CN114288300B

CN114288300B - Application of halofuginone in medicine for treating and preventing fatty liver and obesity

Info

Publication number: CN114288300B
Application number: CN202210110323.0A
Authority: CN
Inventors: 翁建平; 徐索文
Original assignee: University of Science and Technology of China USTC
Current assignee: University of Science and Technology of China USTC
Priority date: 2022-01-29
Filing date: 2022-01-29
Publication date: 2023-04-21
Anticipated expiration: 2042-01-29
Also published as: CN114288300A

Abstract

The invention provides an application of halofuginone in preparing medicines for treating and preventing fatty liver, obesity and related diseases. The invention also provides application of the halofuginone-modified compound in preparing medicaments for treating and preventing fatty liver, obesity and related diseases and a pharmaceutical composition.

Description

Application of halofuginone in medicine for treating and preventing fatty liver and obesity

Technical Field

The invention relates to the technical field of biological medicines, in particular to application of halofuginone in preparing medicines for treating and preventing fatty liver, obesity and related diseases.

Background

Non-alcoholic fatty liver disease (NAFLD), which is a liver metabolic disease in which accumulation of triglyceride-based lipids in hepatocytes is a pathological change, has become one of the most common liver diseases worldwide, and the incidence rate thereof still continues to increase, and in the united states, the number of NAFLD cases is expected to expand from 8310 tens of thousands (about 25% of the total population) in 2015 to 1.09 billion in 2030. Despite the ongoing progress in elucidating the pathogenesis of NAFLD, determining therapeutic targets, and advancing drug development, significant challenges remain unmet and no drug has been approved for the treatment of this disease.

Disclosure of Invention

In view of the above, the present invention provides, in order to be able to solve the above problems at least in part, the use of halofuginone and a compound based on halofuginone modification in the preparation of a medicament for the treatment and prevention of fatty liver, obesity and related diseases.

In order to achieve the above object, an aspect of the present invention provides an application of halofuginone in preparing a medicament for treating and preventing fatty liver, obesity and related diseases.

In another aspect, the invention provides application of a halofuginone-modified compound in preparing medicines for treating and preventing fatty liver, obesity and related diseases.

Wherein the halofuginone modification comprises at least one of: salt formation modification, ester formation modification, amide formation modification, carbamation modification, etherification modification, ring opening modification and cyclization modification.

According to an embodiment of the invention, the related diseases comprise at least one of: insulin resistance, metabolic syndrome, diabetes, hyperglycemia, hyperlipidemia, simple liver steatosis, nonalcoholic steatohepatitis, liver fibrosis, liver cirrhosis, and liver cancer.

According to an embodiment of the present invention, the drug comprises a pharmaceutical formulation formed by dissolution with a drug co-solvent.

In yet another aspect, the invention provides a pharmaceutical composition.

Wherein, the medicine composition comprises halofuginone or a compound based on halofuginone modification and related disease medicines, which are taken together as active ingredients, and one or more pharmaceutically acceptable carriers and/or medicine auxiliary materials.

According to an embodiment of the invention, the carrier comprises a nanoemulsion or a microemulsion.

According to an embodiment of the invention, the pharmaceutical excipients comprise at least one of the following: polyethylene glycol, sodium carboxymethyl cellulose, beta-cyclodextrin.

According to an embodiment of the invention, the related disease drug comprises at least one of: obeticholic acid, pparγ ligand, ACC1/2 inhibitor.

According to embodiments of the present invention, pparγ ligands include pioglitazone, thiazolidinediones.

According to an embodiment of the invention, the ACC1/2 inhibitor comprises PF-05221304, ND-630.

According to the embodiment of the invention, in the application of the halofuginone, the halofuginone can inhibit liver fat deposition, improve fatty liver symptoms and liver function indexes, enhance sugar tolerance, improve insulin resistance and have positive effects on metabolic health.

