Compositions for modulating C-Met and uses for promoting hepatocyte regeneration

Abstract

To provide compositions for regulating C-Met and uses thereof for promoting liver cell regeneration.SOLUTION: The invention discloses compositions for regulating C-Met and applications thereof for promoting liver cell regeneration. The main ingredient of the composition for regulating C-Met comprises Antrodin A and / or DMB (4,7-Dimethoxy-5-methyl-1,3-benzodioxole) which effectively modulates the expression of genes associated with cell regeneration and repair in liver cells (c-Met, Notch etc.) so that repair of damaged liver cells and regeneration of liver cells are effectively promoted.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to compositions related to the treatment of liver disease, and in particular to compositions for modulating C-Met and their use in promoting hepatocytes. [Background technology]

[0002] The liver plays a central role in metabolic reactions in the human body, and most chemicals enter the liver and are metabolized there. This means that the liver is easily affected by harmful substances or their metabolites, impairing liver function. In addition to harmful substances or their metabolites, the liver can also become infected with viruses, leading to hepatitis, cirrhosis, liver cancer, etc.

[0003] Because the early symptoms of liver disease are not obvious, and the liver does not have nerves that sense pain, most patients do not realize that their liver function is abnormal or that they are infected with a virus.Therefore, most patients with liver disease have already progressed to the point where some symptoms appear, making clinical treatment more difficult.Take hepatitis treatment as an example, currently, clinically, there are drug therapies or symptomatic treatments according to the cause of the disease.For example, for hepatitis caused by viruses, antiviral drugs are administered, and for hepatitis caused by fatty liver, weight management is used to slow the progression of hepatitis.However, if hepatitis continues to worsen, it will cause hepatocyte necrosis, causing irreversible damage to liver function, and even if current existing drugs are administered, it is difficult to recover. Summary of the Invention [Problem to be solved by the invention]

[0004] The main object of the present invention is to provide a composition for regulating c-Met, which effectively repairs damaged liver cells and promotes liver cell regeneration by regulating the expression of multiple genes (c-Met, Notch, etc.) related to liver cell repair and regeneration, thereby achieving therapeutic effects on liver injury-related diseases, and to provide an application for promoting liver cells.

[0005] A secondary object of the present invention is to provide a composition for regulating C-Met and its use for promoting hepatocytes, in order to exert the effect of promoting hepatocyte growth. [Means for solving the problem]

[0006] To achieve the above objectives, the present invention discloses a composition for regulating C-Met, which contains an effective amount of DMB (4,7-Dimethoxy-5-methyl-1,3-benzodioxole) as a main ingredient and a carrier, which may be a pharmaceutically acceptable carrier or a food industry acceptable carrier. By administering the composition for regulating C-Met disclosed by the present invention to patients with liver disease, liver damage can be effectively alleviated, thereby achieving therapeutic effects for liver disease.

[0007] In one embodiment of the present invention, the concentration of the DMB compound ranges from 1.25 to 20 ppm.

[0008] In another embodiment of the present invention, the composition for regulating C-Met further comprises an effective amount of Antrodin A, and the concentration thereof is 8.75 ppm or more and less than 140 ppm.

[0009] To achieve a better liver repair effect, the concentration ratio of DMB to Antrodin A is 1:2 to 1:10, and preferably the concentration ratio of DMB to Antrodin A is 1:5 to 1:10.

[0010] Another embodiment of the present invention is to prepare a composition containing DMB compound and / or Antrodin A to promote hepatocyte regeneration, which regulates the expression of multiple genes (c-Met, Notch, etc.) related to hepatocyte repair and regeneration, and reduces intracellular free radicals or oxidative stress, thereby repairing damaged hepatocytes, maintaining the activity of normal hepatocytes, and promoting their regeneration, thereby providing therapeutic effects for diseases related to hepatocyte damage.

