Mygalin: a new anticonvulsant polyamine in acute seizure model and neuroethological schedule

Send Orders for Reprints to [email protected] 122 Central Nervous System Agents in Medicinal Chemistry, 2013, 13, 122-131

Mygalin: A New Anticonvulsant Polyamine in Acute Seizure Model and Neuroethological Schedule

Lívea Dornela Godoy1,2, José Luiz Liberato1,2, Pedro Ismael da Silva Junior3 and Wagner Ferreira dos Santos1,2*

1Neurobiology and Venoms Laboratory, Biology Department, College of Philosophy, Sciences and Literature of Ribeirão Preto, University of São Paulo, Brazil; 2Behavior and Neurosciences Institute (INeC), Campus USP, Brazil; 3Center of Applied Toxicology (CAT-CEPID), Butantan Institute, Brazil

Abstract: Polyamines are compounds that interact with ionotropic receptors, mainly modulating the NMDA receptor, which is strictly related to many neurologic diseases such as epilepsy. Consequently, polyamines rise as potential neuropharmacological tools in the prospection of new therapeutic drugs. In this paper, we report on the biological activity of synthetic polyamine Mygalin, which was tested as an anticonvulsant in model of chemically induced seizures. Male Wistar rats were injected with vehicle, diazepam, MK-801 or Mygalin at different doses followed by Pentylenetetrazole or N-Methyl-D-Aspartate administration. Mygalin presented protection against seizures induced by both NMDA injections and PTZ administration by 83.3% and 16.6%, respectively. Moreover, it prolonged the onset of tonic-clonic seizures induced by PTZ. Furthermore, it was tested in neuroethological schedule evaluating possible side-effects and it presented mild changes in Open Field, Rotarod and Morris Water Maze tests when compared to available anticonvulsant drugs. The mechanism underlying the anticonvulsant effect of Mygalin is noteworthy of further investigation, nevertheless, based on these findings, we hypothesize that it may be wholly or in part due to a possible NMDA receptor antagonism. Altogether, the results demonstrate that Mygalin has an anticonvulsant activity that may be an important tool in the study of prospection of therapeutics in epilepsy neuropharmacology.

Keywords: Anticonvulsant, MK-801, Morris Water Maze, Mygalin, NMDA, Open Field, polyamine, PTZ.

INTRODUCTION

In spite of the growing availability of anticonvulsant drugs for therapeutic use, most patients who present convulsive seizures are treated with conventional anti-epileptic drugs (AEDs) [1]. Only in 70% of the cases seizures are controlled or suppressed by the chronic administration of AEDs, when the diagnosis is well conducted [2]. Therefore, there is a demand for novel studies on compounds either in the basic science or in the pharmacological drug design [3]. Moreover, conventional AEDs usually present undesirable effects such as sedation, motor deficit, chronic toxicity, teratogenesis [4], cognitive deficit and drug interaction, which limit their chronic use [5]. As an alternative, animal toxins, mainly those extracted from arthropods, are rich sources of natural bioactive compounds which show high affinity and selectivity to mammalian central nervous system (CNS). These compounds interact with receptors, transporters and ion channels, modifying neuronal mechanisms [6-8]. Toxins have useful properties to understand synaptic transmission events and the design of novel drugs in basic studies for treating neurological diseases such as Parkinson´s disease, Huntington´s chorea,

*Address correspondence to this author at the Av. Bandeirantes, 3900, FFCLRP/USP – Departamento de Biologia, Zip Code: 14040-901, Ribeirão Preto, São Paulo, Brazil; Tel: +55-16-3602-3657; Fax: +55-16-3602-4886; E-mail: [email protected]

