Abstracts

Rationale: Variants in SCN8A (encoding hNav1.6) can cause the devastating early infantile epileptic encephalopathy-13 (EIEE-13). These variants commonly exhibit a pathologic gain-of-function (GOF) profile that can include a persistent sodium current (INa), which has been proposed as a pharmacological target for reducing seizures and potentially improving comorbidities.PRAX-330 has shown anti-convulsant activity in standard preclinical seizure models and in a mouse model of EIEE-13 (SCN8AD/+ mice). Efficacy was associated with preferential block of persistent INa over peak INa. In addition, preliminary data previously presented suggests PRAX-330 exhibits differentiated inhibition kinetics that might uniquely contribute to its anticonvulsant effects.Here, PRAX-330 was tested against additional EIEE-13 mutants to determine if inhibition would apply to other mutants. We also compared the inhibition kinetics of PRAX-330 with eleven approved drugs and investigational compounds, selected to represent INa blocking antiepileptic drugs (AEDs) and non-AEDs. Methods: Cells stably expressing mutant NaV1.6 were used to assess GOF properties using automated patch clamp recordings. Voltage-dependence of activation and fast inactivation were measured. The presence of enhanced sodium influx was assessed using voltage-protocols for persistent INa (Vm -120mV, 200ms) and ramp INa (Vm -120mV, +600mV/s).The inhibition of persistent and peak INa was studied using wild-type hNav1.6. Voltage protocols were designed to measure inhibition from various membrane voltages (Vm): persistent INa (ATX-II induced), peak INa tonic block (TB, Vm -120mV), and peak INa voltage-dependent block (VDB, Vm SSI V1/2).Binding kinetics were inferred from inhibition kinetics, which were measured using the time dependent loss of INa caused by compound. The apparent binding rate (KON) was measured using the time-dependent inhibition of INa during a variable length conditioning pulse. The apparent unbinding rate (KOFF) was measured using the time-dependent delay in the recovery of INa following an inactivating conditioning pulse. Results: PRAX-330 blocked hNaV1.6 persistent INa with an IC50 of 38nM. This was 100x-1,500x more potent than all other tested INa blocking AEDs and non-AEDs. The selectivity for persistent INa over peak INa (TB/VDB; 408x/3.2x) was greater than all other tested compounds.A panel of EIEE-13 mutant channels were evaluated for GOF features. N1768D (DIV-S6) and R223G (DI-S4) channels expressed a significant persistent INa, while N984K (DII-DIII linker) did not produce elevated persistent INa. However, all mutants generated a GOF ramp INa, which could be blocked by 1µM PRAX-330 (R223G 87 +- 10%, N984K 73 +- 12 %, and N1768D 94 +- 4%).The increased potency of PRAX-330 appears to originate from the rapid development of inhibition (KON = 17s-1µM-1) paired with a moderate dissociation rate (KOFF = 3.2s-1). The KON for PRAX-330 was faster than any of the other tested compounds (>1,500x versus carbamazepine). The KOFF from PRAX-330 was slower than carbamazepine (11x) suggesting a longer, but not excessive residence time. Conclusions: PRAX-330 is a next generation NaV blocker with increased potency and selectivity for persistent INa. PRAX-330 reduced the GOF ramp INa expressed by a panel of EIEE-13 mutants suggesting activity across different mutation carriers. The observed rapid inhibition kinetics suggest PRAX-330 may preferentially inhibit hyperexcitable states without affecting normal neuronal function. Therefore, PRAX-330 is a precision therapeutic for EIEE-13 and is expected to have an improved therapeutic index due to its unique profile. Funding: All work was funded by Praxis Precision Medicines.