Application of Wnt / beta-catenin inhibitor in preparation of medicine for treating myocardial infarction

Abstract

The invention provides an application of a Wnt / beta-catenin inhibitor in preparation of a medicine for treating myocardial infarction, and belongs to the technical field of biological medicines. And the Wnt / beta-catenin inhibitor is KYA1797K, and the Wnt / beta-catenin inhibitor is KYA1797K According to the application disclosed by the invention, the expression of beta-catenin protein is successfully inhibited through a KYA1797K targeted Wnt / beta-catenin pathway, the cardiac function of a myocardial infarction mouse is remarkably improved, and the left ventricular ejection fraction (EF) and the left ventricular shortening fraction (FS) are remarkably recovered, so that the Wnt / beta-catenin inhibitor KYA1797K has a good treatment effect on myocardial infarction. In addition, a myocardial fibrosis area of a KYA1797K treatment group is obviously reduced, which indicates that the KYA1797K has an anti-myocardial fibrosis effect. The development result further proves the potential application value in myocardial infarction treatment.

Description

Technical Field

[0001] This invention relates to the field of biomedical technology, and in particular to the application of a Wnt / β-catenin inhibitor in the preparation of a drug for treating myocardial infarction. Background Technology

[0002] Myocardial infarction is a leading cause of cardiovascular disease-related death. Current treatments primarily include drug therapy, interventional therapy, and surgery. While existing treatment strategies can improve the clinical prognosis of myocardial infarction patients to some extent, their application still has significant limitations. First, the effectiveness of drug therapy is significantly affected by individual patient differences, and some drugs may cause adverse reactions. Second, current treatments have limited effectiveness in intervening in pathological myocardial remodeling and are insufficient to effectively curb the progressive deterioration of cardiac function. Therefore, it is necessary to explore new drugs for the treatment of myocardial infarction.

[0003] Myocardial ischemia and infarction lead to cardiomyocyte death and trigger local inflammatory responses. As inflammation progresses, myocardial fibroblasts are activated, resulting in excessive myocardial fibrosis. Cardiac fibrosis is characterized by the deposition of extracellular matrix proteins in the intercellular spaces of cardiomyocytes following acute and chronic tissue injury events, significantly impairing cardiac function and leading to cardiac tissue remodeling and hardening, including increased ventricular stiffness, impaired systolic function, abnormal electrical conduction, heart failure, and ventricular aneurysm. Early targeting of myofibroblast activation may prevent irreversible remodeling, and anti-fibrotic therapy is a crucial step in breaking the vicious cycle, preventing complications, and improving treatment outcomes.

[0004] Current treatments for myocardial fibrosis following acute myocardial infarction primarily involve strategies targeting various molecules and signaling pathways. Firstly, specific PKM2 inhibitors have been shown to have cardioprotective effects, significantly reducing cardiac fibrosis, improving cardiac function, and decreasing infarct size in an isoproterenol-induced acute myocardial infarction model. Secondly, regulating immune cell signaling pathways is also an important direction; targeting S1P1 has shown to improve cardiac fibrosis and cardiac function in a diabetic model. However, its effectiveness is affected by blood glucose levels and requires cautious application. Furthermore, researchers have innovatively constructed a dual delivery system, using fibrin-specific nanogels to load tissue plasminogen activator (tPA) and the cell contractility inhibitor Y-27632. This system can locally degrade fibrin and inhibit cardiac fibrosis, improving left ventricular ejection fraction and reducing the expression of fibrosis markers such as α-smooth muscle actin. However, the targeting properties and biocompatibility of the nanogels still require further validation. Treatments targeting metabolic pathways have also shown potential in alleviating cardiac fibrosis. Inhibiting lactate signaling can reduce lactate-induced endothelial cell-to-mesenchymal cell transformation, thereby reducing cardiac fibrosis. However, inhibiting lactate signaling may disrupt normal metabolic homeostasis. At the signaling pathway level, modulating the MAPK / ERK signaling pathway may aid cardiac regeneration. In zebrafish models, appropriate activation of the MAPK / ERK pathway promotes regeneration and reduces fibrosis, while inhibition of MEK leads to increased fibrosis. All of these treatments are currently in the preclinical stage, and there are currently no approved direct-acting anti-myocardial fibrosis drugs.

