NMDAR inhibition-independent antidepressant actions of ketamine metabolites.

Major depressive disorder affects around 16 per cent of the world population at some point in their lives. Despite the availability of numerous monoaminergic-based antidepressants, most patients require several weeks, if not months, to respond to these treatments, and many patients never attain sustained remission of their symptoms. The non-competitive, glutamatergic NMDAR (N-methyl-d-aspartate receptor) antagonist (R,S)-ketamine exerts rapid and sustained antidepressant effects after a single dose in patients with depression, but its use is associated with undesirable side effects. Here we show that the metabolism of (R,S)-ketamine to (2S,6S;2R,6R)-hydroxynorketamine (HNK) is essential for its antidepressant effects, and that the (2R,6R)-HNK enantiomer exerts behavioural, electroencephalographic, electrophysiological and cellular antidepressant-related actions in mice. These antidepressant actions are independent of NMDAR inhibition but involve early and sustained activation of AMPARs (α-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid receptors). We also establish that (2R,6R)-HNK lacks ketamine-related side effects. Our data implicate a novel mechanism underlying the antidepressant properties of (R,S)-ketamine and have relevance for the development of next-generation, rapid-acting antidepressants.

Stereochemical and structural effects of (2R,6R)-hydroxynorketamine on the mitochondrial metabolome in PC-12 cells.

BACKGROUND: Impairment in mitochondrial biogenesis and function plays a key role in depression and anxiety, both of which being associated with changes in fatty acid and phospholipid metabolism. The antidepressant effects of (R,S)-ketamine have been linked to its conversion into (2S,6S;2R,6R)-hydroxynorketamine (HNK); however, the connection between structure and stereochemistry of ketamine and HNK in the mitochondrial homeostatic response has not yet been fully elucidated at a metabolic level. METHODS: We used a multi-platform, non-targeted metabolomics approach to study the change in mitochondrial metabolome of PC-12 cells treated with ketamine and HNK enantiomers. The identified metabolites were grouped into pathways in order to assess global responses. RESULTS: Treatment with (2R,6R)-HNK elicited the significant change in 49 metabolites and associated pathways implicated in fundamental mitochondrial functions such as TCA cycle, branched-chain amino acid biosynthetic pathway, glycoxylate metabolic pathway, and fatty acid ß-oxidation. The affected metabolites included glycerate, citrate, leucine, N,N-dimethylglycine, 3-hexenedioic acid, and carnitine and attenuated signals associated with 9 fatty acids and elaidic acid. Important metabolites involved in the purine and pyrimidine pathways were also affected by (2R-6R)-HNK. This global metabolic profile was not as strongly impacted by treatment with (2S,6S)-HNK, (R)- and (S)-ketamine and in some instances opposite effects were observed. CONCLUSIONS: The present data provide an overall view of the metabolic changes in mitochondrial function produced by (2R,6R)-HNK and related ketamine compounds and offer an insight into the source of the observed variance in antidepressant response elicited by the compounds.

GPR55 receptor antagonist decreases glycolytic activity in PANC-1 pancreatic cancer cell line and tumor xenografts.

