Human Reproduction Update 1998, Vol. 4, No. 5 pp. 570—583
`
`© European Society of Human Reproduction and Embryology
`
`Modulation of oestrogenic effects by
`progesterone antagonists in the rat uterus
`
`Kristof Chwaliszl, Klaus Stockemann, Karl-Heinz Fritzemeier and Ulrike Fuhrmann
`
`Research Laboratories of Schen'ng AG, Mullerstrasse 170—178, 13342 Berlin, Germany
`
`TABLE OF CONTENTS
`
`Introduction
`
`Antiprogestins and oestrogen responses
`in the uterus
`
`Oestrogenic effects of anti-progestins in Vitro
`and in vivo
`
`Molecular and morphometric analyses
`Oestrogen-like activities of onapristone
`and mifepristone
`Modulation of oestrogenic effects by
`onapristone and mifepristone
`Oestrogen-like properties of antiprogestins
`Understanding anti-progestin effects on
`oestrogenic responses
`Conclusions
`
`Acknowledgements
`References
`
`570
`
`571
`
`572
`
`573
`
`574
`
`576
`577
`
`580
`581
`
`582
`582
`
`in
`Antiprogestins can modulate oestrogenic effects
`various oestrogen-dependent
`tissues, dependent on
`species, tissue, dose and duration of treatment. Enhanced
`oestrogenic responses to mifepristone and onapristone
`occur in Vitro and in vivo. However, the antiprogestins
`mifepristone, onapristone, and ZK 137 316 can block the
`ability of oestradiol to increase endometrial growth in
`non-human primates. Our purposes were firstly,
`to
`decide whether mifepristone and onapristone had direct
`oestrogenic activity in Vitro and in the uterus of spayed
`and immature rats, and secondly, to discover whether
`antiprogestins exhibit inhibitory effects on oestrogen
`action in the uterus in spayed, oestrogen-substituted rats.
`In
`transactivation
`assays, mifepristone
`induced
`oestrogenic response, whereas onapristone had only
`marginal effects on reporter gene transcription. In
`immature rats, onapristone and mifepristone markedly
`increased uterine weights, and onapristone, but not
`mifepristone significantly enhanced endometrial luminal
`epithelial height, a sensitive oestrogen parameter.
`Conversely,
`in spayed and adrenalectomized rats,
`
`1To whom correspondence should be addressed
`
`neither onapristone nor mifepristone changed uterine
`weights or endometrial morphology, indicating that their
`effects in immature rats were indirect.
`In spayed,
`oestrogen-substituted rats, antiprogestins did not block
`oestradiol-stimulated endometrial growth and luminal
`and glandular epithelium were stimulated more after
`antiprogestin plus oestrogen, than after oestradiol alone.
`All compounds induced compaction of the uterine
`stroma. In spayed rats, onapristone and some other
`130c-configured (type 1) antagonists (ZK 135 569, ZK
`131 535)
`reduced oestradiol-stimulated myometrial
`proliferation and induced an overall uterine weight
`reduction in animals
`treated with oestrogen and
`antiprogestins,
`in comparison with oestradiol-treated
`controls. 13B- configured (type II) antagonists, including
`mifepristone, lilopristone and ZK 112 993, were not
`effective. In the uteri of spayed rats, onapristone was also
`found to enhance the oestradiol-stimulatory effect on
`expression of the oestrogen-dependent proto-oncogene,
`C—fos. In conclusion, antiprogestins do not inhibit, but
`rather enhance, oestrogen-induced uterine glandular
`and luminal epithelium in spayed rats, contrary to their
`effects in primates. The rat model is unsuitable to study
`endometrial antiproliferative effects of antiprogestins in
`primate uteri.
`
`Key words: oestrogen action/progesterone antagonists/
`proliferation/uterus
`
`Introduction
`
`antagonists
`steroidal progesterone
`11l3-aryl-substituted
`(antiprogestins), e.g. mifepristone (RU 486), onapristone
`(ZK 98 299), lilopristone (ZK 98 734), ZK 112 993, and
`other structurally-related compounds bind with high-affinity
`to progesterone receptors (PR) and block progestagenic ef-
`fects both in Vitro and in ViVO (Neefd al ., 1984; Phillibert a
`al, 1985). Mifepnstone and onapristone are the most
`widely-studied
`antiprogestins. Both
`also
`bind
`to
`glucocorticoid (GR) and androgen (AR) receptors and exhibit
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`antiglucocorticoid and anti-androgenic activity in vitro
`(U .Fuhrmann, unpublished results) and in ViVO, onapristone
`being less antiglucocorticoid than mifepristone. These two
`antiprogestins show a very weak binding to human and rat
`oestrogen receptors (ER) (Chwalisz a al, 1995).
