Antidepressant-relevant concentrations of the ketamine metabolite (2 R,6 R)-hydroxynorketamine do not block NMDA receptor function

Abstract

Preclinical studies indicate that (2R,6R)-hydroxynorketamine (HNK) is a putative fast-acting antidepressant candidate. Although inhibition of NMDA-type glutamate receptors (NMDARs) is one mechanism proposed to underlie ketamine's antidepressant and adverse effects, the potency of (2R,6R)-HNK to inhibit NMDARs has not been established. We used a multidisciplinary approach to determine the effects of (2R,6R)-HNK on NMDAR function. Antidepressant-relevant behavioral responses and (2R,6R)-HNK levels in the extracellular compartment of the hippocampus were measured following systemic (2R,6R)-HNK administration in mice. The effects of ketamine, (2R,6R)-HNK, and, in some cases, the (2S,6S)-HNK stereoisomer were evaluated on the following: (i) NMDA-induced lethality in mice, (ii) NMDAR-mediated field excitatory postsynaptic potentials (fEPSPs) in the CA1 field of mouse hippocampal slices, (iii) NMDAR-mediated miniature excitatory postsynaptic currents (mEPSCs) and NMDA-evoked currents in CA1 pyramidal neurons of rat hippocampal slices, and (iv) recombinant NMDARs expressed in Xenopus oocytes. While a single i.p. injection of 10 mg/kg (2R,6R)-HNK exerted antidepressant-related behavioral and cellular responses in mice, the ED50 of (2R,6R)-HNK to prevent NMDA-induced lethality was found to be 228 mg/kg, compared with 6.4 mg/kg for ketamine. The 10 mg/kg (2R,6R)-HNK dose generated maximal hippocampal extracellular concentrations of ∼8 µM, which were well below concentrations required to inhibit synaptic and extrasynaptic NMDARs in vitro. (2S,6S)-HNK was more potent than (2R,6R)-HNK, but less potent than ketamine at inhibiting NMDARs. These data demonstrate the stereoselectivity of NMDAR inhibition by (2R,6R;2S,6S)-HNK and support the conclusion that direct NMDAR inhibition does not contribute to antidepressant-relevant effects of (2R,6R)-HNK.

Keywords: NMDA receptor; antidepressant; depression; hydroxynorketamine; ketamine.

Conflict of interest statement

Conflict of interest statement: T.D.G. has received research funding from Janssen, Roche, and Allergan Pharmaceuticals, and served as a consultant for FSV7 LLC during the preceding 3 years. The authors declare competing financial interests: T.D.G., P.Z., R.M., P.J.M., C.J.T., and C.A.Z. 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. C.A.Z. and R.M. 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 ketamine metabolites in the treatment of depression and neuropathic pain. R.M., P.J.M., C.J.T., and C.A.Z. have assigned patent rights to the US Government but will share a percentage of any royalties that may be received by the Government. P.Z. and T.D.G. have assigned their patent rights to the University of Maryland, Baltimore, but will share a percentage of any royalties that may be received by the University of Maryland, Baltimore. S.F.T. received research support from Janssen, is a consultant for Janssen, is a member of the Scientific Advisory Board for Sage Therapeutics, is a co-founder of NeurOp, Inc., and co-inventor on Emory-owned IP. S.J.M. owns stock in NeurOp, Inc., which is developing NMDAR inhibitors for use in treating neurological disease and disorders. All other authors declare no competing interests.

Figures

Fig. 1.

Metabolism of (R,S)-ketamine to the two hydroxynorketamine (HNK) stereoisomers, (2R,6R)-HNK and (2S,6S)-HNK. The amine group at the chiral center (C2 carbon) of (R)-ketamine and (S)-ketamine undergoes demethylation, producing (R)-norketamine and (S)-norketamine, followed by hydroxylation at the C6 carbon cis to the amine group to give the (2R,6R)- and (2S,6S)-HNKs. (R)-Ketamine selectively forms (2R,6R)-HNK, while (S)-ketamine selectively forms (2S,6S)-HNK. The primary intermediate metabolites, (R)- and (S)-norketamine, are not depicted.

