CN116570601A - Pharmaceutical composition for treating non-alcoholic fatty liver - Google Patents

Abstract

The invention relates to a medicine for treating nonalcoholic fatty liver, belonging to the field of chemical medicine. The invention provides a composition with therapeutic activity for non-alcoholic fatty liver. Compared with the model group, after administration of taurohyodeoxycholic acid for 5 weeks, the formation of lipid droplets in experimental animals is significantly reduced, and the lipid content is significantly reduced, which confirms the Taurohyodeoxycholic acid can reduce lipid deposition in mouse liver caused by high-fat diet; The prevention and treatment of NAFLD with hyodeoxycholic acid provides a solid foundation and helps to promote the clinical application of taurohyodeoxycholic acid.

Description

A kind of pharmaceutical composition for treating non-alcoholic fatty liver

technical field

The invention relates to the application of tauralhyodeoxycholic acid in treating non-alcoholic fatty liver.

Background technique

Non-alcoholic fatty liver disease (NAFLD) is a clinical syndrome characterized by fatty degeneration and fat storage in hepatic parenchymal cells without excessive alcohol consumption. It is by far the most common chronic liver disease. NAFLD is closely related to abnormal glucose and lipid metabolism diseases such as obesity, hyperlipidemia, and type 2 diabetes. The basic course of the disease includes simple fatty liver, steatohepatitis, fatty liver fibrosis, and may eventually develop into fatty liver cirrhosis and fatty liver cancer, which may lead to premature death. There are about 20-40% of NAFLD patients in the world, and the number is increasing.

Intervention of diet and exercise can effectively control NAFLD, and lifestyle intervention is the most important way for the treatment of nonalcoholic fatty liver disease. However, implementation is difficult and often fails to achieve the desired results. At present, the clinical treatment of non-alcoholic fatty liver mainly adopts blood lipid-lowering, blood sugar-lowering, anti-oxidation, and liver-protecting drugs. However, the use of these drugs is limited because of side effects, increased burden on the liver, or poor efficacy. There are currently no approved drugs for the treatment of NAFLD.

Therefore, a drug for highly effective intervention and treatment of non-alcoholic fatty liver is a practical problem urgently needed to be solved clinically.

Contents of the invention

The technical problem to be solved by the present invention is to provide a pharmaceutical composition for treating nonalcoholic fatty liver, which uses taurohyodeoxycholic acid to treat nonalcoholic fatty liver.

Technical solutions

A composition with non-alcoholic fatty liver therapeutic activity, which contains an effective amount of the following compound I or a pharmaceutically acceptable salt thereof:

Further, in the above-mentioned composition having therapeutic activity for non-alcoholic fatty liver, the effective in vivo concentration of compound I is 10-1000 mg/Kg.

Further, in the above-mentioned composition having therapeutic activity for non-alcoholic fatty liver, the effective in vivo concentration of compound I is 50-300 mg/Kg.

Further, by combining the above compound I or a pharmaceutically acceptable salt thereof with at least one excipient selected from diluents, disintegrants, binders, lubricants, surfactants, antioxidants, preservatives and stabilizers Formulations are mixed to prepare mixtures for the preparation of solid pharmaceuticals.

Further, the nonalcoholic fatty liver therapeutic active composition according to claim 1, wherein part of the hydrogen atoms in the compound I contained are replaced by deuterium.

Further, the use of the compound I or its pharmaceutically acceptable salt or the pharmaceutical composition in the preparation of a medicament for preventing and/or treating non-alcoholic fatty liver in mammals.

Further, the pharmaceutical composition can be made into injections, tablets, powders, granules, capsules, oral liquids, ointments, and creams in the preparation of drugs for the prevention and/or treatment of non-alcoholic fatty liver in mammals. form.

Further, the pharmaceutical composition contains pharmaceutically acceptable excipients, including but not limited to diluents, binders, disintegrants, glidants, and , lubricants, flavoring agents, inclusion materials, adsorption materials, etc., are used to prepare oral preparations by preparation methods, including but not limited to granules, powders, tablets, capsules, pills, oral liquids, decoctions, and dripping pills .

Beneficial effect

The present invention further explores the medicinal value of tauralhyodeoxycholic acid in treating non-alcoholic fatty liver. Compared with the model group, after administration of tauralhyodeoxycholic acid for 5 weeks, the formation of lipid droplets in the experimental animals was significantly reduced, and lipids in the liver The content is significantly reduced, confirming that tauralhyodeoxycholic acid can reduce the lipid deposition in the liver of mice caused by a high-fat diet; the medicinal value of tauralhyodeoxycholic acid in the treatment of nonalcoholic fatty liver has been further explored, and the present invention It provides a solid foundation for the clinical application of taurohyodeoxycholic acid in the prevention and treatment of NAFLD, and helps to promote the clinical application of taurohyodeoxycholic acid.

