Apptai mm: T{3335FG9-3.3.02
`Qppasizinn againsi E? Patent (0% E36 $113065 $253138)
`P:a!c:ntv:r:: zkstraéieneaa AB
`Gppasiiiurz by. Cicdtsm Richter Ltd.
`{Em Ref; M2363 I:F:C;\PP
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`«~
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`E
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`$0683.53 3. mamsfi
`MENWJWALTE » aecmsamvmxg
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`SSEBERTSTR. 4
`8?: cars MCINCHEN
`
`23
`
`at
`
`Antiuterotrophic effects of a pure antioestrogen,
`ICI 182,780.: magnetic resonance imaging of
`the uterus in ovariectomized monkeys
`
`3/9’
`
`M. Dukes, D. Miller, A. E. Wakeling and J. C. Wataertnn
`Resezarch iltcpartment i, ICI Pharmaceuticals‘ Aidericy Park. Maccicsfiald, Chcshirs ax :o4T<3, UK.
`
`macswm 30 January 1992
`
`ABSTRACT
`
`ICE 132380 is a potent specific pure antiaestmgen
`which may offer advantagns in breast cancer tram»
`- mam wnlparcd with partiai agcmists like tamoxifen.
`( ‘V11 characterize further the pntency and eflicacy of
`{(31 182,788.
`its effsuts on the uterus of i3¥3I'iEC1Q*
`mizexig oestrogen-trcaied monkcys (Macaca nemw
`trims) have been measured using magnetic rcsonazzce
`imaging (MRI). Quantitative MRI ailuws accurate
`nc:m—invasiw: repetitive measurements at’ andcxmcttiai
`and myametrial vnlume fcliowing hurmanal treat-
`ments? using each animal as is awn contra}. Sing}:
`Em. injcctinns of a iong-acting oii-haseci fmmulatian
`of ICI 18.’-£,’?8£I sustained blnckade of aestradial action
`on the monkey uterus in 3 dmsbdapendent manner
`
`«sf ding
`injectians
`343 weeks. Repeated
`far
`prmsided
`intenra}5
`ICI 382,730/kg
`at
`4«wee:kIy
`increasingly effective blockade ref uterine pro§if”era-
`tion. In a shomacting forrnutazion, ICI 182,,‘?8{} afso
`cernpletcty blacked the trophic aciion at‘ acstradiol,
`administered mnzzurrently, in nvarimtomizcci mom
`krays. Simifarlyg ICI “£82380 caused imsofutian ml? the
`uterus stimulated by prim ircatmnnt with ucstradiol.
`The rate and extent {ref marine inwsvlutian in monkeys
`treated with K?! I82,’?8{} was simiiar is 11131 scan fai-
`{sawing aestmgen witiuirawat These studies deman-
`stratc that {Cl ”i32,'?S{}
`is a {uh}: effective pure
`antioestrogen in a primate.
`Journal sf Endozzrfnafagy (I992) 135, 239-247
`
`!NTR(}I}UC'I'I{}N
`
`KC} 382,780 is a rccamiy described poteni pun: anti-
`crestrogcn (Wakeling, Dukes S: fiawlcr, E980 ). Unfikc
`e, now-stetaidal
`antiaestrogens
`such as
`tam-axifexs,
`L 7'1 I32,‘?8(} has been shown to be devoid of pariiai
`agonist {oestmgcnic} activity in rats and mice and
`may, thcref-ore, nfilxr same advantages fur the treat-
`ment of human breast «mace: (Wakefing as‘: all $99!}.
`A long-iasting antioastrogenic effect nf parenteraliy
`administsereti {(31 182,130, fm-rnulated as 3 suspension
`in araazzhis oil, was dcmcanstrated in rats and in nude
`mice bearing xenografts of human bmast cancer ceils
`(Waketing as at 199)). Thugs studies ware extended
`to examine the delay sf o¢stmgen~in:iuced perinea§
`sweliing in avariectamized monkeys by pretreatment
`with 10 daily doses of (iv 1, 0-5 or I mg ICI 282380,?
`kg. or a single dose: of 10mg,/kg, befmc uestragcn
`treatment. A sustained, dowrelatcd biarckade cf peri-
`mai swelling was noted 1’Waiceiing 2: :21. 2991). ?rc}i-
`minary smdiexs using magnetic resmzance imaging
`(MRI) dam-anstratsci the fcasibiiitgy {sf precise meas-
`urcmcnts of the monkey uterus and its respansc to
`
`endocrine manipuiation (Watcrton, Miller, Dukes &
`Marrefi, 1991). MRI is an idaaal method for such stud-—
`ies since it is quantitative and noninvasive (watertvzm
`e: an’. 299%) and the cemsequmccs of interanimak vari-
`abiiizy are minimiseti by using each animai as its awn
`contml {Watertem Larcombe-Mcfiouall :53’: Miller,
`1992). It greatly reduces, therefore, the number of
`animals required to give a uxaaningful result. This
`paper describes a series of studies undertaken to
`txamins more fuily than phamiacaiagy af ICI l32,'?8{1
`in the ovarieactceméw primate (Macaw aentessrina}.
`
`MATER IALS AMI} METHODS
`
`Quantitative magnetic resnnnnm imaging (MRI)
`
`We have rcpurted previously (Watertun 2: :13. 1991,
`1992} that the appearance of the ccsrpus uteri 9f
`Macaw name;-trfiraa in magnetic resonance: {MR}
`images is way similar by that rcpcrted for women,
`although the conmlnted cervix is: the monkey has a
`rather fiiflhmnt MR appearance fram that in women.
`
`Jwmai sf Eindacrérsmfi-xgy {H92} 135, 239m 24‘? Q E992 mama! of Endaszrimzalogy Ltd
`Eifl22~0?9§!92g’313S~{!239 $82.€3IE},.’i3
`
`P.r.c'n-tea‘ in firm: Srfsain
`
`|nnoPharma Exhibit 10360001
`
`

