Application of a TGF-β receptor inhibitor in the treatment of pulmonary vascular diseases

Abstract

The present invention belongs to the field of pulmonary vascular disease treatment, and specifically discloses a TGF-β receptor inhibitor for the treatment of pulmonary hypertension. In the rat pulmonary hypertension model induced by monocrotaline (MCT), after using the preparation, the right ventricular systolic pressure (RVSP), right ventricular hypertrophy (Fulton index), pulmonary vascular resistance and vascular wall thickness of the rat can be reversed and reduced, and the survival rate of monocrotaline (MCT)-induced pulmonary hypertension rats can be prolonged. Compared with the modeling group, the expression of IL-17, IL-6 and GM-CSF inflammatory factors in the lung tissue of the preparation-treated group was significantly reduced, and the expression of extracellular matrix-related genes CTGF, Fn1 and Col1α1 also decreased. The preparation can significantly alleviate the pulmonary hypertension in rats induced by monocrotaline (MCT), showing good therapeutic effect and application prospects.

Description

Technical field:

[0001] The present invention relates to the field of pulmonary vascular disease treatment, and particularly to the use of a TGF-β receptor inhibitor in the treatment of pulmonary hypertension. Background technology:

[0002] Pulmonary hypertension is a pulmonary vascular disease that can be caused by various cardiopulmonary diseases. It is an abnormal state of persistently elevated pulmonary vascular resistance, with low survival rates and high mortality rates. [1] Pulmonary hypertension is a clinical and pathophysiological syndrome characterized by changes in pulmonary vascular structure or function caused by a variety of heterogeneous diseases (etiologies) and different pathogenesis, leading to increased pulmonary vascular resistance and pulmonary artery pressure, which can then develop into right heart failure or even death. The hemodynamic manifestations are a mean pulmonary artery pressure greater than 20 mmHg, a pulmonary artery wedge pressure ≤15 mmHg, and a pulmonary vascular resistance ≥3 Woods units as measured by right cardiac catheterization. It can be divided into five subtypes. [2] , the present invention is mainly aimed at the application of type I arterial pulmonary hypertension.

[0003] Currently, drug treatments for pulmonary arterial hypertension mainly target three pathways: the prostaglandin pathway, the endothelin pathway, and the nitric oxide (NO) pathway. Approved PAH drug treatments include type 5 phosphodiesterase inhibitors (sildenafil, tadalafil), guanylate cyclase agonists (riociguat), prostacyclins (epoprostenol, treprostinil), and endothelin receptor antagonists (bosentan, ambrisentan). Some large randomized clinical trials have shown that combined drug therapy targeting different pathways can delay disease progression and improve survival compared with monotherapy. [3] .

[0004] Although there are currently a variety of methods for treating patients with pulmonary arterial hypertension, the treatment cycle is long, the cost is high, and the economic and social burdens are heavy.

[0005] Pulmonary vascular endothelial cells can regulate pulmonary artery pressure, and the TGF-β signaling pathway plays an important role in maintaining the normal physiological function and integrity of pulmonary vascular endothelial cells. [4] BMPR2 is a receptor in the TGF-β family pathway. Patients with hereditary pulmonary hypertension often carry a mutated BMPR2 gene. [5,6] In the TGF-β family, in addition to BMP signals, TGF-β1 and Activin A and their downstream genes are also abnormally expressed in pulmonary hypertension. In animal models of pulmonary hypertension, the expression of connective tissue growth factor (CTGF), collagen and fibronectin (Fn1) is increased, resulting in thickening of the blood vessel wall, vascular stenosis, increased pulmonary artery pressure and right ventricular hypertrophy. [7]Although the TGF-β1 receptor ALK5 inhibitors SB525334 and SD-208 can alleviate pulmonary hypertension in monocrotaline-induced rats and inhibit the proliferation of pulmonary vascular smooth muscle, they cannot restore the normal function of pulmonary vascular endothelial cells. [8] Therefore, inhibiting the TGF-β receptor signaling pathway in endothelial cells to obtain new treatments has become a potential research and development direction.

