Ketamine Metabolites Research Articles

Pathophysiology, clinical presentation, and management of ketamine-induced cystitis.

Ketamine is illegally used as a recreational drug in many Asian countries. Long-term ketamine abusers often develop irritable bladder symptoms that gradually develop into more severe urinary frequency and urgency and eventually into a painful ulcerated bladder. These patients typically have reduced functional bladder capacity, increased bladder sensation, detrusor overactivity, severe urgency, urinary incontinence, and bladder contracture. Ketamine metabolites can cause severe inflammation of the urothelium, urothelial barrier deficits, vascular endothelial fibrinoid changes, increased oxidative stress, and bladder wall fibrosis. A decrease in bladder compliance, urinary tract infection, severe bladder pain with a full bladder, and painful micturition are also common symptoms. Finally, with continued abuse of ketamine, hydronephrosis, ureteral stricture, vesicoureteral reflux, and renal failure may develop. Cessation of ketamine is the mainstay of treatment. Lower urinary tract symptoms usually relapse if patients reuse ketamine after stopping. In cases of severe ketamine cystitis, only augmentation enterocystoplasty can relieve bladder pain and restore normal lower urinary tract function. This article reviews the underlying pathophysiology, clinical characteristics, and management of ketamine cystitis.

TRKB interaction with PSD95 is associated with latency of fluoxetine and 2R,6R-hydroxynorketamine.

Brain derived neurotrophic factor (BDNF) and its receptor tropomyosin kinase receptor B (TRKB) are key regulators of activity-dependent plasticity in the brain. TRKB is the target for both slow- and rapid-acting antidepressants and BDNF-TRKB system mediates the plasticity-inducing effects of antidepressants through their downstream targets. Particularly, the protein complexes that regulate the trafficking and synapse recruitment of TRKB receptors might be crucial in this process. In the present study, we investigated the interaction of TRKB with the postsynaptic density protein 95 (PSD95). We found that antidepressants increase the TRKB:PSD95 interaction in adult mouse hippocampus. Fluoxetine, a slow-acting antidepressant, increases this interaction only after a long-term (7days) treatment, while (2R,6R)-hydroxynorketamine (RHNK), an active metabolite of rapid-acting antidepressant ketamine, achieves this within a short treatment regimen (3days). Moreover, the drug-induced changes of TRKB:PSD95 interaction correlate with drug latency in behaviour, observed in mice subjected to an object location memory test (OLM). While silencing of PSD95 by viral delivery of shRNA in hippocampus abolished the RHNK-induced plasticity in mice in OLM, overexpression of PSD95 shortened the fluoxetine latency. In summary, changes in the TRKB:PSD95 interaction contribute to differences observed in drug latency. This study sheds a light on a novel mechanism of action of different classes of antidepressants.

(2R,6R)-Hydroxynorketamine Treatment of Rats Exposed to Repetitive Low-Level Blast Injury.

Many military veterans who experienced blast-related traumatic brain injuries (TBIs) in the conflicts in Iraq and Afghanistan suffer from chronic cognitive and mental health problems, including post-traumatic stress disorder (PTSD). Male rats subjected to repetitive low-level blast exposure develop chronic cognitive and PTSD-related traits that develop in a delayed manner. Ketamine has received attention as a treatment for refractory depression and PTSD. (2R,6R)-hydroxynorketamine [(2R,6R)-HNK] is a ketamine metabolite that exerts rapid antidepressant actions. (2R,6R)-HNK has become of clinical interest because of its favorable side-effect profile, low abuse potential, and oral route of administration. We treated three cohorts of blast-exposed rats with (2R,6R)-HNK, beginning 7-11 months after blast exposure, a time when the behavioral phenotype is established. Each cohort consisted of groups (n = 10-13/group) as follows: 1) Sham-exposed treated with saline, 2) blast-exposed treated with saline, and 3) blast-exposed treated with a single dose of 20 mg/kg of (2R,6R)-HNK. (2R,6R)-HNK rescued blast-induced deficits in novel object recognition (NOR) and anxiety-related features in the elevated zero maze (EZM) in all three cohorts. Exaggerated acoustic startle was reversed in cohort 1, but not in cohort 3. (2R,6R)-HNK effects were still present in the EZM 12 days after administration in cohort 1 and 27 days after administration in NOR testing of cohorts 2 and 3. (2R,6R)-HNK may be beneficial for the neurobehavioral syndromes that follow blast exposure in military veterans. Additional studies will be needed to determine whether higher doses or more extended treatment regimens may be more effective.

