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European Journal of Pharmacology254 (1994) 83-89
Inhibition by topiramate of seizures in spontaneously epileptic rats and D B A / 2 mice Joji Nakamura
a
,,a,
S a c h i k o T a m u r a a, T o m o y u k i K a n d a a, A k i o I s h i i a, K u m a t o s h i T a d a o S e r i k a w a c, J u n z o Y a m a d a c, M a s a s h i S a s a d
I s h i h a r a b,
Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411, Japan b Department of Pharmacology and c Institute for Laboratory Animals, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan d Department of Pharmacology, School of Medicine, Hiroshima University, Hiroshima 734, Japan
(Received 8 July 1993;revised MS received 2 November 1993;accepted 14 December 1993)
Abstract
The effects of topiramate, a novel antiepileptic drug, on tonic and absence-like seizures in spontaneously epileptic rats (SER; z i / z i , tm ~tin) and on sound-induced seizures in DBA/2 mice were investigated. Topiramate (20 and 40 mg/kg i.p.) inhibited
both tonic and absence-like seizures in a dose-dependent manner, whereas phenytoin (20 mg/kg i.p.) and zonisamide (40 mg/kg i.p.) inhibited only the tonic seizures. The inhibitory effects of topiramate on absence-like seizures were antagonized by pretreatment with haloperidol (0.5 mg/kg i.p.), but those on the tonic seizures remained unaffected. Topiramate inhibited sound-induced seizures in DBA/2 mice (EDs0 = 8.6 mg/kg p.o.). These findings suggest that topiramate may be effective for treatment of both convulsive and absence seizures of human epilepsy. The inhibitory effect of topiramate on absence-like seizures in SER may be mediated through the central dopaminergic system. Key words: Topiramate; Spontaneously epileptic rat (SER); Antiepileptic effect; Audiogenic seizure
I. Introduction
Despite the abundance of available antiepileptic drugs, some patients suffer from epileptic seizures which cannot be controlled by conventional antiepileptics. Therefore, novel antiepileptic drugs which can control such refractory epilepsy are highly desirable. Topiramate, a novel anticonvulsant, structurally distinct from known anticonvulsants (Fig. 1), reportedly inhibits convulsions induced by electroconvulsive shock in mice and rats without effects on pentylenetetrazol-, picrotoxin- and bicuculline-induced seizures (Maryanoff et al., 1987; Tanabe et al., 1991). Preliminary studies showed that topiramate is effective in treatment of human refractory partial seizures (Wilder et al., 1988; Aranguiz et al., 1991). However, there is little information regarding the effects of topiramate in animal models of epilepsy. Thus, we investigated the
effect of topiramate on convulsive and absence-like seizures in spontaneously epileptic rats (SER), and on sound-induced seizures in D B A / 2 mice. SER is an epileptic double mutant ( z i / z i , t m / t m ) obtained by mating a heterozygote ( t m / + ) tremor rat, a mutant found in the inbred colony of Kyo:Wistar rats (Yamada et al., 1985), with a homozygous zitter rat ( z i / z i ) found in a Sprague-Dawley colony by Rehm et al. (1982). After 8 - 9 weeks of age, this animal shows tonic convulsions without external stimuli, and with mild stimuli such as hand clap, touch and air blowing. This animal also exhibits absence-like seizures charac-
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Fig. 1. Chemical structure of topiramate.
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J. Nakamura et al. / European Journalof Pharmacology254 (1994)83-89
terized by sudden immobility and staring concomitantly with appearance of 5-7-Hz spike-wave-like complexes in the EEG (Serikawa and Yamada,1986; Sasa et al., 1988a). The tonic and absence-like seizures of this animal appear, respectively to correspond to generalized tonic-clonic seizures ('grand mal' epilepsy) and absence seizures ('petit mal' epilepsy) in humans, because the tonic seizures in the SER are inhibited by phenytoin, whereas the absence-like seizures are inhibited by trimethadione and ethosuximide, and both seizures are blocked by phenobarbital and valproate (Sasa et al., 1988a). The D B A / 2 mouse is a well-known sound-induced seizure model, and shows clonic convulsions induced by sound stimuli at around 3 weeks of age (Schlesinger et al., 1965). These animals were used for evaluation of the effects of topiramate, since the SER and DBA/2 mice are known to be useful for determining the acute and long-term effects of antiepileptic drugs (Chapman et al., 1984; Sasa et al., 1988a; Ujihara et al., 1991).
together with simultaneous behavioral observation, since EEG activity during the tonic and absence-like seizures always changed in accordance with the respective behavioral changes, as described previously (Sasa et al., 1988a). A mild tactile stimulus was given on the back of the animal every 5 min during recording of EEG to induce tonic seizures consistently and to keep the rat awake. Animals were used for evaluation of the effects of drugs after 11 weeks of age when they usually exhibited tonic and absence-like seizures for a total duration of more than 40 and 50 s, respectively, for a 15-min control period. After 15-min control recording, each animal was given a single dose of an anticonvulsant, vehicle, or a combination of topiramate with haloperidol once a day. The EEG of the animals was recorded 15-30, 45-60, 75-90 and 105-120 rain after injection of the drug or vehicle. To examine the antagonistic effects of haloperidol, topiramate was given 15 min after haloperidol administration. The experiments were done between 9:00 and 17:00 h. A higher dose of a drug was administered to the same animal after an interval of at least 1 week for wash-out.
2. Materials and methods
2.1.3. Data analysis The duration of each seizure was measured, and the total duration of the seizures for a 15-rain period was calculated before and after injection of the drug. As for the tonic seizures, the durations of both tactile stimulation-induced and spontaneous convulsions were measured. An appearance of 5-7 Hz spike and wave complex in EEG lasting over 1 s was regarded as an absence-like seizure. The statistical significance of the difference between the means of the total duration in the vehicle-treated group and the drug-treated group as well as those of the topiramate-treated group in the absence and presence of haloperidol pretreatment was determined with Kruskal-Wallis' test or a one-way analysis of variance (ANOVA) followed by ScheffE-type non-parametric test or ScheffE's parametric test, respectively, according to whether a statistically significant difference existed or not in the variances of groups. The statistical differences of variances were tested by Bartlett's test, because these experiments involved three or more groups. In the experiment which determined the duration of action of topiramate, MannWhitney's U-test was used, because the variances of the vehicle- and the topiramate-treated group were not equal according to the F-test. The statistical difference between before (control) and after drug treatment was evaluated using a paired t-test.
