Application of aurora kinase B inhibitor in preparation of medicine for treating pulmonary arterial hypertension

Abstract

The invention provides application of an aurora kinase B inhibitor in preparation of a medicine for treating pulmonary arterial hypertension, and relates to the technical field of medicines. The application of the aurora kinase B inhibitor in preparing the medicine for treating the pulmonary arterial hypertension, provided by the invention, can be used for remarkably improving pulmonary vascular remodeling caused by excessive proliferation and apoptosis resistance of pulmonary artery smooth muscle cells, so that the pulmonary arterial hypertension is effectively relieved; the defect that the occurrence and development of pulmonary vascular remodeling cannot be fundamentally reversed by the conventional targeted therapy is overcome, and the long-term poor prognosis of a patient can be favorably improved. According to the application, a specific inhibitor AZD1152-HQPA of aurora kinase B is adopted to intervene in two representative pulmonary arterial hypertension rat models, and it is found that inhibition of the kinase activity of aurora kinase B has a good curative effect on pulmonary arterial hypertension. The invention clarifies a new application of AZD1152-HQPA in treating pulmonary arterial hypertension, and provides a new medicine for treating pulmonary arterial hypertension by targeting aurora kinase B.

Description

Technical Field

[0001] The present application relates to the field of medical technology, and in particular to the use of an Aurora kinase B inhibitor in the preparation of a drug for treating pulmonary arterial hypertension. Background Art

[0002] Pulmonary hypertension (PH) is a malignant pulmonary vascular disease caused by a variety of etiologies that causes changes in pulmonary vascular structure and / or function, leading to progressive increase in pulmonary vascular pressure, right heart failure, and even death. Clinically, PH can be divided into five categories: arterial PH, PH caused by left heart disease, PH caused by lung disease and / or hypoxia, PH caused by pulmonary artery obstruction, and PH caused by unknown mechanisms and / or multiple factors. According to a number of international registration studies, the global prevalence of PH is about 1%, and the prevalence in the elderly over 65 years old can reach 10%. PH has become one of the major diseases worldwide. Patients with PH are difficult to treat and have a poor prognosis, especially patients with arterial PH have the worst prognosis.

[0003] The pathogenesis of pulmonary hypertension is complex, and the key pathological feature is the irreversible remodeling of pulmonary blood vessels under the stimulation of genetic factors, hypoxia, inflammation, etc. Pulmonary vascular remodeling is jointly participated by multiple cells, among which the excessive proliferation and apoptosis resistance of pulmonary artery smooth muscle cells cause significant thickening of the pulmonary artery media. With the destruction of the elastic fiber plates inside and outside the pulmonary artery, smooth muscle cells can even migrate to the intima, adventitia and distal arterioles, causing full-thickening of the pulmonary artery wall and muscularization of distal arterioles, which plays a key role in the continuous progression of pulmonary vascular remodeling and the progressive increase of pulmonary vascular pressure.

[0004] Commonly used targeted drugs for pulmonary hypertension today include prostacyclin analogs or receptor agonists (such as treprostinil, selexipag), endothelin receptor antagonists (such as macitentan), phosphodiesterase 5 inhibitors (such as sildenafil) and soluble guanylate cyclase agonists (such as riociguat). Although these drugs can temporarily dilate pulmonary vasculature and relieve the clinical symptoms of patients with pulmonary hypertension, they cannot fundamentally reverse the occurrence and development of pulmonary vascular remodeling, which is also the main reason for poor efficacy in patients. Therefore, in-depth analysis of the molecular mechanism of pulmonary vascular remodeling caused by pulmonary artery smooth muscle cell lesions is a key scientific issue to break through the bottleneck of pulmonary hypertension drug research and development, and is of great significance for the clinical prevention and treatment of pulmonary hypertension.

[0005] Aurora kinase B (AURKB) is a member of the Aurora kinase family. It forms a chromosomal passenger complex with three non-enzymatic subunits, INCENP, Survivin, and Borealin, and regulates key cell cycle processes such as mitosis and cytokinesis by mediating serine / threonine phosphorylation of target proteins. Previous studies have shown that Aurora kinase B is abnormally upregulated in a variety of malignant tumors, such as lung cancer, leukemia, breast cancer, glioblastoma, and hepatocellular carcinoma, and is closely associated with poor prognosis in patients, suggesting that this molecule is an important risk factor for proliferative diseases. Highly expressed Aurora kinase B helps tumor cells adapt to the needs of malignant proliferation, and some tumor cells with deleted tumor suppressor genes are highly dependent on Aurora kinase B to maintain survival. AZD1152-HQPA is a specific inhibitor of Aurora kinase B. It has much higher selectivity for Aurora kinase B than its family members Aurora kinase A and other serine / threonine protein kinases. It was developed by AstraZeneca and is used in clinical trials to treat blood tumors such as acute myeloid leukemia and diffuse large B-cell lymphoma, and solid tumors such as small cell lung cancer.

