7-Oxabicyclo[2.2.1]heptyl carboxylic acids as thromboxane A2 antagonists: aza omega-chain analogues

A novel bicyclic prostaglandin analogue, [1S-[1 alpha, 2 alpha (Z), 3 alpha, 4 alpha]]-7-[3-[[[[(1- Oxoheptyl)amino]acetyl]amino]-methyl]-7-oxabicyclo[2.2.1]hept-2- yl]-5-heptenoic acid [-)-7) was found to be a potent and selective thromboxane A2 (TxA2) receptor antagonist. Unlike the related series of omega-chain allylic alcohols, amide 7 and its congeners were uniformly free of direct contractile activity in vitro (bovine coronary) and in vivo (anesthetized guinea pig). Amide 7 was effective in the inhibition of (a) arachidonic acid induced platelet aggregation of human platelet-rich plasma (I50 = 0.18 +/- 0.006 microM), (b) 11,9-epoxymethano-PGH2 induced platelet aggregation of human platelet-rich plasma (I50 = 0.24 microM), (c) 11,9-epoxymethano-PGH2 induced contraction of guinea pig trachea (Kb = 3.0 +/- 0.3 nM) or rat aorta (Kb = 8.8 +/- 1.1 nM), and (d) arachidonic acid induced bronchoconstriction in the anesthetized guinea pig (0.1-1.0 mg/kg iv). Amide 7 inhibited the binding of [5,6-3H2]-[1S- (1 alpha, 2 alpha (Z), 3 alpha, 4 alpha)]-7-[3-[[2-[(Phenyl- amino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5- heptenoic acid to human platelet membranes in a specific and saturable manner with a Kd = 49.6 +/- 1.4 nM.     

Synthesis and pharmacological evaluation of 5,6-exo-epoxy-7-oxabicyclo[2.2.1]heptane derivatives

1 alpha,2 beta(5Z),3 beta(1E,3S),4 alpha,5 alpha,6 alpha]-7-[5,6-Epoxy-3- (3-cyclohexyl-3-hydroxy-3-methyl-1-propenyl)-7-oxabicyclo[2.2.1]-hept-2- yl]-5-heptenoic acid (31) and [1 alpha,2 beta(5Z),3 beta(1E,3S),4 alpha,5 alpha,6 alpha]-7-[5,6-epoxy-3-[3-hydroxy-5-(p-hydroxyphenyl)-1- pentenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (37) were found to be selective TxA2 antagonists at the platelet and pulmonary thromboxane receptors. An efficient stereospecific synthesis of these compounds and a series of structural analogues is described. Compounds 31 and 37 both inhibited the bronchoconstriction induced by arachidonic acid in the anesthetized guinea pig.     

Thromboxane A2 (TP) receptor in the non-pregnant porcine myometrium and its role in regulation of spontaneous contractile activity

