Calcium Channel Receptor Binding Studies for Diltiazem and Its Major Metabolites

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Abstract
Pharmacologically distinct but allosterically interacting calcium channel antagonist binding sites have recently been identified using radiolabeled dihydropyridine derivatives (e.g., [3H]nitrendipine) and the benzothiazepine [3H]diltiazem. Whereas the functional significance of the dihydropyridine calcium channel antagonist receptor is well documented, it remains to be established whether drug interactions with the recognition site for [3H]diltiazem within the slow calcium channel or the allosteric interaction of the diltiazem binding site with the dihydropyridine receptor are of physiological significance. In a study of structure-activity relationships, we therefore examined the effects of diltiazem and five of its analogs on the binding of [3H]diltiazem and [3H]nitrendipine to the rat cerebral cortex. In parallel, we studied the effects of these drugs on the spontaneous myogenic contractions of the rat portal vein a functional test of calcium antagonism. The diltiazem analogs u sed in this study correspond to its major metabolites in humans, i.e., N-desmethyl-MA, desacetyl-(M1), N-desmethyl, desacetyl-(M2), O-desmethyl, desacethyl, desacetyl (M4), N-desmethyl, O-desmethyl, desacetyl-diltiazem (M6). Unlabeled diltiazem inhibited [3H)diltiazem binding at 37.degree.C with a pIC50 [-log IC50 (M)] of 6.87. pIC50 values for M1, MA, M2, M4, and M6 were 6.72, 6.49, 6.03, 5.51, and 5.33, respectively. pIC50 values for these drugs on [3H]diltiazem binding were significantly correlated (p < 0.01) with their pEC50 values for enhancement of [3H]nitrendipine binding to cerebral cortical membranes at 37.degree.C. Maximal enhancement of [3H]nitrendipine binding by diltiazem, M1, MA, M2, M4. and M6 was 73, 50, 9.7, 11, 12, and 52%, respectively. Qualitatively similar results were obtained for drug pIC50 values on [3H]nitrendipine binding in the presence of a constant concentration of verapamil (300nM). Under these conditions, however, a significant enhancement of [3H]nitrendipine binding was seen in the presence of all diltiazem analogs examined. Functionally, diltiazem inhibited the spontaneous contractions of the rat portal vein with a pIC50 of 6.93. The pIC50 in this preparation for the metabolites M1, MA, M2, M4, and M6 were 7.04, 6.31, 6.14, 5.02, and < 4.5, respectively. Thus, within the benzothiazepine series, drug-induced inhibition of phasic activity in the portal vein, a response sensitive to Ca2+ channel antagonism, is significantly correlated with its affinity for the diltiazem calcium channel antagonist recognition site as measured by its competitive inhibition of [3H]diltiazem binding (p < 0.01) or by its allosteric enhancement of [3H]nitrendipine binding (p < 0.05). The magnitude of the allosteric effect on [3H]nitrendpine binding does not appear to be correlated with functional response of calcium antagonism.