Application of golden amide alcohol ester in the preparation of drugs for the prevention / treatment of fatty liver disease and its complications

Abstract

This application provides the use of aurantiamide acetate or a pharmaceutically acceptable salt thereof in the preparation of drugs for the prevention / treatment of fatty liver disease and its complications. Compared with the prior art, this application has the following advantages: This application verifies that aurantiamide acetate can significantly reduce the levels of total cholesterol (TC) and triglycerides (TC) in mice with metabolic-related fatty liver disease, and inhibit the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and low-density lipoprotein (LDL), exhibiting significant lipid-lowering, anti-inflammatory, anti-fibrotic, and hepatoprotective effects. Furthermore, aurantiamide acetate is essentially non-toxic and has good safety, making it suitable for the preparation of drugs for the prevention / treatment of metabolic-related fatty liver disease and its related complications, with broad application prospects.

Description

Technical Field

[0001] This application relates to the field of biomedical technology, specifically to the use of aurora amide alcohol ester in the preparation of drugs for the prevention / treatment of fatty liver disease and its complications. Background Technology

[0002] Metabolic fatty liver disease (MAF) is one of the most prevalent liver diseases globally. In the coming decades, MAF is projected to become a leading cause of end-stage liver disease. MAF affects both adults and children. The overall prevalence of MAF cases is projected to increase by 0% to 30% between 2016 and 2030. A meta-analysis showed an overall MAF prevalence of 29.62% in Asia. The annual incidence of primary liver cancer in the MAF population in Asia is 1.8‰. MAF is a general term encompassing a wide range of liver diseases, from non-alcoholic fatty liver disease, liver fibrosis, and cirrhosis to hepatocellular carcinoma. MAF begins with the abnormal accumulation of triglycerides in the liver, triggering lipotoxicity, endoplasmic reticulum stress, and inflammatory responses, progressing to cirrhosis and even liver cancer. Metabolic steatohepatitis (MSH) is a late-stage form of MAF characterized by hepatic steatosis, inflammation, ballooning degeneration, and / or fibrosis. Despite a deeper understanding of the pathogenesis and therapeutic targets of metabolic-associated fatty liver disease / NASH, no drugs have yet been approved for this indication.

[0003] Aurantiamide acetate is a dipeptide monomeric compound isolated from the traditional Chinese medicine silkworm (Bombyx batryticatus) and the wild plant purslane (Portulaca oleracea L.). In traditional Chinese medicine theory, silkworm (also known as the silkworm pupa) belongs to the liver and lung meridians and has the effects of dispelling wind and calming the mind, resolving phlegm and dissipating nodules, often used for wind-phlegm obstruction syndrome. Purslane, on the other hand, belongs to the liver and large intestine meridians and has the functions of clearing heat and detoxifying, cooling blood and promoting diuresis, and is a classic medicinal material for treating damp-heat jaundice. Traditional Chinese medicine believes that metabolic-related fatty liver disease belongs to the category of "liver stagnation" and "accumulation syndrome", with the core pathogenesis being spleen dysfunction, liver dysfunction, and phlegm and blood stasis. The "phlegm-resolving and nodule-dissipating" effects of silkworm and the "heat-clearing and diuresis-promoting" effects of purslane precisely target the core pathological issue of phlegm and dampness obstruction. As its active ingredient, golden amide alcohol ester inherits the therapeutic concept of "regulating the liver and spleen, eliminating phlegm and removing blood stasis" from the mother medicine. It achieves the modern transformation of "resolving phlegm and dissipating nodules" by inhibiting cathepsin and regulating inflammatory pathways. Summary of the Invention

[0004] In view of the above-mentioned technical limitations, this application proposes the use of golden amide alcohol ester in the preparation of drugs for the prevention / treatment of fatty liver disease and its complications; which overcomes the deficiencies and defects mentioned in the background art.

[0005] To achieve the above objectives, this application adopts the following technical solution:

[0006] The inventive point of this application is to provide the use of aurantiamide acetate or a pharmaceutically acceptable salt thereof in the preparation of a medicament for the prevention / treatment of fatty liver disease and its complications.

[0007] Optionally, in the above applications, the structure of the golden amide alcohol ester is shown in formula (I) below:

[0008]

[0009] Formula (I).

[0010] Golden amide alcohol ester can significantly reduce the levels of total cholesterol (TC) and triglycerides (TC) in mice with metabolic-associated fatty liver disease, and inhibit the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and low-density lipoprotein (LDL), exhibiting significant lipid-lowering, anti-inflammatory, anti-fibrotic, and hepatoprotective effects.

[0011] Optionally, in the above application, the fatty liver disease is selected as metabolic-related fatty liver disease.

