Oxidative stress suppressiOn cOntributes tO antiseizure actiOn Of axitinib and rapamycin in pentylenetetrazOl-induced kindling

Rapamycin and axitinib block different kinases in signaling pathways such as PI3K-Akt-mTOR and BDNF-TrkB, respectively. Both have antiseizure and antioxidative actions, which justify studying the combined effects of these drugs upon seizures and oxidative stress in the chronic model of epilepsy. The investigation aimed to look for the combined effect of rapamycin and axitinib upon pentylenetetrazol (PTZ)-kindled seizures and oxidative stress. Experiments were performed on 300 twoto four-month-old Wistar male rats, which had been kindled daily with PTZ (35.0 mg/kg, i.p.). Malondialdehyde (MDA) level, superoxide dismutase (SOD) activity, and glutathione (GSH) level were determined in brain tissues of kindled rats before and after the treatment. The analysis of antiseizure and antioxidative actions was performed using ED50 of rapamycin and axitinib, with their combined administration using graded dosages of ED50 of each drug. The median effective dose (ED50 ) for rapamycin and axitinib was 0.93 and 4.97 mg/kg, respectively. ED50 of rapamycin when combined with axitinib (2.0 mg/kg) was 0.60 mg/kg, which was reduced by 35.6% when compared with the ed50 administered alone (P < 0.05). The MDA level increased from 152.9±24.8 to 388.3±49.2 nmol/mg of protein (P < 0.05), while SOD activity reduced from 11.14±2.33 to 3.54±1.08 IU/mg of protein (P < 0.05) in brain tissues of the kindled rats. Combined treatment with rapamycin (0.56 mg/kg, i.p.) and axitinib (2.0 mg/ kg, i.p.) resulted in a significant rise in SOD activity (11.09±1.86 IU/mg) and GSH level (7.32±1.34 μg/mg) when compared with the kindled rats (P < 0.05). Combined axitinib and rapamycin therapy have an antiepileptic and antioxidative effect on PTZ-kindled seizures.


introduction
Pharmacologically resistant forms of epilepsy, which are observed in one-third of patients, are a great challenge for antiepileptic treatment [1].
The antiepileptic effect of axitinib on the pentylenetetrazol (PTZ)-induced kindling rat model was shown earlier [2], and suppression of tyrosine kinase type B was identified as a new target [3]. Similarly, Zeng et al. [4] suggested that inhibition of the mammalian target of rapamycin (mTOR) with rapamycin suppressed kainate-induced spontaneous epilepsy. Further, epileptic status initiation with pilocarpine [5,6] and the effect of rapamycin on seizure develop ment was reported [7]. Thus, mTOR pathway inhibitors are recognized as prospective antiepileptic compounds [8].
aim. To prove the antioxidative and antiseizure effects of two drugs (axitinib and rapamycin) and their combination in PTZ-kindled rats. materials and methods experimental animals. Experiments were performed on 300 male Wistar rats (two to four months old) with the initial body weight of 180-270 g. Animals were kept in standard conditions (constant temperature 23°C, relative humidity 60%, 12 h dark/ light cycles; standard diet and tap water were given ad libitum) and were acclimatized to laboratory conditions at least seven days before the experiment. All experiments were carried out following the National Institutes of Health Guidelines for the care and use of laboratory animals and the European Council Directive on 24 November 1986 for Care and Use of Laboratory Animals (86/609/EEC). The experiments were approved by the Odesa National Medical University Bioethics Committee (UBC) (approval No. 3 dated 14/03/2016) before the study. epilepsy model. Kindled seizures were induced, as described previously [17]. PTZ (Sigma Aldrich, St. Louis, MO, USA) was given intraperitoneally (i.p.) daily at a dose of 35.0 mg/kg for 21 days. The severity of seizures was evaluated accordin to the following criteria: 0, absence of symptoms of seizures; 1, facial tremor and separate myoclonic jerks; 2, whole-body clonic seizures; 3, clonic seizures of the whole body with rearings; 4, generalized clonic-tonic seizures with rearings and falling; and 5, repeated seizures as at stage 4 or lethal outcome as a result of seizures. Rats that demonstrated generali zed seizures after both the 20 th and 21 st PTZ injections were taken for further observations and evalua tion effects of compounds.
Study design and experimental group. According to the study design, three main protocols were undertaken (Fig. 1).
