KR20160087037A

KR20160087037A - Pharmaceutical composition and vascular calcification therapeutic drug containing for preventing vascular calcification

Info

Publication number: KR20160087037A
Application number: KR1020150004261A
Authority: KR
Inventors: 국현; 권덕화; 엄광현
Original assignee: 전남대학교산학협력단
Priority date: 2015-01-12
Filing date: 2015-01-12
Publication date: 2016-07-21

Abstract

The present invention relates to a pharmaceutical preparation for preventing or treating vascular calcification, which contains as the active ingredient, RG7112 ([(4S,5R)-2-(4-tert-butyl-2-ethoxyphenyl)-4,5-bis(4-chlorophenyl)-4,5-dimethylimidazol-1-yl]-[4-(3-methylsulphonylpropyl)pyperazin-1-yl]methanone), which is a compound that suppresses MDM2 (Mouse double minute 2 homolog). The compound contained in the preparation of the present invention can suppress the breakdown of HDAC1 by inhibiting the binding of MDM2 and HDAC1 (histone deacetylase 1). The compound of the present invention, RG7112, shows the first successful result among treatments targeting MDM2 to treat diseases related to vascular calcification. According to the present invention, the preparation for inhibiting vascular calcification that contains MDM2 inhibitory material as the active ingredient, can contribute to the treatment of vascular calcification by reducing the expression of genes associated with vascular calcification and inhibiting the action of enzymes that control the generation of vascular calcification.

Description

[0001] The present invention relates to a composition for preventing calcification of blood vessels and a therapeutic agent for vascular calcification comprising the same,

The present invention relates to a composition for preventing calcification of blood vessels and a therapeutic agent for vascular calcification comprising the same.

Vascular calcification refers to the deposition of minerals such as calcium phosphate into bone and soft tissues. Calcification in soft tissues such as blood vessels, kidneys, brain, heart, and lungs is called ectopic calcification and unlike normal calcified areas such as bones and teeth, pathological features of the disease. A wide range of vascular calcification, including coronary arteries, has been observed in patients with end-stage renal disease, and has been reported to increase mortality and cardiovascular outcomes in dialysis patients (Hypertension 38: 938-942, 2001, Kidney Int 65: 1790-1794 , 2004). The most important cause of death in patients with end-stage renal disease is cardiovascular disease. The reason for this is that many risk factors of traditional cardiovascular diseases such as diabetes, hypertension, left ventricular hypertrophy, dyslipidemia and aging are accompanied by many.

MD2 (murine double minute 2) is a protein with the role of E3 ligase in ubiquitination system that break down tumor suppressor protein 53 (p53). The structure of MDM2 contains the RING domain with E3 ligase activity and is expressed in many tissues of humans and mice, and many studies have been reported especially in cancer cells (Honda R and Yasuda H, 2000, Moll UM and Petrenko O, 2003, Mendrysa SM, 2003, Wade M et al., 2012). In order to study the biological functions of MDM2, it is necessary to identify or develop a small molecule modulator that is specific to MDM2 and can regulate its function. To date, Nutlins and RG7112 have been reported to inhibit the activity of MDM2 by interfering with the interaction of MDM2 and p53 (Tovar C et al., 2013). The RG7112 inhibits the activation of MDM2 as an inhibitor of MDM2.

Although MDM2 has been shown to have many studies and roles in most cancer cells, the relevance of cardiovascular disease such as vascular calcification has not been clarified.

Therefore, in the present invention, as a synthesizing material for controlling the activity of MDM2, a small molecule compound RG7112 ([(4S, 5R) -2- (4- tert- butyl-2- ethoxyphenyl) 1-yl] methanone) as a vascular calcification inhibitor in order to provide a novel use of the compound of the present invention do.

Numerous papers and patent documents are referenced and cited throughout this specification. The disclosures of the cited papers and patent documents are incorporated herein by reference in their entirety to better understand the state of the art to which the present invention pertains and the content of the present invention.

