ALLOPURINOL DOES NOT AFFECT THE ANTICONVULSANT ACTIVITY OF CARBAMAZEPINE AND VALPROATE IN MAXIMAL ELECTROSHOCK-INDUCED CONVULSIONS IN MICE

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1 Coyright 2004 by Institute of Pharmacology Polish Academy of Sciences Polish Journal of Pharmacology Pol. J. Pharmacol., 2004, 56, 6772 ISSN ALLOPURINOL DOES NOT AFFECT THE ANTICONVULSANT ACTIVITY OF CARBAMAZEPINE AND VALPROATE IN MAXIMAL ELECTROSHOCK-INDUCED CONVULSIO IN MICE Jolanta Parada-Turska 1,#, Miros³aw Czuczwar 2, Jacek Kiœ 2, Piotr Czuczwar 2, Anna Cioczek 2, Jarogniew uszczki 3, Stanis³aw J. Czuczwar 3,4 Deartment of Rheumatology, Deartment of Pharmacology and Toxicology,! Deartment of Pathohysiology, Medical University, Jaczewskiego 8, PL Lublin, " Isotoe Laboratory, Institute of Agricultural Medicine, Jaczewskiego 2, PL Lublin, Poland Allourinol does not affect the anticonvulsant activity of carbamazeine and valroate in maximal electroshock-induced convulsions in mice. J. PARADA-TURSKA, M. CZUCZWAR, J. KIŒ, P. CZUCZWAR, A. CIO- CZEK, J. USZCZKI, S.J. CZUCZWAR. Pol. J. Pharmacol., 2004, 56, Allourinol, an inhibitor of xanthine oxidase, is indicated in the management of atients with elevated serum and urinary uric acid levels. It was also reorted to be beneficial in atients with eilesy when added to traditional antieiletic drug. Here, we investigated the effect of allourinol uon the electrical seizure threshold and its effect on the rotective efficacy of common antieiletic drugs, carbamazeine (CBZ) and valroate (VPA) against maximal electroshock (MES)-induced convulsions in mice. We found that allourinol administered at doses of 5, 15 or 45 mg/kg, did not affect electrical seizure threshold. When administered acutely or for a rolonged eriod of time (5 times every 24 h), it did not affect anticonvulsant activity of CBZ and VPA in MES. Free lasma concentration of both anticonvulsants was not affected by allourinol given at a dose of 45 mg/kg for 5 days. Thus, our results did not suort suggestions that allourinol can be beneficial as add-on drug in the management of eilesy at least in atients treated with CBZ or VPA. Key words: Allourinol, carbamazeine, valroate, seizures, mice corresondence; jturska@askleios.am.lublin.l

2 J. Parada-Turska, M. Czuczwar, J. Kiœ, P. Czuczwar, A. Cioczek, J. uszczki, S.J. Czuczwar Abbreviations: CBZ carbamazeine, CS # current strength roducing tonic hindlimb extension in 50% of the tested animals, ED # anticonvulsant dose effective in 50% of the tested animals, MES maximal electroshock, VPA valroate INTRODUCTION Allourinol is an inhibitor of xanthine oxidase, the enzyme resonsible for the conversion of hyoxanthine to xanthine and of xanthine to uric acid, the end roduct of urine metabolism in man. Hyeruricemia may be rimary, as in gout, or secondary to diseases, such as acute and chronic leukemia, olycythemia vera, multile myeloma, and soriasis. It may occur due to the use of diuretic agents, during renal dialysis, in the resence of renal damage, during starvation or weight reducing diets. It may occur also in the treatment of neolastic disease with raid resolution of tissue masses. Due to its mechanism of action, allourinol is indicated in the rohylaxis of gout, uric acid and calcium oxalate renal stones as well as rohylaxis of hyeruricemia associated with cancer chemotheray [for reviews see: 1, 3, 14]. Interestingly, allourinol was reorted to be beneficial in atients with eilesy when added to traditional antieiletic drug [6, 16, 18]. However, the lack of clinical efficacy of allourinol as an add-on theray was also described [4, 15]. Anticonvulsant activity of allourinol alone was found also in animal models of convulsions, such as enicillin-induced eiletogenic foci in the rat hiocamus [11], hiocamal-kindled seizures in cats [17], oxygen-induced seizures in mice [7] and in eiletic mutant EL mice [12]. To the best of our knowledge, the interaction of allourinol and antieiletic drugs has never been studied in animal models of eilesy before. This study was devoted to investigate the effect of allourinol uon the electrical seizure threshold and its effect uon the rotective efficacy of conventional antieiletic drugs, carbamazeine (CBZ) and valroate (VPA) against maximal electroshock (MES)-induced convulsions in mice. MATERIALS and METHODS General The exeriments, after accetance by Local Bioethics Committee, were conducted on adult male Swiss mice weighing 2024 g. Each exerimental grou consisted of at least 7 animals. Drugs The following substances were used: allourinol (Sigma, St. Louis, USA), carbamazeine (CBZ; Polfa, Starogard Gdañski, Poland), valroate magnesium (VPA; ICN Polfa, Rzeszów, Poland). VPA