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Michel Gillard - One of the best experts on this subject based on the ideXlab platform.
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pharmacological profile of the novel antiepileptic drug candidate padsevonil interactions with synaptic vesicle 2 proteins and the gabaa receptor
Journal of Pharmacology and Experimental Therapeutics, 2020Co-Authors: Martyn Wood, Laurent Provins, Veronique Daniels, Christian Wolff, Rafal M Kaminski, Michel GillardAbstract:Padsevonil is an antiepileptic drug (AED) candidate synthesized in a medicinal chemistry program initiated to rationally design compounds with high affinity for synaptic vesicle 2 (SV2) proteins and low-to-moderate affinity for the benzodiazepine binding site on GABAA receptors. The pharmacological profile of padsevonil was characterized in binding and electrophysiological experiments. At recombinant SV2 proteins, padsevonil9s affinity for SV2A was higher than that of levetiracetam and brivaracetam (pKi 8.5, 5.2 and 6.6, respectively). Unlike the latter AEDs, both selective SV2A ligands, padsevonil also displayed high affinity for the SV2B and SV2C isoforms (pKi 7.9 and 8.5, respectively). Padsevonil9s interaction with SV2A differed from that of levetiracetam and brivaracetam; it exhibited slower binding kinetics – dissociation t½ 30 min from the human protein at 37°C, compared with SIGNIFICANCE STATEMENT Padsevonil is an antiepileptic drug candidate developed as a single molecular entity interacting with both pre- and postsynaptic targets. Results of in vitro and in vivo radioligand binding assays confirmed this target profile – padsevonil displayed nanomolar affinity for the three synaptic vesicle 2 protein isoforms (SV2A, B and C) and micromolar affinity for the benzodiazepine binding site on GABAA receptors. Furthermore, padsevonil showed higher affinity for, and slower binding kinetics at SV2A than the selective SV2A ligands, levetiracetam and brivaracetam.
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evidence for a differential interaction of brivaracetam and levetiracetam with the synaptic vesicle 2a protein
Epilepsia, 2017Co-Authors: Martyn Wood, Michel GillardAbstract:SummaryObjective Brivaracetam (BRV) and levetiracetam (LEV) are effective antiepileptic drugs that bind selectively to the synaptic vesicle 2A (SV2A) protein. However, BRV differs from LEV in that it exhibits more potent and complete seizure suppression in animal models including in amygdala-kindled mice, where BRV afforded nearly complete seizure suppression. This raises the possibility that aside from potency differences, BRV and LEV may interact differently with the SV2A protein, which is not apparent in radioligand-binding competition studies. In this study, we used a recently identified SV2A allosteric modulator, UCB1244283, that appears to induce conformational changes in SV2A, to probe the binding properties of labeled BRV and LEV. Methods Radioligand binding studies were carried out using [3H]BRV and [3H]LEV. Studies were performed in membranes from both recombinant cells expressing human SV2A protein and human brain tissue. Results The modulator increased the binding of both radioligands but by different mechanisms. For [3H]BRV, the increase was driven mainly by an increase in affinity, whereas for [3H]LEV, the increase was due to an increase in the number of apparent binding sites. Kinetic studies confirmed this differential effect. Significance These studies suggest that LEV and BRV may act at different binding sites or interact with different conformational states of the SV2A protein. It is possible that some of the pharmacologic differences between BRV and LEV could be due to different interactions with the SV2A protein.
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evidence for a differential interaction of brivaracetam and levetiracetam with the synaptic vesicle 2a protein
Epilepsia, 2017Co-Authors: Martyn Wood, Michel GillardAbstract:Brivaracetam (BRV) and levetiracetam (LEV) are effective antiepileptic drugs that bind selectively to the synaptic vesicle 2A (SV2A) protein. BRV differs from LEV in preclinical studies in that it exhibits a more potent and complete seizure protection across animal models. We reported previously that an allosteric modulator of the SV2A protein had differential effects on BRV compared with LEV, suggesting that they act at different sites or with different conformations of the SV2A protein. If this is the case, then we hypothesized that mutations of specific amino acids in the SV2A protein may have differential effects on BRV and LEV binding by the modulator. Mutation of some amino acids identified previously in the binding site of racetams to the SV2A protein had marked effects on binding of both [3 H]BRV and [3 H]LEV (eg, W300F, F277A, G303A, F658A, Y462A, W666A, I663A, D670A, and V661A). However, 3 amino acids were identified (K694, I273, and S294) in which mutation lost the effect of the modulator on [3 H]LEV binding with no effect on the modulation of [3 H]BRV binding. These results confirm that BRV and LEV bind to the human synaptic vesicle 2A protein at closely related sites but interact with these sites in a different way.
