Jarogniew J. uszczki 1, Marek Zuchora 1, Katarzyna M. Sawicka 1, Justyna Koziñska 1, Stanis³aw J. Czuczwar 1,2. Introduction

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1 Pharmacological Reports 2006, 58, ISSN Copyright 2006 by Institute of Pharmacology Polish Academy of Sciences Acute exposure to caffeine decreases the anticonvulsant action of ethosuximide, but not that of clonazepam, phenobarbital and valproate against pentetrazole-induced seizures in mice Jarogniew J. uszczki 1, Marek Zuchora 1, Katarzyna M. Sawicka 1, Justyna Koziñska 1, Stanis³aw J. Czuczwar 1,2 Department of Pathophysiology, Medical University of Lublin, Jaczewskiego 8, PL Lublin, Poland Department of Physiopathology, Institute of Agricultural Medicine, Jaczewskiego 2, PL Lublin, Poland Correspondence: Jarogniew J. uszczki, Abstract: This study examines the effect of acute administration of caffeine sodium benzoate (CAF) on the anticonvulsant action of four conventional antiepileptic drugs (AEDs: clonazepam CZP, ethosuximide ETS, phenobarbital PB and valproate VPA) against pentetrazole (PTZ)-induced clonic seizures in mice. The results indicate that CAF at a dose of 92.4 mg/kg significantly reduced the threshold for PTZ-induced clonic seizures in mice from 69.5 to 51.7 mg/kg (p < 0.05), being ineffective at lower doses of 69.3 and 46.2 mg/kg. Moreover, CAF at doses of 69.3 and 92.4 mg/kg attenuated the protective action of ETS against PTZ-induced seizures, by increasing its median effective dose (ED # ) from to (p < 0.05), and mg/kg (p < 0.01), respectively. In this case, no pharmacokinetic changes in total brain ETS concentrations after systemic ip administration of CAF (at 92.4 mg/kg) were observed, indicating a pharmacodynamic nature of interaction between ETS and CAF in the PTZ-test in mice. In contrast, CAF (at a dose of 92.4 mg/kg reducing the threshold for PTZ-induced seizures) combined with other AEDs (CZP, PB and VPA) did not affect their anticonvulsant action in the PTZ test in mice. Moreover, CAF (92.4 mg/kg) did not alter significantly total brain concentrations of the remaining AEDs (CZP, PB and VPA). The evaluation of potential acute adverse effects produced by AEDs in combination with CAF revealed that neither CAF (up to 92.4 mg/kg) administered alone nor combined with the studied drugs (at doses corresponding to their ED # values in the PTZ-test) affected motor performance of animals in the chimney test. In conclusion, the acute exposure to CAF may diminish the antiseizure protection offered by ETS in epileptic patients. Therefore, patients treated with ETS should avoid CAF. Key words: caffeine, ethosuximide, antiepileptic drugs, pentetrazole, pharmacodynamic interaction Abbreviations: AED antiepileptic drug, CAF caffeine, CZP clonazepam, DZP diazepam, ETS ethosuximide, FPIA fluorescence polarization immunoassay, PB phenobarbital, PTZ pentetrazole, VPA valproate Introduction Overwhelming evidence indicates that caffeine (CAF; 1,3,7-trimethylxantine) has a convulsant action, lowering dose-dependently the seizure threshold in rodents challenged with electrically and chemically induced convulsions [7, 10, 26]. Systemic administration of CAF at doses of mg/kg produced the clonic seizures in mice [25]. Behavioral studies have demonstrated that pretreatment of mice with CAF increased the incidence of tonic convulsions induced by pentetrazole (PTZ) [26], lowered the threshold for electroconvulsions in mice [7, 10], and exerted the proconvulsant activity in the amygdala kindling model of partial seizures [1]. Relatively recently, two clinical re- 652 Pharmacological Reports, 2006, 58,

