`
`in Endocrine Dependent Pathology:
`Basic and Clinical Aspects
`
`Proceedings of an International Symposium,
`Milano, 10-14 April 1994
`
`Editors:
`
`Marcella Motta
`Department of Endocrinology
`University of Milan
`Milan, Italy
`
`Mario Serio
`
`Endocrinology Unit
`Department of Clinical Physiopathology
`University of Florence
`Florence, Italy
`
` 1994
`
`Elsevier
`
`Amsterdam — Lausanne — New York — Oxford — Shannon - Tokyo
`
`|nnoPharma Exhibit 10530001
`
`
`
`@1994 Elsevier Science B.V. All rights reserved.
`
`No part of this publication may be reproduced, stored in a retrieval system or transmitted in any form or
`by any means, electronic, mechanical, photocopying, recording or otherwise without the prior written
`permission of the publisher, Elsevier Science B.V., Permissions Department, P.O. Box 521, 1000 AM
`Amsterdam, The Netherlands.
`No responsibility is assumed by the Publisher for any injury and/or damage to persons or property as
`a matter of products liability, negligence or otherwise, or from use or operation of any methods, products,
`instructions or ideas contained in the material herein. Because of rapid advances in the medical sciences,
`the Publisher recommends that independent verification of diagnoses and drug dosages should be made.
`Special regulations for readers in the USA — This publication has been registered with the Copyright
`Clearance Center Inc. (CCC), 27 Congress Street, Salem, MA 01970, USA. Information can be obtained
`from the CCC about conditions under which photocopies of parts of this publication may be made in the
`USA. All other copyright questions, including photocopying outside the USA should be referred to the
`copyright owner, Elsevier Science B.V., unless otherwise specified.
`
`International Congress Series No. 1064
`ISBN 0-444-81879-0
`
`This book is printed on acid-free paper.
`
`Published by:
`Elsevier Science B.V.
`P.O. Box 211
`-
`1000 AE Amsterdam
`The Netherlands
`
`Library of Congress Cataloging in Publication Data:
`Sex hormones and antihormones in endocine dependent pathology :
`proceedings of an international symposium, Milano. 10-14 April 1994
`I editors, Marcella Motta, Mario Serio.
`p.
`cm. —— (International congress series ; no. 1064)
`Includes bibliographical references and indexes.
`ISBN 0-444-81879-0 (alk. paper)
`2. Prostate-
`1. Breast——Cancer—-Endocrine aspects--Congresses.
`—Cancer-—Endocrine aspects-—Congresses.
`3. Hormones. Sex-
`—Antagon1sts——Therapeutic use——Ccngresses.
`4. Hormones, Sex-
`—Therapeutic useh—Congresses.
`5. Uterus-—D1seases--Endocrine
`aspects——Congresses.
`I. Motta. Marcella.
`II. Serio, Mario.
`III. Series.
`2. Sex
`[DNLM: 1. Neoplasms, Hornone-Dependent—-congresses.
`Hormones—-congresses.
`3. Prostatic Neoplasms——congresses.
`4. Breast Neoplasms-—congresses.
`5. Genital Diseases, Female-
`—congresses.
`H3 EX89 no. 1084 1994 / OZ 200 S518 1994}
`RC280.B6S48
`1994
`616.99'449—-dc20
`DNLM/DLC
`For Library of Congress
`
`94-32151
`CIP
`
`In order to ensure rapid publication this volume was prepared using a method of electronic text processing
`known as Optical Character Recognition (OCR). Scientific accuracy and consistency of style were handled
`by the author. Time did not allow for the usual extensive editing process of the Publisher.
`
`_ Printed in the Netherlands
`
`
`
`
`
`|nnoPharma Exhibit 10530002
`
`
`
`©1994 Elsevier Science B.V. All rights reserved.
`Sex hormones and antihormones in endocrine dependent
`pathology: basic and clinical aspects
`M. Motta and M. Serio, editors,
`
`Pure antioestrogens in breast cancer: experimental and clinical
`observations
`
`R.l. Nicholson‘, J.M.W. Gee‘, C.L. Eaton’, D.L. Manning‘, R.E. Mansel2, A.K.
`Sharmaz, A. Douglas—Jones2, M. Price—'Il1omas3, A. Howell‘, D.J. DeFriend“, N.J.
