Present address of Dr. Sasa: Nagisa Hospital, Hirakata-city, Osaka 573-1183, Japan.
Long-lasting Antiepileptic Effects of Levetiracetam against Epileptic Seizures in the Spontaneously Epileptic Rat (SER): Differentiation of Levetiracetam from Conventional Antiepileptic Drugs
Article first published online: 1 SEP 2005
Volume 46, Issue 9, pages 1362–1370, September 2005
How to Cite
Ji-qun, C., Ishihara, K., Nagayama, T., Serikawa, T. and Sasa, M. (2005), Long-lasting Antiepileptic Effects of Levetiracetam against Epileptic Seizures in the Spontaneously Epileptic Rat (SER): Differentiation of Levetiracetam from Conventional Antiepileptic Drugs. Epilepsia, 46: 1362–1370. doi: 10.1111/j.1528-1167.2005.29604.x
- Issue published online: 1 SEP 2005
- Article first published online: 1 SEP 2005
- Accepted May 2, 2005.
- Long-lasting effects;
- Spontaneously epileptic rat;
- Tonic convulsions;
- Absence-like seizures
Summary: Purpose: Some evidence suggests that levetiracetam (LEV) possesses antiepileptogenic characteristics. The purpose of this study was to investigate the time course of seizure protection by LEV compared with that of phenytoin (PHT), phenobarbital (PB), valproate (VPA), and carbamazepine (CBZ) in the spontaneously epileptic rat (SER). The SER is a double mutant (tm/tm, zi/zi) showing both tonic convulsions and absence-like seizures.
Methods: The effect of single (40, 80, and 160 mg/kg, i.p.) and 5-day (80 mg/kg/day, i.p.) administration of LEV on tonic convulsions and absence-like seizures in SERs were studied. Tonic convulsions induced by blowing air onto the animal's head at 5-min intervals for 30 min and spontaneous absence-like seizures characterized by 5- to 7-Hz spike–wave-like complexes in the cortical and hippocampal EEG were recorded for 30 min. In the single-administration study, observations for seizure activity were performed once before and 3 times (45, 75, and 135 min) after drug administration. In the 5-day administration study, seizure observation was performed 4 times for 30 min (once before and 3 times after drug administration) during the 5-day drug-administration period, and continued once a day until 8 days after the final administration. The antiepileptic effects of 5-day administration of conventional AEDs (PHT, PB, VPA, and CBZ) were examined by using similar methods.
Results: Tonic convulsions and absence-like seizures were inhibited by a single administration of LEV at 80 and 160 mg/kg, i.p., but not significantly at 40 mg/kg, i.p. When LEV was repeatedly administered at 80 mg/kg/day, i.p., for 5 days to SERs, the inhibitory effects on seizures increased with administration time. The number of tonic convulsions and absence-like seizures were significantly reduced to 39.1% and 38.4% compared with previous values, respectively, after 5-day LEV administration. Furthermore, significant inhibition of tonic convulsions was detected ≤3 days after the final administration, and significant inhibition of absence-like seizures was still observed 8 days after the final injection of LEV. This demonstrates long-lasting seizure protection by LEV after cessation of treatment. PHT, PB, VPA, and CBZ inhibited tonic convulsions more potently compared with LEV in SERs. The maximal antiseizure effects of these drugs were reached after the initial administration, with almost the same antiseizure effects observed through day 5, despite continued drug administration. Moreover, a long-lasting treatment effect was not observed with any of these drugs except for PHT and CBZ, both of which showed moderately prolonged antiseizure effects.
Conclusions: These results show that LEV is effective in the treatment of both convulsive and absence-like seizures in SERs after single- and multiple-dose administration. Interestingly, in the 5-day administration study, it was found that the antiepileptic effects for tonic convulsions and absence-like seizures were observed both during the drug-administration period and ≤8 days after the final administration of LEV. This long-lasting effect suggests that LEV may possess an antiepileptogenic effect that it does not share with PHT, PB, VPA, and CBZ.
