Application of loratadine in the preparation of drugs for preventing and treating vascular calcification

Abstract

Loratadine is used in the preparation of a drug for preventing and treating vascular calcification. The structural formula of loratadine is as follows: #imgabs0# The loratadine used in the present invention significantly reduced aortic calcification in mice. These experimental results confirm the effectiveness of loratadine in preventing and treating vascular calcification. The drug of the present invention has low toxicity and high safety.

Description

Technical Field

[0001] The invention belongs to the technical field of biomedicine and relates to the application of loratadine in preparing a medicine for preventing and treating vascular calcification. Background Art

[0002] Atherosclerosis is one of the main pathological factors of cardiovascular disease. It is a chronic inflammatory disease that seriously endangers human health. Almost all patients with cardiovascular disease have a certain degree of vascular calcification in the later stages. Vascular calcification increases the risk of cardiovascular events and all-cause mortality. Vascular calcification refers to the abnormal deposition of calcium salts in the arterial walls. This is a dynamic process regulated by multiple factors. Depending on the site of vascular calcification, it can be divided into intimal calcification and medial calcification. Intimal calcification, as the name suggests, is calcification that occurs in the endothelium of the blood vessels. It is also called atherosclerotic calcification. It is now believed that intimal calcification is an active and self-regulating process. ] . The occurrence and development of intimal calcification is similar to the multi-step process of bone formation, which is controlled by complex enzyme and cellular pathways. Vascular smooth muscle cells are the main cell type involved in vascular calcification. They have the potential to produce mineralized matrix and undergo osteoblast differentiation, leading to calcification deposition. At the same time, intimal calcification is often present in advanced atherosclerotic plaques and develops with the development of atherosclerotic lesions. The site of medial calcification is located in the vascular media, which is often accompanied by diseases such as chronic kidney disease (CKD), diabetes and aging. It will reduce vascular compliance and eventually lead to heart failure or hypertension. Vascular medial calcification is also mainly caused by osteoblast-like differentiation of vascular smooth muscle cells. VSMCs are an important component of the vascular wall, providing structural support, regulating vascular tension and vascular remodeling. VSMCs are one of the main sources of osteoblasts involved in the calcification process. VSMCs can dedifferentiate into osteoblast-like cells, acquire the ability to produce mineralized matrix and cause calcium deposition, which is considered to be the initial stage of vascular calcification.

[0003] Histamine is the main mediator of allergic reactions. It is stored in mast cells or basophils until the cells are stimulated by something that allows it to be released from the cells. Allergic reactions occur when histamine interacts with histamine receptors H1, H2, H3 or H4. Among them, drugs targeting the H1 receptor are most commonly used to treat allergies. Loratadine is an H1 histamine receptor antagonist and a second-generation non-sedating antihistamine that is often used to treat allergic symptoms. It is used for allergic rhinitis, acute or chronic urticaria, allergic conjunctivitis, hay fever and other allergic skin diseases. Loratadine can also inhibit the immune release of inflammatory mediators.

[0004] The invention patent with publication number CN113209098A discloses the use of desloratadine and its pharmaceutically acceptable salt in the preparation of a drug for treating peripheral neuropathy. This patent research mainly explores the application of desloratadine in treating allergic reactions and peripheral neuropathy caused by paclitaxel. In the application, desloratadine and its pharmaceutically acceptable salt can inhibit the increase of allergic mediators and vascular permeability caused by paclitaxel, and at the same time relieve the peripheral nerve pain caused by paclitaxel. We explored the effect of loratadine on blood calcium calcification. The main differences are: (1) First, we explored the test of cell activity of loratadine in HASMC cells; (2) For the prevention and treatment of vascular calcification by loratadine, we mainly explored HASMC cells and aortic rings; (3) The purpose of the research and the research method are also different. (4) Vascular calcification refers to the pathological phenomenon of calcium salts deposited in the blood vessel wall, which mainly occurs in arteries. Vascular permeability refers to the ability of the blood vessel wall to allow water molecules, electrolytes and macromolecules (such as proteins) to pass through. Pathologically increased permeability can lead to edema, inflammation, and other vascular-related pathologies. Vascular calcification and vascular permeability are two closely related vascular pathological features with independent physiological mechanisms. Calcification of the tunica media may lead to increased stiffness, weakening the blood vessels' ability to regulate pressure changes, making endothelial cells more susceptible to damage, and increasing permeability. The use of loratadine to treat calcification has primarily been studied in HASMCs. VSMCs can dedifferentiate into osteoblast-like cells, acquiring the ability to produce mineralized matrix and induce calcium deposition, which is considered the initial stage of vascular calcification.

