KR20230129861A - Composition for preventing, treating or improving vascular calcification comprising MLN4924 - Google Patents

Abstract

The present invention provides a composition for preventing, treating or improving vascular calcification containing MLN4924 (CAS No. 951950-33-7), an inhibitor of E1 (NEDD8 activating enzyme; NAE) activation of Nedd8 (Neural precursor cell expressed developmentally downregulated protein 8). Regarding this, the composition exerts an effect of suppressing calcium accumulation and increased vascular calcification in a vascular calcification model, so it can be effectively used to prevent, treat, or improve vascular calcification.

Description

Composition for preventing, treating or improving vascular calcification comprising MLN4924 {Composition for preventing, treating or improving vascular calcification comprising MLN4924}

The present invention relates to a composition for preventing, treating or improving vascular calcification comprising MLN4924 (CAS No. 951950-33-7), and more specifically, to a Nedd8 (Neural precursor cell expressed developmentally downregulated protein 8) inhibitor or E1 of Nedd8. The present invention relates to a composition that blocks calcium accumulation or vascular calcification containing the NEDD8 activating enzyme (NAE) inhibitor MLN4924.

Vascular calcification refers to the deposition of minerals such as calcium phosphate in bones and soft tissues. Calcification in soft tissues such as blood vessels, kidneys, brain, heart, and lungs is referred to as ectopic calcification, and shows characteristics of a pathological disease differently from areas that are normally calcified, such as bones or teeth. Extensive vascular calcification, including coronary arteries, is commonly observed in patients with chronic kidney disease, and traditional risk factors such as hypertension, diabetes, dyslipidemia, aging, and smoking affect the occurrence of vascular calcification.

Vascular calcification is classified into two types: atherosclerotic calcification and medial artery calcification. In particular, in patients with chronic kidney disease, the concentration of calcium-phosphorus conjugates is increased due to decreased kidney function. If this phenomenon continues, vascular calcification occurs in the vascular smooth muscle cells that make up the medial artery.

Although several drugs such as phosphate binders, active vitamin D analogs, and calcium analogs have been developed to control vascular calcification, vascular calcification is still common in patients with chronic kidney disease. Angina caused by arteriosclerosis is a phenomenon in which cholesterol is deposited in the intima of coronary arteries and excessive proliferation of smooth muscle cells occurs, narrowing blood vessels. As these lesions persist for a long time, calcium is deposited in the area where arteriosclerosis has occurred, hardening the blood vessels and causing calcification.

Several factors and mechanisms may affect the pathogenesis of vascular calcification. Cardiovascular disease, which is associated with vascular calcification, is currently one of the diseases with a high mortality rate. Therefore, in order to treat vascular calcification more effectively, it is necessary to find new mechanisms involved in the occurrence of vascular calcification.

Accordingly, the present inventors inhibited vascular calcification by MLN4924 (CAS No. 951950-33-7), an inhibitor of E1 (NEDD8 activating enzyme; NAE) activation of Nedd8 (Neural precursor cell expressed developmentally downregulated protein 8), which acts in the mechanism causing vascular calcification. The effect was confirmed and the possibility of using this as a therapeutic substance was confirmed.

Accordingly, the purpose of the present invention is to provide a pharmaceutical composition for preventing or treating vascular calcification containing a Nedd8 inhibitor or an E1 inhibitor of Nedd8.

Another object of the present invention is to provide a food composition for preventing or improving vascular calcification containing a Nedd8 inhibitor or an E1 inhibitor of Nedd8.

Another object of the present invention relates to the use of a Nedd8 inhibitor or an E1 inhibitor of Nedd8 to prevent, treat or improve vascular calcification.

The present invention relates to a composition for preventing, treating or improving vascular calcification comprising MLN4924 (CAS No. 951950-33-7). According to the present invention, Nedd8 is expressed in a vascular calcification model induced by treating vascular smooth muscle cells with inorganic phosphate. When treated with MLN4924, an inhibitor of E1 (NEDD8 activating enzyme; NAE) activation of (Neural precursor cell expressed developmentally downregulated protein 8), calcium accumulation and increased vascular calcification can be suppressed.

The present inventors have continuously researched and made diligent efforts to diagnose diseases related to vascular calcification and develop compositions for prevention or treatment. As a result, it was confirmed that vascular calcification was inhibited by MLN4924, an E1 activation inhibitor of Nedd8, in a vascular calcification model induced by treating vascular smooth muscle cells with inorganic phosphate.

Hereinafter, the present invention will be described in more detail.

