British Journal of Cancer (1996) 74, 300-308
`© 1996 Stockton Press All rights reserved 0007-0920/96 $12.00
`
`Pharmacokinetics, pharmacological and anti-tumour effects of the specific
`anti-oestrogen ICI 182780 in women with advanced breast cancer
`
`A Howell‘, DJ DeFriend2, JFR Robertson3, RW Blamey3, L Anderson‘, E Anderson‘, FA
`Sutcliffes and P Walton5
`
`’CRC Department of Medical Oncology, University of Manchester, Christie Hospital, Wilmslow Road, Manchester M20 4BX;
`2Department of Surgery, University Hospital of South Manchester, Nell Lane, West Didsbury, Manchester M20 8LR; ‘Department
`of Surgery, City Hospital, Nottingham NG5 IPB; ‘Tumour Biochemistry Laboratory, Christie Hospital, Wilmslow Road,
`Manchester M20 4BX; 5Zeneca Pharmaceuticals, Alderley Park, Macclesfield, Cheshire SKIO 4 TG, UK.
`
`Summary We have assessed the pharmacokinetics, pharmacological and anti-tumour effects of the specific
`steroidal anti-oestrogen ICI 182780 in 19 patients with advanced breast cancer resistant to tamoxifen. The
`agent was administered as a monthly depot intramuscular injection. Peak levels of ICI 182780 occurred a
`median of 8—9 days after dosing and then declined but were above the projected therapeutic threshold at day
`28. Cm,“ during the first month was 10.5 ng/ml" and during the sixth month was 12.6 ng ml“. The AUCs
`were 140.5 and 206.8 ng day ml" on the first and sixth month of dosing respectively, suggesting some drug
`accumulation. Luteinising hormone (LH) and follicle-stimulating hormone (FSH) levels rose after withdrawal
`of tamoxifen and then plateaued, suggesting no effect of ICI 182780 on the pitu.itary—hypothalamic axis. There
`were no significant changes in serum levels of prolactin, sex horrnone-binding globulin (SHBG) or lipids. Side-
`efl"ects were infrequent. Hot-flushes and sweats were not induced and there was no apparent effect of treatment
`upon the endometrium or vagina. Thirteen (69%) patients responded (seven had partial responses and six
`showed ‘no change’ responses) to ICI 182780, after progression on tamoxifen, for a median duration of 25
`months. Thus ICI 182780, given by monthly depot injection, and at the drug levels described,
`is an active
`second-line anti-oestrogen without apparent negative effects on the liver, brain or genital tract and warrants
`further evaluation in patients with advanced breast cancer.
`
`Keywords: ICI 182780; advanced breast cancer
`
`Half of the patients with advanced breast cancer have
`tumours that either regress or remain stable when treated
`with tamoxifen. Despite initial response all such tumours
`eventually become resistant
`to this anti-oestrogen after a
`median duration of remission of about 18 months (Cole et
`al., 1971; Patterson et al., 1981). Although it acts as an
`oestrogen antagonist with respect to the tumour, tamoxifen is
`oestrogenic with respect to bone (Turken et al., 1989), the
`liver (Bertelli et al., 1988) and the endometrium (Fornander
`et al., 1989). Potential causes of treatment failure may result
`from tamoxifen, or its metabolites (Osborne et al., 1991)
`becoming oestrogenic with respect to the tumour (Howell et
`al., 1990) or from tamoxifen becoming sequestered away
`from the oestrogen receptor
`(ER) and rendered inactive
`(Pavlick et al., 1992).
`A new class of specific anti-oestrogens has been developed
`that produce more complete suppression of the proliferative
`effects of oestrogen upon tumours. Substitution of a long
`side-chain at the 7 alpha position of the oestradiol molecule
`has produced compounds that appear more active as anti-
`oestrogens than the triphenylethylene derivatives such as
`tamoxifen (Wakeling and Bowler, 1987, 1988). The structure
`of the prototype specific anti-oestrogen, ICI 164384, is shown
`in Figure
`1
`together with that of
`ICI
`182780,
`{7a-
`[9(4,4,5,5,pentafluoropentyl-su1phinyl)nonyl]oestra-l ,3,5,( 10)-
`triene-3,17fi-diol} the compound selected for clinical evalua-
`tion because of its greater potency and affinity for the ER
`(Wakeling et al., 1991).
