JP2006298781A - Estrone-3-sulfate transporter activity inhibitor - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide an agent for inhibiting transporter activity of estrone-3-sulfate transporter, a mammary cancer cell proliferation inhibitor and a mammary cancer treating agent unnecessary to be taken into the cell, having excellent drug delivery performance and exhibiting extremely low side actions. <P>SOLUTION: The agent contains one or more components selected from genistein, quercetin, ginkgolide C, theaflavin, theaflavin 3-O-gallate, chalcone, rutin, daidzein, daidzin, flavanone, flavonol, geraldol, hesperetin, hesperidin, ipriflavone, luteolin-7-O-glucoside, methoxychalcone, 4'-metyl-7-methoxy-isoflavone, 5-morin, myricetin, naringenin, naringenin-7-glycoside, naringin, neohesperidin dihydrochalcone, nomilin, primuletin, poncirin, scutellarein, catechin hydrate, and their chemically modified compounds. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  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.

  Membrane proteins that transport substances inside and outside the cell are called transporters. When a specific molecule binds to a transporter embedded in a lipid bilayer, the conformation changes, and the substance is taken up or transported. It is known that In recent years, such transporters, for example, organic anion transporters such as OAT1 that transport organic anionic substances, organic cation transporters such as OCT1 that transport organic cationic substances, peptide transporters such as PEPT1 that transport peptide substances, etc. These genes have been isolated and identified one after another. Some of these transporter genes are ubiquitous in normal tissues and organs throughout the body, but others are known to be localized in specific tissues and organs such as kidney, liver and brain.

On the other hand, estrogens are so-called female hormones that cause estrus in female animals, and are naturally occurring estrone (E ₁ ), estradiol (E ₂ ), estriol (E ₃ ), and estetrol (E ₄ ). Are classified into synthetic estrogens having the same biological activity. With the exception of some synthetic forms such as stilbestrol, estrogens have a steroid structure. The biological action of estrone 0.1 μg is defined as 1 IU (international unit), which is used as a unit of estrogen. The source of secretion is mainly ovarian follicles and corpus luteum, but it is also secreted from the fetal placental system, adrenal gland, testis, etc. during pregnancy. Estrogen secretion from the ovary is governed by gonadotropins secreted by the anterior pituitary gland (downward regulation), but conversely, estrogen also has a feedback effect on the pituitary system (ascending regulation). It is said that the sexual cycle is established by the mutual relationship between the two.

  Furthermore, estrogen acts through its receptor (receptor), and estrogen acts not only on the target tissue, but also on the whole brain, anterior pituitary gland, genitals and mammary gland, and the main physiological functions are endometrium Such as growth of uterine muscle, development of secondary sexual characteristics, mediation of establishment of menstrual cycle, induction of maternal changes during pregnancy, promotion of growth secretion of mammary ducts. 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, osteoporosis, and the like.

  Breast cancer has an abnormal operation of the estrogen receptor system from the time of carcinogenesis, and continues to grow estrogen-dependently 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, but the mechanism is still unclear. Breast cancer is a malignant tumor that arises from the peripheral ducts and acinar epithelium of the epithelial tissue of the mammary gland, and is the leading cancer death among women in the West. Also in Japan, the death rate from breast cancer is on the rise due to recent westernization of eating habits and changes in lifestyle. Prevalence age is in the 40s, risk factors include history of breast cancer, family history, unmarried, elderly first birth, early menarche and late menopause, obesity, radiation exposure, high fat diet, history of benign breast disease, etc. it can. Lymph node metastasis is more frequent in the axilla, subclavian and parasternal, and the number of metastases 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.

