Application of iron chelators in preventing and treating nonalcoholic steatohepatitis

Abstract

The present invention relates to the field of biomedicine, and more specifically to the use of the novel iron chelator CN128 / deferasirox (DFX) in the prevention and treatment of nonalcoholic steatohepatitis. CN128 or deferasirox can significantly reduce liver damage, steatosis, and hepatocellular ballooning in MCD-induced nonalcoholic steatohepatitis.

Description

Technical Field

[0001] The present invention relates to the field of biomedicine, and in particular to application of a novel iron chelator CN128 / deferasirox (DFX) in preventing and treating non-alcoholic fatty liver disease. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) is one of the leading liver diseases prevalent worldwide, with a global prevalence of approximately 25.24% and a continuously increasing prevalence. The NAFLD disease spectrum includes nonalcoholic hepatic steatosis, nonalcoholic steatohepatitis, cirrhosis, and hepatocellular carcinoma. NAFLD not only causes liver disease and mortality but is also closely associated with cardiovascular disease, metabolic syndrome, type 2 diabetes, and malignancies. NASH (nonalcoholic steatohepatitis) is one of the leading causes of cirrhosis, with a 15% to 25% incidence of cirrhosis within 10 to 15 years in NASH patients, posing a serious threat to human health. However, to date, only Zydus Cadila's PPARα / γ agonist, Saroglitazar, has been approved for the treatment of NASH in India. No other NASH treatments have been approved in the United States, Europe, Japan, or other countries and regions. Weight loss through dietary changes, increased exercise, and even surgery remains the primary approach to improving NASH. Therefore, identifying new targets and exploring novel mechanisms for the prevention and treatment of NASH will contribute to the development of novel strategies for its prevention and treatment.

[0003] Studies have shown that a large number of NAFLD patients suffer from iron overload and metabolic abnormalities, resulting in iron overload syndrome. The mouse NASH models constructed by the inventors all showed significant iron overload. Studies have shown that iron overload can produce reactive oxygen species (ROS) through the Fenton reaction and induce lipid peroxidation and cell death, thereby promoting the occurrence and development of NAFLD; in addition, ROS produced by liver iron overload promotes the activation of Kupffer cells, triggering inflammation and fibrosis. Therefore, research and development of drugs that can effectively reduce liver iron accumulation and explore their intrinsic mechanisms for alleviating NASH may become a new strategy for treating NASH.

[0004] The invention of 202010831145.1 "Use of iron chelators in the preparation of drugs for treating or preventing polyomavirus infection" informs that the iron chelator CN128 can be used to treat or prevent polyomavirus infection.

[0005] The invention of 201910088923.X, "Application of iron chelator Deferasirox (DFX) in the treatment of cervical cancer", states that DFX can significantly inhibit the proliferation, division and invasion of cervical cancer cells, inhibit the growth of cervical cancer tumors, and will not cause significant damage to other functions of the body.

[0006] So far, there are no reports on the effects of the new iron chelator CN128 and iron chelator DFX on non-alcoholic fatty liver disease. Summary of the Invention

[0007] The technical problem to be solved by the present invention is to provide an application of an iron chelator CN128 / DFX in targeting non-alcoholic fatty liver disease, providing a basis for new drug research and development and innovative therapies.

[0008] In order to solve the above technical problems, the present invention provides an application of an iron chelator in the preparation of a drug for preventing and treating non-alcoholic steatohepatitis.

[0009] As an improvement of the application of the present invention: the iron chelator is CN128 or Deferasirox.

[0010] As a further improvement of the application of the present invention: CN128 or Deferasirox can significantly reduce liver damage, fatty degeneration, and hepatocyte ballooning in non-alcoholic steatohepatitis induced by MCD diet.

