(R,S)-Ketamine metabolites (R,S)-norketamine and (2S,6S)-hydroxynorketamine increase the mammalian target of rapamycin function

Abstract

Background: Subanesthetic doses of (R,S)-ketamine are used in the treatment of neuropathic pain and depression. In the rat, the antidepressant effects of (R,S)-ketamine are associated with increased activity and function of mammalian target of rapamycin (mTOR); however, (R,S)-ketamine is extensively metabolized and the contribution of its metabolites to increased mTOR signaling is unknown.

Methods: Rats (n = 3 per time point) were given (R,S)-ketamine, (R,S)-norketamine, and (2S,6S)-hydroxynorketamine and their effect on the mTOR pathway determined after 20, 30, and 60 min. PC-12 pheochromocytoma cells (n = 3 per experiment) were treated with escalating concentrations of each compound and the impact on the mTOR pathway was determined.

Results: The phosphorylation of mTOR and its downstream targets was significantly increased in rat prefrontal cortex tissue by more than ~2.5-, ~25-, and ~2-fold, respectively, in response to a 60-min postadministration of (R,S)-ketamine, (R,S)-norketamine, and (2S,6S)-hydroxynorketamine (P < 0.05, ANOVA analysis). In PC-12 pheochromocytoma cells, the test compounds activated the mTOR pathway in a concentration-dependent manner, which resulted in a significantly higher expression of serine racemase with ~2-fold increases at 0.05 nM (2S,6S)-hydroxynorketamine, 10 nM (R,S)-norketamine, and 1,000 nM (R,S)-ketamine. The potency of the effect reflected antagonistic activity of the test compounds at the α7-nicotinic acetylcholine receptor.

Conclusions: The data demonstrate that (R,S)-norketamine and (2S,6S)-hydroxynorketamine have potent pharmacological activity both in vitro and in vivo and contribute to the molecular effects produced by subanesthetic doses of (R,S)-ketamine. The results suggest that the determination of the mechanisms underlying the antidepressant and analgesic effects of (R,S)-ketamine requires a full study of the parent compound and its metabolites.

Figure 1

Structures of (R,S)-ketamine, (R,S)-norketamine and (2S,6S;2R,6R)-hydroxynorketamine. (R,S)-Ketamine undergoes hepatic metabolism to be transformed into (2S,6S;2R,6R)-hydroxynorketamine directly and/or via (R,S)-norketamine as an intermediate.

Figure 2

A representative chromatogram from a 60-min brain sample obtained after a single intraperitoneal injection of (R,S)-ketamine, 40 mg·kg⁻¹ in saline (5 ml·kg⁻¹) (A), after a single intravenous injection of (R,S)-norketamine, 20 mg·kg⁻¹ in saline (5 ml·kg⁻¹) (B), and after a single intravenous injection of (2S,6S)-hydroxynorketamine, 20 mg·kg⁻¹ in saline (5 ml·kg⁻¹) (C). The labeled peaks correspond to (R,S)-ketamine (1), (R,S)-norketamine (2), (2S,6S;2R,6R)-hydroxynorketamine (4a), (2S,6R;2R,6S)-hydroxynorketamine (4b), (2S,5S;2R,5R)-hydroxynorketamine (4c), (2S,4S;2R,5R)-hydroxynorketamine (4d) and (2S,4R;2R,4S)-hydroxynorketamine (4f).

Figure 3

Levels of phospho-active forms of mTOR, ERK1/2, Akt, 4E-BP1 and p70S6K in rat brain (cortex) tissues at different time intervals (0 – 240 min) after administration of either (R,S)-ketamine, (R,S)-norketamine or (2S,6S)-hydroxynorketamine. The mode of administration and dosage protocol of the test compounds are described in the legend of Figure 2. A–C, Representative immunoblots using the indicated primary antibodies. D–F, Scatter plots illustrating the relative levels of phosphorylated and total forms of mTOR, p70S6K and 4E-BP1 in response to (R,S)-ketamine (D), (R,S)-norketamine (E) and (2S,6S)-hydroxynorketamine (F) are shown (n= 3 independent experiments). *, **, P< 0.05, 0.01 (ANOVA) compared with controls.

