ALTERNATIVES TO ANIMAL EXPERIMENTS

Dr.Roopali SomaniP.G Resident

M.R.M.C

Overview

Introduction Need for alternative to animals Laws and regulations Refinement Reduction Replacement In vitro methods In Silico methods Advantages and disadvantages Summary

Introduction

Animals are used in science for: Undergraduates teaching to learn

physiological mechanism, anatomy and effect of various drugs on human body

Postgraduate teaching to show effects of various drugs, to find out the nature of unknown drug and for bioassay

Research to understand the working of body and processes of disease and health

Research to conduct screening for drugs, bioassay and for preclinical testing of new drug

Introduction

Animal models are used to test possibilities that would be difficult or impossible to test using the target species (Humans)

It is mandatory to do extensive toxicological studies in animals before the candidate drug gets approval for clinical trials in humans

“There is no doubt that the best test species for humans are humans. It is not possible to extrapolate animal data directly to humans due to interspecies variation in anatomy, physiology and biochemistry.”

Need for alternatives

In the laboratory an animal maybe Poisoned Deprived of food, water and sleep Applied with skin and eye irritants Subjected to psychological stress Deliberately infected with disease Brain damaged, Paralysed,

Surgically mutilated Irradiated, burned, gassed Force fed and electrocuted

Need for alternatives

Disadvantages of animal experiments

Pain, distress and unethical behaviour to animals

Requirement of skilled manpower

Time consuming protocols

High cost Translation rates of

animal experiments are absymal

Alternatives to animal experiments

Continued but modified use of animals In vitro (test tube) test methods and models

based on human cell and tissue cultures Computerized patient-drug databases and

virtual drug trials Computer models and simulations Computer assisted learning Non-invasive imaging techniques such as MRIs

and CT Scans Microdosing

Laws and regulations

YEAR LAW

1960Prevention of Cruelty to Animals (PCA) Act 1960, amended 1982

1964Committee for the Purpose of Control and Supervision ofExperiments on Animals (CPCSEA)

1972 Wild life protection act

1992Indian National Science Academy (INSA) “Guidelines for care and use of animals in scientific research”, revised 2001

1998

“Breeding of and Experiments on Animals (Control andSupervision) Rules, 1998”, amended 2001, 2006

Laws and regulations

Year Law

2001Indian Council of Medical Research (ICMR) “Guidelines for use of Laboratory animals in Medical Colleges”

2009MCI amendment-Recommends to use alternatives to replace animal experiments

2012Ministry of Health & Family Welfare bans use of animals in educational institutes

2013

University Grants Commission (UGC) “Guidelines fordiscontinuation of dissection and animal experimentation in zoology/life sciences in a phased manner

Continued but modified use of animals

Russel and burch in 1959 proposed that “if animals were to be used in experiments, every effort should be made to replace them with non‑sentient alternatives”

They developed the 3R strategy which includes Refinement- refine experimental methods to

decrease unnecessary pain and trauma to animals

Reduction- reduce the number of animals used in these experiments

Replacement- replace the animal experiments eg- computer simulation models, In-vitro methods, cell culture techniques

Methods of Refinement

Setting the earliest possible end point

Using appropriate analgesics and anaesthetics for painful procedure

Use proper handling technique for animals

Adequate training prior to performing experiment

Ensure drug doses are correct and drugs are not expired

Perform surgeries and procedure aseptically to prevent infection,

Methods of Reduction

Perform pilot studies Design studies to use animals as their

own controls eg- Cross over study Gather data for more than one

experiment concurrently Consult with statistician and use

minimum number of animals Minimise variables such as disease, diet,

stress, genetics Use appropriate species of animals

Replacement

Substitution of insentient material in place of conscious higher animals

Could be relative or absolute Replace higher animals with lower animals Replace live animals with dummies for

teaching and dissection purpose Use computer simulation and in vitro

methods Use cell culture and tissue culture

In vitro models

In vitro biomedical research entails the maintenance of organs, tissues (or fragments of organs and tissues), and cells outside of the body.

