KR101892577B1 - Composition for preventing or treating atherosclerosis comprising reblastatin derivation or pharmaceutical acceptable salts thereof - Google Patents

Abstract

The present invention relates to a composition for preventing or treating atherosclerosis and cardiovascular disease caused by the same comprising a reblastatin derivative or pharmaceutically acceptable salts thereof as active ingredients. The reblastatin derivative or pharmaceutically acceptable salts thereof inhibit secretion of CCL2 by 27-hydroxycholesterol of which its expression is increased by atherosclerosis, movement of monocytes, and the expression of related immune factors and thus has a merit of being variously used as a therapeutic agent for atherosclerosis, health functional food, a treatment supplement, and so on. Moreover, the composition has a function for preventing or treating various cardiovascular diseases which may be caused by a worsening of atherosclerosis and thus has a merit of being used as a therapeutic agent, health functional food, and so on against complications, in particular, ischemic heart disease, stroke, renal failure and ischemic limb disease, caused by atherosclerosis.

Description

TECHNICAL FIELD The present invention relates to a composition for preventing or treating atherosclerosis comprising a levulastatin derivative or a pharmaceutically acceptable salt thereof as an active ingredient,

The present invention relates to a composition for preventing or treating atherosclerosis and a complication due to atherosclerosis, which comprises a levulastatin derivative or a pharmaceutically acceptable salt thereof.

Atherosclerosis is a concept that includes both atherosclerosis and atherosclerosis. It is known as a disease with a high mortality rate globally, and it is a dangerous disease which is the second or third cause of death in Korea.

Low-density lipoprotein cholesterol (LDL) in the blood accumulates and oxidizes in the blood vessel walls, and cholesterol, a component of LDL, is also oxidized in the blood vessels. Oxidized cholesterol causes inflammation, which causes the mononuclear cells to migrate to the vessel wall. Monocytes are then activated in the blood vessel walls by cholesterol oxides, which further amplify inflammation. Through such a series of processes, the monocyte is continuously moved to the inner wall of the blood vessel, resulting in atherosclerosis.

As can be seen from the above, since the movement of monocytes plays a pivotal role in the development and development of atherosclerosis, there is an increasing interest in a method for treating atherosclerosis by inhibiting monocyte migration to the inner wall of blood vessels. In particular, 27-hydroxycholesterol (27-hydroxycholesterol), one of the cholesterol oxides, is the most abundant cholesterol oxide in the atherosclerotic plaque, promoting the migration of inflammatory cells (especially monocytes, T cells) and stimulating macrophages It is known to induce cell death by inducing stress.

On the other hand, in the area where atherosclerosis occurs, a marker such as CD137, CD105, and CD166 is expressed, thereby promoting atherosclerosis. Therefore, these factors are used as a biomarker for confirming the expression of atherosclerosis. In particular, CD137 (4-1BB) is a biomarker that is expressed in a large amount in monocytes that are present in atherosclerosis site, and CD105 (endoglin) is involved in destabilization of monocytes in a site where atherosclerosis occurs as a TGF- Is a known biomarker, and CD166 (Alcam) is a biomarker known to be specifically expressed in atherosclerotic tissue. In particular, a decrease in CD137 in an atherosclerotic animal model attenuates atherosclerosis, and activation of CD137 increases inflammation of the blood vessels. Thus, CD137 is one of the targets for the treatment of atherosclerosis.

Therefore, the inventors of the present invention have studied a compound specifically inhibiting the activity of 27-hydroxycholesterol and thus the monocyte migration, which is important for atherosclerosis, and found that the levulastatin derivative of the present invention has a remarkable activity of 27-hydroxycholesterol Inhibitory effect, and completed the present invention.

