Jarogniew J. Luszczki, Neville Ratnaraj, Philip N. Patsalos, and Stanislaw J. Czuczwar

Transcription

1 Epilepsia, 47(11): , 2006 Blackwell Publishing, Inc. C 2006 International League Against Epilepsy Characterization of the Anticonvulsant, Behavioral and Pharmacokinetic Interaction Profiles of Stiripentol in Combination with Clonazepam, Ethosuximide, Phenobarbital, and Valproate Using Isobolographic Analysis Jarogniew J. Luszczki, Neville Ratnaraj, Philip N. Patsalos, and Stanislaw J. Czuczwar Department of Pathophysiology, Medical University of Lublin, Lublin, Poland; Pharmacology and Therapeutics Unit, Department of Clinical and Experimental Epilepsy, Institute of Neurology, London, United Kingdom; and Department of Physiopathology, Institute of Agricultural Medicine, Lublin, Poland Summary: Purpose: Isobolographic analysis was used to characterize the interactions between stiripentol (STP) and clonazepam (CZP), ethosuximide (ETS), phenobarbital (PB), and valproate (VPA) in suppressing pentylenetetrazole (PTZ)- induced clonic seizures in mice. Methods: The anticonvulsant and acute adverse (neurotoxic) effects of STP in combination with the various conventional antiepileptic drugs (AEDs), at fixed ratios of 1:3, 1:1, and 3:1, were evaluated in the PTZ and chimney tests in mice using the isobolographic analysis. Additionally, protective indices (PI) and benefit indices (BI) were calculated to identify their pharmacological profiles so that a ranking in relation to advantageous combination could be established. Moreover, adverseeffect paradigms were determined by use of the step-through passive avoidance task (long-term memory), threshold for the first pain reaction, grip-strength test (neuromuscular tone), and the hot plate test (acute thermal pain). Brain AED concentrations were also measured so as to ascertain any pharmacokinetic contribution to the pharmacodynamic interactions. Results: All AED combinations comprising of STP and CZP, ETS, PB, and VPA (at the fixed ratios of 1:3, 1:1 and 3:1) were additive in terms of clonic seizure suppression in the PTZ test. However, these interactions were complicated by changes in brain AED concentrations consequent to pharmacokinetic interactions. Thus STP significantly increased total brain ETS and PB concentrations, and decreased VPA concentrations, but was without effect on CZP concentrations. In contrast, PB significantly decreased and VPA increased total brain STP concentrations while CZP and ETS were without effect. Furthermore, while isobolographic analysis revealed that STP and CZP in combination, at the fixed ratios of 1:1 and 3:1, were supraadditive (synergistic; p < 0.05), the combinations of STP with CZP (1:3), ETS, PB, or VPA (at all fixed ratios of 1:3, 1:1, and 3:1) were barely additivity in terms of acute neurotoxic adverse effects in the chimney test. Additionally, none of the examined combinations of STP with conventional AEDs (CZP, ETS, PB, VPA at their median effective doses from the PTZ-test) affected long-term memory, threshold for the first pain reaction, neuromuscular tone, and acute thermal pain. Conclusions: Based on BI values, the combination of STP with PB at the fixed ratio of 1:3 appears to be a particularly favourable combination. In contrast, STP and CZP or ETS (at the fixed ratios of 1:1 and 3:1) were unfavorable combinations. However, these conclusions are confounded by the fact that STP is associated with significant pharmacokinetic interactions. The remaining combinations of STP with PB (1:1 and 3:1), CZP (1:3), ETS (1:3), and VPA (at all fixed ratios of 1:3, 1:1, and 3:1) do not appear to be potential favorable AED combinations. Key words: Stiripentol Antiepileptic drugs Pharmacodynamic interactions Pharmacokinetic interactions Clonazepam Ethosuximide Phenobarbital Valproate Isobolographic analysis. Approximately 30% of patients with epilepsy are refractory to currently available first-line antiepileptic drugs (AEDs) when used as monotherapy regimens (Kwan and Brodie, 2000a, 2000b). Consequently, in these patients Accepted June 28, Address for correspondence and reprint requests to Dr. S.J. Czuczwar, Department of Pathophysiology, Medical University of Lublin, Jaczewskiego 8, PL Lublin, Poland. czuczwarsj@yahoo.com doi: /j x polytherapy, whereby AEDs are prescribed in combination, is undertaken so as to enhance seizure control. Indeed, this approach can provide improved seizure control in 14% of these patients (Kwan and Brodie, 2000a, 2000b). To date, only some two-drug combinations have been proven to be efficacious against specific forms of epilepsy (Stephen and Brodie, 2002). However, with the introduction of newer (second generation) AEDs, into clinical practice, the number of possible combinations has increased exponentially. Presently, there are 15 AEDs 1841

2 1842 J. J. LUSZCZKI ET AL. licensed for clinical use and these may provide 105 various two-drug combinations, of which several can be expected to be favorable in clinical practice. However, the direct testing of anticonvulsant efficacy of all two-drug combinations in patients with refractory epilepsy is not possible because of ethical reasons and/or methodological limitations. Nonetheless, such combinations may be more readily identified and preselected in preclinical studies on animals, and only those, whose anticonvulsant effects offer optimal protection against seizures and, simultaneously, which are devoid of any serious neurotoxic side effects (Löscher and Wauquier, 1996), can be further investigated and verified in the clinical setting. Furthermore, with advances in our understanding of the modes of AED action and of the pathophysiology of seizure initiation, amplification and propagation in the brain, rational polytherapy is increasingly becoming an option. Stiripentol (STP; {4,4-dimethyl-1-[3,4-(methylenedioxy)-phenyl]-1-penten-3-ol}) is a novel AED presently undergoing phase III clinical evaluation. Its primary mode of action is via an effect GABA inhibitory neurotransmission in the brain. STP inhibits GABA metabolism through the blockade of GABA-transaminase activity (Poisson et al., 1984) and reduces