4-methyl esculetin for use in the preparation of a medicament for the prevention or treatment of aortic dissection, a combination and a method for the preparation thereof

Abstract

The application belongs to the technical field of chemical medicines, and particularly relates to application of 4-methyl esculetin in preparation of a medicine for preventing or treating aortic dissection, a combined medicine and a preparation method thereof. 4-methyl esculetin can significantly reduce the expression level of inflammatory factors (such as IL-6) in aortic tissue, inhibit neutrophil-related reactions (such as MPO expression), thereby reducing vascular wall inflammatory reactions; meanwhile, 4-methyl esculetin can also improve aortic structural stability, inhibit aortic expansion and dissection formation, effectively inhibit aortic dissection occurrence and development, improve the survival rate of patients, and has a good application prospect.

Description

Technical Field

[0001] This application belongs to the field of chemical and pharmaceutical technology, and specifically relates to the application of 4-methylaescine in the preparation of drugs for the prevention or treatment of aortic dissection, combination drugs, and preparation methods thereof. Background Technology

[0002] Aortic dissection is a life-threatening cardiovascular emergency characterized by a tear in the aortic intima, allowing blood to enter the media and form a false lumen, leading to delamination or even rupture of the vessel wall. The disease has a rapid onset and progression, and without timely intervention, the mortality rate is extremely high. Epidemiological studies have shown that the occurrence of aortic dissection is closely related to multiple factors, including hypertension, hereditary connective tissue diseases, and atherosclerosis.

[0003] Current clinical treatments mainly include surgical intervention and endovascular repair (such as stent implantation). While these methods can reduce acute-phase mortality to some extent, they primarily target existing structural lesions and are considered "post-operative interventions," failing to fundamentally prevent the disease's progression. Furthermore, surgery is highly invasive, expensive, and carries a certain risk of complications. In recent years, with the deepening research into the pathogenesis of aortic dissection, it has been gradually recognized that this disease is not merely a simple mechanical injury problem, but also involves complex biological processes, including inflammatory responses, vascular smooth muscle cell (VSMC) phenotypic transformation, extracellular matrix (ECM) degradation, and oxidative stress. Among these, inflammatory responses are considered one of the key factors driving the destruction of the aortic wall structure. However, there is currently a lack of effective drug interventions for the progression of aortic disease. Patients, before or in the early stages of the disease, lack effective drug interventions, leading to a persistent state of inflammation and degradation of the aortic wall, which in turn promotes dissection formation.

[0004] Therefore, developing effective drug interventions is of great significance for strategies to prevent or treat aortic dissection. Summary of the Invention

[0005] The purpose of this application is to provide the application of 4-methylesculin in the preparation of drugs for the prevention or treatment of aortic dissection, a combination drug, and a method for its preparation. 4-methylesculin can significantly reduce the expression levels of inflammatory factors (such as IL-6) in aortic tissue and inhibit neutrophil-related responses (such as MPO expression), thereby alleviating vascular wall inflammation. Simultaneously, it can also improve aortic structural stability, inhibit aortic dilation and dissection formation, ultimately improving patient survival rates.

[0006] The first aspect of this application provides the use of 4-methylaepril in the preparation of a medicament for the prevention or treatment of aortic dissection.

[0007] In some alternative implementations, the prevention or treatment of aortic dissection includes intervening in the inflammatory response and / or vascular structural damage during the development of aortic dissection.

[0008] In some alternative implementations, the prevention or treatment of aortic dissection includes prevention or treatment at the stage when the vessel wall structure is gradually damaged but not yet completely destroyed.

[0009] In some alternative embodiments, the drug has at least one of the following functions: 1) Inhibits aortic dilation and / or vascular structure damage; 2) Reduce the level of inflammatory response; 3) Reduce IL-6 and MPO expression levels; 4) Inhibits inflammatory cell infiltration; 5) Improve the survival rate of patients with aortic dissection.

[0010] A second aspect of this application provides a combination drug comprising 4-methylaescin, poloxamer 407, and dimethyl sulfoxide (DMSO).

[0011] In some alternative embodiments, the mass ratio of 4-methylaepril to poloxamer 407 is (0.1-1.5):40.

