TWI306403B - Use of antiprogestings for the induction of apoptosis in a cell - Google Patents

Description

1306403 A7 B7 V. INSTRUCTION DESCRIPTION (1) Field of the Invention The present invention relates to the use of antiprogestin to induce apoptosis. Specifically, the present invention relates to the antiprogestin 11 cold-(4-ethylphenyl)-17 /3 -yl-7-yl;-(1,1,2,2,2-pentafluoroethyl Use of -4,9-dien-3-one or a pharmaceutically acceptable derivative thereof or an analog thereof to induce apoptosis. The present invention further provides for the preparation of an antiprogestin such as breast cancer, which is a cancer therapeutic agent which is a high risk indicator in increments of tumor cells in the S phase of the cell cycle. BACKGROUND OF THE INVENTION Antiprogestin represents a relatively novel and promising agent that has a significant utility in the treatment of hormone-dependent tumors and other diseases. Although antiprogestin was originally created for medical use in non-surgical termination of pregnancy, certain antiprogestogens, such as endocrine therapy for breast cancer that control the progesterone receptor (T. Maudelonde et al.: J. Klijn et al., Hormonal) Manipulation of Cancer: Peptides, Growth Factors and New (Anti) Steroidal Agents, Raven Press, New York, 1987, pp. 55-59). It is also of considerable importance. This new strategy in endocrine therapy is based on the anti-tumor activity of antiprogestin in human hepatocarcinoma cells positive for luteinizing hormone receptors in test tubes and several hormone-dependent breast tumors in living mice and rats. Specifically, the anti-tumor activity of the antiprogestin, onapristone, and methicone (RU 486) has been used in a mouse-dependent MMT-mammary tumor model and in mouse DMBA- and NMU-induced breast tumor models. (MR Schneider et al., Eur. J. Cancer Clin. Oncol., Vol. 25, No. 4, pp. 691-701, 1989; H. Michna et al., Breast Cancer Research and Treatment 14: 275-288, - 4- The paper size applies to the Chinese National Standard (CNS) A4 specification (210 X 297 mm) _ 1306403 A7 _B7__ V. Inventions (2) 1989; H. Michna et al. J. Steroid. Biochem. Vol. 34 , Nos-l-6, pp. 447-453, 1989) conducted research. However, compounds such as mesaconone cannot be recommended as single agents for breast cancer control due to low activity and side effects (D. Perrault et al. J. Clin. Oncol. 1996 Oct, 14(10), pp. 2709- 2712). In addition, the US service of ketone has a strong anti-glucocorticoid side effect (compare LM Kettel et al. Fertil. Steril. 1991 Sep, 56(3), ρρ.402-407; X. Bertagna's Psychoneuroendocrinology 1997; 22 Suppl. Pp. 51-55). Determination of the percentage of tumor cells in each phase of the cell cycle by strong DNA flow cytometry (cf. GM Clark et al. Engl. J. Med. 320, 1989, March, pp. 627-633; LG Dressier et al. Cancer 61 (3), 1988, pp_420-427 and references therein). The different stages of the tumor cell cycle, in particular, the number of tumor cells at certain stages of the cycle, can be important clinical indicators of disease progression and success. In this regard, the number of cells in the S phase of the cell cycle is particularly important. European Patent No. 0 495 825 B1 discloses the use of an antiprogestin (competitive luteinizing hormone antagonist) for the preparation of a breast cancer agent for treating tumor cells in increments of the S phase of the cell as a high risk indicator. This is based on the observation that antiprogestin can actually reduce the tumor cells in the s phase by hindering the development of the GqG! phase in the tumor cell cycle. However, no such efficacy was observed in standard breast cancer therapies such as Tamoxifen, estrogen hormone therapy or oophorectomy. The antiprogestin tested in European Patent No. 0 495 825 B1 is 11-μ-Ν, Ν-didecylamino-phenyl]-Π α-hydroxy-17 /3 -(3-hydroxypropyl )_13 α -mercapto-4,9(10)-estradien-3-one and 11/3-(4-ethylphenyl)-17泠-hydroxy-17α-(C-paper scale for China National Standard (CNS) Α4 Specification (210X297 mm) 1306403 A7 ______B7 V. Description of Invention (3) '1-Alkenyl)-4,9-(10)-Estradiol -3-ketone. 