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Anticonvulsant effects of agomelatine in mice
Epilepsy & Behavior 24 (2012) 324–328
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Epilepsy & Behavior journal homepage: www.elsevier.com/locate/yebeh
Anticonvulsant effects of agomelatine in mice Carlos Clayton Torres Aguiar a, Anália Barbosa Almeida b, 1, Paulo Victor Pontes Araújo b, 1, Germana Silva Vasconcelos b, 1, Edna Maria Camelo Chaves b, 1, Otoni Cardoso do Vale b, 1, Danielle Silveira Macêdo b, 1, Francisca Cléa Florenço de Sousa b, 1, Glauce Socorro de Barros Viana b, 1, Silvânia Maria Mendes Vasconcelos b,⁎ a b
School of Medicine, University of Fortaleza (UNIFOR)/RENORBIO, Rua Desembargador Floriano Benevides Magalhães, 221 3° Andar‐60811-690, Fortaleza, Ceará, Brazil Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Rua Coronel Nunes de Melo, 1127‐60430‐270, Fortaleza, Ceará, Brazil
a r t i c l e
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Article history: Received 3 March 2012 Revised 31 March 2012 Accepted 27 April 2012 Available online 2 June 2012 Keywords: Agomelatine Strychnine Pilocarpine Electroshock stimulation Anticonvulsant effect Picrotoxin Seizure Melatonin
a b s t r a c t Agomelatine is a potent MT1 and MT2 melatonin receptor agonist and a 5-HT2C serotonin receptor antagonist. We analyzed whether agomelatine has anticonvulsant properties. The anticonvulsant activity of agomelatine (25, 50 or 75 mg/kg, i.p.) was evaluated in mouse models of pentylenetetrazole (PTZ-85 mg/kg, i.p.), pilocarpine (400 mg/kg, i.p.), picrotoxin (7 mg/kg, i.p.), strychnine (75 mg/kg, i.p.) or electroshock-induced convulsions. In the PTZ-induced seizure model, agomelatine (at 25 or 50 mg/kg) showed a significant increase in latency to convulsion, and agomelatine (at 50 or 75 mg/kg) also increased significantly time until death. In the pilocarpine-induced seizure model, only agomelatine in high doses (75 mg/kg) showed a significant increase in latency to convulsions and in time until death. In the strychnine‐, electroshock‐ and picrotoxininduced seizure models, agomelatine caused no significant alterations in latency to convulsions and in time until death when compared to controls. Our results suggest that agomelatine has anticonvulsant activity shown in PTZ‐ or pilocarpine-induced seizure models. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Melatonin (N-acetyl-5-methoxytryptamine) is the main hormone related to the regulation of many biological functions including sleep, circadian rhythm, mood, sex maturation, and immune responses [1]. Melatonin has been studied intensively with respect to epilepsy and seizures. Many studies about the effects of melatonin on animal models of epilepsy (pilocarpine, electroshock, pentylenetetrazole, and kainate) have shown that melatonin has a protective effect against seizures [2–4]. Agomelatine (β-methyl-6-chloromelatonin), which is structurally homologous to melatonin, is a potent MT1 and MT2 melatonin receptor agonist as well as a 5-HT2C serotonin receptor antagonist [1,5]. As such and given that many studies have demonstrated the anticonvulsant effects of melatonin, it is possible that agomelatine has anticonvulsant efficacy.
⁎ Corresponding author. Fax: + 55 85 3366 8333. E-mail addresses: [email protected], [email protected] (S.M.M. Vasconcelos). 1 Fax: + 55 85 3366 8333. 1525-5050/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.yebeh.2012.04.134
Despite advances in cellular neurophysiology and biochemical approaches in identifying anticonvulsant substances in vitro, testing does not replace animal models. It is only through animal testing that it is possible to identify compounds that are anticonvulsant and are able to access the relevant brain targets [6]. The models usually used are the chemical and electroshock models, and through them, we can assess possible mechanisms of anticonvulsant action (GABA, glutamate, acetylcholine, glycine, etc.) [7,8]. The objective of this study was to investigate a possible anticonvulsant effect of agomelatine in pentylenetetrazole (PTZ)‐, pilocarpine‐, picrotoxin‐, strychnine‐ and electroshock-induced seizure models.
2. Materials and methods 2.1. Drugs and chemicals Pentylenetetrazole (PTZ, Sigma Chemical Co.), pilocarpine (Sigma Chemical Co.), picrotoxin (Sigma Chemical Co.), and agomelatine (Valdoxan®, Servier) were all dissolved in physiological saline. Fresh drug solutions were prepared on each day of the experiments. Drugs were administered intraperitoneally (i.p.) at a dose of 1 ml per 100 g of animal body weight.
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Table 1 Effects of agomelatine in the seizure model induced by PTZ in mice. Groups
Latency to convulsion (s)
% Animals exhibiting convulsions
Latency to death (s)
% Surviving animals
Control Agomelatine (25 mg/kg) Agomelatine (50 mg/kg) Agomelatine (75 mg/kg)
147.0 ± 16.19 (10) 281.1 ± 32.82 (10)a 249.3 ± 34.65 (8)a 110.8 ± 9.13 (10)b,c
100 100 100 100
155.2 ± 12.48 (9) 342.0 ± 67.7 (10) 561.1 ± 149.2 (10)a 548.4 ± 88.50 (8)a
0 0 10 0
Mice (20–30 g) were injected with agomelatine 25, 50, or 75 mg/kg (i.p.) or saline solution intraperitoneally 30 min before the administration of PTZ (85 mg/kg, i.p.) and then observed for up to 30 min. Results are means ± SEM of the convulsions or death latency. In parentheses, there is the number of animals per group. a, b and c: pb 0.05 when compared to controls, agomelatine 25 or 50 mg/kg, respectively (ANOVA and Tukey test as a post hoc).
stimulation using a pulse generator (ECT Unit 57800‐001; Ugo Basile, Comerio, Italy) (frequency, 60 Hz/s; pulse width, 0.5 ms; shock duration, 0.2 s; current, 13 mA). The end point was the maximum (tonic) extension of the hindlimbs [13].
