identi®ed as a novel orally active and highly selective cyclo-oxygenase-2 (COX-2) inhibitor. 2 In CHO cells stably transfected with human COX isozymes, DFU inhibited the arachidonic aciddependent production of prostaglandin E 2 (PGE 2 ) with at least a 1,000 fold selectivity for COX-2 (IC 50 =41+14 nM) over COX-1 (IC 50 450 mM). Indomethacin was a potent inhibitor of both COX-1 (IC 50 =18+3 nM) and COX-2 (IC 50 =26+6 nM) under the same assay conditions. The large increase in selectivity of DFU over indomethacin was also observed in COX-1 mediated production of thromboxane B 2 (TXB 2 ) by Ca 2+ ionophore-challenged human platelets (IC 50 450 mM and 4.1+1.7 nM, respectively). 3 DFU caused a time-dependent inhibition of puri®ed recombinant human COX-2 with a K i value of 140+68 mM for the initial reversible binding to enzyme and a k 2 value of 0.11+0.06 s 71 for the ®rst order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62+26 mM and 0.06+0.01 s 71 , respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have a 1 : 1 stoichiometry and to dissociate only very slowly (t 1/2 =1 ± 3 h) with recovery of intact inhibitor and active enzyme. The time-dependent inhibition by DFU was decreased by co-incubation with arachidonic acid under non-turnover conditions, consistent with reversible competitive inhibition at the COX active site. 4 Inhibition of puri®ed recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC 50 =63+5 mM at 0.1 mM arachidonic acid). In contrast to COX-2, inhibition was time-independent and rapidly reversible. These data are consistent with a reversible competitive inhibition of COX-1. 5 DFU inhibited lipopolysaccharide (LPS)-induced PGE 2 production (COX-2) in a human whole blood assay with a potency (IC 50 =0.28+0.04 mM) similar to indomethacin (IC 50 =0.68+0.17 mM). In contrast, DFU was at least 500 times less potent (IC 50 497 mM) than indomethacin at inhibiting coagulationinduced TXB 2 production (COX-1) (IC 50 =0.19+0.02 mM). 6 In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 mM), DFU inhibited COX-1 with an IC 50 value of 13+2 mM as compared to 20+1 nM for indomethacin. CGP 28238, etodolac and SC-58125 were about 10 times more potent inhibitors of COX-1 than DFU. The order of potency of various inhibitors was diclofenac4indomethacin*naproxen4nimesulide* meloxicam*piroxicam4NS-398*SC-576664SC-581254CGP 28238*etodolac4L-745,3374DFU. 7 DFU inhibited dose-dependently both the carrageenan-induced rat paw oedema (ED 50 of 1.1 mg kg 71 vs 2.0 mg kg 71 for indomethacin) and hyperalgesia (ED 50 of 0.95 mg kg 71 vs 1.5 mg kg 71 for indomethacin). The compound was also e ective at reversing LPS-induced pyrexia in rats (ED 50 =0.76 mg kg 71 vs 1.1 mg kg 71 for indomethacin). 8 In a sensitive model in which 51 Cr faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no signi®cant e ect was detected after oral...

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Microsomal prostaglandin E synthase-1 (mPGES-1) is an inducible protein recently shown to be an important source of inflammatory PGE 2 . Here we have used mPGES-1 wild type, heterozygote, and null mice to assess the impact of reduction or absence mPGES-1 protein on the production of PGE 2 and other prostaglandins in lipopolysaccharide (LPS)-treated macrophages and mice. Thioglycollate-elicited peritoneal macrophages with mPGES-1 deficiency were found to lose their ability to produce PGE 2 upon LPS stimulation. Resident mPGES-1 ؊/؊ peritoneal macrophages exhibited severely impaired PGE 2 -releasing activity but retained some LPS-inducible PGE 2 production capacity. Both macrophage types showed a 50% decrease in PGE 2 production with removal of one copy of the mPGES-1 gene. In vivo, mPGES-1 deletion abolished the LPS-stimulated production of PGE 2 in spleen, kidney, and brain. Surprisingly, lack of mPGES-1 activity resulted in an 80 -90% decrease in basal, cyclooxygenase-1 (COX-1)-dependent PGE 2 production in stomach and spleen, and a 50% reduction in brain and kidney. Other prostaglandins (thromboxane B 2 , PGD 2 , PGF 2␣ , and 6-keto-PGF 1␣ ) were significantly elevated in stomachs of mPGES-1-null mice but not in other tissues. Examination of mRNA for several terminal prostaglandin synthases did not reveal changes in expression levels associated with mPGES-1 deficiency, indicating that gastric prostaglandin changes may be due to shunting of cyclooxygenase products to other terminal synthases. These data demonstrate for the first time a dual role for mPGES-1 in both inflammatory