`Both compounds have been assessed using in vitro and
`animal models of human breast cancer and compared with
`non-steroidal,
`partial
`agonist
`anti-oestrogens,
`including
`tamoxifen, and also with oestrogen withdrawal. The specific
`anti—oestrogens have shown superiority over these alternative
`
`Correspondence: A Howell
`Received 3 April 1995;
`revised 14 February 1996; accepted 15
`February 1996
`
`methods of oestrogen deprivation with respect to inhibition of
`cell proliferation and oestrogen-induced gene expression. ICI
`164384 and ICI 182780 are up to two orders of magnitude
`more potent than tamoxifen as inhibitors of cell growth in
`vitro. (Wakeling and Bowler, 1987, 1988) and produce a more
`profound blockade of cell division in the G1 phase of the cell
`cycle. The specific anti—oestrogens are more effective than
`tamoxifen in suppressing expression of oestrogen-induced
`genes,
`such as progesterone receptor
`(PgR), pS2 and
`cathepsin D (Nicholson et al., 1994) by breast cancer cells.
`The specific anti—oestrogens have also been shown to produce
`a rapid reduction of intracellular ER levels, possibly as a result
`of inhibition of ER dimerisation and reduction of the ER half-
`life (Fawell et al., 1990; Dauvois et al., 1992). This latter effect
`is in contrast to that of tamoxifen, which has been shown to
`increase ER expression by breast cancer cells
`in vitro
`(Wakeling et al., 1989).
`The experiments outlined above were performed on cell
`lines but similar results were demonstrated when a short-
`acting, propylene glycol-based formulation of ICI 182780 was
`administered by daily intramuscular injection for
`1 week
`before surgery to women with primary breast cancer.
`Compared with pretreatment tumour samples (obtained by
`Tru-cut biopsy),
`those obtained after treatment with the
`specific anti-oestrogen showed reduced proliferation (K167
`expression) and reduced or absent expression of ER, PgR
`and pS2 (DeFriend et al., 1994b; Nicholson et al., 1994).
`Similar clinical experiments with tamoxifen produced no
`change in ER expression, slightly increased PgR expression
`and a reduction in labelling index (Howell et al., 1988;
`Roberston et al., 1991; Clarke et al., 1993; Nicholson et al.,
`1994).
`The aims of the study reported here were to assess the
`long-terrn efficacy and toxicity of the specific anti-oestrogen
`ICI 182780 in patients with advanced breast cancer and to
`evaluate the pharrnacokinetics of the long-acting formulation
`used. Since tamoxifen-resistant breast cancer cell lines have
`been shown to retain sensitivity to specific anti—oestrogens
`
`|nnoPharma Exhibit 1007.0001
`
`

`

`a
`
`on
`
`Study design
`
`|C|18278O and advanced breast cancer
`A Howell et al
`
`301
`
`"'0
`
`b
`
`H0
`
`”’/o
`/(CH2)1oCOlTl(CH2)3CH3
`cu-:3
`
`ICI 164384
`
`OH
`
`”’/x,
`(CH2)9S0(CH2)3CF2CF3
`ICI 182780
`
`Figure 1 Comparison of the structures of ICI 164384 and ICI
`182780.
`
`when grown either in vitro (Lykkesfeld and Sorenson, 1992;
`Brunner et al.,
`l993a,b; Lykkesfeld et al.,
`1994) or as
`xenografts in nude mice in vivo (Gottardis et al., 1989;
`Osborne et al.,
`1991),
`the effects of ICI 182780 were
`evaluated in a group of post-menopausal patients with
`tamoxifen-resistant breast cancer. Since the partial agonist
`activity of tamoxifen on bone density and lipid levels has
`been reported to the beneficial in post-menopausal patients,
`the effects of ICI 182780 at other oestrogen target sites,
`including the hypothalamus/pituitary gland, the liver and the
`endometrium has also been assessed in this study.
`We report that although some drug accumulation occurred
`at the dose level used in this study, administration of ICI
`182780 was associated with a lower than expected incidence
`of side-effects (such as hot flushes and vaginal problems)
`together with a high response rate and long response
`duration in women previously treated with tamoxifen. A
`preliminary report of the early clinical result of this study has
`been published (Howell et al., 1995).
`
`Patients and methods
`
`Patients
`
`Nineteen patients with advanced breast cancer resistant to
`tamoxifen were treated with ICI 182780. The study was
`approved by the ethics committees of each clinical centre.
`Patients were eligible for
`the study if they were post-
`menopausal and age less than 81 years, with histologically
`verified breast cancer. Patients were included if they had been
`treated with tamoxifen as an adjuvant to surgery for more
`than 2 years and then relapsed, or if they had been treated
`with tamoxifen for advanced disease, had a complete or
`partial remission or disease stabilisation (‘no change’) for at
`least 6 months, and subsequently progressed while taking
`tamoxifen. Patients were excluded if
`they had serious
`intercurrent disease, a WHO performance status of greater
`than 2, and a life expectancy of less than 3 months or had
`received previous cytotoxic chemotherapy for advanced
`breast cancer. The characteristics of the patients studied are
`summarised in Table 1. One patient had adjuvant therapy for
`only 9 months and progressed and was thus a protocol
`violation, but is included in the analysis. She had progressive
`disease when treated with ICI 182780.
