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Screening models For immunosuppressants ..pptx
- 1. Pr eclinical Scr eening models f or I mmunosuppr essive agents Guided By Mr. Mukul Tambe Assistant professor Dept. of Pharmacology 5/8/2024 1 Presented By Mr. Rahul Tipare M pharm (Pharmacology)
- 2. •What are immunosuppressant ? •How normal immune response occurs ? •In-vivo preclinical screening model for immunosuppressant •In-vitro preclinical screening model for immunosuppressants •Summary •References 5/8/2024 2 CONTENTS
- 3. IMMUNOSUPPRESSANTS Immunosuppressants are medications that suppress or dampen the activity of the immune system. APPLICATIONS ● Prevent the body from rejecting transplanted organs ● Treat autoimmune disorders where the immune system mistakenly attacks healthy tissues. Example = Multiple sclerosis , Rheumatoid Arthritis Some existing immunosuppressants are Cyclosporine , tacrolimus , sirolimus , abatacept , belatacept 5/8/2024 Reference = Essential of medical pharmacology by K.D. Tripathi 3
- 4. Normal Immune Response & Site of action of Existing Immunosuppressants 5/8/2024 Reference = Essential of medical pharmacology by K.D. Tripathi & Speed pharmacology 4
- 5. in-vivo SCREENING MODELS FOR IMMUNOSUPPRESSENT DRUGS Acute Systemic Anaphylaxis in Rats Anti Anaphylactic Activity (Schultz Dale Reaction) Passive Cutaneous Anaphylaxis Arthrus Type Immediate Hypersensitivity Delayed Type Hypersensitivity 5/8/2024 Reference = Drug Discovery And Evaluation by H. Gerhard Vogal 5
- 6. Acute Systemic Anaphylaxis In Rats Female Sprague Dawley Rat Weight 120 gm 10 mg/kg IM injection of ovalbumin 1ml bordetella pertusis suspension IP route After 11 days 25mg/kg ovalbumin IV route 18 hr prior to challenge Test = 1- 10 mg/kg dexamethasone SC route Control = vehicle Evaluation = shock symptoms and mortality counted 5/8/2024 Reference = Drug Discovery And Evaluation by H. Gerhard Vogal 6
- 7. Modifications of Acute systemic anaphylaxis model In Brown Norway Rats • By Ufkes and Ottenhof ,1984 • Given suspension of trinitrophenyl haptenized ovalbumin together with AIPO4 • Bronchial and cardiovascular function were studied after treatment with Antiallergic agents In Brown Norway Rats • By Elwood et al. ,1992 • Studied the effects of Dexamethasone and Cyclosporin A on Hyperresponsiveness & Inflammatory cell Responses 5/8/2024 Reference = Drug Discovery And Evaluation by H. Gerhard Vogal 7
- 8. Guinea Pig Body weight 300 to 350 gm Anti- Anaphylactic Activity (Schultz Dale Reaction) Sensitized with Alum Precipitated egg Albumin Give injection of 0.125ml egg albumin in each foot pad & 0.5 ml Subcutaneously After 4 weeks, Animals are Sacrificed and ileum is Dissected out 2-3 cm long ileum is Mounted in an Organ bath in tyrode solution at 37°C Contractility of ileum strips is tested by adding standard in one organ bath and Test Compound in Another organ bath Evaluation= The Contractions are Recorded with strain gauges by a Polygraph 5/8/2024 Reference = Drug Discovery And Evaluation by H. Gerhard Vogal 8
- 9. Modifications of Anti Anaphylactic Activity ( Schultz Dale Reaction) In swiss albino mice • By Koppel et.al (1981) • Developed method to induce contraction of mouse trachea by antigen 5/8/2024 Reference = Drug Discovery And Evaluation by H. Gerhard Vogal 9
- 10. Passive Cutaneous Anaphylaxis Antiserum Injected by IM route to shaved dorsal skin of rats After 24 hrs IV Administration of 0.1 ml of mixture of 2.5% Evans blue & 25 mg/ml of egg albumin Test compound is administered with mixture & After 30 min animal is Sacrificed Amount of Evans blue dye that leaked is extracted and determined colorometrically at 620 millimicron wavelength Albino Rats Body weight 120 gm Evaluation = Amount of Evans blue that extracted from passive cutaneous anaphylactic reaction of control group compared to test group 5/8/2024 Reference = Drug Discovery And Evaluation by H. Gerhard Vogal 10
- 11. Modifications of Passive Cutaneous Anaphylaxis model In Rats • By Goose & Blair (1969) • Used Bordetella pertusis as antigen in Passive cutaneous anaphylaxis In Rhesus Monkey • By Patterson et al. (1971) • Tested Passive Cutaneous reactivity to antihuman IgE in Rhesus Monkey 5/8/2024 Reference = Drug Discovery And Evaluation by H. Gerhard Vogal 11
- 12. Arthrus Type Immediate Hypersensitivity Wistar or Sprague Dawley Rat Body weight 220- 280 gm 7 Days prior to Experiment Rats are given 0.5 ml Ovalbumin suspension by IM route Test group 1 hour prior test compound is administered and challenged with 0.1 ml of Ovalbumin in left hind paw Control group treated with solvent alone 3rd Group i.e. Negative Control (Non Sensitized ) treated with solvent Evaluation = The changes in footpad thickness is expressed as percent Change From the vehicle control group . 5/8/2024 Reference = Drug Discovery And Evaluation by H. Gerhard Vogal 12