Can be grown as independent cell lines or preserve the architecture of the entire organ as organ culture and tissue culture

Stem cells are also used as invitro models

Source of tissue for in-vitro methods

Avian- chick embryos Rodents- rats and mice( wild types and transgenic): embryonic, post-natal and adult Human – 1. Neural progenitor cells from aborted foetuses and stem cell lines. 2. Cord blood derived stem cells

Types of in vitro systems- cell culture 1. Cell lines 2. Primary culture 3. Organ architecture preserved

In vitro methods

In vitro Pyrogen test Embryonic stem cell test Local lymph node assay for skin sensitization Clinical skin patch test on human volunteers Neutral red uptake assay Carcinogenicity test Acute toxicity test Repeated dose toxicity test Developmental neurotoxicity test

In vitro pyrogen test

Rabbit pyrogen test is replaced with A. Limulus amoebocyte

lysate(LAL)B. Monocyte activation test

Based on the response of human leukocytes which release inflammatory mediators in response to pyrogen contamination

Limulus amoebocyte lysate(LAL)

Principle- Lipopolysaccharides cause extracellular coagulation of blood( Haemolymph) of horseshoe crab Lumulus polyphemus

Three techniques to perform this test

• Gel clot technique- based on gel formation

• Turbidimetric method-based on development of turbidity after cleavage of endogenous substrate

• Chromogenic method-based on development of color after cleavage of synthetic peptide chromogen complex

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

Embryonic stem cell test

Used for detection of any embryonic toxicity

Principle- the capacity of stem cells(rodent cell line D3) to develop into specialized contracting heart cells in vitro within 10 days is assessed using light microscopic evaluation

End points –1. Inhibition of differentiation 2. Cytotoxic effect on the ES cells 3. Cytotoxic effect on 3T3 fibroblasts

Embryonic stem cell test

Metabolism studies using human microsomal enzymes or cell lines can predict if a non toxic chemical is likely to be metabolized to a toxic form or vice-versa

Positive result classifies the chemical as likely to be hazardous for development and reproduction

Better alternative to study cancer, liver and cardiac toxicity

LOCAL LYMPH NODE ASSAY

Used to test the potential of test compound for skin sensitization

Principle- a test compound is considered as a sensitiser when the lymph node draining the site of chemical application reveals a primary proliferation of lymphocytes as measured by radioactive labelling in test and vehicle groups

Proliferation is proportional to dose applied Stimulation index- ratio of proliferation in test

groups to that of control Index must be atleast 3

Skin patch tests

Corrositex To determine chemical corrosivity. Replaces rabbit test of dermal corrosivity Principle- a unique bio membrane and chemical

detection system which becomes colored when exposed to potentially corrosive substance

Cultured human epidermal keratinocytes mimic human epidermis are used to measure skin irritation and dermal corrosion. Replaced the Draize rabbit skin irritation test

Skin patch tests

Neutral red uptake assay

Alternative to Draize rabbit eye test for screening of chemicals for eye irritation potential

Neutral red penetrates cell membrane and accumulates intracellularly in lysosomes

Alteration of cell surface or lysosomal membrane result in decreased uptake

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

Neutral red uptake assay

Carcinogenicity test

By using cell transformation assays Eg-1. Balb/c3T3 assay 2. Syrian hamster embryo (SHE) These assays are faster, less expensive,

and involve fewer animals Alternative to rodent bioassay and

transgenic mouse model bioassay for carcinogenicity assays

Stem cell models

Can be used for toxicological screening and also as invitro 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

Eg- Genes from a Parkinsons patient were introduced in embryonic stem cells which grew into a model of Parkinsons disease and is used for screening potential drugs

Alzheimers and Diabetes models

Repeated dose toxicity studies

Computerized biokinetic modeling is used as a means of predicting the distribution of chemical among various organs and tissues of the body and also to predict organ specific toxicity

Such predictions are verified quantitatively using cell cultures of specialized tissues

Microorganism based model

Tetrahymena pyriformis—a ciliate protozoan being used to study the effects of anesthetics on metabolism

Salmonella typhimurium—bacteria used in mechanistic studies in genetics as well as the Ames mutagenicity/carcinogenicity test

IN CHEMICO TESTING

The toxic potential of substances can sometimes be detected using relatively simple chemistry based methods and not requiring human cells.Eg- High performance liquid chromatography

Direct peptide reactivity assay- used to assess whether a chemical or cosmetic will cause allergy

The tests works by mimicking a key step in the development of allergies – the binding of proteins found in the skin to the substance.