`

`24% res. DUKES and anthers
`
`{CI £82, 31%;? efiecrs an maxtksy zszerus
`
`We have prtwiausly described mcthndaictgy fur qttan=-
`titative studies {Watcrten 2: at’. 1991), and discnsscd
`the reproducibility and statistical pcwer at‘ the tech-
`niqttes (Waterman at :15. I992}; a brief tiescriptitm is
`provided here.
`
`MR tnstrumentatiuu
`
`The study emptoyed a ‘Biaspec 400/13* instrument
`{Cuford Rcstcarch System; Cmtmtry, UK.) inccsrpca
`rating 3 135'!‘ magnet and pulsed fieici graciitsnts up
`ta iii mT,!m (where T is the Si symbai far Tesla,
`the unit of magnetic: flux density). A radiofrequency
`tcsattator of 208 mm amass diarrtetet was ampinyzzd
`as transmitter and recaiver.
`
`Oblique imaging
`
`to aliew cnmparisons between imaget
`in m-tier
`acquired can different accasions, it is most desirable ta
`mtasure ttommc rather than, say meiometrial height
`or area, since the fenncr avaicis artefacts fttmt tapest-
`timing arrears or shape changes. Iii: cndometriai ml«
`aim: in this specks can be quite smati, iaeiow (M »:::n3
`in chronicaiiy ovariectomizcd animals, rising typicaiiy
`to 1 cm3 foliawing oestrogen stimutatitm. Hence it is
`important tn use MRI methods which aiiaw good
`spatial restaiutien tegetiter with adequate gignal-ta«
`noise ratios. Since tit: uterus is samewiiat eilipsoidai
`with (me Icing axis, oblique imaging aiiaws the use of
`the slice-selected two-ciimansionai Ft>urier—transfnrIn
`
`technique, which takes full advantage (if the better
`rcstxlutian in plane than in slice thickness. Also, the
`orientaticn of the uterus within the pelvis varies from
`day tn day depending both on its size in response
`to aestrogen, and also an other facttxrs, prnsumahiy
`including the wzsiume and dispcxsition of the cements
`of the urinary bladder and tower gut. It is advanta-
`gwus for quantitative studies ta empioy abiique. MRI
`techniques which farce the presxtntaticzm cif than utizrus
`for image analysis ta be simiiar {mm examination ta
`examination, as Shawn in tit: Piatai
`in ctrder to achieve this repmducibit: oblique pics»
`cmatiun, this foiiawing procetitmz: was used in: each
`MRI examination. A muttistice sagittal
`imaga set
`was acquired in a-tdizr
`to cietennine the spatiai
`CE‘!-C!fdiI13.§8S of the uterine cervix and fundus. From
`
`these ca-ardinatcs, MR! parameters were calcuiated
`tr.» aitaw the acquisition of sight ccxntiguous cibiiquc
`siiczes, the thickness. pas-itian and or-iszttatiott of which
`ware forced to depend an the vector connecting the
`ftmdus and mzrvix (Text-fig. 1). Thus, the pmsaatation
`of the uterus in the images is more it-I less independent
`Qf its size, Iocatitm and csrientatian in the petvis.
