Epilepsia, 47(1):10–20, 2006

Blackwell Publishing, Inc.

C 2006 International League Against Epilepsy

Laboratory Research

Pharmacodynamic and Pharmacokinetic Characterization of

Interactions between Levetiracetam and Numerous Antiepileptic
Drugs in the Mouse Maximal Electroshock Seizure Model: An
Isobolographic Analysis

∗ Jarogniew J. Luszczki, †Marta M. Andres, ∗ Piotr Czuczwar, ∗ Anna Cioczek-Czuczwar,

‡Neville Ratnaraj, ‡Philip N. Patsalos, and ∗ †Stanislaw J. Czuczwar
∗ Department of Pathophysiology, Medical University of Lublin, and †Department of Physiopathology, Institute of Agricultural
Medicine, Lublin, Poland; and ‡Pharmacology and Therapeutics Unit, Department of Clinical and Experimental Epilepsy, Institute
of Neurology, London, United Kingdom

Summary: Purpose: Approximately 30% of patients with
epilepsy do not experience satisfactory seizure control with
antiepileptic drug (AED) monotherapy and often require poly-
therapy. The potential usefulness of AED combinations, in terms
of efficacy and adverse effects, is therefore of major importance.
The present study sought to identify potentially useful AED com-
binations with levetiracetam (LEV)
Methods: With isobolographic analysis, the mouse maxi-
mal electroshock (MES)-induced seizure model was investi-
gated with regard to the anticonvulsant effects of carbamazepine
(CBZ), phenytoin, phenobarbital (PB), valproate, lamotrigine,
topiramate (TPM), and oxcarbazepine (OXC), administered
singly and in combination with LEV. Acute adverse effects were
ascertained by use of the chimney test evaluating motor per-
formance and the step-through passive-avoidance task assessing
long-term memory. Brain AED concentrations were determined
to ascertain any pharmacokinetic contribution to the observed
antiseizure effect.
Results: LEV in combination with TPM, at the fixed ratios of
1:2, 1:1, 2:1, and 4:1, was supraadditive (synergistic) in the MES
test. Likewise, the combination of LEV with CBZ (at the fixed
ratio of 16:1) and LEV with OXC (8:1 and 16:1) were supraaddi-
tive. In contrast, all other LEV/AED combinations displayed ad-
ditivity. Furthermore, none of the investigated LEV/AED combi-
nations altered motor performance and long-term memory. LEV
brain concentrations were unaffected by concomitant AED ad-
ministration, and LEV had no significant effect on brain concen-
trations of concomitant AEDs.
Conclusions: These preclinical data would suggest that
LEV in combination with TPM is associated with ben-
eficial anticonvulsant pharmacodynamic interactions. Sim-
ilar, but less profound effects were seen with OXC
and CBZ. Key Words: Levetiracetam—Carbamazepine—
Oxcarbazepine—Topiramate—Antiepileptic drugs—Maximal
electroshock—Pharmacodynamic interactions—Isobolographic
analysis.

Levetiracetam [LEV, [S]-α-ethyl-2-oxo-1-pyrrolidine have a broad spectrum of activity in that it is effective

acetamide] is a novel antiepileptic drug (AED) that is li-
censed for clinical use as adjunctive therapy to treat pa-
tients with intractable partial-onset seizures with or with-
out secondary generalization (1–4). The drug appears to
Accepted July 16, 2005.
Address correspondence and reprint requests to Prof. Dr. S.J.
Czuczwar at Department of Pathophysiology, Medical University
of Lublin, Jaczewskiego 8, PL-20-090 Lublin, Poland. E-mail:
czuczwarsj@yahoo.com
The results of this study were presented in part at the Neurochemical
Conference–Molecular Basis of Neurological Diseases and New Thera-
peutic Strategies, Warsaw, Poland, November 28–29, 2003 [abstract in
Pol J Pharmacol 2003;55:853-854].
against juvenile myoclonic epilepsy (5,6); tonic–clonic,
absence, and myoclonic epilepsy (7–9); photosensitive
epilepsy (10), and atypical absence or atonic seizures (11).
Furthermore, increasing evidence suggests significant ef-
ficacy in children aged 10 years and younger with partial-
onset seizures and in those with myoclonic seizures (12).
That LEV is efficacious as a monotherapy regimen is high-
lighted by the fact that patients with refractory partial
epilepsy can be successfully converted to monotherapy
(13–15). Additionally, LEV has been successfully used
as a first-line drug to treat newly diagnosed patients with
epilepsy (16).

