UA76128C2 - Use of progesterone receptor inhibitors for treating steroid-resistant breast cancer - Google Patents

Abstract

The invention relates to the use of progesterone receptor inhibitors for inhibition of growth-factor-dependency of tumor cells.

Description

Description of the invention

The invention relates to the use of progesterone receptor inhibitors to inhibit the growth factor dependence of tumor 2 cells.

Estradiol and progesterone are involved in the development of breast cancer. However, when diagnosed, only about 1/3 of tumors are found to be dependent on steroid hormones. It is assumed that in most tumors resistant to steroid hormones, the proliferative control of locally acting autocrine and paracrine peptide growth factors is weakened. In this case, invasive tumors with an extremely poor prognosis arise, which are positive for growth factor receptors and resistant to steroid hormones (EPedde et al., Zetip. Opsoi. 19 (1992) 244-253).

Growth factors regulate cell growth by activating intracellular signaling pathways after binding to high-affinity tyrosine kinase receptors on the cell surface. More recent data suggest that breast carcinoma cells can be sensitized by progestins to the 72 mitogenic action of ESE (epidermal growth factor) (Ogozpopu et al., Moi. Epdoshipoiyi. 11 (1997) 1593-1607)

So, for example, progesterone can induce the entry of cells of the TA7O line of human breast carcinoma into

C-phase, which is accompanied by a temporary increase in the activity of cyclin IO and cyclin-dependent kinase 4. However, growth stimulation is limited to one cycle and is accompanied by growth arrest at the C 4/5 transition of the second cycle

IOSgozpopd and others, see above; Myzgome and others, Moi. SeiYi Vioi 13 (1993) 3577-3587). Under the conditions of stopping the cycle by progesterone, cells are sensitive to the proliferative effect of EOR. In addition, it has been shown that progesterone enhances the effect of ESE on T47O cells by stimulating ESEK, Egbr2 and Egr3 and enhances the phosphorylation of signaling molecules on tyrosine. (apde et al., U. Vioi. Spet. 273 (1998), 31308-31316; Kispeg et al., U. Vioi Spet. 273 91998), 31317-31326). In contrast, it has not yet been possible to show the inhibitory effect of ESE on tumor cells by influencing the progesterone receptor. As part of the experiments leading to the present invention, it has now been surprisingly found that progesterone receptor inhibitors, for example, 17-fluoroalkylsteroids, can at least partially inhibit the binding of growth factors such as ESE to tumor cells, especially with tumor cells that have high and/or significant expression of the progesterone receptor. Therefore, the object of this invention is the use of a progesterone receptor inhibitor to obtain an agent that inhibits the binding of growth factors to tumor cells, and especially an agent that inhibits the proliferation of tumor cells or tumors that are formed with the help of growth factors. Progesterone receptor inhibitor in terms of this invention preferably means a substance that competitively inhibits the binding of progesterone to its receptor. In this case, the progesterone receptor inhibitor is preferably selected from "(0) 17"-fluoroalkyl steroids as disclosed, for example, in UUO98/34947. These 17'"-fluoroalkyl steroids have the general M formula I: Which

And Kai

Where is A.B.?

B" means a metal or ethyl group, ime) B2 means a radical of the formula C.EtNo, where n-2, C, 4, 5 or 6, t»1 and tko-2n1, so BZ means free or etherified in the form of a simple or complex ether hydroxyl group,

B" and B? mean each hydrogen atom, together with an additional bond or a methylene group,

Z stands for the ABC steroid ring system or individual formula A, B or C

Ф) име) 60 б5 ши ЖЖ бый Gay v na fi oi sna, F ey 2 more v ha y: 7 i vo z

And in c where 29 means a hydrogen atom, a linear (C4-C1) alkyl group or a branched (C3-C1) alkyl group, or a halide atom,

B" means a hydrogen atom, a linear (Ci-C1)alkyl group or a branched (C3-Cd)alkyl group, or, if 51 means a steroidal ABC ring system A or B, in addition, B9 and Kk" together mean an additional bond bond, c 29 X means an oxygen atom, a hydroxyimino group "M-OH or two hydrogen atoms, Ge)