`Overall control ofgrowth and fill’lCthl’lS in the reproductive
`tract is regulated by oestrogen and progesterone. In the uterus,
`oestrogens
`stimulate endometrial epithelial proliferation,
`control many metabolic events, and are necessary for normal
`uterine growth. Progesterone generally inhibits oestrogen-
`dependent uterine
`epithelial proliferation and involves
`endometrial diflerenfiafion to the secretory type. Specific
`mechanisms proposed for the antiproliferative action of
`progesterone include: (i) a down-regulation of ER in target
`tissues (Hsueh 6 al, 1975, Katzenellenbogen 1980),
`(ii)
`uterine enzyme induction catalysing oestradiol conversion to
`less active metabolites (Tseng and Gurpide, 1975), (iii) a
`decrease in oestrogen-induced specific protein expression
`(Bhakoo dal,
`1977),
`and
`(iv)
`an
`inhibition of
`oestrogen-induced proto-oncogenes (Kirkland a al., 1992,
`Fuhrmann and Stockemann, 1993).
`
`Antiprogestins and oestrogen responses in the
`uterus
`
`Progesterone is a major sex steroid controlling oestrogen
`action in reproductive tracts and other oestrogen-dependent
`tissues. Therefore, it is not surprising that antiprogestins also
`interfere with various oestrogenic responses. The major
`concern of chronic antiprogestins administration in women is
`endometrial hyperplasia due to unopposed oestrogen effects.
`Paradoxically, antiprogestins, including mifepristone (Wolf a
`al., 1989, Slayden a al., 1993, Slayden and Brenner, 1994,
`Heikinheimo a al ., 1996), onapristone (Chwalisz a al ., 1994)
`and the new antiprogestin ZK 137 316 (Slayden a al ., 1997),
`inhibit endometrial proliferation in both ovariectomized and
`intact monkeys. Antiprogestins administered chronically at
`relatively low doses inhibit mitotic activity in endometrial
`epithelium and induce a dose-dependent stromal compaction
`in
`spayed
`and
`intact monkeys
`at high
`oestradiol
`concentrations. Similar endometrial antiproliferative effects
`arise in ovariectomized rabbits where onapristone selectively
`inhibits oestrogen-induced endometrial gland formation
`(Chwalisz 6 al., 1991). Recently, Gemzell-Danielsson a al.
`(1998) found that low-dose mifepristone (0.5 mg daily for 3
`months) delayed endometrial maturation and significantly
`reduced the glandular diameter in premenopausal women.
`These
`effects were
`accompanied by a reduction in
`endometrial glycodelin expression and Dolichus biflorus
`agglutinin (DBA)—lectin binding. Stromal compaction and an
`absence of mitoses arose with 2 mg mifepristone daily for 30
`days (Cameron a al, 1996).
`In monkeys and rabbits
`
`Modulation of oestrogenic effects by antiprogrestins
`
`571
`
`effects were endometrium-specific and
`antiproliferative
`oestrogenic effects in the oviduct and vagina were not
`inhibited by antiprogestins (Chwalisz a al, 1991, Slayden and
`Brenner, 1994). The endometrial antiproliferative effects of
`antiprogestins in the primate endometrium is a most important
`
`property of antiprogestins offering a unique opportunity to
`selectively inhibit oestrogenic effects in the uterus without
`affecting oestrogenic
`response
`in other
`tissues. The
`
`mechanism underlying this endometrial antiproliferative
`effect is still unknown, yet it differs fiom the inhibitory effect
`of a progesterone on the endometrium.
`
`However, uterine oestrogenic responses occur in monkeys
`and women after
`treatment with chronic, high-dose
`antiprogestins. Cystic endometrial hyperplasia occurred
`
`after chronic, high dose oral onapristone (50 mg/kg,
`Schering toxicological study, unpublished data) of intact
`cynomolgus monkeys. Atypical cystic changes also arose
`after chronic treatment of endometriotic women with a
`
`relatively high dose (50 mg daily) of mifepristone (Murphy
`a al., 1995). Recently, Croxatto a al. (1998) reported the
`occurrence of endometrial gland dilation in 34% of women
`treated with 1 mg/day mifepristone for 150 days. The
`significance of this finding is unclear, since no signs of
`
`endometrial hyperplasia were found in this study. A similar
`dilation of endometrial glands was frequently observed in
`monkeys
`after
`chronic
`treatment with
`different
`
`antiprogestins (K.Chwalisz, unpublished data). Interestingly,
`this effect could be observed even within an atrophic
`endometrium accompanied by a drastic reduction of mitotic
`
`activity in the glandular epithelium strongly suggesting that
`antiprogestin-induced gland dilation is due to altered
`glandular
`fluid outflow rather
`than to endometrial
`
`hyperplasia in non-human primates. Moreover, there are
`experimental studies in rats and mice which suggest that both
`mifepristone (Dibbs a al., 1995) and onapristone (Bigsby
`and Young, 1994) may exhibit some oestrogenic-like
`activities by directly interacting with ER
`
`the precise
`that
`indicate
`conflicting results
`These
`mechanism of the divergent antiprogestin effects on the
`endometrium are still poorly understood. These studies also
`show that
`the modulatory impact of oestrogen by
`
`antiprogestins is quite complex, since it may depend on
`species, tissue, antiprogestin dose and type, and duration of
`treatment. We describe the modulatory effect of various
`
`antiprogestins in the non-pregnant rat uterus, since rats are
`widely used to study oestrogenic and anti-oestrogenic
`activities. We specifically address the question of whether
`
`antiproliferative
`or
`oestrogenic
`exert
`antiprogestins
`(anti-oestrogenic) effects in the uteri of castrated rats, and
`
`whether type I (onapristone-type) and type II (mifepristone-
`type) antiprogestins act differently in the rat uterus. The
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`572 KChwalisz et a].
