CN113209097A

CN113209097A - Application of axitinib and analogue in preparation of medicine for resisting cerebral arterial thrombosis

Info

Publication number: CN113209097A
Application number: CN202110624271.4A
Authority: CN
Inventors: 胡富强; 王凯; 袁弘; 孟廷廷; 金翔宇; 周文韬
Original assignee: Zhejiang University ZJU
Current assignee: Zhejiang University ZJU
Priority date: 2021-06-04
Filing date: 2021-06-04
Publication date: 2021-08-06

Abstract

The invention discloses application of axitinib and analogues thereof in preparation of a medicine for resisting cerebral arterial thrombosis. The invention evaluates the therapeutic effect of the axitinib on ischemic stroke, and finds that the axitinib can reduce the middle cerebral artery occlusion and the cerebral ischemic infarction volume of a reperfusion model rat, relieve cerebral edema and improve motor dysfunction; axitinib may also slow down the apoptotic state of mend.3 cerebrovascular cells under oxygen deprivation conditions. Therefore, the axitinib and the analogues thereof can be used for preparing the medicine for resisting ischemic stroke.

Description

Application of axitinib and analogue in preparation of medicine for resisting cerebral arterial thrombosis

Technical Field

The invention belongs to the technical field of biology, and particularly relates to application of axitinib and analogues thereof in preparation of a medicine for resisting cerebral arterial thrombosis.

Background

According to 2016 global disease burden research data published in Lancet Neurology, stroke is the second leading cause of death worldwide (550 ten thousand), and 8010 thousand stroke patients are worldwide. Of the two main categories of stroke (ischemic and hemorrhagic), ischemic stroke accounts for 87% of all stroke-related events. Ischemic stroke is necrosis that occurs as a result of deprivation of oxygen and glucose supplies to a portion of the brain tissue due to blockage of a blood vessel by a thrombus or embolism.

A series of pathophysiological pathways occur in the early stage (0-48 h) of ischemic stroke, including angioedema, Blood Brain Barrier (Brain Blood Barrier) leakage, potential hemorrhagic transformation, astrocyte damage and neuron loss. These events eventually lead to irreparable neuronal damage and the formation of ischemic core regions and surrounding ischemic penumbra regions in the brain (areas of relatively weak ischemia and relatively low infarct). As the ischemic time increases and the disease progresses, the non-necrotic brain tissue in the ischemic penumbra area will gradually transform into infarcted brain tissue in the core area. Therefore, for patients with ischemic stroke in early onset, the main treatment aim is to rescue the brain tissue in the ischemic penumbra area and avoid the enlargement of the ischemic core area and the infarct area.

The ischemic stroke is accompanied by the overexpression of Vascular Endothelial Growth Factor (VEGF). High levels of VEGF can exacerbate early stroke pathological progression, including contributing to blood brain barrier rupture, vascular leakage, and cerebral edema. Furthermore, disruption of the BBB by VEGF increases leakage of glutamic acid and albumin in the blood, activating astrocytes and perturbing K in the brain parenchyma⁺Homeostasis, resulting in excessive neuronal activation and stress.

However, blindly inhibiting or agonizing the levels of VEGF in the brain does not achieve an effective therapeutic effect. Because VEGF has the following beneficial effects in the late cerebral ischemia reperfusion period, such as: increase collateral blood vessel formation, vasodilation, angiogenesis and neuroprotection. The VEGF level in the brain of a patient with ischemic stroke is reasonably regulated according to the condition of the patient, and the VEGF level is the key of a VEGF target drug in treating the ischemic stroke.

