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
- In Vitro Biology, Xenon Pharmaceuticals Inc, Canada
- Chemistry, Xenon Pharmaceuticals Inc, Canada
- In Vivo Biology, Xenon Pharmaceuticals Inc, Canada
- Compound Properties, Xenon Pharmaceuticals Inc, Canada
- Translational Drug Development, Xenon Pharmaceuticals Inc, Canada
- Scientific Affairs, Xenon Pharmaceuticals, Inc, Canada
- Executive Team, Xenon Pharmaceuticals Inc, Canada
Figures
Figure 1 with 2 supplements
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 …
-
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
Figure 1—figure supplement 1
Representative raw traces of voltage-clamp recordings of control and the concentrations close to the NBI-921352 IC50 for all tested sodium channel isoforms in Figure 1.
Note that in some cases currents are outward because the sodium gradients were reversed. This was done to improve assay robustness and reproducibility. See methods for details. We have found that …
Figure 1—figure supplement 2
Potency of NBI-921352 measured form a holding potential approximating the V0.5 of inactivation for NaV1.
6. For the “V0.5” assay the membrane was held at –62 mV for Nav1.6 and the protocol shown above was applied. The solutions used were the same as for hNav1.6 in the standard assay. The protocol …
-
Figure 1—figure supplement 2—source data 1
Quantification of potency with alternate voltage protocol.
- https://cdn.elifesciences.org/articles/72468/elife-72468-fig1-figsupp2-data1-v2.xlsx
Figure 2
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 …
-
Figure 2—source data 1
Quantification inhibition of patient variants.
- https://cdn.elifesciences.org/articles/72468/elife-72468-fig2-data1-v2.xlsx
Figure 3 with 1 supplement
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 …
-
Figure 3—source data 1
Quantification of state dependence of NBI-921352.
- https://cdn.elifesciences.org/articles/72468/elife-72468-fig3-data1-v2.xlsx
Figure 3—figure supplement 1
Representative raw traces of the NaV1.6 currents used to generate the summary data in Figure 3.
(A) Currents from WT hNaV1.6 channels holding at resting state (voltage = –120 mV) before and after ~IC50 concentration of 33 uM NBI-921352 (B) The persistent currents of NaV1.6-N1768D before and …
Figure 4 with 1 supplement
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 …
-
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
Figure 4—figure supplement 1
Results for each tested neuron for the neurons summarized in Figure 4.
For each neuron, the number of action potentials are shown in response to a current injection level of approximately 3 X the rheobase (determined for each neuron individually) before and after …
-
Figure 4—figure supplement 1—source data 1
Quantification of individual neurons.
- https://cdn.elifesciences.org/articles/72468/elife-72468-fig4-figsupp1-data1-v2.xlsx
Figure 5 with 2 supplements
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+…
-
Figure 5—source data 1
Quantification of impact of NBI-921352 on rodent seizures.
- https://cdn.elifesciences.org/articles/72468/elife-72468-fig5-data1-v2.xlsx
Figure 5—figure supplement 1
Dose response and statistical analysis of the NBI-921352 dose groups for which brain and plasma concentration response curves are shown in Figure 5.
In some cases, multiple experimental groups at the same test dose were assessed. In such cases groups at the same dose level were combined here for comparison to vehicle treated animals. Fraction …
Figure 5—figure supplement 2
Comparison of single dose versus sub-chronic dosing for NBI-921352.
Vehicle, day 1 and day 7 (13 doses BID) were run in parallel with Vehicle animals receiving vehicle every day twice a day. Day 1 animals received 12 doses of vehicle BID before a final dosing with …
Figure 6 with 3 supplements
NBI-921352 is more potent than three commonly prescribed NaV inhibitor ASMs.
Brain concentration versus fraction of animals exhibiting is plotted for NBI-921352 versus that for phenytoin, carbamazepine, and lacosamide in the Scn8aN1768D+/- modified 6 Hz model (A), the …
-
Figure 6—source data 1
Quantification of effective brain concentrations of NBI-921352 versus common AEDs.
- https://cdn.elifesciences.org/articles/72468/elife-72468-fig6-data1-v2.xlsx
Figure 6—figure supplement 1
Dose response and statistical analysis of the Carbamazepine dose groups for which brain and plasma concentration response curves are shown in Figures 6 and 7 respectively.
In some cases, multiple experimental groups at the same test dose were assessed. In such cases, groups at the same dose level were combined here for comparison to vehicle treated animals. Fraction …
Figure 6—figure supplement 2
Dose response and statistical analysis of the Phenytoin dose groups for which brain and plasma concentration response curves are shown in Figures 6 and 7 respectively.
In some cases, multiple experimental groups at the same test dose were assessed. In such cases groups at the same dose level were combined here for comparison to vehicle treated animals. Fraction …
Figure 6—figure supplement 3
Dose response and statistical analysis of the Lacosamide dose groups for which brain and plasma concentration response curves are shown in Figures 6 and 7 respectively.
In some cases, multiple experimental groups at the same test dose were assessed. In such cases groups at the same dose level were combined here for comparison to vehicle-treated animals. Fraction …
Figure 7
Rat efficacy compared to acute tolerability for NBI-921352 relative to NaV inhibitor ASMs.
