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COX-2, eicosanoids and the resolution of inflammation

COX-2, eicosanoids and the resolution of inflammation. Paul Colville-Nash Department of Experimental Pathology William Harvey Research Institute. Spector, W.G. & Willoughby, D.A. (1968) Pharmacology of Inflammation. 1500 B.C. Ebers papyrus recommended

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COX-2, eicosanoids and the resolution of inflammation

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  1. COX-2, eicosanoids and the resolution of inflammation Paul Colville-Nash Department of Experimental Pathology William Harvey Research Institute

  2. Spector, W.G. & Willoughby, D.A. (1968) Pharmacology of Inflammation.

  3. 1500 B.C. Ebers papyrus recommended dried leaves of myrtle to expel rheumatic pains from womb

  4. In Roman times in Asia, China, the Americas and Africa, salicylate-containingplants were used

  5. Salicylic acid was synthesized in 1859 by Hermann Kolbe at Marburg University in Germany. His student, von Heyden, adapted the synthesis for industrial production in 1874. In 1876 the anti-rheumatic effect of salicylic acid was demonstrated in a clinical trial.

  6. Aspirin consumption worldwide 15x1012 tablets per year or 45,000 tons per year (information from Bayer AG)

  7. Proposals for Mode of Action of Aspirin-like Drugs • Northover and Subramanian (1961) Inhibit kallikrein • Whitehouse (1962) Interfere with oxidative metabolism • Smith MJH (1966) Stabilise capiliary permeability • Collier HOJ (1969) Block a route to mediator receptors, or block release of an intermediate • McArthur (1971) Displace endogenous anti inflammatory peptides from plasma proteins • Di Rosa et al (1971) Interfere with migration of leukocytes • Barker (1971) Hyperpolarise nerve membranes • Northover (1971, 1973) Inhibit Ca++ uptake or binding to cellular membranes • Chang et al (1972) Inhibit leukocyte phagocytosis • Ignarro (1972) Stabilise lysosomal membranes • Sharma (1972) Inhibit generation of lipoperoxides

  8. 1960s • Prostaglandins are a family of potent lipid mediators derived from arachidonic acid • They are made by all cells in the body except the red blood cells Roles of Prostaglandins in the 1970’s • Pyretic (Feldberg & Gupta, 1973; Milton & Wendlandt, 1973) • Pro-inflammatory(Willis, 1969) • Hyperalgesic(Ferreira, 1972) • Inhibit gastric acid secretion(Robert, 1968) • Contract the uterus(Bergstrom et al .,1968) • Increase renal blood flow(Lonigro et al., 1973)

  9. 1971 Discovered that aspirin and similar drugs inhibit the biosynthesis of prostaglandins proposed that this was their mode of action Indomethacin Inhibition (%) 100 1 1 3 4 80 1 Aspirin 60 3 3 Salicylic acid 40 3 2 1 4 4 20 0 0.1 1.0 10 100 1000 Log concentration (µg/mL) (Vane, 1971)

  10. Clues to COX-2 Flower and Vane (1972) found paracetamol more active on brain COX than on spleen COX. “Our results support the idea that a study of prostaglandins synthetase systems from different systems will lead to aspirin-like drugs with a greater specificity of action.”

  11. Clues to COX-2 “Selective inhibition of prostaglandin production in inflammatory exudates and the gastric mucosa.”Whittle et al. Nature, London (1980) ?COX selectivity to explain the lack of toxicity of salicylate and BW755 on the stomach.

  12. Discovery of COX-2 • Cells may contain two pools of COX, a constitutive COX enzyme and a different COX which is LPS-inducible and the expression of which is sensitive to glucocorticoid inhibition Fu, Masferrer, Seibert, Raz and NeedlemanJ Biol Chem, 1990 • In 1991, Dan Simmons published the structure of a protein encoded by an early response gene which was 60% homologous with COX in ram seminal vesicles Xie et al. Proc. Natl. Acad. Sci. USA88 2692-2696 April 1991

  13. The three dimensional structure of COX-1 has been published by Garavito et al. and that of COX-2 by Browner et al.

  14. Comparison of NSAID Binding Sites COX-2 COX-1 M. Browner, et al, 1998Roche Bioscience

  15. PhysiologicalStimulus InflammatoryStimulus Aspirin-like drugs are anti-inflammatory by inhibition of prostaglandin biosynthesis by COX-2 and are ulcerogenic through inhibition of COX-1 Macrophages/Other Cells COX-1 constitutive COX-2 Induced PGE2 Kidney TXA2 platelets PGI2 endothelium stomach mucosa Proteases PGs Other Inflammatory Mediators Inflammation

  16. Summary of COX Selectivities in WHRI Blood / A549 Assay 100 COX-1 selective COX-2 selective 10 1 (IC50 ratio (COX-2/COX-1) 0.1 0.01 0.001 NS398 Tolmetin etodolac sulindac celecoxib salicylate L745337 diclofenac nimesulide meloxicam Naproxen Aspirin Ibuprofen ketoprofen Flurbiprofen Diflunisal Indomethacin

  17. NSAIDs and Joint Destruction: • NSAIDs clinically efficacious: reduce joint pain and swelling • But • NSAIDs may promote joint damage: • Newman and Ling, Lancet, 1985; • De Brito et al, Br J Rheum, 1986 • Brandt, Am J Med, 1987; • Bottomley et al, Br J Pharm, 1988; • Pettipher et al, Ann Rheum Dis, 1989; • Bulstra et al, Clin Orthop, 1992.

