WO2006057154A1 - Efficacy enhancing agent for anticancer drug - Google Patents

Efficacy enhancing agent for anticancer drug Download PDF

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Publication number
WO2006057154A1
WO2006057154A1 PCT/JP2005/020429 JP2005020429W WO2006057154A1 WO 2006057154 A1 WO2006057154 A1 WO 2006057154A1 JP 2005020429 W JP2005020429 W JP 2005020429W WO 2006057154 A1 WO2006057154 A1 WO 2006057154A1
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Prior art keywords
cells
agent
anticancer
anticancer drug
leukemia
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PCT/JP2005/020429
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French (fr)
Japanese (ja)
Inventor
Yuko Sato
Yukihiko Hara
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Japan as represented by President, International Medical Center of Japan
Mitsui Norin Co., Ltd.
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Publication of WO2006057154A1 publication Critical patent/WO2006057154A1/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/335—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin
    • A61K31/35—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom
    • A61K31/352—Heterocyclic compounds having oxygen as the only ring hetero atom, e.g. fungichromin having six-membered rings with one oxygen as the only ring hetero atom condensed with carbocyclic rings, e.g. methantheline 
    • A61K31/353—3,4-Dihydrobenzopyrans, e.g. chroman, catechin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/33—Heterocyclic compounds
    • A61K31/395—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins
    • A61K31/495—Heterocyclic compounds having nitrogen as a ring hetero atom, e.g. guanethidine or rifamycins having six-membered rings with two or more nitrogen atoms as the only ring heteroatoms, e.g. piperazine or tetrazines
    • A61K31/505—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim
    • A61K31/506—Pyrimidines; Hydrogenated pyrimidines, e.g. trimethoprim not condensed and containing further heterocyclic rings
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K31/00—Medicinal preparations containing organic active ingredients
    • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
    • A61K31/7028—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages
    • A61K31/7034—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin
    • A61K31/704—Compounds having saccharide radicals attached to non-saccharide compounds by glycosidic linkages attached to a carbocyclic compound, e.g. phloridzin attached to a condensed carbocyclic ring system, e.g. sennosides, thiocolchicosides, escin, daunorubicin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K45/00—Medicinal preparations containing active ingredients not provided for in groups A61K31/00 - A61K41/00
    • A61K45/06—Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • A61P35/02—Antineoplastic agents specific for leukemia
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

  • the present invention relates to an agent for enhancing the efficacy of an anticancer agent comprising tea strength techins as an active ingredient, and in particular, effectively enhances the growth inhibitory action of white blood cells.
  • Leukemia is a disease in which leukemia cells in the bone marrow autonomously proliferate, so-called hematologic cancer, which causes anemia, leukocytosis, thrombocytopenia and the like, and is often associated with other organs such as the liver, spleen, and lymph nodes. It is known to create secondary infiltration foci or secondary hematopoietic foci.
  • Cytocinara binoside, brednisolone, daunorubicin and the like have long been used as such chemotherapy for leukemia.
  • daunorubicin (trade name: daunomycin, Meiji Seika Kaisha, Ltd.) is an antibiotic discovered from actinomycetes in 1963 and is widely used for the treatment of acute leukemia and chronic myeloid leukemia during the blast crisis. Due to the high cardiotoxicity of daunorubicin, the amount used is currently limited to 400 mg / m 2 in total.
  • imatinib a selective competitive inhibitor for ABL tyrosine kinase, trade name: Daribec, Novartis Pharma Ltd.
  • Daribec a selective competitive inhibitor for ABL tyrosine kinase
  • imatinib is less likely to cause side effects, it is generally expensive, and the patient's financial burden is heavy. Recently, the emergence of imatinib resistance has been considered as a clinical problem. Imatinib resistance has been reported to appear in approximately 5 to 10% of chronic myelogenous leukemia patients who have not been treated so far, and this resistant clone is not originally induced by imatinib and the patient's body from the beginning. It is thought that it will become more pronounced by taking imatinib, a small amount of clonal strength S that was present in Therefore, the power to reduce the use of imatinib in order to reduce the frequency of appearance of resistant clones has not been resolved yet.
  • catechins which are the main components of tea, are known to have anti-cancer activity, in particular tumor cell growth inhibitory activity, and for example, plant-derived polyphenols are superior to leukemia cells. It exhibits a growth inhibitory action (see Patent Document 1), that epigallocatechin gallate suppresses the survival and proliferation of K-562 cells derived from human chronic myelogenous leukemia (see Non-patent Document 1), and green tea It has been reported that polyphenol-pygallocatechin gallate suppresses the growth of adult T-cell white blood disease (see Non-Patent Document 2). Recently, it has been reported that use of epigallocatechin gallate for chronic lymphocytic leukemia provides a clinically significant inhibitory effect on tumor cell growth (see Non-patent Document 3).
  • Patent Document 1 Japanese Patent Application Laid-Open No. 2001-226276
  • Non-Patent Document 1 Mutation Research, 523-524, p. 33-41, 2003
  • Non Patent Literature 3 Blood, vol. 104, no. 3, p. 788-794, 2004
  • the object of the present invention is to reduce the amount of expensive anticancer drug which causes problems such as emergence of resistance, and to significantly enhance the anticancer activity of the anticancer drug, particularly the leukemia cell proliferation inhibitory activity, To provide an agent for enhancing the efficacy of
  • the present inventors have surprisingly found that using tea strength technics in combination with an anticancer drug results in the same or a large amount of anticancer drug administration. It has been found that the above-mentioned anticancer activity, in particular, tumor cell proliferation inhibitory activity can be obtained.
  • This anti-cancer action in particular tumor cell growth inhibitory action, is not only the power when tea catechins are administered alone, but it is an extremely remarkable action that is extremely unexpected when the anticancer drug is administered alone.
  • particularly remarkable use of a leukemia therapeutic agent as an anticancer agent exerts a remarkable effect of suppressing tumor cell proliferation.
  • tea catechins are useful as an active ingredient of an anticancer drug efficacy enhancer, and reached the present invention.
  • the present invention according to claim 1 is an agent for enhancing the efficacy of an anticancer agent which comprises tea strength techins as an active ingredient.
  • the present invention according to claim 2 is the efficacy enhancer of the anticancer agent according to claim 1, wherein the anticancer agent is a leukemia therapeutic agent.
  • the present invention according to claim 3 is the efficacy enhancer of the anticancer agent according to claim 2, wherein the therapeutic agent for leukemia is imatinib and / or daunorubicin.
  • the present invention according to claim 4 is the anticancer agent according to any one of claims 1 to 3, wherein the tea catechins are at least one selected from the group consisting of strength teinging gallate, epicatheking gallate, gallocatechin gallate and epigallocatechin gallate. Is an efficacy enhancer.
  • the efficacy enhancer of the anticancer agent of the present invention when used in combination with an anticancer agent, causes the tea catechins and the anticancer agent, which are active ingredients, to act synergistically in vivo. Therefore, tea catechins alone or anticancer agents alone are administered.
  • the anticancer effect obtained by the present invention results in an unexpected synergistic effect and exhibits a remarkable tumor cell growth inhibitory effect. As a result, even if the dose of the anticancer drug is reduced, the sufficient anticancer effect can be obtained, so the incidence rate of adverse events possessed by the anticancer drug can be reduced strongly, and the patient can take it safely for a long time. It can contribute significantly to improving the patient's quality of life (QOL).
  • the efficacy enhancer of the anticancer agent of the present invention can reduce the amount used even when used in combination with a molecule-targeted drug such as imatinib, so that medical costs can be saved and the economy can be reduced. It is also possible to increase the effectiveness of the drug and reduce the frequency of appearance of resistant clones.
  • the immunostimulatory action in the patient's body can also be expected by the following mechanism. That is, with imatinib alone, BCR—ABL-positive cells were only put into apoptosis. In combination with the virus, the fraction of cells that undergo necrosis increases, and BCR ABL-positive cells that have fallen into necrosis stimulate the antigen-presenting cells (mainly sickle cells) in the body, and the BCR-ABL antigen becomes dendritic cells. It is thought that it can be presented on the surface of the
  • FIG. 1 shows changes in the number of living cells in culture in each additive drug group of Example 1.
  • FIG. 2 shows the form of cell death in each additional drug group in Example 1.
  • FIG. 3 shows the change in the number of viable cells in culture in each additional drug group in Example 2.
  • Tea catechins used as active ingredients of the efficacy enhancers of the anticancer agent of the present invention include catechins (C), gallocatenins (GC), strengths teken gallate (Cg), gallocatenins gallate (GC g), epicathekin (EC), epigalocatenin. (EGC), epiphytic tequine gallate (ECg) and epigallocatechin gallate (EGCg), which may be used alone or in combination of two or more at a desired mixing ratio.
  • EGCg can be mentioned as a particularly preferred one.
  • These tea catechins are in the form of (+)-body or (one) in one part, or in one part, off.
  • Such tea catechins are mainly leaves obtained from tea plants (Camellia sinensis) belonging to the camellia family, stems, wood parts, barks, roots, fruits, seeds, or two or more of these. It is contained in an extract obtained by extracting a mixture or a powder thereof using water, hot water, an organic solvent, a water-containing organic solvent, or a mixture thereof, and in the present invention, the extract is contained in the present invention. It can be used as tea catechins as it is. In particular, an extract obtained by extracting green tea leaves or dried product thereof with water, hot water, an organic solvent, a water-containing organic solvent, a mixture thereof or the like can be preferably used.
  • the purified product obtained by further purifying the extract can also be used as tea catechins.
  • the Japanese Patent Publication Nos. 1-44234, 2-12474, 2-2275 It can manufacture by the method described in the 5th publication, the Unexamined-Japanese-Patent No. 4-20589, the 5-260907, the 8-109178 publication etc., for example, a tea leaf is extracted with said solvent,
  • the obtained extract can be obtained by purification to a desired degree using an organic solvent fraction, an adsorption resin or the like.
  • tea catechins used in the present invention is not particularly limited, and any of liquids such as the above-mentioned extract and purified product and solid (including powder) can be used.
  • it is good even if it uses a commercial product of tea catechins.
  • commercially available products for example, "Polifone Non” manufactured by Mitsui Norin Co., Ltd., “Saif von Non” manufactured by Taiyo Kagaku Co., Ltd., Itoen Co., Ltd. "Theafran”, etc. can be exemplified.
  • any anticancer agent which is the target of the efficacy enhancer, may be any generally known one, or may be used singly or in combination of two or more. Preferred to be a therapeutic agent.