[0005] In recent years, studies have shown that the Wnt / β-catenin signaling pathway plays a crucial role in regulating cell proliferation, differentiation, and the pathophysiology of various diseases. Currently, research on this pathway mainly focuses on cardiac development and oncological diseases. During myocardial ischemia, activation of the Wnt / β-catenin signaling pathway is a key factor in the development of cardiac fibrosis. Studies have shown that Wnt expression increases significantly during myocardial ischemia, activating the Wnt / β-catenin signaling pathway, promoting the translocation of β-catenin from the cytoplasm to the nucleus, binding to transcription factors TCF / LEF, inducing the transcription of target genes, promoting fibroblast proliferation and differentiation, and ultimately driving cardiac fibrosis. This mechanism has been confirmed in adult models of cardiac hypertrophy and remodeling. Although existing treatment strategies mostly improve cardiac function by inhibiting myocardial fibroblast activation, specific interventions targeting the Wnt / β-catenin pathway remain relatively limited. Some potential methods have been reported in the literature, such as using soluble frizzled-associated proteins (sFRPs) or inhibiting disheveled proteins to block Wnt signaling, thereby regulating left ventricular remodeling. Therefore, targeting the Wnt / β-catenin pathway to inhibit abnormal signaling activity and alleviate cardiac fibrosis may provide a new approach for the treatment of myocardial infarction.

[0006] KYA1797K, as a potent and selective Wnt / β-catenin inhibitor, activates the Axin-GSK3β complex, destabilizing β-catenin and initiating its degradation, thereby inhibiting downstream signaling pathways related to β-catenin. However, there are currently no reports on using the Wnt / β-catenin signaling pathway as a therapeutic target for myocardial fibrosis after acute myocardial infarction, nor are there any existing technologies demonstrating that the Wnt / β-catenin inhibitor KYA1797K has pharmaceutical applications in treating myocardial infarction.

[0007] Based on this, the present invention is proposed. Summary of the Invention

[0008] This invention aims to improve cardiac function after myocardial infarction by targeting the Wnt / β-catenin signaling pathway and inhibiting the activity of β-catenin protein. It also provides a novel use of the Wnt / β-catenin inhibitor KYA1797K in the preparation of drugs for treating myocardial infarction.

[0009] To achieve the above-mentioned objectives, the present invention provides the following technical solution: This invention provides the application of a Wnt / β-catenin inhibitor in the preparation of a drug for treating myocardial infarction, wherein the Wnt / β-catenin inhibitor is KYA1797K.

[0010] Preferably, the drug contains KYA1797K and a solvent.

[0011] Preferably, the solvent comprises 8-12% DMSO, 35-45% PEG300, 4-6% Tween-80, and 40-50% physiological saline.

[0012] Preferably, the concentration of KYA1797K in the solvent is 1~3 mg / mL.

[0013] This invention targets the Wnt / β-catenin pathway with KYA1797K, successfully inhibiting β-catenin protein expression and significantly improving cardiac function in mice with myocardial infarction. Left ventricular ejection fraction (EF) and left ventricular fractional shortening (FS) both significantly recovered, indicating that the Wnt / β-catenin inhibitor KYA1797K has a good therapeutic effect on myocardial infarction. Furthermore, the myocardial fibrosis area in the KYA1797K treatment group was significantly reduced, suggesting its anti-myocardial fibrosis effect. 68 Imaging results of Ga-FAPI-04 showed that the maximum standard uptake value (SUVmax) of mice was significantly reduced after drug treatment, further demonstrating its potential application value in the treatment of myocardial infarction. Attached Figure Description