Fig. 2.

Behavioral effects and tissue concentrations following systemic administration of 10 mg/kg (2R,6R)-HNK to mice. Mice received i.p. injections of vehicle (control, i.e., saline) or (2R,6R)-hydroxynorketamine (HNK) at a dose of 10 mg/kg and were tested in the forced swim test (FST) (A) 1 h and (B) 24 h posttreatment, or were tested in the (C) novelty suppressed feeding test (NSF) 30 min posttreatment [n = 10 mice/treatment; (A) Student’s unpaired t test, t = 2.98, df = 18; (B) Student’s unpaired t test, t = 2.40, df = 18; (C) log-rank, Mantel–Cox test, χ2 = 8.84]. (D) Western blot analysis of hippocampal extracts revealed that levels of mBDNF (Student’s unpaired t test, t = 3.43, df = 20; n = 10–12 mice/treatment), but not pro-BDNF (Student’s unpaired t test, t = 1.22, df = 20; n = 10–12 mice/treatment) were significantly increased 30 min after treatment of mice with (2R,6R)-HNK (10 mg/kg, i.p.) compared with saline [control (CON)]. Total mTOR levels did not change (Student’s unpaired t test, t = 0.19, df = 22; n = 12 mice/treatment), while the ratio of mTOR phosphorylated at Ser2448, to total mTOR increased 30 min posttreatment with (2R,6R)-HNK (Student’s unpaired t test, t = 2.17, df = 22; n = 12 mice/treatment). Concentrations of (2R,6R)-HNK in the (E) plasma and (F) whole brain following systemic administration of (2R,6R)-HNK (10 mg/kg i.p.) to mice (n = 4 mice/treatment/time point). The measured analyte concentrations in the brain were normalized according to tissue weight and are reported as micromoles per kilogram of tissue. (G) Concentrations of (2R,6R)-HNK in the microdialysates from the ventral hippocampus of awake mice collected at a 10-min sampling rate following administration of (2R,6R)-HNK (10 mg/kg, i.p.) corrected for in vivo recovery of 54.8% and for dilution (1:10) of samples collected at low flow rate (0.1 μL/min) with 1 μL/min makeup solvent on the probe outlet (n = 6–7 mice/treatment/time point). (E–G, Insets) Representative chromatograms from the 10-min time point from each assay. Data points and error bars represent mean and SEM, respectively. *P < 0.05 and **P < 0.01.

Fig. 3.

Dose–response relationship for (R,S)-ketamine, (2R,6R)-HNK, and (2S,6S)-HNK to prevent NMDA-induced lethality. Mice received an i.p. injection of ketamine (KET), (2R,6R)- hydroxynorketamine (HNK), or (2S,6S)-HNK. Five minutes after the treatment, mice received an i.p. injection of 250 mg/kg NMDA. (A) Percent lethality at 24 h post-NMDA (n = 6 mice/ dose). (R,S)-ketamine, (2R,6R)-HNK, and (2S,6S)-HNK dose dependently prevented lethality. The effective doses of ketamine, (2R,6R)-HNK, and (2S,6S)-HNK that protected 50% of the population from NMDA-induced lethality (i.e., ED50) were 6.4, 227.8, and 18.63 mg/kg, respectively. (B) Whole-brain measurements following systemic administration of ED50 doses of ketamine (6.4 mg/kg), (2R,6R)-NHK (227.8 mg/kg), and (2S,6S)-HNK (18.63 mg/kg) normalized according to tissue weight (n = 3–4 mice/treatment/time point). Data points and error bars represent mean and SEM, respectively.

Fig. 4.

