The assessment of the efficacy of antiseizure medications (ASMs) in animal models of acute seizures has played a critical role in these drugs’ success in clinical trials for human epilepsy. One of the most widely used animal models for this purpose is the maximal electroshock seizure (MES) model. While there are numerous published reports on the efficacy of conventional ASMs in MES models, there is a need to expand the understanding on the brain concentrations that are needed to achieve optimal levels of efficacy in this model. We assessed the pharmacokinetic/pharmacodynamic (PK/PD) profiles of six ASMs, namely carbamazepine (CBZ), phenytoin (PHT), valproic acid (VPA), lacosamide (LSM), cenobamate (CNB), and retigabine (RTG), using MES models in mice and rats. EC50 values for plasma and the brain were generally higher in mice than rats, with fold differences ranging from 1.3- to 8.6-fold for plasma and from 1.2- to 11.5-fold for brain. Phenytoin showed the largest interspecies divergence. These results suggest that rats may exhibit greater sensitivity to seizure protection in the MES model, likely reflecting species differences in metabolism and brain penetration. These findings highlight the value of considering concentration–response variations and species-specific differences when assessing the efficacy of both conventional ASMs and novel compounds exhibiting anticonvulsant activity.

Background and Purpose Inhibitors of voltage-gated sodium channels (Na(V)s) are important anti-epileptic drugs, but the contribution of specific channel isoforms is unknown since available inhibitors are non-selective. We aimed to create novel, isoform selective inhibitors of Na-v channels as a means of informing the development of improved antiseizure drugs. Experimental Approach We created a series of compounds with diverse selectivity profiles enabling block of Na(V)1.6 alone or together with Na(V)1.2. These novel Na-V inhibitors were evaluated for their ability to inhibit electrically evoked seizures in mice with a heterozygous gain-of-function mutation (N1768D/+) in Scn8a (encoding Na(V)1.6) and in wild-type mice. Key Results Pharmacologic inhibition of Na(V)1.6 in Scn8a(N1768D/+) mice prevented seizures evoked by a 6-Hz shock. Inhibitors were also effective in a direct current maximal electroshock seizure assay in wild-type mice. Na(V)1.6 inhibition correlated with efficacy in both models, even without inhibition of other CNS Na-V isoforms. Conclusions and Implications Our data suggest Na(V)1.6 inhibition is a driver of efficacy for Na-V inhibitor anti-seizure medicines. Sparing the Na(V)1.1 channels of inhibitory interneurons did not compromise efficacy. Selective Na(V)1.6 inhibitors may provide targeted therapies for human Scn8a developmental and epileptic encephalopathies and improved treatments for idiopathic epilepsies.

RationaleNav1.1, 1.2, and 1.6 are transmembrane proteins acting as voltage‐gated sodium channels implicated in various forms of epilepsy. There is a need for knowing their actual concentration in target tissues during drug development.MethodsUnique peptides for Nav1.1, Nav1.2, and Nav1.6 were selected as quantotropic peptides for each protein and used for their quantification in membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra‐high‐performance liquid chromatography–electrospray ionization tandem mass spectrometry.ResultsNav 1.1, 1.2, and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1 ± 0.2, 6.4 ± 1.2, and 4.0 ± 0.6 fmol/μg membrane protein, respectively. In brains, Nav1.2 showed the highest expression, with approximately three times higher (P < 0.003) in rodents than in humans at 3.05 ± 0.57, with 3.35 ± 0.56 in mouse and rat brains and 1.09 ± 0.27 fmol/μg in human brain. Both Nav1.1 and 1.6 expressions were much lower in the brains, with approximately 40% less expression in human Nav1.1 than rodent Nav1.1 at 0.49 ± 0.1 (mouse), 0.43 ± 0.3 (rat), and 0.28 ± 0.04 (humans); whereas Nav1.6 had approximately 60% less expression in humans than rodents at 0.27 ± 0.09 (mouse), 0.26 ± 0.06 (rat), and 0.11 ± 0.02 (humans) fmol/μg membrane proteins.ConclusionsMultiple reaction monitoring was used to quantify sodium channels Nav1.1, 1.2, and 1.6 expressed in stably transfected HEK293 cells and brain tissues from mice, rats, and humans. We found significant differences in the expression of these channels in mouse, rat, and human brains. Nav expression ranking among the three species was Nav1.2 ≫ Nav1.1 > Nav1.6, with the human brain expressing much lower concentrations overall compared to rodent brain.

Rationale Na 1.1, 1.2 and 1.6 are transmembrane proteins acting as voltage gated sodium channels implicated in various forms of epilepsy. There is a need for knowing their actual concentration in target tissues during drug development. Methods Unique peptides for Na 1.1, Na 1.2 and Na 1.6 were selected as quantotropic peptides for each protein and used for their quantification in plasma membranes from stably transfected HEK293 cells and rodent and human brain samples using ultra‐high‐performance liquid chromatography/electrospray ionization tandem mass spectrometry. Results Na 1.1, 1.2 and 1.6 protein expressions in three stably individually transfected HEK293 cell lines were found to be 2.1±0.2, 6.4±1.2 and 4.0±0.6 fmole/µg membrane protein respectively. Na 1.2 showed the highest expression, with approximately 3 times higher (p<0.003) in rodents than in human at 3.05±0.57, 3.35±0.56 in mouse and rat brains and 1.09±0.27 fmole/µg in human, respectively. Both Na 1.1 and 1.6 expressions were much lower than Na 1.2, with approximately 40% less expression in human Na 1.1 when compared with rodents Na 1.1 at 0.49±0.1 (mouse), 0.43±0.3 (rat), and 0.28±0.04 (human); while Na 1.6 was approximately 60% less expression in human when compared to rodents at 0.27±0.09 (mouse), 0.26±0.06 (rat) and 0.11±0.02 (human) fmole/µg membrane proteins. Conclusions MRM was used to quantify sodium channels Na 1.1, 1.2 and 1.6 expressed in stably transfected HEK293 cells and brain tissues from mouse, rat, and human. We found significant differences in the expression of these channels in mouse, rat, and human brains. Na expression ranking among the three species was Na 1.2 >> Na 1.1> Na 1.6, with the human brain expressing much lower concentrations overall in comparison to rodents.

