Influence of MPEP (a selective mGluR5 antagonist) on the anticonvulsant action of novel antiepileptic drugs against maximal electroshock-induced seizures in mice

Progress in Neuro-Psychopharmacology & Biological Psychiatry 65 (2016) 172–178

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Influence of MPEP (a selective mGluR5 antagonist) on the anticonvulsant action of novel antiepileptic drugs against maximal electroshock-induced seizures in mice Dorota Zolkowska a, Maria W. Kondrat-Wrobel b, Magdalena Florek-Luszczki c, Jarogniew J. Luszczki b,d,⁎ a

Department of Neurology, School of Medicine, University of California—Davis, Sacramento, CA, USA Department of Pathophysiology, Medical University of Lublin, Lublin, Poland c Department of Public Health, Institute of Rural Health, Lublin, Poland d Isobolographic Analysis Laboratory, Institute of Rural Health, Lublin, Poland b

a r t i c l e

i n f o

Article history: Received 8 September 2015 Received in revised form 12 October 2015 Accepted 13 October 2015 Available online 21 October 2015 Keywords: Antiepileptic drugs Maximal electroshock-induced seizures Pharmacokinetic/pharmacodynamic interaction MPEP Lamotrigine Oxcarbazepine Pregabalin Topiramate Metabotropic glutamate receptors

a b s t r a c t The aim of this study was to determine the effects of 2-methyl-6-(phenylethynyl)pyridine (MPEP — a selective antagonist for the glutamate metabotropic receptor subtype mGluR5) on the protective action of some novel antiepileptic drugs (lamotrigine, oxcarbazepine, pregabalin and topiramate) against maximal electroshockinduced seizures in mice. Brain concentrations of antiepileptic drugs were measured to determine whether MPEP altered pharmacokinetics of antiepileptic drugs. Intraperitoneal injection of 1.5 and 2 mg/kg of MPEP significantly elevated the threshold for electroconvulsions in mice, whereas MPEP at a dose of 1 mg/kg considerably enhanced the anticonvulsant activity of pregabalin and topiramate, but not that of lamotrigine or oxcarbazepine in the maximal electroshock-induced seizures in mice. Pharmacokinetic results revealed that MPEP (1 mg/kg) did not alter total brain concentrations of pregabalin and topiramate, and the observed effect in the mouse maximal electroshock seizure model was pharmacodynamic in nature. Collectively, our preclinical data suggest that MPEP may be a safe and beneficial adjunct to the therapeutic effects of antiepileptic drugs in human patients. © 2015 Elsevier Inc. All rights reserved.

1. Introduction The important role of the main excitatory neurotransmitter, glutamate and glutamatergic neurotransmission in the pathophysiology of seizures has been demonstrated in various animal seizure models (Barker-Haliski and White, 2015; Werner and Covenas, 2011). Major effort has been devoted toward the development of therapies that target glutamate receptors. Glutamate receptors can be divided into two main classes: ionotropic receptors (including the AMPA, kainate, and NMDA receptors) and metabotropic receptors (Palmada and Centelles, 1998). Metabotropic glutamate receptors (mGluR) are family G-protein-coupled receptors that are the target of interest in the pathophysiology of neuropsychiatric

Abbreviations: MPEP, 2-methyl-6-(phenylethynyl)pyridine; ANOVA, analysis of variance. ⁎ Corresponding author at: Department of Pathophysiology, Medical University, Jaczewskiego 8b, PL 20-090 Lublin, Poland. E-mail addresses: [email protected], [email protected] (J.J. Luszczki).

http://dx.doi.org/10.1016/j.pnpbp.2015.10.005 0278-5846/© 2015 Elsevier Inc. All rights reserved.

disorders (schizophrenia, depression, anxiety and cognitive disorders, pain perception and addiction), neurodegenerative (Alzheimer's, Huntington's and Parkinson's diseases) and neurodevelopmental (fragile X syndrome and autism spectrum disorder) diseases (Golubeva et al., 2015). Molecular cloning identified eight mGluR subtypes (mGluR1–mGluR8) that have been segregated into three receptor groups based on their sequence homology, second messenger mechanisms and pharmacological activity (Conn, 2003; Pin and Acher, 2002; Pin and Duvoisin, 1995). Group I receptors (mGluR1 and mGluR5) are linked to phospholipase C. In neurons they are primarily located postsynaptically and have excitatory effects mediated by ion channels. Groups II (mGluR2 and mGluR3) and III (mGluR4, mGluR6, mGluR7, mGluR8) are coupled to adenyl cyclase, located presynaptically and are involved in reducing neurotransmitter release (Conn, 2003; Schoepp, 2001). Activation of mGluR5 potentiates AMPA and NMDA responses, and modulates the activity of voltage-operated calcium channels (Awad et al., 2000; Sanchez-Prieto et al., 2004; Ugolini et al., 1999). Interestingly the activation of NMDA receptors reverses desensitization of mGluR5 and potentiates its responses (Alagarsamy et al., 1999). Noteworthy, electrophysiological studies have revealed that mGluR5 appears to be the principal mGluR subtype that initiates