[0014] Figure 1 Micro-PET / CT images of three groups of mice (%ID / g represents the percentage injection dose per gram of tissue). A. Injection 18 One hour after F-FDG, micro-PET / CT imaging shows the site of myocardial infarction. 18 F-FDG uptake was significantly reduced (arrow indicated); B. Injection 68 One hour after Ga-FAPI-04 administration, micro-PET / CT imaging showed no significant cardiac abnormalities in either the control group or the sham-operated group. 68 Increased Ga-FAPI-04 uptake, with a maximum standard uptake value (SUVmax) of 0.9, and the location of myocardial infarction in the myocardial infarction group. 68 Ga-FAPI-04 uptake increased significantly (indicated by arrow), with SUVmax at 1.4.

[0015] Figure 2 Four groups of mice were injected 68PET-CT images 1 hour after Ga-FAPI-04 administration (%ID / g is the percentage of injection dose per gram of tissue). 2A. Images on days 1, 7, 14, and 28 in the myocardial infarction (MI) group; 2B. Images on days 1, 7, 14, and 28 in the MI + 5 mg / kg / day KYA1797K group; 2C. Images on days 1, 7, 14, and 28 in the MI + 10 mg / kg / day KYA1797K group; 2D. Images on days 1, 7, 14, and 28 in the MI + 15 mg / kg / day KYA1797K group; 2E. Images on days 1, 7, 14, and 28 in different groups. 68 Ga-FAPI-04 uptake curve.

[0016] Figure 3 The values ​​represent the ventricular ejection fraction (EF) and left ventricular shortening fraction (FS) in mice. 3A: Left ventricular ejection fraction (EF) in mice with myocardial infarction; 3B: Left ventricular shortening fraction (FS).

[0017] Figure 4 Myocardial infarction-related indicators were detected in tissue sections after KYA1797K treatment. A. Immunofluorescence showing β-catenin expression (x75); blue represents 4′,6-diamidinyl-2-phenylindole (DAPI) labeled cell nuclei, red represents β-catenin protein fluorescence signal, and the Merge plot is the superposition of DAPI and β-catenin fluorescence images; B. Immunofluorescence showing FAP expression (x75); blue represents 4′,6-diamidinyl-2-phenylindole (DAPI) labeled cell nuclei, red represents FAP protein fluorescence signal, and the Merge plot is the superposition of DAPI and FAP fluorescence images; C. Immunohistochemistry showing β-catenin expression (x40); β-catenin expression in the 10 mg KYA1797K group was significantly lower than that in the myocardial infarction group; E. Masson staining to assess the degree of fibrosis in mouse heart tissue (×40); the fibrotic area in the 10 mg KYA1797K group was significantly smaller than that in the myocardial infarction group. Detailed Implementation

[0018] The technical solutions provided by the present invention will be described in detail below with reference to the embodiments, but they should not be construed as limiting the scope of protection of the present invention.

[0019] Example 1

[0020] I. Materials

[0021] 1. Laboratory Animals. Twenty-five male C57BL / 6 mice, 8 weeks old and weighing 20–25 g, were provided by Hangzhou Ziyuan Laboratory Animal Technology Co., Ltd. [License No.: SCXK (Zhejiang) 2019-0004]. The mice were housed in a specific-pathogen-free (SPF) environment. Animal research was conducted in accordance with the regulations of Nanjing First Hospital and relevant national regulations on the management and use of laboratory animals.

