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Keywords:

  • Gabapentin;
  • Antiepileptic drugs;
  • Electroshock maximal;
  • Drug interactions;
  • Seizures;
  • Isobolography

Abstract

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Summary:  Purpose: The objective of this study was the isobolographic evaluation of the interactions between the novel antiepileptic drug (AED) gabapentin (GBP) and a number of other AEDs against electroconvulsion-induced convulsions in mice.

Methods: Electroconvulsions were produced by means of an alternating current (ear-clip electrodes, 0.2-s stimulus duration, tonic hindlimb extension taken as the end point). Adverse effects were evaluated with the chimney test (motor performance) and passive-avoidance task (long-term memory). Plasma levels of AEDs were measured by immunofluorescence or high-pressure liquid chromatography.

Results: GBP (≤50 mg/kg) remained ineffective on the electroconvulsive threshold. According to the isobolographic analysis, GBP appears to act synergistically with carbamazepine, valproate, phenytoin, phenobarbital (PB), lamotrigine (LTG), and LY 300164. The pharmacokinetic events may be responsible for the interactions of GBP/PB and GBP/LTG, because only PB and LTG significantly elevated the plasma concentration of this AED. Conversely, GBP did not affect the plasma levels of other AEDs used in this study. No adverse effects were induced by combinations of GBP with these AEDs.

Conclusions: The isobolographic analysis revealed that combinations of GBP with other AEDs generally results in synergistic (supraadditive) interactions.

Gabapentin (GBP), a cyclic analogue of γ-aminobutyric acid (GABA) that was designed as a GABA agonist, in contrast to its maternal compound, readily passes through the blood–brain barrier. The drug has no affinity for the GABAA-receptor complex. In clinical practice, the adjunctive and eventually monotherapeutic use of GBP resulted in significant improvement in patients with both focal and secondarily generalized partial seizures (1). In experimental epilepsy models, the drug protected against pentylenetetrazol (PTZ)-induced tonic, but not clonic convulsions, was ineffective in the maximal electroshock test, and aggravated spike–wave discharges of absence seizures (2).

Lamotrigine (LTG) is a newly developed drug, exerting its anticonvulsive properties primarily through Na+-channel blockade, with a concomitant inhibition of glutamate release, especially under conditions of extreme excitation. In experimental epilepsy, LTG was effective in the maximal electroshock test in mice and genetically epilepsy-prone rats. The drug antagonized PTZ-induced tonic, but not clonic convulsions in mice, protected against photically induced spike–wave jerks, and attenuated generalized amygdala-kindled seizures in rats (2). The spectrum of action of LTG appears broader than that of either phenytoin (PHT) or carbamazepine (CBZ), extending also to absence epilepsy, suggesting a different profile of LTG from that of other Na+-channel blockers. Results indicate that LTG also is superior to PHT and CBZ as regards adverse effects (3). However, its antiglutamate activity occurs at supratherapeutic concentrations (for review, see 4).

Finally, LY 300164 [7-acetyl-5-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3-dioxolo(4,5H)-2,3-benzodiazepine], a potential antiepileptic drug (AED), is a selective noncompetitive antagonist of glutamatergic AMPA (α-amino-3-hydroxy-5-methylisoxazole-4-propionate)/kainate receptors. In preclinical evaluation, it significantly potentiated the antiseizure activity of conventional AEDs against maximal electroshock (5), PTZ-induced seizures (6), and amygdala-kindled seizures (7–9). The AMPA/kainate-receptor antagonists seem to be more advantageous than high-affinity NMDA (N-methyl-d-aspartate)-receptor blockers, at least in regard to less expressed or even the absence of neurotoxic undesired effects (10).

The aim of this study was to assess the influence of GBP on the protection offered by several AEDs, on the basis of isobolographic analysis, allowing us to distinguish between additive and synergistic effects of drug interactions. The adverse effects of such combinations were evaluated in the chimney test (motor coordination) and the passive-avoidance task (an estimate of long-term memory). Finally, we measured the influence of GBP on the plasma concentrations of AEDs, and, reciprocally, the effect of AEDs on the plasma level of GBP, to define a possible involvement of a pharmacokinetic interaction in the obtained results. The results of this study may give some clues as to how to combine drugs to enhance effectiveness without serious adverse activity. About 20% of epilepsy patients cannot be effectively cured with the existing conventional AEDs or their combinations, so there is a need for new AEDs or treatment strategies (1). These new drugs are generally used as adjunctive AEDs, but the experimental background for their effective combinations with conventional AEDs has not been sufficient. Moreover, a question emerges whether combinations of new AEDs or new with potential AEDs may offer more benefits in terms of seizure protection, and this study also deals with this problem.