`
`oestrogen-like effects of antiprogestins are described using
`in-vitro and in-vivo models. In addition,
`the modulatory
`effects of antiprogestins on various parameters of oestrogen
`action,
`including
`C—fOS
`expression,
`uterine
`growth,
`morphology, and morphometry are discussed. Experiments
`were performed on spayed and immature rats, both
`commonly used to study oestrogenic effects. We also wished
`to find out whether rat models are of use in predicting
`antiproliferative endometrial effects of antiprogestins in
`primates. We also examined onapristone and mifepristone
`effects
`on
`uterine ER protein
`concentrations
`in
`ovariectomized rats, in the presence and absence ofoestradiol.
`Immature and adult VVrstar rats (Schering, Berlin, Ger-
`many) were kept in Makrolon cages (type 111) in an air-condi-
`tioned room at a temperature of 22 + 25C and relative
`humidity of 50 + 5%, under a regime of 14 h light: 10 h dark
`cycle (light 6:30—20:30). The animals had free access to the
`standard pellet diet Altromin[
`(Altromin Ltd, Lage, Ger-
`many) and to tap water containing 0.9% saline.
`Figure 1 shows the progesterone antagonists used in this
`study. The 13[3-methyl-substituted (type II) antiprogestins
`used were: mifepristone (RU 486, ZK 95 890: 11[3-[4—(dime-
`thylamino)-phenyl] - 17 [3-hydroxy— 17 [3-(prop- 1 -ynyl)estra-4,
`9-dien-3-one), lilopristone (ZK 98 734, 11[3-[4-(dimethylami-
`no)-phenyl] - 17 [3-hydroxy— 1 7oc-(3 -hydroxyprop- 1 -ynyl)estra-
`4, 9-dien-3 -one). The 13(x-methyl-substituted (type I) antipro-
`gestins used were: onapristine (ZK98 299, 11[3—[4—(dimethy-
`lamino)-phenyl] - 1 7oc-hydroxy- 17[3-(3-hydroxypropyl)- 1 1 [3-
`130c-estra—4, 9-dien-3-one), ZK 131 535 (170c-hydroxy—
`17[3-(3 -hydroxypropl)
`(1 1 [3-[4—1-methylethenyl)phenyl]-
`130c-estra—4, 9-dien-3-one), and ZK 135 695 (11B-[4—(3-fidra-
`nyl)phenyl] - 170c-hydroxy— 17 [3
`(3 -hydroxypropyl)- 13 oc-
`estra-4, 9-dien-3-one). All compounds were from Schering
`AG. For oral administration, the antiprogestins were formu-
`lated in 0.5 ml Myrj[ 53 (ICI, Essen, Germany) saline (85 mg
`Myrj[ 53 in 100 ml 0.9% saline). For so administration, the
`compounds were formulated in 0.2 ml benzylbenzoate + cas-
`tor oil (1:4 v/v). 17[3-oestradiol and the pure anti-oestrogen
`ICI 182 780 (ZK 156 901) were formulated in 0.2 ml benzyl
`benzoate plus castor oil (1:4 v/v).
`
`Oestrogenic effects of anti-progestins in vitro
`and in vivo
`
`Oestrogen-Iike activities of onapristone and
`mifepristone
`
`Effects of onapristone and mifepristone on the
`oestrogen-responsive reporter gene Vthk-L UC in
`MVLN cells expressing human endogenous ER
`
`stably transfected with
`(MCF-7 cells
`MVLN cells
`Vitellogenin-Luciferase-reporter gene
`and Neomycin
`
` $6
`
`2K 98 299
`
`Onapristone
`
`0H
`HUGH
`
`-
`
`
`
` O
`
`ZK 135695
`
`
`
`:
`
`"NOH
`
`0
`
`RU 38 486
`
`Mifepristone
`
`/
`
`.1
`
`0
`
`0H 2/°“
`
`2K 98 734
`
`Lilopristone
`
`OH
`
`O
`
`ZK112 993
`
`2K 131 535
`
`138-methyl-substituted
`antiprogestins (type II)
`
`13a-methyl-substituted
`antiprogestins (Type I)
`
`Figure 1. Structure of 1 3ot- (type 1) and 1 3B-methyl substituted (type
`II) antiprogestins used in the experiments.