Levetiracetam (LEV) is a novel antiepileptic drug (AED) that has broad-spectrum effects on partial and generalized seizures in several animal models of epilepsy (1–3). The clinical effectiveness of LEV has been reported in patients with partial refractory epilepsy (4–7). Although LEV has no effect on acute maximal electroshock and pentylenetetrazol-induced seizures, potent protection has been observed in genetic and chronic epilepsy animal models (1,8). Furthermore, an antiepileptogenic effect of LEV has been reported in the kindling model in rats (8). Recently it was shown that the mechanism of action of LEV differs from that of conventional AEDs. LEV has no direct effect on targets for conventional AEDs, such as Na+ channel (9), T-type Ca2+ channels (9), γ-aminobutyric acid (GABA) (10–14), and glutamate receptors (15). Instead, inhibition of neuronal hypersynchrony (16), N-type Ca2+ channel inhibition (17,18), and binding to SV2A protein (19–21) have been reported for LEV. These results support a novel mechanism of action for LEV, encouraging study of the drug to try to characterize its antiepileptic properties further.
The spontaneously epileptic rat (SER: zi/zi, tm/tm) is a double mutant rat (22) obtained by mating the tremor heterozygous rat (tm/+) with the zitter homozygous rat (zi/zi, autosomal recessive). SER is sterile and exhibits both absence-like seizures and tonic convulsions after age 8 weeks with or without mild mechanical stimulation such as blowing air on to the animal's body (22,23). The tremor rat (tm/tm), showing tremors and absence-like seizures, was found in Kyoto Wister inbred colony by Serikawa and Yamada in 1985 (24). The Zitter rat, found by Rehm et al. (25) in 1982 also exhibits tremors but not epileptic seizures. The SER has a very short life and dies before age 16–18 weeks (26). The mechanism underlying epileptic seizures in SER is thought to include an abnormality of Ca2+ channels, an increase in extracellular glutamate concentrations, enhanced levels of N-acetyl-L-aspartate due to lack of the aspartoacylase gene, and lack of the attractin gene, which is involved in myelin formation (27–31).
The antiseizure profiles of conventional AEDs in SERs are quite similar to their efficacy profile in human epilepsy. Tonic convulsions in SERs are inhibited by phenytoin (PHT), absence-like seizures by ethosuximide (ESM) and trimethadione (TMO), and both types of seizures by valproate (VPA) and phenobarbital (PB) (32). In addition, the SER is a useful model for determining antiepileptic effects after repeated drug administration, as previously reported (26). For these reasons, we evaluated the antiepileptic effects of LEV in SERs and compared these effects with those of PHT, PB, VPA, and CBZ. We observed seizure expression both during the drug-treatment period and after the cessation of the treatment to evaluate the time course of seizure protection.
MATERIALS AND METHODS
A total of 49 male and female SERs, bred at the Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University, were used in these experiments. Each animal was kept in a cage in a room maintained at 23 ± 2°C and 55 ± 5% humidity under a 12-h light/dark cycle (light on at 6). They were provided standard rat chow (F-2; Funabashi Farm, Chiba, Japan) and tap water ad libitum.
All procedures were performed according to the guidelines for use of Laboratory Animals of Hiroshima University School of Medicine.
LEV was kindly supplied by UCB Pharma (Braine-l'Alleud, Belgium). PHT (Dainippon Pharmaceuticals, Osaka, Japan), PB (Sankyo, Tokyo, Japan), VPA (Dainippon Pharmaceuticals), and CBZ (Wako, Tokyo, Japan) were obtained commercially. Other reagents were also obtained commercially.
Under sodium pentobarbital (30 mg/kg) anesthesia, a silver-tipped and an enamel-coated stainless-steel electrode were permanently implanted in the left frontal cortex (∼3.0 mm lateral and 3.0 mm rostral to the bregma on the cranium) and in the left hippocampus (4.0 mm caudal and 2.0 mm lateral to the bregma and 3.0 mm from the cortical surface), according to the brain atlas of Paxinos and Watson (33). A reference electrode was fixed on the frontal cranium. After a 7-day recovery period, each animal was placed in a sound-proof box (40 × 40 × 40 cm) with a small window (11 × 6 cm) to allow behavioral observation. After 30-min habituation, EEG was recorded with a pen-writing polygraph (RM 6200; Nihon Kohden, Tokyo, Japan). Air-puff stimuli were applied to the faces of the SERs every 5 min for 30 min to induce tonic convulsions and to maintain alertness. A 5- to 7-Hz spike–wave complex lasting >1 s was regarded as an absence-like seizure. When the time interval between two spike–wave complexes was <1 s, the complexes were regarded as a single seizure. The numbers and total durations of the absence-like seizures were observed for a period of 30 min.
