J. Steroid Biochem. Molec. Biol. Vol.41, No. 3-8, pp. 339-348, 1992
0960-0760/92$5.00+ 0.00 Copyright© 1992PergamonPress pie
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PROGESTERONE POTENTIAL
ANTAGONISTS: AND
MECHANISM
TUMOR-INHIBITING OF
ACTION
H. MICHNA, l Y. NlsHx~qo,t G. NEEF, 1 W. L. M c G u I R E 2 and M. R. SCHNEIDER l tExperimental Oncology, Research Laboratories of Schering AG, Miillerstrage 170-178, D-1000 Berlin 65, Fed. Rep. Germany and 2Department of Medicine, Division of Oncology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284, U.S.A. Summary--A new approach for the treatment of breast cancer could be the use of progesterone antagonists. These compounds were originally developed for the inhibition of progesterone-dependent processes and have been shown to be effective in inhibition of nidation and interruption of pregnancy. Although the roles of progesterone and the progesterone receptor in control of cell growth remain unclear, it was found in progesterone receptor positive mammary carcinoma cell lines that the antiprogestin, Mifepristone, had an inhibitory effect on cell growth and a growth-inhibiting action on the DMBA-induced mammary carcinoma of the rat. We have shown that the progesterone antagonists, Onapristone and ZK 112993, which possess a reduced antiglucocorticoid activity compared to Mifepristone, exert a strong tumor-inhibiting effect in a panel of hormone-dependent mammary tumor models. The effects of these compounds were in some systems superior to those of tamoxifen or high dose progestins and comparable to ovariectomy. Although prerequisites for their antiproliferative potency are an affinity to the progesterone receptor as well as a sufficient number of available receptors in the tumors, the strong tumor inhibiting potential of the antiprogestins cannot be explained by a classical antihormonal mechanism. Surprisingly, the antitumor activity is evident in spite of elevated serum levels of ovarian and pituitary hormones. It was established by morphometric procedures that treatment with Onapristone triggers differentiation of the mitotically active polygonal tumor epithelial cell towards secretory active glandular structures and acini. All our quantitative light and electron microscopic data indicate that the antitumor action of antiprogestins is accompanied by the initiation of terminal differentiation leading to (apoptotic) cell death. Finally, our flow cytometry studies revealed an accumulation of the tumor cells in the GoG~ phase of the cell cycle, which may result from induction of differentiation since a differentiation-specific G 1 a r r e s t has already been proposed for other stem cell systems. It can be concluded from these data that the progesterone receptor antagonists differ in their mode of action from compounds used in established endocrine treatment strategies for mammary carcinoma. The ability of progesterone antagonists like Onapristone to reduce the number of cells in S-phase may offer a significant clinical advantage, since it is established that the S-phase fraction is a highly significant predictor of disease-free survival among axillary node-negative patients with diploid mammary tumors.
INTRODUCTION I m p r o v e m e n t s in therapy o f breast cancer are sorely needed. In recent years some encouraging results have been observed in experimental models o f breast cancer using progesterone antagonists [1-4], which indicate that these might represent a new class o f c o m p o u n d s offering an innovative strategy in the endocrine therapy o f m a m m a r y carcinomas. Originally, these components were developed to antagonize the
Proceedings of the lOth International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Paris, France, 26-29 May 1991.
effects o f progesterone at the receptor level with regard to fertility control, gynaecology and obstetrics since the progesterone dependence o f nidation and maintenance o f pregnancy implies that competitive progesterone antagonists could find application as contraceptive and abortive c o m p o u n d s [5, 6]. In the laboratories o f R o u s s e l - U c l a f the first progesterone antagonist was discovered [7] and pharmacologically developed [8, 9]. The characteristic structural feature o f this new class of c o m p o u n d s and o f the lead c o m p o u n d R U 38.486 ( = Mifepristone) is a dimethylamino-phenyl g r o u p in the l i f t position o f a 19-nor-steroid (Fig. 1). However, R U 38.486 suffers from the major d r a w b a c k o f having also antiglucocorticoid activity [10, l l]. 339
H. MICHNAet
340
al.
CH3 °
I
~ N "~.w~'~ OH CH3 ~ C ~ C - - C H 3
O"
v
RU38486
Progesterone CH3
O
I
~N.,~,,~.~ CH3 ~
OH
~C~C--CH 3
(CH2)2 - CH2OH
ZK98299
ZK 112 993
Fig. 1. Chemicalstructure of progesteroneand progesteroneantagonists. Therefore, we were looking for compounds with a better dissociation between antiprogestational and antiglucocorticoid activities. Although the two-dimensional representation is superficially similar in appearance to RU 38.486, the 13~methyl compound ZK 98.299 (Fig. 1 [12-15]) represents a new group of progesterone antagonists characterized by an inversion in the junction between the C and D rings. The anticipated alteration in molecular shape has been confirmed by X-ray crystallographic analysis [10]. Interestingly, in our preclinical models for the evaluation of antiglucocorticoid activities, ZK 98.299 shows less activity than RU 38.486 as well as induced no remarkable antiglucocorticoid changes at a dose of 100 mg p.o. in a phase I clinical study (unpublished data). In our research on progesterone antagonists we were furthermore interested in more potent mammary tumor-inhibiting compounds. Therefore, we characterized the antitumor potency of ZK 112.993 (Fig. 1) which differs in its structure from RU 38.486 having a 4-acetyl instead of a 4-dimethylamino group on the l l/~-phenyl ring [10, 16].