2.2. Animals Female Swiss mice (20–30 g) from the Vivarium at Federal University of Cearáwere were used throughout the experiments in five groups of induced seizure models. Each group was constituted by 40 animals, divided in subgroups according to dose of agomelatine (25, 50 or 75 mg/kg) and control group. Animals were kept in plastic cages at 25 °C in 30‐m2 rooms under a controlled 12-hour light/dark cycle with food and water ad libitum. Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals from the U.S. Department of Health and Human Services, Institute of Laboratory Animal Resources, Washington, DC, 1985. The study was approved by the ethics committee for Animal Experiment, Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Brazil, with protocol number 98/10.
2.5. Statistical analysis All results are presented as ±SEM (standard of the error mean). ANOVA was followed by Tukey as the post hoc test. Results were considered significant at p b 0.05.
3. Results In the PTZ‐induced seizure model, agomelatine at doses of 25 or 50 mg/kg caused an increase in latency to convulsion when compared with the control group [F(3.37) = 11.12, p = 0.0023], and only agomelatine in elevated doses (50 or 75 mg/kg) showed an increase in time of death of mice [F(3.36) = 3.873, p = 0.0177]. Ten percent (10%) of the animals that were administered agomelatine at 50 mg/ kg survived compared with the control group (0%), and all animals exhibited convulsions (Table 1). In the pilocarpine-induced seizure model, only a high dose of agomelatine (75 mg/kg) demonstrated an increase in latency to convulsion when compared to the control group [F(3.37) = 8.366, p = 0.0003]. On the other hand, latency to death was increased [F(3.35) = 8.289, p = 0.0003] in animals treated with two doses of agomelatine (50 or 75 mg/kg) when compared to control animals (Table 2). All animals died and 100% exhibited convulsions, with the exception of the group that was pretreated with agomelatine at 75 mg (70% exhibited convulsions). No effect was observed after treatment with agomelatine when compared to the control group in terms of latency to convulsions and time of death in the chemical-induced seizure models (picrotoxin and strychnine) and electroshock stimulation. The only difference occurred in ECS: of animals treated with agomelatine at 25 mg/kg, 20% did not show seizures, and 10% did not die (Tables 3, 4, and 5).
2.3. Pentylenetetrazole (PTZ)‐, pilocarpine‐, picrotoxin‐ and strychnine-induced seizure models Mice were kept individually in transparent mice cages (25 cm× 15 cm× 15 cm) for 30 min to acclimate to their new environment before the commencement of the experiment. The administration of agomelatine (25, 50 or 75 mg/kg, i.p.) or saline (control vehicle) took place 30 min before clonic seizures, and tonic‐clonic convulsions were induced in mice with PTZ (85 mg/kg, i.p.), picrotoxin (7 mg/kg, i.p.), strychnine (75 mg/kg, i.p.), or pilocarpine (400 mg/kg, i.p.). All the animals were observed for convulsions for a period of 30 min after the administration of PTZ, picrotoxin, strychnine, or pilocarpine. Hindlimb extension was taken as tonic convulsion. The onset of tonic convulsion and the number of animals convulsing or not convulsing within the observation period were noted. Agomelatine's ability to prevent or delay the onset of the hindlimb extension exhibited by the animals was taken as an indication of anticonvulsant activity [9–12]. All experiments were carried out between 8:40 a.m. and 4:00 p.m. in a quiet room with room temperature of 22 ± 1 °C. 2.4. Electroshock stimulation (ECS) Animals were pretreated with agomelatine (25, 50 or 75 mg/kg, i.p) or saline (control vehicle, i.p.) 30 min before electroshock
Table 2 Effects of agomelatine in the mouse pilocarpine-induced seizure model. Groups
Latency to convulsions (s)
% Animals exhibiting convulsions
Latency to death (s)
% Surviving animals
Control Agomelatine (25 mg/kg) Agomelatine (50 mg/kg) Agomelatine (75 mg/kg)
648.8 ± 10.56 598.8 ± 28.13 733.4 ± 68.61 1147 ± 148.3
100 100 100 70
640.4 ± 33.04 640.5 ± 33.75 884.1 ± 91.97 927 ± 47.36
0 0 0 0
(10) (10) (10) (10)a,b,c
(10) (10) (8)a,b (10)a,b
Mice (20–30 g) were injected with agomelatine 25, 50, or 75 mg/kg (i.p.) or saline solution intraperitoneally 30 min prior to the administration of pilocarpine (400 mg/kg, sc) and observed over 30 min. Results are means ± SEM of the latency to convulsions or death. In parentheses, there is the number of animals per group. a, b and c: pb 0.05 when compared to controls, agomelatine 25 or 50 mg/kg, respectively (ANOVA and Tukey test as a post hoc).
