stress, inflammation Allergic reactions, parasitic infections, autoimmune diseases Allergic reactions, leukemias, cancers, hypothyroidism Viral infections, some leukemias Viral or fungal infections, tuberculosis, some leukemias, other chronic diseases

Low count may indicate Radiation exposure, drug toxicity, vitamin B12 deficiency, systemic lupus erythematosus (SLE) Drug toxicity, stress

Pregnancy, ovulation, stress, hyperthyroidism Prolonged illness, immunosuppression Bone marrow suppression

5.2 Studies on Different Parameters of Blood

83

Fig. 5.5  Characteristic different blood cells

Principle The white blood cell differential count determines the number of each type of WBCs present in the blood. May-Grunwald-Giemsa staining method is used for differential counting of blood cells. The staining solution contains two dyes i.e. methylene blue (a basic dye) and eosin (an acid dye). The basic dyes carrying a net positive charge and have high affinity towards acidic components of the nucleus and cytoplasm and stain basophil granulocytes and RNA molecules of the cytoplasm of white blood cells. The eosin carrying a net negative charge stains the basic elements of the cell such as haemoglobin or granules of eosinophils. The dye also contains neutral components that stain other cell structure. The Giemsa stain contains azure II, eosin, methyl alcohol and glycerol. It can stain all cellular components. The nuclei of white blood cells and the granules of basophil granulocytes appear in blue because of blue colour of methylene blue, while red blood cells and eosinophil granules appeared red colour because of red colour of eosin. However, cytoplasm of white blood cells appear light blue because presence of RNA molecules in low concentration. Requirements 1. Haemocytometer 2. Microscope 3. Disposable-needle 4. Cover slip 5. May-Grunwald stain (containing acidic eosin, methylene blue, methyl alcohol and glycerol) 6. Giemsa stain (containing azure II, eosin, methyl alcohol and glycerol)

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Experimental Procedure 1. A drop of blood from the finger is placed in the central line of a slide about 1–2 cm from one end. 2. The spreader is placed at an angle of 45° to the slide and then moved back to make contact with the drop. 3. The size of the film should be 3–4 cm in length and film is dried rapidly. A good blood film preparation will be thick at the drop end and thin film at the opposite end. 4. Before staining, the blood smear is air dried slowly and fixed with methyl alcohol for 1 min in order to prevent hemolysis. 5. 20–30 drops of May-Grunwald stain are poured over the slide for about 3 min to the slide covering the surface. 6. After 3 min equal volume of distilled water is added to the stain and left for another 2 min. 7. All the fluid is removed from the glass and Giemsa stain diluted in equal volume of distilled water is added and left on the slide for 20 min. 8. After 20 min, the stain is washed off from the slide by a gentle rinsing with distilled water. 9. The back of the slide is wiped with paper towel to remove excess stain. 10. After drying, the slide is visualised in high power microscopic field. 11. The dry and stained slide without a cover slip is visualised under oil immersion objective (100×). 12. Differential count is carried out by moving the slide in area including the central and peripheral of the smear. 13. A total of 100 white blood cells are counted and recorded it in a table under the following heading: neutrophil, eosinophil, basophil, lymphocyte and monocyte. 14. Then the percentage of each type is calculated and compared it with normal count.

Result and Observation Type of cells Neutrophils

Eosinophils

Characteristic feature Nucleus Multilobed with varying shape and blue violet in colour Cytoplasm Pink, with irregularly distributed red/violet granules Nucleus Bilobed Cytoplasm Pink with regularly distributed yellowish red granules

% of cells

(continued)

5.2 Studies on Different Parameters of Blood Type of cells Basophils

Lymphocytes

Monocytes

Characteristic feature Nucleus Kidney shape Blue violet in colour Cytoplasm Pink with irregularly distributed blue granules Nucleus Big, chromatin-rich, purple/violet in colour Cytoplasm Very thin with blue in colour and without any granules Nucleus Lobular with purple/violet colour Cytoplasm Pale blue-greyish with no visible granules

85 % of cells

Interpretation The percentage distribution of white blood cells is found to be Neutrophils Eosinophils Basophils Lymphocytes Monocytes

5.2.2 Blood Grouping Background Blood grouping system in clinical practice is important because an antigen may in certain circumstances, react with its corresponding antibody and cause harmful clinical effects like haemolytic transfusion reactions and haemolytic disease of newborns referred to as blood incompatibility. The human ABO blood groups were first discovered by Austrian immunologist Karl Landsteiner in 1901. The ABO blood grouping system is the most clinically significant blood grouping system. The ABO blood group is determined by the presence of A and B antigens on the surface of the red blood cells, and of anti-A or anti-B antibodies in the serum. Thus, the red blood cells of blood type A possess antigen A and the serum containing anti-B antibody. Similarly, blood type B has antigen B and anti-A antibody. Blood type AB contains both A and B antigens but no antibodies. Blood type O has no antigens but contains both anti-A and anti-B antibodies. ABO blood grouping is crucial for safe blood transfusion. The determination of an ABO blood group is defined by demonstrating the presence or absence of antigens A and/or B on the surface of human red blood cells and by detecting the presence or absence of anti-A and/or anti-B antibodies in the plasma. According to the ABO blood group system there are four different kinds of blood groups: A, B, AB and O (Table 5.2).

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Table 5.2  Types of blood groups ABO blood group A B AB O

Antigen present on the red blood cells A B A and B Neither A or B

Antibodies regularly present in the serum/ plasma Anti-B Anti-A None Anti-A and anti-B

The Rh blood grouping system was identified by Landsteiner and Wiener who reported that human RBCs were agglutinated by an antibody, common to all rhesus monkeys. This factor was named the Rh factor. Rhesus system (Rh) is the second most important blood group system after ABO blood group system. The Rh factor is an inherited blood protein or antigen present on red blood cells. In Rh system, blood groups are Rh-positive or Rh-negative on the basis of presence or absence of Rh-antigens on the red cell surface. The presence Rh antigen is determined by testing the RBCs with anti-Rh or anti-D. About 85% of the white population and 94% of the black population are reported positive for the Rh antigen. Objective To determine the blood group of the samples. Principle The blood grouping system is based on agglutination reaction and pattern recognition. The ABO antigens are primarily glycoproteins found on the surface of human RBCs. Individuals who lack the A and/or B antigens on their red blood cells generally have naturally occurring antibodies in their plasma that are directed against the missing antigens. Upon mixing red blood cells with antisera, antigens are mixed with corresponding antibodies causing agglutination reaction. The presence of the specific antigen can be determined by the agglutination reaction with a particular antibody. Whereas, absence of agglutination reaction indicates the red blood cells are negative for that particular antigen. The antigen-antibody reaction results in visible agglutination of the red blood cells determining the blood groups A and B and AB.  No agglutination with anti-A, anti-B, determines the blood group as ‘O’. Requirements 1. 2. 3. 4. 5. 6.

Microscope Needle Alcohol Spirit Lamp Cotton Micropipette

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. Antiserum (A, B and D) 7 8. Glass slide Experimental Procedure . The sterilized disposable needle is wiped off with the rectified spirit. 1 2. The finger is also sterilized with rectified spirit by using cotton. Then the finger tip is pricked with the needle. 3. The blood is allowed to ooze out and the blood is sucked by the micropipette. 4. Then three drops of blood are put on a cleaned glass slide and treated with anti-A, antiB and anti-D respectively. 5. The blood is mixed thoroughly with respective antiserum. The slide is left undisturbed for 2 min to allow the reaction to take place. 6. The slide is checked for agglutination reactions and the blood group is determined. Results and Observation Anti-A − − + + − − + +

Anti-B − − − − + + + +

“+”, agglutination; “−”, agglutination

Interpretation (Fig. 5.6) So the blood group is ………..

Fig. 5.6  Analysis of ABO blood group

Anti-D + − + − + − + −

Blood group O +ve O −ve A +ve A −ve B +ve B −ve AB +ve AB −ve

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5.2.3 Total Red Blood Cell Count Background The blood is a type of liquid connective tissue and composed of different cells, metabolic products and cell fragments. The percentage of total blood volume occupied by formed elements, mostly RBCs is called the hematocrit, with a normal range of 38–46% or 40–54% in case of healthy adult females or males respectively. RBCs are non-nucleated cells with a diameter of 7–8 μm with and in doughnut shape. They contain large amount of hemoglobin molecules, which play an important role in the transport of respiratory gases. The average life span of RBCs is about 120  days. Under normal physiological condition the number of RBCs is found to be 4.2 to 5.4 × 106/mm3 of blood for male and for female it is 3.6 to 5.0 × 106/mm3. The deviation from normal range of RBCs reflects a disease condition of the body. Increased in numbers of RBCs is due to congenital heart disease, dehydration, pulmonary fibrosis polycythemia. Decreased in numbers of RBCs is due to anemia, bone marrow failure, erythropoietin deficiency, hemolysis, hemorrhage, leukemia, multiple myeloma, nutritional deficiencies of (Iron, Copper, Folate, Vit. B6, B12). Objective To determine the number of red blood corpuscles (RBCs) of blood sample. Principle RBCs or erythrocytes are circular, biconcave and non-nucleated cells of blood. The mercuric chloride present in Hayem’s solution is a corrosive sublimate and fixes the RBCs of blood. The other ingredient of Hayem’s solution which are served to dilute the blood are isotonic in natureso that RBCs do not burst. Requirements 1. EDTA or heparin 2. Haemocytometer (Neubauer’s counting chamber) 3. Microscope 4. Disposable needle 5. Cotton 6. Rectified spirit 7. RBC pipette 8. Alcohol 70% 9. Lancet. 10. Hayem’s solution (It is prepared by mixing 0.5 g mercuric chloride (HgCl2), 1.0 g sodium chloride (NaCl) and 1.5 g anhydrous sodium sulphate (Na2SO4) in 100 ml distilled water.)

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Experimental Procedure 1. All the components of Haemocytometer are cleaned properly and air dried. 2. One of the fingers is cleaned properly with 70% alcohol and then pricked. 3. The blood is then sucked immediately with the help of RBC pipette up to 0.5 mark. 4. The blood stacking to outer surface of the pipette is wiped out and then Hayem’s solution is sucked up to the 1.0 mark immediately. 5. The pipette is held horizontally and rotated several times so that the blood thoroughly mixes with Hayem’s fluid. 6. The counting chamber is fixed under microscope and the cover slip is placed on it. 7. The tip of the RBC pipette is placed between the cover slip and platform and a few drops of blood is allowed to flow between the cover slip and the counting chamber. 8. The slide is kept as it is for 15 min to settle down RBC on the counting chambers. 9. The number of RBC is counted in the central chamber and calculated. 10. The 40× objective is used, all cells lying on the upper and left lines of any square are included while cells on the lower and right-hand lines are omitted. 11. The cells are counted in five groups of 16 small squares i.e. 80 small squares.

Result and Observation

The total number of red cells / mm 3 = N × 10, 000

Where N is the number of red cells found in 80 squares.

Interpretation The total number of RBC found in the blood sample is ………….. mm3.

5.2.4 Estimation of Haemoglobin (Hb) Background Haemoglobin (Hb) is the respiratory pigment which carries oxygen and gives the blood its characteristic red colour. It is a conjugated protein containing a protein part globulin and a prosthetic group called as haeme. The amount of Hb in red blood cells ordinarily remains constant and is found to be 14–16.5 g/dL of blood in males and 12–15 g/dL of blood in females. The decrease in Hb concentration is called “anaemia”, whereas increase in Hb concentration is called “polycythemia”. The Hb concentration of the blood can be measured spectrophotometrically by either cyanmethaemoglobin method or oxyhaemoglobin (HbO2) method. Objective To calculate the % of haemoglobin in human blood by Sahli’s haemometer.

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Principle Sahli’s method involves conversion of haemoglobin to acid haematin using HCl which is brown in colour. The intensity of the brown colour developed is depended on the concentration of Hb present in the blood sample. This is then diluted to get the colour intensity which matches the standard coloured disc provided in the haemometer. The tube in which the dilution is carried out is calibrated to give % of Hb and Hb in gram per 100 ml blood. Requirements Disposable needle Cotton Rectified spirit Decinormal (N/10) HCl 0% alcohol Distilled water Stirrer Haemometer Haemometers are used to determine the content of haemoglobin in blood. The haemometer consists of two sealed lateral comparison tubes containing acid haematin suspension. A graduated test tube is provided which can fit in the haemometer in between the two side tubes for comparison. A micropipette of 20 μl is provided. The other things provided are a small glass rod stirrer, a small bottle brush, a dropper and a small bottle to contain the decinormal acid solution (Fig. 5.7).

