Full text of DOI:10.1038/sj.bjp.0704606

British Journal of Pharmacology (2002) 135, 1425 ± 1434
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A comparison of the ecacy of carbamazepine and the novel anti-
epileptic drug levetiracetam in the tetanus toxin model of focal
complex partial epilepsy
1,3H.C. Doheny, 2,4M.A. Whittington, 2,5J.G.R. Je€erys & *,1P.N. Patsalos
1Pharmacology and Therapeutics Unit, Department of Clinical and Experimental Epilepsy, Institute of Neurology, Queen
Square, London WC1N 3BG and Department of Physiology and Biophysics, Imperial College School of Medicine at St
Mary's, London
2
1
The tetanus toxin seizure model, which is associated with spontaneous and intermittent
generalized and non-generalized seizures, is considered to re¯ect human complex partial epilepsy.
The purpose of the present study was to investigate and compare the anticonvulsant e€ects of
carbamazepine with that of levetiracetam, a new anti-epileptic drug in this model.
2
One ml of tetanus toxin solution (containing 12 mLD50 ml71 of tetanus toxin) was placed
stereotactically into the rat left hippocampus resulting in generalized and non-generalized seizures.
Carbamazepine (4 mg kg71 h71) and levetiracetam (8 and 16 mg kg71 h71) were administered
during a 7 day period via an osmotic minipump which was placed in the peritoneal cavity.
3
Carbamazepine (4 mg kg71 h71) exhibited no signi®cant anticonvulsant e€ect, compared to
control, when the entire 7 day study period was evaluated but the reduction in generalized seizures
was greater (35.5%) than that for non-generalized seizures (12.6%). However, during the ®rst 2 days
of carbamazepine administration a signi®cant reduction in both generalized seizure frequency (90%)
and duration (25%) was observed. Non-generalized seizures were una€ected. This time-dependent
anticonvulsant e€ect exactly paralleled the central (CSF) and peripheral (serum) kinetics of
carbamazepine in that steady-state concentrations declined over time, with the highest
concentrations achieved during the ®rst 2 days. Also there was a signi®cant 27.3% reduction in
duration of generalized seizures during the 7 day study period (P=0.0001).
4
Levetiracetam administration (8 and 16 mg kg71 h71) was associated with a dose-dependent
reduction in the frequency of both generalized (39 v 57%) and non-generalized (36 v 41%) seizures.
However, seizure suppression was more substantial for generalized seizures. Also a signi®cant dose-
dependent reduction in overall generalized seizure duration was observed.
5
These data provide experimental evidence for the clinical ecacy of levetiracetam for the
management of patients with complex partial seizures. Furthermore, levetiracetam probably does
not act by preventing ictogenesis per se but acts to reduce seizure severity and seizure generalization.
British Journal of Pharmacology (2002) 135, 1425 ± 1434
Keywords: Epilepsy; carbamazepine; levetiracetam; new anti-epileptic drug; tetanus toxin model; complex partial seizures
Abbreviations: CSF, cerebrospinal ¯uid; CNS, central nervous system; EEG, electroencephalograph; HPLC, high performance
liquid chromatography
Introduction
Levetiracetam (Keppra1, ucb LO59, (S)-a-ethyl-2-oxo-pyrro-
lidine acetamide) is a new anti-epileptic drug which has been
recently approved for clinical use in 21 countries world wide,
including the USA and in Europe, as adjunctive treatment for
patients with partial epilepsy that is refractory to other
antiepileptic drugs. In clinical trials, levetiracetam was
associated with a favourable therapeutic index consequent
to its substantial antiepileptic ecacy and good tolerability
pro®le (Kasteleijn-Nolst Trenite et al., 1996; Sharief et al.,
1996; Betts et al., 2000; Cereghino et al., 2000; Ben-
Menachem & Falter, 2000; Shorvon et al., 2000; Grant &
Shorvon, 2000). Pre-clinical studies show no evidence of
carcinogenic, mutagenic or teratogenic potential (Genton &
Van Vleymen, 2000). Furthermore, levetiracetam exhibits
antiepileptogenic e€ects in the rat kindling model of temporal
lobe epilepsy (Loscher et al., 1998). The pharmacokinetic
pro®le of levetiracetam in humans is highly favourable: it is
rapidly and almost completely absorbed, pharmacokinetics
are linear and it is not susceptible to kinetic interaction with
concomitant antiepileptic drugs (Patsalos, 2000). Thus,
levetiracetam appears to be an important addition to the
current range of anti-epileptic drugs.