[0011] The inventors' previous studies have shown that the iron chelator Desferrioxamine (DFO) cannot significantly alleviate NASH caused by MCD (methionine-choline deficient diet). This may be related to the fact that DFO undergoes rapid glucuronidation in the liver to form non-chelated metabolites, resulting in a decrease in its ability to remove iron from the liver. The new iron chelator CN128 introduces a site to reduce the effect of glucuronidation on metabolism, effectively improving the iron chelation efficiency of CN128 in the body, and has the effect of effectively promoting iron excretion and reducing iron accumulation. The iron chelator DFX also has the effect of effectively promoting iron excretion and reducing iron accumulation.

[0012] The details are as follows:

[0013] 1. About CN128:

[0014] During the invention process, the inventors found that the non-heme iron (nonhemeiron) content in the liver tissue of the MCD-induced NASH model increased significantly, and the liver iron accumulation induced by a high-iron diet can significantly promote the development of NASH. Treatment with the new iron chelator CN128 can significantly reduce liver damage, fatty degeneration, and hepatocellular ballooning in MCD diet-induced non-alcoholic fatty liver disease. Furthermore, the inventors found that CN128 can significantly reduce the increased levels of malondialdehyde (MDA) and lipid peroxidation (Lipid ROS) induced by MCD, indicating that CN128 can alleviate the development of NASH by reducing ferroptosis. The results of this study provide a theoretical basis for CN128 to alleviate non-alcoholic fatty liver disease, especially for the research and development of non-alcoholic fatty liver disease drugs.

[0015] The usage and dosage of CN128 for the prevention and treatment of non-alcoholic steatohepatitis are as follows: administer simultaneously with MCD diet feeding, with a dosage of 50 mg / kg / day, orally.

[0016] In summary, the present invention discovered that the novel iron chelator CN128 can be used to prevent and treat non-alcoholic fatty liver disease and has potential clinical application value.

[0017] 2. About DFX

[0018] The inventors discovered that DFX can significantly reduce liver damage, steatosis, and hepatocellular ballooning in MCD-induced NASH. These findings provide a theoretical basis for DFX's potential to alleviate NASH, and in particular, a solid foundation for the development of NASH drugs.

[0019] The usage and dosage of DFX in preventing and treating non-alcoholic steatohepatitis are as follows: administer simultaneously with MCD diet feeding, with a dosage of 50 mg / kg / day, orally.

[0020] In summary, the present invention discovered that the iron chelator DFX can be used to prevent and treat non-alcoholic fatty liver disease and has potential clinical application value. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] The specific embodiments of the present invention are further described in detail below with reference to the accompanying drawings.

[0022] Figure 1-1 Iron accumulation in the liver promotes the development of NASH;

[0023] (A) Nonheme iron content in liver tissue of ND / MCD group;

[0024] (B) Hematoxylin and eosin (H&E) staining (top) and Prussian blue staining (bottom) of liver tissue in the ND / HID group;

[0025] Figure 1-2 CN128 alleviates liver damage in the MCD-NASH mouse disease model;

[0026] (A) Alanine aminotransferase (ALT) levels in serum;

[0027] (B) Serum aspartate aminotransferase (AST) levels.

[0028] Figure 1-3 The effect of CN128 on steatosis, hepatocellular ballooning, and inflammation in the MCD-NASH model;

[0029] (A) H&E staining of liver tissues in each group;

[0030] (B) NAS analysis of (A).

[0031] Figure 1-4 CN128 alleviates iron accumulation, ferroptosis, and lipid peroxidation in the MCD-NASH mouse model;

[0032] (A) Nonheme iron content in liver tissue of each group;

[0033] (B) Malondialdehyde (MDA) content in liver tissue of each group;

[0034] (C) The content of lipid ROS in hepatocytes of each group.

[0035] * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001. Data are expressed as mean ± SEM (n = 4-6 per group), and data were analyzed using t-test or one-way analysis of variance (for more than 2 groups).

[0036] ND: normal diet; MCD: methionine choline-deficient diet; CN128+MCD: MCD diet plus CN128 administration; HID: high-iron diet.

[0037] Figure 2-1 DFX alleviates liver damage in the MCD-NASH mouse disease model;

[0038] (A) Serum alanine aminotransferase (ALT) level;

[0039] (B) Serum aspartate aminotransferase (AST) levels;

[0040] (C) Serum lactate dehydrogenase (LDH) levels.