[0011] The concentration of the DMB is in the range of 1.25 to 20 ppm, and the concentration of the Antrodin A is 8.75 ppm or more and less than 140 ppm. When the composition for promoting hepatocyte regeneration simultaneously contains DMB and Antrodin A, the concentration ratio of DMB to Antrodin A is preferably 1:2 to 1:10, and the concentration ratio of DMB to Antrodin A is preferably 1:5 to 1:10.

[0012] In an embodiment of the present invention, the composition for promoting hepatocyte regeneration may be a food, a health food, or a pharmaceutical composition. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the results of HPLC analysis of Antrodia camphorata mycelium powder. DETAILED DESCRIPTION OF THE INVENTION

[0014] The main components of the composition for regulating c-Met disclosed in the present invention contain Antrodin A and / or DMB (4,7-Dimethoxy-5-methyl-1,3-benzodioxole), which effectively regulate the expression of genes (c-Met, Notch, etc.) related to cell regeneration and repair in hepatocytes, thereby achieving the effect of repairing damaged hepatocytes and promoting hepatocyte regeneration.

[0015] Here, c-Met and Notch are genes involved in the regulatory pathways of hepatocyte repair and regeneration. When hepatocytes are stimulated by hepatotoxic drugs, the expression level of c-Met decreases, and downregulation of c-Met reduces the binding of its ligand HGF, thereby reducing the signaling for hepatocyte repair and regeneration. The expression level of Notch is related to hepatocyte replication and can reduce the ROS content in hepatocytes, thereby exerting a hepatocyte protective effect. In other words, increased expression level of Notch in hepatocytes can promote hepatocyte proliferation and improve liver injury.

[0016] The Antrodin A and DMB disclosed in this invention can be prepared by chemical synthesis or extracted and purified from fungal mycelia. For example, HPLC analysis of Antrodin A (peak appears at a retention time of approximately 76-77 minutes) and DMB (peak appears at a retention time of approximately 32-33 minutes) was performed on powdered Antrodin A mycelia, as shown in Figure 1. The column used was a C18 column (Merck Purospher STAR, 5 μm, 4.5 × 250 mm), with a flow rate of 1.0 mL / min, temperature of 30°C, injection volume of 10 μL, detection time of 120 minutes, and a mobile phase of 0.1% formic acid (A) and 100% acetonitrile (B). The wavelength was 254 nm.

[0017] It is worth noting that Antrodin A and DMB can be extracted and purified from Antrodia camphorata, and while prior art documents state that Antrodia camphorata extracts have cytotoxic activity against cancer cells, the main utility of the composition for regulating C-Met disclosed in the present invention is to protect and repair cells and promote cell regeneration, i.e., the composition for regulating C-Met disclosed in the present invention is not toxic to cells and does not have the ability to kill cells.

[0018] The c-Met and Notch genes disclosed in the present invention are involved in the regulatory pathways of hepatocyte repair and regeneration. Specifically, when hepatocytes are stimulated by hepatotoxic drugs, the expression level of c-Met decreases, and the downregulation of c-Met reduces the binding of its ligand HGF, thereby reducing the signaling for hepatocyte repair and regeneration. The expression level of Notch is related to hepatocyte replication and can reduce the ROS content in hepatocytes, thereby exerting a hepatocyte protective effect. That is, increased expression level of Notch in hepatocytes can promote hepatocyte proliferation and improve liver injury.

[0019] In the following, in order to explain and verify the technical features and effects of the present invention, several examples will be given and further explained with reference to the drawings.

[0020] The cells used in the following examples are readily available to those skilled in the art to which the present invention pertains, and therefore no separate patent deposit is required.

[0021] The Antrodin A and DMB used in the following examples are prepared by chemical synthesis, and the chemical synthesis method is common knowledge in the technical field to which the present invention pertains, so the description thereof is omitted here.

[0022] Example 1: Analysis of Cellular Activity (1) Normal hepatocytes from FL83B mice were collected and divided into several groups (FL83B 1 × 10 4 The cells were divided into groups (1000 cells / well), and the amount of cells in each group was treated under different conditions. After 24 hours of culture, the cell activity of each group was analyzed using a commercially available MTS cell activity assay kit (CellTiter-Glo® One Solution Assay). The cell treatment conditions and the corresponding viability of each group are shown in Tables 1 to 3 below.

