Use of combination of gliflozin family drug and gliptin family drug

Abstract

This invention relates to a pharmaceutical composition for the prevention or treatment of vascular or valvular stenosis. This pharmaceutical composition comprises a gliflozin and a glibenclamide. When the gliflozin and glibenclamide of this invention are administered together, calcification is synergistically reduced compared to administration of each gliflozin and glibenclamide alone. Therefore, the composition according to this invention is very useful for vascular or valvular stenosis.

Description

Technical Field

[0001] The present invention relates to a pharmaceutical composition for preventing or treating vascular or valvular stenosis, comprising an SGLT-2 (sodium-glucose cotransporter 2) inhibitor (such as gliflozin-type drugs) and a DPP-4 (dipeptidyl peptidase-4) inhibitor. Prior Art

[0002] Aortic stenosis is a disease or condition in which the valve that flows from the left ventricle to the aorta (i.e., the aortic valve) fails to open properly when the left ventricle contracts. The pathophysiology of aortic stenosis is complex, involving multiple pathological processes such as lipid retention, oxidation, chronic inflammation, fibrosis, and calcification. Specifically, the condition or disease in which calcification develops in the aortic valve is called calcific aortic valve disease (CAVD). There are currently no approved specific medical treatments for aortic stenosis or CAVD. Therefore, the only treatment options are surgical aortic valve implantation (SAVI) and non-surgical transcatheter aortic valve implantation (TAVI).

[0003] Cardiovascular calcification can exacerbate hypertension, heart failure, acute coronary syndromes, and valvular disease, and lead to various complications. Calcification can occur independently or in conjunction with fibrosis. Numerous epidemiological studies have shown that vascular calcification independently increases mortality. The mechanism of vascular calcification is similar to the normal osteoblastic process during the fetus or after a fracture, and is activated in elderly people, diabetes, chronic renal failure, and chronic inflammatory diseases. Inflammatory responses promote mineral loss from the bones, and free minerals are phagocytosed by abnormal vascular endothelial cells. Calcification also occurs in heart valves (particularly aortic valve calcification), leading to aortic valve narrowing (aortic stenosis).

[0004] The aortic valve calcification process that leads to aortic stenosis begins deep within the aortic valve tissue, near the edges of the attachment sites. In advanced disease, nodules extend through the narrowed surface of the valve. The early stage of calcification is called aortic sclerosis, while the advanced stage, aortic stenosis, occurs when the functional valve area is reduced sufficiently to cause blood flow obstruction.

[0005] There is currently no non-invasive treatment that can cure CAVD.

[0006] However, U.S. Patent No. 9,416,196 discloses that DPP-4 (dipeptidyl peptidase-4) inhibitors, such as gliptin, can treat aortic valve calcification. DPP-4 (dipeptidyl peptidase-4), also known as CD26 (cluster of differentiation 26), is a protein known to be involved in immune regulation, apoptosis, and signal transduction. According to the aforementioned U.S. patent, DPP-4 expression increases when blood vessels and valves calcify, and calcification is significantly reduced when DPP-4 inhibitors are administered. This suggests that DPP-4 inhibitors can be used to treat or prevent vascular or valvular diseases. As DPP-4 inhibitors, several types of gliptins have been revealed, including sitagliptin, vildagliptin, saxagliptin, linagliptin, dutogliptin, gemigliptin, alogliptin, anagliptin, and evogliptin.

[0007] Meanwhile, gliflozin is a general term for a group of drugs called SGLT-2 (sodium-glucose cotransporter 2) inhibitors used to treat type 2 diabetes. Specifically, dapagliflozin, ertugliflozin, empagliflozin, canagliflozin, bexagliflozin, tofagliflozin, ipragliflozin, enavogliflozin, and luseogliflozin have been developed. SGLT-2 inhibitors are drugs that inhibit the reabsorption of sodium and glucose in the renal tubules and increase their excretion from the body. Recently, WO 2021 / 037400 A revealed that dapagliflozin is effective for heart failure with reduced ejection fraction. Summary of the Invention

[0008] However, no drug has been developed to treat stenosis caused by vascular or valvular calcification. The present inventors have endeavored to develop a drug for preventing or treating such stenosis. As a result, the inventors discovered that the combination of gliflozin and gliptin exhibits a synergistic effect in inhibiting vascular or valvular calcification compared to gliptin or gliflozin alone, leading to the completion of the present invention.

