Neuropharmacology and analgesia
Sub-anesthetic concentrations of (R,S)-ketamine metabolites inhibit acetylcholine-evoked currents in α7 nicotinic acetylcholine receptors

https://doi.org/10.1016/j.ejphar.2012.11.023Get rights and content

Abstract

The effect of the (R,S)-ketamine metabolites (R,S)-norketamine, (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine on the activity of α7 and α3β4 neuronal nicotinic acetylcholine receptors was investigated using patch-clamp techniques. The data indicated that (R,S)-dehydronorketamine inhibited acetylcholine-evoked currents in α7-nicotinic acetylcholine receptor, IC50=55±6 nM, and that (2S,6S)-hydroxynorketamine, (2R,6R)-hydroxynorketamine and (R,S)-norketamine also inhibited α7-nicotinic acetylcholine receptor function at concentrations ≤1 μM, while (R,S)-ketamine was inactive at these concentrations. The inhibitory effect of (R,S)-dehydronorketamine was voltage-independent and the compound did not competitively displace selective α7-nicotinic acetylcholine receptor ligands [125I]-α-bungarotoxin and [3H]-epibatidine indicating that (R,S)-dehydronorketamine is a negative allosteric modulator of the α7-nicotinic acetylcholine receptor. (R,S)-Ketamine and (R,S)-norketamine inhibited (S)-nicotine-induced whole-cell currents in cells expressing α3β4-nicotinic acetylcholine receptor, IC50 3.1 and 9.1 μM, respectively, while (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine were weak inhibitors, IC50 >100 μM. The binding affinities of (R,S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine at the NMDA receptor were also determined using rat brain membranes and the selective NMDA receptor antagonist [3H]-MK-801. The calculated Ki values were 38.95 μM for (S)-dehydronorketamine, 21.19 μM for (2S,6S)-hydroxynorketamine and>100 μM for (2R,6R)-hydroxynorketamine. The results suggest that the inhibitory activity of ketamine metabolites at the α7-nicotinic acetylcholine receptor may contribute to the clinical effect of the drug.

Introduction

(R,S)-Ketamine (Fig. 1) is a chiral phencyclidine derivative that was developed as an anesthetic agent (Hirota and Lambert, 2011). (R,S)-Ketamine is extensively transformed by microsomal enzymes into a number of metabolites including (R,S)-norketamine, (R,S)-dehydronorketamine and a series of hydroxynorketamines including (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine (Fig. 1) (Trevor et al., 1983), and the P450 isoforms associated with these transformation have been recently identified (Desta et al., 2012). Initial studies utilizing (R,S)-ketamine, (R,S)-norketamine and (2S,6S;2R,6R)-hydroxynorketamine determined that the CNS activities associated with general anesthesia and recovery were produced by (R,S)-ketamine and (R,S)-norketamine but not (2S,6S;2R,6R)-hydroxynorketamine (Leung and Baillie, 1986). Thus, (R,S)-ketamine was identified as an anesthetic agent, (R,S)-norketamine as an “active” metabolite and (2S,6S;2R,6R)-hydroxynorketamine was considered inactive. When subsequent studies indicated that (R,S)-ketamine and (R,S)-norketamine inhibited the NMDA receptor, this activity became the accepted explanation of the pharmacological effects of (R,S)-ketamine and (R,S)-norketamine (Hirota and Lambert, 2011).

Since the operating hypothesis was centered upon the activity of (R,S)-ketamine and (R,S)-norketamine, most metabolic, pharmacokinetic, pharmacological and clinical studies have concentrated on these compounds. However, while this approach may be valid for anesthetic dosing of (R,S)-ketamine, it does not appear to be applicable to sub-anesthetic dosing of the compound, which is currently being investigated for treatment of neuropathic and acute pain and depression (Hirota and Lambert, 2011). For example, a population-pharmacokinetic analysis of patients receiving low-dose (R,S)-ketamine for the treatment of bipolar depression demonstrated that following the distribution of (R,S)-ketamine and (R,S)-norketamine may not accurately reflect the pharmacodynamics and that (2S,6S)-hydroxynorketamine, (2R,6R)-hydroxynorketamine and (R,S)-dehydronorketamine were major plasma components (Zhao et al., 2012). In addition, a study of clinical response in 67 patients treated with low-dose (R,S)-ketamine for major depressive disorder and bipolar depression indicated plasma concentrations of (R,S)-dehydronorketamine, (2S,5S;2R,5R)-hydroxynorketamine, and (2S,5R;2R,5S)-hydroxynorketamine were associated with lower psychotomimetic or dissociative side effects (Zarate et al., 2012). Thus, the observed clinical responses produced by low dose (R,S)-ketamine may be due to unexplored pharmacological activities of (R,S)-ketamine metabolites.

