7-Nitroindazole enhances dose-dependently the anticonvulsant activities of conventional antiepileptic drugs in the mouse maximal electroshock-induced seizure model

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7-Nitroindazole enhances dose-dependently the anticonvulsant activities of conventional antiepileptic drugs in the mouse maximal electroshock-induced seizure model

Jarogniew J. £uszczki¹, Anna Sacharuk¹, Anna Wojciechowska¹, Marta M. Andres-Mach², Monika Dudra-Jastrzebska², Mohamed Mohamed¹, Katarzyna M. Sawicka¹, Justyna Koziñska¹,

Stanis³aw J. Czuczwar^1,2

Department of Pathophysiology, Medical University of Lublin, Jaczewskiego 8, 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:

7-Nitroindazole (7NI, a nitric oxide synthase [NOS] inhibitor) administered intraperitoneally (ip), 30 min before the test, at doses ranging between 50–200 mg/kg, raised the threshold for electroconvulsions in mice. Linear regression analysis revealed that the doses increasing the threshold by 50% (TID_#) and 100% (TID) over the control value for 7NI were 115.2 and 173.4 mg/kg, respectively. Moreover, 7NI dose-dependently potentiated the anticonvulsant effects of four conventional antiepileptic drugs (AEDs: carbamazepine – CBZ, phenobarbital – PB, phenytoin – PHT, and valproate – VPA) in the mouse maximal electroshock-induced seizure (MES) model. 7NI at 50 mg/kg enhanced only the anticonvulsant effect of PB, whereas the drug at 75 and 100 mg/kg potentiated the antiseizure effects of PB, PHT and VPA, but not those of CBZ against MES-induced seizures. Only 7NI at 150 mg/kg enhanced considerably the antielectroshock action of all studied AEDs in the MES test. Pharmacokinetic evaluation of interactions between 7NI and the investigated AEDs revealed that 7NI (150 mg/kg; ip) did not alter total brain concentrations of conventional AEDs in mice. L-arginine (L-Arg – a natural precursor of NO; administeredip, 500 mg/kg, 60 min before electroconvulsions) did not reverse the activity of 7NI (150 mg/kg), but in contrast, it significantly potentiated the anticonvulsant action of conventional AEDs combined with 7NI (150 mg/kg). Pharmacokinetic increase in total brain AED concentrations was observed for the combinations of L-Arg (500 mg/kg) with 7NI (150 mg/kg) and PHT (by 32%; p < 0.01) or VPA (by 22%; p < 0.05). Neither total brain CBZ nor PB concentrations were altered following the co-administration of L-Arg (500 mg/kg) with 7NI (150 mg/kg). 7NI at doses of 100–200 mg/kg significantly impaired spontaneous ambulatory activity in mice subjected to the Y-maze task. The NOS inhibitor at doses of 50 and 75 mg/kg had no significant effect on locomotor activity of animals, although the number of arm entries within the 5 min of observational time was reduced. Finally, it can be concluded that the enhancement of anticonvulsive efficacy of CBZ, PB, PHT and VPA by 7NI alone or in combination with L-Arg in the MES test, deserves more attention and further neurochemical studies are required to elucidate the exact role of NO in the brain.

Key words:

7-Nitroindazole, L-arginine, nitric oxide, maximal electroshock, electroconvulsive threshold, carbamazepine, phenobarbital, phenytoin, valproate, pharmacodynamic/pharmacokinetic interactions, spontaneous locomotor activity

Pharmacological Reports 2006, 58, 660671 ISSN 1734-1140

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Abbreviations: 7NI – 7-nitroindazole, ADC – arginine decar- boxylase, AED – antiepileptic drug, CBZ – carbamazepine, CNS – central nervous system, CZP – clonazepam, GABA – g-aminobutyric acid, L-Arg – L-arginine, MES – maximal electroshock-induced seizures, MEST – maximal electroshock-induced seizure threshold, NMDA – N-methyl-D- aspartic acid, NO – nitric oxide, NOS – nitric oxide synthase, OXC – oxcarbazepine, PB – phenobarbital, PHT – phenytoin, PIC – picrotoxin, PTZ – pentetrazole, VPA – valproic acid

Introduction

A growing number of recent studies indicates that nitric oxide (NO), a gaseous molecule formed enzy- matically by nitric oxide synthase (NOS; EC 1.14.13.39 ) from L-arginine (L-Arg) with L-citrulline as a co-product [8], plays the role of a neurotransmitter/neuromodulator in the brain [11, 19, 41]. Epilepsy is one of the central nervous system (CNS) diseases in which NO is regarded as an important pathogenic fac- tor involved in the mechanisms underlying seizure initiation and/or propagation. The role of NO in epi- leptogenesis has been examined in a number of in vivo and in vitro studies, however, the obtained results are still contradictory (for review see [61]).

NO has been proposed to function as an endogenous anticonvulsant substance since a decreased syn- thesis of NO in the brain following the administration of NOS inhibitors has resulted in an exacerbation of experimentally-induced convulsions in rats [9, 60].

Moreover, an increase in NO content by intracortical microinjection of sodium nitroprusside inhibited epi- leptiform discharges elicited by penicillin in rats [38].