Kinetic mechanism of NMDA receptor inhibition by the ketamine metabolite 2R,6R-hydroxynorketamine.

2R,6R-hydroxynorketamine (2R,6R-HNK) is a metabolite of the fast-acting dissociative anesthetic ketamine (KET). It is currently in clinical trials for the treatment of major depressive disorder due to its strong safety profile at larger doses. The endogenous therapeutic target of 2R,6R-HNK is unknown. We reported recently that like the parent compound ketamine, 2R,6R-HNK reduces NMDA receptor currents with an open-channel block mechanism which is voltage-dependent, pH-dependent, and competitive with Mg2+, and has ∼100-fold lower efficacy (IC50, 46 µM) than KET (IC50, 0.5 µM) at pH 7.2 and −70 mV. We used electrophysiology, kinetic analyses, and kinetic modeling to examine the effect of 2R,6R-HNK on the reaction mechanism of NMDA receptors. We recorded on-cell single-channel currents from HEK293 cells expressing GluN1/GluN2A receptors (and GFP) with pipettes containing (in mM): 150 NaCl, 2.5 KCl, 1 EDTA, 10 HEPES, pH 7.2 (NaOH), agonists glutamate (1) and glycine (0.1), and applied +100 mV (membrane potential ∼−120 mV). Relative to activity recorded in the absence of 2R,6R-HNK (control, n=7), activity recorded with 50 µM 2R,6R-HNK (n=7) displayed a lower open probability (Po), shorter openings (MOT), and longer closed durations (MCT). Fitting these data with state models allowed us to optimize rate constants for the kinetic model. In the future, we will test these models by simulating macroscopic responses and recording whole cell currents in similar conditions.

NMDA Receptor Activation-Dependent Antidepressant-Relevant Behavioral and Synaptic Actions of Ketamine.

Ketamine is a well-characterized NMDA receptor (NMDAR) antagonist, although the relevance of this pharmacology to its rapid (within hours of administration) antidepressant actions, which depend on mechanisms convergent with strengthening of excitatory synapses, is unclear. Activation of synaptic NMDARs is necessary for the induction of canonical long-term potentiation (LTP) leading to a sustained expression of increased synaptic strength. We tested the hypothesis that induction of rapid antidepressant effects requires NMDAR activation, by using behavioral pharmacology, western blot quantification of hippocampal synaptoneurosomal protein levels, and ex vivo hippocampal slice electrophysiology in male mice. We found that ketamine exerts an inverted U-shaped dose-response in antidepressant-sensitive behavioral tests, suggesting that an excessive NMDAR inhibition can prevent ketamine's antidepressant effects. Ketamine's actions to induce antidepressant-like behavioral effects, up-regulation of hippocampal AMPAR subunits GluA1 and GluA2, as well as metaplasticity measured ex vivo using electrically-stimulated LTP, were abolished by pretreatment with other non-antidepressant NMDAR antagonists, including MK-801 and CPP. Similarly, the antidepressant-like actions of other putative rapid-acting antidepressant drugs (2R,6R)-hydroxynorketamine (ketamine metabolite), MRK-016 (GABAAα5 negative allosteric modulator), and LY341495 (mGlu2/3 receptor antagonist) were blocked by NMDAR inhibition. Ketamine acted synergistically with an NMDAR positive allosteric modulator to exert antidepressant-like behavioral effects and activation of the NMDAR subunit GluN2A was necessary and sufficient for such relevant effects. We conclude rapid-acting antidepressant compounds share a common downstream NMDAR-activation dependent effector mechanism, despite variation in initial pharmacological targets. Promoting NMDAR signaling or other approaches that enhance NMDAR-dependent LTP-like synaptic potentiation may be an effective antidepressant strategy.SIGNIFICANCE STATEMENT The anesthetic and antidepressant drug ketamine is well-characterized as an NMDA receptor (NMDAR) antagonist; though, the relevance and full impact of this pharmacology to its antidepressant actions is unclear. We found that NMDAR activation, which occurs downstream of their initial actions, is necessary for the beneficial effects of ketamine and several other putative antidepressant compounds. As such, promoting NMDAR signaling, or other approaches that enhance NMDAR-dependent long-term potentiation (LTP)-like synaptic potentiation in vivo may be an effective antidepressant strategy directly, or acting synergistically with other drug or interventional treatments.

