[CANCER RESEARCH 54, 408414, January IS, 1994]
`
`Investigation of a New Pure Antiestrogen (ICI 182780) in Women with Primary
`
`Breast Cancer1
`
`David J. DeFriend, Anthony Howell,2 Robert I. Nicholson, Elizabeth Anderson, Mitchell Dowsett, Robert E. Mansel,
`Roger W. Blamey, Nigel J. Bundred, John F. Robertson, Christobel Saunders, Michael Baum, Peter Walton,
`Frances Sutcliffe, and Alan E. Wakeling
`Departments of Surgery [D. J. 0., R. E. M., N. J. 8.], Medical Oncology [A. H.], and Tumour Biochemistry [E. A], Withington and Christie Hospitals, Manchester, UK;
`Department of Surgery, The City Hospital, Nottingham, UK IR. W B., J. E R]; Terwvus Institute for Cancer Research, Cardiffi UK (R. I. N.]; Departments of Surgery [C. S.,
`M. B.] and Biochemistry [M. D.], Royal Marsden Hospital, London, UK; and Zeneca Pharmaceuticals, Macclesfield, UK [R W., F. 3., A. E. W]
`
`ABSTRACT
`
`We have conducted a clinical trial of a novel pure antiestrogen, 7a-
`[9-(4,4,5,S,5-pentafluoropentylsulfinyl)nonyl}estra-1,3,S,(10)-triene-3,I7B-
`diol (ICI 182780), to assess its tolerance, pharmacokinetics, and short term
`biological effects in women with primary breast cancer. Fifty-six patients
`were randomized to either a control group (rt = 19), in which they received
`no preoperative treatment, or a treatment group (n = 37), in which they
`received daily i.m. injections of ICI 182780 at doses of 6 mg (n = 21) or 18
`mg (n = 16) for 7 days prior to primary breast surgery. Serum drug
`concentrations, gonadotropin levels, and sex hormone-binding globulin
`levels were measured during the study period by radioimmunoassay. Ex-
`pression of estrogen receptors (ER), progesterone receptors, the estrogen-
`induced protein p52, and the cell proliferation-related antigen Ki67 was
`determined immunocytochemically in pre- and poststudy tumor samples.
`Treatment with ICI 182780 caused no serious drug-related adverse
`events and had no effect on serum gonadotropin or sex hormone-binding
`globulin levels. Minor adverse events occurred in 5 patients receiving the
`6-mg dose and 3 patients receiving the 18-mg dose. The serum concentra-
`tion of ICI 182780 was dose dependent but showed variation between
`individuals. There was evidence of an approximately 3-fold drug accumu-
`lation over the short treatment period but steady state levels were not
`reached by the end of the 7 days. In patients with ER-positive tumors,
`treatment with ICI 182780 was associated with significant reductions in
`the tumor expression of ER (median ER index, 0.72 before versus 0.02
`after treatment; P < 0.001), progesterone receptor (median progesterone
`receptor index, 0.50 before versus 0.01 after treatment; P < 0.05), and Ki67
`(median Ki67 labeling index, 3.2 before versus 1.1 after treatment; P <
`0.05). Treatment with ICI 182780 also resulted in a significant reduction in
`p82 expression (P < 0.05) but this appeared unrelated to tumor ER status.
`In conclusion, ICI 182780 was well tolerated after short term admin-
`istration and produced demonstrable antiestrogenic effects in human
`breast tumors in viva, without showing evidence of agonist activity. These
`properties identify ICI 182780 as a candidate agent with which to evaluate
`whether a pure estrogen antagonist offers any additional benefit in the
`treatment of human breast cancer over conventional nonsteroidal anties—
`trogens, typified by tamoxifen, which exhibit variable degrees of agonist
`activity.
`
`INTRODUCTION
`
`Estrogen acts as an endocrine growth factor for at least one third of
`human breast cancers. Since endocrine therapy for breast cancer was
`initiated in 1896 by George Beatson (1), a variety of treatment mo—
`dalities have been introduced with the aim of preventing estrogen—
`mcdiated tumor growth (2). Anticstrogens achieve this directly by
`competing with estradiol for binding to the estrogen receptor, through
`which the intracellular effects of estrogens are mediated (3).
`Conventional nonsteroidal antiestrogens,
`typified by tamoxifen,
`compete efficiently for estrogen receptor binding but form a complex
`
`Received 7/9/93; accepted 11/10/93.
