Application of Bamipine Hydrochloride in the Preparation of Medicines for Treating Aortic Valve Calcification

Abstract

The present invention belongs to the technical field of drugs and disease treatment, and specifically relates to the use of bamipine hydrochloride in the preparation of drugs for treating aortic valve calcification. Based on the existing clinical background and basic research theory, the present invention establishes an inducible human valvular interstitial cell calcification model based on commercial osteogenic induction medium, treats human valvular interstitial cells with PAL at a final concentration of 10 μM and 20 μM, uses andrographolide as a positive drug, and performs alizarin red staining to analyze the effect of PAL treatment on inhibiting osteogenic differentiation of human valvular interstitial cells. At the same time, the osteogenic marker genes ALP, RUNX2 and BMP2 are selected, and quantitative PCR, Western blotting, and immunofluorescence staining experiments are used to investigate the inhibitory effect of 10 μM and 20 μM PAL on human valvular interstitial cell calcification from the two dimensions of genes and proteins.

Description

Technical Field

[0001] The invention belongs to the technical field of medicine and disease treatment, and particularly relates to the application of bamipine hydrochloride in preparing medicine for treating aortic valve calcification. Background Art

[0002] Calcific aortic valve disease (CAVD) is one of the common cardiovascular diseases. Similar to atherosclerotic lesions, the pathogenesis of CAVD is related to many factors, such as endothelial damage, lipid accumulation, inflammation and oxidative stress. The characteristics of early CAVD mainly include inflammatory cell infiltration, accumulation of a large number of resident cells in the valve leaflets, accompanied by fibrotic tissue formation and valve thickening. In this process, valvular interstitial cells (VIC) are considered to be direct participants in aortic valve thickening lesions. Calcified nodules begin to appear after early valve tissue damage. Many studies have shown that the osteogenic differentiation of VIC plays a key role in this pathogenesis of aortic valve calcification. Therefore, how to effectively inhibit the abnormal proliferation of VIC, eliminate its accumulation in aortic valve tissue, and especially inhibit the osteogenic differentiation of VIC is the current research focus on alleviating CAVD disease.

[0003] The natural product Palmatine chloride (PAL) (CAS NO.10605-02-4, purchased from Selleck) is a typical isoquinoline alkaloid, which is mainly found in the rhizomes of natural Chinese herbal medicine plants such as Coptis chinensis, Phellodendron chinense and Nandina domestica, and is an effective ingredient in traditional Chinese herbal medicine. It has pharmacological effects such as clearing away heat and detoxifying, and resisting bacteria, viruses, and fungal infections. In addition, PAL also has anti-tumor activity and can significantly inhibit the oxygen uptake of mouse ascites cancer cells. However, the product application of palmatine hydrochloride for the treatment of aortic valve calcification has not been reported. Summary of the invention

[0004] In order to solve the problem that there is no report on the application of bamipine hydrochloride in the treatment of aortic valve calcification in the prior art, the present invention establishes a model of induced human valvular interstitial cell calcification based on commercial osteogenic induction culture medium for research, and provides the application of bamipine hydrochloride in the preparation of drugs for the treatment of aortic valve calcification.

[0005] To achieve the above object, the present invention adopts the following technical solution:

[0006] The invention provides application of bamipine hydrochloride in preparing medicine for treating aortic valve calcification.

[0007] The medicine uses bamipine hydrochloride as an active ingredient and is supplemented with pharmaceutically acceptable carriers or excipients.

[0008] Preferably, the concentration of bamipine hydrochloride in the drug is 10-160 μM.

[0009] Preferably, the pharmaceutical dosage forms include oral dosage forms and injection dosage forms. The oral dosage forms herein refer to oral regular release dosage forms, including ordinary tablets (tablets, enteric-coated tablets, coated tablets, film-coated tablets, sugar-coated tablets, extract tablets, dispersible tablets, scratched tablets), hard capsules, soft capsules (pills), and enteric-coated capsules. Injection dosage forms include injection solutions, intravenous drip injections, injection suspensions, injection sterile powders, intravenous injections, water injections, injection emulsions, powder injections, injections, sterile powder injections, and freeze-dried powder injections.