  • Leukemia is a disease in which leukemia cells in the bone marrow autonomously proliferate, so-called hematological cancers, and is classified as myeloid, lymphoid, monocytic, etc. depending on which lineage of the hematopoietic cells the proliferating cells have. Ru. It is also classified into acute leukemia, chronic leukemia, etc. depending on whether it has a clinically rapid course or a liberal course.
  • any agent that promotes anti-cancer activity such as growth inhibitory activity to cells involved in leukemia belonging to the above-mentioned classification can be used.
  • leukemia caused by the BCR-ABL gene that is, Ph (Philadelphia chromosome) -positive myeloid leukemia cells (for example, TCC S cells (BCR-ABL-positive chronic myeloid leukemia-derived cell line) )
  • BCR-ABL gene is a fusion gene produced by reciprocal translocation between chromosome 9 and chromosome 22, and its gene product is a direct cause of chronic myelogenous leukemia etc. It has been known.
  • leukemia therapeutic agents for example, vinblastine, vincristine, cytosarabinoside, bredonizolone, 6MP, endoxane, adriamycin, daunorubicin, imatinib and the like can be mentioned, among which daunorubicin and Z or imatinib can be mentioned. Is preferred.
  • Daunorubicin (Daunombicin) is an anthracycline antibiotic discovered in 1963 from the actinomycete Streptomyces coerleor uidus strain, and the chemical name is 7-(3-amin o -2, 2, 6 _trideoxy_L_lyxohexosyloxyno _ 9 _acetyl _ 7, 8, 9, 10-tetrahydro-6, 9, 11-tnny droxy-4-methoxy-5, 12-napnthacenezuinon e hydrochloride, also called daunorubicin hydrochloride (brand name: daunomycin, Meiji Seika) It inhibits the synthesis of nucleic acid and is used for the treatment of acute leukemia and acute blast of chronic myelogenous leukemia.
  • daunorubicin The mechanism of action of daunorubicin is to bind to DNA to inhibit nuclear polymerase activity and to cleave the DNA strand by topoisomerase II inhibition. This causes serious damage to all normal cells (such as bone marrow cells, intestinal epithelial cells, and hair root cells) that are synthesizing DNA. It is also known to exert cardiotoxicity in a dose-dependent manner.
  • Imatinib is a product name of Imatinib mesylate (trade name: Daribec, Novartis Pharma Ltd.), which was released in Japan on December 7, 2001, and its chemical name is Benza mide, 4-[ (4-methyl- ⁇ ⁇ ⁇ piperazmyl) methyl J-N- [4-metnyl-3- [[4- (3-pyridinyl) -2-pyrimidinyl] amino] phenyl]-, monomethanesulfo nate, ATP of the ABL gene It attracts attention as a molecular target anticancer agent which specifically inhibits tyrosine kinase activity possessed by BCR-ABL protein which is a product of BCR-ABL gene by competitively binding to the binding site with ATP.
  • BCR-ABL gene is known as a direct onset causative gene such as chronic myelogenous leukemia as described above, specifically suppressing its tyrosine kinase activity is extremely important for leukemia treatment. It is a reasonable treatment.
  • the use form of the efficacy enhancer of the present invention is not particularly limited as long as the tea catechins and the anticancer agent are used in combination with the anticancer agent so as to act cooperatively in vivo.
  • the efficacy enhancer of the present invention and the anticancer agent may be contained in a single preparation, or a preparation containing each agent may be prepared and combined at the time of use.
  • the anticancer agent or the anticancer agent precedes after the efficacy enhancer without prior administration of the efficacy enhancer and the anticancer agent according to the present invention. It may be in a form in which the efficacy enhancer is administered after being administered.
  • the administration method of the efficacy enhancer of the anticancer agent of the present invention may be either oral or parenteral.
  • the efficacy enhancer of the present invention and the anticancer agent are contained in a single preparation, when daunorubicin is used as the anticancer agent, the administration is limited to administration at rest.
  • the form of the efficacy enhancer of the present invention can be, for example, an orally administered agent such as a powder, a granule, a tablet, a pill, a capsule, a solution, a syrup and the like.
  • an orally administered agent such as a powder, a granule, a tablet, a pill, a capsule, a solution, a syrup and the like.
  • preparation for oral administration include: tea catechins, which are active ingredients; if desired, excipients, binders, disintegrants, lubricants, diluents, isotonic agents, etc.
  • the additives conventionally used for oral administration can be appropriately blended and manufactured by formulating them according to known methods.
  • examples of the form of the efficacy enhancer of the present invention for parenteral administration include injections, suppositories and the like.
  • a solubilizing agent, a buffer, a tonicity agent, a stabilizer, a preservative, a soothing agent, etc. may be added to the active ingredient tea catechins, if necessary. It can be manufactured S by appropriately blending additives commonly used in the agent.
  • the dose is effective based on the degree of disease to be treated, patient's age, body weight, type of anticancer agent, dosage, etc.
  • the effective amount of tea catechins (that is, the amount capable of exerting the desired effect of the present invention) which is an ingredient may be appropriately determined so as to be administered.
  • the amount of tea catechins is administered within the range of 0.01 to 100 mg / kg (body weight) per day. Even when the tea strength techin is administered within the above range, a sufficient anti-cancer effect can not be obtained, and a sufficient anti-cancer effect is brought about only by using it in combination with an anticancer agent.
  • the dose of the anticancer drug to be used in combination can be appropriately determined according to the type of the anticancer drug.
  • the weight ratio of the efficacy enhancer and the anticancer drug of the present invention generally depends on the type of the anticancer drug etc.
  • the tea strength techin contained in the efficacy enhancer of the present invention is 1, the leukemia therapeutic agent can be 0. 000001 to:!.
  • tea catechins 1 against 0. 0001 ⁇ ! Preferably 0. 0001 to 0.1.
  • daunorubicin daunorubicin (daunomycin) it is possible to make it 0.01. 1 to 1, preferably 0. 001-0. 1 with respect to 1 tea catechins.
  • the dosage form can be adjusted as appropriate depending on the condition of the patient, either daily administration or intermittent administration.
  • the efficacy enhancer of the anticancer agent of the present invention when used in combination with an anticancer agent, causes tea catechins, which are active ingredients, to act jointly in vivo with the anticancer agent, as shown in the examples below.
  • tea catechins which are active ingredients
  • the efficacy of anticancer agents can be synergistically increased by tea catechins. This not only enables efficient treatment of cancer, but also enables effective treatment for cases in which no effect was obtained by single administration of an anticancer agent. Furthermore, it is possible to reduce the dose of the anticancer drug to one fifth to one tenth.
  • the efficacy enhancer of the present invention can enhance various aspects of the efficacy of the therapeutic agent for leukemia.
  • the efficacy of leukemia therapeutics is enhanced in various ways, such as enhancing the growth suppression of cells involved in leukemia, enhancing cell death, and enhancing tyrosine kinase activity suppression. Therefore, it can be used in various applications in expectation of exerting such various enhancement functions, but in particular, it is preferable to use for the purpose of enhancing the growth suppression of leukemia cells.
  • imatinib which is a leukemia therapeutic agent
  • the combination of power of imatinib which only caused BCR-ABL positive cells to undergo apoptosis, and the efficacy enhancer of the present invention.
  • antigen-presenting cells mainly dendritic cells
  • the combination of imatinib and the efficacy enhancer of the present invention results in an increase in the fraction of cells suffering from necrosis.
  • Production Example 1 Production Method of Tea Catechins
  • Dried green tea leaves 270 kg 1890 liters 80 After extracting with C hot water, add an additional 1350 L of 80. C Extraction was performed with hot water. The extract was cooled to 15 ° C. or less and concentrated with an RO membrane to obtain 500 L of a concentrate. The concentrate was extracted with 1000 L of ethyl acetate, and the ethyl acetate layer was concentrated to 75 kg, which was dried to obtain 15 kg of a crude tea extract.
  • This fraction was subjected to circulation concentration using a centrifugal thin film concentrator (manufactured by Ohkawara Seisakusho Co., Ltd.) to obtain about 20 L of a concentrated solution.
  • the concentrated solution was spray-dried by a spray dryer (manufactured by Two-Piece) to obtain 4.8 kg of tea catechins.
  • Component analysis of the tea catechins obtained in this manner was carried out under the following conditions by high performance liquid chromatography. I did.
  • the composition of tea catechins is shown in Table 1.
  • Test Example 1 Examination of combined effect of imatinib and tea catechins
  • TCC S cells BCR-derived from AML positive chronic myeloid leukemia
  • ECCg green tea-derived epigarocatechin gallate
  • BCR-ABL kinase selective inhibitor molecular target anticancer drug
  • TCC- S cells were cultured at 37 ° C. in the presence of 5% CO at a concentration of 5 ⁇ 3 cells in RPMI-1640 medium containing 10% FCS, and the viable cells were 95%. % Or more
  • TCC-S cells (concentration 2 ⁇ 10 5 1111) 41111 in this state are aliquoted into 14 wells using a 6 ⁇ ⁇ 11 plate, and as shown in Table 2 or FIG. (2) a group to which only imatinib (0. ⁇ ) was added, (3) a group to which only imatinib (0.5 ⁇ ) was added, (4) a group to which only EGCg (50 ⁇ ) was added, (5) A group to which only EGCg (100 ⁇ M) was added, a group to which (6) imatinib (0.
  • a drug-free group a group to which only imatinib (0.5 ⁇ m) was added, a group to which only EGCg (200 ⁇ m) was added, imatinib (0.1 / i M) + EGCg (5 ⁇ m)
  • cytospin using 5 ⁇ 10 2 to 10 3 / slide cells The specimens were prepared and stained with Wright's Giemsa stain and then observed under an optical microscope. The results are shown in Table 3 or Figure 2.
  • the combination addition group of imatinib + EGCg of (6) and (7) the number of cells after 72 hours is only 0.17 ⁇ 10 5 cells / ml, and the addition group (1) to As compared with any of 5), the combination of imatinib and EGCg can be used to obtain a remarkable growth inhibitory effect on TCC-S cells, which is not a simple additive effect but a synergistic effect. It became clear.
  • Test Example 2 Examination of combined effect of daunorubicin and tea catechins
  • Daunomycin which is one of the representative anticancer agents used for white blood disease and solid tumors, using TCC-Y cells (BCR-ABL positive cell line derived from acute lymphocytic leukemia).
  • TCC-Y cells BCR-ABL positive cell line derived from acute lymphocytic leukemia.
  • DNR The tumor cell growth inhibitory effect was examined when combining with EGCg.