*Author for correspondence; E-mail: P.Patsalos@ion.ucl.ac.uk
Current addresses: 3Department of Obstetrics and Gynaecology,
Clinical Science Institute, Galway, Republic of Ireland; 4School of
Biomedical Sciences, University of Leeds, Leeds; 5Department of
Neurophysiology, University of Birmingham Medical School,
Birmingham
The exact mechanism of action of levetiracetam is not
known. Although it signi®cantly decreases spontaneous
substantia nigra pars reticulata neuronal activity, which is

1426
H.C. Doheny et al
Carbamazepine and levetiracetam in focal epilepsy
dependent on strong GABAergic (g-aminobutyric acid) input
from the brain (Loscher et al., 1996), levetiracetam is thought
to exert its anticonvulsant e€ect via non-GABAergic
mechanisms (Margineanu & Wulfert, 1997; Sills et al.,
1997). Furthermore, only the S-enantiomer of levetiracetam
exhibits anticonvulsant properties and the three metabolites
of levetiracetam (representing 27% of an administered
levetiracetam dose) are not pharmacologically active (Klit-
gaard et al., 1998). The ®nding that the anticonvulsant
activity of levetiracetam was stereoselective led to the
identi®cation of a speci®c binding site for levetiracetam in
the rat central nervous system (Noyer et al., 1995). However,
the site remains to be characterized, and its exact role in the
anticonvulsant action of levetiracetam is unknown.
Levetiracetam was originally discovered by random screen-
ing to have potent ecacy against seizures induced in
audiogenic mice (Gower et al., 1992). In contrast to other
established anti-epileptic drugs, levetiracetam has no activity in
the acute maximal electroshock seizure test or the pentylenete-
trazole seizure test, models assumed to predict clinical ecacy
in man (Loscher & Schmidt, 1988). However, levetiracetam, at
doses well below those inducing adverse e€ects, is very
ecacious in animal models of chronic epilepsy, including
genetic (Genetic Absence Epilepsy Rat from Strasbourg,
GAERS) and kindled (amygdala) rats (Gower et al., 1995;
Loscher & Honack, 1993). This selective suppression of
seizures in animal models of chronic epilepsy further suggests
a unique mechanism and pro®le of action for levetiracetam.
days prior to surgery and were allowed free access to normal
laboratory diet (22 F diet, Labsure, Poole, U.K.) and water.
A 12 hour light/dark cycle (light on at 06 : 00 h) was
maintained.
Tetanus toxin
Tetanus toxin, as a lyophilized powder (Wellcome Research
Laboratories, Beckenham, U.K.) was reconstituted under
aseptic conditions using saline to
1200 mLD50 ml71 and stored at 7208C. Aliquots were
subsequently diluted to ®nal concentration of
a stock solution of
a
12 mLD50 ml71 with phosphate bu€ered saline which con-
tained 0.05 M NaH2PO4/NaPO4 (pH 7.4) and 0.2% bovine
serum albumin (Sigma, Poole, Dorset, U.K.) and stored at
4 ± 108C for periods up to 4 weeks.
Surgical procedures
Experimental procedures were licensed under the Animals
(Scienti®c Procedures) Act 1986, and performed in accor-
dance with Home Oce guidelines (U.K.). Rats were
anaesthetized with hypnorm/hypnovel in water (2 : 2 : 4,
3.33 ml kg71 i.p.). One ml of tetanus toxin solution (contain-
ing 12 mLD50 ml71 of tetanus toxin) was placed stereo-
tactically into the left hippocampus (co-ordinates 3.0 mm
posterior, 3.5 mm lateral to bregma and 3.3 mm below the
neocortical surface) according to Paxinos & Watson (1986).
A
single injection of tetanus toxin into the dorsal
Placement of toxin was undertaken manually using a
hippocampus of the rat leads to a chronic epileptic syndrome
in which both generalized and non-generalized seizures occur
spontaneously and intermittently. This model is considered to
re¯ect human complex partial epilepsy, in terms of type of
seizures that occur, the incidence and nature of the
behavioural abnormalities and the fact that anti-epileptic
drugs used in the treatment of complex partial seizures, are
e€ective in this model (Mellanby et al., 1977; 1981; 1984;
1985; Hawkins & Mellanby, 1986; 1987). Carbamazepine,
considered clinically to be the drug of choice in the treatment
of complex partial seizures, is particularly e€ective in this
model (Hawkins et al., 1985).
In the present study we have used the rat tetanus toxin
model of complex partial epilepsy to study the temporal
anticonvulsant actions of levetiracetam. Ecacy was ascer-
tained by measurement of EEG activity using video telemetry
with concurrent measurement of blood and CSF kinetics of
levetiracetam. Because the anticonvulsant action of carbama-
zepine is well documented in this model, we compared the
action of carbamazepine with that of levetiracetam. The
present study demonstrates that levetiracetam has marked
anticonvulsant action at pharmacokinetically determined
levetiracetam concentrations. Pilot data from this study have
been previously published in abstract form (Doheny et al.,
1995; 1997a, b).
Hamilton 7102N syringe over a 1-min period. Subsequently,
bipolar recording electrodes (constructed from twisted Te¯on
coated stainless steel wire (diameter 0.125 mm, Medwire
Corp., New York, U.S.A.) with one tip cut back to 500 ±
750 mm) were placed stereotactically in the right hippocam-
pus. The placement co-ordinates were: 3.1 mm posterior,
2.9 mm lateral and 2.5 mm below the neocortical surface
(Paxinos & Watson, 1986). A third wire was connected to
one of the skull anchor screws and was used to earth the
animal during electroencephalographic recordings. Finally,
the three wires were crimped to contact pins (AWP
Electronics Ltd, Redhill, Surrey, U.K.), wires ®xed to the
skull using dental cement and animal sutured. Post surgery
animals were housed individually in perspex cages with free
access to food and water. A 12 h light/dark cycle was
maintained.
