Epilepsy Research 49 (2002) 189– 202
www.elsevier.com/locate/epilepsyres
Clobazam shows a different antiepileptic action profile from
clonazepam and zonisamide in Ihara epileptic rats
Yoshiki Miura a,*, Shigeru Amano b, Ryuzo Torii c, Nobuo Ihara d
a
Safety Research Laboratories, Department of Safety Pharmacology, Dainippon Pharmaceutical Co., Ltd., 33 -94 Enoki-cho, Suita,
Osaka, Japan
b
College of Medical Technology, Kyoto Uni6ersity, Kyoto, Japan
c
Institute of Laboratory Animals, Shiga Uni6ersity of Medical Science, Otsu, Shiga, Japan
d
Institute of ICR Research, Kyotanabe, Kyoto, Japan
Received 17 August 2001; received in revised form 28 January 2002; accepted 18 February 2002
Abstract
Purpose: Clobazam (CLB, 1,5-benzodiazepine, 1,5-BZP) has been reported to show unique antiepileptic action
profile distinguishing from standard 1,4-BZPs. To further elucidate the action profile of CLB, its effects on the
abnormal circling fits (ACFs) and generalized tonic-clonic convulsions (GTCs) in Ihara epileptic rats (IERs), a
genetically epileptic mutant, were examined in comparison with conventional antiepileptic drugs (AEDs), a 1,4-BZP,
clonazepam (CZP) and a non-BZP, zonisamide (ZNS). Methods: The incidence of ACFs or GTCs in IERs was
recorded automatically by the computer-assisted behavior monitoring system (COBAS N-IV) before, during and after
the drug treatment period for 5 days in each. The drugs were orally administered twice daily. The daily and total
incidences of ACFs or GTCs were calculated every each period in each dose group. The incidences of various
behaviors such as feeding, gnawing and scratching recorded simultaneously were used for evaluating the behavioral
activity (BA). Results: CLB (30 and 60 mg/kg) prevented the appearance of ACFs and GTCs without affecting BAs.
CZP (1 and 3 mg/kg) suppressed the occurrence of ACFs but induced no effect on the incidence of GTCs.
Furthermore, it inhibited BAs at the same doses. ZNS (15 mg/kg) suppressed GTCs but little ACFs without affecting
BA. Conclusion: CLB exhibited a different action profile from CZP and ZNS in a novel epileptic mutant, IERs, and
was expected to be a useful AED superior to 1,4-BZPs in clinical practice. © 2002 Elsevier Science B.V. All rights
reserved.
Keywords: Clobazam; Clonazepam; Zonisamide; Ihara epileptic rat (IER); Generalized tonic-clonic convulsions (GTCs); Abnormal
circling fits (ACFs)
1. Introduction
* Corresponding author. Tel.: +81-6-6337-5942; fax: + 816-6338-7656.
E-mail address: [email protected] (Y.
Miura).
Clobazam (CLB) is a benzodiazepine (BZP)
with a unique structure that the nitrogen atoms
are placed in the 1,5-position of the heterocyclic
ring instead of the 1,4-position observed in the
0920-1211/02/$ - see front matter © 2002 Elsevier Science B.V. All rights reserved.
PII: S 0 9 2 0 - 1 2 1 1 ( 0 2 ) 0 0 0 3 2 - 3
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Y. Miura et al. / Epilepsy Research 49 (2002) 189–202
standard BZPs such as diazepam and clonazepam
(CZP), and the only 1,5-BZP introduced into
clinical practice. Its antiepileptic activity was first
reported in mice by Barzaghi et al. (1973). Gastaut and Low (1979) reported the clinical efficacy
of CLB as adjunctive therapy against all types of
refractory epilepsy. Since then, a lot of animal
studies have shown that CLB has significant and
unique anticonvulsive properties different from
those of 1,4-BZPs (Lucien et al., 1986; Kruse,
1985); its broad anticonvulsive spectrum on convulsions induced by various chemicals and its less
sedative properties as compared with 1,4-BZPs. In
addition, the higher relative potency of CLB
against electroshock-induced seizures than 1,4BZPs suggested that CLB exerted a similar
antiepileptic action profile to conventional nonBZP antiepileptic drugs (AEDs) such as phenytoin (PHT), carbamazepine (CBZ) or zonisamide
(ZNS). Although antiepileptic actions of CLB
have been investigated acutely, its action of repeated treatment in chronic epilepsy models such
as spontaneously epileptic rats with genetic background has been little reported in comparison
with those of 1,4-BZPs and non-BZPs AEDs.