Fig. 5.7  Sahli’s haemometer

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Experimental Procedure 1. The graduated tube is cleaned and dried and filled with N/10 HCl up to 20 mark by means of the dropper. 2. The fingertip is cleaned and pricked lightly with disposable needle. 3. The blood is sucked up to the 20 μl mark of the micropipette and mixed with N/10 HCl solution in graduated tube. 4. The acid haematin solution is taken in a test tube and stirred with the help of glass rod and allowed to stand for a few minute. The acid haematin solution is gradually diluted with N/10 HCl in a drop wise manner. 5. The acid haematin solution is stirred thoroughly after each addition of HCl and its colour is matched with the sealed tubes.

Result and Observation The readings are taken when the colour of the acid haematin solution is matched with that of standard colour. Interpretation The Hb concentration in % is………….. Precautions . The fingertip should be cleaned properly and pricked slightly. 1 2. Experiment should be performed quickly without waste of time so that fresh blood is not allowed to coagulate before transfer to 0.1 N HCl.

5.2.5 Bleeding Time and Clotting Time Background The bleeding time (BT) is a widely used and popular test to explore the haemostasis of the body. Since its initial invention by the French worker Milian in 1901, bleeding time has been considered as a clinically useful test in diagnosis of platelet disorders and assessment of the adequacy of various forms of therapy. Whole blood clotting time (CT) has been used in past to assess both the intrinsic and extrinsic pathways of coagulation. Disorders of coagulation can lead to an increased risk of bleeding (hemorrhage) or clotting (thrombosis). Both BT and CT are used as routine preoperative test in many hospitals. The normal range for bleeding time for human being irrespective of gender is about 1–9 min where as the CT is 5–10 min. Bleeding time is prolonged in purpura which is due to platelet defects. The CT is prolonged in different conditions like vitamin K deficiency, liver diseases, disseminated intravascular coagulation, over dosage of anticoagulants etc.

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Objective To determine the Bleeding time (BT) and Clotting Time (CT). Principle Bleeding time test evaluates how quickly blood clot to stop bleeding. Platelets are tiny cell fragments that circulate in blood. They are the first type of cells that react to any injury in blood vessel. Platelets sealed off the wound and thereby preventing any further blood loss. It is determined by noting time at which blood coming out a small cut and no longer forms a spot on a piece of filter paper when placed in contact with cut surface. The clotting of blood depends upon both extrinsic and intrinsic pathways that work together to form a clot. Bleeding time depends on the integrity of platelets with vessel walls. Whereas clotting time depends on the availability of coagulation factors. Requirements Sterile lancet Cotton Rectified spirit Filter paper Stop watch Capillary glass tubes Experimental Procedure Determination of Bleeding Time by Duke’s Method . The finger tip is sterilized by using rectified spirit and allowed to air dry. 1 2. A deep prick is made by using a sterile lancet so that blood can easily comes out without squeezing. 3. The time when bleeding starts is recorded with the starting the stop-watch. 4. The blood is mopped up by touching the finger tip to the filter paper. 5. This step is repeated at an interval of 15 s by using a fresh portion of the filter paper, till bleeding stops. 6. The time is recorded by stopping the stop-watch (Fig. 5.8). Result and Observation The blood stains on the filter paper are getting smaller and will finally disappear when bleeding stops. The bleeding time is calculated as the interval between the start and end of bleeding. Interpretation The bleeding time (BT) is reported to be... The clotting time (CT) reported to be...

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Fig. 5.8  Determination of bleeding time

Fig. 5.9  Determination of clotting time by observing fibrin formation

Determination of Clotting Time by Capillary Tube Method . The finger tip is sterilized using rectified spirit and allowed to dry. 1 2. A deep prick is made using a sterile lancet, so that blood can easily comes out. 3. When bleeding starts, the time is noted down by starting the stop watch. 4. The blood drop from the finger tip is touched by using one end of the capillary tube kept tilted downwards so that capillary tube gets easily filled up by capillary action. 5. Then, after an interval of 2 min small lengths of the tube are snapped off. 6. Thereafter at an interval of 15 s, small lengths of capillary tube is snapped off till fibrin thread is formed between the snapped ends. 7. When the fibrin thread is first seen, stop watch is stopped and the time is noted down (Fig. 5.9).

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Result and Observation The clotting time is calculated when the fibrin formation is seen first. Clotting time is calculated as the interval between stat of bleeding and formation of first fibrin thread. Interpretation The bleeding time (BT) is reported to be... The clotting time (CT) reported to be...

5.3

Pyrogen Testing

Background Pyrogen is defined as an agent that causes rise in temperature in an animal body. Pyrogenic substance include endotoxin and other bacterial by products which are polysaccharide or proteinaceous in nature. The pharmaceutical products, medical devices, biotherapeutics and cosmetics can induce life threatening fever, if they contain pyrogens. Therefore, it is mandatory for the manufacturing industry to ensure that the pyrogen concentrations do not exceed the specified limits for each product. A number of in vivo and in vitro methods are employed to test the pyrogen content in any pharmaceutical product. The in vivo study mostly involves rabbit pyrogen testing method that surfaced in the 1940s. The in vitro method of pyrogen testing is popularly done by Limulus Amoebocyte Lysate, otherwise known as ‘LAL’ test. Objective To study the pyrogen test of the given sample.

5.3.1 Pyrogen Testing by Animal Model (In Vivo Method) Principle The test involves measuring the rise in temperature of rabbits following the intravenous injection of a test solution and is designed or products that can be tolerated by the test rabbit in a dose not to exceed 10 ml/kg injected intravenously within a period of not more than 10 min. Requirements • Syringes, needles (Disposable) • Glasswares (freed from pyrogens by autoclaving them) • Pyrogen free saline water

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• Test solution: Dissolve test substances in Pyrogen free saline water and warm the liquid to 38 °C before giving injection. • Wooden restrainer to allow normal sitting position of the rabbit • Clinical Thermometer graduated in 0.1 °C/Thermister/Probes • Rabbits (Healthy matured, weighing less than 1.5 kg and maintained in balanced diet and not showing loss of body weight during the week preceding the test) Experimental Procedure It includes (i) Preliminary test (Sham Test) and (ii) Main test. Preliminary Test (Sham Test) . Take fresh rabbits that have not been used during two previous weeks. 1 2. Conditioned them for 1–3 days. 3. After an overnight fast, the animals are examined by injecting 10 ml/kg, i.v. of body weight of a pyrogen free saline solution. 4. Measure the initial temperature of animals at the beginning of the test using thermometer by inserting the thermometer into the rectum of rabbit to a depth of not less than 6 cm. 5. Then Record the temperature of animals after IV injection at an interval of 30 min and continued for 3 h after injection. 6. Any rabbit showing a temperature variance of 0.6 °C or more should not be used for main test. Main Test Animal: Main test is done by taking three groups of rabbits having three rabbits each group. . Select three groups of rabbits (preliminary test passed rabbits). 1 2. Take the overnight fast rabbits and record the temperature of animals using thermometer as done before. Rabbits showing a temperature variance ≥0.2  °C between two successive readings should not be used. 3. After 90 min, give IV injection 10 mL/Kg (Pyrogen free saline solution) inject sample into the marginal vein of the ear of three rabbits not less than 0.5 ml/kg and not greater than 10 ml/kg body wt. 4. Measure the temperature of animals during a period of 3 h at intervals of 30 min. Result and Interpretation 1. If the sum of the group of three rabbits does not exceed 1.4 °C or no individual rabbit shows rise in temperature of 0.6 °C, then the test solution being examined passes the test.

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2. If sum of three rabbits raise in temperature exceeds 1.4 °C or if two or three rabbits show increase in temperature ≥ 0.6 °C, then the test solution being examined does not pass the test. Precaution The study should be carried out in room without disturbances and temperature variance must be ±3 °C.)

5.3.2 Pyrogen Testing by LAL Method (In Vitro Method) Principle The aqueous extract of amoebocytes (blood cells) of the horseshoe crab, Limulus polyphemus is known as LAL (limulus amoebocyte lysate). This test is based on the process of occurrence of coagulation in the hemolymph of horseshoe crab in the presence of bacterial endotoxin or lipopolysaccharides (LPS). The membrane component of Gram negative bacteria forms a gel in presence of LAL which can be used for the detection and quantification of bacterial endotoxins in the sample. The hemolymph of the Limulus polyphemus in presence of LPS, undergoes a number of separate activation processes from pro-enzymes to proteolytic enzymes leading to formation of activated factor C. Factor C further activates the factor B. The factor B turns the gel-forming pro-enzyme into an activated enzyme which forms an insoluble gel. Requirements • Endotoxin reference standard (ERS): It is the freeze dried purified endotoxin of Escherichia coli, which is calibrated in Endotoxin units (EU) by comparison with the International standard. • Control Standard Endotoxin (CSE): CSE is suitably standardized against the ERS which is used for routine bacterial endotoxin testing. • Water BET: Water that gives a negative result under the conditions prescribed in the test for bacterial endotoxins on the preparation under examination. • 0.1  M HCl BET: Prepared from hydrochloric acid using BET with pH adjusted to 6.0–8.0 with 0.1 M sodium hydroxide BET. It also gives a negative result under the conditions of the test. • 0.1 M NaOH BET: Prepared from sodium hydroxide using water BET with pH adjusted to 6.0–8.0 with 0.1 M hydrochloric acid BET. It gives a negative result under conditions of the test. • Lysate: Lysate is the reconstituted lysate of amoebocytes of either of the species of horseshoe crabs; Limulus polyphemus • Water bath or hot block • Pyrogen free Tube

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Experimental Procedure 1. Prepare a series of test solutions with water BET. Adjust the pH to 6.0–8.0 using sterile 0.1 M HCl BET and 0.1 M NaOH BET. 2. Water BET is used as negative control and two positive controls are to be used. 3. One positive control is CSE at 2 λ concentration. 4. Second positive control is test solution spiked with CSE to give 2 λ concentrations (PPC) Result and Observation . Take the pyrogen free reaction tube for each sample in duplicate. 1 2. Take 0.1 ml of lysate and 0.1 ml of positive control sample and 0.1 ml of taking negative control. 3. Repeat the procedure by taking test sample to be tested for pyrogen. 4. Incubate the tubes in water bath or hot block at 37 °C for 1 h. 5. After 1 h invert the tube through 180° and observe for any gelation. Interpretation The given sample complies with endotoxin test if the positive product control gives positive result and the negative as well as the test solution gives negative result. The test is not valid if the product positive control is negative or the negative control is positive or both of these conditions occur.

5.4

Effect of Drug on Central Nervous System

5.4.1 Hypnotic Drugs Background Hypnotic is a drug that induces and/or maintains sleep, similar to normal arousable sleep. The hypnotics are more or less global CNS depressants with quicker onset, shorter duration and steeper dose-response curves. Hypnotics are used to treat insomnia. Hypnotics are classified into two categories as Barbiturates and Non-barbiturates. Objective To evaluate the hypnotic activity of the drug. Principle Hypnotics depress CNS in a relatively non-selective, dose dependent manner producing progressive calming or drowsiness. Upon administration hypnotic drugs induce sleep resembling natural non-rapid eye ball movement (NREM) sleep. The righting reflex is

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considered as one of the ideal method to assess hypnotic activity of drugs in mice. When mice placed on their back or dropped from height, they could restore their normal position, which is known as righting reflex. However, under the influence of CNS depressants like hypnotics, animals fail to show righting reflex activity. Requirements Mice (20–25 g) Pentobarbitone sodium (40 mg/kg) Diazepam (1 mg/kg) Saline (0.9% NaCl) Syringe, Needle Stop watch Experimental Procedure . The mice are weighed and made three groups of three mice each. 1 2. The mice are injected with the following drugs dissolved in water as follows. Group-1: Mice injected with 0.1 ml of saline i.p. Group-2: Mice injected with 0.1 ml of Pentobarbitone sodium (40 mg/kg b.w.) i.p. Group-3: Mice injected with 0.1 ml of Diazepam (1 mg/kg) i.p. 3. The hypnotic activity of the drugs is assessed in terms of loss of righting reflex in mice. 4. The observations are recorded for each mice 15, 30 and 45  min after drug administration. 5. The time of onset of righting reflex in mice is recorded. 6. Next, the time of recovery from sleep is also recorded.

Result and Observation Observation Table Group no. Drug Saline 1 2 3

Animal Time of onset (mean) Time of recovery (mean) 1 2 3 Pentobarbitone sodium 1 2 3 Diazepam 1 2 3

Interpretation From the observation table, it is observed that the mice from Group-1 when placed on their back or dropped from height restore their normal position. On the contrary the mice from

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both Group-2 and Group-3 when placed on their back or dropped from height fails to regain normal posture i.e. they fail to show righting reflex. The activity is correlated with CNS depressant effect of Pentobarbitone sodium and Diazepam.

5.4.2 CNS Depressant Activity Background A drug decreasing CNS activity will produce decrease in spontaneous motor activity in the animals. The CNS depressant activity is assessed using Photoactometer. Objective To study the CNS depressant property of the drug by studying the locomotor activity of mice. Principle The locomotors activity can be an index of wakefulness of mental activity. The locomotors activity is measured by using Photoactometer which consists of a cage of size 30×30 cm. It has a wire mesh at the bottom and has six lights and six photocells. It is arranged in such a way that a single mice can block only one beam, so that when the rays of light falling on photocells are cut off by animals crossing the beam of light, the photocells connected to an electronic automatic counting device will count the number of cut offs (Fig. 5.10).