KR Patent Publication No. 10-2015-0013520 KR Patent Registration No. 10-1672708

1. Ross R. Atherosclerosis-an inflammatory disease. New Engl J Med. 1999: 340 (2): 115-26. 2. Poli G, Biasi F, Leonarduzzi G. Oxysterols in the pathogenesis of major chronic diseases. Redox Biol. 2013; 1 (1): 125-30. 3. Andersson J, Libby P, Hansson GK. Adaptive immunity and atherosclerosis. Clin Immunol. 2010; 134 (1): 33-46. 4. De Meyer I, Martinet W, De Meyer GR. Therapeutic strategies to deplete macrophages in atherosclerotic plaques. Br J Clin Pharmacol. 2012; 74 (2): 246-63. 5. Kwon BS, Oh GT, HJ1, Choi JH, Jung IH, Park JG, Lee MR, Lee MN, Kim B, Yoo JY, Jeong SJ, Kim DY, Park JH, Park HY, Kwack K, Choi BK. CD137 (4-1BB) deficiency reduces atherosclerosis in hyperlipidemic mice. Circulation. 2010; 121 (9): 1124-33. 6. Piao M and Tokunaga O. Significant expression of endoglin (CD105), TGF-1 and TGF R-2 in the atherosclerotic aorta: an immunohistological study. J Atheroscler Thromb. 2006; 13 (2): 8289. 7. Brown AJ, Jessup W, Oxysterols and atherosclerosis, Atherosclerosis, 1999; 142 (1): 1-28. 8. Kim, SM, Lee, S., Kim, K., Bae, SS, Eo SK, Kim, K. 27-Hydroxycholesterol induces recruitment of monocytic cells by enhancing CCL2 production. Biochem Biophys Res Commun. 2013; 442 (3-4): 159-64. 9. Umetani M, Ghosh P, Ishikawa T, Umetani J, Ahmed M, Mineo C, Shaul PW. The cholesterol metabolite 27-hydroxycholesterol promotes atherosclerosis via proinflammatory processes mediated by estrogen receptor alpha. Cell Metab. 2014; 20 (1): 172-82. 10. Son Y, Kim SM, Lee SA, Eo SK, Kim K. Oxysterols induce transition of monocytic cells to phenotypically mature dendritic cell-like cells. Biochem Biophys Res Commun. 2013; 438 (1): 161-8. 11. Shin, JC, Zhu N, Lee D, Kim W, Lee K, Shen YM, Paik SG, Hong YS, Lee JJ. Characterization of tailoring genes involved in the modification of geldanamycin polyketide in Streptomyces hygroscopicus JCM4427 J. Microbiol. Biotechnol. 2008; 18 (6), 1101-8. 12. Kim, W., Lee, K., Hong, Y., Hong, Y., Hong, SS, Rational Biosynthetic Engineering for Optimization of Geldanamycin Analogues. ChemBioChem. 2009; 10 (7) 1243-51. Lee, JS, Paik, SG, Lee, JJ, Hong, YS, Lee, Mutasynthesis of Geldanamycin via the Disruption of a Gene Producing Starter Unit: Generation of Structural Diversity at the Benzoquinone Ring. ChemBioChem. 2007; 8 (13) 1491-4.

An object of the present invention is to provide a composition and a therapeutic aid for the prevention or treatment of atherosclerosis, which comprises a levulastatin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

Still another object of the present invention is to provide a composition for preventing or treating complications due to atherosclerosis, which comprises a levulastatin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

here,

R < ₁ > is H or OH,

R ₂ is H or OH,

R ₃ is a single bond or a double bond.

In order to achieve the above object, the present invention provides a pharmaceutical composition for preventing or treating atherosclerosis, comprising a levulastatin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

The present invention also provides a health functional food composition for preventing or ameliorating atherosclerosis, comprising a levulastatin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

The present invention also provides a therapeutic agent for atherosclerosis comprising a levulastatin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

The present invention also provides a pharmaceutical composition for preventing or treating complications due to atherosclerosis, comprising a levulastatin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

The present invention also provides a health functional food composition for preventing or ameliorating a complication caused by atherosclerosis, which comprises a levulastatin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

here,

R < ₁ > is H or OH,

R ₂ is H or OH,

R ₃ is a single bond or a double bond.

The levulastatin derivative of the present invention or an acceptable salt thereof inhibits the secretion of CCL2 by 27-hydroxycholesterol, which increases expression by atherosclerosis, and the expression of monocyte migration and related immune factors, A health functional food, a therapeutic aid, and the like. Furthermore, it has a function of preventing or treating various complications due to atherosclerosis which may be caused by deepening of atherosclerosis, and it is a therapeutic agent for a complication caused by atherosclerosis, in particular ischemic heart disease, stroke, renal failure and ischemic limb disease It can be used as a functional food for health and the like.

