Levetiracetam’s (Keppra®) binding site and its subsequent identification as the synaptic vesicle protein 2A (SV2A) has opened a new and successful avenue for drug discovery programs at UCB. A number of structurally diverse, selective high affinity SV2A ligands have been identified in binding assays and displayed potent, broad spectrum activity in animal models of epilepsy. Promising preclinical data enabled the identification of brivaracetam as an antiepileptic drug (AED) candidate now in Phase III development for epilepsy. In parallel, intensive research efforts have also been undertaken to further understand the mechanism of action of levetiracetam and other SV2A ligands. A strong correlation between SV2A binding affinity and anticonvulsant potency, initially observed in the audiogenic seizure model, has now been extended to other models of generalized and partial epilepsy. Furthermore, the anticonvulsant efficacy of levetiracetam was significantly reduced in SV2A deficient mice. Several research groups have been working to elucidate the role of SV2A in synaptic vesicle release and cycling, but despite many efforts its function still remains elusive. Recent studies report reduced SV2A expression in brain tissue obtained from both experimental epilepsy models and patients with epilepsy. These observations appear to correlate with the data from SV2A deficient animals which display increased vulnerability to seizures. Furthermore, SV2A deficient mice show rapid development of kindling suggesting accelerated epileptogenesis. SV2A represents an important novel target for AED discovery that is now well validated in a large number of both preclinical and clinical studies.

IDENTIFICATION OF SV2A AS THE BINDING TARGET FOR LEV

Our group has demonstrated that LBS is enriched in purified synaptic vesicle fraction. In fact, [³H]ucb 30889, a derivative of LEV, labeled a synaptic vesicle protein with an approximate molecular mass of 90 kDa, which was consistent with the molecular mass of synaptic vesicle protein 2A (SV2A).¹⁵ Binding studies were then performed in brain membranes obtained from SV2A transgenic mice to determine whether SV2A is indeed necessary for LEV binding. We found that [³H]ucb 30889 binds only to membranes from SV2A (+/+) mice and not to those obtained from SV2A (−/−) mice.¹⁵ Additional in vitro binding experiments performed in brain membranes confirmed that the B_max value for [³H]ucb 30889 is reduced by 50% in SV2A (+/−) when compared to the B_max value obtained with brain membranes of their wild-type littermates.¹⁶ These observations were further corroborated by ex vivo binding experiments. Injection of LEV to both SV2A (+/+) and SV2A (+/−) mice revealed that that SV2A (+/−) mice have half of the protein available for binding as compared to their wild-type littermates.¹⁶ It should be noted, however, that both the radioligand dissociation constants (K_d) in brain membranes and IC₅₀ values of LEV calculated from the ex vivo binding curves were comparable between SV2A (+/+) and (+/−) mice, confirming that SV2A binding properties were not modified in transgenic animals.¹⁶ In fact, the binding characteristics of native SV2A in human and rat brain share very similar binding properties.¹⁷ Furthermore, binding affinities of several SV2A ligands are also comparable when measured in human brain, rat brain and when the recombinant human SV2A protein is expression in Chinese hamster ovary (CHO) cells.¹⁷