2.1. Spontaneously epileptic rats 2.1.1. Animals SER were bred in our institute as reported previously (Serikawa and Yamada, 1986) and kept individually in pail type cages in the animal quarters. Commercial food pellets (F-2, Funabashi Farm, Japan) and water were given ad libitum. Room temperature and relative humidity were kept at 23-25°C and 50-60%, respectively. The room was illuminated for 12 h, from 7:00 to 19:00 h. 2.1.2. E E G recordings Fourteen male and 14 female SER between 11 and 17 weeks old were used. Under chloral hydrate (300 mg/kg i.p.) or sodium pentobarbital anesthesia (40 mg/kg i.p.), bipolar electrodes were chronically implanted in the bilateral frontal cortex and left hippocampus (P, 4.3; L, 2.7; H, 2.4 from bregma) according to the atlas of Paxinos and Watson (1986). An indifferent electrode was placed on the frontal cranium. The animal was placed in a sound-attenuated box (40 X 40 X 40 cm) with a small window for behavior observation, 7-10 days after implantation of the electrodes. Then, an electroencephalogram (EEG) was recorded on a pen-writing electroencephalograph (Nihon Kohden, EEG5109) or a polygraph (Nihon Kohden, RMC-1200) for 15 rain after 60-rain habituation as the control. The duration of tonic and absencelike seizures was measured from the EEG pattern
2.2. DBA / 2 mice
2.2.1. Animals Male D B A / 2 mice (SLC) aged 21-22 days were used. Commercial food pellets (F-2, Funabashi Farm,
J. Nakamura et al. ~European Journal of Pharmacology 254 (1994) 83-89
Japan) and water were given ad libitum. Room temperature and relative humidity were kept at 23-25°C and 50-60%, respectively. The room was illuminated for 12 h, from 7:00 to 19:00 h.
2.2.2. Induction of sound-induced seizure Mice were placed in a cylinder (10 cm diameter X 9 cm high) after drug administration, and exposed to sound (140 dB) for 60 s. Drugs were applied 120 min prior to the application of the sound, since the peak time for anticonvulsive effects in the maximal electroconvulsive shock test for topiramate, zonisamide, phenytoin and phenobarbital was approximately 120 min. The pretreatment time for carbamazepine and valproate was 30 min. 10-20 mice were used for one dose of the drugs, and the protection of the drugs against clonic seizures was examined. The EDs0 values and 95% confidence limits were determined by probit analysis.
85
2.3. Drugs In the SER, topiramate (2,3 : 4,5-bis-O-(1-methylethylidene)-/3-D-fructopyranose sulfamate (R.W. Johnson Pharmaceutical Research Institute, Spring House, PA, USA) and sodium phenytoin (Dainippon Seiyaku, Osaka, Japan) were administered as a solution in a vehicle composed of 40.0% propylene glycol, 10.5% ethanol and 49.5 % distilled water. Zonisamide (Dainippon Seiyaku, Osaka, Japan) was dissolved in distilled water as the sodium salt. An ampule form of haloperidol (Serenace, Dainippon Seiyaku, Osaka, Japan) was used as a non-selective dopamine receptor blocker. This 1-ml ampule contains haloperidol, 5 mg (pH 3.5-4.2). The haloperidol solution was diluted with the same amount of distilled water and used. These drugs were injected intraperitoneally to the SER. In D B A / 2 mice, drugs were administered orally as a 0.3% sodium carboxymethylcellulose solution or sus-
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86
J. Nakamura et aL/ European Journal of Pharmacology 254 (1994) 83-89
pension. The drugs used for D B A / 2 mice were topiramate (R.W. Johnson Pharmaceutical Research Institute, Spring House, PA, USA), phenytoin, carbamazepine, sodium phenobarbital (Wako, Osaka, Japan), zonisamide (Dainippon Seiyaku, Osaka, Japan) and sodium valproate (Kyowa Hakko, Tokyo, Japan).
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3.1. Spontaneously epileptic rats Time after administration (min) Tonic convulsions and absence-like seizures, characterized by the sudden appearance of 5-7-Hz spikewave-like complexes in the E E G of the cerebral cortex and hippocampus, occurred in all twenty-eight SER (Fig. 2). The mean total duration of tonic convulsion (including both spontaneously and tactile stimulationinduced seizures) and absence-like seizures for 15 min in 28 SER was 72.3 + 3.8 and 119.6 + 18.8 s (mean + S.E.M.), respectively. The vehicles had no effects on the tonic and absence-like seizures in SER. The mean total duration of tonic and absence-like seizures for 15-30 min after injection of the vehicle used for topiramate and phenytoin was 83.4 + 9.9% ( P = 0.12) and 126.1 + 17.1% ( P =0.16) ( n = 9 ) of the control value, respectively, whereas the corresponding duration of seizures for distilled water (zonisamide vehicle) was 115.1 + 14.4% ( P = 0.42) and 84.8 + 10.3% ( P = 0.13) (n = 5), respectively.
3.2. Effects of topiramate and other antiepileptic drugs I.p. injection of topiramate at doses of 5, 10 or 20 m g / k g did not induce any overt behavioral or E E G changes. However, topiramate at a dose of 40 m g / k g increased locomotor activity of the SER in the shield box as visually observed, but caused no obvious changes in background EEG. Compared to those of the vehicle, the overall effects of topiramate were highly significant (tonic seizure: Kruskal-Wallis' test P < 0 . 0 1 ; absence-like seizure: Kruskal-Wallis' test P < 0.01). Topiramate at doses of 20 and 40 m g / k g i.p. produced a dose-dependent inhibition of both the tonic and absence-like seizures (Figs. 2, 3 and 4). The inhibitory effects were observed from 15 min after injection and the maximum effects were seen 15-30 min later (Fig. 3). Phenytoin dose dependently inhibited tonic seizures (ANOVA F(3,19) = 9.3, P < 0.01) without affecting absence-like seizures as reported previously (Sasa et al., 1988a) (Fig. 4). Significant inhibition ( P < 0.01) of the tonic seizure was observed with 20 m g / k g of phenytoin, although the absence-like seizure was not affected by 20 mg/kg. The maximum effects were observed
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Time after administration (min) Fig. 3. The time course of the inhibitory effects of topiramate (20 mg/kg i.p.) on the total duration of (A) tonic and (B) absence-like seizures. The percent of control was obtained by dividing each value after drug or vehicle injection by that before injection. Each point and bar represents the mean and S.E.M., respectively (n = 9 for 15-30, 45-60 min; n = 5 for 75-90, 105-120 min). (e) and (o): topiramate and vehicle, respectively.** P < 0.01, as compared with vehicle-injectedgroup.
