A Practical Guide To Pharmacological Biotechnology - Patra 2019

Learning Materials in Biosciences
Jayanta Kumar Patra
Swagat Kumar Das
Gitishree Das
Hrudayanath Thatoi
A Practical
Guide to
Pharmacological
Biotechnology
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Jayanta Kumar Patra • Swagat Kumar Das
Gitishree Das • Hrudayanath Thatoi
A Practical Guide to
Pharmacological
Biotechnology
Jayanta Kumar Patra Swagat Kumar Das
Research Institute of Biotechnology & Medical Department of Biotechnology
Converged Science College of Engineering and Technology,
Dongguk University Biju Patnaik University of Technology
Goyang-si, Gyeonggi-do Odisha
South Korea India
Gitishree Das Hrudayanath Thatoi
Dongguk University Department of Biotechnology
Goyang-si, Gyeonggi-do North Orissa University
South Korea Odisha
India
ISSN 2509-6125 ISSN 2509-6133 (electronic)

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

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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 bio-
chemical 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 chemi-
cals that affect normal or abnormal biochemical function. The field encompasses drug
composition and properties, synthesis and drug design, molecular and cellular mecha-
nisms, organ/systems mechanisms, signal transduction/cellular communication, molecu-
lar 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 regula-
tions, 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 begin-
ning graduate and master students of Pharmacy, Clinical Science, and Biotechnology field
to the techniques of pharmacology. There are few books available on practical pharmacol-
ogy 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 experi-
ments 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
vi 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 Jayanta Kumar Patra

Odisha, India Swagat Kumar Das
Gyeonggi-do, South Korea Gitishree Das
Odisha, India 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
vii
viii Contents
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
Contents ix
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
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
x Contents
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
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 pharmaco-
logical 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.
xi
xii 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 biotechnol-
ogy, upstream and downstream process engineering, biopro-
cess 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 practi-
cal 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 xiii
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 activ-
ities 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 technol-
ogy for mine waste reclamation. Professor Thatoi has han-
dled 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 guid-
ance for their dissertation works. Professor Thatoi has pub-
lished 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.
General Aspects of Pharmacology
Laboratory 1
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 labora-
tory including pharmacology laboratory. The primary objective of laboratory safety stan-
dard 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 hazard-
ous 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 dif-
ferent 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 1

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
2 1 General Aspects of Pharmacology Laboratory
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 poten-
tial 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 3
1.1.3 Drinks, Eatables and Smoking
It is a thumb rule, that any type of drinking and eating is strictly prohibited inside the labo-
ratory, 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, sepa-
rate 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 avoid-
ing 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
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 per-
mission from the in charge authority.
Safety and storage cabinets for flammable chemicals. (https://www.justrite.com/safety-and-storage-

cabinets.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 5
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 radioiso-
topes. Safe disposal of radioisotopes is one of the primary matter of concern for the labora-
tory 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.
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 every-
one 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 loca-
tion 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 7
(a) Rana tigrina
Name of the animal: Rana tigrina

Common English name: Frog
Strain used: Rana esculenta, Rana pipiens and Rana temporaria
Pharmacological uses: 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-complete-

detail/)
(b) Mus musculus (common house rat)
Name of the animal: Mus musculus

Common English name: House mouse
Strain used: Balb-c and Swiss albino.
Pharmacological uses: Widely used in different toxicity studies.
Study related to genetic and cancer research.
Screening of analgesic and anticonvulsants.
Toxicological and teratogenic study.
Common house mouse. (Source: https://en.wikipedia.org/wiki/File:House_mouse.jpg)
(c) Rattus rattus (brown rat)
Name of the animal: Rattus rattus

Common English name: Rat
Strain used: Albino rats of wistar strain, Sprague-Dawley, Wistar Kyoto, Lewis.
Pharmacological uses: 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: Cavia procellus

Common English name: Guinea pig, cavy or domestic cavy
Strain used: Dunkin-Hartley, Peruvian,
Pharmacological uses: 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 9
Guinea pig
(e) Oryctolagus cuniculus (rabbit)
Name of the animal: Oryctolagus cuniculus

Common English name: Rabbit
Strain used: New Zealand white, American Dutch, Himalayan Black
Pharmacological uses: Pyrogen testing.
Irritancy tests.
Pharmacokinetics studies.
Bioassay for different drugs e.g. antidiabetic, hormonal etc.
Rabbit
(f) Mesocricetus auratus (hamster)
Name of the animal: Mesocricetus auratus

Common English Hamster
name:
Strain used: Mesocriceius auratus, Cricetulus griseus
Pharmacological uses: 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-right-

one-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 irre-
spective 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, labo-
ratory 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 11
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 humid-
ity 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 pre-
ferred 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 con-
troller 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 recom-
mended 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 observa-
tion 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.
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 environ-
ment, 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 pro-
viding 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 13
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, uncontami-
nated 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 contain-
ers 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, spe-
cies 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 thera-
pies 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 com-
ponents 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
Fig. 1.2 Sequence of Experimental designing
Fig. 1.3 Steps of experimental design
experiment should include different factors such as hypothesis, controls, statistical analy-
sis 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 15
1.5 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, envi-
ronmental 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

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 chemis-
try of the toxins and their concentration. Several instruments such as UV-visible spectro-
photometer, infrared spectroscopy, gas chromatography, high pressure liquid
chromatography, and immunoassay techniques are used to quantify the toxins. The toxico-
logical 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 17
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-animal-
research. 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 thera-
peutics. 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
Isolated Tissues and Organs
2
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.