  • TCC-Y cells RPMI-16 containing 10% FCS to a concentration of 3 x 10 5 cells / ml Cultivate in 40% medium at 37 ° C in the presence of 5% CO, with 95% or more of viable cells
  • the efficacy enhancer of the anticancer agent of the present invention can be used in combination with the anticancer agent, whereby the tea catechins and the anticancer agent, which are active ingredients, act jointly in vivo. Therefore, tea catechins alone or anticancer agents alone can be administered.
  • the anticancer effect obtained by the present invention results in an unexpected synergistic effect and exhibits a remarkable tumor cell growth inhibitory effect. As a result, even if the dose of the anticancer drug is reduced, sufficient anticancer effect can be obtained, so the incidence rate of adverse events possessed by the anticancer drug can be reduced to an extent that the patient can take it safely for a long time. Can significantly contribute to the improvement of patients' quality of life (QOL).
  • the efficacy enhancer of the anticancer agent of the present invention can reduce the amount used even when used in combination with a molecule-targeted drug such as imatinib, so that medical costs can be saved and the economy can be reduced. It is also possible to increase the effectiveness of the drug and reduce the frequency of appearance of resistant clones.
  • the immunostimulatory action in the patient's body can also be expected by the following mechanism. That is, with imatinib alone, BCR-ABL-positive cells were only put into apoptosis S, and the combination of imatinib and force-techin increased the fraction of cells in necrosis, resulting in necrosis. BCR-ABL It is thought that positive cells can stimulate antigen-presenting cells (mainly dendritic cells) in the body to present the BCR-ABL antigen on the surface of dendritic cells and activate the immunity.
  • antigen-presenting cells mainly dendritic cells

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Abstract

A novel efficacy enhancing agent for anticancer drug that realizes reduction of the usage of expensive anticancer drugs posing the problem of tolerance emergence, etc. and that is capable of strikingly enhancing the antitumor activity, especially leukemic cell multiplication inhibiting activity, of anticancer drug. There are provided an efficacy enhancing agent for anticancer drug, comprising a tea catechin as an active ingredient; such an efficacy enhancing agent for anticancer drug, wherein the anticancer drug is a therapeutic agent for leukemia; such an efficacy enhancing agent for anticancer drug, wherein the therapeutic agent for leukemia is imanitib and/or daunorubicin; and such an efficacy enhancing agent for anticancer drug, wherein the tea catechin is at least one member selected from the group consisting of catechin gallate, epicatechin gallate, gallocatechin gallate and epigallocatechin gallate.

Description

明 細 書  Specification
抗癌剤の効力増強剤  Potentiator of anticancer drug
技術分野  Technical field
[0001] 本発明は、茶力テキン類を有効成分とする抗癌剤の効力増強剤に関し、特に、白 血病細胞の増殖抑制作用を効果的に増強する。  TECHNICAL FIELD [0001] The present invention relates to an agent for enhancing the efficacy of an anticancer agent comprising tea strength techins as an active ingredient, and in particular, effectively enhances the growth inhibitory action of white blood cells.
背景技術  Background art
[0002] 白血病は骨髄の白血病細胞が自律増殖する疾患、いわゆる血液の癌であって、貧 血、白血球増多症、血小板減少症などを起こし、しばしば肝臓、脾臓、リンパ節など の他の臓器に二次的浸潤病巣又は二次的造血病巣を創ることが知られている。  [0002] Leukemia is a disease in which leukemia cells in the bone marrow autonomously proliferate, so-called hematologic cancer, which causes anemia, leukocytosis, thrombocytopenia and the like, and is often associated with other organs such as the liver, spleen, and lymph nodes. It is known to create secondary infiltration foci or secondary hematopoietic foci.
[0003] このような白血病の化学治療薬として古くから使用されているものに、サイトシンァラ ビノシド、ブレドニゾロン、ダウノルビシン等がある。このうち、ダウノルビシン（商品名： ダウノマイシン、明治製菓株式会社)は、 1963年に放線菌から発見された抗生物質 で、急性白血病、慢性骨髄性白血病の急性転化期などの治療に広く用いられている 、ダウノルビシンは心毒性が強いことから、その使用量は総量で 400mg/m2とレ、う 制限を受けているのが現状である。 [0003] Cytocinara binoside, brednisolone, daunorubicin and the like have long been used as such chemotherapy for leukemia. Among them, daunorubicin (trade name: daunomycin, Meiji Seika Kaisha, Ltd.) is an antibiotic discovered from actinomycetes in 1963 and is widely used for the treatment of acute leukemia and chronic myeloid leukemia during the blast crisis. Due to the high cardiotoxicity of daunorubicin, the amount used is currently limited to 400 mg / m 2 in total.
[0004] また最近では、分子標的薬剤であるイマチニブ (ABLチロシンキナーゼに対する選 択的競合阻害剤、商品名：ダリベック、ノバルティスファーマ株式会社)が臨床導入さ れ、その驚異的な奏功性により、慢性骨髄性白血病においては治療概念の大きな変 革をもたらしてレ、る。  [0004] Also, recently, imatinib (a selective competitive inhibitor for ABL tyrosine kinase, trade name: Daribec, Novartis Pharma Ltd.), which is a molecular target drug, has been clinically introduced and its chronic response has led to its remarkable response. In myeloid leukemia, it brings about a major change in the concept of treatment.
イマチニブは副作用の心配は少ないものの、一般的に高価であるため、患者の経 済的負担が大きい。また、最近はイマチニブ耐性の出現が臨床的に問題とされてい る。イマチニブ耐性は、これまで未治療であった慢性骨髄性白血病患者の約 5〜10 %に出現する、と報告されており、この耐性クローンは、イマチニブによって誘導され たものではなぐ最初から患者の体内に存在していた微少のクローン力 S、イマチニブ 服用によって顕性化してくる、と考えられている。従って、耐性クローンの出現頻度を 低下させるために、イマチニブ使用量の減量ィ匕が望まれている力 未だその解決に は至っていない。 [0005] 一方、茶の主要成分であるカテキン類は、抗癌作用、特に腫瘍細胞増殖抑制作用 を有することが知られており、例えば、植物由来のポリフエノール類が白血病細胞に 対して優れた増殖阻害作用を示すこと（特許文献 1参照）や、ェピガロカテキンガレー トがヒト慢性骨髄性白血病由来の K一 562細胞の生存及び増殖を抑制すること（非特 許文献 1参照）や、緑茶ポリフエノールゃェピガロカテキンガレートが成人 T細胞性白 血病の増殖を抑制すること (非特許文献 2参照）が報告されている。最近では、慢性リ ンパ性白血病にェピガロカテキンガレートを使用して、臨床的に顕著な腫瘍細胞増 殖抑制効果が得られる（非特許文献 3参照）との報告もある。 Although imatinib is less likely to cause side effects, it is generally expensive, and the patient's financial burden is heavy. Recently, the emergence of imatinib resistance has been considered as a clinical problem. Imatinib resistance has been reported to appear in approximately 5 to 10% of chronic myelogenous leukemia patients who have not been treated so far, and this resistant clone is not originally induced by imatinib and the patient's body from the beginning. It is thought that it will become more pronounced by taking imatinib, a small amount of clonal strength S that was present in Therefore, the power to reduce the use of imatinib in order to reduce the frequency of appearance of resistant clones has not been resolved yet. On the other hand, catechins, which are the main components of tea, are known to have anti-cancer activity, in particular tumor cell growth inhibitory activity, and for example, plant-derived polyphenols are superior to leukemia cells. It exhibits a growth inhibitory action (see Patent Document 1), that epigallocatechin gallate suppresses the survival and proliferation of K-562 cells derived from human chronic myelogenous leukemia (see Non-patent Document 1), and green tea It has been reported that polyphenol-pygallocatechin gallate suppresses the growth of adult T-cell white blood disease (see Non-Patent Document 2). Recently, it has been reported that use of epigallocatechin gallate for chronic lymphocytic leukemia provides a clinically significant inhibitory effect on tumor cell growth (see Non-patent Document 3).
しかし、カテキン類自体の抗癌作用は、前記した抗癌剤と比べると十分に強いとは いえなかった。  However, the anticancer activity of catechins itself is not sufficiently strong compared to the above-mentioned anticancer agents.
[0006] 特許文献 1 :特開 2001— 226276公報  Patent Document 1: Japanese Patent Application Laid-Open No. 2001-226276
非特許文献 1 : Mutation Research, 523 - 524, p. 33 -41 , 2003  Non-Patent Document 1: Mutation Research, 523-524, p. 33-41, 2003
非特午文献 2 Japanese Journal oi Cancer Research, vol. 9丄 , p. 34— 40, 2000  Non-Japanese Literature 2 Japanese Journal oi Cancer Research, vol. 9 丄, p. 34— 40, 2000
非特許文献 3 : Blood, vol. 104, no. 3, p. 788 - 794, 2004  Non Patent Literature 3: Blood, vol. 104, no. 3, p. 788-794, 2004
発明の開示  Disclosure of the invention
発明が解決しょうとする課題  Problem that invention tries to solve
[0007] 本発明の目的は、高価で耐性出現等の問題のある抗癌剤の使用量を減らし、かつ 、抗癌剤の抗癌作用、特に白血病細胞増殖抑制作用を著しく増強することができる、 新規な抗癌剤の効力増強剤を提供することにある。 [0007] The object of the present invention is to reduce the amount of expensive anticancer drug which causes problems such as emergence of resistance, and to significantly enhance the anticancer activity of the anticancer drug, particularly the leukemia cell proliferation inhibitory activity, To provide an agent for enhancing the efficacy of
課題を解決するための手段  Means to solve the problem
[0008] 本発明者らは上記目的を達成すべく鋭意検討を重ねた結果、驚くべきことに、茶力 テキン類を抗癌剤と併用することにより、抗癌剤を多量に投与したのと同様或いはそ れ以上の抗癌作用、特に腫瘍細胞増殖抑制作用を得られることを見出した。この抗 癌作用、特に腫瘍細胞増殖抑制作用は、茶カテキン類単独投与の場合力 はもちろ ん、抗癌剤を単独投与した場合力 も全く予想外の特段に優れた作用であり、茶カテ キン類が抗癌剤の効力を増強し、その結果もたらされた相乗効果であるものと推測さ れた。 そして、抗癌剤として特に白血病治療剤を用いると、特に顕著な腫瘍細胞増殖抑 制効果が発揮されることをも見出した。 [0008] As a result of intensive studies to achieve the above object, the present inventors have surprisingly found that using tea strength technics in combination with an anticancer drug results in the same or a large amount of anticancer drug administration. It has been found that the above-mentioned anticancer activity, in particular, tumor cell proliferation inhibitory activity can be obtained. This anti-cancer action, in particular tumor cell growth inhibitory action, is not only the power when tea catechins are administered alone, but it is an extremely remarkable action that is extremely unexpected when the anticancer drug is administered alone. Was supposed to enhance the efficacy of the anticancer drug, resulting in the resulting synergistic effect. And, it was also found that particularly remarkable use of a leukemia therapeutic agent as an anticancer agent exerts a remarkable effect of suppressing tumor cell proliferation.
