J. Steroid Biochem. Mulec. Biol. Vol. 43, No. 1-3, pp. 173—l77, 1992
`Printed in Great Britain. All rights reserved
`
`0960-0760/92 $5.00 + 0.00
`Copyright © 1992 Pergamon Press Ltd
`
`ICI 182,780, A NEW ANTIOESTROGEN WITH CLINICAL
`POTENTIAL
`
`ALAN E. WAKELING‘ and JEAN Bowman
`
`Bioscience I and Chemistry 1, [Cl Pharmaceuticals, Mereside Laboratories, Alderley Park,
`Macclesfield, Cheshire SKl0 4'l"G, England
`
`Summary-Previous studies in this laboratory identified a series of 7:1-alkylamide analogues
`of 1719 -oestradiol which are pure antioestrogens. Among this initial lead series of compounds,
`exemplified by ICI 164,384, none was of sufficient in viva potency to merit serious consider-
`ation as a candidate for clinical evaluation. Further structure—activity studies identified a new
`compound, ICI 182,780, 7a-[9-(4,4,5,5,5-pentafluoro-pentylsulphinyl)nonyl]oestra-l,3,5(l0)-
`triene-3,l7[i-diol, with significantly increased antioestrogenic potency. The antiuterotrophic
`potency of [CI 182,780 is more than 10-fold greater than that of ICI 164,384. ICI 182,780 has
`no oestrogen-like trophic activity and,
`like ICI 164,384 is peripherally selective in its
`antioestrogenic efiects. The increased in viva potency of ICI 182,780 was also reflected, in part,
`by intrinsic activity at the oestrogen receptor and in the growth inhibitory potency of ICI
`182,780 in MCF-7 human breast cancer cells. ICI 182,780 was a more efiective 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. Sustained
`antioestrogenic effects of ICI 182,780, following a single parenteral dose of ICI 182,780 in oil
`suspension, were apparent in both rats and pigtail monkeys. In vivo, the antitumour activity
`of ICI 182,780 was demonstrated with xenografts of MCF-7 and Brl0 human breast cancers
`in athymic mice where, over a 1 month period, a single injection of ICI 182,780 in oil
`suspension achieved effects comparable with those of daily tamoxifen treatment. Thus, ICI
`182,780 provides the opportunity to evaluate clinically the potential therapeutic benefits of
`complete blockade of oestrogen effects in endocrine-responsive human breast cancer.
`
`INTRODUCTION
`
`antioestrogens
`partial-agonist
`Nonsteroidal
`like
`tamoxifen
`(ICI
`46,474: Nolvadexi)
`provide excellent palliative treatment for breast
`cancer [1, 2]. However, the diversity of the bio-
`logical actions of such compounds which range
`between full agonist, oestrogen-like trophic
`effects,
`through partial agonism to complete
`blockade of oestrogen action [3], raises the issue
`of whether their clinical efiicacy is in any way
`limited, compared with that which might be
`achieved by complete oestrogen ablation. None
`of the endocrine treatments currently available
`for breast cancer can remove completely the
`trophic influences of endogenous or exogenous
`(e.g. of dietary origin) oestrogens. Potentially,
`antagonist molecules which bind to oestrogen
`
`Proceedings of the Fourth International Congress on
`Hormones and Cancer, Amsterdam, The Netherlands,
`September 1991.
`‘To whom correspondence should be addressed.
`TNo1vadex is a Trade Mark,
`the property of Imperial
`Chemical Industries Plc.
`
`SIM! 43/I-3—M
`
`173
`
`receptors (ER) with high affinity without acti-
`vating any of
`the normal
`transcriptional
`hormone responses, would offer
`the chance
`of achieving complete blockade of oestrogen
`action, whatever the source of the oestrogenic
`stimulus. Such pure antioestrogen molecules
`would be distinctively ditferent from tamoxifen-
`like ligands. The first examples of such com-
`pounds have been described elsewhere [4-6].
