KR100925516B1 - Anticancer composition containing zinc and L-arginine - Google Patents

Abstract

The present invention relates to an anticancer composition comprising zinc and L-arginine as active ingredients in a pharmaceutically effective amount together with a pharmaceutically acceptable carrier or diluent.

Anticancer agent, zinc, L-arginine, apoptosis

Description

Anticancer Composition Comprising Zinc And L-Arginine

Figure 1 shows the effect of the anticancer composition of the present invention on human ovarian adenocarcinoma SKOV-3 as a cell viability through the MTT assay.

Figure 2 shows the effect of the anticancer composition of the present invention on human brain glioblastoma U87 by cell viability through the MTT assay.

Figure 3 shows the effect of the composition containing L- arginine and L- aspartic acid on human ovarian adenocarcinoma SKOV-3 by cell viability through the MTT assay.

Figure 4 shows the effect of the composition containing L- arginine on human ovarian adenocarcinoma SKOV-3 in cell viability through the MTT assay.

The present invention relates to an anticancer composition comprising zinc (Zinc) and L-arginine (L-Arginine). More specifically, the present invention relates to a composition for anticancer, which is useful for treating various types of cancer by containing pharmaceutically effective amount of zinc and L-arginine as active ingredients and a pharmaceutically acceptable carrier or diluent.

Advances in chemotherapy have improved the survival and healing rates of cancer patients, but serious damage to normal cells has been a major problem due to the strong toxicity of anticancer drugs. In order to prevent the side effects of these anticancer drugs, a substance that can specifically inhibit the proliferation of cancer cells is required. Accordingly, many anticancer drugs have been developed. Such anticancer agents include compounds described in WO 96/40142, WO 97/40142, WO 97/13771, WO 95/23141 and the like. However, these compounds can specifically inhibit the proliferation of cancer cells, but because they are chemically synthesized, they can cause many side effects in the human body.

Therefore, it was intended to develop an anticancer agent that can exhibit an excellent anticancer effect while reducing side effects, and for this reason, many anticancer agents with natural ingredients are currently being developed.

The present inventors also studied to develop an anticancer agent made of natural ingredients, and zinc protects normal cells more than cancer cells and induces the death of cancer cells, and when mixed with L-arginine, which is known to have anticancer effects, The effect was found to be high. Accordingly, the present inventors confirmed that the anti-cancer composition containing zinc and L-arginine was prepared and treated to cancer cells, thereby inducing apoptosis and killing cancer cells.

Accordingly, the present invention provides an anticancer composition comprising zinc and L-arginine as active ingredients in a pharmaceutically effective amount together with a pharmaceutically acceptable carrier or diluent.

As used herein, the term "pharmaceutically effective amount" means an amount of the active ingredient which has an improvement or therapeutic effect on the cancer to which the composition of the present invention is applied.

The term "pharmaceutically acceptable carrier or diluent" refers to the transport of the active ingredient from one organ or part of the body to another organ or part of the body and is a liquid or solid filler, diluent, excipient or By pharmaceutically acceptable substance, composition or vehicle such as solvent.

The anticancer composition of the present invention is characterized by containing zinc and L-arginine as active ingredients in a pharmaceutically effective amount together with a pharmaceutically acceptable carrier or diluent. Specifically, the anticancer composition of the present invention comprises 0.001 to 0.025% by weight of zinc and 0.004 to 0.075% by weight of L-arginine, preferably 0.0025 to 0.012% by weight of zinc and 0.0075 to 0.037% by weight of L-. Arginine is mixed and contained together with a pharmaceutically acceptable carrier or diluent. Therefore, the total content of the active ingredient in the anticancer composition of the present invention is 0.005 to 0.1% by weight, preferably 0.01 to 0.05% by weight.

In the present invention, zinc is not limited thereto, but zinc chloride, zinc sulfate, zinc acetate, and the like may be used, and zinc chloride may be preferably used.

In addition, the anticancer composition of the present invention may use a base such as sodium hydroxide, potassium hydroxide, sodium phosphate and potassium phosphate to adjust the pH to 6 to 8.

Hereinafter, it will be described as a representative epithelial ovarian cancer among the many types of cancer to which the anticancer composition of the present invention can be applied. However, one of ordinary skill in the art will see the effects of the anticancer composition of the present invention on epithelial ovarian cancer described herein by way of example, and recognize that the same applies to other types of cancer.

