JP7142886B2 - antifibrotic agent - Google Patents

Description

The present invention relates to antifibrotic agents. More specifically, the present invention relates to an antifibrotic agent capable of suppressing fibrosis of the liver and the like and treating liver cirrhosis or cancer.

In recent years, an increase in the number of liver cancer patients has become a problem. According to a 2012 report by the National Cancer Research Center, the number of deaths from liver cancer in both men and women in Japan ranks fourth among all cancers, reaching as many as 33,000 in 2010. Liver cancer develops on the basis of non-alcoholic steatohepatitis (NASH), which is associated with hepatitis B and C virus infection, heavy drinking and diabetic obesity. That is, liver cancer develops from chronic inflammation and fibrosis of the liver, and regardless of etiology, it develops at an annual rate of 8%. Once liver cancer occurs, recurrence and intrahepatic metastasis are repeated. Liver cirrhosis is a condition in which the liver parenchyma is replaced with extracellular matrix proteins such as type I collagen, resulting in a decrease in functional hepatocytes. In this fibrous liver, the hepatic parenchyma is replaced by myofibroblast (MFB), which is activated by activation of hepatic stellate cells (HSC) whose main function is vitamin A storage under physiological conditions. It is a condition that is Transforming growth factor (TGF)-β and connective tissue growth factor (CTGF) are involved in this transformation, and these factors induce sustained activation of hepatic stellate cells. It has been reported that an increase in myofibroblasts in the and parenchyma is a factor that reduces hepatocyte function and contributes to the development of liver cancer (Non-Patent Documents 1 and 2). Therefore, it is believed that suppression of activation of hepatic stellate cells and control of myofibroblasts will lead to the development of treatments for liver fibrosis and liver cancer.

Various therapeutic or preventive agents for liver diseases have been known so far.
For example, Patent Documents 1 to 3 disclose that a therapeutic/preventive agent for liver disease containing menatetrenone (vitamin K-II) as an active ingredient suppresses cell proliferation of hepatocellular carcinoma and promotes portal vein infiltration after hepatocellular carcinoma treatment. It has been reported that the occurrence of Further, for example, Patent Document 4 reports that menatetrenone can suppress the carcinogenesis of liver cancer derived from chronic liver disease, liver cirrhosis, or hepatitis C virus-induced liver cirrhosis. Further, for example, Patent Document 5 proposes the use of 1,4-naphthoquinone compounds related to proapoptotic activity or antiproliferative activity as anticancer agents.

Nat. Med. , 2001, 17:1668 Hepatology, 2012, 56:769

JP-A-2004-67513 Japanese Patent Application Laid-Open No. 2004-107330 Japanese Patent Application Laid-Open No. 2004-203745 WO 2005/065671 pamphlet Japanese Patent Publication No. 2009-534449

In Patent Documents 1 to 3, it is reported as an active ingredient of a liver disease therapeutic/preventive agent capable of suppressing cell proliferation of hepatocellular carcinoma and suppressing the occurrence of portal vein invasion after hepatocellular carcinoma treatment, and Patent Document 4. Menatetrenone (vitamin K-II), which was reported to be able to suppress the carcinogenesis of liver cancer derived from chronic liver disease, cirrhosis, or hepatitis C viral cirrhosis, has a chemical structure of 2-methyl-1, It is a 3-position derivative of 4-naphthoquinone. In addition, the 1,4-naphthoquinone compound for which the apoptosis-promoting activity and antiproliferative activity were demonstrated in Patent Document 5 is only the 3-position derivative of 2-methyl-1,4-naphthoquinone. Therefore, the anticancer activity of these drugs is considered to be related to the common structure of the 3-position derivative of 2-methyl-1,4-naphthoquinone.

Moreover, these drugs are therapeutic agents for suppressing carcinogenesis or once carcinogenesis occurs, and the treatment or prevention of liver fibrosis (cirrhosis), which is a pre-stage of carcinogenesis, has not been studied at all.

The mechanisms of action for the development of liver fibrosis and liver cancer are different. Liver cancer develops as liver fibrosis progresses, so prevention or treatment of liver fibrosis, which is the primary source of liver cancer, is very effective in preventing liver cancer. However, no drug has been developed that can specifically treat liver fibrosis (cirrhosis).

Against the background of such conventional techniques, development of new therapeutic techniques capable of treating or preventing liver fibrosis is eagerly desired.

An object of the present invention is to provide an antifibrotic agent capable of suppressing activation of hepatic stellate cells, controlling myofibroblasts, and treating or preventing liver fibrosis, cirrhosis, or liver cancer. .

