335
`
`Effects of a non-steroidal pure antioestrogen, ZM 189,154, on
`oestrogen target organs of the rat including bones
`
`M Dukes, R Chester, L Yarwood and A E Wakeling
`Cancer Research Department, Zeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SKID 4T6, UK
`(Requests for offprints should be addressed to A E Wakeling
`
`
`
`Abstract
`
`2 mg/ kg per day achieved maximal uterine atrophy but
`ZM 189,154 ([1RS,2RS]—2—(4—hydroxyphenyl)-2-methy1
`did not affect bone density or growth rate; 10 mg/ kg per
`—1—[9—(4,4,5,5,5—penta—fluoropentyl)sulphinylnonyl]—1,2,
`3,4—tetrahydronaphth—(i—ol)
`is a non—steroidal pure anti—
`day produced a broader spectrum of effects (reduced bone
`oeStrogen. It has a high relative affinity for the oestrogen
`density, increased basal LH, slightly increased growth rate),
`but the magnitude of these was smaller than after ovariec-
`receptor, completely blocks the trophic action ofoestracliol
`(0E2) on the uterus in immature and ovariectomized
`tomy; the 10 mg/kg dose also produced multiple ovarian
`follicular cysts. The failure of ZM 189,154 to achieve
`(OVX) adult rats and, in the latter, also completely blocks
`complete ovariectomy—like effects in intact rats may be due
`the trophic action of OE2 on vagina, bone and growth rate.
`to the action of ovarian factors other than 0E2, or to the
`ZM 189,154 displays no intrinsic oestrogen—agonist activ—
`circulating 0132 levels resulting from the disturbance to
`ity on uterus, vagina, bone, LH secretion or growth rate in
`ovarian function posing too strong a challenge to the
`OVX rats. Differential sensitivity of DEE-regulated pro-
`antagonist.
`cesses was more apparent in intact rats. Daily doses of
`loumal of 1:11er rinulogy [19941 141, 335—341
`06 mg/kg per day of ZM 189,154 blocked ovulation;
`
`
`Introduction
`
`The properties of 7a—alkylamide and 7(1—alkynylsulphinyl
`analogues of oestradiol—17B (0132) which characterize
`them as pure
`antioestrogens have been described
`(Wakeling 8: Bowler 1987, Wakeling ef al 1991). These
`agents are pharmacologically distinct
`from the partial
`agonist antioestrogens such as tamoxifen, notably in their
`capacity to completely block the trophic actions of either
`OE2 or tamoxifen on oestrogen target organs such as the
`uterus and mammary gland in rodents and primates.
`(Wakeling & Bowler 1987, Nicholson rt
`:11
`1.988,
`Wakeling et a] 1991, Dukes at al 1992). However these
`studies also demonstrated that there are significant differ—
`ences in organ sensitivity to the action ofpure antioestro-
`gens; for example in rats, complete inhibition of oestrogen
`action on the uterus can be achieved without affecting LH
`secretion or bone density (Wakeling &’ Bowler 1988,
`Wakeling 1993).
`non—
`In addition to steroidal pure antioestrogens,
`steroidal pure antioestrogens have also been described (von
`Angerer er a] 1990, Sharma 9/ a] 1990, Nishino at a] 1991,
`Day at a] 1991). The activity ofa new agent of this type,
`ZM 189,154,
`(European Patent, EP0124369 B1),
`3
`2—methyltetrahydronaphthalene substituted with a side-
`Chain like that oflCl 182,780 (Fig 1), is reported here to
`illustrate further the range of effects these agents elicit in
`
`oestrogen—dependent tissues. Attention is focussed on the
`differences in dosage needed to affect different oestrogen—
`dependent processes with particular reference to effects on
`bone because of concerns that long term clinical use of
`pure antioestrogens might adversely affect bone density to
`cause an ovariectomy—like onset of osteoporosis (Jordan
`1992).
`
`Materials and Methods
`
`The antioestrogens tamoxifen (ICI 46,474 (trans—l—(4—
`dimethylaminoethoxyphenyl) — 1 ,2 — diphenylbut— 'l — ene]),
`lCl 164.384 (N—n—butyl—N—methyl—'l 1—[3,17—dihydroxy—
`oestra—l,3,5(10)—triene—7-yl]undecanamide)
`and
`2M
`189,154
`([1RSZRS]-2—(4—hydroxyphenyl)—2—methyl-1-
`[9—(4,4,5,5,5—pentafluoropentyl)sulphinylnonyl]—1 ,2,3,4~
`tetrahydronaphth—o—ol) were synthesized in the labora—
`tories of Zeneca Pharmaceuticals.
