NMDAR inhibition-independent antidepressant actions of ketamine metabolites

Abstract

Major depressive disorder affects around 16 per cent of the world population at some point in their lives. Despite the availability of numerous monoaminergic-based antidepressants, most patients require several weeks, if not months, to respond to these treatments, and many patients never attain sustained remission of their symptoms. The non-competitive, glutamatergic NMDAR (N-methyl-d-aspartate receptor) antagonist (R,S)-ketamine exerts rapid and sustained antidepressant effects after a single dose in patients with depression, but its use is associated with undesirable side effects. Here we show that the metabolism of (R,S)-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-related actions in mice. These antidepressant actions are independent of NMDAR inhibition but involve early and sustained activation of AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors). We also establish that (2R,6R)-HNK lacks ketamine-related side effects. Our data implicate a novel mechanism underlying the antidepressant properties of (R,S)-ketamine and have relevance for the development of next-generation, rapid-acting antidepressants.

Extended Data Figure 1. Ketamine’s metabolic transformations in vivo

Ketamine is metabolised in vivo via P450 enzymatic transformations. (i) (R,S)-Ketamine (KET) is selectively demethylated to give (R,S)-norketamine (norKET). (ii) NorKET can be then dehydrogenated to give (R,S)-dehydronorketamine (DHNK). (iii) Alternatively, norKET can be hydroxylated to give the hydroxynorketamines (HNKs). (iv) (R,S)-KET can also be hydroxylated at the 6- position to give either the E-6-hydroxyketamine ((2S,6R;2R,6S)-HK)) or Z-6-hydroxyketamine ((2S,6S;2R,6R)-HK)). (v) Demethylation of (2S,6R;2R,6S)-HK yields the production of (2S,6R;2R,6S)-hydroxynorketamine (HNK). (vi) Demethylation of (2S,6S;2R,6R)-HK further gives (2S,6S;2R,6R)-hydroxynorketamine (HNK).

Extended Data Figure 2. Circulating levels of ketamine and its metabolites following i.p. administration in mice

a, Plasma and b, brain levels of ketamine (KET) and its metabolites following administration of (R,S)-KET (10 mg/kg) in mice. (c-d) Brain levels of c, KET, d, norketamine (norKET) and e, hydroxynorketamine (HNK) following administration of (S)- and (R)-KET. f, Chemical structure of (R,S)-6,6-dideuteroketamine ((R,S)-d₂-KET), which g, displaces [³H]-MK-801 binding with a similar affinity to (R,S)-KET ((R,S)-KET: Ki=799nM (R,S)-d₂-KET: Ki=883nM) (statistical analyses and n numbers see Supplementary Information Table 1).

Extended Data Figure 3. Additional social defeat stress data

a, Chronic social defeat stress and social interaction/avoidance test timeline. (b-c), Administration of (R,S)-ketamine (KET) or MK-801did not affect b, locomotor activity or c, total number of compartmental crosses in the social interaction apparatus. Data are means ± S.E.M. ***p<0.001. SAL, saline (statistical analyses and n numbers see Supplementary Information Table 1).

Extended Data Figure 4. Locomotor effects of (R,S)-ketamine and (R,S)-6,6-dideuteroketamine

After recording baseline activity for 60 min, mice received drug (marked by a vertical dashed line) and locomotor activity was monitored for another 1 hour. (a,b), Administration of (R,S)-KET (10 mg/kg), induced hyper-locomotor responses equally in both male and female mice. (c,d), (R,S)-ketamine (KET) and (R,S)-6,6-dideuteroketamine ((R,S)-d₂-KET) were equally potent in inducing a hyper-locomotor response at the dose of 10 mg/kg. Data are means ± S.E.M. *p<0.05, **p<0.01. SAL, saline (statistical analyses and n numbers see Supplementary Information Table 1).

