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• W1993472744 abstract "SummaryTRA-418 is a novel compound that has been found in our screening for compounds having both thromboxane A2 (TP) receptor antagonistic and prostaglandin I2 (IP) receptor agonistic activities. In the binding assays, TRA-418 showed a 10-fold higher affinity to TP-receptors than IP-receptors. TRA-418 inhibited platelet aggregation induced by the TP-receptor agonist, U-46619 and by arachidonic acid at concentrations lower than those required for inhibition of ADP-induced aggregations. Furthermore, TRA-418 inhibited not only platelet aggregation induced by ADP alone, but also that induced by ADP in the presence of the TP-receptor antagonist, SQ-29548. When the IC50 values of TRA-418 for platelet aggregation were estimated in platelet preparations from monkeys, dogs, cats, and rats using ADP and arachidonic acid as the platelet stimulating agents, it was found that the values estimated in monkey platelets were quite similar to those estimated in human platelets. In ex vivo platelet aggregation in monkeys, TRA-418 exhibited significant inhibitory effects on arachidonic acid-induced aggregation in platelet preparations from monkeys treated at 3 µg kg min−1 or higher doses, where neither a significant decrease in blood pressure nor a significant increase in heart rate was observed. These results are consistent with the fact that TRA-418 has a relatively potent TP-receptor antagonistic activity together with a relatively weak IP-receptor agonistic activity. TRA-418 is a novel compound that has been found in our screening for compounds having both thromboxane A2 (TP) receptor antagonistic and prostaglandin I2 (IP) receptor agonistic activities. In the binding assays, TRA-418 showed a 10-fold higher affinity to TP-receptors than IP-receptors. TRA-418 inhibited platelet aggregation induced by the TP-receptor agonist, U-46619 and by arachidonic acid at concentrations lower than those required for inhibition of ADP-induced aggregations. Furthermore, TRA-418 inhibited not only platelet aggregation induced by ADP alone, but also that induced by ADP in the presence of the TP-receptor antagonist, SQ-29548. When the IC50 values of TRA-418 for platelet aggregation were estimated in platelet preparations from monkeys, dogs, cats, and rats using ADP and arachidonic acid as the platelet stimulating agents, it was found that the values estimated in monkey platelets were quite similar to those estimated in human platelets. In ex vivo platelet aggregation in monkeys, TRA-418 exhibited significant inhibitory effects on arachidonic acid-induced aggregation in platelet preparations from monkeys treated at 3 µg kg min−1 or higher doses, where neither a significant decrease in blood pressure nor a significant increase in heart rate was observed. These results are consistent with the fact that TRA-418 has a relatively potent TP-receptor antagonistic activity together with a relatively weak IP-receptor agonistic activity. Thromboxane A2 (TP) is a potent agent for stimulation of platelet aggregation and induction of vasoconstriction [1Hamberg M. Svensson J. Samuelsson B. Thromboxanes: a new group of biologically active compounds derived from prostaglandin endoperoxides.Proc Natl Acad Sci USA. 1975; 72: 2994-8Crossref PubMed Google Scholar]. Accordingly, TP-receptor antagonists or thromboxane synthase inhibitors were generally expected to be useful as antithrombotic drugs for acute coronary thrombosis and peripheral arterial thrombosis. In fact, TP-receptor antagonists have been demonstrated to exhibit potent antithrombotic effects in several animal models [2Van Der Giessen W.J. Zijlstra F.J. Berk L. Verdouw PD. The effect of the thromboxane receptor antagonist BM 13.177 on experimentally induced coronary artery thrombosis in the pig.Eur J Pharmacol. 1988; 147: 241-8Crossref PubMed Scopus (12) Google Scholar, 3Takiguchi Y. Wada K. Nakashima M. Comparison of the inhibitory effects of the TXA2 receptor antagonist, vapiprost, and other antiplatelet drugs on arterial thrombosis in rats. possible role of TXA2.Thromb Haemost. 