WO2006112330A1

WO2006112330A1 - Inhibitory agent for estrone-3-sulfate transporter activity

Info

Publication number: WO2006112330A1
Application number: PCT/JP2006/307751
Authority: WO
Inventors: Ikumi Tamai; Hikaru Yabuuchi
Original assignee: Genomembrane Co., Ltd.
Priority date: 2005-04-15
Filing date: 2006-04-12
Publication date: 2006-10-26
Also published as: JP2006298781A

Abstract

Disclosed is an agent for inhibiting the transporter activity of an estrone-3-sulfate transporter, an agent for inhibiting the growth of breast cancer cells, or an agent for the treatment of breast cancer, which does not need to be incorporated in cells, has excellent drug delivery property and has few side effects. The agent comprises at least one component selected from genistein, quercetin, ginkgolide C, theaflavin, theaflavin 3-o-gallate, chalcone, rutin, daidzein, daidzin, flavanone, flavonol, geraldol, hesperetin, hespereridine, ipriflavone, luteolin-7-o-glucoside, methoxychalcone, 4'-methyl-7-methoxy-isoflavone, 5-morin, myricetin, naringenin, naringenin-7-glycoside, naringin, neohesperidin dihydrochalcone, nomilin, primuletin, poncirin, scutellarein, catechin hydrate, and chemically modified products thereof.

Description

Specification

Estrone 3-sulfate transporter activity inhibitor

Technical field

The present invention relates to an inhibitor of transporter activity of an estrone-3-sulfate (estrone-3-sulfate) transporter, a breast cancer cell growth inhibitor, and a breast cancer therapeutic agent.

Background art

[0002] Membrane proteins that transport substances inside and outside cells are called transporters. When specific molecules bind to transporters embedded in lipid bilayers, the conformation changes and the substances are taken up. Or they are known to be discharged and transported. In recent years, powerful transporters such as organic transporters such as OAT1 that transport organic organic substances, organic cationic transporters such as OCT1 that transport organic cationic substances, PEPT1 that transport peptide substances, etc. Genes such as peptide transporters have been isolated and identified one after another. Some of these transporter genes are ubiquitous in normal tissues 'organs throughout the body, but others are known to be localized in specific tissues' organs such as kidney, liver and brain.

[0003] On the other hand, estrogen is a so-called female hormone that causes estrus in female animals. Naturally occurring estrone (E), estradiol (E), estriol (E), esthetic

1 2 3 tralol) and synthetic estrogens with similar biological activity. The

Four

With the exception of some synthetic forms such as tilbestrol, estrogen has a steroid structure. Estrone 0 .: The biological action of g is 1 IU (international unit), which is used as an estrogen unit. The secretory source is mainly the ovarian follicle and corpus luteum, but it is also secreted by the fetal placenta, adrenal gland, and testis during pregnancy. The secretion of estrogens, such as ovarian force, is governed by gonadotropins secreted from the anterior pituitary gland (descending sexual regulation), but conversely, estrogen also has a feedback effect on the pituitary system (ascending sex). Regulation), and it is said that the sexual cycle is established by the mutual relationship between the two.

[0004] Furthermore, estrogen acts through its receptor (receptor), and the action of estrogen Is the target tissue, not only the anterior pituitary gland and the mammary gland but also the whole body.The main physiological functions are endometrial proliferation, myometrial development, expression of secondary sexual characteristics, establishment of menstrual cycle. Mediation, induction of maternal changes during pregnancy, and promotion of mammary duct growth and secretion. Estrogens are metabolized mainly in the liver and become conjugated estrogens such as estrone trisulfate and are excreted in the urine. Clinically, it is used for treatment of amenorrhea and menstrual abnormalities, artificial movement of menstruation, menopause, hormone therapy for prostate cancer and breast cancer, and osteoporosis.

[0005] Breast cancer has an abnormal operation of the estrogen receptor system from the time of carcinogenesis, and continues to estrogen-dependent growth at an early stage, but gradually grows out of this control. This suggests that estrogen and its receptor are deeply involved in the development and progression of breast cancer, and its mechanism is still unclear. Breast cancer is a malignant tumor that arises from the peripheral ducts of the epithelial tissue of the mammary gland and the acinar epidermis. Also in Japan, the death rate from breast cancer is on the rise due to the westernization of dietary habits and changes in lifestyle. The most common age is in the 40's. Risk factors include history of breast cancer's family history, unmarried, elderly first birth, early menarche and late menopause, obesity, radiation exposure, high fat diet, and history of benign breast disease. Can do. The number of metastases of lymph node metastasis, which frequently occur in the axilla, under the clavicle, and parasternally, correlates with prognosis. Hematogenous metastases are common in bone, lung, and liver. The main symptom is a breast mass, surface irregularity, hardness, unclear borders, and low mobility.

