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 Pharmaceuticiils, Alderley Park, Macclesfield, Cheshire SK1 0 4T(J, UK
`(Requests for offprints should be addressed to A E Wakeling
`
`Abstract
`
`ZM 189,154 ([1RS,2RS]-2-(4—hydroXyphcnyl)—2-methyl
`-1—[9-(4,4,5,5,5—penta—fluoropentyl)sulphinylnonyl]—1,2,
`3,4—tctrahydronaphth—6—ol)
`is a non—steroidal pure anti-
`oestrogen. It has a high relative affinity for the oestrogen
`receptor, completely blocks the trophic action ofoestradiol
`(OE2) on the uterus in immature and ovariectomized
`(OVX) adult rats and, in the latter, also completely blocks
`the trophic action ofOE2 on vagina, bone and growth rate.
`ZM 189,154 displays no intrinsic oestrogen—ag0nist activ-
`ity on uterus, vagina, bone, LH secretion or growth rate in
`OVX rats. Differential sensitivity of OE:-regulated pro-
`cesses was more apparent
`in intact rats. Daily doses of
`0-6 mg/kg per day of ZM 189,154 blocked ovulation;
`
`2 mg/ kg per day achieved maximal uterine atrophy but
`did not affect bone density or growth rate; 10 mg/kg per
`day produced a broader spectrum of etTects (reduced bone
`density, increased basal LH, slightly increased growth rate),
`but the magnitude of these was smaller than after ovariec-
`tomy; the 10 mg/kg dose also produced multiple ovarian
`follicular cysts. The fiiilure of Z.M 189,154 to achieve
`complete ovariectomy—like cfllects in intact rats may be due
`to the action of ovarian Factors other than OE2, or to the
`circuiating 0132 levels resulting from the disturbance to
`ovarian function posing too strong a challenge to the
`antagonist.
`/oumal (if ltiidrxriiiulogy (1994) 141, 335-341
`
`Introduction
`
`The properties of 7ot—alkylamide and 70t—alkynylsulphinyl
`analogues of oestradiol-1713 (0132) which characterize
`them as pure
`antioestrogens have been described
`(Wakeling 8: Bowler 1987, Wakeling er 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 pniniates.
`(Wakelirig 86 Bowler 1987, Nicholson ct
`til
`1.988,
`Wakelirig et all 1991, Dukes at al 1992). However these
`studies also demonstrated that there are significant dii'fei'—
`enccs in organ sensitivity to the action ofpure antiocstro-
`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,
`Wakeliiig 1993).
`In addition to steroidal pure zintioestrogens, non-
`steroidal pure antioestrogens have also been described (Von
`Angerer er al 1990, Sharma at al 1990, Nishino et L7] 1991,
`Day at al 1991). The activity of a new agent of this type,
`ZM 189,154,
`(European Patent, EP0124369 B1),
`a
`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—de-pendent 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 tarnoxifen (ICI 46,474 (trans—1—(4—
`dimethylziminoethoxyphenyl) — 1 ,2 — diphenylbut— '1 — ene]),
`lCl 164.384 (N—r1—butyl—N—methyl—'11—[3,17—dihydroxy—
`oestra—1,3,5(10)—t1'iene—7-yl]ur1dec2inamide)
`and
`7.M
`189,154
`([1RS,2RS]-2-(4—hydr0xyphenyl)—2—methyl—1-
`[9—(4,4,5,5,5—pentafluoropentyl)sulphinylnonyl]—1 ,2,3,4—
`tetrahydronaphth—6—ol) were synthesized in the labora-
`tories of7.eneca 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
`["1-l|OEZ (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 (Wakelirig er
`
`journal of Endocrinology (1994) 141, 335-341
`0022 0795/94/0141 0335 $08.00/0
`
`© 1994 Journal of Endocrinology Ltd Printed in Great Britain
`
`Astrazeneca Ex. 2027 p. 1
`Mylan Pharms. Inc. v. Astrazeneca AB IPR2016-01326
`
`

`
`336 M DUKES and others
`
`‘ Differential actions of a pure antioestrogen
`
`OH
`
`100
`
`g 20
`
`80
`
`60
`
`,§
`
`EL
`
`E
`.5
`
`1.)