1992; 68: 460-3Crossref PubMed Scopus (20) Google Scholar, 4Kotze H.F. Lamprecht S. Badenhorst P.N. Van Wyk V. Roodt J.P. Alexander K. In vivo inhibition of acute platelet-dependent thrombosis in a baboon model by Bay U3405, a thromboxane A2-receptor antagonist.Thromb Haemost. 1993; 70: 672-5Crossref PubMed Scopus (0) Google Scholar, 5Krupinski K. Ferber H. Breddin H.K. Bielawiec M. The antithrombotic effect of thromboxane receptor antagonist HN 11500 on thrombus formation in laser thrombosis model and platelet function tests.Acta Haematol Pol. 1994; 25: 235-42PubMed Google Scholar, 6Shirakura S. Higo K. Takeda M. Karasawa A. Antithrombotic effects of KW-3635, a thromboxane A2-receptor antagonist, in guinea pigs.Jpn J Pharmacol. 1994; 65: 93-8Crossref PubMed Google Scholar]. Nevertheless, TP-receptor antagonists show only a poor antithrombotic effect in human subjects [7Serruys P.W. Rutsch W. Heyndrickx G.R. Danchin N. Mast E.G. Wijns W. Rensing B.J. Vos J. Stibbe J. Prevention of restenosis after percutaneous transluminal coronary angioplasty with thromboxane A2-receptor blockade. A randomized, double- blind, placebo-controlled trial. Coronary Artery Restenosis Prevention on Repeated Thromboxane-Antagonism Study (CARPORT).Circulation. 1991; 84: 1568-80Crossref PubMed Google Scholar, 8Randomized trial of ridogrel, a combined thromboxane A2 synthase inhibitor and thromboxane A2/prostaglandin endoperoxide receptor antagonist, versus aspirin as adjunct to thrombolysis in patients with acute myocardial infarction. The Ridogrel Versus Aspirin Patency Trial (RAPT).Circulation. 1994; 89: 588-95Crossref PubMed Google Scholar, 9Norris R.M. White H.D. Hart H.H. Williams BF. Comparison of aspirin with a thromboxane antagonist for patients with prolonged chest pain and ST segment depression.N Z Med J. 1996; 109: 278-80PubMed Google Scholar]. Prostaglandin I2 (IP) receptor agonists have demonstrated their potent antiplatelet and antithrombotic effects in experimental animals [10Katsube N. Sakaguchi K. Fujitani B. Aishita H. Anti-platelet and anti-thrombotic effects of OP-41483 alpha-CD, a prostacyclin analogue, in experimental animals.Prostaglandins Leukot Essent Fatty Acids. 1993; 49: 795-804Abstract Full Text PDF PubMed Scopus (0) Google Scholar, 11Saito T. Saitoh S. Asakura T. Kanke M. Owada K. Maruyama Y. Prostacyclin analogue, beraprost, sustains recanalization duration after thrombolytic therapy in acute myocardial infarction model.Int J Cardiol. 1993; 38: 225-33Abstract Full Text PDF PubMed Scopus (0) Google Scholar]. In addition, IP-receptor agonists have been shown to reduce ischemic injury effectively in the acute myocardial infarction and stroke models [12Beitz A. Taube C. Beitz J. Goos H. Graff J. Nohring J. Lindenau K.F. Mest HJ. Influence of iloprost on eicosanoid generation and lipid levels in experimental myocardial ischemia in dogs.Prostaglandins Leukot Essent Fatty Acids. 1989; 35: 141-5Abstract Full Text PDF PubMed Google Scholar, 13Kanayama T. Kimura Y. Tamao Y. Mizogami S. Beneficial effects of a new prostacyclin analogue, KP-10614, on acute myocardial infarction in rats.J Cardiovasc Pharmacol. 1992; 20: 630-7Crossref PubMed Scopus (4) Google Scholar, 14Shima K. Umezawa H. Chigasaki H. Okuyama S. Araki H. Stable prostacyclin improves postischaemic microcirculatory changes in hypertensive rats.Acta Neurochir (Wien). 1995; 137: 89-95Crossref PubMed Scopus (0) Google Scholar]. Despite these useful properties as antithrombotic agents, the clinical application of IP-receptor agonists have been limited to pulmonary hypertension or moderate thrombotic disease such as arterial sclerosis obstruction because of their potent vasodilating effects [15Miyauchi Y. [Treatment of the peripheral vascular diseases with prostaglandin].Nippon Rinsho. 1994; 52: 2182-6PubMed Google Scholar, 16Nagaya N. Uematsu M. Okano Y. Satoh T. Kyotani S. Sakamaki F. Nakanishi N. Miyatake K. Kunieda T. Effect of orally active prostacyclin analogue on survival of outpatients with primary pulmonary hypertension.J Am Coll Cardiol. 