Author response: NBI-921352, a first-in-class, NaV1.6 selective, sodium channel inhibitor that prevents seizures in Scn8a gain-of-function mice, and wild-type mice and rats (2022)

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 for NBI-921352 since equilibration of block after from voltages currents after a strong depolarization as channels and inactivated states and These resurgent currents are in many SCN8A-RES variants and Cummins, 2020; et al., NBI-921352 also inhibited resurgent currents from N1768D channels with apparent IC50 of µM (95% CI: While the resurgent currents be measured with the voltage protocols used to measure inactivated state inhibition, these data indicate that N1768D NaV1.6 resurgent currents are to inhibition by NBI-921352 and that inhibition at similar concentrations. NBI-921352 preferentially inhibited excitatory pyramidal neurons and inhibitory A of NaV1.6 selective inhibitors was to NaV1.1, the voltage-gated sodium channel that is most in inhibitory This allow the selective of excitatory neurons, where NaV1.6 and are believed to be over inhibitory To test this we in glutamatergic pyramidal neurons from mouse and from fast in the of µM NBI-921352 the firing in all three pyramidal neurons tested (Figure The in the under the curves for versus NBI-921352 conditions was evaluated by a two = 3 or cells for each Current > led to a significant reduction of the under the curve in a to the are shown in the data Figure supplement 1 for more Figure with 1 supplement see all Download asset Open asset NBI-921352 inhibits firing in pyramidal neurons but fast-spiking Current versus action-potential in wild-type mouse brain with or µM NBI-921352 or µM was In cortical pyramidal neurons, both NBI-921352 (A) and reduced action-potential In fast-spiking cortical interneurons, treatment with NBI-921352 resulted in a slightly firing while markedly reduced firing The action-potential of neurons in each standard error of the The lower recordings for neurons for each inhibitors of were used for these The in the under the curves for versus NBI-921352 conditions was evaluated by a two = 3 or cells for each a to the are shown in the data Figure supplement 1 for more Figure data 1 Quantification of cortical current Download In NBI-921352 significant on the inhibitory tested (Figure inhibited action-potential firing in both pyramidal neurons and in fast-spiking and the of inhibition was similar in both types of neurons. A and after for all tested neurons is shown in Figure supplement 1. NBI-921352 inhibited electrically induced seizures in mice A selective inhibitor of NaV1.6 to the treatment of disease states by gain of function of NaV1.6 channels. we the ability of NBI-921352 to inhibit electrically induced seizures in mice with a patient-identified GoF variant in the Scn8a gene NaV1.6. N1768D is a variant of NaV1.6 identified in the first reported SCN8A-DEE patient et al., 2012). N1768D NaV1.6 channels have inactivation that results in persistent sodium currents and resurgent currents. NaV1.6 channels are highly expressed in the neurons of the brain, sodium in excitatory neurons leads to seizures. mice bearing the variant were and found to be seizure a mouse with a similar phenotype as that in SCN8A-DEE patients et al., mice seizures at to but seizure onset and is seizure In mice rapidly NBI-921352, it to drug plasma and brain in an efficacious range for or NBI-921352 is more in humans with a of of et al., an of ability to NaV1.6 channels in we designed a of the seizure assay in mice et al., 2001; et al., 2019). A current evoked robust seizures with mice, but not in wild-type Oral administration of NBI-921352 2 to prevented of with in mice in a with a 50% effective of (95% see Figure Figure with 2 supplements see all Download asset Open asset NBI-921352 inhibited electrically induced seizures in of NBI-921352 is plotted versus efficacy in mice in the seizure assay in concentration of NBI-921352 is plotted versus efficacy in mice in the seizure assay in concentration of NBI-921352 is plotted versus efficacy in mice in the seizure assay in open data from that a 2 in the seizure open data from that two doses for these were a 2 in the seizure of NBI-921352 is plotted versus efficacy in wild-type mice in the assay in concentration of NBI-921352 is plotted versus efficacy mice in the assay in concentration of NBI-921352 is plotted versus efficacy mice in the assay in open data from that a 2 in the seizure open data from that two doses for these were a 2 in the seizure of NBI-921352 is plotted versus efficacy in the assay in concentration of NBI-921352 is plotted versus efficacy in the assay in concentration of NBI-921352 is plotted versus efficacy in the assay in open data from that a 2 in the seizure open data from that two doses for these were a 2 in the seizure point the fraction of a with after