, .
`
`Appeal No.: T0336/09-3.3.02
`Opposition against EP Patent (OJ 90 0186.6 / 1250138)
`Patentee: AstraZeneca AB
`Opposition by: Gedeon Richter Ltd.
`Our Ref.: M2363 EP/OPP
`
`-
`voes.us ·& PARTNER
`W\TENTANWALTE • RECHTSANwALTE
`. . SIEBERTSTR. 4
`.
`81875 .MONCHEN
`Antiuterotrophic effects of a pure antioestrogen,
`ICI 182,780: magnetic resonance imaging of
`the uterus in ovariectomized monkeys
`
`23
`
`M. Dukes, D. Miller, A. E. Wakeling and J.C. Waterton
`Research Department 1, ICI Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SKI04TO, U.K.
`
`RECEIVED 30 January 1992
`
`ABSTRACT
`
`ICI 182,780 is a potent specific pure antioestrogen
`which may offer advantages in breast cancer treat-
`. ment compared with partial agonists like tamo:xifen.
`\ characterize further the potency and efficacy of
`(
`1CI 182,780, its effects on the uterus of ovariecto(cid:173)
`mized, oestrogen-treated monkeys (Macaca nemes-
`trina) have been measured using magnetic resonance
`imaging (MRI). Quantitative MRI allows accurate
`non-invasive repetitive measurements of endometrial
`and myometrial volume fo])owing hormonal treat(cid:173)
`ments, using each animal as its own control. Single
`i.m. injections of a long-acting oil-based formulation
`ofICI 182,780 sustained blockade of oestradiol action
`on the monkey uterus in a dose-dependent manner
`
`injections of 4 mg
`for 3- 6 weeks. Repeated
`intervals provided
`ICI 182,780/ kg at 4-weekly
`increasingly effective blockade of uterine prolif era(cid:173)
`tion. In a short-acting formulation, ICI 182, 780 also
`completely blocked the trophic action of oestradiol,
`administered concurrently, in ovariectomized mon(cid:173)
`keys. Similarly, ICI 182,780 caused involution of the
`uterus stimulated by prior treatment with oestradiol.
`The rate and extent of uterine involution in monkeys
`treated with ICI 182,780 was similar to that seen fol(cid:173)
`lowing oestrogen withdrawal. These studies demon(cid:173)
`is a fully effective pure
`strate that ICI 182,780
`antioestrogen in a primate.
`Journal of Endocrinology (1992) 135, 239- 247
`
`INTRODUCTION
`
`(
`
`ICI I 82, 780 is a recently described potent pure anti(cid:173)
`oestrogen (Wakeling, Dukes & Bowler, 1991). Unlike
`non-steroidal antioestrogens such as
`tarnoxifeo,
`~I 182,780 has been shown to be devoid of partial
`agonist (oestrogenic) activity in rats and mice and
`may, therefore, offer some advantages for the treat(cid:173)
`ment of human breast cancer (Wakeling et al. 1991).
`A Jong-lasting antioestrogenic effect of parenterally
`administered ICI 182,780, formulated as a suspension
`in arachis oil, was demonstrated in rats and in nude
`mice bearing xenografts of human breast cancer cells
`(Wakeling et al. 1991 ). Those studies were extended
`to examine the delay of oestrogen-induced perinea)
`swelling in ovariectomized monkeys by pretreatment
`with 10 daily doses of O· I, O· 5 or I mg ICI 182, 780/
`kg, or a single dose of 10 mg/kg, before oestrogen
`treatment. A sustained, dose-related blockade of peri(cid:173)
`neal swelling was noted (Wakeling et al. 1991). Preli(cid:173)
`minary studies using magnetic resonance imaging
`(MRI) demonstrated the feasibility of precise meas(cid:173)
`urements of the monkey uterus and its response to
`
`endocrine manipulation (Waterton, Miller, Dukes &
`Morrell, 1991 ). MRI is an ideal method for such stud(cid:173)
`ies since it is quantitative and non-invasive (Waterton
`et al. 199 I) and the consequences of interanimal vari(cid:173)
`ability are minimized by using each animal as its own
`control (Waterton, Larcombe-McDouall & Miller,
`1992). It greatly reduces, therefore, the number of
`animals requifed to give a meaningful result. Th.is
`paper describes a series of studies undertaken to
`examine more fully the pharmacology of ICI 182, 780
`in the ovariectomized primate (Macaca nemestrina).