15-30 min after injection, and the effect lasted for more than 60 min. Zonisamide dose dependently inhibited tonic seizures (Kruskal-Wallis' test P < 0.01), but 40 m g / k g of the drug did not affect the absence-like seizures. Zonisamide significantly ( P < 0.05) inhibited tonic seizures at 40 m g / k g (Fig. 4). The peak time of effects was seen 15-30 min after the injection.
3.3. Antagonizing effect of haloperidol Haloperidol (0.5 m g / k g i.p.) alone had no significant effects on the absence-like seizures ( P = 0.32) or tonic seizures ( P = 0.32) as reported previously (Ujihara et al., 1991). The mean total duration of tonic and absence-like seizures during 15-min periods were 74.8 + 11.1 and 97.8 + 12.7 s (n = 5) before administration, and 63.2 + 9.2 and 116.2 + 21.7 s 30-45 min after injection of haloperidol. However, when haloperidol was given 15 min before injection of topiramate (20 m g / k g i.p.), the inhibition of the absence-like seizures by
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J. Nakamura et aL / European Journal of Pharmacology 254 (1994) 83-89
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Fig. 4. Effects of topiramate, phenytoin and zonisamide on the total duration of tonic and absence-like seizures. The total duration of both seizures was measured for 15 min before drug injection and 15-30 min after drug injection. Each value is shown as a percentage of the values obtained for 15 min before drug injection. Columns and bars represent mean and S.E.M. (n = 4-9). Solid and open columns represent tonic and absence-like seizures, respectively. *P < 0.05 and ** P < 0.01, as compared with vehicle-injected group.
Table 1 Effects of topiramate and other antiepileptic drugs on sound-induced seizures in DBA/2 mice EDs0 and 95% confidence limits (mg/kg p.o.) Drugs Clonic seizures Tonic convulsions Topiramate Phenytoin Zonisamide Carbamazepine Phenobarbital Valproate
8.6 (6.1-12.0) 1.0 (0.5- 1.6) 31.4 (24.3-42.6) 2.0 (0.8- 4.6) 1.7 (1.0- 2.5) 42.5 (28.6-61.8)
3.5 (2.6-4.4) 0.61 (0.38-0.91) 13.4 (11.2-16.0) 1.0 (0.6- 1.6) 1.3 (0.7- 2.0) 42.5 (28.6-61.8)
When sound stimuli were given for 60 s, abnormal running behavior, clonic seizures and tonic convulsions were induced in 5 0 / 5 0 (100%), 4 4 / 5 0 (88%) and 4 2 / 5 0 (84%) of the animals, respectively. All mice that exhibited tonic convulsions died. Topiramate blocked the sound-induced clonic seizure with an EDs0 of 8.6 m g / k g (Table 1). Tonic convulsions and death did not occur at doses greater than 12.5 m g / k g in any of the 20 animals examined. The EDs0 for inhibiting tonic convulsions was 3.5 m g / k g (Table 1). Phenytoin, zonisamide, carbamazepine, phenobarbital and valproate also dose dependently protected against sound-induced clonic seizures and tonic convulsions (Table 1). Tonic convulsions and death were completely prevented by phenytoin, zonisamide, carbamazepine, phenobarbital and valproate at doses over 3.13, 50, 12.5, 6.25 and 200 m g / k g , respectively.
4. Discussion The present study demonstrated that topiramate inhibits both tonic and absence-like seizures in SER. In our previous study, tonic and absence-like seizures appeared to correspond to generalized tonic-clonic seizures ('grand mal' epilepsy) and simple absence seizures ('petit mal' epilepsy) in human epilepsy, based on the responsiveness of these seizures to the conventional antiepileptic drugs (Sasa et al., 1988a), although
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J. Nakamura et al. / European Journal of Pharmacology 254 (1994) 83-89
the spikes were not usually observed during the tonic seizure except in some cases following wild running. Tonic seizures were inhibited by phenytoin and carbamazepine, whereas absence-like seizures were inhibited by trimethadione and ethosuximide, and both types of seizures were inhibited by phenobarbital, valproate and diazepam (Sasa et al., 1989). Furthermore, chronic administration of phenobarbital inhibited tonic seizures without affecting absence-like seizures, as has been reported for human epilepsy (Serikawa et al., 1990). Since topiramate blocked both types of seizures in SER, it may be effective for treating both convulsive and absence seizures in human epileptic patients. Topiramate 40 mg/kg, phenytoin 20 mg/kg and zonisamide 40 mg/kg inhibited tonic seizures almost completely. The potency of topiramate for inhibition of tonic seizures was similar to that of phenytoin and of zonisamide, since the doses described above were 118, 73 and 171/xmol/kg. However, the latter two anticonvulsants were ineffective to block absence-like seizures of the SER. The effectiveness of topiramate as an anticonvulsant was further supported by its ability to prevent audiogenic seizures in DBA/2 mice, although sound-induced seizures in D B A / 2 mice do not discriminate between the clinical profiles of the antiepileptic drugs (Chapman et al., 1984). The ratio of the EDs0 for impairment of neurological function (rotarod test) to the EDs0 for protection against audiogenic seizures for topiramate exceeded those of phenobarbital and valproate which inhibit both tonic and absence-like seizures in SER (unpublished data). Recently, Hara et al. (1993) have reported that the dopamine content in SER is lower than that of control animals, suggesting that a decrease in dopamine is one of the factors involved in the seizures. We have also demonstrated that dopamine agonists such as apomorphine inhibit both tonic and absence-like seizures in SER (Sasa et al., 1988b). Moreover, thyrotropin-releasing hormone, known to release catecholamines, inhibited the seizures of the SER (Ujihara et al., 1991; Sasa et al., 1989). The inhibitory effects of topiramate on absence-like seizures of the SER were antagonized by haloperidol, a dopamine receptor antagonist. Taken together with these observations, our finding suggests that activation of dopaminergic systems by topiramate is one of the factors involved in the inhibition of these seizures, although the contribution of noradrenergic neurons cannot be completely excluded because of haloperidol possessing a weak al-adrenoceptor antagonistic effect. These findings are also in line with the observation that the anticonvulsant effects of this drug on maximal electroconvulsive shock in mice were attenuated by pretreatment with reserpine (Maryanoff et al., 1987). Since topiramate has no affinity to dopamine D 1 o r
D 2 receptors and no effects on dopamine uptake (Shank et al., 1991), topiramate may enhance dopaminergic activity by promoting the synaptic release of dopamine. The anticonvulsant effect of topiramate on tonic seizures of the SER was not affected by haloperidol. This suggests that the suppressive effect of topiramate on tonic seizures is mainly due to a non-dopaminergic mechanism, although dopamine-mediated anticonvulsant effects may play some role, as thyrotropin-releasing hormone was also effective to inhibit tonic seizures of the SER (Ujihara et al., 1991). We reported that the extracellular concentration of glutamate and aspartate is significantly elevated in the hippocampus of SER (Kanda et al., 1992), and that topiramate restores these elevated levels to normal values (Kanda et al., 1992). This suggests that glutamatergic and aspartatergic neuronal pathways may be involved in the SER seizures in addition to activation of the catecholaminergic system. The anticonvulsant activity of topiramate may simultaneously be related to inhibitory effects on excitatory amino acid neurons. In conclusion, topiramate has antiepileptic effects on both convulsive and absence-like seizures and the inhibitory effect on absence-like seizures in SER may be mediated through the central dopaminergic system.
