Characterization of [3H]Ba 679 BR, a slowly dissociating muscarinic antagonist, in human lung: Radioligand binding and autoradiographic mapping

Abstract

Ba 679 BR [7(S)-(1α,2β,4β,5α,7β)-7-[(hydroxydi(2- thienyl)acetyl)oxy]-9,9-dimethyl-3-oxa-9-azoniatricyclo[3.3.1.02,4]nonane bromide] is a new long-acting muscarinic antagonist developed as a bronchodilator drug. In this study, we have evaluated its affinity, its selectivity, and the distribution of its binding sites in human lung. [3H]Ba 679 BR binds to a homogeneous population of muscarinic receptors in human lung membranes, with affinities in the subnanomolar concentration range. Like ipratropium bromide, Ba 679 BR showed no selectivity in its interactions with rat cerebrocortical M1 receptors (labeled with [3H]telenzepine) or heart M2 and salivary gland M3 receptors [labeled with [N-methyl-3H]scopolamine ([3H]NMS)]. Ba 679 BR displayed 6-20-fold higher affinity, compared with ipratropium bromide. We also studied the rate of Ba 679 BR and ipratropium bromide dissociation from human lung muscarinic receptors, by monitoring [3H]NMS association. Unlike ipratropium bromide (100 nM), which dissociated so quickly that there was little difference in the [3H]NMS association, compared with vehicle-treated membranes, Ba 679 BR (1 nM) had a strong protective effect against [3H]NMS binding (>70%) that lasted for 90 min. Kinetic experiments conducted with [3H]Ba 679 BR confirmed the slow dissociation profile of this compound. The dissociation rate constant (k- 1) for [3H]Ba 679 BR was 3.29 ± 0.18 x 10-3 min-1, corresponding to a half-life of the complex of 212 ± 11 min. Autoradiographic studies revealed that [3H]Ba 679 BR binding sites were densely distributed in alveolar walls and submucosal glands. These results suggest that the slow dissociation profile of Ba 679 BR from human lung muscarinic receptors might be the underlying mechanism by which this drug achieves its long duration of action observed in functional tests.