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. Author manuscript; available in PMC: 2023 Feb 7.
Published in final edited form as: J Psychopharmacol. 2021 Dec 31;36(2):170–182. doi: 10.1177/02698811211064922

Sex-dependent metabolism of ketamine and (2R,6R)-hydroxynorketamine in mice and humans

Jaclyn N Highland a,b, Cristan A Farmer c, Panos Zanos a,d,e, Jacqueline Lovett f, Carlos A Zarate Jr c, Ruin Moaddel f, Todd D Gould a,c,g,h,*
PMCID: PMC9904319  NIHMSID: NIHMS1869947  PMID: 34971525

Abstract

Background:

Ketamine is rapidly metabolized to norketamine and hydroxynorketamine (HNK) metabolites. In female mice, when compared to males, higher levels of (2R,6R;2S,6S)-HNK have been observed following ketamine treatment, and higher levels of (2R,6R)-HNK following the direct administration of (2R,6R)-HNK.

Aims:

Evaluate the impact of sex in humans and mice, and gonadal hormones in mice on the metabolism of ketamine to form norketamine and HNKs and in the metabolism/elimination of (2R,6R)-HNK.

Methods:

In CD-1 mice, we utilized gonadectomy to evaluate the role of circulating gonadal hormones in mediating sex-dependent differences in ketamine and (2R,6R)-HNK metabolism. In humans (34 with treatment-resistant depression and 23 healthy controls) receiving an antidepressant dose of ketamine (0.5 mg/kg i.v. infusion over 40 minutes), we evaluated plasma levels of ketamine, norketamine, and HNKs.

Results:

In humans, plasma levels of ketamine and norketamine were higher in males than females, while (2R,6R;2S,6S)-HNK levels were not different. Following ketamine administration to mice (10 mg/kg i.p.) Cmax and total plasma concentrations of ketamine and norketamine were higher, and those of (2R,6R;2S,6S)-HNK were lower, in intact males compared to females. Direct (2R,6R)-HNK administration (10 mg/kg i.p.) resulted in higher levels of (2R,6R)-HNK in female mice. Ovariectomy did not alter ketamine metabolism in female mice, whereas orchidectomy recapitulated female pharmacokinetic differences in male mice, which was reversed with testosterone replacement.

Conclusion:

Sex is an important biological variable that influences the metabolism of ketamine and the HNKs, which may contribute to sex differences in therapeutic efficacy or side effect burden.

INTRODUCTION

(R,S)-ketamine (ketamine), traditionally used as a drug for anesthesia, has gained attention for its rapid-acting antidepressant efficacy in treatment-resistant depressed patients. A single dose of ketamine often results in a rapid (within hours) reduction of depressive symptoms in individuals suffering from major depressive disorder (Berman et al., 2000), bipolar depression (Diazgranados et al., 2010; Zarate et al., 2012b), and treatment-resistant depression (Fava et al., 2020; Zarate et al., 2006; Murrough et al., 2013). The widespread use of ketamine as a treatment for depression, however, is limited by its dissociative side effects and abuse potential (Krystal et al., 1994; Sassano-Higgins et al., 2016; Zanos et al., 2018). The ketamine metabolite (2R,6R;2S,6S)-hydroxynorketamine (HNK), and predominantly the (2R,6R)-HNK stereoisomer, has been demonstrated to share the antidepressant-relevant effects of ketamine by many (Chou et al., 2018; Fukumoto et al., 2019; Pham et al., 2018; Zanos et al., 2019a; Zanos et al., 2019b; Zanos et al., 2016; Elmer et al., 2020; Lumsden et al., 2019; Aguilar-Valles et al., 2020; Riggs et al., 2020; Casarotto et al., 2021; Wray et al., 2019; Highland et al., 2018; Highland et al., 2021; Rahman et al., 2020; Chen et al., 2020), but not all studies (Yokoyama et al., 2020; Yang et al., 2017). (2R,6R)-HNK lacks the adverse effect burden of ketamine in preclinical studies (Highland et al., 2018; Zanos et al., 2016).

Following its administration, ketamine undergoes rapid and extensive metabolism (Figure 1), primarily catalyzed by the cytochrome P450 (CYP) enzymes in the liver (Adams et al., 1981; Desta et al., 2012; Kharasch and Labroo, 1992). Ketamine first undergoes stereoselective N-demethylation to form (R,S)-norketamine, which is then metabolized to form dehydronorketamine (DHNK) or hydroxylated at the 4, 5, or 6 position to form the (2,4)-, (2,5)- and (2,6)-HNKS, respectively (Desta et al., 2012; Dinis-Oliveira, 2017; Kharasch and Labroo, 1992; Portmann et al., 2010; Woolf and Adams, 1987). Ketamine can also be directly hydroxylated to form the 6-hydroxyketamines and subsequently undergo N-demethylation to form the (2,6)-HNKs (Desta et al., 2012; Portmann et al., 2010). The production of HNKs via either pathway is predominantly catalyzed by CYP2A6, CYP2B6, and CYP3A5 (Desta et al., 2012; Portmann et al., 2010).

Figure 1. Simplified metabolism of ketamine to form the hydroxynorketamines.

Figure 1.

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(R,S)-ketamine (KET) undergoes N-demethylation to form (R,S)-norketamine (norKET) which is further metabolized to form (R,S)-dehydronorketamine (DHNK). (R,S)-norKET can be hydroxylated at the carbon 4, 5, and 6 positions, to form the (2,4)-, (2,5)-, and (2,6)-hydroxynorketamines (HNKs), respectively. Through a minor metabolic pathway, (R,S)-KET can also be directly hydroxylated to form the (2,6)-hydroxyketamines (HKs) which are subsequently N-demethylated to form the (2,6)-HNKs.

