阅读说明：本技术 阿帕替尼作为平滑肌表型转化抑制剂的用途 (Use of apatinib as smooth muscle phenotype transformation inhibitor ) 是由 黄恺 陈敏 邵文超 梁明露 胡立志 李晓光 于 2020-08-05 设计创作，主要内容包括：本发明属于医药领域,涉及阿帕替尼作为平滑肌表型转化抑制剂的用途。本发明提出Apatinib对PDGFRβ有抑制作用,因此Apatinib可以抑制平滑肌表型转换,从而预防和/或治疗血管损伤后再狭窄。此外,Apatinib并无通常心血管药物或其他平滑肌表型转换抑制剂的细胞毒作用,因此可以预期其安全性。其可以作为治疗平滑肌参与的相关疾病如血管再狭窄、肿瘤等疾病的药物而广泛应用。(The invention belongs to the field of medicines, and relates to application of apatinib as a smooth muscle phenotype transformation inhibitor. Apatinib is proposed to have an inhibitory effect on PDGFR beta, so Apatinib can inhibit smooth muscle phenotype switching, thereby preventing and/or treating restenosis after vascular injury. Furthermore, Apatinib has no cytotoxic effect of common cardiovascular drugs or other inhibitors of smooth muscle phenotype switching, and therefore its safety can be expected. It can be widely used as a medicine for treating related diseases involving smooth muscle, such as vascular restenosis, tumor and the like.)

1. An inhibitor of phosphorylation of PDGFR β and of PDGFR β downstream signaling pathways comprising apatinib.

2. The inhibitor according to claim 1, wherein the inhibitor is used for inhibiting smooth muscle phenotype switching.

3. Inhibitor according to claim 2, for use in the prevention and/or treatment of restenosis following vascular injury.

4. The inhibitor according to claim 3, wherein the restenosis after vascular injury comprises PCI restenosis, in-stent restenosis, or restenosis after bypass graft.

5. The inhibitor according to claim 4, wherein the concentration of apatinib in the inhibitor is from 50nM to 200 nM.

6. Use of apatinib in the preparation of an inhibitor of phosphorylation of PDGFR β and of a signaling pathway downstream of PDGFR β.

Technical Field

The invention belongs to the field of medicines, and particularly relates to application of apatinib as a smooth muscle phenotype transformation inhibitor.

Background

Apatinib, having the english name Apatinib and the other name Apatinib mesylate; apatinib mesylate; CS-1160; n- [4- (1-cyanocyclopentyl) phenyl]-2- [ (4-pyridylmethyl) amino]-3-pyridinecarboxamide; YN968D 1; [ Apatinib free form](ii) a APATINIBFREEBASE (YN968D1 FREEBASE). Its molecular weight is 397.47232, molecular formula is C₂₄H₂₃N₅O, CAS number 811803-05-1. Structural formula is

Apatinib has been studied in the past mainly as an inhibitor of the vascular endothelial growth factor receptor 2(VEGFR-2), i.e., to inhibit VEGFR-2. Apatinib can block down-stream signal transduction and inhibit the generation of tyrosine kinase by highly selectively competing the ATP binding site of VEGFR-2 in cells, thereby inhibiting the generation of new blood vessels in tumor tissues and finally achieving the purpose of treating tumors. Therefore, the above drugs are widely used in studies relating to tumors such as gastric cancer.

The phenotypes of Vascular Smooth Muscle Cells (VSMCs) can be classified into a more differentiated contractile phenotype and a less differentiated secretory phenotype, which represent two extreme types of a series of different phenotypes coexisting in the vessel wall and expressing different genes and proteins. The VSMC of normal adult arterial blood vessels predominates in the systolic form, whose main functions are to maintain the elasticity of the blood vessels and to constrict the blood vessels. The contractile VSMC has poor or no proliferation and migration capacity, and the soma is fusiform or banded and contains a large amount of myofilaments and structural proteins; secreted VSMCs, on the other hand, are mainly present in the metaphase and pathological vessels of the embryo, and their main functions are proliferation, migration into the intima, and synthesis of extracellular matrix proteins.

