Activation of adenosine receptors modulates the efflux transporters in brain capillaries and restores the anticonvulsant effect of carbamazepine in carbamazepine resistant rats developed by window-pentylenetetrazole kindling

Journal Pre-proofs Research report Activation of adenosine receptors modulates the efflux transporters in brain capillaries and restores the anticonvulsant effect of carbamazepine in carbamazepine resistant rats developed by window-pentylenetetrazole kindling C. Zavala-Tecuapetla, S. Orozco-Suarez, J. Manjarrez, M. Cuellar-Herrera, A. Vega-Garcia, V. Buzoianu-Anguiano PII: DOI: Reference:

S0006-8993(19)30570-0 https://doi.org/10.1016/j.brainres.2019.146516 BRES 146516

To appear in:

Brain Research

Received Date: Revised Date: Accepted Date:

5 April 2019 15 October 2019 17 October 2019

Please cite this article as: C. Zavala-Tecuapetla, S. Orozco-Suarez, J. Manjarrez, M. Cuellar-Herrera, A. VegaGarcia, V. Buzoianu-Anguiano, Activation of adenosine receptors modulates the efflux transporters in brain capillaries and restores the anticonvulsant effect of carbamazepine in carbamazepine resistant rats developed by window-pentylenetetrazole kindling, Brain Research (2019), doi: https://doi.org/10.1016/j.brainres.2019.146516

This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

© 2019 Published by Elsevier B.V.

Activation of adenosine receptors modulates the efflux transporters in brain capillaries and restores the anticonvulsant effect of carbamazepine in carbamazepine resistant rats developed by window-pentylenetetrazole kindling. Zavala-Tecuapetla Ca*, Orozco-Suarez Sb, Manjarrez Ja, Cuellar-Herrera Mc, VegaGarcia Ab,d, Buzoianu-Anguiano Vb.

a

Laboratory of Physiology of Reticular Formation, National Institute of Neurology

and Neurosurgery. Insurgentes Sur 3877, La Fama, 14269, Mexico City, Mexico. b

Medical Research Unit in Neurological Diseases, Specialty Hospital, National

Medical Center XXI Century, IMSS. Cuauhtemoc 330, Doctores, 06720, Mexico City, Mexico. c

Epilepsy Clinic, Hospital General de México Dr. Eduardo Liceaga. Dr. Balmis 148,

Doctores, 06720, Mexico City, Mexico. d

Department of Physiology, Faculty of Medicine, National Autonomous University

of Mexico, Av. Universidad 3000, C.U., 04510, Mexico City, Mexico.

*Corresponding author: Zavala-Tecuapetla C. Laboratory of Physiology of Reticular Formation, National Institute of Neurology and Neurosurgery. Insurgentes Sur 3877, La Fama, 14269, Mexico City, Mexico. E-mail address: [email protected]

Abstract Up-regulation of efflux transporters in brain capillaries may lead to the decreased therapeutic efficacy of antiepileptic drugs in patients with Drug Resistant Epilepsy. Adenosine receptor activation in brain capillaries can modulate blood-brain barrier permeability by decreasing the protein levels and function of efflux transporters. Therefore, we aimed to investigate whether the activation of adenosine receptors improves convulsions outcome in carbamazepine (CBZ) resistant animals and modulates the protein levels of efflux transporters (P-GP, MRP1, MRP2) in brain capillaries. We employed the window-pentylenetetrazol (PTZ) kindling model to develop CBZ resistant rats by CBZ administration during the post-kindling phase, and tested if these animals displayed subsequent resistance to other antiepileptic drugs. Crucially, we investigated if the administration of a broad-spectrum adenosine agonist (NECA) improves convulsions control in CBZ resistant rats. Of potential therapeutic relevance, in CBZ resistant rats NECA restored the anticonvulsant effect of CBZ. We also evaluated how the resistance to CBZ and the activation of adenosine receptors with NECA affect protein levels of efflux transporters in brain capillaries, as quantified by western blot. While CBZ resistance was associated with the up-regulation of both P-GP/MRP2 in brain capillaries, with the administration of NECA in CBZ resistant rats, we observed a decrease of P-GP and an increase of MRP2 levels, in brain capillaries. Since the activation of adenosine receptors improves the outcome of convulsions probably through the modulation of the efflux transporters protein levels in brain capillaries,

adenosine agonists could be useful as an adjunct therapy for the control of Drug Resistant Epilepsy.

Keywords Drug Resistant Epilepsy; Pentylenetetrazole; Carbamazepine; Brain capillaries; Adenosine receptors; NECA.

Abbreviations PTZ,

pentylenetetrazole;

CBZ,

carbamazepine;

P-GP,

P-glycoprotein;

MRP1/MRP2, Multidrug resistance-associated proteins; A1R, adenosine A1 receptors; A2AR, adenosine A2A receptors.

1. Introduction Temporal Lobe Epilepsy is the most common form of human epilepsy (Engel, 2001) and 20-40% of all cases are resistant to antiepileptic drugs therapy (Kwan and Sander, 2004). Efforts have been made to develop preclinical approaches to model Drug-Resistant Epilepsy to establish the mechanisms and to develop new therapeutic strategies. Animal models, such as the kindling model (a chronic model of Temporal Lobe Epilepsy), have been used to study Drug-Resistant Epilepsy (e.g., phenytoin- or lamotrigine-resistant kindled rats; Löscher, 2011; Löscher and Rundfeldt, 1991; Srivastava and White, 2013).

Classical kindling is a gradual development of seizures in response to a previously subconvulsant stimulus administered in a repeated and intermittent fashion culminating in permanently enhanced seizure susceptibility (Goddard et al. 1969). Recent results in rats show a modified protocol named window-pentylenetetrazole (PTZ) kindling that reduces the number of PTZ administrations necessary to increase brain susceptibility to seizures and establish the fully kindled state (Davoudi et al. 2013). By taking advantage of the smaller number of PTZ injections necessary to develop fully kindled rats, it could be possible to develop drugresistant seizures in these animals immediately after kindling. It has been shown that drug-resistant seizures develop following single exposure to sodium channel blockers (e.g., carbamazepine, CBZ) during the post-kindling phase (Srivastava et al. 2013). Together, both approaches provide a simple and practical animal model that facilitates the study of Drug-Resistant Epilepsy. Of the potential mechanisms underlying Drug-Resistant Epilepsy, the transporter hypothesis is the most studied and establishes that drug-resistance is the result of the up-regulation of efflux transporter proteins in the brain capillaries of the bloodbrain barrier, which prevent drug entry to the brain by actively extruding them from their target location (Sisodiya et al. 2002). Among identified efflux transporters implicated in Drug-Resistant Epilepsy are the ATP-binding cassette (ABC) transporters, specifically P-glycoprotein (P-GP), and the multidrug resistanceassociated proteins (MRP1, MRP2; Aronica et al. 2004; Kwan and Brodie, 2005; Sun et al. 2016; Tishler et al. 1995). Therefore, the search to develop new

therapeutic strategies to downregulate the protein levels of efflux transporters is a rational approach to solve drug-resistance in epilepsy. Adenosine is a purine nucleoside that exerts its actions through four different subtypes of receptors (A1R, A2AR, A2BR and A3R), which are seventransmembrane G-protein-coupled receptors (Fredholm et al. 2011), with a higher density of A1R and A2AR in the brain (Fredholm et al. 2005). Recent findings indicate that activation of adenosine receptors in brain capillaries, either by broad spectrum or selective agonists (mainly A1R or A2AR), decreases P-GP protein levels and function in a time-dependent and reversible manner, both in vitro and in vivo (Kim and Bynoe, 2016), thus modulating blood-brain barrier permeability. In this way, adenosine receptors activation in brain capillaries can be exploited as a new strategy to deliver therapeutic drugs (e.g., antiepileptic drugs) into the brain and have the potential to treat Drug-Resistant Epilepsy, since upregulation of efflux transporters protein levels (like P-GP, MRP1, MRP2) in brain capillaries is an underlying potential mechanism. Therefore, we hypothesize that both approaches -the window-PTZ kindling and post-kindling CBZ administration- might develop CBZ resistant animals and that the activation of adenosine receptors might improve the control of convulsions in CBZ resistant animals probably through the modulation of protein levels of efflux transporters (P-GP/MRPs) in brain capillaries.

2. Results

2.1 Development of fully kindled rats and carbamazepine resistance After administrations of PTZ (35 mg/kg) during the development of the window-PTZ kindling, the rats were more susceptible to convulsions as noted by the progression of convulsive score from stage 1-3 to stage 4-5 convulsions (Fig. 1A). In the kindled group, during the initial four injections the animals presented a maximum mean convulsive score of 2.8 ± 0.16 (Fig. 1A). After a period of no injections (ten sessions), the animals reached a stage 4 or 5 convulsions in response to the last three PTZ-injections, achieving the fully kindled state (convulsive score 4.7 ± 0.07). No-kindled animals presented a maximum mean convulsive score of 1.4 ± 0.10 during the initial four PTZ injections and only presented stage 1-2 convulsions (convulsive score 2.0 ± 0.14) during the windowPTZ kindling developing (Fig. 1A). From a total of 163 animals, 76 rats (47%) were kindled, 57 rats (35%) were nokindled and 30 rats (18%) died during kindling development. Two days (2d) after the last kindling stimulation, carbamazepine administration (CBZ, 40 mg/kg) significantly reduced the convulsive score from 4.5 ± 0.10 to 2.7 ± 0.10 (p < 0.001) in the kindled rats (Fig. 1B). With a second dose of CBZ, 7 days later (day 9), these same rats that previously responded to the anticonvulsant effect of CBZ, were now resistant to the same dose of CBZ (convulsive score 4.2 ± 0.08; Fig. 1B). One week later, we administered a third dose of CBZ (day 16) to verify if resistance to CBZ was permanent in the rats. We observed the same effect as with the second administration (convulsive score 4.3 ± 0.09; Fig. 1B).