(2R,6R)-hydroxynorketamine targeting the basolateral amygdala regulates fear memory

(2R,6R)-Hydroxynorketamine (HNK), a ketamine metabolite, has been proposed as an ideal next-generation antidepressant due to its rapid-acting and long-lasting antidepression-relevant actions. Interestingly, recent studies have shown that (2R,6R)-HNK may have diverse impacts on memory formation. However, its effect on fear memory extinction is still unknown. In the present study, we assessed the effects of (2R,6R)-HNK on synaptic transmission and plasticity in the basolateral amygdala (BLA) and explored its actions on auditory fear memory extinction. Adult male C57BL/6J mice were used in this study. The extracellular electrophysiological recording was conducted to assay synaptic transmission and plasticity. The auditory fear conditioning paradigm was performed to test fear extinction. The results showed that (2R,6R)-HNK at 30 mg/kg increased the number of c-fos-positive cells in the BLA. Moreover, (2R,6R)-HNK enhanced the induction and maintenance of long-term potentiation (LTP) in the BLA in a dose-dependent manner (at 1, 10, and 30 mg/kg). In addition, (2R,6R)-HNK at 30 mg/kg and directly slice perfusion of (2R,6R)-HNK enhanced BLA synaptic transmission. Furthermore, intra-BLA application and systemic administration of (2R,6R)-HNK reduced the retrieval of recent fear memory and decreased the retrieval of remote fear memory. Both local and systemic (2R,6R)-HNK also inhibited the spontaneous recovery of remote fear memory. Taken together, these results indicated that (2R,6R)-HNK could regulate BLA synaptic transmission and plasticity and act through the BLA to modulate fear memory. The results revealed that (2R,6R)-HNK may be a potential drug to treat posttraumatic stress disorder (PTSD) patients.

Enantioselective CE-MS analysis of ketamine metabolites in urine.

The chiral drug ketamine has long-lasting antidepressant effects with a fast onset and is also suitable to treat patients with therapy-resistant depression. The metabolite hydroxynorketamine (HNK) plays an important role in the antidepressant mechanism of action. Hydroxylation at the cyclohexanone ring occurs at positions 4, 5, and 6 and produces a total of 12 stereoisomers. Among those, the four 6HNK stereoisomers have the strongest antidepressant effects. Capillary electrophoresis with highly sulfated γ-cyclodextrin (CD) as a chiral selector in combination with mass spectrometry (MS) was used to develop a method for the enantioselective analysis of HNK stereoisomers with a special focus on the 6HNK stereoisomers. The partial filling approach was applied in order to avoid contamination of the MS with the chiral selector. Concentration of the chiral selector and the length of the separation zone were optimized. With 5% highly sulfated γ-CD in 20mM ammonium formate with 10% formic acid and a 75% filling the four 6HNK stereoisomers could be separated with a resolution between 0.79 and 3.17. The method was applied to analyze fractionated equine urine collected after a ketamine infusion and to screen the fractions as well as unfractionated urine for the parent drug ketamine and other metabolites, including norketamine and dehydronorketamine.

Intranasal (2R, 6R)-hydroxynorketamine for acute pain: Behavioural and neurophysiological safety analysis in mice.

Ketamine is known for its antinociceptive effect and is also used for treatment-resistant depression. However, the efficacy and safety of (2R, 6R)-hydroxynorketamine (HNK), a ketamine metabolite has been sparingly investigated for acute pain management. The current study aims at investigating the antinociceptive effect of intranasal (2R, 6R)-HNK using pre-clinical models of acute pain. Additionally, the behavioural and neurophysiological safety analyses were carried out for the effective time window. Antinociceptive efficacy of (2R, 6R)-HNK was evaluated using the hot plate test and Hargreaves' plantar test. The formalin test was carried out in both the acute and tonic phases. The neurophysiological and behavioural safety analyses were carried out separately for the haemodynamic function, cortical electroencephalography (EEG), and spontaneous behavioural functions. Analgesic effect of (2R, 6R)-HNK was evident by a significant increase in paw-withdrawal latency in both Hargreaves' and hot plate tests. Additionally, the (2R, 6R)-HNK showed a significant ameliorative effect on pain-related behaviour in the second phase of the formalin test. (2R, 6R)-HNK exhibited an anxiolytic effect without causing any significant changes in locomotor activity and haemodynamic parameters. Power spectral density (PSD) analysis of electroencephalogram revealed no significant changes except a comparative increase in the gamma band range. Both the locomotor functions in the open field test and the PSD value of delta wave indicated no sedative effect at the given dose of (2R, 6R)-HNK. The results demonstrated the pain-alleviating effect of (2R, 6R)-HNK without compromising the neurophysiological and behavioural function. Therefore, intranasal (2R, 6R)-HNK is suggested as a safe candidate for further clinical study in the management of acute pain.