[0006] literature:

[0007] [1]Thenappan T, Ormiston ML, Ryan JJ, Archer SL. Pulmonary arterialhypertension: pathogenesis and clinical management. BMJ. 2018; 360: j5492.

[0008] [2]Marc Humbert, Gabor Kovacs, Marius M. Hoeper et al. 2022ESC / ERSGuidelines for the diagnosis and treatment of pulmonary hypertension. European Respiratory Journal, 2022: 2200879

[0009] [3]Ruopp NF, Cockrill BA. Diagnosis and Treatment of Pulmonary ArterialHypertension:A Review.JAMA.2022Apr 12;327(14):1379-1391

[0010] [4] R. Derynck, EHBudi. Specificity, versatility, and control of TGF-betafamily signaling. Sci Signal, 2019, 12(570)

[0011] [5]MarOrriols,Maria Catalina Gomez-Puerto,Peter Ten Dijke.BMP type IIreceptor as a therapeutic target in pulmonary arterial hypertension.Cellularand Molecular Life Sciences,2017,74(16):2979-2995

[0012] [6]Elaine Soon,Alexi Crosby,Mark Southwoodet al.Bone MorphogeneticProtein Receptor Type II Deficiency and Increased Inflammatory CytokineProduction.AGateway to Pulmonary Arterial Hypertension.American Journal ofRespiratory and Critical Care Medicine,2015,192(7):859-872

[0013] [7]Masato Morikawa,Rik Derynck,Kohei Miyazono.TGF-βand the TGF-βFamily:Context-Dependent Roles in Cell and Tissue Physiology.Cold SpringHarbor Perspectives in Biology,2016,8(5):a21873

[0014] [8] M. Thomas, C. Docx, AM Holmeset al. Activin-like kinase 5 (ALK5) mediates abnormal proliferation of vascular smooth muscle cells from patients with familial pulmonary arterial hypertension and is involved in the progression of experimental pulmonary arterial hypertension induced by monocrotaline. Am J Pathol, 2009, 174(2): 380-389 Summary of the invention:

[0015] In order to overcome the deficiencies in the prior art, the present invention provides use of a TGF-β receptor inhibitor in the treatment of pulmonary vascular diseases.

[0016] As an object of the present invention, the present invention provides a TGF-β receptor small molecule inhibitor C 22 H 18 Application of FN7 in improving pulmonary arterial hypertension.

[0017] Preferably, the indicators related to pulmonary hypertension are improved by regulating the TGF-β receptor signaling pathway.

[0018] Preferably, by inhibiting the TGF-β1 receptor ALK5 and the Activin A receptor ALK4, the monocrotaline-induced right ventricular systolic pressure and pulmonary vascular resistance in rats can be reduced, and the survival rate can be significantly improved.

[0019] Preferably, the small molecule compound C 22 H 18 FN7 can inhibit the expression of genes PAI-1, CTGF, Fn1, Col1α1, GM-CSF, IL-6 or α-SMA, and promote the expression of genes CDH5 and Bmpr2.

[0020] Preferably, the pulmonary hypertension rat model is established by induction with monocrotaline, which meets the national new drug approval requirements.

[0021] Beneficial effects:

[0022] This application studies TGF-β receptor inhibitors for alleviating pulmonary vascular diseases such as pulmonary hypertension. This compound is a small molecule compound that inhibits the activation of the serine / threonine protein kinase sites of the TGF-β1 receptor ALK5 and the Activin A receptor ALK4, inhibiting the phosphorylation of smad2 / 3, thereby reducing downstream signal transduction, inhibiting pulmonary vascular endothelial cell proliferation and mesenchymal transition, reducing right ventricular systolic pressure in a rat model of pulmonary hypertension induced by monocrotaline, and significantly improving survival rate. In in vitro experiments, under the activation of Activin A, this compound can inhibit the expression of ALK4 and ALK5 genes in pulmonary vascular endothelial cells, promote the increase of BMPR2 gene expression, and inhibit the expression of PAI-1 and CTGF, downstream genes of TGF-β1 and Activin A. Description of the drawings:

[0023] Figure 1 To construct the experimental flow chart for monocrotaline-induced pulmonary hypertension in rats and the use of compounds.