`
`to measure the relative
`Competitive binding assays
`binding affinity of antioestrogens for rat uterine oestrogen
`receptors were as described elsewhere (Wakeling 85 Slater
`1980) except. that the competitor dilutions were prepared
`in Tris:dimethylformamide (1:1) and mixed together with
`[FT-HOE; (Amersham International, Amersham, UK) with
`cytosol at a ratio of 1 :20.
`The rat uterine weight assay for uterotrophic and
`antiuterotrophic activity has been described (Wakeling er
`
`journal of Endocrinology (1994) 141, 335—341
`0022 0795/94/0141 0335 $08.00/O
`
`© 1994 Journal of Endocrinology Ltd Printed in Great Britain
`
`AstraZeneca Exhibit 2027 p. 1
`InnoPharma Licensing LLC v. AstraZeneca AB IPR2017-00904
`Fresenius-Kabi USA LLC v. AstraZeneca AB IPR2017-01910
`
`

`

`336 M DUKES and others
`
`Differential actions of a pure antioestrogen
`
`OH
`
`100
`
`so
`
`60
`
`40
`
`o
`
`is
`
`i=3
`is
`
`i.)
`g
`
` I
`
`I,I”,
`"’(CH2)9 SO(CH2)3 ng CF3
`ICI 182,780
`
`OH
`
`(CH2)9 SO(CH2)3 CFg CF3
`
` OH
`
`ZM 189,154
`
`FIGURE 1. Structures of the pure antioestrogens ICI 182,780
`and ZM 189,154.
`
`al 1983). Details of doses, routes of administration and
`duration of treatments are reported in the present Figures
`and Tables.
`
`ZM 189,154 and oestradiol benzoate (Sigma Chemical
`Company, Poole, Dorset, UK) were prepared for admin—
`istration by diluting an ethanol stock solution into the
`required volume of arachis oil with gentle warming
`(60 DC). Tamoxifen was prepared for oral administration as
`a dispersion in aqueous 0.5% Tween 80. Dose volumes
`were 0-5 and 0-1 nil/100 g body weight for immature and
`mature rats respectively.
`In uterotrophic/antiuterotrophic studies in ovariectoi
`mized (OVX) rats, ovariectomy was performed at least 2
`weeks before treatment began. For ovulation inhibition
`studies, rats having vaginal smear patterns consistent with
`4—day—oestrous cycles were given either a single dose of
`ZM 189,154 on day 2 or 3 of the cycle, or daily closes on
`days 1 to 4 of the cycle. The rats were then killed by C02
`exposure on the morning of the next scheduled day 1,
`their Fallopian tubes excised and the contents gently
`expressed onto a microscope slide and the number of eggs
`present counted.
`Effects on uterine, ovarian and body weights and plasma
`gonadotrophin concentrations in intact rats were assessed
`after 14 days of dosing, effects on bone parameters were
`assessed after 28 days of dosing,
`this being the shortest
`convenient
`interval
`following ovariectomy at which
`significant
`reductions
`in bone density were readily
`measurable; body, uterine and ovarian weights were also
`monitored in these longer experiments. All the rats used in
`
`journal of Hidnt‘rinolngy (1994) 141, 335-341
`
`g 20
`
`Log“, [Competitor]
`
`FIGURE 2. Competition for binding of5 X 1079 M
`|3H]oestradiolil7[3 (0E2) to rat uterine oestrogen receptor by
`unlabelled 0E2 (C) ZM 189,154 (A), ICI 164,384 (0) and
`tamoxifen (A). Percent inhibition refers to specific binding
`corrected by subtraction from total [3H]OE2 bound, the
`non—specific component recorded in the presence of5 X 1077
`unlabelled 0132. Each point and bar represents the
`meand: S.E.M. of nine observations in three different
`
`experiments. Estimates of competitor concentration which
`reduced [3H]OE2 by 5000 (1C50 values) were calculated by
`linear regression analysis of per cent inhibition versus
`logmkompetitor].
`
`these studies weighed between 240 and 260 g at the start
`of the experiments. At the end of the dosing period, left
`and right femurs were dissected, freed of adherent soft
`tissues, weighed and their volumes determined by
`Archimedes’ principle (by subtraction of the weight of a
`25 ml specific gravity bottle filled with water containing
`each femur, from the sum of the weights of the femur and
`the specific gravity bottle filled only with water)
`to
`estimate gross density. The femurs were then reduced to
`ash and the ash weighed. Gross bone density was calcui
`lated by dividing femur weight by volume; mineral
`density was calculated by dividing femur ash weight by
`volume. One group of rats in each of these studies was
`subjected to ovariectomy on day 1 to provide an estimate
`of
`the maximum antioestrogenic
`effect
`potentially
`attainable.
