Introduction

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In 1988 (Yanagisawa et al.), endothelin-1 (ET-1) was discovered and found to be one of the most potent known vasoconstrictive peptides. ET-1 is a member of a family of 21-amino acid vasoactive peptides, including ET-1, ET-2, and ET-3 from mammals, as well as the sarafotoxins from snake venom. ET receptors are widely distributed in smooth muscle, endothelial, epithelial, central nervous system, and neuroendocrine tissues. Two high-affinity ET receptor subtypes (ET_A and ET_B) have been identified, cloned, and expressed from mammalian tissues, including human (Arai et al., 1990, Sakurai et al., 1990), and are members of the G protein-linked receptor superfamily (Grai and Webb, 1995). The ET_A receptor subtype has higher affinity for ET-1 compared with ET-3 and a nonmammalian member of the ET family sarafotoxin S6c. The ET_B receptor subtype has equal affinity for ET-1, ET-3, and S6c. Stimulation of either ET_A or ET_B receptors causes smooth muscle contraction. In contrast to ET_B receptors on smooth muscle, ET_B receptors located on endothelial cells mediate relaxation of associated vascular smooth muscle through local release of nitric oxide and other relaxant factors. In addition, ET_B receptors located on endothelial cells appear to function as ET-1 clearance receptors (Fukuroda et al., 1994).

We provided the ET_A receptor-selective cyclic pentapeptide antagonist BQ-123 as one of the first pharmacological tools to study the endothelin system (Ihara et al., 1992a). Since its discovery, BQ-123 has been distributed widely to investigators and used to study a number of different disease states. We continued our effort to make more potent, long-lasting, orally active ET receptor antagonists to study the role of ET in pathophysiology. Recently, we developed J-104132 [(+)-(5S,6R,7R)-2-butyl-7-[2-((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3,4-methylenedioxyphenyl)cyclopenteno[1,2-b]pyridine-6-carboxylic; also referred to as L-753,037] as a potent, orally active mixed ET_A/ET_B receptor antagonist.

	Experimental Procedures

Top Abstract Introduction Experimental Procedures Results Discussion References

In Vitro Studies

Binding Assays. In competition studies, high-affinity specific binding of [¹²⁵I]ET-1 was determined using Chinese hamster ovary (CHO) cells expressing cloned human ET_A or ET_B receptors and membrane preparations from frozen human uterus or hippocampus as described previously (Williams et al., 1995). For saturation/reversibility studies, CHO cells expressing cloned human ET_A or ET_B receptors were preincubated for 60 min in the presence and absence of 0.04 or 0.2 nM J-104132 for ET_A and ET_B, respectively. The K_i value for J-104132 was calculated from the relation:
where K_d is the control value and K_d' is the value in the presence of 0.04 or 0.2 nM J-104132 in CHO cells expressing human ET_A and ET_B receptors, respectively.

Phosphatidyl Inositol Hydrolysis Assays. Phosphatidyl inositol hydrolysis assay was performed as described previously (Williams et al., 1995). CHO cells expressing the cloned human ET_A receptor were labeled overnight with myo-[³H]inositol. Cells were harvested the next day with an EDTA-containing solution and resuspended in Krebs-Ringer buffer containing human serum albumin (HSA) and LiCl. Total inositol phosphates were extracted by ion exchange chromatography. Potential agonist activity was assessed by treating the cells with 3 nM J-104132 in the absence of ET-1 and measuring inositol phosphate accumulation in relation to basal levels in these cells.

Rabbit Iliac Artery and Pulmonary Artery Contractile Assays. Male albino rabbits (2-3 kg) were anesthetized with pentobarbital sodium (40 mg/kg i.v.) and exsanguinated. Iliac and pulmonary arteries were isolated. The cleaned segments were cut into spiral preparations. Endothelium was mechanically removed from preparations with the use of wet filter paper. The preparations were then placed in 5-ml organ baths containing modified aerated Krebs-Henseleit solution maintained at 37°C. Mechanical responses were recorded isometrically by a multichannel polygraph (model RMP-6018; Nihon Kohden, Tokyo, Japan). The tissue was equilibrated for at least 60 min with initial tension of 1g and then contracted by adding 50 mM KCl (reference contraction).The concentration-contraction curves for ET-1 or BQ-3020 (ET_B agonist) were obtained by the cumulative addition of these substances to the organ bath. J-104132 or vehicle was added 20 min before the addition of ET-1 or BQ-3020. The experiments also were conducted in the presence and absence of 3% HSA or 3% rat serum albumin (RSA) in rabbit iliac arteries to estimate the effect of plasma protein on the in vitro potency of J-104132. ET-1 and BQ-3020 were dissolved in PBS (pH 7.4) containing 0.05% BSA. J-104132 was dissolved in 100% dimethyl sulfoxide and added to the bath with final concentration of 0.1% dimethyl sulfoxide. The pA₂ value, an index of inhibitory potency, was determined for each individual curve by the equation pA₂ = log (concentration ratio  1)  log [B], where concentration ratio is the ratio of EC₅₀ values with and without the antagonist and [B] is the concentration of the antagonist.