[0024] Figure 2 These are the results of right ventricular pressure testing in model rats induced by monocrotaline for 3 weeks.

[0025] Figure 3 This is a graph of right ventricular pressure measurement.

[0026] Figure 4 The right ventricular output result.

[0027] Figure 5 Ejection fraction indicator.

[0028] Figure 6 Diagram of tricuspid annular systolic displacement.

[0029] Figure 7 is the Fulton index graph.

[0030] Figure 8 Pulmonary vascular resistance diagram.

[0031] Figure 9 This is the survival curve of rats.

[0032] Figure 10 HE staining of rat lung tissue.

[0033] Figure 11 SM22α staining and vascular thickness quantification of rat lung tissue.

[0034] Figure 12 Figure 4 shows the staining and quantification of ALK4 in rat lung tissue.

[0035] Figure 13 The expression diagram of ALK4, Bmpr2 and phosphorylated Smad1 / 5 proteins in rat lung tissue.

[0036] Figure 14 Quantitative expression of ALK4, Bmpr2 and phosphorylated Smad1 / 5 proteins in rat lung tissue.

[0037] Figure 15 This is the gene expression map of rat lung tissue.

[0038] Figure 16 It is a gene expression chart of human pulmonary vascular endothelial cells, where AT-1, AT-2, and AT-4 represent Activin A + TEW (1 nM), Activin A + TEW (2 nM), and Activin A + TEW (4 nM), respectively.

[0039] Figure 17 This is a map of gene expression related to mesenchymal transition in human pulmonary vascular endothelial cells.

[0040] Figure 18 is the structural formula of the compound. Specific implementation method:

[0041] In order to better illustrate the purpose, technical solutions and advantages of the present invention, the present application will be further described below in conjunction with specific embodiments.

[0042] Example 1

[0043] The present invention is based on the pulmonary vascular endothelial TGF-β signaling pathway. First, 170-180 g SD rats were purchased and divided into a control group, an experimental group, and a treatment group, as shown in Table 1. The experimental and treatment groups were intraperitoneally injected with 55 mg / kg of monocrotaline (MCT), while the control group was intraperitoneally injected with an equal volume of PBS solution for 2 weeks.

[0044] Three weeks after monocrotaline induced pulmonary hypertension in rats, the right ventricular systolic pressure of the control group, MCT group, and MCT+PBS group was measured by right cardiac catheterization, which proved that MCT induced an increase in pulmonary artery pressure in rats. Subsequently, the rats in the treatment group were treated with two different concentrations of the compound by weekly gavage (20 mg / kg and 40 mg / kg), and the control group was gavaged with PBS. The rats were gavaged once a week, with the last gavage on the 32nd day, and subsequent experiments were performed on the fifth week.

[0045] The compound is a highly effective, selective, orally bioavailable small molecule inhibitor of TGF-β receptor ALK4 / ALK5 with IC50 of 13nM and 11nM, respectively. It is a small molecule compound with the molecular formula C 22 H 18 FN7, CAS No. 1352608-82-2, structural formula Figure 18As shown, after monocrotaline treatment for 4 weeks, the hemodynamic indicators and gene expression of the four groups of rats were measured. The experimental process is as follows Figure 1 shown.

[0046] Table 1 Experimental groups and treatments

[0047]

[0048]

