Insurmountable antagonism of human mu opioid receptors by buprenorphine is due to hemi-equilibrium

Buprenorphine is an FDA approved drug for the treatment of opioid use disorder and is a long-lasting, low efficacy (partial) agonist of the μ opioid receptor. As a partial agonist, buprenorphine can act as either an agonist or an antagonist depending on the efficiency of the cellular signaling system. Here we investigated the antagonist properties of buprenorphine using a genetically-encoded biosensor to monitor cAMP levels in real time in HEK293 cells expressing a relatively low density of the human μ receptor. Pre-treatment of cells with buprenorphine decreased the maximal response (Emax) of the μ receptor agonists DAMGO and fentanyl, consistent with expectations of a long-lasting partial agonist in these cells. However, buprenorphine pretreatment also reduced the potency of these agonists. In a Schild analysis, the reduction in Emax saturated with increasing concentrations of buprenorphine, while the reduction in potency did not. This unexpected behavior of buprenorphine on potency was mediated through the orthosteric site, as pretreatment with the opioid receptor antagonist naloxone prevented buprenorphine antagonism. Computation simulations using a model of hemi-equilibrium indicated that buprenorphine's slow receptor dissociation could account for both effects of antagonism. In support of this kinetic explanation, buprenorphine antagonism became surmountable with additional agonist incubation time. In this system, buprenorphine appeared to behave both as a competitive and non-competitive (insurmountable) antagonist due to its slow dissociation resulting in partial re-equilibration (hemi-equilibrium). As fast-acting biosensors become more prevalent, it will be important to consider kinetically-mediated effects such as hemi-equilibrium when characterizing the pharmacological properties of antagonists.