Although there are species-related differences in uterine prostanoid receptor subtypes, functional prostanoid receptors in the porcine uterus are similar with those in the human uterus (FP, TP, EP(1), EP(2), EP(3), DP and IP) except for the TP receptor. These similarities promoted us to determine whether TP receptors are present in the non-pregnant porcine uterus. For this purpose, the effects of TP receptor agonists and antagonists were investigated by a contraction study and by a binding study. 9,11-Dideoxy-9 alpha, 11 alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic acid (U46619, 1 nM-10 microM), a stable thromboxane A(2) mimetic, caused tetrodotoxin-resistant contraction in both longitudinal and circular muscles of the uterine cornu. The pEC(50) value in the longitudinal muscle (6.69) was lower than that in the circular muscle (7.62), but the maximum response in the longitudinal muscle was two times larger than that in the circular muscle. The longitudinal and circular muscles of other regions (corpus and cervix) also responded to U46619, and region-related difference in contractile responses was observed only in the longitudinal muscles. 4(Z)-6-(2-o-Chlorophenyl-4-o-hydroxyphenyl-1,3-dioxan-cis-5-yl) hexenoic acid (ICI192605) and 7-[3-[[2-[(phenylamino)carbonyl] hydrazino]methyl]7-oxabicyclo[2.2.1]hept-2-yl]-,[1S-[1 alpha,2 alpha(Z),3 alpha,4 alpha]]-]5-heptenoic acid (SQ29548) inhibited the contractile responses to U46619 competitively. The longitudinal and circular muscles in the cornu contained a single class of [3H]SQ29548 binding site with similar K(d) values (30 nM), but B(max) in the circular muscle (90.9+/-8.6 fmol/mg protein) was two times higher than that in the longitudinal muscle (58.2+/-8.6 fmol/mg protein). The ranking order of competition by TP receptor agonists and antagonists (with pK(i) values in parentheses) was [1S-[1,2(Z),3(1E,3S*),4]]-7-[3-[3-Hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (I-BOP, 7.70)>SQ29548 (7.39)>7-[3-(3-Hydroxy-1-octenyl)bicycle[3.1.1]hept-2-yl]-,[2S-[2 alpha(Z),3 beta(1E,3R*)]]-5-heptenoic acid (CTA(2), 6.55)>7-[3-(3-hydroxy-1-octenyl)-6,6-dimethylbicyclo[3.1.1]hept-2-yl-,[1S-[1 alpha,2 beta(Z),3 alpha(1E,3R*),5 alpha]]-5-heptenoic acid (PTA(2), 6.50)>U46619 (6.41)>7-[5-(3-hydroxy-1-octenyl)-2-oxabicyclo[2.2.1] hept-6yl]-,[1S-[1 alpha,4 alpha,5 alpha(1E,3R*),6 beta(Z)]]-5-heptenoic acid (U44069, 6.34), and this order is consistent with current TP receptors. Treatment with indomethacin (100 nM) and N-tert-butyl-N cent -[(2-cyclohexylamino-5-nitrobenzene) sulfonyl] urea (BM-531, 10 microM) inhibited the spontaneous contractile activities of both longitudinal and circular muscles. The present results indicate that contractile TP receptors are present in the non-pregnant porcine uterus. Therefore, the prostanoid receptor subtypes that exist in the porcine uterus (TP, IP, DP, FP, EP(1), EP(2) and EP(3)) are the same as those present in the human uterus. The distribution of TP receptors in the porcine uterus differed depending on the type of myometrium (longitudinal and circular muscles) and region of the uterus. The endogenous thromboxane A(2)-TP receptor pathway is thought to play a physiological role in regulation of spontaneous contractile activity in the porcine uterus.     

Interphenylene 7-oxabicyclo[2.2.1]heptane thromboxane A2 antagonists. Semicarbazone omega-chains

A series of chiral interphenylene 7-oxabicyclo[2.2.1]heptane semicarbazones 19-26 were prepared and evaluated for their in vitro thromboxane (TxA2) antagonistic activity and in vivo duration of action. The potency of 19-26 was found to highly dependent on the substitution pattern of the interphenylene ring and decreased in the order ortho greater than meta much greater than para. SQ 35,091 (25), [1S-(1 alpha,2 alpha,3 alpha,4 alpha)]-2-[[3-[[[(phenylamino) carbonyl]hydrazono]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]methyl] benzenepropanoic acid, was identified as a potent and long-acting TxA2 antagonist. In human platelet rich plasma SQ 35,091 inhibited arachidonic acid (800 microM) and U-46,619 (10 microM) induced aggregation with I50 values of 3 and 12 nM, respectively. In contrast, no inhibition of ADP (20 microM) induced aggregation was observed at greater than 1000 microM. Receptor binding studies with [3H]-SQ 29,548 showed SQ 35,091 was a competitive antagonist with a Kd value of 1.0 +/- 0.1 nM in human platelet membranes. In vivo SQ 35,091 (0.2 mg/kg po) showed extended protection (T50 = 16 h) from U-46,619 (2 mg/kg iv) induced death in mice. These compounds have for the first time demonstrated that a metabolically stable interphenylene alpha-sidechain can be introduced into a prostanoid-like series of TxA2 antagonists with the maintainance of potent antagonistic activity.     