synaptosomal uptake of GABA (Wegmann et al., 1978), leading to an increase in GABA brain content. STP has, however, no affinity for GABA A and GABA B receptors (Poisson et al., 1984). STP, at relevant clinical concentrations ( µm) markedly increases the mean open duration of GABA A receptordependent chloride channels by a barbiturate-like mechanism (Quilichini et al., 2006). The drug is a racemic mixture of two enantiomers: R(+)-STP and S( )-STP, of which R(+)-STP has a 2.4-fold greater anticonvulsant potency but is also associated with 3-fold faster elimination rate (Shen et al., 1992). Experimental evidence indicates that STP suppresses seizures in various animal models, including maximal electroshock (MES)-, pentylenetetrazole (PTZ)-, bicuculline- and strychnine-induced seizures in rodents (Poisson et al., 1984; Shen et al., 1990, 1992). Moreover, STP protected mice against cocaine-induced clonic seizures (Gasior et al., 1999) and suppressed spike-andwave discharges in a rat genetic model of petit mal epilepsy (Micheletti et al., 1988) and spike-discharges in an alumina gel rhesus monkey model of focal epilepsy (Lockard et al., 1985). In open studies, STP has been reported to decrease the frequency of both partial and generalised seizures, including typical and atypical absences (Martinez-Lage et al., 1984; Farwell et al., 1992, 1993; Perez et al., 1999; Chiron et al., 2000). In the open-label pediatric add-on trial, 49% of patients showed a 50% reduction in seizure frequency and 3 out of 20 children became seizure-free (Perez et al., 1999). This study also reported an improvement in patients with severe myoclonic epilepsy in infancy (SMEI). Subsequently, it was reported that patients with SMEI responded particularly well when STP was used in combination with clobazam (CLB) (Chiron et al., 2000). Furthermore, good responses have been observed when STP was administered in combination with clonazepam (CZP) in multifocal epilepsy in infancy (Chiron et al., 1993, 2000), with valproic acid (VPA) in refractory epilepsy in children (Renard et al., 1993), and with carbamazepine (CBZ) in CBZ-resistant epilepsies (Loiseau et al., 1990). Pharmacokinetic studies have indicated that STP is a potent inhibitor of several cytochrome P450 isoenzymes, as demonstrated in vivo in humans (CYP1A2, 3A4; Tran et al., 1997) and in vitro (CYP1A2, 3A4, 2D6, 2C9, and 2C19) with the use of human liver microsomes (Kerr et al., 1991; Tran et al., 1997). Thus, STP inhibits the metabolism and increases serum concentrations of phenytoin (Levy et al., 1984), CBZ and its primary pharmacologically active metabolite CBZ-epoxide (Kerr et al., 1991; Perez et al., 1999; Cazali et al., 2003), phenobarbital (PB; Levy et al., 1984), primidone (Bebin and Bleck, 1994), CLB and its primary pharmacologically active metabolite norclobazam (Perez et al., 1999; Rey et al., 1999; Chiron et al., 2000; Giraud et al., 2006) and VPA (Levy et al., 1987). Most of these interactions are clinically relevant and require reduction in dosage of the affected drug. Invariably when STP is licensed for clinical use it will be as an add-on therapy in patients with intractable epilepsy. Furthermore, the drug is likely to be used to treat children with SMEI, where it has already been shown to be particularly effective. Consequently, it can be considered appropriate to evaluate its preclinical profile in combination with AEDs that are routinely prescribed to children, namely: CZP, ethosuximide (ETS), PB, and VPA. We have used the mouse PTZ-induced clonic seizure model, a model of myoclonic seizures in humans (Löscher and Schmidt, 1988; Löscher et al., 1991), and the data analysed by isobolography. We also used the chimney test (a measure of motor performance impairment), the stepthrough passive avoidance task (a measure of long-term memory deficits), the grip-strength test (a measure of neuromuscular tone impairment), and the pain threshold and hot plate tests (a measure of antinociception). Finally, in order to ascertain whether the observed interactions were purely pharmacodynamic in nature or that pharmacokinetic interactions also contributed, brain STP, CZP, ETS, PB, and VPA concentrations were also measured. MATERIAL AND METHODS Animals and experimental conditions All experiments were performed on adult male Swiss mice weighing g. The mice were kept in colony cages with free access to food and tap water, under standardised housing conditions (12 h of a light dark cycle, temperature was 21 ± 1 C). After 7 days of adaptation

3 INTERACTIONS OF STP WITH CONVENTIONAL AEDS 1843 to laboratory conditions, the animals were randomly assigned to experimental groups consisting of eight mice. Each mouse participated only in one experiment. All tests were performed between 9.00 a.m. and 2.00 p.m. to minimize confounding effects of circadian rhythms. Procedures involving animals and their care were conducted in accordance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and Polish legislation on animal experimentation. Additionally, all efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data. The experimental protocols and procedures described in this article were approved by the Local Ethics Committee at the Medical University of Lublin (License nr: 420/2003/446/03). Drugs The following AEDs were used in this study: STP (Laboratoires Biocodex, Montrouge Cedex, France), CZP (Polfa, Warszawa, Poland), ETS (Sigma, St. Louis, MO, U.S.A.), PB (Polfa, Krakow, Poland), and VPA (magnesium salt, ICN-Polfa S.A., Rzeszow, Poland). All drugs, except for VPA, were suspended in a 1% solution of Tween 80 (Sigma) in saline, whereas VPA was directly dissolved in saline. All drugs were administered by intraperitoneal (i.p.) injection in a volume of ml/g body weight. Fresh drug solutions were prepared on each day of experimentation and administered as follows: PB and STP 60 min; ETS 45 min; VPA 30 min, and CZP 15 min before seizures and behavioural tests as well as before brain sampling for the measurement of AED concentrations. These pretreatment times were chosen based upon information about their biological activity from the literature (Gasior et al., 1999) and our previous studies (Luszczki et al., 2005a,b). PTZ (Sigma) was dissolved in distilled water and administered subcutaneously (s.c.) into a loose fold of skin in the midline of the neck in a volume of ml/g body weight. PTZ-induced convulsions The anticonvulsant activities of STP and CZP, ETS, PB, and VPA against PTZ-induced clonic seizures were determined after subcutaneous administration of PTZ at its CD 97 (convulsive dose 97, i.e., the dose of PTZ that produced clonic seizures in 97% of mice, which in this study was 100 mg/kg). Following PTZ administration, mice were placed separately into transparent Plexiglas cages ( cm) and observed for 30 min for the occurrence of clonic seizures. Clonic seizure activity was defined as clonus of whole body lasting for over 3 s, with an accompanying loss of righting reflex. The number of animals convulsing out of the total number of mice tested was noted for each treatment regimen. The animals were administered with increasing doses of the AEDs, and the anticonvulsant activity of each drug was evaluated as the ED 50 (median effective dose of an AED, protecting 50% of mice against clonic convulsions). At least four groups of animals were used to estimate each ED 50 value calculated from the respective dose response relationship curve according to Litchfield and Wilcoxon (1949). Similarly, the anticonvulsant activity of a mixture of STP with an AED was evaluated and expressed as ED 50mix, corresponding to the dose of a mixture of both drugs required to protect 50% of animals tested against PTZ-induced clonic convulsions. This experimental procedure has been described in more detail in our earlier studies (Luszczki and Czuczwar, 2004; Luszczki et al., 2005a, 2005b). Chimney test The acute adverse effects of STP and conventional AEDs (CZP, ETS, PB, and VPA) on motor performance impairment were quantified with the chimney test of Boissier et al. (1960). In this test, animals had to climb backwards up a plastic tube (3 cm inner diameter, 25 cm length), and motor impairment was indicated by the inability of the animals to climb backward up the transparent tube within 60 s. The adverse (neurotoxic) effects of AEDs alone were expressed as their median toxic doses (TD 50 in mg/kg), representing the doses, at which the respective AEDs impaired motor coordination in 50% of the animals tested. To evaluate each TD 50 value, at least four groups of animals (each group consisted of eight mice) injected with various doses of the drugs were challenged with the chimney test. A dose response curve was calculated on the basis of the percentage of mice showing motor deficits by means of the log-probit method according to Litchfield and Wilcoxon (1949). Similarly, the combinations of STP with a conventional AED (CZP, ETS, PB, and VPA) were challenged with the chimney test in order to determine TD 50mix values, corresponding to the doses of the mixture of STP and an AED necessary to produce motor impairment in 50% of animals tested. Isobolographic analysis of interactions Interactions between STP and the four conventional AEDs against PTZ-induced seizures and in the chimney test were analyzed according to the methodology previously detailed in our earlier studies, where the precise descriptions of theoretical background with the respective equations showing how to undertake isobolographic calculations have been presented (Luszczki and Czuczwar, 2003, 2004; Luszczki et al., 2003a, 2003b, 2006). In the present study, the isobolographic analysis comprised six stages as follows: 1. Determination of ED 50 or TD 50 values for all AEDs (administered singly) by means of log-probit linear regression analysis according to Litchfield and Wilcoxon (1949). 2. Calculation of purely additive ED 50add s ± S.E.M. (standard errors of the means) or TD 50add s ± S.E.M.

4 1844 J. J. LUSZCZKI ET AL. TABLE 1. Anticonvulsant and acute adverse (neurotoxic) effects of stiripentol (STP) and various conventional AEDs evaluated in pentylenetetrazole (PTZ)-induced clonic seizure model and the chimney test AED ED 50 (mg/kg) n E S.E.M. TD 50 (mg/kg) n T S.E.M. PI CZP ( ) ( ) ETS ( ) ( ) PB 14.8 ( ) ( ) STP ( ) ( ) VPA ( ) ( ) Values are presented as median effective doses (ED 50 s, with 95% confidence limits in parentheses) of AEDs, protecting 50% of animals tested against PTZ-induced clonic seizures or as median toxic doses (TD 50 s, with 95% confidence limits in parentheses) of AEDs, inducing motor impairment in 50% of animals tested in the chimney test. All AEDs were administered i.p.: PB and STP 60 min, ETS 45 min, VPA 30 min, and CZP 15 min prior to the PTZ and chimney tests. PTZ-induced clonic seizures were produced by the subcutaneous injection of PTZ at its CD 97 (100 mg/kg). n, total number of animals used at those doses whose anticonvulsant (n E ) and acute neurotoxic (n T ) effects ranged between 4 and 6 probits; S.E.M., standard error of ED 50 s and TD 50 s; PI, protective index; as a ratio of TD 50 and ED 50 values, describes the margin of safety of therapeutic use of each AED. CZP, clonazepam; ETS, ethosuximide; PB, phenobarbital, STP, stiripentol; and VPA, valproate. for a mixture of the examined AEDs, which is associated with the choice of at least three fixed drug dose ratio combinations (usually, 1:3, 1:1, and 3:1). The ED 50add represents a total additive dose of the drugs in the mixture, providing theoretically a 50% protection against PTZ-induced seizures. The TD 50add corresponds to a total dose of the drugs in the mixture, which impairs theoretically motor performance in 50% of animals challenged with the chimney test. 3. Experimental determination of the ED 50mix s ± S.E.M. or TD 50mix s ± S.E.M. for the corresponding fixed-ratio AED combinations. ED 50mix is an experimentally determined total dose of a mixture of two component drugs, which was administered at a fixed-ratio combination, sufficient for 50% protective effect against PTZ-induced seizures. Analogously, the TD 50mix is an experimentally determined total dose of a mixture of two component drugs, which was administered at a fixed-ratio combination, sufficient for 50% impairment of motor coordination in the chimney test. Generally, to determine the ED 50mix or TD 50mix, both drugs in the mixture (at proportionally raised doses) are administered to animals and a dose response relationship for the mixture (at the fixed ratio) is denoted using the log-probit method. 