[0010] Preferably, the medicine includes tablets, powders, granules, powders and injections.

[0011] Preferably, the concentration of bamipine hydrochloride in the drug is 10-20 μM.

[0012] Preferably, the drug-assisted carrier includes one or more of a binder, a cosolvent, and a dispersant.

[0013] Binders are sticky solid powders or viscous liquids that can aggregate and bind non-sticky or less sticky materials into granules or compress them into shapes, such as polyvidone (PVP), hydroxypropyl methylcellulose (HPMC), sodium carboxymethylcellulose (CMC-Na), and syrup.

[0014] Specifically, ① distilled water: Water itself is non-viscous. When the material contains components that can produce viscosity when in contact with water, distilled water can be used to induce its viscosity and make suitable particles. However, when water is used as a wetting agent, since the material tends to absorb water quickly, it is more likely to be unevenly wetted, and the drying temperature is high, so drugs that are not heat-resistant, easily deteriorate when in contact with water, or are easily soluble in water should not be used. It is best to use low-concentration starch or ethanol instead to overcome the above shortcomings.

[0015] ② Ethanol: For drugs that are sticky in nature but can deteriorate when exposed to water or become too sticky after wetting to make granulation difficult, uneven humidity makes drying difficult or the granules become hard after drying, and the compressed tablets are not easy to disintegrate, etc., ethanol of appropriate concentration can be selected as a wetting agent. The concentration of ethanol depends on the nature of the drug and the ambient temperature, and is generally 30%-70% or higher. And as the concentration of ethanol increases, the viscosity produced after wetting decreases. To a certain extent, ethanol is a dispersant that reduces the viscosity between particles and makes it easier to granulate materials with too strong viscosity. Ethanol is often used as a wetting agent for Chinese medicine extract tablets, but care should be taken to operate quickly to avoid the evaporation of ethanol and the formation of strong sticky lumps.

[0016] ③Polyvidone (PVP): white or milky white powder, non-toxic, high melting point, thermally stable (changes color at 150°C), chemically stable, soluble in water and ethanol to form a viscous colloidal liquid, and a good adhesive.

[0017] Co-solvents Common co-solvents can be divided into three categories: one is certain organic acids and their sodium salts, such as sodium benzoate, sodium salicylate, sodium p-aminobenzoic acid, etc.; one is amide compounds, such as urethane, urea, niacinamide, acetamide, etc.; the other is inorganic salts, such as potassium iodide.

[0018] A dispersant is a surfactant that can quickly wet the surface of drug particles.

[0019] Preferably, the auxiliary materials used in the drug include one or more of starch, dextrin, calcium lactate, citric acid, water and ethanol.

[0020] The present invention also provides a medicine for preventing and treating aortic valve calcification, wherein the medicine is a solution prepared by taking the bamipine hydrochloride as a raw material.

[0021] Preferably, the drug is a solution prepared with bamipine hydrochloride as the main active ingredient and supplemented with water;

[0022] The concentration of the bamipine hydrochloride in the tablet is 10-20 μM.

[0023] Preferably, the treatment of aortic valve calcification is by inhibiting the expression of calcium-related genes. The present invention has found that the above-mentioned drug provided by the present invention can inhibit the expression of calcification-related genes, thereby inhibiting osteoblastic lesions of cells.

