JPH07330599A - Suppressant for resistance to anticancer agent - Google Patents

Abstract

PURPOSE:To obtain the readily available new medicine, containing a specific polyphenol compound, capable of strongly suppressing the manifestation of resistance to anticancer agents in tumorous cells and suppliable in a large amount at a low cost and having high safety without any toxicity. CONSTITUTION:This medicine contains preferably 0.1-80wt.% 10-50C polyphenol compound having 5-20 number of hydroxyl groups {preferably green tea polyphenols such as (+)-catechin of the formula [R<1> and R<2> are each H] and (+)-gallocatechin of the formula (R'' is hydroxyl and R'' is H)}. Furthermore, the compound of the formula is preferably extracted from green tea. The medicine is usually administered in a daily dose of 1mg to 10g expressed in terms of the amount of the green tea polyphenol for an adult in 1 to 4 divided portions.

Description

Detailed Description of the Invention

[0001]

TECHNICAL FIELD The present invention relates to an anticancer drug resistance inhibitor containing a polyphenol compound (particularly tea polyphenol) or a tea extract as an active ingredient. The anticancer drug resistance inhibitor of the present invention can be used particularly in the field of cancer chemotherapy.

[0002]

BACKGROUND OF THE INVENTION Over the past 30 years, cancer chemotherapy has advanced by discovering new anti-cancer agents. With the establishment of combination therapy of anticancer agents, effective treatments have been found for childhood cancers such as Wilms tumor and leukemia, and choriocarcinoma of women, and these are curable cancers. Patients with Burkitt's lymphoma, acute lymphocytic leukemia, Hodgkin's disease, etc. have also been able to return to social life at a significantly higher rate. While such expectations are high for chemotherapy, there are still solid cancers such as lung cancer and colon cancer that hardly respond to chemotherapeutic agents. Furthermore,
Even in the case of the above-mentioned cancers that respond to chemotherapeutic agents, there is a problem that the anticancer agents will eventually become ineffective and resistance to them will increase. 1988
According to US statistics for the year, 49% of cancers diagnosed in a year were intrinsically resistant to chemotherapy from the beginning, and 47% were initially efficacious and had tumors once resolved. It is said to be acquired resistance that recurred later. More than 90% of all cancer deaths are related to chemotherapy ineffectiveness, 61% of which is estimated to be intrinsic resistance and 33% to acquired resistance. These facts show that one of the most important problems that hinders the effects of chemotherapy on cancer is resistance to cytotoxic drugs. As resistance to anticancer drugs, a property of simultaneously exhibiting resistance to various anticancer drugs having little commonalities in action points and structures, that is, multidrug resistance is known. The gene responsible for this multidrug resistance has already been isolated from human cultured cells, and
1 (multidrug-resistance) gene. The human membrane protein (P-glycoprotein) encoded by this multidrug resistance gene MDR1 is a polypeptide consisting of 1,280 amino acids, and acts as a pump for extracellularly discharging the anticancer drug depending on energy. . P-glycoprotein is also associated with resistance to many naturally occurring cytotoxic agents such as vincristine, vinblastine, actinomycin D, gramicidin, adriamycin, colchicine, etoposide (VP-16) and teniposide (VM-26). ing. In order to overcome this multidrug resistance, drugs such as cyclosporine and Ca ²⁺ antagonist verapamil are used, and P
-Attempts have been made to reduce resistance by inhibiting the excretion of intracellular drugs by glycoproteins.

[0003]

[Problems to be Solved by the Invention] However, cyclosporine and Ca ²⁺ antagonists have many side effects. That is, cyclosporine has an immunosuppressive action, and nephrotoxicity may occur, which is dangerous. On the other hand, since Ca ²⁺ antagonists have a strong vasodilatory effect, sympathetic nervous system activity increases with hypotension, and side effects such as headache, palpitations or hot flushes are easily observed, and edema of the lower extremities is often caused. . Therefore, administration of a Ca ²⁺ antagonist such as verapamil is dangerous because it may induce shock due to hypotension or the like. The present inventor, as a result of searching for a substance that can control the expression of anticancer drug resistance and is highly safe for long-term continuous use, surprisingly, a group of polyphenol compounds has an excellent anticancer drug resistance expression. It was found that it has the effect of suppressing the induction. The present invention is based on these findings.