Stage 4/5 convulsions were observed in 100% of animals (58/58) when they received a second (9 d) and third (16 d) injection of CBZ compared to animals (58/58) that were protected at day 2 when they only displayed stage 2 convulsions (Fig. 1C). These animals were considered to be CBZ resistant. CBZ administrations (2-9-16 d) did not significantly modify the stage 2 (Fig. 1D) or stage 4/5 latencies (Fig. 1E), nor stage 4/5 convulsions duration with respect to kindling state (Fig. 1F). In contrast, when vehicle (0.3% DMSO) was administered two days after the last kindling stimulation, it did not modify the convulsive score of the kindled rats (2d; 4.3 ± 0.21; Fig. 1G). When CBZ (40 mg/kg) was administered to these animals on days 9 and 16, the convulsive score was reduced (2.8 ± 0.17 and 2.2 ± 0.54, respectively; p < 0.003; Fig. 1G).

2.2 Effect of administration of antiepileptic drugs on the control of convulsions in CBZ resistant rats In an effort to assess whether CBZ resistant rats would respond to a second antiepileptic drug, they were treated with one of the following drugs: phenytoin (75 mg/kg i.p.), valproic acid (250 mg/kg i.p.) or phenobarbital (25 mg/kg i.p.). Treatment with phenytoin failed to block stage 4/5 convulsions in CBZ resistant rats in response to PTZ administration (Fig. 2A). Phenytoin did not significantly modify the convulsive score (23 d; 4.3 ± 0.18) with respect to CBZ resistant condition (16 d; 4.4 ± 0.20), where 100% of rats (7/7) developed stage 4/5

convulsions (Fig. 2A). Phenytoin did manage to significantly increase the stage 2 convulsions latency (23 d; 141.4 ± 16.39 sec) with respect to the CBZ resistant condition (16 d; 79.9 ± 6.04 sec; p < 0.008; Fig. 2B), but not the latency or duration of stage 4/5 convulsions (Fig. 2C-D). On the other hand, valproic acid effectively blocked the stage 4/5 convulsions in CBZ resistant rats (7/7; Fig. 2A-D). Only 29% of rats (2/7) developed just stage 2 convulsions, where valproic acid significantly reduced the convulsive score (23 d; 0.6 ± 0.37) with respect to the CBZ resistant condition (16 d; 4.4 ± 0.20; p < 0.001; Fig. 2A). Valproic acid did not significantly modify the stage 2 convulsions latency in these animals (Fig. 2B). Finally, phenobarbital not only blocked the stage 4/5 convulsions (6/6) (Fig. 2C-D) but also increased the latency to stage 2 convulsions in CBZ resistant rats (Fig. 2A-B). Phenobarbital significantly reduced the convulsive score (23 d; 2.0 ± 0.63) with respect to CBZ resistant condition (16 d; 4.2 ± 0.17; p < 0.03), where only 67% of rats (4/6) developed stage 2 convulsions (Fig. 2A). Phenobarbital increased the stage 2 convulsions latency (23 d; 445.8 ± 114.6 sec) with respect to the CBZ resistant condition (16 d; 96.0 ± 13.64 sec; p < 0.03; Fig. 2B).

2.3 Protein levels of efflux transporters in rat brain capillaries Before starting with Western blot analysis for quantification of protein levels of efflux transporters, we aimed to confirm P-GP/MRPs and their colocalization with the endothelial cell markers (Giannotta et al. 2013; Park et al. 2009) in each

sample of our capillary preparations (see supporting information, Fig. S1). Also, we provided representative images from immunofluorescence staining of rat brain capillaries that support the results obtained with western blot quantification of the changes of protein levels of efflux transporters (P-GP, MRP1, MRP2), in most of our experimental groups (Fig. 3A).

2.3.1 Changes in protein levels of efflux transporters in brain capillaries from kindled and CBZ resistant rats P-GP. Western blot analysis revealed that the treatment with CBZ significantly decreased P-GP protein levels (28%; 0.72 ± 0.01, p < 0.001) compared to basal levels in the control group (Fig. 3A, B). In the kindled group, P-GP protein levels were increased (27%; 1.27 ± 0.05, p < 0.001) with respect to basal levels (Fig. 3A, B), suggesting that it occurs probably as a result of an epileptogenic process. This increase was also observed in the CBZ resistant group (33%; 1.33 ± 0.03, p < 0.001), but it was not greater than that observed in the kindled group (1.27 ± 0.05) (Fig. 3A, B). MRP1. No significant changes were observed in MRP1 protein levels in the CBZ group (0.86 ± 0.02) (Fig. 3A, C). In the kindled group, MRP1 was significantly lower (28%; 0.72 ± 0.03, p < 0.001) compared to the control group (Fig. 3A, C), and an even greater decrease in the basal protein levels was observed in the CBZ resistant group (80%, 0.20 ± 0.08, p < 0.001), a decrease that was also significant with respect to the kindled group (52%, 0.72 ± 0.03, p < 0.001) (Fig. 3A, C).

MRP2. With respect to MRP2 protein levels, there was no significant change in either CBZ (0.70 ± 0.09) or kindled (1.21 ± 0.22) groups compared to the basal levels (Fig. 3A, D). However, in the CBZ resistant group the MRP2 protein levels increased 48% (1.48 ± 0.05, p < 0.001) compared to the control group, but it was not greater than in the kindled group levels (1.21 ± 0.22) (Fig. 3A, D).

2.3.2. Changes in protein levels of efflux transporters in rat brain capillaries generated by NECA administration P-GP. Treatment with NECA significantly decreased the basal protein levels of PGP at 4 h (NECA-4, 55%; 0.45 ± 0.09, p < 0.001) and 8 h (NECA-8, 80%; 0.20 ± 0.05, p < 0.001) post-treatment, but in the NECA-8 group this decrease was even greater than that observed in the NECA-4 group (25%, p < 0.001) (Fig. 3A, B). These effects seemed to depend on the time of exposure to NECA (Fig. 3A, B). In the CBZ resistant+NECA-8 group, NECA decreased the P-GP protein levels by 33% (0.67 ± 0.04, p < 0.001) with respect to the control group (Fig. 3A, B), by 60% respect to the kindled group (1.27 ± 0.05, p < 0.001) and by 66% compared to the CBZ resistant group (1.33 ± 0.03, p < 0.001) (Fig. 3A, B). MRP1. NECA decreased basal MRP1 protein levels in the NECA-4 (73%; 0.27 ± 0.03, p < 0.001) and NECA-8 (58%; 0.42 ± 0.01, p < 0.001) groups (Fig. 3A, C), but no differences were observed between these groups. In the CBZ resistant+NECA-8 group, there was also a decrease in the basal protein levels (73%; 0.27 ± 0.07, p < 0.001) and by 45% respect to the kindled group (0.72 ±

0.03, p < 0.001), but this decrease was not greater than that observed in the CBZ resistant group (0.20 ± 0.08) (Fig. 3A, C). MRP2. Although there was no significant change in MRP2 protein levels in the NECA-4 group (0.96 ± 0.03) with respect to basal levels (Fig. 3A, D), the NECA-8 group showed a significant increase (160%; 2.60 ± 0.14, p < 0.001) compared to both control and NECA-4 groups, suggesting that a time dependent effect of NECA may exist (Fig. 3A, D). In the CBZ resistant+NECA-8 group, the basal MRP2 protein levels increased (110%; 2.10 ± 0.04, p < 0.001). This increase was also observed with respect to kindled (89%; 1.21 ± 0.22, p < 0.001) and CBZ resistant (62%; 1.48 ± 0.05, p < 0.001) groups (Fig. 3A, D).

2.4 Effect of NECA on the control of convulsions in CBZ resistant rats In CBZ resistant rats, the acute treatment with NECA+CBZ (23d; Fig. 4) was effective not only in blocking stage 4/5 convulsions in 100% of animals (8/8) but also in increasing stage 2 convulsions latency in CBZ resistant rats (5/8) in response to PTZ stimulation (Fig. 4A-D). The convulsive score was significantly reduced in 63% of animals (5/8) in response to PTZ stimulation, showing a mean score from 4.1 ± 0.13 (16 d) to 1.9 ± 0.55 (23 d), respectively (p < 0.001; Fig. 4A). The stage 2 latency increased from 69.9 ± 5.68 sec (16 d) to 132.4 ± 26.32 sec (23 d; p < 0.02; Fig. 4B). Treatment of CBZ resistant rats with NECA only (23d), did not modify the convulsive score (23 d; 4.1 ± 0.17), allowing stage 4/5 convulsions in CBZ resistant

rats (6/6) in response to PTZ stimulation (Fig. 4A). However, NECA did manage to increase both the stage 2 convulsions latency from 71.8 ± 4.67 sec (16 d) to 185.7 ± 40.90 sec (23 d; p < 0.03) and to stage 4/5 convulsions latency from 133.8 ± 40.75 sec (16 d) to 414.0 ± 60.45 sec (23 d; p < 0.04; Fig. 4B-C), although it did not alter the stage 4/5 duration (Fig. 4D). On the other hand, the treatment of CBZ resistant rats with DPCPX+NECA (23 d) did not result in any change of the convulsive score, latency or duration of convulsions, in response to PTZ stimulation (Fig. 4A-D), suggesting a probable participation of A1R on the behavioral effect observed only with NECA administration. Finally, the treatment of CBZ resistant rats with vehicle+CBZ, did not result in any change in convulsive score, latency or duration of convulsions, in response to PTZ stimulation (Fig. 4A-D).

