Target deconvolution studies of (2R,6R)-hydroxynorketamine: an elusive search

Abstract

The off-label use of racemic ketamine and the FDA approval of (S)-ketamine are promising developments for the treatment of depression. Nevertheless, racemic ketamine and (S)-ketamine are controlled substances with known abuse potential and their use is associated with undesirable side effects. For these reasons, research efforts have focused on identifying alternatives. One candidate is (2R,6R)-hydroxynorketamine ((2R,6R)-HNK), a ketamine metabolite that in preclinical models lacks the dissociative and abuse properties of ketamine while retaining its antidepressant-like behavioral efficacy. (2R,6R)-HNK's mechanism of action however is unclear. The main goals of this study were to perform an in-depth pharmacological characterization of (2R,6R)-HNK at known ketamine targets, to use target deconvolution approaches to discover novel proteins that bind to (2R,6R)-HNK, and to characterize the biodistribution and behavioral effects of (2R,6R)-HNK across several procedures related to substance use disorder liability. We found that unlike (S)- or (R)-ketamine, (2R,6R)-HNK did not directly bind to any known or proposed ketamine targets. Extensive screening and target deconvolution experiments at thousands of human proteins did not identify any other direct (2R,6R)-HNK-protein interactions. Biodistribution studies using radiolabeled (2R,6R)-HNK revealed non-selective brain regional enrichment, and no specific binding in any organ other than the liver. (2R,6R)-HNK was inactive in conditioned place preference, open-field locomotor activity, and intravenous self-administration procedures. Despite these negative findings, (2R,6R)-HNK produced a reduction in immobility time in the forced swim test and a small but significant increase in metabolic activity across a network of brain regions, and this metabolic signature differed from the brain metabolic profile induced by ketamine enantiomers. In sum, our results indicate that (2R,6R)-HNK does not share pharmacological or behavioral profile similarities with ketamine or its enantiomers. However, it could still be possible that both ketamine and (2R,6R)-HNK exert antidepressant-like efficacy through a common and previously unidentified mechanism. Given its pharmacological profile, we predict that (2R,6R)-HNK will exhibit a favorable safety profile in clinical trials, and we must wait for clinical studies to determine its antidepressant efficacy.

Figure 1.. (2R,6R)-HNK does not bind nor activate known ketamine binding sites.

Competition binding assays of (2R,6R)-HNK (red) or reference ligands (MK-801 or naloxone, black) versus radioligands ([³H]-MK801, [³H]-DAMGO or [³H]-U69,593) labeling NMDA receptors (A), mu-opioid receptors, MOR, (B) or kappa-opioid receptors, KOR (C), respectively. The curves corresponding to (S)- and (R)-ketamine were published previously[9] and are displayed for reference. All binding assays were performed at the same time in rat whole brain (except cerebellum) membrane suspensions. In vitro signaling elicited by morphine or (2R,6R)-HNK in HEK-293 cells transiently transfected with MOR or KOR (D–F) and cAMP BRET-based biosensor. All data points are mean ± SEM of representative experiments performed in triplicate (experiments were performed 3–6 times to estimate the parameters (Ki, EC50 and Emax) or for statistical evaluation of the main effects of each drug reported in the main text.Figure 2.

Figure 2.. Target deconvolution assays and other suggested targets.

Receptor and enzyme competitive screen at two concentrations (100 nM and 10 μM) of (2R,6R)-HNK (A). Competition binding assays of (2R,6R)-HNK (red) or reference ligands (Substance P, LY341495 (on mGluR2) or pentazocine, black) versus radioligands ([³H]-Substance P, [³H]-LY341495 or [³H]-pentazocine) labeling Tachykinin receptors, NK1R (B), metabotropic glutamate receptors types 2 and 3, mGluR2/3 (C) or sigma-1 receptors (D), respectively. All binding assays were performed in rat whole brain (except cerebellum) membrane suspensions. All data points are mean ± SEM of representative experiments performed in triplicate (experiments were performed 3–6 times to estimate the parameters (Ki, EC50 and Emax) or for statistical evaluation of the main effects of each drug reported in the main text.Figure 3.

Figure 3.. Fast clearance and no brain regional specificity of (2R,6R)-HNK uptake.

