Neuroscience Letters 543 (2013) 12–16
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Parawixin2, a novel non-selective GABA uptake inhibitor from Parawixia bistriata spider venom, inhibits pentylenetetrazole-induced chemical kindling in rats Erica A. Gelfuso a , José L. Liberato a,b , Alexandra O.S. Cunha a , Márcia R. Mortari c , Renê O. Beleboni d , Norberto P. Lopes e , Wagner F. dos Santos a,b,∗ a
Neurobiology and Venoms Laboratory, Department of Biology, College of Philosophy, Sciences and Literature of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil Neuroscience and Behavior Institute INeC, Ribeirão Preto, SP, Brazil c Institute of Biological Sciences, University of Brasilia, Brasilia, DF, Brazil d Department of Biotechnology, University of Ribeirão Preto, Ribeirão Preto, SP, Brazil e Organic Chemistry Laboratory, Department of Physics and Chemistry, College of Pharmaceutical Sciences of Ribeirão Preto, University of São Paulo, Ribeirão Preto, SP, Brazil b
h i g h l i g h t s • • • • •
We investigated the effects of Parawixin2 in kindling acquisition. We used sub-convulsive doses of PTZ until fully kindled animals were observed. We compared the effects of Parawixin2 with DZP and nipecotic acid. Repeated administration of Parawixin2 avoids kindling acquisition. GABA transport inhibition by Parawixin2 is strongly anticonvulsant.
a r t i c l e
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Article history: Received 10 October 2012 Received in revised form 5 February 2013 Accepted 24 February 2013 Keywords: Anticonvulsant GABA transporters Glycine transporters Kindling Parawixin2 Spider venom
a b s t r a c t The aims of the present work were to investigate the effects of the repeated administration of Parawixin2 (2-amino-5-ureidopentanamide; formerly FrPbAII), a novel GABA and glycine uptake inhibitor, in rats submitted to PTZ-induced kindling. Wistar rats were randomly divided in groups (n = 6–8) for different treatments. Systemic injections of PTZ were administered every 48 h in the dose of 33 mg/kg; i.p., that is sufficient to induce fully kindled seizures in saline i.c.v. treated rats in a short period of time (28 days). Treatments in two types of positive controls (diazepam – DZP and nipecotic acid – NA groups) consisted in daily systemic injections of DZP (2 mg/kg; i.p.) or i.c.v. injections of NA (12 g/L), while in experimental groups in daily i.c.v. injections of different doses of Parawixin2 (0.15; 0.075; 0.015 g/L). Seizures were analyzed using the Lamberty & Klitgaard score and kindling was considered as established after at least three consecutive seizures of score 4 or 5. Cumulative seizure scores for each group were analyzed using repeated measures of ANOVA followed by Tukey test. PTZ induced 4 and 5-score seizures after 12 injections in saline treated rats, whereas daily injection of Parawixin2 inhibited the onset of seizures in a dose dependent manner. Also, the challenging administration of PTZ did not raise seizure score in animals treated with the highest dose of Parawixin2 or those treated with DZP or NA. These findings together with previous data from our laboratory show that Parawixin2 could be a useful probe to design new antiepileptic drugs. © 2013 Elsevier Ireland Ltd. All rights reserved.