The present inventors have made extensive efforts to develop a composition for preventing or treating vascular calcification-related diseases. As a result, a compound that inhibits MDM2 (murine double minute 2), RG7112 ([(4S, 5R) -2- (4- tert- ) -4,5-dimethylimidazol-1-yl] - [4- (3-methylsulfonylpropyl) piperazin-1-yl] methanone) as an active ingredient, The present inventors have completed the present invention by confirming that the composition has an inhibitory effect on vascular calcification in vascular smooth muscle cells and vascular tissues.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for preventing or treating vascular calcification-related diseases.

It is another object of the present invention to provide a method for screening a substance for inhibiting vascular calcification.

Other objects and advantages of the present invention will become more apparent from the following detailed description of the invention, claims and drawings.

According to one aspect of the present invention, there is provided a pharmaceutical composition comprising (a) a compound of the following formula (1), which is a substance inhibiting MDM2 (murine double minute 2) activity, or a pharmaceutically acceptable salt thereof; And (b) a pharmaceutically acceptable carrier. The pharmaceutical composition for preventing or treating vascular calcification-related diseases comprises:

Formula 1

(4S, 5R) -2- (4-tert-butyl) -2-methyl-2-pyrrolidone, which is a compound that inhibits MDM2 (murine double minute 2), as a result of efforts to develop a composition for the prevention or treatment of diseases related to angiocalcifications. -2-ethoxyphenyl) -4,5-bis (4-chlorophenyl) -4,5-dimethylimidazol-1-yl] - [4- (3- methylsulfonylpropyl) piperazin- (Methanone) as an active ingredient, and it was confirmed that the composition had an effect of inhibiting calcification of vascular smooth muscle cells and vascular tissues.

In the present invention, it has been experimentally confirmed that HDAC1 is degraded by MDM2 in the vascular calcification model, which is one of the cardiovascular diseases, not the p53-MDM2 complex in the previously reported cancer cells, thereby promoting the calcification of the blood vessels . In other words, RG7112, an inhibitor of MDM2, directly binds to MDM2 to inhibit the binding of MDM2 and p53, as well as to inhibit the degradation of HDAC1 by MDM2 by interfering with the binding of MDM2 and HDAC1.

According to one embodiment of the present invention, the MDM2 activity inhibitory substance binds to the p53-binding domain of MDM2 and inhibits the binding of MDM2 to the subadherent. The MDM2 sub-conditioner is HDAC1 (histone deacetylase 1) or p53.

As used herein, the term " murine double minute 2 inhibitor " means (i) a substance that interacts directly with MDM2, or (ii) an inhibitor of MDM2 activity. The MDM2 inhibitory substance may be various organic or inorganic compounds, peptides, oligonucleotides, natural products or antibodies. In the present invention, the compound RG7112 and its pharmaceutically acceptable salts are meant.

As used herein, the term " interaction "means that a compound that is a MDM2 inhibitor selectively binds to the p53 pocket of MDM2, preferably binds to the p53 pocket-binding domain of MDM2 to modulate the activity of MDM2 , More preferably binding to the p53 pocket binding region of MDM2 to block the binding of HDAC1 (histone deacetylase 1) and MDM2.

For example, the compound of formula 1 (RG7112) binds to the ligand-binding domain (preferably, preferably, p53-binding domain) of MDM2 and binds MDM2 and HDAC1 (histone deacetylase 1), thereby inhibiting degradation of HDAC1 and inhibiting degradation of HDAC1 protein by MDM2-HDAC1 binding. When MDM2 binds to HDAC1, proteolysis of HDAC1 occurs and vascular calcification occurs. The MDM2 inhibitor, RG7112, inhibits the proteolytic activity of HDAC1.

The compound of the formula (1) (RG7112) inhibits vascular calcification, which can be confirmed by measuring the expression of vascular calcification-related proteins or bone formation staining.

According to one embodiment of the present invention, HDAC1 (histone deacetylase 1) is an angiostatic protein whose binding to MDM2 is inhibited by RG7112, which can be confirmed by von kossa staining and calcium assay.