Synthesis of Ketamine Derivatives by Mannich Reactions

Arylcyclohexylamine Ketamine (HCL) has played an important role in Veterinary and Human Medicine as safe and reliable anesthetic agent but asit produces dysphoria several new derivatives of Ketamine have been synthesized till date. In this Research we have Utilized Mannich reaction to Synthesize The ammonia, primary amine and secondary amine are treated with hydrochloric acid and then added to the formaldehyde. Three novel derivatives were synthesized namely2-(2-chlorophenyl)- 6-[(diethyl amino) methyl]-2-(methylamino) cyclohexanone by RXN:102 Mannich Reaction of Ketamine with Di-ethylamine , rxn 113 2-(2chlorophenyl-6-dinitrophenyl hydrazine-methyl2(methylamino) cyclohexanone by Reaction of Ketamine with Di-nitro Phenyl Hydrazine & Rxn: 601. Mannich Reaction of Ketamine with Piperazine to form (2-chlorophenyl)-2-(methylamino)-6-(piperazine -1-yl) methyl cyclohexanone. Other reactions were also undergone to prepare oxazolidine derivatives of ketamine namely Synthesis of ketamine dioxolane derivative:Ketal and Hemiaminal Formation , Rxn: 103 Reaction of Ketamine with Glycerin [6-(2-chlorophenyl)-6-(methylamino)-1, 4-dioxaspiro [4.5] dec-2-yl] methanol and Rxn: 801 Reaction of Ketamine with Ephedrine N ,3,4-trimethyl-2,6 diphenyl-1oxa-4azaspiro [4.5] decan-6-amine derivative was formed. Thin layer chromatography of the synthesized derivatives was performed by using a solvent system of ethyl acetate and chloroform (50:50) and the RF values were calculated. The solubility test proves that compounds obtained from ketamine are polar in nature. All derivatives show similarity in solubility of ketamine. Although the melting points were not exactly comparable to that of ketamine, the range was not more than 5-10°C. This relatively narrow range of melting points proved that these were pure compounds. NMR spectroscopy was performed on all newly synthesized derivatives of ketamine. The notable ones obtained from reactions i.e. Rxn 102, 113, 103, 601and 801. The results of spectroscopy demonstrate that the compounds obtained were completely new species however they were structurally related to ketamine. These derivatives synthesized and confirmed by NMR technology. The derivatives can potentially be formulated for therapeutical purpose. Despite some limitations which are being considered in current drug design, the derivatives have the potential to develop into chemically modified entities that can play a major role in clinical therapeutics. Additional research studies will potentially help to determine the advanced method for high and sophisticated yield of these derivatives. Moreover, synthesis of ketamine metabolites namely N-demethyl compound and N-demethyl-5,6-dehydro analogues is established. However, further studies and modifications of these compounds will open new ventures of drug design and development of clinical implications in health care system.

(2R,6R)-hydroxynorketamine acts through GluA1-induced synaptic plasticity to alleviate PTSD-like effects in rat models

Post-traumatic stress disorder (PTSD) is a debilitating mental disorder with high morbidity and great social and economic relevance. However, extant pharmacotherapies of PTSD require long-term use to maintain effectiveness and have enormous side effects. The glutamatergic system, especially the α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid receptor (AMPAR), is an important target of current research on the mechanism of PTSD. Postsynaptic AMPAR function and expression are known to be increased by (2R, 6R)-hydronorketamine (HNK), the primary metabolite of ketamine. However, whether (2R,6R)-HNK alleviates PTSD-like effects via AMPAR upregulation is yet to be known.In the present study, rats were exposed to single prolonged stress and electric foot shock (SPS&S). Afterwards, gradient concentrations of (2R,6R)-HNK (20, 50, and 100 μM) were administered by intracerebroventricular (i.c.v.) injection. Open field, elevated plus maze, freezing behavior, and forced swimming tests were used to examine PTSD-like symptoms. In addition, the protein levels of GluA1, BDNF and PSD-95 were analyzed using western blotting and immunofluorescence, and the synaptic ultrastructure of the prefrontal cortex (PFC) was observed by transmission electron microscopy.We found that (2R,6R)-HNK changed SPS&S-induced behavioral expression, such as increasing autonomous activity and residence time in the open arm and decreasing immobility time. Likewise, (2R,6R)-HNK (50 μM) increased GluA1, BDNF, and PSD-95 protein expression in the PFC. Changes in synaptic ultrastructure induced by SPS&S were reversed by administration of (2R,6R)-HNK. Overall, we find that (2R,6R)-HNK can ameliorate SPS&S-induced fear avoidance in rats, as well as rat cognates of anxiety and depression. This may be related to GluA1-mediated synaptic plasticity in the PFC.

