Application of AXL inhibitor in preparation of medicine for treating heart failure

Abstract

The invention discloses application of an AXL inhibitor in preparation of a medicine for treating heart failure, and relates to the field of pharmacy. The invention provides application of an AXL inhibitor in a medicine for treating heart failure. The AXL inhibitor can remarkably inhibit abnormal activation of fibroblasts and can remarkably improve the heart function of a mouse with heart failure.

Description

Technical Field

[0001] The present invention relates to the field of pharmacy, and in particular to use of an AXL inhibitor in preparing a medicine for treating heart failure. Background Art

[0002] Heart failure is a clinical syndrome caused by increased intracardiac pressure and / or insufficient cardiac output at rest and / or during exercise, which is caused by abnormal cardiac structure and / or function. Myocardial remodeling is the basic pathological mechanism of heart failure, in which cardiac fibrosis (CF) plays a vital role. Cardiac fibroblasts (CFs) are the main participating cells in myocardial fibrosis. Abnormal proliferation and differentiation of CFs under pathological stimulation leads to excessive production of extracellular matrix (ECM) and collagen deposition, causing increased cardiac stiffness, affecting normal electrical signal conduction, inducing arrhythmias, cardiac structural changes, and systolic and diastolic dysfunction, ultimately leading to heart failure. At present, although many studies have been carried out on the pathogenesis and treatment strategies of myocardial fibrosis, there are still many bottlenecks. The main problem is that the existing treatment methods can only improve the clinical symptoms of patients, but due to the lack of specific treatment methods to reverse or effectively inhibit the development of myocardial fibrosis, the progression of the disease cannot be controlled. Therefore, we need to have a deeper understanding of the core mechanisms that drive the progression of myocardial fibrosis, and on this basis, find new targets for the treatment of myocardial fibrosis, slow the progression of myocardial fibrosis, and thus play a role in treating heart failure.

[0003] The current treatment strategy for myocardial fibrosis is to inhibit the TGF-β signaling pathway and the renin-angiotensin-aldosterone system, but the efficacy of targeted drugs is limited. The drug treatment of myocardial fibrosis is based on diuretics to relieve congestion and neurohormone antagonists to improve the clinical course. Angiotensin-converting enzyme inhibitors (ACEI) or angiotensin receptor blockers (ARB), β-blockers and mineralocorticoid antagonists (MRA) are the main drugs for the clinical treatment of myocardial fibrosis. However, there is currently no sufficient evidence to prove that other drugs can improve myocardial fibrosis. In addition, the anti-fibrotic drugs currently used in clinical practice include antacids, N-acetylcysteine, glucocorticoids, anti-infective drugs, etc., which can only reduce patient mortality to a certain extent. Therefore, new anti-fibrotic drugs, drug targets and prevention and treatment strategies are urgently needed. Summary of the invention

[0004] The present invention finds that AXL inhibitors FC084 and BGB324 can treat symptoms caused by myocardial failure. Therefore, the present invention uses AXL inhibitors FC084 and BGB324 as new targets for anti-heart failure treatment and provides the use of AXL inhibitors FC084 and BGB324 in preparing drugs for treating heart failure.

[0005] The heart failure includes ischemic heart failure and pressure overload heart failure; for example, heart failure caused by disease factors such as chronic myocardial infarction, hypertension, atrial fibrillation and aortic stenosis.

[0006] Another aspect of the present invention provides a medicine, which comprises the AXL inhibitor.

[0007] Compared with the prior art, the present invention has the following technical effects:

[0008] The present invention applies the independently developed AXL specific inhibitor S103822-A32 (FC084 camphorsulfonate tablets) to the treatment of heart failure with fibrosis, and finds that FC084 can inhibit the activity, excessive proliferation, activation, transdifferentiation and extracellular matrix deposition in cardiac fibroblasts, thereby reducing the pathological structural changes of cardiac fibrosis tissue and the expression of fibrosis-related markers, relieving the myocardial fibrosis process in mice, significantly improving the abnormal cardiac function of heart failure mice induced by aortic arch constriction (TAC) and left anterior descending coronary artery ligation (MI), reducing myocardial fibrosis and myocardial hypertrophy, and significantly reducing the infarction area of ​​myocardial infarction mice. Through the application of the AXL inhibitor in drugs for the treatment of heart failure, the AXL activity in cardiac fibroblasts is inhibited, the abnormal activation of fibroblasts is significantly inhibited, and the cardiac function of heart failure mice can be significantly improved.

