“‘
`
`Slgapeal Ni}; Ti}3S6z’l}‘}~3.3.£)2
`(};:pc)si1in:; :1g:3im£EP Palm: {£35 933 6136.53 i25s"3}3§§}
`Patmlea. Astmiieratca M3
`fkymmitian by. Gmicun Richmr Ltd.
`{Int R£f.: M2353 EEPEQW‘
`
`%1f33S3US 3: PM-¥‘i’NEH
`Pfiizztimaviui ~ mfiwkw
`ss&aEsa'§sTa. 4:
`81¢???) MUNCHEN
`
`203
`
`Antiuterotrophic effects of the pure arztioestrogen ICI
`182,780 in adult female mcmkeys (Macaw nemestrimz):
`quantitative magnetic resonance imaging
`
`2313-20
`
`M. Dukes, J. C. Watertcm and A. E. Wakeling
`
`Cancer Research Departxnent, EENECA Pharznaceuticais, Mcresidc, Aldcrley i*a.z-3;, Macclesfield,
`Cheshire sa'..:cm'rc;, UK.
`
`REWSEI1 xawuscmrr RECEIVED 15 Fcbruary I993
`
`A&S’I‘i1ACT
`
`The antiuterotmphic cfiicacy of the pure amioestrcw
`
`(an ICI §S2,’s‘8i} has been demonstrated pr:-wicusly by
`‘zgnetic resonance imaging (MRI) in ouariectomized
`acstrogen»tre:ated monkeys
`(Maczmza nemzsrrina).
`Further charactterizzatinrz cf ihe effects of {CI IBERSE}
`in intact adult female mankeys with nomflai menstrual
`cycles was undertaken to provide an indimticm at‘ its
`p-atantial aciions in prcmcnupausa} wumcn. Changes
`in the volume of uterine tissues war»: measured by
`MRI in early, mid and law cystic. The vahxme of the
`uterus varied up to fivefold between individual
`monkeys but
`serial
`crsbservations
`in
`individuals
`pravided suificieanz precision ta aiiuw aizuurate asses?»
`rrzeems to be made af changes in the endometrium and
`mynmctrium ciuring the amuse cf the menstnzai cynic
`and follszzwixtg ICI 1823851 administration. In c»::m«
`parison with its initial size in untreated monkeys, the
`endwmetrium increased in vcalume by £>£}% and 125%
`in the mid and late czyckz respectively. In cont rain, the
`
`size at‘ the myometrinm decreaseci significantly. by
`15% from cariy In mid cycle and than rawverexi :13
`near its initia! volume in the late cycle. Treatment
`with ICI I82,?80 beginning in the early part of the
`menstruai cycie prevented the growth of the uterus.
`The magnitude and duratim: sf the response was
`dependent on whether €31’ not cevuiaticsn occurred
`during treatmcnt with If?! I$2.’?8f}. In animals rm»
`dared anevulamry, growth of the endametrium was
`blocked compietely by ICI I82,‘?8O and the ‘miume: 01”
`the tissue dcclincd below that prcscm at 111-: star: nf
`the meznstmal cycie. Antiuterrozrophic efiicacy was
`significantiy less in monkeys which mrulated tinting
`treatment with it?! i82;?8{}. The volume: Csf £113.23»-
`matrium was reducexi suhszantialfy by antiuestrugen
`treatment, and the diiference in response between
`ovulatory and ancwuiatary monkeys was less marked
`than that of the eudometrium.
`Iaurnzzf sf Enxiacrinaisgy ($993) 338, 2fl3—2(}‘§
`
`Kxai‘l"i£{}I}UC'l'i(}N
`
`ICI 182380 is a pawn: spccific pure anzioestrogen
`which may prev: superiar ta ca-nvcmianai partial
`agmzist amieestmgens in the ireaamant of breast
`cancer {Wakeiing ex :21. 1993). Previuus
`stuciies
`evaiuateci the antioestrogeni: potency and eificacy of
`ICI 182386 in nvariectomized <:~estmgcn—-treated
`monkeys (Macaw nemesfrimxj by magnetic rescrnance
`imaging (MRI) of the uterus (Waterman er at. 19:91;
`Ifiukes at al‘. 1992). A daily dase of (3-Img ICI
`IS2,?3£3fkg carnpletely bias!-zed
`the utemtrophic
`actitm af emgenous aestradiui-H3. Singk intra«
`muscuiar
`injactians
`at‘ a
`b:mg;«-arzting
`+3iI«-based
`farmuiatien of
`ICI
`i82,‘?8{lI
`in cwariectomézed
`menkeys were 3150 siwwn to inhibit oestrogen acticzn
`in a desmdepcndent sustaincd manner (Dukes at at}.
`
`I992). MRI has been used to ebscrve the uterus
`during the nannal Incmtrual cynic in women (flcmas
`emf. 1986; Haynor er al. 1986; Mcfianhy er al. 1986;
`Wiczyk ex :21. 3988; Barmii er cal. 1991) and the
`response so exogenous endocrine agents {Demas
`22 an‘. 1986; McCarthy as :24‘. 1986; Andrayko am :23.
`1988; Biawin at :25. 199(1). MRI has aiso been usraci
`quantizaiively to measure the changes in volume af
`the endometrium and myometrium during the normal
`manskruai cynic in M. nemastrina (Waterman H as’.
`1992). The MRI studies reparted here were performed
`ts: evaluate {hes antiutcmtraphic affects our? IE?! 132.7788!
`in admit
`femaie monkeys with normal menstrual
`cycles. Such studies are 1-ciwant
`to the pcrtcnzial
`appiicaiicm of ICE }82,‘3’8{} in the treatment of bath
`malignant and benign aestmgen-rasponsive di3££1s¢s
`in pre- and perimmopausal womcn.
`
`Jwrnaf cf Ezsdocrixmfagy (I993) 138., 203~«2{}9 @ £993 Jmzmaf of Emiocrinemgy L16
`IJ£¥22-¥}?§5o“93i{3133~i32U3 $€!2.€)1}!!}
`
`Prrmed £9: Gram firftcfirs
`
`|nnoPharma Exhibit 10570001
`
`

