46 - Ketamine Metabolites in the Treatment of Neuropathic Pain
Lucas Stolle1,2, Joseph P. Pergolizzi1, Irving W. Wainer1
1Spirify Pharma, Naples, Florida, USA. 2University of Oxford, Oxford, Oxfordshire, United Kingdom
Purpose (R,S)-Ketamine is effective in the treatment of peripheral and central pain and is particularly active in neuropathic pain, such as complex regional pain syndrome and treatment resistant migraine headaches. (R,S)-Ketamine is rapidly and extensively metabolized into a wide variety of compounds including hydroxynorketamine (HNK). Initial studies employing multiple murine pain models demonstrated that HNK is a potent analgesic . We demonstrated that a key pharmacological effect of HNK is decreased D-serine concentrations. D-Serine is an essential co-agonist of the NMDA receptor and reduced D-serine production results in reduced NMDA receptor hyper-activation and associated neurotoxicity. We have demonstrated that gabapentin and pregabalin also reduce the cellular production of D-serine . We here report the results of a study designed to create a molecular template for the design of new analgesics using HNK as the model compound . The goal is the optimized attenuation of D-serine production and the introduction of a new paradigm for the treatment of neuropathic pain. Methods The test compounds were (R)-ketamine, (S)-ketamine, (2R,6R)-HNK, (2S,6S)-HNK, (2R,6S)-HNK, (2S,6R)-HNK, (R)-norketamine, (S)-norketamine, (R)-dehydronorketamine, and (S)-dehydronorketamine. The model system was the PC-12 neuronal cell line maintained in vitro using standard techniques. The test compounds were added to the incubation media in escalating concentrations in order to determine the ability of the compounds to reduce intracellular D-serine production, expressed as IC50 values. Intracellular D-serine concentrations were measured using capillary electrophoresis-laser induced fluorescence (CE-LIF) and extra cellular D-serine concentrations were determined using liquid chromatography with mass spectrometric detection. The data was analyzed using Comparative Molecular Field Analysis (CoMFA) employing the molecular structure of the test compounds and the corresponding IC50 values associated with their effect on D-serine concentrations. The models were aligned using 2-chlorobenzyl moiety as a common substructure and steric and electronic molecular fields were sampled on the grid lattice surrounding each structure. The pIC values presenting effects on the intracellular D-serine levels in PC-12 cells of ketamine metabolites were also subjected to 3D-QSAR modeling Results The intracellular D-serine concentrations were reduced by all of the studied compounds, except for (S)-ketamine. The concentrations were reduced by >30%, and (2S,6S)-HNK and (2R,6R)-HNK were the most potent with IC50 values of 0.18 nM and 0.68 nM, respectively. Incubation with (S)-Ketamine increased intracellular D-serine concentration which is consistent with our previous study demonstrating that (S)-ketamine selectively inhibits the ASCT2 transporter, which mediates D-serine cellular export . Incubation with (2S,6S)-HNK, a (S)-ketamine metabolite reduced both intracellular and extra cellular D-serine concentrations indicating that the compound does not affect ASCT2 transport. COMFA and QSAR modeling identified a relationship between molecular structure and effect on D-serine production. The study indicated that the C2 and C6 positions were key interaction points on the cyclohexanone ring of HNK and that hydrophobic and hydrogen bonding capabilities at C2 and hydrogen bonding at C6 are associated with the observed pharmacological activity. Conclusions The study demonstrated that HNK is a template for new drug development aimed at the optimized treatment of neuropathic pain. The computational models developed in our study have been applied to the design of new chemical entities that are under investigation. References  Kroin JS, et al. (2018) Efficacy of ketamine metabolite (2R,6R)-hydroxynorketamine in mice models of pain. Reg Anesth Pain Med 44:111-117.  Singh NS, et al., (2013) Gabapentin and (S)-pregabalin decrease intracellular D-serine in PC-12 cells. Neuroscience Letters 535: 90-94.  Singh NS, et al. (2016) Ketamine metabolites enantioselectively decrease intracellular D-serine concentgrations in PC-12 cells. PloS One 11: e0149499.  Singh NS, et al. (2015) Enantioselective inhibition of D-serine transport by (S)-ketamine. Br J Pharmacol 172: 4546-4559.