Application of lincetinib in preparation of medicine for treating non-alcoholic fatty liver disease

Abstract

The invention relates to application of lincetinib in preparation of a medicine for treating a non-alcoholic fatty liver disease, and belongs to the technical field of biological medicines. The invention discloses the new application of lincetinib in preparation of the medicine for treating the non-alcoholic fatty liver disease for the first time. In a doxorubicin hydrochloride-induced non-alcoholic fatty liver disease animal model, lincetinib is supplemented in an injection manner, so that the lincetinib is effectively proved to be capable of reducing the content of triglyceride, glutamic-pyruvic transaminase and glutamic oxalacetic transaminase in blood by inhibiting the expression of an insulin-like growth factor 1 receptor, rescuing liver function impairment, reducing the content of insulin-like growth factor 1 and reducing the content of insulin-like growth factor 1. And lipid accumulation in serum and liver tissues is strongly reversed, so that the effect of preventing or treating the non-alcoholic fatty liver disease is achieved. Therefore, lincetinib can be used for preparing the medicine for treating the non-alcoholic fatty liver disease, and has important significance in clinical treatment of the non-alcoholic fatty liver disease in the future.

Description

Technical Field

[0001] The invention relates to application of lincitinib in preparing a drug for treating non-alcoholic fatty liver disease, and belongs to the technical field of biomedicine. Background Art

[0002] Insulin-like growth factor 1 receptor (IGF-1R) belongs to the receptor tyrosine kinase family. The protein encoded by this gene binds to insulin-like growth factor (IGF-1), causing phosphorylation of its own tyrosine kinase domain and initiating intracellular signal transduction, regulating cell growth and differentiation. Recent studies have found that IGF-1R is highly expressed in malignant tumor tissues such as colon cancer and liver cancer. By inhibiting the expression of IGF-1R in tumor cells, the IGF-1R inhibitor Linsitinib has become an extremely attractive tumor treatment drug.

[0003] Non-alcoholic fatty liver disease (NAFLD) is a clinical pathological syndrome characterized by diffuse macrovesicular steatosis of hepatocytes. Excessive intake of high-fat foods and long-term use of certain drugs such as chemotherapy drugs can promote the occurrence of NAFLD. NAFLD is the most common chronic liver disease in the world, with a prevalence rate of up to 30% in ordinary adults. In recent years, under the influence of the dual trends of obesity at a young age and population aging, the prevalence of NAFLD has continued to rise. Its prevalence has surged by 68.3% in 15 years. In addition, due to the large population base, a slight increase in the prevalence will bring a large number of new patients. NAFLD patients often have an insidious onset. When they develop into severe fatty hepatitis, they may experience jaundice, poor appetite, nausea and vomiting. In the late stage, they may develop into cirrhosis and liver cancer, which seriously endangers the health of the Chinese people and brings a heavy economic burden to the country, society, and families. It has become a major public health issue facing society today.

[0004] Normally, when a patient is diagnosed with NAFLD, they are often treated clinically through diet control, exercise, and medication, most of which are oral liver-protecting drugs and lipid-lowering drugs. However, existing liver-protecting drugs cannot effectively reverse liver lipid accumulation and fibrosis. Although lipid-lowering drugs can effectively lower lipids, it is necessary to closely monitor changes in liver function during use and be alert to serious adverse reactions such as myopathy and rhabdomyolysis caused by statins. NAFLD patients are often unable to take the drug because of their own abnormal liver function and contraindications to taking the drug.

[0005] Therefore, exploring the role of IGF-1R and its inhibitor lincitinib in the occurrence and development of NAFLD and finding a safe, effective and rapid drug for the clinical treatment of NAFLD have both practical significance and important strategic significance for developing new NAFLD treatment targets. Summary of the invention

[0006] In view of the deficiencies in the prior art, the present invention provides the use of linsitinib in the preparation of a drug for treating non-alcoholic fatty liver disease.

[0007] The technical solution of the present invention is as follows: Application of linsitinib in the preparation of drugs for treating non-alcoholic fatty liver disease.

[0008] Preferably, according to the present invention, the drug for treating non-alcoholic fatty liver disease alleviates the progression of non-alcoholic fatty liver disease by inhibiting the expression of insulin-like growth factor 1 receptor (IGF-1R).

[0009] Preferably, according to the present invention, the drug for treating non-alcoholic fatty liver disease inhibits the progression of non-alcoholic fatty liver disease by reducing the levels of triglycerides, alanine aminotransferase and aspartate aminotransferase and improving lipid accumulation.