[0006] In pulmonary hypertension, the biological behavior of excessive proliferation and apoptosis resistance of pulmonary artery smooth muscle cells is described as a "cancer-like" phenotype. At the same time, the applicant found in previous experiments that the expression level and kinase activity of Aurora kinase B in diseased pulmonary artery smooth muscle cells were significantly increased, suggesting that Aurora kinase B may play an important role. However, there are no reports on the treatment of pulmonary hypertension by targeting Aurora kinase B. Summary of the invention

[0007] The purpose of the present application is to provide an application of an Aurora kinase B inhibitor in the preparation of a drug for treating pulmonary hypertension, so as to solve the problem in the prior art that targeted drugs for pulmonary hypertension cannot effectively improve pulmonary vascular remodeling, leading to a progressive increase in pulmonary vascular pressure.

[0008] To achieve the above objectives, this application adopts the following technical solutions:

[0009] The present application provides the use of an Aurora kinase B inhibitor in the preparation of a drug for treating pulmonary arterial hypertension.

[0010] Preferably, the Aurora kinase B inhibitor includes a substance that inhibits the kinase activity of Aurora kinase B in pulmonary artery smooth muscle cells and / or a substance that reduces the expression level of Aurora kinase B.

[0011] Preferably, the Aurora kinase B inhibitor is AZD1152-HQPA.

[0012] Preferably, the molecular formula of AZD1152-HQPA is C 26 H 30FN7O3, molecular weight is 507.6.

[0013] Preferably, the Aurora kinase B inhibitor is used in combination with other types of drugs for treating pulmonary hypertension, or the Aurora kinase B inhibitor is used alone.

[0014] Preferably, the other types of drugs for treating pulmonary hypertension include: at least one of: prostacyclin analogs, prostacyclin receptor agonists, endothelin receptor antagonists, phosphodiesterase 5 inhibitors and soluble guanylate cyclase agonists.

[0015] The present application also provides a composition for treating pulmonary arterial hypertension, wherein the active ingredients of the composition include an Aurora kinase B inhibitor.

[0016] Preferably, the Aurora kinase B inhibitor is AZD1152-HQPA.

[0017] Preferably, the effective dose of AZD1152-HQPA is 5-30 mg / kg / day.

[0018] Preferably, the composition is in the form of at least one of tablets, solutions, suspensions, emulsions, powders, granules, capsules, microcapsules, microspheres, injections, and liposomes; and the composition also includes pharmaceutical excipients.

[0019] Compared with the prior art, the beneficial effects of this application include:

[0020] The use of the Aurora kinase B inhibitor provided in the present application in the preparation of drugs for the treatment of pulmonary hypertension can significantly improve the pulmonary vascular remodeling caused by excessive proliferation and apoptosis resistance of pulmonary arterial smooth muscle cells, thereby effectively alleviating pulmonary hypertension, overcoming the obstacle that previous targeted therapies could not fundamentally reverse the occurrence and development of pulmonary vascular remodeling, and may help improve the poor long-term prognosis of patients. The examples of the present application use AZD1152-HQPA, a specific inhibitor of Aurora kinase B, to intervene in two representative pulmonary hypertension rat models, and found that inhibiting the kinase activity of Aurora kinase B has a good therapeutic effect on pulmonary hypertension. The present application clarifies the new use of AZD1152-HQPA for the treatment of pulmonary hypertension and provides a new drug targeting Aurora kinase B for the treatment of pulmonary hypertension. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] In order to more clearly illustrate the technical solutions of the embodiments of the present application, the drawings required for use in the embodiments will be briefly introduced below. It should be understood that the following drawings only show certain embodiments of the present application and therefore should not be regarded as limiting the scope of the present application.

[0022] Figure 1This is the immunohistochemical staining of phosphorylated histone H3 in the pulmonary artery of each group of rats in the monocrotaline-induced pulmonary hypertension rat model experimental scheme, scale bar: 50m;

[0023] Figure 2 This is a statistical analysis result of the proportion of phosphorylated histone H3 positive cells in the pulmonary artery of each group of rats in the monocrotaline-induced pulmonary hypertension rat model experimental scheme;

[0024] Figure 3 This is the immunohistochemical staining of phosphorylated histone H3 in the pulmonary artery of each group of rats in the experimental scheme of chronic hypoxia-induced pulmonary hypertension rat model, scale: 50m;

[0025] Figure 4 This is a statistical analysis result of the proportion of phosphorylated histone H3-positive cells in the pulmonary artery of each group of rats in the chronic hypoxia-induced pulmonary hypertension rat model experimental scheme;

[0026] Figure 5 The right ventricular pressure waveforms of rats in each group in the monocrotaline-induced pulmonary hypertension rat model experimental scheme;

[0027] Figure 6 The statistical analysis results of the right ventricular systolic pressure of each group of rats in the monocrotaline-induced pulmonary hypertension rat model experimental scheme;

[0028] Figure 7 The results are statistical analysis of the right ventricular hypertrophy index of each group of rats in the monocrotaline-induced pulmonary hypertension rat model experimental scheme;