One aspect of the present invention is a pharmaceutical composition for preventing or treating vascular calcification comprising a Nedd8 inhibitor or an E1 inhibitor of Nedd8.

In the present invention, the Nedd8 inhibitor or the E1 inhibitor of Nedd8 may be MLN4924.

In the present invention, the Nedd8 inhibitor or the E1 inhibitor of Nedd8 may inhibit the activity of Nedd8 or the E1 activity of Nedd8 or the expression of the gene encoding it.

In the present invention, the vascular calcification may be medial artery calcification or atherosclerotic calcification, for example, medial artery calcification, but is not limited thereto.

In the present invention, the vascular calcification is from the group consisting of chronic kidney disease, arteriosclerosis, end-stage renal disease, hypertension, hyperlipidemia, and aging. It may be caused by one or more selected diseases, for example, it may be caused by chronic kidney disease, but is not limited thereto.

The pharmaceutical composition of the present invention can be used as a pharmaceutical composition containing a pharmaceutically effective amount of MLN4924 and/or a pharmaceutically acceptable carrier.

As used herein, the term “pharmaceutically effective amount” refers to an amount sufficient to achieve the efficacy or activity of the MLN4924 extract described above.

Pharmaceutically acceptable carriers included in the pharmaceutical composition of the present invention are those commonly used in preparation, and include lactose, dextrose, sucrose, sorbitol, mannitol, starch, gum acacia, calcium phosphate, alginate, gelatin, Includes, but is limited to, calcium silicate, microcrystalline cellulose, polyvinylpyrrolidone, cellulose, water, syrup, methyl cellulose, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate, and mineral oil. It doesn't work. In addition to the above ingredients, the pharmaceutical composition of the present invention may further include lubricants, wetting agents, sweeteners, flavoring agents, emulsifiers, suspending agents, preservatives, etc.

The pharmaceutical composition according to the present invention can be administered to mammals, including humans, through various routes. The administration method may be any commonly used method, for example, oral, dermal, intravenous, intramuscular, subcutaneous, etc.

The appropriate dosage of the pharmaceutical composition of the present invention varies depending on factors such as formulation method, administration method, patient's age, body weight, gender, pathological condition, food, administration time, administration route, excretion rate, and reaction sensitivity. Usually, a skilled physician can easily determine and prescribe an effective dosage for the desired treatment or prevention.

The pharmaceutical composition of the present invention is prepared in unit dosage form by formulating it using a pharmaceutically acceptable carrier and/or excipient according to a method that can be easily performed by a person skilled in the art. Alternatively, it can be manufactured by placing it in a multi-capacity container. At this time, the formulation may be in the form of a solution, suspension, or emulsion in an oil or aqueous medium, or may be in the form of an extract, powder, granule, tablet, capsule, or gel (e.g., hydrogel), and may additionally contain a dispersant or stabilizer. .

Another aspect of the present invention is a food composition for preventing or improving vascular calcification comprising a Nedd8 inhibitor or an E1 inhibitor of Nedd8.

In the present invention, the Nedd8 inhibitor or the E1 inhibitor of Nedd8 may be MLN4924.

In the present invention, the Nedd8 inhibitor or the E1 inhibitor of Nedd8 may inhibit the activity of Nedd8 or the E1 activity of Nedd8 or the expression of the gene encoding it.

In the present invention, the vascular calcification may be medial artery calcification or atherosclerotic calcification, for example, medial artery calcification, but is not limited thereto.

In the present invention, the vascular calcification is from the group consisting of chronic kidney disease, arteriosclerosis, end-stage renal disease, hypertension, hyperlipidemia, and aging. It may be caused by one or more selected diseases, for example, it may be caused by chronic kidney disease, but is not limited thereto.

When using the food composition of the present invention as a food additive, the food composition can be added as is or used together with other foods or food ingredients, and can be used appropriately according to conventional methods. In general, when producing a food or beverage, the food composition of the present invention may be added in an amount of 15% by weight or less, preferably 10% by weight or less, based on the raw materials.

There are no special restrictions on the types of foods above. Examples of foods to which the above substances can be added include meat, sausages, bread, chocolate, candies, snacks, confectionery, pizza, ramen, other noodles, gum, dairy products including ice cream, various soups, beverages, tea, drinks, etc. These include alcoholic beverages and vitamin complexes, and include all foods in the conventional sense.

The beverage may contain various flavors or natural carbohydrates as additional ingredients. The above-mentioned natural carbohydrates may include monosaccharides such as glucose and fructose, disaccharides such as maltose and sucrose, natural sweeteners such as dextrin and cyclodextrin, and synthetic sweeteners such as saccharin and aspartame. . The ratio of the natural carbohydrates can be appropriately determined by the selection of a person skilled in the art.

