The Breast (1996) 5, 186-191
`© 1996 Pearson Professional Ltd
`
`ESO TASK FORCE ARTICLE
`
`Models of new antioestrogen action in vivo: primary tumours
`
`E. Anderson, R. Nicholson*, M. Dowsettl and A. Howelli
`
`Clinical Research and iMedical Oncology Departments, Christie Hospital NHS Trust, Manchester, *Tenovus Cancer
`Research Centre, Cardifi”, 7Department ofAcademic Biochemistry, Royal Marsden Hospital, London, UK
`
`S U M MA R Y. We have conducted a clinical trial of the novel pure antioestrogen ICI 182780 to assess its short-term
`biological effects in women with primary breast cancer. The results were compared against those obtained from a similar
`study of tamoxifen. In both studies, the drugs were administered for a short period of time in the interval between first clinic
`attendance and operation. Samples of tumour tissue were obtained both before and after treatment with the antioestrogens.
`Seven days treatment with ICI 182780 caused a significant decrease in tumour proliferation as indicated by the Ki67
`labelling index (Ki67LI). Tamoxifen caused a similar reduction in the Ki67LI after a median of 21 days treatment. In
`oestrogen receptor (ER) positive breast tumours, ICI 182780 caused a profound decrease in the level of receptor protein that
`could be detected immunocytochemically whereas tamoxifen was without effect. This reduction in ER-protein content was
`not reflected in a similar decrease in the mRNA for the receptor. ICI 182780 significantly reduced the expression of two
`oestrogen—regulated genes (the progesterone receptor and pS2) whereas tamoxifen was without effect. Finally, although ICI
`182780 reduced ER expression to almost undetectable levels in some tumours, no other changes suggestive of an endocrine
`insensitive phenotype were apparent.
`In conclusion, ICI 1827 80 produces demonstrable antioestrogenic effects on human primary breast tumours in vivo and is
`without any oestrogen agonist effects. The novel mechanism of action of the new pure antioestrogen, as determined in vitro,
`is reflected in its effects on human primary breast tumours in vivo.
`
`INTRODUCTION
`
`in animals and adjuvant use in women with breast cancer
`is associated with an increased risk of endometrial cancer
`
`the first phase I study of the new specific
`In 1991,
`antioestrogen ICI 182780 in primary breast cancer patients
`was initiated. This trial followed several years developing
`new agents that would be more efficacious than conven-
`tional antioestrogens and with fewer side-effects.
`Conventional
`triphenylethylene-based
`antioestrogens,
`typified by tamoxifen, compete with oestradiol
`(E2) for
`binding to the ER but they also form a complex with the
`receptor that retains some transcriptional activity.‘ Con-
`sequently,
`tamoxifen exhibits a full range of biological
`activity from full oestrogen antagonism to full agonism de-
`pending upon the species, the target tissue and the target
`gene response being studied. Although some of the agonist
`effects of tamoxifen such as reduction in serum cholesterol
`levels and maintenance of bone mineral density are benefi-
`cial, others may be detrimental to patients receiving long-
`term therapy. Tamoxifen stimulates endometrial growth
`
`in some, but not all, studies.“ Moreover, evidence from
`animal studies and from observations of withdrawal re-
`sponses suggests that the agonist properties of tamoxifen
`may stimulate tumour cell growth and is the cause of some
`treatment failures.“ It
`is possible that
`the efficacy of
`tamoxifen and the other conventional non-steroidal anti-
`oestrogens may be compromised compared to that which
`might be achieved by complete oestrogen antagonism.