`
`resistance gene) were cultured in Dulbecco’s modified
`Eagle’s medium (DMEM) without Phenol Red,
`supplemented with 10% fetal calf serum (FCS), 4 mM
`L-glutamine, penicillin,
`and streptomycin. To study
`hormonal effects, MVLN cells were trypsinized, pooled
`and replated onto 96-well dishes at a density of 1.2 \y 104
`cells/well. Cells were cultured in medium supplemented
`with 3% charcoal-stripped FCS in the presence of 10’9 M
`of the anti-oestrogen ICI
`182 780 to reduce high
`background and the appropriate compound. Cells cultured
`in 1% ethanol were used as negative controls for reporter
`gene induction. Transactivation assays were carried out at
`
`least three times. The Luc Assay was performed using the
`Promega kit.
`
`Oestrogenic-like effects of onapristone and mifepristone
`in immature rats
`
`Rats aged 21 days (body weight ~50 g) were randomly
`allocated to nine groups (n = 5/group) and treated so for 3
`consecutive days as follows: group 1, vehicle, group 2,
`oestradiol 0.1 ug/rat, group 3, onapristone 500 ug/rat,
`group 4: ICI 182 780 (ZK 156 901, 500 ug/rat), group 5,
`oestradiol plus ICI 182 780 (0.1 ug/rat and 500 ug/rat
`respectively), group 6, onapristone plus ICI 182 780
`(500 ug/rat each), group 7, onapristone plus oestradiol
`(500 ug/rat and 0.1 ug/rat
`respectively), group 8,
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`mifepristone (RU 486) at 500 ug/rate. Oestradiol doses
`induced a submaximal (50—60%) stimulation of uterine
`
`growth. During autopsy, the uteri were carefiJlly excised
`from surrounding tissue, weighed and placed in Bouin’s
`solution for histology, morphometric
`analysis,
`and
`proliferating cell nuclear antigen (PCNA) staining.
`
`Effects of onapristone and mifepristone on selected
`parameters of oestrogen action in ovariectomized and
`adrenalectomized adult rats
`
`Adult female rats (n = 40, body weight 170—180 g) were
`ovariectomized
`and
`adrenalectomized
`under
`ether
`
`anaesthesia. An additional group of 10 rats remained intact
`until the start of experiment (group 1, intact controls). At 12
`days after surgery the ovariectomized and adrenalectomized
`animals were randomly allocated to four groups and treated
`s.c. for 15 consecutive days with either vehicle (group 2,
`ovariectomized plus adrenalectomized controls), oestradiol
`(0.3 ug/rat, group 3), onapristone, 10 mg/rat (group 4), and
`mifepristone 10 mg/rat (group 5). During autopsy, which
`took place 1 day after cessation of treatment, the uteri and
`vaginae were carefully excised from the surrounding tissue,
`weighed and placed in Bouin’s solution for histological and
`morphometric analysis.
`
`Modulation of oestrogenic effects by onapristone
`and mifepristone
`
`Effects of onapristone on the oestrogen-induced
`expression of c-fos in the rat uterus
`
`Adult female Wistar rats (225—250 g body weight) were
`ovariectomized
`and
`randomly
`allocated
`to
`seven
`experimental groups (six rats per group). At 10 days after
`ovariectomy the animals were treated s.c. as follows: group
`1, vehicle, 0.2 ml, group 2, oestradiol, 3 ug/rat, group 3,
`oestradiol plus progesterone, 3 ug/rat and 3 mg/rat
`respectively, group 4, oestradiol plus onapristone, 3 ug/rat
`and 10 mg/rat respectively, group 5, oestradiol plus
`onapristone plus progesterone, 3 ug/rat, 10 mg/rat, and 3
`mg/rat respectively, group 6, progesterone, 3 mg/rat, group
`7, onapristone, 10 mg/rat. At 2 h after the treatment the
`animals were decapitated and the uteri were removed for
`RNA preparation.
