Sex Hormones and Antihormones
`
`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
`
`GE 1994
`
`Elsevier
`
`Amsterdam — Lausanne — New York — Oxford — Shannon - Tokyo
`
`InnoPharma Exhibit 1053.0001
`
`

`

`@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, PO. Box 521, 1000 AM
`Amsterdam, The Netherlands.
`No responsibility is as3umed 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 hornnnes and antihormunes in endocine dependent pathology :
`proceedings of an international symposium, Milano.
`10—14 April 1994
`/ editors, Marcella Marta, Mario Serin.
`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-—Enducrine aspects-—Congresses.
`3. Hormones, Sex—
`—Antagunists——Therapeut1c use——Ccngresses.
`4. Hormones, Sex-
`—Therapeutic useh-Congresses.
`5. Uterus--Diseases--Endpcrine
`aspects——Cnngresses.
`I. Motta. Marcella.
`II. Seriu, Mario.
`III. Series.
`2. Sex
`[DNLM: 1. Neoplasms, Hormone-Dependent—-congresses.
`Hurmunes—-congresses.
`3. Prostatic Neoplasms—-congresses.
`4. Breast Neoplasms-—congresses.
`5. Genital Diseases, Female-
`-congresses.
`H3 EXSQ no. 1084 1994 / 02 200 3518 19943
`RC280.BBS48
`1994
`618.99'449--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
`
`
`
`
`
`InnoPharma Exhibit 1053.0002
`
`

`

`
`
`
`
`
`@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.I. Nicholsonl, J.M.W. Gee], C.L. Eaton‘, D.L. Manning‘, R.E. Mausel2, A.K.
`Sharmaz, A. Douglas—Jonesz, M. Price—Thomas3, A. Howell", DJ. DeFriend“, N.J.
`Bundred“, B. Anderson“, J.F.R. Robertsons, R.W. Blameys, M. Dowsetté, M. Baumfi,
`P. Walton7 and A.E. W'akeling7
`[Tenovus Cancer Research Centre, Cardiff, UK; 2Departments of Surgery (REM, AS) and Pathology
`(AD-J), UWCM, Cardiff, UK; 3Royal Gwent Hospital, Newport, UK; ‘Withington and Christie Hospitals,
`Manchester, UK; 5Department of Surgery, City Hospital, Nottingham, UK; 6Royal 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.
`
`InnoPharma Exhibit 1053.0003
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`348
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`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 70t 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 10‘9M oestradiol in the presence or absence of a 100-fold excess of ICI
`
`,
`
`OH
`
`
`
`IIII"(CH2)1oCON(CH2)3CH3
`
`H0
`
`QM CH3
`
`OH
`
`HO
`
`“(CH2)QSO(CH2)3CF2CF3
`
`1c: 182,780
`
`Fig. 1. Structures of ICI 164,384 and ICI 182,780.
`
`InnoPharma Exhibit 1053.0004
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`C II
`b
`(a)1looo e num ers
`
`[1000
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`
`349
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`(1 O—9M)
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`+
`lCl 164384
`(10-7M)
`
`
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`7
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`| d
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`rot era Ive n ex
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`1
`
`2
`
`5
`4
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`Days in wlture
`
`6
`
`7
`
`8
`
`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 t 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
`
`InnoPharma Exhibit 1053.0005
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`351
`
`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
`(a) E2
`10911091010 10-9 10a 10-7 105
`d8
`d13 ——>
`
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`
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`
`140
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`
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`
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`
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`
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`
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`
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`
`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
`(10‘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 : SEM of seven replicates.
`
`InnoPharma Exhibit 1053.0007
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`

