KR20090090432A - Pharmaceutical composition for treatment of liver cancer comprising chenodeoxycholic derivative - Google Patents

Abstract

A pharmaceutical composition for treating liver cancer is provided to suppress the proliferation of hepatocelluar carcinoma cell and induce apoptosis. A pharmaceutical composition for treating hepatocellular carcinoma of the chemical formula 1 comprises a chenodeoxycholic acid derivative of the chemical formual 1. The chenodeoxycholic acid derivative suppresses the proliferation of hepatocellular carcinoma and induces apoptosis. The pharmaceutical composition further comprises a carrier, excipient or diluents. A health food for treating hepatocellular carcinoma comprises the chenodeoxycholic acid derivative of the chemical formula 1 as an active ingredient.

Description

Pharmaceutical composition for treatment of liver cancer comprising chenodeoxycholic derivative}

The present invention relates to a pharmaceutical composition for the treatment of liver cancer containing a kenodeoxycholic acid derivative effective as an active ingredient in inhibiting cell proliferation and inducing apoptosis to prevent and treat liver cancer.

Hepatocellular carcinoma (HCC) continues to increase in terms of incidence and mortality, with an incidence of 80% in countries located in Asia and sub-Saharan Africa.

Despite the recent development of diagnostic methods, it is very important to develop effective cancer prophylaxis due to the limited treatment and poor prognosis of HCC, and thus the importance of developing more effective strategies for chemotherapy of liver cancer is highlighted.

Bile acids are polar derivatives of cholesterol that are essential for the absorption of dietary fat and regulate the transcription of genes that control cholesterol homeostasis. Depending on the nature of this chemical structure, several bile acids have distinct physiological effects.

These bile acids are synthesized in the liver and secreted into the bile ducts and digestive tracts, followed by metabolism of primary bile acids such as choline acid and chenodeoxycholic acid (CDCA) by enterobacteriaceae to deoxycholic acid and ursodeoxycholine. Produces secondary bile acids, such as acids and lithocholine acids.

Secondary bile acids form conjugates with glycine or taurine before secreted into bile. These inclusions are also intended to promote fat absorption in the intestine, but are more meaningful for detoxifying bile acids.

On the other hand, apoptosis or predetermined cell death plays an important role in the development of anticancer drugs and cancer treatment.

The cancer suppressor gene egr-1 encodes a transcription factor with a sequence specific DNA binding domain near the c terminus. egr-1 belongs to the immediate early growth response gene family, such as c-fos and c-jun, which make up the AP-1 transcription factor complex.

Many genes such as fibroblast growth factor, platelet-derived growth factor A, multidrug resistance gene MDR1, tumor suppressor gene p53, retinoblastoma susceptibility gene Rb, etc., are regulated by Egr-1.

Egr-1 protein is involved in cellular processes including cell proliferation, cell differentiation and apoptosis. Egr-1 responds instantly and temporarily to environmental signals, including growth factors or cytokines, mechanical stress, hypoxia and ionizing radiation.

In addition, egr-1 plays a regulatory role with a group of immediate early growth response genes encoding subunits of c-fos, c-jun and the transcription factor AP-1. Since c-fos and c-jun are induced by DAN damage, AP-1 is known to be involved in cellular responses induced by DNA damage.

The present inventors have completed the present invention by discovering that cell proliferation is inhibited and apoptosis is induced by treating a henodeoxycholine acid derivative represented by the formula (1) to a liver cancer cell line.

Accordingly, an object of the present invention is to provide a pharmaceutical composition for treating liver cancer, which contains a drug capable of inducing apoptosis and inhibiting the growth of liver cancer cells as an active ingredient.

In order to achieve the above object, the present invention provides a pharmaceutical composition for treating liver cancer containing a kenodeoxycholine acid derivative represented by the formula (1) as an active ingredient:

[Formula 1]

The kenodeoxycholic acid derivatives inhibit proliferation of liver cancer cells; Induces apoptosis of liver cancer cells such as changes in nuclear shape, formation of apoptotic bodies and DNA fragmentation; Alter the cell cycle; Induces p53, a tumor suppressor protein, and also induces p27 depending on the regulation of p21 ^{WAF1 / C1P1} and p53 that is transcriptionally activated by p53; The ratio of Bax: Bcl-2, which is the ratio of pre-apoptosis and anti-apoptosis, is increased; Inhibits expression of COX-2; Since it induces the expression of Egr-1, it is very effective in preventing and treating liver cancer by inhibiting liver cancer cell proliferation and inducing apoptosis.

