ELIOT H. OHLSTEIN, PONNAL NAMBI, DOUGLAS W. P. HAY, MIKLOS GELLAI, DAVID P. BROOKS, JUAN LUENGO, JIA-NING XIANG AND JOHN D.

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1 /98/ $03.00/0 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 286, No. 2 Copyright 1998 by The American Society for Pharmacology and Experimental Therapeutics Printed in U.S.A. JPET 286: , 1998 Nonpeptide Endothelin Receptor Antagonists. XI. Pharmacological Characterization of SB , a High-Affinity and Selective Nonpeptide ET A Receptor Antagonist ELIOT H. OHLSTEIN, PONNAL NAMBI, DOUGLAS W. P. HAY, MIKLOS GELLAI, DAVID P. BROOKS, JUAN LUENGO, JIA-NING XIANG AND JOHN D. ELLIOTT Departments of Cardiovascular, Renal and Pulmonary Pharmacology and Medicinal Chemistry, SmithKline Beecham Pharmaceuticals, King of Prussia, Pennsylvania Accepted for publication April 29, 1998 This paper is available online at 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 Received for publication August 1, ABSTRACT The present study describes the pharmacological profile of ((E)-alpha-[[1-butyl-5-[2-[(2-carboxyphenyl)methoxy]-4-methoxy-phenyl]-1H-pyrazol-4-yl]methlene]-6-methoxy-1,3-benzodioxole-5-propanoic acid) (SB ), a high-affinity, nonpeptide endothelin type A (ET A )-selective receptor antagonist. In human cloned ET A and endothelin type B (ET B ) receptors, SB produced a concentration-dependent displacement of [ 125 I]-endothelin-1 with K i values of 0.13 and 500 nm, respectively. SB elicited concentration-dependent, rightward competitive shifts in the endothelin-1 concentration-response curves in isolated rat aorta and isolated human pulmonary artery (ET A receptor-mediated vascular contraction) with K b values of 1.9 and 1.0 nm, respectively. SB antagonized ET B receptor-mediated vasoconstriction in the isolated rabbit pulmonary artery, as demonstrated by concentration-dependent, rightward shifts in the sarafotoxin S6c concentrationresponse curves (K b 555 nm). SB produced weak functional inhibition of sarafotoxin S6c-mediated endotheliumdependent relaxation (IC 50 7 M). SB (10 M) had no significant effect against contraction produced by several other vasoactive agents and did not significantly influence radioligand binding to a number of diverse receptors. SB ( mg/kg i.v.) dose-dependently inhibited the pressor response to exogenous endothelin-1 in conscious rats. In vivo pharmacokinetic analysis in the rat demonstrated that SB was rapidly absorbed from the GI tract with a bioavailability of 30%. SB had a plasma half-life of 125 min and a systemic clearance of 25.0 ml/min/kg. The present study demonstrates that SB is an antagonist with high affinity for the ET A receptor, while sparing the ET B receptor. SB is a new pharmacological tool that should assist in the elucidation of the role of endothelin in pathophysiology. (Douglas et al., 1995a). The endothelin receptor antagonists RES-701 and PD 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 M, 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. ABBREVIATIONS: ET-1, endothelin-1; ET-3, endothelin-3; S6c, sarafotoxin S6c; ET A receptor, endothelin type A receptor; ET B receptor, endothelin type B receptor; SB , ((E)-alpha-[[1-butyl-5-[2-[(2-carboxyphenyl)methoxy]-4-methoxy-phenyl]-1H-pyrazol-4-yl]methlene]-6- methoxy-1,3-benzodioxole-5-propanoic acid). 650

2 1998 SB , ET Receptor Antagonist 651 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 (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 , would appear to have an attractive compound profile. In this study, we report on the discovery and characterization of SB , the lead compound from a new chemical series of high-affinity nonpeptide endothelin receptor antagonists with such a profile. Materials and Methods 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). [ 125 I]ET-1 binding to membrane preparations was performed as described previously (Nambi et al., 1994). Briefly, assay volumes were 50 l, and the concentrations of membrane proteins were 0.50 and 0.05 g/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 M 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 ( 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; KH 2 PO 4, 1.2; MgSO 4, 1.2; CaCl 2, 2.5; NaHCO 3, 25.0 and dextrose, Tissue baths were maintained at 37 C and aerated continuously with 95% O 2 /5% CO 2,pH 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 M) to produce relaxation of tissues precontracted with norepinephrine (10 M). 