[0029] Figure 8 The right ventricular pressure waveforms of rats in each group in the experimental scheme of chronic hypoxia-induced pulmonary hypertension rat model;

[0030] Fig. 9 The statistical analysis results of the right ventricular systolic pressure of each group of rats in the chronic hypoxia-induced pulmonary hypertension rat model experimental scheme;

[0031] Fig.10 The statistical analysis results of the right ventricular hypertrophy index of each group of rats in the chronic hypoxia-induced pulmonary hypertension rat model experimental program;

[0032] Fig.11 The immunofluorescence co-staining images of PCNA (green) and α-SMA (red) in the pulmonary artery of each group of rats in the experimental scheme of monocrotaline-induced and chronic hypoxia-induced pulmonary hypertension rat model are shown. DAPI (blue) shows the location of the cell nucleus, and the long arrows indicate PCNA and α-SMA double-positive cells. Scale bar: 50m;

[0033] Fig.12The results are statistical analysis of the proportion of PCNA and α-SMA double-positive cells in the pulmonary artery of each group of rats in the monocrotaline-induced and chronic hypoxia-induced pulmonary hypertension rat model experimental scheme;

[0034] Fig.13 Figure 2 shows the TUNEL staining (green) and α-SMA immunofluorescence staining (red) of the pulmonary arteries of rats in each group in the experimental scheme of monocrotaline-induced and chronic hypoxia-induced pulmonary hypertension rat models. DAPI (blue) shows the location of the cell nucleus, and short arrows indicate TUNEL and α-SMA double-positive cells. Scale bar: 50m.

[0035] Fig.14 The results are statistical analysis of the proportion of TUNEL and α-SMA double-positive cells in the pulmonary artery of each group of rats in the monocrotaline-induced and chronic hypoxia-induced pulmonary hypertension rat model experimental scheme;

[0036] Fig.15 HE staining, Verhoeff's van Gieson staining, α-SMA immunohistochemical staining (brown), and α-SMA (green) and CD31 (red) immunofluorescence co-staining of pulmonary arteries of rats in each group in the monocrotaline-induced pulmonary hypertension rat model experimental scheme. Long arrows indicate non-muscularized blood vessels, short arrows indicate partially muscularized blood vessels, and thick arrows indicate completely muscularized blood vessels. Scale bar: 50m.

[0037] Fig.16 The statistical analysis results of the pulmonary artery media thickness of each group of rats in the monocrotaline-induced pulmonary hypertension rat model experimental scheme;

[0038] Fig.17 The results are statistical analysis of the degree of distal pulmonary arteriolar muscularization in each group of rats in the monocrotaline-induced pulmonary hypertension rat model experimental scheme;

[0039] Fig.18 HE staining, Verhoeff's van Gieson staining, α-SMA immunohistochemical staining, and α-SMA and CD31 immunofluorescence co-staining of pulmonary arteries of rats in each group in the experimental scheme of chronic hypoxia-induced pulmonary hypertension rat model. Long arrows indicate non-muscularized blood vessels, short arrows indicate partially muscularized blood vessels, and thick arrows indicate completely muscularized blood vessels. Scale bar: 50m.

[0040] Fig.19 The statistical analysis results of the pulmonary artery media thickness of each group of rats in the chronic hypoxia-induced pulmonary hypertension rat model experimental scheme;

[0041] Fig. 20 These are the statistical analysis results of the degree of distal pulmonary arteriolar muscularization in each group of rats in the chronic hypoxia-induced pulmonary hypertension rat model experimental scheme. DETAILED DESCRIPTION

[0042] As used herein:

[0043] "Prepared from" is synonymous with "comprising." As used herein, the terms "comprising," "including," "having," "containing," or any other variation thereof, are intended to cover a non-exclusive inclusion. For example, a composition, process, method, article, or apparatus that comprises the listed elements is not necessarily limited to only those elements but may include other elements not expressly listed or inherent to such composition, process, method, article, or apparatus.

[0044] The conjunction "consisting of excludes any unspecified element, step, or component. If used in a claim, this phrase renders the claim closed-ended so that it does not include materials other than those described, except for conventional impurities associated therewith. When the phrase "consisting of" appears in a clause of the body of a claim rather than immediately following the subject matter, it limits only the elements described in that clause; other elements are not excluded from the claim as a whole.

[0045] When an amount, concentration, or other value or parameter is expressed as a range, a preferred range, or a range defined by a series of upper preferred values ​​and lower preferred values, this should be understood as specifically disclosing all ranges formed by any pairing of any range upper limit or preferred value with any range lower limit or preferred value, regardless of whether the range is disclosed separately. For example, when a range of "1 to 5" is disclosed, the described range should be interpreted as including ranges "1 to 4", "1 to 3", "1 to 2", "1 to 2 and 4 to 5", "1 to 3 and 5", etc. When a numerical range is described in this article, unless otherwise stated, the range is intended to include its end values ​​and all integers and fractions within the range.