Single-dose study with levetiracetam
Fifteen SERs aged 9–12 weeks (150–250 g, 10 males, five females) exhibiting tonic convulsions were used in this study. LEV at doses of 40, 80, and 160 mg/kg, i.p., was injected and EEGs recorded for duration of 30 min 4 times (before the injection and 45–75, 75–105, and 135–165 min after the injection). The number of tonic convulsions and absence-like seizures induced by the stimuli (air puffs) every 5 min and occurring spontaneously during a 30-min period was recorded. The durations of tonic convulsions and absence-like seizures were measured to obtain the total duration of each seizure time during every 30-min observation period.
Repeated-dose study with levetiracetam
One 12-week-old male and three female SERs were used in this study. LEV (80 mg/kg/day, i.p.) was administered once daily between 10:00 and 11:00 a.m. for 5 successive days. EEG recordings for absence-like seizure and tonic convulsion observation were made 4 times every day as with to those in the single-dose study until 5 days of administration. Thereafter, recordings were made once daily for 30 min between 10 a.m. and noon for the ensuing 8 days after the final drug administration (day 13). Seizure numbers and duration were measured by using the same methods as in the single-dose study.
Effect of repeated-dose treatment with conventional antiepileptic drugs in SERs
Groups of five SERs aged 10–12 weeks were used in this study. PHT (20 mg/kg/day, i.p.), PB (10 mg/kg/day, i.p.), VPA (200 mg/kg/day, i.p.), and CBZ (25 mg/kg/day, i.p.) were injected daily between 10:00 and 11:00 a.m. for 5 days. Doses of PHT, PB, and VPA were selected according to a previous study (32). The dose of CBZ was selected based on previous rat and mouse studies (1). Because a few animals in the PHT and CBZ groups died before termination of the 5-day treatment period, the doses of both AEDs were reduced to 10 mg/kg/day. The number and duration of tonic convulsions induced by air puffs at 5-min intervals were measured for 30 min before and after administration of each drug in the same way as after LEV treatment. Seizure measurement between 45 and 75 min (for PHT and PB) or 15–45 min (for VPA and CBZ) after each injection for 5 days were made between 10:00 and 12:00 a.m. Furthermore seizures were measured at 24 h (day 6), 2, 3, 4, and 5 days after the final administration of each drug. Optimal pretreatment time was selected based on experience reported from previous studies (1,28).
Significant difference in the number and total duration of the seizures for 30 min between pre- and posttreatment times with the drugs was examined by using the paired Student's t test. The data obtained from both male and female SERs were analyzed together, because in a previous study, no sex differences were found in the animals for expression of absence-like seizures and tonic convulsions (26).