The dual role of VEGF in cerebral ischemic diseases is often associated with the cycle of disease progression. How to reasonably utilize the VEGF receptor inhibitor to realize effective treatment of cerebral arterial thrombosis is closely related to the attack stage of cerebral thrombosis, the physicochemical properties of the medicine and the administration time. Bruggen et al opened a human recombinant VEGF antibody (mFlt (1-3) -IgG) and found that intravenous administration of mFlt (1-3) -IgG 12-16h before cerebral ischemia was effective in improving the volume of cerebral infarction in ischemic mice at 1d and 8-12 weeks of ischemia (van Bruggen, N.; et al, VEGF anti-inflammatory recovery diabetes mellitus formation and tissue specimen after infection/recovery injection in the mouse damage.J. Clin. invest.1999,104, 1613-1620). Feng Shu-Qing et al designed small interfering RNA (siRNA-VEGF) to protect oxygen deprivation Induced cerebrovascular endothelial cell damage and found that intraperitoneal injection of VEGF antibody (anti-VEGF Anb) 30min before Ischemia could effectively reduce cerebral infarction volume (Feng, S.Q.; et al, VEGF anti-inflammatory disorders central Ischemia/repeat-Induced in great vitamin inhibiting expression fibrous reticulum Stress-media approach. biol. phase. Bull.2019,42, 692. 702.). The VEGF antibodies listed above all need to be administered before modeling to show significant therapeutic effects, which is essentially a prophylactic treatment against ischemic stroke. Patients with cerebral arterial thrombosis in clinic can be discovered and treated after the patients suffer from the cerebral arterial thrombosis, and the application prospect of the antibody is limited. Zhang et al developed human recombinant VEGF antibodies (rhVEGF165), and intravenous injection after 48h of ischemia improved cerebral infarct volume and motor dysfunction in ischemic rats, but given rhVEGF165 after 1h of ischemia exacerbated blood brain barrier opening and cerebral ischemic injury (Zhang, Z.G.; et al. Bruggen, N.; Chopp, M.VEGF engineogensis and hemodynamics blood-brain barrier leakage in the ischemic brazil.J.Clin.invest.2000, 106, 829-838). The gold time window of the rescue of the ischemic stroke patient is 3-5 h clinically, and the earlier the treatment is received, the more the cerebral infarction volume and the incidence of disability accidents can be reduced. The rhVEGF165 can only exert curative effect after being ischemic for 48 hours, and has slow effect. This has still limited therapeutic value for patients in the early stages of ischemic stroke. Therefore, the development of a method for inhibiting VEGF, which can be used for treating cerebral ischemia in early stage, is more suitable for clinical needs and has higher application value.

Axitinib is a multi-target tyrosine kinase inhibitor developed by the united states of vesper, and can be used to inhibit the activity of the VEGF type two receptor. Currently, axitinib is approved by the FDA as an anti-tumor drug for the treatment of adult patients who have previously failed one of tyrosine kinase inhibitors or cytokine therapy in the advanced Renal Cell Carcinoma (RCC). Other common VEGF receptor selective small molecule inhibitors are vandetanib, cediranib, tivozanib, and the like.

Disclosure of Invention

The invention aims to provide application of axitinib and analogues thereof in preparing a medicine for resisting cerebral arterial thrombosis. The invention discovers that axitinib can reverse the apoptosis state of brain microvascular endothelial cells under the condition of oxygen deprivation through an oxygen deprivation model, and reduce the permeability of a blood brain barrier; through rat cerebral artery occlusion and reperfusion models, the invention discovers that the axitinib can reduce cerebral ischemic infarction volume, improve blood brain barrier function, relieve cerebral edema, improve neurological dysfunction and the like. Therefore, the axitinib and the analogues thereof can be used as the drugs for resisting ischemic stroke.

One of the axitinib and the analogue is a selective small molecule inhibitor of a Vascular Epidermal Growth Factor Receptor (VEGFR), including but not limited to axitinib, vandetanib, cediranib, tivozanib and the like.

The method for preparing the drug for resisting ischemic stroke from the axitinib and the analogue thereof comprises the following steps: firstly, dissolving the axitinib or the analogue thereof in dimethyl sulfoxide to prepare the mother liquor of the axitinib or the analogue thereof in the dimethyl sulfoxide, then mixing the mother liquor of the axitinib or the analogue thereof in required dosage with a surfactant (such as medical Tween-80) according to volume ratio and equal proportion, dropwise adding the mixed liquor into physiological saline, and diluting to the administration concentration.

The prepared axitinib and analogue anti-ischemic stroke medicine thereof can be axitinib and analogue salt thereof or a compound of axitinib and analogue thereof.

Furthermore, the preparation form of the medicine for preparing the axitinib and the analogues thereof for resisting cerebral arterial thrombosis is oral preparation, external preparation, injection or compound preparation.

Furthermore, the medicine for preparing the axitinib and the analogue thereof and resisting the cerebral arterial thrombosis can be prepared from the axitinib and the analogue thereof and a pharmaceutically acceptable carrier.

The medicament is preferably administered after the subject has suffered symptoms of ischemic stroke, and the administration time is preferably within 0 to 48 hours after the onset of the symptoms.

Further, the administration routes of the axitinib and the analogues thereof include, but are not limited to, non-intracranial injection routes such as intravenous injection and intraperitoneal injection.