Ihara epileptic rat (IER) is an epileptic mutant
with generalized tonic-clonic convulsions (GTCs)
spontaneously occurring from about 5 months
after birth. Several lines of studies on IERs developing GTCs have strongly suggested that this
mutant is a novel epileptic animal model for
studying human temporal lobe epilepsy (TLE)
(Amano et al., 1996a,b; Imamoto et al., 1996;
Amano, 1999; Amano et al., 1999; Takamori et
al., 1999). In addition, IERs show a series of
unique behaviors during the developmental process to GTCs according to its aging. Namely, at
around 1 month after birth, the abnormal behaviors such as wild running fits and jumping are
spontaneously observed. Subsequently, from 2 to
3 months after birth, characteristic abnormal circling fits (ACFs) as if chasing rapidly their tails
appear and are repeated for a few months at the
frequency of approximately 10 to 30 attacks per
day. Finally, GTCs occur at the frequency of 1 to
3 attacks per day accompanied with a disappearance of ACFs. GTCs observed in IERs symptomatologically
resemble
the
secondarily
generalized motor seizures usually observed at the
final stage (stage 4 or 5) in the electrically or
chemically kindled rats. However, the developing
process to GTCs in IERs apparently differs from
such experimental kindling animals, and rather it
is seemingly developed under the natural kindling
through repeated excitations of some brain circuit
facilitated by repeated abnormal behaviors including wild running and wet dog shake, and furthermore by ACFs in accordance with aging. At the
present time, although clinical relevance of ACFs
in IERs remains unclear, it is considered at least
that the ACFs reflect the abnormal hyperexcited
behavior involved in the development of GTCs.
In this study, to elucidate the AED profile of
CLB in a chronic epilepsy model as comparing
with those of a 1,4-BZP, CZP and a non-BZP,
ZNS, the effects of 5-days chronic treatment with
the drugs on ACFs, GTCs and the behavioral
activities (BAs) in IERs were examined.
The abstract form of this study has been reported elsewhere (Miura et al., 2001).
2. Materials and methods
2.1. Animals
Male IERs (body weights and age, ACF rats;
410 to 510 g at 4 to 6 months after birth, GTC
rats; 450 to 600 g at 7 to 11 months after birth),
which were maintained at the Institute of Laboratory Animals in Shiga University of Medical Science, were used in our Laboratories. They were
housed individually in the experimental room
with a 12-h light:12-h dark cycle (lights on at
07:00 h), the controlled temperature (239 2 °C)
and the relative humidity (559 5%), and they had
free access to food (CE2, Nihon CLEA) and
water before and during this experiment.
The incidence of ACFs or GTCs for all animals
in the colony was periodically monitored since
birth, and on the basis of the monitoring results
for 10 to 14 days prior to the start of experiments,
the rats with ACFs or GTCs alone satisfying the
following criteria were selected, GTC rats in
which the mean frequency of the seizures is 1 to 3
attacks per day and ACF rats in which the mean
Y. Miura et al. / Epilepsy Research 49 (2002) 189–202
frequency is around 10 to 30 attacks per day.
Each of the rats showing ACFs or GTCs alone
was randomly assigned to one of the groups given
vehicle or different doses of drugs. Each dose
group consisted of four rats.
All experimental procedures were carried out in
compliance with the Internal Guide for the Animal Care and Use at Dainippon Pharmaceutical
Co., Ltd. under the approved protocol.
191
All the vibration signals including ACFs, GTCs
and BAs were stored on a MO disk continuously
for 24 h. The stored recording was exhibited
automatically in a MICROSOFT EXCEL on the WINDOWS system, and provided to the subsequent
calculation analysis of the incidence of each
2.2. ACFs, GTCs and BAs monitoring
Appearance of ACFs, GTCs or BAs during the
pre-treatment, the treatment and the post-treatment periods (5 days in each) was recorded continuously
and
automatically
using
the
computer-assisted behavior monitoring system
originally developed by us (COVAS, Biomedica
Ltd., Osaka, Japan). Briefly, piazoceramic vibration sensor (VM-313A, Biomedica Ltd.) detecting
vibrations of animal cage caused by the movements of animals was attached to the bottom of
the animal cages. The vibration signals were amplified by the charge amplifier (AG2101, NECSanei, Tokyo, Japan) and transferred through the
control box (Biomedica Ltd.) to the PC computer
(NEC, Tokyo, Japan) in which the analyzing software (COVAS N-IV, Biomedica Ltd.) was installed to set up the trigger level for recording the
vibration signal and its recording duration and
for the synchronized videotape recording. When
the signals from the sensor exceeded the trigger
level set up in the computer, the vibration signals
were recorded on the MO disk together with the
time and the date, and synchronously the
videotape recording for the animal behaviors was
run during the period set above. Using this system, the vibration signal patterns of GTCs, ACFs
and other behaviors recorded were very characteristic in each as shown in Fig. 1, and it was easy to
distinguish them each other. Thus, the behaviors
such as feeding, gnawing and scratching recorded
simultaneously with ACFs or GTCs were used as
an indicator of BAs. The BAs were defined as
‘epochs’ triggered by such various behaviors, and
counted as the incidence in each period as well as
ACFs or GTCs.
Fig. 1. Vibration patterns of GTCs, ACFs and other behaviors
provoked in IERs (I) 1, GTCs; 2, rearing and forelimb clonus;
3, repeated GTCs; 4, wet dog shake following GTCs; 5,
immobilization. (II) 1 – 4, ACFs, represented by a series of
large and medium-sized spikes. Rotating more than twice was
usually observed during 1 – 4. A series of signals from the time
when the signal was triggered to the time when the recording
period was terminated was considered one attack of circling
fits. (III) 1, 2, 3, feeding behaviors, low amplitude spikes with
high frequency (1, 2) and medium-sized amplitude (3) were
observed. (IV) 1 – 4: Scratching behaviors, scratching several
times (1), scratching once or twice, medium-sized and low
amplitude spikes with consistent frequency were usually
recorded (2,3). When the scratching ended, the vibration pattern immediately flattened (4).