[0009] The AXL specific inhibitor used in this application is lower in cost than common heart failure drugs such as diuretics, RAAS inhibitors (ACEI, ARB, ARNI), aldosterone receptor antagonists, beta receptor antagonists and positive inotropes in clinical practice. Compared with spironolactone, it has no central nervous system side effects and no obvious renal damage compared with valsartan. Compared with BGB324, the specific AXL inhibitor FC084 developed in the laboratory has a stronger ability to inhibit the activity of AXL. The IC50 of FC084 is 4.1nm, and the IC50 of BGB324 is 60nm, and FC084 can inhibit multiple sites of kinases such as AXL, Mer, Tyro3, ​​IRR and TrkA.

[0010] The application of AXL-specific inhibitor FC084 in the treatment of heart failure has enriched the new indications of FC084 in the clinical phase II as an anticancer drug for inhibiting malignant solid tumors, and developed another medical use of FC084. Based on the protective effect of FC084 in myocardial fibrosis and pulmonary fibrosis, and the therapeutic effect of FC084 on pathological structural remodeling of cardiac tissue, it can be used for the treatment of clinical heart failure patients or in combination with other drugs.

[0011] Experimental results show that administering AXL inhibitors to patients with clinically ischemic and pressure-load heart failure can alleviate myocardial extracellular matrix deposition and fibrosis, improve cardiac systolic and diastolic function, and improve patients' long-term prognosis, which has important clinical significance.

[0012] The concept, specific structure and technical effects of the present invention will be further described below in conjunction with the accompanying drawings to fully understand the purpose, characteristics and effects of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0013] Figure 1 In a specific embodiment of the present invention, an in vitro myocardial fibrosis model is constructed, wherein: Figure 1 A is the result of Western Blot showing that TGF-β1 induces high expression of AXL protein level in primary cardiac fibroblasts (CFs); Figure 1 B is Western Blot showing the protein expression results of fibrosis-related markers FN1 and α-sma in CFs with high expression of AXL; Figure 1 C is the effective inhibitory concentration of BGB324 in the process of TGF-β1-induced myocardial fibrosis verified at the protein level; Figure 1 D is the result of cell viability test of FC084 on the viability of cardiac fibroblasts and IC50;

[0014] Figure 2 It is a flow chart of a test scheme for administering AXL inhibitors FC084 and BGB324 to animals in vivo in a specific embodiment of the present invention;

[0015] Figure 3 In one embodiment of the present invention, the results of showing that the AXL inhibitors BGB324 and FC084 improve ischemic heart failure and cardiac function deterioration in mice caused by MI model are shown. Figure 3 A is a diagram of the experimental scheme for the administration of BGB324, FC084 and the positive drug Spironolactone to treat myocardial infarction in mice; Figure 3 BC: BGB324, FC084 and spironolactone (SPL) improve the ejection fraction (EF%) and fractional shortening (FS%) scores in heart failure mice; Figure 3 D is the morphological diagram of echocardiographic analysis; Figure 3 E is the image of H&E staining to detect the degree of fibrosis 4 weeks after sham operation or myocardial infarction surgery; Figure 3 F is the Masson-stained collagen deposition area image 4 weeks after sham operation or myocardial infarction surgery; Figure 3 G is an immunohistochemical staining image showing the expression of Collagen I 4 weeks after sham surgery or myocardial infarction surgery; Figure 3H is the α-SMA staining image 4 weeks after sham operation or myocardial infarction surgery; Figure 3 I shows the myocardial infarction area of ​​mice detected by TTC staining 4 weeks after myocardial infarction surgery.