Drawings

FIG. 1 schematically shows a schematic of experimental procedure for a C57 mouse according to an embodiment of the present invention;

FIG. 2 (a) is a diagram showing a comparison of body types after administration of the C57 mice according to the embodiment of the present invention;

FIG. 2 (b) shows the body weight change curve of the example of the present invention during the experiment with C57 mice;

FIG. 2 (C) shows a statistical plot of the balance average food intake of the C57 mice fed high fat diet of the examples of the present invention;

FIG. 3 (a) is a graph showing the statistical plot of the mass/weight ratio of white fat (epididymal fat and inguinal fat) of C57 mice for the administration treatment of the example of the present invention;

FIG. 3 (b) is a graph showing statistics of the brown fat mass/body weight ratio of the C57 mice treated with the example of the present invention;

FIG. 3 (C) shows a statistical plot of liver weights of C57 mice treated with the example of the present invention;

FIG. 4 (a) shows the effect of the treatment of the present invention on glucose tolerance in C57 mice;

FIG. 4 (b) is a graph showing the area under the blood glucose concentration curve in the glucose tolerance test of the C57 mice by the administration treatment of the example of the present invention;

FIG. 4 (C) shows the effect of the treatment of the present invention on insulin resistance of C57 mice;

FIG. 4 (d) is a graph showing the area under the blood glucose concentration curve in the insulin resistance test of C57 mice by the administration treatment of the example of the present invention;

FIG. 5 shows results of oil red O staining of liver and liver frozen sections of C57 mice after treatment with the drug according to the examples of the present invention;

FIG. 6 (a) shows the effect of the treatment of the example of the invention on the serum glutamic-oxaloacetic transaminase content of C57 mice;

FIG. 6 (b) shows the effect of the treatment of the example of the present invention on the serum glutamic pyruvic transaminase content of C57 mice;

FIG. 7 (a) shows the effect of the example administration treatment of the present invention on serum triglyceride levels in C57 mice;

FIG. 7 (b) shows the effect of the treatment of the present invention on serum cholesterol levels in C57 mice.

Detailed Description

The present invention will be further described in detail below with reference to specific embodiments and with reference to the accompanying drawings, in order to make the objects, technical solutions and advantages of the present invention more apparent.

Several of the drugs in fatty liver, obesity and related diseases have been shown to be attractive in clinical trials, such as FXR agonists (obeticholic acid), but these findings have been translated into long lasting safety and efficacy. Pparγ ligands (e.g., pioglitazone, a member of the thiazolidinedione family of drugs) have been studied in several trials to improve steatosis, inflammation and hepatocyte balloons, as well as liver fibrosis; however, they are limited by side effects such as weight gain, fluid retention, reduced bone mass, and increased risk of fracture. The ACC1/2 inhibitor PF-05221304 increased the proportion of asymptomatic hypertriglyceridemia patients with increasing doses. The invention provides a new therapeutic drug for treating fatty liver, obesity and related diseases, can obviously improve liver steatosis, reduce serum triglyceride concentration and reduce the level of glutamic-pyruvic transaminase and glutamic-oxaloacetic transaminase which are related indexes of liver toxicity, and has good application value.

The invention provides an application of halofuginone in preparing medicines for treating and preventing fatty liver, obesity and related diseases.

Wherein, the chemical structural formula of the halofuginone is shown in the following formula (1):

the invention also provides application of the halofuginone-modified compound in preparing medicines for treating and preventing fatty liver, obesity and related diseases.

The drug may include a pharmaceutical formulation formed by dissolution using a drug co-solvent. Wherein, the medicine cosolvent can be pharmaceutically acceptable, and the solvent which does not damage the active ingredients of the halofuginone is not particularly limited.

The modified compounds based on halofuginone can be modified for functional groups in halofuginone by methods known in the art, and are included in the present invention as long as they are still pharmaceutically acceptable and do not destroy the active ingredient of halofuginone. Wherein, the halofuginone modification may include, but is not limited to, at least one of: salt formation modification, ester formation modification, amide formation modification, carbamation modification, etherification modification, ring opening modification and cyclization modification.

