Pharmacology of endothelin receptor antagonists ... - Clinical Science

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Clinical Science (2002) 103 (Suppl. 48), 112S–117S (Printed in Great Britain)

Pharmacology of endothelin receptor antagonists ABT-627, ABT-546, A-182086 and A-192621: ex vivo and in vivo studies Jerry L. WESSALE, Andrew L. ADLER, Eugene I. NOVOSAD, Samuel V. CALZADILLA, Brian D. DAYTON, Kennan C. MARSH, Martin WINN, Hwan-Soo JAE, Thomas W. VON GELDERN, Terry J. OPGENORTH and J. Ruth WU-WONG Pharmaceutical Products Division, Abbott Laboratories, Abbott Park, IL 60064, U.S.A.

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Endothelins (ETs), 21-amino-acid peptides involved in the pathogenesis of various diseases, bind to ETA and ETB receptors to initiate their effects. Based on the same core structure, we have developed four small-molecule ET receptor antagonists, ABT-627 (atrasentan), ABT-546, A182086 and A-192621, which exhibit differences in selectivity for ETA and ETB receptors. In this report, we compare the efficacy, potency and pharmacokinetic properties of these four antagonists, including potency in inhibiting ET-1- or Sarafotoxin 6c-induced vessel constriction in isolated arteries and efficacy in antagonizing ET-1-, big ET-1- or Sarafotoxin 6c-induced pressor responses in rats.

INTRODUCTION Endothelin (ET), originally isolated from cultured porcine aortic endothelial cells, is a peptide with 21-aminoacid residues [1]. Three distinct members of the ET family, namely, ET-1, ET-2 and ET-3, have been identified in humans through cloning [2]. Binding of ETs to G-protein-coupled receptors in tissues and cells activates various signalling molecules [3]. Two types of mammalian ET receptors, ETA and ETB, have been characterized. The ETA receptor is selective for ET-1 and ET-2, while ETB receptor binds ET-1, ET-2 and ET-3 with equal affinity [4–7]. ET binding to ETA receptor, which predominates in vascular smooth muscle cells, leads to vasoconstrictive and proliferative responses [8]. The results of ET binding to ETB receptor, which is abundant on vascular endothelial cells and is present to a lesser extent on vascular smooth muscle cells in some tissues and species, are less clearly understood. However, the ETB receptor on the vascular endothelium can mediate vasodilation through

the release of nitric oxide [8], and several lines of evidence support the concept that the ETB receptor is the primary receptor subtype responsible for clearing ET from the circulation [9]. A major advance was made in the ET field with the development of endothelin receptor antagonists [10]. BQ-123 and FR139317 [11,12], two peptidic ETAselective antagonists, are important tools in the investigation of ET-mediated pathophysiology. Following the peptidic compounds, a number of nonpeptide antagonists with improved pharmacokinetics, such as Ro 47-0203 [13], SB 217242 [14], ABT-627 (atrasentan) [15], etc., were developed. Some of these antagonists are being investigated in human clinical trials [16]. Recent evidence suggests that ETA receptor may play a more important pathological role than ETB receptor [8]. However, some tissues express ETB receptor predominantly, which seems to suggest that ETB receptor may also play a pathophysiological role [17]. Thus, ETAselective or ETB-selective antagonists could potentially have their unique utilities. Based on the same chemical

Key words : A-18 2086, A-19 2621, ABT-546, ABT-627, atrasentan, endothelin, endothelin receptor, endothelin receptor antagonist. Abbreviations : ET, endothelin ; S6c, sarafotoxin 6c ; i.v., intravenous ; AUC, area under the curve ; MAP, mean arterial blood pressure. Correspondence : Dr J. R. Wu-Wong, Abbott Laboratories, 5440 Patrick Henry Drive, Santa Clara, CA. 95 054, U.S.A. (e-mail ruth.r.wuwong!abbott.com).

# 2002 The Biochemical Society and the Medical Research Society

ET receptor antagonists sharing same core structure

structural core, we have developed four nonpeptide ET receptor antagonists, ABT-627, ABT-546, A-182086 and A-192621, which exhibit differences in selectivity for ETA and ETB receptors. The purpose of this report is to compare the potency and pharmacokinetic profiles of these four antagonists utilizing in vivo and ex vivo assay systems.

