M Dukes, R Chester, L Yarwood and A E Wakeling
`Cancer Research Department, Zeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
`(Requests for offprints should be addressed to A E Wakeling
`
`
`
`Abstract
`
`ZM 189,154 ([1RS,2RS]-2-(4-hydroxyphenyl)-2-methyl
`2 mg/kg per day achieved maximal uterine atrophy but
`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-6-ol} is a non-steroidal pure anti-
`day produced a broader spectrumofeffects (reduced bone
`density, increased basal LH, slightly increased growthrate),
`oestrogen. It has a high relative affinity for the oestrogen
`but the magnitude of these was smaller than after ovariec-
`receptor, completely blocks the trophic action of ocstradiol
`(OE,) on the uterus in immature and ovariectomized
`tory; 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
`the trophic action of OE, on vagina, bone and growthrate.
`complete ovariectomy-like effects in intact rats may be due
`ZM 189,154 displays no intrinsic oestrogen-agonist activ-
`to the action of ovarian factors ocher than OE;, or to the
`circulating OE, levels resulting from the disturbance two
`ity on uterus, vagina, bone, LH secretion or growth rate in
`OVX rats. Differential sensitivity of OE>-regulated pro-
`ovarian function posing too strong a challenge to the
`cesscs was morc apparent in intact rats. Daily doses of
`antagonist.
`Journal of Endocrinology (1994) 141, 335-341
`0-6 mg/kg per day of ZM 189,154 blocked ovulation;
`
`
`Introduction
`
`The properties of 7a-alkylamide and 7a-alkynylsulphiny!
`analogues of oestradiol-178 (OE,) which characterize
`them as pure
`antioestrogens have been described
`(Wakeling & 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 ofeither
`OE, or tamoxifen on oestrogen target organs such as the
`uterus and mammary gland in rodents and primates.
`(Wakeling & Bowler 1987, Nicholson et
`al 1988,
`Wakeling et af 1991, Dukes et al 1992). However these
`studies also demonstrated that there are significant differ-
`enccs in organsensitivity to the action of pure antioestro-
`gens; for example in rats, complete inhibition of oestrogen
`action on the uterus can be achieved withoutaffecting LH
`secretion or bone density (Wakeling & Bowler 1988,
`Wakcling 1993).
`In addition to steroidal pure antioestrogens, non-
`steroidal pure antioestrogens have also been described (von
`Angerer ef al 1990, Sharmaef al 1990, Nishino et al 1991,
`Day et al 1991). The activity of a new agentofthis type,
`ZM_ 189,154,
`(European Patent, EP0124369 Bl),
`a
`2-methyltetrahydronaphthalenc substituted with a sidc-
`chain like that of TC] 182,780 (Fig 1), is reported here to
`illustrate further the range ofeffects these agents elicit in
`
`oestrogen-dependent tissues. Attention is focussed on the
`differences in dosage needed w affect different oestrogen-
`dependentprocesses withparticular reference to effects on
`bone because of concerns that long term clinical use of
`pure antiocstrogens might adverselyaffect bone density to
`cause an ovariectomy-like onset of osteoporosis {Jordan
`1992).
`
`Materials and Methods
`
`he antioestrogens tamoxifen (ICI 46,474 (trans-1-(4-
`dimethylaminoethoxyphenyl) -1,2-diphenylbut-1- ene]),
`IC] 164,384 (N-n-butyl-N-methyl-1t-[3,17-dihydroxy-
`oestra-1,3,5(10)-triene-7-yljundecanamide)
`and
`7M
`189,154
`({1RS,2RS]-2-(4-hydroxyphenyl)-2-methyl-1-
`[9-(4,4,5,5,5-pentafluoropentyl}sulphinylnonyl]-1,2,3,4-
`tetrahydronaphth-6-ol} were synthesized in che labora-
`tories of Zeneca Pharmaceuticals.