`Bundred", E. Anderson‘, J.F.R. Robertson5, R.W. Blameys, M. Dowsetté, M. Baum“,
`P. Walton7 and A.E. W'akeling7
`1Tenovus Cancer Research Centre, Cardiff, UK; 2Departments of Surgery (REM, AS) and Pathology
`(AD-J), UWCM, Cardifl", UK; 3Royal Gwent Hospital, Newport, UK; ‘Withington and Christie Hospitals,
`Manchester, UK; ‘Department of Surgery, City Hospital, Nottingham, UK,‘ “Royal Marsden Hospital,
`London, UK; and 7Zeneca Pharmaceuticals, Macclesfield, UK
`
`Key words: ICI 182,780, ICI 164,384, oestrogen receptor, oestrogens, tamoxifen
`
`Introduction
`
`The last 10 years have seen the emergence of a new class of pharmacological agents
`termed pure antioestrogens (reviewed in [1]). These compounds, which were
`originally developed by ICI Pharmaceuticals Division in the UK, have the unique
`property of binding to the oestrogen receptor [2] and producing a receptor complex
`which lacks oestrogenic activity [3,4]. If we assume that the action of oestrogens on
`sensitive breast cancers favours cell proliferation and survival, and that they thereby
`act as a driving force for the growth and development of the disease [5], pure
`antioestrogens have the potential to fully negate these activities by producing a state
`of complete oestrogen withdrawal [6].
`The perceived importance of pure antioestrogens, therefore, is as alternatives to
`antihormonal treatments which are designed to reduce the synthesis of oestrogens, but
`which currently fail to nullify oestrogenic signals arising from other sources [7,8],
`and as potential successors to “tamoxifen—like” antioestrogens, which although widely
`and successfully used in the therapy of primary and advanced disease [6], possess
`partial oestrogenic activity [9] which may negate aspects of their effectiveness as
`antitumour agents.
`Since pure antioestrogens are now entering clinical development, the current paper
`seeks to outline some of their basic cellular and antitumour properties on human
`
`Address for correspondence: R.I. Nicholson, Tenovus Cancer Research Centre, University of Wales,
`College of Medicine, Heath Park, Cardiff CF4 AXX, UK.
`
`|nnoPharma Exhibit 10530003
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`
`
`348
`
`breast cancer cells in vitro [1,10] primarily using the lead compound ICI 164,384, and
`to compare this information with data derived from a phase I study of ICI 182,780
`in primary breast cancer patients [11]. In each instance, emphasis will be placed on
`immunohistochemical data as it was our original hope that such an approach would
`facilitate an assessment of the degree to which pure antioestrogens were fulfilling
`their potential as complete antagonists of oestrogen action in clinical breast cancer
`and thereby aid in defining the importance of oestrogens in the regulation of breast
`cancer growth.
`Figure 1 shows the structure of ICI 164,384 and ICI 182,780 which are 70: long-
`chain analogues of oestradiol. The ER binding affinity and potency of ICI 182,780
`are greater than that observed for ICI 164,384 due to the substitution of the amide
`function by a sulphoxide group and the fluorination of the terminal chain [12]. Such
`differences, however, do not alter the intrinsic biological behaviour of the drugs
`which are identical
`to other pure antioestrogens, based on substitutions in the
`oestradiol nucleus [13,14] or nonsteroidal forms [15].
`
`Properties of pure antioestrogens in vitro
`
`One of the most important early observations arising from the functional disablement
`of ER signalling by pure antioestrogens in oestrogen—sensitive human breast cancer
`cell lines was that treated cells frequently became very efficiently growth-arrested
`[10,16,17]. This property is illustrated in Fig. 2a and shows the growth of MCF-7
`cells in lO‘9M oestradiol in the presence or absence of a 100-fold excess of ICI
`
`,
`
`OH
`
`
`
`"""<cH2>1ocoN(cH2>3cH3
`
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`
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`
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`
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`
`HO
`
`""(CH2)9SO(CH2)3CF2CF3
`
`ucn 182,780
`
`Fig. 1. Structures of ICI 164,384 and ICI 182,780.