1 W〇 98/34947 describes 17 heart fluoroalkyl steroids having potent antiprogestin activity and methods for their manufacture. W〇 98/34947 does not discuss or study the 17 α-fluoroalkyl steroids disclosed therein may be involved in apoptosis or cell cycle arrest. It is known that there are agents which hinder the development of GgG in the tumor cells to induce the potential value of cell death, and it is necessary to further confirm an agent having this specific mechanism behavior, such as an antiprogestin. This agent has potential applications for the treatment and prevention of cancers such as breast cancer, which are high risk indicators for tumor cells in the S phase of the cell cycle. OBJECTS OF THE INVENTION Accordingly, it is an object of the present invention to further investigate the inhibition mode of antiprogestin in a hormone-dependent disease such as breast cancer and to provide a method of inducing apoptosis in a given cell. Surprisingly, the inventors found that the antiprogestin 丨丨 point _(4 醯 醯 ) ) _ 17 - - 基 〇 17 〇 : - (1,1,2,2,2-pentafluoroethyl) _Estradiol-4,9-diene-3-ketone (or a pharmaceutically acceptable salt thereof or the like) can be used for induction of apoptosis. The present invention is based on the present invention based on the unexpected antiprogestin 1 _ ( ( ( ( ( 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 羟基 - - _Effective 4,9-diuret-3-one (hereinafter referred to as "antiprogestin (I)") induces apoptosis and death in tumor cells of a standard breast cancer tumor model. It was found that the antiprogestin (1) can induce apoptosis via differentiation at the initiation terminal. Accordingly, the present invention provides the use of an antiprogestin (1) or a pharmaceutically acceptable derivative thereof or an analog thereof for the preparation of a medicament for inducing apoptosis. Start

1306403 V. Description of the invention (terminal differentiation induces apoptosis x society is better than mammalian cells. The cells are better for mammalian cells, and the cells are better: the tumor cells are better, and the tumor is better for breast cancer. Another point is the use of antiprogestin (1) or its medicinal conjugates to prepare a cancer drug with a high risk index for the cell cycle in the s phase of the cell cycle. Progesterone (1) or its similar substance in the test tube induces ##, or fine..." induces the use of cells. This cell is mammalian-, and 'cell is better, human cells are better' Good cancer is preferred. The other aspect of the invention is the method of administering an effective amount of antiprogestin (1) to cells to induce apoptosis. The method can be used in a test tube or in a living body. The cell is preferably a mammalian cell. The human cells are better, and the tumor cells are the best, wherein the tumor is preferably breast cancer. Because of the ability of the antiprogestin (1) or its pharmaceutically acceptable derivative or its analog to induce apoptosis, it can be used for breast cancer, for example. Cell cycle The 8th phase of the increase in tumor cells is a high-risk indicator for the treatment of certain types of cancer. Other types of tumors or hormone-dependent diseases that may be affected and treated by antiprogestin (I)-induced apoptosis may include breast cancer, Nest cancer, endometrial cancer, bone marrow cancer, ovarian infertility, meningitis, meaning diseases that are essentially derived from or affected by hormonal receptors and/or hormonal-dependent pathways. The dose-response effect of antiprogestin (I) in mouse DMBA-induced breast tumor was studied to induce apoptosis-inhibiting tumor growth inhibition efficacy. Compared with the control group, the Chinese National Standard (CNS) A4 specification (210X297 AD) was applied to the paper scale. 1306403 A7 ___B7 V. INSTRUCTIONS (5) Comparison of progesterone Onaps and oophorectomy. This study was performed with anti-progestin (I) at 0.5, 2.0, 5.0 and 10.0 mg/kg per dose. 2 shows the tumor growth inhibition efficacy of antiprogestin (I) induced by murine NMU-induced apoptosis in breast tumors, compared with control group and oophorectomy. The study used 0_5 and 1.0 milligrams per dose. Gram/kg of antiprogestin (1) is carried out. Figure 3 shows the effect of anti-progestogen (I) at 10 mg/kg sc dose on the apoptosis induction of scid mice transfected with human T47D tumor and the effect of tumor growth inhibition, and Comparison of control group and oophorectomy. Figure 4 shows the effect of antiprogestin (I) induced by 10 mg/kg sc dose on MCF-7 human breast cancer model in scid mice and its tumor growth inhibition effect. Comparison with control group and oophorectomy. Figures 5A to 5F show histological data on apoptosis induction in murine NMU-induced breast cancer model (cf. Example 5). In particular, Figure 5A shows treatment with antiprogestin (1) Tumors exhibit ductal and adenoid structures, usually filled with secretory material, compared to the control group (Fig. 5B). Figure 5C shows untreated NMU-embryo breast cancer tissue with high PCNA (proliferating cell nuclear antigen) immune response, comparing