[0005] Currently, no drug is clinically effective in preventing or treating vascular calcification. Furthermore, anticoagulants such as statins and warfarin have the side effect of promoting calcification. Therefore, the search and development of drugs to treat calcification disorders is of great significance. To date, no studies have demonstrated the effectiveness of loratadine in preventing or treating vascular calcification. Summary of the Invention

[0006] The present invention aims to provide an antihistamine drug loratadine for use in a medicine for preventing and treating vascular calcification.

[0007] To achieve the above-mentioned and other related purposes, the present invention provides a technical solution: use of loratadine in the preparation of a drug for preventing and treating vascular calcification.

[0008] The preferred technical solution is: the structural formula of loratadine is as follows:

[0009]

[0010] To achieve the above-mentioned purpose and other related purposes, the present invention provides a technical solution: a pharmaceutical composition for preventing and treating vascular calcification, comprising the aforementioned loratadine and pharmaceutically acceptable excipients.

[0011] The preferred technical solution is that the dosage form of the drug is oral solution, injection, tablet, pill, dispersant, capsule, dripping pill, granule, suspension or emulsion.

[0012] Due to the application of the above technical solution, the present invention has the following advantages compared with the prior art:

[0013] The loratadine of the present invention can significantly inhibit the calcification of smooth muscle cells and the calcification of mouse aortic rings. The new application of the antihistamine loratadine in treating vascular calcification can expand its research in the field of medical technology and provide a new theoretical direction for clinical application. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 These are the results of toxicity tests on HASMC cells at 0μM, 2μM, 5μM, 10μM, and 20μM concentrations of loratadine under normal culture conditions for 24 hours. The results show that loratadine has low cell damage and is highly safe.

[0015] Figure 2 These are the results of toxicity tests on HASMC cells at 0μM, 2μM, 5μM, 10μM, and 20μM loratadine in calcification medium (high phosphate). The results show that even after 6 days of calcification induction, loratadine at a concentration of less than 20μM still causes little damage to cells and is relatively safe.

[0016] Figure 3 The results of Alizarin Red staining of loratadine in the treatment of osteogenic differentiation of smooth muscle cells show that loratadine at 10 μM can have a significant inhibitory effect on the osteogenic differentiation of smooth muscle cells.

[0017] Figure 4 The effect of loratadine on the expression of BMP2 and RUNX2 proteins in smooth muscle cell calcification was shown in Figure 2. The results showed that loratadine could significantly reduce the expression of BMP2 and RUNX2 proteins. *p<0.05, **p<0.01.

[0018] Figure 5 Alizarin red staining of aortic ring calcification in C57BL / 6J mice treated with loratadine. The results showed that loratadine at 10 μM can effectively inhibit aortic ring calcification.

[0019] Figure 6The effect of loratadine on the RUNX2 immunofluorescence intensity of aortic ring calcification in C57BL / 6J mice was studied. The results showed that loratadine could effectively reduce the expression of RUNX2 in phosphate-induced aortic rings. DETAILED DESCRIPTION

[0020] The following describes the implementation of the present invention through specific embodiments. People skilled in the art can easily understand other advantages and effects of the present invention from the contents disclosed in this specification.

[0021] See also Figure 1-6 . It should be noted that the structures, proportions, sizes, etc. illustrated in the drawings of this specification are only used to match the contents disclosed in the specification for people familiar with this technology to understand and read, and are not used to limit the limiting conditions for the implementation of the present invention. Therefore, they have no substantive technical significance. Any modification of the structure, change in the proportional relationship or adjustment of the size should still fall within the scope of the technical content disclosed by the present invention without affecting the efficacy and purpose that can be achieved by the present invention. At the same time, the terms such as "upper", "lower", "left", "right", "middle" and "one" quoted in this specification are only for the convenience of description, and are not used to limit the scope of the implementation of the present invention. Changes or adjustments in their relative relationships should also be regarded as the scope of the implementation of the present invention without substantially changing the technical content.

[0022] Unless otherwise specified, the reagents or materials described in the following examples are commercially available.

[0023] Example 1: Use of loratadine in the preparation of a medicament for preventing and treating vascular calcification

[0024] Use of loratadine (CAS No. 79794-75-5) in preparing a drug for preventing and treating vascular calcification.

[0025] After a large number of experiments, the present invention determined that loratadine prevents and treats phosphate-induced vascular calcification of aortic rings and alleviates the osteogenic differentiation of vascular smooth muscle cells triggered by high phosphate.

[0026] Preferably, loratadine can be used to prepare drugs for treating vascular calcification.