The process of VSMC switching from systolic to secretory is called phenotypic switching of VSMC. Research shows that three signal transduction pathways, namely mitogen-activated protein kinase (MAPK), PI-3-K and cyclic adenosine monophosphate (cAMP), are involved in phenotypic transformation of VSMC through receptors such as VEGFR (VEGFR) and platelet-derived growth factor receptor (PDGFR). Abnormal proliferation and migration of VSMC are common pathological features of occurrence and development of vascular diseases such as hypertension, pulmonary hypertension and the like, and are also important reasons for restenosis after vascular injury, and VSMC phenotypic transformation plays an important role in VSMC proliferation and migration.

According to the difference of two phenotypically expressed proteins of VSMC, corresponding markers can be found when phenotypes are switched. Among them, alpha-smooth muscle actin (alpha-SMA) is predominantly expressed in contractile cells and is an early characteristic marker of VSMC differentiation.

At present, there are several drugs used in scientific research to inhibit restenosis after vascular injury, among which the most common are: angiotensin converting enzyme (abbreviated as ACE) inhibitors spiropril and cilazapril, coating drugs [ such as antithrombotic agents (such as heparin and hirudin), anti-inflammatory drugs, anti-cell proliferation agents (such as rapamycin and paclitaxel) and the like used by drug-coated stents (DES.) existing researches show that Apatinib can play a therapeutic role in tumor diseases by promoting apoptosis and can cause hypertension.

Disclosure of Invention

In order to improve the technical problems, the invention provides an inhibitor for phosphorylation of PDGFR beta and a PDGFR beta downstream signal pathway, which comprises apatinib.

According to an embodiment of the invention, the inhibitor is for inhibiting smooth muscle phenotype switching.

According to an embodiment of the invention, the smooth muscle phenotype switch comprises a switch from contractile to secretory.

According to an embodiment of the invention, the inhibitor is for use in the prevention and/or treatment of restenosis following vascular injury.

According to embodiments of the invention, the post-vascular injury restenosis includes PCI restenosis, in-stent restenosis, post-bypass graft restenosis, and the like.

According to an embodiment of the invention, the concentration of apatinib in the inhibitor is from 50nM to 200 nM.

The invention also provides application of apatinib in preparing inhibitors of PDGFR beta phosphorylation and PDGFR beta downstream signaling pathways.

The invention also provides a method of inhibiting phosphorylation of PDGFR β and PDGFR β downstream signaling pathways comprising administering apatinib to an individual in need thereof.

According to an embodiment of the present invention, the effect of the smooth muscle phenotype switching inhibitor is achieved by inhibiting phosphorylation of PDGFR β and PDGFR β downstream signaling pathway, thereby preventing and/or treating restenosis after vascular injury.

The invention has the following beneficial effects:

the inventors surprisingly found that Apatinib not only can be used as a VEGFR-2 inhibitor, but also has an inhibitory effect on PDGFR beta, so that Apatinib can inhibit smooth muscle phenotype switching, thereby preventing and/or treating restenosis after vascular injury.

Furthermore, Apatinib has no cytotoxic effect of common cardiovascular drugs or other inhibitors of smooth muscle phenotype switching, and therefore its safety can be expected. It can be widely used as a medicine for treating related diseases involving smooth muscle, such as vascular restenosis, tumor and the like.

Drawings

In fig. 1, a is a group of a pseudo operation and a carrier treatment, b is a group of a ligation and a carrier treatment, c is a group of a ligation and an apatinib treatment, and the shape of a blood vessel of a mouse carotid artery after ligation is detected by an oil red-Hematoxylin (HE) staining experiment.