Ketamina - długa droga od anestetyku do prototypowego leku przeciwdepresyjnego. Przegląd potencjalnych mechanizmów działania

Published research studies on the antidepressant activity of ketamine in the last twenty years have significantly changed the way people think about potential new antidepressants and the biological basis of depression. The symptoms of depression may subside for several days after the administration of a dose of ketamine. In contrast, achieving a therapeutic effect with classic antidepressants requires chronic administration. The critical issue for ketamine is understanding the biological basis of its amazing effects. Because one of the main molecular mechanisms of ketamine action is the blockade of NMDA-activated glutamate receptors, a great effort has been directed at understanding the role of the glutamate system in the pathophysiology of depression and the unique antidepressant profile of ketamine. This review discusses the most relevant glutamate hypotheses explaining the molecular and cellular mechanisms of ketamine action. In the first place, phenomena such as the disinhibition of glutamate release and the inhibition of NMDA receptors stimulated by spontaneously released glutamate are discussed, followed by the relationship between the antidepressant effects of ketamine, glutamate, and the functioning of the lateral habenula. The last part of the review discusses the involvement of the individual enantiomers and metabolites of ketamine in its antidepressant activity.

(2R,6R)-hydroxynorketamine (HNK) reverses mechanical hypersensitivity in a model of localized inflammatory pain

The ketamine metabolite (2R,6R)-hydroxynorketamine, or (2R,6R)-HNK, was recently reported to evoke antinociception in response to a noxious thermal stimulus in healthy mice and reverse mechanical hypersensitivity in a murine model of neuropathic pain. This study reports the behavioral effects of (2R,6R)-HNK in male and female C57BL/6J mice exposed to a localized inflammatory pain condition and the broad pharmacological mechanism underlying this effect. Hind paw intraplantar injection of λ-carrageenan (CARR) caused inflammation and mechanical hypersensitivity in mice within 2 h, lasting at least 48 h. Administration of (2R,6R)-HNK (10–30 mg/kg i.p.) 2 h following CARR injection significantly reversed mechanical hypersensitivity within 1 h in male and female mice, and the effect persisted for 24 h following a single dose. The magnitude and timing of the analgesic effect of (2R,6R)-HNK were comparable to the non-steroidal anti-inflammatory drug carprofen. The reversal of hypersensitivity by (2R,6R)-HNK was blocked at 4 and 24 h after administration by pretreatment with the AMPA receptor antagonist NBQX and was not accompanied by changes in locomotor activity. These findings reinforce the growing evidence supporting (2R,6R)-HNK as a novel analgesic in multiple preclinical pain models and further support an AMPAR-dependent mechanism of action. SignificanceThe ketamine metabolite (2R,6R)-HNK reversed mechanical hypersensitivity associated with localized inflammation with onset less than 1 h and duration greater than 24 h, an effect comparable to the NSAID carprofen. Reversal of mechanical hypersensitivity by (2R,6R)-HNK is AMPAR-dependent.

Antinociceptive and Analgesic Effects of (2R,6R)-Hydroxynorketamine.

Commonly used pain therapeutics, such as opioid medications, exert dangerous side effects and lack effectiveness in treating some types of pain. Ketamine is also used to treat pain, but side effects limit its widespread use. (2R,6R)-hydroxynorketamine (HNK) is a ketamine metabolite that potentially shares some beneficial behavioral effects of its parent drug without causing significant side effects. This study compared the profile and potential mechanisms mediating the antinociception activity of ketamine and (2R,6R)-HNK in C57BL/6J mice. Additionally, this study compared the reversal of mechanical allodynia by (2R,6R)-HNK with gabapentin in a model of neuropathic pain. Unlike the near-immediate and short-lived antinociception caused by ketamine, (2R,6R)-HNK produced late-developing antinociception 24 hours following administration. Pharmacological blockade of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors with 2,3-dioxo-6-nitro-7-sulfamoyl-benzo[f]quinoxaline (NBQX) prevented the initiation and expressionof (2R,6R)-HNK antinociception, suggesting the involvement of α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptor-dependent glutamatergic mechanisms in the pain reduction-like responses. Blockade of opioid receptors with naltrexone partially prevented the antinociceptive effect of ketamine but was ineffective against (2R,6R)-HNK. Furthermore, (2R,6R)-HNK did not produce dystaxia, even when tested at doses five times greater than those needed to produce antinociception, indicating a superior safety profile for (2R,6R)-HNK over ketamine. Additionally, (2R,6R)-HNK reversed mechanical allodynia in a spared nerve injury model of neuropathic pain with similar short-term efficacy to gabapentin (within 4 hours) while outperforming gabapentin 24 hours after administration. These findings support the further study of (2R,6R)-HNK as a potentially valuable agent for treating different types of pain and establish certain advantages of (2R,6R)-HNK treatment over ketamine and gabapentin in corresponding assays for pain. SIGNIFICANCE STATEMENT: The ketamine metabolite (2R,6R)-HNK produced antinociception in male and female mice 24 hours after administration via activation of AMPA receptors. The effects of (2R,6R)-HNK differed in time course and mechanism and presented a better safety profile than ketamine. (2R,6R)-HNK also reversed allodynia in SNI-operated animals within 4 hours of treatment onset, with a duration of effect lasting longer than gabapentin. Taken together, (2R,6R)-HNK demonstrates the potential for development as a non-opioid analgesic drug.