`The new class of steroidal antioestrogens, exemplified by
`ICI 164384 and ICI 182780, differ significantly from con-
`ventional agents in both their chemical structure and their
`molecular pharmacology. Both ICI 164384 and ICI 182780
`are derived from E2 substituted at the 70¢ position with an
`alkylarnide or an alkylsulfinyl moiety respectively.7 Both
`ICI 164384 and ICI 182780 bind to the ER with high
`affinity and are complete antagonists in the rat uterus
`oestrogen bioassay. Co—administration of either of the new
`compounds completely inhibits the uterotrophic effects of
` tamoxifen in a dose—dependent manner.7 However, the com-
`Christie Hospital NHS Trust, Wilmslow Road, Manchester M20 4BX, UK
`pound designated ICI 182780 has been chosen for further
`
`186
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`development because of its greater potency and bioavai—
`labilityf‘ ICI 182780 shows a lack of agonist activity in
`several animal model systems including carcinogen—induced
`rat mammary tumours and human breast cancer cells grown
`as xenografts in athymic nude mice. Further studies have
`indicated that both ICI 182780 and ICI 164384 are more
`
`potent and effective than tamoxifen in promoting remissions
`in several models of human breast cancer. Importantly, the
`specific antioestrogens have growth inhibitory effects on
`tumour cells that are resistant to tamoxifen or whose growth
`is stimulated by the non—steroidal antioestrogen.”
`The lack of agonist activity of the new specific anti-
`oestrogens may be related to their mechanism of action
`which is completely different to that of tamoxifen. The
`major effect of ICI 164384 and ICI 182780 is to reduce
`substantially the cellular content of the ER by decreasing
`the half—life of the protein.‘°~“ This reduction in half—life has
`been attributed to inhibition of the energy—dependent nucle-
`ocytoplasmic shuttle of the ER between the cytoplasm and
`the nucleus which blocks re-entry of the receptor into the
`nucleus and promotes its degradation.“ However, dimeri—
`zation of the ER and consequent binding to the ER element
`(ERE) may also be destabilized which further enhances
`receptor degradation.”
`As administration of ICI 182780 in animal studies was
`
`associated with little or no toxicity and as the compound
`appeared to be highly active in several experimental systems,
`a phase I study in primary breast cancer patients was carried
`out in three centres (Manchester, Nottingham and London).
`The aims of the study were three—fold: 1. to determine the
`pharmacokinetics and short—term tolerability of ICI 182780;
`2.
`to discover whether there was evidence of biological
`activity in human primary breast tumours in vivo and; 3.
`to compare the effects of the new compound with those of
`tamoxifen administered in a similar short—terrn protocol.
`The clinical aspects of this study are reported elsewhere.”
`The aim of this report is to summarize the biological actions
`of ICI 182780 on human primary breast
`tumours with
`particular reference as to whether ICI 182780 exerts any
`agonist effects and whether the drug’s novel mechanism of
`action is reflected in its effects in vivo.
`
`THE EFFECTS OF ICI 182780 ON PRIMARY
`BREAST TUMOURS
`
`Fifty—six postmenopausal women with primary breast can-
`cer satisfied the entry criteria [see 13 for details] and partici-
`pated in the study. After giving informed consent,
`the
`patients were randomized to either a control group (n = 19)
`who received no pre—operative treatment, a low dose treat-
`ment group (n = 21) who received 6 mg ICI 182780 per day
`and a high dose treatment group (n = 16) who were treated
`
`Models of new antioestrogen action in vivo
`
`187
`
`with 18 mg ICI 182780 per day. Both the low and the high
`dose treatment groups were given daily i.m. injections of
`ICI 182780 for 7 days prior to surgery. Wherever possible,
`pre—treatment tumour samples were obtained by multiple
`needle core biopsies. Post—treatment samples were obtained
`from the operative specimen at the time of surgery. The
`tumour samples were divided and portions were snap frozen
`in liquid nitrogen for steroid receptor measurement, assay
`of the proliferation-associated antigen Ki67 and mRNA
`extraction. The other portions were fixed in formalin and
`embedded in paraffin wax for subsequent histological
`examination and assay of oestrogen—regulated genes such
`as pS2. All the immunocytochernical assays were carried
`out as previously detailed” and were scored blinded as to
`whether the samples came from treated or control patients.
`The tamoxifen study against which the effects of ICI
`182780 were compared, was carried out on 103 patients
`who presented at the University Hospital of South Man-
`chester with primary breast cancer.” After giving informed
`consent,
`the patients were randomized to receive either
`placebo (n = 44) or tamoxifen (n = 59) at a loading dose of
`4 X 40 mg for 1 day and then 20 mg per day thereafter in
`the interval between clinic attendance and surgery (median
`time = 21 days; range = 6-65 days). Pre- and post—treatment
`tumour samples were obtained and processed as described
`above for the ICI 182780 study. The tamoxifen protocol
`differed from that used for the study of ICI 182780 in that
`the patients received the drug for a variable period of time.
