doi:10.1016/j.neuroscience.2004.12.044 
Copyright © 2005 IBRO Published by Elsevier Ltd.
Antiepileptic action induced by a combination of vigabatrin and tiagabine
Y. Fuetaa, 
, 
, N. Kunugitab and W. Schwarzc
aDepartment of Med. Tech., School of Health Sciences, Univ. Occupat./Environmental Health, Iseigaoka 1-1, Yahatanishi-ku, Kitakyushu 807-8555, Japan
bDepartment of Health Information Science, School of Health Sciences, Univ. Occupat./Environmental Health, Iseigaoka 1-1, Yahatanishi-ku, Kitakyushu 807-8555, Japan
cMax-von-Laue Str. 3, 60438 Frankfurt/Main, Germany
Accepted 21 December 2004.
Available online 12 March 2005.
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Abstract
Vigabatrin, an inhibitor of GABA breakdown by GABA transaminase and of GABA transporter isoform 1 (GAT1), and tiagabine, a highly specific inhibitor of GAT1, have successfully been applied in the treatment of epilepsy. We investigated the effects of individual and combined application of these drugs on GAT1 expressed in Xenopus oocytes, and examined the effects on epileptiform discharges in the CA3 area of brain slices of genetically epileptic El and control ddY mice, and on the occurrence of seizures in El mice. Simultaneous application of vigabatrin and tiagabine inhibited epileptiform discharges induced by high-K+ solution in the brain slices in an antagonistic fashion. The degree of inhibition by tiagabine after pre-treatment with vigabatrin was additive in ddY mice and synergistic in El mice. In Mg2+-free solution, co-treatment by the two drugs produced additive inhibition in slices from both mouse strains, but pre-treatment with vigabatrin produced synergistic inhibition in slices only from ddY mice. In the slices from El mice, a combination of drugs resulted in additive effects in both co- and pre-treatment by the drugs. Although these drugs are also effective in vivo at suppressing seizure occurrence in El mice, the combined application does not show synergistic effects, but rather is antagonistic under the experimental conditions in this particular variant of epilepsy. The synergistic inhibition of epileptiform discharges in brain slices may, in part, have originated from the complex interaction with GAT1. In experiments on the GAT1 expressed in oocytes it could be demonstrated that synergistic inhibition occurs only at low concentration (0.1 nM) of vigabatrin. This illustrates that the oocytes may form a powerful test system for drug screening and investigation of complex drug interactions.
These results present a novel interpretation of synergistic inhibition of certain epileptic discharges using vigabatrin and another drug, and that for successful synergistic treatment of epilepsies carefully designed timed dosage regimens are essential.
Key words: GABA transporter; epilepsy; electrophysiology; antiepileptic medicine; synergism
Abbreviations: ACSF, artificial cerebrospinal fluid; ANOVA, analysis of variance; GABA, gamma-aminobutyric acid; GAT1, GABA transporter isoform 1; NMDA, N-methyl-d-aspartate; ORi, oocyte Ringer’s solution
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Fig. 1. Experimental design for in vitro slice experiments to examine antiepileptic effects induced by a single application of vigabatrin and tiagabine, and a combination of the drugs in vitro in high-K+ and 0-Mg models. Hippocampal slices obtained from epileptic El mice and the mother strain ddY mice were separated into six groups. Open bars represent the perfusion with ACSF, and lined bars represent the perfusion with 8 mM K or Mg-free solution. Closed bars represent the vigabatrin treatment and hatched bars represent the tiagabine treatment. Antiepileptic effects of the following groups were compared at the end of the drug perfusion (timing indicated with the thick arrows). (A) Control group: epileptiform bursting discharges were recorded for 5 min every 0.5 h (thin arrows) for a total time period of 3 h after the induction. (B) Single application: antiepileptic effects of a single application of vigabatrin (B-1) and tiagabine (B-2) were determined after 3 h of treatment with the respective drug. In group B-0, antiepileptic effects of tiagabine were determined during the last 0.5 h of the 3-h period. (C) Simultaneous application: effects of a combination of vigabatrin with tiagabine were determined during a 3-h treatment. (D) Step application: tiagabine was applied at the end of the 2.5-h treatment with vigabatrin.
Fig. 2. Effect of application of 1 μM tiagabine in the presence of different concentrations of vigabatrin on the relative rate of GABA uptake by oocytes with expressed GAT1 (±S.E.M., n given by the numbers above bars). The value of 1 corresponds to a flux rate of 0.58±0.03 pmol/s. Open bars represent uptake in the absence of tiagabine. The relative rate of GABA uptake was different along the vigabatrin concentration for the cells with and without tiagabine (two-way ANOVA). *** P<0.001 compared with 0 nM vigabatrin, + P<0.05; +++ P<0.001 compared with tiagabine at each vigabatrin concentration (one-way ANOVA test followed by Scheffe’s test). A significant difference in the presence and absence of tiagabine was obtained only at a very low concentration of 0.1 and 1 nM vigabatrin. A two-way ANOVA indicated an interaction between tiagabine and vigabatrin only when 0.1 nM concentrations of vigabatrin were used (P<0.0411; see text in details).
Fig. 3. Representative traces of each group in the high-K+ model of El mice. Left traces were interictal-like epileptic discharges for 30 s. Right traces were expanded interictal-like discharges and the scales were indicated.
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Fig. 4. Effects on occurrence frequency of epileptiform discharges in hippocampal slices of ddY and El mice in control group (A), by sole application of vigabatrin or tiagabine (group B), by simultaneous application (tiagabine+vigabatrin; group C), and by tiagabine application 2.5 h after pre-treatment with vigabatrin (vigabatrin>tiagabine; group D). Discharges were induced by perfusion with 8 mM K+ (high-K+) or Mg2+-free (0-Mg) solution. Tiagabine concentration was 20 μM, vigabatrin concentration was 20 μM in the high-K+ model and 100 μM in the 0-Mg model. Antiepileptic effects were determined after 3 h of application as indicated with the thick arrows in the Fig. 1. Data are averages±S.E.M., the numbers of slices used in each group are given at the bottom of each bar. One or two slices from one mouse were provided to the in vitro experiment and the two slices were used in the different protocols. ** P<0.01, *** P<0.001 compared with the control group (one-way ANOVA test followed by Scheffe’s test). See text for details.
Fig. 5. Effects of i.p. injection of different amounts of tiagabine (a) and vigabatrin (b). Score values were determined either 0.5 h or 18 h after each drug injection, respectively. The values are given relative to control animals that were injected with corresponding volumes of pure saline. The value of 1 corresponds to a score value of 4.8±0.1. Each group was made up of six to nine animals. Solid lines are approximations of the concentration dependencies.
Fig. 6. Effect of post-injection of 0.1 mg/kg tiagabine 17.5 h after i.p. injection of different amounts of vigabatrin (filled squares without, open circle with tiagabine injection). Score values are given relative to control animals that were injected with corresponding volumes of pure saline. The single open square represents a value after injection of 0.3 mg/kg tiagabine. Each group was made up of six to 10 animals. Solid lines are an approximation of the concentration dependency.
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