The unbinding kinetics and reversibility of fentanyls and nitazenes by naloxone at the μ opioid receptor

Abstract

The opioid overdose deaths epidemic in North America has reached an alarming height, with fentanyl derivatives (fentanyls) having infiltrated the illicit opioid market to become the largest contributor to these deaths. Notably, during the epidemic, cases of fentanyl overdose displayed a perplexing characteristic, requiring higher or multiple doses of naloxone, the standard FDA-approved competitive opioid antagonist, than heroin overdose deaths. This higher requirement had not been reported for heroin overdoses before fentanyls infiltration. This observation conflicts with the established principle of competitive antagonism which is that antagonist potency should be independent of the agonist used. Overdose cases involving nitazene derivatives (nitazenes) have recently emerged, but limited research exists on their sensitivity to naloxone. To address naloxone sensitivity in vitro, we aimed to investigate the effectiveness of naloxone against a series of fentanyls and nitazenes, which exhibit a wide variability in potency and lipophilicity at the μ opioid receptor, their site of action. To deepen our understanding of ligand-μ opioid receptor molecular interactions, the dissociation half-time (receptor unbinding) of the agonists was studied. Additionally, correlation analyses were performed between lipophilicity, potency, dissociation half-time and sensitivity to naloxone for each agonist. Finally, to further understand the pharmacological characteristics of the μ opioid receptor, we investigated the signalling bias profile of selected fentanyls and nitazenes between the G protein and β-arrestin 2. The findings challenge the reported competitive antagonism mechanism of naloxone, as some fentanyls and nitazenes displayed lower sensitivity to naloxone than the reference compounds DAMGO and morphine. Notably, the half-maximal inhibitory concentration (IC50) of naloxone against carfentanil was 20-fold higher than that against morphine, indicating a significant variation in the potency of naloxone. A strong inverse correlation was revealed between the dissociation half-time of the agonist and its sensitivity to naloxone, while the lipophilicity and potency of the agonist were poorly correlated with its sensitivity to naloxone. Carfentanil and etonitazene displayed a tendency towards β-arrestin 2 signalling and a long dissociation half-time. In conclusion, this research provides insights into the pharmacology of fentanyls and nitazenes and offers critical findings on the competitive nature of naloxone antagonism. The results obtained might have implications for naloxone dosing and/or overdose intervention strategies.