FIG. 1 is a graph showing the results of confirming the inhibitory effect of CCL2 by the levulastatin derivative of the present invention through ELISA.
FIG. 2 is a graph showing the effect of inhibiting monocyte migration by the levulastatin derivative of the present invention through analysis of transwell cell migration. FIG.
FIG. 3 is a graph showing the results of confirming changes in expression of an atherosclerosis-related immune factor by the levulastatin derivative of the present invention through flow cytometry. Specifically, FIG. 3A shows the expression of CD105, FIG. 3B shows the expression of CD137, and FIG. 3C shows the expression of CD166.

The present invention provides a composition for preventing or treating atherosclerosis and atherosclerotic complications, comprising a reblastatin derivative or a pharmaceutically acceptable salt thereof.

Hereinafter, the present invention will be described in detail.

For example, the present invention provides a pharmaceutical composition for preventing or treating atherosclerosis, comprising a levulastatin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

In the present invention, the levulastatin derivative is characterized in that hydrogen or a hydroxyl group is bonded instead of a methoxyl group bonded to carbon number 17 of levulastatin, which is a water-soluble substance, Is structurally different from other ansamycin antibiotics. Especially, there is a chemical structural difference with levulastatin, and it is characterized by exhibiting different activities in vivo. More specifically, the levulastatin derivative may be any one of the following formulas (2) to (5), but is not limited thereto.

[Chemical Formula 1]

here,

R < ₁ > is H or OH,

R ₂ is H or OH,

R ₃ is a single bond or a double bond.

(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

In the present invention, " prevention " means any action that inhibits atherosclerosis by administration of the composition or delays the onset of atherosclerosis.

In the present invention, 'improvement' refers to any action that improves or alleviates symptoms due to atherosclerosis by administration of the composition.

In the present invention, " treatment " means all of the actions of atherosclerosis by administration of the composition.

In the present invention, 'atherosclerosis' is a disease in which cholesterol deposition occurs in the innermost part of a blood vessel, and at the same time, vascular endothelial cell proliferation occurs and blood vessels become narrowed or clogged, causing blood vessels to disturb blood flow to the peripheral blood. Sclerosis or atherosclerosis.

On the other hand, 27-hydroxycholesterol is found in high concentrations in tissues in which atherosclerosis has developed. The 27-hydroxycholesterol increases the secretion of CCL2, thereby increasing the migration of monocytes, Lt; / RTI > Also, 27-hydroxycholesterol increases the expression of CD137. Therefore, the prevention, amelioration or therapeutic effect of atherosclerosis can be confirmed by integrating the experimental results obtained by analyzing the expression changes of the factors and the movement of mononuclear cells. Correlation between the above contents and atherosclerosis can be confirmed more clearly in [Non-Patent Document] of the above-mentioned [Prior Art Document].

In the present invention, the composition comprising the levulastatin derivative is characterized by inhibiting the activity of 27-hydroxycholesterol, which is closely related to atherosclerosis, to prevent, ameliorate or treat atherosclerosis.

In the present invention, the composition may further comprise a pharmaceutically acceptable additive. Examples of the pharmaceutically acceptable additives include starch, gelatinized starch, microcrystalline cellulose, lactose, povidone, colloidal silicon dioxide, hydrogen phosphate Calcium carbonate, lactose, mannitol, sugar, arabic gum, pregelatinized starch, corn starch, powdered cellulose, hydroxypropylcellulose, opaques, starch glycolate sodium, carnauba wax, synthetic aluminum silicate, stearic acid, magnesium stearate, Calcium stearate, white sugar, etc. may be used. The pharmaceutically acceptable additives according to the present invention are preferably included in the composition in an amount of 0.1 to 90 parts by weight, but not limited thereto.