`
`concentrations
`(LH)
`luteinizing hormone
`Plasma
`were assayed by a modification of the double-antibody
`technique described by Niswender er a1 (1969).
`Treatment effects were analysed by comparison of
`group means using Student’s retest.
`
`Results
`
`Interartion wit/1 oestrogen receptor
`
`Competition of ZM 189,154 with [3141—0132 for bind—
`ing to the rat uterus oestrogen receptor was measured
`(Fig 2) and compared with that of tamoxifen and the
`steroidal pure antioestrogen lCl 164,384. Competitive
`
`AstraZeneca Exhibit 2027 p. 2
`
`

`

`M DUKES and others 337
`Differential actions of a pure antioestrogen
`
`
`
`
`
`75
`
`50
`
`
`
`Uterineweight(mg) 25
`
`0
`
`r—I—fi—fi
`0-3
`0-1
`3-0
`10-0
`
`Dose (mg/kg)
`
`FIGURE 4. Antagonism of the uterotrophic effect of tamoxifen
`by ZM 189,154. Immature rats received daily 21 single dose of
`arachis oil vehicle alone (open bar), 10 mg tamoxifen/kg
`orally (solid bar), or the indicated doses of ZM 189,154 s.c.
`together with tamoxifen for 3 days. Points and bars represent
`meansiS.E.M. for a minimum of ten observations in at least
`
`two different experiments. Where no bar is present errors are
`smaller than the symbols.
`
`Effects in O VX mature rats
`
`The trophic and inhibitory eifects of ZM 189,154 on the
`uterus, vagina and growth rate of adult OVX rats were
`measured to determine whether this agent showed the
`differential effects on different oestrogen target organs
`described previously for the steroidal pure antioestrogens
`(Wakeling & Bowler 1988, Wakeling et a] 1991).
`In
`animals treated for 14 days with 0152 alone uterine weight
`increased fourfold compared with ()VX controls, growth
`rate was reduced and full corniflcation of the vagina was
`recorded after 4 days. In contrast, at a daily oral dose of
`10 mg/kg administered alone to OVX rats, ZM 189,154
`had no effect on the uterus, growth rate or vagina (Table
`1) but, given together with 0E2, ZM 189,154 achieved
`72, 96 and 100% blockade of the uterotrophic action of
`OE2 with daily oral doses of 1-5, 4 and 10 mg/kg (Table
`1). However, the lowest dose of ZM 189,154 had little
`effect on OEz—induced vaginal cornification and none of
`the doses reversed the OEZ—induced suppression of body
`weight gain in OVX rats (Table 1),
`Since ZM 189,154 was more potent parenterally than
`orally, the effects of 10 mg/kg s.c. were studied alone and
`in combination with OE2 or tamoxifen. Again, there was
`no evidence for an oestrogenic action of ZM 189,154 on
`the uterus or on growth rate or plasma LH concentration,
`
`lournal of Endocrinology (1994) 141, 335—341
`
`AstraZeneca Exhibit 2027 p. 3
`
`150
`
`100
`
`50
`
`a;
`
`Ej
`
`.C“
`.20U.)
`3Q)
`
`E
`
`eD
`
`0
`
`0-02
`
`0-05
`
`0-1
`
`0-2
`
`0-5
`
`1-0
`
`20
`
`5-0
`
`10-0
`
`Dose (mg/kg)
`
`FIGURE 3. Effects of ZM 189,154 on uterine weight of
`immature rats. Animals received daily a single dose of arachis
`oil vehicle alone (open bar), 0-5 g oestradiol benzoate s.c. alone
`(solid bar), or the indicated doses of ZM 189,154 alone s.c.
`(0 """ C) or orally (A —- A), or oestradiol benzoate together
`with ZM 189,154 s.c. (0—0) or orally (A—A) for 3 days.
`Points and bars represent n1eans:l:s.E.M. for a minimum of 10
`observations in at least two different experiments. Where no
`bar is present errors are smaller than the symbols.
`
`displacement of [3H]—OE2 by ZM 189,154 reflected by
`the parallel displacement curves, allowed calculation of a
`relative binding affinity of0'66 for ZM 189,154 (OE2=1),
`compared with 0-19 and 0-025 for
`ICI 164,384 and
`tamoxifen respectively.
`
`Antiuterotrophit artivity in immature rats
`
`When administered orally or parenterally at doses in the
`range 0-025-10 mg ZM 189,154/kg, the weight of the
`uterus in treated rats was always similar to or less than that
`in vehicle treated immature rats (Fig 3). Co-administration
`of ZM 189,154 together with a maximally effective dose
`of OE2 inhibited the trophic action of OE2 on the
`immature rat uterus in a doseidependent manner (Fig 3).
