Interactions of 1-methyl-1,2,3,4-tetrahydroisoquinoline with lamotrigine, oxcarbazepine, pregabalin, and topiramate in the mouse maximal electroshock-induced seizure model: A type I isobolographic analysis

Epilepsy Research (2010) 89, 207—219

journal homepage: www.elsevier.com/locate/epilepsyres

Interactions of 1-methyl-1,2,3,4tetrahydroisoquinoline with lamotrigine, oxcarbazepine, pregabalin, and topiramate in the mouse maximal electroshock-induced seizure model: A type I isobolographic analysis Jarogniew J. Luszczki a,b,∗, Lucyna Antkiewicz-Michaluk c, Grzegorz Raszewski b, Stanislaw J. Czuczwar a,b a

Department of Pathophysiology, Medical University of Lublin, Jaczewskiego 8, PL 20-090 Lublin, Poland Department of Physiopathology, Institute of Agricultural Medicine, Jaczewskiego 2, PL 20-950 Lublin, Poland c Department of Neurochemistry, Institute of Pharmacology Polish Academy of Sciences, Smetna 12, PL 31-343 Krakow, Poland b

Received 11 March 2009; received in revised form 23 December 2009; accepted 7 January 2010 Available online 1 February 2010

KEYWORDS 1-Methyl-1,2,3,4tetrahydroisoquinoline; Second-generation antiepileptic drugs; Isobolographic analysis; Maximal electroshock; Pharmacodynamic/ pharmacokinetic interaction

Summary The aim of this study was to characterize the anticonvulsant effects of 1-methyl1,2,3,4-tetrahydroisoquinoline (MeTHIQ—–an endogenous parkinsonism-preventing substance) in combination with four second-generation antiepileptic drugs (AEDs: lamotrigine [LTG], oxcarbazepine [OXC], pregabalin [PGB], and topiramate [TPM]) in the mouse maximal electroshock (MES)-induced seizure model by using the type I isobolographic analysis for parallel and non-parallel dose—response relationship curves (DRRCs). Potential adverse-effect profiles of interactions of MeTHIQ with LTG, OXC, PGB and TPM at the fixed-ratio of 1:1 from the MES test with respect to motor performance, long-term memory and skeletal muscular strength were measured along with total brain concentrations of MeTHIQ and TPM. In the mouse MES model, MeTHIQ administered singly had its DRRC parallel to those for OXC and TPM, and simultaneously, non-parallel to those for LTG and PGB. With type I isobolography for parallel DRRCs, the combination of MeTHIQ with TPM at three fixed-ratios of 1:3, 1:1 and 3:1 exerted supra-additive (synergistic) interaction, whereas the combination of MeTHIQ with OXC at the fixed-ratios of 1:3, 1:1 and 3:1 produced additive interaction. Similarly, the type I

∗ Corresponding author at: Department of Pathophysiology, Medical University of Lublin, Jaczewskiego 8, PL 20-090 Lublin, Poland. Tel.: +48 81 718 73 65; fax: +48 81 718 73 64. E-mail addresses: [email protected], [email protected], [email protected] (J.J. Luszczki).

0920-1211/$ — see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.eplepsyres.2010.01.001

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J.J. Luszczki et al. isobolography for non-parallel DRRCs revealed that the combination of MeTHIQ with LTG and PGB at the fixed-ratio of 1:1 produced additive interaction. For all combinations, neither motor coordination, long-term memory nor muscular strength were affected. Total brain concentrations of MeTHIQ and TPM revealed no significant changes in their concentrations when the drugs were combined at the fixed-ratios of 1:3, 1:1 and 3:1. In conclusion, the synergistic interaction of MeTHIQ with TPM at the fixed-ratios of 1:3, 1:1 and 3:1 against MES-induced seizures was pharmacodynamic in nature and thus, it is worthy of consideration in further clinical settings. The combinations of MeTHIQ with LTG, OXC and PGB were neutral in the mouse MES model. © 2010 Elsevier B.V. All rights reserved.

Introduction Overwhelming evidence indicates that 1-methyl1,2,3,4-tetrahydroisoquinoline (MeTHIQ—–an endogenous parkinsonism-preventing substance) exerts neuroprotective activity by antagonizing the behavioral effects of dopaminergic neurodegeneration induced by numerous experimental neurotoxins, including: 1-methyl-4-phenyl1,2,3,6-tetrahydropyridine, 1-methyl-4-phenylpyridine, beta-carbolines, tetrahydroisoquinoline, 1-benzyl-1,2,3,4tetrahydroisoquinoline and rotenone (Tasaki et al., 1991; Kotake et al., 1995, 1996, 2005; Yamakawa and Ohta, 1999; Yamakawa et al., 1999; Antkiewicz-Michaluk et al., 2003, 2004). Moreover, MeTHIQ exhibited neuroprotective effects against glutamate- and kainic acid-induced excitotoxicity by antagonizing neuronal death and calcium influx (Antkiewicz-Michaluk et al., 2006). In radioligand binding studies, MeTHIQ inhibited the binding of [3 H]MK-801 (dizocilpine, an uncompetitive antagonist of the N-methylD-aspartate receptors), showing the properties of the non-competitive N-methyl-D-aspartate receptor antagonist (Antkiewicz-Michaluk et al., 2006). A large body of evidence indicates that MeTHIQ elevated the threshold for electroconvulsions in mice (Luszczki et al., 2006a), and possessed the antielectroshock action in mice at times ranging between 5 and 120 min after its systemic administration (Luszczki et al., 2009a). Additionally, MeTHIQ enhanced the protective action of carbamazepine (CBZ) and valproate (VPA), but not that of phenobarbital (PB) or phenytoin (PHT) against maximal electroshock (MES)induced seizures in mice (Luszczki et al., 2006a). Moreover, the isobolographic analysis of interaction revealed that MeTHIQ synergistically interacted with PB and exerted the additive interaction when concomitantly administered with CBZ, PHT and VPA in the mouse MES model (Luszczki et al., 2009a). Generally, the drugs belonging to the second-generation antiepileptic drugs (AEDs) are characterized by a broader spectrum of anticonvulsant efficacy, lower toxicity and superior pharmacokinetic profiles than classical AEDs (Deckers et al., 2000; Brodie and Schachter, 2001). The aim of this study was to characterize the types of interactions between MeTHIQ and four second-generation AEDs (lamotrigine [LTG], oxcarbazepine [OXC], pregabalin [PGB], and topiramate [TPM]) in the MES-induced seizure test using the type I isobolographic analysis. It is important to note that seizure models in laboratory animals are still the most

important tools in the preclinical search for synergistic interactions between the AEDs. The MES-induced seizure test is used as an experimental model of tonic-clonic seizures and, to a certain extent, of partial convulsions with or without secondary generalization in man (Löscher et al., 1991; Löscher, 2002). The selection of the studied AEDs belonging to the second-generation AEDs was based primarily on their anticonvulsant activity in the mouse MES model. In this study, only the fully active AEDs against MES-induced seizures in mice (i.e., LTG, OXC, PGB and TPM) were combined with MeTHIQ. It is important to note that type I isobolographic analysis requires the clear-cut anticonvulsant activity of all examined drugs which undergo experimental evaluation in the mouse MES test (Luszczki et al., 2009a). Other AEDs, such as tiagabine, vigabatrin, levetiracetam, gabapentin and benzodiazepines were not combined with MeTHIQ in this study because of their virtual inactivity in the mouse MES model (Löscher et al., 1991). Additionally, in the present study, potential acute adverse effects of the combinations of AEDs with MeTHIQ (administered at doses from the MES-induced seizure test) were investigated in relation to motor coordination impairment in the chimney test, long-term memory deficits in the step-through passive avoidance task and skeletal muscular strength in the grip-strength test in mice. Finally, to ascertain whether the observed interactions were purely pharmacodynamic in nature or that pharmacokinetic interactions also contributed, total brain concentrations of MeTHIQ and TPM were measured.