BIOLOGY AND FUNCTION OF SV2

A series of molecular studies described above has led to the identification of SV2A as the molecular correlate of LBS, however the exact role of this protein in synaptic vesicle cycle and neurotransmitter release still remains elusive. SV2 proteins exist as three separate subtypes (SV2A, SV2B and SV2C) and are integral part of secretory vesicles membranes localized in different mammalian secretory organs with the highest expression being observed in the brain.^18,19,20,21 SV2s have 12 transmembrane domains and are members of the major facilitator superfamily (MFS) of membrane transporters.^18,22 LacY, the prototypic member of the MFS proteins, has been shown to exist under two major conformations and the transition between these two conformations would allow the transport of lactose.²³ Similar changes in conformations have been proposed for other members of this family of proteins, which also includes neurotransmitter co-transporters.²⁴ The existence of two major conformations adopted by SV2A proteins in mouse brain was also recently reported by Lynch et al. using Protein Tomography^™.²⁵ One of the conformations has a funnel structure with an opening towards the cytosol and the second displays a “V” shape with the opening directed towards the luminal space. Both conformational states of the protein were present in controls samples and samples treated with LEV indicating that binding of LEV does not induce an obvious conformational change of SV2A and does not stabilize a specific conformational state of the protein.²⁵ This apparent lack of effect of LEV on conformational states of SV2A protein may have been hampered by the limitations of the Protein Tomography^™ technology and its resolution. In that context, as the functional role of SV2A is still largely unknown today, besides its probable involvement in synaptic vesicle cycling and in exo- or endocytosis processes, the downstream consequences of compounds binding to SV2A are not yet elucidated. One could imagine that upon binding to SV2A, each compound could induce or stabilize a conformational change that would be more or less favorable in terms of function, meaning that compounds could show various degree of intrinsic efficacy. If proven correct, this opens the possibility that newer SV2A ligands bearing higher intrinsic efficacy upon binding could either show clinical efficacy at lower levels of SV2A occupancy or even be efficacious in refractory patients.

SV2A is the most studied member of the three SV2 proteins. To date majority of information about its role in neuronal excitability has been obtained in experiments involving knock-out mice.^{21,26,27,28,29} It appears that SV2A is not crucial for vesicle biogenesis or synaptic function, but modulates exocytosis of transmitter-containing vesicles.^26,28 Mice lacking SV2A are characterized by a decrease in the calcium-dependent exocytotic burst, which is a measure of the availability of neurotransmitter vesicles ready to release their content.²⁸ Moreover, lack of SV2A results in decreased action potential-dependent neurotransmission, while action potential-independent neurotransmission remains normal.^26,28 Very recent reports seem to confirm the hypothesis that SV2 proteins play important role in calcium dependent neurotransmission.^30,31,32 Other recent data seem to indicate that SV2A also plays a role in structural changes (volume increase) of synaptic vesicles upon loading with glutamate.³³ Finally, SV2A may influence synaptic vesicle priming regulated by binding of adenine nucleotides.³⁴

VALIDATION OF SV2A AS A DRUG TARGET FOR AEDS

Affinity-Potency Correlation Studies

A key observation initially suggesting that SV2A is the target site for the principal mechanism of action of LEV was the fact that the anticonvulsant potency of selective SV2A ligands strongly correlated with their in vitro binding affinity. Such correlations between the in vitro affinity of ligands and their potency in inhibiting audiogenic seizures in mice have been documented using SV2A from rat cerebral cortex⁹ and human recombinant SV2A expressed in cell lines.¹⁵ Our more recent studies confirmed and extended these initial findings to other animal models of epilepsy, i.e., corneal kindling and absence seizures in genetic absence epilepsy rats from strasbourg (GAERS).³⁵ The anticonvulsant potency of SV2A ligands against audiogenic seizures correlated well with their in vitro binding affinity (r²=0.77; p < 0.001) (Figure 1A). Similar correlation (r²=0.80; p < 0.01) between anticonvulsant activity and SV2A protein in vitro binding was also observed in corneally kindled mice (Figure 1B). Finally, correlation between in vitro SV2A binding and inhibition of spike-wave discharges in GAERS has also been documented (r²=0.72; p < 0.01) (Figure 1C). It is important to note that despite differences in species and epilepsy models, i.e., audiogenic model, corneal kindling and GAERS, the slopes and intercepts of regression lines were not statistically different. These experiments demonstrated the existence of strong correlation between SV2A binding affinity and anticonvulsant potency in three distinct preclinical epilepsy models, which reinforced the significance of this molecular target in the mechanism of action of the tested ligands.³⁵ It also suggested that SV2A-related mechanisms are equally important in protection against seizures irrespectively of the model. It was a surprising finding since different mechanisms and brain regions are likely to be involved in the generation of seizure activity observed in these models. Yet, it appears that SV2A protein is essential for neuronal synchronization fundamentally associated with every type of epileptiform activity. Taken together, these data strongly support the notion that targeting SV2A results in an anticonvulsant activity relevant for both partial and generalized epilepsy, and thereby can provide antiepileptic drug candidates with a potential for broad spectrum clinical efficacy.³⁵

Figure 1

Correlation between binding affinity and protective potency of SV2A ligands against convulsive seizures (audiogenic seizures and corneal kindling) and absence seizures in genetic absence epilepsy rats from strasbourg (GAERS). SV2A binding affinities −log (more...)