`The costs of publication of this article were defrayed in part by the payment of page
`charges. This article must therefore be hereby marked advertisement in accordance with
`18 U.S.C. Sedion 1734 solely to indicate this fact.
`1 Supported by financial assistance from Zencca Phannaceuticals (Macclesfleld, UK).
`2 To whom requests for reprints should be addressed, at Department of Medical
`Oncology, Christie Hospital, Wilmslow Road, Manchester, UK.
`
`with the receptor which retains some transcriptional activity (4). Con-
`sequently, tamoxifen exhibits a range of biological activity from full
`estrogen antagonism to partial agonism, depending upon the species,
`target tissue, and target gene response studied (5, 6).
`Although some of the clinical effects of the agonist (estrogenic)
`activity of tamoxifen, such as reduction of serum cholesterol (7—9)
`and maintenance of bone mineral density (10, 11), may benefit pa-
`tients receiving long term adjuvant treatment, others may be detri-
`mental. Tamoxifen stimulates endometrial growth in animals and its
`use as adjuvant therapy in women with breast cancer has been found
`to be associated with an increased incidence of endometrial carcinoma
`
`in some but not all studies (12—14). Moreover, there is evidence, from
`studies using an animal model of human breast cancer and from
`clinical observations of tumor responses following tamoxifen with-
`drawal at the time of disease progression in patients with advanced
`breast cancer, which suggests that the agonist activity of tamoxifen
`may eventually stimulate breast tumor growth and be a cause of some
`treatment failures (15—17). It is possible, therefore, that the therapeutic
`efficacy of tamoxifen and other conventional nonsteroidal antiestro-
`gens may be compromised, in comparison with that which might be
`achieved by complete estrogen antagonism.
`ICI 182780,3 a 7a-alkylsulfinyl analogue of cstradiol, is a novel
`steroidal anticstrogcn representative of a new class of estrogen an-
`tagonists, which differ significantly in both chemical structure and
`pharmacology from the conventional agents exemplified by tamoxi—
`fen. ICI 182780 is a potent pure antiestrogen; it has been shown to
`exhibit excellent growth-inhibitory effects in animal and in vitro mod-
`els of human breast cancer and appears to have no demonstrable
`intrinsic agonist activity (18). We have conducted a clinical trial to
`investigate the tolerance, pharmacokinetics, and short term biological
`effects of seven daily doses of a short-acting formulation of ICI
`182780 in postmenopausal women prior to surgery for primary breast
`cancer.
`
`PATIENTS AND METHODS
`
`Fifty-six postmenopausal women with primary breast cancer participated in
`the study between October 1991 and November 1992. Patients were considered
`eligible for the study if they were postmenopausal and had histologically or
`cytologically verified primary breast cancer with no overt evidence of metas-
`tases. Patients were excluded from the study if they were older than 75 years,
`or if they had a history of hepatic or renal impairment, insulin-dependent
`diabetes mellitus, or other conditions known to interfere with drug pharmaco-
`kinetics or steroid metabolism. Patients were also ineligible for the study if
`they weighed less than 40 kg or more than 110 kg, or if their baseline
`hematology, clinical chemistry, or urinalysis results were outside the range of
`normal values and considered to be clinically significant.
`
`3 The abbreviations used are: ICI 182780, 7a-[9-(4,4,5,5,5-pentafluoropentylsulfiny1)—
`nonyljcstra-1,3,5,(10)-triene»3,17B-diol; ER, estrogen receptor(s); PgR, progesterone re-
`ceptor(s); SHBG, sex hormone-binding globulin; FSH, follicle—stimulating hormone; LH,
`luteinizing hormone; LI, labeling index; RT, room temperature; PBS, phosphate-buffered
`saline; PAP, peroxidase—antiperoxidase; DAB, diaminobenzidine tetrahydrochloride; ICA,
`immunochemical assay.
`
`408
`
`Downloaded from cancerresaacrjournals.0rg on January 10, 2016. © 1994 American Association for Cancer
`Research.
`
`lnnoPharma Exhibit 1038.0001
`
`

`

`PURI: ANTIESTROGEN IN PRIMARY BREAST CANCER
`
`Study Design. After giving informed consent, patients were randomized to
`either a control group (n = 19),
`in which they received no preoperative
`treatment, or a treatment group (n = 37),
`in which they received daiiy i.m.
