Pharmacological and behavioral divergence of ketamine enantiomers: implications for abuse liability

Abstract

Ketamine, a racemic mixture of (S)-ketamine and (R)-ketamine enantiomers, has been used as an anesthetic, analgesic and more recently, as an antidepressant. However, ketamine has known abuse liability (the tendency of a drug to be used in non-medical situations due to its psychoactive effects), which raises concerns for its therapeutic use. (S)-ketamine was recently approved by the United States' FDA for treatment-resistant depression. Recent studies showed that (R)-ketamine has greater efficacy than (S)-ketamine in preclinical models of depression, but its clinical antidepressant efficacy has not been established. The behavioral effects of racemic ketamine have been studied extensively in preclinical models predictive of abuse liability in humans (self-administration and conditioned place preference [CPP]). In contrast, the behavioral effects of each enantiomer in these models are unknown. We show here that in the intravenous drug self-administration model, the gold standard procedure to assess potential abuse liability of drugs in humans, rats self-administered (S)-ketamine but not (R)-ketamine. Subanesthetic, antidepressant-like doses of (S)-ketamine, but not of (R)-ketamine, induced locomotor activity (in an opioid receptor-dependent manner), induced psychomotor sensitization, induced CPP in mice, and selectively increased metabolic activity and dopamine tone in medial prefrontal cortex (mPFC) of rats. Pharmacological screening across thousands of human proteins and at biological targets known to interact with ketamine yielded divergent binding and functional enantiomer profiles, including selective mu and kappa opioid receptor activation by (S)-ketamine in mPFC. Our results demonstrate divergence in the pharmacological, functional, and behavioral effects of ketamine enantiomers, and suggest that racemic ketamine's abuse liability in humans is primarily due to the pharmacological effects of its (S)-enantiomer.

Figure 1.. Divergent pharmacodynamics of ketamine enantiomers.

Receptor and enzyme competitive screen at two concentrations (100 nM and 10 μM) of S- and (R)-ketamine (A). Competition binding assays of (S)-ketamine (orange) or (R)-ketamine (blue) enantiomers versus radioligands labeling NMDA receptors (B), opioid receptors (C): MOR (solid circles) and KOR receptors (open squares) or sigma receptors (D): Sigma-1 (solid circles) and Sigma-2 (open squares) receptors. All binding assays were performed in rat whole brain (except cerebellum) membrane suspensions. In vitro signaling elicited by morphine or ketamine enantiomers in HEK-293 cells transiently transfected with MOR or KOR (E-G): ketamine enantiomers activate the G-protein (E) but not ß-arrestin signaling (F), and do not inhibit morphine G-protein signaling (G). All data points are mean ± SEM of representative experiments performed in triplicate (experiments were performed 3 to 6 times to estimate the parameters (K_i, EC₅₀ and E_max) reported in the main text.

Figure 2.. Rapid brain uptake, fast clearance and no high affinity target for ketamine enantiomers.

Representative PET images obtained after bolus IV administration of [¹¹C](S)-ketamine (A) or [¹¹C](R)-ketamine (C) at different time windows. After the initial brain uptake, activity remains exclusively in the harderian glands (nonspecific accumulation). In B and D, whole brain time activity curves measured after a bolus administration of either [¹¹C]-ketamine enantiomer preceded by a pretreatment with the cold enantiomer (10 mg/kg IP) or the NMDAR non-competitive antagonists (+)-MK-801 (0.1mg/kg, IP). Data points are mean ± SD of standardized uptake values (SUV, g/ml) obtained from at least two PET images per condition. In E, representative autoradiograms of coronal brain sections of rats injected (IV, 1μCi/g) with radiolabeled [³H](S)-ketamine and [³H](R)-ketamine and euthanized at 40 min post injection. In F, biodistribution of [³H](S)-ketamine and [³H](R)-ketamine 40 min after IV administration. Saturation binding experiments using membrane and cytosolic fractions of rat brain homogenates indicate the lack of a high affinity specific binding (displaceable and saturable) (G-H).

Figure 3.. Functional brain imaging reveals regional differences in the effects of ketamine enantiomers.