FIG. 2 shows the detection of VSMC proliferation using EdU assay with varying concentrations of Apatinib and vehicle (DMSO). Wherein the first column is DAPI stained nuclei, the second column is EdU stained nuclei, and the third column is a fusion map of the first two columns; the first line is a carrier treatment group, the second line is a PDGF-BB and carrier treatment group, the third line is a PDGF-BB and Apatinib treatment group with 50nM, the fourth line is a PDGF-BB and Apatinib treatment group with 100nM, and the fifth line is a PDGF-BB and Apatinib treatment group with 200 nM.

FIG. 3 shows stimulation with Apatinib (50,100,200nM) and vehicle (DMSO), respectively, after treatment of VSMC cells with PDGF-BB. In the figure, Apatinib and a carrier with different concentrations are used for detecting the expression of cyclin and oncostatin through a western blot experiment, and after the VSMC is treated by PDGF-BB, the cyclin expression is increased and the oncostatin expression is reduced; after Apatinib stimulation, cyclin expression is reduced and oncostatin expression is increased.

FIG. 4 shows stimulation with Apatinib (50,100,200nM) and vehicle (DMSO), respectively, after treatment of VSMC cells with PDGF-BB. In the figure, Apatinib and a carrier with different concentrations detect the expression of apoptosis-related protein by a western blot experiment, and no obvious change is seen.

FIG. 5 shows stimulation with Apatinib (50,100,200nM) and vehicle (DMSO), respectively, after treatment of VSMC cells with PDGF-BB. Apatinib was used to detect cell migration using a transwell assay at various concentrations.

FIG. 6 shows stimulation with Apatinib (50,100,200nM) and vehicle (DMSO), respectively, after treatment of VSMC cells with PDGF-BB. Apatinib was used to examine cell proliferation and migration in different concentrations.

In FIG. 7, after ligation of carotid artery of mouse, the mouse was treated with vector and 10 mg/kg. d Apatinib, and in the figure, Apatinib with different concentrations was used to detect the expression of the contractile gene α SMA by immunofluorescence assay.

FIG. 8 shows stimulation with Apatinib (50,100,200nM) and vehicle (DMSO), respectively, after treatment of VSMC cells with PDGF-BB. Apatinib at different concentrations was used to detect PDGFR β phosphorylation in Western blot assays.

Detailed Description

The technical solution of the present invention will be further described in detail with reference to specific embodiments. It is to be understood that the following examples are only illustrative and explanatory of the present invention and should not be construed as limiting the scope of the present invention. All the technologies realized based on the above-mentioned contents of the present invention are covered in the protection scope of the present invention.

Unless otherwise indicated, the raw materials and reagents used in the following examples are all commercially available products or can be prepared by known methods.

The following examples use cell experiments to study the inhibitory effect of the drug on VSMC phenotypic switching.

Example 1

Apatinib used in the test of this example was purchased from MCE, Inc. under model number HY-13342. The phenotypic shift of VSMC in the following experiments was tested using the methods in the following steps, respectively.

And detecting the VSMC cell proliferation condition by using an EdU cell proliferation experiment, and detecting the VSMC cell migration condition by using a cell scratch experiment and a transwell experiment. The detection method is described in Huang D, Wang Y, Wang L, Zhang F, Deng S, Wang R, Zhang Y, Huang K.Poly (ADP-rib) polymerase 1is Induced dependent for transforming growth factor-beta Induced Smad3 activation in vascular smooth cell. PLoS one.2011; e27123. the specific process is as follows:

EdU cell proliferation assay: rat-derived primary cells (VSMC) were seeded in 96-well plates and cells were treated with varying concentrations (50nM,100nM,200nM) of Apatinib and vehicle DMSO for 4h, respectively. After 48h, the four treatments were stimulated with PDGF-BB (30ng/ml) for 48h (control with an equal volume of DSMO), and EdU incorporation was analyzed according to the manufacturer's instructions and photographed with an Olympus cellSens Entry.