The advanced molecular and neurochemical studies have reported that an increase in NO concentration in the brain is associated with the release of the inhibitory neurotransmitterg-aminobutyric acid (GABA) in the cerebral cortex [24], hippocampus [28] and stria- tum [54]. Moreover, NO increases indirectly the brain GABA concentration by inhibiting GABA transaminase, an enzyme responsible for GABA degradation [49]. NO has a significant inhibitory effect on N-methyl-D-aspartate (NMDA) receptor function [37]. Similarly, L-Arg (a natural precursor of NO) has been reported to modulate the experimentally evoked convulsions by inhibiting kainic acid-, pentetrazole (PTZ)-, picrotoxin (PIC)-, and sound-induced seizures in rats and mice [9, 46, 47, 55, 57, 60]. In contrast, several lines of evidence suggest that the activa-

tion of neuronal NOS and increase in NO content in the brain may produce seizures in experimental animals [45, 52]. For example, it has been documented that L-Arg exerted proconvulsant action by enhancing NMDA- and PTZ-induced convulsions in rodents [12, 40]. L-Arg displayed a biphasic action producing both anti- and pro-convulsant effects on PIC-induced seizures in rats [50]. In this case, the 5-min pretreatment with L-Arg at 1000 and 2000 mg/kg delayed the onset of myoclonus and clonic convulsions, whereas L-Arg administered at a dose of 2000 mg/kg; 60 min prior to PIC injection, considerably aggravated the PIC- induced clonic convulsions in rats [50].

The discovery of nitroindazole derivatives, which preferentially and specifically inhibit neuronal NOS activity, was the beginning of a new era in research studies allowing for the examination and clarification of the role of NO in the brain functioning and its involvement in seizure phenomena. So far, most attention has been focused on 7-nitroindazole (7NI) which is considered to be a preferential and selective inhibitor of neuronal NOS [3]. Accumulating experimental evidence indicates that 7NI has anticonvulsant properties by suppressing kainic acid- [22, 44], pilocarpine- [58], PTZ- [17, 23], NMDA- [51], enoxacin- [39], and sound-induced seizures [55] in rodents.

Relatively recently, it has been reported that 7NI at low doses of 50 and 100 mg/kg inhibited PIC-induced seizures, whereas at higher doses of 150 and 200 mg/kg, the NOS inhibitor enhanced the convulsant action of PIC, displaying both anti- and proconvulsant effects in the same experimental model of epilepsy [48]. Proconvulsant properties of 7NI have been also documented in soman-induced seizures in rats, where 7NI enhanced the severity of clonic seizures and increased the lethality produced by soman [25].

Previously, it has been found that 7NI (administered at a dose of 50 mg/kg) enhanced the anticonvulsant action of phenobarbital (PB), but not that of carbamazepine (CBZ), phenytoin (PHT), and valproate (VPA) against maximal electroshock (MES)-induced seizures in mice [5]. Additionally, 7NI augmented the antiseizure effects of two NMDA receptor antagonists:

D,L-(E)-2-amino-4-methyl-5-phosphono-3-pentenoate (CGP 37849) and dizocilpine [4], as well as, it potentiated the antiseizure effect of flurazepam [14] in the MES test in mice. This NOS inhibitor (at 50 mg/kg) potentiated also the antiseizure activity of clonazepam (CZP) and ethosuximide, but not that of VPA and PB against PTZ-induced clonic seizures in mice [7]. Simi-

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larly, in DBA/2 mice, 7NI (at 25 mg/kg) enhanced the anticonvulsant action of CBZ, diazepam, PB, PHT, VPA and lamotrigine against sound-induced seizures [13]. The NOS inhibitor 7NI (at a dose of 50 mg/kg) enhanced the antiseizure effect of PB and CBZ, but not that of PHT, VPA and CZP in amygdala-kindled rats [7]. In contrast, one report has indicated that 7NI (at 100 mg/kg) reduced the anticonvulsant action of PHT, but not that of CBZ in the MES test in mice [4].

Since 7NI at high doses has been shown to produce proconvulsant action in the rat PIC-induced seizure model [48] and reduced the antiseizure effect of PHT in the MES test in mice [4], it was of pivotal impor- tance to assess the dose-response relationship of 7NI in affecting the electroconvulsive threshold in mice.

Additionally, we investigated the effects of 7NI at various increasing doses on the anticonvulsant action of four conventional antiepileptic drugs (AEDs: CBZ, PB, PHT and VPA) in the MES test. To confirm or ex- clude the effect of 7NI on NO content in the brain, we attempted to reverse its inhibitory action on NOS using L-Arg, a natural NO precursor. To determine the acute adverse-effect profile of 7NI, the animals administered the increasing doses of the NOS inhibitor (50–200 mg/kg) were examined in the Y-maze test, evaluating the effect of 7NI on spontaneous ambulatory activity in mice. Finally, the total brain AED concentrations were measured to ascertain whether the observed effects were consequent to pharmacodynamic and/or pharmacokinetic interactions.

Materials and Methods

Animals and experimental conditions

All experiments were performed on adult male Swiss mice weighing 22–26 g. The mice were kept in col- ony cages with free access to food and tap waterad li- bitum, under standardized housing conditions (natural light-dark cycle, temperature of 21 ± 1°C, relative hu- midity of 55 ± 3%). After 7 days of adaptation to laboratory conditions, the animals were randomly as- signed 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 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. Addi- tionally, all efforts were made to minimize animals’

suffering and to use only the number of animals necessary to produce reliable scientific data. The experimental protocols and procedures described hereupon were approved by the Local Ethics Committee at the Medical University of Lublin (Licence no.: 479/04 /509/2004).

Drugs

The following AEDs and agents were used in this study: 7NI (Sigma, St. Louis, MO, USA), L-Arg (RBI, Natick, MA, USA), CBZ (kind gift from Polfa, Starogard, Poland), PB (Polfa, Kraków, Poland), PHT (Polfa, Warszawa, Poland), and VPA – sodium salt (kind gift from ICN-Polfa, Rzeszów, Poland). VPA and L-Arg were dissolved in 0.9% NaCl, whereas the other AEDs and 7NI were suspended in a 1% aqueous solution of Tween 80 (Sigma, St. Louis, MO, USA) and administeredip in a volume of 5 ml/kg of body weight. The control animals received adequate amounts of 0.9% NaCl. Fresh drug solutions or sus- pensions were preparedex tempore on each day of experimentation and administered as follows: PHT – at 120 min, PB and L-Arg – at 60 min, CBZ, VPA and 7NI – at 30 min prior to electroconvulsions, spontaneous ambulatory activity evaluation, and brain sam- pling for the measurement of AED concentrations.

