Use of FEN1 inhibitor in the preparation of a medicament for treating myocardial infarction

Abstract

The present invention belongs to the field of biomedical technology and discloses the application of FEN1 inhibitors in the preparation of drugs for treating myocardial infarction; the FEN1 inhibitors effectively improve the myocardial infarction area, myocardial fibrosis, the phenomenon of myocardial cell death and myocardial inflammation symptoms, weaken the myocardial injury caused by MI, and the FEN1 inhibitors have certain beneficial effects in the treatment of myocardial infarction; the FEN1 inhibitors can effectively improve a series of symptoms induced by myocardial infarction in mice.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to the application of FEN1 inhibitor in the preparation of drugs for treating myocardial infarction. Background Art

[0002] Myocardial infarction (MI) is caused by the acute occlusion of the coronary artery, resulting in persistent and severe acute ischemia of the corresponding myocardium, often accompanied by tissue necrosis and infiltration of inflammatory cells, and is a common clinical heart disease. Research has shown that a variety of inflammatory mediators and biochemical reactions are involved in the process of myocardial infarction, including ischemic injury, oxidative stress injury, complement activation, interleukin release, neutrophil aggregation / infiltration, etc.

[0003] In the prior art, the FEN1 molecule has been mainly studied in the field of tumors. FEN1 inhibitors can be used in combination with some anti-cancer drugs to increase the efficacy of anti-cancer drugs. For example, FEN1 inhibitors are used in combination with cisplatin to treat lung cancer to increase the efficacy.

[0004] Currently, there is no record or report on the research of FEN1 inhibitors in the treatment of myocardial infarction. Therefore, it is necessary to provide a new idea for the subsequent clinical development of new drugs for treating myocardial infarction. Summary of the Invention

[0005] The purpose of the present invention is to solve the deficiencies in the prior art and provide the application of FEN1 inhibitor in the preparation of drugs for treating myocardial infarction.

[0006] To achieve the above purpose, the present invention is realized through the following technical solutions:

[0007] In the first aspect, the present invention provides the application of FEN1 inhibitor in the preparation of drugs for treating myocardial infarction. The English name of the FEN1 inhibitor is FEN1-IN-4, and its alias is 1-(cyclopropylmethyl)-3-hydroxyquinazoline-2,4-dione. Its molecular formula is C12H12N2O3, the molecular weight is 232.24, the CAS number is 1995893-58-7, and the PubChem number is 121231495; its structural formula is as shown in formula (Ⅰ):

[0008]

[0009] In the second aspect, the present invention provides a pharmaceutical composition for treating myocardial infarction, and the active ingredient of the pharmaceutical composition includes the FEN1 inhibitor or a pharmaceutically acceptable salt thereof.

[0010] The term "pharmaceutically acceptable salts" refers to those salts that, within the scope of sound medical judgment, are suitable for contact with the tissues of humans and lower animals without undue toxicity, irritation, allergic response, etc., and are commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable salts are well known in the art.

[0011] In the preparation of drugs for treating myocardial infarction, dosage forms such as injections, oral liquids, powders, etc. can be used; the medicinal dosage is 30 - 40 mg / ㎡ body surface area.

[0012] In this invention, an MI animal model was established by ligating the left anterior descending branch (LAD) of the coronary artery of a mouse heart to observe whether FEN1 in has a therapeutic effect on myocardial infarction. The results showed that FEN1 in has a beneficial effect on myocardial infarction in the mouse heart.

[0013] This invention has the following beneficial effects: The death caused by myocardial infarction is alleviated by the FEN1 inhibitor, and it effectively improves the myocardial infarction area, myocardial fibrosis, the phenomenon of myocardial cell death, and myocardial inflammatory symptoms, weakening the myocardial damage caused by MI. FEN1 in has a certain beneficial effect in the treatment of myocardial infarction. FEN1 in can effectively improve a series of symptoms induced by myocardial infarction in mice, providing a new idea for the subsequent clinical development of new drugs for treating myocardial infarction. Description of the Drawings

[0014] Figure 1 It is a B-ultrasound cardiac function detection diagram of the mouse heart after the treatment of Example 1;