Electoencephalographic (EEG) recordings
One to two days post surgery, when complete recovery from
the surgical procedure occurred, EEG recordings were
commenced. The headstage contacts were connected, via a
slip ring to allow free movement, to a Neurolog System
comprising of an AC preampli®er, an AC/DC ampli®er and
®nally a ®lter (Digitimer, U.K.). The ampli®ed hippocampal
EEG signal was then digitized (1401 plus, Cambridge
Electronic Design, Cambridge, U.K.) and stored on computer
for o€-line analysis using the SPIKE2 software (Cambridge
Electronic Design, Cambridge, U.K.). The animals (six per
study group) were ®lmed continuously during an EEG
recording session using a Panasonic infra-red camera and a
time-lapse video recorder so that seizures could be reviewed
and categorized.
Methods
Animals
Male Sprague-Dawley rats (Harlen Olac, Bicester, U.K.),
weighing 250 ± 350 g, were housed in groups of four for 7 ± 14
British Journal of Pharmacology vol 135 (6)

H.C. Doheny et al
Carbamazepine and levetiracetam in focal epilepsy
1427
Minipump placement
seizure frequency was performed using a two-way non-
parametric analysis of variance. Seizure severity, as demon-
strated by seizure duration, was analysed using a t-test. Data
that were not normally distributed were analysed using the
Mann ± Whitney Rank Sum Test.
Five to seven days after the onset of the tetanus toxin
induced seizure syndrome, rats were re-anaesthetized, as
described above, and an Alzet osmotic minipump (Charles
River, U.K.) containing carbamazepine (n=6; Sigma, Poole,
Dorset, U.K.), constituted in DMSO, propylene glycol and
ethyl alcohol (42.5 : 42.5 : 15, v : v : v), or levetiracetam (n=6;
UCB SA, Chemin du Foriest, Belgium), constituted in saline,
was placed in the peritoneal cavity. Control animals (n=6)
had minipumps implanted that only contained the corre-
sponding drug vehicle.
Results
Behaviour during the seizure syndrome and seizure course
The tetanus toxin-induced seizure syndrome typically began
1 ± 3 days post administration of tetanus toxin, and onset of
seizures was accompanied by a general increase in irritability
and diculty with handling. Seizures themselves produced a
variety of stereotyped behavioural changes, which altered as
the seizure syndrome progressed. The seizures were divided
into two types: non-generalized and generalized. Non-
generalized seizures included behaviours such as immobility,
vibrissal or eyelid twitching and gentle head nodding and
e€ectively corresponded to a partial seizure. During such
seizures the animals were generally not aware of their
surroundings, as determined by their unresponsiveness to
any external stimuli or handling. Generalized seizures
encompassed not only the behaviour described above, but
in addition rearing with or without forelimb clonus or falling
also occurred and this was considered to be equivalent to a
secondarily generalized seizure.
Typically, seizures tended to cluster, with the ®rst cluster
starting 1 ± 2 days post tetanus toxin administration and the
second starting 3 ± 5 days post tetanus toxin administration
and lasting for 7 ± 10 days. Peak seizure frequency occurred
approximately 5 ± 6 days post tetanus toxin administration
(i.e. during the second cluster). Seizure type evolved during
each seizure cluster so that non-generalized seizures evolved
into generalized seizures. This pattern of seizure general-
ization also pertained when the seizure syndrome became
established. Furthermore, as the seizure type evolved, the
duration of the seizures became progressively longer. Thus, at
the beginning of the seizure syndrome the duration of non-
generalized seizures was substantially shorter than that of
generalized seizures. However, as the seizure syndrome
progressed and became established, the di€erence in seizure
duration between non-generalized and generalized seizures
became minimal. Seizures did not exhibit any diurnal pattern,
since there was no signi®cant di€erence in seizure frequency
when the day and night time periods were compared
(mean+s.e.mean seizures; day=655+48.7, night=691+51.3;
P=0.908, Mann ± Whitney rank sum test).
Jugular vein and cisterna magna canulation
In order to determine the peripheral pharmacokinetics
(blood) and the central neuropharmacokinetics (CSF) of
levetiracetam and carbamazepine separate experiments were
undertaken in normal non-seizing rats. Rats were anaesthe-
tized with hypnorm/hypnovel in water (2 : 2 : 4, 3.33 ml kg71
i.p.), an Alzet osmotic minipump was placed into the
peritoneal cavity and catheters implanted in the cisterna
magna, for CSF sampling, and the right jugular vein, for
blood sampling, as previously described (Patsalos et al.,
1992). In these experiments, the minipump was adapted in
that a short coiled tubing (PE 40) was attached to the outlet
of the minipump which represented a dead volume of osmotic
di€usion of 2 days. The length of the coil was established
from pilot experiments and was designed so as to delay drug
delivery into the peritoneal cavity until 2 days after the
placement of the minipump, at which time animals had
recovered from the surgical procedure.