In Vivo Studies

Mouse Lethality Assays. Male ddY mice (7 weeks old) were used in the study. The day before the experiment, mice were divided into groups according to their body weight. Mice weighing more than 30 g were fasted overnight to evaluate effect of fasting on the p.o. potency of J-104132- versus ET-1-induced lethality. A separate group of mice were allowed to access food freely. ET-1 (5 nmol/kg) was injected i.v., and the incidence of death was observed in each mouse. The potency of J-104132 was determined after i.v. and p.o. administration. For i.v. administration, J-104132 was dissolved with equimolar NaOH (0.1 N) and diluted by saline. The pH of the solution was adjusted if it was basic. In the case of p.o. administration, the compound was suspended or dissolved in 0.5% methylcellulose solution. Volume of drug was adjusted to 0.1 ml/10 g b.wt.

Conscious Normotensive Rat Pressor Assays. Big ET-1induced pressor response. Six- to 7-week-old male Sprague-Dawley rats (Charles River Japan, Yokohama, Japan) were anesthetized with sodium pentobarbital (35 mg/kg i.p.) and instrumented with vascular cannulas the day before the experiment according to the previous report (Okada et al., 1994). Before injection of ET antagonist, big ET-1 was administered twice with 1 h between challenges to obtain a steady pressor response. One hour later, ET antagonist was administered orally. Rats were challenged with big ET-1 at 30 min and then every hour up to 8 h and a final challenge at 24 h. The dose of big ET-1, 0.5 nmol/kg, was chosen because this dose elicits a submaximal pressor response in this preparation. J-104132 was administered orally. J-104132 was dissolved in equimolar of NaOH solution (0.1 N) and diluted with saline. The pH of solution was adjusted with 0.1 N hydrochloric acid if necessary.

ET-1-induced pressor response. Male Sprague-Dawley rats (200-400 g) were anesthetized with methohexital (50 mg/kg i.p.) and instrumented with two chronic vascular catheters 12 to 20 h before the experiment. A catheter in the femoral artery was used for direct measurement of blood pressure. A catheter in the femoral vein was used for i.v. administration of J-104132 and pressor agents (ET-1 or methoxamine). Rats were permitted to recover overnight from anesthesia and allowed free access to water. Food was withheld if test compound was to be administered orally. After the appropriate equilibration, rats were challenged with bolus, submaximal doses of ET-1 (0.5 nmol/kg) and methoxamine (50 µg/kg) to ensure patency of catheters and responsiveness of preparation and to determine control response to ET-1. Rats were then administered a single p.o. or i.v. dose of J-104132 (0.1-1.0 mg/kg). Inhibition of pressor responses to methoxamine was used to assess the selectivity of J-104132 for ET receptors versus  adrenergic receptors. J-104132 was dissolved in a 5% saturated sodium bicarbonate/95% saline (i.v.) or water (p.o.) vehicle. The dosing volumes were 2.0 ml/kg i.v. and 5.0 ml/kg p.o.