Epithelial ovarian cancer is the most common type of ovarian cancer as well as the leading cause of malignancy in gynecology. The highest mortality rates in patients with epithelial ovarian cancer are due to the fact that most patients are diagnosed at an advanced stage of cancer due to the latent progression of epithelial ovarian cancer (Ozols, RF, Semin. Oncol., Vol. 22, pp 61-66, (1995)). Although ovarian cancer is treated by surgical removal of the tumor or by aggressive chemotherapy, survival of ovarian cancer patients is only 20-30% due to drug resistance.

To date, little is known about the risk factors and causes of epithelial ovarian cancer. However, many studies have shown that there is a common relationship between the frequency of ovulation and the development of epithelial ovarian cancer, which suggests that young and older women (early menopause and late menopause), Increase in unmarried and unborn women (Franceshi et al., Int. J. Cancer, vol. 49, pp57-60, (1991); Taylor et al., Cancer, vol. 12, p1207-, (1959); Fraumeni et al., J. Natl. Cancer Inst., Vol. 42, p455-, (1969); Dorn et al., Public Health Monogr. 56, PHS Publication No. 590, (1959); Weiss et al., J. Natl. Cancer Inst., vol. 58, p913-, (1977); (Nergri et al., Int. J. Cancer, vol. 49, pp 50-56, (1991)). Oral contraceptives are generally known to have a protective effect against the development of epithelial ovarian cancer (Stanford, JL, Contraception 43, pp543-556, (1991); Negri et al., Int. J. Cancer, vol. 49, pp 50-56, (1991); Franceshi et al. , Int. J. Cancer, vol. 49, pp 61-65, (1991)) A potent theory explaining the development of epithelial ovarian cancer is that the ovarian epithelial surface divides and recovers repeatedly during ovulation (Fathalla, MF, Lancet., Vol. 2, p163-, (1971)) In the process of restoring the epithelial surface, the modified epithelial cells spontaneously mutate, the tumor suppressor genes are inactivated, and the oncogenes are caused by carcinogens. Is easy to be activated.

On the other hand, as a known semen function and anticancer effect, human semen is known to protect the seminal plasma components of sperm after sexual intercourse due to cytotoxicity of lymphocytes (Stities et al., Nature, vol. 253, pp727-729, (1975); James et al., Immunol., Vol. 6, pp61-70, (1985)), have been reported to inhibit the development of humoral immunity and tumor growth in vivo. (Anderson et al., Immunol., Vol. 128, pp535-539, (1985); Michaelis et al., Anticancer Drugs, vol. 11, pp369-376, (2000)), and also in cervical epithelial carcinoma cells Since it affects the production of metalloproteinases (MMP) -2 and MMP-9 mRNA, it is known that semen during sexual activity can affect cervical cancer progression (Jeremias et al., Am J. Obstet.Gynecol., Vol. 181, pp 591-595, (1999). Recently, it has been shown that ribonuclease (BS-RNase) among bovine semen is capable of inducing apoptosis depending on the time and concentration of exposure to human lymphocytes and human tumor cells. BN-RNase is a substance that shows high efficacy against neuroblastoma (NB) cells resistant to chemotherapeutic drugs (Cinatl et al., Anticancer Res., Vol. 20, pp853-859, (2000); Cinatl et. al., Int. J. Oncol., vol 15, pp 1001-1009, (1999)).

In addition, the ecological study of anti-cancer effects of semen, Gjorgov conducted a variety of case-control studies to investigate the correlation between reduced exposure of human semen and the development of breast cancer. For example, we compared the risk of breast cancer in women who used a block contraceptive method (condom) versus women who used a non-blocking method (contraceptives, intra uterine device (IUD), rhythm or fallopian ligation). In contrast, women using blocking contraceptive methods (condoms) were 5.2 times more likely to develop breast cancer (Gjorgov et al., Folia Med., Vol. 40, pp 17-23, (1998)).

The main components of semen exhibiting the anticancer effects shown above include albumin, lactoferrin, transferring factors, immunoglobulins, acid phosphatase, L-carnitine, and L-arginine. , L-Histidine, citric acid, fructose, fructose, magnesium, zinc, prostaglandin and glycerophosphocholine.

As mentioned above, from the prior knowledge of the anticancer effects of epithelial ovarian cancer and semen, we have found that in epithelial ovarian cancer, reduced exposure to human semen during ovulation is a pathological risk factor for the progression of epithelial ovarian cancer. I decided it would be one of Thus, human semen components may contribute to the removal of malignant transformed epithelial cells.

