Appear} Na: Tfll3fis’fl9‘3 3&2
`Qppasizim againm E? Patent :33: £33 (imam $253133)
`Pakmtcs: Miraaneca AB
`Clppasiflm: by. fledmn Richter Ltd.
`flu: Refit M23133 EFKJP?
`
`1-”
`
`E
`
`mews & PARTNER
`WWWALTE : RECHTSAWRLE
`'
`S EBEQTSTR. 4
`,
`5;
`§
`8’! 53’s MUNCHEN
`
`23
`
`a!
`
`Antiuterotrophic effects of a pure antioestrogeng
`ICI 182,780.: magnetic resonance imaging of
`the uterus in ovariectomized monkeys
`
`19/19
`
`M. Dukes, D. Miller, A. E‘ Wakefing and .I. C. Waterfall
`Research Department L, ICI Fhamafleaticals‘ Aidericy Park. Mamicsfield, Chcshirc 5K 3043‘s, UK.
`
`RECEWBD 30 January 1992
`
`
`ABSTRACT
`
`{$182380 is a patent specific pure anticestmgan
`which may nfi‘cr advantagts in breast cancer 1mm
`- mam compared with partiai agenists like tamexifcn.
`( ‘11 characterize further the patency and efficacy of
`1C! 182,788.
`its affects on the uterus of uvariccto»
`mixed: oestrogen-trcaied monkcys (Macaw nemw
`trim) have been meat;qu using magnetic resonazzce
`imaging (MRI). Quantitativc MRI afluws accurate
`nan—invasive: repetitive measuremants cf findamcttifi
`and myametrial valume foliowing hummus! treat—
`ments? using each animal as {as awn cannot Singie
`Em. injectians of a bug-acting niiwhaseci famulatian
`of ICI 182,?80 gustaimd bimkade of aestradiel action
`can the monkey uterus in a domwdapendent manna:
`
`injectiens 0f 4mg
`3~6 weeks. Repeated
`fur
`{CI 382,?3fiflg at $wegkly intervam pmvided
`increasingly affective blockade at“ Marine proiif‘era»
`tion. In a short~acting formutaziom ICI 182,739 aim
`cempletefy blacked the Imphic aciion at“ aestradiol,
`adminisiered mmurrently, in marimwmized mom
`kfiys. Simifazrllsr¥ ICI “182588 causcd invofutian 1317‘ the
`uterus stimulated by prior ircatment with nestradiol.
`The: rate and extent {if Marine invalutian in mankcys
`treated wilh 1C1 ISRRSQ was simifar 3:9 mm scan f0}-
`{sawing Gastroan withfirawat These studies deman—
`stratc that
`ICI 132,?Sfl
`is a {118}: efiactixre pure
`antimstrogen in a primate.
`lawns} sf Endccrfflafagy (1992) 135, 239-247
`
`INTRHHUCTIGN
`
`3C} 382,780 is a rccemiy described potent puts anti»
`aestmgm (Wakeling, Dukes S: Bawlcr, EQQE ). Unfikc
`a, mnsstewidal autimtrogws
`such as
`iamnxifen,
`L ’3 1825380 has been skews £0 be devoid cf pariiai
`agonist {matmgmic} activity in rats and mice and
`may, thcrefore, 9321' mm: advantages fur the: treat-
`ment of human breast cancer (Wakefiing a: at 199i}.
`A long-{3513115 antiaastmgenic effect :1? pamnteraliy
`adminiswmti {(31 182,130, farmulatcd as a suspension
`
`in Exachis mil, was dcmanstrated in rats and in nude
`mice bearing xenografts of human breast cancer sails
`(Waketing a! at. 1991). Thugs studies ware extended
`to examinc the delay 0? mtmgeminfiuced perinea§
`melting in cvariscmmized monkeys by preuwtmem
`w§th 10 daily (insets of 0* 1., 0'5 or I mg ICI 182380;?
`kg; or a single 605%: GF Wing/kg beme matterch
`treatmmL A sustained, dose-relatai blackade 0f peri-
`mai awaiting was noted {Wakeiing at :23. 2991). Frai-
`minary audits using magnetic resaaance imaging
`(MRI) dcmonstratai the feasibiiity {If precise meas-
`urcmcms of the monkey uterus and its mspansc to
`
`endocrine manipuiation (Wanton, Miller, Emit-tea fit
`Mama, [99]). MRI is an idgal method for such stud-—
`ies since it is quantitative: and noninvasive (Waterth
`3: :23. 299%) and the censequmccs of interanimat vari-
`abiiizy are minimised by using each animai as its awn
`comm} {Watermm Larcombe-Mcbouall s52: Miiler,
`1992). It greatly reducca, therefcre, the number of
`animals rcquimd to give a nxaaningful result. This
`paper describes a gems of studies undertaken to
`“amine: more fuily the. pharmacaiagy at” IE1 132388
`in the ovarimmmézed primate (Macaw aenwssrina}.
`
`HATER IALS AND METHODS
`
`Qumimtiw magnetic remnants: inaugng (MRI)
`
`We have repurth praviousty (Watcrtun 2: 51!. 1991,
`1992} mm;
`the appearance of the carpus Mari of
`Macaw namestrfna in magma:
`rcscnaacc: {MR}
`images is wry similar m that regretted fer wamcn,
`anhwgh {he convalan (semi): in the monkey has a
`rather diifibmnt MR appearanca fram that in wamcn.
`
`mem“ 91' fina‘acxa‘rmfagy {1992} 135, 239“ 24?
`MELOWSJQEJBBSfiES? $82,wa
`
`LC} I992 Inumal a? Endacrinulogy Ltd Prmwd is; firm: Edwin
`
`InnoPharma Exhibit 1036.0001
`
`