[0012] In some optional embodiments, the combination drug further includes other pharmaceutically acceptable components, including but not limited to fillers, disintegrants, binders, lubricants, flow aids, surfactants, solubilizers, cosolvents, flavoring agents, colorants, preservatives, antioxidants, buffers, osmotic pressure regulators, emulsifiers, stabilizers, and diluents; in some optional embodiments, the combination drug further includes other pharmaceutically acceptable components, including but not limited to PEG300 and Tween80. A second aspect of this application provides a method for preparing the above-mentioned combination drug, comprising the following steps: It is prepared by mixing 4-methylaescin, poloxamer 407 and dimethyl sulfoxide.

[0013] In some optional embodiments, the preparation method of the above-mentioned combination drug includes the following steps: First, 4-methylesculin is mixed with dimethyl sulfoxide, then diluted with physiological saline to obtain a mixed solution. Then, poloxamer 407 is added to the mixed solution and mixed to obtain the combined drug. In some alternative embodiments, the mixture is diluted 3-5 times with physiological saline to obtain a mixed solution; The mass ratio of 4-methylaescine to poloxamer 407 is (0.1-1.5):40; In some alternative embodiments, the mass concentration of poloxamer 407 in the combination drug is 180-220 g / L.

[0014] In some alternative embodiments, the concentration of 4-methylaescine in the combination drug is 25-27 mM.

[0015] Compared with the prior art, this application has the following advantages: The 4-methylesculin provided in this application is used in the preparation of drugs for the prevention or treatment of aortic dissection. 4-methylesculin can significantly reduce the expression levels of inflammatory factors (such as IL-6) in aortic tissue and inhibit neutrophil-related responses (such as MPO expression), thereby alleviating vascular wall inflammation. Simultaneously, it can also improve aortic structural stability, inhibit aortic dilation and dissection formation, effectively suppressing the development and progression of aortic dissection, improving patient survival rates, and showing promising application prospects.

[0016] The combination drug provided in this application, based on a thermosensitive gel sustained-release system, achieves continuous drug release in the aortic lesion area through local administration, thereby increasing local drug concentration and enhancing therapeutic effect, while reducing the risk of systemic adverse reactions.

[0017] The above description is only an overview of the technical solution of this application. In order to better understand the technical means of this application and to implement it in accordance with the contents of the specification, and to make the above and other objects, features and advantages of this application more obvious and understandable, the following are specific embodiments of this application. Attached Figure Description

[0018] To more clearly illustrate the technical solutions in the specific embodiments of this application or the prior art, the drawings used in the description of the specific embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of this application. For those skilled in the art, other drawings can be obtained from these drawings without creative effort.

[0019] Figure 1 This is a schematic diagram of the drug administration intervention experiment arrangement in Example 2 of this application; Figure 2 This is a comparison diagram of the staining results of the drug administration intervention experiment in Example 3 of this application; Figure 3 This is a gross comparative image of the aortic tissue of mice in the negative control, positive control, and 4-methylesculin experimental group in Example 4 of this application; Figure 4 This is a cross-sectional comparison of the aortic tissue of mice in the negative control, positive control, and 4-methylesculin experimental group in Example 4 of this application; Figure 5 This is a comparison diagram of the changes in aortic arch diameter in mice in Example 4 of this application, including the negative control, positive control, and 4-methylesculin experimental group. Figure 6 This is a comparison of IL-6 and MPO levels in the aortic tissue of mice in the negative control, positive control, and 4-methylesculin experimental group in Example 4 of this application; Figure 7 This is a comparison chart of mouse survival rates in the negative control, positive control, and 4-methylaescin experimental groups in Example 4 of this application. Detailed Implementation

[0020] Exemplary embodiments of this application will now be described in more detail with reference to the accompanying drawings. While exemplary embodiments of this application are shown in the drawings, it should be understood that this application may be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of this application and to fully convey the scope of this application to those skilled in the art.

[0021] It should be understood that the terminology used herein is for the purpose of describing particular exemplary embodiments only and is not intended to be limiting. Unless the context clearly indicates otherwise, the singular forms “a,” “an,” and “described” as used herein may also include the plural forms. The terms “comprising,” “including,” “containing,” and “having” are inclusive and therefore indicate the presence of the stated features, steps, operations, elements, and / or components, but do not exclude the presence or addition of one or more other features, steps, operations, elements, components, and / or combinations thereof. The method steps, processes, and operations described herein are not construed as requiring them to be performed in a particular order described or illustrated unless the order of performance is explicitly indicated. It should also be understood that additional or alternative steps may be used.

[0022] In the description of the embodiments of this application, technical terms such as "first" and "second" are used only to distinguish different objects and should not be construed as indicating or implying relative importance or implicitly specifying the number, specific order, or primary and secondary relationship of the indicated technical features. In the description of the embodiments of this application, "multiple" means two or more, unless otherwise explicitly defined.