The prepared axitinib and the analogue thereof for resisting ischemic stroke can be used for preparing a medicine for recovering blood brain barrier permeability in a cerebral ischemia process and can also be used for preparing a medicine for relieving cerebral vascular injury.

The principle that the axitinib and the analogues thereof are used for treating cerebral ischemia is to inhibit VEGF receptors of intracerebral vascular endothelial cells, slow down the damage of cerebrovascular endothelial cells, promote the expression of claudin-5 and occludin, recover the permeability of a blood brain barrier to a physiological level and regulate the cerebral inflammatory environment of an ischemic patient.

The invention is based on the discovery in the research process that the axitinib can improve the blockage of middle cerebral artery and the cerebral ischemic infarction volume of a reperfusion model rat, relieve cerebral edema and improve motor dysfunction; axitinib may also slow down the apoptotic state of mend.3 cerebrovascular cells under oxygen deprivation conditions. The results of the preliminary study show that the axitinib can play a role in treating early ischemic stroke by improving the function of a blood brain barrier. Therefore, the invention prepares the axitinib and the analogues thereof into the medicine for resisting ischemic stroke, and is used for improving the symptoms of cerebral infarction volume, cerebral edema, motor dysfunction and the like of patients with ischemic stroke.

The invention has the advantages that: compared with the antibody therapy developed by predecessors, the drug administration before onset of disease can achieve the best treatment effect, the drug administration time for preparing the anti-cerebral arterial thrombosis drug from the axitinib and the analogue thereof provided by the invention is 0-48 h after onset of disease, the drug administration time is more in line with the actual condition from onset of disease to treatment of clinical patients, and the application value is high. Compared with antibody therapy, the axitinib and the analogues thereof are clinical medicines, and have high safety of human bodies, low price and easy preparation. The patent provides new treatment application of the axitinib and the analogues thereof, expands the medical application of the axitinib and the analogues thereof, and provides a new treatment means for treating cerebral ischemia; the axitinib and the analogues thereof can effectively reduce the cerebral infarction volume, improve the motor function damage of ischemic rats, recover the blood brain barrier function and reduce the apoptosis of vascular endothelial cells. The non-intracranial administration mode is adopted, so that the safety is high, the administration is convenient and fast, and the compliance of patients is high.

Drawings

FIG. 1: the MTT method measures the activity of the bEnd.3 cells, and the administration of the axitinib can reverse the damage of the bEnd.3 cells induced by oxygen deprivation. (A) Cell survival (n-3) was observed in the OGD group and in the axitinib group at each administered concentration (0.4, 4, 40, 400ng/mL) 5h after oxygen deprivation. (B) Cell survival (n-5) was observed in the OGD group and the axitinib group at each administered concentration (200, 400, 600, 800, 1000ng/mL) 8h after oxygen deprivation.

FIG. 2: the administration of the axitinib relieves the apoptosis of the bEnd.3 induced by hypoxia and glucose deprivation, and the apoptosis state of the bEnd.3 cells of each group is checked under an optical microscope. (A) The cell status of each treatment group (Control group, Control + Axitinib group, OGD group and OGD +400ng/mL Axitinib group) bEnd.3 represents a graph. OGD treatment is carried out for 8h, and the concentration of the administered axitinib is 400 ng/mL. (A) Under an optical microscope, the bEnd.3 forms after being treated by each experimental group under a unit visual field. (B) Number of nuclear staining per unit field of view in immunofluorescence experiments. (mean ± s.e.m, n ═ 4),. P < 0.05; p < 0.01.

FIG. 3: administration of axitinib reduced the amount of apoptosis-related protein expression of glycogen deprivation induced bEnd.3. (A) WB results show the Caspase 3 and Cytochrome protein expression levels in the Control, OGD and OGD +400ng/mL Axitinib groups. (B) Each Caspase 3 protein expression semiquantitative map (n ═ 3) (C) each Cytochrome protein expression semiquantitative map (mean ± s.e.m, n ═ 3).

FIG. 4: the results of immunoblot analysis showed that administration of axitinib alleviated the hypoxia-glucose-deprivation induced deletion of the bEnd.3 Claudin-5. (A) Change in the expression level of Claudin-5 between Con group, OGD group and OGD +400ng/mL Axitinib group. (B) Quantitative graph of each group Image J. mean ± s.e.m, n ═ 3, P < 0.05.