NMU-induced breast cancer tissue treated with antiprogestin (1) with low PCNA immune response (Fig. 5D). Figure 5E shows the apoptosis of the antiprogestin (I)-treated NMU-induced breast cancer tissue compared to the control group (Fig. 5E). Figure 6 shows the effect of tumor growth inhibition with an effective threshold concentration of 1 (Τ9 to 1〇_8 mol/L of antiprogestin (I) on T47D breast cancer cell line (stimulated by estrus hormone), with antiprogestogen Ona Pusitong and pure anti-emotion hormone 11 /9 -Fluor-7 α -{5-[Ν- -8 - This paper scale applies to China National Standard (CNS) Α4 specification (210X297 mm) _ 1306403 A7 ____Β7 V. Invention Description (7) The anti-progestin that is not shown in the following examples (1) The application of different tumor models reveals the cell cycle G〇Gl phase tumor cell accumulation and the number of tumor cells in the cell cycle of 5 and 河2 rivers are significant and biologically A decrease in correlation. This result shows the induction of differentiation. Specific differentiation of Gl has been proposed in other stem cell lines (see JJ Wille Jr" Cancer Res. 1982, 42〇2): 5139 46; RE to (10), j Cell· Biol 1982, 94(2): 400-405). Experimental results obtained in several tumor models revealed that the anti-progestin (1) treatment of polymorphic tumor cells that appear to trigger mitogenic activity differentiates into glandular structures and a large number of secreted products. , and to the spindle-shaped Nibbitic subgroup (necrobiotic subP〇Pulati〇n) (see Example 5' and Figure 5A#〇5B). The size of the tumor, the mitotic index and the malignant grade decreased significantly. The volume ratio of the glandular structure in the tumor and the signs of apoptosis increased compared with the control group. Times (see Example 5, Figure 5Ε and 5F). Without any theoretical limitations, these results symbolize the main mechanism of anti-progesterone (I) anti-tumor effect in the experimental model is the wide-scale direct luteinizing hormone of tumor cells. - Receptor-mediated anti-proliferative potentiation via terminal differentiation associated with terminal cell death. In this way, antiprogestin (1) appears to remove the internal terminal differentiation barrier inherent in malignant tumor cells of progesterone receptor-positive tumor cells. The antiproliferative effect of progesterone (1) appears to be independent of anti-progestin (1) anti-hormone (anti-progesterone) activity. For example, antiprogestin (I) agents, for example, in the case of tumor cells, to hinder GoG, Development of induced apoptosis, a potential use for the treatment and prevention of many diseases. Such agents, including antiprogestogens (1), can be used in the treatment of cell cycles such as breast cancer. The s phase increment of tumor cells is high risk. -10- This paper scale applies Chinese National Standard (CNS) A4 specification (210X 297 mm) 1306403 A7 B7 ____ V. Invention description (8) Target cancer. Therefore, One of the aspects of the present invention is the use of an antiprogestin (I) or a pharmaceutically acceptable derivative thereof or the like for the preparation of a medicament for inducing apoptosis. In a preferred embodiment, the antiprogestin (I) or The use of a pharmaceutically acceptable derivative or the like is a drug for inducing apoptosis of a tumor cell which is preferably a human breast tumor cell. Such drugs are advantageous in the treatment of hormone-dependent diseases such as breast cancer, which are high-risk indicators of tumor cells in the S phase of the cell cycle. The manufacture of the drug can be carried out by methods known in the art. Generally known and customary adjuvants and further suitable carriers or diluents can be used. Suitable adjuvants and carriers may be those recommended in the following cosmetic and related literature: Ullmann's Encyclopedia of Technical Chemistry, Vol. 4, (1953), pp. 1-39; Journal of Pharmaceutical Sciences, Vol. 52 (1963) ), p. 918ff; Hv Czetsch-Lindenwald, “Hilfsstoffe fur Pharmazie und angreanzende Gebiete"; Pharm. Ind. 2, 1961, p.72ff; Dr. HP Fiedler, Lexikon der Hilfsstoffe fiir Pharmazie, Kosmetik und angrenzende Gebiete, Cantor KG , Aulendorf in W u rttemberg, 1971 ° anti-progestin suitable for the purpose of the present invention, preferably antiprogestin (I) or a pharmaceutically acceptable derivative thereof or the like, may be incorporated orally prepared according to known A pharmaceutical composition made by a method of parenteral administration, for example, intraperitoneal, intramuscular 'subcutaneous or dermal surface application. It may also be implanted