[CANCER RESEARCH 51, 3867-3873, August 1, 1991]
`
`A Potent Specific Pure Antiestrogen with Clinical Potential
`Alan E. Wakeling,1 Michael Dukes, and Jean Bowler
`
`Bioscience I ¡A.E. W., M. D.J and Chemistry I fj. B.], ICI Pharmaceuticals, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, United Kingdom
`
`ABSTRACT
`
`Previous studies from this laboratory have described a series of 7a-
`alkylamide analogues of estradiol with pure antiestrogenic activity, ex
`emplified by ICI 164,384. A new compound, 7«-|9-(4,4,5,5,5-pentafluo-
`ropentylsulfinyl)nonyIjestra-1,3,5(
`10)-triene-3,17/3-diol
`(ICI 182,780)
`has now been identified which has significantly increased antiestrogenic
`potency and retains pure estrogen antagonist activity. The antiutero-
`trophic potency of ICI 182,780 in the immature rat was more than 10-
`fold greater than that of ICI 164,384 (50% effective doses of 0.06 and
`0.9 mg/kg, respectively). This order of magnitude increase of in vivo
`potency was also reflected,
`in part, by intrinsic activity at the estrogen
`receptor. The relative binding affinities of ICI 182,780 and ICI 164,384
`were 0.89 and 0.19, respectively, compared with that of estradiol (1.0).
`Similarly, the in vitro growth-inhibitory potency of ICI 182,780 exceeded
`that of ICI 164,384 in MCF-7 human breast cancer cells, where 50%
`inhibitory concentrations of 0.29 and 1.3 HIM,respectively, were recorded.
`ICI 182,780 was a more effective inhibitor of MCF-7 growth than 4'-
`hydroxytamoxifen, producing an 80% reduction of cell number under
`conditions where 4'-hydroxytamoxifen
`achieved a maximum of 50%
`inhibition. This increased efficacy was reflected by a greater reduction of
`the proportion of cells engaged in DNA synthesis in ICI 182,780-treated
`cell cultures compared with tamoxifen-treated cells.
`Sustained antiestrogenic effects, following a single parenteral dose of
`ICI 182,780 in oil suspension, were apparent
`in both rats and pigtail
`monkeys. In vivo, antitumor activity of ICI 182,780 was demonstrated
`with xenografts of MCF-7 and BrlO human breast cancers in nude mice.
`A single injection of ICI 182,780 provided antitumor efficacy equivalent
`to that of daily tamoxifen treatment for at least 4 weeks.
`The properties of ICI 182,780 identify this pure antiestrogen as a
`prime candidate with which to evaluate the potential therapeutic benefits
`of complete estrogen withdrawal
`in endocrine-responsive human breast
`cancer.
`
`of the kind outlined above led us to search
`Considerations
`for novel molecules which would bind ER with high affinity
`without activating any of the normal
`transcriptional
`hormone
`responses
`and consequent manifestations
`of estrogen action.
`Such molecules would be clearly distinguished from tamoxifen-
`like ligands and would be pure antiestrogens. The rationale for
`the design and testing of novel putative pure antagonists
`has
`been described elsewhere (7, 8), as have the first examples of
`such compounds
`(9-11). The prototype pure antiestrogen,
`ICI
`164,384, a 7a-alkylamide
`analogue of estradiol,
`is devoid of
`stimulatory activity and blocks completely the trophic actions
`of estrogens
`and of the partial antiestrogens
`in all estrogen-
`responsive cell and animal models examined to date (see Ref.
`12 for a review).
`In this report we describe some of the properties of a new
`pure antiestrogen,
`ICI 182,780.
`ICI 182,780 is a potent and
`specific inhibitor of estrogen action and demonstrated
`excellent
`growth-inhibitory
`effects in both cell and animal models of
`human breast cancer. Such properties
`identify ICI 182,780 as
`a prime candidate with which to explore the therapeutic poten
`tial of pure antiestrogens
`in the treatment of breast cancer.
`
`MATERIALS AND METHODS
`
`INTRODUCTION
`
`exemplified by tamoxifen [ICI
`antiestrogens,
`Nonsteroidal
`46,474 (Nolvadex)], have been used extensively, and with great
`success,
`in the therapy of breast cancer
`(1, 2). Antiestrogens
`compete with endogenous
`estrogens
`for binding to ER2 but a
`complete description of the mode of action of these molecules
`remains elusive (3, 4). In particular,
`it is difficult
`to account for
`the diversity of biological
`actions which range between full
`agonist, estrogen-like
`trophic effects,
`through partial agonism
`to complete blockade of estrogen action. This diversity was first
`apparent
`in species differences of organ response (5) but re
`markably extends to differential effects of tamoxifen on estro
`gen-responsive
`genes within target cells (6). Because of the
`potential of nonsteroidal
`antiestrogens
`to manifest stimulatory
`activity it remains unclear whether
`their clinical activity is in
`any way limited compared with that which might be achieved
`by complete endocrine ablation.