Extended Data Figure 5. Acute and sustained antidepressant and anti-anhedonic effects of (2R,6R)- and (2S,6S)-hydroxynorketamine

a, A single injection of (2R,6R)-HNK resulted in dose-dependent antidepressant-like responses in the learned helplessness test at the doses of 5-75 mg/kg. b, A single injection of (2S,6S)-hydroxynorketamine (HNK) induced antidepressant-like effects in the learned helplessness test at the dose of 75 mg/kg. c, Administration of (2R,6R)-HNK induced dose-dependent antidepressant effects in the 1- and 24-hour forced-swim test. d, Administration of (2S,6S)-HNK at the dose of 25 mg/kg induced antidepressant effects in the 1- and 24-hour forced-swim test. e, Despite the greater antidepressant efficacy of (2R,6R)-HNK, administration of (2S,6S)-HNK (HNK) results in higher brain hydroxynorketamine levels compared to (2R,6R)-HNK. f, (2R,6R)-HNK manifested dose-dependent antidepressant-like effects in the novelty-suppressed feeding test. g, Similar to (R,S)-ketamine (KET), the antidepressant-like effects of (2R,6R)-HNK in the forced-swim test persisted for at least 3 days post-treatment. h, A single administration of (2R,6R)-HNK reversed chronic corticosterone-induced decreases in sucrose preference. i, A single administration of (2R,6R)-HNK reversed chronic corticosterone-induced decrease in female urine sniffing preference, specifically in mice that developed an anhedonic phenotype. Administration of (2R,6R)-HNK was not associated with changes in j, locomotor activity or k, total compartmental crosses in the social interaction test following chronic social defeat stress. Data are means ± S.E.M. *p<0.05, **p<0.01, ***p<0.001. SAL, saline (statistical analyses and n numbers see Supplementary Information Table 1).

Extended Data Figure 6. (2R,6R)-hydroxynorketamine rapidly increases the frequency and amplitude of AMPA receptor spontaneous excitatory postsynaptic currents in the hippocampus

a, Representative traces of spontaneous excitatory postsynaptic currents (sEPSCs) mediated via AMPA receptors during baseline (before) and 20 min following drug administration (after) in b, example CA1 stratum radiatum interneuron recorded from a rat hippocampal slice. (c-j) Twenty-min exposure of (e,i), (2R,6R)-hydroxynorketamine (HNK), but not (c,g), (R,S)-ketamine (KET) or (d,h), (2S,6S)-HNK increased AMPA sEPSCs frequency and amplitude compared to baseline. Data are means ± S.E.M. *p<0.05, **p<0.01, ***p<0.001 (statistical analyses and n numbers see Supplementary Information Table 1). Abbreviations: FST, forced-swim test; NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione; SAL, saline; SLM, stratum lacunosum-moleculare; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum.

Extended Data Figure 7. Administration of the AMPA receptor antagonist, NBQX, prevents (2R,6R)-HNK-induced increases in gamma oscillations in vivo

a, Administration of (R,S)-ketamine (KET), but not (2R,6R)-hydroxynorketamine (HNK), increased locomotor home-cage activity of mice. Neither (R,S)-KET, nor (2R,6R)-HNK altered cortical b, alpha, c, beta, d, delta or e, theta oscillations in vivo. (f-k) Pre-treatment with the AMPA receptor antagonist, NBQX, did not change the f, locomotor activity, g, alpha, h, beta, j, delta or k, theta oscillations, but it i, prevented (2R,6R)-HNK-induced increases of gamma oscillations in vivo. Data are means ± S.E.M. *p<0.05, **p<0.01 (statistical analyses and n numbers see Supplementary Information Table 1). Abbreviations: NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione; SAL, saline.