`Complete blockade of OEz—induced uterine growth was
`achieved with daily subcutaneous (s.c.) doses of 0-5 mg/kg
`or oral (p.o.) doses of 3—5 mg/kg. Estimates of the dose
`required to reduce uterine weight by 50% (ED502009
`and 0-7 mg/kg, s.c. and p.o. respectively) indicated that
`ZM 189,154 is seven— to eightfold less potent via the oral
`route compared with parenteral administration. Similar
`assays in adult OVX rats and mice confirmed that ZM
`189,154 alone did not stimulate the uterus and did not
`induce vaginal cornification; OEzestimulated growth was
`also blocked by ZM 189,154 (data not shown, ED50 values
`of 1 -3 and 6-2 mg/ kg, p.o. in rats and mice respectively).
`When the immature rat uterus was stimulated by
`treatment with
`tamoxifen
`instead
`of
`013;,
`CO-
`adrninistration of ZM 189,154 antagonized the action of
`tamoxifen in a dose-dependent manner and complete
`blockade of tamoxifen—induced growth was achieved with
`a dose 0f10 mg ZM 189,154/kg (Fig 4).
`
`

`

`338 M DUKES and others
`Differential actions of a pure antioestrogen
`
`TABLE 1. Agonist and antagonist activity of ZM 189,154
`(1‘5—10 mg/kg, orally) and oestradiol (OE2 benzoate;
`0-5 rig/day s.c.). in ovariectomized rats. Values are
`means :1: S.E.M. for groups of five rats treated for 14 days
`
`Weight gain Uterus wt Vaginal
`(g)
`(mg)
`comification1
`
`Treatment
`Ovariectomy
`0122
`ZM 189,154
`(10 mg/kg per day)
`OE2+ZM 189,154
`1-5 mg/kg per day
`4'0 mg/kg per day
`10-0 mg/kg per day
`
`49-0 :1: 2-42
`27-0 i 2-4h
`
`85 :l: 33
`
`342 :: 21b
`
`420 d: 3-8"
`
`80 i 4a
`
`19-8 :l: 4-6b
`20-4 :1; 2'9‘3
`288 :t 3-0“
`
`157 i15“
`91 :1: 21
`
`81 :: 3-
`
`0
`(,0
`
`0
`
`48
`9
`0
`
`HIndicate values which differ significantly, Le. at least P<0-01 (Student's r-tcst)
`ch-r cent total days with pro-oestrous or oestrous smears.
`
`TABLE 2, Agonist and antagonist activity of ZM 189,154
`(10 mg/kg per day s.c.), oestradiol (OE2 benzoate; 0-5 pig/day
`s.c.) and tamoxifen (1 mg/kg per day orally) in ovariectomized
`rats. Values are meansis.E.M. for groups of6 rats treated for
`seven days
`
`Treatment
`Ovaricctomy
`on.
`Tamox1fen
`ZM 189,154
`ZM 189,154+OEP
`ZM 189,154+Tamoxifen
`Tamoxifen l 0132
`
`Weight gain Uterus wt Plasma LH
`(g)
`(g)
`(fig/ml)
`
`34-0 :1: 2-8"
`135 i 3-2h
`0'2 i 26“
`360 21:19“
`258 i 20“
`150 i 0-9h
`34 i 2-8“
`
`173 :1: 10‘
`
`421 :: 27b
`242 i: 10"
`158 i 5‘
`156:1: 10“
`192 :l: 5“
`235 :l: 13"
`
`15-0 :1: 1-3“
`22 :: o-s“
`3-2 i 0'2b
`106 :1: 1-9“
`16-2 :: 3-2“
`131 21:18“
`2-4 :1: 0-5b
`
`
`
`“ cIndicate values whirh differ significantly, is, at least P<0~01 (Student’s t—icst).
`
`whereas both tamoxifen and OE2 significantly reduced
`growth rate and LH concentration and stimulated the
`uterus (Table 2). In combination, ZM 189,154 completely
`reversed the uterotrophic action of OE2 and tamoxifen and
`the suppression of LH, and partially reversed the reduction
`of body weight gain (Table 2).
`
`Efierts in intact adult rats
`
`i. Inhibition of ovulation Single doses of ZM 189,154
`administered on day 2 or 3 of the oestrous cycle inhibited
`ovulation (Table 3). A close of 2 mg ZM 189,154/kg was
`fully effective given on day 2 but not on day 3 ofthe cycle.
`A lower dose of 0-6 mg ZM 189,154/kg administered
`daily on days 1
`to 4 of the cycle also completely inhibited
`ovulation.
