Anticonvulsant activity of melatonin, but not melatonin ...

Behavioural Brain Research 307 (2016) 199–207

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Anticonvulsant activity of melatonin, but not melatonin receptor agonists Neu-P11 and Neu-P67, in mice a ´ Paula Mosinska , Katarzyna Socała b , Dorota Nieoczym b , Moshe Laudon c , Martin Storr d,e , a Jakub Fichna , Piotr Wlaz´ b,∗ a

Department of Biochemistry, Faculty of Medicine, Medical University of Łód´z, Łód´z, Poland Department of Animal Physiology, Institute of Biology and Biochemistry, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Lublin, Poland c Neurim Pharmaceuticals Ltd., Tel-Aviv, Israel d Walter Brendel Center of Experimental Medicine, University of Munich, Germany e Department of Medicine, Division of Gastroenterology, Ludwig Maximilians University of Munich, Munich, Germany b

h i g h l i g h t s • • • • •

Neu-P11 and Neu-P67 have a prolonged duration of action and oral availability, compared to MLT. MLT produced potent anticonvulsant effect in 6 Hz, MEST and PTZ tests. In contrast, neither Neu-P11 nor Neu-P67 affected the seizure threshold in 6 Hz, MEST and PTZ tests. Changes observed in the locomotor activity suggest the interaction of Neu-P11 and Neu-P67 in the CNS that is independent of MT. Effects of MLT have yet to be explored; our studies support its potential as an anticonvulsant therapeutic.

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Article history: Received 3 February 2016 Received in revised form 3 March 2016 Accepted 21 March 2016 Available online 22 March 2016 Keywords: Melatonin receptors Antiepileptic drugs Epilepsy Piromelatine

a b s t r a c t The anticonvulsant activity of melatonin (MLT) have been tested in several in vivo models and against different convulsive stimuli. Although MLT exerts high affinity towards melatonin receptors (MTs), the potential usefulness in the treatment of epilepsy is limited mainly due to its short half-life. Therefore, the purpose of the present study was to compare the anticonvulsant properties of novel MT agonists Neu-P11 and Neu-P67 with MLT in mice. The anticonvulsant activity of tested compounds was evaluated in pentylenetetrazole-(PTZ) and electrically-induced convulsions. The effect of studied compounds on motor coordination and skeletal muscular strength in mice was assessed in the chimney test and grip test, respectively. The locomotor activity after administration of the tested compounds was also evaluated. In the MEST and 6 Hz tests, only MLT (50 and 100 mg/kg, i.p.) significantly increased the seizure threshold. The i.p. administration of MLT (100 mg/kg) and Neu-P67 (200 mg/kg) resulted in a significantly elevated PTZ seizure threshold for forelimbs tonus. The compounds did not affect muscle strength. No alterations in motor coordination were noted. However, the locomotor activity was significantly decreased after administration of all tested compounds. Our study confirms the anticonvulsant potency of MLT and shows that novel synthetic MT agonists Neu-P11 and Neu-P67 have no effect on epileptic seizures in mice. Our data suggest that the activation of MT can be used in the treatment of seizures, but further pharmacological characterization is needed to understand the anticonvulsant activity of MLT and to design efficient MT-targeting antiepileptic drugs. © 2016 Elsevier B.V. All rights reserved.

Abbreviations: 5-HT, serotonin; ANOVA, analysis of variance; CC50 , convulsive current inducing seizure response in 50% of mice; CNS, central nervous system; DMSO, dimethyl sulfoxide; GABA, ␥-aminobutyric acid; GI, gastrointestinal; i.p., intraperitoneally; i.v., intravenously; mCPP, m-chlorophenylpiperazine; MEST, maximal electroshock seizure threshold; MLT, melatonin; MT, melatonin receptor; http://dx.doi.org/10.1016/j.bbr.2016.03.036 0166-4328/© 2016 Elsevier B.V. All rights reserved.