`

`24% to. DUKES and others
`{C} 33.23% afoot: on motto}: merits
`
`We have previously described mothottotogy for quanx
`titativo studies {Watotton 2: at. 3991). and discusscd
`the reproducibility and statistical power of tho toot:-
`niqnos (Waterton er at. [992}; a brief description is
`provided here.
`
`MR instrumentation
`
`The study omptoyed a ‘Biospec 400/23“ instrument
`{Exford Rottearch Systomo, Cottontry, UK.) meow
`rating a 1351‘ magttet and pulsed fieki gtttdionts up
`to it} mTfm (where T is the SI symbol for Tesla,
`the unit of magnetic flux donsity). A tadtoftequency
`resonator of 208 mm amass diamatot was ompioyod
`at transmittor and monitor.
`
`Oblique imaging
`
`to allow oompatisons between imaget
`in order
`acquirod on oifi‘orent occasions, it is most desirable to
`mature volume rather than, say tndometrial height
`or area, since the former avoicts artef‘aots from roposi»—
`timing otters or shape changes. The ondomotrial wk
`om: in this spooks can be quite smaii, beiow (M m3
`in ohronioaily ovariootomizcd animals, rising typicaity
`to 1 mt3 foltowing oestrogen stimttiation. Hence it is
`important to use MRI methods which allow good
`spatial motutiott together with adequate signal-too
`noise ratios. Sins: tho uterus is Somewhat allipsoida}
`with one long axis. oblique imaging allows the use of
`the stioeoolectod twooimonsional Fourierwtransform
`
`technique, which takes full advantage of the better
`rosoltttion in plane than in slioo thickness. Also, the
`orientation of the uterus within the pelvis varies from
`day to day depending both on its size in rotations:
`to oestrogen, and also on other factors. pmsumahly
`including the votome and diswoition of the contents
`of the urinary bladder and tower gut. It is advanta-
`goous for quantitative studies to employ obtique MRI
`techniques which force the presentation of tho atoms
`for image analysis to be simitar from ottaminatiott to
`examination, as shown in this Plato.
`In order to aohiovo this reproducible oblique tattoo
`mtation, tho following procedure Was usod for each
`MRI examination. A mottitiioo tagittal
`image: so:
`was acquired in order
`to determine the spatiai
`coordinates of the: uterine cervix and fundtts. From
`
`those mrdinatos, MRl parameters were oalmtatod
`to altow the acquisition of tight contiguous oblique
`slices, the thicknooo. position and orientation of which
`were forced to depend on the vector connotting the
`furious and cervix (Text—fig. I). Thus, the prosootation
`of the merits in the images is more: or less independent
`of its size,
`location and orientation in tho patios.
`Image matrices were 256 x 256 giving an iii-plane
`restitution of (to tow. Tho signal from adipoto tissue:
`
`looms? of Endocrfooiogy (19923 135. 239-14?
`
`was suppressed, and good contrast between the attr-
`ine tissues was sutured by using poise acqucnoe
`parameters Tu (repetition time} of 3000 ms and TE
`(echo time) of 50 ms.
`
`Quantification of the images
`
`Endometrium and myometrium volumes were:
`obtained by pitta} counting in the ooiique images using
`the irregular region-ofaintomt
`faoititios within the
`programs ‘ttttttmr’ or ‘DISMMR’ (Broker Anaiytische
`Mosstoolmik GmbH, Karlsruho, Germany). Oniy
`tattoos more fondal than the internal at were inducted
`
`in tho analysis. The junction tone was inoludod Iwith
`the myometrium. Any oomribotion to ontiometriat
`voiume from the lumen was oogtootoo since this is.
`very narrow and insignifioant in compariton with the
`ondomotrium. Errors arising from intowbsonror vari—
`ability were minimized stints each of the seven expatri-
`manta was anatyscti by a single obsoon {fifé‘ by
`not, I each by J.B.L.MCD. and by SAIL; see
`Acknowlodgomottts) and our conclusions are based
`on pcmoutago changes, each animal acting as its own
`control. The intraobseroor variabitity cannot be
`groater than the week~to-wook variability, and this is
`5% for the myomtzttittttt
`itt ovarioctomizod females
`{Watcrton e: of. £992). Limitad cormiatians between
`measurements made by MRI and by histology gave
`satisfactory results. sentiment with data proviontly
`reported in the human utorus (Lee, Getseli, “Bali‘e
`or of. 1935).
`
`Animal handling
`
`Mature tomato pigtail monkeys {Macom oemosoioo)
`of Swtttkg body weight were med. Animals wore
`ovariootomizod at least 31 months before their use: in
`those studies. MR! examinations took place. before
`the animals roooivod their morning food, in order to
`minimize: artefaotuai hoist: associatcd with gut
`motion. Anacstttosia was
`induced with {biting
`kctamineg’kg (titular, Park: Davis. Pontypooli
`(3mm, UK.) and maintained with halotttattt:
`(Fluothane,
`ICI
`Pharmaoomioals, Maoolesfiold,
`Choshitoi 13.31.).
`
`Phannacoiogicai protocols
`
`To assess the effects of treatment with ICI 182386,
`each animal was first
`treotod with 5 pg oestraoiol
`benzoate (OB: Sigma Chemioat Company, Poole,
`Botsot, 1.1.31.)ng in araohis oil. 5.2. daily for ’2‘ days
`(day t} to 63:: 6 inclusive}. unless otherwise noted.
`Uterine images wot: rooordod on day {1} immediately
`before the first dose of GB and on day 7’. Animals
`that: received 5 mg progestoronc {fiigma Chanical
`
`InnoPharma Exhibit 1036.0002
`
`