`
`After giving informed consent, all patients participating in
`the study underwent baseline staging investigations before
`commencing treatment with ICI 182780 including X-rays,
`liver ultrasound or computerised tomography (CT) scan and
`isotope bone scan. ICI 182780 was administered as a long-
`acting formulation contained in a castor oil-based vehicle by
`monthly i.m. injection (5 ml) into the buttock. For appraisal
`of drug safety, the first four patients received escalating doses
`of ICI 182780, starting with 100 mg in the first month and
`increasing to 250 mg i.m. from the second month onwards,
`following confirmation of lack of local or systemic drug
`toxicity
`at
`the
`100 mg dose. Patients 5-19 received
`250 mg month“ i.m. from the outset. Treatment with ICI
`182780 was continued until objective tumour progression
`occurred. Patients were seen at intervals of 3-7 days during
`the first month after commencing treatment with ICI 182780
`in order to monitor local and systemic drug tolerability and
`to collect blood samples
`for pharmacokinetic
`studies.
`Thereafter, patients were reviewed at monthly intervals in
`order to evaluate objective tumour response to ICI 182780
`and to further monitor local and systemic drug tolerability.
`Blood samples were taken before commencing treatment with
`ICI
`182780
`and at monthly
`intervals
`thereafter
`for
`measurement of full blood count, clinical biochemistry and
`serum hormone, SHBG and lipid levels. Tumour response to
`therapy was evaluated according to UICC criteria (Hayward
`et a1., 1977). To qualify for the ‘no change’ category, tumour
`growth had to stabilise for more than 6 months (Howell et
`al., 1988; Robertson et al., 1989). Body weight was recorded
`at each monthly review in the majority of patients.
`
`Serum estimations
`
`The concentration of ICI 182780 in serum samples was
`determined by radioimmunoassay (RIA), using antibodies
`raised in sheep to ICI 182780 coupled at the 17-position to
`thyroglobulin and tritiated ICI 182780. The procedure was
`applied after solid base clean up of a diethyl ether/hexane
`extract of serum. The study limit of quantification was
`0.68 ng ml“. The RIA procedure is believed to be specific
`for ICI 182780, since comparative analysis of plasma samples
`from preclinical studies by RIA and high-performance liquid
`chromatography (HPLC) showed a good correlation for ICI
`182780 concentrations. Further,
`ICI
`182780 metabolites
`present
`in these samples were not detected by the RIA.
`Gonadotrophins follicle-stimulating hormone and lutenising
`hormone (FSH and LH) and SHBG were measured by RIA
`in the Regional Radioimmunoassay Laboratory of
`the
`University Hospital of South Manchester. Prolactin was
`measured by immunoradiometric
`assay
`using
`reagents
`supplied by Netria. Total cholesterol levels were determined
`enzymatically
`using
`a
`commercially
`available
`reagent
`(Diamed, Switzerland). Triglyceride levels were determined
`by the glyceryl phosphate oxidase—peroxidase—antiperox-
`idase method using a commercially available kit (Boehringer
`Mannheim, Germany). High-density lipoprotein (HDL)
`cholesterol
`levels were measured after pretreatment of the
`serum samples with buffered magnesium phosphotungstate,
`which selectively precipitates low-density lipoprotein (LDL)
`cholesterol, very low-density lipoprotein (VLDL) cholesterol
`and chylomicrons,
`leaving HDL cholesterol
`in the super-
`natant. Serum levels of LDL cholesterol were calculated
`using the Friedewald equation (Friedewald et al., 1972).
`
`Endometrial assessment
`
`Endometrial thickness was measured in transverse section by
`transabdominal ultrasound using a Siemens Sonoline SL2 with
`a 3.5 MHz sector probe. Baseline and repeat scans at 3 -6
`monthly intervals were performed in five patients. Endometrial
`histology was reviewed on one patient who had a hysterectomy
`for uterine prolapse after 18 months on ICI 182780.
`
`|nnoPharma Exhibit 10070002
`
`

`

`302
`
`IcI182780 and advanced breast cancer
`A Howell et al
`
`No.