The application of the halofuginone or the halofuginone modified compound in preparing medicines for treating and preventing fatty liver, obesity and related diseases is also provided. Wherein, the medicine composition comprises halofuginone or a compound based on halofuginone modification and related disease medicines, which are taken together as active ingredients, and one or more pharmaceutically acceptable carriers and/or medicine auxiliary materials. The pharmaceutical composition can be applied to the preparation of medicines for treating and preventing fatty liver, obesity and related diseases.

According to an embodiment of the invention, the related disease drug comprises at least one of: obeticholic acid, pparγ ligand, ACC1/2 inhibitor. The PPARgamma ligand can comprise pioglitazone and thiazolidinedione medicines. ACC1/2 inhibitors may include PF-05221304, ND-630.

In the present invention, the effective dose of the pharmaceutical co-solvent, carrier or pharmaceutical adjuvant may be specifically determined according to the pharmaceutically acceptable dose.

The dosage of halofuginone or halofuginone-modified-based compounds or pharmaceutical compositions may be determined based on the route of administration, pharmacokinetic parameters of halofuginone, the grade of the associated disease, the health of the subject, and the like.

The effect of halofuginone in fatty liver, obesity and related diseases is schematically illustrated below with reference to FIGS. 1-7. It should be noted that the examples are only specific embodiments of the present invention and are not intended to limit the scope of the present invention.

Preparation of experimental mice: SPF class C57 mice (8 weeks old, male) were purchased from Jiangsu Jiyaokang biotechnology Co., ltd, kept in China university of science and technology animal center, and replaced with sterile litter once a week to obtain food and water freely.

Fig. 1 schematically shows a schematic of experimental procedure of an embodiment of the present invention on a C57 mouse.

As shown in fig. 1, the animals were dosed and treated as follows: the C57 mice were randomly divided into a high-fat feed (60% fat) group and a normal feed group (3:1) after one week of adaptation in the animal house, and after 12 weeks of feeding, the high-fat feed group was divided into 3 groups (vehicle control group, low dose group (halofuginone content 100. Mu.g/kg), high dose group (halofuginone content 250. Mu.g/kg)). Halofuginone hydrobromide is purchased from Shanghai Tao Shu Biotechnology Inc. The medicine is prepared into 30mg/mL mother liquor in dimethyl sulfoxide and stored in a refrigerator at-20 ℃. The mice were intraperitoneally injected at a dose of 100. Mu.g/kg or 250. Mu.g/kg, once every two days, for a period of 12 weeks, at concentrations of 0.03mg/mL and 0.075mg/mL in 0.5% sodium carboxymethylcellulose prior to administration.

During the dosing period, mice were weighed once a week. The mice were harvested after 24 weeks and subjected to subsequent molecular and pathological experiments.

It should be noted that, in this embodiment, all data were processed and statistically analyzed by Graphpad Prism 8.0 software, and the mean value of the samples was analyzed by One-way ANOVA or two-way ANOVA. p <0.05 indicates a statistical difference. * : p <0.05,: p <0.01,: p <0.001,: p <0.0001.

Example 1: effects of halofuginone on mice weight and diet

FIG. 2 (a) shows a comparison of body shapes after administration of the examples of the present invention to C57 mice; FIG. 2 (b) shows the body weight change curve of the example of the present invention during the experiment with C57 mice; FIG. 2 (C) shows a statistical plot of the average food intake on a C57 mouse 3 balance fed high fat diet in accordance with an embodiment of the present invention.

In this example, the body weight of the mice was measured once a week during the experiment for 24 weeks, as shown in fig. 2 (b). As shown in fig. 2 (a), differences in the body type of mice were recorded by photographing at the end point, wherein fig. 2 (a) shows, from left to right, a normal feed group (fed with normal feed, dosed vehicle), a high fat feed group (vehicle control group), a high fat feed group (low dose group, in which halofuginone content is 100 μg/kg), and a high fat feed group (high dose group, in which halofuginone content is 250 μg/kg), respectively. At the last week, mice were placed in a metabolic cage (Promethion) for 4 days, the diet of the mice was recorded, the data source was the daily weight average of food removed from the first day, and the daily average food intake weight of the mice was recorded, as shown in fig. 2 (c).