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evidence for a differential interaction of brivaracetam and levetiracetam with the SV2A protein p5 246
Neurology, 2016Co-Authors: Martyn Wood, Michel GillardAbstract:OBJECTIVE: To investigate the effect of an allosteric modulator of the synaptic vesicle protein 2A (SV2A), 1 on the binding of [ 3 H]levetiracetam (LEV) and [ 3 H]brivaracetam (BRV) to the human SV2A protein. BACKGROUND: The SV2A protein represents the major molecular site of action for BRV and LEV, with BRV exhibiting high affinity and selectivity for this protein, and LEV exhibiting moderate affinity and interacting with other potential anticonvulsant mechanisms. 2 METHOD: Binding studies were performed in membranes (25 μg/well from HEK 293 cells expressing human SV2A protein, 1 175 µg/well from human brain cortex) incubated for 120 min at 4 o C in 0.2 mL buffer with radioligand. Data were analysed in GraphPad PRISM and are mean±SD (N=3). RESULTS: In HEK 293 cell membranes, both [ 3 H]LEV and [ 3 H]BRV bind to a single apparent site. The modulator produced a 2-3-fold increase in affinity, Kd (nM), for [ 3 H]LEV (2500±180 to 900±28) and a 2-fold increase in B max (pmoles/mgPr: from 67±3 to 140±3). On [ 3 H]BRV binding, the modulator produced a 10-fold increase in affinity, Kd (nM), from 190±3 to 20±2 and a 1.3-fold increase in B max from 130±5 to 170±3. These effects of the modulator on the binding of [ 3 H]BRV and [ 3 H]LEV were maintained, albeit to a lesser extent, in native tissue (human cortex). CONCLUSION: The differential effect of the SV2A allosteric modulator on the binding of [ 3 H]LEV and [ 3 H]BRV suggests that they induce or stabilize different conformations of the SV2A protein. Although the functional consequence of this still remains to be elucidated, the distinct interactions of LEV and BRV with the SV2A protein may provide the molecular correlate to the superior pharmacological properties of BRV in animal models of epilepsy. 1.Daniels V et al. Br J Pharmacol 2013;169:1091-1101. 2.Matagne A et al. Br J Pharmacol 2008;154:1662-1671. Study supported by UCB Pharma. Disclosure: Dr. Wood has nothing to disclose. Dr. Gillard holds stock and/or stock options in UCB Pharma.
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brivaracetam rationale for discovery and preclinical profile of a selective SV2A ligand for epilepsy treatment
Epilepsia, 2016Co-Authors: Henrik Klitgaard, Michel Gillard, Karine Leclercq, Alain Matagne, Rafal M Kaminski, Isabelle Niespodziany, Yves Lamberty, Marc Roger De Ryck, Jeanmarie Nicolas, Christian WolffAbstract:Despite availability of effective antiepileptic drugs (AEDs), many patients with epilepsy continue to experience refractory seizures and adverse events. Achievement of better seizure control and fewer side effects is key to improving quality of life. This review describes the rationale for the discovery and preclinical profile of brivaracetam (BRV), currently under regulatory review as adjunctive therapy for adults with partial-onset seizures. The discovery of BRV was triggered by the novel mechanism of action and atypical properties of levetiracetam (LEV) in preclinical seizure and epilepsy models. LEV is associated with several mechanisms that may contribute to its antiepileptic properties and adverse effect profile. Early findings observed a moderate affinity for a unique brain-specific LEV binding site (LBS) that correlated with anticonvulsant effects in animal models of epilepsy. This provided a promising molecular target and rationale for identifying selective, high-affinity ligands for LBS with potential for improved antiepileptic properties. The later discovery that synaptic vesicle protein 2A (SV2A) was the molecular correlate of LBS confirmed the novelty of the target. A drug discovery program resulted in the identification of anticonvulsants, comprising two distinct families of high-affinity SV2A ligands possessing different pharmacologic properties. Among these, BRV differed significantly from LEV by its selective, high affinity and differential interaction with SV2A as well as a higher lipophilicity, correlating with more potent and complete seizure suppression, as well as a more rapid brain penetration in preclinical models. Initial studies in animal models also revealed BRV had a greater antiepileptogenic potential than LEV. These properties of BRV highlight its promising potential as an AED that might provide broad-spectrum efficacy, associated with a promising tolerability profile and a fast onset of action. BRV represents the first selective SV2A ligand for epilepsy treatment and may add a significant contribution to the existing armamentarium of AEDs.
Henrik Klitgaard - One of the best experts on this subject based on the ideXlab platform.
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brivaracetam does not modulate ionotropic channels activated by glutamate γ aminobutyric acid and glycine in hippocampal neurons
Epilepsia, 2017Co-Authors: Isabelle Niespodziany, Alain Matagne, Henrik Klitgaard, Jeanmichel Rigo, Gustave Moonen, Christian WolffAbstract:Brivaracetam (BRV) is a selective, high-affinity ligand for synaptic vesicle protein 2A (SV2A), recently approved as adjunctive treatment for drug-refractory partial-onset seizures in adults. BRV binds SV2A with higher affinity than levetiracetam (LEV), and was shown to have a differential interaction with SV2A. Because LEV was reported to interact with multiple excitatory and inhibitory ligand-gated ion channels and that may impact its pharmacological profile, we were interested in determining whether BRV directly modulates inhibitory and excitatory ionotropic receptors in central neurons. Voltage-clamp experiments were performed in primary cultures of mouse hippocampal neurons. At a supratherapeutic concentration of 100 μm, BRV was devoid of any direct effect on currents gated by γ-aminobutyric acidergic type A, glycine, kainate, N-methyl-d-aspartate, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid. Similarly to LEV, BRV reveals a potent ability to oppose the action of negative modulators on the inhibitory receptors. In conclusion, these results show that BRV contrasts with LEV by not displaying any direct action on inhibitory or excitatory postsynaptic ligand-gated receptors at therapeutic concentrations and thereby support BRV's role as a selective SV2A ligand. These findings add further evidence to the validity of SV2A as a relevant antiepileptic drug target and emphasize the potential for exploring further presynaptic mechanisms as a novel approach to antiepileptic drug discovery.