Concentration–response relationship for (R,S)-ketamine, (2R,6R)-HNK, and (2S,6S)-HNK to inhibit NMDAR fEPSPs in the CA1 field of mouse hippocampal slices. NMDAR-mediated fEPSPs were recorded before and after superfusion of slices with various concentrations of ketamine (KET), (2R,6R)-HNK, and (2S,6S)-HNK. (A–C) Sample recordings of fEPSPs obtained before and during exposure to the slices to KET, (2R,6R)-HNK, or (2S,6S)-HNK are shown. Traces in blue represent baseline potentials. Traces in red, green, and orange represent fEPSPs recorded in the presence of ketamine, (2R,6R)-HNK, or (2S,6S)-HNK, respectively. Traces in gray represent fEPSPs recorded after application of APV. Graphs of changes in fEPSP slope as a function of concentrations of (D) KET and (2R,6R)-HNK and (E) (2S,6S)-HNK. The respective vehicle control values are plotted in blue. Data points and error bars represent mean and SEM, respectively [n = 4–7 slices/test compound concentration; (R,S)-KET and (2R,6R)-HNK control, n = 36; (2S,6S)-HNK control, n = 19 (controls for each concentration were run separately for blinding purposes)]. The IC50 values of ketamine, (2R,6R)-HNK, and (2S,6S)-HNK were found to be 4.5, 211.9, and 47.2 µM, respectively (Table 1).

Fig. 5.

Concentration–response relationship for (R,S)-ketamine and (2R,6R)-hydroxynorketamine (HNK) to inhibit NMDAR mEPSCs in rat hippocampal slice CA1 pyramidal neurons. NMDAR mEPSCs were recorded before and after perfusion of slices with various concentrations of ketamine (KET) and (2R,6R)-HNK. (A) Sample recordings of mEPSCs recorded in the absence (control) and in the presence of different concentrations of KET and (2R,6R)-HNK. (B) Graphs of changes in median EPSC amplitude as a function of compound concentrations. All results were normalized to control, as described in Materials and Methods. Data points and error bars represent mean and SEM, respectively (n = 3–8 neurons/test compound concentration; control, n = 26: controls for each concentration were run separately for blinding purposes). IC50 values were estimated to be 6.4 µM for ketamine and 63.7 µM for (2R,6R)-HNK (Table 1). (C) Cumulative distribution of adjusted amplitudes of mEPSCs recorded in the presence of vehicle (control) or different concentrations of KET and (2R,6R)-HNK. Adjusted amplitude was determined by multiplying every event by its cell’s respective inhibition ratio (postsuperfusion median/presuperfusion median). All events from all cells were pooled together by compound and concentration and then randomized. Subsequently, 300 events were randomly selected from the total pool for each group to generate the cumulative histograms.

Fig. 6.

Concentration-dependent effects of (R,S)-ketamine, (2R,6R)-hydroxynorketamine (HNK), and (2S,6S)-HNK on NMDA-induced whole-cell currents in rat hippocampal slice CA1 pyramidal neurons. (A) Sample recordings of NMDA-induced whole-cell currents with baseline measurements (maximum current following agonist pulse, blue) overlaid with currents in the presence of the maximum concentrations of each test compound [red, ketamine; green, (2R,6R)-HNK; orange, (2S,6S)-HNK]. (B) Concentration–response relationship for inhibition of the whole-cell currents by the test compounds. Data points and error bars represent mean and SEM, respectively (n = 4–13 neurons/test compound concentration; control, n = 24: controls for each concentration were run separately for blinding purposes). The IC50 value for ketamine was calculated to be 45.9 µM.

Fig. 7.

Concentration-dependent inhibition of glutamate NMDAR subtypes by (2R,6R)-hydroxynorketamine (HNK) and (2S,6S)-HNK. Xenopus laevis oocytes coexpressing rat GluN1 with either rat (A) GluN2A, (B) GluN2B, (C) GluN2C, or (D) GluN2D were activated with l-glutamate and glycine (100 μM each) and exposed to increasing concentrations of (2S,6S)-HNK or (2R,6R)-HNK to determine the IC50 for each NMDAR subtype. (2S,6S)-HNK inhibited each NMDAR subtype to a greater degree than its isomeric counterpart (2R,6R)-HNK (Table 1). Data points and error bars represent mean and SEM, respectively (n = 3–20 oocytes/receptor subtype/test compound).