2 Caffeine and conventional AEDs in the PTZ-test Jarogniew J. uszczki et al. ports have documented that cumulative doses of CAF and methylxanthines from the ingested energizing drinks (coffee, tea, etc.) dramatically increased seizure frequency in patients with epilepsy [3, 14]. Although the precise molecular mechanism of the proconvulsant action of CAF remains unclear, this psychoactive compound at micromolar concentrations possesses adenosine receptor antagonizing properties, being a non-specific adenosine A 1 and A 2A receptor antagonist. Moreover, CAF (at millimolar concentrations) due to its phosphodiesterase inhibiting and Ca 2+ releasing properties elicits an increase in camp content and protein kinase A activity in neurons, which may also be responsible for its proconvulsant activity in vivo [4, 9, 28]. Animal studies have previously shown that CAF antagonized the anticonvulsant activity of diazepam (DZP) against PTZ-induced seizures in Wistar rats [27], and inversely, the administration of DZP inhibited the potentiating activities of CAF on PTZ-induced tonic, but not clonic convulsions in ICR mice [13]. Moreover, it has been found that CAF (administered at a convulsive dose of 200 mg/kg) had no effect on the antiseizure activity of ethosuximide (ETS) and valproate (VPA), but significantly reduced the anticonvulsant effects of DZP and phenobarbital (PB) against PTZ-induced clonic seizures in rats [15]. The aim of this study was to determine the effect of acute administration of CAF on the anticonvulsant activity of four conventional antiepileptic drugs (AEDs: clonazepam CZP, ETS, PB and VPA) in PTZ-induced clonic seizures in mice. It is important to note that the PTZinduced clonic seizures in rodents are thought to be an animal model of myoclonic convulsions and, to a certain extent, of absence petit mal seizures in humans [18, 19]. Moreover, potential acute adverse effects produced by AEDs in combination with CAF were evaluated in the chimney test (motor coordination). To ascertain any pharmacokinetic contribution to the observed interactions between CAF and conventional AEDs in PTZ test, the total brain concentrations of all AEDs studied were measured by fluorescence polarization immunoassay (FPIA) method. Materials and Methods Animals and experimental conditions All experiments were performed on male Swiss mice, kept in colony cages with free access to food and tap water under standardized housing conditions (12 h a light-dark cycle, temperature was 22 ± 1 C; relative humidity of 55 ± 5%). After 4 days of adaptation to laboratory conditions, the animals were randomly assigned to experimental groups consisting of 8 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 conformity with current European Community 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 manuscript were approved by the Local Ethics Committee at the Medical University of Lublin (License no. 534/2005/569/2005), and conformed to the Guide for the Care and Use of Laboratory Animals [29]. Drugs The following drugs were used in this study: CAF (caffeine-sodium benzoate [50:50 CAF/benzoate] molecular weight = 194 (caffeine) (sodium benzoate) = 338 g/mol; Sigma, St. Louis, MO, USA), CZP (Polfa, Warszawa, Poland), ETS (Sigma, St. Louis, MO, USA), PB (Polfa, Kraków, Poland), and VPA (magnesium salt, kindly donated by ICN-Polfa S.A., Rzeszów, Poland). All drugs, except for CAF and VPA, were suspended in a 1% aqueous solution of Tween 80 (Sigma, St. Louis, MO, USA), whereas CAF and VPA were directly dissolved in saline. All drugs were injected intraperitoneally (ip) in a volume of ml/g of body weight. Fresh drug solutions were prepared on each day of experimentation and administered as follows: PB 60 min, ETS 45 min, CAF and VPA 30 min, and CZP 20 min before seizure and motor coordination tests, as well as, before brain sampling for the measurement of AED concentrations. These pretreatment times with the AEDs before testing were based upon information about their biological activity from the literature and our previous experiment [24]. PTZ (Sigma, St. Louis, MO, USA) was dissolved in distilled water and administered subcutaneously (sc) into a loose fold of the skin in the midline of the neck in a volume of ml/g. Doses of CAF tested in this study were in the range from 46.2 mg/kg (0.238 mmol/kg) to 92.4 mg/kg (0.476 mmol/kg). They were based on previous experiments evaluating the effects of acute and chronic Pharmacological Reports, 2006, 58,