`
`Effects of onapristone and mifepristone on oestrogen
`receptor (ER) protein in the uterus of ovariectomized
`rats
`
`With the exception of group 1 (intact controls, n = 10), all
`other rats were ovariectomized under ether anaesthesia 14
`
`days prior to the experiment, randomly allocated to nine
`groups and treated s.c. for 3 days as follows: group 2,
`vehicle (n = 25), group 3, oestradiol (0.3 ug/rat/day) plus
`
`Modulation of oestrogenic effects by antiprogrestins
`
`573
`
`vehicle (n = 5), group 4, onapristone (10 mg/rat/day) plus
`vehicle (n = 15), group 5, mifepristone (10 mg/rat/day)
`plus vehicle (n = 15), group 6, oestradiol plus onapristone
`(1 mg/rat, n = 10), group 7, oestradiol plus onapristone (3
`mg/rat/day, n = 10), group 8, oestradiol plus onapristone
`(10 mg/rat/day, n = 10), group 9, oestradiol plus
`mifepristone (10 mg/rat/day, n = 15). The animals were
`killed 24 h after the last treatment and the whole uteri were
`removed for ER measurements.
`
`Effects of various type land type II antiprogestins on
`selected parameters of oestrogenic action in
`ovariectomized, oestradiol-substituted rats
`
`Adult Wistar
`
`rats were ovariectomized under ether
`
`anaesthesia at least 11 days before the experiment. The rats
`were then randomly allocated to treatment and control
`groups and treated for 3 consecutive days with a
`
`in
`s.c.)
`(0.3 ug/rat
`substitution dose of oestradiol
`combination with oral treatment of various antiprogestins
`including onapristone, mifepristone (RU 486), lilopristone
`(ZK 95 734) and ZK 122 993 (1, 3, and 10 mg/rat each).
`During autopsy, which was performed ~24 h after the last
`treatment, the uterine wet weights were measured and the
`uteri were fixed in Bouin’s solution for histological and
`morphometric analysis.
`In a separate experiment
`the
`effects of two additional 130c-conf1gurated compounds
`(ZK 131 535, ZK 135 695) were studied. (see Figure 7 for
`details).
`
`Molecular and morphometric analyses
`
`RNA preparation and Northern blot analysis
`
`Total RNA was prepared according to Maniatis et al.
`(1982). Briefly, RNA was extracted from the uteri by
`immediate homogenization in a buffer containing 4 M
`guanidinium thiocyanate using a Polytron homogenizer
`(Kinematic AG, Littau, Switzerland). Uteri from each
`
`group were pooled for the preparation of each RNA
`sample. RNA was purified by ultracentrifugation through
`5 .7 M CsCl dissolved in SET buffer(10 mM Tris—HCl, pH
`7.4) containing 5 mM EDTA, and 0.1% sodium dodecyl
`sulphate) and precipitated with ethanol. Poly (A)+ RNA
`was
`separated
`from total RNA by
`affinity
`chromatography
`on
`oligo(dT)-cellulose
`columns
`(Pharmacia, Freiburg, Germany). Northern blot analysis
`was carried out according to Maniatis et al. (1982),
`applying 5 ug poly(A)+ RNA per lane. Blots were
`hybridized with [32P]-labelled cDNA probes for c-fos
`(Dianova, Hamburg, Germany). To assure that a constant
`amount of RNA was loaded, the blots were re-hybridized
`with the cDNA probe for 1A (subunit of cytochrome C
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`InnoPharma Exhibit 1089.0004
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`

`574 KChwalisz et a].
`
`
`
`900 n
`
`. E2
`I RU 486
`A ZK 98 299
`
`800 —
`
`700 E
`
`600 -
`
`500 a
`
`400 —
`
`countsx10‘3
`
`
`10‘12
`
`10'11
`
`104°
`
`10‘9
`
`10‘8
`
`10‘7
`
`10'6
`moi/l
`
`Figure 2. Effects of onapristone and mifepristone (RU 486) in the
`transactivation assay. MVLN cells expressing endogenous human
`ER and stably transfected with Vthk-LUC were cultured in the ab-
`sence (EtOH control) and presence of increasing amounts of oestra-
`diol (E2), mifepristone (RU 486) and onapristone (ZK 98 299). Note
`the weak oestrogenic activity of mifepristone.
`
`oxidase; M.Lessl, unpublished data) a ubiquitous mRNA
`in the rat uterus whose expression is not regulated by
`steroid hormones.
`
`Assay for total (cytosolic and nuclear) ER
`
`After excision, rat uteri were deep frozen in liquid nitrogen
`and stored at —805C until use. Receptor determination was
`performed essentially as described earlier (Chwalisz et al .,
`1991). Briefly,
`1 g frozen tissue was broken up in a
`microdismembrator (Braun, Melsungen, Germany). The
`powder was suspended (5 ml/g) in homogenization buffer
`(10 mM Tris—HCl, pH 7.4, 1.5 mM EDTA, 0.4 mM KCl, 5
`mM NaMoO4, 10% glycerol) and was homogenized with a
`Polytron homogenizer. The homogenate was incubated at
`05C for 60 min. During incubation the slurry was gently
`stirred. After that the homogenate was centrifiiged for 90
`min at 100 000 g. Aliquots of the supernatant were diluted
`to a protein concentration of 1 mg/ml and were analysed by
`an ER enzyme immunoassay kit from Abbott Laboratories,
`(Chicago, IL, USA) in accordance with the manufacturer’s
`instructions.