1999; 34: 1188-92Crossref PubMed Scopus (220) Google Scholar]. In this connection, a number of in vitro studies have reported that remarkable synergistic effects on human platelet aggregation can be observed with concomitant use of an IP-receptor agonist and a TP-receptor antagonist [17Parise L.V. Venton D.L. Le Breton GC. Prostacyclin potentiates 13-azaprostanoic acid-induced platelet deaggregation.Thromb Res. 1982; 28: 721-30Abstract Full Text PDF PubMed Google Scholar, 18Sturzebecher S. Witt W. The PGI2-analogue iloprost and the TXA2-receptor antagonist sulotroban synergistically inhibit TXA2-dependent platelet activation.Prostaglandins. 1988; 36: 751-60Crossref PubMed Scopus (15) Google Scholar, 19Bertele V. De Gaetano G. Potentiation by dazoxiben, a thromboxane synthetase inhibitor, of platelet aggregation inhibitory activity of a thromboxane receptor antagonist and of prostacyclin.Eur J Pharmacol. 1982; 85: 331-3Crossref PubMed Google Scholar]. This finding suggests a useful application of an IP-receptor agonist in combination with a TP-receptor antagonist in vivo. In fact, we have confirmed that concomitant use of the IP-receptor agonist, beraprost, at a limited dose effectively potentiates the antithrombotic effects of aspirin and other TP-receptor antagonists without any severe hypotension [20Yamada N. Isogaya M. Ueno Y. Kumagai H. Ochi Y. Nishio S. Synergic effect of beraprost sodium, a PGI2 analogue, and aspirin on canine carotid artery thrombosis model induced by electrical stimulation.Thromb Haemost. 1993; 69: 591Google Scholar]. These results prompted us to screen compounds having a relatively potent TP-receptor antagonistic activity together with a relatively weak IP-receptor agonistic activity. As a result, we have found TRA-418 (Fig. 1) as a novel compound having such dual activities. In the present study, we have investigated the antiplatelet effects of TRA-418 in human and animal platelet preparations. TRA-418, beraprost [21Ohno K. Nagase H. Matsumoto K. Nishiyama H. Nishio S. . Hayashi O Yamamoto S Advances in Prostaglandin Thromboxane and Leukotriene Research. Raven Press, 1985Google Scholar], and ONO-1301 [22Hayashi K. Nagamatsu T. Oka T. Suzuki Y. Modulation of anti-glomerular basement membrane nephritis in rats by ONO-1301, a non-prostanoid prostaglandin I2 mimetic compound with inhibitory activity against thromboxane A2.Synthase Jpn J Pharmacol. 1997; 73: 73-82Crossref PubMed Google Scholar] were synthesized at our laboratory. The active isomer of beraprost, [3H]APS-314d sodium, and [3H]SQ-29548 were purchased from Daiichi Pure Chemicals (Tokyo, Japan). SQ-29548 [23Houston D.S. Shepherd J.T. Vanhoutte PM. Aggregating human platelets cause direct contraction and endothelium-dependent relaxation of isolated canine coronary arteries. Role of serotonin, thromboxane A2, and adenine nucleotides.J Clin Invest. 1986; 78: 539-44Crossref PubMed Google Scholar] and U-46619 were purchased from Cayman Chemical (MI, USA), arachidonic acid sodium salt and ADP from Sigma (MO, USA), collagen from Hormon Chemie (Munich, Germany), thrombin from Yoshitomi Pharmaceutical Industries (Osaka, Japan), epinephrine from Daiichi Pharmaceutical (Tokyo, Japan), 3.8% sodium citrate from Yamanouchi Pharmaceutical (Tokyo, Japan), a low molecular weight heparin sodium, dalteparin, from Kissei Pharmaceutical (Nagano, Japan), and sodium pentobarbital from Dainippon Pharmaceutical (Osaka, Japan). Blood samples were collected from healthy male human volunteers with the approval of the Institutional Ethics Committee of the Pharmaceutical Research Laboratories, Toray Industries, Inc. Written informed consent was obtained from each of the volunteers. The volunteers did not take any drugs for at least within 2 weeks before their participation in this study. Blood samples were also collected from male cynomolgus monkeys (Japan SLC, Shizuoka, Japan), male mongrel dogs (Japan Laboratory Animals, Tokyo, Japan), male ICO cats (Charles River Japan, Yokohama, Japan), and male Wistar rats (Japan SLC) in accordance with the guidelines for the animal care and use established at the Pharmaceutical Research Laboratories, Toray Industries, Inc. Blood was collected from human volunteers by venous puncture. Nine volumes of the collected blood were mixed with one volume of a solution containing (in mmol L−1) 85 trisodium citrate, 65 citric acid, 0.1 indomethacin and 2% glucose solution. Platelet-rich plasma (PRP) was prepared by centrifugation at 120 × g for 10 min at 4 °C. The platelets were washed twice in washing buffer, pH 6.5, containing (in mmol L−1) 115 NaCl, 4.3 K2HPO4, 24.4 Na2HPO4, 5 glucose, 1 EDTA2Na, and 0.01 indomethacin, and resuspended in 10 mmol L−1 Tris buffer, pH 7.4, containing 5 mmol L−1 MgCl2, and 2 mmol L−1 EDTA2Na. The platelets were alternately frozen and thawed three times and then centrifuged at 40 000 × g for 20 min at 4 °C. The membrane preparation was resuspended at 4 °C in assay buffer, pH 7.4, containing 50 mmol L−1 Tris and 5 mmol L−1 MgCl2, and stored at −80 °C until use. Radioligand binding was performed as modified method described previously [24Webb M.L. Liu E.C.K. Monshizadegan H. Hedberg H. Misra R.N. Goldenberg H. Harris DN. Binding and function of a poten new thromboxane receptor antagonist, BMS 180291, in human platelets.J Pharmacol Exp Ther. 1993; 264: 1387-94PubMed Google Scholar]. Briefly, for TP-receptor binding assay, human platelet membranes (10 µg protein) were incubated in assay buffer in the presence of the selective TP-receptor antagonist, [3H]SQ-29548 and TRA-418, for 30 min at 25 °C. For IP-receptor binding assay, human platelet membranes (10 µg protein) were incubated in assay buffer in the presence of the selective IP-receptor agonist, [3H]APS-314d sodium and TRA-418, for 60 min at 4 °C. The bound and free radiolabeled ligand in the reaction mixture were separated by rapid filtration through GF/C filters presoaked in 10 mmol L−1 Tris-HCl buffer. Filters were washed and the residual [3H]SQ-29548 or [3H]APS-314d sodium bound to the filter was determined by liquid scintillation counting. Specific binding was defined as the difference between total binding and non-specific binding, which was determined in the presence of 10 µmol L−1 SQ-29548 or 10 µmol L−1 APS-314d sodium. The rate of dissociation (Kd) and the maximum amount of binding (Bmax) were determined using standard Scatchard plotting techniques according to Web et al. [24Webb M.L. Liu E.C.K. Monshizadegan H. Hedberg H. Misra R.N. Goldenberg H. Harris DN. Binding and function of a poten new thromboxane receptor antagonist, BMS 180291, in human platelets.J Pharmacol Exp Ther. 1993; 264: 1387-94PubMed Google Scholar]. The Kd and Bmax values of [3H]SQ-29548 were 10.2 ± 0.51 nmol L−1 and 5.89 ± 0.62 pmol mg protein−1, respectively (n = 4), and, the Kd and Bmax values of [3H]APS-314d sodium were 14.3 ± 0.52 nmol L−1 and 6.08 ± 0.60 pmole/mg protein, respectively (n = 4). The inhibition constants (Ki values) were calculated according to Chen and Prusoff [25Cheng Y.C. Prusoff WH. Relation between the inhibition constant (Ki) and the concentration of inhibitor which cause 50 percent inhibition of an enzyme reaction.Biochem Pharmacol. 1973; 22: 3099-109Crossref PubMed Scopus (0) Google Scholar] and Hill [26Hill AV. A new mathematical treatment of ionic concentration in muscle and nerve under the action of electric currents, with a theory as to their mode of excitation.J Physiol (Lond). 1910; 40: 109-224Crossref Scopus (190) Google Scholar]. Nine volumes of blood collected from human volunteers or animals, except for rats, were mixed with one volume of 3.8% sodium citrate in a tube. As for blood collected from rats, nine volumes of blood were mixed with one volume of a 1 : 1 mixture of 3.8% sodium citrate and 30 U mL−1 sodium dalteparin. The citrated blood samples were immediately centrifuged at 90–140 g for 10 min at room temperature. The resulting supernatant was obtained as the PRP fraction. The remaining blood was further centrifuged at 1400 × g for 10 min. The resulting supernatant was the platelet-poor plasma fraction. Human PRP was pretreated with TRA-418, SQ-29548, beraprost or ONO-1301 at various concentrations for 1 minute before the addition of U-46619 (2 µmol L−1), arachidonic acid (600 µmol L−1), collagen (1 µg mL−1) or ADP (5 µmol L−1). The platelet stimulation with ADP was carried out in the presence or absence of SQ-29548 (10 µmol L−1). Also, the stimulation with arachidonic acid