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bursting activity and positively modulates mGluR1 and GABA receptors (Besheer and Hodge, 2005; Lanneau et al., 2002). There is little evidence that supports the involvement of mGluR mutations in the pathogenesis of epilepsy (Meldrum and Rogawski, 2007). Epilepsy caused by spontaneous mutations of mGluR is not known to occur in humans or rodents. Although mice with genetically deleted mGluR7 express increased cortical and hippocampal excitability and increased seizure susceptibility (Sansig et al., 2001). Upregulation of group I receptors was reported in the kindling model of epilepsy as detected by biochemical and electrophysiological assays (Akiyama et al., 1992; Keele et al., 2000). Moreover, upregulation of hippocampal mGluR5 and downregulation of group III mGluRs were observed in patients with complex partial seizures (Dietrich et al., 1999; Notenboom et al., 2006). Broad spectrum of mGluRs selective agonists, antagonists and allosteric acting compounds was tested in animal models of epilepsy. Group I agonists such as DHPG [(R,S)-3,5-dihydroxyphenylglycine] and CHPG (2-chloro-5-hydroxyphenylglycine) have convulsant properties. Group I antagonists (AIDA [1-aminoindan-1,5-dicarboxylic acid], LY367385, LY456236, MPEP [2-methyl-6-(phenylethynyl)-pyridine], SIB-1893) and group II agonists are anticonvulsant in a variety of animal models of generalized convulsive and absence-like seizures, as well as in models of complex partial seizures (6 Hz, maximal electroshockinduced seizures, amygdala-kindled seizures) (Alexander and Godwin, 2006; Chapman et al., 1999, 2000; Moldrich et al., 2003; Shannon et al., 2005). DHPG but not AMPA-induced excitation of hippocampal CA1 neurons was inhibited by intravenous MPEP administration; demonstrating that this highly potent noncompetitive mGluR5 antagonist can readily cross the blood–brain barrier following systemic application (Gasparini et al., 1999). Microinfusion of MPEP and the mGluR2 and mGluR3 agonists DCG-IV ((2S,2′R,3′R)-2-(2′,3′-dicarboxycyclopropyl)glycine) into the perirhinal cortex protected against soman-induced seizures or increased latency to onset of seizures in rats (Myhrer et al., 2013a). Recent studies reported that the mGluR1 antagonist AIDA prevented the onset of tonic–clonic seizures caused by acoustic stimulation and the mGluR5 antagonist MPEP reduced the severity of audiogenic seizures (Bashkatova et al., 2015). In the previous study it had been demonstrated that MPEP significantly increased the electroconvulsive threshold in mice, but in a subprotective dose of 1 mg/kg did not affect the anticonvulsant activity of conventional antiepileptic drugs (Zadrozniak et al., 2004). MPEP combined with subprotective doses of valproate and phenobarbital decreased behavioral seizures and the afterdischarge duration in amygdala-kindled seizures in rats (Borowicz et al., 2009). Moreover, MPEP was found to provide significant protection in acute NMDA and glutamate mediated neurodegeneration (O'Leary et al., 2000). The aim of our study was to explore the effects of MPEP on the protective activity of some novel antiepileptic drugs such as lamotrigine, oxcarbazepine, pregabalin and topiramate, in the mouse maximal electroshock-induced seizure model, which is considered as an experimental model of tonic–clonic seizures and, to a certain extent, of partial convulsions with or without secondary generalization in humans (Löscher et al., 1991a). Of note, these antiepileptic drugs are routinely prescribed as the first-line treatment (lamotrigine and oxcarbazepine) or as adjunctive therapy (topiramate) in adult patients with tonic–clonic seizures (National Clinical Guideline Centre, 2012). As regards pregabalin, the drug is also prescribed as an add-on therapy in adults with partial onset seizures with or without secondary generalization (European Medicines Agency, 2009). This is the reason to test these four antiepileptic drugs in our study. The antiepileptic drug combinations with MPEP were also investigated in relation to their possible effects on motor coordination, long-term memory and muscular strength by use of the chimney test, step-through passive avoidance task and grip strength test, respectively. Additionally, total brain concentrations were measured to ascertain whether significant effects of MPEP were related to altered pharmacokinetics of antiepileptic drugs.

