Alternative to Animal Experiment Models

Dr Jayant Rai,
Second Year Resident,
Dept of Pharmacology,
GMC, Surat.
Alternatives to Animal
Experiment Models

Overview
1. Introduction.
2. Animal Experimentation.
3. Historical Overview & Laws.
4. Alternatives to Animal Experiment.
5. Recent trends & Summary.
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Animal Experimentation
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Introduction
What is Animal Experimentation ?
• Use of animals for experimentation in
education, training and research.
• An integral part of the pharmacology
education at medical colleges, pharmacy,
veterinary institutes.
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• An essential part of basic research &
preclinical drug discovery process.
• Being conducted under the strict global
regulatory guidelines across the globe.
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Primary uses of animals in experimentation
Education Under graduate.
Post graduate.
Research Basic Research.
Applied Research.
Cosmetic Testing.
Toxicology Testing.
Xeno-Transplantation.
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Cosmetic testing
General toxicity,
Eye & Skin irritancy,
Phototoxicity & Mutagenicity.
Banned in many countries across the
globe.
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Toxicology Testing
Performed by pharmaceutical companies
or contract animal testing facilities.
New drugs must under go testing prior to
licensing.
Toxicology testing is of two types:
Acute toxicity testing.
Chronic toxicity testing.
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Acute Toxicity
Required for all drugs,
single administration,
up to lethal level,
in at least two species.
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 Chronic Toxicity
Toxic effects on a living organism,
exposed to the substance continuously or
repeatedly.
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Xeno-Transplantation
Transplanting tissues or organs.
To overcome the shortage of human
organs for transplantation.
Recently clinical trials evaluated the
transplantation of pig insulin secreting
cells in to diabetics.
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Source of Animal supply ?
Source for animals in research facilities
vary depending upon location, but most
are supplied by authorized dealers.
Which animals are used ?
Rats, mice, guinea pig, cats, dogs,
monkeys, fishes, birds, hamsters, rabbits,
horses. 12Dr. Jayant Rai

Animals used in Experiments
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Permission to carry out experiment granted
by?
The Institutional Animal Ethics Committee
(IAEC) permit experiments on small
animals.
Committee for the Purpose of Control and
Supervision of Experiments on Animals
(CPCSEA) on large animals.
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• The goal of CPCSEA
guidelines is to promote
the humane care of
animal used in
biomedical and
behavioural research and
testing.
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Historical Overview
• The greatest drug discoveries in the 19th
and 20th centuries were possible due to
the use of animals. Over the last century,
every Nobel Prize for medical research
has been dependent on animal research.
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• Aristotle & Erasistratus were the first to
use live animals in experiments.
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• Raudolf Buchheim
The first Experimental
Pharmacology laboratory in his
own house in 1849 in Germany.
• Ivan Pavlov
Used animals for performing
experiments in late 1800’s.
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• The first animal protection law was
proposed in British parliament at 1822,
promoted by Charles Darwin, Legislation
For The Protection And Regulation Of
Animals.
• The bill for Protection Law Of Animals was
passed in the year 1876.
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• The Animal Welfare Board of India was
set up in accordance with section 4 of the
Prevention of Cruelty To Animal Act
1960.
• In India Sir Ram Nath Chopra made the
beginning in pharmacological research of
traditional drugs.
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• Pioneer in the field of Experimental
Pharmacology of indigenous drugs of
India.
• In-vitro & In-vivo tests for the active
molecules of the drugs.
• Acclaimed as “Father of Indian
Pharmacology”.
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• Animal use in science has been
developed not only in drug discovery but
also in surgical procedures.
• The latest 2012 Nobel laureates in
physiology or medicine also worked on
animals.
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Law Regulating Animal Experiments
YEAR LAW
1960 Prevention of Cruelty to Animals (PCA) Act 1960, amended 1982
1964
Committee for the Purpose of Control and Supervision of
Experiments on Animals (CPCSEA)
1972 Wild life protection act
1992
Indian National Science Academy (INSA) “Guidelines for care
and use of animals in scientific research”, revised 2001
1998
“Breeding of and Experiments on Animals (Control and
Supervision) Rules, 1998”, amended 2001, 2006
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Law Regulating…..
Year Law
2001
Indian Council of Medical Research (ICMR) “Guidelines for use
of Laboratory animals in Medical Colleges”
2009
MCI amendment-Recommends to use alternatives to replace
animal experiments
2012
Ministry of Health & Family Welfare bans use of animals in
educational institutes
2013
University Grants Commission (UGC) “Guidelines for
discontinuation of dissection and animal experimentation in
zoology/life sciences in a phased manner
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Reasons For Developing alternatives
to Animal testing
• Cost-effective.
• Requires less man power & Time-saving.
• Opening up opportunities to explore
responses to existing & emerging therapies.
• Ethical concerns.
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Ethical concerns……
Poisoned,
Deprived of food, water and sleep,
Applied with skin and eye irritants,
Subjected to psychological stress,
Deliberately infected with disease,
Force fed and electrocuted.
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Scientific Support For
Animal Experimentation
• Vital for preventing, curing or alleviating
human diseases.
• The greatest achievements of medicine
have been possible only due to the use of
animals.
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Scientific Support…..
• To effectively model for biomedical
investigations.
• Medical progress would be “severely
maimed by prohibition or severe
curtailing of animal experiments,” and
“catastrophic consequences would ensue”.
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Salient drug discoveries that
involved use of animals
Year Discovery Animal Used Scientist
1901 Diphtheria/tetanus
antitoxin
Horses Emil Adolf von
Behring
1922 Insulin Dogs Frederick,
Banting, Macleod
1939 Prontosil-first
drug for bacterial
infections
Mice Gerhard Domagk
1941 Penicillin Rats Alexander
Fleming
1952 Streptomycin Chicken Selman Waksman
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Alternatives To Animal Experiments
• Test methods that avoid the use of live
animals.
• Guiding principles for more ethical use of
animals in testing are the 3R’s first
described by Russell and Burch in 1959.
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The “ 5 R’s ”
1. Replacement
Use of non-animal methods
over animal methods.
Use of lower species of
animals.
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REPLACEMENT
Dr. Jayant Rai