The present inventors have conducted intensive studies to solve the above problems, and found that a compound having a specific chemical structure that is not a 3-position derivative of 2-methyl-1,4-naphthoquinone activates hepatic stellate cells. It was found to have properties of attenuating the expression of the marker Alpha-Smooth Muscle Actin (αSMA) and/or inducing the expression of cytoglobin (CYGB). Induction of cytoglobin expression is expected to act as a treatment for liver cirrhosis and liver cancer. Cytoglobin is a gas molecule carrier expressed in non-activated hepatic stellate cells under physiological conditions, and also functions as a scavenger of active oxygen. When liver cirrhosis occurs, cytoglobin-negative astrocytes are the main constituents, and the microenvironment becomes hypoxic, accompanied by oxidative stress and persistent inflammation, becoming the home of carcinogenesis. In addition, cancer cells grow in a hypoxic environment while producing vascular endothelial growth factor (VEGF). Therefore, expression of cytoglobin is also considered to be effective in preventing carcinogenesis and growth of cancer cells due to hypoxic environment.

Thus, the present inventors have found that an antifibrotic agent containing a specific compound as an active ingredient can attenuate αSMA expression and/or enhance cytoglobin expression, which can lead to liver fibrosis such as cirrhosis. We have found that it is effective for treatment and prevention, and eventually for treatment and prevention of hepatocellular carcinoma that develops from fibrotic tissues. The present invention has been completed through further studies based on such findings.

That is, the present invention provides inventions in the following aspects.
Section 1. An antifibrotic agent comprising, as an active ingredient, a compound represented by the following general formula (1) and/or a pharmaceutically acceptable salt thereof.

（前記一般式（１）中、Ｒ¹は、水素原子又は炭素数１～３のアルキル基であり、Ｒ²は、水素原子又は置換されていてもよい炭化水素基である。）
項２． 前記一般式（１）で表される化合物が、下記式（ａ）で表される化合物を含む、請求項１に記載の抗線維化剤。

(In general formula (1) above, R ¹ is a hydrogen atom or an alkyl group having 1 to 3 carbon atoms, and R ² is a hydrogen atom or an optionally substituted hydrocarbon group.)
Section 2. The antifibrotic agent according to claim 1, wherein the compound represented by the general formula (1) comprises a compound represented by the following formula (a).

項３． 前記一般式（１）で表される化合物が、下記式（ｂ）で表される化合物を含む、項１又は２に記載の抗線維化剤。

Item 3. Item 3. The antifibrotic agent according to item 1 or 2, wherein the compound represented by the general formula (1) includes a compound represented by the following formula (b).

項４． 肝線維化、肝硬変若しくは肝癌の予防又は治療に使用される、項１又は２に記載の抗線維化剤。

Section 4. Item 3. The antifibrotic agent according to Item 1 or 2, which is used for prevention or treatment of liver fibrosis, liver cirrhosis or liver cancer.

Since the antifibrotic agent of the present invention can suppress the activation of astrocytes and control myofibroblasts, it is effective in preventing or treating fibrosis in tissues such as the liver. It is also effective in preventing or treating diseases such as cancer caused by fibrosis.

1 is a schematic diagram showing the construction of LX-2 cells having the hCOL1A2-mCherry reporter of Experimental Example 1. FIG. 4 is a graph showing the relative fluorescence signal intensity with respect to the number of LX-2 cells in Experimental Example 1. FIG. 4 is a graph showing relative values of mCherry signals of LX-2 cells in Experimental Example 1. FIG. 1 shows micrographs and fluorescence-stained photographs of cells introduced with and without the hCOL1A2-mCherry gene of Experimental Example 1. FIG. FIG. 10 is a diagram showing screening candidate compounds for antifibrotic agents by a cell evaluation system using the hCOL1A2-mCherry reporter in LX-2 cells. 4 is a graph showing the relative values of αSMA and CYGB mRNA expression levels with respect to the concentration of each candidate compound in HHSteC cells of Experimental Example 2. FIG. 3 is a graph showing the relative expression levels of αSMA and CYGB mRNA in HHSteC cells cultured with the addition of each candidate compound of Experimental Example 3. FIG. 10 is a graph showing the relative value of the number of viable cells with respect to the concentration of each compound for HHSteC cells cultured with the addition of each compound of Experimental Example 4. FIG. 4 is a graph showing the relative values of αSMA and CYGB mRNA expression levels with respect to the concentration of each compound in HHSteC cells cultured with the addition of each compound of Experimental Example 4. FIG. 10 is a graph showing the relative expression levels of αSMA and CYGB mRNA in HHSteC cells cultured with the addition of each compound of Experimental Example 5. FIG. 10 is a graph showing the relative values of the expression levels of αSMA, CYGB, PPARγ, and COL1A1 with respect to the concentration of each compound in HHSteC cells cultured with the addition of each compound of Experimental Example 6. FIG. 10 is a graph showing the relative expression levels of αSMA, CYGB, COL1A1, and COL1A2 mRNA with respect to the concentration of each compound in HHSteC cells cultured with the addition of each compound of Experimental Example 7. FIG. 10 is a graph showing serum ALT levels versus C15 dose in each mouse model of Experimental Example 8. FIG. FIG. 10 is a micrograph of Sirius red-stained liver tissue of each mouse model in Experimental Example 8. FIG. 10 is a graph showing the hepatic fibrosis area ratio with respect to the C15 dose calculated from Sirius red staining in the liver tissue of each mouse model in Experimental Example 8. FIG. 10 is a graph showing the relative expression levels of COL1A1 and COL1A2 mRNAs with respect to C15 dose in each mouse model of Experimental Example 8. FIG.

