Intact cell binding for in vitro prediction of sedative and non-sedative histamine H1-receptor antagonists based on receptor internalization

We evaluated changes in the binding properties of sedative and non-sedative histamine H-1-receptor antagonists induced by internalization of H-1 receptors in intact human U373 MG astrocytoma cells. Internalization of H-1 receptors was induced without their degradation by treatment with 0.1 mM histamine for 30 min at 37 degrees C, and then the intact cell binding assay was performed at 4 degrees C. The binding properties of [H-3]mepyramine, a cell-penetrating radioligand for H-1 receptors, were not changed by histamine pretreatment. Displacement curves for sedative H-1-receptor antagonists (diphenhydramine, chlorpheniramine, promethazine, ketotifen, azelastine and oxatomide) against [H-3]mepyramine binding were not changed by histamine pretreatment. In contrast, the displacement curves for non-sedative H-1-receptor antagonists (mequitazine, bepotastine, olopatadine, epinastine, carebastine, desloratadine and fexofenadine) were changed by histamine pretreatment: two types of changes, i.e. a rightward shift in the monophasic curve or an increase in the proportion of the low affinity component of the biphasic curve, were prevented under hypertonic conditions, in which clathrin-mediated receptor internalization is known to be inhibited. Thus, internalization-mediated changes in the binding properties of H-1-receptor antagonists were well correlated with their sedative and non-sedative behaviors, which might confirm their permeability through the biomembrane and possibly the blood brain barrier.