`

`1»
`
`204 M. IJUICE5 and others
`
`Efecrs ef EC? }82,?8{.3 in arm’: menstruating menkegar
`
`MATERIALS AN!) METBGDS
`
`Animal handiieg am} treatment eeieuinlw
`
`Mature female pigtail monkeys (AI. eemeerreze) of
`540 kg body weight with regular menstrual eyeler.
`were used. The ncmnal menstrual cycle in this. species
`is 29-32 days in iength; menstrual bleeding usuaiiy
`lasts 34 days. Daily vegirlaf swabs were taker: to
`detect the «meet of menstrual bleeding and the first
`day cf menetmaiien was rieeigiareri day I ef the
`eycie. Where required, bleed eamples {2 ml) for eaxb-
`sequent hormene analyses were eelleeted daily. MRI
`e:-zaminatiens took place before the animals received
`their mcirning feeti, in order re minimize artefaeteal
`neise associated with get‘. metien. Anaesthesia was
`induced with 8*? mg ketamineikg (VeLlar; Parke
`Daeis, Pentyponi,
`(Sweat, U32.) and waintained
`with haloihane (Fluethane;
`ICI Pharrnaeeuxieals,
`Maoclesfielri, Cheshire, U.l<'..}.
`The timing cal" MRI, drug treatment and bleed
`sampling was scheduled by reference to the firsi clay
`of menstruatien. Te mevide a baseline with which to
`compare the effects at‘ rimg treatment,
`three MRI
`measurements of the uterus were made during each of
`two cycles in ten unzreeteri monkeys (Wetertcm er e3.
`1992}. Images were recorded in early {days 2-—-S}, mid
`(days 12-18} and late (days 21-45} cycle amt sched-
`uled by reference to the diflerenoes in cycle length in
`individuals. Images were reeerdeel at simiiar time»
`paints in menkeys treateel with ECI i82,‘?3€1 beginning
`early in the menstrual cyele (days 2-5). Twe fmmu-
`latiens at‘ If)! 182,‘?8{} were used: 3. sherteacting
`propylene glycol-based solution (F 1) administered
`once daily i.m. for 25 days at a dese ef {H {ee5) or
`{)2 mg {n= 5) {Ci I83,'?8GIkg, and a langecting caster
`eiI~based solution (F2) given as 3 single im. izgeetiee
`el" 235 (R315) er emg (axe) {CI LS2.,?8G}kg.
`
`Quantitative Mill
`
`The methcsd Fer measurement of endemerrium anti
`
`myemezritzm eelume is described in detail elsewhere
`iwalertcrn 2: gr}. 1991, 1992; Dukes er: ml. W92).
`flriefiy,
`the study empleyed a Biespce 40012.3
`instrument {Oxford Research Systems, Cewentry,
`Warwiekshire, U.K.,) te reeerd a series of oblique
`images cf the uterus. For each animal on each
`occasien, sagittal sighting images were used re deter»
`mine the spatial eowardinates of ill: uterine cervix and
`fundns. From these eemrdinates, MR! parameters
`were calculated to allow the aequisizien ef eight
`contiguous ebiique slice images, the thickness, ;>c:ei-
`tier: and orieritatien ml’ which were forced re depend
`on the vector czetmecting the fundus and cervix. This
`procedure allows reprcidueihie presenzatien nf the
`
`Jezerrses’ sf Efldeerinafegy (W93) 133, 3333499
`
`uterus, independent :33‘ its size, location and orienta-
`liun in the pelvis {Dukes er en’. 19%; Waterten er :3}.
`1992}. Image matrices were 256 >-t 2.56 giving an in»
`plane rese-lutirm ef {}~£S:mr11. The appearance all‘ the
`images (see the Plate} results from the choice e-f MR1
`parameters, in particular the magnetic field strength
`of 265 tesla, the eerie time ef Sflme, the repetition
`time ref 3 s, and euppressien {if {he signal fmrn fat.
`To allow comparisons between images acquired an
`difierent eceaeiens,
`tissue eelumes were rneasurexi
`rather than endometrium height er area, since the
`former avoids ariefeets frem repesitienieg errors
`or shape changes. Endometrium and myemetrium
`valumes were obtained by pixel seeming in the
`oblique images rseirig Ilhe irregular reg;ion~ef«imereet
`Facilities within the pregrerns uxrimr or £}ISI‘~lM.R
`(Bruker Analytisehe Messtechnik; Gmhfl, Iiarlsmhe,
`Ciermany). only slices more fundel than the internal
`as were included in the analysis. The junction zone
`was includes: with the myometrium. Any contribution
`in enciurnetrium velume from the lumen was neglected
`since, except during menstruatien, this is very narrew
`and insignificant in eemparisee with {he endeemetrium
`{Drakes er :3}. 1992; Weteriem e: :23’. 19%}.
`Although, in principle, the prescription of regiem
`efiinteresl ear: be subjective, the method empleyed
`minimizes errors, since the presentation cf the uterus
`was the same at each examination (Watenen er ac’.
`I991, I992; Dukes 2: e1. E992). For each animal, the
`same eleserver analysed all imagee. X¥eek~:o-week and
`eyelevteeycle coefficient-5 of variatien were as law as
`5% {Watertxm er :2}. 3992).
`
`Analysis of results
`
`The volumes ef the endemetriem and myemetrium in
`treated and untreated groups were calculated as the
`arithmetic mean {:i‘.: s.e.:.r.} nemalized by reference
`to tissue volumes in individuals at
`the first MRI
`measurement. Differences between group means were
`tested fer significance by the unpaired Student’s
`1-test.
`
`RESULTS
`
`Uterine volume in the enema! menstrual cycle
`
`The wlumes ef the endometrium and myemetrinm
`were measured by MRI in early, mid and late cycle
`in two cycles in each of ten diflerent untreated
`mankeys. {Text-fig. 3}. The mean interval between
`repeat measurements was 4 mcmths
`(range 1-9
`mcmths}. Tissue wlumer. varied widely between indi-
`viduals but less se in repeat measurements in the same
`animal. Typical images reeerdecl in (me menkey at
`
`|nnoPharma Exhibit 1057.0002
`
`