Anesthetized Dog Renal Artery Assays. Mongrel dogs (10-15 kg) were anesthetized with sodium pentobarbital (30 mg/kg i.v.). An endotracheal tube was inserted, and the dog was ventilated with room air at a rate of 20 breaths per min and a tidal volume of 15 to 18 ml/breath. Catheters (PE-260) were placed into the femoral arteries and veins. The catheter placed in the right femoral artery was advanced into the abdominal aorta and positioned just below the bifurcation of the renal arteries to obtain an approximation of renal arterial pressure. The left femoral artery catheter was advanced into the abdominal aorta to measure systemic blood pressure and obtain blood samples. An i.v. infusion of sodium pentobarbital (5 mg/kg/h) was started in the right femoral vein catheter at the beginning of surgery and continued for the entire length of the experiment to maintain a surgical plane of anesthesia. A left flank incision was made, and the left kidney was exposed. The left renal artery was isolated, and an electromagnetic flow probe (Zapeda Instruments, Seattle, WA) was placed around the artery near the abdominal aorta. A 23-gauge bent needle catheter, connected via tubing to a 5-ml syringe filled with heparinized (20 U/ml) saline, was then inserted into the renal artery distal to the flow probe. An infusion of heparinized saline was immediately begun and continued until the start of the ET-1 challenges. A rectal thermometer was inserted into the dog, and body temperature was maintained at approximately 37°C with a heat lamp. The dog was allowed to stabilize for 30 min after the surgery. After stabilization, each dog was given an intrarenal injection of angiotensin II (10 ng/kg) to verify proper placement of the needle catheter and to determine the responsiveness of the preparation. Systemic arterial pressure and renal blood flow were recorded during two 30-min baseline control periods. At the conclusion of the second baseline period, J-104132 treatment was begun. J-104132 was infused for 2 h before the start of the ET-1 challenges to achieve near steady-state plasma levels. At 1 and 2 h after the start of the infusion, blood pressure and renal blood flow were recorded. ET-1 challenges (1, 3, 10, 30, and 100 pmol/min for 30 min at each dose) were administered directly into the renal artery as rising doses at a rate of 75 µl/min. During each of the ET-1 infusion periods, blood pressure and renal blood flow were recorded. To compare efficacy of bolus versus infusion administration, J-104132 was administered as a single i.v. bolus injection (1 mg/kg) 15 min before the first ET-1 challenge in a separate group of animals. The remainder of the protocol was identical with that just described for the infusions. J-104132 was dissolved in a 5% saturated sodium bicarbonate/95% saline (i.v.) or water (p.o.) vehicle. The dosing volume was 2.0 ml/kg i.v. Bosentan was prepared in a vehicle of saline for the i.v. infusion.

Anesthetized Dog Brachial Artery Assays. Female mongrel dogs (approximately 9-14 kg) were anesthetized with sodium pentobarbital (30 mg/kg i.v.), intubated, and allowed to spontaneously breathe room air. The right carotid artery was cannulated for the measurement of systemic blood pressure. The right jugular vein was cannulated for the administration of either J-104132 or vehicle. The right femoral artery was cannulated for the measurement of arterial pressure. The right femoral vein was cannulated for the continuous administration of pentobarbital anesthesia (5 mg/kg/h). A lateral incision was made in the right flank, and the right kidney was exposed by careful dissection, allowing the peritoneum to remain intact. The right renal artery was dissected free, and an ultrasonic flow probe (Transonic Systems, Inc., Itahaca, NY) was placed around it. The left forelimb of the dog was stabilized, and a 5- to 6-cm incision was made above the elbow. The brachial artery was isolated above the major branches, and an ultrasonic flow probe was placed around it. Just distal to the flow probe, a 25-gauge needle bent at 90° was inserted into the brachial artery and affixed with Superglue (3M, St. Paul, MN). The needle was attached via PE 20 tubing to a syringe pump, and normal saline was infused at a rate of 50 µl/min. Arterial blood pressure, renal blood flow, brachial artery flow, and ECG were continuously monitored on a Gould RS3800 physiograph. To determine the potency of J-104132, the animals were divided into three groups: those that received vehicle (5% saturated Na₂HCO₃ in normal saline) and those that received J-104132 at either 0.1 or 0.3 mg/kg/h. Each animal was allowed to stabilize for 45 min before the initiation of the study. After stabilization, the vehicle or J-104132 was administered i.v. into the jugular vein at a rate of 10 or 15 ml/h for 2 h before the first ET-1 challenge. ET-1 was then administered continuously into the brachial artery in step increases in dose ranging from 1 to 300 pmol/min. Each ET-1 dose was infused for 30 min and then switched to the next higher dose of ET-1. The maximal decrease in blood flow was taken when flow reached its nadir. In separate studies, the duration of action for J-104132 was determined for animals that had been divided into three groups: those that received either vehicle or saline and those that received J-104132. After a 45-min stabilization period, each animal received an i.v. bolus of either the vehicle, saline, or J-104132 in a total volume of 5 ml. Ten minutes later and again every hour for 5 h, the animal received a bolus dose of 200 pmol of ET-1 in a total volume of 2 ml into the brachial artery. This dose of ET-1 was found to reproducibly decrease brachial artery flow by 30%.

Plasma Concentration Assay (Oral Bioavailability). Plasma concentration-time profiles of J-104132 were determined in conscious rats aged 7 weeks and weighing from 200 to 300 g.

Expression of Results. Values are expressed as mean ± S.E.M. unless otherwise noted. The differences between control and drug treatment group were analyzed by ANOVA and posthoc multiple comparison analysis performed with a modified t test (Dunnett's). Statistical significant level was set at p < .05.