These drug administration times were based on infor- mation about their biological activity from the litera- ture [36] and were confirmed in our previous experiments [7, 31, 33].

Maximal electroshock seizure threshold (MEST) test

Electroconvulsions were produced by means of an al- ternating current (0.2 s stimulus duration, 50 Hz) de- liveredvia ear-clip electrodes by a generator (Rodent Shocker, Type 221, Hugo Sachs, Freiburg, Germany).

The criterion for the occurrence of seizure activity was the tonic hindlimb extension (i.e., the hind limbs of animals outstretched at 180° to the plane of the body axis). In order to evaluate the threshold for electroconvulsions, at least 4 groups of mice, consisting of 8 animals per group, were challenged with electro- shocks of various intensities to yield 10–30%, 30–50%, 50–70%, and 70–90% of animals with seizures. Then, a current intensity-effect curve was con-

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structed, according to a log-probit method by Litchfield and Wilcoxon [27], from which a median current strength (CS₅₀ in mA) was estimated. Each CS₅₀value represents the current intensity required to induce tonic hindlimb extension in 50% of the mice challenged. After administration of a single dose of 7NI to 4 groups (8 animals per group), the mice were subjected to electroconvulsions. The threshold for electroconvulsions was denoted for various different doses of 7NI, as follows: 50, 75, 100, 150, and 200 mg/kg. The experimental procedure has been described in more detail in our earlier studies [31, 32, 34].

Maximal electroshock seizure (MES) test

The protective activities of conventional AEDs, administered separately and in combination with 7NI, L-Arg, or both agents, were evaluated and expressed as their median effective doses (ED₅₀ in mg/kg) against MES-induced seizures (fixed current intensity of 25 mA, maximum stimulation voltage of 500 V).

The animals were administered different doses of AEDs in order to obtain a variable percentage of pro- tection against MES, allowing the construction of a dose-effect line for each drug administered alone or in combination with 7NI, L-Arg or both agents, according to Litchfield and Wilcoxon [27]. Subse- quently, the ED₅₀ values with their 95% confidence limits were calculated. Each ED₅₀ value represents the dose of an AED required to protect 50% of the animals tested against MES-induced seizures. At least 32 mice were used (8 animals per group) to calculate each ED₅₀ value. The experimental procedure has been described in more detail in our earlier studies [29–31, 33–35].

Measurement of total brain AED concentrations

The animals received an AED + vehicle, a combination of an AED with 7NI or a combination of an AED with 7NI + L-Arg. Mice were killed by decapitation at times chosen to coincide with that scheduled for the MES test and the whole brains of mice were removed from skulls, weighed, and homogenized using Abbott buffer (1:2 weight/volume) in an Ultra-Turrax T8 ho- mogenizer (Staufen, Germany). The homogenates were centrifuged at 10 000 × g for 10 min and the supernatant samples (75 µl) were analyzed by fluorescence polarization immunoassay (FPIA) for CBZ, PB,

PHT or VPA content using a TDx analyzer and rea- gents exactly as described by the manufacturer (Ab- bott Laboratories, North Chicago, IL, USA). All AED concentrations are expressed in µg/ml of brain supernatant as means ± SD of at least 8 determinations.

Statistics

Both CS₅₀(mA) and ED₅₀(mg/kg) values with their respective 95% confidence limits and SE were calculated by computer log-probit analysis [27], and statistically analyzed with one-way analysis of variance (ANOVA) followed by thepost-hoc Bonferroni’s test for multiple comparisons. Simultaneously, the percentage increase in CS₅₀for animals injected with increasing doses of 7NI was calculated. Next, the doses of 7NI and their resultant percentage increases in threshold over the control (vehicle-treated animals) were graphically plotted in rectangular coordinates of the Cartesian plot system and examined with least- squares linear regression analysis according to Glantz and Slinker [20]. From the linear regression equation, doses increasing the threshold by 50% (TID₅₀) and 100% (TID₁₀₀) were calculated. Total brain concentrations of PHT, CBZ, VPA or PB, administered alone or in combination with 7NI and/or L-Arg were statistically analyzed using unpaired Student’st-test. Spon- taneous locomotor activity (general exploration) of the animals evaluated in the Y-maze was analyzed by one-way ANOVA followed by the post-hoc Bonfer- roni’s test for identification of significant treatment effectsvs. control means.

Results

Effect of 7NI on the threshold for electroconvul- sions in mice

Statistical analysis of the data with one-way ANOVA indicated that 7NI (administeredip, at 30 min before the MEST test) dose-dependently increased the threshold for electroconvulsions in mice [F (5, 154) = 31.37;

p < 0.0001]. Bonferroni’s post-hoc test revealed that 7NI at 100, 150 and 200 mg/kg raised significantly the electroconvulsive threshold from 7.3 mA to 9.5 mA (p < 0.05), 12.0 mA (p < 0.001), and 17.2 mA (p < 0.001), respectively (Tab. 1). In contrast, 7NI ad-

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ministered at lower doses of 50 and 75 mg/kg did not affect the electroconvulsive threshold in mice (Tab. 1).

The electroconvulsive threshold for 7NI at doses of 50, 75, 100, 150, and 200 mg/kg was increased by 6.8%, 12.3%, 30.1%, 64.4%, and 135.6%, respectively, as compared to the threshold of control (vehicle-treated) animals (Tab. 1, Fig. 1). The doses of 7NI and their corresponding percentage increases in the threshold were subsequently analyzed with linear regression to calculate the TID₅₀and TID₁₀₀values. The equation of dose-response relationship of 7NI dosevs. increase in the electroconvulsive threshold was: y = 0.0086 x –0.4911 (r²= 0.9439); where y is the threshold increase ratio, x – corresponds to the dose of 7NI, and r²– is the coefficient of determina- tion. As the data points were good-to-fit to the line in our study, we could calculate TID₅₀and TID₁₀₀values which amounted to 115.2 and 173.4 mg/kg (Fig. 1).

