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

Earlier research has shown that subanesthetic doses of (R,S)-ketamine produce rapid antidepressant effects in rodents that are associated with increased phosphorylation of the mammalian target of rapamycin (mTOR) and downstream signalling proteins (pERK1/2, pAkt, p4E-BP1, pp70S6K) and with increased synaptogenesis in the prefrontal cortex. Ketamine is, however, rapidly and extensively metabolised to multiple products including (R,S)-norketamine and diastereomeric hydroxynorketamines such as (2S,6S;2R,6R)-hydroxynorketamine; the contribution of these metabolites to the mTOR-linked molecular effects attributed to the parent drug has been unclear. Paul and colleagues set out to determine whether (R,S)-norketamine and the specific isomer (2S,6S)-hydroxynorketamine have pharmacological activity on the mTOR signalling pathway in vitro and in vivo. The study aimed to measure brain concentrations and mTOR pathway phosphorylation after administration of the parent compound and selected metabolites in male Wistar rats, and to assess concentration-dependent effects on mTOR signalling and serine racemase (m-SR) expression in PC-12 cells. This approach was intended to clarify whether metabolites contribute to molecular events linked to ketamine’s antidepressant and analgesic actions.

In vivo experiments used male Wistar rats housed under standard conditions; for each experimental time point n = 3 animals were used. Animals received a single intraperitoneal injection of (R,S)-ketamine 40 mg/kg, or a single intravenous injection (jugular catheter) of either (R,S)-norketamine 20 mg/kg or (2S,6S)-hydroxynorketamine 20 mg/kg. Rats dosed with (R,S)-ketamine were euthanised at 10, 30, 60, and 240 min postinjection; those given the metabolites were euthanised at 10, 20, 60, and 240 min. Whole brains were collected, one hemisphere used for quantification of drug/metabolite concentrations by liquid chromatography–mass spectrometry and the other prefrontal cortex portion used for Western blot analysis. Brain tissue concentrations were measured after homogenisation, solid-phase extraction and validated LC–MS methods, with limits of quantification reported for each analyte. Western blotting quantified total and phosphorylated forms of mTOR, ERK1/2, Akt, 4E-BP1, p70S6K and monomeric serine racemase (m-SR); densitometry was normalised to β-actin. In vitro studies used rat-derived PC-12 pheochromocytoma cells maintained in RPMI-1640 with serum supplements. To assess de novo m-SR expression, cells were incubated with test compounds for 36 h (concentrations reported below) and analysed by Western blot; these experiments were repeated on three separate days (n = 3). Phosphorylation assays involved serum-starvation followed by incubation with compounds for 1 h (the text also reports 60 min incubations) at concentration ranges: (R,S)-ketamine 0–10,000 nM, (R,S)-norketamine 0–1,000 nM, and (2S,6S)-hydroxynorketamine 0–100 nM. Some PC-12 experiments included a 1 h preincubation with (S)-nicotine (2 μM) before addition of test compounds, and the α7-nAChR antagonist methyllycaconitine (50 nM) was used as a comparator to probe receptor involvement. All cell-based experiments were repeated three times. Statistical analyses used one-way ANOVA with Dunnett post hoc tests; results are reported as mean relative change ± SD and P ≤ 0.05 was considered statistically significant.