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

  Stimulation of breast cancer cell growth by estrone trisulfate involves the following series of processes: uptake of estrone trisulfate into cells, desulfation by estrogen sulfatase, estrone to estradiol by type I 17β-hydroxysteroid dehydrogenase Conversion, nuclear estrogen receptor binding, and gene transcription control (Non-Patent Documents 8 to 11). We have examined the enzymes and receptors involved in the response of breast cancer cells to estrone trisulfate, but the transport mechanism through which ligands such as estrone trisulfate cross the cell membrane is unknown. The log P value of estrone trisulfate is reported to be 1.4, which is significantly lower than that of estrone (4.4) and estradiol (3.9), so that 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 an estrogenic action (Non-patent Document 4, Non-patent document 14, Non-patent document 15). Therefore, a specific transporter seems to be involved in the supply of estrone trisulfate to breast cancer cells exhibiting hormone-dependent growth.

  Among the members of the organic anion transporter (OAT, SLC22A) (Non-Patent Document 16) family and the organic anion transport peptide (OATP, SLC21A, SLCO) (Non-patent Document 17, Non-Patent Document 18) family, estrone trisulfate is transported. It is already known that there is something to do. They are expressed primarily in the liver, kidney, brain, intestine, and other sites, but no expression in breast cancer cells has been reported. Their Km values for estrone trisulfate are in the range of 0.05 μM to 59 μM. In women, plasma levels of estrone trisulfate vary in the range of 1-10 nM (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.

  Recently, Pizzagalli et al. Have suggested the expression of OATP-B, a steroid sulfate transporter in human mammary gland (Non-patent Document 20). However, OATP-B mRNA was not detected in estrogen-dependent MCF-7 cells and T-47D cells, whereas OATP-D and OATP-E mRNA were found in these cells (non-patented). Reference 21). OATP-D and OATP-E exhibit transport activity against estrone 3 sulfate when the gene is transiently expressed in HEK293 cells (Non-patent Document 22). It seems to be involved. However, transporter-mediated uptake of estrone trisulfate in estrogen-dependent cells has not been investigated so far, but uptake in other tissues such as liver, placenta and brain has been reported. In order to fully understand the growth mechanism of breast cancer cells, the mechanism of estrogen supply to such cells must be elucidated.

N Engl J Med 302: 78-90, 1980 J Clin Endocrinol Metab 57: 1125-1128, 1983 Breast Cancer Res Treat 7: 35-44, 1986 Ann N Y Acad Sci 464: 126-137, 1986 J Clin Endocrinol Metab 81: 1460-1464, 1996 J Biol Chem 236: 1043-1050, 1961 J Clin Invest 51: 1020-1033, 1972 Endocrinology 138: 863-870, 1997 Mol Endocrinol 11: 77-86, 1997 Endocrinology 123: 1281-1287, 1988 Breast Cancer 9: 296-302, 2002 J Clin Endocrinol Metab 59: 1128-1132, 1984 Endocrinology 106: 1079-1086, 1980 J Clin Endocrinol Metab 81: 1460-1464, 1996 J Steroid Biochem Mol Biol 73: 225-235, 2000 Pflugers Arch 440: 337-350, 2000 Biochim Biophys Acta 1609: 1-18, 2003 Pflugers Arch 447: 653-665, 2003 Hormone Res 27: 61-68, 1987 J Clin Endocrinol Metab 88: 3902-3912, 2003 J Clin Endocrinol Metab 88: 3902-3912, 2003 Pharm Res 18: 1262-1269, 2001

  As shown in FIG. 1, the following methods (blocks 1 to 4) are known for inhibiting breast cancer cell growth, which blocks upstream signal transduction due to binding of estrogen and its receptor in breast cancer cells. .

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, preventing the conversion of androgens to estrogens, thereby reducing estrogen concentration in the body and suppressing breast cancer growth.

Block 2 (competitive inhibition at estrogen receptor)
Tamoxifen, toremifene: Estrogen receptors such as breast cancer tissues are competitively inhibited with estrogen and exert anticancer activity by exhibiting antiestrogenic activity.

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.

Block 4 (cytotoxic effect using antibody against membrane protein)
Herceptin: Some breast cancer cells highly express HER2 protein. Utilizing the binding between HER2 and an antibody, it exerts an antitumor effect due to an antibody-dependent cytotoxic effect.