Activity detected in serum and brain after bolus i.v. (1 μCi/g) administration of [³H](2R,6R)-HNK in rats (A). Saturation binding experiments using rat serum indicate the lack of high-affinity specific binding in serum proteins (B). Biodistribution of [³H](2R,6R)-HNK 30 min after IV administration, uptake was not blocked in any organ, except the liver, when pretreating the animals with 10 mg/kg of (2R,6R)-HNK (IP) (C). Lack of enrichment in any selected brain region of rats injected (i.v., 1μCi/g) with radiolabeled [³H](2R,6R)-HNK (D). Representative autoradiograms of coronal brain sections of rats injected (IV, 1μCi/g) with radiolabeled[³H](2R,6R)-HNK 30 min after i.v. administration with or without pretreatment with 10 mg/kg of (2R,6R)-HNK (IP) (E). Activity detected in plasma after bolus i.v. (100 μCi/g) administration of [³H](2R,6R)-HNK in rats (F). Whole body autoradiograms of rats injected with radiolabeled [¹⁴C](2R,6R)-HNK 30 min and 4h after bolus i.v. administration (10 mg/kg). All data points are mean ± SEM.

Figure 4.. Changes in metabolic activity induced by (2R,6R)-HNK differ from those produced by (S)-ketamine.

Brain metabolic mapping using [¹⁸F]-FDG PET scanning in rats. [¹⁸F]-FDG uptake images obtained after administration of saline (baseline, n = 4) or (2R,6R)-HNK (10 mg/kg over 40 min, n = 5). They show the average SUVR_WB (standardized uptake value ratio) calculated using the whole brain as a reference region (A,B). Voxel-based parametric mapping analyses revealed significantly increased metabolic activity from baseline values in areas such as the insula, nucleus accumbens, globus pallidus, dorsomedial thalamus, motor, sensory, and visual cortices, and midline cerebellum. Statistical parametric maps of significant decreases of [¹⁸F]-FDG uptake (P < 0.05, paired t test) (C). Regional [¹⁸F]-FDG uptake (SUVR_WB) in the brain regions of animals infused with saline or (2R,6R)-HNK (D). Correlation matrices of the metabolic activity across brain regions of animals infused with saline, (2R,6R)-HNK or (S)-ketamine. The data corresponding to the (S) ketamine dataset was reported in [9]. Full data sets are available in Supplemental Information 6 (E).

Figure 5.. Lack of effects of (2R,6R)-HNK on locomotor activity, CPP and self-administration.

Forced Swimming Test (A): male mice were tested in the forced swimming test 24 hours after a single administration of (2R,6R)-HNK (1, 3 and 10 mg/kg, IP). Plots display immobility time and are representing individual data points with bars indicating mean ± SEM. *** and **** denote statistical significance between groups (P < 0.001 and P < 0.0001, respectively). Acute locomotor activity (B): male mice were and injected with saline or (2R,6R)-HNK (10, 30, and 65 mg/kg, IP) and their locomotor activity was measured. Plots display distance traveled in open field arenas in 5min time bins. CPP: Male mice were injected with (2R,6R)-HNK (0, 10, 30 or 65 mg/kg) or saline on either side of the CPP arena for 6 days and were allowed to explore both sides. Plots in (C) display the distance traveled during the consecutive conditioning sessions. At the end of the conditioning phase the preference for the drug-paired side was quantified as a CPP score (D). In all cases, data points are displayed as mean ± SD of 8 mice per condition or individual data points with bars displaying mean ± SEM. Self-administration: Self-administration training: mean ± SEM number of infusions of saline, (2R,6R)-HNK or (S)-ketamine under the FR1 20-s timeout reinforcement schedule (E). Dose–response curve: mean ± SEM number of infusions for different unit doses of (2R,6R)-HNK or (S)-ketamine under the FR1 20-s timeout reinforcement schedule (F). Data from the (S)-ketamine animals was previously published in [9]. In (G), rats (n=16) were trained to press for heroin and after steady responding it was substituted by either (2R,6R)-HNK or (S)-ketamine. ^$ denotes statistical significance with respect to heroin infusions within the same group (P < 0.05) and * denotes statistical significance between groups (P < 0.01). In all cases data points represent mean ± SEM of number of infusions.