`g
`
`40
`
`Log“, [Competitor]
`
`FIGURE 2. Competition for binding of5 X 1079 M
`|3H]oestradiol—17[3 (OE2) to rat uterine oestrogen receptor by
`unlabelled OE2 (C) ZM 189,154 (A), ICI 164,384 (O) 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 10’7
`unlabelled OE2. Each point and bar represents the
`mean :1: S.E.M. of nine observations in three dilferent
`
`experiments. Estimates of competitor concentration which
`reduced [31-l]OE2 by 50% (ICSD Values) were calculated by
`linear regression analysis of per cent inhibition versus
`logH,[co1npetitor].
`
`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 calcu—
`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
`eflfect 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 t—test.
`
`Results
`
`Interartiorz with oestrogen receptor
`
`Competition of ZM 189,154 with [31-I]—OE2 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 Ex. 2027 p. 2
`
`OH
`
`OH
`
`//,,lh
`”'(eH2)g SO(CH2)3 CF2 CF3
`ICI 132,730
`
`(CH2)9 3O(CH2l3 CF2 CF3
`
`,’
`
`CCH3
`
`ZM 189,154
`
`OH
`
`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 volurne 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 ml/100 g body weight for immature and
`mature rats respectively.
`In uterotrophic/antiuterotrophic studies in ovariecto—
`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 C03
`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 f‘!l(lOCrln()l()gy (1994) 141, 335-341
`
`

`
`100 J
`
`
`
` 501 Uterineweight(mg)
`
`I
`
`_____H."""“*
`
`0 J
`0-02
`0-05
`01
`0-2
`0-5
`1-0
`2-0
`5-0
`10-0
`
`Dose (mg/kg)
`
`FIGURE 3. Effects of ZM 189,154 on uterine Weight of
`immature rats. Animals received daily 21 single dose of arachis
`oil vehicle alone (open bar), 05 g oestradiol benzoate s.c. alone
`(solid bar), or the indicated doses of ZM 189,154 alone s.c.
`(.
`C) or orally (A —- A), or oestradiol benzoate together
`with ZM 189,154 s.c. (C—C) or orally (A—A) for 3 days.
`Points and bars represent meansi S.E.M. for a minimum of1O
`observations in at least two different experiments. Where no
`bar is present errors are smaller than the symbols.
`
`displacement of [31-l]—OE2 by ZM 189,154 reflected by
`the parallel displacement curves, allowed calculation of a
`relative binding aflinity ofO°66 for ZM 189,154 (OE2= 1),
`compared with 0-19 and 0-O25 for
`ICI 164,384 and
`tamoxifen respectively.
`
`Antiuterotrophic activity in inmzature rats
`
`When administered orally or parenterally at doses in the
`range O-O25-10 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 OE;
`inhibited the trophic action of OE2 on the
`immature rat uterus in a dose—dependent manner (Fig 3).
`Complete blockade of OE2—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 73M
`189,154 alone did not stimulate the uterus and did not
`
`induce vaginal cornification; OE2—stimulated growth was
`also blocked by ZM 189,154 (data not shown, EDSO 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 OE2,
`co-
`administration 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 of 10 mg ZM 189,154/kg (Fig 4).
`
`150 —
`
`Differential actions of a pure antioestrogen
`
`M DUKES and others
`
`337
`
`75
`
`50
`
`
`
`Uterineweight(mg) 25
`
`0 l
`0-3
`0- 1
`3-0
`100
`
`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
`meansi S.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 KX mature rats
`
`The trophic and inhibitory effects 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 6: Bowler 1988, Wakeling el al 1991).
`In
`animals treated for 14 days with OE2 alone uterine weight
`increased fourfold compared with OVX controls, growth
`rate was reduced and full cornification 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 OE2, 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 OE2-induced vaginal cornification and none of
`the doses reversed the OE2—ind11ced 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 oi-ZM 189,154 on
`the uterus or on growth rate or plasma Ll-I concentration,
`
`/ournal of Endocrinology (1994) 141, 335-341
`
`Astrazeneca Ex. 2027 p. 3
`
`

`
`338 M DUKES and others
`
`Differential actions of a pure antioestrogen
`
`TABLE 1. Agonist and antagonist activity of ZM 189,154
`(l’5—l0 mg/kg, orally) and oestradiol (OE2 benzoate;
`O-5 pg/day s.c.). in ovariectomized rats. Values are
`meanszlz S.E.M. for groups of five rats treated for 14 days
`
`Weight gain Uterus wt Vaginal
`(g)
`(mg)
`comificationl
`
`Treatment
`Ovariectomy
`015,
`ZM 189,154
`(10 mg/kg per day)
`oE,+z1v1 189,154
`1-5 mg/kg per day
`40 mg/kg per day
`10-0 mg/kg per day
`
`49-0 :1: 2'4“
`27-0 4 2-4“
`
`85 :1: 3“
`342 :t 21“
`
`420 :1: 3'8"
`
`80 :1: 4'
`
`19-8 :1: 4-6"
`204 :1; 2'91’
`28-13 4 30“
`
`157 1 15“
`91 :1: 2'
`81 3-
`
`0
`00
`
`0
`
`48
`9
`0
`
`"’“lndicate values which differ significantly, i.e. at least P<0-01 (Studcntis 1-test).