20 2 Isolated Tissues and Organs
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 21
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 physi-
ological salt solution or drug solution in to an isolated organ (tissues) or for administra-
tion 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
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 con-
trolled 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 ther-
mostatically (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 res-
ervoir 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 tem-
perature 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
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 effi-
cient 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
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/flua-
nisone (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.
2.5 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
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 pre-
vent 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 pro-
viding 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.
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
Katzung BG, Masters SB, Trevor AJ (2012) Basic & clinical pharmacology. McGraw-Hill, New York
Ritter JM, Lewis LD, Mant TGK, Ferro A (2008) A text book of clinical pharmacology and
therapeutics. Hodder Arnold, London
Sierra LM, Gaivao I (eds) (2014) Genotoxicity and DNA repair: a practical approach. Springer
protocols. Humana Press, London
Delhi
Screening of Drugs Using Cell Lines/Isolated
Tissues/Intact Animals 3
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

30 3 Screening of Drugs Using Cell Lines/Isolated Tissues/Intact Animals
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)
1. Healthy, adult male Balb/c mice weighing 28–37 g are maintained in controlled tem-
perature 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
Glucose level Glucose level Glucose level Glucose level % of change in
Mice (0th day) (7th day) (14th day) (28th day) Blood glucose level
Group-1
Group-2
Group-3
Interpretation
The percentage in change in blood glucose level in drug treated mice (Group-3) as com-
pared 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.
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 metab-
olized 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

2-DG6P standard (0.1 mM of 2-DG6P standard is prepared by diluting 2-DG6P with deu-
terated 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).
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.

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 penetrat-
ing 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).
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
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.

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 Treatment with drug Ulcer Index
Un Us Un Us
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.
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)
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 hepatocel-
lular enzymes e.g. ALT and AST are then evaluated from the supernatant by using
standard kit.
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 hepato-
protective 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.
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
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 tis-
sues are kept at −70 °C for estimation of lipid peroxidation.
7. The level of lipid peroxidation (LPx) in liver homogenate is quantified by their reactiv-
ity with thiobarbituric acid in acidic conditions.

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 AST ALT ALP LPx
Group- I
Group- II
Group- III
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, mus-
tard 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.
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.
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 cara-
geenan challenge (Fig. 3.4).

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/ugo-
inflame.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 oxida-
tive 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 oxida-
tive 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
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 pos-
sess 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
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 deter-
mined spectrophotometrically at 540 nm to observe the haemolysis pattern.
9. Then the percentage of haemolysis is calculated by using the following formula.
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%
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
MgCl2 1 mM
CaCl2 1.8 mM
Glucose 25 mM
HEPES acid 10 mM
Adjust pH to 7.4.
Test compound
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 excita-
tion 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.

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 oint-
ments. The eye is supplied by both the sympathetic and parasympathetic nerves. The supe-
rior 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 parasympa-
thetic 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
(Pilocarpine 1%, Adrenaline 0.1%, Atropine 1%, Ephedrine 5%, Cocaine 1%,
Phenylephrine 3%)
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 card-
board 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).

The pupillary size, light reflex and corneal reflex are recorded after 5 min of drug instilla-
tion and observations are tabulated.
Fig. 3.5 Effect of drug on Rabbit’s eye

3.7 Evaluation of Local Anaesthetics 49
Observation Table
Sl. no. Drug Size of pupil Light reflex Corneal reflex
1 Saline Normal Present Present
2 Pilocarpine 1%, Constriction Present Present
3 Atropine 1%, Dilation Absent Absent
4 Ephedrine 5% Dilation Present Present
5 Cocaine 1% Dilation Present Absent
6 Phenylephrine 3% Dilation Present 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.
Requirements
Procaine hydrochloride
Animal holder
Cotton stop watch
Rabbit
. 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.

The corneal reflex for each eye is observed and recorded in the observation table.
Corneal reflex (minutes)
Sample Eye 1 2 3 4 5
Saline Left
Drug Right
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 mem-
branes 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)
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.