BZ means the M radical or the aryl radical, which is optionally substituted by the M group in several positions, while U means a hydrogen atom, a halide atom, -OH, -MO", -Ma, -"CM, -ME Zav, "HO, -СО2НУ, (Си-Сир)Alkoxy, (С.4-Сид)alkanoyloxy, benzoyloxy(С.и-С.од)alkanoyl, (С.4-С.о)hydroxyalkyl or benzoyl group, and Za | 95 are the same or different and, like KU, mean a hydrogen atom or a (C.4-C.od)alkyl group and in the case of radicals -МК 22 also their physiologically compatible salts with acids, and in the case of radicals -СО»ВУ, сч with B? in the sense of hydrogen, as well as their physiologically compatible salts with bases. -

A particularly good example of such progesterone receptor inhibitors is the compound c 11x-(4-acetylphenyl)-17"-hydroxy-17"-(1,1,2,2,2-pentafluoroethyl)estra-4,9-dien-3-one (Compound A below.) In addition

Therefore, other antiprogestins, for example, onapristone Ge (115-(p-(dimethylamino)phenyl|-17'-hydroxy-17-(3-hydroxypropyl)-13 "-estra-4,9-dien-3-one ) are, however, also suitable.

The effect of progesterone receptor inhibitors was found mainly in the case of tumor cells with high or substantial progesterone receptor expression, for example, in the progesterone receptor-positive TA470 breast carcinoma cell line IZagpogisiz et al., Sapseg Kez. 54 (1994), 3668-3877). no) c Progesterone receptor inhibitors inhibit progesterone-induced increase in the expression of growth factors, especially those factors that bind to growth factors of the ECE receptor family, such as, for example, the ECE receptor. The inhibitors particularly preferentially inhibit the binding of ESE to human breast carcinoma cells.

Therefore, in accordance with this invention, progesterone receptor inhibitors can be used in the therapy of tumors in mammals and preferably in humans in order to specifically block the progression of a tumor, especially breast carcinoma from the stage of steroid-dependent growth to the stage of growth factor-dependent development.

At the same time, effective treatment of the tumor at the stage of steroid-dependent growth can occur, for example, with anti-estrogens, which deprives the tumor of the ability to progress at the stage of growth factor-dependent development, which is associated with a significant worsening of the prognosis for the patient. Administration of progesterone receptor inhibitors can also slow down tumor growth at the stage of growth factor-dependent development. For the purpose of this invention, non-steroidal anti-estrogens such as, for example, tamoxifen, nafoxidine, raloxifene and EM800 can be used. The two last mentioned anti-estrogens are representatives of the so-called

ZEKM"vz (selective estrogen receptor modulators); according to the invention, 29 other compounds with the type of action of ZEKM5" can also be used, for example, the compounds mentioned in PCT/EPOO/O5093, and from the latter, in

F! in turn, the compound 5-(4-15-(K5)-4,4,5,5,5-pentafluoropentyl)sulfinyl|Ipentyloxy)phenyl)-6-phenyl-8,9-dihydro-7H-benzocycloheptene-2 -ol.

Examples of steroidal antiestrogens include those compounds disclosed in EP 0348341A, especially faslodex, 60 | those disclosed in MmO98/07740, especially 1T'-fluoro-7'-15-|M-methyl-M-3-(4,4,5,5,5-pentafluoropentylthiopropylamino|pentyl)ester-1,3, 5(10)-triene-3,17"-diol, or those described in MO99/33855, especially 17"-fluoro-7-15-(methyl-(7,7,8,8,9,9, 10,10,10-nonafluorodecyl)amino|pentyl)ester-1,3,5(10)-triene-3,17p-diol.- or 65 their pharmaceutically compatible derivatives or analogs. Aromatase inhibitors with an anti-estrogenic effect can be used in a similar way as anti-estrogens, such as, for example, those listed in EP 0495825 B1 on pages 7-8.