`1 (ERD)
`
`2 (PF)
`
`3 (SAS)
`
`4 (AS)
`
`5 (AR)
`
`6 (FC)
`
`7 (SC)
`
`8 (LH)
`
`9 (NT)
`
`Age at
`entry
`51
`
`75
`
`49
`
`53
`
`68
`
`58
`
`61
`
`55
`
`70
`
`Table I Patient characteristics, tumour receptor status and response to ICI182780
`Duration
`Duration
`of adjuvant
`of tamoxifen Response to
`tamoxifen
`for adv
`tamoxifen
`48
`—
`
`ER”
`0
`
`PR"
`0
`
`Time to
`relapse
`48
`
`Sites of
`disease
`Bone
`
`—
`
`74
`
`45
`
`20
`
`77
`
`—
`
`48
`
`42
`
`-
`
`68
`
`45
`
`20
`
`77
`
`—
`
`—
`
`9
`
`8
`
`—
`
`—
`
`—
`
`—
`
`8
`
`19
`
`—
`
`NC
`
`PR
`
`PR
`
`NC
`
`Breast
`Nodes
`
`Lung
`Pleura
`
`Bone
`
`Bone
`Pleura
`
`Nodes
`
`Bone
`Breast
`
`Breast
`Node
`
`Local
`Bone
`
`Local
`
`99
`
`0
`
`16
`
`74
`
`73
`
`ND
`
`70
`
`95
`
`100
`
`99
`
`22
`
`0
`
`< 5
`
`6
`
`ND
`
`95
`
`80
`
`100
`
`Response to Duration
`182780
`(months)
`PD
`<2
`
`NC
`
`PR
`
`PD
`
`PR
`
`PR
`
`PD
`
`PR
`
`PD
`
`PR
`
`29
`
`12
`
`<2
`
`8
`
`3
`
`<2
`
`25
`
`<2
`
`33 +
`
`10 (MC)
`
`11 (FWT)
`
`12 (MEU)
`
`13 (KG)
`
`14 (IN)
`
`15 (CA)
`
`16 (AC)
`
`17 (LM)
`
`18 (JKJ)
`19 (MB)
`
`64
`
`70
`
`51
`
`62
`
`78
`
`48
`
`64
`
`67
`
`65
`64
`
`201
`
`271
`
`77
`
`~
`
`120
`
`61
`
`68
`
`52
`
`80
`23
`
`-
`
`—
`
`74
`
`—
`
`—
`
`61
`
`67
`
`—
`
`—
`23
`
`8
`
`7
`
`-
`
`12
`
`24
`
`—
`
`—
`
`34
`
`48
`—
`
`NC
`
`NC
`
`PR
`
`NC
`
`NC
`
`Nodes
`Bone
`Breast
`
`Bone
`
`Nodes
`Bone
`
`Bone
`
`Bone
`
`Nodes
`Breast
`
`Breast
`Bone
`
`Bone
`Bone
`
`60
`
`99
`
`90
`
`100
`
`ND
`
`30
`
`70
`
`(l828)"
`29
`
`<5
`
`97
`
`60
`
`0
`
`ND
`
`<5
`
`30
`
`(1)
`29
`
`PD
`
`NC
`
`PD
`
`PR
`
`NC
`
`PR
`
`NC
`
`NC
`NC
`
`<2
`
`33 +
`
`<2
`
`32+
`
`25
`
`9
`
`30+
`30+
`
`adv, advanced disease; PD, progressive disease; NC, no change; PR, partial response; ND, not done. 3% cells positive, immunoassay.
`“Biochemical assay (mol 1").
`
`Statistical analysis
`
`analyses were performed on an Apple
`statistical
`All
`Macintosh personal computer, using the StatView SE
`software programme
`(Abacus Concepts, Berkeley, CA,
`USA). Pharrnacokinectic data were analysed using para-
`metric statistics. Data relating to body weight,
`serum
`gonadotrophin, SHBG and lipid levels were analysed using
`non-parametric statistics. The null hypothesis was rejected at
`a probability level of P<0.05.
`
`Results
`
`Pharmacokinetics
`
`Serum concentrations of ICI 182780 were measured during
`the first month of treatment
`in 15 patients who started
`treatment at the 250 mg dose level and in 11 patients who
`remained on treatment with ICI 182780 during the sixth
`month. In the majority of patients, the measured C,,,,., was
`reached 8 or 9 days after the start of the drug administration.