Inhibitors of voltage-gated sodium channels (Na V s) are important anti-epileptic drugs, but the contribution of specific channel isoforms is unknown since available inhibitors are nonselective. We created a series of compounds with diverse selectivity profiles enabling block of Na V 1.6 alone or together with Na V 1.2. Mice with a heterozygous gain-of-function mutation (N1768D/+) in Scn8a (encoding Na V 1.6) responded with a tonic-clonic seizure to a mild 6 Hz stimulus that was innocuous to wild-type mice. Pharmacologic inhibition of Na V 1.6 in Scn8a N1768D/+ mice prevented seizures. Inhibitors were also effective in a direct current maximal electroshock seizure assay in wild-type mice. Na V 1.6 inhibition correlated with efficacy in both models, even without inhibition of other CNS Na V isoforms. Our data suggest Na V 1.6 inhibition is a driver of efficacy for Na V inhibitor anti-seizure medicines. Selective Na V 1.6 inhibitors may provide targeted therapies for human Scn8a developmental and epileptic encephalopathies and better tolerated treatments for idiopathic epilepsies. Graphical Abstract

Divalent metal transporter 1 (DMT1) cotransports ferrous iron and protons and is the primary mechanism for uptake of nonheme iron by enterocytes. Inhibitors are potentially useful as therapeutic agents to treat iron overload disorders such as hereditary hemochromatosis or β-thalassemia intermedia, provided that inhibition can be restricted to the duodenum. We used a calcein quench assay to identify human DMT1 inhibitors. Dimeric compounds were made to generate more potent compounds with low systemic exposure. Direct block of DMT1 was confirmed by voltage clamp measurements. The lead compound, XEN602, strongly inhibits dietary nonheme iron uptake in both rats and pigs yet has negligible systemic exposure. Efficacy is maintained for >2 weeks in a rat subchronic dosing assay. Doses that lowered iron content in the spleen and liver by >50% had no effect on the tissue content of other divalent cations except for cobalt. XEN602 represents a powerful pharmacological tool for understanding the physiologic function of DMT1 in the gut. SIGNIFICANCE STATEMENT: This report introduces methodology to develop potent, gut-restricted inhibitors of divalent metal transporter 1 (DMT1) and identifies XEN602 as a suitable compound for in vivo studies. We also report novel animal models to quantify the inhibition of dietary uptake of iron in both rodents and pigs. This research shows that inhibition of DMT1 is a promising means to treat iron overload disorders.

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract NBI-921352 (formerly XEN901) is a novel sodium channel inhibitor designed to specifically target NaV1.6 channels. Such a molecule provides a precision-medicine approach to target SCN8A-related epilepsy syndromes (SCN8A-RES), where gain-of-function (GoF) mutations lead to excess NaV1.6 sodium current, or other indications where NaV1.6 mediated hyper-excitability contributes to disease (Gardella and Møller, 2019; Johannesen et al., 2019; Veeramah et al., 2012). NBI-921352 is a potent inhibitor of NaV1.6 (IC500.051 µM), with exquisite selectivity over other sodium channel isoforms (selectivity ratios of 756 X for NaV1.1, 134 X for NaV1.2, 276 X for NaV1.7, and >583 Xfor NaV1.3, NaV1.4, and NaV1.5). NBI-921352is a state-dependent inhibitor, preferentially inhibiting inactivatedchannels. The state dependence leads to potent stabilization of inactivation, inhibiting NaV1.6 currents, including resurgent and persistent NaV1.6 currents, while sparing the closed/rested channels. The isoform-selective profile of NBI-921352 led to a robust inhibition of action-potential firing in glutamatergic excitatory pyramidal neurons, while sparing fast-spiking inhibitory interneurons, where NaV1.1 predominates. Oral administration of NBI-921352 prevented electrically induced seizures in a Scn8a GoF mouse,as well as in wild-type mouse and ratseizure models. NBI-921352 was effective in preventing seizures at lower brain and plasma concentrations than commonly prescribed sodium channel inhibitor anti-seizure medicines (ASMs) carbamazepine, phenytoin, and lacosamide. NBI-921352 waswell tolerated at higher multiples of the effective plasma and brain concentrations than those ASMs. NBI-921352 is entering phase II proof-of-concept trials for the treatment of SCN8A-developmental epileptic encephalopathy (SCN8A-DEE) and adult focal-onset seizures. Editor's evaluation This exciting study reports on the characterization of a novel compound that preferentially targets Nav1.6 voltage-gated sodium channels and shows substantial activity against epilepsy associated SCN8A mutations and seizure activity in a variety of animal models. This compound and approach has significant promise to be translated into a therapeutic for individuals with treatment resistant epilepsy. https://doi.org/10.7554/eLife.72468.sa0 Decision letter Reviews on Sciety eLife's review process Introduction NaV1.6 voltage-gated sodium channels are widely expressed in the brain and are important contributors to neural excitability (Meisler, 2019; Royeck et al., 2008).Mutations in the SCN8A gene result in malfunction of NaV1.6 sodium channels and cause a spectrum of SCN8A-related syndromes in humans, and disruptions of mouse NaV1.6 likewise disrupt normal physiology (Burgess et al., 1995; Gardella and Møller, 2019; Johannesen et al., 2019; Meisler, 2019; Veeramah et al., 2012; Wagnon et al., 2015). Variants of NaV1.6 channels can result in either gain or loss of function. Loss-of-function (LoF) variants in humans are generally associated with autism spectrum disorders with cognitive and developmental delay without epilepsy (Inglis et al., 2020; Liu et al., 2019), but, in some cases, can lead to late-onset seizures.In mice, LoF variants of NaV1.6 lead to motor impairment but increase seizure resistance (Hawkins et al., 2011; Martin