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Y. Miura et al. / Epilepsy Research 49 (2002) 189–202
seizure or behavior in every period before, during
and after drug treatment. Furthermore, they were
collated with the videotape recordings, allowing
us to confirm exactly which kind of behaviors
occurred.
periods for 5 days were analyzed for each group.
As a control group, the vehicle (0.5% tragacanth
solution) not containing drugs was administered
at the volume of 2 ml/kg.
2.3. Circadian 6ariations on the incidence of
GTCs, ACFs and BAs
2.6. Analysis for the drug effects on GTCs, ACFs
and BAs
Prior to the evaluation of drugs on GTCs,
ACFs and BAs in IERs, the circadian variations
of their incidences were measured for 5 days using
16 rats in each. The incidence was amounted
every 1 h during a 1-day period and totaled for 5
days in 16 IERs showing GTCs or ACFs in each.
To determine the effects of drugs on GTCs,
ACFs or BAs, the daily or total incidences of
GTCs, ACFs and the triggered epochs provoked
by various behaviors during the pre-treatment,
treatment or post-treatment period in each group
were measured. The daily incidence was expressed
as the incidence of attacks or epochs per day, and
was compared between the drug-treated group
and the vehicle-treated group at each day. The
total incidence (attacks or epochs per 5 days)
during the treatment or the post-treatment period
in each dose group was expressed as the percentage of the incidence during the pre-treatment
period. The results of the treatment or the posttreatment period in each dose group were compared with those in the vehicle-treated group.
2.4. Drug treatments and the dosage
The following drugs were used: CLB (lot no.
41B001, Aventis Pharma. Deutschland GmbH,
Frankfurt, Germany), zonisamide (ZNS, lot no.
814, Dainippon Pharmaceutical. Co., Ltd., Osaka,
Japan) and clonazepam (CZP, lot no. ACF7408,
WAKO Pure Chemical Industries, Osaka, Japan).
These drugs were suspended in 0.5% (w/v) tragacanth solution to prepare the appropriate doses,
and orally administered twice a day for 5 days at
a volume of 2 ml/kg under the slight ether anesthesia. Doses of drugs sufficiently effective to
show their anticonvulsive actions in experimental
animals were used, 15, 30 and 60 mg/kg for CLB,
15 and 30 mg/kg for ZNS and 1 and 3 mg/kg for
CZP.
2.5. Drug administration schedule
The timing of drug administrations was determined from the result of the circadian variations
for the incidences of GTCs, ACFs and BAs (see
Section 3.2). As the results, the drug at each dose
was orally administered twice a day at 10:30 and
18:30 h during a period of drug treatment for 5
days. The period from 07:00 h. on the day to
07:00 h on the next day was considered a 1-day
observation period (24 h). After establishing the
experimental schedule for 15 days, the daily and
total incidences of GTCs, ACFs or BAs during
the pre-treatment, treatment and post-treatment
2.7. Statistical analysis
Data were expressed as the mean incidence or
percent9 S.E.M. of four animals at each dose of
drugs in the GTCs and ACFs groups, respectively. The statistical analysis was carried out
using the SAS system (version 6.12, SAS Institute
Inc., USA). As for the daily incidences of GTCs,
ACFs or the BAs, significance of difference between each dose group and vehicle group at each
day point through the experimental period (15
days) were analyzed by non-parametric Dunnett’s
test. The drug effects on the total incidences during the treatment and the post-treatment period
were expressed as the percentage of the pre-treatment values, and the significance of difference for
the percentage change between each dose group
and the vehicle-treated group were analyzed in
each period by parametric Dunnett’s multiple
comparison test. The significance level was assumed to be 5% of both sides.
Y. Miura et al. / Epilepsy Research 49 (2002) 189–202
193
Fig. 2. Circadian variations for appearance of GTCs, ACFs and BAs in IERs. The appearance of GTCs, ACFs and the behavioral
activity (BAs) were recorded using the automatic behavior-monitoring system continuously for 5 days in 16 rats showing each GTCs
or ACFs without drug treatment. The period from 07:00 to 19:00 h on the next day was considered a 1-day period. After establishing
the continuous recording for 5 days, the incidences of GTCs and ACFs, and the BAs were amounted every 1 h during a 1-day period
and then totaled for 5 days in each rat, subsequently which was averaged with 16 rats (the mean incidence of GTCs/ACFs/BAs every
each time period for 5 days per rat). A 12-h light:12-h dark cycle in the experimental room was kept under the light period from
07:00 to 19:00 h.
3. Results
3.1. Vibration signal patterns pro6oked by GTCs,
ACFs and other beha6iors
Fig. 1 shows typical recording patterns of the
vibration signals provoked by GTCs, ACFs and
other behaviors in IERs. The recording pattern of
GTCs was very characteristic; initially, very high
amplitude spikes with high frequency (jumping and
clonic convulsion) were shown, followed by high
frequency and low amplitude spikes showing subsequent forelimb clonus with rearing. A series of
these seizures continued for about 20–30 s, and
was followed by immobilization. On the other
hand, the pattern of ACFs was quite different from
that of GTCs; a group of mixed large and mediumsized spikes with moderate frequency was usually
recorded. One attack of ACFs usually consisted of
three or four rotating movements. The duration of
one attack of ACFs was approximately 10 to 20 s.