MATERIALS AND METHODS ET-1, big ET-1 and sarafotoxin 6c (S6c) were purchased from American Peptide Co. (Santa Clara, CA, U.S.A.). All other reagents were of analytical grade.

Animals Male Sprague-Dawley rats were purchased from Charles River (Kingston, NY, U.S.A.). New Zealand white rabbits, male, 2.0–2.5 kg, were purchased from Covance (Kalamazoo, MI, U.S.A.). Male and female dogs used in pharmacokinetic studies were purchased from Marshall Research Animals (North Rose, NY, U.S.A.). Female cynomolgus monkeys were obtained from the Abbott Drug Analysis Colony. All protocols utilizing live animals were approved by Abbott Laboratories’ Institutional Animal Care and Use Committee and were conducted in AAALAC (American Association for Accreditation of Laboratory Animal Care) accredited facilities.

Vessel contraction Antagonism of ET-1- or S6c-induced vasoconstriction by test agents was evaluated using isolated rat aortic rings (mediated by ETA) or rabbit pulmonary artery rings (mediated by ETB) [18]. The experimental details and the analysis of data by Schild analysis to calculate pA values # have been described previously [19].

separate experiments in each group. Difference test of P 0.05 was considered significant.

RESULTS Figure 1 shows that A-182086 produced concentrationdependent, parallel rightward shifts in the S6c (an ETB receptor-selective receptor agonist) concentration– response curve in isolated endothelium-denuded rings of rabbit pulmonary artery (mediated by the vasoconstrictor-type ETB receptor). The EC of S6c-induced &! vasoconstriction was 1.57 nM (n l 6). Schild analysis of the concentration–response curves in the presence of various A-182086 concentrations yielded a pA of # 8.00p0.23 (n l 6). The slope of the regression lines was 1.17p0.27 and r l 0.76. The S6c maximal contraction in the tissue was not significantly affected by A-182086. A182086 (up to concentrations of 10 µM) was devoid of agonist activity. Antagonism of ETA receptor by A-182086 was evaluated in endothelium-intact rat aortic rings in which ET1-induced vasoconstriction is mediated principally by ETA receptor (results not shown). The EC of ET-1&! induced vasoconstriction was 1.10 nM. A-182086 shifted the ET-1 concentration–response relationship to the right in a concentration-dependent manner, and Schild analysis yielded a pA of 8.53p0.26 (n l 6), with a slope of # 0.87p0.14 and r l 0.76. A-182086 alone also did not exhibit any intrinsic agonist activity on baseline contractile tone of endothelium-intact rat aortic rings. Therefore, A-182086 exhibits similar potency for both ETA and ETB receptors in this assay system. The studies were repeated for A-192621. The pA # values of A-192621 were 5.21p0.21 (n l 6) and 8.41p0.33 (n l 5) for ETA and ETB receptors respect-

Pharmacokinetics The pharmacokinetic behaviour of these antagonists was evaluated in male Sprague–Dawley rats (250–300 g), male or female beagle dogs, and female cynomolgus monkeys as described previously [19].

In vivo pseudoefficacy The in vivo efficacy of these antagonists was evaluated in male Sprague-Dawley rats (250–350 g) as described previously [19].

Statistics Statistical analysis of data was performed using StatView II software (Abacus Concepts, Berkely, CA, U.S.A.). Group comparisons were determined by an ANOVA followed by the Fisher’s protected least significant difference test. Unless noted, values are expressed as meanpS.E.M. and n represents the number of animals or

Figure 1 Inhibition of ETB-mediated vasoconstriction in isolated rabbit pulmonary artery by A-182086

S6c alone or with A-182086 at increasing concentrations were tested in isolated rabbit pulmonary artery. The Schild analysis of the data is shown as an inset (n l 6 ; a total of 36 pieces of pulmonary artery rings were prepared from six animals). # 2002 The Biochemical Society and the Medical Research Society

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Table 1

Summary of results from ex vivo and in vivo studies

*A-192621 alone caused elevation of systemic arterial blood pressure ; ND, not determined ; Cmax, maximal concentration. †All values for ABT-627 and ABT-546 have been reported previously [15,18]. Study