et al., 2007). Gain-of-function (GoF) variants in human SCN8A generally result in early-onset SCN8A-related epilepsy syndromes (SCN8A-RES). The most severe of these epilepsy syndromes is SCN8A developmental and epileptic encephalopathy (SCN8A-DEE) (Gardella and Møller, 2019; Hammer et al., 2016; Johannesen et al., 2019). Most SCN8A-RES patients carry de novo heterozygous missense variants that lead to a gain of function of the NaV1.6 channel, although inherited and bi-allelic variants have been reported (Gardella and Møller, 2019; Wengert et al., 2019).SCN8A-DEE patients present early in life with seizure onset usually occurring in the first year of life. After seizure onset, patients begin to miss developmental milestones and display additional symptoms, including cognitive and motor delay, hypotonia, and cortical blindness. SCN8A-DEE individuals are predisposed to early death, including sudden unexplained death in epilepsy (SUDEP). While SCN8A-RESpatients often have treatment-resistant seizures, many can achieve seizure reduction or seizure freedom upon treatment with anti-seizure medicines (ASMs) that non-selectively inhibit voltage-gated sodium channels, like phenytoin (Boerma et al., 2016; Braakman et al., 2017). SCN8A-RES patients may require doses that are higher than those prescribed for most epilepsy patients and, as a result, can be more prone to drug-related adverse events (Boerma et al., 2016; Gardella and Møller, 2019). Even with high doses and multiple ASMs, many patients continue to have uncontrolled seizures as well as extensive comorbidities. The aggressive pharmacotherapy required to protect SCN8Apatients from life-threatening seizures often comes with attendant side effects that would not be tolerated in less severely impacted populations. Existing sodium channel inhibitor ASMs are nonselective, blocking all voltage-gated sodium channel isoforms at similar plasma or brain concentrations. This lack of selectivity likely limits the benefits of sodium channel inhibitors since LoF variants of NaV1.1 are known toimpair inhibitory interneuron function and causegeneralized epilepsy with seizures plus (GEFS+) and SCN1A-DEE (Dravet Syndrome) (Catterall et al., 2010; Claes et al., 2001; Escayg et al., 2000; Gennaro et al., 2003).Thus, inhibiting NaV1.1 may counter the benefit of inhibiting the sodium channels of excitatory neurons. Inhibiting NaV1.4 and NaV1.5 currents is also undesirable since those channels are critical for facilitating contraction of skeletal and cardiac muscles, respectively (Chen et al., 1998; Ptácek et al., 1991; Rojas et al., 1991). We hypothesized that a selective inhibitor of NaV1.6 could provide a safer and more effective treatment for patients with SCN8A-RES and might also be more broadly efficacious in more common forms of epilepsy. An extensive medicinal-chemistry effort produced NBI-921352, the firstpotent and selective inhibitor of NaV1.6 channels (Neurocrine, 2019). We explored the profile of NBI-921352 in vitro, ex vivo and in threepreclinical in vivo rodent seizure models, including electrically induced seizure assays in genetically engineered mice bearing heterozygousScn8a GoFNaV1.6 channels (N1768D), as well as in wild-type mice and rats. Results In vitro NaV potency and selectivity Human NaV channel isoforms hNaV1.1, hNaV1.2, hNaV1.3, hNaV1.4, hNaV1.5, hNaV1.6, and hNaV1.7 were heterologously expressed in HEK-293 cells, and the potency and isoformselectivity of NBI-921352 (Figure 1, Table 1) was determined by automated patch-clamp techniques.NBI-921352 potently inhibitedhNaV1.6 channel currents with an inhibitory concentration 50% (IC50)of 0.051 µM (95% CI: 0.030–0.073 µM; N = 3) calculated from three biological replicates. Inhibition of other human NaV1.X isoforms required higher concentrations of NBI-921352 with IC50’s of 39 µM (95% CI: 31–47 µM; N = 3)for hNaV1.1, 6.9 µM (95% CI: 1.6–12 µM; N = 3) for hNaV1.2, > 30 µM for hNaV1.3, > 30 µM for hNaV1.4, > 30 µM for hNaV1.5, and 14 µM (95% CI: 6.4–22 µM; N = 3) for hNaV1.7. These potencies provide selectivity ratios for hNaV1.6 versus the other hNaV isoforms (IC50 hNaV1.X / IC50 hNaV1.6) of 756 (NaV1.1), 134 (NaV1.2), 276 (NaV1.7) and >583 (NaV1.3, NaV1.4, NaV1.5). Inhibition of NaV1.8 and NaV1.9 was not assessed since both channels arebelieved to be limited to peripheral sensory neurons and are not expected tohave much impact on the efficacy and tolerability of sodium channel inhibitors in epilepsy. Inhibition of NaV1.8 and NaV1.9 might reduce pain perception, but this would not be considered a significant liability for an anti-seizure medicine. Figure 1 with 2 supplements see all Download asset Open asset Potency and isoform selectivity of NBI-921352 for human and mouse NaV channels. Concentration-response curves were generated by automated patch-clamp electrophysiology using the SophionQube. Concentration-response curves were generated for human (A) or mouse (B) NaV channel isoforms heterologously expressed in HEK293 cells. The analysis included only those cells that met pre-specified acceptance criteria for seal quality, current amplitude, and series resistance. Normalized data from all cell recordings at a concentration were grouped together and plotted with GraphPad Prism 8. Details regarding the number of cells analyzed for each NaV channel and concentration can be found in the source data sheet. Error bars indicating the standard error of the mean fraction were plotted for all points, but, in some cases, they were smaller than the data point symbols and, therefore, not visible. The chemical structure of NBI-921352 is shown (C). Figure 1—source data 1 Quantification of potency and isoform selectivity of NBI-921352. https://cdn.elifesciences.org/articles/72468/elife-72468-fig1-data1-v2.xlsx Download elife-72468-fig1-data1-v2.xlsx Table 1 Potency and isoform selectivity of NBI-921352 for human and mouse NaV channels. Note that IC50s for the