Ridogrel inhibits systemic and renal formation of thromboxane A2 and antagonizes platelet thromboxane A2/prostaglandin endoperoxide receptors upon chronic administration to man.

The effects of ridogrel, a dual thromboxane A2 (TXA2) synthase inhibitor and TXA2/prostaglandin (PG) endoperoxide receptor antagonist, on systemic and renal production of prostaglandins and on platelet TXA2/PG endoperoxide receptors was evaluated upon chronic administration (300 mg b.i.d. orally, for 8 and 29 days) to man. Such a medication with ridogrel inhibits the systemic as well as the renal production of TXA2 as measured by the urinary excretion of 2,3-dinor-TXB2 and TXB2 respectively without inducing significant changes in systemic or renal PGI2 production. Simultaneously with the latter effects, the production of TXB2 by spontaneously coagulated whole blood ex vivo is inhibited (greater than 99%) while that of PGE2 and PGF2 alpha is largely increased. Administration of ridogrel causes a three- to five-fold shift to the right of concentration-response curves for U46619 in eliciting platelet aggregation; no tachyphylaxis is observed after 29 days of treatment in this respect. Apart from a reduction of serum uric acid levels with a concomitant increase in urinary uric acid excretion during the first days of treatment, no clinically significant changes in hematological, biochemical, hemodynamic and coagulation parameters occur during the 8 days or 29 days study. The study demonstrates that ridogrel is a potent inhibitor of the systemic as well as renal TXA2 synthase and an antagonist of platelet TXA2/PG endoperoxide receptor in man, covering full activity during 24 h at steady-state plasma level conditions without tachyphylaxis during 29 days of medication. The compound is well tolerated, at least during 1 month of administration.

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.

Production of thromboxane and prostaglandins in human blood in the presence of thromboxane synthase inhibitors: a comparison of RIA and GC/MS determinations.

The radioimmunological determination (RIA) of primary prostaglandins (PGs) in serum and plasma was evaluated with gas chromatography/mass spectrometry (GC/MS). Human blood was stimulated in vitro in the presence or absence of the specific thromboxane synthase inhibitor ridogrel. TxB2, 6-keto-PGF1 alpha, PGE2, PGD2 and PGF2 alpha were determined with RIA and GC/MS on the same samples. For GC/MS analysis, prostanoids were extracted and derivatized to their methoximepentafluorobenzyl-esters-trimethylsilyl-ethers. An excellent correlation was observed between levels of all eicosanoids determined by RIA or GC/MS (r = 0.996-0.999) when plasma was spiked with known amounts of PGs and TxB2 (2-500 ng/ml). In stimulated blood, with or without inhibition of thromboxane synthase, the correlation between RIA and GC/MS values remained good, except for 6-keto-PGF1 alpha. RIA largely overestimated the levels of 6-keto-PGF1 alpha and no significant correlation was found with levels detected by GC/MS. The results demonstrate the importance of corroborating the reliability of RIA with GC/MS.

Determination of 6-keto-PGF1 alpha, 2,3-dinor-6-keto-PGF1 alpha, thromboxane B2, 2,3-dinor-thromboxane B2, PGE2, PGD2 and PGF2 alpha 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.

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 micrograms 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-PGF1 alpha. 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-PGF1 alpha 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. 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.

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

Oxatomide 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.

R 68 070: thromboxane A2 synthetase inhibition and thromboxane A2/prostaglandin endoperoxide receptor blockade combined in one molecule--I. Biochemical profile in vitro.

R 68 070 or (E)-5-[[[(3-pyridinyl)[3-(trifluoromethyl)phenyl]- methylen]amino]oxy] pentanoic acid (Janssen Research Foundation, Belgium) combines specific thromboxane A2 (TXA2) synthetase inhibition with TXA2/prostaglandin endoperoxide receptor blockade in one molecule. In vitro, the compound specifically inhibits the production of TXB2 from [14C] arachidonic acid by washed human platelets (IC50 = 8.2 X 10(-9) M) and by platelet microsomes (IC50 = 3.6 X 10(-9) M), of MDA (IC50 = 1.91 X 10(-8) M) and of TXB2 (IC50 = 1.47 X 10(-8) M) by thrombin-coagulated human platelet-rich plasma (P.R.P.) and whole blood respectively and increases the levels of PGD2, PGE2, PGF2 alpha and 6-keto-PGF1 alpha. The activity of cyclo-oxygenase-, prostacyclin synthetase-, 5-, 12- and 15-lipoxygenase-enzymes are not affected. Additionally, R 68 070 inhibits human platelet aggregation in P.R.P. induced by U 46619 3 X 10(-7) M to 2 X 10(-6) M (IC50 = 2.08 X 10(-6) M to 2.66 X 10(-5) M, collagen 0.5 to 2 micrograms/ml (IC50 = 2.85 X 10(-6) M to 4.81 X 10(-5) M), arachidonic acid 7.5 X 10(-4) M to 2 X 10(-3) M (IC50 = 2.1 X 10(-8) M to 3.3 X 10(-8) M) and the U 46619 (1 X 10(-7) M)-induced accumulation of [32P] phosphatidic acid (IC50 = 5.24 X 10(-7) M) in washed human platelets.(ABSTRACT TRUNCATED AT 250 WORDS)

R 68 070: thromboxane A2 synthetase inhibition and thromboxane A2/prostaglandin endoperoxide receptor blockade combined in one molecule--II. Pharmacological effects in vivo and ex vivo.

