Introduction

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ET-1, a potent and long-acting vasoconstricting peptide, was isolated from cultured medium of porcine endothelial cells in 1988 (Yanagisawa et al., 1988a). After its discovery, two other isopeptides were identified and named ET-2 and ET-3 (Inoue et al., 1989). The receptors corresponding to these isozymes were identified as ET_A and ET_B receptors. ET_A receptors have higher affinity for ET-1 and ET-2, whereas ET_B receptors have nearly equal affinity for ET-1, ET-2 and ET-3 (Arai et al., 1990; Sakurai et al., 1990).

It is reported that the level of circulating ET-1 is elevated in several cardiovascular diseases, such as hypertension, pulmonary hypertension, myocardial infarction, heart failure, renal failure and so on (for reviews, see Miller et al., 1993; Rubanyi and Polokoff, 1994). In addition, ET-1 shows various biological and pathophysiological effects on the cardiovascular system, such as smooth muscle contraction, cell proliferation (smooth muscle cells and mesangial cells, for example), hypertrophy of cardiac myocytes and positive inotropic and chronotropic effects (for reviews, see Rubanyi and Polokoff, 1994; Battistini et al., 1993). Thus ET-1 may be a factor in the pathogenesis of these diseases. The effect of ET-1 described above, which cause the circulatory dysfunction and cardiovascular remodeling, is mainly mediated by ET_A receptors (for review, see Rubanyi and Polokoff, 1994). On the other hand, it is well known that ET_B receptors mediate endothelium-dependent smooth muscle relaxation through the production of endothelium-derived relaxing factor/NO and prostacyclin (De Nucci et al., 1998; Takayanagi et al., 1991), but the pathophysiological role of ET_B receptors is still unclear. Therefore, the ET antagonist, which has ET_A receptor blocking action, is expected to be a therapeutic agent for these diseases.

In 1993 the first orally active ET receptor antagonist, Ro 46-2005, a sulfonamide derivative, was reported (Clozel et al., 1993). After that, bosentan (Clozel et al., 1994), which was modified from Ro 46-2005 and showed higher potency than Ro 46-2005, was reported to have beneficial effects on various cardiovascular models, such as hypertension (Li et al., 1994), pulmonary hypertension (Eddahibi et al., 1995; Chen et al., 1995), cardiac hypertrophy (Kadoura et al., 1996), myocardial ischemia/reperfusion (Wang et al., 1995) and congestive heart failure (Shimoyama et al., 1996; Mulder et al., 1997). However, both compounds are an ET_A/ET_B mixed antagonist and are not satisfied with the potency. Thus, on the basis of the principle that the ET_A receptor antagonist is preferable as a therapeutic agent for cardiovascular diseases, we modified the sulfonamide derivatives and succeeded in finding a novel ET receptor antagonist, T-0201 (fig. 1). In present study, we characterized the pharmacological properties of T-0201 in vitro and in vivo.

View larger version (7K): [in this window] [in a new window]   Fig. 1.   Structure of T-0201.

	Materials and Methods

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Binding studies on membranes. Membranes of CHO cells expressing cloned human ET_A or ET_B receptors were obtained from DuPont NEN through Daiichi Kagaku Yakuhin (Tokyo, Japan). Binding assays were performed in a total volume of 300 µl containing [¹²⁵I]-ET-1 (20 pM); various concentrations of T-0201, bosentan or (+) SB 209670; vehicle (30% DMSO containing 0.1% BSA, for total binding) or 200 nM ET-1 (for nonspecific binding); and incubation buffer (50 mM Tris-HCl, pH 7.4 containing 0.5 mM CaCl₂, 0.1% BSA and 0.05% Tween 20). After 2 hr of incubation at 37°C, bound and free radioligands were separated by filtration using Whatman GF/B glass-fiber filters. The radioactivity was measured with a gamma counter. Specific [¹²⁵I]-ET-1 binding was calculated as the difference between total binding and nonspecific binding. The inhibition constant (K_i) values were calculated from the following equation (Cheng and Prusoff, 1973): K_i = IC₅₀/(1 + [L]/K_d), where IC₅₀ is the 50% inhibitory concentration calculated from the displacement curve, [L] is the concentration of [¹²⁵I]-ET-1 in the tube, and K_d is the equilibrium dissociation constant calculated from Scatchard plot analysis (Scatchard, 1949).

Cell culture. A10 cells (for ET_A receptors; Lin et al., 1991) and GH cells (for ET_B receptors; Mihara and Fujimoto, 1991) were obtained from the American Type Culture Collection through Dainippon Seiyaku (Osaka, Japan). The cells were cultured in Dulbecco's modified Eagle's medium containing 10% FBS, 50 µg/ml streptomycin and 50 U/ml penicillin under humidified 5% CO₂-95% air at 37°C.