MATERIALS AND METHODS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

General

The experiments were carried out on male Swiss mice weighing 20–25 g. The animals were housed in colony cages with free access to food (chow pellets) and tap water. The experimental temperature was 21 ± 1°C, and mice were on a natural light–dark cycle. The experimental groups, consisting of eight to 12 animals, were chosen by means of a randomized schedule. All experiments were done between 10 a.m. and 2 p.m. The experimental procedures in this study were approved by the Local Bioethical Committee of Lublin.

Drugs

Phenytoin (PHT; Sigma, St. Louis, MO, U.S.A.), valproate magnesium (VPA; Polfa, Rzeszów, Poland), carbamazepine (CBZ; Polfa, Starogard, Poland), phenobarbital sodium (PB; Polfa, Cracow, Poland), lamotrigine (LTG; Glaxo Wellcome, Kent, U.K.), LY 300164 (Lilly Research Laboratory, Indianapolis, IN, U.S.A.), and gabapentin (GBP; Parke-Davis Pharmaceutical Research, Plymouth, U.K.) were used in this study. PHT, CBZ, LTG, LY 300164, and GBP were suspended in a 1% solution of Tween 81 (Loba Chemie, Vienna, Austria). VPA and PB were dissolved in sterile water. All drugs were administered intraperitoneally (i.p.), in a volume of 10 ml/kg, PHT, 120 min; PB, LTG, and GBP, 60 min; and VPA, CBZ, and LY 300164, 30 min before electroconvulsions and behavioral tests.

Electroconvulsions

Electroconvulsions were produced with the use of ear-clip electrodes and alternating current (50 Hz) delivered by a Hugo Sachs (type 221; Freiburg, Germany) generator. The stimulus duration was 0.2 s. Tonic hindlimb extension was taken as the end point. The electroconvulsive threshold was evaluated as CS50, which is the current strength (in mA) necessary to produce tonic hindlimb extension in 50% of the animals tested. To estimate the electroconvulsive threshold, at least four groups of mice (eight to 10 animals per group) were challenged with electroshocks of various intensities. Subsequently, an intensity–response curve was calculated on the basis of percentage of mice convulsing. To evaluate the respective 50% effective dose (ED50) values (in mg/kg), mice pretreated with different doses of AEDs were challenged with electroshock of 25 mA, which exceeded the threshold current by ∼4 times. Again, at least four groups of mice, consisting of eight to 10 animals, were used to estimate each ED50 value. A dose–effect curve was constructed, based on the percentage of mice protected.

Chimney test

The effects of AEDs on motor impairment were quantified with the chimney test of Boissier et al. (11). In this test, animals had to climb backward up the plastic tube (3 cm inner diameter, 25 cm length). Motor impairment was indicated by the inability of mice to climb backward up the tube within 60 s, and the results were shown as TD50 (50% toxic dose) values, that is, the respective doses of drugs and their combinations, at which 50% of animals failed to perform the test. The dose ratio in this case was always 1:1. The drugs also were combined at doses used in the electroshock test at dose ratios, gabapentin/an AED was 10:1, except for VPA, with which the dose ratio gabapentin/valproate was 1:3. At these dose ratios, there were synergistic interactions between the drugs in the electroshock test.

Passive-avoidance task

The drug-treated mice were placed in an illuminated box (10 × 13 × 15 cm) connected to a large dark box (25 × 20 × 15 cm), which was equipped with an electric grid floor. Entrance to the dark box was punished by an electric footshock (0.6 mA for 2 s; facilitation of acquisition). The mice that did not enter the dark compartment within 60 s were excluded from the experiment. On the next day (24 h later), the same animals were put into the illuminated box and observed for ≤180 s. The mean time to enter the dark box was subsequently calculated. The control (saline-treated animals) did not enter the dark box within the observation time limit. The dose ratios for the combinations of AEDs were identical to those in the chimney test. According to Venault et al. (12), the step-through passive-avoidance task is recognized as a measure of long-term memory.