`The prototype pure antioestrogen, ICI 164,384,
`N-n-butyl-N-methyl-1 1-(3, 17/3 -dihydroxyoestra-
`1,3,5(l0)-triene-7a-yl)undecanarnide (Fig.
`1),
`is devoid of stimulatory activity and completely
`blocks
`the
`trophic actions of oestrogens,
`and of the partial-agonist antioestrogens,
`in
`all
`oestrogen-responsive
`cell
`and
`animal
`models examined to date (see Ref. [7] for a
`review).
`Here, we describe a new pure antioestrogen,
`ICI 182,780, 7a-[9-(4,4,5,5,5-pentafluoro-pentyl-
`sulphinyl)nonyl]oestra—1,3,5(10)-triene-3,17/3 diol
`(Fig. 1), with a profile of activity which makes
`it a prime candidate for clinical eflicacy studies
`in oestrogen-responsive breast cancer.
`
`|nnoPharma Exhibit 1040.0001
`
`

`

`I74
`
`ALAN E. WAKELING and JEAN Bowman
`
`tage of ICI 182,780, compared with ICI 164,384
`is shown in Fig. 3. Complete blockade of oestro-
`gen action was achieved with a dose of 0.5 mg
`ICI 182,780/kg/day s.c. The uterotrophic action
`of tamoxifen was also blocked in a dose-
`
`dependent manner by co-adminstration of ICI
`182,780 [8].
`In adult female rats increasing daily doses of
`ICI 182,780 reduced the weight of the uterus in
`a dose-dependent fashion (Table 1). At
`the
`highest dose in this study, 1 mg/kg/day, involu-
`tion of the uterus after 14 days was comparable
`with 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, serum LH (Table 1), FSH
`and prolactin concentrations [8] were largely
`unaffected,
`indicating a peripherally selective
`action of ICI 182,780.
`A comparison of the oral and parenteral
`antiuterotrophic potency of ICI 182,780 indi-
`cated that the oral bioavailability of the com-
`pound is relatively poor [8]. It is known that
`many steroids administered orally are subject to
`rapid metabolism by the liver and subsequent
`excretion. A well-established procedure to miti-
`gate such effects is to administer steroids par-
`enterally in oil. Such formulations often have a
`sustained duration of action. This procedure
`was elfective in the case of ICI 182,780 where a
`bolus dose in arachis oil sustained antioestro—
`
`genie activity for in excess of 1 month in both
`rats and monkeys [8].
`
`ER INTERACFION
`
`The competitive inhibition, by ICI 182,780, of
`oestrogen and tamoxifen uterotrophic effects is
`
`.\\\\\\\\\\\\\\\\\\\\\\\\\\\s
`
`
`
`g
`
`E g
`
`E
`
`...
`
`OH
`
`
`
`(CH2l1oC°l|4(C“2)aGHa
`CH3
`
`H0
`
`not 164,384
`
`OH
`
`HO
`
`""'(cH,),so(cH2)3cF2ci=3
`
`ICI 182,780
`Fig. 1. Structure of pure antioestrogens.
`
`OESTROGENIC AND ANTIOESTROGENIC
`ACTIVITY
`
`In rats and mice, ICI 182,780 was devoid of
`uterotrophic activity and, when co-administered
`with oestradiol, completely blocked the mere-
`trophic action of oestradiol in a dose-dependent
`manner (see[8} for rat and Fig. 2 for mouse
`data). The order of magnitude potency advan-
`
`Utedmweight(m3)888
`
`40
`
`20
`
`0
`
`r-:'fi—?r'*'-r*--1
`0.1
`0.3
`1.0
`3.0
`10
`Dosernolkoltoaeenlel
`
`Fig. 2. Efiects of ICI 182,780 on uterine weight of ovari-
`ectomized mice. Groups of 5 adult mice ovariectomized 2
`weeks before treatment received daily, a single dose of
`arachis oil vehicle alone (open bar). 0.5 ug 1713-oestradiol
`benzoate s.c. alone (hatched bar), or the indicated doses of
`ICI 182,780 s.c. together with oestradiol (continuous line),
`for 3 days. The effect of 3mg/kg ICI 182,780 alone is
`indicated by s-.