Description of drawings

Fig. 1 is the HE staining figure of the liver section of each group of mice administered for 5 weeks in Experimental Example 1, wherein A is the control group; B is the model group; C is the administration group;

Fig. 2 is the oil red O staining diagram of the liver slices of mice in each group after administration for 5 weeks in Experimental Example 1, wherein A is the control group; B is the model group; C is the administration group.

Detailed ways

The present invention will be further described below in conjunction with specific embodiments and accompanying drawings 1-2.

In the embodiment of the present invention, ND (Normal diet, normal diet), HFD (High fat diet, high-fat diet); DMSO (dimethyl sulfoxide); TG (triglycerides); TC (total cholesterol).

Example 1, the application of taurohyodeoxycholic acid in the treatment of non-alcoholic fatty liver, using animal experiments to verify the effect of the above drugs, the specific process is as follows.

(1) Experimental animals and reagents

8-week-old healthy C57BL/6 male mice (SPF grade), body weight (20±2) g, provided by the Animal Experiment Center of Shandong First Medical University;

High-fat feed ((basic feed 66%, lard 20%, sucrose 12%, cholesterol 2%) was purchased from Nantong Trophy Technology Co., Ltd., and common feed was provided by Keao Xieli (Tianjin) Feed Co., Ltd.;

Main reagents: taurine hyodeoxycholic acid (purity ≥ 98%) was purchased from Shanghai Yuanye Biotechnology Co., Ltd.; TG detection kit and TC detection kit were purchased from Nanjing Jiancheng Biotechnology Co., Ltd.;

(2) Experimental method

1. Grouping of experimental animals: 24 8-week-old healthy C57BL/6 female mice were adaptively fed for 1 week in an environment with light for 12 hours and a constant temperature of 26°C, and were randomly divided into control group, model group, and drug treatment group. Each group has 8 animals.

2. Establishment and drawing of animal models: control group (administration of 0.2% DMSO-water solution during the modeling period), model group (administration of 0.2% DMSO-water solution during the modeling period), 100mg/Kg THDCA administration group (administration of 0.2% DMSO-water solution during the modeling period) taurine hyodeoxycholic acid gavage), record the body weight of each mouse before administration. Infuse the above-mentioned corresponding doses of drugs or solutions with a gavage volume of 0.1ml/10g/d every day. After gavage, the control group was given ND feeding, and the model group and the treatment group were given HFD feeding. After 12 hours, the mice were sacrificed by cervical dislocation, the eyeballs were quickly removed, blood was collected to separate the serum, and the mice were stored at -80°C. Open the abdominal cavity of the mouse, quickly dissect and remove the liver, take out a size of 0.7cm×0.7cm×0.2cm and fix it with 4% paraformaldehyde for making paraffin sections, and keep the remaining fresh liver tissue at -80°C for TC , TG index detection.

3. Determination of TC and TG content in mouse liver lipid extraction

Cut 50 mg of liver tissue from the same part of different mice, put it into a plastic centrifuge tube containing 0.1 mL of normal saline, mechanically homogenize it in an ice-water bath, and add 0.4 mL of chloroform-methanol (2:1) extract to the homogenate , fully mixed, after standing for 18 hours, centrifuge at 3000/rpm for 10 minutes, it can be seen that the centrifuge tube is divided into three clear upper, middle and lower layers, the upper phase is the water phase, the middle part is the broken tissue mass, and the lower phase is the lipid layer; the lower phase lipid was carefully extracted, stored in a freezer at 4°C, and the content of total cholesterol (TC) and total triglyceride (TG) was measured with a kit.

4. Hematoxylin-eosin staining (H&E) detection of structural changes in mouse liver

Routine paraffin embedding, sectioning, HE staining, and neutral gum sealing. HE staining is used to observe whether the tissue morphology has lesions. The staining steps are as follows: take the modeled mouse liver Tissues are soaked in tissue fixative, and low to high concentrations of alcohol are used as dehydrating agents to gradually remove the water in the tissue block. Dehydrated tissues were embedded in paraffin and sectioned. Before staining, remove the paraffin in the sections with xylene, then stain with Hematoxylin (H) and Eosin (Eosin, E) for 2-5 minutes, use absolute ethanol for color separation and dehydration, xylene is transparent, and after air-drying The slides were sealed with neutral gum, and finally observed and photographed with a microscope.