`
`I
`
`|nnoPharma Exhibit 1053.0004
`
`
`
`(axvooo Cell numbers /1000
`
`we -3:, K3574. cefls
`
`Oestradiol
`(10—9M)
`
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`
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`
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`+
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`(10-7M)
`
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`
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`
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`
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`
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`164
`
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`Days in culture
`
`Fig. 2. Growth and immunohistochemical characterisation of MCF-7 cells. The cells were grown in white
`RPMI tissure, culture medium with 5% DCC-stripped FCS (medium A) containing oestradiol : ICI
`164,384. (a) Cell numbers were assessed using a Coulter Counter; (b,c) Ki67; and ((1) PR assays were
`performed according to the methods of Bouzubar et al. [19] and Walker et al. [20], respectively. The Ki67
`proliferative index was calculated as the proportion of cells showing intense nucleoplasmic and nucleolar
`staining patterns [21]. Results are shown as the mean i SD of six replicates.
`
`164,384. In contrast to the expansion of the cell population that occurs in the
`presence of the steroid, the pure antioestrogen virtually abolishes the growth of the
`MCF-7 cells, allowing at best one doubling of the initial cell number with the cells
`
`|nnoPharma Exhibit 1053.0005
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`then lapsing into a static growth condition. These diverse actions are reflected in the
`growth dynamics of the tumour cells, with several groups showing that while
`oestradiol increases the tumour cell growth fraction and acts to stimulate the passage
`of cells through the cell cycle, pure antioestrogens promote a highly. effective
`‘restriction of the cell cycle approximately 5 h into G1 and hence cause a reduction
`of the proportion of cells undergoing DNA synthesis [16,17]. Immunohistochemically
`this may be visualised using the Ki67 antibody which detects a nuclear antigen that
`is expressed as cells enter S-phase and continues through G2 and M, but is not
`expressed in G1 or G0 [18]. In oestradiol-treated cells on day 4 of culture, many of
`the tumour nuclei are Ki67-positive indicating a high tumour-growth fraction, and
`show intense nucleoplasmic and nucleolar staining patterns, which indicates a high
`proliferative index (Fig. 3a). Following ICI 164,384 treatment strong nuclear staining
`is largely absent and only weak immunostaining or cellular Ki67 negativity remains
`(Fig. 3b). Quantitatively, the effects on Ki67 immunostaining are seen throughout the
`growth curve, with oestradiol
`increasing the growth fraction during exponential
`growth, while the pure antioestrogen progressively decreases their number during the
`treatment period (Fig. 2b, [10]). This effect is more exaggerated using a proliferative
`index based on the intense nuclear staining patterns with no evidence of cell
`proliferation in ICI 164,384-treated cells after day 4 (Fig. 2c). Indeed, on continuous
`exposure to pure antioestrogens there is a complete loss of Ki67 immunostaining (not
`illustrated) as cells pass into a noncycling population [17].
`
`E2 +164
`la) E2
`10-9110-910-1° 1o-9 10* 1o-7 106
`as
`d13 4»
`
`(*3)
`
`20
`
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`
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`
`*pVE2<0.o5
`
`%DeadCells
`
`
`
`Cells/wellx104
`
`‘I40
`
`120
`
`100
`
`50
`
`60
`
`40
`
`E2
`
`1 82
`
`Fig. 4. Influence of pure antioestrogens on MCF—7 cell growth and cell death. (a) Cells were grown for
`8 days in medium A containing oestradiol and then for a further 5 days with oestradiol plus varying doses
`of ICI 164,384. Cell numbers were assessed using a Coulter Counter and the results expressed as mean
`1 SD. (b) Cells were grown for 4 days in medium A supplemented with oestradiol ( 10’9M) or ICI 182,780
`(l0‘7M). The proportion of dead cells was estimated using a Dead/Live EukoLight kit (Molecular Probes
`Inc., USA), which employs the cellular uptake of the red fluorescent nucleic acid chelator ethidium
`bromide to monitor the dead cell population. The results are shown as mean 1 SEM of seven replicates.
`
`|nnoPharma Exhibit 1053.000?
`
`
`
`352
`
`It is noteworthy that the: growth-inhibircrry activity (if pain antianstrngens is not
`3113: restrictad in cytustatic atttiviry; an cnntinnnus expnsura thara at:-:0 E1§3§3'E33t’S to» be
`a cytutnxic cnnipnnentt This is must easily recugniand in unatrngan-girirnnti nails, with
`the. mails baing grown fur 8 days with uestratiiui and than traatad for 9. furthezr 5 days
`with nnatradinl gains variuus deans uf ICI 164,384 {Fig 4a). While 10"3I’v‘1 ICI 164384
`suecbssfuily blncka the. E2-inciunad grnwtir, cnnnantratinns {if that pure antiucstmgen
`uf 104M and about cause a tiecrnas-:-, in the initial cell number. Thi:-3 is rbcugnfised as
`bning a cytutaxic effect uf pure antiuestrogena, evidencari by {ha prnsczn-:5 uf large:
`numbers uf pyknutic nuclei. Quantifinatinn {if the. numbers of dead ceiis swing a twe-
`cniour fluorescence: viability assay shnws. that treatment {if MCF-‘F calls with ICI
`1S2._,?S{) far 4 days significantly elevates the numbers (if dead cnils {etititiiurn brunt-
`idupnsitivca} rrslative tn these uhsarvnri in the prnsunca nf cinstradiol alone (Fig. 413).