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Table 3 Effects of agomelatine in the seizure model induced by picrotoxin in mice. Groups
Latency to convulsion (s)
% Animals exhibiting convulsions
Latency to death (s)
% Surviving animals
Control Agomelatine (25 mg/kg) Agomelatine (50 mg/kg) Agomelatine (75 mg/kg)
1039 ± 50.95 1025 ± 167.3 896.8 ± 59.52 828.9 ± 51.39
100 100 100 100
1088 ± 59.48 1214 ± 121.2 926.8 ± 59.52 875.6 ± 50.77
0 0 0 0
(10) (10) (10) (10)
(10) (10) (10) (10)
Mice (20–30 g) were injected with agomelatine 25, 50, or 75 mg/kg (i.p.) or saline solution intraperitoneally 30 min before the administration of picrotoxin (7 mg/kg, i.p.) and then observed for up to 30 min. Results are means ± SEM of the convulsions or death latency. In parentheses, there is the number of animals per group. (ANOVA and Tukey test as a post hoc).
4. Discussion Melatonin, a hormone that is produced in the pineal gland, has been implicated in many behavioral processes, where it has been shown to have anxiolytic, sedative, and anticonvulsant effects [14]. Melatonin's anticonvulsant activity has been demonstrated against pentylenetetrazole, pilocarpine, L-cysteine and kainate [14–16]. This hormone, in association with an antiepileptic drug (AED), can decrease the frequency of seizures with tonic‐clonic characteristics [17]. Golombek et al. suggest that the effect of melatonin is mediated by central synapses employing gamma-aminobutyric acid (GABA) as an inhibitory transmitter (this possibly depends on the combined effects of membrane ion permeability and increasing chloride ion influx through GABAA-dependent chloride channels). Such data can explain melatonin's anticonvulsant activity [18,19]. Agomelatine, a naphthalene analog of melatonin, is both a human cloned melatonergic MT1 (KI = 0.1 nM) and MT2 (KI = 0.12 nM) receptor agonist and a 5-HT2C-serotonin receptor antagonist (pKi 6.2) [20,21]. Unlike melatonin, as far as we know, there are no articles in the literature reporting research studies about agomelatine's anticonvulsant action. The results of the present research have demonstrated that agomelatine has anticonvulsant action in seizure models induced by pilocarpine and PTZ. Pentylenetetrazole has been a classic GABA receptor antagonist, but neurochemical studies suggest that PTZ binds to the picrotoxin site of the GABA receptor complex and blocks GABA-mediated inhibition. Furthermore, the activation of the NMDA receptor appears to be involved in initiating and generalizing PTZinduced seizures [7,22,23]. In our study, agomelatine in low doses (25 or 50 mg/kg) increased latency to convulsion; however, this protection was not observed with a 75‐mg/kg dose. This suggests that the effect is observed only in lower doses. On the other hand, doses of 50 and 75 mg/kg increased time until death, which is another parameter analyzed in the PTZ model. These findings suggest that agomelatine's main anticonvulsant effect is mediated by GABA receptors because the affinity of agomelatine for the 5-HT2C receptor is in the micromolar range and about 100-fold less than its affinity for melatonin receptors [20]. In line with this evidence, Upton et al. have investigated the role of 5-HT2C receptors in the generation of PTZ-evoked seizures. Serotonin 5-HT2C/2B receptor agonist mCPP (2.5–7 mg/kg, i.p.) and 5-HT2C/2B receptor antagonist SB-206553 (10–20 mg/kg, p.o.) were used for analysis. mCPP produced
anticonvulsant action in the PTZ-induced seizure threshold model, and SB-206553 was able to completely inhibit the anticonvulsant effects of mCPP (2.5 mg/kg, i.p.) in mice. Although activation of 5HT2C receptors appeared to result in anticonvulsant action, SB206553 alone did not lower the threshold to myoclonus, forelimb, and/or hindlimb tonus in mice, thereby indicating that blocking this subtype of receptor was not associated with enhanced susceptibility to generalized seizures [24]. There are similar studies which have evaluated melatonin in PTZinduced seizure models. Solmaz et al. used guinea pigs and found that the anticonvulsant effect of melatonin occurs particularly at high doses (50–160 mg/kg) but that at lower doses, melatonin reduces mortality and reduces the severity of the convulsion increasing the latency period in PTZ-induced seizures [25]. Moezi et al. studied anticonvulsant effects in male mice with melatonin at a dose of 80 mg/kg administered at three different times (5, 15, or 30 min) and three doses of melatonin (20, 40, or 80 mg/kg) in PTZ-induced seizures in mice. This study showed that melatonin was effective only in elevated doses of 40 or 80 mg/kg [22]. Pilocarpine is an agonist of muscarinic ACh receptors expressed particularly in the hippocampus, striatum, and cortex [26]. The seizure is produced by increased activation of ACh receptors [7,27]. In our research, agomelatine has shown an anticonvulsant effect in pilocarpine-induced epilepsy models only at high doses (75 mg/kg). Agomelatine's anticonvulsant effect may be explained by melatonin's effect on the cholinergic system. de Almeida-Paula proposes that melatonin modulates the number of nicotinic acetylcholine receptors via reduction in cyclic AMP accumulation [28]. A study carried out by Lima et al. confirmed this relationship when authors analyzed the effects of the pineal gland (which is responsible for melatonin synthesis) on temporal lobe epilepsy. Wistar male adult rats were submitted to a pinealectomy, and 7 days after surgery, these animals received pilocarpine (350 mg/kg, i.p.). The results indicated that pinealectomy in the pilocarpine model of epilepsy (PME) in rats reduced the latency for the first spontaneous seizure (latent period) and increased the number of spontaneous seizures during the chronic period [29]. The result is similar to what Costa-Lotufo et al. found, demonstrating that melatonin has a weak anticonvulsant activity on pilocarpine-induced seizures in rats, as revealed by an increase in latency to the appearance of the first seizure [30]. Lima et al. demonstrated that melatonin attenuated SE-induced post-lesion and
Table 4 Effects of agomelatine in the mouse strychnine‐induced seizure model. Groups
Latency to convulsions (s)
% Animals exhibiting convulsions
Latency to death (s)
% Surviving animals
Control Agomelatine (25 mg/kg) Agomelatine (50 mg/kg) Agomelatine (75 mg/kg)
158.7 ± 12.77 (10) 131.7 ± 6.69 (20) 127.3 ± 7.17 (10) 142.3 ± 7.7 (10)
100 100 100 100
253.6 ± 32.83 213.5 ± 17.83 293.9 ± 25.41 333.9 ± 53.84
0 0 0 0
(10) (20) (10) (10)
Mice (20–30 g) were administered agomelatine 25, 50, or 75 mg/kg (i.p.) or saline solution intraperitoneally 30 min prior to the administration of strychnine (75 mg/kg, i.p.) and observed over 30 min. Results are means ± SEM of the latency to convulsions or death. In parentheses, there is the number of animals per group. (ANOVA and Tukey test as a post hoc).
