Application of composition comprising atorvastatin, artemisinin and derivatives thereof in relieving and treating metabolism-related fatty liver diseases

Abstract

The invention provides application of a composition in preparation of a medicine for relieving and treating metabolism-related fatty liver diseases. The composition comprises atorvastatin and / or pharmaceutical salt thereof and artemisinin and / or derivatives thereof. The invention provides an application of a composition comprising atorvastatin and / or salts thereof and artemisinin and / or derivatives thereof in relieving and treating metabolism-related fatty liver diseases, and atorvastatin and / or salts thereof and artemisinin and / or derivatives thereof have a synergistic effect in treating the metabolism-related fatty liver diseases. Beta oxidation of fatty acid in liver cells can be synergistically increased, synthesis of glutathione is increased, damage of atorvastatin to liver functions is improved, and fatty degeneration, inflammation and fibrosis of livers of patients with metabolism-related fatty liver diseases are relieved and treated.

Description

Technical Field

[0001] The present invention relates to the field of medical technology, and in particular to an application of a composition comprising atorvastatin, artemisinin and derivatives thereof in alleviating and treating metabolism-related fatty liver disease. Background Art

[0002] Nonalcoholic fatty liver disease (NAFLD) is a group of metabolic diseases characterized by fat accumulation in the liver. Unhealthy lifestyles such as long periods of sitting and lack of exercise, unreasonable dietary structure, and metabolic diseases such as obesity and diabetes are high-risk factors for its onset. International expert consensus recommends renaming NAFLD as metabolic-associated fatty liver disease (MAFLD). NAFLD can progress to nonalcoholic steatohepatitis (NASH), increasing the risk of cirrhosis and hepatocellular carcinoma. The global prevalence of NAFLD is about 25%-30%, and it is as high as 70% in patients with type 2 diabetes and obesity, bringing a serious public health burden. Despite this, there is still a lack of effective treatments.

[0003] Statins are the first-line drugs for treating hyperlipidemia, but their therapeutic effects on NASH and the relationship between benefits and risks are still controversial. Some literature reports that statins can regulate liver lipid metabolism, reduce inflammation and oxidative stress, reduce fibrosis, improve NAFLD / NASH conditions, have greater benefits for improving other metabolic complications, and reduce the mortality rate of patients. However, they will also slightly increase the risk of abnormal glucose metabolism. It is particularly worth noting that statins should still be used with caution in patients with cirrhosis, especially those with decompensated cirrhosis.

[0004] Atorvastatin is a third-generation potent statin. Its application in NAFLD patients has always been a hot topic of research at home and abroad. In early clinical trials, it was found that atorvastatin (10 mg, once a day) in the treatment of NASH patients with dyslipidemia can significantly reduce liver enzyme and blood lipid levels, restore liver enzyme levels to normal, and improve liver density.

[0005] The main concern about the use of statins in patients with NAFLD is the damage to liver function caused by statins. The mechanism of liver damage caused by statins is still unclear. Mitochondrial dysfunction-induced apoptosis may be involved. Even in low-concentration statin culture medium (MEC of 5 or 10 μmol / L), intracellular Ca2+ in HepG2 cells can be observed. 2+ Increased levels lead to changes in mitochondrial permeability, causing mitochondrial dysfunction and increased cell apoptosis.

[0006] Most patients with metabolic-related fatty liver disease also have hyperlipidemia. How to avoid the damage of statins to liver function and provide a drug that can effectively treat NAFLD is one of the hot topics of concern for technicians in this field. Summary of the invention

[0007] The present invention provides an application of a composition comprising atorvastatin, artemisinin and derivatives thereof in alleviating and treating metabolism-related fatty liver disease. The composition can synergistically increase the β-oxidation of fatty acids in hepatocytes, increase the synthesis of glutathione, and improve fatty degeneration, inflammation and fibrosis of the liver in patients with metabolism-related fatty liver disease.

[0008] The present invention provides use of a composition in preparing a medicament for alleviating and / or treating metabolism-related fatty liver disease. The composition comprises atorvastatin and / or a pharmaceutically acceptable salt thereof and artemisinin and / or a derivative thereof.

[0009] In the above application, the pharmaceutically acceptable salt of atorvastatin may be, for example, atorvastatin calcium.

[0010] In the above application, the artemisinin derivative is selected from at least one of artesunate, dihydroartemisinin and artemether. Further, the artemisinin derivative is artesunate.

[0011] In the application described above, the mass ratio of atorvastatin and / or its pharmaceutical salt to artemisinin and / or its derivatives is 1:1-1:5; specifically, it can be one of 1:1, 1:1.5, 1:2, 1:2.5, 1:3, 1:3.5, 1:4, 1:4.5, and 1:5.