3. Discussion This study is the first to investigate how the modulation of adenosine receptors over protein levels of efflux transporters in brain capillaries could have a potential therapeutic value in the treatment of Drug Resistant Epilepsy. Here, we used the window-PTZ kindling as a model to generate CBZ resistant rats. The window-PTZ kindling has the advantage of reducing the number of PTZ injections necessary to increase brain susceptibility and establish the fully kindled state in rats (Davoudi et al. 2013) in contrast to classical kindling (Goddard, 1983).

It is possible to generate CBZ resistance immediately following kindling in these animals, as we observe it from the ninth post-kindling day (9 d) and the CBZ resistance was maintained a week later (16 d), indicating a permanent effect. This drug-resistance was extended to phenytoin (23 d), another sodium channel blocker, indicating that this drug resistance extends to other drugs with a similar mechanism of action (Srivastava and White, 2013). On the other hand, antiepileptic drugs with different mechanisms of action, such as valproic acid (with the broadest range of activity being mainly GABA potentiation and glutamate/NMDA inhibition; Loscher, 2002; Rogawski and Porter 1990) and phenobarbital (which increases the chloride channel open time on GABAA receptors; Loscher and Rogawski, 2012; Macdonald and Olsen, 1994), continue to exert their anticonvulsant effect in CBZ resistant rats. With respect to protein levels of efflux transporters, we observed similar changes as those observed with the classical kindling. There were increased protein levels of PGP and MRP2 in brain capillaries from kindled animals, as observed in the epileptic brain from kindled animals generated by classical kindling (Jing et al. 2010; Liu et al. 2007; van Vliet et al. 2005; Volk et al. 2004; Yao et al. 2012), showing that a few administrations of PTZ results in a similar effect as an upregulation of P-GP/MRP2 protein levels. CBZ resistant animals also showed increased P-GP/MRP2 protein levels in brain capillaries, which coincides with previous results in animal models (Enrique et al. 2017; Volk and Loscher, 2005) and brain tissue of patients with Drug Resistant Epilepsy (Aronica et al. 2004; Dombrowski et al. 2001; Liu et al. 2012; Sisodiya et

al. 2002; Tishler et al. 1995). Up-regulation of P-GP/MRP2 protein levels is probably one of the mechanisms of resistance to CBZ since it is known that it results from activation of the glutamate/NMDA receptor/cyclooxygenase-2 (COX-2) signaling pathway due to high glutamate release induced by epileptic seizures (Bankstahl et al. 2008; Bauer et al. 2008; Luna-Munguia et al. 2015; Zibell et al. 2009). In this study, we found a clear decrease of protein levels of MRP1 in brain capillaries from both kindled and CBZ resistant animals. This is contrary to previous studies that show up-regulation of MRP1 in both brain tissue of epileptic patients (Aronica et al. 2004; Sun et al. 2016) and in conditions of Drug Resistant Epilepsy (Chen et al. 2013; Sisodiya et al. 2001; Wang et al. 2015). In this way, CBZ resistance can be associated with alterations in the protein levels of efflux transporters in rat brain capillaries from the blood-brain barrier, which could affect the brain uptake and extrusion of CBZ and therefore its effectiveness. Some studies support that CBZ might be a substrate for P-GP and/or MRPs (Clinckers et al. 2005; Potschka et al. 2001; Rambeck et al. 2006; Rizzi et al. 2002), while others argue otherwise (Baltes et al. 2007; Luna-Tortós et al. 2008, 2010; Owen et al. 2001). Considering both in vivo and in vitro information, it has been suggested that CBZ is “possibly” transported by P-GP (Zhang et al. 2012). In addition, the major stable active metabolite of CBZ, the carbamazepine-10,11epoxide (Winnicka et al. 2002) has been shown to be a P-GP substrate, suggesting that resistance to CBZ may be attributed to an increased efflux function of P-GP of this metabolite (Zhang et al. 2011).

On the other hand, under physiological conditions, we also found a decrease in PGP protein levels 4 hours after NECA administration, in rat brain capillaries. This decrease was substantial when evaluated after 8 hours, demonstrating that NECA modulation seems to be time dependent. Our findings are in line with the evidence by Kim and Bynoe (2016), who demonstrated that NECA induced a gradual decrease in P-GP protein levels beginning at 2 hours, an effect that was maintained for up to 18 hours, in wild type mouse brains. Additionally, a decrease in MRP1 protein levels was observed and maintained for up to 8 hours, while MRP2 protein levels were increased in a time dependent manner, being observed for up to 8 hours, after NECA administration. We demonstrated for the first time that adenosine receptors activation by NECA affects not only P-GP but also MRPs protein levels in a time dependent manner in rat brain capillaries. Why these modulatory effects on protein levels by adenosine receptors activation are opposite for MRPs subtypes, or what are the mechanisms by which adenosine receptors activation modulate MRPs protein levels are questions that require further study. A possible compensatory change due to decreased P-GP protein levels (Cisternino et al. 2004; Hoffmann and Loscher, 2007) with the up-regulation of MRP2 protein levels after adenosine receptors activation by NECA is other interesting point that needs to be investigated. Another important finding was that in CBZ resistant animals, adenosine receptors activation by NECA (resistant+NECA-8) decreased P-GP and increased MRP2 protein levels in rat brain capillaries compared to the CBZ resistant group. MRP1 decreased, but no more than that observed in the CBZ resistant group. This

suggests that the effects of activating adenosine receptors on MRP1 protein levels may be less sensitive compared with its effects on P-GP or MRP2 efflux transporters in CBZ resistant animals. This demonstrates a clear modulatory effect on protein levels of efflux transporters in brain capillaries by adenosine receptors activation in CBZ resistant rats. This fact might have a therapeutic value in the treatment of Drug Resistant Epilepsy, since the gradual reduction of P-GP or the increase of MRP2 protein levels in the blood-brain barrier could have a positive impact on the efficacy of antiepileptic drugs, such as CBZ. The above statement was confirmed by our behavioral evaluation, where we observed that adenosine receptors activation by NECA restores the anticonvulsant effect of CBZ in CBZ resistant rats, 8 hours after NECA administration. At the blood-brain barrier, activation of adenosine receptors by NECA (a non-selective A1/A2A-R agonist) modulated efflux transporters protein levels in brain capillaries, which regulate the blood-brain barrier permeability in turn and the delivery of drugs into the brain. Thus, the effects observed with the combination of NECA plus CBZ (delay and control of convulsions spreading) could be due in part to this modulation of efflux transporters protein levels, which allows the restoration of the anticonvulsant effect in CBZ resistant rats, probably by facilitating its delivery to the brain. On the other hand, the administration of just NECA in CBZ resistant rats increased only the latencies to convulsions, suggesting that this delay in the evolution of convulsions is probably due to brain activation of adenosine receptors (Adami et al. 1995; Zhang et al. 1990). We observed that the administration of DPCPX (a

selective A1R antagonist), prevented the anticonvulsant behavioral effects generated by systemic NECA administration. Of course, further experiments are required to understand with greater clarity the effect of DPCPX (alone, in combination with CBZ, and in combination with CBZ+NECA) and the activation of adenosine receptors, in the control of convulsions in CBZ resistant rats. The activation of adenosine receptors with selective agonists has been studied through its anticonvulsant effects as a therapeutic option to treat epilepsy and Drug Resistant Epilepsy (Adami et al. 1995; Akula et al. 2007; Barros-Barbosa et al. 2016; Fedele et al. 2006; Gouder et al. 2003; Hargus et al. 2012; Klaft et al. 2016). Specifically, A1R activation has been shown to control CBZ resistant seizure-like activity in neocortex slices from patients with Drug Resistant Epilepsy (Klaft et al. 2016), and to suppress the seizure activity in a mouse model of Drug Resistant Epilepsy (Gouder et al. 2003), as well as to increase the anticonvulsant effects of CBZ (Borowicz et al. 2002; Luszczki et al. 2005; Malhotra and Gupta, 1999). With respect to A2AR activation, several studies confirm a proconvulsive effect in rodents (El Yacoubi et al. 2009; Fukuda et al. 2011; Li et al. 2012; Moschovos et al. 2012; Rombo et al. 2015; Rosim et al. 2011), in such a way that their blockage seems to afford beneficial effects in animal models of epilepsy. In fact, the pharmacological or genetic blockade of A2AR has demonstrated protective effects against amygdala- or PTZ-induced kindled seizures (El Yacoubi et al. 2009; Li et al. 2012; Canas et al. 2018).