The corneal reflex for each eye is observed and recorded in the observation table.
Squeak response (minutes)
Sample Flank 1 2 3 4 5
Saline Left (C side)
Drug Right (T side)
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.
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
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
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 53
Ogu C, Maxa J (2000) Drug interactions due to cytochrome P450. Proc Bayl Univ Med Cent
13:421–423
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
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
Delhi
Vogel HG (2002) Drug discovery and evaluation pharmacological assays. Springer, Berlin
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
Genotoxicity and Toxicological Studies
4
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 pharma-
ceuticals, chemicals, pesticides, biocides, food additives, cosmetics, veterinary drugs for
protection of human and animal. The in vitro genotoxicity test comprises of bacterial

56 4 Genotoxicity and Toxicological Studies
Table 4.1 Commonly used genotoxicity test methods

Type
Sl. of
no. Test method assay Working principle
1 Bacterial reverse In Salmonella typhimurium and Escherichia coli strains are used
mutation test (Ames vitro for bacterial reverse mutation test to detect substitution,
test) 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
2 Mammalian In This test helps in identifying the factors that cause structural
chromosome vitro mutations in chromosomes. Different chromosomal changes
aberration test e.g. polyploidy and duplication are identified using this test
3 Mammalian cell In This test assesses chemical substances that can cause gene
gene mutation test vitro mutations in cell lines. Further, it can detect the different end
or, mouse points of mutation such as thymidine kinase (TK) and
lymphoma test hypoxanthine-guanine phosphor ribosyltransferase (HPRT),
and a transgene of xanthineguanine phosphor
ribosyltransferase (XPRT)
4 Comet assay In This test detects DNA damage in a variety of primary DNA
vivo lesions which other tests can’t identify. This test is also useful
for assessment of hazardous agents which have potential for
genotoxicity or mutagenicity
5 Micronuclei test or, In The mutagen exposure leads to damage and division of cell
chromosome vivo and form smaller micronucleus
aberration test
reverse mutation assay, in vitro mammalian chromosomal aberration test, in vitro mam-
malian 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 erythro-
cyte 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
In Vitro Genotoxicity Assay
4.1 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) 10 g

Citric acid monohydrate (C6H8O7·H2O) 100 g
Potassium phosphate, dibasic, anhydrous (K2HPO4) 500 g
Sodium ammonium phosphate (NaHNH4.PO4·4H20) 175 g
Warm distilled water (about 45 °C) 670 mL
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 dis-
tilled 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 0.0309 g
L-Histidine 0.0244 g
Distilled water 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) 25 mL

NADPa 0.1 M 2 mL
D-Glucose-6-phosphate (1 M) 0.25 mL
KCl (1.65 M)–MgCl2 (0.4 M) salts 1 mL
Liver S-9 fraction (4%) 2 mL
Distilled water 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 0.07654 g
Sterile distilled water 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)

In Vitro Genotoxicity Assay
4.1 59
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.

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 com-
pound 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.
4.2 Mouse Lymphoma Assay
Background
The Mouse Lymphoma Assay (MLA), also known as in vitro mammalian cell gene muta-
tion test is used to study mutations induced by different chemicals or/and their metabo-
lites. 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) phos-
photransferase enzyme that plays an important role in metabolism of pyrimidine nucleo-
tide 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 trifluoro-
thymidine (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 com-
pound. 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 gly-
cine (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) 25 mL

NADPa 0.1 M 2 mL
D-Glucose-6-phosphate (1 M) 0.25 mL
KCl (1.65 M)–MgCl2 (0.4 M) salts 1 mL
Liver S-9 fraction (4%) 2 mL
Distilled water 19.75 mL
4. Methylmethanesulphonate (MMS)
4.2 Mouse Lymphoma Assay 61
5. Cyclophosmphamide
6. Benzo(a)pyrene
7. Test compound
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.

Mutant Frequency (MF) is determined from the plating efficiencies of mutant colonies
(PEM) and plating efficiencies of viable cells (PEV) from the same culture.
PEm
Mutant frequency ( MF ) =
PEv
PEm and PEv are calculated by using the number of colonies and the total number of cells
used for the cloning:
Cm
PEm =
Tm
Cv
PEv =
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 dam-
age 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) 146.1 g

EDTA (100 mM) 37.2 g
Trizma base (10 mM) 1.2 g
NaOH 8 g
dH2O Volume make up to 1000 ml
Electrophoresis Buffer (300 mM NaOH/1 mM EDTA):

Prepare of stock solution:
NaOH (10 N) 200 g NaOH dissolved in 500 ml of dH2O

EDTA (200 mM) 14.89 g of EDTA is dissolved in 200 ml dH2O
Stored at room temperature
For preparation of 1× running electrophoresis buffer
NaOH (10 N) 30 ml

EDTA (200 mM) 5.0 ml
dH2O 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) 48.5 g

dH2O 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.
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 lys-
ing 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 com-
pletely 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 dis-
tilled water to remove excess stain.
8. The cover slip is then placed over it.
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.
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.
In Vitro Teratogenicity Testing
4.4 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 dihy-
drate, made up to 100 ml with distilled water
Penicillin G (benzylpenicillin)
Sodium
5-fluorouracil (Sigma F-6627)
Test compound
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 pos-
sible. 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.

