KR20120061700A - Composition for preventing or treating human breast cancer, comprising talosin A - Google Patents

Abstract

PURPOSE: A talosin A isolated from Actinomyces kitasatospora kifunensis strain and an anti-cancer agent using the same are provided to suppress breast cancer cells and to treat breast cancer with less toxicity. CONSTITUTION: A composition for suppressing breast cancer proliferation and tumor growth contains talocin A of chemical formula 1 or pharmaceutically acceptable salt thereof as an active ingredient. The composition is used for preventing and treating breast cancer. The compound of chemical formula 1 is used in the form of pharmaceutically acceptable salt. The salt is addition salt formed by pharmaceutically acceptable base. The base includes inorganic base or organic base.

Description

Composition for preventing or treating human breast cancer, comprising talosin A}

The present invention inhibits the proliferation of breast cancer cells and effectively suppresses the size of tumors in breast cancer model animals, thereby containing thalosine A or a pharmaceutically acceptable salt thereof, which is useful for the treatment of breast cancer because it is excellent in treating breast cancer and has low toxicity. It relates to a composition to be.

Representative cancer treatments of modern medicine include surgical surgery, biotherapy, radiation therapy, and chemotherapy with chemotherapy. Chemotherapy is a method of inhibiting the proliferation of cancer cells by administering oral or injection of anticancer drugs. The advantage of chemotherapy is that the drug can reach cancer and treat metastatic cancer in any part of the body. Chemotherapy is used as standard therapy in treating metastatic cancer. Of course, chemotherapy can not cure cancer, but it plays an important role in relieving symptoms and extending lifespan. To date, the most widely used anticancer agent among natural anticancer drugs is taxol, which is used to treat breast and ovarian cancers. However, these chemotherapeutic agents have problems such as side effects, anticancer drug resistance, and despite many studies on cancer until now, due to the diversity of the cancer itself and the diversification of the pathogenesis mechanism, the side effects are small and can overcome the resistance. No cancer drugs have been developed.

Currently, the development of cancer prevention substances as a new concept of cancer conquest strategy is urgently required, and the development of such cancer prevention substances can be very useful for inhibiting or treating all diseases as well as cancer. For this reason, as the development of cancer prevention substances is recognized as a new direction of cancer research and attention is focused, the development of cancer prevention substance materials is active not only in the real medicine field but also in the food chemistry field, so the research on natural product materials is active. The situation is made of, and is actually introduced in the food field.

Examples of active substances related to the prevention of reported cancers include β-carotene (Suda, Carcinogenesis, 7, pp711-715, 1986), and ceselin type coumarin (Nishino, Carcinogenesis, 11, pp1557-). 1561, 1990), curcumin (Rao, Carcinogenesis, 14, pp2219-2225, 1993), isolated from Curcuma longa, myristicin (myr) from parsley (Zheng, Carcinogenesis, 13) , pp1921-1923, 1992) and epigallocatechin-3-gallic acid of green tea (Katiyar, Nutrition and Cancer, 18, pp73-73, 1992). In addition, according to the International Reports of Medicinal Chemistry, 19 out of 31 newly approved anticancer drugs from 1983 to 1994, about 61% of drugs were developed separately from natural products or were synthetic compounds derived from semisynthetic derivatives or natural models. Among the 93 anticancer drugs that were developed and used until 1994, six drugs from biological sources such as interferon and interleukin-2 were found, paclitaxol, vincristine and vinbla. 15 drugs from natural sources such as vinblastin, doxorubicin, 25 semisynthetic derivatives of natural products such as etoposide, irinotecan hydrochloride, cisplatin, 33 pure synthetics like amsacrine, cytosine arabinoside, fluorouracil Such as synthetics, but it was the 14 kinds of drugs due to the natural model.

Therefore, the present inventors have completed the present invention by inhibiting the proliferation of breast cancer cells and progressing to the study of Talosin A, which has excellent anticancer effect and low toxicity by inhibiting tumor size in a breast cancer model animal and developing a therapeutic agent using the same. .

It is an object of the present invention to inhibit the proliferation of breast cancer cells, to suppress the tumor size in breast cancer model animals, and because it has excellent anticancer effect and low toxicity, it can be useful for treating breast cancer. To provide its use.

In order to achieve the above object, the present invention provides a composition for inhibiting the growth of breast cancer cells and growth inhibition of tumors, containing as the active ingredient Talosin A or a pharmaceutically acceptable salt thereof represented by the formula (1).

[Formula 1]

The compound represented by Chemical Formula 1 may be used in the form of a pharmaceutically acceptable salt, and as the salt, an addition salt formed by a pharmaceutically acceptable base is useful. As the base, an inorganic base containing an alkali metal or the like may be used, or an organic base such as an amine having a high basicity may be used. Salts in which inorganic bases are used include addition salts such as sodium salts, potassium salts, calcium salts, magnesium salts, and the like. Is an addition salt such as ethanolamine salt, propanolamine salt, ammonium salt, or general tetraalkylamine salt.

