In vitro and in vivo functional profile characterization of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3-carboxamido)morphinan (NAQ) as a low efficacy mu opioid receptor modulator

Abstract

Evidence has shown that downstream signaling by mu opioid receptor (MOR) agonists that recruit β-arrestin2 may lead to the development of tolerance. Also, it has been suggested that opioid receptor desensitization and cyclic AMP overshoot contributes to the development of tolerance and occurrence of withdrawal, respectively. Therefore, studies were conducted with 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(isoquinoline-3-carboxamido)morphinan (NAQ), a MOR selective partial agonist discovered in our laboratory, to characterize its effect on β-arrestin2 recruitment and precipitation of a cyclic AMP overshoot. DAMGO, a MOR full agonist dose-dependently increased β-arrestin2 association with the MOR, whereas NAQ did not. Moreover, NAQ displayed significant, concentration-dependent antagonism of DAMGO-induced β-arrestin2 recruitment. After prolonged morphine treatment of mMOR-CHO cells, there was a significant overshoot of cAMP upon exposure to naloxone, but not NAQ. Moreover, prolonged incubation of mMOR-CHO cells with NAQ did not result in desensitization nor downregulation of the MOR. In functional studies comparing NAQ with nalbuphine in the cAMP inhibition, Ca flux and [S]GTPγS binding assays, NAQ did not show agonism in the Ca flux assay but showed partial agonism in the cAMP and [S]GTPγS assays. Also, NAQ significantly antagonized DAMGO-induced intracellular Ca increase. In conclusion, NAQ is a low efficacy MOR modulator that lacks β-arrestin2 recruitment function and does not induce cellular hallmarks of MOR adaptation and fails to precipitate a cellular manifestation of withdrawal in cells pretreated with morphine. These characteristics are desirable if NAQ is pursued for opioid abuse treatment development.

Characterization of 17-Cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(indole-7-carboxamido)morphinan (NAN) as a Novel Opioid Receptor Modulator for Opioid Use Disorder Treatment

“…To further examine the effects of ligand pretreatment on parameters of DAMGO-stimulated G-protein activation, we also calculated the E max /EC 50 ratio as a measure of receptor activation efficiency as we previously published. 38 This ratio showed a significant decrease in cells treated with DAMGO or morphine, but not in cells treated with any of the low efficacy ligands, compared to vehicle-treated cells (Figure 6C). However, treatment with nalbuphine, NAQ or NAN all slightly decreased this ratio relative to treatment with NTX.…”

“…This result was compared to our previously reported values obtained after treatment of these cells with the same opioid ligands examined above. 38 Both morphine and DAMGO produced an approximate 50% reduction in receptor density ( B max ) whereas the low efficacy opioid ligands did not (Table 3). Interestingly, treatment with NTX, NAQ or NAN produced a significant reduction in the apparent [ 3 H]naloxone binding affinity (Table 3).…”

“…Cellular adaptation to opioids may result in part from downregulation or desensitization of the MOR due to continuous opioid agonist exposure. 38,53,60 mMOR-CHO cells were incubated for 24 h with NAN along with MOR ligands of varying efficacy. Cellular adaptation was quantified as an increase in the EC 50 and/or reduction in the E max value of DAMGO to stimulate [ 35 S]-GTP γ S binding in membranes from ligand-treated cells.…”

“… a Data are mean ± SEM of B max and K D values derived from nonlinear regression analysis of [ 3 H]naloxone saturation binding curves ( n = 4). *, **, ****: p < 0.05, 0.01, 0.0001 different from corresponding value in vehicle-pretreated cells. b Values previously published. 38 …”

“…Further pharmacological studies showed that NAQ antagonizes or minimally activates MOR signaling without producing prototypical MOR agonist adaptations that contribute to tolerance, opioid dependence, or precipitation of cellular withdrawal in both in vitro and in vivo models. 38–41…”