`
`MATERIALS AND METHODS
`
`Quantitative magnetic resonance imaging (MRI)
`We have reported previously (Waterton et al. 1991,
`1992) that the appearance of the corpus uteri of
`Macaca nemestrina in magnetic resonance (MR)
`images is very similar to that reported for women,
`although the convoluted cervix in the monkey has a.
`rather different MR appearance from that in women.
`
`Journal of Endocrinology (1992) 135, 239- 247
`0022-0795/ 92/0135-0239 $02.00/0
`
`(Cl 1992 Journal of Endocrinology Ltd Printed in Great Britain
`
`MYLAN PHARMS. INC. EXHIBIT 1025 PAGE 1
`
`

`
`240 M. DUK.ES and others
`
`·
`
`/Cl 182,780 effects on monkey uterus
`
`We have previously described methodology for quan(cid:173)
`titative studies (Waterton et al. 1991 ), and discussed
`the reproducibility and statistical power of the tech(cid:173)
`niques (Waterton et al. 1992); a brief description is
`provided here.
`
`was suppressed, and good contrast between the uter(cid:173)
`ine tissues was ensured by using pulse sequence
`parameters TR (repetition time) of 3000 ms and Te
`(echo time) of 50 ms.
`
`MR instrumentation
`The study employed a ' Biospec 400/2.3' instrument
`(Oxford Research Systems, Coventry, U.K.) incorpo(cid:173)
`rating a 2.35 T magnet and pulsed field gradients up
`to 10 mT/ m (where T is the SI symbol for Tesla,
`the unit of magnetic flux density). A radiofrequency
`resonator of 208 mm access diameter was employed
`as transmitter and receiver.
`
`ObUque imaging
`In order to allow comparisons between images
`acquired on different occasions, it is most desirable to
`measure volume rather than, say endometrial height
`or area, since the former avoids artefacts from reposi(cid:173)
`tioning errors or shape changes. The endometrial vol(cid:173)
`ume in this species can be quite small, below O· l cm 3
`in chronically ovariectomized animals, rising typically
`to 1 cm3 following oestrogen stimulation. Hence it is
`important to use MRI methods which allow good
`spatial resolution together with adequate signal-to(cid:173)
`noise ratios. Since the uterus is somewhat ellipsoidal
`with one long axis, oblique imaging allows the use of
`the slice-selected two-dimensional Fourier-transform
`technique, which takes full advantage of the better
`resolution in plane than in slice thickness. Also, the
`orientation of the uterus within the pelvis varies from
`day to day depending both on its size in response
`to oestrogen, and aJso on other factors, presumably
`incJuding the volume and disposition of the contents
`of the urinary bladder and lower gut. It is advanta(cid:173)
`geous for quantitative studies to employ oblique MRI
`techniques which force the presentation of the uterus
`for image analysis to be similar from examination to
`examination, as shown in the Plate.
`In order to achieve this reproducible oblique pres(cid:173)
`entation, the following procedure was used for each
`MRI examination. A multislice sagittal image set
`was acquired in order to determine the spatial
`co-ordinates of the uterine cervix and fundus. From
`these co-ordinates, MRI parameters were calculated
`to allow the acquisition of eight contiguous oblique
`slices, the thickness, position and orientation of which
`were forced to depend on the vector connecting the
`fundus and cervix (Text-fig. 1 ). Thus, the presentation
`of the uterus in the images is more or less independent
`of its size, location and orientation in the pelvis.
