Abstracts

Rationale: Voltage-gated sodium channels (Na_V) are important therapeutic targets for anti-epileptic drugs (AEDs) due to their role in action potential initiation and propagation. Variants in human Na_V genes, such as SCN8A encoding hNa_V1.6, are the most common cause of de novo genetic epilepsy and can exhibit gain-of-function profiles leading to neuronal hyperexcitability. Selective Na_V blockade during periods of hyperexcitability (activity dependence) has been proposed as a pharmacological target for reducing pathologic neuronal hyperactivity, while sparing physiological peak I_Na is critical to ensuring normal neuronal function. This study investigates the in vitro effects of PRAX-628 on I_Na expressed by hNa_V1.6.

Methods: Persistent and peak I_Na inhibition was studied using automated patch clamp recordings of Na_V expressed in HEK cells (hNa_V1.6). Voltage protocols measured I_Na inhibition in multiple modes: persistent I_Na (Vm -120mV, 200ms), tonic block (TB; Vm -120mV, 0.2Hz), voltage-dependent block (VDB; Vm inactivation V_1/2), and activity/use dependent block (UDB, Vm -120mV, 10Hz). PRAX-628 was compared to a panel of AEDs, non-AEDs, and investigational compounds. Binding kinetics were inferred from inhibition kinetics. The apparent binding rate (K_ON) was measured using the time-dependent inhibition of I_Na during a variable length conditioning pulse. The apparent unbinding rate (K_OFF) was measured using the time-dependent delay in the recovery of I_Na following an inactivating conditioning pulse.

Results: PRAX-628 exhibited potent activity dependence (UDB IC50 of 200nM, 44x preference to TB) which has been suggested to convey beneficial activity during periods of hyperexcitability. PRAX-628 also blocked hNaV1.6 persistent INa with an IC50 of 128nM (68x preference to TB), which was at least 550x more potent than the other tested INa blocking agents. This profile was different than that of CBZ (persistent INa IC50 of 77,500nM, 30x preference to TB, no UDB observed) and cenobamate (persistent INa IC50 of 71,690nM, 24x preference to TB, UDB 2.3x preference to TB). A similar profile was observed at other tested isoforms and orthologs.  _x000D_ _x000D_ The preference for persistent INa exhibited by PRAX-628 was not retained when compared to activity in the more depolarized VDB assay (0.56x preference to VDB); contrasting with the preferential persistent INa inhibitor PRAX-562 (2.2x preference to VDB). _x000D_ _x000D_ The enhanced activity dependence of PRAX-628 derives from a rapid KON (4.168 s-1*µM-1) and moderate KOFF (1.72 s-1). These kinetics explain the rapid development of peak INa block relative to standard INa blockers such as CBZ and cenobamate (KONs of 0.011 and 0.019 s-1*µM-1, respectively). _x000D_
Conclusions: PRAX-628 is a next generation Na_V blocker with increased potency and activity dependence for peak I_Na as well as greater potency for persistent I_Na. The profile of PRAX-628 may translate to efficacy in epilepsy, and other indications caused by neuronal hyperexcitability, without tolerability issues caused by excessive tonic block of peak I_Na. 

Funding: Praxis Precision Medicines