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Experimental evaluation of anti inflammatory agents

Experimental evaluation of anti inflammatory agents

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Experimental evaluation of anti inflammatory agents

  1. 1. 1Dr. Aditi M. Panditrao
  2. 2. Experimental Evaluation of Anti Inflammatory Agents 2Dr. Aditi M. Panditrao
  3. 3. Introduction • Inflammation – self-defense mechanism • Protective response intended to eliminate the initial cause of cell injury as well as the necrotic cells and tissues resulting from the original insult • Symptom of a wide variety of diseases 3Dr. Aditi M. Panditrao
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  5. 5. Phases of Inflammation • Acute - vasodilatation & increased capillary permeability • Delayed - infiltration of leukocytes and phagocytes • Chronic Proliferative - tissue degeneration and fibrosis 5Dr. Aditi M. Panditrao
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  7. 7. Steps in inflammatory response 1. Recognition of the injurious agent 2. Recruitment of leukocytes 3. Removal of the agent 4. Regulation of the response 5. Resolution 7Dr. Aditi M. Panditrao
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  9. 9. 9Dr. Aditi M. Panditrao
  10. 10. 10 Anti-inflammatory Analgesic Anti-pyretic In vitro In vivo • 3H-Bradykinin receptor binding • 3H-Substance P receptor binding • Assay of PMNLchemotaxis in vitro • PMNL aggregation • Constitutive and inducible cellular arachidonic acid metabolism in vitro • Formation of leukotriene B4 in human white blood cells in vitro • Formation of lipoxygenase products from 14C-arachidonic acid in human PMN in vitro • Formation of eicosanoids from 14C-arachidonic acid in human platelets in vitro • Methods for testing acute and subacute inflammation • Methods for testing the proliferative phase (granuloma formation) • Side effects of anti-inflammatory agents Dr. Aditi M. Panditrao
  11. 11. In Vitro Methods 11Dr. Aditi M. Panditrao
  12. 12. 3H-Bradykinin receptor binding • Bradykinin – inflammatory mediator • Bind to B1 and B2 receptor • B1 receptor – Mast cells, Neutrophils, Macrophages • B2 receptor – Smooth muscle, Sensory nerves, cardiac tissue 12Dr. Aditi M. Panditrao
  13. 13. Hageman factor (XII) Activation Prekallikrein HMW Kininogen LMW Kininogen Kallikrein Kallikrein Bradykinin Kallidin Des Arg Bradykinin Trauma, Tissue injury • Vasodilation • ↑ Vascular Permeability • Bronchoconstriction • Pain B1 • Pain •Histamine release • Recruit Neutrophils • IL-1, TNF-α, etc. INFLAMMATION Role of Bradykinin in Inflammation 13Dr. Aditi M. Panditrao
  14. 14. • Tissue homogenates in incubation buffer • Incubated with • 3H-Bradykinin alone – Total Binding • 3H-Bradykinin + unlabelled Bradykinin (saturated) – Non-specific Binding • 3H-Bradykinin + Test drug (subtract the non-specific binding) • Two types – • Competition studies – increasing concentrations of Test drug • Saturation studies – increasing concentrations of 3H-Bradykinin • Tissues – G. pig ileum, rabbit carotid artery, canine cultured tracheal smooth muscle cells, bovine aortic endothelial cells, human fibroblasts, etc. 14Dr. Aditi M. Panditrao
  15. 15. 3H-Substance P receptor binding • Substance P – inflammatory mediator • Bind to NK1 and NK2 receptor • Direct and Indirect Actions • ↑ Levels – Acute and Chronic Inflammatory processes 15Dr. Aditi M. Panditrao
  16. 16. Trauma, Tissue injury Peripheral Nerves Substance P Direct Actions • Pain • Vasodilation • ↑ Vascular Permeability • Neutrophil recruitment • Activation of Macrophages and Mast cells • Release of ILs, Chemokines NK1, NK2 Indirect Actions • Angiogenesis • Stimulation of Adhesion molecules • Induces Apoptosis? 16Dr. Aditi M. Panditrao
  17. 17. • Tissue homogenates in incubation buffer • Incubated with • 3H-Substance P alone – Total Binding • 3H-Substance P + unlabelled Substance P (saturated) – Non- specific Binding • 3H-Substance P + Test drug (subtract the non-specific binding) • Two types – • Competition studies – increasing concentrations of Test drug • Saturation studies – increasing concentrations of 3H-Substance P • Tissues – Chinese Hamster Ovary (CHO), CHO cells transfected with human ileum NK2 receptor, Guinea pig cortical membranes, human lymphoblasts, 17Dr. Aditi M. Panditrao