3 (for 14 days) treatments with CAF on the antielectroshock activity of PB and VPA in mice [10]. Pentetrazole-induced convulsions Clonic convulsions were induced in mice by the sc administration of PTZ at doses that ranged between 50 and 110 mg/kg. Following the injection of PTZ, the mice were placed separately in transparent Plexiglas cages (25 x 15 x 10 cm) and observed for 30 min for the occurrence of clonic seizures. The 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 convulsive action of PTZ was evaluated as CD 50 (median convulsive dose of PTZ inducing the seizure response in 50% of mice) and CD 97 (convulsive dose 97, i.e., the dose of PTZ that produced clonic seizures in 97% of mice) values. In order to determine the CD 50 and CD 97 values, four or five different doses of PTZ were administered to animals (8 mice per group). Subsequently, a doseresponse relationship curve was calculated from the percentage of mice convulsing according to the logprobit method [8, 17]. The threshold for clonic convulsions was determined for control animals after sc injection of increasing doses of PTZ that ranged between 50 and 110 mg/kg. Subsequently, following ip administration of a constant dose of CAF (46.2 mg/kg; 30 min before PTZ administration) to 4 groups of animals (8 mice per group), increasing doses of PTZ ( mg/kg) were administered sc and the threshold for clonic convulsions was determined. Additionally, increasing doses of CAF (i.e., 69.3 and 92.4 mg/kg) were administered and animals were subjected to the threshold evaluation procedure. The anticonvulsant activities of CZP, ETS, PB and VPA against the PTZ-induced clonic seizures were determined after sc administration of PTZ at its CD 97 (93 mg/kg). The animals were administered 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 [8, 17]. Similarly, the anticonvulsant activity of a mixture of CAF with an AED was evaluated and expressed as ED 50, corresponding to the dose of an AED required to protect 50% of the tested animals against PTZ-induced clonic convulsions. The PTZ-induced clonic seizures have been described in more detail in our earlier reports [22, 24]. Measurement of total brain AED concentrations The animals were administered an AED + vehicle or a combination of CAF (at 92.4 mg/kg) with the respective dose of an AED form the PTZ test. Mice were killed by decapitation at times chosen to coincide with that scheduled for the PTZ test and the whole brains of mice were removed from skulls, weighed, and homogenized using Abbott buffer (2:1 v/w) in an Ultra-Turrax T8 homogenizer (IKA, Staufen, Germany). The homogenates were centrifuged at 10,000 g for 10 min and supernatant samples of 75 µl were put into Abbott system cartridges and placed into a carousel for up to 20 samples. Control samples of a conventional AED were placed at the beginning and end of each carousel for verification of the calibration. The total brain AED concentrations were analyzed by FPIA using a TDx analyzer and reagents exactly as described by the manufacturer (Abbott Laboratories, North Chicago, IL, USA). In case of the estimation of CZP concentrations, an original Abbott reagent for Benzodiazepine was used. The AEDs analyzed were CZP, ETS, PB and VPA and total brain concentrations were expressed in µg/ml (except for brain CZP concentrations expressed in ng/ml) of brain supernatants as the means ± SD of at least 8 separate determinations. Chimney test The effects of CAF alone or in combinations with CZP, ETS, PB and VPA (at doses corresponding to their ED 50 values from the PTZ-test) on motor performance impairment were quantified with the chimney test of Boissier et al. [2]. In this test, animals had to climb backwards up a plastic tube (3 cm inner diameter, 25 cm long), and motor impairment was indicated by the inability of the animals to climb backwards up the transparent tube within 60 s. Statistics Both CD 50 and ED 50 values with their 95% confidence limits were calculated and statistically analyzed by computer-assisted log-probit analysis [8, 17] fol- 654 Pharmacological Reports, 2006, 58,