As a therapeutic agent for vascular calcification, the compound RG7112 of the present invention is the first to show successful results in the treatment of angio-calcification-related diseases targeting MDM2. According to one embodiment of the present invention, the vascular calcification-related diseases are end stage renal failure, atherosclerosis, hypertension, myocardial infarction or thrombosis.

The term "treatment ", as used in the present invention, is also intended to include: 1) a disease that has not yet been diagnosed as having vascular calcification related disease, but which has a disease or disorder in such an animal, preferably a mammal, 2) inhibition of vascular calcification-related diseases, i.e., inhibition of development, and 3) alleviation of vascular calcification-related diseases. For example, when the above-mentioned pharmaceutical composition is administered to a subject in need of treatment of an angi-calcification-related disease in a therapeutically effective amount, the composition moves through the blood vessels and restrains the accumulation of calcium in the blood vessels to restore the function of the blood vessels Vascular calcification can be treated or alleviated. On the other hand, the subject to be administered may be a mammal including a human.

When the composition of the present invention is used for the prevention or treatment of diseases related to vascular calcification, the composition of the present invention may be used as a single therapy, but may be used in combination with other conventional chemotherapy or physical surgery therapies, If the therapy is performed, it is possible to treat the calcification-related diseases more effectively.

The pharmaceutical composition of the present invention can be administered orally or parenterally, and in the case of parenteral administration, it can be administered by intravenous injection, subcutaneous injection, muscle injection, intraperitoneal injection, transdermal administration or the like.

The appropriate dosage of the pharmaceutical composition of the present invention may vary depending on factors such as the formulation method, administration method, age, body weight, sex, pathological condition, food, administration time, administration route, excretion rate, . The daily dosage of the pharmaceutical composition of the present invention is, for example, 0.001-1000 mg / kg. However, the actual dosage of the active ingredient may be determined taking into consideration various factors such as route of administration, body weight, severity, age and sex of the patient, and thus the dose does not limit the scope of the present invention in any form .

The pharmaceutical composition of the present invention may be formulated into a unit dose form by formulating it using a pharmaceutically acceptable carrier and / or excipient according to a method which can be easily carried out by a person having ordinary skill in the art to which the present invention belongs. Or by intrusion into a multi-dose container. The formulations may be in the form of solutions, suspensions, syrups or emulsions in oils or aqueous media, or in the form of excipients, powders, powders, granules, tablets or capsules, and may additionally contain dispersing or stabilizing agents.

According to another aspect of the present invention, the present invention provides a method of screening for an anti-calcification substance comprising the steps of:

(a) contacting MDM2 (murine double minute 2) protein, HDAC1 (histone deacetylase 1) protein and test substance; And

(b) detecting binding of the MDM2 protein and the HDAC1 protein or detecting the HDAC1 protein, wherein when the binding of the MDM2 protein and the HDAC1 protein is decreased or the HDAC1 protein is detected, The test substance is judged to be an inhibitor of vascular calcification.

The method for screening a substance for inhibiting vascular calcification according to the present invention can be carried out by screening a substance which binds to the MDM2 protein and inhibits or reduces the binding of MDM2 protein and HDAC1 protein. The vascular calcification inhibitor is a method for binding the binding motifs of the MDM2 protein to block the binding of both proteins or to modify the structure of the binding motif by binding to the MDM2 protein. . When the binding of the MDM2 protein and the HDAC1 protein is blocked, the HDAC1 protein is not degraded and the vascular calcification does not proceed.

The contacting can be carried out in vitro or in vivo , for example, when the method is (1) a protein-protein interaction-based in vitro screening method, the MDM2 protein, HDAC1 protein and can be carried out by bonding on the in vitro a test substance, (2) a cell-based (cell-based) when the screening is within the contacting with the test substance to the cell containing the MDM2 protein and HDAC1 protein cell MDM2 protein and HDAC1 protein And the like. In this case, it includes a step of measuring the binding of the intracellular MDM2 protein and the HDAC1 protein, and when the binding between the MDM2 protein and the HDAC1 protein is down-regulated, the test substance is used as an angiostatic agent .