1. Introduction Epilepsy is a common and chronic neurological disorder that affects approximately 1% of the global population [3], which is characterized by recurrent and spontaneous seizure activity [16,24],
∗ Corresponding author at: FFCLRP/USP – Department of Biology, Av. Bandeirantes, 3900, Zip Code: 14040901, Ribeirão Preto, SP, Brazil. Tel.: +55 16 3602 3657; fax: +55 16 3602 4886. E-mail address:
[email protected] (W.F. dos Santos). 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2013.02.074
induced by the neuronal hyperactivity in the brain [18]. Kindling is an epilepsy functional model that allows the investigation of epilepsy-related behavioral, neurophysiological and neurochemical changes without gross morphological damage [18,21]. Kindling phenomenon may be described as the repeated stimulation of cerebral structures with sub-threshold electric currents or sub-convulsive doses of chemical convulsants, such as pentylenetetrazole (PTZ), which settle on the appearance and progressive intensification of convulsant activity, culminating in generalized seizures [12]. Moreover, the development of kindling leads to cellular alterations in specific brain areas, such as the hippocampus
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[19] resulting in impairment of learning and memory processes [14]. It has been proposed that kindling-induced epileptogenesis is based upon an enhanced activation of glutamatergic pathways, and/or attenuated activation in GABAergic pathways [18]. Furthermore, behavioral and biochemical data have shown that GABAergic inhibitory activity is reduced in the central nervous system (CNS) of kindled rats as well as in the brains of humans with many types of epilepsy [17,18]. In this context, GABA inhibitory effects may be enhanced either by direct activation of GABAergic receptors or by the blockade of GABA transporters (GATs) present in neuronal and glial membranes surrounding the synaptic cleft [5,23]. For this reason, GABA uptake inhibitors have been widely used as pharmacological tools to elucidate many aspects of neural processes in which GABA is involved. Furthermore, potent anticonvulsant effects were attributed to GAT inhibitors in many animal models of epilepsy, as those drugs prolong the inhibitory GABAergic activity [22,26]. Parawixin2 (2-amino-5-ureidopentanamide; formerly FrPbAII) was isolated from the venom of the spider Parawixia bistriata in the search for novel neuroactive molecules [2]. Parawixin2 is a potent anticonvulsant and anxiolytic agent with mild side-effects when administered intracerebrally in Wistar rats [10,15]. The primary mode of action of this compound is probably related to the inhibition of GABA and glycine re-uptakes, as shown with rat cortical and retina synaptosomes [2]. In terms of GABA neurotransmission and according to Gelfuso et al. [10], Parawixin2 exerts its action preferentially on GABA transporters GAT1 subtype, although it possibly acts on other subtypes, but with lower affinity. Although tested in a variety of acute seizure rat models [10,15], Parawixin2 was not previously studied in chronic models of epilepsy, and therefore, its efficacy as an antiepileptogenic or a disease-modifying drug remains to be analyzed. Based on these facts, the aim of the present work was to evaluate the effects of Parawixin2, a nonselective GABA and glycine uptake inhibitor on the development of rat kindling in comparison with Diazepam and nipecotic acid.
2. Materials and methods 2.1. Animals and surgery Male Wistar rats (200–250 g) obtained from the central vivarium at the University of São Paulo Campus of Ribeirão Preto were used in the assays. The animals were kept in pairs in cages under controlled experimental conditions (25 ◦ C; 50–60% humidity and a 12-h dark/light cycle with lights on at 7:00 a.m.) with free access to rat chow and water ad libitum, except during assays. One day after arrival, animals were anesthetized with sodium thiopental 40 mg/kg (Cristalia, Brazil) for stereotaxic implantation of a stainless steel guide cannula (10 mm) in the right lateral ventricle. The skull was exposed after a single injection of local anesthetics (xylocaine 3% with epinephrine 2%) and stereotaxic coordinates were marked as follows: 0.9 mm posterior to bregma, 1.6 mm lateral from midline and 3.4 mm ventral from the surface of the skull according to the Atlas of Paxinos and Watson [20]. Cannulae were fixed to the skulls of animals with dental acrylate and were sealed with a stainless steel wires to avoid obstruction. The animals were then allowed to rest for 5–7 days to recover from surgery. This work was approved by the Ethics Committee for Experimental Animals at the University Campus (protocol nr: 06.1.91.53.4) that follows the Brazilian Federal Law for Animal Experimentation (Law nr: 11.794/2008) and American Physiological Society and Ethical Guidelines for investigations of
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Fig. 1. Chemical structure of Parawixin2 (2-amino-5-ureidopentanamide) isolated from the Parawixia bistriata spider venom [2].
