A combined medicament for treating atherosclerosis and use thereof

Abstract

The present application relates to the technical field of biological pharmacy, in particular to a combined medicine for treating atherosclerosis and application thereof. The present application provides application of 1,2,3,6-tetra-O-galloyl-beta-D-glucose combined with fenofibrate in preparation of a medicine for treating atherosclerosis. Through synergistic effect of the two, a dual effect of "highly effective lipid-lowering + targeted anti-inflammation" is realized, NLRP3 inflammasome activation is more significantly inhibited, cholesterol level is reduced, expression of VCAM1 and MCP1 is down-regulated to inhibit vascular immune infiltration, aortic root lipid plaque formation is synergistically reduced, a vicious cycle of "lipid accumulation-inflammation activation" in the process of atherosclerosis is highly effectively blocked, and the problem of limited treatment effect of existing single medicine is solved, thereby providing a more optimal treatment scheme for atherosclerosis.

Description

Technical Field

[0001] This invention relates to the field of biopharmaceutical technology, specifically to a combination drug for treating atherosclerosis and its application. Background Technology

[0002] Atherosclerosis is the most serious complication of hyperlipidemia, and its core pathological mechanism is a vicious cycle of lipid accumulation and inflammatory response mediated by oxLDL. Studies have shown that oxLDL can promote intracellular cholesterol accumulation, while activating the NLRP3 inflammasome, upregulating the expression of inflammatory factors such as VCAM1 and MCP1, inducing immune infiltration, accelerating the formation of lipid plaques in blood vessels, and ultimately leading to the progression of atherosclerosis.

[0003] Currently, the core drugs for the clinical treatment of atherosclerosis are mainly lipid-lowering drugs. Fenofibrate, as a classic lipid-lowering drug of the fibrate class, can effectively regulate the blood lipid profile and reduce cholesterol levels, but it lacks anti-inflammatory effects and cannot fundamentally block the synergistic pathological process of inflammation and lipid accumulation. Moreover, the therapeutic effect of single-drug therapy on atherosclerosis is limited.

[0004] Other conventional combination therapy regimens mostly involve the combination of different lipid-lowering drugs (such as statins combined with fibrates). Their focus is still limited to lipid metabolism regulation and lacks targeted anti-inflammatory intervention. Therefore, they cannot block the inflammatory response induced by oxLDL, and the synergistic treatment effect is not ideal.

[0005] Therefore, there is an urgent need to develop combination therapy regimens that have synergistic effects of lowering lipids and reducing inflammation in order to improve the treatment efficacy of atherosclerosis. Summary of the Invention

[0006] To address the above problems, this invention provides a combination drug for treating atherosclerosis and its application.

[0007] This invention is achieved through the following technical solution: A combination drug for treating atherosclerosis, the combination drug comprising 1,2,3,6-tetra-O-galloyl-β-D-glucose and fenofibrate; wherein the mass ratio of 1,2,3,6-tetra-O-galloyl-β-D-glucose to fenofibrate is 1:2~4.

[0008] Preferably, the mass ratio of 1,2,3,6-tetra-O-galloyl-β-D-glucose to fenofibrate is 1:3.

[0009] Preferably, the combined drug can be formulated into a clinically acceptable formulation together with or separately from pharmaceutically acceptable excipients.

[0010] Preferably, the pharmaceutically acceptable excipient is a mixture of corn oil and DMSO.

[0011] Preferably, the volume ratio of corn oil to DMSO is 9:1.

[0012] Preferably, the dosage form of the combined drugs is an oral preparation, an infusion, or an injection.

[0013] Preferably, the combined drug and pharmaceutically acceptable excipients are formulated into a clinically acceptable oral formulation.

[0014] Preferably, when the 1,2,3,6-tetra-O-galloyl-β-D-glucose and fenofibrate are formulated with pharmaceutically acceptable excipients into clinically acceptable formulations, a combination formulation of an injectable and an oral formulation is formed.

[0015] The application of the combined drugs in the preparation of formulations for treating atherosclerosis.

[0016] Compared with the prior art, the present invention has the following beneficial effects: This invention combines TeGG with fenofibrate as the active ingredient to prepare a drug for treating atherosclerosis. Utilizing the complementary targets and synergistic effects of these two drugs, it achieves a dual and highly effective blockade of the pathological process of atherosclerosis, yielding the following significant synergistic effects compared to existing single-drug regimens: 1. Synergistic inhibition of NLRP3 inflammatory activation: The combined use of drugs has a significantly better inhibitory effect on oxLDL-induced NLRP3 inflammasome activation than TeGG or fenofibrate alone, and can more effectively reduce cleaved capsase1 expression and IL-1β secretion, thus blocking the inflammatory response mediated by NLRP3 inflammasome activation at its source.

