Copyright © 2003 by Institute of Pharmacology Polish Journal of Pharmacology Polish Academy of Sciences Pol. J. Pharmacol., 2003, 55, 103–107 ISSN 1230-6002

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INFLUENCE OF LY 300164, AN AMPA/KAINATE ANTAGONIST UPON THE ANTICONVULSANT ACTION OF ANTIEPILEPTIC DRUGS AGAINST AMINOPHYLLINE-INDUCED SEIZURES IN MICE

Mariusz Œwi¹der1, Hubert KuŸniar1, Zdzis³aw Kleinrok1 , Stanis³aw J. Czuczwar2,3,# Department of Pharmacology and Toxicology, Department of Pathophysiology, Medical University, Jaczewskiego 8, PL 20-090 Lublin, !Isotope Laboratory, Institute of Agricultural Medicine, Jaczewskiego 2, PL 20-950 Lublin, Poland

Influence of LY 300164, an AMPA/kainate receptor antagonist, upon the anticonvulsant action of antiepileptic drugs against aminophylline-induced seizures in mice. M. ŒWI¥DER, H. KUNIAR, Z. KLEINROK , S.J. CZU- CZWAR. Pol. J. Pharmacol., 2003, 55, 103–107.

LY 300164 {7-acetyl-3-(4-aminophenyl)-8,9-dihydro-8-methyl-7H-1,3- dioxazolo[4,5-h] [2,3]-}, a novel AMPA/kainate receptor an- tagonist, administered intraperitoneally protected mice against aminophyl- line-induced seizures. At doses up to 0.5 mg/kg, which did not significantly affect the convulsant activity of aminophylline, it potentiated the protective activity of . On the other hand, LY 300164 used at the lowest pro- tective dose of 1.0 mg/kg enhanced anticonvulsant activity of all antiepilep- tic drugs tested in this seizure model. However, LY 300164 neither alone nor combined with antiepileptic drugs, reduced aminophylline-induced mortality.

Key words: antiepileptic drugs, LY 300164, aminophylline-induced sei- zures

 correspondence; e-mail: [email protected] M. Œwi¹der, H. KuŸniar, Z. Kleinrok , S.J. Czuczwar

INTRODUCTION were kept under a natural light-dark cycle. The ex- perimental groups, consisting of 10 animals, were The convulsive action of methylxanthines has selected by means of a randomized schedule. All been known for many years [16, 17]. Clinical data experimental procedures were performed between indicate that aminophylline may induce repetitive 9.00 a.m. and 2.00 p.m. Each mouse was used only generalized seizures in asthmatic patients [11, 15]. once. The experimental procedures described be- However, antiepileptic drugs, such as phenobarbi- low were approved by the Ethics Committee of tal, diphenylhydantoin and diazepam, administered Lublin Medical University. at high doses [18] could partially prevent the con- vulsions. Furthermore, experimental data indicate Aminophylline-induced convulsions that other agents such as carbamazepine, ethosuxi- The mice were injected intraperitoneally (ip) mide, diphenylhydantoin, acetazolamide, trimethadi- • with aminophylline (theophylline2 ethylenediami- one, clonidine and amino-oxyacetic acid have been ne) at the dose of 273 mg/kg, which is the experi- found to be completely ineffective against amino- mentally determined CD97 value for the induction phylline-induced seizures and mortality [7]. On the of clonic convulsions. The animals were then put other hand, aminophylline (and other methylxan- into individual perspex plexiglas cages (25 × 15 × thines) sharply reduced the protective efficacy of 10 cm) and observed for the occurrence of clonic common antiepileptics after acute administration and tonic seizures for 60 min. The endpoint for the [5, 7, 8]. Neither an component of clonic phase was a clonic seizure with a loss of the aminophylline nor a pharmacokinetic interaction ap- righting reflex. Immediately at the end of observa- peared to be responsible for the methylxanthine- tion and also after 2 and 24 h, numbers of animals, induced impairment [5, 6] of the anticonvulsant ac- which stayed alive were recorded. The convulsive tion of antiepileptics. Also, the peripheral effects of test was performed in a quiet room. The control aminophylline were excluded as a cause of this ef- animals were injected with aminophylline on each fect since a peripherally active antago- day of the convulsive test, and the results were re- nist, 8-(p-sulfophenyl)-, was completely producible. devoid of any effect on the protective potency of antiepileptic drugs [2]. Drugs LY 300164 {7-acetyl-3-(4-aminophenyl)-8,9-di- The following antiepileptic drugs were used: hydro-8-methyl-7H-1,3-dioxazolo[4,5-h][2,3]-benzo- valproate (Dipromal, Polfa, Rzeszów, diazepine), an antagonist of AMPA/kainate recep- Poland), carbamazepine (Amizepin), diazepam (Re- tors, potentiated the anticonvulsant activity of an- lanium, both antiepileptics from Polfa, Warszawa, tiepileptic drugs, both against seizures evoked by Poland) and LY 300164 {7-acetyl-3-(4-aminophe- maximal electroshock in mice [9] or amygdala kin- nyl)-8,9-dihydro-8-methyl-7H-1,3-dioxazolo[4,5-h]- dling in rats [1]. Therefore, the aim of the present [2,3]-benzodiazepine}, a novel antagonist of AMPA/ study was to examine the influence of LY 300164 kainate receptor (Lilly Res. Lab., Indianapolis, IN, on the protective effects of diazepam, valproate and USA). Valproate and aminophylline were brought carbamazepine against aminophylline-induced sei- into solution with sterile saline whilst diazepam, zures and mortality in mice. Any beneficial interac- carbamazepine and LY 300164 were suspended in tion could be of importance for management of 1% solution of Tween 80 (Sigma, St. Louis, MO, aminophylline overdose. USA). All drugs were administered ip in a volume of MATERIALS and METHODS 0.01 ml/g of body weight. Valproate magnesium, carbamazepine and LY 300164 were injected 15 min Animals and diazepam – 30 min prior to the test. Doses of The experiments were performed on female valproate refer to its free form. Swiss mice weighing 22–27 g. The animals were Statistics housed in colony cages under standard laboratory conditions with free access to chow pellets (Muri- The data were analyzed by Fischer’s exact gran; Bacutil, Motycz, Poland) and tap water. The probability test and ED50 values of antiepileptics experimental temperature was 21 ± 1°C and mice were computed and statistical analysis of the re-