[0006] 2Z3 of breast cancer is estrogen-dependent (Non-patent Document 1), that is, cell proliferation is controlled by estrogen. The biologically active form of estrogen is estradiol, which is synthesized via two main pathways (Non-patent document 2, Non-patent document 3). One is the aromatase pathway where aromatase converts androgen to estrogen, and the other is the sulfatase pathway where steroid sulfatase converts estrone 3 sulfate to estrone. In patient breast cancer tissues, the activity of sulfatase is 50-200 times that of aromatase, so the sulfatase pathway is a major source of estrone 3 sulfate-derived biological activity estrogen (Non-patent document 3, Non-patent document 3). Reference 4). The circulating plasma concentration of estrone 3 sulfate is about 10 to 20 times that of unconjugated estrogens, and the half life of estrone 3 sulfate is longer than that of estradiol (Non-patent Documents 5 to 7) . Thus, estrone 3 sulfate is thought to play an important role in the progression of breast cancer as a reservoir of active estrogens, even though estrone 3 sulfate itself does not exhibit much biological activity.

[0007] Stimulation of breast cancer cell proliferation by estrone trisulfate involves the following series of processes: uptake of estrone trisulfate into cells, desulfurization oxidation by estrogen sulfatase, type I 17 j8-hydroxysteroid Conversion of estrone to estradiol by dehydrogenase, binding to nuclear estrogen receptor, and regulation of gene transcription (Non-Patent Documents 8 to 11). Well-studied forces of enzymes and receptors involved in breast cancer cell responses to estrone trisulfate The transport mechanism through which ligands such as estrone trisulfate cross the cell membrane is unknown. The logP value for estrone 3 sulfate is reported to be 1.4, which is significantly lower than the values for estrone (4.4) and estradiol (3.9), so estrone and estradiol are diffusible. Nevertheless, it will not be easy to cross the cell membrane by diffusion. (Non-patent document 12). Estrone 3 sulfate was detected in an intact form in the cytoplasmic fraction of MCF-7 cells after incubation with estrone 3 sulfate (Non-patent document 13), and showed estrogenic action (Non-patent document) 4, Non-patent document 14, Non-patent document 15). Thus, a specific transporter appears to be involved in the supply of estrone trisulfate to breast cancer cells that exhibit hormone-dependent growth.

[0008] Organic-on transporter (OAT, SLC22A) (Non-patent document 16) family and organic-on transport peptide (OATP, SLC21A, SLCO) (Non-patent document 17, Non-patent document 18) Some have already been known to transport estrone trisulfate. They are mainly expressed in the liver, kidney, brain, intestine, and other sites. Expression in breast cancer cells has not been reported. Their Km values for estrone 3 sulfate are in the range of 0.05 μΜ to 59 μΜ. In women, plasma levels of estrone 3 sulfate range from 1 to 10 ηΜ (19), so these or other transporters are the first step in hormone-dependent growth. It appears to play a role in the uptake of cancer into cancer cells.

[0009] Recently, Pizzagalli et al. Reported that steroid sulfate transporters in human mammary glands. This suggests the expression of OATP-B (Non-patent Document 20). However, OATP-B mRNA was not detected in estrogen-dependent MCF-7 and T47D cells, whereas OATP-D & t) ATP-E mRNA was found in these cells (non- Patent Document 21). OATP-D¾tJ ^ O ATP-E When the gene is transiently expressed in HEK293 cells, it exhibits transport activity against estrone trisulfate (Non-Patent Document 22) and is involved in the transport of estrone trisulfate in cancer cells It seems to have done. However, the transporter-mediated uptake of estrone trisulfate in estrogen-dependent cells has been reported to be uptake in other tissues such as the liver, placenta and brain, which have not been investigated so far. In order to fully understand the growth mechanism of breast cancer cells, the mechanism of estrogen supply to such cells must be elucidated. Non-Patent Document 1: N Engl J Med 302: 78-90, 1980