- 13. Modifications of Arthrus Type Immediate Hypersensitivity model In swiss albino mice • By Omote et al. (1994) • Sheep Red blood cell suspension used for immunization In Guinea Pigs • By Kamei et al. (1991) • Injection of egg albumin and Freund’s complete adjuvant Subcutaneously into foot pad • Injection repeated 4 times at 7 days intervals . 10 days after last immunization 0.2 ml of 2.5 % egg albumin injected the intensity of arthrus reaction calculated 5/8/2024 Reference = Drug Discovery And Evaluation by H. Gerhard Vogal 13
- 14. 123 Anywhere St. Any City, ST 12345 hello@reallygreatsite.com (123) 456 -7890 Delayed Type Hypersensitivity model Initially Rats are sensitized by 0.5 ml Ovalbumin by IM Route After 7 Days Again 0.1 ml of 0.04 % of Ovalbumin injected in left hind paw Footpad Thickness is measured immediately Sprague Dawley Rat Body weight 150 – 230 mg 5/8/2024 Reference = Drug Discovery And Evaluation by H. Gerhard Vogal 14
- 15. Modifications of Delayed Type Hypersensitivity model In Female CDF1 Mice • By Mizukoshi et al. (1994) • They use Sheep red blood cells for Immunization In swiss albino Mice • By Kemei et. al (1991) • Immunized mice by applying 0.7 ml of 15 % Picryl Chloride solution to skin of the shaved abdomen 5/8/2024 Reference = Drug Discovery And Evaluation by H. Gerhard Vogal 15
- 16. In–vitro SCREENING MODELS FOR IMMUNOSUPPRESANTS Inhibition of Histamine release from Mast cells Chemiluminescence in macrophages 5/8/2024 Reference = Drug Discovery And Evaluation by H. Gerhard Vogal 16
- 17. Inhibition of Histamine release from Mast cells 5/8/2024 Reference = Drug Discovery And Evaluation by H. Gerhard Vogal 17 •Preparation of Mast cell suspension • Wistar rats are Decapitated & exsanguinated • 50ml of HBSS is injected into peritoneal cavity • Peritoneal cells are collected in Centrifuge tube , centrifuged at 2000 rpm & Resuspended in HBSS •Test compound Administration • 1ml test drug is added to the mast cell suspension • Mixture is incubated at 37oC for 15 mins • 3 ml of Allergen is added with equal amount of Calcium Ionophore.
- 18. Inhibition of Histamine release from Mast cells 5/8/2024 Reference = Drug Discovery And Evaluation by H. Gerhard Vogal 18 •Extraction of histamine • 1ml of top layer test compound containing 300mg Nacl & 1.25 ml butanol • The sample is alkanized to extract histamine into butanol by adding 1ml of 3N NaoH • Separation into aqueous and organic phases •Induction of O- phthalaldehyde complexing reaction • 0.1 ml 1N NaoH is added 0.1ml O- phthalaldehyde solution • then complexing reaction is stopped by using 3N HCL • Then Histamine is detected by Fluoroscence detector at wavelength 350 and 450 nm
- 19. Chemiluminescence in Macrophages Chemiluminescence is a chemical reaction that produces light without the involvement of heat. 5/8/2024 Reference = Drug Discovery And Evaluation by H. Gerhard Vogal 19 1.Wistar rats are Decapitated & exsanguinated Macrophages are obtained by flushing peritoneal cavity with 10 ml saline containing heparin 250 IU 1.0.1 ml of macrophage suspension and 0.1 ml of test compound mixed together and incubated at 37oC for 15 mins 1.Then 0.1 ml of 3.5 µm Phorbolester solution is added 1.Luminescence measured in the luminometer 1.The time of maximal count is recorded
- 20. 5/8/2024 20 SUMMARY • Immunosuppressant and their Mechanism of action • Need of immunosuppressants • In-vivo models, different animals , drugs in different doses, molecules act as antigen • In-vitro models, different methods by which we can study antigen antibody reaction outside body • Basic difference in in-vivo, in-vitro, ex-vivo, techniques
- 21. 5/8/2024 21 REFERENCE 1. Essential of Medical Pharmacology, by K.D. Tripathi. 2019. Jaypee The Health Sciences Publisher. New Delhi, London, Panama, Eighth Edition, Page no. 937-945. 2. Drug Discovery and Evaluation Pharmacological Assays, by H. Gerhard Vogal. Springer publication. Berlin, Heidelberg, New York, Barcelona, Hong Kong, London. Second Completely Revised and Enlarged Edition, Page no. 788-805. 3. https://youtu.be/vWh1AJwAIg0?si=kexgn3DNrtlTnuff (Speed Pharmacology Youtube Channel) 4. Carvalho, V.F., Barreto, E.O., Diaz, B.L., Serra, M.F., Azevedo, V., Cordeiro, R.S., Martins, M.A. and e Silva, P.M., 2003. Systemic anaphylaxis is prevented in alloxan-diabetic rats by a mechanism dependent on glucocorticoids. European journal of pharmacology, 472(3), pp.221-227. 5. Baruah, C.C., Gupta, P.P., Nath, A., Patnaik, L.G. and Dhawan, B.N., 1998. Anti-allergic and anti- anaphylactic activity of picroliv—a standardised iridoid glycoside fraction ofpicrorhiza kurroa. Pharmacological research, 38(6), pp.487-492. 6. Naik, S.R., Bhagat, S., Shah, P.D., Tare, A.A., Ingawale, D. and Wadekar, R.R., 2013. Evaluation of anti-allergic and anti-anaphylactic activity of ethanolic extract of Zizyphus jujuba fruits in rodents. Revista Brasileira de Farmacognosia, 23(5), pp.811-818.