Characterization of [3H]ucb 30889 binding to synaptic vesicle protein 2A in the rat spinal cord

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Abstract

The novel antiepileptic drug levetiracetam ((2S-(2-oxo-1-pyrrolidinyl)butanamide, KEPPRA®) possesses a specific binding site in brain, which has very recently been identified as the synaptic vesicle protein SV2A. The aim of this study was to evaluate the presence of a levetiracetam binding site in the spinal cord and compare its properties to that in rat brain. We used [3H]ucb 30889 ((2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide), a levetiracetam analogue, to perform binding assays, photoaffinity labelling and autoradiography experiments, and revealed the presence of SV2A by Western-blot analysis. [3H]ucb 30889 binding kinetics at 4 °C were biphasic and saturation binding curves were compatible with the labelling of a homogenous population of binding sites with a Kd similar to that in brain. Competition curves with ligands known to interact with levetiracetam binding sites and photolabelling experiments indicated that [3H]ucb 30889 labels the same 90 kDa protein in both spinal cord and brain. Levetiracetam binding site was localised in the grey matter of the spinal cord and its expression was not modified in a model of neuropathic pain. This study demonstrates the presence of a specific levetiracetam binding site in the rat spinal cord, which is similar to that found in rat brain.

Introduction

Levetiracetam (2S-(2-oxo-1-pyrrolidinyl)butanamide, KEPPRA®) is a novel antiepileptic drug used as adjunctive therapy for the treatment of refractory partial epilepsy in adults (Hovinga, 2001, Nash and Sangha, 2001). Levetiracetam has a broad spectrum of activity in suppressing seizures as add-on treatment and monotherapy, and is safe and well-tolerated. Levetiracetam also has a favourable pharmacokinetic profile characterised by rapid and nearly complete absorption, very low potential for drug interactions and a prolonged pharmacodynamic effect that permits twice-daily dosing (for review, see Ben-Menachem, 2003). However, its mechanism of action is not fully elucidated. Previous studies revealed that levetiracetam binds saturably, reversibly and stereospecifically to an unidentified binding site in rat brain (Noyer et al., 1995, Gillard et al., 2003). Screening of a large number of known antiepileptic drugs and other neuroactive compounds failed to identify any with affinity for the levetiracetam binding site (Gillard et al., 2003), providing support for the novelty of this site. After identifying the site as a 90 kDa membrane protein enriched in synaptic vesicles (Fuks et al., 2003), the molecular nature of levetiracetam's binding site was recently discovered (Lynch et al., 2004). Levetiracetam and its related analogue ucb 30889 ((2S-(2-[4-(3-azidophenyl)-2oxopyrrolidin-1-yl]butanamide) bind specifically to the synaptic vesicle protein SV2A, which is totally different from the classical targets of established antiepileptic drugs (GABAA receptor, Na+ channels and low-voltage activated Ca2+ currents).

Three SV2 isoforms, called SV2A, SV2B, and SV2C, have been identified and show a unique distribution in brain (Bajjalieh et al., 1992, Bajjalieh et al., 1993, Feany et al., 1992, Janz and Sudhof, 1999). These proteins are twelve transmembrane glycoproteins localised in synaptic vesicles and implicated in the regulation of synaptic vesicle exocytosis (Crowder et al., 1999, Janz et al., 1999). SV2A is the most abundant, being ubiquitous in the central nervous system and expressed in endocrine cells (Buckley and Kelly, 1985, Bajjalieh et al., 1994). Its function remains unknown. However, SV2A knockout (KO) mice, as well as double SV2A/SV2B knockouts, exhibit a severe seizure phenotype, and SV2A seems to have a crucial role in the regulation of vesicle function, but not in vesicle biogenesis or synaptic morphology (Crowder et al., 1999, Janz et al., 1999).

