The Role of Animal Models

in Drug Discovery
 Classification of Animal Models

• Exploratory
to understand a biological mechanism
• Explanatory
to understand a complex biological problem
• Predictive
to discover and quantify the impact of a
treatment
 Animal Models to Humans

• Fidelity
The resemblance of the biological structure in
the animal with the corresponding structure in
humans
• Discriminating ability (predictability)
The similarity between humans and model
species with respect to relevant biological
mechanism is more important than the fidelity
of the model.
 Classification of Disease Models

1. Induced (experimental) disease models

2. Spontaneous (genetic) disease models
3. Transgenic disease models
4. Negative disease models
Mechanism of resistance
5. Orphan disease models
Naturally occurs in a non-human species but
has not yet been identified in humans
 Induced (Experimental) Disease
Models
• Healthy animals initially.
• Condition to be investigated is experimentally
induced. For example, partial hepatectomy
for liver regeneration, streptozotocin-induced
type 1 diabetes
• Unfortunately, few induced models
completely mimic the etiology, course, and
pathology of the target disease in the
humans.
 Spontaneous (Genetic) Disease
Models
• Naturally occurring genetic variants
(mutants) in studying human diseases
For example: nude mice, Snell’s dwarf
mice (lacking pituitary), BB rats

• Concern – the compensatory

mechanisms may differ between
humans and the animal model species
 Transgenic Disease Models

• The advance in genetic engineering and

embryo manipulation technology facilitates
the development of transgenic models.

• Mutations induced by mutagens such as

ethyl-nitroso-urea (ENU) represent another
approach to the generation of new mutants.
 Animal Models in Drug Discovery

• Pharmacodynamics
• Pharmacokinetics
• Toxicology
 Pharmacodynamics

Primary Effect

Secondary Effect
 Pharmacodynamic Primary Effect
Animal Models for Type 2 Diabetes

• Genetic models
db/db mice, ob/ob mice, KK mice, fa/fa
Zucker rats
• Oral Glucose Tolerance Test
Pharmacodynamic Seconday Effect

Adverse effects

Safety Pharmacology
 ICH Topic S7A
Safety Pharmacology Studies for Human
Pharmaceuticals
NOTE FOR GUIDANCE ON SAFETY PHARMACOLOGY
STUDIES FOR HUMAN PHARMACEUTICALS

1. INTRODUCTION

1.1 Objectives Of The Guideline

This guideline was developed to help protect clinical trial
participants and patients receiving marketed products from
potential adverse effects of pharmaceuticals, while avoiding
unnecessary use of animals and other resources. This
guideline provides a definition, general principles and
recommendations for safety pharmacology studies.
www.ich.org
 ICH Guidelines
www.ich.org

• Carcinogenicity Studies
• Genotoxicity Studies
• Toxicokinetics and Pharmacokinetics
• Toxicity Testing
• Reproductive Toxicology
• Pharmacology Studies
• Immunotoxicology Studies
 Genotoxicity
•S2A: Guidance on Specific Aspects of Regulatory
Genotoxicity Tests for Pharmaceuticals

The tripartite harmonised ICH guideline was finalised (Step 4) in July 1995.
This document provides specific guidance and recommendations for
in vitro and in vivo tests and on the evaluation of test results. It  includes
a glossary of terms related to genotoxicity tests to improve consistency in
applications.

• S2B: Genotoxicity: A Standard Battery for

Genotoxicity Testing for Pharmaceuticals

The tripartite harmonised ICH guideline was finalised (Step 4) in July 1997.
This document addresses two fundamental areas of genotoxicity testing:
the identification of a standard set of assays to be conducted for
registration, and the extent of confirmatory experimentation in any
particular genotoxicity assay in the standard battery.
 Concordance of the Toxicity of
Pharmaceuticals in Humans and in Animals
• A multinational pharmaceutical company survey
• Adverse findings of 150 compounds in human clinical
data
and data from preclinical tests in animals
HT* Concordance rate – 71% (rodents + non-rodents)
63% (non-rodents)
43% (rodents)
High concordance rate – cardiovascular (80%)
hematological (91%)
gastrointestinal (85%)
Low concordance rate – neurological (ex. headache,
dizziness)
the only gastrointestinal (nausea)
* HT : human toxicity Regulatory Toxicology and Pharmacology 32:56 (2000)
Time to First Detection of Relevant
Toxicity in Animals
38%

94% detection within one month

Regulatory Toxicology and Pharmacology 32:56 (2000)

Concordance Rate versus Species

The choice of species used might be subject

to more thoughtful consideration.
Regulatory Toxicology and Pharmacology 32:56 (2000)
 Concerns

Though the predictive value of animal studies may seem high, but

 Penicillin is fatal for guinea pigs, but generally well tolerated by

humans
 Aspirin is teratogenic in mice, rats, cats, dogs, monkeys, but
obviously not in pregnant women
 Thalidomide but not cause birth defects in rats or many other
species, but does so in primates

A close phylogenetical relationship or anatomical similarity is

not a guarantee of identical biochemical mechanisms and
parallel physiological response.
 Further Complication

•To which humans?

Often results are obtained from genetically defined and

uniform animal models, but humans are genetically
highly variable, with cultural, dietary and environmental
difference.
 Pharmacokinetics

• A  Adsoprtion (digestive tract, blood-

brain barrier, bioavailability)
• D  Distribution (effector sites)
• M  Metabolism (cytochrome P450
enzymes, metabolites)
• E  Excretion (remove from the body,
kidney (urine) and feces)
Extrapolation from Animals to Humans

Extrapolation :
how data obtained from animal studies reliably
applies to the human
 Pharmacodynamics
 Adverse effects
 Model body size and scaling
 What are the alternatives?