[0004]

Therefore, the present invention relates to an anticancer drug resistance inhibitor containing a polyphenol compound having 10 to 50 carbon atoms and 5 to 20 hydroxyl groups. The present invention also relates to an anticancer drug resistance inhibitor, which comprises tea water or an organic solvent extract.

The present invention will be described in detail below. The polyphenol compound used as an active ingredient in the anticancer drug resistance inhibitor of the present invention is a compound having 10 to 50 carbon atoms and 5 to 20 hydroxyl groups. Preferred polyphenol compounds have a di- or tri-hydroxychroman ring,
For example, general formula (I):

[Chemical 1] (In the formula, R ¹ is a hydrogen atom or a hydroxyl group, and R 1
² is a hydrogen atom or formula

[Chemical 2] A compound represented by the formula (3,4,5-trihydroxyphenylcarbonyl group) and an isomer thereof. Furthermore, the general formula (II):

[Chemical 3] (Wherein R ³ and R ⁴ are the same or different and each is a hydrogen atom or a 3,4,5-trihydroxyphenylcarbonyl group) and isomers thereof can also be mentioned as preferable compounds. . In addition, in this invention, a pure stereoisomer or a mixture thereof can be used.

Among the polyphenol compounds represented by the above general formula (I), a particularly preferred compound is the general formula (I
-1):

[Chemical 4] (In the formula, R ¹ and R ² have the same meanings as described above), or a compound represented by the general formula (I-2):

[Chemical 5] (In the formula, R ¹ and R ² have the same meanings as described above).

In the general formula (I-1), the compound in which R ¹ and R ² are hydrogen atoms is (+) catechin; the compound in which R ¹ is a hydroxyl group and R ² is a hydrogen atom is , (+) Gallocatechin; R ¹ is a hydrogen atom and R ² is a 3,4,5-trihydroxyphenylcarbonyl group is a (+) catechin gallate; R ¹ is a hydroxyl group , R ² is 3, 4,
The compound which is a 5-trihydroxyphenylcarbonyl group is (+) gallocatechin gallate.

In the general formula (I-2), R
^The compound in which ¹ and R ² are hydrogen atoms is (−) epicatechin; the compound in which R ¹ is a hydroxyl group and R ² is a hydrogen atom is (−) epigallocatechin;
The compound in which R ¹ is a hydrogen atom and R ² is a 3,4,5-trihydroxyphenylcarbonyl group is (−) epicatechin gallate; R ¹ is a hydroxyl group and R ² is 3,4. The compound that is a 5,5-trihydroxyphenylcarbonyl group is (−) epigallocatechin gallate.

Among the polyphenol compounds represented by the general formula (II), a particularly preferred compound is the general formula (II-1):

[Chemical 6] (In the formula, R ³ and R ⁴ have the same meanings as described above).

In the general formula (II-1), the compound in which R ³ and R ⁴ are hydrogen atoms is free theaflavin; R ³ is 3,4,5-trihydroxyphenylcarbonyl group, The compound in which R ⁴ is a hydrogen atom is theaflavin monogallate A; the compound in which R ³ is a hydrogen atom and R ⁴ is a 3,4,5-trihydroxyphenylcarbonyl group is theaflavin monogallate B. The compound in which R ³ and R ⁴ are 3,4,5-trihydroxyphenylcarbonyl groups is theaflavin digallate.