into a tissue. The implant may comprise, for example, a biodegradable polymer or a ruthenium film. An inert substance for the synthesis of polydecanes. It can be used as a key agent, a pill, a sugar coating, a capsule, a fine granule, a suppository, and an implant -11 - This paper scale applies to the Chinese National Standard (CNS) A4 Grid (210 X 297 mm) 1306403 V. Description of invention (9), injectable sterile aqueous solution, suspension or lotion, ointment, silicone or by intravaginal (eg, yin π, 哲+) clothing or Intrauterine system (such as 'diaphragm' eclipse) is applied in a special way. For the preparation of oral administration of 荜南丨 ^ ^ d as defined above, the antiprogestin suitable for the purpose of the present invention can be compared with the general known and * With carrier 'such as Arabian rubber, talcum powder, starch, sugar, such as 4 + Ganlu 'methyl cellulose, lactose, gel, surfactant, stearin, Junzhen 'aqueous or non-aqueous excipient, Paraffin derivatives, crosslinkers, 'emulsifiers' lubricants, preservatives and perfumes (such as ether oils), the doctors from the " 〇's anti-progestogen can be dispersed in the particles , as in the microparticles 'composition 0, in order to advance the bioavailability of the active agent, the antiprogestin as defined above for the purposes of the present invention may be as disclosed in PCT/EP/95/02656: VIII, α, 召, or The r_cyclodextrin or its derivative is reacted to form a cyclodextrin water cage. As for the parenteral administration of an antiprogestin suitable for the above object according to the invention, it can be dissolved or suspended in the physiological state of an oil-like surfactant, dispersant or emulsifier, for example, with or without a solubilizing agent. Acceptable in the diluent. The oil that can be used as an example and not as a limit is 'ucking oil, peanut oil, cottonseed oil, soybean oil,! Sesame oil, and sesame oil. The amount administered (e.g., a pharmaceutically effective amount) varies over a wide range and is determined by the condition to be treated and the mode of administration. It can include any amount of effective treatment. The "medical effective amount" is determined by the ability of those skilled in the art. A unit dose can represent from about 01 to 100 milligrams of active agent. If applied to humans|__- Μ This paper scale is suitable for the price of the national standard (heart) iron grid (21. χ 297 public) 12- !306403 V. Description of the invention (10 丨 l l ± daily dose of the agent is about 〇" to gamma, and even milligrams is better '50 mg. The composition of the drug I according to the present invention can be prepared by long-acting injection or implantation according to the need for continuous delivery of the effective agent. Oral administration is a preferred mode of administration. The antiprogestin used in the present invention, particularly the antiprogestin (I), is particularly suitable for oral administration. According to the present invention, at least - progesterone can be formulated, especially antiprogestin (1) or its medicine. An acceptable derivative or analogous substance thereof, which binds to at least one anti-emotional hormone, because many hormone-dependent diseases 1 are breast cancers, which not only display the progesterone receptor, but also exhibit the estrus hormone style. The anti-emotion hormone can simultaneously resist The progestogen (1) may be administered later. The amount of antiprogestin and anti-emotion hormone may be equal or one of the components may be more important than the other, such as antiprogestin: anti-emotion hormone ratio from i: 50 to 50: i, preferably 1:30 to 30:1, 1:15 to 1 5:1 is the best. Examples of anti-emotional hormones suitable for use according to the present invention are non-sterols and anti-emotional hormones, such as tamaoxifen and naf〇xidine and leroxifene, non-slouse (faslodex) and EM80 (an example of a steroid anti-emotional hormone, including European Patent No. 348 341 A and WO 98/07740, especially U-Fluoro- 7α:-{5-[Ν-甲-N-3-(4,4,5,5,5-pentafluoropentylthio-propylamino)-pentyl]-estra-1,3,5(10)-triene-3,17 -diol, or w〇99/33855, especially 11; S-fluoro-7α-{5-[methyl-(7,7,8,8,9,9,丨0,10,1〇 _9-fluoroindolyl)-amino]-phenyl}-estr-1,3,5(10)-triene_3,17-di-diol or a pharmaceutically acceptable derivative thereof or the like. Enzyme inhibitors have anti-emotional hormone potency, as disclosed on pages 7 to 8 of European Patent No. 495 825 B1, -13- This paper silver scale applies to Chinese National Standard (CNS) A4 specification (210X297 mm) 1306403 A7 _B7___ V. INSTRUCTION DESCRIPTION (11) It can also act as an anti-emotional hormone. Another aspect of the invention is the use of antiprogestin (I) or its pharmaceutically acceptable derivative Or its analog preparation for the treatment of tumor cells with an increase in the S phase of the cell cycle as a drug for high-risk cancer. The number of tumor cells in the S phase can be as follows: "Dressier et al." DNA Flow Cytometry and Prognostic Factors in 1331 Frozen Breast DNA flow cytometry as described in Cancer Specimens " Cancer, Vol. 61 (3), 1988, pp. 420-427, also found in McGuire and Dressier "Emerging Impact of Flow Cytometry in Predicting Recurrence and Survival in Breast Cancer Patients, "JNCI, Vol. 75 (3), 1985, pp. 405-409 «S phase high-risk tumor cells show that they are particularly suitable as candidate cells for the use of the present invention. In the case of the antiprogestin (1), the potent antitumor efficacy and the mechanism by which this agent induces apoptosis (see, in particular, Example 5) and the cell cycle arrest form an advantage from standard animal models (see Examples 1 to 4). One of the non-traditional views of the present invention is to provide a method of inducing apoptosis. The cells are preferably mammalian cells, and the human cells are optimal, and the method can be used in a test tube or a living body. Apoptosis induction via induction of terminal differentiation is preferred, for example, to administer antiprogestin (I) or a pharmaceutically acceptable derivative thereof or the like. In this method, an effective amount of the antiprogestin (I) or a pharmaceutically acceptable derivative thereof or the like can be used for the problem cell. In human T47D breast cancer cell lines stimulated by estrus hormone administration, antiprogestin (I) induces complete inhibition of cell growth at an effective threshold concentration of 10_9 to 1 〇·8 mol (see Examples 6 and 6). ). It is particularly surprising that the known antiprogestin, Onaps, does not have the effect of reducing cell growth in this tumor model. Therefore, antiprogestin (1) -14- This paper wave scale is used in China National Standard (CNS) A4 specification (210 X 297 mm) 1306403 A7 _____B7 V. Invention description (12) is superior in efficiency and efficiency as Europe Naps and other antiprogestogens and anti-estrogen hormones such as tamaprofen are even like 11/S-fluoro-7 α -{5·[Ν-曱-N-3-(4,4,5,5 , 5-pentafluoropentylthiopropylamino)-pentyl]triene·3,17--diol (W〇98/〇774〇) pure anti-emotion hormone. The role of antiprogestin (I) in the induction of apoptosis indicates that this antiprogestin (or its pharmaceutically acceptable derivative or its analog) can be used for many symptoms, especially hormone-dependent symptoms that require induction of apoptosis. . Specifically, it can be used for the treatment of, for example, breast cancer, ovarian cancer, endometrial cancer, bone marrow cancer, ovarian infertility, meningitis, meaning, essentially derived from or due to hormonal receptors and/or hormonal dependence The path affects the disease. An antiprogestin such as an antiprogestin (1) can therefore be further used for the preparation of a medicament for the treatment of the aforementioned hormone-dependent disease by inducing apoptosis or death. The invention is further illustrated by way of example. The following examples are not to be construed as limiting. EXAMPLES Example 1: Agents in the DMgA-induced tumor model. Materials and methods: This study used immature female Shipodoli mice (49. 5丨天大: ίο only/group) One dose of oral 10 mg 7,12-dimercaptobenzopyrene (DMBA, Serva/Heidelberg) induced breast tumors. Mice with at least one tumor larger than 15 cm2 were treated with the following substances for four weeks: 丨) solvent control, 2) oophorectomy at the start of treatment, 3) anti-progestin (0.5) of 0.5 mg/kg sc·4) 2 mg/kg sc antiprogestin (1), 5) 5 mg/kg sc anti-15·^ This paper size is applicable to China National Standard (CNS) A4 specification (210X297 mm i------- 1306403 A7 B7 V. Description of invention (13) Progesterone (I), 6) Anti-progestin (1) of 10 mg/kg sc, and 7) Per 5

Further explanation of the DMBA prevention model Statistical analysis of 叼 value i呉. And assessment, see R.G. Metha,