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 concentra-
tions 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
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.
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 fixa-
tion method in biomedical research. Formalin (formaldehyde) is commercially avail-
able 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 concen-
trations 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.
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).

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 symp-
toms 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 Symptoms
Benzodiazepines and other hypnosedatives, alcohol Coma, hypotension, flaccidity
Opioids Coma, pinpoint pupils, hypoventilation
Tricyclic antidepressants, phenothiazines; other drugs Coma, dilated pupils, hyper-reflexia,
with anticholinergic properties tachycardia
Amphetamines, MDMA, anticholinergic agents Restlessness, hypertonia, hyper-reflexia,
pyrexia
Tricyclic antidepressants, phenothiazines, carbon Convulsions
monoxide, monoamine oxidase inhibitors, mefenamic
acid, theophylline, hypoglycaemic agents, lithium,
cyanide
Salicylates Tinnitus, overbreathing, Salicylates
pyrexia, sweating, flushing, usually alert
Table 4.3 Methods of poison elimination

Drug poisoning Method
Salicylates, phenobarbital Alkaline diuresis
Salicylates, methanol, ethylene glycol, lithium, phenobarbital Haemodialysis
Barbiturates, theophylline, disopyramide Charcoal haemoperfusion
Salicylates, theophylline, quinine Gastro-intestinal dialysis
Most anticonvulsants, digoxin Multiple-dose activated charcoal
Table 4.4 Commonly used antidotes for treatment in case of drug overdosing
Overdose drug Antidote
Paracetamol Acetylcysteine i.v., Methionine p. o.
Iron Desferrioxamine
Cyanide 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 chil-
dren. 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 75
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
Corvi R, Madia F (2017) In vitro genotoxicity testing – can the performance be enhanced? Food
Chem Toxicol 106:600–608
Ostling O, Johanson KJ (1984) Microelectrophoretic study of radiation-induced DNA damages in
individual mammalian cells. Biochem Biophys Res Commun 123:291–298
New Delhi
Delhi
Vogel HG (2002) Drug discovery and evaluation pharmacological assays. Springer, Berlin
Experimental Animal Studies
5
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 experi-
mental needs. To minimize risk of injury to the animal and personnel working in the labo-
ratory appropriate physical or chemical restraint should be employed. The blood samples
can be collected from experimental animals by employing the following techniques.

78 5 Experimental Animal Studies
• 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 uncon-
scious 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 vol-
umes 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 effec-
tive 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.
(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, effec-
tive 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 compli-
cations 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, cor-
neal 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
purposes, as a number of diseases and physiological processes alter its characteristic val-
ues 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 High count may indicate Low count may indicate
Neutrophils 60–70 Bacterial infection, Radiation exposure, drug
burns, stress, toxicity, vitamin B12
inflammation deficiency, systemic lupus
erythematosus (SLE)
Eosinophils 1–5 Allergic reactions, Drug toxicity, stress
parasitic infections,
autoimmune diseases
Basophils 0–1 Allergic reactions, Pregnancy, ovulation, stress,
leukemias, cancers, hyperthyroidism
hypothyroidism
Lymphocytes 20–30 Viral infections, some Prolonged illness,
leukemias immunosuppression
Monocytes 2–6 Viral or fungal Bone marrow suppression
infections, tuberculosis,
some leukemias, other
chronic diseases
Fig. 5.5 Characteristic different blood cells
Principle
The white blood cell differential count determines the number of each type of WBCs pres-
ent 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 affin-
ity towards acidic components of the nucleus and cytoplasm and stain basophil granulo-
cytes 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 eosino-
phil 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)
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 objec-
tive (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 fol-
lowing heading: neutrophil, eosinophil, basophil, lymphocyte and monocyte.
14. Then the percentage of each type is calculated and compared it with normal count.

Type of cells Characteristic feature % of cells
Neutrophils Nucleus
Multilobed with varying shape and blue violet in
colour
Cytoplasm
Pink, with irregularly distributed red/violet
granules
Eosinophils Nucleus
Bilobed
Cytoplasm
Pink with regularly distributed yellowish red
granules
(continued)
Type of cells Characteristic feature % of cells

Basophils Nucleus
Kidney shape Blue violet in colour
Cytoplasm
Pink with irregularly distributed blue granules
Lymphocytes Nucleus
Big, chromatin-rich, purple/violet in colour
Cytoplasm
Very thin with blue in colour and without any
granules
Monocytes Nucleus
Lobular with purple/violet colour
Cytoplasm
Pale blue-greyish with no visible granules
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).
Table 5.2 Types of blood groups

ABO blood Antigen present on the red blood Antibodies regularly present in the serum/
group cells plasma
A A Anti-B
B B Anti-A
AB A and B None
O Neither A or B 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. Microscope
2. Needle
3. Alcohol
4. Spirit Lamp
5. Cotton
6. Micropipette
. Antiserum (A, B and D)