The metabolic conversion of ketamine to form HNKs is rapid across species studied, as HNKs have been detected at the earliest time points studied to date (Highland et al., 2021). Namely, HNKs are detected within 2.5–20 min of intraperitoneal (i.p.) or intravenous (i.v.) ketamine injection in rodents, dogs, and horses (Leung and Baillie, 1986; Moaddel et al., 2015; Pham et al., 2018; Tuma et al., 2020; Yamaguchi et al., 2018; Zanos et al., 2019a; Zanos et al., 2016), and at the end of a 40 min i.v. infusion in humans (Kurzweil et al., 2020; Zarate et al., 2012a; Zhao et al., 2012; Farmer et al., 2020). HNKs undergo glucuronide conjugation, which is catalyzed at least partly by the UDP-glucuronosyltransferase UGT2B4 (Moaddel et al., 2010). HNKs are eliminated both in their conjugated and unconjugated forms in urine and bile (Chang and Glazko, 1974; Dinis-Oliveira, 2017; Lankveld et al., 2006; Moaddel et al., 2010; Sandbaumhuter and Thormann, 2018; Turfus et al., 2009).

There is evidence that biological sex impacts the metabolism of ketamine to form the HNKs in mice. In particular, higher levels of the (2R,6R;2S,6S)-HNK metabolite were observed in the brains of female mice, compared to males, following ketamine dosing (Zanos et al., 2016). Additionally, higher levels of (2R,6R)-HNK have also been observed in the plasma and brains of female mice, relative to males, following the direct administration of (2R,6R)-HNK (Highland et al., 2018). However, the sex-dependent differences in the metabolism of ketamine to form (2R,6R;2S,6S)-HNK and in the subsequent metabolism/elimination of (2R,6R)-HNK are not well-defined. Moreover, the mechanisms underlying these differences, including the potential role of gonadal hormones in modulating metabolism, have not been studied in detail. We compared the levels of ketamine, norketamine and (2R,6R;2S,6S)-HNK, following ketamine dosing and levels of (2R,6R)-HNK following its direct administration, in intact male and female mice. We evaluated the activational role of gonadal hormones in mediating sex-dependent pharmacokinetic differences, observing that in intact male mice peak and total plasma concentrations of ketamine were higher and those of (2R,6R;2S,6S)-HNK were lower, compared to females. Gonadectomy prevented the observed sex-dependent metabolism differences in male mice but did not alter ketamine or (2R,6R;2S,6S)-HNK levels in females. Further, testosterone replacement reversed this effect in gonadectomized male mice consistent with a role of testosterone in modulating metabolism. Finally, we assessed whether plasma levels of ketamine, norketamine, and HNKs demonstrated sex-dependent differences in human samples obtained following an antidepressant dose of ketamine finding that plasma levels of ketamine and norketamine were higher in males than females while (2R,6R;2S,6S)-HNK was not different.

MATERIALS AND METHODS

Mouse studies

Animals

Male and female CD-1 mice (Charles Rivers Laboratories; Raleigh, NC, USA), 8–10 weeks old at the time of testing, were habituated to the University of Maryland (Baltimore, MD, USA) animal facility for at least one week prior to testing. Mice were group housed in cages of 4–5 per cage with a constant 12-hour light cycle (lights on/off at 07:00/19:00). Food and water were available ad libitum. Mice were randomized to experimental groups (n=4–5/group). All experiments were performed during the light phase. All studies were approved by the University of Maryland School of Medicine Institutional Animal Care and Use Committee and conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Drugs

(R,S)-ketamine hydrochloride was purchased from Sigma Aldrich (USA). (2R,6R)-HNK hydrochloride was synthesized and characterized at the National Center for Advancing Translation Sciences (National Institutes of Health, Rockville, MD, USA) as previously described (Morris et al., 2017). All compounds were dissolved in saline and administered intraperitoneally (i.p.) in a volume of 7.5 ml/kg.

Surgical procedures

Gonadectomy

Male and female mice were gonadectomized according to previously described methods (Idris, 2012). A surgical plane of anesthesia was induced (3.5–4.5%) and maintained (1.5–3%) with inhaled isoflurane delivered via a precision vaporizer. In male mice, a small incision was made in the midline of the scrotum, the testes were extracted, and the blood supply was clamped and cauterized. The incision was closed with non-absorbable monofilament sutures. In female mice, a small dorsal, midline incision was made in the skin and the fascia gently separated. Small incisions were then made in the abdominal muscle, the ovaries were extracted, and blood supply was clamped and cauterized. The muscle incisions were closed with absorbable monofilament sutures and the skin incisions with non-absorbable monofilament sutures. Sham surgeries were performed in male and female mice as described above, except that testes and ovaries, respectively, were left intact. Mice received carprofen (5 mg/kg, subcutaneous) pre-operatively as well as 24 and 48 h after surgery. All experiments were performed 10 days after surgery (Bowen et al., 2011).

Subcutaneous implants

Testosterone was administered via subcutaneous, testosterone-releasing implants based upon published methods (Bowen et al., 2011; Daan et al., 1975). Lengths of silastic tubing (12 mm length × 2.15 mm outer diameter) were filled with testosterone (12 mg) and sealed with silicone. Control implants were prepared to the same specifications but left empty. Implants were incubated at 37°C in saline overnight (approximately 12 h) prior to implantation. Implants were inserted at the time of gonadectomy or sham surgery. A small, dorsal incision (approximately 5 mm) was made, the implant was carefully inserted subcutaneously, and the incision closed with non-absorbable monofilament sutures.