Parameters

Types

Isolated arteries

Aorta ring (pA2)

ETA ETB

Pharmacokinetics

Plasma half-life (h, oral)

Cmax ( µg/ml)

AUC/i.v. ( µg:h:ml−1)

AUC/oral ( µg:h:ml−1)

Bioavailability (F %)

Agonist-induced pressor response

ED50 (mg/kg) ED50 (mg/kg)

ABT-627†

ABT-546†

A-182086

A-192621

9.04 6.45

8.29 4.57

8.53 8.00

5.2 8.4

Rat Dog Monkey Rat Dog Monkey Rat Dog Monkey Rat Dog Monkey Rat Dog Monkey

6.2 3.5 2.5 3.44 4.67 0.32 7.36 6.45 2.48 8.86 4.59 1.01 60.2 43.2 21.7

2.5 1.3 1.0 1.69 2.47 1.10 3.44 6.25 10.80 3.28 1.66 1.13 47.7 16.2 18.1

8.1 5.0 2.0 2.36 19.04 0.09 10.30 37.34 2.42 11.16 26.67 0.27 54 71 11

5.0 2.0 3.1 0.81 0.38 0.19 3.60 4.07 5.25 2.51 0.45 0.68 34.9 11.0 13.0

ET-1 Big ET-1 S6c Vasodilatory Vasopressor

1 ND ND

10 ND ND

Figure 2 Plasma concentrations of A-182086 after a 5 (i.v.) or 10 (oral) mg/kg dose in the rat (n l 4)

ively. Results for ABT-627 and ABT-546 have been reported previously [15,18]. The average pA values of # these four compounds are summarized in Table 1. These data indicate that ABT-627 and ABT-546 are ETAselective, A-192621 is ETB-selective, while A-182086 is a non-selective antagonist. The pharmacokinetic profile of A-182086 was examined in rat, dog and monkey. In male Sprague– Dawley rats, the pharmacokinetic behaviour of A-182086 following a 5 mg\kg intravenous (i.v.) dose was # 2002 The Biochemical Society and the Medical Research Society

ND 10

No effect* ND

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characterized by an apparent plasma elimination half life of 5.0 h. A-182086 was absorbed from the 10 mg\kg oral dose, with peak plasma concentrations recorded 0.5 h after oral (solution) administration. Peak plasma concentrations averaged 2.36 µg\ml, declining with an apparent elimination half life of 8.1 h. The bioavailability of A182086 was estimated to be 54 % in the rat (Figure 2). Similar studies were performed in the beagle dog and in the cynomolgus monkey, and the results are summarized in Table 1. The pharmacokinetic studies were repeated for A192621 in rat, dog and monkey. Results for ABT-627 and ABT-546 have been reported previously [15,18]. The data from these studies are summarized in Table 1. These data suggest that these four antagonists exhibit good bioavailability in rat, dog and monkey. Measurement of big ET-1- and S6c-induced changes in mean systemic arterial blood pressure in conscious, normotensive rats was used to evaluate the in vivo oral efficacy of A-182086 against ETA and ETB receptors respectively. In Figure 3, A-182086 (1–100 mg\kg) or vehicle was administered orally by gavage. S6c (0.3 nmol\kg, i.v.) was administered as a bolus 1 h after dosing. The pressor response was quantified by calculation of the area under the curve (AUCMAP) of mean arterial blood pressure


Figure 4

Effect of A-19 2621 alone on arterial blood pressure

A-19 2621 (dose as indicated) was administered orally by gavage (A) or in food (B). Blood pressure was determined beginning at 30 min (A) or 3 days (B) after dosing. *P 0.05.