Extended Data Figure 8. Effects of (2R,6R)-hydroxynorketamine on synaptoneurosome protein and protein phosphorylation levels

A single administration of (R,S)-ketamine (KET, 10 mg/kg) or (2R,6R)-hydroxynorketamine (HNK, 10 mg/kg) (a,b), did not alter levels of mTOR or phosphorylated mTOR 1- or 24-hours post-injection in the hippocampus of mice. (b-i), Administration of (R,S)-KET or (2R,6R)-HNK did not alter levels of (c,d), mTOR/phosphorylated mTOR, (e,f), eEF2/phosphorylated eEF2, (g,h), proBDNF/mBDNF, or (i,j), GluA1/GluA2 in the prefrontal cortex of mice. The values for the phosphorylated forms of proteins were normalised to phosphorylation-independent levels of the same protein. Phosphorylation-independent levels of proteins were normalised to GAPDH. Data are means ± S.E.M, and was normalised to the saline-treated control group for each protein. Images are cropped; see Supplementary Fig. 1 for complete blot images *p<0.05 (statistical analyses and n numbers see Supplementary Information Table 1). Abbreviations: eEF2, eukaryotic translation elongation factor 2; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; mBDNF, mature brain-derived neurotrophic factor; mTOR, mammalian target of rapamycin; proBDNF, pro-brain-derived neurotrophic factor; SAL, saline.

Extended Data Figure 9. (2R,6R)-hydroxynorketamine administration does not alter startle amplitude, self-administration drug intake or drug discrimination response rate

a, Startle amplitude as measured in the pre-pulse inhibition task was not affected by administration of (R,S)-ketamine (KET) or (2R,6R)-hydroxynorketamine (HNK). (b,c), Response rate of overall lever pressing per sec in the drug discrimination paradigm was not changed by administration of b, (R,S)-KET, (2R,6R)-HNK or c, phencyclidine (PCP). d, Unlike ketamine, (2R,6R)-HNK did not alter drug intake in the self-administration task in mice. Data are means ± S.E.M. *p<0.05 (statistical analyses and n numbers see Supplementary Information Table 1). SAL, saline.

Figure 1. NMDA receptor inhibition does not explain the antidepressant actions of ketamine

Antidepressant-like responses of a, (R,S)-ketamine (KET) and desipramine (DSP) in the forced-swim test 1- and 24-hours post-treatment. (b-d) Compared to (S)-KET, (R)-KET showed greater and longer-lasting antidepressant-like effects in the b, forced-swim test, c, novelty-suppressed feeding and d, learned helplessness paradigms. (e-g), The alternative NMDAR antagonist MK-801 did not elicit e, 24-hour antidepressant actions in the forced-swim test and (f,g) did not reverse social avoidance induced by chronic social defeat stress. Data are means ± S.E.M. *p<0.05, **p<0.01, ***p<0.001 (statistical analyses and n numbers see Supplementary Information Table 1).

Figure 2. Metabolism of ketamine to (2R,6R)-HNK is necessary and sufficient to exert ketamine-related antidepressant actions

a, Simplified diagram of (R,S)-KET’s metabolism. b, Greater antidepressant-like actions of ketamine in female mice compared to males are associated with (c-e), higher brain levels of e, (2S,6S;2R,6R)-hydroxynorketamine (HNK), but not c, KET, or d, nor-KET. (f-h), Brain levels of f, KET, g, nor-KET and h, (2S,6S;2R,6R)-HNK following administration of (R,S)-KET and 6,6-dideuteroketamine ((R,S)-d₂-KET). (i-j), Effects of (R,S)-KET and (R,S)-d₂-KET in the i, 1- and 24-hours forced-swim and the j, learned helplessness tests. (k-l), Compared to (2S,6S)-HNK, (2R,6R)-HNK manifested greater potency and longer-lasting antidepressant-like effects in the k, forced-swim test and l, learned helplessness paradigms. m, (2R,6R)-HNK reversed chronic social defeat-induced social interaction deficits. Data are means ± S.E.M. *p<0.05, **p<0.01, ***p<0.001 (statistical analyses and n numbers see Supplementary Information Table 1).