Recent studies have demonstrated that mecamylamine has therapeutic efficacy in the treatment of depression and that this effect may be related to its activity as an open-channel non-competitive inhibitor of neuronal nicotinic acetylcholine receptors (Philip et al., 2010). (R,S)-Ketamine is also an open-channel non-competitive inhibitor of homomeric α7 and heteromeric (α4β2, α3β4) nicotinic acetylcholine receptor subtypes (Yamakura et al., 2000; Moaddel et al., 2005), but little is known about the activity of (R,S)-ketamine metabolites at the nicotinic acetylcholine receptor. This study was designed to assess the effect of (R,S)-norketamine, (2S,6S)-hydroxynorketamine, (2R,6R)-hydroxynorketamine and (R,S)-dehydronorketamine on agonist-induced whole-cell current in cells expressing α3β4-nicotinic acetylcholine receptor and α7-nicotinic acetylcholine receptor and their binding affinities to the NMDA receptor. In addition, the binding affinities of (R)- and (S)-dehydronorketamine at the α7-nicotinic acetylcholine receptor were determined.

Section snippets

Materials

(R,S)-Ketamine, (R)-ketamine, (S)-ketamine, (R,S)-norketamine, (R)-norketamine, (S)-norketamine, (R,S)-dehydronorketamine, (R)-dehydronorketamine, (S)-dehydronorketamine, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine were prepared as previously described (Moaddel et al., 2010). Minimum Essential Medium with Earles Salts and l-glutamine (MEM), fetal bovine serum (FBS), penicillin, streptomycin, geneticin were purchased from Gibco (Carlsbad, CA, USA). (±)-Epibatidine dihydrochloride, (

The effect on α7-nicotinic acetylcholine receptor activity

The effect of the test compounds on the function of α7-nicotinic acetylcholine receptors was evaluated using patch-clamp technique in the whole-cell configuration. Initial studies examined their ability to induce current activation at concentrations equal to 10, 50 and 200 nM. No measurable effect was observed at any tested concentration (n=4), as illustrated for (R,S)-dehydronorketamine (Fig. 2).

The ability of the test compounds to affect acetylcholine-induced current was assessed using 280 μM

Discussion

Numerous in vitro studies have determined that (R,S)-ketamine is extensively metabolized by microsomal enzymes producing (R,S)-norketamine (Trevor et al., 1983, Kharasch and Labroo, 1992, Desta et al., 2012), (R,S)-dehydronorketamine (Bolze and Boulieu, 1998, Desta et al., 2012) and a series of diastereomeric hydroxynorketamine metabolites (Adams et al., 1981, Trevor et al., 1983, Woolf and Adams, 1987). The in vitro data were confirmed in studies in healthy volunteers (Turfus et al., 2009) and

Disclosure

Dr. Zarate (CAZ) is listed as a co-inventor on a patent application for the use of ketamine and its metabolites in major depression. CAZ has assigned his rights to the US government but will share a percentage of any royalties that may be received by the government. CAZ, IWW, and RM have submitted a patent, assigned to the US government, for use of ketamine metabolites in treatment of bipolar disorder and major depression.

Acknowledgements

This work was supported by the Intramural Research Programs of the National Institute of Aging (IWW) and the National Institute of Mental Health (CAZ), the Brain & Behavior Research Foundation Bipolar Disorders Award (CAZ), NIA contract number N01AG-3–1009 (GA,LT,LJ), the Foundation for Polish Science (FOCUS and TEAM Programmes) (KJ).

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