KR101796025B1 - Pharmaceutical composition for preventing or treating atherosclerosis comprising sarpogrelate, pravastatin and TNF-inhibitor - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating atherosclerosis comprising sarpogrelate, pravastatin, and a TNF-inhibitor as effective components. In the previous study, the present inventors have reported that the combination of pravastatin and sarpogrelate has a synergistic effect on atherosclerosis. In the present invention, the present inventors have determined whether the pravastatin-sarpogrelate combination has sufficient effects in a mouse model of age-related atherosclerosis, or an additional treatment is needed considering newly involved aging factors and complicated pathophysiological influences in the atherosclerosis development stage. In the above context, by additionally combining an anti-TNF agent that targets TNF known for playing an important role in aging process, the present inventors have verified that the coadministration of pravastatin-sarpogrelate-Etanercept can be an effective therapeutic strategy for preventing or treating age-related atherosclerosis.

Description

TECHNICAL FIELD The present invention relates to a pharmaceutical composition for preventing or treating atherosclerosis, which comprises, as an active ingredient, a pravastatin and a TNF-inhibitor, and a pharmaceutical composition for preventing or treating atherosclerosis comprising sarpogrelate, pravastatin and TNF-

The present invention relates to a pharmaceutical composition for the prevention or treatment of atherosclerosis comprising sarpogrelate, pravastatin and a TNF-inhibitor as an active ingredient.

Statin is a drug that lowers cholesterol by inhibiting 3-hydroxy-3-methylglutaryl coenzyme A reductase. It is used for the treatment of cardiovascular diseases (CVD) . Although statins are widely used to prevent CVD, there are concerns about increasing the risk of diabetes. Pravastatin is more recommended than other statins when using statins in patients with atherosclerosis and high-risk patients with diabetes.

Sapotilylate is a serotonin receptor antagonist, an antiplatelet agent widely used to prevent arterial thrombosis. However, only two studies conducted in rabbits so far have been reported, and little is known about their effect on atherosclerosis. Sapoglirate slowed progression of rabbit atherosclerosis by increasing endothelial nitric oxide synthase (eNOS) and increasing blood viscosity. Since the platelet and clotting systems are important for the progression of atherosclerosis, the combined use of pravastatin and sapogirate was more effective for the prevention of atherosclerosis.

Atherosclerosis is characterized by the accumulation of fat-floating macrophages (foam cells) by a complex inflammatory process that forms atherosclerotic plaques, and by the accumulation of fibrous and smooth muscle cells, It is a major disease. Elevated levels of inflammatory cytokines in the process of senescence play an important role in damaging blood vessels and aggravating atherosclerosis.

Etanercept is a biologic drug that treats inflammatory autoimmune diseases by inhibiting tumor necrosis factor (TNF). Anti-TNF therapy for patients with rheumatoid arthritis (RA) clearly reduces the incidence of heart failure and cardiovascular disease.

Therefore, further studies are needed on the combined administration of sapoglylate, pravastatin and TNF-inhibitor in the prevention and treatment of atherosclerosis related atherosclerosis.

US Open Patent 2012-0039884 (Released on February 16, 2012)

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of atherosclerosis, particularly, atherosclerosis, which comprises sarpogrelate, pravastatin and a TNF inhibitor as an active ingredient.

The present invention provides a pharmaceutical composition for preventing or treating atherosclerosis comprising sarpogrelate, pravastatin and a TNF inhibitor as an active ingredient.

The present invention relates to a pharmaceutical composition for the prevention or treatment of atherosclerosis, comprising as an active ingredient a sapoglylate, pravastatin and a TNF-inhibitor. In a previous study, the present inventors have reported that the combination of pravastatin and sapogyrate shows a synergistic effect on atherosclerosis, but the present invention contemplates whether the combination of pravastatin-saporoglateate exhibits sufficient effect in a model of atherosclerosis of the aging atherosclerosis, The need for additional treatment was determined by considering the complex pathophysiological effects of the involved aging factors and the stage of atherosclerosis. Accordingly, the present inventors have further found that the combination administration of pravastatin-sapogyrylate-etanercept in combination with an anti-TNF agent targeting TNF, which plays an important role in the aging process, is useful for the prevention and treatment of atherosclerosis Strategy.