`injections of ICI 182780 at doses of 6 mg (n = 21) or 18 mg (n = 16) for 7
`days prior to surgery. Two additional patients, randomized to receive ICI
`182780, were Withdrawn from the study before starting treatment because of
`protocol violations. The study was approved by the ethical committees of the
`three participating clinical units.
`All patients randomized to receive ICI 182780 were visited on each treat-
`ment day by an investigator, who administered the trial medication and moni-
`tored local and systemic tolerance. ICI 182780 was administered by i.m.
`injection into the buttock as a short-acting formulation, containing 20 mg/ml
`drug in a propylene glycol-based vehicle. Blood samples were taken before and
`after the study period for measurement of complete blood C0unt, prothrombin
`time, clinical biochemistry, and serum levels of gonadotropins and SHBG.
`During the study period additional blood samples were taken, immediately
`prior to drug administration,
`from patients receiving treatment with ICI
`182780, for measurement of serum drug levels.
`Wherever possible, pretreatment tumor samples were obtained by multiple
`needle core biopsies. Post-treatment tumor samples were obtained from the
`operative specimen at the time of primary breast surgery. Tumor samples were
`divided between immediate snap freezing in liquid nitrogen, for assay of
`steroid hormone receptors and the proliferation—related antigen Ki67, and rou—
`tine fixation in formalin and paraffin wax embedding, for histological assess-
`ment and assay of the estrogen-regulated protein p82.
`Measurement of Serum Gonadotropin and Sex Hormone-binding
`Globulin Levels. Serum levels of gonadotropins (FSH and LH) and SHBG
`were measured by radioimmunoassay in the Regional Radioimmunoassay
`Laboratory of the University Hospital of South Manchester.
`Measurement of Serum Drug Levels. The concentration of ICI 182780 in
`serum samples was determined by radioimmunoassay after solvent extraction.
`Serum samples were added to diethyl ether:hexane (1:1, v/v) and vortex mixed
`for 15 min. After separation the extract was added to a CN Bond-elut column
`which had been preconditioned with methanol and etherzhexane (1:1, V/v).
`After washing of the column with dichloromethane:hexane (1:1, v/v), ICI
`182780 was eluted with methanol, which was reduced to dryness under nitro—
`gen at 50°C. The residue was reconstituted in gelatin PBS containing 0.05%
`Nonidet P-40, to which [3H]ICI 182780 and antisera were then added. The
`samples were incubated at 4"C for 21~24 h, after which a 1% charcoal
`suspension was added for 20 min before centrifugation of the samples at 2200
`rpm for 10 min at 4°C. The resulting supernatant was then added to Beckman
`protein scintillation cocktail and counted for tritium. Concentrations of ICI
`182780 were determined from a calibration curve constructed from calibration
`standards. The performance data for the assay were as follows: limit of detece
`tion, 0.5-1.0 ng/ml; intraassay coefficient of variation, 13%; interassay coef-
`ficient of variation, 15%.
`Immunocytochemistry. The expression of steroid hormone receptors, the
`estrogen-regulated protein p82, and the cell proliferation-related antigen Ki67
`was determined immunocytochemically in pre— and post-treatment
`tumor
`samples.
`Estrogen and Progesterone Receptor Expression. ER and PgR expres—
`sion was measured in snap-frozen tumor samples using commercially available
`kits (ER [CA and PgR ICA; Abbott Laboratories, Diagnostics Division, North
`Chicago, IL), which use a sensitive PAP technique for visualization of the
`receptors. Details of the staining procedure for ER and validity of the results
`have been described previously (19). An identical procedure for staining for
`PgR was followed, substituting the anti—PgR antibody supplied in the Abbott
`PgR lCA kit. Briefly, cryostat sections (5 pm) were mounted on slides treated
`with tissue adhesive and were fixed in 3.7% formaldehyde in 0.01 M PBS for
`15 min, followed by immersion in cold (~20°C) baths of methanol (5 min) and
`acetone (3 min). The slides were then rinsed in PBS and incubated with normal
`goat serum (blocking agent), to prevent nonspecific antibody binding, prior to
`application of the primary antibody for 30 min at RT. Binding of the primary
`antibody was revealed by the indirect PAP procedure (20). This technique uses
`successive applications of goat anti-rat lgG bridging antibody for 30 min at RT,
`rat PAP complexes for 30 min at RT, and finally a solution of the chromogenic
`substrate DAB, containing 0.06% (v/v) hydrogen peroxide, for 6 min. Slides
`were counterstained with hematoxylin, dehydrated, cleared, and mounted for
`examination by light microscopy.