Estimation of the free plasma concentrations of antiepileptic drugs

The animals were administered a vehicle + an AED or GBP with the respective AED. The fixed-drug-ratio combination (gabapentin/an AED) for estimating the free plasma concentrations of AEDs was chosen as 10:1 for all AEDs except VPA, for which fixed-ratio combination was 1:3. Mice were killed by decapitation at times scheduled for the convulsive test, and samples of blood of ∼1 ml were collected into Eppendorf tubes. Samples of blood were centrifuged at 10,500 g (Abbott centrifuge; Irving, TX, U.S.A.) for 3 min, and plasma samples of 70 μl were transferred into system MPS-1 (Amicon, Danvers, MA, U.S.A.) for separation of free from protein-bound microsolutes. Then the MPS-1 tubes were centrifuged at 2,800 g (MPW-360 centrifuge; Mechanika Precyzyjna, Warsaw, Poland) for 10 min, and the filtrate samples of 50 μl were put into Abbott system cartridges. Free plasma levels were estimated by immunofluorescence, by using an Abbott TDx analyzer (Abbott) and expressed in μg/ml as means ± SD of at least eight determinations.

Chromatographic determination of LY 300164, lamotrigine, and gabapentin plasma levels

The plasma levels of LY 300164, LTG, and GBP were measured by high-pressure liquid chromatography (HPLC). The chromatograph (from Laboratorij Pristroje, Praha) was equipped with a 305 micropump (LCP 3001) and an ultraviolet (UV) detector (HP 1050) with a sensitivity setting of 0.1 AUFS (absorbance units full scale) and a time constant of 0.1 s. The Rheodyne 7125 injector valve with a 100-μl sample loop was used for sample injection. For HPLC, a stainless-steel HP ODS column (200 × 4.6 mm) was used at an ambient temperature of 22°C. The mobile phase was methanol/acetonitrile/0.05 M C2H5COOH buffer; 20:20:60 vol/vol (BAKER HPLC grade). The mobile phase flow rate was 1 ml/min. At the times of electroconvulsive seizures, animals were killed, and plasma samples of 200 μl were added to 200 μl of water; 100 μl of methanol/water solution, 1:1; and 50 μl of MeOH/water solution, 1:1 buffer. The solutions were evaporated to dryness under a vacuum system and redissolved in 1 ml of tertbutyl-methyl ether (HPLC, Aldrich), and again evaporated to dryness under a vacuum system. The remains were redissolved in 100-μl mobile phase; samples of 20 μl were then injected into the chromatograph. LY 300164 concentrations were calculated according to the external standard method by using the original Gilson 715 software. The amount of LY 300164 (expressed in micrograms per milliliter of plasma) was determined by comparing the peak area with the peak area of the external standard (CBZ). Stock solutions of LY 300164 serving as internal standards (0.2:0.6:1.2:2.4:4.8 μg/ml) were prepared in mobile phase. They were placed at the beginning and end of each measurement sequence.

The plasma levels of LTG and GBP were evaluated in a similar way. The main difference was that the mobile phase consisted of methanol/acetonitrile/20 mM citric acid/40 mM sodium citrate buffer; 330:90:580 vol/vol for LTG, and 150:150:700 vol/vol for GBP. Moreover, the evaporated solutions were redissolved in 4 ml of tertbutyl-methyl ether, and samples of 50 or 20 μl (for LTG and GBP, respectively), were injected into the chromatograph.

The elution and detection parameters for LY 300164 and LTG were 1 ml/min and 310 nm, respectively. The wave excitation and emission parameters for fluorescent detection of GBP were 270 and 420 nm, respectively. The external standard for LTG was LY 300164, and that for GBP was acenaphten.

Isobolographic analysis

The isobologram method was considered the optimal method to detect synergy (supraadditivity), additive interaction, or antagonism (infraadditivity). In the present study, interactions between drugs, as regards their anticonvulsant efficacy against electroshock test, and motor deficit development, were evaluated isobolographically according to Porreca et al. (13) and Tallarida (14). The isobolographic method is based on a comparison of doses that are determined to be equieffective. To perform the isobolographic analysis, the mixtures of GBP with an AED were coadministered in a number of fixed-ratio combinations (e.g., 1:3, 1:5, 1:7, 1:10 for VPA, or 1:1, 3:1, 5:1, 7:1, 10:1, etc., for the remaining AEDs). From the dose–response curve of the combined drugs, the actual (experimental) ED50exp or TD50exp values with their SEM values were calculated (13,14). The isoboles were drawn by plotting the experimentally determined ED50 or TD50 values of GBP on the X-axis and that of the respective AED on the Y-axis delivered alone or in combination.

Statistics

Both CS50 values for GBP (and their statistical analysis) and ED50 or TD50 values for AEDs alone were calculated according to Litchfield and Wilcoxon (15). The results from the passive-avoidance task were compared with the Kruskal–Wallis test followed by Dunn's test. The plasma concentrations of AEDs were evaluated with unpaired Student's t test.