`In addition, tumour ER and PR content in the tamoxifen
`
`study was calculated as the percentage of tumour cells stained
`positively for the receptor whereas, in the ICI 182780 study,
`receptor measurements were presented as an index which
`combined the percentage of positively stained cells with the
`intensity of staining. The proportion of receptor positive
`tumours identified by each of these methods was not signifi-
`cantly different. Moreover, the changes in the percentage
`of receptor positive cells after antioestrogen treatment
`reflected those of the receptor indices.
`We looked first at the effects of the new antioestrogen
`on tumour cell proliferation as
`indicated by immun-
`ocytochemical detection of the Ki67 proliferation associated
`antigen. Paired samples were available for estimation of the
`Ki67 labelling index (% tumour cells positively stained with
`the Ki67 antibody) in 44 of the 56 subjects in the phase I
`study of ICI 182780. There were no significant differences
`in the pre—treatment Ki67 is between ICI 182780 treated and
`control tumours.” The pure antioestrogen had no effect on
`proliferative activity of ER-negative tumours but, as shown
`in Figure 1,
`it reduced significantly the Ki67LI of ER-
`positive tumours. There was evidence that this effect was
`dose dependent as the decrease in Ki67LI was significant
`only in those treated with the higher (18 mg) dose of ICI
`182780. A similar reduction in Ki67LI was seen after
`
`|nnoPharma Exhibit 1049.0002
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`

`188 The Breast
`
`10
`
`O
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`5
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`
`Ki67LI(%cellsstained)
`
`um
`
`Pre
`
`Post
`
`ICI 182780
`
`Pre
`
`Post
`
`Tamoxifen
`
`Pre— and post-treatrnent Ki67LIs (% tumour cells showing
`Fig. 1
`positive immunostaining) in patients with ER-positive tumours neated
`with ICI 182780 (both doses have been combined) or all patients treated
`with tamoxifen. Columns = interquartile ranges; bars: medians;
`* = P < 0.05 and *** = P < 0.001 by Wilcoxon’s matched-pair signed
`rank test.
`
`tamoxifen treatment (see Fig. 1) except anti-proliferative
`effects were seen in both ER-positive and ER-negative
`tumours (as determined in the pre-treatment sample). This
`is the first demonstration of a reduction in proliferative
`activity of human breast tumours treated with ICI 182780
`in vivo. The absence of a similar effect on ER-negative
`tumours treated with ICI 182780 or on untreated controls
`
`suggests that this reduction was drug related and ER de-
`pendent. In addition, the reduction in Ki67LI appeared to
`be dose dependent because a significant decrease could only
`be demonstrated in the patients treated with the higher
`(18 mg) dose. This may have been due, in part, to the small
`number of patients in the 6 mg dose group who had ER-
`positive tumours as previous studies of ICI 182780 have
`shown clear dose-dependent anti-proliferative effects on
`human breast cancer cell lines in vitro. The effects of ICI
`
`182780 on proliferation were achieved with just 7 days’
`treatment and at serum levels of 5-25 ng/ml” which are
`much lower than the steady state levels reported for tarno—
`xifen and its metabolites (250—450 ng/ml).‘5 Thus, it ap-
`pears that in Vivo, as in vitro, the new pure antioestrogen is
`more potently anti-proliferative than tamoxifen.
`Paired pre- and post-study steroid receptor measurements
`were carried out on 45 (80%) of the 56 participants in the
`ICI 182780 trial. In this study, steroid receptor expression
`was assessed semiquantitatively by determining the per-
`centage of positively stained tumour cells and assessing
`
`the intensity of staining to produce an ER or PR index. In
`the control tumours, we noted that there was a significant
`tendency towards under—estimation of the ER level in the
`pre-treatment biopsy specimens compared to that seen in
`the operative sample. This difference is probably due to
`the difficulties in preserving the receptor content of small
`Tru-cut biopsy samples. Nevertheless, ICI 182780 treat-
`ment caused a profound decrease in the ER index of the
`tumours in a dose dependent manner (Fig. 2A & B). This
`decrease in receptor content was such that some, initially
`ER-positive, tumours appeared ER-negative after treatment
`with the pure antioestrogen. Although it had pronounced
`effects on ER-protein expression, ICI 182780 did not alter
`the amount of ER mRNA that could be extracted from tu-
`
`mours after treatment as measured by Northern analysis
`(Fig. 2C).“ Tamoxifen treatment did not affect either re-
`ceptor protein or mRNA expression in ER-positive breast
`tumours (Fig. 2). The lack of effect of ICI 182780 on
`ER mRNA expression is consistent with its proposed
`mechanism of action where destabilization of the ER dimer,
`
`enhanced ER degradation and a reduced half-life of the ER
`obliterate ER protein content without changing ER mRNA
`transcription.““2 Thus, it appears that at least some of the
`changes described after ICI 182780 treatment in vitro also
`occur in human primary breast tumours in vivo.