`
`Received 4/10/91; accepted 5/21/91.
`The costs of publication of this article were defrayed in part by the payment
`of page charges. This article must
`therefore be hereby marked advertisement
`in
`accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
`' To whom requests for reprints should be addressed.
`2The abbreviations used are: ER. estrogen receptor(s); ICI 164.384, /V-n-butyl-
`yv-meth) I-11-(3,17/3-dihydroxyestra-1,3,5( 10)-triene-7«-yl)undecanamide;
`ICI
`182.780. 7«-[9-(4,4,5,5,5-pentafluoropentylsulfinyl)nonyl]estra-1.3,5(
`10)-triene-
`3,17/i-dioI; OVX, ovariectomized;
`IC50,50% inhibitory concentration; ED50. 50%
`effective dose.
`
`trans-l-(4-ß-
`tamoxifen (ICI 46474;
`Reagents. The antiestrogens
`dimethylaminoethoxyphenyl)-l,2-diphenylbut-l-ene),
`the trans-4'-hy-
`droxy metabolite of tamoxifen,
`ICI 164,384, and ICI 182,780 were
`synthesized in Chemistry Department
`I, ICI Pharmaceuticals.
`Struc
`tures of ICI 164,384 and ICI 182,780 are illustrated in Fig. 1. Stock
`solutions of these agents were prepared in ethanol, stored at 4°C,and
`diluted as required. 17j3-[3H]estradiol, 85-110 Ci/mmol,
`and sodium
`12SI-iodide, IMS 30, were obtained from the Radiochemical Centre,
`Amersham, England. Materials for gonadotropin assays were obtained
`from the National
`Institute for Arthritis, Metabolism, and Digestive
`Diseases, Bethesda, MD except
`for ovine luteinizing hormone
`for
`iodination, supplied by L. E. Reichert, Emory University, Atlanta, GA.
`170-Estradiol benzoate,
`insulin, and materials for flow cytometry (pro-
`pidium iodide, bromodeoxyuridine,
`anti-mouse IgG fluorescein isothio-
`cyanate conjugate; F 0257) were obtained from Sigma Chemical Com
`pany, Poole, Dorset, England, except for purified mouse anti-bromo-
`deoxyuridine monoclonal antibody (No. 7580) which was from Becton
`Dickinson, Mountain View, CA. All materials
`for cell culture were
`from Gibco, Paisley, Scotland, with the exception of Costar
`flasks
`which were from Northumbria Biologicals, Cramlington, England.
`MCF-7 cells were obtained from Dr. C. M. McGrath, Michigan Cancer
`Foundation, Detroit, MI, and BT20 cells were from Dr. J. Taylor,
`Imperial Cancer Research Fund, London, England.
`Estrogen Receptor Binding Assay. The method used for competitive
`binding assays to measure the relative affinity of antiestrogens
`for rat
`uterine ER has been described elsewhere (13) except
`that competitor
`dilutions were prepared in tris:dimethylformamide
`(1:1) (14) and mixed
`together with 17/3-[3H]estradiol and then with cytosol at a ratio of 1:20.
`Cell Proliferation and Flow Cytometry Studies. The methods used for
`MCF-7 cell culture and growth inhibition assays have been detailed
`elsewhere (15). Briefly, cells were cultured in multiwell plates (24-well,
`seeding density 4 x IO4)in minimal essential medium containing phenol
`red,
`insulin (10 ^g/rnl), and 5% charcoal-stripped
`fetal calf serum
`without estradiol. Antiestrogens and/or estradiol were added at 1000-
`fold dilution from ethanol stock, in fresh medium 2 days after seeding.
`Cultures were maintained for 5 days with one further medium change
`and growth was assessed by measurement of total cell protein at the
`3867
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`PURE ANTIESTROGENS
`
`5 x IO6 cells). Mice were
`female nude mice (0.1 ml/approximately
`maintained in a clean environment
`and were given sterile food and
`water. Estrogen supplementation was provided by ethynyl estradiol at
`1 Mg/ml in the water. Antiestrogen treatment was initiated when tumor
`diameter attained a minimum of 0.5 cm. The BrlO tumor at passage
`49 was obtained from Dr. N. Brunner
`(Copenhagen, Denmark) and
`established by implantation of 1-2-mm'
`tumor fragments into the flank
`of anesthetized
`intact adult
`female nude mice. After 3 passages a
`reproducible pattern of growth was established without additional es
`trogen supplementation. Approximately
`two-thirds of animals estab
`lished progressively growing tumors which attained measurable size
`(area, =70 mm2) after 6-7 weeks. Antiestrogen treatment was initiated
`at the time of transplantation. Tamoxifen was administered once daily
`p.o. at a dose of 10 mg/kg (1 ml/100 g body weight of aqueous
`dispersion in 0.5% Tween 80) and ICI 182,780 as a single s.c. injection
`of 5 mg/mouse
`(50 mg/ml
`in arachis oil). Tumor
`size was assessed
`weekly as the product of caliper measurements of the largest diameter
`and the axis perpendicular
`to it.