Alzheimer´s disease, global and retinal ischemia and epilepsy [9]. Among all these bioactive molecules, the peptides and polyamines are more relevant [10, 11, 12]. Polyamines have been widely studied in the last decades, presenting a potential therapeutic use [13]. Several studies have demonstrated that polyamines interact with L-glutamate (L-Glu) receptors, particularly antagonizing the ionotropic type [7, 14, 15]. The NMDA (N-Methyl-D-Aspartate) receptors present polyamine modulating binding sites and are strictly related to neurological function such as long-term potentiation and long-term depression processes that may underlie learning and memory [16]. Consequently, polyamines come as useful tools in the prospection of new drugs as glutamatergic neurotransmission is related to neurological diseases and toxin interaction may have anticonvulsant effects [17]. Thus, polyamines should be tested in in vivo neuroethological models that might underpin new treatments strategies to those diseases [9]. Pereira and co-workers have isolated a bis-acylpolyamine [N1,N8-bis(2,5-dihydroxybenzoyl spermidine)] of 417 Da from the Acanthoscurria gomesiana spider hemolymph. This compound was denominated Mygalin. Previous studies with Mygalin have already demonstrated a potent effect against gram positive and gram negative bacteria in vitro [18]. It has been already reported that molecules that present antibacterial effects may also present neuroactivity, such as

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Neuropharmacological Screening of Mygalin Central Nervous System Agents in Medicinal Chemistry, 2013, Vol. 13, No. 2 123

the neuroprotective effects of 5-S-GAD in oxidative stress-induced apoptosis in the rat retinal ganglion cell line RGC-5 [19, 20]. Considering the neuropharmacological potentialities that polyamines may present, the aim of this study was to investigate the anticonvulsant effect of Mygalin in chemically induced seizure models.

METHODS

Animals and Surgery

Adult male Wistar rats (200-250 g), acquired from the central animal vivarium of University of São Paulo - Ribeirão Preto campus, were kept in a room on a 12h light/dark cycle at the temperature of 25 ± 1 ºC with food and water ad libitum at least two days before any procedure. Animals were intraperitoneally (i.p) anaesthetized with ketamine (80 mg kg-1, Agener União, Brazil) and xilazine (10 mg kg-1, Callier, Spain), their heads were shaved and 2% lidocaine was injected subcutaneously. They were placed in the stereotaxic apparatus (Stoelting, USA) for intracerebroventricular (i.c.v) cannulae implantation following coordinates from bregma [21]. One cannula was attached to the skull with acrylic resin, which was anchored with stainless steel screws. To prevent obstruction, canullae were sealed with stainless steel wire. Rats were allowed to recover for at least 5–7 days. At the end of the experimental assays the rats received anaesthetic thiopental (120 mg/kg; Cristalia, Brazil) overdose, and the position of the cannulae was confirmed. This work was performed according to the Ethical Principles for Animal Experimentation approved by the Ethics Committee of Animal Use (CEUA), University of São

Paulo - Campus Ribeirão Preto (Nr. 09.1.148.53.9), which establishes the animal handling guideline according to federal law 11794 (08/10/2008). All efforts were made throughout the study to minimize animal stress or pain.

Drugs

Through i.p route, we injected Pentylenetetrazole (PTZ, Sigma, USA); diazepam (DZP, Sigma, USA) or MK-801 (Sigma, USA). Mygalin and NMDA (Sigma, USA) treatments were performed via i.c.v route with the aid of a Hamilton syringe moved by an infusion pump (Insight, Brazil), in 0.5 �L/minute flow rate. All drugs were dissolved in sterile isotonic saline except for Mygalin, which was diluted into Milli-Q deionized water – vehicle (Millipore, USA). Mygalin was synthesized at the Center of Applied Toxicology (CAT – CEPID), Butantan/ SP and provided by Dr. Pedro Ismael da Silva Jr. The mygalin was synthesized according to the classical method of peptide chemistry [22]. Briefly, the synthesis was carried out in HBTU solution [23] for the esterification of the carboxyl group of gentisic acid and thus permit the formation of the one carboxamide by formal condensation of two primary amino groups from spermidine with carboxylic group of two molecules of gentisic acid (2,5-dihydroxybenzoic) - available on Ontology of Chemical Entities of Biological Interest (ChEBI) database as Mygalin (CHEBI:64901) [24]. Before biological testing, the sample (15�L) was analyzed to confirm purity degree by direct injection with a solution of 50% acetonitrile in 1:1 acidified water (0.1% formic acid) under a flow of 0.05 mL on LC/MS Finnigan™ Surveyor® MSQ™ Plus (Quadrupole, USA). Analyses were performed with the spectrometer

Fig. (1). Synthetic Mygalin Mass spectra (LC-MS) - m/z de 418.79. Analysis were performed with the spectrometer operating in positive mode on LC/MS Finnigan™ Surveyor® MSQ™ Plus (Quadrupole).