Although these correlations helped in validating the main target for the anticonvulsant properties of selective SV2A ligands, in vitro data do not take into account individual pharmacokinetic properties of compounds such as protein binding, brain penetration and metabolism that may affect the concentration of the drug in the compartment of interest and hence binding to its target. The fact that we observed good correlations between in vitro binding and in vivo efficacy is indicative that most compounds tested were close structural analogues sharing very similar physicochemical and pharmacokinetic properties. Another major limitation of using in vitro binding data is the inability to predict the level of SV2A occupancy that compounds need to reach in vivo in order to afford seizure protection. To address these issues, we performed ex vivo binding experiments in which binding of compounds with selective affinity for SV2A is measured in vitro in brain homogenates from animals that have been administered the compounds prior to being sacrificed. When corrected for experimental conditions (essentially dilution factors), ex vivo binding data are comparable to in vivo binding data as shown in Figure 2 where ex vivo and in vivo binding dose-response curves of LEV are overlapping. The disadvantage of in vivo over ex vivo binding experiments is related to the fact that a substantial amount of radioactive tracer needs to be injected to each animal. Not unexpectedly, we observed a good correlation between SV2A occupancy measured by ex vivo binding and protection against clonic seizures in audiogenic mice as shown in Figure 3 for a series of compounds. However, it appears quite clearly that the doses of compounds needed to occupy 50% of SV2A are generally lower than the doses needed to afford 50% seizure protection (pIC₅₀ > pED₅₀) i.e. a high SV2A occupancy is required to provide pharmacological activity (Figure 3).

Figure 2

Ex vivo and in vivo binding of levetiracetam to SV2A in mouse brain. Increasing doses of levetiracetam were administered ip to mice. Mice were sacrificed 60 min post administration. For ex vivo binding, brains were removed, homogenized and binding using (more...)

Figure 3

Correlation between ex vivo binding affinity and protection against clonic seizures in audiogenic mice. Increasing doses of SV2A ligands were administered ip to mice. Mice were sacrificed 60 min post administration. Brains were removed, homogenized and (more...)

Anticonvulsant Effects of LEV and Their Relation to SV2A Occupancy

Using the same ex vivo binding approach, we explored the relationship between SV2A occupancy and seizure protection in audiogenic mice as a function of time after single dose administration. Results obtained with LEV are depicted in Figure 4. We found that the post-administration time needed for LEV to occupy maximally SV2A was well correlated with the time needed for maximal protection against clonic and tonic seizures. Interestingly, protection afforded against the more severe clonic seizures is lost prior to the decrease in protection against tonic seizures. This suggests that lower amounts of SV2A proteins need to be occupied to prevent later stage less severe (tonic) seizures, while a higher occupancy is needed to prevent both forms of seizures (clonic + tonic). We have also accumulated similar data for other SV2A ligands that display a variety of occupancy-protection patterns. Some compounds quickly occupy SV2A and afford maximal protection against seizures faster than LEV, while other compounds display more rapid decline in occupancy and seizure protection than LEV. This interlink between SV2A occupancy and efficacy in a time dependent manner further strengthen the role of SV2A as the relevant target in the mechanism of action of these drugs.

Figure 4

Time course of SV2A occupancy and seizure protection in audiogenic mice after a single administration of levetiracetam. 210 μmol/kg of levetiracetam was administered ip. SV2A occupancy and protection against tonic and clonic seizures was assessed (more...)