However, it has been proposed that adenosine plays a bi-phasic role in the control of epilepsy, not only lessening convulsive episodes but also bolstering subsequent neuronal damage (neurodegeneration) (Canas et al. 2018; Cunha, 2016). Since convulsions trigger an elevation of the extracellular concentration of adenosine (Berman et al. 2000; During and Spencer, 1992), the prolonged exposure to adenosine results in A1R desensitization after convulsions (Ekonomou et al. 2000; Ochiishi et al. 1999; Rebola et al. 2003), which promotes an increase of susceptibility of the neurons to damage due to loss of A1R capacity to control neurodegeneration (Cunha, 2016). Contrariwise, in chronic epilepsy there is an increase of the density and function of A2AR (Barros-Barbosa et al. 2016; Canas et al. 2018; Rebola et al. 2005), an effect observed in glutamatergic synapses accompanied by an increase in glutamate release (Canas et al. 2018), where glutamate-mediated excitotoxicity becomes a trigger of neurodegeneration (Canas et al. 2018; Cunha, 2016). Thus, the blockade of A2AR, either using genetic deletion of A2AR or selective A2AR antagonists administration can prevent neuronal damage following convulsions (Canas et al. 2018; Rosim et al. 2011), supporting a key role of A2AR in the control of neurodegeneration after a convulsive episode (Canas et al. 2018; Cunha, 2016; Lee et al. 2004; Rosim et al. 2011). Thus, the use of non-selective agonists (NECA) as adjunctive therapy for the control of Drug Resistant Epilepsy presents advantages by having an anticonvulsive effect per se by delaying the evolution of convulsions, as well as by modulating the efflux transporters protein levels (P-GP/MRPs), which results in the

facilitation of the delivery of drugs such as CBZ to the brain and therefore the restoration of its anticonvulsant effect. Another important point is the gradual modulation of NECA over the protein levels of efflux transporters in brain capillaries that has been previously reported allows the blood-brain barrier to recover its permeability, returning to baseline after the effects of NECA end (Bynoe et al. 2015; Kim and Bynoe, 2016). This would allow the temporary access of antiepileptic drugs to control the severity and the evolution of convulsions in Drug Resistant Epilepsy. However, the chronic effects on the regulation of the bloodbrain barrier´s permeability with this kind of adjunctive therapy have not yet been established and should be investigated in greater detail before considering its clinical use to avoid compromising the functionality of the blood-brain barrier and to protect the brain from exposure to toxic compounds. In addition to this, it must be taken into consideration that the use of adenosine receptor agonists has been related to side effects such as cardiovascular peripheral effects, sedative effects and cognition or memory deficits (Borowicz et al. 2004; FDA, 2013; Schindler et al. 2005; Schulz et al. 2012; Stella et al. 1993; Wu et al. 2009) after its systemic administration, so their use as adjunctive therapy needs to be assessed, especially if it would be considered as a chronic treatment for Drug Resistant Epilepsy. Finally, it is important to consider that the activation of adenosine receptors seems to also modulate the blood-brain barrier permeability by regulating the protein levels of molecules involved in tight junction integrity and function (Carman et al. 2011; Kim and Bynoe, 2015), so this modulation could also be part of the observed

effects of NECA on the restoration of CBZ´s anticonvulsant effect in CBZ resistant animals in this study.

3.1 Conclusion This study demonstrates that the window-PTZ kindling is an easy model to develop CBZ resistant animals. CBZ resistance is associated with the up-regulation of PGP/MRP2 proteins in rat brain capillaries from the blood-brain barrier. Interestingly, activation of adenosine receptors by NECA improves the control of convulsions in CBZ resistant rats and also modulates the protein levels of efflux transporters (PGP/MRP2) in brain capillaries. Both effects of activation of adenosine receptors (anticonvulsant action and modulation of blood-brain barrier permeability) are of interest since activation of adenosine receptors could be useful as an adjunct therapy for the control of Drug Resistant Epilepsy as well as new strategy to deliver therapeutic drugs (e.g., antiepileptic drugs) into the brain to treat Drug-Resistant Epilepsy.

4. Experimental procedures 4.1 Animals Male Wistar rats (250–300 g) were maintained in individual cages under controlled environmental conditions (12-h light/dark cycles; temperature, 22 °C) with access to food and water ad libitum. All experiments were performed in accordance with the Mexican Official Norm (NOM-062-ZOO-1999) and with the approval of the

Internal Committee for the Care and Use of Laboratory Animals of the National Institute of Neurology and Neurosurgery M.V.S. (project 25/16). Additionally, all experiments complied with the National Institutes of Health Guide for the Care and Use of Laboratory Animals (NIH Publications No. 80-23, revised 1996). All efforts were made to minimize the number of animals used and their suffering.

4.2 Drugs All drugs were purchased from Sigma (Sigma-Aldrich, St. Louis MO, USA). Pentylenetetrazole (PTZ) and valproic acid sodium salt were dissolved in 0.9% physiological saline solution. Phenytoin, phenobarbital and 8-Cyclopentyl-1,3dipropylxanthine (DPCPX) were freshly dissolved in 0.9% physiological saline solution (pH 11.2 adjusted with 1 M NaOH). Carbamazepine (CBZ) and 5′-(Nethylcarboxamido) adenosine (NECA) were each dissolved in 0.3% dimethyl sulfoxide (DMSO, v/v).

4.3 Window-PTZ kindling To develop the chemical kindling, we followed the protocol proposed by Davoudi et al. (2013). Animals were kindled by intraperitoneal (i.p.) injection of a subthreshold dose of PTZ (35 mg/kg, volume of 1 ml/kg body weight) three times/week (Monday, Wednesday, Friday; Corda et al. 1991). Animals received 4 initial PTZ injections; after that, the rodents did not receive PTZ during the next 10 sessions, and the treatment finished with 3 final PTZ-injections.

After each PTZ injection, behavior was observed for 20 min to score convulsions. The kindling development was classified as follows (modified from Racine, 1972; Corda et al. 1990): 0: no response; 1: ear and facial twitching, sniffing, blinking; 2: nodding or myoclonic body jerks; 3: clonus of one forelimb, bilateral forelimb clonus; 4: rearing with bilateral forelimb clonus; 5: generalized clonic-tonic convulsions, loss of postural control. The maximum response was recorded for each animal. Animals were considered fully kindled after manifestation of three consecutive sessions reaching stage 4 or 5. Animals that were not successfully kindled within 17 sessions were excluded from our study. Kindled rats were used to evaluate the protein levels of efflux transporters by western blot and the rest were tested for CBZ.

4.4 Evaluation of CBZ resistance in kindled rats The aim of these experiments was to determine if the kindled rats were able to generate resistance to CBZ. Behavioral parameters included severity of convulsions, and latency of stage 2 or 4/5 convulsions. Duration was only measured when a stage 4 or 5 convulsion occurred. CBZ resistant group (n=58): kindled rats were treated with a single dose of CBZ (40 mg/kg i.p.) at 2, 9 and 16 days after their last kindling stimulation (post-kindling phase) to obtain CBZ resistant rats. CBZ was administered 60 min prior to stimulation with PTZ (35 mg/kg; Srivastava et al. 2013).

Vehicle group (n=6): kindled rats were handled as described above for the CBZ resistant group, except that on the second day after the last kindling stimulation, they received vehicle (VEH, 0.3% DMSO i.p.) instead of CBZ. From the CBZ resistant group, some rats were used to evaluate the protein levels of efflux transporters by western blot and the rest were tested for other antiepileptic drugs and NECA.

4.5 Effect of antiepileptic drugs in CBZ resistant rats To evaluate if CBZ resistant rats were also resistant to other antiepileptic drugs, one week after the last administration of CBZ (day 23), a second drug trial was performed with one of the following drugs: the phenytoin group (n=7) CBZ resistant rats received phenytoin (75 mg/kg i.p.) 58 min prior to stimulation with PTZ (35 mg/kg i.p.); the valproic acid group (n=7) received valproic acid (250 mg/kg i.p.) 30 min prior to stimulation and the phenobarbital group (n=6) received phenobarbital (25 mg/kg i.p.) 30 min prior to stimulation. Preliminary experiments showed that these doses of antiepileptic drugs were able to reduce convulsions corresponding to stage 2 or higher (Bethmann et al. 2007; Löscher et al. 1993; Post, 2004; Srivastava and White, 2013). Severity, latency and duration of convulsions were also recorded as described above.

4.6 Evaluation of protein levels of efflux transporters

To evaluate the protein levels of efflux transporters (P-GP, MRP1 and MRP2) in the experimental groups of rats, we analyzed the brain capillaries by immunofluorescence and western blot.