`
`ii. Uterine weight Daily s.c. doses of 0-3—2 mg ZM
`189,154/kg for 14 days produced a dose—related reduction
`of uterine weight (Table 4). The maximum regression of
`
`journal of Endocrinology (1994) 141, 335—341
`
`TABLE 3. Inhibition ofovulation by ZM 189,154 in intact rats
`
`Time of
`treatment
`
`No of rats
`ovulating
`
`Ova/ovulating rat
`(Mean :t 5.1).)
`
`Dose
`(mg/kg 5c.)
`7
`1
`2
`1
`2
`0-3
`06
`
`1600 h Day 2
`1600 h Day 2
`1600 h Day 3
`1600 h Day 3
`Days 1
`to 4
`Days 1
`to 4
`
`9/10
`3/5
`0/10
`7/10
`4/10
`4/10
`0/5
`
`14-0 :1: 2-1
`7-7 :1: 4-7
`
`7-7 i 45
`5-3 :t 3-0
`11-3i7'3
`
`'1'»,er 4. Effects of ZM 189,154 given s.c. for 14 or 28 days
`on uterine and ovarian weights and body weight gain in intact
`and ovariectomized rats. Values are means-1:5.EM. for n25 rats
`(14 day treatments) or 10 rats (28 days treatments)
`
`Uterine wt
`("/11 of control)
`
`Ovarian wt
`(% of control)
`
`Body wt gain
`(% of control)
`
`Dose
`(mg/kg per day)
`14 days
`0-3
`(in
`1-0
`1-5
`2-0
`Ovariectomy
`
`28 days
`2-0
`10-0
`Ovariectmriy
`
`752 i 25-1--b
`
`73-7
`(ye-‘1’
`652 i 5-3lb
`47-9 :1: 3-8'1’
`45-2 i 3-3"-
`360
`
`77-5 :1: 5-43
`824 :t 7-6
`745 i108
`706 i 7-2“
`83-2 :1: 9-2
`—
`
`89-6 :1: 17-5b
`
`93-1 :: 17-5b
`72-4 :1; 17-4b
`1000 i 10-9h
`897 i 7-6"
`149-5 i 14-4
`
`811 :t 12-54“-
`83-1 i 59'
`35-0 4 3-23"
`125-5 :1: 13-8J
`119-7 2t 48
`339 i 4'5"h
`
`
`7278 i 33 142-9 i 179
`
`that were significantly (P<1l-()5:
`"blndicatc means (prior to conversion to %)
`Student’s litcst) different from intact and ovariectomimd controls respectively.
`
`the uterus was 86% of that recorded in rats 14 days after
`ovariectomy. Extending the period of dosing to 28 days
`and increasing the dose fivefold to 10 mg ZM 189,154/kg,
`did not significantly increase the extent of uterine atrophy
`compared with the effect ot‘ovariectomy (Table 4).
`
`iii. Ovarian weight and histology At all doses between
`06 and 2 rug/kg per day, ZM 189,154 caused a significant
`20—30% reduction in ovarian weight, but at 10 mg/ kg per
`day mean ovarian weight was slightly, though not signifi—
`cantly, greater than in controls (Table 4). Ovaries from rats
`given 03 mg ZM 189,154/kg for 14 days contained old
`corpora lutea showing signs of vascular congestion and
`degeneration, follicles in various stages of development,
`but no new corpora lutea; one of the old corpora lutea
`contained an entrapped oocyte. Ovaries from rats given
`10 mg ZM 189,154 contained virtually no corpora lutea
`but numerous large irregular cystic follicles.
`In one rat,
`two of the latter showed extensive haemorrhage.
`
`AstraZeneca Exhibit 2027 p. 4
`
`

`

`M DUKES and others
`Differential actions of a pure antioestrogen
`
`339
`
`TABLE 5. Effects of ZM 189,154 on weight of the uterus and on bone density in rats which were
`ovariectomized (OVX) and given ZM 189,154 and/or oestradiol (OE2 benzoate; 0'5 lag/day) for 28
`days. Values are means :t S.E.M., n=5 animals or 11:10 for bone data
`
`Uterus wt
`Bone gross
`Bone mineral
`
`(mg)
`density
`density
`
`Treatment
`Experiment 1.
`Control
`ZM 189,154
`(2 mg/kg per day s.c.)
`OVX
`OVX+ZM 189,154
`Experiment 2.
`Control
`ovx
`OVX+OE2
`OVX+ OE2 +
`+ZM 189,154
`(2 mg/kg per day s.c.)
`Experiment 3.
`Control
`ZM 189,154
`(10 mg/kg per day s.c.)