Non-Patent Document 2: J Clin Endocrinol Metab 57: 1125-1128, 1983

Non-Patent Document 3: Breast Cancer Res Treat 7: 35-44, 1986

Non-Patent Document 4: Ann N Y Acad Sci 464: 126-137, 1986

Non-Patent Document 5: J Clin Endocrinol Metab 81: 1460-1464, 1996

Non-Patent Document 6: J Biol Chem 236: 1043-1050, 1961

Non-Patent Document 7: J Clin Invest 51: 1020-1033, 1972

Non-Patent Document 8: Endocrinology 138: 863-870, 1997

Non-Patent Document 9: Mol Endocrinol 11: 77-86, 1997

Non-Patent Document 10: Endocrinology 123: 1281-1287, 1988

Non-Patent Document 11: Breast Cancer 9: 296-302, 2002

Non-Patent Document 12: J Clin Endocrinol Metab 59: 1128-1132, 1984

Non-Patent Document 13: Endocrinology 106: 1079-1086, 1980

Non-Patent Document 14: J Clin Endocrinol Metab 81: 1460-1464, 1996

Non-Patent Document 15: J Steroid Biochem Mol Biol 73: 225-235, 2000

Non-Patent Document 16: Pflugers Arch 440: 337-350, 2000

Non-Patent Document 17: Biochim Biophys Acta 1609: 1-18, 2003

Non-Patent Document 18: Pflugers Arch 447: 653-665, 2003 Non-Patent Document 19: Hormone Res 27: 61-68, 1987

Non-Patent Document 20: J Clin Endocrinol Metab 88: 3902-3912, 2003

Non-Patent Document 21: J Clin Endocrinol Metab 88: 3902-3912, 2003

Non-Patent Document 22: Pharm Res 18: 1262-1269, 2001

Disclosure of the invention

Problems to be solved by the invention

[0011] As shown in Fig. 1, the following methods (blocks 1 to 4) are used to inhibit the growth of breast cancer cells that block upstream the signal transduction due to binding of estrogen and its receptor in breast cancer cells. It ’s known!

[0012] Block 1 (Inhibition of estrogen synthesis from androgen)

Fadrozole: After menopause, it selectively inhibits aromatase, which acts as a rate-limiting enzyme for estrogen synthesis, and also prevents androgenic activity from converting to estrogen, thereby reducing the in vivo estrogen concentration and suppressing breast cancer growth.

[0013] Block 2 (competitive inhibition at estrogen receptor)

Tamoxifen and toremifene: Estrogen receptors such as breast cancer tissues are competitively inhibited with estrogen and exert anti-breastogenic activity by showing anti-estrogenic activity

[0014] Block 3 (Inhibition of estrogen synthesis from estrone sulfate)

Conjugated estrogens are activated by estrogen sulfatase in the cell and promote cancer cell growth. Inhibits this deconjugating enzyme.

[0015] Block 4 (Cytotoxic effect using antibody to membrane protein)

Herceptin: Some breast cancer cells highly express HER2 protein. Using this binding of HER2 and antibody, it exerts an antitumor effect due to antibody-dependent cytotoxicity.

[0016] In the method of blocks 1 to 4 above for inhibiting the growth of breast cancer cells, an antitumor treatment is required unless an aromatase inhibitor, an estrogen sulfatase inhibitor, or an estrogen competitive inhibitor such as tamoxifen or toremifene is incorporated into breast cancer cells. Ineffectiveness In terms of drug delivery, and when incorporated into cells, there is no problem in terms of side effects. The problem of the present invention is that it is not necessary to be taken up into cells. Estrone trisulfate transporter with excellent drug delivery properties and extremely few side effects The aim is to provide a single transporter activity inhibitor, breast cancer cell growth inhibitor, and breast cancer therapeutic agent.

Means for solving the problem

[0017] The present inventors have intensively studied to solve the above problems, and examined the following block 5 (see Fig. 1) as a method for inhibiting the growth of breast cancer cells.

Block 5 (inhibition of transporter activity)

Anti-tumor effects appear in estrogen-sensitive breast cancer cells by inhibiting the uptake of estrone trisulfate, an estrogen source.

[0018] When the human breast cancer cell line MCF-7 cell line is cultured in the presence of estrone 3 sulfate and many natural organic compounds such as flavonoids as test substances, estrone 3 sulfate expressed on the cell surface It was found that the incorporation of estrone trisulfate into cells via the transporter was inhibited, and the proliferation of MCF-7 cells was suppressed, and the present invention was completed.