[0009] The object of the present invention is to provide a novel composition or method for preventing or treating vascular or valve stenosis.

[0010] In order to achieve the above objectives, in one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating vascular or valve stenosis, which comprises an SGLT-2 inhibitor and a DPP-4 inhibitor.

[0011] Beneficial effects

[0012] The pharmaceutical composition according to the present invention can prevent or treat vascular or valve stenosis. Simple diagram description

[0013] Figure 1a shows a set of photographs showing the degree of calcification in aortic smooth muscle cells induced by treatment with different concentrations of gliptins (DPP-4 inhibitors); FIG1b is a graph showing the degree of calcification quantified by absorbance analysis of the results of FIG1a; Figure 2a shows a set of photographs showing the degree of calcification in aortic smooth muscle cells induced by treatment with different concentrations of gliflozin drugs (SGLT-2 inhibitors); FIG2b is a graph showing the degree of calcification quantified by absorbance analysis of the results of FIG2a; Figure 3a shows a set of photographs comparing the degree of calcification inhibition in calcified aortic smooth muscle cells treated with idagliflozin and dapagliflozin alone and in combination; Figures 3b and 3c show graphs of the degree of calcification quantified by absorbance analysis of the results of Figure 3a; Figure 4a shows a set of photographs comparing the degree of calcification inhibition in calcified aortic smooth muscle cells treated with ipagliptin and empagliflozin alone and in combination; FIG4b shows a graph showing the degree of calcification quantified by absorbance analysis of the results of FIG4a; Figure 5a shows a set of photographs comparing the degree of calcification inhibition in calcified aortic smooth muscle cells treated with ipagliptin and canagliflozin alone and in combination; FIG5b shows a graph showing the degree of calcification quantified by absorbance analysis of the results of FIG5a; Figure 6a shows a set of photographs comparing the degree of calcification inhibition in calcified aortic smooth muscle cells treated with ergliptin and ergliptin alone and in combination; FIG6b shows a graph showing the degree of calcification quantified by absorbance analysis of the results of FIG6a; Figure 7a shows a set of photographs comparing the degree of inhibition of aortic valve calcification when idagliflozin and dapagliflozin were administered alone or in combination in a mouse model of calcification induced by vitamin D3 administration; Figure 7b shows a graph showing the extent of aortic valve calcification in samples administered with idagliflozin and dapagliflozin alone and in combination; FIG8a is a graph showing the extent of renal calcification in samples administered with idagliflozin and dapagliflozin alone and in combination; and FIG8b is a graph showing the degree of vascular calcification in samples to which idagliflozin and dapagliflozin were administered alone or in combination. Implementation Method

[0014] Hereinafter, the present invention will be described in detail.

[0015] The embodiments of the present invention can be modified in various other forms, and the scope of the present invention is not limited to the embodiments described below. Those skilled in the art should fully understand that the embodiments of the present invention are provided to more accurately explain the present invention.

[0016] In addition, unless explicitly stated otherwise, “comprising” an element throughout the specification does not exclude other elements but may include other elements.

[0017] In one aspect of the present invention, the present invention provides a combination of an SGLT-2 inhibitor and a DPP-4 inhibitor for use in preventing or treating vascular or valve stenosis.

[0018] In another aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating vascular or valve stenosis, which comprises an SGLT-2 inhibitor and a DPP-4 inhibitor.

[0019] In another aspect of the present invention, the present invention provides a health functional food composition for preventing or improving valvular stenosis, wherein the health functional food composition comprises an SGLT-2 inhibitor and a DPP-4 inhibitor.

[0020] In another aspect, the present invention provides a method for preventing or treating vascular or valvular stenosis by administering to a subject in need thereof an amount of an SGLT-2 inhibitor and a DPP-4 inhibitor effective to prevent or treat vascular or valvular stenosis.