`
`Dose memo
`
`{Lou scab]
`
`Fig. 3. Comparative antiuterotrophic activity of ICI 164,384
`(A) and ICI 182,780 (0). Experimental procedure as
`described for Fig. 2 except that immature rats were used.
`
`|nnoPharma Exhibit 1040.0002
`
`

`

`Pure antioestrogens
`
`175
`
` Parameter Intact control OVX control 0.03 0.1 0.3 1.0
`
`
`
`
`
`Table 1. Effect of ovanectomy or 14 days’ treatment with ICI 182,780 on uterine and body weight and plasma 1.11 of adult female rats
`182,780 mg/kg/day, s.c.
`
`
`
`Uterine weight (3)
`292.2 3; 33.7
`75.6 :1; 4.2‘
`253.4 -_t 21.2
`180.41 21 1’
`132.2 i 10.8‘
`98.0 :t 6.1‘
`Body weight (g)
`40.0 3‘; 2.5
`64.8 1 1.9‘
`43 6 1 2.5
`44.6 1 1 7
`45.8 1 2.0
`42.6 1 2.1
`
`19.7 -1; 2.2-2.4 10.6L1-I (ng/ml) 2.3 1 0.3 2.1 i 0.2 1.2 1 11.1 1.0 3; 0.1
`
`
`
`
`
`Values are mean :1; SEM, r1 = 5 ‘P < 0.001 cf intact control.
`
`
`
`182,780 with that of other antioestrogens on the
`growth of MCF-7 cells (Fig. 4) showed that it
`was significantly more potent than ICI 164,384
`(XCSO = 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
`(approx. 80% cf 50%, Fig. 4).
`Flow cytometric analysis of cell cycle and
`population distribution of MCF-7 cells treated
`with tamoxifen or ICI 182,780 showed that both
`antioestrogens caused accumulation of cells in
`Go/G, and also reduced the proportion of cells
`capable of continued DNA synthesis. However,
`the maximal efficacy of ICI 182,780 compared
`with that of tamoxifen, when both compounds
`were used at optimum antioestrogenic (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 11M tamoxifen [8].
`
`ANTITUMOUR EFFICACY
`
`The growth of xenografts of MCF-7 human
`breast cancer cells, supported by continuous
`treatment with ethynyl oestradiol, was blocked
`completely for at least 4 weeks by a single s.c.
`injection of 5 mg ICI 182,780 in oil suspension.
`The magnitude of this effect was comparable
`with that in animals treated continuously with a
`high dose of tamoxifen. Similarly, the growth of
`transplants of the Br10 solid human breast
`tumour was also suppressed effectively by ICI
`182,780 [8].
`
`CONCLUSIONS
`
`In comparison with the first reported pure
`antioestrogen ICI 164,384, ICI 182,780 demon-
`strates substantially increased potency. This
`is clearly manifest
`in viva where,
`in anti-
`uterotrophic assays, ICI 182,780 was at least an
`order of magnitude more potent
`than ICI
`164,384 (ED,.,s=0.06 and 0.9 mg/kg, respect-
`ively). In vitro, the intrinsic potency difference
`appears somewhat
`less, for example there is
`
`|nnoPharma Exhibit 1040.0003
`
`consistent with each class of ligand acting
`through an ER-mediated pathway. Formal
`proof of this is provided by the capacity of ICI
`182,780 to compete with [31-I]oestradiol
`for
`binding to rat uterine ER in a concentration-
`dependent manner [8]. IC50 values of 0.83, 0.94
`and 4.8 x 10“M were recorded for oestradiol,
`ICI 182,780 and ICI 164,384, respectively with
`equivalent relative binding affinities of 0.89 and
`0.19 for ICI 182,780 and ICI 164,384, respect-
`ively, compared with oestradiol = 1.