7
8. Glass slide
. 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, anti-
B 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 Anti-D Blood group
− − + O +ve
− − − O −ve
+ − + A +ve
+ − − A −ve
− + + B +ve
− + − B −ve
+ + + AB +ve
+ + − AB −ve
“+”, agglutination; “−”, agglutination
Interpretation (Fig. 5.6)
So the blood group is ………..
Fig. 5.6 Analysis of ABO blood group

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 mer-
curic 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.)
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 solu-
tion 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.
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 oxyhaemo-
globin (HbO2) method.
Objective
To calculate the % of haemoglobin in human blood by Sahli’s haemometer.
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 haemome-
ter 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

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.

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.
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
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 with-
out 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).

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...
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).

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
5.3 Pyrogen Testing 95
• 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)
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 thermom-
eter 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 thermom-
eter 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.
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 polyphe-
mus 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 quantifi-
cation 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 acti-
vates 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
5.4 Effect of Drug on Central Nervous System 97
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)
. 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 nega-
tive 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 posi-
tive 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
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
. 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.

Observation Table
Group no. Drug Animal Time of onset (mean) Time of recovery (mean)
1 Saline 1
2
3
2 Pentobarbitone sodium 1
2
3
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
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
Requirements
Mice (20–25 g)
Drug (Chlorpromazine)
Photoactometer
. The animals are weighed and numbered.

1
2. The photoactometer is switched on and each mouse is placed individually in the activ-
ity 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.

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 mecha-
nisms, without significantly altering consciousness is known as analgesic drug. The anal-
gesic 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
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)
. 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.

Observation Table
Reaction time in seconds after drug administration
Group React time (in (in seconds)
no. Drug seconds) 15 min 30 min 60 min 90 min 120 min
1 Saline
2 Paracetamol
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.
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)

Mice (20–25 g)
Eddy’s hot plate
. 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.

Observation Table
Gr. React time (in Reaction time in seconds after drug administration (in seconds)
no. Drug seconds) 15 min 30 min 60 min 120 min
1 Saline Paw Jump Paw Jump Paw Jump Paw Jump Paw Jump
licking response licking response licking response licking response licking response
2 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.
5.5 Experiment on Cardiovascular System 103
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)
Mice (20–25 g)
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.

Observation Table
Group no. Drug Number of wriths
1 Saline
2 Morphine
3 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 nutri-
ents can be distributed to different parts of the body. The heart rate is controlled by the
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%)
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.
5.5 Experiment on Cardiovascular System 105
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 con-
trol and drug responses.
12. The next drug response is recorded only after the heart rate has returned to the approx-
imate original value.
13. All the observations are tabulated.

Observation Table
Drugs Heart rate Amplitude Tone
Adrenalin (0.01%)
Digoxin (0.05%)
Propanolol (0.2%)
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%)
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.
5.6 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 mus-
cles. 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
. 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.
5.6 Experiments on GI Tract 107
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.

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?
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
New Delhi
Delhi
Clinical Trials
6
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 multidisci-
plinary science that includes professionals of different field such as medicine, pharmacol-
ogy, pharmacy, biomedical science and nursing that evaluate the different aspects of
interaction between drugs on human. This further includes discovery of new drugs, appli-
cation of drugs as therapeutic agents, beneficial and harmful effects of drugs on individu-
als 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

110 6 Clinical Trials
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 physi-
ology 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 loga-
rithm 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 pharmacologi-
cal 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).
*Pharmacodynamics
(Interaction between drugs and
body)
*Pharmacokinetics
Pharmacology (Absorption, distribution,
metabolism, excretion of drugs)
*Evaluation of drug
Therapeutic *Therapeutic trials
evaluation *Pharmacoepidemiology
Clinical Pharmacology *Pharmacovigilance
Control *Rational prescribing

*Official regulations of medicines
*Use and misuse of medicines
*Pharmacoeconomics
Fig. 6.1 Different aspects of clinical pharmacology

6.1 Clinical Pharmacology and Its Genesis 111
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
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. elimi-
nation 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 ves-
sels 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 motil-
ity, 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 hav-
ing 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 influ-
ence 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 113
6.2 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 regula-
tory 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.
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-
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 Selectivity assessed

activity with different in battery of HTS assay
samples
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 therapeu-

tic 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).
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
Computer-aided
design: Protein
DESIGN CYCLE crystallography,
NMR, searches
Biological in 3D databases,
testing Structure-activity de novo design
relationships, QSAR,
molecular modelling
Lead
Optimisation
Candidates for
further drug Investigational
development new drug (IND) New drug
Fig. 6.6 Design cycle for lead optimization desired action

