Studies under non-physiological conditions suggest that long receptor residency time is responsible for the 24-h duration of action of the long-acting muscarinic antagonist (LAMA) tiotropium. We aimed to determine clinically relevant dissociation rates in physiological conditions and evaluate if the differences in onset of action between tiotropium and NVA237, a once-daily dry-powder formulation of the LAMA glycopyrronium bromide in development for chronic obstructive pulmonary disease, and the improved cardiovascular therapeutic index with NVA237 in animal models could be attributed to differences in kinetic rate constants. The binding of radioligand [3H]N-methyl-scopolamine ([3H]NMS) was measured in the presence/absence of several concentrations of unlabeled competitors and data analyzed in a competition kinetic model to provide on/off rates for the competitor. We found shorter dissociation half-lives for NVA237 and tiotropium under physiological (11.4 and 46.2 min, respectively) versus non-physiological conditions (173 and 462 min, respectively). NVA237 had more rapid onset of action (3-4.8x) versus tiotropium, determined in in vitro calcium and rat tracheal strip assay. Simulations suggested that the more rapid onset of NVA237 action could be explained by differences in kinetic parameters. NVA237 had higher equilibrium binding and kinetic selectivity for M3 versus M2 receptors, and faster off-rate from M2 versus M3 receptors versus tiotropium, potentially affording it more favorable therapeutic index. This study suggests that the 24-h duration of action of NVA237 and tiotropium is not solely the result of their slow dissociation from the M3 receptor and highlights the importance of conducting in vitro experiments in conditions reflecting those in vivo.
- Received March 16, 2012.
- Revision received July 6, 2012.
- Accepted July 31, 2012.
- The American Society for Pharmacology and Experimental Therapeutics