  In the method of Blocks 1 to 4 above for inhibiting the growth of breast cancer cells, an anti-tumor effect is exhibited unless an aromatase inhibitor, an estrogen sulfatase inhibitor, or an estrogen competitive inhibitor such as tamoxifen or toremifene is incorporated into the breast cancer cells. Since it is not performed, it cannot be said that there is no problem in terms of drug delivery and in terms of side effects when incorporated into cells. An object of the present invention is to provide an inhibitor of transporter activity of an estrone trisulfate transporter, a breast cancer cell growth inhibitor, or a breast cancer therapeutic agent that does not need to be taken into cells, has excellent drug delivery properties, and has very few side effects. There is.

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)
An anti-tumor effect appears by inhibiting intracellular uptake of estrone trisulfate, which is a source of estrogen, against estrogen-sensitive breast cancer cells.

  When the human breast cancer cultured cell MCF-7 cell line is cultured in the presence of estrone trisulfate and many natural organic compounds such as flavonoids as test substances, the estrone trisulfate transporter expressed on the cell surface is used. It was found that the uptake of all estrone 3 sulfates into cells was inhibited and the growth of MCF-7 cells was suppressed, and the present invention was completed.

  That is, the present invention includes (1) Genistein, Quercetin, Ginkgolide C, Theaflavin, Theaflavin 3-O-gallate, Rutin, Chalcone, Daidzein, Daidin, Flavanone, Flavonol, Geraldol, Hesperetin, Hesperidin, Ipriflavone, Luteolin-7 O-glucoside (Luteolin-7-O-Glucoside), methoxychalcone (Methoxychalcone), 4'-methyl-7-methoxy-isoflavone (4'-metyl-7-methoxy-isoflavone), 5-morin (5-Morin) , Myricetin, naringenin, naringenin-7-glycoside, naringe (Naringin), Neohesperidin Dihydrochalcone, Nomilin, Primuletin, Poncirin, Scutellarein, Catechin Hydrate, and these chemically modified compounds Inhibitors of transporter activity of estrone trisulfate transporter comprising one or more components selected from (2) Genistein, Quercetin, Ginkgolide C, Theaflavin (Theaflavin) ), Theaflavin 3-O-gallate, rutin, chalcone, daidzein, daidzin, flavanone, flavonol, geraldol ( Geraldol), Hesperetin, Hesperetin Hesperidin, Ipriflavone, Luteolin-7-O-Glucoside, Methoxychalcone, 4'-Methyl-7-methoxy-isoflavone (4'-metyl-7) -methoxy-isoflavone), 5-Morin, Myricetin, Naringenin, Naringenin-7-glycoside, Naringin, Neohesperidin dihydrochalcone (Neohesperidin) One or more ingredients selected from Dihydrochalcone, Nomilin, Primuletin, Poncirin, Scutellarein, Catechin Hydrate, and compounds chemically modified from these ingredients Breast cancer cell growth inhibitor and (3) Genistein, Quercetin, Ginkgolay Ginkgolide C, Theaflavin, Theaflavin 3-O-gallate, Rutin, Chalcone, Daidzein, Daidzin, Flavanone ), Flavonol, Geraldol, Hesperetin, Hesperidin, Ipriflavone, Luteolin-7-O-Glucoside, Methoxychalcone 4'-methyl-7-methoxy-isoflavone (4'-metyl-7-methoxy-isoflavone), 5-morin, myricetin, naringenin, naringenin-7-glucoside -7-glycoside), Naringin, Neohesperidin Dihydrochalcone, Nomilin Nomilin, Primuletin, Poncirin, Scutellarein, Catechin Hydrate, and one or more components selected from these chemically modified compounds for breast cancer treatment It relates to the agent.

  According to the present invention, unlike an aromatase inhibitor, an estrogen sulfatase inhibitor, or an estrogen competitive inhibitor such as tamoxifen or toremifene, an estrone trisulfate transporter that does not need to be taken into cells, has excellent drug delivery properties, and has very few side effects. Inhibitors, breast cancer cell growth inhibitors, and breast cancer therapeutic agents can be provided.