Regulation of activator protein-1 by 8-iso-prostaglandin E2 in a thromboxane A2 receptor-dependent and -independent manner

The thromboxane (TX) A(2) receptor (TP) encompasses two alternatively spliced forms, termed the platelet/placental (TP-P) and endothelial (TP-E) type receptors. Experimental evidence suggests that TP activity may be modulated by novel ligands, termed the isoprostanes, that paradoxically act as TP agonists in smooth muscle and TP antagonists in platelet preparations. Here we have investigated whether prototypical isoprostanes 8-iso-prostaglandin (PG)F(2 alpha) and 8-iso-PGE(2) regulate the activity of TP isoforms expressed in Chinese hamster ovary (CHO) cells using activator protein-1 (AP-1)-luciferase activity as a reporter. AP-1-luciferase activity was increased by a TP agonist [9,11-dideoxy-9 alpha,11 alpha-methanoepoxy PGF(2 alpha) (U46619)] in CHO cells transfected with the human TP-P and TP-E receptors, and this response was fully inhibited by TP antagonists [1S-[1 alpha,2 beta(Z),3 alpha,5 alpha]]-7-[3-[[4-iodophenyl)sulfonyl]amino]-6,6-dimethylbicyclo[3.1.1]hept-2-yl]-5-heptenoic acid (I-SAP) and [1S-[1 alpha,2 alpha(Z),3 alpha,4 alpha]]-7-[[2-[(phenylamino) carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1] hept-2-yl]-5-heptenoic acid (SQ 29,548)]. AP-1-luciferase activity was potently (nanomolar concentrations) increased by 8-iso-PGE(2) in CHO TP-P and TP-E cells, and this response was partially inhibited by cotreatment of cells with TP antagonists, whereas 8-iso-PGF(2 alpha) was without effect. Cyclooxygenase inhibitors did not abolish 8-iso-PGE(2) mediated AP-1-luciferase activity, indicating that this response is not dependent on de novo TXA(2) biosynthesis. Interestingly, 8-iso-PGE(2)-mediated AP-1-luciferase activity was near maximal in naive cells between 1 and 10 nM concentrations, and this response was not inhibited by TP antagonist or reproduced by agonists for TP or EP(1)/EP(3) receptors. These observations 1) support a role for novel ligands in the regulation of TP-dependent signaling, 2) indicate that TP-P and TP-E couple to AP-1, 3) provide further evidence that isoprostanes function as TP agonists in a cell-type specific fashion, and 4) indicate that additional targets regulated by 8-iso-PGE(2) couple to AP-1.     

Prostanoid EP3 and TP receptors-mediated inhibition of noradrenaline release from the isolated rat stomach

