Hydroxynorketamines: Pharmacology and Potential Therapeutic Applications.

Hydroxynorketamines (HNKs) are formed in vivo after (R,S)-ketamine (ketamine) administration. The 12 HNK stereoisomers are distinguished by the position of cyclohexyl ring hydroxylation (at the 4, 5, or 6 position) and their unique stereochemistry at two stereocenters. Although HNKs were initially classified as inactive metabolites because of their lack of anesthetic effects, more recent studies have begun to reveal their biologic activities. In particular, (2R,6R)- and (2S 6)-HNK exert antidepressant-relevant behavioral and physiologic effects in preclinical models, which led to a rapid increase in studies seeking to clarify the mechanisms by which HNKs exert their pharmacological effects. To date, the majority of HNK research has focused on the actions of (2R,6R)-HNK because of its robust behavioral actions in tests of antidepressant effectiveness and its limited adverse effects. This review describes HNK pharmacokinetics and pharmacodynamics, as well as the putative cellular, molecular, and synaptic mechanisms thought to underlie their behavioral effects, both following their metabolism from ketamine and after direct administration in preclinical studies. Converging preclinical evidence indicates that HNKs modulate glutamatergic neurotransmission and downstream signaling pathways in several brain regions, including the hippocampus and prefrontal cortex. Effects on other neurotransmitter systems, as well as possible effects on neurotrophic and inflammatory processes, and energy metabolism, are also discussed. Additionally, the behavioral effects of HNKs and possible therapeutic applications are described, including the treatment of unipolar and bipolar depression, post-traumatic stress disorder, chronic pain, neuroinflammation, and other anti-inflammatory and analgesic uses. SIGNIFICANCE STATEMENT: Preclinical studies indicate that hydroxynorketamines (HNKs) exert antidepressant-relevant behavioral actions and may also have analgesic, anti-inflammatory, and other physiological effects that are relevant for the treatment of a variety of human diseases. This review details the pharmacokinetics and pharmacodynamics of the HNKs, as well as their behavioral actions, putative mechanisms of action, and potential therapeutic applications.

(2R,6R)-hydroxynorketamine exerts mGlu2 receptor-dependent antidepressant actions.

Currently approved antidepressant drugs often take months to take full effect, and ∼30% of depressed patients remain treatment resistant. In contrast, ketamine, when administered as a single subanesthetic dose, exerts rapid and sustained antidepressant actions. Preclinical studies indicate that the ketamine metabolite (2R,6R)-hydroxynorketamine [(2R,6R)-HNK] is a rapid-acting antidepressant drug candidate with limited dissociation properties and abuse potential. We assessed the role of group II metabotropic glutamate receptor subtypes 2 (mGlu2) and 3 (mGlu3) in the antidepressant-relevant actions of (2R,6R)-HNK using behavioral, genetic, and pharmacological approaches as well as cortical quantitative EEG (qEEG) measurements in mice. Both ketamine and (2R,6R)-HNK prevented mGlu2/3 receptor agonist (LY379268)-induced body temperature increases in mice lacking the Grm3, but not Grm2, gene. This action was not replicated by NMDA receptor antagonists or a chemical variant of ketamine that limits metabolism to (2R,6R)-HNK. The antidepressant-relevant behavioral effects and 30- to 80-Hz qEEG oscillation (gamma-range) increases resultant from (2R,6R)-HNK administration were prevented by pretreatment with an mGlu2/3 receptor agonist and absent in mice lacking the Grm2, but not Grm3-/-, gene. Combined subeffective doses of the mGlu2/3 receptor antagonist LY341495 and (2R,6R)-HNK exerted synergistic increases on gamma oscillations and antidepressant-relevant behavioral actions. These findings highlight that (2R,6R)-HNK exerts antidepressant-relevant actions via a mechanism converging with mGlu2 receptor signaling and suggest enhanced cortical gamma oscillations as a marker of target engagement relevant to antidepressant efficacy. Moreover, these results support the use of (2R,6R)-HNK and inhibitors of mGlu2 receptor function in clinical trials for treatment-resistant depression either alone or in combination.

Ketamine and Ketamine Metabolite Pharmacology: Insights into Therapeutic Mechanisms.

Ketamine, a racemic mixture consisting of (S)- and (R)-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 N-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.

Ketamine metabolism via hepatic CYP450 isoforms contributes to its sustained antidepressant actions.