ANTIPROLIFERATIVE POTENCY OF PROGESTERONE ANTAGONISTSIN A BIOASSAYON THE MAMMARYGLAND At the moment all the in vitro antiproliferative assays seem not to be reproducible enough
to serve as a screening assay in the search of further compounds[17] and it was recently reported that they were also able to stimulate growth of progesterone receptor positive breast cancer cells[18], thus, exhibiting both antagonist and agonist-like activity depending on the cell culture conditions. In addition, since all tumor models in vivo are time, work and compound intensive, we established a bioassay on the mammary gland for the evaluation and screening of antiproliferative potencies of progesterone antagonists in vivo[19]. In this bioassay the potency of progesterone antagonists to competitively antagonize the specific effects of progesterone on the target organ mammary gland is measured in ovariectomized hormonally substituted rats (Fig. 2). A morphometric analysis of the tubulo-alveolar buds in the inguinal mammary glands revealed a dramatic antiproliferative effect of the progesterone antagonists after as little as 3 days of treatment (Fig. 3). The antiproliferative potency of progesterone antagonists in this bioassay depends on the inhibition of the well-known stimulating effect of progesterone on the development of mammary gland buds [20-23]. So far there is a strong correlation of the antiproliferative potency of these progesterone antagonists in this bioassay and in progesterone receptor positive experimental mammary carcinomas.
Progesterone antagonists
341
Bioassay to evaluate the antiproliferative potency of progesterone antagonists on the mammary gland Control group Ovariectomy (rat, 1009)
End of experiment + Progesterone
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o
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Experimental group with progesterone antagonist Ovariectomy
End of experiment + Progesterone, + Antigestagen
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.
o
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At end of experimentanalysisof the inguinalmammaryglands Fig. 2. Scheme of the experimental design.
ACTIVITY IN ANIMAL M O D E L S OF M A M M A R Y CARCINOMA
In characterizing the mammary carcinoma inhibiting potential of progesterone antagonists a panel of relevant experimental mammary carcinomas was used, including the hormonedependent transplantable M X T ( + ) tumor of the mouse as well as the methylnitrosourea (MNU) and the dimethylbenzanthracen¢ (DMBA) induced mammary carcinoma of the rat [3, 4]. In a first test using a treatment schedule starting immediately after M X T ( + ) tumor implantation (prophylaxis model) for 6 weeks
Onapristone (ZK 98.299, 5 mg/kg) was compared to ZK 112.993 (Smg/kg), tamoxifen (5 mg/kg), high dose estrogen (diethylstilbestrol 2.5 mg/kg) and to ovariectomy (Fig. 4). MXT (+) mammary carcinoma (prophylaxis model) 80 70 -60 oJ E E 50-
o Control & Ovariectomy /o + Tamoxifen 5 mg/kg/d / x DES 2.5 mg/kg/d / O ZK 98299 5 mg/kg/d / t ZK 112993 5 mg/kg/d /
!
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el Fig. 3. Antiproli~rative action of progesterone antagonists on m a m m a r y ~ a n d b u d s i n t h e b i o a s s a y .
1
2
3
Therapy (weeks) Fig. 4. Effects of Onapristone ( = ZK 98.299), ZK t 12.993, ovaricctomy, tamoxifen, diethylstilbestrol ( = D E S ) on growth of the hormone dependent M X T ( + ) mammary carcinoma of the mouse (prophylaxis model). ComPounds were administered 6 times weekly s.c. starting on day 1 after tumor implantation for 6 weeks (n > 9/group, two tumors/mouse: shown are the mean values of the tumor growth curves).
342
H. MICHNAet al.
The strong hormone-dependency of this estrogen and progesterone receptor positive line is demonstrated by the pronounced inhibition of tumor growth by ovariectomy. The ovariectomy as well as all compounds exerted a significant inhibition of tumor area (P < 0.05; Dunnetttest). Both progesterone antagonists induced inhibition comparable to ovariectomy whereas retardation of growth by tamoxifen and diethylstilbestrol (DES) was incomplete. The inhibition of ZK 112.993 was significantly better (P < 0.05, Dunnett-test) than that of tamoxifen, DES and Onapristone. Because of the strong inhibition shown by both progesterone antagonists a treatment schedule was chosen in the next M X T ( + ) experiment starting 3 weeks after tumor implantation. Since the tumors are well established before start of treatment the tumor growth is more difficult to inhibit; in addition, both progesterone antagonists were used in smaller doses (2, 1 and 0.5mg/kg, Fig. 5). Whereas Onapristone caused a dose-dependent growth inhibition comparable to ovariectomy, Z K 112.993 was comparable to ovariectomy at all doses tested (Fig. 5), and was significantly better than the tumor growth inhibition of Onapristone at the two lower doses of 0.5 and 1 mg/kg (P < 0.05, Dunnett-test).
Hormone-dependent MXT (+) mammary carcinoma -Tumor weight2000
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1200
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Fig. 6. Effect of the progesterone antagonist ZK 112.993, tamoxifen, and ovariectomy on mean area of DMBA-induced mammary tumors of the rat (4 weeks therapy, compounds were administered 6 times a week s.c., n > 10/group). Tumor area was termed 100% at start of treatment. To prove the m a m m a r y tumor-inhibiting potential of progesterone antagonists, we used the hormone-dependent D M B A model of the rat. Tumors were treated for 4 weeks when they had reached a certain size which was termed at 100% at start of treatment. As can be seen from the difference in tumor growth between the intact control and ovariectomy, this model is strictly dependent on ovarian hormones (Fig. 6). Whereas tamoxifen in a dose of 6 mg/kg induced tumor retardation which was at the borderline of statistical significance, Z K 112.993 in a dose of 10 mg/kg significantly blocked tumor growth; nevertheless, the inhibition of ovariectomy was superior to Z K 112.993. The MNU-induced tumor model of the rat offers the advantage over the DMBA-induced m a m m a r y tumor of being prolactin-independent [24]. Tumors, induced by a single i.v.-dose of 50 mg/kg M N U , were treated for 6 weeks. Both the progesterone antagonists Onapristone and Z K 112.993 in a dose of 5 mg/kg s.c. led to a significant inhibition of tumor growth (Fig. 7). The effect of Z K 112.993 was dose-dependent and at a dose of 10 mg/kg statistically comparable to the effect of ovariectomy (Fig. 7). These results from a variety of hormonedependent m a m m a r y tumor models demonstrate that progesterone antagonists are potent
Progesterone antagonists Hormone-dependent N M U - M C 40,000 m
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inhibitors of experimental breast cancer, which is in keeping with our previous work on the antitumor potency of Mifepristone, Onapristone, and ZK 112.993 [3,4, 19,25-28]. Their tumor-inhibiting effect was sometimes even better than that of ovariectomy. Compared to the standard treatment for hormone dependent breast cancer, the antiestrogen tamoxifen, progesterone antagonists are superior in the M X T ( + ) model, as well as in the DMBA and MNU-model [25].