[0010] Preferably according to the present invention, in the drug for treating non-alcoholic fatty liver disease, lincitinib is the only active ingredient.

[0011] Preferably according to the present invention, the drug for treating non-alcoholic fatty liver disease comprises lincitinib and a pharmaceutically acceptable carrier.

[0012] Preferably according to the present invention, the dosage form of the drug for treating non-alcoholic fatty liver disease is granules, tablets, capsules, pills or oral liquid preparations.

[0013] Beneficial effects: The present invention discloses for the first time a new use of linsitinib in the preparation of drugs for treating non-alcoholic fatty liver disease. In the animal model of non-alcoholic fatty liver disease induced by doxorubicin hydrochloride, the present invention supplements linsitinib by injection, and effectively confirms that linsitinib can inhibit the expression of insulin-like growth factor 1 receptor, reduce the levels of triglycerides, alanine aminotransferase and aspartate aminotransferase in the blood, save liver function damage, and strongly reverse lipid accumulation in serum and liver tissue, thereby achieving the effect of preventing or treating non-alcoholic fatty liver disease. Therefore, linsitinib can be used to prepare drugs for treating non-alcoholic fatty liver disease, which is of great significance in the future clinical treatment of non-alcoholic fatty liver disease. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 It is the statistical result of the detection of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the serum of mice in each group; In the figure, A is the detection statistical result of alanine aminotransferase (ALT); B is the detection statistical result of aspartate aminotransferase (AST).

[0015] Figure 2 is the result of determination of triglyceride (TG) content in serum and liver tissue of mice in each group; In the figure, A is the statistical result of the detection of triglyceride (TG) in serum; B is the statistical result of the detection of triglyceride (TG) in liver tissue.

[0016] Figure 3 These are the results of Oil Red O staining of liver tissues of mice in each group.

[0017] Figure 4 These are the HE staining results of liver tissues of mice in each group.

[0018] Figure 5 The Sirius red staining of the liver tissues of mice in each group. DETAILED DESCRIPTION

[0019] The technical solution of the present invention is further described below in conjunction with the embodiments, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the embodiments are all common commercially available products unless otherwise specified.

[0020] Doxorubicin HCl (Dox) and linsitinib used in the examples were purchased from Selleck.

[0021] Linsitinib is OSI-906, and its chemical structure is as follows: .

[0022] The alanine aminotransferase (GPT / ALT) activity detection kit, aspartate aminotransferase (GOT / AST) activity detection kit and improved Sirius red staining kit used in the examples were purchased from Beijing Solebow Technology Co., Ltd.

[0023] The liquid sample triglyceride enzymatic assay kit and tissue cell triglyceride enzymatic assay kit used in the examples were purchased from Beijing Pulilai Gene Technology Co., Ltd.

[0024] The modified Oil Red O staining kit used in the examples was purchased from Beyotime Biotechnology Research Institute.

[0025] The C57BL6J mice used in the examples were purchased from Beijing Weitonglihua Company.

[0026] The collection and detection of mouse serum and liver tissue specimens used in the examples were approved by the Animal Ethics Committee of Shandong University.

[0027] Example 1: Construction of a mouse model of non-alcoholic fatty liver disease 1. Animal preparation: 6-week-old C57BL6J mice were placed in the SPF-level animal room of Shandong University and maintained in a normal diet for one week in preparation for subsequent experiments.

[0028] 2. Drug preparation: According to DMSO: H 2 O=5:95 (volume ratio) and mixed evenly to obtain a solvent; doxorubicin hydrochloride (Dox) was added to the solvent and mixed evenly to obtain a Dox solution. The doxorubicin hydrochloride (Dox) can quickly simulate the process of serum and liver lipid accumulation and liver fibrosis in the development of non-alcoholic fatty liver disease (NAFLD), so it can be used to construct a mouse model of non-alcoholic fatty liver disease.

[0029] According to DMSO: PEG300: Tween80: H 2 O=5:30:10:55 (volume ratio) are mixed evenly to obtain a solvent; Linsitinib is added to the solvent, mixed evenly to obtain a Linsitinib solution.

[0030] 3. Drug injection: The mice were divided into four groups. The first group was injected with Dox solution at a dose of 25 mg / kg, recorded as the DOX group; the second group was injected with Linsitinib solution at a dose of 15 mg / kg, recorded as the Linsitinib group; the third group was first injected with Dox solution at a dose of 25 mg / kg, and then injected with Linsitinib solution at a dose of 15 mg / kg, recorded as the Linsitinib+DOX group; the fourth group was injected with DMSO:H 2 O=5:95 (volume ratio) was intraperitoneally injected with an equal volume of solvent into the mice, which was recorded as the Sham group.

