Unique pharmacodynamic properties and low abuse liability of the μ-opioid receptor ligand (S)-methadone

Abstract

(R,S)-methadone ((R,S)-MTD) is a racemic μ-opioid receptor (MOR) agonist comprised of (R)-MTD and (S)-MTD enantiomers used for the treatment of opioid use disorder (OUD) and pain. (R)-MTD is used as an OUD treatment, has high MOR potency, and is believed to mediate (R,S)-MTD's therapeutic efficacy. (S)-MTD is in clinical development as an antidepressant and is considered an N-methyl-D-aspartate receptor (NMDAR) antagonist. In opposition to this purported mechanism of action, we found that (S)-MTD does not occupy NMDARs in vivo in rats. Instead, (S)-MTD produced MOR occupancy and induced analgesia with similar efficacy as (R)-MTD. Unlike (R)-MTD, (S)-MTD was not self-administered and failed to increase locomotion or extracellular dopamine levels indicating low abuse liability. Moreover, (S)-MTD antagonized the effects of (R)-MTD in vivo and exhibited unique pharmacodynamic properties, distinct from those of (R)-MTD. Specifically, (S)-MTD acted as a MOR partial agonist with a specific loss of efficacy at the MOR-galanin 1 receptor (Gal1R) heteromer, a key mediator of the dopaminergic effects of opioids. In sum, we report novel and unique pharmacodynamic properties of (S)-MTD that are relevant to its potential mechanism of action and therapeutic use, as well as those of (R,S)-MTD.

Keywords: NMDAR; computational model; opioid.

Figure 1. Methadone and its enantiomers are MOR agonists.

a, Receptor and enzyme competitive screen at two concentrations (100 nM and 10 μM) of (S)- and (R)-MTD. b, Competition binding assays of (S)-MTD (orange), (R)-MTD (blue), or (R,S)-MTD (black) versus [³H]DAMGO. c-e, Representative slices (c) and analysis from methadone-stimulated [³⁵S]GTPγS autoradiography for CPu (d, upper circle) and NAc (e, lower ellipse). Values are shown as mean ± standard error of the mean. CPu = caudate putamen; MOR = mu opioid receptor; MTD = methadone; NLX = naloxone; NAc = nucleus accumbens. *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 2. Analgesic, cataleptic and differential abuse liability profile of (R,S)-MTD, (R)-MTD, and (S)-MTD.

a-b, Dose response curves of hotplate latency (a) and catalepsy (b) for (R,S)-MTD (black), (R)-MTD (blue) and (S)-MTD (orange), c-e, Lever presses during IVSA training for (R,S)-MTD (100 μg/kg/infusion, c), (R)-MTD (50 μg/kg/infusion, d), and (S)-MTD (500 μg/kg/infusion, e). f, IVSA dose responses for (R,S)-MTD, (R)-MTD, and (S)-MTD. g-k, Representative slices (g) and analysis of receptor occupancy by (R,S)-MTD, (R)-MTD, or (S)-MTD of MORs ([³H]DAMGO, 5nM) in CPU (h, upper circle) and NAc (i, lower ellipse) or NMDARs ([³H]MK-801, 5nM) (j-k). Values are shown as mean ± standard error of the mean. CPu = caudate putamen; ED₅₀ = half maximal effective dose; FR = fixed-ratio schedule; IVSA = intravenous self-administration; MOR = μ opioid receptor; MPE = maximum possible effect; MTD = methadone; NAc = nucleus accumbens; NMDAR = N-methyl-D-aspartate receptor.

Figure 3. VTA-dependent neurochemical and behavioral effects of (R)-MTD and (S)-MTD.

