Thouta, S. | bioRxiv Science

bioRxiv2023

Molecular pharmacology of selective NaV1.6 and dual NaV1.6 and NaV1.2 channel inhibitors that suppress excitatory neuronal activity ex vivo

Sodium channel inhibitors are used to treat neurological disorders of hyperexcitability. However, all currently available sodium channel targeting anti-seizure medications are non-selective among the NaV isoforms which potentially limits efficacy and therapeutic safety margins. XPC-7724 and XPC-5462 represent a new class of small molecule compounds. These compounds target inhibition of the NaV1.6 and NaV1.2 channels in excitatory pyramidal neurons and possess a molecular selectivity of >100 fold against NaV1.1 channels that are dominant in inhibitory cells. This profile will enable pharmacological dissection of the physiological roles of NaV1.2 and NaV1.6 and help to define the role of each channel in disease states. These compounds bind to and stabilize the inactivated-state of the channels, demonstrate higher potency with longer residency times, and slower off-rates than carbamazepine and phenytoin. These compounds possess cellular selectivity ex vivo in inhibiting action potential firing in cortical excitatory pyramidal neurons, whilst sparing fast spiking inhibitory interneurons. XPC-5462 also suppresses epileptiform activity in an ex vivo brain slice seizure model. This class of compounds provides a unique approach for treating neuronal excitability disorders by selectively down-regulating excitatory circuits. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=194 HEIGHT=200 SRC="FIGDIR/small/551643v2_ufig1.gif" ALT="Figure 1"> View larger version (55K): org.highwire.dtl.DTLVardef@70750corg.highwire.dtl.DTLVardef@114756aorg.highwire.dtl.DTLVardef@289009org.highwire.dtl.DTLVardef@1086e23_HPS_FORMAT_FIGEXP M_FIG C_FIG

pharmacology and toxicology↗

bioRxiv2022

Pharmacological determination of the fractional block of Nav channels required to impair neuronal excitability and ex vivo seizures

Voltage-gated sodium channels (Nav) are essential for the initiation and propagation of action potentials in neurons. Of the different channel subtypes, Nav1.1, Nav1.2 and Nav1.6 are prominently expressed in the adult central nervous system (CNS). All three of these sodium channel subtypes are sensitive to block by the neurotoxin tetrodotoxin (TTX), with TTX being almost equipotent on all three subtypes. In the present study we have used TTX to determine the fractional block of Nav channels required to impair action potential firing in pyramidal neurons and reduce network seizure-like activity. Using automated patch-clamp electrophysiology, we first determined the IC50s of TTX on mouse Nav1.1, Nav1.2 and Nav1.6 channels expressed in HEK cells, demonstrating this to be consistent with previously published data on human Nav channels. We then compared this data to the potency of block of Nav current measured in pyramidal neurons from neocortical brain slices. Interestingly, we found that it requires nearly 10-fold greater concentration of TTX over the IC50 to induce significant block of action potentials using a current-step protocol. In contrast, concentrations near the IC50 resulted in a significant reduction in AP firing and increase in rheobase using a ramp protocol. Surprisingly, a 20% reduction in action potential generation observed with 3 nM TTX resulted in significant block of seizure-like activity in the 0 Mg2+ model of epilepsy. Additionally, we found that approximately 50% block in pyramidal cell intrinsic excitability is sufficient to completely block all seizure-like events. These data serve as a critical starting point in understanding how fractional block of Nav channels affect intrinsic neuronal excitability and seizure-like activity. It further suggests that seizures can be controlled without significantly compromising intrinsic neuronal activity and determines the required fold over IC50 for novel and clinically relevant Nav channel blockers to produce efficacy and limit side effects.

neuroscience↗