Experimental Pain in Conscious Animals. Also, every effort was made to avoid unnecessary stress and pain to the animals. 2.2. Parawixin2 and drugs Parawixin2 was obtained by fractionation of low molecular weight compounds of P. bistriata venom in HPLC as described in details by Beleboni et al. [2]. Molecular mass spectral analyses were performed on a Quattro-LC instrument from Micromass (Manchester, UK) and the primary structure of Parawixin2 was obtained by Magnetic Resonance (Fig. 1). The following drugs and concentrations were used in the experiments: nipecotic acid (12 g/animal, Sigma–Aldrich, USA) injected via intracerebroventricular (i.c.v.), diazepam (2 mg/kg, União Química, Brazil) and pentylenetetrazole (PTZ; 33 mg/kg, Sigma–Aldrich, USA) both injected via intraperitoneal (i.p.). All drugs were dissolved in sterile physiological saline (NaCl, 0.9%; w/v), which was used alone as negative control. 2.3. PTZ-induced kindling After recovery from surgery, PTZ administrations were initialized. Animals (n = 6–8, each group) were injected with a single dose of PTZ (33 mg/kg; i.p.) in every other day, in the morning period. Immediately after each injection, animals were observed for 30 min. Convulsive behaviors were scored according to Lamberty & Klitgaard [14] as follows: 0, no response; 1, ear and facial twitching; 2, myoclonic jerks without rearing; 3, myoclonic jerks with rearing; 4, turning over into side position, bilateral clonic–tonic seizures; 5, turning over into back position, generalized clonic and tonic seizures. Rats received PTZ injections until the observation of at least three consecutive seizures of score 4 or 5, when they were considered fully kindled. Cumulative kindling score, average of individual behavioral scores divided by the number of animals, was plotted against time in days. The following treatment groups were included in the analysis: • Group 1: Saline, i.c.v. (daily for 27 days) + PTZ, i.p. (alternated from 1st to 27th day). • Group 2: Diazepam, i.p. (daily for 27 days) + PTZ, i.p. (alternated from 1st to 27th day). • Group 3: Nipecotic acid, i.c.v. (daily for 27 days) + PTZ, i.p. (alternated from 1st to 27th day). • Groups 4–6: Parawixin2, i.c.v. (each dose – daily for 27 days) + PTZ, i.p. (alternated from 1st to 27th day). In the days of PTZ administration, Parawixin2, nipecotic acid and saline were injected 20 min prior to the convulsant, whereas DZP were injected 30 min before the administration of PTZ. One week after the last injection of PTZ, another administration of this convulsant was performed in all rats 15 min after Parawixin2 (0.037, 0.15 and 0.75 g/L) or nipecotic acid (12 g/L) or 30 min after DZP (2 mg/kg). This generally referred to as the challenge dose of
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Mean seizure score
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Fig. 3. Mean area under the curve of each treatment (AUC): (a) PTZ + saline (150 mM), (b) PTZ + DZP (2 mg/kg), (c) PTZ + nipecotic acid (12 g/L), (d) PTZ + Parawixin2 (0.15 g/L), (e) PTZ + Parawixin2 (0.075 g/L) and (f) PTZ + Parawixin2 (0.015 g/L). AUCs were calculated for each animal and then analyzed by one-way ANOVA followed by Tukey test, considering (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001.
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PTZ injections Fig. 2. Cumulative mean seizure score of rats treated with alternate sub-convulsive doses of PTZ. Animals were treated with daily injections of (A) PTZ + saline (0.9%; w/v), PTZ + DZP (2 mg/kg), PTZ + nipecotic acid (12 g/L) and (B) PTZ + Parawixin2 (0.15 g/L), PTZ + Parawixin2 (0.075 g/L), PTZ + Parawixin2 (0.015 g/L). Control animals did not receive PTZ injections (vehicle) and were excluded from the analysis, but were observed during experimental procedures. Repeated measures ANOVA followed by Tukey as the post-test considering significant (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001.
the convulsant and it is delivered to animals in order to check the permanence of the kindled state. 2.4. Statistical analysis Mean scores in each group were submitted to repeated measures analysis of variance (ANOVA). In case of significant effects of treatments, one-way ANOVAs followed by Tukey test, at each time interval, were performed (SPSS for Windows, version 13.0; USA). Furthermore, areas under the curves (AUCs) were compared using one-way ANOVA followed by Tukey test.
