15-hydroxy-11-alpha-9-alpha-(epoxymethano)prosta-5-13-dienoic-acid and sulotroban

In a randomized pilot study we compared the antiplatelet effects of aspirin and BM 13.177 in two groups of 7 patients each undergoing PTCA. As compared with the pretreatment values template bleeding time was prolonged and collagen induced aggregation was inhibited in PRP and WB in all patients. In the course of angiography and PTCA a rise in platelet factor 4 and beta thromboglobulin was observed in both groups, followed by a decrease below the baseline levels. Thromboxane B2 in plasma and serum decreased in the aspirin group but remained unchanged during BM 13.177 treatment. In PRP and WB aggregation induced by U 46 619 was inhibited after ingestion of BM 13.177 but not following ASA. After three months a control coronary angiography was done. There was no difference in regard to the degree of restenosis between both groups. Medication was well tolerated, compliance was good and no side effects were noted.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Angioplasty, Balloon; Arteriosclerosis; Aspirin; Bleeding Time; Blood Platelets; Clinical Trials as Topic; Collagen; Cyclooxygenase Inhibitors; Female; Fibrinolytic Agents; Humans; Male; Middle Aged; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Receptors, Cell Surface; Receptors, Prostaglandin; Receptors, Thromboxane; Sulfonamides; Thromboxane B2; Thromboxanes

BM 13.177, a sulphonamide derivative, prevented platelet aggregation by thromboxane A2 in vitro and selectively inhibited contraction of isolated rabbit femoral arteries induced by two stable endoperoxide analogues. In a double-blind placebo-controlled study, oral BM 13.177 inhibited platelet aggregation induced by arachidonic acid, low dose collagen, and the two stable endoperoxide analogues, and slightly prolonged the bleeding time. Generation of thromboxane or of other prostaglandins was not affected. No side-effects were seen. BM 13.177 appears selectively and safely to block platelet and vessel wall thromboxane receptors and should be useful in elucidating the role of thromboxane A2 in disease.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Blood Platelets; Blood Vessels; Clinical Trials as Topic; Double-Blind Method; Humans; Male; Platelet Aggregation; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Random Allocation; Receptors, Cell Surface; Receptors, Prostaglandin; Receptors, Thromboxane; Sulfonamides; Vasoconstriction

This study aimed to investigate the effects of reactive oxygen species on the hepatic macrophages, the Kupffer cells (KC), and to identify the relevant targets of vasoconstrictors involved in the regulation of intrahepatic microcirculation and therefore portal pressure. The effects of hydrogen peroxide (H2O2), xanthine/xanthine oxidase or a thromboxane (TX) analogue (U46619; 0.1 microM) were tested in sham-operated and fibrotic livers (bile duct ligation over 4 weeks) during isolated rat liver perfusion and in vivo with or without additional KC blockade (gadolinium chloride, 10 mg kg(-1) body weight, 48 and 24 h, i.p.). To investigate downstream mechanisms, a TXA2 antagonist (BM 13.177; 20 microM) or a Rho kinase inhibitor (Y27632; 10 microM) was infused additionally. TXB2 efflux was measured by enzyme-linked immunosorbent assay. The phosphorylation state of moesin (p-moesin), as indicator for Rho kinase activity, was assessed by Western blot analyses. Portal pressure was dose-dependently increased by H2O2 (maximum, 0.5 mM) and, to a lower extent, by xanthine/xanthine oxidase together with catalase. The portal pressure increase by H2O2 was attenuated by previous KC blockade. TXA2 efflux increased after H2O2 infusion and was reduced by KC blockade. The TXA2 antagonist counteracted the H2O2-induced increase in portal pressure. The Rho kinase inhibitor attenuated portal pressure increase after TXA2 analogue or H2O2 infusion. Hepatic levels of p-moesin were increased after H2O2 infusion. Reactive oxygen species increased portal pressure via stimulation of TXA2 production by KCs and a subsequent Rho kinase-dependent contraction of the intrahepatic vasculature. In conclusion, the KCs that are well known to produce H2O2 could also be activated by H2O2. This vicious cycle may best be interrupted at the earliest time point.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Amides; Animals; Gadolinium; Hydrogen Peroxide; Kupffer Cells; Liver Cirrhosis; Male; Microfilament Proteins; Portal Pressure; Pyridines; Rats; Rats, Sprague-Dawley; Reactive Oxygen Species; Sulfonamides; Xanthine; Xanthine Oxidase

Clinical and experimental evidence has shown that myocardial ischemia activates cardiac spinal afferents that mediate sympathoexcitatory reflex responses. During myocardial ischemia, thromboxane A2 (TxA2) is released in large quantities by activated platelets in the coronary circulation of patients with coronary artery disease. We hypothesized that endogenous TxA2 contributes to sympathoexcitatory reflexes during myocardial ischemia through stimulation of TxA2/prostaglandin endoperoxide (TP) receptors. Regional myocardial ischemia was induced by occlusion of a diagonal branch of left anterior descending coronary artery of anesthetized cats. Hemodynamic parameters and renal sympathetic nerve activity were recorded after sinoaortic denervation and bilateral vagotomy. Regional myocardial ischemia evoked significant increases in mean blood pressure (122+/-10 vs. 139+/-12 mmHg, before vs. ischemia), aortic flow (153+/-18 vs. 167+/-20 ml/min), first derivative of left ventricular pressure at 40-mmHg developed pressure (2,736+/-252 vs. 2,926+/-281 mmHg/s), systemic vascular resistance (0.6+/-0.1 vs. 0.9+/-0.12 peripheral resistance units), and renal sympathetic nerve activity (by 22%). The reflex nature of the excitatory responses was confirmed by observing its disappearance after blockade of cardiac nerve transmission with intrapericardial 2% procaine treatment. Moreover, application of U-46619 (2.5-10 microg), a TxA2 mimetic, on the heart caused graded increases in mean arterial pressure and renal nerve activity, responses that were abolished 3 min after local blockade of cardiac neural transmission with intrapericardial procaine. BM 13,177 (30 mg/kg iv), a selective TP receptor antagonist, eliminated the reflex responses to U-46619 and significantly attenuated the excitatory responses during brief (5 min) regional myocardial ischemia. The sympathoexcitatory reflex responses to U-46619 were unchanged by blockade of histamine H1 receptors with pyrilamine and serotonin 5-HT3 receptors with tropisetron, indicating specificity of this TP receptor agonist. These data indicate that endogenous TxA2 participates in myocardial ischemia-mediated sympathoexcitatory reflex responses through a TP receptor mechanism.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Anesthetics, Local; Animals; Cats; Disease Models, Animal; Dose-Response Relationship, Drug; Female; Heart; Hemodynamics; Histamine H1 Antagonists; Indoles; Kidney; Male; Myocardial Ischemia; Pressoreceptors; Procaine; Pyrilamine; Receptors, Thromboxane; Reflex; Serotonin Antagonists; Sulfonamides; Sympathetic Nervous System; Thromboxane A2; Tropisetron; Vagotomy

