Application of MLN4924 and flubendazole in preparation of medicine for treating atherosclerosis

Abstract

The invention discloses an application of MLN4924 and flubendazole in preparation of a medicine for treating atherosclerosis. The MLN4924 and the flubendazole which are low in dosage almost have no cytotoxicity and can be used for treating atherosclerosis by intervening foam cell formation and cholesterol accumulation, and the MLN4924 and the flubendazole are combined for use, so that the effect is stronger, and synergistic interaction is realized.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of atherosclerosis, and particularly relates to application of MLN4924 and flubendazole in preparation of a drug for treating atherosclerosis. BACKGROUND

[0002] Atherosclerosis is a chronic progressive disease with high morbidity and mortality worldwide, and is the main pathological basis of cardiovascular and cerebrovascular diseases. According to the data of the World Health Organization, cardiovascular diseases have ranked first in the global causes of death for many years, and most of them are caused by atherosclerosis. With the population aging and the changes in lifestyle brought by urbanization, the disease burden is becoming increasingly heavy, and the prevalence of major risk factors for atherosclerosis such as hypertension and dyslipidemia continues to rise.

[0003] At present, the drug treatment of atherosclerosis has formed a comprehensive management strategy based on statins, combined with antiplatelet drugs, antihypertensive drugs and hypoglycemic drugs. The core goal 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 medication, such as statin-related myalgia and liver enzyme abnormalities, and the risk of bleeding caused by antiplatelet drugs, seriously affect the medication compliance of patients. SUMMARY

[0004] The purpose of the present application is to provide the application of MLN4924 and flubendazole in preparation of a drug for treating atherosclerosis.

[0005] The application of MLN4924 and flubendazole in preparation of a drug for treating atherosclerosis.

[0006] MLN4924 and flubendazole inhibit the formation of foam cells.

[0007] MLN4924 and flubendazole inhibit the accumulation of cholesterol.

[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 further comprises one or more pharmaceutically acceptable excipients or carriers.

[0011] Preferably, the excipients or carriers include diluents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, adsorption carriers or lubricants.

[0012] Preferably, the pharmaceutical composition can be prepared into tablets, capsules, effervescent tablets, granules, powders, dispersible tablets, oral solutions, pills or injections.

[0013] Advantages of the present application: low-dose MLN4924 and flubendazole have little cytotoxicity, and both can treat atherosclerosis by interfering with foam cell formation and cholesterol accumulation. The combination of MLN4924 and flubendazole has a stronger effect and achieves synergistic effect. BRIEF DESCRIPTION OF DRAWINGS

[0014] Figure 1 Cell viability of THP-1 derived macrophages treated with different concentrations of MLN4924.

[0015] Figure 2 Oil red staining area measurement results of Example 2.

[0016] Figure 3 Cell viability of THP-1 derived macrophages treated with different concentrations of flubendazole.

[0017] Figure 4 Oil red staining area measurement results of Example 4.

[0018] Figure 5 Oil red staining area measurement results of Example 5.

[0019] Figure 6 Measurement results of intracellular cholesterol content of Example 6.

[0020] Figure 7 Measurement results of inhibition of atherosclerosis in a mouse model of Example 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 DESCRIPTION

[0021] In order to facilitate the understanding of the present application, the present application will be described more fully below. However, the present application can be realized in many different forms and is not limited to the embodiments described herein. On the contrary, these embodiments are provided so that the disclosure of the present application can be more thoroughly and completely understood.

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

[0023] Example 1: Low concentration of MLN4924 has no obvious cytotoxicity on THP-1 derived macrophages Human monocyte THP-1 was selected as the research object, and the culture medium was prepared in the proportion of 90% RPMI1640 medium (item number PYG0006, Wuhan Dr. Dec biological Co., Ltd.) + 10% serum (item number FSP500, Suzhou Yikosai Biological Technology Co., Ltd.) + 1% double antibody (item number 2240831, Thermo Fisher, USA), and cultured in an environment of 37°C and 5% carbon dioxide concentration. When the cells were in good condition, PMA (Phorbol 12-myristate 13-acetate) was added to induce differentiation into macrophages for 24 hours, and then different concentrations of MLN4924 were added. After 48 hours of action, CCK-8 cell proliferation and toxicity detection were performed.