Kindling model creation aimed to determine the axitinib and rapamycin median effective dose (ED 50 ), starting with 176 rats that demonstrated fully developed generalized seizures in response to the 20th and 21st PTZ administrations (Fig. 1, protocol  a). Saline i.p. administration was used as a control group. At 24 h after the last injection, all rats of the control group (7 rats) and eight kindled rats were euthanized, and their brains were collected for biochemical analysis (Fig. 1, protocol a). The remaining kindled animals (169 rats) were randomly subdivided for ED50 determination of axitinib (86 rats) and rapamycin (83 rats) (Fig. 1, protocol B). Each group was randomly subdivided into subgroups based on graded doses of drugs. Treatment started within 24 h after the last PTZ administration and was performed for ten days. In 24 h after the 10th administration, two testing trials with PTZ (35.0 mg/kg, i.p.) were performed for ED 50 determination. The number of rats treated with specific doses used in each trial is given in protocols B and C.
The number of rats with the absence of generalized seizures was taken into account as a positive result of the treatment and it was used of ED 50 , and its error calculation. At 24 h after the PTZ administration, eight rats from each group treated with the maximal dosage of drugs were sacrificed for brain tissue biochemical analysis (Fig. 1, protocol B).
The combined effect of axitinib and rapamycin was investigated on another group of kindled rats (117 animals), which demonstrated generalized seizures as a response to the last two kindled PTZ injections ( Fig. 1, protocol C). Animals were randomly assigned to subgroups aimed for three trials of rapamycin ED 50 determination under conditions of axitinib (2.0 mg/kg, i.p.) treatment. Each group was treated daily with different dosages of rapamycin (20, 40, 60, and 80% of its ED 50 as determined in protocol B). Treatment started at 24 h after the last kindled PTZ administration and lasted ten days. At 24 h after the last drug administration, PTZ (35.0 mg/kg, i.p.) was injected, and triple ED 50 of rapamycin was determined. At 24 h after the moment of PTZ administration, eight rats treated with rapamycin (0.56 mg/kg, i.p.) were sacrificed, and their brains were collected for biochemical analysis.
With the aim to control for the effects of the dimethyl sulfoxide (DMSO) solvent, seven kindled rats were assigned additionally for ten days of DMSO ad- Biochemical investigations of oxidative stress. The animals were euthanized in protocols a, B, and C ( Fig. 1) using carbon dioxide, and the brains were removed and maintained over dry ice until the determination of oxidative tissue damage.
The tissue was homogenized in ice-cold (4°C) 0.052 M sodium phosphate buffer (pH 7.0) and 0.4 mM EDTA to produce a 10% homogenate. The homogenate was centrifuged at 10,000 × g at 4°C for 30 min, and the supernatant was separated for further measurements.
Lipid peroxidation was evaluated via measuring thiobarbituric acid (TBA) -active substances [18]. The spectrophotometry was performed at 530 nm. The extinction coefficient of 1.56×10 5 mol/l −1 cm −1 was used to determine the malondialdehyde (MDA) level, and results were expressed as nmol/mg of protein.
The superoxide dismutase activity (SOD, EC 1.15.1.1) was measured using the previously described method Kono [19]. Following this method, the homogenate's supernatant was incubated with nitroblue tetrazolium and hydroxylamine hydrochloride and monitored spectrophotometrically at 560 nm. The percentage inhibition of the rate of nitroblue tetrazolium reduction to 50% of the maximum was then calculated as one unit of SOD activity . Results were expressed as IU/mg of protein.
The glutathione (GSH) level was estimated using the method described by Sedlak and Lindsay [20]. The assay used 5′5dithiobis 2nitrobenzoic acid that binds to the thiol group to give a colored compound detected at 412 nm. The results were expressed in µg/mg of protein.
Protein concentrations were determined by Lowry assay [21], using bovine serum albumin as the standard.
Administration of investigated compounds. In accordance with the study design in protocol B (Fig. 1), axitinib (Sigma Aldrich, St. Louis, MO, USA) was administered in doses of 0.5, 1.5, 4.5, 10.0 mg/kg, i.p. and rapamycin (Pfizer, New Yourk, NY, USA) was administered in doses of 0.1, 0.3, 1.0, 3.0 mg/kg, i.p. Both compounds were dissolved in DMSO and delivered in 0.200.30 ml at 60 min before PTZ administration. In protocol C, rapamycin was administered in dosages of 0.19, 0.37, 0.56 and 0.75 mg/kg combined with axitinib treatment at a dose of 2.0 mg/kg, i.p. (Fig. 1).