5. References Aranguiz, C., J. McJilton, M. Vega and R.E. Ramsay, 1991, Safety and effectiveness of three oral doses of topiramate in the treatment of patients with refractory partial epilepsy, Epilepsia 32, Suppl. 3, 11. Chapman, A.G., M.J. Croucher and B.S. Meldrum, 1984, Evaluation of anticonvulsant drugs in D B A / 2 mice with sound-induced seizures, Arzneim.-Forsch./Drug Res. 34, 1261. Hara, M., M. Sasa, A. Kawabata, T. Serikawa, J. Yamada and S. Takaori, 1993, Decreased dopamine and increased norepinephrine levels in the spontaneous epileptic rat, a double mutant rat, Epilepsia 34, 433. Kanda, T., J. Nakamura, M. Kurokawa, S. Tamura, A. Ishii, K. Ishihara, T. Serikawa, J. Yamada and M. Sasa, 1992, Inhibition of excessive release of excitatory amino acids in hippocampus of spontaneously epileptic rat (SER) by topiramate (KW-6485), Jpn. J. Pharmacol. 58, Suppl. I, 92P. Maryanoff, B.E., S.O. Nortey, J.F. Gardocki, R.P. Shank and S.P. Dodgson, 1987 Anticonvulsant O-alkyl sulfamates. 2,3:4,5-bis-O(1-methylethylidene)-fl-D-fructopyranose sulfamate and related compounds, J. Med. Chem. 30, 880. Paxinos, G. and C. Watson, 1986, The Rat Brain in Stereotaxic Coordinates (Academic Press, Sydney). Rehm, S., P. Mehraein, A.P. Anzil and F. Deerberg, 1982, A new rat mutant with defective overhairs and spongy degeneration of the central nervous system: clinical pathological studies, Lab. Anim. Sci. 32, 70. Sasa, M., Y. Ohno, H. Ujihara, Y. Fujita, M. Yoshimura, S. Takaori, T. Serikawa and J.Yamada, 1988a, Effects of antiepileptic drugs on absence-like and tonic seizures in the spontaneously epileptic rat, a double mutant rat, Epilepsia 29, 505. Sasa, M., H. Ujihara, Y. Fujita, M. Yoshimura, S. Takaori, T.
J. Nakamura et al. / European Journal of Pharmacology 254 (1994) 83-89 Serikawa and J. Yamada, 1988b, Pharmacological studies using genetically defined epilepsy model animal, spontaneously epileptic rat (SER): role of dopaminergic system in controlling absence-like seizure and tonic convulsion, Jpn. J. Pharmacol. 46 (Suppl.), 37P. Sasa, M., H. Ujihara, K. Ishihara, Y. Ohno, Y. Fujita, M. Yoshimura, J. Nakamura, T. Serikawa, J. Yamada and S. Takaori, 1989, Responsiveness of the spontaneously epileptic rat (SER), a double mutant, to antiepileptic drugs, Adv. Neurol. Sci. 33, 909. Schlesinger, K., W. Boggan and D.X. Freedman, 1965, Genetics of audiogenic seizures. 1. Relation to brain serotonin and norepinephrine in mice, Life Sci. 4, 2345. Serikawa, T. and J. Yamada, 1986, Epileptic seizures in rats homozygous for two mutations, zitter and tremor, J. Hered. 77, 441. Serikawa, T., K. Kogishi, J. Yamada, Y. Ohno, H. Ujihara, Y. Fujita, M. Sasa and S. Takaori, 1990, Long-term effects of continual intake of phenobarbital on the spontaneously epileptic rat, Epilepsia 31, 9.
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Shank, R.P., J.L. Vaught, R.B. Raffa and B.E. Maryanoff, 1991, Investigation of the mechanism of topiramate's anticonvulsant activity, Epilepsia 32, Suppl. 3, 7. Tanabe, K., Y. Wang and K. Kimishima, 1991, Anticonvulsant activities and properties of topiramate, J. Yonago Med. Ass. 42, 330. Ujihara, H., R. Renming, M. Sasa, K. Ishihara, Y. Fujita, M. Yoshimura, T. Kishimoto, T. Serikawa, J. Yamada and S. Takaori, 1991, Inhibition by thyrotropin-releasing hormone of epileptic seizures in spontaneously epileptic rats, Eur. J. Pharmacol. 196, 15. Wilder, B.J., J.K. Penry, R.J. Rangel, A. Riela, B.L. Margul and T.B. Marriott, 1988, Topiramate: efficacy, toxicity, and dose/plasma concentrations, Epilepsia 29, 698. Yamada, J., T. Serikawa, J. Ishiko, T. Inui, H. Takeda, Y. Kawai and A. Okaniwa, 1985, Rats with congenital tremor and curled whisker and hair, Exp. Anim. 34, 183.
