(2R,6R)-Hydroxynorketamine is not essential for the antidepressant actions of (R)-ketamine in mice

Abstract

(R,S)-Ketamine has rapid and sustained antidepressant effects in depressed patients. Although the metabolism of (R,S)-ketamine to (2 R,6 R)-hydroxynorketamine (HNK), a metabolite of (R)-ketamine, has been reported to be essential for its antidepressant effects, recent evidence suggests otherwise. The present study investigated the role of the metabolism of (R)-ketamine to (2 R,6 R)-HNK in the antidepressant actions of (R)-ketamine. Antidepressant effects were evaluated using the forced swimming test in the lipopolysaccharide (LPS)-induced inflammation model of mice and the tail suspension test in naive mice. To prevent the metabolism of (R)-ketamine to (2 R,6 R)-HNK, mice were pretreated with cytochrome P450 (CYP) inhibitors. The concentrations of (R)-ketamine, (R)-norketamine, and (2 R,6 R)-HNK in plasma, brain, and cerebrospinal fluid (CSF) samples were determined using enantioselective liquid chromatography-tandem mass spectrometry. The concentrations of (R)-norketamine and (2 R,6 R)-HNK in plasma, brain, and CSF samples after administration of (R)-norketamine (10 mg/kg) and (2 R,6 R)-HNK (10 mg/kg), respectively, were higher than those generated after administration of (R)-ketamine (10 mg/kg). Nonetheless, while (R)-ketamine attenuated, neither (R)-norketamine nor (2 R,6 R)-HNK significantly altered immobility times of LPS-treated mice. Treatment with CYP inhibitors prior to administration of (R)-ketamine increased the plasma levels of (R)-ketamine, while generation of (2 R,6 R)-HNK was almost completely blocked. (R)-Ketamine exerted the antidepressant effects at a lower dose in the presence of CYP inhibitors than in their absence, which is consistent with exposure levels of (R)-ketamine but not (2 R,6 R)-HNK. These results indicate that metabolism to (2 R,6 R)-HNK is not necessary for the antidepressant effects of (R)-ketamine and that unmetabolized (R)-ketamine itself may be responsible for its antidepressant actions.

Fig. 1

Effects of (R)-ketamine ((R)-Ket), (R)-norketamine ((R)-NK), and (2 R,6 R)-hydroxynorketamine ((2 R,6 R)-HNK) in the lipopolysaccharide (LPS)-induced inflammation model and the pharmacokinetic profiles of these compounds. a Experimental schedules for the evaluation of the pharmacological effects of (R)-Ket, (R)-NK, and (2 R,6 R)-HNK. The compounds were administered intraperitoneally (i.p.). b Effects of (R)-Ket, (R)-NK, and (2 R,6 R)-HNK on the LPS-induced increase in the immobility time during the forced swimming test (FST; one-way analysis of variance, F_4,35 = 5.553, P = 0.001). c Effects of (R)-Ket, (R)-NK, and (2 R,6 R)-HNK in the locomotion test (LMT; F_4,35 = 0.356, P = 0.838). b, c Data are represented as the mean ± SEM (n = 8/group). **P < 0.01 compared with Saline, ^##P < 0.01 compared with saline–LPS treatment. d, h Experimental schedules for the pharmacokinetic studies following the intraperitoneal administration of (R)-Ket, (R)-NK, or (2 R,6 R)-HNK. e–g Plasma concentration–time profiles after the administration of (R)-Ket (E), (R)-NK (F), or (2 R,6 R)-HNK (g). Blood was collected sequentially at 5, 15, and 30 min and at 1, 2, and 3 h from the tail vein of each individual mouse. i, j Concentrations of (R)-Ket, (R)-NK, and (2 R,6 R)-HNK in the brain (i) and cerebrospinal fluid (CSF; j) at the time points ((a) 5 min, (b) 15 min) corresponding to their plasma t_max (time at the plasma maximum concentration). e–g, i The plasma and brain levels are represented as the mean ± SEM (n = 3/group). j Each CSF level represents a value derived from a pooled CSF specimen collected from three mice