Bioanalysis of plasma and brain levels

In four separate experiments, ketamine (10 mg/kg, i.p.) was administered to: 1) intact male and female mice, 2) gonadectomized or sham-operated male mice, 3) gonadectomized or sham-operated female mice, and 4) gonadectomized male mice with testosterone-releasing implants, gonadectomized male mice with control implants, and sham-operated male mice with control implants. In two additional experiments, (2R,6R)-HNK (10 mg/kg, i.p.) was administered to 1) intact male or female mice, and 2) gonadectomized or sham-operated male mice. At 10 min, 30 min, 1 h, and 2 h after ketamine treatment, and at 5 min, 10 min, 30 min, 1 h and 2 h after (2R,6R)-HNK treatment, mice were deeply anesthetized (4% isoflurane for approximately 2 min) and then euthanized. Trunk blood was immediately collected into 1.5-ml polypropylene tubes containing 30 μl of disodium EDTA (0.5 M, pH 8.0) and kept on ice until plasma separation (<30 min). Brains were harvested, immediately frozen on dry ice, and stored at −80°C until analysis. Blood was centrifuged at 8000 × g for 6 minutes at 4°C to obtain plasma. Plasma was collected into clean tubes and stored at −80°C until analysis. The concentrations of ketamine, norketamine, and HNK were determined by achiral liquid chromatography-tandem mass spectrometry (LC-MS/MS) using a previously described protocol (Moaddel et al., 2010; Zanos et al., 2016). Values of area under the curve (AUC) for concentration versus time plots were calculated using the linear trapezoidal method.

Human pharmacokinetic study

All participants provided written consent prior to study entry, and this study was approved by the Combined Neuroscience Institutional Review Board of the NIH. Men and women (18 to 65 years old) participated in an inpatient, randomized, placebo-controlled, crossover trial of ketamine conducted on the Mood Disorders Research Unit of the NIH in Bethesda, MD from 2011–2017; all participants were free of additional psychotropic medication for the duration of the study. Thirty-four unmedicated participants with treatment resistant depression (n=22 female, n=12 male; mean age 36±10 years) and 23 healthy controls (HC; n=16 female, n=7 male; mean age 35±11 years) for whom plasma levels of ketamine and its metabolites were obtained as part of a larger study (NCT00088699, National Institutes of Health (NIH) Protocol No. 04-M-0222, sub-study 4) were included in the current analysis. These participants and data have been described elsewhere (Farmer et al., 2020). This was a two-arm, two-period, crossover design. In randomized (1:1) order, 40-minute intravenous infusions of ketamine hydrochloride (0.5 mg/kg) or saline were administered 2 weeks apart in an inpatient setting. Analysis was performed only on blood samples collected from the ketamine treatment. Whole blood samples were collected at baseline, at 40, 80, 120, 230 minutes post-infusion, and 1-day post-infusion using BD vacutainer tubes with sodium heparin and centrifuged at 8000 × g at 4°C for 10 minutes; separated plasma samples were aliquoted and stored at −80°C until analysis. The bioanalytical and pharmacokinetic methods are described in detail elsewhere (Farmer et al., 2020).

Statistical analysis

All experiments and data collection were performed in a blinded and randomized manner. Statistically significant results are indicated with asterisks in the figures (*p<0.05, **p<0.01, ***p<0.001). Data are presented as the mean ± SEM. All statistical tests were two-tailed, and significance was assigned at p<0.05. Statistical analyses of mouse data were performed using GraphPad Prism software (v8; GraphPad Software, Inc.), where differences in concentrations of ketamine and metabolites over time were analyzed using a two-way ANOVA with sex or surgical condition and time as factors, followed by Holm–Šídák post-hoc comparison when significance was reached. Statistical outliers (ROUT, Q=1%) were excluded from analysis. One outlier was excluded from each of the following groups: sham female mouse administered ketamine (120 min); gonadectomized male mouse administered (2R,6R)-HNK (5 min); sham male mouse with vehicle implants administered (2R,6R)-HNK (30 min); gonadectomized male mouse with testosterone-releasing implants administered (2R,6R)-HNK (30 min). Changes in body weight were calculated as the difference in body weight on post-operative day 9 (24 h prior to drug dosing) and the pre-operative body weight and statistically assessed via an unpaired t-test when two groups were compared and via one-way ANOVA when three groups were included. Analyses of the human data were performed in SAS/STAT (v9.4; SAS Institute Inc.). The model of analysis was adjusted to accommodate the data from the human pharmacokinetic study, which had a within-subject design. Covariates were added (age and BMI) and an unstructured residual covariance matrix was used to account for repeated measurement within participant. Study groups (healthy controls and treatment-resistant depression) were combined for analysis of plasma concentrations as there was no group-by-sex interaction. The sex-by-timepoint interaction was decomposed into simple effects of sex at each time point; alpha was not adjusted. The AUC data, which were not repeated measures, were analyzed using an ANCOVA model, adjusting for age and BMI.

RESULTS

Plasma levels of (2R,6R;2S,6S)-HNK are higher and those of ketamine are lower in female, compared to male, mice

Following administration of ketamine (10 mg/kg, i.p) to intact male and female mice, plasma concentrations of ketamine (Figure 2A; effect of sex, F[1,24]=50.9, p<0.0001; effect of time, F[3,24]=188, p<0.0001; interaction, F[3,24]=21.1, p<0.0001; post-hoc comparison, p<0.0001 at 10 min and p=0.049 at 30 min post-treatment) and norketamine (Figure 2B; effect of sex, F[1,24]=6.82, p=0.0153; effect of time, F[3,24]=56.2, p<0.0001; interaction, F[3,24]=0.463, p=0.7109) were lower, while concentrations of (2R,6R;2S,6S)-HNK were higher (Figure 2C; effect of sex, F[1,24]=23.1, p<0.0001; effect of time, F[3,24]=40.2, p<0.0001; interaction, F[3,24]=8.39, p=0.0005; post-hoc comparison, p<0.0001 at 10 min and p=0.0189 at 30 min post-treatment), in females compared to males. Robust, approximately two-fold, sex-dependent differences in the peak plasma concentrations (observed 10 min post-treatment) were observed for ketamine and (2R,6R;2S,6S)-HNK, whereas the Cmax differences in norketamine levels were more subtle (Figure 2). The total plasma concentrations (area under the concentration vs. time curve; AUC) of ketamine and norketamine were approximately two-fold and 1.5-fold greater in males than in females. By contrast, the total plasma concentrations of (2R,6R;2S,6S)-HNK were approximately 1.5-fold greater in females than males.