Cell scratch test: VSMC were seeded into 6-well plates and cultured to 80% density. The cell monolayer was scratched with a 1ml pipette tip. After pre-incubation of cells with Apatinib at various concentrations for 4h, the cells were stimulated with PDGF-BB (30ng/ml) for 48h (control group with equal volume of DSMO), and then cultured in DMEM containing fetal bovine serum at 10% volume concentration. Cells were visualized using an Olympus cellSens entry and wound closure rate was measured using the Image J program.

Cell migration was measured by the Transwell method: VSMC was pretreated with Apatinib for 4h, seeded in the upper air chamber, 500. mu.L DMEM and fetal bovine serum at 10% concentration by volume and PDGF-BB (30ng/ml) were placed in the lower air chamber. After 24h, the cells were fixed in the lower air chamber with 4% by mass of formaldehyde for 20 minutes and stained with 0.1% by mass of crystal violet for 20 minutes. Migrated cells were photographed using the Olympus cellSens channel.

Example 2

Apatinib (10 mg/kg. d) and vehicle (DMSO) were intraperitoneally administered after carotid ligation injury or sham-operated injury in C57BL/6 mice, respectively. After 14 days, the mice were euthanized and the injured blood vessels were subjected to a vascular resection procedure. After paraffin is fixed and embedded by formaldehyde with the mass concentration of 4%, the blood vessel is cut into sections. FIG. 1 shows the detection of the vascular morphology of a mouse after carotid artery ligation by using an oil red-Hematoxylin (HE) staining experiment after the treatment of a sham operation + carrier treatment group, a ligation + carrier treatment group and a ligation + apatinib treatment group. The results are shown in FIG. 1. Wherein, the figure 1is a statistical chart of the average carotid artery thickness of mice in a sham operation + vector treatment group, a ligation + vector treatment group and a ligation + apatinib treatment group from left to right (1a, 1b and 1 c). As can be seen from fig. 1, in comparison with the sham operation + vehicle treatment group, the carotid artery thickness increased after ligation of carotid artery in mice, i.e., vascular injury resulted in excessive proliferation of vascular endothelial cells; and the apatinib can inhibit the hyperproliferation of vascular endothelial cells caused by vascular injury.

Example 3

VSMC cells were treated with PDGF-BB (30ng/ml) and stimulated with Apatinib (50nM,100nM,200nM) and vehicle DMSO at different concentrations, respectively. FIG. 2 shows the detection of VSMC proliferation using EdU assay for Apatinib at different concentrations. In FIG. 2, the first column is the DAPI stained nuclei, the second column is the EdU stained nuclei, and the third column is the fusion map of the first two columns; the first line is a carrier treatment group, the second line is a PDGF-BB and carrier treatment group, the third line is a PDGF-BB and Apatinib treatment group with 50nM, the fourth line is a PDGF-BB and Apatinib treatment group with 100nM, and the fifth line is a PDGF-BB and Apatinib treatment group with 200 nM. As can be seen from fig. 2, after VSMC cells were treated with PDGF-BB, EdU-positive cells increased, and after further treatment with Apatinib, EdU-positive cells decreased, and the amount of EdU-positive cells decreased as the concentration of Apatinib increased. In conclusion, after the VSMC cells are treated by PDGF-BB, the proliferation of the VSMC cells can be promoted, Apatinib can inhibit the cell proliferation caused by PDGF-BB, and the inhibition effect is enhanced along with the increase of the concentration of Apatinib.

Example 4

After VSMC cells were treated with PDGF-BB (30ng/ml), Apatinib (50nM,100nM,200nM) and vehicle DMSO at different concentrations were administered to stimulate the cells, and after proteins were extracted, the expression of cyclin and oncostatin was detected by Western blotting, as shown in FIG. 3. VSMC cells expressed cyclin (PCNA, CyclinD1) and oncostatin (P27, P21) under different treatments. As shown in FIG. 3, after treatment with PDGF-BB, the expression of cyclin was increased and the expression of oncostatin was decreased in VSMC cells; after Apatinib stimulation is given, cyclin expression is reduced, and oncostatin expression is increased; the expression level of the protein related to the apoptosis has no obvious change.

