Effects of three N-(carboxyanilinomethyl) derivatives of p-isopropoxyphenylsuccinimide on the anticonvulsant action of carbamazepine, phenobarbital, phenytoin and valproate in the mouse maximal electroshock-induced seizure model

European Journal of Pharmacology 648 (2010) 74–79

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European Journal of Pharmacology j o u r n a l h o m e p a g e : w w w. e l s ev i e r. c o m / l o c a t e / e j p h a r

Neuropharmacology and Analgesia

Effects of three N-(carboxyanilinomethyl) derivatives of p-isopropoxyphenylsuccinimide on the anticonvulsant action of carbamazepine, phenobarbital, phenytoin and valproate in the mouse maximal electroshock-induced seizure model Jarogniew J. Luszczki a,b,⁎, Janina D. Cioczek a, Sergey L. Kocharov c, Marta Andres-Mach b, Mateusz Kominek a, Dorota Zolkowska d a

Department of Pathophysiology, Medical University of Lublin, Jaczewskiego 8, PL 20-090 Lublin, Poland Isobolographic Analysis Laboratory, Institute of Agricultural Medicine, Jaczewskiego 2, PL 20-950 Lublin, Poland Mndjoyan's Institute of Fine Organic Chemistry, National Academy of Sciences, Azatutyan Avenue 26, RA 375014 Yerevan, Armenia d Department of Neurology, UC Davis School of Medicine, 4635 2nd Avenue, Sacramento, CA 95817, USA b c

a r t i c l e

i n f o

Article history: Received 8 April 2010 Received in revised form 10 July 2010 Accepted 21 August 2010 Available online 6 September 2010 Keywords: Antiepileptic drug Maximal electroshock-induced seizure Pharmacokinetic/pharmacodynamic interaction Succinimide derivative

a b s t r a c t The aim of the study was to determine the influence of N-(ortho-carboxyanilinomethyl)-p-isopropoxyphenylsuccinimide [o-CAMIPPS], N-(meta-carboxyanilinomethyl)-p-isopropoxyphenylsuccinimide [mCAMIPPS], and N-(para-carboxyanilinomethyl)-p-isopropoxyphenylsuccinimide [p-CAMIPPS] on the protective activity of four classical antiepileptic drugs (carbamazepine, phenobarbital, phenytoin and valproate) in the mouse maximal electroshock seizure model. The results indicate that all tested succinimide derivatives administered intraperitoneally at doses of 75 and 150 mg/kg significantly elevated the threshold for electroconvulsions in mice. Succinimide derivatives at a dose of 37.5 mg/kg had no effect on the threshold for electroconvulsions in mice. Furthermore, o-CAMIPPS (37.5 mg/kg) significantly reduced the anticonvulsant activity of carbamazepine, but not that of phenobarbital, phenytoin and valproate in the maximal electroshockinduced seizures in mice. Anticonvulsant efficacy of carbamazepine, phenobarbital, phenytoin and valproate in the maximal electroshock-induced seizures in mice was not changed after administration of m-CAMIPPS or p-CAMIPPS. Pharmacokinetic experiment revealed that o-CAMIPPS significantly increased total brain concentrations of carbamazepine in mice. In conclusion, the reduced anticonvulsant action of carbamazepine by o-CAMIPPS in the maximal electroshock-induced seizures, despite the increased total brain carbamazepine concentrations after combined administration of carbamazepine with o-CAMIPPS, may suggest the antagonistic interaction between drugs. The combinations of m-CAMIPPS or p-CAMIPPS with carbamazepine, phenobarbital, phenytoin and valproate were neutral from a preclinical viewpoint. © 2010 Elsevier B.V. All rights reserved.

1. Introduction Epilepsy, the condition of recurring seizures, is a damaging neurological disorder affecting around 50 million people worldwide. Epileptic seizures occur as a result of imbalance between inhibitory and excitatory neurotransmission in the brain. Despite clear advances in the development of antiepileptic drugs, during recent years about one third of the epilepsy population continue to have seizures (Kwan and Brodie, 2000a). While monotherapy is the most preferable treatment and results in fewer adverse side effects, approximately 40% of patients require treatment with more than one antiepileptic ⁎ Corresponding author. Department of Pathophysiology, Medical University of Lublin, Jaczewskiego 8, PL 20-090 Lublin, Poland. Tel.: + 48 81 718 73 65; fax: + 48 81 718 73 64. E-mail address: [email protected] (J.J. Luszczki). 0014-2999/$ – see front matter © 2010 Elsevier B.V. All rights reserved. doi:10.1016/j.ejphar.2010.08.017

drug (Beghi et al., 2003; Kwan and Brodie, 2000b). Animal based studies allow the comprehensive antiepileptic screening of thousands of newly synthesized substances for potential anticonvulsant activity (White, 2003; White et al., 2002). In experimental epileptology, to detect a substance possessing anticonvulsant activity, a vast number of compounds undergo examination in the initial anticonvulsant screening test in rodents (Stables and Kupferberg, 1997; White et al., 2002). Accumulating experimental evidence based on in vivo screening tests in rodents, indicates that some succinimide derivatives possess clear anticonvulsant properties (Lange et al., 1977; Kaminski and Obniska, 2008; Luszczki et al., 2009b; Zejc et al., 1990). In preclinical studies, numerous succinimide derivatives exerted the anticonvulsant activity in mice at various pretreatment times ranging between 15 and 120 min (Kaminski and Obniska, 2008). For instance, Nmorpholinemethyl derivative of m-bromophenylsuccinimide (Lange et al., 1977), N-pyridyl-substituted succinimides (Zejc et al., 1990),