European Journal of Cancer 36 (2000), pp. 1275-1282 〇 Results: In the untreated control animals, development of tumor growth was observed, with oophorectomy resulting in a significant 90% tumor regression in the animals. Treatment with antiprogestin (I) at doses greater than or equal to 2 mg/kg resulted in significant induction of apoptosis and resulted in inhibition of tumor growth compared to the control group (see Figure 2). ^ There was a significant dose-response relationship. Treatment with 0.5 ng/kg antiprogestin (1) did not significantly prevent tumor growth, with maximal induction of apoptosis at 2 mg/kg and inhibition of growth observed. In this group at 50 ° /. The rats saw complete tumor regression. This experiment tested the highest dose of antiprogestin (I) (1 〇 mg / kg), equivalent to 2 mg / kg. "Olympus with (5 mg / kg) significantly more than a dose of antiprogestin (I )invalid. Conclusion: In the murine DMBA-induced mammary tumor model, antiprogestin (1) strongly induces apoptosis in tumor cells and thus inhibits tumor growth completely in untreated animals. It was found that 2 mg/kg of antiprogestin (1) had the greatest apoptosis effect on tumor cells. Antiprogestin (1) is superior to Onaps in inhibiting tumor growth. Example 2: Tumor-inhibition inhibition of breast cancer model The paper scale is applicable to China National Standard (CNS) A4 specification (21〇X297 mm) 1306403

MATERIALS AND METHODS: Tumors were induced by intravenous injection of mono-NMU (rhomoxylmethyl urea, 5 mg/kg) in lipid-spoken Spoogly rats (purchased from Tierzucht Schbmvalde, 50-55 days old). After 10 days, start with at least one shaped tumor, the following substances are treated for four weeks: 丨) solvent control, 2) oophorectomy at the beginning of treatment, 3) antiprogestin (1), u mg / kg / 曰, 4) Antiprogestin (1), 〇.5 mg/kg/曰, and 5) Onaps, 5 mg/kg/曰. The change in tumor area (% relative to the original tumor size) was measured as a growth inhibition parameter by a double-angle gauge every week. A statistical analysis of the mean difference between the groups was performed using the Kruskal_WaUis_ test. Results • Tumor growth was observed to be observed in untreated control animals, whereas oophorectomy completely inhibited tumor growth. The anti-progestin (I) at a dose of 55 or 1 mg/kg resulted in significant tumor growth inhibition due to apoptosis compared to the control group (see Figure 2). The anti-progestin (1) of Onaps with (5 mg/kg) was significantly lower than the dose (〇·5 mg/kg). Conclusion: In the murine NMU-induced mammary tumor model, anti-progestin (I) completely inhibited tumor growth in untreated animals due to its strong ability to induce tumor cell apoptosis. Both doses (1.0 mg/kg and 0.5 mg/kg) of antiprogestin (I) have significant apoptosis effects on tumor cells. f Example 3: Human T47D1 transferred to Fuhe severe immunodeficient mice: Materials and methods: -17- This paper scale applies to Chinese National Standard (CNS) A4 specification (210X 297 mm)

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Infestive fascination pellets (Innovative Research 〇f America) were used to raise lipids in Fakai combined with severe immunodeficient mice (M&amp;B). The T47D breast cancer cells obtained from the cell culture medium and placed in the matrigel were transplanted into the vicinity of the sputum of the mouse. The treatment was started when the tumor was about 25 cm 2 in size. Continue treatment to tumor development. The experimental groups were: 1) control group (vehicle), 2) nevectomy [3] antiprogestin (I) ' 1 〇 mg / kg s c. The area of the tumor is measured by a rectangular angle clamp. Statistical analysis of the mean planting differences between the groups was performed using the Kruskal-Wallis-test. RESULTS: In the Ding 47D breast cancer model, the nevectomy resulted in a considerable tumor growth inhibition compared to the rapid growth of the control group. Figure 3 clearly shows that administration of 10 ml/kg of antiprogestin (1) to induce apoptosis of tumor cells by sc. The antiprogestin (1) is almost as effective as traditional estrus hormone deprivation therapy (ovectomy). Conclusion: Antiprogestogen (I) is equivalent to the standard estrous hormone considered to be the most effective method in this model for inducing apoptosis and thus inhibiting the growth inhibition of human T47D breast cancer in Fakai complex severe immunodeficiency mice. Deprivation therapy (ovectomy). Example 4: Human MCF-7, which is transferred to a compound severely cancer-deficient mouse, is a painful material and method: Feeding a female lipid Fakai complex with severe immunodeficient mice with Innovative Research 〇f America (Μ &amp;; B) ^ MCF7 breast cancer cells obtained from cell culture medium and placed in matrigd, transplanted into the gas of the mouse - 18 - 'This paper scale is applicable in the SS - 揉 揉 (CNS) A4 specification (21GX297 mm) ) ---- 1306403 V. Description of invention (16) Near the crotch. When the tumor is