Use of Linicitinib in the Preparation of a Medicament for Treating Non-Alcoholic Fatty Liver Disease

Abstract

The present invention relates to the use of linicitinib in the preparation of a medicament for treating non-alcoholic fatty liver disease, and belongs to the technical field of biomedicine. The present invention for the first time discloses a new use of linicitinib in the preparation of a medicament for treating non-alcoholic fatty liver disease. In an animal model of non-alcoholic fatty liver disease induced by doxorubicin hydrochloride, by supplementing linicitinib through injection, it is effectively demonstrated that linicitinib can inhibit the expression of insulin-like growth factor 1 receptor, reduce the contents of triglyceride, alanine aminotransferase and aspartate aminotransferase in the blood, rescue liver function injury, and strongly reverse lipid accumulation in serum and liver tissues, thereby achieving the effect of preventing or treating non-alcoholic fatty liver disease. Therefore, linicitinib can be used in the preparation of a medicament for treating non-alcoholic fatty liver disease, which has important significance in the future clinical treatment of non-alcoholic fatty liver disease.

Description

Technical Field

[0001] The present invention relates to the application of linsitinib in the preparation of a drug for treating non-alcoholic fatty liver disease, belonging to the field of biomedical technology. 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. In recent years, 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 attractive anti-tumor drug.

[0003] Non-alcoholic fatty liver disease (NAFLD) is a clinical and pathological syndrome mainly 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 globally, with a prevalence of up to 30% in the general adult population. In recent years, under the dual trends of low-age obesity and population aging, the prevalence of NAFLD has been continuously rising. Its prevalence has increased sharply by 68.3% in 15 years. Coupled with the large population base, even a slight increase in the prevalence will bring a large number of new patients.

[0004] Usually, when a patient is diagnosed with NAFLD, clinically, dietary control, exercise, and drug treatment are often adopted. Among them, drugs mostly use oral hepatoprotective drugs and lipid-lowering drugs. However, existing hepatoprotective drugs cannot effectively reverse liver lipid accumulation and fibrosis. Although lipid-lowering drugs can effectively reduce lipids, during the medication period, it is necessary to closely monitor changes in liver function and be vigilant against serious adverse reactions such as myopathy and rhabdomyolysis caused by statin drugs. NAFLD patients often cannot take the drugs due to their own abnormal liver function and the existence of medication contraindications.

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

[0006] In view of the deficiencies of the prior art, the present invention provides the application 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:

[0008] The use of linicitinib in the preparation of a medicament for treating non-alcoholic fatty liver disease.

[0009] Preferably according to the present invention, the medicament 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).

[0010] Preferably according to the present invention, the medicament for treating non-alcoholic fatty liver disease inhibits the progression of non-alcoholic fatty liver disease by reducing the contents of triglyceride, alanine aminotransferase and aspartate aminotransferase and improving lipid accumulation.

[0011] Preferably according to the present invention, in the medicament for treating non-alcoholic fatty liver disease, linicitinib is the only active ingredient.

[0012] Preferably according to the present invention, the medicament for treating non-alcoholic fatty liver disease comprises linicitinib and a pharmaceutically acceptable carrier.

[0013] Preferably according to the present invention, the dosage form of the medicament for treating non-alcoholic fatty liver disease is a granule, tablet, capsule, pill or oral liquid preparation.

[0014] Beneficial effects:

[0015] The present invention discloses for the first time a new use of linicitinib in the preparation of a medicament for treating non-alcoholic fatty liver disease. In an animal model of non-alcoholic fatty liver disease induced by doxorubicin hydrochloride, by supplementing linicitinib in an injection manner, it is effectively confirmed that linicitinib can inhibit the expression of insulin-like growth factor 1 receptor, reduce the contents of triglyceride, alanine aminotransferase and aspartate aminotransferase in the blood, rescue liver function injury, and strongly reverse lipid accumulation in serum and liver tissues, so as to achieve the effect of preventing or treating non-alcoholic fatty liver disease. Therefore, linicitinib can be used in the preparation of a medicament for treating non-alcoholic fatty liver disease, which has important significance in the future clinical treatment of non-alcoholic fatty liver disease. Description of the drawings

[0016] Figure 1 are the detection and statistical results of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in the sera of mice in each group;

[0017] In the figure, A is the detection and statistical result of alanine aminotransferase (ALT); B is the detection and statistical result of aspartate aminotransferase (AST).

[0018] Figure 2 are the determination results of the contents of triglyceride (TG) in the sera and liver tissues of mice in each group;

[0019] In the figure, A is the detection and statistical result of triglyceride (TG) in serum; B is the detection and statistical result of triglyceride (TG) in liver tissue.

[0020] Figure 3 It is the Oil Red O staining result of the liver tissue of each group of mice.

[0021] Figure 4 It is the HE staining result of the liver tissue of each group of mice.

