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.     

Biochemical characterization and comparison of rat thromboxane A2/prostaglandin H2 receptors in platelets and cultured aortic smooth muscle cells

Comparison of thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptors in rat cultured vascular smooth muscle cells (VSMC) with those in rat gel-filtered platelets (GFP) was carried out using a receptor-ligand binding assay. The binding of each of three radiolabeled TXA2/PGH2 receptor antagonists [( 3H]S-145,[3H]SQ29,548, and [3H]ONO3708) displayed high affinity and specificity as well as saturable and displaceable binding to a single class of recognition sites with the same maximum number in both VSMC and GFP. The Kd values for [3H]S-145 were almost identical for both VSMC and GFP, whereas the values for [3H]SQ29,548 and [3H]ONO3708 for VSMC were approximately two and six times larger than that for GFP. Kinetic analysis of the binding of each receptor antagonist revealed a smaller K1 value (the association rate constant) for [3H]SQ29,548 and a larger K-1 value (the dissociation rate constant) for [3H]ONO3708 for VSMC compared to GFP, in contrast with almost the same kinetic constants for the [3H]S-145 binding for both cells. Comparison of the inhibitory potencies (Ki values) for [3H]S-145 binding for both VSMC and GFP proved that S-145 had the same affinity for both cells; ONO11120 and BM13177, as well as SQ29,548 and ONO3708, possessed lower affinity for VSMC; and U46619 exhibited higher affinity for VSMC. The rank orders of potency were identical in both cells (S-145 greater than SQ29,548 greater than ONO3708 greater than ONO11120 greater than BM13177), which correlated well with their pharmacological activities. These results suggest a similarity in ligand binding specificity with some differences in the accessibility of the antagonists in the TXA2/PGH2 receptors between platelets and vascular smooth muscles.     

Antithrombotic action of the orally available thromboxane A2 receptor antagonist S-1452 (d-s-145 Ca) in rodents

The thromboxane A₂ (TXA₂) receptor antagonist, S-1452, calcium salt of a steroselectively synthesized d-enantiomer of the racemate S-145, calcium 5(Z)-1R,2S,3S,4S-7-[3-phenylsulfonylaminobicyclo[2.2.1]hept-2-yl]-5-heptenoate dihydrate, was examined for its antithrombotic action in vivo, in mice and rats. Orally administered S-1452 dose-dependently prolonged the bleeding time which was measured by a tail transection method. The effect was observed at doses above 1 mg/kg in both animals. S-1452 clearly prevented U46619-induced sudden death in mice and its ED₅₀ value for the prevention was 0.47 mg/kg at 1 h after oral administration. It was more potent and long-acting than reference compounds such as ONO-3708 and SQ-26548. The preventive effect of S-1452 was also observed in arachidonate-induced sudden death, although much larger doses were required in comparison with the case of the U-46619-induced response (ED₅₀: 4.6 mg/kg p.o., at 30 min; 3.1 mg/kg p.o., at 60 min after S-1452). Aspirin, used as a reference compound, was much less active than S-1452 (ED₅₀: 85.9 mg/kg p.o., at 30 min; 88.2 mg/kg p.o., at 60 min after aspirin). The pharmacologically active moiety of S-1452, d-S-145, was several times more potent than the opposite enantiomer 1-S-145 in the prolongation of the bleeding time in rats, and in the prevention of the U46619-induced sudden death in mice. These stereoselective efficacies suggest that S-1452 causes its antithrombotic action by blocking TXA₂ receptors in vivo. S-1452 significantly reduced collagen-induced thrombocytopenia in mice at doses above 0.1 mg/kg p.o. In rats, the compound improved clearly collagen-induced abnormal electrocardiogram. The minimum effective doses were 1 mg/kg for S-1452, 15 mg/kg for ONO-3708, and 10 mg/kg for OKY-046, 60 min after oral administration. It is concluded that S-1452 is a very potent and orally available antithrombotic compound. © 1993 wiley-Liss, Inc.     