これらの知見から、本発明者らは茶カテキン類が抗癌剤の効力増強剤の有効成分 として有用であると考え、本発明に到達した。  From these findings, the present inventors considered that tea catechins are useful as an active ingredient of an anticancer drug efficacy enhancer, and reached the present invention.
[0009] 即ち、請求項 1記載の本発明は、茶力テキン類を有効成分とする抗癌剤の効力増 強剤である。  [0009] That is, the present invention according to claim 1 is an agent for enhancing the efficacy of an anticancer agent which comprises tea strength techins as an active ingredient.
請求項 2記載の本発明は、抗癌剤が白血病治療剤である請求項 1記載の抗癌剤の 効力増強剤である。  The present invention according to claim 2 is the efficacy enhancer of the anticancer agent according to claim 1, wherein the anticancer agent is a leukemia therapeutic agent.
請求項 3記載の本発明は、白血病治療剤が、イマチニブ及び/又はダウノルビシ ンである請求項 2記載の抗癌剤の効力増強剤である。  The present invention according to claim 3 is the efficacy enhancer of the anticancer agent according to claim 2, wherein the therapeutic agent for leukemia is imatinib and / or daunorubicin.
請求項 4記載の本発明は、茶カテキン類が、力テキンガレート、ェピカテキンガレー ト、ガロカテキンガレート及びェピガロカテキンガレートから選ばれる少なくとも 1種以 上である請求項 1乃至 3記載の抗癌剤の効力増強剤である。  The present invention according to claim 4 is the anticancer agent according to any one of claims 1 to 3, wherein the tea catechins are at least one selected from the group consisting of strength teinging gallate, epicatheking gallate, gallocatechin gallate and epigallocatechin gallate. Is an efficacy enhancer.
発明の効果  Effect of the invention
[0010] 本発明の抗癌剤の効力増強剤は、抗癌剤と組み合わせて用いることにより、有効 成分である茶カテキン類と抗癌剤が生体内で共同的に作用するので、茶カテキン類 単独投与あるいは抗癌剤単独投与により得られる抗癌作用からは予想外の相乗効 果カもたらされ、顕著な腫瘍細胞増殖抑制効果を呈する。その結果、抗癌剤の投与 量を減量しても十分な抗癌効果が得られるため、抗癌剤の持つ有害事象の出現率を 力、なりの程度低減することができ、患者は長期間安心して服用することができ、患者 の QOL (Quality of life)向上に大きく貢献することができる。  [0010] The efficacy enhancer of the anticancer agent of the present invention, when used in combination with an anticancer agent, causes the tea catechins and the anticancer agent, which are active ingredients, to act synergistically in vivo. Therefore, tea catechins alone or anticancer agents alone are administered. The anticancer effect obtained by the present invention results in an unexpected synergistic effect and exhibits a remarkable tumor cell growth inhibitory effect. As a result, even if the dose of the anticancer drug is reduced, the sufficient anticancer effect can be obtained, so the incidence rate of adverse events possessed by the anticancer drug can be reduced strongly, and the patient can take it safely for a long time. It can contribute significantly to improving the patient's quality of life (QOL).
さらに、本発明の抗癌剤の効力増強剤は、イマチニブ等の分子標的薬剤と併用す る場合も、その使用量を従来よりも少なくさせることができるので、医療費の節減が可 能であり、経済的な効果が大きぐまた耐性クローンの出現頻度を低下させることも可 能である。  Furthermore, the efficacy enhancer of the anticancer agent of the present invention can reduce the amount used even when used in combination with a molecule-targeted drug such as imatinib, so that medical costs can be saved and the economy can be reduced. It is also possible to increase the effectiveness of the drug and reduce the frequency of appearance of resistant clones.
そして、本発明の効力増強剤をイマチニブと併用する場合、以下の機序により、患 者体内における免疫賦活作用も期待することができる。即ち、イマチニブのみでは、 BCR— ABL陽性細胞をアポトーシスに陥らせるのみであった力 イマチニブと力テキ ンとの併用により、ネクローシスに陥る細胞分画が増加し、ネクローシスに陥った BCR ABL陽性細胞が体内の抗原提示細胞（主に榭状細胞）を刺激して、 BCR-ABL 抗原を樹状細胞の表面に提示させ、免疫能を賦活させることができる、と考えられる 図面の簡単な説明 When the efficacy enhancer of the present invention is used in combination with imatinib, the immunostimulatory action in the patient's body can also be expected by the following mechanism. That is, with imatinib alone, BCR—ABL-positive cells were only put into apoptosis. In combination with the virus, the fraction of cells that undergo necrosis increases, and BCR ABL-positive cells that have fallen into necrosis stimulate the antigen-presenting cells (mainly sickle cells) in the body, and the BCR-ABL antigen becomes dendritic cells. It is thought that it can be presented on the surface of the
[0011] [図 1]実施例 1の各添加薬剤群における培養中の生細胞数の変化を示す。  FIG. 1 shows changes in the number of living cells in culture in each additive drug group of Example 1.
[図 2]実施例 1の各添加薬剤群における細胞死の形態を示す。  FIG. 2 shows the form of cell death in each additional drug group in Example 1.
[図 3]実施例 2の各添加薬剤群における培養中の生細胞数の変化を示す。  FIG. 3 shows the change in the number of viable cells in culture in each additional drug group in Example 2.
発明を実施するための最良の形態  BEST MODE FOR CARRYING OUT THE INVENTION
[0012] 以下に、本発明について詳細に説明する。  Hereinafter, the present invention will be described in detail.
本発明の抗癌剤の効力増強剤の有効成分として用いられる茶カテキン類としては、 カテキン（C)、ガロカテキン (GC)、力テキンガレート（Cg)、ガロカテキンガレート（GC g)、ェピカテキン (EC)、ェピガロカテキン（EGC)、ェピ力テキンガレート（ECg)及び ェピガロカテキンガレート (EGCg)が挙げられ、これらを単独もしくは 2種以上を所望 とする混合比で組み合わせて用いることができる。このうち、 Cg、 ECg、 GCg及び EG Cgから選ばれる少なくとも 1種以上を用いることが好ましい。さらに、特に好ましいも のとして EGCgを挙げることができる。これら茶カテキン類は、（+ )—体又は（一）一 体のレ、ずれであってもよレ、。  Tea catechins used as active ingredients of the efficacy enhancers of the anticancer agent of the present invention include catechins (C), gallocatenins (GC), strengths teken gallate (Cg), gallocatenins gallate (GC g), epicathekin (EC), epigalocatenin. (EGC), epiphytic tequine gallate (ECg) and epigallocatechin gallate (EGCg), which may be used alone or in combination of two or more at a desired mixing ratio. Among these, it is preferable to use at least one selected from Cg, ECg, GCg and EG Cg. Furthermore, EGCg can be mentioned as a particularly preferred one. These tea catechins are in the form of (+)-body or (one) in one part, or in one part, off.
[0013] このような茶カテキン類は、主にツバキ科に属する茶樹（Camellia sinensis)から 得られる葉、茎、木部、樹皮、根、実、種子のいずれか、あるいはこれらの 2種類以上 の混合物もしくはそれらの粉碎物から、水、熱水、有機溶媒、含水有機溶媒あるいは これらの混合物などを用いて抽出して得られる抽出物に含まれており、本発明にお いては、該抽出物自体を茶カテキン類としてそのまま利用することができる。特に、生 の茶葉あるいはその乾燥物から、水、熱水、有機溶媒、含水有機溶媒、これらの混合 物などを用いて抽出して得られる抽出物を好ましく用いることができる。  [0013] Such tea catechins are mainly leaves obtained from tea plants (Camellia sinensis) belonging to the camellia family, stems, wood parts, barks, roots, fruits, seeds, or two or more of these. It is contained in an extract obtained by extracting a mixture or a powder thereof using water, hot water, an organic solvent, a water-containing organic solvent, or a mixture thereof, and in the present invention, the extract is contained in the present invention. It can be used as tea catechins as it is. In particular, an extract obtained by extracting green tea leaves or dried product thereof with water, hot water, an organic solvent, a water-containing organic solvent, a mixture thereof or the like can be preferably used.
[0014] また、本発明においては、上記したような茶榭から得られる抽出物の他に、該抽出 物をさらに精製して得られる精製物を、茶カテキン類として利用することもできる。 精製物に関しては、特公平 1—44234号公報、同 2— 12474号公報、同 2— 2275 5号公報、特開平 4— 20589号公報、同 5— 260907号公報、同 8— 109178号公 報などに記載された方法により製造することができ、例えば、茶葉を上記の溶媒で抽 出して得られた抽出物を、有機溶媒分画や吸着樹脂などを用いて所望の程度に精 製して得ること力できる。 Further, in the present invention, in addition to the extract obtained from the above-mentioned teacup, the purified product obtained by further purifying the extract can also be used as tea catechins. With regard to refined products, the Japanese Patent Publication Nos. 1-44234, 2-12474, 2-2275 It can manufacture by the method described in the 5th publication, the Unexamined-Japanese-Patent No. 4-20589, the 5-260907, the 8-109178 publication etc., for example, a tea leaf is extracted with said solvent, The obtained extract can be obtained by purification to a desired degree using an organic solvent fraction, an adsorption resin or the like.
そして、本発明で用いる茶カテキン類の形態については、特に限定されず、上記抽 出物、精製物等の液体、固体 (粉末を含む）の別を問わず利用することができる。 尚、本発明においては、茶カテキン類の市販品を用いても良レ、。このような市販品 としては、例えば三井農林 (株)製「ポリフヱノン」、太陽化学 (株)製「サンフヱノン」、 ( 株)伊藤園「テアフラン」などを例示することができる。  The form of tea catechins used in the present invention is not particularly limited, and any of liquids such as the above-mentioned extract and purified product and solid (including powder) can be used. In addition, in the present invention, it is good even if it uses a commercial product of tea catechins. As such commercially available products, for example, "Polifone Non" manufactured by Mitsui Norin Co., Ltd., "Saif von Non" manufactured by Taiyo Kagaku Co., Ltd., Itoen Co., Ltd. "Theafran", etc. can be exemplified.