A to C, (S)- or (R)-ketamine infusions (15mg/kg/h) have differential effects on metabolic activity evaluated as accumulation and trapping of [¹⁸F]FDG in the brains of awake, freely-moving rats. The rats were anesthetized and scanned 40 min after drugs and [¹⁸F]FDG (decay half-life =~110 min) were administered, providing a “snapshot” of metabolic activity during the awake, freely-moving state. Brain-wide voxel-based analysis was used to evaluate differences on activity using a one-way ANOVA. Color shaded areas in B and C represent clusters of voxels (≥100) with significant (p<0.05) increases or decreases in metabolic rate compared to saline infusions. D to F, G-protein signaling induced by ketamine enantiomers in the PFC indicates MOR-mediated signaling by (S)-ketamine. G to K, changes in dopamine tone elicited by infusion of the ketamine enantiomers evaluated as [¹⁸F]-Fallypride (dopamine D2 antagonist) uptake in awake freely moving animals (G). After ketamine and [¹⁸F]fallypride animals were anesthetized and a static PET image was acquired for 30 minutes (G). PET images (I-J) were analyzed using both region of interest-wise and voxel-wise analysis methods. In K, shaded areas represent clusters of voxels (≥100) with significant (p<0.05) increases or decreases in dopamine tone between (S)- and (R)-ketamine. Abbreviations: CPu, Caudate-Putamen; Acb, Nucleus Accumbens; Pit, Pituitary gland.

Figure 4.. Behavioral effects of ketamine enantiomers: locomotor activity, conditioned place preference, and drug self-administration.

(A) Acute locomotor activity: Male mice were placed in an open field arena and injected with saline and increasing doses of (S)-ketamine or (R)-ketamine (5, 10 and 20 mg/kg, IP) every 30 min, with or without pretreatment (10 min) with naltrexone (10 mg/kg, IP). Plots display distance traveled in open field arenas for in 5 min time bins, * denote statistical significance (p<0.05) between (S)-ketamine and (S)-ketamine with naltrexone pretreatment. (B-D) Locomotor sensitization after repeated injections: Male mice were habituated to the arenas for two days (Hab1–2) and then injected with vehicle (saline, black), (S)-ketamine (20 mg/kg, orange), (R)-ketamine (20 mg/kg, blue) for three days (D1–3). On day 8, the mice received increasing doses of (S)-ketamine (5, 10 and 20 mg/kg). Plots display distance traveled in open field arenas for 30 min after each drug. ** denote statistical significance (p<0.01) between (S-) and (R)-ketamine groups. (E-F) CPP: Male mice were injected with one of the ketamine enantiomers (10 mg/kg) or saline on either side of the CPP arena for 6 days and were allowed to explore both sides at the end of the conditioning phase. The preference for the drug-paired side was quantified as a CPP score. Data points are displayed as mean ± SD of 12 mice per condition or individual data points. * denotes statistical significance (p<0.05) from the pre-test condition. (G-M) Self-administration and extinction: (G) Experimental timeline for self-administration and extinction in male and female rats. (H-J) Ketamine self-administration training: mean±SEM number of infusions and active and inactive lever presses under the FR1 20-s timeout reinforcement schedule. (K) Dose response curve: mean±SEM number of infusions for different unit doses of the two enantiomers under the FR1 20-s timeout reinforcement schedule. (L) Progressive ratio: mean±SEM number of infusions and breakpoint (final ratio completed) for (S)-ketamine and (R)-ketamine under the progressive ratio reinforcement schedule. (M) Extinction responding: mean±SEM number of active lever presses during the last retraining session under the FR1 20-s timeout reinforcement schedule (denoted as SA) and during the subsequent extinction session. During the extinction session, lever presses led to contingent presentations of the discrete cue previously paired with (S)-ketamine and (R)-ketamine self-administration training and retraining, but not drug infusions**: significant difference (p < 0.01) between groups. Abbreviations: SA, self-administration; Ext, extinction, (S)-ketamine, (S)-ket; (R)-ketamine, (R)-ket; FR1, fixed ratio 1; NLTX, naltrexone.