[0024] Preferably, the calcium-related genes include BMP2, RUNX2, and ALP. These genes are classic osteogenic differentiation markers, and the high expression of these genes is a key factor in promoting cell calcification.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] The present invention provides the use of bamipine hydrochloride in the preparation of a medicine for treating aortic valve calcification, and provides a new method for treating aortic valve calcification. The present invention is based on the existing clinical background and basic research theory, and establishes a model of induced human valvular interstitial cell calcification based on commercial osteogenic induction culture medium, and uses 10μM and 20μM PAL at final concentrations (final concentration in culture medium) to treat human valvular interstitial cells, and uses andrographolide (Andrographolide, AGP) (CASNo.5508-58-7, Selleck) as a positive drug, and performs alizarin red staining to analyze the effect of PAL treatment on inhibiting osteogenic differentiation of human valvular interstitial cells, and selects osteogenic marker genes ALP, RUNX2 and BMP2, and uses quantitative PCR, Western blotting, and immunofluorescence staining experiments to investigate the inhibitory effect of 10μM and 20μM PAL on human valvular interstitial cell calcification from two dimensions of genes and proteins. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 The cell viability and anti-calcification effect of hVICs treated with PAL in the present invention; wherein, Figure 1 A in it is the chemical structural formula of PAL; Figure 1 B in the figure shows the effect of treatment with 0, 10, 20, 40, 80, and 160 μM PAL on the activity of hVICs; Figure 1 C in the figure is Alizarin Red S staining of hVICs after different treatments: control (normal culture medium), OMI (osteoblast induction medium), OMI+PAL (osteoblast culture medium plus PAL treatment); Figure 1 D is a statistical graph of the results of C; when compared with the control (# vs. control) and OM (* vs. OM), p < 0.05 was considered a significant difference, n = 3.

[0028] Figure 2 The effect of PAL of the present invention on the expression of calcium-related genes / proteins in hVICs induced by OM; wherein, Figure 2 A in the figure is the PCR detection of the mRNA expression levels of osteogenic marker genes RUNX2, BMP2 and ALPL in hVICs after OMI stimulation of hVICs, followed by treatment with 10 μM and 20 μM PAL or without treatment for 48 hours; Figure 2 B in the figure shows the protein expression levels of RUNX2, BMP2 and ALPL in hVICs detected by Western blotting after different PAL treatments for 48 hours; Figure 2 C in the figure shows the protein expression levels of the above genes determined by Western blotting and semi-quantitative analysis; #p<0.05 compared with the control, *p<0.05 compared with OMI. DETAILED DESCRIPTION

[0029] The present invention is described in detail below in conjunction with the accompanying drawings and specific examples, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the following examples are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following examples, unless otherwise specified, can be obtained from commercial sources.

[0030] Example 1: Construction of a human valvular interstitial cell calcification model induced by commercial osteogenic induction medium

[0031] Human valvular interstitial cells (hVICs) with a culture density close to 80% were starved overnight (≥12h) with a 2% volume percentage concentration of FBS+DMEM high-glucose culture medium, and then the starved valvular interstitial cells were induced to calcify with a newly configured osteogenic induction medium. Alizarin red staining was performed 21 days after induction to construct a human valvular interstitial cell calcification model. The constructed human valvular interstitial cell calcification model was used to investigate the effects of different treatments on valvular interstitial cell calcification.

[0032] Among them, osteogenic induction medium: Cyagen, product number: HUXMA-90021.

[0033] 2% FBS+DMEM high-glucose culture medium is DMEM high-glucose culture medium containing 2% FBS in mass percentage.

[0034] Among them, DMEM high-glucose medium was from BOSTER Biotechnology Co., Ltd., catalog number: PYG0073. FBS was bovine serum albumin, Procell, catalog number: 164210.

[0035] The source of human valvular interstitial cells is human primary aortic valve cells. The extraction method can be found in the literature:

[0036] DOI:10.1161 / ATVBAHA.120.314789.

[0037] The specific investigation and test are as follows:

[0038] 1. PAL inhibits the osteogenic differentiation of hVICs induced by commercial osteogenic induction medium (OMI) in vitro

[0039] (1) The human valvular interstitial cell calcification model constructed as in Example 1 was used, and the human valvular interstitial cells were divided into three groups, namely a control group, an OMI group, and an OMI+CDM group.