10
 INTERACTIONS OF LEVETIRACETAM AND AEDS IN THE MES TEST
Although the exact molecular mechanism(s) of anticon-
vulsant action of LEV is (are) unknown, several cellular ef-
fects may account for its antiseizure activity. For example,
it has been reported that LEV reduces voltage-operated
K+ current and inhibits the delayed-rectifier K+ current in
neurons (17), reduces N-type and partially P/Q-type high-
voltage–activated (HVA) Ca2+ currents (18–20), but not
low-voltage–activated Ca2+ currents (21), and suppresses
the inhibitory action of zinc and β-carbolines on GABAA -
and glycine-gated currents (22). Molecular studies involv-
ing transgenic mice suggest that LEV binds to a synaptic
vesicle protein 2A (SV2A), which is involved in vesicle
neurotransmitter exocytosis, and that the affinity of bind-
ing to SV2A significantly correlates with anticonvulsant
potency by a series of LEV derivatives (23). Interestingly,
LEV possesses properties that suggest that not only does
it suppress seizures but that it may also be antiepilep-
togenic (24). Thus whereas LEV was without effect in
the traditional acute maximal electroshock seizure (MES)
and pentylenetetrazole (PTZ) models, which are routinely
used to screen for potential new AEDs (25), it has substan-
tial efficacy in kindled and genetically epileptic animals
(26–30)
Because new AEDs such as LEV are invariably li-
censed, at least in the first instance, for use as adjunctive
therapy in patients with intractable epilepsy and because
30% of patients with epilepsy are prescribed polytherapy
regimens (two or more AEDs) in an attempt to control
their seizures (31–34), the question arises as to which AED
combinations are best, particularly because these patients
often experience numerous problems, including acute and
chronic CNS side effects and idiosyncratic reactions that
are exacerbated by pharmacokinetic or pharmacodynamic
interactions or both (35–37). Indeed, Deckers et al. (38)
highlighted the fact that clinically, no evidence-based data
suggest that any particular AED combination is superior
to another. However, recently Stephen and Brodie (39) re-
ported on their audit of their clinical practice comprising
patients with intractable epilepsy, and they identified the
10 most effective two-AED combinations that were asso-
ciated with seizure freedom for >1 year. When these are
compared with published observations from experiments
on animals by using the MES test, remarkable parallelism
is found between the experimental and clinical observa-
tions and confirms the usefulness of preclinical studies in
general and the method of isobolography in particular as
an aid to preselect potentially efficacious AED combina-
tions for clinical use (40).
Growing evidence, both experimental and clinical, is
found to suggest that LEV is associated with pharmaco-
dynamic interaction and that whereas some of the inter-
actions can be potentially favorable, others are undesir-
able. For example, despite inactivity of LEV in the PTZ
seizure model, its anticonvulsant efficacy was apparent
when LEV was coadministered with topiramate (TPM;
11
41). Anticonvulsant synergy has also been reported be-
tween LEV and diazepam (DZP) in a rat model of status
epilepticus (42). Adverse pharmacodynamic interactions
have been reported in patients between LEV and carba-
mazepine (CBZ; 43) and between LEV and TPM (44).
Thus the present study sought to characterize the types of
interaction between LEV and a variety of conventional and
new AEDs against MES-induced seizures in mice by us-
ing isobolographic analysis. Additionally, we investigated
the various AED combinations in relation to motor impair-
ment by the use of the chimney test and long-term memory
alteration by the use of the step-through passive-avoidance
task. Finally, we measured brain AED concentrations to
ascertain whether the observed effects were consequent to
a pharmacodynamic or a pharmacokinetic interaction or
both.
MATERIALS AND METHODS
Animals and experimental conditions
All experiments were performed on adult male albino
Swiss mice weighing 22–26 g. The mice were kept in
colony cages with free access to food and tap water ad
libitum, under standardized housing conditions (natural
light–dark cycle, temperature of 23 ± 1◦ C, relative humid-
ity of 55 ± 5%). After 7 days of adaptation to laboratory
conditions, the animals were randomly assigned to ex-
perimental groups consisting of eight mice. Each mouse
was used only once. All tests were performed between
9 a.m. and 3 p.m. Procedures involving animals and their
care were conducted in accordance with current European
Community and Polish legislation on animal experimen-
tation. Additionally, all efforts were made to minimize
animal suffering and to use only the number of animals
necessary to produce reliable scientific data. The exper-
imental protocols and procedures described in this arti-
cle were approved by the Local Ethics Committee at the
Medical University of Lublin and complied with the Eu-
ropean Communities Council Directive of 24 November
1986 (86/609/EEC).
Drugs
The following AEDs were used in this study: LEV
(Keppra; UCB Pharma, Braine-l’Alleud, Belgium),
phenytoin (PHT; Polfa Warszawa, Poland), phenobar-
bital (PB; Polfa Krakow, Poland), valproate sodium
(VPA; ICN-Polfa, Rzeszow, Poland), CBZ (Polfa, Starog-
ard, Poland), TPM (Topamax, Cilag AG, Schaffhausen,
Switzerland), lamotrigine (LTG; Lamictal, Glaxo Well-
come, Kent, UK), and oxcarbazepine (OXC, Trileptal,
Novartis Pharma AG, Basel, Switzerland). All drugs ex-
cept VPA were suspended in a 1% solution of Tween 80
(Sigma, St. Louis, MO, U.S.A.) in saline and administered
intraperitoneally (i.p.) in a volume of 5 ml/kg body weight.
VPA was dissolved in 0.9% NaCl. Fresh drug solutions
were prepared ex tempore on each day of experimentation
Epilepsia, Vol. 47, No. 1, 2006
12 J. J. LUSZCZKI ET AL.
and administered as follows: PHT at 120 min; LEV, PB,
TPM, and LTG at 60 min, and OXC, CBZ, and VPA
at 30 min, before electroconvulsions, chimney test, step-
through passive-avoidance task, and brain sampling for
the measurement of AED concentrations. These drug ad-
ministration times were based on their biologic activity
from the literature and confirmed in our previous experi-
ments (40,45–48). The times of peak maximum anticon-
vulsant effects for AEDs were used as reference times in
all experimental tests and for brain AED concentration
determinations.
Maximal electroshock seizure test
Electroconvulsions were produced by means of an al-
ternating current (0.2 s; 25 mA; 500 V; 50 Hz) delivered
via ear-clip electrodes by a generator (Rodent Shocker,
Type 221; Hugo Sachs, Freiburg, Germany). The crite-
rion for the occurrence of seizure activity was the tonic
hindlimb extension. Protective activities of AEDs admin-
istered alone against MES-induced seizures were evalu-
ated as their median effective doses (ED50 values in mg/kg;
with their 95% confidence limits) according to the log-
probit method by Litchfield and Wilcoxon (49). The an-
ticonvulsant activity of a mixture of LEV with an AED
was evaluated and expressed as ED50mix corresponding
to a dose of a mixture necessary to protect 50% of mice
against tonic hindlimb extension in the MES test. The ex-
perimental procedure has been described in more detail in
our earlier studies (40,45–48,50).
Isobolographic analysis
To perform the isobolographic analysis of the interac-
tion between LEV and PHT, VPA, CBZ, PB, LTG, TPM,
and OXC (as regards their anticonvulsant activities against
MES-induced seizures), the AEDs in numerous fixed-
ratio combinations were administered to animals. Sub-
sequently, the experimentally derived ED50mix (± SEM)
values for these mixtures were determined by using the
log-probit method described by Litchfield and Wilcoxon
(49). Moreover, theoretically additive ED50add (±SEM)
values were calculated from the equation presented by
Porreca et al., (51), as follows:
ED50 add = ED50 drug 1 /P1
where P1 is a proportion of the first drug, fully effective
against maximal electroconvulsions (i.e., conventional
and newer AEDs) in the total amount of drug mixture.
It should be noted that for two-drug mixtures, the equa-
tion is true when: P1 + P2 = 1; where P2 is the proportion
of the second drug, virtually ineffective in the MES test
(LEV). The proportions of AEDs in a mixture are based
on a mass quantity of AEDs (for instance, a fixed-ratio
combination of 1:1 comprised equal amounts of LEV and
an AED). This particular kind of isobolographic analysis
(so-called type II) allows the acceptance of mass quantities
of drugs in mixture as a basis to construct the notation of