`

`
`
`~ M. DUKES and othersEjj'2’cr.s of:'(‘! £82,730 in adult mmcrruaring monke}-.v 205
`
`
`
`4'0
`
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`
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`
`I8
`
`[6
`
`I4
`
`I2
`I0
`
`
`
`Volume[cm')
`
`%
`
`’
`
`|
`
`x
`
`I
`
`IN)1_
`
`N_
`
`Isl x5
`
`3_O%
`
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`
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`
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`
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`
`235
`
`238
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`
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`
`217
`
`TEXTJPIGURE l. (a) Endometrium and (£2) myometrium volumes in untreated
`monkeys during the menstrual cycle. For each 0!" tan animals the bars represent
`(From left) tissue volumes measured by magnctic resonance imaging in two
`separate: menstrual cycles, during the early (days 2-5}, mid (days 12-48) and
`lalc (days 21-25) stages of the cycle.
`
`Journal of Endocrinology (1993) 133. 203--209
`
`|nnoPharma Exhibit 1057.0003
`
`

`

`206 M. DUK and others
`
`Eflects of IO’ 182.780 in adult menstruating monkeys
`
`
`
`
`
`
`
`Nonnalizedvolumes(%}
`
`Untreated
`In = 20)
`
`Fl (0-1)
`(n=3)
`
`Fl (02)
`(n = 4)
`
`F2 (2-5)
`in = 3)
`
`F2 (4-0)
`on = 5)
`
`'l"E)(T—FlOURE 2. (a) Endometrium and (b) myornetrium volumes in untreated
`and ICI l82.78D-treated anov-ulatory monkeys. Tissue volumes at mid and late
`cycle were normalized to those recorded at the beginning of the cycle in each
`individual. From the left, values are means i S.l:‘..M. For untreated controls;
`monkeys receiving once daily injections of ICI 182,780 (0-I or 02 mgfkg) (F1)
`for 25 days, beginning on cycle day 3; monkeys receiving a single injection of
`ICI 182,780 (25 or 4 mg/kg) (F2) on day 3.
`
`days 3 (early), 16 (mid) and 24 (late) of the cycle are
`illustrated in the Plate. figs 1-3 and in another case at
`days 2, l6 and 23 in the Plate, figs 7-9. Analysis of the
`data showed that >75% of the variance arises from
`
`Effect of ICI 182,780 on the uterus
`The effects of ICI l82,780 on the appearance of the
`uterus in MR images is shown in the Plate. Plate, figs
`4-6 and 10-12 illustrate the mid corpus of the uterus
`at early. mid and late cycle, in individuals treated
`with the rapid— (F1) or slow- (F2) release Formulations
`of ICI 182.780 respectively, compared with images
`recorded in control cycles in the same individuals
`(Plate,
`figs 1-3 and 7-9). The dramatic shrinkage
`of the endometrium (central high~signal zone)
`is
`immediately apparent;
`the rnyometrial changes are
`less obvious from visual inspection of the images but
`emerge clearly from quantitative image analysis.
`In monkeys treated with ICI 182,780, the magni-
`tude of changes in the volume of the endometrium
`and myometrium was dependent on whether or not
`individual monkeys were rendered anovulatory by the
`treatment. Where serum progesterone concentration
`(not shown) remained below 2 nrnolfl in the second
`half of the menstrual cycle. animals were judged to be
`anovulatory; similarly where progesterone exceeded
`this value and remained elevated for at least ll] days,
`ovulation was assumed to have occurred. By this
`criterion, treatment with 0-] or 0-2 mg ICI l32,780fl<g
`daily (Fl) blocked ovulation in three or four of the
`
`|nnoPharma Exhibit 1057.0004
`
`interanimal variability. Average coefficients of varia-
`tion for volumes measured in dilferent cycies were in
`the range l4—l9% for the endomctrium and 6-10"/E:
`for the myometrium.
`The
`endometrium increased
`
`in
`
`the
`
`in
`
`size
`
`follicular phase of the menstrual cycle in 17 of 20
`cycles (mean increase 60%, P-<0-O01; early vs mid
`cycle volume) and this growth continued in the
`luteal phase in 18 of 20 cycles (mean increase 65%,
`P<0-001; mid vs late cycle volume). Changes in the
`volume of the myometrium were smaller but a
`consistent pattern emerged in which a decrease in
`size was observed in 18 of 20 cycles in the follicular
`phase (mean decrease 16%, P<0~0Ol; early vs mid
`cycle).
`In the luteal phase the volume of the
`myometrium increased in 18 of 20 cycles (mean
`10%. P<0-02; mid vs late cycle volume). The net
`effect of the follicular phase shrinkage and luteal
`phase growth of the myometrium restored mean
`myometrium volume to near that recorded initially
`(94%, P>0-OS compared with early cycle).
`
`Journal of Endocrinology (1993) 138. 203-209
`
`