[0040] The specific grouping and processing methods are as follows:

[0041] Control group: untreated, normal culture medium;

[0042] Untreated group: DMEM high glucose medium + 2% FBS (DMEM high glucose medium is from BOSTER Biotechnology Co., Ltd., catalog number: PYG0073; FBS is bovine serum albumin: Procell Company, catalog number: 164210).

[0043] OMI group; induced by osteogenic medium, Cyagen, catalog number: HUXMA-90021.

[0044] OMI+CDM group: After induction with osteogenic medium, different concentrations of bamipine hydrochloride were added simultaneously.

[0045] Human valvular interstitial cells were treated with different concentrations of bamipine hydrochloride, and IC50 test, CCK8 assay and Alizarin red staining analysis were performed.

[0046] Among them, palmatine hydrochloride (PAL; CAS: 10605-02-4) was purchased from Selleck Company, product catalog number: S2397.

[0047] The specific steps of CCK8 assay and Alizarin red staining analysis are as follows:

[0048] CCK8 assay:

[0049] About 2000 VIC cells were inoculated in each well of a 96-well plate, and three replicate wells were set. After the cells adhered overnight, different treatments were added: ① control group (normal culture medium); ② calcification induction group; ③ different concentrations of PAL + calcification induction group, where the normal culture medium: DMEM high-glucose culture medium + mass percentage concentration of 2% FBS; calcification induction culture medium, Cyagen, catalog number: HUXMA-90021. After 24 hours of treatment, the original culture medium was removed, and serum-free DMEM culture medium containing 10% CCK-8 by volume was added. After incubation in the incubator for 2 hours, the absorbance of each well was measured at a wavelength of 450nm to calculate the cell viability value.

[0050] Alizarin red staining analysis:

[0051] About 5000 VIC cells were inoculated in each well of a 24-well plate, and three replicate wells were set. After the cells adhered overnight, different treatments were added: ① control group (normal culture medium); ② calcification induction group; ③ different concentrations of PAL + calcification induction group, where the normal culture medium: DMEM high glucose culture medium + mass percentage concentration of 2% FBS; calcification induction culture medium, Cyagen, catalog number: HUXMA-90021. After 21 days of treatment, the original culture medium was removed, and Alizarin Red staining solution was added. After staining at room temperature for 30 minutes, the cells were washed 3 times with PBS and then photographed under a microscope.

[0052] The present invention uses CCK-8 method to evaluate the cytotoxicity of PAL to humanVICs (hVICs), and the specific method is as follows: same as above.

[0053] Results Figure 1 B in.

[0054] Figure 1 The results in B showed that cell viability was inhibited as the concentration of PAL increased, and when the concentration of PAL exceeded 40 μM, the viability of hVICs was significantly inhibited.

[0055] Therefore, 10 μM and 20 μM doses of PAL were used in all subsequent in vitro experiments, and andrographolide was used as a positive control. After hVICs were cultured with OMI for 21 days, hVICs were stained with Alizarin Red S to test the degree of cell calcification. The results are shown in Figure 1 C and Figure 1 D in.

[0056] Figure 1 The results showed that different results were observed among the different treatment groups: control group, OMI and OMI plus PAL treatment groups, e.g. Figure 1 C in Figure 1 As shown in D. Compared with the control group, the positive staining of Alizarin Red S after OMI treatment showed a significant difference. Compared with the OMI group, the positive staining of Alizarin Red S induced by OMI in the PAL+OMI group was significantly reduced after PAL treatment. Semi-quantitative analysis showed that the PAL group was reduced by about 2.0 times relative to the OMI group, indicating that PAL treatment can significantly inhibit the calcification of hVICs.

[0057] Among them, the chemical structure of palmatine hydrochloride (PAL) is as follows Figure 1 As shown in A in FIG. , it was purchased from Selleck Company, CAS No.: 10605-02-4; Product Catalog No.: S2397.

