Short communication
Influence of N G -nitro-L-arginine
on the anticonvulsant and acute adverse effects of some newer antiepileptic drugs in the maximal electroshock-induced seizures and chimney
test in mice
Jarogniew J. £uszczki1, Miros³aw Czuczwar2, Piotr Gawlik1,
Gra¿yna Sawiniec-PóŸniak1, Katarzyna Czuczwar1, Katarzyna Sawicka1, Monika Dudra-Jastrzêbska3, Stanis³aw J. Czuczwar1,3
Department of Pathophysiology, Medical University of Lublin, Jaczewskiego 8, PL 20-090 Lublin, Poland
Second Department of Anaesthesiology and Intensive Care, Medical University of Lublin, Staszica 1, PL 20-090 Lublin, Poland
!Department of Physiopathology, Institute of Agricultural Medicine, Jaczewskiego 2, PL 20-950 Lublin, Poland
Correspondence:Jarogniew J. £uszczki, e-mail: jarogniew.luszczki@am.lublin.pl
Abstract:
Overwhelming evidence indicates that nitric oxide (NO) plays an important role in epileptogenesis and seizure activity in the brain.
The results of experimental studies on animals provide, however, discrepant information reporting that NO has both anti- and pro-convulsant action in the brain.
The objective of this study was to determine the effect of N/-nitro-L-arginine (L-NA – a non-specific NO synthase inhibitor) on the anticonvulsant and acute adverse-effect profiles of four second-generation antiepileptic drugs (felbamate [FBM], lamotrigine [LTG], oxcarbazepine [OXC] and topiramate [TPM]) in the maximal electroshock (MES)-induced seizure model and the chimney test in mice.
Results indicated that L-NA (at 40 mg/kg,ip) did not affect significantly the antiseizure activity of all examined drugs. However, the antielectroshock action of FBM and LTG after co-administration of L-NA was attenuated by 36% and 28%. In contrast, the anticonvulsant effects of TPM and OXC were almost unchanged after L-NA administration. Moreover, the NO synthase inhibitor (40 mg/kg,ip) did not enhance the acute adverse-effect profiles of the studied antiepileptic drugs in the chimney test.
In conclusion, the observed reduction of the anticonvulsant effects of FBM and LTG after co-administration of L-NA may suggest a pro-convulsant activity of L-NA and the cooperation of NO with the antiseizure properties of FBM and LTG in the MES test in mice.
Key words:
nitric oxide, maximal electroshock, N/-nitro-L-arginine, felbamate, lamotrigine, oxcarbazepine, topiramate
Pharmacological Reports 2006, 58, 955960 ISSN 1734-1140
Copyright © 2006 by Institute of Pharmacology Polish Academy of Sciences
synthase, OXC – oxcarbazepine, PB – phenobarbital, PIs – protective indices, PHT – phenytoin, PTZ – pentetrazole, TPM – topiramate, VPA – valproate
Introduction
Accumulating evidence indicates that nitric oxide (NO), a gaseous molecule possessing neurotransmit- ter/neuromodulator properties in the brain, plays an important role in the pathophysiology of epilepsy, producing both anti- and proconvulsant effects in various experimental seizure models in rodents [8, 20–22, 24]. NO is produced by the oxidation of L-arginine by NO synthase (NOS – a Ca2+/calmodulin- dependent enzyme), existing in three distinct iso- forms: neuronal (nNOS), inducible (iNOS) and endo- thelial (eNOS) [16, 17]. A considerable progress has been made in the examination of the role of NO in the brain during seizures after discovery of synthetic L-arginine analogues and nitroindazole derivatives, inhibit that inhibit the NOS activity in the brain [16, 17]. It is generally accepted that NG-nitro-L-arginine (L-NA – a non-specific NOS inhibitor) reduces the activity of both, eNOS and nNOS, to the same extent, whereas 7-nitroindazole (7-NI) is considered to be a preferential inhibitor of nNOS activity [1, 16].
Experimental evidence indicates that NG-nitro- L-arginine methyl ester (L-NAME; at a subthreshold dose of 40 mg/kg, ip) attenuated the anticonvulsant effects of valproate (VPA) and phenobarbital (PB), but not those of phenytoin (PHT) and carbamazepine (CBZ) in the maximal electroshock (MES)-induced seizures in mice [6]. In contrast, 7-NI (at a subthresh- old dose of 50 mg/kg,ip) potentiated the antiseizure effects of PB, but not those of CBZ, PHT and VPA against MES-induced seizures [4]. Similarly, it was documented that L-NA (40 mg/kg, ip) impaired the anticonvulsant action of ethosuximide (ETS) by in- creasing its ED50 value by 46% in pentetrazole (PTZ)-induced seizures in mice [7], whereas, 7-NI (50 mg/kg, ip) potentiated the antiseizure effects of ETS, which was associated with a 47% reduction of its ED50value in the PTZ test in mice [5]. Moreover, it has also been reported that molsidomine (MOL,
mice. Relatively recently, we have found that 7-NI at a dose of 50 mg/kg enhanced significantly the anti- convulsant activity of oxcarbazepine (OXC) and po- tentiated the antielectroshock action of FBM, lamo- trigine (LTG) and topiramate (TPM) in mice [13].
