Use of an axl inhibitor in the manufacture of a medicament for treating heart failure

Abstract

This invention discloses the use of AXL inhibitors in the preparation of drugs for treating heart failure, relating to the pharmaceutical field. This invention provides the application of AXL inhibitors in drugs for treating heart failure, wherein the inhibitors can significantly inhibit abnormal fibroblast activation and significantly improve cardiac function in mice with heart failure.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical science, and more particularly to the use of AXL inhibitors in the preparation of drugs for treating heart failure. Background Technology

[0002] Heart failure is a clinical syndrome caused by elevated intracardiac pressure at rest and / or during exercise and / or insufficient cardiac output due to structural and / or functional abnormalities of the heart. Myocardial remodeling is a fundamental pathological mechanism of heart failure, in which cardiac fibrosis (CF) plays a crucial role, with cardiac fibroblasts (CFs) being the main cells involved. Abnormal proliferation and differentiation of CFs under pathological stimulation leads to excessive production of extracellular matrix (ECM) and collagen deposition, resulting in increased cardiac stiffness, impaired normal electrical signal conduction, inducing arrhythmias, cardiac structural changes, and systolic and diastolic dysfunction, ultimately leading to heart failure. Currently, although numerous studies have been conducted on the pathogenesis and treatment strategies of myocardial fibrosis, many bottlenecks remain. The most significant problem is that existing treatments can only improve patients' clinical symptoms, but due to the lack of specific treatments to reverse or effectively inhibit the development of myocardial fibrosis, they cannot control the progression of the disease. Therefore, a deeper understanding of the core mechanisms driving the progression of myocardial fibrosis is needed to identify new targets for treating myocardial fibrosis, slow down its progression, and thus play a role in treating heart failure.

[0003] Current treatment strategies for myocardial fibrosis include inhibiting the TGF-β signaling pathway and the renin-angiotensin-aldosterone system, but the efficacy of targeted drugs is limited. Drug therapy for myocardial fibrosis is based on diuretics to relieve congestion and neurohormone antagonists to improve the clinical course. Angiotensin-converting enzyme inhibitors (ACEIs) or angiotensin receptor blockers (ARBs), beta-blockers, and mineralocorticoid antagonists (MRAs) are currently the main drugs used in the clinical treatment of myocardial fibrosis. However, there is currently insufficient evidence to prove that other drugs can improve myocardial fibrosis. Furthermore, currently used antifibrotic drugs in clinical practice include antacids, N-acetylcysteine, glucocorticoids, and anti-infective drugs, which can only reduce patient mortality to a certain extent. Therefore, there is an urgent need for novel antifibrotic drugs, drug targets, and prevention and treatment strategies. Summary of the Invention

[0004] This invention discovers that AXL inhibitors FC084 and BGB324 can treat symptoms caused by myocardial failure. Therefore, this invention uses AXL inhibitors FC084 and BGB324 as new targets for the treatment of heart failure and provides their use in the preparation of drugs for the treatment of heart failure.

[0005] The heart failure includes ischemic heart failure and stress-induced heart failure; for example, heart failure caused by diseases such as chronic myocardial infarction, hypertension, atrial fibrillation, and aortic stenosis.

[0006] In another aspect, the present invention provides a medicament comprising the aforementioned AXL inhibitor.

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

[0008] This invention applies the AXL-specific inhibitor FC084 (camphor sulfonate tablets) to the treatment of heart failure with fibrosis. It was found that FC084 can inhibit the activity, excessive proliferation, activation, transdifferentiation, and extracellular matrix deposition in cardiac fibroblasts, thereby reducing pathological structural changes in cardiac fibrotic tissue and the expression of fibrosis-related markers. This alleviates the progression of myocardial fibrosis in mice, significantly improves cardiac function abnormalities in mice with aortic arch constriction (TAC) and left anterior descending coronary artery ligation (MI) induced heart failure, reduces myocardial fibrosis and hypertrophy, and significantly reduces the infarct area in mice with myocardial infarction. Through the application of AXL inhibitors in the treatment of heart failure, the activity of AXL in cardiac fibroblasts is inhibited, significantly suppressing abnormal fibroblast activation and significantly improving cardiac function in mice with heart failure.