The kenodeoxycholine acid derivative is preferably contained at 20 μM-100 μM, a concentration that specifically inhibits the proliferation of liver cancer cells.

At this time, if the kenodeoxycholic acid derivative is contained in more than the above content range may cause problems such as liver dysfunction, increased serum cholesterol and diarrhea in the body, if less contained causes problems that are not effective in inhibiting the growth of liver cancer cells Can be.

 The amount and method of applying the pharmaceutical composition for treating liver cancer according to the present invention may vary depending on the formulation and the purpose of use.

In addition, the pharmaceutical composition for treating liver cancer of the present invention may further include a suitable carrier, excipient or diluent commonly used in the preparation of the pharmaceutical composition.

Carriers, excipients or diluents that may be included in the pharmaceutical composition for treating liver cancer of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium Phosphate, calcium silicate, cellulose, methyl cellulose, microcrystalline cellulose, polyvinyl pyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil.

The pharmaceutical composition for treating liver cancer of the present invention is formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, external preparations, suppositories, and sterile injectable solutions, respectively, according to conventional methods. Can be used.

The pharmaceutical composition for treating liver cancer of the present invention may vary depending on the age, sex and weight of the patient, but may be administered once to several times daily in an amount of 50 to 900 mg / kg.

In addition, the dosage of the pharmaceutical composition may be increased or decreased depending on the route of administration, the severity of the disease, sex, weight, age, and the like. Therefore, the above dosage does not limit the scope of the present invention in any aspect.

The pharmaceutical composition for treating liver cancer of the present invention can be administered to mammals such as mice, mice, livestock, humans, and the like by various routes. All modes of administration can be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intrauterine dural or intracerebroventricular injection.

The kenodeoxycholic acid derivative represented by Formula 1, which is an active ingredient of the pharmaceutical composition for treating liver cancer of the present invention, has a 50% lethal dose (LD50) of 5 g / kg or more and is a safe compound.

In addition, the present invention provides a health food for improving liver cancer containing the kenodeoxycholine acid derivative represented by the formula (1) as an active ingredient.

The health food is used with other foods or food additives in addition to such kenodeoxycholic acid derivatives, and may be suitably used according to conventional methods. The mixed amount of the active ingredient can be suitably determined depending on the purpose of use thereof, for example, prophylactic, health or therapeutic treatment.

The effective dose of the kenodeoxycholic acid derivatives contained in the health food can be used in accordance with the effective dose of the pharmaceutical composition, but in the case of prolonged intake for health and hygiene purposes or health control purposes, It may be less than the above range, it is clear that the active ingredient can be used in an amount above the above range because there is no problem in terms of safety.

There is no particular limitation on the kind of the health food, for example, meat, sausage, bread, chocolate, candy, snacks, confectionery, pizza, ramen, other noodles, gums, dairy products including ice cream, various soups, drinks, tea, Drinks, alcoholic drinks, vitamin complexes, etc. are mentioned.

The pharmaceutical composition for treating liver cancer according to the present invention can be very useful for preventing and treating liver cancer by containing Kenodeoxycholic acid represented by Formula 1 as an active ingredient that inhibits proliferation of liver cancer and induces apoptosis. .

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for the purpose of clarifying the contents of the present invention, and the present invention is not limited by the examples.

Example 1 Cell Culture and MTT Analysis

Human liver cancer cell line HepG2 is purchased from American Type Culture Collection (ATCC) and contains 10% (v / v) heat-inactivated fetal bovine serum (FBS), 2 mM glutamine, 100 U / mL penicillin and 100 μg / ml streptomycin. Cultured in DMEM (Dulbecco's Modified Eagle Medium, Gibco BRL) containing. At this time, the culture conditions were incubated at a temperature of 37 ℃ under humidified conditions of 5% CO ₂ and 95% air.

The compound of formula 1 (hereinafter referred to as HS-1200) was synthesized according to a conventionally known method (Cancer Lett., 163: 83-93, 2001), dissolved in ethanol (absolute) and stocked at a concentration of 10 mg / ml. Prepared as a solution and stored at -20 ° C.

[Formula 1]

The final concentration of ethanol in the medium was within 0.1% (50-150 μM) to prevent any effect on cell growth. Cell proliferation was performed using the MTT assay in which MTT [3- (4,5-dimethylthiazol-2-yl) -2,5-diphenyl tetrazolium bromide, Sigma] was converted to MTT-formazan by the mitochondrial enzyme. At this time, HS-1200 was pretreated for 24 hours.

