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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.
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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.
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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.
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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
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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.
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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.
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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).
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