[0046] In these examples, parts and percentages are by mass unless otherwise indicated.

[0047] "Parts by mass" refers to the basic unit of measurement for expressing the mass ratio of multiple components. 1 part can represent any unit mass, such as 1g or 2.689g. If we say that the mass of component A is a parts and the mass of component B is b parts, it means the ratio of the mass of component A to the mass of component B is a:b. Alternatively, it means that the mass of component A is aK and the mass of component B is bK (K is an arbitrary number, indicating a multiple factor). It should not be misunderstood that, unlike the mass parts, the sum of the mass of all components is not limited to 100 parts.

[0048] "And / or" is used to indicate that one or both of the stated situations may occur, for example, A and / or B includes (A and B) and (A or B).

[0049] The embodiments of the present application will be described in detail below in conjunction with specific examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present application and should not be considered as limiting the scope of the present application. If specific conditions are not specified in the examples, they are carried out according to normal conditions or the conditions recommended by the manufacturer. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be purchased commercially.

[0050] Example 1 Inhibition of the kinase activity of Aurora kinase B can effectively improve pulmonary artery pressure and right ventricular remodeling, thereby alleviating pulmonary hypertension

[0051] Experimental animals: 6-8 week old male Sprague-Dawley rats were purchased from Hunan Slake Jingda Experimental Animal Company and housed in an SPF barrier environment at the Department of Experimental Animals of Central South University, with 3 rats per cage at room temperature of 25±2℃, relative humidity of 50%, and 12 hours of light per day. Rats were allowed to drink water and eat freely.

[0052] Experimental design and intervention methods: Experiments targeting Aurora kinase B to treat pulmonary hypertension were carried out in rat models of pulmonary hypertension induced by monocrotaline and chronic hypoxia, respectively.

[0053] Experimental plan 1: After adaptive feeding, 29 rats were randomly divided into 4 groups, namely solvent control group (n=8), AZD1152-HQPA intervention group (n=5), monocrotaline + solvent control group (n=8) and monocrotaline + AZD1152-HQPA intervention group (n=8).

[0054] On the day of the experiment, monocrotaline was first dissolved in saline, and rats in the monocrotaline + solvent control group and the monocrotaline + AZD1152-HQPA intervention group were intraperitoneally injected with a 60 mg / kg dose of monocrotaline solution, and the solvent control group and the AZD1152-HQPA intervention group were intraperitoneally injected with the same volume of saline. Then AZD1152-HQPA was dissolved in a solvent of 5% dimethyl sulfoxide + 40% polyethylene glycol 300 + 5% Tween 80 + 50% pure water to prepare a 6.25 mg / mL clear solution, and rats in the AZD1152-HQPA intervention group and the monocrotaline + AZD1152-HQPA intervention group were intraperitoneally injected with a 25 mg / kg dose of the solution, and rats in the solvent control group and the monocrotaline + solvent control group were intraperitoneally injected with the same volume of solvent. AZD1152-HQPA solution and control solvent were continuously administered for 3 days every 7 days until the model was established on the 21st day.

[0055] Experimental plan 2: After adaptive feeding, 29 rats were randomly divided into 4 groups, namely solvent control group (n=8), AZD1152-HQPA intervention group (n=5), chronic hypoxia + solvent control group (n=8) and chronic hypoxia + AZD1152-HQPA intervention group (n=8).

[0056] On the day of the experiment, rats were treated according to the above-mentioned AZD1152-HQPA intervention method; then the rats in the chronic hypoxia group were placed in a low-pressure hypoxia box (nitrogen was input to maintain an oxygen concentration of 10%) for breeding, and a desiccant was placed in the box for dehumidification; the rats in the control group were placed in a normoxic environment, and AZD1152-HQPA solution and control solvent were given for 3 consecutive days every 7 days until the model was established on the 28th day.

[0057] Detection method:

[0058] 1. Pulmonary artery pressure measurement: In the absence of right ventricular outflow tract obstruction (which is the case in this experiment), the mean pulmonary artery pressure is approximately equal to the right ventricular systolic pressure, so the right ventricular systolic pressure is measured to evaluate the pulmonary artery pressure level in rats.

[0059] First, the thermoplasticity of the PE catheter was used to prepare a right heart catheter with a certain curvature of the opening. After modeling, the rat was anesthetized by intraperitoneal injection of sodium pentobarbital solution at a dose of 30 mg / kg, and it was placed in a supine position and fixed on a mouse board. The skin of the neck was prepared, and the skin and muscles were cut layer by layer, and the right external jugular vein was bluntly separated. The distal end of the blood vessel was ligated with sutures, and a loose knot was tied at the proximal end. A "V"-shaped opening was carefully cut between the two knots with ophthalmic scissors. The inner wall of the blood vessel was gently lifted with a probe, and a PE catheter filled with heparin saline was inserted. Then, the loose knot at the proximal end of the blood vessel was ligated to fix the catheter, and the catheter was connected to the pressure sensor.

