Design, synthesis and biological evaluation of a sulfonylcyanoguanidine as thromboxane A2 receptor antagonist and thromboxane synthase inhibitor

The synthesis and the structure of N-isopropyl-N'-[2-(3'-methylphenylamino)-5-nitrobenzenesulfonyl] urea (14) was drawn from two thromboxane A2 receptor antagonists structurally related to torasemide. Compound 14 showed an IC50 value of 22 nM for the thromboxane A2 (TXA2) receptor of human washed platelets. Compound 14 prevented platelet aggregation induced by arachidonic acid (0.6 mM) and U-46619 (1 microM) with an IC50 value of 0.45 and 0.15 microM, respectively. Moreover, 14 relaxed the rat isolated aorta and guinea-pig trachea precontracted by U-46619, a TXA2 agonist. Its efficacy (IC50) was 20.4 and 5.47 nM, respectively. Finally, 14 (1 microM) completely inhibited TXA2 synthase of human platelets. The pKa value and the crystallographic data of 14 were determined and used to propose an interaction model between the TXA2 antagonists related to torasemide and their receptor.     

Pharmacological actions of ICI 180080, a novel thromboxane receptor antagonist

ICI 180080 (5(Z)-7-[2,2-dimethyl-4-(2-hydroxyphenyl)-1,3-dioxan-cis-5-yl] heptenoic acid) potently inhibited contractions of rat and rabbit aortae and guinea-pig trachea elicited by 11,9-epoxymethano PGH2 (U-46619). This antagonism was selective because contractions of aortae to noradrenaline and 5-hydroxytryptamine and trachea to histamine were not antagonized by ICI 180080. Schild analysis of data obtained from experiments on rabbit aortae indicated that this thromboxane receptor antagonism was competitive (pA2 = 7.50, slope = 1.07). Addition of ICI 180080 to human platelet-rich plasma caused dose-related inhibition of U-46619-induced platelet aggregation. This modification of platelet aggregation was not associated with inhibition of thromboxane synthetase, cyclo-oxygenase or lipoxygenase. ICI 180080 did not modify the primary phase of ADP-induced aggregation of human platelets neither did it affect the platelet inhibitory activity of prostacyclin. When dosed orally to anaesthetized guinea-pigs, ICI 180080 (5-50 mg kg-1) caused dose-related inhibition of U-46619-evoked bronchoconstriction. We conclude that ICI 180080 is a potent, selective, competitive, orally active thromboxane antagonist.     

The thromboxane A2/prostaglandin endoperoxide receptor antagonist activity of CV-4151, a thromboxane A2 synthetase inhibitor

The thromboxane A2/prostaglandin endoperoxide (TXA2/PGH2) receptor antagonist activity of CV-4151, a potent TXA2 synthetase inhibitor, was examined. CV-4151 inhibited guinea pig and human platelet aggregation induced by U-44069 with IC50 values of 1.2 +/- 0.3 X 10(-5) and 1.9 +/- 0.4 X 10(-5) M, respectively, and inhibited the specific binding of [3H]U-46619 to washed guinea pig and human platelets with IC50 values of 1.2 +/- 0.3 X 10(-6) and 5.1 +/- 1.0 X 10(-6) M, respectively. CV-4151 competitively inhibited the contraction of rabbit aortic strips induced by U-44069 with a pA2 value of 5.90. In experiments in mice in vivo, CV-4151 (1 and 10 mg/kg i.v.) significantly inhibited the thrombocytopenia induced by U-44069 in a dose-dependent manner. These results show that CV-4151 has a distinct TXA2/PGH2 receptor antagonist effect, and that this effect together with its inhibition of TXA2 synthetase could be important for the pharmacological action of this compound.     

Characterization of the thromboxane (TP-) receptor subtype involved in proliferation in cultured vascular smooth muscle cells of rat.