`

`Ejfecrs of [C1 £82,780 in adult menstruating monkeys
`
`M. DUKES and others
`
`20?
`
`
`
`200
`
`A100
`
`E3
`
`3‘
`
`E2 E
`
`‘U
`
`0
`
`100
`
`so
`
`0
`
`E
`"4
`E0
`
`2 F
`
`Untreated
`
`F2(4-0)
`r=2(2-5)
`1=2(2-5;
`12-295)
`1:: (0-23
`F1(0-1)
`Fl to-1)
`I69
`233
`219
`217
`174
`225
`235
`(tr) Endometrium and (b) myometrium volumes in untreated and lCl I82,780-treated ovulatory monkeys.
`TEXT-FIGURE 3.
`Tissue volumes at mid and late cycle. normalized to those recorded at the beginning of the cycle in each individual
`monkey, are compared with mean values (it s.ts.M., n-‘-20) for untreated controls. From the left. values are
`means i s.E.M. for untreated controls; monkeys receiving once daily injections of ICI 182,780 {I}! or 0-2 mglkg) {F1} for
`25 days beginning on cycle day 3; monkeys receiving a single injection of ICI 182,780 (26 or 4 mg/kg) (F2) on day 3,
`
`five monkeys respectively, in each dose group. Simi-
`larly. ovulation was blocked in three or five of the six
`monkeys receiving a single injection of 2-5 or 4mg
`ICI 182.780lkg (F2). There was a tendency for the
`her-dose groups to include fewer ovulatory cycles
`‘fit this was not statistically significant with the small
`group sizes employed.
`The mean changes in the volume of the endo-
`metrium and myometrium in ICI 132,780-treated
`animals are presented in Text-figs 2 and 3. Since
`absolute tissue volumes vary widely between indi-
`viduals
`(Te:-ct-fig.
`1),
`the effects of 1C] 182,780
`were assessed by normalizing the volume of tissue(s)
`recorded in each animal at
`the mid and late cycle
`observations to those recorded at the beginning of the
`cycle, prior to treatment, and by comparison with
`similarly normalized observations in the untreated
`controls. In monkeys rendered anovulatory by ICI
`l82.'I'8D (Text-fig. 2), the normal follicular and luteal
`phase growth of the endomctriurn was blocked
`completely and tissue volume at mid and late cycle
`was significantly (P-<0-001)
`less
`than that before
`treatment began in each of the treatment regimens.
`
`Although shrinkage of the endometrium was most
`apparent during the first half of the cycle {mean
`decrease — 61% compared with +60% control,
`P<0-001, at mid cycle). blockade of growth was
`sustained throughout (mean - 67% compared with
`+l25% control. P<0'00l, at
`late cycle). When the
`occurrence of ovulation was accounted for. no sig-
`nificant diflerences emerged between the effects of the
`different forrnulations and doses of ICI 182,780. with
`the exception that
`the 2-5 mg dose (F2) appeared
`slightly less effective (P<O-05) than the 4-0 mg dose in
`the second half of the cycle. The volume of the
`myometrium in [CI 182,780-treated anovulatory
`monkeys (‘Text-fig. 2) decreased more rapidly than in
`controls in each treatment group from early to mid
`cycle {mean - 35% compared with - 16% control.
`P<0vOOl. at mid cycle) and this decline continued in
`the second half of the cycle (mean - 54% compared
`with — 6% control, P-<0-001, at late cycle).
`In monkeys in which treatment did not block
`ovulation (Text-fig. 3), the mean effect of ICI 182330
`on the endometrium in the first half of the cycle
`( — 27% compared with +60% control. P<0-0|] was
`
`Journal ofEm1'a<“r.inol'ogy (1993) 138, 203-209
`
`|nnoPharma Exhibit 1057.0005
`
`