`Competitive binding assays
`to measure the relative
`binding athnity of antioestrogens for rat uterine oestrogen
`receptors were as described elsewhere (Wakeling & Slater
`1980) except that the competitor dilutions were prepared
`in Tris:dimethylformamide (1:1) and mixed together with
`PHJOE, (AmershamInternational, Amersham, UK) wich
`cytosol at a ratio of 1:20.
`The rat uterine weight assay for utcrotrophic and
`antiuterotrophic activity has been described (Wakeling et
`
`Journal of Endocrinology (1994) 141, 335-341
`0022 0795/94/0141 0335 $08.00/0
`
`©) 1994 fournal 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
`
`0
`
`s =
`
`40
`uu
`2 20
`
`100
`
`£80
`= 60
`
`Log,, [Competitor]
`FIGURE 2, Competition for binding of 5 x 107?
`[H]oestradiol-17$ (OE;) to rat uterine oestrogen receptor by
`unlabelled OE, (@) ZM 189,154 (A), ICI 164,384 (OQ) and
`tamoxifen (A), Percent inhibition refers to specific binding
`corrected by subtraction fromtotal (*H]OE, bound, the
`non-specific component recorded in the presence of 5 X 1077
`unlabelled OEF,. Each point and bar represents the
`mean + $.E.M. of nine observations in three different
`experiments. Estimates of competitor concentration which
`reduced [*HJOE, by 50% (ICcq values) were calculated by
`linear regression analysis of per cent inhibition versus
`log, ,[competitor].
`
`OH
`
`4,
`7,Mtg4”
`
`(CHa)g SO(CH2)3 CF2 CF3
`ICI 182,780
`
`OH
` OH
`
`(CHa)g SO(CHa)3 CF2 CF
`
`ZM 189,154
`
`FIGURE 1. Structures of the pure antioestrogens ICI 182,780
`and ZM 189,154.
`
`these studies weighed between 240 and 260gat thestart
`al 1983). Details of doses, routes of administration and
`of the experiments. At the end of the dosing period, Icft
`duration of treatments are reported in the present Figures
`and right femurs were dissected, freed of adherent soft
`and Tables.
`tissues, weighed and their volumes determined by
`ZM 189,154 and oestradiol benzoate (Sigma Chemical
`Archimedes’ principle (by subtraction of the weight of a
`company, Poole, Dorset, UK) were prepared for admin-
`25 ml specific gravity bottle filled with water containing
`istration by diluting an ethanol stock solution into the
`each femur, from the sum ofthe weights of the femur and
`required volume of arachis oil with gentle warming
`the specific gravity bottle filled only with water)
`to
`(60 °C). Tamoxifen was preparedfor oral administration as
`estimate gross density. The fernurs were then reduced to
`a dispersion in aqueous 05% Tween 80. Dose volumes
`ash and the ash weighed. Gross bone density was calcu-
`were 0-5 and 0-1 ml/100 g body weight for immature and
`lated by dividing femur weight by volume; mineral
`mature rats respectively.
`density was calculated by dividing femur ash weight by
`In uterotrophic/antiuterotrophic studies in ovariecto-
`volume, One group of rats in cach of these studies was
`mized (OVX) rats, ovariectomy was performedatleast 2
`subjected to ovariectomy on day 1 to provide an estimate
`weeks before treatment began. For ovulation inhibition
`of
`the maximum antioestrogenic
`effect
`potentially
`studies, rats having vaginal smear patterns consistent with
`attainable.
`4-day-ocstrous cycles were given either a single dose of
`concentrations
`(LH)
`luteinizing hormone
`Plasma
`ZM 189,154 on day 2 or 3 of the cycle, or daily doses on
`were assayed by a modification of the double-antibody
`days 1 to 4 of the cycle. The rats were then killed by CO,
`technique described by Niswender et al (1969).
`exposure on the morning of the next scheduled day 1,
`Treatment effects were analysed by comparison of
`their Fallopian tubes excised and the contents gently
`group means using Student’s t-test.
