- Email: [email protected]
2-Methyl-6-phenylethynyl-pyridine (MPEP), a non-competitive mGluR5 antagonist, differentially affects the anticonvulsant activity of four conventional antiepileptic drugs against amygdala-kindled seizures in rats
Pharmacological Reports 2009, 61, 621–630 ISSN 1734-1140
Copyright © 2009 by Institute of Pharmacology Polish Academy of Sciences
2-Methyl-6-phenylethynyl-pyridine (MPEP), a non-competitive mGluR5 antagonist, differentially affects the anticonvulsant activity of four conventional antiepileptic drugs against amygdala-kindled seizures in rats Kinga K. Borowicz1, Jarogniew J. £uszczki2, Stanis³aw J. Czuczwar2
Department of Pathophysiology, Medical University, Jaczewskiego 8, PL 20-090 Lublin, Poland Department of Physiopathology, Institute of Agricultural Medicine, Jaczewskiego 2, PL 20-950 Lublin, Poland
Correspondence: Kinga K. Borowicz, e-mail: [email protected]
Abstract: 2-Methyl-6-phenylethynyl-pyridine (MPEP), a selective noncompetitive mGluR5 antagonist, influences the action of conventional antiepileptic drugs in amygdala-kindled seizures in rats. MPEP alone (up to 40 mg/kg) did not affect any seizure parameter. Moreover, the common treatment of MPEP with either carbamazepine or phenytoin (administered at subeffective doses) did not result in any anticonvulsant action in kindled rats. However, when combined with subprotective doses of valproate or phenobarbital, MPEP significantly shortened seizure and afterdischarge durations. Importantly, combinations of MPEP with the two antiepileptics did not have the adverse effects of impaired motor performance or long-term memory in rats. Our data indicate that MPEP may positively interact with some conventional antiepileptic drugs in the amygdala-kindling model of complex partial seizures. Key words: MPEP, metabotropic glutamate receptors, conventional antiepileptic drugs, amygdala-kindled seizures
Introduction Several reports have indicated that selective ligands for metabotropic glutamate receptor (mGluR) subtypes have the potential to treat of a wide variety of neurological and psychiatric disorders, including depression, anxiety disorders, schizophrenia, Alzheimer’s disease and epilepsy, among others. All mGluRs have been classified into three groups, which present with different biological, electrophysiological and pharmacological properties. Receptors for group I
mGluRs are positively linked to phospholipase C, while group II and III receptors are negatively coupled to adenylyl cyclase [3]. Disappointing experience with ionotropic, particularly NMDA, receptor antagonists with respect of their usefulness in anticonvulsant therapy [10, 36] have directed researchers’ efforts to modulators of mGluRs. Results from many experimental studies have confirmed the anticonvulsant and neuroprotective actions of group I mGluR antagonists and group II/III mGluR agonists [8]. 2-Methyl-6-phenylethynyl-pyridine (MPEP) and (E)-2methyl-2-styrylpyridine (SIB 1893) are the most Pharmacological Reports, 2009, 61, 621–630
621
well-known representatives of selective noncompetitive antagonists of mGluR5 receptors belonging to group I. They attenuate Ca2+ entry through voltagedependent N- and P/Q- type Ca2+ channels, which causes a reduction in glutamate release [34]. Therefore, it is likely that MPEP and SIB 1893 will present with anticonvulsant properties. Additionally, mGluR5 antagonists significantly decrease the duration of NMDA channels opening [27] and positively modulate mGluR4 receptors [22], which should enhance their anticonvulsant action. However, the latter information has not been confirmed by other authors [15]. Preliminary experiments indicate that mGluR5 antagonists may have therapeutic potential in the treatment of epilepsy, stroke, anxiety, pain, neurodegenerative disease [23] and cocaine withdrawal [26]. In experimental conditions, the selective antagonists of mGlu5 are the most effective against seizures induced by activation of mGlu5 receptors. At higher doses, SIB 1893 and MPEP also block convulsive and non-convulsive primary generalized seizures [9]. SIB 1893 has a proven protective efficacy against soundinduced seizures in DBA/2 and primary generalized non-convulsive seizures in lethargic mice [9]. MPEP has shown anticonvulsive efficacy against clonic seizures induced by pentylenetetrazole. This mGluR5 antagonist does not affect the PTZ-kindling progression; however, it counteracts learning deficits [25] and long-term hippocampal aberrations induced by the PTZ kindling process [24]. Moreover, MPEP exhibits anticonvulsant activity in the 6Hz model of partial seizures [2]. In immature rats, MPEP inhibits behavioral seizures induced by pilocarpine [13] and pentylenetetrazole [21] and reduces the afterdischarges induced by electrical stimulation of the sensorimotor cortical area [16]. In a previous study, it had been demonstrated that SIB 1893 possesses dual proconvulsant (at low doses) and anticonvulsant (at high doses) activity in the electroshock seizure threshold test in mice. SIB 1893, given at ineffective doses, enhances the protective action of valproate against maximal electroshock in mice, the basic model of generalized tonic-clonic convulsions. Moreover, the mGluR5 antagonist does not affect the protection of conventional antiepileptic drugs against pentylenetetrazole-induced convulsions in mice, reflecting myoclonic epilepsy in humans [7, 17, 20]. On the other hand, SIB 1893 reduces amygdala-kindled seizures in rats and potentiates the pro-
622
Pharmacological Reports, 2009, 61, 621–630
tective action of oxcarbazepine in this test, which is the most recommended model of partial complex seizures [5, 17]. The second mGluR5 antagonist, MPEP, increases the electroconvulsive threshold in mice, but it does not affect the efficacy of conventional antiepileptics against the maximal electroshock test [37]. These previous results prompted us to broaden the investigations of MPEP and evaluate its effect on amygdala-kindled seizures and the protective action of four antiepileptics (valproate, carbamazepine, phenobarbital and phenytoin) in this experimental model of epilepsy. We also assessed the influence of MPEP on the brain concentrations of the respective antiepileptics to define the possible involvement of a pharmacokinetic interaction that could explain the obtained results.
Materials and Methods Animals and experimental conditions
All experiments were performed on adult male Wistar rats weighing 200–250 g. The rats were kept in colony cages with free access to food and tap water under standardized housing conditions (natural lightdark cycle; temperature: 21 ± 1°C; relative humidity: 55 ± 5%). After seven days of adaptation to laboratory conditions, the animals were randomly assigned to experimental groups that each consisted of eight rats. All tests were performed between 9:00 a.m. and 2:00 p.m. Procedures involving animals and their care were conducted in conformity 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 listed below conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Local Ethics Committee at the Medical University of Lublin. Surgery and kindling procedure
The rats were anesthetized with pentobarbital (50 mg/kg, ip) and received stereotaxic implantation of one bipolar electrode in the right basolateral amygdala. The
MPEP and antiepileptic drugs in kindled seizures Kinga K. Borowicz et al.