`Image matrices were 256 3': .7556 giving an in-plane
`resoiutinn cf (H5 mm. The signal {mm adipzase tissue
`
`fmasraai of fiidzscrimiegy U992) 13$. 235*---14?
`
`was suppressed, and good contrast between the mar»-
`irze tissues was ensured by using pttise sequence
`paramctcrs. Tu (repetition time} of 3000 ms and Tg
`(ache time} of 50 msi
`
`Qnanttfiaczattam at the imagm
`
`Endametrium and myametrium vcsiumcs warn
`abstained by pixei counting in the oiziique images using
`the irrszguiar region-af»interest faciiities within the
`programs ‘mmmr’ at ‘DISMMR’ (Brake: Anaiytische
`Mcssteacimik Gmbl-L Karisruhe, Germany). Ottty
`siices more fimdal than the interim} cm were inciuded
`
`in the attaiysis. The junction aunt: was itlchldfid with
`the mycemetrium. Any tnantrihutian ti) emiometriai
`voiumc from the lumen was itcgiccttti since this is
`very narrow and insignificant in cmnpariscn with the
`endametrium. Ermrs arising 1‘ram intetobserwar tr:ari—
`abiiity were rninimizazd since each of the seven experi-
`ments was anaiysad by a singie observer {S}? by
`I).M.,
`I each by .i.B.L.Mc1T). and by S.A.B.; see
`Aclcmwiendgements) and £311!‘ aoittziusitms are based
`on pcrcentztgf: citangcs, each animai acting as its awn
`cc-ntroi. The intraoizsemeer variatzitity cannot be
`greater than the week«tc-weak variability, and this is
`5% for the myometrium in mrariactnmized females
`{watcrtan 4:: cu‘. i992). Limitad correiatitms between
`measurements made by MRI and by histology gave
`satisfactury results-.,, contistent with data praviously
`regretted in the human uttarus (Lee, Gerseli, Baife
`at .-:1. 1935).
`
`Animal haunting
`
`Mature femaia pigtaii munkcys {Macaw rtentestriiw)
`of Swtfligg body weight were used. Animals ware
`evariectamizcd at least it mm-itits befare their usa in
`these studies. MRI examinations took placc before
`the animals rcceived their marning feted, in ortier to
`minimize artefactuai
`noistz‘: associated with gut
`motitm. Anaesthesia was
`induced with flfitmg
`kctaminta/kg fiuictalar, Park: Davis. Pontypmli
`Gwcnt, UK.) and maintained with halotitant-.
`(Fluothane,
`ICI Phannaeeuticais, Maocrlesiieid,
`Chesitirei Li.K.).
`
`Pitarmacuiaogicai protocols
`
`To assess the effects of treatment with {Ci I82,?8€i.
`eaah animal was first treamd with 5 its oestradiai
`bcnzoate (OB: Sigma Chemieai Company, P0013,
`B05361, I.LK.),*’it:g in aratthis oi}, s.t‘.. daiiy fat "3 days
`(day t} ten 63:! 6 inciusivc}. unless atiierwisc noted.
`Uterine iinagm wet: reccardcd an day 0 ixnmcdiatciy
`before the first dose cf OB and cm day 7. Animals
`than received 5 mg progwtaranc {Sigma Chemical
`
`|nnoPharma Exhibit 10360002
`
`