[0021] In another aspect of the present invention, the present invention provides a combination comprising an SGLT-2 inhibitor and a DPP-4 inhibitor for use in preparing a medicament for preventing or treating vascular or valve stenosis.

[0022] In another aspect of the present invention, the present invention provides a combination comprising an SGLT-2 inhibitor and a DPP-4 inhibitor for preventing or treating vascular or valve stenosis.

[0023] In another aspect of the present invention, the present invention provides a combination preparation comprising an SGLT-2 inhibitor and a DPP-4 inhibitor for preventing or treating vascular or valve stenosis.

[0024] In this specification, SGLT-2 inhibitors include gliflozin family drugs, and DPP-4 inhibitors include gliptin family drugs. In this specification, gliflozin family drugs may be used to refer to SGLT-2 inhibitors, and DPP-4 inhibitors may be used to refer to gliptin family drugs.

[0025] Throughout this specification, the term "combination" refers to the clinical treatment of preventing or treating vascular or valvular stenosis by administering a gliflozin and a gliptin simultaneously or at different times. The compositions, methods, uses, combinations, and combined preparations described in this specification are all based on the concept of combined use. Specific details of possible combined use are described below. Combined preparations include combinations of individual preparations and complex preparations.

[0026] The following description applies to all forms of the invention, including compositions, methods, uses, combinations and combined preparations.

[0027] Blood vessels include all blood vessels in the body, particularly those associated with the heart, and more specifically, the aorta, vena cava, pulmonary artery, vena cava, and renal arteries. Valve refers to the heart valves, including the aortic valve, pulmonary valve, mitral valve, and tricuspid valve, but preferably refers to the aortic valve. Stenosis may occur in patients with cardiovascular diseases such as heart failure and arteriosclerosis, or renal diseases such as chronic renal failure.

[0028] The above-mentioned vascular or valve stenosis may be accompanied by vascular or valve calcification. Vascular calcification occurs in patients with cardiovascular diseases such as heart failure, arteriosclerosis, or atherosclerosis, or kidney diseases such as renal arteriosclerosis or renal atherosclerosis. Kidney diseases include chronic kidney disease.

[0029] Vascular calcification, or narrowing, that occurs in people with cardiovascular or renal disease can occur in any blood vessel in the body.

[0030] In this context, calcification refers to the accumulation of calcium in the heart, lungs, kidneys, blood vessels, or valves, leading to tissue hardening. As calcification progresses, kidney calcification can lead to kidney disease, blood vessel or valve stenosis, and aortic valve calcification can lead to aortic stenosis.

[0031] Calcification can be either medial or atherosclerotic. Calcified tissue is called calcified tissue. Intimal calcification is associated with atherosclerosis. Atherosclerosis begins with the accumulation of lipid-rich macrophages and T lymphocytes on the inner lining of blood vessels or valves, forming a fatty layer. This is followed by the migration of smooth muscle cells from the medial layer. The chemodynamic substances that stimulate this movement are believed to be produced by nearby endothelial cells, activated phagocytes, and other cells.

[0032] Migrating smooth muscle cells proliferate, accumulate fat, and produce extracellular matrix. Calcification occurs in the center of the atherosclerotic plaque.

[0033] Medial calcification occurs independently of atherosclerosis and intimal calcification. Medial calcification in peripheral arteries is called Münckeberg's sclerosis and is most common in elderly patients with diabetes. Smooth muscle cells and elastin are known to be involved in the development of calcification.

[0034] It has been reported that calcification caused by renal dysfunction mainly occurs in the medial layer, while calcification caused by atherosclerosis or atherosclerosis-related calcification mainly occurs in the intima.

[0035] In addition, in patients with kidney disease, more specifically, in patients with chronic renal failure or chronic kidney disease, the kidney itself will undergo calcification. Therefore, the composition of the present invention can be used to alleviate or treat chronic kidney disease, more specifically, can be used to treat chronic kidney disease accompanied by calcification.