was dissolved in sterile saline. Allourinol and CBZ were susended in a 1% solution of Tween 81 (Loba-Chemie, Vienna, Austria). All drugs were administered intraeritoneally (i) in a volume of 0.1 ml/10 g of body weight at times of their eak activity. Body weight The mice were weighted every 24 h on the laboratory scale for mice with accuracy of 0.1 g. Data were resented as means ± SD; grous consisted of 48 animals. Chimney test The effect of allourinol on motor erformance was evaluated with the chimney test of Boissier et al. [2]. Motor imairment was indicated by the inability of mice to climb backwards u the lastic tube (3 cm i.d., 25 cm length) within 60 s. Electroconvulsions The convulsive threshold was evaluated as CS 50, which is a current strength (in ma) necessary to roduce tonic hindlimb extension in 50% of the tested animals. In order to estimate the threshold, at least four grous of mice were challenged with electroshocks of various intensities. Subsequently, an intensity-resonse curve was calculated on the basis of the ercentage of mice convulsing. CS 50 values with resective 95% confidence limits were calculated. In order to estimate the anticonvulsant ED 50 values (anticonvulsant doses effective in 50% of the tested animals), mice were retreated with different doses of CBZ and VPA and then challenged with MES (25 ma, 50 Hz, 0.2 s) delivered by a Hugo Sachs generator with the use of ear-cli electrodes. CBZ was administered 30 min, and VPA 15 min before MES. ED 50 values with 95% confidence limits were calculated. In order to evaluate the resective ED 50 values, at least four grous of mice, after receiving rogressive doses of the anticonvulsant, 68 Pol. J. Pharmacol., 2004, 56, 6772

3 ALLOPURINOL AND ANTIEPILEPTIC DRUGS were challenged. A dose-effect curve was subsequently constructed on the basis of the ercentage of animals rotected against the convulsions. ED 50 and CS 50 values were calculated according to Litchfield and Wilcoxon [9]. Free lasma concentration of antieiletic drugs Mice were decaitated at times scheduled for the convulsive test. Blood samles were collected and centrifuged at rm for 5 min. Plasma samles of 200 l were transferred to microartition system, MPS-1 (Amicon, Danvers, MA, USA), and centrifuged at 3000 rm for 10 min. Filtrates (50 l) free of rotein-bound microsolutes were ietted into original Abbott system cartridges and the free lasma concentrations of antieiletic drugs were estimated by immunofluorescence, with an Abbott TDx analyzer (Abbott, Irving, TX, USA). Plasma concentrations were exressed in g/ml as means ± SD of at least seven determinations. Statistics Both CS 50 and ED 50 values and their statistical comarisons were calculated by comuter robit analysis, according to Litchfield and Wilcoxon [9]. Body weight data were statistically verified by two-way analysis of variance (ANOVA), followed by the Dunnett s ost-hoc test. Free lasma concentration data were comared by unaired Student s t-test. At least < 0.05 was considered significant. RESULTS Effect of allourinol on body weight and motor erformance Allourinol administered for 5 days at the dose of 45 mg/kg, i, significantly reduced body weight gain. The drug administered at doses of 5 and 15 mg/kg for 5 days was without effect on this arameter (Tab. 1). Allourinol (545 mg/kg) did not affect motor erformance of mice as determined in chimney test (data not shown). Effect of allourinol uon the electrical seizure threshold in mice Allourinol administered at doses of 5, 15 and 45 mg/kg, i, 30 min before electroconvulsions did not affect seizure threshold in mice (Tab. 2). Table 1. Effect of allourinol on the body weight of mice Treatment [mg/kg] Vehicle ± 2.05 Allourinol ± 2.29 Allourinol ± 2.49 Allourinol ± 2.27 Body weight ± SD [g] 1st day 2nd day 3rd day 4th day 5th day ± ± ± ± 2.35* ± ± ± ± 2.18* ± ± ± ± 2.69* Table 2. Effect of allourinol uon the electrical seizure threshold in mice: dose-deendency Treatment [mg/kg] CS # with 95% confidence limits [ma] ± ± ± ± 2.46* Allourinol was administered i at doses of 5, 15 and 45 mg/kg, every 24 h for 5 consecutive days. Data reresent means ± SD. Grous consisted of 48 mice. * at least < 0.05 vs. vehicletreated grou (ANOVA followed by Dunnett s ost-hoc test) Vehicle 8.4 (7.99.0) Allourinol (7.58.7) Allourinol (8.08.8) Allourinol (7.58.6) Allourinol was administered at doses of 5, 15 and 45 mg/kg, i, 30 min before the test. The CS # value (with 95% confidence limits), which is a current strength necessary to roduce convulsions in 50% of the tested animals was calculated by fitting the data by comuterized robit analysis based on the method of Litchfield and Wilcoxon [9]; value of at least < 0.05 was considered statistically significant. not significant Allourinol (15 mg/kg) administered 30, 60 and 120 min before electroconvulsions also did not influence seizure threshold in mice (Tab. 3). Effect of allourinol uon the rotective efficacy of carbamazeine and valroate Allourinol administered i at the dose of 45 mg/kg, 30 min before MES, did not affect the anticonvulsant activity of neither CBZ nor VPA (Tab. 4). Further, when administered every 24 h for 5 consecutive days at doses of 5, 15 and 45 mg/kg, it did not influence the anticonvulsant roerties of both CBZ and VPA (Tab. 4). ISSN

