In our recent studies, we identified compound N-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide (AS-1) as a broad-spectrum hybrid anticonvulsant which showed potent protection across the most important animal acute seizure models such as the maximal electroshock (MES) test, the subcutaneous pentylenetetrazole (s.c. PTZ) test, and the 6-Hz (32 mA) test in mice. Therefore, AS-1 may be recognized as a candidate for new anticonvulsant effective in different types of human epilepsy with a favorable safety margin profile determined in the rotarod test in mice. In the aim of further pharmacological evaluation of AS-1, in the current study, we examined its activity in the 6-Hz (44 mA) test, which is known as the model of drug-resistant epilepsy. Furthermore, we determined also the antiseizure activity in the kindling model of epilepsy induced by repeated injection of pentylenetetrazole (PTZ) in mice. As a result, AS-1 revealed relatively potent protection in the 6-Hz (44 mA) test, as well as delayed the progression of kindling induced by repeated injection of PTZ in mice at doses of 15 mg/kg, 30 mg/kg, and 60 mg/kg. Importantly, the isobolographic analysis showed that a combination of AS-1 and valproic acid (VPA) at the fixed ratio of 1:1 displayed a supra-additive (synergistic) interaction against PTZ-induced seizures in mice. Thus, AS-1 may be potentially used in an add-on therapy with VPA. Moreover, incubation of zebrafish larvae with AS-1 substantially decreased the number, cumulative but not the mean duration of epileptiform-like events in electroencephalographic assay. Finally, the in vitro ADME-Tox studies revealed that AS-1 is characterized by a very good permeability in the parallel artificial membrane permeability assay test, excellent metabolic stability on human liver microsomes (HLMs), no significant influence on CYP3A4/CYP2D6 activity, and moderate inhibition of CYP2C9 in a concentration of 10 μM, as well as no hepatotoxic properties in HepG2 cells (concentration of 10 μM).
Keywords: ADME-Tox properties; Drug-resistant epilepsy; PTZ-kindling model of epilepsy; electrophysiology; isobolographic studies; zebrafish.