[0060] Carefully operate the catheter to move in the external jugular vein. When resistance is felt, rotate the catheter counterclockwise about 135° and continue to try to advance the catheter. If resistance is continuously encountered, adjust the angle again until the resistance disappears and the catheter is successfully inserted into the right atrium. At this time, closely observe the changes in the pressure waveform. The atrial wave should show a sine wave with a pressure of about 5 mmHg and a low frequency. Continue to carefully advance the catheter while constantly fine-tuning the rotation angle. If resistance is encountered and the atrial wave disappears, the catheter must be withdrawn immediately and tried to advance again until it is successfully inserted into the right ventricular sinus. At this time, the pressure waveform should show a ventricular wave, that is, a torsade de pointes wave with a pressure of about 20 mmHg (normal rats) and a faster frequency. After the waveform is stable, the rat pressure waveform is recorded by the biological signal recorder, and the average right ventricular systolic pressure of each rat is calculated using the built-in software.

[0061] 2. Determination of right ventricular remodeling level: The right ventricular hypertrophy index was measured to evaluate the right ventricular remodeling level of rats.

[0062] Rats were killed by cervical dislocation, and the heart and lungs were immediately irrigated thoroughly with heparinized saline through the right ventricle. The heart was removed and dissected, the atria and aorta were removed, and the right ventricular free wall and the left ventricle plus the ventricular septum were separated along the right ventricular surface of the ventricular septum. The blood clots on the surface were rinsed with saline and wiped dry, and the weights were calculated for the right ventricular hypertrophy index (i.e., right ventricular free wall weight / left ventricle + ventricular septum weight).

[0063] 3. Determination of the kinase activity of Aurora kinase B: Phosphorylated histone H3 at the Ser10 site is a conserved downstream modification target of Aurora kinase B and is a recognized marker of the kinase activity of Aurora kinase B. Its increased level reflects the increased kinase activity of Aurora kinase B. Therefore, the kinase activity of Aurora kinase B was determined by immunohistochemical measurement of the expression of phosphorylated histone H3 in the pulmonary vascular wall.

[0064] Preparation of paraffin pathological specimens of rat lung tissue: dissect rat lung tissue, separate the left lower lobe of the lung and fix it in 4% paraformaldehyde for 1 day, replace with new formaldehyde solution and fix it for another day, then trim, dehydrate, make it transparent, and embed it in paraffin to store it as pathological specimens at room temperature, and finally prepare a certain number of 4μm tissue sections for subsequent experiments, with the cross section of the lung lobe being the preferred section.

[0065] Immunohistochemistry: Randomly select slices and bake them at 65℃ for 60 minutes. Immediately place the slices in turpentine and dewax and hydrate them according to the procedure of turpentine I for 10 minutes - turpentine II for 10 minutes - anhydrous ethanol I for 5 minutes - anhydrous ethanol II for 5 minutes - 95% ethanol for 5 minutes - 85% ethanol for 5 minutes - 75% ethanol for 5 minutes - 50% ethanol for 5 minutes - pure water.

[0066] Preheat Tris-EDTA antigen repair solution, place the slices in the repair solution, and place in a 95°C water bath for 30 minutes. Keep the repair solution at a slightly boiling state, then cool naturally to room temperature, and wash with PBS three times, 3 minutes each time. Draw a water blocking circle along the edge of the tissue with an immunohistochemical pen, add an appropriate amount of endogenous peroxidase blocker, incubate at room temperature for 10 minutes, and wash with PBS three times, 3 minutes each time. Add an appropriate amount of PBS solution containing 3% bovine serum albumin + 0.3% TritonX-100, and block and permeabilize the tissue at room temperature for 1 hour. Add an appropriate amount of primary antibody solution (1:2000 phosphorylated histone H3 primary antibody solution), incubate overnight in a 4°C wet box, and wash with PBS five times after rewarming, 3 minutes each time.

[0067] Add an appropriate amount of reaction enhancement solution to the slices, incubate at 37°C for 20 minutes, and wash with PBS three times for 3 minutes each time. Add an appropriate amount of enhanced enzyme-labeled goat anti-mouse / rabbit IgG polymer to the slices, incubate at 37°C for 20 minutes, and wash with PBS five times for 3 minutes each time. Prepare a fresh diaminobenzidine PBS solution at a ratio of 1:20, incubate at room temperature for color development, and observe under a microscope for 30 seconds each until the staining is sufficient. Then add hematoxylin stain to the slices, stain for about 10 seconds, and rinse with running water to return to blue for 10 minutes. Place the slices in anhydrous ethanol for 5 minutes, then in xylene for 10 minutes, thoroughly dehydrate and make them transparent, add an appropriate amount of neutral gum to the slices, seal them with a coverslip, and observe the results under a microscope.

[0068] For phosphorylated histone H3 immunohistochemical staining, the proportion of phosphorylated histone H3-positive cells was quantitatively analyzed to evaluate whether the model was successfully established in the AZD1152-HQPA intervention group. The proportion = number of phosphorylated histone H3-positive cells / number of pulmonary artery wall cells*100%. The proportion of phosphorylated histone H3-positive cells in each rat was the average of the proportions of positive cells in no less than 20 vessels.