Effect of (+)-S-145 calcium salt dihydrate, an orally active antagonist of the thromboxane A2/prostaglandin endoperoxide receptor, on platelet aggregation

The effect of (+)-S-145, (1R, 2S, 3S, 4S)-(5Z)-7-(3-phenylsulfonylaminobicyclo [2.2.1] hept-2-yl) heptenoic acid on human and guinea pig platelet aggregation was examined. (+)-S-145 sodium salt inhibited human platelet aggregation induced by arachidonic acid (AA), 9,11-methanoepoxy-PGH2 (U 46619), collagen, ADP or epinephrine with the IC50 being 0.047-0.146 microM in an in vitro system. When (+)-S-145 calcium salt dihydrate was administered orally to guinea pigs, it inhibited AA-, U-46619- or collagen-induced platelet aggregation dose-dependently with the minimum effective dose being 0.03 mg/kg, and the effective duration being maximally 3 hr. The inhibiting potency and effective duration of (+)-S-145 calcium salt dihydrate after multiple administrations, once a day (0.5 mg/kg) for 7 days, were almost the same as those after a single administration. Although (+)-S-145 sodium salt showed a partial agonist effect (shape change) on platelets in vitro, the effect diminished after pretreatment of the platelets with a lower dose of this compound. These data suggest that (+)-S-145 calcium salt dihydrate is an orally effective potent platelet aggregation inhibitor.     

Characterization of platelet thromboxane A2/prostaglandin H2 receptor by a novel thromboxane receptor antagonist, [3H]S-145

The specific binding sites for S-145, a novel thromboxane A2/prostaglandin H2 (TXA2/PGH2) receptor antagonist with weak partial agonistic activity, were studied in human platelet membranes. [3H]S-145 displayed high affinity and specificity, as well as saturable and displaceable binding, to a single class of recognition sites with the same maximum number of sites (2100 fmol/mg protein) as the other two TXA2/PGH2 receptor antagonists, [3H]SQ29,548 and [3H]ONO3708. Binding of S-145 to the platelet membranes was enhanced by divalent cations (Mg2+ and Ca2+), and the binding affinity in the presence of 20 mM MgCl2 was 0.75 nM, a value which was smaller than those of SQ29,548 (8.7 nM) and ONO3708 (3.7 nM). The rank order of potency (Ki) for a series of TXA2/PGH2 receptor antagonists to displace [3H]S-145 binding to the membranes was correlated with those determined from [3H]SQ29,548 or [3H]ONO3708 binding to the same preparations. Kinetic analysis for the binding of the above radiolabeled antagonist to the crude platelet membranes revealed that the dissociation rate constant (K-1) for S-145 was much smaller than that for other ligands in human, rat and rabbit platelets. The extremely slow dissociation of S-145 from the receptors may explain the long-lasting characteristic of this compound in vivo as well as the abolishment of partial agonistic activity.     

Effect of a novel thromboxane A2 receptor antagonist, S-145, on collagen-induced ECG changes and thrombocytopenia in rodents

The effects of S-145, a newly synthesized thromboxane, A2 (TXA2) receptor antagonist, were studied on collagen-induced changes of electrocardiograms (ECG) in rats and thrombocytopenia in rats and mice. Intravenous injection of collagen induced abnormal ECG changes such as elevation or depression of the ST segment, arrhythmia and in severe cases, cardiac arrest. These changes peaked at 3-5 min and lasted for 10 min. S-145 showed remarkable improvement of the ECG changes by both intravenous and oral administration, and the action lasted over 4 hr with 10 mg/kg, p.o. Reference compounds ONO-3708, dazoxiben and aspirin also improved the ECG changes significantly, but ticlopidine was ineffective. S-145 prevented the collagen-induced thrombocytopenia in rats but did not affect the increase in plasma TXB2 levels. S-145 also prevented collagen-induced thrombocytopenia in mice after either intravenous or oral administration in a dose-dependent manner. The efficacy of S-145 was 4-13 times greater than those of the reference compounds, and the duration of action was over 4 hr with 10 mg/kg, p.o. These results indicate that S-145 is a potent, orally, active and long-lasting TXA2 receptor antagonist, which will be promising as a drug for thromboembolism and ischemic heart disease caused by platelet activation.     