[0027] The drug for treating vascular calcification uses loratadine as an effective ingredient.

[0028] A drug for treating vascular calcification, comprising a pharmaceutically acceptable material of loratadine.

[0029] A preferred embodiment is that the dosage of loratadine is 2-20 μM.

[0030] In a more preferred embodiment, the dosage of loratadine is 10 μM.

[0031] The loratadine of the present invention can significantly inhibit phosphate-induced vascular calcification. The new application of loratadine in treating vascular calcification can expand its research in the field of medical technology and provide a new theoretical direction for clinical application.

[0032] The use of loratadine in the preparation of a drug for preventing and treating vascular calcification, wherein the loratadine (CAS No. 79794-75-5) used has the following structural formula:

[0033]

[0034] 1. Determination of toxicity of loratadine on cells

[0035] (1) Culture 2-10 generations of human aortic smooth muscle cells, prepare a cell suspension in a culture medium containing 10% fetal bovine serum, and seed 5000 cells per well in a 96-well plate. The volume of the cell suspension per well is 200 μL.

[0036] (2) Five groups were set up: normal group, loratadine 2 μM, loratadine 5 μM, loratadine 10 μM, and loratadine 20 μM. Six replicate wells were set up for each drug group, and drug treatment was carried out for 24 h.

[0037] (3) After the incubation period, add 20 μL of MTT solution (5 mg / mL, prepared in double-free medium) to each well. After 4 hours of incubation, terminate the incubation period. Carefully aspirate the medium in each well and add 150 μL of DMSO to each well. Shake the wells at room temperature for 15 minutes to allow the crystals to fully dissolve.

[0038] (4) Select a wavelength of 550 nm, detect the light absorbance of each well on an enzyme-linked immunosorbent assay (ELISA) reader, and record the results.

[0039] 2. Determination of toxicity of loratadine to cells in calcified culture medium

[0040] 1. Preparation of calcification medium

[0041] Calcification of smooth muscle cells was induced by adding appropriate concentrations of inorganic phosphate (Na2HPO4 and NaH2PO4 in a ratio of 1:2) to DMEM. The induction process lasted for 6 days, and fresh medium was replaced every two days.

[0042] 2. Determination of toxicity of loratadine to cells in calcified culture medium

[0043] (1) Culture 2-10 generations of human aortic smooth muscle cells, prepare a cell suspension in a culture medium containing 10% fetal bovine serum, and seed 1000 cells per well in a 96-well plate. The volume of the cell suspension per well is 200 μL.

[0044] (2) Six groups were set up: normal group, calcification group, calcified loratadine 2 μM, calcified loratadine 5 μM, calcified loratadine 10 μM, and calcified loratadine 20 μM. Six replicate wells were set up for each drug group. Culture was carried out under the same culture conditions for 6 days.

[0045] (3) After the incubation period, add 20 μL of MTT solution (5 mg / mL, prepared in double-free medium) to each well. After 4 hours of incubation, terminate the incubation period. Carefully aspirate the medium in each well and add 150 μL of DMSO to each well. Shake the wells at room temperature for 15 minutes to allow the crystals to fully dissolve.

[0046] (4) Select a wavelength of 550 nm, detect the light absorbance of each well on an enzyme-linked immunosorbent assay (ELISA) reader, and record the results.

[0047] 3. Alizarin red staining of osteogenic differentiation of smooth muscle cells treated with loratadine

[0048] (1) Add 500 cells to each well of a 12-well plate and allow the cells to adhere and stabilize overnight.

[0049] (2) Prepare the calcification medium as described in the second part above. In the calcification medium, treat the cells with 5 or 10 μM loratadine.

[0050] (3) Place the cells in an incubator and culture them. Change the culture medium every two days for a total of 6 days.

[0051] (4) After incubation, remove the culture medium and fix the cells with 4% paraformaldehyde for 30 minutes. Wash the cells three times with PBS for 5 minutes each. Stain the cells with Alizarin Red solution for 30 minutes and then wash them three times with acidic PBS for 5 minutes each. Take photos.

[0052] 4. Loratadine reduces the expression levels of BMP2 and RUNX2, key calcification proteins in cells

[0053] (1) Protein extraction: Cells were seeded in 6-well plates. The cells were treated with the following: normal group, calcification group, calcified loratadine 2 μM, calcified loratadine 5 μM, calcified loratadine 10 μM, and calcified loratadine 20 μM. Six days later, the cells were washed twice with PBS. After complete lysis with protein lysis buffer, the cells were centrifuged at 12,000 rpm for 10 min at 4°C. The supernatant was collected and transferred to a -20°C refrigerator for storage.