The pharmacomodulation of sulfonylureas structurally related to torasemide and characterized by a TXA(2)antagonism led to the synthesis of BM-573. This original molecule showed a high affinity (IC(50)1.3 nM) for the TXA(2)receptor of human platelets in comparison with both reference compounds, SQ-29548 (IC(50)21 nM) and sulotroban (IC(50)930 nM). Moreover, this torasemide derivative was found to be a potent inhibitor of human platelet aggregation induced by arachidonic acid (ED(100)=0.13 microM) or by U-46619 (ED(50)=0.24 microM), a TXA(2)agonist. BM-573 relaxed the isolated rat thoracic aorta (ED(50)=28.4 nM) and guinea-pig trachea (ED(50)=17.7 nM) contracted by U-46619. BM-573 (1 microM) completely reduced the platelet production of TXB(2)induced by arachidonic acid. Finally, BM-573 (30 mg/kg, per os) lost the diuretic properties of torasemide in rats.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Antihypertensive Agents; Aorta; Arachidonic Acid; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Guinea Pigs; Humans; Hydrazines; Inhibitory Concentration 50; Models, Chemical; Muscle, Smooth; Platelet Aggregation; Platelet Aggregation Inhibitors; Rats; Rats, Wistar; Receptors, Thromboxane; Sulfonamides; Thromboxane-A Synthase; Torsemide; Trachea; Vasoconstrictor Agents

The present study was undertaken to examine the involvement of platelet-derived serotonin (5-hydroxytryptamine; 5-HT) in thromboxane A2 (TXA2)-induced platelet aggregation. Pharmacological experiments with 5-HT2 and TXA2 inhibitors were conducted on platelet aggregation in platelet-rich plasma from cats. Exogenously added 5-HT, U-46619 (a stable TXA2 analogue) and collagen caused platelet aggregation in a concentration-dependent manner. The combination of low concentrations of 5-HT and U-46619 caused full platelet aggregation, whereas each agent alone, at these concentrations, caused a transient aggregation. 5-HT-induced aggregation was inhibited by ketanserin (0.01-0.3 micromol/l), a 5-HT2 receptor antagonist, in a concentration-dependent manner. Collagen-induced platelet aggregation was also inhibited by ketanserin, whereas the inhibition by indomethacin was modest even at the highest concentration tested (300 micromol/l). U-46619 triggered platelet aggregation in a biphasic manner. Ketanserin inhibited only the second phase of the aggregation. The inhibition of U-46619-induced aggregation by ketanserin occurred at a concentration range similar to that for 5-HT-induced platelet aggregation. Likewise, platelet aggregation induced by the combination of low concentrations of 5-HT and U-46619 was fully inhibited by ketanserin. These data suggest a major involvement of platelet-derived 5-HT in TXA2-dependent aggregation in cat platelets.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Blood Platelets; Cats; Collagen; Cyclooxygenase Inhibitors; Drug Interactions; Female; Indomethacin; Ketanserin; Male; Platelet Aggregation; Platelet Aggregation Inhibitors; Serotonin; Serotonin Antagonists; Sulfonamides; Thromboxane A2

The shape change that occurs when platelets are stimulated with an agonist can be quantitated by monitoring changes in their forward-scatter/side-scatter profile using a flow cytometer. Here we have stimulated platelets in citrated whole blood with several agonists and determined the time-course and extent of the shape change that occurs. In some experiments parallel investigations of shape change and aggregation were performed. Aggregation was measured by monitoring the fall in number of single platelets using a Whole Blood Platelet Counter. Some agents (ADP, PAF, U46619 and 5HT) produced a strong and rapid change in platelet forward-scatter/side-scatter that was maximal within 10 s. Others (A23187 and collagen) produced a strong but slower response. Adrenaline produced only a weak response that was also slow to develop, and PMA did not produce any response. The concentrations of each of ADP, PAF, U46619 and 5HT needed to induce a shape change were lower than those required for aggregation. Selective PAF, TXA2 and 5HT antagonists (WEB 2086, sulotroban and MCI-9042) clearly inhibited both the shape change and the aggregation induced by the appropriate agonist; in each case the effect of the antagonist was to move the dose-response curve to the right. These results are consistent with the shape change and aggregation brought about by each of these agonists being mediated via a single receptor. In contrast, a selective P2T purinoceptor antagonist (ARL 66096) markedly inhibited the aggregation induced by ADP but was found to have little or no effect on shape change. This is consistent with these platelet responses to ADP being mediated by different receptors, with P2T receptors mediating only the aggregation response.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Adenosine Triphosphate; Azepines; Blood Platelets; Humans; In Vitro Techniques; Piperazines; Piperidines; Platelet Activating Factor; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Serotonin; Succinates; Sulfonamides; Thromboxane A2; Triazoles; Vasoconstrictor Agents

This study reports the synthesis, biological evaluation, and application of a new biotinylated derivative 1-[[1S-[1 alpha, 2 alpha (Z),3 alpha, 4 alpha]]-7-[3-[[[[(1-oxocyclohexylpropyl)amino]acetyl]amino] methyl]-7-oxabicyclo [2.2.1]hept-2-yl]-5-heptenoyl]-2-[hexahydro-2'-oxo-1H-thieno[3',4' d] imidazole-4'-pentanoyl]hydrazine (SQB) of the thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor antagonist [1S-[1 alpha,2 alpha(Z),3 alpha,4 alpha]]-7-[3-[[[[(1-oxocyclohexylpropyl)amino]acetyl] amino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (SQ31,491). SQB was synthesized by reacting SQ31,491 with biotin hydrazide, and the product was purified by flash chromatography. It was found that SQB specifically inhibited platelet aggregation in response to U46619 with an IC50 of 275 nM. On the other hand, SQB did not inhibit adenosine diphosphate or A23187-induced aggregation. Competition binding studies revealed that SQB produced a concentration-dependent inhibition of [3H]-[1S-[1 alpha, 2 beta (5Z),3 beta, 4 alpha]]-7-[3-[[2[(phenylamino) carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid ([3H]SQ29,548) specific binding in 3-[(3-cholamidopropyl)dimethylammonio]-1-propane-sulfonate (CHAPS)-solubilized platelet membranes, with a Ki of 220 nM. The shape of the SQB inhibition binding curve was indistinguishable from that produced by the TXA2/PGH2 receptor antagonist BM13.177. Finally, incubation of gel-filtered platelets or platelet-rich plasma with SQB and fluorescein isothiocyanate (FITC)-avidin demonstrated fluorescent labeling of platelet plasma membrane TXA2/PGH2 receptors. Furthermore, this SQB-FITC fluorescent labeling was reduced significantly by co-incubation of the platelets with the TXA2/PGH2 antagonist SQ29,548. Based on the ability of SQB-FITC-avidin to label intact platelets, it can be concluded: (1) that a pool of platelet TXA2/PGH2 receptors resides in the plasma membrane; and (2) that the binding domains for these receptors are oriented at or near the external membrane surface. Collectively, these data demonstrate that SQB is a highly specific probe for TXA2/PGH2 receptors, which should be of significant value for receptor localization studies in platelets and other tissues.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Biotin; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Cell Membrane; Fatty Acids, Unsaturated; Humans; Hydrazines; Microscopy, Fluorescence; Molecular Probes; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Radioligand Assay; Receptors, Prostaglandin; Receptors, Thromboxane; Receptors, Thromboxane A2, Prostaglandin H2; Spectrometry, Fluorescence; Sulfonamides; Thromboxane A2