[0012] In the application as described above, the composition is packaged according to the clinical dosage and prepared into specific dosage units, and the mass of atorvastatin and / or its pharmaceutically acceptable salt in the dosage unit is 5-40 mg. Furthermore, when the pharmaceutically acceptable salt of atorvastatin is atorvastatin calcium, the mass of atorvastatin calcium in the dosage unit is one of 5 mg, 10 mg, 20 mg, 30 mg, and 40 mg.

[0013] In the above application, the composition is packaged according to the clinical dosage and prepared into specific dosage units, and the mass of artemisinin and / or its derivatives in the dosage unit is 10-100 mg. Further, when the derivative of artemisinin is artesunate, the mass of artesunate in the dosage unit is one of 10 mg, 20 mg, 30 mg, 40 mg, 50 mg, 60 mg, 70 mg, 80 mg, 90 mg, and 100 mg.

[0014] In the application as described above, the composition is prepared into a fixed-dose compound medicine according to the clinical dosage, and the fixed-dose compound medicine includes 10-20 mg atorvastatin and / or its pharmaceutical salt and 10-100 mg artemisinin and / or its derivatives. Further, the fixed-dose combination drug includes 10 mg of atorvastatin calcium and 10 mg of artesunate, or includes 10 mg of atorvastatin calcium and 20 mg of artesunate, or includes 10 mg of atorvastatin calcium and 30 mg of artesunate, or includes 10 mg of atorvastatin calcium and 40 mg of artesunate, or includes 10 mg of atorvastatin calcium and 50 mg of artesunate, or includes 20 mg of atorvastatin calcium and 20 mg of artesunate, or includes 20 mg of atorvastatin calcium and 40 mg of artesunate, or includes 20 mg of atorvastatin calcium and 60 mg of artesunate, or includes 20 mg of atorvastatin calcium and 80 mg of artesunate, or includes 20 mg of atorvastatin calcium and 100 mg of artesunate.

[0015] For the above-mentioned applications, the above-mentioned composition can be directly physically mixed and then granulated with pharmaceutical excipients according to conventional pharmaceutical technology to form tablets or capsules; or it can be granulated separately and then pressed into the form of multi-layer tablets.

[0016] In the application as described above, the metabolism-related fatty liver disease includes at least one of metabolism-related fatty liver hepatitis, metabolism-related fatty liver fibrosis, and metabolism-related fatty liver cirrhosis.

[0017] As described above, the drug further comprises one or more pharmaceutically acceptable carriers, excipients or diluents.

[0018] The second aspect of the present invention provides a drug for alleviating and / or treating metabolic-related fatty liver disease, the drug comprising atorvastatin and / or its pharmaceutical salts, artemisinin and / or its derivatives and one or more pharmaceutically acceptable carriers, excipients or diluents.

[0019] In the drug as described above, the mass ratio of atorvastatin and / or its pharmaceutically acceptable salt to artemisinin and / or its derivatives is 1:1-1:5.

[0020] The present invention provides an application of a composition comprising atorvastatin, artemisinin and derivatives thereof in alleviating and treating metabolism-related fatty liver disease. Atorvastatin and / or its salts and artemisinin and / or its derivatives have a synergistic effect in treating metabolism-related fatty liver disease, can synergistically increase the β-oxidation of fatty acids in hepatocytes, increase the synthesis of glutathione, improve the damage of atorvastatin to liver function, and treat fatty degeneration, inflammation and fibrosis of the liver of patients with metabolism-related fatty liver disease. DETAILED DESCRIPTION

[0021] In order to make the purpose, technical solutions and advantages of the present invention clearer, the technical solutions in the embodiments of the present invention will be clearly and completely described below in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0022] Example 1: Effects of a compound of atorvastatin and artemisinin on fatty acid and glutathione content in hepatocytes

[0023] HepG2 liver cancer cells (purchased from the cell bank of the Chinese Academy of Sciences) are selected as research materials. HepG2 liver cancer cells are widely used in pharmacology and toxicology research, and can express many differentiated liver functions, such as the synthesis and secretion of plasma proteins, cholesterol and triglyceride metabolism, lipoprotein metabolism and transport, bile acid synthesis, glycogen synthesis or insulin signaling, etc. Adding free fatty acids (FFA) in the culture medium can cause lipid accumulation in HepG2 liver cancer cells, and the increase in fatty acid content can lead to lipotoxicity, and cause a series of oxidative stress reactions, leading to hepatocyte inflammatory reactions. Glutathione in cells can play an important antioxidant role, and the increase in its content helps cells to cope with the oxidative stress damage caused by fatty acids. Therefore, this embodiment tests the changes in fatty acid content and glutathione content in HepG2 liver cancer cells after adding FFA in the culture medium.