On the other hand, when the HDAC1 protein does not bind to the MDM2 protein, the HDAC1 protein is not degraded, and consequently, the HDAC1 protein can be detected.

Test substances to be analyzed by the screening method of the present invention are organic or inorganic compounds, oligonucleotides, peptides or antibodies. The organic or inorganic compound is a single compound or a mixture of compounds (e.g., a natural extract or a cell or tissue culture). Such test substances can be obtained from a library of synthetic or natural compounds. Methods for obtaining libraries of such compounds are known in the art. Synthetic compound libraries are commercially available from Maybridge Chemical Co., Comgenex (USA), Brandon Associates (USA), Microsource (USA) and Sigma-Aldrich (USA) ) And MycoSearch (USA). The test materials can be obtained by various combinatorial library methods known in the art and include, for example, biological libraries, spatially addressable parallel solid phase or solution phase libraries, deconvolution By the desired synthetic library method, " 1-bead 1-compound " library method, and by synthetic library methods using affinity chromatography screening. Methods for synthesis of molecular libraries are described in DeWitt et al., Proc . Natl . Acad . Sci . USA 90, 6909, 1993; Erb et al. Proc . Natl . Acad . Sci . USA 91, 11422, 1994; Zuckermann et al., J. Med . Chem . 37, 2678, 1994; Cho et al., Science 261, 1303, 1993; Carell et al., Angew . Chem . Int . Ed . Engl . 33,2059,1994; Carell et al., Angew . Chem. Int . Ed . Engl . 33, 2061; Gallop et al., J. Med . Chem . 37, 1233, 1994, and the like.

The binding of the MDM2 protein and the HDAC1 protein can be carried out according to various assay methods and can be carried out in a high throughput manner according to various binding assays known in the art. For example, cell-based assays, in-vitro assays, or yeast-to-hybrid assays can be used.

Meanwhile, in the screening method of the present invention, the test substance, the MDM2 protein and the HDAC1 protein can be labeled with a detectable label. When a test substance labeled MDM2 protein and a HDAC1 protein with a detectable label are used, the occurrence of a bond between the test substance, the MDM2 protein and the HDAC1 protein can be detected by analyzing the signal from the label.

The detection of HDAC1 protein can be performed by Western blotting, ELISA, radioimmunoassay, radioimmunoassay, Ouchterlony immunodiffusion, rocket immunoelectrophoresis, tissue immuno staining, immunoprecipitation assay, complement fixation assay, FACS, , &Lt; / RTI > and the like.

The features and advantages of the present invention are summarized as follows:

(a) The present invention relates to a compound which inhibits mouse double minute 2 homolog (MDM2), RG7112 ([(4S, 5R) -2- (4-tert- butyl-2-ethoxyphenyl) (4-chlorophenyl) -4,5-dimethylimidazol-1-yl] - [4- (3-methylsulfonylpropyl) piperazin-1-yl] methanone as an active ingredient A pharmaceutical composition for preventing or treating diseases is provided.

(b) The compound contained in the composition of the present invention can inhibit the degradation of HDAC1 by blocking the binding of HDAC1 (histone deacetylase 1) with MDM2.

(c) Compound RG7112 of the present invention as an agent for treating vascular calcification presents the first and the most successful results in the treatment of angio-calcification-related diseases targeting MDM2.

(d) The composition for inhibiting vascular calcification comprising an MDM2 action inhibiting substance according to the present invention as an active ingredient can contribute to the treatment of vascular calcification by inhibiting the expression of vascular calcification-related genes and the activity of enzymes that regulate the formation of vascular calcification.