`expressed onto a microscopeslide and the numberof cggs
`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 expennments. All the rats used in
`
`Interaction with oestrogen receptor
`for bind-
`Competition of ZM 189,154 with PH]-OE,
`ing to the rat uterus oestrogen receptor was measured
`(Fig 2) and compared with that of tamoxifen and the
`steroidal pure antioestrogen 1C1 164,384. Competitive
`
`Results
`
`Journal of Endocrinology (1994) 141, 335-341
`
`AstraZeneca Exhibit 2027 p. 2
`
`

`

`M DUKES and others
`Differential actions of a pure antioestrogen
`
`337
`
`150
`
`100
`
`w S
`
`
`
`Uterineweight(mg)
`
`
`
`1:0
`
`20
`
`5-0
`
`0:02 0:05
`
`0-1
`
`02
`
`05
`
`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 alones.c.
`(eo @) or orally (A ——- A), or oestradiol benzoate together
`with ZM 189,154 s.c. (@—@) or orally (A—A) for 3 days.
`Points and bars represent means +5.£.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 [*H]-OE, by ZM 189,154 reflected by
`the parallel displacement curves, allowed calculation of a
`relative binding affinity of 0-66 for ZM 189,154 (OE,=1),
`compared with 0-19 and 0-025 for
`ICI 164,384 and
`tamoxifen respectively.
`
`Antiuterotrophic activity 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-admuinistration
`of ZM 189,154 together with a maximally effective dose
`of OE,
`inhibited the trophic action of OE, on the
`immature rat uterus in a dose-dependent manner(Fig 3).
`Complete blockade of OE,-induced uterine growth was
`achieved with daily subcutancous (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% (ED,,)=0-09
`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 7M
`189,154 alone did not stimulate the uterus and did not
`induce vaginal cornification; OE,-stimulated growth was
`also blocked by ZM 189,154 (data not shown, EDs, 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 OE;,
`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).
`
`75
`
`vi cS
`
`Nwa
`
`
`
`Uterineweight(mg)
`
`0 FT
`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 a single dose of
`arachis oil vehicle alone (open bar), 1:0 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
`means +5$.£.M. for a minimum often observations in at least
`
`two different experiments. Where no baris present errors are
`smaller than the symbols.
`
`Effects in OVX mature rats
`
`Thetrophic 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 & Bowler 1988, Wakeling ef al 1991).
`In
`animals treated for 14 days with OE,alone uterine weight
`increased fourfold compared with OVX controls, growth
`rate was reduced andfull 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 OE,, ZM 189,154 achieved
`72, 96 and 100% blockade of the utcrotrophic action of
`OE, 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 OE,-induced vaginal cornification and none of
`the doses reversed the OE,-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 OE, 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,
`
`fournal of Endocrinology (1994) 141, 335-341
`
`AstraZeneca Exhibit 2027 p. 3
`
`

`

`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 (OE, benzoate;
`0-5 [ig/day s.c.). in ovariectomized rats. Values are
`means +$.£.M. for groups offive rats treated for 14 days
`
`Weight gain Uterus wt Vaginal
`(g)
`(mg)
`cornification'
`
`Treatment
`Ovariectomy
`OE,
`ZM 189,154
`(10 mg/kg per day)
`OE,+ZM 189,154
`1:5 mg/kg per day
`4-0 mg/kg per day
`10-0 mg/kg per day
`
`49-0424
`27-0 4 2-4"
`
`85 + 3°
`
`342421"
`
`42-0 + 3-8"
`
`s04 4
`
`19-8 + 4-6"
`20-4 + 2-9°
`28:8 + 3-0”
`
`1574155
`9142°
`
`81+3°
`
`0
`60
`
`0
`
`48
`9
`0
`
`“Indicate values which differ significantly, i.e. at least P<O-01 (Student’s t-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 (OE, benzoate; 0-5 ug/day
`s.c.) and tamoxifen (1 mg/kg per day orally) in ovariectomized
`rats. Values are means +s.e.M. for groups of 6 rats treated for
`seven days
`
`Treatment
`Ovaricctomy
`OE,
`Tamoxifen
`ZM 189,154
`ZM 189,154+ OE,
`ZM_ 189,154+Tamoxifen
`Tamoxifen | OE,
`
`Weight gain Uterus wt Plasma LH
`(2)
`(g)
`(ng/ml)
`
`34-0 + 2-8?
