KR20050092568A - Asiatic acid derivatives for the therapeutical treatment of hepatic fibrosis and liver cirrhosis - Google Patents

Abstract

The present invention relates to a therapeutic agent for liver fibrosis and cirrhosis containing a derivative of asiatic acid, specifically, to a therapeutic agent for liver fibrosis and cirrhosis comprising an asiatic acid derivative represented by the following Chemical Formula 1, a pharmaceutically acceptable salt thereof, or an ester thereof It is about.

In Formula 1, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are as defined in the detailed description of the invention, respectively.

Description

Asian acid derivatives for the therapeutical treatment of hepatic fibrosis and liver cirrhosis

The present invention relates to a therapeutic agent for liver fibrosis and cirrhosis containing a derivative of asiatic acid, specifically, to a therapeutic agent for liver fibrosis and cirrhosis comprising an asiatic acid derivative represented by the following Chemical Formula 1, a pharmaceutically acceptable salt thereof, or an ester thereof It is about.

[Formula 1]

In Formula 1, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently a hydrogen atom, an oxo group, a C _{1 to} C ₆ lower alkyl group, a benzyl group, a hydroxy group, C _{1 to} C ₆ And a lower alkoxy group, a benzyloxy group and a C _{1 to} C ₆ lower alkylcarbonyloxy group, X is selected from a hydrogen atom, an oxygen atom and a sulfur atom.

The liver is a biological tissue that plays a pivotal role in metabolism in and out of the body. Continued damage to liver tissue caused by various causes, such as chronic drinking, binge drinking, viral hepatitis, and abuse of drugs, leads to chronic liver disease, liver fibrosis and cirrhosis. Liver fibrosis is found in late stages without pain or subjective symptoms, resulting in high mortality and social problems. In Korea, liver disease patients, represented by cirrhosis, have been steadily increasing since the late 70's. In particular, Korea is the number one cause of death among men in their 40s [Statistics Annual Report, Cause of Death, 2001].

 Liver cirrhosis is caused by fibrosis of liver tissue. Liver fibrosis is a condition in which the balance between the synthesis and degradation of connective tissue is lost. It is caused by excessive accumulation of connective tissue in liver tissue and is accompanied by necrosis or inflammation. In particular, hepatic stellate cells (HSCs), which play a role in the storage of vitamin A under normal liver function in the liver, metastasize to myofibroblast form by rapid chronic liver damage and rapidly proliferate into collagen. It is known to advance the process of fibrosis of the liver by increasing the production of extracellular matrix such as proteoglycan, hyaluronan, and the like, and generating excess connective tissues [Friedman et al., Proc. Natl. Acad. Sci. USA, 82: 8681 (1985); Gressner et al., Biochem. Biophys. Res. Commun., 151: 222 (1988); Gressner et al., J. Hepatol., 22: 28 (1995). Proyl-4-hydroxylase (P4H) is known to act as a key enzyme in the synthesis of collagen, a representative component of fibrotic connective tissues, which is an important process in fibrosis [Prockop and Kivirikko, Annu. Rev. Biochem., 64: 403 (1995)]. Therefore, the development of a drug for inhibiting liver fibrosis or development of a liver cirrhosis treatment agent can inhibit P4H, a collagen synthesis enzyme, or a drug capable of inhibiting excessive production of connective tissues represented by collagen of hepatic stellate cells or inhibiting the proliferation of these cells. Although the focus is on development, there is still no development of effective therapeutics.

Asiatic acid and its trisaccharide, Asiaticoside, is a substance isolated from Centella asiatica, first isolated by Bontems in 1941 [Bontems, Bull. Sci. Pharmacol., 49: 186 (1941)], the structure was determined by Polonsky [Polonsky, Compt. Rend., 232: 1878 (1951); Polonsky, Bull. Soc. Chim., 173-80 (1953)]. Extracts of Centella asiatica, including asiatic acid and asiaticosides, have long been used to treat skin modifications caused by cutaneous wounds, chronic ulcers, tuberculosis or leprosy [Boiteau et al., Bull. Soc. Chim., 31: 46-51 (1949)], have been reported to have an effect on dementia and cognitive impairment [European Patent Publication No. 383,171; Jew S et al., Bioorg. Med. Chem. Lett., 10: 119 (2000)].