[0031] 4. Sample collection: After the injection of the above four groups of mice, the mice were cultured for 3 days, and then euthanized and blood was collected from the eyeballs. The blood was centrifuged at 4°C and 1000g for 10 minutes, and the supernatant was collected as serum and frozen at -80°C. The liver was dissected, part of the liver tissue was frozen at -80°C, and part of the liver tissue was fixed with 4% paraformaldehyde at room temperature, and frozen sections and paraffin sections were prepared according to existing methods.

[0032] Example 2: Detection of ALT and AST in serum The sera of the Sham group, DOX group, Linsitinib group and Linsitinib+DOX group obtained in Example 1 were added to a 96-well plate, and then the serum of each group was detected using an alanine aminotransferase (GPT / ALT) activity detection kit and an aspartate aminotransferase (GOT / AST) activity detection kit. The specific detection method was referred to the instructions of the kit. Finally, a standard curve was drawn according to the absorbance detection results at 505 nm, and the levels of ALT and AST in the serum were calculated. The results are shown in FIG. Figure 1 The experimental data were analyzed using GraphPad Prism 6 software, and the t-test was used to analyze the differences among different groups. P<0.05 indicated that the difference was statistically significant, and P<0.01 indicated that the difference was statistically significant.

[0033] Depend on Figure 1 It can be seen that compared with the mice in the Sham group, the ALT and AST levels in the serum of the mice in the DOX group were significantly increased, which indicates that the non-alcoholic fatty liver disease mouse model in Example 1 was successfully established. Compared with the DOX group, the ALT and AST levels in the serum of the mice in the Linsitinib+DOX group injected with Linsitinib at the same time were significantly decreased, that is, Linsitinib can effectively rescue the liver function damage caused by non-alcoholic fatty liver disease mice.

[0034] Example 3. Determination of triglyceride (TG) content in serum and liver tissue 1. The sera of the Sham group, DOX group, Linsitinib group and Linsitinib+DOX group obtained in Example 1 were added to a 96-well plate, and then the serum of each group was detected using a liquid sample triglyceride enzymatic assay kit. The specific detection method was referred to the kit manual. Finally, a standard curve was drawn according to the absorbance detection results at 550 nm to calculate the level of TG in the serum. The results are shown in FIG. Figure 2 As shown in A. The experimental data were analyzed using GraphPad Prism 6 software, and the t-test was used to analyze the differences among different groups. P<0.05 indicated that the difference was statistically significant, and P<0.01 indicated that the difference was statistically significant.

[0035] 2. Weigh the liver tissue accurately, and lyse and homogenize it according to the ratio of 1 mg tissue: 20 μL lysis buffer. After standing, take the supernatant and use the tissue cell triglyceride enzymatic assay kit for detection. The specific detection method refers to the kit instructions. Finally, draw a standard curve based on the absorbance detection results at 550 nm to calculate the TG content in the liver tissue. The results are as follows: Figure 2As shown in B. The experimental data were analyzed using GraphPad Prism 6 software, and the t-test was used to analyze the differences among different groups. P<0.05 indicated that the difference was statistically significant, and P<0.01 indicated that the difference was statistically significant.

[0036] Depend on Figure 2 As shown in A to B, compared with the mice in the Sham group, the TG content in the serum and liver tissue of the mice in the DOX group was significantly increased, which indicates that the non-alcoholic fatty liver disease mouse model in Example 1 was successfully established. Compared with the DOX group, the TG content in the serum and liver tissue of the mice in the Linsitinib+DOX group injected with Linsitinib at the same time was significantly decreased, that is, Linsitinib can effectively improve the lipid accumulation in the serum and liver tissue of mice, thereby inhibiting the progression of non-alcoholic fatty liver disease.

[0037] Example 4: Liver tissue staining 1. Oil Red O staining of liver: The frozen sections of liver tissues of the Sham group, DOX group, Linsitinib group and Linsitinib+DOX group obtained in Example 1 were rewarmed at room temperature for 20 minutes, and the staining washing solution was added for 20 seconds, and then the washing solution was removed by aspiration. Then, Oil Red O staining solution was added dropwise, and the sections were stained for 25 minutes, washed for 30 seconds, stained with hematoxylin for 3 minutes, differentiated with 1% hydrochloric acid alcohol for 1 second, washed with water, immersed in tap water for 3 minutes to turn blue, and observed and photographed under a microscope after sealing. The results are shown in FIG. Figure 3 shown.