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124 Central Nervous System Agents in Medicinal Chemistry, 2013, Vol. 13, No. 2 Godoy et al.

operating in positive mode. Spectra were collected and analyzed by Xcalibur® software 2.0 (Thermo Electron, USA) (Fig. 1). In PTZ-induced seizures model, we used the following Mygalin doses: 0.002; 0.02; 0.2 and 2.0 �g/�L and in NMDA-induced seizures assay 0.0002, 0.002, 0.02, 0.2 and 2.0 �g/�L. In Open Field, Rotarod and Morris Water Maze assays, we used Mygalin at 0.2 and 20 �g/�L.

Anticonvulsant Assessment and Observation Procedures

Rats (n=6) were pretreated with vehicle, MK-801 (39 mg/kg) [25], DZP (2 mg/kg; i.p) or Mygalin doses (via i.c.v), 15 min before the administration of the convulsant drugs PTZ (80 mg/kg, i.p) or NMDA (35 �g/�L, i.c.v). The injection volume for all drugs via i.c.v was 1 �L. The doses of all convulsants were calculated in previous experiments for the dosage producing convulsions in 97% of the animals (CD97) [26].

After the administration of the convulsants, animals were kept individually in transparent observational cages (30 x 20 x 22 cm) and recorded by 90 min (Phillips camera, USA). We used the classification of seizure behaviors according to Lamberty and Klitgaard [27]. Seizures were scored as follows: 0, immobility; 1, head nodding and facial stereotypes; 2, convulsive waves through the body (spasms); 3, myoclonic jerks (with or without rearing) in the upper extremities; 4, clonic convulsions in all extremities, turn over onto sides; 5, tonic–clonic convulsions in all extremities, turn over onto back.

The latencies to the onset of seizures and percentages of animals that presented score 5 seizure were the parameters used to analyze the anticonvulsant effects.

Motor Impairment on Rotarod Test

In order to test the motor function after treatments, rats were submitted to Rotarod test (Ugo Basile, Italy), which consisted of a rotating bar (diameter 5 cm) driven by a motor (speed: 4 rpm). The day before the test, rats were trained to maintain equilibrium on the rotating bar. The training consisted of three subsequent 1 min attempts at 4 rpm only animals that accomplished the entire training were selected to be tested.

Mygalin (2.0 or 20 �g/�L), or vehicle, was administered i.c.v. and animals were placed on the rotating bar every 5-minute intervals (5 to 90 minutes), latencies to falls were registered. Animals that fell off in three subsequent attempts were considered motor impaired.

Spontaneous Locomotor activity on Open Field Test

To evaluate the spontaneous locomotor activity, the rats are placed in the test room for a period of 10 minutes for acclimatization. After that, rats were injected via i.c.v with either Mygalin (2.0 or 20 �g/�L) or vehicle, or diazepam (2 mg/kg, i.p). Subsequently, animals were exposed to a circular acrylic arena (60 cm x 36 cm divided by lines into 12 quadrants) and were monitored. Behaviors were recorded for 20 min and were grouped in four classes modified from Speller and Westby [28] as follows: exploratory (including

locomotor and sniffing exploration), rearing, grooming and inactivity and duration of each category registered. Moreover, we analyzed the spontaneous locomotor activity of the animals by registering lines crossed in four periods of five minutes (0-5; 6-10; 11-15 and 16-20 minutes). The videos were analyzed with the X-Plo-Rat software [29].