`
`" (CH2), 0CON(CH2)3CH3
`
`IC
`
`H3
`
`ICI 164,384
`
`'"•(CH2)9SO(CH2)3CF2CF3
`
`RESULTS
`
`Estrogen Receptor Interaction
`
`ICI 182,780
`Fig. 1. Structure of pure antiestrogens.
`
`and compared with that of controls
`beginning and end of treatment
`treated with ethanol (0.1%) alone. BT-20 cells were treated similarly.
`The measurement of antiestrogen effects on cell cycle and population
`distribution of MCF-7 cells using two parameter
`flow cytometry fol
`lowed the method described previously (16).
`Assays of Estrogenic/Antiestrogenic Effects. The rat uterine weight
`assay for the measurement of estrogenic and antiestrogenic activity has
`been described elsewhere (17). Details of doses, route of administration,
`and duration of treatment are reported in individual figure legends. ICI
`182,780 and 17/3-estradiol benzoate were prepared for administration
`by diluting an ethanol stock solution into the required volume of arachis
`oil with gentle warming (60°C).Tamoxifen was prepared for adminis
`tration p.o. as a dispersion in aqueous 0.5% Tween 80. For immature
`and mature rats the dose volumes were 0.5 and 0.1 ml/100 g body
`weight,
`respectively.
`In studies with intact
`rats, blood samples were
`collected terminally for measurement of luteinizing hormone,
`follicle-
`stimulating hormone, and prolactin. Plasma gonadotropin concentra
`tions were determined by a modification of the double antibody tech
`nique described by Niswender et al. (18).
`In studies with OVX rats, surgical preparation was performed at
`least 2 weeks before treatment began. To measure the duration of action
`of a single large dose of ICI 182,780, OVX rats were treated with a
`daily s.c. dose of 0.5 ^g of estradici benzoate beginning on the day of
`ICI 182,780 administration and continued until vaginal smears showed
`evidence of cornification. At that point
`the experiment was terminated
`and uterine weight was recorded. The arachis oil formulation used in
`these single dose duration of action studies contained
`50 mg ICI
`182,780/ml.
`For studies of the duration of action of ICI 182,780 in monkeys,
`adult female pigtail macaques (Macaca nemestrina) weighing 6-8 kg
`were ovariectomized not less than 6 months before treatment. Prelim
`inary studies established that daily s.c.
`treatment of OVX monkeys
`with 5 ng estradiol benzoate/kg induced permeai swelling in a repro
`ducible manner with individual maxima being attained after 11 days.
`The magnitude of the estrogenic effect was assessed visually on an
`arbitary scale of 1-6. Groups of five monkeys were treated s.c. once
`daily for 10 days with 0.1-1.0 mg/kg ICI 182,780, or with a single dose
`of 10 mg/kg, 10 days before beginning estradiol
`treatment. Perineal
`size was estimated daily and the time taken for initiation of swelling
`(mean score, 2) and attainment of maximum (score, 4-6) was recorded.
`Tumor Growth Inhibition Assays. MCF-7 cells were suspended in
`culture medium (no serum) and inoculated s.c. into the flank of adult
`3868
`
`The capacity of ICI 182,780 to compete with 17/3-[3H]estra-
`diol for binding to rat uterine ER was evaluated and compared
`with that of estradiol and the previously reported pure anties
`trogen ICI 164,384 (Fig. 2). Each antiestrogen displaced 170-
`[3H]estradiol
`in a concentration-dependent
`manner
`and the
`displacement
`curves were parallel
`to that of estradiol.
`IC50
`values of 0.83, 0.94, and 4.48 x 10~8 M were recorded for
`estradiol,
`ICI 182,780, and ICI 164,384, respectively. Relative
`binding affinities calculated from these IC50 values were 0.89
`and 0.19 for ICI 182,780 and ICI 164,384,
`respectively, com
`pared with that of estradiol
`(1).