That is, the present invention relates to (1) Genistein, Quercetin, Ginkgolide C, Theaflavin, Theaflavin 3-0-gallate (Th eaflavin 3-O-gallate), Noretin (Rutin), Canolecon (Chalcone), Daidzein (Daidzein), Daidine (Daidzin), Flavanone (Flavanone), Flavonol, Geraldol (Geraldol), Hesperetin (Hesperidin), flavone (Hesperidin), prione , Luteolin-7-O-darcoside (Luteolin-7-0-Glucoside), methoxycanolone (Methoxychalcone), 4, 1-methinole-1-7-methoxyhiso-isoflavone, 5— Morin (5— Morin), Myricetin (Naringenin), Naringenin (7-glycoside), Naringin (Naringin), Choose from Ohesperidin Dihydrochalcone, Nomilin, Primuletin, Poncirin, Scutellarein, Catechin Hydrate, and chemically modified compounds Inhibitors of the transporter activity of estrone trisulfate transporter with one or more active ingredients as active ingredients, (2) Genistein, Quercetin, Ninkolide C (Ginkgolide C), Theaflavin, Theaflavin 3-O gallate (Theaflavin3- 0-gallate), Rutin, Chalcone, Daidzein, Daidin, Flavanone, Flavonol, Geraldol, Hesperetin, Hesperidin, Ipriflavone, Luteolin-7-O-Dalcoside, Methoxychalcone, 4, 1-methyl 7-methoxy-isoflavone, 5- Morin, Myricetin, Naringen, Naringen 7—Gnorecoside (Naringenin—7-glycoside), Naringin, Neohesperidin Dihydrochalcone, Nomilin, Prep Breast cancer cell growth inhibition containing one or more ingredients selected from muruletin (Primuletin), poncilin (Poncirin), scutellarein (Scutellarei n), force techin hydrate (Catechin Hydrate), and compounds chemically modified from these. (3) Genistein, Quercetin, Ginkgolide C, Theaflavin, Theaflavin 3-O-gallate, Rutin, Power Norrecon (Chalcone), daidzein, daidzin, furanonone, flavonol, geraldol, hesperetin, hesperidin, ipriflavone, luteolin 7— 0—Dalcoside (Luteolin-7-0- Glucoside), Methoxychalcone, 4, 1-Methylolone 7-Methoxy-Isoflavone (4 '-metyl- 7-methoxy-isoflavone), 5- Morin, Myricetin, Naringenin, Naringen-7-glycoside, Naringin ( Naringin, Neohespendin Dihydrocnaicone, Nomilin, Primuletin, Poncirin, Scutellarein, and Catechin Hydrat e, and these The present invention relates to a therapeutic agent for breast cancer comprising one or more components selected from compounds chemically modified as active ingredients.

Brief Description of Drawings

FIG. 1 is a schematic diagram showing a molecular target for breast cancer.

[Fig. 2] Results of inhibition of uptake of estrone trisulfate into breast cancer cells by various test substances It is a figure.

FIG. 3 is a diagram showing the results of suppression of cell growth promotion effect by estrone 3 sulfate by genistein, quercetin, ginkgolide C, theaflavin, and theaflavin 3-O-gallate.

FIG. 4 is a graph showing the results of inhibition of the incorporation of estrone trisulfate into breast cancer cells by various test substances.

BEST MODE FOR CARRYING OUT THE INVENTION

[0021] Inhibitors of transporter activity of the estrone trisulfate transporter of the present invention, breast cancer cell growth inhibitors and breast cancer therapeutic agents include Genistein, Quercetin, Ginkgolide C, Theaflavin (Theaflavin), Theaflavin 3-O-gallate, Rutin, Chalcone, Daidzein, Daidzin, Flavanone, Flavonol, Di Geradolone (Geraldol), Hesperetin, Hesperidin, Ipriflavone, Luteolin-7-O-Dalcoside, Methoxychalcone, 4 , 1-Methyl-7-methoxy-isoflavone (4 '— mety 卜 7-methoxy-isoflavone), 5-Morin (5— Morin), Myricetin (Myricetin) Naringenin, Naringenin 7-glycoside, Naringin, Neohesperidin Dihydrochalcone, Nomilin, Primuletin, Poncillin (Poncirin), scutellarein, and catechin hydrate can be mentioned. Among them, genistein, quercetin, ginkgolide C, theaflavin, theaflavin 3-0-gallate are preferable examples. can do. These may be used alone or in combination of two or more. Furthermore, a compound obtained by chemically modifying these can also be used.

