KR20060136219A - Anti-cancer composition comprising cisplatin and green tea extract - Google Patents

Abstract

The present invention relates to an anticancer composition comprising cisplatin and green tea polyphenol, or cisplatin and EGCG, and to a method for preventing or treating cancer by administering the composition to a patient.

Cis-diamminedichloroplatinum (II): CDDP: cisplatin, cervical cancer, drug resistance, green tea polyphenol (GTP), EGCG, synergistic effect

Description

Anti-cancer composition comprising cisplatin and green tea extract

1 is a result of examining the growth inhibitory effect of cisplatin (A), green tea extract (GTP) (B), EGCG (C).

Figure 2 is a result of examining the synergistic effect of cell growth inhibition by simultaneous administration of cisplatin and green tea extract (GTP) in TC-1 cells. (A) 0.5 uM cisplatin + 1 ug / ml green tea extract (B) 0.5 uM cisplatin + 5 ug / ml green tea extract

Figure 3 is a result of examining the synergistic effect of cell growth by the simultaneous administration of cisplatin and EGCG in TC-1 cells. (A) 0.5 uM cisplatin + 25 uM EGCG (B) 0.5 uM cisplatin + 50 uM EGCG

Figure 4 shows the anti-tumor effects of green tea extract (left) and cisplatin (right) in cervical cancer animal models formed by injecting 5X10 ⁵ TC-1 cells into C57BL / 6 mice.

FIG. 5 shows the results of antitumor effects by simultaneous administration of green tea extract and cisplatin in cervical cancer animal models formed by injecting ⁵ × 10 ⁵ TC-1 cells into C57BL / 6 mice.

The present invention relates to an anticancer composition comprising cisplatin and green tea polyphenol, or cisplatin and EGCG ((-)-epigallocatechin gallate) and use thereof.

Cisplatin ( cis- diamminedichloroplatinum (II): CDDP: cisplatin) is used as an effective chemotherapeutic agent for the treatment of solid cancers such as cervical cancer, colorectal cancer, and lung cancer (Crown JP. Et al., Semin Oncol 2001; 28: 28-37 Jordan P et al., Cell Mol Life Sci 2000; 57: 1229-1235). However, cisplatin can cause cytotoxicity when used at high doses or for prolonged use (Kartalou M et al., 2001; 478: 23-43), and especially in cervical cancer, induction of cancer resistance mechanisms by continuous cisplatin administration (Albets DS et al., An update Semin Oncol. 1991; 18 (1 Suppl 3): 11-24).

One of the problems to be solved in the implementation of chemotherapy with cisplatin is increased resistance. Drug resistance can be attributed to several mechanisms: reduced intracellular drug accumulation, cisplatin inactivation by thiol-containing substances, increased ability to repair DNA damage, HER-2 / neu and PI3-K / It is known that overexpression of Akt and the like and low expression of p53 or activation of apoptosis inhibitors may result in resistance to cisplatin (Siddik ZH et al., 2003; 22 (47): 7265-79).

In order to solve this problem, high-dose chemotherapy has recently been introduced, but new doses such as nephrotoxicity are emerging due to high-dose therapy, and studies to reduce side effects are very urgent (Sardi JE. Et al., Surg Clin North Am 2001). 81: 965-985; McAfee SL. Et al., Curr Opin Obstet Gynecol 1999; 11: 17-21).

There is a growing interest in chemical cancer preventive agents that are readily available to humans to reduce cancer. Chemoprevention is one method of cancer control that slows, prevents, or reverses the incidence of cancer through the administration of natural or chemical compounds in the development of cancer caused by exposure to carcinogens (Tsao AS et al., J Cin 2004; 54: 150-180; Follen M et al., Cancer 2003; 1; 98 (9 Suppl): 2044-2051).

As a chemopreventive agent, studies on the role of green tea extract (polyphenonon E; Poly E or (-)-epigallocthechin-3-gallate (EGCG)) have shown significant progress in clinical and basic studies. EGCG, one of the active ingredients of green tea extract, has been reported to inhibit cervical cancer cell growth through apoptosis and cell cycle arrest (Ahn WS et al., DNA Cell Biol 2003; 22 (3): 217). -224). In addition, theanine in green tea polyphenols (GTP) has been reported to have the effect of protecting normal cells from anticancer toxicity by changing the concentration of intracellular glutamine and reducing cytotoxicity. Sugiyama T et al., Cancer Lett. 2004 30; 212 (2): 177-184.