`
`Estrogenic/Antiestrogenic Effects
`
`to immature
`(s.c.),
`alone, parenterally
`When administered
`female rats ICI 182,780 was devoid of uterotropic activity and,
`when coadministered with estradiol,
`it effectively blocked the
`uterotropic
`action of estradiol
`in a dose-dependent manner
`[ED5o 0.06 mg/kg/day s.c. (Fig. 3/1)]. Complete antagonism of
`estrogen action was achieved with a dose of 0.5 mg ICI 182,780/
`kg/day s.c. The effects of ICI 182,780 administered
`p.o. were
`qualitatively similar but potency was reduced by an order of
`
`100-1
`
`-9
`
`-8
`
`-7
`
`-6
`
`[Competitor]log 10
`to rat uterine
`Fig. 2. Competition for binding of 5 x 10~' M ['H]17/3-estradiol
`estrogen receptor by unlabeled 17fi-estradiol,
`ICI 182.780. and ICI 164,384.
`Percent
`inhibition refers to specific binding corrected by subtraction from total
`17/j-[3H]estradiol bound,
`the nonspecific component
`recorded in the presence of
`5 x 10~7 M unlabeled 17/3-estradiol. Points, mean of 9 observations
`in three
`different experiments; bars, SEM. IC!0 values were calculated by linear regression
`analysis of percentage of inhibition versus Iogi0(competitor].
`
`
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`

`
`240
`
`200
`
`160-
`
`SI
`
`80
`
`40
`
`o-
`0.02
`
`240-
`
`200-
`
`160-1
`
`f-,
`
`80
`
`40-
`
`0.05
`
`01
`
`02
`
`05
`
`.....
`
`I////////7/I
`
`(B)
`
`Pl'RE ,\NTIESTRO(¡I-:SS
`
`fashion (Fig. 5). At
`weight of the uterus in a dose-dependent
`the highest dose in this study, 1 mg/kg/day,
`involution of the
`uterus after 14 days approached
`that
`following ovariectomy.
`Cyclical vaginal cornification was blocked partially (0.1 mg/
`kg/day) or completely (0.3 mg/kg/day)
`but body weight gain
`and serum gonadotropin concentrations were largely unaffected
`(Table 1). The p.o. antiuterotropic
`activity of ICI 182,780 in
`intact rats was substantially less than its parenteral potency; at
`10 mg/kg/day for 14 days an effect approximating
`50% that of
`ovariectomy was recorded.
`administered
`that many steroids
`Following the precedent
`parenterally in oil have a sustained duration of action,
`the effect
`of ICI 182,780 administered
`as a single s.c. bolus dose in oil
`suspension was tested in adult ovariectomized
`rats. The initia
`tion of vaginal cornification and uterine growth by daily admin
`istration of 0.5 ng of estradici benzoate was blocked for more
`than 6 weeks by 10 mg of ICI 182,780. Uterine weights 42 days
`after ICI 182,780 for ovariectomized controls, estrogen-treated
`controls, and ICI 182,780 plus estrogen-treated
`rats were 60.6
`±3.5 (SEM), 311 ±26, and 63.3 ±0.6 mg (n = 5), respectively.
`Similar
`treatment
`of intact adult
`females completely blocked
`
`0.2
`
`05
`
`2Ü¡ SX) ÎÎ.O
`
`Fig. 3. Effects of ICI 182.780 on uterine weight of immature rats. Animals
`received daily, a single dose of arachis oil vehicle alone (D), 0.5 ^g 17^-estradiol
`benzoate s.c. alone (D). or the indicated doses of ICI 182.780 alone (----
`) or
`together with estradici
`(-
`). for 3 days. A. parenteral
`(s.c.) administration: B.
`P.O. administration. Points, means for a minimum of 10 observations
`in at least
`2 different experiments.
`In this and succeeding figures bars on each point represent
`the SEM. Where no bar is present errors were smaller than the symbols.
`
`300-
`
`250-
`
`f
`
`200-1
`
`IS
`
`150'
`
`100-
`
`50-
`
`02
`
`0.5
`
`1.0
`
`2.0
`
`Dose (mg/kg)
`
`50
`
`10.0
`
`effect of tamoxifen by ICI 182,780.
`Fig. 4. Antagonism of the uterotropic
`Immature rats were treated as described in the legend to Fig. 3, except that
`the 0
`represents
`the effect of 1 mg tamoxifen/kg
`alone and
`is the effect of the
`indicated doses of ICI 182.780 together with tamoxifen. Points, mean for at least
`5 observations: oars, SEM.
`
`magnitude compared with s.c. dosing [cf. ED50 0.46 and com
`plete antagonism at 5 mg/kg/day p.o. (Fig. 3fi)]. Similarly,
`the
`uterotropic
`action of tamoxifen was also blocked in a dose-
`dependent manner by coadministration
`of ICI 182,780 (Fig. 4).
`Complete blockade of tamoxifen action required an approxi
`mately 5-fold dose ratio. Similar
`studies of uterotropic
`and
`antiuterotropic
`activity in immature mice and in ovariectomized
`adult rats and mice provided confirmation
`of the pure antago
`nist profile of ICI 182,780 (data not shown).