`Image matrices were 256 x 256 giving an in-plane
`resolution of O· 6 mm. The signal from adipose tissue
`
`Journal of Endocrinology (1992) 135, 239- 247
`
`Quantification of the images
`Endometrium and myometrium volumes were
`obtained by pixel counting in the oblique images using
`the irregular region-of-interest facilities within the
`programs 'uxnmr' or 'DISNMR' (Bruker Analytische
`Messtechnik GmbH, Karlsruhe, Germany). Only
`slices more fundal than the internal os were included
`in the analysis. The junction zone was included with
`the myometrium. Any contribution to endometrial
`volume from the lumen was neglected since this is
`very narrow and insignificant in comparison with the
`endometrium. Errors arising from interobserver vari(cid:173)
`ability were minimized since each of the seven experi(cid:173)
`ments was analysed by a single observer ( 5 / 7 by
`D.M., 1 each by J.B.L.McD. and by S.A.B.; see
`Acknowledgements) and our conclusions are based
`on percentage changes, each animal acting as its own
`control. The intraobserver variability cannot be
`greater than the week-to-week variability, and this is
`5% for the myometrium in ovariectomized females
`(Waterton et al. 1992). Limited correlations between
`measurements made by MRI and by histology gave
`satisfactory results, consistent with data previously
`reported in the human uterus (Lee, Gersell, Balfe
`et al. 1985).
`
`Animal handling
`Mature female pigtail monkeys (Macaca nemestrina)
`of 5- 10 kg body weight were used. Animals were
`ovariectomized at least 11 months before their use in
`these studies. MRI examinations took place before
`the animals received their morning feed, in order to
`minimize artefactual noise associated with gut
`induced with O· 7 mg
`motion. Anaesthesia was
`ketamine/ kg (Vetalar, Parke Davis, Poiltypool,
`Gwent, U.K.) and maintained with halothane
`(Fluothane,
`ICI Pharmaceuticals, Macclesfield,
`Cheshire, U .K .).
`
`Pharmacological protocols
`To assess the effects of treatment with ICI 182,780,
`each animaJ was first treated with 5 µg oestradiol
`benzoate (OB: Sigma Chemical Company, Poole,
`Dorset, U.K.)/ kg in arachis oil, s.c. daily for 7 days
`(day 0 to day 6 incJusive), unless otherwise noted.
`Uterine images were recorded on day 0 immediately
`before the first dose of OB and on day 7. Animals
`then received 5 mg progesterone (Sigma Chemical
`
`MYLAN PHARMS. INC. EXHIBIT 1025 PAGE 2
`
`

`
`/CI 181,780 effects on monkey uterus
`
`· M . DUKES and others 241
`
`Company) s.c., daily for 5 or 6 days. After proges(cid:173)
`terone withdrawal-induced menstruation, animals
`again received OB, together with ICI 182,780. Details
`of the treatment schedules employed and the timing
`of MRI in each experiment are given in the legends to
`Text-figs 2- 5. One experiment used a different (cross(cid:173)
`over) protocol; see legend to Text-fig. 6.
`
`Analysis of the data
`Using our MRI methods to study outbred popula(cid:173)
`tions of this species, we have shown previously
`(Waterton et al. 1992) that interanimal variation in
`the volumes of the uterine tissues greatly exceeds
`week-to-week variation in a single animal, e.g. by a
`factor of 8- 12 in the myometrium, making it benefi(cid:173)
`cial to use each arumal as its own control. However,
`because of extreme endometrial a trophy at day 0, vol-
`("iles of this tissue a.re inaccurate and probably not
`u1eaningful. Hence we have elected to normalize to
`the volumes of endometrial and myometrial tissues
`after 7 days of treatment with 5 µg OB, taken as 100%.
`Except where otherwise stated, results are presented
`as the arithmetic mean ± s .E.M .
`
`RESULTS
`
`An initial treatment with OB alone established the
`extent of uterine response in ovariectomized monkeys.