[0015] Figure 2 It is a diagram of the mouse treatment process in the construction of the mouse myocardial infarction model;

[0016] Figure 3 It is a survival curve diagram of mice after the experiment of FEN1 in improving the death of mice caused by myocardial infarction;

[0017] Figure 4 It is a photographed and quantitatively statistical diagram of the infarct area of mice after the experiment of FEN1 in improving the infarct area of the heart of MI mice;

[0018] Figure 5 It is a Masson staining diagram of the mouse heart after the experiment of FEN1 in improving the cardiac fibrosis of mice caused by myocardial infarction;

[0019] Figure 6 It is a photographed diagram of MitoSOX staining of NRCMs after the experiment of FEN1 knockout improving the production of reactive oxygen species in NRCMs cells caused by hypoxia;

[0020] Figure 7It is a quantitative statistical chart of fluorescence staining after experimental treatment of the improvement of reactive oxygen species production in NRCMs cells caused by hypoxia by FEN1 knockout;

[0021] Figure 8 It is a photographed image of NRCMs MitoSOX staining after experimental treatment of the improvement of reactive oxygen species production in NRCMs cells caused by hypoxia / reoxygenation by FEN1 knockout;

[0022] Figure 9 It is a quantitative statistical chart of fluorescence staining after experimental treatment of the improvement of reactive oxygen species production in NRCMs cells caused by hypoxia / reoxygenation by FEN1 knockout;

[0023] Terms:

[0024] MI: Myocardial infarction

[0025] LAD: Left anterior descending coronary artery

[0026] LV Mass: Left ventricular mass

[0027] FS: Left ventricular fractional shortening

[0028] EF: Ejection fraction

[0029] NRCMs: Primary neonatal rat cardiomyocytes

[0030] TTC: 2,3,5-Triphenyltetrazolium chloride Detailed implementation manners

[0031] The present invention is further described by the following examples, which do not limit the present invention in any way. Without departing from the technical solution of the present invention, any modification or change that is easily achieved by those of ordinary skill in the art to the present invention will fall within the scope of the claims of the present invention.

[0032] The materials used in the following examples are as follows: The FEN1 inhibitor (hereinafter referred to as FEN1 in) was provided by Aladdin (Aladdin Biochemical Technology Co., Ltd., Shanghai, China), and the product number was: CAS No 1995893-58-7. The ICR mice involved were all purchased from the Animal Model Research Institute of Yangzhou University, with an age of 6 weeks, and were raised in the Animal Experiment Center of China Pharmaceutical University. All animal experiments complied with the Guide for the Care and Use of Laboratory Animals and were approved by the Animal Experiment Ethics Committee of China Pharmaceutical University.

[0033] Example 1: Verify whether FEN1 in affects the normal cardiac function of wild-type mice

[0034] Adult wild-type mice were injected with FEN1 in at a dose of 1 mg / kg for seven consecutive days. The control group (WT) was injected with an equal volume of FEN1 in diluent, and the diluent ratio was (50% normal saline + 40% PEG300 + 5% Tween 80 + 5% DMSO). The ICR mice were divided into 4 WT mice and 7 FEN1 in mice groups. Echocardiography was performed to detect cardiac function.

[0035] Echocardiography was performed at the Animal Imaging Center of China Pharmaceutical University using a Vevo 2100 ultrasound system (Visual Sonics, Toronto, Canada). The transducer was 30 MHz, and data such as ventricular size were recorded in M-mode.

[0036] As Figure 1 shown, compared with the control group (WT) mice, there was no change in cardiac function in the FEN1 in group, specifically manifested as no difference in ejection fraction (EF), left ventricular fractional shortening (FS), and left ventricular mass (LV Mass) between the two groups.