`fe
`of intact adult
`Chronic daily parenteral
`(s.c.) treatment
`male rats with increasing doses of ICI 182,780 reduced the
`3869
`
`o-
`
`0.03
`
`0.1
`Dose mg/kg
`
`0.3
`
`1.0
`
`Fig. 5. Effect of ICI 182.780 on the uterus of intact adult rats. Groups of 5
`adult rats with regular 4-day estrous cycles were treated once daily, for 14 days,
`with arachis oil vehicle alone (D) or the indicated doses of ICI 182,780 s.c. Uterine
`weight was also recorded for vehicle-treated animals ovariectomized at the begin
`ning of the study (D).
`
`Table 1 Effect of ICI 1X2,780 on body weight and plasma gonadotropiâ„¢ of adult
`female rats
`Values are mean ±SEM: n = 5. All ICI 182.780 values differ from correspond
`ing OVX controls, at P < 0.001.
`(ng/ml)TreatmentIntact
`
`Gonadotropins
`
`controlOVX
`
`controlICI
`
`wt
`
`gain(g)40.0
`
`±2.564.8
`
`
`
`
`±1.9°43.6
`
`hormone2.4
`
`±0.619.7
`±2.2"2.1
`
`stimulating
`hormone3.0
`
`±0.424.0
`
`±0.5°2.2
`
`±3.33.7
`
`
`±0.7°18.2
`
`182.780(mg/kg)
`0.03
`±2.5
`±0.2
`±8.9
`±0.2
`44.6 ±1.7
`O.I
`1.2 ±0.1
`19.1 ±6.4
`2.5 ±0.6
`45.8 ±2.0
`0.3
`1.0 ±0.1
`2.5 ±0.5
`28.8 ±17.2
`6.0 ±2.2"
`1.0Body
`42.6 ±2.1Luteinizing
`2.3 ±0.3Follicle-
`3.6 ±0.6Prolactin25.3
`°P < 0.001 versus intact control.
`* P < 0.05 versus intact control.
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`
`Table 2 Antiestrogenic action of ICI 182,780 in ovariectomiied estrogen-treated
`monkeys
`
`PURE ANTIESTROGENS
`
`permeaiTreatmentControl
`
`
`
`(estradici benzoatealone)ICI
`
`182,780 10 days: pretreatmentwith0.
`
`
`1 mg/kgs.c.0.5
`
`mg/kgs.c.1.0
`
`mg/kgs.c.10.0
`mg/kg s.c.>2513184123score
`
`182,780 on MCF-7 cells was reversed in a competitive manner
`by estradiol
`(Fig. 7). In the presence of 10~8 M ICI 182,780,
`coincubation with 10"'" M estradiol had no effect but growth
`Days from start of
`
`estrogentreatmentto
`inhibition was reversed partially at 10~9 M and completely by
`reach
`10~8M estradiol. For MCF-7 cells grown in medium containing
`of4-6(maximum)1117234733
`alone provided a moderate,
`red addition of estradiol
`phenol
`concentration-dependent
`growth stimulus (Fig. 7).
`A comparison of the effect of ICI 182,780 with that of other
`antiestrogens
`on the growth of MCF-7 cells (Fig. 8) showed
`that
`it was significantly more potent
`than ICI 164,384 (IC50 =
`0.29 and 1.3 nM, respectively) or 4'-hydroxytamoxifen.
`Also,
`like ICI 164,384,
`the maximum growth-inhibitory
`effect of ICI
`182,780 exceeded that of 4'-hydroxytamoxifen
`[approximately
`80% cf. 50% (Fig. 8)].
`distri
`Flow cytometric analysis of cell cycle and population
`bution of MCF-7 cells treated with tamoxifen or ICI 182,780
`showed that both antiestrogens
`caused accumulation of cells in
`Co/Gìand also reduced the proportion
`of cells capable of
`continued DNA synthesis
`(Table 3). However,
`the maximal
`efficacy of ICI 182,780 compared with that of tamoxifen, when
`both compounds were used at optimum antiestrogenic
`(but not
`cytotoxic concentrations), was much greater. Thus, only 7% of
`cells were still potentially capable of division after 3-5 days of
`treatment with 10 nM ICI 182,780 compared with 37% in
`cultures treated with 4 ßMtamoxifen.
`Human Breast Tumors in Vivo. The effects of ICI 182,780
`were compared with those of tamoxifen in two models of human
`breast cancer grown in nude mice. The growth of xenografts of
`MCF-7 human breast cancer cells, supported
`by continuous
`treatment with ethynyl estradiol, was blocked completely for at
`least 4 weeks by a single s.c. injection of 5 mg of ICI 182,780
`in oil suspension
`(Fig. 9). The magnitude of this effect was
`comparable with that
`in animals
`treated continuously with a
`high dose of tamoxifen (10 mg/kg/day P.O.).
