New hybrid muscarinic agonists: in vitro functional characterization and in vivo analgesic activity evaluation

Background and aim. Several studies demonstrated that activation of muscarinic acetylcholine receptors (mAChRs), in particular the M2 and M4 subtypes, exerts antinociceptive actions (1). So, selective muscarinic agonists could be considered as a good alternative to the classic opioid drugs, whose heavy side effects, such as tolerance and physical dependence, limit their therapeutic application. The aim of this work was to characterize in vitro the muscarinic profile and in vivo the analgesic activity of two different series of bis(ammonio)alkane-type hybrids, incorporating the orthosteric potent and unselective agonist iperoxo into the structure of the M2-selective allosteric modulators W84 (hybrids 6a-9a) and naphmethonium (hybrids 6b-9b)(2,3). Methods. The agonist potency and efficacy of the hybrids were evaluated on isolated electrically stimulated rabbit vas deferens (M1 model), guinea pig left atrium (M2 model) and terminal ileum (M3 model). The analgesic potency was assessed in the writhing test: vehicle or test compounds were administered subcutaneously in mice 30 min before intraperitoneal injection of 0.6% acetic acid. The number of writhes of each mouse was counted from 5 to 10 minutes after acetic acid application. Compound 8b was assayed (0.5mg/kg) also in the presence of opioid antagonist naloxone (1mg/kg s.c.), muscarinic blockers atropine (5mg/kg s.c.) or methylatropine (5mg/kg s.c.) or nicotinic antagonist mecamylamine (1mg/kg s.c.). The incidence of cholinergic peripheral and central unwanted side effects (salivation, lacrimation diarrhea, hypothermia, tremor) was simultaneously evaluated by an observer unaware of the treatment. Potential cardiovascular effects of 8b were finally investigated in anaesthetized rats following intraperitoneal administration of the vehicle or of the hybrid (0.01-0.5 mg/kg). The experiments were carried out in the respect of Italian Law on the use of animals for experimental ends (D.Lgs 26/2014). Results. All the compounds behaved as potent muscarinic agonists, W84 derivatives (a series) being generally less potent than their naphmethonium-related counterparts (b series), with a slight preference towards M1 and M2 sites compared to M3 receptors. This trend was especially evident for compound 8b, which displayed the lowest intrinsic activity in guinea pig ileum. In the writhing test all the compounds, except hybrids 7b and 9b, showed dose-dependent analgesic effects, with hybrids of the b series 15-30 times more potent than their a congeners. Hybrid 8b displayed the most potent analgesic activity (ID50: 0.024 mg/kg) that was antagonised only by muscarinic blockers. Compound 8b was devoid of relevant cholinergic side effects and, as regards cardiovascular parameters, did not evoke hypotension or bradycardia at the threshold analgesic dose (0.01 mg/kg). Conclusion. The strategy to generate selective muscarinic agonists through the combination of potent orthosteric agonist with allosteric modulators yields hybrids with a new advantageous pharmacological profile. Among the newly synthesized hybrids, compound 8b emerged as the most promising derivative, combining the most potent antinociception, seemingly mediated by peripheral muscarinic receptors activation, with the absence of relevant cholinergic side effects. References: Cai et al., (2009), J Neurochem. 111(4): 1000-10. Barocelli et al., (2001), Life Sciences 68: 1775-85. Matera et al., (2014), Eur J Med Chem. 75:222-32