N, newtons; NMDA, N-methyl-D-aspartic acid; NO, nitric oxide; PTZ, pentylenetetrazole; SEM, standard error of the mean; TFMPP, trifluoromethyl-phenylpiperazine. ∗ Corresponding author at: Department of Animal Physiology, Institute of Biology and Biochemistry, Faculty of Biology and Biotechnology, Maria Curie-Skłodowska University, Akademicka 19, PL 20-033 Lublin, Poland. ´ E-mail address: [email protected] (P. Wlaz).

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P. Mosi´ nska et al. / Behavioural Brain Research 307 (2016) 199–207

1. Introduction Melatonin (MLT) is an indole derivative of tryptophan synthesized primarily by parenchymatous cells in the pineal gland. As a secondary source, MLT is also secreted by the gastrointestinal (GI) tract, skin, retina, bone marrow, salivary glands, platelets, and epithelial hair follicles [11]. MLT displays its physiological function as a regulator of circadian rhythms and sleep-wake cycle in humans by interacting with two types of G protein-coupled receptors, namely melatonin receptor (MT)1 and MT2 [29,31,39,40]. Both receptors are widely distributed in various tissues, such as suprachiasmatic nuclei of the hypothalamus, cerebellum, central dopaminergic pathways, coronary blood vessels and aorta, liver, kidney and gallbladder [9]. Treatment with MLT can improve circadian rhythm sleep disorder and insomnia, seasonal affective disorders, GI diseases (for review see Ref. [10]), cancer and cardiovascular diseases [9]. More recently, MLT has been considered to be effective as an add-on option for epilepsy [1,4]. Epilepsy is a common chronic neurological disease associated with various comorbidities, including sleep disorders, depression and anxiety. MLT concentration in the serum of epileptic or febrile epileptic children is lower compared to control group [18], what suggests that MLT may be involved in the pathophysiology of the disease. However, successful treatment of epilepsy with MLT is not easily achieved; the major obstacle is its rapid elimination from the circulation, as the half-life of MLT ranges from 30 min to 45 min in the blood [7]. Various factors affect MLT bioavailability, including uptake and first-pass hepatic metabolism. Two novel MT agonists, Neu-P11 [piromelatine, N-(2-(5methoxy-1H-indol-3-yl)ethyl)-4-oxo-4H-pyran-2-carboxamide] and Neu-P67 possess high affinity at MTs and exert prolonged duration of action compared to MLT. Neu-P11 is a melatonin and serotonin (5-HT1A/1D ) receptor agonist, and 5-HT2B receptor antagonist [24]; Neu-P11 displays ␥-aminobutyric acid (GABA) enhancing properties, but it does not directly interact with GABA receptors. Neu-P11 exhibits a multimodal effect: antineurodegenerative, antidepressant, anxiolytic [44], antidiabetic [36], antihypertensive [20] and antinociceptive [9]. More recently, Neu-P11 has demonstrated its usefulness in myocardial ischemiareoxygenation injury in in vitro models [50], as well as in improving sleep pattern [37] and insulin sensitivity [36]. At present, Neu-P11 is in Phase II Clinical Trial (https://ClinicalTrials.gov Identifier: NCT02615002) for the treatment of Alzheimer’s Disease. Neu-P67, in turn, acts not only at MTs but also exerts an agonistic effect at the 5-HT1A and 5-HT7 site (unpublished data). Both MTs analogs display long half-life and oral availability. The present study was conducted to investigate the effect of Neu-P11 and Neu-P67 on the seizure threshold in three acute seizure tests in mice. Moreover, acute side effects of each compound were evaluated in neuromuscular strength, motor coordination and locomotor activity tests. To further verify the anticonvulsant potential of Neu-P11 and Neu-P67, and allow for direct comparisons, MLT was included in the study and tested in comparable manner with both compounds.