Fura-2-loaded human platelets were used to study Ca2+ release from intracellular compartments, as well as Ca2+ influx from the extracellular space. We investigated the response towards the endoperoxide/thromboxane-receptor agonist. U46619, and the inhibitor of the endoplasmic-reticulum Ca(2+)-ATPase, thapsigargin. U46619 dose-dependently depleted intracellular Ca2+ stores, followed by active sequestration of released Ca2+. Ca2+ influx induced by U46619 largely relies on receptor occupancy. Removing the thromboxane analogue from its receptor by using the endoperoxide/thromboxane-receptor antagonist BM 13177 largely blunted U46619-mediated Ca2+ influx. The Ca(2+)-ATPase inhibitor thapsigargin evoked a gradual rise in intracellular Ca2+, which was potentiated by a preceding activation of platelets with the receptor agonist U46619. This agonist-sensitizing effect also depends on receptor occupancy. Removing U46619 from its receptor by addition of the endoperoxide/thromboxane-receptor antagonist BM13177 suppressed the sensitizing effect completely. Furthermore, interrupting downstream receptor signalling events by raising intracellular levels of cyclic nucleotides (cyclic AMP, cyclic GMP) again suppressed the U46619-sensitizing effect on thapsigargin-induced Ca2+ release. This study indicates that the process of Ca2+ release followed by resequestration in response to a platelet agonist by its own is not sufficient to produce the sensitizing effect. Rather, a continuously occupied receptor triggering sustained downstream signalling events seems to be required for sensitization. The presence of a receptor agonist may induce an increased cycling of Ca2+ between the agonist-responsive and the thapsigargin-dischargeable compartment, leading to faster and more intense accumulation of Ca2+ in the cytosolic compartment after inhibition of the Ca(2+) ATPase. Suggestively, receptor occupancy increases the Ca(2+)-releasing potency of thapsigargin by coupling the thapsigargin-sensitive Ca(2+)-storing compartments with an agonist-responsive compartment that exhibits a high leakage rate in stimulated platelets.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Blood Platelets; Calcium; Cyclic AMP; Cyclic GMP; Cytosol; Epoprostenol; Humans; Intracellular Fluid; Nitroprusside; Platelet Activation; Prostaglandin Endoperoxides, Synthetic; Receptors, Thromboxane; Signal Transduction; Sulfonamides; Terpenes; Thapsigargin; Thromboxane A2

The effects of thromboxane B2 (TxB2) and of two thromboxane mimetics, dl-(9,11), (11,12)-dimethano-TxA2 (ONO 11006) and 9,11-dideoxy-11,9-epoxymethano prostaglandin F2 alpha (U46619) on the cardiac response to adrenergic nerve stimulation in isolated guinea-pig atria were evaluated. All the agonists dose dependently reduced the positive inotropic effect induced by field stimulation, U46619 being the most active. The inhibitory effect of U46619 was reduced by the thromboxane receptor antagonists, sulotroban and AH 23848B. U46619 did not significantly reduce the positive inotropic effect induced by exogenous noradrenaline. However U46619 was unable to modify the tritium overflow induced by field stimulation in preparations preloaded with [3H]noradrenaline. In addition to this influence on adrenergic neurotransmission, U46619 also had a direct positive inotropic effect on cardiac contractility, which was antagonized by AH 23848B. These results indicate that U46619 reduces the cardiac response to sympathetic nerve stimulation and that is also has a direct stimulatory effect on cardiac muscle.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Biphenyl Compounds; Dose-Response Relationship, Drug; Electric Stimulation; Guinea Pigs; Heart Atria; In Vitro Techniques; Male; Norepinephrine; Prostaglandin Endoperoxides, Synthetic; Receptors, Prostaglandin; Receptors, Thromboxane; Sulfonamides; Sympathetic Nervous System; Synaptic Transmission; Thromboxane A2; Thromboxane B2

The effects of KW-3635 (sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)- ethylidene]-6,11-dihydrodibenz[b,e]oxepine-2-carboxylate monohydrate, CAS 127166-41-0) on smooth muscle preparations were examined. In isolated guinea-pig aorta, KW-3635 competitively inhibited the U-46619 (a thromboxane mimetic) induced contractions (pA2 = 7.74), the effect being more potent than those of sulotroban and daltroban. In canine saphenous vein, KW-3635 also antagonized the U-46619-induced contraction (pA2 = 8.11). In this preparation, solutroban and daltroban, but not KW-3635, exhibited intrinsic agonistic action. KW-3635, even at a high concentration of 10(-5) mol/l did not affect the norepinephrine- or KCl-induced contractions of guinea-pig or rat aorta, prostaglandin (PG)E2- or PGF2 alpha-induced contractions of guinea-pig ileum nor the PGE2-induced contraction of rat fundus. KW-3635 at concentrations higher than its thromboxane A2- (TxA2-)antagonistic one, non-competitively inhibited the PGF2 alpha-induced contractions of guinea-pig aorta (pD2' = 6.23), as was the case with daltroban. The inhibitory effect of KW-3635 (3 x 10(-6) mol/l) on U-46619-induced contractions of guinea-pig aorta persisted for longer than 2 h following washout of the tissue, whereas that of daltroban (10(-5) mol/l completely disappeared at 1 h after the washout. In anesthetized guinea-pigs, KW-3635 at doses of 10 to 1000 micrograms/kg (i.v.) inhibited U-46619 (1 microgram/kg i.v.)-induced pressor responses in a dose-dependent manner. The effect of KW-3635 (0.1 to 1 mg/kg i.v.) persisted for longer than 3 h. These results demonstrate that KW-3635 is a potent and specific TxA2 antagonist without agonistic action in vascular smooth muscles. KW-3635 is considered to be a promising candidate for the treatment of patients with disorders mediated via TxA2.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Benzimidazoles; Benzoxepins; Blood Pressure; Cats; Dogs; Female; Guinea Pigs; In Vitro Techniques; Male; Muscle Contraction; Muscle, Smooth; Muscle, Smooth, Vascular; Phenylacetates; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Rabbits; Rats; Rats, Inbred Strains; Species Specificity; Sulfonamides; Thromboxane A2

Injection of U-46619 (9,11-dideoxy-9 alpha, 11 alpha-epoxymethano- prostaglandinF2 alpha; 130 micrograms/kg i.v.) produced sudden death in anesthetized guinea-pigs and rats within 10-15 min. This sudden death is typified by a precipitous drop in mean arterial blood pressure (MABP) and a dramatic decrease in the circulating platelet counts. In guinea-pigs, KW-3635 (sodium(E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)ethylidene]-6,11- dihydrodibenz[b,e]oxepine-2-carboxylate monohydrate, CAS 127166-41-0) at doses of 0.1 mg/kg or greater dramatically protected animals against sudden death induced by injection of U-46619. Pretreatment with KW-3635 (0.3, 1.0 mg/kg i.v.) inhibited the decrease in circulating platelet counts and the decline in blood pressure associated with the i.v. injection of U-46619. Oral administration of KW-3635 (10, 30 mg/kg) also protected the animals from the U-46619-induced sudden death. The effect of KW-3635 was almost the same as that of daltroban, and was more potent than that of sulotroban. In rats, intravenous administration of KW-3635 at doses of 0.3 mg/kg or greater protected against sudden death. In contrast, acetylsalicylic acid a cyclooxygenase inhibitor, did not protect against sudden death induced by U-46619, indicating that the formation of endogenous thromboxane does not play a major role in the lethal effect of U-46619, and that the blockade of the lethal effects of U-46619 is specific for thromboxane receptor antagonists. Our data show that KW-3635 protects guinea-pigs and rats against U-46619-induced sudden death. Therefore, KW-3635 may be useful for the investigation of diseases where thromboxane is involved.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aspirin; Benzimidazoles; Benzoxepins; Blood Pressure; Death, Sudden; Dose-Response Relationship, Drug; Guinea Pigs; Male; Phenylacetates; Platelet Aggregation Inhibitors; Platelet Count; Prostaglandin Endoperoxides, Synthetic; Rats; Rats, Inbred Strains; Sulfonamides; Thromboxane A2; Thromboxanes; Ticlopidine