`
`Immunohistochemistry
`
`sections were prepared from Bouin-fixed
`Tissue
`paraffin-embedded uterine samples. Proliferation was
`studied by using a monoclonal antibody against PCNA
`and by applying the avidin—biotin—peroxidase (ABC)
`technique. After deparaffmization and washing in
`methanol, the slides were incubated in 3% hydrogen
`peroxide and diluted in methanol for 15 min. Non-specific
`binding of avidin/biotin reagents was prevented by using
`a blocking kit (Vector Laboratories, Burlingame, CA,
`USA) for 20 min. Thereafter the slides were incubated
`
`with the specific mouse monoclonal antibody against
`PCNA (DAKO—PCNA, PC Dako, Glostrup, Denmark),
`diluted 1:100, for 60 min at room temperature. The
`sections were then incubated with a biotinylated sheep
`anti-mouse
`immunoglobulin
`(Ig)G (RPN 1061,
`Amersham Life Science, Braunschweig, Germany,
`diluted 1:300) for 60 min at room temperature, and
`followed by incubation with avidin DH—biotinylated
`horseradish peroxidase H complex (Vectastain Elite ABC
`Kit, Vector Laboratories) for 60 min at room temperature
`in accordance with the manufacturer’s instructions.
`
`the sections were developed in a substrate
`Finally,
`solution of 0.05% diaminobenzidine tetrachloride and
`
`0.01% hydrogen peroxide. Slides were then washed in tap
`water, dehydrated in ethanol, cleared in xylene and
`mounted in DPX. Control sections were prepared by
`substituting the primary antibody with unspecific mouse
`IgG.
`
`Morphometric evaluation
`
`Measurements ofthe luminal epithelial height and luminal
`perimeter were performed on haematoxylin/eosin-stained
`paraffin sections, which were sectioned vertically to the
`longitudinal axis using Axioplan II microscope (Carl Zeiss
`GmbH,
`Jena, Germany)
`and Vidas
`2.0
`(Kontron
`Electronics, Eching, Germany) software. A double-sided
`t—test (on = 0.5) was used for the statistical comparison ofthe
`treatment and the corresponding control groups with
`respect to various parameters.
`
`Oestrogen-like activities of onapristone and
`mifepristone
`
`Effects of onapristone and mifepristone in
`transactivation assays in vitro
`
`To determine the oestrogenic activity of mifepristone and
`onapristone, MVLN cells expressing endogenous ER and
`stably
`transfected with VITtk-LUC were
`used.
`ER—mediated activity was investigated by treating the cells
`
`InnoPharma Exhibit 1089.0005
`
`

`

`Modulation of oestrogenic effects by antiprogrestins
`
`575
`
`relative uterine wet weight
`
`200
`
`130
`
`160
`
`120
`
`3:
`.0
`O)
`8 100
`B:
`E
`
`80
`60
`
`14G
`
`
`with increasing amounts of mifepristone and onapristone
`as illustrated in Figure 2. For a positive control of
`ER—meditated reporter gene induction, cells were treated
`with oestradiol. Almost no induction of reporter gene
`expression was observed after onapristone administration
`up to a concentration of 10’6 M. However, a slight increase
`in reporter gene expression occurred after 10’7 M
`mifepristone with a pronounced effect at 10’6 M.
`
`Oestrogen-Iike effects of onapristone and
`mifepristone in immature rats
`
`This experiment was performed to determine whether the
`antiprogestins onapristone and mifepristone exhibit any
`oestrogenic-like
`activity
`in
`immature
`rats. Both
`onapristone (group 3) and mifepristone (group 8),
`significantly (P < 0.05) increased uterine wet weights and
`reached ~50% of the oestrogen stimulation (group 2)
`(Figure 3,
`lower panel)
`in immature rats. However,
`substantial differences in the effects of onapristone (group
`3) and mifepristone (group 8) were observed on uterine
`epithelial height which is a very sensitive parameter of
`oestrogen action in the rodent uterus. Onapristone, but not
`mifepristone, led to a marked increase in epithelial height
`exceeding even the oestradiol effects by >50%. The
`stimulatory effects of onapristone on epithelial height was
`completely blocked by the pure anti-oestrogen ICI 182 780
`(group 6).
`Table I shows the results of the semiquantitative
`evaluation ofthe proliferation marker PCNA. The staining
`for PCNA was most intensive after oestradiol treatment
`
`and was evident in all uterine compartments (luminal and
`glandular
`epithelium,
`endometrial
`stroma
`and
`myometrium). Interestingly, compared with the vehicle
`control group (group 1), onapristone (group 3) moderately
`increased PCNA staining in the glandular epithelium,
`endometrial stroma and the myometrium, but not in the
`luminal
`epithelium.