`

`
`1C? £32383 efe-cts as: make}, uterus
`v M. :3mtes and others
`
`243
`
`Company} s.c., clain far 5 or 6 days? After progcs~
`inane withdrawaldnduwd manstmation, animals
`again received 08, together with ICI 132381}. Details
`af‘ the treatmcm schedules employed and the timing
`a? MRI in each experiment are given in the legends to
`Text-figs 2-5. Una cxpcrimcm used a difl'erent (crusa—
`mer} prams-3!; see legend to Tcxbfig. 6.
`
`Analysis at“ the data
`
`Using our MRi methods ta study cutbred populau
`ticms of this species, w: have Shown prcviausly
`(Watermn et a}. 1992) that interanlmal variation in
`the volumes at" the uterine tissues greatly exceeds
`mk~m~w¢clt variatian in a single animal, mg. by a
`that): of 3-42 in the: myometrium, making it benefi»
`gial to us: each animal as its own Genital Hewmn
`Muse cf” flatten}: endomctrial straphy at £33: {3, val»-
`( “lacs a? this tissm: an: inaccurab: and pmhably nu:
`‘mcaningful. Hence we have elected m normalize: to
`the valumes cf endumetrial and myamctrial tissues
`after ‘? days of treatmem with 5 pg ()3, taken as 100%.
`Elmer): where otherwise stated, results are presented
`as the arithmetic meanztskem.
`
`RESULTS
`
`An initial treatment will: {)3 alum: established the
`
`extent of amine response in ovariectomized mankeys.
`Typical MR images wraparng the avariccmmind
`anti mtmgemtreated uterus
`illustratl:
`the large
`increase in endometrial voluma am: 3’ days 0f GB
`lrealmem (cf. Plate, figs 1 and 2). Texbfigure 2 mm«
`pares the gficcts 3f uncppased acstmgen manner}:
`for 3“ and 14 days on endametrlum and myomem‘um
`volume in ma individual animals. In this and: sumd~
`gsmdics‘ Eastman! efi'ctts an tissue vellum: in indin
`hudual animals are nemalizal to them achicved after
`
`(
`
`? (lays nl‘ 0E treatment, The changes in endomctrium
`mealtime in response to nestmgcn {Menfeld} were pro-
`gartianately much greater than that al‘ myametrium
`(appmx twafald}, Before a 3:11th and}! in these ani«
`mals, m address the effect cf 3C! 1823813,. uterine
`invalutian was inltiatcd by withdrawing OB and treah
`mam far 6 days with pregestamne {Text-fig. 2). Man»
`strual bleeding (not shown) was seen beginning an
`day 24,925 and mminued for 5 t}: 6 days. The endows»
`Mum, and particularly the mynmetriuml had nm
`regressed camplstely m basal 3:335:13 {all day 35 and
`day 0, Texhfigl 2}.
`The affect all" {Cl 182330 on the traphlc: rcspanse {if
`the: uterus to GB was. first addressed using 161 132380
`{emulated as a suspension in arachls ail. This femu-
`latlcm has been demonstrated prmrlously to provide a
`sustained antieszstmgenic cffcc: an the: perinch
`
`(Wakcling 61‘ at. W91} flail}! mama: far :0 days
`with 1 mg K31 182:?80fkg 33:. (tiny 39 to day 48‘ Tux?
`fig. 2}, beginning 3 days helm»: the rcsumptinn at“
