WO2018171279A1

WO2018171279A1 - Use of compound ss-31 in preparation of drug or preparation for treating atherosclerosis and related diseases

Info

Publication number: WO2018171279A1
Application number: PCT/CN2017/118835
Authority: WO
Inventors: 李宽钰; 乔彤
Original assignee: 南京大学
Priority date: 2017-03-24
Filing date: 2017-12-27
Publication date: 2018-09-27
Also published as: CN107041946A

Abstract

Disclosed is the use of compound Szeto-Schiller-31 (SS-31) in the preparation of a drug for treating atherosclerosis and related diseases, such as carotid stenosis, lower extremity arteriosclerosis obliterans and coronary heart disease. Through experiments, it has been found that SS-31 reduces the degree of atherosclerosis, wherein same: significantly decreases the level of arterial ROS in mice, reduces oxidative damage, and promotes the ATP level; significantly reduces the content of macrophages at atherosclerotic plaques, significantly increases the content of smooth muscle cells and collagen content at the plaques, and stabilizes the atherosclerotic plaques; significantly improves the inflammation level; and significantly decreases the expression of the lipid-uptaking protein at aortic plaques. The results show that SS-31 plays a role in the treatment of atherosclerotic diseases and diseases related thereto.

Description

Application of Compound SS-31 in preparing medicines or preparations for treating atherosclerosis and related diseases

Technical field

The invention belongs to the field of chemical medicine and relates to the application of a compound Szeto-Schiller-31 (SS-31) for preparing a medicament for treating atherosclerotic diseases.

Background technique

Atherosclerosis (AS) is the pathological basis of cardiovascular disease, including carotid stenosis, lower extremity arteriosclerosis obliterans and coronary heart disease. The clinical manifestations and pathological features of these diseases are not the same, but they have the common characteristics: the slow development of the lesions, the narrowing of the lumen of the affected arteries, and the lack of blood supply to the distal tissues. So far, there is no ideal preventive drug, which has become a worldwide cause. The most important cause of human death. At present, the pathogenesis of these diseases is still controversial. Genetic, inflammatory, high-fat diet, aging and other complex factors are the common pathogenic factors of these diseases. The pathological mechanisms include vascular endothelial injury and monocyte adhesion migration. Macrophages and macrophages phagocytose lipids to form foam cells and damage metabolic pathways. Atherosclerotic diseases are a serious hazard to human health, which brings great psychological and economic burden to patients, their families and society. The development of drugs for the prevention and treatment of atherosclerosis has received much attention.

Chronic inflammatory conditions and hypercholesterolemia are important features of AS. Arterial endothelial injury is the initial step of AS, and damaged endothelial cells secrete adhesion molecules and cytokines to recruit white blood cells. The persistence of inflammation upregulates the expression of macrophage scavenger receptors, increases ox-LDL uptake, and promotes foam cell formation. Foam cells are the most prominent inflammatory cells in AS plaques. Studies have found that adhesion molecules such as intracellular adhesion molecules, vascular cell adhesion molecules and monocyte chemotactic factors are highly expressed in AS plaques. After adhering to endothelial cells via adhesion molecules, leukocytes are mediated by chemokines and penetrate into the intima of the blood vessels.

Macrophage lipid uptake and discharge imbalance, intracellular lipid accumulation, foam cell formation, is the key to AS plaque formation. CD36 and LOX-1 are oxidative low-density lipoprotein (ox-LDL) receptors, which are lipoproteins on the surface of macrophages; ABCA1 and ABCG1 are lipoproteins on the surface of macrophages, and cholesterol is excreted and receptor HDL Or apolipoprotein A-I binding.

Szeto-Schiller-31 (SS-31) is a novel mitochondrial-targeting compound synthesized by Szeto in 2005. It can act on mitochondrial inner membrane cardiolipin to protect mitochondrial integrity in an animal model of renal ischemia. Reduce or inhibit the production of mitochondria-derived reactive oxygen species (ROS), increase the level of adenosine triphosphate (ATP), and reduce oxidative stress. Obviously these findings are not directly related to anti-atherosclerosis. At present, SS-31 has been applied to many human disease models and has shown certain effects. For example, studies have shown that SS-31 has a protective effect on heart failure and hypertensive cardiomyopathy, although heart failure and hypertensive myocardium The disease and the atherosclerotic disease involved in this application are completely different from the pathogenesis of the disease, the pathophysiological basis, the corresponding therapeutic drugs, therapeutic targets and treatment strategies, and it is impossible to give guidance and enlightenment, but we still want to Boldly try to see if SS-31 has a protective effect on atherosclerotic disease.

