Characterization of Buprenorphine Antagonism at Human Mu Opioid Receptors

Abstract

In 2017 the US government declared a public health emergency to address the national opioid crisis. Despite this focused effort and increased awareness, opioid overdose deaths have since increased by over 50%. Individuals with opioid use disorder have increased risk of opioid related death, and medication assistance can facilitate recovery by reducing opioid cravings and withdrawal effects. One such medication is buprenorphine, an FDA approved drug for the treatment of opioid use disorder sold under the brand Suboxone®, Subutex®, and Zubsolv®. While buprenorphine is clinically described as a partial agonist of the mu opioid receptor (MOR), the literature demonstrates that its pharmacological characteristics are complex. In ongoing studies we are rigorously characterizing the pharmacological actions of buprenorphine at the human MOR.A genetically encoded biosensor was used to monitor cAMP levels in real time in HEK293 cells expressing a low density of the human MOR. In these cells, buprenorphine acted as an antagonist, shifting the concentration response curve of the MOR agonist, DAMGO, to the right (reduced potency) in a concentration‐dependent manner, similar to the action of the MOR antagonist naloxone. Unlike the effect of naloxone, buprenorphine also decreased the maximal response of DAMGO, an effect that saturated with increasing concentrations of buprenorphine. Similar antagonism by buprenorphine was observed when fentanyl was used as the MOR agonist. The effect of buprenorphine to decrease the potency of DAMGO was reduced by washout of buprenorphine. In contrast, the reduction in the maximal response to DAMGO was resistant to washout of buprenorphine. Both effects of buprenorphine (on DAMGO potency and efficacy) were prevented by pre‐treatment with naloxone, followed by rigorous washout. This suggested that buprenorphine’s effects were mediated by a naloxone‐sensitive binding site, presumably the orthosteric site. Given the slow rate of dissociation of buprenorphine binding to MOR in several published studies and the rapid time‐course involved in the cAMP response, we considered the possibility that the observed effects of buprenorphine were due to hemi‐equilibrium conditions. Computations conducted using hemi‐equilibrium equations support the hypothesis that the antagonist effects of buprenorphine in this system are due to hemi‐equilibrium. These kinetics were further supported in high‐expressing hMOR CHO cells, as buprenorphine agonism was resistant to reversal by naloxone or washing. As a frontline treatment for opioid use disorder, an understanding of buprenorphine’s pseudo‐irreversible binding may elucidate buprenorphine’s unique properties and facilitate future drug discovery.