  18. 18. Isolated Tissue Preparations for NK receptors • Inhibition of Substance P-induced endothelium- dependent relaxation of rabbit pulmonary artery, previously contracted with 0.1 μM Noradrenaline • Inhibition of Substance P or Substance P sulfone- induced contractions of guinea pig ileum in the presence of 3 μM Atropine and 3 μM Mepyramine and Indomethacin • Inhibition of Substance P-induced plasma extravasation in guinea-pig bronchi 18Dr. Aditi M. Panditrao
  19. 19. Polymorphonuclear Leukocyte Chemotaxis • Measures the chemotactic effects on the PMN Leukocytes 19Dr. Aditi M. Panditrao
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  22. 22. Polymorphonuclear Leukocyte Chemotaxis • Measures the chemotactic effects on the PMN Leukocytes • Migration rate – number of PMNs in the lower well x 100 number of PMNs in the upper well • Dependent on the concentration of the chemoattractant (e.g., zymosan-activated serum) 22Dr. Aditi M. Panditrao
  23. 23. PMN leukocytes aggregation induced by FMLP • FMLP (formyl-L-methionyl-Lleucyl- L-phenylalanine) • PMNs cell suspensions + test compounds or standard (pentoxiphylline) are dissolved in GBSS • Incubated for 10 min • Serial dilutions of FMLP added • Change in transmittance 23Dr. Aditi M. Panditrao
  24. 24. Constitutive and inducible cellular arachidonic acid metabolism in vitro • Formation of leukotriene B4 in human white blood cells • Formation of lipoxygenase products from 14C- arachidonic acid in human polymorphonuclear neutrophils (PMN) • Formation of eicosanoids from 14C-arachidonic acid in human platelets • Stimulation of inducible prostaglandin pathway in human PMNL 24Dr. Aditi M. Panditrao
  25. 25. COX-1 and COX-2 inhibition • Inhibition studies with recombinant human COX-1 and COX-2 • HPLC assay for oxygenation of radiolabelled arachidonic acid by COX-1 • Determination of the stoichiometry of inhibitor binding • Spectrophotometric assay of recombinant human COX-2 • Whole-cell assays with transfected Chinese hamster (CHO) cells expressing COX-1 and COX-2 25Dr. Aditi M. Panditrao
  26. 26. Induced release of cytokines from human white blood cells • Interleukin-1alpha, IL-1beta, IL-6, IL-8 and TNF-alpha • Cell fraction from whole blood separated • Exposed to LPS (Salmonella abortus equii) with or without test drug • Incubation • Analysis of Cytokines 26Dr. Aditi M. Panditrao
  27. 27. Flow cytometric analysis of intracellular cytokines • Very powerful tool in which individual cells can be simultaneously analyzed for several parameters: • Size, granularity • Expression of surface and intracellular markers • Defined by fluorescent antibodies. • Fluorescent anti-cytokine and anti-chemokine monoclonal antibodies 27Dr. Aditi M. Panditrao
  28. 28. In Vivo Methods 28Dr. Aditi M. Panditrao
  29. 29. • Based on the symptoms observed during the inflammation • Acute/Transient phase: In this phase vasodilation and increased capillary permeability are observed. • Subacute/Delayed phase : Infiltration of leucocytes and phagocytes in blood. • Chronic inflammatory phase: granuloma formation is observed in this phase 29Dr. Aditi M. Panditrao
  30. 30. • Acute and Subacute phase: • Carrageenan induced paw oedema in rats • Croton-oil induced ear edema • Oxazolone induced ear edema • UV-B erythema in guinea pigs • Pleurisy in rats • Vascular permeability • Chronic phase: • Cotton wool granuloma • Glass rod granuloma 30Dr. Aditi M. Panditrao
  31. 31. Paw edema • Most commonly employed method • Various substances used: • Carrageenin, Brewer’s yeast, Formaldehyde, Dextran, Egg albumin, Kaolin, Zymosan • Serotonin, Histamine, Substance P, Cobra Venom • Subplantar region Left Hind Paw (0.05 ml-SC) • Immediately after injection, again 3 and 6 h, and eventually 24 h after challenge 31Dr. Aditi M. Panditrao