Fig. 2

Effects of cytochrome P450 inhibitors (CYPI) on the antidepressant effects and pharmacokinetic profiles of (R)-ketamine ((R)-Ket) in the lipopolysaccharide (LPS)-induced inflammation model. a Experimental schedules for the pharmacokinetic studies following the intraperitoneal administration of (R)-Ket. b–d Plasma concentration–time profiles of (R)-Ket, (R)-norketamine ((R)-NK), and (2 R,6 R)-hydroxynorketamine ((2 R,6 R)-HNK) after the administration of (R)-Ket (3 mg/kg) without (b) and with (c) CYPI pretreatment, and (R)-Ket (10 mg/kg) without CYPI pretreatment (d). Following the administration of (R)-Ket, blood was collected sequentially at 5, 15, and 30 min and at 1, 2, and 3 h from the tail vein of each individual mouse. e, f Effect of CYPI pretreatment on the plasma maximum concentration (C_max; e) and the area under the concentration–time curve from time 0 to 3 h (AUC_0-3h; F) of (R)-Ket, (R)-NK, and (2 R,6 R)-HNK. Pretreatment with CYPI completely blocked (2 R,6 R)-HNK formation to below the lower limit of quantification (BLQ; < 3 ng/mL); therefore, the AUC_0–3h was not calculable (NC) because of the BLQ of the analyte. b–f Data are represented as the mean ± SEM (n = 3/group). g Experimental schedules for the evaluation of the pharmacological effects of (R)-Ket. h Effects of (R)-Ket on the LPS-increased immobility in the forced swimming test (FST) in the presence or absence of CYPI (one-way analysis of variance, F_5,42 = 4.707, P = 0.002). i Effects of (R)-Ket on the locomotion test in the presence or absence of CYPI (LMT; F_5,42 = 0.804, P = 0.553). h, i Data are represented as the mean ± SEM (n = 8/group). **P < 0.01 compared with saline, ^##P < 0.01 compared with saline–LPS treatment, ^@@P < 0.01 compared with CYPI/(R)-ketamine (3 mg/kg)-LPS treatment, ^$$P < 0.01 compared with saline/(R)-ketamine (3 mg/kg)-LPS treatment

Fig. 3

Effects of cytochrome P450 inhibitors (CYPI) on the antidepressant effects and pharmacokinetic profiles of (R)-ketamine ((R)-Ket) in naive ICR mice. a Experimental schedules for the pharmacokinetic studies following the intraperitoneal administration of (R)-Ket. b–d Plasma concentration–time profiles of (R)-Ket, (R)-norketamine ((R)-NK), and (2 R,6 R)-hydroxynorketamine ((2 R,6 R)-HNK) after the administration of (R)-Ket (10 mg/kg, i.p.) without (b) and with (c) CYPI pretreatment and (R)-Ket (30 mg/kg, i.p.) without CYPI pretreatment (d). Following the (R)-Ket administration, blood was collected sequentially at 5, 15, and 30 min and at 1, 2, 4, 8, and 24 h from the tail vein of each individual mouse. e, f Effect of the CYPI pretreatment on the plasma maximum concentration (C_max; e) and the area under the concentration–time curve from time 0 extrapolated to infinite time (AUC_0-∞; f) of (R)-Ket, (R)-NK, and (2 R,6 R)-HNK. Pretreatment with CYPI markedly decreased the (2 R,6 R)-HNK levels to near the lower limit of quantification (LLOQ; 3 ng/mL). b–f Data are represented as the mean ± SEM (n = 3/group). g Experimental schedule for the evaluation of the pharmacological effects of (R)-Ket. h Effects of (R)-Ket on the immobility in the tail suspension test (TST) in the presence or absence of CYPI (one-way analysis of variance, F_4,75 = 3.89, P = 0.006). Data are represented as the mean ± SEM (n = 16/group). **P < 0.01 compared with CYPI, ^##P < 0.01 compared with saline/(R)-ketamine (10 mg/kg) treatment