`Typical MR images comparing the ovariectomized
`and oestrogen-treated uterus illustrate the large
`increase in endometriaJ volume after 7 days of OB
`treatment (cf. Plate, figs J and 2). Text-figure 2 com(cid:173)
`pares the effects of unopposed oestrogen treatment
`for 7 and 14 days on endometrium and myometrium
`volume in two individual animals. In this and succeed-
`i studies, treatment effects on tissue volume in indi(cid:173)
`(
`'-v ,dual animals are normalized to those achieved after
`7 days of OB treatment. The changes in endometrium
`volume in response to oestrogen (> tenfold) were pro(cid:173)
`portionately much greater than those of myometrium
`(approx. twofold). Before a pilot study in these ani(cid:173)
`mals, to address the effect of ICI 182,780, uterine
`involution was initiated by withdrawing OB and treat(cid:173)
`ment for 6 days with progesterone (Text-fig. 2). Men(cid:173)
`strual bleeding (not shown) was seen beginning on
`day 24/ 25 and continued for 5 or 6 days. The endome(cid:173)
`trium, and particularly the myometrium, had not
`regressed completely to basal levels (cf. day 35 and
`day 0, Text-fig. 2).
`The effect of ICI 182,780 on the trophic response of
`the uterus to OB was first addressed using ICI 182,780
`formulated as a suspension in arachis oil. This formu(cid:173)
`lation has been demonstrated previously to provide a
`sustained antioestrogenic effect on the perineum
`
`(Wakeling et al. 1991). Daily treatment for 10 days
`with 1 mg ICI 182,780/ kg s.c. (day 39 to day 48, Text(cid:173)
`fig. 2), beginning 3 days before the resumption of
`daily OB injections (day 42 to day 70, Text-fig. 2),
`completely blocked the uterotrophic action of oestra(cid:173)
`diol for 3-4 weeks (Text-fig. 2). After the effect of
`ICI 182,780 waned (day 70 to day 84, Text-fig. 2)
`progesterone treatment was again used to promote
`uterine involution.
`action
`antiuterotrophic
`The.
`sustained
`of
`ICI 182, 780 confirmed in the above study was investi(cid:173)
`gated further by the intramuscular administration of
`single doses of ICI 182,780 formulated, in solution, in
`a castor oil-based vehicle. The duration of action of
`differing doses of ICI 182, 780 was assessed by MRI
`measurements of endometrium and myometrium vol(cid:173)
`ume immediately before treatment, when ICI 182,780
`was administered i.m. and daily treatment with OB
`commenced, and weekly
`thereafter until uterine
`stimulation was apparent. Treatment effects were
`compared with that of OB alone recorded in each
`animal measured before antioestrogen treatment, as
`described above. The results in Text-fig. 3 confirmed
`that ICI 182,780 blocks the trophic action of OB on
`the endometrium and myometrium and that the
`duration of action of a single i.m. injection of
`ICI 182, 780 was dose-related. The appearance of the
`uterus in MR images recorded before and 2 and 7
`weeks after injection of ICI 182,780 is illustrated in
`the Plate (figs 3-5) and compared with the effect of
`unopposed OB treatment (Plate, fig. 2). Uterotrophic
`response was completely blocked initially (cf. Plate,
`figs 3 and 4 with Plate, fig. 2) but eventually resumed
`(Plate, fig. 5). Estimated by reference to the time from
`the onset of treatment required to reach 50% of the
`unopposed oestrogen control volume, endometrium
`response to OB was blocked for approximately 3, 4
`and 6 weeks, and myometrium for 3, >4 and > 7 weeks
`after 2·5, 4 or 5 mg ICI 182,780/ kg respectively.
`To investigate the effect of repeated treatments with
`the long-acting formulation of ICI 182,780, animals
`treated continuously with OB were given three i.m.
`injections of 4 mg ICI 182,780/ kg at 28-day intervals.
