Abstracts

Levetiracetam (KEPPRA[reg]; LEV), possesses a unique activity profile in animal models of seizure and epilepsy, that correlates a novel mechanism of action different from other antiepileptic drugs (AEDs). LEV binds to a site in the CNS that appears to be distinct from the binding sites of other AEDs and CNS active drugs. Previous reports suggested a correlation between the affinity of a series of LEV analogs for the brain binding site and their potencies in a mouse audiogenic seizure model of epilepsy. This led to an extensive search to identify the protein(s) that comprise this novel binding site. Recent studies revealed that LEV binding was enriched in synaptic vesicles and photoaffinity labelling of purified synaptic vesicles shows that it had a molecular weight of approximately 90 kDa. Using these characteristics, an integral membrane synaptic vesicle protein, SV2, that includes three homologous isoforms, SV2A, B and C, was targeted as a potential binding site candidate. A labelled LEV derivative, [³H]ucb-30889, was utilized to study binding to brain membranes and purified synaptic vesicle fractions from WT and SV2 KO mice, and to heterologously expressed SV2 isoforms. Binding of [³H]ucb-30889 to individual SV2 isoforms was studied using heterologously expressed proteins in COS-7 cells. Anti-seizure activities of LEV and analogues were assessed in sound-susceptible mice (20) by exposing the mice to acoustic stimuli of 90-db, 10 to 20-kHz for 30sec, 60 min following intraperitoneal pre-treatment. LEV and related compounds bind to cloned SV2A (but not significantly to SV2B or SV2C), expressed heterologously in a fibroblast cell line, suggesting that SV2A is the source of the brain binding site. Brain membranes and purified synaptic vesicles from SV2A knockout mice do not bind a tritiated LEV derivative, further supporting that SV2A is responsible for LEV binding. The binding affinities of a series of LEV derivatives to SV2A expressed in fibroblasts correlates strongly to their binding affinities in brain tissue. Finally, there is also a strong correlation between the affinity of LEV derivatives for SV2A expressed in fibroblasts and their potency against seizures in the audiogenic mouse model of epilepsy. These results identify SV2A as the binding site of LEV in the brain and support the hypothesis that the antiepileptic activity of LEV may, in part, be acting through an interaction with the synaptic vesicle protein SV2A. The identification of SV2A as the LEV brain binding site supports previous claims that LEV possesses a mechanism of action that is truly distinct from that of other antiepileptic drugs. (Supported by UCB Pharma)