alternatives to animal experiments
TRANSCRIPT
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ALTERNATIVES TO ANIMAL EXPERIMENTS
Dr.Roopali SomaniP.G Resident
M.R.M.C
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Overview
Introduction Need for alternative to animals Laws and regulations Refinement Reduction Replacement In vitro methods In Silico methods Advantages and disadvantages Summary
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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
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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.”
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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
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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
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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
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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
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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
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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
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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,
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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
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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
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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
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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
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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
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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
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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
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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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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
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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
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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
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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
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Skin patch tests
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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
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Neutral red uptake assay
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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
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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
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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
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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
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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
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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
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Computer aided molecular drug design
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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
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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
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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
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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
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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
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Recent trend
Researchers are working on a “virtual human” which is designed to predict drug metabolism and metabolite interaction with any given organ
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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 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
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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
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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
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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
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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.
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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
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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
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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