[0017] 2. Synergistic inhibition of vascular inflammatory infiltration: Compared with TeGG monotherapy, combination therapy can more significantly downregulate HFD feeding. Ldlr - / - The levels of VCAM1 and MCP1 in the aortic root of mice were significantly reduced, effectively inhibiting vascular immune infiltration and reducing damage to the vascular wall caused by inflammatory factors.

[0018] 3. Synergistic enhancement of lipid-lowering effect: Combined use of drugs can reduce intracellular and in vivo cholesterol levels more significantly, with a lipid-lowering effect that is significantly better than single-drug use, and more effectively improves blood lipid profile and reduces lipid accumulation.

[0019] 4. Synergistic reduction of lipid plaque formation: Combined medication can significantly reduce HFD feeding Ldlr - / - The area of ​​lipid plaques in the aortic root of mice showed a significantly better inhibitory and regressive effect on atherosclerotic plaques than single-drug therapy, effectively delaying the progression of atherosclerosis.

[0020] 5. Achieve dual pathological blockade: For the first time, the precise combination of "lipid-lowering drug (fenofibrate) + oxLDL-targeted anti-inflammatory drug (TeGG)" is achieved, simultaneously blocking the two pathological pathways of oxLDL-mediated lipid accumulation and inflammation activation, breaking the vicious cycle between the two. Attached Figure Description

[0021] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0022] Figure 1 This is a graph showing the detection results of the combined effect of TeGG and fenofibrate at the cellular level in this invention; Figure 1 In the image, A shows the results of IL-1β secretion level detection in cell supernatant; B shows the results of intracellular cholesterol accumulation detection.

[0023] Figure 2 This is a schematic diagram of the animal model of the present invention.

[0024] Figure 3 This is a graph showing the results of animal-level detection of the regulation of NLRP3 inflammasome activation levels by the combined application of TeGG and fenofibrate. Figure 3 In the diagram, A represents the results of caspase 1 activation level detection; B represents the results of serum IL-1β secretion level detection.

[0025] Figure 4 This is a diagram showing the results of the combined application of TeGG and fenofibrate in regulating aortic valve immune infiltration according to the present invention. Figure 4 In the image, A shows the immunofluorescence results of VCAM1 and MCP1 in the aortic valve; B shows the quantitative immunofluorescence statistics of VCAM1 in the aortic valve; and C shows the quantitative immunofluorescence statistics of MCP1.

[0026] Figure 5 The figure shows the results of regulating serum cholesterol levels in mice using the combined application of TeGG and fenofibrate according to the present invention.

[0027] Figure 6 This is a diagram showing the results of the combined application of TeGG and fenofibrate in regulating lipid plaque formation in the aortic root of mice according to the present invention. Figure 6 In the image, A shows the Oil Red Spectrophotometry results of lipid plaques in the aortic root of mice; B shows the quantitative statistical graph of lipid plaques in the aortic root of mice. Detailed Implementation

[0028] To facilitate understanding of the present invention, a more comprehensive description is provided below, along with preferred embodiments. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided to provide a thorough and complete understanding of the disclosure of the present invention.

[0029] Unless otherwise defined, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains. The terminology used in this invention and in its specification is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

[0030] The inventive concept of this invention is as follows: The existing treatment options for atherosclerosis and their limitations are as follows: Fenofibrate, as a mainstream lipid-lowering drug in clinical practice, can effectively improve the lipid profile and reduce lipid levels when used as a single drug. However, it can only intervene in the progression of atherosclerosis from the lipid-lowering level. It does not directly inhibit the activation of NLRP3 inflammasome and its mediated immune infiltration, and it is difficult to block the pathological cycle of "lipid accumulation-inflammatory activation". Therefore, its inhibitory effect on lipid plaques is limited.

[0031] Other conventional combination therapies mostly involve the combination of different lipid-lowering drugs (such as statins + fibrates), which only focus on lipid metabolism regulation and do not have targeted anti-inflammatory target intervention. They still cannot block oxLDL-mediated inflammatory responses, and the synergistic treatment effect is poor.

[0032] Based on this, the present invention provides the application of 1,2,3,6-tetragalloylglucose (TeGG) in combination with fenofibrate in the preparation of drugs for treating atherosclerosis. Through the synergistic effect of the two, a dual effect of "powerful lipid-lowering + targeted anti-inflammatory" is achieved, significantly inhibiting NLRP3 inflammasome activation and reducing cholesterol levels. Simultaneously, it downregulates VCAM1 and MCP1 expression to inhibit vascular immune infiltration, synergistically reducing lipid plaque formation in the aorta and aortic root, and effectively blocking the vicious cycle of "lipid accumulation-inflammatory activation" mediated by oxLDL. This addresses the problem of limited efficacy of existing single-drug treatments and provides a superior treatment option for atherosclerosis.