`3543-2"
`0-2 + 2-6°
`36-0 + 1:9%
`258420"
`15-0 + 0-9"
`3442-8"
`
`173 + 10°
`
`421427°
`242 + 10°
`158+ 5°
`156410"
`192 + 5?
`235 4 13°
`
`
`
`150241-3*
`2240-3°
`32+0-2
`106+ 1:9%
`16243-2°
`13-1 + 1-8"
`24+0-5>
`
`“Indicate values which differ significantly, i.e. at least P<0-01 (Student’s étest).
`
`whereas both tamoxifen and OE, significantly reduced
`growth rate and LH concentration and stimulated the
`uterus (Table 2). In combination, 72M 189,154 completely
`reversed the uterotrophic action of OE, and tamoxifen and
`the suppression of LH,andpartially reversed the reduction
`of body weight gain (Table 2).
`
`Effects 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 dose 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 maximumregression of
`
`Journal of Endocrinology (1994) 141, 335-341
`
`TABLE 3. Inhibition of ovulation by ZM 189,154 in intact rats
`
`Timeof
`treatment
`
`Noofrats
`ovulating
`
`Ova/ovulating rat
`(Mean + s.29.)
`
`Dose
`(ng/kg s.c.)
`
`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
`
`14042-1
`T7447
`
`77445
`5:3 + 3-0
`11-347:3
`
`YVABLE 4. Effects of ZM 189,154 given s.c. for 14 or 28 days
`on utenne and ovarian weights and body weight gain in intact
`and ovariectomized rats. Values are means +5.E.M. for n=5 rats
`(14 day treatments) or 10 rats (28 days treatments)
`
`Uterine wt
`(%of control)
`
`Ovarian wt
`(%of control}
`
`Body wt gain
`(% ofcontrol)
`
`Dose
`(mg/kg per day)
`14 days
`0-3
`(6
`1-0
`15
`2-0
`Ovariectomy
`
`28 days
`2-0
`10-0
`Ovariectomy
`
`75-2481"
`
`737466"
`65-2 + 5-39
`47-9 + 3-87
`45-2 + 3-3"
`36:0
`
`775 45-49
`82:4 + 7-6
`7454 108
`70-6 + 7-2?
`83-24 9-2
`_
`
`89-6 + 17-5"
`
`93-1 £ 17-5"
`72:44 17-4"
`100-0 + 10-9°
`89-7 + 7-6
`149-5 + 14-4
`
`81-1 + 12-57
`83-1 45-9"
`35-0 + 3-2
`125-5 + 13-8*
`119-7448
`33-9 + 4.5%
`
`
`—_—27-8 + 3-3 14294 17-9
`
`that were significantly (P<0-05;
`“Indicate means (prior to conversion to %)
`Student’s (test) different from intact and ovariectomized controls respectively.
`
`the uterus was 86%of that recorded in rats 14 daysafter
`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 of ovariectomy (Table 4).
`
`iii. Ovarian weight and histology Atall 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 wasslightly, though notsignifi-
`cantly, greater than in controls (Table 4). Ovaries from rats
`given 0-3 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 ofthe latter showed extensive haemorrhage.
`
`AstraZeneca Exhibit 2027 p. 4
`
`

`

`M DUKES and others
`Differential actions of a pure antioestrogen
`
`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 (OE, benzoate; 0-5 g/day) for 28
`days. Values are means + s.E.M., #=5 animals or n=10 for bone data
`
`Uterus wt
`Bonegross
`Bone mineral
`
`(mg)
`density
`density
`
`Treatment
`Experiment 1.
`Control
`ZM 189,154
`(2 mg/kg per days.c.)
`OVX
`OVX+ZM 189,154
`Experiment 2.