Materials and methods Animals and experimental conditions All experiments were performed on adult male albino Swiss mice (weighing 22—26 g, 6-week old) purchased from licensed breeder (Dr. T. Gorzkowska, Warszawa, Poland). The mice were kept in colony cages with free access to food and tap water under standardized housing conditions (natural light—dark cycle, temperature of 21 ± 1 ◦ C, relative humidity of 55 ± 3%). After 7 days of adaptation to laboratory conditions, the animals were randomly assigned to experimental groups consisting of 8 mice. Each mouse was used only once. All tests were performed between 9.00 a.m. and 2.00 p.m. Procedures involving animals and their care were conducted in accordance with the Guide for the Care and Use of Laboratory Animals as adopted and promulgated by the National Institutes of Health. Additionally, all efforts were made to minimize animal suffering and to use only the number of animals necessary to produce

Interactions of MeTHIQ with second-generation AEDs in the MES test reliable scientific data. The experimental protocols and procedures described hereupon were approved by the Local Ethics Committee at the Medical University of Lublin (Licenses no.: 15/2006 and 57/2009).

Drugs The following AEDs were used in this study: LTG (Lamictal® , Glaxo Wellcome, Greenford, Middlesex, UK), OXC (Trileptal® , Novartis Pharma AG, Basel, Switzerland), PGB (Lyrica® , Pfizer Limited, Sandwich, Kent, UK), TPM (Topamax® , Cilag AG, Schaffhausen, Switzerland), and MeTHIQ (gift from Dr. J. Boksa, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland). MeTHIQ was dissolved in 0.9% NaCl, whereas the second-generation AEDs were suspended in 1% solution of Tween 80 (Sigma, St. Louis, MO, USA) in saline and administered i.p. in a volume of 5 ml/kg body weight. The control animals received adequate volume of 0.9% NaCl. Fresh drug solutions were administered as follows: MeTHIQ— –at 5 min (Luszczki et al., 2006a, 2009a), OXC at 30 min (Luszczki et al., 2003a), LTG and TPM at 60 min (Luszczki et al., 2003b), and PGB at 120 min (Vartanian et al., 2006), prior to maximal electroconvulsions, chimney, passive avoidance and grip-strength tests, as well as, before brain sampling for the measurement of AED concentrations. The times to the peak of maximum anticonvulsant effects for all AEDs were used as the reference times in all behavioral tests. The route of systemic (i.p.) administration and pretreatment times before testing of the AEDs and MeTHIQ were based upon information about their biological activity from the literature and our previous experiments (Antkiewicz-Michaluk et al., 2001, 2003, 2004; Luszczki et al., 2006a, 2009a).

Maximal electroshock seizure test The protective activities of MeTHIQ, four second-generation AEDs (LTG, OXC, PGB, and TPM) administered separately and their combinations were evaluated and expressed as their median effective doses (ED50 in mg/kg), protecting 50% of mice against MES-induced seizures (fixed current intensity of 25 mA, maximum stimulation voltage of 500 V). Electroconvulsions were produced by a current (0.2 s stimulus duration) delivered via standard auricular electrodes by a Hugo Sachs generator (Rodent Shocker, Type 221, Freiburg, Germany). The criterion for the occurrence of seizure activity was the tonic hindlimb extension. The animals were administered with different drug doses so as to obtain a variable percentage of protection against MES-induced seizures, allowing for the construction of a dose—response relationship curve (DRRCs) for MeTHIQ and second-generation AEDs administered alone, according to Litchfield and Wilcoxon (1949). In the present study, to determine the ED50 values for the examined drugs in the MES test, MeTHIQ was administered at doses ranging between 40 and 70 mg/kg, LTG at doses ranging between 3 and 9 mg/kg, OXC at doses ranging between 8 and 16 mg/kg, PGB at doses ranging between 25 and 150 mg/kg, and TPM at doses ranging between 30 and 60 mg/kg. The anticonvulsant activity of the mixture of MeTHIQ with second-generation AEDs at the fixed-ratios of 1:3, 1:1 and 3:1 (for MeTHIQ + OXC and MeTHIQ + TPM) and at the fixed-ratio of 1:1 (for MeTHIQ + LTG and MeTHIQ + PGB) was evaluated and expressed as median effective doses (ED50 mix values) against MES-induced seizures (for a more detailed information concerning the selection of the fixed-ratio combinations of MeTHIQ with the studied AEDs see next paragraph). This experimental procedure has been described in detail in our earlier studies (Luszczki et al., 2003a, 2006a, 2009a,b).

Isobolographic analysis of interactions To assess the exact nature of interactions between drugs in combination in preclinical studies on animals, an isobolographic

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analysis of interaction was applied according to the method described earlier (Loewe, 1953; Luszczki and Czuczwar, 2003, 2004, 2006; Tallarida, 2006; Luszczki, 2007; Luszczki et al., 2009a,b). The percentage of animals protected against MES-induced seizures per dose of an AED administered alone and the DRRC for each investigated AED in the mouse MES model were fitted using log-probit linear regression analysis according to Litchfield and Wilcoxon (1949). Subsequently, from the respective linear equations the median effective doses (ED50 s) of AEDs administered alone were calculated. To precisely and correctly analyze the experimental data with isobolography, the test for parallelism of DRRCs for MeTHIQ and the investigated AEDs based on the log-probit analysis was used (Luszczki and Czuczwar, 2003, 2004; Luszczki, 2007; Luszczki et al., 2003a, 2009a,b). The test for parallelism was performed according to Litchfield and Wilcoxon (1949), as described in detail in our previous study (Luszczki and Czuczwar, 2006). In this test, MeTHIQ had its DRRC parallel to that of OXC and TPM, and simultaneously, non-parallel to the DRRCs of LTG and PGB (Table 1). This is why the interactions between MeTHIQ and OXC and TPM against MES-induced seizures were analyzed according to the methodology described by Tallarida (2000), and Luszczki et al. (2003a, 2009a), whereas the interactions between MeTHIQ and LTG and PGB against MES-induced seizures were analyzed according to the methodology described by Tallarida (2006), Luszczki (2007), and Luszczki et al. (2009b). It is important to note that the type I isobolographic analysis of interaction considers the parallelism of two DRRCs for the examined AEDs. If two DRRCs are parallel to one another, one can readily evaluate the interaction between drugs for at least three fixed-ratio combinations of 1:3, 1:1 and 3:1. In such a case, one can readily calculate proportions of the drugs in the mixture at the respective fixed-ratio combinations. In contrast, if two DRRCs are non-parallel to each other, one can determine the interaction only for a fixed-ratio of 1:1 because for the fixed-ratio combination of 1:3 the respective doses of the first drug in the mixture are too low, whereas the doses of the second drug in the mixture are too high, producing per se supra- or sub-additive interactions instead of additivity (Tallarida, 2006; Luszczki, 2007). The similar situation is expected for the fixed-ratio combination of 3:1. If the DRRCs are not parallel to one another, the respective doses of the first drug in the mixture are too high and the doses of the second drug in the mixture are too low, producing supra- or sub-additive interactions instead of additivity (Tallarida, 2006; Luszczki, 2007), In the isobolographic analysis of interaction for non-parallel DRRCs, median additive doses of the mixture of MeTHIQ with LTG and PGB—–i.e., doses of the mixture, which theoretically should protect 50% of the animals tested against MES-induced seizures (ED50 add ) were calculated from two equations of additivity presented by Tallarida (2006). For the lower line of additivity the equations at a 50% effect for the combination of MeTHIQ with LTG and PGB were as follows: y = ED50 LTG − [ED50 LTG /(ED50 MeTHIQ /x)p/q ], and y = ED50 PGB − [ED50 PGB /(ED50 MeTHIQ /x)p/q ]; where y is the dose of LTG or PGB; x is the dose of MeTHIQ; p and q are curvefitting parameters (Hill coefficients) for MeTHIQ and LTG and PGB, respectively (Table 1). Similarly, for the upper line of additivity the equations at a 50% effect for the combination of MeTHIQ with LTG and PGB were: y = ED50 LTG [(ED50 MeTHIQ − x)/ED50 MeTHIQ ]p/q and y = ED50 PGB [(ED50 MeTHIQ − x)/ED50 MeTHIQ ]p/q . To calculate the curve-fitting parameters (p and q), probits of response for LTG or PGB and MeTHIQ administered alone were transformed to % effect. It is important to note that when two drugs produce maximal effect but have non-parallel DRRCs, the additivity is represented as an area bounded by two defined curves (lower and upper isoboles of additivity) (Tallarida, 2006). The experimentally derived ED50 values are statistically different if their points are placed outside this region. For supra-additivity (synergy), the experimentally derived ED50 mix points are placed below

