1 DFU (5,5-dimethyl-3-(3-fluorophenyl)-4-(4-methylsulphonyl)phenyl-2(5H)-furanone) was identified 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 acid-dependent production of prostaglandin E-2 (PGE(2)) with at least a 1,000 fold selectivity for COX-2 (IC50=41+/-14 nM) over COX-1 (IC50 > 50 mu M). Indomethacin was a potent inhibitor of both COX-1 (IC50=18+/-3 nM and COX-2 (IC50=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 (TXB2) by Ca2+ ionophore-challenged human platelets (IC50>50 mu M and 4.1+/-1.7 nM, respectively). 3 DFU caused a time-dependent inhibition of purified recombinant human COX-2 with a K-i value of 140+/-68 mu M for the initial reversible binding to enzyme and a k(2) value of 0.11+/-0.06 s(-1) for the first order rate constant for formation of a tightly bound enzyme-inhibitor complex. Comparable values of 62+/-26 mu M and 0.06+/-0.01 s(-1) respectively, were obtained for indomethacin. The enzyme-inhibitor complex was found to have 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 purified recombinant human COX-1 by DFU was very weak and observed only at low concentrations of substrate (IC50=63+/-5 mu M at 0.1 mu M 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 (IC50=0.28+/-0.04 mu M) similar to indomethacin (IC50=0.68+/-0.17 mu M). In contrast, DFU was at least 500 times less potent (IC50>97 mu M) than indomethacin at inhibiting coagulation-induced TXB2 production (COX-1) (IC50=0.19+/-0.02 mu M). 6 In a sensitive assay with U937 cell microsomes at a low arachidonic acid concentration (0.1 mu M), DFU inhibited COX-1 with an IC50 value of 13+/-2 mu M 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 diclofenac>indomethacin similar to naproxen>nimesulide similar to meloxicam similar to piroxicam>NS-398 similar to SC-57666>SC-58125>CGP 28238 similar to etodolac>L-745,337>DFU. 7 DFU inhibited dose-dependently both the carrageenan-induced rat paw oedema (ED50 of 1.1 mg kg(-1) vs 2.0 mg kg(-1) for indomethacin) and hyperalgesia (ED50 of 0.95 mg kg(-1) vs 1.5 mg kg(-1) for indomethacin). The compound was also effective at reversing LPS-induced pyrexia in rats (ED50=0.76 mg kg(-1) vs 1.1 mg kg(-1) for indomethacin). 8 In a sensitive model in which Cr-51 faecal excretion was used to assess the integrity of the gastrointestinal tract in rats, no significant effect was detected after oral administration of DFU (100 mg kg(-1), b.i.d.) for 5 days, whereas chromium leakage was observed with lower doses of diclofenac (3 mg kg(-1)), meloxicam (3 mg kg(-1)) or etodolac (10-30 mg kg(-1)). A 5 day administration of DFU in squirrel monkeys (100 mg kg(-1)) did not affect chromium leakage in contrast to diclofenac (1 mg kg(-1)) or naproxen (5 mg kg(-1)). 9 The results indicate that COX-1 inhibitory effects can be detected for all selective COX-2 inhibitors tested by use of a sensitive assay at low substrate concentration. The novel inhibitor DFU shows the lowest inhibitory potency against COX-1, a consistent high selectivity of inhibition of COX-2 over COX-1 (>300 fold) with enzyme, whole cell and whole blood assays, with no detectable loss of integrity of the gastrointestinal tract at doses >200 fold higher than efficacious doses in models of inflammation, pyresis and hyperalgesia. These results provide further evidence that prostanoids derived from COX-1 activity are not important in acute inflammatory responses and that a high therapeutic index of antiinflammatory effect to gastropathy can be achieved with a selective COX-2 inhibitor.
