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

Abstract

Levetiracetam (2 S-(2-oxo-1-pyrrolidinyl)butanamide, KEPPRA®), 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, [ 3H]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: [ 3H]ucb 30889, (2 S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide. [ 3H]ucb 30889 binds reversibly to specific binding sites in rat brain. Kinetics at 4 °C 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. [ 3H]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 [ 3H]levetiracetam. Competition binding curves with ligands known to interact with levetiracetam binding sites and tissue distribution restricted to the brain indicated that [ 3H]ucb 30889 and [ 3H]levetiracetam bind to the same site. Although levetiracetam binding sites and GABA A (γ-aminobutyric acid) receptors share some ligands such as pentobarbital and pentylenetetrazol, experiments performed with [ 35S]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 [ 3H]ucb 30889 labels specific sites in all brain regions and that this binding is concentration-dependently displaced by levetiracetam.