`daily QB injsctims (day 42 to {la}:
`'31, Tubfig. E),
`campietcly blocked the uzcmtrophic action of aestra»
`dial far 3—3-1 Weeks (Taxbfig. 2). Alia: the. efi'em 0f
`ICI 182380 «lanai {day 78 to day 84, Texhfig. 2)
`pragcstamna treatment was again “sad in gamma:
`marine involutian.
`
`:3?“
`aciian
`antiutemtmphic
`susiaincd
`The
`ICI 182380 canfirmed in than above study was investi-
`gaicd further by i‘ne intramuscular administration of
`single doses 01” 1121 182380 fommlated, in sclution, in
`a castm ail-based vehicle. The duration of action 0?
`difi'cring doses 9!“ ICE 182,?8fi was assessed by MRI
`measuwmems of endometrium and myometrium vol»
`mm: immediately balms zreamzem, when ICII 182380
`“:35 administered £112. and daily LI‘GSCIHERI with Ofi
`cammmced, and weekly thereafter until marine
`stimxxiatiafi was appamm. Tmatmcni effects were
`mmparezi with that «sf QB alom: regarded in cash
`animal measured infers: antidestrogen treatmmtl as
`classified abava. The maults in Text-fig. 3 sanfirmeci
`that {Cl £32580 blacks the Imphic action of GB an
`the endametfium and myametrium and that
`the
`duration of actien m’ a single ilm.
`injectian of
`ICE 282.380 was dawrclated. The apmranuc a!” the
`aim-us in MR images recorded helm-e and 2 mi ’2’
`weeks aflez injection of ICI 182380 is illustrawd in
`the Plate (figs 34} and compamd with the efi'em m“
`unagpmcd GB treatment (Plate, fig. 22) Utemtmphic
`respensc was mmpletcly hleckcci initially {ch Plate,
`figs 3 anti 4 with Plan, fig. 2) but sventually resumzd
`(Plate, fig. 5). Estimated by refercncc m the time: from
`the nasal of treatment required is: reach 50% of the
`nnapposed aestmgen control valuing, enclomezrium
`respunse to OB was blacked fur approximately 3. 4
`and 6 wceks, and myomatrinm far 3, >4 and >7 weeks
`am: 2'5, 4 or 5 mg ICE 182,?30jicg reapectively.
`To investigate the efi'ect of repeated twatmcms with
`the longuacting fomuiation at“ ICI 182580, animals
`treated mntinuously with GE were given three im.
`injections of 4 mg ICI 182,?80/kg at 28~day intervals,
`Uterine wlumes were measured on the first day {if
`treatment 14 days later. and at weekly intervals there:
`after {Text-fig 4a}. Endamatrial growth was sup»
`presseti cempleiely far 2 weeks after the first injectian
`but recwered fully m ocszmgen ventral levels by 4
`week‘s. A semml injectian of ICI 182389 led to a
`ragié invalutlan 0f the eminmctrium for 14 days f0!»
`lowed by a slow rtmvery from anfiaestmgen black-
`ade which was inwmglflc, mashing {ml}; 50% 0f
`aestmgen cantrol immefiiately preceding the third
`injectinn of ICE! 132380. After the third injectian,
`ICI 182380 again caused endomctrial involution and
`antiutcmtmphic activity was sustained for bctwecn é
`
`harm? 9f gnéarrftzaéagp { @912} $33 239d”
`
`InnoPharma Exhibit 1036.0003
`
`