Influence of 7NI on the anticonvulsant activity of conventional AEDs in the MES test in mice

7NI administered systemically (ip) enhanced in a dose-dependent manner the antiseizure effects of conventional AEDs in the MES test in mice (Tab. 2). It was found experimentally that PB was the most sus- ceptible AED to the action of 7NI, since the latter, administered at a dose of 50 mg/kg, significantly enhanced the anticonvulsant action of PB by reducing its ED₅₀from 26.2 to 17.1 mg/kg (p < 0.01; Tab. 2). 7NI at higher doses of 75, 100, and 150 mg/kg, dose- dependently potentiated the anticonvulsant effect of PB (p < 0.001; Tab. 2). In contrast, CBZ was the most re- sistant AED to the action of 7NI in the MES test, because only the highest tested 7NI dose of 150 mg/kg was able to enhance the anticonvulsant action of CBZ against MES-induced seizures. In this case, it was shown that 7NI at 150 mg/kg potentiated significantly the anticonvulsant action of CBZ by reducing its ED₅₀ from 10.8 to 6.4 mg/kg (p < 0.05; Tab. 2). In contrast, 7NI at lower doses (25, 50, 75, and 100 mg/kg) did not modify significantly the antiseizure properties of CBZ in the MES test (Tab. 2). The remaining AEDs tested in the MES test i.e., PHT and VPA were moderately sus- ceptible to the action of 7NI, since the NOS inhibitor at 75, 100 and 150 mg/kg significantly potentiated the antiseizure effects of both AEDs by reducing their ED₅₀ values (Tab. 2). Again, 7NI at lower doses of 25 and 50 mg/kg had no impact on the anticonvulsant effect of PHT and VPA in the MES test in mice (Tab. 2).

Tab. 1. Effect of 7-nitroindazole (7NI) on the electroconvulsive threshold in mice

Treatment

(mg/kg) CS₅₀(mA) SE N TI (%)

Control 7.3 (6.5–8.3) 0.437 32 –

7NI (50) 7.8 (7.0–8.7) 0.399 24 6.8

7NI (75) 8.2 (7.5–9.1) 0.404 32 12.3

7NI (100) 9.5 (8.4–10.7)* 0.618 24 30.1

7NI (150) 12.0 (10.5–13.7)*** 0.825 24 64.4

7NI (200) 17.2 (15.2–19.6)*** 1.069 32 135.6

Data are presented as median current strengths (CS_#values in mA;

with 95% confidence limits in parentheses), necessary to evoke seizure activity (tonic hindlimb extension) in 50% of animals tested. 7NI was givenip, 30 min prior to the test. Statistical evaluation of the data was performed using log-probit method [27] and one-way ANOVA followed by thepost-hoc Bonferroni’s test for multiple comparisons.

SE – standard error of the CS_#; N – number of animals at those CS_#

values whose convulsant effects ranged between 4 and 6 probits.

The threshold for control (vehicle-treated) animals was considered as a baseline (reference) value for calculations of the percentage of the threshold increase (TI) following 7NI administration (the control CS_#value of 7.3 mA was considered as 100%). * p < 0.05 and

*** p < 0.001vs. the control CS_#value

y = 0.0086 x - 0.4911 r²= 0.9439

0.0 0.2 0.4 0.6 0.8 1.0 1.2 1.4 1.6

0 50 100 150 200 250 300

7-Nitroindazole dose (mg/kg)

Thresholdincreaseindex

TID₅₀= 115.2 mg/kg TID₁₀₀= 173.4 mg/kg

Fig. 1. Linear regression analysis of dose-response relationship of 7-nitroindazole (7NI) in the maximal electroshock seizure threshold (MEST) model. The doses of 7NI are plotted (on x-axis) against the threshold increase index (on y-axis) in mice. Analysis of the data was performed with least-squares linear regression analysis according to Glantz and Slinker [20]. The equation of dose-response relationship for 7NI evaluated in the MEST test was y = 0.0086 x – 0.4911 (r= 0.9439); where y – is the decimal fraction of threshold increase, x – corresponds to the drug dose, and r – is the coefficient of deter- mination. Test for homogeneity revealed that points creating the line are homogenous and good-to-fit. The dashed line, parallel to x-axis, represents a dose of 7NI increasing the threshold by 50% (0.5), whereas the dotted line reflects a dose of 7NI increasing the threshold by 100% (1.0). For more details see the Results section

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Influence of L-Arg on the anticonvulsant effects offered by 7NI in combination with conven- tional AEDs in the MES test in mice

L-Arg administered ip at a constant dose of 500 mg/kg did not reverse the effect of 7NI (150 mg/kg) on the anticonvulsant activities of all conventional AEDs (Tab. 3). Surprisingly, L-Arg (500 mg/kg) potentiated the antiseizure activity of the combinations of 7NI (150 mg/kg) with CBZ, PB, PHT and VPA by reducing significantly their ED₅₀ values in the MES test, as compared to the combinations of 7NI with the studied AEDs (Tab. 3). In contrast, L-Arg (500 mg/kg) administered alone had no impact on the anticonvulsant effects of conventional

Tab. 2. Influence of 7-nitroindazole (7NI) on the anticonvulsant activ- ity of conventional antiepileptic drugs (AEDs) against maximal electroshock (MES)-induced seizures in mice