The vibration signals of behaviors such as feeding,
gnawing and scratching were also frequently triggered and recorded. The recorded patterns of these
behaviors could be clearly differentiated from
those of GTCs and ACFs.
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Y. Miura et al. / Epilepsy Research 49 (2002) 189–202
3.2. Circadian 6ariations on the incidences of
GTCs, ACFs and BAs
Fig. 2 shows the circadian variations of incidences for GTCs, ACFs and BAs triggered during
5-days continuous recording period in each IER.
The incidence of GTCs was not differential between the light and dark periods during a 1-day
period, although BAs were remarkably higher
during the dark period than the light period.
Thus, the GTC development was not related with
the circadian rhythm of BAs. In contrast, the
incidence of ACFs showed the circadian rhythm
during a 1-day period similarly to that of BAs;
ACFs were observed more frequently during the
dark period than during the light period.
3.3. Effect of CLB, CZP and ZNS on the daily
incidences of GTCs and the BAs
Upper panel in Fig. 3 shows the daily change of
incidence of GTCs through the experimental period including 5-days treatment with vehicle, CLB
at 60 mg/kg, CZP at 3 mg/kg or ZNS at 15
Fig. 3. Effects of CLB, CZP and ZNS on daily incidences of GTCs and the BAs. The incidence of GTCs or the BAs every 1-day
period was calculated in each rat through the experimental period (15 days). The horizontal and the vertical axis show the
experimental days and the mean daily incidence of GTCs or the mean BAs, respectively. Vehicle or each dose of CLB, CZP and
ZNS was administered orally twice a day during the 5-days period indicated by the horizontal closed column in the figure. The data
were represented as the mean 9 S.E.M. of four rats in each group (, vehicle;
, CLB 60 mg/kg; , CZP 3 mg/kg, , ZNS 15
mg/kg). * P B 0.05, significant differences as compared with the vehicle-treated group at each day (non-parametric Dunnett’s
multiple comparison test).
Y. Miura et al. / Epilepsy Research 49 (2002) 189–202
mg/kg. The mean incidence of GTCs per day at
the first experimental day in each group treated
with vehicle, CLB, CZP or ZNS was 1.39 0.3,
1.8 90.3, 2.0 90.6 and 1.890.3 attacks per day,
respectively, and the values were not statistically
different among groups. In addition, no statistical
differences of the incidences among the groups
were observed at other days during the pre-treatment period. In the vehicle-treated group, the
daily incidence was changed almost consistently
through the experimental period (for 15 days) at
the variation of the incidence from 1.0 to 2.0
attacks per day. As compared with the vehicle
group, significant decreases in the incidences were
observed at the 2nd and 4th days of treatment
(the 7th and 9th experimental day) in the group
treated with CLB 60 mg/kg (at the 2nd day;
1.8 90.6 in the vehicle group vs. 0.89 0.1 attacks
per day in CLB 60 mg/kg group, P =0.0450, at
the 4th day; 2.090.4 in the vehicle group vs.
0.9 90.2 attacks per day in CLB 60 mg/kg group,
P= 0.0413). On the contrary, at the 1st day after
the cessation of treatment (the 11th experimental
day), the significant increase in the incidence was
observed (1.190.1 in the vehicle group vs. 2.39
0.6 attacks per day in CLB 60 mg/kg group,
P= 0.0245). ZNS at 15 mg/kg also induced the
significant reductions of the daily incidence at the
2nd and 4th days of the treatment (0.79 0.3,
P= 0.0387 and 0.690.2 attacks per day, P =
0.0167, respectively) as compared with those in
the vehicle group (see above). In addition, as well
as CLB, the significant increase in the incidence
was observed at the 1st and 3rd days after the
cessation of ZNS treatment (at the 1st and 3rd
day of the post-treatment period; 1.19 0.1 and
1.8 9 0.3 attacks per day in the vehicle group,
3.59 0.9 and 3.59 0.3 attacks per day in ZNS
group, respectively, P=0.0239 and 0.0407). Differing from these effects of CLB and ZNS, in the
group treated with CZP at 3 mg/kg, the daily
incidences of GTCs were little affected during the
treatment and the post-treatment period as compared with those in the vehicle treatment group.
Lower panel in Fig. 3 shows the change of daily
incidence of triggered epochs (daily BAs) in GTC
rats through the experimental period including
5-days treatment with vehicle, CLB at 60 mg/kg,
195
CZP at 3 mg/kg or ZNS at 15 mg/kg. As shown
in the panel, the daily BAs in the group treated
with CLB 60 mg/kg was not statistically different
from that in the vehicle-treated group at any day
through the experimental period (variations of the
mean incidence, 32.8– 55.0 and 28.3–53.0 epochs
per day in the groups of vehicle and CLB 60
mg/kg, respectively). ZNS at 15 mg/kg also did
not affect the BAs in GTC rats at any day during
the experimental period. On the other hand, CZP
exerted a significant inhibition on the daily BAs in
GTC rats during the treatment; the incidences at
the 4th and 5th days of the treatment were significantly lowered (20.59 4.8; P= 0.0238 and 14.39
1.4 epochs per day; P = 0.0114, respectively) in
comparison with those in the vehicle-treated
group (38.59 6.8 and 44.39 8.0 epochs per day,
respectively).