Materials. (±)-(5S,6R,7R)-2-Butyl-7-[2-((2S)-2-carboxypropyl)-4-methoxyphenyl]-5-(3,4-methylenedioxyphenyl)cyclopenteno[1,2-b]pyridine-6-carboxylic acid was synthesized as a racemic mixture and was resolved to the active (+)-enantiomer, J-104132 (Fig. 1), by chiral HPLC at the Drug Discovery Research Laboratories of Banyu Pharmaceuticals (Lynch et al., 1997). BQ-123 (Ishikawa et al., 1992) and BQ-3020 (Ihara et al., 1992b) were also synthesized at Banyu Pharmaceuticals. Bosentan (Ro 47-0203) was synthesized as described in F. Hoffmann LaRoche patent application EP, 510,526, example 55. [¹²⁵I]ET-1 was purchased from Amersham Co. (Des Plaines, IL). ET-1, big ET-1, and S6c were obtained from Peptide Institute (Osaka, Japan).

View larger version (11K): [in this window] [in a new window]   Fig. 1.   Chemical structure of J-104132.

	Results

Top Abstract Introduction Experimental Procedures Results Discussion References

In Vitro Studies

Binding Assays. In competition binding assays, J-104132 potently inhibited [¹²⁵I]ET-1 binding to cloned human ET_A (K_i = 0.034 ± 0.008 nM) or ET_B (K_i = 0.104 ± 0.017 nM) receptors expressed in CHO cells with affinity for ET_A receptors about 3-fold greater than that for ET_B receptors (Table 1). In human tissues (human uterus and hippocampus), where both ET_A and ET_B receptors are present, BQ-123 or S6c was added to the binding reaction mixture to ensure saturation of the ET_A or ET_B receptors, respectively. The affinity of J-104132 for ET_A (uterus: K_i = 0.350 ± 0.158 nM) or ET_B (hippocampus: K_i = 0.343 ± 0.146 nM) receptors in these human preparations was identical. On the other hand, the K_i values for bosentan, used as a reference compound in in vitro studies, were 14.8 ± 3.4 nM for human ET_A and 102 ± 10.6 nM for human ET_B receptors (Table 1). J-104132 has 500- and 1000-fold higher affinity than bosentan at human ET_A and ET_B receptors, respectively. In saturation binding studies, cell preparations were preincubated in the presence or absence of 0.04 or 0.2 nM J-104132 (for human ET_A and ET_B receptors, respectively) before the addition of [¹²⁵I]ET-1. Scatchard analysis indicates that J-104132 reduced the apparent affinity of [¹²⁵I]ET-1 but caused no change in B_max (Fig. 2), and the resulting K_i values were consistent with those obtained in competition-type studies. In these studies, J-104132 was about 7-fold ET_A versus ET_B selective. These results are consistent with a reversible and competitive interaction of J-104132 with ET receptors.

View larger version (14K): [in this window] [in a new window]   Fig. 2.   Saturation binding: J-104132 inhibition of [125I]ET-1 binding to CHO cells expressing cloned human ETA and ETB receptors after 60 min of preincubation. Kd and Bmax values are (A) human ETA control (), 111 and 21.8 pM; and human ETA + 0.04 nM J-104132 (), 213 and 22.4 pM; and (B) human ETB control (), 46.7 and 28.1 pM; and human ETB + 0.2 nM J-104132 (), 105 and 27.4 pM, respectively. The Ki values for J-104132 to ETA and ETB receptors were 0.037 and 0.25 nM, respectively. The Ki value for J-104132 was calculated from the relation: Ki = Kd[Kd'  Kd] × [J-104132], where Kd is the control value and Kd' is the value in the presence of 0.04 or 0.2 nM J-104132.

Phosphatidyl Inositol Hydrolysis Assays. Exposure of CHO cells stably expressing cloned human ET receptors to ET-1 after overnight incubation with myo-[³H]inositol results in a concentration-dependent (EC₅₀ ~ 0.3 nM) accumulation of [³H]inositol phosphates in the presence of LiCl. In this system, J-104132 inhibits the accumulation of [³H]inositol phosphates stimulated by 0.34 nM ET-1 with IC₅₀ values of 0.059 nM in human ET_A/CHO cells (Fig. 3), indicating that the compound is a potent antagonist of functional responses to ET-1. In addition, at a concentration of J-104132 that fully inhibited ET-1-stimutated phosphatidyl inositol hydrolysis (3 nM) and in the absence of ET-1, J-104132 did not enhance phosphatidyl inositol hydrolysis in human ET_A/CHO cells or human ET_B/CHO cells (data not shown), indicating that the compound has no partial ET agonist activity.

View larger version (15K): [in this window] [in a new window]   Fig. 3.   Effect of J-104132 on ET-1-stimulated phosphatidyl inositol hydrolysis in CHO cells expressing cloned human ETA receptors. The inhibitory effects of J-104132 from three separate experiments are shown. Each data point represented the mean ± S.E.M. of three quadruplicate determinations. [ET-1] = 0.3 nM.