The antifibrotic agent of the present invention is characterized by comprising the compounds represented by the above-described general formulas (1) to (7) and pharmaceutically acceptable salts thereof as active ingredients.
The antifibrotic agent of the present invention is described in detail below.

Active Ingredient The antifibrotic agent of the present invention uses a compound represented by the general formula (1) and/or a pharmaceutically acceptable salt thereof as an active ingredient. A compound represented by the general formula (1) is a compound having a 1,4-naphthoquinone skeleton. The antifibrotic agent of the present invention is described below.

In general formula (1), R ¹ is a hydrogen atom or an alkyl group having 1 to 3 carbon atoms. From the viewpoint of more efficiently attenuating the expression of αSMA, R ¹ is preferably an alkyl group having 1 to 3 carbon atoms. Specific examples of the alkyl group include a methyl group, an ethyl group, a 1-propyl group, and an isopropyl group, preferably a methyl group or an ethyl group, and more preferably a methyl group.

In general formula (1), R ² is a hydrogen atom or an optionally substituted hydrocarbon group. The optionally substituted hydrocarbon group may be a linear or branched chain hydrocarbon group, and may be a saturated hydrocarbon or unsaturated hydrocarbon group. The number of carbon atoms in the optionally substituted hydrocarbon group is, from the viewpoint of anti-fibrotic ability, for example 1 to 20, preferably 1 to 10, more preferably 1 to 5, 1 to 4, 1 to 3, 1 to 2 or 1. Examples of hydrocarbon groups having 1 to 20 carbon atoms include alkyl groups having 1 to 20 carbon atoms and alkenyl groups having 2 to 20 carbon atoms.

Examples of alkyl groups having 1 to 20 carbon atoms include methyl, ethyl, n-propyl, 1-methylethyl, n-butyl, 1-methylpropyl, 2-methylpropyl, n-pentyl, 1-methylbutyl, 2,2- dimethylpropyl, 1-ethylpropyl, n-hexyl, 1,1-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1- Ethylbutyl, 1,2,2-trimethylpropyl, n-heptyl, n-octyl, n-nonyl, n-decyl, n-undecyl, n-dodecyl, n-tridecyl, n-tetradecyl, n-pentadecyl, n- hexadecyl, n-heptadecyl, n-octadecyl, n-nonadecyl, n-icosyl and the like.

The alkenyl group having 2 to 20 carbon atoms may have at least one double bond. hexenyl, 1-heptenyl, octenyl, nonenyl, decenyl, undecenyl, dodecenyl, tridecenyl, tetradecenyl, pentadecenyl, hexadecenyl, heptadecenyl, octadecenyl, nonadecenyl, _icosenyl , -CH2CH ₌ C _{( CH3} ) ₂ , CH2CH2C ( _CH3 )= _CH2 .

When the optionally substituted hydrocarbon group is a substituted hydrocarbon group, the substituent is a group other than a hydrocarbon group, and examples thereof include a hydroxyl group, an amino group, a carboxyl group and the like, and are preferred. includes a hydroxyl group. Examples of optionally substituted hydrocarbons include _CH2CH (OH)C( _CH3 )= _CH2 .

From the viewpoint of more efficiently attenuating the expression of αSMA, R ² is preferably a hydrogen atom or an unsubstituted hydrocarbon group. Moreover, from the viewpoint of more strongly inducing the expression of cytoglobin, R ² is preferably a hydrogen atom.

Preferred examples of the compound represented by the general formula (1) include the following formula (a) (Lapachol), formula (b) (2-hydroxy-1,4-naphthoquinone: Lawsone), formula ( c) (2-hydroxy-3-methyl-1,4-naphthoquinone), formula (d) (2-methoxy-3-methyl-1,4-naphthoquinone), formula (e) (2-hydroxy-3-( 2-hydroxy-3-methyl-3-buten-1-yl)-1,4-naphthoquinone).

The compounds represented by formula (1) may be used singly or in combination of two or more.

The salt of the compound represented by the general formula (1) is not particularly limited as long as it is a pharmaceutically acceptable salt. Examples include alkali metal salts such as sodium salts and potassium salts; alkaline earth metal salts; ammonium salts; basic amino acid salts such as arginine, lysine, histidine and ornithine; and amine salts such as monoethanolamine salts and diethanolamine salts. These salts may be used individually by 1 type, and may be used in combination of 2 or more type.