Single-dose study with levetiracetam
All 15 SERs showed tonic convulsions and absence-like seizures characterized by low-voltage fast waves and 5- to 7-Hz spike–wave-like complexes in the EEG (Fig. 1A and B). The mean number and duration of tonic convulsions was 5.4–5.8 times/30 min and 157.4–204.6 s/30 min (n = 5), respectively, and for absence-like seizures, 28.4–53.2 times/30 min and 39.22–77.0 s/30 min (n = 5), respectively (Figs. 2 and 3). When 40, 80, and 160 mg/kg of LEV was administrated to SERs, a significant inhibition of both tonic convulsions and absence-like seizures was observed (see Figs. 2 and 3). The inhibition of tonic convulsions was observed 75–105 min after the administration of 80 and 160 mg/kg. When LEV was administered at a dose of 80 mg/kg, the number of tonic convulsions was significantly reduced from 5.6 ± 0.2 to 3.6 ± 0.5 (64.3%; p < 0.05) and 3.0 ± 0.5 (53.6%; p < 0.01) at 75–105 and 135–165 min after drug administration, respectively (Fig. 2A). Increasing the dose to 160 mg/kg resulted in a significant reduction of the number of the tonic convulsions from 5.8 ± 0.2 to 3.6 ± 0.5 (62.1%; p < 0.05) and 2.2 ± 0.3 (37.9%; p < 0.01) at 75–105 and 135–165 min after the drug administration, respectively (Fig. 2A). The total duration of tonic convulsions was significantly decreased by a dose of 160 mg/kg at 75–105 and 135–165 min after drug injection (p < 0.05; Fig. 2B), although the inhibition was also observed 135–165 min after treatment with 80 mg/kg LEV. Interestingly, more-pronounced effects of LEV were observed 135–165 min after administration of LEV at a dose of 80 and 160 mg/kg, compared with those before 75–105 min after treatment (Fig. 2A and B). The onset of the inhibitory effects of LEV on absence-like seizures appeared earlier than that on the tonic convulsions: the inhibition was observed 15–45 min after the administration of a dose of 80 and 160 mg/kg (Fig. 3A and B). Both number and total duration of absence-like seizures were significantly decreased 15–45, 75–105, and 135–165 min after administration of LEV at 80 and 160 mg/kg, compared with control values (Fig. 3A and B).
Repeated-dose study with levetiracetam
The mean number of tonic convulsions was 5.75 ± 0.22 times/30 min (n = 4) before LEV injection (Fig. 4). When LEV at a dose of 80 mg/kg/day, i.p., was injected once daily for 5 successive days, the mean number of tonic convulsions was significantly reduced at 75–105 and 135–165 min after LEV administration on the first day (Fig. 4). The inhibitory effects on tonic convulsions increased gradually by repeated administration and became more prominent at day 5, the final administration day [45–75 min on day 5; 2.25 ± 0.54 times/30 min (p < 0.05)] (see Fig. 4). Furthermore, significant (p < 0.01) inhibition of the tonic convulsions was observed for 3 days after the final administration of LEV (see Fig. 4). The effect of LEV on the total duration of tonic convulsions was similar to that on the number of tonic convulsions (data not shown).
The mean number of absence-like seizures before LEV administration was 68.50 ± 9.34 times/30 min (n = 4) (Fig. 5). With injection of LEV at 80 mg/kg/day, i.p., an inhibitory effect on the number of absence-like seizures on day 1 was not significant; however, an effect became more prominent at day 5 [45–75 min on day 5; 26.3 ± 4.07 times/30 min (p < 0.05)], the final injection day, in a manner similar to the effect against tonic convulsions (see Fig. 5). Interestingly, the inhibitory effects of LEV on the number of absence-like seizures lasted at least until 8 days after the final administration. Indeed, a significant (p < 0.01) reduction in number and total duration of absence-like seizures was still observed 8 days after the final injection of the drug (day 13; see Fig. 5). The effect of LEV on the duration of absence-like seizures was similar to that on the number of absence-like seizures (data not shown).
Repeated-dose treatment with conventional antiepileptic drugs
As described in the Methods section, at the beginning of treatment with the conventional AEDs, death was observed in the PHT (20 mg/kg, i.p.) and CBZ (25 mg/kg, i.p.) groups. Therefore study doses for the PHT and CBZ groups were reduced to 10 mg/kg in an attempt to avoid further mortality. The effects of each drug on the number of tonic convulsions are shown in Figs. 6–9.
PHT induced a significant inhibition of tonic convulsions 45–75 min after each daily injection. The inhibitory effects of PHT on the number of tonic convulsions were significantly (p < 0.05) different from previous value for 4 days (day 9) after the final administration of drug (Fig. 6). However, this effect rapidly disappeared after drug discontinuation. The inhibition of tonic convulsions by PB was observed 45–75 min after each injection, but no inhibition was detected after termination of treatment (Fig. 7). Unlike the effects of PHT and LEV, the inhibitory effects of PB disappeared 1 day after the final injection (Fig. 7). The effects of VPA on tonic convulsions were similar to those of PB (Fig. 8), and inhibitory effects were not observed 1 day after the final injection of VPA (Fig. 8). Inhibitory effects of CBZ were observed at 15–45 min after each injection (Fig. 9). The inhibition induced by CBZ was detected the next day after each daily administration and after the final drug injection, but not 3 days later (Fig. 9). The effect of these four conventional AEDs on the duration of tonic convulsions was similar to that on the number of tonic convulsions (data not shown).
