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

This rodent study (2014) argues that a full analysis of (R,S)-ketamine’s metabolites is required to understand ketamine’s anti-depressive and analgesic effects.

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.”

Authors: Rajib K. Paul, Nagendra S. Singh, Mohammed Khadeer, Ruin Moaddel, Mitesh Sanghvi, Carol E. Green, Kathleen O’Loughlin, Marc C. Torjman, Michel Bernier & Irving W. Wainer

Summary

(R,S)-KETAMINE, an antidepressant agent used in the treatment of major depressive disorder and bipolar depression, increases the phosphorylation of several proteins and increases the number and function of new spine synapses in the prefrontal cortex.

(R,S)-Ketamine is extensively transformed into (R,S)-norketamine, two diastereomeric hydroxyketamines, a series of diastereomeric hydroxynorketamines, and (R,S)-dehydronorketamine in the rat.

What This Article Tells Us That Is New

Subanesthetic doses of (R,S)-ketamine increase mTOR activity and function in the rat, but the contribution of its metabolites is unknown.

The rapid and extensive metabolic transformation of (R,S)-ketamine is also observed in studies using human microsomal preparations10 and in clinical studies2,3,11. The individual enantiomers of the compound, (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine, lack anesthetic activity but are potent and selective inhibitors of the nAChR.

In this study, male Wistar rats were administered (R,S)-ketamine, (R,S)-norketamine, and (2S,6S)-hydroxynorketamine, and the results were compared with the results obtained in PC-12 cells. The results indicated that (2S,6S)-hydroxynorketamine was the most potent of the three compounds in the suppression of intracellular D-serine levels.

Studies in the Wistar Rat

Male Wistar rats were obtained from Harlan (Livermore, CA) and housed one to three rats per cage, in polycarbonate hanging cages. They had ad libitum access to Harlan Teklad Certified Rodent Chow #2018C and reverse osmosis purified water.

(R,S)-ketamine, (R,S)-norketamine, and (2S,6S)-hydroxynorketamine were administered to rats. After treatment with ketamine, the rats were euthanized with pentobarbital, and the brains were collected and stored frozen at 70°C until analysis.

Brain tissue samples were prepared from drug-free male Wistar rats and used for the determination of ketamine, norketamine, and (2S,6S)-hydroxynorketamine concentrations.

The concentration of (R,S)-ketamine, (R,S)-norketamine, and (2S,6S)-hydroxynorketamine in brain tissue samples was measured using a liquid chromatographic method using mass spectrometric detection, which had been validated for use with clinical samples.

Western blotting was performed on cells and brain tissues and consisted of lysing the tissues with a PRO200 hand homogenizer and then centrifuging the lysates at 14,000g for 20 min at 4°C.

The authors performed Western blotting experiments on phosphorylated forms of ERK1/2, Akt, mTOR, 4E-BP1, p70S6K, and SR using sodium dodecyl sulfate-polyacrylamide gel electrophoresis under reducing conditions and then electrophoretically transferred onto polyvinylidene fluoride membranes (Invitrogen).

Studies in PC-12 Cells

The PC-12 pheochromocytoma cell line was maintained in RPMI-1640 supplemented with 1 mM HEPES buffer, 10% horse serum, 5% fetal bovine serum, 1% sodium pyruvate, 1% L-glutamine, and 1% penicillin/streptomycin. Monomeric SR expression was studied in PC-12 cells using a previously described approach.

PC-12 cells were preincubated with (S)-nicotine for 1 h, followed by the addition of (R,S)-ketamine, (R,S)-norketamine, (2S,6S)-hydroxynorketamine, or methyllycaconitine for an additional 36 h. The m-SR protein level was determined by Western blot analysis.

The effects of (R,S)-ketamine, (R,S)-norketamine, (R,S)-hydroxynorketamine, and methyllycaconitine on pmTOR, pAkt, pERK, p4E-BP1, and p70S6K phosphorylation levels in PC-12 cells were investigated. The same experiment was repeated on 3 separate days (n = 3).

Statistical Analysis

Data are presented as average relative change SD. Graphpad Prism 4 software package was used to carry out statistical analyses.

Brain Tissue Concentrations of ( R,S)-ketamine, ( R,S)-norketamine, and ( 2S,6S)-hydroxynorketamine

After intraperitoneal administration of (R,S)-ketamine, the brain tissue samples contained (R,S)-ketamine, (R,S)-norketamine, and (2S,6S;2R,6R)-hydroxynorketamine as well as four additional diastereomeric hydroxynorketamines.

After intravenous administration of (R,S)-norketamine, the brain tissue samples contained this compound, (2S,6S;2R,6R)-hydroxynorketamine, and three additional diastereomeric hydroxynorketamines. The concentration of (2S,6S)-hydroxynorketamine was the highest in the 10-min sample and was maintained in the 20-min sample.