MECHANISM OF ACTION
Concerning the mechanism of action of progesterone antagonists, an experiment with established M X T ( + ) mammary carcinomas was interesting since after a 3 week therapy, a strong tumor inhibiting potential of Mifepristone and Onapristone (10 mg/kg s.c.) comparable to the effect of ovariectomy was seen. In contrast, treatment with high doses of gestagens like medroxyprogesteroneacetate (MPA 100mg/kg s.c.) or megastroleacetate (Megace 100mg/kg and 25 mg/kg s.c.) were ineffective (Fig. 8). This experiment already favoured our concept that the tumor-inhibiting mechanism of progesterone antagonists is different from that of highdose gestagen treatment.
343
Interestingly, the progesterone antagonists tested so far induce strong activation of pituitary and ovary hormone secretion [2-4], leading to enhanced estrogen and progesterone levels as well as increased uterine weights [2-4]. Therefore, progesterone antagonists induce potent tumor-inhibition in spite of an inhibition of the negative feedback mechanism leading to enhanced ovarian hormones, known to stimulate mammary tumor-growth. However, in ovariectomized mice bearing M X T ( + ) tumors (Fig. 9) Onapristone as well as ZK 112.993 could antagonize completely the pronounced stimulation of tumor growth induced by EB (Fig. 9[26]). In conclusion, these and further in vitro [1] and in rivo [26] data indicate that the tumor-inhibiting mechanism of progesterone antagonists cannot be primarily explained as a progesterone displacement from its receptor, since after ovariectomy the level of circulating progesterone is extremely low [29, and unpublished data]. Keeping in mind that the progesterone receptor content in the MXT tumors is enhanced after estrogen treatment [30, 31], the Therapy 3 weeks of established MXT (+) mammary carcinomas 4 q
_.
r
°1i/ 0 Control ="~== Ovariectomy [
J Tamoxifen 4 mg/kg RU 486 10 mg/kg
[=====1ZK 98299 10 mg/kg l
MPA 100 mg/kg Megace 25 mg/kg Megace 100 mg/kg
Fig. 8. MXT(+) tumor growth pattern in (intact) controls, ovariectomy, ovariectomy substituted with estradiolbcnzoate ( = EB) and simultaneous treatment with either Onapristone or ZK 112.993 (n > 10/group; two tumors/mouse).
344
H. MICHNA et al.
80 70
/
60
E 50 E •
t~
• Control
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/,1 /
• ov EB 0.33 Itg 3xw o ov EB + Onapristone 0.2 mg 6xw • ov Ea + 112.993 0.2 mg 6xw
0
10 20 30 Therapy (days) Fig. 9. MXT-tumor growth pattern in control and ovariectomized mice as we]] as ovariectomized mice substituted with estradiol benzoate (0.33 # g s.c. three times a week) and simultaneous treatment with progesterone antagonists (0.2 m g animal ~ 10 mg/kg s.c. 6 times a week).
strong antitumor activity of these antiprogestins can be explained by a progesterone receptordependent mechanism. Indeed, it could be shown in a set of experiments in vitro [1] and in vivo [26] that the antitumor activity of the progesterone antagonists depends on the availability of a sufficient number of unoccupied progesterone receptors. In detailed quantitative light and electron microscopic studies the mode of antitumor action of progesterone antagonists was compared to ovariectomy: whereas the induction of tumor cell degeneration and necrobiosis is the predominant feature of the mammary tumors after ovariectomy ([4, 32-34], Fig. 10) treatment with the antiprogesterones induces differentiation of mitotically active polygonal tumor cells towards dysplastic glandular structures [4, 34] with a massive sequestering of secretory products (Fig. 10 [4, 34, 35]). Simultaneously, morphometric data indicate that apoptotic cell death--which is a well accepted characteristic sign of terminal differentiation (Fig. l l)--is strongly enhanced [34]. In addition, we elucidated the tumor-inhibiting mechanism analyzing the distribution of tumor cells within the cell cycle in the MXT mammary carcinoma of the mouse and the MNU induced mammary carcinoma of the rat by use of flow cytometry. Treatment of these tumors led to an accumulation of cells in GoG~ phase of the cell cycle together with a significant and biologically relevant reduction in the num-
ber of the cells in the G2M and S phase (Fig. 11). In contrast, use of the conventional endocrine therapies for breast cancer like tamoxifen ([36, 37], Fig. 11) diethylstilbestrol as well as ovariectomy (Fig. 11) displayed no changes in the distribution of cells within the cell cycle, indicating that progesterone antagonists differ in their mechanism of action. Interestingly, There are observations in some stem cell types that hormonal control of G~ and cell differentiation are somehow linked and a differentiation-specific G] arrest has already been proposed [38, 39]. Therefore the accumulation of the tumor cells in GoGl may result from induction of differentiation and thus correlate with all our morphological data. Since it is well accepted that the S-phase fraction is a highly significant predictor of disease-free survival among axillary node-negative patients with diploid mammary tumors [40, 41] the ability of progesterone antagonists like Onapristone and ZK 112.993 to reduce the number of cells in S-phase may offer a significant clinical advantage. Finally, our data stimulate the idea that the mammary carcinoma-inhibiting potential of Blocking of the cell cycle of progesterone antagonists in MNU mammary tumors 8
/
0~ O
to
/ 8
t~
g ~
0- z
Control Ovariectomy ] Tamoxifen 5 mg/kg DES 0.5 mg/kg Fm==I Onapristone 20 mg/kg
i I
i
ZK 112993 20 mg/kg
Fig. 10. Reduced a m o u n t of tumor cells in the S-phase of the cell cycle in M N U m a m m a r y tumors after 6 weeks treatment with Onapristone of Z K 112.993, whereas t a m • x iron, ovariectomy and diethylstilbestrol had no effect on the S-phase fraction.