The compounds represented by the above general formulas (I) and (II) can be obtained by using commercially available products, or by synthesizing them or extracting them from natural products and purifying them. The compounds represented by the general formula (I) and the general formula (II) are mainly known as tea polyphenols, and when they are extracted from a natural product and purified, they are not limited, but tea It is preferable to extract from

The anticancer drug resistance inhibitor according to the present invention can also comprise a tea water extract or an organic solvent extract as an active ingredient. In the present specification, "tea" means tea (Ca
mmellia sinensis, (L) O. Kun
tze) whole plant or part thereof, eg leaves, xylem,
It means a raw or dried product such as root, fruit, etc. as it is or a partially fermented product or a completely fermented product, and these parts can be used alone or in any combination. When using tea leaves as an extraction raw material, there are various forms,
For example, fresh tea leaves to finished tea (dry tea) may be used at any stage of the normal tea-making process, and regardless of the degree of fermentation, fermented tea such as black tea, semi-fermented tea such as oolong tea, and non-fermented tea such as green tea. Any of the fermented teas can be used.

The tea extract, which is an active ingredient of the anticancer drug resistance inhibitor according to the present invention, may contain the above-mentioned tea polyphenols, and thus can be a crude extract of tea. To obtain this crude tea extract, tea can be extracted with warm water (preferably hot water) or with an organic solvent. As the organic solvent, methyl alcohol, ethyl alcohol, n-propyl alcohol,
Lower alcohols such as isopropyl alcohol and butyl alcohol, lower esters such as methyl acetate, ethyl acetate, propyl acetate and butyl acetate, acetone, methyl isobutyl ketone, and the like can be used, and these organic solvents may be used alone or in appropriate combination, and further, Can be used anhydrous or preferably in the hydrated state.

The methods of water extraction and organic solvent extraction include
It is possible to use the method used for usual herbal medicine extraction,
For example, to 1 part by weight of (dry) tea leaves, water or organic solvent 5 to
It is desirable to heat and reflux at a temperature below its boiling point while stirring using 20 parts by weight. The extraction step is usually carried out for 5 minutes to 7 days, preferably 10 minutes to 24 hours, and the extraction time can be shortened by adding an auxiliary means such as stirring if necessary. The water or organic solvent extract can be separated from the insoluble matter by an appropriate method such as filtration or centrifugation. In addition to the hot water extract and the organic solvent extract by the conventional method, the tea extract of the present invention includes the products obtained by further treating these extracts with various organic solvents, adsorbents and the like. If necessary, these extracts can be concentrated or dried to give powder, or can be further purified by crystallization from cold water. The tea extract thus obtained is a polyphenol compound contained in tea (particularly tea leaves), that is, tea polyphenols, for example, tea catechins (catechin, epicatechin, gallocatechin, epigallocatechin, catechin gallate, epicatechin gallate, It contains epigallocatechin gallate, gallocatechin gallate) and tea theaflavins (free theaflavin, theaflavin monogallate A, theaflavin monogallate B, theaflavin digallate) as a mixture, and at the same time contains impurities derived from the raw tea.

The anticancer drug resistance inhibitor according to the present invention can be administered with the above-mentioned polyphenol compound or tea extract as it is, or preferably together with a usual carrier which is pharmaceutically acceptable. The dosage form is not particularly limited and includes, for example, powders, fine granules, granules, tablets,
Capsules, suspensions, emulsions, syrups, extracts, oral agents such as pills, injections, external preparations, ointments,
Parenteral agents such as suppositories can be mentioned. These oral preparations include, for example, gelatin, sodium alginate, starch, corn starch, sucrose, lactose, glucose, mannitol, carboxymethyl cellulose, dextrin, polyvinylpyrrolidone, crystalline cellulose, soy lecithin, sucrose fatty acid ester, talc, magnesium stearate, Excipients such as polyethylene glycol, magnesium silicate, silicic acid anhydride, binders, disintegrants, surfactants,
It can be produced according to a conventional method using a lubricant, a fluidity promoter, a diluent, a preservative, a coloring agent, a fragrance and the like. For example, capsules filled with 1% w / w% of catechin and 99 w / w% lactose mixed therein can be used. The anticancer drug resistance inhibitor of the present invention contains the polyphenol compound in an amount of 0.01 to 99% by weight, preferably 0.1 to 80% by weight. Further, the anticancer drug resistance inhibitor containing the tea extract as an active ingredient can be prepared by appropriately adjusting the polyphenol compound contained therein to fall within the above range. In addition,
When the anticancer drug resistance inhibitor containing a tea extract as an active ingredient is to be prepared as a preparation for oral administration, it is preferable to prepare it using a pharmaceutically acceptable carrier.