But so far, there has been no report on the anti-atherosclerosis and related diseases of SS-31. This application is the first to evaluate the activity of SS-31 in anti-atherosclerosis by model, and found its various regulatory activities and functions in controlling atherosclerosis.

Summary of the invention

Purpose of the invention

The object of the present invention is to discover a new medical use of SS-31 in the preparation of atherosclerosis and related diseases through a disease model associated with atherosclerotic diseases.

Technical solutions

In the present invention, SS-31 is used in an animal model of atherosclerotic disease, and the results show that subcutaneous injection of SS-31 delays the development of mouse AS, blocks the formation of plaque, and stabilizes vulnerable plaque on the other hand. SS-31 can reduce the level of aortic ROS in mice, reduce oxidative damage, increase ATP levels, improve systemic inflammation in mice, and decrease the expression of lipid uptake proteins (CD36 and LOX-1) in mouse aortic plaques. These results indicate that SS-31 can be used clinically as a therapeutic drug for AS or related diseases.

SS-31 itself or its main component is used in the treatment of atherosclerotic diseases including carotid stenosis, lower extremity arteriosclerosis obliterans and coronary heart disease.

Further, SS-31 itself or its use as a main component in the preparation of the following drugs:

(1) Blocking the formation of atherosclerotic plaque and delaying the development of AS;

(2) Stabilizing vulnerable atherosclerotic plaque, preventing unstable angina or / and myocardial infarction;

(3) reduce aortic ROS levels, reduce aortic oxidative damage, increase aortic ATP levels, and improve the physiology and function of the aorta;

(4) reduce the level of aortic or systemic inflammation, block the occurrence and development of AS;

(5) reduce the expression of lipid uptake protein CD36 in aortic plaque, block the occurrence and development of AS;

(6) Decrease the expression of lipid uptake protein LOX-1 in aortic plaque and block the occurrence and development of AS.

Advantages of the invention:

1. The present invention is the first to apply the novel mitochondrial-targeting compound SS-31 for the treatment of atherosclerosis and related diseases, and can be used as a new application for preparing drugs for treating atherosclerosis and related diseases, and has a huge market. Value and social benefits.

2. The present invention provides the use of SS-31 as a main component of a drug in atherosclerotic diseases. SS-31 delays the development of atherosclerotic disease in mice, reduces the formation of plaques, and stabilizes vulnerable plaques.

3, reduce aortic ROS levels, reduce aortic oxidative damage, improve aortic ATP levels, improve the physiology and function of the aorta;

4, reduce the level of aortic or systemic inflammation, block the occurrence and development of AS;

5. Decrease the expression of lipid uptake protein CD36 in aortic plaque and block the occurrence and development of AS;

6. Decrease the expression of lipid uptake protein LOX-1 in aortic plaque and block the occurrence and development of AS.

BRIEF DESCRIPTION OF THE DRAWINGS:

Figure 1 Effect of SS-31 on aortic plaque formation in ApoE ^-/- mice (n=15), data were

Said.

Figure 2: Effect of SS-31 on plaque formation in aortic sinus of ApoE ^-/- mice (n=15), data were

Said.

Figure 3: Effect of SS-31 on plaque components (macrophages) in aortic sinus of ApoE ^-/- mice (n=15), data were

Said.

Figure 4: Effect of SS-31 on plaque components (smooth muscle cells) in aortic sinus of ApoE ^-/- mice (n=15), data were

Said.

Figure 5: Effect of SSS-31 on plaque composition (collagen) in aortic sinus of ApoE ^-/- mice (n=15), data were

Said.

Figure 6: Effect of SS-31 on aortic reactive oxygen species (ROS) in ApoE ^-/- mice.

Figure 7: Effect of SSS-31 on ATP of ApoE ^-/- mouse aorta (n=13), data were

Said.

Figure 8: Effect of SSS-31 on aortic SOD2 protein levels in ApoE ^-/- mice (n=14), data were

Said.

Figure 9: Effect of SSS-31 on total SOD2 enzyme activity in aorta of ApoE ^-/- mice (n=13), data were

Said.

Figure 10: Effect of SS-31 on DNA damage in aortic sinus of ApoE ^-/- mice (n=15), data were

Said.

Figure 11 The effect of SS-31 on CD36 in the aortic sinus region of ApoE ^-/- mice (n=15), the data were

Said.