`
`Ki67 Immunoreactivity. Kio7 immunoreactivity was measured as de-
`scribed previously (21). Briefly, cryostat sections (5 pm) were air dried over-
`night, fixed in acetone at —20°C, and allowed to air dry for an additional 2 h.
`Endogenous peroxidase activity was blocked by incubation with 0.3% (v/v)
`hydrogen peroxide in PBS for 15 min at RT. Slides were then washed in PBS
`and incubated with 10% (v/v) normal rabbit serum in PBS before application
`of the mouse monoclonal antibody Ki67 (Dako Laboratories, Glostrup, Den-
`mark) at a 1:40 dilution for 45 min at RT. Binding of the primary antibody was
`visualized by successive applications of a rabbit anti-mouse bridging antibody
`(1:25 dilution in 10%, v/v, decomplemented human serum in PBS) for 30 min
`at RT, mouse PAP complexes (1:100 in PBS) for 30 min at RT, and finally the
`chromogenic substrate DAB. Slides were counterstained with hematoxylin,
`dehydrated, cleared, and mounted for examination by light microscopy.
`p52 Expression. Expression of p82 was measured in formalin-fixed, par-
`affin—embedded, tumor specimens. Paraffin sections (5 pm) were dewaxed in
`xylene and rehydrated in stepwise dilutions of ethanol. Endogenous peroxidase
`activity was blocked with 10% (v/v) hydrogen peroxide in PBS for 15 min,
`after which the sections were rinsed in PBS and incubated with normal rabbit
`serum for 5 min prior to incubation with a mouse monoclonal anti-p52 anti-
`body (22), at a dilution of 1:400, for 2 h at RT. Binding of the primary antibody
`was visualized by successive applications of biotinylated rabbit anti—mouse
`immunoglobuiins (1:200) for 45 min at RT, streptavidin~horseradish peroxi-
`dase complex for 30 min, and finally the chromogenic substrate DAB. Slides
`were counterstained with hematoxylin, dehydrated, cleared, and mounted for
`examination by light microscopy.
`Evaluation of Staining. In each assay, all specimens had a negative control
`slide (no primary antibody) of an adjacent section to assess the degree of
`nonspecific staining; in addition, a positive control slide of a section from a
`tumor known to express the appropriate antigen was also included. The speci-
`mens were evaluated under a light microscope initially at an ocular magnifi-
`cation of X10, to permit localization of the malignant areas within each section
`and assessment of the heterogeneity of immunostaining within the tumor
`components. All subsequent evaluations were performed using an ocular mag-
`nification of X40.
`
`Estrogen and progesterone receptor expression was assessed semiquantita-
`tively by determining the percentage of tumor cells stained by the primary
`antibody (minimum of 1000 tumor cells evaluated) and assessing the intensity
`of staining using a score of 0 to 3, corresponding to negative, weak, interme-
`diate, and strong staining intensities. The percentage of tumor cells in each of
`these categories was used to calculate an index (I) for each tumor, as follows:
`1 : [(% cells showing an intensity value of 1 X 1) + (% cells showing an
`intensity value of 2 X 2) + (% cells showing an intensity value of 3 X 3)]:‘100.
`Expression of pSZ and the Ki67«related antigen was evaluated by determin-
`ing the percentage of tumor cells stained by the primary antibody in multiple
`representative fields (minimum of 1000 tumor cells evaluated). Assessment of
`staining intensity was not attempted.
`Definition of Steroid Receptor Status. For the purposes of this study,
`tumors were classed as ER/PgR positive or negative according to receptor
`expression in the prestudy tumor sample. Tumors with an ER/PgR index of
`20.05 were considered receptor positive. In cases where ER/PgR expression
`was not evaluable in a prestudy tumor sample, the receptor status was classed
`as unknown.