2. Reduction
Obtain comparable levels
of information from
fewer animals.
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REDUCTION
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To obtain more information from the
same number of animals.
To provide statistically relevant data,
while keeping animal numbers to a
minimum.
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Identify disease
Isolate protein
Find drug
Preclinical testing
GENOMICS, PROTEOMICS & BIOPHARM.
HIGH THROUGHPUT SCREENING
MOLECULAR MODELING
VIRTUAL SCREENING
COMBINATORIAL CHEMISTRY
IN VITRO & IN SILICO ADME MODELS
Potentially producing many more targets
and “personalized” targets
Screening up to 100,000 compounds a
day for activity against a target protein
Using a computer to
predict activity
Rapidly producing vast numbers
of compounds
Computer graphics & models help improve activity
Tissue and computer models begin to replace animal testing
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3. Refinement
Alleviate or minimize
potential pain, suffering
or distress.
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REFINEMENT
Dr. Jayant Rai

4. Rehabilitation
Proper care & medical
attention after
experiment is over.
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5. Responsibility
Monitor the animal &
their wellness in
captive conditions.
Enhance animal welfare and in doing so
improve the quality of science.
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Designing Alternatives
Defining an Alternative.
Developing the Alternative.
Validating the methods.
Acceptance in Scientific Community.
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Types of Alternatives
• In-Vitro Techniques.
• Micro-biological Tests.
• Micro-organisms.
• In-Chemico Testing.
• Computer simulation.
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Types of Alternatives…..
• Epidemiological, Clinical & Genetic
Studies.
• Invertebrate animals & Vertebrate
animals.
• Micro dosing.
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In Vitro Techniques
• In Vitro Pyrogen Test.
• Local Lymph Node Assay.
• Skin Patch Test.
• Neutral Red Uptake Assay.
• Cell Transformation Assay.
• Stem Cell Test.
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Source Of Tissue For
In-vitro Methods
• Avian- chick embryos.
• Rodents- rats and mice: embryonic, post-
natal and adult.
• Human –1. Neural progenitor cells from
aborted foetuses and stem
cell lines.
2. Cord blood derived stem
cells.
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Types Of Cell Culture
1) Cell lines: Mainly two types:
a. Finite cell line: limited culture life spans.
 Divide 20-100 times before extinction.
 Doublings depends on the species, cell
lineage differences, culture conditions
etc.
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b. Continuous cell line:
 Transformed, immortal & tumorigenic.
Capable of growing faster resulting in an
independent culture.
Cells have unlimited life.
2. Primary culture
3. Organ architecture preserved
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A Variety Of Tissue Cultures,
Including Immortalised Cell Lines.
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2. Cell Culture:
A promising alternative.
To create monoclonal antibodies.
Extensively used in cancer research.
Efficiently used to screen highly toxic
compounds at an early stage.
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Applications of Cell Culture
Screening of anti cancer drugs & Cancer
research.
Cell based bioassays.
In vitro screening of several drugs.
Production of anti viral vaccines.
Genetic manipulation & Gene therapy.
Recombinant DNA therapy.
Biotechnology & Molecular biology.
52Dr. Jayant Rai

