We have previously reported the characterization of

We have previously reported the characterization of A-127722, a novel, non-peptide endothelin antagonist that displays very high affinity binding (K=69 pM) with a more than 1000-fold greater affinity for human endothelin ETA than ETB receptor. Although A-127722 is >1000-fold more selective for endothelin ETA receptor, it exhibits a K of 63.3 nM for endothelin ETB receptor. There is a concern that A-127722, at high doses and/or during conditions of chronic dosing, may cause a partial inhibition of the endothelin ETB receptor. To further improve the selectivity of A-127722 towards the endothelin ETA receptor, A-216546 was synthesized. A-216546 retains a high affinity (K=0.46 nM) for the human endothelin ETA receptor with a >25,000-fold greater affinity for endothelin ETA than ETB receptor. Similar binding characteristics are apparent from studies utilizing membranes prepared from MMQ cells (rat pituitary cell line) and porcine cerebellum. Verification that A-216546 is a functional antagonist was demonstrated by the concentration-dependent inhibition of endothelin-1-stimulated arachidonic Ciclopirox release and phosphatidylinositol hydrolysis, and by the lack of any agonist activity in these assays at concentrations up to 1 μM. A comparison of the in vitro potencies between A-216546 and other benchmark endothelin receptor antagonists is shown in Table 1. Clearly, A-216546 is as potent as, or more potent than, most of the endothelin receptor antagonists in the literature, and exhibits a selectivity for endothelin ETA receptor not surpassed by any other known endothelin receptor antagonist. The hallmark activity of endothelin-1 is potent constriction of vascular smooth muscle. The putative pathogenic roles that have been suggested for endothelin-1 usually involve a vasoconstrictive component (Opgenorth, 1995). In this regard, we examined the ability of A-216546 to inhibit endothelin-1-induced contraction of vascular tissue utilizing isolated rat aorta and rabbit pulmonary artery. It is known that, in the rat aorta, the endothelin ETA receptor is the predominant mediator of endothelin-1 activity (Panek et al., 1992), while in the rabbit pulmonary artery, the endothelin ETB receptor mediates the endothelin-1 responses (Fukuroda et al., 1994b). A-216546 produces a parallel and rightward shift of the endothelin-1 concentration–response curve without affecting maximal force generated to yield pA2 values of 8.29 and 4.57 for the aorta and the artery, respectively. These results suggest that A-216546 is highly selective for the endothelin ETA receptor, and, as in the binding and arachidonic acid release assays, A-216546 appears to act as a fully competitive receptor antagonist. In addition to being highly potent and endothelin ETA receptor-selective, the data presented here indicate that A-216546 is orally available in the rat, dog and monkey. In the conscious rat, A-216546 exhibits a dose-related inhibition of the blood pressure response to exogenous endothelin-1 yielding an ED50 of approximately 10 mg/kg for a single oral dose. At 30 mg/kg, nearly 80% of the endothelin-1-induced blood pressure response was antagonized by A-216546, yet no effect was observed on the transient depressor activity of endothelin-1. The in vivo ED50 for the orally administered A-216546 is higher than expected for a compound with a pA2 value of 8.29. The observed disparity between in vitro and in vivo potencies seems to be a general phenomenon for most of the endothelin receptor antagonists, and may be related to the lipophilic nature of these compounds. The carboxylic acid residue of A-216546 is likely to make it susceptible to protein binding, as well. Our previous studies show that most endothelin receptor antagonists exhibit strong binding to plasma proteins, especially serum albumin (Wu-Wong et al., 1996). As a result, when the concentrations of plasma proteins, such as serum albumin, increase, the potency of an antagonist tends to decrease (Wu-Wong et al., 1997a). Reports in the literature on endothelin receptor antagonists utilize various serum albumin concentrations in binding assays. For example, 0.01% human serum albumin or bovine serum albumin was used in the binding assay by Williams et al. (1995)or Sogabe et al. (1993). Bovine serum albumin at 0.1% was used in the assays by Webb et al. (1995)and Reynolds et al. (1995), while 0.5% bovine serum albumin was used by Clozel et al. (1994). In this report, 0.2% bovine serum albumin was used in the receptor binding studies. In comparison, the protein concentration in human blood is 7% and 55–60% of that is serum albumin. When the different protein concentrations in vitro and in vivo are taken into consideration, a higher-than-expected in vivo ED50 for A-216546 may not be too surprising.