Epilepsy Research (2014) 108, 212—222

journal homepage: www.elsevier.com/locate/epilepsyres

Effect of eslicarbazepine acetate in the corneal kindling progression and the amygdala kindling model of temporal lobe epilepsy Heidrun Potschka a, Jonna Soerensen a, Anton Pekcec a, Ana Loureiro b, Patrício Soares-da-Silva b,c,∗ a

Institute of Pharmacology, Toxicology, and Pharmacy, Ludwig-Maximilians-University, Munich, Germany Department of Research and Development, BIAL — Portela & Ca , S.A., S. Mamede do Coronado, Portugal c Department of Pharmacology and Therapeutics, Faculty of Medicine, University of Porto, Porto, Portugal b

Received 10 April 2013; received in revised form 8 October 2013; accepted 21 November 2013 Available online 1 December 2013

KEYWORDS Anticonvulsant drugs; Eslicarbazepine acetate; Eslicarbazepine; (R)-licarbazepine; Corneal kindling; Amygdala kindling

Summary Objective: The present study was aimed at determining the effect of eslicarbazepine acetate (ESL), eslicarbazepine and (R)-licarbazepine administration in the mouse corneal kindling and amygdala kindling models. Methods: NMRI mice were kindled by bilateral corneal stimulation twice daily. In amygdala kindling, mice were stimulated once daily via an implanted depth electrode until 10 generalized seizures were elicited. Maximal electroshocks (MES) were administered via corneal electrodes. Results: The average number of stimulations to reach a fully kindled generalized seizure was markedly increased by ESL. Administration of eslicarbazepine also inhibited the acquisition of kindling, whereas administration of R-licarbazepine did not affect the number of stimulations necessary to induce a specific seizure stage, and did not exert any relevant effect on mean seizure severity during kindling progression. ESL dose-dependently increased the focal seizure threshold and reduced seizure severity in amygdala kindling. Whereas eslicarbazepine treatment increased the afterdischarge threshold in a significant manner, (R)-licarbazepine treatment failed to exert a significant effect on thresholds in fully kindled mice. Administration of ESL and of eslicarbazepine significantly protected mice against MES-induced seizures, whereas that of (R)-licarbazepine failed to provide protection.

∗ Corresponding author at: Department of Research and Development, BIAL, À Avendia da Siderurgia Nacional, 4745-457 S. Mamede do Coronado, Portugal. Tel.: +351 229866100; fax: +351 229866192. E-mail address: [email protected] (P. Soares-da-Silva).

0920-1211/$ — see front matter © 2013 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.eplepsyres.2013.11.017

ESL in kindled seizures

213 Conclusions: These data provide evidence of the anticonvulsant effect of ESL and its active metabolite eslicarbazepine on partial-onset seizures in corneal and amygdala kindling models. Based on an effect of the parent compound and the active metabolite eslicarbazepine, ESL treatment may not merely suppress seizure activity but may also provide a disease-modifying or antiepileptogenic effect. Future studies will be necessary to further evaluate a putative preventive effect, in particular when considering that re-stimulation following wash-out did not indicate a persistent effect. The findings reported here raise doubts on the contribution of (R)-licarbazepine as an active anticonvulsant. © 2013 Elsevier B.V. All rights reserved.

Introduction Eslicarbazepine acetate (ESL) is a once-daily anticonvulsant approved in 2009 by the European Medicines Agency as adjunctive therapy in adults with partial-onset seizures (POS), with or without secondary generalization. The ESL epilepsy clinical program included an initial proof-ofconcept phase II study (Elger et al., 2007) and three subsequent phase III studies in patients refractory to conventional antiepileptic drug (AED) therapy (Ben-Menachem et al., 2010; Elger et al., 2009; Gil-Nagel et al., 2009). Long-term safety and maintenance of therapeutic effect was demonstrated in one-year open-label extensions of these studies (Halasz et al., 2010; Hufnagel et al., 2013; LopesLima et al., 2008). More recently, ESL was approved by the US Food and Drug Administration (FDA) as adjunctive treatment of POS, on the basis of an additional phase III study that included US centers (Sperling et al., 2013). Following oral administration, ESL undergoes extensive first pass hydrolysis to its major active metabolite eslicarbazepine (also known as (S)-licarbazepine) (Almeida et al., 2008a,b; Falcao et al., 2007; Maia et al., 2008; Perucca et al., 2011), which represents approximately 95% of circulating active moieties and is believed to be responsible for its antiseizure effects (Pires et al., 2011; Sierra-Paredes et al., 2011; Torrao et al., 2011) most likely though blockade of voltage-gated sodium channels and type T calcium channels (Brady et al., 2011; Hebeisen et al., 2011). Kindling is based on the phenomenon that repeated application of an electrical stimulus leads to the development of seizures increasing in severity and duration with ongoing constant current stimulations (Goddard et al., 1969; Sato et al., 1990). Whereas the impact of test compounds on thresholds in fully kindled mice can only be determined in animals with an implanted depth electrode, the corneal kindling model can be used as an alternate approach studying the impact on kindling acquisition (Matagne and Klitgaard, 1998). Repeated bilateral transcorneal stimulation of mice induces kindling acquisition with partial and secondarily generalized seizures similar to those induced by conventional kindling through unilateral depth electrodes (Matagne and Klitgaard, 1998; Potschka and Löscher, 1999). Available data that have so far been obtained in the corneal kindling model in mice (Matagne and Klitgaard, 1998; Potschka and Löscher, 1999), indicate that the model seems to be suitable to test drug effects on kindling progression with protocols analogous to those that have already been used in the amygdala kindling model in rats. The amygdala kindling model is characterized by an excellent predictive validity for human temporal lobe epilepsy (Löscher, 2002; McIntyre and Gilby, 2009).