Treatment (mg/kg) ED₅₀(mg/kg) SE N

CBZ + vehicle 10.8 (9.1–12.8) 0.945 16 CBZ + 7NI (25) 10.3 (8.7–12.2) 0.888 16 CBZ + 7NI (50) 9.9 (8.3–11.7) 0.862 24 CBZ + 7NI (75) 8.9 (7.3–10.8) 0.871 16 CBZ + 7NI (100) 8.0 (6.6–9.8) 0.810 32 CBZ + 7NI (150) 6.4 (4.9–8.3)* 0.870 16 PB + vehicle 26.2 (22.4–30.5) 2.066 16 PB + 7NI (25) 21.8 (17.6–27.0) 2.372 16 PB + 7NI (50) 17.1 (13.4–21.9)** 2.136 24 PB + 7NI (75) 13.0 (10.0–17.0)*** 1.757 16 PB + 7NI (100) 7.7 (5.1–11.8)*** 1.668 16 PB + 7NI (150) 3.2 (1.6–6.1)*** 1.066 24 PHT + vehicle 11.2 (9.4–13.2) 0.973 24 PHT + 7NI (25) 10.8 (9.1–12.8) 0.945 16 PHT + 7NI (50) 10.1 (8.6–11.9) 0.851 24 PHT + 7NI (75) 7.5 (6.1–9.3)** 0.810 24 PHT + 7NI (100) 6.4 (4.9–8.4)** 0.874 16 PHT + 7NI (150) 4.7 (3.5–6.3)*** 0.706 16 VPA + vehicle 281.6 (255.5–310.5) 14.01 32 VPA + 7NI (25) 261.0 (239.0–285.1) 11.74 16 VPA + 7NI (50) 244.5 (215.6–277.4) 15.70 16 VPA + 7NI (75) 223.6 (189.0–264.7)* 19.22 24 VPA + 7NI (100) 200.5 (171.5–234.4)** 15.99 16 VPA + 7NI (150) 160.1 (138.5–184.9)*** 11.78 16

Data are presented as median effective doses (ED_#values with their 95% confidence limits), protecting 50% of animals tested against MES-induced seizures. 7NI was administeredip, 30 min before the test, whereas the AEDs were administered ip, as follows: PHT – 120 min, PB – 60 min, CBZ and VPA – 30 min prior to the MES test.

Statistical evaluation of the data was performed with log-probit method and one-way ANOVA followed by thepost-hoc Bonferroni’s test for multiple comparisons [20, 27]. SE – standard error of the ED_#; N – number of animals at the doses whose anticonvulsant effects ranged between 4 and 6 probits. CBZ – carbamazepine; PB – phenobarbital; PHT – phenytoin; VPA – valproate. * p < 0.05;

** p < 0.01; and *** p < 0.001vs. the respective control group (an AED + vehicle-treated animals)

Tab. 3. Influence of L-arginine (L-Arg) on the anticonvulsant action of 7-nitroindazole (7NI) in combination with conventional antiepileptic drugs (AEDs) in the maximal electroshock-induced seizure (MES) test in mice

Treatment (mg/kg) ED₅₀(mg/kg) SE N

CBZ + vehicle 10.8 (9.1–12.8) 0.945 16 CBZ + L-Arg (500) 10.4 (8.9–12.1) 0.806 32 CBZ + 7NI (150) 6.4 (4.9–8.3)* 0.870 16 CBZ + 7NI (150)

+ L-Arg (500) 4.4 (3.4–5.6)*** 0.541 32 PB + vehicle 26.2 (22.4–30.5) 2.066 16 PB + L-Arg (500) 23.6 (18.8–29.5) 2.709 24 PB + 7NI (150) 3.2 (1.6–6.1)*** 1.066 24 PB + 7NI (150)

+ L-Arg (500) 1.1 (0.7–1.9)*** 0.303 16 PHT + vehicle 11.2 (9.4–13.2) 0.973 24 PHT + L-Arg (500) 10.8 (9.1–12.8) 0.945 16 PHT + 7NI (150) 4.7 (3.5–6.3)*** 0.706 16 PHT + 7NI (150)

+ L-Arg (500)

0.9 (0.5–1.6)***^{; # #} 0.266 24

VPA + vehicle 281.6 (255.5–310.5) 14.01 32 VPA + L-Arg (500) 265.9 (243.5–290.4) 11.96 24 VPA + 7NI (150) 160.1 (138.5–184.9)*** 11.78 16 VPA + 7NI (150)

+ L-Arg (500)

94.3 (75.6–117.7)***^{; # #} 10.63 24

Data are presented as median effective doses (ED_#values with their 95% confidence limits), protecting 50% of animals tested against MES-induced seizures. The ED_#values were calculated using log- probit method according to Litchfield and Wilcoxon [27]. Statistical analysis of the data was performed with one-way ANOVA followed by thepost-hoc Bonferroni’s test for multiple comparisons. L-Arg was administeredip, 60 min before the MES test. For more details see the legend to Tab. 2. * p < 0.05 and *** p < 0.001vs. the respective control group (an AED+vehicle-treated animals).p < 0.01vs. the respective 7NI-treated group (an AED + 7NI-treated animals)

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AEDs in the MES test (Tab. 3). Noteworthy, L-Arg (500 mg/kg) combined with 7NI (150 mg/kg) potentiated the anticonvulsant effects of PHT and VPA to a greater extent (p < 0.01) than those produced by CBZ and PB in the MES test in mice (Tab. 3).

Brain AED concentrations

The combination of L-Arg (500 mg/kg) with 7NI (150 mg/kg) considerably increased (by 32%) the total brain concentration of PHT measured with FPIA technique from 0.141 to 0.186 µg/ml (p < 0.01; Tab.