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2. Materials and methods 2.1. Animals Experiments were conducted on adult male albino Swiss mice (weighing 22–26 g). After adaptation to laboratory conditions, animals were randomly assigned to experimental groups each comprised eight mice. Each mouse was used only once and all tests were performed between 08:00 and 15:00 h. The experimental procedures described in this manuscript were approved by the Second Local Ethics Committee at the University of Life Sciences in Lublin (License no.: 15/2013), and complied with the European Communities Council Directive of 24 November 1986 (86/609/EEC). 2.2. Drugs The following drugs were used: 2-methyl-6-(phenylethynyl)pyridine (MPEP — Tocris Cookson Ltd., Bristol, UK), lamotrigine (Lamictal, Glaxo Wellcome, Greenford, Middlesex, UK), oxcarbazepine (Trileptal, Novartis Pharma AG, Basel, Switzerland), pregabalin (Lyrica, Pfizer Limited, Sandwich, Kent, UK) and topiramate (Topamax, Cilag AG, Schaffhausen, Switzerland). All drugs, except for MPEP, were suspended in a 1% solution of Tween 80 (Sigma, St. Louis, MO, USA) in distilled water; MPEP was directly dissolved in distilled water. All drugs were administered intraperitoneally (i.p.), in a volume of 5 ml/kg body weight, at the following pretreatment times: pregabalin — 120 min, lamotrigine and topiramate — 60 min, MPEP and oxcarbazepine — 30 min before electroconvulsions and brain sampling for the measurement of antiepileptic drug concentrations, as reported earlier (Borowicz et al., 2009; Luszczki et al., 2013). 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., 2013). 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 and 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 (MEST) and maximal electroshock seizure (MES) tests, as described elsewhere (Löscher et al., 1991a). 2.3.1. Maximal electroshock seizure threshold test The maximal electroshock seizure threshold test was first used to assess the anticonvulsant effects of MPEP 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 MPEP, the electroconvulsive threshold was calculated according to the log-probit method of Litchfield and Wilcoxon (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. In this test, MPEP was used at four increasing doses of 0.5, 1, 1.5 and 2 mg/kg. 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., 1991a). The antiepileptic drugs administered alone and their combinations with MPEP were tested for their ability to increase the number of animals not responding with tonus (i.e., protected from tonic hind limb

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extension) after stimulation. Again, at least 4 groups of mice, each consisting of 8 animals and treated with a different dose of the antiepileptic drugs alone or in combination with MPEP, 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 by Litchfield and Wilcoxon (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. In the present study, lamotrigine was administered at doses ranging between 2 and 8 mg/kg, oxcarbazepine at doses ranging between 6 and 14 mg/kg, pregabalin at doses ranging between 50 and 175 mg/kg and topiramate at doses ranging between 20 and 70 mg/kg. 2.4. Measurement of total brain antiepileptic drug concentrations Pharmacokinetic evaluation of total brain antiepileptic drug concentrations was performed only for those combinations of MPEP with antiepileptic drugs for which the anticonvulsant effect in the maximal electroshock seizure test was significantly greater than that for control (an antiepileptic drug + vehicle-treated) animals. Thus, the measurements of total brain concentrations of pregabalin and topiramate were undertaken at the doses that corresponded to their ED50 values from the maximal electroshock seizure test. Specifically, mice pretreated with a given antiepileptic drug alone or in combination with MPEP 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. The homogenates were then centrifuged at 10,000 g for 10 min and the supernatant samples of 200 μl were collected and then analyzed for pregabalin content by high pressure liquid chromatography as described previously (Luszczki et al., 2013). Total brain concentration of topiramate was measured by a fluorescence polarization immunoassay using an analyzer (Abbott TDx) and manufacturer-supplied reagent kits (Seradyn, Inc., Indianapolis, IN, USA). Total brain concentrations of pregabalin and topiramate are expressed in μg/g of wet brain tissue as means ± standard error (S.E.M.) of at least 8 separate brain preparations.

