Human Reproduction vol.9 no.11 pp.1991—1996, 1994
`
`The effects of ICI 182,780, a pure anti-oestrogen, on the
`hypothalamic—pituitary—gonadal axis and on
`endometrial proliferation in pre-menopausal women
`
`mice and in cultures of MCF-7 cells (Wakeling et al., 1991).
`It does not demonstrate any oestrogenic activity or effects on
`gonadotrophin secretion in rats (Wakeling et al. , 1991). It shows
`anti-uterotrophic activity in ovariectomized (Dukes et al. , 1992)
`and intact female macaques (Dukes et al. , 1993). This pure anti-
`oestrogenic activity means that it may have clinical potential in
`the treatment of benign and malignant disease that is oestrogen
`dependent in both pre- and post-menopausal women. This paper
`reports the effects of administration of the compound to pre-
`menopausal women on the plasma concentrations of reproductive
`hormones and upon endometrial proliferation.
`
`Materials and methods
`
`Study design
`
`This was an open, randomized, controlled study of seven daily
`i.m.
`injections of ICI 182,780,
`12 mg, compared with no
`treatment, on the plasma concentrations of luteinizing hormone
`
`OH
`
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`
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`
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`E.J.Thomas‘", P.L.Walton2, N.M.Thomasl and
`M.Dowsett3
`
`'Obstetrics and Gynaecology, University of Southampton, Princess
`Anne Hospital, Coxford Road, Southampton $09 4HA,
`2Medical Research Department, Zeneca Pharmaceuticals, Mereside,
`Alderley Park, Macclesfield, Cheshire SK10 4TG and
`3Academic Department of Biochemistry, The Royal Marsden
`Hospital, Fulham Road, London SW3 6]], UK
`
`4To whom correspondence should be addressed
`
`ICI 182,780 has shown pure oestrogen antagonism in vitro
`and in vivo in animals. A total of 17 women with normal
`
`menstrual cycles were administered ICI 182,780, 12 mg daily
`for 7 days in the follicular phase prior to hysterectomy; 11
`normal women were used as controls. Of the 17 patients, three
`(18%) experienced a luteinizing hormone (LH) surge in the
`treatment group compared with five (45%) in the controls
`(P = 0.24), and these patients were only included up to the
`surge. There were no differences in the daily mean plasma
`LH and follicle stimulating hormone concentrations between
`the treatment (n = 17) and control (n = 10) groups. The mean
`plasma oestradiol was higher in the treatment group than
`controls (P < 0.05) on days 5, 6 and 7. However, there was
`no increase in endometrial thickness in the treatment group
`throughout the study. In the control group, endometrial
`thickness increased during the study and was significantly
`higher (P < 0.05) on day 7. There was no ultrasonic evidence
`of ovarian hyperstimulation and no serious adverse events
`reported. This study shows that treatment for 7 days with
`[Cl 182,780 does not cause ovarian hyperstimulation and has
`a potent anti-oestrogenic action on the endometrium. We
`conclude that ICI 182,780 may be a useful compound in the
`treatment of oestrogen-dependent gynaecological disease.
`Key words: anti-oestrogen/endometrium/gonadotrophins/
`oestrogen
`
`Introduction
`
`ICI 182,780 is 7a-[9-(4,4,5,5,5-pentaflu0ro-pentylsulphinyl)
`nonyl]estra—l,3,5(10)-triene-3,l76-diol
`and competes with
`endogenous oestrogen for binding to the oestrogen receptor. It
`is similar to ICI 164,384 (Wakeling and Bowler, 1988) and it
`has been demonstrated to show specific anti-oestrogenic activity
`in vivo in the immature and mature rat (Wakeling et al., 1991).
`The chemical structures of both these compounds are shown in
`Figire 1. It also showed potent anti-tumour activity in vivo in
`xenografts of MCF-7 and Br10 human breast cancers in nude
`
`© Oxford University Press
`
`ICI 182,780
`
`Fig. 1. Structure of ICI 164,384 and [CI 182,780.