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Table 5 Effects of agomelatine in mice electroshock stimulation. Groups
Latency to convulsions (s)
% Animals exhibiting convulsions
Time of convulsions
% Surviving animals
Control Agomelatine (25 mg/kg) Agomelatine (50 mg/kg) Agomelatine (75 mg/kg)
2.44 ± 0.17 2.29 ± 0.12 2.40 ± 0.17 2.59 ± 0.23
100 80 100 100
12.15 ± 0.60 11.73 ± 0.62 11.98 ± 0.54 12.07 ± 0.49
100 90 100 100
(10) (8) (10) (10)
(10) (10) (10) (10)
Mice (20–30 g) were administered with agomelatine 25, 50, or 75 mg/kg (i.p.) or saline solution intraperitoneally 30 min prior to the administration electroshock stimulation using a pulse generator (ECT Unit 57800‐001; Ugo Basile, Comerio, Italy) (frequency, 60 Hz/s; pulse width, 0.5 ms; shock duration, 0.2 s; current, 13 mA). Results are means ± SEM of the latency to convulsions or death. In parentheses, there is the number of animals per group. (ANOVA and Tukey test as a post hoc).
promoted a decrease in the number of seizures in rats with epilepsy caused by the pilocarpine-induced epilepsy model [4]. Picrotoxin acts as blocker of GABAA receptor chloride channel, and the usefulness for screening putative antiepileptic drugs is similar to bicuculline-induced seizures [7]. In our study, agomelatine has not shown anticonvulsant effects in the picrotoxin model. We did not find any research about melatonin in picrotoxin-induced seizure models, only studies where the picrotoxin was used as a drug to operate as a GABAA receptor antagonist in electroshock-induced seizure models [3,31]. It is also possible that the connection in GABAA receptor sites for melatonin is different from picrotoxin. Langebartels et al. evaluated the effects of melatonin on sleep in rats and the contribution of GABAA receptors. Sleep ± wake behavior was assessed in nine rats after intraperitoneal (i.p.) administration of pharmacological doses of melatonin (5 or 10 mg/kg) and later, combined with the administration of the GABAA receptor antagonists picrotoxin (1.5 mg/kg) and melatonin (10 mg/kg). Melatonin failed to attenuate the picrotoxin-induced promotion of wakefulness [3]. All doses of agomelatine were not effective against strychnineinduced seizure models. This is possible because strychnine's convulsive action occurs via postsynaptic inhibition mediated by glycine. Glycine operates as an important inhibitory neurotransmitter, and strychnine acts as a selective, competitive antagonist on all glycine receptors [32]. Melatonin does not directly interact with glycine. The electroshock assay in mice is used primarily as an indication for compounds which are effective in generalized tonic-clonic seizures. Tonic hindlimb extensions are evoked by electric stimuli which are suppressed by antiepileptic drugs [32]. In our study, neither dose of agomelatine was effective as an anticonvulsant when compared to control groups in the ECS model. It is possible that agomelatine did not demonstrate any effects in the ECS test because antiepileptic drugs that work in the ECS model mainly block voltagegated sodium channels. The ECS test may not be sensitive enough to detect new drugs, especially those with different mechanisms of action than commonly used antiepileptic drugs [33]. Borowicz et al. showed that only melatonin (50 mg/kg) administered i.p. in female Swiss mice, 60 min before the test, significantly raised the electroconvulsive threshold, but melatonin (50 mg/kg) injected 30, 120, or 240 min before electroconvulsions did not influence the threshold [3]. The protective action of melatonin (50 mg/kg) in the electroconvulsive threshold test was reversed by picrotoxin or bicuculline. Melatonin at the subconvulsive dose of 25 mg/kg enhanced the anticonvulsive activity of carbamazepine and phenobarbital, but no enhancement was observed in the case of valproate and diphenylhydantoin. This may suggest that the anti-electroshock efficacy of melatonin may depend on GABA, and perhaps on other neurotransmitters [3]. The ECS model is used to evaluate a substance's ability to prevent seizure spread across neural tissue while the PTZ test estimates the capacity to increase the seizure threshold of neural tissue excitation [33]. The results of the present study indicate that agomelatine has an anticonvulsant activity in animal models induced by pilocarpine or PTZ, and this action may be related to GABAertic mechanisms. To the
best of our knowledge, this is the first scientific report on the anticonvulsant activity of agomelatine published in biomedical literature.