Rabbit Iliac Artery and Pulmonary Artery Contractile Assays. In endothelium-denuded rabbit iliac arteries, J-104132 (0.001, 0.01, and 0.1 µM) shifted the concentration-contraction curves for ET-1 to the right in a competitive manner (slope, 0.970.15) with no apparent loss in maximal response of the tissue to ET-1 (Fig. 4A). The pA₂ value for J-104132 was 9.70 ± 0.12 (Table 2). To estimate the effect of plasma protein binding on potency, the antagonistic potency of the test compound in isolated rabbit iliac artery was determined in the presence of physiological concentration (3%) of HSA or RSA. With 3% HSA, J-104132 (0.1 and 1 µM) shifted the concentration-contraction curve for ET-1 in iliac arteries to the right dose-dependently with a pA₂ value of 7.65 ± 0.09 (Table 2). An identical shift in potency for J-104132 was observed in the presence of RSA (pA₂ = 7.89 ± 0.13) (Table 2), indicating that the effect of plasma protein on the potency of J-104132 was similar for HSA and RSA. Thus, in the presence of 3% HSA or RSA, the potency of J-104132 was reduced by a factor of 100. 

View larger version (21K): [in this window] [in a new window]   Fig. 4.   Functional antagonism by J-104132 (, vehicle; , 0.001 µM; , 0.003 µM; , 0.01 µM; , 0.1 µM) of ET-1-stimulated contraction of endothelium-denuded rabbit iliac artery (A, pA2 = 9.70) and BQ-3020 (B, ETB agonist)-stimulated contraction of endothelium-denuded rabbit pulmonary artery (pA2 = 10.14). Each data point represented the mean ± S.E.M. (A, n = 4; B, n = 5 or 6).

In Vivo Studies

Mouse Lethality Assay. In untreated control mice, ET-1 (5 nmol/kg i.v.) causes a 100% death within 5 min due to severe hypertension and ventricular arrhythmia. Pretreatment with i.v. doses of J-104132 protected mice from ET-1-induced lethality in a dose-dependent fashion with ED₅₀ values of 0.045 mg/kg (Table 3). In nonfasted and fasted mice, p.o. pretreatment with J-104132 also effectively protected mice from the ET-1-induced lethality. ED₅₀ values for J-104132 were similar in nonfasted (0.35 mg/kg) and fasted (0.48 mg/kg) mice (Table 3), indicating that the potency of J-104132 was not remarkably affected by food consumption in mice. The ratio of potencies after p.o. and i.v. administration is sometimes used as a crude estimate of p.o. bioavailability. In these experiments, the p.o./i.v. potency ratio was 8 for J-104132, suggesting a good p.o. bioavailability in mice.

Conscious Normotensive Rat Pressor Assays. Big ET-1 (0.5 nmol/kg i.v.)-induced pressor response. Intravenous administration of big ET-1 produced a sustained pressor response that reached its peak 10 to 15 min after injection and returned to the baseline within 1 h. The pressor response elicited by big ET-1 was almost completely inhibited at 30 min after p.o. administration of J-104132 at doses of 1, 3, and 10 mg/kg (Fig. 5). At 1 mg/kg J-104132 p.o., inhibition of the pressor response elicited by big ET-1 gradually recovered within 3 to 4 h, whereas inhibition was present for more than 8 h after the administration of 3 and 10 mg/kg J-104132 p.o. (Fig. 5).

View larger version (26K): [in this window] [in a new window]   Fig. 5.   Effect of the p.o. administration of J-104132 (, vehicle; , 1 mg/kg; , 3 mg/kg; , 10 mg/kg) on big ET-1 (0.5 nmol/kg, i.v.)-induced pressor response in conscious normotensive rats. J-104132 was administered at time zero and big ET-1 was challenged at each time point. Each data point represented the mean ± S.E.M. of peak pressor response after big ET-1 challenges (n = 4-8).

ET-1 (0.5 nmol/kg i.v.)-induced pressor response. In conscious, normotensive rats, dose-related inhibition of ET-1-induced pressor responses was observed after p.o. and i.v. administration of J-104132 (0.1-1 mg/kg) (Fig. 6). J-104132 was selective for ET receptors because it did not affect basal blood pressure or pressor responses to methoxamine (data not shown). Using peak inhibition at each dose, the relative potencies of J-104132 after p.o. administration were three times less than those after i.v. administration. Bosentan had no significant activity when tested at 10 mg/kg i.v. (data not shown).

View larger version (25K): [in this window] [in a new window]   Fig. 6.   Effects of the i.v. and p.o. administrations of J-104132 on ET-1 (0.5 nmol/kg i.v.)-induced pressor response in conscious normotensive rats (n = 4-7). Values are expressed as the mean ± S.E.M. Maximum inhibition by J-104132 on ET-1-elicited pressor responses was obtained 10 and 30 min after i.v. and p.o. administration of J-104132, respectively.

