Receptor reserve reflects differential intrinsic efficacy associated with opioid diastereomers

Structure-activity relationships built around receptor binding or cell-based assays are designed to reveal physiochemical differences between ligands. We hypothesized that agonist receptor reserve may provide a unique approach to distinguish structurally-related agonists exhibiting similar functional characteristics. An intracellular calcium activation assay in Chinese Hamster Ovary (CHO) cells expressing cloned human μ-opioid receptors was developed. We examined two isomers exhibiting indistinguishable receptor binding and in vitro potency profiles. Oxymorphone, a clinically-available congener of codeine has at least two active diastereomeric metabolites (6α- and 6β-oxymorphols) found to be similar for μ-opioid receptor binding affinity (Kd = 15 versus 14 nM) and calcium activation (EC50 = 22 versus 14 nM). Calcium activation was then inhibited in CHO cells in a concentration-dependent manner using the irreversible μ-opioid receptor antagonist, β-funaltrexamine (β-FNA). Under these conditions, ~ 10-fold greater receptor reserve was found for 6α-oxymorphol compared to 6β-oxymorphol. This difference between the oxymorphols corresponded to a rank order of intrinsic efficacy (Emax): DAMGO > oxymorphone = 6α-oxymorphol = oxycodone > 6β-oxymorphol. In addition, 6α-oxymorphol exhibited greater relative potency than the 6β-oxymorphol in mouse tail-flick, hot-plate and phenylquinone writhing antinociceptive assays, regardless of route of administration. Thus the β-FNA/calcium model provides a novel, cell-based approach to distinguish structurally related μ-opioid agonists, and in the specific case of the oxymorphols, receptor reserve differences provided a means to bridge functional in vitro and in vivo models.