Use of benaluopeptin in the preparation of a medicament for treating atherosclerosis

Abstract

The application belongs to the field of new application of drugs, and more particularly relates to application of benalupeptide in preparation of drugs for treating atherosclerosis. The application proves through animal experiments and cell experiments that benalupeptide can significantly reduce lipid deposition in arterial intima, reduce plaque area, and down-regulate expression of intercellular adhesion molecule-1 (ICAM-1), thereby improving vascular endothelial function and delaying progression of atherosclerosis. The application expands the clinical application range of benalupeptide, provides a new treatment strategy for atherosclerosis, and has potential clinical value and good application prospect.

Description

Technical Field

[0001] This invention belongs to the field of new uses of pharmaceuticals, and more specifically, relates to the application of benaglutide in the preparation of drugs for treating atherosclerosis. Background Technology

[0002] Benaglutide is a GLP-1 receptor agonist that improves insulin resistance, lowers blood sugar, and reduces weight. Atherosclerosis is the main pathological basis of cardiovascular and cerebrovascular diseases such as coronary heart disease and stroke. Its pathogenesis is closely related to lipid metabolism disorders, inflammatory responses, vascular endothelial dysfunction, and abnormal proliferation of smooth muscle cells. Currently, statins and PCSK9 inhibitors are commonly used in clinical practice to lower lipids, but they still have limitations in efficacy, adverse reactions, and insufficient improvement in inflammation.

[0003] Currently, the main clinical application of benaglutide is to improve glycemic control in adults with type 2 diabetes. Additionally, benaglutide is used as a weight-loss drug by slowing gastric emptying and inducing a feeling of fullness to reduce food intake. Further research is needed to expand the application range of benaglutide and explore its other pharmaceutical uses. Summary of the Invention

[0004] The purpose of this invention is to provide the application of benaglutide in the preparation of drugs for treating atherosclerosis.

[0005] This invention provides the application of benaglutide in the preparation of drugs for treating atherosclerosis.

[0006] This invention designs an atherosclerosis model in ApoE⁻ / ⁻ mice by partially ligating the left carotid artery in conjunction with a high-fat diet, simulating local plaque formation under low shear stress. Systematic administration of benaglutide, combined with tissue staining and image analysis, validated its inhibitory effect on plaque area. Simultaneously, using a fluid shear force loading system to simulate the state of vascular endothelium in a blood flow environment, combined with Western blot technology, it was found that benaglutide can downregulate the expression of ICAM-1 in endothelial cells, suggesting that it can exert its effects by improving endothelial function and inhibiting inflammatory adhesion responses. This study systematically reveals the anti-atherosclerotic potential of benaglutide from the whole animal to the cellular level, demonstrating its feasibility for practical application.

[0007] Furthermore, the drug is used to reduce lipid deposition in blood vessels.

[0008] Furthermore, the drug is used to inhibit the expression of intercellular adhesion molecule-1 in vascular endothelial cells.

[0009] Furthermore, the drug is made from the benaglutide and pharmaceutically acceptable excipients.

[0010] Furthermore, the pharmaceutically acceptable excipients are natural, semi-natural, or fully synthetic.

[0011] Furthermore, the drug also includes a pharmaceutically acceptable carrier.

[0012] Furthermore, the drug is administered orally or by injection.

[0013] Furthermore, the dosage form of the drug is a solid dosage form or a solution dosage form. The solid dosage form includes granules, tablets, capsules, pills, and drop pills, and the solution dosage form includes oral liquid preparations, oral enemas, and injectable dosage forms.

[0014] Furthermore, the content of benaglutide in the drug is 0.1wt% to 99wt%.

[0015] The present invention has the following beneficial effects: Existing drugs for treating atherosclerosis, such as statins, mainly exert their pharmacological effects by targeting a single point, such as inhibiting cholesterol synthesis. Benaglutide, on the other hand, can achieve multiple effects through multi-target synergy: weight loss, lipid reduction, blood pressure reduction, and anti-inflammation, thus addressing multiple causes of atherosclerosis and achieving "one drug, multiple effects." Furthermore, as the world's first fully human agonist with an amino acid sequence 100% identical to that of natural human GLP-1, it has low immunogenicity, does not increase resting heart rate, and has milder gastrointestinal side effects due to its short half-life and lack of accumulation in the body, providing better safety and tolerability for atherosclerosis patients who require long-term medication. Attached Figure Description

[0016] Figure 1 This is a statistical chart of patch area.

[0017] Figure 2 This is a graph showing the electrophoresis results of the ICAM-1 protein.