The dose of the anticancer drug resistance inhibitor of the present invention varies depending on the type of cancer and the degree of symptoms of the patient and is not particularly limited, but the amount of tea polyphenol is usually about 1 mg to 10 g per adult. Is orally or parenterally administered in 1 to 4 times a day. The tea extract used as an active ingredient in the present invention, as well as the above-mentioned polyphenol compound, are ingredients derived from tea that has long been established in human eating habits. It is very excellent in terms of safety. The polyphenol compound used in the present invention was not particularly toxic.

[0017]

The polyphenol compound used as an active ingredient in the anticancer drug resistance inhibitor of the present invention can inhibit and suppress the development of multidrug resistance. Therefore, the anticancer drug resistance inhibitor of the present invention can maintain or enhance the anticancer action originally possessed by the anticancer drug. Further, the above-mentioned polyphenol compounds are not toxic like cyclosporine and Ca ²⁺ antagonists which have been conventionally used as multidrug resistance inhibitors.

[0018]

The present invention will be described in detail below with reference to examples, but these do not limit the scope of the present invention. Example (1) Measurement of cell number-absorbance curve Adriamycin resistant P388 cells (hereinafter P388 /
(Hereinafter referred to as ADM), the passage cells were collected by centrifugation, washed, and then the number of cells was counted.
^It was adjusted to ⁷ cells / ml. Cell solution is serially diluted to 1 × 10 ² cells / ml, 3 × 10 ² cells
s / ml, 1 × 10 ³ cells / ml, 3 × 10 ³ c
ells / ml, 1 × 10 ⁴ cells / ml, 3 × 1
0 ⁴ cells / ml, 1 × 10 ⁵ cells / ml,
3 × 10 ⁵ cells / ml, 1 × 10 ⁶ cells / ml
The volume was adjusted to 3 × 10 ⁶ cells / ml, and 100 μl was injected into each well of a 96-well plate. Immediately, the MTT assay was performed to determine the cell number-absorbance curve. That is, each well has MTT LabelingR
easy (Cell Proliferation
Kit I; Boehringer Cat. No.
1465007) was dispensed in 10 μl aliquots, the plate was tapped to mix, and the mixture was incubated at 37 ° C. in a CO ₂ incubator for 4 hours. Then, 100 μl of solubilization buffer (10% SDS, 0.01N hydrochloric acid solution) was dispensed into each well, and the cells were disrupted by pipetting 5 to 10 times. It was left to stand overnight in a CO ₂ incubator at 37 ° C. Then, the absorbance at a wavelength of 570 nm was measured with a plate reader at a reference wavelength of 630 nm. The P388 medium used in this example is RPMI 1640 (G
IBCO; 11875-036) 100 ml,
Heat inactivated FBS (GIBCO; 16140-02
2) 10 ml, kanamycin sulfate (Meiji Seika; 1 g /
40 ml, Hanks' solution 0.4 ml and 2-mercaptoethanol (10 mM 2-mercaptoethanol;
This is a medium prepared by adding 0.2 ml of Hanks solution).

(2) Chemical treatment Catechin [(+)-Catechin; Funakoshi Co
de No. 0925: manufactured by EXTRASYNTHESE, France] 1.45 g was dissolved in 10 ml of sterilized water (hereinafter, referred to as CA-100.50-fold solution). CA-1
1 ml of the 00 / 50-fold solution was diluted to 10 ml with sterile water (CA-10 / 50-fold solution). CA-10, 50 times liquid 1m
1 was diluted to 10 ml with sterile water (hereinafter referred to as CA-1.5
It is called a 0-fold solution). Subcultured P388 / ADM was counted by a conventional method. Then 1 in 2 25T flasks
Cells in P388 medium were seeded in an amount of × 10 ⁵ cells / 5 ml. In one flask, 0.1 ml of CA-1 · 50 times solution filtered with a 0.22 μm Millipore filter.
(CA-1: catechin final concentration 0.1 m
M). The remaining one flask was not treated with catechin as a control. 3 for both flasks
The cells were cultured in a CO ₂ incubator at 7 ° C for 2 days.