The inhibitor of transporter activity of the estrone 3 sulfate transporter of the present invention (hereinafter sometimes referred to as “the present inhibitor”) is important in identifying the estrone 3 sulfate transporter. Candidate transporters for estrone 3 sulfate transporters include OAT 1, OAT2, OAT3, OAT4, OATP—A, OATP—B, OATP—C, OATP—D, O ATP-E, OATP-F, OATP-8, NTCP, MRPs, BCRP, etc. You can. As an identification method, cells expressing the candidate estrone 3 sulfate transporter on the cell surface are cultured, and the uptake of the candidate estrone 3 sulfate into the cultured cells is measured in the presence of the inhibitor. Measures the degree of inhibition of the transporter activity of estrone 3 sulfate into cells by the method of 'evaluation' or cell force to express the candidate estrone 3 sulfate transporter on the cell surface. Contact the sulfuric acid with the inhibitor and measure the degree of inhibition of the specific binding of estrone 3 sulfate to the cell membrane fraction by the inhibitor, or express the candidate estrone 3 sulfate transporter on the cell surface A cell membrane fraction is isolated from the cells to be produced, and vesicles are produced from the cell membrane fraction to form isolated cell membrane vesicles. Contacting the inhibitor, if estrone 3 uptake of sulfuric acid into vesicles by the present inhibitor Ku can be mentioned a method for measuring 'assess the degree of inhibition of uptake. Cultivation of cells in the presence of the above estrone 3 sulfate and the present inhibitor uses a growth medium for the cells used, and at least cell proliferation (in the absence of the present inhibitor) under normal cell culture conditions. Contact with the cell membrane fraction of the above estrone 3 sulfate and the inhibitor, or contact with the isolated cell membrane vesicle of the above estrone 3 sulfate and the inhibitor, which is desired to be carried out until significantly recognized. Can be performed by incubation in a growth medium for the cells used or an appropriate buffer.

The above-mentioned inhibitor measures the degree of inhibition of the transporter activity of estrone 3 sulfate into cells by 'measurement of the level of intracellular estrone 3 sulfate' and measures the degree of cell proliferation. 'You can list how to evaluate. When using cultured human breast cancer cell lines such as MCF-7 cells, KPL-1, MKL-F, or cell lines derived from these cell lines, which are described later, The method of measuring and evaluating the degree of cell proliferation can be used, but when using transformed cells expressing the candidate estrone trisulfate transporter, the method of measuring and evaluating the inhibitor concentration in the cell is used. Can be used. In addition, the candidate estrone 3 sulfate transporter gene is expressed in Xenopus laevis oocytes, etc., and changes in membrane potential caused by substrate transport via the transporter are detected using two microelectrodes (current Can also be measured and evaluated by electrophysiological methods. [0024] Cell force that expresses estrone trisulfate transporter on the cell surface As a method of isolating the cell membrane fraction, F. Pietri-Rouxel et al. (Eur. J. Biochem., 247, 1174-1179, 1997) Conventionally known methods such as the method can be used, and as a method for producing vesicles from the cell membrane fraction to obtain isolated cell membrane vesicles, JE Leveled Biol. Chem., 252, 1990-199 7 , 1977) and the like can be used. In addition, as a method for measuring the degree of inhibition of the specific binding of estrone 3 sulfate to the cell membrane fraction by the inhibitor, steady-state binding inhibition using radiolabeled estrone 3 sulfate was used as an equilibrium dissociation constant Kd. As a method for measuring the amount of estrone 3 sulfate incorporated into isolated cell membrane vesicles by this inhibitor or the degree of inhibition of the amount incorporated, I. Tamai et al. ( Biochim. Biophys. Acta, 1512, 273-284, 2001) can be used as a method for measuring intracellular estrone trisulfate, such as a method described in the related art.

[0025] As the cell expressing the estrone 3 sulfate transporter on the cell surface, a transformed cell expressing a candidate estrone 3 sulfate transporter can be used. OAT1, OAT2, OAT3, OAT4, OATP-A, OATP-B, OATP-C, OATP—D, OATP—E, OATP—F, OATP-8, The origins of genes encoding NTCP, MRPs, BCRP are not particularly limited. Examples include humans, inu, ushi, horses, goats, hidges, monkeys, pigs, usagis, rats, mice, etc. The power that can be cited Human preference is preferred.