  Examples of 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 3-O-gallate, Rutin, Chalcone, Daidzein, Daidin, Flavanone, Flavonol, Geraldol Hesperetin, Hesperidin, Ipriflavone, Luteolin-7-O-Glucoside, Methoxychalcone, 4'-methyl-7-methoxy-isoflavone 4'-metyl-7-methoxy-isoflavone), 5-Morin, Myricet (Myricet in), naringenin, naringenin-7-glycoside, naringin, neohesperidin dihydrochalcone, nomilin, primuletin, poncirin , Scutellarein, and catechin hydrate (Catechin Hydrate), among which genistein, quercetin, ginkgolide C, theaflavin, and theaflavin 3-O-gallate can be preferably exemplified. Moreover, these can be used individually by 1 type or in combination of 2 or more types. Furthermore, compounds 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 OAT1, OAT2, OAT3, OAT4, OATP-A, OATP-B, OATP-C, OATP-D, OATP-E, OATP-F, OATP-8, NTCP, MRPs, BCRP and the like can be preferably mentioned. 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. Methods for measuring and evaluating the degree of inhibition of the transporter activity of estrone 3 sulfate into cells and cell membrane fractions isolated from cells that express the candidate estrone 3 sulfate transporter on the cell surface A method of measuring and evaluating the degree of inhibition of the specific binding of estrone 3 sulfate to the cell membrane fraction by contacting with an inhibitor, or from a cell expressing a candidate estrone 3 sulfate transporter to the cell membrane A fraction is isolated, a vesicle is produced from the cell membrane fraction to form an isolated cell membrane vesicle, and estrone trisulfate is added to the isolated cell membrane vesicle. Contacting the present inhibitor may be a method in which the degree of inhibition of uptake or uptake of estrone 3-sulfate into vesicles by the present inhibitor is measured and evaluated. Cell culture in the presence of the above estrone trisulfate and the inhibitor is carried out using a growth medium for the cells used, and at least significant cell growth is observed under normal cell culture conditions (in the absence of the inhibitor). The contact with the cell membrane fraction of the estrone 3 sulfate and the inhibitor, or the contact with the isolated cell membrane vesicle of the estrone 3 sulfate and the inhibitor, The incubation can be performed by incubation in a growth medium or an appropriate buffer.

  As a method for measuring and evaluating the degree of inhibition of the transporter activity of estrone 3 sulfate into cells by the inhibitor, the method for measuring and evaluating the concentration of estrone 3 sulfate in cells or the degree of cell proliferation is measured. -List methods of evaluation. When using human breast cancer cell lines such as MCF-7 cells, KPL-1, MKL-F, or cell lines derived from those cell lines described later, a method for measuring and evaluating the inhibitor concentration in the cells, A method for measuring and evaluating the degree of proliferation can be used, but when a transformed cell expressing a candidate estrone trisulfate transporter is used, a method for measuring and evaluating the inhibitor concentration in the cell is used. can do. In addition, the candidate estrone 3 sulfate transporter gene is expressed in Xenopus oocytes, etc., and changes in membrane potential caused by transport of substrate through the transporter are detected using two microelectrodes (current measurement) It can also be measured and evaluated by electrophysiological methods.

  Examples of a method for isolating a cell membrane fraction from cells expressing the estrone 3 sulfate transporter on the cell surface include the method of F. Pietri-Rouxel et al. (Eur. J. Biochem., 247, 1174-1179, 1997). Conventionally known methods can be used, and methods for preparing vesicles from cell membrane fractions to make isolated cell membrane vesicles include JE Lever et al. (J. Biol. Chem., 252, 1990-1997, 1977). A conventionally known method such as the method of Further, as a method for measuring the degree of inhibition of specific binding of estrone 3 sulfate to the cell membrane fraction by the present inhibitor, the equilibrium dissociation constant Kd is measured from steady state binding inhibition using radiolabeled estrone 3 sulfate. The method can be exemplified, and as a method for measuring the amount of estrone 3 sulfate incorporated into isolated cell membrane vesicles by the present inhibitor or the degree of inhibition of the amount incorporated, I. Tamai et al. (Biochim. Biophys. Acta , 1512, 273-284, 2001), and the like, can be used as a method for measuring intracellular estrone trisulfate.