following stereotaxic coordinates for electrode implantation were used: AP-1.5, L-4.4, V-8.5, according to the brain atlas of Paxinos and Watson [28]. All coordinates were measured from the bregma. Scull screws served as the indifferent reference electrodes. The electrode assembly was attached to the scull by dental acrylic cement. After electrode implantation, the animals were treated with an antibiotic for one week to prevent infection. After a post-operative period of two weeks, stimulation of the amygdala was initiated. Each stimulus consisted of a 1 s train of 50 Hz, 1 ms biphasic square-wave pulses with a current intensity of 500 µA, which were delivered every 24 h, until at least 10 sequential fully kindled stage 5 seizures were elicited. The afterdischarges from the amygdala were recorded prior to and after the stimulation. The seizure severity (SSv) was assessed according to the following modified Racine‘s system [30]: 0 = no seizure response; 1 = immobility, eye closure, ear twitching, twitching of vibrissae, sniffing, facial clonus; 2 = head nodding associated with more severe facial clonus; 3 = clonus of one forelimb; 3.5 = bilateral forelimb clonus without rearing; 4 = bilateral forelimb clonus with rearing; 4.5 = falling on a side (without rearing), loss of righting reflex accompanied by generalized clonic seizures; 5 = rearing and falling on the back accompanied by generalized clonic seizures. Seizure duration (SD) was the duration of limbic seizures (stage 1–2) and motor seizures (stage 3–5). Afterdischarges (AD) were defined as spikes with a frequency of at least 1 Hz and amplitude that was at least two-fold greater than the pre-stimulation baseline present in the EEG recorded from the site of stimulation. Afterdischarge threshold (ADT) was determined after administration of a test drug or its vehicle. Electrical stimulations increasing in steps of about 20% of the previously applied current were given at intervals of 5 min until an afterdischarge lasting at least 3 s was evoked. At the ADT current, seizure severity, seizure duration and afterdischarge duration were recorded and analyzed. Control readings were made two days before and two days after the respective treatments. Subsequently, the mean ± SE was calculated and used for statistical comparisons of the obtained data. Drugs
The following substances and drugs were used in the present study: MPEP (Tocris Cookson Ltd., Bristol,
UK), carbamazepine (CBZ; Polpharma, Starogard, Poland); valproate magnesium (VPA; a gift from ICN-Polfa, Rzeszów, Poland); phenytoin (PHT; Polfa, Warszawa, Poland), and phenobarbital (PB; Polfa, Kraków, Poland). VPA and MPEP were dissolved in distilled water, while CBZ, PHT and PB were suspended in a 1% aqueous solution of Tween 80 (Sigma, St. Louis, MO, USA). All drugs were administered intraperitoneally (ip) in a volume of 0.01 ml/g body weight, PHT – 120 min, PB – 60 min, VPA, CBZ and MPEP – at 30 min before amygdala-kindled seizures and behavioral tests. The pretreatment times corresponded to times of their maximal anticonvulsant activity taken from literature data and confirmed in our previous studies. Rotarod test
Motor coordination was assessed via the rotarod test [11]. The rod (6 cm in diameter) was rotating at 6 rpm. For each training session rats were placed on the rod for 120 s. After that, animals that had been pretreated with antiepileptic drugs or their combinations with MPEP were placed on the rod again. The time it took for each animal to fall was used as a measure of motor impairment. The observation time was always 120 s. Passive avoidance task
Rats were placed in an illuminated box (40 × 40 × 30 cm) connected to a dark box (40 × 40 × 30 cm), which was equipped with an electric grid floor. Entrance into the dark box was punished by an electric footshock (0.8 mA) of 2 s duration. The animals that did not enter the dark compartment were excluded from the experiment. On the next day (24 h later), the same animals were put into the illuminated box and observed up to 180 s. The time period an animal entered the dark box was subsequently noted and the medians with 25 and 75 percentiles were calculated. According to Venault et al. [33], the step-through passive avoidance task may be recognized as a measure of longterm memory. Estimation of brain levels of antiepileptic drugs
Rats were administered with one of the conventional antiepileptic drugs and vehicle or with the respective AED + MPEP. Animals were killed by decapitation at times chosen to coincide with those scheduled for the
Pharmacological Reports, 2009, 61, 621–630
623
seizure test. Brains were removed from skulls, weighed, and homogenized using Abbott buffer (2:1 v/w) in an Ultra-Turrax T8 homogenizer (IKA-WERKE, Stauffen, Germany). The homogenates were centrifuged at 10,000 × g for 10 min The supernatant samples (75 µl) were analyzed by fluorescence polarization immunoassay (FPIA) for PHT, CBZ, VPA or PB content using a TDx analyzer and reagents exactly as described by the manufacturer (Abbott Laboratories, North Chicago, IL, USA). All AED concentrations are expressed in µg/ml of brain supernatants as the means ± SD of at least eight determinations. Statistics
The statistical significances of differences between seizure and afterdischarge durations were calculated by Student‘s t-test for paired replicates. Seizure scores were evaluated with Wilcoxon signed rank test. The results obtained in the step-through passive avoidance task and rotarod test were statistically evaluated using Kruskal-Wallis nonparametric ANOVA followed by post-hoc Dunn’s test. Plasma levels of antiepileptics alone or in combination with MPEP were evaluated with Student‘s t-test for unpaired replicates.
Results
conventional antiepileptic drugs, valproate (at 75 mg/kg) and phenobarbital (15 mg/kg) significantly decreased the afterdischarge duration. Phenytoin (15 mg/kg) markedly decreased the two parameters of seizure and afterdischarge duration. Carbamazepine (10 mg/kg) additionally reduced the seizure severity (Tab. 1–4). In further experiments, MPEP was combined with antiepileptic drugs applied at subprotective doses. Valproate (50 mg/kg) combined with MPEP (10 or 20 mg/kg) markedly reduced the seizure and afterdischarge durations, while with MPEP (5 mg/kg) it shortened only the latter parameter (Fig. 1, Tab. 2). The combined treatment of phenobarbital (10 mg/kg) and MPEP (10 or 20 mg/kg) reduced the seizure and afterdischarge durations (Fig. 2, Tab. 3). However, the combinations of carbamazepine (7.5 mg/kg) and phenytoin (10 mg/kg) with MPEP (10 or 20 mg/kg) did not influence amygdala-kindled seizures (Tab. 4, 5). Rotarod test and passive avoidance task
MPEP (10 and 20 mg/kg), valproate (75 and 50 mg/kg), phenobarbital (10 and 15 mg/kg) and the combinations of MPEP with both antiepileptics did not produce any significant adverse effects with regards to motor- and long-term memory performance (data not shown in Tables). Effect of MPEP on the brain concentrations of valproate and phenobarbital
Influence of MPEP, antiepileptic drugs and their combinations on amygdala-kindled seizures in rats
MPEP (up to 40 mg/kg) did not influence any kindling parameter in the tested animals (Tab. 1). Among the
MPEP (applied at 10 and 20 mg/kg) did not significantly alter the brain levels of valproate (50 mg/kg) and phenobarbital (10 mg/kg). The control concentration of valproate (91.2 ± 5.6) or phenobarbital (10.2 ± 1.3) reached 88.4 ± 6.0 and 9.9 ± 1.6 µg/ml, respectively,
Tab. 1. Influence of MPEP on amygdala-kindled seizures in rats
Treatment (mg/kg)
CADT
ADT
CSSv
SSv
CSD
SD
CAD
AD
MPEP (10)
106.4 ± 24.1
102.3 ± 21.0
5
5
24.8 ± 4.7
25.0 ± 5.4
41.5 ±3.7
41.9 ± 4.2
MPEP (20)
105.5 ± 23.8
108.1 ± 24.8
5
5
23.7 ± 5.3
23.5 ± 4.5
42.9 ±3.3
43.3 ± 3.9
MPEP (40)
108.7 ± 25.0
111.6 ± 26.2
5
5
24.5 ± 4.6
22.7 ± 4.4
43.2 ± 4.0
42.7 ± 3.8
Table data represent the means ± SE or medians with 25 and 75 percentiles of six rats per group. Absence of percentiles indicates identical readings. Control readings were made two days before and after the respective treatments. MPEP was administered 30 min before the test. C – control readings for ADT, SSv, SD and AD, respectively; ADT – afterdischarge duration; SSv – seizure severity; SD – seizure duration; AD – afterdischarge duration
624
Pharmacological Reports, 2009, 61, 621–630
MPEP and antiepileptic drugs in kindled seizures Kinga K. Borowicz et al.
Fig. 1. Effect of valproate (VPA) and their combinations with MPEP on seizure duration (A) and afterdischarge duration (B) in fully kindled rats. SD and AD data are the means ± SE (in seconds). All drugs were applied at their subprotective doses (in parentheses). Control readings were made two days before and after the respective treatments. Statistical analysis of the data was done with the help of the Wilcoxon signed rank test
Tab. 2. Influence of valproate and its combinations with MPEP on amygdala-kindled seizures in rats
Treatment (mg/kg)
CADT
ADT
CSSv
SSv
CSD
SD
CAD
AD
VPA (75)
105.0 ± 29.0
116.4 ± 30.6
5
4.5 (4;5)
28.9 ± 5.0
27.4 ± 4.7
44.0 ± 3.8
37.6 ± 4.2*
VPA (50)
103.3 ± 23.9
112.1 ± 28.6
5
5
30.3 ± 5.7
30.4 ± 5.3
45.2 ± 3.6
VPA (50) + MPEP (20)
101.2 ± 24.0
98.4 ± 27.9
5
5
33.7 ± 6.0
18.9 ± 5.1*** 48.4 ± 4.2
43.4 ± 3.6 27.8 ± 4.4***
VPA (50) + MPEP (10)
105.9 ± 24.2
102.4 ± 28.6
5
5
29.4 ± 5.3
20.3 ± 4.8**
44.3 ± 3.5
25.7 ± 3.9***
VPA (50) + MPEP (5)
106.7 ± 23.6
108.3 ± 29.0
5
5
27.8 ± 5.6
25.7 ± 4.2
46.6 ± 3.7
33.7 ± 3.5***
VPA (50) + MPEP (2.5)
103.5 ± 23.4
106.5 ± 26.2
5
5
29.5 ± 5.4
29.9 ± 4.9
43.1 ± 3.6
39.0 ± 3.6
Table data represent the means ± SE or medians with 25 and 75 percentiles of seven rats per group. Absence of SE or percentiles indicates identical readings. Control readings were made two days before and after the respective treatments. VPA and MPEP were administered 30 min before the seizure test. *** p < 0.001, ** p < 0.01, * p < 0.05 vs. respective controls (Wilcoxon signed rank test). VPA – valproate; C – control readings for ADT, SSv, SD and AD, respectively; ADT – afterdischarge duration; SSv – seizure severity; SD – seizure duration; AD – afterdischarge duration
Pharmacological Reports, 2009, 61, 621–630
625
Fig. 2. Effect of phenobarbital (PB) and their combinations with MPEP on seizure duration (A) and afterdischarge duration (B) in fully kindled rats. SD and AD data are the means ± SE (in seconds). See also the legend to Figure 1
Tab. 3. Effect of phenobarbital and its combinations with MPEP on amygdala-kindled seizures in rats
Treatment (mg/kg)
CADT
ADT
CSSv
SSv
CSD
SD
CAD
AD
PB (15)
102.8 ± 23.4
97.5 ± 21.7
5
5
26.3 ± 5.0
24.8 ± 5.1
42.0 ± 3.7
36.3 ± 4.0**
PB (10)
103.2 ± 23.7
104.3 ± 24.2
5
5 (5;4.5)
27.3 ± 4.2
25.6 ± 5.3
42.7 ± 4.1
41.7 ± 4.8
PB (10) + MPEP (20)
99.5 ± 25.1
96.4 ± 23.7
5
5 (5;4.5)
32.1 ± 5.2
27.5 ± 4.7**
47.6 ± 4.6
32.2 ± 4.2***
PB (10) + MPEP (10)
101.4 ± 24.2
95.4 ± 20.7
5
5
25.7 ± 3.9
21.6 ± 5.0**
43.7 ± 3.8
36.2 ± 3.9**
PB (10) + MPEP (5)
100.8 ± 22.9
101.3 ± 25.5
5
5
28.9 ± 4.7
26.7 ± 5.5
43.3 ± 3.9
40.9 ± 4.2
PB and MPEP were administered 60 min and 30 min before the seizure test, respectively. ** p < 0.01 vs. respective controls (Wilcoxon signed rank test). PB – phenobarbital; C – control readings for ADT, SSv, SD and AD, respectively; ADT – afterdischarge duration; SSv – seizure severity; SD – seizure duration; AD – afterdischarge duration. For more details, see the legend to Table 1
626
Pharmacological Reports, 2009, 61, 621–630
MPEP and antiepileptic drugs in kindled seizures Kinga K. Borowicz et al.
