NMDAR inhibition-independent antidepressant actions of ketamine metabolites

Ketamine can increase BDNF level in the prefrontal cortex, especially in the hippocampus, to exert antidepressant-like effects.

“Ketamine can increase the level of BDNF in the prefrontal cortex, especially in the hippocampus, to exert antidepressant-like effects. 377”

BDNF signaling is an important target of antidepressants.

“an increasing number of studies have shown that BDNF signaling is an important target of antidepressants. 377”

(2R,6R)-HNK has binding affinity Ki and IC50 greater than 100 μM with 24% inhibition at 100 μM, and fails to impact NMDA-evoked currents in CA1 interneurons.

“(2R,6R)-HNK has a binding affinity (Ki) and IC50 greater than 100 μM with 24% inhibition at 100 μM, and fails to impact NMDA-evoked currents in CA1 interneurons (Moaddel et al., 2013; Morris et al., 2017; Zanos et al., 2016).”

The rapidity and magnitude of ketamine's antidepressant effects have been attributed to its ability to rapidly reverse neuroplasticity deficits in animal models.

“The rapidity and magnitude of ketamine's effects have been attributed to its ability to rapidly reverse neuroplasticity deficits in animal models 3,4,10,34 .”

R,R-HNK binds to TRKB, but not to NMDARs, at concentrations that promote neural plasticity, suggesting TRKB may be the hitherto unknown signaling receptor for R,R-HNK.

“Importantly, R,R-HNK binds to TRKB, but not to NMDARs, at concentrations that promote neural plasticity, suggesting that TRKB may be the hitherto unknown signaling receptor for R,R-HNK (Zanos et al., 2016).”

R,R-HNK does not bind to NMDARs at doses that produce antidepressant-like responses in mice.

“and R,R-HNK does not bind to NMDARs at the doses that produce antidepressant-like responses in mice (Zanos et al., 2016).”

R,R-HNK produces antidepressant-like and plasticity-promoting effects without psychotomimetic effects in rodents.

“produces antidepressant-like and plasticity-promoting effects without psychotomimetic effects in rodents (Zanos et al., 2016;Yao et al., 2018)”

In-depth analysis found that ketamine's active metabolite HNK does not block the NMDA receptor, instead displays sustained AMPA receptor activation and lacks ketamine-related side effects.

“However, in-depth analysis of the antidepressant effects of ketamine found that its active metabolite (2R,6R)-hydroxynorketamine (HNK) does not block the NMDA receptor. Instead, HNK displays sustained activation of α-amino-3hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) receptors and lacks ketamine-related side effects81.”

Hydroxynorketamine, a metabolite of ketamine, can exert anti-depressive effects by an NMDAR-independent mechanism.

“Additionally, hydroxynorketamine (HNK), a metabolite of ketamine, can exert its anti-depressive effects by an NMDAR-independent mechanism. 377”

(2R,6R)-HNK does not displace MK-801 binding to NMDARs at 10 μM.

“its inability to displace MK-801 binding to NMDARs (10 μM).”

HNKs, which have antidepressant-like effects in preclinical models, may not require NMDARs for their long-lasting effects on behavior or synaptic plasticity in hippocampus.

“HNKs, which also have antidepressant-like effects in preclinical models, may not require NMDARs for their long-lasting effects on behavior or synaptic plasticity in the hippocampus (Gould et al., 2017; Riggs et al., 2020; Zanos et al., 2016).”

(2R,6R)-HNK has efficacy in rodent models of depression while lacking psychotomimetic side effects and abuse potential.

“The ketamine metabolite (2R,6R)-HNK has received significant attention due to its efficacy in rodent models of depression, while lacking psychotomimetic side effects and abuse potential (Zanos et al., 2016).”

Production of 6-HNKs is important for the full antidepressant-like behavioral effects of ketamine.

“Production of the 6-HNKs is important for the full antidepressant-like behavioral effects of ketamine (Zanos, Highland, Liu, et al., 2019; Zanos et al., 2016; Zheng et al., 2021).”

(2R,6R)-HNK produces antidepressant-like effects and enhancement in AMPAR signaling independent of NMDAR antagonism, suggesting NMDARs do not fully explain ketamine's mechanism of action.

“It appears that (2R,6R)-HNK produces antidepressant-like effects and an enhancement in AMPAR signaling independent of NMDAR antagonism which suggests that NMDARs do not fully explain the mechanism of action of ketamine (Riggs et al., 2020; Zanos et al., 2016).”

Unlike ketamine, (2R,6R)-HNK does not appear to drive self-administration or hyperlocomotion.