Carbamazepine and levetiracetam administration and
blood and CSF sampling
The implanted minipumps were set to deliver into the
peritoneal cavity 4 mg kg71 h71 carbamazepine and 8 or
16 mg kg71 h71 levetiracetam. Two days after surgical
implantation, when complete recovery was achieved, CSF
(30 ml) and blood (50 ml) samples were collected at 30 min
intervals for 9 h (denoted as day 1). On four additional days
(denoted as days 2, 4, 6 and 7), blood and CSF sampling was
undertaken at 30 min intervals for 3.5 h. Blood and CSF
samples were collected in 0.5 ml polypropylene centrifuge
tubes (Tre€ Lab, Degersheim, Switzerland). Blood samples
were centrifuged for 5 min at 11,000 g to separate the cells
from the sera. Sera were transferred to new tubes and sera
and CSF were stored at 7708C until required for analysis.
Carbamazepine and carbamazepine epoxide, the primary
pharmacologically active metabolite of carbamazepine, con-
centrations in sera and CSF were determined using high
performance liquid chromatography (HPLC) with ultraviolet
detection as previously described (Sokomba et al., 1988).
Levetiracetam concentrations were similarly determined using
HPLC with ultraviolet detection by the method of Ratnaraj
et al. (1996).
EEG Activity
The EEG of control animals (minipumps containing drug
vehicle only) showed baseline low frequency ®eld potential
oscillations. The amplitude of the oscillations was dependent
on the activity of the animal so that when the animal was
sleeping the baseline was relatively ¯at while movement
around the recording cage resulted in large amplitude ®eld
potential oscillations in the 4 ± 12 Hz range. Three types of
epileptiform activity were recorded: inter-ictal spikes, poly-
spikes and seizures, and these are similar to those previously
reported (Finnerty & Je€erys, 2000). Inter-ictal spikes and
Data analysis
All data are expressed as mean+s.e.mean, or median+
interquartile range (IQR). Statistical analysis of changes in
British Journal of Pharmacology vol 135 (6)

1428
H.C. Doheny et al
Carbamazepine and levetiracetam in focal epilepsy
polyspikes, were not accompanied by any obvious behaviour-
al changes. Inter-ictal spikes lasted 25 to 100 ms and
consisted of a high frequency oscillation. Polyspikes lasted
0.4 ± 2 s and consisted of a number of inter-ictal spikes
conjoined. The electrographic trace recorded during a seizure
was composed of units which resembled inter-ictal or
polyspikes with the di€erence that seizure activity continued
for longer periods. Furthermore, the normal baseline
hippocampal ®eld potential oscillations, seen when recording
freely moving activity, disappeared leaving a ¯at EEG trace
between inter-ictal type events during a seizure.
Carbamazepine and carbamazepine-epoxide
concentrations in serum and CSF
Carbamazepine was detectable in both the serum and CSF
compartments at 0.5 h after initiation of peritoneal admin-
istration. In contrast, carbamazepine-epoxide was simulta-
neously detected in serum and CSF, somewhat later at 2.5 h
after carbamazepine administration. Cmax values for serum
and CSF carbamazepine and carbamazepine-epoxide were
achieved at 2.5 ± 3.3 h and at 8.0 ± 9.0 h after initiation of
carbamazepine administration. During the subsequent 7 day
period, steady-state serum and CSF concentrations of
carbamazepine and carbamazepine-epoxide declined and the
data for days 1, 2, 4,
6 and 7
after 4 mg kg71 h71
carbamazepine administration are shown in Figure 1.
Levetiracetam concentrations in serum and CSF
Levetiracetam was detectable in the serum compartment at
1.0 h after initiation of intraperitoneal administration.
However, levetiracetam was not detectable in the CSF
compartment until 1.5 h after levetiracetam administration.
Both serum and CSF levetiracetam concentrations rose
linearly and dose-dependently with Cmax values being
achieved at 5.5 ± 9.0 h, at which time steady-state was
achieved. The mean steady-state serum and CSF levetirace-
Figure 1 The temporal relationship between the daily median
number (+ IQR) of generalized (A) and non-generalized (B) seizures
in control (shaded) and carbamazepine (CBZ; non-shaded) treated
rats and CBZ and carbamazepine epoxide (CBZ-E) concentration in
serum and cerebrospinal ¯uid (CSF) in CBZ administrated rats
(4 mg kg71 h71; n=6 per group).
tam concentrations for days 1, 2, 4,
6 and 7 after
8 mg kg71 h71 and 16 mg kg71 h71 levetiracetam administra-
tion are shown in Figures 3 and 4 respectively.
Anticonvulsant effect of carbamazepine
ized seizures, reduction in non-generalized seizures was only
12.6% (P=0.533). Neither reductions achieved statistical
signi®cance. However, statistical signi®cance was achieved for
the reduction in generalized seizures (90% reduction;
P=0.008) when seizure frequency was compared to controls
during the ®rst 2 days of carbamazepine administration.