[0012] Optionally, in the above applications, the complications of fatty liver disease include hyperlipidemia, metabolic-associated steatohepatitis, liver fibrosis, cirrhosis, type 2 diabetes mellitus with insulin resistance as the core, chronic kidney disease, hepatocellular carcinoma, myocardial infarction, stroke, and kidney disease caused by metabolic-associated fatty liver disease combined with type 2 diabetes mellitus.

[0013] Optionally, in the above applications, the dosage form of the drug is selected as tablets, injections, or capsules.

[0014] Optionally, in the above-described applications, the dosage of the golden amide alcohol ester is selected to be 1 mg / kg-10 mg / kg; preferably 10 mg / kg.

[0015] Optionally, in the above-described applications, the drug also includes an atorvastatin preparation that can be used in combination with aureomycin ester; the dosage ratio of aureomycin ester to atorvastatin preparation is selected as 5 mg / kg: 5 mg / kg.

[0016] Compared with the prior art, this application has the following advantages:

[0017] This application verifies that aurantiamide acetate can significantly reduce the levels of total cholesterol (TC) and triglycerides (TC) in mice with metabolic-associated fatty liver disease, and decrease the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), and low-density lipoprotein (LDL). It has significant lipid-lowering, anti-inflammatory, anti-fibrotic, and hepatoprotective effects. Furthermore, aurantiamide acetate is essentially non-toxic and has good safety, making it suitable for the preparation of drugs for the prevention / treatment of metabolic-associated fatty liver disease and its related complications, with broad application prospects. Attached Figure Description

[0018] Figure 1 The following is an example of the effect of aurantiamide acetate (AA, 10 mg / kg) high-dose group, atorvastatin (ATV, 10 mg / kg) treatment group, and control and model groups on mouse body weight compared to one embodiment of this application.

[0019] Figure 2 The image shows the results of HE, Masson's Red, Sirius Red and Oil Red staining of mouse liver in one embodiment of this application. The scale bars in the image are marked from left to right as 0µm, 20µm, 40µm, 60µm, 80µm and 100µm.

[0020] Figure 3 The image shows the results of detecting the levels of ALT, AST, LDL, HDL, TG, and TC in mouse serum in one embodiment of this application (the mice were divided into control group, HFCD model group, HFCD-treated ATV group, and HFCD-treated AA low, medium, and high dose groups).

[0021] Figure 4 The results of detecting IL6 and TNFα levels in mouse serum are shown in one embodiment of this application (mouse grouping and...). Figure 3 same);

[0022] Figure 5 The image shows the results of the detection of the effects of Aurantiamide acetate intervention on the ALT, AST, LDL, HDL, TG, and TC levels in HepG2 and Huh7 cells in one embodiment of this application.

[0023] Figure 6 The image shows the Oil Red staining results of HepG2 and Huh7 cells after intervention with Aurantiamide acetate in one embodiment of this application.

[0024] Figure 7The image shows the ROS fluorescence staining results of HepG2 and Huh7 cells before and after intervention with Aurantiamide acetate, as illustrated in one embodiment of this application.

[0025] Figure 8 The image shows the results of detecting the levels of ALT, AST, LDL, HDL, TG, TC, IL6, and TNFα in mouse serum after combined drug administration, according to one embodiment of this application. Detailed Implementation

[0026] To make the objectives, technical solutions, and advantages of this application clearer, a more detailed description is provided below. However, it should be understood that the description herein is merely for explaining this application and is not intended to limit its scope.

[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs. The terminology used herein is for the purpose of describing particular embodiments only and is not intended to limit the scope of this application. All reagents and instruments used herein are commercially available, and the characterization methods involved can be found in relevant descriptions in the prior art, and will not be repeated here.

[0028] To further understand this application, the following detailed description is provided in conjunction with the preferred embodiments.

[0029] Example 1

[0030] Aurantiamide acetate, CAS: 56121-42-7; purchased from Chengdu Kangbang Biotechnology Co., Ltd.; structural formula shown in formula (I) below.

[0031]

[0032] Equation (Ⅰ).

[0033] Therapeutic effect of aurantiamide acetate on mice with metabolic-associated fatty liver disease:

[0034] 1. Experimental materials:

[0035] Male C57BL / 6 mice, 6-8 weeks old, weighing 18±2g, were provided by the Lanzhou Veterinary Research Institute of the Chinese Academy of Agricultural Sciences. The housing environment was SPF grade, with a temperature of 22±2℃, humidity of 50-60%, and simulated lighting using fluorescent lamps from 7:00 to 19:00. The ventilation rate in the housing was 10-20 times per hour, and the noise level was below 60dB.

[0036] They were housed separately in plastic cages, with free access to food and water for 7 days to allow them to adapt to the environment and undergo quarantine, in preparation for the next stage of the experiment.