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3-cyclohexylsuccinimides (Kaminski and Obniska, 2008), N(anilinomethyl)-p-isopropoxyphenylsuccinimide (Luszczki et al., 2009b), and p-isopropoxyphenylsuccinimide monohydrate (Luszczki et al., 2010) exhibited potent anticonvulsant effects in the maximal electroshock seizure test in mice. Therefore, in the present study, we sought to determine the effects of N-(ortho-carboxyanilinomethyl)-p-isopropoxyphenylsuccinimide (o-CAMIPPS), N-(meta-carboxyanilinomethyl)-p-isopropoxyphenylsuccinimide (m-CAMIPPS) and N-(para-carboxyanilinomethyl)-pisopropoxyphenylsuccinimide (p-CAMIPPS) on the threshold for electroconvulsions and to evaluate their effects on the protective activity of four classical antiepileptic drugs (carbamazepine, phenobarbital, phenytoin and valproate) in the mouse maximal electroshock-induced seizure model. The threshold for electroconvulsions and the maximal electroshock seizure test are both thought to be experimental models of tonic–clonic seizures and, to a certain extent, of partial convulsions with or without secondary generalization in humans (Löscher et al., 1991). These experimental tests provide fast assessment of the antiseizure potential of agents and compounds possessing anticonvulsant properties (Stables and Kupferberg, 1997; White et al., 2002). Furthermore, these tests elucidate the effects of newly synthesized compounds on classical antiepileptic drugs that are currently effective in suppressing tonic–clonic seizures observed in humans (Löscher et al., 1991). Therefore, it was appropriate to use both tests in order to evaluate the anticonvulsive properties of oCAMIPPS, m-CAMIPPS and p-CAMIPPS. Additionally, acute side effects of o-CAMIPPS, m-CAMIPPS and p-CAMIPPS alone and in combination with four classical antiepileptic drugs were investigated regarding impairment of motor coordination, long-term memory and muscular strength utilizing the chimney test, step-through passive avoidance task and grip-strength test, respectively. Finally, total brain antiepileptic drug concentrations were measured with fluorescence polarization immunoassay to ascertain the observed effects as a consequence of the pharmacodynamic and/or a pharmacokinetic interaction.

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Mndjoyan's Institute of Fine Organic Chemistry of the National Academy of Sciences of the Republic of Armenia, Yerevan, Armenia), carbamazepine (a gift from Polpharma, Starogard Gdanski, Poland), phenobarbital (Polfa, Krakow, Poland), phenytoin (Polfa, Warszawa, Poland) and valproate (magnesium salt—kindly donated by ICN-Polfa S.A., Rzeszow, Poland). All drugs, except for valproate, were suspended in a 1% solution of Tween 80 (Sigma, St. Louis, MO, USA) in distilled water, while valproate was directly dissolved in distilled water. All drugs were administered intraperitoneally (i.p.) as a single injection, in a volume of 5 ml/kg body weight. Fresh drug solutions were prepared on each day of experimentation and administered as follows: phenytoin—120 min; phenobarbital, o-CAMIPPS, m-CAMIPPS, p-CAMIPPS—60 min; carbamazepine and valproate—30 min before electroconvulsions, motor coordination, grip-strength and long-term memory tests and before brain sampling for the measurement of antiepileptic drug concentrations. The pretreatment times before testing of the antiepileptic drugs were based upon information about their biological activity from the literature and our previous experiments (Luszczki et al., 2009a,b, 2010). The times to the peak of maximum anticonvulsant effects for all antiepileptic drugs were used as the reference times in all behavioral tests and pharmacokinetic estimation of brain antiepileptic drug concentrations. The pretreatment time (60 min) before testing all tested succinimide derivatives was established in our pilot study as the peak time of maximum anticonvulsant activity (unpublished data). 2.3. Electroconvulsions Electroconvulsions were induced by applying an alternating current (50 Hz; 500 V) via ear-clip electrodes from a rodent shocker generator (type 221; Hugo Sachs Elektronik, Freiburg, Germany). The stimulus duration was 0.2 s. Tonic hind limb extension was used as the endpoint. This apparatus was used to induce seizures in two methodologically different experimental approaches: maximal electroshock seizure threshold test and maximal electroshock seizure test (Löscher et al., 1991).

2. Materials and methods 2.1. Animals and experimental conditions Adult male Swiss mice (weighing 22–26 g) that were kept in colony cages with free access to food and tap water, housed under standardized housing conditions (natural light–dark cycle, temperature of 23 ± 1 °C, relative humidity of 55 ± 5%), were used. After seven days of adaptation to laboratory conditions, the animals were randomly assigned to experimental groups each comprised of eight mice. Each mouse was used only once and all tests were performed between 08:00 a.m. and 03:00 p.m. Procedures involving animals and their care were conducted in accordance with current European Community and Polish legislation on animal experimentation. Additionally, all efforts were made to minimize animal suffering and to use only the number of animals necessary to produce reliable scientific data. The experimental protocols and procedures described in this manuscript were approved by the First Local Ethics Committee at the Medical University of Lublin (License no.: 18/2006) and the Second Local Ethics Committee at the University of Life Sciences in Lublin (License no.: 79/2009), and complied with the European Communities Council Directive of 24 November 1986 (86/609/EEC). 2.2. Drugs The following drugs were used: N-(ortho-carboxyanilinomethyl)-pisopropoxyphenylsuccinimide (o-CAMIPPS), N-(meta-carboxyanilinomethyl)-p-isopropoxyphenylsuccinimide (m-CAMIPPS), and N-(paracarboxyanilinomethyl)-p-isopropoxyphenylsuccinimide (p-CAMIPPS) (o-CAMIPPS, m-CAMIPPS, p-CAMIPPS—synthesized by Dr. S.L. Kocharov,