2. Materials and methods 2.1. Animals Experimentally naïve male albino Swiss mice (Laboratory Animals Breeding, Słaboszów, Poland) weighing 22–30 g were used in all experiments. The total number of animals used in the study was 560. The animals were housed in Makrolon cages under controlled laboratory conditions (22–23 ◦ C, relative humidity, 45–55%, 12 h light/dark cycle, lights on at 6:00 a.m.). A nutritionally-balanced

rodent chow pellets (Agropol S.J., Motycz, Poland) and tap water were available ad libitum. To minimize circadian influences, experiments were performed between 8:00 and 16:00 h, after at least 7 days of acclimatization. Each animal was used only once. The experimental protocol followed the European Communities Council Directive of September 22, 2010 (2010/63/EU) and Polish legislation acts concerning animal experimentations and was approved by the Local Ethics Committee at the Medical University of Lublin (license numbers 37/2013 and 3/2014). 2.2. Drug administration Neu-P11 and Neu-P67 were obtained from Neurim Pharmaceuticals Ltd., Israel. Melatonin was purchased from Tocris Bioscience (MO, USA). Melatonin, Neu-P11 and Neu-P67 were dissolved in 10% dimethyl sulfoxide (DMSO, ICN Biomedicals, Inc., Aurora, OH, USA) in saline and administered intraperitoneally (i.p.) at the dose ranging from 25 to 200 mg/kg. Neu-P11, Neu-P67 and MLT were given 20 min, 20 min, and 15 min before testing, respectively. Control animals received i.p. injection of 10% DMSO. The vehicle had no effects on the observed parameters. Pentylenetetrazole (PTZ, ´ Poland) was dissolved in saline and infused Sigma-Aldrich, Poznan, intravenously (i.v.). The pretreatment schedules and doses of all drugs were selected based on our preliminary studies and available literature [10,24,44,48]. 2.3. The 6 Hz psychomotor seizure threshold test in mice The psychomotor seizure thresholds were examined using square-wave alternating current stimuli (0.2 ms duration pulses at 6 Hz for 3 s) applied via saline-soaked corneal electrodes using a Grass S48 stimulator coupled with a constant current unit CCU1 (both from Grass Technologies, West Warwick, RI, USA). A drop of ocular anaesthetic (1% solution of tetracaine hydrochloride) was applied on the corneas 1 min before the stimulation. Before testing, the electrodes were soaked in 0.9% of saline for good electrical contact. The seizures induced by 6 Hz stimulation were characterized by immobility or stun posture, which was frequently followed by rearing, forelimb clonus, twitching of the vibrissae and elevated or Straub tail [16,48]. The absence of the features listed above or the renewal of normal exploratory behaviour within 10 s after stimulation were considered as lack of seizures. The ‘up-and-down’ method described by Kimball et al. [23] was used in order to choose the current intensity. Each animal was stimulated only once at any given current intensity that was lowered or raised by 0.06 log intervals depending on whether the previously stimulated animal did or did not respond with convulsions, respectively. Data obtained in groups of 18–20 animals were used to determine the threshold current causing 6 Hz-induced seizures in 50% of mice (CC50 with confidence limits for 95% probability). 2.4. Maximal electroshock seizure threshold (MEST) test in mice The electroshock seizures were induced by sine-wave alternating current (maximal output voltage 500 V, 50 Hz for 0.2 s) applied via saline-soaked transcorneal electrodes delivered by a rodent shocker (type 221; Hugo Sachs Elektronik, Freiburg, Germany) as described earler [48]. To minimize the pain, an ocular anaesthetic (1% solution of tetracaine hydrochloride, Sigma-Aldrich) was applied into each eye 1 min before stimulation. Transcorneal electrodes were soaked in 0.9% saline to maximize the conductance. During stimulation mice were manually immobilized and immediately after the stimulation placed in a Plexiglas arena (37 cm × 21 cm × 14 cm) for behavioural observation for the presence or absence of seizure activity. Tonic hindlimb extension served as an endpoint. The thresholds for maximal electroconvulsions