`
`BREAST CANCER CELL GROWTH INHIBITION
`
`ICI 182,780 inhibited 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. The growth
`inhibitory action of ICI 182,780 on MCF-7 cells
`was reversed in a competitive manner by oestro-
`diol [8]. A comparison of the effect of ICI
`
`o 1c11a2,73o
`A lG|164.384
`
`
`
`I 4-hydroxytamoxiten
`
`‘°°
`
`2 so
`
`so
`
`40
`
`20
`
`E3
`
`éC
`§
`8
`
`lC':""""l"'——'-|'_j|
`-10
`-9
`~B
`-7
`-6
`
`[Antioestrogen] log 10
`
`Fig. 4. Effects of ICI 164,384, ICI 182,780 and 4’-hydroxy-
`tamoxifen on the proliferation of MCF-7 human breast
`cancer ails. Cells were plated in 24-well dishes (4 x 10‘/well)
`and cultured for 2 days in MEM with 5% charcoal stripped
`foetal calf serum containing phenol red and insulin but no
`additional oestrogen. 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 antioestrogens added in ethanol (1 pl/ml
`medium). Cells were grown for a further 5 days with fresh
`mediumaddedafter 3days. Cell growthisrepnesentedas the
`difierence between the increase of total protein in control
`and treated wells between day 0 and 5. Points are the mean
`of quadruplicate observations where SEM was <5"/o.
`
`

`

`176
`
`ALAN E. WAKELXNG and JEAN Bowux
`
`only a 4- to 5-fold advantage for ICI 182,780 in
`terms of affinity for ER. The apparent 2-fold
`difference in potency ratio improvement be-
`tween in vitro and in viva assays for the two
`compounds is probably a reflection of differ-
`ences in their distribution and metabolism. The
`
`order of magnitude lower potency between the
`oral and parenteral routes of administration
`suggests strongly that the oral bioavailability of
`ICI 182,780 is relatively low. A common means
`of circumventing the practical constraints con-
`sequent on the poor oral 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, tumour
`growth ceased for at least 1 month after a single
`injection of ICI 182,780 [8].
`Of particular relevance to the therapeutic
`potential of ICI 182,780 are the enhanced
`efficacy compared with 4’-hydroxytamoxifen (or
`tamoxifen) on breast tumour cells and the excel-
`lent antiuterotrophic action achieved without
`affecting body weight and gonadotrophin se-
`cretion. 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 differentially active against peripheral
`and central targets of oestrogen action, a prop-
`erty shared with ICI 164,384 [9]. If translated to
`the clinical setting, this peripheral selectivity of
`action would obviate blockade of central nega-
`tive oestrogen feedback and consequent
`in-
`creases of oestrogen
`production
`in
`the
`premenopausal patient. Also, such selectivity
`would be particularly advantageous in the treat-
`ment of benign uterine and breast pathologies in
`premenopausal patients.
`In respect of the enhanced efficacy of pure
`antioestrogens against
`tumour
`cell growth
`in vitra, fewer of the cells remain in the actively
`proliferating fraction than is the case when
`partial agonists like tamoxifen, 4’-hydroxy-
`tamoxifen or hydroxyclomiphene are used. This
`has been attributed to a residual oestrogenic
`effect of the partial agonists which, although
`small [10, 11], is amplified synergistically by the
`concurrent presence of other breast cell mito-
`gens like insulin [1 1] and IGF-l [12]. The pure
`antioestrogens obviate such effects. The corol-
`lary of these data in the clinical setting is the
`possibility that
`differences of antitumour
`efficacy between tamoxifen and pure antioestro-
`gens may be greater than otherwise anticipated.
`
`In summary, ICI 182,780 offers significant
`advantages compared with pure antioestrogens
`reported previously, particularly with respect to
`in viva potency. Although oral potency appears
`to be limited, probably as a result of poor
`absorption, this disadvantage is offset by the
`sustained antioestrogenic and antitumour ac-
`tivity following parenteral administration of ICI
`182,780 in oil suspension. The data available to
`date for ICI 182,780 presented here, and for ICI
`164,384 [7, 13-15] indicate that pure antioestro—
`gens may find a valuable place in the treatment
`of breast cancer. ICI 182,780 will be used to test
`this proposition.