was performed in the presence or absence of epinephrine (0.5 µmol L−1). Similarly, animal PRP was pretreated with TRA-418 at various concentrations for 1 min before the addition of arachidonic acid (600 µmol L−1) or ADP (10 µmol L−1 in monkey, 20 µmol L−1 in dog and cat and 1 µmol L−1 in rat). The platelet stimulation with ADP in monkey, dog, and cat preparations was carried out in the presence of SQ-29548 (10 µmol L−1). The platelet stimulation with arachidonic acid in dog and cat was performed in the presence of epinephrine (0.5 and 3 µmol L−1, respectively), and that in rat preparations was carried out in the presence of epinephrine (3 µmol L−1) and CaCl2 (3 mmol L−1). Platelet aggregation was monitored by recording transmittance on a four-channel light transmission aggregometer (NBS Haematracer 601, MC Medical, Japan) for 5 min after the addition of a platelet-stimulating agent. For evaluating the effect of the test drugs, % inhibitions of platelet aggregation were calculated from the increases in transmittance observed with the test drugs, on the assumption that no inhibition was observed in the control incubations with vehicle alone. Human PRP was centrifuged at 1000 × g for 8 min at 14 °C in the presence of 0.01 mmol L−1 indomethacin and 0.01 mmol L−1 citric acid to isolate platelets. The platelets were then washed with washing buffer, pH 6.5, and resuspended in 10 mmol L−1 Hepes buffer, pH 7.4, containing 145 mmol L−1 NaCl, 5 mmol L−1 KCl, 1 mmol L−1 MgCl2, and 10 mmol L−1 glucose. Human washed platelets were suspended at 4 × 105 cells µL−1, and stimulated with U-46619 (4 µmol L−1) in the presence of fibrinogen (0.25 mg mL−1) and CaCl2 (1 mmol L−1). Human washed platelets were also suspended at 2 × 105 cells µL−1, and stimulated with thrombin (0.1 U mL−1) in the presence of CaCl2 (1 mmol L−1). Various concentrations of TRA-418, SQ-29548, beraprost or ONO-1301 in aqueous solutions were added to suspensions of washed platelets 1 min before the addition of a platelet-stimulating agent. Platelet aggregation was monitored with the four-channel light transmission aggregometer, as described above. Human PRP was incubated with TRA-418 at various concentrations for 10 min at 37 °C. The reaction was terminated by the addition of trichloroacetic acid in a concentration of 5%. The samples were alternately frozen and thawed three times and centrifuged at 13 000 × g for 30 min at 0 °C. The supernatant was washed three times with water-saturated diethyl ether. The resulting aqueous layer was evaporated to dryness, and the residue was dissolved in assay buffer. Cyclic AMP was determined by the enzyme immunoassay for cyclic AMP (Amersham, Bucks, UK). Cynomolgus monkeys were anesthetized with sodium pentobarbital (35 mg kg−1 i.v.) and given TRA-418 at doses of 3, 10, and 30 µg kg−1 min−1 or ONO-1301 at doses of 0.3, 1, and 3 µg kg min−1 both in a manner of dose escalation by infusion for 30 min for each dose via a catheter inserted into the forearm or saphenous vein. Arterial blood pressure and heart rate were monitored with a polygraph system through a femoral catheter during the infusion period, and the blood pressure and heart rate were recorded at baseline and at the end of the infusion at of each dose. Arterial blood was drawn to examine ex vivo platelet aggregation at baseline and at the end of the infusion at each dose. The collected blood samples were processed to prepare PRP for determination by the light transmission method, as described above. The data are shown as means ± SE. Statistical comparisons between means were performed by one-way anova and Dunnett's test at a significance level of P < 0.05. TRA-418 replaced both the specific TP receptor ligand, the [3H]SQ-29548, and the specific IP receptor ligand, [3H]APS-314d sodium, bound to human platelet membranes. Ki values were shown in Table 1.Table 1Binding affinities of TRA-418 on TP-receptors and IP-receptors in human platelet membraneTP-receptorIP-receptorKi (µmol L−1)0.050 ± 0.00690.43 ± 0.053Slope factor0.97 ± 0.091.96 ± 0.17TP- or IP-receptor binding assay was performed in the presence of [3H]SQ-29548 (10 nmol L−1) or [3H]APS-314d (10 nmol L−1) as