Table 1 shows the duration of generalized and non-
generalized seizure in carbamazepine treated rats compared
to controls. It can be seen that carbamazepine administration
was associated with a signi®cant 27.3% reduction in overall
duration for generalized seizures compared to controls
(P=0.0001). In contrast carbamazepine administration was
associated with a statistically insigni®cant 7.9% reduction in
non-generalized seizure duration (P=0.414).
Figure
1 illustrates the temporal relationship between
generalized and non-generalized seizures and mean steady-
state carbamazepine and carbamazepine-epoxide serum and
CSF concentrations in rats treated with 4 mg kg71 h71
carbamazepine compared with controls. During days 1 and
2 when both serum and CSF carbamazepine and carbama-
zepine-epoxide concentrations were relatively high, carbama-
zepine administration was clearly associated with a signi®cant
reduction in the frequency of generalized (P=0.0001) but not
non-generalized seizures. During days 3 ± 7 the e€ect of
carbamazepine on generalized seizures dissipated and this was
associated with concurrent reduction in carbamazepine and
carbamazepine epoxide concentrations in both the serum and
CSF compartments.
Anticonvulsant effect of levetiracetam
Figure 2 summarizes the anticonvulsant e€ect of carbama-
zepine over the entire 7 day study period. It can be seen that
whilst carbamazepine administration was associated with a
35.5% (P=0.617) reduction in the total number of general-
Figures 3 and 4 illustrate the temporal relationship between
generalized and non-generalized seizures and mean steady-state
British Journal of Pharmacology vol 135 (6)

H.C. Doheny et al
Carbamazepine and levetiracetam in focal epilepsy
1429
levetiracetam serum and CSF concentrations in rats treated with
levetiracetam (8 and 16 mg kg71 h71 respectively) compared
with controls. Levetiracetam administration was clearly asso-
ciated with an overall signi®cant reduction in the frequency of
both generalized and non-generalized seizures during the 7 day
study period. At the lower levetiracetam dose, levetiracetam
reduced the median number of generalized seizures on 6 of the 7
days and non-generalized seizures on 3 of the 7 days (Figure 3).
However, seizure suppression was more substantial for general-
ized seizures compared with non-generalized seizures. Further-
more, at the higher levetiracetam dose, levetiracetam reduced
generalized seizure frequency on each of the 7 days (P=0.0004)
but had little additional e€ect on non-generalized seizures
compared to the lower levetiracetam dose (Figure 4).
levetiracetam administration (Figure 3, 39.1% and 35.8%,
respectively), the reductions did not attain statistical sig-
ni®cance (P=0.071 and P=0.635, respectively). A dose-
dependent reduction in the frequency of generalized (56.7%)
and non-generalized (41.2%) seizures was observed following
16 mg kg71 h71 levetiracetam administration (Figure 4).
However, whilst the decrease in the incidence of generalized
seizures attained statistical signi®cance (P=0.00004), the
decrease in non-generalized seizures did not (P=0.525).
Table 2 shows the duration of generalized and non-general-
ized seizures in levetiracetam treated rats compared to controls.
For generalized seizures, it can be seen that levetiracetam
exhibited a dose-dependent and statistically signi®cant reduc-
tion in overall seizure duration compared to controls (19.9 and
35.7% reduction for 8 and 16 mg kg71 h71 respectively,
P=0.0001 for both doses). In contrast levetiracetam had no
signi®cant e€ect on non-generalized seizure duration.
Figure 5 shows the anticonvulsant e€ect of levetiracetam
over the entire 7 day study period. It can be seen that while
there was a substantial reduction in the total number of
generalized and non-generalized seizures after 8 mg kg71 h71
Discussion
The tetanus toxin model is a chronic model of complex
partial seizures with features very similar to those seen in
human epilepsy namely both non-generalized and generalized
seizures occur over several weeks and seizures are intermittent
in nature. Because the epilepsy is somewhat reversible in that
the animals eventually stop seizing and the accompanying
EEG abnormalities disappear, it is possible to distinguish
between the e€ects of ongoing seizures and the long term
consequences of seizures. Furthermore, since the focal
seizures occur at a consistent rate for 4 ± 6 weeks (Hawkins
& Mellanby, 1987; Je€erys & Williams, 1987), it is possible to
monitor seizure frequency for several days before, during and
after the administration of an anticonvulsant drug. The
model therefore constitutes an excellent experimental model
for the study of not only seizures but also of epilepsy.
The tetanus toxin model used in this study is based on an
adaptation of the model previously described by Mellanby et
al. (1977; 1981). Because of di€erences in the experimental
Figure 2 The overall e€ect of 4 mg kg71 h71 carbamazepine on
generalized and non-generalized seizures compared to control during
a week period. C=control, CBZ=carbamazepine. (n=6 per
group).