In-Vitro Pyrogen Test
• Rabbit Pyrogen Test is replaced with
 Limulus Amoebocyte Lysate (LAL)
 Monocyte Activation Test.
• Based on the response of leukocytes which
release inflammatory mediators in response
to pyrogen contamination.
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Limulus Amoebocyte Lysate
An aqueous extract of blood cells
(amebocyte) from the
horseshoe crab,
Limulus polyphemus.
Testing of parenteral pharmaceuticals and
medical devices that come in contact with
blood or cerebrospinal fluid.
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Monocyte Activation Test
Uses human mononuclear cells obtained from
human volunteers or from blood bank:
Very specific and sensitive.
Detects pro-inflammatory contaminants.
Better than LAL and rabbit pyrogen test.
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• Pyrogen contamination
• Interlukin 1b ELISA
• Read out & Data analysis
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Local Lymph Node Assay
Test the potential of test compound for
skin sensitization.
Proliferation of lymphocytes is
proportional to dose applied.
Stimulation index-ratio of proliferation in
test groups to that of control.
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Skin Patch Tests
• Corrositex
To determine chemical corrosivity.
Principle- A unique bio membrane and
chemical detection system which becomes
coloured when exposed to potentially
corrosive substance.
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• A model of human skin like Epi-Derm
and Epi-Skin.
• Cultured human epidermal keratinocytes
which mimic human epidermis are used
to measure skin irritation and dermal
corrosion.
Replaced the Draize rabbit skin
irritation test .
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Skin patch tests.....
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Neutral Red Uptake Assay
• Alternative to Draize rabbit eye test.
• Neutral red penetrates cell membrane and
accumulates intracellularly in lysosomes.
• NRU assay measures the ability of test
compound to inhibit uptake of neutral red
dye.
• NRU 50 or IC 50 serves as toxicological
end point.
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Cell Transformation Assays.
• Carcinogenicity Testing.
• Eg-1. Balb /c3T3 assay
2. Syrian hamster embryo (SHE)
• Faster, less expensive, & involve fewer
animals.
• Alternative to rodent bioassay and
transgenic mouse model for
carcinogenicity assays.
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Stem Cell Models
For toxicological screening and as in-vitro
models of disease
Disease genes are inserted into embryonic
stem cells, induced to differentiate into
human disease tissue which is used for
screening of drugs.
Alzheimer's and Diabetes models.
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Embryonic Stem Cell Test
• Used for detection of any embryonic
toxicity.
• Can predict if a non toxic chemical is
likely to be metabolized to a toxic form or
vice-versa.
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• Positive result classifies the chemical as
likely to be hazardous for development
and reproduction.
• Better alternative to study cancer, liver
and cardiac toxicity .
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Stem Cells…..
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Microbiological Tests.
Ames test
Detect 80-90% of all carcinogenic chemicals.
Used primarily as a screening system.
Also has a potential as an alternative, after
accurate validation.
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Micro-organisms
Fungi for studies of the metabolism of drugs.
Selected group of fungi have the ability to
metabolize a wide variety of drugs
Cunninghamella elegans
Anti-coagulants, diuretics, anticonvulsants
agents.
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• Tetrahymena pyriformis
A ciliate protozoan
Effects of anesthetics on metabolism.
• Salmonella typhimurium
Bacteria
Used in genetics as well as carcinogenicity
testing.
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• Saccharomyces cerevisiae.
The most popular and important model.
Rapid growth, ease of replica plating mutant
isolation.
Dispersed cells, well defined genetic system.
Highly versatile DNA transformation
system.
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Programmed cell death, cell death
regulators in humans.
Useful in cancer research.
Neurodegenerative diseases like
Alzheimer’s, Parkinson’s and Huntington’s
diseases.
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In Chemico Testing
• Toxic potential of substances can be
detected.
 HPLC (High Performance Liquid
Chromatography ).
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In-Silico Models.
• Quantitative Structure Activity
Relationships.
• Computer Assisted Learning.
• Computer Or Mathematical Analysis.
• Micro-fluidics Chips & DNA Chips.
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Quantitative Structure Activity Relationship
(QSAR).
Mechanistic models.
Aim to predict sensitization from
mechanistic knowledge.
Aimed at predicting from a statistical
perspective.
Ability to generate large databases.
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• Several QSAR systems are available, such
as TOPKAT, DEREK, TOPS-MODE,
Multi-CASE, TIMES-SS.
• The day is not far off when we will be
having an E-cell which resembles a
hypothetical cell.
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Computer Assisted Learning
Range of software packages which
simulate the animal experiments.
Two soft wares are being used in india.
Expharm- developed by JIPMER, India.
X-cology.
79Dr. Jayant Rai