Fig. 5.10 Photoactometer

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Requirements Mice (20–25 g) Drug (Chlorpromazine) Photoactometer Experimental Procedure . The animals are weighed and numbered. 1 2. The photoactometer is switched on and each mouse is placed individually in the activity cage for 10 min. 3. The basal activity score of all animal is noted. 4. Then each mouse is injected with Chlorpromazine at a dose of 3 mg/kg i.p. 5. After 30 min. of injection, the activity is again checked. Result and Observation The percent of decrease in motor activity is calculated. Interpretation Reduction in the motor activity indicates CNS depressant property of the drug.

5.4.3 Analgesic Drugs Background A drug that selectively relieves pain by acting in the CNS or on peripheral pain mechanisms, without significantly altering consciousness is known as analgesic drug. The analgesic activity of a drug can be evaluated by various methods such as tail-flick method, eddy’s hot plate method and acetic acid induced writhing. They are all based upon the change which occurs in the response of the experimental subject to a painful stimulus after dosage with an active compound. The analgesics are broadly classified into Narcotic type and Non-­narcotic type.

5.4.3.1 Screening of Analgesics by Tail-Flick Method Objective To evaluate the analgesic effect of Drug by Tail-flick method. Principle The method is based upon the principle of thermal radiation of heat. The animal is exposed to the noxious stimuli like heat and the time taken to produce response is recorded. The experiment is carried out on an analgesiometer that consist of an electrically heated nichrome wire. The heated nichrome wire acts as noxious stimulus and the time taken to

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produce the effect is noted down. The tail flickering response i.e. withdrawal of tail from heat source is taken as the end point. Requirements Analgesiometer Drug (Paracetamol) Normal saline (0.9% NaCl) Rat (150–200 g) Experimental Procedure . The rats are weighed and divided into two groups (Group-1 and 2). 1 2. The rat is kept in the animal holder and its tail is allowed to rest on a wire which is electrically heated up to 55 °C. 3. The tail flickering response i.e. time taken to withdraw the tail from heat source is recorded as the reaction time. The usual response time is 3–5 s. 4. Now the two group of rats are injected with drugs as follows: Group-1: Rat injected with Normal saline. Group-2: Rat injected with Paracetamol (100 mg/kg bw) 5. Then the procedure is again repeated after administration of drug. 6. The reaction time at 15, 30, 60, 90 and 120  min intervals of drug administration is recorded.

Result and Observation Observation Table Group no. 1 2

Drug Saline Paracetamol

React time (in seconds)

Reaction time in seconds after drug administration (in seconds) 15 min 30 min 60 min 90 min 120 min

Interpretation Increase in the time interval of tail flickering response is taken as analgesic activity.

5.4.3.2 Screening of Analgesics by Eddy’s Hot Plate Method Objective To evaluate the analgesic effect of Drug by hot plate method.

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Principle This method was first described by Eddy and Leimbach in 1953. The method is based upon the principle of thermal radiation of heat. In this method Eddy Hot plate is used which is kept at 55 °C. The animal is placed on hot plate and time taken for jumping from plate is noted before and after administration of drug. Requirements Drug (Morphine sulphate) Normal saline (0.9% NaCl) Mice (20–25 g) Eddy’s hot plate Experimental Procedure . The mice are weighed and divided into two groups (Group-1 and 2). 1 2. The mice are allowed to sit on Eddy’s hot plate maintained at 55 °C. 3. The basal reaction time for hind paw licking or jump response in animals are recorded. 4. The usual response time is 6–8 s. A cut off period of 15 s is observed to avoid damage to paws of the mice. 5. Now the two group of rats are injected with drugs as follows: Group-1: Mice injected with Normal saline. Group-2: Mice injected with Morphine sulphate (5 mg/kg bw, s.c.) 6. Then the procedure is again repeated after administration of drug. 7. The reaction time at 15, 30, 60, 90 and 120  min intervals of drug administration is recorded. A cut off period of 15 s is observed to avoid damage to paws of the mice. Result and Observation Observation Table Gr. no. Drug Saline 1 2

React time (in seconds) Paw Jump licking response

Reaction time in seconds after drug administration (in seconds) 15 min 30 min 60 min 120 min Paw Jump Paw Jump Paw Jump Paw Jump licking response licking response licking response licking response

Morphine sulphate

Interpretation Increase in the time interval of paw licking or Jump response is taken as analgesic activity.

5.4.3.3 Screening of Analgesics by Writhing Test Objective To evaluate the analgesic effect of Drug by acetic-acid induced writhing method.

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Principle Writhing is a painful reaction which can be induced by acetic acid (chemical stimulus) and characterized by clean observable signs such as constriction of abdomen, twining of trunk and extension of hind limbs. Both narcotic and non-narcotic analgesics produce a positive response in writhing method. Requirements Acetic acid (3%) Drug (Morphine, Aspirin) Normal saline (0.9% NaCl) Mice (20–25 g) Experimental Procedure 1. The mice are weighed and divided into three groups (Group-1, 2 and 3 for normal saline, morphine and Aspirin respectively). 2. Acetic acid is administered in the dose of 30 mg/kg to the Group-1 mice injected with normal saline and placed under bell jars for observations for number of wriths over a period of 10 min. 3. The Group-2 and 3 mice are injected with morphine (5 mg/kg) and Aspirin (200 mg/ kg) and after 15 min, acetic acid is administered. 4. The onset and number of wriths are observed for a period of 10 min. Result and Observation Observation Table Group no. 1 2 3

Drug Number of wriths Saline Morphine Aspirin

Interpretation Reduction in number of wriths is taken as analgesic activity.

5.5

Experiment on Cardiovascular System

5.5.1 Effect of Cardiac Stimulants and Depressants Background The main purpose of heart is to pump the blood around the body so that oxygen and nutrients can be distributed to different parts of the body. The heart rate is controlled by the

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autonomic nervous system. The cardiac medications either increase or decrease the force of myocardial contraction. A cardiac stimulant is a substance which acts as a stimulant of the heart via positive chronotropic or inotropic action. Cardiac depressants are little used in medicine, however, some are used to slow the heartbeat in tachycardias and a number of these are often analogues or derivatives of other drugs with optimized activity for this purpose in the heart. Objective To study the effect of cardiac stimulants and depressants on isolated perfused frog’s heart. Principle The heart rate, amplitude ad tones are considered as markers of good health of heart. The heart rate is determined by calculating the contractions per minute. The tachycardia and bradycardia are conditions associated with increased and decreased heart rate. The height of the curve or amplitude is measure of the forceful contraction. Similarly, tone corresponds to partial contraction of muscle at resting condition and is denoted by the base line of curve. Requirements Frog Sherington’s recording drum Kymograph paper Smoking device Starling’s heart lever Ringer’s solution (without calcium chloride) Drugs Adrenalin (0.01%) Digoxin (0.05%) Calcium chloride (0.5%) Propanolol (0.2%) Experimental Procedure 1. The frog is pithed and is dissected followed by careful removal of the heart, 2. After removing the heart, it is immediately transferred to the organ bath containing Ringer’s solution without calcium chloride and perfused through the sinus venosus. 3. A curved needle is inserted in the apex of the heart and then it is attached to a Starling’s heart lever. 4. The contractions are recorded on a kymograph drum moving at a speed so that about 3–4 contractions are recorded per cm.

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5. The normal heart rate is recorded by counting each upstroke (systole) and downstroke (diastole) of the moving drum together as one beat for 1 min. 6. The force of contraction is measured by recording the amplitude or height of the contraction from the baseline with a scale. 7. The tone is measured by observing shift in the baseline and the cardiac rhythm by observing any irregularity in the contractions pattern. 8. About 0.5 ml of the drugs is injected in 1–4 in succession in the tube through which the heart is being perfused and responses are recorded. 9. A control reading (without addition of any drug) is taken before and after each drug response. 10. All the parameters mentioned above should be recorded during the control and drug responses respectively. 11. The heart rate, drug name and the dose is mentioned in the recording during the control and drug responses. 12. The next drug response is recorded only after the heart rate has returned to the approximate original value. 13. All the observations are tabulated.

Result and Observation Observation Table Drugs Adrenalin (0.01%) Digoxin (0.05%) Calcium chloride (0.5%) Propanolol (0.2%)

Heart rate

Amplitude

Tone

Interpretation The cardiac stimulant or depressant effect of drugs on isolated frog’s heart is as follows: Drugs Stimulant/depressant/no effect Adrenalin (0.01%) Digoxin (0.05%) Calcium chloride (0.5%) Propanolol (0.2%)

Precautions 1. When heart stops because of systolic or diastolic arrest, the drum should be stopped and re-started only when the heart is contracting. 2. In case adequate response is not observed use a higher dose.

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Experiments on GI Tract

5.6.1 Muscle Relaxants and Spasmogens Background Many drugs act on the intestine. Adrenergic and cholinergic drugs produce opposite effects on it. These drugs act through their respective receptors. Some drugs act directly on the intestine. This experiment demonstrates the effects of various drugs on the rabbit intestine. Objective To study the effects of spasmogens and relaxants on rabbit’s intestine. Principle Spasmodics are the agents that initiates spasm i.e. increases contractions of smooth muscles. Clinically spasmodic play very important role in treatment of disorders associated with atony of bladder, atony of intestine, post-operative paralytic ileus. However, relaxants are the pharmacological agents that decrease the tone of smooth muscles and induce relaxation. They play valuable role as preanaesthetic agents. Requirements Acetyl choline Barium chloride Atropine sulphate Adrenaline Papavarine Tyrode solution Experimental Procedure . A rabbit is sacrificed and the abdomen is dissected to expose the intestines. 1 2. A part of ileum is taken 10 cm away from ileocaecal valve. 3. An optimal length of tissue (5–6 cm) is cut and a thread is tied to antimesenteric border on both sides. The tissue is placed in a dish containing Tyrode’s solution. 4. Then one end is tied to a fixed point inside organ bath and other point is attached to the lever for recording contractions on the smoked paper pasted over the drum of a kymograph. Lever should be horizontal. 5. The drum is started and the normal pendular movements are recorded for 2 min after wards the drum is stopped. 6. About 10 μg of acetyl choline is added to bath and the effect is recorded for 1 min. Then the drum is stopped.

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7. Then the reparation is washed till activity of intestine is restored to normal. The drum is again started to record the normal pendular movement. 8. In the similar fashion, other drugs such as barium chloride (2 mg), atropine sulphate (5 μg), adrenaline (10 μg) and Papavarine (2 mg) are added to organ bath and the effect is observed for 1 min. Result and Observation Observation Table Drug Force of contraction Acetyl choline Barium chloride Atropine sulphate Adrenaline Papavarine

Interpretation The effect of various drugs on the intestine is as follows: (The acetyl choline, barium chloride and atropine sulphate are spasmogens. Adrenalin and papaverine are relaxants) Precaution 1. Sufficient time should be given for the intestine to recover between two consecutive drug administrations. 2. The readings should be taken before and after giving drugs. 3. The frequency of contraction should be recorded when there is maximum effect of drug. Questions 1. What is righting reflex? 2. How righting reflex study is useful? 3. What is blood DC? 4. What is Bombay blood group? 5. What is the principle behind blood grouping? 6. What is pyrogen? 7. What is LAL? 8. What is the principle of LAL test? 9. What is the percentage of Hb in men and women under normal physiological condition? 10. What are the different methods of pyrogen testing? 11. Give examples of hypnotic drubs?

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2. What is spasmogens? 1 13. What are tachycardia and bradycardia? 14. What are cardiac Stimulants & Depressants? 15. How analgesic activity of any drug is tested?