In the present invention, the composition may be administered orally or parenterally in various clinical formulations. In the case of formulation, a diluent such as a filler, an extender, a binder, a wetting agent, a disintegrant, , And those suitable for use in the art are disclosed in Remington ' s Pharmaceutical Sciences (Mack Publishing Company, Easton PA). Examples of carriers, excipients and diluents that can be contained in the composition include lactose, dextrose, sucrose, oligosaccharide, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, mineral oil and the like.

The solid preparations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, sucrose, Lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate talc are also used. Examples of the liquid preparation for oral administration include suspensions, solutions, emulsions and syrups. In addition to water and liquid paraffin which are commonly used simple diluents, various excipients such as wetting agents, sweeteners, fragrances, preservatives and the like May be included.

The preparation for parenteral administration includes sterilized aqueous solutions, non-aqueous solvents, suspensions, emulsions, freeze-dried preparations, and suppositories. Propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like can be used as the non-aqueous solvent and suspension agent. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like. The parenteral administration can be carried out using an external or intraperitoneal injection, intramuscular injection, subcutaneous injection, intravenous injection, intramuscular injection or intra-thoracic injection.

The composition of the present invention may be administered in a pharmaceutically effective amount.

The term " pharmaceutically effective amount " means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and an effective dosage level will vary depending on the species and severity, age, sex, Activity, sensitivity to the drug, time of administration, route of administration and rate of release, duration of treatment, factors including co-administered drugs, and other factors well known in the medical arts. The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents. And can be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without adverse effect, and can be easily determined by those skilled in the art.

The term " administration " as used in the present invention means to provide a certain composition of the present invention to an individual by any suitable method.

In the present invention, the composition may be administered to a subject by various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine dural or intracerebral injection.

In the present invention, the composition can be used alone or in combination with methods using surgery, radiation therapy, hormone therapy, chemotherapy, and biological response modifiers for the prevention or treatment of atherosclerosis.

As another example, the present invention provides a health functional food composition for preventing or ameliorating atherosclerosis, comprising a levulastatin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

here,

R < ₁ > is H or OH,

R ₂ is H or OH,

R ₃ is a single bond or a double bond.

In the present invention, the term 'health functional food' refers to a food group imparted with added value to function or express the function of the food by using physical, biochemical, biotechnological techniques or the like, or a bioactive rhythm control , Which is designed to express the body's controlling function sufficiently for the prevention and restoration of disease, and should be harmless to the human body when taken over a long period of time.

The health functional food may include food-acceptable food supplementary additives, and may further include suitable carriers, excipients and diluents conventionally used in the production of health functional foods.

When the health functional food composition of the present invention is used as a food additive, the composition may be added as it is, or may be used together with other food or food ingredients, and may be suitably used according to a conventional method. The amount of the active ingredient to be mixed can be suitably determined according to the intended use (prevention, health or therapeutic treatment). In general, the composition of the present invention is added in an amount of not more than 15% by weight, preferably not more than 10% by weight based on the raw material, in the production of food or beverage. However, in the case of long-term intake for the purpose of health and hygiene or for the purpose of controlling health, the amount may be less than the above range, and since there is no problem in terms of safety, the active ingredient may be used in an amount exceeding the above range.

There is no particular limitation on the kind of the health functional food. Examples of health functional foods to which the above substances can be added include dairy products including ice cream, various soups, drinks, tea, drinks, alcoholic beverages and vitamin complexes. And foodstuffs designed to fully express in vivo the function of controlling the body in terms of regulation of bio-defense rhythm of food composition, prevention and recovery of disease, and the like.

In addition to the above, the health functional food composition of the present invention may contain various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and salts thereof, alginic acid and salts thereof, organic acids, protective colloid thickening agents, pH adjusting agents, stabilizers, , Alcohols, carbonating agents used in carbonated drinks, and the like. In addition, the food composition of the present invention may comprise flesh for the production of natural fruit juices, fruit juice drinks and vegetable drinks. These components may be used independently or in combination. Although the ratio of such additives is not critical, it is generally selected in the range of 0.01 to 0.1 parts by weight per 100 parts by weight of the composition of the present invention.