1. The effects of the thromboxane A2 (TxA2)-mimetic, U-46619, on the proliferation of vascular smooth muscle cells (VSMCs) were examined in a clonal smooth muscle cell line, A10, which was derived from foetal rat aorta. 2. [3H]-U-46619 bound to A10 cells of passages 18-20 (p18-20) with two classes of sites. The high affinity site showed a Bmax of 3.0 +/- 1.8 fmol mg-1 protein with a KD value 1.0 +/- 0.1 nM, while the low affinity site showed a Bmax of 43.0 +/- 6.0 fmol mg-1 protein and KD value of 129.0 +/- 7.9 nM. However, [3H]-U-46619 bound to A10 cells from passages 28-30 (p28-30) at a single class of site with a Bmax 111.0 +/- 9.0 fmol mg-1 protein and a KD value of 175.4 +/- 22.0 nM. 3. Cinnamophilin and SQ29548 inhibited specific [3H]-U-46619 binding to p18-20 A10 cells in a concentration-dependent manner with Ki values of 390.0 +/- 3.2 and 4.6 +/- 1.0 nM, respectively at a high affinity site, and 2.6 +/- 0.2 microM and 310.0 +/- 6.4 nM, respectively at the low affinity site. 4. U-46619 produced isometric contractions of rat aorta in a concentration-dependent manner with an EC50 7.0 +/- 1.2 nM. Cinnamophilin and SQ29548 antagonized U-46619-induced aortic contractions with pA2 values 6.3 +/- 0.1 and 8.2 +/- 0.2, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacology of the thromboxane receptor antagonist and thromboxane synthase inhibitor BM-531

BM-531 (N-tert-butyl-N'-[(2-cyclohexylamino-5-nitrobenzene)sulfonyl]urea), a torasemide derivative, is a novel noncarboxylic thromboxane receptor antagonist and thromboxane synthase inhibitor. Indeed, its affinity for human washed platelet TXA2 receptors labeled with [3H]SQ-29548 (IC50 = 0.0078 microM) is higher than sulotroban (IC50 = 0.93 microM) and SQ-29548 (IC50 = 0.021 microM). Moreover, BM-531 is characterized by a potent antiaggregatory property. Indeed, on one hand, in human citrated platelet-rich plasma BM-531 prevents platelet aggregation induced by arachidonic acid (600 microM) (ED100 = 0.125 microM), U-46619, a stable TXA2 agonist (1 microM) (ED50 = 0.482 microM) or collagen (1 microgram/mL) (percentage of inhibition: 42.9% at 10 microM) and inhibits the second wave of ADP (2 microM)-induced aggregation. On the other hand, when BM-531 is incubated in whole blood from healthy donors, the closure time measured by the recently developed platelet function analyser (PFA-100) is significantly prolonged. In addition, at the concentrations of 10 and 1 microM, BM-531 totally prevents the production of TXB2 by human platelets activated by arachidonic acid. Finally, at 10 microM, BM-531 significantly prevents rat fundus contractions induced by U-46619 but not by prostacyclin. These results suggest that BM-531, which is devoid of the diuretic property of torasemide, can be regarded as a promising antiplatelet agent.     

Pharmacological activity of (-)-discretamine, a novel vascular alpha-adrenoceptor and 5-hydroxytryptamine receptor antagonist, isolated from Fissistigma glaucescens.

1. The pharmacological activity of (-)-discretamine, isolated from Fissistigma glaucescens, was determined in rat isolated thoracic aorta, cardiac tissues and ventricular myocytes and guinea-pig isolated trachea. 2. (-)-Discretamine was found to be an alpha 1-adrenoceptor blocking agent in rat thoracic aorta as revealed by its competitive antagonism of noradrenaline (pA2 = 7.20 +/- 0.10)- or phenylephrine (pA2 = 7.60 +/- 0.09)-induced vasoconstriction. It was as potent as phentolamine (pA2 = 7.51 +/- 0.10), but was more potent than yohimbine (pA2 = 6.18 +/- 0.06). Removal of endothelium significantly increased the antagonistic potency of (-)-discretamine on noradrenaline (pA2 = 7.52 +/- 0.09)- or phenylephrine (pA2 = 7.90 +/- 0.09)-induced vasoconstriction. 3. (-)-Discretamine was also an alpha 2-adrenoceptor blocking agent (pA2 = 6.30 +/- 0.15) and a 5-hydroxytryptamine antagonist (pA2 = 6.87 +/- 0.06), both in rat aorta denuded of endothelium. 4. (-)-Discretamine protected alpha-adrenoceptors from alkylation by the irreversible blocking agent, phenoxybenzamine. 5. [3H]-inositol monophosphate formation caused by noradrenaline (3 microM) in rat thoracic aorta was suppressed by (-)-discretamine (10 and 30 microM) and prazosin (3 microM). 6. A high concentration of (-)-discretamine (30 microM) did not affect the contraction induced by the thromboxane receptor agonist U-46619, prostaglandin F2 alpha (PGF2 alpha), angiotensin II, high K+ or endothelin in rat aorta denuded of endothelium. Neither cyclic AMP nor cyclic GMP content of rat thoracic aorta was changed by (-)-discretamine.(ABSTRACT TRUNCATED AT 250 WORDS)     

