Abstracts

Rationale: Gain of function (GoF) mutations in KCNT1 (encoding K_Na1.1, Slack, Slo2.2) cause drug-resistant and severe forms of infantile epilepsy including the devastating epilepsy of infancy with migrating focal seizures (EIMFS) and the autosomal dominant nocturnal frontal lobe epilepsy (ADNFLE). Understanding the role of K_Na1.1 in regulating neuronal activity in health or in GoF epilepsies has been limited by a lack of suitable in vivo tools. We recently described the first small molecule, orally available, selective K_Na1.1 inhibitor (compound 31, PRX-2904; Griffin et al., 2021, PMID: 33859800). Here we expand on in vitro and in vivo profiling including its efficacy in the Kcnt1-P905L (Kcnt1^L/L) mouse model of KCNT1 GoF.

Methods: Automated patch clamp was used to profile PRX-2904 using HEK cells stably expressing human or mouse K_Na1.1 (WT or a panel of GoF mutants) with 70 mM internal sodium to activate the channel. Whole-cell patch clamp recordings from hippocampal CA1 pyramidal neurons in acute brain slices (P16-P30) from WT or Kcnt1L/L mice were performed to evaluate the contribution of K_Na1.1 to intrinsic neuronal excitability. A current injection protocol (-60 pA to +340 pA) was used to determine effects on action potential (AP) firing frequency.

PRX-2904 was tested in Kcnt1L/L mice (P32-40) implanted with ECoG electrodes to monitor interictal spike and seizure frequencies. After establishing a 24 h baseline, Kcnt1L/L mice were dosed with PRX-2904 (30-75 mg/kg) or vehicle, and ECoG recorded for an additional 24 h. The effects of PRX-2904 (10-150 mg/kg) on spontaneous locomotion were tested in outbred CD-1 mice. Brain concentrations of PRX-2904 in satellite mice were measured using mass spectrometry.

Results: PRX-2904 potently inhibited hK_Na1.1-WT (IC50 of 40 nM) and a panel of GoF mutants with IC50 values ranging from 221-1,975 nM. The potency (IC50) for mK_Na1.1-WT was 622 nM and for mK_Na1.1-P905L was 1,012 nM. PRX-2904 (1 or 10 µM) did not significantly affect evoked neuronal AP frequency in WT slices, but reduced firing by 21% (p=0.0015) in Kcnt1^L/L slices. PRX-2904 (30-75 mg/kg) produced robust and sustained decreases in interictal spike and spontaneous seizure frequencies across the dose range tested in Kcnt1^L/L mice. After administration of even the lowest dose (30 mg/kg) which achieved free brain concentrations equivalent to in vitro IC7 of mK_Na1.1-P905L, all mice were seizure-free for 24 h. Dosing PRX-2904 (10-150 mg/kg) achieved free brain concentrations up to in vitro IC58 without affecting spontaneous locomotion in CD-1 mice.

Conclusions: PRX-2904 is a selective inhibitor of K_Na1.1 with activity at human/mouse and WT/mutant channels. In brain slices from Kcnt1^L/L mice, PRX-2904 normalized AP firing, consistent with robust inhibition of interictal spikes and spontaneous seizures in Kcnt1^L/L mice. At doses up to 5-fold higher than the lowest effective dose in Kcnt1^L/L mice, PRX-2904 did not cause changes in locomotion. Future work will evaluate PRX-2904 in additional mouse models of KCNT1 GoF.

Funding: Please list any funding that was received in support of this abstract.: All work was funded by Praxis Precision Medicines.