Major depressive disorder afflicts ~16 percent of the world population at some point in their lives. Despite a number of available monoaminergic-based antidepressants, most patients require many weeks, if not months, to respond to these treatments, and many patients never attain sustained remission of their symptoms. The non-competitive glutamatergic N-methyl-D-aspartate receptor (NMDAR) antagonist, (R,S)-ketamine (ketamine), exerts rapid and sustained antidepressant effects following a single dose in depressed patients. Here we show that the metabolism of ketamine to (2S,6S;2R,6R)-hydroxynorketamine (HNK) is essential for its antidepressant effects, and that the (2R,6R)-HNK enantiomer exerts behavioural, electroencephalographic, electrophysiological and cellular antidepressant actions in vivo. Notably, we demonstrate that these antidepressant actions are NMDAR inhibition-independent but they involve early and sustained α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor activation. We also establish that (2R,6R)-HNK lacks ketamine-related side-effects. Our results indicate a novel mechanism underlying ketamine’s unique antidepressant properties, which involves the required activity of a distinct metabolite and is independent of NMDAR inhibition. These findings have relevance for the development of next generation, rapid-acting antidepressants.

Author Contributions: PZ, RM, PJM, IWW, CJT, CAZ, and TDG were responsible for the overall experimental design. PJM, YF, and CJT synthesised the ketamine metabolites and deuterated ketamine derivatives, and provided mass spectrometer confirmations. Bioanalytical quantitation of ketamine and metabolites were performed by RM, NSS, and KSSD. PZ, PG, and HJP conducted and analysed the results of the behavioural and EEG experiments. XPH supervised and analysed the results of the binding experiments. PY performed the western blot experiments. EXA, MA, JF, and SMT helped design and analyze the electrophysiology experiments, which were conducted by MA, JF, and SMT. GIE and CLM conducted and analysed the results of the i.v. self-administration. PZ and TDG outlined and wrote the paper, which was reviewed by all authors.The authors declare competing financial interests: IWW, RM, and CAZ are listed as co-inventors on a patent for the use of (2R,6R)-hydroxynorketamine, (S)-dehydronorketamine and other stereoisomeric dehydro-and hydroxylated metabolites of (R,S)-ketamine metabolites in the treatment of depression and neuropathic pain. IWW, CAZ, RM, TG, PZ, CT, and PM are listed as co-inventors on a patent application for the use of (2R,6R)-hydroxynorketamine and (2S,6S)-hydroxynorketamine in the treatment of depression, anxiety, anhedonia, suicidal ideation and post-traumatic stress disorders. IWW, CAZ, RM, CT, and PM have assigned their patent rights to the U.S. government but will share a percentage of any royalties that may be received by the government. TG and PZ have assigned their patent rights to the University of Maryland Baltimore but will share a percentage of any royalties that may be received by the University of Maryland Baltimore. All other authors declare no competing interests. HHS Public Access AbstractMajor depressive disorder afflicts ~16 percent of the world population at some point in their lives. Despite a number of available monoaminergic-based antidepressants, most patients require many weeks, if not months, to respond to these treatments, and many patients never attain sustained remission of their symptoms. The non-competitive glutamatergic N-methyl-D-aspartate receptor (NMDAR) antagonist, (R,S)-ketamine (ketamine), exerts rapid and sustained antidepressant effects following a single dose in depressed patients. Here we show that the metabolism of ketamine to (2S,6S;2R,6R)-hydroxynorketamine (HNK) is essential for its antidepressant effects, and that the (2R,6R)-HNK enantiomer exerts behavioural, electroencephalographic, electrophysiological and cellular antidepressant actions in vivo. Notably, we demonstrate that these antidepressant actions are NMDAR inhibition-independent but they involve early and sustained α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptor activation. We also establish that (2R,6R)-HNK lacks ketamine-related side-effects. Our results indicate a novel mechanism underlying ketamine's unique antidepressant properties, which involves the required activity of a dist...

Ketamine, a racemic mixture consisting of ()- and ()-ketamine, has been in clinical use since 1970. Although best characterized for its dissociative anesthetic properties, ketamine also exerts analgesic, anti-inflammatory, and antidepressant actions. We provide a comprehensive review of these therapeutic uses, emphasizing drug dose, route of administration, and the time course of these effects. Dissociative, psychotomimetic, cognitive, and peripheral side effects associated with short-term or prolonged exposure, as well as recreational ketamine use, are also discussed. We further describe ketamine's pharmacokinetics, including its rapid and extensive metabolism to norketamine, dehydronorketamine, hydroxyketamine, and hydroxynorketamine (HNK) metabolites. Whereas the anesthetic and analgesic properties of ketamine are generally attributed to direct ketamine-induced inhibition of -methyl-D-aspartate receptors, other putative lower-affinity pharmacological targets of ketamine include, but are not limited to, γ-amynobutyric acid (GABA), dopamine, serotonin, sigma, opioid, and cholinergic receptors, as well as voltage-gated sodium and hyperpolarization-activated cyclic nucleotide-gated channels. We examine the evidence supporting the relevance of these targets of ketamine and its metabolites to the clinical effects of the drug. Ketamine metabolites may have broader clinical relevance than was previously considered, given that HNK metabolites have antidepressant efficacy in preclinical studies. Overall, pharmacological target deconvolution of ketamine and its metabolites will provide insight critical to the development of new pharmacotherapies that possess the desirable clinical effects of ketamine, but limit undesirable side effects.

