Effects of axitinib upon chronic epileptic activity abstract

Tyrosine-kinase (tyr-kinase) was identified as a principal player for kindled seizures development, and inhibition of tyr-kinase is recognized as a new approach for the epileptic manifestations treatment. The present investigations aims to investigate effects of specific inhibitor of vascular endothelial growth factor receptor (VEGFR)-1, VEGFR-2, and VEGFR-3 generic axitinib upon pentylenetetrazol (PTZ) - induced kindled convulsions, and to compare axitinib effects with diazepam. Wistar rats kindled with PTZ (30,0 i.p.) during 21 days up to a stage of generalized convulsions stage 5, have been used for the observation. Assessment of seizures immediately after the completion of kindling procedure was performed. Axitinib was administered 10 days perorally (p.o.) with PTZ testing dosage injection in 60 min after last epileptogen injection. PTZ was administered to diazepam treated groups in 30 min from the moment of single intraperitoneal (i.p.) diazepam injection. Axitinb prevented generalized convulsions in 5 out of 8 kindled rats in a dose of 5.0 mg/kg, and pronouncement of effect was comparable with such one caused by diazepam in a dose of 1.5 mg/kg. Axitinib in dose of 10.0 mg/kg completely protected generalized convulsions. The conclusion was made that specific antagonist of VEGF axitinib is able to cause anticonvulsive action on chronic PTZ seizures. Observed effectiveness of axitinib is in favor of possibility of tyr-kinase - depended angiogenesis contribution to pathogenesis of PTZ kindling.

Key words: tyrosine-kinase, chronic epileptic activity, antiepileptics.

Introduction

The pathogenesis of epileptic syndrome develops along with the breaking down blood - brain barrier (BBB) function [14, 17]. Among others, remodeling of blood microcirculation on the basis of angiogenesis makes first-line contribution for increasing of permeability of BBB [10]. The absence of pericytes, in newly developed capillaries, is responsible for such increased leakage of their walls [10]. In patients who suffered from chronic intractable temporal lobe epilepsy, significantly higher vessel density in different zones of hippocampi than such one in non-epileptic patients,was shown, and direct positive correlation between vessel density and frequency of seizures was established as well [14].

Vascular endothelial growth factor (VEGF) is responsible for neoangiogenesis, and synthesis of VEGF is promoted by tyrosine - kinase [3]. Antitumor drugs activity is realized via inhibition of tyr- kinase [15, 16] and their antiseizure effectiveness was proved as well [1, 9]. Thus, it was established that blocking of angiogenesis with repeated sunitinib - drug which penetrates BBB, and inhibits tyr-kinase, prevented the development of seizures and hippocampal atrophy in pilocarpine rat model of mesial temporal lobe epilepsy [2]. Exploration of a chemical-genetic approach enabled a conclusion to be reached, that inhibitors of tyr-kinase were regarded as a new avenue for epilepsy treatment [9].

For testing effectiveness of tyr-kinase inhibitors, which poorly penetrate BBB, those models of epileptic syndrome should be explored which are characterized by increased BBB permeability. It was shown that PTZ induced the leakage of BBB on a timely basis [5], while a more stable effect was evident in PTZ kindling as well [8]. Preliminary results gained by [4] were in favor for the suppression of development of PTZ kindling under condition of peroral tyr-kinase inhibitor axitinib administration.

Earlier the critical role played by tyr-kinase in the development of amygdalar ES kindling has been clearly established, and i.c.v. administrations of antibodies to tyr-kinase receptor prevented kindling seizures appearance [9].

One of most selective inhibitors of VEGF receptor tyrosine kinase is axitinib, which engendered its activity against VEGFR-1, VEGFR-2, and VEGFR-3, and in contrast to first-generation inhibitors, axitinib has no substantial inhibitory effect on PDGFRs, B-Raf, c-Kit, and Flt-3 [11, 15]. Axitinib is approved as an anti-angiogenic agent for treating renal cell carcinoma, and it‘s potency in vitro is higher than that of the first-generation VEGFR inhibitors such as sunitinib and sorafenib [3, 15, 16].

That is why the main aim of this work was to investigate effects of axitinib upon PTZ-induced kindled convulsions and compare them with acute PTZ-induced seizures Taking into consideration that PTZ-kindled convulsions are highly sensitive to the antiseizure action of diazepam [7] the effectiveness of axitinib was compared with diazepam.

Materials and Methods

Experiments were performed on male Wistar rats with initial body weight 210-250 g. Animals were kept in standard conditions (constant temperature 23^o C, and relative humidity 60%, 12 hrs dark/light cycles, standard diet and tap water were given ad libitum) and were acclimatized to laboratory conditions for at least 7 days prior to experimentation. At the stage of experimental work planning principles outlined ARRIVE guidelines and the Basel declaration (http://www.basel-declaration.org) including the 3R concept have been taken into consideration.