The Warburg effect is a predominant metabolic pathway in cancer cells characterized by enhanced glucose uptake and its conversion to l-lactate and is associated with upregulated expression of HIF-1α and activation of the EGFR-MEK-ERK, Wnt-ß-catenin, and PI3K-AKT signaling pathways. (R,R')-4'-methoxy-1-naphthylfenoterol ((R,R')-MNF) significantly reduces proliferation, survival, and motility of PANC-1 pancreatic cancer cells through inhibition of the GPR55 receptor. We examined (R,R')-MNF's effect on glycolysis in PANC-1 cells and tumors. Global NMR metabolomics was used to elucidate differences in the metabolome between untreated and (R,R')-MNF-treated cells. LC/MS analysis was used to quantify intracellular concentrations of ß-hydroxybutyrate, carnitine, and l-lactate. Changes in target protein expression were determined by Western blot analysis. Data was also obtained from mouse PANC-1 tumor xenografts after administration of (R,R')-MNF. Metabolomics data indicate that (R,R')-MNF altered fatty acid metabolism, energy metabolism, and amino acid metabolism and increased intracellular concentrations of ß-hydroxybutyrate and carnitine while reducing l-lactate content. The cellular content of phosphoinositide-dependent kinase-1 and hexokinase 2 was reduced consistent with diminished PI3K-AKT signaling and glucose metabolism. The presence of the GLUT8 transporter was established and found to be attenuated by (R,R')-MNF. Mice treated with (R,R')-MNF had significant accumulation of l-lactate in tumor tissue relative to vehicle-treated mice, together with reduced levels of the selective l-lactate transporter MCT4. Lower intratumoral levels of EGFR, pyruvate kinase M2, ß-catenin, hexokinase 2, and p-glycoprotein were also observed. The data suggest that (R,R')-MNF reduces glycolysis in PANC-1 cells and tumors through reduced expression and function at multiple controlling sites in the glycolytic pathway.

Selective GPR55 antagonism reduces chemoresistance in cancer cells.

G protein-coupled receptor 55 (GPR55) possesses pro-oncogenic activity and its function can be competitively inhibited with (R,R')-4'-methoxy-1-naphthylfenoterol (MNF) through poorly defined signaling pathways. Here, the anti-tumorigenic effect of MNF was investigated in the human pancreatic cancer cell line, PANC-1, by focusing on the expression of known cancer biomarkers and the expression and function of multidrug resistance (MDR) exporters such as P-glycoprotein (Pgp) and breast cancer resistance protein (BCRP). Incubation of PANC1 cells with MNF (1µM) for 24h significantly decreased EGF receptor, pyruvate kinase M2 (PKM2), and ß-catenin protein levels and was accompanied by significant reduction in nuclear accumulation of HIF-1α and the phospho-active forms of PKM2 and ß-catenin. Inhibition of GPR55 with either MNF or the GPR55 antagonist CID 16020046 lowered the amount of MDR proteins in total cellular extracts while diminishing the nuclear expression of Pgp and BCRP. There was significant nuclear accumulation of doxorubicin in PANC-1 cells treated with MNF and the pre-incubation with MNF increased the cytotoxicity of doxorubicin and gemcitabine in these cells. Potentiation of doxorubicin cytotoxicity by MNF was also observed in MDA-MB-231 breast cancer cells and U87MG glioblastoma cells, which express high levels of GPR55. The data suggest that inhibition of GPR55 activity produces antitumor effects via attenuation of the MEK/ERK and PI3K-AKT pathways leading to a reduction in the expression and function of MDR proteins.

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

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.

Capillary electrophoresis-laser-induced fluorescence (CE-LIF) assay for measurement of intracellular D-serine and serine racemase activity.

An enantioselective capillary electrophoresis-laser-induced fluorescence (CE-LIF) method for the analysis of D-serine (D-Ser) in cellular matrices has been developed. The assay involves derivatization with FITC followed by CE-LIF using 0.5 mM hydroxyl propyl-ß-cyclodextrin in borate buffer [80 mM, pH 9.3]. The method was able to resolve D-Ser and L-Ser with an enantioselectivity (α) of 1.03 and a resolution (R(s)) of 1.37. Linearity was established from 0.25 to 100.00 µM. The assay was also able to enantioselectively resolve 6 additional amino acid racemates. The method was applied to the determination of intracellular D-Ser concentrations in PC-12, C6, 1312N1, and HepG2 cell lines. This method was used to determine the concentration-dependent increases in D-Ser and associated EC50 values produced by L-Ser and the concentration-dependent decreases in d-Ser and associated IC50 values produced by glycine, a competitive inhibitor of serine racemase (SR). Western blot analysis determined that the PC-12 and C6 cell lines contained monomeric and dimeric forms of SR while the 1321N1 and HepG2 cells contained only the monomeric form. Although the SR dimer has been identified as the active form of the enzyme, all four of the tested cell lines expressed enzymatically active SR.