FIG. 5: immunofluorescence results show that the administration of axitinib alleviates the hypoxia-glucose-deprivation induced deletion of the bEnd.3 Claudin-5. (A) Change in the expression level of Claudin-5 between Con group, OGD group and OGD +400ng/mL Axitinib group. Claudin-5 (red), core (blue). (B) Panel B is a quantitative plot of Image J of panel a (mean ± s.e.m, n is 3). mean ± s.e.m, n ═ 4. P < 0.05; p < 0.01; p < 0.0001.

FIG. 6: axitinib administration alleviated the hypoxia-glucose-deprivation induced depletion of the cend.3 tight junction protein Occludin. (A) The immunofluorescence results showed the expression level of claudin-5, which is a tight junction protein in each treatment group. Occludin (green), nucleus (blue). (B) FIG. B is a quantitative graph of Image J in FIG. A. (mean ± s.e.m, n ═ 4) —,. P < 0.05.

FIG. 7: the axitinib can reduce the permeability of blood brain barrier of an ischemic rat. (A) After the rats are subjected to cerebral ischemia for 19h and Evenesin blue circulates for 5h, the living bodies of the Sham group, the MCAO group and the MCAO +10mg/kg Axitinib group detect the penetration amount of the Evenesin blue in the brain by fluorescence. (B) Fluorescence ROI semiquantitative maps for each group. mean ± s.e.m, n ═ 3, P < 0.05.

FIG. 8: the axitinib can increase the expression level of the tight junction protein of the hemizonal brain tissue of an ischemic rat. (A) Immunoblot analysis, 24h after cerebral ischemia of rats, the expression levels of Claudin-5 and Occludin in semi-dark zone brain tissue of Sham group, MCAO group and MCAO +10mg/kg Axitinib group. (B) Semiquantitative graph of Image J of Claudin-5 for each group of Claudin-5 (C) semiquantitative graph of Image J of Occludin for each group of Claudin. mean ± s.e.m, n ═ 6, P < 0.05.

FIG. 9: the axitinib can reduce the cerebral infarction volume of an ischemic rat. (A) TTC staining results of Sham, MCAO and MCAO +10mg/kg Axitinib groups are presented in the figure. White indicates infarcted area. Red is a normal area. (B) Volume semiquantitative graph of cerebral infarction of each group. mean ± s.e.m, n ═ 6, P < 0.05.

FIG. 10: the axitinib can reduce the volume of edema on the affected side of an ischemic rat and reduce the neurological dysfunction of the ischemic rat. (A) Infarct volume is expressed as (infarct volume/whole brain volume) × 100% (mean ± s.e.m, n ═ 6, × P < 0.05). (B) Representative brain plots for Sham, MCAO and MCAO +10mg/kg Axitinib groups. The right brain volume was significantly higher in the MCAO group than in the left brain. (C) Each group Longa neurological score.

Detailed Description

For the convenience of understanding, the following detailed description will be provided with specific drawings and examples to describe the application of the axitinib and the analogues thereof in preparing the medicine for resisting cerebral arterial thrombosis. It is specifically noted that the specific examples and figures are for illustrative purposes only and it will be apparent to those skilled in the art that, in light of the description herein, various modifications and changes can be made in the invention which are within the scope of the invention.

Example an Acertinib Reversal of apoptosis of brain microvascular endothelial cells under oxygen deprivation conditions

1.1 oxygen sugar deprivation induced vascular endothelial cell injury and drug modulationThe mouse brain microvascular endothelial cell bEnd3 is taken as a research object, and the Oxygen sugar Deprivation (OGD) is adopted to induce vascular endothelial cell injury to be an in vitro model, so that ischemic brain microvascular injury is simulated, and the expression change of relevant indexes of the vascular endothelial cell before and after blood sample deficiency injury is inspected. The specific method comprises the following steps: when the cells are completely fused, the cells are washed 3 times with a sugar-free culture solution HBSS (Hank's balance salt solution, HBSS) preheated to 37 ℃ and cultured in a sugar-free culture medium with an appropriate volume. Placing the cells in an oxygen-deficient closed box, and introducing oxygen-free mixed gas (95% N)₂And 5% CO₂) After about 5min, the inlet and outlet of the anoxic tank are closed, and the anoxic tank is placed at 37 deg.C and 5% CO₂The incubator of (2) for culture stimulation. After several hours of molding, cell samples were collected for subsequent experiments.