EXPHARM
Contains programs on
Effect of drugs on the rabbit eye.
Bio assay of histamine using guinea pig
ileum.
Effect of drugs on the frog heart.
Effect of drugs on dog blood pressure
and heart rate.
Effect of drugs on the ciliary movement
of frog esophagus.
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EXPHARM…..
• The user can conduct experiment and
collect data.
• Each program can be run in two modes-
(a) tutorial mode , (b) examination mode
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X-cology
Content is classified into three sections
Experimental animals
Equipment
To carry out bioassay & experiments
on whole animals.

X-cology.....
• Video demonstrations of different
procedures like isolation and mounting of
animal tissues.
• Screen interactive interface to study the
effects of various drugs on the isolated
tissues.
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Trauma man
• Computer programme
Simulates hemorrhagic, fractures,
amputations and burns.
Is used for military training and training
medical students.
Combat Trauma Patient Simulator similar
to trauma man.
84Dr. Jayant Rai

Computer or Mathematical Analysis
• Translation of biological effect into a
mathematical equation.
Virtual human organs & virtual
metabolism programmes.
Predict drug effects in humans more
accurately then animals can.
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Computers design the molecular structure
of drugs to target specific receptors.
 Eg- Protease inhibitors were designed by
computers and tested in tissue culture and
computer models bypassing animal tests.
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Micro-fluidics & DNA Chips
Multidisciplinary field.
Integrating engineering, physics,
chemistry, biochemistry, nanotechnology,
and biotechnology,
Design of systems in which small
volumes of fluids will be handled.
87Dr. Jayant Rai

Micro-fluidics & DNA Chips…..
Typically, MICRO means one of the
following features:
small volumes (µL, nL, pL, fL)
small size
low energy consumption
effects of the micro domain
88Dr. Jayant Rai

Chips 2 cm wide and contain a series of
tiny chambers containing a sample of
tissue from different parts of the body.
The compartments are linked by micro-
channels through which a blood substitute
flows.
89Dr. Jayant Rai

The test drug is added to the blood
substitute and circulates around the device.
Sensors in the chip feed back information
for computer analysis.
This can be used to study the disease
process and drug metabolism.
90Dr. Jayant Rai