R 68 070 or (E)-5-[[[(3-pyridinyl)[3-(trifluoromethyl)phenyl]- methylen]amino]oxy] pentanoic acid (Janssen Research Foundation, Belgium), a newly developed compound, combining specific thromboxane A2 (TXA2) synthetase inhibition with TXA2/prostaglandin endoperoxide receptor blockade in one molecule, is active in vivo in man and in experimental animals. In man (n = 5), a single oral 400-mg dose of R 68 070 produces deep and protracted inhibition of platelet TXA2 synthetase activity (greater than or equal to 90% for 48 h), increases serum levels of immunoreactive 6-keto-PGF1 alpha, reduces platelet aggregation in P.R.P. induced by U 46619, collagen (greater than 70% for 8 h), arachidonic acid (greater than 90% for 18 h) and prolongs template bleeding times significantly, without affecting plasma coagulation or fibrinolysis. In rats, R 68 070 (1.25 mg/kg orally, -2 h) singly prolongs tail bleeding times as much as a combination of TXA2 synthetase inhibition (dazoxiben 10 mg/kg) and TXA2/prostaglandin endoperoxide receptor blockade (BM 13177 40 mg/kg). In dogs, the compound reduces coronary thrombosis induced by electrical damage (1.25 mg/kg i.v.) and prevents the evolution of occlusion/reperfusion-induced arrhythmias into ventricular fibrillation (2.5 mg/kg i.v.). R 68 070 thus may be an appropriate pharmacological tool to analyze the roles and interactions of agonistic (TXA2, prostaglandin endoperoxides) and antagonistic (PGD2, PGE2, PGI2) metabolites of arachidonic acid in experimental and human pathologies.

Biphasic response of intimal prostacyclin production during the development of experimental atherosclerosis.

Feeding a cholesterol rich diet (0.3%) to rabbits for up to 10 weeks resulted in morphological changes of the vascular wall. Microscopic evaluation of the aorta revealed a lipid infiltration and an intimal thickening containing foam cells, which both became more pronounced as the cholesterol feeding was more prolonged. The intimal prostacyclin production showed a transient increase after 2 weeks, but was significantly decreased after 6 weeks of diet and remained at this low level during the rest of the experiment. No significant changes in formation of thromboxane B2 by the platelets could be observed, whereas the production of 12-HETE was enhanced.

Vitamin C increases the prostacyclin production and decreases the vascular lesions in experimental atherosclerosis in rabbits.

Feeding a cholesterol-rich diet (0.3%) to rabbits resulted in an intimal thickening and lipid infiltration of the aorta. The prostacyclin production by the vascular endothelium was significantly decreased, after a transient increase after 2 weeks of diet. The arachidonic acid metabolism in platelets was hardly changed. Addition of a low dose vitamin C (150 mg/day) to the cholesterol rich diet resulted in decreased lipid infiltration and intimal thickening and the transient increase of the prostacyclin production was postponed to the 4th week. Although this dose of vitamin C could not restore the decreased prostacyclin production observed after 6 weeks diet, a higher dose of vitamin C (600 mg/day), besides its beneficial effect on the lipid infiltration and the intimal thickening in the thoracic aorta, kept the intimal prostacyclin production at normal levels for at least 8 weeks.

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.

Stimulation of prostacyclin production by vitamin C in ram seminal vesicle microsomes: possible mode of action.

The enhancing effect of vitamin C on the conversion of arachidonic acid, endoperoxide G2 and endoperoxide H2 to 6-keto-PGF 1 alpha, the stable metabolite of prostacyclin, by ram seminal vesicle microsomes was further investigated. From the incubations of these substrates with 1-tryptophan, catalase, superoxide dismutase and 15-HPETE it became clear that vitamin C apparently acts mainly through neutralization of the oxidative species formed during the reduction of endoperoxide G2 to endoperoxide H2. Although it has also a more direct stimulating activity on the prostacyclin synthase, a possible interference with hydroperoxy derivatives of arachidonic acid cannot be completely ruled out.

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.

Influence of vitamin C on the metabolism of arachidonic acid and the development of aortic lesions during experimental atherosclerosis in rabbits.

Feeding rabbits a cholesterol-rich diet (0.3%) resulted in morphological changes and a decrease in the prostacyclin production by the aortic endothelium. Addition of vitamin C (0.1%) to the cholesterol-rich diet resulted in a decreased lipid infiltration and intimal thickening. Although there was a tendency to restore the prostacyclin output, vitamin C, in the amount administered, was unable to completely normalise the endothelial PGI2 production.

Vitamin C increases the formation of prostacyclin by aortic rings from various species and neutralizes the inhibitory effect of 15-hydroperoxy-arachidonic acid.

Aortic rings from rats, rabbits and guinea-pigs produce different amounts of 6-oxo-prostaglandin F1 alpha (6-oxo-PGF1 alpha), the stable breakdown product of prostacyclin, i.e. 2760 +/- 195, 160 +/- 10 and 87 +/- 17 pg 6-oxo-PGF1 alpha per mg wet weight in 30 min. Vitamin C enhances the production of 6-oxo-PGF1 alpha by the aortic tissue of these three species, independent of their basal release. This increase was only significant if vitamin C was present in the preincubation as well as in the incubation fluid. 15-Hydroperoxy-arachidonic acid inhibits the production of 6-oxo-PGF1 alpha (IC50:6 microM) and this inhibitory effect was completely neutralized by vitamin C. The increased production of 6-oxo-PGF1 alpha is not due to an increased release of the substrate arachidonic acid. It is suggested that vitamin C enhances the formation of 6-oxo-PGF1 alpha by protecting the cyclo-oxygenase and PGI-synthase.