Pharmacological characteristics of liriodenine, isolated from Fissistigma glaucescens, a novel muscarinic receptor antagonist in guinea-pigs.

1. The pharmacological activities of liriodenine, isolated from Fissistigma glaucescens, were determined in isolated trachea, ileum and cardiac tissues of guinea-pigs. 2. Liriodenine was found to be a muscarinic receptor antagonist in guinea-pig trachea as revealed by its competitive antagonism of carbachol (pA2 = 6.22 +/- 0.08)-induced smooth muscle contraction. It was slightly more potent than methoctramine (pA2 = 5.92 +/- 0.05), but was less potent than atropine (pA2 = 8.93 +/- 0.07), pirenzepine (pA2 = 7.02 +/- 0.09) and 4-diphenylacetoxy-N-methylpiperidine (4-DAMP, pA2 = 8.72 +/- 0.07). 3. Liriodenine was also a muscarinic antagonist in guinea-pig ileum (pA2 = 6.36 +/- 0.10) with a pA2 value that closely resembled that obtained in the trachea. 4. Liriodenine was 10 fold less potent in atrial preparations (left atria, pA2 = 5.24 +/- 0.04; right atria, pA2 = 5.35 +/- 0.09 and 5.28 +/- 0.07 for inotropic and chronotropic effects, respectively) than in smooth muscle preparations. 5. High concentration of liriodenine (300 microM) partially depressed the contractions induced by U-46619, histamine, prostaglandin F2 alpha, neurokinin A, leukotriene C4 and high K+ in the guinea-pig trachea. The inhibitions were characterized by a rightward shift in the concentration-response curves with suppression of their maximal contraction. 6. High concentration of liriodenine (300 microM) did not affect U-46619- or neurokinin A-induced tracheal contraction in the presence of nifedipine (1 microM) or in Ca(2+)-free (containing 0.2 mM EGTA) medium. 7. Neither cyclic AMP nor cyclic GMP content of guinea-pig trachealis was changed by liriodenine (30-300 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Prostaglandin D2 interacts at thromboxane receptor-sites on guinea-pig platelets.

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.     

Differential effects of ganodermic acid S on the thromboxane A2-signaling pathways in human platelets.

Ganodermic acid S (GAS) [lanosta-7,9(11),24-triene-3beta,15alpha-diacetoxy-26-oic acid], isolated from the Chinese medicinal fungus Ganoderma lucidum (Fr.) Karst (Polyporaceae), exerted a concentration-dependent inhibition on the response of human gel-filtered platelets (GFP) to U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxyprostaglandin F2alpha), a thromboxane (TX) A2 mimetic. GAS at 2 microM inhibited 50% of cell aggregation. GAS at 7.5 microM inhibited 80% of Ca2+ mobilization, 40% of phosphorylation of myosin light chain and pleckstrin, 80% of alpha-granule secretion, and over 95% of aggregation. GAS also strongly inhibited U46619-induced diacylglycerol formation, arachidonic acid release, and TXB2 formation. An immunoblotting study of protein-tyrosine phosphorylation showed that GAS inhibited the formation of phosphotyrosine proteins at the steps involving the engagement of integrin alphaIIbbeta3 and aggregation. However, GAS did not inhibit U46619-induced platelet shape change or the inhibitory effect of U46619 on the prostaglandin E1-evoked cyclic AMP level in GFP. It is concluded that GAS inhibits platelet response to TXA2 on the receptor-Gq-phospholipase Cbeta1 pathway, but not on the receptor-G1 pathway.     