Hence, considering the above-mentioned facts, we intended to determine the effect of L-NA (at 40 mg/kg, ip) on the antiseizure efficacy of four second-generation antiepileptic drugs (AEDs: FBM, LTG, OXC and TPM) in the MES-induced seizures in mice. Moreover, the acute adverse-effect profiles of AEDs in combination with L-NA were determined in the chimney test.
Materials and Methods
Animals and experimental conditions
All experiments were performed on adult male albino Swiss mice weighing 22–26 g. The mice were kept in colony cages with free access to food and tap water, under standardized housing conditions (natural light- dark cycle, temperature of 21 ± 1°C, relative humidity of 55 ± 3%). After 7 days of adaptation to laboratory conditions, the animals were randomly assigned to experimental groups consisting of 8 mice. Each mouse was used only once. All tests were performed between 9.00 a.m. and 2.00 p.m. Procedures involv- ing animals and their care were conducted in accor- dance with the European Communities Council Directive of 24 November 1986 (86/609/EEC) and Polish legislation on animal experimentation. Addi- tionally, all efforts were made to minimize animal suf- fering and to use only the number of animals neces- sary to produce reliable scientific data. The experi- mental protocols and procedures described above were approved by the Local Ethics Committee at the Medical University of Lublin (License no. 479/2004/
509/2004).
Drugs
The following drugs were used in this study: L-NA (RBI, Natick, MA, USA), FBM (Taloxa; Schering
Plough, Levallois Perret, France), LTG (Lamictal;
Glaxo Wellcome, Kent, UK), OXC (Trileptal; Novar- tis Pharma AG, Basel, Switzerland), and TPM (Topa- max; Cilag AG, Schaffhausen, Switzerland). All drugs were suspended in a 1% aqueous solution of Tween 80 (Sigma, St. Louis, MO, USA) and adminis- tered ip in a volume of 5 ml/kg body weight. Fresh drug solutions were prepared on each day of experi- mentation and administered as follows: FBM, TPM, and LTG at 60 min, and OXC and L-NA at 30 min, before electroconvulsions and the chimney test. These drug administration times were based on their biologi- cal activity from the literature [20] and were confirmed in our previous experiments [7, 12, 15, 25]. The times of peak maximum anticonvulsant effects for AEDs were used as reference times in the chimney test.
Maximal electroshock seizure (MES) test
Maximal electroconvulsions were produced by an al- ternating current (0.2 s stimulus duration, 50 Hz, fixed current intensity of 25 mA, maximum stimula- tion voltage of 500 V) delivered via ear-clip elec- trodes by a generator (Rodent Shocker, Type 221, Hugo Sachs, Freiburg, Germany). The criterion for the occurrence of seizure activity was the tonic hindlimb extension. The protective activities of second-generation AEDs administered separately and in combination with L-NA were evaluated as their median effective doses (ED50in mg/kg with 95% con- fidence limits) against MES-induced seizures. The animals were administered with different drug doses so as to obtain a variable percentage of protection against MES, allowing the construction of a dose- effect curve for the AEDs administered alone or in combination with L-NA, according to Litchfield and Wilcoxon [10]. Each ED50value represents the dose of a drug required to protect 50% of the animals tested against maximal electroconvulsions. The experimen- tal procedure has been described in more detail in our earlier papers [13, 14].
Chimney test
The effects of second-generation AEDs administered singly or in combination with L-NA, on motor im- pairment were quantified with the chimney test of Boissier et al. [3]. In this test, animals had to climb backward up a plastic transparent tube (3 cm inner di-
ameter, 25 cm length). Motor impairment was indi- cated by the inability of the animals to climb back- ward up the tube within 60 s. The acute adverse ef- fects of the AEDs alone or in combination with L-NA were expressed as median toxic doses (TD50in mg/kg with 95% confidence limits), representing the doses, at which the drugs impaired motor coordination in 50% of the animals tested. To evaluate each TD50 value, at least 4 groups of animals (each group con- sisted of 8 mice) injected with various doses of an AED were challenged with the chimney test. A dose- response curve was calculated on the basis of the per- centage of mice showing motor deficits by means of the log-probit method according to Litchfield and Wilcoxon [10]. The experimental procedure has been described in more detail in our earlier papers [12, 13].