1979). The mice were lifted by the tails so that their forepaws could grasp a wire grid and then they were gently pulled backward by the tail until the grid was released. The maximal force exerted by the mouse before losing grip was recorded and the mean of 3 measurements for each animal was calculated. The muscular strength in mice is expressed in N (newtons) as the means ± S.E.M. of at least 8 determinations. 2.7. Chimney test The acute adverse-effect potentials for the combinations of antiepileptic drugs with MPEP were determined for the antiepileptic drugs administered at doses corresponding to their ED50 values from the maximal electroshock seizure test when combined with MPEP (1 mg/kg). The chimney test of Boissier et al. (Boissier et al., 1960) was used to quantify the adverse effect potential of novel antiepileptic drugs administered in combination with MPEP. In this test, the animals had to climb backwards up a plastic tube (3 cm inner diameter, 30 cm long) and impairment of motor performance was indicated by the inability of the mice to climb backward up the transparent tube within 60 s. 2.8. Statistics The CS50 and ED50 values (with their 95% confidence limits) were calculated by computer log-probit method according to Litchfield and Wilcoxon (Litchfield and Wilcoxon, 1949). After transformation of the respective 95% confidence limits to S.E.M., as described previously (Luszczki et al., 2009), statistical analysis of data from the maximal electroshock seizure threshold and maximal electroshock seizure tests was performed with one-way analysis of variance (ANOVA) followed by the post-hoc Tukey–Kramer test for multiple comparisons. Total brain antiepileptic drug concentrations were statistically compared using the unpaired Student's t-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. The data from the chimney test were statistically analyzed with the Fisher's exact probability test. Differences among values were considered statistically significant if P b 0.05. 3. Results

2.5. Step-through passive avoidance task

3.1. Influence of MPEP upon the threshold for electroconvulsions

Each animal was administered an antiepileptic drug either alone or in combination with MPEP (1 mg/kg), at doses corresponding to their ED50 values from the maximal electroshock seizure test, on the first day before training. The 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 the 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 for up to 180 s. Mice that avoided the dark compartment for 180 s were considered as having remembered the task (Venault et al., 1986). The time the mice took to enter the dark box was noted and the median latencies (retention times) with 25th and 75th percentiles were calculated.

MPEP administered at doses of 1.5 and 2 mg/kg i.p. significantly increased the threshold for electroconvulsions in mice. The thresholds were elevated from 6.32 ± 0.41 mA to 8.58 ± 0.47 mA (P b 0.05) and 9.46 ± 0.48 mA (P b 0.001), by MPEP 1.5 and 2 mg/kg, respectively (F[4115] = 7.895; P b 0.001) (Fig. 1). The experimentally-derived CS50 values for animals receiving MPEP at doses of 0.5 and 1 mg/kg did not significantly differ from that for control animals subjected to the maximal electroshock seizure threshold test (Fig. 1).

2.6. Grip-strength test The effects of combinations of MPEP (1 mg/kg) with different novel 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. (Meyer et al.,

3.2. Effects of MPEP on the protective action of novel antiepileptic drugs in the mouse maximal electroshock seizure model All investigated antiepileptic drugs (lamotrigine, oxcarbazepine, pregabalin and topiramate) administered alone exhibited a clear-cut anticonvulsant activity in the maximal electroshock seizure test in mice (Fig. 2A–D). MPEP at the doses of 0.5 and 1 mg/kg had no impact on the anticonvulsant action of lamotrigine or oxcarbazepine against maximal electroshock-induced seizures in mice (Fig. 2A–B). When MPEP (1 mg/kg) was co-administered with pregabalin and topiramate, it significantly enhanced the anticonvulsant action of the antiepileptic drugs in the maximal electroshock seizure test by reducing the ED50 value of pregabalin from 126.18 ± 11.82 mg/kg to 80.01 ±

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Fig. 1. Effect of MPEP (2-methyl-6-phenylethynyl-pyridine) upon the electroconvulsive threshold in mice. Columns represent median current strengths (CS50 values in mA ± S.E.M.) required to produce tonic hindlimb extension in 50% of animals tested in the maximal electroshock-induced seizure threshold test. MPEP was administered systemically (i.p.) 30 min. before the test. Statistical evaluation of data was performed with log-probit method (Litchfield and Wilcoxon, 1949) followed by one-way analysis of variance with Tukey/Kramer post-hoc test for multiple comparisons. aP b 0.05 and bP b 0.001 vs. control group (vehicle-treated animals).