The present invention also provides a therapeutic agent for atherosclerosis comprising a levulastatin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

In the present invention, the term 'atherosclerosis therapy adjuvant' refers to a drug that can be used as an adjuvant to enhance the effect of the atherosclerosis treatment method commonly used in the art. By using the levulastatin derivative according to the present invention The effect of atherosclerosis therapy used together can be improved.

In addition, the above-mentioned treatment for atherosclerosis can be selected under a general rule to be considered when selecting a treatment for atherosclerosis, such as the hardness of symptoms, the rate of absorption (medication period and drug administration route) to a drug, and the location of occurrence of atherosclerosis.

As another example, the present invention provides a pharmaceutical composition for preventing or treating complications caused by atherosclerosis, comprising a levulastatin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof:

The present invention also provides a health functional food composition for preventing or ameliorating a complication caused by atherosclerosis, which comprises a levulastatin derivative represented by the following formula (1) or a pharmaceutically acceptable salt thereof.

[Chemical Formula 1]

here,

R < ₁ > is H or OH,

R ₂ is H or OH,

R ₃ is a single bond or a double bond.

In the present invention, the atherosclerotic complication refers to a disease caused by atherosclerosis of the atherosclerotic artery resulting in narrowing or clogging of blood vessels and peripheral blood circulation disorder of the blood vessels. For example, when the coronary artery of the heart is clogged, ischemic heart disease such as angina pectoris and myocardial infarction occurs, and if the cerebral artery or carotid artery is blocked, stroke such as cerebral infarction, cerebral hemorrhage, and obstruction of the renal artery and kidney peripheral blood vessels may result in renal failure and ischemic limb disease . Therefore, the atherosclerotic complication of the present invention may be ischemic heart disease, stroke, kidney failure or ischemic limb disease, but it is not limited thereto and may include all of the vascular-related diseases that may be caused by aggravation of atherosclerosis.

The redundant contents are taken into consideration in the complexity of the present specification, and terms not otherwise defined herein have the meanings commonly used in the art to which the present invention belongs.

EXAMPLES Hereinafter, examples, experimental examples and production examples will be described in detail to facilitate understanding of the present invention. However, the following Examples, Experimental Examples and Preparation Examples are intended only to illustrate the contents of the present invention, and the scope of the present invention is not limited to the following Examples, Experimental Examples and Production Examples. The embodiments, examples, and examples of the present invention are provided to enable those skilled in the art to more fully understand the present invention.

Example 1 Synthesis of a Rebrastatin Derivative

1-1. Synthesis of levulastatin derivatives of formulas 2 and 6

The levulastatin derivatives of Formulas 2 and 6 in the following Table 1 were prepared using Streptomyces hygroscopicus AC11 and Streptomyces hygroscopicus AC15, respectively. This is disclosed in detail in [Non-Patent Documents] Nos. 11 and 12 among the above-mentioned [Prior Art Documents].

In brief, each of the AC11 strain and the AC15 strain was cultured in a yeast extract-malt extract medium at 28 占 폚 for 7 days in order to obtain an extract obtained in the form of a culture. Then, the extract was fractionated using silica gel chromatography using CHCl ₃ -MeOH as a mobile phase. As a result, the compounds of Formulas 2 and 6 were finally obtained.

The obtained fractions were analyzed by HPLC (YMC J'sphere ODS-H80, 150 x 4.6 mm id, MeOH-H2O (0.05% acetic acid) gradient, 1 ml / min) using a Waters Delta Prep 3000 system And the structure thereof was finally analyzed. The results are shown in Table 1 below.

1-1. Synthesis of levulastatin derivatives of formulas 3 to 5

To re Blossom statin derivatives shown in Table 1 in the formula is 3 to 5 Actinomycetes Streptomyces high-gloss nose kusu AC2 (Streptomyces hygriscopicus AC2) strain. This is disclosed in detail in [Non-Patent Document] 13 of the above-mentioned prior art documents. On the other hand, the AC2 strain does not produce 3-amino-5-hydroxylbenzoic acid (AHBA).