[0054] (2) The total protein concentration was determined by the BCA method, 30 μg of total protein was quantified, and the protein sample loading volume was calculated.

[0055] (3) Use SDS-polyacrylamide gel electrophoresis at a constant voltage of 124V for about 90 minutes.

[0056] (4) Transfer the protein blot to the NC membrane under a constant voltage of 100 V.

[0057] (5) Obtain the target band according to the protein molecular weight and block with 5% skim milk at room temperature for 1 h.

[0058] (6) After blocking, add the corresponding primary antibody (BMP2, RUNX2) and incubate at 4°C overnight.

[0059] (7) The next day, the primary antibody was recovered and the strips were washed three times with PBST for 8 min each time. The strips were then incubated in the secondary antibody at room temperature.

[0060] (8) Recover the secondary antibody, wash the strips with PBST three times, 8 min each time, and detect the immunoblot using an ECL chemiluminescence kit and imaging equipment.

[0061] 5. Alizarin Red Staining of Phosphate-Induced Aortic Rings of C57BL / 6J Mice by Loratadine

[0062] 1. Induction of arterial ring calcification

[0063] (1) According to experimental needs, the experimental mice were killed and the thoracic aorta was dissected.

[0064] (2) The aorta is dissected and trimmed using a surgical microscope.

[0065] (3) The thin straight part of the aorta was cut into pieces (about 5 mm) and placed in a well plate with complete culture medium overnight.

[0066] (4) According to the experimental needs, the arterial rings were treated experimentally; the groups were set as follows: normal group, calcification group, calcification + loratadine (5 μM), calcification + loratadine (10 μM).

[0067] (4) Add calcification medium and the calcification induction time is 10-15 days.

[0068] (5) After calcification induction, the slices were embedded in OCT and placed in a refrigerator for later use.

[0069] 2. Alizarin red staining of arterial ring

[0070] (1) Thaw frozen sections from the refrigerator at room temperature for 30 minutes.

[0071] (2) Soak the slices in PBS buffer for 30 min and remove OCT.

[0072] (3) Soak the sections in Alizarin Red solution for 30 minutes.

[0073] (4) Wash the sections three times in acidic PBS.

[0074] (5) Observe and take photos under a microscope. The staining results are shown in Figure 5 .

[0075] 6. Immunofluorescence staining of mouse aortic root to detect RUNX2 expression in aortic rings

[0076] (1) Immunofluorescence staining: The frozen sections obtained in the fifth part were taken out of the refrigerator and thawed, and then kept at room temperature for 30 min and soaked in PBS for 5 min.

[0077] (2) Soak in 0.1% Triton-100 for 10 minutes to break the membrane, and then soak in PBS for 5 minutes, twice each time.

[0078] (3) Add 2% BSA for blocking, incubate at room temperature for 10 to 15 minutes, and then pour off.

[0079] (4) Add primary antibody dropwise and incubate at 4°C overnight. Rinse with PBS three times, 10 min each time.

[0080] (5) Add fluorescently labeled secondary antibody and incubate at 37°C for 45 min. Rinse with PBS three times, 10 min each time.

[0081] (6) Add DAPI staining solution, incubate at room temperature for 15 minutes, and then soak and rinse with PBS three times, each time for 10 minutes.

[0082] (7) Sealing: Use anti-fluorescence quenching sealing agent to seal the slides, place them horizontally in the dark for more than 2 hours, and then take photos under a fluorescence microscope after drying. The staining results are shown in the figure. Figure 6 .

[0083] The above results indicate that the loratadine of the present invention can significantly treat high phosphorus-induced vascular cell calcification and mouse aortic ring calcification. Therefore, loratadine has a broad application prospect in the prevention and treatment of vascular calcification.

[0084] The above description is only used to explain the preferred embodiments of the present invention and is not intended to limit the present invention in any form. Therefore, any modifications or changes made to the present invention under the same inventive spirit should still be included in the scope of protection intended by the present invention.

Claims

1. Use of loratadine in the preparation of drugs for preventing and treating vascular calcification.

2. Use of loratadine according to claim 1 in the preparation of a drug for preventing and treating vascular calcification, characterized in that: The structural formula of the loratadine is as follows: 。 3. The use of loratadine according to claim 1 in the preparation of a drug for preventing and treating vascular calcification, characterized in that : The drug includes pharmaceutically acceptable excipients.

4. The use of loratadine according to claim 1 in the preparation of a drug for preventing and treating vascular calcification, characterized in that: The dosage form of the drug is oral solution, injection, tablet, dispersant, capsule, dripping pill, granule, suspension or emulsion.

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