`The
`stimulatory
`effects
`of
`mifepristone (group 7) on PCNA staining were less
`pronounced and were restricted to the endometrial stroma
`and the myometrium.
`
`4o
`
`20
`
`o
`
`comml
`
`E2
`
`ONA
`
`lCl
`
`E2+lCl ONA+|Cl EZ+ONA RU486
`
`
`luminal epithelial height
`40 7
`
`35 J
`
`30 7
`
`25 —
`
`g 20 7
`15 7
`
`1o —
`
`5 7
`
`o 7
`
`77
`control
`(1)
`
`E2
`(2)
`
`ONA
`(3)
`
`ICl
`(4)
`
`E2+ICI ONA+ICI E2+ONA RU486
`(5)
`(6)
`(7)
`(8)
`
`group
`
`Figure 3. Effects of onapristone and mifepristone on uterine wet
`weights (upper panel) and luminal epithelial height lower panel in
`immature rats. 21 day old rats were treated s.c. for 3 consecutive days
`with either the vehicle (group 1)‘, 0.1 ug/rat oestradiol (E2, group 2)‘,
`500 ug/rat onapristone (group 3)‘, 500 ug/rat 1C1 182 780 (anti-oes-
`trogen, group 4)‘, oestradiol plus 1C1 182 780 (500 ug/rat each, group
`5)‘, onapristone plus 1C1 182 780 (same doses; group 6)‘, oestradiol
`plus onapristone (group 7, same doses); and mifepristone (RU 486',
`500 ug/rat, group 8). Note a significant (P < 0.05) increase in uterine
`weights after both onapristone (group 3, upper panel) and mifepri-
`stone (group 8, upper panel) treatment, and a marked luminal epithe-
`lial hypertrophy after oestradiol (group 2), onapristone alone (group
`3, lower panel), and onapristone plus oestradiol treatment (group 7,
`lower panel) compared with vehicle-treated control rats (group 1).
`
`Effects of onapristone and mifepristone on selected
`parameters of oestrogen action in ovariectomized
`and adrenalectomized adult rats
`
`The aim of this study was to determine whether the
`antiprogestins onapristone and mifepristone exhibit any
`oestrogen-like
`activities
`in
`ovariectomized
`and
`adrenalectomized rats,
`i.e.
`in the total absence of
`
`In contrast to
`oestrogen and progesterone secretion.
`immature
`rats, neither onapristone (group 4) nor
`
`mifepristone (group 5) increased the uterine wet weights
`or luminal epithelial height (Figure 4) after s.c. treatment
`for 15 days, when compared with the vehicle control
`group (group 2, ovariectomized and adrenalectomized
`rats). There was also no stimulatory effect on vaginal
`weights (Figure 4,
`lower panel). The morphological
`evaluation did not reveal any oestrogenic effect in the
`uterus or vagina after either antiprogestin (data not
`shown).
`
`InnoPharma Exhibit 1089.0006
`
`

`

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`
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`
`
`
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`
`
`
`ins
`
`1A
`
`Figure 5. Effects of onapristone (ONA) and progesterone (P) on the
`oestrogen-induced expression of C—fos in the rat uterus 2 h after treat-
`ments. Adult female ovariectomized rats were treated s.c. with the
`
`vehicle (OVX), oestradiol (E2, 3 pg), 3 ug oestradiol plus 3 mg pro-
`gesterone (E2+P),
`3 ug oestradiol plus 10 mg onapristone
`(E2+ONA)', 3 ug oestradiol plus 10 mg onapristone plus 3 mg pro-
`gesterone (E2+P+ONA)', 3 mg progesterone (P), and 10 mg onapri-
`stone (ONA). Note the inhibition of oestradiol-induced C—fos expres-
`sion by progesterone (E2+P) and a strong expression after a com-
`bined oestradiol plus onapristone treatment (E2+ONA). Note also
`lack of C—fos expression after onapristone alone.
`
`exerted a pronounced stimulatory effect on C—fos
`expression, which was
`substantially
`inhibited
`by
`progesterone and slightly enhanced by onapristone
`(Figure 5). Neither progesterone nor onapristone showed
`any stimulatory effects on C—fOS expression when
`administered alone. However,
`the inhibitory effect of
`progesterone was competely blocked by onapristone.