Other Ingredients The antifibrotic agent of the present invention may contain other pharmacologically active ingredients in addition to the active ingredients described above, depending on the type of disease to be treated.

In addition to the active ingredient, the antifibrotic agent of the present invention contains, if necessary, pharmaceutically acceptable carriers and additives in order to prepare desired dosage forms and formulation forms. good too. Examples of such carriers and additives include diluents, excipients, binders, disintegrants, lubricants, suspending agents, solubilizers, stabilizers, sweeteners, coloring agents, flavoring agents, and flavoring agents. agents, surfactants, humectants, preservatives, pH adjusters, buffers, thickening agents and the like.

Dosage form The dosage form of the antifibrotic agent of the present invention is not particularly limited, and may be appropriately set according to the mode of administration and the like. Specific dosage forms of the antifibrotic agent of the present invention include liquid preparations such as injections, syrups, cell suspensions, and liposome preparations; tablets, hard capsules, soft capsules, granules, powders, Examples include solid formulations such as pills. In the case of an injection, it may be in the form of a powder for preparation (for example, a freeze-dried powder) to be dissolved in physiological saline or the like before use.

Administration target The antifibrotic agent of the present invention has a preventive or therapeutic effect for diseases expected to have a preventive or therapeutic effect by suppressing the activation of hepatic stellate cells in vivo, or a preventive or therapeutic effect by controlling myofibroblasts. It is applied and used for diseases where is expected.
The antifibrotic agent of the present invention is also effective in preventing or treating liver fibrosis or cirrhosis, or in preventing or treating liver cancer caused by liver fibrosis or cirrhosis.

Furthermore, the antifibrotic agent of the present invention is effective in preventing and treating not only the liver but also fibrosis in other tissues, and is also effective in preventing and treating cancers originating from fibrotic tissues. is. That is, the antifibrotic agent of the present invention can also be used for the prevention or treatment of fibrosis or cancer in tissues other than the liver. Tissues (other than liver) targeted for prevention or treatment of fibrosis with the antifibrotic agent of the present invention include, for example, tissues such as lung, breast, heart, stomach, intestine, skin, and bone marrow.
Cancers (other than liver cancer) include, for example, lung cancer, breast cancer, stomach cancer, colon cancer, tongue cancer, thyroid cancer, kidney cancer, prostate cancer, uterine cancer, ovarian cancer, osteosarcoma, chondrosarcoma, rhabdomyosarcoma, and the like. is mentioned.

In the antifibrotic agent of the present invention, the target organism for administration may be any organism in which fibrosis is suppressed and desired, and in addition to humans, rats, hamsters, guinea pigs, mice, cows, sheep, pigs, and goats. , monkeys, rabbits and other mammals.

Administration method Examples of administration forms of the antifibrotic agent of the present invention include parenteral administration such as topical administration, subcutaneous administration, intraperitoneal administration, intramuscular administration, intravenous administration, rectal administration, and intradermal administration; and may be appropriately set according to the type of disease to be applied. A preferred mode of administration of the antifibrotic agent of the present invention is oral administration.

The dosage of the antifibrotic agent of the present invention may be appropriately set according to the type of disease to be applied, the age, sex, body weight, degree of symptoms, dosage form, etc. of the subject of administration. The total amount of the compound represented by the general formula (1) and/or a salt thereof is about 500 to 5000 μg per day, and may be administered in one or several divided doses.

EXAMPLES The present invention will be described in more detail below with reference to Examples, but the present invention is not limited to these Examples. In the drawings, "*" indicates P<0.05, "**" indicates P<0.01, and "***" indicates P<0.001.

Experimental example 1
(Screening for antifibrotic substances)
In order to screen candidate compounds for anti-fibrotic substances, a lentivirus incorporating the mCherry gene downstream of the human collagen type I (COL1A2) gene promoter was prepared, and hCOL1A2-mCherry reporter was applied to human hepatic stellate cell line LX-2 cells. was introduced (Fig. 1).

The resulting recombinant LX-2 cells were transferred to a 96-well plate containing Dulbecco's modified Eagle's medium (DMEM) or DMEM medium supplemented with supplements (FGF-2 (fibroblast growth factor-2), ScienCell) at 1×. The cells were seeded at a cell density of 10 ⁴ , 2×10 ⁴ or 4×10 ⁴ cells/well and cultured at 37° C. for 72 hours. FGF-2 has a COL1A2 expression suppressing effect. After culturing, mCherry signal intensity was measured (excitation wavelength, fluorescence wavelength). FIG. 2 shows a graph representing the relative fluorescence signal intensity with respect to the cell number of LX-2 cells, and FIG. 3 shows the relative Graphs representing signal intensities are shown. From the graph in FIG. 2, the signal intensity increased as the number of cells increased, and from the graph in FIG. 3, the addition of FGF2-containing supplements with COL1A2 expression-suppressing action reduced the mCherry signal in any cell number. confirmed that the hCOL1A2 gene was correctly introduced into the LX-2 cells.