`However, samples were not available between day 2 and day
`8. The profile was quite flat between days 2 and 8, supported
`by preclinical data in dogs where the Cm, was seen on day 1
`or 2. Following both the
`100 mg and 250 mg doses,
`continuous release of drug from the ICI 182780 slow release
`formulation was shown throughout the one month dosing
`interval. The profiles of the serum concentration of ICI
`
`182780 are shown in Figure 2. Comparison of data after the
`first and sixth monthly 250 mg doses of ICI 182780 showed
`that the mean exposure to the drug increased slightly after
`multiple dosing. Mean Cm,
`(which occurred on day 7)
`increased from 10.5 ng ml" to 12.8 ng ml", accompanied
`by increases in mean end-of-month concentrations from
`3.1 ng ml“
`to
`5.6 ng ml"
`and AUC values
`from
`140.5 ng day ml" to 206.8 ng day ml“ for the first and
`sixth months respectively in the 11 patients studied. Multiple
`dosing produced a 1.2-fold increase in Cm, and a 1.5-fold
`increase in AUC, indicating a degree of accumulation at the
`250 mg dose level. This greater exposure was not associated
`with any increased side-efl'ects or irritancy (see below). There
`was no significant difference in the median Cm, and AUC
`between responders and non-responders to treatment (Table
`II). After 6 months of treatment there was no significant
`difference between Cm, and AUC for patients who had a
`partial reponse (PR) compared with those with a no change
`(NC) response.
`
`Eflects on hormones and lipids
`
`The serum levels of FSH, LH, prolactin and SHBG, before
`and during treatment with ICI 182780, are shown in Figure
`3. The median levels of FSH and LH before starting
`treatment with ICI 182780 were below the normal range for
`post-menopausal women, whereas the median SHBG level
`was above the normal range, both possibly related to the
`
`|nnoPharma Exhibit 1007.0003
`
`

`

`11.5
`11
`10.5
`
`Concentration
`
`(ngm|’1)
`
`0
`
`2
`
`4
`
`6 81012141618202224262830
`
`Time (days)
`
`Figure 2 Mean serum concentrations of ICI 182780 during the
`first and sixth months of treatment. T, Profile at entry; - - -,
`profile month 6.
`
`agonist activity of previous treatment with tamoxifen. During
`the first 3 months of administration of ICI 182780, there was
`significant
`increases in the serum concentration of FSH
`(median pre- and post-treatment values 26 and 52 IU 1"
`respectively; P<0.05, Wilcoxon’s matched-pairs signed-rank
`test) and LH (median pre- and post-treatment values 26 and
`42 IU 1“ respectively; P<0.005). Thereafter, no further
`significant overall changes occurred in serum gonadotrophin
`levels but wide variation between individual patients were
`observed, refleeted in the broad interquartile ranges seen in
`Figure 3. Serum SHBG levels showed an overall trend to
`decrease following treatment with the specific anti-oestrogen,
`falling from a median level of 100 mmol l" pretreatment to
`55 mmol 1“ after 8 months of treatment (P=NS. Figure 3c).
`
`Ic|182780 and advanced breast cancer
`A Howell et al
`%
`303
`
`This overall reduction appeared to result predominantly from
`four patients who continued tamoxifen up to the time of
`starting ICI 182780. The remaining patients,
`including 11
`others on tamoxifen and four who had stopped tamoxifen
`some time before entry, showed very little change in serum
`SHBG levels during treatment. Serum prolactin levels
`remained within the normal
`range, and did not change
`significantly throughout the treatment period. There were no
`significant changes in serum levels of total cholesterol, LDL
`cholesterol, HDL cholesterol and triglyceride (Figure 3) for
`the 12 patients treated in the South Manchester Breast Unit
`during treatment with ICI 182780.
`
`Side-effects
`
`No serious drug-related adverse events occurred in any of
`the 19 patients treated with ICI 182780. Minor systemic
`adverse
`events were
`reported
`by
`two
`patients
`and
`comprised a transient bloodstained vaginal discharge and
`a subjective feeling of living in a ‘dream-like state’ (similar
`to one she had while taking tamoxifen) in one patient and
`alteration of body odour (noticed by her husband for a 1
`month period), possibly associated with increased hair
`greasiness,
`in the other. Administration of the pure anti-
`oestrogen was not associated with any alteration in the
`frequency of night sweats or hot flushes, if already present,
`and none were initiated. None of the patients reported
`vaginal dryness or altered libido despite direct questioning
`at each monthly out-patient attendence. The long-acting
`formulation of ICI 182780 used in this study appeared well
`tolerated locally at the site of injection despite the relatively
`large volume (5 ml) administered. One patient developed
`bruising
`over
`the
`buttock
`and
`a
`second
`developed
`tenderness at
`the injection site following drug administra-
`tion on one occasion each, and a third patient had local
`erythema
`at
`the
`injection site on one occasion. No
`clinically
`significant
`changes
`in
`full blood count
`or
`unexpected changes in the biochemical profile occurred in
`any of the patients participating in the study.
