Introduction

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A critical need in the search for endothelin-based therapeutics is clarification of the physiological and pathophysiological functions of the endothelin receptor subtypes. Effects associated with ET_A receptor activation include vasoconstriction, mitogenic activity, electrolyte excretion, microvascular permeability and release of biological mediators (Ohlstein et al., 1995). In contrast, the ET_B receptor is associated with both vasodilator and vasoconstrictor actions, as well as effects on neuronal function (Ohlstein et al., 1995). On the basis of the in vivo effects of ET_B receptor agonists, such as S6c and IRL-1620, studies from a number of laboratories have suggested the presence of two ET_B receptor subtypes, one mediating vasodilation and the other mediating vasoconstriction (Warner et al., 1993a,b; MacLean et al., 1994; Ohlstein et al., 1994a; Douglas et al., 1995b). These ET_B receptor subtypes have been tentatively designated as ET_B1 and ET_B2 (Douglas et al., 1995a). The endothelin receptor antagonists RES-701 and PD 142893 have both been reported to be ET_B1-selective antagonists in functional assays (Warner et al., 1993a; Douglas et al., 1995b). These compounds show no functional antagonist activity, even at concentrations up to 10 µM, against responses apparently mediated by the ET_B2 receptor (Warner et al., 1993a; Douglas et al., 1995a; Hay et al., 1996). In radioligand binding studies using cloned human ET_B receptors, however, these antagonists have K_i values in the submicromolar range. Accordingly, on the basis of pharmacological functional studies, the cloned human ET_B receptor most closely resembles the ET_B1-mediated response functionally linked to vasodilation. However, inasmuch as the available pharmacological data suggest the presence of heterogeneous functional ET_B receptor subtypes, confirmation by conventional protein purification/molecular cloning is necessary before their existence can be firmly established.

Although the recent identification of peptide and nonpeptide receptor antagonists represents an important milestone in endothelin research, it is likely that elucidation of the role of ET-1 in the pathophysiology of diseases will require the clinical testing of high-affinity, structurally distinct compounds with a range of selectivities for the ET_A and the ET_B receptors and their subtypes. We have reported recently on the discovery and characterization of a high-affinity mixed ET_A/ET_B receptor antagonist, SB 217242 (Ohlstein et al., 1996). However, a high-affinity ET_A-selective antagonist that exerts reduced effects on the ET_B1 receptor mediating vasodilation, while maintaining the inhibition of the ET_B2 vasoconstrictor effects demonstrated by SB 217242, would appear to have an attractive compound profile. In this study, we report on the discovery and characterization of SB 234551, the lead compound from a new chemical series of high-affinity nonpeptide endothelin receptor antagonists with such a profile.

	Materials and Methods

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Membrane preparation and radioligand binding. Chinese hamster ovary cells stably transfected with cloned human ET_A and ET_B receptors were cultured and cell membranes prepared as reported previously (Nambi et al., 1994). [¹²⁵I]ET-1 binding to membrane preparations was performed as described previously (Nambi et al., 1994). Briefly, assay volumes were 50 µl, and the concentrations of membrane proteins were 0.50 and 0.05 µg/tube for human ET_A and ET_B receptors, respectively. The concentrations of the radioligands were 30 to 1000 pM for saturation-binding and 300 pM for competition-binding experiments using the cloned human ET_A and ET_B receptors. Nonspecific binding was measured in the presence of 1 µM unlabeled ET-1. The incubations (performed for 60 min at 30°C) were stopped by dilution with cold buffer and filtration through Whatman GF/C filters presoaked in 0.1% bovine serum albumin. The filters were washed three times (5 ml each time) and counted using a gamma counter.