Figure 12: Effect of SS-31 on LOX-1 in the aortic sinus region of ApoE ^-/- mice (n=15), data were

Said.

Figure 13: Effect of SS-31 on ABCA1 in aortic sinus region of ApoE ^-/- mice (n=15), data were

Said.

detailed description:

Effect of SS-31 on atherosclerosis in ApoE-/- mice

1 Materials and methods

1.1 Experimental grouping and specimen collection

Male 8-week-old ApoE ^-/- mice (genetic background: C57BL/6) were purchased from the Institute of Model Animals of Nanjing University. ApoE ^-/- mice were housed in SPF-class animal rooms and fed a high-fat diet. High-fat diet formula: 0.2% cholesterol and 20% fat mixed with conventional feed. ApoE ^-/- mice were randomly divided into control group (P), low-dose drug group (M1, 1 mg/kg/d) and high-dose drug group (M3, 3 mg/kg/d), with 30 rats in each group. Group P was injected subcutaneously with 5 mL·kg ^-1 ·d ^-1 saline, and M1 group was injected subcutaneously with SS-31 5 mL·kg ^-1 ·d ^-1 (SS-31 powder was dissolved in physiological saline at a concentration of 0.2 mg·mL ^-1 , Shanghai Qiang Yao Biotechnology Co., Ltd. synthesis, dose reference), M3 group subcutaneous injection of SS-31 5mL·kg ^-1 · d ^-1 (concentration 0.6mg·mL ^-1 ). After 12 weeks, the mice were anesthetized by intraperitoneal injection of pentobarbital (40 mg·kg ^-1 ), blood was taken through the inferior vena cava, the anesthetic dose (80 mg·kg ^-1 ) was added, and the neck dislocation was sacrificed, and the heart and aorta were collected. Cardiac specimens were fixed in 4% paraformaldehyde for 24 hours, embedded in OCT or embedded in paraffin, and 10 slices of 6μm thick paraffin or frozen sections were continuously cut in the microtome. The aortic sinus paraffin sections were stored at room temperature for use. Frozen sections of aortic sinus were placed in propylene glycol for 2 min, stained in oil red O staining tank for 16 h, 85% propylene glycol was differentiated for 1 min, hematoxylin staining for 3 min, and the film was mounted and photographed under an optical microscope (ZEISS, Germany) as soon as possible. Gross aureus red staining was performed in each group of mouse aorta (see the reference for specific procedures). Superoxide dismutase 2 (SOD2) protein levels were detected by aorta, total SOD activity was detected by aorta, and ATP levels were detected by aorta. .

1.2 blood sample processing

Blood samples were taken overnight at 4 ° C, centrifuged at 2500 g for 20 min, and the supernatant was taken to obtain serum samples. The serum biochemical analyzer (Beckman Coulter AU542) was used to detect the content of serum triglyceride (TG) and total cholesterol (TC). Enzyme-linked immunosorbent assay (ELISA) kit (Wuhan Bude Bioengineering Co., Ltd.) detects intracellular adhesion molecule-1 (ICAM-1) and monocyte chemotaxis Factor-1 (Monocyte chemoattractant protein, MCP-1), interleukin (IL-6) and C-reactive protein (CRP) levels.

1.3DHE staining, ATP detection and SOD activity detection

The aorta was embedded in OCT immediately after excision. After cryopreservation, a 6 μm thick slice was sliced on a glass slide. The fluorescent probe (Dihydroethidium, DHE, 10 μM, Sigma-Aldrich, USA) was incubated at 37 ° C for 30 min in the dark. Focusing microscope (ZEISS HB050, ZEISS, Germany) observed fluorescence, and fluorescence strongly reacted with ROS levels. Immediately after the aorta was isolated, the ATP level and total SOD activity were measured using the ATP test kit (Shanghai Biyuntian Biotechnology Co., Ltd.) and the SOD test kit (Nanjing Institute of Bioengineering).

1.4Western blotting

After the aorta was lysed by lysate (Thermo Fisher Scientific, UK), the protein concentration determined by BCA method, 35 μg of total protein extract was electrophoresed on a 12% SDS-PAGE gel, and transferred to a nitrocellulose transfer membrane (Nitrocellulose). Membranes, NC) Post-antibody (SOD2, 1:1000 dilution, Abcam, UK; Tubulin, 1:2000 dilution, Sigma-Aldrich, USA) overnight at 4°C, secondary antibody (goat anti-rabbit or goat anti-mouse, 1:10000) Dilute) for 1 h. Immediately after incubation of the NC membrane with the ECL chemiluminescent substrate (Shanghai Biyuntian Biotechnology Co., Ltd.), a Western blotting chemiluminescence detection system (Thermo Fisher Scientific, UK) was used for exposure shooting. Image J software was used to detect SOD2 content.