[0018] Figure 3 This is a graph showing the electrophoresis results of GAPDH protein. Detailed Implementation

[0019] The present invention will now be described in detail with reference to the accompanying drawings and specific embodiments, but this should not be construed as limiting the invention. Unless otherwise specified, the technical means used in the following embodiments are conventional means well known to those skilled in the art, and the materials, reagents, etc. used in the following embodiments are commercially available unless otherwise specified.

[0020] Benaglutide's CAS number is 123475-27-4.

[0021] Example 1: Animal experiments verify the anti-atherosclerotic effect of benaglutide.

[0022] 1. Animal grouping: Eight-week-old male ApoE⁻ / ⁻ mice were selected and randomly divided into two groups after one week of acclimatization: the model control group (CON) and the benaglutide intervention group (BN). All mice were housed separately under the same environmental conditions.

[0023] 2. Surgical modeling: All mice were anesthetized with afotin injection, and partial ligation of the left carotid artery was performed. The specific steps were as follows: the occipital artery, superior thyroid artery, and external carotid artery of the left common carotid artery were separated and ligated, leaving only the internal carotid artery patent, to create a stable low-shear stress blood flow environment to accelerate local plaque formation.

[0024] 3. After surgery, all mice were allowed free access to a high-fat diet (containing 1.25% cholesterol and 40% fat) for 3 weeks. The benaglutide intervention group received subcutaneous injections of benaglutide solution (150 μg / kg / day, divided into three equal doses) at 8:00, 14:00, and 20:00 daily, while the model control group received an equal volume of sterile saline simultaneously.

[0025] 4. Sample Collection and Processing: Samples were collected 3 weeks later. After anesthesia and euthanasia, the heart was perfused with physiological saline, and the left carotid artery was quickly removed. The tissue was directionally embedded in OCT embedding medium, flash-frozen on dry ice, and stored at -20°C. 5μm cross-sectional sections were continuously cut using a cryostat and attached to anti-detachment slides.

[0026] 5. Histochemical staining: After the sections were warmed to room temperature, the procedure was performed according to the Oil Red O staining kit instructions. The sections were fixed with the kit's internal fixative, incubated with the dedicated staining working solution in the dark, and stained with differentiation solution for differentiation. Finally, the cell nuclei were counterstained with hematoxylin, and the sections were mounted with glycerol gelatin.

[0027] 6. Image Analysis and Results: After 24 hours of mounting, the sections were scanned panoramically under a 40x objective lens using a digital pathology slide scanner. ImageJ software was used to manually delineate the vascular intima boundary, and the plaque area (Oil Red O positive red area) and lumen area were quantitatively analyzed.

[0028] from Figure 1 As can be seen, compared with the model group, the benaglutide group showed a reduction in aortic plaque area in Oil Red O staining. The ratio of plaque area to lumen area in the benaglutide group was reduced by about 10% compared with the model group, indicating that it has an inhibitory effect on vascular lipid deposition.

[0029] Example 2: Benalutin's effect on improving vascular endothelial function.

[0030] I. Experimental Methods.

[0031] Human umbilical vein endothelial cells (Huvecs) were seeded at a rate of 50,000 cells per well into µ-Slide I 0.4 Luer cell slides (ibid). After the cells had fully adhered, the slide inlets and outlets were connected to a tubing system pre-filled with culture medium. The cells were divided into a control group (NC) and a treatment group (NC+BN). The control group received 12 mL of DMEM culture medium, while the treatment group received 12 mL of DMEM culture medium with a benaglutide concentration of 100 nmol / L.

