Preclinical experimentation: An overview

Description of Preclinical pharmacology, laboratory animals, handling technique and route of administration, legal regulation, in vivo & in vitro experimentation

1. Dr. Subhash R. Yende Asst. Professor Gurunanak College of Pharmacy, Nagpur Preclinical Experimentation : An overview

2. Basic terminology Animals Used in Preclinical Pharmacology Legal Regulation Handling and other techniques In vivo & in vitro Experimentation Dr. Subhash R. Yende 2 Content

3. Pharmacology Toxicology Clinical Pharmacology Preclinical Pharmacology In vivo and in vitro experimentation Dr. Subhash R. Yende 3 Basic terminology

4. Drug Discovery and development NCE/Herbal Drug Preclinical Studied (Physicochemical Parameters, ADME, Toxicity test, Screening for Activity) IND/ FDA review Clinical Trials (Phase I,II,III) (Product Formulation, Manufacturing and control) NDA (submission, FDA review, Approval and FDA action) Post Marketing (Phase IV Trials, Adverse reaction reporting ) 4 Dr. Subhash R. Yende

5. Rats ( Rattus norvegicus ) Albino Wistar , Sprague Dawley Mice ( Mus musculus ) Swiss Albino Rabbit ( Oryctolagus cuniculus ) Guinea Pig ( Cavia porcellus ) Zebra fish Frog ( Rana tgrina ) Monkey Dog Transgenic animals Dr. Subhash R. Yende 5 Animals Used in Preclinical Pharmacology

6. CPCSEA (Committee for the purpose of Control and Supervision of Experiments on Animals) Section 15 of the Prevention of Cruelty to Animals (PCA) Act, 1960 Ensure that animals are not subjected to unnecessary pain or suffering before, during or after performance of experiments on them. Provide guidelines for Breeding of and Experiments on Animals IAEC (Institutional Animal Ethics Committee) Constitute as per Rule 13 of the Breeding of and Experiments on Animals(Control and Supervision) Rules, 1998. Operated in accordance with the procedures specified by the CPCSEA Scrutinize and give approval to project proposals for experimentation on animals Dr. Subhash R. Yende 6 Legal Regulation

7. Dr. Subhash R. Yende 7 Handling and Route of administration

8. 8 Dr. Subhash R. Yende

9. Blood collection Cardiac puncture Retro-orbital Tail vein Marginal Ear Vein Euthanasia ( Scarification) Technique Head Blown Cervical Dislocation Anesthesia Dr. Subhash R. Yende 9 Technique used

10. Toxicity testing In vivo experimentation In vitro experimentation Histopathology Experimentation 10 Dr. Subhash R. Yende

11. Dr. Subhash R. Yende 11 ‘ All substances are poisons; the right dose differentiates a poison and a remedy’ - Paracelsus Acute toxicity test - The adverse effects occurring within a short time of administration of single dose or multiple doses given within 24 h. Use for determination of LD 50 of the compound. As per OECD Guideline AOT- 423 Observations- Behavioural changes and Mortality Toxicity testing

12. The subacute toxicity test involves daily or frequent exposure to the compound over a period up to about 90 days. Chronic toxicity testing is usual to expose the animals to the chemical for the greater part of the life span. (6 -12 month or up to 2 year) Observations- Biochemical organ function tests: blood glucose, protein, bilirubin , serum levels of hepatic enzymes etc. Physiological measurements: food consumption and body weigh Metabolic studies: Urine and faeces Haematological information: Quantification of blood cells Histopathological studies: Dr. Subhash R. Yende 12

13. Locomotor activity Screening of anti Epileptic drugs MES /PTZ/Strychnine induced Screening of anti Alzheimer’s drugs Scopolamine-induced amnesia in mice. Dr. Subhash R. Yende 13 In vivo Experimentation

14. Estimation of AchE activity in rat brain Estimation of SGOT, SGPT, Bilirubin level, NF Kb, TNF-Alpha Determination of cell viability by MTT (3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyltetrazolium bromide) assay method (anti cancer activity) Cell culture and cell line study Liver cell lines HepG2, Hep3B, HBG, and HepaRG (liver toxicity) pancreatic carcinoma (PANC-1), colon carcinoma (RKO), breast adenocarcinoma (MCF-7, ER-positive), brain neuroblastoma (IMR32 ) Dr. Subhash R. Yende 14 In vitro Experimentation

15. Microscopic identification and examination of tissue samples Dr. Subhash R. Yende 15 Histopathology

16. Rat and mice are mostly used as experimental animal Experimental protocol should be approved from IAEC and CPCSEA Acute toxicity test determine toxicity profile and lethal dose of drug In vivo, in vitro and histopathological studies are used for preclinical screening of drug Dr. Subhash R. Yende 16 Summary

17. Ghosh , M.N. “Fundamentals of experimental Pharmacology” Medhi B, Prakash A. ‘Practical Manual of experimental and clinical pharmacology’ Vogel H. G, Drug discovery and evaluation. CPCSEA guideline for laboratory animals, Indian Journal of Pharmacology, 2003,35. S. K. Kulkarni , Handbook of Experimental Pharmacology, Vallabh Prakashan Dr. Subhash R. Yende 17 Reference Books

18. THANK YOU Dr. Subhash Ramchandra Yende Mobile No. 9028665370 E mail- [email protected] 18 Dr. Subhash R. Yende