[0069] 4. Statistical analysis: All statistical analyses were performed in R (version: 4.3.2) and GraphPad Prism 8.0.

[0070] The Shapiro-Wilk test and Bartlett test were used to determine whether continuous variables were normally distributed and had homogeneous variance. Continuous variables that were normally distributed and had homogeneous variance were expressed as mean ± standard deviation, and the differences between groups were compared using Student's t test; otherwise, they were expressed as median (interquartile range), and the differences between groups were compared using Mann-Whitney U test. Categorical variables were expressed as number (percentage), and the differences between groups were compared using chi-square test or Fisher's exact test. P < 0.05 (two-sided test) was considered statistically significant.

[0071] 5. Experimental Results

[0072] (1) AZD1152-HQPA administration effectively inhibited the kinase activity of Aurora kinase B

[0073] The expression level of phosphorylated histone H3 in rat lung tissue sections was detected by immunohistochemistry, and it was found that the proportion of phosphorylated histone H3 positive cells in the pulmonary arteries of monocrotaline rats and chronic hypoxia rats treated with AZD1152-HQPA was significantly lower than that in the pulmonary hypertension rat model treated with the control solvent, proving that the kinase activity of Aurora kinase B was reduced. Figures 1 to 4 shown.

[0074] (2) Inhibition of the kinase activity of Aurora kinase B significantly improved pulmonary artery pressure and right ventricular remodeling in a rat model of pulmonary hypertension

[0075] The rat right heart catheter detection and cardiac anatomical weighing showed that the right ventricular systolic pressure and right ventricular hypertrophy index of monocrotaline and chronic hypoxia rat models were significantly higher than those of normal control rats, while AZD1152-HQPA significantly reduced the right ventricular systolic pressure and right ventricular hypertrophy index of pulmonary hypertension rat models and had no significant effect on normal rats. Figures 5 to 10 shown.

[0076] Based on the results of Example 1, it can be seen that the administration of AZD1152-HQPA successfully inhibited the kinase activity of Aurora kinase B, thereby effectively improving pulmonary artery pressure and right ventricular remodeling, and relieving pulmonary hypertension.

[0077] Example 2 Inhibition of the kinase activity of Aurora kinase B alleviates pulmonary vascular remodeling by reversing excessive proliferation and apoptosis resistance of pulmonary artery smooth muscle cells

[0078] On the basis of the experiment in Example 1, the effect of inhibiting the kinase activity of Aurora kinase B on pulmonary vascular remodeling caused by pulmonary artery smooth muscle cell lesions was further studied.

[0079] Detection method:

[0080] 1. Detect the expression level of PCNA, a proliferation marker of pulmonary artery smooth muscle cells, by immunofluorescence staining. PCNA is a protein that is recognized to be upregulated during cell proliferation and can indirectly reflect the proliferation of pulmonary artery smooth muscle cells.

[0081] Randomly select slices, bake them in the usual way, dewax and hydrate them, and then use an immunohistochemistry pen to draw a water-blocking circle along the edge of the tissue. Add an appropriate amount of tissue autofluorescence quencher A solution to the tissue, incubate at room temperature for 30 minutes, and wash with PBS three times, 3 minutes each time. Add an appropriate amount of PBS solution containing 3% bovine serum albumin + 0.3% TritonX-100, block and permeabilize the tissue at room temperature for 1 hour. Add an appropriate amount of primary antibody dilution solution (1:200 α-SMA and 1:200 phosphorylated histone H3 mixed primary antibody solution), incubate overnight in a 4℃ wet box, and wash with PBS five times, 3 minutes each time. Add an appropriate amount of fluorescent dye-coupled secondary antibody solution (generally 1:200 dilution) of the corresponding species of the primary antibody, incubate at room temperature for 2 hours in the dark, and wash with PBS five times, 3 minutes each time. Add an appropriate amount of tissue autofluorescence quencher B solution, incubate at room temperature for 5 minutes, and wash the slices with running water for 10 minutes. Add wash-free DAPI dye solution and anti-fluorescence quencher, fix the coverslip, and finally observe under an upright fluorescence microscope.

[0082] The ratio of double positive cells of PCNA and α-SMA, a marker of pulmonary artery smooth muscle cells, was quantitatively analyzed. The ratio = number of double positive cells / number of α-SMA positive cells*100%. The ratio of double positive cells in each rat was the average of the ratios of double positive cells in no less than 20 vessels.

[0083] 2. Detect the apoptosis level of pulmonary artery smooth muscle cells by TUNEL staining. TUNEL staining is one of the main methods recognized for detecting cell apoptosis.