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. Human lung tissue, from organ donors with no known history of respiratory disorders, was obtained from the International Institute for the Advancement of Medicine (Exton, PA) and the National Disease Research Interchange (Philadelphia, PA). Lungs were received within 24 hr of removal. A glass probe was placed in the pulmonary artery, and 4-mm ring preparations were prepared; the endothelium was removed as described above. The preparations were then placed in 10-ml organ baths containing Krebs solution and connected via silk suture to Grass FT03C force-displacement transducers. Mechanical responses were recorded isometrically by MP100WS/Acknowledge data acquisition system (BIOPAC Systems, Goleta, CA). Tissues were equilibrated under 2 gm resting load for 1 hr, and washed every 15 min with fresh Krebs solution, before the start of each experiment. To test for viability, after the equilibration period and before construction of cumulative concentration-response curves, tissues were exposed to phenylephrine (10 M) for human pulmonary artery or to KCl (60 mm) for rat aorta and rabbit pulmonary artery. After plateau of this reference contraction, tissues were washed several times over 60 min. In experiments examining the effects of receptor antagonists, tissues were exposed to the compound for 30 min before the addition of contractile agonists. Only one agonist concentrationresponse curve was generated per tissue, and paired tissues were used for evaluation of receptor antagonists. Experiments evaluating ET B -mediated vasodilation in norepinephrine (1 M)-precontracted isolated rabbit saphenous vein were performed as described previously (Douglas et al., 1995b). Agonist-induced responses were expressed as a percentage of the reference contraction obtained at the beginning of the experiment. Geometric mean EC 50 values were calculated from linear regression analyses of data. Using nonlinear least-squares regression, concentration-response curves were analyzed by fitting the experimental data to the following logistic equation: R R max Cn H n EC H 50 C n (1) H where R is the response, C is the concentration of agonist, EC 50 is the concentration of agonist required to produce a half-maximal response, n H is the Hill coefficient and R max is the maximal response. Schild analysis was performed on concentration-response curves initially fitted using nonlinear least-squares regression (Arunlakshana and Schild, 1956). Where appropriate, antagonist affinities were calculated and expressed as K b, where K b [antagonist]/(dose ratio 1). In vivo hemodynamics. Male Sprague-Dawley rats ( 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. On the day of experimentation, animals received a bolus i.v. dose of 100 pmol/kg ET-1 ( ED 75 dose). Thirty minutes later, immediately prior to a repeat dose of ET-1, either saline vehicle or SB (0.1 1 mg/kg) was administered. Repeat doses of ET-1 were administered every 30 min over a subsequent 4-hr period. Pharmacokinetics of SB in rats. The pharmacokinetic evaluation of SB 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 was administered as a 2-hr intraduodenal infusion (in saline vehicle) via the duodenal cannula at a rate of 50 g/kg/hr (total dose, 6 mg/kg). Blood samples (110 l) 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 as an i.v. infusion. SB was administered via the femoral vein cannula at a rate of 0.20 g/hr/kg for 2 hr.