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Table 1 Anticonvulsant effects of MeTHIQ, lamotrigine (LTG), oxcarbazepine (OXC), pregabalin (PGB), and topiramate (TPM) administered singly against maximal electroshock (MES)-induced seizures in mice. Drug

ED50

n

CFP

p/q

MeTHIQ LTG OXC PGB TPM

56.75 ± 3.06 5.00 ± 0.78 12.08 ± 0.89 74.00 ± 21.07 46.16 ± 3.11

24 16 8 24 24

8.770 (p) 2.810 (q1 ) 39.215 (q2 ) 1.593 (q3 ) 5.337 (q4 )

— 3.121 0.224 5.505 1.643

Test for parallelisma

MeTHIQ MeTHIQ MeTHIQ MeTHIQ

S.R. = 1.287 S.R. = 1.040 S.R. = 2.226 S.R. = 1.048

f f f f

Non-parallel Parallel Non-parallel Parallel

vs. vs. vs. vs.

LTG OXC PGB TPM

ratio ratio ratio ratio

S.R. = 1.177 S.R. = 1.100 S.R. = 1.411 S.R. = 1.112

S.R. < f ratio S.R., the examined two DRRCs are parallel S.R. > f ratio S.R., the examined two DRRCs are non-parallel Results are presented as median effective doses (ED50 values in mg/kg ± S.E.M.) of MeTHIQ, LTG, OXC, PGB and TPM administered singly against MES-induced seizures in mice. The drugs were administered systemically (i.p.), as follows: MeTHIQ, 5 min; OXC, 30 min; LTG and TPM, 60 min; PGB, 120 min before the MES-induced seizure initiation. n, total number of animals used at doses whose expected anticonvulsant effects ranged between 4 and 6 probits (16% and 84%); CFP, (q and p) curve-fitting parameters; p/q, ratio of p and q1 , q2 , q3 or q4 values; S.R., slope function ratio for the respective two-drug combinations (i.e., SLTG /SMeTHIQ , SMeTHIQ /SOXC , SPGB /SMeTHIQ , and STPM /SMeTHIQ ); f ratio S.R., factor for slope function ratio for the respective two-drug combinations. Test for parallelism of two DRRCs was performed according to Litchfield and Wilcoxon (1949). a All detailed calculations required to perform the test for parallelism of two DRRCs were presented in Appendix to the paper by Luszczki and Czuczwar (2006).

the area bounded by the lower and upper isoboles of additivity, and for sub-additivity (antagonism)—–above this region (Tallarida, 2006). Proportions of MeTHIQ and LTG and PGB in the mixture were calculated only for the fixed-ratio combination of 1:1, and the mixtures of MeTHIQ with LTG and PGB were administered to animals. Evaluation of the experimentally derived ED50 mix at the fixed-ratio of 1:1 was based upon the dose of the mixture protecting 50% of animals tested against MES-induced seizures in mice. Interactions between MeTHIQ and OXC and TPM against MESinduced seizures were analyzed according to the methodology previously detailed in our earlier studies (Luszczki and Czuczwar, 2003, 2004, 2006; Luszczki et al., 2003a, 2006b, 2009a). Based on the ED50 values denoted previously for MeTHIQ and the AEDs administered alone, the median additive doses of mixtures of MeTHIQ with OXC and TPM (ED50 add s—–i.e., doses of the two-drug mixtures, which theoretically should protect 50% of the animals tested against MES-induced seizures) for three fixed-ratio combinations of 1:3, 1:1 and 3:1 were calculated from the equations of additivity presented by Loewe (1953), as follows: x/ED50 MeTHIQ + y/ED50 OXC = 1; and x/ED50 MeTHIQ + y/ED50 TPM = 1; where x is the dose of MeTHIQ and y is the dose of OXC or TPM, respectively, co-administered as a mixture that exerts the desired effect (50% effect for ED50 ). Subsequently, proportions of the AEDs in the mixture were calculated and the respective mixtures of MeTHIQ with OXC and TPM at three fixed-ratios were administered to animals. The anticonvulsant effects offered by MeTHIQ and OXC and TPM in combination, at three fixed-ratios of 1:3, 1:1 and 3:1 in the mouse MES model, were evaluated and expressed as the experimentally derived ED50 mix values, corresponding to the doses of two-drug mixture, sufficient for the 50% protective effect against MES-induced seizures in mice. Finally, to determine the separate doses of MeTHIQ, LTG, OXC, PGB and TPM in the mixture, the ED50 mix values were multiplied by the respective proportions of AEDs (denoted for purely additive mixture). Further details regarding these concepts and all required equations allowing the calculation of S.E.M. for ED50 add values have been published elsewhere (Tallarida, 2006; Luszczki, 2007; Luszczki et al., 2009a,b).

Measurement of total brain concentrations of MeTHIQ and antiepileptic drug Pharmacokinetic evaluation of total brain drug concentrations was performed only for those combinations of MeTHIQ with an AED, producing synergistic interaction in the mouse MES test. Thus, the measurement of total brain concentrations of MeTHIQ and TPM was undertaken at the drug doses corresponding to the fixed-ratios of 1:3, 1:1 and 3:1 in the MES test. Mice were killed by decapitation at times chosen to coincide with that scheduled for the MES test and whole brains were removed from skulls, weighed, harvested and homogenized using Abbott buffer (2:1, v/w; Abbott Laboratories, North Chicago, IL, USA) in an Ultra-Turrax T8 homogenizer (IKAWerke, Staufen, Germany). The homogenates were centrifuged at 10,000 × g for 10 min and the supernatant samples (75 ␮l) were analyzed by fluorescence polarization immunoassay (FPIA) using a TDx analyzer and reagents (TPM) exactly as described by the manufacturer (Seradyn Inc., Indianapolis, IN, USA). Brain concentrations of MeTHIQ were determined by highperformance liquid chromatography (HPLC) using a Dionex HPLC system (Sunnyvale, CA, USA) comprising of a quaternary pump P 580, vacuum degasser and UV—vis detector (UVD 340S). The mobile phase consisting of 0.08 M triethylammonium phosphate buffer solution (pH 3.6) and acetonitrile in the ratio of 90:10 (v/v) and chromatographic separation was achieved by use of a Zorbax SB-C18 5 ␮m column (Agilent Technologies, Santa Clara, CA, USA). Chromatography was performed at ambient temperature using a flow-ratio of 1.2 ml/min. The column eluate was monitored at 215 nm with a sensitivity of 0.01 absorbance units full scale (AUFS). To detect MeTHIQ concentrations, brain homogenate samples were prepared for analysis as follows: 200 ␮l brain homogenate was pipetted into a 2.5-ml plastic centrifugal filter devices (Millipore Corporation, Billerica, MA, USA), to which a 200 ␮l of 0.08 M triethylammonium phosphate buffer solution and a 400 ␮l acetonitrile were added and vortex-mixed for 1 min. After centrifugation (at 10,000 × g for 10 min) the organic layer was removed and a 20 ␮l of the aqueous phase was injected into the HPLC system. Quantitation was achieved by use of chromatographic peak height and that

Interactions of MeTHIQ with second-generation AEDs in the MES test was linearly related over the range of 0.4—20 ␮g/ml of MeTHIQ. The within-batch and between-batch precision was <8% and <7%, respectively. Total brain concentrations of MeTHIQ and TPM were expressed in ␮g/ml of brain supernatants as means ± S.D. of at least 10 separate brain preparations.