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  33. 33. • Biphasic Response: 1st Phase – Hyperemia – Histamine, Serotonin, Kinins 2nd Phase – Edema – Prostaglandins, Chemokines • % Inhibition – Baseline volume – Volume at time x 100 Baseline volume 33Dr. Aditi M. Panditrao
  34. 34. Ear edema • Croton oil, Tetradecanoyl phorbol acetate, Xylol, Oxazolone • Topical application • Whole ear, punched discs, caliper, other ear 34Dr. Aditi M. Panditrao
  35. 35. Ultraviolet B erythema in guinea pigs • Erythema – earliest sign of inflammation • Depilated skin exposed to UV-B rays for 20-30 secs. • Degree of erythema estimated after 2 hours visually • 0 = no erythema • 1 = mild erythema • 2 = moderate erythema • 3 = strong erythema • 4 = very strong erythema 35Dr. Aditi M. Panditrao
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  37. 37. • Pure measure of the vasodilatory phase • No accompanying exudation or edema • Drawbacks: • Variable response • Accurate measurement of response • Other sites/animals - • Guinea pig ear • Sprague Dawley rats • Hairless mice (skin thickness) 37Dr. Aditi M. Panditrao
  38. 38. Pleurisy test • Exudative Inflammation • Histamine, Bradykinin, Prostaglandins, Dextran, Antigens, Microbes, irritants like turpentine and carrageenan etc. • Fluid extravasation, leukocyte migration, lysosomal enzyme activities etc. 38Dr. Aditi M. Panditrao
  39. 39. Vascular permeability • Inhibitory activity of drugs against increased vascular permeability • Extravasation of dye into surrounding tissue 39Dr. Aditi M. Panditrao
  40. 40. Cotton wool granuloma • Foreign body granuloma • Sterile Cotton pellets embedded subcutaneously • Back, Axilla, Groin • After 7 days – Pellet + Granuloma removed • Weight of granulomatous tissue = Dry weight of granuloma cotton – initial weight of cotton pellet 40Dr. Aditi M. Panditrao
  41. 41. 41Dr. Aditi M. Panditrao
  42. 42. Glass Rod granuloma • Foreign body granuloma • Glass peices embedded subcutaneously • Back, Axilla, Groin • After 20-40 days – Granuloma + Connective removed entirely • Glass rod can be removed • Tensile strength and wet weight of granulomatous tissue can be measured 42Dr. Aditi M. Panditrao
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  44. 44. Side Effects of Anti- inflammatory Agents 44Dr. Aditi M. Panditrao
  45. 45. Ulcerogenic effect in rats • Gastric irritation • 48 hrs fasting • 3, 5, 7 hrs after drug administration – sacrificed • Check gastric lining for hyperemia and ulcers 45Dr. Aditi M. Panditrao
  46. 46. Measurement of gastric mucosal damage by intragastric inulin • Inulin in the gastric wall – measure of gastric damage • Given intragastric • 3H-inulin radioactivity is expressed as disintegrations/min/ml of plasma or per gram of tissue 46Dr. Aditi M. Panditrao
  47. 47. Determination of blood loss • Microbleeding which is not detected by observation of overt ulcers • Feces, Stomach and intestinal contents • Heme - spectrophotometry 47Dr. Aditi M. Panditrao
  48. 48. Specific Disease Process 48Dr. Aditi M. Panditrao
  49. 49. Adjuvant Arthritis in Rats • Mimics human rheumatoid arthritis • Freund’s Adjuvant – Mycobacterium butyricum • Delayed hypersensitivity response to mycobacterial antigen • Subplantar region of left hind paw 49Dr. Aditi M. Panditrao
  50. 50. • Day 1 Day 4 Day 7 Local Peak Systemic reaction disease • Parameters : • Paw edema, weight, grip strength , deformity measured • Evaluation of chronically administered drug against inflammation • On Day 5 and 21: Plethysmometer 50Dr. Aditi M. Panditrao
  51. 51. • On Day 21: ears: absence of nodules and redness . . . . . . . . . 0 presence of nodules and redness . . . . . . . . 1 nose: no swelling of connective tissue . . . . . . . . . 0 intensive swelling of connective tissue . . . 1 tail: absence of nodules . . . . . . . . . . . . . . . . . . . . . 0 presence of nodules . . . . . . . . . . . . . . . . . . . . . 1 forepaws: absence of inflammation . . . . . . . . . . . . 0 inflammation of at least 1 joint . . . . . . . 1 hind paws: absence of inflammation . . . . . . . . . . . 0 slight inflammation . . . .. . . . . . . . . . . . . 1 moderate inflammation . . . . . . . . . . . . . 2 marked inflammation . . . . . . . . . . . . . . . 3 51Dr. Aditi M. Panditrao