4.6.1 Isolation of brain capillaries For evaluation in brain capillaries, the following groups (n=6 per group) were included: Control: control rats, treated with saline solution only. CBZ: rats receiving only three administrations of carbamazepine (40 mg/kg i.p.) one time per week for 3 weeks. NECA-4: rats treated with a single administration of NECA (1 mg/kg i.p.) and sacrificed 4 hours later. NECA-8: rats treated with a single administration of NECA (1 mg/kg i.p.) and sacrificed 8 hours later. Kindled: fully kindled rats. Resistant: CBZ resistant rats. Resistant+NECA-8: CBZ resistant rats treated with a single administration of NECA (1 mg/kg i.p.) and sacrificed 8 hours later. Capillaries from rat brains (2-3 animals per preparation) were freshly isolated using a modified protocol based on a previous study (Nakagawa et al. 2009; Tontsch and

Bauer, 1989). The isolation procedure was carried out with freshly prepared sucrose buffer (1 M HEPES, 1 M sucrose, pH 7.4) maintained at 4 °C. Additionally, all material was previously rinsed out with a 1% BSA-PBS solution. Animals were euthanized by overdose of pentobarbital and decapitated. Brains were immediately put in ice-cold sucrose buffer. The next step was to remove the meninges, choroid plexus and cerebellum from the brains. Then the tissue was mashed and gently homogenized with sucrose buffer aliquots. The homogenate was centrifuged at 1000 g for 10 min at 4°C. The supernatant was discarded and the capillary pellet was again resuspended in sucrose buffer and treated as above. A series of centrifugation steps (4 more times) were performed until the dense white layer of myelin in the upper part of the pellet was removed. After that, the capillary pellet was resuspended in sucrose buffer and the capillary suspension was filtered through a 70 µm filter (MACS Miltenyi Biotec). The filtrate was collected, resuspended and filtered through a 30 µm filter (MACS Miltenyi Biotec). The capillaries that were bound to the filter were collected by gentle agitation with sucrose buffer. This filtrate was centrifuged (1000 g for 10 min at 4°C) and the resultant pellet was resuspended in sucrose buffer. The resulting pellet (almost exclusively containing brain capillaries) was used for immunohistochemistry or Western blot analysis.

4.6.2 Immunofluorescence Freshly isolated rat brain capillaries were fixed on poly-L-lysine-coated slides for 24 h at 4°C. After washing with 0.12 M PBS, brain capillaries were blocked with 1%

normal goat serum (Vector Laboratories, Inc.) in 0.12 M PBS. Later, brain capillaries were incubated with primary antibodies: mouse anti-mdr1 (3H-2833, Santa

Cruz

Biotechnology),

mouse

anti-MRP1

(sc-59605;

Santa

Cruz

Biotechnology) and rabbit anti- MRP2 (sc-20766; Santa Cruz Biotechnology) all diluted in 0.12 M PBS (1:200) for 48 h at 4 °C. Brain capillaries were washed with PBS and incubated for 2 h at room temperature, with the corresponding fluorochrome-conjugated secondary antibody (1:200): Alexa Fluor 488 (A21121 or A11008, Molecular Probes, Life Technologies). Immunostainings were coverslipped with Vectashield mounting medium (Vector Laboratories, Inc.) and evaluated using a Nikon Ti Eclipse inverted confocal microscope equipped with an A1 imaging system, both controlled from the proprietary software NIS Elements v.4.50. Imaging was performed using a 20x (dry, NA 0.8) objective lens, as specified in the text. The dye was excited in a sequential mode using the built-in laser lines: 488 nm. The corresponding fluorescence was read in the range of 500550 nm, using the manufacturer-provided filter sets. Images were acquired and analyzed using NIS Elements v.4.50.

4.6.3 Western blot analysis Rat brain capillaries were homogenized in lysis buffer containing EDTA (5 mM) plus protease inhibitor cocktail (cOmplete, ROCHE, Germany). Samples were centrifuged at 12,000 g for 30 min at 4 °C. Pellets were resuspended, and protein concentrations were determined according to the Bradford method (Thermo Scientific). Protein samples (15 μg) were separated by 6% SDS–PAGE gels (80 V)

and electrotransferred from the gel to nitrocellulose membranes (3 h, 90 V, room temperature; Bio-Rad) using a transfer buffer (0.192 M Glycine, 0.025 M Tris Base, 20% Methanol, pH 8.3). Membranes were then blocked with blocking buffer for 2 h (Odyssey blocking buffer PBS, for P-GP detection; 10% milk powder, for MRP1 and MRP2 detection). After blocking, the membranes were incubated overnight at 4 °C, with the appropriate primary antibody (1:1000): mouse monoclonal anti-P-GP (170 kDa; ab3083, Abcam), mouse monoclonal anti-MRP1 (190 kDa, MRPm5; sc59605, Santa Cruz Biotechnology), or rabbit polyclonal anti- MRP2 (190 kDa, H300; sc-20766, Santa Cruz Biotechnology). Membranes were then washed (3 times, 5 min) and incubated for 2 h with the corresponding secondary antibody: goat anti-mouse/IRDye 800CW (1:20 000; LICOR 926-32210) for P-GP immunofluorescence detection, and horseradish peroxidase-conjugated secondary antibody

(1:10

000;

Vector

Laboratories

Inc.)

for

MRP1

and

MRP2

chemiluminescence detection. Proteins were detected through visualization of the immunoreaction using an enhanced fluorescence system (Odyssey CLx imaging system LICOR) or a chemiluminescence solution (LuminataTM Crescendo Western HRP substrate; Millipore, USA). To determine the ratio between each efflux transporter and β-actin, the membranes were stripped and reprobed with mouse monoclonal anti-β-actin antibody (C4 sc47778; Santa Cruz Biotechnology) to ascertain equal loading of protein. Briefly, membranes were stripped with ReBlot Plus Strong Antibody Stripping Solution (cat. 2504, Millipore, USA) by gentle agitation for 25 min. Membranes were then washed (2 times, 5 min) and were blocked with blocking buffer for 2 h (10% milk

powder). After blocking, the membranes were incubated overnight at 4 °C, with the primary antibody (1:1000): mouse monoclonal anti-β-actin (43 kDa; C4 sc-47778; Santa Cruz Biotechnology). Finally, membranes were washed (2 times, 5 min) and incubated for 2 h with the corresponding secondary antibody (1:10 000). β-actin was detected as previously explained for efflux transporters. The intensity of the bands was analyzed by densitometric analysis (ImageJ) and calculated according to the reference bands of β-actin. The results were expressed as ratios of efflux transporters/β-actin (P-GP/β-actin, MRP1/β-actin, MRP2/β-actin), and then normalized relative to the values measured in the control groups.

4.7 Effect of NECA in CBZ resistant rats To evaluate whether adenosine receptors modulation by NECA (a non-selective A1/A2A-R agonist) could affect the anticonvulsant effect of CBZ on CBZ resistant rats, we prepared the following experimental groups: NECA+CBZ (n=8): one week after the last administration of CBZ (day 23), CBZ resistant rats were administered with NECA (1 mg/kg i.p. 8 h prior to stimulation with PTZ 35 mg/kg i.p.) and CBZ (40 mg/kg i.p. 1 h before of PTZ), respectively. NECA (n=6): these animals were treated as above, except that CBZ resistant rats were only treated with NECA. DPCPX+NECA (n=6): these animals were treated as NECA+CBZ group, except that CBZ resistant rats were treated with selective A1R antagonist, DPCPX (1 mg/kg i.p. 5 min prior to administration of NECA) instead of CBZ.

Vehicle+CBZ (n=6): these animals were treated as NECA+CBZ group, except that CBZ resistant rats were treated with vehicle (0.3% DMSO i.p.) instead of NECA. For NECA administration, the dose was chosen based on a previous study demonstrating protection against PTZ-induced lethal convulsions in rats by i.p. administration (Adami et al. 1995). The treatment time was chosen taking into account the decrease in the protein levels of P-GP that starts at 2 hours in brain capillaries from Wild-type mice treated with NECA, which started to show drug accumulation in the brain 4 h later (Kym and Bynoe, 2016). The dose of DPCPX was based on previous studies in rat (Malhotra et al. 1996; Malhotra and Gupta, 1997). Severity, latency and duration of convulsions were also recorded as described above.

4.8 Statistical Analysis GraphPad Prism 5.01 (GraphPad software, La Jolla, USA) was used for data analysis and graph preparation. Data are presented as the mean ± S.E.M. For all analyses, differences were considered significant at p<0.05. The results of the progression of convulsions during kindling development or between different administrations of CBZ in the severity, latency and duration of convulsions were analyzed using a one-way ANOVA with repeated measures and Tukey´s post hoc test for multiple comparisons. Comparison between the CBZ resistant condition (16 d) and drug treatment (23 d) was performed for each drug using Student’s t-test for paired replicates. We analyzed changes in protein levels of efflux transporters at

rat brain capillaries by western blot, between the different groups evaluated, by a one-way ANOVA followed by Tukey´s post hoc test for multiple comparisons.

Conflict of interest None.

Acknowledgments The authors would like to thank Dr. Vadim Perez Koldenkova for his advice with the confocal microscopy and collecting images.

Funding This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.

References Adami, M., Bertorelli, R., Ferri, N., Foddi, M.C., Ongini, E., 1995. Effects of repeated administration of selective adenosine A1 and A2A receptor agonists on pentylenetetrazole-induced convulsions in the rat. Eur. J. Pharmacol. 294(2-3), 383-389.