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.
6.4 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 informa-
tion described in the general investigational plan. However, adverse event is termed
serious in case of life-threatening adverse event, inpatient hospitalization or prolonga-
tion of existing hospitalization, a persistent or significant incapacity or substantial dis-
ruption 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, medi-
cal 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 119
6.5 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 car-
ried 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 Targeted disease Unethical practice
1 Tuskegee Effects of syphilis in Subjects were not informed about their
syphilis trials humans disease and were denied treatment
(1932–1972)
2 Manhattan Effects of radiation The participants included pregnant women,
Project disabled children, hospital patients, and
(1940–1960) enlisted military personnel and most of them
had no knowledge of the experiments
3 Thalidomide Control nausea in the early Patients were however not informed that the
disaster, 1950 stages of pregnancy drug was investigational, in the testing phase
of the regulatory process
4 Gene therapy, Gene therapy for Ornithine Failure to disclose in the informed consent
1999 Trans Carbamylase documentation the risks involved in the use
of adenoviral vectors
5 TeGenero Rheumatoid arthritis and B Deviations from the approved protocol were
clinical trial, cell chronic lymphocyte alleged. The dose was to be administered at
2006 leukemia 2-h intervals, but was apparently done at
10-min intervals.
6 Bial clinical Treatment of anxiety and Unethical conduct of the trial include the
trial, 2016 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
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 arti-
cle 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 synop-
sis 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 participa-
tion. They might have also faced retaliatory response from senior members of a hierar-
chy 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.
6.6 Ethics in Clinical Research 121
• 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 com-
promise 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 implement-
ing 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 con-
stituted 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.
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 monitor-
ing 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.
6.6 Ethics in Clinical Research 123
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 justi-
fies 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
consent from individual participant is necessary and it should be replaced by permis-

sion of any other person.
20. 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.
21. 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.
22. 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.
23. 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.
24. 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.
25. 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 institu-
tions, 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 125
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
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
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
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
Ritter JM, Lewis LD, Mant TGK, Ferro A (2008) A text book of clinical pharmacology and thera-
peutics. Hodder Arnold, London
New Delhi
Delhi
IPR and Ethics in Animal Studies
7
7.1 Introduction
Intellectual property rights (IPR) are legal and official strategies that are assigned to pro-
tect 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.

