PRX-2904 Is a Small Molecule Selective Inhibitor of KNa1.1 (KCNT1) That Reverses the ECoG Phenotype of a KCNT1 Gain-of-Function Mouse Model
Abstract number :
Submission category :
7. Anti-seizure Medications / 7E. Other
Submission ID :
Presentation date :
12/5/2021 12:00:00 PM
Published date :
Nov 22, 2021, 12:55 PM
Archana Jha, PhD - Praxis Precision Medicines; Melody Li - The Florey Institute of Neuroscience and Mental Health; Nikola Jancovski - The Florey Institute of Neuroscience and Mental Health; Liam Scott - Praxis Precision Medicines; Robert Hatch - The Florey Institute of Neuroscience and Mental Health; Marion Wittmann - Praxis Precision Medicines; Steven Petrou - The Florey Institute of Neuroscience and Mental Health; Kristopher Kahlig - Praxis Precision Medicines; Zoë Hughes - Praxis Precision Medicines
Rationale: Gain of function (GoF) mutations in KCNT1 (encoding KNa1.1, 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 KNa1.1 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 KNa1.1 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 (Kcnt1L/L) mouse model of KCNT1 GoF.
Methods: Automated patch clamp was used to profile PRX-2904 using HEK cells stably expressing human or mouse KNa1.1 (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 KNa1.1 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 hKNa1.1-WT (IC50 of 40 nM) and a panel of GoF mutants with IC50 values ranging from 221-1,975 nM. The potency (IC50) for mKNa1.1-WT was 622 nM and for mKNa1.1-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 Kcnt1L/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 Kcnt1L/L mice. After administration of even the lowest dose (30 mg/kg) which achieved free brain concentrations equivalent to in vitro IC7 of mKNa1.1-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 KNa1.1 with activity at human/mouse and WT/mutant channels. In brain slices from Kcnt1L/L mice, PRX-2904 normalized AP firing, consistent with robust inhibition of interictal spikes and spontaneous seizures in Kcnt1L/L mice. At doses up to 5-fold higher than the lowest effective dose in Kcnt1L/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.