`

`16:? }32,?88 sf:-ct: an mmlcey I-stems
`
`~
`
`res.
`
`i:3L”iiES and mhers
`
`243
`
`Company} s.c., daily far 5 er 6 days. After pmgcs—-
`terone withdrawal~indu::¢d menstruation, animals
`again received 08, together with ICI 132380. Details
`cf‘ the treatment schedules employed 3.116 the timing
`cal‘ MRI in each experiment are given in the legends to
`Text-figs 2-—5. Gm: cxpcrimcnt used a :iifl'er::nt (er-ass»
`met} grratocol; see legend to 'I‘c3:t~—fig. 6.
`
`Analysis at‘ the data
`
`Using our MR3 methods to study outbred puopula-«
`ticms of this species, Wt: have shown prcviclusly
`(Watertcm ex :23. 1992) that interanimal variation in
`the volumes at" the uterine tissues greatly exceeds
`wee:k-ta-wszel‘< variatitm in a single animal, mg. by a
`factcv: of 842 in the mycemetrium, making it bcnefi-
`cial to use each animal as its awn control. Hawewmr,
`la-ecause cf’ mctrcme an-domctrial atmphy at flay £3, val»-
`( "Vacs at” this tissue are inaccm-at: and probably nut
`‘meaningful. Hence we txavc elected In normalizc to
`the volumes af endometrial and tnyumetrial tissues
`after ‘? days of treatment with 5 pg 033, taken as 100%.
`Ezwept where otherwise stated, results are presented
`as the arithmetic meanziasgm.
`
`RESULTS
`
`An initial treatment with {)8 alum: established the
`
`extent of uterine response in ovariectomized mcmlceys.
`Typical MR images mmparing the uvaricctontiztd
`anti acstrcsgerx-treated uterus
`illustrate the large
`incrtsase in enclotnetrial voluma after 3’ days of GB
`treatment (cf. Plate, figs l and 2). 'l"ext~figure 2 mm-«
`pares the ¢fl“ccts at‘ uncppascd ucstrogen treatment
`for ? and 14 days on endczmctrlum and myometrium
`volume in two individual animals. In this am} su%d~
`gstudics, treatment efictts an tissuc vnlume in indi-»
`~-udual animals are nermalizatl to those achieved after
`
`(
`
`'3‘ days an!‘ OE treatxnent. The changes in endamctrium
`wlurne in response to an-estrogen iktenfsld} were pre-
`purti-rzmately much greater than than of mynmetrium
`(approx. twafold}. Before a pilot sstutiy in these ani-
`mals, ms address the effect cf ICI lS2,?8{). uterine
`imrolutian was initiated by withdrawing OB and treat«
`man: 5:): 6 days with pmgestemnc {Text-fig. 2). Men»
`strual bleeding {not shown) was seen beginning an
`{lay 2¢l;’25 and continued for 5 es: 6 days. The ancients»
`mum, and particularly the mzmmetrium, had not
`regressed camplstely ta basal ltwels (cf. day 35 and
`day 0, Text-fig, 2}.
`The cffcctt at“ {Cl 132380 «on the traphic: rcspanse at’
`the: uterus to GB was first addressed using ICI 132380
`fm-mulatezi as a suspension in arachis ail. This t‘ar::r1u—
`latlcm has been demornstrated grrevlously to provide a
`sustained antieestmgenic effect an the perincum
`
`(Wakcling at at‘. 1991}. Baily trexatmcnt for £0 days
`with 1 mg K31 l82,'?80fkg s.c':. (clay 39 to day 48, T:xt~
`fig. 2}, beginning 3 days before the rcsumptinn at“