FIG. 1 shows the results of experiments in which the mutual binding between HDAC1 and MDM2 is confirmed to be weakened by RG7112, an inhibitor of MDM2,
FIG. 2 shows that high phosphate solution (Pi) induces vascular calcification in rat vascular smooth muscle cells. At this time, protein expression of HDAC1 was decreased, but protein expression was restored by RG7112, an inhibitor of MDM2,
FIG. 3 shows that calcitization induced by vascular calcification was induced by a high phosphate solution (Pi) in rat vascular smooth muscle cells, but inhibition of vascular calcification by RG7112, an inhibitor of MDM2, was confirmed by a calcium assay.
FIG. 4 shows that vascular calcification was induced by a high phosphate solution (Pi) in rat vascular smooth muscle cells, but inhibition of vascular calcification by dose-dependent RG7112 inhibitor of MDM2 was confirmed by von kossa staining.
FIG. 5 shows that when the high dose vitamin D was injected subcutaneously in rats, the calcification of the artery was induced, but when the RG7112 inhibitor of MDM2 was treated, the vein calcification was completely blocked,
FIG. 6 is a graph showing that protein expression of HDAC1 is reduced in rats induced by vascular calcification by injecting high-dose vitamin D, and then protein expression of HDAC1 is restored when RG7112, an inhibitor of MDM2, is treated.

Hereinafter, the present invention will be described in more detail with reference to Examples. It is to be understood by those skilled in the art that these embodiments are only for describing the present invention in more detail and that the scope of the present invention is not limited by these embodiments in accordance with the gist of the present invention .

Example

Experimental Method

Cultured primary vascular smooth muscle cells

Aortic vascular smooth muscle cells were cultured in 7-8-week-old (250-300 g) rats after the aortic dissection. The collagenase solution (0.2% collagenase Type I, Elastase II, BSA, trypsin inhibitor, 15 mM HEPES, Ham's F12 medium) and incubated at 37 DEG C for 30 minutes with shaking. After the culture medium is settled, endothelial cells are scraped off and the outer membrane is peeled off and then cut into dissection culture medium (penicillin, streptomycin, Ham's F12 media). The dissection culture medium was discarded, and the mixture was placed in a collagenase solution and incubated at 37 ° C for 90 minutes with shaking. The collagenase reaction was stopped by adding DMEM containing 10% (v / v) fetal bovine serum (FBS) to the cell supernatant, followed by incubation at 37 ° C. in the incubator, and the next day, cells were replaced with 10% FBS / give.

Induction of vascular calcification

To the primary cultured vascular smooth muscle cells, add 200 mM phosphoric acid solution (pH 7.4) so that the final concentration is 2 mM phosphoric acid solution. After the first 200 mM phosphate solution is replaced with 0day, replace the medium every two days and add the phosphate solution. At this time, 0.1 μM or 0.5 μM RG7112 should be treated together with phosphate solution. At 6 days, cell culture medium was discarded, washed with 1 × PBS, and 0.6 N HCl was added to measure calcium deposition, and the cells were reacted overnight in a refrigerated state. The next day, the HCl from which the calcium was dissolved was transferred to a new tube, centrifuged at 10,000 rpm for 3 minutes, and then 5 μl of the sample to be measured including the calcium standard solution was added to the 96-well plate. Then, 200 μl of the calcium- After the reaction, the absorbance at 570 nm was measured. At this time, the calcium result is corrected to the protein concentration of each cell. After removing the HCl from the cells, the cells were lysed by adding 0.1% NaOH / 0.1% SDS solution to the remaining cells, and the cells were incubated for 30 minutes in the cold state after breaking the cell membrane. Proteins were then separated by centrifugation at 14,000 rpm for 15 minutes.

Induction experiment of rat vascular calcification

6-8 week old C57BL / 6 mice were purchased from Samtako (Korea). Vitamin D solution (5x10 ⁵ IU) was prepared for injection as follows. Vitamin D (4.575 mg, Cholecalciferol, Sigma) in 70 μl ethanol was mixed with 500 μl cremophor (alkamuls EL-620, Sigma) for 15 minutes at room temperature. The solution was mixed with 6.2 ml sterilized water containing 250 mg dextrose And finally mixed for 15 minutes. Mice were randomly assigned to six experimental groups: vehicle + vehicle (n = 3) (used as a control), vitamin D ₃ + Vehicle treated group (n = 3), vehicle + RG7112 (50 mg / kg / day) treated group (n = 5), vitamin D 3 + RG7112 (50 mg / kg / day) group (n = 5). Mice were dosed with vitamin D at a dose of ⁵ x 10 ⁵ IU / kg body weight for 3 days and sacrificed 9 days after the first injection. RG7112 was dissolved in DMSO solution, diluted in physiological saline, 200 μl per day for 9 days from the day of vitamin D administration at a dose of 50 mg / kg / day, and 200 μl of vehicle was intraperitoneally injected.