The mean days that the mean number of tonic convulsion increased/returned to 50% of baseline levels after treatment discontinuation are shown in Table 1. The days until the return to 50% level of the baseline in LEV treatment were 4.0 ± 0.4 days (n = 4), whereas those of conventional AEDs were <1.8 days (see Table 1). LEV was clearly different from conventional AEDs by a long-lasting antiseizure effect after termination of treatment.
|AEDs||Return to 50% of baseline (days ± 95% confidence interval)|
|Levetiracetam||4.0 ± 0.4|
|Phenytoin||1.8 ± 0.5|
|Carbamazepine||1.5 ± 0.3|
LEV was found to be effective in inhibiting both convulsive and absence-like seizures in SERs by single and 5-day multiple administrations. The inhibitory effects on both seizure types gradually increased by repeated administration. Furthermore, the antiseizure effects of LEV were strikingly long-lasting, being observed <8 days after cessation of 5-day prolonged administration. Such long-lasting seizure protection appears not to reflect an accumulation of the drug in plasma or tissue, because plasma half-life (t1/2) of LEV is 2–3 h in rat (8,12,34,35). Tmax of LEV in plasma is 0.25–0.7 h (8,12,34,35). The time to peak concentration of LEV in the brain is slightly delayed (12,34); however, almost all drug in the brain disappears within 24 h after a single administration, as studied by using [14C]LEV. Furthermore, one major (ucb L057) and two minor metabolites of LEV have no effects on epileptic seizures (36). Both plasma and brain LEV concentrations reach steady state easily (37), and the steady state is preserved for a long time. No accumulation of the drug has been observed by multiple administration, because LEV does not induce hepatic enzymes and is not metabolized in the liver (38,39). Pharmacokinetic differences were not observed between single- and repeated-administration studies. Therefore after cessation of treatment, the drug rapidly disappears from tissue. Instead, it could be considered that the long-lasting seizure inhibition by LEV is due to antiepileptogenic effects of the drug. Biologic changes induced by LEV may occur duringthe administration period (from day 1 to day 5), and could remain after drug cessation (from day 6 to day 8 for tonic convulsions and from day 6 to day 13 for absence-like seizures). Antiepileptogenic effects of LEV in addition to antiepileptic effects have been reported in the amygdala-kindling rat model (8). Indeed, LEV was reported to suppress kindling acquiring in this model. Together with the observation of the present study, it appears that the drug may modulate the epileptogenic process, potentially inducing expression of inhibitory factors via effects on gene expression. In that context, it is interesting that the present study observed that the antiseizure effect of LEV did not reach maximum on the first day, but increased with repeated administration (Fig. 4).