In this regard, the present inventors studied human semen components affecting cancer cells in vitro, and as a result, zinc protects normal cells more than cancer cells and induces death of cancer cells. In the conventional cancer treatment, it was found that the anticancer effect is increased when it is mixed with L-arginine, which is known to delay the growth and metastasis of tumors and cancer cells by promoting the anticancer activity of cytotoxic T cells and natural killer cells. Accordingly, the present inventors prepared anticancer compositions containing zinc and L-arginine and treated them with human ovarian adenocarcinoma SKOV-3 and human glioblastoma U87, resulting in high cell death killing cancer cells. Confirmed. In other words, as a result of confirming the cancer cell lines of SKOV-3 and U87 as an anticancer effect, the anticancer composition of the present invention containing zinc and L-arginine as active ingredients induces cell death to reduce cell viability of cancer cells. 1 and 2, and as a comparative experiment, a composition containing L-arginine and L-Aspartic acid, or a composition containing L-arginine, whose anticancer effects are known. Treatment with the cancer cell line did not effectively reduce the cell viability of cancer cells (see FIGS. 3 and 4).

In connection with the above results, the anticancer composition of the present invention may be useful for treating various kinds of cancers including but not limited to: bladder, breast, intestine, kidney, liver, lung (including small cell lung cancer) Carcinomas such as, brain, esophagus, gallbladder, ovary, pancreas, stomach, cervix, thyroid, prostate, and skin (including squamous cell carcinoma); Hematopoietic tumors of the lymphatic system, including leukemia, acute lymphocytic leukemia, acute lymphocytic leukemia, B-cell lymphoma, T-cell lymphoma, Hodgkin's lymphoma, non-Hodgkin's lymphoma, hairy cell lymphoma and Burkitt's lymphoma; Hematopoietic tumors of the myeloid line, including acute and chronic myeloid leukemia, myelodysplastic syndrome and promyelocytic leukemia; Tumors of mesenchymal origin, including fibrosarcoma and rhabdomyosarcoma; Tumors of the central and peripheral nervous system, including astrocytoma, neuroblastoma, glioma and schwannoma; And other tumors including melanoma, normal carcinoma, teratoma, osteosarcoma, pigmentosa, keratinous blastoma, thyroid follicular cancer and Kaposi's sarcoma.

In a preferred embodiment, the anticancer composition of the present invention is useful for suppressing or treating lung cancer, brain cancer, breast cancer, colon cancer or epithelial ovarian cancer, and particularly preferably for suppressing or treating epithelial ovarian cancer.

The anticancer composition of the present invention can be used alone, but radiation therapy or chemotherapy (cell growth arrest or cytotoxic substance, antibiotic-type substance, alkylating agent, anti-metabolic substance, hormonal agent, immune agent, interferon-type substance, cycloox Cigenase inhibitors (eg, COX-2 inhibitors), metallomatrix protease inhibitors, teromerase inhibitors, tyrosine kinase inhibitors, anti-growth factor receptor materials, anti-HER substances, anti-EGFR substances, anti-angiogenic substances In combination with other anticancer therapies, such as farnesyl transferase inhibitors, ras-raf signal conduction pathway inhibitors, cell cycle inhibitors, other cdk inhibitors, tubulin binding agents, topoisomerase I inhibitors, topoisomerase II inhibitors, and the like. Can be used.

In addition, the anticancer composition of the present invention is one or more chemotherapeutic agents optionally contained in liposome preparations (e.g., taxanes, taxane derivatives, encapsulated taxanes, CPT-11, camptothecin derivatives, anthracycline glycosides, doxorubicin, Rubicin, epirubicin, etoposide, navelbine, vinblastine, carboplatin, cisplatin, esturamustine, celecoxib, schgen SU-5416, schgen SU-6668, herceptin, etc.). It may be.

When formulating such combinations in constant doses, the anticancer compositions of the present invention are used within the following dosage ranges and other pharmaceutically active substances within the approved dosage ranges. In addition, the anticancer composition of the present invention can be used sequentially with known anticancer agents when the combination agent is inappropriate.

An anticancer composition of the present invention containing the active ingredient and a pharmaceutically acceptable carrier or diluent is usually formulated according to conventional methods and administered in a pharmaceutically suitable form. That is, the anticancer composition of the present invention may be administered in various dosage forms, such as tablets, capsules, dragees or film-encapsulated tablets, oral forms of solutions or suspensions, intramuscular, intravenous and / or intrathecal and / or spinal cord injections or It may be formulated and administered in the parenteral form of infusion.