[0037] 2. Experimental Methods:

[0038] This experiment established a mouse model of metabolic-related fatty liver disease using a 60% high-fat and choline-deficient diet (HFCD) to study the therapeutic effect of Aurantiamide acetate on this model mouse. The control group was fed a normal diet.

[0039] 2.1 Experimental Grouping:

[0040] Mice were randomly divided into a control group, a model group, an atorvastatin treatment group (Atorvastatin, ATV, 10 mg / kg), a low-dose Aurantiamide acetate group (1 mg / kg), a medium-dose Aurantiamide acetate group (5 mg / kg), and a high-dose Aurantiamide acetate group (10 mg / kg), with 6 mice in each group.

[0041] In cell experiments, HepG2 and Huh7 cell lines were used, and a final concentration of 0.75 mM was prepared by mixing sodium oleate and sodium palmitate in a molar ratio of 2:1 for 72 hours to simulate a non-alcoholic fatty liver model.

[0042] 2.2 Administration method:

[0043] After 6 weeks of rearing in an SPF-grade environment, drug administration began. The control and model groups received only 0.5% CMCNa injections starting from week 6 of HFCD rearing. The low-dose, medium-dose, and high-dose Aurantiamide acetate groups received 1 mg / kg, 5 mg / kg, and 10 mg / kg, respectively, prepared as a suspension in 0.5% CMCNa and administered via intraperitoneal injection once daily. The atorvastatin treatment group received 10 mg / kg via gavage. All groups received continuous administration for 28 days. The normal control and model control groups received the corresponding volume of solvent. Aurantiamide acetate was purchased from Chengdu Kangbang Biotechnology Co., Ltd. (CAS: 56121-42-7). ALT, AST, LDL, HDL, TG, and TC reagent kits were purchased from Nanjing Jiancheng Biotechnology Co., Ltd., ROS reagent kits were purchased from Shanghai Beyotime Biotechnology Co., Ltd., and Oil Red staining reagent kits were purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0044] 2.3 Experimental Methods:

[0045] 2.3.1 Serum TC, TG, IL-6, TNFα, ALT, AST, HDL, and LDL were measured using the kit:

[0046] Blood was collected via ocular sampling. After standing at room temperature for 2 hours, the blood was centrifuged at 3000 rpm for 5 minutes, and the supernatant was collected and tested according to the instructions of the different reagent kits.

[0047] 2.3.2 Preparation of mouse liver pathological sections:

[0048] Rat livers were fixed with 4% paraformaldehyde, then embedded in paraffin and sectioned. They were then stained with HE, Masson's Red, and Sirius Red. In addition, Oil Red O staining was performed on frozen pathological sections of fresh tissue.

[0049] 2.3.3 Cellular biochemical detection:

[0050] Following the kit instructions, after collecting the cells, they were lysed and then analyzed using a 96-well plate. The results were read using a full-spectrum microplate reader, and the data were then analyzed.

[0051] 2.3.4 Cell ROS detection:

[0052] A metabolic-related fatty liver disease model was constructed using 6-well plates and cell crawling. Cells were pretreated using this method and then observed and photographed under a fluorescence microscope.

[0053] 3. Experimental Data Detection and Processing:

[0054] On day 28 after administration, mice were fasted for 8 hours, blood was collected from their eyeballs, serum was separated, and the left lobe of the liver was fixed in 10% formaldehyde solution for later use.

[0055] 3.1 Serum marker measurement:

[0056] The levels of total cholesterol (TC), triglycerides (TG), IL-6, TNFα, and the activities of alanine aminotransferase (ALT), aspartate aminotransferase (AST), high-density lipoprotein (HDL), and low-density lipoprotein (LDL) were measured in mice.

[0057] 3.2 Liver pathological and histological observation:

[0058] Rat livers were fixed in 4% neutral formaldehyde, embedded in paraffin, sectioned, dewaxed in xylene, dehydrated in graded ethanol, stained with hematoxylin and eosin (HE) as usual, dehydrated in ethanol, cleared in xylene, mounted with resin, and observed under a microscope.

[0059] 3.3 Statistical Analysis:

[0060] Data processing was performed using Graphpad 8.0 software, including one-way ANOVA.

[0061] 4. Experimental Results:

[0062] 4.1 Changes in body weight and pathological indicators:

[0063] Mouse weight as Figure 1 As shown, preliminary experiments revealed widespread fat accumulation in the livers of mice after 6 weeks of HFCD diet, but no obvious fibrosis or hepatocyte damage was observed. Therefore, at week 6 (13 weeks of age), while continuing the HFCD diet, mice were given intraperitoneal injections of different concentrations of Aurantiamide acetate, and it was found that their weight gain trend was significantly slower compared to HFCD alone.