Figure 3 Antagonism of S6c-induced (0.3 nmol/kg, i.v. bolus) dilatory and/or pressor responses by A-182086 in conscious rats

(A) Blood pressure responses to exogenous S6c were determined at 1 h after dosing. (B) and (C) The dilatory and pressor responses were quantified by calculation of the AUCMAP measured over time and where MAP was expressed as the percent change from baseline pressure (i.e. average pressure measured over 30 min prior to the S6c challenge). *P 0.05. p.o., per os (oral). (MAP) measured over time, where MAP was expressed as the percent change from baseline pressure (i.e. average pressure measured over 30 min prior to S6c challenge). A-182086 exhibited a dose-dependent inhibition of both the depressor and pressor responses induced by S6c, and statistically significant inhibition was achieved at doses of

3–100 mg\kg for the depressor response, and 10– 100 mg\kg for the pressor response. The ED values are &! determined as 3 and 10 mg\kg for the depressor and pressor responses respectively. A-182086 (1–100 mg\kg, administered orally by gavage) also exhibited a dose-dependent inhibition of the peak pressor response to big ET-1 (0.5 nmol\kg, i.v.), and statistically significant inhibition was achieved at doses of 10–100 mg\kg (results not shown). The ED is &! determined as 10 mg\kg. The in vivo efficacy studies were repeated for A192621 in the rat with ET-1 for the assessment of its potency against ETA, and with S6c for ETB. As predicted, A-192621 inhibited the ET-1-induced dilatory response (mediated by ETB), but failed to inhibit the ET-1-induced pressor response (mediated by ETA) ; A-19 2621 was potent in blocking both the dilatory and pressor responses induced by S6c (mediated by ETB) with an ED value of 30 mg\kg. One interesting observation &! was that A-192621 alone, administered orally either by gavage or in food, caused elevation of arterial blood pressure (Figure 4). The results for ABT-627 and ABT# 2002 The Biochemical Society and the Medical Research Society

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546 have been reported previously [15,19]. The ED &! values of the four compounds from the in vivo studies are summarized in Table 1. These results indicate that these compounds are efficacious in inhibiting agonist-induced pressor and\or depressor responses as predicted by their selectivity towards the ETA or ETB receptor.

DISCUSSION We have reported the development and characterization of A-127722 (ABT-627 or atrasentan) and A-216546 (ABT-546), two novel, nonpeptide ET antagonists that are highly selective for ETA, previously [15,19]. Based on the same core structure, we have developed A-192621 [20], an ETB-selective antagonist, and A-182086 [21], a non-selective antagonist. Data clearly demonstrate that these four compounds exhibit distinct characteristics in term of their selectivity towards the two subtypes of ET receptors. ABT-627 and ABT-546 are selective for ETA, while A-192621 is selective for ETB, and A-182086 is a ‘ balanced ’ ETA\ ETB antagonist. To determine the efficacy of these compounds, we examined the ability of the antagonists to inhibit agonist-induced constriction of vascular tissue utilizing isolated rat aorta and rabbit pulmonary artery. It is known that, in the rat aorta, ETA is the predominant mediator [18], while in the rabbit pulmonary artery, ETB mediates agonist-induced responses [22]. All four compounds produce a parallel and rightward shift of the agonist (either ET-1 or S6c) concentration–response curve without affecting maximal force, and appear to act as fully competitive receptor antagonists. The potency and selectivity of these four compounds from this study is consistent with that observed in the receptor binding study. The pharmacokinetic studies indicate that all four compounds are orally available in the rat, dog and monkey. In addition, in the conscious rat, both ABT-627 and ABT-546 exhibit a dose-related inhibition of the blood pressure response to exogenous ET-1 with ED &! values of 1 and 10 mg\kg respectively. In the same animal model, A-182086 inhibits the pressor response induced by big-ET-1 (mediated by ETA) with an ED value of &! 10 mg\kg, and both dilatory and pressor responses induced by S6c (mediated by ETB) with ED values of &! 3 and 10 mg\kg respectively. As predicted by its selectivity toward the ETB receptor, A-192621 inhibits both dilatory and pressor responses induced by S6c mediated by ETB with an ED value of 30 mg\kg, and &! failed to inhibit the ET-1-induced pressor response mediated by ETA. A-192621 alone caused elevation of arterial blood pressure and an elevation in the plasma ET-1 level [23], suggesting that the ETB receptor is likely to be the clearance receptor for ET-1, and is involved in # 2002 The Biochemical Society and the Medical Research Society

maintaining arterial blood pressure in the conscious normotensive rat. In summary, this paper show that four compounds derived from the same core structure are highly potent, orally available ET receptor antagonists with different selectivity toward the two ET receptor subtypes.

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