Morris Water Maze (MWM)

Rats were submitted to a training period of four consecutive days sessions, six trainings per day in the MWM in order to evaluate possible cognitive impairments in spatial learning and memory [30]. Groups were divided in naïve (animals that did not undergo surgery), vehicle or Mygalin (0.0002 or 2.0 �g/�L). The water maze apparatus consisted of a white polyethylene circular pool (diameter 140 cm, depth 50 cm), filled with water (23 ± 1 ºC) and milk up to 25 cm to the boards and divided in four quadrants considering the southwest quadrant as the goal quadrant. A hidden platform was placed in the center of the goal quadrant. Each training trial consisted of placing the animals on randomly chosen quadrants of the pool, except for the goal quadrant. Animals were allowed to explore the pool for 90 seconds, or until they found the hidden platform. If they did not find the platform after the 90 seconds, they were gently placed over it for 30 seconds. The six attempts and the latencies to find the platform in the training periods were recorded. On the last day, after the last training attempt, the platform was removed from the pool for the probe trial session when the occupancy in each quadrant of the pool was quantified.

Statistical Analyses

In the analyses of latencies to the onset of tonic-clonic seizure, behaviors in Open Field test and Probe Trial in MWM it was used one-way ANOVA followed by Student Newman-Keuls post-test. Also in MWM test, each day was analyzed by one-way ANOVA of repeated measures followed by post-test Student Newman-Keuls.

Data of all days were submitted to two-way ANOVA of repeated measures followed by Student Newman-Keul post-test (to evaluate groups and trial effects in spatial learning and memory). In the analysis of crossed lines in Open Field Test it was used two-way ANOVA followed by Bonferroni post-test (comparing treatments and time effect).

For median-seizure score analysis, we used non-parametric test of Kruskal-Wallis followed by Dunn´s post-test analysis. The death frequencies and seizure frequencies were submitted to Chi-square test. We considered p < 0.05 as statistically significant.

RESULTS

PTZ Chemical Acute Seizure Model

It was observed in groups pre-treated with 0.02, 0.2 and 2.0 �g/�L Mygalin 16.66% protection against tonic-clonic seizures. Meanwhile, the dose of 0.002 �g/�L did not protect the animals against tonic-clonic seizure in this experimental model (all animals achieved score 5) therefore, it presented no statistically significant difference from vehicle treatment group. Animals that were treated with diazepam presented a


statistically significant difference compared to vehicle. No animal of this group presented tonic-clonic seizures (�2=10.00,1 p< 0.001) (Table 1).

Table 1. Protection against tonic-clonic seizures and death induced by PTZ i.p injection (80 mg/kg).

Treatment % Protection against Tonic-Clonic

Seizure (n)

Vehicle (i.c.v) 0 (0/5)

Diazepam (2 mg/kg; i.p) 100 (5/5)**

Mygalin (0.002 �g/�L; i.c.v) 0 (0/6)

Mygalin (0.02 �g/�L; i.c.v) 16.66 (1/6)

Mygalin (0.2 �g/�L; i.c.v) 16.66 (1/6)

Mygalin (2.0 �g/�L; i.c.v) 16.66 (1/6)

** p< 0.001 compared to vehicle group in Chi-square test. PTZ – Pentylenetetrazole. i.p - intraperitoneally. i.c.v - intracerebroventricular.

When the latencies to the onset of the tonic-clonic seizures of animals pre-treated with 0.002 �g/�L Mygalin (dose that did not protect animals from tonic-clonic seizures) were compared to controls we observed that there was a significant increase in the latency [F(4,28)= 5.032; p< 0.01)] (Fig. 2).

Fig. (2). Mygalin effect on mean latencies to the onset of tonic-clonic seizures (score 5) in PTZ convulsant acute model. Mean latencies were submitted to one-way ANOVA analysis, followed by Student Newman-Keuls post-test. * p<0.01 compared to vehicle group.

NMDA Chemical Acute Seizure Model

When tested in NMDA model, positive controls diazepam and MK-801 presented 75 and 100% of protection against tonic-clonic seizures, respectively. Mygalin also presented a significant protection against tonic-clonic seizure, even though as an inverted dose-response curve, as the lower doses produced a higher protection effect. The

0.0002 �g/�L Mygalin dose protected 83.3% of animals against tonic-clonic seizures (� 2= 10.37,1 p< 0.001), while 0.002, 0.02 and 0.2 �g/�L doses protected 66.7% (� 2=7.467,1 p< 0.01) and 2.0 �g/�L dose protected 50% (� 2=5.091,1 p< 0.05) (Table 2).

Table 2. Protection against tonic-clonic seizures induced by NMDA i.c.v injection.

