Stereoselective inhibition of rat brain cyclooxygenase by dexketoprofen.

Although it has been assumed that the effects of nonsteroidal antiinflammatory drugs (NSAIDs) are mainly the result of their action on local synthesis of prostaglandins, there is growing evidence to suggest that they may also exert a central analgesic action. Some authors have suggested that inhibition of prostaglandin synthesis in the brain could contribute to the analgesic action. The effect of dexketoprofen trometamol (tromethamine salt of the enantiomer (+)-S-ketoprofen) on prostaglandin synthesis was investigated in rat brain fragments and in cyclooxygenase preparations from rat brain microsomes. Effects of the (-)-R-enantiomer and the racemic mixture were also evaluated. Significant levels of prostaglandin F2 alpha (PGF2 alpha) were synthesized in rat brain fragments after 10 min of incubation at 37 degrees C. Dexketoprofen was found to be a potent inhibitor of this PGF2 alpha production in rat brain (IC50 = 6.2 nM), and it completely suppressed PGF2 alpha production at 1 microM concentration. In addition, inhibition of PGF2 alpha synthesis by dexketoprofen was highly stereoselective since the enantiomer (-)-R-ketoprofen was significantly less potent (IC50 = 294 nM); with this enantiomer, even at high concentrations such as 1 microM, less than 60% inhibition was achieved. These results correlated with those obtained in the study of racemic ketoprofen and its enantiomers on cyclooxygenase activity of rat brain microsomes, where dexketoprofen also inhibited enzymatic activity stereoselectively. IC50 values obtained for dexketoprofen, (-)-R-ketoprofen, and rac-ketoprofen were 3.5 microM, 45.3 microM, and 5.8 microM, respectively. The above results could be related to the potent analgesic effect of dexketoprofen observed in vivo, which was also stereoselective. Taken together, these findings suggest that prostaglandin synthesis inhibition in rat brain by dexketoprofen could be associated, at least in part, with the analgesic effect of this NSAID.

Stereoselective inhibition of inducible cyclooxygenase by chiral nonsteroidal antiinflammatory drugs.

The stereoselective inhibition of inducible cyclooxygenase (COX-2) by chiral nonsteroidal antiinflammatory drugs (NSAIDs)--ketoprofen, flurbiprofen, and ketorolac--has been investigated. The activity and inhibition of COX-2 was assessed in three different in vitro systems: guinea pig whole blood, lipopolysaccharide (LPS)-stimulated human monocytes, and purified preparations of COX-2 from sheep placenta. The results were compared with the inhibition of constitutive cyclooxygenase (COX-1) in three parallel in vitro models: clotting guinea pig blood, human polymorphonuclear leukocytes, and purified COX-1 from ram seminal vesicles. In the whole blood model, both isoenzymes were inhibited by S-enantiomers with equal potency but S-ketoprofen was the most active on COX-2 (IC50 = 0.024 mumol/L). In contrast, both isoenzymes were inhibited less than 40% by all three R-enantiomers at high concentration (> 1 mumol/L). The inhibition of COX by the R-enantiomers may be attributed to contamination with the S-enantiomers (approximately 0.5%). A significant degree of enantioselectivity in COX-2 inhibition was also observed in intact cells. The S-enantiomers inhibited COX-2 from monocytes with IC50 values in the range of 2 to 25 nmol/L, being 100 to 500-fold more potent than the corresponding R-enantiomers. Finally, S-ketoprofen inhibited COX-2 from sheep placenta (IC50 = 5.3 mumol/L) with slightly less potency than S-ketorolac (IC50 = 0.9 mumol/L) and S-flurbiprofen (IC50 = 0.48 mumol/L), whereas the R-enantiomers were found to be essentially inactive (IC50 > or = 80 mumol/L). It is concluded that the chiral NSAIDs studied here inhibit with comparable stereoselectivity both COX-2 and COX-1 isoenzymes, and that the inhibition of COX-2 previously observed for racemic NSAIDs should be attributed almost exclusively to their S-enantiomers.

Effect of manoalide on human 5-lipoxygenase activity.