104 Pol. J. Pharmacol., 2003, 55, 103–107 LY300164 AND ANTIEPILEPTIC DRUGS AGAINST AMINOPHYLLINE-INDUCED SEIZURES sults was performed by computer probit analysis, seizures, 36 exhibited the tonic phase and 36 died according to Litchfield and Wilcoxon [10]. within the 60 min observation period.

RESULTS Influence of LY 300164 on the clonic phase of aminophylline-induced seizures Effect of aminophylline at its CD97 upon seizure activity LY 300164 (up to 0.5 mg/kg) remained without effect against aminophylline (273 mg/kg)-induced Aminophylline evoked clonic and tonic sei- clonic seizures in mice. However, LY 300164 (1.0 zures in a dose-dependent manner, and its CD97 for mg/kg) significantly decreased the number of clo- the induction of clonic phase was 273 mg/kg (data nic (Fig. 1) and tonic (result not shown in Figure 1) not shown). The latency to the onset of convulsions convulsions in comparison with the control ani- varied from 15 to 30 min, with a mean ± SE of 25 ± mals. As can be seen from Figure 1, LY 300164 4 min. Mice showed accelerated respiration, occa- showed a graded dose-response effect on the epi- sional tremors, incoordination and mild hyperactiv- sodes of clonic convulsions, and its ED50 value ity prior to the onset of seizures. Out of 40 mice in- against aminophylline-induced seizures was 0.8 jected with aminophylline at CD97, 38 had clonic (0.5–1.2) mg/kg (Fig. 1).

A B

100 100 DZA 75 75 DZA + LY 0.25 DZA + LY 0.5 50 LY 300164 50 DZA + LY 1.0

25 25 % of mice protected % of mice protected 0 0 0 1 2 3 4 5 mg/kg 0.0 2.5 5.0 7.5 mg/kg LY 300164 DZA

C D

100 100

75 CBZ 75 VPA CBZ + LY 0.5 VPA + LY 0.25 50 CBZ + LY 1.0 50 VPA + LY 0.5 VPA + LY 1.0 25 25 % of mice protected % of mice protected 0 0 0 5 10 15 mg/kg 0 100 200 300 mg/kg CBZ VPA

Fig. 1. Effect of LY 300164 upon the protective activity of antiepileptic drugs, was expressed as a percentage of mice protected against aminophylline-induced seizures. The control groups of animals were injected with aminophylline on each day of the convul- sive test. Aminophylline was administered at its CD'% dose (the dose of drug necessary to induce clonic seizures in 97% of animals), which was 273 mg/kg. DZA – diazepam; CBZ – carbamazepine; VPA – valproate magnesium. All drugs were administered ip in volu- me of 0.01 ml/g of body weight, VPA, CBZ and LY 300164 – 15 min, DZA – 30 min prior to the test. The ED#s of DZA + LY 300164 (0.5 mg/kg), DZA + LY 300164 (1.0 mg/kg), VPA + LY 300164 (1.0 mg/kg) were statistically significantly different vs. the respec- tive ED#s of DZA, CBZ, and VPA at least at p < 0.05

