Use of mln4924 and flubendazole in the manufacture of a medicament for treating atherosclerosis

Abstract

The application discloses application of MLN4924 and flubendazole in preparation of drugs for treating atherosclerosis. Low-dose MLN4924 and flubendazole have little cytotoxicity, and both can treat atherosclerosis by interfering with the formation of foam cells and cholesterol accumulation; when MLN4924 and flubendazole are used in combination, the effect is stronger, and synergy is achieved.

Description

Technical Field

[0001] This invention belongs to the field of atherosclerosis technology, specifically relating to the application of MLN4924 and flubendazole in the preparation of drugs for treating atherosclerosis. Background Technology

[0002] Atherosclerosis is a chronic, progressive disease characterized by high morbidity and mortality worldwide, and it is the most significant pathological basis for cardiovascular and cerebrovascular diseases. According to the World Health Organization, cardiovascular disease has consistently been the leading cause of death globally, with the vast majority of these deaths caused by atherosclerosis. With the aging population and lifestyle changes brought about by urbanization, the disease burden is becoming increasingly heavy, with the prevalence of major risk factors for atherosclerosis, such as hypertension and dyslipidemia, continuing to rise.

[0003] Currently, drug treatment for atherosclerosis has evolved into a comprehensive management strategy based on statins as the cornerstone, combined with antiplatelet drugs, antihypertensive drugs, and hypoglycemic agents. The core objective of these drugs is to control risk factors and stabilize plaques to prevent rupture or erosion, thereby significantly reducing the risk of major adverse cardiovascular events such as myocardial infarction and stroke. However, the side effects of long-term use, such as statin-related myalgia, abnormal liver enzymes, and the bleeding risk associated with antiplatelet drugs, severely impact patient adherence. Summary of the Invention

[0004] The purpose of this invention is to provide the use of MLN4924 and flubendazole in the preparation of a medicament for treating atherosclerosis.

[0005] Application of MLN4924 and flubendazole in the preparation of drugs for treating atherosclerosis.

[0006] MLN4924 and flubendazole inhibit foam cell formation.

[0007] MLN4924 and flubendazole inhibit cholesterol accumulation.

[0008] A drug for treating atherosclerosis, the active ingredients of which are MLN4924 and flubendazole.

[0009] Preferably, the mass ratio of MLN4924 to flubendazole is 1:4.

[0010] Preferably, it also includes one or more pharmaceutically acceptable excipients or carriers.

[0011] Preferably, the excipients or carriers include diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption promoters, surfactants, adsorbent carriers, or lubricants.

[0012] Preferably, the pharmaceutical composition can be formulated into dosage forms such as tablets, capsules, effervescent tablets, granules, powders, dispersible tablets, oral liquids, pills, or injections.

[0013] The beneficial effects of this invention are as follows: low doses of MLN4924 and flubendazole have almost no cytotoxicity and can both treat atherosclerosis by intervening in foam cell formation and cholesterol accumulation. The combined use of MLN4924 and flubendazole has a stronger effect and achieves synergistic effect. Attached Figure Description

[0014] Figure 1 To evaluate the cellular activity of different concentrations of MLN4924 on THP-1-derived macrophages.

[0015] Figure 2 The results of the oil red staining area measurement in Example 2 are shown.

[0016] Figure 3 To investigate the cellular activity of different concentrations of flubendazole on THP-1-derived macrophages.

[0017] Figure 4 The results of the oil red staining area measurement in Example 4 are shown.

[0018] Figure 5 The results of the oil red staining area measurement in Example 5 are shown.

[0019] Figure 6 The results of intracellular cholesterol content determination are shown in Example 6.

[0020] Figure 7 The results of the inhibition of atherosclerosis in the mouse model of Example 7 are shown in Figure 7. A is the plaque area calculated by Oil Red O staining of the mouse aorta; B is the absolute plaque area calculated by HE staining of the plaque section at the root of the mouse aorta. Detailed Implementation

[0021] To facilitate understanding of the present invention, a more comprehensive description will be given below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0022] The MLN4924 used in the following examples was purchased from MedChemexpress, catalog number: HY-70062; and flubendazole was purchased from MCE, catalog number: HY-B0294.

