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.
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We thank A. Keller for his assistance with the qEEG experiments, M. K. Lobo for assistance with the social defeat experiments, B. Alkondon for the rat hippocampal slice preparation and biocytin processing, V. Meadows and S. Krimmel for assistance with behavioural experiments and manuscript review, E. Pereira for critical comments on the manuscript and figures, D. Luckenbaugh for assistance with statistical analysis, and C. Moore for the small molecule X-ray crystallography. Research was supported by NIMH grants MH099345 and MH107615 to T.D.G. and MH086828 to S.M.T., and the NIA (R.M., I.W.W.), NIMH (C.A.Z.), and NCATS (C.J.T.) NIH intramural research programs. Receptor binding profiles and Ki determinations were supported by the NIMH Psychoactive Drug Screening Program, Contract HHSN-271-2008-025C, to B. L. Roth in conjunction with J. Driscoll. Initial synthesis of the ketamine metabolites used in this study was supported by NIA Contract HHSN271201000008I to I.W.W.
The authors declare competing financial interests: I.W.W., R.M., and C.A.Z. are listed as co-inventors on a patent for the use of (2R,6R)-hydroxynorketamine, (S)-dehydronorketamine and other stereoisomeric dehydro and hydroxylated metabolites of (R,S)-ketamine metabolites in the treatment of depression and neuropathic pain. They have assigned their rights in the patent to the U.S. government but will share a percentage of any royalties that may be received by the government. I.W.W., C.A.Z., R.M., T.G., P.Z., C.T., and P.M. are listed as co-inventors on a patent application for the use of (2R, 6R)-hydroxynorketamine and (2S, 6S)-hydroxynorketamine in the treatment of depression, anxiety, anhedonia, suicidal ideation and post-traumatic stress disorders. I.W.W., C.A.Z., R.M., C.T., and P.M. have assigned their rights in this patent to the U.S. government but will share a percentage of any royalties that may be received by the government. T.G. and P.Z. have assigned their rights in this patent to the University of Maryland but will share a percentage of any royalties that may be received by the University of Maryland.
Extended data figures and tables
Ketamine is metabolised in vivo via P450 enzymatic transformations. i, (R,S)-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)-HNK. vi, Demethylation of (2S,6S;2R,6R)-HK further gives (2S,6S;2R,6R)-HNK.
Extended Data Figure 2 Circulating levels of ketamine and its metabolites following i.p. administration in mice.
a, b, Plasma (a) and brain (b) levels of ketamine and its metabolites after administration of (R,S)-KET (10 mg kg−1) in mice. c, d, Brain levels of KET (c), norKET (d) and HNK (e) following administration of (S)- and (R)-KET. f, g, Chemical structure of (R,S)-6,6-dideuteroketamine ((R,S)-d2-KET) (f), which displaces [3H]MK-801 binding with a similar affinity to (R,S)-KET ((R,S)-KET: Ki = 799 nM; (R,S)-d2-KET: Ki = 883 nM) (g). See Supplementary Table 1 for statistical analyses and n numbers. Source data
a, Chronic social defeat stress and social interaction/avoidance test timeline. b, c, 24 h after administration, neither (R,S)-KET nor MK-801 affected locomotor activity (b) or total number of compartmental crosses in the social interaction apparatus (c). Data are mean ± s.e.m. ***P < 0.001. See Supplementary Table 1 for statistical analyses and n numbers. Source data
After recording baseline activity for 60 min, mice received drug (marked by a vertical dashed line) and locomotor activity was monitored for another 1 h. a, b, Administration of (R,S)-KET (10 mg kg−1), induced hyperlocomotor responses equally in both male and female mice. c, d, (R,S)-KET and (R,S)-d2-KET were equally potent in inducing a hyperlocomotor response at the dose of 10 mg kg−1. Data are mean ± s.e.m. *P < 0.05, **P < 0.01 (see Supplementary Table 1 for statistical analyses and n numbers). Source data
Extended Data Figure 5 Acute and sustained antidepressant and anti-anhedonic effects of (2R,6R)- and (2S,6S)-HNK.