Effect of ( R,S)-ketamine, ( R,S)-norketamine, and ( 2S,6S)-hydroxynorketamine on the In Vivo Phosphorylation of mTOR and Related Proteins in Rat Brain Tissue

Western blot analysis of rat brain tissue showed that the phosphorylation of mTOR, 4E-BP1, p70S6K, ERK1/2, and Akt increased with time after administration of (R,S)-ketamine, but the phosphorylation of p4EBP1, pERK1/2, and pAkt did not reach statistical significance.

(R,S)-norketamine increased the levels of phosphorylated forms of mTOR, pp70S6K, and p4E-BP1 in the 20- and 60-min samples more than the levels of (R,S)-ketamine.

(2S,6S)-hydroxynorketamine increased pmTOR, p4E-BP1, and pp70S6K levels, similar to (R,S)-ketamine, in the 20- and 60-min samples, but did not reach statistical significance.

Effect of ( R,S)-ketamine, ( R,S)-norketamine, and ( 2S,6S)-hydroxynorketamine on the Expression of m-SR in PC-12 Cells

Treatment of PC-12 cells with (R,S)-ketamine, (R,S)-norketamine, and (2S,6S)-hydroxynorketamine produced concentration-dependent increases in the expression of the m-SR protein, with maximum at 600, 10, and 0.05 nM, respectively.

Effect of ( R,S)-ketamine, ( R,S)-norketamine, and ( 2S,6S)-hydroxynorketamine on the Phosphorylation of mTOR and Related Proteins in PC-12 Cells

PC-12 cells were incubated for 60 min with a range of concentrations of (R,S)-ketamine, (R,S)-norketamine, and (2S,6S)-hydroxynorketamine. All three compounds produced significant and concentration-dependent increases in the phosphorylation of various signaling intermediates. Cell treatment with (R,S)-ketamine (400 to 600 nM) increased protein phosphorylation, which gradually lost significance at concentrations greater than 2,000 nM. Incubation with (R,S)-norketamine (1 to 250 nM) increased pmTOR levels, which lost significance at concentrations greater than 250 nM.

Effect of ( S)-nicotine on the Expression of m-SR and the Phosphorylation of mTOR and Related Proteins in PC-12 Cells Treated with ( R,S)-ketamine, ( R,S)-norketamine, ( 2S,6S)-hydroxynorketamine, and Methyllycaconitine

We have shown that the selective 7 -nAChR antagonist methyllycaconitine increases the expression of m-SR in PC-12 cells, whereas the selective 7 -nAChR agonist (S)-nicotine attenuates the response to methyllycaconitine.

Discussion

The effects of (R,S)-ketamine in the rat were studied using subanesthetic doses, and the results demonstrated that (2S,6S)-hydroxynorketamine contributes to the effects produced by the administration of subanesthetic doses of (R,S)-ketamine.

In this study, the administration of (R,S)-ketamine, (R,S)-norketamine, and (2S,6S)-hydroxynorketamine to male Wistar rats increased the expression of several proteins in the prefrontal cortex. This activation of the mTOR signaling pathway was associated with the rapid antidepressant effects of (R,S)-ketamine.

We recently reported that the nAChR antagonists methyllycaconitine and (R,S)-dehydroxynorketamine increased the de novo protein synthesis of m-SR in 1321N1 astrocytoma cells via the mTOR pathway.

Incubation of PC-12 cells with (R,S)-ketamine, (R,S)-norketamine, (2S,6S)-hydroxynorketamine, and methyllycaconitine produced significant increases in the levels of phosphorylated mTOR, 4E-BP1, p70S6K, ERK1/2, and Akt and that of m-SR protein. Treatment with (S)-nicotine attenuated the response produced by ketamine and its metabolites, and the relative potencies of the test compounds reflect their activities at the nAChR. The data indicate that the observed pharmacological effect is initiated by inhibition of the nAChR.

Ketamine and its metabolites stimulate multiple signaling pathways, including the phosphatidylinositide 3-kinase/Akt pathway, which in turn regulates the translation of SR mRNA into monomeric SR protein. Ketamine and its metabolites also promote the activity of the mTOR complex 2, which mediates mTORC1 activation.

A single subanesthetic dose of (R,S)-ketamine relieved depression-like behaviors in rats suffering from neuropathic pain, but not spared nerve injury – induced hypersensitivity. The higher doses of (R,S)-ketamine required to achieve analgesia might negate or perhaps even decrease phosphorylation of the proteins associated with the mTOR pathway and protein expression.

m-SR was decreased in PC-12 cells after incubation with voltage-gated calcium channel 2 inhibitors, and SR inhibitors are being explored for the treatment of some central nervous system disorders.

The results of the study suggest that (R,S)-ketamine produces therapeutic effects via a combination of independent but interrelated pharmacological effects at the nAChR. These effects are reflected in the contradictory effects produced by methyllycaconitine and (R,S)-dehydroxynorketamine in 1321N1 and PC-12 cells.