Statistical analysis. The values of biochemical measurements were compared using one-way ANOVA and Tukey's honestly significant differen ce (HSD) post hoc test. Data were presented as a mean value (M) and standard error of the mean (SEM). P values < 0.05 were considered significant. To avoid the influence of outliers, only observations falling between the median±3.0 standard deviations of the sample were included in the dataset. The Shapiro Wilk test for normality was used. Calculation of ED 50 and its error was performed using https:// www.aatbio.com/tools/ed50-calculator/. For comparison, the ED 50 t-test for two means was used with a significance level (α) = 0.05. Linear regression was employed for dose-dependence studies. For statistical analysis, the program SPSS Statistics (IBM, New York, NY, USA) was used.

results and discussion
Behavioral characteristics of the seizures in kindled rats (protocol A). Seizures starting from the third to sixth injection and that were progressive in their development. The moment of kindling completion and inclusion of animals into the experimental group was recognized as generalized seizures induced with the two last PTZ administrations.
Administrations of rapamycin at the dosage of 0.1 mg/kg, i.p., prevented seizures in one out of 11 rats (9.1%). A ten times larger dosage (1.0 mg/ kg) prevented seizure fits in five out of nine (55.6%), while the highest dosage (3.0 mg/kg, i.p.) effectively protected seven out of ten (70.0%) rats from generali zed seizures. The ED 50 of rapamycin was 0.94±0.09 mg/kg (Fig. 2, B).
Searching for combined effects of drugs (protocol C). Rapamycin treatment in a dosage of 40% from the ED 50 (0.37 mg/kg) given under conditions of daily axitinib administration (40% of ED 50 , 2.0 mg/ kg) prevented generalized seizure fits in three out of nine (33.3%) rats. A two times larger dosage (80% of ED 50 , 0.75 mg/kg) effectively protected nine out of ten (90.0%) rats from generalized seizures.
The data obtained in this study favored the dosedependent effectiveness of axitinib and rapamycin against PTZ-kindled generalized seizure fits. Also, combined administration revealed a better effect against behavioral seizures and metabolic indices of oxidative stress. The increased effectiveness was proved with the significant reduction of the ED 50 of rapamycin when combined with axitinib and a significant increase of SOD activity and GSH level compared with the corresponding values in kindled rats. In addition, the reduction of MDA observed in rats treated with both axitinib and rapamycin was more pronounced than in rats with separate drug administration.
Hence, the obtained data lead to the assumption that ROS production by itself represents the mechanism of kindled seizure development. Such an assumption is in concordance with Zhu et al. [11], who reported excessive MDA production and decreased enzymatic activities of SOD and glutathione peroxidase (GSHPX) in the hippocampus in fully PTZ kindled mice. Suppression of ROS production might also underlie or significantly contribute to the combined effect of the investigated drugs' antiseizure action. Such an assumption corresponds with data on reducing MDA, SOD, and GSHPX caused by rapamycin in the model of testicular torsion-detorsion injury [22]. Antioxidant effects caused by rapamycin/mTOR blockade have been shown under different pathologic conditions [23,24].
For axitinib, a more subtle antioxidant action, if any, might be assumed as far as oxidative stress mediated genotoxic effects are recognized as the primary mechanism of antitumor activity, realized in concordance with the targeting of receptors of vascular endothelial growth factor (VEGFR) 1-3 [25].
Besides VEGFRtargeted effects also include oxidative DNA damage, leading to mitotic catastrophe and a cellular senescence program. Nevertheless, it was shown that the analogous inhibitor of tyrosine kinase B, sunitinib, might even act as an antioxidant by ameliorating lipid peroxidation and increasing the GSH level in cisplatintreated mice [25].
Hence, considering that the expression of brain derived neurotrophic factor (BDNF) is linked with ROS production, the presence of a functional interaction between mTOR and BDNFTrk [26,27] creates the basis for the synergy of the antiseizure action of rapamycin and axitinib. Our study findings suggest the combined usage of these two drugs for the blockade of two different kinase signaling pathways -PI3KAktmTOR and BDNFTrkB -to improve the effectiveness of epilepsy treatment.
Conclusions. The study data favor the role played by ROS production in brain tissue for the development of PTZ-induced kindled seizures. The increased effectiveness of axitinib and rapamycin regarding the reduction of oxidative stress manifestations might contribute to their improved seizure control.

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