  18. A role in the resolution of inflammation? Cyclooxygenase (COX) A key enzyme in prostaglandin synthesis Two isoforms: COX-1, constitutive, maintenance of physiological processes COX-2, inducible, major target for new generation NSAIDs with reduced side effects e.g. less injurious to normal gastric mucosa BUT COX-2 associated with wound healing phase of gastric ulcers COX-2 selective inhibitors delay healing of gastric ulcers in rodents

  19. Willoughby, D.A. (1975). Annals of Rheumatic Disease, 34.

  20. Summary • Initial peak in COX-2 protein expression at 2hours, associated with maximal PGE2 synthetic activity ex vivo and raised PGE2 levels in exudates • Second greater peak in COX-2 protein expression at 48hours during resolution associated with minimal PGE2 synthetic activity ex vivo and minimal levels of PGE2 in exudates • Non-selective and selective COX-2 inhibitors exacerbate inflammation if given during resolution phase of carrageenan pleurisy • Second peak of COX-2 protein expression associated with raised levels of PGD2 and 15deoxyD12-14PGJ2, these prostanoid's levels reduced on treatment with COX inhibitors • Replacement of these prostanoids during COX inhibitor treatment reverses exacerbation of inflammation at 48hours

  21. Inducible cyclooxygenase (COX-2) may have anti-inflammatory properties Derek Gilroy, Paul Colville-Nash, Dean Willis, Joanne Chivers, Mark Paul-Clark and Derek Willoughby Department of Experimental Pathology William Harvey Research Institute Barts and The London, Queen Mary’s School of Medicine and Dentistry Charterhouse Square London, EC1M 6BQ. United Kingdom Nature Medicine, 5, p698, 1999.

  22. Leukocyte NF-B activity EMSA 6h 48h l l A A Time (h) e e 0 0 l l e e 5 5 R R p p R R 3 6 24 48 c S N c S N NF-B

  23. ) 1.5 200 6 0 1 ( *** ) s l l m l *** 1.0 e ( c e t y 100 a r d o t u 0.5 a x m E m a l f 0.0 0 n 24h con PDTC 24h con PDTC I ) 1.5 200 6 0 1 ( *** ) l s m l *** l ( e 1.0 c e t y a 100 r d o u t x 0.5 a E m m a l f 0.0 0 n I 24h con MG132 24h con MG132

  24. *** ***

  25. Possible role for NF-kB in the resolution of inflammation Toby Lawrence, Derek W Gilroy, Paul Colville-Nash and Derek A Willoughby Department of Experimental Pathology William Harvey Research Institute Barts and The London, Queen Mary’s School of Medicine and Dentistry Charterhouse Square London, EC1M 6BQ. United Kingdom Nature Medicine, 7(12), p1291, December 2001.

  26. RelA (p65) Caspase 3 RHD NLS TA1 TA2 Apoptosis overrides survival signals through a caspase-mediated dominant-negative NF-B loop. Levkau et al. Nat. Cell Biol. 1:227 (1999) Ravi et al. Cancer Res. 58:882 (1998) Kang et al. J. Biol. Chem. 276:24638 (2001)

  27. Endogenous 15deoxy12-14PGJ2 regulates leukocyte apoptosis in vivo ** ** * *

  28. NSAID 15deoxyD12-14PGJ2 X X X X X X X Proinflammatory cytokines (e.g.IL-1b, TNFa, IL-6) Matrix metalloproteases (e.g. MMP-1, MMP-2) Inflammatory cell adhesion molecules (e.g. ICAM-1) Inflammatory enzyme systems (e.g. iNOS)

  29. COX-1, COX-2, and COX-3 and the future treatment of chronic inflammatory disease • Derek A Willoughby, Adrian R Moore and Paul Colville-Nash • Department of Experimental Pathology, William Harvey Research Institute • Barts and The London, Queen Mary’s School of Medicine and Dentistry • Charterhouse Square, London, EC1M 6BQ. United Kingdom • Lancet, 355 (9204), p646, 2000. • Treatment during disease flare with NSAIDs beneficial • Treatment during periods of disease remission perhaps less desirable • Options: a) Stop NSAID treatment during remission - difficult • b) Replace endogenous mechanisms - not available yet • c) Block reactivated pro-inflammatory systems

  30. Alternative approaches

  31. iPLA2 cPLA2 sPLA2 Arachidonic acid Cytochrome p450 5-, 12- & 15 lipoxygenase PGG2 COOH O O O2H Cyclooxygenase PGH2 PGD2 COOH O OH hPGD2S O TxA2 TxA2 synthase COOH OH COOH O O OH mPGE2S/cPGE2S O PGF2 synthase OH PGI2 synthase dehydration PGE2 PGF2 O PGI2 OH 15deoxy12-14PGJ2 COOH COOH COOH COOH O OH OH OH OH O OH OH Arachidonic acid release and metabolism

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