[0009] The AXL-specific inhibitor used in this application is less expensive than commonly used heart failure drugs such as diuretics, RAAS inhibitors (ACEIs, ARBs, ARNIs), aldosterone receptor antagonists, β-receptor antagonists, and positive inotropic agents. Compared to spironolactone, it has no central nervous system side effects, and compared to valsartan, it causes no significant kidney damage. Compared to BGB324, the laboratory-developed specific AXL inhibitor FC084 exhibits stronger AXL inhibition. FC084 has an IC50 of 4.1 nm, while BGB324 has an IC50 of 60 nm. Furthermore, FC084 can inhibit multiple sites of kinases, such as AXL, Mer, Tyro3, ​​IRR, and TrkA.

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

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

[0012] The following will further explain the concept, specific structure, and technical effects of the present invention in conjunction with the accompanying drawings, so as to fully understand the purpose, features, and effects of the present invention. Attached Figure Description

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

[0014] Figure 2 This is a flowchart of an experimental protocol for treating animals with the AXL inhibitor FC084 and BGB324 in a specific embodiment of the present invention.

[0015] Figure 3 This is a specific embodiment of the present invention, demonstrating the results of the AXL inhibitors BGB324 and FC084 improving ischemic heart failure and cardiac function deterioration in mice induced by MI. Figure 3 A is a diagram of the experimental protocol for treating myocardial infarction in mice with BGB324, FC084, and the positive control drug Spironolactone. Figure 3 BC results showed that BGB324, FC084, and spironolactone (SPL) improved ejection fraction (EF%) and shortened ejection fraction (FS%) in mice with heart failure. Figure 3 D represents the echocardiographic morphology diagram; Figure 3 E is an image of fibrosis detected by H&E staining 4 weeks after sham surgery or myocardial infarction surgery; Figure 3 F is a Masson staining image of collagen deposition area 4 weeks after sham surgery or myocardial infarction surgery; Figure 3 G is an immunohistochemical staining image showing Collagen I expression 4 weeks after sham surgery or myocardial infarction surgery; Figure 3H represents α-SMA staining images 4 weeks after sham surgery or myocardial infarction surgery; Figure 3 I represents the infarct area of ​​a mouse heart as determined by TTC staining 4 weeks after myocardial infarction surgery.

[0016] Figure 4 This is a specific embodiment of the present invention, showing the results of BGB324 and FC084 improving cardiac function in mice with TAC-induced cardiac overload-induced heart failure. Figure 4 A is a morphological diagram showing the effects of AXL inhibitors BGB324 and FC084, as well as the positive control drug Valsartan, on heart size. Figure 4 B represents the analysis results of the effects of AXL inhibitors FC084 and BGB324 on cardiac function in mice with TAC-induced heart failure detected by small animal echocardiography. Figure 4 C-4D represents the quantization results of LVEF and LVFS; Figure 4 E represents the heart-to-body weight ratio in mice; Figure 4 F represents the ratio of mouse heart weight to tibia length; Figure 4 G is an H&E staining image 8 weeks after sham surgery or aortic ligation surgery; Figure 4 HI is a Masson staining image 8 weeks after sham surgery or aortic ligation. Figure 4 J is an image of Collagen I immunohistochemical staining 8 weeks after sham surgery or aortic ligation. Figure 4 K represents α-SMA staining images 8 weeks after sham surgery or aortic ligation. Figure 4 Representative images of WGA staining in WT and AXL inhibitor-injected mice 8 weeks after aortic ligation surgery were obtained to analyze cardiomyocyte area.

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

[0018] The present invention will now be described in detail with reference to 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 providing a clearer and more explicit definition of the scope of protection of the present invention.

[0019] The following provides a brief overview of one or more aspects to offer a basic understanding of them. This overview is not an exhaustive summary of all conceived aspects, nor is it intended to identify key or decisive elements of all aspects, nor to define the scope of any or all aspects. Its sole purpose is to present some concepts of one or more aspects in a simplified form to prepare for the more detailed descriptions that follow.