[0023] As can be seen from the results in Table 1 below, when the concentration of Antrodin A was 140 ppm, it was toxic to hepatocytes, resulting in a decrease in hepatocyte viability, while when the concentration was 8.75 to 70 ppm, it effectively maintained and improved the activity and viability of normal hepatocytes. As can be seen from the results in Table 2, the addition of DMB at various concentrations not only did not cause toxicity to normal hepatocytes, but also promoted hepatocyte growth.

[0024] Comparing the results in Tables 1 and 3, it can be seen that under the same concentration of Antrodin A, the viability of normal hepatocytes when DMB is also added is higher than that when Antrodin A alone is added. This indicates that when the concentration of Antrodin A does not cause cytotoxicity, the addition of DMB helps improve cell activity; and when the concentration of Antrodin A causes cytotoxicity, the addition of DMB can protect hepatocytes from the cytotoxic effects caused by Antrodin A, effectively improving cell activity and viability.

[0025] [Table 1]

[0026] [Table 2]

[0027] [Table 3]

[0028] Example 2: Analysis of Cellular Activity (II) Normal hepatocytes from FL83B mice were collected and divided into several groups (FL83B 1 × 10 4 The cells were divided into groups (cells / well) and each group was first treated with 10 mM acetaminophen for 24 hours to induce hepatocyte damage. The acetaminophen was then removed, and the cells were then treated with different concentrations of Antrodin A and / or DMB for 24 hours. The cell activity of each group was then analyzed using a commercially available MTS cell activity assay kit (CellTiter-Glo® One Solution Assay). The treatment conditions and corresponding viability of each group of cells are shown in Tables 4 to 6 below.

[0029] As can be seen from the results in Tables 4 and 5, the addition of Antrodin A or DMB can help repair damaged liver cells and increase liver cell activity. When the concentration of DMB is lower than that of Antrodin A, it has a good effect on repairing damaged liver cells.

[0030] Next, as can be seen from the results in Table 6, when damaged hepatocytes were treated with Antrodin A and DMB simultaneously, the repair effect on the hepatocytes was superior to that of treatment with Antrodin A alone, and when the concentration ratio of DMB to Antrodin A was 1:2 to 1:10, it could help improve the activity and viability of damaged hepatocytes. Here, when the concentration ratio of DMB to Antrodin A was 1:5 to 1:10, the repair effect on damaged hepatocytes was higher.

[0031] [Table 4]

[0032] [Table 5]

[0033] [Table 6]

[0034] Example 3: Analysis of gene expression The gene expression in the cells treated with different concentrations of Antrodin A and DMB in Example 2 was analyzed using a Real-time qPCR kit, and the results are shown in Tables 7 to 9 below.

[0035] As can be seen from the results in Tables 7 to 9, both Antrodin A and DMB help injured hepatocytes increase the expression levels of c-Met and Notch, thereby accelerating the repair and regeneration of injured hepatocytes. When cells were treated with Antrodin A and DMB simultaneously, the expression levels of c-Met and Notch in injured hepatocytes were significantly higher than when treated with Antrodin A alone, indicating that DMB has a strong ability to regulate c-Met and Notch.

[0036] [Table 7]

[0037] [Table 8]

[0038] [Table 9]

[0039] To summarize the results of Examples 1 to 3, the composition for regulating C-Met disclosed in the present invention not only does not cause cytotoxicity to hepatocytes, but also has the effect of repairing damaged hepatocytes and promoting hepatocyte regeneration. Therefore, when an effective amount of the composition for regulating C-Met is administered to a patient with liver disease, it can treat liver damage and improve liver function.