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were adapted according to an ‘up-and-down’ method [23]. In this method, current intensity was lowered or raised by 0.06 log intervals depending on whether the previously stimulated animal did or did not exert seizure activity, respectively. Each mouse was stimulated only once at any given current intensity. Experimental groups comprised 18–21 animals. Obtained data was used to determine the threshold current causing an endpoint in 50% of mice (CC50 with confidence limits for 95% probability).

located on the sides and spaced at 1.5 cm intervals. Mice pretreated with, Neu-P11, Neu-P67 or vehicle 15 min before the experiment, or MLT 10 min prior to the test, were placed individually in the actimeter and allowed to freely explore the arena for 10 min. The number of crosses of the infrared beams was counted by the device within 10 min of the test.

2.5. The intravenous pentylenetetrazole (PTZ) seizure threshold test in mice

The results are expressed as means ± SEM and were analyzed using one-way analysis of variance (ANOVA) followed by the Tukey’s post-hoc test for multiple comparison. Data obtained from the MEST test and the 6 Hz seizure threshold test were analyzed according to Kimball et al. [23] and are presented as CC50 values with 95% confidence limits. Bartlett’s test was used to verify the homogeneity of variances. The results from the chimney test were analyzed with the Fisher’s exact probability test. p value was considered statistically significant when p < 0.05. All statistical calculations were performed with GraphPad Prism version 5.03 for Windows (GraphPad Software, San Diego, CA, USA).

Briefly, mice were placed in a cylindrical plastic restrainer (12 cm long, 3 cm inner diameter) and a 27-gauge needle (Sterican® , B. Braun Melsungen, Melsungen, Germany) was inserted into the lateral tail vein [48]. The needle was attached by polyethylene tubing (PE20RW, Plastics One Inc., Roanoke, VA, USA) to a 5 ml plastic syringe, which was mounted on a syringe pump (model Physio 22, Hugo Sachs Elektronik–Harvard Apparatus GmbH, March-Hugstetten, Germany). The 1% aqueous solution of PTZ, was administered into the lateral tail vein of unrestrained animals. The correct needle placement in the tail vein was verified by the appearance of blood in the tubing. PTZ solution was infused at a constant rate of 0.2 ml/min. Endpoints recorded to determine seizure thresholds were as follows: (1) the initial myoclonic twitch, (2) the generalized clonus with loss of righting reflex and (3) the onset of tonic forelimb extension. The time elapsed from the start of PTZ infusion to the onset of all three seizure stages was measured. The seizure thresholds were calculated separately for each endpoint according to the formula: threshold dose of PTZ (mg/kg) = (infusion duration (s) × infusion rate (ml/s) × PTZ concentration (mg/ml) × 1000)/body weight (kg). Seizure thresholds were expressed as the amount of PTZ (mg/kg) ± SEM needed to produce the first sign of each endpoint. Tonic convulsions were often lethal for mice. All surviving animals were euthanized immediately after the end of the infusion. 2.6. The grip-strength test in mice The effects of the studied compounds on skeletal muscle strength was measured in the grip-strength test [48] in mice (11–13 animals/group). The grip-strength apparatus (BioSeb, Chaville, France) consisted of a steel wire grid (8 × 8 cm) connected to an isometric force transducer. Animals were lifted by the tail so that they could grasp the grid with their forepaws. Subsequently, the animals were pulled backward gently by the tail until they released the grid. The maximal grip strength value (in newtons, N) of the animal was displayed on the screen. The procedure was repeated three times and the mean force exerted by each mouse was recorded. The mean force was normalized to body weight and expressed in N/g ± SEM. 2.7. Chimney test The chimney test was used to assess the acute adverse effects of Neu-P11, Neu-P67 and MLT on motor performance in mice (12 animals/group). In this test, the inability of an animal to climb backward up through a Plexiglas tube (inner diameter 3 cm, length 30 cm) within 60 s was an indication of motor impairment [5]. 2.8. Locomotor activity The IR Actimeter system (Panlab/Harvard Apparatus, Barcelona, Spain) was used to monitor the spontaneous locomotor activity of mice. IR Actimeter system consisted of a square arena surrounded by a 25 × 25 cm frame, containing a total of 16 × 16 infrared beams