11.90 mg/kg (F[2,69] = 3.466; P = 0.037) and topiramate from 49.34 ± 4.31 mg/kg to 30.53 ± 4.69 mg/kg (F[2,77] = 4.229; P = 0.018) (Fig. 2C–D). MPEP at the dose of 0.5 mg/kg did not affect the anticonvulsant action of pregabalin or topiramate against maximal electroshock-induced seizures in mice (Fig. 2C–D). 3.3. Effect of MPEP on total brain antiepileptic drug concentrations As determined by the high pressure liquid chromatography, total brain concentration of pregabalin (80 mg/kg) administered alone was 21.24 ± 1.72 μg/g of wet brain tissue and did not significantly differ from that determined for the combination of pregabalin (80 mg/kg) with MPEP (1 mg/kg), which was 24.64 ± 1.95 μg/g of wet brain tissue. Similarly, as determined by the fluorescence polarization immunoassay method, total brain concentration of topiramate (30.5 mg/kg) administered separately was 3.73 ± 0.47 μg/g of wet brain tissue and did not significantly differ from that determined for the combination of topiramate (30.5 mg/kg) with MPEP (1 mg/kg), which was 4.07 ± 0.52 μg/g of wet brain tissue. Since MPEP at 1 mg/kg did not significantly affect the anticonvulsant potential of lamotrigine and oxcarbazepine in the maximal electroshock seizure test in mice, the total brain concentrations of these two drugs were not measured with the high pressure liquid chromatography method. 3.4. Effects of MPEP and its combination with novel antiepileptic drugs on motor performance, long-term memory and muscular strength MPEP administered alone at a dose of 1 mg/kg did not affect motor coordination in mice subjected to the chimney test (Table 1). Similarly, MPEP at 1 mg/kg did not affect muscular strength in mice in the gripstrength test or alter long-term memory in animals challenged with the step-through passive avoidance task (Table 1). When MPEP (1 mg/kg) was administered in combination with lamotrigine, oxcarbazepine, pregabalin or topiramate at doses corresponding to their ED50 values from the maximal electroshock seizure test, motor performance as assessed by the chimney test, skeletal muscular strength of the animals, as assessed by the grip-strength test or long-term memory as determined in the passive avoidance test were not significantly affected (Table 1). 4. Discussion The anticonvulsant effects of MPEP had been previously reported in a variety of animal studies. MPEP was found to be effective in animal models of generalized convulsive and absence-like seizures (maximal electroshock, pentylenetetrazole and 6 Hz seizure models), in absence epilepsy model (lethargic mice) and soman-induced seizures (Barton et al., 2003; Chapman et al., 2000; Myhrer et al., 2013b; Zadrozniak et al., 2004). The major purpose of the present study was to characterize