Figure 3. Activation of AMPA receptors is necessary for the acute antidepressant effects of (2R,6R)-HNK

a, (2R,6R)-hydroxynorketamine (HNK) does not displace [³H]-MK-801 binding. (b-c), (R,S)-ketamine inhibited, but (2S,6S)-HNK and (2R,6R)-HNK did not inhibit b, currents evoked by application of NMDA to stratum radiatum interneurons in rat hippocampal slices, quantified as c, percent inhibition (↓: 30-sec pulse). (d-e), Normalised d, fEPSP slope and e, amplitude from stimulation of the Schaffer collateral pathway in rat hippocampal slices. f, Representative field-potential traces in the same hippocampal slice before (baseline) and 60 min after application of (2R,6R)-HNK. (g-h), Pre-treatment with the AMPA receptor inhibitor NBQX 10 minutes prior to (R,S)-ketamine (KET) and (2R,6R)-HNK prevented their antidepressant-like actions in the g, 1-hour or h, 24-hours forced-swim test. i, Representative EEG spectrograms for 10-min prior (baseline) and 1-hour after administration of (R,S)-ketamine or (2R,6R)-HNK (indicated by a dashed line). j, Normalised gamma power changes following administration of (R,S)-KET, (2R,6R)-HNK, or vehicle. Data are means ± S.E.M. *p<0.05, **p<0.01, ***p<0.001(statistical analyses and n numbers see Supplementary Information Table 1).

Figure 4. Activation of AMPA receptors is required for the sustained antidepressant effects of (2R,6R)-HNK

(a-h) Protein and protein phosphorylation levels from hippocampal synaptoneurosomes. (a,b) A single administration of (R,S)-ketamine (KET) or (2R,6R)-hydroxynorketamine (HNK) decreased phosphorylation of eEF2, a, 1 hour and b, 24 hours post-injection. (c,d), While administration of (2R,6R)-HNK or (R,S)-KET did not alter the levels of proBDNF or mBDNF c, 1 hour post-injection, it increased mBDNF levels. d, 24 hours post-treatment. (e,f), (R,S)-KET and (2R,6R)-HNK did not change levels of GluA1 and GluA2 at e, 1-hour but did f, increase at 24-hours post-treatment. (g,h), Administration of the AMPA receptor inhibitor NBQX, 30 min prior to the 24-hour forced-swim test prevented the antidepressant effects of both (R,S)-KET and (2R,6R)-HNK administered 23.5 hours prior to NBQX. Data are means ± S.E.M. Images cropped; see Supplementary Fig. 1 for complete blot images. *p<0.05, **, p<0.01, ***p<0.001 (statistical analyses and n numbers see Supplementary Information Table 1). Abbreviations: : eEF2, eukaryotic translation elongation factor 2; FST, forced-swim test; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; mBDNF, mature brain-derived neurotrophic factor; NBQX, 2,3-dihydroxy-6-nitro-7-sulfamoyl-benzo[f]quinoxaline-2,3-dione; proBDNF, pro-brain-derived neurotrophic factor; SAL, saline.

Figure 5. (2R,6R)-HNK lacks ketamine-related side effects

(a,b) After recording baseline activity for 1 hour, mice received drug (dashed line) and locomotor activity was monitored for 1 hour. a, Administration of (2S,6S)-hydroxynorketamine (HNK) dose-dependently changed locomotor activity, while administration of b, (2R,6R)-HNK did not. c, (2S,6S)-HNK, but not d, (2R,6R)-HNK, induced motor in-coordination in the rotarod. Unlike (R,S)-KET, (2R,6R)-HNK administration did not induce e, pre-pulse inhibition deficits, (f,g), (R,S)-KET-associated discriminative stimulus, or h, self-administration. Data are means ± S.E.M. *p<0.05, **, p<0.01, ***p<0.001, KET vs saline (SAL); for panel c, * (R,S)-KET, ^# (2S,6S)-HNK (statistical analyses and n numbers see Supplementary Information Table 1).