4). In contrast, 7NI (150 mg/kg) combined with PHT did not affect the brain concentration of the latter drug (Tab. 4). Similarly, the co-application of L-Arg

(500 mg/kg) with 7NI (150 mg/kg) significantly raised (by 22%) the brain concentration of VPA from 59.8 to 73.16 µg/ml (p < 0.05; Tab. 4), whereas 7NI (150 mg/kg) co-administered with VPA had no impact on the total brain VPA concentrations (Tab. 4).

Pharmacokinetic evaluation of the remaining interactions with FPIA technique, for CBZ and PB with 7NI and L-Arg, revealed that neither the combination of L-Arg (500 mg/kg) with 7NI (150 mg/kg) nor 7NI (150 mg/kg) injected alone affected the total brain concentrations of CBZ and PB in mice (Tab. 4).

Spontaneous locomotor activity testing

In the Y-maze test, 7NI administered alone (ip, 30 min. before the test) reduced in a dose-dependent manner locomotor activity of animals. One-way ANOVA followed by thepost-hoc Bonferroni’s test revealed that mean numbers of arm entries of animals following 7NI administration differed significantly from those observed in untreated animals [F (5, 42) = 30.23;

p < 0.0001]. The mean number of arm entries within the 5-min of observational period for control (vehicle-treated) animals was 24.25 ± 0.94, whereas for animals administered with 7NI at doses of 100, 150 and 200 mg/kg, the mean numbers of arm entries were 17.88 ± 1.42, 12.0 ± 1.32, and 6.19 ± 1.01, respectively (Fig. 2). In contrast, 7NI alone at doses of 50 and 75 mg/kg did not affect significantly spontaneous locomotor activity of animals in the Y-maze test and the mean numbers of arm entries were 22.25

± 1.38 and 21.38 ± 1.45, respectively (Fig. 2).

Tab. 4. Influence of 7-nitroindazole (7NI) on the brain AED concen- trations

Treatment (mg/kg) Brain concentration (µg/ml) CBZ (4.4) + vehicle 0.926 ± 0.085

CBZ (4.4) + 7NI (150) + L-Arg (500) 0.993 ± 0.095 CBZ (6.4) + vehicle 1.579 ± 0.223 CBZ (6.4) + 7NI (150) 1.435 ± 0.243 PB (1.1) + vehicle 1.425 ± 0.122 PB (1.1) + 7NI (150) + L-Arg (500) 1.464 ± 0.168 PB (3.2) + vehicle 2.165 ± 0.122 PB (3.2) + 7NI (150) 2.198 ± 0.108 PHT (0.9) + vehicle 0.141 ± 0.019

PHT (0.9) + 7NI (150) + L-Arg (500) 0.186 ± 0.023**  32%

PHT (4.7) + vehicle 0.484 ± 0.093 PHT (4.7) + 7NI (150) 0.452 ± 0.074 VPA (94.3) + vehicle 59.80 ± 4.36

VPA (94.3) + 7NI (150) + L-Arg (500) 73.16 ± 4.44*  22%

VPA (160.1) + vehicle 68.84 ± 4.44 VPA (160.1) + 7NI (150) 73.64 ± 5.00

Data are presented as the means ± SD of at least 8 separate brain preparations and expressed in µg/ml of brain supernatant. The obtained results were statistically analyzed with unpaired Student’s t-test. Brain AED concentrations were determined with fluorescence polarization immunoassay. For more details see the legend to Tab. 2 and 3. – increase in brain AED concentrations. * p < 0.05;

** p < 0.01vs. the respective AED + vehicle-treated group

0 4 8 12 16 20 24 28

Control 7NI (50) 7NI (75) 7NI (100) 7NI (150) 7NI (200)

**

***

[mg/kg]

Numberofarmentries

Fig. 2. Effect of 7-nitroindazole (7NI) on spontaneous locomotor ac- tivity in the Y-maze test in mice. Columns represent mean numbers of arm entries of the animals in the Y-maze task ± SE as the error bars.

7NI was administeredip 30 min. before the testing session. The observational time was set up to 5 min. Statistical analysis of the data was performed with one-way ANOVA followed by thepost-hoc Bon- ferroni’s test. ** p < 0.01 and *** p < 0.001vs. control group

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Discussion

Results presented herein indicate that 7NI in a dose- dependent manner increased the threshold for electroconvulsions exerting a clear-cut anticonvulsant effect against electrically induced seizures in mice. Our ob- servations are partly in agreement with those reported by Baran et al. [4], who have found that 7NI at 100 mg/kg elevated the threshold for electroconvulsions in mice. As mentioned above, 7NI at lower doses of 50–100 mg/kg has been found to produce the anticonvulsant effects by decreasing PIC-induced convulsions in rats, whereas at higher doses of 150 and 200 mg/kg, the NOS inhibitor enhanced PIC- induced seizures in rats exerting proconvulsant effect [57]. This biphasic action of 7NI on PIC-induced seizures in rats and the findings that NOS activity in the brain decreased 30–60 min after administration of 7NI at 150 and 200 mg/kg, but not at lower doses of 50 and 100 mg/kg [57], allowed the authors to claim that 7NI at high doses had proconvulsant properties in animals. In contrast, we found that 7NI at doses of 150 and 200 mg/kg had a clear-cut anticonvulsant effect against MEST-induced seizures in mice. So, the observed discrepancy between effects observed after 7NI administration at high doses of 150 and 200 mg/kg may result either from inter-species differences between mice and rats or seizure models used in both experiments. Noteworthy, the PIC- induced seizures were evoked after ip administration of PIC at a dose of 5 mg/kg [57]. However, the authors did not evaluate a pharmacokinetic profile of PIC in the presence of high doses of 7NI. It is likely that 7NI may dose-dependently increase the PIC concentration in the brain and thus, enhance the seizure activity induced by PIC in animals, providing simultaneously a false evidence that 7NI at high doses has proconvulsant properties.