For example, when the compositions of the present invention are formulated as oral solids, diluents (e.g. lactose, dextrose, sucrose, cellulose, corn starch or potato starch), active agents (e.g. Silica, talc, stearic acid, magnesium or calcium stearate and / or polyethylene glycol), binders (e.g., starch, arabic gum, gelatin methylcellulose, carboxymethylcellulose or polyvinyl pyrrolidone), disintegrants (E.g., starch, alginic acid, alginate or sodium starch glycolate), formal mixtures, dyes, sweeteners, wetting agents (e.g. lecithin, polysorbates, laurylsulfate) and drugs commonly used in pharmaceuticals It may contain a chemically inert substance. These agents can be prepared by known methods, for example by means of mixing, granulating, tableting, dragging or film-coating processes.

In addition, when formulated as a liquid dispersion for oral administration such as syrups, emulsions and suspensions, it may contain natural gums, agar, sodium alginate, pectin, methylcellulose, carboxymethylcellulose or polyvinyl alcohol together with the active ingredient. .

In addition, when formulated as a suspension or solution for intramuscular injection, it may contain sterile water, olive oil, ethyl oleate, glycol (eg propylene glycol) and, if necessary, a suitable amount of lidocaine hydrochloride with the active ingredient. Can be.

In addition, when formulated as a solution for intravenous injection or infusion, it may contain sterile water with the active ingredient or preferably in the form of a sterile, aqueous, isotonic saline solution or may contain carrier propylene glycol.

The therapeutically effective amount of the anticancer composition of the present invention may vary depending on the age, sex, site of application, frequency of administration, time of administration, dosage form, type of adjuvant, etc., but in the case of injection, it is generally 50 to 200 mg, preferably 100 To 150 mg is administered 1 to 5 times a day, and in the case of oral administration, it is generally administered 250 to 1,000 mg, preferably 300 to 500 mg 1 to 5 times a day.

The invention will be more specifically illustrated by the following examples and experimental examples. However, these examples and experimental examples are only embodiments of the present invention and do not limit the scope of the present invention.

<Example 1>

Injection

0.01% by weight of zinc chloride (Sigma, U.S.A.)

L-arginine (Ajinomoto, Japan) 0.03 wt%

10N sodium hydroxide pH7 adjustment amount

100% of sterile water

The ingredients were combined in the amounts shown to prepare a vial (100 mg).

<Example 2>

Injection

0.01% by weight of zinc sulfate (Sigma, U.S.A.)

L-arginine (Ajinomoto, Japan) 0.03 wt%                     

10N sodium hydroxide pH7 composition

100% composition of sterile water

The ingredients were combined in the amounts shown to prepare a vial (100 mg).

<Example 3>

refine

0.01% by weight of zinc chloride (Sigma, U.S.A.)

L-arginine (Ajinomoto, Japan) 0.03 wt%

Lactose 30% by weight

Magnesium Stearate 5% by weight

10% by weight sodium starch glycolate

10N sodium hydroxide pH7 composition

100% composition of sterile water

The ingredients were mixed in the indicated contents, stirred for 1 hour while maintaining at 30 to 60 ° C, cooled to room temperature, and compressed into tablets according to a conventional method for preparing tablets to prepare 350 mg of the composition.

<Example 4>

refine

0.01% by weight of zinc sulfate (Sigma, U.S.A.)

L-arginine (Ajinomoto, Japan) 0.03 wt%

Lactose 30% by weight

Magnesium Stearate 5% by weight

10% by weight sodium starch glycolate

10N sodium hydroxide pH7 composition

100% composition of sterile water

The ingredients were mixed in the indicated contents, stirred for 1 hour while maintaining at 30 to 60 ° C, cooled to room temperature, and compressed into tablets according to a conventional method for preparing tablets to prepare 350 mg of the composition.

Experimental Example

Measurement of Cell Viability According to Treatment of Anticancer Composition of the Present Invention

In order to investigate the effects of the anticancer composition of the present invention on the growth and survival of cancer cells, the cell viability was measured by treating the cancer cell line with a composition prepared by varying the content of the active ingredient. Cell lines were SKOV-3 (human ovarian adenocarcinoma, ATCC No .: HTB-77) and U87 (human brain glioblastoma, ATCC No .: HTB-14).