From dose response and kinetic experiments as shown in Figure 2 and 4, it appears that LEV needs to occupy nearly 90% of SV2A in order to protect against clonic seizures in audiogenic mice. It is also clear that there is a threshold level of occupancy that needs to be reached to afford seizure protection and that minute variations around this threshold are sufficient to prevent or not the occurrence of a seizure. To illustrate this, a group of 10 mice were treated with LEV and 2 h post-administration 6 mice were protected against sound elicited clonic seizures and 4 were not. We measured SV2A occupancy in each mouse and found quite surprisingly that the individual SV2A receptor occupancy was nearly identical for all animals. We were expecting that in protected mice, the level of SV2A occupancy would be higher than in non protected mice (reflecting inter-individual variations due to slight differences in drug administration and/or pharmacokinetics). This might be explained by the fact that a seizure is not a gradual, but a binary response and therefore even small differences in SV2A occupancy might be sufficient to be either above or below the threshold for a seizure to happen. This is in agreement with the rather steep dose response curves observed for LEV in showing protection against audiogenic¹ or corneally kindled seizures in mice.³⁶

To further ascertain whether high occupancy of SV2A is also needed to afford seizure protection in patients, we have predicted SV2A occupancy by LEV in the brain of human patients based on affinity and available pharmacokinetic data. We used the following equation describing a single bimolecular interaction between a ligand and a receptor:

$$\text{SV}2\text{A\hspace{0.17em}Occupancy}=100\%\times \frac{[\text{Levetiracetam}]}{[\text{Levetiracetam}]+Ki}$$

[Levetiracetam] refers to the free concentration of LEV in the brain. This was approximated by using total plasma concentrations measured in healthy volunteers and patients (UCB data on file, see ref. ³⁷) corrected for plasma protein binding of 10%.³⁸ Brain to plasma ratios of 0.4 to 1 were considered based on studies in rodents.³⁹ Ki is the equilibrium dissociation constant (affinity) of LEV for SV2A protein. We took the value of 8 μM that was measured at 37°C in brain tissue. We first validated this approach by predicting, in the same way, the SV2A occupancy in mouse brain 60 min after administration using total plasma concentrations corrected for protein binding, and using a brain to plasma ratio of 0.4 as reported for mouse.³⁹ The predicted SV2A occupancy related to administered dose is shown in Figure 2 and it compares quite satisfactorily with the experimental data obtained from ex vivo and in vivo binding experiments.

LEV’s total plasma concentrations at a clinically active daily dose of 1g are ranging from 6–8 μg/mL (C_min) to 15–20 μg/mL (C_max) (UCB data on file, see ref. ³⁷). Plasma concentrations have been shown to be linearly proportional to the administered dose³⁸ and can be adjusted accordingly. Using the above equation and assuming a brain to plasma ratio of 1 the predicted SV2A occupancy by LEV in patients varies from 80 to 93% at C_min and C_max for a 1g daily dose and from 92 to 98% for a 3g daily dose. With a less favorable brain to plasma ratio of 0.5, the predicted values are 67% (C_min) and 87% (C_max) for a 1g daily dose or 86% (C_min) and 98% (C_max) for a 3g daily dose. These predicted levels of SV2A occupancy in patients are remarkably close to those measured in animal models of epilepsy at active pharmacological doses and confirm that high SV2A occupancy is needed by current SV2 ligands to adequately afford protection against seizure.

Pharmacology and Phenotyping of SV2A Transgenic Animals in Seizure Models

Direct evidence that the in vivo anticonvulsant activity of LEV is indeed mediated by SV2A has been lacking until recently.¹⁶ Genetically engineered knock-out animals, that lack a given drug target, are frequently used to proof in vivo selectivity of pharmacological agents and to demonstrate the lack of therapeutic activity in the absence of the molecular target. Similar prove of concept evidence could have been obtained by testing LEV in SV2A (−/−) homozygous mice. However, these animals suffer from severe seizures starting very early in their development and do not survive beyond 2–3 weeks after birth, which precludes their use in pharmacological in vivo experiments.^26,29 Therefore, we decided to use SV2A (+/−) mice, which are deficient in the SV2A protein, but develop normally after birth. First, we demonstrated by video-EEG monitoring that SV2A (+/−) mice do not display any overt epileptic phenotype, but rather show pro-epileptic traits such as decreased seizure thresholds and accelerated kindling development. The pro-epileptic phenotype of SV2A (+/−) mice was observed in kindling models, pilocarpine, kainate, pentylenetetrazol (iv) and 6 Hz models, but not in the MES model.¹⁶ Interestingly, the pro-epileptic phenotype of SV2A (+/−) in a range of different experimental seizure models appeared as a “mirror image” of the unique pharmacological profile of LEV in the same models.³