Daily administration of Parawixin2 induced a dose-dependent anticonvulsant effect. In this regard, significant differences between PTZ + saline group and the highest dose of Parawixin2 were observed at the 6th day of PTZ administration, whereas rats treated with the intermediate dose of Parawixin2 (0.075 g/L) exhibited lower seizure score after 8–13th PTZ injections (p < 0.05). Similar results were found for both nipecotic acid and DZP, decreasing seizure score at the 6th PTZ injection and at 7th injection, respectively. Also, at the 8th injection of PTZ, seizure mean score of rats treated with nipecotic acid, DZP, 0.015 and 0.075 g/L Parawixin2 were reduced (p < 0.05). The analysis of AUCs of all treatments revealed that animals pre-treated with Parawixin2 (0.15 g/L), DZP or nipecotic acid had significantly lower AUCs then PTZ + saline injected animals or the lowest dose of Parawixin2 [F(5,29) = 20.23; p < 0.001] (Fig. 3). Finally, the analysis of mean seizure score of rats after the challenge administration of PTZ before anticonvulsant administration showed that none of the two lower doses of Parawixin2 (0.075 and 0.015 g/L) inhibited seizure onset. In contrast, we observed that rats treated with Parawixin2 (0.15 g/L), DZP and nipecotic acid did not present convulsive seizures [F(5,29) = 41.67; p < 0.001] (Fig. 4).
3. Results Chronic administration of a sub-convulsive dose of PTZ (33 mg/kg, i.p.) on alternate days for a period of 27 days progressively increased seizures score, producing kindling in PTZ + saline-treated rats (Fig. 2A and B). In this case, the first myoclonic jerks (score 2 seizures) were observed after the 3rd administration, and kindling criteria were reached after 12 injections. DZP, nipecotic acid and the highest dose of the Parawixin2 (0.15 g/L) suppressed the progression of PTZ kindling (Fig. 2A and B). Statistical analyses revealed significant effects of treatment [F(5,9) = 21.083; p < 0.001], number of injections [F(13,17) = 22.765; p < 0.0001], and treatment-versus-number of injections interaction [F(65,77) = 3.328; p < 0.001]. One-way ANOVA analyses showed a significant effect of treatments in all analyzed periods [F(5,29) varying from 2.715 to 30.296; p < 0.05]. Post hoc analysis indicated that rats treated with Parawixin2 (0.15 g/L), DZP and nipecotic acid had lower seizure score in all assessed periods as compared with PTZ + saline injected group and the lowest dose of Parawixin2 (0.015 g/L) (p < 0.05).
Fig. 4. Mean seizure score of rats treated with the challenging administration of PTZ sub-convulsive dose. Animals were treated with (a) PTZ + saline (150 mM), (b) PTZ + DZP (2 mg/kg), (c) PTZ + nipecotic acid (12 g/L), (d) PTZ + Parawixin2 (0.15 g/L), (e) PTZ + Parawixin2 (0.075 g/L) and (f) PTZ + Parawixin2 (0.015 g/L). Data were analyzed by one way ANOVA followed by Tukey as the post-test considering significant (**) p < 0.01 and (***) p < 0.001.