Anesthetized Dog Renal Artery Assays. ET-1 (1-100 pmol/min) infusion directly into the renal artery of the anesthetized dog produces a dose-related decrease in renal blood flow. On the systemic (i.v. infusion) administration of J-104132 (0.01-0.3 mg/kg/h), the ET-1 dose-response curve was shifted to the right in a dose-related manner. J-104132 administered at 0.03 mg/kg/h (i.v.) shifted the ET-1 dose-response curve approximately 12-fold, whereas a larger dose of bosentan (1.0 mg/kg/h) was required to produce a similar shift in the ET-1 dose-response curve (Fig. 7A). Therefore, J-104132 is approximately 30-fold more potent than bosentan in this assay.

View larger version (20K): [in this window] [in a new window]   Fig. 7.   Effects of i.v. infusion of J-104132 (, vehicle; , 0.01 mg/kg/h; , 0.03 mg/kg/h; , 0.1 mg/kg/h; , 0.3 mg/kg/h) and bosentan (, 1 mg/kg/h) on ET-1-induced renal and forelimb blood flow reductions. A, ET-1 (1-100 pmol/min for 30 min at each dose into the renal artery)-induced renal blood flow reduction (n = 3-9). B, ET-1(1-300 pmol/min for 30 min at each dose into the brachial artery)-induced forelimb blood flow reduction (n = 5 or 6) in anesthetized dogs. Each data point represented the mean ± S.E.M.

Anesthetized Dog Brachial Artery Assays. Figure 7B depicts the effect of i.v. infusion of J-104132 on reductions in brachial artery flow produced with intrabrachially administered ET-1. At a dose of 0.3 mg/kg/h, J-104132 led to a 15- to 20-fold shift in the dose-response curve compared with the vehicle. On the other hand, intrabrachial infusion of ET-1 reduced not only brachial artery flow but also renal blood flow. Equal or greater effects of J-104132 to shift the ET-1 dose-response curve in the renal vascular bed were observed. A dose of 0.3 mg/kg/h shifted the dose-response curve to ET-1 in this vascular bed by more than 20-fold (data not shown). A second aspect of this study was to assess whether there were any significant hemodynamic effects of J-104132. Over the time course of 4 h, there were no marked alterations in heart rate, although systemic arterial pressure was observed to slowly decline by approximately 18% over the entire protocol period at the 0.3 mg/kg/h dosage. The functional duration of action of J-104132 was also assessed in this preparation. In the absence of J-104132, a bolus dose of 200 pmol of ET-1 caused a 28 ± 6% reduction in brachial artery flow. This reduction in brachial flow was reproducible through six challenges over 5 h. With this background, animals were administered J-104132 as an i.v. bolus over 2 min at doses of either 0.3 or 1.0 mg/kg. ET-1 challenges were commenced 10 min after J-104132 challenges. J-104132 dose dependently antagonized the ET-1-induced reduction in blood flow. At 10 min after dosing, the reduction in flow was 8 ± 2% and 2 ± 2% at 0.3 and 1.0 mg/kg, respectively. However, at 2 h after dosing, ET-1 led to a 25 ± 3% reduction in flow at the 0.3 mg/kg dose, which was not different from the vehicle. The functional duration at 1.0 mg/kg was longer. At 3 and 4 h, the reduction in flow was 22 ± 3% and 31± 2%, respectively. These results indicate an functional duration of action of 3 to 4 h in this model after i.v. administration of 1 mg/kg J-104132.

Plasma Concentration Assay (Oral Bioavailability)

The plasma concentrations of J-104132 in rats after the administration of i.v. doses of 0.3, 1, and 3 mg/kg decreased multiexponentially during the first 4 h; thereafter, there were slight to moderate increases in concentration at 4 to 10 h after the drug administration (Fig. 8). This plasma concentration-time profile indicates possible enterohepatic circulation of the compound and prevents determination of an accurate plasma half-life for J-104132 in the rat. The CLp ranged from 16.7 to 21.3 ml/min/kg (Table 4). The V_d and MRT ranged from 0.72 to 1.27 liters/kg and from 0.76 to 1.12 h, respectively (Table 4).

View larger version (18K): [in this window] [in a new window]   Fig. 8.   Plasma concentration-time profiles of J-104132 (, 0.3 mg/kg; , 1 mg/kg; , 3 mg/kg; , 10 mg/kg) in conscious normotensive rats after (A) i.v. (n = 3) or (B) p.o. (n = 5) dosing. Each value represented the mean ± S.D.