3.4. Effects of CLB, CZP and ZNS on the daily
incidences of ACFs and the BAs
Upper panel in Fig. 4 shows the daily change of
incidence of ACFs through the experimental period including 5-days treatment with vehicle, CLB
at 60 mg/kg, CZP at 3 mg/kg or ZNS at 15
mg/kg. The incidence of ACFs at the first experimental day in the group treated with vehicle,
CLB, CZP or ZNS was 25.3 9 5.2, 21.39 3.4,
21.39 4.1 and 21.39 2.1 attacks per day, respectively, and those were not statistically different
among groups. Additionally, there were no statistical differences of the incidences at other days
during the pre-treatment period among groups.
The daily incidence of ACFs in the vehicle group
was little changed through the experimental period with the variation of the mean incidence
from 20.8 to 28.3 attacks per day. As compared
with those in the vehicle group, significant decreases in the daily incidence were observed at the
2nd day of the treatment with CLB at 60 mg/kg
(26.597.0 in the vehicle group vs. 3.39 2.6 attacks per day in CLB 60 mg/kg group, P=
0.0104). In addition, although not statistically
significant, the moderate inhibition was observed
at the 1st, 3rd, 4th and 5th day during the treatment. CZP suppressed ACFs more potently than
CLB; CZP at 3 mg/kg caused the significant and
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Y. Miura et al. / Epilepsy Research 49 (2002) 189–202
Fig. 4. Effects of CLB, CZP and ZNS on daily incidences of ACFs and the BAs. The incidence of ACFs or the BAs every 1-day
period was calculated in each rat through the experimental period (15 days). The horizontal and the vertical axis show the
experimental days and the mean daily incidence of ACFs or the mean BAs, respectively. Vehicle or each dose of CLB, CZP and ZNS
was administered orally twice a day during the 5-days period indicated by the horizontal closed column () in the figure. The data
were represented as the mean 9S.E.M. of four rats in each group (, vehicle; , CLB 60 mg/kg, , CZP 3 mg/kg, , ZNS 15
mg/kg). *, P B0.05, **P B0.01: significant differences as compared with the vehicle-treated group at each day (non-parametric
Dunnett’s multiple comparison test).
marked suppression at all days during the treatment period (6.391.2, 5.19 0.6, 3.89 0.9, 4.89
1.4 and 6.0 9 1.2 attacks per day at the 1st, 2nd,
3rd, 4th and 5th day of the treatment, respectively). However, there was no effect on the incidence after the cessation of treatment. On the
other hand, ZNS showed no effect on the daily
incidence of ACFs at any day during the treatment and the post-treatment period.
The effects of 5-days treatment with vehicle,
CLB, CZP or ZNS on the mean daily BAs in
ACF rats through the experimental period were
shown in lower panel of Fig. 4. In the group
treated with vehicle, the BAs were varied in the
range of 40.0 9 12.0 to 53.0 9 8.0 epochs per day
through the experimental period, showing almost
consistent daily incidence of behavioral epochs.
The daily BAs in ACF rats treated with CLB 60
mg/kg was varied in the range of 29.39 3.1 to
56.59 12.8 epochs per day through the experimental period. Compared with those in the vehicle group, the mean BAs at any day in the group
treated with CLB were not statistically different
from that in the vehicle-treated group. As well,
Y. Miura et al. / Epilepsy Research 49 (2002) 189–202
ZNS treatment also did not affect the mean daily
BAs in ACF rats at any day through the experimental period. On the other hand, CZP at 3
mg/kg caused significant decreases in the daily
BAs at the 2nd and 3rd days of the treatment in
ACF rats as well as in GTC rats (at the 2nd day,
40.898.5 in vehicle group vs. 17.39 2.0 epochs
per day; P=0.0045, at the 3rd day, 48.09 5.4 in
vehicle group vs. 21.094.6 epochs per day in
CZP 3 mg/kg group; P = 0.0265).
3.5. Effects of CLB, CZP and ZNS on the Total
Incidences of GTCs and ACFs
As shown in Fig. 5, the effects of 5-days treat-
197
ment with CLB, CZP or ZNS on GTCs and
ACFs were compared as total incidence for 5 days
between the pre-treatment period and the treatment or the post-treatment period. Upper panel in
Fig. 5 represents the effects of CLB, CZP or ZNS
at various doses on GTC incidence. In the vehicletreated group, the total incidence of GTCs during
the pre-treatment, the treatment and the posttreatment periods was 7.89 0.3, 8.0 9 0.9
(103.19 10.7% of the pre-treatment) and 7.09 0.7
(90.69 9.4%) attacks per 5 days, respectively. The
total incidence during the pre-treatment period in
each group of CLB 30 and 60 mg/kg was 6.89 0.6
and 9.8 9 1.5 attacks per 5 days, respectively.