[0036] Treatable stenosis according to the present invention is not particularly limited, but includes clinically diagnosed stenosis and may include early stages of sclerosis that are not clinically diagnosed. It may include the stage where symptoms of fibrosis and / or calcification are recognized, preferably the stage where symptoms of calcification are recognized. In the present invention, "treating" includes reducing the degree of stenosis in the treated tissue, thereby slowing the progression of stenosis or reversing the symptoms of stenosis to a normal state. When referring to slowing the progression of stenosis, it means that the progression of stenosis is significantly slowed, as a relative measure, compared to when not receiving the combination treatment according to the present invention (e.g., when no treatment is given or when only a typical gliflozin or gliptin is administered alone). Slowing the progression of stenosis can mean, for example, a slowing of 1%, 2%, 3%, 5%, 10%, 15%, 20%, 30%, 40%, or 50%, and the degree of stenosis progression can be quantitatively assessed, for example, by blood flow velocity, degree of calcification, etc.

[0037] When stenosis is accompanied by calcification, treatment includes not only completely reversing the calcification to normal tissue, but also slowing the progression of calcification and reducing the extent of existing calcification. When referring to slowing the progression of calcification, this refers to a significant slowing of calcification progression relative to when not receiving the combination therapy according to the present invention (e.g., when no treatment is given or when only a typical gliflozin or gliptin is administered alone). Slowing the progression of calcification includes, for example, slowing the rate of increase in the amount of calcium deposits or the area or weight of calcified tissue, as indicators of calcification, by more than 1%, more than 2%, more than 3%, more than 5%, more than 10%, more than 15%, more than 20%, more than 30%, more than 40%, or more than 50%. Reducing the extent of calcification includes reducing the area of ​​calcified tissue by 1% or more, 2% or more, 3% or more, 5% or more, 10% or more, 15% or more, 20% or more, 30% or more, 40% or more, or 50% or more, based on the area of ​​calcified tissue before the start of combined administration of a gliflozin and gliptin according to the present invention.

[0038] Methods for quantifying the area of ​​calcified tissue can be performed, for example, by quantifying the area of ​​calcium deposits stained using Alizarin Red S (ARS) or von Kossa (VK) staining. In the present invention, "prevention" can include alleviating calcification before it occurs or preventing its progression.

[0039] In the present invention, gliflozins are not particularly limited to SGLT-2 inhibitors, but may include one or more gliflozins selected from the group consisting of dapagliflozin, ertugliflozin, empagliflozin, canagliflozin, bexagliflozin, tofagliflozin, ipragliflozin, enavogliflozin, and luseogliflozin. Gliptins are not particularly limited to DPP-4 inhibitors, but may include one or more gliptins selected from the group consisting of evogliptin, linagliptin, saxagliptin, and sitagliptin.

[0040] In one specific embodiment of the present invention, the gliflozin-type drug may be dapagliflozin, erpagliflozin, empagliflozin or canagliflozin, and in another specific embodiment, the gliflozin-type drug may be dapagliflozin.

[0041] In one embodiment of the present invention, the gliptin drug can be ipragliptin, linagliptin, sitagliptin or saxagliptin, and in another embodiment, the gliptin drug can be ipragliptin.

[0042] In the present invention, gliflozins and gliptins can be included in a composition or administered in a therapeutically effective amount to prevent vascular or valve calcification. Pharmaceutical compositions or health functional food compositions according to the present invention include not only single-administration formulations of a physical mixture of the gliflozin and gliptin, but also formulations prepared as separate administration formulations that are then combined at the time of administration.

[0043] It is desirable to determine the dosage of gliflozins and gliptins so that they are administered in a therapeutically effective amount within a range that is not harmful to the subjects.

[0044] The dosage of these drugs can be determined based on body weight, age, sex, health status, diet, administration frequency, administration method, excretion and severity of the disease, and those skilled in the art can determine the appropriate amount. For example, gliflozin and gliptin drugs can be administered at a dose of 0.01 to 500 mg per day, and can be administered once or more per day, for example, twice, three times or four times.

[0045] Furthermore, their administration cycle can be determined daily, on a number of days selected from 2 to 7 days, or on an irregular basis. The duration of administration can be short-term, ranging from 2 days to 1 month, medium-term, from over 1 month to 3 months, long-term, from over 3 months to 1-3 years, or even for the remainder of one's life. Obviously, the dosage and administration cycle may vary depending on the number of days of administration.