Recently, the same international drug has been filed in the United States and Korea as these compounds exhibit hepatocellular protective effect [Korean Patent Publication No. 1998-42830; US Patent No. 6,274,559]. However, according to the experimental results introduced in these documents, although there are no hepatic fibrotic or medicinal effects for the treatment of liver cirrhosis, studies on liver fibrosis therapy are required. Therefore, in the present invention, the hepatic stellate cells that play a key role in the liver fibrosis process to clarify the effect of inhibiting the liver fibrosis of asiatic acid derivatives.

The inventors of the present invention have repeatedly studied studies on collagen synthesis, inhibition of cell proliferation, expression of genes related to liver fibrosis using hepatic stellate cells, which are the main cells of hepatic fibrosis, with respect to the derivatives of Asiatic acid having the structure of Chemical Formula 1, The present invention has been found to be useful for inhibiting and treating liver fibrosis.

It is an object of the present invention to provide a liver fibrosis and liver cirrhosis therapeutic agent comprising the asiatic acid derivative of Formula 1 as an active ingredient.

The present invention relates to the use in the treatment of liver fibrosis and cirrhosis, comprising the asiatic acid derivative represented by the following formula (1), a pharmaceutically acceptable salt or ester thereof.

[Formula 1]

In Formula 1, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently a hydrogen atom, an oxo group, a C _{1 to} C ₆ lower alkyl group, a benzyl group, a hydroxy group, C _{1 to} C ₆ And a lower alkoxy group, a benzyloxy group and a C _{1 to} C ₆ lower alkylcarbonyloxy group, X is selected from a hydrogen atom, an oxygen atom and a sulfur atom.

In the compound represented by Formula 1 according to the present invention, preferably, R ₁ and R ₂ are independently a hydrogen atom, an oxo group, a hydroxy group, a C ₁ -C ₃ lower alkoxy group, a benzyloxy group, and C _1- A C ₃ lower alkylcarbonyloxy group; R ₃ and R ₄ are independently selected from oxo group, hydroxyl group, C ₁ -C ₃ lower alkoxy group and C ₁ -C ₃ lower alkylcarbonyloxy group; R ₅ is selected from a hydrogen atom, a hydroxy group, a C ₁ -C ₃ lower alkoxy group and a C ₁ -C ₄ lower alkylcarbonyloxy group; R ₆ is selected from a hydrogen atom and a C _{1 to} C ₃ lower alkyl group; X is the case of compounds which are hydrogen or oxygen atoms.

In the compound represented by Formula 1 according to the present invention, the most preferable compounds are specifically expressed as follows:

Methyl 2α, 3β, 23-trihydroxyurse-12-ene-28-oate,

2α, 3β, 23-triacetyloxyurse-12-ene-28-oxyic acid,

Methyl 2α-methyloxy-3β, 23-diacetyloxyurse-12-ene-28-oate,

3β, 23-diacetyloxyurse-2-oxo-12-ene-28-oxyic acid,

3β-hydroxy-23-trimethylacetyloxyurse-12-ene-28-aux acid,

Methyl 2α-methyloxyurse-3-one-12-ene-23-al-28-oate,

Methyl 2α, 3β, 23-trimethyloxyurse-12-ene-28-oate,

2α, 3β, 23-triacetyloxyurse-11-oxo-12-ene-28-aux acid,

2α, 3β, 23-trihydroxyurse-11-oxo-12-ene-28-oxy acid,

Methyl 2α-benzyloxy-3β, 23-diacetyloxyurth-12-ene-28-oate.

The compound represented by Chemical Formula 1 according to the present invention is a known compound already known, and a preparation method thereof is also known [Korean Patent Registration No. 69547].

However, the present invention is characterized by the use of the compound represented by the formula (1) in the treatment of liver fibrosis and cirrhosis.

In administering the compound represented by Formula 1 as a medicament, a suitable method, for example, oral, oral, sublingual, rectal, intravaginal, intranasal, topical or parenteral (intravenous, intracavernous, intramuscular, subcutaneous and Administration), including in vitro.