Progesterone antagonists
Fig. 11 (a, b)--legend overleaf
345
Fig. 11. Ultrastructure of the M X T ( + ) mammary carcinoma. (a) Grown in an intact control mouse: polygonal, undifferentiated cell populations with the ultrastructural characteristics of metabolic activation ( x 8.810). (b) Tumor from an ovariectomized mouse, displaying necrobiotic tumor cells with autophagic reactions, sequestered cell processes and typically signs of leucocyte invasion ((3 = Granulocyte) adherent to the endothelial cell (x8.810). (c) Therapy of established, tumors for 2 weeks with Onapristone (50 mg/kg/d). Note: Arrangement of tumor cells to dysplastic, highly secretory active glandular acini with the formation of numerous intercellular junctional complexes ( x 8.810). (d) Typical feature of induction of cell death by apoptosis after treatment with Onapristone: nuclear fragmentation in the mammary tumor epithelial cells giving the typical apoptotic bodies (x 10.800). 346
Progesterone antagonists
progesterone antagonists may be due to an ability to eliminate an intrinsic block in terminal differentiation--which is the natural process to cell death in non-tumorigenic cells.
17. 18.
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in G0G I. Breast Cancer Res. Treat. 17 (1990) 155-156. Scott R. E., Hoed B. J., Wille J. J., Florine D. L., Krawisz B. R. and Kankatsu Y.: Coupling of proadipocyte growth arrest and differentiation. II. A cell cycle model for the physiological control of cell proliferation. J. Cell. Biol. 94 (1982) 400--405. Wille J. J., Maercklein P. B. and Scott R. E.: Neoplastic transformation and defective control of cell proliferation and differentiation. Cancer Res. 42 (1982) 5139-5146. Clark G. M., Dressier L. G,, Owens M. A., Pounds G., Oldaker T. and McGuire W. L.: Prediction of relapse or survival in patients with node-negative breast cancer by DNA flow cytometry. N. Engl. J. Med. 320 (1989) 627--633. McGuire W. L. and Dressier L. G.: Emerging impact of flow cytometry in predicting recurrence and survival in breast cancer patients. J N C I 75 (1985) 405-410.
0960-0760/92$5.00+ 0.00 Copyright© 1992PergamonPress pie
Printed in Great Britain. All rights reserved
PROGESTERONE POTENTIAL
ANTAGONISTS: AND
MECHANISM
TUMOR-INHIBITING OF
ACTION
H. MICHNA, l Y. NlsHx~qo,t G. NEEF, 1 W. L. M c G u I R E 2 and M. R. SCHNEIDER l tExperimental Oncology, Research Laboratories of Schering AG, Miillerstrage 170-178, D-1000 Berlin 65, Fed. Rep. Germany and 2Department of Medicine, Division of Oncology, The University of Texas Health Science Center at San Antonio, 7703 Floyd Curl Drive, San Antonio, TX 78284, U.S.A. Summary--A new approach for the treatment of breast cancer could be the use of progesterone antagonists. These compounds were originally developed for the inhibition of progesterone-dependent processes and have been shown to be effective in inhibition of nidation and interruption of pregnancy. Although the roles of progesterone and the progesterone receptor in control of cell growth remain unclear, it was found in progesterone receptor positive mammary carcinoma cell lines that the antiprogestin, Mifepristone, had an inhibitory effect on cell growth and a growth-inhibiting action on the DMBA-induced mammary carcinoma of the rat. We have shown that the progesterone antagonists, Onapristone and ZK 112993, which possess a reduced antiglucocorticoid activity compared to Mifepristone, exert a strong tumor-inhibiting effect in a panel of hormone-dependent mammary tumor models. The effects of these compounds were in some systems superior to those of tamoxifen or high dose progestins and comparable to ovariectomy. Although prerequisites for their antiproliferative potency are an affinity to the progesterone receptor as well as a sufficient number of available receptors in the tumors, the strong tumor inhibiting potential of the antiprogestins cannot be explained by a classical antihormonal mechanism. Surprisingly, the antitumor activity is evident in spite of elevated serum levels of ovarian and pituitary hormones. It was established by morphometric procedures that treatment with Onapristone triggers differentiation of the mitotically active polygonal tumor epithelial cell towards secretory active glandular structures and acini. All our quantitative light and electron microscopic data indicate that the antitumor action of antiprogestins is accompanied by the initiation of terminal differentiation leading to (apoptotic) cell death. Finally, our flow cytometry studies revealed an accumulation of the tumor cells in the GoG~ phase of the cell cycle, which may result from induction of differentiation since a differentiation-specific G 1 a r r e s t has already been proposed for other stem cell systems. It can be concluded from these data that the progesterone receptor antagonists differ in their mode of action from compounds used in established endocrine treatment strategies for mammary carcinoma. The ability of progesterone antagonists like Onapristone to reduce the number of cells in S-phase may offer a significant clinical advantage, since it is established that the S-phase fraction is a highly significant predictor of disease-free survival among axillary node-negative patients with diploid mammary tumors.