Example 5

After VSMC cells were treated with PDGF-BB (30ng/ml), Apatinib (50nM,100nM,200nM) and vehicle DMSO at different concentrations were administered to stimulate the cells, and after protein extraction, expression of apoptotic proteins (Caspase 3, cleaved Caspase 3, Bcl-2, Bax) was detected by Western blotting, as shown in FIG. 4. As can be seen from FIG. 4, Apatinib at different concentrations had no significant effect on the expression of apoptosis-related proteins.

Example 6

VSMC cells were treated with PDGF-BB (30ng/ml) and stimulated with Apatinib (50nM,100nM,200nM) and vehicle DMSO at different concentrations, respectively, to detect cell migration using the transwell assay. The results are shown in FIG. 5. As is clear from FIG. 5, PDGF-BB promotes cell migration, while Apatinib inhibits cell migration, and the inhibitory effect increases with increasing concentration.

Example 7

After the treatment of VSMC with PDGF-BB (30ng/ml), different concentrations of Apatinib (50nM,100nM,200nM) and vehicle DMSO were administered to stimulate VSMC, and the proliferation and migration of VSMC were examined by the scratch assay, as shown in FIG. 6. From FIG. 6, it is seen that PDGF-BB promotes the rate of closure of cell scars, Apatinib inhibits the promotion, and that Apatinib concentration increases to enhance the inhibition of closure of cell scars.

Example 8

After the carotid artery of a mouse is respectively ligated, the mouse is treated by a mouse vector DMSO and 10 mg/kg. d Apatinib, the expression condition of the contractile gene alpha SMA is detected by an immunofluorescence experiment, a tissue section is incubated overnight at 4 ℃ by using an SM alpha-actin primary antibody (the volume ratio is 1: 100), and then incubated for 2h at 37 ℃ by using a FITC-conjugated fluorescent secondary antibody. Nucleic acids were stained with DAPI at 37 ℃ for 15 min. The sections were finally visualized using an Olympus cellSens entry. Carotid morphology was observed and the results are shown in FIG. 7. In fig. 7, the first column is the sham-operated plus empty treatment group, the second column is the operated plus empty treatment group, the third column is the operated plus Apatinib treatment group, and the first row is immunofluorescent staining of carotid artery α SMA, the second row is DAPI staining of carotid artery nuclei, and the third row is a fused image of the first two rows. As can be seen from FIG. 7, after the mouse was ligated to the carotid artery, the carotid artery wall was thickened and the expression level of the contractile gene α SMA in the carotid artery was decreased; after further treatment with Apatinib, the carotid wall thickness decreased and the expression level of the contractile gene α SMA increased. After the blood vessel is damaged, the blood vessel wall is thickened, and the expression quantity of the contraction gene alpha SMA is reduced.

Example 9

After the VSMC cells were treated with PDGF-BB, they were stimulated with Apatinib (50nM,100nM,200nM) and vehicle DMSO at different concentrations, respectively, and the PDGFR β phosphorylation level was detected by Western blotting, as shown in FIG. 8. FIG. 8 shows that PDGFR β phosphorylation was increased after treatment of VSMC with PDGF-BB; PDGFR β phosphorylation levels decreased after further treatment with Apatinib, and decreased with increasing Apatinib concentration. Thus, PDGF-BB promotes phosphorylation of PDGFR β, while Apatinib inhibits phosphorylation of PDGFR β, and this inhibition increases with increasing Apatinib concentration.

From the results of the experiments, Apatinib was able to directly inhibit the phenotypic shift of VSMC.

The embodiments of the present invention have been described above. However, the present invention is not limited to the above embodiment. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.