neuronal sodium channels, NaV 1.1, NaV 1.2, and NaV1.6, have been more accurately defined than those for non-neuronal sodium channels. Explicit IC50’s for NaV1.3, NaV1.4, and NaV1.5 were not determined since the inhibition at the highest concentration tested (30 µM) was <50%. IC50’s are the mean of 3 separate biological replicates of the IC50 determinations for each channel. The error is shown as the 95% confidence interval of the mean IC50. None of the other tested isoforms displayed IC50’s within the 95% confidence interval of the NaV1.6 IC50 and the confidence intervals were well separated as well. NaV1.6NaV1.1NaV1.2NaV1.3NaV1.4NaV1.5NaV1.7Human IC50 (µM)0.051396.9> 30> 30> 301495% CI0.030–0.07331–471.6–12---6.4–22Human Selectivity hNaV1.X / hNaV1.61756134> 583> 583> 583276Mouse IC50 (µM)0.0584111Mouse Selectivity mNaV1.X / mNaV1.61709191 Since we intended to evaluate in vivo effects of NBI-921352 in mouse seizure models, we also assessed the potency of NBI-921352 in the mouse NaV isoforms that are most highly expressed in the brain, NaV1.6, NaV1.1, and NaV1.2. The potency and selectivity in mouse NaV channels closely paralleled that seen in the human orthologues with IC50’s of 0.058 µM (95% CI: 0.046–0.070 µM; N = 3) for mNaV1.6, 41 µM (95% CI: 30–52 µM; N = 3) for mNaV1.1, and 11 µM (95% CI: 8.2–14 µM; N = 3) for mNaV1.2. Selectivity ratios (IC50 mNaV1.X / IC50 mNaV1.6) were 709 (NaV1.1), and 191 (NaV1.2). These data indicate that NBI-921352 potently inhibits both human and mouse NaV1.6 channels, and that it does so at concentrations ≥ 134 fold lower than for any of the other channel isoforms tested. NBI-921352 inhibited patient-identified variants of NaV1.6 channels Patients with SCN8A-RES carry missense variants in the NaV1.6 channel. A great number of variants have been identified, with a range of biophysical defects. Since most variants are de novo, many have been identified in only one or a few patients. For this reason, we determined the effectiveness of NBI-921352 to inhibit nine patient identified variants spread across the channel (Figure 2, Table 2; Gardella and Møller, 2019; Wagnon and Meisler, 2015). The nine variants studied have all been identified in SCN8A-RES patients and are in Domains II, III, IV, and the C-terminus. Inhibition of the mutant channel constructs was evaluated by automated patch-clamp electrophysiological techniques after transient transfection of the human NaV1.6 variant construct of interest into Expi293F cells. All the variants were sensitive to inhibition by NBI-921352. Observed IC50s for inhibition were 0.051 µM (WT mean from Figure 1), 0.031 µM (95% CI: 0.027–0.037 µM)(T767I), 0.021 µM (95% CI: 0.017–0.026 µM)(R850Q), 0.032 µM (95% CI: 0.029–0.036 µM)(N984K), 0.035 µM (95% CI: 0.029–0.043 µM)(I1327V), 0.039 µM (95% CI: 0.031–0.050 µM)(N1466K), 0.34 µM (95% CI: 0.26–0.44 µM)(R1617Q), 0.055 µM (95% CI: 0.046–0.064 µM)(N1768D), 0.068 µM (95% CI: 0.054–0.085 µM)(R1872W), and 0.035 µM (95% CI: 0.029–0.041 µM)(N1877S). We found that eight of the nine variants were inhibited with a potencysimilar to that of the wild-type channel, with most being slightly more potently inhibited. Only one variant, R1617Q, required markedly higher concentrations of NBI-921352 for inhibition, with an IC50for inhibition 6.6-fold higher than that of the wild-type NaV1.6 channel. The reduced potency for R1617Qis consistent with the variant residing in the predicted binding site of NBI-921352 in the domain IV voltage sensor, see discussion. Figure 2 Download asset Open asset Comparison of NBI-921352 potency on human wild-type NaV1.6 and patient-identified variants of NaV1.6. All constructs were transiently transfected into Expi293F cells and evaluated by automated patch-clamp electrophysiology using the SophionQube. The voltage-clamp methods were identical to those used for evaluation of the wild-type channels. The error bars indicate the 95% confidence interval of the fitted IC50 generated in Prism. The horizontal dotted line is at the IC50 for wild type NaV1.6 (51 nM, see Figure 1). The gray shaded band indicates the 95% confidence range for the IC50 for wild-type NaV1.6. Only two variants have fitted IC50’s outside of the 95% confidence interval for the wild-type channel IC50. R850Q was slightly more potently inhibited and R1617Q was less potently inhibited than the wild-type channel. R1617Q is near the proposed binding site for NBI-921352. Figure 2—source data 1 Quantification inhibition of patient variants. https://cdn.elifesciences.org/articles/72468/elife-72468-fig2-data1-v2.xlsx Download elife-72468-fig2-data1-v2.xlsx Table 2 Comparison of NBI-921352 potency on human wild-type NaV1.6 and patient-identified gain-of-function variants of NaV1.6. IC50s corresponding to Figure 2 are shown in the table and were calculated as indicated for Table 1. The 95% confidence intervals are those determined by the fit of the IC50 in Prism. WTT767IR850QN984KI1327VN1466KR1617QN1768DR1872WN1877ShNaV1.6 IC50 (µM)0.0510.0310.0210.0320.0350.0390.3490.0540.0670.03495% CI0.030–0.0730.027–0.0370.017–0.0260.029–0.0350.029–0.0420.031–0.0490.28 to0.400.047–0.0650.053–0.0850.029–0.040Fold changeWT / Variant-0.60.40.60.70.86.81.11.30.7 NBI-921352 is a state-dependent inhibitor Many small molecule inhibitors of NaV channels bind preferentially to open and or inactivated states (Bean et al., 1983; Courtney et al., 1978; Strichartz, 1976). NaV inhibitors that are structurally similar to NBI-921352 are known to act by binding to the VSD4 in the ‘UP’, position and hence stabilize inactivated states (Ahuja et al., 2015; McCormack et al., 2013). Charge movement in VSD4 has been linked to the voltage dependence of fast inactivation (Ahern et al., 2016). It is likely that the anionic aryl sulfonamide headgroup of NBI-921352 interacts with the fourth arginine in S4 of VSD4 and prevents return of VSD4 to the rested position and recovery from inactivation as seen for related