The upper part of FIG. 4 shows photomicrographs of cells introduced with the hCOL1A2-mCherry gene (hCOL1A2 pro-mCherry) and cells without introduction (no introduction), and the lower part of FIG. Fluorescence signals of cells with no introduction (hCOL1A2 pro-mCherry) and cells with no introduction (No introduction) are shown. From the photograph in FIG. 4, fluorescence signals derived from the mCherry reporter gene were detected in the cells transfected with the hCOL1A2-mCherry gene.

Next, using recombinant LX-2 cells in which the introduction of the hCOL1A2-mCherry gene was confirmed, candidate compound screening for antifibrotic substances was performed. As candidate compounds, 1880 kinds of compounds owned by industry-academia collaboration at Osaka University were used. In screening, recombinant LX-2 cells were seeded in a 96-well plate containing DMEM medium at 1×10 ⁴ cells/well and cultured at 37° C. for 24 hours. Then, the candidate compound was added to 10 μM, and cultured at 37° C. for 3 days. After culturing, the fluorescent signal of the cells was measured, and those with a supplement effect of 50% or more were selected.

FIG. 5 shows 10 hit compounds as a result of screening. Further, specific compounds are shown in Table 1 below.

Experimental example 2
(Influence of screening compounds on expression of αSMA and cytoglobin)
HHSteC cells (human hepatic stellate cells, ScienCell) were seeded at 2×10 ⁵ cells/well in a 6-well plate containing serum-free SteCM medium containing penicillin and streptomycin and cultured at 37° C. for 24 hours. Thereafter, candidate compounds C1 to C10 shown in FIG. 5 were prepared and added to 0.3 to 30 μM in serum-free DMEM medium containing 0.1% DMSO, and cultured at 37° C. for 3 days. As a control, a serum-free DMEM medium containing 0.1% DMSO without these compounds was added and cultured under the same conditions as above. After culturing, the mRNA expression levels of αSMA (α-Smooth Muscle Actin) and cytoglobin (CYGB) were measured.

The αSMA and cytoglobin mRNA expression levels were measured as follows. First, cultured HHSteC cells were collected. Next, total RNA was extracted from HHSteC cells, and expression levels of αSMA and cytoglobin mRNA were measured by (real-time) RT-PCR. Also, relative values of the expression levels of αSMA and cytoglobin when each compound was added were determined, with the mRNA expression levels of αSMA and cytoglobin in the control being set to 1. Table 2 shows the nucleotide sequences of the primers used in PCR.

FIG. 6 shows a graph showing the relative mRNA levels of αSMA and cytoglobin (CYGB) relative to the concentrations of the candidate compounds. The graph in FIG. 6 showed that αSMA activity was most suppressed and cytoglobin (CYGB) activity was most increased when C2 (Lapachol) was used.

Experimental example 3
(Effect of selected compounds on expression of αSMA and cytoglobin)
Further, among the compounds used in Experimental Example 2, C2: Lapachol and C3: Citroptene were used, and αSMA and The expression level of cytoglobin was measured. Assuming that the mRNA expression levels of αSMA and cytoglobin in the case where no candidate compound was added (S-) were 1, respectively, the relative values of the expression levels of αSMA and cytoglobin by each compound C2 and C3 were determined.

FIG. 7 shows a graph showing the relative expression levels of αSMA and cytoglobin (CYGB) under each condition. From the graph in FIG. 7, C3:citropten did not significantly suppress the expression of αSMA compared to S-, and did not significantly induce the expression of cytoglobin (CYGB). On the other hand, C2: Lapachol significantly suppressed αSMA expression and significantly induced cytoglobin (CYGB) expression compared to S-.

Experimental example 4
(Influence of 1,4-naphthoquinone compounds on expression of αSMA and cytoglobin)
Next, C2: 1,4-naphthoquinone compounds (C11 to C18) shown in Tables 3 and 4 below were prepared as analogues of Lapachol.
HHSteC cells (human hepatic stellate cells, ScienCell) were seeded at 2×10 ⁵ cells/well in a 6-well plate containing serum-free SteCM medium containing penicillin and streptomycin and cultured at 37° C. for 24 hours. Thereafter, candidate compounds (C11 to C18) were prepared and added to 0.3 to 30 μM in serum-free DMEM medium containing 0.1% DMSO, and cultured at 37° C. for 2 days. As a control, a serum-free DMEM medium containing 0.1% DMSO without these compounds was added and cultured under the same conditions as above. After culturing, the number of viable cells and the mRNA expression levels of αSMA and cytoglobin were measured.
The number of cells was measured using CCK8 (Dojindo Laboratories), and the number of cells in the control was set to 1, and the relative number of cells to which each candidate compound was added was obtained.
In addition, the αSMA and cytoglobin mRNA expression levels were measured under the same conditions as in Experimental Example 2 described above. Taking the mRNA expression levels of αSMA and cytoglobin in the control as 1, the relative values of the expression levels of αSMA and cytoglobin when each compound was added were determined.