[0022] Figure 5 It is the Sirius red staining of the liver tissue of each group of mice. Detailed implementation mode

[0023] The technical solution of the present invention will be further described below in conjunction with embodiments, but the protection scope of the present invention is not limited thereto. The reagents and materials involved in the embodiments are all ordinary commercially available products without special instructions.

[0024] Doxorubicin HCl (Dox) and Linsitinib used in the embodiments were both purchased from Selleck.

[0025] Linsitinib, that is, OSI-906, has the following chemical structural formula:

[0026] .

[0027] The kits for detecting the activity of alanine aminotransferase (GPT / ALT), aspartate aminotransferase (GOT / AST) and the improved Sirius red staining kit used in the embodiments were all purchased from Beijing Solarbio Science & Technology Co., Ltd.

[0028] The liquid sample triglyceride enzymatic determination kit and the tissue cell triglyceride enzymatic determination kit used in the embodiments were both purchased from Beijing PrimeTest Gene Technology Co., Ltd.

[0029] The improved Oil Red O staining kit used in the examples was purchased from Beyotime Institute of Biotechnology.

[0030] The C57BL6J mice used in the embodiments were all purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd.

[0031] The collection and detection of mouse serum and liver tissue specimens used in the embodiments were approved by the Animal Ethics Committee of Shandong University.

[0032] Example 1. Construction of a non-alcoholic fatty liver disease mouse model

[0033] 1. Animal preparation: Place 6-week-old C57BL6J mice in the SPF-level animal house of Shandong University and keep them in a stable state with normal diet for one week for subsequent experiments.

[0034] 2. Drug preparation: Mix DMSO:H2O = 5:95 (volume ratio) evenly to obtain a solvent; add doxorubicin hydrochloride (Dox) to the solvent and mix evenly to obtain a Dox solution. This doxorubicin hydrochloride (Dox) can quickly simulate the processes of serum and liver lipid accumulation and liver fibrosis during the occurrence and development of non-alcoholic fatty liver disease (NAFLD), so it can be used to construct a non-alcoholic fatty liver disease mouse model.

[0035] Mix DMSO:PEG300:Tween80:H2O = 5:30:10:55 (volume ratio) evenly to obtain a solvent; add linsitinib to the solvent and mix evenly to obtain a linsitinib solution.

[0036] 3. Drug injection: Divide the mice into four groups. The first group intraperitoneally injects the Dox solution into the mice at a dose of 25 mg / kg, denoted as the DOX group; the second group intraperitoneally injects the linsitinib solution into the mice at a dose of 15 mg / kg, denoted as the Linsitinib group; the third group first intraperitoneally injects the Dox solution into the mice at a dose of 25 mg / kg, and then intraperitoneally injects the linsitinib solution into the mice at a dose of 15 mg / kg, denoted as the Linsitinib + DOX group; the fourth group intraperitoneally injects the same volume of solvent into the mice according to DMSO:H2O = 5:95 (volume ratio), denoted as the Sham group.

[0037] 4. Sample collection: After the above four groups of mice are injected, continue to culture them for 3 days, then euthanize the mice, take blood from the eyeballs, centrifuge at 4°C and 1000g for 10 minutes, collect the supernatant as serum, and store it at -80°C; dissect the liver, freeze part of the liver tissue at -80°C, and fix part of the liver tissue with 4% paraformaldehyde at room temperature, and prepare frozen sections and paraffin sections according to the existing method.

[0038] Example 2. Detection of alanine aminotransferase (ALT) and aspartate aminotransferase (AST) in serum

[0039] The sera of the Sham group, DOX group, Linsitinib group, and Linsitinib+DOX group obtained in Example 1 were added to 96-well plates, and then the sera of each group were detected using a glutamic pyruvic transaminase (GPT / ALT) activity detection kit and a glutamic oxaloacetic transaminase (GOT / AST) activity detection kit. The specific detection method was referred to the kit instructions. Finally, a standard curve was plotted based on the absorbance results detected at 505 nm, and the levels of ALT and AST in the sera were calculated. The results are as Figure 1 shown. Among them, the experimental data were analyzed using GraphPad Prism 6 software, and the t-test was used for the differential analysis of different groups. P<0.05 indicates that the difference is statistically significant, and P<0.01 indicates that the difference has significant statistical significance.

[0040] It can be Figure 1 seen that compared with the mice in the Sham group, the levels of ALT and AST in the sera of the mice in the DOX group were significantly increased, indicating that the non-alcoholic fatty liver disease mouse model in Example 1 was successfully constructed. Compared with the DOX group, the levels of ALT and AST in the sera of the mice in the Linsitinib+DOX group injected with Linsitinib simultaneously were significantly decreased, that is, Linsitinib can effectively rescue the liver function injury caused by non-alcoholic fatty liver disease mice.