`ovx
`
`386 :1: 333
`
`
`135 :: 8b
`111 :1: 6“
`104 i 3‘
`
`411 :1: 46”
`101 :1: 3”
`475 i 7“
`
`100 i at
`
`369 :1: 48“
`
`125 i 4b
`99 i 51'
`
`1-612 :1: 0-0072
`
`0-742 :1: 0-0093
`
`1604 i 00053
`1569 i 0008“
`1582 i 0-006C
`
`1600 i 0003“
`1532 i 0007b
`1591 3: 0-007‘
`
`1532 i 0006b
`
`1629 i 0004*
`
`1580 i 0-004b
`1-571 :1: 0-007b
`
`0730 i 0007"
`0685 i 0010‘
`0-701 i 0-008C
`
`0730 i 00042
`0652 i 0-010b
`0738 i 0-010a
`
`0684 i 0‘006b
`
`0766 i 00053
`
`0-727 i 0-005b
`0704 :1: 04009b
`
`HIndicate values which differ significantly, is. at least P<()-()l (Student’s I—test).
`
`iv. Body weight gain Ovariectomy significantly in—
`creased growth with average daily weight gain increasing
`from 206 g in controls to 296 g in OVX rats. In contrast,
`doses of ZM 189,154 up to 2 nag/kg per day tended to
`reduce weight gain slightly (Table 4). However,
`the
`highest dose of10 mg ZM 189,154/kg administered for 28
`days did produce an ovariectomyelike effect, but of smaller
`magnitude than that caused by ovarian ablation.
`
`v. Plasma LH At doses up to 1-5 mg/kg per day for
`14
`days, meani S.E.M.
`plasma LH concentrations
`(253 :1: 021 ng/ ml) were comparable with those in intact
`control rats (218:1:0-12 ng/ ml). In rats given 10 mg/kg
`for 28 days, LH was elevated (4-53 :1:0-97 ng/ml) to about
`half the extent seen in OVX rats (994:1: 1-33 ng/rnl).
`
`vi. Bone density Ovariectomy significantly reduced
`both the gross and mineral density of femur bone. after 28
`days; the meanis.E.M. reduction was 35 iii-5% in gross
`density and 8-8 :1: 09% in mineral density (Table 5).
`Treatment with 2mg ZM 189,154/kg did not reduce
`either gross or mineral bone density in intact animals, and
`in OVX rats did not increase bone density. Oestrogen
`treatment prevented ovariectomy—induced uterine regres—
`sion and bone loss (Table 5, experiment 2). Administration
`of 2 mg ZM 189,154/kg together with OE2 completely
`blocked this protective effect of OE2 (Table 5, experiment
`2) indicating a complete blockade of OF.2 action on the
`bones as well as the uterus in OVX rats.
`
`rats, 10 mg ZM 189,154/kg per day did
`In intact
`produce significant reductions in bone density (Table 5,
`experiment 3): gross and mineral density were reduced
`30% and 51%, respectively, compared with reductions of
`3-6 and 81% in OVX rats.
`
`Discussion
`
`The use of the steroidal pure antioestrogen ICI 182,780
`in the therapy of breast cancer (Wakeling ct al 1991)
`may confer advantages when compared with the well—
`established use of partial agonists like tamoxifen. For
`example, the development of resistance due to oestrogen—
`like activity, as has been seen with tamoxifen, is unlikely
`to occur (Wakeling 1993). However, a possible undesir—
`able consequence of pure antioestrogen therapy is an
`adverse effect on bone mineral metabolism leading to
`induction or exacerbation of osteoporosis (Jordan 1992). In
`this respect the oestrogenic activity of tamoxifen is ben—
`eficial, particularly for
`long—term adjuvant
`therapy of
`breast cancer
`(Jordan 1992). Earlier studies with ICI
`182,780 in intact adult female rats showed clear differences
`between the susceptibility of different oestrogen target
`organs to its antioestrogenic action (Wakeling et a] 1991).
`For example, at doses which produced an ovariectomy—
`like regression of the uterus, no effect was seen on
`gonadotrophin secretion or on the rate of growth of the
`animals. Also, there was a differential between the dose of
`
`journal of Endocrinology (1994) 141, 335—341
`
`AstraZeneca Exhibit 2027 p. 5
`
`

`

`340 M DUKES and others
`
`Differential actions of a pure antioestrogen
`
`ICI 182,780 required to block cyclical vaginal cornifi—
`cation completely and that producing maximal
`anti—
`uterotrophic effects. More recently it was also shown that
`a dose ofICI 182,780 tenfold greater than that required to
`reduce uterine weight by 50% did not affect
`the gross
`density of femur bone in intact adult female rats ('Wakeling
`1993).
