Fig. 1. A–C Effects of tiagabine (TGB), vigabatrin (VGB), and their combinations on the threshold for electroconvulsions. Doses of TGB, VGB and mixtures of their combinations (VGB:TGB at fixed-ratios of 1:3, 1:1 and 3:1) were plotted graphically on the X-axis, whereas the percentage of threshold increase over the threshold for control animals was placed on the Y-axis, respectively. Least-squares linear regression analysis was used to determine the equations of DRRCs between AED doses and their corresponding percentage of threshold increase values. Subsequently, from the respective equations, the TID₂₀ values for TGB, VGB, and their combinations (i.e., the drug doses increasing the threshold for electroconvulsions by 20% over control threshold) were calculated. The TID₂₀ value for TGB was 4.4 mg/kg (A), and that for VGB was 226.2 mg/kg (B). The TID₂₀ values for the combinations of VGB with TGB at fixed-ratios of 1:3, 1:1 and 3:1 were 68.5 mg/kg, 126.1 mg/kg, and 186.5 mg/kg, respectively (C).

Fig. 2. Isobologram illustrating the interactions for the combinations of vigabatrin (VGB) with tiagabine (TGB) in the maximal electroshock seizure threshold (MEST) test. The TID₂₀ values for VGB and TGB are placed on the graph, on X- and Y-axes, respectively. The isobole of additivity is shown as a solid line drawn between the aforementioned TID₂₀ values of VGB and TGB, which connects the X- and Y-axes. The dotted lines represent on the isobologram the theoretically calculated 95% confidence limits of TID_{20 add} values as the error bars, whereas the data obtained experimentally (TID_{20 mix}) are presented as the open points (o) with the respective 95% confidence limits as the error bars. Since S.E.M. can be readily transformed in 95% confidence limits (and vice versa), all the TID₂₀ values are presented on the graph with their 95% confidence limits. The experimental TID_{20 mix} values of the mixture of VGB and TGB, for the fixed-ratio combinations of 1:3, 1:1 and 3:1, were plotted close to the theoretical isoboles of additivity and thus, indicating additive interactions in the MEST test.

Fig. 3. Isobologram illustrating the interactions for the combinations of vigabatrin (VGB) with tiagabine (TGB) against pentylenetetrazole (PTZ)-induced seizures. The ED₅₀ values for VGB and TGB are placed on the graph, on the abscissa (X) and ordinate (Y), respectively. The straight line connecting these both ED₅₀ values represents the theoretical line of additivity for a continuum of different fixed dose ratios. The open points (o) depict the experimentally derived ED_{50 mix} values (with 95% confidence limits as the error bars) for total doses expressed as the proportion of VGB and TGB that produced a 50% anticonvulsant effect. The dotted lines represent on the isobologram, theoretically additive 95% confidence limits of ED_{50 add} values. The experimentally derived ED_{50 mix} values, for the fixed-ratios of 1:3 and 1:1 were found to be significantly below the theoretical isoboles of additivity, indicating supra-additive (synergistic) interactions. The combination of VGB and TGB at the fixed-ratio of 3:1 was plotted close to the line of additivity, indicating additive interactions. For more details see the legend to Fig. 2. p < 0.05 and p < 0.01 for the fixed-ratio combinations of 1:3 and 1:1 vs. the respective ED_{50 add} values.

Interactions between vigabatrin (VGB) and tiagabine (TGB) in the maximal electroshock seizure threshold (MEST) test — an isobolographic analysis

Data are presented as doses of two-drug mixture increasing threshold for electroconvulsions by 20% (TID₂₀ in mg/kg ± S.E.M.). Least-squares linear regression equations were used to determine the TID₂₀ values from the respective DRRCs of VGB and TGB in combinations at 3 fixed-ratios of 1:3, 1:1 and 3:1. For the mixtures of VGB with TGB, the TID₂₀ values were either theoretically calculated from the equation of additivity (TID_{20 add}) or experimentally derived (TID_{20 mix}) from the MEST test in mice. Statistical evaluation of data was performed with unpaired Student's t-test. The AEDs were administered systemically (i.p.) at times corresponding to their peaks of maximum anticonvulsant activity, i.e., VGB at 240 min, and TGB at 15 min before the MEST-induced seizures. FR — fixed-ratio combination; N — total number of animals at those doses whose expected anticonvulsant effects were between 4 and 6 probits, denoted for the experimental mixture of drugs (N_mix) and theoretically calculated (N_add) from the equation of additivity; VGB_add, TGB_add, VGB_mix, and TGB_mix — doses of VGB and TGB consisting the mixture at the TID_{20 add} and TID_{20 mix} values.

Interactions between vigabatrin (VGB) and tiagabine (TGB) against pentylenetetrazole (PTZ)-induced seizures in mice — an isobolographic analysis

Results are presented as median effective doses (ED₅₀ in mg/kg ± S.E.M.) of drug mixtures, determined either experimentally (ED_{50 mix}) or theoretically calculated (ED_{50 add}) from the line of additivity. VGB_add, TGB_add, VGB_mix, and TGB_mix — doses of VGB and TGB consisting the mixture at the ED_{50 add} and ED_{50 mix} values. The PTZ-induced clonic seizures were produced by the s.c.-injection of PTZ at its CD₉₇ (100 mg/kg). Statistical evaluation of data was performed with unpaired Student's t-test. Significantly different at p < 0.05, and at p < 0.01 vs. the respective ED_{50 add}. For more details consult the legend to Table 1.

Effects of the combinations of vigabatrin (VGB) and tiagabine (TGB) on motor coordination and neuromuscular tone in the chimney and grip-strength tests

Data are presented as the percentage of motor coordination impairment in animals challenged with the chimney test (8 animals per group) and as means of neuromuscular strength (in Newtons  ± S.D. of at least 24 determinations) from the grip-strength test. The AEDs were administered systemically (i.p.) at doses corresponding to their TID_{20 mix} and ED_{50 mix} values from the MEST and PTZ tests; VGB — at 240 min and TGB — at 15 min before the tests. Results from the chimney test were statistically analyzed with Fisher's exact probability test, whereas the statistical analysis of data from the grip-strength test was performed with one-way ANOVA followed by the post-hoc Bonferroni's test.

Effects of vigabatrin (VGB), tiagabine (TGB), and their combinations on the acute nociceptive effects in the hot-plate test

Data are presented as means of maximum possible antinociceptive effects (MPAE in % ± S.E.M.) in animals (8 mice per group) challenged with the hot-plate test. The AEDs were administered systemically (i.p.) at doses corresponding to their TID_{20 mix} and ED_{50 mix} values from the MEST and PTZ tests; VGB — at 240 min and TGB — at 15 min before the test. Statistical evaluation of data was performed with one-way ANOVA followed by the post-hoc Bonferroni's test for multiple comparisons. For more details regarding the calculation of % MPAE see Methods section.

p < 0.05, p < 0.01 and p < 0.001 vs. control (vehicle-treated) animals.