In vitro endothelin receptor antagonist activity. Male Sprague-Dawley rats (300-325 g) or New Zealand White rabbits (2-3 kg) were euthanized with sodium pentobarbital (100 mg/kg i.p.). Rat thoracic aortae and rabbit pulmonary arteries were excised, cleaned of adherent tissue and the vascular endothelium denuded by gently rubbing the intimal surface of the vessel with a stainless steel probe (Ohlstein et al., 1989). Rabbit saphenous veins were prepared as described previously (Douglas et al., 1995b). Isolated vessels were cut into 4-mm rings and suspended in 10-ml organ bath chambers containing Krebs-bicarbonate solution (mM: NaCl, 112.0; KCl, 4.7; KH₂PO₄, 1.2; MgSO₄, 1.2; CaCl₂, 2.5; NaHCO₃, 25.0 and dextrose, 11.0. Tissue baths were maintained at 37°C and aerated continuously with 95% O₂/5% CO₂, pH = 7.35. Resting tensions of 1 gm for rat aorta, rabbit pulmonary artery and saphenous vein were maintained throughout the experiments. Endothelial integrity was assessed pharmacologically in terms of the ability of ACh (0.1 µM) to produce relaxation of tissues precontracted with norepinephrine (10 µM). Tissues were equilibrated for 1 hr before the start of experiments, and isometric tensions were recorded on Beckman R-611 dynographs using Grass FTO3c force-displacement transducers.

(1)

In vivo hemodynamics. Male Sprague-Dawley rats (300-350 g) were prepared with chronic indwelling catheters, following the protocol of Ohlstein et al., (1994b). Briefly, rats were anesthetized with sodium methohexitone (100 mg/kg i.p.) and catheters placed in the abdominal aorta (to record systemic arterial blood pressure) and vena cava (for i.v. bolus administration of drugs) via the left femoral artery and vein, respectively. All tubing was tunneled under the skin and exited at the midscapular region. Tubing was filled with a dextrose:heparin solution (0.5 g/ml dextrose and 1000 units/ml heparin) to prevent obstructive thrombus formation. After completion of the surgery, animals were housed in Plexiglas cages under a 12-hr light-dark cycle with access to standard laboratory chow and drinking water ad libitum. Animals were allowed at least 3 days to recover from surgical intervention before undergoing experimentation.

Pharmacokinetics of SB 234551 in rats. The pharmacokinetic evaluation of SB 234551 was performed as described previously (Ohlstein et al., 1996). Briefly, male Sprague-Dawley rats (350 gm) were surgically prepared with indwelling cannulas in the vena cava, femoral artery and duodenum. SB 234551 was administered as a 2-hr intraduodenal infusion (in saline vehicle) via the duodenal cannula at a rate of 50 µg/kg/hr (total dose, 6 mg/kg). Blood samples (110 µl) were collected from the femoral arterial cannula at various time intervals over 1440 min. The animals received approximately 2 ml of heparinized blood from untreated donor rats upon completion of this leg of the study. One week later, rats were crossed over to receive SB 234551 as an i.v. infusion. SB 234551 was administered via the femoral vein cannula at a rate of 0.20 µg/hr/kg for 2 hr.

Pharmacokinetic analysis. SB 234551 was isolated from rat plasma by liquid-liquid extraction and was quantitated by reverse-phase HPLC with MS/MS detection performed on an API III tandem triple quadrupole mass spectrometer (Perkin Elmer Sciex Instruments, Rochester, NY). The assay provided a lower limit of quantification of 5 ng/ml based on 0.05 ml plasma and was linear up to 1000 ng/ml. Plasma concentration-time profiles were analyzed using noncompartmental methods. The area under the plasma concentration-time curve (AUC) was estimated by a combination of linear and log trapezoidal methods. Plasma clearance was calculated as dose/AUC. Bioavailability was determined as dose-normalized AUC (intraduodenal)/dose-normalized AUC (intravenous). The apparent terminal half-life was estimated by least-squares linear regression analysis of the log-transformed concentration-time data.

Calculations and statistics. Values are expressed as mean ± S.E.M., and n represents the number of animals or separate experiments studied in a particular group. Statistical analysis was conducted using ANOVA or two-tailed Student's t test for paired samples, where appropriate, P  .05 being accepted as significant.