The effects of KW-3635 (sodium (E)-11-[2-(5,6-dimethyl-1-benzimidazolyl)- ethylidene]-6,11-dihydrodibenz[b,e] oxepine-2-carboxylate monohydrate, CAS 127166-41-0) on platelet aggregation were examined. In human washed platelets, KW-3635 shifted the concentration-aggregation curves for U-46619, a thromboxane A2 (TxA2) mimetic, to the right. The pA2 value for KW-3635 was 8.8 +/- 0.10, while those for sulotroban and daltroban were 6.31 +/- 0.18 and 7.75 +/- 0.07, respectively. In human platelet rich plasma (PRP), KW-3635 at 10(-8) mol/l to 10(-6) mol/l inhibited the aggregations induced by U-46619 (1 mumol/l) or collagen (1.5 micrograms/ml). However, KW-3635 at up to 10(-5) mol/l did not affect the primary phase of platelet aggregation induced by adenosine diphosphate or epinephrine. KW-3635 at 10(-5) mol/l did not affect the antiaggregatory effects of the prostaglandins PGI2, PGE1 and PGD2. These results indicate that KW-3635 is a potent and selective TxA2 receptor antagonist. The TxA2 antagonistic effects of KW-3635 were compared with that of daltroban in PRP from various animals species. The effects of KW-3635 on platelet aggregation were species-dependent and KW-3635 exhibited the most prominent activity in human platelets. The activities of KW-3635 in mouse and rabbit PRP were much less potent. In PRP from guinea-pigs, dogs, cats and rats, KW-3635 exhibited moderate anti-aggregatory effects. In the guinea-pig PRP, KW-3635 at 10(-7) mol/l to 3 x 10(-6) mol/l inhibited both the platelet aggregation and the concomitant adenosine triphosphate secretion in a concentration-dependent manner, the effect being more potent than those of sulotroban and daltroban. In the experiments on the platelet aggregation ex vivo in guinea-pigs, KW-3635 at oral doses of 3 and 10 mg/kg inhibited the aggregations induced by U-46619 (1, 3 mumol/l), collagen (3, 6, 9 micrograms/ml) and arachidonate (50, 100 mumol/l). The effects lasted for longer than 7 h following oral administration. These results indicate that KW-3635 is a specific and orally active TxA2 receptor antagonist. KW-3635 is expected to be a drug useful for the treatment of patients with thrombotic disorders.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Triphosphate; Adult; Animals; Benzimidazoles; Benzoxepins; Cats; Dogs; Drug Interactions; Epinephrine; Guinea Pigs; Humans; In Vitro Techniques; Mice; Middle Aged; Phenylacetates; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Prostaglandins; Rabbits; Rats; Receptors, Prostaglandin; Receptors, Thromboxane; Sulfonamides; Thromboxane A2

Conformational analyses on thromboxane A2 (TxA2), its receptor agonist, U-46619, and its receptor antagonist, sulotroban, were carried out by molecular mechanics (MMFF) or molecular orbital (MNDO) methods. Two kinds of putative active conformations of TxA2 and the agonist were proposed on the basis of these results by referring to the hairpin conformation hypothesis. From the superposition of stable conformers of sulotroban on those conformers, the molecular structural requirements for potent TxA2 receptor antagonism were elucidated. S-145 in which these requirements are satisfied was a very potent TxA2 antagonist.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Bridged Bicyclo Compounds; Bridged-Ring Compounds; Computer Simulation; Fatty Acids, Monounsaturated; Models, Molecular; Molecular Conformation; Prostaglandin Endoperoxides, Synthetic; Receptors, Prostaglandin; Receptors, Thromboxane; Stereoisomerism; Structure-Activity Relationship; Sulfonamides; Thromboxane A2

The effects of SK&F 95587 (4[2-(benzenesulfonamido)-ethyl] phenoxyacetic acid), a thromboxane (TX) receptor blocking agent, on bronchoconstrictor responses were investigated in paralyzed, anesthetized, mechanically ventilated cats. Intravenous injections of the TXA2 receptor mimics, U-46619 [(15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta5Z,13E-dienoic acid] and U-44069 (9,11-dideoxy-11 alpha,9 alpha-epoxymethano PGF2 alpha), produced dose-related increases in transpulmonary pressure and lung resistance and decreases in dynamic compliance. After administration of SK&F 95587, 5 mg/kg i.v., bronchoconstrictor responses to U-46619 and U-44069 were reduced markedly, whereas airway responses to prostaglandin (PG)F2 alpha, serotonin, PGD2 or the PGD2 metabolite, 11 beta-PGF2 alpha, were not altered. The duration of action of SK&F 95587 was greater than 3 hr, and the blockade was overcome when 10-fold larger doses of the TXA2 mimics were administered. Bronchoconstrictor responses to platelet-activating factor (PAF) were blocked by SK&F 95587 and by the novel PAF receptor antagonist, BN 50730. BN 50730 also blocked the fall in systemic arterial pressure in response to PAF. However, BN 50730 did not influence airway responses to U-46619, PGF2 alpha, PGD2 or serotonin and had no effect on baseline bronchomotor tone or arterial pressure. The PAF receptor antagonism with BN 50730 was overcome when 10-fold larger doses of PAF were administered and the dose-response curves for changes in lung resistance and dynamic compliance were shifted to the right in a parallel manner. The present data suggest that SK&F 95587 has selective TX receptor blocking activity, and that BN 50730 has selective PAF receptor blocking properties in the airways of the cat. The present data also provide support for the hypothesis that bronchoconstrictor responses to PAF are mediated by specific receptors, which are coupled to a phospholipase and, when activated, result in the release of TXA2 and contraction of airway smooth muscle.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Azepines; Bronchoconstriction; Cats; Dose-Response Relationship, Drug; Female; Lung; Male; Platelet Activating Factor; Platelet Aggregation Inhibitors; Platelet Membrane Glycoproteins; Prostaglandin Endoperoxides, Synthetic; Receptors, Cell Surface; Receptors, G-Protein-Coupled; Receptors, Prostaglandin; Receptors, Thromboxane; Respiration; Sulfonamides; Tetrazoles; Thienopyridines; Time Factors; Triazoles