The marine natural product manoalide (MLD) has been described to inactivate phospholipase A2 (PLA2) from several sources as well as to inhibit synthesis of eicosanoids in human polymorphonuclear leukocytes (HPMNL). MLD also reduces chemically-induced inflammation in vivo. In this investigation we have examined the effect of MLD on A23187-induced generation of leukotriene B4 (LTB4) and thromboxane B2 (TXB2) in HPMNL as well as 5-lipoxygenase (5-LO) activity from HPMNL sonicated preparations. In the intact cell system, MLD inhibited with similar potency biosynthesis of LTB4 and TXB2 (IC50 1.7 and 1.4 microM, respectively). In order to discern if inhibition of 5-LO is involved in the effect of MLD, we examined the action of this compound on 5-LO activity from 10,000 x g and 100,000 x g supernatants of sonicated HPMNL homogenates. The enzymatic activity was not affected at concentrations of MLD up to 50 microM. These data indicate that MLD is not a direct inhibitor of 5-LO activity from HPMNL and support the hypothesis that its anti-inflammatory action could be related with a reduction of eicosanoid biosynthesis via inhibition of PLA2.

[Effect of PAF (platelet-activating factor) on hemostasis. Studies on endothelial cells and platelets]. / Influencia del PAF (factor activador de las plaquetas) en la hemostasia. Estudios en células endoteliales y plaquetas.

PURPOSE: To evaluate the direct action of PAF on the pro-coagulant activity of cultured vascular endothelial cells; to analyse by photometric methods the thrombogenic effect of PAF on platelets, and to assess platelet deposition on vascular endothelium. MATERIAL AND METHODS: Human endothelial cells were isolated from umbilical cord vein and incubated for three minutes at 22 degrees C with different PAF concentrations (10(9) M to 10(-7) M) in order to assess the influence of this lipidic mediator on the procoagulant activity of the cells. The effect of PAF on platelet aggregation was assessed by aggregation studies using arachidonic acid (AA) and different PAF and Lyso-PAF concentrations (10(-8) M to 10(-4) M). Serotonin (5-HT) release was tested in platelet rich plasma (PRP) samples highly sensitive to PAF. PRP samples were incubated for 30 minutes at 22 degrees C with 100 microCi 3H-5-HT. Platelets were activated with 10(-7) M to 10(-9) M PAF concentrations, the percentage of 3H-5-HT released into the extra-platelet medium being calculated. Baumgartner's continuous perfusion model was used to study platelet deposition on the vascular endothelium. The morphometric evaluation was carried out by a planimeter assembled to a data processor with a programme devised for analysing the platelet-subendothelium interaction. RESULTS: Evaluation of the procoagulant activity on the cell surface: The expression of the procoagulant activity showed no variations with respect to the controls under different concentrations of PAF. Platelet aggregation and release studies: Normal values of platelet aggregation (80% to 100%) were found when using AA, however, there were great case to case variations under different PAF concentrations. Serotonin release was less marked than aggregation itself. The use of Lyso-PAF failed to elicit platelet aggregation and serotonin release in any case. Morphometric evaluation: The results attained showed that perfusion carried out with 10(-8) M PAF concentration showed contact, adhesiveness and thrombus formation figures similar to those of control perfusion. CONCLUSIONS: Human platelets are not too sensitive to PAF activity, only high PAF concentrations being capable of inducing platelet aggregation and 5-HT release with ample variability. This suggests the existence of a heterogeneous platelet population with PAF receptors. Low PAF concentrations do not modify the haemostatic function, and only those PAF concentrations inducing maximal release and aggregations could reduce the interaction of platelets with vascular subendothelium and the formation of thrombi.

Stereoselective cyclooxygenase inhibition in cellular models by the enantiomers of ketoprofen.

The pharmacological activity of rac-ketoprofen and its enantiomers was investigated in vitro using different cellular models. The effect of these compounds on arachidonic acid metabolism was assessed by measuring the inhibition of prostanoid generation under the action of several agonists. Thus, we have evaluated the inhibition of (1) thromboxane B2 synthesis in rabbit platelets and human polymorphonuclear leukocytes (PMNs), (2) prostaglandin E2 synthesis in three cultured cells, namely human umbilical vein endothelial cells (HUVEC), human keratinocytes, and mouse macrophage-like P388D1 cells. The IC50 values found for (+)-(S)-ketoprofen were in the range between 0.1 nM and 0.8 microM, being slightly lower in all models than those found for rac-ketoprofen (0.4 nM-3 microM). On the other hand (-)-(R)-ketoprofen showed inhibition of cyclooxygenase only at concentrations two or three orders of magnitude higher than those required for the (+)-(S) enantiomer. These results, obtained with cell types of relevance for inflammatory processes and with compounds of high optical purity, demonstrate that the prostanoid biosynthesis inhibition caused by the drug rac-ketoprofen is exclusively due to its dextrorotatory enantiomer.