[0023] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.

[0024] In the description of the embodiments in this application, the term "and / or" is merely a description of the relationship between related objects, indicating that three relationships can exist. For example, A and / or B can represent: A existing alone, A and B existing simultaneously, and B existing alone. Additionally, the character " / " in this document generally indicates that the preceding and following related objects have an "or" relationship.

[0025] In the description of the embodiments of this application, the term "multiple" refers to two or more (including two), similarly, "multiple sets" refers to two or more (including two sets), and "multiple pieces" refers to two or more (including two pieces).

[0026] Those skilled in the art can understand the specific meaning of the above terms in the embodiments of this application according to the specific circumstances.

[0027] The following explanations of some terms used in this application are provided to help those skilled in the art to understand them.

[0028] Current treatment strategies primarily focus on mechanical repair (such as surgery or endovascular treatment), which essentially only passively intervene in blood vessels that have already suffered structural damage, failing to effectively regulate the inflammatory response and structural damage of the aortic wall in the early stages of the disease. Compared to ordinary vascular diseases, aortic dissection involves a complex pathological process, including inflammatory cell infiltration, cytokine release, neutrophil activation, and vascular wall instability. Existing drugs struggle to simultaneously and effectively regulate these multiple key processes, resulting in limited treatment efficacy.

[0029] Currently, there is a lack of novel small molecule compounds that have a clear protective effect against aortic dissection, which to some extent limits the development of drug treatment for aortic dissection.

[0030] The first aspect of this application provides the use of 4-methylaepril in the preparation of a medicament for the prevention or treatment of aortic dissection.

[0031] During the development of aortic dissection, inflammatory cells (such as neutrophils and macrophages) infiltrate the vessel wall and release various inflammatory factors (such as IL-6) and proteolytic enzymes (such as matrix metalloproteinases), thereby accelerating the degradation of the extracellular matrix and weakening the structural stability of the vessel wall. Simultaneously, myeloperoxidase (MPO) and other active substances released by neutrophils can further exacerbate oxidative stress, creating a vicious cycle that ultimately leads to aortic dilation and even dissection. 4-Methylesacin can significantly reduce the expression levels of inflammatory factors (such as IL-6) in aortic tissue and inhibit neutrophil-related responses (such as MPO expression), thereby alleviating the inflammatory response of the vessel wall. Furthermore, it can improve aortic structural stability, inhibit aortic dilation and dissection formation, and ultimately improve patient survival rates.

[0032] This application expands the range of small molecule drugs that can be used to intervene in aortic dissection, providing new options for drug treatment of this disease.

[0033] In some alternative implementations, the prevention or treatment of aortic dissection includes intervening in the inflammatory response and / or vascular structural damage during the development of aortic dissection.

[0034] In some alternative embodiments, the prevention or treatment of aortic dissection includes intervention during the stage when the vessel wall structure is gradually damaged but not yet completely destroyed. This invention, by intervening at a critical stage of disease progression (week 2 of BAPN feeding), allows the drug to act on the aortic wall where damage is gradually forming but before severe structural destruction has occurred, thereby more effectively delaying or blocking the development of aortic dissection. This invention features a more rational timing control strategy.

[0035] In some alternative embodiments, the drug has the function of inhibiting aortic dilation and / or vascular structural damage.

[0036] In some alternative embodiments, the drug has the function of reducing the level of inflammatory response.

[0037] In some alternative embodiments, the drug has the function of reducing the expression levels of IL-6 and MPO.

[0038] In some alternative embodiments, the drug has the function of inhibiting the infiltration of inflammatory cells.

[0039] In some alternative embodiments, the drug has the function of improving the survival rate of patients with aortic dissection.

[0040] This invention targets key pathological steps in the development of aortic dissection, such as inflammatory response and vascular wall structural damage. By reducing the expression of the inflammatory factor IL-6 and the neutrophil-related marker MPO, it inhibits the inflammatory response and improves vascular wall stability. The approach of this invention has a more clearly defined pathway of action in inflammation regulation and structural protection.

[0041] A second aspect of this application provides a combination drug comprising 4-methylaepril and a sustained-release agent.