the ligand, respectively. Values are means ± SE of four determinations. Open table in a new tab TP- or IP-receptor binding assay was performed in the presence of [3H]SQ-29548 (10 nmol L−1) or [3H]APS-314d (10 nmol L−1) as the ligand, respectively. Values are means ± SE of four determinations. TRA-418 inhibited in vitro platelet aggregation, regardless of the platelet-stimulating agent used, in a manner dependent on the concentration of the test drug. The IC50 values of TRA-418 estimated in the platelet aggregations induced by U-46619, arachidonic acid, and collagen were about 1/4 of those estimated in the aggregations induced by ADP, ADP + SQ-29548, and arachidonic acid + epinephrine (Table 2).Table 2Effects of TRA-418, SQ-29548, beraprost and ONO-1301 on in vitro platelet aggregation in human PRPAggregating AgentIC50 (μM) TRA-418SQ-29548BeraprostONO-1301Arachidonic acid0.56 ± 0.010.018 ± 0.00030.0059 ± 0.00020.18 ± 0.003Arachidonic acid +epinephrine2.6 ± 0.670.64 ± 0.0430.11 ± 0.0345.2 ± 0.17Collagen0.54 ± 0.0070.025 ± 0.0090.0076 ± 0.00090.17 ± 0.014U-466190.64 ± 0.110.021 ± 0.0030.0076 ± 0.00070.17 ± 0.011ADP1.8 ± 0.54>100.0057 ± 0.0010.17 ± 0.003ADP +SQ-295482.1 ± 0.14—0.005 ± 0.00020.22 ± 0.003Platelet aggregation was induced by arachidonic acid (600 μM) in the absence or presence of epinephrine (0.5 μM), by collagen (1 μg mL−1), by U-46619 (4 μM) or by ADP (5 μM) in the absence or in the presence of SQ-29548 (10 μM). Values are means ± S.E. of 3-4 determinations. Open table in a new tab Platelet aggregation was induced by arachidonic acid (600 μM) in the absence or presence of epinephrine (0.5 μM), by collagen (1 μg mL−1), by U-46619 (4 μM) or by ADP (5 μM) in the absence or in the presence of SQ-29548 (10 μM). Values are means ± S.E. of 3-4 determinations. The TP-receptor antagonist, SQ-29548, inhibited the platelet aggregations induced by U-46619, arachidonic acid, arachidonic acid + epinephrine, and collagen (Table 2). However, SQ-29548 did not show any clear inhibition of ADP-induced primary aggregation, but inhibited secondary aggregation. Concentration-dependent inhibitions of platelet aggregation were also observed with the IP-receptor agonists, beraprost and ONO-1301, regardless of the platelet-stimulating agent (Table 2). Unlike the case of TRA-418, these IP-receptor agonists inhibited platelet aggregation induced by any of the platelet-stimulating agents, except for arachidonic acid in the presence of epinephrine, to similar extents in the same concentration range. The IC50 values of these compounds estimated in the system induced by arachidonic acid in the presence of epinephrine were more than 10-fold of those estimated in the systems induced by the other platelet-stimulating agents. Table 3 shows the IC50 values of TRA-418 estimated in monkey, dog, cat, and rat PRP in terms of the arachidonic acid- and ADP-induced platelet aggregations. The IC50 value estimated in monkey PRP was quite similar to that estimated in human PRP.Table 3Effects of TRA-418 on in vitro platelet aggregation in monkey, dog, cat and rat PRPAggregating AgentIC50 (μM) MonkeyDogCatRatArachidonic acid0.56 ± 0.022.6 ± 0.51.1 ± 0.22.9 ± 0.6ADP2.7 ± 0.711 ± 1.017 ± 4.017 ± 4.0Platelet aggregation was induced by arachidonic acid (600 μM) or ADP (5 μM). Values are means ± S.E. of 4−7 determinations. Open table in a new tab Platelet aggregation was induced by arachidonic acid (600 μM) or ADP (5 μM). Values are means ± S.E. of 4−7 determinations. TRA-418, beraprost and ONO-1301 inhibited U-46619- and thrombin-induced platelet aggregation in human washed platelets in a concentration-dependent manner (Table 4). The IC50 value of TRA-418 for U-46619-induced platelet aggregation was approximately 1/7 less than that for thrombin-induced platelet aggregation. In contrast, neither beraprost nor ONO-1301 showed any remarkable differences in the IC50 values for U-46619-induced aggregation and for thrombin-induced aggregation. SQ-29548 inhibited the U-46619-induced aggregation, but not the thrombin-induced aggregation.Table 4Effects of TRA-418, SQ-29548, beraprost and ONO-1301 on in vitro platelet aggregation in human washed plateletsAggregating