Figure 2. Plasma concentrations of ketamine and metabolites following ketamine administration to male and female mice.

Figure 2.

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Plasma concentrations of (A) ketamine (KET), (B) norketamine (norKET), and (C) (2R,6R;2S,6S)-hydroxynorketamine (HNK) following a single intraperitoneal injection of ketamine (10 mg/kg) to intact male (M) and female (F) mice measured at 10, 30, 60 and 120 minutes. Data are the mean ± SEM. n=4 per sex and time point. *Indicates male vs. female comparison; *p<0.05, **p<0.01, ***p<0.001. Inset area under the concentration vs. time curve (AUC).

Gonadectomy alters metabolism of ketamine in male, but not female mice

To determine whether gonadal hormones mediated the observed sex-dependent differences in the metabolism of ketamine, male and female mice underwent gonadectomy 10 days prior to ketamine dosing. In female mice, ovariectomy did not significantly alter plasma levels of ketamine (Figure 3A; effect of gonadectomy, F[1,23]=2.29, p=0.144; effect of time, F[3,23]=248, p<0.0001; interaction, F[3,23]=1.20, p=0.333), norketamine (Figure 3B; effect of gonadectomy, F[1,24]=0.912, p=0.349; effect of time, F[3,24]=50.1, p<0.0001; interaction, F[3.24]=0.732, p=0.543), or (2R,6R;2S,6S)-HNK (Figure 3C; effect of gonadectomy, F[1,24]=0.838, p=0.369; effect of time, F[3,24]=154.8, p<0.0001; interaction, F[3,24]=0.197, p=0.897), compared to sham-operated controls.

Figure 3. Gonadectomy alters plasma levels of ketamine and hydroxynorketamine following ketamine treatment in male, but not female, mice.

Figure 3.

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(A-C) Plasma concentrations of (A) ketamine (KET), (B) norketamine (norKET), and (C) (2R,6R;2S,6S)-hydroxynorketamine (HNK), following a single intraperitoneal injection of ketamine (10 mg/kg) to female mice 10 days after sham surgery or gonadectomy (GDX) measured at 10, 30, 60 and 120 minutes. (D-F) Plasma concentrations of (D) KET, (E) norKET, and (F) (2R,6R;2S,6S)-HNK following a single intraperitoneal injection of ketamine (10 mg/kg) to male mice 10 days after sham surgery or gonadectomy (GDX). Data are the mean ± SEM. n=3–4 per surgical condition and time point. *Indicates sham vs. GDX comparison; *p<0.05, **p<0.01, ***p<0.001. Inset area under the concentration vs. time curve (AUC).

In male mice, orchidectomy resulted in significantly lower plasma levels of ketamine (Figure 3D; effect of gonadectomy, F[1,22]=7.17, p=0.0138; effect of time, F[3,22]=72.3, p<0.0001; interaction, F[3,22]=2.18, p=0.119) and higher levels of (2R,6R;2S,6S)-HNK (Figure 3F;effect of gonadectomy, F[1,22]=8.98, p=0.0067; effect of time, F[3,22]=19.0, p<0.0001; interaction, F[3,22]=6.37, p=0.0029; post-hoc comparison, p=0.0003 at 10 minutes) in the first 10 min post-treatment, compared to sham-operated controls. Orchidectomy did not significantly alter levels of norketamine (Figure 3E; effect of gonadectomy F[1,22]=0.424, p=0.522; effect of time, F[3,22]=18.9, p<0.0001; interaction, F[3,22]=0.388, p=0.763). Relative to sham-operated controls, orchidectomy resulted in approximately 1.4-fold and 1.5-fold lower peak and total plasma ketamine levels, respectively (Figure 3D). Peak (2R,6R;2S,6S)-HNK plasma levels were approximately 2-fold higher in orchiectomized males, while total (2R,6R;2S,6S)-HNK plasma concentrations were approximately 1.2-fold higher in gonadectomized males, relative to sham-operated controls (Figure 3F). Gonadectomy increased post-operative weight gain (measured as the difference between body weight on post-operative day 9 and pre-operative body weight) in female mice but attenuated post-operative weight gain in male mice (Table 1).

Table 1.

Effect of gonadectomy on post-operative weight gain in mice.

Figure Group Weight change
(g; mean ± SEM)		 Statistical test and outcome
Figure 2A–D sham females
GDX females		 2.1 ± 0.26
3.4 ± 0.35		 t-test,
t[30]=2.93		 effect of GDX,
p=0.0065
Figure 2E–H sham males
GDX males		 0.65 ± 0.21
−1.0 ± 0.21		 t-test,
t[28]=5.60		 effect of GDX,
p<0.0001
Figure 3B sham males
GDX males		 2.28 ± 0.28
0.81 ± 0.40		 t-test,
t[38]=2.99		 effect of GDX,
p=0.0049
Figure 4 sham males with control implants
GDX males with control implants
GDX+T males		 1.1 ± 0.21
−0.27 ± 0.21
0.68 ± 0.17		 one-way ANOVA;
F[2,68]=6.99		 effect of surgery,
p=0.0017
Holm–Šídák post-hoc comparisons
 sham with control implant vs. GDX with control implant, p=0.0013
sham with control implant vs. GDX+T, p=0.176
GDX with control implant vs. GDX+T,
p=0.0430
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Post-operative weight gain was assessed as the difference in body weight on post-operative day 9 (24 h prior to treatment) and the pre-operative body weight. Abbreviations: GDX, gonadectomized/gonadectomy; sham, sham-operated; GDX+T, gonadectomized male mice with testosterone-releasing implants.