Differential effects on human platelet function of thromboxane A2 (TXA2) synthetase inhibition singly and of TXA2 synthetase inhibition combined with TXA2/prostaglandin endoperoxide receptor antagonism were revealed, using ridogrel as a probe. Ridogrel combines selective TXA2 synthetase inhibition with TXA2/prostaglandin receptor antagonism in one molecule: in washed human platelets, the compound reduces the production of TXB2 (IC50 = 1.3 X 10(-8) M) and increases that of PGF2 alpha, PGE2, PGD2 from [14C]arachidonic acid. Additionally, at higher concentrations (Ki = 0.52 X 10(-6) M), it selectively antagonizes the breakdown of inositol phospholipids, subsequent to stimulation of TXA2/prostaglandin endoperoxide receptors with U 46619. The latter happens in a competitive way with fast receptor association-dissociation characteristics. At low concentrations (1 X 10(-9)-1 X 10(-7) M) producing single TXA2 synthetase inhibition, ridogrel reduces the collagen-induced formation of TXB2 by washed platelets, but enhances [32P]phosphatidic acid (PA) accumulation and [3H]5-hydroxytryptamine (5-HT) release. At higher concentrations (1 X 10(-6)-1 X 10(-5) M) which additionally block U 46619-induced [32P]PA accumulation, ridogrel inhibits the [32P]PA accumulation and release of [3H]5-HT by human platelets stimulated with collagen. These observations, corroborated by results obtained with OKY 1581, sulotroban, indomethacin and human serum albumin, suggest a causal role for prostaglandin endoperoxides in the stimulation by TXA2 synthetase inhibition of platelet reactions to collagen. They reinforce the concept that TXA2 synthetase inhibition-induced reorientation of cyclic endoperoxide metabolism, away from TXA2 into inhibitory prostanoids, requires additional TXA2/prostaglandin endoperoxide receptor antagonism to achieve optimal anti-platelet effects.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Blood Platelets; Collagen; Humans; In Vitro Techniques; Indomethacin; Inositol Phosphates; Methacrylates; Platelet Activating Factor; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Serotonin; Sulfonamides; Thromboxane A2; Thromboxane-A Synthase

1. Administration of arachidonic acid caused dose-dependent vasoconstriction in the isolated rat kidney perfused in situ with Krebs-Henseleit solution. 2. Inhibition of cyclo-oxygenase with indomethacin or meclofenamate reduced the renal vasoconstrictor effect of arachidonic acid. 3. The renal vasoconstrictor effect of arachidonic acid was unaffected by CGS-13080 at concentrations that effectively reduced thromboxane A2 (TxA2) synthesis by platelets and the kidney. 4. The endoperoxide/TxA2 receptor antagonist, SQ 29,548, abolished the renal vasoconstrictor effect of arachidonic acid and of U46619, an endoperoxide analogue. In contrast, SQ 29,548 did not affect the renal vasoconstrictor response to angiotensin II, prostaglandin E2 or F2 alpha. 5. These data suggest that the vasoconstrictor effect of arachidonic acid in the isolated kidney of the rat is mediated by its metabolites, including the prostaglandin endoperoxides.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Anti-Arrhythmia Agents; Arachidonic Acids; Bridged Bicyclo Compounds, Heterocyclic; Fatty Acids, Unsaturated; Hydrazines; In Vitro Techniques; Kidney; Male; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandin-Endoperoxide Synthases; Prostaglandins G; Prostaglandins H; Rats; Rats, Inbred Strains; Receptors, Prostaglandin; Receptors, Thromboxane; Sulfonamides; Thromboxane A2; Thromboxane-A Synthase; Thromboxanes; Vasoconstriction

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 16,16-Dimethylprostaglandin E2; Animals; Eicosanoids; Gastric Mucosa; Hydrogen Peroxide; Male; Prostaglandin Endoperoxides, Synthetic; Rats; Stomach; Stomach Ulcer; Sulfonamides

1. BW A868C, a novel compound, behaved as a simple competitive antagonist in a human washed platelet aggregation assay. Anti-aggregatory concentration-effect curves to BW 245C were displaced in a parallel manner. The shifts accorded with a Schild plot slope of unity and a pKB of 9.26. 2. Inhibition of platelet aggregation by prostaglandin D2 (PGD2) was antagonized with a similar potency, as were the relaxation effects of BW 245C and PGD2 in the rabbit jugular vein. BW A868C can, therefore, be classified as a DP-receptor antagonist. 3. Actions of BW A868C at other prostaglandin receptors (IP, EP1, EP2, TP and FP) were excluded at concentrations up to 1,000 times higher than the DP-receptor affinity. 4. Analyses of BW 245C- and PGD2-mediated effects were complicated by additional agonist receptor interactions which were revealed by BW A868C. In rabbit jugular vein a resistant phase of agonism was detectable, indicating that both agonists exerted effects through another receptor (possibly EP2). Also, PGD2, in addition to its anti-aggregatory effect on platelets, demonstrated a pro-aggregatory action in the presence of BW A868C. 5. The contractile effects of PGD2 in guinea-pig tracheal strips were resistant to 10 microM BW A868C indicating that they were not mediated through DP-receptors. 6. To our knowledge this is the first account of a well-classified competitive antagonist at the DP-receptor. Its potency and selectivity make it an important new tool in prostanoid receptor classification and identification.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Blood Platelets; Blood Vessels; Guinea Pigs; Humans; Hydantoins; In Vitro Techniques; Jugular Veins; Male; Muscle Contraction; Platelet Aggregation; Prostaglandin D2; Prostaglandin Endoperoxides, Synthetic; Rabbits; Receptors, Prostaglandin; Sulfonamides; Trachea

The stable PGI2-analogue iloprost and the TXA2-receptor antagonist sulotroban were investigated for possible cooperative effects on platelet function and experimental thrombus formation in guinea pigs and rats. Iloprost and sulotroban inhibit intravascular platelet aggregation in guinea pigs and rats induced by the stable endoperoxide U 46.619 and collagen, with iloprost being the more potent and (for collagen) more efficacious drug. Combinations of both compounds show synergistic or additive effects on in vivo platelet function. Thrombus formation in rats induced by vascular damage is strongly reduced by combining doses of iloprost and sulotroban (BM 13.177) which given alone are ineffective. These results suggest a cooperative enhancement of antiplatelet and antithrombotic effects for combinations of iloprost and sulotroban. In view of disadvantages of currently used platelet inhibitors this cooperativity may offer a new approach in antiplatelet therapy.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Cardiovascular Agents; Collagen; Drug Synergism; Epoprostenol; Fibrinolytic Agents; Guinea Pigs; Iloprost; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Rats; Sulfonamides; Thrombocytopenia; Thrombophlebitis; Thromboxanes

The stable PGI2-analogue iloprost and the TXA2-receptor antagonist sulotroban (BM 13177) were investigated for possible synergistic effects on platelet aggregation in human platelet rich plasma in vitro. Iloprost and sulotroban synergistically inhibited U 46619, collagen, and the second wave of ADP-induced platelet aggregation. Iloprost and sulotroban at concentrations showing little or no inhibition alone resulted, in combination, in marked or complete inhibition of U 46619 or collagen induced aggregation. Combination of iloprost 10(-10) M, which had no effect on the concentration-response curve (CRC) to U 46619, with sulotroban 5 x 10(-6) M, which shifted the CRC to U 46619 by a factor of 3 to the right, resulted in a rightward shift of the U 46619 CRC by a factor of 4.5. To attain a 4.5-fold shift with either compound alone, a concentration of 5 x 10(-10) M iloprost or 10(-5) M sulotroban was required. A similar mutual enhancement of inhibitory effects was seen for combinations of the PGI2-analogue cicaprost (ZK 96.480) with sulotroban or the TXA2-receptor antagonist SQ 29548 with iloprost. When the TXA2-dependent part of collagen-induced aggregation was fully inhibited by sulotroban, the concentrations of iloprost necessary for 90% inhibition were reduced by a factor of 2.5 - 3. In the presence of acetylsalicylic acid, the synergistic action of sulotroban and iloprost was reduced and merely additive effects against U 46619-induced platelet aggregation were found, suggesting that the release of endogenous TXA2 plays an important role for the synergistic effect of the two compounds. The combination of a PGI2-analogue and a TXA2-antagonist may lead to a safer and more effective control of platelet activation than with either compound alone.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Aspirin; Bridged Bicyclo Compounds, Heterocyclic; Collagen; Drug Synergism; Epoprostenol; Fatty Acids, Unsaturated; Humans; Hydrazines; Iloprost; In Vitro Techniques; Platelet Aggregation; Platelet Aggregation Inhibitors; Prostaglandin Endoperoxides, Synthetic; Prostaglandins, Synthetic; Receptors, Prostaglandin; Receptors, Thromboxane; Sulfonamides

Substances shifting the balance between thromboxane (TX) and prostacyclin (PGI2) in favour of PGI2 could be of therapeutic value for arrhythmia treatment. This seems to be independent of the pathogenetic mechanism. The TX receptor antagonist BM 13177, the lipoxygenase inhibitor esculetin were effective in ouabain and aconitine induced arrhythmias while the PGI2 formation stimulating substance nafazatrom was only effective in aconitine induced arrhythmia. BM 13177 and esculetin could also counteract the arrhythmogenic effect of PAF. TX synthetase inhibition by HOE 944 was ineffective or partially effective, resp..