[0041] Figure 2-2DFX significantly alleviated steatosis, hepatocyte ballooning, and inflammatory markers in the MCD-NASH mouse model;

[0042] (A) Hematoxylin and eosin (H&E) staining of liver tissues in each group;

[0043] (B) NAS analysis of (A);

[0044] Figure 2-3 DFX alleviates iron accumulation, ferroptosis, and lipid peroxidation in the MCD-NASH mouse model;

[0045] (A) Nonheme iron content in liver tissue of each group;

[0046] (B) MDA content in liver tissue of each group.

[0047] Figure 3 DFO is less effective in treating MCD-induced NASH.

[0048] (A) Serum alanine aminotransferase (ALT) level;

[0049] (B) Serum aspartate aminotransferase (AST) levels;

[0050] (C) Hematoxylin and eosin (H&E) staining of liver tissues in each group;

[0051] (D) Triglyceride content in liver tissue of each group.

[0052] * represents P < 0.05, ** represents P < 0.01, *** represents P < 0.001. Data are expressed as mean ± SEM (n = 4-6 per group), and data were analyzed using t-test or one-way analysis of variance (for more than 2 groups).

[0053] ND: normal diet; MCD: methionine choline deficient diet; DFX+MCD: MCD diet and simultaneous administration of DFX; DETAILED DESCRIPTION

[0054] The present invention will be further described below with reference to specific embodiments, but the protection scope of the present invention is not limited thereto.

[0055] 1. Iron chelating agent CN128

[0056] (1) The results are as follows Figure 1-1 As shown. The detection of non-heme iron in liver tissue of ND group and MCD group showed that there was significant iron accumulation in liver tissue of NASH model ( Figure 1-1A). In addition, compared with a normal diet, iron accumulation in the liver increased significantly after 8 weeks of high-iron diet feeding, and significant lipid accumulation was observed in the liver, indicating that iron accumulation in the liver can promote the development of NASH ( Figure 1-1 B). Therefore, developing drugs that can effectively reduce liver iron accumulation and exploring their intrinsic mechanisms of alleviating NASH may become a new strategy for treating NASH.

[0057] (2) In order to explore the therapeutic effect of iron chelators on NASH, the inventors used a new iron chelator CN128 to treat MCD-NASH. The results showed that CN128 can effectively reduce liver damage in NASH models ( Figure 1-2 A&B), lipid degeneration, inflammation (1-3A&B), iron accumulation in liver tissue (1-4A), and reduced ferroptosis levels (1-4B&C), thereby alleviating the development of NASH.

[0058] 1. Materials and Methods

[0059] 1.1 Experimental Animals

[0060] Eight-week-old SPF-grade C57BL / 6 male mice were purchased from Shanghai Slack Laboratory Animal Co., Ltd. and housed in an SPF environment. After acclimating to a standard diet of AIN-76A (Research Diets, Inc.) for 2 weeks, they were randomly divided into groups based on body weight, with six mice per group.

[0061] 1.2 Drugs and treatment

[0062] CN128 was dissolved in normal saline (concentration of 5 g / L) and administered simultaneously with MCD diet feeding. The CN128 dosage was 50 mg / kg / day, orally.

[0063] 1.3 Construction of mouse NASH model

[0064] "CN128+ND" control group: mice were fed with ND diet for 4 weeks and simultaneously orally administered CN128 (dose: 50 mg / kg / day). After 4 weeks, the mice were anesthetized with pentobarbital (70 mg / kg), blood was collected from the heart, serum was separated, and liver tissue was collected.

[0065] “Vehicle+ND” control group: CN128 was replaced with placebo (equal volume of normal saline), and the rest were the same as the “CN128+ND” control group.