1
Table 1 Comparison of seizure duration of generalized and non-generalized seizures in carbamazepine treated and control rats during
the 7 day period of drug administration
Carbamazepine
(4 mg kg71 h71
)
Control
Generalized
Non-generalized
Generalized
Non-generalized
Rat
1
Mean (+s.e.mean)
Mean (+s.e.mean)
130.4
(4.9)
138.3
(14.9)
95.4
(3.7)
103.0
(4.1)
88.5
110.3
(4.5)
55.0
113.5
(7.4)
94.3
(8.5)
76.7
(6.3)
93.4
(2.0)
38.6
(2.7)
89.0
(9.6)
97.0
(3.0)
45.0
(5.0)
97.0
2
(14.5)
86.0
(1.2)
73.3
(21.9)
89.3
(18.9)
95.6
(6.4)
3
(3.0)
83.5
4
(0.5)
111.0
(13.0)
40.0
5
(19.0)
98.0
(6.5)
6
(5.0)
Overall
mean
(+s.e.mean)
110.7
5.6
83.4
6.2
80.5
4.0
76.8
8.1
Seizure duration is in seconds.
British Journal of Pharmacology vol 135 (6)

1430
H.C. Doheny et al
Carbamazepine and levetiracetam in focal epilepsy
Figure 3 The temporal relationship between the daily median
number (+IQR) of generalized (A) and non-generalized (B) seizures
in control (shaded) and levetiracetam (non-shaded) treated rats and
levetiracetam concentration in serum and cerebrospinal ¯uid (CSF) in
levetiracetam administrated rats (8 mg kg71 h71; n=6 per group).
Figure 4 The temporal relationship between the daily median
number (+IQR) of generalized (A) and non-generalized (B) seizures
in control (shaded) and levetiracetam (non-shaded) treated rats and
levetiracetam concentration in serum and cerebrospinal ¯uid (CSF) in
levetiracetam administrated rats (16 mg kg71 h71; n=6 per group).
protocol it was necessary to further characterize the model
and also to further develop, characterize and validate the
EEG monitoring of the resultant seizures. The model
adaptations made in the present study include: (1) The site
of tetanus toxin administration; unilateral versus bilateral
and dorsal versus ventral hippocampus; (2) Amount of
unknown and may be the consequence of di€erences in
anatomical and physiological di€erences between di€erent
batches of animals and also variation in di€erent aliquots of
tetanus toxin.
In
this
tetanus
toxin
model,
carbamazepine
(4 mg kg71 h71) exhibited no signi®cant anticonvulsant e€ect,
compared to control, when the entire 7 day study period was
evaluated but the reduction in generalized seizures was
greater (35.5%) than that for non-generalized seizures
(12.6%). However, when the ®rst 2 days of carbamazepine
administration are compared to those of vehicle controls,
carbamazepine was associated with a signi®cant reduction in
both generalized seizure frequency (90%) and duration
(25%). Non-generalized seizures were una€ected. Thus,
carbamazepine exhibited a time-dependent anticonvulsant
e€ect and this exactly paralleled the central (CSF) and
peripheral (serum) kinetics of carbamazepine and carbama-
zepine-epoxide in that steady-state concentrations declined
over time, with the highest concentrations achieved during
the ®rst 2 days (Figure 1). The decline in carbamazepine
tetanus
toxin
administered
(12 mLD50 ml71
versus
6 mLD50 ml71); (3) Route of drug administration, continuous
infusion via an intraperitoneal osmotic minipump over 7 days
versus three times a day oral administration until a 7 day
seizure remission period had occurred; (4) Timing of drug
administration, 7 ± 9 days after the induction of seizures
versus ®rst day of seizure occurrence. Even though there are
di€erences in the procedures for inducing the seizure model,
the time course of electroencephalographic features and
indeed the behavioural manifestations were almost identical
in the two studies (Mellanby et al., 1977; 1981). A notable
observation is that in comparison to generalized seizures,
non-generalized seizures exhibited considerable variation in
baseline values between studies (Figures 2 and 5). However,
the exact reason for this, which is typical of this model, is
concentrations is attributable to autoinduction,
a well
British Journal of Pharmacology vol 135 (6)

H.C. Doheny et al
Carbamazepine and levetiracetam in focal epilepsy
1431
Table 2 Comparison of seizure duration of generalized and non-generalized seizures in levetiracetam treated and control rats during
the 7 day period of drug administration
Levetiracetam
(8mg kg71 h71
Levetiracetam
(16mg kg71 h71
)
Control
)
Control
Generalized Non-generalized Generalized Non-generalized Generalized Non-generalized Generalized Non-generalized
Mean (+s.e.mean) Mean (+s.e.mean) Mean (+s.e.mean) Mean (+s.e.mean)
Rat
1
119.3
20.6
(1.2)
22.9
(2.1)
21.3
(0.7)
21.3
(1.1)
39.6
(5.5)
32.1
(3.3)
63.6
21.5
(2.4)
16.6
(1.7)
28.9
(2.3)
36.7
(0.8)
25.6
(2.1)
33.1
(2.0)
156.0
107.0
(3.0)
111.0
96.0
(10.6)
94.0
(4.9)
126.6
(9.7)
85.8
(12.3)
93.3
(10.3)
87.8
(1.9)
88.6
(4.1)
(1.4)
75.6
(3.8)
43.9
(1.9)
55.1
(1.3)
82.9
(8.0)
48.9
(2.5)
(10.2)
126.9
(8.2)
131.4
(5.9)
110.7
(3.5)
118.3
(5.0)
(1.7)
123.0
(6.3)
128.6
(11.5)
84.4
(7.9)
83.8
(8.9)
99.3
(7.0)
2
117.0
(12.5)
86.4
(17.5)
116.3
(3.0)
104.5
(6.8)
108.4
(3.8)
(17.4)
103.0
(9.6)
113.0
(1.5)
120.0
(2.7)
111.5
(10.6)
3
4
5
6
152.0
(6.9)
Overall mean
(+s.e.mean)
102.1
(3.9)
25.1
(1.1)
65.7
(2.3)
26.4
(1.0)
130.8
(3.0)
105.1
(4.9)
104.8
(4.2)
105.4
(4.4)
Seizure duration is in seconds.
described characteristic of carbamazepine (McNamara et al.,
1979; Sumi et al., 1987; Wedlund & Levy, 1983).