[0038] 2. Sirius red staining of liver: The paraffin sections of liver tissues of the Sham group, DOX group, Linsitinib group and Linsitinib+DOX group obtained in Example 1 were routinely dewaxed to water, and Sirius red staining solution was dripped for 15 min. The sections were quickly rinsed with distilled water to remove excess staining solution, and then quickly dehydrated with a series of ethanol starting from 75%, and xylene was transparent. After sealing with neutral gum, the sections were observed under a microscope and photographed. The results are shown as follows: Figure 4 shown.

[0039] 3. HE staining of liver: The paraffin sections of liver tissues of the Sham group, DOX group, Linsitinib group and Linsitinib+DOX group obtained in Example 1 were routinely dewaxed to water, stained with hematoxylin staining solution for 3 min, rinsed with running water, and the degree of staining was observed under a microscope; differentiated with 1% hydrochloric acid alcohol for 1 s, washed with water, immersed in tap water for 3 min to turn blue, and the color was observed under a microscope; eosin staining solution was added dropwise, stained for 3 min, washed with distilled water, and observed under a microscope, and a series of ethanol was quickly dehydrated starting from 75%, xylene was transparent, and neutral gum was used for sealing and observation under a microscope and photography. The results are as follows Figure 5 shown.

[0040] Depend on Figures 3-5 It can be seen that compared with the Sham group, the fatty lesions and fibrosis changes in the liver of mice in the DOX group were significantly aggravated, which further illustrates that the non-alcoholic fatty liver disease mouse model in Example 1 was successfully constructed. Compared with the DOX group, the TG content in the liver tissue of mice in the Linsitinib+DOX group was significantly decreased, the macrovesicular fatty lesions in the hepatocytes were significantly improved, and the liver fibrosis level was significantly improved, that is, Linsitinib can effectively improve the lipid accumulation in the liver tissue of mice, inhibit the occurrence of fibrosis, and thus inhibit the progression of non-alcoholic fatty liver disease.

[0041] In summary, the present invention discloses for the first time a new use of linsitinib in the preparation of drugs for the treatment of non-alcoholic fatty liver disease. In the animal model of non-alcoholic fatty liver disease induced by doxorubicin hydrochloride, the present invention supplements linsitinib by injection, and effectively confirms that linsitinib can inhibit the expression of insulin-like growth factor 1 receptor, reduce the levels of triglycerides, alanine aminotransferase and aspartate aminotransferase in the blood, save liver function damage, and strongly reverse lipid accumulation in serum and liver tissue, thereby achieving the effect of preventing or treating non-alcoholic fatty liver disease. Therefore, linsitinib can be used to prepare drugs for the treatment of non-alcoholic fatty liver disease, which is of great significance in the future clinical treatment of non-alcoholic fatty liver disease.

[0042] The above-described embodiments are only preferred specific implementation schemes of the present invention and are not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. Application of linsitinib in the preparation of drugs for the treatment of non-alcoholic fatty liver disease.

2. The use of linsitinib as claimed in claim 1 in the preparation of a drug for treating non-alcoholic fatty liver disease, characterized in that: The drug for treating non-alcoholic fatty liver disease alleviates the progression of non-alcoholic fatty liver disease by inhibiting the expression of insulin-like growth factor 1 receptor.

3. The use of linsitinib in the preparation of a drug for treating non-alcoholic fatty liver disease according to claim 1, characterized in that: The drug for treating non-alcoholic fatty liver disease inhibits the progression of non-alcoholic fatty liver disease by reducing the content of triglycerides, alanine aminotransferase and aspartate aminotransferase and improving lipid accumulation.

4. The use of linsitinib in the preparation of a drug for treating non-alcoholic fatty liver disease according to claim 1, characterized in that: In the drug for treating non-alcoholic fatty liver disease, lincitinib is the only active ingredient.

5. The use of linsitinib in the preparation of a drug for treating non-alcoholic fatty liver disease according to claim 1, characterized in that: The drug for treating non-alcoholic fatty liver disease comprises lincitinib and a pharmaceutically acceptable carrier.

6. The use of linsitinib in the preparation of a drug for treating non-alcoholic fatty liver disease according to claim 1, characterized in that: The dosage form of the drug for treating non-alcoholic fatty liver disease is granules, tablets, capsules, pills or oral liquid preparations.

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