Our findings indicate that 7NI potentiated the antiseizure action of four conventional AEDs (CBZ, PHT, PB and VPA) by reducing dose-dependently their ED₅₀values in the MES test. Moreover, it was shown that pretreatment with L-Arg (500 mg/kg) did not reverse the effects mediated by 7NI, but it enhanced significantly the anticonvulsant properties of 7NI combined with conventional AEDs. The present study confirms previously published report [5] on the ability of 7NI to potentiate the anticonvulsant activity of PB against MES-induced seizures in mice, and ex-

tends this finding showing that 7NI enhanced the antiseizure effects of the other AEDs studied in a dose- dependent manner. Interestingly, Baran et al. [4] have documented that 7NI at a dose of 100 mg/kg reduced significantly the protective action of PHT and thus, exerted a proconvulsant effect on MES-induced seizures. In contrast, in our study, 7NI in a dose range between 75–150 mg/kg considerably increased the antiseizure action of PHT in the MES test. The observed discrepancy in 7NI action following its systemic administration at a dose of 100 mg/kg, one can try to explain by different 7NI pretreatment times. In our study, 7NI was administered at 30 min before electroconvulsions, and this pretreatment time was considered to be the time to peak effect of 7NI [5, 13, 36, 48]. Noteworthy, in the study by Baran et al. [4], 7NI was administeredip at 90 min prior to electroconvulsions, and probably that fact was responsible for a proconvulsant effect of 7NI, reducing the anticonvulsant effect of PHT in the MES test in mice. Moreo- ver, the authors have documented that 7NI at 100 mg/kg did not modify the anticonvulsant action of CBZ [7], and this finding is identical with our ob- servations showing that 7NI at 100 mg/kg had no impact on the anticonvulsant properties of CBZ in the MES test in mice. Again, it is unclear why the results concerning the interaction of 7NI with PHT in the MES test are contradictory and those for the combination of 7NI with CBZ are in agreement with our findings.

Interestingly, the present study indicated that 7NI at 100 mg/kg increased significantly the threshold for electroconvulsions in mice, but the NOS inhibitor did not enhance the anticonvulsant effects of CBZ against MES-induced seizures in mice. This phenomenon is very important showing evidently that drugs applied at doses raising the threshold for electroconvulsions do not necessarily have to potentiate the anticonvulsant activity of co-administered AEDs in preclinical studies. Similar situation was observed in our recent experiments in which we examined interactions between 2-phosphonomethyl-pentanedioic acid (a glutamate carboxypeptidase II inhibitor) and conventional AEDs. It was found experimentally that 2-phosphonomethyl-pentanedioic acid at a dose of 150 mg/kg (that significantly increased the electroconvulsive threshold in mice) had no significant effect on the anticonvulsant activity of CBZ, PHT and PB in the MES test in mice [34]. So, one can conclude that despite the 7NI-evoked increase in electroconvulsive threshold, the NOS inhibitor was unable to enhance the anticon-

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vulsant action of CBZ in the MES test. Perhaps, 7NI interacted antagonistically with CBZ in the suppression of MES-induced seizures and, therefore, 7NI at 100 mg/kg probably produced no significant effect on CBZ. This hypothesis could, at least in part, explain the lack of effect of 7NI on the anticonvulsant activity of CBZ in the MES test, although some advanced neurochemical studies are required to elucidate the exact characteristics of interactions between 7NI and CBZ in the MES test in mice.

Relatively recently, we have reported that 7NI at 50 mg/kg potentiated significantly the anticonvulsant effect of oxcarbazepine (OXC) in the MES test in mice, by reducing its ED₅₀ value from 11.2 to 6.6 mg/kg (p < 0.01) [30]. Surprisingly, OXC and CBZ have similar chemical structure and 7NI at 50 mg/kg preferentially and selectively enhanced the antiseizure effect of OXC, but not that of CBZ in the MES test in mice. At present, it is difficult to explain which molecular mechanism(s) of action is(are) responsible for the observed differences in the interaction profile of OXC and CBZ with 7NI in the MES test in mice. Perhaps, other unknown as yet mechanisms of action of these AEDs contribute to the observed differences. On the other hand, accumulating evidence indicates that OXC and CBZ, despite their chemical and structural similarities, are quite different AEDs with diverse anticonvulsant profiles in experimental studies (for review see [53]). Our findings seem to confirm the existence of such differences between OXC and CBZ (for more details see discussion in [30]).

Pharmacokinetic evaluation of interactions between 7NI and the studied AEDs revealed that the NOS inhibitor, administered at the highest tested dose of 150 mg/kg, did not alter the total brain concentrations of conventional AEDs, indicating a pharmacodynamic nature of the observed interactions. Noticea- bly, in this study, the total brain concentrations of AEDs were estimated instead of their free plasma lev- els because previous findings indicated that only the evaluation of AED concentrations in biophase of experimental animals (i.e., brain tissue or cerebrospinal fluid) provided the exact pharmacokinetic assessment of the interactions between AEDs [10, 35]. Moreover, it is important to note that 7NI due to its preferential inhibition of neuronal NOS, did not produce changes in the vascular system [3, 42], therefore, the pretreatment with 7NI alone was unlikely to modulate the action of AEDs by altering their penetration into the

brain. Interestingly, it was found that L-Arg (500 mg/kg) increased the total brain concentrations of PHT and VPA, and thus, it potentiated pharmacoki- netically the antiseizure effect offered by the combinations of PHT and VPA with 7NI. In contrast, the total brain concentrations of CBZ and PB were not affected by L-Arg, although it potentiated the anticonvulsant action produced by the combinations of CBZ and PB with 7NI. Considering this fact, it is unclear which mechanism(s) of action of L-Arg is(are) responsible for an increase in total brain PHT and VPA concentrations in mice following the administration of 7NI. This observation requires addi- tional extensive studies to evaluate the exact role of 7NI, L-Arg, and NO in the brain.