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brivaracetam rationale for discovery and preclinical profile of a selective SV2A ligand for epilepsy treatment
Epilepsia, 2016Co-Authors: Henrik Klitgaard, Michel Gillard, Karine Leclercq, Alain Matagne, Rafal M Kaminski, Isabelle Niespodziany, Yves Lamberty, Marc Roger De Ryck, Jeanmarie Nicolas, Christian WolffAbstract:Despite availability of effective antiepileptic drugs (AEDs), many patients with epilepsy continue to experience refractory seizures and adverse events. Achievement of better seizure control and fewer side effects is key to improving quality of life. This review describes the rationale for the discovery and preclinical profile of brivaracetam (BRV), currently under regulatory review as adjunctive therapy for adults with partial-onset seizures. The discovery of BRV was triggered by the novel mechanism of action and atypical properties of levetiracetam (LEV) in preclinical seizure and epilepsy models. LEV is associated with several mechanisms that may contribute to its antiepileptic properties and adverse effect profile. Early findings observed a moderate affinity for a unique brain-specific LEV binding site (LBS) that correlated with anticonvulsant effects in animal models of epilepsy. This provided a promising molecular target and rationale for identifying selective, high-affinity ligands for LBS with potential for improved antiepileptic properties. The later discovery that synaptic vesicle protein 2A (SV2A) was the molecular correlate of LBS confirmed the novelty of the target. A drug discovery program resulted in the identification of anticonvulsants, comprising two distinct families of high-affinity SV2A ligands possessing different pharmacologic properties. Among these, BRV differed significantly from LEV by its selective, high affinity and differential interaction with SV2A as well as a higher lipophilicity, correlating with more potent and complete seizure suppression, as well as a more rapid brain penetration in preclinical models. Initial studies in animal models also revealed BRV had a greater antiepileptogenic potential than LEV. These properties of BRV highlight its promising potential as an AED that might provide broad-spectrum efficacy, associated with a promising tolerability profile and a fast onset of action. BRV represents the first selective SV2A ligand for epilepsy treatment and may add a significant contribution to the existing armamentarium of AEDs.
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targeting SV2A for discovery of antiepileptic drugs
Epilepsia, 2010Co-Authors: Rafal M Kaminski, Michel Gillard, Henrik KlitgaardAbstract:Levetiracetam's (Keppra) binding site and its subsequent identification as the synaptic vesicle protein 2A (SV2A) enabled the discovery of high affinity SV2A ligands with promising anticonvulsant properties. Among these, brivaracetam was selected for further development. SV2A represents an important antiepileptic drug target validated in both preclinical and clinical studies. For an expanded treatment of this topic see Jasper's basic mechanisms of the epilepsies. 4th ed. (Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado-Escueta AV, eds) published by Oxford University Press (available on the National Library of Medicine Bookshelf [NCBI] at www.ncbi.nlm.nih.gov/ books).
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proepileptic phenotype of SV2A deficient mice is associated with reduced anticonvulsant efficacy of levetiracetam
Epilepsia, 2009Co-Authors: Rafal M Kaminski, Michel Gillard, Karine Leclercq, Alain Matagne, Etienne Hanon, Genevieve Lorent, Donald Dassesse, Henrik KlitgaardAbstract:Summary Purpose: Synaptic vesicle protein 2A (SV2A) constitutes a distinct binding site for an antiepileptic drug levetiracetam (Keppra). In the present study we characterized SV2A (+/−) heterozygous mice in several seizure models and tested if the anticonvulsant efficacy of levetiracetam is reduced in these mice. Methods: Seizure thresholds of male SV2A (+/−) mice and their wild-type littermates were assessed in pilocarpine (i.p.), kainic acid (s.c.), pentylenetetrazol (i.v.), 6-Hz and maximal electroshock models. Kindling development was compared in amygdala and corneal kindling models. Ex vivo binding of levetiracetam to SV2A was also performed. Results: Long-term electroencephalography (EEG) monitoring and behavioral observations of SV2A (+/−) mice did not reveal any spontaneous seizure activity. However, a reduced seizure threshold of SV2A (+/−) mice was observed in pilocarpine, kainic acid, pentylenetetrazol, and 6-Hz models, but not in maximal electroshock seizure model. Accelerated epileptogenesis development was also demonstrated in amygdala and corneal kindling models. Anticonvulsant efficacy of levetiracetam, defined as its ability to increase seizure threshold for 6 Hz electrical stimulation, was significantly reduced (approx. 50%) in the SV2A (+/−) mice, consistently with reduced binding to SV2A in these mice. In contrast, valproate produced the same anticonvulsant effect in both SV2A (+/+) and SV2A (+/−) mice. Discussion: The present results evidence that SV2A is involved in mediation of the in vivo anticonvulsant activity of levetiracetam, in accordance with its previously proposed mechanism of action. Furthermore, the present data also indicate that even partial SV2A deficiency may lead to increased seizure vulnerability and accelerated epileptogenesis.