[0016] Figure 4 In a specific embodiment of the present invention, BGB324 and FC084 improve the cardiac function of mice with cardiac overload heart failure induced by TAC, wherein: Figure 4 A is a morphological diagram showing the effects of AXL inhibitors BGB324, FC084 and the positive drug Valsartan on heart size; Figure 4 B is the analysis result of the effect of AXL inhibitors FC084 and BGB324 on the cardiac function of mice in TAC model-induced heart failure detected by small animal echocardiography; Figure 4 C-4D is the quantitative results of LVEF and LVFS; Figure 4 E is the result of the heart-to-body ratio of mice; Figure 4 F is the result of the ratio of mouse heart weight to tibia length; Figure 4 G is the H&E staining image 8 weeks after sham operation or aortic ligation surgery; Figure 4 HI is the Masson staining image 8 weeks after sham operation or aortic ligation surgery; Figure 4 J is the image of Collagen I immunohistochemical staining 8 weeks after sham operation or aortic ligation surgery; Figure 4 K is the α-SMA staining image 8 weeks after sham operation or aortic ligation surgery; Figure 4 L is a representative image of WGA staining of WT and AXL inhibitor-injected mice 8 weeks after aorta ligation surgery to analyze the cardiomyocyte area.

[0017] Figure 5 In a specific embodiment of the present invention, the AXL inhibitor FC084 reduces the expression of cardiac fibrosis-related markers in ischemic heart failure induced by the MI model; wherein, Figure 5 A is Western Blot detection of the expression of Fn1 and α-SMA proteins in mouse myocardial fibrosis tissue and the degree of fibroblast activation; Figure 5 BF is qRT-PCR detection of the mRNA levels of Fn1, Collagen 1α1, Collagen 3α1, CNN2 and ACTA2 in the hearts of mice with ischemic heart failure treated with FC084 and BGB324. DETAILED DESCRIPTION

[0018] The present invention is described in detail below in conjunction with the accompanying drawings so that the advantages and features of the present invention can be more easily understood by those skilled in the art, thereby making a clearer and more definite definition of the protection scope of the present invention.

[0019] A brief summary of one or more aspects is given below to provide a basic understanding of these aspects. This summary is not an exhaustive overview of all conceived aspects, and is neither intended to identify the key or critical elements of all aspects nor to define the scope of any or all aspects. Its only purpose is to give some concepts of one or more aspects in a simplified form as a prelude to a more detailed description that will be given later.

[0020] Axl (also known as Ark, Ufo and Tyro-7) is a member of the TAM receptor tyrosine kinase family. Its signaling pathway not only plays a key role in tumor occurrence, progression, and metastasis, but also mediates drug resistance and immune escape, making Axl a highly potential target for tumor treatment. FC084 is a highly selective AxL receptor tyrosine kinase inhibitor that inhibits AxL to affect its downstream signaling pathways, inhibits tumor cell proliferation and metastasis, and reshapes the tumor microenvironment, reducing immunosuppression and immune escape. Non-clinical studies have shown that FC084CSA, in combination with PD-1 / PD-L1 monoclonal antibodies and chemotherapy drugs, can produce significant synergistic anti-tumor effects. Phase 1 clinical studies have shown that FCO84CSA tablets are well tolerated and safe in patients with advanced malignant solid tumors, supporting the study of FCO84CSA tablets in combination with immunotherapy or other treatment options. As research continues to deepen, the application scope of FCO84 will gradually expand, and major breakthroughs are expected to be achieved in the treatment of many non-small cell lung cancers and other solid tumors with "undruggable" target mutations (such as STK11, KRAS, KEAP1, etc.).

[0021] Since there is no effective means of preventing and treating myocardial ischemic heart failure and pressure overload heart failure in clinical practice, the present invention has found through research that receptor tyrosine kinase AXL has the effect of inducing and aggravating myocardial fibrosis, and receptor tyrosine kinase AXL is a risk factor in the process of myocardial fibrosis and an effective target in the process of heart failure.

[0022] The experimental preparations used in the following examples of the present invention include:

[0023] AXL inhibitor FC084 is a drug developed by the Fourth Affiliated Hospital of Harbin Medical University and is in Phase II clinical trials; its structural formula is as follows: (MW = 637.72)

[0024]

[0025] Inhibitor BGB324: Shanghai MedChemExpress Company (HY-15150; MCE; Shanghai, China), its structural formula is shown below:

[0026]

[0027] Positive control drug-spironolactone (SPL): Shanghai MedChemExpress (HY-B0561; MCE; Shanghai, China);

[0028] Positive control drug - Valsartan: Shanghai MedChemExpress Company (HY-18204; MCE; Shanghai, China).