Epilepsia, Vol. 47, No. 1, 2006
fixed-ratio combinations. Thus the proportions of AEDs
in a mixture were calculated as ratios of mass quantity of
the constituent AEDs (for example, for the fixed-ratio of
1:16, the mixture comprised 1 mg of an active AED and
16 mg of a virtually ineffective drug, making 17 mg of a
two-drug mixture, in which the first AED was present in
the proportion of 1/17 = 0.059, and the second AED, in
the proportion of 16/17 = 0.941). Similarly, for the fixed
ratio of 1:4, the proportion of a fully active AED was
1/5 = 0.2, whereas the proportion of LEV was 4/5 = 0.8.
In brief, in the type II of isobolographic analysis, the fixed-
ratio combination describes how many times a dose of a
virtually ineffective drug prevails in the dose of a fully
effective drug in the mixture (for the fixed ratio of 1:16,
the LEV dose was 16-fold the dose of the second AED in
the mixture, whereas for the fixed ratio of 1:4, the LEV
dose was only fourfold the dose of the second AED in
the mixture). Subsequently, the theoretic amount of pure
additive (ED50 add ) mixture at a fixed ratio of 1:16 is calcu-
lated as follows: ED50 of an AED/P1 . For example, for the
combination of CBZ with LEV, ED50 add = 10.2/0.059 =
173.4 mg/kg (Table 2), and analogously, the ED50 add value
for the mixture of CBZ with LEV at a fixed ratio of 1:4
amounted to 10.2/0.2 = 51 mg/kg (Table 2).
A more detailed description and the theoretical back-
ground relating to isobolographic analysis, including
equations showing how to calculate ED50 add values and
their SEM (in case one of the investigated AEDs is vir-
tually ineffective against MES) has been presented in our
previous studies (45,52). In brief, from the equation of ad-
ditivity presented by Loewe (53), one can determine the
relative potency of each drug present in a mixture, as fol-
lows: x/X + y/Y = 1; where x and y are doses of drugs in
mixture that exert a desired effect (usually 50% effect); X
and Y are doses of drugs used separately, which produce
the same effect (50% effect); x/X and y/Y are relative po-
tencies of respective drugs used in the mixture. Hence, the
relative potency ratio represents a quotient of a dose of an
AED present in mixture producing a desired 50% effect
and its ED50 value, when the drug is administered sepa-
rately. In the situation in which one of the examined AEDs
is ineffective in a mixture, the equation of additivity is ex-
pressed in a shorter form, as follows: x/X = 1; where x/X is
the relative potency of a fully active AED. Because LEV
was virtually ineffective against MES-induced seizures,
one could not determine its ED50 for the drug used sepa-
rately, and thus the expression of y/Y had to be ignored.
Simultaneously, because of limitations of the approach,
one could not denote the relative potency values for LEV
in various fixed-ratio combinations.
Moreover, the equation of additivity presented by
Loewe (53) allows the classification of interactions be-
tween AEDs as supraadditive, if its value is lower than 1;
sub-additive, if its value is >1, and purely additive with
the value equals to 1 (53,54). Unfortunately, this definition
 INTERACTIONS OF LEVETIRACETAM AND AEDS IN THE MES TEST
does not take into consideration the existence of variance
related with experimental drug dose-response relation de-
termination. In isobolography, the value denoted from the
equation of additivity is called the interaction index, and it
describes the power and characteristics of the interaction
(54,55). Generally, it is conventionally accepted that inter-
action index values <0.7 are predictive for supraadditive
interactions; whereas values >1.3 reflect subadditive in-
teractions, and values ranging between 0.7 and 1.3 are
reflective of additive interactions (45,54,56). However,
the classification of interactions based only on interac-
tion indices without any statistical tests is not appropri-
ate in isobolographic preclinical studies. Indeed, recently
there has been substantial discussion concerning this as
well as the inappropriate use of classification of inter-
actions based on overlapped 95% confidence limits for
ED50 add and ED50mix values in isobolographic studies (57).
Only the use of unpaired Student’s t test provides reliable
statistical evaluation of data in interaction studies based
on fixed-ratio combinations using isobolographic analysis
(51,55,57).
Despite these limitations, in this study we compared the
doses of the various AEDs in terms of separate drug doses
of LEV and fully effective AEDs composing both exper-
imentally derived (ED50mix ) and theoretically calculated
(ED50 add ) values (Tables 2 and 3). Although the separate
doses of fully effective AEDs seem to be more meaning-
ful for comparing the efficacy of drugs in the MES test, it
must always be borne in mind that the antiseizure effect
of a two-drug mixture is the consequence of the action of
both drugs.
Measurement of brain AED concentrations
The fixed-ratio AED combination of 1:1 was used to
determine brain AED concentrations for PB + LEV, TPM
+ LEV, CBZ + LEV, OXC + LEV, PHT + LEV, VPA
+ LEV, and LTG + LEV. Mice were killed by decapita-
tion at times chosen to coincide with that scheduled for
the MES test. Brains were removed from skulls, weighed,
and homogenized by using distilled water (2:1 wt/vol) in
an Ultra-Turrax T8 homogenizer (IKA-WERKE, Staufen,
Germany). The homogenates were centrifuged at 10,000 g
for 10 min. The supernatant samples (75 µl) were analyzed
by fluorescence polarization immunoassay for PHT, CBZ,
VPA, PB, or TPM content by using a TDx analyzer and
reagents exactly as described by the manufacturers (Ab-
bott Laboratories, North Chicago, IL, U.S.A., and Opus
Diagnostics, Fort Lee, NJ, U.S.A.). OXC and LTG were
quantified by high-performance liquid chromatography
(HPLC) as described previously (40,46).
LEV concentrations were determined by the HPLC
method described by Ratnaraj et al. (58). The limit of
detection of the method is 1 µM, and the within-batch
13
and between-batch precisions are 2–5% and 3–6%, re-
spectively.
All AED concentrations are expressed in µg/ml of brain
supernatants as means ± SD of at least eight determina-
tions.
Chimney test
The effects of combinations of LEV with PHT, VPA,
CBZ, PB, OXC, TPM, and LTG at doses corresponding
to the ED50mix values for the fixed ratios of 1:8 (for PB
+ LEV), 1:4 (for TPM + LEV), 1:16 (for CBZ + LEV,
OXC + LEV, and PHT + LEV), 1:1 (for VPA + LEV), and
1:32 (for LTG + LEV) on motor performance impairment
were quantified with the chimney test of Boissier et al.
(59). In this test, animals had to climb backward up a
plastic tube (3 cm inner diameter, 25 cm length), and motor
impairment was indicated by the inability of the animals
to climb backward up the transparent tube within 60 s.
Step-through passive-avoidance task
The effects of combinations of LEV with PHT, VPA,
CBZ, PB, OXC, TPM, and LTG on long-term memory im-
pairment were quantified with the step-through passive-
avoidance task of Venault et al. (60). In this test, the ani-
mals were placed in an illuminated box (10 × 13 × 15 cm)
connected to a larger dark box (25 × 20 × 15 cm) equipped
with an electric grid floor. Entrance of the animals to the
dark box was punished by an adequate electric footshock
(0.6 mA for 2 s). On the following day (24 h later), the
pretrained animals were placed again into the illuminated
box and observed ≤180 s. The time that the mice took
to enter the dark box was noted, and the median laten-
cies with 25th and 75th percentiles were calculated. Mice
that avoided the dark compartment for 180 s were consid-
ered to remember the task. The experimental procedure
has been described in more detail in our earlier studies
(61,62).
Statistics
The experimentally derived ED50 values with their SEM
were calculated by computer log-probit analysis accord-
ing to Litchfield and Wilcoxon (49) as described previ-
ously (45,52). In isobolography, the experimentally de-
rived ED50mix values for the mixture of LEV with an AED
were statistically compared with their respective theoret-
ically additive ED50 add values by the use of unpaired Stu-
dent’s t test, according to the method described by Porecca
et al., (51) and Tallarida (55). Total brain AED concen-
trations were statistically analyzed by using the unpaired
Student’s t test. Qualitative variables from the chimney
test were compared by use of the Fisher’s exact probabil-
ity test, whereas the results obtained in the step-through
passive-avoidance task were statistically evaluated by
Epilepsia, Vol. 47, No. 1, 2006
14 J. J. LUSZCZKI ET AL.