[0015] 本発明において、効力増強剤の対象である抗癌剤は、一般的に知られているもの であれば何でも良ぐまた 1種または 2種以上組み合わせて用いても良いが、特に白 血病治療剤であることが好ましレ、。  [0015] In the present invention, any anticancer agent, which is the target of the efficacy enhancer, may be any generally known one, or may be used singly or in combination of two or more. Preferred to be a therapeutic agent.
白血病は、骨髄の白血病細胞が自律増殖する疾患、いわゆる血液の癌であって、 増殖細胞がいずれの系統の造血細胞の特徴を有するかにより、骨髄性、リンパ性、 単球性等に分類される。また、臨床的に急速な経過を迪るカ \或いは寛恕な経過を 迪るかにより、急性白血病、慢性白血病等に分類される。  Leukemia is a disease in which leukemia cells in the bone marrow autonomously proliferate, so-called hematological cancers, and is classified as myeloid, lymphoid, monocytic, etc. depending on which lineage of the hematopoietic cells the proliferating cells have. Ru. It is also classified into acute leukemia, chronic leukemia, etc. depending on whether it has a clinically rapid course or a liberal course.
[0016] 本発明の効力増強剤がその効力を増強する白血病治療剤としては、上記した分類 に属した白血病に関与する細胞に、増殖阻害作用等の抗癌作用を促進させるもの であれば何であっても良いが、中でも、 BCR— ABL遺伝子に起因する白血病、即ち 、 Ph (フィラデルフィア染色体）陽性骨髄性白血病細胞（例えば、 TCC S細胞（BC R— ABL陽性慢性骨髄性白血病由来株化細胞） )や Ph陽性急性リンパ性白血病に 関与する細胞に対し、優れた抗癌作用を発揮するものが好ましい。 BCR— ABL遺 伝子とは、第 9番染色体と第 22番染色体との間の相互転座によって生ずる融合遺伝 子であり、その遺伝子産物は慢性骨髄性白血病などの直接の発症原因となることが 知られている。  [0016] As a therapeutic agent for leukemia in which the efficacy enhancer of the present invention enhances its efficacy, any agent that promotes anti-cancer activity such as growth inhibitory activity to cells involved in leukemia belonging to the above-mentioned classification can be used. Among them, leukemia caused by the BCR-ABL gene, that is, Ph (Philadelphia chromosome) -positive myeloid leukemia cells (for example, TCC S cells (BCR-ABL-positive chronic myeloid leukemia-derived cell line) ) And those that exert excellent anti-cancer effects on cells involved in Ph-positive acute lymphoblastic leukemia. The BCR-ABL gene is a fusion gene produced by reciprocal translocation between chromosome 9 and chromosome 22, and its gene product is a direct cause of chronic myelogenous leukemia etc. It has been known.
このような白血病治療剤としては、例えば、ビンブラスチン、ビンクリスチン、サイトシ ンァラビノシド、ブレドニゾロン、 6MP、エンドキサン、アドリアマイシン、ダウノルビシ ン、イマチニブなどを挙げることができ、中でもダウノルビシン及び Z又はイマチニブ が好ましい。 As such leukemia therapeutic agents, for example, vinblastine, vincristine, cytosarabinoside, bredonizolone, 6MP, endoxane, adriamycin, daunorubicin, imatinib and the like can be mentioned, among which daunorubicin and Z or imatinib can be mentioned. Is preferred.
[0017] ダウノルビシン（Daunombicin)は、 1963年に放線菌 Streptomyces coerleor uidus株から発見されたアントラサイクリン系の抗生物質で、化学名は 7— (3 -amin o— 2, ό , 6 _trideoxy_L_lyxohexosyloxyノ _ 9 _acetyl_ 7， 8, 9, 10— tetr ahydro— 6, 9, 11— tnny droxy— 4— methoxy― 5 , 12— napnthacenezuinon e hydrochlorideであり、塩酸ダウノルビシン（商品名：ダウノマイシン、明治製菓）と も呼ばれている。核酸の合成を阻害し、急性白血病、慢性骨髄性白血病の急性転化 時などの治療に用いられている。  [0017] Daunorubicin (Daunombicin) is an anthracycline antibiotic discovered in 1963 from the actinomycete Streptomyces coerleor uidus strain, and the chemical name is 7-(3-amin o -2, 2, 6 _trideoxy_L_lyxohexosyloxyno _ 9 _acetyl _ 7, 8, 9, 10-tetrahydro-6, 9, 11-tnny droxy-4-methoxy-5, 12-napnthacenezuinon e hydrochloride, also called daunorubicin hydrochloride (brand name: daunomycin, Meiji Seika) It inhibits the synthesis of nucleic acid and is used for the treatment of acute leukemia and acute blast of chronic myelogenous leukemia.
ダウノルビシンの作用機序は、 DNAと結合して核ポリメラーゼ活性を阻害し、トポィ ソメラーゼ II阻害により DNA鎖を切断することである。このため、 DNA合成を行なつ ている全ての正常細胞 (骨髄細胞、腸管上皮細胞、毛根細胞など）に重篤な障害を もたらす。また、容量依存性に心毒性を発揮することも知られている。  The mechanism of action of daunorubicin is to bind to DNA to inhibit nuclear polymerase activity and to cleave the DNA strand by topoisomerase II inhibition. This causes serious damage to all normal cells (such as bone marrow cells, intestinal epithelial cells, and hair root cells) that are synthesizing DNA. It is also known to exert cardiotoxicity in a dose-dependent manner.
[0018] イマチニブ（Imatinib)は、 2001年 12月 7日に本邦で発売になったメシル酸ィマチ ニブ（商品名：ダリベック、ノバルティスファーマ株式会社）のことで、化学名は Benza mide, 4— [ (4— methyl—丄一 piperazmyl) methyl J— N— [4— metnyl— 3— [ [ 4— (3— pyridinyl)— 2— pyrimidinyl] amino] phenyl]― , monomethanesulfo nateであり、 ABL遺伝子の ATP結合部位に ATPと競合的に結合することにより、 B CR— ABL遺伝子の産物である BCR— ABLタンパク質が有するチロシンキナーゼ 活性を特異的に阻害する分子標的抗癌剤として注目を浴びている。 BCR— ABL遺 伝子は、前記したように、慢性骨髄性白血病などの直接の発症原因遺伝子として知 られていることから、そのチロシンキナーゼ活性を特異的に抑制することは、白血病 治療上、極めて理にかなつた治療法である。 [0018] Imatinib (Imatinib) is a product name of Imatinib mesylate (trade name: Daribec, Novartis Pharma Ltd.), which was released in Japan on December 7, 2001, and its chemical name is Benza mide, 4-[ (4-methyl- 丄 一 丄 piperazmyl) methyl J-N- [4-metnyl-3- [[4- (3-pyridinyl) -2-pyrimidinyl] amino] phenyl]-, monomethanesulfo nate, ATP of the ABL gene It attracts attention as a molecular target anticancer agent which specifically inhibits tyrosine kinase activity possessed by BCR-ABL protein which is a product of BCR-ABL gene by competitively binding to the binding site with ATP. Since the BCR-ABL gene is known as a direct onset causative gene such as chronic myelogenous leukemia as described above, specifically suppressing its tyrosine kinase activity is extremely important for leukemia treatment. It is a reasonable treatment.
[0019] 本発明の効力増強剤の利用形態は、茶カテキン類と抗癌剤が生体内で共同的に 作用するように、抗癌剤と組み合わせて用いられるのであれば、特に限定されない。 例えば、単一の製剤中に本発明の効力増強剤と抗癌剤とを含有させても良いし、 各剤を含有した製剤をそれぞれ調製し、それらを使用時に組み合わせるといった形 態であっても良い。また、本発明の効力増強剤と抗癌剤とを時間的に同時に投与す る必要もなぐ効力増強剤が先に投与された後に抗癌剤が、あるいは抗癌剤が先に 投与された後に効力増強剤が投与される形態でも構わない。 The use form of the efficacy enhancer of the present invention is not particularly limited as long as the tea catechins and the anticancer agent are used in combination with the anticancer agent so as to act cooperatively in vivo. For example, the efficacy enhancer of the present invention and the anticancer agent may be contained in a single preparation, or a preparation containing each agent may be prepared and combined at the time of use. In addition, the anticancer agent or the anticancer agent precedes after the efficacy enhancer without prior administration of the efficacy enhancer and the anticancer agent according to the present invention. It may be in a form in which the efficacy enhancer is administered after being administered.
[0020] また、本発明の抗癌剤の効力増強剤の投与方法は、経口的、非経口的のいずれ でも良い。ただし、単一の製剤中に本発明の効力増強剤と抗癌剤とを含有させる場 合に、抗癌剤としてダウノルビシンを使用する場合は、静中投与に限られる。  In addition, the administration method of the efficacy enhancer of the anticancer agent of the present invention may be either oral or parenteral. However, in the case where the efficacy enhancer of the present invention and the anticancer agent are contained in a single preparation, when daunorubicin is used as the anticancer agent, the administration is limited to administration at rest.
経口的に投与する場合の本発明の効力増強剤の形態は、例えば、粉末剤、顆粒 剤、錠剤、ピル、カプセル剤、液剤、シロップ剤等の経口投与剤とすることができる。 経口投与剤とする場合の製造例を挙げると、有効成分である茶カテキン類に、所望 により、賦形剤、結合剤、崩壊剤、滑沢剤、希釈剤、等張化剤等、一般の経口投与 剤に慣用されている添加剤を適宜配合し、公知の方法に従って製剤化することにより 製造すること力 Sできる。  When orally administered, the form of the efficacy enhancer of the present invention can be, for example, an orally administered agent such as a powder, a granule, a tablet, a pill, a capsule, a solution, a syrup and the like. Examples of preparation for oral administration include: tea catechins, which are active ingredients; if desired, excipients, binders, disintegrants, lubricants, diluents, isotonic agents, etc. The additives conventionally used for oral administration can be appropriately blended and manufactured by formulating them according to known methods.
一方、非経口的に投与する場合の本発明の効力増強剤の形態は、例えば、注射 剤、坐剤等を挙げることができる。注射剤とする場合の製造例を挙げると、有効成分 である茶カテキン類に、所望により、溶解補助剤、緩衝剤、等張化剤、安定剤、保存 剤、無痛化剤等、一般の注射剤に慣用されている添加剤を適宜配合することにより 製造すること力 Sできる。  On the other hand, examples of the form of the efficacy enhancer of the present invention for parenteral administration include injections, suppositories and the like. In the case of preparation as an injection, for example, a solubilizing agent, a buffer, a tonicity agent, a stabilizer, a preservative, a soothing agent, etc. may be added to the active ingredient tea catechins, if necessary. It can be manufactured S by appropriately blending additives commonly used in the agent.