compounds (Ahuja et al., 2015). For these reasons, we expect that binding will be encouraged by VSD4 residing in the UP, inactivated, state. We designed our voltage-clamp protocols to encourage high occupancy of inactivated states by holding at depolarized membrane potentials (–45 mV for NaV1.1, NaV1.2, NaV1.3, NaV1.4, and NaV1.6). The isoforms that inactivate at the most negative membrane potentials (NaV1.5 and NaV1.7) accumulate excess slow inactivation at –45 mV, reducing the signal size. For these two isoforms, the membrane potential was held at –60 mV to preserve robust assay performance while fully inactivating the channels. Our investigation into the state dependence of NBI-921352 did not attempt to rigorously differentiate binding to open channels versus fast inactivated or fast inactivated versus slow inactivated states. To query the state dependence of NBI-921352, we measured the apparent potency with two different voltage protocols that favor either the closed (rested) state or inactivatedstates (Figure 3). Holding the membrane potential at –120 mV induces most channels to reside in the resting state. Brief depolarizations to measure NaV1.6 current enabled the determination of an IC50of 36 µM (95% CI: 29–47 µM) for rested-state channels. Holding the membrane potential at –45 mV encourages channels to transition into inactivated states. Brief hyperpolarizations allow rapid recovery from inactivation for channels that are not bound to drug followed by a short 20ms test pulse to –20 mV to measure currents from unbound channels (see Materials and methods for details). Measuring the ability of NBI-921352 to inhibit reopening of activated channels leads to an apparent IC50 of 0.051 µM (Figures 1 and 3). Thus, NBI-921352 strongly prefers inactivated channels, inhibiting them at concentrations more than 750-fold less than those needed to inhibit rested or ‘peak’ sodium currents. Figure 3 with 1 supplement see all Download asset Open asset NBI-921352 is a state-dependent inhibitor of NaV1.6 and preferentially targets inactivated channels. Concentration-response curves were generated for human WT and N1788D channel isoforms heterologously expressed in HEK293 cells. The analysis included only those cells that met pre-specified acceptance criteria for seal quality, current amplitude, and series resistance. Normalized data from all cell recordings at a concentration were grouped together and plotted with GraphPad Prism 8. Details regarding the number of cells analyzed for each NaV channel isoform and concentration can be found in the source data sheet. Error bars indicating the standard error of the mean fraction were plotted for all points. The blue dotted line indicates the concentration-response curve for wild-type NaV1.6 from Figure 1. When NaV1.6 channels were equilibrated with NBI-921352 at voltages that allow equilibration with inactivated states (–45 mV), the compound provided potent inhibition, as seen in Figure 1. NBI-921352 also exhibited potent block of NaV1.6 when measuring persistent or resurgent sodium current using distinct voltage protocols (see Materials and methods and text). Forcing channels to the rested, closed state by hyperpolarizing to –120 mV resulted in very weak inhibition. Current evoked from very negative potentials is sometimes referred to as ‘peak current’. The 95% confidence intervals for the IC50’s reported in the results are those provided for the error of the fit by Prism. Figure 3—source data 1 Quantification of state dependence of NBI-921352. https://cdn.elifesciences.org/articles/72468/elife-72468-fig3-data1-v2.xlsx Download elife-72468-fig3-data1-v2.xlsx At more hyperpolarized potentials, potency for all isoforms will tend to be somewhat less. Nonetheless potency on NaV1.6 at a more physiologic potential (–62 mV) is shown in Figure 1—figure supplement 2 where the push towards inactivated states is not so strong. NBI-921352 remains potent (IC500.053 µM) in this assay, suggesting that potency and selectivity under physiologic conditions will remain high. NBI-921352 inhibited persistent and resurgent currents from mutant NaV1.6 channels The state-dependent nature of inhibition is also revealed in other types of voltage-clamp protocols, including those designed to measure persistent or resurgent sodium currents. Some drugs or candidate drugs, like PRAX-330 and Riluzole, have been touted based on their preference for persistent currents, but this appears to be a feature of many of the compounds in the NaV inhibitor class since they are generally poor inhibitors of closed/rested state channels and bind preferentially to activated channels (Colombo et al., 2013; Mason et al., 2019; Wengert and Patel, 2021). Apparent differences in persistent current selectivity are largely driven by differential kinetics and concentration dependences in combination with the electrophysiological protocols chosen for the measurements. Elevated persistent and or resurgent currents are believed to underlie or contribute to the pathology of many sodium channel related pathologies (Mason et al., 2019; Pan and Cummins, 2020; Potet et al., 2020; Tidball et al., 2020; Zaman et al., 2019). In most conditions, normal NaV1.6 channels inactivate rapidly and nearly completely after opening. Persistent currents result from channels that are not stably inactivated – a common phenotype for epilepsy-inducing variants in NaV1.6, including N1768D (Tidball et al., 2020; Wagnon et al., 2015). We found that NBI-921352 inhibited N1768D NaV1.6 persistent currents (measured as the non-inactivating current 10ms after initiation of the depolarizing test pulse) with a similar potency as for activated wild-type NaV1.6 channels with an IC50 of 0.059 µM (95% CI: 0.044–0.082 µM) (Figure 3). A more traditional approach to measuring persistent currents is to step from a very hyperpolarized voltage (for example –120 mV) to a strong depolarization for 50ms or longer. This