TABLE 1. Anticonvulsant activity of the various AEDs in the Isobolographic analysis of interactions between LEV
mouse maximal electroshock seizure model and numerous AEDs in the MES test
AED ED50 (mg/kg) N SEM Isobolographic evaluation of data revealed that CBZ
combined with LEV at the fixed ratio of 1:16 exerted
CBZ 10.2 (8.8–11.9) 16 0.797 supraadditive (synergistic) interaction in the MES test
LTG 5.6 (4.2–7.5) 8 0.836
OXC 11.9 (10.3–13.7) 24 0.857 (Table 2; Fig. 1). The remaining fixed ratios tested be-
PB 22.5 (19.8–25.6) 22 1.480 tween CBZ and LEV (i.e., 1:1, 1:2, 1:4, and 1:8) were in-
PHT 10.8 (8.9–13.1) 32 1.077 different against electroconvulsions (Table 2). Likewise,
TPM 46.8 (39.7–55.1) 24 3.898
VPA 249.2 (229.0–271.0) 24 10.70 all the fixed-ratio combinations evaluated for PB and LEV
(1:1, 1:2, 1:4, and 1:8), PHT and LEV (1:1, 1:2, 1:4, 1:8,
Data are presented as median effective doses (ED50 values with and 1:16), as well as VPA and LEV (1:1, 2:1, 5:1, 10:1,
95% confidence limits in parentheses) of AEDs that protect 50% of
animals against maximal electroshock–induced seizures. N, number and 20:1) were indifferent in the MES test (Table 2). Sim-
of animals between 4 and 6 probits; SEM, standard error of ED50 ; ilarly, all the fixed-ratio combinations between LTG and
CBZ, carbamazepine; LTG, lamotrigine; OXC, oxcarbazepine; PB, LEV (1:1, 1:2, 1:4, 1:8, 1:16, and 1:32) were also indiffer-
phenobarbital; PHT, phenytoin; TPM, topiramate; VPA, valproate.
ent (Table 3). In contrast, combinations of OXC and LEV
(at the fixed ratios of 1:8 and 1:16), as well as TPM and
LEV (at four fixed ratios of 2:1, 1:1, 1:2, and 1:4) were
using Kruskal–Wallis nonparametric analysis of variance
observed to be supraadditive (Table 3; Figs. 2 and 3).
followed by post hoc Dunn’s test.
Brain AED concentrations
Brain concentrations of CBZ, LTG, OXC, PB, VPA,
RESULTS
PHT, and TPM were not significantly different when these
AED anticonvulsant effects against maximal AEDs were administered alone compared with when ad-
electroconvulsions in mice ministered in combination with LEV at a fixed ratio of
With the exception of LEV, all other the AEDs inves- 1:1 (Table 4). Similarly, LEV brain concentrations were
tigated, when administered singly, were associated with not significantly different when LEV was administered in
significant anticonvulsant activity in the MES test, and combination with these AEDs compared with when LEV
their ED50 values are presented in Table 1. was administered alone (Table 5).

