Cyclooxygenase (COX) is the key enzyme in the synthesis of prostaglandins and thromboxanes, and these COX products are important mediators of pain, fever and inflammation. Inflammatory effects result both from direct actions of prostaglandins (on the microvasculature, on nociceptive afferents and on temperature-regulating centres in the hypothalamus) and, indirectly, by synergy with other inflammatory mediators including bradykinin, histamine, activated component and platelet activating factor. There are two isoforms of the enzyme, namely a constitutive form (COX-1) that is present in many tissues such as platelets, stomach, lungs, kidneys etc and an inducible form (COX-2) that is expressed during inflammation as a result of stimulation by cytokines, nitric oxide and growth factors. Currently there is a great interest in developing inhibitors specific for COX-2 and it is expected that it will lack the adverse effects caused by the inhibition of COX-1 enzyme. The present study was undertaken to investigate the inhibition of COX-1 and COX-2 in pain, inflammatory processes and platelet aggregation based on in-vitro and in-vivo assays used to classify the inhibitors of COX-1 andCOX-2.
Analgesic study of COX-1 and -2 inhibitors was carried out using two experimental models; (i) tail immersion method and (ii) acetic- acid induced writhing test in mice in which pain was induced by thermal and chemical stimuli respectively. The results show that all COX-1 and COX-2 inhibitors possess significant analgesic effect in the doses of 1,10 and 20 mg/kg to various degrees. The COX.1 inhibitors such as indomethacin (ED50= 10 mg/kg), flurbiprofen (ED50 = 10 mg/kg) and resveratrol (ED50 = 17 mg/kg) were more potent whereas velarylsalicylate (ED50 = 48 mg/kg) was less potent. Similarly COX-2 inhibitors like nimesulide (ED50 = 19 mg/kg), NS-398 (ED50 = 10 mg/kg) and etodolac (ED50 = 24 mg/kg) significantly reduced the pain. The results were comparable in both experimental models and the relative order of potency was indomethacin> flurbiprofen > NS-398 > resveratrol > nimesulide > etodolac > valerylsalicylate.
Anti-inflammatory studies were conducted using carrageen an-induced rat and mice paw edema tests. In rats, edema was measured by plethysmometery whereas, in mice edema was measured by comparing the difference between the weight of normal and edematous hind paws. In all these tests COX inhibitors were administered orally at different doses ranging from 1-20 mg/kg. The maximum inhibition of edema was found at 20 mg in case of COX-1 inhibitors but for COX-2 inhibitors maximum response was observed at 10 mg/kg, which W3S comparable to that produced by aspirin (150 mg/kg). The ED50 values for COX-1 and -2 inhibitors in rat paw edema tests were obtained as flurbiprofen; ED50 = 6 mg/kg, resveratrol; ED50 = 20 mg/kg, valerylsalicylate; ED50 = 50 mg/kg, nimesulide; ED50 = 5 mg/kg, NS398; ED50 = 5 mg/kg and etodolac; ED50 = 13 mg/kg. Indomethacin (ED50= 12" 4mg/kg) was also effective in inhibiting both rat and mice paw edema. These. results are indicative of underlying mechanisms and the role of COX-2 inc inflammation since paw edema was significantly (P < 0.05) suppressed by COX-2 inhibitors. The in-vitro anti-platelet effects were determined by tribidometric method using Chronology Dual-channel Lumi-aggregomerter. Aggregation was induced by various platelet agonists like epinephrine (epi= 20 µM), platelet activating factor (PAF= 800 nM) and arachidonic acid (AA= 1,6 mM) and then blocked by COX-1 and -2 inhibitors in a concentration dependent fashion, We found that sub threshold concentrations of these agonists (epinephrine= 1 µM and 5-HT= 2 µM ), (epinephrine= 1 µM and PAF= 5 nM), (PAF= 5 nM and 5 HT= 2 µM), (PAF=5 nM and AA= 0,2 mM), (epinephrine= 1µM and AA= 0.2mM) when added together in platelet suspension they show synergism followed by marked aggregation.
We also examined the signaling mechanisms involved in the synergistic interaction of various platelet agonists. First we determined the involvement of cyclooxygenase responsible for TXA2 formation in platelets, a potent platelet agonist. The results of COX-1 inhibitors show that they inhibit platelet aggregation with comparatively much lower IC50 values, whereas COX-2 inhibitors also suppressed platelet aggregation mediated by the synergistic interaction of various platelet agonists as they also exert inhibitory effects on TXB2. We found that all of the COX inhibitors diminished platelet aggregation at comparatively less IC50 values against the synergism mediated by various platelet agonists than the agonists alone. The effectiveness of different inhibitors which act at different steps of the signaling cascade indicating the involvement multiple signaling pathways responsible for the synergistic interaction of two agonists. Infact recent data support the view of complexity of such interactions.
To investigate the signaling pathways involved in the synergistic interaction among each pair of platelet agonists we used various receptor blockers (yohimbine, α2 -adrenergic blocker; methysergide, 5-HT2 receptor antagonist; WEB 2086, PAF receptor antagonist; SQ 29, 548, (TXA2 receptor blocker) and cell signaling inhibitors (U73122, PLC inhibitor; PO 98059, MAP kinase inhibitor; herbimycin A, TLCK inhibitor, SNAP; NO donor, and chelerythrine chloride, PKC inhibitor).Our results show that the synergism mediated by these agonists is due to the activation of Gi/PLC, Gq/PLC, Ca2+, MAP kinase and mainly the COX pathway.
As our results suggested that platelet aggregation (synergism) is mediated by the pair of each agonist was due to the activ8tion of COX pathway in platelet, we further confirmed the results by determining the production of TXA2 using platelet homogenate by radio-thin layer chromatography (TLC) method and found that subthreshold concentrations of platelet agonists alone showed a little effect on TXA2 formation but when used together they stimulated platelet TXA2 formation up to many folds.
We also determined the in-vivo anti-platelet effects of COX inhibitors by arachidonic acid (AA) induced shock and death in rabbits. Arachidonic acid when injected intravenously, in the dose of 2 mg/k(1, it causes pulmonary embolism and leads to sudden death. The pretreatment of rabbits with intraperitcmeal (Lp) injection of COX inhibitors (50 mg/kg) completely prevented death or lethality in rabbits induced by AA (P < 0.001). Results show that the potency of COX-inhibitors was found higher in in-vivo anti platelet activity as compared to in-vitro effects. The rank order of potency was obtained as flurbiprofen > resveratrol > indomethacin> nimesulide > etodolac > valerylsalicylate.
These results are indicative of various pharmacological actions of COX-1 and COX-2 inhibitors such as analgesia, anti-inflammatory, anti-platelet (in-vivo and in-vitro) activities and inhibition of TXA2 formation in platelets. These beneficial properties further enhance the utility of these inhibitors in the treatment of diseases where COX, AA, PAF and other inflammatory mediators play an important role in various pathological conditions.