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Anti-Arrhythmia Agents; Arachidonic Acids; Arrhythmias, Cardiac; Epoprostenol; Guinea Pigs; Heart; Iloprost; Ouabain; Prostaglandin Endoperoxides, Synthetic; Reference Values; Sulfonamides

In order to tag the TXA2/PGH2 receptor of human platelets, we synthesized azido-BSP (= 4-[2-(4-azido-benzenesulfonylamino)-ethyl]phenoxyacetic acid), a photolabile derivative of the specific TXA2/PGH2 receptor antagonist sulotroban (= BM 13.177). If protected from UV light, azido-BSP competitively inhibited the shape change of human washed platelets stimulated by the TXA2 mimetic U 46619. Schild analysis revealed a pA2 = 6.7 (apparent KD = 0.2 mumol/l). Irreversible inhibition of the U 46619-induced platelet activation was achieved by irradiating for 5 min with UV light of 254 nm a platelet suspension containing azido-BSP. After subsequent washing, the platelets were stimulated with U 46619, ADP or PAF. Under these conditions azido-BSP inhibited the shape change, aggregation and [3H]serotonin release induced by U 46619 but not the shape change induced by ADP or PAF. The concentrations of azido-BSP which blocked the U 46619-induced [3H]serotonin release and the aggregation were 0.5 mumol/l and 1.0 mumol/l, respectively, whereas even 50.0 mumol/l of azido-BSP only partially inhibited the U 46619-stimulated shape change. Obviously, increasing numbers of thromboxane receptors have to be blocked in order to inhibit the [3H]serotonin release, the aggregation and the shape change. Even at an azido-BSP concentration equal to 250 times the apparent dissociation constant, enough receptor sites remained active to allow U 46619 to induce the shape change. In sulotroban was added prior to irradiation, the blocking effect of azido-BSP decreased with increasing concentrations of sulotroban. These results show that azido-BSP is a specific and high affinity ligand of the TXA2/PGH2 receptor and that it covalently links to the receptor under irradiation. Azido-BSP is a new tool to identify and characterize the TXA2/PGH2 receptor.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Affinity Labels; Azides; Blood Platelets; Fibrinolytic Agents; Humans; Kinetics; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins H; Receptors, Prostaglandin; Receptors, Thromboxane; Sulfonamides; Thromboxane A2

The pro-ulcerogenic actions of the thromboxane mimetic, U-46619 on the rat gastric mucosa have been investigated, utilizing a novel technique which allows administration directly into the left gastric artery. Local intra-arterial infusion of U-46619 (100-500 ng/kg/min for 10 min) induced dose-dependent macroscopic damage in both the corpus and antral regions, characterized as vasocongestion, disruption and haemorrhage, with deep penetrating ulcers in the antral mucosa. Vascular congestion, epithelial cell and glandular disruption was observed histologically in both corpus and antral regions. Local intra-arterial infusion of lower doses of U-46619 (25-100 ng/kg/min) significantly disrupted the mucosa in the presence of 10% ethanol in a concentration which itself did not induce macroscopic damage. The damaging actions of U-46619 were substantially reduced by pretreatment with the thromboxane-receptor antagonist, BM 13,177 (5mg/kg i.v.) or 16,16-dimethyl PGE2 (5 micrograms/kg s.c.). These findings support the role of endogenous thromboxane A2 as a local mediator of gastric injury.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; 16,16-Dimethylprostaglandin E2; Animals; Arteries; Drug Synergism; Ethanol; Gastric Mucosa; Infusions, Intra-Arterial; Male; Prostaglandin Endoperoxides, Synthetic; Rats; Rats, Inbred Strains; Receptors, Prostaglandin; Receptors, Thromboxane; Stomach; Stomach Ulcer; Sulfonamides

The purpose of this study was to evaluate the pharmacology and pharmacodynamics of the thromboxane receptor antagonists, BM 13.177 and BM 13.505, for prevention of U 46619-induced sudden death in anesthetized male Sprague-Dawley rats. The injection of U 46619 (100 micrograms/kg i.v.) produced sudden death typically between 5 and 15 min. Administration of 0.01 mg/kg BM 13.505 (i.v.) 0.1 h prior to the U 46619 challenge did not protect against sudden death, while doses of 0.03 mg/kg or greater protected completely (100% survival). A dose of 1 mg/kg BM 13.505 afforded protection to 2 h but not to 24 h, while a single dose of 30 mg/kg administered 24 h prior to the U 46619 challenge provided complete protection against the lethal event. Administration of BM 13.177 (30 mg/kg, i.v. or i.p.) blocked the effects of U 46619 when administered 0.1 h before the challenge, but not when given 2 or 3 h prior to the challenge with U 46619. Pretreatment with indomethacin did not block the effects of U 46619, indicating that formation of endogenous thromboxane does not play a major role in the lethal effects of U 46619, and that blockade of the lethal effects of U 46619 was specific for thromboxane receptor antagonists. These data demonstrate that BM 13.177 and BM 13.505 prevented sudden death produced by the injection of U 46619. At comparable doses of 30 mg/kg, the duration of action for BM 13.505 was was significantly greater than for BM 13.177. These data suggest that BM 13.177 and BM 13.505 may be useful for the investigation of diseases where thromboxane is involved.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Anti-Arrhythmia Agents; Death, Sudden; Dose-Response Relationship, Drug; Electrocardiography; Male; Phenylacetates; Prostaglandin Endoperoxides, Synthetic; Rats; Rats, Inbred Strains; Respiration; Sulfonamides; Thromboxanes

The balance between the eicosanoids seems to be involved in the biochemical regulation of cardiac rhythm. Under several pathophysiological conditions thromboxane (TX) could be an important factor for the genesis of cardiac arrhythmias. Shifting the balance between the arrhythmogenic TX and the antiarrhythmic prostacyclin (PGI2) in favour of PGI2 by substances influencing the arachidonic acid cascade might by useful for the treatment of arrhythmias. PAF induced toxic arrhythmogenicity seems to be mediated by TX. Therefore the suppression of TX influence can antagonize these arrhythmias, too.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Arrhythmias, Cardiac; Epoprostenol; Fatty Acids, Unsaturated; Guinea Pigs; Male; Platelet Activating Factor; Prostaglandin Endoperoxides, Synthetic; Rats; Rats, Inbred Strains; Sulfonamides; Thromboxanes