TABLE 2. Isobolographic characterization of the interaction between various conventional AEDs and levetiracetam (LEV) in the
mouse maximal electroshock seizure model

AED-combination FR AEDmix + LEVmix = ED50mix (mg/kg) Nmix AEDadd b + LEVadd = ED50 add (mg/kg) Nadd

CBZ + LEV 1:1 9.5 9.5 19.0 ± 1.51 16 10.2 10.2 20.4 ± 1.59 14
CBZ + LEV 1:2 9.9 19.7 29.6 ± 1.95 24 10.2 20.4 30.6 ± 2.39 14
CBZ + LEV 1:4 9.1 36.4 45.5 ± 4.91 24 10.2 40.8 51.0 ± 3.98 14
CBZ + LEV 1:8 9.7 77.5 87.2 ± 7.63 8 10.2 81.6 91.8 ± 7.16 14
CBZ + LEV 1:16 7.3 116.3 123.6 ± 12.24a 16 10.2 163.2 173.4 ± 12.72 14
PB + LEV 1:1 26.2 26.2 52.4 ± 3.70 20 22.5 22.5 45.0 ± 2.96 20
PB + LEV 1:2 21.8 43.5 65.3 ± 7.12 16 22.5 45.0 67.5 ± 4.44 20
PB + LEV 1:4 23.8 95.2 119.0 ± 9.45 16 22.5 90.0 112.5 ± 7.40 20
PB + LEV 1:8 21.2 169.9 191.1 ± 21.49 16 22.5 180.0 202.5 ± 13.32 20
PHT + LEV 1:1 10.9 10.9 21.8 ± 1.74 8 10.8 10.8 21.6 ± 2.16 30
PHT + LEV 1:2 10.9 21.8 32.7 ± 2.66 16 10.8 21.6 32.4 ± 3.24 30
PHT + LEV 1:4 10.5 42.2 52.7 ± 4.79 16 10.8 43.2 54.0 ± 5.40 30
PHT + LEV 1:8 10.5 83.7 94.2 ± 7.44 16 10.8 86.4 97.2 ± 9.71 30
PHT + LEV 1:16 10.1 160.8 170.9 ± 10.77 16 10.8 172.8 183.6 ± 17.27 30
VPA + LEV 1:1 267.1 267.1 534.2 ± 18.84 16 249.2 249.2 498.4 ± 21.45 22
VPA + LEV 2:1 258.3 129.1 387.4 ± 18.11 16 249.2 124.6 373.8 ± 16.09 22
VPA + LEV 5:1 274.7 55.0 329.7 ± 12.66 8 249.2 49.8 299.0 ± 12.87 22
VPA + LEV 10:1 273.8 27.4 301.2 ± 11.59 8 249.2 24.9 274.1 ± 11.80 22
VPA + LEV 20:1 272.5 13.6 286.1 ± 10.95 8 249.2 12.5 261.7 ± 11.26 22

Data are presented as median effective doses (ED50 ± SEM) protecting 50% of animals tested against electroconvulsions. The ED50 values were
either experimentally determined from the mixture of two AEDs (ED50mix ) or theoretically calculated from the equation of additivity (ED50 add ).
Statistical evaluation of data was performed by using unpaired Student’s t test. FR, fixed ratio of drug dose combinations; AEDmix , dose of an AED in
the experimentally derived mixture; LEVmix , dose of LEV in the experimental mixture; AEDadd , dose of an AED in purely additive mixture; LEVadd ,
theoretically calculated dose of LEV in purely additive mixture for the respective fixed ratio; N, total number of animals at those doses whose expected
effects were between 4 and 6 probits, denoted for the experimental mixture of drugs (Nmix ) and theoretically calculated (Nadd ) from the equation of
additivity; CBZ, carbamazepine; PB, phenobarbital; PHT, phenytoin; VPA, valproate.
a p < 0.01 vs. the respective ED
50 add , indicating supraadditive (synergistic) interaction.
b The doses of conventional AEDs in purely additive mixture are constant for all fixed-ratio combinations.

Epilepsia, Vol. 47, No. 1, 2006

 INTERACTIONS OF LEVETIRACETAM AND AEDS IN THE MES TEST 15

TABLE 3. Isobolographic characterization of the interaction between various new AEDs and levetiracetam (LEV) in the mouse
maximal electroshock seizure model
ED50mix ED50 add
AED-combination FR AEDmix + LEVmix = (mg/kg) Nmix AEDadd c + LEVadd = (mg/kg) Nadd

LTG + LEV 1:1 5.6 5.6 11.2 ± 1.67 8 5.6 5.6 11.2 ± 1.67 6
LTG + LEV 1:2 5.2 10.4 15.6 ± 2.11 16 5.6 11.2 16.8 ± 2.51 6
LTG + LEV 1:4 5.7 22.6 28.3 ± 3.24 24 5.6 22.4 28.0 ± 4.19 6
LTG + LEV 1:8 5.7 46.0 51.7 ± 4.79 24 5.6 44.8 50.4 ± 7.54 6
LTG + LEV 1:16 5.2 82.9 88.1 ± 7.78 16 5.6 89.6 95.2 ± 13.40 6
LTG + LEV 1:32 4.9 157.9 162.8 ± 15.16 24 5.6 179.2 184.8 ± 26.79 6
OXC + LEV 1:1 11.6 11.6 23.2 ± 1.52 16 11.9 11.9 23.8 ± 1.72 22
OXC + LEV 1:2 10.1 20.2 30.3 ± 2.84 24 11.9 23.8 35.7 ± 2.57 22
OXC + LEV 1:4 10.8 43.0 53.8 ± 4.72 16 11.9 47.6 59.5 ± 4.29 22
OXC + LEV 1:8 8.7 69.7 78.4 ± 8.54b 16 11.9 95.2 107.1 ± 7.7 22
OXC + LEV 1:16 7.8 125.3 133.1 ± 14.84a 8 11.9 190.4 202.3 ± 13.72 22
TPM + LEV 4:1 39.4 9.8 49.2 ± 6.37 24 46.8 11.7 58.5 ± 4.87 22
TPM + LEV 2:1 28.7 14.3 43.0 ± 6.18a 24 46.8 23.4 70.2 ± 5.85 22
TPM + LEV 1:1 29.8 29.8 59.6 ± 7.98a 32 46.8 46.8 93.6 ± 7.80 22
TPM + LEV 1:2 27.3 54.5 81.8 ± 13.79a 32 46.8 93.6 140.4 ± 11.70 22
TPM + LEV 1:4 33.1 132.4 165.5 ± 23.95b 16 46.8 187.2 234.0 ± 19.50 22

Data are presented as ED50 values ± SEM, protecting 50% of animals tested against electroconvulsions.
LTG, lamotrigine; OXC, oxcarbazepine, TPM, topiramate.
a p < 0.01 and b p < 0.05 vs. the respective ED c
50 add values, indicating supraadditive (synergistic) interactions. The doses of second-generation
AEDs in purely additive mixture are constant for all fixed-ratio combinations. For more details, see Table 2 legend.