References Badyal D (2008) Practical manual of pharmacology. Jaypee Brothers Medical Publishers, New Delhi BM Swamy V, Jayaveera KN, Reddy V (2014) Experimental pharmacology and toxicology. S. Chand & Company, New Delhi Goyal RK (2017) Practical in pharmacology. B. S. Shah Prakashan, Ahmedabad Kale SR, Kale RR (2017) Practical pharmacology and toxicology. Nirali Prakashan, Mumbai Katzung BG, Masters SB, Trevor AJ (2012) Basic & clinical pharmacology. McGraw-Hill, New York Medhi B (2017) Practical manual of experimental and clinical pharmacology. Jaypee Brothers Medical Publishers, New Delhi Ritter JM, Lewis LD, Mant TGK, Ferro A (2008) A text book of clinical pharmacology and therapeutics. Hodder Arnold, London Salmon DM (2014) Practical pharmacology for the pharmaceutical sciences. Wiley, Chichester Sierra LM, Gaivao I (eds) (2014) Genotoxicity and DNA repair: a practical approach. Springer protocols. Humana Press, London Singhal KC (1997) Pharmacology laboratory manual. CBS Publishers & Distributor, New Delhi Thatoi HN, Dash S, Das SK (2017) Practical biotechnology, principle and protocols. I.K. International, New Delhi Tripathi KD (2013) Essentials of medical pharmacology. Jaypee Brothers Medical Publishers, New Delhi Turner RA, Hebborn P (eds) (1971) Screening methods in pharmacology. Academic, New York Vogel HG (ed) (2002) Drug discovery and evaluation pharmacological assays. Springer, Berlin Woolley A (2008) A guide to practical toxicology. Informa Healthcare USA, New York

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Clinical Trials

6.1

Clinical Pharmacology and Its Genesis

Pharmacology is the science of drugs and it deals with interaction of chemical molecules or any single chemical substance exogenously administered to living systems which can produce a biological response. In a broader sense, the field of pharmacology is classified into two types such as (i) non-clinical and (ii) clinical pharmacology. The non-clinical pharmacology mostly dealt with animal models where as the clinical pharmacology study involves systematic evaluation of drugs in human. Clinical pharmacology is a multidisciplinary science that includes professionals of different field such as medicine, pharmacology, pharmacy, biomedical science and nursing that evaluate the different aspects of interaction between drugs on human. This further includes discovery of new drugs, application of drugs as therapeutic agents, beneficial and harmful effects of drugs on individuals and society, and the deliberate misuse of drugs. Clinical pharmacology is considered as rather a young discipline since it is originated around middle of the twentieth century and its objective is to optimise drug therapy. It is difficult to find who first coined the term ‘Clinical Pharmacology’ as opinions differ between countries. However, Louis Losgagna, a professor of medicine at Johns Hopkins University in America is commonly known as father of Clinical pharmacology. Different activities such as clinical pharmacology services, training and research are included under clinical pharmacology study. Clinical pharmacology provides a scientific basis for a rational, safe and effective use of drug along with evaluation of new medicines for their safety and efficacy. The clinical pharmacology plays its role in several dimensions such as (i) critical evaluation of new and old therapies, (ii) drug utilisation studies and pharmacoepidemiological services, (iii) pharmacogenetics, (iv) rational introduction and use of new drug into health care system, (v) therapeutic drug monitoring, clinical drug toxicology evaluation, (vi) pharmacovigilance etc. The different aspects of clinical

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­ harmacology study have been summarized in Fig. 6.1. Clinical pharmacology has been p integral to experimental medicine, particularly using drugs to probe mechanisms of physiology and disease. Pharmacodynamics Pharmacodynamics can be defined as what drugs do to the body that involves receptor binding, chemical interactions and post receptor effects. It refers to the relationship between drug concentration at the site of action and the resulting therapeutic effect. It also involves study of time course and therapeutic efficiency of the drug and intensity of the adverse effects. Maximum effect (Emax) of a drug can be evaluated by plotting the logarithm of concentration against its pharmacological action (effect) (Fig. 6.2). EC50 or 50% effective concentration values of a drug is considered as the basis for determination of potency of drugs. Therefore, lower EC50 value, more potent is the drug. Normally, body functions through different chemotransmitters, hormones, receptors, enzymes, different carrier molecules, DNA etc. Drugs act by altering the control systems of body by selectively binding to some specialised constituents of the cell and thereby altering their function and contributed its physiological activity. The detailed mechanism of action by which drugs produce their effect is studied in pharmacodynamics study. The dose response relationship i.e. the relationship between drug dose and their pharmacological effect is also evaluated using pharmacodynamics. Further, this study also provides a scientific rationale of using drug to counteract specific pathophysiological indications against particular diseases (Fig. 6.3).

Pharmacology

Therapeutic evaluation Clinical Pharmacology

Control

Fig. 6.1  Different aspects of clinical pharmacology

*Pharmacodynamics (Interaction between drugs and body) *Pharmacokinetics (Absorption, distribution, metabolism, excretion of drugs) *Evaluation of drug *Therapeutic trials *Pharmacoepidemiology *Pharmacovigilance *Rational prescribing *Official regulations of medicines *Use and misuse of medicines *Pharmacoeconomics

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Fig. 6.2  Drug concentration at the receptor site vs therapeutic effect (as percentage of maximal effect). (Source: https://www.ashp.org/-/media/store%20files/p2418-sample-chapter-1.pdf)

Fig. 6.3  Mechanism of drug action

Pharmacokinetics Pharmacokinetics is the field of science that includes the study of drug absorption, distribution, metabolism, and excretion. Pharmacokinetics study also referred as ‘what the body does to the drug’. The clinical pharmacokinetics study applies the principles of pharmacokinetic in safe and efficient management of drugs for individual patient. The

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objective of the clinical pharmacokinetics is enhancing efficacy and decreasing toxicity of a drug. The pharmacokinetics of a drug determines the onset, duration, and intensity its therapeutic effect. However, several factors influence the pharmacokinetics of a drug, viz patient-­related factors and chemical properties of drug. The patient-related factors include renal function, genetic makeup, sex, age etc. The knowledge of pharmacokinetic properties of drugs helps in correct adjusting the dosage regimen more precisely. The pharmacokinetic principles can also be applied to individualize pharmacotherapy also known as therapeutic drug monitoring. A fundamental concept in pharmacokinetics is drug clearance, i.e. elimination of drugs from the body. Absorption Absorption is considered as an important factor for all routes of administration of drugs except for intravenous drugs. Drugs need to cross the membranes and enter the blood vessels to express their therapeutic effect. Some drugs are transported through membrane pores and some drugs can cross the membrane by diffusion. However, some drugs are transported by carrier mediated transport. Absorption of drug is determined by different properties e.g. dosage forms, pH, food, presence of other drugs, antacids, intestinal motility, enzyme metabolism etc. Distribution After reaching the systemic circulation, drugs distributed itself into different sites of the body viz. body fluid, blood, plasma, bone, fat and organs like heart, liver and kidneys having enriched blood supply. However, binding of protein to the free drug decreases its concentration in the circulation thereby limiting its distribution inside the body. Metabolism The modification of drugs by enzymes of the body is termed as metabolism. It results into formation of water-soluble compounds which can be easily from the body. The therapeutic effect and toxicity of drugs are largely depended upon metabolism. Drug metabolism is mostly carried out in liver by different reactions like conjugation or functionalisation. Several other factors like genetics, environmental factors, age and disease states also influence the rates of drug metabolism. Excretion After metabolism of drugs in the liver, most of the drugs are be transported to bile and thereby excreted in faeces. Hence, drugs are mostly eliminated by urine, faeces. However, other drug excretion routes include saliva, sweat, breast milk etc. Drugs can be excreted through expired air which is affected by respiration rates and cardiac output.

6.3 Drug Development and Clinical Trials

6.2

113

 ational and International Agencies and Their Role N in Clinical Pharmacology

Under the aegis of World Health Organization (WHO) and the United Nations Educational, Scientific and Cultural Organization (UNESCO), the Council for International Organizations of Medical Sciences (CIOMS) was established in 1949 to lay guidelines and ethical principles for conducting of biomedical research involving human subjects as per the Declaration of Helsinki. Several initiatives have been taken post Word War-II era for protecting the human rights and these were embodied in the World Medical Association’s Declaration of Helsinki in 1964. Other regulatory functions are carried out by specialized governmental agencies such as national medicines regulatory authorities (NMRA) such as the US Food and Drug Administration (US FDA) and EMEA (in Europe). These regulatory authorities advise and coordinate and inspect the clinical research programmes to follow Good Manufacturing Practice (GMP), Good Laboratory Practice (GLP) and Good Clinical Practice (GCP). In few countries, governments, directly or through their specialized agencies, are also involved in taking decisions to ensure that only effective, safe, good quality medicines are used to treat their citizens and also for developing national treatment guidelines. In some countries, separate department for clinical pharmacology has been developed in university hospital for the manifold interests of clinical pharmacology teaching, research and clinical service.

6.3

Drug Development and Clinical Trials

Development of drugs with high chemotherapeutic index and site specific action is of prime importance. Therefore, designing of drug employing rational approach to minimize the trial and error is the primary aim of drug development. Drug design seeks to explain: (a) Physico-chemical properties of the biological compounds and molecular interaction of the bioactive molecules involved. (b) Interaction of the drugs specifically with the protoplasm to elicit a particular pharmacological response of the drug. (c) The metabolism and excretion of the drugs by the organism. (d) To deduce the structural activity relationship (SAR) between bioactivities with chemical structure of the compound.

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Factors Governing Drug-Design Factors governing drug design include: • Type and strain of animal used for experimental study and types of clinical screening of the new drugs. • Relationships between chemical structure of the drug and its associated biological activities. • Quantitative structure-activity relationships (QSARs) vary to an appreciable extent in depth and sophistication based on the nature of evaluation of structure or activity. • Introduction of functional groups in a molecule leads to interactions with important functional groups of biochemical components of living organisms.

Process of Drug Design Drug design is considered as an integrated approach and it involves various steps, namely: chemical synthesis, evaluation for activity-spectrum, toxicological studies, metabolism of the drug (Fig. 6.4). However, the drug design can be broadly divided into four steps, namely • • • •

Target identification and selection Target validation Lead identification Lead optimization (Fig. 6.5)

Identification of biological targets One of the common biological targets for drug discovery is either enzymes or the receptors which are mostly involved in different physiological response by regulating hormones and other endogenous effectors. The other type of biological target may be nucleic acids. Validation of biological targets Validation of selected and identified target is necessary to confirm whether the correct target has been identified or not. The validation process utilises the reliable and suitable animal models with latest techniques in gene targeting and expression are all essential to the. Validation also helps researchers to identify any secondary target that the drug may bind to, which may lead to any sort of unwanted or adverse reaction. Ideally the drug candidate should be such that it binds to a single target only. Lead Identification A compound having requisite structure with functional groups and exhibiting desired biological activity is regarded as a lead compound. The lead compound possessed possi-

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Compounds from synthetic programme, combinatorial library, chemical library, natural product source In vitro high throughput screening (HTS) evaluation of human/mammal receptor, enzyme assay, receptor system

NO

Active

Reconfirmation of activity with different samples

Selectivity assessed in battery of HTS assay

Active

NO

Animal model of therapeutic target

Pharmacokinetics, formulation and acute toxicology study

Approval for clinical development Fig. 6.4  Flowchart for evaluation of new chemical entities

bility of further development by suitable modification of structure to enhance its therapeutic potential. Therefore, several high-throughput screening techniques are being utilized to identify lead compounds from synthesized or natural compounds. Lead optimization Lead optimization can be carried out by different techniques such as: Structure-Based Drug Design (SBDD), Quantitative Structure-Activity Relationship (QSAR) and Computer-Assisted Drug Design (CADD). A huge amount of data can be generated using the above techniques which can be utilized in optimization of lead compound (Fig. 6.6).

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Fig. 6.5  Steps of drug development

Target Identification

Target Validation

Lead Identification

Lead Optimization

Clinical Trials

Lead structure/s

Biological concept

Series design, synthesis design

Syntheses

DESIGN CYCLE Biological testing

Structure-activity relationships, QSAR, molecular modelling

Computer-aided design: Protein crystallography, NMR, searches in 3D databases, de novo design

Lead Optimisation Candidates for further drug development

Investigational new drug (IND)

Fig. 6.6  Design cycle for lead optimization desired action

New drug

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117

Clinical Trials Clinical trial helps in screening, diagnosis along with assessment of safety and efficacy of new drug molecule. In this study, information regarding the toxicity aspects of the new molecule will be assessed in human. The clinical trial study consists of four phases, such as Phase 1, Phase 2, Phase 3 and Phase 4. Phase 1 The clinical trial Phase 1 study is done on a small group of 10–30 healthy human volunteers to assess the safety, side effects along with the dose of the new molecule is determined. Generally, the safety, tolerability, pharmacokinetics (PK) and pharmacodynamics (PD) aspects of the new molecule is evaluated in this phase. Phase I clinical trials generally last from several months to a year. Phase 2 Phase 2 studies are generally carried out on a comparatively larger population of about 20–300 or so as compared to Phase I trials. Phase 2 study further evaluates the safety of the new drug molecule with the target disease. In this phase, the drug’s efficacy and safety, metabolism and PK are also evaluated. The Phase 2 also involves placebo therapy. This phase can take about 2 years to complete. Phase 3 Phase 3 studies are carried out in a multiple sites in a randomized manner in diseased population at larger scale. This phase covers a large group of individuals of about 300– 5000 or more with target disease and is quite expensive. This phase is considered as a therapeutic confirmatory phase since all the parameters of phase 2 are confirmed in this phase. This phase also compare the new therapeutic approach of the new drug molecule with that of the standard drug regimen. Different combinations of drugs are also tested in this phase. After successful completion of this phase, regulatory submissions are generally made for “NEW DRUG APPLICATION (NDA)”. This phase may take somewhere about 3–6 years to complete. Phase 4 Phase 4 is also known as post marketing surveillance trial. This phase of study is carried out, once the drug is released to the market. This also involves pharmacovigilance study. The company continues monitoring of the drug to evaluate the side effects and safety of the drug along with assessing its long term benefit or risks. The rationale behind this phase is to check for any new adverse or serious reaction which was not detected in the earlier phases and may be observed in this phase.