“there is evidence that (2R,6R)-HNK does not elicit the same desire to self-administer or hyperlocomotion effects as ketamine”

Preclinical research initially suggested that conversion to (2S,6S;2R,6R)-HNK was necessary for ketamine's antidepressant-like effects and that (2R,6R)-HNK could have its own antidepressant effects in mice.

“Preclinical research first suggested that metabolism to (2S,6S;2R,6R)-HNK was essential for ketamine's antidepressantlike effects and that (2R,6R)-HNK could exert its own antidepressant effects in mice [45,46].”

A metabolite of R-ketamine lacking dissociative properties has demonstrated robust antidepressant-like effects in rodents.

“a metabolite of R-ketamine lacking dissociative properties has also demonstrated robust antidepressant-like effects in rodents”

2R,6R-HNK was reported to have antidepressant-like effects and no side effects in rodents.

“2R,6R-HNK was reported to have antidepressant-like effects and no side effects in rodents.44”

Ketamine increased expression of AMPA receptor GluA1 and GluA2 subunits in the mouse hippocampus.

“Furthermore, ketamine increased expression of AMPA receptor GluA1 and GluA2 subunits in the mouse hippocampus.44,101”

In mice, ketamine administration was not noted to affect mTOR phosphorylation in hippocampal or cortical tissue and rapamycin did not block ketamine-induced antidepressant effects.

“In mice, ketamine administration was not noted to affect mTOR phosphorylation in hippocampal or cortical tissue44,96 and rapamycin did not block ketamine-induced antidepressant effects.96”

Ketamine may act through neuroactive metabolites, particularly hydroxynorketamines, rather than through NMDAR blockade.

“Lastly, there is an exciting recent hypothesis that ketamine may exert its antidepressant actions not by blocking NMDARs, but rather by its neuroactive metabolites, particularly the hydroxynorketamines (HNKs).”

Deuterating ketamine to reduce its hydrolysis eliminates its behavioral actions in mice, suggesting NMDAR inhibition is not sufficient for antidepressant effects.

“Zanos et al. [153] reported that the behavioral actions of ketamine in mice could be eliminated by deuterating the molecule to greatly reduce its rate of hydrolysis, suggesting that ketamine's inhibition of NMDARs is not sufficient for its antidepressant actions.”

Acute hydroxynorketamine application to hippocampal slices potentiates SC-CA1 synaptic transmission via increased presynaptic glutamate release probability.

“Acute application of HNKs to hippocampal brain slices also produces a direct potentiation of SC-CA1 synaptic transmission [153], mediated by an increase in the probability of presynaptic glutamate release [161].”

Direct hydroxynorketamine administration mimics ketamine's behavioral actions in mice, promotes EEG gamma oscillations, increases AMPAR and BDNF expression, and decreases hippocampal eEF2 phosphorylation.

“Direct administration of HNKs mimicked the behavioral actions of ketamine in mice, promoted EEG gamma oscillations, promoted expression of AMPARs and BDNF, and decreased phosphorylation of eEF2 levels in the hippocampus, providing plenty of mechanisms that could be triggered independently from NMDAR inhibition.”

Ketamine and GABA A receptor negative allosteric modulators promote gamma oscillations in the prefrontal cortex at doses that reverse chronic stress behavioral consequences.

“Indeed, ketamine and GABA A receptor negative allosteric modulators share an ability to promote gamma oscillations in the PFC at doses that reverse the behavioral consequences of chronic stress [152,153].”

AZD6765, mGluR2/3 antagonists, alpha5 subunit-selective GABA A receptor negative allosteric modulators, and the ketamine metabolite (2R,6R)-HNK all increase EEG power in the gamma frequency band.

“such as AZD6765 [149], mGluR 2/3 antagonists [150], alpha5 subunit-selective GABA A R-NAMs [151, 152], and the ketamine metabolite (2 R,6 R)-HNK [153], all of which enhance EEG power in the gamma frequency band.”

Recent evidence suggests that ketamine metabolite norketamine is responsible for antidepressant effects through AMPA receptor activation.

“In the case of ketamine, recent evidence suggests that one of its metabolites, norketamime (HNK) is actually responsible for the antidepressant effects of this drug, through activation of glutamate AMPA receptor (GluA-R) (Zanos et al 2016).”

The stereoselective engagement of protein targets is a feature of numerous chemical probes and drugs.

“The stereoselective engagement of protein targets is a feature of numerous chemical probes and drugs15–19”

Ketamine has antidepressant effects in many antidepressant-relevant tests in experimental animals.