2.3.1. Maximal electroshock seizure threshold test The maximal electroshock seizure threshold test was first used to assess the anticonvulsant effects of succinimide derivatives administered alone. In this test, at least 4 groups of control mice, each consisting of 8 animals, were challenged with currents of varying intensities ranging between 5 and 8 mA so that 10–30%, 30–50%, 50– 70% and 70–90% of animals exhibited the endpoint. After establishing the current intensity–effect curve (i.e., current intensity in mA vs. percentage of mice convulsing) for each dose of succinimide derivatives tested, the electroconvulsive threshold was calculated according to the log-probit method of Litchfield and Wilcoxon (1949). The electroconvulsive threshold was expressed as the median current strength value (CS50 in mA) predicted to produce tonic hind limb extension in 50% of the animals tested. This experimental procedure was performed for various increasing doses of tested succinimide derivatives (37.5, 75 and 150 mg/kg), until the threshold for electroconvulsions of succinimide derivatives-injected animals was statistically different from that of the control animals. Only doses of o-CAMIPPS, m-CAMIPPS, p-CAMIPPS that did not significantly affect the seizure threshold in the maximal electroshock seizure threshold test were selected for testing in combination with four classical antiepileptic drugs in the maximal electroshock seizure test (see the following discussion). This approach allowed us to rule out any contribution of the intrinsic anticonvulsant efficacy of succinimide derivatives in the effects observed in combination with the antiepileptic drugs in the maximal electroshock seizure test. This experimental procedure has been described in more detail in our earlier studies (Luszczki et al., 2009a,b).

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2.3.2. Maximal electroshock seizure test In the maximal electroshock seizure test, mice were challenged with a current of the fixed intensity (25 mA) that was 4–5-fold higher than the CS50 value in vehicle-treated control mice (Löscher et al., 1991). These parameters of stimulation (maximal electroshock) typically result in all mice responding with tonic hind limb extension immediately after stimulation. The antiepileptic drugs administered alone and their combination with o-CAMIPPS, m-CAMIPPS, pCAMIPPS were tested for their ability to increase the number of animals not responding with tonus (i.e., protected from tonic hind limb extension) after stimulation. Again, at least 4 groups of mice, each consisting of 8 animals and treated with a different dose of the antiepileptic drug alone or in combination with succinimide derivatives, were challenged with a current of 25 mA to yield 10–30%, 30– 50%, 50–70% and 70–90% of animals protected from tonic seizures. After constructing a dose–effect curve (i.e., dose in mg/kg vs. percentage of mice protected), the protective median effective dose (ED50) value of the antiepileptic drug tested was calculated according to a log-probit method (Litchfield and Wilcoxon, 1949). Each ED50 value represented a dose of the antiepileptic drug (in mg/kg) predicted to protect 50% of mice tested against maximal electroshock-induced extension of the hind limbs. o-CAMIPPS, m-CAMIPPS and p-CAMIPPS were tested for its ability to affect the anticonvulsive potency of antiepileptic drugs. As mentioned earlier, all tested succinimide derivatives were administered in doses that per se had no effect on seizure threshold in the maximal electroshock seizure threshold test. In this experimental protocol, an increase in the anticonvulsant potency of the antiepileptic drug tested in combination with o-CAMIPPS, m-CAMIPPS or p-CAMIPPS would be reflected by a lower ED50 value of the test antiepileptic drug (i.e., lower dose of test drug was necessary to protect 50% of mice challenged). In contrast, a decrease in the anticonvulsant potency of the antiepileptic drug tested in combination with o-CAMIPPS, m-CAMIPPS or p-CAMIPPS would be reflected by a higher ED50 value of the test antiepileptic drug (i.e., higher dose of test drug was necessary to protect 50% of mice challenged). In the present study, carbamazepine and phenytoin were administered at doses ranging between 6 and 14 mg/kg, phenobarbital at doses ranging between 10 and 24 mg/kg and valproate at doses ranging between 125 and 275 mg/kg. This experimental procedure has been described in detail in our earlier studies (Luszczki et al., 2009a,b).

drug concentrations are expressed in μg/ml of brain supernatants as means ± S.D. of at least eight separate brain preparations. 2.5. Chimney test Potential adverse effects of classical antiepileptic drugs administered in combination with o-CAMIPPS, m-CAMIPPS or p-CAMIPPS were evaluated using the chimney test of Boissier et al. (1960). In this test, mice were placed at the bottom of a plastic, transparent tube (3 cm inner diameter, 30 cm long) and had to climb backwards up to the top of the tube. Impairment of motor performance was indicated by the inability of the mice to climb out of the tube within 60 and the results were expressed as the percentages of animals that failed to perform the task. Combinations of classical antiepileptic drugs, administered at doses corresponding to their ED50 values from the maximal electroshock seizure test, with 37.5 mg/kg o-CAMIPPS, mCAMIPPS or p-CAMIPPS were evaluated for potential acute adverseeffects. This experimental procedure has been described in detail in our earlier studies (Luszczki et al., 2009a,b). 2.6. Grip-strength test The effects of combinations of 37.5 mg/kg o-CAMIPPS, m-CAMIPPS or p-CAMIPPS with classical antiepileptic drugs at doses corresponding to their ED50 values from the maximal electroshock seizure test on skeletal muscular strength in mice were quantified by the grip-strength test of Meyer et al. (1979). The time before the commencement of the grip-strength test (after drug administration) was identical to that for the maximal electroshock seizure test. The grip-strength apparatus (BioSeb, Chaville, France) was equipped with a wire grid (8 × 8 cm) connected to an isometric force transducer (dynamometer). The mice were lifted by the tails so that their forepaws could grasp the grid. The mice were then gently pulled backward by the tail until the grid was released. The maximal force exerted by the mouse before losing grip was recorded. The mean of three measurements for each animal was calculated and subsequently, the mean maximal force of eight animals per group was determined. The muscular strength in mice is expressed in N (Newtons) as means ± S.E. of at least eight determinations. This experimental procedure has been described in detail in our earlier studies (Luszczki et al., 2009a,b).

