Beetens J

Ketoconazole inhibits the biosynthesis of leukotrienes in vitro and in vivo.

Ketoconazole inhibits in vitro (IC50:2.6 X 10(-5) M) the formation of 5-HETE and LTB4 by isolated, carrageenin-elicited rat peritoneal PMN leukocytes, challenged with the Ca2+-ionophore A23187 in the presence of [14C]-arachidonic acid ([14C]-AA). The relative potency of various compounds tested in this respect is NDGA greater than nafazatrom greater than phenidone greater than ketoconazole greater than BW 755C. In contrast to the other compounds studies, ketoconazole in vitro, up to 1 X 10(-4) M, has no effect on the fatty acid cyclo-oxygenase or the 12-lipoxygenase-mediated metabolism of [14C]-AA by isolated human platelets; however, it stimulates the 15-lipoxygenase activity in phenylhydrazine-induced rabbit reticulocytes. After oral administration (10-40 mg/kg, -2 hr), ketoconazole inhibits in a dose-dependent way, the leukotriene-mediated anaphylactic bronchoconstriction in guinea pigs. This study demonstrates that ketoconazole is a comparatively specific and orally active inhibitor of the 5-lipoxygenase activity bearing on the production of leukotrienes derived from arachidonic acid.

Platelet-mediated vascular permeability in the rat: A predominant role for 5-hydroxytryptamine

The intradermal injection in rat skin of washed, thrombin-activated platelets produces an increase in vascular permeability, the intensity of which increments with the platelet concentration. Pretreatment of the recipient animals with serotonergic antagonists, including the specific 5-HT2 receptor blocker ketanserin, potently inhibits the platelet-mediated and the 5-HT-induced vascular defect. Amine depletion of platelets or skin tissues with reserpine reduces the response to platelets. Platelet prostanoid and lipoxygenase derivatives play no major role in the vascular response to platelet. The permeability increase induced by exogenous 5-HT and by activated platelets is reduced by alpha 1-adrenergic stimulation with noradrenaline or phenylephrine and by beta 2-stimulation with terbutaline or isoprenaline, and is potentiated by adenosine; this points to a modulation of permeability by blood flow changes and to a direct beta-adrenergic effect at the endothelial cell membrane. This study demonstrates a predominant role for 5-HT in the platelet-mediated vascular permeability increase in a sensitive species like the rat.

5-Hydroxytryptamine and arachidonic acid metabolites modulate extensive platelet activation induced by collagen in cats in vivo.

1 The pathways contributing to the platelet adhesion/aggregation reaction elicited by collagen microfibrils, administered to cats in vivo, were analysed. 2 The intra‐aortic infusion of collagen (100 μg kg−1 in 1 min) caused an extensive activation of platelets, as evidenced by the time‐dependent drop of free platelet numbers in whole blood, and the increases of 5‐hydroxyindoles (5‐HI), 5‐hydroxytryptamine (5‐HT) and thromboxane B2 (TXB2) levels in plasma, prepared from effluent venous blood sampled from the inferior caval vein. 3 5‐HT2 receptor blockade with ketanserin (0.63 mg kg−1 i.v., 10 min) and cyclo‐oxygenase inhibition with aspirin (10 mg kg−1 i.v., 10 min) slightly attenuated the peak reduction of free platelets in whole blood in response to collagen without affecting changes in plasma 5‐HI. Aspirin, but not ketanserin, reduced the collagen‐induced changes in plasma TXB2, prostaglandin E2 (PGE2) and 6K‐PGF1α. 4 Dual TXA2 synthetase inhibition/TXA2‐prostaglandin endoperoxide receptor antagonism with ridogrel (5 mg kg−1 i.v., 10 min) halved the drop in free platelets, reduced the release of platelet 5‐HI, inhibited the increase in plasma TXB2 and elevated that of 6K‐PGF1α and PGE2 in response to collagen. 5 Combined treatment with ketanserin and aspirin reduced the collagen‐induced drop of free platelets and the release of platelet 5‐HI to a similar extent as ridogrel alone; plasma prostanoids were affected as with aspirin alone. 6 Combined administration of ketanserin and ridogrel virtually eliminated the collagen‐induced platelet adhesion/aggregation response and release of 5‐HI; prostanoids were affected as with ridogrel alone. 7 The results indicate that the interplay between 5‐HT and arachidonic acid metabolites is causally involved in the platelet reaction to activation induced by collagen in cats in vivo.