As shown in fig. 2 (a) to 2 (c), both the low dose group (100 μg/kg of halofuginone content) and the high dose group (250 μg/kg of halofuginone content) significantly reduced the body weight of the mice and the food intake of the mice was increased, as compared with the vehicle control group.

Example 2: effects of halofuginone on fat weight and liver weight in mice

FIG. 3 (a) is a graph showing the statistical plot of the mass/weight ratio of white fat (epididymal fat and inguinal fat) of C57 mice for the administration treatment of the example of the present invention; FIG. 3 (b) is a graph showing statistics of the brown fat mass/body weight ratio of the C57 mice treated with the example of the present invention; FIG. 3 (C) shows a statistical plot of liver weights of C57 mice for the administration treatment of the examples of the present invention.

In this example, after the corresponding treatment for 24 weeks, the white fat (epididymal fat and inguinal fat) and the brown fat at the scapula were completely removed from each group of mice, weighed, and the fat mass/weight ratio of the mice was calculated, and the statistical results were shown in fig. 3 (a) and 3 (b).

Experimental results: both the low dose group (100. Mu.g/kg of halofuginone content) and the high dose group (250. Mu.g/kg of halofuginone content) significantly reduced the white fat content compared to the vehicle control group, with no effect on brown fat content.

In this example, after the end of 24 weeks of the corresponding treatment for each group of mice, the livers of the mice were simultaneously removed and weighed as shown in fig. 3 (c).

Experimental results: the liver weight of the mice in the low dose group (halofuginone content 100. Mu.g/kg) was significantly reduced compared to the vehicle control group.

Example 3: effects of halofuginone on glucose tolerance and insulin resistance in mice

In this example, glucose tolerance and insulin tolerance experiments were performed at week 22 and week 23, respectively.

The sugar tolerance test steps can be as follows: the mice were placed in a clean feed-free cage one night before the experiment, fasted for 16 hours, a glucose solution of 0.5g/mL was prepared before the experiment, the weight of the mice was weighed, the fasting blood glucose was measured for 0min before glucose injection, and glucose was then injected, wherein the injection dose may be 2g/kg, and the blood glucose concentration after glucose injection (15 min, 30min, 60min, 90min, 120 min) was measured.

The insulin resistance test steps can be as follows: the body weight of the mice was measured before the experiment, 0.2U/mL insulin solution was prepared on the day of the experiment, and the mice were placed in a clean cage on the day of the experiment, and fasted for 4 hours. The fasting blood glucose was measured for 0min before insulin injection, followed by insulin injection at a dose of 0.5U/kg, and the blood glucose concentration was measured after glucose injection (15 min, 30min, 45min, 60min, 90min, 120 min).

FIG. 4 (a) shows the effect of the treatment of the present invention on glucose tolerance in C57 mice; FIG. 4 (b) is a graph showing the area under the blood glucose concentration curve in the glucose tolerance test of the C57 mice by the administration treatment of the example of the present invention; FIG. 4 (C) shows the effect of the treatment of the present invention on insulin resistance of C57 mice; FIG. 4 (d) is a graph showing the statistics of the area under the blood glucose concentration curve in the insulin resistance test of C57 mice by the administration treatment of the example of the present invention.

Experimental results: as shown in fig. 4 (a) to 4 (d), both the low dose group (halofuginone content 100 μg/kg) and the high dose group (halofuginone content 250 μg/kg) were able to increase glucose tolerance and improve insulin resistance in mice, as compared to the vehicle control group.

Example 4: effects of halofuginone on liver steatosis

FIG. 5 shows liver and liver ice-cold section O staining results of C57 mice after treatment with the inventive examples.

In this example, after 24 weeks of corresponding treatment on each group of mice, the whole liver was removed, as shown in fig. 5, after photographing and weighing of the liver, the liver lobules at the same position were cut and put into paraformaldehyde for fixation overnight, PBS was washed the next day and put into 30% sucrose for dehydration, and the liver was embedded in a frozen embedding medium the third day, and the embedded liver was continuously sliced in a frozen microtome. The slices are stored at-80 ℃, and are taken out and rewarmed for 20min at room temperature when in use. Then dyeing is performed.