`
`the
`Statistical Analysis. With the exception of serum drug level data,
`results have been analyzed using nonparametric statistics. Comparison of pre-
`and post-treatment samples within specified patient groups has been performed
`using Wilcoxon’s matched—pairs signed-rank test. Differences between speci-
`fied patient groups have been analyzed using the Mann-Whitney U test (for
`analyses involving two specified groups) or the Kruskal-Wallis test (for analy-
`ses of three or more specified groups). The null hypothesis was rejected at a
`probability level (P) of $0.05.
`
`RESULTS
`
`Patient Demography. The details of patients in the three treatment
`groups are summarized in Table 1. The three groups were well
`matched with respect to patient age, tumor size, T stage, and axillary
`lymph node status. However, the group treated with ICI 182780 at the
`
`409
`
`Downloaded from cancerresaacrjournals.0rg on January 10, 2016. © 1994 American Association for Cancer
`Research.
`
`InnoPharma Exhibit 1038.0002
`
`

`

`PURE ANTIESTROGEN IN
`
`PRIMARY BREAST CANCER
`
`prior to undergoing wide local excision of her primary breast tumor,
`from which she made an uneventful postoperative recovery. Because
`of concern about satisfactory tumor clearance, the patient underwent
`a second operation 3 weeks later (i.e., 21 days after completing
`treatment with ICI 182780). The surgery was again uneventful but the
`patient collapsed 2 days postoperatively with a fatal pulmonary em-
`bolus. There had been no changes in the patient’s platelet count or
`coagulation parameters following treatment with ICI 182780 and in
`the view of the surgeon concerned this event was unrelated to the use
`of the drug.
`Pharmacokinetics. The mean serum drug levels versus time
`achieved in patients receiving the 6-mg and 18-mg daily doses of ICI
`182780 are shown in Fig. 1. The serum concentration of ICI 182780
`was dose dependent but showed some variation between individual
`patients. An approximately 3-fold drug accumulation was seen over
`the 7—day dosing period, although steady state serum levels were not
`reached by the end of the study.
`Endocrinology. Paired pre- and poststudy endocrinology measure-
`ments were available for 33 (59%) of the 56 subjects. No significant
`changes in the serum levels of LH, FSH, or SHBG were seen in any
`of the treatment groups during the study period (data not shown).
`Steroid Receptor Expression. Paired pre- and poststudy measure—
`ments of ER and PgR expression were available for 45 (80%) of the
`56 subjects, of which 28 (62%) were ER positive and 20 (45%) were
`PgR positive, as measured in prestudy tumor samples. Among patients
`receiving the 6-mg dose of ICI 182780, 40% had ER-positive tumors,
`compared to 86% of patients receiving the 18-mg dose and 63% of
`controls. Although these differences were not statistically significant,
`they could potentially have affected the distribution of other ER—
`related parameters within the treatment groups.
`Among the ER-positive tumors, there were no significant differ-
`ences between the treatment groups with respect to the prestudy ER or
`PgR values. 0f the 16 tumors identified as ER negative in prestudy
`needle core biopsy specimens, 2 were identified as weakly ER posi—
`tive in poststudy samples (ER index values of 0.05 and 0.15) and 2
`were identified as PgR positive (PgR index values of 0.55 and 0.70).
`ER Expression. Among the patients in the control group with
`ER-positive tumors, there was a significant tendency for the prestudy
`needle core biopsy sample to underestimate the level of ER expres-
`sion, as measured in the poststudy surgical tumor specimen (median
`pre- and poststudy ER indices of 0.50 and 0.95, respectively; P < 0.05,
`Wilcoxon matched-pairs signed-rank test) (Fig. 2A). Despite this find-
`
`7g /%2%a S
`
`tudy Day
`
`Serum
`[ICI 182780]
`(no/ml)
`
`30
`
`25
`
`2°
`
`15
`
`10
`
`Fig. 1. Serum concentrations of ICI 182780 during the 7—day treatment period. Col-
`umns, means; bars, 2 SEM. Patients received daily i.m. injections of 6 mg (I) or 18 mg
`(a) ICI 182780. Numbers below columns, number of drug level measurements available
`on individual treatment days.