`tumor
`The growth of transplants
`of the BrlO human breast
`was also suppressed effectively by ICI 182,780. Mice implanted
`with a 1-2-mm1 tumor mass were given a single 5-mg s.c.
`injection of ICI 182,780 on the day of implantation
`or daily
`treatment
`for 8 weeks with tamoxifen
`(10 mg/kg/day
`P.O.).
`Tumor measurements
`(Fig. 10) showed a substantial
`and sus
`tained reduction of tumor growth in ICI 182,780-treated mice
`
`[ICI 182.780] log 10
`
`Fig. 6. Effects of ICI 182.780 on the proliferation of MCF-7 and BT-20 human
`breast cancer cells. Cells were plated in 24-well dishes (4 x 104/well) and cultured
`for 2 days in minimal essential medium with 5% charcoal-stripped
`fetal calf
`serum containing phenol red and insulin but no additional estrogens. One dish
`was assayed for total protein (Lowry) as day 0 control; remaining dishes received
`fresh medium with (treated) or without
`(control) the indicated concentrations of
`ICI 182,780 added in ethanol (l ¿ig/mlmedium). Cells were grown for a further
`5 days with fresh medium added after 3 days. Cell growth is represented as the
`difference between the increase of total protein in control and treated wells
`between day 0 and day 5. Points, mean of quadruplicate observations where SEM
`was less than 5%.
`
`cyclical vaginal cornification for at least 3 weeks and regressed
`the uterus
`(24% cf.
`intact control weight at 21 days after
`treatment).
`activity of ICI 182,780 was also measured
`The antiestrogenic
`in OVX pigtail macaques. Maximum swelling is attained after
`11 days of estrogen treatment
`(5 ¿¿g/kg/day).Pretreatment
`of
`monkeys with 0.1, 0.5, or 1 mg ICI 182,780/kg/day
`s.c. for 10
`days prior to estrogen replacement produced an increasing delay
`in the onset of perineal
`swelling, of the order of 1, 2, and 5
`weeks, respectively (Table 2). Administration
`of a single dose
`of 10 mg ICI 182,780/kg
`s.c.
`in oil suspension delayed the
`onset of perineal
`swelling by 3 weeks and the attainment
`of
`maximum swelling by in excess of 4 weeks (Table 2).
`
`140-1
`
`120-
`
`100
`
`20-
`
`&gQ
`
`. 3
`
`O
`
`10 12
`
`10 •" 10-'°
`
`10 9
`
`[Estradiol] log 10
`
`Breast Cancer Growth Inhibition
`
`ICI 182,780 was an
`Human Breast Cancer Cells in Vitro.
`effective inhibitor of the growth of ER-positive MCF-7 human
`breast cancer cells but was without effect on the growth of ER-
`negative BT-20 human breast cancer cells (Fig. 6). ICI 182,780
`was fully effective at 10~9M on MCF-7 cells grown in medium
`containing phenol
`red but without added estradiol. Cell death
`was not observed in either MCF-7 or BT-20 cells exposed to
`IO'5 M ICI 182,780. The growth-inhibitory
`action of
`ICI
`
`Fig. 7. Effects of 17/i-estradiol on the growth of MCF-7 cells in the absence
`and presence of 10~"M ICI 182.780. The experimental procedure was as described
`for Fig. 6.
`3870
`
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`
`
`Research.
`
`MYLAN PHARMS. INC. EXHIBIT 1008 PAGE 4
`
`

`
`O ICI 182,780
`
`A ICI 164,384
`
`•4-hydroxytamoxifen
`
`PURE ANTIESTROGENS
`
`in which the amide moiety was
`tion of the 7«side chain,
`replaced by other polar groups and the terminal alkyl function
`was fluorinated
`(10), produced the pentafluoropentylsulfinyl
`compound ICI 182,780.
`(Fig. 2) and as a specific and
`In receptor binding studies
`reversible inhibitor of MCF-7 breast cancer cell growth (Figs.
`6-8),
`ICI 182,780 demonstrated
`an approximately
`5-fold in
`crease of intrinsic potency compared with ICI 164,384. This
`increased potency was clearly manifest
`in vivo where,
`in the rat
`antiuterotropic
`assay,
`ICI 182,780 [ED50 = 0.06 mg/kg (Fig.
`3)] was at least an order of magnitude more potent
`than ICI
`
`100-1
`
`80-
`
`60-
`
`40-
`
`20 J
`
`o-
`
`Q 8 Co
`
`>
`
`oo
`
`.
