J. Steroid Biochem. Molec. Biol. Vol. 43, No. 1-3, pp. 173-177, 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 I, ICI Pharmaceuticals, Mereside Laboratories, Alderley Park,
`Maoclesfield, Cheshire SKl04TG, England
`
`SnmInary—Previous studies in this laboratory identified a series of 7a-alkylamide analogues
`of 176-oestradiol which are pure antioestrogens. Among this initial lead series of compounds,
`exemplified by ICI 164,384, none was of sufficient in vivo 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-penty1su1phiny1)nonyl]oestra-1,3,5(10)-
`triene-3,l7fl-diol, with significantly increased antioestrogenic potency. The antiuterotrophic
`potency of ICI 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 effects. 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 efi'ective 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 efiects 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.
`
`J. Steroid Btochem. Molec. Biol. Vol. 43, No. 1-3, pp. 173-177, 1992 0960-0760/92 $5.00 + 0.00 Printed in Great Britmn. All rights rtscrved Copyright © 1992 Pergamon Press Ltd ICI 182,780, A NEW ANTIOESTROGEN WITH CLINICAL POTENTIAL ALAN E. WAKELING* and JEAN BOWLER Bioscience I and Chemistry I, ICI Pharmaceuticals, Mereside Laboratories, Alderley Park, Macclesfield, Cheshire SKI0 4TG, England Summary--Previous studies in this laboratory identified a series of 7a-alkylamide analogues of 17/~-oestradiol which are pure antioestrogens. Among this initial lead series of compounds, exemplified by ICI 164,384, none was of sufficient/n vivo 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(10)- triene-3,171/-diol, with significantly increased antioestrogenic potency. The antiuterotrophic potency of ICI 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 effects. The increased/n vivo 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 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. 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 Nonsteroidal partial-agonist antioestrogens like tamoxifen (ICI 46,474: Nolvadexi') provide excellent palliative treatment for breast cancer [I, 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 efficacy 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. tNolvadex is a Trade Mark, the property of Imperial Chemical Industries Plc. 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 different 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-11-(3, 17fl-dihydroxyoestra- 1,3,5(10)-triene-7~-yl)undecanamide (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, 70~-[9-(4,4,5,5,5-pentattuoro-pentyl- sulphinyl)nonyl]oestra-l,3,5(10)-triene-3,171t diol (Fig. 1), with a profile of activity which makes it a prime candidate for clinical efficacy studies in oestrogen-responsive breast cancer. stub 43/J.~M 173
`
`INTRODUCTION
`
`antioestrogens
`partial-agonist
`Nonsteroidal
`like
`tamoxifen
`(ICI
`46,474: Nolvadexf)
`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.
`1’Nolvadex is a Trade Mark,
`the property of Imperial
`Chemical Industries Plc.
`
`SIM! 43/I-3—M
`
`173
`
`receptors (ER) with high afiinity 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 difierent 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-l 1-(3, 1 7B -dihydroxyoestra-
`1,3,5(l0)-triene-7a-yl)undecanamide (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-pentyb
`sulphinyl)nonyl]oestra-1,3,5(l0)-triene-3,l7fi diol
`(Fig. 1), with a profile of activity which makes
`it a prime candidate for clinical eflicacy studies
`in oestrogen-responsive breast cancer.
`
`MYLAN PHARMS. INC. EXHIBIT 1009 PAGE 1
`
`

`
`| 74 ALAN E. WAKELING and JEAN BOWIJ~ OH HO~CON(CH2)3CH3 I CH3 ICI 164,384 OH HO~gs.qO(CHa)aCF2CF3 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 utero- 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- 140- 120- ~10O- 411- 20- 0:1 03 110 3~0 lC) Dou mo~o [Loom] Fig. 2. Effects of ICI 182,780 on uterine weight of ovari- eotomized mice. Groups of 5 adult mice ovariectomized 2 weeks before treatment received daily, a sinsle dose of arachis oil vehicle alone (open bar), 0.5 #g 17~-oestradiol benzoate s.c. alone (hatched bar), or the indicated doses of ICI 182,780 s.c. together with cestradiol (continuous line), for 3 days. The effect of 3 mg/kg ICI 182,780 alone is indicated by *. 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 [81. 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 effective 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 INTERACTION The competitive inhibition, by ICI 182,780, of oestrogen and tamoxifen uterotrophic effects is 300- | 200- v I 100. 0- o.61 o.~ oh 0~3 1:o 3'.0 ld.0 Doee mo~ [t.og ~] Fig. 3. Comparative antiuterotrophic activity of ICI 164,384 (&) and ICI 182,780 (@). Experimental procedure as described for Fig. 2 except that immature rats were used.
`
`MYLAN PHARMS. INC. EXHIBIT 1009 PAGE 2
`
`