1.2 administration of Asitinib to increase the viability of bEnd.3 cells under oxygen deprivation injury

The viability of the bned.3 cells at different time of OGD after addition of different concentrations of axitinib was determined by thiazole blue colorimetry (MTT assay) (the concentration of axitinib administered was 0.4, 4, 40, 200, 400, 600, 800, 1000 ng/mL). The well-grown bEnd.3 cells were harvested at 1X 10 per well⁴Inoculating the seeds into a 96-well plate at a certain density, and incubating for 24 hours in a carbon dioxide incubator. And when the cell fusion degree reaches 70-80%, replacing the cell culture medium with an HBSS solution containing a series of concentration of axitinib, and carrying out OGD treatment for several hours. While the control group was cultured under normal conditions for the same period of time. After the incubation, 200. mu.L of fresh culture medium was added to each well, and 20. mu.L of MTT solution (5mg/mL) was added thereto for further incubation for 4 hours. After incubation, the liquid in each well was carefully discarded, and then 200 μ L DMSO was added to dissolve to form blue (or blue-violet) formazan crystals (formazan) insoluble in water. The 96-well culture plate added with DMSO is shaken in an air bath oscillator for 30min, and an enzyme-labeling instrument is used for measuring the absorbance at the wavelength of 570nm, wherein the control group is a normal culture group. Cell viability (cell viability) was calculated according to the formula: cell viability (%) ═ A_treat/A_controlX 100% where A_treatTo treat the absorbance of the wells, A_controlThe absorbance of the normal culture group was obtained.

The results are shown in fig. 1, the average survival rates of the bEnd.3 cells of the control groups were 63.55% and 25.41% in the case of 5h (A) and 8h (B), respectively, and the average survival rates of the bEnd.3 cells of the control groups were 3978% and 25.41% in the case of the administration of axitinib, to some extent, wherein the average survival rates of the bEnd.3 cells of the axitinib administration groups in the case of 5h and 8h in the case of the oxygen-deprivation treatment were 75.97% and 32.37%, respectively, at a dose of 400 ng/ml.

1.3 administration of Asitinib reverses the apoptotic State of bEnd.3 cells under oxygen deprivation Damage

The treatment groups were examined under light microscopy for apoptotic status. Sterilized coverslips were placed into 24-well plates for seeding of vascular endothelial cells. Cells were incubated in 5% CO₂Culturing in an incubator, and carrying out different treatments on cells: the experimental groups are Con group, OGD group and OGD +400ng/mL Axitinib group. Con group was cultured under normal conditionsAnd (4) cells, and after the last two groups are treated by OGD or medicines for 8 hours, observing the cell state of each group under an optical microscope.

The results in fig. 2 show that the normal control group bned.3 cells exhibited a good fusiform shape and the cells exhibited wavy streaks as a whole. After 8h of OGD treatment, the bned.3 cells per field were more apoptotic and deficient, and the cells shriveled and separated into single cells (fig. 2A). And the Axitinib administration treatment can effectively reduce the apoptosis of the bEnd.3 cells and relieve the phenomena of shrinkage and separation of the cells. Results of the number of nuclear staining within the immunofluorescence unit field of view also suggest that Axitinib can effectively reduce OGD-induced apoptosis of bend.3 cells (fig. 2B).

1.4 administration of Asitinib reverses the bEnd.3 apoptosis-related proteins Caspase-3 and cyctochrome C expression level

Western-Blot is adopted to detect the expression quantity of Caspase-3 and Cyctochrome C protein. bEnd.3 cells at 2X 10⁵The density of each well was seeded on 6-well plates. After 7 days of culture, the dosing dose of axitinib was 400ng/mL (dissolved in 2. mu.L DMSO), and both the OGD group and the dosing group were subjected to OGD treatment for 8h, while the control group was cultured for the same time under normal medium conditions.

According to the results shown in FIG. 3, after 8h of OGD treatment, the expression level of the apoptosis-related protein Cyctochrome C in the bEnd.3 cells is increased, while the expression level of the intact Caspase-3(35kDa) is reduced, which indicates that the expression level of the cleavage protein Caspase-3(17kDa) with the apoptosis activity is increased. And the administration of the axitinib can reduce Cyctochrome C and improve the expression quantity of complete Caspase-3(35 kDa).