In addition to the above, the food composition of the present invention contains various nutrients, vitamins, electrolytes, flavors, colorants, pectic acid and its salts, alginic acid and its salts, organic acids, protective colloidal thickeners, pH adjusters, stabilizers, preservatives, glycerin, and alcohol. , may contain carbonating agents used in carbonated drinks, etc. In addition, the food composition of the present invention may contain pulp for the production of natural fruit juice, fruit juice drinks, and vegetable drinks. These ingredients can be used independently or in combination. The proportions of these additives can also be appropriately selected by those skilled in the art.

The present invention provides a composition for preventing, treating or improving vascular calcification containing MLN4924 (CAS No. 951950-33-7), an inhibitor of E1 (NEDD8 activating enzyme; NAE) activation of Nedd8 (Neural precursor cell expressed developmentally downregulated protein 8). Regarding this, the composition exerts an effect of suppressing calcium accumulation and increased vascular calcification in a vascular calcification model, so it can be effectively used to prevent, treat, or improve vascular calcification.

Figure 1 is a graph showing the change in calcium accumulation by treatment with different concentrations of MLN4924 (CAS No. 951950-33-7) in vascular smooth muscle cells treated with inorganic phosphate according to an embodiment of the present invention.
Figure 2 is a photograph of Western blot results confirming changes in the expression level of Runx2 protein, a vascular calcification marker, in vascular smooth muscle cells treated with inorganic phosphate according to an embodiment of the present invention.
Figure 3 is a photograph showing the results of confirming the degree of vascular calcification caused by MLN4924 concentration in vascular smooth muscle cells treated with inorganic phosphate according to an embodiment of the present invention using alizarin red S staining.

Hereinafter, the present invention will be described in more detail through the following examples. However, these examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Throughout this specification, “%” used to indicate the concentration of a specific substance means (weight/volume)% for solid/solid, (weight/volume)% for solid/liquid, and Liquid/liquid is (volume/volume)%.

Example 1: Cell culture and vascular calcification induction

The aorta was isolated from a 6-7 week old Sprague Dawley rat, cultured in Ham's F12 medium containing collagenase, vascular smooth muscle cells were isolated, and 10% fetal bovine serum (FBS) was added. ) was cultured using Dulbecco's modified Eagle's medium containing (Dulbecco's modified Eagle's medium) as a general medium.

Compared to the control group cultured with the general medium, the experimental group that induced vascular calcification was treated with 4 mM inorganic phosphate in the vascular smooth muscle cells in the general medium, and the medium was changed after 48 hours based on the time of first treatment with inorganic phosphate. Then, the cells were treated with inorganic phosphoric acid again, and after 72 hours, vascular smooth muscle cells were collected and prepared as an experimental group as a vascular calcification model.

Example 2: Confirmation of calcium accumulation in vascular calcification model

The vascular calcification model prepared according to the method in Example 1 was analyzed for changes in the degree of calcium deposition due to treatment with MLN4924 (CAS No. 951950-33-7).

Specifically, after seeding vascular smooth muscle cells in a 24-well plate, the experimental group was treated with 4 mM inorganic phosphate and 10 and 100 nM of MLN4924, respectively, according to the method of Example 1. In order to confirm the amount of calcium accumulated in the collected vascular smooth muscle cells, they were immersed in 0.6 N HCl and reacted under refrigerated conditions for 24 hours. The amount of calcium was measured using a calcium assay kit (QuantiChrom calcium assay kit), and the amount of protein was measured. It was measured using a protein analysis kit (BCA protein assay kit). Afterwards, the amount of calcium accumulation was corrected by the amount of protein.

As can be seen in Figure 1, the amount of calcium accumulated in vascular smooth muscle cells increased by inorganic phosphate treatment was decreased in a concentration-dependent manner by MLN4924 treatment.

Example 3: Confirmation of expression level of vascular calcification marker by MLN4924

With respect to the vascular calcification model prepared according to the method in Example 1, the degree to which protein expression of Runx2, a vascular calcification marker, was suppressed by MLN4924 treatment was analyzed through Western blot.

Specifically, vascular smooth muscle cells were treated with 4 mM inorganic phosphate and 1 uM of MLN4924, respectively, according to the method of Example 1, and the cells were harvested using trypsin-EDTA and then incubated in 1% NP-40 buffer (150 mM NaCl, 1 uM % NP-40, 50mM Tris-HCl (pH 8.0), 10mM NaF, 1mM Na _{3 VO4} , 1mM EDTA, 1mM DTT, 1mM PMSF, protease inhibitor) were added and sonicated and reacted for 30 minutes. . After centrifugation at 13,000 rpm for 15 minutes, the supernatant was recovered, and the protein was quantified using a kit (BCA protein assay kit).