Antagonistic action of AA-2414 on thromboxane A2/prostaglandin endoperoxide receptor in platelets and blood vessels

AA-2414, (+/-)-7-(3,5,6-trimethyl-1,4-benzoquinon-2-yl)-7-phenylheptanoi c acid, inhibited the aggregation of guinea pig platelets induced by a prostaglandin endoperoxide (PGH2) analogue, U-44069 and the specific binding of another analogue, [3H]U-46619 to washed guinea pig platelets with IC50 values of 3.1 x 10(-7) and 8.2 x 10(-9) M, respectively. AA-2414 competitively inhibited the contraction of rabbit aorta and pig coronary arteries induced by U-44069 with pA2 values of 8.3 and 9.0, respectively. AA-2414 also inhibited the contraction of rabbit aorta induced by PGF2 alpha (pA2: 7.8) and the contraction of pig coronary arteries induced by PGF2 alpha, PGD2 and 9 alpha,11 beta-PGF2 with pA2 values of 7.8, 8.6 and 7.8, respectively. But, AA-2414 had no effect on the antiaggregatory effect of PGD2 on the aggregation of guinea pig platelets. In experiments with guinea pigs ex vivo, AA-2414 (0.1-1 mg/kg, p.o.) dose-dependently inhibited the platelet aggregation induced by U-44069; the inhibition at a dose of 1 mg/kg was 100% at 1 hr and was 89% even at 24 hr after the administration. The thromboxane (TX) A2/PGH2 receptor antagonistic action of AA-2414 was stereospecific. These results show that AA-2414 is a potent, orally active and long acting TXA2/PGH2 receptor antagonist. In addition, AA-2414 has PGF2 alpha, PGD2 and 9 alpha,11 beta-PGF2 antagonistic effects.     

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.     

Effects of ONO-3708, an antagonist of the thromboxane A2/prostaglandin endoperoxide receptor, on platelet aggregation and thrombosis

The beneficial effects of an antagonist of the thromboxane A2/prostaglandin endoperoxide receptor, 7-[2 alpha,4 alpha-(dimethylmethano)-6 beta-(2-cyclopentyl-2 beta- hydroxyacetamido)-1 alpha-cyclohexyl]-5(Z)-heptenoic acid (ONO-3708) on thrombosis were examined. ONO-3708 at 0.1-3 microM inhibited the human platelet aggregation induced by thromboxane A2, prostaglandin H2, collagen, ADP (secondary phase) and epinephrine (secondary phase) without affecting prostanoid synthesis and the content of cyclic AMP in platelets. The in vivo effects, on coronary thrombosis in this case, were examined in two canine models. ONO-3708, 3 to 300 micrograms/kg i.v., prevented dose dependently the coronary thrombosis induced by partial obstruction of the coronary artery. ONO-3708, 3 micrograms/kg per min i.v., significantly prevented electrically stimulated coronary thrombosis without affecting systemic blood pressure and heart rate. These results indicate that the thromboxane A2/prostaglandin endoperoxide receptor could play an important role in the pathogenesis of thrombosis and that ONO-3708 may have therapeutic advantages in preventing thrombosis.     

Effects of KW-3635, a novel dibenzoxepin derivative of a selective thromboxane A2 antagonist, on human, guinea pig and rat platelets.