[0022] 2. Main reagents and instruments. KYA1797K was purchased from MCE Corporation in China; FAPI-04 was purchased from ABX Corporation in the United States; 18 F-FDG was purchased from Senko (Nanjing) Pharmaceutical Technology Co., Ltd.; FAP and β-catenin antibodies were purchased from Abcam, UK; HRP-labeled goat anti-rabbit secondary antibody, environmentally friendly dewaxing solution, bovine serum albumin (BSA), tissue autofluorescence quencher, DAPI staining reagent, antifluorescence quenching mounting medium, citrate antigen retrieval solution (pH 6.0), universal tissue fixative, Masson staining kit, histochemical reagent kit, DAB chromogenic agent, 20×Tris-EDTA antigen retrieval solution, normal rabbit serum (concentrated), and hematoxylin and eosin (H&E) staining solution were all purchased from Servicebio, Wuhan; an upright fluorescence microscope was purchased from Nikon, Japan; a constant staining kit was purchased from China National Pharmaceutical Group Chemical Reagent Co., Ltd.; the main instruments included an upright fluorescence microscope (Nikon, Japan), an iQS-TS fully automated labeling module, and... 68 Ge / 68 Ga generator (ITG GmbH, Germany), microPET / CT instrument (Inveon, Siemens GmbH, Germany), high performance liquid chromatography (HPLC) instrument (LC-20AT, Shimadzu Corporation, Japan), Zorbax Rax-C18 column (Jiangsu Hanbang Technology Co., Ltd.), electronic balance (BS-110S, Sartorius GmbH, Germany).

[0023] II. Experimental Methods

[0024] 1. 68 Preparation and quality control of Ga-FAPI-04. The first step of this implementation plan is the preparation of Ga-FAPI-04. 68 Ga-FAPI-04 was tested, and its quality was ensured to meet the requirements of subsequent research. The specific steps are as follows: 1) Equipment preparation: Install the iQS-TS automation module and equip it with the corresponding piping system to facilitate the automated handling of reactants.

[0025] 2) Reactant injection: Inject the FAPI precursor into the reaction flask and connect it. 68Ge / 68 A Ga generator ensures the smooth progress of the reaction.

[0026] 3) Execute the production protocol: Operate in accordance with the production protocol of the automation module to ensure the stability and consistency of reaction conditions.

[0027] 4) Product Testing: Visually inspect the clarity of the product to ensure it is a colorless and clear solution. Measure the pH value to ensure it is within the appropriate range. Detect the radiochemical purity using high-performance liquid chromatography (HPLC) to ensure it is greater than 99%.

[0028] 5) Stability test: After incubation at 37°C for 4 hours in a PBS and FBS system, the radiochemical purity of the labeled substance was tested to ensure that it remained greater than 99% and no obvious free radicals were observed. 68 Ga radioactive peak.

[0029] 2. Establishment and Grouping of a Mouse Model of Myocardial Infarction. The second step of this implementation plan is to establish a mouse model of myocardial infarction and group it into groups for subsequent evaluation of drug treatment. The specific steps are as follows: 1) Anesthesia and surgery: Mice were anesthetized with 2% isoflurane, the heart was exposed, and the coronary artery was ligated 1-2 mm below the lower edge of the left atrial appendage. The outer surface of the anterior wall of the ventricle turned pale, confirming the successful establishment of myocardial infarction.

[0030] 2) Model validation: using 18 F-FDG PET / CT imaging was used to confirm the successful establishment of the myocardial infarction model. On the first day after the infarction model was established, mice were subjected to 18F-FDG imaging using microPET / CT and injected with 3.7 MBq of [unclear - possibly a specific drug or solution]. 18 F-FDG, scan after 60 minutes to obtain %ID / g data.

[0031] 3) Drug preparation: KYA1797K was dissolved in a solvent mixture consisting of 10% DMSO, 40% PEG300, 5% Tween-80 and 45% physiological saline to prepare solutions of different concentrations (1 mg / ml, 2 mg / ml and 3 mg / ml).

[0032] 4) Grouping: Mice were randomly divided into the following groups: Myocardial infarction (MI) group (solvent-treated control); MI + KYA1797K low-dose group (5 mg / kg / day); MI + KYA1797K medium-dose group (10 mg / kg / day); MI + KYA1797K high-dose group (15 mg / kg / day).

[0033] 5) Administration method: All mice were given intraperitoneal injection for 28 days. The MI group was injected intraperitoneally with 100 μL of solvent mixture, while the other groups were injected intraperitoneally with 100 μL of KYA1797K solution at concentrations of 1 mg / ml, 2 mg / ml and 3 mg / ml, respectively.