• Micro-fluidics is of two types:
i. Passive Micro-fluidics.
ii. Active Micro-fluidics.
 Passive Micro-fluidics: passive fluid
control techniques like capillary forces
play their role in moving, mixing,
separating or otherwise processing.
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Active Micro-fluidics: refers to the
defined manipulation of the working fluid
by active (micro) components such as
micro-pumps or micro-valves.
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DNA Chips
• DNA chips (microarrays): Early biochips
were based on the idea of a DNA microarray ,
e.g., the Gene Chip DNA array.
A piece of glass, plastic or silicon substrate
Pieces of DNA (probes) are affixed in a
microscopic array.
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In-Silico Models.....
Include models of diabetes,
Asthma and drug absorption.
Require verification in animal and human
tests before licensing.
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Major Barrier to In-silico Models
Resources not available in local languages,
Difficulty finding resources,
Lack of money.
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Epidemiologic, Clinical Studies &
Genetic Monitoring
• Epidemiologic…
A valuable tool in Evidence-based
Medicine.
Identify risk factors & determine
treatment.
Reveals unbiased relationships.
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• Genetic Monitoring
To detect changes in species abundance
or diversity.
An important tool in minimizing animal
use.
• Clinical Trial
About vaccines or new therapies or new
ways of using known treatments.
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Invertebrate Animals
• Used to replace the laboratory animals.
• The most used invertebrate species are
Drosophila melanogaster, a fruit fly
Caenorhabditis elegans, a nematode
worm.
Hydra, a cnidarian.
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Drosophila melanogaster
A classic model used for detecting
mutagenicity.
Teratogenicity.
Reproductive toxicity.
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Invertebrates…..
Short life cycle
Studied in large numbers,
A distinct advantage over the vertebrates.
Have a potential as alternatives to use of
conventional animals.
Fruit flies can be useful to identify
pharmacologically active compounds.

Vertebrate Animals
A recent vertebrate model, the Zebra fish
has proven to a very good model for
toxicity testing.
Produced specific tissue, organ and
behavioral toxicity.
Used and validated in large scale high
throughput screens for various
psychotropic drugs.
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Vertebrates…..
Zebra fishes
Develops rapidly, are small, inexpensive
to maintain.
Chemical administration can be done
directly or by micro-injection.
The morphological and molecular basis of
tissue and organ development are
identical or similar to other vertebrates
including man.
102Dr. Jayant Rai

Micro-dosing
• Enable potential new drugs to be tested
safely in relevant species using ultra
sensitivity of accelerator mass
spectrometry.
• A ‘micro-dose’ is defined as less than one
hundredth of the pharmacological dose
up to a maximum of 100 µg.
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Micro-dosing…..
To determine ADME.
Analysed using an accelerator mass
spectrometer (AMS).
Early metabolism data can be obtained
before going into human phase 1 trials.
Allows testing in relevant species.
104Dr. Jayant Rai

Organisations Researching &
Funding Alternatives.
• Center for Alternative Animal Testing
(CAAT).
• Interagency Coordinating Committee on the
Validation of Alternative Methods
(ICCVAM).
• Davis Center for Animal Alternatives.
• European Center for Validation of Alternative
Methods (ECVAM).
105Dr. Jayant Rai

Recent Trend
• “Virtual Human”.
• “Digital Frog”.
• Researchers have developed a novel
computer software which identifies genetic
changes that allow bacteria to develop
resistance to new experimental drugs.
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Recent…..
• Duke University researcher’s used the
software to predict a constantly evolving
infectious bacterium’s countermoves to
one of these new drugs, before the drug is
even tested in patients.
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Recent…..
• An Indian-origin scientist Mrs Sangeeta
Bhatia working at MIT, has been awarded
Heinz award, who has developed artificial
human Micro-livers for drug testing.
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Summary
• Animal ethics is an issue as important as
the human welfare.
• Various alternatives to animal use have
been suggested, which need to be
implemented in an effective manner.
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• For this
various computer models,
bioinformatics tools,
in vitro cell cultures,
enzymatic screens,
model organisms.
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• Use of modern analytical techniques, data
acquisition and statistical procedures to
analyze the results of alternative protocols
can provide dependable outcomes.
• These integrated approaches would result
in minimum involvement of animals in
scientific procedures.
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References
• Biomedical Research. G.Jagadeesh, Sreekant Murthy,
Y.K.Gupta, Amitabh Prakash. 1st Edition.
• Fundamentals of Experimental Pharmacology.
M.N.Ghosh. 6th Edition.
• Animal Use In Pharmacology Education And
Research: The Changing Scenario. Dinesh K. Badyal
and Chetna Desai. Indian Journal of Pharmacology,
Vol. 46, No. 3, May-June, 2014, pp. 257-265.
112Dr. Jayant Rai

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