[0046] The dosage ratio of the gliflozin drug and the gliptin drug can be determined by one skilled in the art as a therapeutically effective amount and, for example, can be determined within the range of 1:1000 to 1000:1, 1:500 to 500:1, 1:100 to 100:1, 1:50 to 50:1, 1:10 to 10:1, 1:5 to 5:1, and 1:2 to 2:1 based on the weight of the active ingredients for a period of time.

[0047] In the present invention, the gliflozin and gliptin can be administered simultaneously or at different times. Simultaneous administration not only includes co-administration in a physically mixed form, but also includes administration at different times within 6, 5, 4, 3, 2, or 1 hour, when drug-drug interactions may occur. The pharmaceutical composition or health functional food composition according to the present invention includes not only a single-administration formulation of a physically mixed gliflozin and gliptin, but also compositions prepared as separate formulations and then combined at the time of administration. When the composition of the present invention is provided as a separate formulation, they can be administered simultaneously or at different times as described above. The term "pharmaceutical composition" in the present invention is not intended to be limited to a physically mixed composition, but rather refers to a pharmaceutical composition.

[0048] Even if the drugs are administered at different times, the blood drug distribution can be adjusted so that the gliflozin drug and the gliptin drug interact in the blood, thereby achieving the effect of co-administration of the drugs. For example, they can be administered at different times ranging from more than 6 hours to 48 hours.

[0049] For co-administration, the gliflozin drug and the gliptin drug can be formulated and used independently of each other. Commercially available formulations containing each drug as an active ingredient for vascular or valvular stenosis and other indications can also be used.

[0050] For co-administration, the active ingredients of the gliflozin and gliptin drugs can be formulated together as a combination preparation to include both in one preparation.

[0051] When the drug is formulated independently or as a combined preparation, the preparation can be formulated for oral administration, such as tablets, capsules, powders, granules, or suspensions, or can be formulated into parenteral injections, but is preferably formulated for oral administration. When formulated, these preparations may further include one or more pharmaceutically acceptable additives. These additives may include at least one selected from the group consisting of excipients (diluents), binders, disintegrants, lubricants, and colorants.

[0052] The compositions according to the present invention can be administered alone or in combination with surgery, hormonal therapy, drug therapy, and biomodulators to prevent or treat vascular or valve calcification.

[0053] For co-administration, a health functional food composition containing the active ingredients of the gliflozin class of drugs and the active ingredients of the gliptin class of drugs can be formulated.

[0054] There are no particular limitations on the other ingredients that may be included in the health functional food composition of the present invention. These may include, for example, various herbal extracts, food additives, or natural carbohydrates, such as those found in traditional foods. The composition may further include food additives commonly used in the art, such as flavorings, colorants, fillers, stabilizers, and the like.

[0055] Obviously, in the health functional food composition, the gliflozin and the gliptin are provided as separate preparations and then used together.

[0056] For the above-mentioned health functional food composition, the dosage, administration cycle and administration days can be applied as described above.

[0057] Hereinafter, the present invention will be described in detail by way of the following examples.

[0058] However, the following examples are only used to illustrate the present invention, and the content of the present invention is not limited thereto.

[0059] Example 1: In vitro cell testing

[0060] Experimental methods:

[0061] 1. Human aortic smooth muscle cells (HASMCs) were seeded at 1x104 cells per well in a 48-well plate.

[0062] 2. Culture the cells for 3 days until the cell density reaches approximately 80%.

[0063] 3. After 3 days, check whether the cell density is approximately 80%.

[0064] 4. Prepare osteogenic medium (OM) to induce calcification in HASMCs. The medium is composed of 10% fetal bovine serum (FBS), 1% penicillin-streptomycin, and 3 mM calcium chloride (CaCl2) in Dulbecco's Modified Essential Medium (DMEM) high glucose.

[0065] 5. Solutions were prepared by adding DPP-4 inhibitors (imigliptin, sitagliptin, linagliptin, saxagliptin) and SGLT-2 inhibitors (dapagliflozin, canagliflozin, empagliflozin, erpagliflozin) alone or in combination at different concentrations to the OM prepared above.