Chimney test The effects of the studied AEDs administered alone and in combination with MeTHIQ (administered at doses corresponding to their ED50 mix values at the fixed-ratio of 1:1 from the MES-induced seizure test) on motor coordination impairment were quantified with the chimney test of Boissier et al. (1960). In the chimney test, animals had to climb backwards up the plastic tube (3 cm inner diameter, 30 cm length). Motor impairment was indicated by the inability of the animals to climb backward up the transparent tube within 60 s. Data were presented as a percentage of animals that failed to perform the chimney test. This experimental procedure has been described in detail in our earlier studies (Luszczki and Czuczwar, 2004; Luszczki et al., 2006a,b).

Step-through passive avoidance task On the first day before training, each animal received either the studied AEDs administered alone or the respective combinations of MeTHIQ with the studied AEDs, at doses corresponding to their ED50 mix values at the fixed-ratio of 1:1 from the MESinduced seizure test. Subsequently, animals were placed in an illuminated box (10 cm × 13 cm × 15 cm) connected to a larger dark box (25 cm × 20 cm × 15 cm) equipped with an electric grid floor. Entrance of animals to the dark box was punished by an adequate electric footshock (0.6 mA for 2 s). The animals that did not enter the dark compartment were excluded from subsequent experimentation. On the following day (24 h later), the pre-trained animals did not receive any treatment and were placed again into the illuminated box and observed up to 180 s. Mice that avoided the dark compartment for 180 s were considered to remember the task. The time that the mice took to enter the dark box, was noted and the median latencies (retention times) with 25th and 75th percentiles were calculated. The step-through passive avoidance task gives information about ability to acquire the task (learning) and to recall the task (retrieval). Therefore, it may be regarded as a measure of long-term memory (Venault et al., 1986). This experimental procedure has been described in detail in our earlier studies (Luszczki et al., 2003c).

Grip-strength test The effects of the studied AEDs administered alone and in combination with MeTHIQ (administered at doses corresponding to their ED50 mix values at the fixed-ratio of 1:1 from the MES-induced seizure test) on muscular strength in mice were quantified by the gripstrength test of Meyer et al. (1979). The grip-strength apparatus (BioSeb, Chaville, France) comprised a wire grid (8 cm × 8 cm) connected to an isometric force transducer (dynamometer). The mice were lifted by the tails so that their forepaws could grasp the grid. The mice were then gently pulled backward by the tail until the grid was released. The maximal force exerted by the mouse before losing grip was recorded. The mean of 3 measurements for each animal was calculated and subsequently, the mean maximal force of 8 animals per group was determined. The skeletal muscular strength in mice was expressed in N (Newton) as means ± S.E.M. of at least 8 determinations. This experimental procedure has been described in detail in our earlier study (Luszczki et al., 2009a,b).

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Statistics The ED50 and ED50 mix values (with their respective 95% confidence limits) for MeTHIQ, second-generation AEDs administered alone and their combinations at the fixed-ratios of 1:3, 1:1 and 3:1 (for MeTHIQ + OXC and MeTHIQ + TPM) and at the fixed-ratio of 1:1 (for MeTHIQ + LTG and MeTHIQ + PGB) in the MES-induced seizure test were calculated by computer-assisted log-probit analysis according to Litchfield and Wilcoxon (1949). In the isobolographic analysis for parallel DRRCs, the experimentally derived ED50 mix values for the mixture of MeTHIQ with OXC and TPM at 3 fixed-ratios of 1:3, 1:1 and 3:1 were statistically compared with their respective theoretical additive ED50 add values by using the unpaired Student’s t-test, according to the method described by Tallarida (2000). Similarly, in the isobolographic analysis for non-parallel DRRCs, the experimentally derived ED50 mix values for the mixture of MeTHIQ with LTG and PGB at the fixed-ratio of 1:1 were statistically compared with their respective theoretical additive ED50 add values for lower and upper lines of additivity by using the unpaired Student’s t-test with Welch’s corrections, according to the method described by Tallarida (2006). Total brain concentrations of MeTHIQ and TPM were statistically analyzed using the unpaired Student’s t-test. Qualitative variables from the chimney test were compared by using the Fisher’s exact probability test. Median retention times obtained in the passive avoidance task were statistically evaluated using Kruskal—Wallis nonparametric ANOVA. The results from the grip-strength test were verified with one-way ANOVA. Differences among values were considered statistically significant if P < 0.05.

Software used Microsoft’s Excel spreadsheet was used to perform calculations and to graphically illustrate the results in form of isobolograms. This spreadsheet was programmed to compute all calculations automatically and determine the DRRCs of MeTHIQ and the AEDs administered alone and in combination from the log-probit linear regression analysis according to Litchfield and Wilcoxon (1949). The theoretically additive ED50 add values and their S.E.M. at the fixedratio combinations of 1:3, 1:1, and 3:1 (for parallel DRRCs), as well as, the lower and upper ED50 add values and their S.E.M. at the fixed-ratio combination of 1:1 (for non-parallel DRRCs), were also calculated with this programmed spreadsheet. All statistical tests were performed using commercially available GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA, USA).

Results Anticonvulsant effects of MeTHIQ and LTG administered separately and in combination in the mouse MES model MeTHIQ administered alone (i.p., 5 min before the test) at doses ranging between 50 and 70 mg/kg produced a clear anticonvulsant effect that increased from 25% to 87.5% against MES-induced seizures. The equation of DRRC for MeTHIQ (y = 12.304x − 16.580; Fig. 1A), allowed the determination of the ED50 value for MeTHIQ, which was 56.75 ± 3.06 mg/kg (Table 1). Similarly, LTG administered singly (i.p., 60 min before the test) at doses ranging between 3 and 8 mg/kg produced a definite antiseizure activity that increased from 12.5% to 87.5% in the mouse MES model. The equation of DRRC for LTG (y = 5.244x + 1.335; Fig. 1A), allowed the determination of the ED50 value for LTG that amounted to 5.00 ± 0.78 mg/kg (Table 1). The test for paral-