`Control
`OVX
`OVX+ OE,
`OVX+ OE, +
`+ZM 189,154
`(2 mg/kg perdays.c.)
`Experiment 3.
`Control
`ZM. 189,154
`(10 mg/kg per days.c.)
`OVX
`
`386 + 33°
`
`
`135+8°
`1114+6°
`104 + 3°
`
`411+ 46°
`101 + 3°
`475 47°
`
`100 + 3°
`
`369 + 48°
`
`12544?
`99 + 5°
`
`1-612 + 0-007*
`
`0-742 + 0-009"
`
`1-604 + 0-005*
`1-569 + 0-008°
`1-582 + 0-006°
`
`1-600 + 0-003°*
`1-532 + 0-007"
`1-591 + 0-007*
`
`1-532 + 0-006"
`
`1-629 + 0-004?
`
`1-580 + 0-004"
`1-571 £ 0-007"
`
`0-730 + 0:007*
`0-685 + 0:010°
`0-701 + 0-008°
`
`0-730 + 0-004?
`0-652 + 0-010"
`0-738 + 0-010*
`
`0-684 + 0-006"
`
`0-766 + 0-005"
`
`0-727 + 0-005"
`0-704 + 0-009"
`
`“Indicate values which differ significantly, i.e. at least P<O-O1 (Student’s é-test).
`
`iv. Body weight gain Ovariectomy significantly in-
`creased growth with average daily weight gain increasing
`from 2-06 g in controls to 2:96 g in OVXrats. 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 of 10 mg ZM 189,154/kg administered for 28
`days did produce an ovariectomy-like 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, mean+s.e.m.
`plasma LH concentrations
`(2:5340-21 ng/ml) were comparable with those in intact
`control rats (2-18+0-12 ng/ml). In rats given 10 mg/kg
`for 28 days, LH was elevated (4-53 0-97 ng/ml) to about
`half the extent seen in OVX rats (9-94 + 1-33 ng/ml).
`
`vi. Bone density Ovariectomy significantly reduced
`both the gross and mineral density of femur boneafter 28
`days; the mean+3.B.M. reduction was 3-5 £0-5% in gross
`density and 88+0-9% 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-induceduterine regres-
`sion and boneloss (Table 5, experiment 2). Administration
`of 2mg ZM 189,154/kg together with OF, completely
`blocked this protective effect of OE, (Table 5, experiment
`2) indicating a complete blockade of QE, action on the
`bones as well as the uterus in OVX rats.
`
`rats, 10mg ZM 189,154/kg per day did
`In intact
`produce significant reductions in bone density (Table 5,
`experiment 3): gross and mineral densicy were reduced
`3-0% and 5-1%, 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 et al 1991)
`may confer advantages when compared with the well-
`established use of partial agonists lke tamoxifen. For
`example, the developmentofresistance due to oestrogen-
`like activity, as has becn seen with tamoxifen, is unlikely
`to occur (Wakcling 1993). However, a possible undesir-
`able consequence of purc antioestrogen therapy is an
`adverse effect on bone mineral metabolism leading to
`induction or exacerbation ofosteoporosis (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 showedcleardifferences
`between the susceptibility of different oestrogen target
`organs to its antioestrogenic action (Wakeling et al 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
`
`inhibition of the preovulatory LH surge, but may also
`ICI 182,780 required to block cyclical vaginal cornifi-
`cation completely and that producing maximal anti-
`involve inhibition of follicular maturation or of pituitary
`uterotrophic effects. More recently it was also shownthat
`priming for
`the positive feedback response to
`the
`a dose of ICI 182,780 tenfold greater than that required to
`preovulatory oestrogen surge. Comparison of the smallest
`reduce uterine weight by 50% did not affect
`the gross
`doses that block ovulation (0:3-0-6 mg/kg per day) with
`the dose at which elevation of basal LH concentrations
`density of femur bonein intact adult female rats (Wakeling
`was seen (10 mg/kg per day) suggests a margin of about
`1993).