[0033] The beneficial effects of the present invention will be illustrated below through specific embodiments.

[0034] 1,2,3,6-Tetragalloylglucose (TeGG) was purchased from MedChemExpress, catalog number HY-111832. Fenofibrate was purchased from Sigma, product number F6020; The RPMI 1640 was purchased from Procell Systems, part number PM150110; oxLDL was purchased from Yeasen Biotechnology, catalog number 20605ES05.

[0035] Example 1: Validation of the synergistic effect of TeGG and fenofibrate at the cellular level Cell Culture and Treatment: This invention uses the human monocytic leukemia cell line (THP-1) as an in vitro experimental model. Cells are seeded in RPMI 1640 complete medium containing 10% fetal bovine serum and cultured routinely in a cell culture incubator at 37°C, 5% CO2, and saturated humidity. Cell morphology and growth status are observed daily. Cells in the logarithmic growth phase and in good condition are induced to differentiate with phorbol ester (100 nM) for 24 hours, and then the medium is replaced with fresh medium for subsequent experiments.

[0036] The TeGG + Fenofibrate combined treatment group (TeGG + Fenofibrate group) involved treating THP-1 cells with 10 μM TeGG after induced differentiation to establish a pre-protective effect. After treatment, 50 μg / mL oxLDL was added to stimulate the cells, inducing abnormal activation of the NLRP3 inflammasome and intracellular cholesterol accumulation, thus constructing an in vitro cell model. After oxLDL stimulation ended, 50 μM fenofibrate was added for combined treatment. The experiment also included a TeGG-only treatment group (TeGG group), a fenofibrate-only treatment group (Fenofibrate group), a blank control group (Control group), and a model control group (PBS group). Cell culture conditions, treatment durations, and procedures were kept consistent across all groups to ensure comparability and reliability of the experimental results. The TeGG-only treatment group (TeGG group) was treated with 10 μM TeGG first, then stimulated with 50 μg / mL oxLDL, without adding fenofibrate; the fenofibrate-only treatment group (Fenofibrate group) was not treated with TeGG, then stimulated with 50 μg / mL oxLDL, followed by 50 μM fenofibrate; the blank control group (Control group) was cultured without TeGG, fenofibrate, and oxLDL, and under standard conditions; the model control group (PBS group) was treated with equal volumes of PBS instead of TeGG (pretreatment stage) and fenofibrate (treatment stage), then stimulated with 50 μg / mL oxLDL.

[0037] ① Intracellular cholesterol content determination: THP-1 cells from each group were collected after treatment, and lysis, extraction and color development were performed according to the standard procedure in the kit instructions. The total intracellular cholesterol level was quantitatively detected using a commercial intracellular cholesterol detection kit to reflect the intervention effect of TeGG, fenofibrate alone and in combination on oxLDL-induced intracellular cholesterol accumulation.

[0038] ② Determination of IL-1β content in cell supernatant: Cell culture supernatant was collected from each group, and the operation was strictly carried out in accordance with the instructions of the human IL-1β ELISA kit. The absorbance value was measured by microplate reader and the IL-1β concentration was calculated. The IL-1β release level was used as the key evaluation indicator for NLRP3 inflammasome activation.

[0039] Experimental results are as follows Figure 1 As shown, in the oxLDL-induced THP-1 cell model, TeGG pretreatment significantly inhibited oxLDL-mediated NLRP3 inflammasome overactivation, as evidenced by a significant decrease in IL-1β secretion levels in the cell supernatant. Simultaneously, TeGG effectively improved oxLDL-induced lipid metabolism disorders and significantly reduced abnormal intracellular cholesterol accumulation. Compared with TeGG alone or fenofibrate alone, the combination of TeGG and fenofibrate treatment was more effective in inhibiting NLRP3 inflammasome activation and reducing intracellular cholesterol accumulation.

[0040] Example 2: Animal model validation of the synergistic effect of TeGG and fenofibrate Establishing a high-fat diet for 16 weeks of HFD feeding Ldlr - / - A mouse model of atherosclerosis was established, with intervention starting from week 4 of modeling. Both TeGG and fenofibrate were administered using a mixture of corn oil and DMSO (9:1 volume ratio), maintaining consistent dosage volumes. The TeGG + TeGG + Fenofibrate group received TeGG (10 mg / kg) daily via intraperitoneal injection and fenofibrate (30 mg / kg) daily via gavage. A model group (Vehicle group), a control group, a TeGG monotherapy group (10 mg / kg), and a fenofibrate monotherapy group (30 mg / kg) served as controls. Intervention continued until the end of the experiment. Figure 2 As shown.

