Screening models for immunomodulators
- 1. NETAJI SUBHAS CHANDRA BOSE INSTITUTE OF PHARMACY URMISTHA SARKAR M.PHARM 1ST YEAR
- 2. IMMUNITY is the balanced state of multicellular organisms having adequate biological defence to fight infection, disease, or other unwanted biological invasion, while having adequate tolerance to avoid allergy, and autoimmune diseases. IMMUNOMODULATORS are the agents that modulates the immune system by suppress or stimulate the immune response.Therefore these are divided into two type: IMMUNOSUPPRESSANT IMMUNOSTIMULANT
- 3. •Acute systemic anaphylaxis in rats •Passive cutaneous anaphylaxis •Delayed type hypersensitivity •Arthus type immediate hypersensitivity •Collagen type II induced arthritis in rats
- 4. PRINCIPLE:Anaphylaxis is defined as a serious allergic reaction that is rapid in onset and may cause death. It is influenced by cellular events in mast cells and basophiles resulting in the release of mediators. RATIONALE:Rats are immunized with ovalbumin and Bordetella pertussis suspension as adjuvant. After 11 days the animals are challenged by intravenous injection of ovalbumin. The shock symptoms can be inhibited by corticosteroids and intravenous disodium cromoglycate. ANIMAL:Female Sprague-Dawley rats weighing 120 g PROCEDURE: Ten–20 animals are used for each group. Firstly immunized by i.m. injection of 10 mg/kg highly purified ovalbumin. Simultaneously 1 ml of Bordetella pertussis suspension (2 × 1010 organisms) is injected intraperitoneally.
- 5. Eleven days later the animals are challenged by intravenous injection of 25 mg/kg highly purified ovalbumin 18 hours prior to challenge; test- dexomethasone 1-10mg/kg s.c injection control- vehicle EVALUATION:The shock symptoms are scored and mortality counted. Results after treatment are compared with untreated controls. Pretreatment with corticosteroids or disodium cromoglycate can inhibit death and ameliorate shock symptoms. Statistical calculation is performed using the χ2-test.
- 6. PRINCIPLE: Passive cutaneous anaphylaxis is a immune reaction of the immediate type. By passive immunization of rats in the skin with rat anti- ovalbumin serum and a challenge 2 days later with ovalbumin at the same skin area antigen-antibody complexes are formed in the mast cells inducing release of mediators. This results in vasodilatation, increase in permeability of the vessel walls and leakage of plasma. RATIONALE: To make the allergic reaction visible, Evan’s blue dye is administered along with the antigen. Evan’s blue dye is attached to the albumin fraction of plasma, producing a blue spot. This blue spot indicates that an anaphylactic reaction has taken place in the skin. ANIMAL: Male albino wister rat weighing 100 g. PROCEDURE: Antiserum are injected intradermally in to the shaved dorsal skin of rats.
- 7. After 24hr each animal is challenged with the i.v administration of 0.1 ml of 2.5% evans blue dye containing 25 mg/ml of egg albumin Test compound also administered along with antigen After 30 mins animals are sacrificed. Amount of evans blue dye that leaked at the site of reaction are extracted and determined colorimetrically at 620 mµ wavelength. EVALUATION: The amount of Evans blue extracted from passive cutaneous anaphylactic reaction is taken as 100 percent. Percent inhibition of passive cutaneous anaphylactic reaction in the rats treated with the test compound is calculated.
- 8. PRINCIPLE & RATIONALE: Delayed type hypersensitivity is a reaction of cell mediated immunity and becomes visible only after 16–24 h .The same methods as for testing immediate type hypersensitivity can be used. ANIMAL: albino wister rat weighing 150g PROCEDURE: 7 days prior,rats are sensitized by i.m administration of 0.5 ml of ovalbumin. After 7 days again 0.1 ml of 0.04% ovalbumin injected in the left hind paw. Foot pad thickness is measured immediately and after 24h later.
- 9. PRINCIPLE: Antigen antibody induced reaction leading to an inflammatory factors that characterized by edema, hemorrhage and vasculities. RATIONALE: Arthus reaction of the immediate type becomes maximal 2–8 h after challenge. ANIMAL: Albino wister rat of both sex weighing about 220 g. PROCEDURE: 7 days prior,rats are sensitized by i.m administration of 0.5 ml of ovalbumin 1st GROUP (TREATED GROUP): 1hr prior test compound are administered and challenged with 0.1 ml of ovalbumin in the left hind paw.
- 10. 2nd GROUP (POSITIVECONTROL): Sensitized animal treated with solvent alone. 3rd GROUP (NEGATIVECONTROL): Non sensitized animal treated with solvent alone. EVALUATION: The change in footpad thickness is expressed as the percent change from the vehicle control group. Comparison of experimental group to positive control is evaluated statistically using Student’s t-test.The footpad thickness can be measured by calipers.
- 11. PRINCIPLE: The demonstration of antibodies to collagen in patients with rheumatic polyarthritis suggests that autoimmunity may contribute to the pathophysiology of synovitis and joint destruction. RATIONALE:Because of the similarities of the symptoms in rats to human disease the test is considered to be useful to detect anti- inflammatory and immunosuppressive properties of test compounds. ANIMAL: Six to 12 maleWistar rats with an initial weight of about 120 g are used for each group. PROCEDURE: On day 1, each rat receives intradermal injection total of 0.5 mg collagen in 0.5 ml, equally divided, in 5 sites. one at the base of each appendage and one in the nape of the neck.
- 12. Seven days post-immunization, the animals receive identical booster injections. Control animals receive only the incomplete Freund’s adjuvant diluted with 0.1 M acetic acid. The volume of both hind paws is measured plethysmographically on day 20. From days 20–40, the animals receive the test compounds p.o. once a day. On day 41, the paw volumes are recorded again. EVALUATION: The paw volumes of treated animals are recorded plethysmographically. The increase versus day 20 is calculated. The increase is compared with that of controls or animals treated with a standard drug. Otherwise, arthritic scores can be determined.
- 13. Vogel.H.G, Drug Discovery and Evaluation, second edition, Chapter:I, Antiarthrotic and immunmodulatory activity, page no: 797-806