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Anticonvulsant effects of agomelatine in mice Carlos Clayton Torres Aguiar a, Anália Barbosa Almeida b, 1, Paulo Victor Pontes Araújo b, 1, Germana Silva Vasconcelos b, 1, Edna Maria Camelo Chaves b, 1, Otoni Cardoso do Vale b, 1, Danielle Silveira Macêdo b, 1, Francisca Cléa Florenço de Sousa b, 1, Glauce Socorro de Barros Viana b, 1, Silvânia Maria Mendes Vasconcelos b,⁎ a b
School of Medicine, University of Fortaleza (UNIFOR)/RENORBIO, Rua Desembargador Floriano Benevides Magalhães, 221 3° Andar‐60811-690, Fortaleza, Ceará, Brazil Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Rua Coronel Nunes de Melo, 1127‐60430‐270, Fortaleza, Ceará, Brazil
a r t i c l e
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Article history: Received 3 March 2012 Revised 31 March 2012 Accepted 27 April 2012 Available online 2 June 2012 Keywords: Agomelatine Strychnine Pilocarpine Electroshock stimulation Anticonvulsant effect Picrotoxin Seizure Melatonin
a b s t r a c t Agomelatine is a potent MT1 and MT2 melatonin receptor agonist and a 5-HT2C serotonin receptor antagonist. We analyzed whether agomelatine has anticonvulsant properties. The anticonvulsant activity of agomelatine (25, 50 or 75 mg/kg, i.p.) was evaluated in mouse models of pentylenetetrazole (PTZ-85 mg/kg, i.p.), pilocarpine (400 mg/kg, i.p.), picrotoxin (7 mg/kg, i.p.), strychnine (75 mg/kg, i.p.) or electroshock-induced convulsions. In the PTZ-induced seizure model, agomelatine (at 25 or 50 mg/kg) showed a significant increase in latency to convulsion, and agomelatine (at 50 or 75 mg/kg) also increased significantly time until death. In the pilocarpine-induced seizure model, only agomelatine in high doses (75 mg/kg) showed a significant increase in latency to convulsions and in time until death. In the strychnine‐, electroshock‐ and picrotoxininduced seizure models, agomelatine caused no significant alterations in latency to convulsions and in time until death when compared to controls. Our results suggest that agomelatine has anticonvulsant activity shown in PTZ‐ or pilocarpine-induced seizure models. © 2012 Elsevier Inc. All rights reserved.
1. Introduction Melatonin (N-acetyl-5-methoxytryptamine) is the main hormone related to the regulation of many biological functions including sleep, circadian rhythm, mood, sex maturation, and immune responses [1]. Melatonin has been studied intensively with respect to epilepsy and seizures. Many studies about the effects of melatonin on animal models of epilepsy (pilocarpine, electroshock, pentylenetetrazole, and kainate) have shown that melatonin has a protective effect against seizures [2–4]. Agomelatine (β-methyl-6-chloromelatonin), which is structurally homologous to melatonin, is a potent MT1 and MT2 melatonin receptor agonist as well as a 5-HT2C serotonin receptor antagonist [1,5]. As such and given that many studies have demonstrated the anticonvulsant effects of melatonin, it is possible that agomelatine has anticonvulsant efficacy.
⁎ Corresponding author. Fax: + 55 85 3366 8333. E-mail addresses: [email protected], [email protected] (S.M.M. Vasconcelos). 1 Fax: + 55 85 3366 8333. 1525-5050/$ – see front matter © 2012 Elsevier Inc. All rights reserved. doi:10.1016/j.yebeh.2012.04.134
Despite advances in cellular neurophysiology and biochemical approaches in identifying anticonvulsant substances in vitro, testing does not replace animal models. It is only through animal testing that it is possible to identify compounds that are anticonvulsant and are able to access the relevant brain targets [6]. The models usually used are the chemical and electroshock models, and through them, we can assess possible mechanisms of anticonvulsant action (GABA, glutamate, acetylcholine, glycine, etc.) [7,8]. The objective of this study was to investigate a possible anticonvulsant effect of agomelatine in pentylenetetrazole (PTZ)‐, pilocarpine‐, picrotoxin‐, strychnine‐ and electroshock-induced seizure models.
2. Materials and methods 2.1. Drugs and chemicals Pentylenetetrazole (PTZ, Sigma Chemical Co.), pilocarpine (Sigma Chemical Co.), picrotoxin (Sigma Chemical Co.), and agomelatine (Valdoxan®, Servier) were all dissolved in physiological saline. Fresh drug solutions were prepared on each day of the experiments. Drugs were administered intraperitoneally (i.p.) at a dose of 1 ml per 100 g of animal body weight.
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Table 1 Effects of agomelatine in the seizure model induced by PTZ in mice. Groups
Latency to convulsion (s)
% Animals exhibiting convulsions
Latency to death (s)
% Surviving animals
Control Agomelatine (25 mg/kg) Agomelatine (50 mg/kg) Agomelatine (75 mg/kg)
147.0 ± 16.19 (10) 281.1 ± 32.82 (10)a 249.3 ± 34.65 (8)a 110.8 ± 9.13 (10)b,c
100 100 100 100
155.2 ± 12.48 (9) 342.0 ± 67.7 (10) 561.1 ± 149.2 (10)a 548.4 ± 88.50 (8)a
0 0 10 0
Mice (20–30 g) were injected with agomelatine 25, 50, or 75 mg/kg (i.p.) or saline solution intraperitoneally 30 min before the administration of PTZ (85 mg/kg, i.p.) and then observed for up to 30 min. Results are means ± SEM of the convulsions or death latency. In parentheses, there is the number of animals per group. a, b and c: pb 0.05 when compared to controls, agomelatine 25 or 50 mg/kg, respectively (ANOVA and Tukey test as a post hoc).
stimulation using a pulse generator (ECT Unit 57800‐001; Ugo Basile, Comerio, Italy) (frequency, 60 Hz/s; pulse width, 0.5 ms; shock duration, 0.2 s; current, 13 mA). The end point was the maximum (tonic) extension of the hindlimbs [13].