[0042] The 4-methylaescine used in this application is a coumarin-type small molecule compound, which is a white solid powder at room temperature and has good chemical stability. Its structural formula is: Regarding solubility, this compound is readily soluble in organic solvents such as dimethyl sulfoxide (DMSO), ethanol, and dimethylformamide, but has low solubility in water, typically requiring formulation with organic solvents or solubilizing systems. In animal experiments, 4-methylaescin is often administered in a complex solvent system composed of DMSO, PEG300, and Tween 80, or in suspension form. Given its poor water solubility and rapid clearance in vivo, local sustained-release administration helps increase its effective concentration in target tissues, thereby enhancing the drug's efficacy.

[0043] In some alternative embodiments, the sustained-release agent includes a thermosensitive gel; In some alternative embodiments, the sustained-release agent includes poloxamer 407.

[0044] In some optional embodiments, the mass ratio of 4-methylaepril to the sustained-release agent is (0.1-1.5):40. For example, the mass ratio of 4-methylaepril to the sustained-release agent can be selected as 0.1:40, 0.2:40, 0.3:40, 0.4:40, 0.5:40, 0.6:40, 0.7:40, 0.8:40, 0.9:40, 1:40, 1.2:40, 1.3:40, 1.4:40, or 1.5:40.

[0045] In some optional embodiments, the combined drug further includes a solvent; In some alternative embodiments, the solvent includes an organic solvent; In some alternative embodiments, the organic solvent includes at least one of dimethyl sulfoxide (DMSO).

[0046] In some optional embodiments, the combination drug further includes other pharmaceutically acceptable components, including but not limited to fillers, disintegrants, binders, lubricants, flow aids, surfactants, solubilizers, cosolvents, flavoring agents, colorants, preservatives, antioxidants, buffers, osmotic pressure regulators, emulsifiers, stabilizers, and diluents; in some optional embodiments, the combination drug further includes other pharmaceutically acceptable components, including but not limited to PEG300 and Tween80. This application provides a combination drug, which, based on the mass of 4-methylaepril, has a commonly administered dose range of 10-300 mg / kg in mice and exhibits significant dose-dependent pharmacokinetic characteristics, and is mainly eliminated by liver metabolism.

[0047] A second aspect of this application provides a method for preparing the above-mentioned combination drug, comprising the following steps: It is prepared by mixing 4-methylaepril, a sustained-release agent, and a solvent.

[0048] In some optional embodiments, the preparation method of the above-mentioned combination drug includes the following steps: First, 4-methylesculin is mixed with dimethyl sulfoxide, then diluted with physiological saline to obtain a mixed solution. Then, poloxamer 407 is added to the mixed solution and mixed to obtain the combined drug. In some optional embodiments, the mixture is diluted 3-5 times with physiological saline to obtain a mixed solution; for example, it can be selectively diluted 3.5 times, 4 times, 4.5 times, or 5 times with physiological saline to obtain a mixed solution.

[0049] The mass ratio of 4-methylaescine to poloxamer 407 is (0.1-1.5):40; for example, the mass ratio of 4-methylaescine to the sustained-release agent can be selected as 0.1:40, 0.2:40, 0.3:40, 0.4:40, 0.5:40, 0.6:40, 0.7:40, 0.8:40, 0.9:40, 1:40, 1.2:40, 1.3:40, 1.4:40, or 1.5:40.

[0050] In some optional embodiments, the mass concentration of poloxamer 407 in the combination drug is 180-220 g / L. For example, the mass concentration of poloxamer 407 in the combination drug can be selected as 180 g / L, 185 g / L, 188 g / L, 190 g / L, 192 g / L, 195 g / L, 198 g / L, 200 g / L, 203 g / L, 205 g / L, 208 g / L, 210 g / L, 213 g / L, 215 g / L, 218 g / L, or 220 g / L.

[0051] In some optional embodiments, the concentration of 4-methylaepril in the combination drug is 25-27 mM; for example, the concentration of 4-methylaepril in the combination drug can be selected as 25 mM, 25.5 mM, 26 mM, 26.5 mM, or 27 mM.

[0052] This application also provides an animal model of aortic dissection: a mouse model induced by β-aminopropionitrile (BAPN) combined with angiotensin II (Ang II).

[0053] The basic principle of this model is as follows: β-aminopropionitrile is a lysyl oxidase inhibitor that can inhibit the cross-linking of collagen and elastin, leading to a decrease in the structural strength of the blood vessel wall; angiotensin II can increase the mechanical stress of the blood vessel wall by raising blood pressure and inducing inflammatory response; the combined effect of the two can significantly promote the destruction of the aortic wall structure, thereby inducing the formation of aortic dissection.