“Moreover, antidepressant effects of ketamine have been demonstrated in many antidepressant-relevant tests in experimental animals (e.g. 9, 10–14); also see 15.”

Ketamine, similar to negative allosteric modulators of alpha5-containing GABAA receptors, enhances gamma band EEG power, which is hypothesized to be directly related to cortical disinhibition.

“Notably, ketamine 13, similar to negative allosteric modulators of alpha5-containing GABAA receptors 47, enhance gamma band electroencephalography power, which is hypothesized to be directly related to cortical disinhibition 49–52”

Following ketamine administration, (2S,6S;2R,6R)-HNK is the major HNK metabolite found in plasma and brain of mice, as well as plasma of humans.

“Following ketamine administration, (2S,6S;2R,6R)-HNK is the major HNK metabolite found in the plasma and brain of mice 13, as well as the plasma of humans 120.”

Animal studies show MK-801 does not exert sustained antidepressant actions, though it does have acute actions in some studies.

“Similar to these human data, animal studies show that the NMDAR channel-blocking antagonist MK-801 does not exert sustained antidepressant actions, though it does have acute actions in some studies 9, 10, 13, 103.”

Preclinical rodent studies have indicated rapid-acting antidepressant behavioral actions of (S)-ketamine in mice.

“Nevertheless, we note that pre-clinical rodent studies have also indicated rapid-acting antidepressant behavioral actions of (S)-ketamine in mice 13, 116–118.”

The superiority of (R)-ketamine is not related to a U-shaped dose response, as it is superior to (S)-ketamine with up to a 30-fold range of doses in multiple mouse tests.

“The superiority of (R)-ketamine does not seem to be related to a U-shaped dose response of the drugs, as it has been shown superior to (S)-ketamine with up to a 30-fold range of doses in multiple mouse tests of antidepressant efficacy 13, 118.”

Administration of equal antidepressant-relevant doses of (R)- and (S)-ketamine in mice did not yield different brain levels, indicating superiority not due to greater brain exposure.

“Importantly, administration of equal, antidepressant-relevant, doses of (R)- and (S)-ketamine in mice did not yield different levels of these enantiomers in the brain of mice 13, indicating that the antidepressant superiority of (R)-ketamine in rodent models is not due to greater brain exposure.”

It is unlikely that ketamine exerts its full antidepressant actions solely via inhibition of the NMDAR, at least in rodents.

“These data indicate that it is unlikely that ketamine exerts its full antidepressant actions solely via inhibition of the NMDAR, at least in rodents.”

(S)-ketamine has approximately 4-fold greater affinity/potency at inhibiting NMDAR compared to (R)-ketamine.

“(S)-ketamine has ~4-fold greater affinity/potency at inhibiting the NMDAR compared to its (R)-ketamine enantiomer 13, 111–115.”

(S)-ketamine's antidepressant behavioral actions require higher doses compared to (R)-ketamine.

“These findings were subsequently replicated by Zanos et al. (2016), who showed that (S)-ketamine’s antidepressant behavioral actions require higher doses compared to those of (R)-ketamine 13.”

In mice, the greater antidepressant behavioral effect in females was associated with higher brain levels of (2S,6S;2R,6R)-HNK, but not ketamine or norketamine levels.

“In mice, this behavioral effect was associated with higher brain levels of (2S,6S;2R,6R)-HNK, but not ketamine or norketamine levels 13”

AMPAR containing GluA1 and/or GluA2 subunit upregulation was observed in mPFC and hippocampal synaptoneurosome fractions at 24 hours post-injection, indicating rapid-triggered and sustained recruitment of AMPAR in synapse, consistent with synaptic strengthening.

“AMPAR containing GluA1 and/or GluA2 subunit upregulation was also observed in mPFC 75 and hippocampal 13 synaptoneurosome fractions at 24 hours post-injection, indicating a rapid-triggered and sustained recruitment of AMPAR in the synapse, consistent with synaptic strengthening.”

Greater antidepressant behavioral responses of a single administration of ketamine have been observed in female compared to male rats and mice.

“In addition, greater antidepressant behavioral responses of a single administration of ketamine have been observed in female compared to male rats and mice 13, 124, 125.”

A dose of 20 mg/kg (2R,6R)-HNK was capable of reversing anhedonia following chronic social defeat stress in mice.

“Indeed, a dose of 20 mg/kg was capable of reversing anhedonia following chronic social defeat stress in mice 13.”

At relevant concentrations (10 mg/kg or brain Cmax = ~10 μmol/kg in mice), (2R,6R)-HNK does not appear to inhibit the NMDAR.

