alternative methods of animal toxicity.pptx

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1. ALTERNATIVE METHODS TO ANIMAL TOXICITY TESTING Saif Imtiyaz M Pharm Pharmacology Jamia Hamdard

2. PRESENTATION OUTLINES Introduction…………………………………………….. 01- 02 The Rs Principles……………………………………… 03 Need For Alternatives To Animals…………………. 04 Alternative Methods…………………………...……… 05 Different alternative methods……………………….. 06- 22 References……………………………………………… 23

3. Introduction Animal models have been used to develop human biochemistry, physiology, pharmacology, endocrinology, and toxicity. Every year, 10-100 million animals are used for testing.  Animals used experimentation distributed among zebra- fish to primates . Vast majority of animals are sacrificed at end of research programme.  The use of animals can be further subdivided according to the degree of suffering Minor animal suffering :- observing animals in behavioral studies, single blood sampling , immunization without adjuvants, etc. Moderate animal suffering :- repeated blood sampling , recovery from general anesthesia , etc. Severe animal suffering :- LD50% test , starvation vaccine potency tests, etc. 1

4. Alternative methods to animals testing are the development and implementation of test method that avoid use of live animals or use of less animals in method. The council directive on protection of animals used for experiments and scientific purpose in Article 23 “The commission and member states should encourage research into development and validation of alternative methods which could provide the same level of information as that obtained in experiment using animals but which involves less animal”. 2

5. The 5 R’s Principle Alternative methods able to do: Reduce Refine Replace ; collectively called as “The 3Rs Principle”. The 3Rs principles were defined in 1959 by W.M.S Russel and R.L Bruch. They provide a strategy for rational and stepwise approach to minimizing animals use and suffering in experiments without compromising the quality and quantity of scientific work being undertaken. Reuse & Rehabilitate- The 4 th & 5 th R of Research implies addition of ‘responsibility ’ to the original three R’s, reflects integrity, honesty, and scientific correctness in appropriate and reasonable use of laboratory animals. 3

6. Needs for Alternative Methods Because in laboratory animals may be : Invitro testing human cell lines have been useful in securing relevant information for human risk assessment thereby opening up opportunities to explore responses to existing and emerging therapies human cell lines can be used for the tumor and some other chronic disease. 4

7. Alternative Techniques The term “ alternative ” is used to refer to those techniques or methods that replace the use of laboratory animals altogether reduce the number of animals required or techniques to minimize the level of stress endured by the animal. It is not possible to replace whole animal models with in vitro systems to evaluate drug effects on major organ systems. However, techniques can greatly reduce the number of animals needed, and refined protocols can improve the design efficiency and quality of studies , and lessen stress and discomfort experienced by lab animals The field of alternative study particularly in vitro toxicology has evolved into a respected discipline and is attracting competent and motivated scientist around the world. 5

8. Alternative to Animal Experiments In silico [computer modelling technique] 6

9. 1. Continued But Modified Use Of Animals Russel and Burch developed 3R’s strategy which include : 7

10. Methods Of Refinement Providing relief [pain and distress] by giving drugs like analgesics, anesthetics, tranquillizers and sedatives. By changing procedure 8

11. Methods Of Reduction 9

12. 10

13. 2. Invitro Models 11 In vitro testing is the scientific analysis of the effects of chemical substances on cultured bacteria or mammalian cells, organ, tissues, enzymes receptors enzymes .  In vitro (literally 'in glass') testing methods are employed primarily : ̵ ̵ to identify potentially hazardous chemicals to confirm the lack of certain toxic properties in the early stages of the development of potentially useful new substances such as therapeutic drugs, agricultural chemicals and food additives. In vitro testing methods can be more useful and cost-effective than toxicology studies in living animals (which are termed in vivo or "in life" methods). 