2.4. Measurement of total brain antiepileptic drug concentrations

2.7. Step-through passive avoidance task

Pharmacokinetic evaluation of total brain antiepileptic drug concentration was performed only for those combinations of succinimide derivatives with antiepileptic drugs for which the anticonvulsant effect in the maximal electroshock seizure test was significantly different than that of control (an antiepileptic drug +vehicle-treated) animals. Thus, the measurement of total brain concentration of carbamazepine was undertaken at the dose that corresponded to the ED50 value of carbamazepine from the maximal electroshock seizure test. Specifically, mice pretreated with carbamazepine alone or in combination with o-CAMIPPS were decapitated at times reflecting the peak of maximum anticonvulsant effects for the drugs in the maximal electroshock seizure test. The whole brains of mice were removed from skulls, weighed, harvested and homogenized using Abbott buffer (1:2 weight/volume; Abbott Laboratories, North Chicago, IL, USA) in an Ultra-Turrax T8 homogenizer (IKA Werke, Staufen, Germany). The homogenates were then centrifuged at 10,000 ×g for 10 min and the supernatant samples of 100 μl were collected and then analyzed for antiepileptic drug content. Total brain concentrations of carbamazepine were measured by a fluorescence polarization immunoassay using an analyzer (Abbott TDx) and manufacturer-supplied reagent kits (Abbott Laboratories, North Chicago, IL, USA). Total brain antiepileptic

The effects of classical antiepileptic drugs applied alone at doses equal to their ED50 values against the maximal electroshock seizure test, or in combination with 37.5 mg/kg of succinimide derivatives (o-CAMIPPS, m-CAMIPPS or p-CAMIPPS) on long-term memory were evaluated. Each animal was administered an antiepileptic drug either alone or in combination with succinimide derivatives, on the first day before training. The pretreatment time of drugs in the passive avoidance task was identical to that of the maximal electroshock seizure test. Subsequently, animals were placed in an illuminated box (10×13×15 cm) connected to a larger dark box (25×20×15 cm) equipped with an electric grid floor. Entrance of pretreated animals to the dark box was punished by an adequate electric footshock (0.6 mA for 2 s). The animals that did not enter the dark compartment were excluded from subsequent experimentation. On the following day (24 h later), the pre-trained animals were placed again into the illuminated box and observed up to 180 s. Mice that avoided the dark compartment for 180 s were considered to remember the task. The time that the mice took to enter the dark box was noted and the median latencies (retention times) with 25th and 75th percentiles were calculated. The step-through passive avoidance task gives information about ability to acquire a task (learning) and to recall a task (retrieval). Therefore, it may be regarded as a measure of long-term

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memory (Venault et al., 1986). This experimental procedure has been described in detail in our earlier studies (Luszczki et al., 2005). 2.8. Statistics Both CS50 and ED50 values with their 95% confidence limits were calculated by computer log-probit analysis according to Litchfield and Wilcoxon (1949). Subsequently, the respective 95% confidence limits were transformed to S.E. as described previously (Luszczki et al., 2009a). Statistical analysis of data from the maximal electroshock seizure threshold test was performed with one-way analysis of variance (ANOVA) followed by the post-hoc Tukey–Kramer test for multiple comparisons among four CS50 values. Statistical analysis of data from the maximal electroshock seizure test was performed either with Litchfield and Wilcoxon method for a comparison between two ED50 values or with one-way ANOVA followed by the post-hoc Tukey– Kramer test for multiple comparisons among three ED50 values. Total brain antiepileptic drug concentrations were statistically compared using the unpaired Student's t-test. Qualitative variables from the chimney test were compared by use of the Fisher's exact probability test. The results obtained in the step-through passive avoidance task were statistically evaluated using Kruskal–Wallis nonparametric ANOVA. The results from the grip-strength test were verified with one-way ANOVA. Differences among values were considered statistically significant if P b 0.05. All statistical tests were performed using commercially available GraphPad Prism version 4.0 for Windows (GraphPad Software, San Diego, CA, USA). 3. Results 3.1. Influence of o-CAMIPPS, m-CAMIPPS, and p-CAMIPPS on the threshold for electroconvulsions All tested succinimide derivatives administered systemically (i.p., 60 min prior to the test) at doses of 75 and 150 mg/kg significantly elevated the threshold for electroconvulsions in mice. o-CAMIPPS Table 1 Effect of N-(o-carboxyanilinomethyl)-p-isopropoxyphenylsuccinimide (o-CAMIPPS), N-(m-carboxyanilinomethyl)-p-isopropoxyphenylsuccinimide (m-CAMIPPS), and N(p-carboxyanilinomethyl)-p-isopropoxyphenylsuccinimide (p-CAMIPPS) on the threshold for electroconvulsions in mice. Treatment (mg/kg) Vehicle o-CAMIPPS (37.5) o-CAMIPPS (75) o-CAMIPPS (150) F (3;84) = 4.136; P = 0.0087 Vehicle m-CAMIPPS (37.5) m-CAMIPPS (75) m-CAMIPPS (150) F (3;92) = 12.65; P b 0.0001 Vehicle p-CAMIPPS (37.5) p-CAMIPPS (75) p-CAMIPPS (150) F (3;84) = 9.28; P b 0.0001

CS50 (mA)

n

5.66 ± 0.37 6.75 ± 0.53 7.75 ± 0.47a 8.20 ± 0.60b

16 24 24 24

6.40 ± 0.42 7.20 ± 0.45 8.36 ± 0.44a 12.26 ± 1.08c

24 32 32 8

5.66 ± 0.37 6.51 ± 0.41 8.09 ± 0.48a 9.87 ± 0.85c

16 24 24 24

Data are presented as median current strengths (CS50 values ± S.E.), required to produce tonic hind limb extension in 50% of animals tested in the maximal electroshock-induced seizure threshold test. o-CAMIPPS, m-CAMIPPS and p-CAMIPPS were administered i.p. 60 min before the test. Statistical evaluation of the data was performed with log-probit method (Litchfield and Wilcoxon, 1949) and one-way ANOVA followed by the post-hoc Tukey–Kramer test for multiple comparisons. n— number of animals tested at those current strength intensities, whose seizure effects ranged between 16% and 84%; F—F-statistics from one-way ANOVA; P—probability from one-way ANOVA. a P b 0.05 vs. the control (vehicle-treated) animals. b P b 0.01, vs. the control (vehicle-treated) animals. c P b 0.001 vs. the control (vehicle-treated) animals.