The postganglionic sympathetic nerves of the isolated rat stomach were electrically stimulated twice at 1 Hz for 1 min. Prostaglandin E(2) and ONO-AE-248 (16S-9-deoxy-9beta-chloro-15-deoxy-16-hyfroxy-17,17-trimethylene-19,20-didehydro prostaglandin F(2)) (an EP(3) receptor agonist) reduced the evoked noradrenaline release, while ONO-DI-004 (17S-2,5-ethano-6-oxo-17,20-dimethyl prostaglandin E(1)) (an EP(1) receptor agonist), ONO-AE1-259-01 (11,15-O-dimethyl prostaglandin E(2)) (an EP(2) receptor agonist) and ONO-AE1-329 [16-(3-methoxymethyl)phenyl-omega-tetranor-3,7-dithia prostaglandin E(1)] (an EP(4) receptor agonist) had no effect. U-46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy prostaglandin F(2alpha)) and I-BOP (7-[3-[3-hydroxy-4-(4-iodophenoxy)-1-butenyl]-7-oxabicyclo[2,2,1] hept-2-yl]-,[1S[1alpha,2alpha(Z),3beta(1E,3S)4alpha]]-5-heptenoic acid) (TP receptor agonists) also reduced the noradrenaline release and these inhibitory effects were abolished by SQ-29548 (7-[3-[[2-[(phenylamino) carbonyl] hydrazino]methyl]-7-oxabicyclo[2,2,1]hept-2-yl][1S(1alpha,2alpha(Z), 3alpha,4alpha]-5-heptenoic acid) (a TP receptor antagonist). The inhibitory effect of U-46619, but not ONO-AE-248, was abolished by pertussis toxin. These results suggest that the prostanoid EP(3) and TP receptors mediate the inhibition of gastric noradrenaline release; TP, but not EP(3), receptor-mediated inhibition is mediated by a pertussis toxin-sensitive mechanism in rats.     

9,11-epoxy-9-homoprosta-5-enoic acid analogues as thromboxane A2 receptor antagonists

A novel bicyclic prostaglandin analogue, (1S)-[1 alpha, 2 alpha(Z),3 alpha(1E,3S*,4R*),4 alpha]-7-[3-(3-hydroxy-4-phenyl-1-pentenyl)-7- oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (4), was found to be a potent and selective thromboxane A2 (TxA2) receptor antagonist. Alcohol 4 was the only member in a series of allylic alcohols which did not display direct contractile activity in the rat stomach strip model. Alcohol 4 was effective in the inhibition of (a) arachidonic acid induced platelet aggregation of human platelet-rich plasma (I50 = 0.65 +/- 0.1 microM); (b) 11,9-epoxymethano-PGH2 induced contraction of guinea pig trachea (pA2 = 8.0 +/- 0.2) or rat aorta (pA2 = 8.1 +/- 0.2); and (c) arachidonic acid induced bronchoconstriction in the anesthetized guinea pig (1 mg/kg iv). A radioiodinated analogue of 4 bound in a specific and saturable manner to human platelet membranes with a Kd = 2.3 +/- 0.9 nM. Modification of the alpha-chain, in an attempt to minimize in vivo metabolism, resulted in TxA2 receptor antagonists of reduced in vitro potency.     

9,11-Epoxy-9-homo-14-thiaprost-5-enoic acid derivatives: potent thromboxane A2 antagonists

A novel bicyclic prostaglandin analogue, (1S)-[1 alpha,2 alpha(Z),3 alpha,4 alpha]-7-[3-[(hexylthio)methyl]-7- oxabicyclo [2.2.1]hept-2-yl]-5-heptenoic acid ((-)-10), and its cogeners were found to be potent antagonists at the TxA2 receptor. Compound (-)-10 was the only stereoisomer out of eight possible structures that was active. Thioether (-)-10 was 30-40-fold more potent than another TxA2 antagonist, BM 13.177, in inhibiting arachidonic acid (AA) induced aggregation of human platelet-rich plasma. Compound (-)-10 was effective (I50 = 0.5 +/- 0.4 microM) in inhibiting 9,11-azo-PGH2-induced (0.1 microgram/mL) contraction of guinea pig tracheal spirals. The bronchoconstriction in anesthetized guinea pigs induced by AA was also effectively antagonized by (-)-10 (1 mg/kg, iv); however, in this assay (-)-10 exhibited some direct agonist activity. Radioligand binding studies in washed (human) platelets revealed that (-)-10 is one of the most potent ligands for the PGH2/TxA2 receptor yet described (Kd = 1.6 +/- 0.4 nM).     

Characterization of rat glomerular thromboxane A2 receptors: comparison to rat platelets.

