Evidence for a distinct Ca2+ antagonist receptor for the novel benzothiazinone compound HOE 166

The pharmacological and binding properties of the novel enantiomerically pure benzothiazinone (R)-(+)-3,4-dihydro-2-isopropyl-4-methyl-2-[2-[4-[4-[2-(3,4,5-tri- methoxyphenyl)-ethyl]-piperazinyl]-butoxyl-phenyl]-2H-1,4- benzothiazine-3-one dihydrochloride (HOE 166), are described. HOE 166 stereoselectively inhibited KCl-but not noradrenaline-induced contractions of guinea-pig pulmonary arteries, rabbit aorta, rat mesenteric artery preparations and k-strophantin-induced enhancement of guinea-pig papillary muscle contraction in a dose-dependent manner. KCl-induced smooth muscle contraction was inhibited by HOE 166 with IC50-values of approximately 70 nM (5-11 times less potent than nifedipine, 2-16 times more potent than verapamil), the respective S-(-)-enantiomer being approximately 10-fold less potent. HOE 166 decreased the upstroke velocity of the slow action potential in partially depolarized guinea-pig papillary muscle at similar concentrations than nifedipine. To investigate possible interactions with the calcium channel, HOE 166 and its S-(-)-enantiomer were characterized by radioligand binding studies in heart, brain and skeletal muscle transverse-tubule membranes. HOE 166 was a 4-15 times more potent inhibitor of reversible (+)-[3H]PN200-110, (-)-[3H]desmethoxyverapamil and d-cis [3H]diltiazem binding compared to its pharmacologically less active (S)-(-)-enantiomer, with IC50 values in the low nanomolar range. Extensive equilibrium and kinetic studies suggest that HOE 166 exerts its Ca2+-antagonistic effect by binding to a Ca2+-channel-associated drug receptor which is distinct from the 1,4-dihydropyridine, phenylalkylamine or benzothiazepine-selective domain. This HOE 166-selective site is, however, allosterically linked to the other sites of the Ca2+ antagonist receptor complex. We conclude that HOE 166 is a novel calcium antagonist.