`daily OB injections (day 42 to {la}; ‘F0, Tcxbfig. E},
`campletcly blockesd the utemtrophic: action of €‘:rE5£1‘a~
`die! fur 3-4 weeks (Taxbfig. 2). Altar the effect at’
`IC1182,?8O waned {clay ‘E0 to day 84, Texhfig. 2)
`prtxgcstcrnne treatment was again used to pmmate
`marine involution.
`
`tat‘
`actian
`antiuterotmpliic
`sustainad
`The
`ICI 18238!) mnfirmed in than abmse. study was investi-
`gatcd further by the intramuscular administration of
`single doses osf‘ 11131 182380 fonnulated, in solution, in
`3 castm ail-based vehicle. The duration of action of
`differing doses 91‘ ICE l82,’?S0 was assessed by MRI
`measumnents of enclometrium and myometrium vol»
`umc immediately befare treatrnent, when ICII l82._,?80
`was administered i.m. and daily Lrcatrusant with 03
`cummcncted, and weekly thereafter until marine
`stimulation was appamnt. Trcatxrxcnt effects were
`compareti with that at’ QB alom: ram-rdcd in cash
`animal measured before: antioestrogen traatmantl as
`dascribcd abavc. The results in Text-fig. 3 sunfinned
`that XCSI £32380 blacks the trcxphla action of GB an
`the endametrium and myametrium and that
`the
`duration of action at’ a single hm.
`injectian of
`ICE 282.380 was daswreiated. The appattaranaze :32" the
`uterus in MR images recorded before and 2 amt ’?
`weeks afte: injection of ICI 182380 is ill"-ustratad in
`the Plate (fig 3-§} and compared with the effect at‘
`uflflppflstti GB treatmcnt (Plate, fig. 22) Utemtrophic
`response was caompletely blocked initially (cf. Plate,
`figs 3 amt 4 with Plate, fig. 2) but eventually resumed
`(Plate, fig. 5). Estimated by refercnce to the time from
`the cmset of treatment required ta reach 50% of‘ the
`nnapposed oestrogen control volume, enclometrium
`raspunsa to 0B was blacked far appmximately 3. 4
`and 45 wceks, and myomctrinm far 3, >4 and 22-’? weeks
`after 2-5, at ca: 5 mg ICE I82,*?3i3/'icg respectively.
`Ta investigate the effect of repeated tnaatmcnts with
`the long-acting fonnuiation cat“ ICI 132,780, animals
`treated mntinuously with GE were given three im.
`injections of at mg ICI 182,33!)/kg at 28~day intervals.
`Uterine volumes were measured on the first day of
`treatment, 14 days later, and at weekly intervals thare:
`after {Text-fig‘ 4:2}. Endametrial growth was sup-
`pressant cempletely far 2 weeks after the first injeczticm
`but rccmsered fully tc: uestmgen wntml levels by 4
`weekk. A semnci injection of ICI 182380 led to a
`ragtici inwlutlon cf the emltmxctrium for I4 days lb!»
`lowed by a slow recovery from antiaestmgen black-
`ade which was incemplctc, mashing enlzg 50% af
`eestrogen control immediately preceding the third
`injectiun of ICJI 1-"$2380. After the third injectian,
`ICI 182330 again caused enclomctrial involution and
`antiutcratrophic; activity was sustained for bctween at
`
`Jaurzmf cf Ensierrfxzafiagp { 1992} 335, 239---24‘?
`
`|nnoPharma Exhibit 10360003
`
`