[0026] The method for expressing a gene or cDNA encoding a transporter as described above in a cell is not particularly limited, but Davis et al. (BASIC METHODS IN MOLECULAR BIOL OGY, 1986) and Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANU AL, 2nd Ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), for example, as described in many standard laboratory manuals such as calcium phosphate transfer, DEAE-dextran. Mediated transfusion, transvection, microinjection, cationic lipid mediated transfection, electoporation, open-cell introduction, scrape loading, ballistic introduction, infection, etc. Expression by introducing a transporter gene into a host cell by gene transfer method The ability to transport transporter genes Among them, it is more preferable to express the transporter in the cell by infecting the host cell with an expression vector containing the transporter gene, even though it is preferable to introduce the expression vector containing the above into the host cell.

[0027] Examples of the above expression vectors include adenoviral vectors (Science, 252, 431-434, 1991) used for transient expression in all cells including non-dividing cells (other than blood cells), division, and the like. Retroviral vectors used for long-term expression in cells (Microbiology and Immunology, 158, 1-23, 1992) and adeno-associated viral vectors that can be introduced into non-pathogenic, non-dividing cells and used for long-term expression ( Curr. Top. Microbiol. Immunol, 158, 9 7-129, 1992), Papova Winores vector such as SV40, virus vector such as vaccinia Winores vector etc., ability to specifically mention ribosome etc. It is not limited to these. Among these, an adenovirus vector capable of gene expression in cells with high efficiency is particularly preferable. In addition, a transporter gene can be introduced into these expression vectors by a conventional method. For example, an expression vector can be constructed by inserting a transporter gene or the like downstream of an appropriate promoter in these expression vectors. it can. In addition to the transporter gene and marker gene, the expression vector regulates the expression of the enhancer, terminator, etc. in addition to IRES (ribosome-binding site in the mRNA) to regulate the expression level of the transporter per cell. Including control array.

[0028] Host cells that express the transporter include insect cells such as Drosophila S2 and Spodoptera Sf9, Vero cells, HeLa cells, CHO cells, WI-38 cells, BHK cells, COS 7 cells, MDCK cells, C127 Cells, HKG cells, human kidney cell lines, CV-1 cells, LLC—MK2 cells, MDBK cells, MRC-5 cells, Caco-2 cells, HT29 cells, human lymphoblasts, Xenopus laevis, etc. And dhfr-deficient strains, HGPRT-deficient strains, and wabain resistant strains. Specifically, CHO—K1 (Chinese hamster ovary cells: ATCC CCL61), BHK (Nomster kidney cells: ATCC CCL 10), COS-7 (CV- Origin, SV—40 cells: ATCC CRL1651), Vero Cells (African green monkey kidney cells: ATCC CCL81) and human lymphoblasts (IM-9, ATCC CCL159) can be shown in column f. [0029] Since the transporter is a membrane protein expressed on the cell membrane surface, neutralizing antibodies against the estrone trisulphate transporter may selectively inhibit the target against extracellular force. Once the trisulfate transporter can be identified, neutralizing antibodies against the estrone trisulfate transporter can be generated. Specific antibodies that can be specifically recognized include monoclonal antibodies, polyclonal antibodies, single chain antibodies, humanized antibodies, chimeric antibodies, bifunctional antibodies that can simultaneously recognize two epitopes, and the like. These antibodies are produced by administering cells expressing the estrone trisulfate transporter or the membrane fraction thereof to animals (preferably non-humans) using conventional protocols. For example, monoclonal antibodies The preparations include antibodies produced by continuous cell line cultures, the Hypridoma method (Nature 256, 495-497, 1975), the Trioma method, the human B cell Hypridoma method (Immunol ogy Today 4, 72 1983) and EBV-hybridoma method (MONOCLONAL ANTIBODIE S AND CANCER THERAPY, 77-96, Alan R. Liss, Inc., 1985).

[0030] The breast cancer cell growth inhibitor of the present invention can be used for identification of an estrone trisulfate transporter and also as a breast cancer therapeutic agent because it can suppress the growth of breast cancer cells. As an identification method, a cell expressing a candidate estrone trisulfate transporter on the cell surface is cultured in the presence of the breast cancer cell growth inhibitor of the present invention, and the method for measuring and evaluating the degree of cell proliferation is exemplified. be able to.