Figure 1 shows that administration of triple combination of pravastatin, sapogirilate and etanercept in a 60 week old aged low density lipoprotein receptor knockout (LDLr KO) mouse reduces atherosclerotic lesions. (A) Oil Red O-Represents the frontal area of the stained aorta. (B) Oil Red O-This is the result of quantifying the stained plaque area. The error bars represent standard errors, and significance was analyzed using Student's t- test (* P <0.05, n = 6-7 / group). NFD, normal fat diet; HFD, high fat diet (Western type diet); NS, no significance; PRA, pravastatin; SAR, sapoglylate; ETA, etanercept.
Figure 2 shows the results of histochemical analysis of atherosclerotic aortic root lesions. LDLr KO mice were treated with either (a) normal fat diet or (b) high fat diet (Western type diet) intake for 12 weeks and (c) etanercept administration for 12 weeks, or (d) combination administration of pravastatin and sapogirate (e) pravastatin, a combination of sapoglylate and etanercept. (A) Oil Red O quantification, (B) intercellular adhesion molecule 1 (ICAM-1), (C) monocyte / macrophage-2 (MOMA- Results of quantification of immunostained atherosclerotic plaque. The scale bar represents 600 μm and the error bars represent standard errors. Significance was analyzed by Student's t- test (* P <0.05, ** P <0.01, n = 6-7 / group). NFD, normal fat diet; HFD, high fat diet (Western type diet); NS, no significance; PRA, pravastatin; SAR, sapoglylate; ETA, etanercept.
FIG. 3 shows that the administration of pravastatin-sapogyrylate-etanercept triple combination results in a significant inhibition of TNF expression. Serum was isolated and (A) tumor necrosis factor (TNF) and (B) interleukin-6 (IL-6) were measured. The error bars represent standard error and significance was analyzed by Student's t- test (* P <0.05, ** P <0.01, n = 6-7 / group). NFD, normal fat diet; HFD, high fat diet (Western type diet); NS, no significance; PRA, pravastatin; SAR, sapoglylate; ETA, etanercept.

Accordingly, the present inventors believe that, in the treatment of pravastatin-saporoglate, an additional blockade of TNF may provide a synergistic effect in the treatment of atherosclerosis-related atherosclerosis, and that pravastatin, sapoglylate and Etanercept were administered in combination to confirm the therapeutic effect, and the present invention was completed.

The present invention provides a pharmaceutical composition for preventing or treating atherosclerosis comprising sarpogrelate, pravastatin and a TNF inhibitor as an active ingredient.

Preferably, the TNF inhibitor may be Etanercept, Infliximab, adalimumab or Golimumab, and most preferably Etanercept, however, the TNF inhibitor may be an etanercept, infliximab, adalimumab or golimumab, But is not limited to.

Preferably, the atherosclerosis is but is not limited to aging atherosclerosis.

Preferably, the pharmaceutical composition comprises at least one of intercellular adhesion molecule 1 (ICAM-1), monocyte / macrophage-2 (MOMA-2) and tumor necrosis factor (TNF) Can be reduced.

Preferably, the pharmaceutical composition may reduce total cholesterol and low-density lipoprotein (LDL) cholesterol levels.

The pharmaceutical composition of the present invention can be administered by any conventional method selected from the group consisting of injections, granules, powders, tablets, pills, capsules, suppositories, gels, suspensions, emulsions, .

In addition, the pharmaceutical composition may contain a pharmaceutically acceptable carrier in addition to the above-mentioned composition. Such a pharmaceutically acceptable carrier is usually used in the preparation of pharmaceuticals, and includes lactose, dextrose, sucrose, sorbitol, But are not limited to, mannitol, starch, acacia, calcium phosphate, alginate, gelatin, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methylcellulose, methylhydroxybenzoate, Talc, magnesium stearate, mineral oil, and the like, but is not limited thereto. In addition, the pharmaceutical composition may further include a lubricant, a wetting agent, a sweetening agent, a flavoring agent, an emulsifying agent, a suspending agent, a preservative, etc. as an additive.

The pharmaceutical composition may be administered orally or parenterally, for example, intravenously, subcutaneously, or intraperitoneally, depending on the severity of the symptoms. The dose may be determined depending on the patient's body weight, age, sex, Health status, diet. Administration time, administration method, excretion rate, severity of disease, etc., and may be administered once to several times per day.

In one embodiment of the invention, parenteral administration of pravastatin 40 mg / kg BW and 50 mg / kg BW of saprolyrate was administered orally five times per week, and the amount of etanercept 5 mg / kg BW was administered three times per week But is not limited to.

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail with reference to the following examples. However, the following examples are intended to illustrate the contents of the present invention, but the scope of the present invention is not limited to the following examples. Embodiments of the present invention are provided to more fully describe the present invention to those skilled in the art.

The following experimental examples are intended to provide experimental examples that are commonly applied to the respective embodiments according to the present invention.

