Potency, dissociation kinetics and reversibility of fentanyls and
nitazenes by naloxone at the μ opioid receptor
Abstract
Background and Purpose: Fentanyls and nitazenes are μ 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 μ receptor of several fentanyl and nitazene analogues
and compared them to morphine and DAMGO. Experimental Approach: In vitro
assays of G protein activation and signalling and arrestin recruitment
were performed. AtT20 cells expressing μ 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 μ opioid receptors, to assess Gi protein activation and
β-arrestin 2 recruitment. Key Results: The rate of agonist dissociation
from the μ 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 Implications: With 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.