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4. Discussion We have identified Parawixin2, from the venom of the spider P. bistriata, which inhibits both GABA and glycine uptakes in synaptosomes from rat cerebral cortices and retina [2]. Previous works showed that i.c.v. administration of Parawixin2 induced marked anticonvulsant effects against several models of chemically induced acute seizures [10,15]. In this work we have analyzed the effects of chronic administration of Parawixin2 in the progression of PTZ-induced kindling, and we demonstrated that Parawixin2 effectively blocked behavioral expression of these seizures. Development of PTZ-induced kindling appears to involve changes in inhibitory transmission as previous studies show strong association of seizures with loss of GABAergic neurons and decrease in the number of GABAA receptor binding sites [8,13]. GABAA receptor agonists phenobarbital and DZP suppress the development of kindling, whereas the effects of GABAB receptor agonists are dependent on age and dose [25]. Paradoxically, the blockade of GABAB receptor, delays the onset of seizures and ameliorate cognitive and memory impairments followed by PTZ kindling [11]. To date, four subtypes of GATs have been cloned, GAT 1–4, of which GAT-1 is the most predominant in brain neurons [4]. The expression of transporter subtypes has been investigated. According to Dalby and Nielsen [7], GABA transporters may exert different roles in the kindling-induced epileptogenesis. Specific inhibitors of GAT-1, such as tiagabine and nipecotic acid suppress seizures of animals submitted to focal kindling, secondarily generalized seizures and clonic seizures, but they are ineffective against tonic convulsions induced by electroshock [7]. Conversely, GAT non-specific inhibitors block convulsions induced by electroshock, but they are ineffective against clonic convulsions induced by chemical convulsants and their effectiveness in focal kindling seizures is apparently proportional to the ratio between affinities for GAT-1/GAT-3 [6]. The effects of Parawixin2 on kindling progression could be attributed to the blockade of GABAergic transporters, which is thought to be the main mode of action of Parawixin2 [2]. The blockade of GABA transporters may lead to an increase in the concentration of the released GABA in the synaptic cleft and thus, a prolonged inhibitory effect. The advantage of using GABA transport inhibitors as a therapeutic strategy is that these agents may cause less untoward side-effects, since they act on a concentration of GABA released under physiological conditions [1,9]. In order to compare the effectiveness of Parawixin2, we chose to use two drugs that enhance GABAergic inhibitory effects, diazepam and nipecotic acid. In this regard, we showed that Parawixin2 blocked seizure progression in 100% of rats treated with 0.15 g/L, approximately 0.14 nmol, whereas the dose of nipecotic acid needed was 12 g/L, approximately 46.5 nmol a far higher dose. This difference in efficiency was previously reported by Liberato et al. [15] in rats submitted to limbic seizures elicited by the GABAergic blockade of the Area tempestas in the deep pyriform cortex. According to these authors the microinjection of nipecotic acid into the Substantia nigra pars reticulata does not suppress limbic seizures elicited by the microinjection of GABA antagonists into the A. tempestas. In contrast, Parawixin2 efficiently blocks these seizures. Moreover, Parawixin2 blocks chemically induced seizures, whereas, non-specific GABA transport inhibitors fail [10]. The present findings, together with previous data, might corroborate with the initial hypothesis of a non-selective action of Parawixin2 over GABA transporters, although it might preferentially block GAT1 subtype. Therefore, our main hypothesis is that, since our administration of Parawixin2 was performed in via i.c.v. and GAT1 subtype is widely expressed in the limbic structures that generate kindling behaviors, our molecule is preferentially blocking these types of transporters and increasing GABA concentration in the synaptic cleft. Another point is the action of Parawixin2 on