Akula, K.K., Dhir, A., Kulkarni, S.K., 2007. Systemic administration of adenosine ameliorates pentylenetetrazol-induced chemical kindling and secondary behavioural and biochemical changes in mice. Fundam. Clin. Pharmacol. 21(6), 583-594. Aronica, E., Gorter, J.A., Ramkema, M., Redeker, S., Ozbas-Gerceker, F., van Vliet, E.A., Scheffer, G.L., Scheper, R.J., van der Valk, P., Baayen, J.C., Troost, D., 2004. Expression and cellular distribution of multidrug resistancerelated proteins in the hippocampus of patients with mesial temporal lobe epilepsy. Epilepsia 45(5), 441-451. Baltes, S., Gastens, A.M., Fedrowitz, M., Potschka, H., Kaever, V., Loscher, W., 2007. Differences in the transport of the antiepileptic drugs phenytoin, levetiracetam and carbamazepine by human and mouse P-glycoprotein. Neuropharmacology 52(2), 333-346. Bankstahl, J.P., Hoffmann, K., Bethmann, K., Löscher, W., 2008. Glutamate is critically involved in seizure-induced overexpression of P-glycoprotein in the brain. Neuropharmacology 54(6), 1006-1016. Barros-Barbosa, A.R., Ferreirinha, F., Oliveira, Â., Mendes, M., Lobo, M.G., Santos, A., Rangel, R., Pelletier, J., Sévigny, J., Cordeiro, J.M., Correia-deSá, P., 2016. Adenosine A2A receptor and ecto-5'-nucleotidase/CD73 are upregulated in hippocampal astrocytes of human patients with mesial temporal lobe epilepsy (MTLE). Purinergic Signal. 12(4), 719-734.

Bauer, B., Hartz, A.M., Pekcec, A., Toellner, K., Miller, D.S., Potschka, H., 2008. Seizure- induced up-regulation of P-glycoprotein at the blood-brain barrier through glutamate and cyclooxygenase-2 signaling. Mol. Pharmacol. 73(5), 1444-1453. Berman, R.F., Fredholm, B.B., Aden, U., O’Connor, W.T., 2000. Evidence for increased dorsal hippocampal adenosine release and metabolism during pharmacologically induced seizures in rats. Brain Res. 872, 44-53. Bethmann, K., Brandt, C., Löscher, W., 2007. Resistance to phenobarbital extends to phenytoin in a rat model of temporal lobe epilepsy. Epilepsia 48(4), 816826. Borowicz, K.K., Łuszczki, J., Czuczwar, S.J., 2002. 2-Chloroadenosine, a preferential agonist of adenosine A1 receptors, enhances the anticonvulsant activity

of

carbamazepine

and

clonazepam

in

mice.

Eur.

Neuropsychopharmacol. 12(2), 173-179. Borowicz, K.K., Swiader, M., Wielosz, M., Czuczwar, S.J., 2004. Influence of the combined treatment of LY 300164 (an AMPA/kainate receptor antagonist) with adenosine receptor agonists on the electroconvulsive threshold in mice. Eur. Neuropsychopharmacol. 14(5), 407-412. Bynoe, M.S., Viret, C., Yan, A., Kim, D.G., 2015. Adenosine receptor signaling: a key to opening the blood-brain door. Fluids Barriers CNS. Sep 2, 12, 20. Canas, P.M., Porciúncula, L.O., Simões, A.P., Augusto, E., Silva, H.B., Machado, N.J., Gonçalves, N., Alfaro, T.M., Gonçalves, F.Q., Araújo, I.M., Real, J.I.,

Coelho, J.E., Andrade, G.M., Almeida, R.D., Chen, J.F., Köfalvi, A., Agostinho, P., Cunha, R.A., 2018. Neuronal Adenosine A2A Receptors Are Critical Mediators of Neurodegeneration Triggered by Convulsions. eNeuro 26, 5(6), 0385-18, 1-12. Carman, A.J., Mills, J.H., Krenz, A., Kim, D.G., Bynoe, M.S., 2011. Adenosine receptor signaling modulates permeability of the blood-brain barrier. J. Neurosci. 31(37), 13272-13280. Chen, Y.H., Wang, C.C., Xiao, X., Wei, L., Xu, G., 2013. Multidrug resistanceassociated protein 1 decreases the concentrations of antiepileptic drugs in cortical extracellular fluid in amygdale kindling rats. Acta Pharmacol. Sin. 34(4), 473-479. Cisternino, S., Mercier, C., Bourasset, F., Roux, F., Scherrmann, J.M., 2004. Expression, up-regulation, and transport activity of the multidrug-resistance protein Abcg2 at the mouse blood-brain barrier. Cancer Res. 64(9), 32963301. Clinckers, R., Smolders, I., Meurs, A., Ebinger, G., Michotte, Y., 2005. Quantitative in vivo microdialysis study on the influence of multidrug transporters on the blood–brain barrier passage of oxcarbazepine: concomitant use of hippocampal

monoamines

as

pharmacodynamic

markers

for

the

anticonvulsant activity. J. Pharmacol. Exp. Ther. 314(2), 725-731. Corda, M.G., Giorgi, O., Longoni, B., Orlandi, M., Biggo, G., 1990. Decrease in the function of the gamma-aminobutyric acid-coupled chloride channel produced

by the repeated administration of pentylenetetrazol to rats. J. Neurochem. 55(4), 1216-1221. Corda, M.G., Orlandi, M., Lecca, D., Carboni, G., Frau, V., Giorgi, O., 1991. Pentylenetetrazol-induced kindling in rats: effect of GABA function inhibitors. Pharmacol. Biochem. Behav. 40(2), 329-333. Cunha, R.A., 2016. How does adenosine control neuronal dysfunction and neurodegeneration?. J. Neurochem. 139(6), 1019-1055. Davoudi, M., Shojaei, A., Palizvan, M.R., Javan, M., Mirnajafi-Zadeh, J., 2013. Comparison between standard protocol and a novel window protocol for induction of pentylenetetrazol kindled seizures in the rat. Epilepsy Res. 106(1-2), 54-63. Dombrowski, S.M., Desai, S.Y., Marroni, M., Cucullo, L., Goodrich, K., Bingaman, W., Mayberg, M.R., Bengez, L., Janigro, D., 2001. Overexpression of multiple drug resistance genes in endothelial cells from patients with refractory epilepsy. Epilepsia 42(12), 1501-1506. During, M.J., Spencer, D.D., 1992. Adenosine: a potential mediator of seizure arrest and postictal refractoriness. Ann. Neurol. 32, 618-624. Ekonomou, A., Sperk, G., Kostopoulos, G., Angelatou, F., 2000. Reduction of A1 adenosine receptors in rat hippocampus after kainic acid-induced limbic seizures. Neurosci. Lett. 21, 284(1-2), 49-52.

El Yacoubi, M., Ledent, C., Parmentier, M., Costentin, J., Vaugeois, J.M., 2009. Adenosine A2A receptor deficient mice are partially resistant to limbic seizures. Naunyn Schmiedebergs Arch. Pharmacol. 380, 223-232. Engel, J.Jr., 2001. Mesial temporal lobe epilepsy: what have we learned? Neuroscientist 7(4), 340-352. Enrique, A., Goicoechea, S., Castano, R., Taborda, F., Rocha, L., Orozco, S., Girardi, E., Bruno-Blanch, L., 2017. New model of pharmacoresistant seizures induced by 3-mercaptopropionic acid in mice. Epilepsy Res. 129, 816. Fedele, D.E., Li, T., Lan, J.Q., Fredholm, B.B., Boison, D., 2006. Adenosine A1 receptors are crucial in keeping an epileptic focus localized. Exp. Neurol. 200(1), 184-190. Food and Drug Administration U.S., 2013. FDA warns of rare but serious risk of heart attack and death with cardiac nuclear stress test drugs Lexiscan (regadenoson) and Adenoscan (adenosine). Nov 20. Fredholm, B.B., Chen, J.F., Cunha, R.A., Svenningsson, P., Vaugeois, J.M., 2005. Adenosine and brain function. Int. Rev. Neurobiol. 63, 191-270. Fredholm, B., IJzerman, A., Jacobson, K., Linden, J., Müller, C., 2011. International Union of Basic and Clinical Pharmacology. LXXXI. Nomenclature and classification of adenosine receptors-an update. Pharmacol. Rev. 63(1), 134.

Fukuda, M., Suzuki, Y., Hino, H., Morimoto, T., Ishii, E., 2011. Activation of central adenosine A2A receptors lowers the seizure threshold of hyperthermiainduced seizure in childhood rats. Seizure. 20, 156-159. Giannotta, M., Trani, M., Dejana, E., 2013. VE-cadherin and endothelial adherens junctions: active guardians of vascular integrity. Dev. Cell. 16, 26(5), 441454. Goddard, G.V., 1983. The kindling model of epilepsy. Trends Neurosci. 6, 275-279. Goddard, G.V., McIntyre, D.C., Leech, C.K., 1969. A permanent change in brain function resulting from daily electrical stimulation. Exp. Neurol. 25(3), 295330. Gouder, N., Fritschy, J.M., Boison, D., 2003. Seizure suppression by adenosine A1 receptor activation in a mouse model of pharmacoresistant epilepsy. Epilepsia 44(7), 877-885. Hargus, N.J., Jennings, C., Perez-Reyes, E., Bertram, E.H., Patel, M.K., 2012. Enhanced actions of adenosine in medial entorhinal cortex layer II stellate neurons in temporal lobe epilepsy are mediated via A(1)-receptor activation. Epilepsia 53(1), 168-176. Hoffmann, K., Löscher, W., 2007. Upregulation of brain expression of Pglycoprotein in MRP2-deficient TR(-) rats resembles seizure-induced upregulation of this drug efflux transporter in normal rats. Epilepsia 48(4), 631645.