The experimental ED50 (TD50) values for a mixture of drugs were compared with the respective theoretical additive ED50s (TD50s). If the actual ED50exp (TD50exp) is not different from the respective theoretical additive ED50add or TD50add, then the effect of the drug administration is additive; otherwise, if the mixture ED50exp (TD50exp) is statistically lower than the theoretical additive ED50add (TD50add) value, a synergistic interaction between drugs occurs. The statistical evaluation of ED50exp (TD50exp) versus the respective additive values was performed according to Porreca et al. (13), who recommended Student's t test. That is why the respective ED50add(TD50 add) or ED50exp(TD50exp) values are shown with the SEM. The following equations show how to calculate ED50add and SEM values. From equations described by Porreca et al. (1990), one can determine the (ED50)add value according to

  • image(1)

where P1 is a proportion of drug1 (effective against electroshock) in the total amount of drug mixture. It is obvious that, for the mixture of two drugs, the expression is true when

  • image(2)

where P2 is a proportion of drug2 (ineffective against electroshock).

Again, following the equations in the article of Porreca et al. (13), one can calculate

  • image(3)

where SEM is the standard error of the mean of respective ED50 values.

This equation describes the dependence of SEM(ED50) on an ED50 value:

  • image(4)

where logarithms are to base 10.

Additionally, we applied a log-probit method elaborated by Litchfield and Wilcoxon (15), which has widely been adapted for the pharmacologic studies on the dose–effect response of drug applications. The authors presented an analysis of results of drug–dose effects obtained in animal studies, showing equations permitting calculation of SEM[log(ED50)].

  • image(5)

where s is the difference between two log doses whose expected effects differ by 1 probit, N' is the total number of animals tested between the log dose limits corresponding to expected probits 4 and 6, and sqrt is a square root of the expression in parentheses.

To calculate s we used an algebraic transformation of another equation presented by Litchfield and Wilcoxon (15):

  • image(6)

Another description of this equation also is acceptable:

  • image(7)

where S is a slope function obtained directly from log-probit analysis of data according to Litchfield and Wilcoxon (15). Consequently,

  • image(8)

Finally, transforming all equations presented here, the SEM(ED50)add for a fixed proportion of drugs was determined and is presented as

  • image(9)

RESULTS

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

Effects of gabapentin on the electroconvulsive threshold

GBP (25 and 50 mg/kg), administered i.p. 60 min before the test, did not affect the electroconvulsive threshold in mice. The AED applied at doses of 100 and 200 mg/kg increased the threshold from 6.1 (5.4–6.9) mA to 8.0 (7.0–9.1) and 16.2 (14.2–18.4) mA (Table 1).

Table 1.  Influence of gabapentin on the electroconvulsive threshold in mice
Treatment (mg/kg)CS50 (mA)
  1. CS50 (in mA; 95% confidence limits in parentheses) is a current strength necessary to produce convulsions in 50% of the animals tested. Gabapentin (GBP) was administered i.p. 60 min before the test.

  2. a  p < 0.01, bp < 0.001, versus respective vehicle. Calculation of CS50 values and their statistical analysis was performed according to Litchfield and Wilcoxon (15).

Vehicle6.1 (5.4–6.9)
GBP (25)6.2 (5.5–7.1)
GBP (50)6.9 (6.2–7.8)
GBP (100)8.0 (7.0–9.1)a
GBP (200)16.2 (14.2–18.4)b

Isobolographic analysis of the protection offered by gabapentin combined with studied antiepileptic drugs in electroshock-induced seizures in mice

It was impossible to evaluate the ED50 for GBP against electroshock in mice. There was apparently a bell-shaped response, with the maximal protection being 50% at the dose of 300 mg/kg (result not shown). The experimentally assessed ED50 values with 95% confidence limits (15) for CBZ, PHT, PB, VPA, LTG, and LY 300164 were 10.0 (8.4–11.9), 10.4 (9.4–11.5), 21.1 (19.3–25.2), 249 (223–267), 5.1 (3.7–6.6), and 3.9 (3.1–4.8) mg/kg, respectively (results not shown).