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SV2A protein is a broad spectrum anticonvulsant target functional correlation between protein binding and seizure protection in models of both partial and generalized epilepsy
Neuropharmacology, 2008Co-Authors: Rafal M Kaminski, Michel Gillard, Karine Leclercq, Alain Matagne, Benoit Kenda, Philippe Michel, Patrice Talaga, Henrik KlitgaardAbstract:Abstract SV2A, a synaptic vesicle protein, has been recently identified as a binding target for levetiracetam (Keppra®). The specific mechanism by which SV2A binding leads to seizure protection has not yet been fully elucidated. However, a functional correlation between SV2A binding affinity and anticonvulsant potency has been observed in the mouse audiogenic seizure model. The present study was undertaken to test whether similar correlations exist in rodent models of partial and generalized epilepsies. As expected, there was a high degree of correlation between anticonvulsant potency and SV2A binding affinity in the mouse audiogenic seizure model (r2 = 0.77; p
Rafal M Kaminski - One of the best experts on this subject based on the ideXlab platform.
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pharmacological profile of the novel antiepileptic drug candidate padsevonil interactions with synaptic vesicle 2 proteins and the gabaa receptor
Journal of Pharmacology and Experimental Therapeutics, 2020Co-Authors: Martyn Wood, Laurent Provins, Veronique Daniels, Christian Wolff, Rafal M Kaminski, Michel GillardAbstract:Padsevonil is an antiepileptic drug (AED) candidate synthesized in a medicinal chemistry program initiated to rationally design compounds with high affinity for synaptic vesicle 2 (SV2) proteins and low-to-moderate affinity for the benzodiazepine binding site on GABAA receptors. The pharmacological profile of padsevonil was characterized in binding and electrophysiological experiments. At recombinant SV2 proteins, padsevonil9s affinity for SV2A was higher than that of levetiracetam and brivaracetam (pKi 8.5, 5.2 and 6.6, respectively). Unlike the latter AEDs, both selective SV2A ligands, padsevonil also displayed high affinity for the SV2B and SV2C isoforms (pKi 7.9 and 8.5, respectively). Padsevonil9s interaction with SV2A differed from that of levetiracetam and brivaracetam; it exhibited slower binding kinetics – dissociation t½ 30 min from the human protein at 37°C, compared with SIGNIFICANCE STATEMENT Padsevonil is an antiepileptic drug candidate developed as a single molecular entity interacting with both pre- and postsynaptic targets. Results of in vitro and in vivo radioligand binding assays confirmed this target profile – padsevonil displayed nanomolar affinity for the three synaptic vesicle 2 protein isoforms (SV2A, B and C) and micromolar affinity for the benzodiazepine binding site on GABAA receptors. Furthermore, padsevonil showed higher affinity for, and slower binding kinetics at SV2A than the selective SV2A ligands, levetiracetam and brivaracetam.
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brivaracetam rationale for discovery and preclinical profile of a selective SV2A ligand for epilepsy treatment
Epilepsia, 2016Co-Authors: Henrik Klitgaard, Michel Gillard, Karine Leclercq, Alain Matagne, Rafal M Kaminski, Isabelle Niespodziany, Yves Lamberty, Marc Roger De Ryck, Jeanmarie Nicolas, Christian WolffAbstract:Despite availability of effective antiepileptic drugs (AEDs), many patients with epilepsy continue to experience refractory seizures and adverse events. Achievement of better seizure control and fewer side effects is key to improving quality of life. This review describes the rationale for the discovery and preclinical profile of brivaracetam (BRV), currently under regulatory review as adjunctive therapy for adults with partial-onset seizures. The discovery of BRV was triggered by the novel mechanism of action and atypical properties of levetiracetam (LEV) in preclinical seizure and epilepsy models. LEV is associated with several mechanisms that may contribute to its antiepileptic properties and adverse effect profile. Early findings observed a moderate affinity for a unique brain-specific LEV binding site (LBS) that correlated with anticonvulsant effects in animal models of epilepsy. This provided a promising molecular target and rationale for identifying selective, high-affinity ligands for LBS with potential for improved antiepileptic properties. The later discovery that synaptic vesicle protein 2A (SV2A) was the molecular correlate of LBS confirmed the novelty of the target. A drug discovery program resulted in the identification of anticonvulsants, comprising two distinct families of high-affinity SV2A ligands possessing different pharmacologic properties. Among these, BRV differed significantly from LEV by its selective, high affinity and differential interaction with SV2A as well as a higher lipophilicity, correlating with more potent and complete seizure suppression, as well as a more rapid brain penetration in preclinical models. Initial studies in animal models also revealed BRV had a greater antiepileptogenic potential than LEV. These properties of BRV highlight its promising potential as an AED that might provide broad-spectrum efficacy, associated with a promising tolerability profile and a fast onset of action. BRV represents the first selective SV2A ligand for epilepsy treatment and may add a significant contribution to the existing armamentarium of AEDs.