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the anticonvulsant effects of MPEP in combination with four novel antiepileptic drugs. We found that intraperitoneal injection of 1.5 and 2 mg/kg of MPEP significantly elevated the threshold for electroconvulsions in mice and the compound in a dose of 1 mg/kg significantly enhanced the anticonvulsant activity of pregabalin and topiramate, but not that of lamotrigine or oxcarbazepine in the maximal electroshock seizure test in mice. Electrophysiological studies confirm that MPEP acts as a highly potent non-competitive inhibitor of mGluR5 through a novel allosteric site and additionally affects human NMDA1A 2B (10 μM), NMDA1A 2A (100 μM) and kainate Glu6-(IYQ) (100 μM) receptor subtypes (Gasparini et al., 1999; Lea and Faden, 2006). The anticonvulsant potency of MPEP varies in different seizure models. Noteworthy, MPEP was found to be the most potent in inhibiting seizures induced by selective mGluR5 agonists and was less efficacious in suppressing seizures induced by non-selective mGluR5 and mGluR1 agonists. Weaker potency was observed in the sound-induced seizure model in DBA/2 mice (Chapman et al., 2000). Interestingly, MPEP microinfused into the perirhinal cortex of rats exerts full protection against soman-induced seizures and increased latency to onset of seizures. Additionally, MPEP significantly inhibited pseudocholinesterase activity in the brain (Myhrer et al., 2013a). Reduced levels of acetylcholinesterase and pseudocholinesterase in the brains of rats treated with MPEP were found to be positively correlated with latency to seizure onset or protection against seizures. When given systematically 20 min after seizure onset, the combination of MPEP (30 mg/kg) with HI-6 (1-[([4(aminocarbonyl)pyridino]methoxy)methyl]-2-[(hydroxyimino)methyl]pyridinium) and procyclidine successfully terminated seizures and reduced mortality in soman-induced seizures (Myhrer et al., 2013a). MPEP (200 mg/kg) administered at 2 h from induction of pilocarpine status epilepticus effectively terminated behavioral seizures, but did not prevent delayed neuronal loss. Combinations of MPEP with low doses of MK801 (0.1 mg/kg) or with MK801 (0.1 mg/kg) and diazepam (0.5 mg/kg) administered at 1 h time point terminated behavioral and EEG seizure signs within 2 min and were subsequently neuroprotective (Tang et al., 2007). It has been previously reported that MPEP alone produced dosedependent protection in the mouse maximal electroshock seizure model with ED50 of 72.4 mg/kg and protective index (P.I.) of 2. Increased potency of MPEP was observed in the 6 Hz seizure model with ED50 value of 17.4 mg/kg and P.I. of 8.3 (Barton et al., 2003). Our results indicating that MPEP elevated, in a dose-dependent manner, the threshold for electroconvulsions in mice are consistent with data obtained previously in the study by Zadrozniak et al. (Zadrozniak et al., 2004). It is important to note that MPEP at the same sub-protective dose (the dose that by itself did not significantly affect the threshold for electroconvulsions) of 1 mg/kg did not provide any protection in the maximal electroshock seizure model when combined with classical antiepileptic drugs: valproate, carbamazepine, phenytoin and phenobarbital (Zadrozniak et al., 2004). Similarly, in our study MPEP at the sub-protective dose of 1 mg/kg had no significant impact on the protective action of oxcarbazepine or lamotrigine in the mouse maximal electroshock seizure model. MPEP in doses of up to 40 mg/kg was ineffective in the amygdala kindling model in rats. Moreover, combination of MPEP (10–20 mg/kg) with sub-protective doses of carbamazepine and phenytoin did not influence seizures or afterdischarge duration in kindled rats (Borowicz et al., 2009). Both, carbamazepine and lamotrigine, have been shown to exert their anticonvulsant activity predominantly via fast inactivation of voltage-gated sodium channels and are considered “classical” sodium channel blocking antiepileptic drugs (Rogawski and Loscher, 2004). Oxcarbazepine is a structural derivative of carbamazepine and acts primarily by blocking voltage-gated sodium channels that reduce high-frequency repetitive firing of neurons (Macdonald and Greenfield, 1997; McLean et al., 1994; White, 1999). In addition, oxcarbazepine and its active metabolite monohydroxy derivative inhibit N-type calcium channels and reduce glutamatergic

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Fig. 2. A–D. Effect of MPEP on the anticonvulsant action of four novel antiepileptic drugs against maximal electroshock-induced seizures in mice. Left panel: dose–response relationships for protective activity of novel antiepileptic drugs alone and in combination with MPEP in the mouse maximal electroshock-induced seizure model. Antiepileptic drugs were administered i.p.: pregabalin (PGB) — 120 min, lamotrigine (LTG) and topiramate (TPM) — 60 min, MPEP and oxcarbazepine (OXC) — 30 min before electroconvulsions. Data points indicate percentage of animals protected. Each point represents eight mice. Right panel: columns represent median effective doses (ED50 in mg kg/kg ± S.E.M.) of lamotrigine, oxcarbazepine, pregabalin and topiramate protecting 50% of animals tested against maximal electroshock-induced hind limb extension. Statistical analysis of data was performed with one-way ANOVA followed by the post hoc Tukey–Kramer test for multiple comparisons. aP b 0.05 vs the respective control group (an antiepileptic drug + vehicle-treated animals).

Table 1 Effects of MPEP and its combination with four novel antiepileptic drugs on long-term memory in the passive avoidance task, motor performance in the chimney test and muscular strength in the grip-strength test in mice. Treatment (mg/kg)

Retention time (s)

Grip-strength (N)

Motor coordination impairment (%)

Vehicle MPEP (1.0) + vehicle Lamotrigine (3.74) + MPEP (1.0) Oxcarbazepine (8.71) + MPEP (1.0) Pregabalin (80.01) + MPEP (1.0) Topiramate (30.53) + MPEP (1.0)

180 (180; 180) 180 (180; 180) 180 (180; 180) 180 (180; 180) 180 (180; 180) 180 (180; 180)

0.887 ± 0.037 0.852 ± 0.040 0.900 ± 0.039 0.910 ± 0.044 0.880 ± 0.051 0.923 ± 0.041

0 0 12.5 0 37.5 0

Results from the passive avoidance task, assessing long-term memory in mice, are presented as median retention times (in seconds (s); with 25th and 75th percentiles in parentheses). Results from the grip-strength test, assessing skeletal muscular strength in mice, are presented as mean grip-strengths (in newtons (N) ± S.E.M.). Data from the chimney test, assessing motor performance in mice, are presented as percentage (%) of animals showing motor coordination impairment. Each experimental group consisted of 8 mice. Statistical analysis of data from the passive avoidance task was performed with nonparametric Kruskal–Wallis ANOVA test. The results 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.