(R,S)-ketamine (ketamine) has rapid and sustained antidepressant (AD) efficacy at sub-anesthetic doses in depressed patients. A metabolite of ketamine, including (2R,6R)-hydroxynorketamine ((6)-HNKs) has been reported to exert antidepressant actions in rodent model of anxiety/depression. To further understand the specific role of ketamine's metabolism in the AD actions of the drug, we evaluated the effects of inhibiting hepatic cytochrome P450 enzymes on AD responses. We assessed whether pre-treatment with fluconazole (10 and 20 mg/kg, i. p.) 1 h prior to ketamine or HNKs (10 mg/kg, i. p.) administration would alter behavioral and neurochemical actions of the drugs in male BALB/cJ mice with a highly anxious phenotype. Extracellular microdialysate levels of glutamate and GABA (Gluext, GABAext) were also measured in the medial prefrontal cortex (mPFC). Pre-treatment with fluconazole altered the pharmacokinetic profile of ketamine, by increasing both plasma and brain levels of ketamine and (R,S)-norketamine, while robustly reducing those of (6)-HNKs. At 24 h post-injection (t24 h), fluconazole prevented the sustained AD-like response of ketamine responses in the forced swim test and splash test, as well as the enhanced cortical GABA levels produced by ketamine. A single (2R,6R)-HNK administration resulted in prevention of the effects of fluconazole on the antidepressant-like activity of ketamine in mice. Overall, these findings are consistent with an essential contribution of (6)-HNK to the sustained antidepressant-like effects of ketamine and suggest potential interactions between pharmacological CYPIs and ketamine during antidepressant treatment in patients.

Sex-dependent metabolism of ketamine and (2R,6R)-hydroxynorketamine in mice and humans.

BACKGROUND: Ketamine is rapidly metabolized to norketamine and hydroxynorketamine (HNK) metabolites. In female mice, when compared to males, higher levels of (2R,6R;2S,6S)-HNK have been observed following ketamine treatment, and higher levels of (2R,6R)-HNK following the direct administration of (2R,6R)-HNK. AIM: The objective of this study was to evaluate the impact of sex in humans and mice, and gonadal hormones in mice on the metabolism of ketamine to form norketamine and HNKs and in the metabolism/elimination of (2R,6R)-HNK. METHODS: In CD-1 mice, we utilized gonadectomy to evaluate the role of circulating gonadal hormones in mediating sex-dependent differences in ketamine and (2R,6R)-HNK metabolism. In humans (34 with treatment-resistant depression and 23 healthy controls) receiving an antidepressant dose of ketamine (0.5 mg/kg i.v. infusion over 40 min), we evaluated plasma levels of ketamine, norketamine, and HNKs. RESULTS: In humans, plasma levels of ketamine and norketamine were higher in males than females, while (2R,6R;2S,6S)-HNK levels were not different. Following ketamine administration to mice (10 mg/kg i.p.), Cmax and total plasma concentrations of ketamine and norketamine were higher, and those of (2R,6R;2S,6S)-HNK were lower, in intact males compared to females. Direct (2R,6R)-HNK administration (10 mg/kg i.p.) resulted in higher levels of (2R,6R)-HNK in female mice. Ovariectomy did not alter ketamine metabolism in female mice, whereas orchidectomy recapitulated female pharmacokinetic differences in male mice, which was reversed with testosterone replacement. CONCLUSION: Sex is an important biological variable that influences the metabolism of ketamine and the HNKs, which may contribute to sex differences in therapeutic antidepressant efficacy or side effects.

Hydroxynorketamine Pharmacokinetics and Antidepressant Behavioral Effects of (2,6)- and (5R)-Methyl-(2R,6R)-hydroxynorketamines.

(R,S)-Ketamine is rapidly metabolized to form a range of metabolites in vivo, including 12 unique hydroxynorketamines (HNKs) that are distinguished by a cyclohexyl ring hydroxylation at the 4, 5, or 6 position. While both (2R,6R)- and (2S,6S)-HNK readily penetrate the brain and exert rapid antidepressant-like actions in preclinical tests following peripheral administration, the pharmacokinetic profiles and pharmacodynamic actions of 10 other HNKs have not been examined. We assessed the pharmacokinetic profiles of all 12 HNKs in the plasma and brains of male and female mice and compared the relative potencies of four (2,6)-HNKs to induce antidepressant-relevant behavioral effects in the forced swim test in male mice. While all HNKs were readily brain-penetrable following intraperitoneal injection, there were robust differences in peak plasma and brain concentrations and exposures. Forced swim test immobility rank order of potency, from most to least potent, was (2R,6S)-, (2S,6R)-, (2R,6R)-, and (2S,6S)-HNK. We hypothesized that distinct structure-activity relationships and the resulting potency of each metabolite are linked to unique substitution patterns and resultant conformation of the six-membered cyclohexanone ring system. To explore this, we synthesized (5R)-methyl-(2R,6R)-HNK, which incorporates a methyl substitution on the cyclohexanone ring. (5R)-Methyl-(2R,6R)-HNK exhibited similar antidepressant-like potency to (2R,6S)-HNK. These results suggest that conformation of the cyclohexanone ring system in the (2,6)-HNKs is an important factor underlying potency and that additional engineering of this structural feature may improve the development of a new generation of HNKs. Such HNKs may represent novel drug candidates for the treatment of depression.