The specific effects of U 46619 (9,11-dideoxy,9 alpha-11 alpha-methanoepoxyprostaglandin F2 alpha), thromboxane A2-prostaglandin H2 (TxA2/PGH2) analogue, on human platelet shape change, myosin light-chain phosphorylation, serotonin release, fibrinogen receptor exposure, and platelet aggregation were measured and compared with binding of [3H]U 46619 to platelets. Shape change and myosin light-chain phosphorylation were found to be saturable and dose dependent, having effective concentration producing 50% of the maximum response (EC50) values of 0.035 +/- 0.005 and 0.057 +/- 0.021 microM, respectively (mean +/- SE). These two effects were competitively inhibited by specific antagonists of TxA2/PGH2 receptors (BM 13177, PTA-OH, and 1.PTA-OH) indicating that they are receptor mediated. Binding of [3H]U 46619 showed two components. Occupancy of high-affinity binding sites [dissociation constant (Kd) = 0.041 +/- 0.009 microM, maximum binding site (Bmax) = 19.4 +/- 5.3 fmol/10(7) platelets, with 1,166 +/- 310 sites/platelet; n = 12] correlated with platelet shape change and myosin light-chain phosphorylation. We propose that a second component with an apparent Kd of 1.46 +/- 0.47 microM (n = 12) represents a second, low-affinity site. Mean EC50 values for U 46619-induced serotonin release, platelet aggregation, and fibrinogen receptor exposure were 0.54 +/- 0.13. 1.31 +/- 0.34 and 0.53 +/- 0.21 microM, respectively. Therefore, the platelet release reaction was not directly correlated with occupancy of high-affinity receptors but could be related to the second binding component of U 46619. Fibrinogen receptor exposure and platelet aggregation caused by U 46619 appeared to be events mediated by the release of adenosine diphosphate from platelet-dense granules.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Blood Platelets; Humans; Kinetics; Myosin-Light-Chain Kinase; Platelet Aggregation Inhibitors; Platelet Membrane Glycoproteins; Prostaglandin Endoperoxides, Synthetic; Serotonin; Sulfonamides

The anti-aggregatory prostanoid, prostaglandin D2 (PGD2) does not completely inhibit ADP-induced aggregation of guinea-pig platelets and thus produces a bell-shaped dose-inhibition curve. The nature of this bell-shaped curve has now been investigated in guinea-pig platelet-rich plasma. Two selective thromboxane receptor antagonists, 13-aza-prostanoic acid (13-AZA; 16-64.4 microM) and BM 13.177 (5.9-29.8 microM), converted PGD2 to a full inhibitor of aggregation in a dose-related manner. The putative platelet PGD2 receptor antagonist, N-0164 (75 microM) also converted PGD2 to a full inhibitor of platelet aggregation. In contrast to 13-AZA and BM 13.177, higher concentrations of N-0164 (380 and 760 microM) caused a dose-related rightward shift of the PGD2 dose-inhibition curve. The thromboxane receptor antagonism of N-0164 was confirmed in studies in which the dose-aggregation curve to U-46619, a thromboxane mimetic, was competitively antagonized with a pA2 value of 4.67 and a slope of 1.13, comparable to that of 13-AZA. The results show that N-0164 acts as both a platelet PGD2 and thromboxane-receptor antagonist in both human and guinea-pig platelet-rich plasma. The results further indicate that PGD2 can interact at thromboxane receptors in guinea-pig platelets.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Diphosphate; Animals; Blood Platelets; Guinea Pigs; Male; Organophosphonates; Organophosphorus Compounds; Platelet Aggregation; Prostaglandin D2; Prostaglandin Endoperoxides, Synthetic; Prostaglandins D; Prostanoic Acids; Receptors, Cell Surface; Receptors, Prostaglandin; Receptors, Thromboxane; Sulfonamides

The pharmacokinetics and pharmacodynamic effect on platelet activation of a single 800 mg oral dose of BM 13.177 have been investigated in 8 male volunteers. BM 13.177 disappeared from plasma with a terminal elimination half-life of 0.85 h. 52% of the dose was excreted unchanged in urine. Assuming complete absorption, total clearance was calculated to be 741.3 ml/min and renal clearance to range from 310.4 to 396.9 ml/min. The pharmacodynamic studies were performed ex vivo/in vitro in studies were performed ex vivo/in vitro in platelets stimulated either with methyl mercury chloride or with U 46619. Methyl mercury chloride is a platelet activator that requires TXA2 formation from endogenous arachidonic acid, whereas U 46619 is a stable PGH2 analogue and thromboxane mimetic at the platelet TXA2/PGH2 receptor. A close correlation between the plasma concentration-time profile of BM 13.177 and inhibition of platelet shape change or aggregation was demonstrated.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adult; Blood Platelets; Fibrinolytic Agents; Humans; Kinetics; Male; Methylmercury Compounds; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Serotonin; Sulfonamides; Time Factors

The antiaggregatory and antisecretory effects of two newly developed thromboxane receptor antagonists, BM-13,177 and SQ-29,548, were studied in an in vivo model of platelet activation. Arterial platelet count and whole blood ATP concentrations were measured continuously on-line in the arterial blood of anesthetized rabbits. Injections of collagen decreased peripheral platelet count by 25% of initial value. ATP concentrations increased 50 to 100 nM during collagen challenge. SQ-29,548 and BM-13,177 dose-dependently reduced platelet loss to about 50% of that observed in vehicle treated animals. Injection of arachidonic acid (AA) or 9,11-methanoepoxy-PGH2 resulted in sudden death of the animals associated with a 67 to 69% decrease in platelet count and a marked release of ATP. Pretreatment with SQ-29,548 or BM-13,177 increased survival rates from 0 to 100%, and reduced or totally inhibited ATP secretion and decreases in platelet count. In contrast, the thromboxane synthetase inhibitor, dazoxiben, was effective in inhibiting AA induced sudden death, but was without any effect when 9,11-methanoepoxy-PGH2 was used as the challenging agent. We conclude that SQ-29,548 and BM-13,177 are effective in antagonizing the effects of subsequent conversion to thromboxane A2. BM-13,177 and SQ-29,548 are generally considered as specific antagonists of endoperoxide/thromboxane receptors on platelets and smooth muscles in vitro. Preliminary clinical studies with BM-13,177 showed a marked inhibition of ex vivo platelet aggregation in human volunteers and patients (18, 19). These reports encourage further study of thromboxane receptor antagonists as effective anti-thrombotic drugs in the experimental and clinical setting.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenosine Triphosphate; Animals; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Blood Pressure; Bridged Bicyclo Compounds, Heterocyclic; Collagen; Fatty Acids, Unsaturated; Fibrinolytic Agents; Hydrazines; Imidazoles; Kinetics; Male; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Rabbits; Receptors, Prostaglandin; Receptors, Thromboxane; Sulfonamides; Thromboxane-A Synthase; Thromboxanes