[0029] Example 1: Experiment on receptor tyrosine kinase AXL as a risk factor in the progression of heart failure and aggravating the progression of heart failure

[0030] 1.1. Establishment of in vitro myocardial fibrosis model: Primary cardiac fibroblasts were isolated from 3-7 day old Kunming neonatal mice using type II collagenase, and after adherence to the wall, TGF-β1 was used to induce cardiac fibroblasts to become fibrotic.

[0031] 1.2. Western blot was used to detect primary cardiac fibroblasts treated with Control and TGF-β1, n = 3, and the test results were as follows Figure 1 As shown in A, the protein results show that compared with the Control group, the expression of fibrosis-related markers FN1 and α-SMA in the primary cardiac fibroblast group treated with TGF-β1 increased, and the expression of receptor tyrosine kinase AXL was significantly upregulated.

[0032] 1.3. Primary fibroblasts of neonatal rats were isolated and transfected with AXL overexpression plasmid, n=3, and the Western blot results were as follows: Figure 1 As shown in B, Figure 1 As shown in B, compared with the pcDNA3.1 group, the expression of fibrosis-related markers FN1 and α-SMA in primary cardiac fibroblasts transfected with AXL increased, showing an increase in the differentiation of fibroblasts into myofibroblasts, indicating that AXL promotes the fibrotic change of fibroblasts.

[0033] 1.4. After TGF-β1 induced fibrosis in primary cardiac fibroblasts using Western blot, different concentrations of AXL-specific inhibitor BGB324 were used to detect the expression of fibrosis-related markers. The results were as follows: Figure 1 As shown in Figure C, the experimental results show that 100 ng / mL of BGB324 is the best concentration. AXL-specific inhibitor BGB324 can inhibit the activation of fibroblasts into myofibroblasts.

[0034] 1.5. Different concentrations of FC084 were administered to fibroblasts. Figure 1As shown in D, the effect of FC084 on the proliferation and viability of fibroblasts was displayed by CCK8, and the optimal concentration of the drug was evaluated.

[0035] It can be seen from Example 1 that the present invention discovered for the first time that AXL is a risk factor in the progression of heart failure. High expression of AXL promotes the occurrence and development of myocardial fibrosis. However, using AXL-specific inhibitors to inhibit AXL activity in cardiac fibroblasts can significantly inhibit abnormal fibroblast activation and significantly improve the heart of heart failure mice, including left ventricular ejection fraction and short-axis shortening rate.

[0036] Example 2: AXL expression during heart failure and its specific inhibitors BGB324 and FC084 improve the treatment of ischemic heart failure caused by myocardial infarction (MI)

[0037] 2.1. Establishment of a mouse model of ischemic heart failure induced by myocardial infarction (MI)

[0038] Eight-week-old male C57BL / 6 mice were anesthetized with 1% sodium pentobarbital and connected to a ventilator with a tidal volume of 0.8-0.9 ml at a rate of 110 times / min. The left anterior descending artery (LAD) was ligated with 7 / 0 at 1 / 3 of the line from the lower edge of the auricle to the apex of the heart to establish a mouse animal model of heart failure caused by myocardial infarction.

[0039] Model mice were intraperitoneally injected with 100 μL of BGB324 (20 mg / kg) and FC084 (20 mg / kg) and the positive control drug spironolactone (10 mg / kg), respectively. The process was as follows Figure 2 and Figure 3 As shown in A, according to the different drugs injected intraperitoneally into mice, the mice were divided into Sham group, MI group, MI+BGB324 (20 mg / kg) group, MI+FC084 (20 mg / kg) group and TAC+control drug spironolactone (SPL) (10 mg / kg) group.

[0040] 2.2. The cardiac function of mice was evaluated by measuring the thickness of the left ventricular wall of mice. One week after myocardial infarction surgery, the inhibitor BGB324 (20 mg / kg), FC084 (20 mg / kg) and the positive control drug spironolactone (10 mg / kg) were intraperitoneally injected for three weeks, 3 times a week. Four weeks later, the cardiac function of mice was evaluated by measuring the thickness of the left ventricular wall of the mice after anesthesia using small animal echocardiography. The quantitative results of mouse ultrasound LVEF and LVFS (n=10 / group) and the morphological analysis of echocardiography are shown in the figure Figure 3As shown in BD, it can be seen that the left ventricular ejection fraction, short-axis shortening rate and left ventricular posterior wall thickness increased, and the left ventricular inner diameter decreased, indicating that the injection of BGB324 (20 mg / kg), FC084 (20 mg / kg) and spironolactone (10 mg / kg) can significantly improve the heart function of mice with heart failure.