The Western-blotting method comprises the following steps: protein was extracted, samples containing the same concentration of total protein were separated on 8-15% sodium dodecyl sulfate-polyacrylamide gel (SDS-PAGE), blotted (U ═ 100V, 1h), blocked with 5% skim milk powder (formulated with 1 x PBS) for 1h, washed, incubated with corresponding primary and secondary antibodies, exposed, and developed. Immunoblot analysis the following primary antibodies were used: caspase 3(1:1000, Rabbit Polyclonal Antibody, Cell signalling Technology, 9662), Cyctochrome C (1:1000, Rabbit Polyclonal Antibody, Cell signalling Technology, 4280), beta-actin (1:1000, Rabbit Polyclonal Antibody, Union).

Example inhibition of Down-Regulation of brain microvascular endothelial cell Tight-connexin under conditions of oxygen sugar deprivation by Diacetinib

The oxygen sugar deprivation damage constructed as described in 1.1 of example one was modeled. The barrier function of the blood brain barrier is mainly controlled by the tight junction proteins Claudin-5 and Occludin on brain microvascular cells. Therefore, this patent primarily investigates the protective effects of axitinib administration on OGD-induced claudin damage of bned.3 cells.

2.1 immunoblotting method for detecting expression quantity of Claudin-5 tight junction protein of each treatment group

bEnd.3 cells at 2X 10⁵The density of each well was seeded on 6-well plates. After 7 days of culture, the dosing dose of axitinib was 400ng/mL (dissolved in 2. mu.L DMSO), and both the OGD group and the dosing group were subjected to OGD treatment for 8h, while the control group was cultured for the same time under normal medium conditions. And detecting the expression quantity of claudin-5 protein by Western-Blot. The procedure is as in example 1.4. Immunoblot analysis the following primary antibodies were used: claudin-5(1:1000, Mouse Monoclonal Antibody, Invitrogen, 35-2500).

According to the results in FIG. 4, the expression level of the bEnd.3 cell Claudin-5 is remarkably reduced after OGD treatment for 8 h. And the administration of the axitinib can obviously reverse the down-regulation phenomenon of the expression level of the bEnd.3 cell Claudin-5 caused by the OGD treatment induction and promote the recovery of the barrier function of the cell.

2.2 immunofluorescence method for detecting expression level of Claudin-5 and Occludin of each treatment group

Sterilized coverslips were placed into 24-well plates for seeding of vascular endothelial cells. Cells were incubated in 5% CO₂Culturing in an incubator, and carrying out different treatments on cells: the experimental groups were Con group, OGD group and OGD +400ng/ml Axitinib group. The control group was cultured under normal conditions, and the latter two groups were treated with OGD or drug for 8h and fixed with 4% polymethine casein. Before staining, the cells were washed 3 times with PBS buffer, blocked with 3% Bovine Serum Albumin (BSA), and then addedAfter dilution with Claudin-5 antibody (1: 150, monoclonal antibody, Invitrogen, 35-2500) and Occludin antibody (1:100, polyclonal antibody, Abclone) were added, and the mixture was incubated overnight at 4 ℃. The next day, the cells were washed with PBS buffer 3 times, added with AlexaFlour488 fluorescent secondary antibody (1: 200, St. next, Shanghai, China) and incubated for 4h in the dark, washed with PBS buffer 3 times, and then stained with Hoechst, a nuclear dye, for 7 min. And finally washing for 3 times by using PBS buffer solution, sealing and detecting by using a laser confocal microscope.

According to the results shown in FIG. 5 and FIG. 6, the immunofluorescence results show that the expression levels of bEnd.3 cell Claudin-5 and Occludin are significantly lower than those of the control group after OGD treatment for 8 hours. Compared with the OGD treatment group, the treatment of the axitinib administration can up-regulate the expression level of Claudin-5 and Occludin and promote the recovery of the barrier function of brain microvascular cells.

Example Triaxitinib administration restores blood brain barrier function in rats with acute ischemic stroke

3.1 construction of animal model for blocking and refilling middle cerebral artery of rat