We also demonstrated for the first time a functional involvement of SV2A in mediation of the anticonvulsant effect of LEV, which was indeed reduced in SV2A (+/−) mice.¹⁶ This was illustrated by its failure to produce the same degree of increase in the threshold for induction of 6 Hz seizures in SV2 (+/−) mice as in their wild-type littermates (Figure 5). In contrast, valproate, which has SV2A-unrelated mechanisms of action⁹, produced the same magnitude of threshold increase in both genotypes (Figure 5). We decided to use an unbiased approach for this comparison and ascertain whether the same doses of LEV and valproate would produce the same magnitude of threshold increase for 6 Hz seizures in both SV2A (+/+) and SV2A (+/−) mice. Remarkably, this was true only in the case of valproate. A low dose of valproate produced an increase in the threshold for 6 Hz seizures which was comparable between SV2A (+/+) and SV2A (+/−) mice (Fig 5). A higher dose of valproate afforded further increase of the threshold, which again was almost identical in the two genotypes. Similarly, a low dose of LEV produced comparable increases in the seizure threshold in both genotypes. However, in contrast to valproate, a higher dose of LEV failed to provide an additional threshold increase in the SV2A (+/−) mice, while the threshold was further increased in SV2A (+/+) mice. In fact, a higher dose of LEV produced a 50% higher increase in threshold in wild-type mice compared to the increase obtained in SV2 (+/−) mice. It is important to remember that SV2A (+/−) mice still express 50% of the SV2A protein, thus occupancy of SV2A by LEV may afford some protection against seizures. Furthermore, SV2A (+/−) mice have a significantly reduced threshold for 6 Hz seizures and at lower stimulation currents it might have been somewhat easier to elevate the threshold with the same dose of LEV, which in fact has the same SV2A affinity in both genotypes. The difference in the effects of LEV on seizure threshold became significant only at the dose that occupied nearly all of the SVA2 binding sites in both genotypes, but since SV2A (+/−) have 50% less sites available for LEV binding, the degree of seizure protection was also reduced by approximately 50%.

Figure 5

Current-responses for seizures induced by 6 Hz electrical stimulation after treatment with levetiracetam (LEV) in SV2A (+/+) mice (panel A) and SV2A (+/−) mice (panel B). Points representing the percentage of animals (n ≥ 8 per group) (more...)

CHANGES IN SV2A EXPRESSION IN PRECLINICAL MODELS AND HUMAN EPILEPSY

Discovery of SV2A as the binding target for LEV prompted investigations of the potential role of SV2A in the pathophysiology of epilepsy. Van Vliet et al.⁴⁰ has used immunohistochemistry and Western blot analysis to study SV2A expression patterns during epileptogenesis and chronic epilepsy. Hippocampal samples from autopsy controls, patients who died from status epilepticus and pharmacoresistant temporal epilepsy (TLE) patients were analyzed. Additionally, SV2A expression was assessed in the hippocampus of rats at different stages of epileptogenesis in a post status epilepticus model. A remarkable consistency has been observed between human and rat samples. Namely, SV2A expression was significantly decreased in the hippocampus of TLE patients with hippocampal sclerosis and also in the mossy fiber terminals during the latent and chronic phase of epileptogenesis in rats.⁴⁰ Based on these results it is not possible to establish a clear cause-effect relationship between reduced expression of SV2A and development of epilepsy, but these data are very consistent with the above described pro-epileptic phenotype of SV2A-deficnt mice and accelerated epileptogenesis observed in these animals.¹⁶ Since SV2A is the binding site of LEV and the drug shows reduced efficacy in SV2A-deficient animals¹⁶ the data of van Vliet et al.,⁴⁰ may also explain an apparent lack of efficacy of LEV in some patients with TLE. It is conceivable that reduced expression of the target for LEV, namely the SV2A protein, could underline the cause of non-responsiveness to the drug in some patients. This hypothesis can be verified by comparison of SV2A expression levels assessed by positron emission tomography (PET) with patient response or non-response to LEV. It is plausible that SV2A expression changes may be responsible for different responses to LEV, because pharmacogenetics studies failed to identify SV2A genetic variants that influence response to LEV.⁴¹ Future SV2A PET ligands may also allow studying occupancy-efficacy relationships to obtain full translation of the data we have obtained in animal models to human epilepsy.