[0024] Experimental method: (1) THP-1 cells were plated in a 96-well plate, and PMA (20 ng / ml) was added to induce for 24 hours; (2) Different concentrations of MLN4924 (0, 0.25 μM, 0.5 μM, 0.75 μM, 1 μM) were added for 24 hours; (3) 0.5 ml of CCK-8 reagent was added to 9.5 ml of culture medium and mixed well for standby; (4) The culture medium in the 96-well plate was aspirated, 100 ml of CCK-8 reagent working solution was added to each well, and after 2 hours of incubation in the incubator, the M5 enzyme label instrument was used to detect the absorbance at 450 nm.

[0025] Statistical analysis used one-way ANOVA, and the experimental results are shown in Figure 1 , and the results showed that low-dose MLN4924 (≤1 μM) had no obvious cytotoxicity to THP-1 derived macrophages.

[0026] Example 2: MLN4924 inhibits the formation of foam cells PMA was added to THP-1 cells to induce differentiation into macrophages, and after 24 hours, MLN4924 (1 μM) was added to the experimental group, and after 24 hours of action, oxLDL was added to the experimental group and the control group, and after 24 hours, oil red staining was performed.

[0027] Experimental method: (1) THP-1 cells were plated in a 12-well plate with cell slides, and PMA (20 ng / ml) was added to induce for 24 hours; (2) After 24 hours of treatment with MLN4924 (1 μM), oxLDL (20 μg / ml) was added and incubated for 24 hours; (3) The culture medium was aspirated and washed once with PBS; (4) 10% neutral formaldehyde fixation for 10 minutes; (5) Add 60% isopropanol (diluted with PBS) and wash slightly; (6) Dilute 0.5% oil red O solution with water at a ratio of 3:2, stand for 10 minutes, and then filter with a qualitative filter paper at a medium speed; (7) Stain for 30 minutes, add 60% isopropanol and wash slightly; (8) Stain the nucleus with hematoxylin and rinse with tap water; (9) Take out the slide, seal it with glycerol gelatin, and then take a photo under a microscope; (10) Use Image J software to quantitatively analyze the oil red staining area.

[0028] Statistical analysis uses one-way ANOVA, and the experimental results are shown in Figure 2 After adding MLN4924 before the action of oxLDL, the oil red staining area of macrophages is significantly reduced compared with the control group. The above results suggest that MLN4924 inhibits the formation of foam cells.

[0029] Example 3: Low-concentration flubendazole has no obvious cytotoxicity on THP-1 derived macrophages Human mononuclear cells THP-1 were selected as the research object, and the culture medium was prepared at a ratio of 90% RPMI1640 medium + 10% serum + 1% double antibody. The cells were cultured in an environment with a temperature of 37°C and a carbon dioxide concentration of 5%. When the cells were in good condition, different concentrations of flubendazole were added after being induced by PMA for 24 hours to differentiate into macrophages. After 48 hours of action, CCK-8 cell proliferation and toxicity detection were performed.

[0030] Experimental method: (1) THP-1 cells were plated in a 96-well plate, and PMA (20 ng / ml) was added for induction for 24 hours; (2) Different concentrations of flubendazole (0, 0.25 μM, 0.5 μM, 0.75 μM, 1 μM) were added for 24 hours; (3) Add 0.5 ml of CCK-8 reagent to 9.5 ml of culture medium and mix well for standby; (4) The culture medium in the 96-well plate was aspirated, 100 ml of CCK-8 reagent working solution was added to each well, and the absorbance at 450 nm was detected using an M5 enzyme label instrument after 2 hours of incubation in the incubator.

[0031] Statistical analysis uses one-way ANOVA, and the experimental results are shown in Figure 3 The results show that low-dose flubendazole (≤1 μM) has no obvious cytotoxicity on THP-1 derived macrophages.

[0032] Example 4: Flubendazole inhibits the formation of foam cells 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.

