Click to download
A potential role for dissociation rate from the M3 muscarinic receptor in the clinical onset of action of NVA237 and tiotropium Inhaled M3 muscarinic receptor antagonists are important bronchodilators used in the treatment of chronic obstructive pulmonary disease (COPD). The onset of action of tiotropium in the clinic is relatively slow, taking around 2 h to achieve maximal bronchodilation (Donohue et al., 2002). Recent studies with NVA237, an inhaled formulation of glycopyrrolate, show the potential for a faster onset of action (Overend et al., 2008). We have investigated whether these potential differences in onset of action can be explained by differences in the kinetic rate constants of the two drugs. In addition, we have examined the onset of functional antagonism in a cell-based in vitro calcium assay. l-[N-methyl-3H]Scopolamine methyl chloride was used to label the M3 receptor expressed in CHO-cell membranes. Competition kinetic experiments were carried out in the presence of unlabelled M3 receptor antagonist as previously described by Sykes et al., 2009. The assay was initiated by addition of membranes at specific time points and the plate was harvested at time point 0 using a manual Packard harvester. Data was analysed using GraphPad Prism software to give the kon and koff of the unlabelled compound. The kinetics of M3 receptor antagonists are shown in Table 1.
Table 1. The kinetic parameters of NVA237 and tiotropium. Data are mean ± s.e. mean (n ≥ 3). Concentrations of tiotropium and NVA237 at 30-fold their Ki were employed for studying the onset of action of these compounds in a CHO-M3 cell based in-vitro calcium assay. Compound was added to the cells at various time intervals before addition of an EC80 of methacholine at time 0. The ability of each compound to inhibit the subsequent calcium response was then monitored using a FLIPR machine. All experiments were run at 37°C in HBSS. At 30-fold Ki, both NVA237 and tiotropium were able to fully inhibit the subsequent EC80 of methacholine. The rate at which the antagonists achieved full inhibition was, however, different. The t½ of inhibition by NVA237 was 6.1 ± 2.1 min (n = 3), whereas the t½ for tiotropium was 29.4 ± 4.2 min (n = 3). The dissociation rate constant for NVA237 was 4.1-fold faster than that for tiotropium meaning it will be 4 times faster to achieve equilibrium with the M3 receptor (at equivalent concentrations). This was confirmed in the in vitro calcium assay, where the onset of action of NVA237 was 4.8-fold faster than tiotropium. These differences in dissociation rate suggest that NVA237 will have a more rapid onset of action than tiotropium in the clinic.
Donohue et al, (2002) Chest 122;47-55. |
|||||||||||||||||||
