www.nature.com/npp ARTICLE N-Methyl-D-aspartate receptor antagonist d-methadone produces rapid, mTORC1-dependent antidepressant effects Manoela V. Fogaça1, Kenichi Fukumoto1, Tina Franklin 1, Rong-Jian Liu1, Catharine H. Duman1, Ottavio V. Vitolo2 and Ronald S. Duman1 Currently available antidepressants have a delayed onset and limited efficacy, highlighting the need for new, rapid and more efficacious agents. Ketamine, an NMDA receptor antagonist, has emerged as a new rapid-acting antidepressant, effective even in treatment resistant patients. However, ketamine induces undesired psychotomimetic and dissociative side effects that limit its clinical use. The d-stereoisomer of methadone (dextromethadone; REL-1017) is a noncompetitive NMDA receptor antagonist with an apparently favorable safety and tolerability profile. The current study examined the rapid and sustained antidepressant actions of d-methadone in several behavioral paradigms, as well as on mTORC1 signaling and synaptic changes in the medial prefrontal cortex (mPFC). A single dose of d-methadone promoted rapid and sustained antidepressant responses in the novelty-suppressed feeding test (NSFT), a measure of anxiety, and in the female urine sniffing test (FUST), a measure of motivation and reward. D-methadone also produced a rapid reversal of the sucrose preference deficit, a measure of anhedonia, in rats exposed to chronic unpredictable stress. D-methadone increased phospho-p70S6 kinase, a downstream target of mTORC1 in the mPFC, and intra- mPFC infusion of the selective mTORC1 inhibitor rapamycin blocked the antidepressant actions of d-methadone in the FUST and NSFT. D-methadone administration also increased levels of the synaptic proteins, PSD95, GluA1, and Synapsin 1 and enhanced synaptic function in the mPFC. Studies in primary cortical cultures show that d-methadone also increases BDNF release, as well as phospho-p70S6 kinase. These findings indicate that d-methadone induces rapid antidepressant actions through mTORC1-mediated synaptic plasticity in the mPFC similar to ketamine. Neuropsychopharmacology (2019) 44:2230–2238; https://doi.org/10.1038/s41386-019-0501-x INTRODUCTION activates mammalian target of rapamycin complex 1 (mTORC1) Major depressive disorder (MDD) is a debilitating illness that signaling that is involved in protein synthesis and synaptogenesis affects ~17% of the US population and is estimated to be the [9–12]. Ketamine produces a rapid (30 min) and transient (back to second leading cause of disability worldwide by 2020 [1, 2]. baseline by 2 h) activation of mTORC1 signaling, but a sustained Approximately a third of MDD patients do not respond adequately increase in synaptic number and function (2 h to 7 days) that is to currently available monoamine reuptake inhibitor medications. associated with the rapid and sustained antidepressant actions of Moreover, these drugs require several weeks to produce an ketamine [10, 13]. antidepressant response, a significant limitation for patients Although ketamine has the potential to revolutionize the experiencing personal suffering and at risk of suicide [3]. These treatment of MDD, it also has significant side effects, including issues highlight the urgent need for new drugs that address the dissociative and psychotomimetic actions, as well as abuse limitations of currently available pharmacological agents (i.e., potential that limit its clinical use [14]. In this regard, drugs that relatively low efficacy and time lag for therapeutic response). act as NMDA channel blockers but lack ketamine-like side effects The discovery that a single dose of ketamine, an N-methyl-D- are promising therapeutic candidates to treat MDD. A novel aspartate (NMDA) receptor antagonist, produces rapid antidepres- compound that is in current development is the d-stereoisomer of sant effects (within hours) in MDD subjects, including those methadone (d-methadone or dextromethadone). Methadone is a considered treatment resistant, represents a major advance and a synthetic racemic mixture that is available for clinical use to treat novel therapeutic target [4, 5]. Previous work indicates that moderate to severe pain and opioid dependence due to its μ- ketamine rapidly enhances the function of glutamatergic synapses opioid receptor agonism [15]. Both d- and l-isomers bind non- in the brain, notably the medial prefrontal cortex (mPFC), competitively to the MK-801-labeled site of the NMDAR with low promoting post-synaptic α-amino-3-hydroxy-5-methyl-4-isoxazo- micromolar IC50 values similar to that of ketamine, memantine, lepropionic (AMPA)-mediated calcium influx and release of brain- and dextromethorphan, known NMDAR antagonists [15]. derived neurotrophic factor (BDNF) by principal pyramidal D-methadone has similar, low micromolar affinity at the different neurons [6–8]. Extracellular BDNF, in turn, binds to tropomyosin GluN2 subunits (2A-2D) of the NMDA receptor, with slightly higher receptor kinase B (TrkB) at the post-synaptic membrane and affinity for the GluN2B subunit [16]. However, studies have shown 1Department of Psychiatry, Yale University School of Medicine, 