about 25 square centimeters in size, open f treatment to tumor development. The experimental group was: _照组&quot;ut knife removal, 3) antiprogestin (1), 1〇mg/male' · The area of the abdomen was measured with a 规 gauge clip. Kru secrets, is used to conduct statistical analysis between groups. </ RTI> Results: In the (10) occupational cancer model, 1 nest resection resulted in a considerable tumor growth inhibition compared with the control group. Figure 4 clearly shows that a • administration of 10 mg/kg of antiprogestin (1) induces apoptosis of tumor cells. Antiprogestogen (1) is equivalent to standard estrus hormone deprivation therapy (ie, nest resection). </ RTI> Conclusion: Antiprogestogen (1) is equivalent to standard estrous hormone deprivation therapy (ovarian resection) in the human MCFm cancer that is transferred to the Fakai complex severe immunodeficient mice and thus inhibits its growth. Example 5 Proliferation and TUNFJ. Public Gas, Village Materials and Methods: - Intravenous injection of female N. sinensis (p-immediate methylurea, % mg/kg) into a female Spergdori (purchased from Tierzucht Sch〇nwaMe, 5〇55 days old) induced tumors. Rats with at least one tumor larger than 15 square millimeters were treated for seven days with the following materials: 丨) solvent control, 2) ovarian resection at the beginning of treatment, and 3) anti-progestin (3) per day of 3 mg/kg s.c. Tumors were examined at the end of the treatment, placed in formalin and embedded in paraffin. The histology of the tumor was removed and the proliferation index and apoptosis induction analysis were performed. This paper scale applies to the Chinese National Standard (CNS) A4 specification (21〇Χ297 mm) -19- 1306403 A7 B7 V. INSTRUCTIONS (17) Histology: Tissue slides were fixed with haematoxilin and analyzed by microscopy. Proliferation index: PCNA performance was determined to measure the proliferation index. Proliferating cell nuclear antigen (PCNA) is a 36 kD nuclear cycle associated with the cell cycle. Nuclear PCNA immunity exists in the proliferative zone of normal tissues. A monoclonal antibody that recognizes the epitopes of coagulation and migration and is used to study its cellular distribution. TUNEL (attenuation test): The biochemical marker of apoptosis is the degradation of genetic DNA, an irreversible result of cell death. The characteristic DNA splits as a result of activation of the nucleosidase, which selectively cleaves DNA located between the units of the nucleosome. The DNA strand break was detected by enzymatic characterization of the 3'-OH end of luciferin-dUTP using a terminal deoxynucleotidyl transfer acid. (TUNEL, erminal Deoxynucleotidyl Transferase-Mediated dUTP liick End Labeling, cf. Gavrieli et al. J. Cell. Biol. 1 19, 493, 1992). The incorporated luciferin is detected by reacting with an anti-luciferin antibody alkaline phosphatase and then reacting with an alkaline phosphate substrate. Results: Histology: After treatment with antiprogestin (I), tissue sections of NMU tumors showed mixed ducts and glandular cells, which were usually filled with secretory material (Fig. 5A). In addition, the volume ratio of glandular structures in the cells was compared with the control group (Fig. 5B). In addition, mammary tumors of animals treated with antiprogestogen (I) showed differentiation characteristics of the morphology.

Proliferation index: PCNA has a high immune response in untreated NMU-induced breast cancer tissues (Fig. 5C: untreated control group). Treatment of murine NMU-anti-progestin (I)-induced breast cancer tissue to induce differentiation has reduced -20- This paper scale applies Chinese National Standard (CNS) A4 specification (210 X 297 mm) 1306403 A7 —__B7____ V. Invention Description (18) Number of cells in the PCNA immune response (Fig. 5D). These data show that the proliferative index is reduced by the treatment of breast cancer with antiprogestin to induce differentiation. TUNEL (apoptosis): Figure 5E shows the comparison of apoptosis induced by antiprogestin (I) in NMU-induced breast cancer tissue compared to the untreated control group (Fig. 5F). Obviously, antiprogestin (I) alone induces NMU-induced apoptosis in breast cancer tissues and thus inhibits tumor growth. Example 6: Antiprogestin in test tubes (1) Anti-proliferative activity materials and methods for T47D cell lines: T47D cells were grown in charcoal-treated serum with 〇·1ηΜΕ2 (estradiol) plus antiprogestin in a medium change 6 Days to add. It is then fixed and stained with crystal violet, the absorbance is recorded and the values are normalized to R.B. Lichtner, J.