This study was designed to characterize rat glomerular thromboxane A2 (TxA2) receptors and compare them to rat platelet TxA2 receptors. The radioligand binding characteristics of the receptors were characterized using [125I][1S-(1 alpha,2 beta(5Z),3 alpha-(1E,3R*),4 alpha]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo- [2.2.1]heptan-2yl]-5-heptenoic acid ([125I]BOP), a TxA2 agonist. Equilibrium binding with [125I]BOP, as well as competitive binding assays between [125I]BOP and 13-azapinane TxA2 receptors antagonists, were performed in rat glomerular membranes (RGM) and washed rat platelets (WRP). [125I]BOP identified a single class of TxA2 receptor sites in glomerular membranes with a Kd of 318 +/- 55 pM and a Bmax of 260 +/- 62 fmol/mg protein (n = 14). [125I]BOP was displaced by the TxA2 agonist 15S-hydroxy-11 alpha,9 alpha(epoxymethano)-prosta-5Z,13E-dienoic acid (U-46,619) (IC50 = 22 +/- 6 nM, n = 3), the antagonist SQ-29,548 (IC50 = 41 +/- 7 nM, n = 4), and stereoselectively by the antagonists (-)-9-chlorobenzyl-6-fluoro-1,2,3,4-tetrahydrocarbazol-1-yl acetic acid (L-657,925) (IC50 = 0.27 +/- 0.04 nM, n = 3) and (+)-9-chlorobenzyl-6-fluoro-1,2,3,4-tetrahydrocarbazol-1-yl acetic acid (L-657,926) (IC50 = 124 +/- 0 nM, n = 2). The ability of six 13-azapinane TxA2 antagonists to compete with [125I]BOP was evaluated. The rank orders for the 13-azapinanes showed no significant correlation between RGM and WRP.(ABSTRACT TRUNCATED AT 250 WORDS)     

Thromboxane prostanoid receptor in human airway smooth muscle cells: a relevant role in proliferation

Thromboxane A(2) has been implicated as a mediator of bronchial hyperresponsiveness in asthma. Modulating agents are currently marketed in Japan and under clinical evaluation in the US, but full characterization of the thromboxane A(2) receptor and the signaling pathways that link it to the proliferative events taking place during airways structural remodeling has not been achieved. Here, we report that the presence of mRNA for both alpha and beta isoforms of the thromboxane A(2) receptor in smooth muscle cells from human bronchi correlates with protein expression evaluated by radioligand binding of the antagonist, SQ29,548 ([1S-[1alpha,2alpha(Z),3alpha,4alpha]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic-acid) (K(d)=3.4 nM+/-44%CV, coefficient of variation, B(max)=41 fmol/mg prot+/-38%CV). The receptor is functional, as the agonist, U46619 (9,11-dideoxy-9alpha,11alpha-methanoepoxy-prosta-5Z,13E-dien-1-oic-acid), induced a concentration-dependent Ca(2+) transient (EC(50)=0.12 microM+/-27%CV). Furthermore, U46619 concentration dependently increased DNA synthesis and markedly potentiated the epidermal growth factor mitogenic effect. Both events were specifically inhibited by SQ29,548, independently from transactivation of the epidermal growth factor receptor and partially sensitive to pertussis toxin.     

Antagonistic actions of S-145 on vascular and platelet thromboxane A2 receptors

We studied the actions of a potent thromboxane A2/prostaglandin in H2 (TP) receptor antagonist, (+/-)-(5Z)-7-[3-endo-[(phenylsulfonyl)amino]bicyclo [2.2.1]hept-2-exo-yl]heptenoic acid (S-145) on vascular and platelet receptors in the pig. S-145 showed almost the same affinity for both receptors in ligand binding studies with [3H]U46619 or [3H]SQ29, 548. The binding affinity of S-145 was 5-8 times higher than that of SQ29,548, a well-characterized TP receptor antagonist. However, S-145 inhibited U46619-induced contractions of pig coronary arteries with an IC50 value 52.5 times lower than that of SQ29,548, and was approximately equipotent with SQ29,548 in inhibiting U46619-induced secondary aggregation of pig platelets. Detailed kinetic studies on [3H]S-145 binding revealed that the apparent discrepancy between the pharmacological potency of S-145 in platelet and vascular systems was not due to tissue selectivity, but its small association constants for both receptors.     

