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- MATERIALS AND METHODS
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.
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- MATERIALS AND METHODS
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.