Materials. All solutions were prepared daily. Endothelin isopeptides and BQ-123 were purchased from American Peptide Co. (Santa Clara, CA). [¹²⁵I]ET-1 (2200 Ci/mmol) was obtained from New England Nuclear (Boston, MA). All other chemicals were of the highest grades available. SB 234551, SB 209670, SB 217242, PD 156707, L-749,329, BQ-788 and bosentan were synthesized in the Department of Medicinal Chemistry, SmithKline Beecham Pharmaceuticals (King of Prussia, PA). RES-701 (Matsuda et al., 1993) was kindly provided by Kyowa Hakko Kogyo (Tokyo, Japan).

	Results

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Radioligand binding to human ET_A/ET_B receptors. Competition of [¹²⁵I]-ET-1 binding to recombinant human ET_A and ET_B receptors by unlabeled ET-1, ET-3 and the subtype-selective ligands S6c, BQ-123, and SB 234551 is shown in figure 1. Whereas ET-1 displayed similar affinities for ET_A and ET_B receptors (IC₅₀ = 0.3 and 0.2 nM, respectively), ET-3 had approximately 500-fold lower affinity for ET_A receptors, with IC₅₀ values of 100 and 0.2 nM for ET_A and ET_B, respectively (fig. 1). Similarly, S6c had an IC₅₀ of 0.5 nM for the ET_B receptor (fig. 1, bottom panel), whereas BQ-123 displayed an IC₅₀ of 50 nM for the ET_A receptor (fig. 1, top panel). In addition, BQ-123 displayed weak affinity for the cloned human ET_B receptors, and S6C displayed weak affinity toward the cloned human ET_A receptor.

View larger version (28K): [in this window] [in a new window]   Fig. 1.   Competitive binding of [125I]ET-1 to human cloned ETA (top panel) and ETB (bottom panel) receptors by ET-1 (), ET-3 (), S6c (), BQ-123 () and SB 234551 ().

In vitro functional activity. SB 234551 (10-1000 nM) produced concentration-dependent, parallel rightward shifts in the ET-1 concentration-response curves in the isolated rat aorta (fig. 2). The contractile response elicited by ET-1 in this tissue is mediated by the ET_A receptor (Ohlstein et al., 1996). The K_b value for inhibition of ET-1-induced contraction for SB 234551 was 1.9 ± 0.1 nM (fig. 2). Schild analysis of these concentration-response curves yielded a slope of the regression line of 0.97, which was not significantly different from unity. ET-1-induced maximal contraction in the isolated rat aorta was not significantly affected by SB 234551. No agonist activity was observed with SB 234551 at the highest concentration studied (10 µM).

View larger version (22K): [in this window] [in a new window]   Fig. 2.   Concentration-response curves for inhibition by SB 234551 of ETA-mediated contraction by ET-1 in rat isolated aorta. SB 234551 , control; , 10 nM; , 100 nM; , 1 µM) was added to baths 30 min before initiation of the ET-1 concentration-response curves. Results are expressed as a percentage of the response to KCl (60 mM) and are represented as the mean ± S.E.M; n = 6.

View larger version (18K): [in this window] [in a new window]   Fig. 3.   Effect of SB 234551 on ET-1 concentration-response curves in human pulmonary artery. SB 234551 (, control; , 10 nM) was added to baths 30 min before initiation of the ET-1 concentration-response curves. Responses are expressed as a percentage of the contraction produced by KCl (100 mM) added at the start of the experiment and are represented as the mean ± S.E.M.; n = 4.

View larger version (7K): [in this window] [in a new window]   Fig. 4.   Representative tracing of S6c-mediated endothelium-dependent vasodilation in rabbit isolated saphenous veins. The blood vessel was precontracted with norepinephrine (1 µM).

Inhibition of endothelin-induced pressor responses in conscious normotensive rats. Changes in mean arterial pressure in response to bolus i.v. injections of a submaximal dose of ET-1 (100 pmol/kg, the approximate ED₇₅) were measured at 30-min intervals before and after bolus i.v. administration of vehicle or SB 234551 (0.1, 0.1 and 1.0 mg/kg) in conscious, chronically catheterized male Sprague-Dawley rats. Unlike responses in anesthetized rats, the change in blood pressure after the bolus injection of ET-1 is of brief duration (3-5 min) and repeatable in short intervals without tachyphylaxis, as illustrated by the values for the vehicle-treated group in figure 5. Basal mean arterial blood pressure averaged 111 ± 2.5 mmHg for all rats and was not statistically different between groups; neither was it altered by the injection of various doses of SB 234551. The change in blood pressure after injections of 100 pmol/kg ET-1 was 35 ± 2 mmHg during the control period. SB 234551 (0.1-1 mg/kg) inhibited the pressor response to ET-1 in a dose-dependent manner (fig. 5). Maximal inhibition (80%) was observed with a dose of 1.0 mg/kg at 30 min after dosing, and the response to ET-1 gradually returned to control levels by 4 hr.