INTRODUCTION I m p r o v e m e n t s in therapy o f breast cancer are sorely needed. In recent years some encouraging results have been observed in experimental models o f breast cancer using progesterone antagonists [1-4], which indicate that these might represent a new class o f c o m p o u n d s offering an innovative strategy in the endocrine therapy o f m a m m a r y carcinomas. Originally, these components were developed to antagonize the
Proceedings of the lOth International Symposium of the Journal of Steroid Biochemistry and Molecular Biology, Recent Advances in Steroid Biochemistry and Molecular Biology, Paris, France, 26-29 May 1991.
effects o f progesterone at the receptor level with regard to fertility control, gynaecology and obstetrics since the progesterone dependence o f nidation and maintenance o f pregnancy implies that competitive progesterone antagonists could find application as contraceptive and abortive c o m p o u n d s [5, 6]. In the laboratories o f R o u s s e l - U c l a f the first progesterone antagonist was discovered [7] and pharmacologically developed [8, 9]. The characteristic structural feature o f this new class of c o m p o u n d s and o f the lead c o m p o u n d R U 38.486 ( = Mifepristone) is a dimethylamino-phenyl g r o u p in the l i f t position o f a 19-nor-steroid (Fig. 1). However, R U 38.486 suffers from the major d r a w b a c k o f having also antiglucocorticoid activity [10, l l]. 339
H. MICHNAet
340
al.
CH3 °
I
~ N "~.w~'~ OH CH3 ~ C ~ C - - C H 3
O"
v
RU38486
Progesterone CH3
O
I
~N.,~,,~.~ CH3 ~
OH
~C~C--CH 3
(CH2)2 - CH2OH
ZK98299
ZK 112 993
Fig. 1. Chemicalstructure of progesteroneand progesteroneantagonists. Therefore, we were looking for compounds with a better dissociation between antiprogestational and antiglucocorticoid activities. Although the two-dimensional representation is superficially similar in appearance to RU 38.486, the 13~methyl compound ZK 98.299 (Fig. 1 [12-15]) represents a new group of progesterone antagonists characterized by an inversion in the junction between the C and D rings. The anticipated alteration in molecular shape has been confirmed by X-ray crystallographic analysis [10]. Interestingly, in our preclinical models for the evaluation of antiglucocorticoid activities, ZK 98.299 shows less activity than RU 38.486 as well as induced no remarkable antiglucocorticoid changes at a dose of 100 mg p.o. in a phase I clinical study (unpublished data). In our research on progesterone antagonists we were furthermore interested in more potent mammary tumor-inhibiting compounds. Therefore, we characterized the antitumor potency of ZK 112.993 (Fig. 1) which differs in its structure from RU 38.486 having a 4-acetyl instead of a 4-dimethylamino group on the l l/~-phenyl ring [10, 16].
ANTIPROLIFERATIVE POTENCY OF PROGESTERONE ANTAGONISTSIN A BIOASSAYON THE MAMMARYGLAND At the moment all the in vitro antiproliferative assays seem not to be reproducible enough
to serve as a screening assay in the search of further compounds[17] and it was recently reported that they were also able to stimulate growth of progesterone receptor positive breast cancer cells[18], thus, exhibiting both antagonist and agonist-like activity depending on the cell culture conditions. In addition, since all tumor models in vivo are time, work and compound intensive, we established a bioassay on the mammary gland for the evaluation and screening of antiproliferative potencies of progesterone antagonists in vivo[19]. In this bioassay the potency of progesterone antagonists to competitively antagonize the specific effects of progesterone on the target organ mammary gland is measured in ovariectomized hormonally substituted rats (Fig. 2). A morphometric analysis of the tubulo-alveolar buds in the inguinal mammary glands revealed a dramatic antiproliferative effect of the progesterone antagonists after as little as 3 days of treatment (Fig. 3). The antiproliferative potency of progesterone antagonists in this bioassay depends on the inhibition of the well-known stimulating effect of progesterone on the development of mammary gland buds [20-23]. So far there is a strong correlation of the antiproliferative potency of these progesterone antagonists in this bioassay and in progesterone receptor positive experimental mammary carcinomas.
Progesterone antagonists
341
Bioassay to evaluate the antiproliferative potency of progesterone antagonists on the mammary gland Control group Ovariectomy (rat, 1009)
End of experiment + Progesterone
3 rag/rat
.
o
g
Io.,,
g
,o
I
Experimental group with progesterone antagonist Ovariectomy
End of experiment + Progesterone, + Antigestagen
3 mg/rat 1 mg/rat
.
o
g
Io.,,
g
,o
I
At end of experimentanalysisof the inguinalmammaryglands Fig. 2. Scheme of the experimental design.
ACTIVITY IN ANIMAL M O D E L S OF M A M M A R Y CARCINOMA
In characterizing the mammary carcinoma inhibiting potential of progesterone antagonists a panel of relevant experimental mammary carcinomas was used, including the hormonedependent transplantable M X T ( + ) tumor of the mouse as well as the methylnitrosourea (MNU) and the dimethylbenzanthracen¢ (DMBA) induced mammary carcinoma of the rat [3, 4]. In a first test using a treatment schedule starting immediately after M X T ( + ) tumor implantation (prophylaxis model) for 6 weeks
Onapristone (ZK 98.299, 5 mg/kg) was compared to ZK 112.993 (Smg/kg), tamoxifen (5 mg/kg), high dose estrogen (diethylstilbestrol 2.5 mg/kg) and to ovariectomy (Fig. 4). MXT (+) mammary carcinoma (prophylaxis model) 80 70 -60 oJ E E 50-
o Control & Ovariectomy /o + Tamoxifen 5 mg/kg/d / x DES 2.5 mg/kg/d / O ZK 98299 5 mg/kg/d / t ZK 112993 5 mg/kg/d /
!