[0021] 本発明の抗癌剤の効力増強剤を癌治療の目的として用いる場合、その投与量は、 治療目的の疾患の程度、患者の年齢、体重、抗癌剤の種類、投与量等に基づいて、 有効成分である茶カテキン類の有効量 (即ち本発明の所望の効果を発揮しうる量)が 投与されうるように適宜決定すればよい。通常は、茶カテキン類の量が 1日当たり 0. 01〜100mg/kg (体重）の範囲内となるように投与される。尚、茶力テキン類を上記 範囲内で投与しても、十分な抗癌効果を得ることはできず、抗癌剤と併用することに より初めて十分な抗癌作用がもたらされる。  When the agent for enhancing the efficacy of the anticancer agent of the present invention is used for the purpose of cancer treatment, the dose is effective based on the degree of disease to be treated, patient's age, body weight, type of anticancer agent, dosage, etc. The effective amount of tea catechins (that is, the amount capable of exerting the desired effect of the present invention) which is an ingredient may be appropriately determined so as to be administered. Usually, the amount of tea catechins is administered within the range of 0.01 to 100 mg / kg (body weight) per day. Even when the tea strength techin is administered within the above range, a sufficient anti-cancer effect can not be obtained, and a sufficient anti-cancer effect is brought about only by using it in combination with an anticancer agent.
また、併用する抗癌剤の投与量は、抗癌剤の種類に応じて適宜定めることができる そして、本発明の効力増強剤と抗癌剤の重量比は、抗癌剤の種類等にもよるが、 一般に白血病治療薬の場合、本発明の効力増強剤に含まれる茶力テキン類を 1とす ると、白血病治療薬は 0. 000001〜：!とすることができる。特に、イマチニブの場合は 、茶カテキン類 1に対し 0. 0001〜：！、好ましくは 0. 0001〜0. 1とすること力 Sできる。 また、ダウノルビシン (ダウノマイシン）の場合は、茶カテキン類 1に対し 0. 0001〜1、 好ましくは 0. 001-0. 1とすること力 sできる。 In addition, the dose of the anticancer drug to be used in combination can be appropriately determined according to the type of the anticancer drug. And, the weight ratio of the efficacy enhancer and the anticancer drug of the present invention generally depends on the type of the anticancer drug etc. In the case where the tea strength techin contained in the efficacy enhancer of the present invention is 1, the leukemia therapeutic agent can be 0. 000001 to:!. In particular, in the case of imatinib, tea catechins 1 against 0. 0001 ~! , Preferably 0. 0001 to 0.1. Moreover, in the case of daunorubicin (daunomycin), it is possible to make it 0.01. 1 to 1, preferably 0. 001-0. 1 with respect to 1 tea catechins.
投与形態は、患者の病状に応じて、連日投与又は間欠投与のいずれでもよぐ適 宜調節することができる。  The dosage form can be adjusted as appropriate depending on the condition of the patient, either daily administration or intermittent administration.
[0022] 本発明の抗癌剤の効力増強剤は、抗癌剤と併用されることにより、後述の実施例で 示されるように、有効成分である茶カテキン類が抗癌剤と共に生体内で共同的に作 用して、抗癌剤の効力を茶カテキン類によって相乗的に増加させることができる。こ れにより癌の効率的な治療が可能となることはもちろん、さらに、抗癌剤の単独投与 では効果が得られなかった症例に対しても有効な治療が可能となる。さらにまた、抗 癌剤の投与量を 5分の 1〜 10分の 1にまで減少させることが可能である。  [0022] The efficacy enhancer of the anticancer agent of the present invention, when used in combination with an anticancer agent, causes tea catechins, which are active ingredients, to act jointly in vivo with the anticancer agent, as shown in the examples below. Thus, the efficacy of anticancer agents can be synergistically increased by tea catechins. This not only enables efficient treatment of cancer, but also enables effective treatment for cases in which no effect was obtained by single administration of an anticancer agent. Furthermore, it is possible to reduce the dose of the anticancer drug to one fifth to one tenth.
[0023] 特に、本発明の効力増強剤は、白血病治療剤の様々な効力を、種々の面から増強 すること力 sできる。例えば、白血病に関与する細胞の増殖抑制を増強したり、細胞死 を増強したり、チロシンキナーゼ活性抑制を増強するなど、種々の形で白血病治療 剤の効力を増強する。従って、このような様々な増強機能の発揮を期待して、種々の 用途で用いることができるが、特に、白血病細胞の増殖抑制を増強する目的で使用 するのが好ましい。  [0023] In particular, the efficacy enhancer of the present invention can enhance various aspects of the efficacy of the therapeutic agent for leukemia. For example, the efficacy of leukemia therapeutics is enhanced in various ways, such as enhancing the growth suppression of cells involved in leukemia, enhancing cell death, and enhancing tyrosine kinase activity suppression. Therefore, it can be used in various applications in expectation of exerting such various enhancement functions, but in particular, it is preferable to use for the purpose of enhancing the growth suppression of leukemia cells.
さらに、白血病治療剤であるイマチニブを単独で投与した際には、 BCR— ABL陽 性細胞をアポトーシスに陥らせるのみであった力 イマチニブと本発明の効力増強剤 とを併用することにより、患者体内における免疫賦活作用を期待することができる。即 ち、ネクローシスに陥った BCR— ABL陽性細胞は、体内の抗原提示細胞（主に樹状 細胞）を刺激して、 BCR— ABL抗原を榭状細胞の表面に提示させ、免疫能を賦活さ せることが知られているが、イマチニブと本発明の効力増強剤とを併用する結果、ネ クローシスに陥る細胞分画が増加するからである。  Furthermore, when imatinib, which is a leukemia therapeutic agent, is administered alone, it is possible to use the combination of power of imatinib, which only caused BCR-ABL positive cells to undergo apoptosis, and the efficacy enhancer of the present invention. Can be expected to have an immunostimulatory effect in Specifically, necrosed BCR-ABL-positive cells stimulate antigen-presenting cells (mainly dendritic cells) in the body to present the BCR-ABL antigen on the surface of sickle cells, thereby enhancing immunity. It is known that the combination of imatinib and the efficacy enhancer of the present invention results in an increase in the fraction of cells suffering from necrosis.
実施例  Example
[0024] 以下に製造例、試験例を挙げ、本発明をさらに詳しく説明する。ただし、本発明は これに限定されるものではない。  Hereinafter, the present invention will be described in more detail by way of production examples and test examples. However, the present invention is not limited to this.
[0025] 製造例 1 :茶カテキン類の製造方法 Production Example 1: Production Method of Tea Catechins
乾燥した緑茶葉 270kgを 1890Lの 80。C熱水で抽出した後、さらに 1350Lの 80。C 熱水で抽出をおこなった。この抽出液を 15°C以下に冷却した後、 RO膜により濃縮し 、濃縮液 500Lを得た。この濃縮液を 1000Lの酢酸ェチルで抽出し、酢酸ェチル層 を 75kgにまで濃縮し、これを乾燥させて 15kgの粗茶抽出物を得た。 Dried green tea leaves 270 kg 1890 liters 80. After extracting with C hot water, add an additional 1350 L of 80. C Extraction was performed with hot water. The extract was cooled to 15 ° C. or less and concentrated with an RO membrane to obtain 500 L of a concentrate. The concentrate was extracted with 1000 L of ethyl acetate, and the ethyl acetate layer was concentrated to 75 kg, which was dried to obtain 15 kg of a crude tea extract.
このようにして得られた粗茶抽出物 12kgを原料とし、 22Lの 10%メタノール水溶液 に溶解し、架橋スチレン系合成吸着樹脂（三菱化成 (株)製「HP_ 20」 ) 300ccに茶 抽出成分を吸着させた。 280Lの 10%メタノール水溶液を通液することにより未吸着 成分を除いた後、 280Lの 40%メタノール水溶液を通液することにより茶カテキン画 分を溶出させた。続いて 600Lの 70%メタノール水溶液を通液することにより不溶成 分を洗浄'除去し、茶力テキン画分約 240Lを回収した。この画分を、遠心薄膜濃縮 装置（(有)大河原製作所製）により循環濃縮をおこない、濃縮液約 20Lを得た。この 濃縮液をスプレードライヤー（二口製）により噴霧乾燥し、茶カテキン類 4. 8kgを得た このようにして得られた茶カテキン類の成分分析は、高速液体クロマトグラフィーに より下記の条件でおこなった。茶カテキン類の成分組成を表 1に示す。  Using 12 kg of the crude tea extract thus obtained as a raw material, it is dissolved in 22 liters of a 10% aqueous methanol solution, and the extracted components are adsorbed on 300 cc of a cross-linked styrenic synthetic adsorption resin ("HP 20" manufactured by Mitsubishi Kasei Corp.) I did. After removing the non-adsorbed components by passing 280 L of a 10% aqueous methanol solution, the tea catechin fraction was eluted by passing 280 L of a 40% aqueous methanol solution. Subsequently, insoluble components were removed by washing with 600 L of 70% aqueous methanol solution to remove approximately 240 L of tea strength tekin fraction. This fraction was subjected to circulation concentration using a centrifugal thin film concentrator (manufactured by Ohkawara Seisakusho Co., Ltd.) to obtain about 20 L of a concentrated solution. The concentrated solution was spray-dried by a spray dryer (manufactured by Two-Piece) to obtain 4.8 kg of tea catechins. Component analysis of the tea catechins obtained in this manner was carried out under the following conditions by high performance liquid chromatography. I did. The composition of tea catechins is shown in Table 1.
[0026] (高速液体クロマトグラフィーの条件） (Conditions of high performance liquid chromatography)
カラム ： Mightysil (関東化学 (株)製）  Column: Mightysil (Kanto Chemical Co., Ltd.)
移動相 A液：ァセトニトリル：燐酸水溶液 = 10： 400の溶液  Mobile phase A solution: Acetonitrile: phosphoric acid aqueous solution = 10: 400 solution
移動相 B液：メタノール：ァセトニトリル：燐酸水溶液 = 200 : 10 : 400の溶液 検出 ： UV230nm  Mobile phase B: Methanol: acetonitrile: phosphoric acid aqueous solution = 200: 10: 400 solution Detection: UV 230 nm
カラム温度： 40°C  Column temperature: 40 ° C
サンプル温度：室温  Sample temperature: room temperature
サンプノレ量： 10 μ ΐ  Amount of sampling: 10 μ ΐ
流速 ： lml/ min.  Flow rate: lml / min.