CONCLUSION

SV2A constitutes the unique binding site for LEV and plays an important role in synaptic vesicle function. Affinity-potency correlations in several models of partial and generalized epilepsy indicate that SV2A is a broad spectrum anticonvulsant target. Anticonvulsant activity of LEV is closely linked with occupancy and availability of SV2A, whereas SV2A deficiency leads to increased seizure vulnerability and accelerated epileptogenesis. Taken together, existing experimental data prove that SV2A plays crucial role in mediation of the anticonvulsant action of LEV in vivo and indicate that the SV2A protein represents an important and well validated target for the discovery of novel AEDs. Finally, the finding that SV2A protein is as a clinically validated target for epilepsy triggered further discovery programs exploring therapeutic potential for SV2A ligands with different binding properties.

REFERENCES

1.

Gower AJ, Noyer M, Verloes R, Gobert J, Wülfert E. ucb L059, a novel anti-convulsant drug: pharmacological profile in animals. Eur J Pharmacol. 1992;222:193–203. [PubMed: 1451732]

2.

Löscher W, Hönack D. Profile of ucb L059, a novel anticonvulsant drug, in models of partial and generalized epilepsy in mice and rats. Eur J Pharmacol. 1993;232:147–158. [PubMed: 8467854]

3.

Klitgaard H, Matagne A, Gobert J, Wülfert E. Evidence for a unique profile of levetiracetam in rodent models of seizures and epilepsy. Eur J Pharmacol. 1998;353:191–206. [PubMed: 9726649]

4.

Löscher W, Hönack D, Rundfeldt C. Antiepileptogenic effects of the novel anticonvulsant levetiracetam (ucb L059) in the kindling model of temporal lobe epilepsy. J Pharmacol Exp Ther. 1998;284:474–479. [PubMed: 9454787]

5.

Margineanu DG, Matagne A, Kaminski RM, Klitgaard H. Effects of chronic treatment with levetiracetam on hippocampal hyperexcitability developing after pilocarpine-induced status epilepticus in rats. Brain Res Bull. 2008;77:282–285. [PubMed: 18722515]

6.

Niespodziany I, Klitgaard H, Margineanu DG. Levetiracetam inhibits the high-voltage-activated Ca²⁺ current in pyramidal neurones of rat hippocampal slices. Neurosci Lett. 2001;306:5–8. [PubMed: 11403944]

7.

Lukyanetz EA, Shkryl VM, Kostyuk PG. Selective blockade of N-type calcium channels by levetiracetam. Epilepsia. 2002;43:9–18. [PubMed: 11879381]

8.

Rigo J-M, Hans G, Nguyen L, et al. The anti-epileptic drug levetiracetam reverses the inhibition by negative allosteric modulators of neuronal GABA- and glycine-gated currents. Brit J Pharmacol. 2002;136:659–672. [PMC free article: PMC1573396] [PubMed: 12086975]

9.

Noyer M, Gillard M, Matagne A, Henichart JP, Wulfert E. The Novel Antiepileptic Drug Levetiracetam (Ucb L059) Appears to Act Via a Specific Binding Site in CNS Membranes. Eur J Pharmacol. 1995;286:137–146. [PubMed: 8605950]

10.

Klitgaard H. Levetiracetam: the preclinical profile of a new class of antiepileptic drugs. Epilepsia. 2001;42(Supplement 4):13–18. [PubMed: 11564119]

11.

Klitgaard H, Verdru P. Levetiracetam: the first SV2A ligand for the treatment of epilepsy. Expert Opin Drug Disc. 2007;2:1537–1545. [PubMed: 23484603]

12.

Kenda BM, Matagne AC, Talaga PE, Pasau PM, Differding E, Lallemand BI, Frycia AM, Moureau FG, Klitgaard HV, Gillard MR, Fuks B, Michel P. Discovery of 4-substituted pyrrolidone butanamides as new agents with significant antiepileptic activity. J Med Chem. 2004;47:530–49. [PubMed: 14736235]

13.