The mechanism of action on the effects of green tea Polyohenols (EGCG, Polyphenon E) on the living body is prostaglandin (PGs) in the microsome of skin cells induced by PMA (phorbol 12-myristate 13-acetate). ) Blocking the increase (Zhao BL., Et., Mol Biol Int. 1989; 14: 175-185). Inhibit or block the production of orotidine 5-phosphate decarboxylase (ODC) (Martinez J., Biochem Pharmacol. 2000; 59: 865-870) or inhibit free radical formation (Cloutier MM, Lung 1995; 173: 307). 19) and is known to inhibit protein kinase C and cell proliferation (Iwai K., Int J Food Sci Natr. 2001; 52: 443-51). In addition, EGCG and glutamyl pyruvic transaminase increase the phase II enzymes GST (glutathione-s-trasferase), GSH peroxidase, catalase, etc. Tumor promoters and hormones are known to block the binding of receptors.

In previous experiments, we used ovarian cancer cell lines to investigate the anticancer effects of EGCG associated with apoptosis and cell cycle.In OVC-treated SKOV-3 and OVCAR-3 cell lines, the G (1) phase and PA-1 It was confirmed that the cell cycle was stopped in the G (1) / S phase and the increase of apoptosis related genes (Bax, PCNA, Bcl-X) was confirmed by EGCG treatment (Huh SW et al., Gynecol Oncol. 2004; 94; 94 (3): 760-708).

Based on the above studies, green tea active ingredients known to have anticancer effects were applied to cancer treatment using the existing anticancer agent cisplatin, to solve the problems of cisplatin-induced toxicity and drug resistance, and to increase the therapeutic activity.

Therefore, the present inventors have confirmed that the combination of cisplatin with an active green tea active ingredient, specifically polyphenol or EGCG, has a more synergistic effect at the single administration of each component, and completed the present invention.

One object of the present invention is to provide an anticancer composition comprising cisplatin and green tea polyphenol.

It is another object of the present invention to provide an anticancer composition comprising cisplatin and EGCG ((-)-epigallocatechin gallate).

Another object of the present invention is to provide a method for preventing or treating cancer by administering the composition to a patient.

In one embodiment, the present invention relates to an anticancer composition having a synergistic effect of cisplatin comprising green tea polyphenols or EGCG ((-)-epigallocatechin gallate).

As used herein, the term "synergistic effect" means a higher effect than the additive effect of a single component when two or more components are used in combination, and in the present invention, the activity separated from the existing anticancer agent and green tea. The synergistic anticancer effect when used with ingredients, specifically polyphenols or EGCG, was elucidated.

The inventors have looked at the anticancer effects of applying cisplatin in combination with the green tea active ingredient on a cellular and biometric level. The combination of the two different components may be accompanied by a decrease in activity and further side effects, but according to the present inventors, it was confirmed that when the green tea active ingredient, specifically polyphenol or EGCG, was used together with cisplatin, it showed synergistic anticancer activity. . Cisplatin is a chemical that can cause serious toxicity and resistance, but since green tea active ingredients derived from natural products have little toxicity, its application is more widespread.

In one specific embodiment, the present invention relates to an anticancer composition comprising cisplatin and green tea polyphenols.

In another specific embodiment, the present invention relates to an anticancer composition comprising cisplatin and EGCG ((-)-epigallocatechin gallate).