`352
`
`
`
`Qualitomutry Value FISH
`
`It i5 notcwcrtiiy {has {hc grcwth-inhihitcry activity (if can: anticcctrcgccc is act
`3113: restrictcd m cymstatic activiiy; {in ccntinuccs cxpcsurc thcrc alsc apgcars in be.
`a cytctcxic ccmpcnentl This is mcst easily reccgnicccl it: ccatrcgcn-girimcd cells, with
`thc mils ccng grown far 8 days wizh cesircdicl and than trcatvad fcr a fufihcr 5 clays
`with ccctradicl {plus varicuc dcacc cf ICI 164,384 {Fig 4a). While lfl‘alvi ICI 164384
`succcssfclly blcckc zhc EZ-incicccd grcwtii, cccccntraticns cf thc purc aminestmgcii
`cf MAM and Show cause a dccrcasc in the initial cell number. This; is rcccgniscd as
`izcing a cytctcxic effect cf pure anticastrogcnc, evidenccti lay {he prcccncc cf large:
`numbers cf pylmctic miclci. Qcantiflcaiicn cf thc numbers of dead cclls swing a twa-
`cclour fluaresceacc viabiliiy assay shcws that treatment cf MGR”? cclls with ICI
`132;?39 far 4 days significantly clcvaics thc numbcrc cf dead cclls {cthidicm incum-
`idc»pcsitivc} rclativc tc ihcsc chacrvcfi if; the ptcccncc cf ccctradicl alone (Fig. zlb).
`The: gmwihvinhiijiiory activity if purc anticcstmgcns an human braaat cancer cell
`
`ICI 182335 {13416) ct 1C1 164,334 (1&4M).
`
`
`
`-E2 E2 182 16¢
`
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`Fig. 5. inflccmc cf 3C! $64,384 on p32 mRNsx in ccszrcgcc-primcd cclic. Cells wcrc grcwn for 3' days
`it: meiiium a ccntairxing ccstmdicl (1cm; and {hm far the sgccificd iimcc with 82 pics {CI iififigc
`(11341“), RNA cxtracticn and Ncnhcm analysis far 982 wcrc gerfcrmcd according tc thc pmcccurc cf
`Magring at all {22]. The insert shows a PCR {21:} cyciea} cf p32 (Icwcrl and actin (upper) ciDNA
`fcllcwing :cvcrce transcriptica cf their mRNA. The calls were treatcd for 3? days with ccctx‘adicl { M‘QM},
`
`InnoPharma Exhibit 1053.0008
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`

`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 p52 immunostaining that is
`Similarly,
`induced by oestradiol (Fig. 3e) is largely absent following ICI 164,384 treatment
`(Fig. 31). 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 10'7M 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
`
`' bcI-2 HScore value
`2
`
`1.5
`
`0.5
`
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`
`
`
`1.5
`
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`
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`(1 o-SM)
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`(1 (WM)
`
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`
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`(1 o-SM)
`
`—E2
`
`152
`(1 ON)
`
`Fig. 6. Influence of ICI 182,780 on TGF—oc and bc1—2 immunostairiing in MCF-7 cells. Cells were grown
`for 8 days in Medium A (—132) supplemented with oestradiol or ICI 182,780. TGF—OL and bc1—2 assays
`were performed according to the methods of Nicholson et al. [25] and Gee et a1. [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 i SD of f0ur replicates.
`
`InnoPharma 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-oc, 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 bc1-2 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 [1,10]. 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 10‘12M. 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) [10,26,27]. 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 [17,28—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
`
`InnoPharma Exhibit 1053.0010
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`700
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`2
`
`ER-ICA HScore
`
`60
`
`40
`
`20
`
`4-OHT (1 WM)
`
`1 64 (1 O‘7M)
`
`1.5
`
`0.5
`
`4-OHT (WM)
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`O
`
`1
`
`2
`
`3
`
`4
`
`5
`
`6
`
`7
`
`8
`
`Days in Culture
`
`Days in Culture
`
`Fig. 7. Growth and immunohistochemical 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 a1. [20] and H score values calculated as in Fig. 6. The results are shown as mean : 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
`
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`356
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`maintain cell growth and viability. In thismodel, 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 antihormonal 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 561postmenopausal 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. 86
`and 90). No short-term effects of ICI 182,780 or tamoxifen were recorded on TGF-oc
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`