Gonadectomy alters metabolism of (2R,6R)-HNK in male mice

Following the direct administration of (2R,6R)-HNK (10 mg/kg, i.p.) to intact male and female mice, females had higher plasma levels of (2R,6R)-HNK at 5 and 10 min post-treatment (Figure 4A; effect of sex, F[1,34]=61.7, p<0.0001; effect of time, F[4,24]=462, p<0.0001, interaction, F[4,34]=16.8, p<0.0001; post-hoc comparison, p<0.0001 at 5 and 10 min post-treatment). Peak plasma levels (observed 5 min post-treatment) were approximately 1.3-fold higher in females, while concentrations 10 min post-treatment were approximately two-fold higher in females, compared to males (Figure 4A). Total plasma concentrations of (2R,6R)-HNK were approximately two-fold higher in female mice, relative to males (Figure 4A, inset).

Figure 4. Plasma concentrations of (2R,6R)-hydroxynorketamine following direct administration to intact male and female, and gonadectomized male, mice.

Figure 4.

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Plasma concentrations of (2R,6R)-hydroxynorketamine (HNK) following a single intraperitoneal injection (10 mg/kg) to (A) intact male (M) and female (F) mice, and (B) to gonadectomized or sham-operated male mice measured at 5, 10, 30, 60 and 120 minutes. Data are the mean ± SEM. n=3–5 per sex or surgical condition and time point. *Indicates sham vs. GDX comparison; *p<0.05, **p<0.01, ***p<0.001. Inset: values of the area-under-the-concentration vs. time curve (AUC) for male and female mice.

To determine if gonadectomy also altered (2R,6R)-HNK levels following its direct administration, male mice underwent orchiectomy or sham surgery 10 days prior to (2R,6R)-HNK dosing. Similar to the effects observed following ketamine dosing, orchidectomy resulted in higher plasma levels of (2R,6R)-HNK following its direct administration (Figure 4B; effect of gonadectomy, F[1,29]=13.7, p=0.0009; effect of time, F[4,29]=206, p<0.0001; interaction, F[4,29]=4.84, p=0.0041; post-hoc comparison, p=0.0011 at 5 min post-treatment and p=0.0009 at 10 min post-treatment). Plasma levels were approximately 1.3-fold higher at their peak, 5 min post-treatment, and approximately 1.6-fold higher 10 min post-treatment, in orchiectomized male mice, relative to sham-operated controls (Figure 4B). Following its direct administration, total plasma concentrations of (2R,6R)-HNK was approximately 1.4-fold greater in gonadectomized males compared to sham-operated controls (Figure 4B, inset). Consistent with the earlier experiment (Figure 3 and see Table 1), orchidectomy resulted in attenuated post-operative weight gain in this cohort of male mice (Table 1).

Testosterone modulates the metabolism of ketamine in male mice

To evaluate whether circulating testosterone modulated the metabolism of ketamine, male mice underwent orchidectomy or sham surgery, and approximately half of the orchiectomized males received testosterone-releasing subcutaneous implants to replace circulating testosterone. Sham-operated controls and the remaining orchiectomized males received control subcutaneous implants. Ten days after the surgery, ketamine (10 mg/kg, i.p.) was administered and the plasma levels of ketamine, norketamine, and (2R,6R;2S6S)-HNK were determined (Figure 5). Consistent with the previous experiment (Figure 4), orchidectomy resulted in lower plasma levels of ketamine (Figure 5A; effect of surgical group, F[2,46]=4.61, p=0.0150; effect of time, F[3,46]=124, p<0.0001; interaction, F[6,46]=2.42, p=0.0404; post-hoc comparison, gonadectomized vs. sham-operated males with control implants, p=0.0003 at 10 min) and higher plasma levels of (2R,6R;2S6S)-HNK (Figure 5C; effect of surgical group, F[2,48]=30.0, p<0.0001; effect of time, F[3,48]=80.7, p<0.0001; interaction, F[6,48]=5.62, p=0.0002; post-hoc comparison, gonadectomized vs. sham-operated males with control implants, p<0.0001 at 10 min, p=0.0013 at 30 min, p=0.0239 at 1 h), compared to sham-operated controls. Testosterone replacement reversed the effect of gonadectomy, resulting in plasma levels of ketamine that were indistinguishable from sham-operated controls but higher than gonadectomized males with control implants (Figure 5A; effect of surgical group, F[2,46]=4.61, p=0.0150; effect of time, F[3,46]=124, p<0.0001; interaction, F[6,46]=2.42, p=0.0404; post-hoc comparison, gonadectomized males with testosterone-releasing vs. control implants, p=0.0017 at 10 min) and levels of (2R,6R;2S6S)-HNK that were indistinguishable from sham-operated controls but lower compared to orchiectomized males with control implants (Figure 5C; effect of surgical group, F[2,48]=30.0, p<0.0001; effect of time, F[3,48]=80.7, p<0.0001; interaction, F[6,48]=5.62, p=0.0002; post-hoc comparison, gonadectomized males with testosterone releasing vs. control implants, p<0.0001 at 10 min, p=0.0029 at 30 min, p=0.0367 at 1 h). No statistically significant differences were detected in the levels of norketamine between experimental groups (Figure 5B). Post-operative weight gain was attenuated in orchiectomized males with control implants compared to sham-operated males, and this effect was reversed by testosterone replacement (Table 1). There was no statistical difference in post-operative weight gain between sham-operated males and orchiectomized males with testosterone replacement (Table 1).

Figure 5. Testosterone modulates the metabolism of ketamine in male mice.

Figure 5.