Pharmacology of L-655,240 (3-[1-(4-chlorobenzyl)-5-fluoro-3-methyl-indol-2-yl]2,2-dimethylpro pan oic acid); a potent, selective thromboxane/prostaglandin endoperoxide antagonist

L-655,240 (3-[1-(4-chlorobenzyl)-5-fluoro-3-methyl-indol-2-yl]2,2-dimethylpropa noic acid) has been studied in vitro on the guinea-pig tracheal chain, pulmonary artery and thoracic aorta ring and shown to be a potent, competitive antagonist of contractions induced by the prostaglandin endoperoxide analogue, U-44069 (pA2 values 8.0, 8.4 and 8.0 respectively). Selectivity on the guinea-pig trachea was indicated by non-competitive antagonism of contractions induced by prostaglandin D2 and minimal activity against contractions induced by leukotriene D4, prostaglandin F2 alpha, serotonin, histamine and acetylcholine. L-655,240 was a potent inhibitor of the aggregation of washed human platelets induced by U-44069 (IC50 value 7 X 10(-9) M) and inhibited aggregation of human platelet rich plasma induced by U-44069, U-46619, thromboxane A2 and collagen but not ADP or platelet activating factor. In vivo i.v. L-655,240 administered to guinea-pigs inhibited bronchoconstriction induced by i.v. U-44069 and arachidonic acid (ED50 values 0.09 and 0.23 mg kg-1) but not histamine, acetylcholine or serotonin. When administered to rhesus monkeys (3 and 10 mg/kg p.o.), L-655,240 inhibited ex vivo platelet aggregation induced by U-44069 but not ADP. It is concluded that L-655,240 is a potent, selective, orally active thromboxane/prostaglandin endoperoxide antagonist.     

Electromechanical characterization of cinnamophilin, a natural thromboxane A2 receptor antagonist with anti-arrhythmic activity, in guinea-pig heart

BACKGROUND AND PURPOSE:Cinnamophilin, a thromboxane A(2) receptor antagonist, has been identified as a prominent anti-arrhythmic agent in rat heart. This study aimed to determine its electromechanical and anti-arrhythmic effects in guinea-pig hearts. EXPERIMENTAL APPROACH: Microelectrodes were used to study action potentials in ventricular papillary muscles. Fluo-3 fluorimetric ratio and whole-cell voltage-clamp techniques were used to record calcium transients and membrane currents in single ventricular myocytes, respectively. Intracardiac electrocardiograms were obtained and the anti-arrhythmic efficacy was determined from isolated perfused hearts. KEY RESULTS: In papillary muscles, cinnamophilin decreased the maximal rate of upstroke (V(max)) and duration of action potential, and reduced the contractile force. In single ventricular myocytes, cinnamophilin reduced Ca(2+) transient amplitude. Cinnamophilin decreased the L-type Ca(2+) current (I(Ca,L))(IC(50)=7.5 microM) with use-dependency, induced a negative shift of the voltage-dependent inactivation and retarded recovery from inactivation. Cinnamophilin also decreased the Na(+) current (I(Na)) (IC(50)=2.7 microM) and to a lesser extent, the delayed outward (I(K)), inward rectifier (I(K1)), and ATP-sensitive (I(K,ATP)) K(+) currents. In isolated perfused hearts, cinnamophilin prolonged the AV nodal conduction interval and Wenckebach cycle length and the refractory periods of the AV node, His-Purkinje system and ventricle, while shortening the ventricular repolarization time. Additionally, cinnamophilin reduced the occurrence of reperfusion-induced ventricular fibrillation. CONCLUSIONS AND IMPLICATIONS: These results suggest that the promising anti-arrhythmic effect and the changes in the electromechanical function induced by cinnamophilin in guinea-pig heart can be chiefly accounted for by inhibition of I(Ca,L) and I(Na).     