As a result, as shown in Figure 1 HS-1200 inhibited the growth of HepG2 cells in a concentration-dependent manner.

Example 2 Nuclear Staining Analysis (Hockest Staining)

Hoechst staining was performed to examine whether inhibition of cell proliferation by HS-1200 is associated with apoptosis induction.

HepG2 cells prepared as in Example 1 were washed with PBS and fixed with 3.7% paraformaldehyde (Sigma Chemical Co.) dissolved in PBS for 10 minutes at room temperature. Fixed cells were washed with PBS and stained for 20 minutes at room temperature using 4 μg / ml hoist 33342. Again washed with PBS twice and analyzed by fluorescence microscope.

As a result, as shown in Fig. 2a, no change in nuclear structure could be observed in the control group, whereas in the experimental group treated with HS-1200, apoptosis characteristics such as formation of nuclei with a chromatin condensation and apoptosis bodies were observed in a concentration-dependent manner. Could.

Example 3 DNA Fragmentation Assay

DNA fragmentation, which is another hallmark of apoptosis, was analyzed.

After 24 hours of HS-1200 treatment, HepG2 cells were washed twice with cold PBS, using lysis buffer [5 mM Tris-HCl (pH 7.5), 5 mM ethylenediaminetetraacetic acid, 0.5% tryptone X-100]. And resuspended at 4 ° C. for 30 minutes.

After centrifugation at 27,000xg for 15 minutes, the supernatant was treated with RNase, followed by protease K digestion, phenol / chloroform / isoamyl alcohol (25: 24: 1, v / v / v) extraction and isopropanol precipitation It was.

DNA was separated through 1.5% agarose gel, stained with 0.1 μg / ml ethidium bromide (EtBr, Sigma) and visualized using ultraviolet light.

As a result, a typical ladder pattern of internucleosome DNA fragmentation was observed by HS-1200 treatment as shown in FIG. 2B. Thus, it was determined that HS-1200 induces apoptosis cell death.

Example 4 Cell Cycle Analysis

HepG2 cells pretreated with HS-1200 for 24 hours were treated with trypsin, washed with PBS and fixed at 4 ° C. for 30 minutes with 75% ethanol. Prior to analysis, cells were washed again with PBS, suspended in cold propidium iodide (PI, Sigma) solution and incubated in the dark for 30 minutes at room temperature.

Flow cytometry analysis was performed using a FACScan flow cytometry system (Becton Dickinson).

As a result, continuous accumulation of G0 / G1 cycle and apoptotic cells can be observed as shown in FIG. 3B, and apoptotic cells can be detected in a concentration-dependent manner as shown in FIG. 3A.

Example 5 Western Blot Analysis

After collecting and separating HepG2 cells prepared as in Example 1, protein concentration was quantified according to the method described by the manufacturer using Bio Rad Protein Assay (BioRad Lab.).

Homogenized whole cell extract from HepG2 cells treated with HS-1200 for 24 hours was gel loaded buffer [125 mM Tris-HCl, 4% sodium dodecyl sulfate (SDS), 10% 2-mercaptoethanol and 0.2% Bromophenol blue, pH 6.8] and the ratio was 1: 1 and boiled for 5 minutes (Cancer Letters, 229: 49-57, 2005).

That is, the same amount of protein was electrophoresed on a 10% SDS-polyacrylamide gel and transferred to a nitrocellulose membrane (Schleicher & Schuell) by electroblot. Blots were obtained from Santa Cruz Biotechnology Inc. primary antibodies such as p53 (SC-126), p21 (SC-817), p27 (SC-1641), cyclin D1 (SC-246), cdk2 (SC -260), Cyclin A (SC-239), E2F-1 (SC-193), Bax (SC-493), Bcl-2 (SC-509), COX-2 (SC-19999), Egr-1 ( Probe for 1 hour with an antibody against SC-110, incubated with diluted enzyme-binding secondary antibody, peroxidase-labeled donkey anti-rabbit immunoglobulin and peroxidase-labeled sheep anti-mouse immunoglobulin, It was visualized by enhanced chemiluminescence according to Amersham Corp.).

The primary antibodies were purchased from Santa Cruz Biotechnology Inc., and the secondary antibodies peroxidase-labeled donkey anti-rabbit immunoglobulin and peroxidase-labeled sheep anti-mouse immunoglobulin were purchased from Amersham.

