AN UPDATE ON CYCLOOXYGENASE ENZYME AS THERAPEUTIC AGENTS

1. AN UPDATE ON CYCLOOXYGENASE ENZYME AS THERAPEUTIC AGENTS Dr. Kripa Shanker Gupta Post Grad Student 2nd Yr MAMC, N. Delhi

2. History • 1930 – Goldbatt : extraction of PGs from semen Von Euler : BP & smooth muscle contraction • 1950s – Purification of Prostaglandins • 1971 – John Vane : Aspirin acts through COX • 1980s – COX – constitutively expressed and unregulated. • Late 80s – COX induced by Inflammatory cytokines • 1991 – Second isoform of COX : COX – 2 • 2002 – 3rd variant : COX – 3 ?

3. EICOSANOID FACTS • 20-carbon compounds • Include prostaglandins, prostacyclins, thromboxanes, leukotrienes • Physiological effects at very low concentrations • Many of their effects mediated by cyclic AMP or calcium second messengers • Unlike hormones, not transported in the blood • Local mediators that act where synthesized or in adjacent cells

4. Dietary linoleic acid metabolism Arachidonic acid esterification Membrane phospholipids  Cell Activation Events: mechanical trauma, cytokines growth factors Zyflo  Arachidonic acid  Anti-inflammatory glucocorticoids Lipooxygenase (LOX) Cyclooxygenase (COX)  Aspirin, Indomethacin, Ibuprofen NSAIDs Phospholipase A2 (PLA2) GC induce lipocortin that inhibits PLA2 Prostaglandins and thromboxanes (Cyclic/ring product) Leukotrienes (Linear product) Aspirin inhibits irreversibly Indomethacin forms a salt bridge in the binding site Ibuprofen competes for substrate binding Zyflo competes with AA for binding Figure1. Liberation of arachidonic acid and its metabolism to prostaglandins/ thromboxanes or to leukotrienes

5. aspirin indomethacin ibuprofen O2 Cyclooxygenase PGG2 2GSH Hydroperoxidase GSSG Arachidonic Acid (6) derived from membrane phospholipids X Prostaglandin endoperoxide synthase PGH2 central intermediate (Head of pathway) Figure3. Conversion of arachidonic acid to PGH2

6. Thromboxane Synthase Prostacyclin Synthase PGI2 platelet disaggregation (Inhibits aggregation); vasodilation  stomach acid secretion TXA2 platelet aggregation (Together) vasoconstriction platelets endothelial cells Isomerase (many cells) Isomerase (CNS, mast & fat cells) PGE2 wake (Eye opener), pain, fever, inflammation, renal arteriolar dilation PGD2 Sleep (Drowsy) Reductase fat cells 15-deoxy-12,14-PGJ2 induce fat cell differentiation (Jumbo) PGH2 central intermediate (Head of pathway) PGF2 labor induction (Fetus) Figure3. Conversion of PGH2 to prostaglandins and thromboxane of the “2-series”

7. Cyclooxygenase Enzyme • Earlier tissue homogenates used as source of enzymatic activity • Present in all cells • Purified enzyme by amide gel electrophoresis • Classified as integral microsomal membrane protein • Different PG synthesis rate and activity reported

8. Evidence of COX isoforms • Autoinactivation rates of COX, inhibition of NSAIDS and time course profile of PGE2 and PGF2alpha synthesis Activating platelets – within minutes PG Synthesis Mitogen stimulated fibroblasts – hours • Steroids inhibited the IL-1 induced COX activity but not basal COX activity • Cell Growth Studies – genes products inducible in-vitro exhibiting COX similarity • Western blot hybridization c DNA probe – 2 different mRNA 4 kbps and 2.8 kbps

9. Discovery of COX - 2 • Studies on cell division • Mitogen activated early genes activity in fibroblasts • Swiss 3T3 cells – sequence encoded new inducible gene • Mouse TIS10 cDNA expression increased prostaglandin E2 activity of which suppressed by NSAIDS

10. Structural Details • Both are dimer bound to microsomal membrane • 4 domains • Dimerization domain • Membrane binding domain • Catalytic domain – differ in structure • Terminal signal peptide domain – differ in length • Post/co translational glycosylation

14. A. Physiological stimulus B. Inflammatory stimulus (tissue injury, chronic arthritis) macrophages/other cells clotting, parturition, gastrointestinal and renal protection COX-2 induced by cytokines (e.g., TNF) COX-1 constitutive Proteases Prostaglandins especially PGE2 Other inflammatory mediators (histamine, etc) Prostacyclin endothelium-anticlotting stomach mucosa:  H+,  HCO3-,  mucus PGE2 Kidney: arteriolar dilation; Na+/H2O excretion TXA2 platelet aggregation PGF2 parturition Inflammation, redness, swelling, pain Figure 8. Actions of two known isoforms of cyclooxygenase (COX).