Treatment % Protection against Tonic-Clonic

Seizure (n)

Vehicle (i.c.v) 0 (8/8)

Diazepam (2mg/kg; i.p) 75 (6/8)***

Mygalin (0.0002 �g/�L; i.c.v) 100 (6/6)***

Mygalin (0.002 �g/�L; i.c.v) 83.3 (5/6)***

Mygalin (0.02 �g/�L; i.c.v) 66.7 (4/6)**

Mygalin (0.2 �g/�L; i.c.v) 66.7 (4/6)**

Mygalin (2.0 �g/�L; i.c.v) 50 (3/6)

*** p< 0.001, ** p< 0.01 and * p< 0.05 compared to vehicle group in Chi-square test. i.p - intraperitoneally. i.c.v - intracerebroventricular.

Although we observed the protection against seizure score 5 in Mygalin groups and diazepam pre-treatments, only the antagonist MK-801 reduced to a statistical significance the median score [H= 14.83; p< 0.01)] (Fig. 3).

Fig. (3). Median of seizures scores of the groups in NMDA convulsant acute model. Analyzed with Kruskal-Wallis followed by Dunn’s post-test. * p < 0.01 compared to vehicle group.

The latency to the onset of tonic-clonic seizure analysis revealed that animals treated with Mygalin did not differ from the control group (data not shown).

Rotarod Test

The Rotarod test was performed in order to analyze possible motor impairment effect after Mygalin treatments.

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The 0.2 �g/�L Mygalin pre-treatment reduced the latencies in three consecutive falls from the apparatus in 20% of animals starting from 25 minutes after the Mygalin i.c.v injection. Also 20 �g/�L Mygalin pre-treatment reduced latencies in three consecutive falls from the apparatus in 40 % of animals, starting from the period of 20 minutes.

Open Field Test

We evaluated the spontaneous locomotor activity in the different treatments (Fig. 4). No difference was observed between treatments in time spent in rearing [F(3,20)= 1.230; p= 0.348], but there was a significant difference in inactivity, exploration and grooming of animals treated with Mygalin. In the inactivity behavior, it was observed that animals injected with 0.2 �g/�L Mygalin spent most of the time inactive in the Open Field when compared to all other treatments [F(3,20)= 5.490; p< 0.05)], and that treatment with both 0.2 �g/�L and 20 �g/�L Mygalin rats spent more time grooming when compared to diazepam treatment [F(3,20)=4.527; p< 0,05)]. In exploration, it was observed that diazepam group explored significantly less the maze when compared to vehicle [F(3,20)= 4.200; p< 0.05)]. The line-crossed analysis demonstrated no differences between treatments [F(3,12)= 77.85; p= 0.7538] (Fig. 5).

Fig. (5). Analysis of the lines crossed, through periods grouped 1-5, 6-10, 11-15 and 16-20 minutes in Open Field test. Treatments with vehicle, diazepam (2 mg/kg) and 0.2 and 2.0 �g/�L Mygalin in the Open Field. The means were submitted to one-way ANOVA analysis followed by Student Newman-Keuls post-test.

Morris Water Maze Test

On the first and second days (the initial period when animals are exposed to the maze) the four groups presented a similar performance in the mean latency, [F(3,12) =1.029; p= 0,9568] [F(3,12) = 0.7324; p= 0.5524] respectively (Fig. 6).

Fig. (4). Analysis of the general activity of animals treated with vehicle, diazepam (2 mg/kg) or Mygalin 0.2 and 2 �g/�L doses in the Open Field test. The means were submitted to one-way ANOVA followed by Student Newman-Keuls post-test. * p <0.05 compared to DZP group, # p <0.05 compared to vehicle group and a for p <0.05 compared to Mygalin group 0.2 �g/�L.

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On the third day, we detected difference between Mygalin and control groups, as Mygalin-treated rats presented no difference from previous trials while naïve and vehicle-treated animals reduced the escape latency. Analysis showed that 0.0002 �g/�L Mygalin was statistically different from both control groups [F(3,12) = 7.450; p=0.0045]. On the fourth day, naïve and vehicle-treated animals continued to reduce the escape latency, but also did Mygalin-treated animals [F(3,12) = 3.392; p= 0.0365]. Therefore, on the last day, there was no statistically significant difference between groups.