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Plasma concentrations of (A) ketamine (KET), (B) norketamine (norKET), and (C) (2R,6R;2S6S)-hydroxynorketamine (HNK) following a single intraperitoneal injection of ketamine (10 mg/kg) to sham-operated male mice with control implants (SHAM), gonadectomized male mice with control implants (GDX), and gonadectomized male mice with testosterone-releasing implants (GDX+T) measured at 10, 30, 60 and 120 minutes. Inset: area under the curve (AUC). Data points and error bars represent mean and SEM, respectively. n=4–5 per surgical condition and time point. *Indicates SHAM vs. GDX comparison; **p<0.01, ***p<0.001. #Indicates GDX vs. GDX+T, ##p<0.01, ###p<0.001. Inset: values of area under the concentration vs. time curve (AUC).

Effects of sex on ketamine, norketamine, and (2R,6R;2S,6S)-HNK levels in humans

Following an intravenous infusion of ketamine (0.5 mg/kg over 40 min) to human subjects, males had higher plasma levels of ketamine (Figure 6A; t[51]=-3.08, p=0.00329 at 80 min; t[51]=-2.75, p=0.00824 at 120 min; t[51]=-3.29, p=0.00181 at 230 min) and norketamine (Figure 6B; t[51]=-2.49, p=0.0160 at 80 min; t[51]=-2.87, p=0.00593 at 120 min; t[51]=-4.016, p=0.000195 at 230 min), compared to females, similar to findings observed in mice (Figure 2). The plasma AUCs for ketamine and norketamine were approximately 1.2-fold and 1.3-fold higher, respectively, in males compared to females (ketamine, Figure 6A; t[52]=-2.35, p=0.0226; norketamine, Figure 6B, t[50]=-2.47, p=0.0171). Although mouse studies revealed a robust sex-dependent difference in the levels of (2R,6R;2S,6S)-HNK following ketamine treatment (Figure 2C), this effect was not observed in the human samples (Figure 6C). While in mice only (2R,6R;2S,6S)-HNK was detectable, in humans additional HNK metabolites were also measurable as previously reported (Farmer et al., 2020). Human plasma levels of both (2R,6R;2S,6S)-HNK (Figure 6C) and (2R,4R;2S,4S;2R,6S;2S,6R)-HNK (Figure 6D) were similar between the sexes. While levels of (2R,4S;2S,4R; 2R,5R;2S,5S)-HNK were initially higher in female subjects (Figure 6E; t[51]=2.00, p=0.0507 at 40 min post-infusion), levels similar between both sexes at later sampling time points, and no statistically significant differences were detected in the AUCs. We performed an analysis of the plasma AUCs to evaluate whether the sex-dependent differences observed in humans may be dependent on female hormonal status. When the following groups were excluded from analysis: (1) females who were on hormonal birth control (n=8), (2) post-menopausal females (n=6), and (3) both females who were on hormonal birth control and post-menopausal females, the sex-dependent differences in plasma AUCs for ketamine (females on birth control excluded, t[44]=-2.31, p=0.0256; post-menopausal females excluded, t[46]=-2.19, p=0.0334; both excluded, t[38]=-2.15, p=0.0382) and norketamine (females on birth control excluded, t[43]=-2.79, p=0.00783; post-menopausal females excluded, t[44]=-2.25, p=0.0292; both excluded, t[37]=-2.55, p=0.0151) were not meaningfully changed.

Figure 6. Plasma concentrations of ketamine and metabolites following intravenous ketamine infusion to humans.

Figure 6.

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Plasma concentrations of (A) ketamine (KET), (B) norketamine (norKET), (C) (2R,6R;2S,6S)-hydroxynorketamine (HNK), (D) (2R,4R;2S,6S) and (2S,6R;2R,6S)-HNK, and (E) (2R,4S;2S,4R) and (2S,5S;2R,5R)-HNK following an intravenous infusion of ketamine (0.5 mg/kg over 40 min) to male (M; n=19) and female (F; n=38) human subjects measured at 40, 80, 120, 230 minutes, and one day post-infusion. Data are the estimated marginal mean ± SEM. *p<0.05, **p<0.01, ***p<0.001. Inset area under the concentration vs. time curve (AUC).

DISCUSSION

We identified that female mice have higher circulating levels of (2R,6R;2S,6S)-HNK following ketamine treatment (Figure 2) and higher levels of (2R,6R)-HNK following the direct administration of (2R,6R)-HNK (Figure 4A), consistent with previous studies (Zanos et al., 2016; Highland et al., 2018). After ketamine treatment, female mice also produced lower plasma levels of ketamine and norketamine, compared to males (Figure 2). In contrast with the present data, one earlier study reported that female rats produced higher levels of ketamine and norketamine following ketamine administration, compared to males (Saland and Kabbaj, 2018), while a second study reported no sex-dependent differences in the levels of (R)-ketamine, (R)-norketamine, or (2R,6R)-HNK in C57BL/6 mice following (R)-ketamine administration (Chang et al., 2018). The apparent discrepancies could be, at least in part, accounted for by species- or strain-specific activity of different CYP isoforms. Of note, CYP3A, which is a major participant in the metabolism of ketamine, exhibits species-specific differences (Martignoni et al., 2006), and there is evidence that multiple CYP isoforms exhibit strain differences and/or strain-specific sex differences in mice (Lofgren et al., 2004). Overall, our data are consistent with sex-dependent differences in both the phase I metabolism of ketamine to (2R,6R)-HNK and (2S,6S)-HNK, and in the phase II metabolism and/or elimination of (2R,6R)-HNK and (2S,6S)-HNK in mice. In particular, the data suggest that in female mice ketamine is metabolized more rapidly and/or extensively to (2R,6R;2S,6S)-HNK, and (2R,6R;2S,6S)-HNK is eliminated more slowly than in males. Evidence suggests that production of (2R,6R;2S,6S)-HNK is required for the full antidepressant actions of ketamine (Zanos et al., 2016; Zanos et al., 2019a). Thus, the increased antidepressant potency of ketamine in female mice (Zanos et al., 2016; Carrier and Kabbaj, 2013; Franceschelli et al., 2015; Sarkar and Kabbaj, 2016; Saland et al., 2016; Saland et al., 2017) may be explained by increased production of (2R,6R;2S,6S)-HNK in females. However, we note that the relationship between HNK levels and antidepressant response following ketamine administration in humans requires further clarification, as some studies have reported a negative correlation between plasma levels of (2R,6R)- or (2R,6R;2S,6S)-HNK and antidepressant response to ketamine (Farmer et al., 2020; Grunebaum et al., 2019).