Briefly, the AC2 strain was first cultured in 100 ml actinomycetes cultured YEME medium at 28 ° C for 3 days. After that, the culture medium of the medium was transferred to 2 L of YEME medium and further cultured at 28 ° C for 1 day. After that, 700 mg of 3-aminobenzoic acid was added, followed by further cultivation at 28 ° C for 7 days Respectively. Then, the culture medium was repeatedly extracted twice with ethyl acetate (EtOAc), and then the extract was filtered to remove insolubles. The filtered extract was then concentrated and the concentrate was partitioned between EtOAc and H ₂ O to give an organic phase extract. Next, the organic phase extract was fractionated using silica gel chromatography using CHCl ₃ -MeOH as a mobile phase, and the resulting compound was finally obtained as a compound of Formulas 3 to 5.

The obtained fractions were analyzed by HPLC (YMC J'sphere ODS-H80, 150 x 4.6 mm id, MeOH-H2O (0.05% acetic acid) gradient, 1 ml / min) using a Waters Delta Prep 30 00 system And the structure thereof was finally analyzed. The results are shown in Table 1 below.

name constitutional formula Levulastatin

(2) 17-dimethoxyl-levulastatin

(3) 18-dihydroxy-17-dimethoxyl-levulastatin

Formula 4 18-dihydroxy-17-O-dimethyl-levulastatin

Formula 5 Diene-18-dihydroxy-17-O-dimethyl-levulastatin

6 15-hydroxy-17-dimethoxyl-levulastatin

The compounds of the formulas (2) to (5), which were found to be effective for the prevention or treatment of excellent atherosclerosis through inhibition of the activity of 27-hydroxycholesterol through the following Experimental Examples 1 to 3, Respectively.

Experimental Example  1. Through enzyme-linked immunosorbent assay (ELISA) Levulastatin  Identification of inhibitory effects of CCL2 (chemokine (C-C motif) ligand 2) on derivatives

An experiment was conducted to confirm the inhibitory effect of the levulastatin derivative of the present invention on atherosclerosis-suppressing effect of CCL2, which promotes the migration of monocytes, using ELISA. THP-1 monocyte cells (ATCC ^ⓡ TIB-202 ^TM ) used in the present invention were purchased from ATCC (American Type Culture Collection), and THP-1 cells were cultured according to the provided manual. The following experiments were carried out using BD OptEIA ™ Hunan MCP-1 ELISA Kit (Cat # 559017) from BD Biosciences.

Specifically, in this experiment, THP-1 cells were divided into 7 experimental groups and 27-hydroxycholesterol (27OH, manufactured by Santa Cruz Biotechnology) and 1 μg / ml of the compounds of Formulas 2 to 6 One was treated together and then cultured. In addition, one test group was cultured without any treatment as a negative control group, and the other test group was treated with only 27-hydroxycholesterol as a positive control group and then cultured. Cultivation was performed for 48 hours after drug treatment. After that, the culture medium and cells were separated by centrifugation. 100 μL of the separated culture medium was put into a microplate coated with CCL2 antibody as a primary antibody and cultured for 2 hours. Subsequently, the medium was removed and CCL2 bound to a microplate was incubated with an antibody with a secondary antibody, horseradish peroxidase, for 1 hour. The primary and secondary antibodies used were the antibodies provided in the ELISA kit.

Then, the antibody was removed, and a series of steps of treating the substrate solution provided in the kit used in the present experiment was performed, and then the measurement was performed using a microplate reader. The measured absorbance was then substituted into the standard curve provided in the kit used for the experiment to calculate the CCL2 concentration. The results are shown graphically in FIG. (P <0.001; ***, P <0.001;###)

As shown in Fig. 1, in the positive control group, it was confirmed that the secretion amount of CCL2 in the macrophage was remarkably increased by the treatment with 27-hydroxycholesterol. When the compounds of the formulas (2) to (5) were subsequently treated, it was confirmed that the amount of CCL2 secretion was reduced to a level similar to that of the negative control. However, when the compound of Chemical Formula 6 was treated, it was confirmed that the level of CCL2 secretion was not decreased unlike the case of treating the compounds of Chemical Formula 2 to 5, and maintained at a level similar to that of positive control CCL2. This shows that the levulastatin derivative of the present invention specifically inhibits CCL2 secretion in macrophages by the activity of 27-hydroxycholesterol.