A unilateral cerebral artery occlusion model of a rat prepared by a wire-embolization method according to a rat cerebral artery occlusion and reperfusion (MCAO/R) model established according to the literature causes focal ischemic stroke. Specifically, male adult Wistar rats weighing 270-290g were selected. 0.33ml/100g of 10% chloral hydrate was intraperitoneally injected into anesthetized rats, the rats were fixed in the supine position, the skin was incised in the middle of the neck, the tissues of each layer were bluntly separated, the right common carotid artery was exposed, and the external carotid artery and the internal carotid artery were isolated. A slipknot is tied at the proximal end of the common carotid artery. The common carotid artery is threaded through double lines, the line at the far end is tied into a dead knot, and the near end is tied into a loose knot fixing thread for use. The artery clamp closes the internal carotid artery, a small opening is cut between two lines on the common carotid artery, a line bolt is inserted from the small opening to the internal carotid artery direction, the line bolt is lightly tied by a fixing line, the artery clamp is loosened, the line bolt is continuously and lightly pushed to the internal carotid direction until the line bolt mark position (about 18mm +/-0.5 mm), and the line bolt is fastened and fixed. After 1 hour, the thread plug is completely pulled out, the fixing thread is tied off, the slipknot of the common carotid artery is loosened, the blood supply of the common carotid artery to the internal carotid artery is recovered, and the neck of the rat is sutured. The body temperature of the rats was maintained at 37 ℃ until the rats were awake after surgery. According to the requirement of experimental design, the behavioral evaluation is carried out after a plurality of times of ischemia reperfusion, and then the brain is taken and the sample is collected. The Sham group (Sham group) was given the same dose of saline while the right common carotid artery was ligated and the others were left untreated.

3.2 methods of administering axitinib

The method for preparing the drug for resisting ischemic stroke from the axitinib comprises the following steps: firstly, dissolving the axitinib in dimethyl sulfoxide (DMSO) to prepare 50mg/ml of an axitinib DMSO mother solution, mixing the required dose of the axitinib DMSO mother solution with a surfactant (such as medical Tween-80) according to an equal proportion of a volume ratio, dropwise adding the mixed solution into physiological saline, and diluting to a dosing concentration. After the common carotid artery is inserted into the wire plug, the tail vein is injected with the solution of axitinib (the administration dose can be 0.1mg/kg, 1mg/kg, 10mg/kg and 100 mg/kg). The following examples each illustrate the administration of 10mg/kg of Astinib, and evaluate the therapeutic effect of Astinib on ischemic stroke.

3.3 treatment with Asitinib, significant decrease in England Permeability of Evans blue

The exudation of microvessels was examined by tail vein injection of Evans blue (2% wt/vol in PBS, 3 mL/kg). After MCAO/R molding for 19h, the rat tail is injected with Evans blue. Circulating for 5h, performing mouse left ventricle PBS perfusion, fixing 4% PFA, taking out the complete brain, and qualitatively observing Ewensky blue distribution intensity in brain tissue by a small animal living body imager.

As shown in fig. 7, compared with the control group, the fluorescence intensity of the brain on the ischemic side of the ischemic rat (right side of fig. 7A) was significantly enhanced, indicating that the amount of evans blue permeated in the brain was significantly increased, the permeability of the brain-blood barrier on the ischemic side was improved, and the barrier function of the blood-blood barrier was seriously damaged. And the group administered with the axitinib can improve the penetration of the Evans blue in the brain on the ischemic side and recover the barrier function of the blood brain barrier.

3.4 treatment with AxitinibUp regulating the expression level of brain tight junction protein in ischemic rat

After MCAO/R molding for 24h, and after anesthesia, brain was collected. After the brain is soaked in cold 0.32M sucrose physiological saline solution for a little time to clean residual blood stains on the surface of the brain, the brain tissue of the ischemic penumbra is taken for carrying out Western blotting, and the expression quantity of each group of Claudin-5 is analyzed. Coronal sections were taken 3 mm and 9 mm from the anterior frontal lobe and 6mm thick brain tissue blocks were taken. Approximately 2mm of sagittal suture line along the brain block, 4 mm of tissue between the two hemispheres was cut from top to bottom. This structure is fed by the anterior cerebral artery and discarded. The remaining left hemibrain block was cut at 2mm from the sagittal suture, at an angle of 30 ° relative to the sagittal section. The lateral cortex is the ischemic core region and the medial cortex is the ischemic penumbra. The semi-dark band of brain tissue was homogenized and lysed with RIPA buffer supplemented with protease inhibitor cocktail and PMSF. Western blotting was carried out in accordance with the method described in example II 1.4.

As shown in the results of fig. 8, compared to Sham group, brain tissue Claudin-5 and Occludin were significantly downregulated in ischemic penumbra in rats with cerebral ischemia, whereas axitinib administration reversed ischemia-induced downregulation of Claudin-5 and Occludin, promoting restoration of blood brain barrier function from pathological to near physiological in ischemic rats.