As used herein, the term “anticancer” means a tendency or ability to inhibit or kill the proliferation of cancer cells. When administered with green tea polyphenols or EGCG than when cisplatin alone, it was confirmed that in cancer cell lines, cell growth was inhibited and cell death increased. In addition, it was confirmed that the tumor of the mouse was significantly reduced. In particular, significant synergistic anticancer effects were observed when EGCG was used in combination with cisplatin. TC-1 cells expressing E6 and E7 genes were treated with cisplatin and EGCG in combination. Cell growth decreased by 32.2% when treated with 0.5 uM of cisplatin alone on day 5, and decreased by 5.0% when treated with 25 uM of EGCG alone.However, cell growth when combined with 0.5 uM of cisplatin and 25 uM of EGCG. Showed a significant decrease of 75.2%. In addition, EGCG 50 uM alone was reduced by 18.1%, but a combination of 0.5 uM cisplatin and EGCG 50 uM showed a significant decrease of 81.2%.

When cisplatin is administered with green tea polyphenols or EGCG, it exhibits better anticancer effects than chemotherapeutic agents alone, and is particularly useful for the treatment of cancers having cisplatin resistance. In addition, the use of a small dose of cisplatin reduces the risk of inducing toxicity and the use of the composition is desirable when long term treatment is required.

Cancers that can be prevented or treated with the compositions of the present invention are solid cancers. Solid cancers include, but are not limited to, colorectal cancer, small intestine cancer, rectal cancer, anal cancer, esophageal cancer, pancreatic cancer, gastric cancer, kidney cancer, cervical cancer, breast cancer, lung cancer, ovarian cancer, and the like. In particular, it is effective in the treatment of cervical cancer. Cervical cancer can be caused by a variety of causes, especially in the case of cervical cancer caused by HPV infection is very effective.

The composition comprises a pharmaceutically acceptable carrier and can be formulated for human or veterinary use.

Pharmaceutical compositions for oral administration may be presented as discrete units such as capsules or tablets; Powder or granules; Solvents, syrups or suspensions (in aqueous or non-aqueous liquids; edible blowing agents or whips; or emulsions).

Suitable excipients for tablets or hard gelatin capsules are lactose, corn starch or derivatives thereof, stearic acid or salts thereof. Suitable excipients for use in soft gelatin capsules are, for example, vegetable oils, waxes, fats, semi-solids or liquid polyols. Excipients that can be used to prepare solvents and syrups are, for example, water, polyols and sugars. To prepare suspending agents, oils such as vegetable oils can be used to provide oil-in-water or water-in-oil suspensions. Pharmaceutical compositions adapted for transdermal administration may be presented as separate patches to enable intimate contact with the epidermis of the recipient for a long time.

Pharmaceutical compositions for parenteral administration include aqueous and non-aqueous sterile injectable solutions which may contain antioxidants, buffers, bacteriostats and solutes (which are substantially isotonic with the recipient's blood); And aqueous and non-aqueous sterile suspending agents which may include suspending agents and thickening agents. Excipients that can be used in the injectable solvents include, for example, water, alcohols, polyols, glycerin and vegetable oils. Such compositions may be presented in unit-dose (single) or multi-dose (several) containers, such as sealed ampoules and vials, and immediately before use, sterile liquid carriers such as injectable water. Can be stored under freeze-drying conditions requiring only the addition of. Immediate injection solutions and suspensions can be prepared from sterile powders, granules and tablets.

In another aspect, the invention relates to a method of preventing or treating cancer by administering the composition to a patient.

As used herein, the term "patient" refers to humans and animals with cancer or at risk of cancer, such as cattle, horses, sheep, pigs, goats, camels, antelopes, and dogs. By administering to a patient a composition comprising cisplatin and green tea polyphenol of the present invention or a composition comprising cisplatin and EGCG, cancer can be effectively prevented or treated above.

As used herein, the term "prevention" means any action that inhibits or delays the onset of cancer by administration of the composition. As used herein, the term "treatment" means any action that improves or beneficially alters cancer symptoms by administration of the composition.

As used herein, the term "administration" means introducing a predetermined substance into a patient by any suitable method, and the route of administration of the composition of the present invention may be administered through any general route as long as it can reach the target tissue. . Intraperitoneal administration, intravenous administration, intramuscular administration, subcutaneous administration, intradermal administration, oral administration, topical administration, nasal administration, pulmonary administration, rectal administration, but is not limited thereto. In addition, the pharmaceutical composition may be administered by any device in which the active agent may migrate to the target cell.

