Abstracts

Rationale: Although a large number of anti-seizure medications (ASMs) are available, they have multiple side effects causing a decline in the quality of life of patients with epilepsy. In order to create an ASM that has a wider margin between therapeutic effect and adverse effect than that of existing ASMs, E2730 was discovered by in vivo phenotypic screening, then the mechanism of action was identified as an uncompetitive and selective inhibitor of γ-aminobutyric acid transporter 1 (GAT-1). Here, we describe the preclinical in vivo characteristics of E2730.

Methods: Anti-seizure effects of E2730 were evaluated in several animal model of epilepsy: corneal kindling, pharmacoresistant 6 Hz psychomotor seizure, amygdala kindling, fragile X syndrome (Fmr1 knockout mice), and Dravet syndrome (Scn1a^+/- mice) models. Effects of E2730 on motor coordination was assessed in accelerating rotarod tests. Effects of E2730 on basal level and high potassium-induced increase of extracellular γ-aminobutyric acid (GABA) concentration in mouse hippocampus were examined by microdialysis technique. In the microdialysis assay, effects of tiagabine, a noncompetitive GAT-1 inhibitor, were also examined as a reference compound.

Results: In the corneal kindling model in mice, E2730 had a potent anti-seizure effect with a 50% effective dose (ED₅₀) of 7.9 mg/kg. In the pharmacoresistant 6 Hz psychomotor seizure model in mice, E2730 showed dose-dependent anti-seizure effect with an ED₅₀ value of 17 mg/kg. In the amygdala kindling model in rats, E2730 showed dose-dependent anti-seizure effect from 10 to 50 mg/kg. In fragile X syndrome model mice, E2730 reduced the incidence of wild-running, tonic-clonic seizure, and respiratory arrest with ED₅₀ values of 19.1, 17.1, and 16.8 mg/kg, respectively. In Dravet syndrome model mice, E2730 significantly elevated the temperature of myoclonic jerk initiation (10 and 20 mg/kg) and generalized tonic-clonic seizure initiation (20 mg/kg) compared with the vehicle-treated group. In an accelerating rotarod test, E2730 did not influence motor coordination of mice at doses up to 200 mg/kg. The 50% toxic dose (TD₅₀) was 350 mg/kg. In the microdialysis assay in mouse hippocampus, E2730 at 10, 30, and 100 mg/kg did not increase basal extracellular GABA concentration, however significantly enhanced high potassium-induced increase of GABA concentration at 30 and 100 mg/kg. On the other hand, tiagabine at 3, 10, and 30 mg/kg significantly increased basal GABA concentration and also significantly enhanced high potassium-induced increase of GABA concentration at 10 and 30 mg/kg.

Conclusions: E2730 had broad efficacy in a variety of animal models of epilepsy with wide margin against effect on motor coordination. The unique characteristics of uncompetitive mode of GAT-1 inhibition by E2730 was demonstrated in microdialysis assay. E2730 increased extracellular GABA concentration in mouse hippocampus selectively in activated condition induced by high potassium, whereas noncompetitive GAT-1 inhibitor tiagabine increased GABA concentration both in physiological basal and activated conditions.

Funding: Please list any funding that was received in support of this abstract.: Eisai Co., Ltd.