`

`»
`
`298
`
`M. uuxrzs and anthers
`
`Effects of’ EC! £82,780 in adult menttreaatirrg rrraniceyr
`
`seen in the anawlatcsry mczmltcys
`less than that
`but not significantly {PNII-03} ac. Hawevar,
`the
`mean respmtsc czmcealecl Widb clilfcrenccs between
`iriflividual monkeys with five {Isl the seven animals
`slmwing shrinkage (mean ~« 3S;l:5‘3r‘£:} whilst
`the
`remaining two mtmkeys experienced net gruwth at"
`the errdsametriunt (+l{)3% and +23%). In the latter
`animals, the gmwth {hf endometrium resumezl in the
`lute:-zl phase of the cycle l§+‘}"0%) at a rate similar in:
`that
`in untreated monkeys {+55%}. Mean tirsac
`Vfliflmfi remained, htrwever, lea-:3 than that in untreated
`cnntrols at the and at‘ the cm}: (P<l}~0l), but grtater
`than that
`in anovulatery monkeys (P<‘(}-D01). A
`similar pattern at" change was observed in the
`myametrium (’I“ext«fig. 3). At mid cycle the mean
`valumc af‘
`the tissue was
`reduced significantly
`{M 3'}'%: P<{}-{ll cnmparccl with central) and this
`effect was mt different
`tram that
`in atmvulawry
`animals (W 35‘i»’a}+ However, as was the case for the
`cnciomelrium,
`the myometrium did recover in the
`luteal phase (+18%) but ms: to the volume seen in
`controls. All
`the late cycle ubservaticm,
`ttfects of
`lreatnleat an that: myrzmstrium were significantly lest;
`{.P<f{l'{3t} in avulatary than in aria-xrulawry monkeyt.
`
`DISCUSSIGN
`
`MRI pravidas an accurate anal rapraclucible means emf
`measuring the changes in rcslume: of the uterus in
`response to aestrcsgerr treatment
`in uvariactemizcd
`rrmnkeys {Watertcm £2 at 199l) and has been used to
`evaluate the antiuterotraphic acticm eel“ lift l3?:..7"3{)
`(Dukes e: mi 1992). This techniqm: has been applied
`previously In rtctermine the changers in volurne of
`the enclometrium and myometrium during the can rs:
`of the narmal rttertstruat cycle at‘ M. nemwrrfna
`(‘Waterton er at‘. 1992) and in this repair: to measure
`the affmzts of treatment with ICE l82,?8{l can these
`changes.
`At the time of menstruation, uterine size, in partic-
`ular that of the enclometrtum, would be expected to
`reach a minimum and to increase during the fallicular
`phase {if the cycle and, in a lesser extent, in the lutcai
`phase. To address this question, MRI measurements
`were made at early, mitt anti lair: cycle in each of two
`normal cyclas in ten manlcrzys. In the early part :22” the
`cycle (days 2-53, the vulum: cf the endumrztriuzm and
`myomctrium in individuals varied over a tviczle range
`{up ti) fivrzfurlci), but tissue volumes were much lass
`variable in repetitive measurements in the same
`individual ("I‘ext—fig. I), A clear pattern at‘ change
`emerged during the menstrual cycle in which signifl—
`cant grawth of the endometrium was seen in both the
`fallicular and lttlfial phases. Perhaps surprisingly, the
`mean increase in size of the tznclnmetrfium was similar
`
`fauna? qf ilrreirrcrirrtxlugy {I953} 138. 263-209
`
`comparing the first and second half of the cycle.
`Clastradinl is ctmsiclered ta be thr: majrir mitagan for
`the uterus and it was thus anticipated that that largest
`change in the: endvzamstrium would occur in the falli-
`crtlar phase at‘ the cycle. ‘Hr-zawever, a propcrtionately
`similar increase in the valume at" the endametrium
`
`was seen in the luteal phase ef the cycle, MRI cannot
`be used is deterrnine whether the effects cl‘ {Estrogen
`and progettcrone on the size of the enrirtmetrium
`can be attrihuted mainly In epithelial hyperplasia as
`opposed to lrypertmphy. Changes in the size of the
`myumctrium during thc menstrual cycle were quanti—
`tatively much smaller than them: of the endrxmetrium
`and‘ in the follicular phase, qualitatively different
`{Text-fig. 1}. Remarkably, the myometrium (increased
`in size when the enclotnetriurzt was gnawing, mean
`myomclrium volume being smaller in mid cycle than
`at rncnstruaticn. This may indicate that the threslwid
`of sensitivity of the myamctrium to oestrngtzns is
`higher than that of the endamctrium andior that the
`rate at which the myometrium responds to changes
`in lmrtrtttrtal status is less: than that far the critic»
`meztriurn. Hawcvcr
`S.)‘{il¢§ mechanisms,
`such as
`ht:~r:none:«imtuced changes;
`in muscle trzane, may
`influence: the apparent Sim (if the uterus and are net
`accrcruntesd for in these MRI images.
`Initial studies at‘ the effettts cf ICI 182380 em-
`played a daily daring regimen with the antioestrogen
`fnrtnuiated in propylene glycnl whistle (F1) to provide
`rapid release in viva. it was Shawn previously that the
`chaztcrs dcesrzs cif till I32,'?3{l {G-l——l}~2 mglkg per day}
`achieve complete and rapidly reversible bleckade: of
`exogerwus oestrogen stimulation at‘ the uterus in the
`cmariecttxmized mcmkeys {Dukes as at 1992). Daily
`treatment during the menstrual cycle prtrsvided clear
`evidence that
`ICI £32,780 also has a substantial
`antiaestmgenit: effect an the uterus in intact monkeys
`{Text-figs 2 and 3}. The magnitude of the anti«
`uterotrophir: effect was strazmgly influenced by the
`cencurrent endocrine effects of
`It'll
`l82,';'30,
`in
`particular by whether the mszutkeys ovulated during
`the treatment pfififld. In anomlatory monkeys, not
`only was the rrrmnal
`increase in volume at‘ the
`endamclrium in the faflicular phasrz absent, the ends»
`mctréum actually clocrcasad in size to: volume mm»
`parable to thasc siren in tlvariecmmized munkezys after
`oestrogen withdrawal (Watarton at ac’. 199i; Iliukes
`at all. 1992}, indicating that treatment achieved cam-
`pletc lzalczckade of this trophic: actien of cndcgenmts
`mstrugens. This blockade of nrsstregrzrt arztian ml the:
`endcemetrium was sustained in the second half of
`
`the cycle. An aestmger: wrtlxdrawal-—like actian an the
`myarnctriun: was also recorded, where the normal
`small tie.-crease in volume rat" the mynmetrium in the
`follicular phase was significantly amplified by ICE
`l82.,’?8{l, and also became: similar in magnitude ta that
`
`|nnoPharma Exhibit 1057.0006
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`