4 Caffeine and conventional AEDs in the PTZ-test Jarogniew J. uszczki et al. lowed by one-way ANOVA with the post-hoc Dunnett s test for multiple comparisons [11, 21]. The experimentally derived 95% confidence limits of CD 50 and ED 50 values were transformed into SE according to the method presented in our previous studies [20, 21, 23]. Total brain AED concentrations administered alone or in combinations with CAF were statistically analyzed using the unpaired Student s t-test. Qualitative variables from the chimney test were compared with Fisher s exact probability test. A p value of at least < 0.05 was accepted as statistically significant. Results Effect of acute administration of CAF on the threshold for PTZ-induced seizures in mice CAF administered ip in a dose dependent manner reduced the threshold for PTZ-induced seizures in mice (Tab. 1). Statistical evaluation of the data revealed that CAF at a dose of 92.4 mg/kg lowered significantly the threshold for PTZ-induced seizures from Tab. 1. Effect of acute administration of caffeine (CAF) upon the threshold for pentetrazole (PTZ) induced seizures in mice Treatment (mg/kg) CD # of PTZ (mg/kg) N SE Control 69.5 ( ) CAF (46.2) 62.4 ( ) CAF (69.3) 57.5 ( ) CAF (92.4) 51.7 ( )* Values are presented as median convulsive doses (CD # ) of PTZ, necessary to induce clonic seizures in 50% of animals tested. CAF was administered ip at 30min prior to the sc administration of PTZ. Calculations of CD # values were performed using a computerassisted log-probit method [8, 17]. The CD # values were statistically analyzed with one-way ANOVA followed by the post-hoc Dunnett s test for multiple comparisons [11, 21]. N total number of animals at those doses of PTZ whose convulsant effects were between 4 and 6 probits. SE standard error of the CD # values. * p < 0.05 vs. control group (saline-treated animals) Effect of acute CAF administration on the anticonvulsant activity of conventional AEDs against PTZ-induced seizures in mice ED 50 of ETS (mg/kg) ** ETS + saline ETS + CAF(92.4) ETS + CAF(69.3) ETS + CAF(46.2) Fig. 1. Effect of caffeine (CAF) on the anticonvulsant activity of ethosuximide (ETS) in the pentetrazole (PTZ)-induced seizures in mice. Columns represent median effective doses of ETS (ED # in mg/kg; with 95% confidence limits as the error bars) protecting 50% of animals against PTZ-induced clonic seizures in mice. Statistical evaluation of the data was performed using log-probit method [8, 17], followed by one-way ANOVA with the post-hoc Dunnett s test for multiple comparisons [11, 21]. ETS was administered ip at 45 min, and CAF at 30min prior to the test. PTZ was administered sc at a dose of 93 mg/kg, which was its CD '% (a dose of PTZ evoking clonic convulsions in 97% of animals). * p < 0.05, and ** p < 0.01 vs. control group (ETS alone-treated animals) CAF administered alone at a dose of 92.4 mg/kg, considerably decreased the antiseizure effects of ETS, increasing by 55% its ED 50 from ( ) mg/kg to ( ) mg/kg (p < 0.01; Fig. 1). Likewise, CAF administered at 69.3 mg/kg markedly reduced the effect of ETS against PTZ-induced seizures, raising by 43% the ED 50 of ETS from ( ) mg/kg to ( ) mg/kg (p < 0.05; Fig. 1). Only, CAF at a dose of 46.2 mg/kg had no significant impact on the antiseizure effect offered by ETS against PTZ-induced seizures, although, in this case, a 16% increase in ED 50 value for ETS was observed (Fig. 1). In contrast, the systemic (ip) administration of CAF (at 92.4 mg/kg) did not affect significantly the antiseizure activities of other AEDs under study (CZP, PB, and VPA; Tab. 2). * 69.5 ( ) mg/kg to 51.7 ( ) mg/kg (p < 0.05; Tab. 1). Similarly, CAF at lower doses of 46.2 and 69.3 mg/kg also reduced the threshold for PTZinduced seizures, although the reduction was moderate and did not attain statistical significance (Tab. 1). Brain AED concentrations CAF administered at a dose of 92.4 mg/kg did not affect significantly total brain concentrations of ETS, PB and VPA (Tab. 4). Since the FPIA technique was Pharmacological Reports, 2006, 58,