If proteins bind to the substance then it is very unlikely that it will cause an allergic reaction

In silico models

Computer aided molecular drug design Quantitative structure activity

relationships Computer assisted learning Computer or mathematical analysis Microfluidic chips DNA chips Organ on chip Human on chip

Computer aided molecular drug design

Computer assisted learning (CAL)

CAL deals with a range of software packages which simulate the animal experiments

Two softwares are curently used in india

Expharm- developed by JIPMER, India

X-cology

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 The user can conduct experiment and collect

data Each program can be run in two modes-

a) tutorial mode , (b) examination mode

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

Content is classified into three sections Experimental animals Equipment Experimental technique – procedure to

carry out bioassay and experiments on whole animals

Trauma man

Computer programme Simulates hemorrhaging, fractures,

amputations and burns Is used for military training and training

medical students Combat Trauma Patient Simulator similar

to trauma man

Computer or mathematical analysis

Translation of biological effect into a mathematical equation.

Virtual human organs and virtual metabolism programmes can now predict drug effects in humans more accurately then animals can.

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

Recent trend

Researchers are working on a “virtual human” which is designed to predict drug metabolism and metabolite interaction with any given organ

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 targetsand “personalized” targets

Screening up to 100,000 compounds aday for activity against a target protein

Using a computer topredict activity

Rapidly producing vast numbersof compounds

Computer graphics & models help improve activity

Tissue and computer models begin to replace animal testing

Microfluidic chips

Chips 2 cm wide and contain a series of tiny chambers each containing a sample of tissue from different parts of the body.

The compartments are linked by microchannels through which a blood substitute flows

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

Micro dosing studies

A ‘microdose’ is defined as less than one hundredth of the proposed pharmacological dose up to a maximum of 100 µg

Can be measured in any biological sample including plasma and urine 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

Quantitative structure activity relationships

Computer programs which can predict the toxicity of new chemicals or drugs based on their similarity to more established compounds.

Principle that similar chemicals should have similar biological properties.

Greater computer power and the ability to generate large databases have facilitated the development of these methods and a wide range of models now exist that cover a variety of toxicities

Advantages

Alternative scientific tests are often more reliable than animal tests.

The use of human tissue in toxicity testing is more accurate than the animal models.

Cruelty-free products are more environmentally friendly.

Summary

Ethical concerns and dismal rate of translation with animal experiments have led to development of alternate methods

3R concept- Reduce- reduce the number of animals used Refine- refine the experimental procedure Replace- wherever feasible replace the animal

experiment

Summary In vitro methods In chemico In silico

Pyrogen tests • LAL • Monocyte activation

test Teratogenicity • Embryonic stem cell

test Skin sensitizer • Local lymph node

assaySkin irritation • Corrositex • Epiderm • Episkin • Skin ethic RHE Eye irritation • Neutral red uptake

assayCarcinogenicity • Cell transformation

assaysStem cell models • LUHMES for

parkinsons

HPLCDirect peptide reactivity assay

CAL- EXPHARM X-Cology Trauma man Computer aided

molecular drug design Microfluidic chips Quantitative structure

activity relation ships

Human studies Microdosing studies

References

Fundamentals of Experimental Pharmacology. M.N.Ghosh. 6th edition.

Practical Manual of Pharmacology. Dinesh Badyal. 1st edition.

A Review on Alternatives to Animal Testing Methods in Drug Development. Ranganatha N, I. J. Kuppast. International Journal of Pharmacy and Pharmaceutical Sciences.

Animal use in pharmacology education and research: The changing scenario. Dinesh K. Badyal, Chetna Desai. Indian Journal Of Pharmacology