`The gruwth-—i:1i::ibitory activity Uf pars antiunstrugens on human brauat cancer cell
`
`Oensitomutry Value
`
`PB!‘-I
`
`-E2 E2 ‘£32 16$
`
`2!‘:
`
`Ch
`
`24!‘:
`
`$031 " 4:!
`
`Fig. 5. Influence us‘ 3:?! $64,384 an p32 n1RN:'>t in ncstrngen-primed cssiis. Cells wart: gmwn for 3' days
`in rnetiiurn :4 cnntairxing nastradin} (1{I””NI) and than fer the specified times with E2 pins {CI ii:-4,384
`(1U""M.}. RNA axtractiun and Nerthern analysis far 932 were giarfcrrned accnrding tn the pmcatiure bf
`Manning at a1. [22]. The insert shows a PCR (213 cycles} :33“ {:52 {lower} and actin (upper) <:1')NA
`fnllnwziug taverns t1'3fi3(21‘i§3{i0-ii uf their mRNA. The Celts were treated for Ir‘ days with nestsadiui { §i}‘gb¢§},
`{CI 1$2;}‘3£} {1{}‘7194) in‘ IC1 164,334 (1{}'?M).
`
`|nnoPharma Exhibit 1053.0008
`
`
`
`353
`
`lines is preceded by changes in the expression of several oestrogen—regulated genes,
`with,
`for example,
`the high levels of nuclear PR immunostaining induced in
`oestradiol—treated MCF—7 cells (Fig. 3c) being rapidly reversed by the presence of ICI
`164,384 (Fig. 3d).
`Indeed, examination of the percentage of PR—positive cells
`throughout oestradiol and ICI 164,384 treatment, shows that not only does the pure
`antioestrogen block oestradiol~induced PR levels, but that it also obliterates all PR
`signalling after 4 days of culture (Fig. 2d). Such cells are no longer responsive to
`progesterone.
`the substantial increase in cytoplasmic pS2 immunostaining that is
`Similarly,
`induced by oestradiol (Fig. 3e) is largely absent following ICI 164,384 treatment
`(Fig. 30. Any residual pS2 staining tends to be present towards the outer cell
`membrane in small secretory vesicles. Once excluded, however, the cells remain
`negative with no evidence of pS2 production within the endoplasmic reticulum.
`Predictably, the decrease in oestrogen—regulated proteins often corresponds to a fall
`in their mRNA levels and on Northern analysis it may be seen that following the
`administration of l0‘7M ICI 164,384 to oestradiol-primed cells, pS2 mRNA levels
`rapidly decline (Fig. 5). GAPDH-corrected densitometer readings indicate that within
`2 days pS2 mRNA levels are very low and by day 5 they are undetectable. Even after
`reverse transcription of the mRNA and PCR the pS2 CDNA is barely visible in ICI
`
`(a)
`
`2
`
`TGFa HSeore value
`
`'
`2
`
`bcl—2 HScore value
`
`1.5
`
`0-5
`
`1_5
`
`* pVE2<0.05
`
`0.5
`
`E2
`(1 0'9M)
`
`-E2
`
`182 ‘
`(1 O'7M)
`
`,
`
`E2
`(1 O'9M)
`
`.E2
`
`152
`(1 O'7M)
`
`Fig. 6. Influence of ICI 182,780 on TGF—oc and bcl—2 immunostaining in MCF—7 cells. Cells were grown
`for 8 days in Medium A (—E2) supplemented with oestradiol or ICI 182,780. TGF—0L and bcl—2 assays
`were performed according to the methods of Nicholson et al. [25] and Gee et al. [24], respectively. H
`score values were calculated as the sum of the fraction of cells staining at intensity readings 1+, 2+ and
`3+ [20]. The results are shown as mean : SD of four replicates.