ISSN 1230-6002 105 M. Œwi¹der, H. KuŸniar, Z. Kleinrok , S.J. Czuczwar

Influence of LY 300164 upon the protec- seizures in mice [7]. Moreover, novel antiepileptic tive activity of antiepileptic drugs against drugs (e.g. lamotrigine, vigabatrine) did not protect clonic phase of aminophylline-induced mice against aminophylline seizures and some of them could even shorten the latency period to onset seizures and postconvulsive mortality of convulsions [13]. LY 300164 (0.5 mg/kg), when applied together Aminophylline has been shown to produce de- with diazepam, significantly reduced its ED50 value creased cerebral blood flow, respiratory alkalosis, from 3.3 (2.5–4.2) to 1.8 (1.2–2.7) mg/kg. More- systemic hypotension, and cardiac tachyarrhyth- over, LY 300164 (1 mg/kg) co-administered with mia. Such systemic effects undoubtedly are not the diazepam and carbamazepine also potentiated their only factors playing an important role in mice mor- activity [ED50 values were reduced from 3.3 to 1.1 tality following aminophylline administration. In (0.7–1.9) kg/mg and from 7.4 (6.0–9.0) to 3.6 fact, it might be due to the combined effects of di- (2.1–5.9) mg/kg, respectively]. Also, LY 300164 at rect action of methylxanthine on the central nerv- the dose of 1 mg/kg reduced the ED50 value of val- ous system (the earlier onset of seizures or mortal- proate from 185 (160–215) to 139 (113–171) mg/kg, ity in the control groups as trigger mechanism) and being without effect at lower doses (Fig. 1). the accompanying metabolic and cardiovascular Interestingly, the AMPA/kainate receptor an- abnormalities (occurring after some hours) [3]. In- tagonist and antiepileptic drugs reduced the mortal- terestingly, a combination of some conventional ity rate, evaluated after the first and second hour antiepileptics and a b-blocker produced a significant following the injection of the convulsant. Neither protection against aminophylline-induced mortality antiepileptics or the AMPA/KA receptor antagonist [4, 14]. alone nor their combinations influenced this para- In conclusion, our study indicates that in ami- meter when assessed after 24 h (results not shown). nophylline intoxication, the basic problem is not to stop seizure activity, but to overcome long-term mortality. Common antiepileptics and LY 300164 DISCUSSION diminished seizures, however, they were complete- ly ineffective against aminophylline-induced mor- This study has shown that LY 300164 protected tality. Therefore, further studies are needed to fully mice from clonic seizures induced by aminophyl- elucidate the mechanisms of aminophylline epilep- line. Moreover, at the highest dose of 1.0 mg/kg, it togenosity and to establish an algorhythm for clini- significantly enhanced the protective activity of the cal treatment of the patients suffering from amino- studied antiepileptics against the clonic phase of phylline intoxication. aminophylline-induced convulsions. The AMPA/KA receptor antagonist reduced ED s of carbamazepine 50 Acknowledgments. The generous gifts of LY 300164 and diazepam by over 50% or valproate by about from Mrs. Margaret H. Niedenthal (Eli-Lilly, Indianapolis, 25%. Similarly, Czuczwar et al. [7] indicated that IN, USA) and valproate magnesium (Polfa, Rzeszów, Po- some NMDA receptor antagonists afforded protec- land) are greatly appreciated. Dr. Mariusz Œwi¹der is a re- tion against aminophylline-induced clonic seizure cipient of the Fellowship for Young Researches awarded by activity. Neither antiepileptic drug tested nor LY The Foundation for Polish Science. This study was sup- 300164, administered alone or combined with an- ported by the grant No. P05F 018 10 from the State Com- mittee for Scientific Research, Warszawa, Poland. tiepileptics, reduced mortality assessed after 24 h. The previous studies indicated poor efficacy of conventional antiepileptic drugs against amino- REFERENCES phylline-induced convulsions in mice [12]. Also, 1. Borowicz K.K., Kleinrok Z., Czuczwar S.J.: The Czuczwar et al. [7] proved that ethosuximide, di- AMPA/kainate receptor antagonist, LY 300164, in- phenylhydantoin, trimethadione and carbamazepine creases the anticonvulsant effects of diazepam. Nau- were totally ineffective in aminophylline-induced nyn-Schmied. Arch. Pharmacol., 2000, 361, 629–635. 2. clonic seizures. However, the present results show Borowicz K.K., Kozicka M., Kleinrok Z., Czuczwar that carbamazepine protected against aminophylli- S.J.: Influence of aminophylline and 8-(p-sulfophe- nyl)theophylline on the anticonvulsive action of di- ne-induced convulsions, but at lower doses. Carba- phenylhydantoin, phenobarbital, and valproate against mazepine used at higher doses (20–50 mg/kg) was maximal electroshock-induced convulsions in mice. totally ineffective against aminophylline-induced J. Neural Transm.-Gen. Sect., 1993, 93, 157–163.

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