Effects of LEV administered singly and in
combination with the various AEDs on motor
performance and long-term memory
None of the examined combinations of LEV and the
various AEDs (administered at doses corresponding to the
ED50mix values for the fixed ratios, where LEV was present
in a maximal dose in mixture) was associated with motor
deficit in animals challenged with the chimney test. Like-
wise, these combinations did not affect long-term memory
in the step-through passive-avoidance task (Table 6).
DISCUSSION
The major findings of this isobolographic study are as
follows:

14
Carbamazepine dose (mg/kg)

12

10

8
1:1 2:1
4:1
8:1 **
6
16:1
4

0
0 20 40 60 80 100 120 140 160

Levetiracetam dose (mg/kg)

FIG. 1. Isobologram illustrating the synergistic interaction between levetiracetam (LEV) and carbamazepine (CBZ) in the mouse maximal
electroshock seizure test. LEV and CBZ doses are shown plotted graphically on the X- and Y-axes, respectively. The heavy line is parallel
to the X-axis, representing the ED50 value for CBZ, and defines the theoretical dose-additive line for a continuum of different fixed-dose
ratios. The dotted lines represent 95% confidence limits for CBZ. The solid circles (•) depict the experimentally derived ED50mix values
for total doses of mixtures expressed as proportions of LEV and CBZ that produced median anticonvulsant effects. The 95% confidence
limits of the experimentally determined ED50mix values are presented horizontally and vertically in the form of crosses. The ED50mix for the
mixture of LEV + CBZ at the fixed ratio of 16:1 is significantly below the theoretical line of additivity, indicating a supraadditive (synergistic)
interaction (∗∗ p < 0.01). In contrast, the remaining ED50mix values at the fixed ratios of 1:1, 2:1, 4:1, and 8:1 are close to the line of
additivity, indicating additive interactions.

Epilepsia, Vol. 47, No. 1, 2006

16 J. J. LUSZCZKI ET AL.

16

14

Oxcarbazepine dose (mg/kg)

12

10
1:1 *
8
2:1
4:1 **
6 8:1
16:1
4

0
0 20 40 60 80 100 120 140 160 180

Levetiracetam dose (mg/kg)

FIG. 2. Isobologram illustrating the synergistic interaction between levetiracetam (LEV) and oxcarbazepine (OXC) in the mouse maximal
electroshock seizure test. LEV and OXC are shown plotted graphically on the X- and Y-axes, respectively. The ED50mix values for the
mixture of LEV+OXC at the fixed ratios of 8:1 and 16:1 are significantly below the theoretical line of additivity, indicating supraadditivity (∗ p
< 0.05 and ∗∗ p < 0.01). In contrast, the remaining ED50mix values at the fixed ratios of 1:1, 2:1, and 4:1 are close to the line of additivity,
indicating additivity. For more details, see Fig. 1 legend.

1. Despite the inactivity of LEV in the MES test, it was 4. Brain AED concentrations were not significantly
associated with supraadditive interactions when ad- different between animals administered AEDs in
ministered in combination with TPM [for all the ex- combination with LEV (at a fixed ratio of 1:1)
amined fixed ratios (2:1, 1:1, 1:2, and 1:4) except compared with those in which AEDs were admin-
for 4:1]; istered singly, indicating pharmacodynamic inter-
2. CBZ in combination with LEV (at the fixed ratio of actions between LEV and other AEDs in the MES
1:16) and OXC with LEV (at fixed ratios of 1:8 and test.
1:16) were similarly associated with supraadditive
interactions in the MES test; Because TPM is associated with multiple molecular
3. None of the studied AED combinations were asso- mechanisms of action (for review, see 63), it is highly
ciated with concurrent alteration in motor perfor- possible that LEV may potentiate its anticonvulsant effect
mance or long-term memory; and via one or more mechanisms. For example, it could be

60
1:4

50
Topiramate dose (mg/kg)

40

*
30 ** **
**
20 1:1 4:1
1:2
2:1
10

0
0 20 40 60 80 100 120 140 160 180
Levetiracetam dose (mg/kg)
FIG. 3. Isobologram illustrating the synergistic interaction between levetiracetam (LEV) and topiramate (TPM) in the mouse maximal
electroshock seizure test. LEV and TPM doses are shown plotted graphically on the X- and Y-axes, respectively. The ED50mix values for
the mixture of LEV + TPM at the fixed ratios of 1:2, 1:1, 2:1, and 4:1 are significantly below the theoretical line of additivity, indicating
supraadditivity (∗ p < 0.05 and ∗∗ p < 0.01). Only the fixed ratio of 1:4 is close to the line of additivity, indicating additivity. For more details,
see Fig. 1 legend.