Synthesis and in vitro activity of various derivatives of a novel thromboxane receptor antagonist, (+/-)-(5Z)-7-[3-endo-[(phenylsulfonyl)amino]bicyclo[2.2.1] hept-2-exo-yl]heptenoic acid

Several sulfonyl derivatives (13a-t) of (+/-)-(5Z)-7-(3-endo-aminobicyclo[2.2.1]hept-2-exo-yl)heptenoic acid (VI) were synthesized via its methyl ester 10. Sulfonylation of 10 with 11a-t followed by saponification yielded 13a-t. Inhibitory concentrations (IC50) of the corresponding sodium salts 14a-t for platelet aggregation were measured with rat washed platelets (WP) and rabbit platelet-rich plasma (PRP). IC50 values of some derivatives for contraction of the rat aorta were also measured. The IC50 values for rat WP increased from 2.9 to 26 nM in the order of 14a, 14c, 14d, and 14b for derivatives with an arylsulfonyl residue, depending on the number of of intervening methylene groups. Methyl derivative 14e exhibited a higher IC50 value than n-hexyl derivative 14f. Substitution with a p-methyl, p-fluoro-, or p-chloro group in 14a retained or slightly reduced its IC50 value, while a p-n-pentyl or p-oxycarbonyl group augmented it significantly. The representative 14a suppressed (15S)-15-hydroxy-11,9-(epoxymethano)prosta-5(Z),13(E)-dienoic acid (U-46619) induced aggregation of human WP with an IC50 value of 7.7 nM, which corresponds well to the IC50 value of 3 nM obtained for each displacement by 14a of [3H]-U-46619 or (5Z,15 xi)-9 alpha, 11 alpha-(dimethylmethano)-15-hydroxy-16-(3-[125I]iodo- 4-hydroxyphenyl)-17,18,19,20-tetranor-13-aza-11a-carbathrombo-5-en oic acid [( 125I]-PTA-OH) bound to human WP. Synthesis of thromboxane A2 (TxA2) in human WP stimulated by thrombin was not inhibited by 14a at a concentration up to 10 microM. From these observations, the corresponding acid 13a (S-145) was concluded to be a potent TxA2 receptor antagonist.     

9,11-Epoxy-9-homo-14-oxaprosta-5-enoic acid derivatives. Novel inhibitors of fatty acid cyclooxygenase

A novel bicyclic prostaglandin analogue, [1R-[l alpha,2 beta (5Z),3 beta,4 alpha]]-7-[3-[(hexyloxy)methyl]- 7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (1), and cogeners were found to be potent inhibitors of fatty acid cyclooxygenase. Compound 1 was the only stereoisomer out of eight possible structures that was active. Ether 1 was 20 times more potent than indomethacin (IND) in inhibiting arachidonic acid (AA) induced aggregation of human platelet-rich plasma. Compound 1 was also more potent than IND in several in vivo assays, AA-induced sudden death in the conscious mouse (2 times) and AA-induced bronchoconstriction in the anesthetized guinea pig (16-45 times).     