`
`Pure antioestrogens 175 Table 1. Effect of ovanectomy or 14 days' treatment with ICI 182,780 on utenne and body weight and plasma LH of adult female rats 182,780 mg/kg/day, s.c. Parameter Intact control OVX control 0.03 0.1 0.3 1.0 Uterine weight 0g) 292.2+33.7 75.6-+4.2* 253.4=1=21.2 180.4+21 I* 132.2_+ 10.8" 98.0+6.1" Body weight (g) 40.0 _+ 2.5 64.8 _+ 1.9" 43 6 _+ 2.5 44.6 _+ I 7 45.8 _+ 2.0 42.6 _+ 2.1 LH (ng/ml) 2.4 + 0.6 19.7 -4- 2.2* 2.1 -+ 0.2 1.2 + 0.1 1.0 -+ 0.1 2.3 + 0.3 Values are mean _+ SEM, n ffi 5 *P < 0.001 cf intact control. 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 [3H]oestradiol for binding to rat uterine ER in a concentration- dependent manner [8]. ICso values of 0.83, 0.94 and 4.8 x 10-SM 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 oestra- diol [8]. A comparison of the effect of ICI 20- o ICI 182,780 Q~ ~ • ICI 164,384 - I I I I I - 10 -9 -8 -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 cells. Cells were plated in 24-weU dishes (4 x 104/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 #l/ml medium). 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 treatted wells between day 0 and 5. Points are the mean of quadruplicate observations where SEM was <5%. 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 (ICs0= 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 G0/Gm 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 #M 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 Brl0 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 rive where, in anti- uterotrophic assays, ICI 182,780 was at least an order of magnitude more potent than ICI 164,384 (EDs0s ffi 0.06 and 0.9mg/kg, respect- ively). In vitro, the intrinsic potency difference appears somewhat less, for example there is
`
`MYLAN PHARMS. INC. EXHIBIT 1009 PAGE 3
`
`

`
`176 ALAN E. WAKELING and JEAN BOWI.,iiR 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 /n vitro and in vivo 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 vitro, 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 [11] and IGF-I [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 vivo 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. Acknowledgements--We thank B. Curry, L. Green, E. Monk, J. Morrell, E. Newboult, J. Pittam, S. Peters, R Stevenson, J. Tattershall and L Yarwood for their assistance with these studies. REFERENCES 1 Litherland S. and Jackson I M.' AnUoestrogens m the management of hormone-dependent breast cancer. Cancer Treat. Rev. 15 0988) 183-194. 2. Early Breast Cancer Tnallists' Collaboratwe 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 antzestrogens as anUtumour agents Endocrine Rec. 11 (1990) 578~10. 4 Wakehng A. E and Bowler J' Steroidal pure anu- oestrogens. J. Endocr 112 (1987) R7-R10 5. Wakehng A E. and Bowler J.: Biology and mode of action of pure antioestrogens. J. Steroid Bwchem. 30 (1988) 141-147. 6. Bowler J., Ldley T. J, Pittam J. D and Wakehng A. E • Novel steroidal pure antlestrogens. Steroids 54 (1989) 71-99 7. Wakehng A. E' Sterotdal pure antlestrogens. In Regulatory Mechanisms in Breast Cancer (F.~lited by M. Lippman and R. Dickson). Kluwer Academic Publishers, Boston (1990) pp. 239-257. 8. Wakeling A. E., Dukes M. and Bowler J.' A potent specific anuestrogen with chmcal potential Cancer Res 51 (1991) 3867-3873. 9. Wakeling A. E and Bowler J' Novel anUoestrogens without partial agonlst acuwty. J. Steroid Biochem. 31 (1988) 645~553 10 Katzenellenbogen B S., Kendra K. L, Norman M. J and Berthols 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. E.: Effect of antioestrogens on the proliferation of MCF-7 human breast cancer cells. J. Molec. Endocr. l (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 ceils. J. Biol. Chem. 265 (1990) 21172-21178. 13. Gottardis M. M., Jiang S.-Y., Jeng M.-H. and Jordan V. C.: Inhibition of tamoxifen-stimulated growth of an
`
`MYLAN PHARMS. INC. EXHIBIT 1009 PAGE 4
`
`

`
`Pure antioestrogens 177 MCF-7 tumour variant in athymic rmce by novel steroidal antiestrogens. Cancer Res. 49 (1989) 4090-4093. 14. Gottardis M. M., Ricchio M. E., Satyaswaroop P. G. and Jordan V. C.: Effect of steroidal and nonsterotdal antiestrogens on the growth of a tamoxifen-sumulated human endometrial carcinoma (EnCal01) in athymic mice. Cancer Res..$0 (1990) 3189-3192. 15. Nicholson R. I., 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.
`
`MYLAN PHARMS. INC. EXHIBIT 1009 PAGE 5