4 J. Parada-Turska, M. Czuczwar, J. Kiœ, P. Czuczwar, A. Cioczek, J. uszczki, S.J. Czuczwar Table 3. Effect of allourinol uon the electrical seizure threshold in mice: time-deendency Time [min] CS # with 95% confidence limits [ma] Vehicle (7.07.8) Allourinol (7.88.7) Allourinol (7.77.9) Allourinol (7.78.6) Allourinol was administered at the dose of 15 mg/kg, i, 30, 60 and 120 min before the test. The CS # value (with 95% confidence limits), which is a current strength necessary to roduce convulsions in 50% of the tested animals, was calculated by fitting the data by comuterized robit analysis based on the method of Litchfield and Wilcoxon [9]; value of at least < 0.05 was considered statistically significant. not significant Table 4. Effect of allourinol uon the rotective efficacy of carbamazeine (CBZ) and valroate (VPA) against maximal electroshock (MES)-induced convulsions in mice Treatment CBZ Allourinol 1 45mg/kg + CBZ CBZ Allourinol 5 5mg/kg + CBZ Allourinol 5 15mg/kg + CBZ Allourinol 5 45mg/kg + CBZ VPA Allourinol 1 45mg/kg + VPA VPA Allourinol 5 5mg/kg + VPA Allourinol 5 15mg/kg + VPA Allourinol 5 45mg/kg + VPA ED # of antieiletic drug with 95% confidence limits [mg/kg] 9.6 ( ) 9.2 ( ) 10.1 ( ) 9.7 ( ) 9.2 ( ) 11.2 ( ) ( ) ( ) ( ) ( ) ( ) ( ) Effect of allourinol uon free lasma concentration of carbamazeine and valroate Allourinol administered at doses of 5, 15 and 45 mg/kg, i, every 24 h for 5 consecutive days did not affect the free lasma concentration of CBZ and VPA in mice (Tab. 5). - - Allourinol was administered i, once (1 ) or once daily for 5 consecutive days (5 ). The last injection of allourinol was given 30 min before MES. The ED # value (with 95% confidence limits), which is anticonvulsant dose effective in 50% of the tested animals was calculated by fitting the data by comuterized robit analysis based on the method of Litchfield and Wilcoxon [9]; value of at least < 0.05 was considered statistically significant. CBZ carbamazeine; VPA valroate; not significant Table 5. Effect of allourinol uon free lasma concentration of carbamazeine (CBZ) and valroate (VPA) in mice Treatment Vehicle + CBZ Allourinol 5 45mg/kg + CBZ Vehicle + VPA Allourinol 5 45mg/kg + VPA DISCUSSION Drug lasma concentrations [ g/ml] 2.62 ± ± ± ± 6.48 We found that allourinol administered at doses of 5, 15 or 45 mg/kg did not affect electrical seizure threshold in mice. However, the anticonvulsant effect of allourinol was reorted in other animal models of seizures. Wada et al. [17] assessed the effect of allourinol administered at doses of 5 and 50 mg/kg, i, on seizures kindled from the feline hiocamus and observed that allourinol at the higher dose significantly reduced the behavioral seizure stage, but not afterdischarge duration. Exeriments erformed on rats with electrodes imlanted into the dorsal art of the left hiocamus showed that allourinol injected i at 25 or 50 mg/kg decreased the activity of the enicillininduced eiletogenic foci [11]. Prolonged exosure to hyerbaric oxygen has toxic effect on the central nervous system manifested by grand mal seizures. It was shown that allourinol delayed the onset of oxygen-induced seizures in mice [7]. More recently, antieiletic effect of allourinol was reorted in EL mice, a mutant model of secondarily generalized seizures. In that study, allourinol was chronically given er os at the dose of 75 mg/kg [12]. The mechanism of antieiletic activity of allourinol is not clear, however, its effect on the rate of sueroxide formation was usually considered [7, 12]. In this study, we investigated the effect of allourinol uon the anticonvulsant activity of antieiletics in MES in mice. MES-induced seizures in Allourinol was administered i at dose of 45 mg/kg, i, every 24 h for 5 consecutive days (5 ). The last injection of allourinol was given 30 min before blood collection. Data reresent means ± SD. Grous consisted of 7 mice. Significance was acceted at < 0.05 vs. resective vehicle-treated grou (Student s t-test). CBZ carbamazeine; VPA valroate; not significant 70 Pol. J. Pharmacol., 2004, 56, 6772