`

`242 M, DUKES and others
`
`ICI £82,780 efiizcts on monkey uterus
`
`Internal 05
`
`
`
`TEXT-FIGURVE 1. 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 ICI 182,780 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 182,780, was characteristic of a
`competitive
`reversible
`antiocstrogenic
`action of
`ICI 182,780. This was investigated further using a
`propylene glycol formulation of ICI I32,'!'80 which
`provides rapid release of ICI 182,780 in viva. Ovari-
`ectomized monkeys were first treated for 14 days with
`OB alone to establish control oestrogen response by
`MRI of the uterus at 0. 7 and I4 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
`eiicctive 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 cndometrium and
`myornetriurn resumed at a rate similar to that in the
`control experiment.
`
`Journal of Endocrinology (1992) I35. 239247
`
`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
`ICI 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 O3 alone for 7 days
`and then rewived either continued treatment for a
`
`further 2 weeks with OB together with daily i.m. injec-
`tions of 0-2 mg ICI 182,780/lcg in propylene glycol
`solution or propylene glycol vehicle alone. Three
`weeks later the experiment was repeated but each ani-
`mal was crossed-over 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 0B 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
`perinea] swelling studies (Walceling el al. 1991) served
`
`|nnoPharma Exhibit 1036.0004
`
`

`

`
`
`M. DUKES and others 243
`(Cl !82,780 efiecis on monkey uterus
`
`| 200
`
`ta)
`
`I50
`
`I00
`
`P‘
`A 50
`35
`-3C
`
`3
`
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`35
`49
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`T2‘
`
`133
`
`Days of experiment
`
`‘1’EXT~FIGUl-IE 2. {a} Endometriurn and (b) myometrium volumes in
`two ovariectomized monkeys treated once daily with oestradiol
`benzoate (OB) or OB together with ICI 182,780. Percentage values
`were calculated by reference to the volume of the endometrium and
`rnyometrium in each animal after the initial treatment with 5 pg OB / kg
`s.c. alone for 1’ days. Open bars (days 0-H and days 42-83) repre-
`sent once daily s.c. injection of 5 pg OB/kg s.c. The closed bar indicates
`once daily treatment for 10 days with 1 mg ICI 182.780/kg s-c. in
`arachis oil suspension (days 39-48). involution of the uterus was
`induced by withdrawal of O3 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 GB (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 myometrium 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 endornetrium and myome-
`triurn recorded in control studies where each animal
`
`Journal’ of Endocrinology (I992) I35, 239-247
`
`|nnoPharma Exhibit 1036.0005
`
`

`

`244 M. EJURES and whats
`
`[CI I82,?3E} efikttx an monkey uterus
`
`
`
`262313
`
`543 mg
`
`5-0 mg
`
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`
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`33
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`
`TEX?-F££}UflE 3. Buration of the antiutermrogahfiaz effact ea‘ singfz
`iniacticens (sf {CI t82,’?S!} an the {:2} enciccrrxctrium and {£2} myormatrium
`in ovariecmmizeé monkeys. Percentage values (mean 3:3. E1.M.,
`:2 m
`341) ware calculatcsd by reference to the volume 0f‘ the zndomzw
`trinm and myometrium in mach anima! after an initial control mat-
`ment with 3 pg Ofifkg 53:. alarm far 1? days and subscqnant
`wixhdrawai, as describcd fur Text-fig. 1. A single i.m. injaxiian of
`2*S. 4% at 5 mg iCI I82.‘380g’3:g in castor oil soluticen was adminiy
`tered, tcxgeihzr with daiiy QB Iraatmam which ezominucd until sig-
`nificant enda-mania! growth was observed. Endamctrium and
`mymnetrium as-oiume was measured immediatcfiy befure antioestrw
`gen injcctian (weak 0) and at wacekiy intervals thereaftcr.
`
`was exposed to GB aécme. Gcneraiiy, the ‘con0trc3l*
`volume was that rescorcied afta: 7‘ days cf" treatment
`with eestradiol Mona. in the: firs! study (Text-fig. 2}
`the start <3f,cmtrad§aI traatment was delayad anti}
`afier the first 3 days 0!’ {CI i32,’?80 treatment so facili-
`tate initial hffickade cf ecstmgan rccepters by the
`anticcstrogen, and continued until substantial uterine
`grawih was absawexi. This study (Taxbfig. I and the
`
`Pkue) cmxfirmed that Itifi 182380 is an effective a:1ti~
`utercz-trophic agcnt in the. primate anti tha: a hung»
`mating effeci was sustained folmwing a singie eil~
`depot injection.
`Alternative soluble Farrnulatiorm ware: mught ms
`facilitate othzr investigations of ICI £82,730 (um
`mported hers), far example, phannacokinetic and
`tcaxicalogicai studics, directed tccwards its preclinical
`
`Jwrcazzf sag!‘ Emiacrimtcfagy ( I992} 135, .7.3§«~2-4?
`
`|nnoPharma Exhibit 1036.0006
`
`