Based on these findings we selected the corneal kindling model, which avoids surgical implantation, for the evaluation of ESL administration on the development of kindling epileptogenesis in the mouse. The amygdala kindling model, which allows repeated stimulation and threshold determination, was used to further evaluate ESL potency on generation and spread of partial onset seizures in fully kindled mice. Moreover, we aimed to compare the efficacy of treatment with the parent compound with that of its main metabolite eslicarbazepine as well as the (R)enantiomer, (R)-licarbazepine, in order to further explore the stereoselectivity of these enantiomers as anticonvulsants. This comparison is of particular interest as ESL is mainly, metabolized to eslicarbazepine, whereas oxcarbazepine (OXC) forms both metabolites eslicarbazepine and the (R)-enantiomer (Bialer and Soares-da-Silva, 2012). Thus, a comparison of the efficacy in seizure and chronic epilepsy models allows novel conclusions about the relative contribution of ESL as well as the ESL and/or OXC metabolites. Both ESL and eslicarbazepine produced a significant inhibitory effect on kindling progression, but (R)licarbazepine was found to be ineffective in preventing kindling progression. Similar findings were observed in the amygdala kindling model. For such a reason ESL, eslicarbazepine and (R)-licarbazepine were tested in maximal electroshock (MES) test in the mouse. Mice were used for the experiments as the metabolic pathways in this species reflect the metabolism in humans (Alves et al., 2008; Bialer and Soares-da-Silva, 2012; Hainzl et al., 2001).

Materials and methods Animals NMRI mice were purchased at a body weight of 20—35 g (Harlan Winkelmann Versuchstierzucht, Borchen, Germany; Elevage Janvier, Le Genest-Saint-Isle, France; or HarlanInterfauna, Barcelona, Spain) were used in experiments (as detailed in supplementary materials and methods). All animal care and handling was conducted in accordance with the European Directive number 86/609 and in compliance with the German Animal Welfare Act.

Corneal kindling procedure Female mice were stimulated twice daily (interstimulation interval 6—7 h) on twelve consecutive days as described earlier (Potschka and Löscher, 1999) and detailed in supplementary materials and methods. Vehicle (30% DMSO in Aq.

214

H. Potschka et al.

dest) or the compounds dissolved in 30% DMSO were administered intraperitoneally (i.p.) twice daily. Treatment was continued during the 12-day corneal stimulation procedure with administrations 15 min before the kindling stimulations in the morning and afternoon. The injection volume was 10 ml/kg.

vehicle injections (30% DMSO; volume of administration 10 ml/kg or 20 ml/kg). The rotarod test was performed in groups of 9—10 animals. The rotarod test was considered successfully completed when an individual animal succeeded to stay on the rotating rod for at least 1 min in one of three subsequent trials.

Electrode implantation and amygdala kindling procedure Fifteen animals were used to determine the optimal coordinates for electrode implantation for the present study. Thirty-six female mice were implanted and fully kindled as described in supplementary materials and methods. Determination of afterdischarge threshold was repeated at least two times to prove reproducibility before animals were used for drug testing.

Maximal electroshock (MES) test Male NMRI mice (n = 10 per group) were administered MES (50 mA, rectangular current: 0.6 ms pulse width, 0.4 s duration, 50 Hz) via corneal electrodes connected to a constant current shock generator (Ugo Basile 7801) and the number of animals exhibiting tonic convulsions was documented (Swinyard et al., 1952). ESL, eslicarbazepine and (R)-licarbazepine were evaluated at 25 mg/kg, administered p.o. 60 min before the test. The effects were compared with a vehicle (0.2% HPMC in distilled water) control group. Satellite groups of male mice were administered ESL, eslicarbazepine and (R)-licarbazepine under the same experimental conditions as those subjected to the MES test with the exception that the satellite animals did not receive the shocks.

Experiments in fully kindled mice In all experiments in fully kindled mice, the afterdischarge threshold was determined. Seizure duration was the time period of limbic and/or motor seizures. Afterdischarge duration was defined as the period of high amplitude spiking (at least 1 Hz frequency and twice the pre-stimulation amplitude) in the electroencephalogram (EEG) of the amygdala electrode, including the time of stimulation. Eslicarbazepine acetate, eslicarbazepine and (R)-licarbazepine were dissolved in the vehicle solution (30% DMSO in Aq. dest.). The compounds were administered intraperitoneally (i.p.) in a volume of 10 ml/kg bodyweight. In experiments with the highest dosage (300 mg/kg), the volume of administration was increased to 20 ml/kg bodyweight. The test compounds were administered 15 min before threshold determination. Data obtained after administration of these substances were compared with data obtained in the same mice after i.p. injection of vehicle (30% DMSO) 2—3 days before the drug experiment. Statistical significance of seizure data in the kindling model was calculated by the Wilcoxon Signed Rank test for paired replicates. The significance of differences in the number of animals showing seizure activity in response to electrical stimulation up to a maximum of 1200 ␮A were analyzed using Fisher’s exact test. Satellite groups of mice administered the tests substances under the same experimental conditions as those subjected to amygdala kindling with the exception that animals without electrode implantation and without stimulation were used for pharmacokinetics experiments as detailed in supplementary materials and methods.

Rotarod test Mice were handled and trained for rotarod testing until all animals reproducibly completed the trial under control conditions. The test was considered successfully completed once the animals maintained their equilibrium on the rod rotating at a constant speed of 15 rpm for 3 min (rotarod apparatus from Ugo Basile 7650; Comerio, Italy). The impact of treatment with the compounds eslicarbazepine acetate, eslicarbazepine and (R)-licarbazepine on rotarod performance was tested in these mice. The rotarod test was performed before drug injection and 15 min following administration. Additional groups received respective

Analyses of eslicarbazepine acetate and metabolite concentrations Brain and plasma concentrations of eslicarbazepine acetate, eslicarbazepine and (R)-licarbazepine were determined using a validated enantioselective LC—MS/MS assay, as previously described (Loureiro et al., 2011) as detailed in supplementary materials and methods.