[0031] The therapeutic agent for breast cancer of the present invention can also be used as a prophylactic agent for breast cancer. When used as a therapeutic agent for pharmaceuticals, a normal pharmaceutically acceptable carrier, binder, stabilizer, enhancer. Various preparation co-components such as a dosage form, a diluent, a pH buffer, a disintegrant, a solubilizer, a solubilizer, and an isotonic agent can be added. These therapeutic agents can be administered orally or parenterally, such as powders, granules, tablets, capsules, syrups, suspensions, etc. Alternatively, for example, a dosage form such as a solution, emulsion, suspension or the like can be administered parenterally by injection. When administered parenterally, it can be administered topically. In the case of preparations for oral administration, various conventional organic or inorganic carrier substances are used as pharmacologically acceptable carriers. Excipients such as pumps, lubricants such as talc and magnesium stearate, binders such as hydroxypropylcellulose and polybulurpyrrolidone, disintegrants such as carboxymethylcellulose, etc. Solvents such as physiological saline alcohol, solubilizing agents such as polyethylene glycol and propylene glycol, suspending agents such as stearyltriethanolamine, sodium lauryl sulfate and lecithin, isotonic agents such as glycerin and D-manntol Agents, phosphates, acetates, buffers such as kenates, etc. can be blended. If necessary, formulation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be combined. In the case of preparations administered parenterally, water-soluble solvents such as distilled water and physiological saline, solubilizing agents such as sodium salicylate, isotonic agents such as sodium chloride sodium, glycerin, D-mannitol, Stabilizers such as human serum albumin, preservatives such as methylparaben, and narcotics such as benzyl alcohol can be added. The dosage of the therapeutic agent can be appropriately selected depending on the patient's weight and age, dosage form, symptoms, etc. For example, when administered to an adult, the active ingredient is usually about 0.001 to 500 mg as a single dose, It is preferable to administer 1 to 50 mg once to 3 times a day.

[0032] Hereinafter, the present invention will be described more specifically with reference to examples, but the technical scope of the present invention is not limited to these examples.

Example 1

[0033] (Inhibition of uptake activity of estrone 3 sulfate)

In the presence of excess estrone 3 sulfate, the inhibitory effects of various test substances on [ ³ H] estrone 3 sulfate uptake by MCF-7 cells (ATCC-HTB22) were examined. MCF-7 cells containing 125 mM NaCl, 4.8 mM KC1, 5.6 mM D-glucose, 1.2 mM CaCl, 1.2 mM KH PO, 1.2 mM MgSO and 25 mM Hepes, p

2 2 4 4

It was suspended in the transport medium prepared in H7.4. Cell suspension in transport medium and a solution containing unlabeled ImM estrone trisulfate, 3.6 nM [ ³ H] estrone trisulfate and 30 μL of each test compound individually for 10 minutes at 37 ° C. And started transportation. When appropriate, remove 150 μL of the mixture and centrifuge through a layer of silicone oil (SH550, Toray Dow Corning Co.) and liquid paraffin of density 1.03 (Wako Pure Chemical Industries). The cells were separated from the transport medium by each Cell pellets were solubilized in 3N KOH and neutralized with HC1. Thereafter, the associated radioactivity was measured with a liquid scintillation force counter using Clearsol 1 (manufactured by Nacalai Tesque) as the liquid scintillation fluid. The result is shown in figure 2. As a result, when genistein, quercetin, ginkgolide C, theaflavin, and theaflavin 3-O-gallate were used, it was found that the uptake activity of estrone 3 sulfate was strongly inhibited.

Example 2

[0034] (Inhibition of cell growth promoting effect by estrone trisulfate)

Inhibition of the cell growth promoting effect of estrone 3 sulfate on MCF-7 cells (ATCC—HTB22) was examined using genistein, quercetin, ginkgolide C, theaflavin, and theaflavin 3-O-gallate. We examined whether this could be achieved by suppressing intracellular accumulation of estrone 3 sulfate. MCF-7 cells 8 × 10 ³ ZO. Seed in 96-well plate at a concentration of 32 cm ² and cultured for 1 day, then estrone 3 sulfate (E 3S; Sigma Chemi cals) was present at a concentration of ΙΟΟηΜ In the absence or absence of 30 μΜ bromosulfophthalein (BSP: Sigma Chemicals) as a positive control and 30 μΜ each dissolved in 0.3% dimethyl sulfoxide (DMSO). Medium containing genistin, quercetin, ginkgolide, theaflavin, and theaflavin 3- 3-gallate were added, and cultured for another 3 days. Thereafter, the amount of cells was measured. The results are shown in Figure 3. As is clear from FIG. 3, cell growth promoting effects were observed in the presence of estrone 3 sulfate alone as a control and in the presence of estrone 3 sulfate dissolved in 0.3% DMSO. On the other hand, 30 μΜ of gestin, quercetin, ginkgolide C, theaflavin, and theaflavin 3-O-gallate dissolved in 0.3% DMSO in the presence of 100 nM estrone 3 sulfate (E 3S) were added. In the same manner as in the case of supplementing with BSP, cell growth was inhibited compared to the control.