Odessa National Medical University Bioethic Committee (UBC) approval (No17) dated 19/03/2013 was obtained before the start of the study.

Kindled convulsions were induced as described previously [6]. PTZ (—Sigma Aldrich”) was given intraperitoneally (i.p.) daily in a dose of 30.0 mg/kg for 21 days. The severity of convulsions was evaluated according to the following criteria: 0, absence of symptoms of seizures; 1, facial tremor and separate myoclonic jerks; 2, whole-body clonic convulsions; 3, clonic convulsions of the whole body with rearings; 4, generalized clonic-tonic convulsions with rearings and falling; and 5, repeated convulsions as at stage 4 or lethal outcome as a result of a seizure fit. Those rats, which failed into generalized fits, as a response to both 20-th and 21-th PTZ injections, were taken for further observations and screening effects of compounds.

Table 1 - Effect of axitinib and diazepam® upon 30.0 mg/kg i.p. PTZ-induced kindling

Statistics derived by Kruscall-Wallis and Mann-Whitney U tests.

I.p. administration of diazepam® to kindled animals (0.5; 1.5 and 5.0 mg/kg) resulted in a dosedependent seizure-protecting effect that was statistically significant for two doses, 1.5 and 5.0 mg/kg (Table 1), p<0.05 as compared with controls. Both doses prevented stage 4 seizures in prevalent majority of rats in each groups (7 out of 8 and 6 out of 7 correspondently).

Axitinib (—Sigma Aldrich”) was administered in doses of 1.0 - 10.0 mg/kg, per orally (p.o.), and diazepam® (“Calmpose”,“Ranbaxy Diagnostics”, India) was injected in doses of 0.5 - 5.0 mg/kg, i.p. Both compounds were dissolved in 5.0% methyl cellulose (Methocel, —Sigma Aldrich”) and axitinib was administered in 60 min before PTZ during last 10 days of kindled PTZ injections and convulsions were

estimated after 21-t PTZ injection. Diazepam® was administered in 30 min before 21-t injection of PTZ. Acute seizures were observed after 10 daily administrations of a axitinib (5.0 and 10.0 mg/kg, p.o.), and PTZ was administered in 60 min after the last (10-th) axitinib administration, while diazepam was administered in 30 min before testing usage of PTZ. Control animals were treated with Methocel only.

Data were analyzed by nonparametric analysis of variance ANOVA (Kruskall-Wallis test), followed by a Mann-Whitney test when appropriate; p<0.05 was accepted as a defining statistical difference.

Results

Behavioral characteristics of the convulsions in kindled rats

Repeated i.p. administration of PTZ (30.0 mg/kg) resulted in progressive development of seizure manifestations, starting from the third to sixth injection. At the moment of completion of kindling - after the 21-t PTZ administration, prevalent number of rats (two thirds) demonstrated generalized clonic - tonic fits. Those rats with generalized seizures were used for further investigations.

Effects of axitinib and diazepam

P.o. administration of axitinib in doses of 1.0; 5.0 and 10.0 mg/kg resulted in a dose-dependent decrease in the severity of kindled convulsions. Statistical significance was achieved at a dosage of 5.0 mg/kg (Table 1) at which dose 5 of 8 animals were protected against stage 4-5 seizures (p<0.05) as compared with controls. When administered in a dose of 10.0 mg/kg, axitinib® completely prevented development of stage 4-5 convulsions.

Discussion

Hence, gained data revealed that axitinib caused anticonvulsive action on the PTZ - kindled seizures in rats, which was pronounced in the prevention of generalized clonic-tonic fits. It should be stressed that axitinib poorly crosses BBB [13] and BBB is poorly penetrable for other inhibitors of tyr-kinase, depending upon constellation of transport proteins P-glycoprotein (ABCB1) and breast cancer resistance protein (ABCG2) [12]. That is why gained data, showing effectiveness of axitinib used in rather low doses upon kindled seizures, are in favor for the increasing of BBB permeability as a result of PTZ kindling establishment.

Such BBB deteriorations might be connected with different mechanisms of PTZ kindling development. Thus appearance of BBB-impermeable contrast agent in the parenchyma of the diencephalon, hippocampus and cerebral cortex, observed in 15 min after PTZ-induced general seizures in mice, was explained by increased NO production and induction BBB failure [5]. Angiogenesis, as another mechanism of BBB leakage in kindled rats, is much longer with the time of its manifestation and obviously is not contributive to acute PTZ convulsions. Hence, it might be assumed that, in the course of PTZ kindling, the pronouncement of BBB deterioration is progressively increased in parallel to assumed angiogenesis, which explains the effectiveness of axitinib.

Conclusion. Obtained data and analysis of mechanisms of axitinib effects are in favor of a possible role played by VEGF and remodeling of vessels in PTZ - kindled seizures development.

Financial support: Ministry of HealthCare of Ukraine.

Conflict of interests: there are any conflicts.

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