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modulation of the conformational state of the SV2A protein by an allosteric mechanism as evidenced by ligand binding assays
British Journal of Pharmacology, 2013Co-Authors: Veronique Daniels, Martyn Wood, Karine Leclercq, Rafal M Kaminski, Michel GillardAbstract:Background and Purpose Synaptic vesicle protein 2A (SV2A) is the specific binding site of the anti-epileptic drug levetiracetam (LEV) and its higher affinity analogue UCB30889. Moreover, the protein has been well validated as a target for anticonvulsant therapy. Here, we report the identification of UCB1244283 acting as a SV2A positive allosteric modulator of UCB30889. Experimental Approach UCB1244283 was characterized in vitro using radioligand binding assays with [3H]UCB30889 on recombinant SV2A expressed in HEK cells and on rat cortex. In vivo, the compound was tested in sound-sensitive mice. Key Results Saturation binding experiments in the presence of UCB1244283 demonstrated a fivefold increase in the affinity of [3H]UCB30889 for human recombinant SV2A, combined with a twofold increase of the total number of binding sites. Similar results were obtained on rat cortex. In competition binding experiments, UCB1244283 potentiated the affinity of UCB30889 while the affinity of LEV remained unchanged. UCB1244283 significantly slowed down both the association and dissociation kinetics of [3H]UCB30889. Following i.c.v. administration in sound-sensitive mice, UCB1244283 showed a clear protective effect against both tonic and clonic convulsions. Conclusions and Implications These results indicate that UCB1244283 can modulate the conformation of SV2A, thereby inducing a higher affinity state for UCB30889. Our results also suggest that the conformation of SV2A per se might be an important determinant of its functioning, especially during epileptic seizures. Therefore, agents that act on the conformation of SV2A might hold great potential in the search for new SV2A-based anticonvulsant therapies.
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targeting SV2A for discovery of antiepileptic drugs
Epilepsia, 2010Co-Authors: Rafal M Kaminski, Michel Gillard, Henrik KlitgaardAbstract:Levetiracetam's (Keppra) binding site and its subsequent identification as the synaptic vesicle protein 2A (SV2A) enabled the discovery of high affinity SV2A ligands with promising anticonvulsant properties. Among these, brivaracetam was selected for further development. SV2A represents an important antiepileptic drug target validated in both preclinical and clinical studies. For an expanded treatment of this topic see Jasper's basic mechanisms of the epilepsies. 4th ed. (Noebels JL, Avoli M, Rogawski MA, Olsen RW, Delgado-Escueta AV, eds) published by Oxford University Press (available on the National Library of Medicine Bookshelf [NCBI] at www.ncbi.nlm.nih.gov/ books).
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proepileptic phenotype of SV2A deficient mice is associated with reduced anticonvulsant efficacy of levetiracetam
Epilepsia, 2009Co-Authors: Rafal M Kaminski, Michel Gillard, Karine Leclercq, Alain Matagne, Etienne Hanon, Genevieve Lorent, Donald Dassesse, Henrik KlitgaardAbstract:Summary Purpose: Synaptic vesicle protein 2A (SV2A) constitutes a distinct binding site for an antiepileptic drug levetiracetam (Keppra). In the present study we characterized SV2A (+/−) heterozygous mice in several seizure models and tested if the anticonvulsant efficacy of levetiracetam is reduced in these mice. Methods: Seizure thresholds of male SV2A (+/−) mice and their wild-type littermates were assessed in pilocarpine (i.p.), kainic acid (s.c.), pentylenetetrazol (i.v.), 6-Hz and maximal electroshock models. Kindling development was compared in amygdala and corneal kindling models. Ex vivo binding of levetiracetam to SV2A was also performed. Results: Long-term electroencephalography (EEG) monitoring and behavioral observations of SV2A (+/−) mice did not reveal any spontaneous seizure activity. However, a reduced seizure threshold of SV2A (+/−) mice was observed in pilocarpine, kainic acid, pentylenetetrazol, and 6-Hz models, but not in maximal electroshock seizure model. Accelerated epileptogenesis development was also demonstrated in amygdala and corneal kindling models. Anticonvulsant efficacy of levetiracetam, defined as its ability to increase seizure threshold for 6 Hz electrical stimulation, was significantly reduced (approx. 50%) in the SV2A (+/−) mice, consistently with reduced binding to SV2A in these mice. In contrast, valproate produced the same anticonvulsant effect in both SV2A (+/+) and SV2A (+/−) mice. Discussion: The present results evidence that SV2A is involved in mediation of the in vivo anticonvulsant activity of levetiracetam, in accordance with its previously proposed mechanism of action. Furthermore, the present data also indicate that even partial SV2A deficiency may lead to increased seizure vulnerability and accelerated epileptogenesis.
Martyn Wood - One of the best experts on this subject based on the ideXlab platform.
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pharmacological profile of the novel antiepileptic drug candidate padsevonil interactions with synaptic vesicle 2 proteins and the gabaa receptor
Journal of Pharmacology and Experimental Therapeutics, 2020Co-Authors: Martyn Wood, Laurent Provins, Veronique Daniels, Christian Wolff, Rafal M Kaminski, Michel GillardAbstract:Padsevonil is an antiepileptic drug (AED) candidate synthesized in a medicinal chemistry program initiated to rationally design compounds with high affinity for synaptic vesicle 2 (SV2) proteins and low-to-moderate affinity for the benzodiazepine binding site on GABAA receptors. The pharmacological profile of padsevonil was characterized in binding and electrophysiological experiments. At recombinant SV2 proteins, padsevonil9s affinity for SV2A was higher than that of levetiracetam and brivaracetam (pKi 8.5, 5.2 and 6.6, respectively). Unlike the latter AEDs, both selective SV2A ligands, padsevonil also displayed high affinity for the SV2B and SV2C isoforms (pKi 7.9 and 8.5, respectively). Padsevonil9s interaction with SV2A differed from that of levetiracetam and brivaracetam; it exhibited slower binding kinetics – dissociation t½ 30 min from the human protein at 37°C, compared with SIGNIFICANCE STATEMENT Padsevonil is an antiepileptic drug candidate developed as a single molecular entity interacting with both pre- and postsynaptic targets. Results of in vitro and in vivo radioligand binding assays confirmed this target profile – padsevonil displayed nanomolar affinity for the three synaptic vesicle 2 protein isoforms (SV2A, B and C) and micromolar affinity for the benzodiazepine binding site on GABAA receptors. Furthermore, padsevonil showed higher affinity for, and slower binding kinetics at SV2A than the selective SV2A ligands, levetiracetam and brivaracetam.