Protective Indices (PIs)
Protective index (PI) for each second-generation AED (FBM, LTG, OXC and TPM), administered alone or in combination with L-NA, was calculated by divid- ing a given TD50value, evaluated in the chimney test, by the respective ED50value determined in the MES 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 coordina- tion or other neurotoxic manifestations) in preclinical studies [11].
Statistics
The ED50and TD50values with their respective 95%
confidence limits were calculated and statistically analyzed by computer-assisted log-probit method ac- cording to Litchfield and Wilcoxon [10].
Results
Effect of L-NA upon the anticonvulsant action of FBM, LTG, OXC and TPM against MES-induced seizures
The investigated AEDs (FBM, LTG, OXC, and TPM) administered alone exhibited a clear-cut anticonvul- sant activity in the MES test in mice and their ED50
L-NA and second-generation AEDs in the MES test in mice
Jarogniew J. £uszczki et al.
values are presented in Table 1. Noteworthy, it was previously reported that L-NA at 40 mg/kg did not al- ter the threshold for electroconvulsions in mice, hence, the NOS inhibitor exertedper se neither anti- nor proconvulsive action in mice [27]. The co- administration of L-NA at 40 mg/kg with FBM was associated with a 36% attenuation of the anticonvul- sant efficacy of FBM in the MES test (Tab. 1). Like- wise, a 28% reduction of the antiseizure effects was documented for LTG following the administration of L-NA (40 mg/kg) (Tab. 1). In the case of TPM, a 9%
increase in its ED50value was observed in the MES test after administration of L-NA (Tab. 1). In contrast, L-NA (at 40 mg/kg) co-administered with OXC caused a 4% reduction of the ED50value of the latter drug in the MES test (Tab. 1). Statistical analysis of the data re- vealed no significant changes in the ED50 values of AEDs following the administration of L-NA.
values are presented in Table 1. Noteworthy, it was previously reported that L-NA at 40 mg/kg did not al- ter the threshold for electroconvulsions in mice, hence, the NOS inhibitor exertedper se neither anti- nor proconvulsive action in mice [27]. The co- administration of L-NA at 40 mg/kg with FBM was associated with a 36% attenuation of the anticonvul- sant efficacy of FBM in the MES test (Tab. 1). Like- wise, a 28% reduction of the antiseizure effects was documented for LTG following the administration of L-NA (40 mg/kg) (Tab. 1). In the case of TPM, a 9%
increase in its ED50value was observed in the MES test after administration of L-NA (Tab. 1). In contrast, L-NA (at 40 mg/kg) co-administered with OXC caused a 4% reduction of the ED50value of the latter drug in the MES test (Tab. 1). Statistical analysis of the data re- vealed no significant changes in the ED50 values of AEDs following the administration of L-NA.
Effect of L-NA on the acute adverse effects of AEDs in the chimney test
All studied AEDs (FBM, LTG, OXC, and TPM) ad- ministered alone exhibited clear-cut motor impair- ment in the chimney test in mice and their TD50val-
Similarly, a 21% increase in motor coordination defi- cits in the chimney test was observed for the combina- tion of L-NA with LTG (Tab. 1). L-NA at 40 mg/kg slightly enhanced OXC-produced impairment of mo- tor coordination in the chimney test and the TD50 value of OXC was lowered by 12% (Tab. 1). In the case of TPM, the co-administration of the AED with L-NA was associated with a 9% reduction of its TD50 value (Tab. 1). In all cases, no significant changes in the TD50 values of AEDs following the administra- tion of L-NA were observed.
Protective indices (PIs)
Simultaneous evaluation of TD50and ED50values for all AEDs allowed the calculation of their PIs, describ- ing the safety and tolerability profile of AEDs admin- istered singly (Tab. 1). The highest PI was associated with TPM (16.2), whereas OXC had the lowest value (6.47). LTG and FBM had PI values of 7.47 and 7.74, respectively (Tab. 1). L-NA (40 mg/kg) co-administered with FBM, LTG, OXC and TPM was responsible for the reduction in PI value of all AEDs studied. The PI values were lower than those denoted for each AED alone, showing that the gap between the anticonvul- sant doses of the AEDs in combination with L-NA protecting the animals against MES-induced seizures and the doses producing motor impairment in the chimney test is narrow (Tab. 1).