Clinical studies have demonstrated that a single sub-anesthetic dose of the dissociative anesthetic ketamine induces rapid and sustained antidepressant actions in treatment-resistant patients. Although this finding has been met with enthusiasm, ketamine’s widespread use is limited by its abuse potential and dissociative properties. Recent preclinical research has focused on unraveling the molecular mechanisms underlying the unique antidepressant actions of ketamine in an effort to develop novel pharmacotherapies, which will mimic ketamine’s antidepressant actions but lack its undesirable effects. Here, we review hypotheses for the mechanism of action of ketamine as an antidepressant, including direct synaptic or extra-synaptic (GluN2B-selective) NMDAR inhibition, selective inhibition of NMDARs localized on GABAergic interneurons, and the role of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPAR) activation. We also discuss links between ketamine’s antidepressant actions and downstream mechanisms regulating synaptic plasticity, including brain-derived neurotrophic factor (BDNF), eukaryotic elongation factor 2 (eEF2), mechanistic target of rapamycin (mTOR), and glycogen synthase kinase-3 (GSK-3). Mechanisms that do not involve direct inhibition of the NMDAR, including a role for ketamine’s (R)-ketamine enantiomer and hydroxynorketamine (HNK) metabolites, specifically (2R,6R)-HNK, are also discussed. Proposed mechanisms of ketamine’s action are not mutually exclusive and may act in a complementary fashion to exert the acute changes in synaptic plasticity, leading to sustained strengthening of excitatory synapses, which are necessary for antidepressant behavioral actions. Understanding the molecular mechanisms underpinning ketamine’s antidepressant actions will be invaluable for the identification of targets, which will drive the development of novel, effective, next-generation pharmacotherapies for the treatment of depression.

The main challenge in treating opioid addicts is to maintain abstinence due to the affective consequences associated with withdrawal which may trigger relapse. Emerging evidence suggests a role of the neurohypophysial peptide oxytocin (OT) in the modulation of mood disorders as well as drug addiction. However, its involvement in the emotional consequences of drug abstinence remains unclear. We investigated the effect of 7-day opioid abstinence on the oxytocinergic system and assessed the effect of the OT analogue carbetocin (CBT) on the emotional consequences of opioid abstinence, as well as relapse. Male C57BL/6J mice were treated with a chronic escalating-dose morphine regimen (20-100 mg/kg/day, i.p.). Seven days withdrawal from this administration paradigm induced a decrease of hypothalamic OT levels and a concomitant increase of oxytocin receptor (OTR) binding in the lateral septum and amygdala. Although no physical withdrawal symptoms or alterations in the plasma corticosterone levels were observed after 7 days of abstinence, mice exhibited increased anxiety-like and depressive-like behaviors and impaired sociability. CBT (6.4 mg/kg, i.p.) attenuated the observed negative emotional consequences of opioid withdrawal. Furthermore, in the conditioned place preference paradigm with 10 mg/kg morphine conditioning, CBT (6.4 mg/kg, i.p.) was able to prevent the stress-induced reinstatement to morphine-seeking following extinction. Overall, our results suggest that alterations of the oxytocinergic system contribute to the mechanisms underlying anxiety, depression, and social deficits observed during opioid abstinence. This study also highlights the oxytocinergic system as a target for developing pharmacotherapy for the treatment of emotional impairment associated with abstinence and thereby prevention of relapse.