`like ZM
`antioestrogens
`The availability of pure
`189,154, differing substantially in chemical structure from
`the steroidal antagonists exemplified by ICI 182,780,
`provides an opportunity to examine further the selectivity
`of action of pure antioestrogens. ZM 189,154 is a non—
`steroidal agent selected for further evaluation from a series
`of naphthalene and tetrahydronaphthalene derivatives (EP
`0124369 B1) substituted with long amidoalkyl or sulphi—
`nylalkyl side—chains analogous to those attached to steroid
`derivatives described previously by this laboratory (Bowler
`et al 1989). Like ICI 182,780, ZM 189,154 has high
`affinity for
`the oestrogen receptor
`(Fig 2), completely
`blocks the trophic action of exogenous OE2 or tamoxifen
`on the immature rat uterus in a dose—dependent manner
`and has no intrinsic agonist activity (Figs 3 and 4). The
`antiuterotrophic potency of ZM 189,154 is comparable
`with that of [CI 182,780. In OVX rats, ZM 189,154 is
`devoid of oestrogenic effects on the uterus, vagina, growth
`rate, LH secretion and bone density (Tables 1, 2 and 5). In
`contrast,
`tamoxifen,
`like OEE,
`substantially reduced
`growth rate and plasma LH (Table 2). When coe
`administered with 0132 (or tamoxifen) to OVX rats, ZM
`189,154 completely blocked its trophic effects on uterus,
`vagina and bone and its suppression of LH secretion but
`only partially reversed its suppression of growth rate
`(Tables 1, 2 and 5).
`Differing target organ sensitivity to ZM 189,154 was
`more apparent in intact rats. Thus, whereas a single dose of
`2 mg/kg was sufficient
`to block ovulation completely
`(Table 3) and, on repeated administration, to cause uterine
`atrophy of about 90% of that following ovariectomy (Table
`4), the same dose had no effect on growth rate (Table 4)
`or bone density (Table 5). However, at a higher dose of
`10 mg ZM 189,154/kg, growth rate and LH secretion
`increased (Table 4) and bone density decreased (Table 5)
`as in OVX rats, but to a lesser extent. Differential effects
`onM 189,154 were also recorded on the ovary (Table 4).
`At doses of 0-3—2 mg/kg, ovarian weight was reduced
`whereas, at
`a daily dose of 10 mg/kg, ovarian size
`increased. Histological examination suggested that
`the
`reduction in ovarian weight was due to the presence of
`fewer corpora lutea consistent with blockade of ovulation:
`there was no evidence of marked follicular stimulation.
`
`However in the rats given 10 mg ZM 189,154/kg per day,
`follicular hyperstimulation and ovulation inhibition were
`apparent. The fact that for acute inhibition of ovulation a
`dose of 2 mg/kg was needed, whereas daily doses of as
`little as 06 mg/kg also completely blocked ovulation
`suggests that the effect of the latter is not simply due to
`
`Journal of Endocrmo/ogy (1994) 141, 335—341
`
`inhibition of the preovulatory LH surge, but may also
`involve inhibition of follicular maturation or of pituitary
`priming for
`the positive feedback response to
`the
`preovulatory oestrogen surge. Comparison of the smallest
`doses that block ovulation (08—06 mg/ kg per day) with
`the dose at which elevation of basal LH concentrations
`
`was seen (10 mg/kg per day) suggests a margin of about
`20—fold between these
`two
`processes;
`even
`acute
`inhibition of ovulation, which may be more directly
`related to inhibition of OEZ—mediated positive feedback
`on LH release,
`is achieved at
`lower doses than are
`needed to completely reverse OEzemediated negative
`feedback on gonadotrophin secretion. Differences in end
`organ
`sensitivity
`to ZM 189,154 probably reflect
`differing oestradiol
`thresholds rather than drug access,
`though the latter may be
`a
`factor
`in relation to
`hypothalamic effects.
`Differences in apparent sensitivity to ZM 189,154
`between OVX and intact rats were also observed: bone
`
`density in intact rats was not affected by a daily dose of
`2 mg ZM 189,154/kg but the same dose reversed the
`bone—sparing action of oestradiol in OVX animals (Table
`5); even at 10 mg/kg per day, ZM 189,154 did not reduce
`uterine weight to the same extent as ovariectorny (Table
`5), and the 90% antiuterotrophic effect achieved in intact
`rats is
`to be contrasted with the 100% effect seen in
`
`oestrogen~treated OVX rats. These diiferences may
`simply be due to diiferent levels and temporal patterns
`of circulating oestradiol between intact and oestrogen—
`treated OVX rats, exaggerated by the effects of ZM
`189,154 on ovarian function (prolonged or enhanced
`follicular activity). However, it is possible that substances
`other than oestradiol, secreted by the ovary, could also
`contribute to differences between effects in OVX and
`intact rats.