Chemicals Eslicarbazepine acetate [(−)-(S)-10-acetoxy-10,11-dihydro5H-dibenzo/b,f/azepine-5-carboxamide], eslicarbazepine [(+)-(S)-10,11-dihydro-10-hydroxy-5H-dibenzo/b,f/azepine -5-carboxamide], (R)-licarbazepine [(−)-(R)-10,11-dihydro10-hydroxy-5H-dibenzo/b,f/azepine-5-carboxamide], 10,11-dihydrocarbamazepine (used as internal standard), and oxcarbazepine, were all synthesized in the Laboratory of Chemistry, BIAL — Portela & Ca , S.A., with purities >99.5%. Sodium pentobarbital was obtained from Sigma (St. Louis, MO) and dissolved in saline (0.9%, w/v) for i.p. administrations.

Statistical analysis Data are expressed as mean ± SEM. Significance of differences between groups in the corneal kindling were calculated by Kruskal—Wallis followed by Dunn’s post hoc test. Statistical significance of seizure data in the amygdala kindling model was calculated by the Wilcoxon Signed Rank test for paired replicates. Values that did not lie within three standard deviations of the mean were excluded from analysis (one animal from the (R)-licarbazepine 200 mg/kg group in the amygdala kindling). The significance of differences in the number of animals showing seizure activity in response to electrical stimulation up to a maximum of 1200 ␮A were analyzed using Fisher’s Exact Probability test. In the MES test the number of tonic convulsions and the number of deaths were recorded and data were statistically analyzed using

ESL in kindled seizures

215

A

Fisher’s Exact Probability tests. Differences were considered to be statistically significant when P < 0.05.

5

Progression of corneal kindling following the washout phase During the restimulation phase no significant differences were observed in mean seizure severity when comparing groups with prior treatment with the vehicle control group. A 100% seizure response (i.e. all mice exhibiting at least a score 3 seizure) was observed at day 3 and 4 of the

4 3

*

2 1

* **

0 0

*

*

*

*

2

* 4

*

* Vehicle ESL 30 mg/kg ESL 100 mg/kg

** * 6

8

10

12

Days of stimulation

B 5 Seizure severity

Twice daily treatment with ESL 30 mg/kg and 100 mg/kg exhibited an inhibitory effect on acquisition of kindling (Fig. 1A). As compared to the vehicle control group mean seizure severity proved to be significantly lower in the 100 mg/kg ESL treatment group at almost all stimulation sessions of the first seven days. The number of stimulations necessary to induce a seizure with a severity score of 5 and 6 was significantly increased in mice that received administrations of ESL 30 mg/kg and 100 mg/kg (Fig. 2D). The lowest dosage of ESL used in the present study (10 mg/kg) exhibited no significant effect on kindling acquisition. In the 100 mg/kg ESL treatment group one animal did not show a generalized seizure during the 12-day treatment phase. Whereas kindling acquisition was rather constant in the control group, animals which received 100 mg/kg ESL more often exhibited focal seizures or even no seizure activity although they already showed generalized seizure activity at a previous stimulation session. Twice daily treatment with eslicarbazepine 100 mg/kg exhibited an inhibitory effect on acquisition of kindling (Figs. 1B and 2B and E). As compared to the vehicle control group mean seizure severity proved to be significantly lower in the 100 mg/kg eslicarbazepine treatment group at all stimulation sessions of the first three days. The lower dosage of eslicarbazepine used in the present study (30 mg/kg) exhibited no significant effect on kindling acquisition. When treatment was terminated at day twelve 100% of the vehicletreated animals and of all treated animals had reached the kindling criterion, i.e. at least one generalized seizure (score 4—6). Twice daily treatment with (R)-licarbazepine 30 mg/kg and 100 mg/kg exhibited no inhibitory effect on acquisition of kindling (Fig. 1C). When seizure severity scores were compared between (R)-licarbazepine treated animals and control animals, no significant differences were determined except for one stimulation session, i.e. the stimulation in the afternoon of day 8. The number of stimulations necessary to induce a seizure with a severity score of 3, 4, 5, and 6 did not differ between treated mice and control mice (Fig. 2C). During the course of the experiment no adverse effects on motor performance were obvious in ESL, eslicarbazepine and (R)-licarbazepine treated animals.

4 3 2 1

* **

0 0

2

* * *

Vehicle Eslicarbazepine 30 mg/kg Eslicarbazepine 100 mg/kg

4

6

8

10

12

Days of stimulation

C 5 Seizure severity

Acquisition of kindling following corneal stimulation

Seizure severity

Results

4 3 2 1

Vehicle R-Licarbazepine 30 mg/kg R-Licarbazepine 100 mg/kg

0 0

2

4

6

8

10

12

Days of stimulation Figure 1 Effect of different dosages of twice daily eslicarbazepine acetate (ESL), eslicarbazepine and (R)-licarbazepine on acquisition of kindling, i.e. development of seizure severity upon repeated transcorneal stimulation. Data are mean values ± SEM shown for each stimulation session. Experiments were started with 15 mice per group. Due to mortality with ongoing stimulations animal numbers decreased to 9—13 mice per group at the end of the 12-day stimulation and treatment phase. *P < 0.05 compared to vehicle group.

216

H. Potschka et al.

Figure 2 Effect of different dosages of twice daily eslicarbazepine acetate (ESL), eslicarbazepine and (R)-licarbazepine on the number of stimulations to reach the respective seizure score. Data are mean values ± SEM. *P < 0.05 compared to vehicle group.

restimulation phase in all groups except for the group with prior 100 mg/kg ESL treatment. The latter group reached a 100% seizure response for a first time with the second stimulation at the fourth restimulation day.