“Important for the mechanism of action of (2R,6R)-HNK as an antidepressant (and thus ketamine’s action) is the fact that at relevant concentrations (i.e., 10 mg/kg or brain Cmax = ~10 μmol/kg in mice), (2R,6R)-HNK does not appear to inhibit the NMDAR.”

(2R,6R)-HNK exerts more potent and longer-lasting antidepressant actions compared with (2S,6S)-HNK.

“In accordance with the findings that (R)-ketamine is a more potent antidepressant compared to the (S)-ketamine 13, 116, 117, 127, the (2R,6R)-HNK metabolite, which is solely produced via the metabolism of (R)-ketamine, exerts more potent and longer-lasting antidepressant actions compared with the (2S,6S)-HNK enantiomer, which is produced via the metabolism of (S)-ketamine.”

At 10 μM, (2R,6R)-HNK does not functionally inhibit NMDARs localized at stratum radiatum interneurons in hippocampal slices, compared to ~50% inhibition by ketamine at this concentration.

“In addition, at 10 μM concentration, (2R,6R)-HNK does not functionally inhibit the NMDARs localized at stratum radiatum interneurons in hippocampal slices, compared to ~50% inhibition by ketamine at this concentration 13.”

Quantitative EEG measurements in humans, rats, and mice revealed ketamine-induced increases in gamma-band power, which indicates activation of fast ionotropic excitatory receptors, including AMPARs.

“Quantitative EEG measurements in humans 101, as well as rats e.g. 135 and mice e.g. 13 revealed ketamine-induced increases in gamma-band power, which, in addition to a putative measure of cortical disinhibition, also indicates activation of fast ionotropic excitatory receptors, including AMPARs 136–138.”

Metabolism of ketamine to (2S,6S;2R,6R)-HNK is necessary for its antidepressant action in rodent tests.

“There is evidence that metabolism of ketamine to (2S,6S;2R,6R)-HNK is necessary for its antidepressant action in rodent tests 13.”

(2R,6R)-HNK did not induce NMDAR inhibition-mediated side effects in mice in open-field, rota-rod, and pre-pulse inhibition tests at doses up to 125, 125, and 375 mg/kg respectively, in contrast to antidepressant dose of 10 mg/kg.

“However, (2R,6R)-HNK did not induce any NMDAR inhibition-mediated side effects in mice in the open-field test (locomotor activity; doses up to 125 mg/kg), the rota-rod test (motor incoordination; doses up to 125 mg/kg) and the pre-pulse inhibition test (sensory dissociation; doses up to 375 mg/kg) 13, 130, in contrast to the antidepressant dose of 10 mg/kg.”

Pre-treatment with AMPAR antagonist NBQX prevents antidepressant-like actions of ketamine in forced-swim test, tail-suspension test, novelty-suppressed feeding test, learned helplessness test, and stress-induced sucrose preference deficits.

“Indeed, pre-treatment with the AMPAR antagonist NBQX prevents the antidepressant-like actions of ketamine in the forced-swim test 9, 13, 117, 139, 140, tail-suspension test 117, 141, novelty-suppressed feeding test 142, learned helplessness test 141 and stress-induced sucrose preference deficits 117, 143.”

Deuteration at C6 did not change ketamine's binding affinity for NMDAR but dramatically decreased in vivo metabolism to HNK; this manipulation prevented ketamine's antidepressant actions in mice.

“This was shown by chemically altering ketamine via deuteration at the C6 position, which did not change its binding affinity for the NMDAR, but dramatically decreased its in vivo metabolism to (2S,6S;2R,6R)-HNK. This manipulation prevented ketamine’s antidepressant actions in mice 13”

(2R,6R)-HNK treatment in mice induces an acute and transient increase in high frequency gamma power, similar to ketamine.

“Similar to ketamine, (2R,6R)-HNK treatment in mice induces an acute and transient increase in high frequency gamma power 13.”

The effect of (2R,6R)-HNK on AMPAR-mediated excitatory post-synaptic potentials appears independent of any possible NMDAR inhibition by (2R,6R)-HNK, since the NMDAR antagonist AP-5 was present in the vehicle wash solution.

“This effect appears independent of any possible NMDAR inhibition by (2R,6R)-HNK, since the NMDAR antagonist AP-5 was present in the vehicle wash solution 13.”

Acute AMPAR activation is required for the gamma power increase and the rapid and sustained antidepressant actions of (2R,6R)-HNK in mice.