`(a)
`
`Difference in ER HScore
`
`(b)
`
`Difference in ER HSeore
`
`(C)
`
`Difference in PR HScore
`
`357
`
`p<o.o1
`['—_I
`
`Control
`Icl182
`Tamoxifen
`Control
`[01182
`101182
`(n=10)
`16mg(nx12)
`(n=15)
`(nzlm
`6mg (n=a)
`13mg (n=|2)
`Difference in p82 Score Difference in Ki67 scare
`
`
`
`p<0.00| .
`
`
`Control
`(n=1o)
`
`IO! 1 62
`8mg (n=a)
`
`lc|182
`15mg (n=12)
`
`leference In PR HScore
`
`(9)
`
`~1
`
`
`Control
`Icnsz
`Tamoxifen
`'
`(n=|0)
`1emg(n=|2)
`(n=i5)
`
`Control
`(n27)
`
`Icnez
`iemg(n=a)
`
`Tamoxifen
`(n=15)
`
`.
`
`Control
`("=10)
`
`louse
`emg(n=e)
`
`Icnaz
`ramg(n=12)
`
`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—regulated 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
`
`InnoPharma Exhibit 1053.0013
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`

`358
`
`1.2
`
`1.0
`
`0.8
`
`a)
`L-
`
`8
`(I)
`I 0.6
`E
`L“
`
`0.4
`
`0.2
`
`0.0
`
`‘ "2
`
`1-0
`
`0.8
`
`(n
`‘—
`
`8
`U)
`I 0.6
`E
`Kl
`
`0.4
`
`0.2
`
`0.0
`
`‘
`
`r——‘——T———-r—
`Pre
`Post
`
`r———v_-—fi—‘
`Pre
`Post
`
`10° \ 20
`
`is
`
`mE.
`
`E
`
`g
`2
`E 10
`E
`s
`,‘2K
`
`‘[
`
`5
`
`60
`
`o 0
`
`513
`
`NU
`
`U)
`)
`a 40
`
`20
`
`o
`
`r————1————1——
`Pre
`Post
`
`\ 2
`o A
`
`r———r—.———r——
`Pre
`905‘
`
`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
`
`InnoPharma Exhibit 1053.0014
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`

`359
`
`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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`. Kluwer, 1991;239—257.
`2. Wilson APM, Weatherill PJ, Nicholson RI, Davies P, Wakeling AE. J Steroid Biochem 1990;35:
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`Wakeling AE, Bowler J. J Steroid Biochem 1988;31:645—653.
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`Manning DL, Nicholson RI, Eaton CL. In: Recent Advances in Endocrinology and Metabolism, vol
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`Nicholson RI. Curr Opin Invest Drugs 1993;2z1259—1268.
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`'
`11. DeFriend DL, Howell A, Nicholson RI et al. Cancer Res 1994;54:1—7.
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`15. von Angerer E, Knebel N, Kager M, Ganss B. J Med Chem 1990;33:2635—2640.
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`18. van Dierendonck JH, Wijsman JI—I, Keijzer R, van de Velde CJ, Cornelisse CJ. Am J Pathol
`1991;138:1165—1172.
`
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`I
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`g
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`IO, Elston CW, Robertson JFR, Blarney RW. Br. J.
`
`19. Bouzubar N, Walker KJ, Griffiths K,
`'
`Cancer 1989;59:943—947.
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`23. Bates SE, Davidson NE, Valverius EV, Freter CE, Dickson RB, Tam JP, Kudlow JE, Lippman ME,
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`v
`24. Gee JMW,. Robertson JFR, Hoyle HB, Kyme SR, McClelland 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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`*
`27. Dauvois S, Danielian PS, White R, Parker MG. Proc Natl Acad Sci USA 1992;89:4037—4041.
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`'
`35. Wakeling AE. Breast Cancer Res Treat 1993;5:1—9.
`
`‘
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`InnoPharma Exhibit 1053.0016
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