5 Tab. 2. Effect of caffeine (CAF) on the anticonvulsant effects of conventional AEDs against PTZ-induced clonic seizures in mice Tab. 4. Influence of CAF on total brain concentrations of conventional AEDs in mice Treatment (mg/kg) ED # (mg/kg) CZP + saline ( ) CZP + CAF (92.4) ( ) PB + saline 11.4 ( ) PB + CAF (92.4) 12.0 ( ) VPA + saline ( ) VPA + CAF (92.4) ( ) Data are presented as median effective doses of AEDs (ED # with 95% confidence limits in parentheses) protecting 50% of animals tested against PTZ-induced clonic seizures in mice. PTZ was administered sc at a dose of 93 mg/kg, which was its CD '% (a dose of PTZ evoking clonic convulsions in 97% of animals). Statistical evaluation of the data was performed using log-probit method [8, 17]. All drugs were administered ip: phenobarbital (PB) 60min, valproate (VPA) and caffeine (CAF) 30min, and clonazepam (CZP) 20min prior to PTZ Tab. 3. Effects of caffeine (CAF), conventional AEDs and their combinations on motor performance in the chimney test in mice Treatment (mg/kg) Brain concentrations (µg/ml) or (ng/ml) CZP (3.7) + vehicle ± 1.85 CZP (3.7) + CAF (92.4) ± 2.01 ETS (198.3) + vehicle ± 13.8 ETS (198.3) + CAF (92.4) ± 18.7 PB (12.0) + vehicle 6.59 ± 0.62 PB (12.0) + CAF (92.4) 6.71 ± 0.58 VPA (169.2) + vehicle ± 9.96 VPA (169.2) + CAF (92.4) ± 8.75 Results are presented as means ± S.D. of at least 8 determinations and expressed as µg/ml (or ng/ml for CZP) of brain supernatants. The drugs were administered ip at times chosen to coincide with those scheduled for the PTZ test: PB at 60min, ETS 45 min, VPA and CAF at 30min, and CZP at 20min before brain sampling. These test times represent the times of maximum anticonvulsant effect of the AEDs. Statistical evaluation of the data was performed with unpaired Student s t-test. CAF caffeine, CZP clonazepam, ETS ethosuximide, PB phenobarbital, VPA valproate Treatment (mg/kg) Motor deficit (%) CAF (92.4) + saline 10 CAF (69.3) + saline 0 ETS (198.3) + saline 0 ETS (198.3) + CAF (92.4) 30 ETS (182.3) + CAF (69.3) 20 ETS (148.5) + CAF (46.2) 20 CZP (0.037) + saline 0 CZP (0.037) + CAF (92.4) 0 PB (12.0) + saline 0 PB (12.0) + CAF (92.4) 0 VPA (169.2) + saline 0 VPA (169.2) + CAF (92.4) 20 Data are presented as the percentage of animals showing motor impairment in the chimney test. The impairment of motor coordination was indicated as the inability of the mice to climb backwards up the transparent tube within 60s. Motor deficits were calculated from the groups of 10mice. Statistical analysis of the data using Fisher s exact probability test revealed no significant impairment of motor coordination in mice. For more details consult the legend to Tab. 2 not sensitive enough to detect the concentration of CZP at mg/kg, the drug at the dose of 3.7 mg/kg (i.e., 100-fold higher) was subjected to the FPIA determination. In this case, the total brain CZP concentrations were not changed following ip administration of CAF at a dose of 92.4 mg/kg (Tab. 4). Effects of CAF, conventional AEDs and their combinations on motor performance in the chimney test in mice In the chimney test, ETS (198.3 mg/kg) combined with CAF (92.4 mg/kg) produced a 30% impairment of motor coordination in animals. In this case, three out of 10 mice were unable to perform correctly the test and climb backwards up the plastic tube within 60 s (Tab. 3). Moreover, both drugs administered alone produced no significant motor impairment in the chimney test in mice (Tab. 3). Likewise, none of the remaining AEDs (VPA, CZP and PB) alone and in combination with CAF (92.4 mg/kg) altered significantly motor performance in the chimney test in mice (Tab. 3). Discussion Results presented herein indicate clearly that CAF at 92.4 mg/kg significantly lowered the threshold for clonic convulsions induced by PTZ. CAF at lower doses of 69.3 and 46.2 mg/kg also decreased the threshold for PTZ-induced clonic seizures although the data analyzed with one-way ANOVA did not attain statistical significance. It is important to note that CAF at 92.4 mg/kg had no impact on the threshold for 656 Pharmacological Reports, 2006, 58,