17. COX 1 Variants COX -1 V 1 (COX – 3) PCOX – 1A PCOX – 1B COX – 1 SNPs Other COX – 2 Variants COX -2 V 1 PCOX – 2 B COX – 2 SNPs Other Newer isoforms ( Variants )

18. Isoforms

19. COX – 3 : a theory without evidence? • acetaminophen as a "COX-3" inhibitor or even a "selective COX-3 inhibitor" in rat studies • induced enzyme appearing 48 hours after the start of the inflammation • COX – 1 gene with intron -1 changing protein folding and active site confirmation • Expressed more in brain cells

20. Nonsteroidal Anti-inflammatory Drugs(NSAIDs) • Common therapeutic indications • Common adverse effects • Different pharmacokinetics and potency • Different chemical families • Common mechanism of action (cyclooxygenase inhibition) • Different selectivities to COX I and II Similarities more striking than Differences

21. Common Therapeutic Uses • Analgesic(CNS and peripheral effect) may involve non-PG related effects • Antipyretic (CNS effect) • Anti-inflammatory (except acetaminophen) rheumatic fever, rheumatoid arthritis, other rheumatological diseases: due mainly to PG inhibition. • Dysmenorrhoea • Prophylaxis of diseases due to platelet aggregation (CAD, post-op DVT) • PDA Closure • Pre-eclampsia and hypertension of pregnancy (?excess TXA2) • Some are Uricosuric

23. Non Selective COX inhibitors Non competitive Aspirin Competitive Phenylbutazone Ibuprofen Naproxen Diclofenac Piroxicam Ketorolac Analgesic with Antipyretic without anti inflammatory action Paracetamol Metamizol Nefopam Preferential COX – 2 inhibitors Nimesulide Meloxicam Nabumetone Selective COX -2 inhibitors Celcoxib Rofecoxib Valdecoxib Etoricoxib Parecoxib Lumoracoxib COX inhibitors

24. COOH O O O O O O COOH OH O C C C C C C CH3 CH3 CH3 CH3 CH3 CH3 O CH3 CH2 O C Ser  Acetylated Cyclooxygenase (inactive) Figure4. Structure and mechanism of action of aspirin CH2 OH Ser  Cyclooxygenase (active)

25. Need of Selective COX -2 Inhibitors • inhibition of COX-2 - anti-inflammatory effects • inhibition of COX-1 - recognized toxicities of NSAIDs, • a) peptic ulcers and the associated risks of bleeding, perforation and obstruction;

26. NSAID Loss of PGI2 induced inhibition of LTB4 mediated endothelial adhesion and activation of neutrophils Loss of PGE2 and PGI2 mediated inhibition of acid secretion and cytoprotective effect ↑ Leukocyte-Endothelial Interactions Capillary Obstruction Proteases + Oxygen Radicals Ischemic Cell Injury Endo/Epithelial Cell Injury Mucosal Ulceration

27. Endoscopic Picture of Gastric Ulcer10

28. Need of Selective COX -2 Inhibitors • inhibition of COX-2 - anti-inflammatory effects • inhibition of COX-1 - recognized toxicities of NSAIDs, • a) peptic ulcers and the associated risks of bleeding, perforation and obstruction; • b) prolonged bleeding time; and, • c) renal insufficiency . • inflamed tissues target without disturbing the homeostatic functions of prostaglandins in noninflamed organs. • Theoretically, selective COX-2 inhibition should preserve the anti-inflammatory efficacy

29. What is Selectivity • Ratio of COX – 1 / COX – 2 inhibition activity • COX -1 activity : ability to inhibit TXB 2 production from platelets • COX – 2 activity : ability to inhibit PGI 2 production from monocytes in response to stimuli

31. Rise of COXIBS • Large scale trials showed equal efficacy and lower GI side effects • Market taken by a storm • Celecoxib and then Rofecoxib became billion dollar drugs • Extensive chronic usage in Inflammatory disorders like RA, Osteoarthritis and other Inflammatory disorders • Newer Applications : Adenomas , AD

34. Risk of Hospitalization of Upper GI Bleeding in High Risk Patients 5 4 3.3 3 Adjusted Odds Ratio of Risk 2.1 2 1.3 1.0 1 0 Non-use Celecoxib Rofecoxib NSAIDs Comparison of GI Benefits Adapted from Cardiovascular Safety of Celecoxib & Risk-Benefit Assessment - Celebrex, Pfizer, 2/16/05

35. Big Questions • Does COX-2 serve a physiological function(s)? • COX -2 in macula densa – response to Na+ restriction • Role in ovulation and fertility • Brain – temperature control • Does COX-1 serve an inflammatory function(s)? • No clear cut evidence – dubious gene knock out studies

36. Correlation with GI symptoms • Theoretically COX – 2 inhibitors should be free of side effects • Increase in selectivity ratio should decrease the GI side effects.

38. Disturbing Reports from the long term trials of the Coxibs CLASS AND VIGOR TRIALS RISE IN CVS EVENTS

39. Celecoxib clinical trials • Four Main Trials • CLASS • APC • PreSAP • ADAPT

40. CLASS Trial • Purpose • GI toxicity • Pain alleviation efficacy • Patients • 8000 patients • Average age of 60 y.o. • With Osteoarthritis or Rheumatoid Arthritis • Without history of GI disease

41. CLASS Trial:Celecoxib vs. NSAIDs

42. CLASS Trial:Serious CV Events Adapted from COX-2 CV Safety - Celecoxib, Dr. James Witter, MD, PhD. 2/16/05

43. NO RISK CLASS Trial:Conclusions • GI risk is lower than NSAIDs • No significant difference between NSAIDs and Celecoxib for CV risk

44. APC Trial • Purpose • Reduce probability of adenomatous polyps (reduction of colon cancer) • Patients • 2000 patients • Average age of 60 y.o. • Prior adenomas

45. APC Trial: Baseline Characteristics

46. 0

47. Adapted from a New England Journal of Medicine article: “Cardiovascular Risk Associated with Celecoxib in a Clinical Trial for Colorectal Adenoma Prevention”, March 17, 2005.

48. APC Trial: Death Rates Adapted from Celecoxib in Adenoma Prevention - The APC Trial, Dr. Ernest Hawk, MD, MPH, NIH, 2/15/2005

49. 0

50. RISK APC Trial:Conclusions • Early trial suspension • Planned duration of 3-5 years • Stopped early but followed patients for about 3 years • Fair trial results • Significant difference between Celebrex CV risk and placebo CV risk