[0054] The specific operational steps typically include: selecting young mice (such as 3-week-old C57BL / 6J mice); adding β-aminopropionitrile to the drinking water and feeding them continuously for 4 weeks; subsequently, continuously infusing angiotensin II via a micro-osmotic pump to achieve stable drug administration; and observing aortic dilation, dissection formation, and animal survival over a certain period of time. This model can effectively simulate the pathological characteristics of human aortic dissection.

[0055] In some optional embodiments, a mouse aortic dissection model was established by feeding 3-week-old mice with an aqueous β-aminopropionitrile (BAPN) solution for 4 weeks combined with angiotensin II-induced dissection. Two weeks after feeding the mice with the BAPN solution, they were treated with 4-methylesculin. In this embodiment, week 2 was preferably used as the drug intervention window for the mouse aortic dissection model.

[0056] This application relates to the use of 4-methylesculin in the preparation of drugs for the prevention or treatment of aortic dissection. The inventors have discovered that 4-methylesculin exhibits a dose-dependent effect in vivo, and its effective dosage can be adjusted according to the route of administration and site of action. In some alternative embodiments, under systemic administration conditions, the commonly used dosage (e.g., oral or intraperitoneal injection) ranges from 5 to 50 mg / kg; while under the preferred local administration conditions of this application, the systemic dosage can be significantly reduced.

[0057] In some optional embodiments, the combination drug of this application uses a thermosensitive gel (such as Pluronic® F127) as a carrier to deliver the drug locally to the target tissue, achieving sustained drug release in the aortic lesion area, thereby increasing local drug concentration and enhancing therapeutic effect while reducing the risk of systemic adverse reactions. This invention has significant advantages in improving drug utilization efficiency.

[0058] In other alternative embodiments, the combination drug of this application or the preparation of a drug containing 4-methylesculin for the prevention or treatment of aortic dissection may also be administered via different routes of administration (such as injection, oral administration or other sustained-release systems).

[0059] In a preferred embodiment, the effective dosage of the combination drug of this application, based on the mass of 4-methylaepril, when administered topically is approximately 3-10 mg / kg (converted to mouse body weight). For example, mice can be treated with doses of 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, or 10 mg / kg, all of which can achieve the technical effect of inhibiting the occurrence and development of aortic dissection as described in this application. Therefore, those skilled in the art can reasonably adjust the dosage of 4-methylaepril according to specific experimental conditions and administration methods, and the range may include any of the above specific values, or a range between any two values.

[0060] This application also provides the use of 4-methylaepril in the prevention or treatment of aortic dissection.

[0061] In a preferred embodiment, the effective dosage of 4-methylaepril for local administration is approximately 3-10 mg / kg (based on mouse body weight). For example, mice can be treated with doses of 3 mg / kg, 4 mg / kg, 5 mg / kg, 6 mg / kg, 7 mg / kg, 8 mg / kg, or 10 mg / kg, all of which achieve the technical effect of inhibiting the development of aortic dissection as described in this application. Therefore, those skilled in the art can reasonably adjust the dosage of 4-methylaepril according to specific experimental conditions and administration methods, and the range may include any of the above specific values, or a range between any two values.

[0062] This invention achieves effective intervention in aortic dissection at a relatively low dosage level (approximately 5 mg / kg based on the effective content of 4-methylesculin), while most small molecule drugs in the prior art require higher doses to achieve similar effects. Therefore, this invention has certain advantages in reducing drug dosage and improving safety.

[0063] Finally, the invention verified its technical effectiveness through multi-dimensional results such as improved vascular morphology, reduced inflammatory markers, and improved survival rate, indicating that the technical solution not only has a basis for action at the molecular level, but also can improve disease outcomes at the overall level, and has good application prospects.

[0064] Unless otherwise defined, the technical terms used in the following embodiments have the same meaning as commonly understood by those skilled in the art to which this application pertains. Unless otherwise specified, the experimental reagents used in the following embodiments are all conventional biochemical reagents; the raw materials, instruments, and equipment used in the following embodiments can all be obtained through commercial purchase or by existing methods; the amounts of experimental reagents used are, unless otherwise specified, the amounts used in conventional experimental operations; and the experimental methods used are, unless otherwise specified, conventional methods. It should be further noted that the following descriptions are merely exemplary and not intended to limit the scope of this application. Moreover, the comparative examples below are selected to compare with the technical solutions of this application to demonstrate the advancement of the technical solutions of this application, and do not necessarily represent prior art in this technical field.