Example dosing with tetraacetatinib reduces cerebral infarction volume in rats in the early stages of ischemic stroke, and mitigates disease progression

4.1 treatment with Asitinib to reduce cerebral infarction volume in ischemic rats

After 24h of cerebral ischemia reperfusion, after anesthesia, taking out the brain. The brain was then cut into 8 coronal sections of 2mm from the tip of the frontal lobe to a 16mm thick area anterior to the cerebellum. Placing into 1% 2,3, 5-triphenyltetrazolium chloride (2,3, 5-triphenyltetrazolium chloride, TTC), keeping the tinfoil paper in the dark, placing into a 37 deg.C water bath, turning over once brain slice after 5min to uniformly dye the brain slice, taking out after 5min, transferring into 4% paraformaldehyde, storing at 4 deg.C, and scanning brain slice after 24 hr. The surviving portion of the brain slice was red and the dead portion was pale white. Infarct area and total area of each brain section were measured using Image J. Infarct volume was calculated by multiplying the increased infarct area per section by the section thickness (2mm), and the results were expressed as (infarct volume/whole brain volume) × 100%.

As shown in fig. 9A, TTC staining results suggested that there were a large number of white infarcted areas in the ischemic side brain of the ischemic rats compared to the normal group. Whereas, the administration of axitinib can reduce white infarcted areas of the affected brain. The semi-quantitative (fig. 9B) results suggest that pre-administration of axitinib may effectively reduce the cerebral infarct volume from 11.87% to 6.84%.

4.2 treatment of Anciltinib to improve cerebral edema in ischemic rats

After 24h of cerebral ischemia reperfusion, after anesthesia, taking out the brain. After the brain was immersed in cold physiological saline for a few hours to wash the surface of the brain from remaining blood stains, the brain was placed on a brain mold and photographed, and the volume of the half area of the brain on the ischemic side and the non-ischemic side was measured using Image J. The degree of cerebral edema was evaluated as the ratio of the volume of the cerebral ischemic side to the cerebral half area on the non-ischemic side.

As shown in the results of fig. 9B, there was a significant edema in the affected side of the ischemic rats, with a significantly higher volume of the right brain than the left brain. And the administration of the axitinib can obviously reduce the volume of the brain on the affected side and improve the edema phenomenon. Semi-quantitative results (fig. 9A) show that axitinib administration can reduce the volume of lateral cerebral edema to near normal physiological state.

4.3 treatment of Asitinib for improving neurological dysfunction in ischemic rats

The neurological dysfunction is one of important indexes for evaluating ischemic stroke injury. After 24h of reperfusion treatment of rats, neuro-dysfunctional behaviours were examined according to Longa neurological score, and neurological deficits were graded in 5 grades:

0 minute: normal, without neurological deficit; 1 minute: the right (paralyzed) anterior paw can not be fully extended, but does not have obvious circumgyration when walking and slight neurological deficit; and 2, dividing: the rat turns to the right side, walks slowly and suffers from moderate neurological deficit; and 3, dividing: the rat body is inclined towards the right side, slowly rotates for a small circle, and is severely damaged in nerve function; and 4, dividing: the patient can not walk spontaneously and the consciousness is lost.

As shown in fig. 9C, compared to the cerebral ischemia group, the axitinib administration significantly reduced the neurological impairment caused by ischemia, and the Longa neurological score of the axitinib administration group was significantly reduced compared to the model group.

Claims

1. Application of axitinib and analogues in preparing medicine for resisting cerebral arterial thrombosis is provided.

2. The use according to claim 1, wherein the axitinib and analogs in the medicament are selective small molecule inhibitors of the vascular epidermal growth factor receptor, including but not limited to axitinib, vandetanib, cediranib, tizozanib.

3. The use according to claim 1 or 2, wherein the axitinib and the analogue in the medicament are salts of axitinib and the analogue or complexes thereof.

4. The use of claim 1, wherein the medicament is formulated as an oral, topical, injectable or combination formulation.

5. The use according to claim 1, wherein the medicament is prepared from axitinib and analogues and a pharmaceutically acceptable carrier.

6. The use of claim 1, wherein the drug is administered within 0-48 hours after the onset of ischemic stroke.

7. The use of claim 1, wherein the drug prepared from axitinib and the like is administered by a route selected from the group consisting of, but not limited to, intravenous (i.v.), intraperitoneal (i.p.) and non-intracranial injection.