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administered at doses of 75 and 150 mg/kg significantly increased the electroconvulsive threshold from 5.66 mA to 7.75 (P b 0.05) and 8.20 mA (P b 0.01), respectively (Table 1). m-CAMIPPS increased the electroconvulsive threshold from 6.40 mA to 8.36 (P b 0.05) and 12.26 mA (P b 0.001), respectively (Table 1). p-CAMIPPS increased the electroconvulsive threshold from 5.66 mA to 8.09 (P b 0.05) and 9.87 mA (P b 0.001), respectively (Table 1). All tested succinimide derivatives at a lower dose of 37.5 mg/kg did not significantly affect the control CS50 values (Table 1). 3.2. Effects of o-CAMIPPS, m-CAMIPPS and p-CAMIPPS on the protective action of carbamazepine, phenobarbital, phenytoin and valproate in the mouse maximal electroshock seizure model All investigated classical antiepileptic drugs (carbamazepine, phenobarbital, phenytoin and valproate) administered alone exhibited a clear anticonvulsant activity in the maximal electroshock seizure test in mice and their ED50 values are presented in Table 2. Co-administration of o-CAMIPPS in the dose of 37.5 mg/kg with carbamazepine, significantly diminished the anticonvulsant action of the latter drug in the maximal electroshock seizure test. o-CAMIPPS reduced the ED50 value of carbamazepine from 10.77 mg/kg to 14.16 mg/kg (by 24%; P b 0.05; Table 2). In contrast, o-CAMIPPS administered at the dose of 18.75 mg/kg had no significant impact on the antiseizure action of carbamazepine against maximal electroshock-induced seizures (Table 2).

Table 2 Effect of N-(o-carboxyanilinomethyl)-p-isopropoxyphenylsuccinimide (o-CAMIPPS), N-(m-carboxyanilinomethyl)-p-isopropoxyphenylsuccinimide (m-CAMIPPS), and N(p-carboxyanilinomethyl)-p-isopropoxyphenylsuccinimide (p-CAMIPPS) on the protective activity of four classical antiepileptic drugs against maximal electroshockinduced seizures in mice. Treatment (mg/kg)

ED50 (mg/kg)

n

Carbamazepine + vehicle Carbamazepine + o-CAMIPPS (18.75) Carbamazepine + o-CAMIPPS (37.5) F (2;61) = 3.696; P = 0.0305 Carbamazepine + vehicle Carbamazepine + m-CAMIPPS (37.5) Carbamazepine + vehicle Carbamazepine + p-CAMIPPS (37.5) Phenobarbital + vehicle Phenobarbital + o-CAMIPPS (37.5) Phenobarbital + vehicle Phenobarbital + m-CAMIPPS (37.5) Phenobarbital + vehicle Phenobarbital + p-CAMIPPS (37.5) Phenytoin + vehicle Phenytoin + o-CAMIPPS (37.5) Phenytoin + vehicle Phenytoin + m-CAMIPPS (37.5) Phenytoin + vehicle Phenytoin + p-CAMIPPS (37.5) Valproate + vehicle Valproate + o-CAMIPPS (37.5) Valproate + vehicle Valproate + m-CAMIPPS (37.5) Valproate + vehicle Valproate + p-CAMIPPS (37.5)

10.77 (9.07–12.79) 11.58 (10.07–13.31) 14.16 (12.40–16.16)a

16 24 24

0.95 0.82 0.96

10.54 8.96 10.22 8.11 20.05 20.80 23.25 21.78 23.25 24.10 10.77 11.92 10.90 10.11 10.90 10.09 289.0 266.0 263.9 235.8 263.9 281.3

16 24 16 8 16 32 16 16 16 16 16 32 32 24 32 24 16 24 16 16 16 24

0.88 0.43 0.80 0.84 1.60 2.01 1.83 2.37 1.83 1.51 0.95 0.93 0.86 0.73 0.86 0.95 11.36 11.96 11.38 11.74 11.38 11.70

(8.96–12.41) (8.16–9.84) (8.77–11.91) (6.61–9.94) (17.15–23.44) (17.20–25.15) (19.93–27.12) (17.59–26.97) (19.93–27.12) (21.31–27.26) (9.07–12.79) (10.23–13.89) (9.34–12.72) (8.77–11.66) (9.34–12.72) (8.40–12.13) (267.5–312.2) (243.5–290.4) (242.5–287.2) (213.9–260.0) (242.5–287.2) (259.2–305.2)

S.E.

Results are presented as median effective doses (ED50 in mg/kg, with 95% confidence limits in parentheses) of antiepileptic drugs, protecting 50% of animals tested against maximal electroshock-induced hindlimb extension. All antiepileptic drugs were administered i.p.: phenytoin—120 min, phenobarbital—60 min, carbamazepine and valproate—30 min prior to the maximal electroshock-induced seizure test. o-CAMIPPS, m-CAMIPPS, and p-CAMIPPS were administered i.p. at 60 min before the maximal electroshock-induced seizure test. Statistical analysis of data was performed either with log-probit method for single comparisons or with one-way ANOVA followed by the post-hoc Tukey–Kramer test for multiple comparisons. n—total number of animals used at those doses whose anticonvulsant effects ranged between 4 and 6 probits; S.E.— standard error of ED50s. F—F-statistics from one-way ANOVA; P—probability from oneway ANOVA. a P b 0.05 vs. the respective control (an antiepileptic drug + vehicle-treated) animals.