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evidence for a differential interaction of brivaracetam and levetiracetam with the synaptic vesicle 2a protein
Epilepsia, 2017Co-Authors: Martyn Wood, Michel GillardAbstract:SummaryObjective Brivaracetam (BRV) and levetiracetam (LEV) are effective antiepileptic drugs that bind selectively to the synaptic vesicle 2A (SV2A) protein. However, BRV differs from LEV in that it exhibits more potent and complete seizure suppression in animal models including in amygdala-kindled mice, where BRV afforded nearly complete seizure suppression. This raises the possibility that aside from potency differences, BRV and LEV may interact differently with the SV2A protein, which is not apparent in radioligand-binding competition studies. In this study, we used a recently identified SV2A allosteric modulator, UCB1244283, that appears to induce conformational changes in SV2A, to probe the binding properties of labeled BRV and LEV. Methods Radioligand binding studies were carried out using [3H]BRV and [3H]LEV. Studies were performed in membranes from both recombinant cells expressing human SV2A protein and human brain tissue. Results The modulator increased the binding of both radioligands but by different mechanisms. For [3H]BRV, the increase was driven mainly by an increase in affinity, whereas for [3H]LEV, the increase was due to an increase in the number of apparent binding sites. Kinetic studies confirmed this differential effect. Significance These studies suggest that LEV and BRV may act at different binding sites or interact with different conformational states of the SV2A protein. It is possible that some of the pharmacologic differences between BRV and LEV could be due to different interactions with the SV2A protein.
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evidence for a differential interaction of brivaracetam and levetiracetam with the synaptic vesicle 2a protein
Epilepsia, 2017Co-Authors: Martyn Wood, Michel GillardAbstract:Brivaracetam (BRV) and levetiracetam (LEV) are effective antiepileptic drugs that bind selectively to the synaptic vesicle 2A (SV2A) protein. BRV differs from LEV in preclinical studies in that it exhibits a more potent and complete seizure protection across animal models. We reported previously that an allosteric modulator of the SV2A protein had differential effects on BRV compared with LEV, suggesting that they act at different sites or with different conformations of the SV2A protein. If this is the case, then we hypothesized that mutations of specific amino acids in the SV2A protein may have differential effects on BRV and LEV binding by the modulator. Mutation of some amino acids identified previously in the binding site of racetams to the SV2A protein had marked effects on binding of both [3 H]BRV and [3 H]LEV (eg, W300F, F277A, G303A, F658A, Y462A, W666A, I663A, D670A, and V661A). However, 3 amino acids were identified (K694, I273, and S294) in which mutation lost the effect of the modulator on [3 H]LEV binding with no effect on the modulation of [3 H]BRV binding. These results confirm that BRV and LEV bind to the human synaptic vesicle 2A protein at closely related sites but interact with these sites in a different way.
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synthesis and preclinical evaluation of 11c ucb j as a pet tracer for imaging the synaptic vesicle glycoprotein 2a in the brain
The Journal of Nuclear Medicine, 2016Co-Authors: Nabeel Nabulsi, Shu-fei Lin, Joel Mercier, Soheila Najafzadeh, Stephane Carre, Daniel Holden, Mariechristine Vandergeten, Anand K Deo, Nathalie Price, Martyn WoodAbstract: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 11C-UCB-J ((R)-1-((3-(11C-methyl-11C)pyridin-4-yl)methyl)-4-(3,4,5-trifluorophenyl)pyrrolidin-2-one) in nonhuman primates, including whole-body biodistribution. Methods:11C-UCB-J was prepared by C-11C-methylation of the 3-pyridyl trifluoroborate precursor with 11C-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 (VT) was estimated using the 1-tissue-compartment model. Target occupancy was assessed using the occupancy plot; the dissociation constant (Kd) was determined by fitting self-blocking occupancies to a 1-site model, and the maximum number of receptor binding sites (Bmax) values were derived from baseline VT and from the estimated Kd and the nondisplaceable distribution volume (VND). Results:11C-UCB-J was synthesized with greater than 98% purity. 11C-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, 11C-UCB-J displayed high uptake and fast kinetics. VT was high (∼25–55 mL/cm3) 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 Kd estimate of 3.4 nM and Bmax of 125–350 nM, in good agreement with the in vitro inhibition constant (Ki) of 6.3 nM and regional Bmax in humans. Whole-body biodistribution revealed that the liver and the brain are the dose-limiting organs for males and females, respectively. Conclusion:11C-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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evidence for a differential interaction of brivaracetam and levetiracetam with the SV2A protein p5 246
