Composition for treating vascular or cardiac valvular calcification, containing thiamine derivative

Abstract

The present invention relates to: a pharmaceutical composition for preventing or treating vascular or cardiac valvular calcification, containing a thiamine derivative or a pharmaceutically acceptable salt thereof; and a food composition for alleviating vascular or cardiac valvular calcification, containing a thiamine derivative or a salt thereof, and the compositions of the present invention can be effectively used for a use of preventing, treating or alleviating vascular or cardiac valvular calcification.

Claims

1. A method for preventing or treating vascular calcification or valvular calcification in a subject, the method comprising: a step of administering to the subject a pharmaceutical composition comprising a thiamine derivative represented by chemical formula 1, below, or a pharmaceutically acceptable salt thereof, ##STR00019## wherein, ##STR00020## R.sub.1 is selected from the group consisting of  and R.sub.2 is selected from the group consisting of ##STR00021##

2. The method of claim 1, wherein R.sub.1 is ##STR00022## and R.sub.2 is ##STR00023##

3. The method of claim 1, wherein the vascular or valvular calcification is caused by at least one disease or condition selected from the group consisting of a valvular disease, hyperlipidemia, aging, estrogen deficiency, angina, heart failure, kidney disease, uremia, diabetes, an inflammatory disease, and a cardiovascular disease.

4. The method of claim 1, wherein the vascular calcification is medial calcification or atherosclerotic calcification.

5. The method of claim 1, wherein the valvular calcification is aortic valve calcification.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 shows calcification inhibitory effects by fursultiamine (F) and allithiamine (A) according to an example of the present disclosure.

(2) FIG. 2 shows the quantification of calcified calcium in order to investigate the calcification inhibitory effects by fursultiamine and allithiamine according to an example of the present disclosure.

(3) FIG. 3 shows the results of Von Kossa staining executed in order to investigate the calcification inhibitory effect (in vivo) by fursultiamine according to an example of the present disclosure.

(4) FIG. 4 shows the quantification of calcium deposited in blood vessels in order to investigate the calcification inhibitory effect (in vivo) by fursultiamine according to an example of the present disclosure.

MODE FOR CARRYING OUT THE INVENTION

(5) Hereinafter, the present disclosure will be described in more detail with reference to examples. These examples are provided only for the purpose of illustrating the present disclosure in more detail, and therefore, according to the purpose of the present disclosure, it would be apparent to a person skilled in the art that these examples are not construed to limit the scope of the present disclosure.

Preparation Example 1: Primary Culture of Vascular Smooth Muscle Cells (VSMCs)

(6) The surrounding tissues were removed from the thoracoabdominal aorta isolated from male cattle, and endothelial cells that line blood vessels were removed. The cleaned blood vessels were divided into small fragments, which were then aligned on a culture dish containing Dulbecco's modified Eagle's medium (DMEM, Low Glucose, Thermo Fisher Scientific, USA) supplemented with 20% fetal bovine serum (FBS, Hyclone, Australia). Thereafter, culturing was performed in a cell incubator at 37° C. and 5% CO2 for 3-4 weeks. The vascular smooth muscle cells stretching out around the vascular tissues were subjected to primary culture with media changed every two days.

Preparation Example 2: Calcification Induction in Vascular Smooth Muscle Cells

(7) The vascular smooth muscle cells cultured in Preparation Example 1 above were stabilized by subculture in DMEM (high glucose) supplemented with penicillin/streptomycin antibiotics and 10% FBS. Thereafter, the cells were inoculated at 4×10.sup.5 in 6-well culture dishes, and on the next day of inoculation, the cells were treated with inorganic phosphate (Pi) or both inorganic phosphate and thiamine (fursultiamine or allithiamine; 100 or 200 μM). The cells were cultured for 10 days while media and drugs were changed every 48 hours.

Test Example 1: Investigation of Calcification of Vascular Smooth Muscle Cells

(8) After the induction of calcification by the method in Preparation Example 2 above, the media were removed from the culture dishes, followed by washing with phosphate buffer saline (PBS) three times. Then, 4% paraformaldehyde was added, and the cells were fixed at 4° C. for 15 minutes. After the fixation, the cells were washed with primary distilled water for 5 minutes two times, treated with 5% silver nitrate, and then irradiated with ultraviolet light for 20-30 minutes. Thereafter, the cells were washed with primary distilled water two or three times, and then were determined for a degree of calcification through an optical microscope (BX53 microscope, Olympus, Japan).