[0023] Example 1: Low concentrations of MLN4924 showed no significant cytotoxicity to THP-1-derived macrophages.

[0024] Human monocytes THP-1 were selected as the research object. The culture medium was prepared as follows: 90% RPMI 1640 medium (product number PYG0006, Wuhan Boster Biological Co., Ltd.) + 10% serum (product number FSP500, Suzhou EcoSy Biotechnology Co., Ltd.) + 1% penicillin antibody (product number 2240831, Thermo Fisher Scientific, USA). The cells were cultured at 37℃ and 5% carbon dioxide. When the cells were in good condition, the inducing agent PMA (Phorbol 12-myristate 13-acetate) was added to induce differentiation into macrophages for 24 hours. Then, different concentrations of MLN4924 were added and treated for 48 hours. CCK-8 cell proliferation and cytotoxicity were then detected.

[0025] Experimental methods:

[0026] (1) THP-1 cells were seeded into 96-well plates and PMA (20 ng / ml) was added for induction for 24 hours;

[0027] (2) Add different concentrations of MLN4924 (0, 0.25μM, 0.5μM, 0.75μM, 1μM) and treat for 24 hours;

[0028] (3) Add 0.5 ml of CCK-8 reagent to 9.5 ml of culture medium and mix well for later use;

[0029] (4) Aspirate the culture medium from the 96-well plate, add 100ml of CCK-8 reagent working solution to each well, and incubate for 2 hours. Then use an M5 microplate reader to detect the absorbance at 450nm.

[0030] Statistical analysis was performed using one-way ANOVA; experimental results are shown below. Figure 1 The results showed that low-dose MLN4924 (≤1μM) had no significant cytotoxicity on THP-1-derived macrophages.

[0031] Example 2: MLN4924 inhibits foam cell formation

[0032] THP-1 cells were induced to differentiate into macrophages by adding PMA. After 24 hours, the experimental group was treated with MLN4924 (1 μM). After 24 hours, the experimental group and the control group were treated with oxLDL. Oil Red staining was performed after 24 hours.

[0033] Experimental methods:

[0034] (1) THP-1 cells were seeded into 12-well plates with cell spreaders and PMA (20 ng / ml) was added for 24 hours to induce induction.

[0035] (2) After treatment with MLN4924 (1 μM) for 24 hours, oxLDL (20 µg / ml) was added and incubated for 24 hours.

[0036] (3) Discard the culture medium and wash once with PBS;

[0037] (4) Fix with 10% neutral formaldehyde for 10 minutes;

[0038] (5) Add 60% isopropanol (diluted with PBS) and wash briefly;

[0039] (6) Dilute 0.5% Oil Red O solution with water at a ratio of 3:2, let stand for 10 minutes, and then filter with medium-speed qualitative filter paper;

[0040] (7) After staining for 30 minutes, wash briefly with 60% isopropanol;

[0041] (8) Stain the nucleus with hematoxylin and rinse with tap water;

[0042] (9) Remove the slide, mount it with glycerin gelatin, and take a picture under a microscope;

[0043] (10) Use Image J software to perform quantitative statistics on the area stained with oil red.

[0044] Statistical analysis was performed using one-way ANOVA; experimental results are shown below. Figure 2 After adding MLN4924 before oxLDL treatment, the Oil Red staining area of ​​macrophages was significantly reduced compared to the control group. These results suggest that MLN4924 inhibits foam cell formation.

[0045] Example 3: Low concentrations of flubendazole showed no significant cytotoxicity to THP-1-derived macrophages.

[0046] Human monocytes THP-1 were selected as the research object. The culture medium was prepared as follows: 90% RPMI 1640 medium + 10% serum + 1% penicillin and antibiotics. The cells were cultured at 37℃ and 5% carbon dioxide. When the cells were in good condition, the inducing agent PMA was added to induce differentiation into macrophages for 24 hours. Then, different concentrations of flubendazole were added and treated for 48 hours. CCK-8 cell proliferation and toxicity were then detected.