`

`242 M, DUKES and others
`
`1C! 182,780 efiizcrs on monkey uterus
`
`Internal 05
`
`
`
`TEXT-nouns l. Prescribed presentation of the uterus in the oblique
`image set. The slice width is automatically adjusted to accommodate
`variations in the length of the uterus.
`
`and 5 weeks. The volume of the myometrium varied
`significantly less than that of the endometrium during
`the course of this experiment (cf. Text-fig. 4a and b)
`and myometrial growth was blocked completely from
`the first dose of [CI 182,730 to the end of the
`experiment.
`The gradual recovery of uterine response to oestro-
`gen after treatment with long-acting oil-depot form u-
`lations of
`[CI l82,780, was characteristic of a
`competitive
`reversible
`antioestrogcnic
`action of
`[CI 182,780. This was investigated further using a
`propylene glycol formulation of [CI I32J80 which
`provides rapid release of ICI 182,780 in viva. Ovari-
`ectomized monkeys were first treated for 14 days with
`DB alone to establish control oestrogen response by
`MRI of the uterus at 0. 7 and 14 days (Text-fig. 5].
`After hormone withdrawal,
`this
`treatment was
`repeated, but with the addition of daily i.m. injections
`of 0-1 or 1-0 mg ICI 182,780/kg for the first '7 days
`of the experimental period. Both doses were equally
`reflective and the data presented in Text-fig. 5 demon-
`strate that blockade of oestrogen stimulation occurred
`only during the 1-day antioestrogen treatment period
`since. during the second week of (unopposed) oestro-
`gen treatment, the growth of both endometrium and
`myornetriurn resumed at a rate similar to that in the
`control experiment.
`
`Journal of Endocrinology {1992) 135. 2397247
`
`Each of the studies described above employed con-
`current treatment with OB and [CI 182.780 to block
`the onset of uterine growth. The capacity of
`1C] 182,780 to produce involution of the uterus in the
`oestrogen-treated monkey noted in the repeat dose
`study with the long-acting formulation was investiga-
`ted furthcr. Using a cross-over design, each of two
`animals was first treated with 03 alone for 7 days
`and then received either continued treatment for a
`
`further 2 weeks with OB together with daily i.m. injec-
`tions of 0-2 mg [C] 182,780/lcg in propylene glycol
`solution or propylene glycol vehicle alone. Three
`weeks later the experiment was repeated but each ani-
`mal was crossedaover to the alternative treatment pro-
`tocol. The rate and extent of involution of both
`
`endometriurn and myometrium during the period
`when animals received ICI 182,780 and OB were the
`same as those following oestrogen withdrawal (Text.
`fig. 6), as was the time of onset and duration of oestro—
`gen-withdrawal bleeding.
`
`DISCUSSION
`
`An initial study (Text-fig. 2) employing a dosing
`regime similar to that described previously for the
`perineal swelling studies (Wakeling e: at. 1991) served
`
`InnoPharma Exhibit 1036.0004
`
`

`

`
`
`M. DUKES and others 243
`1C1 182,780 efl'eclr on monkey uterus
`
`200
`
`[a]
`
`I:®-|—___'_‘lE
`
`ISO
`
`100
`
`8
`
`G
`
`é
`
`
`
` §Tissuevolume("Aofvolumeonday7) E
`
`50
`
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`
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`
`I4
`
`* I-T—r—T—fi—hr-fi—r—i—IF—i
`35
`49
`63
`77
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`
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`
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`
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`
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`
`63
`
`77
`
`133
`
`Days at experiment
`
`Tex-emotion 2. (a) Endometrium and (b) myometrium volumes in
`two ovariectomized monkeys treated once daily with oestradiol
`benzoate (OB) or OB together with [(31 182,780. Percentage values
`Were calculated by reference to the volume of the endometrium and
`myometrium in each animal alter the initial treatment with 5 pg OB / kg
`so. alone for 7 days. Open bars (days 0—H and days 42—83) repre-
`sent once daily s.c. injection of 5 pg OB/kg so. The closed bar indicates
`once daily treatment for 10 days with 1 mg lCI 182.780/kg s-c. in
`arachis oil suspension (days 39-48). lnvolution of the utems was
`induced by withdrawal of 03 and once daily treatment with 5 mg
`progesterone for 5 days (hatched bars, days I448 and days 83—87)-
`Experiment days designated by reference to the first injection of OB (day
`0).
`
`to illustrate the utility of MRI to provide accurate
`sequential data on the changes in the primate uterus
`following hormone treatment. Repeated measure-
`ments of endometrium and myometrlum volume in
`individual animals allowed each animal to act as its
`own control. The extensive series of studies reported
`
`here was completed with only 15 animals using a non-
`invasive technique entirely analogous to those now
`available in the clinic.
`
`ICI 182,780 treatment effects were calculated by
`reference to the volume of endometrium and myomc-
`triurn recorded in control studies where each animal
`
`Journal of Endocrinology ( I992) I35, 239-247
`
`InnoPharma Exhibit 1036.0005
`
`

