Use of nintedanib or a salt thereof for the manufacture of a medicament for the treatment of hyperlipidemia

Abstract

The application belongs to the field of metabolic disease treatment, and provides application of nintedanib or a salt thereof in preparation of a drug for treating hyperlipidemia. It is found for the first time that nintedanib can significantly up-regulate the expression of ATGL by inhibiting the ubiquitination and degradation of ATGL by MKRN2, thereby producing a significant lipid-lowering effect. The drug has low cytotoxicity, a wide therapeutic window, and good development potential.

Description

Technical Field

[0001] This application pertains to the field of metabolic disease treatment, specifically, it provides the use of nintedanib or its salts in the preparation of medicaments for treating hyperlipidemia. Background Technology

[0002] Adipose triglyceride lipase (ATGL) is the rate-limiting enzyme in the catabolism of triglycerides, primarily responsible for hydrolyzing triglycerides (TG) into diglycerides and free fatty acids (FFAs). ATGL is widely expressed in various tissues throughout the body, including the liver, adipose tissue, and muscle. In the body, ATGL-mediated lipolysis is tightly coupled with fatty acid β-oxidation. Moderate upregulation of ATGL expression can accelerate the breakdown and utilization of fatty acids, reduce intracellular lipid accumulation, thereby decreasing the assembly and secretion of very low-density lipoprotein (VLDL), ultimately leading to a decrease in circulating lipid levels. Previous studies have shown that ATGL expression levels are relatively insufficient in patients with hyperlipidemia and non-alcoholic fatty liver disease; therefore, finding safe and effective ATGL expression promoters has significant clinical value.

[0003] Nintedanib is a known multi-target tyrosine kinase inhibitor that primarily inhibits VEGFR, PDGFR, and FGFR. Clinically, it is mainly used to treat idiopathic pulmonary fibrosis and certain malignant tumors. To date, no existing technology has publicly demonstrated that nintedanib can upregulate the expression of the ATGL gene, nor is it considered to have a lipid-lowering effect. Summary of the Invention

[0004] In response to the above situation, this application provides the use of nintedanib or its salt in the preparation of a drug for treating hyperlipidemia.

[0005] On the other hand, this application provides the use of nintedanib or its salts in the preparation of medicaments for treating hypertriglyceridemia.

[0006] On the other hand, this application provides nintedanib or its salts for the preparation of a treatment for non-alcoholic fatty liver disease.

[0007] Furthermore, the salt is an ethanesulfonate.

[0008] Furthermore, the drug upregulates the expression of triglyceride lipase.

[0009] Furthermore, the drug inhibits the degradation of ATGL by MKRN2.

[0010] Furthermore, the drug lowers triglyceride or cholesterol levels in the blood.

[0011] Furthermore, the drug is an oral preparation.

[0012] In addition to oral formulations, other pharmaceutically acceptable dosage forms, such as injections, such as aqueous solutions, powders for injection, or topical formulations, may also be used in this application after appropriate validation.

[0013] Furthermore, the drug includes pharmaceutically acceptable excipients.

[0014] The pharmaceutically acceptable excipients include, but are not limited to, fillers, binders, sustained-release agents, controlled-release agents, lubricants, antioxidants, preservatives, flavor maskers, taste enhancers, solvents, solubilizers, suspending agents, pH adjusters, osmotic pressure adjusters, coating agents, capsules, etc., preferably excipients suitable for oral formulations. The selection of excipients can be made by those skilled in the art using common knowledge in the field.

[0015] Furthermore, the drug is in the form of a soft capsule.

[0016] The hyperlipidemia or hypertriglyceridemia described in this application is not limited to conditions caused by Mycobacterium tuberculosis infection; hyperlipidemia or hypertriglyceridemia caused by other reasons can also be treated with the drugs described in this application.

[0017] Furthermore, the pharmaceutically acceptable salt is ethanesulfonate.

[0018] The chemical formula of nydanib described in this application is as follows:

[0019]

[0020] The drugs used clinically are mainly in the form of ethanesulfonate salts. Other salts, conjugates, hydrates, and variants of these drugs are also within the scope of protection of this application.