`
`410
`
`Table 1 Patient characteristics
`Treatment group
`ICI 182780,
`lCI 182780,
`
`Control
`6 mg/day
`18 mg/day
`n
`19
`21
`16
`
`Age (years)
`Median
`Range
`
`Tumor size (cm)
`Median
`Range
`Tumor T stage
`T2
`T3
`
`Histological type
`[LC
`[DC
`
`Histological grade
`I
`[l
`Ill
`
`Axillary lymph node status
`Positive
`Negative
`Not known
`
`ER status
`Positive
`Negative
`Not known
`
`61
`51—74
`
`2.2
`0.55.5
`
`18 (95%)
`1 (5%)
`
`66
`49—74
`
`2.5
`1.5—5.0
`
`17 (81%)
`4 (19%)
`
`1 (6%)
`17 (94%)
`
`2 (10%)
`18 (90%)
`
`6 (35%)
`4 (24%)
`7 (41%)
`
`10 (59%)
`7 (41%)
`2
`
`10 (63%)
`6 (37%)
`3
`
`2 (11%)
`5 (28%)
`11 (61%)
`
`9 (45%)
`11 (55%)
`1
`
`6 (40%)
`9 (60%)
`6
`
`63
`54~71
`
`2.3
`0.7—4.5
`
`15 (94%)
`1 (6%)
`
`4 (25%)
`12 (75%)
`
`5 (42%)
`5 (42%)
`2 (16%)
`
`8 (53%)
`7 (47%)
`1
`
`12 (86%)
`2 (14%)
`2
`
`PgR status
`9 (64%)
`4 (25%)
`7 (44%)
`Positive
`5 (36%)
`12 (75%)
`9 (56%)
`Negative
`
`
`
`3 5Not known 2
`
`6-mg dose contained a greater proportion of histological grade III,
`steroid receptor-negative tumors than did the control and 18—mg dose
`groups.
`Drug Tolerability. Treatment with ICI 182780 caused no serious
`drug—related adverse events, and no patients were withdrawn from the
`study because of drug toxicity. Minor systemic adverse events were
`reported by 5 patients receiving the 6-mg dose and 3 patients receiving
`the 18-mg dose (Table 2). The majority of these events were consid-
`ered to be unrelated to the trial
`treatment,
`in the opinion of the
`investigating clinician. In addition, the short-acting formulation of ICI
`182780 used in this study was well tolerated locally at the site of
`injection, with only 1 patient developing any significant local reaction.
`No clinically significant changes in the laboratory parameters meas—
`ured (biochemical profile, complete blood count, prothrombin time,
`and urinalysis) were seen in patients receiving ICI 182780.
`One death occurred among the patients randomized to receive ICI
`182780. The patient received 7 days of treatment at the 18-mg dose
`
`Table 2 Adverse events reported for patients treated with ICI 182780
`
`
`Dose (mg)
`Adverse event
`Duration
`6
`Headache
`6 h
`6
`Dyspepsia
`6
`Headache
`6
`Headache
`6
`Headache
`Hypertension
`
`3 h
`5 h
`3 h
`16 h
`
`Severity
`Mild
`Moderate
`Moderate
`Mild
`Mild
`Moderate
`
`Relationship
`to drug
`No opinion
`Probably unrelated
`Probably unrelated
`Probably unrelated
`Probably unrelated
`Definitely unrelated
`
`18
`
`18
`18
`
`Facial swelling
`Vaginal spotting
`Hyperglycemia
`Headache
`Headache
`
`2.5 h
`
`No opinion
`Mild
`No opinion
`Mild
`Probably unrelated
`Moderate
`5 h
`Probably related
`Moderate
`
`1 day Definitely unrelated Moderate
`
`
`Downloaded from cancerresaacrjournals.org on January 10, 2016. © 1994 American Association for Cancer
`Research.
`
`InnoPharma Exhibit 1038.0003
`
`

`

`PURE ANTIESTROGEN IN PRIMARY BREAST CANCER
`
`A)
`
`ER
`index
`
`2.0
`
`1.5
`
`1.0
`
`0.5
`
` 0.0
`Pre
`Post
`Pre
`Post
`Controls
`ICI 182780
`
`B)
`
`2.0
`
`PgR
`index
`
`1.5
`
`1.0
`
`0.5
`
`0.0
`
`Pre
`Post
`Controls
`
`Pre
`Post
`ICI 182780
`
`Fig. 2. Pre- and poststudy tumor ER expression (A) and PgR expression (B) in controls
`and treated patients with ER-positive tumors. Columns, interquanile ranges; bars, medi-
`ans.