Amygdala kindling Administration of ESL dose-dependently increased the focal seizure threshold (ADT), effects being significant at 200 and 300 mg/kg ESL (Fig. 3). It needs to be noted that in 1 out of 10 animals receiving 200 mg/kg ESL and in 7 out of 13 animals receiving 300 mg/kg ESL no seizure activity was observed until the maximum stimulation current of 1200 ␮A (Supplementary Table 1). In these instances 1200 ␮A were used for calculation and further data analysis. In response to 200 and 300 mg/kg ESL, threshold increases reached >289% and >1319%, respectively. Statistical analysis of seizure parameters was performed for the data obtained from all animals in which seizure activity was observed. Seizure duration and afterdischarge duration recorded at ADT were not significantly altered in the ESL-treated group. At threshold stimulation seizure severity was reduced in a dose-dependent manner by administration of ESL with a significant difference to the vehicle control experiment at 200 and 300 mg/kg ESL. The mean plasma and brain concentrations of ESL and its metabolites eslicarbazepine, (R)-licarbazepine and oxcarbazepine 15 min after a single i.p. administration of 100, 200 or 300 mg/kg ESL in the mouse are depicted in Table 1. Eslicarbazepine was the main entity in plasma followed by

ESL and oxcarbazepine; (R)-licarbazepine was not found in detectable amounts. The concentration of ESL and eslicarbazepine in plasma represented, respectively, 8.5—14.1% and 84.6—89.7% of circulating entities. In brain, eslicarbazepine was found to be the main entity followed by ESL and oxcarbazepine, (R)-licarbazepine remaining at non-detectable concentrations. However, the concentration of ESL and eslicarbazepine in brain represented, respectively, 37.3—44.8% and 51.5—62.7% of entities in brain. Because ESL concentrations in brain were surprisingly high, it was felt worthwhile to administer eslicarbazepine in an attempt to differentiate effects from ESL and eslicarbazepine. Following administration of eslicarbazepine the focal seizure threshold (ADT) increased significantly at 200 mg/kg (Fig. 3). In response to 200 mg/kg eslicarbazepine threshold increases reached 47%. Statistical analysis of seizure parameters was performed for the data obtained from all animals in which seizure activity was observed. Afterdischarge duration recorded at ADT was not significantly altered following treatment with eslicarbazepine. Seizure severity was significantly reduced following administration of 200 mg/kg eslicarbazepine. The brain levels of eslicarbazepine 15 min after the administration of 200 mg/kg eslicarbazepine were 78.7 ± 13.1 nmol/g (Table 1), which was similar to the sum of ESL (28.0 mol/g) and eslicarbazepine (37.5 nmol/g) in brain after the administration of 200 mg/kg ESL (Table 1). After the administration of 200 mg/kg eslicarbazepine, ESL and (R)-licarbazepine were not detectable in brain or plasma, and eslicarbazepine represented 91% of entities in brain; 9% was

ESL in kindled seizures

217

Figure 3 Experiments in fully kindled mice: effects of increasing doses of ESL (100, 200 and 300 mg/kg, i.p.) on (A) focal seizure threshold (afterdischarge threshold, ADT), (B) on seizure duration (SD1) at threshold stimulation, (C) on after-discharge duration (ADD1) at threshold stimulation and (D) on seizure severity (Racine score), and effects of eslicarbazepine and (R)-licarbazepine (both at 200 mg/kg, i.p.) on (E) focal seizure threshold (afterdischarge threshold, ADT), (F) on seizure duration (SD1) at threshold stimulation, (G) on after-discharge duration (ADD1) at threshold stimulation and (H) on seizure severity (Racine score). *P < 0.05 compared to vehicle group.

218

H. Potschka et al.

Table 1 Plasma and brain concentrations of eslicarbazepine acetate (ESL), eslicarbazepine and (R)-licarbazepine after 15 or 60 min administration (ip or po) of ESL, eslicarbazepine and (R)-licarbazepine. Values are means ± SEM of 3—5 determinations per group. mg/kg

ESL

Eslicarbazepine

R-Licarbazepine

Oxcarbazepine

100 100 100

13.3 ± 2.3 ND ND

92.9 ± 4.7 208.4 ± 23.9 ND

ND ND 279.9 ± 20.5

1.4 ± 0.1 4.7 ± 0.9 3.1 ± 0.3

100 100 100

7.5 ± 0.5 ND ND

14.6 ± 1.5 37.5 ± 4.4 ND

ND ND 55.9 ± 5.11

ND 0.4 ± 0.3 ND

100 100 100

ND ND ND

168.1 ± 10.1 202.1 ± 14.3 ND

ND ND 183.4 ± 36.0

8.7 ± 0.8 9.6 ± 0.6 6.1 ± 0.8

100 100 100

ND ND ND

67.7 ± 4.0 84.0 ± 1.08 ND

ND ND 85.4 ± 14.9

11.9 ± 1.2 11.1 ± 0.6 3.0 ± 0.9

200 200 200 300

21.9 ± 11.3 ND ND 22.9 ± 4.1

236.2 ± 72.1 479.2 ± 66.6 2.9 ± 0.4 282.8 ± 17.2

ND 5.3 ± 0.9 591.6 ± 43.6 ND

4.5 ± 0.9 14.4 ± 2.3 10.6 ± 1.0 5.7 ± 0.6

200 200 200 300

28.0 ± 10.6 ND ND 37.3 ± 3.2

37.5 ± 8.7 78.7 ± 13.1 ND 58.7 ± 4.0

ND ND 90.9 ± 2.7 ND

2.7 ± 0.3 8.4 ± 1.5 5.2 ± 0.4 4.1 ± 0.5

ND ND ND ND ND ND ND ND ND

8.7 ± 1.5 22.1 ± 2.4 84.3 ± 5.1 11.0 ± 1.4 29.4 ± 4.6 54.8 ± 12.7 ND 0.3 ± 0.1 06 ± 0.1

ND ND ND ND 0.3 ± 0.1 0.6 ± 0.2 9.4 ± 0.7 23.6 ± 2.5 42.6 ± 5.0

1.0 ± 0.1 4.6 ± 4.4 13.8 ± 2.6 1.5 ± 0.2 4.2 ± 0.6 9.4 ± 2.7 1.0 ± 0.1 2.5 ± 0.2 4.8 ± 0.4