Epilepsia, Vol. 47, No. 1, 2006

 INTERACTIONS OF LEVETIRACETAM AND AEDS IN THE MES TEST 17

TABLE 4. Brain concentrations of the various AEDs TABLE 6. The effect of various AEDs administered singly and
administered singly or in combination with levetiracetam in combination with levetiracetam (LEV) on motor
(LEV) performance and long-term memory
Treatment (mg/kg) Brain concentration (µg/ml) Treatment Animals Retention
(mg/kg) FR impaired (%) time (s)
CBZ (9.5) + vehicle 2.33 ± 0.31
CBZ (9.5) + LEV (9.5) 2.46 ± 0.40 Vehicle + LEV (300)a - 0 180 (180; 180)
LTG (5.6) + vehicle 0.26 ± 0.02 CBZ (7.3) + LEV (116.3) 1:16 0 180 (180; 180)
LTG (5.6) + LEV (5.6) 0.27 ± 0.02 PB (21.2) + LEV (169.9) 1:8 0 180 (180; 180)
OXC (11.6) + vehicle 2.05 ± 0.18 PHT (10.1) + LEV (160.8) 1:16 0 180 (175; 180)
OXC (11.6) + LEV (11.6) 2.11 ± 0.16 VPA (267) + LEV (267) 1:1 12.5 172.8 (136.8; 180)
PB (26.2) + vehicle 12.07 ± 0.98 LTG (4.9) + LEV (157.9) 1:32 0 180 (180; 180)
PB (26.2) + LEV (26.2) 11.98 ± 1.29 OXC (7.8) + LEV (125.3) 1:16 0 180 (180; 180)
PHT (10.9) + vehicle 1.61 ± 0.34 TPM (33.1) + LEV (132.4) 1:4 0 180 (180; 180)
PHT (10.9) + LEV (10.9) 1.77 ± 0.32
TPM (29.8) + vehicle 4.31 ± 0.30 Data are expressed as percentage of animals that failed to perform
TPM (29.8) + LEV (29.8) 4.42 ± 0.33 the chimney test and as median retention time (with 25th and 75th
VPA (267) + vehicle 88.01 ± 7.14 percentiles in parentheses) during which the animals avoided entering
VPA (267) + LEV (267) 92.79 ± 8.39 the dark compartment in the step-through passive-avoidance task. The
combinations of LEV and the various AEDs were administered at doses
Data are presented as mean ± SD of at least eight determinations. corresponding to the ED50mix values for the fixed ratios, whereas LEV
Statistical evaluation of data was performed with unpaired Student’s t was present in a maximal dose in mixture. Moreover, the effect of LEV
test. administered alone at 300 mg/kg (a ) on motor coordination and/or
CBZ, carbamazepine; LTG, lamotrigine; OXC, oxcarbazepine; PB, long-term memory was determined to exclude any potential adverse
phenobarbital; PHT, phenytoin; TPM, topiramate; VPA, valproate. effects produced by the drug itself. Statistical analysis of data from the
chimney test was performed by using the Fisher’s exact probability
test, whereas the results from the step-through passive-avoidance task
readily envisaged that the modulation of the function of were analyzed by using the Kruskal–Wallis nonparametric ANOVA test
SV2A by LEV, which is involved in vesicle exocytosis of followed by Dunn’s post hoc test. PHT was administered at 120 min
before testing, whereas LEV, PB, TPM, and LTG were administered at
neurotransmitters, combined with the inhibition of excita- 60 min, and CBZ, OXC, and VPA were administered at 30 min before
tory neurotransmission via α-amino-3-hydroxy-5-methyl- testing. These test times represent the time of maximal anticonvulsant
4-isoxazoleproprionic acid (AMPA)/kainate receptors by effect of the AEDs. FR, fixed-ratio combination; CBZ, carbamazepine;
PB, phenobarbital; PHT, phenytoin; VPA, valproate; LTG, lamotrigine;
TPM (64,65) could synergistically suppress MES-induced OXC, oxcarbazepine; TPM, topiramate.
seizures. Another synergistic mechanism that could be en-
visaged entails the inhibition of N- and P/Q-types HVA
Ca2+ channels by LEV combined with the inhibition of
neuronal L-type HVA Ca2+ channels mediated by TPM MES test. The remaining fixed ratios tested for these AED
(66). combinations were indifferent (Tables 2 and 3). The ob-
Additionally, it was found that the combinations of CBZ served synergistic interactions between LEV and CBZ and
with LEV (at the fixed ratio of 1:16) and OXC with LEV OXC may probably be attributable to the fact that LEV (at
(at fixed ratios of 1:8 and 1:16) were supraadditive in the doses >100 mg/kg) can significantly increase the thresh-
old for electroconvulsions in mice (67). Conversely, ad-
ditional mechanisms could include an effect of LEV on
TABLE 5. Brain levetiracetam (LEV) concentrations after
N- and P/Q-types HVA Ca2+ channels and an effect of
LEV administration, singly and in combination with the
various AEDs CBZ and OXC on Na+ -channel blockade or activation
of the adenosinergic inhibitory neurotransmitter system
Treatment (mg/kg) Brain concentration (µg/ml) (68). In contrast to CBZ and OXC, LTG and PHT (two
LEV (5.6) + vehicle 0.46 ± 0.06 AEDs whose main mechanism of action is also related
LEV (5.6) + LTG (5.6) 0.42 ± 0.05 with Na+ channel blockade) did not interact synergisti-
LEV (9.5) + vehicle 1.74 ± 0.12 cally with LEV in the MES test, even if the dose of LEV
LEV (9.5) + CBZ (9.5) 1.84 ± 0.18
LEV (10.9) + vehicle 1.75 ± 0.19 considerably elevated the electroconvulsive threshold in
LEV (10.9) + PHT (10.9) 1.76 ± 0.21 animals.
LEV (11.6) + vehicle 1.87 ± 0.13 Surprisingly, high LEV doses did not potentiate the an-
LEV (11.6) + OXC (11.6) 1.94 ± 0.20
LEV (26.2) + vehicle 5.26 ± 0.57 ticonvulsant activities of LTG, PB, VPA, and PHT in the
LEV (26.2) + PB (26.2) 5.19 ± 0.40 MES test. The maximal fixed ratios tested in the present
LEV (29.8) + vehicle 5.82 ± 0.52 study were chosen on the bases that they would moder-
LEV (29.8) + TPM (29.8) 5.64 ± 0.71
LEV (267) + vehicle 61.16 ± 4.78 ately increase the threshold for electroconvulsions in mice.
LEV (267) + VPA (267) 59.47 ± 3.65 Thus the maximal fixed ratio tested for TPM was 1:4, for
PB, 1:8; for OXC, CBZ, and PHT, it was 1:16; and for
Data are presented as mean ± SD of at least eight determinations.
Statistical evaluation was performed with unpaired Student’s t test. For LTG, it was 1:32. The one exception was VPA, which was
more details, see Table 4 legend. tested at the fixed ratio of 1:1.