`
`Effects of onapristone and mifepristone on ER
`protein in the uterus of ovariectomized rats
`
`This experiment was performed to determine whether
`onapristone and mifepristone modulate the synthesis of ER
`in spayed rats. The results of this experiment are presented
`in Figure 6. Relatively high ER levels were measured in
`ovariectomized animals (group 2). Oestrogen treatment
`reduced ER synthesis by ~50% (group 3). Surprisingly,
`onapristone (group 4), but not mifepristone (group 5)
`treatment alone (10 mg/rat/day each) increased ER protein
`levels markedly. The effect ofmifepristone alone (group 5)
`was comparable to that of oestradiol
`(group 3). A
`combined oestradiol plus onapristone (groups 6—8) or
`mifepristone
`treatment
`(group
`9)
`increased ER
`concentrations beyond oestradiol treatment level alone
`(group 3). The effects ofonapristone were dose-dependent.
`
`InnoPharma Exhibit 10890007
`
`576 KChwalisz et a].
`
`relative uterine wet weight *
`
`300
`
`N8mgI100gb.w. 8O o
`
`luminal e ithelial hei ht
`p
`g
`
`100
`80
`60
`40
`
`um
`
`20
`
`00o
`relative vaginal wet weight
`iu
`
`mg/1009b.w.
`
`.#O)OO iii
`No
`
`|
`
`| o
`
`intact
`(1)
`
`OVX+ADX
`(2)
`
`E2
`(3)
`
`ONA
`(4)
`
`RU486
`(5)
`
`group
`
`Figure 4. Effects of onapristone (ONA) and mifepristone (RU) on
`uterine wet weights (upper panel), luminal epithelial height (middle
`panel) and vaginal weights (lower panel) in ovariectomized and adre-
`nalectomized adult rats. The animals were treated s.c. for 15 consecu-
`
`tive days with either the vehicle (OVX+ADX', group 2), oestradiol
`(0.3 ug/rat', group 3), onapristone, (10 mg/rat, group 4), or mifepri-
`stone (10 mg/rat, group 5). The intact animals (intact, group 1) were
`treated with the vehicle). The effects of oestradiol treatment (group 3)
`differed significantly (P < 0.05) from the ovariectomized and adrena-
`lectomized control group (group 2) and were similar to those seen in
`intact controls. Note that neither parameter of oestrogen action was
`significantly (P < 0.05) influenced by the antiprogestins onapristone
`and mifepristone. *Values are significantly different (P < 0.05) from
`ovariectomized and adrenalectomized controls (group 2).
`
`Modulation of oestrogenic effects by
`onapristone and mifepristone
`
`Effects of onapristone on the oestrogen-induced
`expression of c-fos in the rat uterus
`
`The aim of this ex-vivo experiment was to determine
`whether the antiprogestin onapristone modulates the
`oestrogen-dependent C—fOS expression in the presence and
`absence of progesterone treatment. The design of this
`experiment was established in a pilot study in which a
`single injection of oestradiol resulted in the expected
`transient increase in C—fOS peaking 2 h after injection (data
`not shown). Therefore, this particular time period was
`selected for the present experiment. Oestradiol treatment
`
`

`

`Modulation of oestrogenic effects by antiprogrestins
`
`577
`
`Table l. Proliferating cell nuclear antigen (PCNA) staining in the uterus of immature rats treated so. with oestradiol, onapristone
`(ONA), mifepristone (RU 486) and onapristone plus the pure anti-oestrogen ICI 182 780 (ICI)
`
`Treatment
`
`Vehicle
`
`Oestradiol
`
`ONA
`
`Luminal epithelium
`Glandular epithelium
`Stroma
`
`Myometrium
`
`+
`+
`+
`
`+
`
`+++
`+++
`+++
`
`+++
`
`+
`++
`++
`
`++
`
`ICI
`—
`_
`
`_/+
`_/+
`
`Oestradiol + ICI
`—
`_
`
`ONA + ICI
`—
`_
`
`+
`+
`
`+
`+
`
`RU 486
`+
`+
`
`+/++
`+/++
`
`— = no staining; + = low staining; ++ = medium staining; +++ = strong staining.
`
`intact OVX
`
`E2
`
`RU
`
`ONA
`1o
`
`§ .
`
`§ O
`h8ER(fmolmg/protein) M0)88
`
`ONA+E2
`3
`
`1
`
`1a
`
`RU+EZ
`10
`
`mg
`
`Figure 6. Effects of onapristone (ONA) and mifepristone (RU) on
`the total oestrogen receptor (ER) protein concentrations in the uterus
`of ovariectomized, oestradiol (E2)-substituted (0.3 ug/rat/day) rats.
`The ER protein was measured in uterine homogenates using an en-
`zyme immunoassay kit (Abbott). The uteri of each group were
`pooled prior to the homogenization procedure. Note that onapristone
`markedly increased ER concentrations when administered alone,
`and dose-dependently increased ER concentrations in combination
`with oestradiol.
`
`9C,E). Pronounced subnuclear vacuolization was observed
`
`after a combined treatement with