The anticonvulsant e€ect of carbamazepine observed in the
present study further validates this model of complex partial
seizures, particularly since carbamazepine is e€ective clini-
cally for the management of patients with complex partial
seizures (Porter, 1987). However, the maximum anticonvul-
sant e€ect of carbamazepine was much greater than that
previously reported (Hawkins et al., 1985). This can be
attributed in part not only to the di€erence in the mode of
model induction with the tetanus toxin (discussed above) but
perhaps more signi®cantly to the carbamazepine blood
concentrations achieved. Thus, whilst carbamazepine mean
steady-state serum concentrations of 29 and 11 mmol l71 were
achieved for days 1 and 2 respectively (Figure 1), Hawkins et
al. (1985) achieved serum concentrations of approximately
6 ± 9 mmol l71. The clinical therapeutic (target) range for
carbamazepine is 20 ± 50 mmol l71 (Patsalos, 2001). Hawkins
et al. (1985) administered carbamazepine by mouth on a
three times a day basis, with rough allowances made for any
volume of drug not completely ingested by the rats. Not only
is this approach inecient and time consuming but also
stressful to the animals, which in turn can in¯uence seizure
threshold. Perhaps, using an intraperitoneal osmotic mini-
pump, particularly for drugs with a relatively short half-life,
is a more reliable method for continuous drug administra-
tion, and indeed our animals did not exhibit any signs
indicative of overt stress. A notable disadvantage of the
osmotic minipump, however, is that it has a ®xed volume and
therefore limited duration of drug delivery.
Levetiracetam administration (8 and 16 mg kg71 h71) was
associated with an overall signi®cant reduction in the
frequency of both generalized and non-generalized seizures
during the 7 day study period. However, seizure suppression
was more substantial for generalized seizures compared with
non-generalized seizures. Furthermore, the e€ect of levetir-
acetam was dose-dependent so that after 8 mg kg71 h71
levetiracetam administration, generalized and non-generalized
seizure frequency was reduced by 39.1 and 35.8% and after
16 mg kg71 h71 levetiracetam the equivalent reductions were
Figure 5 The overall e€ect of 8 mg kg71 h71 (A) and
16 mg kg71 h71 (B) levetiracetam on generalized and non-generalized
seizures compared to control during a 1 week period. C=control,
LEV=levetiracetam. (*=P50.05, NPANOVA; n=6 per group).
British Journal of Pharmacology vol 135 (6)

1432
H.C. Doheny et al
Carbamazepine and levetiracetam in focal epilepsy
56.7 and 41.2%. However, the decrease in the frequency of
non-generalized seizures was not statistically signi®cant.
Levetiracetam also exhibited a signi®cant dose-dependent
reduction in overall generalized seizure duration compared to
controls (Table 2) but was without signi®cant e€ect in
relation to non-generalized seizure duration. These observa-
tions suggest that whereas levetiracetam does not e€ect
ictogenesis per se it does reduce seizure severity and,
particularly, seizure generalization.
impaired (Bekenstein & Lothman, 1993; Sloviter, 1991;
Whittington & Je€erys, 1994).
Levetiracetam exhibits a wide spectrum of anticonvulsant
e€ects in various animal models of generalized seizures in rats
and mice (Gower et al., 1992; 1995; Margineanu & Wulfert,
1995). Levetiracetam also retards the development of kindling
induced in mice by subthreshold doses of pentylenetetrazole
(Gower et al., 1992) and signi®cantly reduces the severity and
duration of both focal and secondary generalized seizures and
also of afterdischarge duration in amygdala-kindled rats
(Loscher et al., 1996). However, the exact mechanism of
action of levetiracetam is unknown, although levetiracetam
has recently been shown to inhibit high-voltage activated
Ca2+ currents in hippocampal pyrimidal neurons (Niespod-
ziany et al., 2000). A direct action on GABA is considered
unlikely (Margineanu & Wulfert, 1995; Sills et al., 1997;
Loscher et al., 1996) particularly since levetiracetam has no
signi®cant anity for the GABAA receptor complex (Noyer
et al., 1995) and extracellular concentrations of GABA in the
frontal cortex or the hippocampus are una€ected by
levetiracetam (Tong & Patsalos, 2001). Furthermore, since
levetiracetam in the present study has been observed to have
signi®cant ecacy in the tetanus toxin model, these data
further suggest perhaps an indirect e€ect on GABA by
levetiracetam. Interestingly such an indirect action on GABA
by levetiracetam was suggested from a recent in vitro study,
which reported that levetiracetam reversed the inhibitory
action of negative allosteric modulators on both GABA and
glycine-gated currents in hippocampal neurons (Rigo et al.,
2000). Indeed the study by Rigo et al. (2000) is particularly
noteworthy in relation to the ecacy of levetiracetam in the
tetanus toxin model since tetanus toxin induced seizures are
the consequence of a block, in part, on the presynaptic
release of both GABA and glycine.