[0041] 2.3. Representative images of Masson, H&E and immunohistochemical staining of BGB324, FC084 and spironolactone-treated mice with ischemic heart failure (Scale bar, 50 μm) Figure 3 E- Figure 3 As shown in F. Masson staining results showed that BGB324 (20 mg / kg), FC084 (20 mg / kg) and spironolactone (10 mg / kg) could reduce the area of ​​collagen deposition in the heart of mice with heart failure. H&E staining results showed that BGB324 (20 mg / kg), FC084 (20 mg / kg) and spironolactone (10 mg / kg) could improve the disordered myocardial structure accompanied by inflammatory cell infiltration during myocardial infarction. α-SMA and Collagen I immunohistochemical staining results showed that BGB324 and FC084 could reduce the extracellular matrix deposition and the transformation of fibroblasts to myofibroblasts caused by myocardial infarction. In the figure, extracellular matrix proteins such as FN1, CollagenⅠ and α-SMA are important indicators for evaluating myocardial fibrosis, and α-SMA is involved in the contraction of smooth muscle and is currently considered to be a marker of myofibroblasts.

[0042] 2.4. TTC staining was performed on mice treated with BGB324, FC084 and spironolactone 3 weeks after myocardial infarction surgery to obtain the following results: Figure 3 I shows a representative image of TTC staining in mice (Scale bar, 2 mm). The results show that BGB324, FC084 and spironolactone can significantly reduce the infarct area in mice with ischemic heart failure.

[0043] Example 3: A trial of the AXL specific inhibitors BGB324 and FC084 in the treatment of aortic ligation (TAC)-induced cardiac overload heart failure

[0044] 3.1. Establishment of a mouse model of cardiac overload heart failure induced by aortic ligation (TAC)

[0045] Eight-week-old male C57BL / 6 mice were anesthetized with 1% sodium pentobarbital, connected to a ventilator with a tidal volume of 0.8-0.9 ml at a rate of 110 times / min, and the aortic arch was narrowed to simulate a heart failure mouse animal model with increased cardiac afterload.

[0046] Model mice were intraperitoneally injected with 100 μL of BGB324 (20 mg / kg) and FC084 (20 mg / kg) and the positive control drug valsartan (10 mg / kg), respectively. The process was as follows Figure 2 As shown, according to the different drugs injected intraperitoneally into the mice, the mice were divided into Sham group, TAC group, TAC+BGB324 group, TAC+FC084 group and TAC+control drug valsartan group.

[0047] 3.2. Heart morphology of mice in each group Figure 4 As shown in A, the heart becomes enlarged in the TAC model, but after treatment with BGB324, FC084 and valsartan, the heart morphology is significantly smaller than that of the simple TAC model mice, indicating that the therapeutic effects of BGB324 and FC084 on pressure overload heart failure are similar to those of the commonly used clinical drug valsartan.

[0048] 3.3. Ventricular wall thickness is an important indicator for evaluating the cardiac function of mice. Small animal echocardiography was used to measure the thickness of the left ventricular wall of mice after anesthesia. The results are as follows: Figure 4 As shown in BD, it can be seen that after treatment with BGB3244, FC0844 and valsartan, the cardiac function of mice with pressure overload heart failure was improved, and cardiac function indicators such as LVEF and LVFS were significantly restored. The left ventricular ejection fraction, short-axis shortening rate and left ventricular posterior wall thickness increased, and the left ventricular inner diameter decreased.

[0049] 3.4. Myocardial fibrosis and cardiomyocyte hypertrophy are important features of cardiac remodeling. Cardiomyocyte hypertrophy and heart failure in different treatment groups were evaluated by measuring heart weight / body weight (HW / BW) and heart weight / tibia length (HW / TL). Figure 4 As shown in EF, the results show that compared with the control group, AXL-specific inhibitors FC084 and BGB324 can significantly reduce TAC-induced myocardial fibrosis and cardiomyocyte hypertrophy.