[0066] For gliptin alone, the drug was given at 10, 25, 50, and 100 M concentrations (except saxagliptin, which was treated with 2, 5, 10 and 20 M concentration), for gliflozin alone, the drug was treated at 10, 20, 30 and 40 The concentration of M was treated.

[0067] To compare the effect of the combination therapy, ipragliptin alone was administered at 10 and 25 M concentrations, and dapagliflozin alone at 1, 5, 10, 20, 30, and 40 M concentration, and the other gliflozins except dapagliflozin were treated at 10, 20, 30 and 40 The cells were treated with 100 μg / mL of 4% PEG-100 μg / mL of water, and the combination treatment was performed with each concentration combination.

[0068] 6. Add drug solution to the appropriate wells for each group.

[0069] 7. Every other day, prepare a solution containing drugs in OM and add it to the wells.

[0070] 8. After 12 days of treatment with the drug solution, alizarin red staining was performed to determine the degree of calcification.

[0071] 9. For quantitative comparative analysis of calcification, dispense 10% cetylpyridinium chloride solution into each well.

[0072] 10. Incubate the reaction on a shaker for 30 minutes to allow the Alizarin Red solution to elute from each well.

[0073] 11. Measure the absorbance at 540 nm using a reader to compare the degree of calcification. The results were normalized to OM without any drug treatment as a control.

[0074] result:

[0075] Results for gliptins and gliflozins, both alone and in combination, are shown in the figure.

[0076] In the figure, Evo represents ipagliptin, Lina represents linagliptin, Sita represents sitagliptin, Saxa represents saxagliptin, Dapa represents dapagliflozin, Empa represents empagliflozin, Cana represents canagliflozin, Ertu represents erpagliflozin, and OM represents the control without drug treatment in the cell culture medium.

[0077] The absorbance was measured and normalized to the control along with the staining results.

[0078] Figures 1a and 1b show the results of treatment with gliptins (DPP-4 inhibitors) at each concentration, demonstrating that they inhibit calcification in a concentration-dependent manner.

[0079] Figures 2a and 2b show the results of treatment with gliflozin (SGLT-2 inhibitor) at each concentration, demonstrating that they inhibit calcification in a concentration-dependent manner.

[0080] Figures 3a to 6b show the extent of calcification inhibition with DPP-4 inhibitors and SGLT-2 inhibitors, alone and in combination.

[0081] When idagliflozin and dapagliflozin were treated together, a synergistic anti-calcification effect was demonstrated compared with either treatment alone.

[0082] When empagliflozin and empagliflozin were treated together, a synergistic anti-calcification effect was demonstrated compared with either treatment alone.

[0083] When ipragliptin and canagliflozin were treated together, a synergistic anti-calcification effect was demonstrated compared with either treatment alone.

[0084] When ipragliptin and erpagliflozin were treated together, a synergistic anti-calcification effect was demonstrated compared with either treatment alone.

[0085] Example 2: In vivo mouse model test

[0086] Experimental methods:

[0087] Preparation of vitamin D3 solution

[0088] 1. Prepare a vitamin D3 solution by dissolving an accurate weight of vitamin D3 in a solution of 30% PEG400, 0.5% Tween80, 5% propylene glycol, and 64.5% saline (6.5 x 104 IU / ml (1.625 mg / ml)) and ultrasonicating for 1 hour.

[0089] 2. Preparation of Gliptin and Gliflozin

[0090] All materials were accurately weighed and dissolved or dispersed in 0.5% CMC sodium salt to prepare 1 mg / kg / 100 μl of ipragliptin and 2 mg / kg / 100 μl of ipragliptin. l's dapagliflozin.

[0091] Animal (mice) preparation

[0092] Animals: C57BL / 6 mice (OrientBio, Seongnam, South Korea)

[0093] Gender:Male

[0094] Age at arrival: C57BL / 6 (6 weeks)

[0095] Animals were housed in filter-top polycarbonate cages (10 animals per cage) in a sterile cleanroom with controlled temperature (25°C) and humidity (45–55%). The light-dark cycle was 12 h:12 h, and sterilized food and water were provided ad libitum.