`
`In summary, in OVX rats ZM 189,154 is an effective
`and complete antagonist of the action of a maximally
`effective dose of exogenous 0E2 on uterus, vagina,
`bones and LH secretion and it partially reverses the
`action of OE2 on growth rate. A broadly similar pattern
`of effects is seen in intact animals, but the magnitude of
`reversal of oestrogen—dependent
`effects
`tends
`to be
`somewhat smaller than that achieved by ovariectomy,
`possibly because of concomitant oestrogen~like actions of
`other ovarian factors that do not operate through the
`oestrogen
`receptor,
`or
`because
`rising
`endogenous
`oestrogen concentrations resulting from disturbance to
`ovarian function pose too strong a challenge to the
`antagonist.
`Issues of differential
`tissue
`sensitivity in
`relation to dose and endogenous oestrogen concentration
`clearly
`require
`careful
`consideration when
`pure
`antioestrogens such as ZM 189,154 and ICI 182,780 are
`used as
`research tools
`to probe different aspects of
`oestrogen action. Such issues are also likely to be very
`important in the future therapeutic application of pure
`antioestrogens (Wakeling 1993).
`
`AstraZeneca Exhibit 2027 p. 6
`
`

`

`Differential actions of a pure ant/oestrogen
`
`M DUKES and others
`
`341
`
`References
`
`von Angerer E, Knebel N, Kager M & Ganss B 1990 1—(Aminoalkyl)—
`2-pheny1indoles as novel pure estrogen antagonists. journal of
`Medicinal Chemistry 33 2636—2640.
`Bowler], Lilley T], Pittam ]D & Wakeling AE 1989 Novel steroidal
`pure antiestrogens. Steroids 54 71—99.
`Day BW, Magarian RA, ]ain PT, Pento ]T, Mousissian GK 8e Meyer
`KL 1991 Synthesis and biological evaluation of a series of
`1,1—dichloro-2,2,3-triarylcyclopropanes as pure antiestrogens. journal
`ofMedicinal Chemistry 34 842—851.
`Dukes M, Miller D, Wakeling AE & Waterton ]C 1992
`Antiuterotrophic efiects ofa pure antioestrogen, ICI 182,780:
`magnetic resonance imaging of the uterus in ovariectomized
`monkeys. journal ofEndom'nology 135 239—247.
`]ordan VC 1992 The strategic use of antiestrogens to control the
`development and growth of breast cancer. Cancer 70 977—982.
`Nicholson RI, Gotting KE, Gee] 81 Walker K] 1988 Actions of
`oestrogens and antioestrogens on rat mammary gland development:
`relevance to breast cancer prevention. journal of Steroid Biochemistry
`30 95—103,
`Nishino Y, Schneider MR, Michna H & von Angerer E 1991
`Pharmacological characterization of a novel oestrogen antagonist,
`ZK 119010, in rats and micejournal ofEndocrinology 130
`409—414.
`
`Niswender GD, Midgley AR, Monroe SE & Reichert LE 1969
`Immunoassay for rat luteinizing hormone with antiovine LH serum
`and ovine Ll-l. Proceedings ofthe Societyfor Experimental Biology and
`Medicine 128 807—811.
`
`Sharma AP, Saeed A, Durani S & Kapil RS 1990 Structure—activity
`relationship of antiestrogens. Phenolic analogues of 2,3—diaryl—2H—1—
`benzopyrans.journal ofMedicinal Chemistry 33 3222—3229.
`Wakeling AE 1993 The future of new pure antiestrogens in clinical
`breast cancer. Breast Cancer Research and Treatment 25 1—9.
`
`Wakeling AE & Bowler] 1987 Steroidal pure antioestrogens. journal Qf
`Endocrinology 112 R7—R10.
`Wakeling AE & Bowler] 1988 Novel antioestrogens without partial
`agonist activity, journal of Steroid Biochemistry 31 645—653.
`Wakeling AE, Dukes M & Bowler] 1991 A potent specific pure
`antiestrogen with clinical potential. Cancer Research 51 3867—3873.
`Wakeling AE, O’Connor KM & Newboult E 1983 Comparison of the
`biological efiects of tamoxifen and a new antioestrogen (LY 117018)
`on the immature rat uterus. journal of Endocrinology 99 447—453.
`Wakeling AE & Slater SR 1980 Estrogen—receptor binding and
`biologic activity of tamoxifen and its metabolites. Cancer Treatment
`Reports 64 741—744.
`
`Received 4 October 1993
`
`Accepted 1 February 1994
`
`loumal of Endocrinology (1994) 141, 335—341
`
`AstraZeneca Exhibit 2027 p. 7
`
`