Against these pre-treatment values, there was a
Fig. 5. Effects of CLB, CZP and ZNS on total incidences of GTCs and ACFs. The total incidences of GTCs and ACFs during the
pre-treatment, the treatment and the post-treatment period with vehicle, CLB, CZP or ZNS in each rat were calculated (attacks or
epochs per 5 days). Then, the change in the treatment or the post-treatment periods from the pre-treatment period was expressed
as the percentage of the value in the pre-treatment period. The data were represented as the mean 9S.E.M. of four rats in each dose
group. Open ( ), shaded ( ) and closed columns ( ) represent the pre-treatment, the treatment and the post-treatment period,
respectively, in each dose group of each drug. The vertical axis shows the mean% of the incidences of GTCs and ACFs in the
pre-treatment period. *, PB 0.05, **, P B0.01: significant differences as compared with the values during the treatment period in
the vehicle-treated group, c PB0.05, significant difference as compared with the value during the post-treatment period in the
vehicle-treated group (parametric Dunnett’s multiple comparison test).
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Y. Miura et al. / Epilepsy Research 49 (2002) 189–202
moderate (not significant) reduction of the incidence (to 5.0 attacks per 5 days, 74.995.1%,
P = 0.0529) in the group treated with CLB at 30
mg/kg, and a significant reduction (to 5.3 attacks
per 5 days, 55.595.5%, P = 0.0169) at 60 mg/kg
as compared with the vehicle-treated group
(103.1%, see above). The reduced total incidence
of GTCs by the 5-days treatment with CLB at 30
or 60 mg/kg was almost returned to the pre-treatment levels after the cessation of treatment (6.39
1.3; 92.3916.9% and 9.090.7 attacks per 5 days;
98.0 914.4%, respectively). In the case of CZP
treatment, the total incidence of GTCs during the
pre-treatment period in the groups of CZP 1 and
3 mg/kg was 10.090.9 and 9.09 1.7 attacks per 5
days, respectively. The incidence of GTCs during
the treatment with CZP at 1 and 3 mg/kg appeared to be slightly increased; to 13.89 3.8 attacks per 5 days, 132.89 26.2% of the
pre-treatment and to 13.39 3.6 attacks per 5
days, 146.8920.5%, respectively. However, these
values were not detected as statistically significant
changes compared with the vehicle-treated group
(P =0.206 and 0.162, respectively), indicating that
CZP did not affect the total incidence of GTCs by
the 5-days treatment. ZNS at 15 and 30 mg/kg
exhibited a dose-related and potent suppression of
the total incidence of GTCs against the pre-treatment values as shown by decreases from 10.09
2.1 to 4.59 1.2 and 7.591.3 to 1.09 0.5 attacks
per 5 days, respectively, and the significant reductions of the% of the pre-treatment incidence were
observed as compared with that in the vehicle
group (103.1 9 10.7% in the vehicle group vs.
43.9 92.1 and 17.99 6.9%, P = 0.0475 and
0.0068, respectively). Furthermore, the total GTC
incidence markedly suppressed by the treatment
with ZNS 30 mg/kg was significantly increased
during the post-treatment period (90.59 9.4 in the
vehicle group vs. 161.797.3% in ZNS 30 mg/kg
group, P = 0.0359).
Lower panel in Fig. 5 shows the effects of
5-days treatment with CLB, CZP or ZNS at
various doses on the total incidence of ACFs
during the treatment and the post-treatment periods. In the vehicle-treated group, the ACF incidence during the treatment and the post-treatment
periods was changed from 129920.7 to 124.09
28.2 (93.39 7.7%) and to 135.0923.5 attacks per
5 days (104.99 6.9%), respectively. On the other
hand, during the treatment with CLB, the total
incidence of ACFs was suppressed in the dose-dependent manner; although the effect was little
observed at the dose of 15 mg/kg, the incidence
was significantly reduced from 114.5917.9 to
63.89 13.1 attacks per 5 days (55.19 4.0%, P=
0.0021) at 30 mg/kg and from 120.5 9 22.6 to
52.09 6.9 attacks per 5 days (44.89 4.6%, P=
0.0007) at 60 mg/kg, as compared with the change
in the vehicle-treated group. And, the significant
changes were almost returned to the pre-treatment
incidence after the cessation of treatment. As well
as CLB, CZP at 1 and 3 mg/kg also dose-dependently decreased the total incidences of ACFs
from the pre-treatment incidence (114.59 6.0 to
67.39 5.3 and 105.3 9 13.4 to 25.5 9 3.7 attacks
per 5 days, respectively), and the% of the pretreatment incidence was 59.09 4.7 and 24.1 9
0.7%, respectively. These % values showed
statistically significant differences from that in the
vehicle-treated group (93.39 7.7% in vehicle, P =
0.0007 and 0.00003 at CZP 1 and 3 mg/kg, respectively). The suppressed total incidence of ACFs by
CZP treatment was almost returned to the pretreatment incidence without a rebound phenomenon in the post-treatment period. However,
ZNS did not significantly affect the total incidence
of ACFs during 5-days treatment (P = 0.3609),
although slight decrease in the incidence was observed from 110.0 9 7.0 to 87.0 9 16.5 attacks per
5 days by the treatment (77.99 10.9%)
4. Discussion
IER is a genetically epileptic mutant showing
GTCs that recurs spontaneously from approximately 5 months after birth. It has been strongly
suggested that this mutant is a novel epileptic
model with spontaneous limbic seizures for studying human TLE through the symptomatological,
electroencephalographic and neuropathological
studies (Amano et al., 1996a,b; Imamoto et al.,
1996; Amano et al., 1997; Amano, 1999;
Takamori et al., 1999; Amano et al., 1999)
Y. Miura et al. / Epilepsy Research 49 (2002) 189–202
demonstrating the following results, (1) the generalized seizures of the rats began with the face and
head myoclonus followed by rearing and GTCs as
usually observed in the limbic seizure models; (2)
the GTCs occurred spontaneously without any
artificial stimuli; (3) sustained spike discharges
generalized into the hippocampus, amygdala and
neocortex
were
electroencephalographically
recorded during the GTCs; and (4) there was
sprouting of mossy fibers into the inner molecular
layer of the dentate gyrus in the hippocampus.