[0065] Example 1 This embodiment provides a method for preparing a combination drug, including the following steps: 4-Methylaescine was dissolved in dimethyl sulfoxide to prepare a 100 mM stock solution. An appropriate amount of this stock solution was taken and poloxamer 407 (trade name Pluronic® F127) was added. The solution was then diluted with physiological saline to a final concentration of 200 g / L for poloxamer 407 and 26 mM for 4-methylaescine. The mixture was thoroughly stirred and incubated overnight at 4°C to ensure complete dissolution, yielding a clear, low-viscosity, flowable solution, which was the combined drug.

[0066] The combined drug has temperature-responsive gel properties: at low temperature (4°C), the system is a clear solution that is easy to inject; when the temperature rises to body temperature (about 37°C), the poloxamer 407 molecules in the system self-assemble to form a stable three-dimensional network structure, which rapidly transforms the solution into a semi-solid gel, thereby achieving in-situ retention and sustained release of the drug.

[0067] Example 2 To demonstrate the efficacy of the combination drug prepared in Example 1 of this application for the prevention or treatment of aortic dissection, mice were used as experimental subjects to construct a mouse model of thoracic aortic dissection.

[0068] In this embodiment, a mouse thoracic aortic dissection model was induced by feeding C57BL / 6J mice with β-aminopropionitrile aqueous solution for 4 weeks combined with angiotensin II-induced dissection.

[0069] A schematic diagram of the drug administration intervention experiment arrangement is shown below. Figure 1 As shown, the specific operation includes the following steps: The body weight of 3-week-old mice was measured in advance. The drug dosage was 1 g / kg mouse body weight / day. The aortic dissection model group was given β-aminopropionitrile (BAPN group), and the negative control group was given H2O (H2O group). The body weight was measured once a week and the drug concentration was updated.

[0070] Four weeks after feeding mice with β-aminopropionitrile aqueous solution, the mice were weighed and angiotensin II sustained-release pumps were prepared using a micro osmotic pump (Alzet Model 1007D) at a dosage of 1000 ng / kg mouse body weight / day.

[0071] Mice were first anesthetized by intraperitoneal injection of tribromoethanol (50 mg / kg mouse body weight). The mice were placed in a left lateral decubitus position, and the skin on the back of the neck, slightly to the right, was incised down to the superficial fascia, approximately 0.5 cm wide. Using straight-tipped forceps, a sac-like space was bluntly dissected and opened up to the mouse's tail. The angiotensin II sustained-release pump was placed into this sac-like space, with the outlet facing the tail. The wound was then sutured.

[0072] Mice were fed β-aminopropionitrile and their arterial blood flow was examined by ultrasound on days 7, 14, 21, and 28. Simultaneously, tissue samples were taken to observe the aortic dissection. Finally, the progression of aortic dissection was determined by gross observation and hematoxylin-eosin staining.

[0073] For aortic sampling in mice, the mice were first euthanized using overdose anesthesia or cervical dislocation, and then fixed in a supine position on a dissecting board. After disinfecting the skin of the chest and abdomen with 75% alcohol, the skin and thoracic and abdominal cavities were cut along the midline of the abdomen with tissue scissors to fully expose the heart and thoracic cavity. The heart was gently lifted with forceps, and the upward-arching aortic arch was identified and separated at the base of the heart. Using fine microscissors, the connecting vascular branches and surrounding connective tissue were carefully cut along the aortic arch, completely freeing the aortic arch to the thoracic aorta segment. After removal, the aorta was immediately placed in pre-cooled physiological saline or PBS to wash away any residual blood, and then fixed according to the requirements of subsequent experiments. The entire operation required gentle and rapid movements to avoid traction damage to the vessel wall. After sampling, the aorta was grossly photographed and measured.

[0074] In the hematoxylin-eosin staining method, mouse aortic tissue was fixed in 4% paraformaldehyde, then routinely embedded in paraffin and sectioned. After dewaxing to water, the sections were stained with hematoxylin for 10 minutes, rinsed with running water, and then differentiated with 1% hydrochloric acid ethanol for a few seconds, followed by blue reversion with running water. Subsequently, they were stained with eosin for 3 minutes and quickly rinsed with running water. Finally, the sections were sequentially dehydrated with graded ethanol, cleared with xylene, and mounted with neutral resin, allowing observation of the aortic wall structure under a microscope.