4. Statistical comparison of the experimentallyderived ED 50mix or TD 50mix values with their corresponding theoretically additive ED 50add or TD 50add values was undertaken by use of the unpaired Student s t-test, according to Porreca et al. (1990) and Tallarida (2000). 5. Graphical illustration of the examined interactions as isobolograms, which are a simple form of visualization of interactions. 6. Finally, to determine the separate doses of AEDs in the mixture, the ED 50mix or TD 50mix values were multiplied by the respective proportions of AEDs (denoted for purely additive mixture). To simplify the notation and nomenclature of interactions in isobolography, the drug doses were administered at fixed-ratio combinations (e.g., 1:3, 1:1, and 3:1). The fixed drug dose ratios are usually presented in form of natural numbers (1:3, 1:1, 3:1) and they reflect fractions of ED 50 values denoted for the drugs used separately. For example, the mixture at the fixed ratio of 1:3 is comprised 1 / 4 of the ED 50 of the first drug and 3 / 4 of the ED 50 of the second drug. Thus, the isobolographic notation of fixed ratio combinations comprises of numerators of fractions of ED 50 values for AEDs used in the mixture. For instance, in the present study, the ED 50 values for STP and CZP administered alone in the PTZ test were mg/kg and mg/kg, respectively (Table 1). Hence, the mixture of STP with CZP at the fixed ratio of 1:3 comprised STP at ( 1 / 4 of mg/kg = mg/kg) and CZP ( 3 / 4 of mg/kg = mg/kg). Thus, the ED 50add value for the fixed ratio of 1:3 was mg/kg (Table 2). Noteworthy, in this two-drug mixture, CZP prevailed over STP with respect to its pharmacological (antiseizure) activity against PTZ-induced clonic seizures, but it did not exceed quantitatively in the mixture. Analogously, the two-drug mixture for the combination of 1:1 in the PTZ test comprised STP ( 1 / 2 of mg/kg = mg/kg) and CZP ( 1 / 2 of mg/kg = mg/kg), where the drugs were combined in equieffective (isoeffective) doses. Thus, the ED 50add value for the fixed-ratio combination of 1:1 was mg/kg (Table 2). Likewise, the fixed-ratio combination of 3:1 comprised STP ( 3 / 4 of mg/kg = mg/kg) and CZP ( 1 / 4 of mg/kg = mg/kg), producing the ED 50add of mg/kg (Table 2). In this combination, STP clearly prevailed over CZP in the mixture. All the above-mentioned drug doses for the respective fixed-ratio combinations were primarily considered as additive because they were directly calculated from the equation of additivity. Further details regarding these concepts have been published by Luszczki and Czuczwar (2003, 2004) and Luszczki et al., (2003a,b, 2006). Furthermore, in the present study, the protective index (PI) for each AED was calculated by dividing a given

5 INTERACTIONS OF STP WITH CONVENTIONAL AEDS 1845 TABLE 2. Isobolographic characterization of the interactions between stiripentol and various conventional AEDs in the mouse PTZ-induced clonic seizure model AED-combination FR STP + AED = ED 50mix (mg/kg) n mix ED 50add (mg/kg) = STP + AED n add STP + CZP 1: ± ± STP + CZP 1: ± ± STP + CZP 3: ± ± STP + ETS 1: ± ± STP + ETS 1: ± ± STP + ETS 3: ± ± STP + PB 1: ± ± STP + PB 1: ± ± STP + PB 3: ± ± STP + VPA 1: ± ± STP + VPA 1: ± ± STP + VPA 3: ± ± Data are presented as median effective doses (ED 50 s ± S.E.M.) protecting 50% of animals tested against PTZ-induced clonic seizures. The clonic phase of PTZ-induced seizures was produced by the s.c.-injection of PTZ at its CD 97 (100 mg/kg). The ED 50 s were either experimentally determined from the mixture of two AEDs (ED 50mix ) or theoretically calculated from the equation of additivity (ED 50add ). Additionally, the actual doses of STP and conventional AEDs that comprised the mixtures, at all fixed-ratio combinations and for both ED 50mix and ED 50add values, are presented in separate columns (as STP and AED). Statistical evaluation of the data was performed by using unpaired Student s t-test according to Porecca et al. (1990) and Tallarida (2000). 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 (n mix) and theoretically calculated (n add) from the equation of additivity; STP, stiripentol; CZP, clonazepam; ETS, ethosuximide; PB, phenobarbital; and VPA, valproate. TD 50 value, evaluated in the chimney test, by the respective ED 50 value, determined in the PTZ test. The PI is considered an index of the margin of safety and tolerability between anticonvulsant doses and doses of AEDs exerting acute adverse effects (e.g., sedation, ataxia, impairment of motor coordination, or other neurotoxic manifestations; Löscher and Nolting, 1991). Subsequently, the isobolographic parameter benefit index (BI), was calculated for all combinations examined as a quotient of PI mix and PI add of respective fixed-ratio combinations. PI mix is experimentally determined from the corresponding TD 50mix and ED 50mix values, whereas PI add is theoretically calculated from the equation of additivity for the PTZ and chimney tests as a ratio of TD 50add and ED 50add values. BI determines the margin of safety and tolerability of drugs in combinations, considering both, the anticonvulsant and acute neurotoxic effects produced by the mixture of two drugs. Drug combinations, whose BI value is greater than the border value of 1.3, can be considered as favourable combinations. Drug combinations, whose BIs range between 0.7 and 1.3 are considered to be neutral, whereas those, whose BI is lower than the border value of 0.7 are considered unfavourable (Luszczki et al., 2003a, 2003b; 2005c). Measurement of total brain AED concentrations Brain AED concentrations were determined only in mice that were administered STP + an AED at the fixedratio combination of 1:1. This fixed-ratio combination was chosen because it comprised both AEDs being present at maximally equieffective doses. Mice