`

`£}fi'ee£e ef EC! 182, E88} 5:: admit meesmmrizxg meekeys
`
`2.1. ounces and others
`
`209
`
`seen feiiewing oestrogen withdrawai in evarieetm
`mixer} mankeys. This shrinkage es? the myametrium
`ceetirmeti in the lutmi phase,
`The alternative eiifibased tang-azzting formuiatien
`(P2) of {CI 182,780, shown previeusiy to susiain
`blockade 01' lbs: uterotmphic: action if ecstradisi in
`cnaarieetemizeci monkeys fer appreximateiy 1 month
`(Dukes at am‘. 3992), produced cfiecw an the uterus
`simfiar to these ef the short-acting Fonmziation.
`These previous phannacelegicai findings are cniireiy
`eensistent with the findings in the present study with
`respect ten the duration «pf autism, the apparent dese-
`respense, and the longer sustained blockade ei‘
`myemetrial than enctamezrial gmwzh. In monkeys
`rendered anevulatery, the entimerotrephie efiieei of
`the 4%) mgfkg dase was indistinguishable {ram that
`of the shat:-acting feneulalione Hewever, the Iewex
`2-S mgfkg dust did not fully bimrk the trophic acxiun
`(_\f_endegexmus oestregens on the emiuxneitium in the
`mend ha?!‘ of the cycie, but éid cause progressive
`myametrial shrinkage. This prevides further evidence
`that the threshold at‘ sensitivity cf the myemetrium to
`uestmgens is higher khan that of the endametrium.
`With both fcrnnulations, the antiutemtrophic e£¥i~
`ceey of BL‘! 182,?88 in monkeys that mmiaked during
`treatment was more variable and generally smailer
`than in anovulamry animals, but slit! suhstamial in
`the majority {five of aeeen} of individuals {Text-figs 2
`and 31
`Gverell, ef the total of 22 treated ‘eyeies’, 15 were
`anevuiatary. With both formuiaticms there was same
`evidence that
`inhibixion of emmien was dese~
`
`related, but there were insufficicnt data to eetablish
`statistically significant ctifierenees. The reasons for the
`variability between individuals in their susceptibility
`to bteckede ef ovulation are net understood.
`
`These studies were mmpleted using nan-invasive
`1: methods with fewer than 2%) animals. In practice
`sfieh infarmaticm is unebtainahie in terminal experi»
`meme. Far example, it is estimated that detection cf‘
`myometrial shrinkage at the same time as cndemetrial
`gmwth by ten-nine! methods at the same sig1ificam:e
`icvel weuid have required ewe: 3“S{? animals. Thus,
`MRI may be a particularly sensitive and peewerful
`tea} for efinica} studies in endocrine phannaecaiegy.
`The elinieai usefulness of ICI 182380 remains in be
`determined, but its effeet in a case es?‘ spontaneceus
`aéenomyosis
`in a mcmkey has been described
`(Waterten er a1. 1993}.
`
`ACKN(}WLEI}(II£MENTS
`
`iitzarrocrks, I). Miiler,
`We thank 8.1- A. Breen, M.
`J. S. W. Morreii, 3. B. Larcumbe-Mebauail, D. Priest