FIG. 8 shows a graph showing the relationship between the concentration of each compound and the relative number of HHSteC cells cultured by adding each compound at that concentration. From FIG. 8, it was found that the C13 and C14 compounds decreased the number of viable cells when the concentration exceeded a certain level, and exhibited cytotoxicity when the concentration was 30 μM or higher. Other compounds showed no cytotoxicity even at a concentration of 30 μM.

FIG. 9 shows a graph showing the relationship between the concentration of each compound and the relative expression levels of αSMA and CYGB mRNA in HHSteC cells cultured with the addition of each compound at that concentration. From FIG. 9, even if any compound was added, attenuation of αSMA expression was observed when the concentration of the compound was 1 μM compared to when it was 0.3 μM. Among them, C12, C13, C15, C16, and C17 tended to attenuate αSMA expression as the concentration further increased. Among them, C12 (vitamin K2) is a compound reported to have an anticancer effect, but it was also found to have an effect of attenuating αSMA expression, ie, an antifibrotic effect. In addition, C11 (vitamin K1) is a compound that has a structure very similar to C12 and has a common structure of a 3-position derivative of 2-methyl-1,4-naphthoquinone, and, like C12, acts to attenuate αSMA expression. That is, it was found to have anti-fibrosis ability. On the other hand, the compounds of C15, C16, C17 and C18 having the structure of the general formula (1) have a characteristic structure of a 3-position derivative of 2-methyl-1,4-naphthoquinone common to C11 and C12. However, it was unexpectedly found to have an action to attenuate αSMA expression, that is, an anti-fibrotic ability. In particular, C17 had a remarkable tendency to attenuate αSMA expression even compared to C12. Therefore, from the viewpoint of the effect of attenuating αSAM expression, C15, C16 and C17, especially C17, are considered to be particularly potent antifibrotic substances with weak cytotoxicity and high antifibrotic activity.

Moreover, an increase in the expression level of cytoglobin was observed when any of the compounds was added. The expression level of cytoglobin generally tended to increase as the concentration of the compound increased. Among them, C12 (vitamin K2) is a compound reported to have an anticancer effect, but it was also found to have an effect of increasing the expression level of cytoglobin, that is, to have an antifibrotic effect. In addition, C11 (vitamin K1) is a compound having a common structure of a 3-position derivative of 2-methyl-1,4-naphthoquinone, which is very similar in structure to C12. It has been found to have an augmenting action or antifibrotic potential. On the other hand, the compounds of C15, C16, C17 and C18 having the structure of the general formula (1) have a characteristic structure of a 3-position derivative of 2-methyl-1,4-naphthoquinone common to C11 and C12. Unexpectedly, it was found to have the effect of increasing the expression level of cytoglobin, that is, the anti-fibrotic ability. In particular, C15 (Lawsone) showed a remarkable tendency to increase the expression level of cytoglobin even compared to C12. Therefore, from the viewpoint of the effect of increasing the expression level of cytoglobin, C15 (Lawsone) is considered to be a particularly potent anti-fibrotic substance with weak cytotoxicity and high anti-fibrotic activity.

Experimental example 5
(Influence of 1,4-naphthoquinone compound on expression of αSMA and cytoglobin)
Using C2: Lapachol, C15: Lawsone, and C12: Vitamin K2, the expression levels of αSMA and cytoglobin were measured under the same conditions as in Experimental Example 2 above. A control (Veh) to which these compounds were not added was also tested in the same manner as described above. Assuming that the control αSMA and cytoglobin mRNA expression levels were 1, the relative values of the αSMA and cytoglobin expression levels under each condition were determined. FIG. 10 shows a graph showing the relative expression levels of αSMA and cytoglobin (CYGB) under each condition.

Also, a graph showing the relative mRNA levels of αSMA and cytoglobin (CYGB) relative to the concentration of C2 obtained in Experimental Example 2 (Fig. 6), and the concentrations of C12 and C15 obtained in Experimental Example 4. In the graph (Fig. 9) showing the relative mRNA levels of αSMA and cytoglobin (CYGB) relative to Concentrations for enhancing up to 2-fold are summarized in Table 5 below. From FIG. 10 and Table 5, it was found that C2 has a particularly high ability to suppress αSMA expression, and that ability is exhibited at a lower concentration. It was also found that C15 has a particularly high ability to induce cytoglobin (CYGB) expression, and that ability is exhibited at a lower concentration.