were killed by decapitation at times chosen to coincide with that scheduled for the PTZ-test and whole brain were removed from skulls, weighed, and homogenized using Abbott buffer (2:1 vol/weight; Abbott Laboratories, North Chicago, IL, U.S.A) in an Ultra-Turrax T8 homogeniser (Staufen, Germany). The homogenates were centrifuged at 10,000g for 10 min and the supernatant samples (75 µl) were analyzed by fluorescence polarization immunoassay (FPIA) using a TDx analyser and reagents (CZP, ETS, PB, and VPA) exactly as described by the manufacturer (Abbott Laboratories). For the quantitation of CZP, the benzodiazepine assay kit was used. Total brain concentrations were expressed in µg/ml (except for CZP, whose concentrations were expressed in ng/ml) of brain supernatants as means ± S.D. of at least eight separate brain preparations. Brain STP concentrations were determined by highperformance liquid chromatography (HPLC) using an automated Gilson (Anachem) HPLC system comprising of a 234 autosampler, two 306 pumps and a UV 155 variable wavelength detector set at 215 nm. The mobile phase was constituted as phosphate (50 mm) and acetonitrile (25:75) and chromatographic separation was achieved by use of an ESA RP-C18 3 µm column (ESA Analytical Ltd. Buckinghamshire, U.K.). Brain homogenate samples were prepared for analysis as follows: 50-µl brain homogenate were pipetted into a 1.5-ml plastic tube to which was added 100-µl acetonitrile and vortex-mixed for 1 min. Subsequently, samples were centrifuged for 5 min, and 90 µl of the supernatant were transferred into an autosampler vial from which 10 µl were injected automatically into the column. Quantitation was achieved by use of chromatographic peak areas and these were linearly related over the range µg/ml STP. The within-batch and betweenbatch precision was <4% and <6%, respectively. Light-dark, step-through passive avoidance task Each animal was administered an AED (STP, CZP, ETS, PB, and VPA) either singly or in combination at the fixed

6 1846 J. J. LUSZCZKI ET AL. ratio of 1:1 from the PTZ test on the first day before training. This fixed-ratio combination was chosen because it comprised both AEDs being present at maximally equieffective doses. The time before the commencement of the training session (after drug administration) was identical to that for the PTZ test. Subsequently, animals were placed in an illuminated box ( cm) connected to a larger dark box ( 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 pretrained animals 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; 2005b). Pain threshold test The pain threshold was assessed as a minimal exposing time to an electrical stimulation required to induce the first pain reaction in animals. This test was performed under conditions identical to the experiment testing the step-through passive avoidance task. The animals were placed separately on the grid surface connected with a current generator. Afterwards, each mouse was exposed to an electric footshock (0.6 ma), and the time to induce the first pain reaction in animals was measured and expressed as the latency (in s). For ethical reasons, the animals were exposed to the current impulse up to 10 s. In the event that the mouse did not express any reactions to the stimulus of 10 s, the test was terminated and the animal was assigned this cutoff latency. It has to be stressed that the first pain reaction was considered as the endpoint and after displaying some reactions the animals were immediately set free from the stimulation. To evaluate the latency to pain reaction, the testing took place at times chosen to coincide with that scheduled for the PTZ-test. The first pain reaction of animals observed in our study was expressed as: jerks of fore and hind limbs with or without squeaking; violent seeking of escape with a tendency to jump out; wild running. The reaction of control animals to an electric stimulus was instantaneous and the latency did not exceed 2 s. All testing was undertaken using unanaesthetized mice. A more detailed description of the use of these procedures has been reported by Luszczki et al. (2003c, 2005b). Hot plate test The hot plate test, a standard model used to determine the antinociceptive efficacy of compounds with respect to acute thermal nociception, was conducted according to that described by Eddy and Leimbach (1953). The device consisted of an electrically heated surface and an open Plexiglas tube (17 cm high 22 cm diameter) to confine the animals to the heated surface (Ugo Basile, Varese, Italy sponsored by a SCSR grant No.: 2P05D05126). The temperature was set at 56.0 ± 0.1 C. Mice were placed on the hot plate, and the time until either licking of the fore or hind paws, shaking the hind paws or jumping off occurred was recorded by a stopwatch. Animals were tested once before baselines were taken and this trial served as the control reaction time for the animals and mice showing a reaction time greater than 12 s were excluded from the subsequent test. The predrug latencies were between 6 and 9 s. Subsequently, the animals were administered AEDs alone or in combinations and at times to peak of anticonvulsant activities of AEDs, which coincided with those from the PTZ test, were subjected to the hot plate test. A maximum cutoff of 30 s was chosen so as to avoid tissue damage to animals and mice not responding within 30 s were removed and assigned a score of 30 s. The maximum possible antinociceptive effect (MPAE) was defined as the lack of a nociceptive response during the exposure to the heat stimulus, and the percentage of MPAE was calculated according to the formula: [(T 1 T 0 )/(T 2 T 0 )] 100, where T 0 and T 1 were the latencies obtained before and after drug administration, and T 2 was the cutoff time of 30 s. Grip-strength test The effects of AEDs (STP, CZP, ETS, PB, and VPA) administered singly or in combination at the fixed ratio of 1:1 from the PTZ test on mouse neuromuscular strength were quantified by the grip-strength test. This fixed-ratio combination was chosen because it comprised both AEDs being present at maximally equieffective doses. The time before the commencement of the grip-strength test (after drug administration) was identical to that for the PTZ test. The grip-strength apparatus (BioSeb, Chaville, France sponsored by a SCSR grant No.: 2P05D05126) comprised a wire grid (8 8 cm) connected to an isometric force transducer (dynamometer). The mice were lifted by the tail 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 three measurements for each animal was calculated and subsequently, the mean maximal force of eight animals per group was determined. The grip-strength test was used to determine the effects of AEDs on neuromuscular strength in mice, which was expressed in Newtons as means ± S.D. of at