`
`1991
`
`InnoPharma Exhibit 1061.0001
`
`
`
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`
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`
`
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`

`

`E.) .Thomas at al.
`
`(LH), follicle stimulating hormone (FSH), oestradiol and sex
`hormone binding globulin (SHBG) and on endometrial thickness
`as determined by ultrasound, in normal pre—menopausal female
`patient volunteers. A total of 30 volunteers were recruited and
`it was planned to randomize with a 2:1 ratio between those
`receiving active agent and controls. Three patients withdrew prior
`to starting the study and were substituted randomly. This resulted
`in 19 patients being randomized to receive ICI 182,780 and 11
`to receive no treatment. All patients started the study between
`day S and day 9 of the menstrual cycle and then underwent
`hysterectomy between days 12 and 18 of the cycle. Plasma was
`sampled on each day of the study for the estimation of the
`concentrations of LH, FSH, oestradiol, progesterone and SHBG.
`Vaginal ultrasound was performed to measure endometrial
`thickness immediately before the study and on the fourth and
`seventh days of treatment. Ethical approval was given by the Joint
`Ethics Committee of Southampton and South-West Hampshire
`Health Authority and Southampton University, and all volunteers
`gave written consent.
`
`Volunteers
`
`Women aged <50 years with menstrual cycles of between 21
`and 42 days who were scheduled for hysterectomy for either
`menorrhagia or fibroids were asked to join the study. All patients
`had had a normal dilatation and curettage, including endometrial
`histology within the past 2 years, had received no exogenous
`steroids for 3 months prior to starting the study and had no severe
`intercurrent illness.
`
`Plasma assays
`
`All samples from a single subject were run in the same assay.
`LH and FSH were measured using the radioimmunoassay
`technique described by Ferguson et al. (1982). For LH, the intra—
`assay coefficient of variation (CV) was 5.8% and the inter-assay
`CV 7.8%. For FSH, the intra-assay CV was 4.2% and the inter-
`assay CV 6.5%. Plasma oestradiol was measured by
`radioimmunoassay [DPC Coat-a-Count; Diagnostic Products
`(UK) Ltd, Abingdon, UK]. Analyses were initially conducted
`on samples
`from three
`treated patients using lipidex
`chromatography separation prior to assay using a system which
`separated ICI 182,780 from oestradiol
`(chloroformzhexane:
`methanol, 50:50: 1). No significant difference was found between
`the results obtained with or without chromatography and the
`analyses were therefore conducted without this step. The intra-
`assay CV was 4.9% and the inter-assay CV 8.8%. Plasma SHBG
`was assayed with the Farmos immunoradiometric assay method
`(Organon Teknika, Cambridge, UK). The intra—assay CV was
`3.2% and the inter-assay CV 5.3%. Plasma progesterone was
`measured with the DPC Coat—a—Count radioimmunoassay.
`
`Vaginal ultrasound
`
`Vaginal ultrasonography was performed with an Ultramark 4
`ultrasound scanner with a 5 MHZ probe (ATL, Stevenage, UK).
`Endometrial thickness was measured three times on each occasion
`and the mean value calculated. The measurements were taken
`
`from the external edge of each echogenic interface between the
`endometrium and the myometrium (Santolaya, 1992). Follicular
`volume was measured using the average of three diameters
`1 992
`
`(Thomas et al. , 1986). The ultrasonographer was unaware of
`the allocation to either treatment or control.
`
`Data analysis
`The endocrine data were not assumed to have a normal
`
`distribution and have been logarithmically transformed for
`analysis, as this has been shown to normalize the distribution
`of the plasma concentrations of oestradiol and gonadotrophins
`(Kletzky et al., 1975). Comparisons within groups were
`performed with repeated analysis of variance (ANOVA) and
`between groups with multivariate analysis. For clarity, the data
`are presented in Figures 2 and 3 as arithmetic means and standard
`deviation. Endometrial thickness was assumed to have a normal
`
`distribution and ANOVA was used to compare the two groups.