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−1. b, A single injection of (2S,6S)- HNK induced antidepressant-like effects in the learned helplessness test at the dose of 75 mg kg−1. c, Administration of (2R,6R)-HNK induced dose-dependent antidepressant effects in the 1- and 24-h FST. d, Administration of (2S,6S)-HNK at the dose of 25 mg kg−1 induced antidepressant effects in the 1- and 24-h FST. 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 NSF test. g, Similar to (R,S)-KET, the antidepressant-like effects of (2R,6R)-HNK in the FST persisted for at least 3 days after 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 locomotor activity (j) or total compartmental crosses in the social interaction test after chronic social defeat stress (k). Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (see Supplementary Table 1 for statistical analyses and n numbers). Source data
Extended Data Figure 6 (2R,6R)-HNK rapidly increases the frequency and amplitude of AMPAR spontaneous excitatory postsynaptic currents in the hippocampus.
a, Representative traces of spontaneous excitatory postsynaptic currents (sEPSCs) mediated via AMPARs during baseline (before) and 20 min after drug administration. b, Example CA1 stratum radiatum interneuron recorded from a rat hippocampal slice. c–j, Twenty-minute exposure of (2R,6R)-HNK (e, i), but not (R,S)-(KET) (c, g) or (2S,6S)-HNK (d, h), increased AMPA sEPSCs frequency and amplitude compared to baseline. Data are mean ± s.e.m. *P < 0.05, **P < 0.01, ***P < 0.001 (see Supplementary Table 1 for statistical analyses and n numbers). SLM, stratum lacunosum-moleculare; SO, stratum oriens; SP, stratum pyramidale; SR, stratum radiatum. Source data
Extended Data Figure 7 Administration of the AMPAR antagonist, NBQX, prevents (2R,6R)-HNK-induced increases in gamma oscillations in vivo.
a, Administration of (R,S)-KET, but not (2R,6R)- HNK, increased locomotor home-cage activity of mice. b–e, Neither (R,S)-KET nor (2R,6R)-HNK altered cortical alpha (b), beta (c), delta (d) or theta (e) oscillations in vivo. f–k, Pre-treatment with the AMPAR antagonist, NBQX, did not change the locomotor activity (f), alpha (g), beta (h), delta (j) or theta (k) oscillations, but it prevented (2R,6R)-HNK-induced increases of gamma oscillations in vivo (i). Data are mean ± s.e.m. *P < 0.05, **P < 0.01 (see Supplementary Table 1 for statistical analyses and n numbers). Source data
Extended Data Figure 8 Effects of (2R,6R)-hydroxynorketamine on protein and protein phosphorylation levels in synaptoneurosomes.
a, b, A single administration of (R,S)-KET (10 mg kg−1) or (2R,6R)- HNK (10 mg kg−1), did not alter levels of mTOR or phosphorylated mTOR in the hippocampus 1 h (a) or 24 h (b) after injection. c–j, Administration of (R,S)-KET or (2R,6R)-HNK did not alter levels of mTOR/phosphorylated mTOR (c, d), eEF2/phosphorylated eEF2 (e, f), proBDNF/mBDNF (g, h), or GluA1/GluA2 (i, j), in the prefrontal cortex of mice. The values for the phosphorylated forms of proteins were normalized to phosphorylation-independent levels of the same protein. Phosphorylation-independent levels of proteins were normalized to GAPDH. Data are mean ± s.e.m, and were normalized to the saline-treated control group for each protein. Images are cropped; see Supplementary Fig. 1 for complete blot images. *P < 0.05 (see Supplementary Table 1 for statistical analyses and n numbers). Source data
Extended Data Figure 9 (2R,6R)-HNK administration does not alter startle amplitude, drug discrimination rate or self-administration drug intake.
a, Startle amplitude as measured in the pre-pulse inhibition task was not affected by administration of (R,S)-KET or (2R,6R)-HNK. b, c, Response rate of overall lever pressing per sec in the drug discrimination model was not changed by administration of (R,S)-KET, (2R,6R)-HNK (b) or PCP (c). d, Unlike ketamine, (2R,6R)-HNK did not alter drug intake in the self-administration task in mice. Data are mean ± s.e.m. *P < 0.05 (see Supplementary Table 1 for statistical analyses and n numbers). Source data
This file contains (1) Supplementary Table 1, which includes the details for the number of animals and the results of the statistical analyses in each experiment; (2) Supplementary Figure 1, with the complete blot images for Figure 4 and Extended Data Figure 8; (3) the Supplementary Methods for the synthesis of (2R,6R)-HNK, (2S,6S)-HNK, and D-Ket, in addition to the analytical data that supports the synthetic steps and the X-ray crystallographic data that confirms the absolute and relative conformation for (2S,6S)-HNK and (2R,6R)-HNK. (PDF 7157 kb)
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Zanos, P., Moaddel, R., Morris, P. et al. NMDAR inhibition-independent antidepressant actions of ketamine metabolites. Nature 533, 481–486 (2016). https://doi.org/10.1038/nature17998
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