15 min post dose (ip) Plasma nmol/ml ESL Eslicarbazepine R-Licarbazepine Brain nmol/g ESL Eslicarbazepine R-Licarbazepine 60 min post dose (ip) Plasma nmol/ml ESL Eslicarbazepine R-Licarbazepine Brain nmol/g ESL Eslicarbazepine R-Licarbazepine 15 min post dose (ip) Plasma nmol/ml ESL Eslicarbazepine R-Licarbazepine ESL Brain nmol/g ESL Eslicarbazepine R-Licarbazepine ESL 60 min post dose (po) Plasma nmol/ml ESL ESL ESL Eslicarbazepine Eslicarbazepine Eslicarbazepine R-Licarbazepine R-Licarbazepine R-Licarbazepine

10 25 50 10 25 50 10 25 50

ND = not detectable.

oxcarbazepine. The focal seizure threshold was not significantly affected after the administration of 200 mg/kg (R)-licarbazepine (Fig. 3). Statistical analysis of seizure parameters was performed for the data obtained from all animals in which seizure activity was observed. Seizure duration and afterdischarge duration recorded at ADT was not significantly altered following the administration of (R)licarbazepine. The brain levels of (R)-licarbazepine 15 min after the administration of 200 mg/kg (R)-licarbazepine were 90.9 ± 2.7 nmol/g (Table 1), which was similar to the amount of eslicarbazepine (78.7 nmol/g) in brain after the

administration of 200 mg/kg eslicarbazepine (Table 1). After the administration of 200 mg/kg (R)-licarbazepine, ESL and eslicarbazepine were not detectable in brain or plasma, and (R)-licarbazepine represented 95% of entities in brain; oxcarbazepine represented 5% of entities in brain. Except for one animal in the group receiving 200 mg/kg eslicarbazepine acetate, all animals successfully completed the rotarod test following administration of 100 or 200 mg/kg eslicarbazepine acetate, eslicarbazepine or (R)-licarbazepine. The administration of 20 ml/kg 30% DMSO resulted in motor impairment with a failure to complete

ESL in kindled seizures

A

219

EslicarbazepineR-Licarbazepine

ESL

MES-induced seizures (% reduction)

0

10 mg/kg 25 mg/kg 50 mg/kg

-50

* * -100

*

*

*

Figure 4 Effect of different dosages of eslicarbazepine acetate (ESL), eslicarbazepine and (R)-licarbazepine against maximal electroshock (MES) induced seizures. Data are percent change from control. *P < 0.05 compared to vehicle group.

the rotarod test in 40% of the animals. This vehicle solution was used in further drug experiments with 300 mg/kg eslicarbazepine acetate; at this dose level a failure to complete the rotarod test was observed in 80% of the animals (Supplementary Table 2). Maximal electroshock (MES) test Because there is lack of information on the anticonvulsant effects of (R)-licarbazepine, namely in mice—the most representative species with respect to the metabolism of both ESL and OXC in humans, it was felt worthwhile to check how this entity performs in the MES-induced seizures. Data from the literature indicates that ED50 values for ESL and eslicarbazepine are 23.0 ± 1.9 and 27.8 ± 3.2 mg/kg, respectively (Pires et al., 2011). In this set of experiments mice received oral doses of 10, 25 and 50 mg/kg (R)-licarbazepine, eslicarbazepine, ESL or vehicle (0.2% HMPC and 5 ml/kg) with a pretreatment time of 60 min before stimulation. Treatment with 25 mg/kg ESL and with 25 mg/kg eslicarbazepine significantly protected mice against MES induced seizures, whereas administration of (R)-licarbazepine (25 mg/kg) failed to provide significant protection (10% reduction in seizure frequency). A complete protection of mice against MES induced seizures was obtained when ESL, eslicarbazepine and (R)-licarbazepine were administered at 50 mg/kg (Fig. 4). In mice treated with ESL or eslicarbazepine, eslicarbazepine was the circulating entity with similar levels in both treatment groups, whereas (R)-licarbazepine was the main entity in mice treated with (R)-licarbazepine. The exposure to eslicarbazepine in eslicarbazepine-treated animals was similar to the exposure of (R)-licarbazepine in (R)-licarbazepinetreated mice (Table 1).

Discussion The present study was performed to determine the effect of administration of ESL, eslicarbazepine and (R)-licarbazepine in the mouse corneal and amygdala kindling models. Moreover, pharmacokinetic data were obtained for the pretreatment times and administration routes used in the present study. As previously described for mice (Hainzl et al., 2001; Alves et al., 2008), ESL was extensively