< Experimental Example >

One. Atherosclerosis

The male C57BL / 6J low density lipoprotein receptor knockout (LDLr KO) mouse (B6.129S7- Ldlr ^tm1Her / J) was obtained from Prof. Oh Taek Taek of Ewha Womans University (Seoul, Korea) Respectively. The mice were fed a standard laboratory diet without water restriction. LDLr KO mice (age, 60 weeks; n = 6 / group) were randomly divided into 5 groups. Four groups received high-fat, high-cholesterol diet (D12108; Research Diets Inc., New Brunswick, NJ, USA). The fifth group received standard laboratory diets. The HFD group received either peritoneal administration of etanercept alone (5 mg / kg body weight [BW], 3 times / week), saponylate (50 mg / kg BW) and pravastatin / kg BW) were administered orally five times per week, or the three drugs, sapoglylate, pravastatin and etanercept, were combined in the same manner as described above, whereas the control and vehicle groups received only 0.5% carboxymethyl cellulose cellulose (CMC; Sigma, St. Louis, Mo., USA) was administered with PBS (Welgene, Daejeon, Korea). Animal studies were approved by the Animal Experimental Ethics Committee at the Catholic University Sacred Heart Campus (2014-020-02).

2. Histochemical analysis and immunohistochemical analysis

After 12 weeks of diet, tissues and blood were collected from the mice. The heart and aorta were perfused with PBS under anesthesia. To measure atherosclerotic plaque, the thoracoabdominal aorta was fixed with 4% paraformaldehyde (Sigma). The heart containing the aortic root was obtained, washed and placed in paraformaldehyde at 4 ° C for 24 hours, embedded in a Tissue CellPath OCT matrix (CellPath Ltd., Newtown, UK) for histochemical analysis and immunohistochemistry . The atherosclerotic burden of the thoracic abdominal aorta was quantitated by en face Oil Red O-staining. The aorta was pinned to a black-silicon plate in a 60-mm dish at room temperature, rinsed twice with PBS, and dehydrated with propylene glycol. The dish was allowed to dry and the tissue was allowed to react for 2 hours at room temperature in Oil Red-O dissolved in propylene glycol (Sigma). The tissues were washed 3 to 4 times with 85% propylene glycol to stain, and each dish was rinsed with PBS. The aorta was photographed using a stereomicroscope (AxioCam, Carl Zeiss, Inc., Zena, Germany) equipped with a CCD-camera and immersed in PBS. Images were imaged using imaging software (National Institutes of Health, Bethesda, MD, USA) And the occupied surface area of Oil Red O-stained lesion was measured. A series of frozen sections (5 μm, CM1950, Leica) were prepared at a total of three cusps in the aortic root, and atherosclerotic lesions were analyzed at the three sites. To measure atherosclerotic plaque size, some of the sections were stained with Oil Red O. The remaining sections were used for immunohistochemical analysis of atherosclerotic plaque composition. The air-dried slices were fixed in ice-cold acetone and blocked with 10% bovine serum albumin (Bovogen Biologicals, Keilor East VIC, Australia) and incubated with intercellular adhesion molecule 1 (ICAM-1; (Santa Cruz Biotechnology, Santa Cruz, CA, USA) or anti-monocyte / macrophage-2 (MOMA-2; Abcam, Cambridge, MA, USA). The sections were reacted with appropriate HRP secondary antibodies and visualized according to the manufacturer's instructions using DAB kit (Dako, Carpentaria, CA, USA). All sections were contrasted with Harris hematoxylin (YD Diagnostics, Seoul, Korea). Morphological results were obtained using a slide scanner (APERIO CS2; Leica).

3. Serum lipid analysis and cytokine analysis

For histological analysis, blood was collected from the back of the eyes of each group of mice (n = 6 / group). Serum was separated and stored at -80 ° C. Total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol and low-density lipoprotein (LDL) cholesterol were measured by Hitachi 7020 (Hitachi, Tokyo, Japan). Levels of serum interleukin (IL) -6 (Max; BioLegend Inc., San Diego, CA, USA) and tumor necrosis factor (TNF) (Max; BioLegend Inc., San Diego, Calif. And measured according to the manufacturer's instructions using an enzyme-linked immunoassay (ELISA) kit.