Example 3

[0035] (Inhibition of estrone trisulfate uptake activity)

In the same manner as in Example 1, the inhibitory effect of various test substances on the uptake activity of estrone 3 sulfate was examined. The results are shown in Fig. 4. As a result, chalcone, daidzein, daidzin, flavanone, flavonol, dizalraldol, hesperetin, hesperzin, ypriflavone, Luteolin 7-O-Dalcoside, Methoxychalcone, 4'-Methyl-1-7-methoxy-Isoflavone, 5-Morine, Myricetin, Naringen, Naringen 7-Dalcoside, Naringin, Neohesperidin Hyde mouth chalcone, nomiline, primuletin, poncillin, scutellarein, and force tekin hydrate were found to have a strong inhibitory action on estrone trisulfate uptake.

Industrial applicability

According to the present invention, unlike estrogen competitive inhibitors such as aromatase inhibitors, estrogen sulfatase inhibitors, tamoxifen and toremifene, it has excellent drug delivery properties that do not need to be taken into cells and has very few side effects. Can provide a transporter inhibitor, a breast cancer cell growth inhibitor, and a breast cancer therapeutic agent.

Claims

The scope of the claims

[1] Genistein, Quercetin, Ginkgolide C, Theaflavin, Theaflavin 3-O-gallate, Rutin, Chalcone , Daidzein, Daidzin, Flavanone, Flavonol, Geraldol, Hesperetin, Hesperidin, Ipriflavone, Luteolin 1—O-Dalcoside (Luteolin-7-0-Glucoside), 4, Methoxychalcone, 4, 1-Methylolone 7-Methoxy-isoflavone (4'—mety 卜 7-methoxy-isoflavone), 5-—Molin ( 5- Morin), Myricetin, Naringenin, Naringenin 7-glycoside, Naringin, Neohesperidin dinoid One or more selected from canorecon (Neohesperidin Dihydrochalcone), nomilin, primuletin, poncilin, scutellarein, force techin hydrate (Ca techin Hydrate), and compounds chemically modified from these An inhibitor of the transporter activity of the estrone trisulfate transporter, comprising as an active ingredient.

[2] Genistein, Quercetin, Ginkgolide C, Theaflavin, Theaflavin 3-O-gallate, Rutin, Chalcone , Daidzein, Daidzin, Flavanone, Flavonol, Geraldol, Hesperetin, Hesperidin, Ipriflavone, Luteolin 1—O-Dalcoside (Luteolin-7-0- Glucoside), Methoxychalcone, 4, 1-Methylolone 7-Methoxy-1-isoflavone, 5—Molin ( 5- Morin), Myricetin, Naringenin, Naringenin 7-glycoside, Naringin, Neohesperidin dinoiduro One or more selected from canorecon (Neohesperidin Dihydrochalcone), nomilin, primuletin, poncilin, scutellarein, force techin hydrate (Ca techin Hydrate), and compounds chemically modified from these A breast cancer cell growth inhibitor comprising the above ingredients as an active ingredient.

[3] Genistein, Quercetin, Ginkgolide C, Theaflavin, Theaflavin 3-O-gallate, Theaflavin 3-O-gallate, Rutin, Chalcone, Daidzein, Daidzin, Flavanone, Flavonol ), Geraldol, Hesperetin, Hesperidin, Ipriflavone, Luteolin-7-O-Dalcoside, Methoxychalcone ), 4, 1-methylol 7-methoxy-isoflavone (4'-methy 卜 7-methoxy-isoflavone), 5- Morin (5- Morin), myricetin (Myricetin), Naringenin, Naringen I 7—Gnorecoside (Naringenin-7-glycoside), Naringin (Naringin), Neohesperidin Dihydrochalcone (Neohesperidin Dihydrochalcone), Nomilin (Primilet) in), Poncirin, Scutellarein, Catechin Hydrate, and a compound force obtained by chemically modifying these. A therapeutic agent for breast cancer comprising one or more selected components as active ingredients.