5 ALLOPURINOL AND ANTIEPILEPTIC DRUGS rodents rovide a reliable model of generalized tonic-clonic convulsions [10]. Moreover, this exerimental model was roved to be suitable for the evaluation of anticonvulsant efficacy of drugs used in combination [5]. We found that allourinol administered acutely or for 5 days did not influence the activity of both commonly used antieiletics, CBZ and VPA. Neither dose nor route of administration seems to be resonsible for the lack of anticonvulsant activity of allourinol in our study. The drug was administered i acutely or for 5 days at doses of 5, 15 and 45 mg/kg. Similar doses were used in other studies in which anticonvulsant effect was observed [11, 17]. Moreover, the highest used dose of 45 mg/kg significantly reduced body weight gain in mice, suggesting that this dose was high enough to roduce biological effect in animals. Recently, reduction of body weight due to allourinol administration was reorted in chicken [8]. Interestingly, there are numerous reorts showing that allourinol is beneficial in atients with eilesy when added to a traditional antieiletic drug. In a double-blind, randomized, lacebocontrolled, cross-over study erformed in 80 atients with eiletic seizures refractory to standard antieiletic drugs, allourinol administered at doses of mg/kg significantly reduced total and secondarily generalized seizures by 10.5 and 27.9%, resectively [18]. In another study conducted by Tada et al. [16], allourinol was used as add-on theray in 31 atients with intractable eilesy. Its administration for a short time was beneficial in 17 atients, 8 atients were seizure-free, 8 had 75% decrease in seizure frequency, and 1 had greater than 50% decrease. Allourinol was found to be most effective in atients with localizationrelated eilesy, esecially in secondarily generalized tonic-clonic seizures. Following 1 year long follow-u, its effectiveness still continued in 8 of 14 atients who exhibited initial imrovement [16]. In 38 children aged from 4 months to 10 years with rogressive history of the disease, frequent severe seizures and ineffective conventional antieiletic drug treatment, allourinol given at daily doses of 4 to 5 mg/kg, was reorted to have ositive effects in 10 of 28 atients [6]. On the other hand, Sander and Patsalos [15] assessed the effect of allourinol in a 12 week add-on uncontrolled study in a series of 12 atients with intractable eilesy. They observed weekly seizure frequencies to be reduced in 4 atients and increased in 5 atients. Since these changes were not significant it was concluded that differences were robably attributed to the random variation of seizures [15]. Similar conclusion was drawn by Coola and Pascotto [4] on the basis of a doubleblind, lacebo-controlled cross-over trial with allourinol as add-on theray in childhood refractory eilesy conducted on 17 atients, receiving allourinol and matched lacebo for 12 weeks at 2 doses, 10 mg/kg/day during the first week and 15 mg/kg/day thereafter, with a washout eriod of 2 weeks between treatment hases. The total number of seizures was reduced by 5098% in 4 atients (23.5%) and by 2549% in another 4 (23.5%). However, the number of seizures remained unchanged in 5 atients (29.4%) and worsened in 4 (23.5%). Adverse side effects were generally mild and transient, suggesting that allourinol is well-tolerated. The final conclusion of this study was, that a mean follow-u of 10 months of the resonders did not show any relevant efficacy of allourinol as an adjuvant theray for refractory eilesy, even at high doses [4]. Allourinol did not affect free lasma concentration of CBZ and VPA in mice. Similarly, no influence of allourinol on lasma concentration of antieiletic drugs was reorted in humans [18]. On the other hand, it was found that the reeated administration of allourinol at doses of 20 and 50 mg/kg significantly retarded the elimination of henytoin from the circulation and dramatically decreased the urinary excretion of -hydroxy-henytoin, a major metabolite of henytoin. However, a single administration of allourinol did not give rise to these effects [13]. In conclusion, our results showed that allourinol affected neither electrical seizure threshold nor antieiletic efficacy of CBZ or VPA in maximal electroshock model in mice. Thus, our results do not suort suggestions that allourinol can be useful as add-on drug in the management of eilesy at least in atients treated with CBZ or VPA. Acknowledgment. This study was suorted by a grant No. PW 263/02 from Medical University in Lublin, Poland. REFERENCES 1. Blair B, Fabrizio M: Pharmacology for renal calculi. Exert Oin Pharmacother, 2000, 1, ISSN

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