I~, 4o
/+1 ,+
¢, 4000 f r~
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/
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5
6
//+"
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ZK 112993:0,1 mg
2000 i ~
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1000 - - O
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t
el Fig. 3. Antiproli~rative action of progesterone antagonists on m a m m a r y ~ a n d b u d s i n t h e b i o a s s a y .
1
2
3
Therapy (weeks) Fig. 4. Effects of Onapristone ( = ZK 98.299), ZK t 12.993, ovaricctomy, tamoxifen, diethylstilbestrol ( = D E S ) on growth of the hormone dependent M X T ( + ) mammary carcinoma of the mouse (prophylaxis model). ComPounds were administered 6 times weekly s.c. starting on day 1 after tumor implantation for 6 weeks (n > 9/group, two tumors/mouse: shown are the mean values of the tumor growth curves).
342
H. MICHNAet al.
The strong hormone-dependency of this estrogen and progesterone receptor positive line is demonstrated by the pronounced inhibition of tumor growth by ovariectomy. The ovariectomy as well as all compounds exerted a significant inhibition of tumor area (P < 0.05; Dunnetttest). Both progesterone antagonists induced inhibition comparable to ovariectomy whereas retardation of growth by tamoxifen and diethylstilbestrol (DES) was incomplete. The inhibition of ZK 112.993 was significantly better (P < 0.05, Dunnett-test) than that of tamoxifen, DES and Onapristone. Because of the strong inhibition shown by both progesterone antagonists a treatment schedule was chosen in the next M X T ( + ) experiment starting 3 weeks after tumor implantation. Since the tumors are well established before start of treatment the tumor growth is more difficult to inhibit; in addition, both progesterone antagonists were used in smaller doses (2, 1 and 0.5mg/kg, Fig. 5). Whereas Onapristone caused a dose-dependent growth inhibition comparable to ovariectomy, Z K 112.993 was comparable to ovariectomy at all doses tested (Fig. 5), and was significantly better than the tumor growth inhibition of Onapristone at the two lower doses of 0.5 and 1 mg/kg (P < 0.05, Dunnett-test).
Hormone-dependent MXT (+) mammary carcinoma -Tumor weight2000
t: 2: 3: 4: 5: 6: 7: 8:
1800 1600 1400 vE
Control Ovariectomy
ZK ZK ZK ZK ZK ZK
9 8 2 9 9 2 mg/kg/d 9 8 2 9 9 1 mg/kg/d 9 8 2 9 9 0.5 mg/kg/d 112993 2 mg/kg/d 112993 1 mg/kg/d 112993 0.5 mg/kg/d
1200
1000 u
E
800
o
16OO
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i
i
i
i
i
1
2
3
4
5
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6
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Fig. 5. Effects of progesterone antagonists and of ovariectomy on growth of established, hormone-dependent MXT(+) mammary tumors of the mouse (mean values). Compounds were administered 6 times weekly s.c. starting 3 weeks after tumor implantation for 3 weeks (n > 9/group; two tumors/mouse).
DMBA - induced MC (rat) p ...... 300
-
.0
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,0'
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~) "~
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"
I
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/ '
," "¢
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Fig. 6. Effect of the progesterone antagonist ZK 112.993, tamoxifen, and ovariectomy on mean area of DMBA-induced mammary tumors of the rat (4 weeks therapy, compounds were administered 6 times a week s.c., n > 10/group). Tumor area was termed 100% at start of treatment. To prove the m a m m a r y tumor-inhibiting potential of progesterone antagonists, we used the hormone-dependent D M B A model of the rat. Tumors were treated for 4 weeks when they had reached a certain size which was termed at 100% at start of treatment. As can be seen from the difference in tumor growth between the intact control and ovariectomy, this model is strictly dependent on ovarian hormones (Fig. 6). Whereas tamoxifen in a dose of 6 mg/kg induced tumor retardation which was at the borderline of statistical significance, Z K 112.993 in a dose of 10 mg/kg significantly blocked tumor growth; nevertheless, the inhibition of ovariectomy was superior to Z K 112.993. The MNU-induced tumor model of the rat offers the advantage over the DMBA-induced m a m m a r y tumor of being prolactin-independent [24]. Tumors, induced by a single i.v.-dose of 50 mg/kg M N U , were treated for 6 weeks. Both the progesterone antagonists Onapristone and Z K 112.993 in a dose of 5 mg/kg s.c. led to a significant inhibition of tumor growth (Fig. 7). The effect of Z K 112.993 was dose-dependent and at a dose of 10 mg/kg statistically comparable to the effect of ovariectomy (Fig. 7). These results from a variety of hormonedependent m a m m a r y tumor models demonstrate that progesterone antagonists are potent
Progesterone antagonists Hormone-dependent N M U - M C 40,000 m
30,000
1: Control 2: Ovariectomy 3:98299 5 mg/kg/d 4:112993 5 mg/kg/d 5:112993 10 mg/kg/d
E
~ 2o,ooo e-
._~ 10,000
•
0
1
2
3
4
5
Fig. 7. Effect of progesterone antagonists and ovaricctomy on the weight of established MNU-induced mammary tumors of the rat. Compounds were administered 6 times weekly s.c. for 6 weeks.
inhibitors of experimental breast cancer, which is in keeping with our previous work on the antitumor potency of Mifepristone, Onapristone, and ZK 112.993 [3,4, 19,25-28]. Their tumor-inhibiting effect was sometimes even better than that of ovariectomy. Compared to the standard treatment for hormone dependent breast cancer, the antiestrogen tamoxifen, progesterone antagonists are superior in the M X T ( + ) model, as well as in the DMBA and MNU-model [25].