`like ZM
`antioestrogens
`The availability of pure
`20-fold between these
`two
`processes;
`even
`acute
`189,154, differing substantially in chemical structure from
`inhibition of ovulation, which may be more directly
`the steroidal antagonists exemplified by ICI 182,780,
`related to inhibition of OE,-mediated positive feedback
`provides an opportunity to examine further the selectivity
`on LH release,
`1s achieved at
`lower doses than are
`of action of pure antioestrogens. ZM 189,154 is a non-
`needed to completely reverse OE,-mediated negative
`feedback on gonadotrophinsecretion. Differences in end
`steroidal agent selected for further evaluation fromaseries
`of naphthalene and tetrahydronaphthalene derivatives (EP
`organ
`sensitivity
`to ZM 189,154 probably reflect
`0124369 B1) substituted with long amidoalkyl or sulphi-
`differing oestradiol
`thresholds rather than drug access,
`nylalkyl side-chains analogous to those attached to steroid
`though the latter may be
`a
`factor
`in relation to
`hypothalamic effects.
`derivatives descnbed previously by this laboratory (Bowler
`et al 1989). Like ICI 182,780, ZM 189,154 has high
`Differences in apparent sensitivity to ZM 189,154
`between OVX and intact rats were also observed: bone
`affinity for
`the oestrogen receptor
`(Fig 2), completely
`blocks the trophic action of exogenous OE, or tamoxifen
`density in intact rats was not affected by a daily dose of
`on the immature rat uterus in a dose-dependent manner
`2mg ZM 189,154/kg but the same dose reversed the
`and has no intrinsic agonist activity (Figs 3 and 4). The
`bone-sparing action ofoestradiol in OVX animals (Table
`antiuterotrophic potency of ZM 189,154 is comparable
`5); even at 10 mg/kg per day, 72M 189,154 did not reduce
`with that of ICI 182,780. In OVX rats, ZM 189,154 is
`utcrine weight to the same extent as ovariectomy (Table
`devoid of cestrogenic cffects on the uterus, vagina, growth
`5), and the 90% antiuterotrophic effect achieved in intact
`rats is
`to be contrasted with the 100% effect scen in
`rate, LH secretion and bone density (Tables 1, 2 and 5). In
`oestrogen-treated OVX rats. These differences may
`contrast,
`tamoxifen,
`like OE,
`substantially reduced
`growth rate and plasma LH (Table 2). When co-
`simply be due to different levels and temporal patterns
`administered with OE, (or tamoxifen) to OVX rats, ZM
`of circulating oestradiol between intact and oestrogen-
`189,154 completely blocked its trophic effects on uterus,
`treated OVX rats, exaggerated by the effects of 7M
`vagina and bone andits suppression of LH secretion but
`189,154 on ovarian function (prolonged or enhanced
`only partially reversed its suppression of growth rate
`follicular activity). However, it is possible that substances
`(Tables 1, 2 and 5).
`other than oestradiol, secreted by the ovary, could also
`contribute to differences between effects in OVX and
`Differing target organ sensitivity to ZM 189,154 was
`intact rats.
`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 (lable 5). However, at a higher dose of
`10mg ZM 189,154/kg, growth rate and LH secretion
`increased (Table 4) and bone density decreased (Table 5)
`as in OVXrats, but to a lesser extent. Differential effects
`of ZM 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 duc to the presence of
`fewer corpora lutea consistent with blockade of ovulation;
`there was no evidence of marked follicular stimulation.
`Howeverin therats given 10 mg ZM 189,154/kgper 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 doscs of as
`little as 0-6 mg/kg also completely blocked ovulation
`suggests that the effect of the latter is not simply due to
`
`In summary, in OVX rats ZM 189,154 is an effective
`and complete antagonist of the action of a maximally
`effective dose of exogenous OE, on uterus, vagina,
`bones and LH secretion and it partially reverses the
`action of OE, 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),
`
`Journal of Endocrinology (1994) 141, 335-341
`
`AstraZeneca Exhibit 2027 p. 6
`
`

`

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