We examined the binding of [3H]U-46619, a thromboxane A2 agonist, to human and guinea pig platelets and the binding of [3H]SQ 29,548, a thromboxane A2 antagonist, to human, rat and guinea pig platelets. KW-3635 (sodium (E)-11-[2-(5,6-dimethyl-1- benzimidazolyl)ethylidene]-6,11-dihydrodibenz[b,e]oxepin-2-c arboxylate monohydrate) concentration-dependently inhibited the [3H]U-46619 binding to human and guinea pig platelets with inhibition constants of 1.2 nM and 2.7 nM, respectively. KW-3635 also potently inhibited the [3H]SQ 29,548 binding to human and guinea pig platelets with inhibition constants of 1.9 nM and 3.2 nM, respectively. In contrast, KW-3635 was less active against thromboxane A2/prostaglandin H2 receptors in rat platelets with an inhibition constant of 97 nM. KW-3635 at 10(-5) M did not antagonize various receptors including prostaglandin E2, prostaglandin I2 and neurotransmitters. In addition, 10(-5) M KW-3635 did not alter the prostaglandin D2-induced cAMP accumulation in EBTr cells. KW-3635 was inactive towards thromboxane synthase, cyclooxygenase and prostaglandin I2 synthase up to 10(-5) M. KW-3635 slightly inhibited 5-lipoxygenase with an IC50 value of 71 microM. These data indicate that KW-3635 is a potent and selective non-prostanoic thromboxane A2 antagonist, and it can recognize the species differences in thromboxane A2/prostaglandin H2 receptors.     

Quinones. 4. Novel eicosanoid antagonists: synthesis and pharmacological evaluation

A new series of omega-phenyl-omega-quinonylalkanoic acids and related compounds was synthesized. The compounds were tested for their inhibitory effects on U-44069-induced contraction of the rabbit aorta. (+/- )-7-(3,5,6-Trimethyl-1,4-benzoquinon-2-yl)-7-phenylheptanoic acid (4d) (AA-2414) with pA2 value of 8.28 was one of the most potent compounds. Compound 4d inhibited U-46619-induced contraction of the guinea pig lung (pA2 = 8.29) and U-44069-induced aggregation of the guinea pig platelet (IC50 = 3.5 x 10(-7) M). Compound 4d displaced the binding of [3H]U-46619 to guinea pig platelets (IC50 = 7.4 x 10(-9) M). Compound 4d also showed very potent inhibitory effects with an MED of 0.3 mg/kg (po) on U-46619-, LTD4-, PAF-, or IgG1-induced bronchoconstriction in guinea pigs. The enantiomers of 4d were prepared. The R-(+) isomer 8a was active in both in vitro and in vivo tests, but the S-(-) isomer 8b was much less active. We concluded that the antiasthmatic effects of 4d were based mainly on the TXA2 receptor antagonistic action. In addition, compound 4d showed potent inhibitory effects on PGD2-, PGF2 alpha-, and 11-epi-PGF2 alpha-induced contraction of the guinea pig tracheal strips. The diverse inhibitory effects might be expressed in terms of eicosanoid-antagonistic activity.     

Effects of the new antiplatelet agent 2-methyl-3-(1,4,5,6-tetrahydronicotinoyl)pyrazolo[1,5-a]pyridine on platelet aggregation and thrombosis in experimental animals.

Antiplatelet and antithrombotic effects of KC-764 (2-methyl-3-(1,4,5,6-tetrahydronicotinoyl)pyrazolo[1,5-a]pyridine, CAS 94457-09-7) were studied. KC-764 inhibited arachidonic acid (AA)- and collagen-induced platelet aggregation with IC50s of 1.0 x 10(-8)-2.8 x 10(-7) mol/l for humans, rabbits, guinea pigs and dogs, and IC50s of 3.9 x 10(-6)-3.7 x 10(-5) mol/l for mice and rats in vitro. KC-764 inhibited AA- and collagen-induced aggregation with ID50s of 0.04-0.09 mg/kg p.o. in rabbits and dogs, and ID50 of 13.0 mg/kg p.o. in rats. These antiaggregatory activities of KC-764 were stronger than those of acetyl-salicylic acid (ASA), indometacin, cilostazol and ticlopidine. KC-764 inhibited the production of thromboxane B2 (TXB2) in rabbit platelet microsomes, washed platelets and reconstituted platelet rich plasma (RPRP) with IC50s of 2.9 x 10(-6) mol/l, 2.8 x 10(-7) mol/l and 4.3 x 10(-8) mol/l, respectively. The in vitro inhibitory activity of KC-764 on AA-induced platelet aggregation was more potent when RPRP was used rather than washed platelet suspension containing 30% rabbit plasma. ASA did not show such an augmentation. KC-764 prevented collagen- and AA-induced thrombosis at more than 1 mg/kg p.o. and more than 0.1 mg/kg i.v. in mice and rabbits. KC-764 showed the wider margin of dose between antiplatelet action and prolongation of bleeding time in rabbits than ASA and indometacin. These results indicated that KC-764 was a potent antithrombotic drug to prevent TXB2 production and less possible to induce untoward actions as compared with ASA or indometacin.     