6 Caffeine and conventional AEDs in the PTZ-test Jarogniew J. uszczki et al. electroconvulsions after both acute and chronic (for 14 days) treatments in mice [10]. Moreover, CAF at 92.4 and 69.3 mg/kg significantly reduced the anticonvulsant activity of ETS dose-dependently increasing its ED 50 values against PTZ-induced seizures in mice. In contrast, the anticonvulsant properties of other AEDs under study (CZP, PB and VPA) remained without statistically significant change following the systemic (ip) administration of CAF in the PTZ-test in mice. Interestingly, in adult Wistar rats, it has been reported that CAF (at 200 mg/kg) had no effect on the anticonvulsant action of ETS and VPA, but it significantly reduced the antiseizure action of DZP and PB against PTZ-induced clonic seizures [15]. In the light of this fact, our results obtained in mice are contradictory, especially, with respect to the antiseizure effects of ETS and PB in the PTZ-test. These evident discrepancies may be explained through the inter-species differences (rats vs. mice). Moreover, different doses of CAF used in both studies should be borne in mind. For instance, in our study, the animals were administered CAF at a dose significantly decreasing the threshold for PTZ-induced seizures (92.4 mg/kg), whereas in the study by Kulkarni et al. [15], rats were administered with CAF at a convulsant dose of 200 mg/kg. In our study, PTZ was administered at its CD 97, which was 93 mg/kg, whereas Kulkarni et al. [15] have used PTZ at a dose of 70 mg/kg. Additionally, Kulkarni et al. [15] have evaluated the anticonvulsant effects of AEDs by calculating their ED 100 values, whereas in our study we determined their median effective doses (ED 50 values). From the pharmacological point of view, the calculation of ED 50 values is a standard procedure during preclinical evaluation of drug dose-response effects. Noteworthy, the examination of dose-response relationship curves between drug doses and their resultant pharmacological (biological) effects is based on statistical presumptions concerning normal (Gausian) distribution [8, 27]. Thus, the ED 50 values calculated from the log-probit regression and/or Hill equations are determined in approximation with their corresponding 95% confidence limits. On the other hand, the calculation of 100% effective dose (ED 100 ) has practically no sense because according to the normal distribution equation the ED 100 value tends towards infinity (+ ) [8, 27]. In the light of this fact, the results presented by Kulkarni et al. [15] cannot be compared directly with those presented in our study. Tab. 5. Caffeine (CAF) content of popular drinks and beverages Drinks CAF (mg/240 ml)* SOFT DRINKS Red Bull 80.0 Socko 80.0 SoBe No Fear 79.0 KMX Orange 75.0 SoBe Adrenaline Rush 75.0 Rockstar 75.0 Tab Energy 72.4 XTC Power Drink 70.0 Pepsi 55.5 Coke 45.6 Shasta Cola 44.4 Dr. Pepper 41.0 Mr. Pibb 40.0 Pepsi-Cola 37.5 Cherry Coke 34.0 Coca-Cola 34.0 Sprite 0 7-Up 0 Fresca 0 OTHER BEVERAGES Coffee, espresso 400 Coffee, drip Coffee, brewed Coffee, instant Tea, black 60 Tea, iced 47 Dannon Coffee Flavored Yogurt 45 Lipton Tea Nestea Iced Tea 34 Tea, green 30 Slim-Fast Cappuccino Shake 26.6 Hot cocoa 14 Coffee, decaffeinated, brewed 3 4 Coffee, decaffeinated, instant 2 3 Detailed lists of products containing CAF one can find in the Internet. For more details consult: ; ; *CAF content was originally expressed in mg per (U.S.) ounces, however, in this table, ounces have been converted to ml Pharmacological Reports, 2006, 58,

7 Moreover, it has been reported previously that CAF antagonized the effect of DZP on PTZ-induced tonic seizures in mice, having simultaneously no impact on PTZ-induced clonic seizures in mice [13]. Our observation, showing that CAF did not alter significantly the anticonvulsant activity of CZP in PTZ-induced clonic seizures in mice seems to confirm these findings. There were no significant changes in the antiseizure effects produced by DZP and CZP in mice after co-administration of CAF with PTZ. It is worth noting that the studied AEDs (at doses corresponding to their ED 50 values in the PTZ-test) in combination with CAF did not affect motor coordination in the chimney test in mice. Moreover, lack