B 1995 Stockton Press All rights reserved 0007-1188/95 $12.00 X Effects of the novel anti-inflammatory compounds, N- [2-(cyclohexyloxy)-4-nitrophenyl] methanesulphonamide (NS-398) and 5-methanesulphonamido-6-(2,4-difluorothio- phenyl)-1-indanone (L-745,337), on the cyclo-oxygenase activity of human blood prostaglandin endoperoxide synthases Maria R. Panara, Anita Greco, Giovanna Santini, Maria G. Sciulli, Maria T. Rotondo, Roberto Padovano, Maria di Giamberardino, Francesco Cipollone, Franco Cuccurullo, Carlo Patrono & 'Paola Patrignani Departments of Pharmacology and Medicine, University of Chieti 'G. D'Annunzio' School of Medicine, 66013 Chieti, Italy 1 We have evaluated the selectivity of ketoprofen and two novel nonsteroidal anti-inflammatory drugs, N-[2-(cyclohexyloxy)-4-nitrophenyl]methanesulphonamide (NS-398) and 5-methanesulphonamido-6-(2,4- difluorothiophenyl)-1-indanone (L-745,337), in inhibiting the cyclo-oxygenase activity of prostaglandin endoperoxide synthase-2 (PGHS-2) vs PGHS-1 in human blood monocytes and platelets, respectively. 2 Heparinized whole blood samples were drawn from healthy volunteers pretreated with aspirin, 300 mg 48 h before sampling, to suppress the activity of platelet PGHS-1 and incubated at 37°C for 24 h with increasing concentrations of the test compounds in the presence of lipopolysaccharide (LPS, 10 yig ml-'). Immunoreactive PGE2 levels were measured in plasma by a specific radioimmunoassay as an index of the cyclo-oxygenase activity of LPS-induced monocyte PGHS-2. 3 The effects of the same inhibitors on platelet PGHS-1 activity were assessed by allowing whole blood samples, drawn from the same subjects in aspirin-free periods, to clot at 37°C for 1 h in the presence of the compounds and measuring immunoreactive thromboxane B2 (TXB2) levels in serum by a specific radioimmunoassay. 4 Under these experimental conditions, ketoprofen enantioselectively inhibited the cyclo-oxygenase activity of both PGHS-1 and PGHS-2 with equal potency (IC50 ratio: approx. 0.5 for both enantiomers), while L-745,337 and NS-398 achieved selective inhibition of monocyte PGHS-2 (IC50 ratio: > 150). L- 745,337 and NS-398 did not affect LPS-induced monocyte PGHS-2 biosynthesis to any detectable extent. 5 We conclude that L-745,337 and NS-398 are selective inhibitors of the cyclo-oxygenase activity of human monocyte PGHS-2. These compounds may provide adequate tools to test the contribution of this novel pathway of arachidonate metabolism to human inflammatory disease. Keywords: Prostaglandin endoperoxide synthases; human blood monocytes; human platelets; L-745,337; NS-398; ketoprofen Introduction The conversion of arachidonic acid to prostaglandin H2 (PGH2) is catalysed by prostaglandin endoperoxide synthase (PGHS) which exhibits both cyclo-oxygenase and peroxidase activities (DeWitt, 1991). PGH2 is further metabolized by other enzymes to various prostanoids (prostaglandins, pros- tacyclin and thromboxane A2). Two isozymes of PGHS are known, referred to as PGHS-1 and PGHS-2 (Smith, 1992). PGHS-1 is a constitutive enzyme present in almost all cell types (Simmons et al., 1991). Thus, PGHS-1 is the major iso- form of gastrointestinal tissue (DeWitt & Smith, 1988). Pros- tanoid production by PGHS-1 is involved in physiological functions such as vascular homeostasis, control of kidney function and gastric cytoprotection (Smith, 1992). PGHS-2 is induced in a more restricted cell-specific fashion by mitogenic and inflammatory stimuli (Kujubu et al., 1991; Fletcher et al., 1992; O'Banion et al., 1992; Lee et al., 1992; O'Sullivan et al., 1992a,b; Hempel et al., 1994; Patrignani et al., 1994). The cyclo-oxygenase activity of PGHS is inhibited by as- pirin and related nonsteroidal anti-inflammatory drugs (NSAIDs) (Vane, 1971). Inhibition of prostanoid formation by NSAIDs is the basis for their therapeutic actions as well as their side-effects. In fact, it has been suggested that the anti- inflammatory action of NSAIDs is due to inhibition of PGHS- 2, whereas the toxic effects on the stomach and kidney are due to inhibition of the constitutive isozyme, PGHS-1 (Vane, 1994). Since PGHS-1 and PGHS-2 are structurally distinct proteins with only 60% homology (Jones et al., 1993), the development of drugs that selectively inhibit the cyclo-oxyge- nase activity of PGHS-2 might lead to a new generation of anti-inflammatory drugs with increased tolerability (Vane, 1994). Recently, new anti-inflammatory agents such as N-[2- (cyclohexyloxy)-4-nitrophenyl] methanesulphonamide (NS- 398) and 5-methanesulphonamido-6-(2,4-difluorothiophenyl)- 1-indanone (L-745,337) have been developed that produce fewer gastrointestinal lesions in experimental animals than conventional NSAIDs (Futaki et al., 1993; Chan et al., 1994; Chan et al., 1995; Masferrer et al., 1994). Thus, the aim of our study was to evaluate the selectivity of L-745,337 and NS-398 in comparison with ketoprofen in inhibiting the cyclo-oxyge- nase activity of PGHS-2 vs PGHS-1 in human blood mono- cytes and platelets, respectively. The whole blood assay which we have recently developed (Patrignani et al., 1994) has distinct advantages vis-a-vis other assay systems (Meade et al., 1993; Mitchell et al., 1993): (1) it can be used in vitro in the pre- clinical assessment of inhibitor molecules as well as ex vivo during phase I/II studies; (2) it compares clinically relevant target cells; (3) it takes into account the variable degree of protein binding of different inhibitors. 1 Author for correspondence at: Cattedra di Farmacologia I, Universita di Chieti 'G.D'Annunzio', Palazzina delle Scuole di Specializzazione, Via dei Vestini, 31, 66013 Chieti, Italy. British Journal of Pharmacology (1995) 116, 2429-2434