[0049] At the end of the third week of MCT induction, rats were anesthetized with 2% sodium barbital intraperitoneally. We used right cardiac catheterization to measure the right ventricular systolic pressure (RVSP) of the control group, MCT group, and MCT+PBS group. Compared with the control group, the right ventricular systolic pressure of the rats in the MCT group and MCT+PBS group was significantly increased, and there was no difference between the two groups. Figure 2 As shown in the figure, it was proved that MCT induced pulmonary hypertension model in rats successfully. Then the rats were treated with two concentrations of the compound. The right ventricular pressure was detected after 2 weeks. The right ventricular pressure of the high-dose treatment group was reduced, which was statistically significant compared with the experimental group. Figure 3 As shown. Rats were anesthetized with 7% chloral hydrate at a dose of 0.5 mL / 100 g, and right ventricular output was detected using an animal ultrasound instrument. Ultrasound detection showed that the hemodynamic indicators of the model group rats, such as right ventricular output (CO), ejection fraction (EF), and tricuspid annular plane systolic excursion (TPASE), were significantly reduced, while these hemodynamic indicators were significantly improved in the treatment group, such as Figure 4 、 5 As shown in Figure 6. The right ventricle of the rat was cut out according to the anatomical part of the heart using surgical instruments. The right ventricle, left ventricle and ventricular septum were weighed and the ratio between the two was calculated. The pulmonary vascular resistance was calculated according to the formula right ventricular systolic pressure × body weight ÷ right ventricular output. The Fulton index and pulmonary vascular resistance were also increased in the modeling group. After using the compound, the Fulton index and pulmonary vascular resistance decreased, as shown in Figure 6. Figure 7 and Figure 8 As shown. Starting from the intraperitoneal injection of monocrotaline in rats, the survival status of each group of rats was recorded daily, and the recorded values ​​were entered into Graphpad Prism8. The Logrank statistical test results were viewed using CurveComparison. The survival rates of rats in the treatment group and the model group were significantly different. This compound can prolong the survival rate of rats induced by monocrotaline, as shown in Figure 2. Figure 9 shown.

[0050] The routine HE staining procedure is as follows: after the rats are intubated, the lung tissue is cut and placed in a 4% paraformaldehyde solution. Three rats are selected from each group, and the longest section of the lung tissue is taken for staining. Microscope observation is selected at 10× and 40× magnifications; SM22α immunohistochemical staining is performed routinely. The lung sections are the same as those in the control group. Figure 10 ImageJ software was used to calculate the percentage of vascular wall thickness of each rat. In terms of histology, HE staining and SM22α immunohistochemical staining showed that the pulmonary vessels in the model group were significantly narrowed and had plexiform lesions, while the pulmonary vascular stenosis in the treatment group was significantly alleviated. Figure 11 、 12 As shown, we also used ALK4 immunohistochemical staining, ALK4 immunohistochemical routine staining, lung sections were the same Figure 11 Quantitative analysis was performed using ImageJ software. The expression of ALK4 in the vascular endothelium of the model group was significantly increased, while that in the treatment group was decreased, and the expression was statistically significant. Figure 12 shown.

[0051] In the present invention, the protein expression in rat lung tissue was detected by western blot. Three rats were selected from each group. The lung tissue was ground into a homogenate using a tissue grinder, and protease and phosphatase inhibitors were added to the lysate. After the protein concentration was quantified, the sample was loaded with a uniform protein concentration. ALK4 and Bmpr2 antibodies were used to detect protein levels, and the bands were quantitatively analyzed using ImageJ software. The expression of ALK4 and Bmpr2 in lung tissue was detected by western blot protein blotting technology. The expression of ALK4 in the modeling group was significantly increased, and the expression of ALK4 in the lung tissue of the treatment group was reduced after treatment with the inhibitor. Bmpr2 is also a member of the TGFβ family. Its normal expression plays an important role in maintaining the integrity and normal function of endothelial cells. In the modeling group, the expression of Bmpr2 was significantly decreased, while the expression level of the protein was restored in the treatment group. Figure 13 、 14 shown.

[0052] The lung tissues of rats in each group were ground, RNA was extracted using the Trizol method, and reverse transcribed into cDNA. qPCR was used to detect gene expression in lung tissues. The expression of TGF-β downstream connective tissue-related target genes CTGF, Fn1, and Col1α1 increased in the modeling group, while the expression of these genes decreased significantly in the treatment group. In addition, the expression of inflammatory factors such as IL-6, IL-17, and GM-CSF also decreased. Figure 15The above results show that in the monocrotaline-induced pulmonary hypertension model in rats, the inhibitor can significantly improve the rats' hemodynamic indicators, relieve vascular stenosis, reduce the gene and protein expression of ALK4 on the surface of lung tissue and pulmonary vascular endothelial cells, restore Bmpr2 gene expression and phosphorylation of downstream signaling molecules, and reduce the expression of connective tissue factor and inflammatory factors.