[0066] "CN128+MCD" experimental group: Mice were fed an MCD diet for 4 weeks to establish an MCD-NASH model, and CN128 (dose: 50 mg / kg / day) was orally administered simultaneously. After 4 weeks, the mice were anesthetized with pentobarbital (70 mg / kg), blood was collected from the heart, serum was separated, and liver tissue was collected.

[0067] "Vehicle+MCD" experimental group: CN128 was replaced with placebo (equal volume of normal saline), and the rest were the same as the "CN128+MCD" experimental group.

[0068] 1.4ALT / AST Detection and Analysis

[0069] After the collected blood was left at room temperature for 2 hours, it was centrifuged at 5000 rpm for 10 minutes to collect serum. ALT / AST levels were measured using an ALT / AST kit.

[0070] The results are as follows Figure 1-2 As shown in the data, compared with the placebo group, CN128 administration significantly alleviated the ALT / AST content in the MCD-NASH model.

[0071] 1.5 Hematoxylin and eosin (H&E) staining

[0072] (1) Dewaxing of paraffin sections: sequentially place the sections in xylene I for 10 min, xylene II for 10 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, 95% alcohol for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min, 70% alcohol for 5 min, and wash with distilled water.

[0073] (2) Hematoxylin staining of cell nuclei: Stain sections with Harris hematoxylin for 3-8 minutes, wash with tap water, differentiate with 1% hydrochloric acid alcohol for a few seconds, rinse with tap water, turn blue with 0.6% ammonia solution, and rinse with running water.

[0074] (3) Eosin staining of cytoplasm: Slice into eosin staining solution and stain for 1-3 minutes.

[0075] (4) Dehydration and mounting: Dehydrate the sections in 95% alcohol I for 5 min, 95% alcohol II for 5 min, anhydrous ethanol I for 5 min, anhydrous ethanol II for 5 min, xylene I for 5 min, and xylene II for 5 min. Remove the sections from the xylene and allow them to dry slightly. Mount the sections with neutral gum. Examine under a microscope and collect and analyze images.

[0076] The results are as follows Figure 1-3 As shown in the results, CN128 administration significantly alleviated steatosis, hepatocellular ballooning and inflammation levels in the MCD-NASH model compared with the placebo group.

[0077] 1.6 Detection of non-heme iron content in liver tissue

[0078] (1) Weigh approximately 0.1 g of liver tissue into a 1.5 mL centrifuge tube, add 1 mL of tissue digestion solution (3 M hydrochloric acid, 300 mL / L; 0.61 M (10%), 99.735 g / L trichloroacetic acid), digest at 65°C for 72 h, and then dilute to 1.5 mL with tissue digestion solution. Centrifuge at 10,000 g for 10 minutes, and collect the supernatant for subsequent analysis.

[0079] (2) Add the iron colorimetric working solution (iron colorimetric stock solution: saturated sodium acetate solution: distilled water (V / V / V) = 1:5:5) to a 96-well plate (200 μL per well), add 10 μL of distilled water or 10 μL of tissue digestion solution or 10 μL of 500ug / dL iron standard solution or 10 μL of sample digestion solution, mix thoroughly, develop color at room temperature for 10 minutes, and measure the absorbance at 535nm using a microplate reader. Calculate the non-heme iron content based on the OD value. Calculation formula: Tissue non-heme iron (μg / g tissue wet weight) = OD value / tissue weight × (1.5-0.25 × tissue weight) × (1 / iron standard solution OD value × 4.77)

[0080] The results are as follows Figure 1-4 As shown in A, CN128 administration significantly reduced MCD diet-induced hepatic iron accumulation compared with the placebo group.

[0081] 1.7 MDA content detection

[0082] (1) Liver tissue homogenate was lysed and centrifuged (12000 rpm, 10 min), and the supernatant was used for subsequent analysis;

[0083] (2) The MDA working solution (TBA diluent: TBA storage solution: antioxidant = 3000 μl: 1000 μl: 60 μl) was prepared using the Biyuntian lipid oxidation (MDA) detection kit (S0131S).