View larger version (22K): [in this window] [in a new window]   Fig. 5.   Duration of action of i.v. bolus injection of SB 234551 in the conscious rat. SB 234551 (, 0.1 mg/kg; , 0.3 mg/kg; , 1 mg/kg) or saline vehicle () was administered i.v. at time zero, and repeat doses of endothelin-1 were administered every 30 min. The values at time zero are the means of responses to three consecutive injections of 100 pmol/kg ET-1 at 30-min intervals. Data are presented as group means ± S.E.M. of percent change from the control response (100%) for each rat. The data (absolute values) were evaluated using an ANOVA for repeated measures. To compare the values with their own controls, Student's paired t test was used. A value of P < .05 was accepted as statistically significant (n = 5).

Oral pharmacokinetics of SB 234551 in conscious normotensive rats. Disposition kinetics and oral performance of SB 234551 were assessed in conscious male Sprague-Dawley rats. These investigations involved cross-over studies in multiple-cannulated rats to quantify the extent of absorption (intraduodenal infusion) and pharmacokinetic linearity. The plasma concentration-time profile demonstrated that SB 234551 was rapidly absorbed after intraduodenal infusion. The pharmacokinetic parameters are summarized in table 4. The systemic plasma clearance of SB 234551 was 25.0 ml/min/kg, and the intraduodenal bioavailability was 30%. The terminal plasma half-life values after i.v. and intraduodenal routes were similar: approximately 129 and 125 min, respectively.

	Discussion

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The development of high-affinity and subtype-selective receptor antagonists is expanding our understanding of the role of endothelin in pathophysiology. We have recently reported the development of a novel indane series of compounds that are high-affinity antagonists of both ET_A and ET_B receptors (Ohlstein et al., 1994a,b, 1996). Although the prototype molecule, SB 209670, is a high-affinity, selective endothelin receptor antagonist, there is only a 50-fold difference between the affinity at the ET_A receptor and that at the ET_B receptor. It is still not clear what is the optimal endothelin receptor subtype selectivity for endothelin-based therapeutics. On the basis of current understanding, however, it appears that receptors that are defined as ET_B1 mediate vasodilation and can be regarded as "beneficial," whereas stimulation of the purported ET_B2 receptors produces undesired effects such as smooth muscle contraction (Douglas et al., 1995a,b). This hypothesis has to be clarified, so compounds that differentiate between the vasodilator ET_B1 and the vasoconstrictor ET_B2 receptors would help to delineate further the role of endothelin in the etiology of cardiovascular and other diseases. Such compounds would also provide information on the optimal profile for a therapeutically useful endothelin receptor antagonist for specific disorders.