[0021] Beneficial effects:

[0022] This invention is the first to discover that nintedanib can significantly upregulate ATGL expression by inhibiting the ubiquitination and degradation of ATGL by MKRN2, thereby producing a significant lipid-lowering effect. This drug exhibits low cytotoxicity, a broad therapeutic window, and good development potential. Attached Figure Description

[0023] Figure 1 The images show BODIPY immunofluorescence patterns indicating abnormal intracellular lipid droplet accumulation and Western blot analysis of decreased ATGL expression levels following Mycobacterium tuberculosis infection.

[0024] Figure 2 This is a statistical analysis chart showing that after Mycobacterium tuberculosis infection, serum ATGL levels decrease, TG levels increase, and FFAs degradation decreases.

[0025] Figure 3 Western blot analysis of the full-length MKRN2 and its three truncated portions (designed based on the N-terminal, C-terminal, and M-terminal ubiquitin domains) binding with and degrading ATGL.

[0026] Figure 4 Western blot analysis of nintedanib treatment inhibiting MKRN2 binding to Ag85A and MKRN2 ubiquitin activity.

[0027] Figure 5 Western blot analysis of MKRN2 and ATGL in BMDM macrophages after nintedanib treatment.

[0028] Figure 6 Statistical analysis of serum ATGL, TG and FFAs in a hyperlipidemic model using nintedanib.

[0029] Figure 7 Immunohistochemical staining image of ATGL expression in lung tissue of a mouse model of hyperlipidemia and Oil Red staining image of frozen sections for detecting lipid droplet accumulation. Detailed Implementation

[0030] Example 1: Abnormal accumulation of intracellular lipid droplets and decreased ATGL expression after Mycobacterium tuberculosis infection.

[0031] *M. bovis* strain in logarithmic growth phase was used to infect BMDM macrophages of 6-8 week old mice with a multiplicity of infection (MOI) of 10, and cells were collected at different time points (6 h, 12 h, and 24 h) post-infection. Lipid droplets in THP-1 macrophages were stained with BODIPY, *M. bovis* was labeled with LAM antibody, and observed using immunofluorescence microscopy. Immunoblot analysis 24 h after *M. bovis* infection was used to detect the protein expression levels of lipid metabolism-related proteins ATGL and PLIN2, as well as lipid synthesis-related protein SREBP1.

[0032] Results: Imaging results showed that, compared with the uninfected control group (Mock), the number of lipid droplets and bacteria in the M. bovis infected group was significantly increased. Figure 1 Part A). Further quantitative analysis of fluorescence intensity showed that the content of total neutral lipids and the number of bacteria in the cells increased significantly with the extension of infection time. Figure 1 (Parts B and C). ATGL in the infected group was significantly lower than in the Mock group, while PLIN2 and SREBP1 were not significantly different between the two groups ( Figure 1 (Parts D and E).

[0033] Example 2: After Mycobacterium tuberculosis infects the body, serum ATGL levels decrease, TG levels increase, and FFA degradation decreases.

[0034] Serum samples were collected from 40 individuals who tested positive for tuberculosis, and serum samples were collected from 60 individuals who tested positive for tuberculosis as a negative control. The levels of ATGL, TG, and FFAs in the serum were measured.

[0035] Results: The test results confirmed that 82.5% of tuberculosis-positive individuals had serum ATGL levels lower than normal. Figure 2 Parts A and B); 92.5% of tuberculosis-positive individuals had serum TG levels higher than normal ( Figure 2 Parts C and D); 85% of tuberculosis-positive individuals had serum FFA levels lower than normal ( Figure 2 (Parts E and F of the study). These results preliminarily indicate that after Mycobacterium tuberculosis infects the body, it can significantly interfere with the host's lipid metabolism balance, reduce the expression of ATGL protein, inhibit lipid catabolism, thereby increasing triglyceride levels, reducing the breakdown and utilization of fatty acids, and leading to hyperlipidemia.

[0036] Example 3: MKRN2 ubiquitin ligase degradation of ATGL assay

[0037] Twenty-four hours after transfecting HEK-293T cells with full-length MKRN2, three truncated variants, and ATGL plasmids, total cellular protein was extracted, and Western blot was used to detect the ubiquitination and degradation of ATGL by full-length MKRN2 and three truncated variants.

[0038] Results: Design drawings of the full length and three truncated sections of MKRN2 ( Figure 3 In part A of the unloaded group, ATGL interacts with MKRN2, and the C-terminal ubiquitous domain plays a major role. Figure 3 (Part B); MKRN2 can ubiquitinate and degrade ATGL, with the C-terminal ubiquitin domain playing a major role ( Figure 3 Part C).