Ketamine Metabolites Enantioselectively Decrease Intracellular D-Serine Concentrations in PC-12 Cells.

D-Serine is an endogenous NMDA receptor co-agonist that activates synaptic NMDA receptors modulating neuronal networks in the cerebral cortex and plays a key role in long-term potentiation of synaptic transmission. D-serine is associated with NMDA receptor neurotoxicity and neurodegeneration and elevated D-serine concentrations have been associated with Alzheimer's and Parkinsons' diseases and amyotrophic lateral sclerosis. Previous studies have demonstrated that the ketamine metabolites (rac)-dehydronorketamine and (2S,6S)-hydroxynorketamine decrease intracellular D-serine concentrations in a concentration dependent manner in PC-12 cells. In the current study, PC-12 cells were incubated with a series of ketamine metabolites and the IC50 values associated with attenuated intracellular D-serine concentrations were determined. The results demonstrate that structural and stereochemical features of the studied compounds contribute to the magnitude of the inhibitory effect with (2S,6S)-hydroxynorketamine and (2R,6R)-hydroxynorketamine displaying the most potent inhibition with IC50 values of 0.18 ± 0.04 nM and 0.68 ± 0.09 nM. The data was utilized to construct a preliminary 3D-QSAR/pharmacophore model for use in the design of new and more efficient modulators of D-serine.

Enantioselective inhibition of d-serine transport by (S)-ketamine.

BACKGROUND AND PURPOSE: Patients with major depressive disorder receiving racemic ketamine, (R,S)-ketamine, experience transient increases in Clinician-Administered Dissociative States Scale scores and a coincident drop in plasma d-serine levels. The results suggest that (R,S)-ketamine produces an immediate, concentration-dependent pharmacological effect on d-serine plasma concentrations. One potential source of this effect is (R,S)-ketamine-induced inhibition of the transporter ASCT2, which regulates intracellular d-serine concentrations. In this study, we tested this hypothesis by examining the effect of (S)- and (R)-ketamine on ASCT2-mediated transport of d-serine in PC-12 and 1321N1 cells and primary neuronal cells in culture. EXPERIMENTAL APPROACH: Intracellular and extracellular d-serine levels were determined using capillary electrophoresis-laser-induced fluorescence and liquid chromatography-mass spectrometry respectively. Expression of ASCT2, Asc-1 and serine racemase was determined utilizing Western blotting. KEY RESULTS: (S)-Ketamine produced a concentration-dependent increase in intracellular d-serine and reduced extracellular d-serine accumulation. In contrast, (R)-ketamine decreased both intracellular and extracellular d-serine levels. The ASCT2 inhibitor, benzyl-d-serine (BDS), and ASCT2 gene knockdown mimicked the action of (S)-ketamine on d-serine in PC-12 cells, while the Asc-1 agonist d-isoleucine reduced intracellular d-serine and increased extracellular d-serine accumulation. This response to d-isoleucine was not affected by BDS or (S)-ketamine. Primary cultures of rat neuronal cells expressed ASCT2 and were responsive to (S)-ketamine and BDS. (S)- and (R)-ketamine increased the expression of monomeric serine racemase in all the cells studied, with (S)-ketamine having the greatest effect. CONCLUSIONS AND IMPLICATIONS: (S)-Ketamine decreased cellular export of d-serine via selective inhibition of ASCT2, and this could represent a possible source of dissociative effects observed with (R,S)-ketamine.

Activation of ß2-adrenergic receptor by (R,R')-4'-methoxy-1-naphthylfenoterol inhibits proliferation and motility of melanoma cells.