Binding studies on cultured cells. Binding studies were performed as Clozel et al. described previously (Clozel et al., 1989) with minor modification. Briefly, A10 cells (1 × 10⁵ cells/well) or GH cells (5 × 10⁴ cells/well) were seeded in 24-well plates and cultured for 1 or 2 days. The cells were washed 2 times with binding buffer (Hank's balanced salt solution containing 0.1% BSA) and were incubated at 37°C with [¹²⁵I]-ET-1 (20 pM) in the presence of various concentrations of T-0201, vehicle (30% DMSO containing 0.1% BSA, for total binding) or 200 nM ET-1 (for nonspecific binding) in binding buffer in a total volume of 500 µl. After 2 hr of incubation, the cells were washed with ice-cold binding buffer and were lysed in 1 N NaOH. The lysate was neutralized with the same volume of 1 N HCl, and then the radioactivity was measured with a gamma counter (ARC-360, Aloka, Japan). Specific [¹²⁵I]-ET-1 binding was calculated as the difference between total binding and nonspecific binding.

Saturation binding. A10 and GH cells were used to perform saturation binding studies with 10, 20, 50, 75, 100 and 200 pM [¹²⁵I]-ET-1 in the absence or the presence of 0.3 nM (in A10 cells) or 30 nM (in GH cells) T-0201. Experimental conditions were the same as those described above.

Isolated tissue contraction. Male Sprague-Dawley (SD) rats (9-11 weeks old; Charles River Japan, Yokohama, Japan) were anesthetized with ether, and male Japanese White rabbits (2.5-3.5 kg; Kitayama Labs, Nagano, Japan) were anesthetized with sodium pentobarbital (60 mg/kg i.v.). The thoracic aorta, trachea (rat) or pulmonary artery (rabbit) was isolated, cleared of adhering fat and connecting tissue and cut into 4-mm rings. The endothelium or epithelium was denuded by gentle rubbing of the intimal surface with cotton swab. The rings were suspended in a 10-ml organ bath containing Krebs-bicarbonate solution (in mM): 118 NaCl, 4.7 KCl, 2.5 CaCl₂, 1.2 KH₂PO₄, 1.2 MgSO₄, 25 NaHCO₃, 11 glucose, which was bubbled with 95% O₂-5% CO₂ at 37°C. The ring segment was attached to an isometric transducer (UL-10, Minebea, Tokyo, Japan) connected to a carrier amplifier (AP-601G, Nihon Kohden), and isometric tension was recorded by a pen recorder (NC6625, Graphtec, Tokyo, Japan). The resting tension was adjusted to 1.5 g (aorta) or 1.0 g (trachea and pulmonary artery). After a 60-min equilibration period, the maximal contraction induced by 40 mM (aorta), 50 mM (trachea) or 80 mM (pulmonary artery) KCl was determined as the reference contraction. The rings were then washed and stretched to a resting tension again. The preparations were incubated with various concentrations of T-0201 or vehicle (DMSO) for 20 min, and then ET-1 (aorta and pulmonary artery) or S6c (trachea) was applied cumulatively (0.1-300 nM). In any given experimental set, preparations from the same rat or rabbit were allotted for vehicle and for one of the concentrations of T-0201. The results were expressed as a percentage of the KCl-induced reference contraction. The pA₂ value, as the potency of antagonism, was determined using the equation pA₂ = log(concentration ratio  1)  log[B] where the concentration ratio is the ratio of the EC₅₀ value with to that without the antagonist, and [B] is the concentration of the antagonist (Arunlakshana and Schild, 1956).

Measurement of blood pressure in anesthetized rats. Male SD rats 10 to 11 weeks old were anesthetized with thiobutabarbital sodium (100 mg/kg i.p.). After tracheal intubation, the rats were artificially ventilated with room air using a rodent ventilator (SN-480-7, Shinano, Tokyo, Japan) at a volume of 1 ml/100 g b.wt. and a respiration rate of 70 strokes/min. The left femoral artery was cannulated and connected to a pressure transducer (TP-400T, Nihon Kohden, Tokyo, Japan) for measuring blood pressure. The right femoral vein was cannulated for administration of drugs. After stabilization of blood pressure, atropine (0.4 mg/kg i.v.) and mecamylamine (3 mg/kg i.v.) were injected to inhibit autonomic nervous reflexes, and then various doses of T-0201 (0.01-10 mg/kg) or vehicle (saline) were administered i.v. as a bolus. Five minutes later, big ET-1 (1 nmol/kg) was injected i.v.