The composition of the present invention is administered in a pharmaceutically effective amount.

As used herein, the term “pharmaceutically effective amount” means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment, and the effective dose level refers to the disease type and severity, age, sex, It can be determined according to the activity of the drug, sensitivity to the drug, the time of administration, the route of administration and the rate of release, the duration of treatment, factors including the concurrent drug and other factors well known in the medical field. The compositions of the present invention may be administered in a single or multiple doses. Taking all of the above factors into consideration, it is important to administer an amount that can obtain the maximum effect in a minimum amount without side effects, and can be easily determined by those skilled in the art.

Hereinafter, preferred examples are provided to aid in understanding the present invention. However, the following examples are merely provided to more easily understand the present invention, and the present invention is not limited by the following examples.

Example 1 Cell Lines and Cell Culture

Human lung cancer cell line TC-1 (T-C. Wu, Johns Hopkins Medical Institution, Baltimore, MD) expressing HPV16 (Human papilloma virus 16) E6, E7 genes was used. Cell culture medium was 10% FBS (Gibco, Rockville, MD, USA), streptomycin / penicillin (Gibco, Rockville, MD, USA), L-glutamine in RPMI 1640 (Gibco, Rockville, MD, USA). (Gibco, Rockville, MD, USA), 2.2 mg / ml sodium bicarbonate (Sigma, St. Louis, MO, USA), 0.4 mg / ml G418-disulfate (Duchefa, Haarlem, Netherland) Used. TC-1 cell lines were cultured in a 37 ° C, CO2 incubator.

Example 2: Confirmation of Cell Growth Inhibition

TC-1 cells were administered with experimental and control groups. The experimental group was administered with cisplatin (Dong-A Pharmaceutical), GTP or EGCG (Yukihiko Hara, Mutsui Norin Co., Fujied, Japan) alone, with cisplatin and GTP, and with cisplatin and EGCG. , The control group was not treated. TC-1 cell lines were seeded in 96 wells at 1 × 10 ⁴ cells / well and incubated for 24 hours. Cisplatin at 0.1, 0.5 and 2.5 uM concentrations per well was treated with GTP at 1, 5 and 25 ug / ml concentrations and EGCG at 25, 50 and 100 uM concentrations alone. Cisplatin and GTP were treated with 0.1 uM cisplatin and 1 ug / ml GTP, 0.1 uM cisplatin and 5 ug / ml GTP, 0.5 uM cisplatin and 1 ug / ml GTP and 0.5 ug cisplatin and 5 ug / ml Cisplatin. Cisplatin and EGCG co-treated groups were treated with cisplatin 0.1 uM and EGCG 25 uM, cisplatin 0.1 uM and EGCG 50 uM, cisplatin 0.5 uM and EGCG 25 uM and cisplatin 0.5 uM and EGCG 50 uM. MTT assays were performed after 1, 2, 3, 4 and 5 days. 20 μl of MTT solution (Sigma, St, Louis, MO) was added per well, and then incubated for 4 hours in a CO ₂ incubator. After removing all the supernatant, the absorbance was measured at 595 nm with ELISA-Reader (Spectromax 250, Molecular Devices) by adding DMSO (Sigma, St. Louis, Mo.) at 100 ul / well. Absorbance was measured three times and averaged.

 After treatment with cisplatin, GTP and EGCG alone in TC-1 cells expressing E6 and E7 genes, they were cultured for 5 days and examined for cell growth inhibition effect. Treatment with cisplatin 0.1, 0.5 and 2.5 uM on day 5 decreased cell growth to 20.3, 32.2 and 52.5%, respectively, compared to the control without cisplatin (FIG. 1A) and treated with GTP 1, 5 and 25 ug / ml. In one case, cell growth was decreased to 14.0, 15.8, and 90.8%, respectively, compared to the control group without GTP (Fig. 1B), and 5.0, 18.1, respectively, compared to the control group without EGCG treatment at EGCG 25, 50, and 100 uM. , At a rate of 91.6% (FIG. 1C).