When administered to rats at 1, 3, and 10 mg/kg p.o., the absorption of J-104132 was rapid. C_max values were 178, 477, and 1754 ng/ml reached at 0.25, 0.4, and 0.75 h, respectively. Thereafter, the concentrations decreased gradually (Fig. 8; Table 4), showing a long plasma duration of J-104132. Furthermore, there were slight increases in concentrations at 4 to 6 h after the drug administration, consistent with enterohepatic circulation of J-104132. MRT was 2.8 to 3.1 h. Bioavailabilities of 34% and 44% were obtained at 1 and 3 mg/kg, respectively (Table 4).

	Discussion

Top Abstract Introduction Experimental Procedures Results Discussion References

We provided the first pharmacological cyclic pentapeptide antagonist BQ-123 in 1992 as a selective ET_A receptor antagonist (Ihara et al.), as well as the first potent, selective ET_B receptor antagonist, BQ-788 (Ishikawa et al.) in 1994. These antagonists have been very useful tools to investigate the role of ET in physiological and pathological processes. However, the use of these peptides is limited by their short duration of action and lack of p.o. bioavailability. The present study describes the characterization of a nonpeptide ET receptor antagonist, J-104132, which is an orally active and one of the most potent ET antagonists with both ET_A and ET_B receptors blocking activities. In radioligand-binding assay, K_i values for J-104132 are 0.03 and 0.1 nM at cloned human ET_A and ET_B receptors, respectively, indicating that an affinity for ET_A is only 3-fold greater than that for ET_B receptor. The affinity of J-104132 for ET_A and ET_B receptors is 500- and 1000-fold greater than that of bosentan, respectively. In 1994, SB 209670 (Ohlstein et al.) was reported to be the most potent nonpeptide mixed ET_A/ET_B receptor antagonist identified so far; that is, its K_i values to cloned human ET_A and ET_B receptors are 0.2 and 18 nM, respectively. J-104132 has approximately 10-fold greater affinity for ET_A receptors and approximately 200-fold greater affinity for ET_B receptors compared with those of SB 209670. We also reported on a potent, orally active, mixed ET_A/ET_B antagonist, L-754,142 (Williams et al., 1995). In the present study, J-104132 is 3- and 30-fold higher in affinity for ET_A and ET_B receptors, respectively, compared with L-754,142.

With regard to reversibility of J-104132 interaction with ET receptors, the preincubation of ET receptors with J-104132 reduced the affinity of [¹²⁵I]ET-1 binding without affecting the B_max values in binding assay. In addition, J-104132 shifted the response curve for ET-1 to the right without depression of the maximal response in isolated vessels with either ET receptor subtype. These results indicate the reversibility and competitive manner of the J-104132 interaction with ET receptors.

To assess the selectivity of J-104132 for ET receptors, J-104132 was also tested at concentrations as large as 10 µM in a variety of radioligand competition binding assays using more than 30 different human receptors or cloned receptors expressed in cultured cells. As a result, J-104132 was found to have no binding affinity for any of the receptors (i.e., A₁, A_2A, AT₁, BK₂, ₁, ₂, ₁, ₂, D₃, M₁, M₂, M₃, M₄, M₅, NK₁, NK₂, BK₁, 5-HT_1a, TXA₂, NPY₁, NPY₂, oxytocin) (data not shown). These results indicate J-104132 is highly selective for ET receptors.

J-104132 inhibited ET-1-induced phosphatidyl inositol hydrolysis in human ET_A/CHO cells, produced parallel rightward shifts in the ET-1 concentration-response curves in endothelium-denuded rabbit iliac arteries, and shifted the concentration-response curve for BQ-3020 to the right in endothelium-denuded rabbit pulmonary arteries. Together, these results demonstrate that J-104132 is a potent antagonist of functional responses mediated via ET_A and ET_B receptors in several different preparations. The selectivity of J-104132 for ET receptors was confirmed by the observations that J-104132 did not affect KCl- and norepinephrine-induced contractions in iliac arteries.