[0037] Example 2: Effect on mice with myocardial infarction model

[0038] 2.1 Construction of a mouse myocardial infarction model

[0039] Adult male ICR mice were depilated on the chest. Using 1% chloral hydrate saline aqueous solution, the mice were anesthetized by intraperitoneal injection. After detecting no response by pinching the toes, the ventilator was turned on and the parameters were set (respiratory ratio 2:1, tidal volume 6 - 8 mL, frequency 70 times / min). The tracheal intubation was inserted into the trachea of the mouse along the glottis. Observing the respiratory condition of the mouse, if the chest rise and fall were consistent with the ventilator frequency, the intubation was successful and the MI surgery could be performed. The mice were placed in the right lateral position, the local skin was disinfected, the intercostal skin was incised, the muscles between the third and fourth intercostals were bluntly separated, the pericardium was opened to expose the left anterior descending branch (LAD) of the coronary artery of the heart, and the LAD was quickly ligated with 6-0 sterile silk thread to form myocardial ischemia in the anterior wall of the left ventricle. The chest cavity was quickly closed with a purse-string suture method, and the chest was squeezed to prevent pneumothorax. The adult male ICR mice were randomly divided into 2 groups, namely the MI group and the MI + FEN1 in group.

[0040] The diagram of the mouse treatment process is as Figure 2 shown.

[0041] 2.2 FEN1 in improves mouse death caused by myocardial infarction

[0042] First, a mouse myocardial infarction model was constructed by LAD constriction (Example 2). Adult male ICR mice were randomly divided into 2 groups, the MI group and the MI + FEN1 in group.

[0043] AsFigure 3 As shown, the mortality rate of mice in the MI+FEN1 in group was significantly lower than that of mice in the MI group. The above experimental results suggest that FEN1 in can significantly improve the death of mice caused by myocardial infarction and increase the survival rate of mice.

[0044] 2.3 FEN1 in improves the cardiac infarction area in MI mice

[0045] Two days after myocardial infarction modeling (hereinafter referred to as MI-2d), the mice were sacrificed by cervical dislocation under isoflurane anesthesia. The hearts were removed and the left and right ventricles were kept intact. The residual blood was squeezed out and the hearts were rinsed clean in sterile physiological saline. The hearts were sectioned with a mold and stained with TTC to observe and quantify the infarction area.

[0046] As Figure 4 shown, LAD constriction induced an increase in the cardiac infarction area in mice, while FEN1 in alleviated the myocardial infarction caused by MI. The above experimental results suggest that FEN1 in reduces the increase in the cardiac infarction area in mice caused by MI surgery. Statistical method for data: The data are expressed as mean ± SEM (standard error of the mean) values. Unpaired two-tailed Student's t-test was used for comparison between the two groups. After one-way ANOVA for multiple groups followed by Tukey multiple comparison test or two-way ANOVA followed by Bonferroni multiple comparison test, a p-value < 0.05 was considered statistically significant.

[0047] 2.5 FEN1 in improves cardiac fibrosis in mice caused by myocardial infarction

[0048] Five days after myocardial infarction modeling (hereinafter referred to as MI-5d), the mice were sacrificed by cervical dislocation under isoflurane anesthesia. The hearts were removed and the left and right ventricles were kept intact. The residual blood was squeezed out and the hearts were rinsed clean in sterile physiological saline. One group cut the heart tissue at the same site of each group of mice along the long axis of the heart, fixed it in 10% neutral formalin solution for 24 hours, and observed the collagen structure of the pathological tissue by Masson staining.

[0049] As Figure 5 shown, LAD constriction induced a significant increase in the myocardial fibrosis area in the hearts of mice, while FEN1 in alleviated the myocardial fibrosis caused by MI. The above experimental results suggest that FEN1 in can significantly improve the myocardial fibrosis caused by myocardial infarction in mice.

[0050] Example 3:

[0051] FEN1 knockout improves the production of reactive oxygen species in NRCMs cells caused by hypoxia

[0052] To verify the experimental results of FEN1 in reducing the infarct area in vitro, MitoSOX staining was used to observe the cellular oxidative damage of NRCMs cells.

[0053] Culture and treatment of NRCMs cells: NRCMs cells were cultured in 24-well plates. After transient transfection with the overexpression plasmid or knockout plasmid of FEN1 for 24 h, hypoxia treatment was carried out for 8 h. Then, the cells were fixed with 4% paraformaldehyde for 15 minutes and rinsed twice with ddH2O. Then, 500 μL of MitoSOX reactive oxygen species probe staining solution was added to each well, and the plate was placed in a 37 °C constant temperature incubator for dark staining for 25 minutes. Then, the cells were rinsed twice with ddH2O, and the plate was placed under an inverted fluorescence microscope (Carl ZEISS, AxioVert.A1, Dublin, CA, U.S.A.) for observation and photography.