`Uterine volumes were measured on the first day of
`treatment, 14 days later, and at weekly intervals there(cid:173)
`after (Text-fig. 4a). Endometrial growth was sup(cid:173)
`pressed completely for 2 weeks after the first injection
`but recovered fully to oestrogen control levels by 4
`weeks. A second injection of ICI 182, 780 led to a
`rapid involution of the endometrium for 14 days fol(cid:173)
`lowed by a slow recovery from antioestrogen block(cid:173)
`ade which was incomplete, reaching only 50% of
`oestrogen control immediately preceding the third
`injection of ICI 182,780. After the third injection,
`ICI 182, 780 again caused endometrial involution and
`antiuterotrophic activity was sustained for between 4
`
`Journal of Endocrinology ( 1992) 135, 239- 247
`
`MYLAN PHARMS. INC. EXHIBIT 1025 PAGE 3
`
`

`
`242 M . DUKES and others
`
`·
`
`IC/ 182,780 effects on monkey uterus
`
`8
`
`•
`
`Fund us
`
`0
`
`6
`
`0
`
`0
`
`Internal os
`
`0
`
`Cervix
`
`3
`
`2
`
`TEXT-FJOURE I. Prescribed presentation of the uterus in the obliqu~
`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(cid:173)
`gen after treatment with long-acting oil-depot formu(cid:173)
`lations of ICI 182,780, was characteristic of a
`competitive reversible antioestrogenic action of
`ICI 182,780. This was investigated further using a
`propylene glycol formulation of ICI 182,780 which
`provides rapid release of ICI 182,780 in vivo. Ovari(cid:173)
`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 14 days (Text-fig. 5).
`After hormone withdrawal,
`this
`treatment was
`repeated, but with the addition of daily i.m. injections
`of O· 1 or l · 0 mg I CI 182, 780 / kg for the first 7 days
`of the experimental period. Both doses were equally
`effective and the data presented in Text-fig. 5 demon(cid:173)
`strate that blockade of oestrogen stimulation occurred
`only during the 7-day antioestrogen treatment period
`since, during the second week of (unopposed) oestro(cid:173)
`gen treatment, the growth of both endometrium and
`myometrium resumed at a rate similar to that in the
`control experiment.
`
`Journal of Endocrinology (1992) 135, 239- 247
`
`Each of the studies described above employed con(cid:173)
`current treatment with OB and ICI 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(cid:173)
`ted further. Using a cross-over design, each of two
`animals was first treated with OB alone for 7 days
`and then received either continued treatment for a
`further 2 weeks with OB together with daily i.m. injec(cid:173)
`tions of 0·2 mg ICI 182,780/ kg in propylene glycol
`solution or propylene glycol vehicle alone. Three
`weeks later the experiment was repeated but each ani(cid:173)
`mal was crossed-over to the alternative treatment pro(cid:173)
`tocol. The rate and extent of involution of both
`endometrium and myometrium during the period
`when animals received ICI 182,780 and OB were the
`same as those following oestrogen withdrawal (Text(cid:173)
`fig. 6), as was the time of onset and duration of oestro(cid:173)
`gen-withdrawal bleeding.
`
`DISCUSSION
`
`An initial study (Text-fig. 2) employing a dosing
`regime similar to that described previously for the
`perinea} swelling studies (Wakeling et al. 1991) served
`
`MYLAN PHARMS. INC. EXHIBIT 1025 PAGE 4
`
`

`
`IC/ 182,780 effects on monkey uterus
`
`· M . DUKES and others 24;
`
`200
`
`(a)
`
`150
`
`100
`
`50
`
`......
`,_
`>. "' ~
`
`c
`0
`u
`E
`:l
`~ 200
`....
`
`0
`
`- 14
`
`0
`
`14
`
`35
`
`49
`
`63
`
`77
`
`133
`
`150
`
`0
`~
`~
`0
`E
`:l
`0
`>
`u
`:l
`~
`f:: 100
`
`c-·
`
`(
`
`50
`
`0
`
`~.----~~.~------11'1:~--.---~~,~----~~-.~-'fJ----,
`- 14
`0
`14
`35
`49
`63
`77
`133
`Days or experiment
`
`TEXT-FIGURE 2. (a) Endometrium 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 cndometrium and
`myometrium in each animal after the initial treatment with 5 µg OB/kg
`s.c. alone for 7 days. Open bars (days 0-14 and days 42-83) repre(cid:173)
`sent once daily s.c. injection of 5 µg OB/kg s.c. The closed bar indicates
`once daily treatment for JO days with I mg ICI 182,780/ kg s.c. in
`aracbis oil suspension (days 39-48). Involution of the uterus was
`induced by withdrawal of OB and once daily treatment with 5 mg
`progesterone for 5 days (batched bars, days 14- 18 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(cid:173)
`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(cid:173)
`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·
`trium recorded in control studies where each animal
`
`Journal of Endocrinology (1992) 135, 239- 247
`
`MYLAN PHARMS. INC. EXHIBIT 1025 PAGE 5
`
`

`
`244 M. DUKES and others
`
`·
`
`/Cl 182,780 effects on monkey uterus
`
`2·5mg
`
`4·0mg
`
`S·Omg
`
`100
`
`(a)
`
`80
`
`60
`
`40
`
`-(cid:173)r--
`
`>(cid:173)OS
`~ 20
`0
`u e ::>
`~ o_.__._.......__ ................... _._~~ ........ _.__._ ........ ..__--. ....... _._ ......................................... ___.