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Safety Assessment in Clinical Trials

The most important aspect of the drug-development process is ensuring patient safety during and post clinical trials. Both clinical trials and clinical practice must be carried out as per patient illness and needs of the patient. Therefore, monitoring of patient safety at all levels of drug development process is of utmost importance. Few important aspects of drug safety assessment during clinical trials are as follows: 1. Adverse event Any unfavourable and unintended sign, symptom, disease or adverse event associated with the use of a drug can be termed as adverse event. 2. Adverse drug reaction An adverse event may happen due to use of medicinal product is termed as an adverse drug reaction (ADR). The adverse reaction can be categorized into three types such as suspected, unexpected and serious. Suspected adverse reactions are related to adverse event caused due to the particular drug. The adverse event or suspected adverse reaction is considered “unexpected” if it is not listed in brochure with the risk information described in the general investigational plan. However, adverse event is termed serious in case of life-threatening adverse event, inpatient hospitalization or prolongation of existing hospitalization, a persistent or significant incapacity or substantial disruption of the ability to conduct normal life functions, or a congenital anomaly or birth defect. 3. Safety concerns The detail pharmacokinetics and pharmacodynamics information of the drug or pharmacologically related drugs can be useful in the identification and exploration of major safety concerns of the drug under clinical trial. Any unexpected life threatening adverse reactions must be reported more rapidly to FDA. Casualty assessment must be carried out against any suspected adverse reactions. 4. FDA system of managing risks A product gets approved when benefits the associated with its intended use outweigh risks. 5. Post marketing safety Post marketing surveillance for new drug is carried out once a particular drug has been released into the market. Post marketing surveillance involves evaluating safety aspect of a new drug by reporting databases, monitoring prescription, recording health status, checking patient registries and recording linkage between health databases. 6. Sources of safety information and Safety evaluation The safety of a new drug can be evaluated by collecting information from different sources e.g. adverse drug reaction reports, clinical and epidemiological studies, medical literature, pharmaceutical companies, regulatory authorities of across the globe and ­morbidity, mortality databases, non-clinical, post marketing surveillance and safety profile of other drugs.

6.5 Ethics in Clinical Trials

6.5

119

Ethics in Clinical Trials

Testing of novel chemicals for use in therapy or diagnosis is an essential part of clinical pharmacology. The preclinical trials part of the evaluation for safety and efficacy, are carried out in experimental animals. But, the drugs developed for human use are tested on human volunteers. The time window for development of new drug from discovery phase to final distribution in the market takes about 15 years or more which is extremely capital intensive and risky which could result into unethical practices in the recruitment of human volunteers in clinical trials and further experiment. Few important unethical clinical trials conducted on human beings are (i) Tuskegee syphilis trials, (ii) Manhattan Project, (iii) Thalidomide disaster, (iv) Gene therapy, (v) TeGenero clinical trial, (vi) Bial clinical trial etc. (Table 6.1). Clinical trial is necessary to find out the safety of a drug against a particular disease so that health professionals will know how to prevent and treat illness effectively. However, there are some ethical concerns about clinical trials as the use of human participants in research would give valuable knowledge that will benefit others. The ethical codes and guidance help in delimiting research involving human participants. The Nuremberg Code, the Declaration of Helsinki, the Belmont Report, and the U.S. Code of Federal Regulations Table 6.1  Few infamous human clinical trials Sl. no. Name and year Tuskegee 1 syphilis trials (1932–1972) 2 Manhattan Project (1940–1960) 3

Thalidomide disaster, 1950

4

Gene therapy, 1999

5

TeGenero clinical trial, 2006

6

Bial clinical trial, 2016

Targeted disease Effects of syphilis in humans Effects of radiation

Unethical practice Subjects were not informed about their disease and were denied treatment

The participants included pregnant women, disabled children, hospital patients, and enlisted military personnel and most of them had no knowledge of the experiments Control nausea in the early Patients were however not informed that the stages of pregnancy drug was investigational, in the testing phase of the regulatory process Gene therapy for Ornithine Failure to disclose in the informed consent Trans Carbamylase documentation the risks involved in the use of adenoviral vectors Rheumatoid arthritis and B Deviations from the approved protocol were cell chronic lymphocyte alleged. The dose was to be administered at leukemia 2-h intervals, but was apparently done at 10-min intervals. Treatment of anxiety and Unethical conduct of the trial include the motor disorders associated administration of the experimental drug to with Parkinson’s Disease, all six volunteers simultaneously, instead of and chronic pain associated one receiving a test dose and being checked with cancer for adverse reactions, before giving it to the others

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provide guidance for researchers to respect and protect the rights and welfare of human research participants. A protocol that is not suitable scientifically or clinically should not be conducted and hence not to be approved by the ethical committee. Few important issues accompanying clinical trials are as follows: • Risk-Benefit Balance Determination of risk-benefit balance is the most difficult ethical issues. It generally compares the risk and potential benefits of the drug use which is more beneficial for society. The investigator must ensure that the potential benefit outweighs the risk of harm associated with the study. Both risk of harm and potential benefits are highly dependent on the phase of a clinical trial. Therefore, development of the protocol should address the different concerns viz. third party review, pre-clinical information, test article manufacturing, clinical rationale, study objective, study design – treatment, study design – outcome, study design-randomisation, participant availability, resources etc. • Informed Consent Process The participant of the clinical trial process must give their voluntary consent on the basis of their preference and choice. The informed consent document contains a synopsis of the clinical trial protocol, its purpose, treatment, risk of harm, potential benefits, alternative treatments and voluntary participation. • Secondary Analysis of Clinical Database The analysis of previously created research data sets is referred as secondary analysis. • Vulnerable Participants The vulnerable participants are referred to those individuals who have been unduly influenced by justified or unjustified expectation of benefits associated with participation. They might have also faced retaliatory response from senior members of a hierarchy in case of refusal to participate and hence are unwillingly have participated in the clinical trial study. • Privacy and Confidentiality The confidentiality must be kept confidential and investigators must take adequate and necessary steps to ensure confidentiality of personal information of participants for all stages of the clinical research study. • Safety Monitoring The clinical trial study must incorporate a plan to assess the safety of participants. Policies and procedures must be reviewed so that safety monitoring of plan provides adequate protection for participants. • Participant Recruitment Procedures The participant must be selected from a patient pool at the study site by referring participants from other clinics or by advertisement or by directly approaching or screening the public. • Qualification of Investigator and Research Staff For carrying out clinical trials, investigators must have requisite qualification and experience. They should provide evidence of such qualifications and updated curriculum vitae or other relevant documentation requested by the sponsor.

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• Financial Conflict of Interest Conflict of interest situation may arise where an individual and corporation interests interfere with a professional obligation. Financial conflicts of interest may lead to compromise of truthfulness of research. • Clinical Trial Insurance and Indemnity The contract to compensate an individual for loss or damage is called as indemnity. The indemnity provides a legal protection to participant in event of any unforeseen adverse circumstance. • Essential Clinical Trial Documents The essential documents enable anyone to evaluate the trial and quality of the generated data. By looking at the essential documents, the ethical committee can verify whether the clinical trial is as per the compliance of the investigator, sponsor and monitor with Good Clinical Practice (GCP) and applicable regulatory authority requirements. • Clinical Trial Registration It is mandatory to register before any clinical trials as it provide complete information regarding the past research and whether it was successful or unsuccessful. • Dissemination of Trial Results All the stake holders of the clinical trial study i.e. the sponsor, investigator and institution have an ethical responsibility to publicly disseminate the results of clinical research in a timely manner.

6.6

Ethics in Clinical Research

Ethics refers to moral principles governing human character and conduct. Experimentation on human being is subject to ethical standards that promote respect for all and protect their health and rights. Ethics in clinical research focuses largely on identifying and implementing the acceptable conditions for exposure of some individuals to risks and burdens for the benefit of society at large. In the recent past, a considerable progress has been occurred in human biomedical research and biotechnology. For simultaneous smooth progress in research and prevention of human exploitation as test subject, it becomes mandatory that every research proposal on involving human subjects be cleared by an appropriately constituted ethical committee. Ethical guidelines for clinical research were formulated only after discovery of inhumane behaviour with participants during research experiments. Few important aspects of addressing ethical issues in planning and conduct of research are as follows. 1. Scientific and social value and respect for rights Patients, health professionals, researchers, policy-makers, public health officials, pharmaceutical companies and others rely on the results of research activities and decisions that affect individual and public health, welfare. Therefore it should be ensured that the outcome of the proposed studies are scientifically sound, build on an adequate knowledge base and are likely to generate important information.

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2. Research conducted in low-resource settings In order to ensure an overall fair distribution of the benefits and burdens of the research, the government and other relevant stakeholders, should provide local health infrastructure to the population or community where the research will be conducted. 3. Equitable distribution of benefits and burdens in the selection of individuals and groups of participants in research Sponsors, researchers, governmental authorities, research ethics committees and other stakeholders must ensure that the benefits and burdens of research are equitably distributed. Those who are unlikely to get any benefit from the research should not bear any share of the risks or burdens of the research participation. 4. Potential individual benefits and risks of research The researcher, sponsor and the research ethics committee must ensure that risks to participants are minimized and appropriately balanced in relation to the prospect of potential individual benefit and the social and scientific value of the research. 5. Choice of control in clinical trials The research participants in the control group for a trial of a diagnostic, therapeutic, or preventive intervention should receive an established effective intervention. Placebo can be used as a comparator in case of unavailability of effective intervention for the condition under study. 6. Caring for participants’ health needs Researchers and sponsors must take adequate steps for addressing participants’ health needs during research and after the research is concluded. 7. Community engagement Researchers, sponsors, health authorities and relevant institutions should engage potential participants and communities in a meaningful participatory process design, development, implementation, design of the informed consent process and monitoring of research. 8. Collaborative partnership and capacity-building for research and research review Health related research involving human participants need to be ensured that the research finding should be reviewed ethically and scientifically by competent and independent research ethics committees and should be conducted by competent research authorities. Further, researchers and sponsors should contribute to capacity building for research and review. 9. Individuals capable of giving informed consent Researchers should seek and obtain consent from the potential participant after providing relevant information about the research and ascertaining that the potential participant has adequate understanding of the material facts to give their free and informed consent to participate in research, or to decline to do so. 10. Modifications and waivers of informed consent Research involving humans must be carried out after obtaining participant’s individual informed consent or that of a legally authorized representative from a research ethics committee.

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11. Collection, storage and use of biological materials and related data Biological materials and its related data including health, employment records, must be collected and stored properly. The authorities must have a governance system to obtain authorization for future use of these materials in research. The rights and welfare of individuals from whom the materials are collected must not be adversely affected. 12. Collection, storage and use of data in health related research The data for human health related research must be collected and stored properly and for use of these data specific informed consent for a particular use or broad informed consent for unspecified future use must be obtained from the person from whom the data were originally obtained. 13. Reimbursement and compensation for research participants The participants for specific research should be reimbursed for costs incurred during the research including travel costs. Further, reasonable compensation must be given for their inconvenience and time spent. However, the compensation may be monetary or non-monetary. 14. Treatment and compensation for research-related harms The participants of specific research should get free treatment and rehabilitation for any physical, psychological or social harm caused due to participation in health-­ related research. Further, legitimate compensation must be provided to the participant for their lost wages. 15. Research involving vulnerable persons and groups Research ethics committees must ensured specific protections for vulnerable individuals and groups when considered for research so that the rights and welfare of these individuals and groups can be safeguarded. 16. Research involving adults incapable of giving informed consent Adults are not capable of giving informed consent and are therefore can’t protect their own interest. Hence they must be included in health related research unless a good scientific reason justifies their exclusion. 17. Research involving children and adolescents Children and adolescents have different physiologic condition and different health needs and therefore required special consideration by research ethics committees. They must be included in health related research unless a good scientific reason justifies their exclusion. 18. Women as research participants Women have different physiologies and health needs and justified their inclusion in health related research. However, permission for participation in research must only be given after getting their individual informed consent. 19. Pregnant and breastfeeding women as research participants Both pregnant and breast feeding mothers have distinctive physiologic condition and health needs and research involving them must be promoted. However, informed

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21.

22.

23.

24.

25.