MECHANISM OF ACTION
Concerning the mechanism of action of progesterone antagonists, an experiment with established M X T ( + ) mammary carcinomas was interesting since after a 3 week therapy, a strong tumor inhibiting potential of Mifepristone and Onapristone (10 mg/kg s.c.) comparable to the effect of ovariectomy was seen. In contrast, treatment with high doses of gestagens like medroxyprogesteroneacetate (MPA 100mg/kg s.c.) or megastroleacetate (Megace 100mg/kg and 25 mg/kg s.c.) were ineffective (Fig. 8). This experiment already favoured our concept that the tumor-inhibiting mechanism of progesterone antagonists is different from that of highdose gestagen treatment.
343
Interestingly, the progesterone antagonists tested so far induce strong activation of pituitary and ovary hormone secretion [2-4], leading to enhanced estrogen and progesterone levels as well as increased uterine weights [2-4]. Therefore, progesterone antagonists induce potent tumor-inhibition in spite of an inhibition of the negative feedback mechanism leading to enhanced ovarian hormones, known to stimulate mammary tumor-growth. However, in ovariectomized mice bearing M X T ( + ) tumors (Fig. 9) Onapristone as well as ZK 112.993 could antagonize completely the pronounced stimulation of tumor growth induced by EB (Fig. 9[26]). In conclusion, these and further in vitro [1] and in rivo [26] data indicate that the tumor-inhibiting mechanism of progesterone antagonists cannot be primarily explained as a progesterone displacement from its receptor, since after ovariectomy the level of circulating progesterone is extremely low [29, and unpublished data]. Keeping in mind that the progesterone receptor content in the MXT tumors is enhanced after estrogen treatment [30, 31], the Therapy 3 weeks of established MXT (+) mammary carcinomas 4 q
_.
r
°1i/ 0 Control ="~== Ovariectomy [
J Tamoxifen 4 mg/kg RU 486 10 mg/kg
[=====1ZK 98299 10 mg/kg l
MPA 100 mg/kg Megace 25 mg/kg Megace 100 mg/kg
Fig. 8. MXT(+) tumor growth pattern in (intact) controls, ovariectomy, ovariectomy substituted with estradiolbcnzoate ( = EB) and simultaneous treatment with either Onapristone or ZK 112.993 (n > 10/group; two tumors/mouse).
344
H. MICHNA et al.
80 70
/
60
E 50 E •
t~
• Control
40
E 30 I20
o Ovariectomy
I
/,1 /
• ov EB 0.33 Itg 3xw o ov EB + Onapristone 0.2 mg 6xw • ov Ea + 112.993 0.2 mg 6xw
0
10 20 30 Therapy (days) Fig. 9. MXT-tumor growth pattern in control and ovariectomized mice as we]] as ovariectomized mice substituted with estradiol benzoate (0.33 # g s.c. three times a week) and simultaneous treatment with progesterone antagonists (0.2 m g animal ~ 10 mg/kg s.c. 6 times a week).
strong antitumor activity of these antiprogestins can be explained by a progesterone receptordependent mechanism. Indeed, it could be shown in a set of experiments in vitro [1] and in vivo [26] that the antitumor activity of the progesterone antagonists depends on the availability of a sufficient number of unoccupied progesterone receptors. In detailed quantitative light and electron microscopic studies the mode of antitumor action of progesterone antagonists was compared to ovariectomy: whereas the induction of tumor cell degeneration and necrobiosis is the predominant feature of the mammary tumors after ovariectomy ([4, 32-34], Fig. 10) treatment with the antiprogesterones induces differentiation of mitotically active polygonal tumor cells towards dysplastic glandular structures [4, 34] with a massive sequestering of secretory products (Fig. 10 [4, 34, 35]). Simultaneously, morphometric data indicate that apoptotic cell death--which is a well accepted characteristic sign of terminal differentiation (Fig. l l)--is strongly enhanced [34]. In addition, we elucidated the tumor-inhibiting mechanism analyzing the distribution of tumor cells within the cell cycle in the MXT mammary carcinoma of the mouse and the MNU induced mammary carcinoma of the rat by use of flow cytometry. Treatment of these tumors led to an accumulation of cells in GoG~ phase of the cell cycle together with a significant and biologically relevant reduction in the num-
ber of the cells in the G2M and S phase (Fig. 11). In contrast, use of the conventional endocrine therapies for breast cancer like tamoxifen ([36, 37], Fig. 11) diethylstilbestrol as well as ovariectomy (Fig. 11) displayed no changes in the distribution of cells within the cell cycle, indicating that progesterone antagonists differ in their mechanism of action. Interestingly, There are observations in some stem cell types that hormonal control of G~ and cell differentiation are somehow linked and a differentiation-specific G] arrest has already been proposed [38, 39]. Therefore the accumulation of the tumor cells in GoGl may result from induction of differentiation and thus correlate with all our morphological data. Since it is well accepted that the S-phase fraction is a highly significant predictor of disease-free survival among axillary node-negative patients with diploid mammary tumors [40, 41] the ability of progesterone antagonists like Onapristone and ZK 112.993 to reduce the number of cells in S-phase may offer a significant clinical advantage. Finally, our data stimulate the idea that the mammary carcinoma-inhibiting potential of Blocking of the cell cycle of progesterone antagonists in MNU mammary tumors 8
/
0~ O
to
/ 8
t~
g ~
0- z
Control Ovariectomy ] Tamoxifen 5 mg/kg DES 0.5 mg/kg Fm==I Onapristone 20 mg/kg
i I
i
ZK 112993 20 mg/kg
Fig. 10. Reduced a m o u n t of tumor cells in the S-phase of the cell cycle in M N U m a m m a r y tumors after 6 weeks treatment with Onapristone of Z K 112.993, whereas t a m • x iron, ovariectomy and diethylstilbestrol had no effect on the S-phase fraction.