PgH2 analogs as potential antiplatelet derivatives.

Previous observations implicating PgH2 as a direct activator of platelets suggested that derivatives of U46619, a well-characterized TxA2 receptor agonist having structural homology with PgH2, might possess antiplatelet activity. The present work describes the synthesis of [1S-(1 alpha,2 beta,3 alpha,4 alpha)]-3-[(tetrahydropyranyloxy)methyl]- 2-[2-[(triphenylmethyl)oxy]ethyl]-5-oxabicyclo[2.2.1]heptane (14) a potentially useful intermediate for the synthesis of various epoxymethano derivatives. The latter was converted to [1S-(1 alpha,2 beta (Z),3 alpha,4 alpha)]-7-[3-[[2- [(phenylamino)carbonyl]-hydrazino]methyl]-5-oxabicylo[2.2.1]hept-2 - yl]-5-heptenoic acid (23), an epoxymethano derivative of PgH2 containing a hydrazide lower side chain as previously used in the TxA2 antagonist, SQ 29,548. The intermediate 14 was also converted to [1S-(1 alpha,2 beta (Z),3 alpha,4 alpha)]-7- [3-[(hexylamino)methyl]-5-oxabicyclo[2.2.1]hept-2-yl]-5-heptenoic acid (25) which contained a simple aza side chain as used in earlier antagonists. Derivatives 23 and 25 appeared to be specific antagonists of the human platelet TxA2 receptor as evidenced by their inhibition of U46619 (1.5 microM) induced aggregation of human platelet rich plasma (IC50 = 22 and 7 microM, respectively), while having little effect on ADP (2 microM) induced aggregation at much higher concentrations. In addition, one of these derivatives, the bicycloamine 25, was shown to compete for [3H]U46619 binding to washed human platelets with an IC50 value of 25 microM, supporting the notion that these derivatives were acting at the thromboxane receptor. However, the potency of these derivatives was less than for previously reported TxA2 antagonists, suggesting that simple linear combinations of functionality from molecules active at the human platelet thromboxane receptor will be of limited predictive value.     

Comparison of vascular and platelet thromboxane A2/prostaglandin H2 receptors in the pig

We compared the properties of vascular and platelet thromboxane A₂/prostaglandin H₂ receptors in the pig. The binding profiles of U46619, several prostaglandins and thromboxane A₂/prostaglandin H₂ receptor antagonists to the aorta and platelet receptors were almost the same irrespective of whether the agonist ([³H]U46619) or the antagonist ([³H]SQ29,548) radioligand was used, except that the receptor density in the latter was 4 times higher than that in the aorta. The antagonists suppressed U46619-induced contraction of pig coronary artery and secondary aggregation of platelets at potencies comparable to their K₁ values in the binding experiments. On the other hand, the responses of the artery specimens to U46619 and the prostaglandins differed from those of the platelets. Thus, the binding sites in the vascular and platelet receptors seem to be the same or quite similar, but there seem to be different mechanism(s) leading the agonistic binding signal to final responses.     