Wherein the dyeing step may include, but is not limited to, the following steps:

the first step: the sections were washed 2-3 times with PBS for 5min each, lifted up with filter paper to dryness, and then circled with a histochemical pen.

And a second step of: the sections were set up and blotted with filter paper after washing with 60% isopropanol for 10s and decanting.

And a third step of: 0.3% oil red O staining solution submerges the tissue sections for 1min.

Fourth step: the supernatant was removed by washing with 60% isopropyl alcohol for 10 s.

Fifth step: washing with PBS was performed once (slow washing was performed), the supernatant was decanted and blotted with paper.

Sixth step: glycerogelatin (preheated at 60-70 ℃) is sealed, dropped on a glass slide and covered with a cover slip. After completion, the microscope is photographed.

Experimental results: as shown in fig. 5, both the low dose group (halofuginone content 100 μg/kg) and the high dose group (halofuginone content 250 μg/kg) were able to reduce liver fat deposition in mice compared to vehicle control group; among them, the low dose group (100. Mu.g/kg) had a better effect of alleviating fatty liver compared with the above.

Example 5: effects of halofuginone on serum glutamic-oxaloacetic transaminase (AST) and serum glutamic-pyruvic transaminase (ALT) levels in mice

FIG. 6 (a) shows the effect of the treatment of the example of the invention on the serum glutamic-oxaloacetic transaminase content of C57 mice; FIG. 6 (b) shows the effect of the treatment of the example of the present invention on the serum glutamic pyruvic transaminase content of C57 mice.

In this example, the detection of serum glutamic-oxaloacetic transaminase (AST) and serum glutamic-pyruvic transaminase (ALT) levels in mice was delegated to the detection by wuhansai wile biotechnology limited.

Experimental results: as shown in fig. 6 (a) and 6 (b), both the serum glutamic-oxaloacetic transaminase content and the serum glutamic-pyruvic transaminase content of C57 mice were lower in the low dose group (halofuginone content 100 μg/kg) and the high dose group (halofuginone content 250 μg/kg) compared to the vehicle control group. Indicating that halofuginone can obviously down regulate the elevation of liver AST and ALT caused by high-fat feed feeding and reverse liver function injury.

Example 6: effects of halofuginone on mouse serum Triglyceride (TG) content and serum Cholesterol (CHO) content

FIG. 7 (a) shows the effect of the example administration treatment of the present invention on serum triglyceride levels in C57 mice; FIG. 7 (b) shows the effect of the treatment of the present invention on serum cholesterol levels in C57 mice.

In this example, the mouse serum Triglyceride (TG) content and serum Cholesterol (CHO) content assays were commissioned for the detection by wuhansai wilfordii biotechnology limited.

Experimental results: as shown in fig. 7 (a) and 7 (b), both the low dose group (halofuginone content 100 μg/kg) and the high dose group (halofuginone content 250 μg/kg) were lower in serum Triglyceride (TG) and serum Cholesterol (CHO) levels in C57 mice than the vehicle control group. Indicating that halofuginone can significantly down regulate serum TG and CHO content.

Weight loss due to dietary and lifestyle intervention reduces the transport of metabolic substrates to the liver and can improve all features of non-alcoholic fatty liver, including liver fibrosis. The application of the halofuginone provided by the invention can effectively reduce the weight increase of mice caused by high-fat diet without restricting diet, inhibit liver fat deposition, improve fatty liver symptoms and liver function indexes, enhance sugar tolerance, improve insulin resistance and have positive effects on metabolic health.

The foregoing description of the embodiments has been provided for the purpose of illustrating the general principles of the invention, and is not meant to limit the invention thereto, but to limit the invention thereto, and any modifications, equivalents, improvements and equivalents thereof may be made without departing from the spirit and principles of the invention.

Claims

1. Application of halofuginone in preparing medicine for treating and preventing non-alcoholic fatty liver disease is provided.

2. The use of claim 1, wherein the medicament comprises a pharmaceutical formulation formed by dissolution with a pharmaceutical co-solvent.