converted to eslicarbazepine, as has been observed in humans (Almeida et al., 2008a,b; Falcao et al., 2007; Maia et al., 2008; Perucca et al., 2011). Administration of ESL as well as of its 10-hydroxy metabolite eslicarbazepine significantly retarded corneal kindling development and significantly increased the afterdischarge threshold in fully kindled mice. These data indicate that administration of both compounds inhibits seizure initiation and can protect against focal seizure activity. At selected dosages thereof, ESL and eslicarbazepine exerted an effect on seizure severity. These data give evidence that the administration of the compounds can cause interference with seizure progression by inhibiting propagation of activity from the focus. The doses needed to significantly increase the seizure threshold in amydgala-kindled mice were substantially higher than the doses needed to increase the threshold in the MES test. This is in line with the standard anticonvulsants carbamazepine, phenytoin and valproate, which need higher doses to be effective in the kindling test than in the MES test (Löscher et al., 1993; Matagne and Klitgaard, 1998). Repeated bilateral transcorneal stimulation of mice induces kindling acquisition with seizures similar to those induced by amygdala kindling via depth electrodes in rats (Potschka and Löscher, 1999). When considering presumable commonalities between the amygdala kindling model in rats and the corneal kindling model in mice, the data may indicate that ESL and eslicarbazepine possess comparable features as drugs which proved to retard amygdala kindling like the clinically established drugs valproate, phenobarbital, lamotrigine, levetiracetam, or topiramate (Löscher et al., 1998; Silver et al., 1991; Stratton et al., 2003). Given the lack of an inhibitory effect of carbamazepine on progression of amygdala kindling (Silver et al., 1991), the present data could indicate that ESL and eslicarbazepine possess a superior profile in this respect as compared to carbamazepine. The effects in this study may for example be mediated through different or additional mechanisms to use-dependent neuronal sodium channel blockade. On the other hand, this may merely reflect differences between the kindling models and the species used, i.e. between the rat amygdala kindling model and the mouse corneal kindling model. In this respect, it is important to keep in mind that seizure initiation and spread differs in both models with origin in the limbic forebrain (with unilateral stimulation) vs. presumable spread from the cornea (bilateral stimulation) to limbic brain regions. The latter mode of stimulation may promote drug effects which are based on an inhibition of neuronal activity propagation. In this context, it would be of specific interest to determine the efficacy of further clinically established antiepileptic drugs in the corneal kindling model in order to allow a comparison with the effects of ESL and eslicarbazepine administration. Based on the present data, administration of ESL and eslicarbazepine has the potential to retard kindlinginduced epileptogenesis. The observed effects can reflect an anticonvulsant effect on partial onset seizures. Diseasemodifying or antiepileptogenic effects may also be involved. However, it needs to be considered that there was no evidence for a persistent effect when kindling was continued following wash-out.

220 As already described above, ESL is only hydrolysed to eslicarbazepine in humans and mice (Bialer and Soaresda-Silva, 2012), whereas oxcarbazepine serves as a prodrug of both 10-hydroxy metabolites, i.e. eslicarbazepine and (R)-licarbazepine, which appear in the plasma and urine in a 4:1 ratio (Schutz et al., 1986; Volosov et al., 1999). In this context, it is of specific interest to compare the efficacy following the administration of both enantiomers. Administration of eslicarbazepine resulted in significant effects in both kindling models not only retarding kindling progression but also increasing the afterdischarge threshold in fully kindled mice. In apparent contrast, administration of (R)-licarbazepine failed to produce respective effects in both models. These data are in accordance with the finding that treatment with eslicarbazepine was more potent in the MES test than treatment with the (R)-enantiomer. Altogether, the findings reported here raise doubts on the contribution of (R)-licarbazepine as an active anticonvulsant. Alhough (R)-licarbazepine is endowed with effects as an active voltage-gated sodium channel (VGSC) blocker (Hebeisen et al., 2011), this may not suffice to guarantee efficacy of this entity as an anticonvulsant. Considerable differences in the pharmacodynamic properties between eslicarbazepine and (R)-licarbazepine have been observed. The affinity of (R)-licarbazepine for VGSCs in the resting state versus the inactive was found to be 4-fold that for eslicarbazepine (Hebeisen et al., 2011), which may favor a better efficacy of the latter over the former. Another remarkable difference between eslicarbazepine and (R)-licarbazepine is concerned with the potency of (R)-licarbazepine as a blocker of voltage-gated potassiumchannels, namely KV 7.2 currents, whereas eslicarbazepine is devoid of effect (Soares-da-Silva et al., 2011). In fact, activation of voltage-gated potassium-channels (KV 7/M) during the initial stages of an action potential discharge suppresses later action potentials and inhibition of channel activity strongly enhances repetitive firing (Brown and Passmore, 2009). KV 7.2 knock-out mice have a reduced electroconvulsive threshold and increased sensitivity to convulsing agents (Peters et al., 2005). Finally, administration of eslicarbazepine, but not of (R)-licarbazepine, was effective in retarding kindling development during bilateral corneal stimulation in mice (Pekcec et al., 2011). This potential for eslicarbazepine treatment to elicit antiepileptogenic effects, in contrast to (R)-Licarbazepine, may relate to the ability of eslicarbazepine to effectively inhibit high and low affinity Cav 3.2 inward currents (Brady et al., 2011). An excellent predictive validity of amygdala kindling has been substantiated by comparison of experimental and clinical data (Löscher, 2002). Based on this comparison, data from amygdala kindled animals predict an efficacy of a test compound for treatment of partial seizures. In consideration of this knowledge, our data predict a clinical effect of ESL and its 10-hydroxy metabolite, eslicarbazepine, on partial onset seizures. This fits well with the outcome of the clinical experience with ESL in epilepsy treatment also considering data from the initial proof-of-concept phase II study (Elger et al., 2007) as well as three subsequent phase III studies in patients refractory to conventional antiepileptic drug (AED) therapy (Ben-Menachem et al., 2010; Elger et al., 2009; GilNagel et al., 2009). Long-term safety and maintenance of

H. Potschka et al. therapeutic effect was demonstrated in one-year open-label extensions of these studies (Halasz et al., 2010; Hufnagel et al., 2013). In conclusion, the data presented here provides evidence for an anticonvulsant effect of ESL treatment on partial-onset seizures, indicating that it may interfere with seizure progression by inhibiting propagation of activity from the focus. However, disease-modifying effects may also be involved. Administration of ESL may not merely cause suppression of seizure activity but may also provide a disease-modifying or antiepileptogenic effect. Future studies will be necessary to further evaluate a putative preventive effect, in particular when considering that restimulation following wash-out did not indicate a persistent effect. Eslicarbazepine was confirmed as an active metabolite that thus seems to significantly contribute to beneficial effects observed after administration of the parent compound ESL. The findings reported here raise doubts on the contribution of the OXC metabolite (R)-licarbazepine as an active anticonvulsant.