[0027] [表 1] 茶カテキン類の組成 [0027] [Table 1] Composition of tea catechins
(重量％)  (% By weight)
Figure imgf000011_0001
Figure imgf000011_0001
[0028] 試験例 1：イマチニブと茶カテキン類の併用効果の検討 Test Example 1: Examination of combined effect of imatinib and tea catechins
腫瘍細胞として TCC S細胞（BCR— ABL陽性慢性骨髄性白血病由来）を用い て、緑茶由来のェピガロカテキンガレート（EGCg)と分子標的抗癌剤（BCR— ABL キナーゼ選択的阻害剤）であるイマチニブとを併用した場合の腫瘍細胞増殖抑制効 果を検討した。  Using TCC S cells (BCR-derived from AML positive chronic myeloid leukemia) as tumor cells, green tea-derived epigarocatechin gallate (EGCg) and molecular target anticancer drug (BCR-ABL kinase selective inhibitor) with imatinib The effect of suppressing tumor cell growth was investigated when using in combination.
[0029] (試験方法）  [0029] (Test method)
TCC— S細胞を:!〜 3X105個 Zmlの濃度となるように 10%FCSを含む RPMI—1 640培地を用いて、 37°C、 5% CO存在下で培養し、生細胞％が 95%以上の状態 TCC- S cells were cultured at 37 ° C. in the presence of 5% CO at a concentration of 5 × 3 cells in RPMI-1640 medium containing 10% FCS, and the viable cells were 95%. % Or more
2  2
で、細胞数が指数関数的に増加している時期（exponential phase)にあることを確 認した。  So, we confirmed that it was in the exponential phase when the number of cells increased exponentially.
次に、この状態にある TCC— S細胞（濃度 2X105個 1111)41111を6\^11 plateを 用いて 14wellに分注し、表 2又は図 1に示すように、（1)薬剤を添カ卩しない群、 （2)ィ マチニブ（0. ΙμΜ)のみ添加した群、（3)イマチニブ（0. 5 μΜ)のみ添加した群、（ 4) EGCg (50 μΜ)のみ添加した群、（5) EGCg (100 μΜ)のみ添加した群、（6)ィ マチニブ（0. ΙμΜ) +EGCg (50 μ Μ)を併用添加した群、及び（7)イマチニブ（0. 1 μ M) +EGCg (100 μ Μ)を併用添加した群の各群に分け、それぞれに対応して 薬剤を添加した。 Next, TCC-S cells (concentration 2 × 10 5 1111) 41111 in this state are aliquoted into 14 wells using a 6 \ ^ 11 plate, and as shown in Table 2 or FIG. (2) a group to which only imatinib (0.ΙμΙ) was added, (3) a group to which only imatinib (0.5 μΜ) was added, (4) a group to which only EGCg (50 μΜ) was added, (5) A group to which only EGCg (100 μM) was added, a group to which (6) imatinib (0. μμM) + EGCg (50 μM) was added in combination, and (7) imatinib (0.1 M) + EGCg (100) It divided into each group of the group which co-added and added (microbe), and the medicine was added according to each.
上記の各群について、培養開始時 (Ohr)、培養開始から 24, 48, 72時間後（24hr 、 48hr、 72hr)の生細胞数を、トリパンブルー染色法（trypanblue dye exclusion method)で計測した。その結果を表 2又は図 1に示す。 [0030] [表 2] For each of the above groups, the number of viable cells at the start of culture (Ohr) and 24, 48 and 72 hours after the start of culture (24 hr, 48 hr, 72 hr) was counted by trypan blue dye exclusion method. The results are shown in Table 2 or Figure 1. [0030] [Table 2]
Figure imgf000012_0001
Figure imgf000012_0001
[0031] また、薬剤無添加群、イマチニブ（0· 5 μ Μ)のみ添加した群、 EGCg (200 μ Μ) のみ添加した群、イマチニブ（0. 1 /i M) +EGCg (5 μ Μ)併用添加群（上記の（6) 及び（7) )の間で、細胞死の形態に差異があるかを検討するために、 5 Χ 102〜103 個/ slideの細胞を用いてサイトスピン標本を作製し、ライト'ギムザ染色法で染色した 後に、光学顕微鏡下で観察した。その結果を表 3又は図 2に示す。 In addition, a drug-free group, a group to which only imatinib (0.5 μm) was added, a group to which only EGCg (200 μm) was added, imatinib (0.1 / i M) + EGCg (5 μm) In order to examine whether there is a difference in the form of cell death between the combined addition groups ((6) and (7) above), cytospin using 5 × 10 2 to 10 3 / slide cells The specimens were prepared and stained with Wright's Giemsa stain and then observed under an optical microscope. The results are shown in Table 3 or Figure 2.
[0032] [表 3]  [Table 3]
Figure imgf000012_0002
Figure imgf000012_0002
(試験結果） (Test results)
まず、表 2又は図 1に示されるように、（1)薬剤無添加群の検体における TCC S 細胞の数は、培養開始から増加を続け、 72時間後には 7. 32 X 105個/ mlに達した のと比較して、（2)のイマチニブ単独添加群や（4)及び（5)の EGCg単独添加群で は軽度の増殖抑制が見られた。また、 （3)のイマチニブ単独添加群は 0. 25 X 105個 /mlとかなりの増殖抑制が見られた。 First, as shown in Table 2 or FIG. 1, (1) the number of TCC S cells in the sample without drug addition continues to increase from the start of the culture, and after 72 hours, 7. 32 x 10 5 cells / ml. In the (2) imatinib alone addition group and in (4) and (5) EGCg alone addition groups compared to There was mild growth suppression. In addition, (3) imatinib alone added group showed significant growth suppression with 0.25 × 10 5 cells / ml.
これに対し、（6)及び（7)のイマチニブ + EGCg併用添加群の場合は、 72時間後 の細胞数がわずか 0. 17 X 105個/ mlと、上記した添加群（1)〜（5)のいずれと比 ベても少なかったことから、イマチニブと EGCgとの併用により TCC— S細胞の顕著 な増殖抑制効果を得ることができ、これは単なる相加効果ではなく相乗効果であるこ とが明らかとなった。 On the other hand, in the case of the combination addition group of imatinib + EGCg of (6) and (7), the number of cells after 72 hours is only 0.17 × 10 5 cells / ml, and the addition group (1) to As compared with any of 5), the combination of imatinib and EGCg can be used to obtain a remarkable growth inhibitory effect on TCC-S cells, which is not a simple additive effect but a synergistic effect. It became clear.
また、特に、（6)及び（7)の併用添加群ではいずれも、イマチニブの添加量が（3) のイマチニブ添加群の 5分の 1であるにも拘らず、顕著に少なかったことから、少量の EGCg (50 μ Μ)を、イマチニブと併用することにより、イマチニブ使用量を 0. 5 μ Μ 力、ら 0. 1 μ Μに減量し得ることも明らかとなった。  Moreover, in particular, in all of the combination addition groups of (6) and (7), although the addition amount of imatinib was one fifth of that of the imatinib addition group of (3), it was significantly smaller. It was also revealed that the use of a small amount of EGCg (50 μM) in combination with imatinib could reduce the amount of imatinib used to 0.5 μM, or 0.1 μM.
[0034] また、表 3又は図 2に示されるとおり、イマチニブ単独添加（0. 5 μ Μ)群では典型 的なアポトーシスを示す細胞（Apoptotic cells :細胞核か小球状する）が 64· 12% と圧倒的多数であった力 イマチニブ（0. Ι β Μ) +EGCg (50 β Μ)併用添加群で は、アポトーシスを示す細胞は 15· 24%に減少し、変形した細胞（deformed cell 1. 0%)ネクローシス（Pyknosis)を示す細胞（Pyknotic cells)が 2· 44%と増カロし ていた。 In addition, as shown in Table 3 or FIG. 2, 64.12% of cells (Apoptotic cells: cell nuclei or spheroids) showing typical apoptosis were obtained in the imatinib single addition (0.5 μM) group. In the group that added imatinib (0. β β Μ) + EGCg (50 β Μ) in combination, the cells showing apoptosis were reduced to 15 24%, and the cells that were apoptotic were deformed (deformed cell 1. 0). %) Cells (Pyknotic cells) showing necrosis (Pyknosis) had increased their calories to 2.44%.
このこと力 、イマチニブを単独で投与した際には、 BCR—ABL陽性細胞をアポト 一シスに陥らせるのみであつたのに対し、イマチニブと本発明の効力増強剤とを併用 することにより、ネクローシスに陥る細胞が増加し、患者体内における免疫賦活作用 を期待することができることが明らかとなった。  This fact was that when imatinib was administered alone, BCR-ABL-positive cells were only put into apoptosis, while using the combination of imatinib and the efficacy enhancer of the present invention resulted in necrosis. It has become clear that the number of cells falling into the lungs can be increased, and the immunostimulatory action can be expected in the patient's body.
[0035] 試験例 2：ダウノルビシンと茶カテキン類の併用効果の検討  Test Example 2: Examination of combined effect of daunorubicin and tea catechins
TCC— Y細胞（BCR—ABL陽性急性リンパ性白血病由来株化細胞）を用いて、白 血病や固形腫瘍に用いられる代表的な抗ガン剤の一つであるダウノマイシン (ダウノ ルビシン (DNR)の商品名。以下、 DNRと略記する。）を EGCgと併用した場合の腫 瘍細胞増殖抑制効果を検討した。  Daunomycin (DNR), which is one of the representative anticancer agents used for white blood disease and solid tumors, using TCC-Y cells (BCR-ABL positive cell line derived from acute lymphocytic leukemia). (Trade name) (hereinafter abbreviated as DNR) The tumor cell growth inhibitory effect was examined when combining with EGCg.
