Binding characteristics of [3H]ucb 30889 to levetiracetam binding sites in rat brain

Levetiracetam (2S-(2-oxo-1-pyrrolidinyl)butanamide, KEPPRA (R)), a novel antiepileptic drug, has been shown to bind to a specific binding site located in brain (levetiracetam binding site [Eur. J. Pharmacol. 286 (1995) 137]). However, [H-3]levetiracetam displayed only micromolar affinity for these sites making it an unsuitable probe for further characterization. The present study describes the binding properties of an analogue of levetiracetam: [H-3]ucb 30889, (2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide. [H-3]ucb 30889 binds reversibly to specific binding sites in rat brain. Kinetics at 4 degreesC were biphasic with half-times of association and dissociation of, respectively, 3 and 4 min for the fast component and 47 and 61 min for the slow component. [3 H]ucb 30889 saturation binding curves were compatible with the labelling of a homogenous population of binding sites having a B-max of 4496 +/- 790 fmol/mg protein (mean +/- S.D., n = 5) and a K-d of 62 +/- 20 nM (mean +/- S.D., n = 5), a 20-fold increase in affinity compared to [H-3]Ievetiracetam. Competition binding curves with ligands known to interact with levetiracetam binding sites and tissue distribution restricted to the brain indicated that [3 H]ucb 30889 and [3 H]levetiracetam bind to the same site. Although levetiracetam binding sites and GABA(A) (gamma-aminobutyric acid) receptors share some ligands such as pentobarbital and pentylenetetrazol, experiments performed with [S-35]TBPS (tert-butyl-bicyclo[2.2.2]phosporothionate), a probe for the GABA(A) Cl- channel do not support the hypothesis that levetiracetam binding sites are part of the GABA(A) receptor complex. Preliminary autoradiography studies in rat brain revealed that [3 H]ucb 30889 labels specific sites in all brain regions and that this binding is concentration-dependently displaced by levetiracetam. (C) 2003 Elsevier B.V. All rights reserved.