[0024] 1. Determination of fatty acid content

[0025] HepG2 liver cancer cells with a passage number of less than 20 were selected and inoculated in a 96-well black culture plate at a seeding density of 3×10 5 / mL. 24h after inoculation, HepG2 liver cancer cells were divided into a control group, a high-fat model group and 9 drug-treated groups, with 6 wells in each group. The control group cells used 1% bovine serum albumin plus culture medium; the high-fat model group used complete culture medium containing 1% bovine serum albumin and 1mM / L free fatty acids; the drug-treated group used complete culture medium containing 1% bovine serum albumin, 1mM / L free fatty acids and 5μM / L of different test drugs shown in Table 1.

[0026] After 24 hours of culture, the cells were rinsed twice with preheated PBS buffer, and 50 μg / mL Nile red solution was added at a volume of 100 μL / well in a light-proof environment, and then treated in a 37°C incubator for 10 minutes. The Nile red solution was aspirated, and the cells were rinsed twice with warm PBS buffer. Then the 96-well plate was turned upside down on absorbent paper and waited to dry. Finally, the fluorescence intensity was detected using a multifunctional microplate reader at an excitation wavelength of 488 nm and an emission wavelength of 550 nm. The whole process was performed under light-proof conditions. The fluorescence intensity of the control group was taken as 1, and the relative fluorescence intensity of the high-fat model group and the drug-treated group was calculated. The higher the relative fluorescence intensity, the higher the fatty acid content. The measurement results are shown in Table 1.

[0027] 2. Determination of glutathione content

[0028] HepG2 liver cancer cells with a passage number of less than 20 were selected and inoculated in 12-well plates at a density of 3×10 5 / mL. 24h after inoculation, HepG2 liver cancer cells were divided into a control group, a high-fat model group and 9 drug-treated groups, with 4 wells in each group. The control group cells used 1% bovine serum albumin plus culture medium; the high-fat model group used complete culture medium containing 1% bovine serum albumin and 1mM / L free fatty acids; the drug-treated group used complete culture medium containing 1% bovine serum albumin, 1mM / L free fatty acids and 5μM / L of different test drugs shown in Table 1.

[0029] After 24 hours of culture, cells were collected and rinsed with PBS three times and counted, and then the instructions of the Bio-Tech GSH Detection Kit were followed as follows: Collect cells by centrifugation and aspirate the supernatant. Add 3 times the volume of the cell pellet and fully vortex the protein removal reagent M solution. Then use liquid nitrogen and a 37°C water bath to quickly freeze and thaw the sample twice, and let it stand on ice for 5 minutes. Centrifuge at 10,000g for 10 minutes at 4°C. Take the supernatant for the determination of total glutathione or store it at -80°C. Add samples or standards in a 96-well plate in turn, then add 150 μl of total glutathione detection working solution, mix well, and incubate at 25°C or room temperature for 60 minutes to determine the total glutathione level.

[0030] Take part of the above sample for total glutathione content to be measured, add 20 μl of diluted GSH scavenging auxiliary solution per 100 μl of sample, and immediately vortex mix. Then add GSH scavenging working solution per 100 μl of sample, add 4 μl of GSH scavenging reagent working solution, immediately vortex mix, react at 25°C for 60 minutes, and then use it for the determination of oxidized glutathione.

[0031] The preparation steps of the standard and sample were the same for detecting the total glutathione and oxidized glutathione content, and then the absorbance value was detected at 412nm. The total glutathione and oxidized glutathione levels were calculated by the standard curve, and the formula for calculating reduced glutathione was: reduced glutathione = total glutathione - oxidized glutathione × 2. The obtained glutathione levels were normalized by the number of cells to calculate the difference in glutathione content between different groups.

[0032] Table 1 Comparison of fatty acid content and glutathione content in hepatocytes

[0033]

[0034]

[0035] Note: The ratios of artemisinin compounds and statins when used together are all mass ratios. # indicates p<0.01 for the model group versus the normal group; * indicates p<0.01 for the drug-treated group versus the model group.

[0036] According to the data provided in Table 1, artemisinin compounds alone or in combination with statins can reduce the lipid content in fatty acid-induced hepatocytes, and the effect of combined use is generally better than that of single use, among which the combination of artesunate and atorvastatin has the best effect. Artemisinin compounds can also increase the content of glutathione in hepatocytes, but atorvastatin reduces the content of glutathione compared with the model group, which may be the reason for its liver damage. The combination of artemisinin compounds and atorvastatin increases the content of glutathione more, among which the combination of artesunate and atorvastatin has the highest glutathione content.

