Karine Leclercq

SV2A protein is a broad-spectrum anticonvulsant target: functional correlation between protein binding and seizure protection in models of both partial and generalized epilepsy.

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 (r(2)=0.77; p<0.001). A similar correlation was also observed in the mouse corneal kindling (r(2)=0.80; p<0.01) and in the rat model of generalized absence epilepsy (GAERS) (r(2)=0.72; p<0.01). Moreover, there were no significant differences between the slopes and intercepts of regression lines in these models. Interestingly, the protective potencies in these three epilepsy models were also well correlated with each other. As such, protective doses of a given SV2A ligand in one model could be easily predicted based on the data obtained in another model. Taken together, these results support the concept that SV2A protein is an important target for both partial and generalized epilepsies and thereby relevant for the generation of new antiepileptic drugs with potential broad-spectrum efficacy.

Binding characteristics of brivaracetam, a selective, high affinity SV2A ligand in rat, mouse and human brain: Relationship to anti-convulsant properties

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.

Proepileptic phenotype of SV2A-deficient mice is associated with reduced anticonvulsant efficacy of levetiracetam

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.

Brivaracetam: Rationale for discovery and preclinical profile of a selective SV2A ligand for epilepsy treatment

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.

Electrical, Molecular and Behavioral Effects of Interictal Spiking in the Rat

OBJECTIVE Epilepsy is a disease characterized by chronic seizures, but is associated with significant comorbidities between seizures including cognitive impairments, hyperactivity, and depression. To study this interictal state, we characterized the electrical, molecular, and behavior effects of chronic, neocortical interictal spiking in rats. METHODS A single injection of tetanus toxin into somatosensory cortex generated chronic interictal spiking measured by long-term video EEG monitoring and was correlated with motor activity. The cortical pattern of biomarker activation and the effects of blocking MAPK signaling on interictal spiking and behavior were determined. RESULTS Interictal spiking in this model increases in frequency, size, and becomes repetitive over time, but is rarely associated with seizures. Interictal spiking was sufficient to produce the same molecular and cellular pattern of layer 2/3-specific CREB activation and plasticity gene induction as is seen in the human interictal state. Increasing spike frequency was associated with hyperactivity, demonstrated by increased ambulatory activity and preferential circling toward the spiking hemisphere. Loud noises induced epileptic discharges, identical to spontaneous discharges. Treatment with a selective MAPK inhibitor prevented layer 2/3 CREB activation, reduced the frequency of epileptic discharges, and normalized behavioral abnormalities, but had no effect on seizures induced by electrical kindling. INTERPRETATION These results provide insights into the development of interictal epileptic spiking, their relationship to behavior, and suggest that interictal and ictal activities utilize distinct molecular pathways. This model, that parallels recent observations in humans, will be useful to develop therapeutics against interictal spiking and its behavioral comorbidities.

Genetic background of mice strongly influences treatment resistance in the 6 Hz seizure model

The 6 Hz model of focal seizures has been increasingly used to identify anticonvulsant compounds with potential activity against therapy‐resistant epilepsy, but the protective response to anticonvulsants in this model could be dependent on experimental conditions and selection of mouse strains.

Low potency and limited efficacy of antiepileptic drugs in the mouse 6 Hz corneal kindling model

Corneal kindling is a useful alternative to electrically induced amygdala or hippocampal kindling, which requires advanced surgical and EEG techniques that may not be easily available in many laboratories. Therefore the first aim of this study was to evaluate whether repeated 6 Hz corneal stimulation in mice would lead to an increased and persistent seizure response as described for higher frequency (50/60 Hz) corneal kindling. Male NMRI mice stimulated twice daily (except weekends) for 3 s with 6 Hz electrical current at 44 mA displayed robust kindling development, i.e., a progressive increase in seizure severity. The majority of the animals (about 90%) developed a fully kindled state, defined as at least 10 consecutive stage 3-5 seizures within 5 weeks of corneal stimulation. Afterwards, the fully kindled state was maintained for at least 8 weeks with only two days of stimulations per week. Next, the protective efficacy of four mechanistically different antiepileptic drugs (AEDs; clonazepam, valproate, carbamazepine and levetiracetam) was assessed and compared between 6 Hz and 50 Hz fully kindled mice. All tested AEDs showed a relatively lower potency in the 6 Hz kindling model and a limited efficacy against partial seizures was observed with carbamazepine and levetiracetam. We can conclude that 6 Hz kindling may be more advantageous than the previously described 50/60 Hz corneal kindling models due to its robustness and persistence of the fully kindled state. Furthermore, the observed low potency and limited efficacy of AEDs in 6 Hz fully kindled mice suggest that this model could be a useful tool in the discovery of novel AEDs targeting treatment resistant epilepsy.

