Dose-dependent inhibition of platelet cyclooxygenase-1 and monocyte cyclooxygenase-2 by meloxicam in healthy subjects.

We evaluated whether therapeutic blood levels of meloxicam are associated with selective inhibition of monocyte cyclooxygenase (COX)-2 in vitro and ex vivo. Concentration-response curves for the inhibition of monocyte COX-2 and platelet COX-1 were obtained in vitro after the incubation of meloxicam with whole blood samples. Moreover, 11 healthy volunteers received placebo or 7.5 or 15 mg/day meloxicam, each treatment for 7 consecutive days, according to a randomized, double-blind, crossover design. Before dosing and 24 h after the seventh dose of each regimen, heparinized whole blood samples were incubated with lipopolysaccharide (10 microgram/ml) for 24 h at 37 degrees C, and prostaglandin E2 was measured in plasma as an index of monocyte COX-2 activity. The production of thromboxane B2 in whole blood allowed to clot at 37 degrees C for 60 min was assessed as an index of platelet COX-1 activity. The administration of placebo did not significantly affect plasma prostaglandin E2 (21. 3 +/- 7.5 versus 19.1 +/- 4 ng/ml, mean +/- S.D., n = 11) or serum thromboxane B2 (426 +/- 167 versus 425 +/- 150 ng/ml) levels. In contrast, the administration of 7.5 and 15 mg of meloxicam caused dose-dependent reductions in monocyte COX-2 activity by 51% and 70%, respectively, and in platelet COX-1 activity by 25% and 35%, respectively. Although the IC50 value of meloxicam for inhibition of COX-1 was 10-fold higher than the IC50 value of COX-2 in vitro, this biochemical selectivity was inadequate to clearly separate the effects of meloxicam on the two isozymes after oral dosing as a function of the daily dose and interindividual variation in steady-state plasma levels.

Effects of flurbiprofen and flurbinitroxybutylester on prostaglandin endoperoxide synthases.

The aim of our study was to evaluate the selectivity of flurbiprofen and flurbinitroxybutylester for inhibition of the cyclooxygenase activity of prostaglandin endoperoxide synthase-2 vs. prostaglandin endoperoxide synthase-1 in human blood monocytes and platelets, respectively. In whole blood, flurbiprofen was approximately 10-fold more potent that flurbinitroxybutylester to inhibit the cyclooxygenase activity of platelet prostaglandin endoperoxide synthase-1 (IC50 microM: 0.90 +/- 0.27 vs. 10.70 +/- 5, mean +/- S.D., P < 0.05). In contrast, the 2 compounds were equipotent to inhibit prostaglandin endoperoxide synthase-2 cyclooxygenase activity in whole blood (IC50 microM: 0.90 +/- 0.25 vs. 0.80 +/- 0.35) or isolated monocytes (IC50 microM: 0.03 +/- 0.02). Neither flurbiprofen nor flubinitroxybutylester (0.28-112 microM) affected prostaglandin endoperoxide synthase isozyme expression by lypopolysaccharide-stimulated monocytes. In whole blood, flurbinitroxybutylester was slowly converted to flubiprofen and this in turn could influence the extent of inhibition of the cyclooxygenase activity of prostaglandin endoperoxide synthase-1. In conclusion, the addition of a nitroxybutyl moiety to flurbiprofen seems to reduce its capacity to inhibit the cyclooxygenase activity of prostaglandin endoperoxide synthase-1. Whether this effect will result in a reduced risk of gastrointestinal toxicity remains to be studied in man.

Induction of prostaglandin endoperoxide synthase-2 in human monocytes associated with cyclo-oxygenase-dependent F2-isoprostane formation.

1. The isoprostane 8-epi-prostaglandin (PG)F2 alpha is produced by free radical-catalyzed peroxidation of arachidonic acid. It may also be formed as a minor product of the cyclo-oxygenase activity of platelet PGH synthase (PGHS)-1. We investigated 8-epi-PGF2 alpha production associated with induction of the human monocyte PGHS-2 and its pharmacological modulation. 2. Heparinized whole blood samples were drawn from healthy volunteers, 48 h following oral dosing with aspirin 300 mg to suppress platelet cyclo-oxygenase activity. One ml aliquots were incubated with lipopolysaccharide (LPS: 0.1-50 micrograms ml-1) for 0-24 h at 37 degrees C. PGE2 and 8-epi-PGF2 alpha were measured in separated plasma by radioimmunoassay and enzyme immunoassay techniques. 3. Levels of both eicosanoids were undetectable (i.e. < 60 pg ml-1) at time 0. LPS induced the formation of PGE2 and 8-epi-PGF2 alpha in a time- and concentration-dependent fashion, coincident with the induction of PGHS-2 detected by Western blot analysis of monocyte lysates. After 24 h at 10 micrograms ml-1 LPS, immunoreactive PGE2 and 8-epi-PGF2 alpha averaged 10,480 +/- 4,643 and 295 +/- 140 pg ml-1 (mean +/- s.d., n = 6), respectively. 4. Dexamethasone and 5-methanesulphonamido-6-(2,4-difluorothiophenyl)-1-indano ne (L-745,337), a selective inhibitor of the cyclo-oxygenase activity of PGHS-2, reduced PGE2 and 8-epi-PGF2 alpha production in response to LPS. 5. Isolated monocytes produced PGE2 and 8-epi-PGE2 alpha in response to LPS (10 micrograms ml-1) in a time-dependent fashion. Monocyte PGE2 and 8-epi-PGF2 alpha production was largely prevented by dexamethasone (2 microM) and cycloheximide (10 micrograms ml-1) in association with suppression of PGHS-2 but not of PGHS-1 expression. 6. We conclude that the induction of PGHS-2 in human monocytes is associated with cyclo-oxygenase-dependent generation of the vasoconstrictor and platelet-agonist 8-epi-PGF2 alpha.

Effects of the novel anti-inflammatory compounds, N-[2-(cyclohexyloxy)-4-nitrophenyl] methanesulphonamide (NS-398) and 5-methanesulphonamido-6-(2,4-difluorothio-phenyl)-1-inda none (L-745,337), on the cyclo-oxygenase activity of human blood prostaglandin endoperoxide synthases.

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-indano ne (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 degrees C for 24 h with increasing concentrations of the test compounds in the presence of lipopolysaccharide (LPS, 10 micrograms ml-1). 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 degrees 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.