2.9. Statistical analysis

3. Results 3.1. Effects of Neu-P11, Neu-P67 and MLT in the 6 Hz psychomotor seizure test in mice To evaluate the potential influence of Neu-P11, Neu-P67 and MLT on partial psychomotor seizure, we used the 6 Hz psychomotor seizure threshold test. As depicted in Fig. 1., Neu-P11 and NeuP67 (both at 100 and 200 mg/kg, i.p.) did not alter the 6 Hz seizure threshold (Fig. 1A and B, respectively). In contrast, acute administration of MLT (50 and 100 mg/kg, i.p.) resulted in a significant augmentation in current intensity necessary to induce psychomotor seizure in the 6 Hz seizure test (Fig. 1C). Consequently, MLT raised the CC50 value from 11.05 mA in control group to 14.79 mA when injected at a dose of 50 mg/kg and 19.28 mA at the dose of 100 mg/kg [Tukey’s multiple comparison test: p < 0.001 for both studied doses of MLT]. 3.2. Effects of Neu-P11, Neu-P67 and MLT in the MEST test in mice The effect of Neu-P11, Neu-P67 and MLT on the threshold in the MEST test is shown in Fig. 2. Neither Neu-P11 nor Neu-P67 at the doses of 100 and 200 mg/kg (i.p.) produced any significant changes in the seizure threshold values (p > 0.05). A post-hoc test revealed that MLT was ineffective at the dose of 25 mg/kg, while at the doses of 50 and 100 mg/kg it significantly raised the CC50 value from 8.51 mA in control group to 9.62 mA and 11.32 mA, respectively [Tukey’s multiple comparison test: p < 0.05 for 50 mg/kg and p < 0.001 for 100 mg/kg]. 3.3. Effects of Neu-P11, Neu-P67 and MLT on the seizure threshold in the i.v. PTZ test in mice The i.p. administration of Neu-P11 (100 and 200 mg/kg) did not produce any significant effect on the doses of PTZ necessary to produce the first apparent sign of each endpoint: myoclonic twitch, the onset of generalized clonus or the elicitation of tonic extension of the forelimbs (Fig. 3A, B and C, respectively). The i.p. administration of Neu-P67 did not change the threshold for myoclonic twitch (Fig. 4A) and generalized clonus (Fig. 4B), independently of doses used in the study. However, at the dose of 200 mg/kg Neu-P67 significantly altered forelimb extension (Fig. 4C). The results of the PTZ test for MLT are shown in Fig. 5. No statistically significant effect was observed in myoclonic twitch (Fig. 5A). In contrast, at the highest dose tested (100 mg/kg) MLT caused an

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Fig. 1. Effect of Neu-P11 (A), Neu-P67 (B) and melatonin (C) on the seizure threshold in the 6 Hz seizure test in mice. Neu-P11 and Neu-P67 were injected i.p. 20 min before the test; melatonin was administered i.p. 15 min prior to the test. Control animals received vehicle. Each experimental group consisted of 18–20 animals. Data are presented as CC50 value in mA (with upper 95% confident limits) which indicates current intensity predicted to produce convulsions in 50% of mice. ***p < 0.001 as compared to the CC50 value of the control group (one-way ANOVA followed by Tukey’s post-hoc test).

Fig. 2. Effect of Neu-P11 (A), Neu-P67 (B) and melatonin (C) on maximal electroshock seizure threshold. Neu-P11 and Neu-P67 were injected i.p. 20 min before the test; melatonin was administered i.p. 15 min prior to the test. Control animals received vehicle alone. Each experimental group consisted of 18–21 animals. Figure shows data for CC50 (in mA) values with upper 95% confident limits. Each CC50 value indicates current intensity predicted to induce convulsions in 50% of mice. *p < 0.05; ***p < 0.001 vs. control (one-way ANOVA followed by Tukey’s post-hoc test).