`
`|nnoPharma Exhibit 1053.0009 A
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`354
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`164,384 and ICI 182,780 treated cells (Fig. 5 insert), indicating that pure antioestro-
`gens can produce a rapid and complete shutdown of oestrogen-regulated gene
`function.
`
`A number of the changes in gene expression may directly contribute to the
`mechanism of action of the drugs, with ICI 182,780 promoting decreases in
`immunodetectable TGF-ot, an oestrogen—inducible mitogenic growth factor [23], and
`the bcl—2 protein, a factor which has been implicated in the protection of cells against
`programmed cell death [24]. In each instance, while these proteins are readily
`detectable in a high proportion of cells treated by oestradiol, their levels are lowered
`by oestrogen withdrawal and further reduced by the pure antioestrogen (Fig. 6a,b).
`This is especially evident for the bcl—2 protein, with oestradiol-related immunostaining
`(Fig. 3g) being largely abolished by ICI 182,780 (Fig. 3h). Indeed, bcl—2 positivity
`is a relatively rare event following the administration of pure antioestrogens and, in
`line with its role in cell survival, its absence is often associated with the presence of
`pyknotic nuclei (Fig. 3h) and corresponding elevated ethidium bromide staining (not
`illustrated).
`initially on oestrogen-induced
`The inhibitory actions of pure antioestrogens,
`transcriptional events and subsequently on cell proliferation and survival, consistently
`exceed the effects which may be achieved by antioestrogens with partial oestrogen-
`like activity and also, somewhat surprisingly, surpass the effects of oestrogen
`withdrawal [l,10]. This property is illustrated in Fig. 7a where ICI 164,384 may be
`seen to severely impair the growth of MCF-7 cells in tissue culture medium which
`lacks phenol red and where the 5% foetal calf serum has been extensively stripped
`of its endogenous oestrogens by charcoal absorption, a procedure which reduces the
`level of endogenous oestradiol to below l0‘”M. The degree of inhibition promoted
`by ICI 164,384 exceeds that achieved by an equivalent dose of 4—hydroxytamoxifen -
`(4—OHT). These activities are reflected in the tumour cell growth fraction, as
`determined by Ki67 immunostaining [10]. The additional growth-inhibitory activity
`of pure antioestrogens appears specific for ER signalling, in that it is reversible by
`oestradiol and is observed only in ER—positive cell lines (not illustrated). It is also
`seen under a wide variety of growth conditions, including residual growth in serum-
`free medium, where the levels of endogenous oestrogens are extremely low (Fig. 7b).
`As a biological paradox, the additional antitumour activity of pure antioestrogens
`in ER—positive cells may,
`in part, reside in their ability to reduce the cellular
`concentration of ER, with ICI 164,384 causing a rapid decline in ER positivity (Figs.
`3i,j and 7c,d) [l0,26,27]. This property contrasts with the increases in ER levels
`which are seen on either oestrogen withdrawal or 4-OHT treatment (Fig. 7c,d). The
`potential importance of this action is emphasised by recent cell and molecular biology
`studies which have not only shown that the ER occupied by either relatively small
`amounts of oestrogens, or antioestrogens with partial oestrogen-like activity, can
`interact with the signalling pathways of several growth factors to amplify mitogenic
`responses in breast cancer cells [l7,28—30], but also that growth factors can
`sometimes aid ER-induced transcriptional activity in the apparent absence of the
`ligand [31,32] and that, where examined,
`these actions are susceptible to pure
`
`|nnoPharma Exhibit 10530010
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`700
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`Cell numbers] 1000
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`700
`
`Cellnumbers/1000
`
`355
`
`600
`
`500
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`400
`
`300
`
`100
`
`0
`
`minus E2
`
`-
`_
`4 OHT (107M)
`
`O
`
`1
`
`2
`
`3
`
`4
`
`164 (10"M)
`5
`6
`7
`
`8
`
`600
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`500
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`400
`
`20
`
`°
`
`100
`
`0
`
`.