[0050] 3.5. Masson, H&E, immunohistochemical staining, and WGA staining (Scale bar, 50 μm) were performed on mice with pressure overload heart failure induced by TAC model after 8 weeks of BGB324, FC084, and valsartan treatment. The staining results are shown in Figure 4 G- Figure 4As shown in Figure L, Masson staining results showed that BGB324, FC084 and valsartan treatment could reduce collagen deposition in the hearts of mice with heart failure. H&E staining results showed that TAC-induced myocardial structural disorder accompanied by inflammatory cell infiltration could be improved by BGB324, FC084 and valsartan treatment. Immunohistochemical staining for α-SMA and Collagen I expression showed that BGB324, FC084 and valsartan treatment could reduce collagen deposition and fibroblast-to-myofibroblast transformation caused by myocardial infarction. WGA staining experiments showed that BGB324, FC084 and valsartan significantly improved myocardial hypertrophy caused by TAC.

[0051] 3.6. Western Blot was used to show the levels of fibrosis-related proteins in the hearts after BGB324, FC084 and valsartan treatment to detect the degree of fibroblastic cell proliferation. Figure 5 As shown in A, by detecting the expression of related markers FN1 and α-SMA protein in mouse myocardial fibrosis tissue, it can be seen that BGB324, FC084 and valsartan treatment can significantly inhibit the expression of fibrosis-related markers in mice with ischemic heart failure. qRT-PCR was used to detect the mRNA levels of fibrosis-related markers FN1, Collagen1α1, Collagen3α1, CNN2 and ACTA2 in the hearts of mice treated with BGB324, FC084 and valsartan, as shown in Figure 5 As shown in BF, n=6, the results show that treatment with BGB324, FC084 and valsartan significantly reduced the mRNA levels of fibrosis-related markers in mouse cardiac fibroblasts.

[0052] It can be seen from the above examples that the AXL inhibitors FC084 and BGB324 have good effects on treating ischemic heart failure and pressure overload heart failure and reduce myocardial fibrosis as compared with the control group, and have the functions of improving the relaxation and contraction of the patient's heart and improving cardiac dysfunction, and reducing the degree of fibrosis, and their effects are comparable to those of positive drugs for treating heart failure. Echocardiography showed that FC084 and BGB324 restored the cardiac function of mice. FC084 and BGB324 reduce the expression of fibrosis-related markers by specifically inhibiting the expression of AXL, and exert anti-heart failure effects. Pathological tissue staining showed that FC084 inhibited collagen deposition and cardiac remodeling in mouse heart tissue, slowing down the pathological process of myocardial fibrosis. Similarly, FC084 and BGB324 inhibited the fibrosis marker protein fibronectin (FN1), type I collagen (CollagenⅠ) and myofibroblast marker (α-SMA). The above research results show that AXL inhibitors FC084 and BGB324 can improve the myocardial fibrosis process of mice with heart failure caused by aortic arch constriction (TAC) and left anterior descent of the coronary artery ligation (MI), restore the cardiac function of mice, inhibit the pathological remodeling of mouse heart tissue and the massive deposition of extracellular matrix. It clarifies the role of FC084 and BGB324 in the process of ischemic heart failure and pressure overload heart failure, effectively improves the prognosis of patients, and provides a new theoretical basis for the clinical application and combination therapy of FC084 and BGB324, which can be used for the treatment of heart failure and has clinical significance.

[0053] The above embodiments are merely illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Anyone familiar with the art may modify or alter the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or alterations made by a person of ordinary skill in the art without departing from the spirit and technical concept disclosed by the present invention shall still be covered by the claims of the present invention.

Claims

1. Use of AXL inhibitors in the preparation of drugs for treating heart failure.

2. The use according to claim 1, wherein the AXL inhibitor is FC084 or BGB324, and the structure of BGB324 is: The structure of FC084 is:

3. The use according to claim 1, wherein the heart failure comprises ischemic heart failure and cardiac overload heart failure. 4 . A medicament comprising the AXL inhibitor according to any one of claims 1 to 3 .

Images