[0033] Experimental methods: (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. (2) After treatment with flubendazole (1 μM) for 24 hours, oxLDL (20 µg / ml) was added and incubated for 24 hours; (3) Discard the culture medium and wash once with PBS; (4) Fix with 10% neutral formaldehyde for 10 minutes; (5) Add 60% isopropanol (diluted with PBS) and wash briefly; (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; (7) After staining for 30 minutes, wash briefly with 60% isopropanol; (8) Stain the nucleus with hematoxylin and rinse with tap water; (9) Remove the slide, mount it with glycerin gelatin, and take a picture under a microscope; (10) Use Image J software to perform quantitative statistics on the area stained with oil red.

[0034] Statistical analysis was performed using one-way ANOVA; experimental results are shown below. Figure 4 When 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.

[0035] Example 5: A 1:4 mixture of MLN4924 and flubendazole effectively inhibited foam cell formation. 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.

[0036] Experimental methods: (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. (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. (3) Discard the culture medium and wash once with PBS; (4) Fix with 10% neutral formaldehyde for 10 minutes; (5) Add 60% isopropanol (diluted with PBS) and wash briefly; (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; (7) After staining for 30 minutes, wash briefly with 60% isopropanol; (8) Stain the nucleus with hematoxylin and rinse with tap water; (9) Remove the slide, mount it with glycerin gelatin, and take a picture under a microscope; (10) Use Image J software to perform quantitative statistics on the area stained with oil red.

[0037] Statistical analysis was performed using one-way ANOVA; experimental results are shown below. Figure 5 Treatment 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.

[0038] Example 6: A 1:4 mixture of MLN4924 and flubendazole can effectively inhibit cholesterol accumulation. 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.

[0039] Experimental methods: (1) THP-1 cells were seeded into 6-well plates and PMA (20 ng / ml) was added for 24 hours to induce induction; (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. (3) Aspirate the culture medium, add 0.5 ml PBS to each well to scrape 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 for another 2 times; (4) Add the lysate to the cell precipitate, and perform ultrasonic disruption under ice water bath conditions (power 300 W, 3 seconds / time, interval 15 seconds, 3 minutes), and directly measure the lysed liquid without centrifugation; (5) Total cholesterol test: the determination system is 2.5 μl lysate (add 2.5 μl distilled water to the blank hole, add 2.5 μl calibration to the calibration hole, and add 2.5 μl lysate to the sample hole), 250 μl working solution (Nanjing Jiancheng total cholesterol test kit, item number A111-1-1), 3 replicates of empty group are set in each group, mix well, incubate at 37°C for 10 minutes, wavelength 510 nm, and measure the absorbance value of each well by an enzyme label instrument.

[0040] Statistical analysis uses one-way ANOVA, and the experimental results are shown in Figure 6 , the content of intracellular cholesterol is significantly lower than that of the control group and the MLN4924 and flubendazole single action groups after treatment with the 1:4 mixture of MLN4924 and flubendazole, and the 1:4 mixture of MLN4924 and flubendazole can synergistically inhibit the accumulation of cholesterol.

[0041] Example 7 Inhibition of the atherosclerosis process in a mouse model by the 1:4 mixture of MLN4924 and flubendazole To further study the function of MLN4924 and flubendazole in the atherosclerosis process, we selected the currently recognized ideal mouse model ApoE- / - mouse for studying atherosclerosis. We randomly divided eight-week-old ApoE- / - mice into four groups, and injected 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) into three experimental groups, respectively, and injected 200 μL / kg PBS into the control group (twice a week, for 12 weeks of continuous injection), and performed high-fat feeding to establish an atherosclerosis model.

[0042] Experimental method: (1) After the mice were anesthetized and sacrificed after high-fat diet induction, the organs were removed; (2) The whole aorta was taken out and the fat tissue around the aorta was removed; (3) The mouse aorta was cut longitudinally, and the intimal surface was placed flat; (4) 0.5 ml 4% paraformaldehyde was added for fixation at 4°C overnight; (5) 5 ml PBS was used to flush for 2 hours; (6) After removing the adventitia of the aorta, PBS was used for flushing; (7) Dehydrate in 5 ml propylene glycol for 2 minutes at room temperature; (8) Stain in 5 ml 0.5% oil red O for 2 hours at room temperature; (9) Wash in 85% propylene glycol (diluted with PBS) for 4 times successively; (10) Wash in PBS; (11) Scan and take pictures after fixing the aorta; (12) Calculate the proportion of the area of the plaque stained by oil red O to the whole area of the aorta; use Image J software for quantitative analysis; use single factor analysis of variance to calculate p value for statistical analysis.