approach is not viable for NBI-921352 since equilibration of block after from such negative, non-physiological, voltages takes several seconds. Resurgent currents occur after repolarizing following a strong depolarization as channels redistribute between closed, open, and inactivated states (Raman and Bean, 1997). These resurgent currents are enhanced in many SCN8A-RES variants (Pan and Cummins, 2020; Raman et al., 1997). NBI-921352 also effectively inhibited resurgent currents from N1768D channels with apparent IC50 of 0.037 µM (95% CI: 0.025–0.060 µM). While the resurgent currents cannot be measured with the same voltage protocols used to measure inactivated state inhibition, these data indicate that N1768D NaV1.6 resurgent currents are susceptible to inhibition by NBI-921352 and that inhibition occurs at similar concentrations. NBI-921352 preferentially inhibited excitatory pyramidal neurons and spared inhibitory interneurons A primary goal of creating NaV1.6 selective inhibitors was to spare NaV1.1, the voltage-gated sodium channel that is most prevalent in inhibitory interneurons. This should allow the selective targeting of excitatory neurons, where NaV1.6 and NaV1.2 are believed to be dominant, over inhibitory interneurons. To test this hypothesis, we performed current-clamp experiments in glutamatergic pyramidal neurons from mouse layer five neocortex and from fast spiking interneurons in the same region. Application of 0.250 µM NBI-921352 decreased the maximum firing rate in all three pyramidal neurons tested (Figure 4A). The difference in the cumulative area under the curves (AUC) for control versus NBI-921352 treated conditions was evaluated by a paired two tailed t-test (N = 3 or 4 cells for each point). Current injection levels > 160 pA led to a significant reduction of the cumulative area under the input output curve (p < 0.05 in a paired two-tailed t-tests) relative to the control condition. Specific p values are shown in the data transparency Excel file. See Figure 4—figure supplement 1 for more individual neuron comparisons. Figure 4 with 1 supplement see all Download asset Open asset NBI-921352 inhibits firing in pyramidal neurons but spares fast-spiking interneurons. Current input versus action-potential output evaluations in wild-type mouse brain slices treated with vehicle or 0.25 µM NBI-921352 (A & B), or 100 µM carbamazepine (C & D) was plotted. In cortical pyramidal neurons, both NBI-921352 (A) and carbamazepine (C) reduced action-potential spiking. In fast-spiking cortical interneurons, treatment with NBI-921352 resulted in a trend toward slightly increased firing frequency (B), while carbamazepine markedly reduced firing (D). The main upper panels compare average action-potential count of 3–4 neurons in each condition±the standard error of the mean. The lower panels show recordings for individual representative neurons for each condition. No inhibitors of synaptic inputs were used for these experiments. The difference in the cumulative area under the curves (AUC) for control versus NBI-921352 treated conditions was evaluated by a paired two tailed t-test (N = 3 or 4 cells for each point). *Indicates a p < 0.05 relative to the control condition. Specific p values are shown in the data transparency Excel file. See Figure 4—figure supplement 1 for more individual neuron comparisons. Figure 4—source data 1 Quantification of cortical neuron current clamp input output. https://cdn.elifesciences.org/articles/72468/elife-72468-fig4-data1-v2.xlsx Download elife-72468-fig4-data1-v2.xlsx In contrast, NBI-921352 had no significant effect on the fast-firing inhibitory interneurons tested (Figure 4B). Carbamazepine significantly inhibited action-potential firing in both pyramidal neurons and in fast-spiking interneurons and the degree of inhibition was similar in both types of neurons. A before and after comparison for all tested neurons is shown in Figure 4—figure supplement 1. NBI-921352 inhibited electrically induced seizures in Scn8aN1768D/+ mice A selective inhibitor of NaV1.6 should lend itself to the treatment of disease states caused by pathologic gain of function of NaV1.6 channels. Hence, we examined the ability of NBI-921352 to inhibit electrically induced seizures in mice with a patient-identified GoF variant in the Scn8a gene encoding NaV1.6. N1768D is a variant of NaV1.6 identified in the first reported SCN8A-DEE patient (Veeramah et al., 2012). N1768D NaV1.6 channels have impaired voltage-dependent inactivation gating that results in persistent sodium currents and enhanced resurgent currents. Because NaV1.6 channels are highly expressed in the neurons of the brain, increased sodium flux in excitatory neurons leads to seizures. Genetically modified mice bearing the same variant (Scn8aN1768D/+) were created and found to be seizure prone, producing a mouse model with a similar phenotype as that observed in SCN8A-DEE patients (Wagnon et al., 2015).Some Scn8aN1768D/+ mice develop spontaneous seizures at age p60 to p100, but seizure onset and frequency is quite variable, making spontaneous seizure studies challenging. In addition, mice rapidly clear NBI-921352, making it extremely difficult to maintain drug plasma and brain levels in an efficacious range for chronic or subchronic dosing experiments. NBI-921352 is more stable in humans with a half-life of elimination of approximately 8.5 hr (Beatch et al., 2020). As an alternative means of assessing NBI-921352’s ability to engage NaV1.6 channels in vivo, we designed a modified version of the 6 Hz psychomotor seizure assay in Scn8aN1768D/+ mice (Barton et al., 2001; Focken et al., 2019). A mild current stimulation (12 mA) evoked robust generalized tonic-clonic seizures (GTC) with hindlimb extensionin Scn8aN1768D/+ mice, but not in wild-type littermates. Oral administration of NBI-921352 2 hr prior to electrical stimulation prevented induction of GTC with hindlimb extension