Moreover, the experimentally evaluated ED50 values (EDexp) for the combinations of GBP and CBZ (3:1, 5:1, 7:1, 10:1, 30:1) were significantly lower than the theoretically calculated additive ED50 for the mixture (EDadd), thus strongly indicating the synergistic interaction between the two drugs. The combined treatment of GBP and PHT (1:1, 3:1, 5:1, 7:1, 10:1) resulted in synergy, because the EDexp values were significantly lower than the respective EDadd values (Fig. 1). GBP and PB apparently acted synergistically when the drugs were injected in fixed fractions of dose ratios 5:1, 7:1, 10:1 (Table 2). Similarly, synergy was found when GBP was coadministered with VPA in fixed proportions of 1:3, 1:5, 1:7, 1:10 (Table 3), LTG (3:1, 5:1, 7:1, 10:1, 20:1, 40:1), and LY 300164 (5:1, 7:1, 10:1, 20:1, 50:1; Table 4).

image

Figure 1. Isobologram displaying fixed-ratio drug interactions between gabapentin (GBP) and phenytoin (PHT) in the electroshock test. The 50% effective dose (ED50) value for PHT is plotted on the y-axis. The heavy line is parallel to the x-axis, representing the ED50 value for PHT, and defines the theoretical dose-additive line for a continuum of different fixed-dose ratios. Dotted lines relate to ratio of drug doses and represent on the graph the 1:1, 3:1, 5:1, 7:1, and 10:1 fixed-dose ratios of GBP and PHT. Solid symbols (·), the experimentally derived points for total doses expressed as the proportion of GBP and PHT that produce an equivalent 50% effect. The experimental points for the GBP/PHT mixture were found to be significantly below the theoretical additive line, indicating supraadditive (synergistic) interactionversus theoretical additive points. The standard errors (SEM) for experimental and additive points are represented by the error bars. Level of significance for 1:1 fixed-ratio combination is significant at p < 0.01, whereas all remaining fixed-ratio combinations of GBP and DPH (3:1, 5:1, 7:1, and 10:1) are significant at p < 0.001.

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Table 2.  Effect of the combinations of gabapentin with carbamazepine, phenytoin, or phenobarbital against electroshock in mice
  1. Data are presented as ED50 values ± SEM.

  2. F, dose ratio (GBP/an antiepileptic drug); EDadd, theoretical additive ED50; EDexp, experimental ED50 of the respective mixture; GBP, gabapentin; CBZ, carbamazepine; PHT, diphenylhydantoin; PB, phenobarbital; ND, not determined.

  3. a  p < 0.01; bp < 0.001 versus the respective EDadd value. Calculations of EDs were made according to Litchfield and Wilcoxon (15), and isobolography was performed according to Porreca et al. (13).

F1:13:15:17:110:130:1
GBP + CBZ      
 EDadd20.1 ± 1.340.2 ± 2.560.2 ± 3.880.4 ± 5.0110.4 ± 6.9312.1 ± 19.6
 EDexp20.4 ± 1.532.1 ± 1.0a41.8 ± 1.3a55.8 ± 1.5a60.6 ± 2.4b84.8 ± 2.3b
GBP + PHT      
 EDadd20.8 ± 1.041.6 ± 2.162.3 ± 3.183.2 ± 4.2114.3 ± 5.7ND
 EDexp14.4 ± 1.6a26.6 ± 1.3b34.8 ± 1.3b30.1 ± 1.3b40.6 ± 1.4bND
GBP + PB      
 EDadd42.2 ± 3.784.4 ± 7.4126.3 ± 11.1168.8 ± 14.8321.8 ± 20.4ND
 EDexp42.3 ± 3.073.3 ± 3.676.34 ± 3.5a57.5 ± 2.8b73.6 ± 3.8bND
Table 3.  Effect of the combinations of gabapentin with valproate against electroshock in mice
  1. Data are presented as ED50 values ± SEM.

  2. F, dose ratio (GBP/VPA); EDadd, theoretical additive, EDexp, experimental ED50 of the respective mixture; GBP, gabapentin; VPA, valproate.

  3. a  p < 0.001; bp < 0.05 versus the respective EDadd value. See also footnotes of Table 2.

F1:31:51:71:101:20
GBP + VPA     
 EDadd325.1 ± 17.2292.7 ± 15.5278.6 ± 14.8268.2 ± 14.2256.0 ± 13.6
 EDexp166.1 ± 32.4a169.3 ± 40.5a170.1 ± 42.2b164.3 ± 40.8b183.4 ± 36.6
Table 4.  Effect of the combinations of gabapentin with lamotrigine or LY 300164 against electroshock in mice
FGBP + LTGGBP + LY 300164
EDaddEDexpEDaddEDexp
  1. Data are presented as ED50 values ± SEM.

  2. F, dose ratio (GBP/an antiepileptic drug); EDadd, theoretical additive ED50; EDexp, experimental ED50 of the respective mixture; GBP, gabapentin; LTG, lamotrigine; ND, not determined.

  3. a  p < 0.01; bp < 0.001 versus the respective EDadd value. See also legend of Table 2.