Disclosure This study was sponsored by BIAL — Portela & Ca , S.A. All authors were involved in the design or conduct of the study, the collection, management or analysis of the data, and the preparation or review of the manuscript. H. Potschka, has received research grants from BIAL — Portela & Ca , S.A., the sponsor of the studies. A. Loureiro and P. Soares-da-Silva were employees of BIAL — Portela & Ca , S.A. at the time of the studies.

Acknowledgments We confirm that we have read the Journal’s position on issues involved in ethical publication and affirm that this report is consistent with those guidelines. This study was supported by BIAL — Portela & Ca , S.A. We thank Heidrun Zankl, Christian Rathert and Carmen Meyer for their excellent technical assistance.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/ j.eplepsyres.2013.11.017.

References Almeida, L., Minciu, I., Nunes, T., Butoianu, N., Falcao, A., Magureanu, S.A., Soares-da-Silva, P., 2008a. Pharmacokinetics, efficacy, and tolerability of eslicarbazepine acetate in children and adolescents with epilepsy. Journal Clinical Pharmacology 48, 966—977. Almeida, L., Potgieter, J.H., Maia, J., Potgieter, M.A., Mota, F., Soares-da-Silva, P., 2008b. Pharmacokinetics of eslicarbazepine acetate in patients with moderate hepatic impairment. Europeran Journal Clinical Pharmacology 64, 267—273. Alves, G., Figueiredo, I., Castel-Branco, M., Lourenco, N., Falcao, A., Caramona, M., Soares-da-Silva, P., 2008. Disposition of

ESL in kindled seizures eslicarbazepine acetate in the mouse after oral administration. Fundamental & Clinical Pharmacology 22, 529—536. Ben-Menachem, E., Gabbai, A.A., Hufnagel, A., Maia, J., Almeida, L., Soares-da-Silva, P., 2010. Eslicarbazepine acetate as adjunctive therapy in adult patients with partial epilepsy. Epilepsy Research 89, 278—285. Bialer, M., Soares-da-Silva, P., 2012. Pharmacokinetics and drug interactions of eslicarbazepine acetate. Epilepsia 53, 935—946. Brady, K., Hebeisen, S., Konrad, D., Soares-da-Silva, P., 2011. The effects of eslicarbazepine, R-licarbazepine, oxcarbazepine and carbamazepine on ion transmission Cav 3.2 channels. Epilepsia 52 (Suppl. 6), 260. Brown, D.A., Passmore, G.M., 2009. Neural KCNQ (Kv7) channels. British Journal of Pharmacology 156, 1185—1195. Elger, C., Bialer, M., Cramer, J.A., Maia, J., Almeida, L., Soaresda-Silva, P., 2007. Eslicarbazepine acetate: a double-blind, addon, placebo-controlled exploratory trial in adult patients with partial-onset seizures. Epilepsia 48, 497—504. Elger, C., Halasz, P., Maia, J., Almeida, L., Soares-da-Silva, P., 2009. Efficacy and safety of eslicarbazepine acetate as adjunctive treatment in adults with refractory partial-onset seizures: a randomized, double-blind, placebo-controlled, parallel-group phase III study. Epilepsia 50, 454—463. Falcao, A., Maia, J., Almeida, L., Mazur, D., Gellert, M., Soaresda-Silva, P., 2007. Effect of gender on the pharmacokinetics of eslicarbazepine acetate (BIA 2-093), a new voltage-gated sodium channel blocker. Biopharmaceutics & Drug Disposition 28, 249—256. Gil-Nagel, A., Lopes-Lima, J., Almeida, L., Maia, J., Soaresda-Silva, P., 2009. Efficacy and safety of 800 and 1200 mg eslicarbazepine acetate as adjunctive treatment in adults with refractory partial-onset seizures. Acta Neurologica Scandinavica 120, 281—287. Goddard, G.V., McIntyre, D.C., Leech, C.K., 1969. A permanent change in brain function resulting from daily electrical stimulation. Experimental Neurology 25, 295—330. Hainzl, D., Parada, A., Soares-da-Silva, P., 2001. Metabolism of two new antiepileptic drugs and their principal metabolites S(+)and R(−)-10,11-dihydro-10-hydroxy carbamazepine. Epilepsy Research 44, 197—206. Halasz, P., Cramer, J.A., Hodoba, D., Czlonkowska, A., Guekht, A., Maia, J., Elger, C., Almeida, L., Soares-da-Silva, P., 2010. Longterm efficacy and safety of eslicarbazepine acetate: results of a 1-year open-label extension study in partial-onset seizures in adults with epilepsy. Epilepsia 51, 1963—1969. Hebeisen, S., Brady, K., Konrad, D., Soares-da-Silva, P., 2011. Inhibitory effects of eslicarbazepine acetate and its metabolites against neuronal voltage-gated sodium channels. Epilepsia 52 (Suppl. 6), 257—258. Hufnagel, A., Ben-Menachem, E., Gabbai, A.A., Falcao, A., Almeida, L., Soares-da-Silva, P., 2013. Long-term safety and efficacy of eslicarbazepine acetate as adjunctive therapy in the treatment of partial-onset seizures in adults with epilepsy: Results of a 1-year open-label extension study. Epilepsy Research 103, 262—269. Lopes-Lima, J., G-NA, Maia, J., Almeida, L., Soares-da-Silva, P., 2008. Long-term treatment of partial epilepsy with eslicarbazepine acetate (ESL): results of a one-year open-label extension of study BIA-2093-303. Epilepsia 49 (Suppl. 7), 441—442. Löscher, W., 2002. Animal models of epilepsy for the development of antiepileptogenic and disease-modifying drugs. A comparison of the pharmacology of kindling and post-status epilepticus models of temporal lobe epilepsy. Epilepsy Research 50, 105—123. Löscher, W., Hönack, D., Rundfeldt, C., 1998. Antiepileptogenic effects of the novel anticonvulsant levetiracetam (ucb LO59) in the kindling model of temporal lobe epilepsy. Journal of Pharmacology and Experimental Therapeutics 284, 474—479.

