Localization and photoaffinity labelling of the levetiracetam binding site in rat brain and certain cell lines

Fuks, B. B.; Gillard, Michel; Maury, Philippe; Lynch, Berkley A.; Vertongen, Pascale; Leprince, Pierre; Klitgaard, Henrik; Chatelain, Pierre

doi:10.1016/j.ejphar.2003.08.033

Cited by 57 publications

(45 citation statements)

References 22 publications

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“…However, it is well established that the drug binds in a reversible and stereoselective way to specific binding sites found in the brain and in neuronal cell lines, including rat pheochromocytoma PC12 cells (Fuks et al, 2003), and recently identified as the synaptic vesicle protein SV 2A (Lynch et al, 2004). The functional consequences of this binding remain, however, currently unclear.…”

mentioning

confidence: 98%

“…To confirm the specificity of LEV effect on IP 3 -dependent Ca 2ϩ release, we studied the effect of 1 M LEV on [Ca 2ϩ ] i response elicited by 1 M BK in NIH-3T3 cells since fibroblasts do express B 2 receptors (Faussner et al, 1991) but lack LEV binding sites (Fuks et al, 2003). In these cells, LEV did not induce any change either in basal [Ca 2ϩ ] i (131.1 Ϯ 9.9 versus 124.5 Ϯ 5.7 nM, respectively, in control and LEVtreated cells) or in the [Ca 2ϩ ] i response elicited by BK (1 M) (Fig.…”

Section: Lev Does Notmentioning

confidence: 99%

“…To address this hypothesis, we studied, by means of single-cell Fura-2 video imaging, the effect of LEV on the [Ca 2ϩ ] i response to BK and ATP, two neurotransmitters that induce an IP 3 -dependent increase in [Ca 2ϩ ] i (Fatatis et al, 1994) and participate in epileptogenesis. The study was performed in PC12 cells, a neuron-like cell line with a high expression of LEV-binding sites (Fuks et al, 2003).…”

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confidence: 99%

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The Antiepileptic Drug Levetiracetam Decreases the Inositol 1,4,5-Trisphosphate-Dependent [Ca²⁺]_iIncrease Induced by ATP and Bradykinin in PC12 Cells

Cataldi¹,

Lariccia

Secondo

et al. 2005

J Pharmacol Exp Ther

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The present study explores the hypothesis that the new antiepileptic drug levetiracetam (LEV) could interfere with the inositol 1,4,5-trisphosphate (IP 3 )-dependent release of intracellular Ca 2ϩ initiated by G q -coupled receptor activation, a process that plays a role in triggering and maintaining seizures. We assessed the effect of LEV on the amplitude of [Ca 2ϩ ] i response to bradykinin (BK) and ATP in single Fura-2/acetoxymethyl ester-loaded PC12 rat pheochromocytoma cells, which express very high levels of LEV binding sites. LEV dose-dependently reduced the [Ca 2ϩ ] i increase, elicited either by 1 M BK or by 100 M ATP (IC 50 , 0.39 Ϯ 0.01 M for BK and 0.20 Ϯ 0.01 M for ATP; Hill coefficients, 1.33 Ϯ 0.04 for BK and 1.38 Ϯ 0.06 for ATP). Interestingly, although the discharge of ryanodine stores by a process of calcium-induced calcium release also took place as part of the [Ca 2ϩ ] i response to BK, LEV inhibitory effect was mainly exerted on the IP 3 -dependent stores. In fact, the drug was still effective after the pharmacological blockade of ryanodine receptors. Furthermore, LEV did not affect Ca 2ϩ stored in the intracellular deposits since it did not reduce the amplitude of [Ca 2ϩ ] i response either to thapsigargin or to ionomycin. In conclusion, LEV inhibits Ca 2ϩ release from the IP 3 -sensitive stores without reducing Ca 2ϩ storage into these deposits. Because of the relevant implications of IP 3 -dependent Ca 2ϩ release in neuron excitability and epileptogenesis, this novel effect of LEV could provide a useful insight into the mechanisms underlying its antiepileptic properties.

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confidence: 98%

Section: Lev Does Notmentioning

confidence: 99%

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confidence: 99%

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The Antiepileptic Drug Levetiracetam Decreases the Inositol 1,4,5-Trisphosphate-Dependent [Ca²⁺]_iIncrease Induced by ATP and Bradykinin in PC12 Cells

Cataldi¹,

Lariccia

Secondo

et al. 2005

J Pharmacol Exp Ther

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“…Binding assays were performed as previously described (17)(18)(19). The selectivity of UCB-J was assessed by its ability to interact with various receptors, transporters, enzymes, and ion channels (.55 targets) when tested at a concentration of 10 mM.…”

Section: In Vitro Binding Assaysmentioning

confidence: 99%

Synthesis and Preclinical Evaluation of¹¹C-UCB-J as a PET Tracer for Imaging the Synaptic Vesicle Glycoprotein 2A in the Brain