Experimenters' sex modulates mouse behaviors and neural responses to ketamine via corticotropin releasing factor.

We show that the sex of human experimenters affects mouse behaviors and responses following administration of the rapid-acting antidepressant ketamine and its bioactive metabolite (2R,6R)-hydroxynorketamine. Mice showed aversion to the scent of male experimenters, preference for the scent of female experimenters and increased stress susceptibility when handled by male experimenters. This human-male-scent-induced aversion and stress susceptibility was mediated by the activation of corticotropin-releasing factor (CRF) neurons in the entorhinal cortex that project to hippocampal area CA1. Exposure to the scent of male experimenters before ketamine administration activated CA1-projecting entorhinal cortex CRF neurons, and activation of this CRF pathway modulated in vivo and in vitro antidepressant-like effects of ketamine. A better understanding of the specific and quantitative contributions of the sex of human experimenters to study outcomes in rodents may improve replicability between studies and, as we have shown, reveal biological and pharmacological mechanisms.

Group II metabotropic glutamate receptor blockade promotes stress resilience in mice.

Stress is a leading risk factor for the onset and recurrence of major depression. Enhancing stress resilience may be a therapeutic strategy to prevent the development of depression in at-risk populations or its recurrence in depressed patients. Group II metabotropic glutamate receptor (mGlu2/3) antagonists have been recognized for antidepressant-like actions in preclinical models, but have not been evaluated for prophylactic effects. We assessed the role of mGlu2/3 in modulating stress resilience using subtype-specific knockout mice lacking mGlu2 (Grm2-/-) or mGlu3 (Grm3-/-), and pharmacological manipulations of mGlu2/3 activity during or prior to the induction and reinstatement of stress-induced behavioral deficits. Grm2-/-, but not Grm3-/-, mice exhibited reduced forced-swimming test immobility time and were resilient to developing inescapable shock (IES)-induced escape deficits. Grm2-/- mice were also resilient to developing corticosterone (CORT)-induced escape deficits and chronic social defeat stress-induced anhedonia. Pharmacological blockade of mGlu2/3 with the antagonist LY341495 during stress prevented the development of IES- and CORT-induced escape deficits, while activation with the agonist LY379268 increased susceptibility to escape deficits. Prophylactic treatment with the LY341495, both systemically and via microinjection into the medial prefrontal cortex (mPFC), up to 7 days before IES, prevented both the induction of escape deficits and their reinstatement by brief re-exposure to IES up to 20 days after treatment. Overall, blockade of mGlu2/3 enhanced stress resilience and deletion of mGlu2, but not mGlu3, conferred a stress-resilient phenotype, indicating that prophylactic treatments reducing mGlu2 activity may protect against stress-induced changes underlying the development or recurrence of stress-induced disorders, including depression.

(R)-Ketamine exerts antidepressant actions partly via conversion to (2R,6R)-hydroxynorketamine, while causing adverse effects at sub-anaesthetic doses.

BACKGROUND AND PURPOSE: (R)-Ketamine (arketamine) may have utility as a rapidly acting antidepressant. While (R)-ketamine has lower potency than (R,S)-ketamine to inhibit NMDA receptors in vitro, the extent to which (R)-ketamine shares the NMDA receptor-mediated adverse effects of (R,S)-ketamine in vivo has not been fully characterised. Furthermore, (R)-ketamine is metabolised to (2R,6R)-hydroxynorketamine (HNK), which may contribute to its antidepressant-relevant actions. EXPERIMENTAL APPROACH: Using mice, we compared (R)-ketamine with a deuterated form of the drug (6,6-dideutero-(R)-ketamine, (R)-d2 -ketamine), which hinders its metabolism to (2R,6R)-HNK, in behavioural tests predicting antidepressant responses. We also examined the actions of intracerebroventricularly infused (2R,6R)-HNK. Further, we quantified putative NMDA receptor inhibition-mediated adverse effects of (R)-ketamine. KEY RESULTS: (R)-d2 -Ketamine was identical to (R)-ketamine in binding to and functionally inhibiting NMDA receptors but hindered (R)-ketamine's metabolism to (2R,6R)-HNK. (R)-Ketamine exerted greater potency than (R)-d2 -ketamine in several antidepressant-sensitive behavioural measures, consistent with a role of (2R,6R)-HNK in the actions of (R)-ketamine. There were dose-dependent sustained antidepressant-relevant actions of (2R,6R)-HNK following intracerebroventricular administration. (R)-Ketamine exerted NMDA receptor inhibition-mediated behaviours similar to (R,S)-ketamine, including locomotor stimulation, conditioned-place preference, prepulse inhibition deficits, and motor incoordination, with approximately half the potency of the racemic drug. CONCLUSIONS AND IMPLICATIONS: Metabolism of (R)-ketamine to (2R,6R)-HNK increases the potency of (R)-ketamine to exert antidepressant-relevant actions in mice. Adverse effects of (R)-ketamine require higher doses than those necessary for antidepressant-sensitive behavioural changes in mice. However, our data revealing that (R)-ketamine's adverse effects are elicited at sub-anaesthetic doses indicate a potential risk for sensory dissociation and abuse liability.