(3) IC ₅₀ measurement The drug-treated cells (CA-1 treated) and control cells in the above (2) were collected by centrifugation and the number of cells was counted by a conventional method. After washing the cells three times with Hank's solution, P388 medium cell solution (1 × 10 ⁵ cells / ml) was prepared for each sample. 100 μl / well (= 10 ⁴ c) of drug-treated cells or control cells were added to the wells of a 96-well microplate.
(ells / well). ADM (Adriamycin; Doxorubicin-HCl; SIGM
A, Code No. D-4035) 200 nM, 6
00 nM, 2 μM, 6 μM, 20 μM, 60 μM, 20
2 ml each of 0 μM and 600 μM physiological saline solutions were prepared. 1/20 volume of each ADM solution prepared to each concentration was added to the cell culture medium [addition amount 5 μl / 100 μl medium], and the mixture was cultured at 37 ° C. for 2 days in a CO ₂ incubator. Then, add MTT Labeling Re to each well.
agent (Cell Proliferation
Kit I; Boehringer Cat. No. 1
465007) was dispensed in 10 μl aliquots, the plate was tapped to mix, and the mixture was incubated at 37 ° C. in a CO ₂ incubator for 4 hours. Then, add solubilization buffer (1
100 μl of 0% SDS 0.01N hydrochloric acid solution) was dispensed, and the cells were disrupted by pipetting 5 to 10 times.
Furthermore, it was left to stand overnight in a CO ₂ incubator at 37 ° C. Then, use a plate reader to set the reference wavelength to 630 nm.
The absorbance at a wavelength of 570 nm was measured.

From this, the relative cell number was calculated by dividing each cell number by the cell number under the condition that the control (without catechin treatment) ADM was not added. The relative cell number at each adriamycin concentration was plotted on the graph, and the adriamycin concentration at which the relative cell number was 0.5 (50%) was determined as IC ₅₀ . The results are shown in Fig. 1. In FIG. 1, the results of the control experiment are shown by ●, and the results of the catechin-treated cells are shown by ■. The IC ₅₀ for ADM in the control experiment was 1953 nM, while the IC ₅₀ for the catechin treatment experiment was 1151 nM and 4
It has decreased by 1.1%. This indicates that the resistance of P388 / ADM to ADM was suppressed by catechin treatment. This indicates that the efficacy of ADM can be restored by co-administering catechin to cancer cells that have acquired resistance to ADM.

[0022]

INDUSTRIAL APPLICABILITY The polyphenol compound or tea extract used as an active ingredient in the present invention strongly suppresses the resistance of anticancer agents expressed in tumor cells. Further, the polyphenol compound and the tea extract used as active ingredients in the present invention are components of tea that has been drunk since ancient times, and tea is an easily available and practically suitable raw material. Since the main component is a natural product that is consumed in a considerable amount in daily life, its safety is high, there is no concern about side effects on the human body, and cyclosporin and Ca ²⁺ antagonists that have been tried to overcome multidrug resistance have been tried. No toxicity was seen. Therefore, the present invention provides a novel and effective anticancer drug resistance inhibitor at a low cost and in a large amount.

[Brief description of drawings]

FIG. 1 is a graph showing the results of comparing the suppression of adriamycin resistance between cells treated with a polyphenol compound according to the present invention and cells not treated with a polyphenol compound.

Claims (3)

[Claims] 1. The number of carbon atoms is 10 to 50 and the number of hydroxyl groups is 5.
20. An anticancer drug resistance inhibitor, comprising: 20 to 20 polyphenol compounds. 2. A compound selected from the group consisting of epigallocatechin gallate, epicatechin gallate, epigallocatechin, epicatechin and isomers thereof, free theaflavin, theaflavin monogallate A, theaflavin monogallate B and theaflavin digallate. The anticancer drug resistance inhibitor according to claim 1, which comprises at least one of: 3. An anticancer drug resistance inhibitor comprising tea water or an organic solvent extract as an active ingredient and a pharmaceutically acceptable carrier.