Brain pharmacokinetics: After intraperitoneal (R,S)-ketamine 40 mg/kg, brain concentrations peaked at 137 ± 6 μM/g at 10 min and fell to 0.6 ± 0.1 μM/g by 240 min. Multiple metabolites including (R,S)-norketamine and diastereomeric hydroxynorketamines were detected; at 60 and 240 min the combined hydroxynorketamine isomers exceeded (R,S)-norketamine levels. Following intravenous (R,S)-norketamine 20 mg/kg, peak brain levels were 88 ± 8 μM/g at 10 min, declining to 1.0 ± 0.1 μM/g at 240 min, with hydroxynorketamine metabolites appearing and exceeding norketamine at later time points. Intravenous administration of (2S,6S)-hydroxynorketamine 20 mg/kg yielded only that compound in brain, with a peak of 127 ± 4 μM/g at 10 min; levels were maintained at 20 min and about 10% of peak remained at 240 min. In vivo phosphorylation (rat prefrontal cortex): Intraperitoneal (R,S)-ketamine produced a time-dependent increase in phosphorylated mTOR (pmTOR) and pp70S6K of about ~2.5-fold, with maxima at 30–60 min that reached statistical significance (P < 0.05). Increases in p4E-BP1 (~1.5-fold), pERK1/2 (~2-fold) and pAkt (~1.3-fold) did not reach significance. Intravenous (R,S)-norketamine induced markedly larger increases in pmTOR (reported as ~15- and ~25-fold) in 20- and 60-min samples together with significant increases in pp70S6K, p4E-BP1 and pERK1/2 (pERK1/2 rise ~6-fold); pAkt was not affected. Administration of (2S,6S)-hydroxynorketamine produced time-dependent increases in pmTOR (~2-fold), p4E-BP1 (~2-fold) and pp70S6K (~2.5-fold) significant at 20 and 60 min; pERK1/2 and pAkt tended to increase but did not reach significance. PC-12 cell results — m-SR expression: Treatment with all three compounds produced concentration-dependent increases in monomeric serine racemase. Maximal m-SR expression occurred at different potencies: (2S,6S)-hydroxynorketamine maximal at ~0.05 nM, (R,S)-norketamine at ~10 nM, and (R,S)-ketamine at ~600 nM. ANOVA indicated significant increases at 600 and 1,000 nM for (R,S)-ketamine, at 10 and 25 nM for (R,S)-norketamine, and between 0.05 and 0.25 nM for (2S,6S)-hydroxynorketamine. PC-12 cell results — phosphorylation of mTOR pathway: Incubation for 60 min with the compounds produced concentration-dependent increases in phosphorylation of mTOR, 4E-BP1, p70S6K, ERK1/2 and Akt. (R,S)-Ketamine elicited significant phosphorylation increases at 400–600 nM with relative increases of ~1.5–3-fold over baseline, losing significance at >2,000 nM. (R,S)-Norketamine produced significant effects from 1 to 250 nM, with 25 nM generating ~2.5–5-fold increases. (2S,6S)-Hydroxynorketamine was active at 0.01–10 nM, with 0.5 nM producing ~2.8–4.2-fold increases; effects diminished at concentrations above ~1 nM. The relative potencies across assays were therefore (2S,6S)-hydroxynorketamine > (R,S)-norketamine > (R,S)-ketamine. Role of α7-nAChR: Preincubation with the α7-nAChR agonist (S)-nicotine attenuated the increases in m-SR and in phosphorylation of mTOR pathway proteins produced by ketamine, its metabolites and by methyllycaconitine; nicotine alone had no effect. These data are presented as supporting receptor-mediated initiation of the signalling cascade.

Paul and colleagues interpret the data to indicate that both (R,S)-norketamine and (2S,6S)-hydroxynorketamine are pharmacologically active and contribute to the mTOR pathway activation observed after subanesthetic (R,S)-ketamine. In vivo rat results showed that administration of the parent drug and the two metabolites increased phosphorylation of mTOR and downstream translational regulators; in vitro PC-12 data demonstrated that these compounds stimulate mTOR-dependent signalling and increase de novo synthesis of monomeric serine racemase, with a potency rank order of (2S,6S)-hydroxynorketamine > (R,S)-norketamine > (R,S)-ketamine. The authors propose a mechanistic link between antagonism of the homomeric α7-nicotinic acetylcholine receptor and activation of mTOR signalling: inhibition of basal α7-nAChR activity by the parent drug and metabolites appears to stimulate ERK and Akt pathways and thereby promotes mTOR complex function and cap-dependent protein synthesis, as reflected by increased phosphorylation of 4E-BP1 and p70S6K and by increased m-SR translation. The attenuation of effects by (S)-nicotine is cited as evidence for nAChR involvement. Paul and colleagues also note that phosphorylation of Akt on Ser473 suggests possible mTORC2 engagement upstream of mTORC1, but acknowledge that how mTORC2 is regulated in this context remains unclear. Although the study did not assess behavioural outcomes or directly measure synaptogenesis, the investigators link their molecular findings to hypotheses about ketamine’s rapid antidepressant actions while distinguishing those molecular effects from mechanisms relevant to analgesia. They suggest that dosing regimens used for analgesia (higher or continuous doses) could have different time- and concentration-dependent effects on the mTOR pathway than the single subanesthetic doses associated with antidepressant-like molecular changes. An alternative or additional mechanism discussed is regulation of serine racemase activity and consequent reductions in intracellular D-serine, which could indirectly decrease NMDA receptor activity and contribute to therapeutic effects. Key limitations reported within the extracted text include the lack of behavioural or synaptogenesis data in this study and the rapid in vivo conversion among compounds that complicates attribution of effects solely to the parent drug; these considerations motivate the authors’ conclusion that understanding ketamine’s antidepressant and analgesic mechanisms requires study of both the parent compound and its metabolites. The investigators also highlight that some prior assumptions—that hydroxynorketamines lack pharmacological activity—are challenged by their findings showing potent, selective actions at α7-nAChR and consequent mTOR pathway activation.