Matagne A, Margineanu DG, Kenda B, Michel P, Klitgaard H. Anti-convulsive and anti-epileptic properties of brivaracetam (ucb 34714), a high-affinity ligand for the synaptic vesicle protein, SV2A. Brit J Pharmacol. 2008;154:1662–1671. [PMC free article: PMC2518465] [PubMed: 18500360]

14.

Matagne A, Margineanu DG, Potschka H, Löscher W, Michel P, Kenda B, Klitgaard H. Profile of the new pyrrolidone derivative seletracetam (ucb 44212) in animal models of epilepsy. Eur J Pharmacol. 2009;614:30–37. [PubMed: 19383493]

15.

Lynch BA, Lambeng N, Nocka K, Kensel-Hammes P, Bajjalieh SM, Matagne A, Fuks B. The synaptic vesicle protein SV2A is the binding site for the antiepileptic drug levetiracetam. Proc Nat Acad Sci USA. 2004;101:9861–9866. [PMC free article: PMC470764] [PubMed: 15210974]

16.

Kaminski RM, Gillard M, Leclercq K, Hanon E, Lorent G, Dassesse D, Matagne A, Klitgaard H. Proepileptic phenotype of SV2A-deficient mice is associated with reduced anticonvulsant efficacy of levetiracetam. Epilepsia. 2009;50:1729–1740. [PubMed: 19486357]

17.

Gillard M, Chatelain P, Fuks B. Binding characteristics of levetiracetam to synaptic vesicle protein 2A (SV2A) in human brain and in CHO cells expressing the human recombinant protein. Eur J Pharmacol. 2006;536:102–108. [PubMed: 16556440]

18.

Bajjalieh SM, Peterson K, Shinghal R, Scheller RH. SV2, a Brain Synaptic Vesicle Protein Homologous to Bacterial Transporters. Science. 1992;257:1271–1273. [PubMed: 1519064]

19.

Buckley K, Kelly RB. Identification of a transmembrane glycoprotein specific for secretory vesicles of neural and endocrine cells. J Cell Biol. 1985;100:1284–1294. [PMC free article: PMC2113776] [PubMed: 2579958]

20.

Feany MB, Lee S, Edwards RH, Buckley KM. The synaptic vesicle protein SV2 is a novel type of transmembrane transporter. Cell. 1992;70:861–867. [PubMed: 1355409]

21.

Janz R, Südhof TC. SV2C is a synaptic vesicle protein with an unusually restricted localization: anatomy of a synaptic vesicle protein family. Neuroscience. 1999;94:1279–1290. [PubMed: 10625067]

22.

Saier MH Jr, Beatty JT, Goffeau A, Harley KT, Heijne WH, Huang SC, Jack DL, Jähn PS, Lew K, Liu J, Pao SS, Paulsen IT, Tseng TT, Virk PS. The major facilitator superfamily. J Mol Microbiol Biotechnol. 1999;1:257–279. [PubMed: 10943556]

23.

Holyoake J, Sansom MS. Conformational change in an MFS protein: MD simulations of LacY. Structure. 2007;15:873–884. [PubMed: 17637346]

24.

DeFelice LJ. Transporter Structure and Mechanism. Trends Neurosci. 2004;27:352–359. [PubMed: 15165740]

25.

Lynch BA, Matagne A, Brannstrom A, von Euler A, Jansson M, Hauzenberger E, Soderhall JA. Visualization of SV2A Conformations in Situ by the Use of Protein Tomography. Biochem Biophys Res Commun. 2008;375:491–495. [PubMed: 18692481]

26.

Crowder KM, Gunther JM, Jones TA, Hale BD, Zhang HZ, Peterson MR, Scheller RH, Chavkin C, Bajjalieh SM. Abnormal neurotransmission in mice lacking synaptic vesicle protein 2A (SV2A). Proc Nat Acad Sci USA. 1999;96:15268–15273. [PMC free article: PMC24809] [PubMed: 10611374]

27.