[0084] Randomly select slices, bake, dewax and hydrate in the usual way. Prepare 20g / mL proteinase K solution and incubate the slices at room temperature for 20 minutes to permeabilize the tissue. For the positive control sample, add DNAse to lyse the tissue cell genome; for the negative control sample, do not add recombinant terminal deoxyribonucleotidyl transferase in the next step. Add balance solution to each group of samples and positive and negative control samples, incubate at room temperature for 30 minutes, and prepare TUNEL labeling solution in the dark according to the system of 34μL pure water + 10μL 5x balance solution + 5μL BrightGreen tetramethylrhodamine-deoxyuridine triphosphate dye + 1μL recombinant terminal deoxyribonucleotidyl transferase. Add labeling solution to the above groups of samples, cover the labeling solution with sealing film to ensure uniform distribution of the reagent, incubate in a 37℃ wet box for 60 minutes, and wash twice with PBS for 3 minutes each time. Immunofluorescence staining of α-SMA was performed according to the immunofluorescence staining method. Add wash-free DAPI dye solution and anti-fluorescence quencher, fix the coverslip, and finally observe under an upright fluorescence microscope.

[0085] The TUNEL fluorescence signal and the ratio of α-SMA double-positive cells were quantitatively analyzed. The ratio = number of double-positive cells / number of α-SMA positive cells*100%. The ratio of double-positive cells in each rat was the average of the ratios of double-positive cells in no less than 20 vessels.

[0086] 3. HE staining to detect the general morphology of the pulmonary artery

[0087] Randomly select slices, bake, dewax and hydrate as usual. Add appropriate amount of hematoxylin stain to the slices, stain for 10 minutes, differentiate in 1% hydrochloric acid ethanol solution for about 3 seconds, and place in running water to rinse and blue for 10 minutes. Place the slices in 70% and 80% ethanol solutions for 2 minutes, then add appropriate amount of eosin stain, stain for about 10 seconds, and place in 95% ethanol solution for color separation. At this time, observe the depth of HE staining under a microscope. If the eosin staining is too dark, place it in 80% ethanol solution to wash and stain again. If it is too light, use eosin stain to re-stain. Conventionally dehydrate, make it transparent, and seal the slices, and observe the results under a microscope.

[0088] 4. Verhoeff's Van Gieson staining and α-SMA immunohistochemical staining to detect the media thickness of the pulmonary artery

[0089] Randomly select slices, bake, dewax and hydrate as usual. Place the slices in a resorcinol fuchsin solution and stain at room temperature for 2 hours, being careful not to dry the slices. Rinse the slices in running water for 1 minute, then place them in a hydrochloric acid differentiation solution for about 3 seconds until the black dye on the surface of the tissue dissolves, and then place them in running water for 10 minutes. Add van Gieson dye to the slices, stain for 30 seconds, then quickly place them in a 95% ethanol solution for 5 seconds and then place them in anhydrous ethanol. Dehydrate, make them transparent, and seal the slices as usual, and observe the results under a microscope.

[0090] This staining method can well display the elastic plates inside and outside the pulmonary artery, so it is suitable for observing the pulmonary artery media. The media thickness was quantitatively analyzed using Image J software, media thickness = (vessel diameter - lumen diameter) / vessel diameter * 100%, and the media thickness of each rat was the average of no less than 20 blood vessels.

[0091] 5. Detection of the degree of muscularization of distal pulmonary arterioles by immunofluorescence staining

[0092] According to the immunofluorescence staining method, the immunofluorescence co-staining of pulmonary artery endothelial cell markers CD31 and α-SMA was performed. The degree of muscularization of distal pulmonary arterioles can be divided into three categories: complete muscularization (CD31-positive intima wrapped with complete α-SMA-positive media), partial muscularization (partially complete media wrapped outside the intima) and non-muscularization (no media wrapped outside the intima). The degree of muscularization of pulmonary arterioles in each rat was the average value of no less than 50 vessels, and the three categories were expressed as percentages. The complete muscularization of pulmonary arterioles in each group of rats was statistically analyzed.

[0093] 6. The statistical analysis method is the same as in Example 1.

[0094] 7. Experimental Results

[0095] (1) Inhibition of the kinase activity of Aurora kinase B significantly reduces the proliferation of pulmonary artery smooth muscle cells and promotes their apoptosis

[0096] like Fig.11 , Fig.12 , Fig.13 and Fig.14 As shown in the figure, the PCNA positive ratio of pulmonary artery smooth muscle cells in the monocrotaline and chronic hypoxia rat models was significantly higher than that in the control rats, and the TUNEL positive ratio was significantly lower than that in the control rats, indicating that the pulmonary artery smooth muscle cells in the pulmonary hypertension rat model showed pathological characteristics of excessive proliferation and apoptosis resistance. After the administration of AZD1152-HQPA, the proliferation level of pulmonary artery smooth muscle cells in the pulmonary hypertension rat model was significantly reduced, and the apoptosis level was significantly increased.