[0020] Axl (also known as Ark, Ufo, and Tyro-7) is a member of the TAM receptor tyrosine kinase family. Its signaling pathway plays a crucial role not only in tumorigenesis, progression, and metastasis but also mediates drug resistance and immune escape, making Axl a highly promising target for cancer therapy. FC084 is a highly selective AxL receptor tyrosine kinase inhibitor that affects downstream signaling pathways by inhibiting AxL, thereby suppressing tumor cell proliferation and metastasis, remodeling the tumor microenvironment, and reducing immunosuppression and immune escape. Non-clinical studies have shown that FC084CSA, in combination with PD-1 / PD-L1 monoclonal antibodies and chemotherapy drugs, produces a significant synergistic anti-tumor effect. Phase 1 clinical trials have shown that FCO84CSA tablets are well-tolerated and safe in patients with advanced malignant solid tumors, supporting the inclusion of FCO84CSA tablets in studies for combination with immunotherapy or other treatment regimens. As research continues, the application scope of FCO84 will gradually expand, and it is expected to achieve major breakthroughs for many non-small cell lung cancers and other solid tumors with "undruggable" target mutations (such as STK11, KRAS, KEAP1, etc.).

[0021] Currently, there are no effective prevention and treatment methods for ischemic heart failure and stress-overload heart failure in clinical practice. This invention, through research, has discovered that receptor tyrosine kinase AXL can induce and exacerbate myocardial fibrosis. Receptor tyrosine kinase AXL is a risk factor in the progression of myocardial fibrosis and an effective target in the progression of heart failure.

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

[0023] Inhibitor BGB324: Shanghai MedChemExpress Co., Ltd. (HY-15150; MCE; Shanghai, China), its structural formula is shown below:

[0024] ;

[0025] Positive control drug - spironolactone (SPL): Shanghai MedChemExpress Co., Ltd. (HY-B0561; MCE; Shanghai, China);

[0026] Positive control drug - Valsartan: Shanghai MedChemExpress Co., Ltd. (HY-18204; MCE; Shanghai, China).

[0027] Example 1: Experiment on the role of receptor tyrosine kinase AXL as a risk factor in the progression of heart failure and its aggravation.

[0028] 1.1 Establishment of an in vitro model of myocardial fibrosis: Primary cardiac fibroblasts were isolated from 3-7 day old Kunming suckling mice using type II collagenase. After adhesion, TGF-β1 was used to induce fibrosis in the cardiac fibroblasts.

[0029] 1.2. Western blot analysis was performed on primary cardiac fibroblasts treated with Control and TGF-β1 (n=3). The results are as follows: Figure 1 As shown in Figure A, the protein results indicate that, compared to the Control group, the expression of fibrosis-related markers FN1 and α-SMA was increased in the TGF-β1-treated primary cardiac fibroblast group, and the expression of receptor tyrosine kinase AXL was significantly upregulated.

[0030] 1.3. After isolating primary fibroblasts from neonatal mice, they were transfected with the AXL overexpression plasmid (n=3). The Western blot results are as follows: Figure 1 As shown in B, by Figure 1 As shown in Figure B, compared with the pcDNA3.1 group, the expression of fibrosis-related markers FN1 and α-SMA in primary cardiac fibroblasts transfected with AXL was increased, indicating an increased differentiation of fibroblasts into myofibroblasts, suggesting that AXL promotes fibrotic changes in fibroblasts.

[0031] 1.4. Western blot analysis was performed on TGF-β1-induced fibrosis in primary cardiac fibroblasts. Different concentrations of the AXL-specific inhibitor BGB324 were then used to detect the expression of fibrosis-related markers. The results are as follows: Figure 1 As shown in Figure C, experimental results indicate that the optimal concentration of BGB324 is 100 ng / mL. The AXL-specific inhibitor BGB324 can inhibit the activation of fibroblasts into myofibroblasts.

[0032] 1.5. Different concentrations of FC084 were administered to fibroblasts, as follows: Figure 1 As shown in Figure D, the effect of FC084 on fibroblast proliferation and viability was demonstrated using CCK8 assays, and the optimal drug concentration was evaluated.

[0033] As can be seen from Example 1, this invention discovers 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, by using AXL-specific inhibitors to inhibit AXL activity in cardiac fibroblasts, abnormal fibroblast activation can be significantly inhibited and the heart of mice with heart failure can be significantly improved, including left ventricular ejection fraction and fractional shortening.

[0034] Example 2: Trial of AXL expression and its specific inhibitors BGB324 and FC084 improving the treatment of ischemic heart failure caused by myocardial infarction (MI) during the progression of heart failure.

[0035] 2.1 Establishing a mouse model of ischemic heart failure induced by myocardial infarction (MI).

[0036] 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 breaths / min. A mouse model of myocardial infarction-induced heart failure was established by ligating the left anterior descending artery (LAD) with 7 / 0 ligation at one-third of the distance from the lower edge of the auricle to the apex of the heart.