Pharmacological profile and therapeutic potential of BM-573, a combined thromboxane receptor antagonist and synthase inhibitor

BM-573 (N-terbutyl-N'-[2-(4'-methylphenylamino)-5-nitro-benzenesulfonyl]urea), a torsemide derivative, is a novel non-carboxylic dual TXA2 synthase inhibitor and receptor antagonist. The pharmacological profile of the drug is characterized by a higher affinity for the thromboxane receptor than that of SQ-29548, one of the most powerful antagonists described to date, by a complete prevention of human platelet aggregation induced by arachidonic acid at a lower dose than either torsemide or sulotroban, and by a significantly prolonged closure time measured by the platelet function analyser (PFA-100). Moreover, at the concentrations of 1 and 10 microM, BM-573 completely prevented production of TXB2 by human platelets activated by 0.6 mM of arachidonic acid. BM-573 prevents rat fundus contraction induced by U-46619 but not by prostacyclin or other prostaglandins. Despite possessing a chemical structure very similar to that of a diuretic torsemide, BM-573 has no diuretic activity. BM-573 does not prolong bleeding time and, unlike some of the other sulfonylureas, has no effect on blood glucose levels. In vivo, BM-573 appears to have antiplatelet and antithrombotic activities since it reduced thrombus weight and prolonged the time to abdominal aorta occlusion induced by ferric chloride. BM-573 also relaxed rat aorta and guinea pig trachea precontracted with U-46619. In pigs, BM-573 completely antagonized pulmonary hypertensive effects of U-46619 and reduced the early phase of pulmonary hypertension in models of endotoxic shock and pulmonary embolism. Finally, BM-573 protected pigs from myocardial infarction induced by coronary thrombosis. These results suggest that BM-573 should be viewed as a promising therapeutic agent in the treatment of pulmonary hypertension and syndromes associated with platelet activation.     

U-46619, a selective thromboxane A2 mimetic, inhibits the release of endogenous noradrenaline from the rat hippocampus in vitro

Possible roles of thromboxane A2 (TXA2) in the release mechanism of hippocampal noradrenaline (NA) were examined in vitro. Slices or crude synaptosomes prepared from the rat hippocampus were superfused with modified Krebs-Ringer solution. Application of 20 mM KCl for 5 min increased the release of NA from the slices, and this release was consistently reproduced. Application of U-46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy prostaglandin F2alpha), a specific TXA2 mimetic, just before the second KCl (20 mM) stimulation decreased the KCl-evoked NA release in a concentration-dependent manner (10-100 microM). This U-46619 (50 microM)-induced inhibition of NA release was abolished by 10 microM SQ29548, a specific TXA2 receptor antagonist. In experiments with hippocampal crude synaptosomes, however, KCl (20 and 40 mM)-evoked release of NA was not attenuated by U-46619 (100 microM). Furthermore, the inhibitory effect of U-46619 (50 microM) in the sliced preparations was not modified by 100 microM (-)-bicuculline, a GABA(A)-receptor antagonist. The present results indicate that U-46619 inhibits the release of NA from the rat hippocampus by activation of TXA2 receptors. Activation of TXA2 receptors probably excites an unidentified but not GABAergic neuron system, thereby inhibiting the NA release from the rat hippocampus.     

Competitive antagonism at thromboxane receptors in human platelets.