As a result, expression of the tumor suppressor protein p53 was induced at 25 and 50 μM (see FIG. 4A), and cell cycle regulators Cycline A, Cycline D1, cdk2 and E2F-1 were concentration-dependently inhibited (see FIG. 4B). p21, which is dependent on the regulation of p21 ^{WAF1 / C1P1} and p53, which is transcriptionally activated by p53, was induced in HepG2 cells treated 24 hours with 50 μM HS-1200 (see FIG. 4A).

To elucidate the apoptosis mechanism in HepG2 cells initiated by HS-1200, the apoptosis protein in p53 signaling was examined. The Bcl-2 family proteins, including the pre-apoptosis group and the anti-apoptosis group, play an important role in p53 dependent apoptosis, whereas the pre-apoptosis protein Bax rises markedly after HS-1200 treatment, whereas the anti-apoptosis protein As Bcl-2 was decreased, it was confirmed that HS-1200 changed the Bax: Bcl-2 ratio in HepG2 cells (see FIG. 4C).

In addition, COX-2 protein expression was significantly reduced as shown in Figure 5b, Egr-1 protein expression was induced as shown in Figure 6b.

Example 6 RT-PCR Analysis

Total RNA was isolated according to previously known methods (Cancer Lett., 199: 157-167, 2003). Single stranded cDNA was synthesized from 2 μg of total RNA using M-MLV reverse transcriptase (Gibco-BRL).

mRNA was amplified by PCR using SEQ ID NOs: 1 and 2, primer pairs for COX-2, SEQ ID NOs: 3 and 4, primer pairs for EGR-1, and SEQ ID NOs: 5 and 6, primer pairs for GAPDH.

PCR reactions were 1 × (3 min at 94 ° C.); 35 X (45 sec at 94 ° C., 45 sec at 58 ° C., 1 min at 72 ° C.); And 1 × (10 min at 72 ° C.), the amplification products obtained by PCR were separated by electrophoresis on 1% agarose gel and visualized by EtBr staining.

As a result, COX-2 mRNA expression was significantly reduced as shown in Figure 5a, Egr-1 mRNA expression was induced in 30 minutes after HS-1200 treatment as shown in Figure 6a.

Example 7 Egr-1 siRNA Transfection

Control-non-targeting siRNA (Cat No. D-001210-01-05) and Egr-1 siRNA were purchased from Dharmacon Research. The sequence of the siRNA double stranded target Egr-1 is shown in SEQ ID NOs: 7 and 8.

HepG2 cells were aliquoted into 6 well plates containing antibiotic-free medium for 24 hours prior to transfection and transfected at 70% confluence. Transfection was treated with 4 μl of Lipofectamine 200 (Invitrogen) using Egr-1 siRNA 80 nM mixed in serum depleted OPTI-MEM (Invitrogen).

Six hours after transfection, the medium was exchanged with DMEM containing 10% FBS without antibiotics. After 24 hours transfection, cells were treated with HS-1200 for 4 hours and protein analyzed.

As a result, as shown in FIG. 7, Egr-1 siRNA completely silenced the Egr-1 gene and significantly inhibited the expression of p53, p21 ^{WAF1 / C1P1} and p27. In addition, silencing of the Egr-1 gene also inhibited the expression of COX-2.

Example 8 Acute Toxicity Test

HS-1200 was suspended in 0.5% methylcellulose solution and injected once intravenously into 6 week old SPF-based SD rats (3 per group) at a dose of 1.0 g / kg / 15 mL.

The mortality, clinical symptoms, and weight changes of the animals were examined, and hematological and blood biochemical tests were performed. The necropsy was performed to observe the abdominal and thoracic organ abnormalities.

As a result, no significant clinical symptoms or dead animals were noted in all animals treated with HS-1200, and no toxic changes were observed in weight changes, blood tests, blood biochemical tests, and autopsy findings.

Hereinafter, examples of pharmaceutical compositions and health foods according to the present invention will be described.

< Formulation example  1> Powder  Produce

Powder was prepared by mixing 50 mg of HS-1200, 100 mg of lactose and 10 mg of talc and filling into an airtight bag.

< Formulation example  2> Preparation of Tablet

Tablets were prepared by mixing 50 mg of HS-1200, 100 mg of corn starch, 100 mg of lactose, and 2 mg of magnesium stearate, followed by compression according to a conventional method for preparing tablets.