Glibenclamide inhibits thromboxane-mediated vasoconstriction by thromboxane receptor blockade

Because sulfonylureas, such as glibenclamide, are used to treat Type 2 diabetes and because this disease is associated with various cardiovascular complications that may be mediated by thromboxane (TX), this study was designed to characterize the role of glibenclamide on TX-mediated contractions in isolated ring segments of bovine coronary arteries and rabbit aortas. A series of TXA(2) analogs [9,11 Dideoxy-9alpha, 11alpha-methanoepoxy prostaglandin F(2alpha) (U46619), [1S-(1alpha, 2beta(5Z),3alpha(1E, 3R*),4alpha)]-7-[3-(3-hydroxy-4-(4'-iodophenoxy)-1-butenyl)-7-oxabicyclo [2.2.1]heptan-2-yl]-5-heptenoic acid (I-BOP), carbocyclic TXA(2) (CTA(2)) and 9,11-dideoxy-9alpha,11alpha-epoxymethano prostaglandin F(2alpha) (U44069)], endothelin and phenylephrine contracted both types of blood vessels. Glibenclamide (10 microM) inhibited the contraction to each of the TX agonists but had no effect on endothelin- or phenylephrine-induced contractions. We hypothesized that this effect was due to a direct effect to block the vascular smooth muscle cell TX receptor. Receptor binding studies were performed in rabbit vascular smooth muscle cells and indicated that glibenclamide (10 microM) inhibited (125)I-BOP binding by more than 80%. The inhibition constants or K(i) for glibenclamide was 0.53 microM. These studies provide the first evidence that the ability of glibenclamide to inhibit TX-mediated contractions occurs independent of the vascular K(ATP) channel and is, instead, mediated by the blockade of the vascular TX receptor.     

Benextramine acts as an irreversible noncompetitive antagonist of U46619-mediated contraction of the rat small mesenteric artery

We have studied the effects of benextramine on the U46619 (11,9-epoxymethano-15S-hydroxy-prosta-5Z,13E-dienoicacid)-mediated contraction of the rat isolated small mesenteric artery. U46619 (10 nM-10 μM) produced a concentration-dependent contraction of the small mesenteric artery. The selective prostanoid TP receptor antagonist, SQ 30,741 (1S-[1,2(5Z),3,4]]-7-[[[[[(oxaheptyl)amino]acetyl]amino]-methyl]-7-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid; 1 μM), produced a parallel, rightward shift of the U46619 curve with an associated pA₂ value of 7.43 ± 0.09. Treatment of tissues with 100 μM benextramine depressed the maximum response to U46619 in a time-dependent manner. However, neither SQ 30,741 (10 μM) nor U46619 (10 μM) incubation significantly protected against this effect. Thus, benextramine acts as an irreversible noncompetitive antagonist of U46619. The mechanism of this action is not yet clear.     

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.     

Characterization of the thromboxane receptor mediating prostacyclin release from cultured endothelial cells.

The thromboxane A2 (TXA2) mimetic, 9,11-dideoxy-11,9-epoxymethano-prostaglandin F 2 alpha (U46619), mobilized calcium in the bovine aortic endothelial cell line AG4762 and stimulated release of prostacyclin from these cells. The U46619-stimulated release of prostacyclin could be inhibited by TXA2 antagonists with the order of potency [Is-[1 less than a, 2 less than b(5z), 3 less than b, 4 less than a]]-7-[3-[[2-[(phenylamino)carbonyl]hydrazino]methyl]-7-oxabicyclo- [2.2.1]hept-2-yl]-5- heptenoic acid (SQ29548) greater than 4-[2-(4-chlorobenzene-sulphonamido) ethyl]phenylacetic acid (BM13505) greater than 4-[2-(phenylsulphonamido)-ethyl]phenoxyacetic acid (BM13177), which was consistent with release being mediated by a TXA2 (TP) receptor. The TP receptor ligands, [3H]SQ29548 and 9,11-dimethylmethano-16(3-[125I]iodo-4-hydroxyphenyl)-13,14-dih ydr o-13-aza- 15-omega-o-tetranor-thromboxane ([125I]-PTA-OH), both appeared to bind to a homogenous population of sites in AG4762 cell membranes. The affinities of [3H]SQ29548 and [125I]PTA-OH were approximately 10 nM and approximately 0.3 nM, respectively, and the density of sites labelled by either ligand was approximately 25 fmol/mg protein. Under conditions where equilibrium was approached, the specific binding of [3H] SQ29548 or [125I]PTA-OH was displaced by SQ29548, BM13505 and BM13177 with the same order of potency and similar apparent affinities as in the functional assay, suggesting that these binding sites represent bona fide TP receptors.     