The composition of the present invention can be usefully used for the prevention or treatment of breast cancer.

Meanwhile, the composition for inhibiting breast cancer cell proliferation and tumor growth inhibition of the present invention may be administered in the form of oral delivery, parenteral delivery or parenteral insertion.

The composition of the present invention is preferably administered parenterally.

Talosin A according to the present invention can be administered systemically or topically, the administration can be oral or parenteral administration and can be used in the form of a general pharmaceutical formulation. That is, the compound of formula 1 of the present invention may be administered in various dosage forms, orally and parenterally (preferably as an injection), in actual clinical administration, and when formulated, commonly used fillers, extenders, binders, wetting agents. And diluents or excipients such as disintegrants and surfactants. Solid form preparations for oral administration include tablets, pills, powders, granules, capsules, and the like, which form at least one excipient such as starch, calcium carbonate , Sucrose or lactose, gelatin, etc. Liquid preparations for oral administration include suspensions, solutions, emulsions, and syrups, and may include various excipients, such as wetting agents, sweeteners, fragrances, and preservatives, in addition to commonly used simple diluents such as water and liquid paraffin. have. Formulations for parenteral administration include sterile aqueous solutions, non-aqueous solvents, suspensions, emulsions and the like. As the non-aqueous solvent and suspending agent, propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate and the like can be used.

An effective dose of the compound of formula 1 of the present invention may be appropriately selected in consideration of sex, age, patient's condition, etc., but is generally 10 to 1000 mg / day, preferably 20 to 500 mg / day to an adult, It can be administered in divided doses 1 to 3 times a day.

Since talosin A inhibits the proliferation of breast cancer cells and effectively suppresses the tumor size in breast cancer model animals, it is excellent in treating breast cancer and is less toxic. Therefore, the composition containing talosin A or a pharmaceutically acceptable salt thereof is used for breast cancer. It can be useful for prevention and treatment.

1 shows the result of purified HPLC analysis of thalosin A.
Figure 2 shows the results of measuring the breast cancer cell proliferation inhibitory activity of Experimental Example 1.
Figure 3 shows the breast cancer preventive effect of thalosin A as a result of Experimental Example 2.
Figure 4 shows the effect of treatment of breast cancer of thalosin A as a result of Experimental Example 2.

Hereinafter, the present invention will be described in more detail with reference to Examples.

However, the following examples are illustrative of the present invention, and the content of the present invention is not limited by the examples.

< Manufacturing example  1> Preparation of Talosin A

<1-1> Talosin A Production

1 vial of conserved talosin A-producing strain (Kitasatospora chifunansis KCCM-10710P) spore fluid was thawed at room temperature, inoculated into a 500 ml Erlenmeyer flask containing 100 ml of the seed medium, and then in a 30 ° C. shaker incubator. Primary seed culture was performed for 24 hours at 250 rpm. After completion of the primary seed culture, 3 ml of the culture solution was inoculated into a 500 ml Erlenmeyer flask containing 100 ml of the secondary seed culture medium, followed by secondary seed culture for 24 hours at 250 rpm in a 30 ° C shake incubator. 4 L of the main culture medium was prepared, filled into a 7 L fermentor, sterilized at 121 ° C. for 20 minutes, and cooled at room temperature (see Table 1). The culture temperature was adjusted to 30 ° C., the aeration rate was 0.5 VVM, and the stirring speed was operated at 500 rpm. 200 ml of the secondary seed culture was inoculated into each fermenter and incubated for 6 days. After the incubation in the 7 L fermentor was completed, 3.1 to 3.5 L of the culture solution was recovered per fermenter, and the culture supernatant was recovered by centrifugation at 7,000 rpm for 20 minutes to remove the cells, and stored in a 4 ° C. refrigerator.

Composition of medium for production of talosin A Spawn culture (1st, 2nd) Fermenter Main Culture Glucose 20 g Glucose 20 g Soluble starch 20 g Soluble starch 50 g Soybean meal 10 g Soybean meal 35 g Yeast extract 2 g Yeast extract 5 g K ₂ HPO ₄ 0.5 g K ₂ HPO ₄ 10 g NaCl 2 g NaCl 2 g CaCO ₃ 1 g CaCO ₃ 4 g Distilled water 1,000 ml Distilled water 1,000 ml SAG (defoamer) 0.5 g

