Pharmacological analysis of the interaction of antimuscarinic drugs at M(2) and M(3) muscarinic receptors in vivo using the pithed rat assay.

Muscarinic receptor antagonists form the mainstay of the therapeutic options for airway, bladder, and gastrointestinal smooth muscle disorders. Both M(2) and M(3) muscarinic receptors are involved in mediating smooth muscle contractility, although the relative functional contribution of each subtype, especially in the disease state, is unclear. Because the potency and selectivity of compounds for a given receptor in an in vivo setting can be dissimilar to that observed in an in vitro system, we developed an in vivo assay to simultaneously determine the absolute potency and selectivity of muscarinic receptor antagonists at M(2) and M(3) receptors using the pithed rat. Methacholine (MCh)-induced bradycardia and depressor responses were used as surrogate functional endpoints for M(2) and M(3) receptor activation, respectively. The influence of the muscarinic antagonists, tolterodine, oxybutynin, darifenacin, Ro 320-6206, solifenacin, or tiotropium on the MCh-induced responses were studied. The estimated DR(10) values (dose producing a tenfold shift in the MCh curve) of tolterodine, oxybutynin, darifenacin, Ro 320-6206, solifenacin, and tiotropium for the M(2) muscarinic receptor-mediated bradycardia were 0.22, 1.18, approximately 2.6, 0.025, 0.40, and 0.0026 mg/kg, respectively, and 0.14, 0.18, 0.11, 3.0, 0.18, and 0.0017 mg/kg, respectively, for the M(3) muscarinic receptor-mediated depressor response. In a separate set of experiments, a single intravenous dose of tiotropium was administered before a MCh curve at 1, 3, 6, or 9 h to determine if tiotropium exhibited time-dependent selectivity for the M(3) receptor as has been reported from in vitro studies. The results indicate a slight preference of tiotropium for the M(3) receptor at later time points. The pithed rat assay may serve useful for elucidating the functional contribution of M(2) and M(3) receptors to the in vivo pharmacological effects of antagonists in disease animal models.