European Journal of Pharmacology254 (1994) 83-89
Inhibition by topiramate of seizures in spontaneously epileptic rats and D B A / 2 mice Joji Nakamura
a
,,a,
S a c h i k o T a m u r a a, T o m o y u k i K a n d a a, A k i o I s h i i a, K u m a t o s h i T a d a o S e r i k a w a c, J u n z o Y a m a d a c, M a s a s h i S a s a d
I s h i h a r a b,
Pharmaceutical Research Laboratories, Kyowa Hakko Kogyo Co., Ltd., 1188 Shimotogari, Nagaizumi-cho, Sunto-gun, Shizuoka 411, Japan b Department of Pharmacology and c Institute for Laboratory Animals, Faculty of Medicine, Kyoto University, Sakyo-ku, Kyoto 606, Japan d Department of Pharmacology, School of Medicine, Hiroshima University, Hiroshima 734, Japan
(Received 8 July 1993;revised MS received 2 November 1993;accepted 14 December 1993)
Abstract
The effects of topiramate, a novel antiepileptic drug, on tonic and absence-like seizures in spontaneously epileptic rats (SER; z i / z i , tm ~tin) and on sound-induced seizures in DBA/2 mice were investigated. Topiramate (20 and 40 mg/kg i.p.) inhibited
both tonic and absence-like seizures in a dose-dependent manner, whereas phenytoin (20 mg/kg i.p.) and zonisamide (40 mg/kg i.p.) inhibited only the tonic seizures. The inhibitory effects of topiramate on absence-like seizures were antagonized by pretreatment with haloperidol (0.5 mg/kg i.p.), but those on the tonic seizures remained unaffected. Topiramate inhibited sound-induced seizures in DBA/2 mice (EDs0 = 8.6 mg/kg p.o.). These findings suggest that topiramate may be effective for treatment of both convulsive and absence seizures of human epilepsy. The inhibitory effect of topiramate on absence-like seizures in SER may be mediated through the central dopaminergic system. Key words: Topiramate; Spontaneously epileptic rat (SER); Antiepileptic effect; Audiogenic seizure
I. Introduction
Despite the abundance of available antiepileptic drugs, some patients suffer from epileptic seizures which cannot be controlled by conventional antiepileptics. Therefore, novel antiepileptic drugs which can control such refractory epilepsy are highly desirable. Topiramate, a novel anticonvulsant, structurally distinct from known anticonvulsants (Fig. 1), reportedly inhibits convulsions induced by electroconvulsive shock in mice and rats without effects on pentylenetetrazol-, picrotoxin- and bicuculline-induced seizures (Maryanoff et al., 1987; Tanabe et al., 1991). Preliminary studies showed that topiramate is effective in treatment of human refractory partial seizures (Wilder et al., 1988; Aranguiz et al., 1991). However, there is little information regarding the effects of topiramate in animal models of epilepsy. Thus, we investigated the
effect of topiramate on convulsive and absence-like seizures in spontaneously epileptic rats (SER), and on sound-induced seizures in D B A / 2 mice. SER is an epileptic double mutant ( z i / z i , t m / t m ) obtained by mating a heterozygote ( t m / + ) tremor rat, a mutant found in the inbred colony of Kyo:Wistar rats (Yamada et al., 1985), with a homozygous zitter rat ( z i / z i ) found in a Sprague-Dawley colony by Rehm et al. (1982). After 8 - 9 weeks of age, this animal shows tonic convulsions without external stimuli, and with mild stimuli such as hand clap, touch and air blowing. This animal also exhibits absence-like seizures charac-
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Fig. 1. Chemical structure of topiramate.
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terized by sudden immobility and staring concomitantly with appearance of 5-7-Hz spike-wave-like complexes in the EEG (Serikawa and Yamada,1986; Sasa et al., 1988a). The tonic and absence-like seizures of this animal appear, respectively to correspond to generalized tonic-clonic seizures ('grand mal' epilepsy) and absence seizures ('petit mal' epilepsy) in humans, because the tonic seizures in the SER are inhibited by phenytoin, whereas the absence-like seizures are inhibited by trimethadione and ethosuximide, and both seizures are blocked by phenobarbital and valproate (Sasa et al., 1988a). The D B A / 2 mouse is a well-known sound-induced seizure model, and shows clonic convulsions induced by sound stimuli at around 3 weeks of age (Schlesinger et al., 1965). These animals were used for evaluation of the effects of topiramate, since the SER and DBA/2 mice are known to be useful for determining the acute and long-term effects of antiepileptic drugs (Chapman et al., 1984; Sasa et al., 1988a; Ujihara et al., 1991).
together with simultaneous behavioral observation, since EEG activity during the tonic and absence-like seizures always changed in accordance with the respective behavioral changes, as described previously (Sasa et al., 1988a). A mild tactile stimulus was given on the back of the animal every 5 min during recording of EEG to induce tonic seizures consistently and to keep the rat awake. Animals were used for evaluation of the effects of drugs after 11 weeks of age when they usually exhibited tonic and absence-like seizures for a total duration of more than 40 and 50 s, respectively, for a 15-min control period. After 15-min control recording, each animal was given a single dose of an anticonvulsant, vehicle, or a combination of topiramate with haloperidol once a day. The EEG of the animals was recorded 15-30, 45-60, 75-90 and 105-120 rain after injection of the drug or vehicle. To examine the antagonistic effects of haloperidol, topiramate was given 15 min after haloperidol administration. The experiments were done between 9:00 and 17:00 h. A higher dose of a drug was administered to the same animal after an interval of at least 1 week for wash-out.
2. Materials and methods
2.1.3. Data analysis The duration of each seizure was measured, and the total duration of the seizures for a 15-rain period was calculated before and after injection of the drug. As for the tonic seizures, the durations of both tactile stimulation-induced and spontaneous convulsions were measured. An appearance of 5-7 Hz spike and wave complex in EEG lasting over 1 s was regarded as an absence-like seizure. The statistical significance of the difference between the means of the total duration in the vehicle-treated group and the drug-treated group as well as those of the topiramate-treated group in the absence and presence of haloperidol pretreatment was determined with Kruskal-Wallis' test or a one-way analysis of variance (ANOVA) followed by ScheffE-type non-parametric test or ScheffE's parametric test, respectively, according to whether a statistically significant difference existed or not in the variances of groups. The statistical differences of variances were tested by Bartlett's test, because these experiments involved three or more groups. In the experiment which determined the duration of action of topiramate, MannWhitney's U-test was used, because the variances of the vehicle- and the topiramate-treated group were not equal according to the F-test. The statistical difference between before (control) and after drug treatment was evaluated using a paired t-test.