In the case of oral, oral or sublingual administration of the compound represented by Formula 1 for the purpose of treating or preventing liver fibrosis and cirrhosis, the dosage is generally 0.1 to 400 mg based on an adult patient having an average weight of 70 kg. / Day. Thus, in general adult patients, a single dose or several doses, once or several times a day, may be formulated into tablets or capsules containing pharmaceutically acceptable excipients or carriers to contain 0.05-200 mg of the active compound. Is taken in one dose.

In formulations for parenteral administration, the effective compound is administered in an amount of 0.01 to 100 mg, in practice the physician determines the most appropriate dosage for each patient depending on the age, weight and characteristics of the patient. The above dosages exemplify the average case and may be high or low depending on individual differences, which is within the scope of the present invention.

In human application, the compound represented by Formula 1 may be administered alone, but may be generally administered in admixture with a pharmaceutical carrier selected in consideration of the mode of administration and standard phamaceutical practice. For example, the compounds may be in the form of tablets containing starch or lactose, in the form of capsules or ovules containing alone or excipients, or elixirs or suspensions containing chemicals that flavor or color. Oral, oral or sublingual.

Such liquid preparations may be suspending agents (e.g., semisynthetic glycerides such as methylcellulose, witepsol or mixtures of apricot kernel oil and PEG-6 esters or PEG-8 and caprylic / capric Pharmaceutically acceptable additives such as glyceride mixtures such as mixtures of glycerides). It can also be injected parenterally, for example, intravenously, intracavernosally, intramuscularly, subcutaneously and intratracheally. It is also most preferred to use the compound as a sterile aqueous solution for parenteral administration, in which the solution is combined with other substances (e.g. salt or mannitol, glucose) in order to have isotonicity with the blood. Same monosaccharides).

Therefore, the medicament of the present invention is a compound represented by the formula (1) or pharmaceutically acceptable salts thereof, or esters thereof as an active ingredient, it contains a pharmaceutically acceptable diluent or excipient.

Hereinafter, the specific configuration and operation of the present invention will be described with reference to Examples and Formulation Preparation Examples, but the scope of the present invention is not limited only to the following Examples.

Example 1 Inhibitory Effects of Cell Proliferation on Activated Hepatic Stellate Cells

As liver fibrosis progresses, the proliferation of activated hepatic stellate cells is promoted and the synthesis of constituents of extracellular matrix by these cells is activated. Therefore, the effect of inhibiting cirrhosis can be confirmed by observing the effect of inhibiting cell proliferation on hepatic stellate cells. .

HSC-T6, an activated hepatic stellate cell line in rats, was sold from Friedman, MD (Mount Sinai Medical Center, NY).

HSC-T6 cells were cultured in 96-well plates for 24 hours using DMEM (Dulbeccos modified Eagles Medium) with 5% Fetal bovine serum (FBS) as a culture medium, and then changed to fetal calf-free medium. Next, asiatic acid derivatives were treated at various concentrations. After 24 hours of drug treatment, the cell proliferation rate was measured by MTS measurement using Promega's MTS solution kit (Cell Titer 96 non-radioactive cell proliferation assay kit). That is, after the MTS solution was added and reacted for 90 minutes at 37 ℃ and the absorbance was measured at 490 nm, the results are shown in FIG.

As shown in Figure 1, most of the derivatives of Asiatic acid having a structure of the formula (1) characterized by the present invention showed the inhibitory activity of the growth of hepatic stellate cells in a concentration-dependent manner.

Example 2 Inhibitory Effect of Asian Acid Derivatives on Collagen Synthesis in Hepatic Stellate Cells

Collagen is an important component of liver connective tissue. However, as described above, excessive amounts of collagen accumulate in liver tissue when liver fibrosis is induced or when liver fibrosis progresses to cirrhosis. In this process, activated hepatic stellate cells are known to be responsible for excessive collagen accumulation. Therefore, in this example, the effect of the collagen in the activated hepatic stellate cells of asiatic acid derivatives was examined.

In this example, the collagen synthesis inhibitory effect was measured by the following method. The effect of the derivatives of asiatic acid on collagen synthesis by quantifying the amount of hydroxyproline, an amino acid component that appears specifically in collagen in HSC-T6 cells identical to the cell line used in Example 1, accounting for about 10% of collagen Learned.