[0075] Example 3 Immunohistochemical staining of mouse aortic sections To screen the time window for drug intervention and verify the results of drug intervention, immunohistochemical staining experiments were performed on paraffin sections of aortic specimens from mice with dissection models at different time points in Example 2: Baked paraffin sections at 68°C for 1 hour. Then, sequentially immerse the sections in xylene I and xylene II for 10 minutes each, followed by immersion in 100%, 95%, 80%, and 70% ethanol for 2 minutes each, and finally rinse with distilled water for 5 minutes. Immerse the sections in preheated citrate buffer and microwave on high to above 95°C for 15 minutes to retrieval antigens. Afterward, allow them to cool naturally to room temperature and rinse three times with PBS for 5 minutes each time. Add 3% hydrogen peroxide solution to the sections and incubate at room temperature in the dark for 10 minutes to quench endogenous peroxidase activity, followed by rinsing three times with PBS. Discard the PBS and add sufficient 5% BSA blocking solution to the tissue, blocking at room temperature for 30 minutes. Discard the blocking solution, do not wash, and directly add IL-6 and MPO primary antibodies diluted 1:100, ensuring complete coverage of the tissue. Incubate overnight at 4°C or 2 hours at 37°C, then wash three times with PBS for 5 minutes each time. Add HRP-labeled goat anti-rabbit secondary antibody working solution diluted 1:100 and incubate at room temperature for 60 minutes. Then wash three times with PBS, 5 minutes each time. Next, add freshly prepared DAB chromogenic solution and monitor the color development process under a microscope. When a brown signal appears in the target area but the background remains unstained, immediately rinse with tap water or PBS to terminate the reaction. Counterstain the cell nuclei with hematoxylin for 2 minutes, then rinse with tap water to restore blue color. Subsequently, rapidly dehydrate the cells sequentially with 70%, 80%, 95%, and 100% ethanol, and then clear twice with xylene. Finally, mount the slide with neutral resin and observe the acquired images under a microscope.

[0076] Determining the time window for drug intervention in a mouse model of aortic dissection Mice were sampled at different time points during the aortic dissection modeling process in Example 2, and immunohistochemical staining was performed on the aortic tissue to label F4 / 80 positive cells in the tissue. Figure 2 As shown in the figure, the H2O group is the negative control group, and the BAPN group is the aortic dissection model group. Figure 2 The yellow substance marked by the middle arrow is F4 / 80, a macrophage marker. Macrophages are the main cell type that secretes the inflammatory factors IL-1β and IL-6. Macrophages appear during aortic dissection formation and their number gradually increases as the disease progresses. Figure 2 The staining results showed that F4 / 80 positive cells appeared in the third week of modeling, and the number of positive cells gradually increased with the progression of the disease. This indicates that key node cells leading to the development of aortic dissection appeared from the third week after feeding mice with β-aminopropionitrile aqueous solution, and the number of cells increased over time, eventually reaching its peak in the tissue where aortic dissection was formed. Therefore, the second week after feeding mice with β-aminopropionitrile aqueous solution can be considered as a time window for intervention in aortic dissection formation in mice.

[0077] Example 4 In this embodiment, in order to conduct more targeted in vivo intervention experiments on aortic dissection tissue, it is preferable to administer the drug by incubating it with a drug gel at the aortic arch of mice.

[0078] An equal volume of dimethyl sulfoxide and physiological saline was mixed using Example 1, and then poloxamer 407 was added to bring the final concentration of poloxamer 407 in the solution to 20% (w / v). The mixture was thoroughly stirred and incubated overnight at 4°C to ensure complete dissolution, yielding the comparative drug. In this embodiment, the combination drug prepared according to Example 1 of this application was used for intervention, and the intervention effect was observed by comparing it with a positive control group and a negative control group. The experimental procedure is as follows: Specifically, the procedure involved randomly grouping mice weighing 9.5–12 grams. During the second week of feeding β-aminopropionitrile, the mice underwent open-chest surgery. During the surgery, the combination drug prepared in Example 1 was injected at a dose of 20 μL / mouse into the aortic arch of the experimental group mice, while the control drug was injected at a dose of 20 μL / mouse into the aortic arch of the positive control group mice. This was done only once throughout the entire animal model period, forming a gel and releasing slowly (the experimental group experienced slow release of 4-methylesculin). After the open-chest surgery, the mice continued to be fed an aqueous solution of β-aminopropionitrile. After four weeks of β-aminopropionitrile feeding, a sustained-release pump containing angiotensin II (1000 ng / kg mouse body weight / day) was implanted subcutaneously into the mice, allowing it to act in the mice for 24–48 hours. Six three-week-old mice were used as a negative control group and fed sterile water for four weeks. The aorta of the mice was observed after the experiment, and the results are as follows: Figure 3 and Figure 4 As shown. Among them, Figure 3 The gross aortic tissue of mice in the negative control, positive control, and 4-methylesaefoliate experimental groups is shown. Figure 4 The images show the aortic tissue cross sections of mice in the hematoxylin-eosin staining negative control, positive control, and 4-methylesculin experimental group. Figure 3 and Figure 4 Findings: Compared with the positive control group, the aorta of mice in the 4-methylesculin experimental group was closer to that of mice in the negative control group, with no abnormal tissue proliferation or tearing.