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glycine transporters [2]. Since Parawixin2 is also a glycine uptake inhibitor [2] and glycine is recognized as an important inhibitory neurotransmitter, an implication of glycine uptake inhibition on anticonvulsant effects of Parawixin2 cannot be excluded here. To our knowledge there are no reports on drugs with these modes of action, leaving Parawixin2 as a unique compound with in vivo activity as anticonvulsant with potential disease modifying effects. 5. Conclusion In this work we showed that Parawixin2 is an alternative tool that can be used as a probe to design novel therapeutic agents, since it blocks the establishment of kindling phenomenon. In this same context, we conclude that the blockade of GABA transporters may represent a rational strategy to prevent neuronal disorders which involve alterations in GABAergic function. Conflicts of interest The authors declare no conflicts of interest. Acknowledgements This work was supported by FAPESP (São Paulo Research Foundation, scholarship process nr: 04/14151-1; E.A.G.), CAPES and CNPq from Brazilian government. Authors are grateful to Mr Amauri Ramos Pinhal and Jose Carlos Tomaz for their technical assistance. References [1] K.E. Andersen, J. Lau, B.F. Lundt, H. Petersen, P.O. Hunsfeldt, P.D. Suzdak, M.D.B. Swedberg, Synthesis of novel GABA uptake inhibitors. Part 6: Preparation and evaluation of N- asymmetrically substituted nipecotic acid derivatives, Bioorg. Med. Chem. 9 (2001) 2773–2785. [2] R.O. Beleboni, R. Guizzo, A.C. Fontana, A.B. Pizzo, R.O.G. Carolino, L. Gobbo-Neto, N.P. Loppes, J. Coutinho-Netto, W.F. Santos, Neurochemical characterization of a neuroprotective compound from Parawixia bistriata spider venom that inhibits synaptosomal uptake of GABA and glycine, Mol. Pharmacol. 69 (2006) 1998–2006. [3] D.E. Blum, New drugs for persons with epilepsy, Adv. Neurol. 76 (1998) 57–81. [4] M.A.R.C. Daemen, G. Hoogland, J.M. Cijntje, G.H. Spincemaille, Upregulation of the GABA-transporter GAT-1 in the spinal cord contributes to pain behavior in experimental neuropathy, Neurosci. Lett. 444 (2008) 112–115. [5] N.O. Dalby, GABA-level increasing and anticonvulsant effects of three different GABA uptake inhibitors, Neuropharmacology 39 (2000) 2399–2407. [6] N.O. Dalby, Inhibition of ␥-aminobutyric acid uptake: anatomy, physiology and effects against epileptic seizures, Eur. J. Pharmacol. 479 (2003) 127–137. [7] N.O. Dalby, E.B. Nielsen, Comparison of the preclinical anticonvulsant profiles of tiagabine, lamotrigine, gabapentin and vigabatrin, Epilepsy Res. 28 (1997) 63–72. [8] P. Follesa, A. Tarantino, S. Floris, A. Mallei, S. Porta, S. Tuligi, E. Cagetti, M. Caddeo, A. Mura, M. Serra, G. Biggio, Changes in the gene expression of GABA receptor subunit mRNAs in the septum of rats subjected to pentylenetetrazole-induced kindling, Mol. Brain Res. 70 (1999) 1–8. [9] A. Gadea, A.M. Lopez-Colome, Glial transporters for glutamate, glycine, and GABA III GABA transporters, J. Neurosci. Res. 64 (2001) 461–468. [10] E.A. Gelfuso, A.O.S. Cunha, M.R. Mortari, J.L. Liberato, K.H. Paraventi, R.O. Beleboni, J. Coutinho-Netto, N.P. Lopes, W.F. Santos, Neuropharmacological profile of FrPbAII, purified from the venom of the social spider Parawixia bistriata (Araneae, Araneidae), in Wistar rats, Life Sci. 80 (2007) 566–572. [11] D. Getova, W. Froestl, N.G. Bowery, Effects of GABA receptor antagonism on the development of pentylenetetrazole-induced kindling in mice, Brain Res. 809 (1998) 182–188. [12] G.V. Goddard, Development of epileptic seizures through brain stimulation at low intensity, Nature 214 (1967) 1020–1021. [13] D. Kondziella, A. Bidar, B. Urfjell, O. Sletvold, U. Sonnewald, The pentylenetetrazole-kindling model of epilepsy in SAMP8 mice: behavior and metabolism, Neurochem. Int. 40 (2002) 413–418. [14] Y. Lamberty, H. Klitgaard, Consequences of pentylenotetrazole-induced kindling on spational memory and emotional responding in the rat, Epilepsy Behav. 1 (2000) 256–261.
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