<1-2> Talosin A Tablet

The culture supernatant was passed through HP-20 resin for about 12 hours to attach talosin A material, followed by 5 l (25 ml flow rate) with 25% methanol, 5 l with 50% methanol, and then 1 l with 100% methanol. For elution of delosin A, 7 L of a solvent in which methanol and acetone were mixed at a 3: 1 ratio was poured. TLC analysis of each eluted fraction confirmed the presence of thalosin A substance, and recovered only the thalosin A active fraction. TLC analysis conditions were methylene chloride: methanol = 10: 1. After HP-20 chromatography, silica gel chromatography was performed to facilitate the removal of impurities. The active fractions recovered from HP-20 chromatography were concentrated under reduced pressure, all solvents were removed and a small amount of methanol and 20 g of silica gel were mixed and ground in a mortar and pestle. It was loaded into a 400 mL capacity silica gel resin and poured into methylene chloride + methanol (20: 1) solvent (4 mL / min). After doubling the volume of the column, methylene chloride + methanol (10: 1) solvent was aliquoted in 50 ml volumes with twice the column volume at the same flow rate. Each preparative sample was confirmed by TLC analysis and a sample containing talosin A was recovered separately. High-purity talosin A was recovered through a delosin A crystallization process using temperature variables and solubility difference.

The purified Talosin A purified product was crystallized while reducing the volume in a reduced pressure concentrator controlled at 45 ℃. Purity of Talosin A was about 90% or more by HPLC analysis (see FIG. 1).

< Experimental Example  1> Measurement of proliferation inhibitory activity of breast cancer cells

The estrogen-independent mouse breast cancer cell line MDA-MB-231 cell line was distributed from the Korea Cell Line Bank and passaged at intervals of 3 to 4 days in a 100 mm cell culture dish. DMEM with 10% bovine serum (fetal bovine serum) was used as a culture medium, and cultured in a 37 ° C., 5% CO ₂ incubator. Genistein and thalosin A were added to the cell culture to 0, 10, 20, 40 and 80 μM and then cultured for 7 days to evaluate the effect on cell proliferation. The effect of test substance on cell proliferation was evaluated by DNA analysis according to the method of West et al. (1985). After the culture was removed, the solution was washed twice with a washing solution, and then stored in 2.7 ml of cell disruption solution for 30 minutes to induce cell disruption. After cell disruption, the pH was corrected to 7, and Hoechest-33258 staining solution was added to each well, and then 200 μl of the supernatant was transferred to a black 96-well plate, followed by DNA spectrometry (excitation, 350 nm; emission 455 nm). Was measured. The results are shown in FIG.

< Experimental Example  2> Measurement of breast cancer growth inhibition in breast cancer model animals

<2-1> Experimental Animals and Breeding Environment

Five-week-old BALB / c mouse females were purchased from Orient Bio and used in the experiment after one week of acclimation. The obtained animals were separated into five cages and purified and tested at a temperature of 20-25 ° C., a relative humidity of 40-60%, and an illumination time of 12 hours (07:00 lit-19: 00 off). Feed and water were freely ingested. At this time, AIN-93G, a phytoestrogens-free diet, was used to minimize the effects of phytoestrogens.

<2-2> Breast Cancer Induction and Tumor Size Measurement

The experiment was conducted by dividing 30 mice into 8 groups, and the number of experimental animals was set to 5 mice per group. To investigate breast cancer preventive effects of talosin A, DMSO, genistein (750 μg / g) and talosin A (750 μg / g and -Δ-, 1500 μg / g) in AIN-93G feed without phytoestrogens ) Was fed 5 days before cancer cell transplantation. To investigate the cancer treatment effect of talosin A, DMSO, genistein (750 μg / g) and talosin A (750 μg / g and 1500 μg / g) were added to AIN-93G feed without phytoestrogens. Feeding was given 5 weeks after cancer cell transplantation. Breast cancer was induced by inoculating MDA-MB-231 tumor cells in 4 regions of the trunk with 106 cells of athymic mice in all experimental groups.

All animals were examined once a day at regular intervals for changes in general symptoms, death, and death. Tumor length was measured using a digital vernier caliper, and tumor size was calculated using the formula length / 2 × width / 2 × 3.14. The results are shown in FIGS. 3 and 4.

Claims (4)

A composition for inhibiting breast cancer cell proliferation and growth inhibition of tumors, which comprises thalosine A represented by Formula 1 or a pharmaceutically acceptable salt thereof as an active ingredient.
[Formula 1]

The method according to claim 1,
The composition is a composition for inhibiting breast cancer cell proliferation and growth inhibition of tumors used for the prevention and treatment of breast cancer. The method according to claim 1,
The composition is a composition for inhibiting breast cancer cell proliferation and tumor growth inhibition administered in the form of oral delivery, parenteral delivery or parenteral insertion. The method according to claim 1,
The composition is a composition for inhibiting breast cancer cell proliferation and growth inhibition of the tumor administered in the form of an injection.

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