7 INTERACTIONS OF STP WITH CONVENTIONAL AEDS 1847 least 24 determinations (three measurements for each of eight animals per group). Statistics The ED 50 and TD 50 values with their 95% confidence limits were calculated by computer-assisted log-probit analysis according to Litchfield and Wilcoxon (1949). The obtained 95% confidence limits were transformed to S.E.M. as described previously (Luszczki et al., 2003a,b 2006). Statistical evaluation of isobolographic interactions was performed by the use of Student s t-test in order to detect the differences between the experimentally derived (ED 50mix or TD 50mix ) and theoretical additive (ED 50add or TD 50add ) values, according to Porreca et al. (1990) and Tallarida (2000). Total brain AED concentrations were statistically analyzed using the unpaired Student s t-test. The results from the step-through passive avoidance task and the pain threshold test were statistically analyzed using Kruskal-Wallis nonparametric ANOVA test followed by the post hoc Dunn s test. The data from the grip-strength and hot plate tests were statistically analyzed using oneway ANOVA followed by the post hoc Bonferroni s test. Results were considered statistically significant if p < RESULTS Anticonvulsant and acute adverse-effect (neurotoxic) profiles of AEDs All five AEDs (CZP, ETS, PB, STP, and VPA) administered alone exhibited a clear-cut anticonvulsant activity in the mouse PTZ test. The AED ED 50 values are shown in Table 1. Similarly, in the chimney test, it was possible to determine TD 50 values for all the AEDs (Table 1). Simultaneous evaluation of TD 50 and ED 50 values for all AEDs allowed the determination of their PIs, describing the safety and tolerability profile of AEDs administered singly (Table 1). The highest PI was associated with CZP (317.1), whereas VPA had the lowest value (2.84). PB, STP, and ETS had PIs that ranged between 2.89 and 8.39 (Table 1). Isobolographic characteristic of interactions between STP and CZP, ETS, PB, and VPA against PTZ-induced clonic convulsions Statistical evaluation of data revealed that the combinations of STP with CZP, ETS, PB, and VPA for all fixed ratios of 1:3, 1:1, and 3:1 were additive in suppressing PTZ-induced clonic seizures. However, with isobolography, it was observed that all combinations tested displayed a tendency towards subadditivity (antagonism) at the fixed ratios of 1:1 and 3:1 (Fig. 1A D, Table 2). In contrast, the AED combinations at the fixed ratio of 1:3 showed a tendency towards supraadditivity (synergy; for the combination of STP with PB; Fig. 1C), additivity (for the combination of STP with CZP; Fig. 1A), or subadditivity (antagonism; for the combinations of STP with ETS and VPA; Fig. 1B, D) in the PTZ test (Table 2). Isobolographic analysis of interactions between STP and CZP, ETS, PB, and VPA in the chimney test It was observed that STP combined with CZP at the fixed ratio of 1:1 and 3:1 exerted supraadditive (synergistic) interaction in the chimney test (p < 0.05; Table 3; Fig. 2A). Only, the combination of STP with CZP at the fixed-ratio of 1:3 displayed additivity (Table 3, Fig. 2A). In contrast, STP combined with ETS, PB, and VPA (at the fixed-ratios of 1:3, 1:1, and 3:1) exerted additive interactions (Table 3; Fig. 2B D). However, the combination of STP with ETS at all fixed ratios tested displayed a tendency towards supraadditivity (synergy; Fig. 2B). The interactions between STP and PB showed a tendency towards subadditivity (antagonism; Fig. 2C). Similarly, the coadministration of STP with VPA at the fixed ratios of 1:3 and 3:1 resulted in a tendency towards subadditivity (Fig. 2D). Only, the combination of STP with VPA at the fixed ratio of 1:1 displayed a tendency towards supraadditivity in the chimney test (Table 3; Fig. 2D). Isobolographic parameter evaluation In the present study, only one combination, namely STP and PB at a fixed ratio of 1:3, out of 12 fixed-ratio combinations investigated could be considered to be favorable, having a BI value of 1.39 and therefore higher than the border value of 1.3. In this case, the tendency towards supraadditivity (synergy) in the PTZ test associated with a tendency towards subadditivity (antagonism) in terms of acute motor coordination impairment in the chimney test fulfilled the criterion of an advantageous combination. The remaining BI values for the combination of STP and PB at fixed ratios of 1:1 and 3:1 were 0.89 and 0.92, respectively, indicating neutral combinations (Table 4). In contrast, the combination of STP with CZP at the fixed ratios of 1:1 and 3:1 may be considered unfavorable since their BI values were lower than the border value of 0.7 (Table 4). In these cases, supraadditivity (synergy) in the chimney test was associated with a tendency towards subadditivity in the PTZ test. Only, STP combined with CZP at the fixed ratio of 1:3, displayed a neutral combination (Table 4). The BI values calculated for the combination of STP with VPA ranged between 0.77 and 0.84, indicating that these combinations were at best neutral (Table 4). Moreover, the combination of STP with ETS at the fixed ratios of 1:1 and 3:1 were unfavorable combinations because of their tendency towards supraadditivity in the chimney test and simultaneously, a tendency to subadditivity in the PTZ-test. Only, the combination of STP with ETS at the fixed ratio of 1:3 was neutral with a BI value (0.74) marginally greater than the border value of 0.7 (Table 4).

