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

Abstract

The opioid crisis is a significant public health issue with more than 115 people dying from opioid overdose per day in the United States. The aim of the present study was to characterize the in vitro and in vivo pharmacological effects of 17-cyclopropylmethyl-3,14β-dihydroxy-4,5α-epoxy-6α-(indole-7-carboxamido)morphinan (NAN), a μ opioid receptor (MOR) ligand that may be a potential candidate for opioid use disorder treatment that produces less withdrawal signs than naltrexone. The efficacy of NAN was compared to varying efficacy ligands at the MOR, and determined at the δ opioid receptor (DOR) and κ opioid receptor (KOR). NAN was identified as a low efficacy partial agonist for G-protein activation at the MOR and DOR, but had relatively high efficacy at the KOR. In contrast to high efficacy MOR agonists, NAN did not induce MOR internalization, downregulation, or desensitization, but it antagonized agonist-induced MOR internalization and stimulation of intracellular Ca²⁺ release. Opioid withdrawal studies conducted using morphine-pelleted mice demonstrated that NAN precipitated significantly less withdrawal signs than naltrexone at similar doses. Furthermore, NAN failed to produce fentanyl-like discriminative stimulus effects in rats up to doses that produced dose- and time-dependent antagonism of fentanyl. Overall, these results provide converging lines of evidence that NAN functions mainly as a MOR antagonist and support further consideration of NAN as a candidate medication for opioid use disorder treatment.

Keywords: NAN; Opioid use disorder; mixed function ligand; naltrexamine; opioid antagonist; μ opioid receptors.

Figure 1.

Food and Drug Administration approved compounds for the treatment of opioid use disorder or opioid overdose (A) together with previously reported (B) and recently reported (C) MOR selective antagonists.

Figure 2.

Concentration-effect curves of (A) DAMGO, NAQ, NAN, nalbuphine, and NTX in mMOR-CHO cells and (B) DAMGO, NAQ, and NAN in mouse thalamus. Data are presented as mean values ± SEM from at least four independent experiments. Net agonist stimulated [³⁵S]-GTPγS binding was 167.58 ± 9.01 and 221.13 ± 5.73 fmol/mg and the basal binding in the absence of the agonist was 32.54 ± 1.77 and 125.19 ± 4.13 fmol/mg in mMOR-CHO cells and thalamus, respectively.

Figure 3.

Concentration effect curves of DAMGO in the absence and presence of 5 nM (A) NTX, (B) NAN in mMOR-CHO cells (upper panels) and mouse thalamus (lower panels). Data are represented as mean values ± SEM from at least four experiments. Net agonist stimulated [³⁵S]-GTPγS binding was 169.31 ± 5.85 and 279.53 ± 4.84 fmol/mg and the basal binding in the absence of the agonist was 33.07 ± 0.82 and 140.05 ± 3.94 fmol/mg in mMOR-CHO cells and thalamus, respectively. B_max value obtained from the saturation binding assay for mMOR-CHO cells was 1.76 ± 0.50 pmol/mg.

Figure 4.

Ca²⁺ flux assays in Gα_qi5 transfected hMOR-CHO cells. (A) MOR full agonist DAMGO concentration-dependently increased intracellular Ca²⁺ level, whereas no apparent agonism was observed for NAN. (B) NAN antagonized DAMGO (500 nM)-induced intracellular Ca²⁺ increase. The EC₅₀ of DAMGO = 36.32 ± 1.85 nM and the IC₅₀ of NAN = 50.29 ± 1.62 nM. Data are presented as mean values ± SEM (n = 3).

Figure 5.

NAN (s.c.) in opioid-withdrawal assays in chronic morphine-exposed mice (n = 5): (A) Wet-dog shakes, (B) paw tremors, and (C) escape jumps. The first column in each figure represents placebo-pelleted mice, while the second to the fifth columns represent morphine-pelleted mice. *** P < 0.001, * P < 0.05, compared to 1 mg/kg naltrexone (NTX, s.c.).

Figure 6.

Effects of ligand pretreatment of mMOR-CHO treated cells on mMOR-stimulated [³⁵S]GTPγS binding. (A) E_max values of DAMGO. (B) EC₅₀ values of DAMGO. (C) Receptor efficiency (E_max/EC₅₀). Data are presented as mean values ± SEM (n = 4). Statistics: values without any of the same letter designations are p < 0.05 different from each other by ANOVA with Tukey’s post hoc test.

Figure 7.

NAN inhibited etorphine-induced MOR internalization in N2A-HA-rMOR-N2A cell line. Cells were incubated with mouse anti-HA.11 antibody for 1 h, treated with or without ligands as indicated, fixed with 4% PFA, and immunostained with Alexa Fluor 594 (Red) goat anti-mouse IgG (see Methods). Panel labels: Control showed HA-rMOR in plasma membranes. Etorphine, treatment with 1 μM etorphine for 15 min induced HA-rMOR endocytosis. NAN, treatment at 10 μM for 30 min had no effect on HA-rMOR distribution. NAN+Etorphine, treatment with NAN inhibited HA-rMOR internalization induced by etorphine. This experiment was performed twice with two different HA-rMOR-N2A clonal cells with similar results. Results from one of the clonal cells (H38) are presented.

Figure 8.

Effects of NAN (32 mg/kg, SC) and NAQ (1–32 mg/kg, SC) to produce fentanyl-appropriate responding in rats trained to discriminate 0.04 mg/kg, SC fentanyl from saline. Abscissa: drug dose in milligrams per kilogram. Ordinate: top panels: percent fentanyl-appropriate responding; bottom panels: response rate in responses/s. The symbols above saline (S) or fentanyl (F) show percent fentanyl-appropriate responding on all training days preceding test days. Each point represents the mean ± SEM of 6 (3 female and 3 male) rats.

Figure 9.

Potency (left panels) and time course (right panels) of NAN pretreatment on the discriminative stimulus effects of 0.056 mg/kg fentanyl in rats. Left abscissa: drug dose in milligrams per kilogram. Right abscissa: NAN pretreatment time (min) before fentanyl administration. Top ordinate: percent fentanyl-appropriate responding. Bottom ordinate: rates of responding in responses per sec. Each point represents the mean ± SEM of 6 (3 female and 3 male) rats. Filled points denote statistically significance compared to vehicle pretreatment (p < 0.05).

Figure 10.

Chemical structure of NAN with atom notation were derived from the complex after molecular dynamics simulations (A). Root-mean-square deviation (rmsd) of the protein backbone atoms of the three systems relative to the respective starting structures (B). Binding modes of NAN_MOR (C), NAN_KOR (D), and NAN_DOR (E) complexes with key amino acid residues in the binding site after 10 ns MD simulations. Protein shown as cartoon model in light-pink (MOR), light-blue (KOR), and light-green (DOR); NAN and key amino acid residues shown as stick model. Carbon atoms: NAN_MOR complex (magentas); NAN_KOR complex (cyan); NAN_DOR complex (green); surface models in yellow represent the “address” domain of MOR, KOR, and DOR.