Experimental example 6
(Effect of Lawsone (C15) on the expression of αSMA and cytoglobin)
HHSteC cells (human hepatic stellate cells, ScienCell) were seeded at 2×10 ⁵ cells/well in a 6-well plate containing serum-free SteCM medium containing penicillin and streptomycin and cultured at 37° C. for 24 hours. Thereafter, the compounds ICG-001 and C15 shown in Table 6 were prepared and added to 0.3 to 12 μM in serum-free DMEM medium containing 0.1% DMSO, and cultured at 37° C. for 2 days. As a control, a serum-free DMEM medium containing 0.1% DMSO without these compounds was added and cultured under the same conditions as above. After culturing, the mRNA expression levels of αSMA (α-Smooth Muscle Actin) and cytoglobin (CYGB) were measured.

The αSMA and cytoglobin mRNA expression levels were measured as follows. First, cultured HHSteC cells were collected. The mRNA expression levels of αSMA, cytoglobin, peroxisome proliferator-activated receptor γ (PPARγ), and COL1A1 were measured. Regarding the measurement of the mRNA expression levels of αSMA, cytoglobin, PPARγ, and COL1A1, αSMA and cytoglobin were measured in the same manner as in Experimental Example 2, and PPARγ and COL1A1 were measured using the primers shown in Table 7. RT-PCR was performed in the same manner as in 2. Taking the mRNA expression levels of αSMA, cytoglobin, PPARγ, and COL1A1 in the control as 1, the relative values of the mRNA expression levels of αSMA, cytoglobin, PPARγ, and COL1A1 when each compound was added were determined.

FIG. 11 shows graphs showing αSMA relative mRNA levels, cytoglobin (CYGB) relative mRNA levels, PPARγ relative mRNA levels, and COL1A1 relative mRNA levels with respect to the concentration of each compound. The graph in FIG. 11 shows that C15 exerts the αSMA expression-suppressing ability, PPARγ expression-suppressing ability, and COL1A1 expression-suppressing ability in particular at lower concentrations than ICG-001.

Experimental example 7
(Effect of Lawsone (C15) on TGFβ1 signaling)
HHSteC cells (human hepatic stellate cells, ScienCell) were seeded at 1×10 ⁵ cells/well in a 12-well plate containing serum SteCM medium containing penicillin and streptomycin and cultured at 37° C. for 24 hours. Thereafter, Lawsone (C15) was prepared and added to 3 or 10 μM in serum-free DMEM medium containing 0.1% DMSO, and 3 hours after the addition, transforming growth factor β1 (transforming growth factor β1), a cytokine that promotes fibrosis, was added. hTGFβ1) was added at 2 ng/ml and cultured at 37° C. for 2 days. As a control, culture was performed under the same conditions as above without addition of compound C15 and hTGFβ1.

After culturing, the mRNA expression levels of αSMA, cytoglobin, COL1A1 and COL1A2 were measured. The expression levels of αSMA, cytoglobin, and COL1A1 mRNA were measured in the same manner as in Experimental Example 6. COL1A2 was measured in the same manner as in Experimental Example 6, except that the primers shown in Table 8 were used. Performed by PCR. Taking the mRNA expression levels of αSMA, cytoglobin, COL1A1, and COL1A2 in the control as 1, the relative values of the mRNA expression levels of αSMA, cytoglobin, COL1A1, and COL1A2 when each of the predetermined amounts of C15 and hTGFβ1 were added were determined.

FIG. 12 shows a graph showing the relative mRNA amounts of αSMA, cytoglobin (CYGB), COL1A1, and COL1A2 with respect to the concentration of each compound. The graph in FIG. 12 showed that C15 suppressed the expression of αSMA, COL1A1 and COL1A2 promoted by hTGFβ1, and promoted the expression of cytoglobin (CYGB) suppressed by hTGFβ1.

Experimental example 8
(Evaluation of fibrosis-suppressing ability of Lawsone (C15) by hepatic fibrosis mouse model)
Eight-week-old ICR mice (7-week-old mice were acclimatized after purchase) were injected intraperitoneally with physiological saline supplemented with thioacetamide (TAA), which induces hepatic fibrotic lesions, twice a week for a total of 15 times. A liver fibrosis mouse model was prepared by intraperitoneal administration. The dose of TAA was 100 μg/g body weight for the 1st time, 200 μg/g body weight for the 2nd to 5th times, 300 μg/g body weight for the 6th to 9th times, and 400 μg/g body weight for the 10th to 15th times. From the 13th administration of TAA, Lawsone (C15) in 10% DMSO/90% olive oil was administered orally four times in 8 days in parallel. A single dose of Lawsone was 10 or 100 mg/kg body weight. As a control, it was orally administered to a liver fibrosis mouse model under the same conditions except that Lawsone (C15) was not included (0 mg/kg body weight (VEH)). Further, instead of TAA, except that CNT (mixed solvent of 1 v / v% DMSO and 99 v / v% corn oil) was used, intraperitoneal administration was performed under the same conditions, and a single dose of Lawsone was 100 mg / kg. Oral administration models were also prepared under the same conditions except that body weight or 0 mg/kg body weight (VEH) was used.