(R,R')-4'-methoxy-1-naphthylfenoterol [(R,R')-MNF] is a highly-selective ß2 adrenergic receptor (ß2-AR) agonist. Incubation of a panel of human-derived melanoma cell lines with (R,R')-MNF resulted in a dose- and time-dependent inhibition of motility as assessed by in vitro wound healing and xCELLigence migration and invasion assays. Activity of (R,R')-MNF positively correlated with the ß2-AR expression levels across tested cell lines. The anti-motility activity of (R,R')-MNF was inhibited by the ß2-AR antagonist ICI-118,551 and the protein kinase A inhibitor H-89. The adenylyl cyclase activator forskolin and the phosphodiesterase 4 inhibitor Ro 20-1724 mimicked the ability of (R,R')-MNF to inhibit migration of melanoma cell lines in culture, highlighting the importance of cAMP for this phenomenon. (R,R')-MNF caused significant inhibition of cell growth in ß2-AR-expressing cells as monitored by radiolabeled thymidine incorporation and xCELLigence system. The MEK/ERK cascade functions in cellular proliferation, and constitutive phosphorylation of MEK and ERK at their active sites was significantly reduced upon ß2-AR activation with (R,R')-MNF. Protein synthesis was inhibited concomitantly both with increased eEF2 phosphorylation and lower expression of tumor cell regulators, EGF receptors, cyclin A and MMP-9. Taken together, these results identified ß2-AR as a novel potential target for melanoma management, and (R,R')-MNF as an efficient trigger of anti-tumorigenic cAMP/PKA-dependent signaling in ß2-AR-expressing lesions.

What is hydroxynorketamine and what can it bring to neurotherapeutics?

(R,S)-Ketamine was initially developed as an anesthetic agent and its pharmacological properties were determined on the basis of this clinical use. However, pharmacological studies in rat led to the development of the 'Ketamine Paradigm', whereby (R,S)-ketamine and its N-demethylated metabolite (R,S)-norketamine were deemed the active compounds whereas the other ketamine metabolites were considered inactive. Recent in vivo and in vitro studies with (2S,6S)-hydroxynorketamine, a previously identified 'inactive' metabolite, have demonstrated that this compound is an active and selective inhibitor of the α7 subtype of the nicotinic acetylcholine receptor and that this activity contributes to the pharmacological responses associated with the antidepressant activity of (R,S)-ketamine. Thus, it appears that it is necessary to reassess the 'Ketamine Paradigm' in regards to the use of sub-anesthetic doses of (R,S)-ketamine in the treatment of treatment-resistant depression.

Gabapentin and (S)-pregabalin decrease intracellular D-serine concentrations in PC-12 cells.

The effects of gabapentin (GBP) and (S)-pregabalin (PGB) on the intracellular concentrations of d-serine and the expression of serine racemase (SR) in PC-12 cells were determined. Intracellular d-serine concentrations were determined using an enantioselective capillary electrophoresis assay with laser-induced fluorescence detection. Increasing concentrations of GBP, 0.1-20µM, produced a significant decrease in d-serine concentration relative to control, 22.9±6.7% at 20µM (*p<0.05), with an IC(50) value of 3.40±0.29µM. Increasing concentrations of PGB, 0.1-10µM, produced a significant decrease in d-serine concentration relative to control, 25.3±7.6% at 10µM (*p<0.05), with an IC(50) value of 3.38±0.21µM. The compounds had no effect on the expression of monomeric-SR or dimeric-SR as determined by Western blotting. The results suggest that incubation of PC-12 cells with GBP and PGB reduced the basal activity of SR, which is most likely a result of the decreased Ca(2+) flux produced via interaction of the drugs with the α(2)-δ subunit of voltage-gated calcium channels. d-Serine is a co-agonist of the N-methyl d-aspartate receptor (NMDAR) and reduced d-serine concentrations have been associated with reduced NMDAR activity. Thus, GBP and PGB may act as indirect antagonists of NMDAR, a mechanism that may contribute to the clinical effects of the drugs in neuropathic pain.

Nicotinic acetylcholine receptor antagonists alter the function and expression of serine racemase in PC-12 and 1321N1 cells.