Figure 4

Expression of monomeric serine racemase (m-SR) protein in PC-12 cells after 36 h incubation with different concentrations of (R,S)-ketamine (Ket, 0 – 10 μM) (A), (R,S)-norketamine (NK, 0 – 1 μM) (B), and (2S,6S)-hydroxynorketamine (HNK, 0 – 0.1 μM) (C). A–C, Representative Western blot analysis with primary antibodies raised against serine racemase and β-actin. D, Scatter plots illustrating the relative levels of m-SR in response to 600 nM (R,S)-ketamine (Ket), 10 nM (R,S)-norketamine (NK) and 0.05 nM (2S,6S)-hydroxynorketamine (HNK) after quantification and normalization with β-actin (n= 3 independent experiments). *, **, P< 0.05, 0.01 (ANOVA) compared with control cells.

Figure 5

Effect of (R,S)-ketamine on the levels of phospho-active forms of mTOR, Akt, ERK1/2, p70S6K and 4E-BP1 in PC-12 cells. A, Cells were treated with different concentrations of (R,S)-ketamine (0 – 10 μM) for 1 h and processed for Western blot analysis. (B), Scatter plots illustrating the relative ratio of phosphorylated versus total forms of mTOR, Akt, ERK1/2, p70S6K and 4E-BP1 in response to cell treatment with 600 nM of (R,S)-ketamine are shown (n = 3 independent experiments). ** P< 0.01 (ANOVA) compared with control cells.

Figure 6

Effect of (R,S)-norketamine on the levels of phospho-active forms of mTOR, Akt, ERK1/2, p70S6K and 4E-BP1 in PC-12 cells. A, Cells were treated with different concentrations of (R,S)-norketamine (0 – 1 μM) for 1 h and processed for Western blot analysis. (B), Scatter plots illustrating the relative ratio of phosphorylated versus total forms of mTOR, Akt, ERK1/2, p70S6K and 4E-BP1 in response to cell treatment with 25 nM of (R,S)-norketamine are shown (n = 3 independent experiments). *, ** P< 0.05, 0.01 (ANOVA) compared with control cells.

Figure 7

Effect of (2S,6S)-hydroxynorketamine on the levels of phospho-active forms of mTOR, Akt, ERK1/2, p70S6K and 4E-BP1 in PC-12 cells. A, Cells were treated with different concentrations of (2S,6S)-hydroxynorketamine (0 – 0.1 μM) for 1 h and processed for Western blot analysis. (B), Scatter plots illustrating the relative ratio of phosphorylated versus total forms of mTOR, Akt, ERK1/2, p70S6K and 4E-BP1 in response to cell treatment with 0.5 nM of (R,S)-hydroxynorketamine are shown (n = 3 independent experiments). *, **, P< 0.05, 0.01 (ANOVA) compared with control cells.

Figure 8

Effects of (R,S)-ketamine (Ket, 1 μM), (R,S)-norketamine (NK, 10 nM), (2S,6S)-hydroxynorketamine (HNK, 0.1 nM) and methyllycaconitine (MLA, 50 nM) with or without nicotine (2 μM) on the levels of monomeric serine racemase (m-SR) protein in PC-12 cells. A, Representative immunoblots of m-SR and β-actin. B, Scatter plot illustrating the relative m-SR levels after quantification and normalization with β-actin (n = 3 independent experiments). *, P< 0.05 (ANOVA) compared with control cells.

Figure 9

Effects of (R,S)-ketamine, (R,S)-norketamine, (2S,6S)-hydroxynorketamine and methyllycaconitine with or without nicotine on the levels of phospho-active forms of mTOR, Akt, ERK1/2, p70S6K and 4E-BP1 in PC-12 cells. A, Cells were treated with (R,S)-ketamine (Ket, 1 μM), (R,S)-norketamine (NK, 10 nM), (2S,6S)-hydroxynorketamine (HNK, 0.1 nM) or methyllycaconitine (MLA, 50 nM) with or without nicotine (2 μM) for 1 h and then processed for Western blot analysis. A, Representative immunoblots. B–F, Scatter plots illustrating the relative ratio of phosphorylated versus total forms of mTOR (B), Akt (C), ERK1/2 (D), p70S6K (E) and 4E-BP1 (F) are shown (n= 3 independent experiments). *, **, P< 0.05, 0.01 (ANOVA) compared with control cells.

Figure 10

Schematic representation of the modulation of serine racemase expression functioning via mTOR and ERK pathways. Abbreviations: Ket, (R,S)-ketamine; NorKet, (R,S)-norketamine; HNK, (2S,6S)-hydroxynorketamine; nAchR, nicotinic acetylcholine receptor; mTORC1, mammalian target of rapamycin complex 1; mTORC2, mammalian target of rapamycin complex 2; SR, serine racemase.