`Serial endometrial ultrasound examinations were per-
`
`Table II Results of Cm“ and AUC during months 1 and 6 according to response categories. There were no
`significant differences in drug kinetics between responders and non-responders
`On entry
`A! month 6
`
`C,,,,,,,
`(ngm1"1)
`4.4“
`1.6“
`5.5
`9.7
`29.9
`5.6
`
`AUC
`(ngdaymfl)
`53.1“
`25.1“
`105.7
`138.2
`289.3
`36.7
`
`Cm“
`(ngml")
`
`AUC
`(ng day mfl)
`
`15.8
`
`243.7
`
`Response
`Progressive disease
`
`No change
`
`Partial response
`
`Patient
`1
`4
`7
`9
`11
`13
`
`Median
`
`2
`12
`15
`17
`18
`19
`
`Median
`
`3
`5
`6
`8
`10
`14
`16
`
`Median
`
`5.6
`
`1.8”
`7.2
`9.0
`9.5
`10.3
`11.0
`
`9.3
`
`2.9“
`17.4
`7.7
`5.9
`14.8
`9.1
`4.4
`
`7.7
`
`79.4
`
`23.2“
`143.6
`107.8
`125.5
`183.2
`137.8
`
`131.6
`
`56.2“
`188.4
`118.3
`118.7
`206.6
`134.6
`72.8
`
`118.7
`
`7.5
`12.2
`15.8
`14.9
`10.2
`17.2
`
`12.8
`
`9.9
`17.6
`
`10.0
`9.1
`13.5
`12.0
`
`11.0
`
`135.8
`179.3
`201.7
`297.6
`156.1
`308.0
`
`190.9
`
`139.5
`203.0
`
`175.2
`191.7
`266.0
`190.8
`
`191.0
`
`“Patients 1-4 received 100 mg dose at entry and 250 mg dose from month 2 onwards.
`
`MYLAN PHARMS. INC. EXHIBIT 1006 PAGE 4
`
`|nnoPharma Exhibit 1007.0004
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`

`

`lc|182780 and advanced breast cancer
` A Howell et al
`304
`
`CHOL
`
`TRIG
`
`HDLC
`
`LDLC
`
`6 5
`
`-
`'_ 4
`
`E3
`E 2
`
`4
`
`_ 3
`'_
`
`E2
`E
`
`4
`6 810121416
`‘Fume (months)
`
`0
`
`0
`
`2
`
`6 810121416
`4
`'|'1me(months)
`
`0 2 4 6 810121416
`Time(months)
`
`FSH
`
`SHBG
`
`200
`
`150
`
`100
`
`50
`
`0
`
`0
`
`2
`
`Prolactin
`
`12107
`
`5
`
`400 13
`350
`300
`
`.I_ 250
`S200
`"150
`100
`50
`0
`
`4 6 810121416
`Time(months)
`
`0
`
`2
`
`4
`
`6 810121416
`
`Time(months)
`
`0
`
`2
`
`4 6 810121416
`Time (months)
`
`2o
`0
`
`0 2
`
`4 6 810121416
`'|'Ime (months)
`
`1o
`
`4
`
`9121210 8 3 6 6
`8
`7
`7
`I 5
`°
`5
`E
`4
`3
`
`21o
`
`Time (months)
`
`LH
`
`7019191714 9
`
`7
`
`5 6
`
`0
`
`2
`
`4 6 810121416
`
`0
`
`0
`
`2
`
`3
`
`'1
`—
`E 2
`E
`
`1
`
`100
`
`so
`
`7- 60
`2 40
`
`6°
`so
`
`‘T_ 40
`2 3o
`2o
`
`10
`0
`
`Figure 3 Median and interquartile ranges of lipids and hormones during treatment with ICI 182780. Numbers above the curves
`refer to the numbers of patients tested. Twelve patients were tested for the four lipids (CHOL, cholesterol; TRIG, triglyceride;
`HDLC, high-density lipoprotein; LDLC, low-density lipoprotein), 19 for the LH, FSH and SHBG and 13 for prolactin. Numbers
`decline because patients go off study after progression.
`
`(mm)
`
`Endometrialthickness
`
`12
`
`15
`
`18
`
`1'1me (months)
`
`Figure 4 Serial ultrasound estimations of endometrial thickness
`in five patients. Thickened endometrium compared with normal
`post-menopausal women was thought to be due to treatment with
`tamoxifen. No significant change occurred up to 15 months of
`treatment with ICI 182780.