[0054] As Figure 6 and Figure 7 shown, hypoxia can significantly induce the production of reactive oxygen species in NRCMs cells, mainly manifested by an increase in MitoSOX-stained clusters. In the hypoxia + sg FEN1 group (sg FEN1 represents transfection with the FEN1 knockdown plasmid constructed using the CRISPR-Cas9 technique, and NC represents transfection with the V2 plasmid vector as a control, as shown in the figure), the production rate of reactive oxygen species in NRCMs cells was significantly lower than that in the hypoxia group. In the hypoxia + FEN1 ov group (FEN1 ov represents transfection with the overexpression plasmid of FEN1, and CON represents transfection with the vector pcDNA3.1 of the overexpression plasmid, as shown in the figure), the production rate of reactive oxygen species in NRCMs cells was significantly higher than that in the hypoxia group. The above experimental results suggest that FEN1 knockout can improve the production of reactive oxygen species in NRCMs cells caused by ischemia, and it is also confirmed in vitro experiments that FEN1 in can weaken the cardiac damage caused by myocardial infarction. Data statistical method: The data are expressed as mean ± SEM (standard error of the mean) values. Unpaired two-tailed Student's t-test was used for comparison between two groups. After one-way comparison of multiple groups by ANOVA followed by Tukey multiple comparison test or two-way ANOVA followed by Bonferroni multiple comparison test, a p-value < 0.05 was considered statistically significant.

[0055] FEN1 knockout improves the production of reactive oxygen species in NRCMs cells caused by hypoxia / reoxygenation

[0056] To further verify the experimental results of FEN1 in reducing the infarct area in vitro, MitoSOX staining was used to observe the production of reactive oxygen species in NRCMs cells under hypoxia / reoxygenation treatment.

[0057] Culture and treatment of NRCMs cells: NRCMs cells were cultured in 24-well plates. After transiently knocking out the plasmid for 24 h, the cells were treated with hypoxia for 45 min, and then cultured normally for 8 h. The cells were fixed with 4% paraformaldehyde for 15 minutes and rinsed twice with ddH2O. Then, 500 μL of MitoSOX reactive oxygen species probe staining solution was added to each well, and the plate was placed in a 37 °C constant temperature incubator for dark staining for 25 minutes. The cells were rinsed twice with ddH2O again, and then the plate was placed under an inverted fluorescence microscope (Carl ZEISS, Axio Vert.A1, Dublin, CA, U.S.A.) for observation and photography.

[0058] As Figure 8 and Figure 9 shown, hypoxia / reoxygenation can significantly induce the production of reactive oxygen species in NRCMs cells, mainly manifested by an increase in MitoSOX-stained clusters. Compared with the hypoxia group of NRCMs cells, the production rate of reactive oxygen species in the hypoxia / reoxygenation + sgFEN1 group of NRCMs cells was significantly decreased. Knockout of FEN1 can improve the production of reactive oxygen species in NRCMs cells caused by hypoxia / reoxygenation treatment, and it was also confirmed in vitro experiments that FEN1 in can attenuate the cardiac injury caused by ischemia-reperfusion. Data statistics method: The data are expressed as mean ± SEM (standard error of the mean) values. Unpaired two-tailed Student's t-test was used for comparison between two groups. After one-way ANOVA for multiple groups followed by Tukey's multiple comparison test or two-way ANOVA followed by Bonferroni's multiple comparison test, a p-value < 0.05 was considered statistically significant.

[0059] The above shows and describes the basic principles, main features and advantages of the present invention. However, the above are only specific embodiments of the present invention, and the technical features of the present invention are not limited thereto. Any other implementation manner obtained by those skilled in the art without departing from the technical solution of the present invention should be covered within the patent scope of the present invention.

Claims

1. Use of FEN1 inhibitor in the preparation of a medicament for treating myocardial infarction, characterized in that, The chemical name of the FEN1 inhibitor is 1-(cyclopropylmethyl)-3-hydroxyquinazoline-2,4-dione, and its structural formula is as shown in Formula (I):

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