This study, firstly, presented that the GTC incidence in IERs did not show the apparent circadian rhythm differing from the BAs, which were
observed more frequently during the dark period
than during the light period as observed usually in
the normal rodents. This was consistent well with
the result in the early study by Amano et al.
(1997) that the circadian rhythm of GTC occurrence in IERs was not apparent. Thus, it was
evident that GTCs were induced in no relation to
the sleep-awake cycle in IERs.
The results of chronic 5-days treatment with
CLB, CZP and ZNS in IERs revealed that the
antiepileptic action of CLB on GTCs was similar
to that of ZNS but clearly distinguished from
CZP. Namely, although ZNS was more potent
than CLB, both drugs dose-relatedly and significantly reduced the daily and total incidences of
GTCs during their treatments. In contrast, CZP
could not prevent the appearance of GTCs at all.
As mentioned above, GTCs in IERs have been
positioned at least as an animal model of human
TLE. Therefore, the significantly suppressive efficiencies of CLB and ZNS against GTCs in IERs
strongly suggest their usefulness for protecting
against human TLE. In fact, in spite that human
TLE had been indicated to be refractory to any
existing AEDs, CLB has been reported to exhibit
the significant improvement in patients with partial epilepsy including TLE resistant to treatment
with conventional AEDs (Allen et al., 1983;
Koeppen et al., 1987; Schmidt et al., 1986; Remy,
1994). As well, ZNS has also been demonstrated
to show the excellent efficacy against the several
types of partial epilepsy in human (Oguni et al.,
1988; Yagi and Seino, 1992; Schmidt et al., 1993;
Leppik et al., 1993). These evidence suggested that
199
the efficacies of CLB and ZNS against GTCs in
IERs could be predictive for the clinical efficacies
of both drugs against TLE. On the other hand,
CZP showed no effect on the occurrence of GTCs
in IERs. This result was the same as that in our
previous study (Miura et al., 1999), in which no
suppressive effect of CZP given once daily for 5
days on GTCs in IERs was obtained, although
the drug administration schedule was different
from that in this study. In general, 1,4-BZPs such
as diazepam and CZP have been used clinically
for acute treatment given intravenously against
status epilepticus and for chronic treatment
against only particular seizure types; absence
seizures, the Lennox– Gastaut syndrome and myoclonic epilepsy (Sato, 1989). Thus, 1,4-BZPs fundamentally seem to be less efficacious against
partial epilepsies such as TLE than conventional
non-BZP AEDs in clinical practice (Sato, 1989;
Löscher and Schmidt, 1988). Accordingly, it is
considered that no suppressive effect of CZP on
GTCs in IERs may reflect its less clinical efficacy
against partial epilepsy including TLE. Thus, clinical futures of CLB, CZP and ZNS against human
TLE were fitted to their efficacies against GTCs in
IERs, supporting the predictability of IERs for
the novel drugs against refractory human limbic
seizures in TLE. On the other hand, there were
some animal studies demonstrating that 1,4-BZPs
including CZP produced significant decrease in
the durations of both behavioral and EEG
seizures in the complete amygdala kindled-rats
(Sato et al., 1990; Löscher et al., 1986). Although
these results in IERs and amygdala kindled rats
appear to be a discrepancy for the efficacy of
CZP, it is assumed to be derived from distinct
developmental mechanisms of GTCs between
IERs and amygdala kindled-rats. In other words,
amygdala kindled rats had been proposed as an
animal model for complex partial seizure with
secondarily generalization via known epileptogenic focus, on the other hand, IER is a TLE-like
seizure model with generalization via unknown
focus.
Another finding for the drug effects on GTCs
in IERs was the ‘rebound phenomenon’ of the
seizure incidence after the cessation of treatment
with ZNS. Such phenomenon was also observed
200
Y. Miura et al. / Epilepsy Research 49 (2002) 189–202
at the 1st day after the cessation of treatment
with CLB at 60 mg/kg, although not reflected to
the effect on the total incidence of the posttreatment period. In fact, the evidence of such
phenomenon following rapid withdrawal of ZNS
or CLB has been also noted in human since
before (Allen et al., 1983; Remy, 1994; Yagi and
Seino, 1992; Leppik et al., 1993). Thus, our data
on rebound phenomenon of ZNS and CLB in
GTCs would support the clinical future of these
AEDs.