`

`2%: M. ravines and when
`[CI 182339 efikctx m mankey uterus
`
`
`262313
`
`1 (a)
`
`5N3 mg
`
`5% mg
`
`
`
`l
`
`‘
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`Winks after ICI 182330 traalmml
`
`11123456?
`
`mf-FEfiURE 3* mutation of the amimermmghfic aim: of sing}:
`infiammns (3f ICI $238!} an thc: {:3} endemtfium and {b} myomztrium
`in ovariecmmizeé monkays. Percentage values (me—an 3:31am, :1 w
`34) war: talcuiatcsd by reference to the volume 0? the macaw»
`mam and myometrium in each animal after an initial control mat-
`mcm with 5 pg ()Bjkg 53:. glam: far ’3" days and subscqnant
`withdrawal; as (Merit-ad {as Text-fig. l. A single 11m. injeciian or
`25, «t m 5 mg ICI ISRJSflgkg in cast-or oil solutien was admin}?
`tered, mgflhzr with daiiy QB treatment: which continued anti! sig-
`nificant endumetriai growth was observed. Endommium and
`myametrium Winnie was. meat;qu immediatew befure antioestm-
`gen injectian (weak (I!) and at with}! intervals thereaftcn
`
`was c3»:ch to GB akme. Generaiiy, the ‘contml’
`volume was that receded after 7“ days cf” treatment
`with oestradiol Moms. in the: firs! study (“Text-fig. 2}
`th: start efficstradhl twatme was dslayad anti}
`afier the firs} 3 days 02' {CI 182,?8fl tmatmem so facil§~
`tazc initial himkad: cf 0331703311 recepters by the
`antimtmgen, and continued until substantial uterine
`gmwih was abszzwcd. This study (Taxi—fig. I and the
`
`93am) annfirmed that ICi 182380 is an effective anti~
`utemtmphic agant in the. primate anti than a lung»
`lasting effeci was sustaintd folinwing a singie (kiln
`depot injaaiion.
`Alternative soluble Famulmiom ware: mught m
`facilitate othzr investigatians of ICI £82389 (um
`mported hers), far example, pharmacokinetic and
`taxicalogicai studics, directed {awards its preclinical
`
`fwrwf sf Wrinm‘agy (1992} 335, 23%24?
`
`InnoPharma Exhibit 1036.0006
`
`

`

`M swims and others
`ICI 182330 we“: as: manta}: mng
`
`
`24:
`
`140 m «t
`
`120
`
`ms
`
`80
`
`60
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`8
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`at:£236
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`“5st after fim injeccian a!“ K11 {82,730
`
`mmmcwna 4+ Efi‘est of reputed dam ar ICI 132339 at:
`this (a) endomtrium and {£3} myamcirium a!“ avarificiov
`mm aestmgcmtreatcd monkcys, Pamemage mines (mean
`$ flaw, 32:13) was: calculatcd by «farm tn the vnlume
`cf that endomtfium and mynmctfium in each animal aftcr an
`initial mum} treatment with 5 lg OBfkg it. alone far ‘2
`days- and subsequent Withdrale as described fur Tmtwfig. L
`Singing: im. injections of 4 mg ICE} 182,780,!kg were repeated at
`23—day intervals (arrows) in animals receiving once daily injcc»
`{inns of 5 1.1g GBfkg. Tissua mealtimes were measured imme»
`fiiatcly baffle, and 14 days aft-3n than first ICI 182380 kinetic-n
`and at weekly intervals thereafter.
`
`evaluaiian. The phamacoiogy of Castor oii- and pro-
`pyicne glymI-hascd solarium, pmtmypic 0f hug and
`Ebert-acting formulations
`for
`in}.
`injection,
`is
`reparted here. For the formeu the duratien 0f an:iaes«
`traganic blockade 0f the uterus was evaluated fallow
`ing single Lm. injections at" 2'5, 4 a: 5 mg ICI 182,780,}
`
`kg {Texvfig 3). The results showefi that a time of
`4 mgf‘kg mast CLUBer appmximates that requimd w
`sustain himkade of matrogen actien for I month, a
`closing interval likely it: be clinicain commian in
`thtrapeutic stuciies in breast cancgr patients, This was
`canfinned by giving three sumive dases m" 4mg
`
`JWflfif «cf Wawiswe‘ogy US$32} 135, 23mm
`
`InnoPharma Exhibit 10360007
`
`