Drugs. T-0201, bosentan and (+)SB 209670 were synthesized at the Lead Optimization Research Laboratory, Tanabe Seiyaku Co., Ltd. (Saitama, Japan). Thiobutabarbital sodium was synthesized at Tanabe R&D Service Co., Ltd. (Saitama, Japan). For the binding study, T-0201, bosentan and (+)SB 209670 were dissolved in 0.1% BSA containing 30% DMSO. T-0201 was dissolved in 100% DMSO for the isolated tissue contraction study, in saline for i.v. administration and in 0.25% CMC for oral administration. ET-1 and S6c were obtained from Peptide Institute (Osaka, Japan). [¹²⁵I]-ET-1 was purchased from Amersham Co. (Des Plaines, IL). Atropine sulfate was obtained from Wako Junyaku (Osaka, Japan). BSA and mecamylamine were purchased from Sigma Chemical Co. (St. Louis, MO). Culture reagents were from Gibco BRL (Tokyo, Japan). All other chemicals were of analytical grade.

Data analysis. All results were expressed as mean ± S.E.M. Statistical analysis was performed by one-way analysis of variance and the Bonferroni/Dunn test or Scheffé's test. Differences were considered statistically significant at a value of P < .05.

	Results

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Binding studies. In binding assays, T-0201 inhibited the specific binding of [¹²⁵I]-ET-1 to human cloned ET_A and ET_B receptors expressed in CHO cells in a concentration-dependent manner (fig. 2; table 1). The K_i value of T-0201 was 0.015 ± 0.004 nM for human cloned ET_A receptors. In contrast, T-0201 weakly inhibited [¹²⁵I]ET-1 binding to human cloned ET_B receptors (K_i = 41 ± 21 nM). Binding experiments were also performed for bosentan and (+)SB 209670 (table 1). The respective K_i values for human cloned ET_A receptors were 81 ± 26 nM and 0.51 ± 0.16 nM, and those for human cloned ET_B receptors were 140 ± 26 nM and 6.2 ± 2.9 nM.

View larger version (17K): [in this window] [in a new window]   Fig. 2.   Effect of T-0201 on [125I]-ET-1 binding to the membrane preparation of human cloned ETA (; n = 7) and ETB receptors (; n = 6) expressed on CHO cells. Results are expressed as mean ± S.E.M.

View larger version (23K): [in this window] [in a new window]   Fig. 3.   Typical illustrations of saturation curve (upper) and Scatchard plot of [125I]-ET-1 saturation binding (lower). Panels A and C) Specific binding of [125I]-ET-1 to A10 cells in the absence () or the presence of 0.3 nM T-0201 (). Panels B and D) Specific binding of [125I]-ET-1 to GH cells in the absence () or the presence of 30 nM () T-0201.

In vitro functional studies. T-0201 exhibited no agonistic action at any concentration on the isolated tissue preparation used in these studies. In the endothelium-denuded rat aorta rings, T-0201 (1, 3 and 10 nM) shifted the concentration-response curve of ET-1-induced contraction to the right in a concentration-dependent manner without affecting maximal response (fig. 4A). The pA₂ value of T-0201 analyzed by Schild plot was 9.0 ± 0.2 (slope = 1.22 ± 0.14). In rat tracheal rings, T-0201 (0.1, 1 and 10 µM) also inhibited the S6c-induced contraction (fig. 4B), but the pA₂ value of 6.8 ± 0.3 (slope = 0.97 ± 0.15) was lower than that in the rat aorta. Similar experiments were performed for bosentan (data not shown); its pA₂ values were 7.4 ± 0.2 (slope = 0.87 ± 0.13) for the rat aorta and 7.3 ± 0.3 (slope = 0.99 ± 0.12) for the rat trachea, respectively.

View larger version (18K): [in this window] [in a new window]   Fig. 4.   Effect of T-0201 (, vehicle; , 1 nM; , 3 nM; , 10 nM; , 0.1 µM; , 1 µM; , 10 µM) or of its vehicle on the concentration-response curves of ET-1-induced contraction of the isolated endothelium denuded rat aorta (panel A, n = 5) and S6c-induced contraction of the isolated epithelium denuded rat trachea (panel B, n = 4). Values are expressed as mean ± S.E.M.

View larger version (23K): [in this window] [in a new window]   Fig. 5.   Effect of T-0201 (, 1 µM; , 10 µM; , 100 µM) or of its vehicle on concentration-response curve of ET-1-induced contraction of the isolated endothelium-denuded rabbit pulmonary artery (n = 4). Values are expressed as mean ± S.E.M.