[0058] Human valvular interstitial cells are primary human aortic valve cells. For the extraction method, please refer to the literature: DOI:10.1161 / ATVBAHA.120.314789. The English name of human valvular interstitial cells is human VIC, hVICs.

[0059] The source of andrographolide was positive control drug purchased from Selleck Company, CAS No.: 5508-58-7; Product Catalog No.: S2261.

[0060] Osteogenic induction medium (OMI) was purchased from Prosai Company, catalog number: PD-014.

[0061] Alizarin Red S was purchased from Cyagen Biosciences, catalog number: ALIR-10001.

[0062] 2. PAL significantly inhibited the calcification of hVICs by inhibiting the expression of hVICs osteogenic markers induced by OMI

[0063] In order to further study the effect of PAL treatment on the expression of osteogenic marker genes / proteins in hVICs, the present invention uses PCR to detect the expression levels of osteogenic marker genes RUNX2, BMP2 and ALPL in hVICs after different treatments of OMI stimulation. The specific method is as follows: human valvular interstitial cells (hVICs) are divided into 3 groups, namely, control group, OMI group and OMI+PAL group.

[0064] The specific grouping and processing methods are as follows:

[0065] Control group: no calcification induction treatment was performed and cells were cultured in normal medium;

[0066] OMI group; induced by osteogenic medium;

[0067] OMI+CDM group: After induction with osteogenic medium, different concentrations of bamipine hydrochloride were added simultaneously.

[0068] Western blotting was used to detect the expression levels of RUNX2, BMP2, and ALPL in the OMI group and OMI+CDM group after treatment. Figure 2 .

[0069] The results showed that compared with the control group, OMI treatment significantly increased the expression levels of RUNX2, BMP2 and ALPL (#p<0.05); and compared with the OMI-induced group, PAL treatment significantly reversed the upregulation of OMI-induced hVICs osteogenic genes (*p<0.05) ( Figure 2 A).

[0070] Western blotting and semi-quantitative analysis also showed that PAL treatment significantly inhibited the osteogenic differentiation of hVICs induced by OMI compared with the OMI-induced group ( Figure 2 B in 2 and C in 2).

[0071] The above results indicate that PAL can significantly inhibit the calcification of hVICs by inhibiting the expression of osteogenic-specific markers in hVICs.

[0072] In summary, the present invention is based on the existing clinical background and basic research theory, and establishes a model of induced human valvular interstitial cell calcification based on commercial osteogenic induction medium. Human valvular interstitial cells are treated with PAL at a final concentration of 10 μM and 20 μM (final concentration in culture medium). Andrographolide (AGP) (CAS No. 5508-58-7, Selleck) is used as a positive drug, and Alizarin red staining is performed to analyze the effect of PAL treatment on inhibiting osteogenic differentiation of human valvular interstitial cells. At the same time, osteogenic marker genes ALP, RUNX2 and BMP2 are selected, and quantitative PCR, Western blotting, and immunofluorescence staining experiments are used to investigate the inhibitory effects of 10 μM and 20 μM PAL on human valvular interstitial cell calcification from two dimensions of genes and proteins, respectively.

[0073] The problem to be solved by the present invention is how to effectively treat or prevent aortic valve calcification in an individual.

[0074] According to the above-mentioned needs, in one aspect of the present invention, the present invention provides use of bamipine hydrochloride in the preparation of a compound for preventing and / or treating aortic valve calcification in an individual.

[0075] Based on this, the present invention provides the use of bamipine hydrochloride in the preparation of a drug for treating aortic valve calcification, providing a new method for preventing and treating aortic valve calcification. The drug is based on bamipine hydrochloride as an active ingredient and is supplemented with a pharmaceutically acceptable carrier.

[0076] The present invention provides a drug for preventing and treating aortic valve calcification disease with bamipine hydrochloride as an active ingredient, and the drug can be prepared into oral dosage forms, injection dosage forms, inhalation dosage forms and other pharmaceutical dosage forms. Specific pharmaceutical dosage forms include injections, solutions, powders, tablets, pills, aerosols, capsules, and emulsifiers. The drug is prepared by mixing the above-mentioned bamipine hydrochloride with different carriers according to different dosage forms, and processing it into a dosage form that can be used by animals through the preparation process of the corresponding dosage form.