(9) As can be confirmed in FIG. 1, the calcification (black dots) was increased in the vascular smooth muscle cells treated with only inorganic phosphate (Pi), but the calcification (black dots) was inhibited in the vascular smooth muscle cells treated with both fursultiamine (F) and allithiamine (A) at 100 and 200 μM for each (Pi+F100 μM, Pi+F200 μM, Pi+A100 μM, and Pi+A200 μM).

Test Example 2: Quantification of Calcium in Vascular Smooth Muscle Cells

(10) After the induction of calcification by the method in Preparation Example 2 above, the media were removed from the culture dishes, followed by washing with PBS. Then, the cells were decalcified by treatment with 0.6 N HCl at 4° C. for 24 hours, The supernatant was recovered for the use in the quantification of calcium, and proteins were extracted from the cells by using 0.1 N NaOH/0.1% SDS, and quantified by using the BCA method.

(11) The absorbance of a calcium sample (standard) was measured at 612 nm by using a Quantichrom™ calcium analysis kit (DICA-500, BioAssay System, USA), and the calcium concentration was determined by using Equation 1, and then, the value thereof was divided (corrected) by the quantified amount of proteins, thereby finally quantifying calcium.

(12) $\begin{array}{cc}\mathrm{Calcium}\mathrm{concentration}=\frac{O{D}_{\mathrm{SAMPLE}}\setminus -{\mathrm{OD}}_{\mathrm{BLANK}}}{\mathrm{Slope}}\left(\mathrm{mg}/\mathrm{dL}\right).& \left[\mathrm{Equation}1\right]\end{array}$

(13) (OD.sub.SAMPLE and OD.sub.BLANK represent the OD.sub.612nm values of calcium sample and blank (water), respectively; and the slope represents the inclination value on the measured absorbance graph)

(14) As can be confirmed from FIG. 2, the calcium amount was increased by inorganic phosphate in the vascular smooth muscle cells treated with only inorganic phosphate (vehicle), but the calcium amount increased by inorganic phosphate was decreased by treatment with both fursultiamine and allithiamine at 100 and 200 μM for each (*p<0.01 vs Control; **p<0.01 vs Vehicle).

Test Example 3: In Vivo

(15) C57BL/6J mice aged 8-9 weeks were grouped into 5 groups of 7 mice each, and four groups (one group being used as a control) underwent the induction of vascular calcification by subcutaneous administration of a high dose (9.0×10.sup.5 IU) of vitamin D3 (Vit. D3) once a day for a total of 3 days.

(16) Out of these, three groups were subjected to oral administration of 10 mg/kg and 30 mg/kg fursultiamine and 50 mg/kg dichloroacetate (DCA, positive control), respectively, from three days before the first administration of vitamin D3, once a day for a total of 13 days.

(17) 3-1. Von Kossa Staining

(18) First, on the 10th day after the administration of vitamin D3, the mice were sacrificed and the abdomen was opened, and then the aorta was separated by cleanly removing surrounding tissues of the aorta, and fixed with 4% paraformaldehyde (PFA) for 24 hours. The fixed aorta was washed, immersed in a 5% silver nitrate solution, and incubated under UV light for 30 minutes.

(19) As can be confirmed from FIG. 3, the aorta was mostly darkly colored due to an increase in vascular calcification in the vitamin D3 groups, whereas the colored parts were significantly reduced in the DCA group (positive control) compared with the vehicle group. The colored parts were also reduced (recovered) dose-dependently in the fursultiamine groups of the present disclosure, and especially, the degree of recovery was excellent in the 30 mg/kg administration group compared with the DCA administration group (positive control).

(20) 3-2. Calcium Quantification

(21) Additionally, on the 10th day after the administration of vitamin D3, the mice were sacrificed and the abdomen was opened, and then the aorta was separated by cleanly removing surrounding tissues and frozen in liquid nitrogen. The frozen tissues were thawed, weighed after the removal of moisture, and then subjected to decalcification with a 0.6 N HCl solution. The colorimetric Ca quantification kit (Bioassay DICA-500) was used to quantify calcium in the 0.6 N HCl solution used in the decalcification, and the calcium quantification was corrected by using the tissue weight.

(22) As can be confirmed from FIG. 4, the amount of calcium was increased by about 10 times in the vitamin D3 groups compared with the control group with no vitamin D3 injection, but the amount of calcium in the aorta, which increased by vitamin D3, was significantly decreased in the DCA group (positive control). The amount of calcium was also decreased dose-dependently in the fursultiamine groups of the present disclosure, and the degrees of decrease were excellent compared with the DCA group (positive control).