In Probe trial one-way ANOVA analysis, we found that in Mygalin-treated groups, animals spent a short period in the goal quadrant, scattering the time almost homogenously along the four quadrants (Fig. 7), 2.0 �g/�L Mygalin [F(3,16)= 0.1788; p= 0.9092] 0.0002 �g/�L Mygalin, [F(3,16) = 1.342; p= 0.2959]. In another hand, naïve and vehicle-treated rats created defined routes to the platform and kept swimming for a significantly long time in the goal quadrant [F(3,16) = 37.39; p< 0.0001] and [F(3,16) = 8.278; p=0.0015]. The acquisition curves for rats, which give the means from six trials from each day (Fig. 8). The rats learned to locate the hidden platform with progressively shorter latencies over the course of the study. As expected, naïve and vehicle-treatment rats learned to locate the hidden platform efficiently until the end of the study, while the Mygalin-treated rats were less efficient. A two-way ANOVA of repeated-measures on escape latencies indicated a significant effect for groups [F(3,80) = 22.41; p<0.0001], for trials [F(12,80) = 42.48; p<0.0001] and for their interaction [F(4,80) = 14.01; p<0.0001], suggesting differences between

groups in the ability to find the platform over the course of four days.

DISCUSSION

Anticonvulsant Activity in PTZ Acute Chemical SeizureModel The PTZ model has proved to be a good predictor of clinical efficacy of generalized spike-wave epilepsies of the absence type, and most common efficacy is related to agents that reduce the calcium flow through T-type calcium channels or that enhance chloride flow through GABA receptors [31]. These results demonstrated that the polyamine Mygalin in the higher doses (0.02, 0.2 and 2.0 �g/�L) has an anticonvulsant effect of 16.66% against the seizures induced by PTZ. It was also observed that although the Mygalin dose (0.002 �g/�L) protects animals from tonic-clonic seizure, it delayed the onset of the tonic-clonic seizure induced by that GABAergic antagonist. It may be suggested that these effects may be more related to a reduction of the excitatory transmission due to Mygalin antagonistic modulation in the glutamatergic system, correcting the imbalance generated by chemical inhibition in GABAergic system with PTZ injection. Cremer and co-workers [32] have analyzed the balance between excitatory and inhibitory neurotransmission in PTZ chronic model. They assessed the density of ligant sites of GABA, adenosine and L-Glu receptors in Wistar rats injected with PTZ and observed that there is an increase of 20-25% density in MK-801 ligant sites in NMDA receptor. Therefore, as there is a compensatory mechanism by increasing NMDA ligant sites density, those sites might be an interesting pharmacological target to explore in association with polyamines in this anticonvulsant model.

Fig. (6). Escape latency analysis on each day of training of naïve, vehicle, 0.0002 and 2.0 �g/�L Mygalin groups in Morris Water Maze. The means were submitted to one-way ANOVA of repeated measures followed by Student Newman-Keuls post-test. * p <0.05 compared to naïve group and # p <0.05 compared to vehicle group.

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Fig. (7). Probe Trial analysis of naïve, vehicle, 0.0002 and 2.0 �g/�L Mygalin groups in the Morris Water Maze. The means were submitted

to one-way ANOVA of repeated measures followed by Student Newman-Keuls post-test. *** p <0.0001 and ** p <0.001 target quadrant

compared to other three quadrants.

Fig. (8). Two-way ANOVA of repeated measures escape latency analysis over the days of naïve, vehicle, Mygalin 0.0002 and 2.0 �g/�L

groups in Morris Water Maze, followed by Bonferroni post-test. * p <0.05, *** p <0.0001 compared to naïve; ### p <0.0001 compared to

vehicle; aa compared to Mygalin 2.0 �g/�L.

Anticonvulsant Activity in NMDA Acute Chemical

Seizure Model

Glutamatergic receptors are closely involved in neuropathologies and are of considerable interest in the development of compounds that antagonize those types of receptors, particularly the NMDA receptor due to anticonvulsant effects [33].