[0064] Liver pathological and histological changes such as Figure 2 As shown, after 10 weeks of HFCD feeding, HE staining revealed numerous fat vacuoles, inflammatory cell infiltration, and hepatocyte necrosis in the mouse liver. Masson and Sirius Red staining showed extensive fibrosis in the liver tissue, and Oil RED O staining revealed that the vacuoles contained numerous fat droplets. After 4 weeks of treatment with aurantiamide acetate, the fat vacuoles, inflammatory cell infiltration, and hepatocyte necrosis were significantly improved, the degree of fibrosis was significantly reduced, and the accumulation of lipid droplets in the liver tissue was also significantly improved. This result indicates that aurantiamide acetate has a significant therapeutic effect on liver damage caused by HFCD-induced metabolic-associated fatty liver disease in animal models, and can be used to treat lipid droplet accumulation and liver damage caused by metabolic-associated fatty liver disease, and may even alleviate a certain degree of fibrosis (×200 original magnification, scale bar 50 μm).

[0065] 4.2 Serum biochemical properties of mice:

[0066] like Figure 3 , 4 As shown, compared with the control group, mice on the HFCD 10-week diet showed a significant increase in TG, TC, ALT, AST, LDL, TNFα, and IL-6, all typical blood biochemical manifestations of metabolic-related fatty liver disease. Starting from week 6 of the HFCD diet, after 4 weeks of treatment with different concentrations of Aurantiamide acetate, all indicators decreased, with the 10 mg / kg group showing the most significant decrease.

[0067] 4.3 Biochemical characteristics of HepG2 and Huh7 cells:

[0068] like Figure 5 As shown, a metabolic-related fatty liver disease model was constructed in HepG2 and Huh7 cells for 48 hours using a mixture of sodium palmitate and sodium oleate at molar concentrations. It was found that TG, TC, and LDL showed an increasing trend in the model group. After intervention with different concentrations of Aurantiamide acetate for 48 hours, all indicators decreased (groups included control group, solvent control group, model group, 6.25µM concentration group, 12.5µM concentration group, and 25µM concentration group; the solvent refers to the solvent used to dissolve sodium oleate and sodium palmitate, and this solvent group was added to avoid the solvent affecting the model; multiple concentration groups refer to the 0.75µM modeling solution obtained by mixing the two as described in section 2.1 above, which was then prepared by mixing the solvent separately).

[0069] 4.4 Oil Red staining of HepG2 and Huh7 cells:

[0070] like Figure 6 As shown, HepG2 and Huh7 cell lines were stained using an Oil Red O cell-specific staining kit. It was found that the lipid droplet accumulation was significantly reduced in the Aurantiamide acetate treatment group compared to the model group (×200 original magnification, scale bar 50 μm).

[0071] 4.5 ROS Detection:

[0072] like Figure 7 As shown, the HepG2 and Huh7 cell lines exhibited significantly elevated reactive oxygen species (ROS) levels. However, after intervention with Aurantiamide acetate, ROS levels showed a significant decreasing trend, indicating that Aurantiamide acetate can improve cellular oxidative stress (×200 original magnification, scale bar 50 μm).

[0073] Example 2

[0074] Drugs used for the prevention / treatment of fatty liver disease and its complications also include atorvastatin preparations that can be used in combination with aureomycin; the dosage ratio of aureomycin to atorvastatin preparations is selected as 5 mg / kg: 5 mg / kg.

[0075] 1. Experimental Groups:

[0076] Mice were randomly divided into a control group, a model group, a high-dose Aurantiamide acetate group (10 mg / kg), a combination therapy of atorvastatin alone (10 mg / kg), and a combination therapy of aurantiamide acetate (5 mg / kg), with 6 mice in each group.

[0077] 2. The administration method, experimental method, and experimental data detection are the same as those in items 2.2, 2.3, and 3 of Example 1, respectively.

[0078] 3. The biochemical properties of mouse serum were tested according to section 4.2 of Example 1:

[0079] like Figure 8 As shown, compared with the control group, mice on the HFCD 10-week diet showed a significant increase in TG, TC, ALT, AST, LDL, TNFα, and IL-6, all typical blood biochemical manifestations of metabolic-related fatty liver disease. Starting from week 6 of the HFCD diet, after 4 weeks of treatment in the combination therapy group, all indicators decreased, and the treatment effect was significantly better than the high-dose auramine alcohol ester alone group and the combination therapy alone group.

[0080] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. The use of aurora amide alcohol ester or a pharmaceutically acceptable salt thereof in the preparation of a medicament for treating fatty liver disease, characterized in that, The fatty liver disease was selected as metabolic-related fatty liver disease.

2. The application according to claim 1, characterized in that, The dosage form of the drug may be tablets, injections, or capsules.

3. The application according to claim 1 or 2, characterized in that, The drug also includes an atorvastatin preparation that can be used in combination with aureomycin ester; the dosage ratio of aureomycin ester to atorvastatin preparation is selected as 5 mg / kg: 5 mg / kg.

Images