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Figure 1 (A)—(D) Log-probit dose—response relationship curve (DRRC) analysis of MeTHIQ and lamotrigine (LTG), oxcarbazepine (OXC), pregabalin (PGB) and topiramate (TPM) administered alone and in combination against maximal electroshock (MES)-induced seizures in mice. Doses of MeTHIQ and four second-generation AEDs administered alone and in combination at the fixed-ratio of 1:1 (for MeTHIQ with LTG and PGB) and at three fixed-ratios of 1:3, 1:1 and 3:1 (for MeTHIQ with OXC and TPM) were transformed to logarithms, whereas the protective effects offered by the AEDs and MeTHIQ against MES-induced seizures were transformed to probits according to Litchfield and Wilcoxon (1949). Linear regression equations of DRRCs for MeTHIQ and the AEDs (LTG, OXC, PGB or TPM) administered alone and in combinations are presented on the graphs (A—D); where y, is the probit of response; x, is the logarithm (to the base 10) of an AED dose or a dose of the mixture of MeTHIQ with an AED; and r2 , coefficient of determination. Points constructing DRRCs on each graph reflect the number of groups of animals (8 mice per group) used in this study. Test for parallelism revealed that the experimentally determined DRRCs for MeTHIQ and LTG and PGB (administered alone) are not parallel to one another (A and C), whereas those for MeTHIQ and OXC and TPM are parallel (B and D). For more details see Table 1.

lelism of DRRCs between MeTHIQ and LTG revealed that the drugs had their DRRCs non-parallel to one another (Table 1; Fig. 1A). The combination of MeTHIQ with LTG at the fixedratio of 1:1 exerted the antiseizure activity in the MES test and the experimentally derived ED50 mix value from the DRRC for the mixture of both AEDs (y = 6.655x − 4.368; Fig. 1A) was 25.57 ± 2.55 mg/kg (Table 2).

Anticonvulsant effects of OXC administered separately and in combination with MeTHIQ in the mouse MES model OXC administered alone (i.p., 30 min before the test) at doses ranging between 10 and 14 mg/kg produced the definite anticonvulsant activity that increased from 12.5% to 87.5% in the mouse MES model. The equation of DRRC for OXC (y = 15.586x − 11.863; Fig. 1B), allowed the determination of its ED50 value that amounted to 12.08 ± 0.89 mg/kg (Table 1). The test for parallelism of DRRCs between MeTHIQ and OXC revealed that the drugs had their DRRCs parallel to one another (Table 1; Fig. 1B). The combination of

MeTHIQ with OXC at the fixed-ratio of 1:3 exerted the antiseizure activity in the MES test and the experimentally derived ED50 mix value from the DRRC for the mixture of both drugs (y = 18.897x − 19.819; Fig. 1B) was 20.58 ± 0.89 mg/kg (Table 3). Similarly, MeTHIQ combined with OXC at the fixedratio of 1:1 produced the antiseizure activity in the MES test and thus, the ED50 mix value calculated from the DRRC for the mixture of both drugs (y = 13.648x − 15.373; Fig. 1B) was 31.10 ± 1.51 mg/kg (Table 3). The last tested combination of MeTHIQ with OXC in the mouse MES model at the fixedratio of 3:1 exerted also the definite anticonvulsant effect and the ED50 mix value calculated directly from the DRRC for the mixture of both drugs (y = 18.750x − 25.125; Fig. 1B) was 40.43 ± 1.75 mg/kg (Table 3).

Anticonvulsant effects of PGB administered separately and in combination with MeTHIQ in the mouse MES model PGB administered alone (i.p., 120 min before the test) at doses ranging between 25 and 150 mg/kg produced the def-

Results are presented as median effective doses (ED50 values in mg/kg ± S.E.M.) for two-drug mixtures, determined either experimentally (ED50 mix ) or theoretically calculated (ED50 add ) from the equations of additivity (Tallarida, 2006), protecting 50% of the animals against MES-induced seizures. The actual doses of MeTHIQ, LTG and PGB that comprised the mixtures at the fixed-ratio of 1:1 for the ED50 mix and ED50 add values are presented in separate columns. MeTHIQ, dose of MeTHIQ in the mixture; AED, dose of LTG or PGB in the mixture; nmix , total number of animals used at those doses whose expected anticonvulsant effects ranged between 16% and 84% (i.e., 4 and 6 probits) for the experimental mixture; n add , total number of animals calculated for the additive mixture of the drugs examined (nadd = n MeTHIQ + n LTG − 4) or (nadd = n MeTHIQ + n PGB − 4). Statistical evaluation of data was performed with unpaired Student’s t-test with Welch’s correction. a ED 50 add value calculated from the equation for the lower line of additivity. b ED 50 add value calculated from the equation for the upper line of additivity.

3.44 56.79 36 44 36 44 19.25 ± 4.00 30.42 ± 20.63 24 16 25.57 ± 2.55 51.58 ± 6.88 MeTHIQ + LTG MeTHIQ + PGB

23.50 22.39

2.07 29.19

17.69 13.20

1.56 17.22

42.50 ± 6.44 100.33 ± 18.20

39.06 43.54

AED MeTHIQ nadd ED50 add b AED MeTHIQ nadd ED50 add a AED MeTHIQ nmix ED50 mix Combination

Table 2 Isobolographic analysis of interactions (for non-parallel DRRCs) of MeTHIQ with lamotrigine (LTG) and pregabalin (PGB) at the fixed-ratio of 1:1 against maximal electroshock (MES)-induced seizures.

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213

inite anticonvulsant activity that increased from 12.5% to 75% in the mouse MES model. The equation of DRRC for PGB (y = 2.332x + 0.641; Fig. 1C), allowed the determination of its ED50 value that amounted to 74.00 ± 21.07 mg/kg (Table 1). The test for parallelism of DRRCs between MeTHIQ and PGB revealed that the drugs had their DRRCs non-parallel to each other (Table 1; Fig. 1C). In this case, the combination of MeTHIQ with PGB at the fixed-ratio of 1:1 exerted the antiseizure activity in the MES test and the experimentally derived ED50 mix value from the DRRC for the mixture of both drugs (y = 6.093x − 5.434; Fig. 1C) was 51.58 ± 6.88 mg/kg (Table 2).

Anticonvulsant effects of TPM administered separately and in combination with MeTHIQ in the mouse MES model TPM administered singly (i.p., 60 min before the test) at doses ranging between 40 and 60 mg/kg produced the definite antiseizure activity that increased from 25% to 87.5% in the mouse MES model. The equation of DRRC for TPM (y = 9.858x − 11.407; Fig. 1D), allowed the determination of the ED50 value for TPM that amounted to 46.16 ± 3.11 mg/kg (Table 1). The test for parallelism of DRRCs between MeTHIQ and TPM revealed that the drugs had their DRRCs parallel to one another (Table 1; Fig. 1D). The combination of MeTHIQ with TPM at the fixed-ratio of 1:3 exerted the antiseizure activity in the MES test and the experimentally derived ED50 mix value from the DRRC for the mixture of both drugs (y = 8.923x − 8.661; Fig. 1D) was 33.96 ± 3.10 mg/kg (Table 3). Similarly, MeTHIQ combined with TPM at the fixedratio of 1:1 produced the antiseizure activity in the MES test and thus, the ED50 mix value calculated from the DRRC for the mixture of both drugs (y = 9.296x − 9.530; Fig. 1D) was 36.56 ± 2.61 mg/kg (Table 3). The last tested combination of MeTHIQ with TPM in the mouse MES model at the fixedratio of 3:1 exerted also the definite anticonvulsant effect and the ED50 mix value calculated directly from the DRRC for the mixture of both drugs (y = 13.811x − 17.308; Fig. 1D) was 41.23 ± 1.98 mg/kg (Table 3).

Isobolographic analysis of interaction between MeTHIQ and LTG in the mouse MES model The isobolographic analysis of interaction for non-parallel DRRCs revealed that the mixture of MeTHIQ with LTG at the fixed-ratio of 1:1 exerted additive interaction in the MES test in mice (Fig. 2A). The experimentally derived ED50 mix value for this fixed-ratio combination was 25.57 ± 2.55 mg/kg, whereas the additively calculated ED50 add values were 19.25 ± 4.00 mg/kg (for the lower ED50 add ) and 42.50 ± 6.44 mg/kg (for the upper ED50 add ; Table 2). Thus, the ED50 mix value did not significantly differ from the ED50 add values (Table 2; Fig. 2A).