In this report, we describe the pharmacological characterization of SB 234551, the lead compound from a new series of pyrazole endothelin receptor antagonists. SB 234551 has a unique receptor selectivity profile; binding studies indicate that the compound has approximately 5000-fold more affinity for the cloned human ET_A receptor than for the cloned human ET_B receptor (table 1). There was a good correlation between the affinity of SB 234551 for recombinant human ET_A receptors from the results of radioligand binding and functional studies: in the former the K_i for SB 234551 was 0.13 nM, and the K_b values for inhibition of ET-1-mediated vasoconstriction in the isolated rat aorta and human pulmonary artery were 1.9 and 1.0 nM, respectively. These data indicate that SB 234551 is one of the highest-affinity endothelin receptor antagonists yet reported in human vascular tissue. In contrast, it is interesting to note that in human pulmonary artery, neither bosentan nor PD 156707 produced significant inhibition of ET-1-mediated vasoconstriction, despite high affinity in the cloned human ET_A receptor. In particular, convincing evidence exists that the responses in human large pulmonary artery appear to be mediated predominantly, if not exclusively, via ET_A receptors: 1) S6c, the ET_B receptor ligand, has no effect on basal vascular tone (Hay et al., 1993), and 2) ET-1-induced responses are inhibited potently by BQ-123 (Hay et al., 1993; Buchan et al., 1994). The lack of activity of bosentan or PD 156707 has not been explained, but it may indicate the presence of an ET_A receptor subtype that has differential sensitivity to the available endothelin receptor antagonists. Alternatively, it may be due to differences in antagonist affinities, which have also been reported for cultured human pulmonary smooth muscle cells (Hatakeyama et al., 1994) and in functional studies in this same tissue (Buchan et al., 1994). Nonetheless, the present data support the interpretation that ET-1 produces vascular contraction of the human pulmonary artery by stimulating the ET_A receptor, an effect antagonized significantly by SB 234551.

Numerous reports have demonstrated that multiple ET_B receptor subtypes exist (Warner et al., 1993a,b; Sudjarwo et al., 1993; Karaki et al., 1994, MacLean et al., 1994; Douglas et al., 1995b; Gellai et al., 1996; McCulloch and MacLean, 1995, 1996). For example, an ET_B receptor found on the vascular endothelium has been associated with endothelium-dependent vasorelaxation. This receptor has been termed "ET_B1-like." In the isolated rabbit saphenous vein, SB 234551 weakly inhibits ET_B-mediated release of endothelium-dependent vasodilation (IC₅₀ = 7 µM), in contrast to SB 209670 (3 nM), BQ-788 (300 nM) and RES 701 (300 nM) (table 3). The other ET_B receptor, designated "ET_B2-like," has been implicated in endothelin-mediated vasoconstriction (Warner et al., 1993a,b; Douglas et al., 1995b; Hay et al., 1996). The rabbit pulmonary artery possesses ET_B2-like receptors, and SB 234551 produces functional inhibition of the ET_B2-like receptor, as demonstrated by antagonism of S6c-mediated contraction of the isolated rabbit pulmonary artery (K_b = 555 nM). Although the affinity of SB 234551 in this tissue is modest, experiments with SB 209670 and SB 217242 also show a disparity between inhibition of rabbit and human ET_B receptor-mediated constriction, the latter two compounds being more potent in human bronchial ET_B receptors (Hay et al., 1996).

The functional roles of ET_A and ET_B receptors have been studied previously in the canine kidney (Brooks et al., 1994, 1995). In these studies renal vasoconstriction was shown to be mediated by ET_A receptors, and ET_B receptor stimulation inhibited sodium reabsorption. Furthermore, the ET_B1 receptor subtype was shown to mediate tubular sodium reabsorption, because RES-701 antagonized ET_B1-mediated natriuresis induced by S6c or ET-1 (in the presence of BQ-123). In addition, ET_B1 receptors may induce renal vasodilation. The in vivo demonstration of the ET_B1 receptor sparing activity of SB 234551 has been demonstrated in this same model (Brooks et al., 1998). The i.v. infusion of SB 234551 (30 µg/kg/min) in anesthetized dogs was demonstrated to inhibit the vasoconstrictor responses to exogenously administered ET-1 and significantly to increase renal plasma flow and urinary sodium excretion (Brooks et al., 1998). These data indicate that SB 234551 unmasks ET_B1 receptor-induced renal vasodilation and inhibition of sodium reabsorption.

In summary, this is the first report on the pharmacological characterization of SB 234551, the lead molecule from a new pyrazole series of endothelin receptor antagonists. SB 234551 is a high-affinity antagonist of ET_A/ET_B2-mediated functional responses. The optimal receptor profile (i.e., ET_A vs. ET_B) for the most therapeutically useful endothelin receptor antagonist is not yet known, but it is likely that different diseases require compounds with different receptor subtype profiles. However, because both ET_A and ET_B2 receptors are involved in vasoconstriction, an antagonist with high affinity for these receptors might be predicted to provide beneficial effects.