  As a cell that expresses the estrone 3 sulfate transporter on the cell surface, a transformed cell that expresses a candidate estrone 3 sulfate transporter can be used. The OAT1, OAT2, OAT3, OAT4, OATP-A, OATP-B, OATP-C, OATP-D, OATP-E, OATP-F, OATP-8, NTCP, MRPs used for the production of such transformed cells The origin of the gene encoding BCRP is not particularly limited, and examples thereof include humans, dogs, cows, horses, goats, sheep, monkeys, pigs, rabbits, rats, mice and the like. Is preferred.

  A method for expressing a gene or cDNA encoding the transporter as described above in a cell is not particularly limited, but Davis et al. (BASIC METHODS IN MOLECULAR BIOLOGY, 1986) and Sambrook et al. (MOLECULAR CLONING: A LABORATORY MANUAL, 2nd Ed. , Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989), as described in many standard laboratory manuals such as calcium phosphate transfection, DEAE-dextran mediated transfection, transvection, By introducing the transporter gene into the host cell by microinjection, cationic lipid-mediated transfection, electroporation, transduction, scrape loading, ballistic introduction, infection, etc. Expression However, it is preferable to introduce an expression vector containing a transporter gene into the host cell, and in particular, the transporter can be expressed in a cell by infecting the host cell with an expression vector containing the transporter gene. More preferred.

  Examples of the expression vector include adenoviral vectors (Science, 252, 431-434, 1991) used for transient expression in all cells including non-dividing cells (other than blood cells), and in dividing cells. Retrovirus vectors used for long-term expression (Microbiology and Immunology, 158, 1-23, 1992) and adeno-associated virus vectors used for long-term expression (Curr. Top. Microbiol. Immunol., 158, 97-129, 1992), virus vectors such as SV40 and vaccinia virus vectors, as well as liposomes, and the like. It is not something. Among these, an adenovirus vector capable of expressing genes in cells with high efficiency is particularly preferable. In addition, the 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 is an IRES (ribosome binding site in the mRNA) for normalizing the expression level of the transporter per cell, as well as a control sequence that regulates the expression of enhancers, terminators, etc. May be included.

  Examples of host cells that express the transporter include insect cells such as Drosophila S2, Spodoptera Sf9, Vero cells, HeLa cells, CHO cells, WI-38 cells, BHK cells, COS-7 cells, MDCK cells, C127 cells, and 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 and other oocytes, and their dhfr deficiency Strains, HGPRT-deficient strains, ouabain resistant strains, and the like. Specifically, CHO-K1 (Chinese hamster ovary cell: ATCC CCL61), BHK (hamster kidney cell: ATCC CCL10), COS-7 (CV-1 Origin, SV-40 cell: ATCC CRL1651), Vero cell (Africa) Examples thereof include midisal kidney cells (ATCC CCL81) and human lymphoblast cells (IM-9, ATCC CCL159).

  Since the transporter is a membrane protein expressed on the surface of the cell membrane, neutralizing antibodies against the estrone trisulfate transporter may selectively inhibit the target from outside the cell. From this, the inhibitor inhibits estrone trisulfate trans Once the porter 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 on the membrane surface and cell membrane fractions thereof to animals (preferably other than humans) using conventional protocols. The preparation involves the hybridoma method (Nature 256, 495-497, 1975), the trioma method, the human B cell hybridoma method (Immunology Today 4, 72, 1983) and EBV resulting in antibodies produced by continuous cell line cultures. -Any method such as the hybridoma method (MONOCLONAL ANTIBODIES AND CANCER THERAPY, 77-96, Alan R. Liss, Inc., 1985) can be used.

  The breast cancer cell growth inhibitor of the present invention can be used for identification of an estrone trisulfate transporter and as a breast cancer therapeutic agent because it can inhibit the growth of breast cancer cells. Examples of the identification method include a method of culturing cells expressing the candidate estrone trisulfate transporter on the cell surface in the presence of the breast cancer cell growth inhibitor of the present invention, and measuring and evaluating the degree of cell proliferation. it can.