1:110.0 ± 1.47.7 ± 0.77.8 ± 0.86.5 ± 0.3
3:120.0 ± 2.911.6 ± 1.2a15.6 ± 1.612.9 ± 0.3
5:130.0 ± 4.317.4 ± 1.2a23.3 ± 2.516.4 ± 0.5a
7:140.0 ± 5.820.9 ± 0.9a31.2 ± 3.319.4 ± 0.8a
10:155.0 ± 8.031.8 ± 1.2a42.9 ± 4.524.3 ± 0.9a
20:1104.2 ± 15.132.9 ± 0.7a81.2 ± 8.634.7 ± 0.8b
40:1208.2 ± 44.335.7 ± 0.8bNDND
50:1NDND162.5 ± 17.287.1 ± 0.7a
60:1305.0 ± 44.369.4 ± 1.3bNDND

Dark-avoidance acquisition and retention testing

GBP coadministered with other AEDs in the dose ratio of 10:1, or 1:3 dose (in the case of the mixture with VPA), did not affect the performance of mice in the passive-avoidance task (Table 5).

Table 5.  Effects of antiepileptic drugs, LY 300164, and their combinations with gabapentin on the retention of a passive-avoidance task in mice
Treatment (mg/kg)Median (25, 75 percentiles)
  1. Kruskal–Wallis test followed by Dunn's post hoc test were used for statistical analysis of the data. Experimental groups consisted of 10 mice. See also Materials and Methods and legends of Tables 2–4.

Vehicle180 (180, 180)
CBZ (5.52)180 (180, 180)
GBP (55.2)180 (158, 180)
CBZ (5.52) + GBP (55.2)180 (150, 180)
PHT (3.69)180 (180, 180)
GBP (36.9)180 (160, 180)
PHT (3.69) + GBP (36.9)180 (153, 180)
PB (6.69)180 (180, 180)
GBP (66.9)177.5 (154, 180)
PB (6.69) + GBP (66.9)180 (135, 180)
VPA (124.5)180 (154, 180)
GBP (41.5)180 (155.5, 180)
VPA (124.5) + GBP (41.5)180 (153, 180)
LTG (2.89)180 (131, 180)
GBP (28.9)180 (157.5, 180)
LTG (2.89) + GBP (28.9)180 (129, 180)
LY 300164 (2.21)180 (163, 180)
GBP (22.1)180 (166.5, 180)
LY 300164 (2.21) + GBP (22.1)180 (142, 180)

Chimney test

The experimentally evaluated TD50 values (TDexp) for the combinations of GBP with other AEDs, in the fixed fraction of dose ratio 1:1, were not significantly lower than the theoretically calculated additive TD50 values for the respective mixtures (TDadd), suggesting additivity between drugs in motor-deficit development (Table 6 ). Antiepileptics (AEDs) also were combined in dose ratios used for the calculation of ED50 values in the electroshock test. For GBP and VPA, the ratio was 1:3, whereas that for GBP and the remaining AEDs was 10:1. In no case did AEDs alone or combined produce impairment of motor performance in mice (Table 7).

Table 6.  Additive and experimental TD50 values of combinations of gabapentin with antiepileptic drugs in the chimney test in mice
MixtureTDadd ± SEMTDexp± SEM
  1. Data are presented as median toxic dose (TD50) values ± SEM. TDadd, theoretical additive TD50; TDexp, experimental TD50 of the respective mixture; GBP, gabapentin; LTG, lamotrigine; PHT, phenytoin; CBZ, carbamazepine; PB, phenobarbital; VPA, valproate. Drugs were administered at a fixed ratio of 1:1, based on the respective TD50 values. TD50 values were calculated according to Litchfield and Wilcoxon (15). Calculations of SEM and statistical comparison were performed according to the method of Tallarida (14).

LTG + GBP122.9 ± 40.280.9 ± 18.3
PHT + GBP144.7 ± 40.277.7 ± 24.6
CBZ + GBP139.4 ± 40.2165.7 ± 24.9
LY 300164 + GBP112.2 ± 40.2157.6 ± 21.6
PB + GBP109.7 ± 40.273.3 ± 13.0
VPA + GBP281.6 ± 40.9306.4 ± 79.3
Table 7.  Effects of antiepileptic drugs alone or combinations of gabapentin with the AEDs on the motor performance of mice in the chimney test
Treatment (mg/kg)Mice impaired (%)
  1. The results of the chimney test are expressed as a percentage of animals showing motor impairment. Each experimental group consisted of 10 animals. The Fisher's exact probability test was used for statistical comparisons. For abbreviations, refer to Table 6.