Experimental Example  2. Transwell  Cell migration analysis ( Transwell  cell migration assay Levulastatin  Identification of inhibitory effect of derivatives on monocyte migration

Experiments were carried out to confirm the effect of the levulastatin derivative of the present invention on the inhibition of atherosclerosis, in particular the inhibition of the migration of monocytes, through a transwell permeable support (Costar). THP-1 monocyte cells (ATCC ^ⓡ TIB-202 ^TM ) used in the present invention were purchased from ATCC (American Type Culture Collection), and THP-1 cells were cultured according to the provided manual.

Specifically, in this experiment, THP-1 cells were divided into 7 experimental groups and 27-hydroxycholesterol (27OH, manufactured by Santa Cruz Biotechnology) and 1 μg / ml of the compounds of Formulas 2 to 6 One was treated together and then cultured. In addition, one test group was cultured without any treatment as a negative control group, and the other test group was treated with only 27-hydroxycholesterol as a positive control group and then cultured. Cultivation was performed for 48 hours after drug treatment. After that, the medium and cells were separated by centrifugation, and the separated medium was dispensed into the lower chamber. On the other hand, 5 × 10 ⁵ THP-1 cells contained in 100 μl of BSA (0.1%) were dispensed into the upper chamber of the transwell. After incubation for 3 hours in a CO2 incubator, the upper chamber was removed. The number of cells migrated in the separated chamber was measured using a Vi-Cell cell counter. The results are shown in Fig. (P &lt;0.001; ***, P &lt;0.001;###)

As shown in FIG. 2, it was confirmed that the migration of mononuclear cells was significantly increased by treatment with 27-hydroxycholesterol in the positive control group. It was confirmed that the treatment of the compounds of the formulas (2) to (5) thereafter markedly decreased the migration of mononuclear cells and decreased to a level similar to or lower than that of the negative control group. However, when the compound of Chemical Formula 6 was treated, the mobility of monocytes was not decreased and the mobility of monocytes was maintained at a level similar to that of the positive control, unlike the case of treating the compounds of Chemical Formulas 2 to 5. This indicates that the levulastatin derivative of the present invention specifically inhibits mononuclear migration by the activity of 27-hydroxycholesterol.

Experimental Example  3. Flow cell  Analyzer Levulastatin  Derivative Atherosclerosis  relation Immune factor  Analysis of expression changes

Using FACS (Fluorescence activated cell sorter) as a flow cytometry analyzer, the effect of the levulastatin derivative of the present invention on the inhibition of atherosclerosis, in particular, the expression of CD105, CD137 and CD166, which are related immune factors expressed by atherosclerosis Experiments were performed. THP-1 monocyte cells (ATCC ^ⓡ TIB-202 ^TM ) used in the present invention were purchased from ATCC (American Type Culture Collection), and THP-1 cells were cultured according to the provided manual.

Specifically, in this experiment, THP-1 cells were divided into 7 experimental groups and 27-hydroxycholesterol (27OH, manufactured by Santa Cruz Biotechnology) and 1 μg / ml of the compounds of Formulas 2 to 6 After treatment with either one, the cells were cultured. In addition, one test group was cultured without any treatment as a negative control group, and the other test group was treated with only 27-hydroxycholesterol as a positive control group and then cultured. Cultivation was performed for 48 hours after drug treatment. The cultured cells were then separated and washed with PBS. The washed cells were suspended in the primary antibody solution for CD105, CD137 and CD166, and left as such for 2 hours under the temperature condition of 4 占 폚. After washing the cells with PBS, the cells were suspended in 500 μl of paraformalin (1%). Then, each cell was analyzed with a flow cytometer (FACS canto II, BD). Results obtained for CD105 are shown in Fig. 3A, CD137 is shown in Fig. 3B, and CD166 is shown in Fig. 3C. On the other hand, cat #: SC-20532, cat #: SC-367393 and cat #: SC-25624 purchased from Santa Cruz Biotechnology were used as primary antibodies against CD105, CD137 and CD166.