2.2. Animals Female Swiss mice (20–30 g) from the Vivarium at Federal University of Cearáwere were used throughout the experiments in five groups of induced seizure models. Each group was constituted by 40 animals, divided in subgroups according to dose of agomelatine (25, 50 or 75 mg/kg) and control group. Animals were kept in plastic cages at 25 °C in 30‐m2 rooms under a controlled 12-hour light/dark cycle with food and water ad libitum. Experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals from the U.S. Department of Health and Human Services, Institute of Laboratory Animal Resources, Washington, DC, 1985. The study was approved by the ethics committee for Animal Experiment, Department of Physiology and Pharmacology, Faculty of Medicine, Federal University of Ceará, Brazil, with protocol number 98/10.
2.5. Statistical analysis All results are presented as ±SEM (standard of the error mean). ANOVA was followed by Tukey as the post hoc test. Results were considered significant at p b 0.05.
3. Results In the PTZ‐induced seizure model, agomelatine at doses of 25 or 50 mg/kg caused an increase in latency to convulsion when compared with the control group [F(3.37) = 11.12, p = 0.0023], and only agomelatine in elevated doses (50 or 75 mg/kg) showed an increase in time of death of mice [F(3.36) = 3.873, p = 0.0177]. Ten percent (10%) of the animals that were administered agomelatine at 50 mg/ kg survived compared with the control group (0%), and all animals exhibited convulsions (Table 1). In the pilocarpine-induced seizure model, only a high dose of agomelatine (75 mg/kg) demonstrated an increase in latency to convulsion when compared to the control group [F(3.37) = 8.366, p = 0.0003]. On the other hand, latency to death was increased [F(3.35) = 8.289, p = 0.0003] in animals treated with two doses of agomelatine (50 or 75 mg/kg) when compared to control animals (Table 2). All animals died and 100% exhibited convulsions, with the exception of the group that was pretreated with agomelatine at 75 mg (70% exhibited convulsions). No effect was observed after treatment with agomelatine when compared to the control group in terms of latency to convulsions and time of death in the chemical-induced seizure models (picrotoxin and strychnine) and electroshock stimulation. The only difference occurred in ECS: of animals treated with agomelatine at 25 mg/kg, 20% did not show seizures, and 10% did not die (Tables 3, 4, and 5).
2.3. Pentylenetetrazole (PTZ)‐, pilocarpine‐, picrotoxin‐ and strychnine-induced seizure models Mice were kept individually in transparent mice cages (25 cm× 15 cm× 15 cm) for 30 min to acclimate to their new environment before the commencement of the experiment. The administration of agomelatine (25, 50 or 75 mg/kg, i.p.) or saline (control vehicle) took place 30 min before clonic seizures, and tonic‐clonic convulsions were induced in mice with PTZ (85 mg/kg, i.p.), picrotoxin (7 mg/kg, i.p.), strychnine (75 mg/kg, i.p.), or pilocarpine (400 mg/kg, i.p.). All the animals were observed for convulsions for a period of 30 min after the administration of PTZ, picrotoxin, strychnine, or pilocarpine. Hindlimb extension was taken as tonic convulsion. The onset of tonic convulsion and the number of animals convulsing or not convulsing within the observation period were noted. Agomelatine's ability to prevent or delay the onset of the hindlimb extension exhibited by the animals was taken as an indication of anticonvulsant activity [9–12]. All experiments were carried out between 8:40 a.m. and 4:00 p.m. in a quiet room with room temperature of 22 ± 1 °C. 2.4. Electroshock stimulation (ECS) Animals were pretreated with agomelatine (25, 50 or 75 mg/kg, i.p) or saline (control vehicle, i.p.) 30 min before electroshock
Table 2 Effects of agomelatine in the mouse pilocarpine-induced seizure model. Groups
Latency to convulsions (s)
% Animals exhibiting convulsions
Latency to death (s)
% Surviving animals
Control Agomelatine (25 mg/kg) Agomelatine (50 mg/kg) Agomelatine (75 mg/kg)
648.8 ± 10.56 598.8 ± 28.13 733.4 ± 68.61 1147 ± 148.3
100 100 100 70
640.4 ± 33.04 640.5 ± 33.75 884.1 ± 91.97 927 ± 47.36
0 0 0 0
(10) (10) (10) (10)a,b,c
(10) (10) (8)a,b (10)a,b
Mice (20–30 g) were injected with agomelatine 25, 50, or 75 mg/kg (i.p.) or saline solution intraperitoneally 30 min prior to the administration of pilocarpine (400 mg/kg, sc) and observed over 30 min. Results are means ± SEM of the latency to convulsions or death. In parentheses, there is the number of animals per group. a, b and c: pb 0.05 when compared to controls, agomelatine 25 or 50 mg/kg, respectively (ANOVA and Tukey test as a post hoc).