< Formulation example  3> Preparation of capsule

50 mg of HS-1200, 100 mg of corn starch, 100 mg of lactose and 2 mg of magnesium stearate were mixed according to a conventional capsule preparation method, and filled into gelatin capsules to prepare capsules.

< Formulation example  4> Preparation of Injection

50 mg of HS-1200, a sterile distilled water dose and a pH adjuster dose for injection were prepared in the amounts of the above components per ampoule (2 mL) according to a conventional injection method.

< Formulation example  5> Liquid  Produce

200 mg of HS-1200, 10 g of isomerized sugar, 5 g of mannitol and an appropriate amount of purified water were dissolved in purified water according to a conventional method for preparing a liquid, and lemon flavor was added appropriately. The solution was prepared by sterilization by adjusting the total amount to 100 ml and then filling the brown bottle.

Preparation Example 6 Preparation of Health Food

HS-1200 20 mg, vitamin mixture proper amount (70 μg of vitamin A acetate, 1.0 mg of vitamin E, 0.13 mg of vitamin B, 0.15 mg of vitamin B 2, 0.5 mg of vitamin B 6, 0.2 μg of vitamin B 12, vitamin C 10 mg, 10 μg biotin, 1.7 mg nicotinic acid amide, 50 μg folic acid, 0.5 mg calcium pantothenate and an appropriate amount of mineral mixture (1.75 mg ferrous sulfate, 0.82 mg zinc oxide, 25.3 mg magnesium carbonate, 15 mg potassium monophosphate, diphosphate 55 mg of calcium, 90 mg of potassium citrate, 100 mg of calcium carbonate, 24.8 mg of magnesium chloride) were mixed, and then granules were prepared and health food was prepared according to a conventional method.

Figure 1 shows the growth inhibitory activity of liver cancer cells by HS-1200 treatment,

Figure 2a is a fluorescence picture showing the nuclear structural modification of liver cancer cells according to HS-1200 treatment,

Figure 2b shows the DNA fragmentation pattern of liver cancer cells according to HS-1200 treatment,

Figure 3a is an analysis of apoptotic cells in liver cancer cells treated with HS-1200,

Figure 3b shows the cell cycle change in liver cancer cells treated with HS-1200,

Figure 4a shows the protein expression patterns of p53, p21 and p27 in liver cancer cells treated with HS-1200,

Figure 4b shows the expression pattern of the cell cycle regulatory protein in liver cancer cells treated with HS-1200,

Figure 4c shows the protein expression patterns of Bax and Bcl-2 in liver cancer cells treated with HS-1200,

5A and 5B show expression patterns of COX-2 mRNA and COX-2 protein in liver cancer cells treated with HS-1200, respectively.

6A and 6B show expression patterns of Egr-1 mRNA and Egr-1 protein in liver cancer cells treated with HS-1200, respectively.

Figure 7 shows the expression pattern of the protein according to Egr-1 siRNA treatment in liver cancer cells treated with HS-1200.

<110> Pusan National University Industry-University Cooperation Foundation <120> Pharmaceutical composition for treatment of liver cancer          comprising chenodeoxycholic derivative <130> dp-ul-2008-0005 <160> 6 <170> KopatentIn 1.71 <210> 1 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> sense primer for COX-1 <400> 1 tgcccagctc ctggcccgcc gctt 24 <210> 2 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for COX-1 <400> 2 gtgcatcaac acaggcgcct cttc 24 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> sense primer for EGR-1 <400> 3 ccctaagctg gaggagatga tg 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for EGR-1 <400> 4 tcatgctcac taggccactg ac 22 <210> 5 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> sense primer for GAPDH <400> 5 cggagtcaac ggatttggtc gtat 24 <210> 6 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> antisense primer for GAPDH <400> 6 agccttctcc atggtggtga agac 24  

Claims (4)

A pharmaceutical composition for treating liver cancer containing a kenodeoxycholine acid derivative represented by the following Chemical Formula 1 as an active ingredient: [Formula 1]

The pharmaceutical composition for treating liver cancer according to claim 1, wherein the kenodeoxycholine acid derivative inhibits liver cancer cell proliferation and induces apoptosis. The pharmaceutical composition for treating liver cancer according to claim 1 or 2, wherein the kenodeoxycholine acid derivative is contained at 20 μM-100 μM, which is a concentration that specifically inhibits the proliferation of liver cancer cells. Health foods for improving liver cancer containing a kenodeoxycholine acid derivative represented by the following formula (1) as an active ingredient: [Formula 1]

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