Abstracts

RATIONALE: Levetiracetam (KEPPRA[reg]), a novel antiepileptic drug with a high safety margin, has been shown to bind to a specific binding site located preferentially in rat brain (levetiracetam binding site or LBS: Noyer et al., [italic]Eur. J. Pharmacol.[/italic] 1995, 286:137-146). However, [³H]levetiracetam displayed only micromolar affinity for these sites making it unsuitable for further characterization. The present study describes the binding properties of [³H]ucb 30889, (2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide, a new, more potent, analogue of levetiracetam.
METHODS: [³H]ucb 30889 binding experiments were conducted on crude rat brain membranes at 4[degree]C. Incubation time for equilibrium studies was 120 min. For kinetic and competition studies, [³H]ucb 30889 was used at a concentration of 1.3 nM in 0.5 ml of a Tris-HCl (pH 7.4) buffer containing 2 mM Mg²⁺. Localization of the LBS in brain substructures was assessed by autoradiography on 25 [mu]m thick slices incubated under similar conditions. Non-specific binding was determined by the inclusion of 1 mM levetiracetam in the assay.
RESULTS: [³H]ucb 30889 binds reversibly and with high affinity to binding sites in rat cerebral cortex. Binding kinetics were biphasic: half-times for association and dissociation were respectively, 3 [plusminus] 2 min and 4 [plusminus] 1 min for the fast component (25 to 50% of the sites), and 47 [plusminus] 13 min and 61 [plusminus] 15 min for the slow component. Saturation binding curves were compatible with a homogenous population of binding sites with a Bmax of 3713 [plusminus] 407 fmol/mg prot and a K[sub]d[/sub] of 30 [plusminus] 8 nM. pIC[sub]50[/sub] values for a variety of analogues and other compounds known to interact with the LBS, such as pentylenetetrazol or bemegride, were identical whether obtained with [³H]ucb 30889 or [³H]levetiracetam. Sites labeled by [³H]levetiracetam and [³H]ucb 30889 also have the same tissue distribution (CNS only). Preliminary autoradiography binding studies in rat brain revealed that [³H]ucb 30889 labels specific sites diffusely localized throughout the brain which can be dose-dependently inhibited by levetiracetam.
CONCLUSIONS: Competition binding curves and tissue distribution of specific binding indicates that [³H]ucb 30889 and [³H]levetiracetam bind to the same site (LBS) present in variable densities throughout the brain. Furthermore, [³H]ucb 30889 displays a 30 fold higher affinity for LBS than [³H]levetiracetam. Its biphasic kinetics could be related to site heterogeneity negative cooperativity or isomerization although the first hypothesis is not supported by equilibrium data.
[Supported by: UCB S.A. Pharma Sector]; (Disclosure: Salary - UCB S.A.)