Abstracts

Rationale: Benzodiazepines have been shown to potentiate the anticonvulsant activity of levetiracetam (LEV) in animal models. A medicinal chemistry program was therefore initiated to rationally design compounds having high affinity for the synaptic vesicle 2A (SV2A) protein and low-to-moderate affinity for the benzodiazepine binding site (BZD) on the GABAA receptor complex. Consequently, padsevonil (PSL; UCB0942) was selected as a new antiepileptic drug candidate. Here we describe the in vitro pharmacological interaction of PSL with SV2 proteins and the BZD site. Methods: [3H]PSL (2.55 Ci mmol-1) was custom-labeled (Aptuit, Kansas, USA) and [3H]Flunitrazepam (Flu) was purchased from GE Healthcare (Gent, Belgium). Human brain tissue was obtained from ABS Inc (Wilmington, USA). Recombinant human SV2A, SV2B, SV2C proteins were expressed in HEK 293 cells and rat GABAA subunits (α1,β2,γ2; α2,β2,γ2; α5,β2,γ2) in COS-7 cells. Membranes were prepared and binding assays performed according to Gillard et al. (Eur J Pharmacol 2011;664:36–44). For in vivo studies, mice received vehicle or PSL (i.p.; n=6) and central SV2A occupancy was determined using [3H]UCB 30889 and BZD occupancy using [3H]Flu (Li et al., Neuropharmacol 2006;51:168–72). Results: In membranes expressing recombinant SV2 A, B, and C proteins, PSL displayed high affinity for all three subtypes (Table 1). In contrast, LEV and BRV displayed low affinity for the SV2B and SV2C proteins labeled by [3H]PSL. In membranes expressing recombinant GABAA receptor subtypes, PSL showed moderate affinity for the α1,β2,γ2 (Table 1) and α5,β2,γ2 subtypes (pKi 6.4 and 6.4, respectively, at 4°C) and low affinity for the α2,β2,γ2 subtype ( 3H]PSL displayed monophasic association/dissociation kinetics at 37°C with dissociation half-lives of 5-30 min for the SV2 isoforms; those of [3H]BRV and [3H]LEV to the SV2A protein are too fast to determine (Gillard et al., 2011). The Bmax for [3H]PSL was 9.3±1.6 pmol/mg in human cortex (Table 2) compared to 4 for [3H]BRV at 37°C (Gillard et al., 2011), consistent with labeling of additional sites such as SV2B and SV2C. In in vivo occupancy studies, marked brain occupancy of SV2A was seen at low doses of PSL (ED50 0.4 µmol/kg, 0.2 mg/kg), while brain BZD occupancy was only seen at higher doses (ED50 180 µmol/kg, 60 mg/kg). Conclusions: PSL is a first-in-class dual SV2 and BZD ligand with high and moderate binding affinities, respectively. This profile was maintained in in vivo occupancy studies. Additionally, PSL interacts with the SV2 proteins differently from BRV and LEV: firstly it binds to SV2B and SV2C with similar high affinity as SV2A; secondly it has a higher Bmax than BRV or LEV; thirdly it has slower kinetics. How these unique properties contribute to the preclinical and clinical profile of PSL remains to be determined. Funding: UCB Pharma-sponsored.