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consent from individual participant is necessary and it should be replaced by permission of any other person. Research in disasters and disease outbreaks The natural as well as orchestrated disasters involving earthquakes, tsunamis or military conflicts, and disease outbreaks put a devastating health impact on a larger affected population and hence required health related research as a disaster response. However, the ethical principles for clinical research must be upholded as per the guidelines. Cluster randomized trials Cluster randomized trials (CRTs) involves groups of individuals (clusters), communities, hospitals, or units of a health facility that are randomized to different interventions. The ethical principles that govern all health-related research with humans are applicable to CRTs and it should determine the affected research participants, their informed consent for the said research and necessary permission to carry out such research. Use of data obtained from the online environment and digital tools in health-related research In today’s internet world, with the help of the different online and other digital tools, there always looms a threat that the personal information of the participant involved in a health-related research may be revealed or else inferred when they are published. So necessary steps should be taken to ensure the privacy of the individuals. Requirements for establishing research ethics committees and for their review of protocols The decision to carry out any particular health related research should be determined by a research ethical committee. Therefore all research proposals must be submitted to research ethical committee for approval. Research ethical committee must include multidisciplinary membership in order to competently review the proposed research. Public accountability for health-related research Researchers, sponsors, research ethics committees, funders, editors and publishers have an obligation for research and its results. Therefore, public accountability is necessary for the social and scientific value of health related research. Conflicts of interest Generally, the aim of the health-related research is to generate information or data necessary to promote people’s health, in ethically ways. The conflicts of interest can be defined as the conflicts between the goal of health-related research and secondary interests like financial gain scientific recognition of the researchers, research institutions, sponsors, research ethics committees, and policy-makers. Conflicts of interest can influence the research questions and methods and therefore it is necessary to develop and implement policies and procedures to identify, mitigate, eliminate, or manage such conflicts of interest.

References

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Questions 1. What is clinical pharmacokinetics? 2. What is pharmacodyanamics? 3. What are the different stages of clinical trials? 4. What is placebo study? Why it is important? 5. What is the importance of post marketing survey in clinical trial study? 6. What is adverse drug reaction (ADR)? 7. What are the different types of ADR? 8. What is TDM (Therapeutic drug monitoring)? 9. What is cluster randomized trials (CRTs)? 10. What are the important steps of drug development? 11. What are QSAR and SAR studies? 12. What are the safety concerns of ADR? 13. What are the important aspects of drug safety assessment? 14. What do you understand by ethics in clinical trial? 15. What are the ethical concerns of clinical trial? 16. What are the important aspects of ethical issues in clinical research?

References Badyal D (2008) Practical manual of pharmacology. Jaypee Brothers Medical Publishers, New Delhi BM Swamy V, Jayaveera KN, Reddy V (2014) Experimental pharmacology and toxicology. S. Chand & Company, New Delhi Clinical pharmacology in research, teaching and health care: considerations by IUPHAR, the International Union of Basic and Clinical Pharmacology (2010) J Basic Clin Pharmacol Toxicol 107:531–559 Ethical guidelines for biomedical research on human subjects (2000) Indian Council of Medical Research, New Delhi Fan J, de Lannoy IA (2014) Pharmacokinetics. Biochem Pharmacol 87(1):93–120 Goyal RK (2017) Practical in pharmacology. B. S. Shah Prakashan, Ahmedabad https://www.ashp.org/-/media/store%20files/p2418-sample-chapter-1.pdf. Accessed on 10 Dec 2018 International ethical guidelines for biomedical research involving human subjects (2002) Council for International Organizations of Medical Sciences, Geneva Kale SR, Kale RR (2017) Practical pharmacology and toxicology. Nirali Prakashan, Mumbai Katzung BG, Masters SB, Trevor AJ (2012) Basic & clinical pharmacology. McGraw-Hill, New York Medhi B (2017) Practical manual of experimental and clinical pharmacology. Jaypee Brothers Medical Publishers, New Delhi Nautiyal N, Rastogi R, Gamperl HJ (2015) Drug safety assessment in clinical trials: concepts and issues. Int J Pharm Sci Res 6:4159–4167 Ratain MJ, Plunkett WK Jr (2003) Principles of pharmacokinetics. In: Kufe DW, Pollock RE, Weichselbaum RR et al (eds) Holland-Frei Cancer Medicine, 6th edn. B.C. Decker, Hamilton

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Ritter JM, Lewis LD, Mant TGK, Ferro A (2008) A text book of clinical pharmacology and therapeutics. Hodder Arnold, London Salmon DM (2014) Practical pharmacology for the pharmaceutical sciences. Wiley, Chichester Sierra LM, Gaivao I (eds) (2014) Genotoxicity and DNA repair: a practical approach. Springer protocols. Humana Press, London Singhal KC (1997) Pharmacology laboratory manual. CBS Publishers & Distributor, New Delhi Thatoi HN, Dash S, Das SK (2017) Practical biotechnology, principle and protocols. I.K. International, New Delhi Tripathi KD (2013) Essentials of medical pharmacology. Jaypee Brothers Medical Publishers, New Delhi Turner RA, Hebborn P (eds) (1971) Screening methods in pharmacology. Academic, New York Vogel HG (ed) (2002) Drug discovery and evaluation pharmacological assays. Springer, Berlin Woolley A (2008) A guide to practical toxicology. Informa Healthcare USA, New York

7

IPR and Ethics in Animal Studies

7.1

Introduction

Intellectual property rights (IPR) are legal and official strategies that are assigned to protect the conceptions of the mind such as creations, art and literature works, and various designs (Dutfield 2005). It is also included the marks that are imprinted on different products inorder to differentiate them from similar products sold in the market (Dutfield 2005). However over years, the concept of intellectual property (IP) is overextended to inculcate not particularly the copyrights, industrial designs, patents, and trademarks; but it also includes the geographical signs, rights of the plant breeders’, profession secrets, along with the rights of the layout-designs of different integrated circuits (Dutfield 2003, 2005). Under the IP law, imperceptible possessions including creations; works related to fictional and artistic; various designs; and idioms; symbols and signs etc. can be safe guard (Dutfield 2005; http://www.innovaccess.eu/definition-ip). This type of protection are possible due to different types of IP rights such as patents, trademark, designs, copyright etc. (http://www. innovaccess.eu/definition-ip).

7.2

Intellectual Property Rights and Its Different Categories

IPRs are generally described “imperceptible,” but these are nonetheless alike to other legal property rights which can also be vended, rented, given to someone as gift, licensed, and permit its holder a distinct rights to use or exploit it commercially (Nambisan 2017a). Basing on the concept, IPR has been categorized as the various types such as Patents that safeguard the new inventions; Copyrights that safeguard the works related to art and literature; different types of trademarks, trade secrets; recorded (industrial) design; Outline designs of different integrated circuits and geographical locations.

© Springer Nature Singapore Pte Ltd. 2019 J. K. Patra et al., A Practical Guide to Pharmacological Biotechnology, Learning Materials in Biosciences, https://doi.org/10.1007/978-981-13-6355-9_7

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Apart from the copyright types, which is universal in nature, all other IPRs are based on the specific region and are limited to the specific geographical locations in which it has been awarded (Nambisan 2017a). These are also monopoly rights which require the approval of the right’s proprietor for its uses. IPRs are usually approved by the specified authority for a restricted time period, excluding the trademarks and topographical signs that has an infinite life period with renewal at regular intervals, however the trade secrets have infinite life span and doesn’t require any renewal (Nambisan 2017a). The significant properties of the different forms of IPR are summarized in the Table 7.1.

7.2.1 Patents Which Protect Inventions A patent is assigned to a work and its owner possess the right to prevent others from misusing the invention (whether products or processes) that are new, that involve a creative step and are susceptible to industrial applications (http://www.innovaccess.eu/types-of-ip). This control is approved for a precise field, in a distinct nation and for a maximum period of 20 years in a condition that the invention is to be fully disclosed and it’s all detail technical features to be published (https://ocpatentlawyer.com/four-types-intellectual-propertyprotect-idea/; http://www.innovaccess.eu/types-of-ip). Basically there are two types of patents i.e. utility type and design type. The utility patent types need to be applied to the United States Patent and Trademark Office and its term is generally for a period of 20 years and is non-provisional in type. Whereas the design patent need to be registered with the United States Patent and Trademark Office immediately otherwise its idea will be dedicated to public after 1 year of its marketing.

7.2.2 Copyrights Which Protect Works of Literature and Art Copyright is a lawful term that defines about the specific rights that is assigned to its original designer for its original fictional, melodic or imaginative works which allow them to control their subsequent use (http://www.innovaccess.eu/types-of-ip). Copyright protection usually exists autonomously of any registration or prior examination. Most types of products are protected by a copyright, basically the images and words on the packaging of the products, its label, the product itself along with its style and its composition recipe together with the webpage are protected with a copyright (https://ocpatentlawyer.com/ four-types-intellectual-property-protect-idea/). The benefits of a copyright registration are that it is low-priced to secure, and the law allows us to demand attorney fees from the infringers (https://ocpatentlawyer.com/four-types-intellectual-property-protect-idea/). Copyrights protect original works of authorship that are fixed in a palpable medium of appearance (https://ocpatentlawyer.com/four-types-intellectual-property-protect-idea/).

7.2 Intellectual Property Rights and Its Different Categories

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Table 7.1  Types of Intellectual Property Rights (IPRs) and their significant properties Type of IPR Patent Copyright and related Rights

What does it cover Inventions

Forms of creativity primarily concerned with mass communication, e.g., books, paintings or drawings, music, poems Traditional Cultural Music, musical instruments, stories, Expressions (TCEs) art, handicrafts, words, names and insignia, performances, textiles, carpet, jewelry designs, forms of architecture Signs that individualizes the goods Trademark (t), of a given enterprise and Service Marks, distinguishes them from the goods Collective Marks, of its competitors and Certification Marks Industrial Designs Formal or ornamental appearance for mass produced items (original ornamental or nonfunctional features that result from design activity) Integrated Circuits Layout designs (topographies) of integrated designs Geographical Names and symbols which indicate Indications (GI) a certain geographical origin of a given product—applied to products whose quality and characteristics are attributable to their geographical origin Appellations of Special kind of indication of source Origin (AO) Confidential business information Protection against unfair competition, which provides an enterprise a competitive edge (manufacturing or Trade secrets industrial secrets and commercial secrets)

Term of the IPR 20 years from the date of filing Life of the author and not less than 50 years after the death of the author

International conventions Paris Convention Berne Convention

Undertaken in cooperation with UNESCO

Usually 10 years, renewed indefinitely on payment of additional fees Usually 15 years, renewal is usually subject to payment of renewal fees

Madrid Agreement

Hague Agreement

Usually 15 years Indefinitely

TRIPS Agreement

Indefinitely

Lisbon Agreement

Indefinitely, until public disclosure of the secret occurs

Reproduced with permission from Nambisan (2017a) Source: WIPO (2004). WIPO intellectual property handbook, Reprinted 2008. Retrieved from http:// www.wipo.int/edocs/pubdocs/en/intproperty/489/wipo_pub_489.pdf

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7.2.3 Trade Marks A trademark is an emblem or mark which is given by a particular business or a company or enterprise to differentiate its own products or services from others in the market ­(http:// www.innovaccess.eu/types-of-ip). Generally a trade mark includes a particular sign or design prepared graphically with some figures, special design or letters, words, numbers and shape and are impregnated or labeled on the product itself, or its covering/packaging and which is useful to distinguish different products of different companies or manufactures (http://www.innovaccess.eu/types-of-ip).

7.2.4 Trade Secrets All inventions usually start out as a trade secret for the inventor. Inventors have an intuitive desire to keep their ideas and invention as a top secret affair and they attain this by using various type of IPRs such as patents, trademarks, copyrights etc. (https://ocpatentlawyer. com/four-types-intellectual-property-protect-idea/). A trade secret title protects any piece of knowledge (e.g. formula, pattern, device or compilation of information) that are not known to the public and provides is sole owner to get complete benefit out of it by keeping it as a secret (https://ocpatentlawyer.com/four-types-intellectual-property-protect-idea/). Trade secrets are concerned about the undisclosed or exclusive material of various profitable value. These are not protected by explicit legal laws as compared to other type of IPs, however, there are few less powerful laws and employment contracts which are useful in some specific cases (http://www.innovaccess.eu/types-of-ip). The level of defense assigned to the trade secrets are however different from place to place or country to country (http://www.innovaccess.eu/types-of-ip).

7.3

Importance of IPR in Drug Development

Generally, the pharmaceutical industry sector give a special care to the IP policy, since it is considered as a significant issue for a number of controversies worldwide about the association between the IPRs, R&D inducements, estimating and contact to a number of medicines (Cockburn 2009). Basically, IPRs are assumed to have two types of major field of influence in the pharmaceutical sectors that includes the estimation and contact or access of various commodities and the debate is mainly based on the linkage between different types of IPRs (such as patent rights in particular); elimination of opponents and the accessibility and estimating the values of newly made medicines and secondly, the problem of the R&D inducements, which is responsible for providing the actual value for discovery, development and marketing of newly made drugs or medicines through the IPR laws (Cockburn 2009).

