Downloaded 296 times
More Related Content
Similar to Preclinical screening of antiallergics
Preclinical screening of antiallergics
- 1. Submitted by, Miss. Prajitha.P FirstM.Pharm, Pharmacology Devaki Amma Memmorial College of Pharmacy, Malappuram 1
- 2.
- 3.
- 4.
- 5. IN VITRO METHODS Inhibition of histamine release from mast cells cell Mitogen induced lymphocyte proliferations Inhibition of T cell proliferation PFC (Plague forming colony) test in vivo Inhibition of dihydro-orotate dehydrogenase 5
- 6. IN VIVOMETHODS Acute systemic anaphylaxis in rats Anti-anaphylactic activity in guinea pigs ( Schultz- dale reaction) Delayed type of hypersensitivity Passive cutaneous anaphylaxis in rats Arthus type immediate hypersensitivity Spontaneous autoimmune diseases in animals 6
- 7. Reversed passivearthus reaction Adjuvant arthritis in rats Collagen type 2 induced arthritis in rats Proteoglycan induced progressive polyarthritis in mice Pristance induced arthritis in mice Experimental autoimmune thyroiditis Porcine cardiac myosin induced autoimmune myocarditis in rats 7
- 8.
- 9. Rats aresensitized by ovalbumin and bordetella pertusis After 11 days they are challenged by i.v. ovalbumin leading to shock It is counteracted by corticosteroids and i.v. disodium cromoglycate 9
- 10. Methodology: Female sprague dawleyrats (120-150g) Injected i.m with 10mg/kg of ovalbumin simultaneously With bordetella pertusis suspension i.p IgE Ab migrates to surface of mast cells & basophilic granulocytes 11days 10
- 11. Iv injection of25mg/kg ovalbumin 18 hrs before given with corticosteroids and disodium cromoglycate Ag-Ab complex forms on the surfaceof mast cell release histamine, 5-HT, etc SHOCK MORTALITY(80%) counteract 11
- 12. Symptoms ofshock scored and mortality recorded, compared with untrated animals. 12
- 13. Guinea pigsare sensitized against egg albumin 3week release histamine Contraction of smooth muscle 13
- 14. Methodology guinea pigs(f/m),300-400g alumprecipitated egg albumin egg albumine(1mg/ml) in saline stirred kept in room temp 0.125 ml alum egg albumine to foot pad and 0.5ml SC 14
- 15. 4 weeks Killed usinganaesthetic ether Ileum dissected out & cleaned Mounted on organ bath(tyrode soln @ 37°C (stabilize for 15mnts) barium chloride The tissue respond to spasmogen are selected 3 bath selected 15
- 16. Test gps stdgps Control gp Administered ovalbumin Contractions recorded on physiograph 16
- 17. 3.Passive cutaneous anaphylaxisin rats it is Ag-Ab reaction: immediate type skin is passively immunized with rat ovalbumin serum & challenged after 2 days with ovalbumin at same site. Ag-Ab complex in mast cell release histamine and other mediators 17
- 18. Cause vasodilation,increased vascular permeability, leakage of plasma. To make the allergic reaction clear, inject Evans blue dye with the Ag with get attached to the albumin fraction of the plasma. Ag –Ab reaction spot appears as blue spot, so can confirm the occurrence of allergy 18
- 19. methodology: male wistar rats(250g) they are adrenalectomized 3 days Egg albumin 1mg/ml injected on foot pad 8days Animals are bled and antiserum is collected Diluted such a way that it should produce a wheal of 15-20mm diameter on rat skin in preliminary test 19
- 20. It isinjected intradermaly shaved dorsal skin of rts 100g. 24 hrs 0.1ml 2.5% Evans blue dye containing 25mg/ml egg albumin Test drug introduced IV: simultan.with Ag- 1 hr before challenging Dye soln. 20
- 21. 30 minsacrificed using ether anaesthesia. At Passive cutaneous anaphylactic reaction site Evans blue is leaked Extracted and determined colorimetrically 620nm Amount of Evans blue leaked for control gp taken as 100%, and calculating the % inhibition in the extravasation of dye in the rats in treated animals. Compared with std & control gps 21
- 22. 3.Arthus type immediatehypersensitivity reaction There is an Ag-Ab complex after the injection of antigen. But if the person or the animal is previously immunized with the antibody( i.e, it have circulating Ab) there is precipitation of allergic reaction. It is due to the activation of complement and PMN cell get activate and leads to inflammation 22
- 23. Methodology: in thisanimals are challenged with ovalbumin suspension(in paraffin oil)+pertusis vaccine in saline soln. mixed together to get emulsion. Wistar rats of 250-300g are selected Injected with emulsion After 7 days a gp of 8 animals (s,t c) are housed in a cage with diet and optional water till 1 hr before the arthus reaction 23
- 24. Each gpthen injected with purified ovalbumin in paw Swelling reaches max in few hrs Thickness measured with calipers or plethymometric method % reduction inthickness of foot pad compared with std and control gp 24
- 25.
- 26. Inhibition of histaminerelease from mast cells. Asthma hypersensitivity reactions Mediators are released from mast cells (histamin) Here; calcium ionophore induces mast cell to release histamin 26
- 27. Procedure: wistar ratare decapitated and exsanguinated 50ml Hank’s balanced salt soln (HBSS) injected to peritonial cavity Abdominal wall opened and collected fluid containing peritonial cell centrifuge at 2000rpm Resuspend in HBSS 1ml test drug added to mast cell suspension incubate @ 37ºC Cell made to 3ml with HBSS 27
- 28. Equal vol.of caciumionophore ia added Control soln 1. Spontaneous histamin release mast cells & soln used to determine base line 2. Histamin release: mast cell+ calcium ionophpre 3. Test compound control: soln+ compound to test the compound for native fluorescence 4. Solution control: only soln to determine the base line incubate @ 37ºC 28
- 29. From the suspension:1mi of top layer transferred to a tube containing 300mg Nacl+1.25ml butanol + NaOH mechanical shaking 1ml of the top layer pipetted to 5ml tube containing n-heptane +0.12N HCL mixed by inverting Organic layer aq.layer 29
- 30. To thetwo layer add 100µl 0.2% phthaldehyde soln. + 100µl NaOH 2mnts reaction is stopped by the addition of 50µl 3N Hcl Total sample added to auto sample vial Histamine concentration is determined by fluorescence detector using excitation wave length 350nm & emission wavelength 450nm 30
- 31.