Neurology, 2016Co-Authors: Martyn Wood, Michel GillardAbstract:OBJECTIVE: To investigate the effect of an allosteric modulator of the synaptic vesicle protein 2A (SV2A), 1 on the binding of [ 3 H]levetiracetam (LEV) and [ 3 H]brivaracetam (BRV) to the human SV2A protein. BACKGROUND: The SV2A protein represents the major molecular site of action for BRV and LEV, with BRV exhibiting high affinity and selectivity for this protein, and LEV exhibiting moderate affinity and interacting with other potential anticonvulsant mechanisms. 2 METHOD: Binding studies were performed in membranes (25 μg/well from HEK 293 cells expressing human SV2A protein, 1 175 µg/well from human brain cortex) incubated for 120 min at 4 o C in 0.2 mL buffer with radioligand. Data were analysed in GraphPad PRISM and are mean±SD (N=3). RESULTS: In HEK 293 cell membranes, both [ 3 H]LEV and [ 3 H]BRV bind to a single apparent site. The modulator produced a 2-3-fold increase in affinity, Kd (nM), for [ 3 H]LEV (2500±180 to 900±28) and a 2-fold increase in B max (pmoles/mgPr: from 67±3 to 140±3). On [ 3 H]BRV binding, the modulator produced a 10-fold increase in affinity, Kd (nM), from 190±3 to 20±2 and a 1.3-fold increase in B max from 130±5 to 170±3. These effects of the modulator on the binding of [ 3 H]BRV and [ 3 H]LEV were maintained, albeit to a lesser extent, in native tissue (human cortex). CONCLUSION: The differential effect of the SV2A allosteric modulator on the binding of [ 3 H]LEV and [ 3 H]BRV suggests that they induce or stabilize different conformations of the SV2A protein. Although the functional consequence of this still remains to be elucidated, the distinct interactions of LEV and BRV with the SV2A protein may provide the molecular correlate to the superior pharmacological properties of BRV in animal models of epilepsy. 1.Daniels V et al. Br J Pharmacol 2013;169:1091-1101. 2.Matagne A et al. Br J Pharmacol 2008;154:1662-1671. Study supported by UCB Pharma. Disclosure: Dr. Wood has nothing to disclose. Dr. Gillard holds stock and/or stock options in UCB Pharma.
Alain Matagne - One of the best experts on this subject based on the ideXlab platform.
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brivaracetam does not modulate ionotropic channels activated by glutamate γ aminobutyric acid and glycine in hippocampal neurons
Epilepsia, 2017Co-Authors: Isabelle Niespodziany, Alain Matagne, Henrik Klitgaard, Jeanmichel Rigo, Gustave Moonen, Christian WolffAbstract:Brivaracetam (BRV) is a selective, high-affinity ligand for synaptic vesicle protein 2A (SV2A), recently approved as adjunctive treatment for drug-refractory partial-onset seizures in adults. BRV binds SV2A with higher affinity than levetiracetam (LEV), and was shown to have a differential interaction with SV2A. Because LEV was reported to interact with multiple excitatory and inhibitory ligand-gated ion channels and that may impact its pharmacological profile, we were interested in determining whether BRV directly modulates inhibitory and excitatory ionotropic receptors in central neurons. Voltage-clamp experiments were performed in primary cultures of mouse hippocampal neurons. At a supratherapeutic concentration of 100 μm, BRV was devoid of any direct effect on currents gated by γ-aminobutyric acidergic type A, glycine, kainate, N-methyl-d-aspartate, and α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid. Similarly to LEV, BRV reveals a potent ability to oppose the action of negative modulators on the inhibitory receptors. In conclusion, these results show that BRV contrasts with LEV by not displaying any direct action on inhibitory or excitatory postsynaptic ligand-gated receptors at therapeutic concentrations and thereby support BRV's role as a selective SV2A ligand. These findings add further evidence to the validity of SV2A as a relevant antiepileptic drug target and emphasize the potential for exploring further presynaptic mechanisms as a novel approach to antiepileptic drug discovery.
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brivaracetam rationale for discovery and preclinical profile of a selective SV2A ligand for epilepsy treatment
Epilepsia, 2016Co-Authors: Henrik Klitgaard, Michel Gillard, Karine Leclercq, Alain Matagne, Rafal M Kaminski, Isabelle Niespodziany, Yves Lamberty, Marc Roger De Ryck, Jeanmarie Nicolas, Christian WolffAbstract:Despite availability of effective antiepileptic drugs (AEDs), many patients with epilepsy continue to experience refractory seizures and adverse events. Achievement of better seizure control and fewer side effects is key to improving quality of life. This review describes the rationale for the discovery and preclinical profile of brivaracetam (BRV), currently under regulatory review as adjunctive therapy for adults with partial-onset seizures. The discovery of BRV was triggered by the novel mechanism of action and atypical properties of levetiracetam (LEV) in preclinical seizure and epilepsy models. LEV is associated with several mechanisms that may contribute to its antiepileptic properties and adverse effect profile. Early findings observed a moderate affinity for a unique brain-specific LEV binding site (LBS) that correlated with anticonvulsant effects in animal models of epilepsy. This provided a promising molecular target and rationale for identifying selective, high-affinity ligands for LBS with potential for improved antiepileptic properties. The later discovery that synaptic vesicle protein 2A (SV2A) was the molecular correlate of LBS confirmed the novelty of the target. A drug discovery program resulted in the identification of anticonvulsants, comprising two distinct families of high-affinity SV2A ligands possessing different pharmacologic properties. Among these, BRV differed significantly from LEV by its selective, high affinity and differential interaction with SV2A as well as a higher lipophilicity, correlating with more potent and complete seizure suppression, as well as a more rapid brain penetration in preclinical models. Initial studies in animal models also revealed BRV had a greater antiepileptogenic potential than LEV. These properties of BRV highlight its promising potential as an AED that might provide broad-spectrum efficacy, associated with a promising tolerability profile and a fast onset of action. BRV represents the first selective SV2A ligand for epilepsy treatment and may add a significant contribution to the existing armamentarium of AEDs.