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Treatment (mg/kg)

Brain concentration (μg/ml)

Additionally, all tested succinimide derivatives co-administered with classical antiepileptic drugs had no significant impact on skeletal muscular strength of the animals, as assessed by the grip-strength test (Table 4).

Carbamazepine (14.2) + vehicle Carbamazepine (14.2) + o-CAMIPPS (37.5)

3.31 ± 0.59 4.22 ± 0.62a

4. Discussion

Table 3 Brain concentrations of carbamazepine administered singly or in combination with N-(o-carboxyanilinomethyl)-p-isopropoxyphenylsuccinimide (o-CAMIPPS).

Data are presented as mean concentrations (in μg/ml ± S.D. of 8 determinations) of carbamazepine in the brain of experimental animals. Statistical evaluation of data was performed with unpaired Student's t-test. Brain tissue samples were taken at times scheduled for the maximal electroshock-induced seizure test and the total brain antiepileptic drug concentrations were quantified using fluorescence polarization immunoassay. For more details see the legend to Table 2. a P b 0.01 vs. the control group (carbamazepine + vehicle-treated animals).

Moreover, o-CAMIPPS did not change the ED50 values in the maximal electroshock seizure test when co-administered with phenobarbital, phenytoin or valproate (Table 2). Neither m-CAMIPPS nor p-CAMIPPS had significant effect on the anticonvulsant activity of any tested classical antiepileptic drugs in the maximal electroshock seizure test (Table 2). 3.3. Influence of o-CAMIPPS on total brain antiepileptic drug concentrations As determined by the fluorescence polarization immunoassay method, 37.5 mg/kg o-CAMIPPS significantly increased total brain concentration of co-administered carbamazepine at a dose of 14.2 mg/kg (by 27%; Pb 0.01; Table 3). 3.4. Effects of o-CAMIPPS, m-CAMIPPS and p-CAMIPPS in combination with various antiepileptic drugs on motor performance, long-term memory and skeletal muscular strength of animals in the chimney, step-through passive avoidance and grip-strength tests All tested succinimide derivatives (o-CAMIPPS, m-CAMIPPS and p-CAMIPPS) administered in combination with carbamazepine, phenobarbital, phenytoin and valproate at doses equal to their ED50 values against the maximal electroshock seizure did not affect motor performance in mice subjected to the chimney test (Table 4). Furthermore, none of the combinations studied impaired longterm memory as determined in the passive avoidance test (Table 4).

The results indicate that all succinimide derivatives tested in the current study elevated, in a dose-dependent manner, the threshold for electroconvulsions in mice. o-CAMIPPS at the sub-protective dose of 37.5 mg/kg (the dose that by itself did not significantly affect the threshold for electroconvulsions) diminished the anticonvulsant activity of carbamazepine against maximal electroshock-induced seizures in mice with no significant effect on the antielectroshock action of phenobarbital, phenytoin or valproate. It should be stressed that a similar situation has been documented for niguldipine (a calcium channel inhibitor). It has been found that niguldipine at a dose of 5 mg/kg significantly elevated the threshold for electroconvulsions in mice and reduced the anticonvulsant action of carbamazepine and phenobarbital, but not that of phenytoin and valproate in the mouse maximal electroshock-induced seizure model (Borowicz et al., 1997). o-CAMIPPS co-administered with carbamazepine significantly increased brain concentrations of the latter drug, thus, suggesting an antagonistic interaction. In this study, total brain antiepileptic drug concentrations were verified with fluorescence polarization immunoassay technique. As reported in literature, only total brain antiepileptic drug concentrations provide the exact classification and characterization of interactions between antiepileptic drugs (Cadart et al., 2002; Luszczki et al., 2003). It should be mentioned that o-CAMIPPS could affect total brain concentrations of the remaining antiepileptic drugs (phenobarbital, phenytoin and valproate). However, the concentrations of phenobarbital, phenytoin and valproate were not measured in this study because o-CAMIPPS did not affect the anticonvulsive activity of these classical antiepileptic drugs against maximal electroshock-induced seizures in mice. Moreover, m-CAMIPPS and p-CAMIPPS did not affect the protective action of four classical antiepileptic drugs against maximal electroshock-induced seizures in mice. Since total brain carbamazepine concentrations were significantly elevated after administration of o-CAMIPPS in mice, one could suppose that the

Table 4 Effects of N-(o-carboxyanilinomethyl)-p-isopropoxyphenylsuccinimide (o-CAMIPPS), N-(m-carboxyanilinomethyl)-p-isopropoxyphenylsuccinimide (m-CAMIPPS), and N-(pcarboxyanilinomethyl)-p-isopropoxyphenylsuccinimide (p-CAMIPPS) and its combinations with four classical antiepileptic drugs on long-term memory, muscular strength and motor performance in mice. Treatment (mg/kg)

Retention time (s)

Grip-strength (N)

Motor coordination impairment (%)

Vehicle o-CAMIPPS (37.5) + vehicle m-CAMIPPS (37.5) + vehicle p-CAMIPPS (37.5) + vehicle Carbamazepine (14.2) + o-CAMIPPS (37.5) Carbamazepine (9.0) + m-CAMIPPS (37.5) Carbamazepine (8.1) + p-CAMIPPS (37.5) Phenobarbital (20.8) + o-CAMIPPS (37.5) Phenobarbital (21.8) + m-CAMIPPS (37.5) Phenobarbital (24.1) + p-CAMIPPS (37.5) Phenytoin (11.9) + o-CAMIPPS (37.5) Phenytoin (10.1) + m-CAMIPPS (37.5) Phenytoin (10.1) + p-CAMIPPS (37.5) Valproate (266.0) + o-CAMIPPS (37.5) Valproate (235.8) + m-CAMIPPS (37.5) Valproate (281.3) + p-CAMIPPS (37.5)