The in vivo potency of J-104132 was assessed using challenges with exogenous ET-1 or big ET-1. Intravenous injection of J-104132 potently inhibited ET-1 (5 nmol/kg)-induced lethality in mice, and its ED₅₀ value was 0.045 mg/kg. Furthermore, p.o. potency of this compound was examined in this assay using fasted and nonfasted mice. The ED₅₀ values were 0.48 and 0.35 mg/kg, respectively, suggesting that J-104132 is a potent, orally active ET antagonist. Because the ED₅₀ values were similar in fasted and fed mice, the p.o. potency of J-104132 was not affected by food consumption. In big ET-1-induced pressor response in conscious rats, J-104132 induced almost complete inhibition at dose of 1 mg/kg p.o. and showed marked inhibition at 8 h after administration of J-104132 at doses of 3 and 10 mg/kg p.o. A mixed ET_A/ET_B receptor antagonist can completely inhibit ET-1-induced depressor and pressor responses, whereas an ET_A-selective antagonist will not inhibit a component of pressor response and does not block the depressor response (McMurdo et al., 1993). J-104132 showed typical ET_A/ET_B inhibition patterns to ET-1-induced responses in rats, and the inhibitory potency of J-104132 after i.v. dosing on pressor response was more than 160-fold more potent than that of bosentan. In addition, the p.o. administration of J-104132 at 1 mg/kg nearly eliminated the pressor response to ET-1 in this model. Therefore, J-104132 is a potent orally active ET receptor antagonist with a long duration of action in in vivo animal studies. Relatively high plasma concentrations of J-104132 after i.v. or p.o. administration of 1 or 3 mg/kg were observed for 10 h in rats. However, the plasma T_1/2 could not be calculated because there appeared to be enterohepatic circulation of the drug.

In anesthetized dogs, ET-1 was administered directly into renal artery or brachial artery to generate dose-response (blood flow) curves, and the inhibitory potency of J-104132 after i.v. infusion was quantified. J-104132 shifted the dose-response curve for ET-1 after infusion into renal artery to the right in a dose-dependent manner, and its inhibitory effect was about 30-fold more potent than that for bosentan. In the case of infusion of ET-1 into brachial artery, the amount of ET-1 reaching the kidney is markedly less because the ET-1 must pass through the lungs before reaching the kidney. Because the lungs are a major site of ET-1 removal from the circulation, it is assumed that reduced amounts of ET-1 are reaching the kidney compared with the brachial artery. Despite this, ET-1 was able to cause a significant reduction in renal blood flow, which was antagonized by J-104132. These results not only indicate that the renal arteries are more sensitive to ET-1 than arteries in the forelimb but also suggest that J-104132 is more potent in the renal vascular bed than the forelimb. From in vitro and in vivo studies, J-104132 is highly selective for ET receptors, is orally active, and is one of the most potent mixed ET_A/ET_B antagonist.

Recently, several orally active ET_A selective, such as TBC-11251 (Wu et al., 1997), T-0201 (Hoshino et al., 1998), and SB 234551 (Ohlstein et al., 1998) or ET_A/ET_B mixed antagonists, such as bosentan (Clozel et al., 1994), SB 209670 (Ohlstein et al., 1994), and L-754,142 (Williams et al., 1995), have been developed as part of continuing efforts to investigate physiological and pathophysiological roles of the ET system, as well as to develop new therapeutic agents. In this regard, efficacy with ET receptor antagonists has been demonstrated in preclinical models and in patients with congestive heart failure (Kiowski et al., 1995, Love et al., 1996) and hypertension (Krum et al., 1998).

When ET-1 or big ET-1 is administered to humans, potent, long-lasting vasoconstrictor and pressor activities are observed. At present, it is well known that these effects of ET-1 or big ET-1 are mediated through both ET_A and ET_B receptors present on vascular smooth muscle. An ET_A receptor antagonist, BQ-123, produced not only inhibition of forearm vasoconstriction to brachial infusion of ET-1 but also progressive forearm vasodilatation in human (Haynes and Webb, 1994). Furthermore, systemic i.v. administration of a mixed ET_A/ET_B receptor antagonist, TAK-044, produced reductions in the blood pressure in healthy people (Haynes et al., 1996). These results indicate that endogenous ET-1 is involved in the maintenance of the major resistance vascular tone through the activation of both ET_A and ET_B receptors in humans and animals. In patients with essential hypertension, bosentan (mixed ET_A/ET_B receptor antagonist) resulted in a significant reduction in diastolic pressure, which was similar to the reduction with enalapril compared with placebo (Kyum et al., 1998). Bosentan significantly improved hemodynamics (i.e., reduced left ventricular ejection fraction, elevated resting pulmonary capillary wedged pressure and/or cardiac index) due to systemic and venous vasodilatation in patients with chronic severe congestive heart failure. From these reports, in recent years, the role of ET-1 in the pathophysiology of several cardiovascular diseases is being brought to light. However, the participation of ET receptor subtypes in various disorders has yet to be clearly defined and may depend on the model. Recently, results with many nonpeptide orally active highly selective ET_A receptor antagonists or mixed ET_A/ET_B receptor antagonists have been published. Presently, it is not known whether ET_A-selective or mixed ET_A/ET_B antagonists will be optimal for the treatment of different diseases.

In conclusion, J-104132 is a selective, potent, orally active antagonist of ET_A and ET_B receptors and therefore an excellent pharmacological tool to explore the therapeutic utility of a mixed ET_A/ET_B receptor antagonist.