[0037] The model mice were intraperitoneally injected with 100 μL of BGB324 (20 mg / kg) and FC084 (20 mg / kg), respectively, along with the positive control drug spironolactone (10 mg / kg). The procedure was as follows: Figure 2 and Figure 3 As shown in Figure A, mice were divided into four groups based on the different drugs injected into their peritoneal cavity: 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.

[0038] 2.2. Cardiac function in mice was evaluated by measuring left ventricular wall thickness. One week after myocardial infarction surgery, mice were intraperitoneally injected with the inhibitors BGB324 (20 mg / kg), FC084 (20 mg / kg), and the positive control drug spironolactone (10 mg / kg) for three weeks, three times a week. Four weeks later, small animal echocardiography was used to measure the left ventricular wall thickness after anesthesia to evaluate cardiac function. Quantitative results of LVEF and LVFS in mice (n=10 / group) and echocardiographic morphological analysis are shown below. Figure 3 As shown in BD, the left ventricular ejection fraction, fractional shortening, and posterior wall thickness increased, while the left ventricular diameter decreased, indicating that injections of BGB324 (20 mg / kg), FC084 (20 mg / kg), and spironolactone (10 mg / kg) significantly improved cardiac function in mice with heart failure.

[0039] 2.3. Representative images (scale bar, 50 μm) of mice with ischemic heart failure treated with BGB324, FC084, and spironolactone were obtained by Masson, H&E, and immunohistochemical staining. Figure 3 E- Figure 3 As shown in Figure F, Masson staining results showed that treatment with BGB324 (20 mg / kg), FC084 (20 mg / kg), and spironolactone (10 mg / kg) all reduced the area of ​​collagen deposition in the hearts of mice with heart failure. H&E staining results showed that BGB324 (20 mg / kg), FC084 (20 mg / kg), and spironolactone (10 mg / kg) all improved the myocardial structural disorder accompanied by inflammatory cell infiltration during myocardial infarction. Immunohistochemical staining results with α-SMA and Collagen I showed that both BGB324 and FC084 could alleviate extracellular matrix deposition and fibroblast transformation into myofibroblasts caused by myocardial infarction. In the figure, extracellular matrix proteins such as FN1, Collagen I, and α-SMA are important indicators for assessing myocardial fibrosis, and α-SMA is involved in smooth muscle contraction and is currently considered a marker of myofibroblasts.

[0040] 2.4. Mice that underwent BGB324, FC084, and spironolactone treatment for myocardial infarction 3 weeks after surgery were subjected to TTC staining to obtain the following results: Figure 3 Image I shows a representative TTC staining image of mice (Scale bar, 2 mm). The results indicate that BGB324, FC084, and spironolactone can significantly reduce the infarct area in mice with ischemic heart failure.

[0041] Example 3: Trial of AXL-specific inhibitors BGB324 and FC084 in treating heart failure caused by aortic ligation (TAC) with cardiac overload.

[0042] 3.1. Establishing a mouse model of cardiac overload-induced heart failure induced by aortic ligation (TAC).

[0043] 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 breaths / minute to narrow the aortic arch and simulate a mouse model of heart failure with increased cardiac afterload.

[0044] The model mice were intraperitoneally injected with 100 μL of the positive control drug valsartan (10 mg / kg) and BGB324 (20 mg / kg) and FC084 (20 mg / kg), respectively. The procedure was as follows: Figure 2As shown, mice were divided into four groups based on the different drugs injected into their peritoneal cavity: Sham group, TAC group, TAC+BGB324 group, TAC+FC084 group, and TAC+control drug valsartan group.

[0045] 3.2. The morphological images of the hearts of mice in each group are shown below. Figure 4 As shown in Figure A, the heart becomes enlarged in the TAC model. However, after treatment with BGB324, FC084, and valsartan, the heart morphology is significantly smaller than that of the TAC model mice alone, indicating that the therapeutic effects of BGB324 and FC084 on stress overload type heart failure are similar to those of the clinically commonly used drug valsartan.

[0046] 3.3. Ventricular wall thickness is an important indicator for evaluating cardiac function in mice. Small animal echocardiography was used to measure the left ventricular wall thickness in mice after anesthesia. The results are as follows: Figure 4 As shown in BD, after treatment with BGB3244, FC0844 and valsartan, the cardiac function of mice with heart failure due to pressure overload was improved, and cardiac function indicators such as LVEF and LVFS were significantly restored. The left ventricular ejection fraction, fractional shortening, and thickness of the left ventricular posterior wall increased, while the left ventricular diameter decreased.