`

`246 M. DUKES and others
`
`[CI 182.780 ejects on make): uterus
`
`20"
`
`(a)
`
`r
`
`{m
`
`I:
`
`-
`
`[50
`
`100
`
`8
`
`D
`
`§
`
`50
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`o
`
`(b)
`
`D
`
`7
`
`42
`
`49
`
`56
`
`63
`
`Days
`
`of experiment
`
`
`
`
`
`TissuevolumeIf%ofvolumeonday7}
`
`TEXT-FIGURE 5. Effect of short—term
`treatment with [Cl 182,780 on
`(a) endomctrium and (b) myomctn'um in ovariectunized oestro-
`gen-treated monkeys. Percentage values were calculated by reference
`to the volume of the endometrium and myomctrium in each ani-
`mal after an initial control troatment with 5 pg 0B/kg s.c. alone for
`7 days and subsequent withdrawal, as described for Text—fig. 1.
`Open bars (days 0—14, days 42—63) represent one: daily treatment
`with 5 [lg OB/kg so The closed bar indicates treatment once
`daily with 0-! mg (filled symbols: n=2) or 1 mg (open symbols:
`n=3) [C] [32.780/k3 Lot. in propylene glycol solution for ‘7 days
`(days 42—49). involution of the uterus follom'ng initial oostrogen
`treatment was induced by withdrawal of DB and once daily treat-
`ment with 5 mg progesterone for 5 days (hatched bar. days
`14-18). Experiment days designated by reference to the first injection
`of on (day 0).
`
`1C1182,780/mg at 4-weckly intervals (Text-fig. 4).
`Interestingly, in that study the increasing delay of the
`onset of uterine growth after the second and third
`doses
`indicated a
`cumulative biological
`efi‘ect.
`However, estimates of concentration of drug in the
`
`serum did not indicate that drug accumulation was
`responsible for this increased efficacy (F. ‘Sutclifi‘e,
`unpublished studies). To demonstrate that the sus-
`tained action of ICI 182,780 in oil reflects a slow
`release of active drug. the effect was compared with
`
`Journal of Endocrfnafogy (1992) I355 239447
`
`InnoPharma Exhibit 1036.0008
`
`