  2. GBP, gabapentin; LTG, lamotrigine; PHT, phenytoin; VPA, valproate; CBZ, carbamazepine; PB, phenobarbital.

LY 300164 (2.21)0
GBP (22.1)0
GBP (22.1) + LY 300164 (2.21)20
LTG (2.89)0
GBP (28.9)0
GBP (28.9) + LTG (2.89)10
PHT (3.69)0
GBP (36.9)0
GBP (36.9) + PHT (3.69)0
VPA (124.6)0
GBP (41.5)0
GBP (41.5) + VPA (124.6)10
CBZ (5.52)0
GBP (55.2)0
GBP (55.2) + CBZ (5.52)10
PB (6.69)0
GBP (66.9)10
GBP (66.9) + PB (6.69)30

Influence of gabapentin on the plasma levels of antiepileptic drugs and the studied antiepileptic drugs on the plasma concentration of gabapentin

GBP applied at its highest doses did not affect the plasma levels of CBZ, VPA, PB, PHT, LTG, or LY 300164 (Table 8). In contrast, plasma concentrations of GBP were significantly enhanced by PB and LTG, but not by the remaining AEDs (Table 9).

Table 8.  Influence of gabapentin on plasma levels of antiepileptic drugs
Treatment (mg/kg)Plasma levels
  1. Values are the means in μg/ml of plasma ± SD of eight determinations. Blood samples were taken at times scheduled for the convulsive test. Plasma levels of carbamazepine (CBZ), phenytoin (PHT), phenobarbital (PB), and valproate (VPA) were evaluated by immunofluorescence, and that of lamotrigine (LTG) and LY 300164 by high-pressure liquid chromatography. Unpaired Student's t test was used for statistical evaluation of the data. See also legends of Tables 2–4.

CBZ (5.52)0.46 ± 0.09
CBZ (5.52) + GBP (55.2)0.51 ± 0.15
PHT (3.69)0.32 ± 0.12
PHT (3.69) + GBP (36.9)0.33 ± 0.03
PB (6.69)5.13 ± 0.39
PB (6.69) + GBP (66.9)5.09 ± 0.29
VPA (124.6)100.53 ± 19.65
VPA (124.6) + GBP (41.5)119.81 ± 26.10
LTG (2.89)1.02 ± 0.21
LTG (2.89) + GBP (28.9)0.99 ± 0.15
LY 300164 (2.21)3.49 ± 0.52
LY 300164 (2.21) + GBP (22.1)3.88 ± 0.52
Table 9.  Influence of antiepileptic drugs on plasma level of gabapentin
Treatment (mg/kg)Plasma levels
  1. Values are the means in μg/ml of plasma ± S.D. of eight determinations. Blood samples were taken at times scheduled for the convulsive test. Plasma level of gabapentin (GBP) were evaluated by high-pressure liquid chromatography. Unpaired Student's t test was used for statistical evaluation of the data.

  2. CBZ, carbamazepine; PHT, phenytoin; PB, phenobarbital; VPA, valproate; LTG, lamotrigine.

  3. a  p < 0.01, bp < 0.05 versus the respective control group. See also legends of Tables 2–4.

GBP (55.2)11.32 ± 1.80
GBP (55.2) + CBZ (5.52)12.06 ± 2.33
GBP (36.9)9.28 ± 1.93
GBP (36.9) + PHT (3.69)8.49 ± 1.05
GBP (66.9)19.75 ± 2.44
GBP (66.9) + PB (6.69)23.33 ± 1.95a
GBP (41.5)10.82 ± 0.90
GBP (41.5) + VPA (124.6)10.96 ± 1.07
GBP (28.9)11.24 ± 1.03
GBP (28.9) + LTG (2.89)12.80 ± 1.33b
GBP (22.1)7.01 ± 0.98
GBP (22.1) + LY 300164 (2.21)7.07 ± 0.83

DISCUSSION

  1. Top of page
  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES

This study demonstrates that GBP (≤50 mg/kg) remained ineffective on the electroconvulsive threshold in mice. According to isobolographic analysis, GBP appears to act synergistically with CBZ, VPA, PHT, PB, LTG, and LY 300164, because experimentally evaluated ED50 values (EDexp) for the respective combinations applied in various proportions were significantly lower than the theoretically calculated additive ED50s for the mixtures of GBP with an AED (EDadd). Although the pharmacokinetic events apparently participate in the interactions between GBP and PB or LTG, the remaining combinations appear to be dependent on pharmacodynamic events. It is worth mentioning that only PB and LTG significantly elevated the plasma concentration of the GABA analogue. Conversely, GBP did not affect the free plasma concentrations of AEDs used in the present study. Because pharmacokinetic studies were performed for only some drug ratios, it is impossible to exclude entirely a possibility of pharmacokinetic interactions for all evaluated drug ratios.