Isobolographic analysis of interaction between MeTHIQ and OXC in the mouse MES model The isobolographic analysis of interaction for parallel DRRCs revealed that all three fixed-ratio combinations of MeTHIQ

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Figure 2 (A)—(D) Isobolograms showing interactions between MeTHIQ and lamotrigine (LTG), oxcarbazepine (OXC), pregabalin (PGB) and topiramate (TPM) against maximal electroshock (MES)-induced seizures in mice. The median effective dose (ED50 ) for MeTHIQ is plotted graphically on X-axis, whereas the ED50 of the examined AEDs (LTG, OXC, PGB and TPM) is placed on Y-axis (A—D). The solid lines on the X- and Y-axes represent the S.E.M. for the AEDs administered alone. The open circles () depict the experimentally derived ED50 mix s (±S.E.M.) and theoretically calculated ED50 add s (±S.E.M.) for total dose expressed as the proportion of MeTHIQ and an AED that produced 50% protection of animals against MES-induced seizures. The S.E.M. values are presented as horizontal and vertical error bars for the ED50 mix s and ED50 add s. (A) Interactions between MeTHIQ and LTG. The lower and upper isoboles of additivity represent the curves connecting the ED50 values for MeTHIQ and LTG administered alone. The dotted line starting from the point (0; 0) corresponds to the fixed-ratio of 1:1 for the combination of MeTHIQ with LTG. The diagonal dashed line connects the ED50 for MeTHIQ and LTG on the X- and Y-axes. The points A and A depict the theoretically calculated ED50 add values for both, lower and upper isoboles of additivity. The point M represents the experimentally derived ED50 mix value for total dose of the mixture expressed as proportions of MeTHIQ and LTG that produced a 50% anticonvulsant effect (50% isobole) in the mouse MES model. The sum of X- and Y-coordinates, for each point placed on the isobologram, corresponds to the respective ED50 values. The point S reflects the ED50 add value denoted theoretically from the Loewe’s equation for the fixed-ratio combination of 1:1. The experimentally derived ED50 mix value is placed between the points S and A , within the area of additivity bounded by two isoboles of additivity, indicating additive interaction between MeTHIQ and LTG in the mouse MES model. The X- and Y-coordinates for all points presented on the isobologram are as follows: A (17.69; 1.56), A (39.06; 3.44), S (28.38; 2.50), and M (23.50; 2.07). (B) Interactions between MeTHIQ and OXC. The straight line connecting the ED50 values of MeTHIQ and OXC on the graph represents the theoretical line of additivity for a continuum of different fixed dose ratios. The dotted lines starting from the point (0; 0) correspond to the fixed-ratios of 1:3, 1:1, and 3:1 for the combination of MeTHIQ with OXC. The experimental ED50 mix values of the mixture of MeTHIQ with OXC for all the fixed-ratios of 1:3, 1:1, and 3:1 are placed close to the theoretical line of additivity, indicating the additive interaction. Points M1, M2, and M3 on the graph reflect the ED50 mix s at the fixed-ratios of 1:3, 1:1 and 3:1, respectively. Similarly, the points A1, A2, and A3 illustrate the ED50 add s at the fixed-ratios of 1:3, 1:1 and 3:1, respectively. The X- and Y-coordinates for all points presented on the isobologram are as follows: M1 (12.56; 8.02), M2 (25.64; 5.46), M3 (37.75; 2.68), A1 (14.19; 9.06), A2 (28.38; 6.04), and A3 (42.56; 3.02). (C) Interactions between MeTHIQ and PGB. The lower and upper isoboles of additivity represent the curves connecting the ED50 values for MeTHIQ and PGB administered alone. The points A and A depict the theoretically calculated ED50 add values for both, lower and upper isoboles of additivity. The point M represents the experimentally derived ED50 mix value for total dose of the mixture expressed as proportions of MeTHIQ and PGB that produced a 50% anticonvulsant effect (50% isobole) in the mouse MES model. The experimentally derived ED50 mix value is placed between the points S and A , within the area of additivity bounded by two isoboles of additivity, indicating additive interaction between MeTHIQ and PGB in the mouse MES model. The X- and Y-coordinates for all points presented on the isobologram are as follows: A (13.20;

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215

Table 3 Isobolographic analysis of interactions (for parallel DRRCs) of MeTHIQ with oxcarbazepine (OXC) and topiramate (TPM) in the mouse maximal electroshock (MES)-induced seizure model. Combination

FR

ED50 mix

nmix

MeTHIQ

MeTHIQ + OXC

1:3 1:1 3:1

20.58 ± 0.89 31.10 ± 1.51 40.43 ± 1.75

16 24 16

12.56 25.64 37.75

MeTHIQ + TPM

1:3 1:1 3:1

33.96 ± 3.10** 36.56 ± 2.61*** 41.23 ± 1.98***

16 24 24

9.87 20.16 32.44

AED

ED50 add

nadd

MeTHIQ

AED

8.02 5.46 2.68

23.25 ± 1.02 34.42 ± 1.59 45.58 ± 2.31

28 28 28

14.19 28.38 42.56

9.06 6.04 3.02

24.09 16.40 8.79

48.81 ± 2.45 51.46 ± 2.18 54.10 ± 2.42

44 44 44

14.19 28.38 42.56

34.62 23.08 11.54

Data are presented as median effective doses (ED50 values in mg/kg ± S.E.M.) protecting 50% of animals tested against MES-induced seizures. The ED50 values were either experimentally determined from the mixture of two AEDs (ED50 mix ) or theoretically calculated (ED50 add ) from the equation of additivity (Loewe, 1953). The actual doses of MeTHIQ and an AED (OXC or TPM) that comprised the mixtures at all three fixed-ratio combinations for both ED50 mix and ED50 add values are presented in separate columns. MeTHIQ, dose of MeTHIQ in the mixture; AED, dose of OXC or TPM in the mixture. Statistical evaluation of data was performed by using unpaired Student’s t-test. FR, fixed-ratio of drug dose combinations; n, total number of animals used at those doses whose expected anticonvulsant effects ranged between 4 and 6 probits, denoted for the experimental mixture of drugs (nmix ) and theoretically calculated (nadd = n MeTHIQ + n OXC − 4) or (nadd = n MeTHIQ + n TPM − 4) from the equation of additivity. ** P < 0.01 vs. the respective ED 50 add values. *** P < 0.001 vs. the respective ED 50 add values.

with OXC at 1:3, 1:1, and 3:1 exerted additive interaction in the mouse MES model (Table 3; Fig. 2B). The ED50 mix value for the mixture of MeTHIQ with OXC at the fixed-ratio of 1:3 was 20.58 ± 0.89 mg/kg and it did not significantly differ than the corresponding ED50 add of 23.25 ± 1.02 mg/kg (Table 3; Fig. 2B). Similarly, the experimentally derived ED50 mix value for the combination of MeTHIQ with OXC at the fixed-ratio of 1:1 was 31.10 ± 1.51 mg/kg and did not considerably differ than that for the corresponding ED50 add , which was 34.42 ± 1.59 mg/kg (Table 3; Fig. 2B). The mixture of MeTHIQ with OXC at the fixed-ratio of 3:1 additively protected the animals against MES-induced seizures, and the ED50 mix value was 40.43 ± 1.75 mg/kg, which did not significantly differ when compared to the ED50 add of 45.58 ± 2.31 mg/kg (Table 3; Fig. 2B).