`‘Per 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; O-5 pg/day
`s.c.) and tamoxifen (1 mg/kg per day orally) in ovariectomized
`rats. Values are meansi S.E.M. for groups 056 rats treated for
`seven days
`
`Weight gain Uterus wt Plasma LH
`(g)
`(g)
`(ng/ml)
`
`Treatment
`Ovaricctomy
`012,,
`Tamoxifen
`2M 189,154
`2M 189,154+OE;,_
`[M 189,154+Ta1noxifen
`Tainoxifen 1 0133
`
`34-0 :1: 2'8“
`13-5 4 3-2“
`0-2 4 2-6"
`360 21:19‘
`258 i 2-0"
`15-0 zt 0-9"
`34 i 2-8“
`
`173 :1: 10“
`421 :1: 27*‘
`242 1 10“
`158 i 5‘
`156 i 111"
`192 :1: 5‘
`235 :t 13"
`
`15-0 :1: 1-3"
`2-2 :1: 0-3“
`3-2 1: 02“
`10-6 :1: 19‘
`16-2 :: 3-2“
`13-1 :1:1-8“
`2-4 :1: 0-51’
`
`“ "Tnclic.m- valuec which differ significantly, ie. at least ]"<()-U1 (Student‘s t—1t-st).
`
`whereas both tamoxifen and 0132 significantly reduced
`growth rate and LH concentration and stimulated the
`uterus (Table 2). In combination, ZM 189,154 completely
`reversed the uterotrophic action ofOE2 and tamoxifen and
`the suppression ofLl-1, and partially reversed the reduction
`of body weight gain (Table 2).
`
`Efiects 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—2mg 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 of ovulation by ZM 189,154 in intact rats
`
`Time of
`treatment
`
`No of rats
`ovulating
`
`Ova/ovulating rat
`(Mean i 5.1).)
`
`Dose
`(mg/kg sc.)
`—
`1
`2
`1
`2
`0-3
`0-6
`
`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 :: 2-1
`7-7 :: 4-7
`
`7-7 i 45
`5-3 :t 3-0
`11-3i7'3
`
`TABLE 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: S.E.M. for 11:5 rats
`(14 day treatments) or 10 rats (28 days treatments)
`
`Uterine wt
`("/0 of control)
`
`Ovarian wt
`(% of control)
`
`Body wt gain
`(‘/0 ofcontrol)
`
`Dose
`(mg/kg per day)
`14 days
`0-3
`06
`1-0
`1-5
`2-0
`Ovariectomy
`
`28 days
`2-0
`100
`Ovariectomy
`
`752 i 8-1””
`73-7
`<1-0*"
`«,5-2 15-3”’
`47-0 1: 3-8'“
`45-2 i 33''’
`30-0
`
`77-5 :1: 5-4“
`82-4 :1: 7-0
`74-5110-8
`70-6 1 7-2‘
`83-2 2: 9-2
`—
`
`89-6 :1:17-5“
`931 :1: 17-5“
`72-4117-4“
`1000 4 10-9“
`89-7 1 7-0"
`149-5 :1: 14-4
`
`35-0 4 3-2“
`33-914?“
`27-?» i 33
`
`83-1 1 5-9‘
`119-7: 48
`1
`
`81-1 1 12-5-1-
`125-5:1:13-8“
`142-9 :E 17")
`
`that were significmrly (P<11-()5;
`"blIlLl1.L'il.tC means (prior to conversion to %)
`Sl.udL'nt’s 1—t<:st) different fi'om intact and ovariectomi7ed cnmrnls 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 ofovariectomy (Table 4).
`
`111. Ovarian weight and histology At all doses between
`0-6 and 2 mg/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, greatcr than in controls (Table 4). Ovaries from rats
`given 0-3 mg ZM 189,154/kg for 14 days contained old
`corpora lutca 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 haemorrhagc.