`

`ICI 18‘.?.?&{2 wear: as: encmisey xmznss
`
`M. 1Z>U§i2E$ and others
`
`24:
`
`140 W §
`
`12!}
`
`me
`
`30
`
`60
`
`3
`
`3
`
`£15)
`
`
`
`G
`
`asC!
`
` STmucvatum:1%of-mlum:anday7)
`
`3
`
`8
`
`*3
`
`2
`
`3v-‘n5t'§'?89Ii3IE12l3
`weak:-. after first injcccian G1‘ K11 182,730
`
`‘!"Ex“£“~'Fi£3UlitE 4+ Effect of‘ repeated doses at‘ ICTI 132,‘}'3fl at:
`this (:2) endomaefium and {£3} mynmcirium af avariaciw
`mmc’: aestmgcn-treated monkcys, Percentage vaiues (mean
`3.: S.Ei.M.,. a=e3) mt: caiculaicd by rcfcrrcncz: tn the wlume
`at’ the «endomctrium and mynmctrium in each animal aftcr an
`initial mantra} treatment with 5 ;:.g Ofifkg 5.1:. alone fur ‘F
`days. and subsequent withdrawal: as described for T::;r.t~fig. 1.,
`Singing im. injections of 4 mg EC? I82,?8{l,/kg were repeated at
`23-day intervals (arrows) in animals receiving once daily inject»
`{inns of 5 gag (}B,.’kg. Tissue: miumes were measured imme-
`ciiatcly befbrc, and 14 days aftcr, tit: firs: ICI 182380 injratztim:
`and at weekly intervals thereafter.
`
`mralnaiian. The phamzacaiogy of caster oii- and pro-
`pyicnc glyml-hasad scluticns, prmatypic sf hang» and
`shat’:-acting fannulations
`for man.
`injection,
`is
`reported here. For the former, the duration of am‘.ic~es—
`traganic blockade of the uterus was evaiuated fallow-
`ing singie i.m. injections cf 26, 4 at 5 mg ICI i82,7Sfi,;‘
`
`kg {’f‘ext«fig, 3). The results showeri that a chase of
`4 mgfkg mast cinseiy agipmximales that requirsd £0
`sustain hfcrckade of oestrogen action for 1 month, a
`chasing interval likely to» be clinicaily convenient in
`thnarapeutic studies in breast canccr patients. This was
`ccsnfirmeti by giving three successive dases -:11" Mug
`
`Jflffflflf of fitsfacrinaafogy {W12} 135, 239424?
`
`|nnoPharma Exhibit 1036.000?
`
`

`

`246 M. DUKES and others
`
`[CI 182,780 ejects on morrkey uterus
`
`3°°
`
`(at
`
`p F
`
`l
`
`j
`
`[50
`
`I00
`
`8
`
`TissuevolumeIf%ofvolumeonday7} §D
`
`
`
`
`
`E
`
`50
`
`0
`
`D
`
`7
`
`42
`
`49
`
`56
`
`63
`
`Days
`
`of experiment
`
`TEXT-FIGURE 5. Effect of sl1ort—term
`treatment with [Cl 182,780 on
`(a) endometrium and (b) myometfiurn in ovariectomized oestro-
`gen-treated monkeys. Percentage values were calculated by reference
`to the volume of the endometrium and myometrium in each ani-
`mal after an initial control treatment with 5 pg OB/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 ug OB/kg so The closed bar indicates treatment once
`daily with 0-! mg (filled symbols: n=2) or I mg (open symbols:
`n=3) [C] I32.780/kg Lot. in propylene glycol solution for 7 days
`(days 42-49). involution of the uterus following initial oestrogen
`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).
`
`ICI 182,780/mg at 4-weekly 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
`efiect.
`However, estimates of concentration of drug in the
`
`serum did not indicate that drug accumulation was
`responsible for this increased eflicacy (F. Sutclitfe,
`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) [35, 239447
`
`|nnoPharma Exhibit 1036.0008
`
`