[0096] Guidelines: All animals were handled in accordance with the regulations of the Institutional Animal Care and Use Committee of Daegu University of Korean Medicine (Gyeongsan, South Korea).

[0097] 4. In vivo Drug Administration: Mouse Model of Vitamin D-Induced Calcification

[0098] After one week of adaptation, the mice were randomly divided into 18 groups.

[0099] All groups of mice received subcutaneous injections of vitamin D3 (6.5 x 105 IU / kg) every 24 hours for three days. Simultaneously, all groups, except the untreated group (disease), received oral administration of 0.5% CMC, and gliptin and gliflozin, either alone or in combination, depending on their concentration, with one-hour intervals. Seven days later, the intact heart, kidneys, and aorta were excised and isolated under anesthesia. The excised heart was stored in buffered formalin until further histopathological and histomorphological examination.

[0100] 5. Aortic Valve Calcification Analysis

[0101] Each heart sample was transversely sectioned one at a time around the origin / root region of the ascending aorta. Histological sections were then prepared from all 180 samples received. All cross-sectional heart-aortic valve sections were refixed in 10% neutral buffered formalin for 24 hours to prepare histological samples. After embedding the samples in paraffin, two serial sections, 3 to 4 μm thick, were prepared from each paraffin block and stained with hematoxylin and eosin (HE) for general histopathology or von Karolinska (VK) for calcium deposition.

[0102] For histopathological analysis, the mean calcified aortic valve area (% / mm2) was calculated from VK staining. For this analysis, the histopathologist was blinded to group distribution. Histopathological examinations were performed on one section, cross-section, and heart-aortic valve histology area per group, totaling 180 C57BL / 6 mouse heart samples, and statistical analysis was performed.

[0103] 6. Renal Calcification and Aortic Calcification Analysis

[0104] The left kidney was used for analysis of each renal sample, and the descending aorta was used for analysis of the aorta. The excised left kidney and descending aorta were each diluted with chilled saline to 10 times the tissue volume. The tissues were homogenized using a homogenizer and stored in 0.6 N HCl at 4°C for 24 hours to separate the calcium from the tissue. The supernatant was then separated using a refrigerated centrifuge, and the amount of calcium in the supernatant was measured. For accurate measurement, quantification was performed based on the amount of protein.

[0105] result:

[0106] Results for gliptins and gliflozins, both alone and in combination, are shown in the figure.

[0107] Figures 7a and 7b show the extent of inhibition of aortic valve calcification when ipragliptin and dapagliflozin were administered alone and in combination in a mouse model of vitamin D3-induced calcification. When ipragliptin and dapagliflozin were treated together, a synergistic anti-calcification effect was demonstrated compared to either treatment alone.

[0108] Figures 8a and 8b show the extent of inhibition of renal and aortic calcification when idagliflozin and dapagliflozin were administered alone and in combination in a mouse model of vitamin D3-induced calcification.

[0109] As shown in the aortic valve calcification results, when idagliflozin and dapagliflozin were treated together, a synergistic anti-calcific effect was demonstrated compared with either treatment alone.

[0110] Cana: Canagliflozin Dapa: Dapagliflozin Empa:Empagliflozin Ertu: Ergliflozin Evo:Idaglitin Lina: Linagliptin OM:Control Sita: Sitagliptin Saxa: saxagliptin

Claims

1. The use of a combination of a gliflozin family drug and a gliptin family drug for the preparation of a medicine for the prevention or treatment of: kidney disease with renal calcification; vascular stenosis with vascular calcification; or valvular stenosis with valvular calcification, wherein, The glitin drug is evogliptin, and the gliflozin drug is selected from any one of the group consisting of dapagliflozin, ertugliflozin, empagliflozin, and canagliflozin.

2. The use as described in claim 1, wherein the valvular stenosis includes an aortic valve stenosis.

3. The use as described in claim 2, wherein the aortic stenosis includes calcific aortic valve disease (CAVD).

4. The use as described in claim 1, wherein the glibenclamide drug and the glitin drug are administered together.

5. The use as described in claim 1, wherein the glibenclamide and the gliptin are administered at different times.

6. The use as described in claim 1, wherein the combination is provided in the form of a combination formulation.

Images