[0041] Example 3. Determination of triglyceride (TG) content in sera and liver tissues

[0042] 1. The sera of the Sham group, DOX group, Linsitinib group, and Linsitinib+DOX group obtained in Example 1 were added to 96-well plates, and then the sera of each group were detected using a triglyceride enzymatic assay kit for liquid samples. The specific detection method was referred to the kit instructions. Finally, a standard curve was plotted based on the absorbance results detected at 550 nm, and the level of TG in the sera was calculated. The results are as Figure 2 shown in A. Among them, the experimental data were analyzed using GraphPad Prism 6 software, and the t-test was used for the differential analysis of different groups. P<0.05 indicates that the difference is statistically significant, and P<0.01 indicates that the difference has significant statistical significance.

[0043] 2. The liver tissues were accurately weighed, lysed and homogenized according to 1 mg tissue: 20 μL lysis buffer, and the supernatant was taken after standing. It was detected using a triglyceride enzymatic assay kit for tissue cells. The specific detection method was referred to the kit instructions. Finally, a standard curve was plotted based on the absorbance results detected at 550 nm, and the content of TG in the liver tissues was calculated. The results are as Figure 2As shown in B. Among them, the experimental data were analyzed using GraphPad Prism 6 software, and the t-test was used for the differential analysis of different groups. P<0.05 indicates that the difference is statistically significant, and P<0.01 indicates that the difference has significant statistical significance.

[0044] As can be seen from Figure 2 A to B in the figure, compared with the mice in the Sham group, the TG content in the serum and liver tissues of the mice in the DOX group was significantly increased, indicating 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 serum and liver tissues of the mice in the Linsitinib+DOX group injected with Linsitinib simultaneously was significantly decreased, that is, Linsitinib can effectively improve the lipid accumulation in the serum and liver tissues of mice, and thus inhibit the progression of non-alcoholic fatty liver disease.

[0045] Example 4. Liver tissue staining

[0046] 1. Oil red O staining of the liver: The frozen sections of the 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, the staining washing solution was added and then aspirated after 20 s, and then the oil red O staining solution was dropped. After staining for 25 minutes, it was washed for 30 s, the nucleus was stained with hematoxylin for 3 min, differentiated with 1% hydrochloric acid alcohol for 1 s, washed with water, soaked in tap water for 3 min for blue return, and observed and photographed under the microscope after mounting. The results are as Figure 3 shown.

[0047] 2. Sirius red staining of the liver: The paraffin sections of the liver tissues of the Sham group, DOX group, Linsitinib group, and Linsitinib+DOX group obtained in Example 1 were dewaxed to water routinely, dropped with Sirius red staining solution for 15 min, quickly rinsed with distilled water to remove the excess staining solution, dehydrated quickly with a series of ethanol starting from 75%, made transparent with xylene, and observed and photographed under the microscope after mounting with neutral gum. The results are as Figure 4 shown.

[0048] 3. HE staining of the liver: The paraffin sections of the liver tissues of the Sham group, DOX group, Linsitinib group, and Linsitinib+DOX group obtained in Example 1 were dewaxed to water routinely, dropped with hematoxylin staining solution for 3 min, rinsed with running water, and the staining degree was observed under the microscope; differentiated with 1% hydrochloric acid alcohol for 1 s, washed with water, soaked in tap water for 3 min for blue return, and the color was observed under the microscope; dropped with eosin staining solution, stained for 3 min, observed under the microscope after washing with distilled water, dehydrated quickly with a series of ethanol starting from 75%, made transparent with xylene, and observed and photographed under the microscope after mounting with neutral gum. The results are as Figure 5 shown.

[0049] It can be seen from Figures 3 - 5 that compared with the Sham group, the fatty liver lesions and fibrosis changes in the livers of mice in the DOX group were significantly aggravated, which further indicated 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 tissues of mice in the Linsitinib + DOX group was significantly decreased, the macrovesicular steatosis in hepatocytes was significantly improved, and the liver fibrosis level was significantly improved, that is, Linsitinib could effectively improve the lipid accumulation in the liver tissues of mice, inhibit the occurrence of fibrosis level, and thus inhibit the progression of non-alcoholic fatty liver disease.

[0050] In summary, 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 non-alcoholic fatty liver disease animal model induced by doxorubicin hydrochloride, by supplementing linsitinib by injection, it was effectively confirmed that linsitinib could inhibit the expression of insulin-like growth factor 1 receptor, reduce the contents of triglyceride, alanine aminotransferase and aspartate aminotransferase in the blood, rescue liver function injury, and strongly reverse the lipid accumulation in serum and liver tissues, so as to achieve 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 has important significance in the future clinical treatment of non-alcoholic fatty liver disease.

[0051] The above embodiments are only preferred specific implementation schemes of the present invention and are not used to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. The use of linsitinib in the preparation of a drug for treating non-alcoholic fatty liver disease, characterized in that: Lincitinib is the only active ingredient in the drug for treating non-alcoholic fatty liver disease; 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.

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 comprises lincitinib and a pharmaceutically acceptable carrier.

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 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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