Isobolographic analysis of interaction between MeTHIQ and PGB in the mouse MES model The isobolographic analysis of interaction for non-parallel DRRCs revealed that the mixture of MeTHIQ with PGB at the fixed-ratio of 1:1 exerted additive interaction in the MES test in mice (Fig. 2C). The experimentally derived ED50 mix value for this fixed-ratio combination was 51.58 ± 6.88 mg/kg, whereas the additively calculated ED50 add values were 30.42 ± 20.63 mg/kg (for the lower ED50 add ) and 100.33 ± 18.20 mg/kg (for the upper ED50 add ; Table 2). Thus, the ED50 mix value did not significantly differ from the ED50 add values (Table 2; Fig. 2C).

Isobolographic analysis of interaction between MeTHIQ and TPM in the mouse MES model The isobolographic analysis of interaction for parallel DRRCs revealed that all three fixed-ratio combinations of MeTHIQ with TPM at 1:3, 1:1, and 3:1 exerted supra-additive (synergistic) interaction in the mouse MES model (Table 3; Fig. 2D). The ED50 mix value for the mixture of MeTHIQ with TPM at the fixed-ratio of 1:3 was 33.96 ± 3.10 mg/kg which was significantly less than the corresponding ED50 add of 48.81 ± 2.45 mg/kg (P < 0.01; Table 3; Fig. 2D). Similarly, the experimentally derived ED50 mix value for the combination of MeTHIQ with TPM at the fixed-ratio of 1:1 was 36.56 ± 2.61 mg/kg and considerably lower than that for the corresponding ED50 add , which was 51.46 ± 2.18 mg/kg (P < 0.001; Table 3; Fig. 2D). The mixture of MeTHIQ with TPM at the fixed-ratio of 3:1 supra-additively protected the animals against MES-induced seizures, and the ED50 mix value was 41.23 ± 1.98 mg/kg, which was significantly reduced compared to the ED50 add of 54.10 ± 2.42 mg/kg (P < 0.001; Table 3; Fig. 2D).

Total brain concentrations of MeTHIQ and TPM Bi-directional evaluation of total brain drug concentrations revealed that MeTHIQ (co-administered with TPM at doses corresponding to the ED50 mix values at the fixed-ratios of 1:3, 1:1 and 3:1 from the MES test) did not affect total brain concentrations of TPM in experimental animals (Table 4), and

17.22), A (43.54; 56.79), S (28.38; 37.00), and M (22.39; 29.19). (D) Interactions between MeTHIQ and TPM. The straight line connecting the ED50 values of MeTHIQ and TPM on the graph represents the theoretical line of additivity for a continuum of different fixed dose ratios. The experimental ED50 mix values of the mixture of MeTHIQ with TPM for all the fixed-ratios of 1:3, 1:1, and 3:1 are placed significantly below the theoretical line of additivity, indicating the supra-additive (synergistic) interaction. Points M1, M2, and M3 on the graph reflect the ED50 mix s at the fixed-ratios of 1:3, 1:1 and 3:1, respectively. Similarly, the points A1, A2, and A3 illustrate the ED50 add s at the fixed-ratios of 1:3, 1:1 and 3:1, respectively. The X- and Y-coordinates for all points presented on the isobologram are as follows: M1 (9.87; 24.09), M2 (20.16; 16.40), M3 (32.44; 8.79), A1 (14.19; 34.62), A2 (28.38; 23.08), and A3 (42.56; 11.54). Asterisks indicate statistical significance at **P < 0.01 and ***P < 0.001 vs. the respective ED50 add values.

216 Table 4

J.J. Luszczki et al. Total brain concentrations of MeTHIQ and topiramate (TPM) administered singly or in combination.

FR

Treatment (mg/kg)

Total brain concentration (␮g/ml)a

1:3

TPM (24.09) + vehicle TPM (24.09) + MeTHIQ (9.87) MeTHIQ (9.87) + vehicle MeTHIQ (9.87) + TPM (24.09)

5.969 6.395 2.044 2.125

± ± ± ±

0.696 0.642 0.304 0.268

1:1

TPM (16.40) + vehicle TPM (16.40) + MeTHIQ (20.16) MeTHIQ (20.16) + vehicle MeTHIQ (20.16) + TPM (16.40)

4.521 4.872 3.753 4.046

± ± ± ±

0.374 0.508 0.454 0.441

3:1

TPM (8.79) + vehicle TPM (8.79) + MeTHIQ (32.44) MeTHIQ (32.44) + vehicle MeTHIQ (32.44) + TPM (8.79)

3.376 3.749 6.799 6.932

± ± ± ±

0.356 0.487 0.472 0.553

Data are presented as means (±S.D.) and expressed as ␮g/ml of brain supernatants of 10 determinations (n = 10). Estimation of total brain concentrations of TPM was performed with fluorescence polarization immunoassay (FPIA), whereas total brain concentrations of MeTHIQ were measured with high-pressure liquid chromatography (HPLC). Statistical evaluation of data was performed with unpaired Student’s t-test. Brain tissue samples were taken at times scheduled for the MES test. FR, fixed-ratio combination of MeTHIQ with TPM. a Concentrations relate to that drug shown first in the respective treatment column.

inversely, TPM had no impact on total brain concentrations of MeTHIQ in mice (Table 4). Total brain concentrations of LTG, OXC and PGB combined with MeTHIQ were not measured and pharmacokinetically verified in the present study because MeTHIQ and the AEDs did not synergistically interact in the MES test in mice.

Effects of MeTHIQ in combination with LTG, OXC, PGB, and TPM on motor performance, long-term memory and skeletal muscular strength in the chimney, passive avoidance and grip-strength tests When MeTHIQ and each of four second-generation AEDs (LTG, OXC, PGB, TPM) were co-administered at doses corresponding to their ED50 mix values at the fixed-ratio of 1:1 from the MES-induced seizure test, motor performance of animals as assessed by the chimney test was unaffected (Table 5). Furthermore, none of the combinations studied (MeTHIQ with LTG, OXC, PGB, TPM) impaired long-term memory as determined in the passive avoidance test (Table 5). Similarly, MeTHIQ concomitantly administered with the AEDs had no significant impact on skeletal muscular strength of the animals as assessed by the grip-strength test (Table 5). Moreover, it was found that the control (vehicle-treated) mice and animals receiving MeTHIQ or AEDs alone (at doses corresponding to their ED50 values from the MES-induced seizures) did not show any sings of impaired motor coordination, long-term memory or muscular skeletal strength, as assessed in the chimney, passive avoidance and grip-strength tests, respectively (Table 5).

Discussion The results presented herein indicate that MeTHIQ synergistically interacted with TPM and exerted additive interaction when combined with OXC, PGB and LTG in the mouse MES model. Previously, it has been reported that MeTHIQ syner-

gistically interacted with PB and, simultaneously, produced additive interaction when concomitantly administered with CBZ, PHT, and VPA in the MES test (Luszczki et al., 2009a). Evaluation of total brain concentrations of MeTHIQ and TPM revealed that both drugs interacted pharmacodynamically because no changes in total brain concentrations of MeTHIQ and TPM were observed in this study. It is worthy of mentioning that the pharmacokinetic evaluation of total brain drug concentrations was performed bi-directionally by measuring both MeTHIQ and TPM concentrations. Previously, it has been documented that only total brain concentrations of AEDs provided full insight into the nature of isobolographic interaction between drugs (Luszczki et al., 2003d). This is why, only total brain drug concentrations, but not free plasma concentrations of TPM and MeTHIQ were measured with FPIA and HPLC, respectively. To scientifically explain the selective enhancement of the anticonvulsant action of TPM and PB by MeTHIQ, leading to the synergistic interaction between the drugs, one should consider their molecular mechanisms of action. As regards the anticonvulsant effect of TPM, the drug: (a) inhibits voltage-sensitive sodium channels (Taverna et al., 1999; Shank et al., 2000); (b) potentiates ␥-aminobutyric acid (GABA)-mediated inhibitory neurotransmission through binding to a novel site on the GABAA receptor complex (White et al., 2000); (c) blocks excitatory neurotransmission mediated by ␣-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)/kainate receptors through a negative modulatory effect on calcium-permeable AMPA/kainate receptors (Gibbs et al., 2000; Gryder and Rogawski, 2003; Braga et al., 2009); (d) inhibits neuronal L-type high-voltageactivated calcium channels (Zhang et al., 2000); (e) weakly inhibits the carbonic anhydrase isoenzymes CA II and CA IV, and through the modulation of pH influences voltageand receptor-gated ion channels (Dodgson et al., 2000; Shank et al., 2005); (f) binds to phosphorylation sites on AMPA/kainate receptors and thereby exerts an allosteric modulatory effect on channel conductance (Angehagen et

