Direct measurement of muscarinic agents in the central nervous system of mice using ex vivo binding.

Muscarinic agents produce a range of side effects including hypothermia and tremor. Although these responses can be used to estimate the in vivo activity of these muscarinic agents in the central nervous system (CNS), the approach is limited by compensatory feedback mechanisms and the difficulty of equating degree of receptor occupancy to effect. We have developed an ex vivo assay to measure the potency and penetration of muscarinic agents into the CNS. The muscarinic antagonists scopolamine and N-methylscopolamine dose dependently inhibited the ex vivo binding of [3H]oxotremorine-M to homogenates of mouse whole brain membranes. Following intraperitoneal administration these compounds had ED50 values of 2.6 and 26 mg/kg respectively, which were comparable to the doses which inhibited RS86 induced hypothermia in mice. Three muscarinic agonists RS86, pilocarpine and arecoline also demonstrated CNS activity in this assay with ED50 values of 11, 23 and 220 mg/kg. RS86 and pilocarpine additionally showed good penetration into the CNS with estimated values of 1.5 and 0.31% of the administered dose. These values were comparable with the ability of these compounds to induce a centrally mediated hypothermic response. These studies demonstrate a simple, quick and reliable biochemical means of assessing a muscarinic agent's potency and penetration within the CNS.