[0047] Experimental methods:

[0048] (1) THP-1 cells were seeded into 96-well plates and PMA (20 ng / ml) was added for induction for 24 hours;

[0049] (2) Add different concentrations of flubendazole (0, 0.25 μM, 0.5 μM, 0.75 μM, 1 μM) and treat for 24 hours;

[0050] (3) Add 0.5 ml of CCK-8 reagent to 9.5 ml of culture medium and mix well for later use;

[0051] (4) Aspirate the culture medium from the 96-well plate, add 100ml of CCK-8 reagent working solution to each well, and incubate for 2 hours. Then use an M5 microplate reader to detect the absorbance at 450nm.

[0052] Statistical analysis was performed using one-way ANOVA; experimental results are shown below. Figure 3 The results showed that low-dose flubendazole (≤1 μM) had no significant cytotoxicity on THP-1-derived macrophages.

[0053] Example 4: Flubendazole inhibits foam cell formation

[0054] THP-1 cells were induced to differentiate into macrophages by adding PMA. After 24 hours, the experimental group was treated with flubendazole (1 μM). After 24 hours, the experimental group and the control group were treated with oxLDL. Oil Red staining was performed after 24 hours.

[0055] Experimental methods:

[0056] (1) THP-1 cells were seeded into 12-well plates with cell spreaders and PMA (20 ng / ml) was added for 24 hours to induce induction.

[0057] (2) After treatment with flubendazole (1 μM) for 24 hours, oxLDL (20 µg / ml) was added and incubated for 24 hours;

[0058] (3) Discard the culture medium and wash once with PBS;

[0059] (4) Fix with 10% neutral formaldehyde for 10 minutes;

[0060] (5) Add 60% isopropanol (diluted with PBS) and wash briefly;

[0061] (6) Dilute 0.5% Oil Red O solution with water at a ratio of 3:2, let stand for 10 minutes, and then filter with medium-speed qualitative filter paper;

[0062] (7) After staining for 30 minutes, wash briefly with 60% isopropanol;

[0063] (8) Stain the nucleus with hematoxylin and rinse with tap water;

[0064] (9) Remove the slide, mount it with glycerin gelatin, and take a picture under a microscope;

[0065] (10) Use Image J software to perform quantitative statistics on the area stained with oil red.

[0066] Statistical analysis was performed using one-way ANOVA; experimental results are shown below. Figure 4When flubendazole was added before oxLDL treatment, the Oil Red staining area of ​​macrophages was significantly reduced compared with the control group. These results suggest that flubendazole inhibits the formation of foam cells.

[0067] Example 5: A 1:4 mixture of MLN4924 and flubendazole effectively inhibited foam cell formation.

[0068] THP-1 cells were induced to differentiate into macrophages by adding PMA. After 24 hours, MLN4924 (1 μM), flubendazole (1 μM), and a 1:4 mixture of MLN4924 and flubendazole (0.2 μM MLN4924 plus 0.8 μM flubendazole) were added to each group. After 24 hours of treatment, oxLDL was added to each group to induce the formation of foam cells. Oil Red staining was performed after 24 hours.

[0069] Experimental methods:

[0070] (1) THP-1 cells were seeded into 12-well plates with cell spreaders and PMA (20 ng / ml) was added for 24 hours to induce induction.

[0071] (2) After treating with MLN4924 (1 μM), flubendazole (1 μM), and a 1:4 mixture of MLN4924 and flubendazole (0.2 μM MLN4924 plus 0.8 μM flubendazole) for 24 hours, oxLDL (20 µg / ml) was added and incubated for 24 hours.

[0072] (3) Discard the culture medium and wash once with PBS;

[0073] (4) Fix with 10% neutral formaldehyde for 10 minutes;

[0074] (5) Add 60% isopropanol (diluted with PBS) and wash briefly;

[0075] (6) Dilute 0.5% Oil Red O solution with water at a ratio of 3:2, let stand for 10 minutes, and then filter with medium-speed qualitative filter paper;

[0076] (7) After staining for 30 minutes, wash briefly with 60% isopropanol;

[0077] (8) Stain the nucleus with hematoxylin and rinse with tap water;

[0078] (9) Remove the slide, mount it with glycerin gelatin, and take a picture under a microscope;

[0079] (10) Use Image J software to perform quantitative statistics on the area stained with oil red.