Preclinical studies indicate that (2R,6R)-hydroxynorketamine (HNK) is a putative fast-acting antidepressant candidate. Although inhibition of NMDA-type glutamate receptors (NMDARs) is one mechanism proposed to underlie ketamine’s antidepressant and adverse effects, the potency of (2R,6R)-HNK to inhibit NMDARs has not been established. We used a multidisciplinary approach to determine the effects of (2R,6R)-HNK on NMDAR function. Antidepressant-relevant behavioral responses and (2R,6R)-HNK levels in the extracellular compartment of the hippocampus were measured following systemic (2R,6R)-HNK administration in mice. The effects of ketamine, (2R,6R)-HNK, and, in some cases, the (2S,6S)-HNK stereoisomer were evaluated on the following: (i) NMDA-induced lethality in mice, (ii) NMDAR-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 field of mouse hippocampal slices, (iii) NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) and NMDA-evoked currents in CA1 pyramidal neurons of rat hippocampal slices, and (iv) recombinant NMDARs expressed in Xenopus oocytes. While a single i.p. injection of 10 mg/kg (2R,6R)-HNK exerted antidepressant-related behavioral and cellular responses in mice, the ED50 of (2R,6R)-HNK to prevent NMDA-induced lethality was found to be 228 mg/kg, compared with 6.4 mg/kg for ketamine. The 10 mg/kg (2R,6R)-HNK dose generated maximal hippocampal extracellular concentrations of ∼8 µM, which were well below concentrations required to inhibit synaptic and extrasynaptic NMDARs in vitro. (2S,6S)-HNK was more potent than (2R,6R)-HNK, but less potent than ketamine at inhibiting NMDARs. These data demonstrate the stereoselectivity of NMDAR inhibition by (2R,6R;2S,6S)-HNK and support the conclusion that direct NMDAR inhibition does not contribute to antidepressant-relevant effects of (2R,6R)-HNK.

Traditional pharmacological treatments for depression have a delayed therapeutic onset, ranging from several weeks to months, and there is a high percentage of individuals who never respond to treatment. In contrast, ketamine produces rapid-onset antidepressant, anti-suicidal and anti-anhedonic actions following a single administration to depressed patients. Proposed mechanisms of ketamine’s antidepressant action include N-methyl-D-aspartate receptor (NMDAR) modulation, GABAergic interneuron disinhibition, and direct actions of its hydroxynorketamine (HNK) metabolites. Downstream actions include activation of mechanistic target of rapamycin (mTOR), deactivation of glycogen synthase kinase-3 and eukaryotic elongation factor 2 (eEF2), enhanced brain-derived neurotrophic factor (BDNF) signaling, and activation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptors (AMPARs). These putative mechanisms of ketamine action are not mutually exclusive and may complement each other to induce potentiation of excitatory synapses in affective-regulating brain circuits, which results in amelioration of depression symptoms. We review these proposed mechanisms of ketamine action in the context of how such mechanisms are informing the development of novel putative rapid-acting antidepressant drugs. Such drugs that have undergoing pre-clinical, and in some cases clinical, testing include the muscarinic acetylcholine receptor antagonist scopolamine, GluN2B-NMDAR antagonists (i.e., CP-101,606, MK-0657), (2R,6R)-HNK, NMDAR glycine site modulators (i.e., 4-chlorokynurenine - pro-drug of the glycineB NMDAR antagonist 7-chlorokynurenic acid), NMDAR agonists (i.e. GLYX-13 (rapastinel)), metabotropic glutamate receptor 2/3 (mGluR2/3) antagonists, GABAA receptor modulators, and drugs acting on various serotonin receptor subtypes. These ongoing studies suggest that the future acute treatment of depression will typically occur within hours, rather than months, of treatment initiation.

Currently approved antidepressant drug treatment typically takes several weeks to be effective. The noncompetitive N-methyl-D-aspartate (NMDA) receptor antagonist ketamine has shown efficacy as a rapid-acting treatment of depression, but its use is associated with significant side effects. We assessed effects following blockade of the glycine B co-agonist site of the NMDA receptor, located on the GluN1 subunit, by the selective full antagonist 7-chloro-kynurenic acid (7-Cl-KYNA), delivered by systemic administration of its brain-penetrant prodrug 4-chlorokynurenine (4-Cl-KYN) in mice. Following administration of 4-Cl-KYN, 7-Cl-KYNA was promptly recovered extracellularly in hippocampal microdialysate of freely moving animals. The behavioral responses of the animals were assessed using measures of ketamine-sensitive antidepressant efficacy (including the 24-hour forced swim test, learned helplessness test, and novelty-suppressed feeding test). In these tests, distinct from fluoxetine, and similar to ketamine, 4-Cl-KYN administration resulted in rapid, dose-dependent and persistent antidepressant-like effects following a single treatment. The antidepressant effects of 4-Cl-KYN were prevented by pretreatment with glycine or the a-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptor antagonist 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione (NBQX). 4-Cl-KYN administration was not associated with the rewarding and psychotomimetic effects of ketamine, and did not induce locomotor sensitization or stereotypic behaviors. Our results provide further support for antagonism of the glycine B site for the rapid treatment of treatment-resistant depression without the negative side effects seen with ketamine or other channelblocking NMDA receptor antagonists.