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Binding characteristics of brivaracetam, a selective, high affinity SV2A ligand in rat, mouse and human brain: Relationship to anti-convulsant properties
European Journal of Pharmacology, 2011Co-Authors: Michel Gillard, Bruno Fuks, Karine Leclercq, Alain MatagneAbstract:Brivaracetam is a novel synaptic vesicle protein 2A (SV2A) ligand reported to be 10 fold more potent than levetiracetam in animal models of epilepsy. This study reports the binding profile of brivaracetam in the brain of several species in relation to its anticonvulsant properties. The affinity, kinetics and selectivity of brivaracetam and its tritiated form [(3)H]ucb 34714 have been determined by in vitro binding experiments in rat, human and mouse brain and on recombinant human SV2A. Brivaracetam and levetiracetam ex vivo binding to SV2A and anticonvulsant activities in audiogenic mice were compared in relation to dose and time. Brivaracetam bound selectively with 20 fold higher affinity than levetiracetam to SV2A. [(3)H]ucb 34714 bound reversibly and with high affinity to an homogenous population of binding sites in rat and human brain and to human SV2A expressed in CHO cells. The binding sites labeled by [(3)H]ucb 34714 in brain had the pharmacological characteristics of SV2A and no specific binding could be detected in the brain of SV2A(-/-) knock-out mice. The time- and dose-dependency of brivaracetam and levetiracetam for binding to brain SV2A and for providing seizure protection in audiogenic mice correlated well; brivaracetam being more potent and faster than levetiracetam. Brivaracetam is a potent and selective SV2A ligand. From its affinity and pharmacokinetics, simulations predicted that at therapeutically relevant doses, brivaracetam should occupy more than 80% of SV2A in human brain, in line with levels of occupancy observed in pre-clinical models of epilepsy.
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proepileptic phenotype of SV2A deficient mice is associated with reduced anticonvulsant efficacy of levetiracetam
Epilepsia, 2009Co-Authors: Rafal M Kaminski, Michel Gillard, Karine Leclercq, Alain Matagne, Etienne Hanon, Genevieve Lorent, Donald Dassesse, Henrik KlitgaardAbstract:Summary Purpose: Synaptic vesicle protein 2A (SV2A) constitutes a distinct binding site for an antiepileptic drug levetiracetam (Keppra). In the present study we characterized SV2A (+/−) heterozygous mice in several seizure models and tested if the anticonvulsant efficacy of levetiracetam is reduced in these mice. Methods: Seizure thresholds of male SV2A (+/−) mice and their wild-type littermates were assessed in pilocarpine (i.p.), kainic acid (s.c.), pentylenetetrazol (i.v.), 6-Hz and maximal electroshock models. Kindling development was compared in amygdala and corneal kindling models. Ex vivo binding of levetiracetam to SV2A was also performed. Results: Long-term electroencephalography (EEG) monitoring and behavioral observations of SV2A (+/−) mice did not reveal any spontaneous seizure activity. However, a reduced seizure threshold of SV2A (+/−) mice was observed in pilocarpine, kainic acid, pentylenetetrazol, and 6-Hz models, but not in maximal electroshock seizure model. Accelerated epileptogenesis development was also demonstrated in amygdala and corneal kindling models. Anticonvulsant efficacy of levetiracetam, defined as its ability to increase seizure threshold for 6 Hz electrical stimulation, was significantly reduced (approx. 50%) in the SV2A (+/−) mice, consistently with reduced binding to SV2A in these mice. In contrast, valproate produced the same anticonvulsant effect in both SV2A (+/+) and SV2A (+/−) mice. Discussion: The present results evidence that SV2A is involved in mediation of the in vivo anticonvulsant activity of levetiracetam, in accordance with its previously proposed mechanism of action. Furthermore, the present data also indicate that even partial SV2A deficiency may lead to increased seizure vulnerability and accelerated epileptogenesis.
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visualization of SV2A conformations in situ by the use of protein tomography
Biochemical and Biophysical Research Communications, 2008Co-Authors: Berkley Lynch, Alain Matagne, Annika Brannstrom, Anne Von Euler, Magnus Jansson, Elenor Hauzenberger, Arvid J SoderhallAbstract:The synaptic vesicle protein 2A (SV2A), the brain-binding site of the anti-epileptic drug levetiracetam (LEV), has been characterized by Protein Tomography™. We identified two major conformations of SV2A in mouse brain tissue: first, a compact, funnel-structure with a pore-like opening towards the cytoplasm; second, a more open, V-shaped structure with a cleft-like opening towards the intravesicular space. The large differences between these conformations suggest a high degree of flexibility and support a valve-like mechanism consistent with the postulated transporter role of SV2A. These two conformations are represented both in samples treated with LEV, and in saline-treated samples, which indicates that LEV binding does not cause a large-scale conformational change of SV2A, or lock a specific conformational state of the protein. This study provides the first direct structural data on SV2A, and supports a transporter function suggested by sequence homology to MFS class of transporter proteins.