1.5Masson special dyeing

The tissue sections of the mice were dewaxed, hematoxylin staining for 4 min, Masson's modified trichrome dyeing for 8 min, bright green staining for 8 min, 0.2% ammonium acetate wash to no dye shedding, dehydration, transparency, sealing, observation under light microscope And take pictures. Image J software was used to analyze the amount of collagen (blue) in the plaque.

1.6 Immunohistochemistry (IHC)

Tissue sections of mice were dewaxed, antigen-repaired, 3% H ₂ O ₂ inhibited endogenous peroxidase, primary antibody (1:200 dilution) was incubated for 1.5 h at room temperature, and secondary antibody (1:200 dilution) was incubated for 30 min at room temperature. DAB coloration, hematoxylin counterstaining. After the staining was completed, it was observed under an optical microscope and photographed. CD68 (macrophage molecular marker), α-SMA (smooth muscle cell molecular marker) antibody was purchased from Abcam, UK; CD36 antibody was purchased from Proteintech Group, USA; LOX-1 antibody was purchased from Santa Cruz Biotech, USA; ABCA1 antibody was purchased from US Signalway Antibody LLC; secondary antibody (goat anti-rabbit or oxygen anti-mouse) purchased from Santa Cruz Biotech, USA. The intensity of the IHC positive region was analyzed using Image J software.

1.7 data processing

Statistical analysis was performed using SPSS 22.0 software. Measurement data by mean ± standard deviation

It is indicated that the Student's t test is used for comparison between groups. p < 0.05 indicates that the difference was statistically significant.

2 results

2.1SS-31 can delay the development of ApoE ^-/- mouse AS

The P group ApoE ^-/- mice were similar in weight to the administration group (M1 and M3) at 8 and 20 weeks, and the serum TC and TG in the administration group were similar to the P group at 20 weeks, as shown in Table 1. Gross aortic red staining of the aorta revealed a significant reduction in plaque area in the ApoE ^-/- mice of the M1 and M3 groups, as shown in Figure 1, and quantified on the right. Oil red staining of the frozen section of the aortic sinus area showed that the plaque size of the ApoE ^-/- mice in the M1 and M3 groups was significantly reduced, as shown in Fig. 2, and the quantified map below. The plaque composition changes were studied by CD68, α-SMA immunohistochemistry and Masson special staining. As shown in Fig. 3, the immunohistochemical positive areas of CD68 in M1 and M3 groups were significantly reduced, and the quantitative map was below; Fig. 4 is α-SMA. Immunohistochemistry showed a significant increase in smooth muscle cells at the plaques of ApoE ^-/- mice in the M1 and M3 groups, with quantified maps below; Figure 5 is a special staining of Masson, blue area is quantified below, and ApoE ^{-/- is} small in M1 and M3 groups. Collagen increased significantly at the plaque of the mouse. The above results indicate that the plaque area of the SS-31 group is significantly reduced and the plaque is more early and more stable. Therefore, SS-31 can be used to block the formation of atherosclerotic plaque and delay the development of AS.

2.2SS-31 reduces aortic oxidative stress levels in ApoE ^-/- mice and increases aortic ATP synthesis

The level of oxidative stress at the plaques of AS increased, while SS-31 as an antioxidant reduced the level of oxidative stress, so we examined the ROS levels and ATP levels in the aorta of each mouse. Figure 6 is a DHE staining of aortic frozen sections. The red positive areas of ApoE ^-/- mice in the M1 and M3 groups were significantly reduced, indicating a decrease in ROS levels and a decrease in oxidative stress in ApoE ^-/- mice. Aortic ATP results showed a significant increase in aortic ATP levels in the M1 and M3 groups, as shown in Figure 7. SOD is the main protein for ROS clearance in cells, including SOD1 in cells and SOD2 in mitochondria. SOD2 plays a major role. Aortic Western blotting showed no change in SOD2 protein levels, as shown in Figure 8, below which is a quantitative map; but the total aortic SOD activity was significantly increased, as shown in Figure 9. Increased ROS levels impair DNA, and 8-OHDG immunohistochemistry was used to detect arterial DNA damage. The results showed that the 8-OHDG-positive area of mice in M1 and M3 groups was significantly reduced, as shown in Figure 10, and the upper part was 8-OHDG immunized. Histochemical image, below is a quantitative map of positive areas. The above results indicate that SS-31 reduces the level of arterial oxidative stress and can therefore be used to reduce aortic oxidative damage and further expansion of atheromatous plaques.