`
`thank B. Curry, L. Green,
`Acknawledgementswwe
`E. Monk, J. Morrell, E. Newboult, J. Pittam, S. Peters,
`R Stevenson, J. Tattershall and L Yarwood for their
`assistance with these studies.
`
`REFERENCES
`
`l Litherland S. and Jackson I M: Antioestrogens in the
`management of hormone-dependent breast cancer.
`Cancer Treat. Rev. 15 (1983) 183-194.
`2. Early Breast Cancer Tnallists’ Collaborative Group.
`Treatment of Early Breast Cancer. Worldwide Evidence
`1985-1990. Oxford University Press, Oxford, Vol.
`1
`(1990).
`3 Jordan V. C. and Murphy C. S.. Endocrine pharma-
`cology of antiestrogens as antituinour agents Endocrine
`Rev. 11 (1990) 578-610.
`4 Wakeling A. E and Bowler J‘ Steroidal pure anti-
`oestrogens. J. Endorr 112 (1987) R7—Rl0
`5. Wakeling A E. and Bowler J.: Biology and mode of
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`(1988) 141-147.
`6. Bowler J., Lilley T. J , Pittam J. D and Wakeling A. E '
`Novel steroidal pure antiestrogens. Steroids 54 (1989)
`71-99
`In
`7. Wakeling A. E- Steroidal pure antiestrogens.
`Regulatory Mechanisms in Breast Cancer (Edited by
`M. Lippman and R. Dickson). Kluwer Academic
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`8. Wakeling A. E., Dukes M. and Bowler J." A potent
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`51 (1991) 3867-3873.
`9. Walceling A. E and Bowler J ' Novel antioestrogens
`without partial agonist activity. J. Steroid Biochem. 31
`(1988) 645-653
`10 Katzenellenbogen B S., Kendra K. L, Norman
`M. J and Berthois Y.: Proliferation, hormonal respon-
`siveness, and estrogen receptor content of MCF—7
`human breast cancer cells grown in the short-term and
`long-term absence of estrogens. Cancer Res. 47 (1987)
`4355-4360.
`11. Wakeling A. E., Newboult E. and Peters S. 13.: Effect of
`aiitioestrogens on the proliferation of MCF-7 human
`breast cancer cells. J. Malec. Endocr. 2 (1989) 225-234.
`12. Stewart A. J., Johnson M. D., May F. E. B. and Westley
`B. R.: Role of insulin-like growth factors and the type
`I insulin-like growth factor receptor in the estrogen-
`stimulated proliferation of human breast cancer cells.
`J. Biol. Chem. 265 (I990) 21172-—2ll78.
`13. Gottardis M. M., Jiang S.-Y., Jeng M.-H. and Jordan
`V. C.: Inhibition of tamoxifen-stimulated growth of an
`
`|nnoPharma Exhibit 1040.0004
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`Pure antioestrogens
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`177
`
`in athymic IIIICB by novel
`MCF-7 tumour variant
`steroidal
`antiestrogens. Cancer Res.
`49
`(1989)
`4090-4093.
`.Gottardis M. M., Riechio M. E., Satyaswaroop
`P. G. and Jordan V. C.: Effect of steroidal and
`nonsteroidal
`antiestrogens on the growth of
`a
`tamoxifen-stimulated human endometrial carcinoma
`
`(EnCal0l) in athymic mice. Cancer Res. 50 (1990)
`3189-3192.
`. Nicholson R. 1., Walker K. J., Bouzubar N., Wills R. J.,
`Gee J. M. W., Rushmere N. K. and Davies P.: Estrogen
`deprivation in breast cancer. Clinical, experimental and
`biological aspects. Ann. N. Y. Acad. Sci. 595 (1990)
`316-327.
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`|nnoPharma Exhibit 1040.0005
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