The effects of cell growth inhibition on the combination of cisplatin and GTP on TC-1 cells expressing E6 and E7 genes were examined. Treatment of cisplatin and GTP with TC-1 decreased cell growth as compared to treatment alone. Treatment with cisplatin 0.5 uM alone on day 5 decreased 32.2% and 14.0% GTP 1 ug / ml, respectively, compared to the control group, but when cisplatin 0.5 uM and GTP 1 ug / ml were treated alone It showed a 40.5% reduction rate higher than the treatment group (FIG. 2A). In addition, when treated with GTP 5 ug / ml alone was reduced 15.8% and when combined treatment with cisplatin 0.5 uM and GTP 5 ug / ml showed a higher inhibition rate of 36.9% than the group alone (FIG. 2B).

The effects of cell growth inhibition on the combination of cisplatin and EGCG on TC-1 cells expressing E6 and E7 genes were examined. Treatment with cisplatin 0.5 uM alone on day 5 resulted in a 32.2% reduction in cell growth compared to the control group, and treatment with EGCG 25 uM alone decreased 5.0%, but combined with cisplatin 0.5 uM and EGCG 25 uM Showed a significant decrease of 75.2% (FIG. 3A). In addition, when treated with EGCG 50 uM alone, cell growth was reduced by 18.1%, and when combined with 0.5 uM cisplatin and EGCG 50 uM, the cell growth was markedly reduced to 81.2% (FIG. 3B).

The MTT results were averaged three times, expressed as standard deviation, and the test results were analyzed by non-paired Student's t-test, and considered to be significant when p <0.05.

Example 3: Synergistic Anticancer Effect by Simultaneous Administration of GTP and Cisplatin in Mice

C57 / BL6 mice were inoculated subcutaneously in the right flank with 5 × 10 ⁵ TC-1 cells. About 14 days after tumors can be touched by hand (approximately 8 to 9 mm), 10 mice per group are injected intravenously with GTP at 100 mg / kg bw concentrations for 4 weeks, once a week or 5, 10 or 20 mg. Cisplatin was injected intraperitoneally at a concentration of / kg bw. In addition, in order to examine the synergistic anticancer effect when cisplatin and GTP were administered in combination, GTP was injected intravenously at a concentration of 100 mg / kg bw for 4 weeks once a week, and at the same time 5, 10 mg / kg bw of cisplatin Intraperitoneal injection. One week later, GTP, cisplatin, or GTP and cisplatin were reinjected into mice by the method and amount of the first injection. Tumor size was measured twice a week and expressed as mean and standard deviation (SD) of tumor size (*, p value <0.05).

As a result, 100 mg / kg b.w. In case of treatment with concentration, tumor size decreased by 17.6, 17.1, 15.7, and 11.6% at 4 days, 11 days, 15 days and 22 days, respectively. 5 mg / kg b.w. Treatment with cisplatin at the concentration reduced tumor size by 17.9, 23.4, 30.3, 26.1%, and 10 / kg b.w. Treatment with cisplatin at the concentration reduced tumor size by 19.2, 37.9, 51.1, and 55.4% (FIG. 4). On the other hand, when cisplatin was administered in combination with GTP, tumor size was significantly reduced than when administered alone. 100 mg / kg b.w. of GTP and 5 mg / kg b.w. In the case of simultaneous administration of cisplatin at a concentration, tumor size decreased by 9.6, 24.8, 34.7, and 33.1% compared to the control group, and 100 mg / kg b.w. of GTP and 10 mg / kg b.w. Concurrent administration of cisplatin at a concentration reduced tumor size by 14.3, 41.3, 53.3, and 61.3% compared to the control group (FIG. 5).

Cisplatin and green tea polyphenol of the present invention, or an anticancer composition comprising cisplatin and EGCG can effectively prevent and treat cancer, and in particular, to treat cancers having low toxicity and side effects and having cisplatin resistance. Is effective in

Claims (5)

An anticancer composition comprising cisplatin and green tea polyphenol. An anticancer composition comprising cisplatin and EGCG ((-)-epigallocatechin gallate). The composition of claim 1 or 2, wherein the cancer is a solid cancer. The composition of claim 3, wherein the cancer is cervical cancer. The composition of claim 4 which is cervical cancer caused by HPV infection.

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