[0084] (3) Sample determination: Add 0.1 ml of homogenate, lysate, or PBS solution to a centrifuge tube as a blank control, add 0.1 ml of standard solution of different concentrations (1, 2, 5, 10, 20, 50 μM) to prepare a standard curve, add 0.1 ml of sample for determination; then add 0.2 ml of MDA detection working solution. After mixing, heat at 100°C or in a boiling water bath for 15 minutes. Cool to room temperature in a water bath and centrifuge at 1000 g for 10 minutes at room temperature. Take 200 μl of supernatant and add it to a 96-well plate. Measure the absorbance at 532 nm using a microplate reader. Calculate the MDA content based on the OD value.

[0085] The results are as follows Figure 1-4 As shown in B, compared with the placebo group, CN128 administration significantly reduced the MDA content in the liver tissue of the MCD-NASH model.

[0086] 1.8 Lipid ROS Detection

[0087] The "ND" control group / mice were fed an ND diet for 2 days, then anesthetized with pentobarbital (70 mg / kg), and hepatocytes were isolated. The "CN128+MCD" experimental group: Mice were fed an MCD diet for 2 days and simultaneously orally administered CN128 (dose: 50 mg / kg / day). They were anesthetized with pentobarbital (70 mg / kg), and hepatocytes were isolated. The "vehicle+MCD" experimental group: CN128 was replaced with a placebo (equal volume of normal saline), and all other conditions were the same as the "CN128+MCD" experimental group. Hepatocytes were isolated. The isolated hepatocytes were stained with Lipid ROS fluorescent dye, and the Lipid ROS content was measured by flow cytometry.

[0088] The results are as follows Figure 1-4 As shown in C, compared with the placebo group, CN128 administration significantly reduced the content of Lipid ROS in the liver tissue of the MCD-NASH model.

[0089] Description: The statistical method of the present invention: using GraphPad Statistical analysis was performed using Statistical Software 7.01 (San Diego, USA) and presented as mean ± standard error of the mean (SEM). Data were analyzed by two-tailed t-test or one-way ANOVA. ns indicates no significant difference; *P < 0.05, **P < 0.01, ***P < 0.001.

[0090] result:

[0091] Since studies have reported that NASH models have iron overload, the inventors constructed an MCD-NASH model and showed that the iron content in the liver tissue of the NASH model increased significantly ( Figure 1-1 A), and liver iron accumulation promotes the development of NASH ( Figure 1-1 B). Therefore, targeting iron accumulation may be an effective approach for treating NASH. However, traditional medical iron chelators rapidly undergo glucuronidation in the liver, forming non-chelated metabolites, which impair their ability to remove iron from the liver. The inventors used the novel iron chelator CN128 (50 mg / kg / 24 h) to treat NASH in a MCD model.

[0092] The inventors established an MCD-NASH model and administered CN128 or placebo daily. The results showed that compared with the placebo group, CN128 significantly alleviated liver damage (ALT, AST) in the MCD-NASH model ( Figure 1-2 A, B), fatty degeneration, hepatocyte ballooning, inflammation and fibrosis ( Figure 1-3A, B). At the same time, the inventors' research showed that CN128 can significantly alleviate the increase in lipid peroxidation levels in liver tissue of NASH models, indicating that CN128 can alleviate the development of NASH by reducing ferroptosis ( Figure 1-4 B, C). In summary, the novel iron chelator CN128 can significantly prevent and treat MCD diet-induced nonalcoholic steatohepatitis.

[0093] 2. Iron chelator DFX:

[0094] 1. Materials and Methods

[0095] 1.1 Experimental Animals

[0096] Same as above (i.e., same as the corresponding content of "1. Iron chelating agent CN128").

[0097] 1.2 Drugs and treatment

[0098] DFX stock solution was dissolved in dimethyl sulfoxide (DMSO) at a concentration of 150 mM.

[0099] In the in vivo experiment, DFX was administered simultaneously with MCD diet feeding at a dose of 50 mg / kg / day, orally.