It is postulated that neuropathic pain bears several similarities to epileptic seizures, including a therapeutic benefit from anticonvulsant drugs (Swerdlow, 1984). It was furthermore recently shown that levetiracetam induces an anti-hyperalgesic effect in chronic pain models (Ardid et al., 2003) and decreases neuropathic pain in patients (Price, 2004). It was therefore tempting to hypothesize that levetiracetam may act on a site expressed in the spinal cord. However, the presence of the levetiracetam binding site in the rat spinal cord has not been established. Only mRNA expression of SV2A (Bajjalieh et al., 1993, Bajjalieh et al., 1994) or immunostaining with a monoclonal antibody that does not distinguish between the three isoforms (Booj et al., 1989, Wang et al., 2000, Roosen et al., 2001, Brooke et al., 2004) have been described.

The synthesis of the new radioligand [3H]ucb 30889 with a 20-fold higher affinity for the levetiracetam binding site than tritiated levetiracetam permitted further analysis of levetiracetam binding sites in the brain (Gillard et al., 2003), and in the spinal cord. By using [3H]ucb 30889, we report here the existence of a levetiracetam binding site in the rat spinal cord. Binding and photoaffinity labelling experiments, autoradiography and Western-blot studies were performed to characterize it and compare its properties to the brain sites.

Section snippets

Drugs and radioligands

Levetiracetam (2S-(2-oxo-1-pyrrolidinyl)butanamide), ucb L060 (2R-(2-oxo-1-pyrrolidinyl)butanamide), ucb 30889 (2S-(2-[4-(3-azidophenyl)-2oxopyrrolidin-1-yl]butanamide) and ucb 34714 (2S-(2-[4R-(2-oxo-4-propylpyrrolidin-1-yl]butanamide)) were synthesized at UCB (Braine-l'Alleud, Belgium). [3H]ucb 30889 (32 Ci/mmol) was custom labelled by Amersham Biosciences (Roosendaal, The Netherlands). Bemegride, chlordiazepoxide, and pentobarbital were purchased from Sigma-Aldrich (Bornem, Belgium).

Binding kinetics and saturation binding isotherms

In the rat spinal cord, [3H]ucb 30889 binding is reversible (Fig. 1). Binding kinetics at 4 °C are biphasic with a fast component having, respectively, half-times of association (at a radioligand concentration of 1.12 nM) and dissociation of 2.8 ± 0.5 and 2.6 ± 0.6 min and a slow component of 38.5 ± 9.2 and 42.6 ± 14.4 min (n = 3). Binding kinetic constants are indicated in Table 1. Saturation binding curves of [3H]ucb 30889 were compatible with the labelling of a homogeneous population of binding sites (

Discussion

The results presented here demonstrate that the levetiracetam binding site is abundant in the rat spinal cord. This site which has never been described in the literature before exhibits similar characteristics to that present in the cerebral cortex. The affinity of various drugs and levetiracetam analogues was equivalent in both tissues showing that [3H]ucb 30889 was indeed labelling the same site. Radioligand affinity found in rat spinal cord (52 ± 14 nM) was comparable to that obtained in rat

Acknowledgement

We thank Michaëlla Grossman for her technical expertise.

References (34)

  • M. Noyer et al.

    The novel antiepileptic drug levetiracetam (ucb L059) appears to act via a specific binding site in CNS membranes

    Eur. J. Pharmacol.

    (1995)
  • R.A. Pyle et al.

    Phosphorylation of synaptic vesicle protein 2 modulates binding to synaptotagmin

    J. Biol. Chem.

    (2000)
  • A.E. Schivell et al.

    Isoform-specific, calcium-regulated interaction of the synaptic vesicle proteins SV2 and synaptotagmin

    J. Biol. Chem.

    (1996)
  • A.E. Schivell et al.

    SV2A and SV2C contain a unique synaptotagmin-binding site

    Mol. Cell. Neurosci.

    (2005)
  • S.M. Bajjalieh et al.

    SV2, a brain synaptic vesicle protein homologous to bacterial transporters

    Science

    (1992)
  • S.M. Bajjalieh et al.

    Brain contains two forms of synaptic vesicle protein 2

    Proc. Natl. Acad. Sci. U. S. A.

    (1993)
  • S.M. Bajjalieh et al.

    Differential expression of synaptic vesicle protein 2 (SV2) isoforms

    J. Neurosci.

    (1994)
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