`
`ing, treatment with ICI 182780 caused a significant reduction in the
`median ER index of ER-positive tumors, from 0.73 before treatment
`to 0.02 after treatment (P < 0.001). When the patients receiving ICI
`182780 were further subdivided according to the dose received, sig—
`nificant reductions in the median ER indices of ER—positive tumors
`were evident at both the 6-mg and 18-mg dose levels, decreasing from
`0.60 to 0.06 in the 6-mg dose group (P < 0.05) and from 0.73 to 0.01
`in the 18—mg group (P < 0.01).
`PgR Expression. There was a similar, although nonsignificant,
`tendency for the prestudy needle core biopsy specimen to underesi-
`mate the expression of PgR in untreated controls with ER-positive
`tumors (median pre— and poststudy PgR indices of 0.7 and 1.3, re-
`spectively; P = 0.07, Wilcoxon matched-pairs signed—rank test) (Fig.
`23). In patients with ER-positive tumors treated with ICI 182780,
`there was a significant reduction in the median PgR index, from 0.50
`before treatment to 0.01 after treatment (P < 0.05). The reduction in
`PgR expression did not achieve statistical significance when the ef-
`fects of individual dose levels of ICI 182780 were analyzed separately.
`pSZ Expression. Paired pre- and poststudy measurements of p82
`were available for 37 (66%) of the 56 patients (Fig, 3). There were no
`significant differences between the treatment groups with respect to
`the prestudy level of p82 expression (median p82 expression of 24%
`for controls versus 7% for treatment group; P = 0.76, Mann-Whitney
`U test) and there was no significant change in p82 expression between
`the pre- and poststudy tumor samples in the control group. Treatment
`
`with ICI 182780 resulted in a significant reduction in p82 levels
`(median pre- and post-treatment p82 expression of 7% and 1%, re-
`spectively; P < 0.05, Wilcoxon matched—pairs signed-rank test). How—
`ever,
`there was no relationship with increasing drug dose and no
`difference according to tumor ER status prior to treatment.
`Ki67 Labeling Index. Paired pre— and post-treatment measure-
`ments of the Ki67 LI were available for 44 (79%) of the 56 subjects
`(Fig. 4). There were no significant differences between the treatment
`groups with respect to the prestudy tumor Ki67 LI. Among invasive
`ductal carcinomas, there was a trend for the pretreatment Ki67 L1 to
`increase with increasing histological grade. This increase was signifi—
`cant between grades I and III (median Ki67 LI of 1.8% versus 5.5%,
`respectively; P < 0.05, Mann-Whitney U test). In addition, the median
`Ki67 LI was higher among ER-negative than ER-positive tumors
`(6.0% versus 3.6%) but this difference was not statistically significant
`(P = 0.7; Mann-Whitney U test).
`Following treatment with ICI 182780 there was a significant re-
`duction in the median Ki67 LI of ER-positive tumors, from 3.2%
`before treatment
`to 1.1% after treatment (P < 0.05%; Wilcoxon
`matched-pairs signed—rank test). However, there were no significant
`changes in the Ki67 LI of ER-negative or control tumors. When the
`ER-positive tumors were further subdivided according to the dose of
`ICI 182780 received, a significant reduction in the Ki67 LI was
`evident only with patients treated with the 18—mg daily dose, for
`whom the median Ki67 LI decreased from 4.0% before treatment to
`
`1.1% after treatment (P < 0.05). With patients receiving the 6—mg dose
`
`100
`
`p32
`expression 30
`(°/o cells
`stained)
`
`60
`
`40
`
`20
`
`0
`
`
`
`Post
`Pre
`Control
`
`Post
`Pre
`ICI 182780
`
`Fig. 3. Pre- and poststudy tumor p82 expression in controls and treated patients
`irrespective of ER status. Columns, interquartile ranges; bars, medians.
`
`10
`
`Ki 67 LI
`("/0)
`
`
`
`Post
`Pre
`Control
`
`Post
`Pre
`ICI 182780
`
`Fig. 4. Pre- and poststudy tumor Ki67 L1 in controls and treated patients with ER«
`positive tumors. Columns, interquartile ranges; bars, medians.
`
`411
`
`Downloaded from cancerres.aacrjournals.0rg on January 10, 2016. © 1994 American Association for Cancer
`Research.