As mentioned above, different action profile
on GTCs was found between CLB and CZP in
spite that both drugs work through the same
mechanism of action, i.e. enhancement of
GABAergic activity by the increase in the frequency of chloride-channel opening through
BZP binding sites on GABA receptor complex
(Shorvon, 1989; Nakamura et al., 1996; Haefely,
1983). At the present time, the exact mechanism
explicable for the different profile between both
drugs remains unknown. However, several studies indicated that CLB showed the same potent
anticonvulsant effects in animal models of chemically induced seizures as well as PHT, CBZ or
valproate (VPA), and that the effects of CLB
were more specific and seemingly superior to the
activity of 1,4-BZPs (Kruse, 1985; Shenoy et al.,
1982). In addition, the higher relative potency of
CLB against electroshock-induced seizures than
1,4-BZPs was also observed. Thus, it is likely
that CLB exhibits some antiepileptic mechanism
like PHT, CBZ or VPA’s action in addition to
the GABAergic mechanism that has been proposed since before.
In addition to GTCs, IERs exhibit a characteristic abnormal behavior, ACFs, which also recurs spontaneously at the frequency of 10– 30
attacks per day during a regular period of
months prior to the GTC development. The
clinical relevance of ACFs in IERs can not be
made exactly clear at the present time, because
there was still no report approaching the characteristics of ACFs. During the early period after
birth, before ACFs are induced, other abnormal
behaviors such as wild running fits, wet dog
shaking and jumping appear. It is considered
that a series of abnormally excited behaviors in-
cluding ACFs during the developmental process
to GTCs accompanied by aging in IERs is incorporated into a series of behavioral process as
the natural kindling leading to the development
of generalized motor seizures. Preliminary study
for EEGs in IERs showing ACFs did not find
any generation of focal spike discharges or generalized seizure discharges in the hippocampus,
amygdala and neocortex during the behavior
(unpublished observation). Thus, although it is
yet uncertain to define ACFs as the focal
seizures prior to the development of generalized
convulsions in IERs, it may be possible to position ACFs at least as a hyperexcited behavior
stimulating repeatedly some brain circuits presumably linked to the GTC development.
The appearance of ACFs in IERs showed apparently the circadian rhythm differing from
GTCs; ACFs appeared more frequently during
the dark period than during the light period in
a parallel fashion with BAs. This result confirmed the previous observation that the circling
occurred mostly during dark period with a peak
between 20:00 and 22:00 (Amano et al., 1997).
This result together with that in GTCs could
justify the number (twice a day) and the timing
(10:30 and 6:30 h) of drug administrations in
this study.
Such ACFs were apparently suppressed by
CLB and CZP in the dose-related manner, but
resistant to ZNS treatment. The suppressive potency of CLB against ACFs was stronger than
that against GTCs although weaker than that of
CZP against ACFs. Thus, CLB and CZP
showed specifically the suppressive efficiencies
against ACFs, suggesting that these two drugs
will exhibit the improved efficacy clinically
against the hyperexcited behaviors during interictal period or prior to the seizure generalization. Furthermore, since it has been known that
CLB would share the similar mechanism of action to CZP; enhancement of GABAergic activity, and ZNS did not with such an action, it is
likely that ACFs in IERs are specifically suppressed by the drugs with the GABAergic activity-enhancing mechanism but not by the drugs
without such mechanism. Further pharmacological evaluation of various kinds of drugs on
ACFs will be interested in.
Y. Miura et al. / Epilepsy Research 49 (2002) 189–202
It has been reported that the most prominent
problems except for epileptic seizures in TLE
patients were the emotional disturbance, hyperemotionality; fear, anxiety and/or psychosis (Bear,
1979; Dodrill and Batzel, 1986). In animals also,
several investigators have reported the hyperemotional behaviors such as increased defensive response and threat vocalizations that accompany
at various stages of kindling in rats or cats
(Adamec, 1990; Depaulis et al., 1997; Kalynchuk,
2000). Based on these reports, one of the interpretations may make it possible to position ACFs as
a hyperemotional behavior in the pre-epileptic
condition, although the behavioral expression
(phenotype) of ACFs is different from those
behaviors.
Finally, the BAs in IERs with GTCs or ACFs
were little affected by CLB or ZNS, but significantly depressed by CZP. The results of CZP on
BAs in IERs in this study supported the general
fact that the most common side effects of 1,4BZPs in clinical practice were sedation, drowsiness and ataxia (Sato, 1989). Conversely, little
suppression of BAs by CLB or ZNS in IERs
observed in this study strongly suggested that
CLB or ZNS should exhibit less sedative influence
and could be used more safely than CZP in
clinical practice.
In conclusion, this study revealed the following
findings in IERs, (1) CLB showed the significant
inhibition of GTCs and ACFs but little sedative
effect on BAs; (2) ZNS showed the potent suppression against GTCs without affecting ACFs
and BAs; and (3) CZP showed the potent suppression against ACFs and BAs but no effect on
GTCs. Thus, CLB exhibited the different action
profile from CZP and ZNS in IERs as shown by
its broad action spectrum against ACFs and
GTCs without affecting BAs. Therefore, it was
expected that CLB would be a useful AED superior to 1,4-BZPs and non-BZP in clinical use.
Acknowledgements
We wish to thanks to K. Ishibashi, K. Terada,
Y. Nose and M. Masui for technical assistance
and animal maintenance.
201
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