Augmented potentiation of renal vasoconstrictor responses by thromboxane A2 receptor stimulation in the alloxan-diabetic rat

Dose-response curves were obtained to bolus injections of noradrenaline (NA) and 5-hydroxytryptamine (5-HT) in blood and Krebs-perfused kidneys of male Wistar rats. Vasoconstrictor responses to both NA and 5-HT were significantly attenuated in blood-perfused kidneys of alloxan-treated 14 day diabetic rats compared with non-diabetic animals. Responses to low doses of NA were also significantly attenuated in Krebs-perfused kidneys from diabetic rats but responses to 5-HT were augmented. Dose-dependent potentiation of vasoconstrictor responses to NA and 5-HT in Krebs-perfused kidneys of both non-diabetic and diabetic rats occurred during infusion of the thromboxane A2 (TxA2)-mimetic U46619 [15S)-hydroxy-11 alpha, 9 alpha-(epoxymethano) prosta-5Z, 13E-dienoic acid). The potentiation by U46619 (11 ng mL-1) was inhibited in both groups during infusion of the thromboxane receptor antagonist AH23848 [( 1 alpha(Z), 2 beta, 5 alpha]-(+/-)-7-[5[[(1,1'-biphenyl)-4-yl]methoxyl]-2-(4- morpholinyl)-3-oxocyclopentyl]-4-heptenoic acid). Infusion of 5-HT in Krebs-perfused kidneys of non-diabetic rats, causing a rise in perfusion pressure of similar magnitude to that produced by infusion of 111ng mL-1 U46619, did not significantly affect responses to bolus injections of NA. Potentiation of vasoconstrictor responses to low concentrations of 5-HT by U46619 was significantly greater in Krebs-perfused kidneys of diabetic rats than kidneys from non-diabetic animals. Activation of vascular TxA2 receptors augments the vasoconstrictor effects of 5-HT in Krebs-perfused diabetic rat kidneys to a greater extent than in non-diabetic kidneys.     

Evaluation of the effects of anti-thromboxane agents in platelet-vessel wall interaction

We evaluated the capacity of anti-aggregating agents to influence thromboxane A(2) and prostacyclin formation, arachidonic acid-endoperoxide redirection, platelet aggregation and vessel tone, in isolated rabbit aorta incubated with homologous platelets. Picotamide (N,N'bis(3-pyridinylmethyl)-4-methoxy-isophthalamide), the only dual thromboxane A(2)-synthase inhibitor/receptor antagonist in clinical use, inhibited arachidonic acid-induced platelet aggregation with low potency, increased 180-fold by aorta presence. It inhibited thromboxane A(2) formation in platelets and, in aorta presence, increased prostacyclin formation. Ozagrel (OKY-046, (E)-3-(4-(1-imidazolylmethyl)phenyl)-2-propenoic acid), a pure thromboxane A(2)-synthase inhibitor, behaved similarly to picotamide, although the aorta caused a higher (600-fold) shift. The potency of the antagonist SQ 29,548 (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) was unaffected by aorta. In coincubation experiments, arachidonic acid-challenge increased thromboxane A(2)-dependent vessel tone; picotamide increased prostacyclin and reduced thromboxane A(2) formation and vasoconstriction. Ozagrel mimicked picotamide; aspirin (acetylsalicylic acid) reduced aorta contractility, thromboxane A(2) and prostacyclin formation. SQ 29,548 reduced vasoconstriction without affecting eicosanoids. We demonstrate the importance of redirection of eicosanoids in the mechanism of action of thromboxane A(2) inhibitors/antagonists within platelet-vascular wall interactions. These findings bear relevance in the development of novel anti-thrombotic drugs.