3 652 Ohlstein et al. Vol. 286 Pharmacokinetic analysis. SB was isolated from rat plasma by liquid-liquid extraction and was quantitated by reversephase 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 concentrationtime 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. All experiments were performed in accordance with the guidelines of the Animal Care and Use Committee, SmithKline Beecham Pharmaceuticals and AALAC. 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). [ 125 I]ET-1 (2200 Ci/mmol) was obtained from New England Nuclear (Boston, MA). All other chemicals were of the highest grades available. SB , SB , SB , PD , 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 Radioligand binding to human ET A /ET B receptors. Competition of [ 125 I]-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 is shown in figure 1. Whereas ET-1 displayed similar affinities for ET A and ET B receptors (IC and 0.2 nm, respectively), ET-3 had approximately 500-fold lower affinity for ET A receptors, with IC 50 values of 100 and 0.2 nm for ET A and ET B, respectively (fig. 1). Similarly, S6c had an IC 50 of 0.5 nm for the ET B receptor (fig. 1, bottom panel), whereas BQ-123 displayed an IC 50 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. SB was a high-affinity ligand at the cloned human ET A receptor, with approximately 5000-fold higher affinity for this receptor than for the human cloned ET B receptor: K i 0.13 nm and 500 nm, respectively (fig. 1). The affinities of SB and other nonpeptide endothelin receptor antagonists are summarized in table 1. These data demonstrate that SB is one of the highest-affinity nonpeptide receptor antagonists yet described for the cloned human ET A receptor but has low affinity for the cloned human ET B receptor. In vitro functional activity. SB ( 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 was nm (fig. 2). Schild analysis of these Fig. 1. Competitive binding of [ 125 I]ET-1 to human cloned ET A (top panel) and ET B (bottom panel) receptors by ET-1 ( ), ET-3 ( ), S6c (}), BQ-123 (Œ) and SB (E). TABLE 1 Comparison of in vitro affinities of endothelin receptor antagonists for inhibition of [ 125 I]-ET-1 binding in cloned human ET A and ET B receptors Compound Cloned Human ET A K i (nm) Cloned Human ET B K i (nm) SB SB SB Bosentan 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 No agonist activity was observed with SB at the highest concentration studied (10 M). Characterization of ET-1-induced contraction in isolated human pulmonary artery was performed. The responses to ET-1 in the human large pulmonary artery are mediated predominantly, if not exclusively, via ET A receptor activation (Hay et al., 1993). In human isolated large pulmonary arteries, ET-1 (1 300 nm) produced a concentration-dependent contraction with an EC 50 of 5 nm (fig. 3). SB (10 nm) elicited high-affinity, surmountable antagonism of ET-1- induced contractions with a K b of 1.0 nm. The relative affinities of SB and other nonpeptide

4 1998 SB , ET Receptor Antagonist 653 TABLE 2 Effects of various endothelin receptor antagonists against contractions produced by ET-1 in isolated human pulmonary artery Compound K b (nm) SB (10 nm) SB (10 nm) SB (30 nm) Bosentan No effect (1 M) BQ (3 M) PD No effect (100 nm) L-749, (10 nm) Results are expressed as K b (nm); n 3to6. Fig. 2. Concentration-response curves for inhibition by SB of ET A -mediated contraction by ET-1 in rat isolated aorta. SB F, control; {, 10nM;, 100 nm; E, 1 M) 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. Fig. 3. Effect of SB on ET-1 concentration-response curves in human pulmonary artery. SB (F, 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. endothelin receptor antagonists were evaluated against ET- 1-induced contractions in human pulmonary artery (table 2). The affinity of SB was similar to those for SB and L-749,329 (K b 0.62 and 2.2 nm, respectively), approximately 5-fold greater than that of SB (K b 5.3 nm) and approximately 200-fold greater than that of BQ-123 (K b 221 nm; table 2). It is interesting to note that in the isolated human pulmonary artery, both bosentan (1 M) and PD (100 nm) had no significant effect on ET-1 induced contraction. SB produced functional inhibition of ET B2 receptor-mediated vasoconstriction, as demonstrated by antagonism of S6c-mediated contraction in the isolated rabbit pulmonary artery. S6c produces contractile responses in this tissue via ET B receptor activation (Warner et al., 1993a,b; Ohlstein et al., 1994a). SB (10 M) produced inhibition in the S6c concentration-response curves, with a K b of nm (table 3). Comparison with other endothelin receptor antagonists shows that SB had about the same functional blockade for the ET B2 receptor in rabbit pulmonary artery as SB (K b 353 nm) and had approximately 35-fold less affinity at this receptor than SB SB had at least 2-fold more affinity than either bosentan or BQ-788, whereas RES-701 (10 M) had no affinity for this receptor. S6c produces ET B1 -mediated vasodilation in the rabbit saphenous vein (Douglas et al., 1995b). ET-3 produced similar vasodilation responses in the isolated rabbit saphenous vein (data not shown). SB was a weak antagonist of ET B1 receptor-mediated vasodilation, as demonstrated by its limited ability to inhibit ET-3-mediated vasodilation (IC 50 7 M) in the isolated endothelium-intact rabbit saphenous vein (fig. 4; table 3). Thus an approximately 3600-fold difference was apparent between the K b value for inhibition of the ET A receptor-mediated functional response, with respect to inhibition of the ET B receptor-mediated functional response (table 3). In contrast, both SB and SB produced greater inhibition of ET B2 receptor-mediated dilation in the rabbit saphenous vein, with respective IC 50 values of 4 and 70 nm (table 3). Furthermore, bosentan was a weak inhibitor of ET B2 receptor-mediated dilation, with an IC 50 value of 2 M. The selectivity of SB was evaluated in several in vitro assays. SB (10 M), a concentration four orders of magnitude greater than the K i or K b at the ET A receptor, TABLE 3 Comparison of in vitro functional affinities of various endothelin receptor antagonists against contractions produced by S6c in the isolated rabbit pulmonary artery, or S6c-mediated dilation in the rabbit saphenous vein Compound ET B1 - mediated Dilation IC 50 (nm) ET B2 -mediated Constriction K b (nm) SB SB SB Bosentan BQ RES ,000 All compounds were evaluated at 10 M; n 4to5.