HSC-T6 cells were incubated for 24 hours in Petridish, and then, asiatic acid derivatives were treated at various concentrations for 24 hours. The cells were collected and ground by ultrasonication three times for 20 seconds to prepare a cell homogenate, and 12N hydrochloric acid was added to the homogenate to make a final 6N hydrochloric acid solution. After acid hydrolysis at 110 ° C. for 18 hours, the solution was evaporated to dryness. 1.2 mL of 50% isopropanol solution and 0.2 mL of 5.6 mM chloramine-T solution were added and reacted at room temperature for 10 minutes. Then, 1 mL of Ehrlich reaction was developed for 30 minutes at 60 ° C., and the absorbance at 558 nm was measured. Measured. The measurement result is shown in FIG.

As shown in FIG. 2, the derivatives of asiatic acid differed depending on their chemical structure, but most of the compounds inhibited collagen synthesis of hepatic stellate cells by reducing the amount of hydroxyproline in hepatic stellate cells. Among these compounds, especially compounds such as zlx-i-85, zlx-i-87, zlx-i-89, zlx-i-92 inhibited collagen synthesis ability of activated hepatic stellate cells by about 50%. In addition, when the hydroxyl groups of R ₁ , R ₂ and R ₃ of the compound represented by the formula (1) is substituted, the collagen synthesis inhibitory effect was high, the substitution of the carboxy group of R ₄ did not significantly affect the activity. The above results show that Asian acid derivatives can be used to treat these diseases by inhibiting the accumulation of excess collagen produced during liver fibrosis or cirrhosis.

Example 3 Effects of Asian Acid Derivatives on Hepatic Fibrosis-related Gene Expression

Expression of genes involved in collagen synthesis and degradation was confirmed using Reverse Transcription-Polymerase Chain Polymerization (RT-PCR).

The cell line used HSC-T6 cells, the same hepatic stellate cell lines of Example 1 above. Silibinin was treated as a positive control, and asiatic acid derivatives were treated at concentrations that showed no cytotoxicity. Subsequent to the subunits and subunits of procollagen, TGF β-1, TIMP-1, TIMP-2, MMP-2 and P4H, genes involved in collagen synthesis and degradation, 24 hours after the treatment of asiatic acid derivatives in HSC-T6 cells MRNA expression level was measured by RT-PCR.

Easy blue solution (Intron) was used for the isolation of total RNA, and the first cDNA was synthesized from 2 mg of total RNA using oligo-dT primer and M-MLV reverse transcriptase (Promega). The resulting PCR product was quantified using Bio-Imeager's analysis software (TINA 2.0) based on the amount of β-actin, and the results are shown in FIGS. 3A and 3B.

As shown in Figures 3a and 3b, most of the Asian acid derivatives treated in this example inhibited the mRNA expression of TIMP-1 and TIMP-2, which are inhibitors of collagen degrading enzymes, and also important enzymes in the collagen production process. The expression of phosphorus P4H was reduced. These results indicate that Asian acid derivatives increase the expression of substances at the gene level that inhibit collagen, which is excessively produced during the progression of hepatic fibrosis or cirrhosis, and increase collagenase activity. Therefore, it has been confirmed that the asiatic acid derivatives can be very useful in treating liver fibrosis or cirrhosis.

Formulation Preparation Example 1: Tablet

2.5 mg active ingredient

Lactose BP 151.0 mg

Starch BP 30.0 mg

Pregelatinized Corn Starch BP 15.0 mg

The active ingredient is sieved and mixed with lactose, starch and pregelatinized corn starch, then a suitable volume of purified water is added and granulated into a powder. The granules were dried, mixed with magnesium stearate, and compressed to obtain tablets.

Formulation Preparation Example 2 Capsule

2.5 mg active ingredient

Starch 1500 96.5 mg

Magnesium Stearate BP 1.0 mg

The active ingredient was sieved and mixed with excipients and then filled into gelatin capsules to obtain capsules.