Target deconvolution studies of (2R,6R)-hydroxynorketamine: an elusive search.

The off-label use of racemic ketamine and the FDA approval of (S)-ketamine are promising developments for the treatment of depression. Nevertheless, racemic ketamine and (S)-ketamine are controlled substances with known abuse potential and their use is associated with undesirable side effects. For these reasons, research efforts have focused on identifying alternatives. One candidate is (2R,6R)-hydroxynorketamine ((2R,6R)-HNK), a ketamine metabolite that in preclinical models lacks the dissociative and abuse properties of ketamine while retaining its antidepressant-like behavioral efficacy. (2R,6R)-HNK's mechanism of action however is unclear. The main goals of this study were to perform an in-depth pharmacological characterization of (2R,6R)-HNK at known ketamine targets, to use target deconvolution approaches to discover novel proteins that bind to (2R,6R)-HNK, and to characterize the biodistribution and behavioral effects of (2R,6R)-HNK across several procedures related to substance use disorder liability. We found that unlike (S)- or (R)-ketamine, (2R,6R)-HNK did not directly bind to any known or proposed ketamine targets. Extensive screening and target deconvolution experiments at thousands of human proteins did not identify any other direct (2R,6R)-HNK-protein interactions. Biodistribution studies using radiolabeled (2R,6R)-HNK revealed non-selective brain regional enrichment, and no specific binding in any organ other than the liver. (2R,6R)-HNK was inactive in conditioned place preference, open-field locomotor activity, and intravenous self-administration procedures. Despite these negative findings, (2R,6R)-HNK produced a reduction in immobility time in the forced swim test and a small but significant increase in metabolic activity across a network of brain regions, and this metabolic signature differed from the brain metabolic profile induced by ketamine enantiomers. In sum, our results indicate that (2R,6R)-HNK does not share pharmacological or behavioral profile similarities with ketamine or its enantiomers. However, it could still be possible that both ketamine and (2R,6R)-HNK exert antidepressant-like efficacy through a common and previously unidentified mechanism. Given its pharmacological profile, we predict that (2R,6R)-HNK will exhibit a favorable safety profile in clinical trials, and we must wait for clinical studies to determine its antidepressant efficacy.

Blood-based biomarkers of antidepressant response to ketamine and esketamine: A systematic review and meta-analysis.

(R,S)-ketamine (ketamine) and its enantiomer (S)-ketamine (esketamine) can produce rapid and substantial antidepressant effects. However, individual response to ketamine/esketamine is variable, and there are no well-accepted methods to differentiate persons who are more likely to benefit. Numerous potential peripheral biomarkers have been reported, but their current utility is unclear. We conducted a systematic review/meta-analysis examining the association between baseline levels and longitudinal changes in blood-based biomarkers, and response to ketamine/esketamine. Of the 5611 citations identified, 56 manuscripts were included (N = 2801 participants), and 26 were compatible with meta-analytical calculations. Random-effect models were used, and effect sizes were reported as standardized mean differences (SMD). Our assessments revealed that more than 460 individual biomarkers were examined. Frequently studied groups included neurotrophic factors (n = 15), levels of ketamine and ketamine metabolites (n = 13), and inflammatory markers (n = 12). There were no consistent associations between baseline levels of blood-based biomarkers, and response to ketamine. However, in a longitudinal analysis, ketamine responders had statistically significant increases in brain-derived neurotrophicfactor (BDNF) when compared to pre-treatment levels (SMD [95% CI] = 0.26 [0.03, 0.48], p = 0.02), whereas non-responders showed no significant changes in BDNF levels (SMD [95% CI] = 0.05 [-0.19, 0.28], p = 0.70). There was no consistent evidence to support any additional longitudinal biomarkers. Findings were inconclusive for esketamine due to the small number of studies (n = 2). Despite a diverse and substantial literature, there is limited evidence that blood-based biomarkers are associated with response to ketamine, and no current evidence of clinical utility.