Mouse, rat, and dog bioavailability and mouse oral antidepressant efficacy of (2R,6R)-hydroxynorketamine.

BACKGROUND: (R,S)-ketamine has gained attention for its rapid-acting antidepressant actions in patients with treatment-resistant depression. However, widespread use of ketamine is limited by its side effects, abuse potential, and poor oral bioavailability. The ketamine metabolite, (2R,6R)-hydroxynorketamine, exerts rapid antidepressant effects, without ketamine's adverse effects and abuse potential, in rodents. METHODS: We evaluated the oral bioavailability of (2R,6R)-hydroxynorketamine in three species (mice, rats, and dogs) and also evaluated five candidate prodrug modifications for their capacity to enhance the oral bioavailability of (2R,6R)-hydroxynorketamine in mice. Oral administration of (2R,6R)-hydroxynorketamine was assessed for adverse behavioral effects and for antidepressant efficacy in the mouse forced-swim and learned helplessness tests. RESULTS: (2R,6R)-hydroxynorketamine had absolute bioavailability between 46-52% in mice, 42% in rats, and 58% in dogs. Compared to intraperitoneal injection in mice, the relative oral bioavailability of (2R,6R)-hydroxynorketamine was 62%, which was not improved by any of the candidate prodrugs tested. Following oral administration, (2R,6R)-hydroxynorketamine readily penetrated the brain, with brain to plasma ratios between 0.67-1.2 in mice and rats. Oral administration of (2R,6R)-hydroxynorketamine to mice did not alter locomotor activity or precipitate behaviors associated with discomfort, sickness, or stereotypy up to a dose of 450 mg/kg. Oral (2R,6R)-hydroxynorketamine reduced forced-swim test immobility time (15-150 mg/kg) and reversed learned helplessness (50-150 mg/kg) in mice. CONCLUSIONS: These results demonstrate that (2R,6R)-hydroxynorketamine has favorable oral bioavailability in three species and exhibits antidepressant efficacy following oral administration in mice.

A Negative Allosteric Modulator for α5 Subunit-Containing GABA Receptors Exerts a Rapid and Persistent Antidepressant-Like Action without the Side Effects of the NMDA Receptor Antagonist Ketamine in Mice.

New antidepressant pharmacotherapies that provide rapid relief of depressive symptoms are needed. The NMDA receptor antagonist ketamine exerts rapid antidepressant actions in depressed patients but also side effects that complicate its clinical utility. Ketamine promotes excitatory synaptic strength, likely by producing high-frequency correlated activity in mood-relevant regions of the forebrain. Negative allosteric modulators of GABA-A receptors containing α5 subunits (α5 GABA-NAMs) should also promote high-frequency correlated electroencephalogram (EEG) activity and should therefore exert rapid antidepressant responses. Because α5 subunits display a restricted expression in the forebrain, we predicted that α5 GABA-NAMs would produce activation of principle neurons but exert fewer side effects than ketamine. We tested this hypothesis in male mice and observed that the α5 GABA-NAM MRK-016 exerted an antidepressant-like response in the forced swim test at 1 and 24 h after administration and an anti-anhedonic response after chronic stress in the female urine sniffing test (FUST). Like ketamine, MRK-016 produced a transient increase in EEG γ power, and both the increase in γ power and its antidepressant effects in the forced swim test were blocked by prior administration of the AMPA-type glutamate receptor antagonist 2,3-dioxo-6-nitro-1,2,3,4-tetrahydrobenzo[f]quinoxaline-7-sulfonamide (NBQX). Unlike ketamine, however, MRK-016 produced no impairment of rota-rod performance, no reduction of prepulse inhibition (PPI), no conditioned-place preference (CPP), and no change in locomotion. α5 GABA-NAMs, thus reproduce the rapid antidepressant-like actions of ketamine, perhaps via an AMPA receptor (AMPAR)-dependent increase in coherent neuronal activity, but display fewer potential negative side effects. These compounds thus demonstrate promise as clinically useful fast-acting antidepressants.