8 1848 J. J. LUSZCZKI ET AL. FIG. 1. Isobolograms showing interactions between stiripentol and various conventional AEDs against PTZ-induced clonic seizures The median effective dose (ED 50 ) for stiripentol (STP) is plotted graphically on the x-axis, whereas the ED 50 values of the various AEDs (clonazepam [CZP], ethosuximide [ETS], phenobarbital [PB] and valproate [VPA]) are plotted on the y-axis (A D). The solid lines on the x and y axes represent the 95% confidence limits (CLs) for the AEDs administered alone. The straight line connecting these two ED 50 values on each graph represents the theoretical line of additivity for a continuum of different fixed dose ratios, whereas the dashed lines represent on each isobologram the theoretical additive 95% CLs of ED 50 add s. The open circles (o) depict the experimentally derived ED 50 mix s (with 95% CLs as the error bars) for the total dose expressed as the proportion of STP and an AED that produced a 50% anticonvulsant effect. Alternatively, all 95% CLs of the experimental ED 50 mix s are presented horizontally and vertically in the shape of a cross. (A) Interactions between STP and CZP. The experimental ED 50 mix of the mixture of STP+CZP for the fixed ratio of 1:3 and 1:1 are close to the theoretical line of additivity, indicating additivity. Only the combination of STP+CZP at the fixed ratio of 3:1 is placed above this line, and thus displaying the tendency towards subadditivity. (B) Interactions between STP and ETS. The experimental ED 50 mix s of the mixture of STP+ETS, for the fixed ratios of 3:1, 1:1, and 3:1 indicate a tendency towards subadditivity. (C) Interactions between STP and PB. The experimental ED 50 mix for the fixed ratio of 1:3 is placed below the line of additivity, indicating a tendency towards supraadditivity. The other fixed ratio combinations (1:1 and 3:1) are above the line of additivity and thus displaying a tendency towards subadditive interactions. (D) Interactions between STP and VPA. All experimental ED 50 mix s of the mixture of STP+VPA indicate a tendency towards subadditivity. Brain AED concentrations STP (142.3 mg/kg) coadministered with ETS (95.4 mg/kg) resulted in a significant 39% increase in total brain ETS concentration (p < 0.001; Table 5). Similarly, STP (133.7 mg/kg) combined with PB (8.9 mg/kg) resulted in a significant 20% increase in total brain PB concentration (p < 0.001; Table 5). In contrast, STP (at mg/kg) coadministered with VPA (82.1 mg/kg) significantly reduced total brain VPA concentration (16%; p < 0.01; Table 5). In combination with CZP, STP had no significant effect on total brain CZP concentrations (Table 5). While neither CZP nor ETS coadministered with STP affected total brain STP concentrations (Table 6), PB (8.9 mg/kg) and VPA (82.1 mg/kg) coadministration were associated with a 21% decrease (p < 0.001) and a 124% increase (p < 0.001) in STP concentrations, respectively (Table 6). Effects of STP administered alone and in combinations with CZP, ETS, PB, and VPA on long-term memory, threshold for the first pain reaction, neuromuscular strength and acute thermal pain When STP was coadministered with CZP, ETS, PB, and VPA, at the fixed ratio of 1:1, the threshold for the first pain reaction was unaffected (Table 7). Furthermore, none of the studied combinations impaired long-term memory as determined in the passive avoidance task, the median retention times being 180 s (Table 7). Similarly these combinations had no effect on neuromuscular strength, as assessed by the grip-strength test (Table 8), and did not lengthen the latency to the acute thermal pain in animals challenged with the hot plate test (Table 8). Moreover, STP administered alone at a dose of mg/kg, being its ED 50 value from the PTZ test, did not affect long-term

9 INTERACTIONS OF STP WITH CONVENTIONAL AEDS 1849 TABLE 3. Isobolographic characterization of the interactions between stiripentol and various conventional AEDs in the chimney test AED-combination FR STP + AED = TD 50 mix (mg/kg) n mix TD 50 add (mg/kg) = STP + AED n add STP + CZP 1: ± ± STP + CZP 1: ± a ± STP + CZP 3: ± a ± STP + ETS 1: ± ± STP + ETS 1: ± ± STP + ETS 3: ± ± STP + PB 1: ± ± STP + PB 1: ± ± STP + PB 3: ± ± STP + VPA 1: ± ± STP + VPA 1: ± ± STP + VPA 3: ± ± Results are presented as median toxic doses (TD 50 s ± S.E.M.) producing motor impairment in 50% of animals tested in the chimney test. The TD 50 values were either experimentally determined from the mixture of two AEDs (TD 50 mix ) or theoretically calculated from the equation of additivity (TD 50 add ). Statistical evaluation of the data was performed by using unpaired Student s t-test according to Porecca et al. (1990) and Tallarida (2000). For more details see the legend of Table 2. a p < 0.05 versus the respective TD 50 add value. memory, threshold for the first pain reaction, neuromuscular strength and acute thermal pain in animals (Tables 7 and 8). DISCUSSION STP is a new AED currently undergoing phase III clinical evaluation with particular efficacy in childhood epilepsies including SMEI, absence seizures (typical and atypical) and multifocal epilepsy in infancy (Martinez-Lage et al., 1984; Farwell et al., 1992, 1993; Renard et al., 1993; Perez et al., 1999; Chiron et al., 2000). Furthermore, patients appear to respond particularly well to STP when used in combination with CZP and VPA. We sought, therefore, to characterize the interaction profile of STP when administered in combination with AEDs typically prescribed to children (CZP, ETS, PB, and VPA) using the mouse PTZ induced seizure model and various behavioral and adverse effect paradigms and pharmacokinetic evaluation. Isobolography was used to determine the equieffective doses of AEDs so that the observed interactions could be classified in terms of supraadditive (synergistic), subadditive (antagonistic), indifferent, or additive interactions (Berenbaum, 1989; Greco et al., 1995; Tallarida, 2000; Luszczki and Czuczwar, 2003, 2004). The present study shows that STP in combination with CZP, ETS, PB, and VPA was associated with additive interactions for all fixed ratios tested (1:3, 1:1, and 3:1) in the mouse PTZ-induced clonic seizure model. However, with the exception of CZP, these data are complicated by