[0036] (試験方法）  [0036] (Test method)
TCC— Y細胞を:!〜 3 X 105個/ mlの濃度となるよう 10%FCSを含む RPMI—16 40培地を用いて、 37°C、 5% CO存在下で培養し、生細胞％が 95%以上の状態で TCC-Y cells: RPMI-16 containing 10% FCS to a concentration of 3 x 10 5 cells / ml Cultivate in 40% medium at 37 ° C in the presence of 5% CO, with 95% or more of viable cells
2  2
、細胞数が指数関数的に増加している時期（exponential phase)にあることを確認 した。次に、この状態にぁる丁〇〇一¥細胞（濃度1 105個/1111)21111を、 5本のファ ルコンチューブ（Falcon tube)に分注し、表 4及び図 3に示すように、（1)薬剤を添 加しない群、（2)EGCg(10 xM)のみ添加した群、（3)DNR(0. 05 μΜ)のみ添加 した群、（4)DNR(0. 5μΜ)のみ添加した群、及び（5)DNR(0. 05 μΜ) +EGCg (50 μ Μ)を併用添加した群の各群に分け、それぞれに対応して薬剤を添加した。 上記の条件下で 72時間培養した後、生細胞数をトリパンブルー染色法 (trypanbl ue dye exclusion method)で計測した。その結果を表 4又は図 3に示す。 It was confirmed that the number of cells was in the exponential phase (exponential phase). Next, the Aru Ding hundred one ¥ cells (concentration of 1 105 cells / 1111) 21111 in this state, dispensed into five files Le Con tubes (Falcon Tube) min, as shown in Table 4 and Figure 3 (1) No drug addition group, (2) Group added with only EGCg (10 x M), (3) Group added with only DNR (0. 05 μm), and (4) Group added only with DNR (0.5 μm). And (5) DNR (0. 05 μm) + EGCg (50 μm) were separately added to each group, and drugs were added correspondingly. After culturing for 72 hours under the above conditions, the number of viable cells was counted by trypan blue dye exclusion method. The results are shown in Table 4 or Figure 3.
[表 4] [Table 4]
TCC Y細胞に対する DNRと EGC gとの增殖抑制効果  Growth inhibitory effect of DNR and EGC g on TCC Y cells
Figure imgf000014_0001
(試験結果）
Figure imgf000014_0001
(Test results)
表 4及び図 3から明らかなように、（2)EGCg(10/iM)単独添加群では、 72時間後 も TCC Y細胞が観察されたが、 （3)の DNR(0. 05 μΜ)単独添加群ではかなりの 増殖抑制が見られ、また、（4)の DNR(0. 5 / M)単独添加群では顕著な細胞増殖 抑制が観察された。  As apparent from Table 4 and FIG. 3, in the (2) EGCg (10 / iM) -only addition group, TCC Y cells were observed even after 72 hours, but DNR (0. 05 μΜ) alone of (3) was observed. Significant growth suppression was observed in the addition group, and remarkable cell growth suppression was observed in the DNR (0.5 / M) single addition group of (4).
これに対し、 (5)DNR(0. 05 iM) +EGCg併用添加群の場合は、上記した添カロ 群（1)〜（4)のいずれと比べても少なかったことから、 DNRと EGCgとの併用により T CC S細胞の顕著な増殖抑制効果を得ることができ、これは単なる相加効果ではな く相乗効果であることが明らかとなった。  On the other hand, in the case of the (5) DNR (0. 05 iM) + EGCg combined addition group, compared with any of the above-mentioned additional caro groups (1) to (4) described above, the DNR and EGCg It was found that a significant inhibitory effect on T CC S cells can be obtained by the combined use of C.sub.CCS cells, which is not merely an additive effect but a synergistic effect.
また、特に、（5)DNR(0. 05μΜ) +EGCg併用添加群の場合は、 DNRの添加量 力 4)の 10分の 1にも拘らず、（4)と同様の細胞増殖抑制効果が見られたことから、 少量の EGCg (50 μΜ)を DNRと併用することにより、 DNR使用量を 0. 5 μ Μ力ら 0 . 05に減量し得ることも明らかとなった。 In addition, in the case of the (5) DNR (0. 05 μm) + EGCg combined addition group, the same cell growth inhibitory effect as (4) is obtained despite the fact that it is 1/10 of the addition amount of DNR 4). As seen, using a small amount of EGCg (50 μm) in combination with DNR results in a DNR usage of 0.5 μm 0 It also became clear that it could be reduced to 05.
産業上の利用可能性 Industrial applicability
本発明の抗癌剤の効力増強剤は、抗癌剤と組み合わせて用レ、ることにより、有効 成分である茶カテキン類と抗癌剤が生体内で共同的に作用するので、茶カテキン類 単独投与あるいは抗癌剤単独投与により得られる抗癌作用からは予想外の相乗効 果カもたらされ、顕著な腫瘍細胞増殖抑制効果を呈する。その結果、抗癌剤の投与 量を減量しても十分な抗癌効果が得られるため、抗癌剤の持つ有害事象の出現率を 力なりの程度低減することができ、患者は長期間安心して服用することができ、患者 の QOL (Quality of life)向上に大きく貢献することができる。  The efficacy enhancer of the anticancer agent of the present invention can be used in combination with the anticancer agent, whereby the tea catechins and the anticancer agent, which are active ingredients, act jointly in vivo. Therefore, tea catechins alone or anticancer agents alone can be administered. The anticancer effect obtained by the present invention results in an unexpected synergistic effect and exhibits a remarkable tumor cell growth inhibitory effect. As a result, even if the dose of the anticancer drug is reduced, sufficient anticancer effect can be obtained, so the incidence rate of adverse events possessed by the anticancer drug can be reduced to an extent that the patient can take it safely for a long time. Can significantly contribute to the improvement of patients' quality of life (QOL).
さらに、本発明の抗癌剤の効力増強剤は、イマチニブ等の分子標的薬剤と併用す る場合も、その使用量を従来よりも少なくさせることができるので、医療費の節減が可 能であり、経済的な効果が大きぐまた耐性クローンの出現頻度を低下させることも可 能である。  Furthermore, the efficacy enhancer of the anticancer agent of the present invention can reduce the amount used even when used in combination with a molecule-targeted drug such as imatinib, so that medical costs can be saved and the economy can be reduced. It is also possible to increase the effectiveness of the drug and reduce the frequency of appearance of resistant clones.
そして、本発明の効力増強剤をイマチニブと併用する場合、以下の機序により、患 者体内における免疫賦活作用も期待することができる。即ち、イマチニブのみでは、 BCR—ABL陽性細胞をアポトーシスに陥らせるのみであった力 S、イマチニブと力テキ ンとの併用により、ネクローシスに陥る細胞分画が増加し、ネクローシスに陥った BCR 一 ABL陽性細胞が体内の抗原提示細胞（主に樹状細胞）を刺激して、 BCR— ABL 抗原を樹状細胞の表面に提示させ、免疫能を賦活させることができる、と考えられる  When the efficacy enhancer of the present invention is used in combination with imatinib, the immunostimulatory action in the patient's body can also be expected by the following mechanism. That is, with imatinib alone, BCR-ABL-positive cells were only put into apoptosis S, and the combination of imatinib and force-techin increased the fraction of cells in necrosis, resulting in necrosis. BCR-ABL It is thought that positive cells can stimulate antigen-presenting cells (mainly dendritic cells) in the body to present the BCR-ABL antigen on the surface of dendritic cells and activate the immunity.

Claims

請求の範囲 The scope of the claims
[1] 茶力テキン類を有効成分とする抗癌剤の効力増強剤。  [1] An agent for enhancing the efficacy of an anticancer agent comprising tea strength techins as an active ingredient.
[2] 抗癌剤が白血病治療剤である請求項 1記載の抗癌剤の効力増強剤。 [2] The agent for enhancing the efficacy of the anticancer agent according to claim 1, wherein the anticancer agent is a therapeutic agent for leukemia.
[3] 白血病治療剤が、イマチニブ及び/又はダウノルビシンである請求項 2記載の抗癌 剤の効力増強剤。 [3] The agent for enhancing the efficacy of the anticancer agent according to claim 2, wherein the therapeutic agent for leukemia is imatinib and / or daunorubicin.
[4] 茶カテキン類が、力テキンガレート、ェピカテキンガレート、ガロカテキンガレート及 びェピガロカテキンガレートから選ばれる少なくとも 1種以上である請求項 1乃至 3記 載の抗癌剤の効力増強剤。  [4] The agent for enhancing the efficacy of the anticancer drug according to any one of claims 1 to 3, wherein the tea catechins are at least one selected from strength tequingalate, epicathenic gallate, gallocatechin gallate and epigallocatechin gallate.
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Cited By (2)

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CN102232960A (en) * 2010-04-23 2011-11-09 复旦大学 Epigallocatechin gallate (ECG) and daunorubicin (DNR) composition and use thereof
JP2022533567A (en) * 2019-05-07 2022-07-25 雲南大葉帝紅生物科技有限公司 Use of combined use of epigallocatechin gallate and a tyrosine kinase inhibitor for the manufacture of a therapeutic drug for cancer

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JPH1036260A (en) * 1996-07-18 1998-02-10 Mitsui Norin Kk Enhancement of effect of anticancer agent
JPH11246402A (en) * 1997-12-26 1999-09-14 Japan Found Cancer Res Telomerase inhibitor
JP2001226276A (en) * 2000-02-16 2001-08-21 Natl Inst Of Advanced Industrial Science & Technology Meti New antileukemic cell agent
WO2003037344A1 (en) * 2001-11-02 2003-05-08 Shire Biochem Inc. Pharmaceutical compositions for the treatment of leukemia comprising dioxolane nucleosides analogs

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Publication number Priority date Publication date Assignee Title
JPH1036260A (en) * 1996-07-18 1998-02-10 Mitsui Norin Kk Enhancement of effect of anticancer agent
JPH11246402A (en) * 1997-12-26 1999-09-14 Japan Found Cancer Res Telomerase inhibitor
JP2001226276A (en) * 2000-02-16 2001-08-21 Natl Inst Of Advanced Industrial Science & Technology Meti New antileukemic cell agent
WO2003037344A1 (en) * 2001-11-02 2003-05-08 Shire Biochem Inc. Pharmaceutical compositions for the treatment of leukemia comprising dioxolane nucleosides analogs

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN102232960A (en) * 2010-04-23 2011-11-09 复旦大学 Epigallocatechin gallate (ECG) and daunorubicin (DNR) composition and use thereof
CN102232960B (en) * 2010-04-23 2013-08-21 复旦大学 Epigallocatechin gallate (ECG) and daunorubicin (DNR) composition and use thereof
JP2022533567A (en) * 2019-05-07 2022-07-25 雲南大葉帝紅生物科技有限公司 Use of combined use of epigallocatechin gallate and a tyrosine kinase inhibitor for the manufacture of a therapeutic drug for cancer
JP7336777B2 (en) 2019-05-07 2023-09-01 雲南大葉帝紅生物科技有限公司 Use of a combination of epigallocatechin gallate and a tyrosine kinase inhibitor for the manufacture of a therapeutic drug for cancer

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