[0100] 1.3 Construction of mouse NASH model

[0101] "DFX+ND" control group: mice were fed with ND diet for 4 weeks and simultaneously orally administered DFX (dose: 50 mg / kg / day). After 4 weeks, the mice were anesthetized with pentobarbital (70 mg / kg), blood was collected from the heart, serum was separated, and liver tissue was collected.

[0102] "Vehicle+ND" control group: CN128 was replaced with placebo (equal volume of normal saline), and the rest was the same as the "DFX+ND" control group.

[0103] "DFX+MCD" experimental group: Mice were fed an MCD diet for 4 weeks to establish an MCD-NASH model, and DFX (dose: 50 mg / kg / day) was orally administered simultaneously. After 4 weeks, the mice were anesthetized with pentobarbital (70 mg / kg), blood was collected from the heart, serum was separated, and liver tissue was collected.

[0104] "Vehicle+MCD" experimental group: CN128 was replaced with placebo (equal volume of normal saline), and the rest was the same as the "DFX+MCD" experimental group.

[0105] 1.4ALT / AST / LDH Detection and Analysis

[0106] Same as above.

[0107] The results are as follows Figure 2-1As shown in the results, compared with the placebo group, DFX administration significantly alleviated the ALT / AST / LDH levels in the MCD-NASH model.

[0108] 1.5 Hematoxylin and eosin (H&E) staining

[0109] Same as above.

[0110] The results are as follows Figure 2-2 As shown in the results of the present study, compared with the placebo group, DFX administration significantly alleviated the steatosis, hepatocellular ballooning and inflammation levels in the MCD-NASH model.

[0111] 1.6 Detection of non-heme iron content in liver tissue

[0112] Same as above.

[0113] The results are as follows Figure 2-3 As shown in A, DFX administration significantly reduced MCD diet-induced hepatic iron accumulation compared with the placebo group.

[0114] 1.7 MDA content detection

[0115] Same as above.

[0116] The results are as follows Figure 2-3 As shown in B, compared with the placebo group, DFX administration significantly reduced the MDA content in liver tissue of the MCD-NASH model.

[0117] 2 Results

[0118] The inventors established an MCD-NASH model and administered DFX or placebo daily. The results showed that compared with the placebo group, DFX significantly alleviated liver damage (ALT, AST, LDH) in the MCD-NASH model ( Figure 2-1 AC), fatty degeneration, hepatocellular ballooning, inflammation level ( Figure 2-2 A, B). Further studies have shown that DFX treatment significantly reduces iron accumulation in MCD-NASH liver tissue ( Figure 2-3 A), and DFX can significantly reduce the level of lipid peroxidation ( Figure 2-3 B) This suggests that DFX may alleviate the development of NASH by reducing iron accumulation and ferroptosis induced by lipid peroxidation. In summary, the iron chelator DFX can significantly prevent and treat diet-induced nonalcoholic steatohepatitis.

[0119] In addition, it should be emphasized that the inventors found that: referring to the above experimental method, in vivo experiments using the commonly used iron chelator DFO showed that DFO has a poor therapeutic effect on NASH ( Figure 3 ). Therefore, not all iron chelators can be used to prevent and treat nonalcoholic steatohepatitis.

[0120] Finally, it should be noted that the above examples are merely specific embodiments of the present invention. Obviously, the present invention is not limited to the above examples and is subject to numerous variations. All variations that can be directly derived or conceived by a person of ordinary skill in the art from the disclosure of the present invention are considered to be within the scope of protection of the present invention.

Claims

1. Use of an iron chelator in the preparation of a drug for preventing and treating nonalcoholic steatohepatitis, characterized in that: The iron chelator was CN128, and the nonalcoholic steatohepatitis was caused by MCD, where MCD stands for methionine-choline deficient diet.

2. The use according to claim 1, characterized in that: CN128 significantly reduces liver damage, fatty degeneration, hepatocellular ballooning, inflammation and fibrosis in MCD diet-induced non-alcoholic steatohepatitis; significantly alleviates the increased level of lipid peroxidation in liver tissue and alleviates the development of NASH by reducing ferroptosis.

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