180 180 180 180 180 180 180 180 180 180 180 180 180 180 180 180

102.5 ± 5.54 104.9 ± 5.79 103.1 ± 5.79 103.9 ± 5.64 103.9 ± 5.29 101.9 ± 5.15 102.7 ± 5.39 103.6 ± 6.10 100.6 ± 5.18 100.5 ± 5.72 102.9 ± 5.69 102.7 ± 5.49 101.2 ± 5.60 101.9 ± 5.78 100.8 ± 5.78 102.9 ± 5.26

0 0 0 0 0 0 0 0 0 0 0 0 0 25 25 12.5

(180; (180; (180; (180; (180; (180; (180; (180; (180; (180; (180; (180; (180; (180; (180; (180;

180) 180) 180) 180) 180) 180) 180) 180) 180) 180) 180) 180) 180) 180) 180) 180)

Results are presented as: 1) median retention times (in seconds (s); with 25th and 75th percentiles in parentheses) from the passive avoidance task, assessing long-term memory in mice; 2) mean grip-strengths (in Newtons (N) ± S.E.) from the grip-strength test, assessing muscular strength in mice; and 3) percentage (%) of animals showing motor coordination impairment in the chimney test in mice. Each experimental group consisted of 8 mice. Statistical analysis of data from the passive avoidance task was performed with nonparametric Kruskal–Wallis ANOVA test, whereas those from the grip-strength test were analyzed with one-way ANOVA. The Fisher's exact probability test was used to analyze the results from the chimney test. All drugs were administered i.p. at times scheduled from the maximal electroshock-induced seizures and at doses corresponding to their ED50 values against maximal electroconvulsions in mice (for more details see the legend to Table 2).

J.J. Luszczki et al. / European Journal of Pharmacology 648 (2010) 74–79

total brain concentrations of the remaining antiepileptic drugs were also increased despite no significant effect in the mouse maximal electroshock-induced seizure model. It is highly likely that combinations of m-CAMIPPS and p-CAMIPPS with carbamazepine, phenobarbital, phenytoin and valproate might involve pharmacokinetic interactions, although the effects in the mouse maximal electroshock-induced seizure model were neutral. Comparing the effects produced by o-CAMIPPS, m-CAMIPPS or p-CAMIPPS with those reported earlier for N-(anilinomethyl)-pisopropoxyphenylsuccinimide and p-isopropoxyphenylsuccinimide monohydrate, it is noteworthy, that the carboxyanilinomethyl group substituted to a different location (ortho-, meta- or para-) modified the physico-chemical properties of the tested compounds, thus, changing their pharmacological profile in vivo. It is important to note that o-CAMIPPS at a dose of 37.5 mg/kg significantly decreased the anticonvulsant action of carbamazepine, whereas neither m-CAMIPPS, pCAMIPPS, N-(anilinomethyl)-p-isopropoxyphenylsuccinimide nor p-isopropoxyphenylsuccinimide monohydrate altered the anticonvulsive activity of carbamazepine in the maximal electroshock-induced seizures test in mice (Luszczki et al., 2009b, 2010). Additionally, o-CAMIPPS, mCAMIPPS and p-CAMIPPS did not change anticonvulsive activity of phenobarbital, phenytoin and valproate in the maximal electroshockinduced seizures test. In comparison, previously tested N-(anilinomethyl)-p-isopropoxyphenylsuccinimide potentiated the anticonvulsant action of phenobarbital and valproate and p-isopropoxyphenylsuccinimide monohydrate significantly enhanced the anticonvulsant action of phenytoin and valproate in the maximal electroshock-induced seizures test in mice (Luszczki et al., 2009b, 2010). It has been reported that the interaction between N-(anilinomethyl)-p-isopropoxyphenylsuccinimide and valproate was affected by a significant pharmacokinetic increase in total brain valproate concentration (Luszczki et al., 2009b), whereas, p-isopropoxyphenylsuccinimide monohydrate interaction with valproate was pharmacodynamic in nature because p-isopropoxyphenylsuccinimide monohydrate had no impact on the total brain valproate concentration. The mechanism of interaction between different succinimide derivatives and classic antiepileptic drugs is unknown, at present, and more advanced molecular, neurochemical and electrophysiological studies are required. The evaluation of acute adverse effects in this study indicates that oCAMIPPS, m-CAMIPPS and p-CAMIPPS applied at a dose of 37.5 mg/kg in combination with four classical antiepileptic drugs had no impact on long-term memory, muscular strength and motor performance of the animals tested. In summary, results obtained in this study revealed that chemical substitution of the carboxyanilinomethyl group to different location (ortho-, meta- or para-) of p-isopropoxyphenylsuccinimide does not create valuable compounds with no added benefit which would be valuable addition to classical antiepileptic drugs. Despite promising dose-dependent elevation of the threshold for electroconvulsions in mice, o-CAMIPPS, m-CAMIPPS and p-CAMIPPS did not express favourable interactions when co-administered with classical antiepileptic drugs. Therefore, o-CAMIPPS, m-CAMIPPS and p-CAMIPPS are unlikely to be useful in clinical settings based on the classical antiepileptic drugs. Acknowledgments This study was supported by grants from the Medical University of Lublin and the Institute of Agricultural Medicine (Lublin, Poland). The

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authors are grateful for the generous gifts of valproate from ICN-Polfa S.A. (Rzeszow, Poland) and carbamazepine from Polpharma S.A. (Starogard Gdanski, Poland). Professor J.J. Luszczki is a recipient of the Fellowship for Leading Young Researchers from the Ministry of Science and Higher Education (Warszawa, Poland).