The inhibitory effects of three prostanoid analogues, EP 045, EP 092 and pinane thromboxane A2 (PTA2), on the aggregation of human platelets in vitro have been investigated. In diluted platelet-rich plasma (PRP), EP 045 (20 microM) and EP 092 (1 microM) completely inhibited irreversible aggregation responses to thromboxane A2 (TXA2), prostaglandin H2 (PGH2) and five chemically stable thromboxane mimetics, including 11,9-epoxymethano-PGH2 and 9,11-azo-PGH2. Reversible aggregation produced by the prostanoid analogue, CTA2, was also inhibited. The block of the stable agonist action was surmountable. In plasma-free platelet suspensions EP 045 and EP 092 were more potent antagonists. Schild analysis indicated a competitive type of antagonism for EP 045 (affinity constant of 1.1 X 10(7) M-1); the nature of the EP 092 block is not clear. Primary aggregation waves induced by ADP, platelet activating factor (Paf) and adrenaline were unaffected by EP 045 and EP 092, whereas the corresponding second phases of aggregation were suppressed. Aggregation and 5-hydroxytryptamine (5-HT) release induced by either PGH2 or 11,9-epoxymethano-PGH2 were inhibited in a parallel manner by EP 045. Inhibition of thromboxane biosynthesis is not involved in these effects. EP 045 and EP 092 did not raise adenosine 3':5'-cyclic monophosphate (cyclic AMP) levels in the platelet suspensions. In plasma-free platelet suspensions PTA2 produced a shape change response which could be blocked by EP 045. PTA2, therefore, has a thromboxane-like agonist action. The block of the aggregatory action of 11,9-epoxymethano-PGH2 by PTA2 appears to be mainly due to competition at the thromboxane receptor. However, PTA2 produced a slight rise in cyclic AMP levels; this could be due to a very weak stimulant action on either PGI2 or PGD2 receptors present in the human platelet. Functional antagonism by PTA2 may therefore augment its thromboxane receptor blocking activity. The results are discussed in terms of (a) the specificity of antagonism produced by EP 045, EP 092 and PTA2, (b) the validity of affinity constant determinations for receptor antagonists when aggregation is the biological response, and (c) the characteristics of the human platelet thromboxane receptor in comparison with those of thromboxane receptors in smooth muscle.     

GR32191, a highly potent and specific thromboxane A2 receptor blocking drug on platelets and vascular and airways smooth muscle in vitro.

1. The thromboxane A2 (TP)-receptor blocking activity and specificity of action of GR32191 ([1R-[1 alpha(Z),2 beta,3 beta,5 alpha]]-(+)-7-[5-([1,1'-biphenyl] -4-ylmethoxy)-3-hydroxy-2-(1-piperidinyl)cyclopentyl]-4-heptoni c acid has been evaluated in human platelets and various smooth muscle preparations, both vascular and non-vascular, from a range of species including man. 2. Utilising a platelet counting method to assess aggregation the drug was found to antagonise, in a surmountable manner, human platelet aggregation produced by the TP-receptor agonists, U-46619, EP171 and SQ26655, in whole blood and physiological buffer, with pA2 values of approximately 8.3 and 8.7 in the two media respectively. In the presence of GR32191 the rate of aggregation induced by U-46619 was slowed. 3. The effect of GR32191 upon U-46619-induced platelet shape change and aggregation in platelet-rich plasma was evaluated utilising a turbidometric technique. Both shape change and aggregation were antagonised by GR32191. At relatively high concentrations of the drug a slowing of aggregation and shape change to U-44619 was seen and an unsurmountable antagonism became apparent. 4. The action of GR32191 upon human platelets was specific with platelet aggregation induced by adenosine 5'-diphosphate, platelet activating factor, vasopressin and adrenaline and the inhibitory effects of prostacylin (PGI2), prostaglandin D2 (PGD2) and N-ethylcarboxamide-adenosine (NECA) being unaffected by concentrations of the drug as high as 10 microM. Furthermore, at concentrations of up to 100 microM, the drug itself produced no shape change or aggregation, of human platelets. 5. GR32191 also specifically and potently antagonised in a competitive, surmountable manner the contractile actions of U-46619 upon human vascular smooth muscle and antagonised U-46619-induced contractions of vascular and airways smooth muscle preparations from rat, dog, guinea-pig and rabbit with varying potency. This is discussed in terms of possible heterogeneity of TP-receptors. 6. GR32191 therefore represents a highly potent and specific TP-receptor blocking drug. This profile of action, coupled to its long duration of effect in man described elsewhere, make it an ideal drug tool for elucidating the physiological and pathophysiological role of thromboxane A2.     