221 Löscher, W., Rundfeldt, C., Honack, D., 1993. Pharmacological characterization of phenytoin-resistant amygdala-kindled rats, a new model of drug-resistant partial epilepsy. Epilepsy Research 15, 207—219. Loureiro, A.I., Fernandes-Lopes, C., Bonifacio, M.J., Wright, L.C., Soares-da-Silva, P., 2011. Hepatic UDP-glucuronosyltransferase is responsible for eslicarbazepine glucuronidation. Drug Metabolism and Disposition: The Biological Fate of Chemicals 39, 1486—1494. Maia, J., Almeida, L., Falcão, A., Soares, E., Mota, F., Potgieter, J.H., Potgieter, M.A., Soares-da-Silva, P., 2008. Effect of renal impairment on the pharmacokinetics of eslicarbazepine acetate. International Journal of Clinical Pharmacology and Therapeutics 46, 119—130. Matagne, A., Klitgaard, H., 1998. Validation of corneally kindled mice: a sensitive screening model for partial epilepsy in man. Epilepsy Research 31, 59—71. McIntyre, D.C., Gilby, K.L., 2009. Kindling as a model of human epilepsy. The Canadian Journal of Neurological Sciences 36 (Suppl. 2), S33—S35. Pekcec, A., Potschka, H., Soares-da-Silva, P., 2011. Effects of eslicarbazepine acetate and its metabolites in the corneal kindling model of epilepsy. Epilepsia 52 (Suppl. 6), 257. Perucca, E., Elger, C., Halasz, P., Falcao, A., Almeida, L., Soaresda-Silva, P., 2011. Pharmacokinetics of eslicarbazepine acetate at steady-state in adults with partial-onset seizures. Epilepsy Research 96, 132—139. Peters, H.C., Hu, H., Pongs, O., Storm, J.F., Isbrandt, D., 2005. Conditional transgenic suppression of M channels in mouse brain reveals functions in neuronal excitability, resonance and behavior. Nature Neuroscience 8, 51—60. Pires, N., Palma, N., Loureiro, A.I., Bonifacio, M.J., Wright, L.C., Soares-da-Silva, P., 2011. Effects of eslicarbazepine acetate, eslicarbazepine, carbamazepine and oxcarbazepine in the maximal electroconvulsive shock test in the mice. Epilepsia 52 (Suppl. 6), 118. Potschka, H., Löscher, W., 1999. Corneal kindling in mice: behavioral and pharmacological differences to conventional kindling. Epilepsy Research 37, 109—120. Sato, M., Racine, R.J., McIntyre, D.C., 1990. Kindling: basic mechanisms and clinical validity. Electroencephalography and Clinical Neurophysiology 76, 459—472. Schutz, H., Feldmann, K.F., Faigle, J.W., Kriemler, H.P., Winkler, T., 1986. The metabolism of 14C-oxcarbazepine in man. Xenobiotica; The Fate of Foreign Compounds in Biological Systems 16, 769—778. Sierra-Paredes, G., Sierra-Marcuno, G., Loureiro, A.I., Wright, L.C., Soares-da-Silva, P., 2011. Effects of eslicarbazepine acetate on acute and chronic latrunculin A-induced seizures and extracellular amino acid levels in the mouse hippocampus. Epilepsia 52 (Suppl. 6), 119. Silver, J.M., Shin, C., McNamara, J.O., 1991. Antiepileptogenic effects of conventional anticonvulsants in the kindling model of epilepsy. Annals of Neurology 29, 163—356. Soares-da-Silva, P., Bulling, A., Hebeisen, S., Konrad, D., 2011. The effects of eslicarbazepine, R-licarbazepine and carbamazepine on ion transmission through Kv 7.2 channels. Epilepsia 52, 258—259. Sperling, M., Harvey, J., Biraben, A., Galimberti, C., Kowacs, P., Hong, S.B., Cheng, H., Blum, D., Nunes, T., Soares-da-Silva, P., 2013. Adjunctive eslicarbazepine acetate in patients with refractory partial-onset seizures: efficacy results of a 12 week randomized placebo-controlled study. In: 67th Annual Meeting of the American Epilepsy Society, Washington, D.C. Stratton, C.S., Large, C.H., Cox, B., Davies, G., Hagan, R.M., 2003. Effects of lamotrigine and levetiracetam on seizure development in a rat amygdala kindling model. Epilepsy Research 53, 95—106.

222 Swinyard, E.A., Brown, W.C., Goodman, L.S., 1952. Comparative assays of antiepileptic drugs in mice and rats. The Journal of Pharmacology and Experimental Therapeutics 106, 319—330. Torrao, L., Machado, R., Pires, N., Palma, N., Bonifacio, M.J., Wright, L.C., Soares-da-Silva, P., 2011. Effects of eslicarbazepine acetate, eslicarbazepine, carbamazepine and

H. Potschka et al. oxcarbazepine in the 6-HZ psychomotor seizure model in the mice. Epilepsia 52 (Suppl. 6), 118—119. Volosov, A., Xiaodong, S., Perucca, E., Yagen, B., Sintov, A., Bialer, M., 1999. Enantioselective pharmacokinetics of 10hydroxycarbazepine after oral administration of oxcarbazepine to healthy Chinese subjects. Clinical Pharmacology and Therapeutics 66, 547—553.

Effect of eslicarbazepine acetate in the corneal kindling progression and the amygdala kindling model of temporal lobe epilepsy.

The present study was aimed at determining the effect of eslicarbazepine acetate (ESL), eslicarbazepine and (R)-licarbazepine administration in the mo...
803KB Sizes 0 Downloads 0 Views