2.1. Spontaneously epileptic rats 2.1.1. Animals SER were bred in our institute as reported previously (Serikawa and Yamada, 1986) and kept individually in pail type cages in the animal quarters. Commercial food pellets (F-2, Funabashi Farm, Japan) and water were given ad libitum. Room temperature and relative humidity were kept at 23-25°C and 50-60%, respectively. The room was illuminated for 12 h, from 7:00 to 19:00 h. 2.1.2. E E G recordings Fourteen male and 14 female SER between 11 and 17 weeks old were used. Under chloral hydrate (300 mg/kg i.p.) or sodium pentobarbital anesthesia (40 mg/kg i.p.), bipolar electrodes were chronically implanted in the bilateral frontal cortex and left hippocampus (P, 4.3; L, 2.7; H, 2.4 from bregma) according to the atlas of Paxinos and Watson (1986). An indifferent electrode was placed on the frontal cranium. The animal was placed in a sound-attenuated box (40 X 40 X 40 cm) with a small window for behavior observation, 7-10 days after implantation of the electrodes. Then, an electroencephalogram (EEG) was recorded on a pen-writing electroencephalograph (Nihon Kohden, EEG5109) or a polygraph (Nihon Kohden, RMC-1200) for 15 rain after 60-rain habituation as the control. The duration of tonic and absencelike seizures was measured from the EEG pattern
2.2. DBA / 2 mice
2.2.1. Animals Male D B A / 2 mice (SLC) aged 21-22 days were used. Commercial food pellets (F-2, Funabashi Farm,
J. Nakamura et al. ~European Journal of Pharmacology 254 (1994) 83-89
Japan) and water were given ad libitum. Room temperature and relative humidity were kept at 23-25°C and 50-60%, respectively. The room was illuminated for 12 h, from 7:00 to 19:00 h.
2.2.2. Induction of sound-induced seizure Mice were placed in a cylinder (10 cm diameter X 9 cm high) after drug administration, and exposed to sound (140 dB) for 60 s. Drugs were applied 120 min prior to the application of the sound, since the peak time for anticonvulsive effects in the maximal electroconvulsive shock test for topiramate, zonisamide, phenytoin and phenobarbital was approximately 120 min. The pretreatment time for carbamazepine and valproate was 30 min. 10-20 mice were used for one dose of the drugs, and the protection of the drugs against clonic seizures was examined. The EDs0 values and 95% confidence limits were determined by probit analysis.
85
2.3. Drugs In the SER, topiramate (2,3 : 4,5-bis-O-(1-methylethylidene)-/3-D-fructopyranose sulfamate (R.W. Johnson Pharmaceutical Research Institute, Spring House, PA, USA) and sodium phenytoin (Dainippon Seiyaku, Osaka, Japan) were administered as a solution in a vehicle composed of 40.0% propylene glycol, 10.5% ethanol and 49.5 % distilled water. Zonisamide (Dainippon Seiyaku, Osaka, Japan) was dissolved in distilled water as the sodium salt. An ampule form of haloperidol (Serenace, Dainippon Seiyaku, Osaka, Japan) was used as a non-selective dopamine receptor blocker. This 1-ml ampule contains haloperidol, 5 mg (pH 3.5-4.2). The haloperidol solution was diluted with the same amount of distilled water and used. These drugs were injected intraperitoneally to the SER. In D B A / 2 mice, drugs were administered orally as a 0.3% sodium carboxymethylcellulose solution or sus-
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86
J. Nakamura et aL/ European Journal of Pharmacology 254 (1994) 83-89
pension. The drugs used for D B A / 2 mice were topiramate (R.W. Johnson Pharmaceutical Research Institute, Spring House, PA, USA), phenytoin, carbamazepine, sodium phenobarbital (Wako, Osaka, Japan), zonisamide (Dainippon Seiyaku, Osaka, Japan) and sodium valproate (Kyowa Hakko, Tokyo, Japan).
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3.1. Spontaneously epileptic rats Time after administration (min) Tonic convulsions and absence-like seizures, characterized by the sudden appearance of 5-7-Hz spikewave-like complexes in the E E G of the cerebral cortex and hippocampus, occurred in all twenty-eight SER (Fig. 2). The mean total duration of tonic convulsion (including both spontaneously and tactile stimulationinduced seizures) and absence-like seizures for 15 min in 28 SER was 72.3 + 3.8 and 119.6 + 18.8 s (mean + S.E.M.), respectively. The vehicles had no effects on the tonic and absence-like seizures in SER. The mean total duration of tonic and absence-like seizures for 15-30 min after injection of the vehicle used for topiramate and phenytoin was 83.4 + 9.9% ( P = 0.12) and 126.1 + 17.1% ( P =0.16) ( n = 9 ) of the control value, respectively, whereas the corresponding duration of seizures for distilled water (zonisamide vehicle) was 115.1 + 14.4% ( P = 0.42) and 84.8 + 10.3% ( P = 0.13) (n = 5), respectively.
3.2. Effects of topiramate and other antiepileptic drugs I.p. injection of topiramate at doses of 5, 10 or 20 m g / k g did not induce any overt behavioral or E E G changes. However, topiramate at a dose of 40 m g / k g increased locomotor activity of the SER in the shield box as visually observed, but caused no obvious changes in background EEG. Compared to those of the vehicle, the overall effects of topiramate were highly significant (tonic seizure: Kruskal-Wallis' test P < 0 . 0 1 ; absence-like seizure: Kruskal-Wallis' test P < 0.01). Topiramate at doses of 20 and 40 m g / k g i.p. produced a dose-dependent inhibition of both the tonic and absence-like seizures (Figs. 2, 3 and 4). The inhibitory effects were observed from 15 min after injection and the maximum effects were seen 15-30 min later (Fig. 3). Phenytoin dose dependently inhibited tonic seizures (ANOVA F(3,19) = 9.3, P < 0.01) without affecting absence-like seizures as reported previously (Sasa et al., 1988a) (Fig. 4). Significant inhibition ( P < 0.01) of the tonic seizure was observed with 20 m g / k g of phenytoin, although the absence-like seizure was not affected by 20 mg/kg. The maximum effects were observed
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Time after administration (min) Fig. 3. The time course of the inhibitory effects of topiramate (20 mg/kg i.p.) on the total duration of (A) tonic and (B) absence-like seizures. The percent of control was obtained by dividing each value after drug or vehicle injection by that before injection. Each point and bar represents the mean and S.E.M., respectively (n = 9 for 15-30, 45-60 min; n = 5 for 75-90, 105-120 min). (e) and (o): topiramate and vehicle, respectively.** P < 0.01, as compared with vehicle-injectedgroup.