[0097] (2) Inhibition of the kinase activity of Aurora kinase B significantly reduced pulmonary artery medial thickening and distal pulmonary arteriolar muscularization

[0098] To further reveal the role of Aurora B in pulmonary vascular remodeling, especially its role in pulmonary artery media remodeling dominated by pulmonary artery smooth muscle cells, the general morphology of rat pulmonary arteries was evaluated by HE staining, the changes in the thickness of rat pulmonary artery media were evaluated by Verhoeff's vanGieson staining and α-SMA immunohistochemical staining, and the degree of muscularization of rat distal pulmonary arterioles was evaluated by immunofluorescence co-staining of α-SMA and CD31.

[0099] like Figures 15 to 20 As shown in the results, compared with the control rats, monocrotaline and chronic hypoxia rats showed excessive thickening of the pulmonary artery wall, relative stenosis or even occlusion of the lumen, and obvious muscularization of the distal arterioles, while AZD1152-HQPA effectively reversed these pathological phenomena. In normal rats, AZD1152-HQPA had no significant effect on the morphology of pulmonary vessels.

[0100] like Fig.15 and Fig.18 As shown in the figure, Verhoeff's van Gieson staining can clearly show the inner and outer elastic plates and the middle media layer of the pulmonary artery. Based on this staining, the pulmonary artery media thickness of each group of rats was quantitatively analyzed, confirming that the media thickness of the pulmonary hypertension rat model was significantly increased compared with the control rats, and AZD1152-HQPA intervention significantly reduced the media thickness of the pulmonary hypertension rat model. Fig.16 and Fig.19 shown.

[0101] α-SMA and CD31 immunofluorescence co-staining can simultaneously display the intima and media of the pulmonary artery, thus reflecting whether the distal pulmonary arterioles of rats are muscularized. Most of the arterioles in normal control rats and rats treated with AZD1152-HQPA alone were in a non-muscularized state. Almost all of the arterioles in the pulmonary hypertension rat model were muscularized to varying degrees, while the degree of muscularization of the arterioles in pulmonary hypertension rats treated with AZD1152-HQPA was significantly reduced, mainly reflected in the decrease in the proportion of fully muscularized arterioles, such as Fig.17 and Fig. 20 shown.

[0102] Based on the results of Example 2, it can be seen that inhibiting the kinase activity of Aurora kinase B significantly reverses the pathological process of excessive proliferation and apoptosis resistance of pulmonary artery smooth muscle cells, thereby effectively alleviating the progression of pulmonary vascular remodeling in pulmonary hypertension.

[0103] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present application, rather than to limit it. Although the present application has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present application.

[0104] In addition, those skilled in the art will appreciate that, although some embodiments herein include certain features included in other embodiments but not other features, the combination of features of different embodiments is meant to be within the scope of the present application and form different embodiments. For example, in the above claims, any one of the claimed embodiments may be used in any combination. The information disclosed in this background technology section is intended only to deepen the understanding of the overall background technology of the present application and should not be regarded as an admission or in any form of implication that the information constitutes prior art known to those skilled in the art.

Claims

1. Use of Aurora kinase B inhibitors in the preparation of drugs for the treatment of pulmonary arterial hypertension.

2. Use of the Aurora kinase B inhibitor according to claim 1 in the preparation of a drug for treating pulmonary hypertension, characterized in that: The Aurora kinase B inhibitors include substances that inhibit the kinase activity of Aurora kinase B in pulmonary artery smooth muscle cells and / or substances that reduce the expression level of Aurora kinase B.

3. Use of the Aurora kinase B inhibitor according to claim 1 in the preparation of a drug for treating pulmonary hypertension, characterized in that: The Aurora kinase B inhibitor is AZD1152-HQPA.

4. Use of the Aurora kinase B inhibitor according to claim 3 in the preparation of a medicament for treating pulmonary hypertension, characterized in that: The molecular formula of AZD1152-HQPA is C 26 H 30 FN7O3.

5. Use of the Aurora kinase B inhibitor according to claim 1 in the preparation of a medicament for treating pulmonary hypertension, characterized in that: The Aurora kinase B inhibitor is used in combination with other types of drugs for treating pulmonary hypertension, or the Aurora kinase B inhibitor is used alone.

6. Use of the Aurora kinase B inhibitor according to claim 5 in the preparation of a drug for treating pulmonary hypertension, characterized in that: The other types of drugs for treating pulmonary hypertension include: at least one of prostacyclin analogs, prostacyclin receptor agonists, endothelin receptor antagonists, phosphodiesterase 5 inhibitors and soluble guanylate cyclase agonists.

7. A composition for treating pulmonary hypertension, characterized in that: The active ingredient of the composition includes an Aurora kinase B inhibitor.

8. The composition for treating pulmonary hypertension according to claim 7, characterized in that: The Aurora kinase B inhibitor is AZD1152-HQPA.

9. The composition for treating pulmonary hypertension according to claim 8, characterized in that: The effective dose of AZD1152-HQPA is 5-30 mg / kg / day.

10. The composition for treating pulmonary hypertension according to claim 7, characterized in that: The composition may be in the form of at least one of tablets, solutions, suspensions, emulsions, powders, granules, capsules, microcapsules, microspheres, injections, and liposomes; the composition may also include pharmaceutical excipients.

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