14. Various Test In Vitro Methods In vitro pyrogen test Embryonic stem cell test Carcinogenicity test Neurotoxicity test Source Of Tissue For In Vitro Methods 12

15. 3. Tissue Culture Techniques Tissue culture is in vitro maintenance and propagation of isolated cells, tissues or organs in an appropriate artificial environment. APPLICATION OF ANIMAL CELL CULTURE 13

16. 4. In silico [Computer Modelling] Techniques Without animal dissection computer generated stimulation are used to predict the various possible biological and toxic effects of a chemical or potential drug candidate. VARIOUS TYPES IN SILICO MODELS 14

17. A. Computer Aided Drug Design [CADD] It  is the inventive process of finding new medications based on the knowledge of a biological target. It involves the design of molecules that are complementary in shape and charge to the biomolecular target with which they interact and therefore will bind to it. It is used to predict the receptor binding site for a potential drug molecule. CADD works to identify the probable binding site and hence avoid testing of unwanted chemicals having no biological activity. Computational approach to discover, develop and analyze drugs. 15

18. Drug design with the help of computers may be used at any of the following stages of drug discovery: hit identification using virtual screening (structure- or ligand-based design) hit-to-lead optimization of affinity and selectivity (structure-based design, QSAR, etc.) lead optimization: optimization of other pharmaceutical properties while maintaining affinity. Advantages of CADD Time Cost Accuracy information about the disease screening is reduced Database screening less manpower is required 16

19. Categories of software 17

20. B. Quantitative Structure Activity Relationship A quantitative structure-activity relationship (QSAR) is a mathematical relationship which correlates measurable or calculable molecular properties to some specific biological activity in terms of an equation 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. QSAR has been widely used in medicinal chemistry as support in drug’s discovery and development process as well as in study of harmful and poisonous substances in toxicological chemistry. QSAR attempts to find consistent relationship between biological activity and molecular properties, so that these “rules” can be used to evaluate the activity of new compounds 18

21. ADVANTAGES OF QSAR It gives quantifying the relationship between structure and activity with their physiochemical property basis. Possible to make predictions of designed compounds before the chemical synthesis of novel analogues. It may help to understand the interactions between functional group of designed molecules and their activity of target enzyme or protein. DISADVANTAGES OF QSAR Due to biological data experimental error it may give false correlations. If training set of molecule is less, the data may not reflect the complete property and it cannot be used to predict the most active compounds. In some 3D QSAR study ligands binding receptor or protein may not be available 19

22. C. Organ On Chips It is a multichannel 3-D micro fluidic cell culture chip which simulates the activities, mechanisms, physiological response of entire organs. These micro devices are translucent , they provide a window to watch inner workings of human organs. Organ-on-chips that contain human cells grown in a state- of the-art system to mimic the structure and function of human organ and organ system. The chips can be used instead of animals in disease research, drug testing and toxicity testing and have been shown to replicate human physiology , diseases and drug responses more accurately than crude animal experiments. 20

23. How will these organs on chips help the pharmaceutical industries?? Replace animal models. Help the laboratories reach the stage of clinical trials. Comparison studies of drugs on human and animals. To study the effect of drug on its main action of site and also other organs. Study of toxicity of drugs and cosmetics. To study about cancer cells and produce new drugs in cancer treatment. Ensure better regulatory decision-making. Develop vaccines and drugs to counter bioterrorism threats. 21

24. D. HET-CAM (Hen's egg- chorioallantoic membrane) TEST The fresh fertile white leghorn eggs are used. The eggs are held in optimized incubation condition. On day 10 inner egg membrane is removed , after careful removal the living vascular Chorio Allantoic-Membrane is exposed. The test substance is dropped over CAM in a volume of 0.2- 0.3 ml and irrigated after 20 sec. with 5ml warm water. The CAM, the blood vessels, including capillary system, and albumin are examined and scored for irritant effects 0.5, 2, 5min after test compound application. 22

25. References Fundamentals of Experimental Pharmacology. M.N.Ghosh . 7th edition, 2020. Practical Manual of Pharmacology. Dinesh Badyal . 1st edition, 2021. Alternative Methods to Animal Experiments in Toxicity Testing. Nuhoğlu Öztürk , Zeyno & Aksoy, Abdurrahman.  7th International Congress on Veterinary and Animal Sciences (2022). Animal Use In Pharmacology Education And Research: The Changing Scenario. Dinesh K. Badyal and Chetna Desai. Indian Journal of Pharmacology, May-June, 2014. 23