`
`Astrazeneca Ex. 2027 p. 4
`
`

`
`Differential actions of a pure antioestrogen
`
`M DUKES and others
`
`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; O-5 pg/day) for 28
`days. Values are means :t S.E.M., rz=5 animals or n= 10 for bone data
`
`Uterus wt
`(mg)
`
`Bone gross
`density
`
`Bone mineral
`density
`
`Treatment
`Experiment 1.
`Control
`ZM 189,154
`(2 mg/kg per day so)
`OVX
`OVX+ZM189,154
`Experiment 2.
`Control
`ovx
`OVX -+- OE2
`OVX + 0132 +
`+ 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: 33“
`
`sh
`135
`111 :t 6”
`104i3‘
`
`411 :1: 46“
`101 1 3“
`475 :1: 7‘
`
`100 :t 3b
`
`369 :1: 48“
`
`125 i 4”
`99 i 5“
`
`1-612 10-007*
`
`0-742 1 0-0093
`
`1-604 1 0-005“
`1-569 1 0-008“
`1-582 1 0-006“
`
`1-600 1 0-003“
`1-532 1 0007*’
`1-5911 0-007*
`
`1-532 1 0-006"
`
`1-629 1 0-0041
`
`1-580 1 0-004*’
`1-5711 0-007*’
`
`0-730 1 0-0073
`0-685 1 0-010“
`0-701 1 0-008‘
`
`0-730 1 0-004*
`0-652 1 0010*’
`0-738 1 0-0101
`
`0-684 1 0-006*’
`
`0-766 1 0-0053
`
`0-727 1 0-005"
`0-704 1 0-009*‘
`
`‘fllndicatc values which differ significantly, i.e. at least P<(l-(ll (Studcntls I—te5t).
`
`iv. Body weight gain Ovariectomy significantly in-
`creased growth with average daily weight gain increasing
`from 2106 g in controls to 296 g in OVX rats. In contrast,
`doses of ZM 189,154 up to 2 mg/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 ovariectomy—like effect, but ofsmaller
`magnitude than that caused by ovarian ablation.
`
`v. Plasma LH At doses up to 1-5 mg/kg per day for
`14
`days, mean:t S.E.M.
`plasma LH concentrations
`(253 :1: 021 ng/ ml) were comparable with those in intact
`control rats (2-18:l:O-12 ng/ml). In rats given 10 mg/ kg
`for 28 days, LH was elevated (4-53 :l:0-97 ng/ml) to about
`half the extent seen in OVX rats (9-94:1: 1-33 ng/ml).
`
`v1. Bone density Ovariectomy significantly reduced
`both the gross and mineral density of femur bone after 28
`days; the meani S.E..M. reduction was 3'5 :l:0'5% in gross
`density and 8-8:120-9% in mineral density (Table 5).
`Treatment with 2 mg 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 OE‘, Completely
`blocked this protective eflect ofOE2 (Table 5, experiment
`2) indicating a complete blockade of OE2 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 8-1% in OVX rats.
`
`Discussion
`
`The use of the steroidal pure antioestrogen ICI 182,780
`in the therapy of breast cancer (Wakeling er 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-
`lilce activity, as has been seen with tamoxifen, is unlikely
`to occur (Wakeling 1993). However, :1 possible undesir-
`able consequence of pure antioestrogen therapy is an
`adverse effect on bone mineral metabolism leading to
`induction or exacerbation ofosteoporosis (Jordan 1992). ln
`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 ('\X/'akeling et ai 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
`
`/ournal of Endocrinology (1994) 141, 335-341
`
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`

`
`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
`availability of pure
`The
`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 OE3 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 OE2,
`substantially reduced
`growth rate and plasma LH (Table 2). When co—
`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 close 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
`
`ofZM 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 0-6 mg/kg also completely blocked Ovulation
`suggests that the effect of the latter is not simply due to
`
`Iournal of Endocrino/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 (06-06 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 OE2—mediated positive feedback
`on LH release,
`is achieved at
`lower doses than are
`
`needed to completely reverse OE2—mediated 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 ovariectomy (Table
`5), and the 90% antiuterotrophic effect achieved in intact
`rats is
`to be contrasted with the 100% effect seen in
`
`oestrogemtreated OVX rats. These differences may
`simply be due to different 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 OE2 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 oestrogemlike 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 (\X/akeling 1993).
`
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`
`

`
`Differential actions of a pure antioestrogen
`
`M DUKES and others
`
`341
`
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`Received 4 October 1993
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`Accepted 1 February 1994
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`/oumal of Endocrinology (1994) 141, 335-341
`
`Astrazeneca Ex. 2027 p. 7