Example 1: In vascular smooth muscle cells RG7112 Of arterial calcification

1-1. HDAC1 and MDM2 Combination blocking effect

MDM2 binds to HDAC1 to induce vascular calcification due to proteolytic degradation of HDAC1. In this study, we investigated whether MDM2 inhibitor RG7112 modulates proteolytic activity of HDAC1. After overexpression of MDM2-HA in 293T cells, RG7112 was treated, HA was pulled out by immunoprecipitation method, and the binding of MDM2 and HDAC1 was confirmed using Western. As a result, as shown in FIG. 1, MDM2 and HDAC1 strongly bind to each other. When RG7112 is treated, MDM2 and HDAC1 bind weakly. That is, the MDM2 inhibitor, RG7112, acts to block vascular calcification by inhibiting HDAC1 protein degradation.

1-2. HDAC1 Inhibition of vascular calcification

Primary vascular smooth muscle cells were pretreated with 2 mM phosphoric acid solution and 0.1 μM RG7112, and the expression of HDAC1 protein was confirmed by Western blotting (WB). The primary cultured VSMCs were treated with 2 mM phosphoric acid solution and 0.1 μM RG7112 followed by addition of 1% NP-40 buffer (150 mM NaCl, 1% NP-40, 50 mM Tris-HCl pH 8.0, 1 mM EDTA, DTT, 10 mM NaF, 1 mM Na ₃ VO ₄ , 1 mM PMSF, protease inhibitor) were added to each well and the vascular smooth muscle cells were collected and reacted with sonication for 15 minutes. After centrifugation at 13,000 rpm for 15 minutes, the supernatant was recovered and proteins were quantified using BCA protein reagent (Thermo Scientific). 50 μg of the protein was subjected to SDS-PAGE electrophoresis and then transferred to a polyvinylidene fluoride (PVDF membrane) to obtain a primary antibody (α-HDAC1 (1: 1000, Millipore) and α- -actin (1: 1000, Santa Cruz) for one day. Subsequently, the cells were reacted with a secondary antibody (anti-mouse IgG, horseradish peroxidase (HRP) -linked antibody, 1: 5000, Cell signaling company) for 1 hour and washed three times with TBST buffer. After washing, proteins were identified using a Western Chemiluminescent HRP substrate from Immobilion (Millipore, USA). As a result, HDAC1 expression was reduced in the group inducing vascular calcification by the phosphoric acid solution, whereas reduction of HDAC1 expression was blocked in the group treated with the phosphoric acid solution and RG7112 (FIG. 2).

1-3. RG7112 Blockage of vascular calcification

Primary vascular smooth muscle cells were cultured in 2 mM phosphate solution and 0.5? After RG7112 treatment, calcium deposition in blood vessels was confirmed by Ca assay method. The cultured vascular smooth muscle cells were treated with 2 mM phosphoric acid solution and 0.5? RG7112 was treated for 6 days and then the medium was suctioned. 0.6 N HCl was added and reacted for one day. 5? The supernatant was placed in a 96-well plate and the A and B solutions in the QuantiChrom Calcium assay kit (Bioassay) were mixed 1: 1 and 200 μl each, and then the absorbance at 570 nm was measured. The remaining cells are 200? 0.1 N NaOH / 0.1% SDS solution was added and the vascular smooth muscle cells were collected and reacted with sonication for 15 minutes. After centrifugation at 13,000 rpm for 15 minutes, the supernatant was recovered and proteins were quantified using BCA protein reagent (Thermo Scientific). Fig. 3 shows that calcium deposition was increased in vascular smooth muscle cells when inducing vascular calcification with phosphate solution, while calcium deposition was inhibited in the group treated with RG7112.

