Abstracts

Rationale: Epileptic encephalopathy (EE) is associated with de novo Na_V variants in Na_V1.6 and Na_V1.2 that commonly result in the gain-of-function (GOF) of sodium current (I_Na) leading to neuronal hyperexcitability, premature action potential generation, and network synchronization. The spectrum of GOF changes typically includes an abnormally enhanced persistent I_Na, which has been proposed as a pharmacological target for reducing seizures and potentially improving other comorbidities in EE. Previous reports with PRAX-330 (GS-458967, GS967) have demonstrated protection from seizures in mouse models of Na_V1.6 or Na_V1.2 EE with efficacy being associated with a preferential block of persistent I_Na over peak I_Na. The current study extends the biophysical characterization of PRAX-330 to multiple modes of I_Na inhibition, with a focus on determining potency, selectivity for persistent I_Na and the speed of I_Na block. Methods: The inhibition of persistent and peak I_Na by PRAX-330 was studied using automated patch clamp recordings of human Na_V1.6 or Na_V1.2 expressed in a HEK cells. Voltage protocols were designed to measure I_Na inhibition in multiple modes: persistent I_Na (V_m -120mV, 200ms), tonic block (TB, V_m -120mV), voltage-dependent block (VDB, V_m SSI V_1/2), and use-dependent block (UDB, 50Hz). PRAX-330 was compared to a panel of Na_V blocking AEDs, including lamotrigine, phenytoin, carbamazepine, and lacosamide. Off-target activity was estimated using a broad CEREP binding panel consisting of 80 targets, including various ion channels, transporters, and receptors. Results: PRAX-330 demonstrated potent inhibition of toxin induced persistent I_Na for both hNa_V1.6 and hNa_V1.2 with IC_50s of 38nM and 45nM, respectively. This activity was more potent than the inhibition of peak I_Na in either the TB or VDB paradigms. For hNav1.6, PRAX-330 exhibited 400x selectivity for persistent I_Na over peak I_Na TB (IC₅₀ of 15,500nM) and retained a 2.7x preference over peak I_Na VDB (IC₅₀ of 101 nM). In contrast, approved Na_V blocking AEDs were far less potent for persistent I_Na (IC_50s: lamotrigine 78,480nM, phenytoin 108,800nM, carbamazepine 77,490nM, and lacosamide >300,000nM) and none demonstrated preference for persistent I_Na over peak I_Na VDB (lamotrigine 0.5x, phenytoin 0.5x, carbamazepine 0.6x, and lacosamide 0.3x). PRAX-330 also exhibited selective inhibition of hNa_V1.2 persistent I_Na over peak I_Na VDB (3.5x). PRAX-330 induced UDB of hNav1.6 peak I_Na between 2.5x and 5x more rapidly than the other Na_V AEDs (tau of 59 ms^-1 versus between 141ms^-1 and 295ms^-1). In all assays, the I_Na inhibition induced by PRAX-330 recovered quickly and completely. PRAX-330 did not exhibit significant activity in the CEREP panel of radioligand binding assays. Conclusions: Compared to currently approved Na_V blocking AEDs, the novel I_Na inhibitor PRAX-330 is significantly more potent and selective for Navs. Moreover, PRAX-330 exhibits greater selectivity for persistent I_Na compared to peak I_Na and more rapid inhibition kinetics. Therefore, PRAX-330 is being developed as a precision therapeutic for EE caused by Na_V1.6 or Na_V1.2 GOF and is expected to have an improved therapeutic index due to its unique profile. Funding: All work was funded by Praxis Precision Medicines.