`Comparison of the changes in oestrogen—regulated gene
`expression caused by ICI 182780 with those induced by
`tamoxifen suggests that the new drug exerts a greater anti-
`oestrogenic effect in vivo. In the patients treated with ICI
`182780, there was a significant reduction in the median PR
`index from 0.5 before treatment
`to 0.01 afterwards
`
`(P<0.05; Fig. 3A). There was no evidence of a dose-
`dependent effect as separate analysis of the two doses of ICI
`182780 did not reach statistical significance. In contrast,
`tamoxifen increased the PR content of some ER-positive
`tumours although, for the group as a whole, this increase
`was not significant (Fig. 3A). The expression of another
`oestrogen_regulated gene, pS2, was also reduced by ICI
`182780 at both the protein and the mRNA levels (Fig. 3B
`& C). As in the case of PR, tamoxifen treatment increased
`
`pS2 expression in some primary breast tumours although
`overall this alteration was not significant. The effects of ICI
`182780 on a third oestrogen—regulated gene pLIV—1 were
`rather less pronounced than its effects on PR or pS2 al-
`though there was a trend for pLIV—1 mRNA expression to
`be reduced after treatment with the pure antioestrogen (data
`not shown).
`As both the PR and pS2 genes are regulated by
`oestrogen,"’“‘ the levels of their respective protein products
`would be expected to fall
`in the presence of oestrogen
`antagonism. This is, indeed, the case in the present study
`of ICI 182780 where the decrease in the levels of PR
`
`and pS2 expression is consistent with the formation of a
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`|nnoPharma Exhibit 10490003
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`Models of new antioestrogen action in vivo
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`
`(A) Pre- and post-treatment PR measurements in patients with
`Fig. 3
`ER-positive tumours treated with ICI 182780 or tamoxifen; * = P < 0.05
`and nsd = no significant difference by Wilcoxon’s matched-pair signed-
`rank test; (B) Pre— and post-treatment pS2 measurements in patients
`treated with ICI 182780 or tamoxifen; * = P < 0.05 and nsd = no
`significant difference by Wilcoxon’s test; (C) levels of pS2 mRNA
`measured following no treatment — control, low and high dose ICI
`182780 (182 low and 182 high respectively) or tamoxifen (Tarn)
`treatment; * = P < 0.05 and nsd = no significant difference by Mann-
`Whitney U test. Columns = interquartile ranges; bars = medians.
`
`of PR or pS2 and the rather small effects of ICI 182780
`on expression of its mRNA are not surprising. Tamoxifen’s
`effects on PR and pS2 expression are entirely consistent
`with its oestrogen agonist properties. This agonism appears
`to be due to the failure of tamoxifen to block all of the
`
`transcription activating functions of the ER. However, there
`is also evidence to suggest
`that
`the agonist activity of
`tamoxifen could be enhanced further by the interaction of
`other signal
`transduction pathways with the tamoxifen:
`ER complex.” As the pure antioestrogens obliterate cellular
`ER protein content, it has been suggested that opportunities
`for this type of interaction should not occur resulting in
`a more complete oestrogen antagonism?“ Taken together,
`the differential actions of ICI 182780 and tamoxifen on
`
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`
`(A) Pre— and post—treatment ER measurements in patients with
`Fig. 2
`ER—positive tumours treated with ICI 182780 or tamoxifen; *** =
`P < 0.001 and nsd: no significant difference by Wilcoxon’s matched-pair
`signed-rank test; (B) percentage change in ER protein content following
`no treatment — control, low and high dose ICI 182780 (182 low and 182
`high respectively) or tamoxifen (Tam) treatment; *** = P < 0.001 and
`nsd = no significant difference by Mann—Whitney U test; (C) levels of
`ER mRNA measured after treatment with ICI 182780 or tamoxifen; nsd
`= no significant difference by Mann—Whitney U test. Columns =
`interquartile ranges; bars = medians.