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Table 3 Effects of agomelatine in the seizure model induced by picrotoxin in mice. Groups
Latency to convulsion (s)
% Animals exhibiting convulsions
Latency to death (s)
% Surviving animals
Control Agomelatine (25 mg/kg) Agomelatine (50 mg/kg) Agomelatine (75 mg/kg)
1039 ± 50.95 1025 ± 167.3 896.8 ± 59.52 828.9 ± 51.39
100 100 100 100
1088 ± 59.48 1214 ± 121.2 926.8 ± 59.52 875.6 ± 50.77
0 0 0 0
(10) (10) (10) (10)
(10) (10) (10) (10)
Mice (20–30 g) were injected with agomelatine 25, 50, or 75 mg/kg (i.p.) or saline solution intraperitoneally 30 min before the administration of picrotoxin (7 mg/kg, i.p.) and then observed for up to 30 min. Results are means ± SEM of the convulsions or death latency. In parentheses, there is the number of animals per group. (ANOVA and Tukey test as a post hoc).
4. Discussion Melatonin, a hormone that is produced in the pineal gland, has been implicated in many behavioral processes, where it has been shown to have anxiolytic, sedative, and anticonvulsant effects [14]. Melatonin's anticonvulsant activity has been demonstrated against pentylenetetrazole, pilocarpine, L-cysteine and kainate [14–16]. This hormone, in association with an antiepileptic drug (AED), can decrease the frequency of seizures with tonic‐clonic characteristics [17]. Golombek et al. suggest that the effect of melatonin is mediated by central synapses employing gamma-aminobutyric acid (GABA) as an inhibitory transmitter (this possibly depends on the combined effects of membrane ion permeability and increasing chloride ion influx through GABAA-dependent chloride channels). Such data can explain melatonin's anticonvulsant activity [18,19]. Agomelatine, a naphthalene analog of melatonin, is both a human cloned melatonergic MT1 (KI = 0.1 nM) and MT2 (KI = 0.12 nM) receptor agonist and a 5-HT2C-serotonin receptor antagonist (pKi 6.2) [20,21]. Unlike melatonin, as far as we know, there are no articles in the literature reporting research studies about agomelatine's anticonvulsant action. The results of the present research have demonstrated that agomelatine has anticonvulsant action in seizure models induced by pilocarpine and PTZ. Pentylenetetrazole has been a classic GABA receptor antagonist, but neurochemical studies suggest that PTZ binds to the picrotoxin site of the GABA receptor complex and blocks GABA-mediated inhibition. Furthermore, the activation of the NMDA receptor appears to be involved in initiating and generalizing PTZinduced seizures [7,22,23]. In our study, agomelatine in low doses (25 or 50 mg/kg) increased latency to convulsion; however, this protection was not observed with a 75‐mg/kg dose. This suggests that the effect is observed only in lower doses. On the other hand, doses of 50 and 75 mg/kg increased time until death, which is another parameter analyzed in the PTZ model. These findings suggest that agomelatine's main anticonvulsant effect is mediated by GABA receptors because the affinity of agomelatine for the 5-HT2C receptor is in the micromolar range and about 100-fold less than its affinity for melatonin receptors [20]. In line with this evidence, Upton et al. have investigated the role of 5-HT2C receptors in the generation of PTZ-evoked seizures. Serotonin 5-HT2C/2B receptor agonist mCPP (2.5–7 mg/kg, i.p.) and 5-HT2C/2B receptor antagonist SB-206553 (10–20 mg/kg, p.o.) were used for analysis. mCPP produced
anticonvulsant action in the PTZ-induced seizure threshold model, and SB-206553 was able to completely inhibit the anticonvulsant effects of mCPP (2.5 mg/kg, i.p.) in mice. Although activation of 5HT2C receptors appeared to result in anticonvulsant action, SB206553 alone did not lower the threshold to myoclonus, forelimb, and/or hindlimb tonus in mice, thereby indicating that blocking this subtype of receptor was not associated with enhanced susceptibility to generalized seizures [24]. There are similar studies which have evaluated melatonin in PTZinduced seizure models. Solmaz et al. used guinea pigs and found that the anticonvulsant effect of melatonin occurs particularly at high doses (50–160 mg/kg) but that at lower doses, melatonin reduces mortality and reduces the severity of the convulsion increasing the latency period in PTZ-induced seizures [25]. Moezi et al. studied anticonvulsant effects in male mice with melatonin at a dose of 80 mg/kg administered at three different times (5, 15, or 30 min) and three doses of melatonin (20, 40, or 80 mg/kg) in PTZ-induced seizures in mice. This study showed that melatonin was effective only in elevated doses of 40 or 80 mg/kg [22]. Pilocarpine is an agonist of muscarinic ACh receptors expressed particularly in the hippocampus, striatum, and cortex [26]. The seizure is produced by increased activation of ACh receptors [7,27]. In our research, agomelatine has shown an anticonvulsant effect in pilocarpine-induced epilepsy models only at high doses (75 mg/kg). Agomelatine's anticonvulsant effect may be explained by melatonin's effect on the cholinergic system. de Almeida-Paula proposes that melatonin modulates the number of nicotinic acetylcholine receptors via reduction in cyclic AMP accumulation [28]. A study carried out by Lima et al. confirmed this relationship when authors analyzed the effects of the pineal gland (which is responsible for melatonin synthesis) on temporal lobe epilepsy. Wistar male adult rats were submitted to a pinealectomy, and 7 days after surgery, these animals received pilocarpine (350 mg/kg, i.p.). The results indicated that pinealectomy in the pilocarpine model of epilepsy (PME) in rats reduced the latency for the first spontaneous seizure (latent period) and increased the number of spontaneous seizures during the chronic period [29]. The result is similar to what Costa-Lotufo et al. found, demonstrating that melatonin has a weak anticonvulsant activity on pilocarpine-induced seizures in rats, as revealed by an increase in latency to the appearance of the first seizure [30]. Lima et al. demonstrated that melatonin attenuated SE-induced post-lesion and
Table 4 Effects of agomelatine in the mouse strychnine‐induced seizure model. Groups
Latency to convulsions (s)
% Animals exhibiting convulsions
Latency to death (s)
% Surviving animals
Control Agomelatine (25 mg/kg) Agomelatine (50 mg/kg) Agomelatine (75 mg/kg)
158.7 ± 12.77 (10) 131.7 ± 6.69 (20) 127.3 ± 7.17 (10) 142.3 ± 7.7 (10)
100 100 100 100
253.6 ± 32.83 213.5 ± 17.83 293.9 ± 25.41 333.9 ± 53.84
0 0 0 0
(10) (20) (10) (10)
Mice (20–30 g) were administered agomelatine 25, 50, or 75 mg/kg (i.p.) or saline solution intraperitoneally 30 min prior to the administration of strychnine (75 mg/kg, i.p.) and observed over 30 min. Results are means ± SEM of the latency to convulsions or death. In parentheses, there is the number of animals per group. (ANOVA and Tukey test as a post hoc).