Effects of ONO-3708, an antagonist of the thromboxane A2/prostaglandin endoperoxide receptor, on blood vessels

The pharmacological properties of a novel thromboxane A2/prostaglandin endoperoxide receptor antagonist, ONO-3708, on blood vessels were examined in vitro and in vivo. ONO-3708, 10 microM, inhibited the rabbit aorta contractions induced by thromboxane A2, prostaglandin H2, 11,9-epoxymethano-prostaglandin H2 (U-46619) or prostaglandin F2 alpha without affecting the contractions induced by angiotensin II, serotonin or norepinephrine. ONO-3708, at a concentration of 1 to 100 nM, appeared to be a competitive inhibitor of the contractile responses of the canine basilar artery to 9,11-epithio-11,12-methano-thromboxane A2 (STA2), U-46619 and PGF2 alpha, and a non-competitive inhibitor of the contractile responses to 15-hydroperoxy-eicosatetraenoic acid (15-HPETE). In in vivo studies, ONO-3708 (10 and 100 micrograms/kg per min i.v.) ameliorated the decrease in diameter of the basilar artery induced by the i.v. infusion of STA2 (0.1 microgram/kg per min) in cats. Furthermore, infusion of ONO-3708 (10 and 30 micrograms/kg per min i.v.) prevented the cerebral vasospasm in an experimental subarachnoid hemorrhage model in dogs. These results indicate that ONO-3708 is a potent antagonist of the thromboxane A2/prostaglandin endoperoxide receptor in vitro and in vivo and may be of therapeutic use in preventing cerebral vasospasm.     

Z-335, a thromboxane A2 receptor antagonist, suppresses the progression of arachidonic acid-induced hind limb gangrene in rats

We have developed a new rat model of gangrenous peripheral vascular disease with vascular injury and occlusive thrombi. Rat hind limb gangrene was induced by injecting arachidonic acid (2 mg/leg) into the femoral artery. Using this model, we evaluated the effect of a thromboxane A2 receptor antagonist, Z-335, on the progression of hind limb gangrene. Z-335 (10 mg/kg/d, p.o.) ameliorated arachidonic acid-induced hind limb gangrene. In contrast, daltroban (10 mg/kg/d, p.o.) and cilostazol (100 mg/kg/d, p.o.) tended to improve the hind limb gangrene but their effects failed to reach statistical significance. Z-335 (10 mg/kg, p.o.) inhibited U-46619-induced, but not collagen-induced, platelet aggregation in rat whole blood. Daltroban (10 mg/kg, p.o.) showed a tendency to inhibit U-46619-induced platelet aggregation. Cilostazol (100 mg/kg, p.o.) did not inhibit U-46619- or collagen-induced platelet aggregation. Histopathological examination of the injured paws showed that Z-355 (10 mg/kg, p.o.) had partly inhibited the formation of occlusive thrombi. These results indicate that the thromboxane A2 receptor antagonist Z-335 is effective against arachidonic acid-induced hind limb gangrene in rats. Our experiments suggest that Z-335 may be beneficial in the prevention of gangrenous peripheral vascular disease.     

Effects of STA(2), a thromboxane A(2) mimetic, in inducing airflow obstruction and airway microvascular leakage in guinea pigs

BACKGROUND: U-46619, a thromboxane A(2) (TXA(2)) mimetic, is shown to cause airway microvascular leakage, although the effects is weak when comparing with that to induce bronchoconstriction in guinea pigs. OBJECTIVE: In order to know the airway effect of TXA(2) more accurately, we have examined the effects of STA(2), a TXA(2) mimetic with higher affinity to TXA(2) (TP) receptors than U-46619, to induce airway microvascular leakage and airflow obstruction. METHODS: Anesthetized and ventilated guinea pigs were i.v. given STA(2) (3-30 nmol/kg) or U-46619 (3-100 nmol/kg) 1 min after i.v. Evans blue dye. STA(2)- and U-46619-induced increases in lung resistance (R(L)) was measured for 6 min. The amount of extravasated Evans blue dye in the lower airways was, then, examined as an index of leakage. In selected animals, specific TP receptor antagonists (10 microg/kg S-1452 or 10 mg/kg ONO-3708) were pretreated i.v. RESULTS: Both STA(2) and U-46619 induced significant increases in leakage and airflow obstruction. However, STA(2) induced a slow and significantly less increase in R(L) but caused a significantly greater increase in extravasation of Evans blue dye compared to U-46619. Specific TP receptor antagonists completely abolished both airway effects induced by STA(2) and U-46619. CONCLUSION: Our present results have supported a possibility that TXA(2) induces microvascular leakage as well as bronchoconstriction in the airways.     