• Reduction - fewer animals

• Refinement - less painful
procedures
• Replacement - alternative
techniques
 3Rs
• Reduction
reduction in the number of animals used for a
particular purpose
• Refinement
refinement of procedures to minimise
suffering
• Replacement
replacement of animal use wherever possible
The 4th R
The 4th R

• Responsibility
 Reduction alternatives
Good planning of studies
• Rational and efficient use of animals
• no wasting
• pilot studies
• screening tests
• Proper statistical design
• Use of inbred starins (for some study types)
 Refinement alternatives
• Minimized potential for pain or distress
• Enhanced animal well-being
• Improved housing conditions and
experimental techniques
 Replacement alternatives (1)
• Efficient use of existing information
• In silico methods (computer simulations, mathematical
models, QSAR)
• ”Read-across”, grouping of chemicals
• In vitro methods: isolated organs
tissue slices
tissue cultures
cell cultures
subcellular fractions
• Lower organisms
• Early stages of development
 Replacement alternatives (2)

In vitro methods are not primarily “replacements” of in

vivo methods. Typically these methods have different
roles in research, and they are complementary for each
others.

Depending on the objective of the study in vitro

methods may be the most appropriate methods for
certain areas of interest, because they can more
accurately provide the information required (e.g.
cellular and molecular events).
 Replacement alternatives (3)

• Absolute replacement: no need to use animals (cell

lines, human or invertebrate cells and tissues)
• no need to test for skin irritation if pH <2.0 or >11.5
• no need to test for eye irritation if the chemical is a
skin irritant
• Relative replacement: humane killing of animals to
provide cells or tissues for in vitro studies
• Partial replacement: e.g. use of non-animal methods as
prescreens in toxicity testing
 Replacement alternatives (4)

• Direct replacement: e.g. human or guinea pig skin is

used in vitro to replace guinea pig tests in vivo
• Indirect replacement: e.g. Limulus amoebocyte lysate
(LAL) test or a test based on whole human blood is
used to replace rabbit pyrogen tests
 Limulus amoebocyte lysate (LAL) test

Horseshoe grab (Limulus polyphemus)

 MTT cytotoxicity test in cell culture
Principle:
Formazan crystals are
formed by mitochondrial
succinate
dehydrogenase in living
and early apoptotic
cells, but not in dead
cells.
MTT
(3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl tetrazolium bromide)

http://www.ib.amwaw.edu.pl/home/dslado/video/mtt.html
Current use of replacement alternatives

• Safety testing of chemicals

• Local toxicity
• Genotoxicity
• Screening tests
• Quality control and potency testing of vaccines
• Production of monoclonal antibodies
 Skin corrosion and skin irritation tests

In vitro In vivo
• Skin corrosion: “artificial” • Corrosivity test on rabbit
human skin cultures skin
 Eye irritation tests

In vitro In vivo
• Eye irritation: • Draize test in rabbit’s eye
• HET-CAM (hen’s egg chorio-
allantoic membrane) test

HET-CAM = hen’s egg chorio-allantoic membrane

• BCOP (bovine corneal opacity) test
• ICE (isolated chicken eye) test
 Production of monoclonal antibodies
In vitro In vivo
• Cell culture • Mouse ascites method
 Pyrogen tests
In vitro In vivo
• Limulus test • Rabbit pyrogen test

Horseshoe grab (Limulus Limulus Amebocyte Lysate

polyphemus) Test Kit

• Human whole blood test

 Analysis of biologically active compounds

In vitro In vivo
• Pregnancy test (immune assay) • Frog pregnancy bioassay

• Bioassays in chicken, rats and

• Hormones, vitamins (HPLC, mice
immune assays)
• Convulsion test (mouse), blood
• Insulin determination glucose determination (mouse,
rabbit)
 Advantages of in vitro tests

• Controlled testing conditions

• Lack of systemic effects
• Reduction of variability between experiments
• Testing is fast (and cheap)
• Small amount of test material is required
• Limited amount of toxic waste is produced
• Human cells and tissues can be used
• Transgenic cells carrying human genes can be used
• Reduction of testing in animals
 Limitations of in vitro tests

• General toxicity profile of a chemical cannot be

assessed
• In vivo dose-responses cannot be obtained (for human
risk assessment)
• Systemic effects cannot be evaluated
• Interactions between tissues and organs cannot be
tested
• Pharmacokinetics cannot be evaluated
• Specific organ sensitivity cannot be assessed
• Chronic effects cannot be tested
No relevant replacement alternatives

• Pharmacokinetics / toxicokinetics
• Systemic toxicity
• Organ systems toxicity (CNS, respiratory,
cardiovascular, gastrointestinal etc.)
• Immunotoxicity
• Male and female reproduction toxicity (fertility tests,
developmental toxicity tests, peri- and postnatal
toxicity tests)
• Subchronic and chronic toxicity
• Carcinogenicity tests
Stepwise in vitro and in vivo testing

Skin / eye irritation and corrosivity testing (OECD 404, 405)

1. Existing data on human and laboratory animals
2. Toxicity based on physico-chemical properties + QSAR
3. Toxicity in vitro
• positive result  classification +
• negative or equivocal result  in vivo testing
4. Toxicity in vivo
• first one or few animals
• positive  classification +
• negative  more animals  classification -