[0080] Statistical analysis was performed using one-way ANOVA; experimental results are shown below. Figure 5Treatment with a 1:4 mixture of MLN4924 and flubendazole significantly reduced the Oil Red staining area of ​​macrophages compared to the control group and the groups treated with either MLN4924 or flubendazole alone. The experimental results are shown in […]. Figure 5 The above results suggest that a 1:4 mixture of MLN4924 and flubendazole can synergistically inhibit the formation of foam cells.

[0081] Example 6: A 1:4 mixture of MLN4924 and flubendazole can effectively inhibit cholesterol accumulation.

[0082] THP-1 cells were induced to differentiate into macrophages by adding PMA. After 24 hours, MLN4924 (1 μM), flubendazole (1 μM), and a 1:4 mixture of MLN4924 and flubendazole (0.2 μM MLN4924 plus 0.8 μM flubendazole) were added to each group. After 24 hours of treatment, oxLDL was added to each group to induce the formation of foam cells. The intracellular cholesterol content was measured after 24 hours.

[0083] Experimental methods:

[0084] (1) THP-1 cells were seeded into 6-well plates and PMA (20 ng / ml) was added for 24 hours to induce induction;

[0085] (2) After treating with MLN4924 (1 μM), flubendazole (1 μM), and a 1:4 mixture of MLN4924 and flubendazole (0.2 μM MLN4924 plus 0.8 μM flubendazole) for 24 hours, oxLDL (20 µg / ml) was added and incubated for 24 hours.

[0086] (3) Discard the culture medium, add 0.5 ml of PBS to each well, scrape off the cells and collect them into an EP tube, centrifuge at 1000 rpm for 10 minutes, discard the supernatant, repeat this operation, and wash the cells with PBS twice more.

[0087] (4) Add lysis buffer to the cell pellet and sonicate under ice-water bath conditions (300W power, 3 seconds / time, 15 seconds interval, 3 minutes). The lysed liquid is directly measured without centrifugation.

[0088] (5) Total cholesterol test: The test system consists of 2.5 μl lysis buffer (2.5 μl distilled water for blank wells, 2.5 μl calibrator for calibration wells, and 2.5 μl lysis buffer for sample wells) plus 250 μl working solution (Nanjing Jiancheng Total Cholesterol Test Kit, catalog number A111-1-1). Each group has 3 replicates. Mix well, incubate at 37°C for 10 minutes, and measure the absorbance of each well using an ELISA reader at a wavelength of 510 nm.

[0089] Statistical analysis was performed using one-way ANOVA; experimental results are shown below. Figure 6After treatment with a 1:4 mixture of MLN4924 and flubendazole, the intracellular cholesterol content was significantly reduced compared with the control group and the groups treated with MLN4924 or flubendazole alone. The 1:4 mixture of MLN4924 and flubendazole can synergistically inhibit cholesterol accumulation.

[0090] Example 7: Inhibition of atherosclerosis in a mouse model by a 1:4 mixture of MLN4924 and flubendazole.

[0091] To further investigate the functions of MLN4924 and flubendazole in the process of atherosclerosis, we selected the ApoE- / - mouse, a currently recognized ideal mouse model for studying atherosclerosis. Eight-week-old ApoE- / - mice were randomly divided into four groups. The experimental groups were injected with 5 mg / kg MLN4924, 5 mg / kg flubendazole, and a 1:4 mixture of MLN4924 and flubendazole (1 mg / kg MLN4924 plus 4 mg / kg flubendazole), respectively. The control group was injected with 200 μL / kg PBS (twice a week for 12 weeks). All mice were fed a high-fat diet to establish the atherosclerosis model.

