Ligand‐Dependent Reversal Kinetics of Mu Opioid Receptor Agonists by the Novel Antagonist, Methocinnamox

Abstract

Naloxone (NLX) is a competitive antagonist at the mu opioid receptor and is currently the only pharmacological intervention available to treat an opioid overdose. Unfortunately, NLX is limited by its surmountability and its rapid metabolism/clearance can lead to a reemergence of narcotic effects (renarcotization). Methocinnamox (MCAM) is a long‐acting antagonist at mu receptors both in vivo and in vitro. In addition to its orthosteric antagonism, we have found that MCAM differentially modulates orthosteric properties (affinity and/or efficacy) of mu agonists via an allosteric mechanism (Zamora, Smith, et al. (2021). Pharmacology Research & Perspectives. https://doi.org/10.1002/prp2.887). To further explore the interactions between MCAM and the human mu opioid receptor, we compared the rate by which MCAM and NLX reverse cAMP inhibition by the mu receptor agonists DAMGO and fentanyl. Treatment with either DAMGO or fentanyl (~ 30 x EC50) inhibited forskolin (FSK)‐stimulated intracellular cAMP levels. When administered 20 min after agonist, NLX (400 nM; ~100 x Ki) reversed DAMGO‐ and fentanyl‐mediated inhibition of cAMP production at a similar rate (25.4% ± 2.5 min‐1 vs 26.8% ± 5.5 min‐1, respectively). Since reversal by NLX was driven mostly by dissociation of the agonists, the similar rate of reversal by NLX indicate that DAMGO and fentanyl dissociate from the receptor at similar rates. Reversal by MCAM (100 nM; ~ 100 x Ki) however was ligand‐dependent. The rate of reversal of DAMGO‐mediated inhibition by MCAM was 11.7% ± 1.3 min‐1, whereas reversal of fentanyl was slower (4.1% ± 0.9 min‐1; P<0.001). The ligand‐dependence of MCAM’s reversal kinetics on DAMGO vs fentanyl’s inhibition of cAMP production is consistent with the reported allosteric action of MCAM, and may be explained by modulation of ligand dissociation by MCAM. Together, these data suggest in addition to its orthosteric antagonism, MCAM may allosterically modulate the binding of mu agonists.