Tab. 4. Effect of carbamazepine and its combinations with MPEP on amygdala-kindled seizures in rats
Treatment (mg/kg)
CADT
ADT
CSSv
SSv
CSD
CBZ (10)
116.4 ± 33.0
117.1 ± 30.1
5
3.5 (3;4)*
24.4 ± 4.5
CBZ (7.5)
114.8 ± 33.9
108.6 ± 32.4
5
4.5 (4;5)
CBZ (7.5) + MPEP (10)
115.3 ± 30.4
113.0 ± 29.7
5
CBZ (7.5) + MPEP (20)
44.7 ± 3.5
46.5 ± 3.8
5
SD
CAD
AD
20.9 ± 4.3**
39.1 ± 5.0
33.0 ± 5.1**
26.1 ± 3.4
25.0 ± 5.0
40.3 ± 4.8
40.0 ± 4.3
4.5 (4;5)
26.3 ± 3.8
25.6 ± 4.4
40.9 ± 3.2
38.5 ± 5.7
4 (3.5.5)
27.3 ± 3.6
26.7 ± 3.4
41.3 ± 4.0
42.7 ± 3.6
CBZ and MPEP were administered 30 min before the seizure test. *** p < 0.001, ** p < 0.01, * p < 0.05 vs. respective controls (Wilcoxon signed rank test). CBZ – carbamazepine; C – control readings for ADT, SSv, SD and AD, respectively; ADT – afterdischarge duration; SSv – seizure severity; SD – seizure duration; AD – afterdischarge duration. For more details, see the legend to Table 1
Tab. 5. Effect of phenytoin and its combinations with MPEP on amygdala-kindled seizures in male and female rats
Treatment (mg/kg)
CADT
ADT
CSSv
SSv
CSD
SD
CAD
AD
PHT (15)
99.2 ± 19.4
104.2 ± 23.5
5
4 (3;5)
28.7 ± 4.8
23.9 ± 4.0**
40.2 ± 5.1
34.7 ± 4.8**
PHT (10)
98.5 ± 19.9
100.7 ± 20.0
5
4.5 (4;5)
29.4 ± 4.6
26.0 ± 4.3
41.5 ± 5.2
39.9 ± 6.4
PHT (10) + MPEP (10)
100.1 ± 20.8
97.6 ± 21.2
5
4 (3.5;5)
27.8 ± 4.4
26.4 ± 5.0
41.8 ± 5.6
38.3 ± 5.5
PHT (10) + MPEP (20)
97.9 ± 20.2
94.8 ± 22.6
5
4.5 (4;5)
29.9 ± 4.2
27.5 ± 4.6
42.0 ± 5.2
42.7 ± 6.0
DPH was administered 120 min and MPEP 30 min before the seizure test. ** p < 0.01 vs. respective controls (Wilcoxon signed rank test). PHT – phenytoin; C – control readings for ADT, SSv, SD and AD, respectively; ADT – afterdischarge duration; SSv – seizure severity; SD – seizure duration; AD – afterdischarge duration. For more details, see the legend to Table 1
in the presence of MPEP (10 mg/kg). MPEP (20 mg/kg) changed the concentration of valproate from 102.7 ± 6.5 to 99.3 ± 6.2 µg/ml and that of phenobarbital from 9.5 ± 1.5 to 9.3 ± 1.7 µg/ml (data not shown in Tables).
Discussion The present results demonstrate that the co-administration of MPEP with valproate or phenobarbital (administered at subprotective doses) produced significant protective effects against kindled-seizures in rats in terms of seizure and afterdischarge durations.
However, the increment of MPEP dose from 10 to 20 mg/kg did not enhance the final anticonvulsant effect of the studied combinations. Since the mGluR5 antagonist did not alter the brain concentrations of the two antiepileptics, the revealed interactions seem to be of a pharmacodynamic nature. In electrophysiological studies, MPEP completely blocks mGluR5 receptors with an IC50 of 36 nM and inhibits mGluR6 and NMDA1A/2B at concentrations 10 µM. However, it has no significant effect on human mGluR1 ( 30 µM) or mGluR2,3,4,7,8 ( 100 µM) [15]. According to some authors, the brain levels of MPEP applied at pharmacologically active doses are far below the concentrations needed to inhibit NMDA receptors in vitro [14]. If so, MPEP may be considered as a selective mGluR5 antagonist. Pharmacological Reports, 2009, 61, 621–630
627
Literature on the action of mGluR ligands in experimental models of epilepsy is rather scarce. In several studies, MPEP has been shown to have the greatest efficacy against convulsions induced by selective mGluR5 agonists. Weaker effects have been observed in sound-induced convulsions in DBA/2 mice and maximal electroshock in mice [9]. It is also known that mGluR5 antagonists may increase the anticonvulsant potential of some antiepileptic drugs and substances. For instance, MPEP combined with MK801 (0.1 mg/kg) and diazepam (0.5 mg/kg) could stop both behavioral and electrical status epilepticus (under EEG monitoring) within a few minutes after the administration [32]. Our previous data indicate that although MPEP increases the electroconvulsive threshold in mice, it fails to affect the protective action of conventional antiepileptic drugs against maximal electroshock [37]. Interestingly, in similar experimental conditions, the less active mGluR5 antagonist, SIB 1893 [23], decreases the electroconvulsive threshold, but potentiates the action of valproate [7, 20]. The kindling model of temporal lobe epilepsy is the only animal model that predicts the unfavorable clinical activity of competitive NMDA antagonists [18]. Experiments conducted in the same model have confirmed the anticonvulsant potential of some AMPA/kainate receptor antagonists [4, 6]. These results indicate the utility of this model in the search for more effective and less toxic glutamate receptor ligands. Nevertheless, results obtained by Löscher et al. [19] do not support a significant anticonvulsant potential for the group I mGlu receptor antagonists, 3-ethyl-2-methyl-quinolin-6-yl-(4-methoxycyclohexyl)methanone methanesulfonate (EMQMCM) and ([(2methyl-1,3-thiazol-4-yl)ethynyl]pyridine) (MTEP), in amygdala-kindled rats. On the other hand, in our previous experiments, SIB 1893 enhances the anticonvulsant efficacy of oxcarbazepine [5]. In light of these results, the positive interactions between MPEP and valproate or phenobarbital appear quite promising, also in the aspect of adverse effects. In contrast to most NMDA receptor antagonists, mGluR5 antagonists do not induce significant motor or long-term memory impairment. Our results confirm previous observations made by Mares and Mikulecka [21] who reported that at anticonvulsant doses, MPEP does not affect motor performance in immature rats. It should be also mentioned that MPEP decreases NMDA or glutamate-mediated neuronal toxicity [27].
628
Pharmacological Reports, 2009, 61, 621–630
This feature would be beneficial for antiepileptic therapy. Further, MPEP presents with antidepressant-like and anxiolytic-like effects, probably mediated through neuropeptide Y and norepinephrine signaling [12, 29, 35]. According to Smolders et al. [31], combined anticonvulsant-antidepressant activities of MPEP may result from locally elicited increases in hippocampal DA and/or 5-HT levels. Such properties may be important in the antiepileptic strategy, since mood disorders and anxiety-panic disorders are the most frequent co-morbid diseases in epileptic patients [1]. In conclusion, MPEP may positively interact with some antiepileptic drugs against amygdala-kindled convulsions, and this finding may initiate a novel strategy in the treatment of complex partial seizures. This observation, supported by a possibility of the beneficial influence of MPEP on mood disorders and anxiety accompanying epilepsy, justifies further investigations with this mGluR5 antagonist. Perhaps chronic treatment with MPEP will be more advantageous and reveal its complete anticonvulsant potential.
