Reply to “Letter to the Editor regarding ‘Unique pharmacodynamic properties and low abuse liability of the μ-opioid receptor ligand (S)-methadone’”

We want to thank Pappagallo et al. [1] for their commentary about our recent article [2] which delved into the pharmacodynamic divergence of (R)- and (S)-methadone. Based on our experimental findings, we concluded that (S)-methadone, like (R,S)-methadone and (R)-methadone, is an agonist at the μ opioid receptor (MOR) but with much lower potency. Importantly, (S)-methadone exhibits a unique pharmacodynamic effect by acting as a MOR antagonist when MOR is complexed with the galanin 1 receptor (Gal₁R). Since our previous studies showed that MOR-Gal₁R heteromers mediate the dopaminergic effects of opioids [3], this specific antagonist property of (S)-methadone explains the low efficacy of (R,S)-methadone at activating the dopaminergic system and at eliciting euphoric effects. Accordingly, at sufficient doses/concentrations, (S)-methadone counteracts the locomotor activating and dopamine releasing effects of (R)-methadone. While (S)-methadone may act as an antagonist at the N-methyl-D-aspartate receptor (NMDAR), the drug concentration needed to achieve this effect is higher than what is needed to interact with MORs in vivo. As such, we stated “The currently assumed role of NMDAR blockade in the purported antidepressant effects of (S)-MTD should be reframed in the context of its MOR agonistic properties.” Pappagallo et al. [1] disagreed with this statement. We address their comments below.

Our hot plate findings are a key component of the MOR agonist effects of (S)-methadone, but they are not the only evidence we provided. (S)-methadone also produces hypothermia and partial catalepsy [2], two prototypical effects of MOR agonists in rats. We also used [³⁵S]GTPγS binding in rat brain sections to show that (S)-methadone increases GPCR activity in the striatum at a concentration that does not interact with NMDARs, and that this response was blocked by pretreatment with the preferential MOR antagonist naltrexone. Since the [³⁵S]GTPγS assay is specific to GPCRs and does not involve ion channel activation or inhibition, we conclude that this activation is mediated by opioid receptors, most likely MORs. We also used BRET in transfected cells to show that (S)-methadone acts as an agonist at MOR. Finally, we found that 30 mg/kg (S)-methadone, a dose used to produce antidepressant-like effects in rats (10–40 mg/kg) [4, 5], occupies nearly 80% of striatal MORs without significant occupancy of NMDARs, using an assay which has previously shown occupancy by ketamine at these receptors [6]. Thus, we used diverse and independent approaches to confirm that (S)-methadone binds to and activates MORs at doses/concentrations that do not bind NMDARs in vivo.