As shown in FIG. 3, it was confirmed that the expression of CD105, CD137 and CD166 was increased by the treatment with 27-hydroxycholesterol in the positive control group, and it was confirmed that the expression was increased more than 8 times. The expression of CD105, CD137 and CD166 was markedly decreased in the case of treatment with the compounds of the formulas (2) to (5) and then decreased to a level similar to that of the negative control. However, when the compound of Chemical Formula 6 was treated, it was confirmed that the expression of CD105, CD137, and CD166 was still maintained at a high level as in the positive control, unlike the case of treating the compounds of Chemical Formulas 2 to 5. This shows that the levulastatin derivative of the present invention specifically inhibits the expression of CD105, CD137 and CD166 by the activity of 27-hydroxycholesterol.

In summary, the levulastatin derivatives or acceptable salts thereof of the present invention inhibit the secretion of CCL2, the migration of mononuclear cells and the expression of related immune factors by 27-hydroxycholesterol, which is increased in expression by atherosclerosis, A therapeutic agent for atherosclerosis, a health functional food, a therapeutic aid, and the like. Furthermore, it has a function of preventing or treating various complications due to atherosclerosis which may be caused by deepening of atherosclerosis, and it is a therapeutic agent for a complication caused by atherosclerosis, in particular ischemic heart disease, stroke, renal failure and ischemic limb disease It can be used as a functional food for health and the like.

Hereinafter, formulation examples of the pharmaceutical composition including the levulastatin derivative of the present invention or an acceptable salt thereof will be described, but the present invention is not intended to be limited thereto but is specifically described.

Formulation Example 1. Preparation of pharmaceutical preparations

1. Manufacturing of powder

The levulastatin derivative or its acceptable salt 20 mg

Lactose 100 mg

Talc 10 mg

The above components can be mixed and filled in an airtight container to prepare powders.

2. Preparation of tablets

The levulastatin derivative or its acceptable salt 10 mg

Corn starch 100 mg

Lactose 100 mg

Magnesium stearate 2 mg

After mixing the above components, tablets may be prepared by tableting according to a conventional method for producing tablets.

3. Preparation of capsules

The levulastatin derivative or its acceptable salt 10 mg

Crystalline cellulose 3 mg

Lactose 14.8 mg

Magnesium stearate 0.2 mg

The above components may be mixed and filled in a gelatin capsule according to a conventional method for preparing a capsule, thereby preparing a capsule.

4. Preparation of injections

The levulastatin derivative or its acceptable salt 10 mg

180 mg mannitol

Sterile sterilized water for injection 2974 mg

Na ₂ HPO ₄ 2H ₂ O 26 mg

(2 ml) per 1 ampoule according to the usual injection preparation method.

5. Manufacture of liquids

The levulastatin derivative or its acceptable salt 20 mg

10 g per isomer

5 g mannitol

Purified water quantity

Each component is added to and dissolved in purified water according to a conventional liquid preparation method, and the lemon flavor is added in an appropriate amount, then the above components are mixed, and then purified water is added. The total amount of the liquid agent added to the purified water is adjusted to 100 ml, and the liquid agent can be finally prepared by filling it in a brown bottle and sterilizing it.

Claims (9)

delete A pharmaceutical composition for preventing or treating atherosclerosis comprising a compound represented by any one of the following formulas (2) to (5):
(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

. 3. The method of claim 2,
Wherein said atherosclerosis is atherosclerosis or arteriosclerosis. 2. A pharmaceutical composition for the prevention or treatment of atherosclerosis according to claim 1, wherein said atherosclerosis is atherosclerosis or atherosclerosis.
3. The method of claim 2,
The pharmaceutical composition for preventing or treating atherosclerosis, wherein the composition inhibits the activity of 27-hydroxycholesterol.
A health functional food composition for preventing or ameliorating atherosclerosis, comprising a levulastatin derivative represented by any one of the following formulas (2) to (5) or a pharmaceutically acceptable salt thereof:
(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

.
A therapeutic agent for atherosclerosis comprising a levulastatin derivative represented by any one of the following formulas (2) to (5) or a pharmaceutically acceptable salt thereof:
(2)

(3)

[Chemical Formula 4]

[Chemical Formula 5]

.

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