`
`InnoPharma Exhibit 1038.0004
`
`

`

`PURE AN'I‘IES'I'ROGEN IN
`
`PRIMARY BREAST CANCER
`
`there was a smaller and nonsignificant decrease in the Ki67 LI, from
`l.8% before treatment to 0.8% after treatment (P = 0.3).
`
`DISCUSSION
`
`This study is the first investigation of short term administration of
`ICI 182780 to women with primary breast cancer. We have found the
`short-acting formulation of ICI 182780 used in this study to be well
`tolerated at the two doses studied over a 7-day treatment period and to
`be associated with minimal local or systemic toxicity. These findings
`are in agreement with earlier studies in healthy human volunteers,
`who received single doses of 2—60 mg of the short-acting formulation
`of ICI 182780 by i.m. injection.4 The two drug dose levels used in the
`present study were selected to be within the expected dose range
`required to produce antiestrogenic efficacy and were derived from the
`results of previous studies in primates.4
`The most frequent adverse event observed in the present study was
`mild to moderate headache. The absence of comparable adverse event
`data from the control group makes it difficult to assess whether these
`events were drug related. This difficulty is compounded because the
`patient population in this study included women who had only re-
`cently been informed of their diagnosis of breast cancer and of their
`need for breast surgery and who might therefore be expected to exhibit
`a number of stress-related symptoms, including headaches. A daily
`placebo injection would have been a better comparison for the control
`group but was rejected prior to the study was started, on ethical
`grounds.
`The adverse events observed in this study were mostly considered
`to be unrelated to administration of 1C] 182780, in the opinion of the
`attending physician. However, similar adverse events, particularly
`headaches, were also reported in healthy volunteer studies with the
`same short-acting drug formulation.4 It
`is possible,
`therefore, that
`these adverse events were related either to the drug itself or to the
`propylene glycol-based vehicle used in the short-acting formulation.
`This question will be addressed in future studies which are planned
`with a different, long-acting, formulation of ICI 182780 contained in
`a castor oil-based vehicle.
`
`More serious toxicity has not been seen in any of the previous
`human volunteer or animal studies, and in particular there has been no
`evidence of altered coagulation or thrombogenicity after treatment
`with ICI 182780, even when it was administered at much higher doses
`than were used in the present study. It therefore seems unlikely that the
`fatal pulmonary embolus that occurred in a patient who received the
`18-mg dose of ICI 182780 was drug related. However, since such
`complications are rare following breast operations, it is important that
`blood thrombogenicity be appropriately monitored in future studies
`with ICI 182780. Conventional nonsteroidal antiestrogens
`like
`tamoxifen do have a potentially thrombogenic effect via reduction of
`antithrombin 111 levels (7, 23—25), although this effect has not been
`shown to be clinically significant and has been attributed to the
`agonist activity of these drugs, which does not appear to be shared by
`ICI 182780 (18). However, the pure antagonist profile of activity of
`ICI 182780 in human subjects will need to be confirmed in future
`clinical studies.
`
`Measurement of serum drug levels in this study showed that there
`was some interpatient variation in the serum concentration of ICI
`182780 achieved. The mean pre-dose concentration was dose depen-
`dent and increased over the 7-day dosing period, showing an approxi—
`mately 3-fold accumulation. However, the serum concentrations of
`ICI 182780 failed to reach a plateau by day 7, indicating that steady
`state drug levels were not achieved by the end of the study period.
`
`4 Zeneca Pharmaceuticals, unpublished observations.
`
`412
`
`Animal studies have demonstrated considerable interspecies variabil-
`ity in the elimination half—life of ICI 182780, with a half-life of about
`4 h in rats and 2 days in dogs after i.m. administration.4
`No attempt was made in the present study to assess the distribution
`and metabolism of ICI 182780. Animal studies have shown that the
`
`drug is rapidly released from the injection site and is distributed to
`most tissues, with the exception of the brain and spinal cord, within 2
`h after i.m. dosing. All species studied produce a large number of
`metabolites, which have yet to be identified. The principal route of
`drug elimination appears to be in bile.4
`The maximum serum drug levels achieved in this short term study
`were on the order of 10 nM, which is 100 times lower than the typical
`serum level of tamoxifen achieved with standard doses of 20 mg p.o.
`daily. However, because the ER binding affinity of ICI 182780 is
`approximately 100 times