[0041] The model group (Vehicle group) was fed a high-fat diet and received equal volumes of solvent via intraperitoneal injection and gavage.

[0042] The blank control group (Control group) was fed a normal diet and received no drug intervention.

[0043] The TeGG single-drug group (10 mg / kg) consisted of a high-fat diet, intraperitoneal injection of TeGG (10 mg / kg), and gavage with an equal volume of solvent.

[0044] The fenofibrate monotherapy group (30 mg / kg) consisted of: high-fat diet, gavage of fenofibrate (30 mg / kg), and intraperitoneal injection of an equal volume of solvent.

[0045] ① Detection of caspase1 expression in the aortic root: The expression levels of total caspase1 and cleaved-capsase1 in the aortic root were detected by Western blot to assess the effect of drugs on the activation level of NLRP3 inflammasome.

[0046] ② Detect serum IL-1β levels: Measure using a commercially available IL-1β kit according to the manufacturer's instructions to assess the effect of the drug on the secretion level of inflammatory factors.

[0047] ③ Detection of VCAM1 and MCP1 levels in the aortic root: The expression of VCAM1 and MCP1 in aortic root sections was observed by immunofluorescence technology to assess the effect of drugs on immune infiltration in the aortic root.

[0048] ④ Detect serum cholesterol levels: Use a commercially available cholesterol test kit according to the manufacturer's instructions to measure and assess the effect of the drug on serum cholesterol.

[0049] ⑤ Detection of lipid plaque formation in the aortic root: Oil red staining of aortic root sections was performed to quantitatively analyze the area of ​​lipid plaques and assess the effect of drugs on lipid plaque formation in the aortic root.

[0050] Results: In a mouse model of atherosclerosis, TeGG alone could inhibit the activation of the NLRP3 inflammasome in the aortic root, such as... Figure 3 As shown, it simultaneously reduces VCAM1 and MCP1 recruitment and inhibits immune infiltration in the aortic root, such as Figure 4 As shown; however, fenofibrate alone was not significantly effective in inhibiting NLRP3 inflammasome activation and immune infiltration. Compared to single-drug therapy, the combination of TeGG and fenofibrate showed superior inhibitory effects on NLRP3 inflammasome activation. Although both TeGG and fenofibrate alone could reduce serum cholesterol levels in atherosclerotic mice, the combined effect was more significant, as shown in... Figure 5 As shown. Furthermore, the combination of TeGG and fenofibrate is significantly more effective than single-agent therapy in inhibiting lipid plaque formation in the aortic root, such as... Figure 6As shown.

[0051] The technical features of the above embodiments can be combined in any way. For the sake of brevity, not all possible combinations of the technical features in the above embodiments are described. However, as long as there is no contradiction in the combination of these technical features, they should be considered to be within the scope of this specification.

[0052] The embodiments described above are merely illustrative of several implementations of the present invention, and while the descriptions are specific and detailed, they should not be construed as limiting the scope of the invention. Those skilled in the art can make various modifications and improvements without departing from the concept of the present invention, and these all fall within the scope of protection of the present invention. Therefore, the scope of protection of this invention should be determined by the appended claims.

Claims

1. A combination drug for treating atherosclerosis, characterized in that, The combination of drugs includes 1,2,3,6-tetra-O-galloyl-β-D-glucose and fenofibrate; The mass ratio of 1,2,3,6-tetra-O-galloyl-β-D-glucose to fenofibrate is 1:2~4.

2. The combined medication according to claim 1, characterized in that, The mass ratio of 1,2,3,6-tetra-O-galloyl-β-D-glucose to fenofibrate is 1:

3.

3. The combined medication according to claim 1, characterized in that, The combined drugs can be formulated into clinically acceptable formulations together or separately with pharmaceutically acceptable excipients.

4. The combined medication according to claim 3, characterized in that, The pharmaceutically acceptable excipient is a mixture of corn oil and DMSO.

5. The combination drug according to claim 4, characterized in that, The volume ratio of corn oil to DMSO is 9:

1.

6. The combination drug as described in claim 3, characterized in that, The dosage form of the combined drugs is an oral preparation, an infusion, or an injection.

7. The combination drug as described in claim 6, characterized in that, The combined drug and pharmaceutically acceptable excipients are formulated into a clinically acceptable oral formulation.

8. The combination drug as described in claim 6, characterized in that, When the 1,2,3,6-tetra-O-galloyl-β-D-glucose and fenofibrate are formulated with pharmaceutically acceptable excipients into clinically acceptable formulations, a combination formulation of an injectable and an oral formulation is formed.

9. The use of the combined drug as described in any one of claims 1 to 8 in the preparation of an agent for treating atherosclerosis.

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