Western blot analysis demonstrated that PC-12 cells express monomeric and dimeric forms of serine racemase (m-SR, d-SR) and that 1321N1 cells express m-SR. Quantitative RT-PCR and functional studies demonstrated that PC-12 cells express homomeric and heteromeric forms of nicotinic acetylcholine receptors (nAChR) while 1321N1 cells primarily express the α7-nAChR subtype. The effect of nAChR agonists and antagonists on SR activity and expression was examined by following concentration-dependent changes in intracellular d-Ser levels and SR protein expression. Incubation with (S)-nicotine increased d-Ser levels, which were attenuated by the α7-nAChR antagonist methyllycaconitine (MLA). Treatment of PC-12 cells with mecamylamine (MEC) produced a bimodal reduction of d-Ser reflecting MEC inhibition of homomeric and heteromeric nAChRs, while a unimodal curve was observed with 1321N1 cells, reflecting predominant expression of α7-nAChR. The nAChR subtype selectivity was probed using α7-nAChR selective inhibitors MLA and (R,S)-dehydronorketamine and α3ß4-nAChR specific inhibitor AT-1001. The compounds reduced d-Ser in PC-12 cells, but only MLA and (R,S)-dehydronorketamine were effective in 1321N1 cells. Incubation of PC-12 and 1321N1 cells with (S)-nicotine, MEC and AT-1001 did not affect m-SR or d-SR expression, while MLA and (R,S)-dehydronorketamine increased m-SR expression but not SR mRNA levels. Treatment with cycloheximide indicated that increased m-SR was due to de novo protein synthesis associated with phospho-active forms of ERK1/2, MARCKS, Akt and rapamycin-sensitive mTOR. This effect was attenuated by treatment with the pharmacological inhibitors U0126, LY294002 and rapamycin, which selectively block the activation of ERK1/2, Akt and mTOR, respectively, and siRNAs directed against ERK1/2, Akt and mTOR. We propose that nAChR-associated changes in Ca(2+) flux affect SR activity, but not expression, and that MLA and (R,S)-dehydronorketamine bind to allosteric sites on the α7-nAChR and promote multiple signaling cascades that converge at mTOR to increase m-SR levels.

Screening approach, optimisation and scale-up for chiral liquid chromatography of cathinones.

Screening approaches adopted in pharmaceutical companies for chiral LC method development may be quite complicated and sophisticated in order to guarantee a high success rate. However in other environments it may be of more value to assess how simple a screen might be used to still have a good chance of achieving success. The genuine need to develop chiral separations for the former 'legal-high' drug mephedrone and related cathinones of topical interest presented a good opportunity to develop this theme. In initial work on mephedrone itself, no chiral separation was observed on Chirobiotic V, Cyclobond I 2000 DNP, Whelk-O1 and AmyCoat using reversed phase mobile phases. However, using normal phase solvents, chiral separation was observed on all the chiral stationary phases (CSP) used except Chiralcel OJ-H. Of the chiral separations observed on RegisPack, RegisCell and Whelk-O1, some optimisation work was carried out on the latter two which had showed the greatest enantioselectivity. Following optimisation, the best enantioselectivity (1.59) and enantioresolution (5.90) was found with a 250 mm × 4.6 mm I.D. Whelk-O1 column using a propan-2-ol (IPA)-hexane-trifluoroacetic acid (TFA)-triethylamine (TEA) (10:90:0.05:0.05, v/v/v/v) mobile phase. Subsequent screening on other cathinones was restricted to RegisPack, RegisCell and Whelk-O1 or equivalent phases with two mobile phases and this gave a very good success rate. Indeed it was possible to separate all six cathinones on one column, RegisCell, with one mobile phase, propan-2-ol-hexane-TFA (15:85:0.1, v/v/v) but obviously it had been necessary to go through the 3-column screen to arrive at this finding. While Whelk-O1 was not so successful, ease of optimisation on this phase was again a feature. To illustrate the applicability of these separations, it was shown that, as a basis for semi-preparative work, the optimsed mephedrone separation on Whelk-O1 could be scaled-up to a 2000µl injection of a 1.0 mg ml(-1) solution in mobile phase (2.0mg on-column) while still using the 250 mm × 4.6 mm I.D. analytical column.