  The breast cancer therapeutic agent of the present invention can also be used as a preventive agent for breast cancer, and when used as a pharmaceutical therapeutic agent, a normal pharmaceutically acceptable carrier, binder, stabilizer, excipient, Various preparation ingredients such as 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, and can be administered orally, for example, in the form of powders, granules, tablets, capsules, syrups, suspensions, etc., or 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 directly locally. In the case of preparations for oral administration, various conventional organic or inorganic carrier substances are used as pharmacologically acceptable carriers. For example, tablets include excipients such as lactose and starch, talc, and magnesium stearate. Lubricants such as hydroxypropylcellulose and polyvinylpyrrolidone, disintegrants such as carboxymethylcellulose, etc. can be blended. Solvents such as physiological saline alcohol, polyethylene glycol, propylene Solubilizing agents such as glycol, suspending agents such as stearyltriethanolamine, sodium lauryl sulfate, lecithin, isotonic agents such as glycerin and D-mannitol, buffering agents such as phosphate, acetate and citrate Etc. can be blended. If necessary, formulation additives such as preservatives, antioxidants, colorants, sweeteners and the like can be blended. For preparations for parenteral administration, water-soluble solvents such as distilled water and physiological saline, solubilizing agents such as sodium salicylate, isotonic agents such as sodium chloride, glycerin and D-mannitol, human serum albumin, etc. Stabilizers, preservatives such as methylparaben, and narcotics such as benzyl alcohol can be blended. 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, preferably It is desirable to administer 1 to 50 mg once to 3 times a day.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, the technical scope of this invention is not limited to these illustrations.

(Inhibition of estrone 3 sulfate uptake activity)
In the presence of excess estrone 3 sulfate, the inhibitory effect of various test substances on the uptake of [ ³ H] estrone 3 sulfate by MCF-7 cells (ATCC-HTB22) was examined. MCF-7 cells were treated with 125 mM NaCl, 4.8 mM KCl, 5.6 mM D-glucose, 1.2 mM CaCl ₂ , 1.2 mM KH ₂ PO ₄ , 1.2 mM MgSO ₄ and 25 mM Hepes. It was suspended in a transport medium containing and adjusted to pH 7.4. The cell suspension in transport medium and a solution containing unlabeled 1 mM estrone trisulfate, 3.6 nM [ ³ H] estrone trisulfate and 30 μM each test compound were individually incubated for 10 minutes at 37 ° C. for transport. Started. When appropriate, remove 150 μL of the mixture and transport by centrifugal filtration through a layer of silicone oil (SH550, Toray Dow Corning Co.) mixture and 1.03 density liquid paraffin (Wako Pure Chemical Industries). Cells were separated from the medium. Each cell pellet was solubilized in 3N KOH and neutralized with HCl. Thereafter, the associated radioactivity was measured with a liquid scintillation counter using Clearsol-1 (manufactured by Nacalai Tesque) as the liquid scintillation fluid. The results are shown in FIG. As a result, it was found that when genistein, quercetin, ginkgolide C, theaflavin, and theaflavin 3-O-gallate were used, the uptake activity of estrone 3 sulfate was strongly inhibited.

(Suppression 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. Whether it was achieved by suppressing intracellular accumulation of estrone trisulfate was examined. MCF-7 cells were seeded in a 96-well plate at a concentration of 8 × 10 ³ cells / 0.32 cm ² and cultured for 1 day, and then in the presence of 100 nM estrone 3 sulfate (E ₁ 3S; manufactured by Sigma Chemicals). Or, in the absence, 30 μM bromosulfophthalein (BSP: Sigma Chemicals) as a positive control and 30 μM genistein, quercetin, and ginkgolide dissolved in 0.3% dimethyl sulfoxide (DMSO), respectively. Medium containing C, theaflavin, and theaflavin 3-O-gallate was added, respectively, and further cultured for 3 days. Thereafter, the amount of cells was measured. The results are shown in FIG. As is clear from FIG. 3, a cell growth promoting effect was 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, when 30 μM genistein, quercetin, ginkgolide C, theaflavin, and theaflavin 3-O-gallate dissolved in 0.3% DMSO were added in the presence of estrone trisulfate (E ₁ 3S) at a concentration of 100 nM, BSP As in the case of adding, cell growth was inhibited compared to the control.