`
`Results
`
`All 30 volunteers completed the study. The mean :1: SD age for
`those randomized to treatment was 41.5 :1: 4.2 years, and this
`was not significantly different from that of 43 :1: 3.3 years for
`those in the control group. There was also no significant
`difference in the mean :t: SD weight in the treatment (66.7 :l:
`5.4 kg) compared with the control group (64.3 :t: 7.1 kg). The
`median for the day of the cycle on which the study commenced
`was 7 (range 5—9) in the control group and also day 7 (range
`6—8) in the treatment group. Analysis of the ultrasound and
`endocrine results showed that one volunteer in the treatment group
`had an unluteinized follicular cyst at the start of the study and
`another had elevated plasma LH and FSH with a static oestradiol
`and a low plasma progesterone concentration, suggesting incipient
`ovarian failure. These two were removed from further analyses.
`This left 17 volunteers randomized to receive treatment and 11
`
`controls. There were no serious adverse events or significant side-
`effects reported in the treatment group. Hysterectomy was
`performed within 4 days of stopping the treatment.
`
`LH surge
`
`Three patients in the treatment group (18%) and five (45%) in
`the control group experienced an LH surge during the 7—day
`period of the study, verified by plasma LH concentrations and
`raised plasma progesterone concentrations in the subsequent days
`(P = 0.24, Fisher’s Exact test). The data for each individual
`in each group are displayed in Figure 2. In the treatment group,
`the LH surges occurred on days 2 and 3 of the treatment period,
`with none appearing after that. In the control group, one surge
`appeared on day 1, one on day 3, one on day 4 and two on day
`5 of the study. Because the LH surge induces such a large
`transformation in the function of the follicle we have eliminated
`
`the data from the beginning of the surge onwards from further
`analysis so that all comparisons are in the follicular phase of the
`cycle. This means that, in the treatment group, 17 patients were
`evaluated on day l, 15 on day 2 and 14 from then onwards. In
`the control group, because one patient had started the surge on
`day 1, this left 10 evaluable patients on days 1 and 2, eight on
`days 3 and 4 and six from then onwards.
`
`Plasma LH, FSH and oestradiol
`
`Figure 3 compares the geometric mean concentrations of plasma
`LH, FSH and oestradiol
`in the treatment group against
`the
`
`
`
`
`
`
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`InnoPharma Exhibit 1061.0002
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`|C|182J80
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`CONTROL
`
`1C1 182,780 in pre—menopausal women
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`PLASMAPROGESTERONENMOL\L
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`PLASMAOESTRADIOLPMOL\L
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`DAYS OF TREATMENT
`
`Fig. 2. Comparison of the individual results for the concentration of plasma LH, oestradiol and progesterone in those volunteers who
`experienced an LH surge in the treatment (n = 3) and control (n = 5) groups.
`
`controls. Using multivariate analysis, there were no statistically
`significant differences in the concentrations of LH or FSH
`between the two groups for any of the days of the study. Using
`repeated ANOVA there was no difference within the groups in
`the plasma concentration of LH throughout the study. However,
`within both groups 016 decrease in plasma FSH concentration
`over the study was statistically significant (treatment group:
`P = 0.01; control group: P = 0.04). Plasma oestradiol
`concentrations significantly increased in both groups (treatment:
`P < 0.0001; control: P < 0.05, using ANOVA) throughout
`the treatment period. Oestradiol concentrations were significantly
`higher on days 5, 6 and 7 in the treatment group compared to
`controls (P < 0.02, multivariate ANOVA). The median (range)
`of plasma oestradiol in the treatment group was 919 (689— 1661)
`pmol/l on day 6 and l 140 (965-— 1801) pmol/l on day 7. Plasma
`
`oestradiol was higher on day 7 than on day 6 in 12 of the treatment
`group and five of the controls, suggesting that follicular growth
`was continuing in the majority of volunteers. If all the values
`for plasma oestradiol are summed to produce an area under the
`curve, then the distribution is significantly higher in the treatment
`group (P < 0.00001, Wilcoxon) than in the controls.