References: 1. Akanuma N, Hara E, Adachi N, Hara K, Koutroumanidis M: Psychiatric comorbidity in adult patients with idiopathic generalized epilepsy. Epilepsy Beh, 2008, 13, 248–251. 2. Barton ME, Peters SC, Shannon HE: Comparison of the effect of glutamate receptor modulators in the 6 Hz and maximal electroshock seizure models. Epilepsy Res, 2003, 56, 17–26. 3. Bordi F, Ugolini A: Group I metabotropic glutamate receptors: implications for brain diseases. Prog Neurobiol, 1999, 59, 55–79. 4. Borowicz KK, Duda AM, Kleinrok Z, Czuczwar SJ: Interaction of GYKI 52466, a selective non-competitive antagonist of AMPA/kainate receptors, with conventional antiepileptic drugs in amygdala-kindled seizures in rats. Pol J Pharmacol, 2001, 53, 101–108. 5. Borowicz KK, £uszczki JJ, Czuczwar SJ: SIB 1893, a selective mGluR5 receptor antagonist, potentiates the anticonvulsant of oxcarbazepine against amygdala-kindled convulsions. Pol J Pharmacol, 2004, 56, 459–464. 6. Borowicz KK, £uszczki J, Szadkowski M, Kleinrok Z, Czuczwar SJ: Influence of LY 300164, an antagonist of AMPA/kainate receptors, on the anticonvulsant activity of clonazepam. Eur J Pharmacol, 1999, 380, 67–72. 7. Borowicz KK, Piskorska B, £uszczki J, Czuczwar SJ: Influence of SIB 1893, a selective mGluR5 receptor antagonist, on the anticonvulsant activity of conventional antiepileptic drugs in two models of experimental epilepsy. Pol J Pharmacol, 2003, 55, 735–740.
MPEP and antiepileptic drugs in kindled seizures Kinga K. Borowicz et al.
8. Bruno V, Copani, A, Bonanno L, Knöpfel T, Kuhn R, Roberts PJ, Nicoletti F: Activation of group III metabotropic glutamate receptors is neuroprotective in cortical cultures. Eur J Pharmacol, 1996, 310, 61–66. 9. Chapman AG, Nanan K, Williams M, Meldrum BS: Anticonvulsant activity of two metabotropic glutamate group I antagonists selective for the mGlu5 receptor: 2-methyl-6-(phenylethynyl)-pyridine (MPEP), and (E)-6methyl-2-styryl-pyridine (SIB 1893). Neuropharmacology, 2000, 39, 1567–1574. 10. Czuczwar SJ, Turski WA, Kleinrok Z: Interactions of excitatory amino acid antagonists with conventional antiepileptic drugs. Metab Brain Dis, 1996, 11, 143–152. 11. Dunham NW, Miya TS: A note on a simple apparatus for determining neurological deficits in rats and mice. J Am Pharm Assoc, 1957, 46, 208–209. 12. Heidbreder CA, Bianchi M, Lacroix LP, Faedo S, Perdona E, Remelli R, Cavanni P, Crespi F: Evidence that the metabotropic glutamate receptor 5 antagonist MPEP may act as an inhibitor of the norepinephrine transporter in vitro and in vivo. Synapse, 2003, 50, 269–76. 13. Jesse CR, Savegnago L, Rocha JB, Nogueira CW: Neuroprotective effect caused by MPEP, an antagonist of metabotropic glutamate receptor mGluR5, on seizures induced by pilocarpine in 21-day-old rats. Brain Res, 2008, 10, 1198–1197. 14. Kuhn R, Pagano A, Stoehr N, Vranesic I, Flor PJ, Lingenhöhl K, Spooren W et al.: In vitro and in vivo characterization of MPEP, an allosteric modulator of the metabotropic glutamate receptor subtype 5: review article. Amino Acids, 2002, 23, 207–211. 15. Lea PM, Faden AI: Metabotropic glutamate receptor subtype 5 antagonists MPEP and MTEP. CNS Drug Rev, 2006, 2, 149–166. 16. Lojkova D, Mares P: Anticonvulsant action of an antagonist of metabotropic glutamate receptors mGluR5 MPEP in immature rats. Neuropharmacology, 2005, 49, 219–229. 17. Löscher W: Animal models of epilepsy for the development of antiepileptogenic and disease-modifying drugs. A comparison of the pharmacology of kindling and post-status epilepticus models of temporal lobe epilepsy. Epilepsy Res, 2002, 50, 105–123. 18. Löscher W: Pharmacology of glutamate receptor antagonists in the kindling model of epilepsy. Neuropharmacology, 1998, 54, 721–741. 19. Löscher W, Dekundy A, Nagel J, Danysz W, Parsons CG, Potschka H: MGlu1 and mGlu5 receptor antagonists lack anticonvulsant efficacy in rodent models of difficult-to-treat partial epilepsy. Neuropharmacology, 2006, 50, 1006–1015. 20. £uszczki J, Borowicz KK, Czuczwar SJ: SIB 1893 possesses pro- and anticonvulsant activity in the electroshock seizure threshold test in mice. Pol J Pharmacol, 2002, 54, 517–520. 21. Mares P, Mikulecka A: MPEP, an antagonist of metabotropic glutamate receptors, exhibits anticonvulsant action in immature rats without a serious impairment of motor performance. Epilepsy Res, 2004, 60, 17–26. 22. Mathiesen JM, Svendsen N, Bräuner-Osborne H, Thomsen C, Ramirez MT: Positive allosteric modulation of the hu-
23. 24.
25.
26.
27.
28. 29.
30. 31.
32.
33.
34.
35.
man metabotropic glutamate receptor 4 (hmGluR4) by SIB 1893 and MPEP. Br J Pharmacol, 2003, 138, 1026–1030. Micheli F: Methylphenylethynylpyridine (MPEP) Novartis. Curr Opin Investig Drugs, 2000, 1, 355–359. Nagaraya RY, Becker A, Reymann KG, Balschun D: Repeated administration of group I mGluR antagonists prevents seizure-induced long-term aberrations in hippocampal synaptic plasticity. Neuropharmacology, 2005, 49, 179–187. Nagaraja RY, Grecksch G, Reyman KG, Schroeder H, Becker A: Group I metabotropic glutamate receptors interfere in different ways with pentylenetetrazole seizures, kindling, and kindling-related learning deficits. Naunyn Schmiedebergs Arch Pharmacol, 2004, 370, 26–34. Neugebauer V, Zinebi F, Russel R, Gallagher JP, Shinnick-Gallagher P: Cocaine and kindling alter the sensitivity of group II and III metabotropic glutamate receptors in the central amygdala. J Neurophysiol, 2000, 84, 759–770. O’Leary DM, Movsesyan V, Vicini S, Faden AI: Selective mGluR5 antagonists MPEP and SIB-1893 decrease NMDA or glutamate-mediated neuronal toxicity through actions that reflect NMDA receptor antagonism. Br J Pharmacol, 2000, 131, 1429–1437. Paxinos G, Watson C: The Rat Brain in Stereotaxic Coordinates, 2nd ed. Academic Press, Sydney, 1986. Pilc A, K³odzinska A, Brañski P, Nowak G, Palucha A, Szewczyk B, Tatarczyñska E et al.: Multiple MPEP administration evoke anxiolytic- and antidepressant-like effects in rats. Neuropharmacology, 2002, 43, 181–187. Racine RJ: Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol, 1972, 32, 281–294. Smolders I, Clinckers R, Meurs A, De Bundel D, Portelli J, Ebinger G, Michotte Y: Direct enhancement of hippocampal dopamine or serotonin levels as a pharmacodynamic measure of combined antidepressant-anticonvulsant action. Neuropharmacology, 2008, 54, 1017–1028. Tang FR, Chen PM, Tang YC, Tsai MC, Lee WL: Two-methyl-6-phenylethynyl-pyridine (MPEP), a metabotropic glutamate receptor 5 antagonist, with low doses of MK801 and diazepam: a novel approach for controlling status epilepticus. Neuropharmacology, 2007, 53, 821–831. Venault P, Chapouthier G, De Carvalho LP, Simiand J, Morre M, Dodd RH, Rossier J: Benzodiazepines impair and beta-carbolines enhance performance in learning and memory task. Nature, 1986, 321, 864–866. Wang SJ, Sihra TS: Noncompetitive metabotropic glutamate 5 receptor antagonist (E)-2-methyl-6-styrylpyridine (SIB 1893) depresses glutamate release through inhibition of voltage-dependent Ca2+ entry in rat cerebrocortical nerve terminals (synaptosomes). J Pharmacol Exp Ther, 2004, 309, 951–958. Wieroñska JM, Smia³owska M, Brañski P, Gasparini F, K³odzinska A, Szewczyk B, Palucha A et al.: In the amygdala anxiolytic action of mGlu5 receptors antagonist MPEP involves neuropeptide Y but not GABA-A signaling. Neuropsychopharmacology, 2004, 29, 514–521.
Pharmacological Reports, 2009, 61, 621–630
629
36. Wojtal K, Borowicz KK, B³aszczyk B, Czuczwar SJ: Interactions of excitatory amino acid receptor antagonists with antiepileptic drugs in three basic models of experimental epilepsy. Pharmacol Rep, 2006, 58, 587–598. 37. Zadro¿niak M, Sêkowski A, Czuczwar SJ, Borowicz KK: Effect of MPEP, a selective mGluR5 antagonist, on
630
Pharmacological Reports, 2009, 61, 621–630
the antielectroshock activity of conventional antiepileptic drugs. Pol J Pharmacol, 2004, 56, 595–598.