[0037] Example 2: Therapeutic effects of atorvastatin and artesunate on metabolic dysfunction-related steatohepatitis

[0038] 5-week-old male C57BL / 6 mice were randomly divided into a normal group (10 mice) and a model group (40 mice) according to body weight. The mice in the model group were fed a high-fat diet (HFD) and 15% fructose water (w / w), and the normal mice were fed a normal diet. After 16 weeks of feeding, baseline body weight and serum ALT were measured. The mice in the model group were randomly divided into 4 groups according to body weight and serum ALT levels, namely, the model control group, artesunate + atorvastatin calcium group (20+10 mg / kg), artesunate group (20 mg / kg), and atorvastatin calcium group (10 mg / kg), with 10 animals in each group. The mice in each group were given the vehicle or compound by PO, once a day, for 8 consecutive weeks.

[0039] After administration, the animal body weight and food intake were recorded twice a week. At the end of the experiment, serum was taken for blood biochemistry testing, and the liver was taken for liver pathological analysis. After 24 weeks of HFD feeding induction, compared with the normal group animals, the body weight, serum ALT and AST, serum TC and FFA, serum HDL and LDL, liver, liver-to-body ratio, liver TG and TC, liver fibrosis-related gene expression, NAS score and fibrosis score of the model group mice were significantly increased, indicating that the mouse NASH model was successfully established. After 8 weeks of treatment, the ALT, AST and NAS scores of the drug-treated group mice were tested, and the test results are shown in Table 2.

[0040] Table 2 NAS score results of liver function and liver pathology

[0041] Group ALT AST NAS score Normal group 33±6 54±5 0.5±0.17 Model Group <![CDATA[450±48 # ]]> <![CDATA[317±38 # ]]> <![CDATA[5.9±0.23 # ]]> Artesunate group <![CDATA[182±22 * ]]> <![CDATA[127±9 * ]]> <![CDATA[3.12±0.25 * ]]> Artesunate + Atorvastatin Calcium <![CDATA[156±21 * ]]> <![CDATA[112±16 * ]]> <![CDATA[2.8±0.33 ** ]]> Atorvastatin calcium <![CDATA[221±42 * ]]> <![CDATA[192±32 * ]]> <![CDATA[3.2±0.13 * ]]>

[0042] In Table 2, # indicates that the model group is compared with the normal group, p < 0.05; * indicates that the drug-treated group is compared with the model group, p < 0.05; ** indicates that the drug-treated group is compared with the model group, p < 0.01.

[0043] According to Table 2, compared with the model group, the test drug artesunate combined with atorvastatin can significantly improve liver function and liver NAS score, and the effect is better than artesunate and atorvastatin alone.

[0044] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. Use of a composition in the preparation of a medicament for alleviating and / or treating metabolic-related fatty liver disease, characterized in that: The composition comprises atorvastatin and / or a pharmaceutically acceptable salt thereof and artemisinin and / or a derivative thereof.

2. The use according to claim 1, characterized in that: The mass ratio of atorvastatin and / or its pharmaceutically acceptable salt to artemisinin and / or its derivatives is 1:1-1:

5.

3. The use according to claim 1, characterized in that: The artemisinin derivative is selected from at least one of artesunate, dihydroartemisinin and artemether.

4. The use according to claim 1, characterized in that: The composition is packaged according to the clinical dosage and prepared into dosage units of specific specifications, and the mass of atorvastatin and / or its pharmaceutically acceptable salt in the dosage unit is 5-40 mg.

5. The use according to claim 1, characterized in that: The composition is packaged according to the clinical dosage and prepared into dosage units of specific specifications, and the mass of artemisinin and / or its derivatives in the dosage unit is 10-100 mg.

6. The use according to claim 1, characterized in that: The composition is prepared into a fixed-dose compound medicine according to a clinical administration dose, and the fixed-dose compound medicine comprises 10-20 mg atorvastatin and / or a pharmaceutically acceptable salt thereof and 10-100 mg artemisinin and / or a derivative thereof.

7. The use according to any one of claims 1 to 6, characterized in that: The metabolism-related fatty liver disease includes at least one of metabolism-related fatty liver hepatitis, metabolism-related fatty liver fibrosis, and metabolism-related fatty liver cirrhosis.

8. The use according to any one of claims 1 to 6, characterized in that: The medicament also includes one or more pharmaceutically acceptable carriers, excipients or diluents.

9. A drug, characterized in that The medicine is used for alleviating and / or treating metabolism-related fatty liver disease, and comprises atorvastatin and / or its pharmaceutically acceptable salt, artemisinin and / or its derivatives and one or more pharmaceutically acceptable carriers, excipients or diluents.

10. The drug according to claim 9, characterized in that The mass ratio of atorvastatin and / or its pharmaceutically acceptable salt to artemisinin and / or its derivatives is 1:1-1:5.

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