In vitro activity: ICA-121431 is a potent, highly selective small molecule inhibitor of the human Nav1.3 and Nav1.1 voltage gated sodium channels with IC50 of 19 nM. It exhibit up to 1,000-fold selectivity for human Nav1.3/Nav1.1 (ICA-121431, IC50, 19 nM) vs. other TTX-sensitive or resistant (i.e., Nav1.5) sodium channels, in other words, it has little or no activity against human Nav1.5 or Nav1.7 channels. Voltage-gated sodium channels initiate action potentials in brain neurons, and sodium channel blockers are used in therapy of epilepsy.

Kinase Assay: ICA-121431 is a potent, highly selective small molecule inhibitor of the human Nav1.3 and Nav1.1 voltage gated sodium channels with IC50 of 19 nM. It exhibit up to 1,000-fold selectivity for human Nav1.3/Nav1.1 (ICA-121431, IC50, 19 nM) vs. other TTX-sensitive or resistant (i.e., Nav1.5) sodium channels

Cell Assay: Coverslips containing HEK-293 cells expressing unmodified and mutated Navchannels were placed in a recording chamber on the stage of an inverted microscope and perfused (~1 mL/min) with an extracellular solution containing 138 mM NaCl, 2 mM CaCl2, 5.4 mM KCI, 1 mM MgCl2, 10 mM glucose, and 10 mM Hepes (pH 7.4), with NaOH. Recording patch pipettes were filled with an intracellular solution containing 135 mM CsF, 5 mM NaCl, 2 mM MgCl2, 10 mM EGTA, and 10 mM Hepes (pH 7.3) with NaOH and had a resistance of 1–2 MΩ. All recordings were made at room temperature (22–24 °C) using AXOPATCH 200B amplifiers and PCLAMP software (Molecular Devices). Nav currents were measured using the whole-cell configuration of the patch clamp technique. All compounds were dissolved in DMSO to make 10-mM stock solutions, which were then diluted into extracellular solution to attain the final concentrations desired. The final concentration of DMSO (<0.3%) was found to have no significant effect on sodium currents. Test compound effects were evaluated using a protocol in which cells depolarized from a holding potential of –120 mV (or –140 mV for Nav1.5) to a membrane potential that inactivated half of the available channels for 8 s (determined empirically for each channel type) followed after a 20-ms recovery at –120 mV by a test pulse to 0 mV to assess magnitude of inhibition. Concentration response data were analyzed using nonlinear least-squares fit of the Logistic Equation (GraphPad Prism 5) to provide IC50 determinations.