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Screening models for inflammatory drugs

Screening methods for inflammatory drugs.

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Screening models for inflammatory drugs

  1. 1. SeminarOn Screening Methods For Inflammatory Drugs By: underguidance of: SHAIKH ZOHRAMEENA MRs.ANURADHA M.PHARMA (Pharmacology) 1st year
  2. 2.  Inflammation is a universal host defensive process involving a complex network of cell- cell, cell-mediator and tissue interaction.  Inflammation is response to variety of harmful stimuli physical, chemical, traumatic antigen challenge, infectious agents and ionizing radiations.
  3. 3. Factors Exogenous: Physical, chemical, mechanical, nutritional and biological etc. Endogenous: immunological reactions, neurological and genetical disorders. Types of inflammation Acute inflammation- short lasting. Chronic inflammation- may persists for week, months and year. Principle components of inflammatory response Increased blood flow. Increase capillary permeability. Increased migration of leucocytes into effected area.
  4. 4.  Inflammation disease cover a broad spectrum of conditions including auto immune diseases. Examples- Rheumatoid Arthritis, Osteoarthritis, Inflammatory bowel disease, Multiple sclerosis, Asthma, Chronic obstructive pulmonary disease, Allergic Rhinitis, Infectious disease, various types of Cancers and Cardiovascular diseases.  Inflammation is regulated by a large number of pro and anti inflammatory Medication such as histamine, PG (PGT2 and Prostacyclin), leukotriene (LTB4), serotonin, bradykinin, cytokines (IL-1, IL-6, IL-8, IL-n, TNF-a), Reactive oxygen species, growth factors, lysosomes, contents of neutrophils, Adipokines (leptin, adiponectins, resistin).
  5. 5.  COX assay.  Mast cell degranulation.  Inhibition of NO production induced by TNF- a in mouse macrophages.  Measurement of NO production in mouse macrophages.  Adhesion assays.  Platelet- neutrophils adhesion.  Cell based reporter gene assay.  FMLP- induced O2 generation by polymorphonuclear cells (PMNs).  FMLP- induced adhesion of PMN of HUVEC.
  6. 6.  UV- B induced erythema in guinea pigs.  Carrageen induced paw edema model.  Plural exudation method.  Cotton pellet induced granuloma.  Adjuvant arthritis.  Papaya latex induced arthritis.  Candida albicans induced arthritis in mice.  Air pouch model.  Croton oil induced ear edema in mice.  Arachidonic acid induced ear edema in mice.  Dextran sulphate sodium induced colitis in mice.
  7. 7.  COX-1 10ml of sample solution added to 19ml of 0.1M of L-adrenaline, dihydrogen tartrate and 10mM of hematin. After adding 0-2 units of COX-1 it is pre incubated for 5 mins by adding 10 mL of 10% formic acid. The PGE2 concentration is measured with a PGE2 enzyme immune assay.  COX-2 Assay mixture consists of 100mm rod phosphate, 1mm of hematin gelation, 2.5mL of compound in DMSO. It is pre incubated for 15 mins at 22 C and the 20 mL of solution of 1mM arachidonic acid and 1mM TPMD in assay buffer is added. The absorbance at 400nm is measured over the fast 36 sec and % inhibition calculated. The enzyme inhibition of TPMD in absence of COX-2 is also observe and subtracted from activity in presence of COX-2
  8. 8.  Heparinized Tyrode’s solution is injected into the peritoneal cavity of exsanguinated rat (Sprague dawley after abdominal massage, the cells in peritoneal fluid are harvested and separated through 38% Bovine Serum Albumin. Cells are washed and suspended in Tyrode’s solution with 0.1% BSA.  The cell suspension is pre incubated with test drugs at 37 C for 3 min. fifteen mins after addition of compound 48/80 (standard compound for mast cell degranulation), glucuronidase (1mM phenolphthalein-D-glucuronide in 0.1 M acetic acid buffer, pH 4.5 is used as a substrate, absorbance monitored at 550nm after alkalization) and histamine (0.2% o-phthalaldehyde condensation in pH 12.5 fluorescence is monitored at 350/450 nm after acidification) in the supernatant are determined.
  9. 9. o The total content is measured after treatment of the cell suspension with Triton X-100. o The percentage release determined is the index of anti- inflammatory activity.
  10. 10.  Thrombin activated human platelets are incubated with drug at 20 C for 10 mins, and mixed with neutrophils at a ratio of 10:1.  Neutrophil with two or more (number positives) and one or no adherent platelets (number negatives) are counted as index of activity.  The test drug block the adhesion with respect to controls.
  11. 11.  Animal used- Albino guinea pigs  Test drug is administered 30 mins before exposure.  Duration of exposure to UV- B: 20 sec.  Area of exposure: Depilated skin.  Degree of erythema estimated after 2 hours visually on scale of 0-4.  Model can be use as a pure measure of the vasodilatory phase in the inflammation reaction.
  12. 12.  Rat paw edema (acute and subacute phases)  Drugs: carageenan (1%, 0.1 ml SC), indomethacin 2 mg/kg IP.  Site: subplantar region of left hind paw.  Biphasic o 1- release of histamines, 5 HT and kinins. o 2- release of prostaglandins. Parameter : paw volume upto the ankle joint is measured in treated and untreated groups before and after carrageenan.
  13. 13.  For measurements of activity of anti- inflammatory drugs on proliferative components of subacute and chronic inflammatory processes  Procedure : Sterile cotton pellets (wt- 5mg-50mg). • SC sites of back, axilla and groin. duration: 1-14 days • Pellet along with granuloma removed and weighed.
  14. 14. Weight of granulomatous tissue = Dry weight of granuloma cotton – initial weight of cotton pellet. Drugs effective: corticosteroids.