Plasma concentration versus efficacy data is shown for the rat DC-MES assay for NBI-921352 (A), carbamazepine (B), phenytoin (C), and lacosamide (D). The vertical dotted lines indicate the lowest …
-
Figure 7—source data 1
Quantification of effective and tolerated plasma concentrations of NBI-921352 versus common AEDs.
- https://cdn.elifesciences.org/articles/72468/elife-72468-fig7-data1-v2.xlsx
Tables
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 …
| NaV1.6 | NaV1.1 | NaV1.2 | NaV1.3 | NaV1.4 | NaV1.5 | NaV1.7 | |
|---|---|---|---|---|---|---|---|
| Human IC50 (µM) | 0.051 | 39 | 6.9 | > 30 | > 30 | > 30 | 14 |
| 95% CI | 0.030–0.073 | 31–47 | 1.6–12 | - | - | - | 6.4–22 |
| Human Selectivity hNaV1.X / hNaV1.6 | 1 | 756 | 134 | > 583 | > 583 | > 583 | 276 |
| Mouse IC50 (µM) | 0.058 | 41 | 11 | ||||
| Mouse Selectivity mNaV1.X / mNaV1.6 | 1 | 709 | 191 |
Table 2
Comparison of NBI-921352 potency on human wild-type NaV1.6 and patient-identified gain-of-function variants of NaV1.6.
| WT | T767I | R850Q | N984K | I1327V | N1466K | R1617Q | N1768D | R1872W | N1877S | |
|---|---|---|---|---|---|---|---|---|---|---|
| hNaV1.6 IC50 (µM) | 0.051 | 0.031 | 0.021 | 0.032 | 0.035 | 0.039 | 0.349 | 0.054 | 0.067 | 0.034 |
| 95% CI | 0.030–0.073 | 0.027–0.037 | 0.017–0.026 | 0.029–0.035 | 0.029–0.042 | 0.031–0.049 | 0.28 to0.40 | 0.047–0.065 | 0.053–0.085 | 0.029–0.040 |
| Fold changeWT / Variant | - | 0.6 | 0.4 | 0.6 | 0.7 | 0.8 | 6.8 | 1.1 | 1.3 | 0.7 |
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Cell line (H. sapiens) | Expi293F | Thermo Fischer | cat# A14527 | SCN8A mutant transient transfections |
| Cell line (H. sapiens) | FreeStyle 293 F | Thermo Fischer | cat# R710-07 | Stable transfections |
| Strain, strain background (M. musculus) | C57BL/6 J male Scn8aN1768D/+ (stock#400690)x C3HeB/FeJ female (strain#000658).Both Male and Female heterozygous Scn8aN1768D/+ micewere tested | Licensed from Miriam Meisler, Univ. of Michigan.Wagnon et al., 2015 | Colony maintained at The Jackson Laboratory | |
| Strain, strain background (M. musculus) | CF-1, male | Charles River | Code: 023 | |
| Strain, strain background(R. norvegicus) | Sprague Dawley, male | Envigo | Code: 002 | |
| Chemical compound, drug | NBI-921352 | US Patent #10246453 B2 | Compound ID #101 | Synthesized at Xenon Pharmaceuticals |
| Chemical compound, drug | Carbamazepine | Sigma-Aldrich | C4024 | |
| Chemical compound, drug | Phenytoin | Sigma-Aldrich | D4505 | |
| Chemical compound, drug | Lacosamide | Toronto Research Chemicals | L098500 | |
| Gene (H. sapiens) | SCN1A | GenBank | NM_006920 | |
| Gene (H. sapiens) | SCN2A | GenBank | NM_021007 | |
| Gene (H. sapiens) | SCN3A | GenBank | NM_0069220 | |
| Gene (H. sapiens) | SCN4A | GenBank | NM_000334 | |
| Gene (H. sapiens) | SCN5A | GenBank | NM_198056 | |
| Gene (H. sapiens) | SCN8A | GenBank | NM_014191 | |
| Gene (H. sapiens) | SCN9A | GenBank | NM_002977 | |
| Gene (M. musculus) | SCN1A | GenBank | NM_018733.2 | |
| Gene (M. musculus) | SCN2A | GenBank | NP_001092768.1 | |
| Gene (M. musculus) | SCN8A | GenBank | NM_001077499 | |
| Gene (H. sapiens) | SCN1B | GenBank | NM_199037 | |
| Gene (H. sapiens) | FGF13 | GenBank | NM_033642 | |
| Gene (H. sapiens) | CNTN1 | GenBank | NM_001843 | |
| Recombinant DNA reagent | pcDNA4/TO (vector) | Thermo Fischer | cat #V102020 | Vector for SCNxA genes |
| Recombinant DNA reagent | pcDNA6/TR(regulatory vector for tetracycline repressor protein) | Thermo Fischer | cat#V102520 | Vector to generate inducible FreeStyle 293 F and Expi293F |
| Recombinant DNA reagent | pcDNA3.1 (+)(vector) | Thermo Fischer | cat#V79020 | Vector for SCN1B gene |
| Recombinant DNA reagent | pcDNA3.1/Hygro(+) | Thermo Fischer | cat# V87020 | Vector for FGF13 and CNTN1 genes |