Participation of thromboxane A(2) in the cough response in guinea-pigs: antitussive effect of ozagrel

1. The purpose of this study was to investigate the involvement of thromboxane A(2) (TXA(2)) in the cough response in a guinea-pig cough model. Here, we describe results obtained using a selective TXA(2) synthetase inhibitor, ozagrel, and a selective TXA(2) agonist, U-46619. 2. Guinea-pigs were anaesthetized and exposed to an aerosol of capsaicin (100 microM) to elicit coughing. The number of coughs was 20.0+/-5.8 during capsaicin provocation (5 min), but only 2. 8+/-0.4 during a 5-min inhalation of phosphate-buffered saline (PBS) (P:<0.05). 3. TXB(2) levels in BAL were 101.4+/-8.0 and 58.4+/-8.7 pg ml(-1) following capsaicin and PBS inhalation, respectively (P:<0. 01), but there was no intergroup difference in the cell populations in BAL. 4. Inhalation of U-46619 did not induce a cough response by itself at concentrations of 100 ng ml(-1) to 10 microg ml(-1). However, it caused a 2 fold increase in the number of capsaicin-induced coughs. 5. To explore the source of the TXA(2), BAL cells were stimulated with capsaicin and the supernatants collected for analysis. The TXB(2) concentration in BAL was increased dose-dependently, indicating that TXA(2) is released from BAL cells in response to capsaicin. 6. Ozagrel was administered orally 1 h before a 5 min capsaicin provocation and the number of coughs was counted during the capsaicin inhalation. Ozagrel decreased the number of coughs dose-dependently (ED(50) value, 26.3 mg kg(-1)). 7. These results show that TXA(2) modulates the capsaicin-induced cough response by increasing capsaicin-sensitivity.     

Pharmacokinetic and pharmacodynamic profiles of vapiprost, a selective, long-lasting thromboxane receptor antagonist, after single and multiple oral administration to healthy volunteers.

A selective thromboxane A2 (TXA2) receptor blocking agent, vapiprost, was orally administered to healthy male Japanese volunteers to investigate the pharmacokinetic and pharmacodynamic properties. The time-profile of vapiprost concentration in plasma was determined and the effects of the drug on platelet aggregation in platelet-rich plasma (PRP) induced by a stable TXA2 receptor agonist U-46619, adenosine diphosphate (ADP) and collagen, and platelet aggregation in whole blood induced by U-46619 ex vivo were simultaneously examined and compared. In the single-dose study (5, 10, and 20 mg/man) the plasma concentrations of the drug were fitted well to a one-compartment open model with a first-order absorption. The area under plasma concentration-time curve (AUC) and maximum plasma concentration (Cmax) showed dose-related increases, whereas the mean elimination half-lives (t1/2) remained approximately constant within the range of 0.99-1.1 hour. The drug was hardly recovered unchanged in urine. The platelet aggregation in PRP induced by collagen or U-46619 and the secondary aggregation by ADP were inhibited; that induced by U-46619 was the most specifically and completely inhibited at 2 hours after administration of any dose. The duration for maintaining the significant inhibition tended to depend on the dose and ranged from 24 to 36 hours after administration, which was much longer than expected from the plasma concentration of drug. The time-profile of inhibiting whole blood platelet aggregation that was induced by U-46619 was almost parallel to that of platelet aggregation in PRP by the same aggregant. The bleeding time was slightly prolonged 2 and 8 hours after administrations of 10 and 20 mg, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Synthesis and thromboxane A2/prostaglandin H2 receptor antagonistic activity of phenol derivatives.