`

`M. ouxes and others
`lClr 182,780 efi’ecrr on monkey uterus
`
`24',
`
`'= :1m I:
`
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`(a)
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`so
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`s
`_i
`2
`‘5
`
`g 0
`g
`0"” m
`s
`.i
`
`50
`
`action of oestradiol was confined strictly to the period
`of IC] 182,780 treatment. Endometrial and myo-
`metrial growth resumed immediately after cessation
`of ICI 182,730 treatment. These studies also indicated
`that a daily dose of 0-1 mg ICI l82,780/kg was
`sufficient to block the action of exogenous OB given
`at a dose (511g OB/kg daily) which sustains serum
`oestradiol concentrations of the same order as those
`
`measured in women in the proliferative phase of the
`menstrual cycle.
`The cross—over study illustrated in Text-fig. 6 dem-
`onstrated that [Cl 182,780 not only blocks oestrogen
`action when administered concurrently with 03
`(Text-figs 2-5), but will also cause involution of a
`prestimulated uterus in the continuing presence of
`oestradiol. The similarity of this action. compared
`with that of withdrawing exogenous oestrogen, is con-
`sistent with the View that [Cl 182.780 is a fully effec-
`tive pure antioestrogen in the primate.
`Finally.
`thesc
`studies
`revealed a differential
`response to oestradiol between the myometrium and
`endometrium, where the endometrium appeared more
`sensitive, as reflected by a more rapid recovery from
`antioestrogen blockade; see for example Text figs 3—5.
`An analogous dilferential response is seen in intact
`monkeys where, early in the menstrual cycle when
`oestradiol concentration is low, endomctrial prolifera-
`tion is
`accompanied by myometrial
`involution
`(Waterton e! at. l992).
`
`0
`
`r—Hfi—fi—r—fi—hr—rfi—Wfi
`~23
`-l4
`0
`I4
`28
`42
`.56
`
`ACKNOWLEDGEMENTS
`
`Days of experiment
`
`TEXT-FIGURE 6. Comparison of oestrogen withdrawal and
`[Cl ISZJBO treatment on (a) endometrium and (b)
`myomclrium in two ovariectomized oestrogen—treated
`monkeys. Percentage values were calculated by reference
`.the volume of the endometriurn and myometrium in
`(
`“each animal after an initial control treatment with 5 pg
`OB/kg s.c. alone for 7 days and subsequent withdrawal as
`described for Text-fig. I. Open bars represent once daily
`treatment with 5 pg OB/kg s.c. Solid bars represent once
`daily injections of 0-2 mg [C] 182.730/kg im. in propylene
`glycol solution and dotted bars propylene glycol vehicle
`alone. Thus. each animal was treated first with OB alone
`(days 0—6, and days 42—48). followed for l6 days (7—22 or
`49-64} by either OB together with [Cl 182.780 (squares.
`upper treatment sequence. days) or vehicle alone (circles.
`lower treatment sequence). Three weeks later the experi-
`ment was repeated but each animal crossed-over to the
`alternative treatment protocol. Experiment days desig-
`nated by reference to the first injection of OH (day 0).
`
`that following injection of a propylene glycol solution
`which is known to be cleared rapidly (A. Wakeling &
`M. Dukes. unpublished studies). The results in Text-
`fig. 5 showed clearly that blockade of the uterotrophic
`
`We thank M. Horrocks. J. S. W. Morrell. D. Priest,
`J. Tattersall, J. B. Larcombe-McDouall and S. A.
`Broen for excellent technical support in this work.
`
`REFERENCES
`
`Lee. J. K. T.. Gerscll. D. 1.. Balfe. D. M.. Worthington. .l. L...
`Pious. D. St Gapp. G- (I985). The uterus: in vitro
`Mil-anatomic correlation of normal and abnormal specimens.
`Radiology 151. 175-179.
`Wakeling. A. 5.. Dukes. M. Jr. Bowler. J. (1991). A potent specific
`pure antioestrogen with clinical potential. Cancer Research 5|.
`3867—3873.
`Waterton. J. C.. Miller. 0.. Dukes. M. d: Morrell. J. S. W. (1991).
`Oblique NMR imaging of the uterus in macaques: uterine
`response to estrogen stimulation. Magnetic Resonance in
`Medicine 20. 228—239.
`Waterton. J. (2.. lemombc-McDouall. J. B. at Miller. D. (1992).
`Quantitative MRI of the prostate and uterus in monkeys.
`Magnetic Resonance in Medicine (In Press.)
`
`Journal of Endocrinology (1992) 335. 239- 247
`
`InnoPharma Exhibit 10360009
`
`

`

`
`
` IC'1182,780 effects on monkey uterusFLA-rte ‘ M m.er and others
`
`
`
`
`
`DESCRIPTION OF PLATE
`
`Magnetic resonance images (MRI) of the monkey uterus. Ezteh illugtralion is a detail.
`representing 3-4 x 34 em From one slice of the eight recorded “I catch measurement.
`The appearance at mid-corpus uteri is Shmvn as follows. Ovariecttimized control (Fig. I).
`after 7 days oftreatmcnt with oeslradiol benzoate (DB: 5 pgfltg xx.) ( Fig. 2) and 0 (Fig. 3}.
`l4 (Fig. 4) and 49 (Fig. 5) days ttfler :1 single i.rrt. injeclion of 5 mg lCl lSZJSOfrng. in
`easier oil solution. together with daily oeslradiel treatman Four concentric zones of
`MR signal intensity are seen: high central signal of the cndometrium; medium-low signal.
`myomelrial junction zone: medium-high signal of the myometriem: and no signal.
`peripheral adipose tissue. The high signal intensity seen at the left
`in Figs 3 and S is urine
`in the bladder.
`
`[Huang p 243}
`
`errtml H!-Eflthil‘l‘flfllhlg‘l' (WU?) [35‘ 33‘) 347
`
`InnoPharma Exhibit 1036.0010
`
`