(Inhibition of estrone 3 sulfate uptake activity)
In the same manner as in Example 1, the inhibitory action of estrone trisulfate uptake activity by various test substances was examined. The results are shown in FIG. As a result, chalcone, daidzein, daidzin, flavanone, flavonol, geraldol, hesperetin, hesperzin, ipriflavone, luteolin-7-O-glucoside, methoxychalcone, 4′-methyl-7-methoxy-isoflavone, 5-morin (5- Morin), myricetin, naringenin, naringenin-7-glucoside, naringin, neohesperidin dihydrochalcone, nomiline, primuletin, poncillin, scutellarein, and catechin hydrate were found to have a strong inhibitory action on the uptake activity of estrone trisulfate.

It is a schematic diagram which shows the molecular target of breast cancer. It is a figure which shows the result of the uptake | capture inhibition to the breast cancer cell of the estrone 3 sulfate by various test substances. It is a figure which shows the result of the suppression by the genistein, quercetin, ginkgolide C, theaflavin, and theaflavin 3-O-gallate of the cell growth promotion effect by estrone 3 sulfate. It is a figure which shows the result of the uptake | capture inhibition to the breast cancer cell of the estrone 3 sulfate by various test substances.

Claims (3)

  Genistein, Quercetin, Ginkgolide C, Theaflavin, Theaflavin 3-O-gallate, Rutin, Chalcone, Daidzein ), Daidin, Flavanone, Flavonol, Geraldol, Hesperetin, Hesperidin, Ipriflavone, Luteolin-7-O-glucoside (Luteolin-7-) O-Glucoside), Methoxychalcone, 4'-methyl-7-methoxy-isoflavone, 5-Morin, Myricetin, Naringenin ( Naringenin, Naringenin-7-Glycoside, Naringin, Neohesperi One or more types selected from Neohesperidin Dihydrochalcone, Nomilin, Primuletin, Poncirin, Scutellarein, Catechin Hydrate, and compounds chemically modified from these An inhibitor of the transporter activity of the estrone trisulfate transporter, comprising as an active ingredient.   Genistein, Quercetin, Ginkgolide C, Theaflavin, Theaflavin 3-O-gallate, Rutin, Chalcone, Daidzein ), Daidin, Flavanone, Flavonol, Geraldol, Hesperetin, Hesperidin, Ipriflavone, Luteolin-7-O-glucoside (Luteolin-7-) O-Glucoside), Methoxychalcone, 4'-methyl-7-methoxy-isoflavone, 5-Morin, Myricetin, Naringenin ( Naringenin, Naringenin-7-Glycoside, Naringin, Neohesperi One or more types selected from Neohesperidin Dihydrochalcone, Nomilin, Primuletin, Poncirin, Scutellarein, Catechin Hydrate, and compounds chemically modified from these A breast cancer cell growth inhibitor comprising the above ingredients as active ingredients.   Genistein, Quercetin, Ginkgolide C, Theaflavin, Theaflavin 3-O-gallate, Rutin, Chalcone, Daidzein ), Daidin, Flavanone, Flavonol, Geraldol, Hesperetin, Hesperidin, Ipriflavone, Luteolin-7-O-glucoside (Luteolin-7-) O-Glucoside), Methoxychalcone, 4'-methyl-7-methoxy-isoflavone, 5-Morin, Myricetin, Naringenin ( Naringenin, Naringenin-7-Glycoside, Naringin, Neohesperi One kind selected from Neohesperidin Dihydrochalcone, Nomilin, Primuletin, Poncirin, Scutellarein, and catechin hydrate (Catechin Hydrate), and compounds chemically modified from these A breast cancer therapeutic agent comprising the above ingredients as active ingredients.