`
`thickness
`formed in five responding patients. Endometrial
`was greater than the expected <2 mm usually found in post-
`menopausal women,
`in all patients. The thickness of the
`endometrium remained unchanged in all patients during
`treatment with ICI 182780 (Figure 4). Endometrial histology
`was reviewed on one patient who had a hysterectomy. This
`was
`reported as
`showing an atrophic post-menopausal
`pattern with cystic change. The glands were lined by
`flattened and cuboidal epithelium. There was no mitotic
`activity, epithelial ectoplasia or polyp formation. There were
`
`Table III Response rate and durations of response to ICI 182780 in
`relation to duration of previous treatment with tamoxifen
`Response
`ICI182780
`
`Durations (months)
`
`Number
`(%)
`
`Partial
`
`7 (37)
`
`25+ (PRl9)“, 33+ (NC8), 32+ (PR24)
`25+ (A67), 12 (A74), 8 (A20), 3 (A77)
`
`No change
`
`6 (32)
`
`29+ (NC8), 33+ (A74), 23+ (A61), 30+
`(NC48), 30+ (A23), 9 (NC34)
`
`Progression
`
`6 (31) All patients progressed in <8 weeks
`(A48, A45, NC7, PR8, A9, NCl2)
`“Letters in brackets indicate response to tamoxifen when given for
`advanced disease (PR, partial
`remission; NC, no change; PD,
`progressive disease) or if given as an adjuvant therapy (A). The
`numbers in the brackets
`indicate duration of treatment with
`tamoxifen.
`
`no significant changes in body weight during treatment with
`ICI 182870. Mean body weight (kg:s.d.) was 63.8i14.0
`(n=l3) at
`the beginning of treatment, 64.9il5.8 (n=ll)
`after 6 months, 64.5:l7.2 (n=9) after 10 months and
`64.21183 (n=9) after 16 months of treatment with ICI
`182780.
`
`Response
`
`All 19 patients are evaluable for response to ICI 182780
`(Table III). Six patients were unresponsive de novo and
`showed objective evidence of disease progression within 2
`months of commencing treatment. The remaining 13 patients
`(69%) all
`responded to treatment with the specific anti-
`oestrogen for a median duration of 25 months. Seven patients
`(37%) showed PR5 for 33+, 32+, 25+, 25,
`I2, 8 and 3
`months, and six patients (32%) showed NC responses for
`33 + , 30+ , 30+ , 29, 23 and 9 months. Thus five patients are
`still in remission and continuing treatment with ICI 182780
`after 30-33 months. Responses have been observed in six of
`the nine women who progressed while receiving tamoxifen as
`
`MYLAN PHARMS. INC. EXHIBIT 1006 PAGE 5
`
`|nnoPharma Exhibit 1007.0005
`
`

`

`IcI182780 and advanced breast cancer
`A Howell et at
`
`305
`
`treatment for advanced breast cancer as well as in seven of
`the ten women who relapsed after treatment with tamoxifen
`as adjuvant
`therapy. There appeared to be no association
`between duration of treatment with tamoxifen and subse-
`quent response to ICI 182780 (Table III).
`
`Discussion
`
`investigation of long-term
`This study represents the first
`administration of the specific anti-oestrogen, ICI 182780, to
`patients with breast cancer and demonstrates that predicted
`therapeutic levels of ICI 182780, as judged from animal
`experiments (Wake1ing et al., 1991; Dukes et al., 1993) and
`our previous short phase I study (DeFriend et al., 1994b) can
`be achieved and maintained for 1 month following a single
`i.m. injection of the long-acting formulation used. Treatment
`with ICI 182780 was associated with minor effects on serum
`hormones and lipid levels, produced few side-effects and
`resulted in a high response rate after tamoxifen failure,
`together with a median reponse duration of 25 months.
`Pharrnacokinetic data concerning the release characteris-
`tics of the drug into the serum in this study were found to be
`similar to those previously demonstrated in adult female
`monkeys (Dukes et al., 1993). From studies on inhibition of
`endometrial proliferation in the monkey and inhibition of
`tumour proliferation in a previous phase I study,
`it was
`predicted that serum levels of ICI 182780 in the range of 2-
`3 ng ml" were consistent with a therapeutic effect in patients
`with advanced breast cancer. However, a direct pharmaco-
`kinetic—pharrnacodynamic link is not proven with the few
`patients studied to date. Serum drug concentrations in excess
`of this were observed with the 250 mg dose used in the
`present study for most of the first and all of the sixth month.
`However,
`there was evidence of drug accumulation after
`multiple dosing, such that after 6 months treatment there was
`an 80% increase in mean end of month drug levels and a
`50% increase in the AUC compared with data from month 1.
`These data suggest
`that lower doses of the drug may be
`effective
`in maintaining therapeutic serum drug levels,
`although further clinical studies are required to confirm this
`hypothesis.
`Previous animal studies with ICI 182780 have shown that
`the activity of specific anti-oestrogens may vary between
`different oestrogen target sites
`(Wakeling, 1993).