3.4. Effects of Neu-P11 and Neu-P67 on muscular strength and motor coordination in mice

increase in seizure threshold for both the generalized clonus and forelimb tonus (p < 0.05 and p < 0.001, respectively) (Fig. 5B and C).

To determine the effects of MT receptor agonists and MLT on neuromotor functions of the muscles, a grip-strength test was performed. As presented in Fig. 6., Neu-P11 and Neu-P67 (both at 100 and 200 mg/kg, i.p.), and MLT (25, 50 and 100 mg/kg, i.p.) did


Fig. 3. Effects of Neu-P11 on seizure threshold in the i.v. PTZ test. Neu-P11 was administered 20 min prior to seizure testing; control animals received vehicle alone. Each bar represents the mean (+ SEM; n = 12–13 mice/dose) dose of i.v. PTZ (in mg/kg) necessary to induce the myoclonic twitch (A), generalized clonus (B) and forelimb tonus (C).

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Fig. 4. Effects of Neu-P67 on seizure threshold in the i.v. PTZ test. Neu-P67 was administered 20 min prior to seizure testing; control animals received vehicle alone. Each bar represents the mean (+ SEM; n = 10–12 mice/dose) dose of i.v. PTZ (in mg/kg) necessary to induce the myoclonic twitch (A), generalized clonus (B) and forelimb tonus (C). *p < 0.05 vs. control (one-way ANOVA followed by Tukey’s post-hoc test).

3.5. Effects of Neu-P11, Neu-P67 and MLT on locomotor activity in mice not affect muscle strength in mice (p > 0.05). Likewise, there were no significant changes in motor coordination, as assessed in the chimney test [Fisher’s test: p > 0.05 for all studied groups; data not shown].

Data obtained in spontaneous locomotor activity test are presented in Fig. 7. Both compounds, Neu-P11 (Fig. 7A) and Neu-P67 (Fig. 7B) significantly reduced the activity counts measured within 10 min after administration (100 and 200 mg/kg, i.p.) (p < 0.001

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Fig. 5. Effects of melatonin on seizure threshold in the i.v. PTZ test. Melatonin was administered 15 min prior to seizure testing; control animals received vehicle alone. Each bar represents the mean (+ SEM; n = 11–13 mice/dose) dose of i.v. PTZ (in mg/kg) necessary to induce the myoclonic twitch (A), generalized clonus (B) and forelimb tonus (C). *p < 0.05; ***p < 0.001 vs. control (one-way ANOVA followed by Tukey’s post-hoc test).

vs control group). Similarly, MLT (Fig. 7C) at the highest dose (100 mg/kg, i.p.) resulted in decreased activity counts (p < 0.01 vs control group). 4. Discussion In the present study, we investigated the anticonvulsant activity of MT agonists, Neu-P11 and Neu-P67, in three models of acute

Fig. 6. Effect of Neu-P11 (A), Neu-P67 (B) and melatonin (C) on neuromuscular strength measured by grip strength test. Data are presented as mean + SEM of grip strength in newtons/gram of mouse body weight (N/g). Each experimental group consisted of 11–13 mice.

seizures, and evaluated their effects on neuromuscular strength and locomotor activity in mice. The experiment was designed to determine whether Neu-P11 and/or Neu-P67 affect the epileptic course in a similar manner to MLT. The results of our study indicate that neither Neu-P11 nor Neu-P67 changed the seizure threshold in PTZ, 6 Hz and MEST models of seizures. Even though Neu-P11 and Neu-P67 failed to affect the skeletal muscular strength in the grip strength test, the significant changes observed in the locomotor activity test suggest their possible interaction with receptors in the central nervous system (CNS). In contrast, the majority of tests confirmed the anticonvulsant properties of MLT.