In addition, vascular smooth muscle cells were treated with 2 mM phosphoric acid solution and RG7112 by concentration, and vascular calcification was confirmed by von kossa staining. Vascular smooth muscle cells were treated with 2 mM phosphoric acid solution, 0.1 μM, 0.3 μM and 1 μM RG7112 for 6 days, and then the medium was suctioned and fixed with 10% formalin for 30 minutes. After washing with sterile distilled water, the cells were reacted with 5% silver nitrate at room temperature for 30 minutes and then washed with sterilized distilled water. After reacting with 5% sodium thiosulfate at room temperature for 2 minutes, it was washed with distilled water and photographed with a microscope. In Fig. 4, calcified nodule formation was significantly increased in the vascular calcification-induced vascular calcification of the vascular smooth muscle cells, whereas calcineurized nodule formation was significantly reduced in the RG7112-treated samples.

Example 2: In vascular calcification induction model mice RG7112 Confirming the effect of inhibiting vascular calcification by

2-1. Inhibition of vascular calcification

We examined the effect of MDM2 inhibitor, RG7112, on the vascular calcification effect in animals injected with high dose vitamin D. To induce vascular calcification, vitamin D (5x10 ⁵ IU / Kg) was diluted in physiological saline to 7-8 week old mice (C57BL / 6, Samtaco) and subcutaneously injected with 150 μl each. After 3 days of subcutaneous injection, the calcification of the blood vessels was induced for 6 days. The phenotype was analyzed after intraperitoneal administration of RG7112 (50 mg / kg / day) for 200 days in total for 9 days. The aorta was extracted from each mouse and fixed in a 10% formaldehyde solution. After fixed for one day, vascular calcification was observed by alizarin red S staining using histological method. As a result, vascular calcification was induced in the group treated with vitamin D and vehicle , Vitamin D and RG7112 treated group (Fig. 5).

2-2. HDAC1 Protein expression

After subcutaneous injection of vitamin D, 200 μl of RG7112 (50 mg / kg / day) was administered intraperitoneally for a total of 9 days, and the aorta was extracted from each mouse to isolate the protein and the protein expression of HDAC1 was confirmed by Western blot.

As shown in FIG. 6, in the group treated with vitamin D and vehicle, protein expression of HDAC1 was decreased, but in the group treated with vitamin D and RG7112, no decrease in HDAC1 protein expression was observed. This is because the MDM2 inhibitor RG7112 blocked the induction of vascular calcification by inhibiting proteolysis of HDAC1. These results demonstrate that MDM2 inhibitors such as RG7112 can be used as agents to treat angio-calcification.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the same is by way of illustration and example only and is not to be construed as limiting the scope of the present invention. Accordingly, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims

(a) a compound of the following general formula (1), or a pharmaceutically acceptable salt thereof, which is an inhibitor of MDM2 (murine double minute 2) activity as an active ingredient; And (b) a pharmaceutically acceptable carrier. A pharmaceutical composition for preventing or treating vascular calcification-related diseases,

Formula 1

The pharmaceutical composition of claim 1, wherein the MDM2 activity inhibitor binds to a p53-binding domain of MDM2 to inhibit MDM2 binding to a sub-regulator.

3. The pharmaceutical composition according to claim 2, wherein the MDM2 sub-conditioner is HDAC1 (histone deacetylase 1) or p53.

The pharmaceutical composition according to claim 1, wherein the disease associated with vascular calcification is end stage renal failure, atherosclerosis, hypertension, myocardial infarction or thrombosis.

A method of screening for an angiostatic inhibitor comprising the steps of:
(a) contacting MDM2 (murine double minute 2) protein, HDAC1 (histone deacetylase 1) protein and test substance; And
(b) detecting binding of the MDM2 protein and the HDAC1 protein or detecting the HDAC1 protein, wherein when the binding of the MDM2 protein and the HDAC1 protein is decreased or the HDAC1 protein is detected, The test substance is judged to be an inhibitor of vascular calcification.