Prostaglandins, Leukotrienes and Essential FattyAcids (1998) 59(1 ), 55-62 © HarcourtBrace& Co. Ltd 1998 Systematic pharmacological approach to the characterization of NSAIDs M. Lora, ~ J.-B. Denault, ~ R. Leduc, ~ A. J. de Brum-Fernandes ~'2 ~Department of Pharmacology Faculty of Medicine, Universite de Sherbrooke, Canada 2Department of Medicine (Rheumatic Diseases Unit), Faculty of Medicine, Universit6 de Sherbrooke, Canada Summary Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used for the treatment of inflammatory diseases. NSAIDs inhibit cyclooxygenase (COX), the rate limiting enzyme responsible for the conversion of arachidonic acid into prostaglandins. Recent studies have shown the existence of two isoforms of cyclooxygenase: COX-l, now often referred to as the constitutive form, and COX-2, an inducible form which is the major isoenzyme involved in prostaglandin synthesis in inflammation and other pathological situations. Since inhibition of prostaglandin production in tissues where they play a physiological role leads to important side effects, a COX-2 preferential inhibitor would present therapeutical advantages. In the present study, we evaluated the inhibitory properties of cyclooxygenase inhibitors on human COX-1 and COX-2 using a heterologous expression system. We investigated instantaneous inhibition and pre-incubation inhibition as well as time recovery of cyclooxygenase activity assays with the aid of four NSAIDs: mefenamic acid, indomethacin, aspirin and NS-398. Our results demonstrate that instantaneous inhibition assays have little correlation with clinical results. Inhibition assays using pre-incubation with the drugs tested, however, more closely resemble the data from in vivo studies. Cyclooxygenase recovery assays enabled better characterization of simple competitive inhibitors, competitive reversible time-dependent inhibitors and irreversible time-dependent inhibitors. The data illustrate the usefulness of our system in allowing a better determination of the pharmacological characteristics of NSAIDs as well as permitting a comparison among different drugs. INTRODUCTION Cyclooxygenase (COX §, E.C. 1.14.99.1) is the first enzyme involved in the synthesis of prostaglandins, catalyzing the formation of prostaglandin H2 (PGH2) from arachidonic acid. The unstable PGH2 is the common precursor of prostaglandins and thromboxane. Cyclooxygenase (COX) is the primary target of non-steroidal anti-inflammatory drugs (NSMDs), and this action is believed to be respon- sible for their anti-inflammatory action. 1 Unfortunately, inhibition of prostaglandin production in organs such as the stomach and kidney, where they play important physiological roles, 2-3 can result in gastric lesions and nephrotoxicity, important side effects that limit the use of these drugs. Received 15 October 1997 Accepted 17 December 1997 Correspondence to: Artur J. de Brum-Fernandes, MD, PhD, 3001, 12th Avenue Nord, Rheumatic Diseases Unit, Sherbrooke, PQ, Canada, J1H 5N4. Tel: 819 564 5261; Fax: 819 569 5265; E-mail: a.fernan@courrier.usherb.ca Recent studies have shown the existence of two isoforms of COX. The first isoenzyme described, cyclooxygenase-1 (COX-I) is constitutive and produces prostaglandins which have physiological functions such as protecting the stomach and kidney from damage. 2-3 A more recently discovered isoenzyme, cyclooxygenase-2 (COX-2) is induced by inflammatory stimuli such as cytokines 4-8 a nd produces prostaglandins which contribute to the pain and swelling of inflammation. The cDNAs encoding COX-1 and COX-2 have been cloned from mouse 9-11 as well as human sources 12-15and expressed in vitro. 1°<3,~6<9 The differences in primary structures between COX-1 and COX-2 suggest that this could lead to divergent affinities for fatty acid substrates and NSAIDs. It has been shown that murine COX-1 and COX-2 differ in their sensitivity to inhibition by some NSAIDs, 1~ as do human COX-1 and COX-2. 2° In this context, selective inhibition of COX-2 could provide an anti-inflammatory action without the side-effects of the currently available NSMDs.11,2>22 55