The absorbance of the untreated control group described in Steroid Biochem. Mol. Biol. 1999, 71; 181-189. The TUNEL assay was performed in a similar manner to Example 5 above. The only difference was that the analysis used microscopy slides to replace tissue sections. Results: _ In this T47D cell line test, antiprogestin (1) was as low as 1〇-9 To 1 (T8 Moh/L effective threshold concentration shows effective tumor growth inhibitory activity, while antiprogestogen Onaps does not show any inhibition a even pure estrus hormone 11) 8-fluoro-7〇 :-{5-[&gt;1-Methyl-1^3-(4,4,5,5,5-pentafluoropentylthio-propylamino)-pentyl]- although -1,3,5 (10) - Tris--3,17/3-diol 98/07740) was also significantly less effective than antiprogestin (I) (see Figure 6). Conclusion: -21 - -----1 This paper is suitable for the S-painter standard (CNS) A4 specification (210 X 297 mm) 1306403 A7 B7

V. INSTRUCTIONS (19) The antiprogestin (I) of the present invention induces complete inhibition of growth of estrus hormone-stimulated T47D cells at a very low concentration and is thus superior in efficacy and efficiency to other tests such as Onopus Antiprogestin and pure anti-oenophotic hormone vermiculite-fluorine_7 α-{5-[Ν-mercapto-N-3-(4,4,5,5,5-pentafluoropentylthio-propylamine ))-pentyl]-estr-1,3,5(10)-triene-3,17-diol. -22- This paper scale applies to Chinese National Standard (CNS) A4 specification (210 X 297 mm)

Claims (1)

I3〇64Q3&gt;G125781 Patent Application Chinese Patent Application Range Replacement Amendment (97 years i丨H A8 B8 VI. Patent scope 1. A pharmaceutical composition for inducing apoptosis, point-(4-acetamidine) Phenyl) 1.7 million-trans group]^..." 4,9-diene-3 ketone. '2. The pharmaceutical group according to the scope of the patent application is caused by terminal differentiation. 3_According to the scope of patent application The medical combination of the first or second item of mammalian cells. D The antiprogestin 11 is a five-gas ethyl)-carving agent in which the apoptosis inducer, wherein the cell is a medical composition according to claim 3, wherein the cell is a human cell. 5. A pharmaceutical composition according to claim 2 or 2 of the patent application, tumor cells. 6. According to the fifth application of the patent application scope, the mammalian cell is a breast cancer drug, wherein the tumor is 7. The pharmaceutical composition according to the middle (4) (4), the rape treatment: the tumor in the S phase of the cell cycle Cells as a high-risk indicator: cancer 0 8. According to the scope of the patent, the medical (four) compound of the patent scope, wherein the disease is cancer. 9. A method for inducing a cell, wherein an effective amount of an antiprogestin (4-ethyl phenyl) _17 _ _ _ 17 α ^, cut-five gas ethyl bud _ 4, 9-diene The -3 -ketone system is administered to cells in vitro. 10. The method according to the scope of the patent application 笫9ii 夕*·^T π Target 弟 y, wherein the cell is a mammalian tumor cell. 11. The method according to claim 9 or H), wherein the cell is a breast cancer cell. 1306403 Patent application scope A8 B8 C8 D8 12·- anti-progestogen U^(4_乙醯笨基) l7卜经基_i7叫, m2. pentaoxyethyl) female_4,9·di diluted·3_ Use for the preparation of a medicament for inducing a cell&gt; 13. According to the use of item 12 of the scope of patent application, the induction of apoptosis is caused by the initiation of differentiation. 'Thin 14. According to the patent application range 12 or 13 cells. The use of the item, wherein the cells are breastfeeding 15. According to the scope of the patent application, the use of the human shell is in which the mammalian cells are human cells. 16. According to the patent application range 12 or 13 cells. Use of the item 'where the cell is a tumor Π. According to the π &lt; use of the scope of claim 16 of the patent, wherein the sputum 18 · according to the scope of the patent application of the 12th item - "Tian" For the treatment of tumor cancer in increments of the S phase of the cell cycle. Slip,,, and field cells as high-risk indicators 19. According to the application of the scope of claim 18, the disease is breast cancer. -2-