Received: October 20, 2008; in revised form: June 9, 2009.
Copyright © 2009 by Institute of Pharmacology Polish Academy of Sciences
2-Methyl-6-phenylethynyl-pyridine (MPEP), a non-competitive mGluR5 antagonist, differentially affects the anticonvulsant activity of four conventional antiepileptic drugs against amygdala-kindled seizures in rats Kinga K. Borowicz1, Jarogniew J. £uszczki2, Stanis³aw J. Czuczwar2
Department of Pathophysiology, Medical University, Jaczewskiego 8, PL 20-090 Lublin, Poland Department of Physiopathology, Institute of Agricultural Medicine, Jaczewskiego 2, PL 20-950 Lublin, Poland
Correspondence: Kinga K. Borowicz, e-mail: [email protected]
Abstract: 2-Methyl-6-phenylethynyl-pyridine (MPEP), a selective noncompetitive mGluR5 antagonist, influences the action of conventional antiepileptic drugs in amygdala-kindled seizures in rats. MPEP alone (up to 40 mg/kg) did not affect any seizure parameter. Moreover, the common treatment of MPEP with either carbamazepine or phenytoin (administered at subeffective doses) did not result in any anticonvulsant action in kindled rats. However, when combined with subprotective doses of valproate or phenobarbital, MPEP significantly shortened seizure and afterdischarge durations. Importantly, combinations of MPEP with the two antiepileptics did not have the adverse effects of impaired motor performance or long-term memory in rats. Our data indicate that MPEP may positively interact with some conventional antiepileptic drugs in the amygdala-kindling model of complex partial seizures. Key words: MPEP, metabotropic glutamate receptors, conventional antiepileptic drugs, amygdala-kindled seizures
Introduction Several reports have indicated that selective ligands for metabotropic glutamate receptor (mGluR) subtypes have the potential to treat of a wide variety of neurological and psychiatric disorders, including depression, anxiety disorders, schizophrenia, Alzheimer’s disease and epilepsy, among others. All mGluRs have been classified into three groups, which present with different biological, electrophysiological and pharmacological properties. Receptors for group I
mGluRs are positively linked to phospholipase C, while group II and III receptors are negatively coupled to adenylyl cyclase [3]. Disappointing experience with ionotropic, particularly NMDA, receptor antagonists with respect of their usefulness in anticonvulsant therapy [10, 36] have directed researchers’ efforts to modulators of mGluRs. Results from many experimental studies have confirmed the anticonvulsant and neuroprotective actions of group I mGluR antagonists and group II/III mGluR agonists [8]. 2-Methyl-6-phenylethynyl-pyridine (MPEP) and (E)-2methyl-2-styrylpyridine (SIB 1893) are the most Pharmacological Reports, 2009, 61, 621–630
621
well-known representatives of selective noncompetitive antagonists of mGluR5 receptors belonging to group I. They attenuate Ca2+ entry through voltagedependent N- and P/Q- type Ca2+ channels, which causes a reduction in glutamate release [34]. Therefore, it is likely that MPEP and SIB 1893 will present with anticonvulsant properties. Additionally, mGluR5 antagonists significantly decrease the duration of NMDA channels opening [27] and positively modulate mGluR4 receptors [22], which should enhance their anticonvulsant action. However, the latter information has not been confirmed by other authors [15]. Preliminary experiments indicate that mGluR5 antagonists may have therapeutic potential in the treatment of epilepsy, stroke, anxiety, pain, neurodegenerative disease [23] and cocaine withdrawal [26]. In experimental conditions, the selective antagonists of mGlu5 are the most effective against seizures induced by activation of mGlu5 receptors. At higher doses, SIB 1893 and MPEP also block convulsive and non-convulsive primary generalized seizures [9]. SIB 1893 has a proven protective efficacy against soundinduced seizures in DBA/2 and primary generalized non-convulsive seizures in lethargic mice [9]. MPEP has shown anticonvulsive efficacy against clonic seizures induced by pentylenetetrazole. This mGluR5 antagonist does not affect the PTZ-kindling progression; however, it counteracts learning deficits [25] and long-term hippocampal aberrations induced by the PTZ kindling process [24]. Moreover, MPEP exhibits anticonvulsant activity in the 6Hz model of partial seizures [2]. In immature rats, MPEP inhibits behavioral seizures induced by pilocarpine [13] and pentylenetetrazole [21] and reduces the afterdischarges induced by electrical stimulation of the sensorimotor cortical area [16]. In a previous study, it had been demonstrated that SIB 1893 possesses dual proconvulsant (at low doses) and anticonvulsant (at high doses) activity in the electroshock seizure threshold test in mice. SIB 1893, given at ineffective doses, enhances the protective action of valproate against maximal electroshock in mice, the basic model of generalized tonic-clonic convulsions. Moreover, the mGluR5 antagonist does not affect the protection of conventional antiepileptic drugs against pentylenetetrazole-induced convulsions in mice, reflecting myoclonic epilepsy in humans [7, 17, 20]. On the other hand, SIB 1893 reduces amygdala-kindled seizures in rats and potentiates the pro-
622
Pharmacological Reports, 2009, 61, 621–630
tective action of oxcarbazepine in this test, which is the most recommended model of partial complex seizures [5, 17]. The second mGluR5 antagonist, MPEP, increases the electroconvulsive threshold in mice, but it does not affect the efficacy of conventional antiepileptics against the maximal electroshock test [37]. These previous results prompted us to broaden the investigations of MPEP and evaluate its effect on amygdala-kindled seizures and the protective action of four antiepileptics (valproate, carbamazepine, phenobarbital and phenytoin) in this experimental model of epilepsy. We also assessed the influence of MPEP on the brain concentrations of the respective antiepileptics to define the possible involvement of a pharmacokinetic interaction that could explain the obtained results.
Materials and Methods Animals and experimental conditions
All experiments were performed on adult male Wistar rats weighing 200–250 g. The rats were kept in colony cages with free access to food and tap water under standardized housing conditions (natural lightdark cycle; temperature: 21 ± 1°C; relative humidity: 55 ± 5%). After seven days of adaptation to laboratory conditions, the animals were randomly assigned to experimental groups that each consisted of eight rats. All tests were performed between 9:00 a.m. and 2:00 p.m. Procedures involving animals and their care were conducted in conformity 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 listed below conformed to the Guide for the Care and Use of Laboratory Animals and were approved by the Local Ethics Committee at the Medical University of Lublin. Surgery and kindling procedure
The rats were anesthetized with pentobarbital (50 mg/kg, ip) and received stereotaxic implantation of one bipolar electrode in the right basolateral amygdala. The
MPEP and antiepileptic drugs in kindled seizures Kinga K. Borowicz et al.