et al. 2016

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The synaptic vesicle glycoprotein 2A (SV2A) is found in secretory vesicles in neurons and endocrine cells. PET with a selective SV2A radiotracer will allow characterization of drugs that modulate SV2A (e.g., antiepileptic drugs) and potentially could be a biomarker of synaptic density (e.g., in neurodegenerative disorders). Here we describe the synthesis and characterization of the SV2A PET radiotracer 11 C-UCB-J ((R)-1-((3-( 11 C-methyl-11 C)pyridin-4-yl)methyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-2-one) in nonhuman primates, including whole-body biodistribution. Methods: 11 C-UCB-J was prepared by C-11 C-methylation of the 3-pyridyl trifluoroborate precursor with 11 C-methyl iodide via the Suzuki-Miyaura cross-coupling method. Rhesus macaques underwent multiple scans including coinjection with unlabeled UCB-J (17, 50, and 150 μg/kg) or preblocking with the antiepileptic drug levetiracetam at 10 and 30 mg/kg. Scans were acquired for 2 h with arterial sampling and metabolite analysis to measure the input function. Regional volume of distribution (V T ) was estimated using the 1-tissue-compartment model. Target occupancy was assessed using the occupancy plot; the dissociation constant (K d ) was determined by fitting self-blocking occupancies to a 1-site model, and the maximum number of receptor binding sites (B max ) values were derived from baseline V T and from the estimated K d and the nondisplaceable distribution volume (V ND ). Results: 11 C-UCB-J was synthesized with greater than 98% purity. 11 C-UCB-J exhibited high free fraction (0.46 ± 0.02) and metabolized at a moderate rate (39% ± 5% and 24% ± 3% parent remaining at 30 and 90 min) in plasma. In the monkey brain, 11 C-UCB-J displayed high uptake and fast kinetics. V T was high (∼25-55 mL/cm 3 ) in all gray matter regions, consistent with the ubiquitous expression of SV2A. Preblocking with 10 and 30 mg/kg of levetiracetam resulted in approximately 60% and 90% occupancy, respectively. Analysis of the self-blocking scans yielded a K d estimate of 3.4 nM and B max of 125-350 nM, in good agreement with the in vitro inhibition constant (K i ) of 6.3 nM and regional B max in humans. Whole-body biodistribution revealed that the liver and the brain are the dose-limiting organs for males and females, respectively. Conclusion: 11 C-UCB-J exhibited excellent characteristics as an SV2A PET radiotracer in nonhuman primates. The radiotracer is currently undergoing first-in-human evaluation.

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“…Further characterization of the binding site led to its classification as an integral membrane protein of widespread distribution in brain, localized in neurons and enriched in the synaptic vesicle membrane fraction (11,12). SDS͞PAGE on the LEV-binding protein from brain membranes cross-linked to a tritiated photoaffinity LEV derivative determined that the protein has an apparent molecular mass of Ϸ90 kDa (12).…”

mentioning

confidence: 99%

The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam

Lynch¹,

Lambeng²,

Nocka³

et al. 2004

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

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Here, we show that the synaptic vesicle protein SV2A is the brain binding site of levetiracetam (LEV), a new antiepileptic drug with a unique activity profile in animal models of seizure and epilepsy. The LEV-binding site is enriched in synaptic vesicles, and photoaffinity labeling of purified synaptic vesicles confirms that it has an apparent molecular mass of Ϸ90 kDa. Brain membranes and purified synaptic vesicles from mice lacking SV2A do not bind a tritiated LEV derivative, indicating that SV2A is necessary for LEV binding. LEV and related compounds bind to SV2A expressed in fibroblasts, indicating that SV2A is sufficient for LEV binding. No binding was observed to the related isoforms SV2B and SV2C. Furthermore, there is a high degree of correlation between binding affinities of a series of LEV derivatives to SV2A in fibroblasts and to the LEV-binding site in brain. Finally, there is a strong correlation between the affinity of a compound for SV2A and its ability to protect against seizures in an audiogenic mouse animal model of epilepsy. These experimental results suggest that SV2A is the binding site of LEV in the brain and that LEV acts by modulating the function of SV2A, supporting previous indications that LEV possesses a mechanism of action distinct from that of other antiepileptic drugs. Further, these results indicate that proteins involved in vesicle exocytosis, and SV2 in particular, are promising targets for the development of new CNS drug therapies.

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Localization and photoaffinity labelling of the levetiracetam binding site in rat brain and certain cell lines

Cited by 57 publications

References 22 publications

The Antiepileptic Drug Levetiracetam Decreases the Inositol 1,4,5-Trisphosphate-Dependent [Ca²⁺]_iIncrease Induced by ATP and Bradykinin in PC12 Cells

The Antiepileptic Drug Levetiracetam Decreases the Inositol 1,4,5-Trisphosphate-Dependent [Ca²⁺]_iIncrease Induced by ATP and Bradykinin in PC12 Cells

Synthesis and Preclinical Evaluation of¹¹C-UCB-J as a PET Tracer for Imaging the Synaptic Vesicle Glycoprotein 2A in the Brain

The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam

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Localization and photoaffinity labelling of the levetiracetam binding site in rat brain and certain cell lines

Cited by 57 publications

References 22 publications

The Antiepileptic Drug Levetiracetam Decreases the Inositol 1,4,5-Trisphosphate-Dependent [Ca2+]iIncrease Induced by ATP and Bradykinin in PC12 Cells

The Antiepileptic Drug Levetiracetam Decreases the Inositol 1,4,5-Trisphosphate-Dependent [Ca2+]iIncrease Induced by ATP and Bradykinin in PC12 Cells

Synthesis and Preclinical Evaluation of11C-UCB-J as a PET Tracer for Imaging the Synaptic Vesicle Glycoprotein 2A in the Brain

The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam

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Product

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The Antiepileptic Drug Levetiracetam Decreases the Inositol 1,4,5-Trisphosphate-Dependent [Ca²⁺]_iIncrease Induced by ATP and Bradykinin in PC12 Cells

The Antiepileptic Drug Levetiracetam Decreases the Inositol 1,4,5-Trisphosphate-Dependent [Ca²⁺]_iIncrease Induced by ATP and Bradykinin in PC12 Cells

Synthesis and Preclinical Evaluation of¹¹C-UCB-J as a PET Tracer for Imaging the Synaptic Vesicle Glycoprotein 2A in the Brain