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transmission (Ambrosio et al., 2002; Calabresi et al., 1995; McLean et al., 1994; Stefani et al., 1995; Waldmeier et al., 1996). Electrophysiological studies using mixtures of phenytoin, carbamazepine and lamotrigine have discovered that these drugs bind to a common recognition site on the sodium channel and they have greater affinity to the inactivated channel state rather than open or closed (Kuo, 1998). Noteworthy, lamotrigine suppresses postsynaptic AMPA receptors and reduces glutamate release (Lee et al., 2008). Differences in molecular mechanisms of action are one probable explanation for MPEP's incapability to influence anticonvulsant action of lamotrigine and oxcarbazepine. In the case of pregabalin, neurochemical studies have revealed that the drug binds with high affinity and specificity to the alpha2delta subunit of P/Q-type voltage-gated calcium channels (Dooley et al., 2002, 2007; Stahl et al., 2013; Taylor et al., 2007). Studies using living cultured rat hippocampal neurons revealed that pregabalin, at therapeutically relevant concentrations, reduces the emptying of neurotransmitter vesicles from presynaptic sites resulting in a decreased release of neurotransmitters including glutamate, norepinephrine, substance P and calcitonin gene-related peptide (Micheva et al., 2006; Taylor et al., 2007). Although the effects on GABA do not contribute to the pharmacological action of pregabalin and gabapentin (a second-generation antiepileptic drug), activation of the GABA synthesizing enzyme glutamic acid decarboxylase may be the underlying mechanism of antiepileptic activity (Silverman et al., 1991; Taylor et al., 2007). Of note, gabapentin at the antiseizure doses elevated GABA turnover in rats and patients with epilepsy (Löscher et al., 1991b; Petroff et al., 1996). Since pregabalin is both structurally and pharmacologically related to gabapentin, it is highly likely that the drug also affects GABA turnover. Therapeutic activity of topiramate has been associated with several cellular mechanisms but the relevance of its pharmacological actions to broad-spectrum antiseizure activity is uncertain. Topiramate inhibits voltage-gated sodium channels and neuronal Ltype high-voltage-activated calcium channels, potentiates GABAmediated inhibitory neurotransmission, blocks the excitatory effects mediated by AMPA/kainate receptors; and weakly inhibits carbonic anhydrase isoenzymes. It has also been suggested that the pharmacological effects of topiramate are mediated indirectly by the modification of channel phosphorylation state instead of a direct action (Meldrum and Rogawski, 2007; Shank and Maryanoff, 2008). Considering the broad spectrum of pharmacological actions of topiramate and pregabalin, a variety of molecular mechanism may underlay enhanced anticonvulsant activity of their combinations with MPEP. The modulatory effects on glutamatergic and GABA-ergic neurotransmission may be considered the mechanisms potentially relevant to seizure protection. Whether the described actions of pregabalin and topiramate on glutamatemediated neurotransmission contribute to their antiseizure activity in combination with MPEP remains to be determined. More advanced neurochemical and electrophysiological studies are required to explain such interactions. Many clinically used antiepileptic therapies have significant side effects that disable their use in clinical settings. MPEP administered alone or in combination with four novel antiepileptic drugs did not affect motor coordination, muscular strength or performance in the standard variant of the stepthrough passive avoidance task in exposed animals. Our results confirm previous observations that MPEP at anticonvulsant doses did not impair motor coordination or performance in the passive avoidance task in adult rats (Borowicz et al., 2009). Studies conducted in immature rats revealed anxiolytic-like effects of MPEP without influence on habituation, a form of non-associative learning expressed as a decrease of orienting reactions during repeated exposures to open field, and further reaffirmed the lack of negative effects on motor performance (Mikulecka and Mares, 2009). Besides being effective in a variety of seizure and status epilepticus models, MPEP decreases NMDA and glutamate-mediated neuronal toxicity (O'Leary et al., 2000). In the focal cerebral ischemia model, MPEP appears to be neuroprotective when applied early