2.3SS-31 improves systemic inflammation in ApoE ^-/- mice

AS is a chronic inflammatory disease, and ICAM-1 and MCP-1 are the major inflammatory factors in AS, promoting the adhesion of monocytes/macrophages to endothelial cells and migration to the inner membrane. The serum levels of ICAM-1 and MCP-1 in the M1 and M3 mice were significantly reduced (see Table 1). Macrophages that migrate to the endothelium secrete pro-inflammatory factors, including IL-6, IL-1β and Tumor necrosis factor alfa (TNF-α). These inflammatory factors mediate systemic inflammatory responses, such as activation of acute phase proteins encoded by the liver gene, including CRP and serum amyloid A (SAA). Serum IL-6 was significantly decreased in the ApoE ^-/- mice of the M1 and M3 groups, and CRP was slightly decreased, as shown in Table 1. The above results indicate that SS-31 can improve the systemic inflammation level of ApoE ^-/- mice, which is beneficial to delay the further development of atheroma.

2.4SS-31 reduces the expression of aortic lipid uptake protein in ApoE ^-/- mice

Foam cell formation is the key to the emergence of early plaques in AS. Excessive intake of Oxidized low-density lipoprotein (ox-LDL), excessive cholesterol esterification, and impaired cholesterol release lead to accumulation of cholesterol esters, formation of lipid droplets, and the cells gradually transform into foam cells. CD36 (Cluster of differentiation 36) and LOX-1 (Lectin-like ox-LDL receptor-1) are ox-LDL receptors and are important lipid uptake proteins. ABCA1 (ATP-binding cassette A1) is a lipid. Excrete protein. As shown in Figures 11-13, the expression of CD36 and LOX-1 in the aortic plaques of the ApoE ^-/- mice in the M1 and M3 groups was significantly decreased, while the expression of ABCA1 was unchanged. These results indicate that SS-31 reduces the expression of aortic CD36 and LOX-1, inhibits ox-LDL uptake, prevents lipid accumulation, and reduces foam cell formation. Therefore, SS-31 can be used to block the occurrence and development of AS.

Table 1 mouse body weight and serum lipid index.

Note: Data are used

Indicates that *p<0.05 and n=15.

Claims

Use of Compound SS-31 for the preparation of a medicament or formulation for the treatment of atherosclerotic diseases.
The use of the compound SS-31 according to claim 1 for the preparation of a medicament or preparation for treating atherosclerotic diseases, characterized in that the atherosclerotic disease comprises carotid stenosis, lower extremity arteriosclerosis obliterans and coronary heart disease.
The use of the compound SS-31 according to claim 1 or 2 for the preparation of a medicament or a preparation for treating atherosclerotic diseases, characterized in that the compound SS-31 is used for preparing atherosclerotic plaque formation and delaying AS. Development of applications on drugs or preparations.
The use of the compound SS-31 according to claim 1 or 2 for the preparation of a medicament or preparation for treating atherosclerotic diseases, characterized in that the compound SS-31 is used for preparing stable vulnerable atherosclerotic plaque and preventing instability The application of drugs or preparations for angina pectoris and myocardial infarction.
The use of the compound SS-31 according to claim 1 or 2 for the preparation of a medicament or preparation for treating atherosclerotic diseases, characterized in that the compound SS-31 is prepared for alleviating systemic oxidative stress and chronic inflammation of the body The application of the drug or formulation of the process.
Use of the compound SS-31 according to claim 1 or 2 for the preparation of a medicament or preparation for treating atherosclerotic diseases, characterized by the use of the compound SS-31 for the preparation of a medicament or preparation for pathological characteristics of vascular aging.
Compound SS-31 is useful in the preparation of drugs or agents that reduce aortic ROS levels, reduce aortic oxidative damage, increase aortic ATP levels, and increase aortic physiology and function.
Compound SS-31 is useful in the preparation of a medicament or formulation for reducing the level of aortic or systemic inflammation, arresting the development and development of AS.
Compound SS-31 is useful in the preparation of a drug or preparation for reducing the expression of lipid uptake protein CD36 at aortic plaque, arresting the development and development of AS.
Compound SS-31 is useful in the preparation of a drug or formulation for reducing the expression of lipid uptake protein LOX-1 at aortic plaque, arresting the development and development of AS.