[0079] The maximum diameter of the aortic arch is an important indicator for assessing the progression of aortic dissection. During the experiment, the aortic arch diameter of mice at different time points was measured using a VisualSonics Vevo 2100 ultrasound imaging system. The results are as follows: Figure 5 As shown, the results indicate that the aortic arch diameter in the 4-methylescin experimental group was closer to that of the negative control group and significantly smaller than that of the positive control group. These experimental results demonstrate that 4-methylescin effectively inhibits the development of aortic dissection.

[0080] Inflammation-related markers in aortic tissue were detected using immunohistochemistry, and the results are as follows: Figure 6 As shown, compared with the positive control group, the expression levels of IL-6 and MPO in the blood vessel wall of mice were significantly reduced after 4-methylescin intervention, indicating that the drug can inhibit the release of inflammatory factors and neutrophil infiltration, thereby reducing the inflammatory response of the blood vessel wall.

[0081] The survival rate of mice is an important indicator reflecting the therapeutic effect of drugs on aortic dissection. At the beginning of the experiment, there were 6 negative controls, 12 positive controls, and 11 mice in the 4-methylesculin experimental group. At the end of the experiment, the survival rate of mice in the positive control group was only 42%, the survival rate of mice in the negative control group was 100%, and the survival rate of mice in the 4-methylesculin-treated group increased to 91%. Specific results are as follows... Figure 7 As shown.

[0082] The aortic arch of mice was treated with medication during the second week. Compared with the positive control group, from Figures 3 to 7 It is evident that the mice in the 4-methylesaefoliate experimental group showed significant improvements in vascular morphology, vascular diameter, and survival rate in mice with aortic dissection.

[0083] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of this application, and are not intended to limit them. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features therein. Such modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of this application.

Claims

1. The use of a 4-methylesculin in the preparation of a drug for the prevention or treatment of aortic dissection.

2. The application according to claim 1, characterized in that, The prevention or treatment of aortic dissection includes intervening in the inflammatory response and / or vascular structural damage during the development of aortic dissection.

3. The application according to claim 1, characterized in that, The prevention or treatment of aortic dissection includes prevention or treatment during the stage when the vascular wall structure is gradually damaged but not yet completely destroyed.

4. The application according to claim 1, characterized in that, The drug has at least one of the following functions: 1) Inhibits aortic dilation and / or vascular structure damage; 2) Reduce the level of inflammatory response; 3) Reduce IL-6 and MPO expression levels; 4) Inhibits inflammatory cell infiltration; 5) Improve the survival rate of patients with aortic dissection.

5. A combination drug, characterized in that, The combination drug includes 4-methylaescin, poloxamer 407, and dimethyl sulfoxide; The mass ratio of 4-methylaescine to poloxamer 407 is (0.1-1.5):

40.

6. A method for preparing the combination drug according to claim 5, characterized in that, Includes the following steps: It is prepared by mixing 4-methylaescin, poloxamer 407 and dimethyl sulfoxide.

7. The method for preparing the combination drug according to claim 6, characterized in that, Includes the following steps: First, 4-methylesculin is mixed with dimethyl sulfoxide, then diluted with physiological saline to obtain a mixed solution. Then, poloxamer 407 is added to the mixed solution and mixed to obtain the combined drug. The mass ratio of 4-methylaescine to poloxamer 407 is (0.1-1.5):

40.

8. The method for preparing the combination drug according to claim 6 or 7, characterized in that, The mass concentration of poloxamer 407 in the combined drug is 180-220 g / L.

9. The method for preparing the combination drug according to claim 6 or 7, characterized in that, The concentration of 4-methylaescine in the combined drug is 25-27 mM.

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