Jing, X., Liu, X., Wen, T., Xie, S., Yao, D., Liu, X., Wang, G., Xie, L., 2010. Combined

effects

of

epileptic

seizure

and

phenobarbital

induced

overexpression of P-glycoprotein in brain of chemically kindled rats. Br. J. Pharmacol. 159(7), 1511-1522. Kim, D.G., Bynoe, M.S., 2015. A2A Adenosine Receptor Regulates the Human Blood-Brain Barrier Permeability. Mol. Neurobiol. 52(1), 664-678. Kim, D.G., Bynoe, M.S., 2016. A2A adenosine receptor modulates drug efflux transporter P-glycoprotein at the blood-brain barrier. J. Clin. Invest. 126(5), 1717-1733. Klaft, Z.J., Hollnagel, J.O., Salar, S., Calişkan, G., Schulz, S.B., Schneider, U.C., Horn, P., Koch, A., Holtkamp, M., Gabriel, S., Gerevich, Z., Heinemann, U., 2016. Adenosine A1 receptor-mediated suppression of carbamazepineresistant seizure-like events in human neocortical slices. Epilepsia 57(5), 746-756. Kwan, P., Sander, J.W., 2004. The natural history of epilepsy: an epidemiological view. J. Neurol. Neurosurg. Psychiatry 75(10), 1376-1381. Kwan, P., Brodie, M.J., 2005. Potential role of drug transporters in the pathogenesis of medically intractable epilepsy. Epilepsia 46(2), 224-235. Lee, H.K., Choi, S.S., Han, K.J., Han, E.J., Suh, H.W., 2004. Roles of adenosine receptors in the regulation of kainic acid-induced neurotoxic responses in mice. Mol. Brain Res. 125, 76-85.

Li, X., Kang, H., Liu, X., Liu, Z., Shu, K., Chen, X., Zhu, S., 2012. Effect of adenosine A2A receptor antagonist ZM241385 on amygdala-kindled seizures and progression of amygdala kindling. J. Huazhong Univ. Sci. Technol. Med. Sci. 32, 257-264. Liu, X., Yang, Z., Yang, J., Yang, H., 2007. Increased P-glycoprotein expression and decreased phenobarbital distribution in the brain of pentylenetetrazolekindled rats. Neuropharmacology 53(5), 657-663. Liu, J.Y., Thom, M., Catarino, C.B., Martinian, L., Figarella-Branger, D., Bartolomei, F., Koepp, M., Sisodiya, S.M., 2012. Neuropathology of the blood-brain barrier and pharmaco-resistance in human epilepsy. Brain 135(Pt 10), 31153133. Löscher, W., 2002. Basic pharmacology of valproate: a review after 35 years of clinical use for the treatment of epilepsy. CNS Drugs 16(10), 669-694. Löscher, W., 2011. Critical review of current animal models of seizures and epilepsy used in the discovery and developmentof new antiepileptic drugs. Seizure 20(5), 359-368. Loscher, W., Rogawski, M.A., 2012. How theories evolved concerning the mechanism of action of barbiturates. Epilepsia 53 Suppl 8, 12-25. Löscher, W., Rundfeldt, C., 1991. Kindling as a model of drug-resistant partial epilepsy: selection of phenytoin-resistant and nonresistant rats. J. Pharmacol. Exp. Ther. 258(2), 483-489.

Löscher, W., Rundfeldt, C., Hönack, D., 1993. Pharmacological characterization of phenytoin-resistant amygdala-kindled rats, a new model of drug-resistant partial epilepsy. Epilepsy Res. 15(3), 207-219. Luna-Munguia, H., Salvamoser, J.D., Pascher, B., Pieper, T., Getzinger, T., Kudernatsch, M., Kluger, G., Potschka, H., 2015. Glutamate mediated upregulation of the multidrug resistance protein 2 in porcine and human brain capillaries. J. Pharmacol. Exp. Ther. 352(2), 368-378. Luna-Tortos, C., Fedrowitz, M., Loscher, W., 2008. Several major antiepileptic drugs are substrates for human P-glycoprotein. Neuropharmacology 55(8), 1364-1375. Luna-Tortos, C., Fedrowitz, M., Löscher, W., 2010. Evaluation of transport of common antiepileptic drugs by human multidrug resistance-associated proteins (MRP1, 2 and 5) that are overexpressed in pharmacoresistant epilepsy. Neuropharmacology 58(7), 1019-1032. Luszczki, J.J., Kozicka, M., Swiader, M.J., Czuczwar, S.J., 2005. 2-Chloro-N6cyclopentyladenosine enhances the anticonvulsant action of carbamazepine in the mouse maximal electroshock-induced seizure model. Pharmacol. Rep. 57(6), 787-794. Macdonald, R.L., Olsen, R.W., 1994. GABAA receptor channels. Annu. Rev. Neurosci. 17, 569-602.

Malhotra, J., Gupta, Y.K., 1997. Effect of adenosine receptor modulation on pentylenetetrazole-induced seizures in rats. Br. J. Pharmacol. 120(2), 282288. Malhotra, J., Gupta, Y.K., 1999. Effect of adenosinergic modulation on the anticonvulsant effect of phenobarbitone and carbamazepine. Methods Find. Exp. Clin. Pharmacol. 21(2), 79-83. Malhotra, J., Seth, S.D., Gupta, S.K., Gupta, Y.K., 1996. Adenosinergic mechanisms in anticonvulsant action of diazepam and sodium valproate. Environ. Toxicol. Pharmacol. 15, 1(4), 269-277. Moschovos, C., Kostopoulos, G., Papatheodoropoulos, C., 2012. Endogenous adenosine induces NMDA receptor-independent persistent epileptiform discharges in dorsal and ventral hippocampus via activation of A2 receptors. Epilepsy Res. 100(1-2), 157-167. Nakagawa, S., Deli, M.A., Kawaguchi, H., Shimizudani, T., Shimono, T., Kittel, A., Tanaka, K., Niwa, M., 2009. A new blood-brain barrier model using primary rat brain endothelial cells, pericytes and astrocytes. Neurochem. Int. 54(3-4), 253-263. Ochiishi, T., Takita, M., Ikemoto, M., Nakata, H., Suzuki, S.S., 1999. Immunohistochemical analysis on the role of adenosine A1 receptors in epilepsy. Neuroreport. 10, 3535-3541.

Owen, A., Pirmohamed, M., Tettey, J.N., Morgan, P., Chadwick, D., Park, B.K., 2001. Carbamazepine is not a substrate for P-glycoprotein. Br. J. Clin. Pharmacol. 51(4), 345-349. Park, S.L., Kim, Y.M., Ahn, J.H., Lee, S.H., Baik, E.J., Moon, C.H., Jung, Y.S., 2009. Cadmium stimulates the expression of vascular cell adhesion molecule-1 (VCAM-1) via p38 mitogen-activated protein kinase (MAPK) and JNK activation in cerebrovascular endothelial cells. J. Pharmacol. Sci. 110(3), 405-409. Post, R.M., 2004. Neurobiology of seizures and behavioral abnormalities. Epilepsia 45 (Suppl 2), 5-14. Potschka, H., Fedrowitz, M., Löscher, W., 2001. P-glycoprotein and multidrug resistance-associated protein are involved in the regulation of extracellular levels of the major antiepileptic drug carbamazepine in the brain. Neuroreport 12(16), 3557-3560. Racine, R.J., 1972. Modification of seizure activity by electrical stimulation. II. Motor seizure. Electroencephalogr. Clin. Neurophysiol. 32(3), 281-294. Rambeck, B., Jurgens, U.H., May, T.W., Pannek, H.W., Behne, F., Ebner, A., Gorji, A., Straub, H., Speckmann, E.J., Pohlmann-Eden, B., Loscher, W., 2006. Comparison of brain extracelular fluid, brain tissue, cerebrospinal fluid and serum concentrations of antiepileptic drugs measured intraoperatively in patients with intractable epilepsy. Epilepsia 47(4), 681-694.