AgentIC50 (μM) TRA-418SQ-29548BeraprostONO-1301U-466190.053 ± 0.00210.24 ± 0.0380.007 ± 0.00180.050 ± 0.0060Thrombin0.38 ± 0.052>100.010 ± 0.00280.048 ± 0.0004Platelet aggregation was induced by U-46619 (4 μM) in the presence of fibrinogen (0.25 mg mL−1) and CaCl2 (1 mM) or by thrombin (0.1 U mL−1) in the presence of CaCl2 (1 mM). Values are means ± S.E. of 3 determinations. Open table in a new tab Platelet aggregation was induced by U-46619 (4 μM) in the presence of fibrinogen (0.25 mg mL−1) and CaCl2 (1 mM) or by thrombin (0.1 U mL−1) in the presence of CaCl2 (1 mM). Values are means ± S.E. of 3 determinations. Incubation of human PRP with TRA-418 resulted in concentration-dependent increases in cAMP content in platelets (Fig. 2). TRA-418 was administered to monkeys by infusion rates of 3, 10, and 30 µg kg−1 min−1 in a dose escalation manner. The infusion at each dose was performed for 30 min. Similarly, the IP-receptor agonist, ONO-1301, was administered by infusion consecutively at 0.3, 1, and 3 µg kg−1 min−1 in a dose escalation manner. As seen in Fig. 3, complete inhibitions of ex vivo arachidonic acid-induced platelet aggregation were observed with platelets from animals treated with TRA-418 at 3 µg kg−1 min−1 and the higher doses. With respect to ex vivo ADP-induced platelet aggregation, about 50% and almost complete inhibition were seen with platelets from animals treated with TRA-418 at 10 and 30 µg kg−1 min−1, respectively. Furthermore, TRA-418 did not cause any significant changes in blood pressure or heart rate in the dose range examined (Fig. 3). In contrast, infusion of ONO-1301 caused a dose-dependent inhibition of ex vivo ADP-induced platelet aggregation. With respect to the arachidonic acid-induced platelet aggregation, the infusion of ONO-1301 resulted in inhibitions of not greater than 50% even at the highest dose examined. Furthermore, the infusion of ONO-1301 caused decreases in blood pressure accompanied by the elevation of heart rate in a dose-dependent manner. In this study, we have examined the pharmacological profile of TRA-418 in terms of its antiplatelet effects. TRA-418 had about 10-fold higher affinity to TP-receptors than to IP-receptors, as shown by the Ki values determined in the binding assays using human platelet membranes. From the platelet aggregation studies, TRA-418 inhibited thromboxane A2 mediated platelet aggregation such as arachidonic acid and U-46619 aggregation. Furthermore, in an in vitro vascular smooth muscle assay, TRA-481 completely antagonized U-46619 induced vascular contraction in a competitive inhibitory manner (data not shown). From these results, we concluded that TRA-418 possesses a TP-receptor antagonist activity. TRA-418 inhibited the ADP-induced aggregation in the presence of SQ-29548 at the concentrations required for the inhibitions in the absence of SQ-29548. Furthermore, TRA-418 stimulated cAMP production in the platelet. The inhibitory effect of TRA-418 on the ADP induced platelet aggregation was exhibited at a much lower concentration than the concentration that increased cAMP production. This discrepancy also had been reported for other IP agonists (i.e. iloprost and beraprost) [27Nogimori K. Kajikawa N. Nishio S. Yajima M. Effects of TRK-100, a stable prostacyclin analogue, on regulation of cyclic AMP metabolism in platelets.Prostaglandins. 1989; 37: 205-12Crossref PubMed Scopus (14) Google Scholar, 28Nishio S. Matsuura H. Kanai N. Fukatsu Y. Hirano T. Nishikawa N. Kameoka K. Umetsu T. The in vitro and ex vivo antiplatelet effect of TRK-100, a stable prostacyclin analog, in several species.Jpn J Pharmacol. 1988; 47: 1-10Crossref PubMed Google Scholar, 29Armstrong R.A. Jones R.L. MacDermet J. Wilson NH. Prostaglandin endoperoxide analogues which are both thromboxane receptor antagonists and prostacyclin.Br J Pharmacol. 