[0032] Start the pump, adjust the parameters to achieve a flow rate of 4.34, and incubate continuously in an incubator at 37°C and 5% CO2 for 24 hours. After 24 hours, stop the pump, remove the cell slide, and slowly inject 200 μL of pre-cooled sterile PBS from the inlet end using a pipette to rinse the channel. Then, aspirate the waste liquid from the outlet end. Repeat twice. After aspirating the PBS, add 120 μL of potent RIPA lysis buffer containing protease inhibitors and phosphatase inhibitors. Add the lysis buffer from one end using a pipette and immediately gently and repeatedly pipette the buffer into the channel several times to ensure that the lysis buffer is in full contact with all cells. Incubate the entire slide on ice for ten minutes. Then, aspirate the lysis buffer with a pipette and transfer it into a 1.5 mL EP centrifuge tube. Insert the ultrasonic probe vertically and gently into the centrifuge tube, start the ultrasonic instrument, perform pulse processing, and after the ultrasonic treatment is completed, let it stand on ice for two minutes, centrifuge at 13000 rpm for 15 minutes at 4°C, aspirate the supernatant and transfer it to a new 1.5 ml EP centrifuge tube, add loading buffer at a volume ratio of 1:4, and heat in a metal bath at 95°C for 10 minutes. Preparation of SDS-PAGE gel: First, pour the separating gel, flatten the upper layer with anhydrous ethanol, pour off the water layer after solidification, then pour the stacking gel and immediately insert the comb. Install the solidified gel into the electrophoresis tank, add 1x electrophoresis buffer, add protein pre-stained marker to the first well using a micropipette, and add protein samples from the control group and the drug group to the subsequent wells in sequence. First, use a constant voltage of 80V to compress the sample into a thin line in the stacking gel. When the band enters the separating gel, switch to a constant voltage of 120V until the bromophenol blue indicator reaches near the bottom of the gel. Prepare the "sandwich": In the transfer buffer, assemble the membrane on the transfer plate in the following order (from negative to positive): sponge → filter paper → gel → NC membrane → filter paper → sponge. Gently roll the membrane with a glass rod to ensure that there are no air bubbles between each layer. Place the "sandwich" in the transfer tank, add the pre-cooled transfer buffer and ice box, and transfer the membrane at a constant current of 400mA in an ice bath for 60 minutes. After transfer, remove the membrane and rinse it once with TBST. Block it in a general protein-free blocking buffer at room temperature for 1 hour with shaking. Cut the membrane according to the protein content. Put the high protein content membrane into the primary antibody ICAM-1 / CD54 Monoclonal antibody (Cat No. 60299-1-Ig, Proteintech) dilution buffer, and put the low protein content membrane into the GAPDH antibody dilution buffer. Incubate overnight at 4°C. After incubation, wash the membrane three times with TBST on a shaker for 10 minutes each time to remove unbound primary antibody. Put the membrane into the secondary antibody Goat Anti-Mouse IgG HRP (Cat No. M21001, Abmart) dilution buffer and incubate at room temperature with shaking for 1 hour. Wash the membrane three times with TBST on a shaker for 10 minutes each time to completely remove unbound secondary antibody.

[0033] Development: Mix equal volumes of chemiluminescent substrate solution A and solution B. Place the membrane face up in the dark chamber and evenly drop the mixed ECL reagent, ensuring it covers the entire membrane. Incubate for 1-2 minutes, absorb excess liquid with filter paper, wrap the membrane with plastic wrap, and place it in the dark chamber of the chemiluminescence imager. Adjust the exposure time to capture a clear image with moderate signal intensity and a clean background. Use ImageJ software to analyze the target band.

[0034] II. Experimental Results.

[0035] High expression of ICAM-1 is a marker event for atherosclerotic plaque formation, and the results are as follows: Figure 2 As shown, the expression of intercellular adhesion molecule-1 (ICAM-1) in the benaglutide group was downregulated, suggesting that it can improve vascular endothelial function and reduce endothelial cell dysfunction.

[0036] The electrophoresis results of GAPDH protein are as follows: Figure 3 As shown, the bands are in the same position, have similar brightness, and the background is clean with no extraneous bands, indicating that the sample loading is balanced and the internal control is qualified.

[0037] It should be noted that when numerical ranges are mentioned in the claims of this invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. To avoid redundancy, the present invention describes preferred embodiments.

[0038] Although preferred embodiments of the invention have been described, those skilled in the art, upon learning the basic inventive concept, can make other changes and modifications to these embodiments. Therefore, the appended claims are intended to be interpreted as including both the preferred embodiments and all changes and modifications falling within the scope of the invention.

[0039] Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Application of benaglutide in the preparation of drugs for treating atherosclerosis.

2. The application according to claim 1, characterized in that, The drug is used to reduce lipid deposition in blood vessels.

3. The application according to claim 1, characterized in that, The drug is used to inhibit the expression of intercellular adhesion molecule-1 in vascular endothelial cells.

4. The application according to claim 1, characterized in that, The drug is made from the benaglutide and pharmaceutically acceptable excipients.

5. The application according to claim 4, characterized in that, The pharmaceutically acceptable excipients are natural, semi-natural, or fully synthetic.

6. The application according to claim 4, characterized in that, The drug can be administered orally or by injection.

7. The application according to claim 4, characterized in that, The drug is available in either a solid dosage form or a solution dosage form. The solid dosage forms include granules, tablets, capsules, pills, and drop pills, while the solution dosage forms include oral liquid preparations, oral enemas, and injectable dosage forms.

8. The application according to claim 4, characterized in that, The content of benaglutide in the drug is 0.1wt% to 99wt%.

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