(2R,6R)-hydroxynorketamine rapidly potentiates optically-evoked Schaffer collateral synaptic activity

(2R,6R)-hydroxynorketamine (HNK) is a metabolite of ketamine that exerts rapid and sustained antidepressant-like effects in preclinical studies. We hypothesize that the rapid antidepressant actions of (2R,6R)-HNK involve an acute increase in glutamate release at Schaffer collateral synapses. Here, we used an optogenetic approach to assess whether (2R,6R)-HNK promotes glutamate release at CA1-projecting Schaffer collateral terminals in response to select optical excitation of CA3 afferents. The red-shifted channelrhodopsin, ChrimsonR, was expressed in dorsal CA3 neurons of adult male Sprague Dawley rats. Transverse slices were collected four weeks later to determine ChrimsonR expression and to assess the acute synaptic effects of an antidepressant-relevant concentration of (2R,6R)-HNK (10 μM). (2R,6R)-HNK led to a rapid potentiation of CA1 field excitatory postsynaptic potentials evoked by recurrent optical stimulation of ChrimsonR-expressing CA3 afferents. This potentiation is mediated in part by an increase in glutamate release probability, as (2R,6R)-HNK suppressed paired-pulse facilitation at CA3 projections, an effect that correlated with the magnitude of the (2R,6R)-HNK-induced potentiation of CA1 activity. These results demonstrate that (2R,6R)-HNK increases the probability of glutamate release at CA1-projecting Schaffer collateral afferents, which may be involved in the antidepressant-relevant behavioral adaptations conferred by (2R,6R)-HNK in vivo. The current study also establishes proof-of-principle that genetically-encoded light-sensitive proteins can be used to investigate the synaptic plasticity induced by novel antidepressant compounds in neuronal subcircuits.This article is part of the Special Issue on ‘Ketamine and its Metabolites’.

Comparative metabolomic analysis in plasma and cerebrospinal fluid of humans and in plasma and brain of mice following antidepressant-dose ketamine administration

Subanesthetic-dose racemic (R,S)-ketamine (ketamine) produces rapid, robust, and sustained antidepressant effects in major depressive disorder (MDD) and bipolar disorder (BD) and has also been shown to effectively treat neuropathic pain, complex regional pain syndrome, and post-traumatic stress disorder (PTSD). However, to date, its mechanism of action remains unclear. Preclinical studies found that (2 R,6 R;2 S,6 S)-hydroxynorketamine (HNK), a major circulating metabolite of ketamine, elicits antidepressant effects similar to those of ketamine. To help determine how (2 R,6 R)-HNK contributes to ketamine’s mechanism of action, an exploratory, targeted, metabolomic analysis was carried out on plasma and CSF of nine healthy volunteers receiving a 40-minute ketamine infusion (0.5 mg/kg). A parallel targeted metabolomic analysis in plasma, hippocampus, and hypothalamus was carried out in mice receiving either 10 mg/kg of ketamine, 10 mg/kg of (2 R,6 R)-HNK, or saline. Ketamine and (2 R,6 R)-HNK both affected multiple pathways associated with inflammatory conditions. In addition, several changes were unique to either the healthy human volunteers and/or the mouse arm of the study, indicating that different pathways may be differentially involved in ketamine’s effects in mice and humans. Mechanisms of action found to consistently underlie the effects of ketamine and/or (2 R,6 R)-HNK across both the human metabolome in plasma and CSF and the mouse arm of the study included LAT1, IDO1, NAD+, the nitric oxide (NO) signaling pathway, and sphingolipid rheostat.

Analgesic and Antinociceptive Effects of (2R,6R)‐hydroxynorketamine (HNK) in Mice