`mmus E2
`
`164 (10‘7M)
`
`0123456789101112131415
`
`Days in Culture
`
`Days In Cultuie
`
`(C)
`
`100
`
`as ER-ICA + cells
`
`(d)
`
`2
`
`ER-ICA HScore
`
`300 200
`4-OHT (1 o'7M) 0.5
` 80
`
`60
`
`40
`
`20
`
`0
`
`4-OHT (1 O‘7M)
`
`164 (10'7M)
`
`1.5
`
`0
`
`164 (10'7M)
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
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`6
`
`7
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`1
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`2
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`3
`
`4
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`5
`
`6
`
`7
`
`8
`
`Days in Culture
`
`Days in Culture
`
`Fig. 7. Growth and immunohistochernical characterisation of MCF-7 cells. Cells were grown in medium
`A (a,c,d) or DCCM serum—free medium (b) in the presence or absence of ICI 164,384 or 4—OHT. Cell
`numbers were assessed by Coulter Counter. The ER assays were performed according to the method of
`Walker et al. [20] and H score values calculated as in Fig. 6. The results are shown as mean x SD of 6
`replicates.
`
`antioestrogens.
`It may be more appropriate, therefore, to visualise the ER as part of a central
`signalling pathway that can be influenced by both oestrogens and growth factors to
`
`|nnoPharma Exhibit 10530011
`
`
`
`356
`
`maintain cell growth and viability. In thissmodel, it can be envisaged that the partial,
`or even total, removal of oestradiol or other oestrogens may not prevent all ER-
`mediated signalling. In order to negate the remainder, there would be a need to either
`remove the ER or interfere with growth factor actions, fortuitously both of which are
`achieved by pure antioestrogens and represent a new pharmacological affront to
`tumour cells. Projection of this model to the clinical situation would suggest that
`since current antihorrnonal methods fail to achieve total oestrogen deprivation and do
`not obliterate ER levels and growth factor signalling [5—7,33,34], a potential exists
`for pure antioestrogens to improve the outcome of endocrine therapy in such
`important areas as the rate and duration of remission and the prevention and treatment
`of endocrine—resistant states [6,35].
`
`Properties of pure antioestrogens in clinical breast cancer
`
`Although clinical trials with pure antioestrogens are in their infancy and consequently
`little is known about their clinical properties, in late 1991 a phase I study of ICI
`182,780 was initiated [11]. The purpose of the study was to assess the safety and
`pharmacokinetic properties of the drug and to investigate its biological effects on
`tumour tissue. The latter was achieved by measuring a number of immunohistochemi—
`cally—determined end points on pretreatment needle core biopsies from newly
`diagnosed primary breast cancer patients and comparing the results with identical
`measurements performed on the post—treatment operative specimen removed at the
`time of primary surgery. In total 56,postmenopausal women were randomised to
`either a control group, which received no preoperative treatment, or a treatment
`group, which received daily intramuscular injections of ICI 182,780 at doses of 6 mg
`or 18 mg for 7 days prior to mastectomy.
`Figure 8a shows the dose effect of the pure antioestrogen on ER expression in
`patients whose tumours were initially classified as ER positive. Although both doses
`produced a significant reduction in ER levels, the largest decrease was observed at
`18 mg/day. Indeed, in 11 tumours with initially medium and high ER values, nine
`showed low or absent levels of the receptor in the post—treatment sample (Fig. 9a).
`Comparison of the data with similar measurements derived from short—term tamoxifen
`treated patients (20 mg b.i.d.) showed that although tamoxifen produced a suppression
`of ER levels, the effects were not as great as those induced by the highest dose level
`of ICI 182,780 (Fig. 8b).
`Examination of PR levels in these samples revealed a divergence of response to
`the antioestrogens, with ICI 182,780 (18 mg/day) inhibiting PR expression (Fig. 8c),
`while tamoxifen promoted some increase in its tissue concentration (Fig. 8d). In
`initially ER-positive and PR~positive tumours, ICI 182,780 (18 mg/day) caused a fall
`in PR levels in the majority of patients (Fig. 9b). Indeed, in several patients tumours
`were negative for both of these steroid receptors following the 7-day treatment period.
`pS2 showed similar responses to PR, although the effects appeared blunted (Figs. 8e
`and 9c). No short—term effects of ICI 182,780 or tamoxifen were recorded on TGF—0t
`
`|nnoPharma Exhibit 10530012
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`(3)
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`Difference in ER Hscore
`
`(b)
`
`Difference in ER Hseore
`
`(C)
`
`Difference in PR Hscore
`
`357
`
`p<o.o1
`r-mu
`
`Control
`(n=1o)
`
`I01 1 52
`umg (n=a)
`
`lC|182
`15mg (n=12)
`
`Control
`(n=10)
`
`lC|152
`16mg (nx12)
`
`Tamoxifen
`(n=15)
`
`-1
`
`Contro|
`(n=m)
`
`“H182
`emg (n=a)
`
`W182
`13mg (n=I2)
`
`Difference In PR Hscore
`
`(9)
`
`Difference in p82 Score
`
`Difference in Ki67 scare
`
`p<o.oo1 .