Rebola, N., Coelho, J.E., Costenla, A.R., Lopes, L.V., Parada, A., Oliveira, C.R., Soares-da-Silva, P., de Mendonça, A., Cunha, R.A., 2003. Decrease of adenosine A1 receptor density and of adenosine neuromodulation in the hippocampus of kindled rats. Eur. J. Neurosci. 18(4), 820-828. Rebola, N., Porciúncula, L.O., Lopes, L.V., Oliveira, C.R., Soares-da-Silva, P., Cunha, R.A., 2005. Long-term effect of convulsive behavior on the density of adenosine A1 and A2A receptors in the rat cerebral cortex. Epilepsia. 46, (Suppl 5), 159-165. Rizzi, M., Caccia, S., Guiso, G., Richichi, C., Gorter, J.A., Aronica, E., Aliprandi, M., Bagnati, R., Fanelli, R., D'Incalci, M., Samanin, R., Vezzani, A., 2002. Limbic seizures induce P-glycoprotein in rodent brain: functional implications for pharmacoresistance. J. Neurosci. 22(14), 5833-5839. Rogawski, M.A., Porter, R.J., 1990. Antiepileptic drugs: pharmacological mechanisms

and

clinical

efficacy

with

consideration

of

promising

developmental stage compounds. Pharmacol. Rev. 42(3), 223-286. Rombo, D.M., Newton, K., Nissen, W., Badurek, S., Horn, J.M., Minichiello, L., Jefferys, J.G., Sebastiao, A.M., Lamsa, K.P., 2015. Synaptic mechanisms of adenosine A2A receptor-mediated hyperexcitability in the hippocampus. Hippocampus. 25, 566-580. Rosim, F.E., Persike, D.S., Nehlig, A., Amorim, R.P., de Oliveira, D.M., Fernandes, M.J., 2011. Differential neuroprotection by A1 receptor activation and A2A

receptor inhibition following pilocarpine induced status epilepticus. Epilepsy Behav. 22, 207-213. Schindler, C.W., Karcz-Kubicha, M., Thorndike, E.B., Müller, C.E., Tella, S.R., Ferré, S., Goldberg, S.R., 2005. Role of central and peripheral adenosine receptors in the cardiovascular responses to intraperitoneal injections of adenosine A1 and A2A subtype receptor agonists. Br. J. Pharmacol. 144(5), 642-650. Schulz, S.B., Klaft, Z.J., Rosler, A.R., Heinemann, U., Gerevich, Z., 2012. Purinergic P2X, P2Y and adenosine receptors differentially modulate hippocampal gamma oscillations. Neuropharmacology 62(2), 914-924. Sisodiya, S.M., Lin, W.R., Squier, M.V., Thom, M., 2001. Multidrug-resistance protein 1 in focal cortical dysplasia. Lancet 357(9249), 42-43. Sisodiya, S.M., Lin, W.R., Harding, B.N., Squier, M.V., Thom, M., 2002. Drug resistance in epilepsy: expression of drug resistance proteins in common causes of refractory epilepsy. Brain 125(Pt 1), 22-31. Srivastava, A.K., White, H.S., 2013. Carbamazepine, but not valproate, displays pharmacoresistance in lamotrigine-resistant amygdala kindled rats. Epilepsy Res. 104(1-2), 26-34. Srivastava, A.K., Alex, A.B., Wilcox, K.S., White, H.S., 2013. Rapid loss of efficacy to the antiseizure drugs lamotrigine and carbamazepine: a novel experimental model of pharmacoresistant epilepsy. Epilepsia 54(7), 11861194.

Stella, L., Berrino, L., Maione, S., de Novellis, V., Rossi, F., 1993. Cardiovascular effects of adenosine and its analogs in anaesthetized rats. Life Sci. 53(10), 755-763. Sun, Y., Luo, X., Yang, K., Sun, X., Li, X., Zhang, C., Ma, S., Liu, Y., Yin, J., 2016. Neural overexpression of multidrug resistance-associated protein 1 and refractory epilepsy: a meta-analysis of nine studies. Int. J. Neurosci. 126(4), 308-317. Tishler, D.M., Weinberg, K.I., Hinton, D.R., Barbaro, N., Annett, G.M., Raffel, C., 1995. MDR1 gene expression in brain of patients with medically intractable epilepsy. Epilepsia 36(1), 1-6. Tontsch, U., Bauer, H.C., 1989. Isolation, characterization, and long-term cultivation of porcine and murine cerebral capillary endothelial cells. Microvasc. Res. 37(2), 148-161. van Vliet, E.A., Redeker, S., Aronica, E., Edelbroek, P.M., Gorter, J.A., 2005. Expression of multidrug transporters MRP1, MRP2, and BCRP shortly after status epilepticus, during the latent period, and in chronic epileptic rats. Epilepsia 46(10), 1569-1580. Volk, H.A., Löscher, W., 2005. Multidrug resistance in epilepsy: rats with drugresistant seizures exhibit enhanced brain expression of P-glycoprotein compared with rats with drug-responsive seizures. Brain 128(Pt 6), 13581368.

Volk, H.A., Potschka, H., Löscher, W., 2004. Increased expression of the multidrug transporter P-glycoprotein in limbic brain regions after amygdala-kindled seizures in rats. Epilepsy Res. 58(1), 67-79. Wang, C., Hong, Z., Chen, Y., 2015. Involvement of p38 MAPK in the Drug Resistance of Refractory Epilepsy through the regulation multidrug resistance-associated protein 1. Neurochem. Res. 40(7), 1546-1553. Winnicka, R.I., Topacinski, B., Szymczak, W.M., Szymanska, B., 2002. Carbamazepine poisoning: elimination kinetics and quantitative relationship with carbamazepine 10, 11-epoxide. J. Toxicol. Clin. Toxicol. 40(6), 759765. Wu, C., Wong, T., Wu, X., Sheppy, E., Zhang, L., 2009. Adenosine as an endogenous regulating factor of hippocampal sharp waves. Hippocampus 19(2), 205-220. Yao, D., Liu, L., Jin, S., Li, J., Liu, X.D., 2012. Overexpression of multidrug resistance-associated protein 2 in the brain of pentylenetetrazole-kindled rats. Neuroscience 227, 283-292. Zhang, G., Franklin, P.H., Murray, T.F., 1990. Anticonvulsant effect of Nethylcarboxamidoadenosine against kainic acid-induced behavioral seizures in the rat prepiriform cortex. Neurosci. Lett. 13, 114(3), 345-350. Zhang, C., Zuo, Z., Kwan, P., Baum, L., 2011. In vitro transport profile of carbamazepine, oxcarbazepine, eslicarbazepine acetate, and their active metabolites by human P-glycoprotein. Epilepsia 52(10), 1894-1904.

Zhang, C., Kwan, P., Zuo, Z., Baum, L., 2012. The transport of antiepileptic drugs by P-glycoprotein. Adv. Drug. Deliv. Rev. 64(10), 930-942. Zibell, G., Unkruer, B., Pekcec, A., Hartz, A.M., Bauer, B., Miller, D.S., Potschka, H., 2009. Prevention of seizure-induced up-regulation of endothelial Pglycoprotein by COX-2 inhibition. Neuropharmacology 56(5), 849-855.

Figure captions Fig. 1. Progression of window-PTZ kindling and development of CBZ resistant animals. (A) Convulsive score versus number of assay after application of PTZ (35 mg/kg i.p.) (mean ± SEM). The window-PTZ kindling progressed from stage 1-3 to stage 4-5 convulsions. Fully kindled rats developed stage 4-5 convulsions during the three last administrations of PTZ, though there was a subgroup of rats that only showed stage 1-3 convulsions (no-kindled rats). *p < 0.05, significantly different from the first assay at kindled group. (B-G) Effects on kindled rats by CBZ (40 mg/kg, i.p.) administration at 2, 9 and 16 days after the last kindling stimulation. Convulsive score (B), % animals with stage 4/5 convulsions (C), stage 2 latency (D), stage 4/5 latency (E) and stage 4/5 duration (F). In (G), the effect of vehicle (VEH) administration (0.3% DMSO v/v i.p.) 2 days after last kindling stimulation on convulsive score is shown. Data represent mean ± SEM. *p < 0.05, significantly different from kindled condition.

Fig. 2. Effects of other antiepileptic drugs (23 d) such as phenytoin (PHE, 75 mg/kg i.p.), valproic acid (VPA, 250 mg/kg i.p.) and phenobarbital (PB, 25 mg/kg i.p.) on convulsive score (A), stage 2 latency (B), latency (C) and duration (D) of stage 4/5 convulsions in CBZ resistant rats (16 d). Data represent mean ± SEM. #p < 0.05, significantly different from the CBZ resistant condition (16 d).

Fig. 3. Protein levels of efflux transporters in freshly isolated rat brain capillaries from

animals

under

different

drug-conditions.

Representative

images

of

immunofluorescence labeling of efflux transporters (A) and quantitative analysis of western blot bands of P-GP (B), MRP1 (C) and MRP2 (D) efflux transporters in brain capillaries. β-actin was used as a loading control and the histogram of the densitometry quantification shows the ratio of efflux transporters/β-actin intensity. Rat brain capillaries are from a single preparation (pooled tissue from 3 rats). Data are expressed as the means ± SEM. *p < 0.05 significantly different from the control group; ɸp < 0.05 significantly different from the NECA-4 group; #p < 0.05 significantly different from the kindled group;

p

< 0.05 significantly different from

the resistant group. Scale bar: 40 µm.

Fig. 4. Effect of NECA on the anticonvulsant efficacy of CBZ in CBZ resistant rats. Striped bars represent behavioral responses to PTZ in CBZ resistant rats (16 d) while open bars represent behavioral responses to PTZ in CBZ resistant rats exposed to drug treatment (23 d). Convulsive score (A), stage 2 latency (B), latency (C) and duration of stage 4/5 convulsions (D), were evaluated. Vehicle (VEH, 0.3% DMSO i.p.). Data represent mean ± SEM. different from the CBZ resistant condition (16 d).

@p

< 0.05 significantly

Highlights  Carbamazepine resistance was associated with the up-regulation of Pglycoprotein and multidrug resistance-associated protein-2 levels in rat brain capillaries.  NECA modified the protein levels of efflux transporters in rat brain capillaries under physiological conditions.  Carbamazepine resistant animals treated with NECA showed decreased protein levels of P-glycoprotein and increase of multidrug resistanceassociated protein-2 in brain capillaries.  NECA

restored

the

anticonvulsant

effect

of

carbamazepine

at

carbamazepine resistant rats.  Adenosine receptors activation could be useful as an adjunct therapy for the control of Drug Resistant Epilepsy.