Epilepsia, Vol. 47, No. 1, 2006

18 J. J. LUSZCZKI ET AL.
Results from our study concerning the synergistic in-
teraction between LEV and TPM are generally in agree-
ment with those reported by Sills et al. (41). In their study,
by using the MES and PTZ-tests in mice, LEV (50 and
250 mg/kg) in combination with TPM (25 and 125 mg/kg)
was associated with a potent anticonvulsant effect, sup-
pressing tonic–clonic seizures in the MES test, whereas
LEV (50 and 250 mg/kg) in combination with TPM
(125 mg/kg) protected the animals against PTZ-induced
seizures, despite the inactivity of the two drugs when ad-
ministered alone (41). Furthermore, it has been observed
that LEV (200 mg/kg) in combination with DZP (1 mg/kg)
was synergistic with regard to control of status epilepti-
cus induced by perforant-path stimulation in rats (42). In-
terestingly, the synergistic interaction of LEV with TPM
occurred at lower doses of LEV (>15 mg/kg; Fig. 3) than
those for the interactions with CBZ (>110 mg/kg; Fig. 1)
and OXC (>70 mg/kg; Fig. 2). This observation is very
important because it suggests that synergy for the interac-
tion of LEV with TPM is more powerful than that for the
interactions between LEV and CBZ and LEV and OXC.
It should be clearly stated that supraadditivity (synergy)
identified by isobolographic analysis reflects an activity of
two AEDs in mixture to provide the same seizure protec-
tion at lower doses than each individual drug administered
alone, but not an ability to provide a more complete seizure
suppression or enhanced efficacy of AEDs. The latter is the
parameter of interest when selecting specific AED com-
binations for the treatment of intractable epilepsy.
In the clinical setting, adverse pharmacodynamic inter-
actions have been reported between LEV and CBZ (43)
and between LEV and TPM (44), and more recently, Kelly
et al. (69) reported that in their clinical experience, the
most frequently discontinued combinations due to un-
desirable adverse effects were those of LEV with CBZ
and LTG. In the present study, is noteworthy that LEV
had no significant effect on the adverse-effect profiles
of the various AEDs investigated with regard to motor
performance (chimney test) and long-term memory (step-
through passive-avoidance task). However, it should be
highlighted that the evaluation of acute neurotoxic effects
in this study was performed only at doses of LEV and
the various AEDs that corresponded to the ED50mix at the
maximal fixed-ratio combination tested. Moreover, it is
notable that the median toxic dose (TD50 ) of LEV with re-
spect to the impairment of motor coordination of animals
in the rotarod test, denoted in our previous study, was 1,601
(1,324–1,935) mg/kg (67). Furthermore, we have reported
that LEV (administered at a constant dose of 150 mg/kg)
potentiated the acute neurotoxic effects of TPM and CBZ
in the rotarod test in mice, reducing considerably their
TD50 values from 423 to 246 mg/kg and from 53.6 to 37.3
mg/kg, respectively (67). The other AEDs tested in combi-
nation with LEV in the rotarod (i.e., PHT, VPA, PB, LTG,
OXC, and felbamate) produced no significant impairment
Epilepsia, Vol. 47, No. 1, 2006
of motor coordination in animals (67). Furthermore, be-
cause neither brain TPM nor CBZ concentrations were af-
fected by LEV nor were brain LEV concentrations affected
by TPM and CBZ administration, the observed enhanced
neurotoxicity was considered to be the consequence of
pharmacodynamic interactions. Similarly, in the present
study, brain AED concentration measurements (Tables 4
and 5) reveal that LEV not only did not affect brain CBZ,
LTG, OXC, PB, PHT, TPM or VPA, but that these AEDs
did not affect brain LEV concentrations when adminis-
tered in combination. Therefore the synergistic anticon-
vulsant efficacy between LEV and TPM, CBZ, and OXC
can be considered to be the consequence of a pharma-
codynamic, and not a pharmacokinetic, interaction. In-
deed the lack of a pharmacokinetic interaction between
these four AEDs with LEV would concur with its clini-
cal profile (70–72). It should be highlighted that although
in this study, brain AED concentrations were measured
at only one fixed-ratio combination (1:1), higher doses
would not be expected to interact pharmacokinetically,
because LEV is devoid of pharmacokinetic characteris-
tics that are associated with such interactions (70–73).
We recently reported that LEV at much higher doses than
those investigated in the present study (150 mg/kg) had no
effect on brain CBZ (37.3 mg/kg) or TPM (246.2 mg/kg)
concentrations, and conversely, brain LEV concentrations
were unaffected by CBZ and TPM administration (67).
Although clinical evidence suggests that CBZ and OXC
have different pharmacologic profiles (68), it is notewor-
thy that both AEDs interacted synergistically with LEV
in the MES test, and this is in line with the fact that these
two AEDs have very similar mechanisms of action.
In conclusion, our preclinical data would suggest that
LEV in combination with TPM is associated with ben-
eficial anticonvulsant pharmacodynamic interactions and
that these data are in line with other anticonvulsant phar-
macodynamic interactions that have been reported with
LEV in other animal models. Notably, LEV also was as-
sociated with beneficial anticonvulsant pharmacodynamic
interactions with OXC and CBZ, but these were less pro-
found at the doses tested in this study.
Acknowledgment: This study was supported by grants from
Institute of Agricultural Medicine, Lublin, Poland, and Medi-
cal University, Lublin, Poland. Dr. J.J. Luszczki is a recipient
of the Fellowship for Young Researchers from the Foundation
for Polish Science. The kind gifts of valproate sodium from
ICN-POLFA Rzeszow S.A. (Poland) and carbamazepine from
POLFA Starogard (Poland) are greatly appreciated. We express
our thanks to Mr W. Zgrajka (Institute of Agricultural Medicine,
Lublin, Poland) for the skilful determination of the brain con-
centrations of LTG and OXC.
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