`
`transcriptionally inactive ICI l82780:ER complex and sub-
`sequent down—regulation of the ER protein.'°“2 We did find,
`however,
`that pS2 was expressed in some ER—negative
`tumours and that
`its expression was decreased in these
`tumours as well as in those that were ER-positive. This lack
`of association with ER positivity may be due to the fact that
`analysis of pS2 expression was carried out on a separate
`piece of tumour tissue from that used for estimation of
`steroid receptor expression and proliferation. The control
`of pLIV—l gene expression is rather more complex than that
`
`

`

`190 The Breast
`
`oestrogen—regulated gene expression in human primary
`breast tumours in vivo, suggest that the new drug is more
`effective and does not have the agonist effects associated
`with conventional antioestrogens.
`The final issue that we wished to address in this phase I
`study of ICI 182780 was whether the decrease in tumour ER
`content was accompanied by other changes suggestive of
`the development of an endocrine insensitive phenotype.
`This question was raised by the finding that there is a
`strict inverse relationship between ER and expression of the
`epidermal growth factor receptor (EGFR) in clinical breast
`tumour samples.“ Those tumours containing EGFR or its
`ligand transforming growth factor on (TGFOL) are frequently
`unresponsive to endocrine therapyfm Furthermore, sup-
`pression of ER expression by other agents such as sodium
`butyrate or phorbol ester results in a concomitant rise in
`tumour cell EGFR content.”-24 Accordingly, the expression
`of EGFR and TGFOL was determined immunocytochemically
`on a subset of tumour samples taken before and after treat-
`ment with ICI 182780.25 Figure 4 shows that neither dose of
`the pure antioestrogen changed the EGFR index even
`though ER expression was reduced in all cases. Likewise,
`no effects of ICI 182780 on expression of TGFot could be
`detected. Although the number of cases examined in each
`treatment group is small, it is reassuring to note the lack of
`effect of ICI 182780 on either EGFR or TGFot expression.
`These results suggest that, at least in the short term, treat-
`ment with the pure antioestrogen does not induce changes
`characteristic of resistance to endocrine therapy.
`From the results of this first phase I study of ICI 182780
`in human primary breast tumours in vivo it
`is evident
`that this new drug is more potently antioestrogenic than
`
`2.5
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`ll\1/
`
`PM
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`Page
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`Pre
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`Post
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`PM
`
`Post
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`
`Control
`
`102 low
`
`182 high
`
`(A) Pre and post-treatrnent measurements of EGFR content in
`Fig. 4
`control ER-positive tumours or those receiving the low (182 low) and
`high (182 high) doses of ICI 182780; (B) Pre- and post-treatment
`measurements of TGFot content in control ER-positive tumours or those
`receiving the low and high doses of ICI 182780. There were no
`significant differences between the pre- and post-treatment values for any
`of the treatment groups (Wilcoxon's matched-pair signed-rank test).
`
`tamoxifen without any evidence of oestrogen agonism.
`Furthermore, the novel mechanism of ICI 182780 appears
`to be reflected in its effects on human breast tumour tissue
`in vivo.
`
`Acknowledgements
`
`The above study was a collaboration between the Christie Hospital NHS
`Trust, the City Hospital, Nottingham and the Royal Marsden Hospital,
`London. In addition to the authors,
`the following people participated:
`David DeFriend, Ian Laidlaw, Robert Clarke, Richard MacClelland, Julia
`Gee, David Manning, Christobel Saunders, Robert Mansel, Michael Baum,
`Roger Blarney, John Robertson, Peter Walton and Alan Wakeling.
`The European School of Oncology gratefully acknowledges an educa-
`tional grant from Zeneca Pharmaceuticals which made the meeting possible.
`
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`|nnoPharma Exhibit 1049.0005
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