`... 0 100
`u e ::>
`~ 80
`~
`
`~
`
`(b)
`
`"' .: 60
`
`(
`
`40
`
`20
`
`o..__._...._ ....... _._...._ ....... ~ ............................. .___.__._.......__ ................. ....._ ................ __.
`0 I 2 3 4 5 6 7
`0 I 2 3 4
`0 I 2 3 4
`Weeks after ICI 182.780 treatment
`
`TEXT-FIGURE 3. Duration of the antiutcrotrophic effect of single
`injections of ICI 182,780 on the (a) endometrium and (b) myometrium
`in ovariectomizcd monkeys. Percentage values (mean± s .E.M., n =
`3-4) were calculated by reference to the volume of the endome(cid:173)
`trium and myometrium in each animal after an initial control treat(cid:173)
`ment with 5 µg OB/ kg s.c. alone for 7 days and subsequent
`withdrawal, as described for Text-fig. I . A single i.m . injection of
`2·5, 4 or 5 mg ICI 182,780/kg in castor oil solution was adminis(cid:173)
`tered, together with daily OB treatment which continued until sig(cid:173)
`nificant cndometrial growth was observed. Endometrium and
`myometrium volume was measured immediately before antioestro(cid:173)
`gen injC(:tion (week 0) and at weekly intervals thereafter.
`
`was exposed to OB alone. Genera1ly, the 'control'
`volume was that recorded after 7 days of treatment
`with oestradiol alone. In the first study (Text-fig. 2)
`the start of. oestradiol treatment was delayed until
`after the first 3 days of ICI 182, 780 treatment to facili(cid:173)
`tate initial blockade of oestrogen receptors by the
`antioestrogen, and continued until substantial uterine
`growth was observed. This study (Text-fig. 1 and the
`
`Plate) confirmed that ICI 182,780 is an effective anti(cid:173)
`uterotrophic agent in the primate and that a long(cid:173)
`lasting effect was sustained following a single oil(cid:173)
`depot injection.
`Alternative soluble formulations were sought to
`facilitate other investigations of ICI 182,780 (not
`reported here), for example, phannacokinetic and
`toxicological studies, directed towards its preclinical
`
`Journal of Endocrinology ( 1992) 135, 239- 247
`
`MYLAN PHARMS. INC. EXHIBIT 1025 PAGE 6
`
`

`
`ICI 182,780 effects on monkey uterus
`
`· M . DUKES and others 24!
`
`140
`
`J.
`(a) f
`
`120
`
`100
`
`80
`
`60
`
`40
`
`~
`
`;::-
`>.
`"'
`'O
`c
`0 e 20
`::I
`0
`.... 0
`>
`0
`~ ., 80
`E
`::I
`0
`> .,
`::I
`::'.!
`i=
`
`(b)
`
`60
`
`40
`
`20
`
`0
`
`II 12 13
`7 8 9 10
`3 4 5 6
`0 2
`Weeks after first injection of ICI 182,780
`
`TEXT-FIGURE 4. Effect of repeated doses of JCJ 182,780 on
`the (a) endometrium and (b) myometrium of ovariecto(cid:173)
`mized oestrogen-treated monkeys. Percentage values (mean
`± s.E.M., n = 6) were calculated by reference to the volume
`of the endomctrium and myometrium in each animal after an
`initial control treatment with S µg OB/kg s.c. alone for 7
`days and subsequent withdrawal, as described for Text-fig. I.