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(15 min) rather than late (135 min) as a post-injury treatment (Bao et al., 2001). The results of our study suggest that the anticonvulsant effects of MPEP with pregabalin and topiramate are pharmacodynamic in nature as MPEP did not significantly alter total brain concentrations of either drug in experimental animals. Noteworthy in our study, as reported earlier, total brain concentrations of antiepileptic drugs were verified with fluorescence polarization immunoassay (topiramate) and high pressure liquid chromatography (pregabalin) techniques. Only comparisons of total brain concentrations provide the exact classification and characterization of interactions between antiepileptic drugs in preclinical studies (Cadart et al., 2002; Luszczki et al., 2003). 5. Conclusions In conclusion, our study suggests that the co-administration of MPEP with novel antiepileptic drugs such as pregabalin and topiramate, would be a promising treatment when applied in clinical settings, especially in patients with tonic–clonic seizures or partial convulsions with or without secondarily generalization. Moreover, our data provide pivotal information on the beneficial effects of MPEP in combination with novel antiepileptic drugs such as pregabalin and topiramate with no exacerbation of side effects. Our data suggest that further neurochemical and electrophysiological studies are needed to confirm that MPEP might be considered as a safe and efficacious supplementary therapeutic agent in epilepsy treatment. Disclosure of conflicts of interest. The authors have no disclosures to declare. Acknowledgments This study was supported by grants from the Medical University (DS 474/2013) and Institute of Rural Health (12120/2013) (Lublin, Poland). The authors express their thanks to Dr. G. Raszewski (Institute of Rural Health, Lublin, Poland) for the skillful determination of the brain concentrations of pregabalin. The authors are grateful to M. Depciuch, M. Garbal, M. Grzywacz and A. Kopiec (Medical University, Lublin, Poland) for their technical assistance. References Akiyama, K., Daigen, A., Yamada, N., Itoh, T., Kohira, I., Ujike, H., et al., 1992. Longlasting enhancement of metabotropic excitatory amino acid receptor-mediated polyphosphoinositide hydrolysis in the amygdala/pyriform cortex of deep prepiriform cortical kindled rats. Brain Res. 569, 71–77. Alagarsamy, S., Marino, M.J., Rouse, S.T., Gereau, R.W., Heinemann, S.F., Conn, P.J., 1999. Activation of NMDA receptors reverses desensitization of mGluR5 in native and recombinant systems. Nat. Neurosci. 2, 234–240. Alexander, G.M., Godwin, D.W., 2006. Metabotropic glutamate receptors as a strategic target for the treatment of epilepsy. Epilepsy Res. 71, 1–22. Ambrosio, A.F., Soares-Da-Silva, P., Carvalho, C.M., Carvalho, A.P., 2002. Mechanisms of action of carbamazepine and its derivatives, oxcarbazepine, BIA 2-093, and BIA 2-024. Neurochem. Res. 27, 121–130. Awad, H., Hubert, G.W., Smith, Y., Levey, A.I., Conn, P.J., 2000. Activation of metabotropic glutamate receptor 5 has direct excitatory effects and potentiates NMDA receptor currents in neurons of the subthalamic nucleus. J Neurosci. 20, 7871–7879. Bao, W.L., Williams, A.J., Faden, A.I., Tortella, F.C., 2001. Selective mGluR5 receptor antagonist or agonist provides neuroprotection in a rat model of focal cerebral ischemia. Brain Res. 922, 173–179. Barker-Haliski, M., White, H.S., 2015. Glutamatergic mechanisms associated with seizures and epilepsy. Cold Spring Harb. Perspect. Biol. 5, a022863. Barton, M.E., Peters, S.C., Shannon, H.E., 2003. Comparison of the effect of glutamate receptor modulators in the 6 Hz and maximal electroshock seizure models. Epilepsy Res. 56, 17–26. Bashkatova, V.G., Sudakov, S.K., Prast, H., 2015. Antagonists of metabotropic glutamate receptors prevent the development of audiogenic seizures. Bull. Exp. Biol. Med. 159, 1–3. Besheer, J., Hodge, C.W., 2005. Pharmacological and anatomical evidence for an interaction between mGluR5- and GABA(A) alpha1-containing receptors in the discriminative stimulus effects of ethanol. Neuropsychopharmacology 30, 747–757.

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