[0039] Example 4: MKRN2 ubiquitin ligase activity inhibition assay

[0040] Six hours after transfecting HEK-293T cells with three plasmids carrying different tags (Ag85A, MKRN2, and ATGL), the cells were treated with 1 μM Nintedanib for 24 hours. Total cellular protein was extracted, and Western blot analysis was performed to detect the interaction between Ag85A and MKRN2 and the ubiquitination and degradation of ATGL by MKRN2.

[0041] Results: Compared with the control group without a host, the interaction between Ag85A and MKRN2 was significantly reduced in the Nintedanib treatment group, and the ubiquitination and degradation of ATGL were inhibited. Figure 4 This indicates that Nintedanib can inhibit the ubiquitination and degradation of ATGL by MKRN2.

[0042] Example 5: Detection of ATGL protein stability in BMDM macrophages

[0043] After infecting M. bovis (MOI=3) with BMDM cells for 4 hours, the cells were treated with 1 μM Nintedanib for 24 hours. Total cellular protein was extracted, and the levels of ATGL and MKRN2 proteins were detected by Western blot.

[0044] Results: Compared with the DMSO control group, the Nintedanib treatment group showed increased ATGL protein levels and significantly inhibited MKRN2 protein levels. Figure 5 This indicates that Nintedanib can effectively inhibit the degradation of ATGL by MKRN2 under infectious conditions.

[0045] Example 6: Nintedanib replenishes ATGL levels, reduces TG levels, and increases FFA levels in the serum of a mouse model of hyperlipidemia following tuberculosis infection.

[0046] Female C57BL / 6N mice were infected with Mycobacterium tuberculosis via nasal instillation. Seven days after infection, mice were given oral administration of the compound nintedanib (60 mg / kg / day) for four weeks. Serum samples were collected to detect the levels of ATGL, TG, and FFAs in the serum.

[0047] Results: In the nintedanib treatment group, the serum ATGL level returned to normal compared with the challenge group. Figure 6 Part A); TG content decreased to normal levels ( Figure 6 Part B); FFAs levels rise to normal levels due to increased ATGL expression enhancing lipid metabolism and breakdown ( Figure 6 (Part C of the study). These preliminary results indicate that nintedanib treatment can enhance host lipid metabolism, increase ATGL protein expression, promote lipid catabolism, and enhance the breakdown and utilization of fatty acids, thereby reducing triglyceride levels in the body and treating / preventing hyperlipidemia.

[0048] Example 7: Observation of nintedanib's regulation of in vivo tissue ATGL and lipid droplet staining in a mouse model of hyperlipidemia following tuberculosis infection.

[0049] Female C57BL / 6N mice were infected with Mycobacterium tuberculosis via nasal instillation. Seven days after infection, they were given oral administration of the compound nintedanib (60 mg / kg / day) for four weeks. Lung tissue was fixed for two weeks. One portion was used for paraffin embedding and sectioning, and after dewaxing, ATGL monoclonal antibody was used to stain the lung tissue for ATGL. The other portion was used for Octe embedding and frozen sectioning, and stained with a modified Oil Red O kit. Both stained tissues were then analyzed under a microscope.

[0050] Results: The lung tissue of mice in the nintedanib treatment group showed a significantly higher level of ATGL antibody-positive staining compared to the challenge group. Figure 7 Part A); the staining of oil red lipid droplets was significantly reduced ( Figure 7 Part B).

Claims

1. The use of nintedanib or its salts in the preparation of drugs for treating hyperlipidemia.

2. Use of nintedanib or its salts in the preparation of drugs for treating hypertriglyceridemia.

3. Nintedanib or its salts in the preparation of treatments for non-alcoholic fatty liver disease.

4. The application according to any one of claims 1-3, wherein the salt is an ethanesulfonate.

5. The application according to any one of claims 1-3, wherein the drug upregulates the expression of triglyceride lipase.

6. The application according to any one of claims 1-3, wherein the drug inhibits the degradation of ATGL by MKRN2.

7. The application according to any one of claims 1-3, wherein the drug reduces blood triglyceride or cholesterol levels.

8. The application according to any one of claims 1-7, wherein the drug is an oral preparation.

9. The application according to claim 8, wherein the medicament comprises pharmaceutically acceptable excipients.

10. The application according to claim 8, wherein the drug is a soft capsule.

Images