Binding characteristics of [3H]ucb 30889 to levetiracetam binding sites in rat brain
Introduction
Levetiracetam (2S-(2-oxo-1-pyrrolidinyl)butanamide, KEPPRA®) is a novel antiepileptic drug efficacious as adjunctive therapy in the treatment of partial-onset epileptic seizures in adults Hovinga, 2001, Nash and Sangha, 2001. Compared to other antiepileptics, levetiracetam has favorable pharmacokinetics (Patsalos, 2000), and does not interact with cytochromes P450 (Nicolas et al., 1999). It displays a unique pharmacological profile as it is devoid of anticonvulsant activity in the acute maximal electroshock and maximal pentylenetetrazol tests yet shows potent protection in electrically and pentylenetetrazol kindled animals Klitgaard, 2001, Klitgaard et al., 1998.
The biochemical target and the mechanism(s) of action of levetiracetam remain to be defined. There is little or no effects of levetiracetam on GABA (γ-aminobutyric acid), glutamate or glycine levels in brain nor on the activity of GABA transaminase or glutamic acid decarboxylase Sills et al., 1997, Tong and Patsalos, 2001. Levetiracetam does not modulate voltage-gated Na+ or T-type Ca2+ currents (Zona et al., 2001) but partially inhibits N-type Ca2+ current in hippocampal neurones (Niespodziany et al., 2001). Levetiracetam has also been reported to antagonise negative modulators (Zn2+ and β-carbolines) of the GABAA and glycine receptors, thereby increasing Cl− fluxes (Rigo et al., 2002). Using tritiated levetiracetam we identified, several years ago, a specific binding site in rat brain that was named levetiracetam binding site (Noyer et al., 1995). Levetiracetam binding sites are unique and do not correspond to any known receptor or channel that might be involved in neuroexcitability. Only a few drugs, such as chlordiazepoxide, pentylenetetrazol, bemegride and pentobarbital competed with levetiracetam for its binding sites. Although these drugs are known to interact with the GABAA receptor complex, GABA, benzodiazepines and carbolines did not modulate levetiracetam binding.
There is a good correlation between the affinity of levetiracetam analogues for levetiracetam binding sites and their potency in suppressing tonic seizures in audiogenic sensitive mice. However, the rather low affinity of labelled levetiracetam for levetiracetam binding sites (close to 1 μM) has thwarted all efforts to further characterise this binding site.
In this paper, we describe the binding characteristics of [3H]ucb 30889 ((2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide) (Fig. 1), a new radioligand with higher affinity for levetiracetam binding sites that circumvents the limitations encountered using tritiated levetiracetam. Furthermore, we demonstrate that this ligand can be used in autoradiography studies.
Section snippets
Drugs and radioligands
Levetiracetam (2S-(2-oxo-1-pyrrolidinyl)butanamide), piracetam (2-(2-oxo-1-pyrrolidinyl)acetamide), ucb 30889 ((2S)-2-[4-(3-azidophenyl)-2-oxopyrrolidin-1-yl]butanamide) and analogues were synthesised at UCB (Braine-l'Alleud, Belgium). Bemegride, bicuculline, carbamazepine, chlordiazepoxide, ethosuximide, felbamate, gabapentin, pentobarbital, phenytoin, picrotoxin, vigabatrin and zonisamide were purchased from Sigma-Aldrich (Bornem, Belgium). Pentylentetrazol was bought from Acros Organics
Binding kinetics
[3H]ucb 30889 (Fig. 1) binding is reversible with binding kinetics that are temperature dependent. In rat cerebral cortex, at 4 °C, kinetics are biphasic (Fig. 2) with a fast component having, respectively, half-times of association (at a radioligand concentration of 1 nM) and dissociation of 3±2 and 4±1 min and a slow component having half-times of association and dissociation of 47±13 and 61±15 min. At 25 °C, only one fast component with a half-time of 2±2 min remains (Fig. 2) and the total
Discussion
This article describes a new radioligand with higher affinity for levetiracetam binding sites than [3H]levetiracetam and its use to further characterise these binding sites. Previous results showed that the chiral carbon of levetiracetam (S-stereoisomer) is responsible for a marked 1000-fold stereoselectivity in binding affinity (ucb L060, the R-enantiomer virtually does not bind to the levetiracetam binding sites; Noyer et al., 1995). Based on these observations and confirmed by other pairs of
Acknowledgements
We thank Christy Van Der Perren and Michel Legrand for their technical expertise and Dr. Henrik Klitgaard for critically reading the manuscript.
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