Progesterone antagonists
Fig. 11 (a, b)--legend overleaf
345
Fig. 11. Ultrastructure of the M X T ( + ) mammary carcinoma. (a) Grown in an intact control mouse: polygonal, undifferentiated cell populations with the ultrastructural characteristics of metabolic activation ( x 8.810). (b) Tumor from an ovariectomized mouse, displaying necrobiotic tumor cells with autophagic reactions, sequestered cell processes and typically signs of leucocyte invasion ((3 = Granulocyte) adherent to the endothelial cell (x8.810). (c) Therapy of established, tumors for 2 weeks with Onapristone (50 mg/kg/d). Note: Arrangement of tumor cells to dysplastic, highly secretory active glandular acini with the formation of numerous intercellular junctional complexes ( x 8.810). (d) Typical feature of induction of cell death by apoptosis after treatment with Onapristone: nuclear fragmentation in the mammary tumor epithelial cells giving the typical apoptotic bodies (x 10.800). 346
Progesterone antagonists
progesterone antagonists may be due to an ability to eliminate an intrinsic block in terminal differentiation--which is the natural process to cell death in non-tumorigenic cells.
17. 18.
REFERENCES 19. 1. Bardon S., Vignon F., Montcourrier P. and Rochefort H.: Steroid receptor-mediated cytotoxicity of an antiestrogen and an antiprogestin in breast cancer cells. Cancer Res. 47 (1987) 1441-1448. 2. Bakker G. H., Setyono-Han B., de Jong F. H. and Klijn J. G. M.: Mifepristone in treatment of experimental breast cancer in rats. In Hormonal Manipulation of Cancer: Peptides, Growth Factors and New (Anti)Steroidal Agents (Edited by J. G. M. Klijn, R. Paridaens and J. A. Foekens). Raven, New York (1987). 3. Schneider M. R., Michna H., Nishino Y. and E1 Etreby M. F.: Antitumor activity of the progesterone antagonists ZK 98.299 and RU 38.486 in the hormone-dependent MXT mammary tumor model of the mouse and the DMBA- und MNU-induced mammary tumor model of the rat. Eur. J. Cancer Clin. Oncol. 25 (1989) 691-701. 4. Michna H., Schneider M. R., Nishino Y. and El Etreby M. F.: Antitumor activity of the antiprogesterones ZK 98.299 and RU 38.486 in hormone dependent rat and mouse mammary tumors: mechanistic studies. Breast Cancer Res. Treat. 14 (1989) 275-288. 5. Csapo A. J.: Progesterone block. Am. J. Anat. 98(1956) 273~91. 6. Csapo A. J.: The "seesaw" theory of the regulatory mechanism of pregnancy. Am. J. Obstet. Gynec. 121 (1975) 578-581. 7. Herrman W., Wyss R., Riondel A., Philibert D., Teuseh G., Sakiz E. and Baulieu E. E.: Effet d'un steroid antiprogesterone cbez la femme interruption du cycle menstruel et de la grossesse au debut. C.R. Acad. Sci. Paris 294 0982) 933-938. 8. Baulieu E. E. and Ulmann A.: Antiprogesterone activity of RU 486 and its contragestive and other applications. Human Reprod. 1 0986) 107-1 l0 and references cited therein. 9. Philibert D., Moguilewsky M., Mary J., Lecaque D., Tournemine C., Secchi J. and Deraedt R.: Pharmacological profile of RU 486 in animals. In The Antiprogestin Steroid RU 486 and Human Fertility Control (Edited by E. E. Baulieu and S. Segal). Plenum, New York (1987) pp. 49-68. 10. Henderson D.: Antiprogestational and antiglucocorticoid activities of some novel I lfl-aryl substituted steroids. In Pharmacology and Clinical Uses of Inhibitors of Hormone Secretion and Action (Edited by B. J. A. Furr and A. E. Wakeling). Bailliere Tindall, London (1987) pp. 184-21 I. 11. Klijn J. G. M., de Jong F. H., Bakker G. H., Lamberts S. W. J., Rodenburg C. J. and Alexieva-Figusch J.: Antiprogestins, a new form of endocrine therapy for human breast cancer. Cancer Res. 49 (1989) 2851-2856. 12. Neef G., Beier S., Elger W., Henderson D. and Wiechert R.: New Steroids with antiprogestational and antiglucocorticoid activities. Steroids 44 (1984) 349-372. 13. Neef G., Saner G., Seeger A. and Wiechert R.: Synthetic variations of the progesterone antagonist RU 38 486. Tetrahedron Lett. 25 (1984) 3425-3428. 14. Wiecbert R. and Neef G.: Synthesis of antiprogestational steroids. J. Steroid Biochem. 27 (1987) 851-858. 15. Elger W., Beier S., Chwalisz K., F/ihnrich M., Hasan S. H., Henderson D., Neef G. and Rohde R.: Studies on the mechanisms of action of progesterone antagonists. J. Steroid Biochem. 25 (1986) 835-845. 16. Neef G., Cleve G., Ottow E., Sceger A. and Wiechert SB 41/3-8--K
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34. Gehring S., Michna H., Kiihnel W., Nishino Y. and Schneider M. R.: Morphometrical and histochemical studies on the differentiation potential of progesterone antagonists in experimental mammary carcinomas. Acta Endocr. 124 (1991) 177. 35. Gehring S., Michna H., Nishino Y., Schneider M. R. and Kiihnel W.: Differenzierung von Mammacarcinomen durch Antigestagene: Morphometrisehe, lektinhistochemische und ultrastrukturelle Aspekte. Verh. Anat. Ges. 172 (1991) 92. 36. Briinner W., Bronzert D., Vindulov L. L., Rygaard K., Spang-Thomson M. and Lippmann M. E.: Effect on growth and cell cycle kinetics of estradiol and tamoxifen on MCF-7 human breast cancer cells grown in vitro and in nude mice. Cancer Res, 49 (1989) 1515-1520. 37. Michna H., Schneider M. R., Nishino Y., El Etreby M. F. and McGuire W. L.: Progesterone antagonists block the growth of experimental mammary tumors
38.
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