following stereotaxic coordinates for electrode implantation were used: AP-1.5, L-4.4, V-8.5, according to the brain atlas of Paxinos and Watson [28]. All coordinates were measured from the bregma. Scull screws served as the indifferent reference electrodes. The electrode assembly was attached to the scull by dental acrylic cement. After electrode implantation, the animals were treated with an antibiotic for one week to prevent infection. After a post-operative period of two weeks, stimulation of the amygdala was initiated. Each stimulus consisted of a 1 s train of 50 Hz, 1 ms biphasic square-wave pulses with a current intensity of 500 µA, which were delivered every 24 h, until at least 10 sequential fully kindled stage 5 seizures were elicited. The afterdischarges from the amygdala were recorded prior to and after the stimulation. The seizure severity (SSv) was assessed according to the following modified Racine‘s system [30]: 0 = no seizure response; 1 = immobility, eye closure, ear twitching, twitching of vibrissae, sniffing, facial clonus; 2 = head nodding associated with more severe facial clonus; 3 = clonus of one forelimb; 3.5 = bilateral forelimb clonus without rearing; 4 = bilateral forelimb clonus with rearing; 4.5 = falling on a side (without rearing), loss of righting reflex accompanied by generalized clonic seizures; 5 = rearing and falling on the back accompanied by generalized clonic seizures. Seizure duration (SD) was the duration of limbic seizures (stage 1–2) and motor seizures (stage 3–5). Afterdischarges (AD) were defined as spikes with a frequency of at least 1 Hz and amplitude that was at least two-fold greater than the pre-stimulation baseline present in the EEG recorded from the site of stimulation. Afterdischarge threshold (ADT) was determined after administration of a test drug or its vehicle. Electrical stimulations increasing in steps of about 20% of the previously applied current were given at intervals of 5 min until an afterdischarge lasting at least 3 s was evoked. At the ADT current, seizure severity, seizure duration and afterdischarge duration were recorded and analyzed. Control readings were made two days before and two days after the respective treatments. Subsequently, the mean ± SE was calculated and used for statistical comparisons of the obtained data. Drugs
The following substances and drugs were used in the present study: MPEP (Tocris Cookson Ltd., Bristol,
UK), carbamazepine (CBZ; Polpharma, Starogard, Poland); valproate magnesium (VPA; a gift from ICN-Polfa, Rzeszów, Poland); phenytoin (PHT; Polfa, Warszawa, Poland), and phenobarbital (PB; Polfa, Kraków, Poland). VPA and MPEP were dissolved in distilled water, while CBZ, PHT and PB were suspended in a 1% aqueous solution of Tween 80 (Sigma, St. Louis, MO, USA). All drugs were administered intraperitoneally (ip) in a volume of 0.01 ml/g body weight, PHT – 120 min, PB – 60 min, VPA, CBZ and MPEP – at 30 min before amygdala-kindled seizures and behavioral tests. The pretreatment times corresponded to times of their maximal anticonvulsant activity taken from literature data and confirmed in our previous studies. Rotarod test
Motor coordination was assessed via the rotarod test [11]. The rod (6 cm in diameter) was rotating at 6 rpm. For each training session rats were placed on the rod for 120 s. After that, animals that had been pretreated with antiepileptic drugs or their combinations with MPEP were placed on the rod again. The time it took for each animal to fall was used as a measure of motor impairment. The observation time was always 120 s. Passive avoidance task
Rats were placed in an illuminated box (40 × 40 × 30 cm) connected to a dark box (40 × 40 × 30 cm), which was equipped with an electric grid floor. Entrance into the dark box was punished by an electric footshock (0.8 mA) of 2 s duration. The animals that did not enter the dark compartment were excluded from the experiment. On the next day (24 h later), the same animals were put into the illuminated box and observed up to 180 s. The time period an animal entered the dark box was subsequently noted and the medians with 25 and 75 percentiles were calculated. According to Venault et al. [33], the step-through passive avoidance task may be recognized as a measure of longterm memory. Estimation of brain levels of antiepileptic drugs
Rats were administered with one of the conventional antiepileptic drugs and vehicle or with the respective AED + MPEP. Animals were killed by decapitation at times chosen to coincide with those scheduled for the
Pharmacological Reports, 2009, 61, 621–630
623
seizure test. Brains were removed from skulls, weighed, and homogenized using Abbott buffer (2:1 v/w) in an Ultra-Turrax T8 homogenizer (IKA-WERKE, Stauffen, Germany). The homogenates were centrifuged at 10,000 × g for 10 min The supernatant samples (75 µl) were analyzed by fluorescence polarization immunoassay (FPIA) for PHT, CBZ, VPA or PB content using a TDx analyzer and reagents exactly as described by the manufacturer (Abbott Laboratories, North Chicago, IL, USA). All AED concentrations are expressed in µg/ml of brain supernatants as the means ± SD of at least eight determinations. Statistics
The statistical significances of differences between seizure and afterdischarge durations were calculated by Student‘s t-test for paired replicates. Seizure scores were evaluated with Wilcoxon signed rank test. The results obtained in the step-through passive avoidance task and rotarod test were statistically evaluated using Kruskal-Wallis nonparametric ANOVA followed by post-hoc Dunn’s test. Plasma levels of antiepileptics alone or in combination with MPEP were evaluated with Student‘s t-test for unpaired replicates.
Results
conventional antiepileptic drugs, valproate (at 75 mg/kg) and phenobarbital (15 mg/kg) significantly decreased the afterdischarge duration. Phenytoin (15 mg/kg) markedly decreased the two parameters of seizure and afterdischarge duration. Carbamazepine (10 mg/kg) additionally reduced the seizure severity (Tab. 1–4). In further experiments, MPEP was combined with antiepileptic drugs applied at subprotective doses. Valproate (50 mg/kg) combined with MPEP (10 or 20 mg/kg) markedly reduced the seizure and afterdischarge durations, while with MPEP (5 mg/kg) it shortened only the latter parameter (Fig. 1, Tab. 2). The combined treatment of phenobarbital (10 mg/kg) and MPEP (10 or 20 mg/kg) reduced the seizure and afterdischarge durations (Fig. 2, Tab. 3). However, the combinations of carbamazepine (7.5 mg/kg) and phenytoin (10 mg/kg) with MPEP (10 or 20 mg/kg) did not influence amygdala-kindled seizures (Tab. 4, 5). Rotarod test and passive avoidance task
MPEP (10 and 20 mg/kg), valproate (75 and 50 mg/kg), phenobarbital (10 and 15 mg/kg) and the combinations of MPEP with both antiepileptics did not produce any significant adverse effects with regards to motor- and long-term memory performance (data not shown in Tables). Effect of MPEP on the brain concentrations of valproate and phenobarbital
Influence of MPEP, antiepileptic drugs and their combinations on amygdala-kindled seizures in rats
MPEP (up to 40 mg/kg) did not influence any kindling parameter in the tested animals (Tab. 1). Among the
MPEP (applied at 10 and 20 mg/kg) did not significantly alter the brain levels of valproate (50 mg/kg) and phenobarbital (10 mg/kg). The control concentration of valproate (91.2 ± 5.6) or phenobarbital (10.2 ± 1.3) reached 88.4 ± 6.0 and 9.9 ± 1.6 µg/ml, respectively,
Tab. 1. Influence of MPEP on amygdala-kindled seizures in rats
Treatment (mg/kg)
CADT
ADT
CSSv
SSv
CSD
SD
CAD
AD
MPEP (10)
106.4 ± 24.1
102.3 ± 21.0
5
5
24.8 ± 4.7
25.0 ± 5.4
41.5 ±3.7
41.9 ± 4.2
MPEP (20)
105.5 ± 23.8
108.1 ± 24.8
5
5
23.7 ± 5.3
23.5 ± 4.5
42.9 ±3.3
43.3 ± 3.9
MPEP (40)
108.7 ± 25.0
111.6 ± 26.2
5
5
24.5 ± 4.6
22.7 ± 4.4
43.2 ± 4.0
42.7 ± 3.8
Table data represent the means ± SE or medians with 25 and 75 percentiles of six rats per group. Absence of percentiles indicates identical readings. Control readings were made two days before and after the respective treatments. MPEP was administered 30 min before the test. C – control readings for ADT, SSv, SD and AD, respectively; ADT – afterdischarge duration; SSv – seizure severity; SD – seizure duration; AD – afterdischarge duration
624
Pharmacological Reports, 2009, 61, 621–630
MPEP and antiepileptic drugs in kindled seizures Kinga K. Borowicz et al.
Fig. 1. Effect of valproate (VPA) and their combinations with MPEP on seizure duration (A) and afterdischarge duration (B) in fully kindled rats. SD and AD data are the means ± SE (in seconds). All drugs were applied at their subprotective doses (in parentheses). Control readings were made two days before and after the respective treatments. Statistical analysis of the data was done with the help of the Wilcoxon signed rank test
Tab. 2. Influence of valproate and its combinations with MPEP on amygdala-kindled seizures in rats
Treatment (mg/kg)
CADT
ADT
CSSv
SSv
CSD
SD
CAD
AD
VPA (75)
105.0 ± 29.0
116.4 ± 30.6
5
4.5 (4;5)
28.9 ± 5.0
27.4 ± 4.7
44.0 ± 3.8
37.6 ± 4.2*
VPA (50)
103.3 ± 23.9
112.1 ± 28.6
5
5
30.3 ± 5.7
30.4 ± 5.3
45.2 ± 3.6
VPA (50) + MPEP (20)
101.2 ± 24.0
98.4 ± 27.9
5
5
33.7 ± 6.0
18.9 ± 5.1*** 48.4 ± 4.2
43.4 ± 3.6 27.8 ± 4.4***
VPA (50) + MPEP (10)
105.9 ± 24.2
102.4 ± 28.6
5
5
29.4 ± 5.3
20.3 ± 4.8**
44.3 ± 3.5
25.7 ± 3.9***
VPA (50) + MPEP (5)
106.7 ± 23.6
108.3 ± 29.0
5
5
27.8 ± 5.6
25.7 ± 4.2
46.6 ± 3.7
33.7 ± 3.5***
VPA (50) + MPEP (2.5)
103.5 ± 23.4
106.5 ± 26.2
5
5
29.5 ± 5.4
29.9 ± 4.9
43.1 ± 3.6
39.0 ± 3.6
Table data represent the means ± SE or medians with 25 and 75 percentiles of seven rats per group. Absence of SE or percentiles indicates identical readings. Control readings were made two days before and after the respective treatments. VPA and MPEP were administered 30 min before the seizure test. *** p < 0.001, ** p < 0.01, * p < 0.05 vs. respective controls (Wilcoxon signed rank test). VPA – valproate; C – control readings for ADT, SSv, SD and AD, respectively; ADT – afterdischarge duration; SSv – seizure severity; SD – seizure duration; AD – afterdischarge duration
Pharmacological Reports, 2009, 61, 621–630
625
Fig. 2. Effect of phenobarbital (PB) and their combinations with MPEP on seizure duration (A) and afterdischarge duration (B) in fully kindled rats. SD and AD data are the means ± SE (in seconds). See also the legend to Figure 1
Tab. 3. Effect of phenobarbital and its combinations with MPEP on amygdala-kindled seizures in rats
Treatment (mg/kg)
CADT
ADT
CSSv
SSv
CSD
SD
CAD
AD
PB (15)
102.8 ± 23.4
97.5 ± 21.7
5
5
26.3 ± 5.0
24.8 ± 5.1
42.0 ± 3.7
36.3 ± 4.0**
PB (10)
103.2 ± 23.7
104.3 ± 24.2
5
5 (5;4.5)
27.3 ± 4.2
25.6 ± 5.3
42.7 ± 4.1
41.7 ± 4.8
PB (10) + MPEP (20)
99.5 ± 25.1
96.4 ± 23.7
5
5 (5;4.5)
32.1 ± 5.2
27.5 ± 4.7**
47.6 ± 4.6
32.2 ± 4.2***
PB (10) + MPEP (10)
101.4 ± 24.2
95.4 ± 20.7
5
5
25.7 ± 3.9
21.6 ± 5.0**
43.7 ± 3.8
36.2 ± 3.9**
PB (10) + MPEP (5)
100.8 ± 22.9
101.3 ± 25.5
5
5
28.9 ± 4.7
26.7 ± 5.5
43.3 ± 3.9
40.9 ± 4.2
PB and MPEP were administered 60 min and 30 min before the seizure test, respectively. ** p < 0.01 vs. respective controls (Wilcoxon signed rank test). PB – phenobarbital; C – control readings for ADT, SSv, SD and AD, respectively; ADT – afterdischarge duration; SSv – seizure severity; SD – seizure duration; AD – afterdischarge duration. For more details, see the legend to Table 1
626
Pharmacological Reports, 2009, 61, 621–630
MPEP and antiepileptic drugs in kindled seizures Kinga K. Borowicz et al.