`
`Endometn'al thickness
`
`Figure 3 compares the mean endometrial mickness in millimetres
`for the treatment group versus controls. The mean i SD
`endometrial thickness in the control group was 7.2 :t 1.7 mm
`on day 1, 8.2 :1: 1.3 mm on day 3 and 9.7 :1: 1.2 mm on day
`7. For the treatment group the values for the same days are 6.2 :1:
`2, 6.4 :e 1.3 and 6.7 :l: 2.1 mm respectively. There is no
`significant increase in thickness in the treatment group, but there
`1 993
`
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`
`Fig. 3. Comparison of the mean (iSD) plasma concentrations of LH, FSH, oestradiol and the endometrial thickness of the treatment group
`(0, n = 17) and the controls (V, n = 10).
`
`is a significant increase in the control group (P < 0.03, ANOVA)
`throughout the study. The thickness on day 7 is significantly less
`in the treated volunteers than the controls (P < 0.05, multivariate
`ANOVA) in spite of the plasma oestradiol concentration being
`higher.
`
`Follicular growth
`
`Only four of the women in the control group had interpretable
`ultrasonic determination of follicular growth, and therefore a
`comparison between the two groups was not possible. In the
`treatment group there was no evidence of multiple folliculogenesis
`during the administration of the drug. The mean :t SD follicular
`volume was 0.9 :l: 0.2 ml on day l, 1.2 :l: 0.5 ml on day 4
`and 1.9 :l: 0.5 ml on day 7, and these increases were significant
`(P < 0.0001, ANOVA). In 12 of the 14 women exposed to ICI
`182,730 there was an increase in follicular volume on day 7 in
`comparison to day 4.
`
`SHBG
`
`in the mean plasma SHBG
`There were no differences
`concentrations for any day in the treatment group compared with
`controls.
`
`1 994
`
`Discussion
`
`This study reports on the administration of a pure anti-oestrogen
`to women with regular menstrual cycles. The results showed that
`after 7 days administration of ICI 182,780 there was no detectable
`increase of LH and FSH secretion and no ultrasonic evidence
`
`of ovarian hyperstimulation. It appears that the LH surge may
`be suppressed by the anti-oestrogen after 3 days of treatment and
`that follicular growth continues. In spite of the continued secretion
`of oestradiol
`there is no increase in endometrial
`thickness,
`demonstrating the powerful oestrogen antagonism of
`the
`compound.
`The exact mechanism of action of ICI [82,780 is not clear.
`It binds to the oestrogen receptor with an affinity of 0.89
`compared to that of oestradiol. This is greater than the 0.19
`affinity of ICI 164,384, which is another example of this series
`of compounds on which many non—clinical studies have reported,
`confirming pure oestrogen antagonism (Wakeling et al., 1991).
`Investigations with ICI 164,384 have shown that
`it prevents
`binding of the occupied receptor to DNA cellulose (Wilson et
`al. , 1990). In transient-transfection studies, the addition of the
`antibody MP16 restored the ability of receptors occupied by ICI
`164,384 to bind to DNA. This antibody restores high-affinity
`binding to DNA to mutant receptors that are defective for
`
`InnoPharma Exhibit 1061.0004
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`

`

`dimerlzation, and it was concluded that these antagonism of
`oestradiol prevented receptor dimerization. This hypothesis was
`given further support by the report that ICI 164,384 causes a
`rapid loss of oestrogen receptor expression in the mouse uterus
`(Gibson et al. , 1991), and it was also shown to decrease the half—
`life of the oestrogen receptor from ~ 5 to < l h (Dauvois et al.,
`1992), The authors concluded that this decrease in half-life was
`probably a result of the inability of the occupied receptors to form
`homodimers. This inability is likely to prevent binding to DNA
`and thus it was postulated that it was through this mechanism
`that the anti—oestrogenic activity was propagated (Fawell et al.,
`1990).