Determination of 6-keto-PGF1α, 2,3-dinor-6-keto-PGF1α, thromboxane B2, 2,3-dinor-thromboxane B2, PGE2, PGD2 and PGF2α in human urine by gas chromatography—negative ion chemical ionization mass spectrometry

A method for quantification of 6-keto-PGF1 alpha, 2,3-dinor-6-keto-PGF1 alpha TXB2, 2,3-dinor TXB2, PGE2, PGD2 and PGF2 alpha in human urine samples, using gas chromatography-negative ion chemical ionization mass spectrometry, is described. Deuterated analogues were used as internal standards. Methoximation was carried out in urine samples which were subsequently applied to phenylboronic acid cartridges, reversed-phase cartridges and thin-layer chromatography. The eluents were further derivatized to pentafluorobenzyl ester trimethylsilyl ethers for final quantification by gas chromatography-mass spectrometry. The overall recovery was 77% for tritiated 6-keto-PGF1 alpha and 55% for tritiated TXB2. Urinary levels of prostanoids were determined in a group of six volunteers before and after intake of the thromboxane synthase inhibitor Ridogrel, and related to creatinine clearance.

Arachidonic acid metabolites, ADP and thrombin modulate occlusive thrombus formation over extensive arterial injury in the rat.

Platelet-dependent occlusive thrombosis at sites of deep vessel wall injury elicited by electrical stimulation of rat carotid arteries was significantly reduced by thromboxane A2 (TXA2) synthetase inhibition and/or TXA2/prostaglandin endoperoxide receptor antagonism (ridogrel 1.25 mg/kg i.v.; dazoxiben 5 mg/kg i.v.; sulotroban 20 mg/kg i.v.), by inhibition of ADP-dependent platelet responses (ticlopidine 3 x 200 mg/kg orally) and by anticoagulation (heparin 250 U/kg i.v.; warfarin 1.25 mg/kg i.p.). This points to an involvement of arachidonic acid metabolites, ADP and thrombin as modulators of the thrombotic process. The antithrombotic effect of ridogrel (IC50 = 0.22 mg/kg i.v.) was abolished by cyclooxygenase inhibition (suprofen 5 mg/kg i.v.) but enhanced by cAMP phosphodiesterase inhibition (HL 725 6 micrograms/kg/min i.v.), demonstrating the importance of platelet inhibitory prostanoids such as PGD2, and prostacyclin formed after TXA2 synthetase inhibition. High doses of ridogrel (1.25 mg/kg i.v.) producing additional TXA2/prostaglandin endoperoxide receptor antagonism were more effective than lower doses (0.16 mg/kg i.v.) providing TXA2 synthetase inhibition alone. The antithrombotic effect of ridogrel, when combined with ticlopidine or heparin, exceeded that of the single compounds, pointing to interactions between arachidonic acid metabolites, ADP and thrombin in the formation of occlusive thrombosis at sites of arterial injury.

Suppression of PAF-induced bronchoconstriction in the guinea-pig by oxatomide: mechanism of action.

SummaryOxatomide potently (ED50 0.9 mg/kg orally, −2 h) attenuates the reduction of pulmonary tidal volume elicited by PAF (250 ng/kg i.v.) in anaesthetized, ventilated and propranolol-treated guinea-pigs. The increase of the pulmonary inflation pressure elicited by PAF (40 ng/kg i. v.) in such animals, ventilated at a fixed tidal volume, is also significantly reduced by the compound, but substantially higher doses (5 mg/kg i. v., −15 min) are required. The potency of oxatomide in the latter respect (50.4% reduction) is equivalent to that of ketotifen at 5 mg/kg i.v. (55% reduction).In spontaneously breathing, anaesthetized guinea-pigs, oxatomide (5 mg/kg i. p., −1 h) significantly reduces the increase in pulmonary resistance, but not the reduction in dynamic compliance, elicited by PAF (30, 60, 90 ng/kg i. v.), suggesting a pharmacological interference mainly with PAF-induced processes in the larger airways. Changes in arterial blood pressure, haemoconcentration, thrombocytopenia and leukopenia induced by PAF in vivo, contraction of guinea-pig lung parenchymal strips, production of superoxide anion by alveolar macrophages, aggregation and release of ATP by platelets challenged with PAF in vitro are not affected by the compound.These observations suggest that oxatomide attenuates the PAF-induced pulmonary reactions by inhibiting the release and/or the effect of allergic mediators elicited by the phospholipid rather than by a direct antagonism at the PAF receptors.

Gas chromatographic—mass spectrometric determination of 11-dehydrothromboxane B2 in human urine

The extension of a method for the determination of thromboxane B2 (TxB2), 2,3-dinor-TxB2, 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha) and 2,3-dinor-6-keto-PGF1 alpha to quantify 11-dehydro-TxB2 in the same urinary sample is described. After phenylboronic acid and C18 column chromatography, 11-dehydro-TxB2, which is present in urine as the lactone and its corresponding hydroxy acid, was quantitatively converted into its lactone form for a thin-layer purification step and pentafluorobenzyl esterification. Quantification of eicosanoids was achieved by analysing their trimethylsilyl ethers with gas chromatography and negative-ion chemical ionization mass spectrometry. The overall recovery from urine for tritiated 11-dehydro-TxB2 was 80%. The detection limit was 10 pg/ml. The method was applied to the determination of these eicosanoids in volunteers and in patients suffering from acute myocardial infarction.