The main cyclooxygenase-dependent arachidonic acid derivatives produced by monocytes and macrophages have been shown to be thromboxane A2 and prostaglandin E2. The immunomodulatory effects of thromboxane A2 were examined using a specific thromboxane synthase inhibitor (dazoxiben), a thromboxane A2 analog (U46619), and a thromboxane A2 receptor blocker (BM13.177). Dazoxiben inhibited lymphocyte proliferation in response to mitogens (PHA and OKT3), but also reoriented cyclic endoperoxide metabolism towards the production of prostaglandin E2. Prostaglandin E2 has been shown previously to inhibit mitogen-induced lymphocyte proliferation. U46619, a stable thromboxane A2 analog, slightly enhanced lymphocyte responses to mitogens in the presence of dazoxiben and in the presence of a cyclooxygenase inhibitor (indomethacin). This occurred at concentrations of U46619 which are probably supraphysiological in view of the short half-life of natural thromboxane A2. Finally, the thromboxane A2 receptor blocker BM13.177 did not have any effect on mitogen-induced lymphocyte proliferation. It is concluded that thromboxane A2 has no or minimal modulatory effects on lymphocyte proliferative responses to mitogens and that the effect of thromboxane A2 synthase inhibition is rather due to reorientation of cyclic endoperoxide metabolism, resulting in increased prostaglandin E2 production.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Antibodies, Monoclonal; Cyclooxygenase Inhibitors; Dinoprostone; Humans; Imidazoles; Indomethacin; Lymphocyte Activation; Lymphocytes; Phytohemagglutinins; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Prostaglandins E; Receptors, Prostaglandin; Receptors, Thromboxane; Sulfonamides; Thromboxane A2; Thromboxane-A Synthase; Thromboxanes

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Aspirin; Blood Platelets; Collagen; Humans; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandins H; Serotonin; Sulfonamides; Thrombin; Thromboxane A2; Thromboxanes

BM 13.177 (0.1-100 microM) produced a concentration-dependent reduction of the platelet shape change, aggregation and (3H)serotonin release induced by the stable PGH2 analogues U 46619 and U 44069 or exogenous and endogenous arachidonic acid, the latter mobilized by hydrogen peroxide or collagen. BM 13.177 (100 microM) did not inhibit the primary platelet activation by ADP, serotonin, thrombin or collagen in washed platelets or citrated PRP that had been pre-treated with ASA (acetylsalicylic acid). The formation of TXB2 triggered by 100 microM hydrogen peroxide or 10 microM arachidonic acid was not influenced by BM 13.177 (10 microM). In spiral strips of rat and rabbit aorta, BM 13.117 markedly reduced the vasoconstriction triggered by U 46619 and PGF2 alpha. BM 13.177 did not inhibit the K+-or noradrenaline-induced constriction. The concentration/response curves of the U 46619-stimulated platelet shape change and of the vasoconstriction induced by U 46619 and PGF2 alpha were shifted in parallel to the right by BM 13.177, implicating a competitive antagonism. The pAx values were about the same in these models which indicates that BM 13.177 does not differentiate between the thromboxane receptors in human platelets and rabbit aorta. In mice, BM 13.177 prevented in a dose-dependent fashion the sudden death and the symptoms of respiratory depression and shock induced by i.v. injections of U 46619 or arachidonic acid. BM 13.177 did not exert partial agonist activity in the in vitro and in the animal models.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aorta; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Dinoprost; Humans; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Prostaglandins F; Rats; Receptors, Cell Surface; Receptors, Prostaglandin; Receptors, Thromboxane; Serotonin; Sulfonamides; Thromboxanes; Vasoconstriction

BM 13.177 (4-[2-(benzenesulfonamido)-ethyl]-phenoxyacetic acid) is a representative of a new class of sulfonamidophenylcarboxylic acids which possess platelet-inhibitory and anti-thrombotic activity and inhibits the contraction of rabbit aorta stimulated by PG endoperoxides and TXA2. BM 13.177 5 mg/kg body weight p.o. protected rabbits from arachidonate-induced sudden death and greater than or equal to 10 mg/kg dose-dependently reduced the experimental thrombus formation induced in the rabbit aorta by perivascular administration of silver nitrate. In guinea-pigs, the collagen-induced bronchoconstriction was inhibited in a dose- and time-dependent fashion. The formation of TXA2 and the TXA2-induced platelet aggregation and smooth muscle contraction are probably crucial events in these experimental models. The protective effect of BM 13.177 may, therefore, be due to the TXA2-antagonizing effect of BM 13.177, which has been conclusively demonstrated in human platelets (PATSCHEKE and STEGMEIER, Thrombosis Res., 33, 277-288 (1984). The antagonism of TXA2 is supported by the observation that BM 13.177 also specifically inhibits the contraction of isolated arterial strips from rabbits which were stimulated with the thromboxane A2 mimetic U 46619. Schild-plot with a slope close to unity suggests a competitive type of antagonism. BM 13.177 exhibited neither anti-inflammatory nor ulcer-inducing activity of cyclooxygenase inhibitors. Furthermore it did not block the TXB2 formation in spontaneously clotting blood from rabbits and did not inhibit the release of prostacyclin-like activity from rabbit aortas. The lack of toxicological effects in long-term toxicity studies in rat and dog, together with the absence of objective and subjective side effects in the first human studies have encouraged us to initiate clinical trials in order to evaluate the therapeutic benefit of this new approach in humans.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Epoprostenol; Female; Fibrinolytic Agents; Guinea Pigs; Male; Muscle Contraction; Muscle, Smooth, Vascular; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Rabbits; Sulfonamides; Thrombosis; Thromboxane A2; Thromboxane B2; Thromboxanes

The mode of action of BM 13.177 (4-[2-(benzenesulfonamido)-ethyl] phenoxyacetic acid), a new anti-aggregating and anti-thrombotic agent, was studied in human washed platelets and citrated PRP. With ASA-treated platelets, BM 13.177 (0.1 - 100 microM) did not inhibit the shape change and the aggregation induced by ADP, serotonin, adrenaline, thrombin, or collagen. Therefore, BM 13.177 is neither an antagonist of ADP, serotonin, adrenaline, thrombin, or collagen nor a common pathway inhibitor like PGE1, or an inhibitor of the platelet interactions during aggregation. However, BM 13.177 (greater than or equal to 0.1 microM) produced a dose-dependent reduction of shape change, aggregation and release of [3H]serotonin induced by the stable PGH2 analogues U 46619 and U 44069 in ASA-treated platelets or ASA-treated citrated PRP. In untreated platelets, BM 13.177 inhibited platelet activation by U 46619 or U 44069 and by exogenous arachidonic acid or by endogenous arachidonic acid mobilized by hydrogen peroxide. Consequently, the ADP- and adrenaline-induced secondary aggregation and [3H]serotonin release in citrated PRP and the major effects of collagen were also inhibited. In washed platelets treated with 10 microM arachidonic acid or 100 microM hydrogen peroxide, the formation of TXB2 was not inhibited by 10 microM BM 13.177. However, the TXB2 formation after stimulation with 1,200 microM hydrogen peroxide was partially reduced by BM 13.177 to the same extent as by PGE1. This reduction may be due to the absence of a secondary release of arachidonic acid from phospholipids if the platelets were prevented from activation by BM 13.177 or PGE1. Arachidonic acid and hydrogen peroxide also induced the shape change, aggregation and release of washed platelets when thromboxane formation was inhibited by dazoxiben. Under these conditions, BM 13.177 was able to abolish the platelet response which was due to accumulating prostaglandin endoperoxides. These results show that BM 13.177 acts as a selective antagonist of TXA2 and prostaglandin endoperoxides. Its inhibitory effect on platelet function does not depend on an inhibition of either the primary release of arachidonic acid or the activities of cyclooxygenase or thromboxane synthetase.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Arachidonic Acid; Arachidonic Acids; Blood Platelets; Collagen; Dose-Response Relationship, Drug; Epinephrine; Humans; Hydrogen Peroxide; Imidazoles; Indomethacin; Platelet Aggregation; Prostaglandin Endoperoxides, Synthetic; Prostaglandins E; Serotonin; Sulfonamides; Thromboxane A2; Thromboxane B2; Thromboxanes