[0040] Example 4: Safety Test In this example, a total of 29 subjects with chronic hepatitis B were randomly divided into two groups: a control group (n=14) administered the C-Met modulating composition but not the antiviral drug entecavir, and a treatment group (n=15) administered the antiviral drug entecavir and the C-Met modulating composition.

[0041] The C-Met-modulating composition administered in this example contained 614.01 ppm DMB and 3937.39 ppm Antrodin A, i.e., the ratio of Antrodin A to DMB in the C-Met-modulating composition administered in this example was approximately 6.5:1. The drug administration period was 48 weeks, and treatment efficacy and virologic relapse were monitored and recorded at 12, 24, 36, 48, 60, and 72 weeks after the start of treatment. The results are shown in Tables 10 and 11 below, with blank spaces indicating no data. Furthermore, treatment efficacy and virologic relapse status were analyzed for patients with or without hepatitis B virus e antigen during and after treatment in the treatment group. The results are shown in Table 12, with blank spaces indicating no data. In Tables 10 to 12, virological relapse is defined as a hepatitis B DNA level exceeding 2000 IU / mL on two consecutive occasions at least 3 months after the end of treatment with a nucleotide anti-hepatitis B virus drug. Virologic relapse is defined as an increase in ALT to more than twice the normal level and a hepatitis B DNA level exceeding 2000 IU / mL after the end of treatment with a nucleotide anti-hepatitis B virus drug.

[0042] In addition, blood and urine were collected from the treatment and control groups, and the following values ​​were measured: AST, ALT, bilirubin, alkaline phosphatase (ALK-P), r-GT (Gamma-glutamyl transferase), AFT (Alpha-Fetoprotein), PT (Prothrombin Time), APTT (Activated Partial Thromboplastin Time), albumin, globulin, renal BUN, creatinine, fasting blood glucose (GlucoseAC), glycosylated hemoglobin (HbA1c), white blood cells, red blood cells, hemoglobin, hematocrit (HCT), red blood cell distribution width (RDW), platelets, mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH), mean corpuscular hemoglobin concentration (MCHC), mean platelet volume (MPV), and urine specific gravity. The serum lipids (g / L), urine pH, and urothelial cell count were detected, and each detection index of the two groups was further analyzed. The results are shown in Tables 13 to 41.

[0043] The above results indicate that long-term administration of the composition for regulating C-Met disclosed in the present invention will not adversely affect the health of patients with viral hepatitis. In other words, the composition for regulating C-Met disclosed in the present invention is a reliably safe hepatoprotective ingredient and can be used as an adjuvant to antiviral drugs.

[0044] [Table 10]

[0045] [Table 11]

[0046] [Table 12]

[0047] [Table 13]

[0048]

Table 14

[0049]

Table 15

[0050] Table 16

[0051]

Table 17

[0052]

Table 18

[0053]

Table 19

[0054] Table 20

[0055] Table 21

[0056] Table 22

[0057] Table 23

[0058] Table 24

[0059] Table 25

[0060] Table 26

[0061] Table 27

[0062] Table 28

[0063] Table 29

[0064]

Table 30

[0065] Table 31

[0066] Table 32

[0067] Table 33

[0068] Table 34

[0069] Table 35

[0070] Table 36

[0071] Table 37

[0072] Table 38

[0073] Table 39

[0074] Table 40

[0075] Table 41

Claims

1. A composition for promoting expression of c-Met, comprising: The composition contains DMB (4,7-Dimethoxy-5-methyl-1,3-benzodioxole) and Androdin A, The composition, wherein the concentration of the Androdin A is 53.33 to 70 ppm, and the concentration of the DMB is 10 to 26.67 ppm.

2. A composition for promoting expression of Notch, comprising: The composition contains DMB (4,7-Dimethoxy-5-methyl-1,3-benzodioxole) and Androdin A, the concentration of Androdin A is 35 ppm and the concentration of DMB is 5 ppm; or A composition, wherein the concentration of Androdin A is 72.7 ppm and the concentration of DMB is 7.3 ppm.

3. The composition according to claim 1 or 2, which is a composition for liver protection.

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