In vivo studies. In anesthetized rats, exogenous big ET-1 (1 nmol/kg i.v.) increased mean blood pressure, and maximal response was 65.3 ± 5.8 mmHg observed within 20 min in vehicle group. When T-0201 was injected as an i.v. before big ET-1 administration, it had no effect on the base-line pressure. T-0201 (0.1, 1 and 10 mg/kg i.v.) significantly and dose-dependently inhibited the big ET-1-induced pressor response by 48%, 72% and 97%, respectively (fig. 6).

View larger version (23K): [in this window] [in a new window]   Fig. 6.   Effect of the i.v. administration of T-0201 (0.01, 0.1, 1 and 10 mg/kg) or vehicle on big ET-1 (1 nmol/kg i.v.)-induced pressor response in the anesthetized rats (n = 6). Base values were 72.7 ± 3.2 (vehicle), 71.0 ± 3.3 (0.01 mg/kg), 71.8 ± 1.4 (0.1 mg/kg), 70.2 ± 2.6 (1 mg/kg) and 72.2 ± 2.0 mmHg (10 mg/kg), respectively. Values are expressed as mean ± S.E.M. ***P < .001 compared with vehicle.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Effect of the p.o. administration of T-0201 (0.01-10 mg/kg) or vehicle on big ET-1 (1 nmol/kg i.v.)-induced pressor response in the anesthetized rats (n = 6-8). Base values were 68.0 ± 4.1 (vehicle), 72.0 ± 5.6 (0.01 mg/kg), 63.8 ± 2.1 (0.1 mg/kg), 68.7 ± 2.3 (1 mg/kg) and 61.3 ± 3.6 mmHg (10 mg/kg), respectively. Values are expressed as mean ± S.E.M. ***P < .001 compared with vehicle.

View larger version (40K): [in this window] [in a new window]   Fig. 8.   Duration of the inhibitory effect of orally administered T-0201 (hatched column, 0.1 mg/kg; solid column, 1 mg/kg) or vehicle (open column) on big ET-1 (1 nmol/kg i.v.)-induced pressor response in the anesthetized rats (n = 6). Base values of vehicle, T-0201 0.1 mg/kg and T-0201 1 mg/kg groups follow. 1 hr: 64.8 ± 3.9, 62.7 ± 4.0 and 58.8 ± 3.1 mmHg; 4 hr: 66.0 ± 4.6, 70.2 ± 3.8 and 58.0 ± 5.3 mmHg; 8 hr: 68.3 ± 4.6, 66.3 ± 2.3 and 67.7 ± 2.3 mmHg; 24 hr: 57.7 ± 7.7, 63.3 ± 2.3 and 67.7 ± 3.8 mmHg, respectively. Values are expressed as mean ± S.E.M. **P < .01 and ***P < .001 compared with vehicle.

	Discussion

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In this study, we demonstrated the pharmacological profile of T-0201, a novel nonpeptide ET-antagonist: 1) In the binding study, T-0201 has higher affinity for ET_A receptors. 2) In the isolated tissue, T-0201 has potent ET_A antagonistic action. 3) T-0201 has orally active, bioavailable and long-lasting properties in vivo.

In the binding study using human cloned ET-receptors, expressed on CHO cell membrane, T-0201 showed about 3000-fold higher affinity for ET_A receptors (K_i = 0.015 nM) than for ET_B receptors (K_i = 41 nM). The K_i value of bosentan was 81 nM for ET_A receptors and 140 nM for ET_B receptors, and the K_i values of (+)SB 209670 were 0.51 nM and 6.2 nM, respectively. Thus T-0201 shows higher affinity and selectivity for ET_A than do bosentan and (+)SB 209670, which are known as ET_A/ET_B mixed antagonists (Clozel et al., 1994; Ohlstein et al., 1994). On the other hand, A10 and GH cells exclusively express ET_A and ET_B receptors, respectively (Lin et al., 1991; Mihara and Fujimoto, 1991). In competitive binding study with these cells, T-0201 also showed higher selectivity for ET_A receptors (K_i = 0.10 nM and 5.8 nM, respectively).

In the saturation binding studies, T-0201 increased the K_d values of [¹²⁵I]-ET-1 binding to both A10 and GH cells but did not affect the values of B_max, which suggests that T-0201 shows competitive antagonism.

Table 2 shows the specificity of T-0201 to the ET receptor, which we demonstrated using many kinds of receptors and enzymes. T-0201 at a concentration of 5 or 50 µM did not affect the radioligands binding or enzyme activities in any of 26 receptors and seven enzymes except the ET receptor. Thus T-0201 was specific for the ET receptor, especially for the ET_A receptor.