Additionally, we demonstrated that testosterone modulates the metabolism of ketamine and (2R,6R)-HNK in mice. We found that ovariectomy did not alter the levels of ketamine or its metabolites in female mice but that orchidectomy resulted in lower levels of ketamine and higher levels of (2R,6R;2S,6S)-HNK after ketamine treatment in males (Figure 3). An earlier study investigated the impact of the estrous cycle on ketamine’s metabolism in rats, but reported no differences between the levels of ketamine, norketamine, or DHNK measured in diestrus and proestrus female rats (Saland and Kabbaj, 2018), which is consistent with the lack of effect of ovariectomy in female mice observed here. Further, we demonstrated that testosterone replacement reversed the effect of orchidectomy in male mice (Figure 5), consistent with circulating testosterone mediating the observed sex-dependent metabolism differences. However, circulating male gonadal hormones may only partly explain the differences in ketamine metabolism. The differences in peak ketamine plasma levels and in total ketamine and (2R,6R;2S,6S)-HNK plasma levels were more robust when comparing intact females versus males (2-fold reduction in peak ketamine levels, 2-fold reduction in ketamine AUC, and 1.5-fold increase in (2R,6R;2S,6S)-HNK AUC) than for gonadectomized versus sham-operated males (1.4-fold reduction in peak ketamine levels, 1.5-fold reduction in ketamine AUC, and only 1.2-fold reduction in (2R,6R;2S,6S)-HNK AUC). Similarly, when comparing total plasma exposure following direct administration of (2R,6R)-HNK, more robust differences were observed for intact females versus males (2-fold) than for gonadectomized vs. sham-operated males (1.4-fold), although both females and gonadectomized males had similar (1.3-fold) increases in peak (2R,6R)-HNK concentrations compared to intact or sham-operated males, respectively.

Experiments were conducted 10 days after gonadectomy. It is possible that activating effects of circulating gonadal hormones persist to some degree greater than 10 days post-gonadectomy and, thus, it remains possible that gonadectomy may result in more robust metabolism changes after a more prolonged period prior to treatment. It is also possible that organizational effects of gonadal hormones, that are initiated during earlier stages in development and not altered by gonadectomy in adulthood, may partially contribute to metabolism differences. Furthermore, we note that gonadectomy resulted in changes in body weight compared to sham-operated controls, particularly a reduction in weight gain in male mice and an increase in weight gain in females (Table 1). The decreased weight gain observed in gonadectomized males, potentially due to reduced body fat (Krotkiewski et al., 1980), may itself result in pharmacokinetic changes. Finally, we note that, in the experiments with intact female mice, we did not control for estrous cycle as an experimental variable. However, intact female mice housed in our animal facility, where they are housed in cages with a closed air ventilation system and not exposed to the odors of male mice, do not cycle (unpublished data). Therefore, the intact females included in these experiments were expected to have relatively consistent levels of circulating gonadal hormones.

Our data indicate that testosterone, mediates metabolism of ketamine to (2R,6R;2S,6S)-HNK, potentially via multiple mechanisms. First, because both testosterone (Maenpaa et al., 1993; Krauser et al., 2004; Sohl et al., 2009; Kandel et al., 2017) and ketamine (Desta et al., 2012) undergo CYP3A4-mediated metabolism, it is possible that circulating testosterone attenuates metabolism of ketamine to form (2R,6R;2S,6S)-HNK via competition for the available enzyme. A second possibility, not mutually exclusive with the former, is that testosterone alters the activity and/or expression of enzymes involved in the metabolism of ketamine and (2R,6R)-HNK. Sex-dependent differences have been observed for several of the CYP450 enzymes involved in ketamine’s metabolism to HNKs, including increased CYP2A6, CYP2B6, and CYP3A4 expression and activity in females relative to males, in both humans and rodents (Yang et al., 2012; Waxman and Holloway, 2009; Gochfeld, 2017). Evidence suggest that sex-dependent differences in CYP450 activity are modulated by the gonadal hormones, including testosterone, via effects on growth hormone release from the pituitary gland (reviewed in Waxman and Holloway, 2009). Thus, a testosterone-dependent attenuation of the activity and/or expression of CYP2A6, CYP2B6, or CYP3A4 could underlie decreased metabolism of ketamine to form (2R,6R;2S,6S)-HNK in intact males, relative to females or gonadectomized controls.

Competition with testosterone may also underlie the changes in phase II metabolism or elimination of (2R,6R)-HNK. The HNKs undergo glucuronide conjugation, which is partly catalyzed by the glucuronosyltransferase isoform UGT2B4 (Moaddel et al., 2010). While testosterone has not been reported as a substrate for this specific isoform (Barre et al., 2007), it does undergo glucuronide conjugation catalyzed by other glucuronosyltransferase isoforms, predominantly UGT2B17 (Turgeon et al., 2001; Sten et al., 2009). If UGT2B17 also contributes to the glucuronidation of (2R,6R)-HNK, then competition with testosterone may also underlie the lower (2R,6R)-HNK levels following its direct administration to intact males, compared with females or gonadectomized males. Additionally, it is possible that testosterone modulates the phase II metabolism or elimination of (2R,6R)-HNK by altering the expression and/or activity of the enzymes involved in this metabolic process. In particular, it has been reported that males have higher expression of UGT2B17, in addition to UGT2B28 and UGT2A3 (Yang et al., 2012), which may additionally contribute to these differences. However, the contribution of UGT2B17, or other isoforms, to the glucuronidation of (2R,6R)-HNK remains unknown.

