The main finding of our study is that intravenous levetiracetam treatment transiently attenuates the behavioral response associated with pilocarpine-induced status epilepticus in a dose-dependent fashion, but does not significantly change the electrographic appearance of ictal activity. Earlier treatment has a more prolonged behavioral effect than delayed treatment. Moreover, pretreatment with intravenous LEV significantly delayed the onset of convulsive activity after administration of systemic pilocarpine. In addition, treatment with high doses of LEV attenuated pilocarpine-status-induced neuronal injury in hippocampus as assayed by analysis of DNA fragmentation.
We chose to use a well-characterized chemoconvulsant model of status epilepticus to examine the effect of acute intravenous administration of LEV on ongoing seizure activity, and used clinical observation of ictal behavior as our primary endpoint to assess efficacy. Because pilocarpine induces a highly stereotyped behavioral response that progresses through a series of well defined clinical stages of severity, we were able to detect partial responses to treatment, which we captured as “time in grade”. Based on the hypothesis that intravenous LEV would reduce seizure intensity, we chose to define a reduction in severity grade from Grade 3 to Grade 2 as a response. Moreover, we were able to quantify the duration of reduction to allow statistical analysis of the effect and dose comparison. Using this approach we established a clear dose-response relationship between LEV dose and duration of grade reduction. Our results complement Mazarati's finding in the self-sustaining status epilepticus model that intravenous injection of LEV 10 minutes after perforant path stimulation shortened seizure duration at doses of 200 mg/kg or greater, and in combination with diazepam, LEV suppressed seizures immediately [13
Importantly, we found that the dose response curve is shifted to the left when LEV is administered 10 minutes, as compared to 30 minutes, after clinical seizure onset. A similar finding in a study of diazepam treatment of pilocarpine-induced seizures [33
] supports the clinical observation that earlier treatment is more likely to suppress status epilepticus in patients [34
We also examined the effect of pretreatment with intravenous LEV on the latency to onset of pilocarpine-induced seizures and confirmed that pretreatment significantly delayed the appearance of initial behavioral changes [9
]. We therefore speculate that LEV raises the threshold for occurrence of status epilepticus and may have a role in preventing status in individuals who are predisposed to this serious clinical condition.
Although the behavioral effect of intravenous LEV treatment after the onset of seizures was incomplete, i.e. seizures were not fully terminated, we found clear evidence of a reduction in seizure-induced neuronal injury as assayed by staining for DNA fragmentation. Gibbs found that LEV administration early after seizure onset protected against mitochondrial dysfunction in the self-sustaining status epilepticus model, suggesting one possible mechanism of LEV-mediated neuroprotection in status epilepticus [35
The mechanism of behavioral attenuation in the absence of change in EEG remains unclear. Whereas many antiepileptic drugs target Na+
channels, T-type Ca++
channels, the GABAergic systems, or glutamate receptors. LEV has an atypical anticonvulsant profile in animal models where it is active against audiogenic seizures and seizures induced by kindling stimulation, but not against maximal electroconvulsive shock or pentylenetetrazole-induced seizures. The mechanism of action of LEV remains incompletely defined. LEV binds to the synaptic vesicle protein SV2A with high affinity [23
]. SV2A is a glycoprotein that exists in all synaptic vesicles membranes and plays an important role in synaptic vesicle cycling and neurotransmitters release into the synaptic cleft. An SV2A mouse knockout model manifests abnormal neurotransmission that results in early development failure, severe seizures and death [37
]. It is possible that transient LEV-mediated modulation of SV2A function could underlie the transient behavioral response we observed even in the absence of a detectable effect on electrographic discharge.
Relatively high doses of LEV were required to attenuate ictal behavioral activity in our study in comparison to the doses used to block spontaneous seizures or kindling seizures, and relative to doses used clinically. Mazarati [13
] and Gibbs [35
] also found a relatively high dose requirement in their studies of LEV activity against self-sustaining status epilepticus. We did not measure LEV serum levels in our study. It is possible that rodents metabolize LEV rapidly, especially in the setting of status, leading to reduced bioavailability. In rats the elimination half-life of LEV in serum is between 1.8–2.8 h [38
] whereas in humans the serum half-life is 6–8 h [39
], demonstrating a substantial species difference in pharmacokinetics. Additional pharmacokinetic studies will be required to address this issue. Interestingly, while LEV is rapidly absorbed and transported across the blood-brain barrier, there is a significant delay between Tmax (serum)
and Tmax (CSF)
(0.25–0.50 h vs. 1.33–1.92 h) [38
]. Because the efficacy of treatment of SE is highly dependent on the duration of SE prior to treatment, it is possible that a high LEV dose is required to achieve adequate CSF concentrations in a timely manner. It is also possible that the systemic chemoconvulsant model we used dictates the need for unusually high doses of LEV. In particular, the chemoconvulsant remains in circulation throughout the treatment period, presenting an ongoing challenge to homeostatic mechanisms. Moreover, because LEV does not appear to act as a specific neurotransmitter antagonist, its unique mechanism of action may impose a higher dose requirement to achieve efficacy. This notion is supported indirectly by the relatively high dose requirement in clinical use, usually expressed in grams per day rather than milligrams per day as with many anticonvulsant drugs.
We have shown that intravenous LEV, administered at high doses, reduces the intensity of the behavioral response to pilocarpine in the lithium-pilocarpine model of status epilepticus. We have also demonstrated that intravenous LEV, again at high dose, reduces the severity of neuronal injury in hippocampus after Li-Pilo SE. It is therefore surprising that we were unable to demonstrate significant change in the ictal EEG pattern recorded from animals with Li-Pilo SE treated with LEV. This paradox focuses attention on the mechanism of the behavioral and protective effects of LEV. While a specific biochemical effect of LEV might mediate the behavioral and neuroprotective responses we observed, an alternative hypothesis is that LEV acts downstream of the cerebral cortex to modify the expression of epileptic activity. If true, this hypothesis would raise the concern that treatment of status with intravenous LEV may convert convulsive SE into non-convulsive SE by acting to dissociate cortical structures from sub-cortical output pathways. Additional studies will be required to address these issues.