The polyamine Mygalin in our study has shown to be a neuroactive molecule which protected the animals in the chemical convulsant model with NMDA. It was also demonstrated that Mygalin reduced the median seizure score

proximate to MK-801 scores. Also, Mygalin protected significantly the animals against tonic-clonic seizures, achieving 83.3% protection with the lowest dose. Previous studies with polyamines isolated from spiders in epilepsy model observed the protection of tonic-clonic seizures in DBA/2 mice [34].

Acypolyamines are small policationic structures that act on ion channels associated to selective ionic receptors [35]. Many neurotoxins isolated from spider and wasp venoms that are glutamatergic receptors antagonists have polyamine structure [10, 36, 9] once that they probably act blocking the

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channel as non-competitive ligants in sites inside the ionic channel after the agonist binding [14]. Our results strongly indicate that the bis-acypolyamine Mygalin has a plausible neuromodulation effect on L-Glu neurotransmission, possibly acting in NMDA receptor in antagonistic way. As an example of molecule that acts on NMDA receptors and which has drawn attention is the memantine. This drug is a clinically well tolerated uncompetitive NMDA receptor antagonist with strong voltage-dependency and rapid blocking/unblocking kinetics. It is approved by FDA and it is currently used mainly in the treatment of dementia, but is also studied as an alternative treatment for chronic pain, diskynesia, Parkinson’s disease, sclerosis, glaucoma, depression and has many other therapeutic applications. Memantine, which uncompetitively moderate affinity for NMDA receptor antagonist, could be useful in the treatment of several CNS disorders associated with disturbances in glutamatergic transmission. Its anticonvulsant effectiveness was demonstrated in several animal models of generalized seizures and data indicate that combination therapy might represent a promising therapeutic approach in epilepsy pharmacology treatment [37, 38]. In the present study, we observed a Mygalin inverted dose-response curve in NMDA anticonvulsant model, presenting a gradual reduction in the effect in 83.3, 66.7 and 50% as doses increased. This effect known as hormesis, is frequently encountered [39], and can possibly be explained by polyamine lack of selectivity to ionotropic receptors [15] as they have been found to modulate several neurotransmitter receptors, such as GABA and nicotinic receptors [40]. This lack of selectivity also possibly reflected a different activation of NMDA receptor subunits that (such as NR2) may control either the stimulatory as the inhibitory effect of espermine site at NMDA receptors [41]. Mygalin did not reduce latencies to the onset of tonic-clonic seizures in this model, and this was also observed in Jorotoxin, another spider-derived polyamine, when studied in mice in NMDA convulsant acute model [42]. Besides the fact that both studies did not observe difference in latency, both polyamines protected animals from tonic-clonic seizure.

Rotarod Performance, Exploratory Behavior and Spontaneous Locomotor Activity

Although MK-801 has an interesting anticonvulsant activity, it causes unpleasant side-effects such as ataxia and stereotyped behaviors [5]. Mygalin treatment altered rotarod performance, as 20 �g/�L Mygalin (47.96 nmols) caused 40% motor impairment, but this dose was one folder higher compared to the highest dose (2 �g/�L) in anticonvulsant assays. Comparatively, Jorotoxin, a widely studied polyamine, caused 100% ataxia in the animals when 47 nmols are injected i.c.v [42]. In Open Field test, it was observed that with 0.2 �g/�LMygalin treatment, animals remained more inactive, even more than diazepam-treated animals. But with the higher dose (2.0 �g/�L), a significant difference in inactivity was not detected. However, we observed no modification with Mygalin treatment on the exploratory behavior in locomotion

or elevation compared to vehicle, and animals were responsive to different stimulus. Therefore, it was assumed that Mygalin treatment did not present sedation as diazepam, in which group animals explored significant less the Open Field than 2.0 �g/�L Mygalin treatment. In previous studies with polyamines, such as studies with the Jorotoxin, polyamines usually caused an increase in motor activity and abnormal behavior such as limb stretching [42]. We have only observed what may possibly be called as an altered behavior an increased grooming. Polyamines, that usually present espermidine moiety in their structure, are linked to an arousal of stereotyped behaviors [43]. Although we observed some modifications in the neuroethologic models, these are considered mild when compared to other AEDs and other polyamines.