Custer KL, Austin NS, Sullivan JM, Bajjalieh SM. Synaptic vesicle protein 2 enhances release probability at quiescent synapses. J Neurosci. 2006;26:1303–1313. [PMC free article: PMC6674579] [PubMed: 16436618]

28.

Xu T, Bajjalieh SM. SV2 modulates the size of the readily releasable pool of secretory vesicles. Nat Cell Biol. 2001;3:691–698. [PubMed: 11483953]

29.

Janz R, Goda Y, Geppert M, Missler M, Südhof TC. SV2A and SV2B function as redundant Ca²⁺ regulators in neurotransmitter release. Neuron. 1999;24:1003–1016. [PubMed: 10624962]

30.

Yao J, Nowack A, Kensel-Hammes P, Gardner RG, Bajjalieh SM. Cotrafficking of SV2 and synaptotagmin at the synapse. J Neurosci. 2010;30:5569–5578. [PMC free article: PMC2866018] [PubMed: 20410110]

31.

Wan Q-F, Zhen-Yu Zhou Z-Y, Thakur P, Vila A, Sherry DM, Janz R, Heidelberger R. SV2 acts via presynaptic calcium to regulate neurotransmitter release. Neuron. 2010;66:884–895. [PMC free article: PMC2913707] [PubMed: 20620874]

32.

Chang WP, Südhof TC. SV2 renders primed synaptic vesicles competent for Ca²⁺-induced exocytosis. J Neurosci. 2009;29:883–897. [PMC free article: PMC2693337] [PubMed: 19176798]

33.

Budzinski KL, Allen RW, Fujimoto BS, Kensel-Hammes P, Belnap DM, Bajjalieh SM, Chiu DT. Large structural change in isolated synaptic vesicles upon loading with neurotransmitter. Biophys J. 2009;97:2577–2584. [PMC free article: PMC2770603] [PubMed: 19883601]

34.

Yao J, Bajjalieh SM. Synaptic vesicle protein 2 binds adenine nucleotides. J Biol Chem. 2008;283:20628–20634. [PMC free article: PMC2475693] [PubMed: 18524768]

35.

Kaminski RM, Matagne A, Leclercq K, Gillard M, Michel P, Kenda B, Talaga P, Klitgaard H. 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. 2008;54:715–720. [PubMed: 18207204]

36.

Matagne A, Klitgaard H. Validation of corneally kindled mice: a sensitive screening model for partial epilepsy in man. Epilepsy Res. 1998;31:59–71. [PubMed: 9696301]

37.

Perruca E, Gidal BE, Baltès E. Effects of antiepileptic co-medication on levetiracetam pharmacokinetics: a pooled analysis of data from randomized adjunctive therapy trials. Epilepsy Res. 2003;53:47–56. [PubMed: 12576167]

38.

Patsalos PN. Pharmacokinetic profile of levetiracetam: toward ideal characteristics. Pharmacol Ther. 2000;85:77–85. [PubMed: 10722121]

39.

Benedetti MS, Coupez R, Whomsley R, Nicolas JM, Collart P, Baltes E. Comparative pharmacokinetics and metabolism of levetiracetam, a new anti-epileptic agent, in mouse, rat, rabbit and dog. Xenobiotica. 2004;34:281–300. [PubMed: 15204700]

40.

van Vliet EA, Aronica E, Redeker S, Boer K, Gorter JA. Decreased expression of synaptic vesicle protein 2A, the binding site for levetiracetam, during epileptogenesis and chronic epilepsy. Epilepsia. 2009;50:422–433. [PubMed: 18717715]

41.

Lynch JM, Tate SK, Kinirons P, Weale ME, Cavalleri GL, Depondt C, Murphy K, O’Rourke D, Doherty CP, Shianna KV, Wood NW, Sander JW, Delanty N, Goldstein DB, Sisodiya SM. No major role of common SV2A variation for predisposition or levetiracetam response in epilepsy. Epilepsy Res. 2009;83:44–51. [PubMed: 18977120]

42.

Gillard M, Fuks B, Michel P, Vertongen P, Massingham R, Chatelain P. Binding characteristics of [3H]ucb 30889 to levetiracetam binding sites in rat brain. Eur J Pharmacol. 2003;478:1–9. [PubMed: 14555178]