Created with BioRender.com. a-f, acute locomotor activation schematics (a, e) and analysis with (R,S)-MTD (b), (R)-MTD (c), or (S)-MTD (d) alone, or pretreatment of (S)-MTD before (R)-MTD (f). Data shown as the average of the square root of centimeters traveled per ten minutes. Asterisks are compared to saline; pound symbols are compared to (R)-MTD alone. g-l, psychomotor sensitization schematics (g, k) and analysis with (R,S)-MTD (h), (R)-MTD (i), or (S)-MTD alone (j), or pretreatment of (S)-MTD before (R)-MTD (I). Asterisks are comparison between D1 and D2; pound symbols are comparison between D1 and D3. m-o, effect of intracranial perfusion of (R)-MTD and (S)-MTD in the VTA on somato-dendritic dopamine release from in vivo microdialysis experiments. Values represent mean dopamine concentrations as a percentage of baseline ± standard error of the mean (average of 5 samples before the enantiomer administration). The rectangles in the x axis indicate the period of corresponding enantiomer perfusion. In o, co-perfusion of both enantiomers, with (S)-MTD (100 μM) beginning 20 min before (R)-MTD (10 μM). p, Analysis of [³⁵S]GTPγS recruitment by R-MTD (1 μM) with or without preincubation of S-MTD (1 μM or 10 μM) in the VTA. Values are shown as mean ± standard error of the mean. D1, 2, 3 = day 1, 2, or 3; Hab = habituation, MTD = methadone, VTA = ventral tegmental area. *,^# P < 0.05, **P < 0.01, ***,^### p < 0.001.

Figure 4. MOR-Gal₁R heteromer-dependent loss of efficacy of (S)-MTD.

a-f, BRET experiments in HEK-293T cells cotransfected with MOR fused to RLuc and the α subunit of the Gi protein fused to YFP (schematically shown in a), g-l. CODA-RET experiments in HEK-293T cells cotransfected with MOR fused to nRLuc, Gal₁R fused to cRLuc and Gi-YFP (schematically shown in g). In b and h, representative experiments with concentration-responses of (R,S)-MTD (black), (R)-MTD (blue), and (S)-MTD (orange); values represent the mean ± standard error of the mean of triplicates; in c-d and i-j, corresponding E_max and EC₅₀ values from 6 independent experiments with triplicates, shown as dots and presented with the mean ± standard error of the mean or median with interquartile ranges, respectively; asterisks are compared to (R)-MTD values. In e and k, representative experiments of the effect of increasing concentrations of (S)-MTD on BRET and CODA-RET values obtained with (R)-MTD at 100 nM; values represent the mean ± SEM of triplicates; in f and I, corresponding BRET and CODA-RET values of the effect of (R)-MTD (100 nM) in the presence and absence of (S)-MTD (1 μM) from 7 and 9 independent experiments with triplicates, shown as dots and presented with the mean ± standard error of the mean; asterisks are compared to basal values, m-n, Schematic 2D representation of (R)- and (S)-MTD. Grey arrows represent groups of the ligand located toward the conserved protonated amine (left) and toward the -CO-CH2-CH3 moiety (right). The phenyl groups of methadone are depicted by either blue (R-) or orange (S-) arrows. Docking and MD-simulated models (Supplementary Fig. 6) of (R)- and (S)-MTD bound to the MOR. The phenyl rings, in a “V” shaped conformation, point up to interact with H299^6.52 and W320^7.35 in (R)-MTD, and point down to interact with W295^6.48 in (S)-MTD. o-p, Previous results show that MOR forms homodimers via the TM 5/6 interface in the absence of Gal₁R or via the TM 4/5 interface in the presence of Gal₁R. MD simulations show that the TM 4/5 triggers an inward movement of TM 5 and, importantly, the inward movement of V238^5.42 (Supplementary Fig. 8). Thus, in the TM 5/6 interface the phenyl ring of (S)-MTD (flexible arrows) partially restricts the conformation of W295^6.48 (flexible ellipses), whereas in the TM4/5 interface V238^5.42 restricts the conformation of the phenyl ring (single arrow) and in consequence W295^6.48 (single ellipse) in the inactive conformation. BRET = bioluminescence resonance energy transfer; CODA-RET = Complemented donor-acceptor resonance energy transfer; E_max = maximal response; EC₅₀ = half maximal effective concentration; Gal₁R = galanin 1 receptor; MD = molecular dynamics; MOR = mu opioid receptor; MTD = methadone. *P < 0.05, **P < 0.01, ***P < 0.001