Tab. 4. Effect of carbamazepine and its combinations with MPEP on amygdala-kindled seizures in rats
Treatment (mg/kg)
CADT
ADT
CSSv
SSv
CSD
CBZ (10)
116.4 ± 33.0
117.1 ± 30.1
5
3.5 (3;4)*
24.4 ± 4.5
CBZ (7.5)
114.8 ± 33.9
108.6 ± 32.4
5
4.5 (4;5)
CBZ (7.5) + MPEP (10)
115.3 ± 30.4
113.0 ± 29.7
5
CBZ (7.5) + MPEP (20)
44.7 ± 3.5
46.5 ± 3.8
5
SD
CAD
AD
20.9 ± 4.3**
39.1 ± 5.0
33.0 ± 5.1**
26.1 ± 3.4
25.0 ± 5.0
40.3 ± 4.8
40.0 ± 4.3
4.5 (4;5)
26.3 ± 3.8
25.6 ± 4.4
40.9 ± 3.2
38.5 ± 5.7
4 (3.5.5)
27.3 ± 3.6
26.7 ± 3.4
41.3 ± 4.0
42.7 ± 3.6
CBZ and MPEP were administered 30 min before the seizure test. *** p < 0.001, ** p < 0.01, * p < 0.05 vs. respective controls (Wilcoxon signed rank test). CBZ – carbamazepine; C – control readings for ADT, SSv, SD and AD, respectively; ADT – afterdischarge duration; SSv – seizure severity; SD – seizure duration; AD – afterdischarge duration. For more details, see the legend to Table 1
Tab. 5. Effect of phenytoin and its combinations with MPEP on amygdala-kindled seizures in male and female rats
Treatment (mg/kg)
CADT
ADT
CSSv
SSv
CSD
SD
CAD
AD
PHT (15)
99.2 ± 19.4
104.2 ± 23.5
5
4 (3;5)
28.7 ± 4.8
23.9 ± 4.0**
40.2 ± 5.1
34.7 ± 4.8**
PHT (10)
98.5 ± 19.9
100.7 ± 20.0
5
4.5 (4;5)
29.4 ± 4.6
26.0 ± 4.3
41.5 ± 5.2
39.9 ± 6.4
PHT (10) + MPEP (10)
100.1 ± 20.8
97.6 ± 21.2
5
4 (3.5;5)
27.8 ± 4.4
26.4 ± 5.0
41.8 ± 5.6
38.3 ± 5.5
PHT (10) + MPEP (20)
97.9 ± 20.2
94.8 ± 22.6
5
4.5 (4;5)
29.9 ± 4.2
27.5 ± 4.6
42.0 ± 5.2
42.7 ± 6.0
DPH was administered 120 min and MPEP 30 min before the seizure test. ** p < 0.01 vs. respective controls (Wilcoxon signed rank test). PHT – phenytoin; C – control readings for ADT, SSv, SD and AD, respectively; ADT – afterdischarge duration; SSv – seizure severity; SD – seizure duration; AD – afterdischarge duration. For more details, see the legend to Table 1
in the presence of MPEP (10 mg/kg). MPEP (20 mg/kg) changed the concentration of valproate from 102.7 ± 6.5 to 99.3 ± 6.2 µg/ml and that of phenobarbital from 9.5 ± 1.5 to 9.3 ± 1.7 µg/ml (data not shown in Tables).
Discussion The present results demonstrate that the co-administration of MPEP with valproate or phenobarbital (administered at subprotective doses) produced significant protective effects against kindled-seizures in rats in terms of seizure and afterdischarge durations.
However, the increment of MPEP dose from 10 to 20 mg/kg did not enhance the final anticonvulsant effect of the studied combinations. Since the mGluR5 antagonist did not alter the brain concentrations of the two antiepileptics, the revealed interactions seem to be of a pharmacodynamic nature. In electrophysiological studies, MPEP completely blocks mGluR5 receptors with an IC50 of 36 nM and inhibits mGluR6 and NMDA1A/2B at concentrations 10 µM. However, it has no significant effect on human mGluR1 ( 30 µM) or mGluR2,3,4,7,8 ( 100 µM) [15]. According to some authors, the brain levels of MPEP applied at pharmacologically active doses are far below the concentrations needed to inhibit NMDA receptors in vitro [14]. If so, MPEP may be considered as a selective mGluR5 antagonist. Pharmacological Reports, 2009, 61, 621–630
627
Literature on the action of mGluR ligands in experimental models of epilepsy is rather scarce. In several studies, MPEP has been shown to have the greatest efficacy against convulsions induced by selective mGluR5 agonists. Weaker effects have been observed in sound-induced convulsions in DBA/2 mice and maximal electroshock in mice [9]. It is also known that mGluR5 antagonists may increase the anticonvulsant potential of some antiepileptic drugs and substances. For instance, MPEP combined with MK801 (0.1 mg/kg) and diazepam (0.5 mg/kg) could stop both behavioral and electrical status epilepticus (under EEG monitoring) within a few minutes after the administration [32]. Our previous data indicate that although MPEP increases the electroconvulsive threshold in mice, it fails to affect the protective action of conventional antiepileptic drugs against maximal electroshock [37]. Interestingly, in similar experimental conditions, the less active mGluR5 antagonist, SIB 1893 [23], decreases the electroconvulsive threshold, but potentiates the action of valproate [7, 20]. The kindling model of temporal lobe epilepsy is the only animal model that predicts the unfavorable clinical activity of competitive NMDA antagonists [18]. Experiments conducted in the same model have confirmed the anticonvulsant potential of some AMPA/kainate receptor antagonists [4, 6]. These results indicate the utility of this model in the search for more effective and less toxic glutamate receptor ligands. Nevertheless, results obtained by Löscher et al. [19] do not support a significant anticonvulsant potential for the group I mGlu receptor antagonists, 3-ethyl-2-methyl-quinolin-6-yl-(4-methoxycyclohexyl)methanone methanesulfonate (EMQMCM) and ([(2methyl-1,3-thiazol-4-yl)ethynyl]pyridine) (MTEP), in amygdala-kindled rats. On the other hand, in our previous experiments, SIB 1893 enhances the anticonvulsant efficacy of oxcarbazepine [5]. In light of these results, the positive interactions between MPEP and valproate or phenobarbital appear quite promising, also in the aspect of adverse effects. In contrast to most NMDA receptor antagonists, mGluR5 antagonists do not induce significant motor or long-term memory impairment. Our results confirm previous observations made by Mares and Mikulecka [21] who reported that at anticonvulsant doses, MPEP does not affect motor performance in immature rats. It should be also mentioned that MPEP decreases NMDA or glutamate-mediated neuronal toxicity [27].
628
Pharmacological Reports, 2009, 61, 621–630
This feature would be beneficial for antiepileptic therapy. Further, MPEP presents with antidepressant-like and anxiolytic-like effects, probably mediated through neuropeptide Y and norepinephrine signaling [12, 29, 35]. According to Smolders et al. [31], combined anticonvulsant-antidepressant activities of MPEP may result from locally elicited increases in hippocampal DA and/or 5-HT levels. Such properties may be important in the antiepileptic strategy, since mood disorders and anxiety-panic disorders are the most frequent co-morbid diseases in epileptic patients [1]. In conclusion, MPEP may positively interact with some antiepileptic drugs against amygdala-kindled convulsions, and this finding may initiate a novel strategy in the treatment of complex partial seizures. This observation, supported by a possibility of the beneficial influence of MPEP on mood disorders and anxiety accompanying epilepsy, justifies further investigations with this mGluR5 antagonist. Perhaps chronic treatment with MPEP will be more advantageous and reveal its complete anticonvulsant potential.