Interactions of MeTHIQ with second-generation AEDs in the MES test

217

Table 5 Effects of MeTHIQ, lamotrigine (LTG), oxcarbazepine (OXC), pregabalin (PGB), topiramate (TPM), and their combination at the fixed-ratio of 1:1 on long-term memory in the passive avoidance task, muscular strength in the grip-strength test, and motor performance in the chimney test in mice. Treatment (mg/kg)

Motor performance (%)

Retention time (s)

Grip-strength (N)

Vehicle MeTHIQ (56.8) + vehicle LTG (5.0) + vehicle OXC (12.1) + vehicle PGB (74.0) + vehicle TPM (46.2) + vehicle MeTHIQ (23.50) + LTG (2.07) MeTHIQ (25.64) + OXC (5.46) MeTHIQ (22.39) + PGB (29.19) MeTHIQ (20.16) + TPM (16.40)

100 100 100 100 100 100 100 100 100 100

180 180 180 180 180 180 180 180 180 180

86.5 86.3 88.8 87.8 87.2 87.1 84.8 83.8 85.2 87.1

(180; (180; (180; (180; (180; (180; (180; (180; (180; (180;

180) 180) 180) 180) 180) 180) 180) 180) 180) 180)

± ± ± ± ± ± ± ± ± ±

4.53 4.01 4.44 4.39 4.69 4.21 4.25 4.35 4.51 4.01

Results are presented as: (1) percentage of animals without impairment of motor coordination in the chimney test in mice; (2) median retention times (in s; with 25th and 75th percentiles in parentheses) from the passive avoidance task, assessing long-term memory in mice; (3) mean strengths (in Newton ± S.E.M.) from the grip-strength test, assessing skeletal muscular strength in mice. Each experimental group consisted of 8 mice. Statistical analysis of data from the passive avoidance task was performed with nonparametric Kruskal—Wallis ANOVA, whereas the results from the grip-strength test were analyzed with one-way ANOVA. The Fisher’s exact probability test was used to analyze the results from the chimney test. All drugs were administered i.p. at times scheduled from the MES test, and at doses corresponding to their ED50 values (when administered alone) and ED50 mix values at the fixed-ratio of 1:1 (when administered in combination with MeTHIQ) against MES-induced seizures in mice (for more details see legend to Tables 1—3).

al., 2004); (g) inhibits GABAA receptor-mediated depolarizing responses enhancing conductance of some types of potassium channels (Herrero et al., 2002). With respect to PB, the drug by facilitating GABA-mediated inhibition through the allosteric modulation of neuronal postsynaptic GABAA receptors (Brodie and Schachter, 2001; Mathers et al., 2007), hyperpolarizes the postsynaptic neuronal cell membrane, and thus, disrupts epileptiform transmission (Twyman et al., 1989). Moreover, PB at relatively low concentrations inhibits responses mediated by AMPA receptors (Ko et al., 1997). Thus, one can suggest that MeTHIQ, as a non-competitive NMDA receptor antagonist (AntkiewiczMichaluk et al., 2006; Pietraszek et al., 2009), enhances the anticonvulsant effects of TPM and PB, whose common anticonvulsant mechanisms of action are related to the activation of GABA-mediated inhibitory neurotransmission in the brain (White et al., 2007; Latini et al., 2008). Moreover, the reduction of the excitatory neurotransmission evoked by the blockade of AMPA/kainate receptors produced by TPM and PB should be borne in mind while considering the common anticonvulsant mechanism that might be responsible for the synergistic interaction between MeTHIQ and TPM and PB in terms of seizure suppression in the mouse MES model. Although this hypothesis is speculative, it can readily explain the observed synergistic interaction between drugs. On the other hand, one can ascertain that the blockade of neuronal calcium and sodium channels is not responsible for the synergistic interaction between MeTHIQ and TPM or PB, because as documented herein, LTG, OXC and PGB, whose main anticonvulsant effects are mediated by the inhibition of calcium and sodium channels, did not synergistically interact with MeTHIQ. Moreover, PHT and CBZ two classical AEDs that also inhibit sodium channels, did not produce synergistic interaction when concomitantly administered with MeTHIQ in the MES test in mice (Luszczki et al., 2009a). To unequivocally elucidate the exact molecular mechanisms of action underlying the synergistic interaction

between MeTHIQ and TPM and PB, more advanced neurochemical and electrophysiological studies are required. Noteworthy, the additive interaction between MeTHIQ and LTG, OXC or PGB may also be favorable from a clinical point of view because of the reduction of drug doses required to stop seizure activity. The reduction of drug doses in polytherapy is an important factor contributing to the decrease of adverse effects observed in patients receiving AEDs in monotherapy in high effective, but poorly tolerated doses (Schmidt, 1996; Deckers et al., 2000). The evaluation of potential acute adverse effects for the combinations of MeTHIQ with the studied second-generation AEDs (at doses corresponding to the ED50 mix values at the fixed-ratio of 1:1), in the grip-strength, step-through passive avoidance, and chimney tests, provided evidence that the drugs in combination produced no acute adverse effects in animals, at the same pretreatment times and at dose ranges as determined in the MES-induced seizure test. It should be stressed that the fixed-ratio combination of 1:1 was preferentially chosen to investigate the side effects because MeTHIQ and the AEDs were present in the mixture at equi-effective (equivalent) doses, producing the same effects against MES-induced seizures. Based on this preclinical study one can conclude that MeTHIQ synergistically interacted with TPM in the MESinduced seizure test and the observed interaction was pharmacodynamic in nature. Thus, the combination might become a new therapeutic option for patients with epilepsy, if the results from this study could be extrapolated into further clinical trials. The combinations of MeTHIQ with the remaining AEDs studied (i.e., LTG, OXC and PGB), although exerted additive interaction in the MES-induced seizure test, they also could be beneficial because of the reduced doses of the AEDs without losing the antiseizure effects when combined with MeTHIQ. Nevertheless, more advanced studies are required to assess the effectiveness and usefulness of these combinations in further clinical settings.

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Conflicts of interest Professor S.J. Czuczwar has received support from UCB Pharma and Sanofi-Aventis as a speaker. The remaining authors have no conflicts of interest to disclose.

Acknowledgments This study was supported by grants from the Institute of Agricultural Medicine (Lublin, Poland) and Medical University of Lublin (Lublin, Poland). The authors thank to Dr. J. Boksa (Department of Chemistry, Institute of Pharmacology, Polish Academy of Sciences, Krakow, Poland) for synthesis and kind supply of MeTHIQ. Professor J.J. Luszczki is a Recipient of the Fellowship for Leading Young Researchers from the Ministry of Science and Higher Education (Warszawa, Poland).

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