< Example  1> Aging LDLr  From the KO mouse Atherosclerosis  Exhibit antagonistic effects on progress Pravastatin  And Sapphire grill  Double-dose combination administration and Pravasta Administration of tin-sapoglyrilate-etanercept triple combination

The combination of pravastatin and sapoglirate was effective in improving atherosclerosis in young mice, but its activity appeared to be incomplete considering the underlying complex mechanisms of atherosclerosis and atherosclerosis. Thus, the present inventors have confirmed that the addition of etanercept to pravastatin and saporoglurate prescription can improve the therapeutic effect of pravastatin and sapogirillate. As a result of Oil Red-O staining, it was found that the combination of pravastatin (40 mg / kg) and sapoglyrate (50 mg / kg) in combination with pravastatin-sapogyrylate-etanercept (20 mg / kg) Lt; RTI ID = 0.0 &gt; HFD-induced &lt; / RTI &gt; Although not statistically significant, plaques tended to decrease further in the etanercept-additional triple combination administration group. However, the etanercept alone treatment group did not show atherosclerotic plaque area reduction.

< Example  2> Aorta Vicinal  Plaque area reduction, monocyte / macrophage invasion, ICAM-1 expression and TNF  The anti- Atherosclerosis  Effect on effects

Next, we examined the plaque generation by performing Oil Red-O staining at the aortic root of LDLr KO mice, confirming ICAM-1 expression by specific antibodies and monocyte / macrophage infiltration through MOMA-2 expression Normal diet, HFD or HFD + medication. (FIG. 2A), ICAM-1 (FIG. 2B) and MOMA-2 (FIG. 2C) -stained plaque areas were significantly reduced in the triple combination administration group compared to the etanercept alone administration group or the double combination administration group . In addition, TNF levels were significantly reduced (Fig. 3A), but there was no difference in IL-6 (Fig. 3B) in the combination administration of pravastatin, sapogrilate and etanercept.

< Example  3> Pravastatin - Sapphire grill - Etanercept  Decreased total cholesterol and LDL cholesterol only in triple combination

After 12 weeks of HFD and drug administration in aged mice, the weight gain between the groups did not differ significantly (Table 1). After 12 weeks of drug administration, serum lipid levels were analyzed. Compared with the HFD control, total cholesterol and LDL levels were lowered only when pravastatin-sapogyrylate-etanercept triple combination was administered (Table 2). Compared with the control group, lipid levels were not significantly different in the combination group of pravastatin and sapogirillate in combination with etanercept alone. The mean change in total cholesterol in the triple combination group was -30% on average and the mean change in LDL level in the triple combination group was -33% on average.

weight NFD HFD HFD
+ ETA
+ 5MPK HFD
+ PRA 40 MPK
+ SAR 50 MPK HFD
+ PRA 40 MPK
+ SAR 50 MPK
+ ETA 5MPK 60 weeks 47.01 + - 3.742 47.13 + - 2.574 47.50 + - 3.315 47.11 ± 2.906 47.44 + 2.879 72 weeks 43.40 + - 4.477 49.10 ± 5.655 49.40 ± 3.928 50.84 + 4.999 45.94 + 4.555

Error bars represent SEM, and significance was analyzed by Student's t-test (N = 6-7 / group) compared to the HFD group.

NFD, normal fat diet; HFD, high fat diet; PRA, pravastatin; SAR, sapoglylate; ETA, etanercept.

Milligrams
/ deciliter NFD HFD HFD
+ ETA
+ 5MPK HFD
+ PRA 40 MPK
+ SAR 50 MPK HFD
+ PRA 40 MPK
+ SAR 50 MPK
+ ETA 5MPK Total cholesterol 474.0 + - 75.67 1989 ± 211.7 1740 + 172.9 1804 ± 136.5 1376 ± 90.79 * Triglyceride 229.0 + - 53.29 929.3 ± 146.9 858.5 ± 196.0 878.4 ± 126.3 879.6 ± 43.21 HDL cholesterol 70.00 + - 4.290 95.60 +/- 8.518 84.00 7.394 91.60 ± 6.013 100.4 ± 9.196 LDL cholesterol 171.2 ± 32.92 665.5 + 82.18 570.0 + - 55.83 553.6 ± 49.91 441.6 + - 39.56 *

The error bars represent SEM, and significance was analyzed by Student's t-test as compared to the HFD group (* P <0.05, N = 6-7 / group).

NFD, normal fat diet; HFD, high fat diet; PRA, pravastatin; SAR, sapoglylate; ETA, etanercept.

Claims (5)

A pharmaceutical composition for the prevention or treatment of aging atherosclerosis, which comprises sarpogrelate, pravastatin and etanercept as an active ingredient. delete delete The pharmaceutical composition of claim 1, wherein the pharmaceutical composition is selected from the group consisting of intercellular adhesion molecule 1 (ICAM-1), monocyte / macrophage-2 (MOMA-2) and tumor necrosis factor &Lt; / RTI &gt; TNF). &Lt; / RTI &gt; The pharmaceutical composition according to claim 1, wherein the pharmaceutical composition reduces total cholesterol and low-density lipoprotein (LDL) cholesterol levels.

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