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...

5-Lipoxygenase-activating protein (FLAP) is an 1 S-kDa integral membrane protein which is essential for cellular leukotriene (LT) synthesis, and is the target of LT biosynthesis inhibitors. However, the mechanism by which FLAP activates S-LO has not been determined. We have expressed high levels of human FLAP in Spodopteru frugiperda (39) insect cells infected with recombinant baculovirus, and used this system to demonstrate that FLAP specifically binds ['2sI]L-739,059, a novel photoaffinity analog of arachidonic acid. This binding is inhibited by both arachidonic acid and MK-886, an LT biosynthesis inhibitor which specifically interacts with FLAP. These studies suggest that FLAP may activate 5-LO by specifically binding arachidonic acid and transferring this substrate to the enzyme.5-Lipoxygenase-activating protein; 5-Lipoxygenase; Arachidonic acid; Photoaffinity labeling; Baculovirus

No abstract

No abstract

No abstract

The highly potent, selective, and brain-penetrant metabotropic glutamate subtype 5 (mGlu5) receptor antagonists 3-(5-pyridin-2-yl-2H-tetrazol-2-yl)benzonitrile (47) and 3-fluoro-5-(5-pyridin-2-yl-2H-tetrazol-2-yl)benzonitrile (48) are reported. Compound 47 is active in the rat fear-potentiated startle (FPS) model of anxiety with ED(50) = 5.4 mg/kg (po) when dosed acutely. In this model the anxiolytic effects of 47 rapidly tolerate on repeated dosing.

MK-0591 (3-[1-(4-chlorobenzyl)-3-(t-butylthio)-5-(quinolin-2-yl-methoxy)- indol-2-yl]-2,2-dimethyl propanoic acid, previously L-686,708) is a potent inhibitor of leukotriene (LT) biosynthesis in intact human and elicited rat polymorphonuclear leukocytes (PMNLs) (IC50 values 3.1 and 6.1 nM, respectively) and in human, squirrel monkey, and rat whole blood (IC50 values 510, 69, and 9 nM, respectively). MK-0591 had no effect on rat 5-lipoxygenase. MK-0591 has a high affinity for 5-lipoxygenase activating protein (FLAP) as evidenced by an IC50 value of 1.6 nM in a FLAP binding assay and inhibition of the photoaffinity labelling of FLAP by two different photoaffinity ligands. Inhibition of activation of 5-lipoxygenase was shown through inhibition of the translocation of the enzyme from the cytosol to the membrane in human PMNLs. MK-0591 was a potent inhibitor of LT biosynthesis in vivo, first, following ex vivo challenge of blood obtained from treated rats and squirrel monkeys, second, in a rat pleurisy model, and, third, as monitored by inhibition of the urinary excretion of LTE4 in antigen-challenged allergic sheep. Inhibition of antigen-induced bronchoconstriction by MK-0591 was observed in inbred rats pretreated with methysergide, Ascaris-challenged squirrel monkeys, and Ascaris-challenged sheep (early and late phase response). These results indicate that MK-0591 is a potent inhibitor of LT biosynthesis both in vitro and in vivo indicating that the compound will be suitable for assessing the role of leukotrienes in pathological situations.

No abstract