References: 1. Akanuma N, Hara E, Adachi N, Hara K, Koutroumanidis M: Psychiatric comorbidity in adult patients with idiopathic generalized epilepsy. Epilepsy Beh, 2008, 13, 248–251. 2. Barton ME, Peters SC, Shannon HE: Comparison of the effect of glutamate receptor modulators in the 6 Hz and maximal electroshock seizure models. Epilepsy Res, 2003, 56, 17–26. 3. Bordi F, Ugolini A: Group I metabotropic glutamate receptors: implications for brain diseases. Prog Neurobiol, 1999, 59, 55–79. 4. Borowicz KK, Duda AM, Kleinrok Z, Czuczwar SJ: Interaction of GYKI 52466, a selective non-competitive antagonist of AMPA/kainate receptors, with conventional antiepileptic drugs in amygdala-kindled seizures in rats. Pol J Pharmacol, 2001, 53, 101–108. 5. Borowicz KK, £uszczki JJ, Czuczwar SJ: SIB 1893, a selective mGluR5 receptor antagonist, potentiates the anticonvulsant of oxcarbazepine against amygdala-kindled convulsions. Pol J Pharmacol, 2004, 56, 459–464. 6. Borowicz KK, £uszczki J, Szadkowski M, Kleinrok Z, Czuczwar SJ: Influence of LY 300164, an antagonist of AMPA/kainate receptors, on the anticonvulsant activity of clonazepam. Eur J Pharmacol, 1999, 380, 67–72. 7. Borowicz KK, Piskorska B, £uszczki J, Czuczwar SJ: Influence of SIB 1893, a selective mGluR5 receptor antagonist, on the anticonvulsant activity of conventional antiepileptic drugs in two models of experimental epilepsy. Pol J Pharmacol, 2003, 55, 735–740.
MPEP and antiepileptic drugs in kindled seizures Kinga K. Borowicz et al.
8. Bruno V, Copani, A, Bonanno L, Knöpfel T, Kuhn R, Roberts PJ, Nicoletti F: Activation of group III metabotropic glutamate receptors is neuroprotective in cortical cultures. Eur J Pharmacol, 1996, 310, 61–66. 9. Chapman AG, Nanan K, Williams M, Meldrum BS: Anticonvulsant activity of two metabotropic glutamate group I antagonists selective for the mGlu5 receptor: 2-methyl-6-(phenylethynyl)-pyridine (MPEP), and (E)-6methyl-2-styryl-pyridine (SIB 1893). Neuropharmacology, 2000, 39, 1567–1574. 10. Czuczwar SJ, Turski WA, Kleinrok Z: Interactions of excitatory amino acid antagonists with conventional antiepileptic drugs. Metab Brain Dis, 1996, 11, 143–152. 11. Dunham NW, Miya TS: A note on a simple apparatus for determining neurological deficits in rats and mice. J Am Pharm Assoc, 1957, 46, 208–209. 12. Heidbreder CA, Bianchi M, Lacroix LP, Faedo S, Perdona E, Remelli R, Cavanni P, Crespi F: Evidence that the metabotropic glutamate receptor 5 antagonist MPEP may act as an inhibitor of the norepinephrine transporter in vitro and in vivo. Synapse, 2003, 50, 269–76. 13. Jesse CR, Savegnago L, Rocha JB, Nogueira CW: Neuroprotective effect caused by MPEP, an antagonist of metabotropic glutamate receptor mGluR5, on seizures induced by pilocarpine in 21-day-old rats. Brain Res, 2008, 10, 1198–1197. 14. Kuhn R, Pagano A, Stoehr N, Vranesic I, Flor PJ, Lingenhöhl K, Spooren W et al.: In vitro and in vivo characterization of MPEP, an allosteric modulator of the metabotropic glutamate receptor subtype 5: review article. Amino Acids, 2002, 23, 207–211. 15. Lea PM, Faden AI: Metabotropic glutamate receptor subtype 5 antagonists MPEP and MTEP. CNS Drug Rev, 2006, 2, 149–166. 16. Lojkova D, Mares P: Anticonvulsant action of an antagonist of metabotropic glutamate receptors mGluR5 MPEP in immature rats. Neuropharmacology, 2005, 49, 219–229. 17. Löscher W: Animal models of epilepsy for the development of antiepileptogenic and disease-modifying drugs. A comparison of the pharmacology of kindling and post-status epilepticus models of temporal lobe epilepsy. Epilepsy Res, 2002, 50, 105–123. 18. Löscher W: Pharmacology of glutamate receptor antagonists in the kindling model of epilepsy. Neuropharmacology, 1998, 54, 721–741. 19. Löscher W, Dekundy A, Nagel J, Danysz W, Parsons CG, Potschka H: MGlu1 and mGlu5 receptor antagonists lack anticonvulsant efficacy in rodent models of difficult-to-treat partial epilepsy. Neuropharmacology, 2006, 50, 1006–1015. 20. £uszczki J, Borowicz KK, Czuczwar SJ: SIB 1893 possesses pro- and anticonvulsant activity in the electroshock seizure threshold test in mice. Pol J Pharmacol, 2002, 54, 517–520. 21. Mares P, Mikulecka A: MPEP, an antagonist of metabotropic glutamate receptors, exhibits anticonvulsant action in immature rats without a serious impairment of motor performance. Epilepsy Res, 2004, 60, 17–26. 22. Mathiesen JM, Svendsen N, Bräuner-Osborne H, Thomsen C, Ramirez MT: Positive allosteric modulation of the hu-
23. 24.
25.
26.
27.
28. 29.
30. 31.
32.
33.
34.
35.
man metabotropic glutamate receptor 4 (hmGluR4) by SIB 1893 and MPEP. Br J Pharmacol, 2003, 138, 1026–1030. Micheli F: Methylphenylethynylpyridine (MPEP) Novartis. Curr Opin Investig Drugs, 2000, 1, 355–359. Nagaraya RY, Becker A, Reymann KG, Balschun D: Repeated administration of group I mGluR antagonists prevents seizure-induced long-term aberrations in hippocampal synaptic plasticity. Neuropharmacology, 2005, 49, 179–187. Nagaraja RY, Grecksch G, Reyman KG, Schroeder H, Becker A: Group I metabotropic glutamate receptors interfere in different ways with pentylenetetrazole seizures, kindling, and kindling-related learning deficits. Naunyn Schmiedebergs Arch Pharmacol, 2004, 370, 26–34. Neugebauer V, Zinebi F, Russel R, Gallagher JP, Shinnick-Gallagher P: Cocaine and kindling alter the sensitivity of group II and III metabotropic glutamate receptors in the central amygdala. J Neurophysiol, 2000, 84, 759–770. O’Leary DM, Movsesyan V, Vicini S, Faden AI: Selective mGluR5 antagonists MPEP and SIB-1893 decrease NMDA or glutamate-mediated neuronal toxicity through actions that reflect NMDA receptor antagonism. Br J Pharmacol, 2000, 131, 1429–1437. Paxinos G, Watson C: The Rat Brain in Stereotaxic Coordinates, 2nd ed. Academic Press, Sydney, 1986. Pilc A, K³odzinska A, Brañski P, Nowak G, Palucha A, Szewczyk B, Tatarczyñska E et al.: Multiple MPEP administration evoke anxiolytic- and antidepressant-like effects in rats. Neuropharmacology, 2002, 43, 181–187. Racine RJ: Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr Clin Neurophysiol, 1972, 32, 281–294. Smolders I, Clinckers R, Meurs A, De Bundel D, Portelli J, Ebinger G, Michotte Y: Direct enhancement of hippocampal dopamine or serotonin levels as a pharmacodynamic measure of combined antidepressant-anticonvulsant action. Neuropharmacology, 2008, 54, 1017–1028. Tang FR, Chen PM, Tang YC, Tsai MC, Lee WL: Two-methyl-6-phenylethynyl-pyridine (MPEP), a metabotropic glutamate receptor 5 antagonist, with low doses of MK801 and diazepam: a novel approach for controlling status epilepticus. Neuropharmacology, 2007, 53, 821–831. Venault P, Chapouthier G, De Carvalho LP, Simiand J, Morre M, Dodd RH, Rossier J: Benzodiazepines impair and beta-carbolines enhance performance in learning and memory task. Nature, 1986, 321, 864–866. Wang SJ, Sihra TS: Noncompetitive metabotropic glutamate 5 receptor antagonist (E)-2-methyl-6-styrylpyridine (SIB 1893) depresses glutamate release through inhibition of voltage-dependent Ca2+ entry in rat cerebrocortical nerve terminals (synaptosomes). J Pharmacol Exp Ther, 2004, 309, 951–958. Wieroñska JM, Smia³owska M, Brañski P, Gasparini F, K³odzinska A, Szewczyk B, Palucha A et al.: In the amygdala anxiolytic action of mGlu5 receptors antagonist MPEP involves neuropeptide Y but not GABA-A signaling. Neuropsychopharmacology, 2004, 29, 514–521.
Pharmacological Reports, 2009, 61, 621–630
629
36. Wojtal K, Borowicz KK, B³aszczyk B, Czuczwar SJ: Interactions of excitatory amino acid receptor antagonists with antiepileptic drugs in three basic models of experimental epilepsy. Pharmacol Rep, 2006, 58, 587–598. 37. Zadro¿niak M, Sêkowski A, Czuczwar SJ, Borowicz KK: Effect of MPEP, a selective mGluR5 antagonist, on
630
Pharmacological Reports, 2009, 61, 621–630
the antielectroshock activity of conventional antiepileptic drugs. Pol J Pharmacol, 2004, 56, 595–598.
Received: October 20, 2008; in revised form: June 9, 2009.