Brivaracetam does not alter spatial learning and memory in both normal and amygdala-kindled rats

Several antiepileptic drugs (AEDs) may induce memory deficits when tested in preclinical models at doses that exert significant protection against seizures. Brivaracetam (BRV) is a novel high-affinity SV2A ligand also displaying inhibitory activity at neuronal voltage-gated sodium channels. In the present study we have investigated the effects of BRV, at doses that exerted marked anticonvulsant effects in kindled rats, upon cognitive functioning and memory in both normal and amygdala-kindled rats using place learning version of Morris water maze. In addition the effect of BRV on long-term potentiation (LTP) in rat hippocampal slices has been investigated. BRV (2.1, 6.8 or 21.0mg/kg i.p.) was injected daily, 60min before each session. Results indicated that in both normal and amygdala-kindled rats BRV did not alter the latency to find the hidden platform or swimming speed during the four consecutive days of learning. Similarly, the time spent in the target quadrant, used as a further independent index of spatial memory, was not modified by BRV treatment. Likewise, BRV did not affect the LTP induction in CA1 hippocampal region when tested at 3-30microM concentration range, which had been demonstrated to significantly reduce epileptiform activity in slice models. Based on the results of the present study it can be expected that BRV will not have detrimental effects on hippocampal-dependent cognitive functions in patients with epilepsy.

Cross-species pharmacological characterization of the allylglycine seizure model in mice and larval zebrafish

Treatment-resistant seizures affect about a third of patients suffering from epilepsy. To fulfill the need for new medications targeting treatment-resistant seizures, a number of rodent models offer the opportunity to assess a variety of potential treatment approaches. The use of such models, however, has proven to be time-consuming and labor-intensive. In this study, we performed pharmacological characterization of the allylglycine (AG) seizure model, a simple in vivo model for which we demonstrated a high level of treatment resistance. (d,l)-Allylglycine inhibits glutamic acid decarboxylase (GAD) - the key enzyme in γ-aminobutyric acid (GABA) biosynthesis - leading to GABA depletion, seizures, and neuronal damage. We performed a side-by-side comparison of mouse and zebrafish acute AG treatments including biochemical, electrographic, and behavioral assessments. Interestingly, seizure progression rate and GABA depletion kinetics were comparable in both species. Five mechanistically diverse antiepileptic drugs (AEDs) were used. Three out of the five AEDs (levetiracetam, phenytoin, and topiramate) showed only a limited protective effect (mainly mortality delay) at doses close to the TD50 (dose inducing motor impairment in 50% of animals) in mice. The two remaining AEDs (diazepam and sodium valproate) displayed protective activity against AG-induced seizures. Experiments performed in zebrafish larvae revealed behavioral AED activity profiles highly analogous to those obtained in mice. Having demonstrated cross-species similarities and limited efficacy of tested AEDs, we propose the use of AG in zebrafish as a convenient and high-throughput model of treatment-resistant seizures.

Lack of synaptic vesicle protein SV2B protects against amyloid-β25–35-induced oxidative stress, cholinergic deficit and cognitive impairment in mice

SV2B is a synaptic protein widely distributed throughout the brain, which is part of the complex vesicle protein machinery involved in the regulation of synaptic vesicle endocytosis and exocytosis, and therefore in neurotransmitters release. The aims of the present work were twofold: (1) phenotype SV2B knockout mice (SV2B KO) in a battery of cognitive tests; and (2) examine their vulnerability to amyloid-β25-35 (Aβ25-35) peptide-induced toxicity. SV2B KO mice showed normal learning and memory abilities in absence of Aβ25-35 injection. SV2B KO mice were protected against the learning deficits induced after icv injection of an oligomeric preparation of amyloid-β25-35 peptide, as compared to wild-type littermates (SV2B WT). These mice failed to show Aβ25-35-induced impairments in a number of cognitive domains: working memory measured by a spontaneous alternation procedure, recognition memory measured by a novel object recognition task, spatial reference memory assessed in a Morris water-maze, and long-term contextual memory assessed in a inhibitory avoidance task. In addition, SV2B KO mice were protected against Aβ25-35-induced oxidative stress and decrease in ChAT activity in the hippocampus. These data suggest that SV2B could be a key modulator of amyloid toxicity at the synaptic site.