Fig. 7. The influence of i.p. administration of Neu-P11 (A), Neu-P67 (B) and melatonin (C) on locomotor activity. Neu-P11 and Neu-P67 was administered 15 min before the test; melatonin was injected 10 min prior to the test. Control animals received vehicle alone. Data are presented as mean + SEM. Each experimental group consisted of n = 7–10 animals. *p < 0.05; **p < 0.01; ***p < 0.001 vs. control (one-way ANOVA followed by Tukey’s post-hoc test).

During the last decade, a significant portion of literature put a special interest on the association between MLT and epilepsy. The anticonvulsant activity of MLT was tested in various laboratory animals and against different convulsive stimuli, e.g., penicillin [49], glutamate, bicuculline, pilocarpine, kainate, 3-mercaptopropionic acid, N-methyl-d-aspartate (NMDA), L-cysteine [28] or amygdala kindling [22]. All the studies ensured the anticonvulsant and neuroprotective activity of MLT after its acute administration. Similarly, in male gerbils, chronic subcutaneous administration of MLT

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for 10 weeks significantly reduced the number and severity of PTZ-induced seizures [8]. Our findings are consistent with the above-mentioned studies and confirm that MLT has anticonvulsant activity in experimental animal models of acute seizures. In patients with intractable and refractory epilepsy, MLT appears to influence seizure by reducing its frequency and severity [17,33]. In addition, exogenous MLT administration exerted beneficial effects on neuronal damage and behavioural complications concomitant with epilepsy [34,41]. Finally, MLT significantly improves sleep deprivation and synchronizes disturbed daytime behaviour—common features which exacerbate the epileptogenic activity [2,12,21]. Taken together, the results of animals’ studies and clinical trials vote for MLT as an attractive anti-epileptic treatment. Given the poor pharmacokinetic profile of MLT, several MLT agonists with modified properties and prolonged-release are currently of therapeutic interest in the management of epilepsy e.g., agomelatine, ramelteon or tasimelteon [1,15,19]. Recently, a number of studies verified the usefulness of another MLT agonist, Neu-P11, which exhibits a promising neuroprotective effect [6], attenuates insulin resistance [36] and hypertension [20,32], and produces antinociception [9,24]; however, as far as we know, there are no articles in the literature so far discussing the effects of Neu-P11, and its sister compound—Neu-P67, in animal models of epilepsy. Surprisingly, the pretreatment with Neu-P11 and Neu-P67 did not prevent from seizures. Both MT analogs neither increased nor decreased the seizure threshold in the 6 Hz, MEST and PTZ tests. In contrast, in each animal model of seizures MLT significantly increased seizure threshold. In general, genetic abnormalities, prolonged acute symptomatic seizures, up-regulation of immune responses, and diverse brain insults, such as stroke, infections, tumours, cortical dysplasia, traumatic brain injury or neurodegeneration, constitute a potential cause of epileptogenesis [13,25]. However, the instability between inhibitory (GABA-ergic) and excitatory (glutamate-mediated) neurotransmission is also a potential trigger of epilepsy development. Inasmuch as MLT acts upon the central synapses where it associates with GABAA , voltage-gated calcium channel, nitric oxide (NO) and 5-HT-dependent pathways, other anticonvulsant drugs may additionally target NMDA receptors or voltage-gated sodium channel [14,27]. It is reasonable to think that by targeting MTs, MLT as well as Neu-P11 and Neu-P67 should all induce similar anticonvulsant effect, in particular in the PTZ seizure threshold test, in which a classical GABAA receptor antagonist that binds to the picrotoxin site of the GABAA receptor complex and blocks GABA-mediated inhibition through NMDA receptor is used [1,14]. Unfortunately, we observed that neither Neu-P11 nor Neu-P67 stimulated MTs and thus did not mediate the inhibition of GABA-dependent actions. There is a possibility that both Neu-P11 and Neu-P67 activate MTs but to the extent that does not affect the seizure threshold in the PTZ test; it may be thus hypothesized that the dosages used in the study were too low to generate the anticonvulsant effect. We may also speculate that GABA-related effects triggered by MLT differ from those of Neu-P11 and Neu-P67 either because of up- or downregulation of specific signalling pathways, or formation of homo-/heterodimers with other receptors. However, this hypothesis needs further investigation. Worth mentioning, MLT deficiency via pinealectomy causes greater levels of neurodegeneration, which support the notion about the neuroprotective role of MLT [3]. Surprisingly, the study by Buendia et al. [6] also proved the efficacy of Neu-P11 in neuroprotection. The pro-survival effect was mediated by MT receptors, and particularly through the Jak/Stat, Akt and Erk1/2 cellular pathways [6]. This is the only study available that presents, at least in part, the molecular explanation of neuroprotective effect of Neu-P11. To date, only a few studies evaluated the relation between 5-HT subtypes and seizure occurrence. Stean et al. [38] demonstrated