15-30 min after injection, and the effect lasted for more than 60 min. Zonisamide dose dependently inhibited tonic seizures (Kruskal-Wallis' test P < 0.01), but 40 m g / k g of the drug did not affect the absence-like seizures. Zonisamide significantly ( P < 0.05) inhibited tonic seizures at 40 m g / k g (Fig. 4). The peak time of effects was seen 15-30 min after the injection.
3.3. Antagonizing effect of haloperidol Haloperidol (0.5 m g / k g i.p.) alone had no significant effects on the absence-like seizures ( P = 0.32) or tonic seizures ( P = 0.32) as reported previously (Ujihara et al., 1991). The mean total duration of tonic and absence-like seizures during 15-min periods were 74.8 + 11.1 and 97.8 + 12.7 s (n = 5) before administration, and 63.2 + 9.2 and 116.2 + 21.7 s 30-45 min after injection of haloperidol. However, when haloperidol was given 15 min before injection of topiramate (20 m g / k g i.p.), the inhibition of the absence-like seizures by
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J. Nakamura et aL / European Journal of Pharmacology 254 (1994) 83-89
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Fig. 4. Effects of topiramate, phenytoin and zonisamide on the total duration of tonic and absence-like seizures. The total duration of both seizures was measured for 15 min before drug injection and 15-30 min after drug injection. Each value is shown as a percentage of the values obtained for 15 min before drug injection. Columns and bars represent mean and S.E.M. (n = 4-9). Solid and open columns represent tonic and absence-like seizures, respectively. *P < 0.05 and ** P < 0.01, as compared with vehicle-injected group.
Table 1 Effects of topiramate and other antiepileptic drugs on sound-induced seizures in DBA/2 mice EDs0 and 95% confidence limits (mg/kg p.o.) Drugs Clonic seizures Tonic convulsions Topiramate Phenytoin Zonisamide Carbamazepine Phenobarbital Valproate
8.6 (6.1-12.0) 1.0 (0.5- 1.6) 31.4 (24.3-42.6) 2.0 (0.8- 4.6) 1.7 (1.0- 2.5) 42.5 (28.6-61.8)
3.5 (2.6-4.4) 0.61 (0.38-0.91) 13.4 (11.2-16.0) 1.0 (0.6- 1.6) 1.3 (0.7- 2.0) 42.5 (28.6-61.8)
When sound stimuli were given for 60 s, abnormal running behavior, clonic seizures and tonic convulsions were induced in 5 0 / 5 0 (100%), 4 4 / 5 0 (88%) and 4 2 / 5 0 (84%) of the animals, respectively. All mice that exhibited tonic convulsions died. Topiramate blocked the sound-induced clonic seizure with an EDs0 of 8.6 m g / k g (Table 1). Tonic convulsions and death did not occur at doses greater than 12.5 m g / k g in any of the 20 animals examined. The EDs0 for inhibiting tonic convulsions was 3.5 m g / k g (Table 1). Phenytoin, zonisamide, carbamazepine, phenobarbital and valproate also dose dependently protected against sound-induced clonic seizures and tonic convulsions (Table 1). Tonic convulsions and death were completely prevented by phenytoin, zonisamide, carbamazepine, phenobarbital and valproate at doses over 3.13, 50, 12.5, 6.25 and 200 m g / k g , respectively.
4. Discussion The present study demonstrated that topiramate inhibits both tonic and absence-like seizures in SER. In our previous study, tonic and absence-like seizures appeared to correspond to generalized tonic-clonic seizures ('grand mal' epilepsy) and simple absence seizures ('petit mal' epilepsy) in human epilepsy, based on the responsiveness of these seizures to the conventional antiepileptic drugs (Sasa et al., 1988a), although
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the spikes were not usually observed during the tonic seizure except in some cases following wild running. Tonic seizures were inhibited by phenytoin and carbamazepine, whereas absence-like seizures were inhibited by trimethadione and ethosuximide, and both types of seizures were inhibited by phenobarbital, valproate and diazepam (Sasa et al., 1989). Furthermore, chronic administration of phenobarbital inhibited tonic seizures without affecting absence-like seizures, as has been reported for human epilepsy (Serikawa et al., 1990). Since topiramate blocked both types of seizures in SER, it may be effective for treating both convulsive and absence seizures in human epileptic patients. Topiramate 40 mg/kg, phenytoin 20 mg/kg and zonisamide 40 mg/kg inhibited tonic seizures almost completely. The potency of topiramate for inhibition of tonic seizures was similar to that of phenytoin and of zonisamide, since the doses described above were 118, 73 and 171/xmol/kg. However, the latter two anticonvulsants were ineffective to block absence-like seizures of the SER. The effectiveness of topiramate as an anticonvulsant was further supported by its ability to prevent audiogenic seizures in DBA/2 mice, although sound-induced seizures in D B A / 2 mice do not discriminate between the clinical profiles of the antiepileptic drugs (Chapman et al., 1984). The ratio of the EDs0 for impairment of neurological function (rotarod test) to the EDs0 for protection against audiogenic seizures for topiramate exceeded those of phenobarbital and valproate which inhibit both tonic and absence-like seizures in SER (unpublished data). Recently, Hara et al. (1993) have reported that the dopamine content in SER is lower than that of control animals, suggesting that a decrease in dopamine is one of the factors involved in the seizures. We have also demonstrated that dopamine agonists such as apomorphine inhibit both tonic and absence-like seizures in SER (Sasa et al., 1988b). Moreover, thyrotropin-releasing hormone, known to release catecholamines, inhibited the seizures of the SER (Ujihara et al., 1991; Sasa et al., 1989). The inhibitory effects of topiramate on absence-like seizures of the SER were antagonized by haloperidol, a dopamine receptor antagonist. Taken together with these observations, our finding suggests that activation of dopaminergic systems by topiramate is one of the factors involved in the inhibition of these seizures, although the contribution of noradrenergic neurons cannot be completely excluded because of haloperidol possessing a weak al-adrenoceptor antagonistic effect. These findings are also in line with the observation that the anticonvulsant effects of this drug on maximal electroconvulsive shock in mice were attenuated by pretreatment with reserpine (Maryanoff et al., 1987). Since topiramate has no affinity to dopamine D 1 o r
D 2 receptors and no effects on dopamine uptake (Shank et al., 1991), topiramate may enhance dopaminergic activity by promoting the synaptic release of dopamine. The anticonvulsant effect of topiramate on tonic seizures of the SER was not affected by haloperidol. This suggests that the suppressive effect of topiramate on tonic seizures is mainly due to a non-dopaminergic mechanism, although dopamine-mediated anticonvulsant effects may play some role, as thyrotropin-releasing hormone was also effective to inhibit tonic seizures of the SER (Ujihara et al., 1991). We reported that the extracellular concentration of glutamate and aspartate is significantly elevated in the hippocampus of SER (Kanda et al., 1992), and that topiramate restores these elevated levels to normal values (Kanda et al., 1992). This suggests that glutamatergic and aspartatergic neuronal pathways may be involved in the SER seizures in addition to activation of the catecholaminergic system. The anticonvulsant activity of topiramate may simultaneously be related to inhibitory effects on excitatory amino acid neurons. In conclusion, topiramate has antiepileptic effects on both convulsive and absence-like seizures and the inhibitory effect on absence-like seizures in SER may be mediated through the central dopaminergic system.