in Scn8aN1768D/+ mice in a dose-dependent manner with a 50% effective dose (ED50) of 15 mg/kg (95% CI 9.6–23 mg/kg, see Figure 5A). Figure 5 with 2 supplements see all Download asset Open asset NBI-921352 inhibited electrically induced seizures in rodents. Dose of NBI-921352 is plotted versus efficacy in Scn8aN1768D+/- mice in the modified 6 Hz psychomotor seizure assay in A. Plasma concentration of NBI-921352 is plotted versus efficacy in Scn8aN1768D+/- mice in the modified 6 Hz psychomotor seizure assay in B. Brain concentration of NBI-921352 is plotted versus efficacy in Scn8aN1768D+/- mice in the modified 6 Hz psychomotor seizure assay in C.Green open triangles represent data from animals that received a single dose 2 hr before testing in the seizure assay. Red open triangles represent data from animals that received two daily doses (once every 12 hr) for 6 days. On day 7, these animals were given a final dose (the 13th dosing) 2 hr before testing in the seizure assay. Dose of NBI-921352 is plotted versus efficacy in wild-type mice in the DC-MES assay in D. Plasma concentration of NBI-921352 is plotted versus efficacy inwild-type mice in the DC-MES assay in E. Brain concentration of NBI-921352 is plotted versus efficacy inwild-type mice in the DC-MES assay in F. Blue open circles represent data from animals that received a single dose 2 hr before testing in the seizure assay. Red open circles represent data from animals that received two daily doses (once every 12 hr) for 6 days. On day seven these animals were given a final dose (the 13th dosing) 2 hr before testing in the seizure assay. Dose of NBI-921352 is plotted versus efficacy inwild-type rats in the DC-MES assay in G. Plasma concentration of NBI-921352 is plotted versus efficacy inwild-type rats in the DC-MES assay in H. Brain concentration of NBI-921352 is plotted versus efficacy inwild-type rats in the DC-MES assay in I. Black open diamonds represent data from animals that received a single dose 2 hr before testing in the seizure assay. Red open diamonds represent data from animals that received two daily doses (once every 12 hr) for 6 days. On day 7, these animals were given a final dose (the 13th dose) 2 hr before testing in the seizure assay. Each point represents the fraction of animals exhibiting a GTC with hindlimb extension after

NBI-921352 (formerly XEN901) is a novel sodium channel inhibitor designed to specifically target NaV1.6 channels. Such a molecule provides a precision-medicine approach to target SCN8A-related epilepsy syndromes (SCN8A-RES), where gain-of-function (GoF) mutations lead to excess NaV1.6 sodium current, or other indications where NaV1.6 mediated hyper-excitability contributes to disease (Gardella and Møller, 2019; Johannesen et al., 2019; Veeramah et al., 2012). NBI-921352 is a potent inhibitor of NaV1.6 (IC500.051 µM), with exquisite selectivity over other sodium channel isoforms (selectivity ratios of 756 X for NaV1.1, 134 X for NaV1.2, 276 X for NaV1.7, and >583 Xfor NaV1.3, NaV1.4, and NaV1.5). NBI-921352is a state-dependent inhibitor, preferentially inhibiting inactivatedchannels. The state dependence leads to potent stabilization of inactivation, inhibiting NaV1.6 currents, including resurgent and persistent NaV1.6 currents, while sparing the closed/rested channels. The isoform-selective profile of NBI-921352 led to a robust inhibition of action-potential firing in glutamatergic excitatory pyramidal neurons, while sparing fast-spiking inhibitory interneurons, where NaV1.1 predominates. Oral administration of NBI-921352 prevented electrically induced seizures in a Scn8a GoF mouse,as well as in wild-type mouse and ratseizure models. NBI-921352 was effective in preventing seizures at lower brain and plasma concentrations than commonly prescribed sodium channel inhibitor anti-seizure medicines (ASMs) carbamazepine, phenytoin, and lacosamide. NBI-921352 waswell tolerated at higher multiples of the effective plasma and brain concentrations than those ASMs. NBI-921352 is entering phase II proof-of-concept trials for the treatment of SCN8A-developmental epileptic encephalopathy (SCN8A-DEE) and adult focal-onset seizures.

Nav1.7 is an extensively investigated target for pain with a strong genetic link in humans, yet in spite of this effort, it remains challenging to identify efficacious, selective, and safe inhibitors. Here, we disclose the discovery and preclinical profile of GDC-0276 (1) and GDC-0310 (2), selective Nav1.7 inhibitors that have completed Phase 1 trials. Our initial search focused on close-in analogues to early compound 3. This resulted in the discovery of GDC-0276 (1), which possessed improved metabolic stability and an acceptable overall pharmacokinetics profile. To further derisk the predicted human pharmacokinetics and enable QD dosing, additional optimization of the scaffold was conducted, resulting in the discovery of a novel series of N-benzyl piperidine Nav1.7 inhibitors. Improvement of the metabolic stability by blocking the labile benzylic position led to the discovery of GDC-0310 (2), which possesses improved Nav selectivity and pharmacokinetic profile over 1.

We describe the synthesis and biological evaluation of a series of novel aryl sulfonamides that exhibit potent inhibition of NaV1.5. Unlike local anesthetics that are currently used for treatment of Long QT Syndrome 3 (LQT-3), the most potent compound (-)-6 in this series shows high selectivity over hERG and other cardiac ion channels and has a low brain to plasma ratio to minimize CNS side effects. Compound (-)-6 is also effective inshortening prolonged action potential durations (APDs) in a pharmacological model of LQT-3 syndrome in pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Unlike most aryl sulfonamide NaV inhibitors that bind to the channel voltage sensors, these NaV1.5 inhibitors bind to the local anesthetic binding site in the central pore of the channel.