128 7 IPR and Ethics in Animal Studies
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 misus-
ing 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 techni-
cal features to be published (https://ocpatentlawyer.com/four-types-intellectual-property-
protect-idea/; http://www.innovaccess.eu/types-of-ip).
Basically there are two types of patents i.e. utility type and design type. The utility pat-
ent 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 immedi-
ately 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 origi-
nal 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 protec-
tion 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 129
Table 7.1 Types of Intellectual Property Rights (IPRs) and their significant properties
International
Type of IPR What does it cover Term of the IPR conventions
Patent Inventions 20 years from the Paris
date of filing Convention
Copyright and Forms of creativity primarily Life of the author Berne
related Rights concerned with mass and not less than Convention
communication, e.g., books, 50 years after the
paintings or drawings, music, death of the author
poems
Traditional Cultural Music, musical instruments, stories, Undertaken in
Expressions (TCEs) art, handicrafts, words, names and cooperation
insignia, performances, textiles, with UNESCO
carpet, jewelry designs, forms of
architecture
Trademark (t), Signs that individualizes the goods Usually 10 years, Madrid
Service Marks, of a given enterprise and renewed Agreement
Collective Marks, distinguishes them from the goods indefinitely on
and Certification of its competitors payment of
Marks additional fees
Industrial Designs Formal or ornamental appearance Usually 15 years, Hague
for mass produced items (original renewal is usually Agreement
ornamental or nonfunctional subject to payment
features that result from design of renewal fees
activity)
Integrated Circuits Layout designs (topographies) of Usually 15 years
integrated designs
Geographical Names and symbols which indicate Indefinitely TRIPS
Indications (GI) a certain geographical origin of a Agreement
given product—applied to products
whose quality and characteristics
are attributable to their geographical
origin
Appellations of Special kind of indication of source Indefinitely Lisbon
Origin (AO) Agreement
Protection against Confidential business information Indefinitely, until
unfair competition, which provides an enterprise a public disclosure
Trade secrets competitive edge (manufacturing or of the secret occurs
industrial secrets and commercial
secrets)
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
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 131
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-the-
pharmaceutical-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,
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 manufac-
ture 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 133
• 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 contro-
versy (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 Publication
number Ref. no. and title Inventors date
US5843780 [Thomson 1998] Primate embryonic Thomson, J.A. 1998/12/01
stem cells
WO9730151 [Dani et al. 1997] Cytokine expressed Dani, C., Chambers, 1997/08/21
by DIA/LIF-deficient embryonic stem I.P., Beuhr, M.L.,
cells for the inhibition of Smith, A.G.
differentiation
US6875608 [Chambers and Smith 2005] Chambers, I.P., Smith, 2005/04/05
Pluripotency determining factors and A.G.
use thereof
US2005255573 [Smith and Burdon 2005a] Propagation Smith, A.G., Burdon, 2005/11/17
and/or derivation of embryonic stem T.G.
cells
US2005196859 [Smith and Burdon 2005b] Smith, A.G., Burdon, 2005/09/08
Propagation and/or derivation of T.G.
embryonic stem cells
EP0662512 [Terstappen et al. 1995] Human Terstappen, L.W., 1995/07/12
hematopoietic stem cells Loken, M. R., Huang,
S., Olweus, J., Lund-
Johnsson, F.
US5840580 [Terstappen et al. 1998] Phenotypic Terstappen, L. W., 1998/11/24
characterization of hematopoietic stem Loken, M. R., Huang,
cells S., Olweus, J., Lund-
Johansen, F.
WO9818486 [Rao et al. 1998] Use of leptin to Rao, D.D., Sitnicka, E., 1998/05/07
stimulate hematopoiesis Bartelmez, S.H.,
Hagen, F.S.
WO03076613 [Ema et al. 2003] Protein sustaining Ema, H., Nakauchi, H., 2003/09/18
undifferentiated stem cells as such Osawa, K.
US2005063961 [Friedlander et al. 2005] Ema, H., Nakauchi, H., 2005/03/24
Hematopoietic stem cells and methods Osawa, K.
of treatment of neovascular eye
diseases therewith
MXPA05000972 [Dasilva 2005] Hematopoietic stem Dasilva, K. 2005/09/02
cells and methods of treatment of
neovascular eye diseases therewith
MXPA05011752 [Otani 2006] Hematopoietic stem cells Otani, A. 2006/06/06
and methods of treatment of
neovascular eye diseases therewith
JP06042663 [Nakatsuji et al. 2006] Discrimination Naatsuji, N., Tada, T., 2006/02/16
marker of ES cell Tada, M.
WO04072226 [Nakatsuji et al. 2004] Marker for the Naatsuji, N., Tada, T., 2004/08/26
undifferentiated state of cell and Tada, M.
composition and method for separation
and preparation of stem cells
JP05110565 [Yamanaka 2005] Agent for keeping Yamanaka, N. 2005/04/28
differentiation/pluripotency
WO06025802 [Robson et al., 2006] Method for Robson, P., Rodda, D., 2006/03/09
maintaining pluripotency of stem/ Ng Huck, H.
progenitor cells
Reproduced with permission from Rendon and Blake (2007)
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.
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 experi-
ments 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.
7.6 Risk Assessment and Management in IPR 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/important-
ethical-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-ethical-
considerations-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/intellectual-
property-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 man-
agement of the IP risk factors in a (Svendsen et al. 1997). It is formerly about the certifica-
tion, 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-
property-risk-assessment-top-down-vs-bottom-o-connell; https://www.ipeg.com/ip-risk-
management-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 struc-
tural process and the circumstances under which a study is planned, performed, moni-
tored, recorded, archived and reported (https://www.pharmatutor.org/articles/
good-laboratory-practices-global-strategy-plan-action-public-health-innovation-intellec-
tual-property; Dolan 2007). The prime goal of GLP is to defend the steadiness, and reli-
ability 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 manage-
ment 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 139
Table 7.3 Relation of probable risk groups to Biosafety Levels, Practices, and Equipment
Risk Laboratory
group Biosafety level Laboratory type practices Safety equipment
1 Basic – Biosafety Basic teaching, GMT None; open bench work
Level 1 research
2 Basic – Biosafety Primary health GMT plus Open bench plus BSC for
Level 2 services; protective clothing, potential aerosols
diagnostic biohazard sign
services,
research
3 Containment – Special As Level 2 plus BSC and/or other primary
Biosafety Level 3 diagnostic special clothing, devices for all activities
services, controlled access,
research directional airflow
4 Maximum Dangerous As Level 3 plus Class III BSC, or positive
containment – pathogen units airlock entry, pressure suits in conjunction
Biosafety Level 4 shower exit, special with Class II BSCs,
waste disposal 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-innovation-
intellectual-property).
The summary of the GLP principles are as below (https://www.pharmatutor.org/arti-

cles/good-laboratory-practices-global-strategy-plan-action-public-health-innovation-
intellectual-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 141
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
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from natural products. J Ethnopharmacol 51:17–27
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Biotechnol 24:251–254
Chambers IP, Smith AG (2005) US6875608
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Cockburn IM Intellectual Property Rights and pharmaceuticals: challenges and opportunities for
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Crespi RS (2005) Ethico-legal issues in biomedicine patenting: a patent professional viewpoint. Sci
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Dani C, Chambers IP, Beuhr ML, Smith AG (1997) WO9730151
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Learning Materials in Biosciences

More information about this series at http://www.springer.com/series/15430

ISSN 2509-6125 ISSN 2509-6133 (electronic)

