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Pharmacology of the Urotensin-II Receptor Antagonist ACT-058362: First Demonstration of a Pathophysiological Role of the Urotensin System

Urotensin-II (U-II) is a cyclic peptide now described as the most potent vasoconstrictor known. U-II binds to a specific G-protein coupled receptor, formerly the orphan receptor GPR14, now renamed urotensin receptor (UT receptor), and present in mammalian species. ACT-058362 (1-[2-(4-benzyl-4-hydroxy-piperidin-1-yl)-ethyl]-3-(2-methyl-quinolin-4-yl)-urea sulfate salt) is a new potent and specific antagonist of the human UT receptor. ACT-058362 antagonizes the specific binding of 125 I-Iabeled U-II on natural and recombinant cells carrying the human UT receptor with a high affinity in the low nanomolar range and a competitive mode of antagonism, revealed only with prolonged incubation times. ACT-058362 also inhibits U-II induced calcium mobilization and mitogen-activated protein kinase (MAPK) phosphorylation. The binding inhibitory potency of ACT-058362 is more than 100-fold less on the rat than on the human UT receptor, which is reflected in a pD’ 2 of 5.2 for inhibiting contraction of isolated rat aortic rings induced by U-II. In functional assays of short incubation times, ACT-058362 behaves as an apparent non-competitive inhibitor. In vivo, intravenous ACT-058362 prevents the no-reflow phenomenon, which follows renal artery clamping in rats, without decreasing blood pressure, and prevents the subsequent development of acute renal failure and the histological consequences of ischemia. In conclusion, the in vivo efficacy of the specific UT receptor antagonist ACT-058362 reveals a role of endogenous U-II in renal ischemia. As a selective renal vasodilator, ACT-058362 may be effective in other renal diseases. The study was performed in male Wistar rats weighing 200 to 300 g. The rats were handled according to the "Position of the American Heart Association on Research Animal Use" adopted November 11, 1984, by the American Heart Association. All rats were housed in climate-controlled conditions with a 12-h light/dark cycle and free access to normal pelleted rat chow and drinking water. The experiments were performed after an adaptation period of at least one week. Rats were anesthetized with 150 mg/kg, i.p. thiobutabarbital-Na (Inactin, Altana Pharma, Konstanz, Germany) and placed on a thermostatically controlled heating table to maintain body temperature at 36–38 ° C. A tracheotomy tube was put in place and a catheter was inserted into the left jugular vein for drug infusion. A polyethylene cannula was placed in the right carotid artery and connected to a pressure transducer (MLT1050 precision BP transducer, AD Instruments, Hastings, UK) for recording of arterial blood pressure. Through a midline abdominal incision, the right kidney was removed and a Doppler flow probe was placed around the left renal artery for measurement of renal blood velocity. The probe was connected to a pulsed Doppler flowmeter (Triton Technology, San Diego, CA, USA). Tracings were recorded on a PowerLab (IOX Data acquisition, Emka Technologies, Paris, France) connected to a Dell Dimension 733R computer with the Datanalyst software (version 1.5, Emka Technologies, Paris, France). After a stabilization period, mean arterial blood pressure (MAP), heart rate (HR), and renal blood flow were continuously recorded. After baseline measurements, rats randomly received a continuous infusion of ACT-058362 (10 mg/kg/h, i.v.) or vehicle (saline, 2 ml/kg/h). This rate of infusion resulted in plasma concentrations of around 5 µ M. Thirty minutes after starting infusion, renal ischemia was induced by a 45-min clamping of the left renal artery with a snare as stock solutions Dilutions were made using 0.1% BSA on the of each experiment. Norepinephrine bitartrate, 5-hydroxytryptamine hydrochloride, L-phenylephrine hydrochloride and acetylcholine chloride were obtained from Sigma KCl, HEPES, BSA Fraction V, DMSO and EDTA were obtained from DMEM and PBS were from Laboratories and Invitrogen and Ham’s F12 medium was from Scatchard analysis was performed on human UT receptors expressed on membrane preparations from recombinant CHO cells to determine human U-II and ACT-058362 binding kinetics. Initial experiments were performed at room temperature with incubation time of 8 h where the binding of 125 I-U-II reached a plateau. The experimental curve in the Scatchard analysis was linear and compatible with one binding site with an apparent K D of 50 pM and B max of 2.4 pM corresponding to 3600 receptors per cell. However, ligand-dissociation experiments indicated the presence of two binding sites for 125 I-U-II: a fast dissociation site with k off in the order of 0.02 min − 1 , and a slow dissociation site with a k off 0.0015 min − 1 (data not shown). The very low k off rate of the slow dissociation site indicated that the time required to reach binding equilibrium may not have been sufficient in the initial binding studies where an incubation time of 8 h was used.