Sex Differences in the Pharmacokinetics of Low-dose Ketamine in Plasma and Brain of Male and Female Rats

Abstract

Recent work from our group and others has revealed a higher sensitivity of female rodents to the antidepressant-like effects of the N-methyl d-aspartate receptor antagonist ketamine strongly influenced by circulating estrogen and progesterone levels. However, in the absence of any preclinical studies of pharmacokinetic sex differences using low-dose ketamine in rats, it is unclear whether the effects of sex and hormonal milieu on ketamine's behavioral actions are influenced by differences in ketamine metabolism between male and female rats. Therefore, this work examined whether sex and hormonal status affect ketamine metabolism and distribution in male and female rats using a low antidepressant-like dose selectively effective in females. Intact male rats and female rats in either diestrus (low estrogen, progesterone) or proestrus (high estrogen, progesterone) were administered low-dose ketamine, and their plasma and brains were collected to analyze levels of ketamine and its metabolites norketamine (NK) and dehydronorketamine. Females exhibited greater concentrations of ketamine and NK over the first 30 min following treatment in both brain and plasma, largely accounted for by slower clearance rates and longer half-lives. Interestingly, despite the impact of ovarian hormones on behavioral sensitivity to ketamine, no appreciable differences in pharmacokinetic parameters existed between proestrus and diestrus female rats. This work is the first to demonstrate sex differences in ketamine pharmacokinetics in rats, and suggests that while sex differences in metabolism may influence the amount of ketamine and NK reaching target areas in the brain, the impact of circulating hormone levels here is negligible.

Fig. 1.

Pharmacokinetics experimental design and sample preparation workflow. AUC, area under the curve; DHNK, dehydronorketamine; HCl, hydrochloric acid; HPC, hippocampus; K, ketamine; LC-MS/MS, liquid chromatography-tandem mass spectrometry; MeOH, methanol; mPFC, medial prefrontal cortex; NK, norketamine; PK, pharmacokinetic.

Fig. 2.

Calibration curves for ketamine and metabolite standards in blank plasma and brain matrices. Linearity (R² > 0.9995) was observed for ketamine (K), norketamine (NK), and dehydronorketamine (DHNK) calibrants (Cal) within a range of 1–500 ng/ml for plasma (a) and 5–1000 ng/g for brain (b). Response depicted as the ratio of the peak area (AUC) of each analyte to that of the internal standard (IS). Data expressed as mean of three technical replicates ±S.E.M.

Fig. 3.

Plasma concentration-time profiles of ketamine and its metabolites in male and cycling female rats. (a and b) Females displayed greater levels of ketamine (K) rapidly upon intraperitoneal administration at 5 (P = 0.0367) and 10 (P = 0.0385) min postdose, relative to their male counterparts, regardless of hormonal status. (c and d) Higher concentrations of the norketamine (NK) metabolite were also subsequently observed in female rats at 30 (P = 0.0013)- and 90-min (P = 0.0111) time points compared with males, in an estrous-cycle independent manner. (e) However, dehydronorketamine (DHNK) was significantly elevated in male compared with female rats 10 (P = 0.0328) and 30 (P = 0.0031) min after ketamine administration. (f) Estrous cycle had minimal influence on DHNK levels in females. (g–j) Superimposed K, NK, and DHNK concentrations reveal greater metabolite-to-parent concentrations in males than in females through the first 30 min posttreatment, despite their overall lower levels of K and NK during this time. *P < 0.05 vs. male, **P < 0.01 vs. male.

Fig. 4.

Brain concentration-time profiles of ketamine and norketamine in male and cycling female rats. (a and e) Concentrations of ketamine (K) were ∼2-fold greater in female compared with male rats 5–10 min (P < 0.0001) following administration in the hippocampus (HPC) and medial prefrontal cortex (mPFC), regardless of estrous cycle stage (b and f). (c and g) These differences were more pronounced for its metabolite norketamine (NK), whose levels in females were significantly higher 5–60 min posttreatment in both regions (P < 0.0001) compared with those in males. (d and h) While NK levels in the HPC were higher in diestrus than in proestrus females 5 min after ketamine treatment (P = 0.0413), hormonal status had minimal effect on distribution of the parent drug and its metabolite within the HPC and mPFC. Data are expressed as mean ± S.E.M. (n = 3 to 4/group/time point). **P < 0.0001 vs. male, *P < 0.05 vs. proestrus.

Fig. 5.

Metabolite ratios and brain distribution of ketamine and norketamine in male and cycling female rats. (a) Ratio of norketamine to ketamine area under the concentration-time curve (AUC) values in plasma and brain of male (n = 3 to 4/time point) and female (n = 7 to 8/time point) rats (brain regions were averaged due to overall similarity). (b and c) Cumulative brain-to-plasma AUC ratios for ketamine and norketamine, respectively, over time in male and female rats. Data expressed as ratios of AUC_0-t values calculated using pooled group data from biologic replicate measurements at each time point—plasma (ng/ml) and brain tissue (ng/g) concentrations were converted into micromolar (µM) units beforehand for direct comparison between analytes. AUC_0-180 ratio: total analyte exposure across all time points measured for each analysis, depicted by gray vertical bar. N.Q., not quantifiable, ketamine detected in brain samples < lower limit of quantitation 90–180 min; here, AUC_0-60 represents total exposure up to this time point, indicated by a dashed vertical bar for comparison.