Twenty-four hours after the final administration, blood was collected from the mice, and serum ALT levels were measured as hepatocellular injury markers. A graph depicting serum ALT levels versus C15 dose is shown in FIG.

In addition, the liver was excised and the hepatic fibrosis area ratio was measured by Sirius red staining. FIG. 14 shows a photomicrograph of liver tissue stained with Sirius red (Collagen I is stained). In FIG. 14, the upper photographs represent hematoxylin-eosin staining, and the lower photographs represent Sirius Red staining. FIG. 15 shows a graph showing the ratio of hepatic fibrosis area to C15 dose calculated from Sirius red staining.

Furthermore, the expression levels of COL1A1 and COL1A2 mRNA in liver tissue were measured. The expression levels of mouse COL1A1 and mouse COL1A2 mRNAs were measured in the same manner as in Experimental Example 6, except that the primers shown in Table 9 were used. Taking the mRNA expression levels of αSMA, cytoglobin, COL1A1, and COL1A2 in the control (0 mg/kg of C15 with CNT added) as 1, the relative values of the mRNA expression levels of αSMA, cytoglobin, COL1A1, and COL1A2 when C15 was added were calculated. asked. FIG. 16 shows a graph showing the relative expression levels of COL1A1 and COL1A2 mRNAs with respect to C15 dosage.

From FIGS. 13 to 16, it was examined whether Lawsone (C15) exerts fibrosis-suppressing ability in liver fibrosis mouse model (method of stepwise administration of thioacetamide). A significant decrease in markers (serum ALT value), a significant decrease in fibrosis area ratio (Sirus red staining area), and a significant decrease in COL1A1 and COL1A2 expression were observed. In other words, it was proved that Lawsone (C15) has an anti-fibrotic effect at the biological level. In addition, liver fibrosis was not induced in the CNT administration group, and serum ALT level, liver fibrosis area ratio, and COL1A1 and COL1A2 expression before administration of C15 (0 mg / kg) and after administration (100 mg / kg) no change was seen.

Experimental example 9
Using epoxy beads (trade name FG beads ^(R) , Tamagawa Seiki Co., Ltd.), Lawsone (C15) was chemically reacted with the epoxy beads according to the protocol of the product to prepare Lawsone-magnetic beads. One million HHSteC cells were lysed with Lysis Buffer (Promega), centrifuged at 10,000 g, and the supernatant (protein solution) was recovered. A protein solution corresponding to 1 mg of protein and 0.1 mg of Lawsone-magnetic beads were reacted in a 150 mM KCl solution at 4° C. overnight. After washing the Lawsone-magnetic beads after the reaction, proteins adsorbed to the Lawsone-magnetic beads were extracted using a sample buffer for western blotting. Extracted proteins were separated by SDS-PAGE and visualized by silver staining. After silver staining, a band specifically observed in the Lawsone-magnetic bead-derived extract was isolated and subjected to mass spectrometry analysis. As a result, the protein that specifically bound to Lawsone (C15) was identified as HSD17B4, which has never been reported to bind to vitamin Ks.

SEQ ID NO: 1 is the forward primer for αSMA.
SEQ ID NO: 2 is the reverse primer for αSMA.
SEQ ID NO:3 is the forward primer for cytoglobin.
SEQ ID NO: 4 is a reverse primer for cytoglobin.
SEQ ID NO:5 is the forward primer for PPARγ.
SEQ ID NO:6 is the reverse primer for PPARγ.
SEQ ID NO:7 is the forward primer for COL1A1.
SEQ ID NO: 8 is the reverse primer for COL1A1.
SEQ ID NO:9 is the forward primer for COL1A2.
SEQ ID NO: 10 is the reverse primer for COL1A2.
SEQ ID NO: 11 is the forward primer for mouse COL1A1.
SEQ ID NO: 12 is the reverse primer for mouse COL1A1.
SEQ ID NO: 13 is the forward primer for mouse COL1A2.
SEQ ID NO: 14 is the reverse primer for mouse COL1A2.

Claims (5)

An αSMA activity inhibitor and/or cytoglobin activity enhancer containing a compound represented by the following formula (a) and/or a pharmaceutically acceptable salt thereof as an active ingredient (however, when used for antifibrosis and liver except when used for cancer).

An anti-fibrotic agent (except when used for liver cancer) containing a compound represented by the following formula (b) and/or a pharmaceutically acceptable salt thereof as an active ingredient.

3. The antifibrotic agent according to claim 2, which is used for prevention or treatment of liver fibrosis or liver cirrhosis. An antifibrotic agent comprising, as an active ingredient, a compound represented by the following formulas (c), (d) and/or (e) and/or a pharmaceutically acceptable salt thereof.

5. The antifibrotic agent according to claim 4, which is used for prevention or treatment of liver fibrosis, liver cirrhosis or liver cancer.

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