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that 5-HT analogs with high affinity to 5-HT1A , 5-HT1B and 5-HT1D subtypes produced marked dose-dependent increase in seizure threshold measured in MEST test in rats. In line, the intrahippocampal administration of the 5-HT1A receptor agonist increased the seizure threshold in hippocampal-kindled cats [26,46], whereas in rats, it retarded the development of amygdala kindling [47]. Furthermore, the activation of 5-HT2 receptors led to the anticonvulsant action of trifluoromethyl-phenylpiperazine (TFMPP) [45] and m-chlorophenylpiperazine (mCPP) [35] in MEST test in rodents. Even more curiously, it has been shown that 5-HT participates in seizure activation and propagation and that the anticonvulsant action of MLT is partially mediated by serotonergic mechanism [30,42,43]. Even though Neu-P11 acts on 5-HT1A/1D , whereas NeuP67 binds specifically to 5-HT1A and 5-HT7 receptor subtypes, the effect generated by both compounds through the activation of 5HT receptors remains unknown. Albeit, the same MEST test as mentioned above was incorporated in our study, the observations showed an opposite action of Neu-P11 and Neu-P67 on the seizure threshold. Our experiment diverges from earlier studies in many aspects, e.g., animal species included and route of administration used and therefore no conclusion can be made; however, the MT–5HT crosstalk is an important aspect of the pharmacology of the Neu-P compounds. 5. Conclusion Administration of MLT resulted in a significant reduction in the number and intensity of the seizures in three acute seizure mouse models. In contrast, our experiments did not show any anticonvulsant effect of Neu-P11 or Neu-P67. Taken together, our study provides additional support for MTs being an attractive target for the development of anticonvulsant drugs, but also proves that their pharmacology still needs further investigation. Furthermore, the neuroprotective action of Neu-P11 and Neu-P67 and their interaction with endogenous systems, like serotonergic should be taken into account in further research designed for the development of CNS-oriented therapeutics. Conflicts of interest Moshe Laudon is an employee of Neurim Pharmaceuticals Ltd., and provided Neu-P11 and Neu-P67 without any financial support. All other authors declare that they have no conflicts of interest. Author contributions P.M., J.F., P.W. designed the research study, K.S., D.N., P.W. performed the research, M.S., M.L. provided necessary tools and reagents, P.M., K.S. analyzed the data, P.M., J.F. wrote the manuscript, P.M., K.S., D.N., M.L., M.S., J.F., P.W. approved final version of the manuscript. Acknowledgements Supported by the Medical University of Lodz (Project “UMED Grants” 63/2014-2015 to P.M. and 503/1-156-04/503-01 to J.F.) and National Science Center (UMO-2013/11/B/NZ7/01301 and UMO2014/13/B/NZ4/01179 to J.F.) and by Funds for Statutory Activity of Maria Curie-Skłodowska University, Lublin, Poland. References [1] C.C. Aguiar, A.B. Almeida, P.V. Araujo, G.S. Vasconcelos, E.M. Chaves, O.C. do Vale, D.S. Macedo, F.C. de Sousa, G.S. Viana, S.M. Vasconcelos, Anticonvulsant effects of agomelatine in mice, Epilepsy Behav. 24 (2012) 324–328.

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