`
`
`Control
`(n=I0)
`
`lCl182
`1emg(n=I2)
`
`Tamoxifen
`(n=15)
`
`'
`
`Control
`(n=7)
`
`Icnaz
`1emg(n=a)
`
`Tamoxifen
`(n=15)
`
`7
`
`Control
`(n=10)
`
`louse
`cmg(n=e)
`
`Icnaz
`1amg(n=I2)
`
`Fig. 8. Influence of ICI 182,780 and tamoxifen on clinical breast cancer immunostaining. The data were
`calculated as differences in assay values between pre— and poststudy samples. H scores were determined
`as in Fig. 6. The results are shown as median values with interquartile ranges.
`
`or the bcl-2 protein (not illustrated). In contrast, ICI 182,780 (18 mg/day) produced
`a significant reduction in the median Ki67 labelling index of ER—positive tumours
`(Fig. 8f), with individual Ki67 values decreasing in the majority of ER—positive
`tumours examined (Fig. 9d).
`
`Clinical vs. in vitro data: the future
`
`In many respects the tissue responses of clinical breast cancer to pure antioestrogens
`appear similar to those induced in human breast cancer cells in culture. This is
`encouraging due to the pronounced antitumour activity of pure antioestrogens in vitro.
`In both instances the drugs are clearly pharmacologically active and promote
`decreases in oestrogen—regu1ated gene expression, with no evidence of oestrogen-like
`activity. They also instigate a substantial fall in oestrogen receptor levels, generating
`in many instances ER negativity. This represents a novel action for this class of
`
`|nnoPharma Exhibit 10530013
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`
`
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`Post
`
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`Fig. 9. Influence of ICI 182,780 on clinical breast cancer immunostaining. Pre- and poststudy assay results
`are shown for each ‘patient. H scores were determined as in Fig. 6.
`
`antihormonal drug and may have important implications for growth factor signalling
`through the ER. In each instance, decreases in Ki67 immunostaining were recorded
`and provide preliminary evidence that treatment with ICI 182,780 may achieve a
`
`|nnoPharma Exhibit 10530014
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`
`359
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`desired end point of antioestrogen therapy in breast cancer, that is an inhibition of
`cell proliferation.
`In the future it will be important to titrate out the antioestrogen dose against the
`types of parameter described in the cell culture studies and extend the treatment
`period in patients with biopsiable disease. Only when the maximum reduction in
`oestrogen-regulated genes and ER has been established will the full potential of pure
`antioestrogens have been met in clinical breast cancer and the role of oestrogens
`delineated. Having achieved this goal, their actions should be compared to other
`treatments designed to interfere with the production of oestrogens or their cellular
`activity. Such studies would establish whether pure antioestrogens pass existing
`thresholds of response to antihormonal measures and the importance of partial vs.
`complete oestrogen withdrawal.
`
`Acknowledgements
`
`The authors wish to thank the Tenovus Organisation for their generous financial
`‘ support.
`
`References
`
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`Wakeling AE, Bowler J. J Steroid Biochem 1988;31:645-653.
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`Nicholson RI. Curr Opin Invest Drugs 1993;2:1259-1268.
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`'
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`|nnoPharma Exhibit 10530015
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`360
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`i
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`g
`
`IO, Elston CW, Robertson JFR, Blarney RW. Br. J.
`
`19. Bouzubar N, Walker KJ, Griffiths K,
`'
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`v
`24. Gee JMW, Robertson JFR, Hoyle HB, Kyme SR, McClel1and RA, Ellis 10, Blarney RW, Nicholson
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`RW. Cancer Res 1994;54.
`‘
`26. Gibson MK, Nemmer LA, Beckman WC Jr, Davis VL, Curtis SW, Korach KS. Endocrinology
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`‘
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`
`29. Vignon F, Bouton M-M, Rochefort H. Biochem Biophys Res Commun 1987;146:l502—l508.
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`'
`35. Wakeling AE. Breast Cancer Res Treat 1993;5:1—9.
`
`‘
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