The ®nding that levetiracetam has a speci®c binding site in
the rat CNS (Noyer et al., 1995) needs comment. The site
appears to be speci®cally con®ned to the synaptic plasma
membranes of the hippocampus, cortex, cerebellum and
striatum, as no speci®c binding was observed in a range of
peripheral tissues. Levetiracetam binding anity to hippo-
campal membranes is modest (Kd=780+115 nm) but is of
high binding capacity (Bmax=9.1+1.2 pmol mg71 protein).
Similar Kd and Bmax values were obtained with the cortex,
cerebellum and striatum. Since the Kd of levetiracetam for
this binding site is approximately 1000 fold less than the
concentrations achieved in the CSF compartment in the
present study, it can be concluded that this binding site is
unlikely to play a signi®cant role in the anticonvulsant action
of levetiracetam in this model.
The kinetic pro®les of levetiracetam correlated well with
the observed anticonvulsant pro®le. Steady-state levetirace-
tam concentrations in serum and CSF were achieved by the
second day of sampling and these concentrations were
maintained for the entire study period. Unlike carbamaze-
pine, levetiracetam exhibited no evidence of autoinduction.
Both serum and CSF levetiracetam concentrations increased
dose-dependently and essentially linearly and con®rm pre-
vious data (Doheny et al., 1999; Tong & Patsalos, 2001). As
this is the ®rst study whereby the administration of
levetiracetam was by osmotic minipump and other studies
of the anticonvulsant ecacy and pharmacokinetics of
levetiracetam comprised of single dose or multiple dose
administration by intraperitoneal injection, there are no
comparable data to compare directly. However, from the
levetiracetam serum and CSF concentrations achieved in the
present study the levetiracetam doses used (4 mg kg71 h71
and 8 mg kg71 h71; Figures 3 and 4) can be considered
equivalent to 40 mg kg71 and 60 mg kg71 levetiracetam i.p.,
respectively (Doheny et al., 1999; Tong & Patsalos, 2001).
Consequently, the levetiracetam doses used in the present
study can be considered relevant, since in animal models of
epilepsy, the e€ective doses at which 50% of animals respond
(ED50) ranges from 5 to 30 mg kg71 (Gower et al., 1992) and
the ED50 for suppression of secondarily generalized seizures
in the amygdala-kindled model of epilepsy is 40 mg kg71
(Loscher & Honack, 1993). Furthermore, the levetiracetam
doses used in the present study are comparable to those used
clinically where levetiracetam at doses up to 60 mg kg71 are
used to treat patients with epilepsy (Patsalos, 2000).
Tetanus toxin is a potent clostridal neurotoxin, which
blocks preferentially the presynaptic release of GABA and
glycine, the two major inhibitory neurotransmitters of the
CNS. Indeed, several studies have reported on the decrease in
GABA release and of the associated reduction in the size of
IPSPs (Whittington & Je€erys, 1994; Jordan & Je€erys, 1992;
Empson & Je€erys, 1993). These e€ects are considered to be
the consequence of the cleavage of synaptobrevin by tetanus
toxin (Schiavo et al., 1992). However, the epileptogenic e€ect
of tetanus toxin far outlasts the impairment of GABA release
(Whittington
&
Je€erys, 1994). Furthermore, synaptic
In conclusion this study demonstrates that whilst levetir-
acetam signi®cantly reduces the frequency of both generalized
and non-generalized seizures, its e€ect is substantially greater
on generalized seizures. Furthermore, levetiracetam acted to
reduce the severity and duration of seizure generalization.
inhibition in the ipsilateral focus (at the site of injection of
tetanus toxin) drops to 10% of controls during the ®rst 2
weeks, but there is a subsequent recovery. In contrast, the
loss of synaptic inhibition in the contralateral mirror focus
never drops below 50% (Empson
&
Je€erys, 1993;
Whittington & Je€erys, 1994). Thus, these studies suggest a
more complex mechanism of action of the tetanus toxin and/
or the induced focus than just the blockade of inhibitory
(GABA and glycine) neurotransmission. A further mechan-
ism may relate to inhibitory neurones not being recruited by
the hippocampal network, either because they are less
excitable or because the synapses that excite them are
This work was supported in part by a Brain Research Trust
Student Fellowship awarded to Helen C. Doheny. We are grateful
to UCB SA, Chemin du Foriest, Belgium for the supply of
levetiracetam and !,2,2-dimethyl-5-oxo-1-pyrrolidine acetamide.
British Journal of Pharmacology vol 135 (6)

H.C. Doheny et al
Carbamazepine and levetiracetam in focal epilepsy
1433
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