7.3 Importance of IPR in Drug Development

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In the last decade, considerable resurrection of interest and pursuit have been carried out on drug discovery and development based on traditional knowledge, ethnobiology and natural products, both in the public and private sectors (Boyd 1996). It is apparent that behind any successful development of natural drug discovery, there is always a successful venture of cooperation and collaboration of a number of international groups that work together and there is always a chance of complexity, increase in the expenses, long time period and a number of uncertainties (Boyd 1996). Considering this hurdles in developing a natural product based drug or medicine, there should be a communal understanding and a mutual perspective about the perceptions of IP and IPR (Boyd 1996). It is considered that there is lots of risks including a lengthy procedure with longer time period and requiring too much of money in developing a new and effective drug. It has been reported that it take around 12–15  years and a investimate of about US$750  million for a pharmaceutical company to commercialize a newly made drug from the laboratory to the patients to use it for curing a disease (Atkinson and Jones 2009). It is also a fact that not all the compounds tested in the R&D laboratory of a pharmaceutical company are developed and commercialized as drugs, it is estimated that only one out of every 5–10 thousand compounds tested and finally approved after a series of tests and clinical trials as drugs for human use (Atkinson and Jones 2009). Keeping all these constraints into account, the pharmaceutical companies are highly cautious and tried their level best to keep their process and the contents as top secret and protect them through patents till they expire. It is strongly believed that the patents and IP rights rendered to the pharmaceutical companies are highly crucial for the continuation of research and deployment works leading to the formulation of new and innovative modern drugs to be used for human care for the betterment of the society (Atkinson and Jones 2009). Different types of IPR involved in pharmaceutical industry includes, patents, industrial designs, trademarks, copyright, and trade Secrets (Atkinson and Jones 2009; https://www.iipta.com/role-of-ipr-in-thepharmaceutical-industry/).

7.3.1 A New Drug as a New IP A newly formulated medicine/drug can only be considered as a new product that can obtain IPRs if it’s completely new in the sense of its chemical structure/configuration and detailed medicinal uses are not known before or not published before prior to its discovery and claim by any company. Furthermore, the IP holder shares same rights and advances as that of any owner of any kind of patentable invention (Boyd 1996).

7.3.2 Safeguard of IP in Finding a New Medicine of Natural Origin IP encompassing the development of a newly formulated medicine or drug should satisfy certain standard mandatory criteria such as originality, usefulness and unobviousness,

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before it can be considered for registration as a patent (Boyd 1996). Equally, both a precise legal explanation of originator and development of importance, in agreement with the IP laws of a given country, are essential to the rationality, and therefore the possible price, if any, of the patent within the given country (Boyd 1996).

7.3.3 V  arious Phases in the Natural Product Based Discovery of Drugs Where ‘Inventive’ Contributions Might Occur Novel or theoretical contributions takes place any time during the process of natural drug discovery. It includes, selection and classification of different organism in the study; standardizing and applying the most effective screening technique; separation, cleansing and physical explanation of the active compounds; finding of original, undetectable and valuable organic entities; and, formulation of synthetic analogs or imitative compounds. To succeed as a creative or theoretical role to any of these steps, the input must be a more specific, tactical and newly made task; and, the input should be active, and focused precisely to the specific patentable discovery as claimed by the organization (Boyd 1996).

7.3.4 M  ost Important Steps in the Natural Based Drug Development Procedure The advancement of a development process for novel medicine that leads to the manufacture of highly valuable marketable drug is a tremendously lavish and complicated process that might take long time and huge amount of money. The important steps in the natural product based drug discovery process includes • Bioproduction of majority of compound for expansion • Pharmacological prescription preparation and development, study, constancy, control of their quality • Toxicology related studies, pharmacokinetics of various drugs, and their process metabolism • Research on trial drug applications • And, various stages of clinical trials (Phase I, II, III, etc.). Most of the medications that move in to the pipeline, eventually fall out of the line due to any of many possibly stubborn glitches that it might has come across (Boyd 1996). The most luxurious and disastrous failures of natural product based drug discovery includes the following points (Boyd 1996). • Those drugs in which terminal problems such as lack of satisfactory effectiveness. • Surprising toxicities that might occur at the time of advanced clinical testing.

7.4 Patenting Cells, Cell Lines and Animals

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• Normally, most of the drugs get rejected during the preclinical testing and research most before reaching the advanced clinical tests.

7.3.5 Importance of IPR in Drug Development Most of the factors are necessary during the process of drug development and the most important factor is the IPR. IPR provides safety and water tight protection to the designed and developed drug of a company by patents and trade secret. It provides economic growth and effectiveness to the company by providing sole rights for manufacture and marketing of the drugs. It helps in safeguarding a standard and guarantees quality to the consumers. It produces solutions to various global challenges related to the drug. And at last, it inspire the innovation made by the company and also reward the entrepreneurs (https://www.iipta. com/role-of-ipr-in-the-pharmaceutical-industry/).

7.4

Patenting Cells, Cell Lines and Animals

It is presently recognized throughout the world, that a patent can only be accepted if it satisfies the following points i.e. it should be original and novel; it should have a wide industrial applications; and above all, all the related issues such as methodology, literature, results etc. should be clearly disclosed (Rendon and Blake 2007). During the year 1980s, in the time of the development and flourish of the recombinant DNA technology, a few of the initial patent applications related to biotechnology that includes studies on different cells and the cell lines with altered genetic material were filed (Table 7.2). And ever since then, the patenting of human genes been accepted to the patent authorities and policy makers in many countries, however it again gave rise to various social and ethical controversy (Caulfield and Brownsword 2006; Caulfield and von Tigerstrom 2006; Crespi 2005; Rendon and Blake 2007). However according to the belief of some, if the human genetic material and their alteration will take place then it would definitely affect the integrity and self-respect of the human race (Caulfield and von Tigerstrom 2006; Rendon and Blake 2007) and debate the following points that: • Genes and related components are occurred by the natural process and thus it should not be controlled by any single person or a group. • Genes are, thus, categorised as discovered entities not invented components. • Genes are regarded as already existed components neither they are new nor innovative. • Thus, separation and copying of gene cannot be considered as a separate and innovative technique in this post-genomic era.

Table 7.2  Patents related to human stem cells and pluripotency genes Publication number US5843780 WO9730151

US6875608

US2005255573

US2005196859

EP0662512

Ref. no. and title [Thomson 1998] Primate embryonic stem cells [Dani et al. 1997] Cytokine expressed by DIA/LIF-deficient embryonic stem cells for the inhibition of differentiation [Chambers and Smith 2005] Pluripotency determining factors and use thereof [Smith and Burdon 2005a] Propagation and/or derivation of embryonic stem cells [Smith and Burdon 2005b] Propagation and/or derivation of embryonic stem cells [Terstappen et al. 1995] Human hematopoietic stem cells

Inventors Thomson, J.A. Dani, C., Chambers, I.P., Beuhr, M.L., Smith, A.G.

1997/08/21

Chambers, I.P., Smith, A.G.

2005/04/05

Smith, A.G., Burdon, T.G.

2005/11/17

Smith, A.G., Burdon, T.G.

2005/09/08

Terstappen, L.W., Loken, M. R., Huang, S., Olweus, J., LundJohnsson, F. US5840580 [Terstappen et al. 1998] Phenotypic Terstappen, L. W., characterization of hematopoietic stem Loken, M. R., Huang, cells S., Olweus, J., LundJohansen, F. Rao, D.D., Sitnicka, E., WO9818486 [Rao et al. 1998] Use of leptin to stimulate hematopoiesis Bartelmez, S.H., Hagen, F.S. WO03076613 [Ema et al. 2003] Protein sustaining Ema, H., Nakauchi, H., undifferentiated stem cells as such Osawa, K. Ema, H., Nakauchi, H., US2005063961 [Friedlander et al. 2005] Hematopoietic stem cells and methods Osawa, K. of treatment of neovascular eye diseases therewith MXPA05000972 [Dasilva 2005] Hematopoietic stem Dasilva, K. cells and methods of treatment of neovascular eye diseases therewith MXPA05011752 [Otani 2006] Hematopoietic stem cells Otani, A. and methods of treatment of neovascular eye diseases therewith JP06042663 [Nakatsuji et al. 2006] Discrimination Naatsuji, N., Tada, T., marker of ES cell Tada, M. WO04072226 [Nakatsuji et al. 2004] Marker for the Naatsuji, N., Tada, T., Tada, M. undifferentiated state of cell and composition and method for separation and preparation of stem cells JP05110565 [Yamanaka 2005] Agent for keeping Yamanaka, N. differentiation/pluripotency [Robson et al., 2006] Method for Robson, P., Rodda, D., WO06025802 maintaining pluripotency of stem/ Ng Huck, H. progenitor cells

Reproduced with permission from Rendon and Blake (2007)

Publication date 1998/12/01

1995/07/12

1998/11/24

1998/05/07

2003/09/18 2005/03/24

2005/09/02

2006/06/06

2006/02/16 2004/08/26

2005/04/28 2006/03/09

7.5 Ethics in Laboratory Animal Studies

135

At this stage, when the authorities clash with the issues on different patents on human gene and related issues, the scientific advancements in the field of stem cell research are further exaggerating the issues even more. In the year, 1987, the United States Patent and Trademark Office (USPTO) proclaimed that “now considers non-naturally occurring, nonhuman, multi-cellular living organisms, including animals, to be patentable subject matter” ­(https://www.lotempiolaw. com/2012/06/blog-2/animal-patents/). The first patent concerning an animal is issued to the Harvard University in the year 1988 by the USPTO.  This patent is related to a genetically modified mouse the “Oncomouse” which develops cancers that mirror the human diseases for its application in pharmaceutical drug discovery sectors. And due to this reason, the USA was considered as the first to claim a patent for an animal (https:// www.lotempiolaw.com/2012/06/blog-2/animal-patents/).

7.5

Ethics in Laboratory Animal Studies

Scientific research using the animals’ has played a major part in the studies on a number of scientific and medical cases during the last 100 years and till date it is the most trusted technology in the understanding the more complex issues of various human and animal diseases (Festing and Wilkinson 2007). Successful developments in the invention and discovery of modern drugs and mode of treatments are only made possible due to the enormous animal trials and tests (Festing and Wilkinson 2007). Normally, scientists around the world, accept controls on the use of animals in research and clinical trials as none of them want to use animals unnecessarily causing harm to them and thus they made a provision to use the animal for research within an ethical framework (Festing and Wilkinson 2007). International guidelines for biomedical research concerning animals, include scientific justification and ethical acceptability (Svendsen et al. 1997). The use of laboratory animals causing uncomfort to the animals are to be considered for review and approval by a committee of members of ethics in order to regulate their use (Svendsen et al. 1997). There are three vital issues in the management of laboratory animal as below (Allan and Blackshaw 1986). • The morals of using the laboratory animals for various tests and experiments. • The wellbeing and happiness of the laboratory animals to be used for various experiments. • The methodical legitimacy of the particular species and its quantity to be used in a single experiment.

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7.5.1 Ethical Considerations It included the following points. Animal Rights – A distinction should be drawn between the concepts of animal welfare and animal rights (Allan and Blackshaw 1986). Animal Experimentation  – These different views raise the vital issue of whether it is appropriate to use animals in scientific investigations and, if so, what ethical responsibilities we have to provide for their welfare (Allan and Blackshaw 1986). Alternatives to Animal Experiments – It is significant to deliberate the replacements which may be appropriate to use in place of, or in combination with, animal experiments (Balls 1983, Allan and Blackshaw 1986). Obligations of the Research Worker – It is the caring use and care of all animal life that defines an ethical and careful society.

7.5.2 Welfare Considerations: The Animal and the Environment The environment is vital to the management of laboratory animal and the welfare of the animal and it is measured throughout the breeding-holding phase and the experimental phase. (Allan and Blackshaw 1986). The Breeding-Holding Phase  – The laboratory animals used for experimental purposes have been selected and bred for many generations under laboratory conditions, and need a well-controlled environment to keep them healthy. The design of animal housings must be considered on the basis of the variety of animal species kept and their differing age groups (Allan and Blackshaw 1986). The Experimental Phase – The wellbeing of the laboratory animals used in the experiments are solely the responsibility of the research worker. Attention are given to the handling and limitation, experimental processes (e.g., anesthesia, blood collection), and euthanasia. (Allan and Blackshaw 1986).

7.5.3 Ethics in Animal Experimentation In order to avert unwarranted suffering, ethical attentions in case of the animal studies are verymuch important. Generally, the research protocol are reviewed by the animal ethics committees before an experiment on the laboratory animals are conducted, (https://www. enago.com/academy/important-ethical-considerations-animal-studies/). Basically such committee follows three main thumb rules as follows.