`Single i.m. injections of 4 mg ICI 182,780/ kg were repeated at
`28-day intervals (arrows) in animals receiving once daily injec(cid:173)
`tions of 5 µg OB/ kg. Tissue volumes were measured imme(cid:173)
`diately before, and 14 days after, the first ICI 182,780 injection
`and at weekly intervals thereafter.
`
`evaluation. The pharmacology of castor oil- and pro(cid:173)
`pylene glycol-based solutions, prototypic of long- and
`short-acting formulations
`for
`i.m.
`injection,
`is
`reported here. For the former, the duration of antioes(cid:173)
`trogenic blockade of the uterus was evaluated follow(cid:173)
`ing single i.m. injections of2·S, 4 or 5 mg ICI 182,780/
`
`kg (Text-fig. 3). The results showed that a dose of
`4 mg/ kg most closely approximates that required to
`sustain blockade of oestrogen action for I month, a
`dosing interval likely to be clinically convenient in
`therapeutic studies in breast cancer patients. This was
`confirmed by giving three successive doses of 4 mg
`
`Journal of Endocrinology (1992) 135, 239-247
`
`MYLAN PHARMS. INC. EXHIBIT 1025 PAGE 7
`
`

`
`246 M . DUKES and others
`
`·
`
`IC/ 182,780 effects on monkey uterus
`
`-
`
`200
`
`(a)
`
`15-0
`
`100
`
`SQ
`
`(h)
`
`.....
`>.
`"'
`"O
`~
`0
`u
`E
`:J
`0 >
`....
`c
`0
`u e 100
`0 >
`u
`:I .,, .,,
`i=
`
`0
`
`. :J
`
`50
`
`0
`
`/
`-,~~-..~~_,, ~~--...~~--...~~--~~~
`
`0
`
`7
`
`42
`Days of experiment
`
`49
`
`56
`
`63
`
`TEXT·FIOURE 5. Effect of short-term treatment with ICI 182,780 on
`(a) endometrium and (b) myometrium in ovariectomized oestro(cid:173)
`gen-treated monkeys. Percentage values were calculated by reference
`to the volume of the endometrium and myometrium in each ani(cid:173)
`mal after an initial control treatment with 5 µg 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 once daily treatment
`with S µg OB/ kg s.c. The closed bar indicates treatment once
`daily with O· l mg (filled symbols: n = 2) or I mg (open symbols :
`n = 3) ICI 182, 780 /kg i.m. in propylene glycol solution for 7 days
`(days 42-49). Involution of the uterus following initial oestrogen
`treatment was induced by withdrawal of OB and once daily treat(cid:173)
`ment with S mg progesterone for S days (hatched bar, days
`14-18). Experiment days designated by reference to the first injection
`of OB (day 0).
`
`ICI 182,780/ mg at 4-weekly interva)s (Text-fig. 4).
`Interestingly, in that study the increasing deJay of the
`onset of uterine growth after the second and third
`doses
`indicated a cumulative biologica) effect.
`However, estimates of concentration of drug in the
`
`serum did not indicate that drug accumulation was
`responsible for this increased efficacy (F. Sutcliffe,
`unpublished studies). To demonstrate that the sus(cid:173)
`tained action of ICI 182,780 in oil reflects a slow
`reJease of active drug, the effect was compared with
`
`Journal of Endocrinology (1992) 135, 239-247
`
`MYLAN PHARMS. INC. EXHIBIT 1025 PAGE 8
`
`

`
`u NI
`
`100
`
`(a)
`
`50
`
`.......
`"
`~ "Q
`8
`j
`~ ... 0
`..
`) 100
`-s
`!!
`JI ....
`
`~
`~
`
`0
`
`(
`
`(b)
`
`IC! 182,780 effects on monkey uterus
`
`· M. DUKES and others 24~
`
`action of oestradiol was confined strictly to the period
`of ICI 182,780 treatment. Endometrial and myo(cid:173)
`metrial growth resumed immediately after cessation
`oflCI 182,780 treatment. These studies also indicated
`that a daily dose of 0· 1 mg ICI 182,780/ kg was
`sufficient to block the action of exogenous OB given
`at a dose (5-µg 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(cid:173)
`onstrated that ICI 182, 780 not only blocks oestrogen
`action when administered concurrently with OB
`(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(cid:173)
`sistent with the view that ICI 182,780 is a fully effec(cid:173)
`tive pure antioestrogen in the primate.