5 654 Ohlstein et al. Vol. 286 Fig. 4. Representative tracing of S6c-mediated endothelium-dependent vasodilation in rabbit isolated saphenous veins. The blood vessel was precontracted with norepinephrine (1 M). did not significantly affect radiolabeled ligands binding to a number of receptors with diverse ligands, including CGRP, IL-8, NK-1, NK-2, NK-3, arginine vasopressin and thrombin. However, SB produced weak inhibition of radioligand binding of [ 125 I]angiotensin II to rat adrenal cortex (K i 5.2 M) and angiotensin II-induced vasoconstriction in the rabbit aorta (K b 2.9 M). Nevertheless, this degree of affinity was over three orders of magnitude lower at the AT 1 receptor than at the ET A receptor, and it is unlikely that this activity contributes significantly to the compound s pharmacological activity. 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 ED 75 ) were measured at 30-min intervals before and after bolus i.v. administration of vehicle or SB (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 mmhg for all rats and was not statistically different between groups; neither was it altered by the injection of various doses of SB The change in Fig. 5. Duration of action of i.v. bolus injection of SB in the conscious rat. SB (F, 0.1 mg/kg;, 0.3 mg/kg;, 1 mg/kg) or saline vehicle (E) 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). blood pressure after injections of 100 pmol/kg ET-1 was 35 2 mmhg during the control period. SB (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. Oral pharmacokinetics of SB in conscious normotensive rats. Disposition kinetics and oral performance of SB were assessed in conscious male Sprague-Dawley rats. These investigations involved crossover studies in multiple-cannulated rats to quantify the extent of absorption (intraduodenal infusion) and pharmacokinetic linearity. The plasma concentration-time profile demonstrated that SB was rapidly absorbed after intraduodenal infusion. The pharmacokinetic parameters are summarized in table 4. The systemic plasma clearance of SB 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 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 , 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 , the lead compound from a new series of pyrazole endothelin receptor antagonists. SB 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 TABLE 4 Pharmacokinetic parameters for SB in Sprague-Dawley Rats Parameters Intraduodenal Intravenous Total dose (mg/kg) Plasma clearance (ml/min/kg) Bioavailability (%) 30 NA Terminal half-life (min) NA not applicable.

6 1998 SB , ET Receptor Antagonist 655 between the affinity of SB for recombinant human ET A receptors from the results of radioligand binding and functional studies: in the former the K i for SB 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 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 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 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 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 endotheliumdependent vasorelaxation. This receptor has been termed ET B1 -like. In the isolated rabbit saphenous vein, SB weakly inhibits ET B -mediated release of endothelium-dependent vasodilation (IC 50 7 M), in contrast to SB (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 produces functional inhibition of the ET B2 -like receptor, as demonstrated by antagonism of S6cmediated contraction of the isolated rabbit pulmonary artery (K b 555 nm). Although the affinity of SB in this tissue is modest, experiments with SB and SB 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 has been demonstrated in this same model (Brooks et al., 1998). The i.v. infusion of SB (30 g/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 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 , the lead molecule from a new pyrazole series of endothelin receptor antagonists. SB 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. Acknowledgments The authors thank A. Gao, A. Konalian-Beck, S. Atkinson, M. Darcy and D. Shah for synthesis of the compounds used in this study; M. Pullen and M. Luttmann for excellent technical assistance; Dr. Kei-lei Fong for assisting in the pharmacokinetic studies and Dr. Stephen Douglas for critical reading of the manuscript. References Arunlakshana O and Schild HO (1956) Some quantitative uses of drug antagonists. 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