Abstracts

Rationale: Brivaracetam (BRV, ucb 34714), is an investigational antiepileptic drug which displays a ten fold higher affinity than levetiracetam (LEV; Keppra^®) for the synaptic vesicle protein 2A (SV2A). In agreement with the established relationship between SV2A affinity and antiepileptic activity, BRV is likewise more potent and provides a more complete seizure suppression than LEV in several animal models of epilepsy. Here, we report the binding characteristics of BRV in mouse, rat and human brain as well as on cloned SV2 proteins. Methods: [³H]BRV (8 Ci/mmol) was custom labeled by GE Healthcare. Its binding characteristics were determined in vitro in brain homogenates from rat and human (obtained from ABS) and on cloned human SV2 proteins as described for [³H]ucb 30889 in Gillard et al. (Eur. J. Pharmacol. 536, 102-108, 2006). Ex vivo binding of BRV was measured, post i.p. administration, in brain tissue from mice primed for susceptibility to audiogenic seizures and compared to seizure protection.Results: At 4°C, [³H]BRV binding to rat brain and cloned human SV2A was reversible with biphasic kinetics. Association half-times in rat brain were 6 and 63 min for the fast and slow components compared to 1 and 16 min for cloned SV2A. Corresponding dissociation half-times were 17/139 min and 1/20 min in rat brain and cloned SV2A respectively. However, saturation curves indicated that [³H]BRV labeled an homogeneous population of binding sites with high affinity in rat and human brain (K_D = 62 ± 8 nM and 55 ± 7 nM respectively) and in cells expressing human SV2A (K_D = 152 ± 40 nM). These values are in good agreement with the values measured previously for BRV by competition experiments (80-100 nM) and represent a 10 to 25 fold increased affinity compared to LEV. In human brain, [³H]BRV labeled the same number of sites as [³H]ucb 30889, a selective SV2A radioligand (Gillard et al., Eur. J. Pharmacol. 478, 1-9, 2003). These sites had the characteristics of SV2A as determined by the affinity of a selection of compounds known to bind to this protein. No specific binding of [³H]BRV was observed in cells expressing SV2B or SV2C proteins. Similarly, BRV at 10 µM did not bind to any of more than 40 other tested targets including GPCR, ion channels and enzymes. Ex vivo binding experiments performed in sound susceptible mice brain revealed that BRV occupied 50% of SV2A sites after an ip dose of 8 µmol/kg compared to a dose of 200 µmol/kg needed for LEV which is consistent with its higher affinity. Maximal SV2A occupancy by BRV occurred 5 min after ip administration. SV2A occupancy decayed with a terminal half-life close to 90 min. This binding kinetic closely paralleled that of clonic and tonic seizure protection afforded by BRV in sound susceptible mice at the same dose.Conclusions: BRV is a reversible and selective SV2A ligand that binds with high affinity in rodent and human brain. BRV rapidly penetrates into mice brain where it binds to SV2A with a 25 fold lower IC₅₀ than LEV.