Formulation Preparation Example 3: Injection

Active ingredient 800 g / mL

Dilute hydrochloric acid BP until pH 3.5

Injectable sodium chloride BP up to 1 mL

The active ingredient was dissolved in an appropriate volume of sodium chloride BP for injection, and the pH of the resulting solution was adjusted to pH 3.5 with dilute hydrochloric acid BP, followed by volume control and mixing thoroughly with sodium chloride BP for injection. The solution was filled into a 5 ml Type 1 ampoule of clear glass, encapsulated under an upper grid of air by dissolving the glass, and then autoclaved at 120 ° C. for at least 15 minutes to obtain an injection.

As described above, the amino acid derivatives of Formula 1 inhibit the proliferation of activated hepatic stellate cells, which are the main cells of liver fibrosis, inhibit the collagen synthesis ability of these cells, and excessively during the progression of liver fibrosis or cirrhosis. By inhibiting the resulting collagen at the gene level and collagen degradation also increases at the gene level it can prevent the progression of liver fibrosis or liver fibrosis. Therefore, the pharmaceutical composition containing an asiatic acid derivative can be effectively used for the prevention and treatment of liver fibrosis and cirrhosis.

1 is a graph showing the effect of inhibiting cell proliferation of asiatic acid derivatives on activated hepatic stellate cells (HSC-T6 cells) of rats.

Figure 2 is a graph showing the effect of hydroxyproline production by hepatic stellate cells of asiatic acid derivatives.

Figure 3a shows the effect on the expression of hepatic fibrosis-related genes in HSC-T6 cells by asiatic acid derivatives as RT-PCR product agarose gel electrophoresis band.

Figure 3b is a quantitative analysis of the RT-PCR product shown in Figure 3a using the analysis software using the amount of β-actin (actin) as a reference material.

Claims (3)

The agent for preventing and treating liver fibrosis or cirrhosis, characterized in that it comprises asiatic acid derivative represented by the following formula (1) and a pharmaceutically acceptable salt thereof or ester thereof: [Formula 1] In Formula 1, R ₁ , R ₂ , R ₃ , R ₄ , R ₅ and R ₆ are each independently a hydrogen atom, an oxo group, a C _{1 to} C ₆ lower alkyl group, a benzyl group, a hydroxy group, C _{1 to} C ₆ And a lower alkoxy group, a benzyloxy group and a C _{1 to} C ₆ lower alkylcarbonyloxy group, X is selected from a hydrogen atom, an oxygen atom and a sulfur atom. The compound of claim 1, wherein R ₁ and R ₂ are independently selected from a hydrogen atom, an oxo group, a hydroxyl group, a C ₁ -C ₃ lower alkoxy group, a benzyloxy group, and a C ₁ -C ₃ lower alkylcarbonyloxy group; R ₃ and R ₄ are independently selected from oxo group, hydroxyl group, C ₁ -C ₃ lower alkoxy group and C ₁ -C ₃ lower alkylcarbonyloxy group; R ₅ is selected from a hydrogen atom, a hydroxy group, a C ₁ -C ₃ lower alkoxy group and a C ₁ -C ₄ lower alkylcarbonyloxy group; R ₆ is selected from a hydrogen atom and a C _{1 to} C ₃ lower alkyl group; X is a hydrogen atom or an oxygen atom, liver fibrosis or liver cirrhosis prevention and treatment agent. According to claim 1, wherein the compound represented by the formula (1) Methyl 2α, 3β, 23-trihydroxyurse-12-ene-28-oate, 2α, 3β, 23-triacetyloxyurse-12-ene-28-oxyic acid, Methyl 2α-methyloxy-3β, 23-diacetyloxyurse-12-ene-28-oate, 3β, 23-diacetyloxyurse-2-oxo-12-ene-28-oxyic acid, 3β-hydroxy-23-trimethylacetyloxyurse-12-ene-28-aux acid, Methyl 2α-methyloxyurse-3-one-12-ene-23-al-28-oate, Methyl 2α, 3β, 23-trimethyloxyurse-12-ene-28-oate, 2α, 3β, 23-triacetyloxyurse-11-oxo-12-ene-28-aux acid, 2α, 3β, 23-trihydroxyurse-11-oxo-12-ene-28-aux acid, and A agent for preventing and treating liver fibrosis or cirrhosis, characterized in that it is selected from methyl 2α-benzyloxy-3β, 23-diacetyloxyurth-12-ene-28-oate.