RationaleNa(v)1.6 is a transmembrane voltage gated sodium channel implicated in various forms of epilepsy. Modulation of its activity in epilepsy animal models can be accomplished using inhibitors which may result in changes in its expression. There is a need to generate reliable quantitative measurements of Na(v)1.6 expression in animal models. This research explores the feasibility of quantifying Na(v)1.6 expression in mouse brains using targeted multiple reaction monitoring (MRM) mass spectrometry. MethodsA combination of in silico tryptic Na(v)1.6 peptides and MRM transitions were used to select target peptides. This was followed by a simple proteomic work-up including plasma membrane isolation, trypsin-based proteolysis and ultra-high-performance/electrospray ionization tandem mass spectrometry (UHPLC/ESI-MS/MS) to detect the presence of Na(v)1.6 in induced HEK293 cells. The unique Na(v)1.6 peptide, DSLFIPR, was selected as probe for quantifying Na(v)1.6 levels in brains from C57BL/6J wild-type mice as well as two kinds of mutants including Scn8a(N1768D/+) and heterozygous null Scn8a(+/-) mice using isotope dilution targeted mass spectrometry. ResultsThe feasibility of using targeted MRM for quantifying Na(v)1.6 expression in mice brains was demonstrated. Expression of Na(v)1.6 in brains (hippocampi) from wild-type and mutant Scn8a(N1768D/+) mice were found to be around 0.40 fmol/g. Mutant null Scn8a(+/-) heterozygous mice, on the other hand, showed levels of 0.22 fmol/g as expected based on this particular mutation which only generates 50% of the expression in wild-type mice. Na(v)1.6-overexpressed HEK293 cells showed 3.7 fmol/g of Na(v)1.6 expression, suitable for screening new compounds for Na(v)1.6 blocking activity. ConclusionsThe results of the present feasibility study support the use of DSLFIPIR for quantification of Nav1.6 in brain tissues using UHPL/ESI-MS/MS.

Nonselective antagonists of voltage-gated sodium (NaV) channels have been long used for the treatment of epilepsies. The efficacy of these drugs is thought to be due to the block of sodium channels on excitatory neurons, primarily NaV1.6 and NaV1.2. However, these currently marketed drugs require high drug exposure and suffer from narrow therapeutic indices. Selective inhibition of NaV1.6, while sparing NaV1.1, is anticipated to provide a more effective and better tolerated treatment for epilepsies. In addition, block of NaV1.2 may complement the anticonvulsant activity of NaV1.6 inhibition. We discovered a novel series of aryl sulfonamides as CNS-penetrant, isoform-selective NaV1.6 inhibitors, which also displayed potent block of NaV1.2. Optimization focused on increasing selectivity over NaV1.1, improving metabolic stability, reducing active efflux, and addressing a pregnane X-receptor liability. We obtained compounds 30-32, which produced potent anticonvulsant activity in mouse seizure models, including a direct current maximal electroshock seizure assay.

Herein, we report the discovery and optimization of a series of orally bioavailable acyl sulfonamide NaV1.7 inhibitors that are selective for NaV1.7 over NaV1.5 and highly efficacious in in vivo models of pain and hNaV1.7 target engagement. An analysis of the physicochemical properties of literature NaV1.7 inhibitors suggested that acyl sulfonamides with high fsp3 could overcome some of the pharmacokinetic (PK) and efficacy challenges seen with existing series. Parallel library syntheses lead to the identification of analogue 7, which exhibited moderate potency against NaV1.7 and an acceptable PK profile in rodents, but relatively poor stability in human liver microsomes. Further, design strategy then focused on the optimization of potency against hNaV1.7 and improvement of human metabolic stability, utilizing induced fit docking in our previously disclosed X-ray cocrystal of the NaV1.7 voltage sensing domain. These investigations culminated in the discovery of tool compound 33, one of the most potent and efficacious NaV1.7 inhibitors reported to date.

The sodium channel NaV1.7 has emerged as a promising target for the treatment of pain based on strong genetic validation of its role in nociception. In recent years, a number of aryl and acyl sulfonamides have been reported as potent inhibitors of NaV1.7, with high selectivity over the cardiac isoform NaV1.5. Herein, we report on the discovery of a novel series of N-([1,2,4]triazolo[4,3-a]pyridin-3-yl)methanesulfonamides as selective NaV1.7 inhibitors. Starting with the crystal structure of an acyl sulfonamide, we rationalized that cyclization to form a fused heterocycle would improve physicochemical properties, in particular lipophilicity. Our design strategy focused on optimization of potency for block of NaV1.7 and human metabolic stability. Lead compounds 10, 13 (GNE-131), and 25 showed excellent potency, good in vitro metabolic stability, and low in vivo clearance in mouse, rat, and dog. Compound 13 also displayed excellent efficacy in a transgenic mouse model of induced pain.

Selective block of Na(V)1.7 promises to produce non-narcotic analgesic activity without motor or cognitive impairment. Several Na(V)1.7-selective blockers have been reported, but efficacy in animal pain models required high multiples of the IC50 for channel block. Here, we report a target engagement assay using transgenic mice that has enabled the development of a second generation of selective Nav1.7 inhibitors that show robust analgesic activity in inflammatory and neuropathic pain models at low multiples of the IC50. Like earlier arylsulfonamides, these newer acylsulfonamides target a binding site on the surface of voltage sensor domain 4 to achieve high selectivity among sodium channel isoforms and steeply state-dependent block. The improved efficacy correlates with very slow dissociation from the target channel. Chronic dosing increases compound potency about 10-fold, possibly due to reversal of sensitization arising during chronic injury, and provides efficacy that persists long after the compound has cleared from plasma.