`Finally,
`these 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 differential response is seen in intact
`monkeys where, early in the menstrual cycle when
`oestradiol concentration is low, endometriaJ prolifera(cid:173)
`tion
`is accompanied by myometrial
`involution
`(Waterton et al. 1992).
`
`50
`
`0
`
`-28
`
`-14
`
`28
`0
`14
`Days of experiment
`
`42
`
`56
`
`ACKNOWLEDGEMENTS
`
`TEXT-FIGURE 6. Comparison of oestrogen withdrawal and
`ICI 182,780 treatment on (a) endometrium and (b)
`myometrium in two ovariectomizcd oestrogen-treated
`monkeys. Percentage values were calculated by reference
`1the volume of the endometrium and myometrium in
`(
`~ach animal after an initial control treatment with 5 µg
`08/ kg s.c. alone for 7 days and subsequent withdrawal as
`described for Text-fig. I. Open bars represent once daily
`treatment with 5 µg OB/ kg s.c. Solid bars represent once
`daily injections of O· 2 mg ICI 182,780/kg i.m. 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 16 days (7- 22 or
`49-64) by either OB together with ICI 182,780 (squares,
`upper treatment sequence, days) or vehicle alone (circles,
`lower treatment sequence). Three weeks later the experi(cid:173)
`ment was repeated but each animal crossed-over to the
`alternative treatment protocol. Experiment days desig(cid:173)
`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. Wakeling &
`M. Dukes, unpublished studies). The results in Text(cid:173)
`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.
`Breen for excellent technical support in this work.
`
`REFERENCES
`
`Lee, J. K. T., Gerscll, D. J .• Balfe, D. M., Worthington, J. L.,
`Picus, D. & Gapp, G. (1985). The uterus: in vitro
`MR-anatomic correlation of nonnal and abnormal specimens.
`Radiology 157, 175- 179.
`Wakeling, A. E., Dukes, M. & Bowler, J . (1991). A potent specific
`pure antioestrogen with clinical potential. Cancer Research 51,
`3867- 3873.
`Waterton, J.C., Miller, D., 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.
`Waterton, J.C., Larcombe-McDouall, J. B. & Miller, D . (1992).
`Quan.titative MRI of the prostate and uterus in monkeys.
`Magnetic Resonance in Medicine (In Press.)
`
`Journal of Endocrinology (1992) 135, 239· 247
`
`MYLAN PHARMS. INC. EXHIBIT 1025 PAGE 9
`
`

`
`PLATE
`
`JC/ 182,780 effect.~ on monkey U/erus
`
`M . nuKF-'i and others
`
`2
`
`3
`
`4
`
`DESCRIPTlON OF PLATE
`
`s
`
`Magnetic resonance images (MRI) of the monkey uterus. Each illustration is a detail.
`representing 3·4 x 3·4 cm from one slice of the eight recorded <tt euch measurement.
`The appearance at mid-corpus uteri is shown as follows. Ovaricctomi7.ed control (Fig. I).
`after 7 days of treatment with oestradiol benzoatc (08: 5 µg/ kg s.c.) (Fig. 2) and 0 (fig. 3).
`14 (fig. 4) and 49 (Fig. 5) days after a single i.m. injection of 5 mg IC I 182.780/ mg. in
`castor oil solution. together with daily oestradiol treatment. Four concentric zones of
`MR signal intensity arc seen: high central signal of the endomctrium: medium-low signal.
`myometrial junction zone: medium-high signal of the myometrium: and no signal.
`peripheral adipose tissue. T he high signal intensity seen at the left in rigs 3 and 5 is urine
`in the bladder.
`
`I Facing p. 2411)
`
`J1111m11/ o( f.'11tl<>ai110/ogy ( 199:? > 1 3~. :?.W :?4 7
`
`MYLAN PHARMS. INC. EXHIBIT 1025 PAGE 10