`

`M. ounces and 0th:
`ICI l82,780 eject: on monkey uterus
`
`24'.
`
`I00
`
`(gr)
`
`8
`
`ca
`
`§
`
`
`
`Tissuevtjjaef%ol‘volumeonday7)
`
`lb)
`
`
`
`action of oestradiol was confined strictly to the period
`of lCl 182,780 treatment. Endometrial and myo-
`rnetrial 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
`suflicient to block the action of exogenous OB given
`at a dose (5-pg 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 lB2,780 not only blocks oestrogen
`action when administered concurrently with 03
`(Text-figs 2-5), but will also cause involution of a
`prestirnulated 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,
`these
`studies
`revealed a differential
`response to oestradiol between the myometrium and
`endornetrium, 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, cndornetrial prolifera-
`tion is
`accompanied by myometrial
`involution
`(Waterton er al. i992).
`
`0
`*23
`-I4
`0
`I4
`28
`42
`56
`
`ACKNOWLEDGEMENTS
`
`Days of experiment
`
`TEXT’-FIGURE 6. Comparison of oestrogen withdrawal and
`lCl l82.780 treatment on (it) endometrium and (I1)
`rnyometriurn in two ovariectomized oestrogen-treated
`monkeys. Percentage values were calculated by reference
`.the volume of the endometrium and rnyornetrium 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 lCl 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 03 together with [Cl l8Il,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 OB [day 0).
`
`that following injection of a propylene glycol solution
`which is known to be cleared rapidly (A. Walceling &
`M. Dukes. unpublished studies). The results in Text-
`fig. 5 showed clearly that blockade of the uterotrophic
`
`We thank M. Horroclcs, J. S. W. Morrell, D. Priest,
`J. Tattersall, J. B. Larcombe-McDouall and S. A.
`Breen for excellent technical support in this work.
`
`REFERENCES
`
`Lee, J. K. T.. Gerscll. D. .l.. Balfe. D. M.. Worthington. J. I...
`Pious. D. & Gapp. (3. (I985). The uterus: in vitro
`MR-anatomic correlation of normal and abnormal specimens.
`Radr'ol'ogy I57, I75-179.
`Walreling. A. E.. Dukes. M. Jr. Bowler. J. (1991). A potent specific
`pure antioestrogen with clinical potential. Cancer Research 5|.
`3867-3873.
`Wsterton, J. C.. Miller. [1, Dukes, M. & 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.
`Walertqn. J. C.. Larcornbe-McDouall. J. B. & Miller. D. (1992).
`Quantitative MRI of the prostate and uterus in monkeys.
`Magnetic Resonance in Medicine (In Press.)
`
`Journal of Endocrinology (1992) £35. 239- 247
`
`|nnoPharma Exhibit 1036.0009
`
`

`

`I-L/vrta
`
`ICI 182,78!) cjfferm an riinnkuy uterus
`
`- M nLn<r;~: and others
`
`DESCRIPTION OF PLATE
`
`Magnetic resonance images (MRI) of the monkey uterus. Each illustration is at detail.
`representing 3-4 >< 34 cm from one slice of the eight recorded at each measurement.
`The appearance at mid-mrptis uteri is Shawn as follows. Ovetrieetninized control (Fig. I ).
`after 7 days oftreatmcnt with oestradio1benzoate(OB: 5 pgfkg s.c.) ( Fig. 2):.1nd 0 (Fig. 3}.
`M (Fig. 4) and 49 (Fig. 5) days after :1 single i.m. injection of 5 mg lCl l82.780,¢‘ntg, in
`castor oil solution. together with daily oeslradiol lfC&IIl‘I1Cnl. 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 myometrit.-m: and no signal.
`peripheral adipose tissue. The high signal intensity seen at the left
`in Figs 3 and S is urine
`in the bladder.
`
`i+;,._»{1-.g P 343}
`
`Jnnriml u!"I-}:m'm‘1mn!ug‘i' (WU?) [35, 3.10 347
`
`|nnoPharma Exhibit 1036.0010
`
`