bidirectional pharmacokinetic interactions. Thus, in combination with STP, brain ETS concentrations increased by 39%; brain STP concentrations were unaffected. Brain PB concentrations decreased by 21% when administered in combination with STP while concomitant STP concentrations increased by 20%. Finally, STP in combination with VPA resulted in a 16% decrease in brain VPA concentrations and a concurrent 2.2-fold increase in brain STP concentrations. Overall, these data would suggest that STP in combination with ETS resulted in primarily a subadditive (antagonistic) interaction, which was secondarily masked by a pharmacokinetic increase in total brain ETS concentration and thus, a final additive isobolographic characteristic. In case of the combination of STP with PB, the 21% decrease in brain STP concentrations was probably compensated by the concomitant 20% increase in brain PB concentrations, resulting in isobolography the additive interaction. In contrast, theoretically the 2.2-fold increase in brain STP concentrations that occurred during VPA coadministration should be associated with a synergistic interaction between STP and VPA. However, in the present study, the experimentally derived ED 50 mix value at the fixed ratio of 1:1 for the mixture of STP with VPA did not differ significantly from that calculated from the equation of additivity (ED 50 add ), and thus, indicating additive interaction. In such a case, one can hypothesize that STP combined with VPA produced primarily a subadditive (antagonistic) interaction. Clearly, STP is associated with significant pharmacokinetic interactions (Tables 5 and 6) and confirms that seen clinically, and indeed this characteristic has complicated the undertaking of placebo-controlled adjunctive-therapy trial because it is very difficult to separate the effects of STP per se from that of changes in plasma concentrations of concomitantly administered AEDs. The increase in brain PB (20%) concentrations when STP was coadministered (Table 5) can be explained on the basis of inhibition of PB hepatic metabolism by STP (Levy et al., 1984). A similar hepatic inhibition can explain the increase in brain ETS (39%) concentrations, although this interaction has not been previously reported. Surprisingly, brain VPA concentrations decreased (16%; Table 5) when

10 1850 J. J. LUSZCZKI ET AL. FIG. 2. Isobolograms showing interactions between stiripentol and various conventional AEDs in the chimney test. The median toxic dose (TD 50 ) for stiripentol [STP] is plotted graphically on the x-axis, whereas the TD 50 values of the various AEDs (clonazepam [CZP], ethosuximide [ETS], phenobarbital [PB], and valproate [VPA]) are plotted on the y-axis (A D). The solid lines on the x and y axes represent the 95% confidence limits (CLs) for the AEDs administered alone. The straight line connecting these two TD 50 values on each graph represents the theoretical line of additivity for a continuum of different fixed dose ratios, whereas the dashed lines represent on each isobologram the theoretical additive 95% CLs of TD 50 add s. The open circles (o) depict the experimentally derived TD 50 mix s (with 95% CLs as the error bars) for the total dose expressed as the proportion of STP and an AED that produced 50% motor coordination impairment. Alternatively, all 95% CLs of the experimental TD 50 mix s are presented horizontally and vertically in the shape of a cross. (A) Interactions between STP and CZP. The experimental TD 50 mix s of the mixture of STP+CZP, for the fixed ratios of 3:1 and 1:1 are significantly below the theoretical line of additivity, indicating supraadditive (synergistic) interactions (at p < 0.05). Only the combination of STP+CZP at the fixed ratio of 1:3 is placed close to the line of additivity. (B) Interactions between STP and ETS. The experimental TD 50 mix s of the mixture of STP+ETS, for the fixed ratios of 3:1, 1:1, and 3:1 are plotted near to the line of additivity, and thus indicating a tendency towards supraadditivity. (C) Interactions between STP and PB. The experimental TD 50 mix for the fixed ratios of 1:3, 1:1, and 3:1 are placed above the line of additivity, indicating a tendency towards subadditivity. (D) Interactions between STP and VPA. The experimental TD 50 mix of the mixture of STP+VPA at the fixed ratio of 1:1 is placed below the line of additivity, and thus indicating a tendency towards supraadditivity (synergy). The other fixed-ratio combinations (1:3 and 3:1) are above the line of additivity and thus displaying a tendency towards subadditive interactions. coadministered with STP, appears to contradict previous reports that VPA plasma concentrations are increased by STP coadministration. However, since both STP (99%) and VPA (90%) are significantly bound to plasma proteins (Perucca and Kupferberg, 2002), the observed reduction in brain VPA concentrations may be the consequence of a plasma protein binding displacement interaction. Finally, our data would suggest for the first time that STP is associated with bidirectional pharmacokinetic interactions whereby brain STP concentrations are increased (124%) and decreased (21%) by VPA and PB, respectively (Table 6). These data can be readily explained on the basis that VPA is a potent inhibitor and that PB is a potent inducer of hepatic cytochrome P450 metabolism. The lack of a pharmacokinetic interaction between STP and CZP requires further comment. For the brain CZP analysis, it was necessary to employ a 100-fold higher dose (2.3 mg/kg) compared to that indicative of the PTZ test (0.023 mg/kg). The reason for this is because the FPIA method used to measure CZP concentrations was not sensitive enough to measure CZP concentrations that occurred after mg/kg CZP administration. Since brain STP concentrations were unaffected by 2.3 mg/kg CZP coadministration one would expect that at a much lower dose (0.023 mg/kg) brain concentrations would be similarly unaffected. However, at the higher CZP dose used in the present study, it is very possible that a pharmacokinetic interaction, whereby brain CZP concentrations were affected by STP, was masked since relative doses are critical with regards to competitive inhibitory interactions, as could theoretically occur between STP and CZP.