Stiripentol inhibits spike-and-wave discharges in animal models of absence seizures: A new mechanism of action involving T-type calcium channels

Abstract

Objective: Stiripentol (STP; Diacomit®) is an antiepileptic drug indicated for Dravet syndrome that has been identified as a γ-aminobutyric acid (GABAergic) positive allosteric modulator. Dravet syndrome is characterized by multiple seizure types: generalized tonic-clonic, focal, myoclonic, and absence seizures. In addition to its antiepileptic effects on tonic-clonic seizures, STP has also been reported to reduce the frequency of atypical absence seizures in patients. Our study focused on STP potential effects on absence seizures, to better characterize its full spectrum of mechanisms of action.

Methods: STP effects on absence seizures were quantified by electroencephalographic recording in two animal models: rats treated with a low dose of pentylenetetrazol (20 mg/kg ip) and rats from the WAG/Rij strain. In addition, we characterized STP effects on T-type calcium channel activity. Peak currents were recorded with manual patch clamp on cells transfected with cDNA encoding for the human isoform for Ca_v 3.1, Ca_v 3.2, and Ca_v 3.3.

Results: STP administered before pentylenetetrazol almost completely abolished the generation of spike-and-wave discharges (SWDs) at the dose of 300 mg/kg. At this dose, STP also statistically significantly decreased SWD cumulated duration and number in WAG/Rij rats. Its antiepileptic effect was maintained in WAG/Rij rats, whose seizures were aggravated by the GABA agonist THIP (gaboxadol hydrochloride). Furthermore, electrophysiological recordings showed that STP inhibits T-type calcium channel peak activity, with a higher specificity for the Ca_v 3.3 subtype.

Significance: In addition to its previously characterized anticonvulsive properties, these data highlight a new mechanism of action of STP on abnormal thalamocortical activity. This strong antiabsence effect on seizures is correlated with an inhibition of T-type calcium channels. This new mechanism of action could be implicated in the specificity of STP therapeutic effects in Dravet syndrome.

Keywords: Dravet syndrome; absence seizures; epilepsy; spike-and-wave discharges; stiripentol; thalamocortical oscillations.

FIGURE 1

(A) Stiripentol (STP) decreased spike‐and‐wave discharge (SWD) duration at the dose of 150 mg/kg and suppressed SWD occurrence at 300 mg/kg. Representative electroencephalographic traces depict cortical activity recordings 10–15 min after administration of (a) STP (300 mg/kg ip), (b) pentylenetetrazol (PTZ; 20 mg/kg sc), (c) STP (150 mg/kg ip) and PTZ (20 mg/kg sc), and (d) STP (300 mg/kg ip) and PTZ (20 mg/kg sc). Scale bar = 1 s; amplitude = .2 mV. (B) STP suppressed SWDs induced after PTZ administration. Quantification of SWDs in 20‐min periods after PTZ (20 mg/kg ip) and vehicle (veh) or STP (150, 300 mg/kg) is shown. Data are represented as the mean and SEM; n = 9, 7, 10/group; *p < .05 compared to vehicle, two‐way analysis of variance for repeated measures (factor treatment and factor period), followed by a Holm–Sidak multiple comparison test for parametric data. (C) Quantification of SWDs in a 40‐min period after PTZ and STP administration. Mean values are represented as stars; n = 9, 7, 10/group; *p < .05 compared to vehicle, Kruskal–Wallis nonparametric comparison followed by a post hoc Dunn test. LV, liquid vehicle

FIGURE 2

(A) Stiripentol (STP) decreased spike‐and‐wave discharge (SWD) duration at the dose of 150 mg/kg and suppressed SWD occurrence at 300 mg/kg in the WAG/Rij rat. Representative electroencephalographic traces depict cortical activity recordings 10–15 min after administration of (a) vehicle, (b) STP (150 mg/kg ip), and (c) STP (300 mg/kg ip). Scale bar = 1 s; amplitude .2 mV. (B) Quantification of SWDs in a 40‐min period after STP administration. Mean values are represented as stars; n = 7/group; *p < .05, **p < .01, ***p < .001, compared to vehicle, one‐way analysis of variance for repeated measures followed by a Holm–Sidak multiple comparison test for parametric data. LV, liquid vehicle

FIGURE 3

(A) Gaboxadol hydrochloride (THIP) increased spike‐and‐wave discharges (SWDs) in the WAG/Rij rat, and stiripentol (STP) retained its suppressive effect on SWDs at the dose of 300 mg/kg. Representative electroencephalographic traces depict cortical activity recordings 10–15 min after administration of (a) vehicle, (b and b’) THIP, or (c) THIP and STP. Scale bar = 1 s; amplitude = .2 mV. (B) Quantification of SWDs in a 40‐min period after THIP and STP administration. Mean values are represented as stars; n = 10/group; *p < .05, ***p < .001 compared to vehicle, paired t test. LV, liquid vehicle

FIGURE 4

Stiripentol (STP) inhibits voltage‐dependent calcium channels. (A) Representative current traces from Chinese hamster ovary cells expressing Ca_v3.1 and Ca_v3.3 or human embryonic kidney cells expressing Ca_v3.2. Green = 5 µmol·L^–1; red = 50 µmol·L^–1. (B) Concentration–effect curve for the inhibition of maximal current induced by STP at steady state. Each point represents the mean ± SEM (n = 3 cells per concentration)