Abstracts

Rationale: Epileptic encephalopathy (EE) can be caused by mutations in voltage-gated sodium channels, including SCN2A and SCN8A (encoding NaV1.2 and NaV1.6, respectively). Mutations in SCN8A have been reported to increase persistent sodium current (INa) in hippocampal pyramidal neurons, which enhances neuronal excitability and network synchronization, likely underlying seizures in these patients, and is thought to contribute to other co-morbidities.PRAX-330 is a novel INa inhibitor with a preference for inhibiting persistent INa over peak INa. This profile differs from approved INa targeting anti-epileptic drugs (AEDs), which do not display preference for persistent INa (e.g. carbamazepine, CBZ). Treatment with PRAX-330 reduces seizures in a mouse model of NaV1.6 EE (Scn8aD/+) with selective inhibition of persistent INa observed using isolated neurons. The effect of PRAX-330 on the neuronal firing in brain slices is not currently known. Methods: Action potential (AP) firing was investigated using whole-cell patch-clamp recordings from hippocampal CA1 pyramidal neurons in brain slices from C57BL/6 mice in the absence and presence of PRAX-330 or CBZ. Firing was induced by current injections (-60 to +340pA, 400ms). Following baseline subtraction, APs were identified using a 50mV threshold and areas under the curve from individual input-output relationships were calculated. AP amplitude was calculated relative to AP threshold (50V/s). AP adaption (amplitude and threshold) across a train of APs was determined at the 200pA current step by comparing the first:last AP fired. Results: PRAX-330 and CBZ were tested relative to their respective potency for inactivated hNaV1.6, which was defined as the voltage dependent block (VDB) of peak INa. The VDB-IC50 was approximately 100nM for PRAX-330 and 45,000nM for CBZ. At these concentrations (1x VDB-IC50) PRAX-330 and CBZ significantly reduced the AP firing of CA1 pyramidal neurons by 10% and 35%, respectively. CBZ had a dramatic enhancement in AP adaptation across an AP train, where the AP amplitude and threshold of the first:last AP fired were reduced by 40% compared to baseline. CBZ also lead to a slowing and then cessation of firing in a majority of neurons tested during the train. This profile suggests a robust inhibition of peak INa by CBZ. In contrast, PRAX-330 induced a moderate reduction in AP amplitude and threshold with firing sustained during the current injection. Moreover, increasing the concentration of PRAX-330 to 1,000nM (10x-VDB-IC50) reduced AP firing by 35%, which was similar to the effect of CBZ. However, at this higher concentration, PRAX-330 only produced a 10% reduction in AP amplitude and threshold compared to baseline and neurons did not demonstrate firing cessation during an AP train. These data are consistent with a lower inhibition of peak INa compared to CBZ. No changes in the kinetics of the 1st AP fired were observed for either compound. Conclusions: PRAX-330 reduced the intrinsic excitability of hippocampal CA1 neurons with only a moderate reduction in action potential amplitude and threshold, suggesting selective inhibition of persistent INa relative to peak INa. In contrast, a similar reduction in intrinsic excitability by CBZ was associated with substantial reductions in AP amplitude, threshold and firing cessation suggesting robust inhibition of peak INa. Therefore, PRAX-330 may be better tolerated compared to CBZ due to enhanced selectivity for persistent INa. Funding: All work was funded by Praxis Precision Medicines.