and COX-1-mediated PGE 2 production and suggest an interdependence of prostanoid production with tissue-specific alterations of prostaglandin levels in the absence of mPGES-1. Prostaglandins (PG)1 are lipid metabolites of arachidonic acid that are synthesized by a two-step reaction catalyzed by a cyclooxygenase and a terminal prostaglandin synthase. The cyclooxygenase product PGH 2 serves as common precursor to all five major prostanoids (TXA 2 , PGE 2 , PGD 2 , PGI 2 , and PGF 2␣ ). The physiological roles of PG are both diverse and complex, with effects on kidney ion transport, vascular homeostasis, gastrointestinal protection and motility, pregnancy and parturition, sleep, and immune function (1-3). In particular, the primary mediators of pain and inflammation are PGE 2 and PGI 2 (4), whereas pyresis is mediated by PGE 2 through the EP3 receptor (5).Two major cyclooxygenase isoforms are known, each with distinct roles. Expression patterns suggest that the constitutively expressed COX-1 plays housekeeping functions, whereas the inducible COX-2 is implicated in inflammatory processes. Exceptions to this paradigm have been uncovered with observations of constitutive COX-2 expression in several neuronal structures of the brain and in the kidney (6 -9). Thus, although COX-2 plays a pivotal role in inflammation, it also serves housekeeping functions. Indeed, the phenotype of the COX-2 null mice is indicative of COX-2 roles during kidney dev...

Both nonsteroidal anti-inflammatory drugs, such as ibuprofen, and the prototypical selective cyclooxygenase (Cox)-2 inhibitors DuP-697 and NS-398 block the inhibition of Cox-1 by aspirin in vitro. However, clinical studies have shown that the Cox-2 selective drugs (or coxibs) rofecoxib and etoricoxib, at therapeutic doses, do not interfere with the antiplatelet effect of aspirin, in contrast to ibuprofen. Here, we have evaluated the relative potential of ibuprofen and various coxibs to interfere with the inactivation of Cox-1 by aspirin by using purified enzyme and calcium ionophoreactivated human platelets. The irreversible inactivation of Cox-1 by aspirin can be antagonized by ibuprofen and coxibs, albeit with widely different potencies. The rank order of potencies for this process (ibuprofen > celecoxib > valdecoxib > rofecoxib > etoricoxib) parallels that obtained for the inhibition of Cox-1-mediated thromboxane B 2 production by calcium ionophore-stimulated platelets. The antagonism of aspirin therefore likely involves a competition at the enzyme active site. The EC 50 value for the antagonism against 10 M aspirin for each drug is Ϸ10-to 40-fold lower than the corresponding IC 50 value for inhibition of platelet Cox-1 activity, consistent with the much weaker initial binding of aspirin to Cox-1 as compared with arachidonic acid. These results show that a low affinity for Cox-1 and a high degree of Cox-2 selectivity confers a low potential to block aspirin inhibition of platelet Cox-1, consistent with the results of clinical studies. The two cyclooxygenase (Cox) isozymes (or prostaglandin H synthases), which share Ϸ60% sequence identity, perform the first committed steps in the prostaglandin pathway by catalyzing the oxygenation of arachidonic acid to PGG 2 (Cox activity) and the reduction of PGG 2 to PGH 2 (peroxidase activity) (1-3). Cox-1 is constitutively expressed in most cell types, including platelets, whereas Cox-2 is absent from most healthy tissues but is induced by proinflammatory or proliferative stimuli. Cox-1 plays a role in the production of prostaglandins involved in protection of the gastric mucosal layer and thromboxanes (TX) in platelets. Cox-2 generally mediates elevations of prostaglandins associated with inflammation, pain, and pyresis (2). Nonsteroidal antiinflammatory drugs (NSAIDs) such as aspirin and ibuprofen are generally nonselective inhibitors of Coxs. This lack of selectivity has been linked to their propensity to cause gastrointestinal side effects. The new Cox-2 selective inhibitors, or coxibs, show the same antiinflammatory, analgesic, and antipyretic effects as nonselective NSAIDs but have reduced side-effect profiles (4).The use of low-dose aspirin (50-325 mg͞day) is currently indicated for cardiovascular prophylaxis (5). The mechanism of this cardioprotective effect is because of the irreversible inactivation of platelet Cox-1, resulting in a reduced production of the proaggregatory TXA 2 . As platelets are anuclear, the function of the platelets is inhibited for the rema...