“Importantly, administration of the AMPAR antagonist NBQX prior to (2R,6R)-HNK abolished the gamma power oscillation increases, as well as the acute and sustained antidepressant effects of this metabolite in mice, indicating that acute AMPAR activation is required for gamma power increase and its rapid and sustained antidepressant actions 13.”

Administration of the AMPAR antagonist NBQX prior to (2R,6R)-HNK abolishes the gamma power oscillation increases, as well as the acute and sustained antidepressant effects of this metabolite in mice.

“Importantly, administration of the AMPAR antagonist NBQX prior to (2R,6R)-HNK abolished the gamma power oscillation increases, as well as the acute and sustained antidepressant effects of this metabolite in mice, indicating that acute AMPAR activation is required for gamma power increase and its rapid and sustained antidepressant actions 13.”

(2R,6R)-HNK induces an increase in AMPAR-mediated excitatory post-synaptic potentials recorded from the CA1 region of hippocampal slices following stimulation of Schaffer collateral axons.

“In line with an AMPAR activation-dependent mechanism of ketamine’s antidepressant action, (2R,6R)-HNK induces an increase in AMPAR-mediated excitatory post-synaptic potentials recorded from the CA1 region of hippocampal slices following stimulation of Schaffer collateral axons, suggesting an enhancement of excitatory synaptic transmission 13 (see Figure 1).”

Blockade of the AMPAR with NBQX 23.5 hours after (2R,6R)-HNK administration abolishes its antidepressant actions at 24 hours post-injection.

“Consistent with a mechanistic model where the sustained activity of synaptic AMPARs is required for the long-lasting antidepressant actions of (2R,6R)-HNK, it was shown that similar to ketamine 140 blockade of the AMPAR (with NBQX) 23.5 hours after (2R,6R)-HNK administration abolished its antidepressant actions at 24 hours post-injection 13.”

(2R,6R)-HNK, at 10 μM, a concentration which did not alter NMDAR EPSCs, increases the frequency of AMPAR-mediated excitatory post-synaptic currents recorded from CA1 stratum radiatum interneurons, which receives glutamatergic inputs from the Schaffer collaterals.

“In support of this, (2R,6R)-HNK, at the same concentration which did not alter NMDAR EPSCs (i.e., 10 μM), increases the frequency of AMPAR-mediated excitatory post-synaptic currents recorded from CA1 stratum radiatum interneurons, which receives glutamatergic inputs from the Schaffer collaterals 13.”

MK-801 failed to induce long-lasting antidepressant actions in several animal tests.

“However, MK-801 failed to induce long-lasting antidepressant actions in several animal tests 9, 10, 13, 103.”

Ketamine and (2R,6R)-HNK administration increases synaptoneurosomal BDNF protein levels 24 hours post-injection in the hippocampus of mice.

“In addition, ketamine and (2R,6R)-HNK administration increases synaptoneurosomal BDNF protein levels 24 hours post-injection in the hippocampus of mice 13.”

Protein synthesis through the eEF2 kinase/BDNF translation pathway might be involved in the antidepressant actions of (2R,6R)-HNK.

“(2R,6R)-HNK administration also induced a decrease in hippocampal eEF2 phosphorylation 1 and 24 hours post-treatment, concomitant with increased BDNF levels at 24 hours 13, suggesting that protein synthesis through the eEF2 kinase/BDNF translation pathway might be involved in the antidepressant actions of this metabolite.”

(2R,6R)-HNK administration induces a decrease in hippocampal eEF2 phosphorylation 1 and 24 hours post-treatment, concomitant with increased BDNF levels at 24 hours.

“(2R,6R)-HNK administration also induced a decrease in hippocampal eEF2 phosphorylation 1 and 24 hours post-treatment, concomitant with increased BDNF levels at 24 hours 13, suggesting that protein synthesis through the eEF2 kinase/BDNF translation pathway might be involved in the antidepressant actions of this metabolite.”

Ketamine exerts NMDAR inhibition-independent antidepressant actions, and these effects require the metabolism of ketamine to the (2S,6S;2R,6R)-HNK metabolite.

“Indeed, it was shown that ketamine exerts NMDAR inhibition-independent antidepressant actions, and that these effects require the metabolism of ketamine to the (2S,6S;2R,6R)-HNK metabolite 13.”

The (2R,6R)-HNK metabolite is sufficient to induce antidepressant actions, similar to those observed following ketamine administration, in animal tests.

“Moreover, the (2R,6R)-HNK metabolite is sufficient to induce antidepressant actions, similar to those observed following ketamine administration, in animal tests.”