1986; 87: 543-51Crossref PubMed Scopus (0) Google Scholar]. From these results, including the binding assays, we concluded that TRA-418 possesses an IP-receptor agonistic activity. In human washed platelets, TRA-418 inhibited the U-46619-induced aggregation at concentrations lower than those for the inhibitions in PRP. The IC50 value in washed platelets was one-twelfth of that in PRP. In contrast, the IC50 value of beraprost in washed platelets was the same as that in PRP. These data are reasonably interpreted to mean that TRA-418, but not beraprost, has relatively high plasma proteins binding. The relatively high plasma protein binding might cause discrepancies that Ki values were extremely smaller than IC50 values in platelet aggregation studies. In the platelet aggregation experiments, it is noteworthy that TRA-418 inhibited the arachidonic acid-induced aggregation in the presence of epinephrine. The IP-receptor agonists, beraprost and ONO-1301, required remarkably higher concentration to inhibit the arachidonic acid-induced aggregation in the presence than in the absence of epinephrine, as evidenced of 20–30-fold higher IC50 values. These findings are in agreement with a previous report that IP-receptor agonists are relative insensitive to epinephrine-mediated platelet aggregation [30Zahavi M. Zahavi J. Relative insensitivity of epinephrine induced platelet aggregation to prostacyclin.Thromb Res. 1986; 44: 119-24Abstract Full Text PDF PubMed Google Scholar]. Furthermore, even the TP-receptor antagonist, SQ-29548, required remarkably higher concentration to inhibit the arachidonic acid-induced aggregation in the presence rather than in the absence of epinephrine. In the case of TRA-418, the IC50 value in the presence of epinephrine was only 4-fold of that in the absence of epinephrine. This smaller difference in IC50 seen with TRA-418 may be due to the increased effect of the TP-receptor activity by the IP-receptor antagonistic activity. Although the detailed mechanism for this remains to be clarified, these results suggest the usefulness of TRA-418 as an antithrombotic agent. In order to design ex vivo the experiment, we tried to find the animal what had a similar sensitivity to TRA-418 like as human, employing platelet function assay. The results showed that there were huge species differences among the animals. The most potent inhibitory effects were observed with PRP from monkeys and the inhibitory effects which observed in monkey PRP were quite similar to those observed in human PRP. From this evidence, the ex vivo experiment was carried out in monkeys. In the ex vivo study, the arachidonic acid-induced aggregation was completely inhibited by the infusion of TRA-418 even at the lowest dose examined. Infusion of TRA-418 also caused dose-dependent inhibitions of the ADP-induced platelet aggregation without any significant changes in blood pressure and heart rate even at the highest dose examined. These results suggested that the increased effect of the relatively potent TP-receptor antagonistic activity with the relatively weak IP-receptor agonistic activity of TRA-418 might result in less potent cardiovascular effects as compared to other IP agonists. In conclusion, TRA-418 has been shown to possess dual activities comprised of a TP-receptor antagonistic activity and an IP-receptor agonistic activity. The TP-receptor antagonistic and IP-receptor agonistic activities were demonstrated in in vitro platelet aggregation studies. The ex vivo experiment clearly illustrated the beneficial properties of the relatively weak IP-receptor agonistic activity and potent TP antagonist effect to avoid decreases in blood pressure. Thus, compounds having a relatively potent TP-receptor antagonistic activity together with a relatively weak IP-receptor agonistic activity may be useful as antithrombotic agents for treatment of thrombosis in the brain and heart in clinical settings. Authors sincerely thank Mr K. Hoshi and Mr T. Takeda who took a major role in the large-scale synthesis of TRA-418 and determination of adequate salt form of TRA-418 and to Dr William J Cady, PharmD for revision of English language." @default.
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• W1993472744 title "TRA-418, a novel compound having both thromboxane A2 receptor antagonistic and prostaglandin I2 receptor agonistic activities: its antiplatelet effects in human and animal platelets" @default.
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