Article Figures & Data
Figures
- Fig. 1.
Vixotrigine is a voltage-dependent Nav blocker. (A) Voltage protocols to investigate voltage-dependent inhibition of Nav1.x subtypes. Holding potentials were –90 and –60 mV (or –90 and –70 mV for Nav1.5). A conditioning prepulse to –120 mV was applied 20 milliseconds prior to test pulses to 0 or 10 mV. Peak currents were measured during the 20-millisecond test pulse. (B) Vixotrigine inhibition of Nav1.7 is dependent on holding potential. The concentration-response relationship shifts left at depolarized potentials (–60 mV) relative to –90 mV. Values are means ± S.D. for all wells recorded at each concentration (n = 4–6 wells/concentration). (C) Plot of voltage-dependent IC50 shifts for Nav1.1–1.8. Orange points are IC50 values for holding potentials of –90 mV. Arrowheads indicate IC50 values for holding potentials of –60 or –70 mV. A decrease in IC50 concentration was seen for Nav1.1–1.7.
- Fig. 2.
Vixotrigine is a use-dependent Nav blocker. (A) Voltage protocols to demonstrate use-dependent inhibition of Nav1.x. From a holding potential of –60 mV (–70 mV for Nav1.5), a series of 25 depolarizing steps at 10 Hz evoked sodium currents. The peak amplitude of the 25th pulse was used to calculate use-dependent block. (B) Vixotrigine inhibition of Nav1.7 is use-dependent. The concentration-response curve for vixotrigine shifts left for the 25th pulse (blue) relative to the first pulse (gray). Values are means ± S.D. for all wells recorded at each concentration (n = 4–6 wells/concentration). (C) Plot of use-dependent IC50 shifts for Nav1.1–1.8, with the same format as Fig. 1B. Vixotrigine use-dependent IC50 values decreased for all Nav subtypes.
- Fig. 3.
Nav selectivity profiles for vixotrigine, carbamazepine, PF-05089771, and A-803467. (A) Five-point concentration-response curves for tonic block at –60 mV (–70 mV Nav1.5), top panels; use-dependent block (25th pulse at 10 Hz from –60 mV), lower panels. All compounds were compared over the same concentration range for human Nav1.1–1.8. Points are means ± S.D. for all wells recorded at each concentration (n = 4–6 wells/concentration for each drug). (B) Use-dependent Nav1.1–1.8 IC50 values for each compound that is presented in (A). IC50 values (<50% block) beyond the concentration range are plotted at >100 on the y-axis. Vixotrigine Nav1.1–1.8 IC50 values are within one log unit, whereas PF-05086771 shows both sub-micromolar and out-of-range IC50 values. (C) Use-dependent Nav1.1–1.8 percent peak current blocked at 10 µM for vixotrigine, PF-05089771, A-803467, and at 100 µM for carbamazepine. Yellow line denotes ±5% peak current block.
- Fig. 4.
Onset of slow-inactivation states and block by vixotrigine, lacosamide, lamotrigine, and carbamazepine. (A) Voltage protocol to examine slow inactivation. Variable lengths of 0-mV depolarizing pulses followed by a 5-millisecond recovery pulse to relieve fast inactivation. (B) Plot of conditioning pulse duration vs. channel availability relative to 2.5-millisecond conditioning pulses. Each Nav subtype shows similar time-dependent onset of slow inactivation, with a large loss of channel availability after 1-second conditioning pulses. (C) Channel availability vs. conditioning pulse duration for Nav1.1, 1.2, 1.6, and 1.7 in the presence of Nav blockers. Black curves show channel availability in vehicle. Vixotrigine, carbamazepine, and lamotrigine block occurs before significant slow inactivation. Lacosamide block develops after depolarizations >320 milliseconds. (D) Data from (C) plotted as difference relative to vehicle channel availability at each conditioning pulse duration. Curve minima are the times with the largest differences relative to vehicle. Points are means ± S.D. for all wells tested at each concentration (n = 4–10 wells/condition).
- Fig. 5.
Recovery from slow inactivation. (A) Voltage protocol to examine recovery from slow inactivation. Slow inactivation was induced with 1-second, 0-mV conditioning pulses followed by variable hyperpolarizing recovery times. (B) Comparison of slow-inactivation recovery kinetics for Nav subtypes. Nav availability recovers to >90% after 1-second recovery pulses. (C) Pharmacological modulation of slow-inactivation recovery. Channel availability plotted as a function of recovery pulse duration for Nav1.1, 1.2, 1.6, and 1.7. Vixotrigine prolongs recovery from slow inactivation similarly to lacosamide, whereas carbamazepine block recovers to baseline within ∼100 milliseconds. Points are means ± S.D. for all wells tested at each concentration (n = 4–11 wells/condition).
- Fig. 6.
Recovery from fast inactivation. (A) Voltage protocol to examine recovery from fast inactivation. Fast inactivation was induced with 10-millisecond, 0-mV conditioning pulses followed by variable hyperpolarizing recovery times. (B) Comparison of fast-inactivation recovery kinetics for Nav subtypes. Nav availability recovers to >90% after 10- to 40-millisecond recovery pulses. (C) Pharmacological modulation of slow-inactivation recovery. Channel availability plotted as a function of recovery pulse duration for Nav1.1, 1.2, 1.6, and 1.7. Vixotrigine prolongs recovery from fast inactivation similarly to slow inactivation. Points are means ± S.D. for all wells tested at each concentration (n = 4–11 wells/condition).
- Fig. 7.
Vixotrigine effect on DRG neuron sodium currents. (A) Vixotrigine shifts steady-state sodium current inactivation curves left at 10 µM. Significant differences from control were found after test pulses between –95 and –35 mV (n = 8 neurons vehicle, n = 8 vixotrigine). (B) Carbamazepine shifts steady-state sodium inactivation curves left at 100 µM (n = 5). Significant differences from control were found between –85 and –55 mV. (C) Carbamazepine did not significantly shift DRG inactivation curves relative to control at 11 µM (n = 9). Asterisk denotes voltage range significanlty different from control p<0.05.
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Characterization of Vixotrigine