`In the
`present study, we have attempted to obtain preliminary data
`on the effects of long-term administration of ICI 182780 on
`the pituitary gland/hypothalamus, the liver and the endome-
`trium. Serum gonadotrophin levels significantly increased
`during the first 3 months of treatment with ICI 182780 and
`then remained stable thereafter. This change is in contra-
`distinction to that
`seen with the triphenylethylene anti-
`oestrogen,
`tamoxifen, which reduces serum levels of FSH
`and LH in post-menopausal patients
`because of
`its
`oestrogenic effect on the pituitary gland and hypothalamus
`(Willis et al., 1977). All but four patients in the present study
`were treated with tamoxifen up until
`treatment with ICI
`182780 was initiated. Levels of FSH and LH at this time were
`below the range for normal post-menopausal women, which
`is attributable to previous therapy with tamoxifen. The rise of
`gonadotrophins during the first 3 months of treatment with
`ICI 182780 may therefore have been a passive effect caused
`by cessation of
`tamoxifen rather
`than an active anti-
`oestrogen effect of the new agent. The fact
`that gonado-
`trophin levels rose to well within post-menopausal values and
`then remained stable would support the view that ICI 182780
`is without effect on gonadotrophin levels. This apparent lack
`of activity of the specific anti-oestrogen on the hypothalamus
`and pituitary gland is further supported by the findings that
`ICI 182780 did not
`initiate or exacerbate hot flushes or
`sweats in the present study, nor did it produce significant
`changes in serum prolactin levels.
`Treatment with tamoxifen has been reported to increase
`serum levels of SHBG secondary to a probable oestrogenic
`
`effect of tamoxifen on the liver (Sakai et al., 1978). The
`levels of SHBG in some of our patients before starting
`therapy with ICI 182780 were high, consistent with the
`oestrogenic
`effect of prior
`treatment with tamoxifen.
`Following commencement of treatment with ICI 182780, a
`slight decline in SHBG levels occurred, consistent with
`tamoxifen withdrawal, but thereafter median levels of SHBG
`remained in the middle of
`the normal
`range for our
`laboratory, suggesting that the specific anti-oestrogen may
`have little effect on SHBG synthesis in the liver. A lack of
`efl‘ect of the compound on the liver is further suggested by
`evaluation of lipid changes. Tamoxifen is known to reduce
`levels of cholesterol and LDL cholesterol and is associated
`with an increase in triglycerides and HDL cholesterol
`consistent with an oestrogenic effect on the liver (Bertelli
`et al., 1988; Love et al. 1990). None of these changes
`reported during treatment with tamoxifen were observed
`during the present study. This would further suggest that
`ICI 182780 is peripherally selective with respect to the liver.
`However we cannot explain why there was not the expected
`changes in lipids as patients came off tamoxifen.
`The lack of apparent adverse effects of ICI 182780 seen in
`the present study would, if confirmed in future larger trials,
`give the specific anti-oestrogen potential advantages over
`currently available second-line endocrine agents. The ob-
`served lack of effect of ICI 182780 on body weight over a
`period of study that ranged from 6 to 16 months would give
`the new agent a potential advantage over megestrol acetate,
`the most widely used second-line endocrine therapy in breast
`cancer, which resulted in weight gain of > 5% of body weight
`in 25% of patients in one study (Willemse et al., 1990) and a
`median weight gain of 9 lbs after 180 days of treatment in
`another (Cruz et al., 1990). Overall, 83% of patients reported
`side-effects during treatment with megestrol acetate (Willemse
`et al., 1990).
`the
`tamoxifen,
`troublesome side-effects of
`The most
`current
`first-line endocrine therapy of choice,
`are the
`inception or exacerbation of hot flushes and sweats and the
`initiation of vaginal discharge. As already stated, ICI 182780
`did not induce or exacerbate hot flushes or sweats in the
`present study and furthermore did not cause symptoms of
`vaginal dryness or altered libido. Since the vaginal discharge
`experienced by 10-33% of patients during tamoxifen therapy
`is thought to be related to the oestrogenic activity of the
`drug, we expected ICI 182780 to be associated with vaginal
`dryness and irritation; the fact that this did not occur further
`suggests that ICI 182780 may be peripherally selective with
`respect to oestrogen target-site activity.
`thickness were
`Serial measurements of endometrial
`obtained from five patients during treatment with ICI
`182780. Before commencing treatment with the specific anti-
`oestrogen, the endometrial thickness in all five patients was
`greater
`than that
`found in the nonnal post-menopausal
`uterus. We assume that the overall ‘thickening’ we detected
`was related to previous tamoxifen therapy as this phenom-
`enon has been widely reported. During the relatively short
`duration of the present study, there was no further increase in
`thickening during treatment with ICI 182780. However there
`was also no decline in thickness as might be expected
`following treatment with a specific anti-oestrogen. Whether
`this finding reflected a true increase in endometrial thickness
`or an additional swelling of the myometrium, which has also
`been described in women taking tamoxifen (Cohen et al.,
`1994),
`is n