Antinociceptive effects of potent, selective and brain penetrant muscarinic M4 positive allosteric modulators in rodent pain models

Analgesic properties of orthosteric agonists of the muscarinic M-4 receptor subtype have been documented in literature reports, with evidence from pharmacological and in vivo receptor knock out (KO) studies. Constitutive M-4 receptor KO mice demonstrated an increased response in the formalin pain model, supporting this hypothesis. Two novel positive allosteric modulators (PAM) of the M-4 receptor, Compounds 1 and 2, were characterized in rodent models of acute nociception. Results indicated decreased time spent on nociceptive behaviors in the mouse formalin model, and efficacy in the mouse tail flick assay. The analgesic-like effects of Compounds 1 and 2 were shown to be on target, as the compounds lacked any activity in constitutive M-4 KO mice, while retaining activity in wild type control littermates. The analgesic-like effects of Compounds 1 and 2 were significantly diminished in KO mice that have selective deletion of the M-4 receptor in neurons that co-express the dopaminergic D-1 receptor subtype, suggesting a centrally-mediated effect on nociception. The opioid antagonist naloxone did not diminish the effect of Compound 1, indicating the effects of Compound 1 are not secondarily linked to opioid pathways. Compound 1 was evaluated in the rat, where it demonstrated analgesic-like effects in tail flick and a subpopulation of spinal nociceptive sensitive neurons, suggesting some involvement of spinal mechanisms of nociceptive modulation. These studies indicate that M-4 PAMs may be a tractable target for pain management assuming an appropriate safety profile, and it appears likely that both spinal and supraspinal pathways may mediate the antinociceptive-like effects.