Progesterone receptor inhibitors can be administered by conventional routes, such as topically, topically, subcutaneously, enterally, or parenterally. For enteral administration, tablets, coated tablets, capsules, pills, suspensions or solutions are particularly suitable, which can be prepared in the usual way with the addition of additional substances and fillers known in the field of galenic preparations. For local or topical application, for example, vaginal suppositories or transdermal systems such as skin patches are suitable. Subcutaneous administration can be carried out by injection of an oily solution.

A standard dose may contain, for example, 0.1-100 mg of an active compound or active compounds (progesterone receptor inhibitor or 70 inhibitors). When administered to a person, the daily dose of the active compound or active compounds is about 0.1-400mg, preferably about 10-10Omg and, in particular, about 5Omg.

In addition, the invention should be explained with the help of the following examples and figures.

Fig. 1 represents the antiproliferative effect of the studied substances on cells of the TA47O breast carcinoma line.

Fig. 2 represents the amount of progesterone receptor (RK) and estrogen receptor (EC) protein in cells of the line

TA7O breast carcinoma.

Fig.3 represents the transcriptional activity of the progesterone receptor in T470 cells.

Fig. A4 presents the analysis by Sketchhard of ESE binding to T47O cells as a function of the presence of the test substances.

Fig.5 represents the dependence of ESE binding to T470 cells on the presence of the tested substances.

Example

Materials and methods.

Materials: 125|-ECE (100 mCi/mmol) was obtained from Ategepat Wysnieg. Compound A, hydrotamoxifen (4-OH-Tat), Ha 2M182780 and estradiol were synthesized (at the Institute of Chemistry of Pharmaceutical Agents) by Zspegipda AS according to known methods.

Cell lines

Cells of the TA47O human breast carcinoma line, positive for estrogen (EC) and progesterone (RE) receptors, were used (Egeake et al., VVKS, 101(1981), 1131-1138). ee)

Growth studies

Tumor cells were cultured at a density of 5,000 cells/well in 9b-well plates for 6 days in s milk medium with the addition of 1095 bovine serum, 200 ONM insulin and 0.1 nM estradiol in the presence of the COMPOUNDS indicated in each case, and cell growth was assessed by staining crystal violet.

The amount of proteinRkKiEKk i-th

The amounts of RK and EC in cell lysates were determined using assays of binding of the steroid to - radioactively labeled progesterone or estradiol according to the methods described | (Bigtapp et al. (Sopigaseriiop, 54 (1996), 243-251)).

Binding of 129I-ESE to tumor cells «

T470 cells pretreated with E5020 were incubated for 2 hours with 729I-EOE at 42C. Non-specific binding was in all cases less than 1095 of total binding. c) c Transactivation analysis "» T47O0 cells were transfected for a short time with MTM-G OS (Sa et al., EMVO .., 9:2237-40)| and cultivated in the absence or presence of NM K5BO20. In an RC-mediated antagonism experiment, transiently transfected T47O cells were treated with K5BO20 and, in addition, increasing concentrations of compound A or KI486.

After 24 hours, a test with luciferase was performed. - 2. Results b Fig. 1 demonstrates the anti-proliferative effect of various tested substances. T47O0 cells) were cultured in the presence (upper cross-hatched band) or in the absence (lower cross-hatched band) of OM1nM E" and increasing concentrations of compound A (d), onapristone (sh), 2K191703 (g) or 4-OH-Tat ( diamond). In the case of T47O cells, compound A exhibits a pronounced antiproliferative effect in extremely low concentrations.

Figure 2 shows the amounts of RK and EC protein in T470 cells. Fig. 3 shows the transcriptional activity of REK in T47O cells, on the basis of which these cells were briefly transfected with MTMU-H OS and cultured (a) in the absence (Co) or in the presence of NM KBO20. In the test for PK-mediated antagonism, short-term transfected T47O cells were treated with 0.1 mM K5BO20 and 22 compound A or BC468 in increasing concentrations (b).