GBP increases GABA synthesis, turnover, and nonvesicular release, although the drug acts mainly by decreasing neuronal calcium influx through a specific auxiliary subunit of voltage-dependent calcium channels (1). Conversely, the mechanism of action of VPA, CBZ, PB, PHT, and LTG is associated mostly with their ability to delay the recovery from inactivation of sodium channels. However, in contrast to CBZ, PHT possibly binds to different types of the α-subunit of the sodium channel (16). VPA and PB also potentiate GABA-mediated inhibition, and PB also may antagonize glutamate-induced excitation (1). Therefore, different mechanisms of action may underlie the nature of the synergistic interaction between GBP and CBZ, VPA, PHT, LTG, or LY 300164.

Moreover, according to the isobolographic analysis, synergy in respect to AED effectiveness was accompanied by additivity as regards motor impairment. The combinations displaying clear-cut synergy in the electroshock test did not affect the performance of mice in the chimney test. Interestingly, in kindled rats, GBP displayed a separation between motor impairment and anticonvulsive effects of at least factor 3.5 (2). In a murine model of acute cocaine toxicity, GBP showed a significant separation between anticonvulsive and side-effect profiles as well (17). This phenomenon may be responsible for the encouraging results obtained in the chimney test. It also should be underlined that in our study, the concomitant treatment of GBP with other AEDs did not produce long-term memory deficits.

In clinical practice, AEDs are usually given in doses that are effective. However, the risk of adverse effects during polytherapy increases with the enhancement of AED concentrations (18). To avoid undesired effects associated with two-drug therapy, one of them should be used in the lowest effective dose, or both AEDs should be given in the middle ranges of doses (1.5 therapy; 19,20).

Gabapentin also has been combined with conventional AEDs in a model of reflex epilepsy–sound-induced seizures in DBA/2 mice (21). In a nonprotective dose of 2.5 mg/kg, GBP increased the protective efficacy of CBZ, diazepam (DZP), PHT, PB, and VPA. With DZP, PB, and VPA, the potentiation of their anticonvulsant effect was the most expressed. The therapeutic indexes of the AEDs alone were worse than those of combined treatments. The pharmacokinetic mechanism may be excluded because GBP did not significantly affect the plasma concentration of AEDs. Interestingly, GBP (in a subprotective dose of 25 mg/kg against maximal electroshock in mice) also has been combined with conventional AEDs and decreased the ED50 values of CBZ (by 28%), PHT (by 52%), PB (by 58%), and VPA (by 28%; 22,23). Again, no pharmacokinetic interactions have been noted with regard to conventional AEDs. Although the procedure run in these three articles (21–23) does not allow distinguishing between additive and synergistic interactions, these results at least may point to such a possibility, and this was clearly verified in the present study.

It is noteworthy that the dose ratio may be critical for the final outcome of an interaction between AEDs. This is evident from the present results that in some dose ratios, the interactions were simply additive (e.g., GBP/CBZ, 1:1), and in many, very significantly synergistic. Results from other studies also point to this problem. For instance, Gordon et al. (24) reported on the considerable reduction of the ED50 value of felbamate (FBM) against maximal electroshock in mice by non-protective doses of conventional AEDs. In contrast, a total failure for FBM in nonprotective doses to affect the ED50 values of conventional AEDs was reported (25). In our opinion, this must be considered by the clinicians when introducing drug combinations in epilepsy patients.

According to Deckers et al. (16), there is a controversy about rational polytherapy whether to combine AEDs working through similar or completely different mechanisms. The present results seem to support the latter possibility because GBP, acting mainly on voltage-dependent calcium channels (1), was combined with the drugs exerting their anticonvulsant effects primarily through other mechanisms. A very potent interaction between GBP and LTG or LY 300164 also must be accentuated, although the former is at least partially dependent on the pharmacokinetic interaction.

Acknowledgment: We are grateful to Lilly Research Laboratories (Indianapolis, IN, U.S.A.), Polfa (Cracow), and Polfa (Rzeszów) for the generous gifts of LY 300164, phenobarbital sodium, and valproate magnesium.

Dr. Swiader is the recipient of a fellowship for young researchers from the Foundation for Polish Science. This study was supported by a KBN grant 4PO5A 033 19.

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  2. Abstract
  3. MATERIALS AND METHODS
  4. RESULTS
  5. DISCUSSION
  6. REFERENCES
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