[0092] Experimental methods:

[0093] (1) Mice were euthanized after being induced by a high-fat diet and their organs were removed;

[0094] (2) Remove the entire aorta and remove the surrounding adipose tissue;

[0095] (3) Cut the mouse aorta longitudinally and lay it flat with the intima side down;

[0096] (4) Add 0.5 ml of 4% paraformaldehyde and fix at 4°C overnight;

[0097] (5) Rinse with 5 ml PBS for 2 hours;

[0098] (6) Rinse with PBS after removing the aortic adventitia;

[0099] (7) Dehydrate in 5 ml of propylene glycol at room temperature for 2 minutes;

[0100] (8) Stain in 5 ml of 0.5% Oil Red O at room temperature for 2 hours;

[0101] (9) Wash 4 times with 85% propylene glycol (diluted with PBS);

[0102] (10) Rinse with PBS;

[0103] (11) Scanning and photographing the aorta after fixation;

[0104] (12) Calculate the proportion of the area of ​​the Oil Red O stained plaque to the total area of ​​the aorta; use Image J software for quantitative analysis; use one-way ANOVA to calculate the p-value for statistical analysis.

[0105] Phenotypic analysis was performed on the four groups of mice: Figure 7 In Figure A, the plaque area was calculated from the mouse aorta using Oil Red O staining. The aortic plaque area in the MLN4924 and flubendazole 1:4 mixed administration group was significantly lower than that in the control group and the MLN4924 and flubendazole single administration groups, indicating that the combination of MLN4924 and flubendazole can synergistically inhibit the formation of atherosclerotic plaques.

[0106] Experimental methods:

[0107] (1) Mice were euthanized after being induced by a high-fat diet, and the root tissue of the aortic plaque was separated and fixed in 10% formalin for 48 hours.

[0108] (2) After fixing the tissue block, place it into the embedding box;

[0109] (3) Dehydrate the tissue with alcohol gradients: 70%, 80%, 90% I, 90% II, 95% I, 95% II, 100% I, and 100% II for 10 minutes each;

[0110] (4) Clear the tissue with xylene: xylene I and xylene II for 5 minutes each;

[0111] (5) Immerse the tissue in paraffin: 40 minutes each in paraffin bath I and paraffin bath II;

[0112] (6) Embedding: The tissue is made into a wax block;

[0113] (7) Slice, with a thickness of 4 μm;

[0114] (8) Overnight baking in a wafer oven;

[0115] (9) Dewaxing the sections: 10 minutes each for xylene I and xylene II; rehydration: 5 minutes each for 100% I and 100% II alcohol, and 3 minutes each for 95%, 90%, 80%, and 75% alcohol.

[0116] (10) Rinse with water 3 times, 2 minutes each time;

[0117] (11) Stain the nucleus with hematoxylin for 10 minutes;

[0118] (12) Rinse with tap water for 2 minutes;

[0119] (13) Stain with eosin for 2 minutes;

[0120] (14) Rinse with tap water for 2 minutes;

[0121] (15) 90% and 100% alcohol for 2 minutes each; xylene I and xylene II for 4 minutes each;

[0122] (16) Neutral resin sealing;

[0123] (17) Observation and photography were performed using a Nikon Bx60 microscope connected to a Nikon DP70 camera; quantitative analysis was performed using ImageJ software; statistical analysis was performed using one-way ANOVA to calculate p-values.

[0124] Figure 7 In Figure B, the absolute area of ​​the plaque in the aortic root of mice was calculated by HE staining. The plaque area in the aortic root of mice in the MLN4924 and flubendazole 1:4 mixed administration group was significantly lower than that in the control group and the MLN4924 and flubendazole single administration groups, indicating that the combination of MLN4924 and flubendazole can synergistically inhibit the occurrence of atherosclerosis.

[0125] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the invention patent. It should be noted that those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the protection scope of the present invention. Therefore, the protection scope of this invention patent should be determined by the appended claims.

Claims

1. The application of MLN4924 and flubendazole in the preparation of drugs for treating atherosclerosis, characterized in that, The mass ratio of MLN4924 to flubendazole is 1:4.

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