[0043] Phenotype analysis was performed on four groups of mice: Figure 7 A is the plaque area calculated by oil red O staining of the aorta of mice. The plaque area of the aorta of mice in the group administered with MLN4924 and flubendazole at a ratio of 1:4 was significantly lower than that of the control group and the groups administered with MLN4924 and flubendazole alone, indicating that the combination of MLN4924 and flubendazole can synergistically inhibit the formation of atherosclerotic plaques.

[0044] Experimental method: (1) After the mice were induced by high-fat diet and euthanized, the aortic plaque root tissues were isolated and fixed with 10% formalin for 48 hours; (2) After the fixed tissues were trimmed, they were placed in embedding boxes; (3) The tissues were dehydrated by alcohol gradient: 70%, 80%, 90% I, 90% II, 95% I, 95% II, 100% I, 100% II, each for 10 minutes; (4) The tissues were transparentized with xylene: xylene I, xylene II, each for 5 minutes; (5) The tissues were immersed in wax: wax tank I, wax tank II, each for 40 minutes; (6) Embedding, the tissues were made into wax blocks; (7) Sectioning, the thickness was 4 μm; (8) Overnight baking; (9) Dewaxing of the sections: xylene I, xylene II, each for 10 minutes; rehydration: 100% I, 100% II alcohol, each for 5 minutes, 95%, 90%, 80%, 75%, each for 3 minutes; (10) Water washing for 3 times, each for 2 minutes; (11) Nucleus staining with hematoxylin for 10 minutes; (12) Tap water washing for 2 minutes; (13) Eosin staining for 2 minutes; (14) Tap water washing for 2 minutes; (15) 90%, 100% alcohol each for 2 minutes; xylene I, xylene II each for 4 minutes; (16) Neutral resin sealing; (17) Nikon Bx60 microscope connected with Nikon DP70 camera was used for observation and photographing; Image J software was used for quantitative analysis; single factor variance analysis was used for statistical analysis to calculate p value.

[0045] Figure 7 The absolute plaque area was calculated by HE staining of plaque sections of the root of the mouse aorta. The plaque area of the mouse aorta root 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.

[0046] The above-described embodiments only express several embodiments of the present application, which are described in detail and specifically, but cannot be understood as the limitation of the scope of the patent. It should be noted that for ordinary skilled in the art, several modifications and improvements can be made without departing from the concept of the present application, which are all within the protection scope of the present application. Therefore, the protection scope of the patent of the present application should be subject to the appended claims.

Claims

1. Use of MLN4924 and flubendazole in the preparation of a medicament for treating atherosclerosis.

2. Use of MLN4924 and flubendazole according to claim 1 for the preparation of a medicament for the treatment of atherosclerosis, characterized in that, MLN4924 and flubendazole inhibit the formation of foam cells.

3. Use of MLN4924 and flubendazole for the manufacture of a medicament for the treatment of atherosclerosis according to claim 1, characterized in that, MLN4924 and flubendazole inhibit the accumulation of cholesterol.

4. A medicament for treating atherosclerosis, characterized by, The active ingredients are MLN4924 and flubendazole.

5. The medicament for treating atherosclerosis according to claim 4, wherein The mass ratio of the MLN4924 and flubendazole is 1:

4.

6. The medicament for the treatment of atherosclerosis according to claim 4 or 5, wherein One or more pharmaceutically acceptable excipients or carriers are also included.

7. The medicament for treating atherosclerosis according to claim 6, wherein The excipients or carriers include diluents, excipients, fillers, binders, humectants, disintegrants, absorption promoters, surfactants, adsorptive carriers or lubricants.

8. The medicament for treating atherosclerosis according to claim 4 or 5, wherein The pharmaceutical composition can be prepared into tablets, capsules, effervescent tablets, granules, powders, dispersible tablets, oral solutions, pills or injections.

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