of any significant changes in total brain AED concentrations revealed that the observed interactions between CAF and conventional AEDs in the PTZ-test were pharmacodynamic in nature. On the other hand, we did not entirely exclude the existence of an interaction between CAF and PTZ, since neither CAF nor PTZ concentrations were measured in the brains of experimental animals. Nevertheless, considering the effects produced by CZP, PB and VPA in combination with CAF and PTZ, one can ascertain that the appearance of any pharmacokinetic changes in the brain CAF and PTZ concentrations is unlikely, since the combination of CAF with PTZ had no impact on the anticonvulsant action of these AEDs. Noticeably, Kulkarni et al. [15] did not verify pharmacokinetically plasma or brain AED concentrations in experimental animals. Therefore, no correct conclusions about the exact characteristics of interactions between CAF and conventional AEDs in rats could be formed from their study. One can try to explain the antagonistic pharmacodynamic interaction between CAF and ETS, by considering molecular mechanisms of action of both drugs. With respect to the ETS s mechanisms of action, the experimental evidence indicates that the drug preferentially binds to the inactivated state of lowthreshold T-type Ca 2+ channels and selectively inhibits the pathological firing without any effects on normal neuronal activity [5, 12]. Moreover, it has been found that ETS decreases the Ca 2+ -activated K + current in thalamo-cortical neurons [5], and partially reduces the non-inactivating Na + current [16]. All these changes in Na +,K +, and Ca 2+ currents following the ETS administration are responsible for disrupting thalamo-cortical synchronized activity of neurons during spike and wave discharges in vivo [6]. As already mentioned, CAF at micromolar concentrations possesses adenosine receptor antagonizing properties, being a non-specific adenosine A 1 and A 2A receptor antagonist. Moreover, CAF (at millimolar concentrations) due to its phosphodiesterase inhibiting and Ca 2+ releasing properties increases camp content and A-kinase activity in neurons providing its proconvulsant activity in vivo [4, 9, 26]. Hence, taking into account molecular mechanisms of action of both drugs, CAF and ESM, it is difficult to explain the observed antagonistic interaction between these drugs in the PTZ-test. One can hypothesize that CAF by releasing the intracellular Ca 2+ from neurons was able to remove partially the blockade of low-threshold T-type Ca 2+ channels in the thalamo-cortical neurons evoked by ETS. Thus, the latter drug could not offer full protection against PTZ-induced seizures, and higher doses of ETS were required to provide the protection in 50% of animals in the PTZ test. Although this working hypothesis may plausibly explain the antagonistic interaction between CAF and ETS, the existence of other molecular mechanisms, underlying this interaction, should be borne in mind. To elucidate the exact nature of interaction between CAF and ETS, further more advanced neurochemical and electrophysiological studies are required. In conclusion, the results of this experimental study indicate that the acute administration of CAF antagonized the antiseizure effects of ETS on PTZ-induced clonic seizures in mice. If the experimental data can be extrapolated into clinical settings, our results suggest that epileptic patients medicated with ETS (especially, children and adolescents) should avoid CAFcontaining beverages (Tab. 5), because CAF may diminish the antiseizure protection offered by ETS, and thus, it may contribute to the generation of seizure attacks in patients after ingestion of CAF. Acknowledgments: This study was supported by a grant from the Medical University of Lublin. The kind gift of valproate magnesium from ICN-Polfa Rzeszów S.A. (Poland) is greatly appreciated. References: 1. Albertson TE, Joy RM, Stark LG: Caffeine modification of kindled amygdaloid seizures. Pharmacol Biochem Behav, 1983, 19, Pharmacological Reports, 2006, 58,

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