Abstracts

Rationale: The voltage gated sodium channel Na_V1.6 is a critical mediator of neuronal excitability in the brain.  Gain of function mutations in SCN8A, the gene encoding Na_V1.6, cause seizure syndromes in both humans (EIEE13) and mice.  Multiple animal seizure models have also demonstrated upregulation of Na_V1.6 channel expression.  Isoform non-selective inhibitors of voltage gated sodium channels have demonstrated utility in controlling seizures, but are dose limited by narrow therapeutic indices.  We reasoned that Na_V1.6 selective inhibitors could provide an improved safety profile by avoiding block of off-target sodium channels like Na_V1.5 (cardiac) and Na_V1.1 (inhibitory interneurons).  Methods: We developed a novel class of selective sodium channel inhibitors that are potent on Na_V1.6 but highly selective against Na_V1.5 and Na_V1.1.  We assessed these compounds in the mouse maximal electroshock seizure assay (MES) in wild type CF-1 mice after a single oral dose of test compound or after 7 or 14 days of b.i.d. dosing.  After the MES assay, animals were euthanized and the concentration of compound in plasma and brain tissue was measured by mass spectrometry.  Total mRNA and protein levels of Na_V1.6 and Na_V1.1 in the hippocampus of mice treated with vehicle or a representative test article (XPC264) were assessed by qPCR and western blot, respectively.  The selectivity and sensitivity of the assays were evaluated using tissue from heterozygous SCN1A and SCN8A knockout mice. Results: Oral gavage with multiple novel selective inhibitors, effectively prevented MES induced tonic clonic seizures with hind-limb extension.  Administration of XEN901, XPC264, or XEN195, b.i.d. for 7 to 14 days increased the fraction of animals protected from seizures at doses that provided partial protection when given once.  That is, after repeat dosing, lower doses of compound were required to prevent tonic clonic seizures.  This effect was not related to compound accumulation, since neither plasma nor brain levels increased after multiple days of dosing.  We found instead that repeated dosing of a representative compound, XPC264, lead to an approximately 50% reduction of the Na_V1.6 protein levels in brain tissue (p +/- mice, validating the assay conditions. Daily dosing with non-selective, inhibitors of Na_V channels carbamazepine and phenytoin did not affect efficacy or Na_V1.6 protein levels. Conclusions: Oral gavage of mice with novel Na_V1.6 selective inhibitor XPC264 effectively prevents seizures in the MES assay and repeated dosing enhances the number of animals protected by low doses, apparently by reducing Na_V1.6 protein levels in the brain. We expect that selective inhibitors of Na_V1.6 such as XPC264, and XEN901 represent a potential improved class of sodium channel targeted AED’s. Drug induced reduction of Na_V1.6 protein levels may be an effective means of improving seizure control. Funding: All authors are employees of Xenon Pharmaceuticals Inc.