Chronic pain can be challenging to treat and increases the risk of developing psychological disorders such as depression. Identifying novel treatment modalities that effectively alleviate pain is essential to improve clinical treatment and rehabilitation for patients with pain conditions. Ketamine is an effective analgesic for many types of pain. However, its widespread use is limited by its side effect profile and requirement for intranasal/intravenous administration under medical supervision. (2R,6R)‐hydroxynorketamine (HNK) is a ketamine metabolite that lacks the psychotomimetic effects of its parent drug but retains ketamine’s anti‐stress effects. Therefore, it is natural to question whether (2R,6R)‐HNK may also possess analgesic activity.Administration of (2R,6R)‐HNK produced antinociception in healthy mice exposed to a noxious, painful stimulus 24 hours after injection. The dose response for the delayed‐yet‐persistent antinociception revealed an inverted U shape with significant antinociception at doses of 10‐18 mg/kg for both sexes and 30 mg/kg in female mice. Mice pretreated with different receptor antagonists to examine the potential mechanism for (2R,6R)‐HNK mediated antinociception revealed a mechanism dependent on AMPA receptors and not opioid receptors. In contrast, ketamine antinociception was not dependent on AMPA receptors and partially dependent on opioid receptors. These results demonstrate that both (2R,6R)‐HNK and the parent drug ketamine produce antinociception but work via different neural mechanisms.In separate studies, (2R,6R)‐HNK administration reversed mechanical allodynia associated with localized inflammatory pain induced in mice by injecting λ‐carrageenan into the hind paw. The onset for this analgesia‐like activity was less than 1 hour with a duration greater than 24 hours following a single administration. (2R,6R)‐HNK was effective at reversing mechanical allodynia at doses of 10 & 30 mg/kg in both male and female animals. These results demonstrate (2R,6R)‐HNK exhibits great promise for treating inflammatory pain in addition to other pain types. Overall, these data suggest that (2R,6R)‐HNK may be a safe alternative therapy for pain that could be made widely available to patients and support the need for continued investigation and development of (2R,6R)‐HNK as a novel non‐opioid pain treatment.

P390. Synaptic Mechanisms by Which (2R,6R)-Hydroxynorketamine Promotes Hippocampal-Dependent Plasticity and Behavior

Ketamine can rapidly alleviate symptoms of treatment-resistant major depression within hours of a single administration. While this finding offers a new and promising trajectory for novel therapeutic development, identifying rapid antidepressant mechanisms of action have proven especially difficult. The goal of the current study was to investigate the cellular actions of (2R,6R)-hydroxynorketamine (HNK) – a metabolite of ketamine that has been shown preclinically to exert rapid antidepressant-like effects without dissociative-like properties and abuse potential.

Norketamine, the Main Metabolite of Ketamine, Induces Mitochondria-Dependent and ER Stress-Triggered Apoptotic Death in Urothelial Cells via a Ca2+-Regulated ERK1/2-Activating Pathway.

Ketamine-associated cystitis is characterized by suburothelial inflammation and urothelial cell death. Norketamine (NK), the main metabolite of ketamine, is abundant in urine following ketamine exposure. NK has been speculated to exert toxic effects in urothelial cells, similarly to ketamine. However, the molecular mechanisms contributing to NK-induced urothelial cytotoxicity are almost unclear. Here, we aimed to investigate the toxic effects of NK and the potential mechanisms underlying NK-induced urothelial cell injury. In this study, NK exposure significantly reduced cell viability and induced apoptosis in human urinary bladder epithelial-derived RT4 cells that NK (0.01–0.5 mM) exhibited greater cytotoxicity than ketamine (0.1–3 mM). Signals of mitochondrial dysfunction, including mitochondrial membrane potential (MMP) loss and cytosolic cytochrome c release, were found to be involved in NK-induced cell apoptosis and death. NK exposure of cells also triggered the expression of endoplasmic reticulum (ER) stress-related proteins including GRP78, CHOP, XBP-1, ATF-4 and -6, caspase-12, PERK, eIF-2α, and IRE-1. Pretreatment with 4-phenylbutyric acid (an ER stress inhibitor) markedly prevented the expression of ER stress-related proteins and apoptotic events in NK-exposed cells. Additionally, NK exposure significantly activated JNK, ERK1/2, and p38 signaling and increased intracellular calcium concentrations ([Ca2+]i). Pretreatment of cells with both PD98059 (an ERK1/2 inhibitor) and BAPTA/AM (a cell-permeable Ca2+ chelator), but not SP600125 (a JNK inhibitor) and SB203580 (a p38 inhibitor), effectively suppressed NK-induced mitochondrial dysfunction, ER stress-related signals, and apoptotic events. The elevation of [Ca2+]i in NK-exposed cells could be obviously inhibited by BAPTA/AM, but not PD98059. Taken together, these findings suggest that NK exposure exerts urothelial cytotoxicity via a [Ca2+]i-regulated ERK1/2 activation, which is involved in downstream mediation of the mitochondria-dependent and ER stress-triggered apoptotic pathway, consequently resulting in urothelial cell death. Our findings suggest that regulating [Ca2+]i/ERK signaling pathways may be a promising strategy for treatment of NK-induced urothelial cystitis.