`and 3. Tattershaii fer expert technical assistance.
`
`REFERENCES
`
`Azxdreykue J. 3,... Biumaefelé, 21., Mat.-sisal], L, 3%.. Mamet‘ S. E.,
`Hricak. H. &. Ia-flea, R. B. (1933). Use ef an aganisxie anaieg
`ef gonad~::rtronpin~reIeaeing hertmzvm {rzafarelizfl ta treat
`ieiomyemasz eeeessment by magmttie resrmancc imaging.
`American fezzreef cf Qbszerriex and Gyeeeefegy 1.58, 9&3»-910.
`Bartuli, 1 M. Meuiin. Ga, I3eLanne:.a L., Chagnaud, C. 8:
`Kaebarian, H. £1991}. The nouns! merus an magnetixt
`ntsaenansze imaging and variations associated with the
`hurmunal 33312:. Szsrgieef-Radfefogie Astazwmy !3, 213420.
`flames, B. E‘, Hrieein, H. & Jaflc. R. B. (19825; Utefine
`imaging: eflbcte :31‘ haemona! siimuiation. Redieiegy 159,
`323-125.
`Dukes, M., Mitten 3)., Wakeling, A. E. & Waterion, J. (3.
`(I992). Améulemiraphic efieats Inf 2! pure amiacstmgen, ICE
`i81,?S{}: magnetic resonance imaging of the uterus in
`evariectomizeccd monkeys. Jaermzf ef Endecrinefegy 135.
`33?-34?.
`Haynes, 3}. Fr‘... Meek, L. A, S-aulee, M. IL, Simmer; W. F5.
`Mezstana, M. A. at Mass, in 9:. {I936}. Changiag appearance
`ef the nermafl elem: timing the menstrual cycle. Radiefegy
`16!, 459-462.
`McCarthy, 8., Tauber. C. & Gare. I. (1986), Female pelvic
`anatomy: MR assessment of variatione during the meosmza-.1
`cycle and with use at“ cm} eemraeepzives. Reeiaiegy I63}.
`3 19-123.
`
`Wakding‘ A“ E. Bakes. M. &. fiewien 1, {}9}9i}u A. potent
`specific mm: anti-Brlrstragen with eiiriistal patentiee Center
`Research 51, 3367-38?3.
`Wataflon. J. (2., fircen. 8* Aw mk:-.3, 1%.. Horrecics. M. 8:
`Wadswonh, P. {Z993}. A case of asienemyesis diagnesed by
`magnetic reeenanee imaging and treated with the name! pure
`anticecstre-gen ICI 38333:). Leiwrasezy Aefmszi Science {In
`Pme}
`Waierton, 3. £1. La:ce:nbe»?vic£>ouel1. 3. E. 8: Mifics. $3. {$992}.
`Quantitative MRI of the prostate and uterus in memkeys.
`Megrxezie Reseneme in Medfeiee 28. 34491.
`Watering, J. 172., Miilez, 1)., Dukes, M. S: Mam-:11, J. S. W,
`{ESE1}. Qbiique MMR imaging ef the meme in macaques:
`uterine response: It‘: estrogen etimufimiun. Magaesie Reeeneeee
`:31 Medicine 20, 223~239.
`Wieeyk. FE. Pt. hens, C. 1... Richards, Cm L, Chef. M. 3.,
`(369631, E. 3.. Rabiuewit: J. G. & Laufer, N. {£983}
`Comparison ui‘ magnetic resunsxnee imaging and uitrascnnd in
`evaluating feltieelax and eetiemetrial development ihxeughetst
`the wane? cycle. Fertffiry wee‘ Sierflizy 4:9. 969-—9?S.
`Kama. M., McCarthy, 8., Scam. L., Lange, R, Levy; (3., Velte.
`3. 8: Camila. F. (39%). Munimring therapy with a
`genadotropin-reieaszng humane anelag: utiliiy ef MR
`imaging. Raeieiegy ['35, 5£l3~S(36.
`
`Jeunraf if Erscfeerinofeg}-' {1993} 338,, 2€13««209
`
`|nnoPharma Exhibit 1057.000?
`
`