`In the treatment group there were no LH surges after the first
`3 days. This was different from the control group in which there
`were surges on days 4 and 5. Temporally, it would be expected
`that LH surges should occur with increasing frequency as the
`follicular phase develops, and this suggests that the use of ICI
`182,780 may suppress the LH surge if it has not already been
`initiated by the beginning of treatment. However, this study only
`extended to day 16 of the menstrual cycle, and it is possible that
`a number of LH surges could have occurred spontaneously after
`that time. Therefore, the possibility that ICI 182,780 suppresses
`the LH surge requires more detailed study. There was no increase
`in LH or FSH secretion as a result of administration of ICI
`
`182,780, and the pattern of decreasing FSH secretion was as
`expected for a normal follicular phase of the menstrual cycle for
`both groups (Thomas et al. , 1986). This was unexpected, as we
`had postulated that as the compound was a potent anti-oestrogen,
`there would be an increase in LH and FSH secretion similar to
`
`that seen post-menopausally.
`There are no other studies reporting the result of the,
`administration of pure anti-oestrogens to women with which to
`compare these results.
`In the human,
`tamoxifen does not
`necessarily stimulate LH and FSH secretion,
`the response
`appearing to be variable (Tajima, 1984; Tajima and Fukushima,
`1983). Clomiphene citrate does appear to increase LH and FSH
`secretion in the human (Martikainen et a1. , 1991), although these
`responses can be variable (Randall and Templeton, 1991). The
`variability for tamoxifen and Clomiphene could be explained by
`them being both oestrogenic and anti-oestrogenic. However, ICI
`182,780 is a pure anti-oestrogen and we have no obvious
`explanation why it did not cause an increase in gonadotrophin
`secretion. It is possible that it takes longer than 7 days before
`this effect becomes apparent. Another explanation is that ICI
`182,780 does not act centrally, at least in this treatment schedule,
`although this would contradict our hypothesis that it may suppress
`the LH surge. It is possible, however, that the compound may
`act by controlling the stimulus for the surge of LH at the pituitary
`rather than the hypothalamic level. This possibility would not
`then conflict with the lack of central action on the tonic release
`of LH and FSH. More detailed studies are needed and ICI
`
`182,780 provides a useful experimental tool for the investigation
`of the role of oestradiol in the control of LH and FSH secretion.
`
`Follicular growth continued in spite of exposure to ICI 182,780.
`There was no luteinization of these follicles as determined by
`plasma progesterone concentrations, but this may also have been
`because no patient was studied beyond day 16 of the cycle. The
`plasma oestradiol concentration was higher in the treatment group
`
`[Cl 182,780 in pre-menopausal women
`
`and this did not appear to be mediated by increased gonadotrophin
`stimulation. An explanation for this may have been that there
`was change in the ratio of bioactive to immunoactive FSH as
`a result of ICI 182,780. It also is possible that the anti-oestrogenic
`effect of the compound alters granulosa and thecal cell function,
`leading to an increased secretion of oestradiol. Oestradiol has
`been reported as increasing FSH-stimulated steroidogenesis in
`cultured marmoset granulosa cells (Shaw and Hodges, 1992).
`However, this observation, while verifying that oestradiol may
`have paracrine actions, does not help to explain how ICI 182,780
`may alter oestradiol secretion by granulosa cells. It may also be
`that there were some smaller follicles that were also contributing
`to the secretion of oestradiol that were not consistently identified
`using ultrasound. Because this study showed that follicular growth
`was continuing in 12 of the 14 volunteers given ICI 182,780,
`longer studies are needed to observe whether the follicles continue
`to grow or whether they become atretic. Investigation of the fate
`of the follicles in the treatment group over 4-5 weeks will be
`vital for determining the long-term use of the compound.
`Continued follicular growth or the initiation of a follicular cyst
`will limit its therapeutic value. On the other hand, atresia of the
`follicle with no fimher stimulation of folliculogenesis will provide
`a suitable environment for long-term use.