GF) Figure 4 shows the Scatchard analysis of 722I-ESE binding to T470 cells. Cells were cultured for 48 hours in the presence of 20 nM K5O20 with or without 20 nM compound A and then washed. After that, ESE binding was determined in the concentration range of 0.25-150ng/ml ESE by incubation for 2 hours at 492°C. The insets show the number of bound ligands relative to the logarithm of the free ligand concentration.

Clearly, it was possible to block the increase in ESE binding caused by K5BO20 (middle figure) relative to the control (upper figure) when compound A was added (lower figure).

Figure 5 shows binding of 729I-ESE to intact T470 cells. To this end, cells were treated for 48 h with 2 or 20 nM K5BO20 and compound A or onapristone or compound A alone. Here it can also be seen that compound A blocks the enhancement of ESE binding to T470 cells induced by K5020. A similar, albeit much weaker, effect was also found in the case of onapristone.

Discussion

The above results show that the growth of T470 cells, which is stimulated by estradiol, was effectively blocked by compound A in intensive and substantial contact with RK.

Using transactivation assays, it was possible to show that RK in T47O cells showed transcriptional activity and could be blocked by compound 1.

Stimulation of T47O0 cells with K5020 led to a 2-3-fold increase in the expression of the ECE receptor, which was blocked by compound A. At the same time, the binding of ECE to cells increased 2-3 times and could be prevented by compound A and less effectively onapristone Increased binding of ESE to cells treated with K5BO20 could be caused by increased expression of the ESE receptor or increased heterodimer formation between the ESE receptor and egrB2.

These results indicate the interaction between the signaling systems of RK and growth factor in human breast carcinoma cells. By using antiprogestins, the progression of tumor cells from the stage of steroid-dependent growth to the stage of growth factor-dependent development is inhibited or prevented.

Claims (2)

The formula of the invention 1. Application of the compound of the general formula I in Izh-y; () dk EZ ts li o where B" means a methyl or ethyl group, B? means a radical of the formula C.EtpNo, where n means 2, 3, 4, 5 or 6, t"1 and ttvo-2p1, (ee) ВZ means a free, ether or ester hydroxyl group, si B and 5 each means a hydrogen atom, together they mean an additional bond or a methylene group, - ЗИ means a steroid ring system ABC of the specific formula A, B or C ee , ke, det | - y o g N u, dO - ts m ve i? A Z ' -i same (22) po ; - z 50 ra IC e) m : Е V F) ime) 60 b5 ne , and po r m t EE s where 25 means a hydrogen atom, a linear (C--Cl)alkyl group or a branched (C3-Cd)alkyl group, or a 75 halogen atom, B" means a hydrogen atom, a linear (Ci-C1-alkyl group) or a branched (C3-Cd-alkyl group, or, if 5 represents a steroidal ABC ring system A or B, in addition to this 25 Its B" represents together an additional bond, X represents an oxygen atom, a hydroxyimino group "M-OH or two hydrogen atoms, BZ represents a radical M or aryl radical, which is optionally substituted by group M in several pol , where M means a hydrogen atom, a halogen atom, -OH, -MO», -Maz, -SM, -MA Zarya, -МНВЗОв, -СО, (С4-Со)Alkoxy, (C.-C.)alkanoyloxy, benzoyloxy(C.1-C.0)alkanoyl, (C.4-C.0)hydroxyalkyl or benzoyl group, and c bza and Eb are the same or different and, like C, denote a hydrogen atom or (C4-Co)alkyl group, and in the case of -MAZaB95 radicals, also their physiologically compatible salts with acids, and in the case of -SOVU radicals, with VZ in the meaning of hydrogen, also their physiologically compatible salts with bases, for the preparation of a medicinal product for treatment of steroid-resistant breast cancer. 2. Application according to claim 1, where the compound is co 11 β-(4-acetylphenyl)-17 β-hydroxy-17 gu, -(1,1,2,2,2-pentafluoroethyl)estra-4,9-diene -3-one with C. Application according to claim 1 or 2, where the binding of the ESE growth factor to cancer cells "- mammary gland" is inhibited. (Se) m. - with . and? -I (22) - with 50 IR e) F) ime) 60 b5