5. Oil Red O staining (Oil Red O) detection of lipid droplet accumulation in mouse liver

The modeled mouse liver tissues of the control group, model group, and drug-administered group were respectively soaked in tissue fixative, dehydrated with gradient sucrose solution, and frozen into sections. The frozen sections of the mouse liver were placed at room temperature, and washed three times with PBS. Oil red staining solution was used to stain the section for 10 min. Pour off the oil red staining solution, rinse with 60% isopropanol for 3 times, and then lightly wash with distilled water for 3 times. Finally, fix with gelatin. Observe and take pictures under a microscope.

(3) Experimental results

Table 1. The effect of taurourhyodeoxycholic acid on reducing liver lipid content in mouse non-alcoholic fatty liver model (mean±SEM, n=8)

Compared with the control group #P<0.05##P<0.01; compared with the model group *P<0.05**P<0.01

1. Changes in TC and TG content

Analysis of Table 1 found that compared with the control group, the levels of TC and TG in the liver of the mice in the model group were significantly increased; compared with the model group, the levels of TC and TG in the liver of the mice in the drug treatment group were significantly lower. Taurohyodeoxycholic acid can significantly reduce the levels of TC and TG in the liver of NAFLD mice.

2. HE staining observation and analysis

As shown in Figure 2, the HE staining results of the liver showed that the liver cells in the control group were normal in morphology, no obvious degeneration and necrosis were seen, and no obvious inflammatory cells or fibrous tissue hyperplasia were seen in the portal area; the liver tissue of the model group was almost covered with fat vacuoles Replacement, ballooning degeneration of hepatocytes, inflammatory cell infiltration in the portal area, no fibrous tissue hyperplasia; fat vacuoles distributed in the liver tissue in the treatment group, no obvious inflammatory cells or fibrous tissue hyperplasia. It can be concluded that taurourhyodeoxycholic acid can better improve the degree of hepatic steatosis in NAFLD mice.

3. Oil red O staining results of frozen liver sections

No lipid droplets were formed in the control group, and a large number of red lipid droplets appeared in the liver cells of the mice in the model group, and the lipid content was significantly higher than that in the control group. Compared with the model group, after 5 weeks of administration of taurohyodeoxycholic acid, the formation of lipid droplets was significantly reduced, and the lipid content was significantly reduced. Taurohyodeoxycholic acid reduces lipid deposition in the liver of mice induced by a high-fat diet.

The above-mentioned embodiment is a preferred embodiment of the present invention, but the embodiment of the present invention is not limited by the above-mentioned embodiment, and any other changes, modifications, substitutions, combinations, Simplifications should be equivalent replacement methods, and all are included in the protection scope of the present invention.

Claims (8)

1. A composition with non-alcoholic fatty liver therapeutic activity, characterized in that, the composition contains effective dose of the following compound I or its pharmaceutically acceptable salt: 2. The composition with therapeutic activity of non-alcoholic fatty liver as claimed in claim 1, characterized in that, in the composition with therapeutic activity of non-alcoholic fatty liver, the effective in vivo concentration of compound I is 10-1000 mg /Kg. 3. The composition with therapeutic activity of non-alcoholic fatty liver as claimed in claim 1, characterized in that, in the composition with therapeutic activity of non-alcoholic fatty liver, the effective concentration of compound I in the body is 50-300 mg /Kg. 4. the composition with nonalcoholic fatty liver therapeutic activity as claimed in claim 1, is characterized in that, by compound I or its pharmaceutically acceptable salt and at least one is selected from diluent, disintegrant, Excipients in binders, lubricants, surfactants, antioxidants, preservatives and stabilizers are mixed to prepare mixtures for the preparation of solid pharmaceuticals. 5. The composition with non-alcoholic fatty liver therapeutic activity as claimed in claim 1, wherein some or all of the hydrogen atoms in compound I are replaced by deuterium. 6. The composition having non-alcoholic fatty liver therapeutic activity according to claim 1, characterized in that said composition is used in the preparation of medicines for preventing and/or treating non-alcoholic fatty liver in mammals. 7. the composition with nonalcoholic fatty liver therapeutic activity as claimed in claim 1, is characterized in that, described composition can be made into the medicament of preventing and/or treating nonalcoholic fatty liver in mammals Various forms of injection, tablet, powder, granule, capsule, oral liquid, ointment, and cream. 8. The composition with nonalcoholic fatty liver therapeutic activity as claimed in claim 1, further comprising pharmaceutically acceptable adjuvants, including but not limited to diluents, adhesives, disintegrants, flow aids Agents, lubricants, flavoring agents, inclusion materials, adsorption materials, etc., are used to prepare oral preparations by preparation methods, including but not limited to granules, powders, tablets, capsules, pills, oral liquids, decoctions, drops pill.