`The data showed that,
`in spite of continued oestradiol
`stimulation, there was no significant endometrial growth in the
`ICI 182,780 group. The rate of growth in the control group was
`similar to that reported in normal women in the follicular phase
`(Bakos et a1. , 1993). This verifies a potent anti-oestrogenic
`activity in vivo and agrees with the data of Dukes er a1. (1992,
`1993), who showed no growth of the endometrium in both the
`intact and the ovariectomized monkey. This observation provides
`support
`for the use of [CI 182,780 to treat disorders of
`endometrial proliferation,
`such as endometrial carcinoma,
`endometriosis and dysfunctional uterine bleeding. Its use in the
`treatment of these disorders will depend on the side-effects
`encountered in the long term, specifically, the occurrence of hot
`flushes and bone demineralization, as well as acceptable effects
`on folliculogenesis.
`In conclusion, this study demonstrated that ICI 182,780 is well
`tolerated during short-term use. It did not cause an increase in
`LH or FSH secretion and may suppress the LH surge. There
`was no evidence of ovarian hyperstimulation although follicular
`growth continued. There appeared to be a potent anti-oestrogenic
`effect on the endometrium in vivo.
`
`Acknowledgement
`
`We are grateful for the laboratory assistance of Miss Anita Smith and
`Mrs Debbie Doody and for the help of the medical staff of the Princess
`Anne Hospital.
`
`References
`
`Bakos,0., Lundkvist,0. and Bergh, T. (1993) Transvaginal sonograph’ic
`evaluation of endometrial growth and texture in spontaneous ovulatory
`cycles—a descriptive study. Hum. Reprod., 8, 799—806.
`(1992)
`Dauvois,S., Danielian,P.S., White,R.
`and Parker,M.G.
`Antiestrogen 1C1 164,384 reduces cellular estrogen receptor content
`by increasing its
`turnover. Proc. Natl. Acad. Sci. USA, 89,
`4037—4041.
`
`1995
`
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`gmz‘11Joqmaooq110AltswAtunsutqdoHSLILIOf1'2/310'S{121H‘t10[pJOJXO'dQILIInq/flduq{not}papeommoq
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`InnoPharma Exhibit 1061.0005
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`

`E.J.Thomas er al.
`
`Dukes,M., Miller,D., Wakeling,A.E. and Waterton,J.C. (1992) Anti-
`uterotrophic effects of a pure anti-oestrogen, 1C1 182,780: magnetic
`resonance imaging of the uterus in ovariectomised monkeys. J.
`Enclacrinol.. 135, 239—247.
`Dukes,M., Watenon.J.C. and Wakeling,A.E. (1993) Antiutertrophic
`effects of the pure anti-oestrogen, 1C1 182,780,
`in adult female
`monkeys: quantitative magnetic resonance imaging. J. Endocrinol.,
`138, 203 —210.
`and
`Fawe11,S.E., White,R., Hoare,S., Sydenham,M.. Page,M.
`Parker,M.G. (1990) Inhibition of estrogen receptor—DNA binding
`by the ‘pure‘ antiestrogen ICI 164,384 appears to be mediated by
`impaired receptor dimerization. Proc. Natl. Acad. Sci. USA, 87,
`6883—6887.
`
`Fergison.1(.. Hayes.M. and Jeffcoate,S.L. (1982) Astandardised multi-
`centre procedure for plasma gonadotrophin radioimmunoassay. Arm.
`Biochem, 19, 358.
`Gibson,M.K., Nemmers,L.A., Beckman,W.C., Jr., Davis,V.L.,
`Curtis,S.W. and Korach,1(.S. (1991) The mechanism ofICI 164,384
`antiestrogenicity involves the rapid loss of estrogen receptor in uterine
`tissue. Endocrinology, 129, 2000—2010.
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`
`Received on April 27, 1994; accepted on July 12, 1994
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`1996
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