Potency, dissociation kinetics and reversibility of fentanyls and nitazenes by naloxone at the μ opioid receptor

Background and PurposeFentanyls and nitazenes are opioid receptor agonists responsible for a large number of opioid overdose deaths. Here, we compared the potency, dissociation kinetics and antagonism by naloxone at the receptor of several fentanyl and nitazene analogues and compared them to morphine and DAMGO. Experimental ApproachIn vitro assays of G protein activation and signalling and arrestin recruitment were performed. AtT20 cells expressing receptors were loaded with a membrane potential dye and changes in fluorescence used to determine agonist potency, dissociation kinetics and susceptibility to antagonism by naloxone. BRET experiments were undertaken in HEK293T cells expressing opioid receptors, to assess Gi protein activation and {beta}-arrestin 2 recruitment. Key ResultsThe rate of agonist dissociation from the receptor varied, with morphine, DAMGO, alfentanil and fentanyl dissociating rapidly whereas isotonitazene, etonitazene, ohmefentanyl and carfentanil dissociated slowly. Slowly dissociating agonists were more resistant to antagonism by naloxone. For carfentanil, the slow rate of dissociation was not due to G protein receptor kinase-mediated arrestin recruitment as its rate of dissociation was not affected by inhibition of GRKs with Compound 101. The in vitro relative potencies of fentanyls and nitazenes compared to morphine were much lower than that previously observed in in vivo experiments. Conclusions and ImplicationsWith fentanyls and nitazenes, that slowly dissociate from the opioid receptor, antagonism by naloxone is pseudo competitive. In overdoses involving fentanyls and nitazenes higher doses of naloxone may be required for reversal than those normally used to reverse heroin overdose. What is already knownO_LI"Fentanyls" and "nitazenes" are potent agonists at the opioid receptor. C_LI What does this study addO_LISome fentanyls and nitazenes dissociate slowly and are less sensitive to naloxone antagonism C_LI What is the clinical significanceO_LIMore naloxone may be required to reverse overdoses involving fentanyls and nitazenes C_LI