[0047] 3.4. Myocardial fibrosis and cardiomyocyte hypertrophy are important features of cardiac remodeling. Cardiomyocyte hypertrophy and heart failure in different treatment groups were assessed by measuring heart weight / body weight (HW / BW) and heart weight / tibia length (HW / TL) ratios. Figure 4 As shown in the EF diagram, the results indicate that, compared with the control group, the AXL-specific inhibitors FC084 and BGB324 can significantly reduce TAC-induced myocardial fibrosis and cardiomyocyte hypertrophy.

[0048] 3.5. Mice with stress overload-induced heart failure induced by a TAC model after 8 weeks of treatment with BGB324, FC084, and valsartan underwent Masson staining, H&E staining, immunohistochemical staining, and WGA staining (scale bar, 50 μm). The staining results are as follows: Figure 4 G- Figure 4As shown in Figure L, Masson staining results indicated that treatment with BGB324, FC084, and valsartan reduced 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 was improved by treatment with BGB324, FC084, and valsartan. Immunohistochemical staining for α-SMA and Collagen I expression showed that treatment with BGB324, FC084, and valsartan reduced collagen deposition and fibroblast-to-myofibrillar transformation caused by myocardial infarction. WGA staining experiments showed that BGB324, FC084, and valsartan significantly improved TAC-induced myocardial hypertrophy.

[0049] 3.6. Western blot analysis was performed to visualize the levels of fibrosis-related proteins in the heart after treatment with BGB324, FC084, and valsartan, in order to detect the degree of fibroblast formation. Results are as follows: Figure 5 As shown in Figure A, by detecting the expression of FN1 and α-SMA proteins, relevant markers in mouse myocardial fibrosis tissue, it was found that treatment with BGB324, FC084, and valsartan significantly inhibited the expression of fibrosis-related markers in mice with ischemic heart failure. The mRNA levels of fibrosis-related markers FN1, Collagen1α1, Collagen3α1, CNN2, and ACTA2 in the hearts of mice treated with BGB324, FC084, and valsartan were detected by qRT-PCR. Figure 5 As shown in BF, n=6, the results indicate that treatment with BGB324, FC084, and valsartan significantly reduced the mRNA levels of fibrosis-related markers in mouse cardiac fibroblasts.

[0050] As demonstrated by the above examples, both AXL inhibitors FC084 and BGB324 showed good therapeutic effects on ischemic heart failure and stress-overload heart failure compared to the control group, effectively reducing myocardial fibrosis. They improved cardiac diastolic and systolic function and alleviated cardiac dysfunction, mitigating the degree of fibrosis. Their efficacy was comparable to that of positive drugs for treating heart failure. Echocardiography showed that FC084 and BGB324 restored cardiac function in mice. FC084 and BGB324 exerted their anti-heart failure effect by specifically inhibiting AXL expression and reducing the expression of fibrosis-related markers. Pathological staining showed that FC084 inhibited collagen deposition and cardiac remodeling in mouse cardiac tissue, slowing the pathological progression of myocardial fibrosis. Similarly, FC084 and BGB324 inhibited fibrosis marker proteins fibronectin (FN1), type I collagen (Collagen I), and myofibroblast markers (α-SMA). The above research results show that the AXL inhibitors FC084 and BGB324 can improve the myocardial fibrosis process in mice with heart failure caused by aortic arch stenosis (TAC) and left anterior descending coronary artery ligation (MI), restore cardiac function in mice, inhibit pathological remodeling of cardiac tissue and excessive deposition of extracellular matrix, clarify the role of FC084 and BGB324 in the progression of ischemic heart failure and stress overload heart failure, effectively improve patient prognosis, and provide a new theoretical basis for the clinical application and combination therapy of FC084 and BGB324. They can be used for the treatment of heart failure and have clinical significance.

[0051] The above embodiments are merely illustrative of the principles and effects of the present invention and are not intended to limit the invention. Any person skilled in the art can 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 those skilled in the art without departing from the spirit and technical concept disclosed in the present invention should still be covered by the claims of the present invention.

Claims

1. Use of an AXL inhibitor in the preparation of a drug for treating heart failure with fibrosis, wherein the heart failure includes ischemic heart failure and cardiac overload heart failure, and the AXL inhibitor is BGB324, whose structure is as follows: .

Images