5. References Aranguiz, C., J. McJilton, M. Vega and R.E. Ramsay, 1991, Safety and effectiveness of three oral doses of topiramate in the treatment of patients with refractory partial epilepsy, Epilepsia 32, Suppl. 3, 11. Chapman, A.G., M.J. Croucher and B.S. Meldrum, 1984, Evaluation of anticonvulsant drugs in D B A / 2 mice with sound-induced seizures, Arzneim.-Forsch./Drug Res. 34, 1261. Hara, M., M. Sasa, A. Kawabata, T. Serikawa, J. Yamada and S. Takaori, 1993, Decreased dopamine and increased norepinephrine levels in the spontaneous epileptic rat, a double mutant rat, Epilepsia 34, 433. Kanda, T., J. Nakamura, M. Kurokawa, S. Tamura, A. Ishii, K. Ishihara, T. Serikawa, J. Yamada and M. Sasa, 1992, Inhibition of excessive release of excitatory amino acids in hippocampus of spontaneously epileptic rat (SER) by topiramate (KW-6485), Jpn. J. Pharmacol. 58, Suppl. I, 92P. Maryanoff, B.E., S.O. Nortey, J.F. Gardocki, R.P. Shank and S.P. Dodgson, 1987 Anticonvulsant O-alkyl sulfamates. 2,3:4,5-bis-O(1-methylethylidene)-fl-D-fructopyranose sulfamate and related compounds, J. Med. Chem. 30, 880. Paxinos, G. and C. Watson, 1986, The Rat Brain in Stereotaxic Coordinates (Academic Press, Sydney). Rehm, S., P. Mehraein, A.P. Anzil and F. Deerberg, 1982, A new rat mutant with defective overhairs and spongy degeneration of the central nervous system: clinical pathological studies, Lab. Anim. Sci. 32, 70. Sasa, M., Y. Ohno, H. Ujihara, Y. Fujita, M. Yoshimura, S. Takaori, T. Serikawa and J.Yamada, 1988a, Effects of antiepileptic drugs on absence-like and tonic seizures in the spontaneously epileptic rat, a double mutant rat, Epilepsia 29, 505. Sasa, M., H. Ujihara, Y. Fujita, M. Yoshimura, S. Takaori, T.
J. Nakamura et al. / European Journal of Pharmacology 254 (1994) 83-89 Serikawa and J. Yamada, 1988b, Pharmacological studies using genetically defined epilepsy model animal, spontaneously epileptic rat (SER): role of dopaminergic system in controlling absence-like seizure and tonic convulsion, Jpn. J. Pharmacol. 46 (Suppl.), 37P. Sasa, M., H. Ujihara, K. Ishihara, Y. Ohno, Y. Fujita, M. Yoshimura, J. Nakamura, T. Serikawa, J. Yamada and S. Takaori, 1989, Responsiveness of the spontaneously epileptic rat (SER), a double mutant, to antiepileptic drugs, Adv. Neurol. Sci. 33, 909. Schlesinger, K., W. Boggan and D.X. Freedman, 1965, Genetics of audiogenic seizures. 1. Relation to brain serotonin and norepinephrine in mice, Life Sci. 4, 2345. Serikawa, T. and J. Yamada, 1986, Epileptic seizures in rats homozygous for two mutations, zitter and tremor, J. Hered. 77, 441. Serikawa, T., K. Kogishi, J. Yamada, Y. Ohno, H. Ujihara, Y. Fujita, M. Sasa and S. Takaori, 1990, Long-term effects of continual intake of phenobarbital on the spontaneously epileptic rat, Epilepsia 31, 9.
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Shank, R.P., J.L. Vaught, R.B. Raffa and B.E. Maryanoff, 1991, Investigation of the mechanism of topiramate's anticonvulsant activity, Epilepsia 32, Suppl. 3, 7. Tanabe, K., Y. Wang and K. Kimishima, 1991, Anticonvulsant activities and properties of topiramate, J. Yonago Med. Ass. 42, 330. Ujihara, H., R. Renming, M. Sasa, K. Ishihara, Y. Fujita, M. Yoshimura, T. Kishimoto, T. Serikawa, J. Yamada and S. Takaori, 1991, Inhibition by thyrotropin-releasing hormone of epileptic seizures in spontaneously epileptic rats, Eur. J. Pharmacol. 196, 15. Wilder, B.J., J.K. Penry, R.J. Rangel, A. Riela, B.L. Margul and T.B. Marriott, 1988, Topiramate: efficacy, toxicity, and dose/plasma concentrations, Epilepsia 29, 698. Yamada, J., T. Serikawa, J. Ishiko, T. Inui, H. Takeda, Y. Kawai and A. Okaniwa, 1985, Rats with congenital tremor and curled whisker and hair, Exp. Anim. 34, 183.