Morris Water Maze Performance

The NMDA receptor is involved in many important functions in brain, including neural plasticity, learning and memory [44]. Morris and co-workers [45] demonstrated that i.c.v injection of a competitive antagonist NMDA known as AP5 caused selective impairment in spatial learning in rats, correlated to the blockage of long-term potentiation (LTP) in hippocampus. Overall, although apparently NMDA competitive antagonists are effective as anticonvulsants and neuroprotectors, those compounds usually present neurobehavioral toxicity [46]. During Mygalin anticonvulsant tests, we detected that it was effective, mostly in NMDA acute chemical seizure model. Therefore, as it may present antagonic modulation in NMDA-induced seizure, we decided to investigate the behavior effects of Mygalin on MWM. We observed that, similar to NMDA antagonists, Mygalin might have caused mild spatial memory deficit in rats submitted to the aquatic maze. It was more evident in the lowest dose, which had the highest efficacy in protecting the animals from tonic-clonic seizures. It was statistically significant on the third day when we observed that the escape mean latency did not reduce as the control animals. In the probe trial it is observed that Mygalin-treated animals did not show preference for the platform quadrant, swimming in circles almost equally in all quadrants. Possibly, the rats did not use a cognitive mapping to find the hidden platform, also many of them, even having found the platform, did not stay for the 30 seconds – they often threw themselves again into the water. It was probably used procedural strategy in which they eventually found the platform not distinguishing it as unique place [47]. This lack of persistence of finding platform and the neglect of rest in it was first reported as an anxiety reduction in PTZ kindling model by Lamberty and Klitgaard [27].

Functional Implications

Our data are supported by other studies that report anticonvulsant polyamines in acute chemical seizure model. Other polyamines as Argiotoxin, Jorotoxin and fractions extracted from Agelenopis aperta spider, studied in pharmacological screenings, have demonstrated to be


effective in blocking or attenuating seizures in rats submitted to convulsant models [48-51].

The most successful strategies in developing anticonvulsant drugs are either enhancing the GABAergic transmission or reducing the glutamatergic transmission through ionic channel modulation [9, 52]. Complementary to that, the new perspective in anticonvulsant drugs is related to multiple action mechanism with good pharmacological profile [53].

CONCLUSIONS

This is the first neurobiological characterization of Mygalin. The present study demonstrated the interesting effect in protecting animals from tonic-clonic seizures in acute chemical convulsant models. The distinct effect on the assays suggests that it may be involved in NMDA receptor modulation, which is related to seizure induction and it also suggests that mild side-effects may be correlated to this modulation.

To conclude, Mygalin seems to be a valuable compound with interesting pharmacological activity and may have potential either as a tool in studying of animal models of epileptic seizures or as a lead in anticonvulsant drug development.

CONFLICT OF INTEREST

We assure this article has never been submitted or published elsewhere and the results obtained are showed with full approval of all authors. Therefore we declare no conflict of interest.

ACKNOWLEDGEMENTS

This work was supported by the Excellence Post-graduation Program from Brazilian National Council for Research (Capes-PROEX), São Paulo State Research Foundation (FAPESP) and also Brazilian National Research Council (CNPq) providing a scholarship to Ms. Lívea Dornela Godoy (Pibic nº116720/2009-3) and to José Luiz Liberato (nº 143019/2008-3). We also thank Mr. Alberth Carreño Gonzalez and Ms. Luisa Lange Canhos for a critical reading of this manuscript.

ABBREVIATIONS

AEDs = Anti-epileptic drugs

ANOVA = Analysis of variance

AP5 = (2R)-amino-5-phosphonovaleric acid

DBA/2 = Dilute Brown non Agouti 2

DZP = Diazepam

GABA = Gama-Aminobutyric acid

i.c.v. = Intracerebroventricular

i.p. = Intraperitonial

JSTX-3 = Jorotoxin-3

L-Glu = L-glutamate

LTD = Long-term depression

LTP = Long-term potentiation

MWM = Morris Water Maze

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Received: June 18, 2013 Revised: July 03, 2013 Accepted: July 03, 2013

Mygalin: a new anticonvulsant polyamine in acute seizure model and neuroethological schedule

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