Consideration of possible structural similarities between thromboxane A2 and the hydroquinone form of (R)-(+)-7-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-7- phenylheptanoic acid (R-(+)-AA-2414) led to the development of a new series of thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor antagonists, namely 7-(4-fluorophenyl)-7-(2-hydroxyphenyl)heptanoic acids (I). These compounds were found to be potent TXA2/PGH2 receptor antagonists. Compounds having either a carbonyl or a hydroxymethyl group at the para-position of the phenolic hydroxy group exhibited most potent activities in this series. Compounds 14, 15, 18, and 26 inhibited the specific binding of [3H]U-46619 to guinea pig platelet membranes (IC50 = 4.4, 80, 32, and 13 nM, respectively), and also inhibited U-46619-induced human platelet aggregation (IC50 = 310, 69, 79, and 78 nM, respectively). Comparison of the UV spectra of the compounds with a carbonyl group at the para-position of phenolic hydroxy group revealed that the activity tended to increase in accordance with a decrease in the torsional angle between the carbonyl group and the phenol ring. These results suggested that the spacial location of the carbonyl and hydroxymethyl oxygen are important for significant increase in activity and that the carbonyl and hydroxymethyl oxygen at the para-position of the phenolic hydroxy group might interact with one of the TXA2/PGH2 receptor sites.     

Pharmacological actions of S-145, a novel thromboxane A2 antagonist, in various smooth muscles

Antagonistic actions of S-145 ((+-)-5(Z)-7-[3-endo[(phenylsulfonyl)amino]bicyclo[2.2.1] hept-2-exo-yl]heptenoic acid) against U-46619, a thromboxane A2 mimic, were studied using isolated thoracic aorta of the rat and the trachea, lung parenchyma and ileum of the guinea pig. S-145 as well as SQ-29548 and ONO-3708 inhibited the contraction of aorta induced by U-46619 in a concentration-dependent manner. The IC50 value of each compound was 1.4, 14.5 and 52.6 nM. S-145 also inhibited contractions of the aorta induced by high concentrations of PGE1, PGE2 and PGF2 alpha, but failed to affect the responses to K+, Ca2+, NE, 5-HT, and angiotensin II. Contractions of trachea and lung parenchyma of the guinea pig induced by U-46619 were concentration-dependently inhibited by S-145, but those induced by histamine and leukotriene D4 were not affected. Ileac contractions by PGE2 and PGF2 alpha were not inhibited by S-145. The (+)-isomer of S-145 was more potent and the (-)-isomer was less potent than S-145 for antagonistic action against U-46619. These results suggest that S-145 is a potent and specific antagonist to the thromboxane A2 receptor; and in the aorta, the thromboxane A2 receptor may respond to high concentrations of PGs.     

Photoaffinity receptor antagonist for human platelet thromboxane A2/prostaglandin H2 receptors

9,11-Dimethylmethano -11,12-methano-16-(3-azido-4-iodophenoxy)-13,14- dihydro-13-aza-15 alpha beta-omega-tetranor TXA2 (I-PTA-PON3) was synthesized and evaluated as a potential photoaffinity probe of the human platelet thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor. I-PTA-PON3 inhibited the aggregation of washed human platelets induced by the TXA2 mimetic U46619 [(15S)-hydroxy-11 alpha, 9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid]. Schild analysis of the data revealed a Kd of 9.5 nM and a slope not significantly different from -1. Equilibrium binding studies using [125I]PTA-OH, a TXA2/PGH2 receptor antagonist, showed that I-PTA-PON3 plus photolysis resulted in a 52% reduction in the number of binding sites (1252 +/- 202/platelet) compared to the nonphotolyzed group (2557 +/- 293/platelet) (N = 5, P less than 0.05) with no significant change in the Kd. Repetition of the incubation with I-PTA-PON3 and photolysis a second time resulted in a further 77% (578 +/- 163 binding sites/platelet) reduction in the number of binding sites. Incubation of washed human platelets with I-PTA-PON3 (163 nM) followed by photolysis and removal of the non-covalently bound I-PTA-PON3 resulted in no change in the EC50 value for the TXA2 mimetic, U46619, when compared to controls that were either exposed to I-PTA-PON3 and not photolyzed or exposed only to photolysis. The second photolysis of I-PTA-PON3 resulted in a significant 42% increase in the EC50 value of U46619-induced aggregation compared to the non-photolyzed group (N = 4, P less than 0.05). These results suggest that I-PTA-PON3 is a useful probe for the study of TXA2/PGH2 receptors and that spare TXA2/PGH2 receptors may exist in the platelet.