`"ÔJ
`O
`
`i
`-10
`
`\
`-9
`
`r
`-7
`
`-6
`
`log 10
`[Antiestrogen]
`Fig. 8. Effects of different antiestrogens on the growth of MCF-7 cells. The
`experimental procedure was as described for Fig. 6. Points, mean derived from
`three or more different experiments with quadruplicate
`observations
`in each.
`SEM was less than 4%.
`
`Table 3 Effects of antiestrogens on population distribution of MCF-7 human
`breast cancer cells
`cellsCyclingTreatmentControlTamoxifen0.4
`% of
`
`300 n
`
`200-
`
`100--
`
`II1
`
`-1-G2 +M81211101314791010
`+ G2 +M26711810223211NoncyclingGo/G,10414452501777798482S
`
`1234
`
`Control
`
`1234
`
`1234
`
`Tamoxifen
`
`ICI 182,780
`
`Weekly tumor measurement
`Fig. 9. Effect of ICI 182,780 and tamoxifen on the growth of established MCF-
`7-derived tumors in nude mice. Columns, means (n Ì5) of tumor area normalized
`by reference to initial area preceding the 4-week treatment period: bars, SEM.
`All animals received continuous ethynyl oliatimi
`(1 ng/ml
`in drinking water).
`Additionally,
`tamoxifen-treated
`animals were dosed daily (10 mg/kg p.o.) and
`ICI 182,780 once (5 mg/mouse s.c.) at the beginning of the 4-week measurement
`period.
`
`140-1
`
`120-
`
`•Control
`* Tamoxifen
`o IC1182,780
`
`CM
`
`—100-
`E
`-§-BOH
`•
`
`60H
`
`40-
`
`20-
`
`2Ã(cid:143)
`
`o K
`
`50
`
`60
`
`70
`
`80
`
`90|
`
`100
`
`110
`
`120
`
`130
`
`Days post implantation
`Fig. 10. Effect of ICI 182,780 and tamoxifen on the growth of BrlO human
`breast tumors in nude mice. Groups of 10 female nude mice bearing transplants
`of BrlO received either no treatment
`(control), daily tamoxifen (10 mg/kg p.o.)
`for 8 weeks beginning on the day of transplantation,
`or a single dose of ICI
`182,780 (5 mg/mouse s.c.). Values are mean tumor area, (n = 6-8) for all tumors
`attaining measurable size by day 50 postimplantation:
`bars. SEM. Approximately
`3 months postimplantation
`all mice were ovariectomized (arrow) and tumor
`measurements were continued for 1 month further.
`
`M1
`x IO'6
`
`X IO'6M2
`
`x 10~6M4
`
`x 10-'MICI
`182,7800.4
`x I0~9
`M1
`
`x 10"'M2
`
`x IO'9M4
`
`x 10"'M10
`x 10-' MGo/G,564135302747131046S
`
`to that of high-dose tamoxifen treatment. Note that 2
`similar
`weeks after the end of tamoxifen treatment
`tumor growth rate
`showed evidence of a return to control
`level whereas, even 3
`months after a single dose of ICI 182,780,
`tumor growth rate
`remained below that of control. Ovariectomy
`of all animals
`after 3 months demonstrated
`the estrogen sensitivity of these
`tumors (Fig. 10).
`
`DISCUSSION
`
`exemplified by
`The discovery of novel steroidal antiestrogens
`ICI 164,384 (9, 10) provided for the first
`time pure estrogen
`antagonists which have shed new light on the physiology (11,
`19-22) and the molecular mode of action of estrogens
`and
`antiestrogens
`(23-26). Among the initial series of 7«-alkylam-
`ide analogues of 17/3-estradiol described previously (10) none
`was of sufficient potency in vivo to merit serious consideration
`as a candidate for clinical use. We therefore sought
`to identify
`more potent compounds which retained the potentially advan
`tageous properties of ICI 164,384. For breast cancer treatment,
`these included high affinity for ER (27),
`the absence of estro-
`genie activity (9, 11-14), and more effective antiproliferative
`action on breast cancer cells than classical
`tamoxifen-like par
`tial agonist antiestrogens
`(22-24). Further
`synthetic modifica-
`
`3871
`
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`cancerres.aacrjournals.org Downloaded from
`
`on December 28, 2015. © 1991 American Association for Cancer
`Research.
`
`MYLAN PHARMS. INC. EXHIBIT 1008 PAGE 5
`
`

`
`PURE ANTIESTROGENS
`
`164,384 [ED50 = 0.9 mg/kg; see Ref. 15]. The apparent 2-fold
`difference in potency ratio improvement
`between in vitro and
`in vivo assays for the two compounds
`is likely a reflection of
`differences in distribution and metabolism. Both in vitro and in
`vivo studies were consistent with a competitive
`interaction
`between ICI 182,780 and estradiol
`for binding to ER. The
`absence of a significant estrogenic activity of ICI 182,780 was
`clearly apparent
`in rodent uterotropic
`assays (e.g., Fig. 3) and
`in its capacity to block completely the stimulatory
`action of
`tamoxifen (Fig. 4).