  52. 52. • Day 5 : Primary Lesions • Day 21 : Secondary Lesions • Arthritic Index : Sum of scores of each animal • Total % change = percent inhibition of the injected paw on day 5 + percent inhibition of the non-injected paw on day 21 + percent change of the arthritic index 52Dr. Aditi M. Panditrao
  53. 53. Schultz-Dale Reaction • Anti-anaphylactic activity • Guinea pigs are sensitized against egg albumin. • Challenge after 3 weeks causes in isolated organs release of mediators, e.g. histamine, which induce contraction in isolated ileum 53Dr. Aditi M. Panditrao
  54. 54. Adhesion Assays • VCAM-1,ICAM-1 and E-selectin • HUVEC ,HDMEC cell lines • Cell based ELISA – assay • Endothelial Cell – Neutrophil adhesion • Platelet – Neutrophil adhesion 54Dr. Aditi M. Panditrao
  55. 55. References • Mahesh G, Ramkanth S, Saleem MTS. Anti-inflammatory drugs from medicinal plants- A comprehensive review. International Journal of Review in Life Sciences 2011; 1(1): 1-10. • Paschapur MS, Patil MB, Kumar R, Patil SR. Evaluation of anti-inflammatory activity of ethanolic extract of Borassus flabellifer L. male flowers (inforescences) in experimental animals. Journal of Medicinal Plant Research 2009; 3(2): 49-54. • Kumar V, Bhat ZA, Kumar D, Khan NA, Chashoo IA. Evaluation of anti-inflammatory potential of leaf extracts of Skimmia anquetilia. Asian Pacific Journal of Tropical Biomedicine 2012; 627-630. • Lavanya R, Maheshwari US, Harish G, Raj BJ, Kamali S, Hemamalani D, Varma BJ, Reddy UC. Investigation of In-vitro anti- inflammatory, anti-platelet and antiarthritic activities in the leaves of Anisomeles malabarica Linn. Research Journal of Pharmaceutical, Biological and Chemical Sciences 2010; 1(4): 745-752. • Talhouk RS, El-Jouni W, Baalbaki R, Gali-Muhtasib H, Joanna K, Talhouk SN. Anti- inflammatory bioactivities in water extract of Centaurea ainetensis. Journal of Medicinal Plants Reserch 2008; 2(2): 24-33. • Ghani A. Medicinal plants of Bangladesh with chemical constituents and uses. 2nd ed. Dhaka: Asiatic Society of Bangladesh; 2003, p. 345. • Kumar S, Kumar D, Deshmukh RR, Lokhande PD, More SN, Rangari VD. Antidiabetic potential of Phyllanthus reticulatus in alloxan-induced diabetic mice. Fitoterapia 2008; 79: 21-23. • Winter CA, Risley EA, Nuss GW. Carrageenan induced oedema in hind paws of the rats as an assay for anti-inflammatory drugs. Proceedings of the Society for Experimental Biology and Medicine; 111: 544-557. • Okokon JE, Udoh AE, Frank SG, Amazu LU. Anti-inflammatory and analgesic activities of Melanthera scandens. Asian Pacific Journal of Tropical Biomedicine 2012; 1: 144-148. • Manning, D. C., Snyder, S. H., Kachur, J. F., Miller, R. J. & Field, M. (1982) Nature (London) 299, 256- 259. 55Dr. Aditi M. Panditrao
  56. 56. References • Das S, Datta R, Nandy S. Phytochemical screening and evaluation of anti- inflammatory activity of methanolic extract of Abroma augusta Linn. Asian Pacific Journal of Tropical Disease 2012; S114-S117. • Georgewill OA, Georgewill UO, Nwankwoala RNP. Anti-inflammatory effects of Morninga oleifera Lam extract in rats. Asian Pacific Journal of Tropical Medicine 2010; 3: 133-135. • Georgewill OA, Georgewill UO. Evaluation of the anti-inflammatory activity of extract of Vernonia amygdalina. Asian Pacific Journal of Tropical Medicine 2010; 3: 150-51. • Rosa MD, Giround JP, Willoghby DA. Studies of the acute inflammatory response induced in rats in different sites by Carrageenan and turpentine. Journal of Pathology 1971; 104: 15-29. • Sheetal S. Chaudhari, Sanjay R. Chaudhari, Machindra J. Chavan. Analgesic, anti-inflammatory and anti-arthritic activity of Cassia uniflora Mill. Asian Pacific Journal Tropical Biomedicine 2012; 2: 181-86. • Khan H, Khan MA, Muhammad N, Ashraf N, Gul F, Tariq SA. Anti- inflammatory and antioxidant activity of Joshanda partially mediated through inhibition of lipoxygenase. Phytopharmacology 2012; 3: 19-28 56Dr. Aditi M. Panditrao
  57. 57. Thank You!! 57Dr. Aditi M. Panditrao

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