Alternatives to animal experiments

1. Alternatives to animal experiments Dr.Roopali Somani P.G Resident M.R.M.C

2. Overview Introduction Need for alternative to animals Laws and regulations Refinement Reduction Replacement In vitro methods In S ilico methods Advantages and disadvantages Summary

3. 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

4. Introduction Animal models are used to test possibilities that would be difficult or impossible to test using the target species (Humans) I t 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 .”

5. Need for alternatives In the laboratory an animal maybe P oisoned D eprived of food, water and sleep A pplied 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

6. 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

7. Alternatives to animal experiments Continued but modified use of animals I n vitro (test tube) test methods and models based on human cell and tissue cultures C omputerized patient-drug databases and virtual drug trials C omputer models and simulations Computer assisted learning N on-invasive imaging techniques such as MRIs and CT Scans M icrodosing

8. Laws and regulations 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

9. Laws and regulations 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

10. 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 R efinement - refine experimental methods to decrease unnecessary pain and trauma to animals R eduction - reduce the number of animals used in these experiments R eplacement - replace the animal experiments eg - computer simulation models, In-vitro methods, cell culture techniques

11. Methods of Refinement

12. 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

13. 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

14. 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

15. 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 Cell lines Primary culture Organ architecture preserved

16. 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

17. In vitro pyrogen test Rabbit pyrogen test is replaced with Limulus amoebocyte lysate(LAL) Monocyte activation test Based on the response of human leukocytes which release inflammatory mediators in response to pyrogen contamination

18. 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

19. 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

20. 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 – Inhibition of differentiation Cytotoxic effect on the ES cells Cytotoxic effect on 3T3 fibroblasts

21. 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 P ositive result classifies the chemical as likely to be hazardous for development and reproduction Better alternative to study cancer, liver and cardiac toxicity

22. 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

23. Skin patch tests Corrositex To determine chemical corrosivity . R eplaces 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

24. Skin patch tests

25. 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

26. Neutral red uptake assay

27. 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

28. 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 P arkinsons patient were introduced in embryonic stem cells which grew into a model of P arkinsons disease and is used for screening potential drugs Alzheimers and Diabetes models

29. Repeated dose toxicity studies C omputerized 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

30. 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

31. 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

32. In silico models Computer aided molecular drug design Quantitative structure activity relationships Computer assisted learning C omputer or mathematical analysis Microfluidic chips DNA chips Organ on chip Human on chip

33. Computer aided molecular drug design

34. 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

35. 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

36. X- cology video demonstrations of different procedures like isolation and mounting of animal tissues S creen 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

37. 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

38. 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 - P rotease inhibitors were designed by computers and tested in tissue culture and computer models bypassing animal tests

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

40. 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

41. 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

42. Micro dosing studie s 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). E arly metabolism data can be obtained before going into human phase 1 trials. Allows testing in relevant species

43. Quantitative structure activity relationships C omputer 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

44. 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.

45. 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

46. Summary In vitro methods In chemico In silico Pyrogen tests LAL Monocyte activation test Teratogenicity Embryonic stem cell test Skin sensitizer Local lymph node assay Skin irritation Corrositex Epiderm Episkin Skin ethic RHE Eye irritation Neutral red uptake assay Carcinogenicity Cell transformation assays Stem cell models LUHMES for parkinsons HPLC Direct 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

47. References Fundamentals of Experimental P harmacology. M.N.Ghosh . 6 th edition. Practical Manual of Pharmacology. Dinesh Badyal . 1 st edition. A Review on Alternatives to Animal T esting M ethods in Drug Development. R anganatha 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