Neuropharmacology and Analgesia
Binding characteristics of brivaracetam, a selective, high affinity SV2A ligand in rat, mouse and human brain: Relationship to anti-convulsant properties

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Abstract

Brivaracetam is a novel synaptic vesicle protein 2A (SV2A) ligand reported to be 10 fold more potent than levetiracetam in animal models of epilepsy. This study reports the binding profile of brivaracetam in the brain of several species in relation to its anticonvulsant properties.

The affinity, kinetics and selectivity of brivaracetam and its tritiated form [3H]ucb 34714 have been determined by in vitro binding experiments in rat, human and mouse brain and on recombinant human SV2A. Brivaracetam and levetiracetam ex vivo binding to SV2A and anticonvulsant activities in audiogenic mice were compared in relation to dose and time.

Brivaracetam bound selectively with 20 fold higher affinity than levetiracetam to SV2A. [3H]ucb 34714 bound reversibly and with high affinity to an homogenous population of binding sites in rat and human brain and to human SV2A expressed in CHO cells. The binding sites labeled by [3H]ucb 34714 in brain had the pharmacological characteristics of SV2A and no specific binding could be detected in the brain of SV2A−/− knock-out mice. The time- and dose-dependency of brivaracetam and levetiracetam for binding to brain SV2A and for providing seizure protection in audiogenic mice correlated well; brivaracetam being more potent and faster than levetiracetam.

Brivaracetam is a potent and selective SV2A ligand. From its affinity and pharmacokinetics, simulations predicted that at therapeutically relevant doses, brivaracetam should occupy more than 80% of SV2A in human brain, in line with levels of occupancy observed in pre-clinical models of epilepsy.

Introduction

Close to 50 million patients worldwide are suffering from epilepsy, a disease characterized by recurrent spontaneous seizures. About two third of them will be relieved by current therapies. However, despite the introduction over the past fifteen years of several new antiepileptic drugs, one third of patients will show resistance to treatment. While, in general, these newer antiepileptic drugs have improved properties with respect to pharmacokinetic and safety profiles they still fail to provide complete relief of the symptoms, thereby leaving an important medical need unmet (French and Pedley, 2008, Meldrum and Rogawski, 2007).

Levetiracetam (Keppra®), is a novel antiepileptic drug believed to primarily exert its activities through a unique mechanism of action involving binding to SV2A, a protein present in synaptic vesicles (for review see Klitgaard and Verdru, 2007).

Following the discovery of SV2A as the target of levetiracetam (Lynch et al., 2004), a target-based rational drug discovery program was initiated with the purpose of identifying more potent SV2A ligands that provided more complete seizure suppression than levetiracetam. This program led to the identification of brivaracetam (ucb 34714) as a compound displaying at least 10-fold higher affinity than levetiracetam for SV2A (Kenda et al., 2004). Brivaracetam has been extensively evaluated and compared to levetiracetam in a wide variety of in vitro and in vivo preclinical models of seizures and epilepsy. This assessment has demonstrated that brivaracetam exerts a more potent seizure protection than levetiracetam and, in several models, a more complete seizure suppression (Matagne et al., 2008).

Brivaracetam, is currently in phase III clinical studies following positive results in phase IIa trials that showed high efficacy in patients suffering from photosensitive epilepsy (Kasteleijn-Nolst Trenite et al., 2007) and in phase IIb trials that showed efficacy and high tolerability as an adjunctive treatment in adult patients with refractory partial-onset epilepsy (Malawska and Kulig, 2008, van Paesschen and Brodsky, 2007).

Since brivaracetam is differentiated from levetiracetam not only in potency but also in efficacy in several preclinical models of epilepsy, it has been postulated that either additional mechanisms of action for brivaracetam might exist besides binding to SV2A, or alternatively that the binding of brivaracetam to SV2A might lead to a qualitatively different functional response (Matagne et al., 2008). In that context, brivaracetam but not levetiracetam has been shown to inhibit voltage-dependent Na+ channels in rat cortical neurons albeit at high concentrations (Zona et al., 2010).

To further explore the possibility that there may be additional targets for brivaracetam, we extensively investigated the binding properties of radiolabeled and unlabeled brivaracetam in human, mouse and rat brain and to human recombinant SV2A protein expressed in CHO cells. We also performed ex vivo binding experiments in audiogenic mice in order to gain better insight into the pharmacodynamics of brivaracetam and the relationship existing between SV2A binding and protection against seizure. Finally, from the results obtained in this study and others, we extrapolated and simulated SV2A occupancy by brivaracetam in human epilepsy patients.

Section snippets

Chemicals

Levetiracetam (2S-(2-oxo-1-pyrrolidinyl)butanamide), brivaracetam (2S-2-[(4R)-2-oxo-4-propylpyrrolidin-1-yl]butanamide; ucb 34714) and analogs were synthesized at UCB (Braine-l'Alleud, Belgium). Bemegride, bicuculline and chlordiazepoxide were purchased from Sigma-Aldrich (Bornem, Belgium). Pentylentetrazol was purchased from Acros Organics (Pittsburgh, USA). [3H]ucb 34714 (8 Ci/mmol) and [3H]ucb 30889 (2S-2-[4-(3-azidophenyl)-2-oxo-1-pyrrolidinyl]butanamide) (32 Ci/mmol) were custom synthesized

Binding of brivaracetam to SV2A in rat and human cerebral cortex

Brivaracetam possesses two chiral centers (see Fig. 1). Initial studies performed in rat brain using [3H]ucb 30889 as a radioligand showed that brivaracetam had respectively 5 and 3200 fold higher affinity for SV2A than ucb 34713 and ucb-100230-1, two of its diastereoisomers (Fig. 2 and Table 1). The affinity of brivaracetam for rat SV2A (80 nM) was about 15 fold higher than that of levetiracetam (1250 nM) and this was further confirmed in human cerebral cortex with affinity values for

Discussion

Using [3H]ucb 30889 to label SV2A in rat and human brain (Gillard et al., 2003, Gillard et al., 2006), we found that brivaracetam binds to SV2A in both species with high nanomolar affinity, representing a 15- to 30-fold improvement over levetiracetam. As previously observed for levetiracetam (Gillard et al., 2006), brivaracetam displayed identical affinity for rat and human SV2A.

Brivaracetam contains two chiral centers (see Fig. 1). The chiral center linking the butanamide moiety to the

Disclosure statement

The authors, other than being UCB SA employees or former UCB SA employee, declare having no other conflict of interest.

Acknowledgments

The authors wish to thank Mrs. Christy Van Der Perren, Mrs. Bernadette Wantier and Mr. Philippe Motte for their skillful assistance in the binding studies and Mrs. Zita Povegliano for conducting the in vivo studies.

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    Current address: GlaxoSmithKline Biologicals, B-1330 Rixensart-Belgium.

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