The cells were placed in 96-well plates to 3 × 10 ³ cells / well and incubated for 12 hours. As culture medium, DMEM (Dulbecco's modified) was added 10% (v / v) of heat-inactivated FBS (fetal bovine serum), 100 μg / ml of streptomycin, 100 U / ml of penicillin and 100 μg / ml of L-glutamine. Eagle's medium, Life Technology, Inc., USA). Each cell line was cultured and treated with a composition prepared by varying the content of the active ingredient in the same manner as in Examples 1 and 2, and then, at 37 ° C., 5% of carbon dioxide and 95% of oxygen were supplied for 24 hours. Incubated and then cell viability was measured.

Cell viability was measured by known MTT (3- (4,5-dimethylthiazole-2-yl) 2,5-diphenyl-2H-tetrazolium bromidine) assay (Hansen, MB et al., J Immunol.Methods, 172, 203-210 (1989). 20 μl MTT solution [MTT at 10 mg / ml in (phosphate buffered saline, phosphate buffered saline)] was added to each well and the plate was incubated at 37 ° C. for 4 hours. After removing the medium, formazan crystals were dissolved in 200 µl of DMSO (dimethyl sufoxide, dimethyl sulfoxide) and added to each well. This was shaken for 10 minutes at room temperature and mixed and measured at 540 nm using a Bio-Rad Model 3550 Microplate Reader (Microplate Reader, Richmond, CA.). At this time, DMEM-FBS and MTT were added and the wells without addition of the composition were used as a control. Experimental results are shown in FIGS. 1 and 2.

As can be seen from FIGS. 1 and 2, the viability of cell lines SKOV-3 and U87 treated with a composition containing zinc and L-arginine decreased with increasing content of the active ingredients zinc and L-arginine. It was. In FIG. 1, the survival rate of the composition containing zinc chloride and L-arginine is reduced to 50% or less when the content of the component is about 0.008% by weight or more, and the survival rate is 20 when the content of the component is about 0.01% by weight or more. Decreased below%. In addition, in the composition containing zinc sulfate and L-arginine, the survival rate was reduced to 50% or less when the content of the component was about 0.004% or more, and the survival rate was 20% or less when the content of the component was about 0.01% by weight or more. Reduced to. In FIG. 2, the survival rate of the composition containing zinc chloride and L-arginine is reduced to 50% or less when the content of the component is about 0.005% or more, and the survival rate is 20 when the content of the component is about 0.008% or more. Decreased below%.

Comparative Experimental Example

Measurement of cell viability following treatment of a composition containing L-arginine and L-aspartic acid or a composition containing L-arginine

 A cancer cell comprising a composition containing L-arginine and L-aspartic acid (Ajinomoto, Japan) or a composition containing L-arginine as a known component of anticancer effects for comparison with the survival rate of cancer cells treated with the anticancer composition of the present invention. Treatments were performed weekly to determine cell viability. SKOV-3 was used as the cell line, and the culture of these cells, the method of treating the composition with the cancer cell line, and cell viability were measured in the same manner as in the experimental example. Experimental results are shown in FIGS. 3 and 4.

As can be seen from Figures 3 and 4, the survival rate of the cell line SKOV-3 treated with the composition, although the survival rate decreases with increasing L-arginine and L-aspartic acid content or L-arginine content The survival rate was over 50%.

That is, when compared with the survival rate of the cancer cells treated with the anticancer composition of the present invention, the results of the comparative example did not show a reduction in the effective survival rate of cancer cells.

As described above, the anticancer composition of the present invention containing zinc and L-arginine as active ingredients has an effect of inducing apoptosis and killing cancer cells.

Claims (6)

An anticancer composition comprising zinc and L-arginine as active ingredients in a pharmaceutically effective amount together with a pharmaceutically acceptable carrier or diluent. The anticancer composition according to claim 1, wherein the pharmaceutically effective amount of the active ingredient is 0.001 to 0.025 wt% zinc and 0.004 to 0.075 wt% L-arginine. The composition of claim 1, wherein zinc in the active ingredient is zinc chloride; Zinc sulfate; And zinc acetate composition selected from the group consisting of. The method of claim 1, wherein the application of the composition is lung cancer; Brain cancer; Breast cancer; Colon cancer; And epithelial ovarian cancer. The anticancer composition according to claim 4, wherein the object of application of the composition is epithelial ovarian cancer. The method according to any one of claims 1 to 5, The anticancer composition is an anticancer composition, characterized in that the formulation for oral administration or formulation for parenteral administration.

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