`potential of ICI
`Of particular
`relevance to the therapeutic
`182,780 are two observations
`reported here: (a) the enhanced
`efficacy compared with 4'-hydroxytamoxifen
`(or tamoxifen) on
`breast
`tumor
`cells (Fig. 8; Table 3); and (b)
`the excellent
`antiuterotropic
`action (Figs. 3-5; Table 2) achieved without
`affecting body weight and gonadotropin
`secretion (Table 1).
`The castration-like uterine involution achieved in intact animals
`in the absence of an effect on the latter indices of hypothalamic-
`pituitary function indicates
`that
`ICI 182,780 might be differ
`entially active against peripheral and central
`targets of estrogen
`action, a property shared with ICI 164,384 (15). If translated
`to the clinical setting,
`this peripheral selectivity of action would
`obviate blockade of central negative estrogen
`feedback and
`consequent
`increases of estrogen production
`in the premeno-
`pausal patient. With respect
`to the enhanced efficacy of pure
`antiestrogens
`against
`tumor cell growth in vitro we have shown
`previously for ICI 164,384 ( 16,28-30)
`and here for ICI 182,780
`(Table 3) that fewer of the cells remain in the actively prolifer
`ating fraction than is the case when partial agonists like tamox
`ifen, 4'-hydroxytamoxifen,
`or hydroxyclomiphene
`are used.
`This has been attributed
`to a residual
`stimulatory
`estrogenic
`effect of the partial agonists which, although small (16, 31), is
`amplified synergistically by the concurrent
`presence of other
`breast cell mitogens
`like insulin (16) and insulin-like growth
`factor 1 (32). The pure antiestrogens
`obviate such effects. The
`corollary of these data in the clinical setting is the possibility
`that differences of antitumor
`efficacy between tamoxifen and
`pure antiestrogens may be greater
`than otherwise anticipated.
`The order of magnitude lower potency between the p.o. and
`parenteral
`routes of administration
`(Fig. 3) suggests strongly
`that
`the p.o. bioavailability of ICI 182,780 is relatively low. A
`common means of circumventing the practical constraints
`con
`sequent on the poor p.o. bioavailability of steroids
`is to use
`parenteral
`depot
`formulations with an extended duration
`of
`action. The utility of this approach was demonstrated with ICI
`182,780 dispersed in arachis oil. Thus, single s.c. injections of
`ICI 182,780 in ovariectomized,
`estrogen-treated
`rats and mon
`keys (Table 2) provided extended antiestrogenic
`activity.
`of ICI
`The potential
`efficacy of "oil depot"
`formulations
`studies.
`182,780 was demonstrated
`in nude mouse antitumor
`The antitumor action of a single parenteral dose of ICI 182,780
`on MCF-7 xenografts was similar
`to that achieved by daily
`administration
`of a high dose of tamoxifen over a 4-week period
`(Fig. 9). Tumor growth ceased in ICI 182,780-treated
`animals
`but no significant
`tumor
`involution was seen, an effect consist
`ent with previous observations
`in this
`(33) and other
`(34)
`laboratories where estrogen withdrawal
`failed to precipitate
`tumor
`regression. The absence of superior
`efficacy of
`ICI
`182,780, compared with that of tamoxifen,
`is consistent with
`the fact that
`tamoxifen lacks significant
`in vivo tumor growth-
`stimulatory action, and high doses of tamoxifen did not cause
`tumor
`regression in short-term studies with this tumor model
`(34). The effect of ICI 182,780 in vivo is therefore consistent
`
`than a cytotoxic action. Others, using
`with a cytostatic rather
`the same model system, have reported tumor
`involution during
`tamoxifen treatment
`to the extent
`that
`tumors almost disap
`peared after 3-4 weeks (35). This has been attributed
`to an
`alteration in cell death rate (35). In the current
`study (Fig. 9),
`3-4 weeks of continuous
`high dose tamoxifen treatment
`pro
`duced a significant decrease (P < 0.05) of mean tumor volume,
`compared with that at
`the start of treatment,
`but not
`tumor
`disappearance. A recent study of the effects of estrogen with
`drawal on MCF-7 tumors in nude mice has also demonstrated
`tumor
`regression associated with both cessation of cell prolif
`eration
`and the activation
`of programmed
`cell death (36).
`Kyprianou et al. (36) attribute
`interlaboratory
`differences
`in
`apparent
`response
`to hormone withdrawal
`to variations
`in
`MCF-7 cell phenotype where some but not all sublines have
`lost the capacity to initiate apoptosis. Whether differences exist
`between pure