[0034] 3. PET-CT imaging of a mouse model of myocardial infarction. The third step in this implementation plan is to perform PET-CT imaging on a mouse model of myocardial infarction to evaluate the efficacy of drug treatment. The specific steps are as follows: 1) Imaging time points: Imaging is performed on days 1, 7, 14, and 28 after myocardial infarction modeling.

[0035] 2) Injection 68 Ga-FAPI-04 assessment of fibrosis degree: Mice were injected with 3.7 MBq via the tail vein. 68 Ga-FAPI-04, micro-PET / CT scan performed 60 minutes later.

[0036] 3) Image reconstruction and analysis: After image reconstruction, the region of interest (ROI) is delineated to obtain %ID / g data.

[0037] 4. Echocardiography. The fourth step in this implementation plan is to quantitatively assess the cardiac function of mice using echocardiography. The specific steps are as follows: 1) Anesthesia: Mice were anesthetized by inhalation of 1.5-2.0% isoflurane.

[0038] 2) Equipment used: The Vevo 2100 ultrasound instrument (VisualSonics, Canada) and M-mode echocardiography were used to assess cardiac function.

[0039] 3) Data analysis: The left ventricular ejection fraction (EF) and left ventricular shortening fraction (FS) are automatically analyzed and calculated to assess the heart's pumping efficiency and systolic function.

[0040] 5. Pathological Examination. The fifth step in this implementation plan is to conduct pathological examination to assess morphological changes in myocardial tissue and the expression of β-catenin protein. The specific steps are as follows: 1) Tissue sampling: Paraffin-embedded tissue from the hearts of mice in the control and experimental groups was taken and serially sectioned.

[0041] 2) Staining method: HE staining: used to assess morphological changes in tissues.

[0042] Masson staining: to observe the deposition of collagen fibers in the myocardium.

[0043] Immunohistochemical staining: β-catenin antibody was used to detect its expression in myocardial tissue.

[0044] 3) Microscopic observation: After staining, the slides are dehydrated, cleared, and mounted with neutral resin, and then images are acquired and analyzed under a microscope.

[0045] 6. Immunofluorescence staining. The sixth step in this protocol is to use immunofluorescence staining to observe the effect of KYA1797K on the expression of myocardial infarction-related proteins. The specific steps are as follows: 1) Section processing: After dewaxing, the sections are subjected to antigen retrieval. After retrieval, the sections are allowed to cool naturally and placed in PBS (pH 7.4) for washing 3 times, 5 minutes each time.

[0046] 2) Serum blocking: Add BSA for serum blocking, and the blocking time is 30 minutes.

[0047] 3) Antibody incubation: Add an appropriate concentration of primary antibody and incubate overnight at 4°C. The next day, wash the slides with PBS to remove unbound primary antibody.

[0048] 4) Secondary antibody incubation: Add the appropriate secondary antibody and incubate at room temperature for 50 minutes under dark conditions.

[0049] 5) Nuclear staining: Add DAPI staining solution to stain the nuclear cells and incubate for 10 minutes.

[0050] 6) Microscopic observation: The slides were mounted using anti-fluorescence quenching mounting medium and images were acquired under a fluorescence microscope.

[0051] 7. Statistical Analysis: The final step in this implementation plan is to conduct statistical analysis to verify the reliability of the experimental results. The specific steps are as follows: 1) Data analysis software: GraphPad Prism 9 software was used for data analysis.

[0052] 2) Data Representation: Quantitative data that conforms to a normal distribution are represented as... ±s represents.

[0053] 3) Intergroup comparisons: One-way ANOVA was used for comparisons among multiple groups, and t-tests were used to compare differences between groups for two independent samples.

[0054] 4) Statistical significance: p < 0.05 is considered statistically significant.

[0055] III. Results

[0056] 1. 68 Preparation and quality control of Ga-FAPI-04. 68Ga-FAPI-04 labeling took 15 minutes, producing a colorless, clear solution with a labeling yield of (89.0±5.0)%. HPLC analysis showed the product had a radiochemical purity greater than 99%. After incubation at 37°C for 4 hours in a PBS and FBS system, the radiochemical purity of the labeled product remained >99%, with no obvious free radicals observed. 68 Ga radioactive peak.