Influence of SQ 29,548 on vasoconstrictor responses in the mesenteric vascular bed of the cat

The effects of SQ 29,548 on vasoconstrictor responses were investigated in the feline mesenteric vascular bed. Injections of the thromboxane (TX) A2 mimics, U46619 and U44069, caused dose-related increases in mesenteric arterial perfusion pressure. After administration of SQ 29,548, 0.5 mg/kg i.v, vasoconstrictor responses to U46619 and U44069 were reduced markedly whereas responses to prostaglandin (PG) F2 alpha, angiotensin II, vasopressin and BAY K 8644, an agent which enhances calcium entry, were not altered. The duration of the TXA2 receptor blockade was greater than 2 h and SQ 29,548 had no significant effect on mesenteric vasodilator responses to PGE2, isoproterenol, nitroglycerin, acetylcholine or bradykinin. SQ 29,548, at a dose of 0.5 mg/kg i.v., significantly reduced the response to TXB2, which had modest vasoconstrictor activity in the mesenteric vascular bed. However, when the dose of SQ 29,548 was reduced to 0.05 mg/kg i.v., responses to TXB2 were not altered, whereas responses to U46619 were significantly decreased. SQ 29,548 had no significant effect on vasoconstrictor responses to norepinephrine or to sympathetic nerve stimulation. The TXA2 receptor antagonist blocked the vasoconstrictor component of the biphasic response to the PG precursor, arachidonic acid, and the endoperoxide, PGH2. The results of these studies suggest that SQ 29,548 is a specific TX receptor antagonist in the mesenteric vascular bed, that the vasoconstrictor component of the biphasic response to arachidonic acid and PGH2 is due to formation of TXA2, and that endogenously formed TXA2 does not modulate adrenergic responses in the mesenteric circulation of the cat.     

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.     

ONO NT-126 is a potent and selective thromboxane A2 antagonist in human astrocytoma cells

Stimulation of thromboxane A2 (TXA2) receptors in human astrocytoma cells (1321N1) results in activation of phospholipase C via a pertussis toxin-insensitive G-protein. In the present study, the potency of a new TXA2 receptor antagonist, ONO NT-126, was examined with regard to receptor binding and phosphoinositide hydrolysis in human astrocytoma cells and was compared to that of the other known TXA2 antagonists. [3H]SQ29548 binding to membranes was inhibited by ONO NT-126 and the other TXA2 antagonists with Ki values (nM) of 0.09, 2.18, 8.35 and 25.9 for ONO NT-126, S-145, SQ29548 and ONO3708, respectively. STA2 and U46619, TXA2 receptor agonists, also inhibited [3H]SQ29548 binding with Ki (nM) of 25.1 and 233.5, respectively. STA2 and U46619 stimulated phosphoinositide hydrolysis in a concentration-dependent manner with EC50 values of 43.6 nM for STA2 and 1.2 microM for U46619, respectively. STA2 (1 microM)-induced phosphoinositide hydrolysis was also inhibited by TXA2 antagonists. The Ki values of TXA2 antagonists for the inhibition of phosphoinositide hydrolysis (nM) were 0.10 for ONO NT-126, 3.31 for S-145, 8.31 for SQ29548 and 19.49 for ONO3708 all of which were similar to those for receptor binding. The results indicate that ONO NT-126 is a potent and selective antagonist of TXA2 receptors in human astrocytoma cells.