An absence of long-lasting pharmacologic effect of CLB has previously been reported in SERs (40). In the present study, we also examined the duration of anticonvulsant effect of conventional AEDs (PHT, PB, VPA, and CBZ) after termination of 5-day repeated-dose treatment. At the beginning of this study, death was observed in the PHT (20 mg/kg/day) and CBZ (25 mg/kg/day) groups. Therefore the doses of PHT and CBZ were reduced to a dose of 10 mg/kg/day. Death was not observed in the previous PHT study at 20 mg/kg/day (32). However, SERs may be sensitive to some drugs, and furthermore SERs have a relatively short life, and death was observed at 13 weeks in a previous study devoid of drug administration (22). We cannot exclude the possibility that the SER rats are metabolically and pharmacologically different from other rat species. Because SERs have a long deletion on chromosome 10q (30), some enzymes or proteins for the gene coded in that deleted region must be very low or depleted. The anticonvulsive effects of the four conventional AEDs tested were very strong and potent compared with that of LEV, and maximal seizure protection was observed on the first treatment day. PB and VPA strongly inhibited tonic seizures; however, these inhibitory effects were not observed the day after the last injection; they were effective in inhibiting seizures only a few hours after repeated administration. These results suggest that PB and VPA directly act on the neurons and/or neural networks involved in seizures to inhibit them but do not have antiepileptogenic effects. However, Silver et al. (41) reported the antiepileptogenic effect of PB and VPA on electrical-kindling development in rats. The difference between these results may be attributed to the difference in the animal models: kindling and genetic models. In contrast to the effects of PB and VPA, the inhibitory effects of PHT and CBZ on tonic convulsions lasted for 4 and 1 day(s) after the final administration of the 5-day regimen, respectively. However, these effects disappeared rapidly compared with those of LEV. These relatively long-acting effects of CBZ are considered to be due to a direct action of the drug, because they appears to correlate to the relatively long t1/2 of the drug and the fact that the main metabolite of the drug (CBZ-10,11-epoxide) also has antiseizure effects (42,43). The plasma half-life of CBZ was reported to be ∼8 to 15 h in rats (44,45), although reports of shorter half-life also exist (43,46–48). Although the possibility that PHT may have antiepileptogenic effects cannot be completely excluded, it seems unlikely, because the drug has been reported not to inhibit seizure development in the rat amygdala kindling model (49). The plasma half-life of PHT after oral administration was reported to be 6.98 ± 0.60 h (50) or 3.60 ± 0.48 h (51) in rats.
Some reports show a long-lasting pharmacologic effect of LEV in humans (52–54). LEV is rapidly and almost completely absorbed after single and multiple oral administrations in humans (39). Plasma peak levels are generally reached within 1 h after administration (39,55). The half-life of LEV in humans ranges between 7 and 8 h (39,55). Kasteleijn-Nolst et al. (52) showed that LEV had a long-lasting effect on the suppression of photosensitive epilepsy. Inhibition of photosensitive epileptiform discharges by single oral administration of LEV lasted >6 h and ≤30 h in some patients (52). Sohn et al. (53) observed a suppressive effect of LEV on cortical excitability in healthy humans. These effects persisted until 6 h and were still present at 24 h. These observations are consistent with our results and support the conclusion that LEV induces long-lasting seizure suppression devoid of relation to its pharmacokinetic profile.
The antiepileptic mechanism of LEV is not fully elucidated; however, it does not involve effects on traditional targets, such as Na+ channels (9), T-type Ca2+ channels (9), the GABAergic systems (10–14), or glutamate receptors (15). LEV does, however, inhibit neuronal hypersynchrony (16) and N-type Ca2+ channels (17,18). More recently, a LEV-binding protein SV2A (synaptic vesicle protein 2A) was discovered (19–21). This is a membrane glycoprotein common to all synaptic vesicles (56–61). The biophysiologic role of SV2A remains to be determined. However, SV2A has an important functional role in the modulation of the synaptic vesicle cycle and neurotransmitter release into the synaptic cleft, because mice lacking SV2A fail to grow, experience severe seizures, and die within 3 weeks (62). A clear correlation was found between the affinity of LEV and its analogues to SV2A and the potency of their seizure protection in the mouse audiogenic model of epilepsy (20,63). It could be speculated that this novel mechanism may contribute to the potential antiepileptogenic properties of LEV, as reflected in the long-lasting seizure suppression observed in the present study.
In conclusion, this study observed that LEV exhibits unique characteristics that are seen as a long-lasting effect on tonic convulsions and absence-like seizures in SERs, supporting the idea that the drug may possess antiepileptogenic properties.
Acknowledgment: This work was supported by a grant from UCB S.A. Belgium. We thank Dr. Henrik Klitgaard (UCB S.A.) for his advice and levetiracetam supply, and Drs. Alan Matagne and Etienne Hanon (UCB S.A.) for their assistance with the statistical analysis.
- 15Levetiracetam: no relevant effect on ionotropic excitatory glutamate receptors. Epilepsia 2000;41(suppl 7):35., , , et al.
- 40Effects of clobazam on epileptic seizures in spontaneous epileptic rat (SER). Jpn J Pharmacol Ther 2000;28: 259–65., , , et al.