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137

• The animal experiments must be substituted wherever possible by other methods such as mathematical modeling, or an in vitro biological system. • There must be a decrease in the number of animals used. Only the number required to get consistent data must be used in an experiment. A thorough literature search must be done previously to prevent duplicating experiments. • The study must be developed to minimize its overall impact on the animals used. Besides, there should also be a local animal care committee which should safeguard the health, hygiene and care of the laboratory animals used in the study (https://www.enago. com/academy/important-ethical-considerations-animal-studies/). All researchers conducting the animal experiments should also be qualified in handling the particular species in the study. Their pain or discomfort should be minimized and taken care of. Anesthesia should be used as required and repeated surgical procedures on the same animal should be avoided wherever possible (https://www.enago.com/academy/importantethical-considerations-animal-studies/). The humane treatment of the test animals should be combined into the study protocols and aseptic techniques should be used whenever possible. Only skilled people should perform surgical procedures and anesthetization of the animals used in the study (https://www.enago.com/academy/important-ethicalconsiderations-animal-studies/).

7.6

Risk Assessment and Management in IPR

7.6.1 IP Risks There are both rewards and risks connected with IP. Assumed the growing importance of IP across most industry sectors, many organizations are facing a diverse range of IP related risks. However, the IP risks are of different types and thus are categorized into different categories that includes the copyrights, trademarks, patents; the center of the IP related danger; the impact and probability of the IP risk, the topographical nature of the IP risk like generic or specific in nature, etc. (https://www.linkedin.com/pulse/intellectualproperty-risk-assessment-top-down-vs-bottom-o-connell).

7.6.2 IP Risk Management IP risk administration deals with the various methods, techniques and tools for the management of the IP risk factors in a (Svendsen et al. 1997). It is formerly about the certification, assessment, and grade of the IP related risks along with the coordinated and cost-effective uses of various resources in order to cut the chance and/or the influence of these IP related risks to the group. The two basic components of IP risk management are IP risk assessment and IP risk mitigation ­(https://www.linkedin.com/pulse/intellectual-

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property-risk-assessment-top-down-vs-bottom-o-connell; https://www.ipeg.com/ip-riskmanagement-how-to-deal-with-it-part-1/). Risk assessment is concerned with the documentation, quantification and position of the IP linked risks a group faces. Once the revelation to IP linked risk has been identified, quantified and prioritized; IP risk mitigation actions for the organization’s contact to the risk can be planned. There are different methods such as the contingency, avoidance, reduction, prevention, etc. which organizations employ to lessen the IP related risks.

7.7

Good Laboratory Practices in IPR

Good Laboratory Practice (GLP) is an excellence scheme that is concerned with the structural process and the circumstances under which a study is planned, performed, monitored, recorded, archived and reported (https://www.pharmatutor.org/articles/ good-laboratory-practices-global-strategy-plan-action-public-health-innovation-intellectual-property; Dolan 2007). The prime goal of GLP is to defend the steadiness, and reliability of various safety tests (nonclinical) for a number of chemicals (Nambisan 2017b). Basically, these safety related tests are intended for generating different data factors that are usually related to physicochemical properties, toxicity properties (nonclinical) etc. and for their utilization by the governing experts in order to make appropriate decision on the risk/safety of any product Nambisan 2017b). Initially the GLP policies were made for toxicity antalysis and are only concerned to those laboratories dealing with animal experiments, but now a days GLP is followed by all the laboratories which are regulated by various authorities such as the food and the drug administration (FDA). GLP is now made mandatory for clinical trials (Nambisan 2017b). Similarly, handling of different types of microbes in the laboratory also comes under the GLP and safe handling is verymuch essential to ensure the health of the working person, the community and above all the environment (Nambisan 2017b). Some of the common GLP as pointed out by Nambisan (2017b) are as follows • All tests should be conducted by qualified personnel inside the allotted area in a laboratory. • Each experimental project need to have a principal investigator who should take whole responsibility of the testes and experiments conducted in a laboratory. • Quality Assurance Unit should audit the overall experimental results. • All laboratory experiments need to be carried out as per the written and filed management approved Standard Operating Procedures (SOPs). The SOPs should include the strategies, management, equipment action, technical action, and analytical procedures. • All control and test samples and working chemicals need to be properly lebelled, marked and dated with info concerning its source, pureness, constancy, concentration, storage settings, and expiration date.

7.8 Principles of GLP

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Table 7.3  Relation of probable risk groups to Biosafety Levels, Practices, and Equipment Risk group Biosafety level Basic – Biosafety 1 Level 1 2 Basic – Biosafety Level 2

3

Containment – Biosafety Level 3

4

Maximum containment – Biosafety Level 4

Laboratory type Basic teaching, research Primary health services; diagnostic services, research Special diagnostic services, research Dangerous pathogen units

Laboratory practices GMT

Safety equipment None; open bench work

GMT plus Open bench plus BSC for protective clothing, potential aerosols biohazard sign

As Level 2 plus special clothing, controlled access, directional airflow As Level 3 plus airlock entry, shower exit, special waste disposal

BSC and/or other primary devices for all activities

Class III BSC, or positive pressure suits in conjunction with Class II BSCs, double-ended autoclave (through the wall), filtered air

BSC biological safety cabinet, GMT good microbiological techniques Reproduced with permission from Nambisan (2017b)

• The equipment need to be properly maintained, standardised, and must be intended to meet the required analytical procedures. In order to safeguard the safety of a laboratory, a comprehensive laboratory guideline has been prepared by the World Health Organization (WHO) and the National Institutes of Health (NIH) (Nambisan 2017b). Both the concerned authorities have recommended for cataloging of different biological agents based on their possibility of causing damage to the human and environment (Table  7.3). Basically, four types of biosafety levels are endorsed by the authorities in order to deal with the highly risk organisms (https://www. studymode.com/essays/Assg-75515575.html; Nambisan 2017b).

7.8

Principles of GLP

The various pronciples of the GLP includes the following points such as organization of laboratory and the laboratory personnel, maintenance of quality, facilities in the laboratory, equipment, reagents and materials used in the laboratory, tests and the reference items, safety procedures, results and data interpretation etc. (https://www.pharmatutor.org/articles/ good-laboratory-practices-global-strategy-plan-action-public-health-innovationintellectual-property).

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The summary of the GLP principles are as below (https://www.pharmatutor.org/articles/good-laboratory-practices-global-strategy-plan-action-public-health-innovationintellectual-property; Nambisan 2017a, b). • Management of the society and employees –– Management of various duties –– Sponsor duties –– Duty of the principal investigator –– Duty of the laboratory scientists and researchers • Quality assurance program –– Quality Assurance Staffs • Different services –– Services for Test and Reference Items –– Equipment, reagents and materials • Test structures –– Physical/Chemical –– Biological • Items for the tests and for reference purpose • Standard working procedures • Presentation of the Study –– Study Plan –– Conduct of Study • Interpretation of the outcome • Storage of data and report preparation.

Related Questions 1. What are the different forms of IPR as per TRIPS agreement? 2. Write the importance of ethical issues in clinical research. 3. What are the characteristic features of hazard? 4. What are the different steps of risk assessment? 5. What is the need for Biosafety? 6. What do you understand by Biosafety level? 7. Classify microbes into different groups of risk groups as per NIH guidelines. 8. What is bioethics? 9. What are the Rights of Patentee? 10. What are the different steps of patent application? 11. What is HACCP? 12. How patent is different from trade secret? 13. Why IPR is important for new drug development? 14. What are GLP and GMP?

References

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5. What is the importance of GLP? 1 16. What are the principles of biosafety? 17. What are the patentable ingredients in biotechnology? 18. Whether plants can be patented? 19. Plant Breeder’s Rights? 20. What is the validity period of Patent?

References Allan DJ, Blackshaw JK (1986) Ethics, welfare, and laboratory animal management. In: Fox MW, Mickley LD (eds) Advances in animal welfare science 1986/87. The Humane Society of the United States, Washington, DC, pp 1–8 Atkinson JDM, Jones R (2009) Intellectual property and its role in the pharmaceutical industry. Future Med Chem 1:1547–1550 Balls M (1983) Alternatives to experimental animals. Vet Rec 113(7):398–401 Boyd MR (1996) The position of Intellectual Property Rights in drug discovery and development from natural products. J Ethnopharmacol 51:17–27 Caulfield T, Brownsword R (2006) Human dignity. a guide to policy making in the biotechnology era? Nat Rev Genet 7:72–76 Caulfield T, von Tigerstrom B (2006) Gene patents, health care policy and licensing schemes. Trends Biotechnol 24:251–254 Chambers IP, Smith AG (2005) US6875608 Cockburn Iain M 2009 Chapter 5, Intellectual Property Rights and pharmaceuticals: challenges and opportunities for economic research. In: The economics of intellectual property. World Intellectual Property Organization, pp 150–179 Cockburn IM Intellectual Property Rights and pharmaceuticals: challenges and opportunities for economic research. http://www.wipo.int/edocs/pubdocs/en/wipo_pub_1012-chapter5.pdf. Accessed on 24 Oct 2018 Crespi RS (2005) Ethico-legal issues in biomedicine patenting: a patent professional viewpoint. Sci Eng Ethics (1, 1):117–136 Dani C, Chambers IP, Beuhr ML, Smith AG (1997) WO9730151 Dasilva K (2005) MXPA05011752 Dolan K (2007) Laboratory animal law: legal control of the use of animals in research. Blackwell, Oxford Dutfield G. 2003. Ethics and biosafety|Intellectual Property Rights. In: Encyclopedia of applied plant sciences, pp 284–289 Dutfield G (2005) Intellectual Property Rights and gene-based technologies for animal production and health. In: Makkar HPS, Viljoen GJ (eds) Applications of gene-based technologies for improving animal production and health in developing countries. Springer, Dordrecht, pp 449–520 Ema H, Nakauchi H, Osawa K (2003) WO03076613 Festing S, Wilkinson R (2007) The ethics of animal research. Talking point on the use of animals in scientific research. EMBO Rep 8(6):526–530 Friedlander, M., Otani, A., Da Silva, K., Moreno, S. H.: US2005063961 (2005) http://www.innovaccess.eu/definition-ip. Accessed on 23 Oct 2018 https://ocpatentlawyer.com/four-types-intellectual-property-protect-idea/. Accessed on 23 Oct 2018

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https://www.enago.com/academy/important-ethical-considerations-animal-studies/. Acessed on 25 Oct 2018 https://www.iipta.com/role-of-ipr-in-the-pharmaceutical-industry/. Accessed on 24 Oct 2018 https://www.innovationpolicyplatform.org/content/types-ipr. Accessed on 23 Oct 2018 https://www.ipeg.com/ip-risk-management-how-to-deal-with-it-part-1/. Accessed on 25 Oct 2018 https://www.linkedin.com/pulse/intellectual-property-risk-assessment-top-down-vs-bottom-oconnell. Accessed on 25 Oct 2018 https://www.lotempiolaw.com/2012/06/blog-2/animal-patents/. Accessed on 24 Oct 2018 https://www.pharmatutor.org/articles/good-laboratory-practices-global-strategy-plan-action-publichealth-innovation-intellectual-property. Accessed on 25 Oct 2018 https://www.studymode.com/essays/Assg-75515575.html. Acessed on 17 Dec 2018 Nakatsuji N, Tada T, Tada M (2004) WO04072226 Nakatsuji N, Tada T, Tada M (2006) JP06042663 Nambisan P (2017a) Relevance of Intellectual Property Rights in biotechnology. In: Nambisan P (ed) An introduction to ethical, safety and Intellectual Property Rights issues in biotechnology. Academic/Elsevier, London, pp 291–309 Nambisan P (2017b) Laboratory biosafety and good laboratory practices. In: Nambisan P (ed) An introduction to ethical, safety and Intellectual Property Rights issues in biotechnology. Academic/ Elsevier, London, pp 253–271 Otani A (2006) MXPA05011752 Rao DD, Sitnicka E, Bartelmez SH, Hagen FS (1998) WO9818486 Rendon EM, Blake DJ (2007) Patenting human genes and stem cells. Recent Pat DNA Gene Seq 1:25–34 Robson P, Rodda D, Ng Huck H (2006) WO06025802 Smith AG, Burdon TG (2005a) US2005255573 Smith AG, Burdon TG (2005b) US2005196859 Svendsen O, Sandoe P, Thorn NA (1997) Laboratory animal science, welfare and ethics in pharmacology and toxicology. Pharmacol Toxicol 80:3–5 Terstappen LW, Loken MR, Huang S, Olweus J, Lund-Johnsson F (1995) EP0662512 Terstappen LW, Loken MR, Huang S, Olweus J, Lund-Johansen F (1998) US5840580 Thomson JA (1998) Primate embryonic stem cells. US5843780 WIPO (2004) WIPO intellectual property handbook, Reprinted 2008. Retrieved from http://www. wipo.int/edocs/pubdocs/en/intproperty/489/ WTO (1994) Agreement on trade-related aspects of Intellectual Property Rights, Articles 15 and 39 (2), World Trade Organization, Geneva. http://www.wto.org/english/tratop_e/trips_e/t_agm0_e. htm Yamanaka N (2005) JP2005110565