Discussion
Results presented herein indicated that L-NA at a subthreshold dose of 40 mg/kg did not significantly affect the anticonvulsant activity of four newer (second-generation) AEDs (FBM, LTG, OXC and TPM) against MES-induced seizures in mice. Moreo- ver, we showed that L-NA did not potentiate the acute adverse effects produced by the newer AEDs in the chimney test. Considering both, the anticonvulsant and acute adverse effects of AEDs administered alone and in combination with L-NA, one can ascertain that L-NA reduced the PI values of the newer AEDs.
As mentioned in the Introduction, we have reported that MOL enhanced the anticonvulsant activity of Treatment (mg/kg) ED# (mg/kg) TD# (mg/kg) PI
FBM + vehicle 50.5 (40.0–63.8) 391 (315–487) 7.74 FBM + L-NA (40) 68.5 (55.1–85.2) 331 (249–440) 4.83 LTG + vehicle 4.7 (3.1–7.0) 35.1 (19.8–62.1) 7.47 LTG + L-NA (40) 6.0 (5.4–6.8) 27.7 (17.8–43.2) 4.62 OXC + vehicle 11.2 (9.2–13.6) 72.5 (65.0–80.9) 6.47 OXC + L-NA (40) 10.8 (9.3–12.7) 64.0 (55.4–74.0) 5.93 TPM + vehicle 41.4 (32.4–53.0) 669 (554–808) 16.2 TPM + L-NA (40) 45.1 (37.1–55.0) 611 (452–825) 13.5
Data are presented as median effective doses (ED# , protecting 50% of animals against MES-induced seizures) and median toxic doses (TD# , producing motor coordination impairment in 50% of animals tested) with 95% confidence limits in parentheses. Calcula- tions of the ED# and TD# values and their statistical analysis were performed with computer-assisted log-probit method according to Litchfield and Wilcoxon [10]. The protective indices (PIs) of AEDs administered alone or in combinations, as quotients of their respec- tive TD# and ED# values from the chimney and MES tests, deter- mine the margin of safety and tolerability of drugs in combinations, considering both, their anticonvulsant and acute adverse effects.
FBM felbamate, LTG lamotrigine, OXC oxcarbazepine, TPM topiramate
FBM by reducing its ED50value by 41% in the MES test in mice [15]. Since MOL (a donor of NO) potenti- ated the anticonvulsant efficacy of FBM, and L-NA (a NOS inhibitor) alleviated its antiseizure effects in the MES test, one can conclude that NO plays an im- portant role in the antiseizure effects of FBM and probably LTG in the MES test. Relatively recently, we have documented that 7-NI (50 mg/kg,ip) potentiated the antiseizure effects of OXC, FBM, LTG and TPM in the MES test by reducing their ED50 values by 41%, 16%, 53%, and 40%, respectively [13]. So, comparing the results presented in this study (after L-NA co-administration) with those reported previ- ously (after 7-NI co-administration), one can ascertain that L-NA reduces, whereas 7-NI potentiates the an- tielectroshock action of the newer AEDs in the MES test in mice. A similar situation has been observed in PTZ-induced seizures in mice. It was documented that L-NA (40 mg/kg,ip) impaired the anticonvulsant action of ETS by increasing its ED50 value by 46%
[7], whilst, 7-NI (50 mg/kg, ip) potentiated the anti- convulsant effects of ETS in the PTZ-test in mice, which was associated with a 47% reduction of the ED50 value of ETS [5]. Interestingly, the anticonvul- sant effects of VPA and PB after co-administration of L-NA or 7-NI were unchanged in the PTZ-test [5, 7].
At present, it is impossible to explain the observed discrepancy in the interaction profiles of 7-NI and L-NA with conventional and newer AEDs in the MES and PTZ tests. It was hypothesized that another un- known as yet mechanism of action of the agent unre- lated to the NOS inhibition and the decrease in NO content in the brain participated in the 7-NI-induced potentiation of the antiseizure effects of AEDs [7, 28].
Details concerning the influence of NOS inhibitors on the regulation of cerebral blood flow in physiological and pathological conditions as well as non-specific ef- fects of 7NI on seizure activity have been presented elsewhere [9, 18, 19, 23, 26, 28].
In conclusion, our results confirm indirectly that NO had the anticonvulsant action and the reduction of NO content in the brain through the inhibition of NOS activity by L-NA impaired the anticonvulsant effects of FBM and LTG in the MES test. This hypothesis could, at least in part, explain the observed alleviation of antiseizure effects after co-administration of L-NA with newer AEDs. The lack of significant effects of L-NA on the anticonvulsant and acute adverse-effect profiles of newer AEDs deserves our attention from a preclinical point of view.
Acknowledgment:
This study was supported by a grant from the Medical University of Lublin.
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Received:
February 21, 2006; in revised form: April 21, 2006.