[0077] The carrier refers to a conventional drug carrier in the pharmaceutical field, including but not limited to any one of ethanol, liquid polyethylene glycol, isopropanol, Tween-80, glycerol, sesame oil, water, saline, buffer, micropowder silica gel, cellulose, stearic acid, cellulose derivatives, starch, sucrose, and lactose.

[0078] It should be noted that the drug can be prepared into a suitable dosage form according to actual needs and can be prepared into the drug according to the preparation method corresponding to the corresponding drug dosage form.

[0079] Let's take the solution as an example to illustrate its effect. The specific research is as follows:

[0080] A solution with bamipine hydrochloride as an active ingredient is composed of the following raw materials: 10.0 mg bamipine hydrochloride and 100 mL normal saline. This concentration is converted according to the 20 μM cell concentration, and the usage is expanded 10 times for animals.

[0081] The preparation method of the solution comprises the following steps:

[0082] 10.0 mg of bamipine hydrochloride is dissolved in 100 mL of normal saline, mixed, and packaged to obtain a solution for preventing and treating aortic valve calcification.

[0083] We evaluated the treatment of aortic valve calcification in individuals using the solution prepared above, and the specific steps are as follows:

[0084] The human valvular interstitial cell calcification model cells constructed in Example 1 were treated with the solution prepared above, hVICs were stained with Alizarin Red S to test the degree of cell calcification, and the expression levels of RUNX2, BMP2, and ALPL were detected by Western blotting. The specific methods were the same as in Example 1.

[0085] The results show that the prepared solution can significantly inhibit the calcification of human valvular interstitial cells (hVICs), and can significantly inhibit the calcification of human valvular interstitial cells (hVICs) by inhibiting the expression of osteogenic specific markers in human valvular interstitial cells (hVICs).

[0086] From the above results, it can be seen that the solution with bamipine hydrochloride as the active ingredient provided by the present invention has the effect of inhibiting the calcification of human valvular interstitial cells (hVICs). The results show that the bamipine hydrochloride provided by the present invention has the ability to treat and can provide a material basis for the preparation of drugs for the prevention and treatment of aortic valve calcification.

[0087] It should be noted that when the present invention involves a numerical range, it should be understood that the two endpoints of each numerical range and any numerical value between the two endpoints can be selected. In order to avoid redundancy, the present invention describes a preferred embodiment.

[0088] Although preferred embodiments of the present invention have been described, those skilled in the art may make additional changes and modifications to these embodiments once they understand the basic inventive concepts, and all such changes and modifications fall within the scope of the present invention.

Claims

1. Use of bamipine hydrochloride in the preparation of a drug for treating aortic valve calcification, characterized in that: The drug contains bamipine hydrochloride as an active ingredient and is supplemented with a pharmaceutically acceptable carrier or excipient; The concentration of the bamipine hydrochloride in the drug is 10 to 160 μM.

2. The use according to claim 1, characterized in that: The pharmaceutical dosage forms include oral dosage forms and injection dosage forms.

3. The use according to claim 2, characterized in that: The medicine includes tablets, powders, granules, powders and injections.

4. The use according to claim 3, characterized in that: The concentration of bamipine hydrochloride in the drug is 10-20 μM.

5. The use according to claim 1, characterized in that: The drug-assisted carrier includes one or more of a binder, a cosolvent, and a dispersant; The auxiliary materials used in the medicine include one or more of starch, dextrin, calcium lactate, citric acid, water and ethanol.

6. The use according to claim 1, characterized in that: The method for treating aortic valve calcification is by inhibiting the expression of calcium-related genes.

7. The use according to claim 6, characterized in that: The calcium-related genes include BMP2, RUNX2, and ALP.

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