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Table 5 Effects of agomelatine in mice electroshock stimulation. Groups
Latency to convulsions (s)
% Animals exhibiting convulsions
Time of convulsions
% Surviving animals
Control Agomelatine (25 mg/kg) Agomelatine (50 mg/kg) Agomelatine (75 mg/kg)
2.44 ± 0.17 2.29 ± 0.12 2.40 ± 0.17 2.59 ± 0.23
100 80 100 100
12.15 ± 0.60 11.73 ± 0.62 11.98 ± 0.54 12.07 ± 0.49
100 90 100 100
(10) (8) (10) (10)
(10) (10) (10) (10)
Mice (20–30 g) were administered with agomelatine 25, 50, or 75 mg/kg (i.p.) or saline solution intraperitoneally 30 min prior to the administration electroshock stimulation using a pulse generator (ECT Unit 57800‐001; Ugo Basile, Comerio, Italy) (frequency, 60 Hz/s; pulse width, 0.5 ms; shock duration, 0.2 s; current, 13 mA). Results are means ± SEM of the latency to convulsions or death. In parentheses, there is the number of animals per group. (ANOVA and Tukey test as a post hoc).
promoted a decrease in the number of seizures in rats with epilepsy caused by the pilocarpine-induced epilepsy model [4]. Picrotoxin acts as blocker of GABAA receptor chloride channel, and the usefulness for screening putative antiepileptic drugs is similar to bicuculline-induced seizures [7]. In our study, agomelatine has not shown anticonvulsant effects in the picrotoxin model. We did not find any research about melatonin in picrotoxin-induced seizure models, only studies where the picrotoxin was used as a drug to operate as a GABAA receptor antagonist in electroshock-induced seizure models [3,31]. It is also possible that the connection in GABAA receptor sites for melatonin is different from picrotoxin. Langebartels et al. evaluated the effects of melatonin on sleep in rats and the contribution of GABAA receptors. Sleep ± wake behavior was assessed in nine rats after intraperitoneal (i.p.) administration of pharmacological doses of melatonin (5 or 10 mg/kg) and later, combined with the administration of the GABAA receptor antagonists picrotoxin (1.5 mg/kg) and melatonin (10 mg/kg). Melatonin failed to attenuate the picrotoxin-induced promotion of wakefulness [3]. All doses of agomelatine were not effective against strychnineinduced seizure models. This is possible because strychnine's convulsive action occurs via postsynaptic inhibition mediated by glycine. Glycine operates as an important inhibitory neurotransmitter, and strychnine acts as a selective, competitive antagonist on all glycine receptors [32]. Melatonin does not directly interact with glycine. The electroshock assay in mice is used primarily as an indication for compounds which are effective in generalized tonic-clonic seizures. Tonic hindlimb extensions are evoked by electric stimuli which are suppressed by antiepileptic drugs [32]. In our study, neither dose of agomelatine was effective as an anticonvulsant when compared to control groups in the ECS model. It is possible that agomelatine did not demonstrate any effects in the ECS test because antiepileptic drugs that work in the ECS model mainly block voltagegated sodium channels. The ECS test may not be sensitive enough to detect new drugs, especially those with different mechanisms of action than commonly used antiepileptic drugs [33]. Borowicz et al. showed that only melatonin (50 mg/kg) administered i.p. in female Swiss mice, 60 min before the test, significantly raised the electroconvulsive threshold, but melatonin (50 mg/kg) injected 30, 120, or 240 min before electroconvulsions did not influence the threshold [3]. The protective action of melatonin (50 mg/kg) in the electroconvulsive threshold test was reversed by picrotoxin or bicuculline. Melatonin at the subconvulsive dose of 25 mg/kg enhanced the anticonvulsive activity of carbamazepine and phenobarbital, but no enhancement was observed in the case of valproate and diphenylhydantoin. This may suggest that the anti-electroshock efficacy of melatonin may depend on GABA, and perhaps on other neurotransmitters [3]. The ECS model is used to evaluate a substance's ability to prevent seizure spread across neural tissue while the PTZ test estimates the capacity to increase the seizure threshold of neural tissue excitation [33]. The results of the present study indicate that agomelatine has an anticonvulsant activity in animal models induced by pilocarpine or PTZ, and this action may be related to GABAertic mechanisms. To the
best of our knowledge, this is the first scientific report on the anticonvulsant activity of agomelatine published in biomedical literature.
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