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Binding kinetics of a novel muscarinic antagonist, [3H]GSK573719: A comparison to [3H]tiotropium. GSK573719 is a potent, pan-active muscarinic cholinergic receptor (mAChR) antagonist that demonstrates slow functional reversibility in vitro and long duration of action in vivo when administered directly to the lungs. GSK573719 may be suitable for use as a once-daily anti-bronchoconstrictive agent for COPD and is being progressed into humans. In this report (funded by GlaxoSmithKline) we describe the pharmacological characterisation of the binding kinetics of [3H]GSK573719 and [3H]tiotropium to the human mAChR subtype M2 or M3 stably expressed in chinese hamster ovary (CHO) cells. Radioligand binding experiments were conducted using either [3H]GSK573719 or [3H]tiotropium exposed to membrane fragments obtained from CHO cells expressing the human recombinant M2 or M3 mAChR at 37°C in binding buffer (50mM HEPES, pH 7.4). Non-specific binding was determined by addition of 10μM atropine. Saturation, association, and dissociation binding studies were performed to determine receptor binding kinetics at the M2 and M3 mAChR (equilibrium dissociation constant (K D), total number of receptors (B max), association rate (k on), dissociation rate (k off), and dissociation half-life (t1/2 )). Dissociation was initiated by a 1:20 dilution in binding buffer (containing 10µM atropine). All data shown are mean ± SEM, n=4.
Table 1. The receptor saturation binding parameters for [3H]GSK573719 and [3H]tiotropium at human M2 and M3 receptors. Specific binding data from saturation experiments were fitted to a one affinity site model and this analysis resulted in pK D and B max values shown in Table 1. [3H]GSK573719 and [3H]tiotropium both exhibited sub-nM affinity for M2 and M3 mAChRs. A comparable affinity for the M3 receptor was exhibited for both these radioligands whilst [3H]GSK573719 had a marginally greater selectivity for the M3 over the M2 receptor (˜5-fold) compared with [3H]tiotropium (˜3-fold). The B max values for [3H]GSK573719 and [3H]tiotropium at both the human M2 and M3 mAChR were similar suggesting that both radioligands were labelling the same population of receptors.
Table 2. The receptor binding kinetic parameters for [3H]GSK573719 and [3H]tiotropium at human M2 and M3 receptors. Comparable values of k on were obtained for the two radioligands at each receptor subtype although [3H]GSK573719 and [3H]tiotropium associated more rapidly with the M2 mAChR than with the M3 mAChR (Table 2). [3H]GSK573719 and [3H]tiotropium dissociated faster from the M2 mAChR than the M3 mAChR, however dissociation of [3H]GSK573719 from the M2 mAChR was faster (˜4-fold) than that of [3H]tiotropium. The t 1/2 values for dissociation of [3H]tiotropium were much longer at both receptors. In summary, [3H]GSK573719 exhibited a high affinity for both the M2 and M3 mAChR with a marginally greater selectivity for M3 over the M2 sub-type when compared with [3H]tiotropium. The slow dissociation kinetics of [3H]GSK573719 at the M3 mAChR are consistent with a long duration of action.
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