[0057] 2. The myocardial infarction model was successfully established. On the first day after modeling in mice, the myocardial infarction site in the myocardial infarction group was [data missing]. 18 F-FDG intake was significantly reduced ( Figure 1 A), and 68 The uptake of Ga-FAPI-04 increased significantly. Figure 1 B). HE staining results showed that the myocardial fiber structure in the infarcted area was destroyed, the striations disappeared, the eosinophilicity was increased (in deep red), the cell nuclei disappeared or shrunken, and there was a large number of neutrophil infiltration, which contrasted with the neatly arranged and clearly striationed structure of normal myocardium.

[0058] 3. Effects of KYA1797K treatment on cardiac function in mice and evaluation using micro-PET / CT imaging ( Figure 2 On day 14 after treatment, the myocardial infarction group... 68 Ga-FAPI-04 uptake reached its peak in the MI group, MI + 5 mg / kg / day KYA1797K group, MI + 10 mg / kg / day KYA1797K group, and MI + 15 mg / kg / day KYA1797K group. 68 The maximum standard intake values ​​(SUVmax) of Ga-FAPI-04 were (2.38 ± 0.55)%ID / g, (2.00 ± 0.44)%ID / g, (1.30 ± 0.21)%ID / g, and (1.14 ± 0.23)%ID / g, respectively. Further comparative analysis showed a significant difference in intake between the MI+5 mg / kg / day KYA1797K group and the MI+10 mg / kg / day KYA1797K group (t=3.322, P=0.0091), while there was no statistically significant difference in intake between the MI+10 mg / kg / day KYA1797K group and the MI+15 mg / kg / day KYA1797K group (t=1.607, P=0.160). Figure 2 (A~E). On day 28 after myocardial infarction modeling, the left ventricular ejection fraction (EF) and left ventricular shortening fraction (FS) of mice in the myocardial infarction group were significantly reduced. Figure 3 A, Figure 3B) indicates impaired cardiac pumping efficiency and systolic function. However, after drug treatment, both EF and FS recovered (F values: 50.63 and 29.93, respectively, both P < 0.001). These results indicate that KYA1797K showed some efficacy in treating myocardial infarction in mice, with the optimal dose of 10 mg / kg / day. Therefore, subsequent treatment involved dividing the mice into a sham surgery group, a myocardial infarction group, and a 10 mg KYA1797K group.

[0059] 4. Effects of KYA1797K on the expression of myocardial infarction-related proteins and its therapeutic effect. Immunofluorescence results showed that the expression levels of FAP and β-catenin were significantly increased in the myocardial infarction group, while the expression of both FAP and β-catenin was significantly decreased in the KYA1797K treatment group (F values: 105.4 and 39.20, respectively, both P < 0.001). Figure 4 A, Figure 4 B). Immunohistochemistry showed that β-catenin protein exhibited a strong positive signal in the infarct area, and the positive reaction of β-catenin was significantly weakened in the KYA1797K treatment group (F=30.15, P<0.001). Figure 4 C, Figure 4 D). Masson staining results showed that the fibrotic area was significantly smaller in the KYA1797K treatment group compared to the myocardial infarction group (F=184.6, P<0.001). Figure 4 E, Figure 4 F) indicates that the drug has an anti-myocardial fibrosis effect.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. The use of a Wnt / β-catenin inhibitor in the preparation of a drug for treating myocardial infarction, characterized in that, The Wnt / β-catenin inhibitor is KYA1797K.

2. The application as described in claim 1, characterized in that, The drug contains KYA1797K and a solvent.

3. The application as described in claim 2, characterized in that, The solvents include 8-12% DMSO, 35-45% PEG300, 4-6% Tween-80, and 40-50% physiological saline.

4. The application as described in claim 3, characterized in that, The concentration of KYA1797K in the solvent is 1~3 mg / mL.

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