30 ug of the quantified protein was taken and electrophoresed on SDS-PAGE, then transferred to polyvinylidene fluoride (PVDF membrane) and incubated with a primary antibody (α-Runx2 (1:1000, Abcam) specific to the protein to be detected. , α-b-Actin (1:1000, Santa Cruz)) was added and reacted under refrigerated conditions for one day. Afterwards, it was reacted with secondary antibody (anti-mouse IgG or anti-rabbit IgG, horseradish peroxidase (HRP)-linked antibody, 1:5000, Cell signaling) at room temperature for 1 hour and washed at least three times with 1xTBST buffer. Then, the detection amount of the target protein was confirmed using a standard substrate (Western Chemiluminescent HRP substrate).

As can be seen in Figure 2, the expression level of Runx2 increased in the inorganic phosphate treatment group (Pi) compared to the control group, and the expression level of Runx2 was confirmed to decrease again in the MLN4924 treatment group (Pi+MLN4924) compared to the inorganic phosphate treatment group. Therefore, MLN4924 was confirmed to be an inhibitor of Runx2. It was observed to have an effect of suppressing expression.

Example 4: Confirmation of vascular calcification inhibition effect by MLN4924

The effect of inhibiting vascular calcification by treatment with different concentrations of MLN4924 was confirmed for the vascular calcification model prepared in Example 1.

Specifically, after inoculating vascular smooth muscle cells in a 24-well plate, the experimental group was treated with 4 mM inorganic phosphate and 10 and 100 nM of MLN4924, respectively, according to the method of Example 1. Afterwards, the cells were fixed with 10% formalin at room temperature for 1 hour, the formalin was removed, washed three times with PBS, the cells were slightly dried, and then 40 mM alizarin red S (pH 4.2) dye, which stains calcium salts, was applied. was added and reacted for 20 minutes. After staining, the cells were washed with tertiary distilled water to remove non-specific dye staining the cells.

As can be seen in Figure 3, calcium salts stained red were observed in the inorganic phosphate treated group (Pi) compared to the control group, and the degree of red color decreased again in the MLN4924 treated group (Pi+MLN4924) compared to the inorganic phosphoric acid treated group. Since it was confirmed that MLN4924 was observed to have the effect of suppressing the progression of calcification.

Claims (10)

A pharmaceutical composition for preventing or treating vascular calcification, comprising a Nedd8 inhibitor or an E1 (NEDD8 activating enzyme; NAE) inhibitor of Nedd8. The pharmaceutical composition for preventing or treating vascular calcification according to claim 1, wherein the Nedd8 inhibitor or the E1 inhibitor of Nedd8 is MLN4924 (CAS No. 951950-33-7). The pharmaceutical composition for preventing or treating vascular calcification according to claim 1, wherein the Nedd8 inhibitor or the E1 inhibitor of Nedd8 inhibits the activity of Nedd8 or the E1 activity of Nedd8 or the expression of the gene encoding it. The pharmaceutical composition for preventing or treating vascular calcification according to claim 1, wherein the vascular calcification is medial artery calcification or atherosclerotic calcification. The method of claim 1, wherein the vascular calcification is caused by chronic kidney disease, arteriosclerosis, end-stage renal disease, hypertension, hyperlipidemia, and aging. A pharmaceutical composition for preventing or treating vascular calcification, which is caused by one or more diseases selected from the group. A food composition for preventing or improving vascular calcification comprising a Nedd8 inhibitor or an E1 (NEDD8 activating enzyme; NAE) inhibitor of Nedd8. The food composition for preventing or improving vascular calcification according to claim 6, wherein the Nedd8 inhibitor or the E1 inhibitor of Nedd8 is MLN4924 (CAS No. 951950-33-7). The food composition for preventing or improving vascular calcification according to claim 6, wherein the Nedd8 inhibitor or the E1 inhibitor of Nedd8 inhibits the activity of Nedd8 or the E1 activity of Nedd8 or the expression of the gene encoding it. The food composition for preventing or improving vascular calcification according to claim 6, wherein the vascular calcification is medial artery calcification or atherosclerotic calcification. The method of claim 6, wherein the vascular calcification consists of chronic kidney disease, arteriosclerosis, end-stage renal disease, hypertension, hyperlipidemia, and aging. A food composition for preventing or improving vascular calcification, which is caused by one or more diseases selected from the group.