To evaluate whether sex-dependent metabolism differences also occur in humans, we compared the levels of ketamine, norketamine, and HNKs in plasma samples obtained from male and female subjects who received an antidepressant dose of ketamine (0.5 mg/kg, 40 min infusion) (Farmer et al., 2020). In the human samples, higher levels of ketamine and norketamine were observed in males compared to females (Figure 6A–B), consistent with what we observed in mice (Figure 2A–B). However, unlike in mice (Figure 2C), human plasma levels of (2R,6R;2S,6S)-HNK were similar between the sexes (Figure 6C). Human plasma levels of (2R,4R;2S,4S;2S,6R;2R,6S)-HNK (Figure 6D) were also similar between the sexes. While levels of (2R,4S;2S,4R;2S,5S;2R,5R)-HNK were initially higher in female subjects (at 40 min post-infusion), levels were similar between both sexes at later sampling time points (Figure 6E). The human samples were obtained following a 40-min intravenous ketamine infusion, whereas the mouse samples were obtained following an acute intraperitoneal injection. It is unknown whether sex-dependent differences in HNK levels may be observed at earlier times, such as during the infusion period, in humans. Nonetheless, the higher plasma levels of ketamine and norketamine observed in the plasma of male subjects suggests that the metabolism/elimination of ketamine and norketamine may be faster in females, compared to males, consistent with the observations in mice.

In humans, the observed sex-dependent differences in plasma AUC of ketamine and norketamine were not meaningfully changed when females who were on hormonal birth control and/or post-menopausal women were excluded from analysis, consistent with our hypothesis that testosterone, rather than estrogen or progesterone, modulates the sex-dependent metabolism differences. We note, however, that the study design did not allow us to determine whether testosterone levels were correlated with differences in ketamine and metabolite levels in humans, and further studies are required to definitely determine the mechanisms underlying the observed metabolism differences.

While the effect of biological sex on ketamine’s metabolism and antidepressant efficacy has not been studied extensively in humans, several studies have reported sex-dependent differences in the levels of ketamine and its metabolites following an infusion of ketamine (Zarate et al., 2012a) or in ketamine’s antidepressant outcomes or adverse effects, though the results have been mixed (Rybakowski et al., 2017; Coyle and Laws, 2015; Freeman et al., 2019; Niciu et al., 2014). Zarate et al. (2012a) observed significantly higher plasma levels of (2R,6R;2S,6S)-HNK, (2R,5R;2S,5S)-HNK, and DHNK in female patients, compared to males, while male subjects had significantly higher levels of the 6-hydroxyketamines. A prior human study reported that there was no statistically significant difference in plasma levels of ketamine between males and females, although only a single time point was evaluated and the study combined samples collected under various experimental conditions (Morgan et al., 2006). It has been reported that the antidepressant response to ketamine is more prevalent among males compared to females (Rybakowski et al., 2017), and a meta-analysis indicated that male sex was predictive of sustained antidepressant response 1-week after ketamine treatment (Coyle and Laws, 2015). However, other studies have reported no significant differences in the antidepressant response to ketamine between the sexes (Freeman et al., 2019; Niciu et al., 2014). Although a study of recreational ketamine users reported that females were more likely to experience discontinuation symptoms, including anxiety and dysphoria (Chen et al., 2014), following an antidepressant infusion of ketamine, one clinical trial found no significant difference in the frequency of adverse events between males and females (Freeman et al., 2019), while a second study reported that dissociative side effects were more prevalent in males (Derntl et al., 2019). An earlier human study found sex differences in the cognitive and anesthetic effects of ketamine infusion, suggesting greater sensitivity in males, have been reported (Morgan et al., 2006). Overall, these sex-dependent differences in ketamine and HNK levels are an important variable that should be considered in future studies.

ACKNOWLEDGEMENTS

This work was supported by NIH R01-MH107615 and RAI145211A, and VA Merit Awards 1I01BX004062 and 101BX003631-01A1 to TDG, and by the NIA (RM), NIMH (CAZ), and NCATS (CJT) NIH intramural research programs. The contents of this manuscript do not represent the views of the U.S. Department of Veterans Affairs or the United States Government.

Declaration of interest/funding:

RM and CAZ 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 in the treatment of depression and neuropathic pain. JNH, PZ, RM, CAZ, and TG are listed as co-inventors on patents or patent application for the pharmacology or use of (2R,6R)-hydroxynorketamine, (2S,6S)-hydroxynorketamine, and molecular variants relevant to the treatment of depression, anxiety, anhedonia, suicidal ideation and post-traumatic stress disorders. RM, PM, CAZ, and CT have assigned their patent rights to the U.S. government but will share a percentage of any royalties that may be received by the government. JNH, PZ and TG 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. This work was supported by NIH R01-MH107615 and RAI145211A, and VA Merit Awards 1I01BX004062 and 101BX003631-01A1 to TDG, and by the NIA (RM), NIMH (CAZ), and NCATS (CJT) NIH intramural research programs.

Footnotes

DECLARATION OF COMPETING INTERESTS

RM and CAZ 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 in the treatment of depression and neuropathic pain. JNH, PZ, RM, CAZ, and TG are listed as co-inventors on patents or patent application for the pharmacology or use of (2R,6R)-hydroxynorketamine, (2S,6S)-hydroxynorketamine, and molecular variants relevant to the treatment of depression, anxiety, anhedonia, suicidal ideation and post-traumatic stress disorders. RM, PM, CAZ, and CT have assigned their patent rights to the U.S. government but will share a percentage of any royalties that may be received by the government. JNH, PZ and TG 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.

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