[0053] In the in vitro experiment, we first cultured human pulmonary vascular endothelial cells at a rate of 8×10 5 The cell density was inoculated into a 12-well plate, and human pulmonary vascular endothelial cells were stimulated with different concentrations of ActivinA (50 ng / mL and 100 ng / mL), and 1 nM, 2 nM and 4 nM compounds were added to the corresponding wells. After 48 hours, the cells were collected, RNA was extracted with Trizol, and converted into cDNA. Real-time quantitative PCR was used to detect gene expression. The results showed that the expression of ALK4, ALK5, CTGF and IL-6 in human pulmonary vascular endothelial cells stimulated by ActicinA (50 ng / mL and 100 ng / mL) was significantly increased, and the expression of Bmpr2 was decreased, and there was a certain concentration dependence. After the addition of the compound (1 nM, 2 nM and 4 nM), the expression of ALK4, ALK5, CTGF and IL-6 decreased in a dose-dependent manner with the increase of the compound concentration, and the expression of Bmpr2 increased in a dose-dependent manner, as shown in FIG. Figure 16 shown.

[0054] We also detected the expression of endothelial-mesenchymal transition-related genes. After treatment with ActivinA and IL-17, the expression of α-SMA in human pulmonary vascular endothelial cells increased, while the expression of endothelial marker gene CDH5 decreased. The inhibitor can significantly reduce the expression of α-SMA and restore the expression of CDH5. Figure 17 The above results indicate that the inhibitor can restore the phenotypic transformation of human pulmonary vascular endothelial cells stimulated by Activin A and IL-17, maintain the normal expression of marker genes of pulmonary vascular endothelial cells, reduce the expression of inflammatory factors and connective tissue factors, and restore the balance between the pro-proliferative Activin A signal and the anti-proliferative BMP signal.

[0055] The above experimental results show that in the monocrotaline (MCT)-induced rat model of pulmonary hypertension, the inhibitor can reduce right ventricular systolic pressure, Fulton index, and pulmonary vascular resistance, as well as increase right ventricular output and tricuspid annular systolic excursion. Histologically, it alleviates vascular wall thickening and reduces the gene expression of inflammatory factors IL-6 and GM-CSF in lung tissue. In vitro, it can also inhibit the expression of ALK4, ALK5, CTGF, and IL-6 in human pulmonary vascular endothelial cells stimulated by Activin A and IL-17, restore the gene expression of Bmpr2, inhibit endothelial cell-mesenchymal transition, and maintain the phenotype of pulmonary vascular endothelial cells. In summary, the inhibitor has a significant effect in the treatment of monocrotaline-induced pulmonary hypertension in rats and is effective in the treatment of pulmonary vascular disease.

[0056] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit the scope of protection of the present invention. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention may be modified or replaced by equivalents without departing from the essence and scope of the technical solutions of the present invention.

Claims

1. Use of C22H18FN7, a small molecule inhibitor of TGF-β signaling receptor, for the preparation of a drug for treating pulmonary arterial hypertension, characterized in that: The CAS number of C22H18FN7, a small molecule inhibitor of TGF-β signaling receptor ALK4, is 1352608-82-2. In rats with pulmonary hypertension induced by monocrotaline, it reduces right ventricular systolic pressure and pulmonary vascular resistance and significantly prolongs survival rate.

2. The use according to claim 1, characterized in that: It improves pulmonary hypertension by regulating the TGF-β receptor signaling pathway, reduces the expression of IL-17, IL-6 and GM-CSF in lung tissues of rats with pulmonary hypertension induced by monocrotaline, and reduces the expression of extracellular matrix-related genes PAI-1, CTGF and Fn1; at the same time, it inhibits the expression of IL-6, CTGF and α-SMA genes in endothelial cells, and restores the expression of CDH5 and Bmpr2 genes in endothelial cells.

3. The use according to claim 1, characterized in that: The pulmonary hypertension rat model was established by inducing monocrotaline, which meets the national new drug approval requirements.

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