Library of Congress Control Number: 2019933573

© Springer Nature Singapore Pte Ltd. 2019

Gyeonggi-do, South Korea Jayanta Kumar Patra

1 General Aspects of Pharmacology Laboratory ���������������������������������������������� 1

3 Screening of Drugs Using Cell Lines/Isolated Tissues/Intact Animals���������� 29

5.4 Effect of Drug on Central Nervous System������������������������������������������������ 97

7.6 Risk Assessment and Management in IPR�������������������������������������������������� 137

Jayanta Kumar Patra M.Sc. Ph.D., PDF, is currently

Swagat Kumar Das B.Pharm., M.Tech., Ph.D., is presently

Gitishree Das M.Sc. Ph.D., PDF, is currently working as

Hrudayanath Thatoi M.Sc., M.Phil., Ph.D., is currently

1.1 General Safety Features in a Pharmacology Laboratory

1.1.1 Clothing and Footwear

© Springer Nature Singapore Pte Ltd. 2019 1

Laboratory aprons or lab coats and lab shoes

1.1.3 Drinks, Eatables and Smoking

1.1.4 Personal Safety and Hygiene

1.1.5 Handling Chemicals

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

1.1.6 Laboratory Work Place

1.1.8 House Keeping

Fig. 1.1 Warning symbols used in laboratory

1.1.11 Laboratory Safety Signs

1.2  ses and Management of the Laboratory Animals

Different kinds of animals are used in a pharmacology laboratory for experimentation

(a) Rana tigrina

Name of the animal: Rana tigrina

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

(b) Mus musculus (common house rat)

Name of the animal: Mus musculus

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

(c) Rattus rattus (brown rat)

Name of the animal: Rattus rattus

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

(d) Cavia procellus (Guinea pig)

Name of the animal: Cavia procellus

(e) Oryctolagus cuniculus (rabbit)

Name of the animal: Oryctolagus cuniculus

(f) Mesocricetus auratus (hamster)

Name of the animal: Mesocricetus auratus

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

1.3 Animal House Facility, Care

1.3.1 Environmental Aspects

1.3.2 Physical Facilities

1.3.2.1 Building Materials

1.3.2.2 Door, Windows, Floor

1.3.2.3 Drains, Walls, Ceilings

1.3.2.4 Storage Area

1.3.2.5 Sanitizing Facility

1.3.2.6 Experimental Area

1.3.3 Animal Care

1.3.3.1 Animal Housing System

1.3.3.2 Activity, Food, Bedding, Water

1.3.3.3 Hygiene and Cleanliness

1.3.3.4 Waste Disposal

1.3.3.5 Record Keeping

Gyeonggi-do, South Korea Jayanta Kumar Patra

1 General Aspects of Pharmacology Laboratory �� 1

3 Screening of Drugs Using Cell Lines/Isolated Tissues/Intact Animals�� 29

5.4 Effect of Drug on Central Nervous System�� 97

7.6 Risk Assessment and Management in IPR�� 137

1.1 General Safety Features in a Pharmacology Laboratory

1.1.1 Clothing and Footwear

1.1.3 Drinks, Eatables and Smoking

1.1.4 Personal Safety and Hygiene

1.1.5 Handling Chemicals

1.1.6 Laboratory Work Place

1.1.8 House Keeping

1.1.11 Laboratory Safety Signs

1.2 ses and Management of the Laboratory Animals

1.3 Animal House Facility, Care

1.3.1 Environmental Aspects

1.3.2 Physical Facilities

1.3.2.1 Building Materials

1.3.2.2 Door, Windows, Floor

1.3.2.3 Drains, Walls, Ceilings

1.3.2.4 Storage Area

1.3.2.5 Sanitizing Facility

1.3.2.6 Experimental Area

1.3.3 Animal Care

1.3.3.1 Animal Housing System

1.3.3.2 Activity, Food, Bedding, Water

1.3.3.3 Hygiene and Cleanliness

1.3.3.4 Waste Disposal

1.3.3.5 Record Keeping

1.4 Experimental Design

2.1 Basic Instruments Used for Isolated Tissue Experiments

2.2 Organ Baths

2.3 Intestinal Muscle Preparations

2.4 Skeletal Muscle Preparations

2.5 Cardiac Muscle Preparations

3.1 Evaluation of Antidiabetic Agents

3.1.1 Antidiabetic Evaluation of Drug in Induced Diabetic Mice Model

3.1.2 Glucose Uptake Assay

3.2 Evaluation of Antiulcer Activity

3.3 Evaluation of Hepatoprotective Drugs

3.3.1 In vitro Assessment of Hepatoprotective Effect

3.3.2 In vivo Evaluation of Hepatoprotective Effect

3.4 Evaluation of Anti-inflammatory Agents

3.5 Evaluation of Antioxidant Activity

3.6 Effect of Drug in Rabbit Eye

3.7 Evaluation of Local Anaesthetics

3.7.1 Evaluation of Local Anaesthetics

3.7.2 Evaluation of Local Anaesthetics