34 Park Street, New Haven, CT 06520, USA and 2Relmada Therapeutics Inc., 880 Third Ave, 12th floor, New York, NY 10022, USA Correspondence: Ronald S. Duman ([email protected]) Received: 21 June 2019 Revised: 16 August 2019 Accepted: 20 August 2019 Published online: 27 August 2019 © American College of Neuropsychopharmacology 2019 N-Methyl-D-aspartate receptor antagonist d-methadone produces rapid,. MV Fogaça et al. 2231 that d-methadone has 10–30-fold lower affinity for the μ and δ- Behavior studies opioid receptor subtypes compared with l-methadone [17, 18] and Sucrose preference test. At day 18 of CUS the animals were does not produce typical opioid-induced effects in humans at habituated to a palatable solution of 1% sucrose for 48 h, during doses predicted to exert antidepressant activity [15]. On the which the position of the bottle was counterbalanced across days. contrary, the l-isomer accounts for µ-opioid effects of methadone On the day of the test, rats were water deprived for 6 h and then [16–20]. D-methadone (REL-1017) is currently in clinical develop- presented with pre-weighed identical bottles of 1% sucrose and ment in a Phase 2a study for the treatment of individuals with water for 1 h. Sucrose and water consumption were determined depression who have not responded to traditional antidepressants by measuring the change in the volume weight of fluid consumed. (NCT03051256). Sucrose preference was calculated as the ratio of the volume of Two Phase 1 studies, a single ascending dose (SAD) study and a sucrose versus total volume of sucrose and water consumed multiple ascending dose (MAD) study, showed that d-methadone during the test. is well tolerated in humans and does not induce dissociative or psychotomimetic adverse events that are observed with ketamine Female urine sniffing test (FUST). Animals were habituated to [15]. In addition, a preliminary preclinical study showed that a the presence of a cotton-tipped applicator in the home cage for single dose (10–40 mg/kg) of d-methadone induces an 60 min as previously described [23]. Then, each animal was antidepressant-like effect in the rat forced swim test (FST), similar exposed for 5 min to a cotton-tipped applicator infused with water to ketamine [21]. The current study extends this work by and, 45 later, with fresh urine collected from female rats (9–14- investigating the effects of d-methadone in different rodent week-old). The test was video recorded and the total time spent models of depression and antidepressant response, sniffing the cotton-tipped applicator, water or urine was mTORC1 signaling, and synaptic number and function in rat measured. mPFC. Our findings highlight the participation of fast cortical mTORC1 signaling and synaptic changes in the antidepressant Novelty suppressed feeding test (NSFT). Animals were food actions of d-methadone. Moreover, the selected behavioral deprived for 18 h and placed in a red-light room containing an readouts are associated with distinct brain circuits and behavioral open field (76.5 × 76.5 × 40 cm, Plexiglas) with one pellet of food domains affected in depression (e.g., anhedonia and reward in the center. The latency to feed was measured up to 15 min. circuit; anxiety and fear circuit), thus increasing the translational Home cage food intake was measured right after the test during relevance of our findings. 20 min. 1234567890();,: Locomotor activity (LMA). The animals were placed in clean home MATERIALS AND METHODS cages with no bedding and activity determined by an infrared Animals automated tracking system and recorded as beam breaks (Med Male Sprague-Dawley rats (260–300 g) from Jackson Laboratories Associates) during 30 min. (New Haven, USA) were paired-housed at the Ribicoff Facility (Connecticut Mental Health Center, USA) in a temperature- Western blot controlled room (23 ± 2 °C) with a 12/12-h light–dark cycle (lights For brain tissue collection, rats were decapitated and the mPFC on: 7:00 a.m./lights off: 7:00 p.m.). Animals were single housed and whole hippocampus removed. Tissues were homogenized to 3–5 days prior the drug treatments. Rats received food and water protein extraction of the synaptosome fraction as previously ad libitum throughout the study period, except when food or described [7, 10]. For primary cortical culture, cells were scrubbed water deprivation was used as stressors in the CUS or when from the wells, collected into RIPA buffer and sonicated. animals were submitted to the novelty-suppressed feeding test Homogenates were submitted to western blot analysis and the (NSFT). Procedures were conducted in compliance with the bands quantified as previously described [7, 10] (primary National Institute of Health (NIH) guidelines for the care and use antibodies: rabbit anti-phospho-p70S6K, rabbit anti-p70S6K, rabbit of laboratory animals and were approved by the Yale Animal Care anti-phospho-p44-42 ERK, rabbit anti-p44-42 ERK, rabbit anti- and Ethics Committee. phospho-4EBP1, rabbit anti-phospho-mTOR, rabbit anti-mTOR, rabbit anti-GluA1, rabbit anti-Synapsin1, rabbit anti-PSD95, 1:1000, Drug administration Cell Signaling.