sq-29548 and sulotroban

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

In this study we examined the thromboxane A(2)(TXA(2)) receptor antagonist property of BM-531 (N-tert -butyl- N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, on platelet function. The drug affinity for human washed platelet TXA(2)receptors labelled with [(3)H]SQ-29,548 has been determined (IC50: 0.0078 microM) and demonstrated to be higher than sulotroban (IC50: 0.93 microM) and SQ-29,548 (IC50: 0.021 microM). The antiaggregatory potency has been confirmed since we demonstrated that BM-531 prevented platelet aggregation in human citrated platelet-rich plasma induced by arachidonic acid (600 microM) (ED100: 0.125 microM), U-46619, a stable TXA(2)agonist (1 microM) (ED50: 0.482 microM) and collagen (1 microg mL(-1)) (% of inhibition: 42.9% at 10 microM) and inhibited the second wave of ADP (2 microM). Moreover, when BM-531 was incubated in whole blood from healthy donors, the closure time measured by the recently developed platelet function analyser (PFA-100(trade mark)) was significantly prolonged. These results suggest that BM-531 can be regarded as a novel non-carboxylic TXA(2)antagonist with a powerful antiplatelet potency.

Topics: Adenosine Diphosphate; Binding, Competitive; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Dose-Response Relationship, Drug; Fatty Acids, Unsaturated; Hemostasis; Humans; Hydrazines; Inhibitory Concentration 50; Molecular Structure; Platelet Aggregation; Platelet Aggregation Inhibitors; Receptors, Thromboxane; Sulfonamides; Sulfonylurea Compounds; Time Factors; Torsemide

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

1. Exposure of human resuspended platelets in vitro for 30 min to the potent thromboxane A2 (TP)-receptor blocking drug GR32191, followed by its removal by dilution-dissociation, reduced the degree of subsequent binding to 2 nM [3H]-GR32191 by almost 50%. Exposure for longer periods (60 min) led to a further reduction. However, no change in the Kd of the radioligand was observed. 2. This effect of GR32191 could not be explained by persistent binding of drug to platelets since a dilution-dissociation stage, designed to remove all drug, was included prior to measurement of binding. 3. Using an alternative TP-receptor radioligand, [3H]-SQ29,548, to monitor receptor number, a reduction in Bmax was observed after GR32191 pre-treatment; the Kd value of the radioligand remained unchanged. 4. The effect was not a common property of TP-receptor blocking drugs since pre-exposure of platelets in vitro for 30 min to BM13.177 or SQ29,548 did not produce a fall in subsequent Bmax to [3H]-SQ29,548. 5. While the mechanism behind this apparent down-regulation of platelet TP-receptor is unknown, it may explain the long duration of action of GR32191 upon platelets in man which persists in the absence of detectable drug in the plasma.

Topics: Binding, Competitive; Biphenyl Compounds; Blood Platelets; Bridged Bicyclo Compounds, Heterocyclic; Down-Regulation; Fatty Acids, Unsaturated; Heptanoic Acids; Humans; Hydrazines; In Vitro Techniques; Ligands; Platelet Aggregation Inhibitors; Radioligand Assay; Receptors, Thromboxane; Sulfonamides

1. Canine jugular and femoral veins were studied to determine the possible importance of thromboxane (TXA2) and prostaglandin endoperoxides (prostaglandin H2, PGH2) in mediating bradykinin(BK)-induced contraction. 2. Isolated vein rings incubated in modified Krebs solution contracted to TXA2/PGH2 analogs SQ26655 and U44069 with potency of contraction exceeding that for BK. The potency ranking for both veins was SQ26655 greater than U44069 greater than BK greater than PGF2 alpha greater than TXB2 much greater than PGD2. 3. The cyclo-oxygenase inhibitors indomethacin (3 x 10(-7) M) and flufenamic acid (10(-5) M) reduced BK contractions without affecting those induced by noradrenaline (NA). 4. TXA2/PGH2 receptor antagonists SQ29548 (10(-8) M) and BM13177 (10(-6) M) strongly inhibited BK-induced tension. The action of antagonists was reversible with negligible influence on NA-elicited contraction. Selective removal of endothelium had no effect on BK-induced contraction or the action of the antagonists. 5. The thromboxane synthase inhibitors dazoxiben (10(-4) M) and CGS 12970 (10(-5) M) had no significant inhibitory effect on BK-induced tension. 6. These results suggest that in canine jugular and femoral vein, the action of BK is largely dependent upon stimulation of the cyclo-oxygenase pathway to produce PGH2 and possibly TXA2, which can activate a smooth muscle TXA2/PGH2 receptor to elicit vasoconstriction.

Topics: Animals; Bradykinin; Bridged Bicyclo Compounds, Heterocyclic; Dogs; Endothelium, Vascular; Fatty Acids, Unsaturated; Female; Hydrazines; Imidazoles; In Vitro Techniques; Muscle Contraction; Muscle, Smooth, Vascular; Prostaglandin Endoperoxides; Prostaglandin Endoperoxides, Synthetic; Prostaglandin H2; Prostaglandin-Endoperoxide Synthases; Prostaglandins H; Pyridines; Receptors, Prostaglandin; Sulfonamides; Thromboxane-A Synthase; Thromboxanes; Veins

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

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

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