ET-1 is a potent constrictor of smooth muscle. It has been reported that the ET-1-induced smooth muscle contraction is mediated mainly by ET_A receptors (for review, see Rubanyi and Polokoff, 1994). However, recent reports indicate that the contraction mediated by ET_B receptors is also observed in some smooth muscle, including rat trachea (Henry, 1993) and rabbit pulmonary artery (Panek et al., 1992). In order to clarify the functional effect of T-0201, we also investigated the effect of T-0201 on the contractile response of isolated tissues, using the rat aorta for ET_A-mediated contraction (Panek et al., 1992) and the rat trachea for ET_B-mediated contraction. Pretreatment of T-0201 shifted the concentration-response curve of ET-1 to the right in the rat aorta without affecting the maximal response (pA₂ = 9.0). On the other hand, higher concentrations of T-0201 were needed to inhibit the S6c-induced contraction in the rat trachea (pA₂ = 6.9). Thus, in isolated tissues, T-0201 showed higher selectivity for ET_A receptors. In contrast, bosentan showed similar pA₂ values in the rat aorta (ET_A) and trachea (ET_B), in which the pA₂ values were 7.4 and 7.3, respectively, a result that suggests low selectivity between ET_A and ET_B receptors. All these results were consistent with that in the binding study. Therefore, although both T-0201 and bosentan are sulfonamide derivatives, there are apparent differences in their potency and selectivity to ET_A receptors.

A recent report suggested that ET_A and ET_B receptors coexist on the vascular smooth muscle of rabbit pulmonary artery and that both receptors cause contractile response (LaDouceur et al., 1993). Another report showed that the ET-1-induced contraction was not completely inhibited by individual treatment with BQ-123, an ET_A receptor antagonist, or BQ-788, an ET_B receptor antagonist, but was completely inhibited by the combination of these two antagonists (Fukuroda et al., 1994). These investigators eventually concluded that blockade of both receptors would be necessary for the inhibition of some ET_A/ET_B composite types of responses. T-0201 was an ET_A-selective antagonist as described above, but considering the result of the rat trachea experiments, the high concentration (0.1-10 µM) of T-0201 was also able to inhibit ET_B-mediated contraction. Thus the inhibitory effect of the high concentration of T-0201 (1-100 µM) on the ET-1-induced contractile response in the rabbit pulmonary artery may be due to the antagonism of both ET_A and ET_B receptors.

ET-1 is converted from big ET-1 by endothelin-converting enzyme (ECE) in vivo (Yanagisawa et al., 1988a). When ET-1 is administered i.v. to rats, a biphasic response, consisting of first a transient depressor response and then a pressor response, is observed (Yanagisawa et al., 1988a; Yanagisawa et al., 1988b). In contrast to ET-1, the i.v. administration of big ET-1 produced only the pressor response. The pressor response induced by exogenous big ET-1 is considered to be mediated mainly by ET_A receptors in the rat (Haleen et al., 1993). The i.v. administration of T-0201 at a lower dose of 0.1 mg/kg significantly inhibited the pressor response to exogenous big ET-1 (1 nmol/kg i.v.). This result indicates that T-0201 has a potent antagonistic activity in vivo as well as in vitro. The fact that T-0201 has no effect on ECE activity (data not shown) suggests that the inhibitory effect of T-0201 on the pressor response to exogenous big ET-1 is based on its ET receptor antagonistic action. Administered p.o., T-0201 produced a significant inhibition of the big ET-1-induced pressor response at doses (0.1 mg/kg) similar to that used in i.v. administration. These results indicate that T-0201 has high oral activity and high bioavailability.

In addition, having a long duration of action is often useful in therapeutic agents for chronic diseases such as hypertension, heart failure and renal failure. In order to clarify the duration of action of T-0201, we examined the inhibitory effect of T-0201 (0.1 and 1 mg/kg p.o.) on the pressor response to exogenous big ET-1 at 1, 4, 8 and 24 hr after administration. The maximal inhibitory effect of T-0201 was obtained 1 hr after dosing at both doses, which suggests that the duration of action depended on the dose of T-0201. These results indicate that T-0201 has a long-lasting effect in vivo.

In summary, T-0201 is a potent, orally active, long-lasting ET antagonist with high selectivity to ET_A receptors. Therefore, T-0201 may be a valuable tool in understanding the role of ETs in physiological and pathophysiological processes. Further, it should be considered as a therapeutic agent for various diseases to which endogenous ETs contribute.