`

`an
`
`2103
`
`M. 12:61:13.5 and mhers
`
`Efects cf1CI 182.?8{? Er: min}: menssmarshg mankeys:
`n
`
`I3ESCRI!”I‘!ON ()1? PLATE
`
`Magnetic: remnant: (MR) images of the monkey utarus.
`Each illustration is a email, represanting 3-4 x 3-4 cm,
`fmm am: slice cf 11:: eight utcorded at each mcasurcment.
`The appearasxca at mid-mtgus utcri is Shawn as fofilows.
`Tap raw: images abtaimzd 3: day 3 (Fig? 1), day 16 (Fig.
`2} and sziay 24 (Fig. 3} of an untreated menstrual cycle.
`Second raw: imagszs ubtained at day 3 (Fig. 4}, day 15
`(Fig. 5} and day 24 (Fig. 6) in the same animai during a
`cynic in which {H mg K31 lS2,?$43fkg was injevcted fiaiiy
`from day 3. Third row: imagea obtained at day 2 (Fig. 3'),
`day E6 (Fig. 3) and day 23 (Fig. 9} at‘ an umreatcd
`mensirual cycie. Batmm row: images obtained at day 2
`
`(Fig. 10}, day 16 (Fig. 11) and clay 23 (Fig 12} in the
`same: animal timing 3 crycie in which 4 mg 1C1 I82,?8{Ifkg
`sustained-rckease famiuiaiian was injectad an day 2. The
`cycies shown in that tap three rnaws were ovmlatarjs‘. Fm:
`at five canccntris: macs sf MR signal intensity are men:
`annual sigzai void, znenstruai haemm-rhage (91:13: during
`menstruation); high signal, endamctrium; medium»-Iszrw
`sigma}, myommial junctinn mac; medium-high sigma},
`myomctriurn; and nor sigxal. peripheral adipose tisaua.
`The area (aften crascenhshapeti} at’ high sigma} intensity
`secs: at the 12!: in Figs ‘RN12 is mine in thc bladder. At:
`nvary is partialiy visible {hottum right) in Figs ‘E-ii.
`
`fact:-mi! {Ff Endacrinafagy {I993} 13$, 203-2139
`
`|nnoPharma Exhibit 1057.0008
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`

`

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`
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`=
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`|nnoPharma Exhibit 1057.0009
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