References Beghi, E., Gatti, G., Tonini, C., Ben-Menachem, E., Chadwick, D.W., Nikanorova, M., Gromov, S.A., Smith, P.E., Specchio, L.M., Perucca, E., BASE Study Group, 2003. Adjunctive therapy versus alternative monotherapy in patients with partial epilepsy failing on a single drug: a multicentre, randomised, pragmatic controlled trial. Epilepsy Res. 57, 1–13. Boissier, J.R., Tardy, J., Diverres, J.C., 1960. A simple novel method to explore tranquilizer activity: the chimney test (in French). Med. Exp. Basel 3, 81–84. Borowicz, K.K., Gasior, M., Kleinrok, Z., Czuczwar, S.J., 1997. Influence of isradipine, niguldipine and dantrolene on the anticonvulsive action of conventional antiepileptics in mice. Eur. J. Pharmacol. 323, 45–51. Cadart, M., Marchand, S., Pariat, C., Bouquet, S., Couet, W., 2002. Ignoring pharmacokinetics may lead to isoboles misinterpretation: illustration with the norfloxacin– theophylline convulsant interaction in rats. Pharm. Res. 19, 209–214. Kaminski, K., Obniska, J., 2008. Synthesis and anticonvulsant properties of new 1-(2pyridinyl)-3-substituted pyrrolidine-2, 5-dione derivatives. Acta Pol. Pharm. 65, 457–465. Kwan, P., Brodie, M.J., 2000a. Early identification of refractory epilepsy. N. Engl. J. Med. 342, 314–319. Kwan, P., Brodie, M.J., 2000b. Epilepsy after the first drug fails: substitution or addition? Seizure 9, 464–468. Lange, J., Rump, S., Galecka, E., Ilczuk, I., Lechowska-Postek, M., Rabsztyn, T., 1977. Synthesis and properties of new cyclic derivatives of succinic acid with anticonvulsant activity. Pharmazie 32, 82–84. Litchfield, J.T., Wilcoxon, F., 1949. A simplified method of evaluating dose–effect experiments. J. Pharmacol. Exp. Ther. 96, 99–113. Löscher, W., Fassbender, C.P., Nolting, B., 1991. The role of technical, biological and pharmacological factors in the laboratory evaluation of anticonvulsant drugs. II. Maximal electroshock seizure models. Epilepsy Res. 8, 79–94. Luszczki, J.J., Swiader, M., Czuczwar, M., Kis, J., Czuczwar, S.J., 2003. Interactions of tiagabine with some antiepileptics in the maximal electroshock in mice. Pharmacol. Biochem. Behav. 75, 319–327. Luszczki, J.J., Wojcik-Cwikla, J., Andres, M.M., Czuczwar, S.J., 2005. Pharmacological and behavioral characteristics of interactions between vigabatrin and conventional antiepileptic drugs in pentylenetetrazole-induced seizures in mice: an isobolographic analysis. Neuropsychopharmacology 30, 958–973. Luszczki, J.J., Antkiewicz-Michaluk, L., Czuczwar, S.J., 2009a. Isobolographic analysis of interactions between 1-methyl-1, 2, 3, 4-tetrahydroisoquinoline and four conventional antiepileptic drugs in the mouse maximal electroshock-induced seizure model. Eur. J. Pharmacol. 602, 298–305. Luszczki, J.J., Kocharov, S.L., Czuczwar, S.J., 2009b. N-(anilinomethyl)-p-isopropoxyphenyl-succinimide potentiates the anticonvulsant action of phenobarbital and valproate in the mouse maximal electroshock-induced seizure model. Neurosci. Res. 64, 267–272. Luszczki, J.J., Kocharov, S.L., Czuczwar, S.J., 2010. Effect of p-isopropoxyphenylsuccinimide monohydrate on the anticonvulsant action of carbamazepine, phenobarbital, phenytoin and valproate in the mouse maximal electroshock-induced seizure model. Pharmacol. Rep. 62, 194–202. Meyer, O.A., Tilson, H.A., Byrd, W.C., Riley, M.T., 1979. A method for the routine assessment of fore- and hindlimb grip strength of rats and mice. Neurobehav. Toxicol. 1, 233–236. Stables, J.P., Kupferberg, H.J., 1997. The NIH Anticonvulsant Drug Development (ADD) Program: preclinical anticonvulsant screening project. In: Avanzini, G., Regesta, G., Tanganelli, P., Avoli, M. (Eds.), Molecular and Cellular Targets for Anti-epileptic Drugs. John Libbey, London, pp. 191–198. Venault, P., Chapouthier, G., de Carvalho, L.P., Simiand, J., Morre, M., Dodd, R.H., Rossier, J., 1986. Benzodiazepine impairs and beta-carboline enhances performance in learning and memory tasks. Nature 321, 864–866. White, H.S., 2003. Preclinical development of antiepileptic drugs: past, present, and future directions. Epilepsia 44, 2–8. White, H.S., Woodhead, J.H., Wilcox, K.S., Stables, J.P., Kupferberg, H.J., Wolf, H.H., 2002. Discovery and preclinical development of antiepileptic drugs, In: Levy, R.H., Mattson, R.H., Meldrum, B.S., Perucca, E. (Eds.), Antiepileptic Drugs, fifth ed. Lippincott Williams & Wilkins, Philadelphia, pp. 36–48. Zejc, A., Obniska, J., Wilimowski, M., Rutkowska, M., Witkowska, M., Barczynska, J., Kedzierska-Gozdzik, L., Wojewodzki, W., Orzechowska-Juzwenko, K., Plawiak, T., 1990. Synthesis and anticonvulsant properties of some arylsuccinate methylpyridylimides. Pol. J. Pharmacol. Pharm. 42, 69–77.