Relatively recently, there has appeared a hypothesis proposed by Smith et al. [55], explaining the lack of reversal of the 7NI effect by L-Arg co- administered. Noticeably, the administration of 7NI produced a reduction of NO content in the brain, which was associated with concomitant reduction of citrulline concentration and accumulation of L-Arg in the brain. An accumulation of L-Arg caused by the blockade of the NOS pathways could lead to the alter- native metabolism of L-Arg by arginine decarboxy- lase (ADC; EC 4.1.1.19) into agmatine [26, 43]. Since agmatine has the anticonvulsant action in the MES [56] and PTZ [18] models of seizures, no doubts exist that it can enhance the antiseizure effects of conventional AEDs. Also agmatine itself can inhibit NOS activity [2], potentiating the effect exerted by NOS inhibitors. The above-mentioned facts taken together with observation that 7NI at high doses produces a complete inhibition of neuronal NOS [52] may be one of the possible explanations, why in the present study, L-Arg administrated with 7NI did not reverse the effect offered by 7NI, but inversely, potentiated the anticonvulsant activities of conventional AEDs co-administered with 7NI and L-Arg.

In order to evaluate acute adverse (neurotoxic) effects produced by 7NI administered at increasing doses of 50–200 mg/kg on locomotor activity, the Y- maze test in mice was used. Results indicated that 7NI reduced dose-dependently spontaneous ambulatory activity of animals by decreasing the mean numbers of arm entries within the 5-min of observational period. This fact speaks against the separate and combined use of 7NI at high doses (100–200 mg/kg) as an anticonvulsant agent. Moreover, a slight reduction of spontaneous ambulatory activity was observed for

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AEDs administered at their ED₅₀ values determined in the MES test. In this case, statistical analysis of the data revealed no significant decrease in ambulatory activity in mice examined in the Y-maze test (results not shown). Previously, it has been documented that 7NI at a dose of 120 mg/kg significantly suppressed locomotor activity in mice subjected to the open-field test with electronic measurement [16]. The impaired locomotor activity has also been observed in animals administered 7NI at doses of 20 and 50 mg/kg [59]. In this case, the NOS inhibitor reduced significantly total distance traveled by animals and total time spent on moving in the open-field test in mice with electronic measurement [59]. Likewise, 7NI at doses of 40, 80 and 160 mg/kg produced (in a dose-dependent manner) locomotor deficits in the pole test in mice [1]. In DBA/2 mice, 7NI at 160 mg/kg evoked severe ataxia with loss of righting reflexes [55]. Similarly, the reduced locomotor activity has been observed in rats administered 7NI at a dose of 120 mg/kg and examined in the open-field and elevated plus-maze tests [62]. In contrast, some experimental studies have indicated that 7NI at a dose of 50 mg/kg produced no significant motor deficits in the chimney test in mice and rats [5–7]. Moreover, the NOS inhibitor administered at doses of 15, 30, 60 and 90 mg/kg did not alter locomotor activity in the open-field test in rats [63].

In the light of the above-mentioned facts, our results are generally in agreement with those reporting that 7NI attenuated spontaneous locomotor activity in animals. Noteworthy, in our study, 7NI at doses of 50 and 75 mg/kg reduced spontaneous ambulatory activity of animals, although the data did not attain statistical significance. However, 7NI at doses of 100, 150 and 200 mg/kg considerably suppressed locomotor activity in mice. Considering the fact that 7NI administered alone at a dose of 120 mg/kg impaired spontaneous ambulatory activity in rats [62], it is highly likely that the NOS inhibitor at 200 mg/kg also induced acute neurotoxic effects in rats in the PIC-induced seizures [57]. Hence, the results presented by Vanaja and Ekambaram [57] may be related to general depression and intoxication of the CNS by 7NI and PIC, but not to the specific NO-dependent changes in the brain.

Nevertheless, both phenomena should be considered when explaining the effects produced by 7NI and PIC in rats. On the other hand, 7NI at low doses of 50 and 75 mg/kg potentiated the anticonvulsant effects of PB, PHT and VPA, by reducing their ED₅₀in the MES test

and did not affect significantly spontaneous ambulatory activity of animals in the Y-maze task.

It has to be emphasized that 7NI at doses of 100, 150 and 200 mg/kg suppressed MEST-induced seizures and simultaneously produced deficits in locomotor activity in animals. Hence, the question arises whether the antiseizure effects produced by 7NI in the MES and MEST tests are evoked by some specific mechanisms of action of 7NI related to changes in NO content in the brain or rather they are attributable to a general depression of the CNS which should be borne in mind while interpreting its effects from the Y-maze test in mice.

Based on our preclinical study, one can conclude that 7NI by increasing the electroconvulsive threshold in mice has a potent antiseizure potential against electroconvulsions in the dose range between 100–200 mg/kg.

The 7NI-induced enhancement of the anticonvulsant efficacy of four conventional AEDs in the MES test and the lack of any pharmacokinetic changes evoked by 7NI in total brain AED concentrations provide evidence of the favorable pharmacodynamic interactions between the investigated drugs. The suppression of spontaneous ambulatory activity of animals following the systemic (ip) administration of 7NI at high doses speaks against its use as an anticonvulsant agent in preclinical studies. Since the effects produced by 7NI were not reversed by the pretreatment with L-Arg, more advanced studies are necessary to elucidate this phenomenon.

Acknowledgment:

This study was supported by a grant from Medical University of Lublin. The authors are grateful for the generous gifts

of valproate sodium salt from ICN Polfa S.A. (Rzeszów, Poland) and carbamazepine from Polfa (Starogard, Poland).

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Received:

March 21, 2006; in revised form: 17 July, 2006