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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...

Prostaglandin (PG) E(2) is a potent inducer of cortical and trabecular bone formation in humans and animals. Although the bone anabolic action of PGE(2) is well documented, the cellular and molecular mechanisms that mediate this effect remain unclear. This study was undertaken to examine the effect of pharmacological inactivation of the prostanoid receptor EP(4), one of the PGE(2) receptors, on PGE(2)-induced bone formation in vivo. We first determined the ability of EP(4)A, an EP(4)-selective ligand, to act as an antagonist. PGE(2) increases intracellular cAMP and suppresses apoptosis in the RP-1 periosteal cell line. Both effects were reversed by EP(4)A, suggesting that EP(4)A acts as an EP(4) antagonist in the cells at concentrations consistent with its in vitro binding to EP(4). We then examined the effect of EP(4) on bone formation induced by PGE(2) in young rats. Five- to 6-week-old rats were treated with PGE(2) (6 mg/kg/day) in the presence or absence of EP(4)A (10 mg/kg/day) for 12 days. We found that treatment with EP(4)A suppresses the increase in trabecular bone volume induced by PGE(2). This effect is accompanied by a suppression of bone formation indices: serum osteocalcin, extent of labeled surface, and extent of trabecular number, suggesting that the reduction in bone volume is due most likely to decreased bone formation. The pharmacological evidence presented here provides strong support for the hypothesis that the bone anabolic effect of PGE(2) in rats is mediated by the EP(4) receptor.