0013-7227/98/$03.00/0
`Endocrinology
`Copyright © 1998 by The Endocrine Society
`
`Vol. 139, No. 5
`Printed in USA.
`
`Comparison of the Effects of the New Orally Active
`Antiestrogen EM-800 with ICI 182 780 and Toremifene
`on Estrogen-Sensitive Parameters in the
`Ovariectomized Mouse
`
`CELINE MARTEL, CLAUDE LABRIE, ALAIN BELANGER, SYLVAIN GAUTHIER,
`YVES MERAND, XUN LI, LOUIS PROVENCHER, BERNARD CANDAS, AND
`FERNAND LABRIE
`
`Laboratory of Molecular Endocrinology, CHUL Research Center, Quebec, Canada G] V 4G2
`
`ABSTRACT
`inhibitory effect on this parameter under the experimental conditions
`used. Comparable effects were observed when estrogen receptor pro-
`The nonsteroidal antiestrogen EM-800 is approximately 10-fold
`tein levels were measured by enzyme immunoassay. After oral ad-
`more potent than ICI 182 780, the most potent known steroidal an-
`ministration, EM-800 exerted maximal 83% and 88% inhibitions of
`tiestrogen, at inhibiting estrone—stimulated uterine weight in ovari-
`uterine and vaginal weight, respectively, whereas maximal inhibi-
`ectomized mice (half-maximal inhibitory daily sc doses of 0.2 and 2.0
`tions limited to 51% and 67% were achieved with toremifene. This
`ug, respectively). At maximal doses, however, both compounds lead
`to a similar maximal 90% inhibition of estrone-stimulated uterine
`limited inhibition by toremifene ofthe stimulatory effect of estrone on
`uterine and vaginal weight is probably due to the intrinsic estrogenic
`weight. A 10-fold higher activity of EM-800 compared with ICI 182
`activity of the compound. The present data also show that the ste-
`780 was also observed on estrone-stimulated vaginal weight, with
`roidal antiestrogen ICI 182 780 has less than 3% the activity of
`maximal inhibitions of 96% and 90%, respectively, achieved by the
`EM-800 when administered by the oral route. In fact, EM-800 ad-
`two compounds. In addition, EM-800 injected sc or administered
`ministered orally is 2- to 3-fold more potent than ICI 182 780 injected
`orally led to a marked loss of uterine and vaginal estrogen receptor
`sc. (Endocrinology 139: 2486—2492, 1998)
`levels measured by binding assay, whereas ICI 182 780 exerted no
`
`
`STROGENS are recognized to play the predominant role
`in breast cancer development and growth (1). As the
`first step in the action of estrogens in target tissues is binding
`to the estrogen receptor (ER), a logical approach for the
`treatment of estrogen-sensitive breast cancer is the use of
`antiestrogens, or compounds that block the interaction of
`estrogens of all sources with the ER. Tamoxifen, the only
`antiestrogen widely used for the treatment of breast cancer
`in women, behaves as a mixed agonist/ antagonist of estro-
`gen action (2), thus potentially limiting its therapeutic po-
`tential for the treatment of breast cancer in women.
`
`(3—5), 113-
`Recently, 7a-alkyl derivatives of estradiol
`amidoalkoxyphenyl estradiols (6), or estradiol 7a-alkyl de-
`rivatives possessing additional changes designed to increase
`their affinity for the ER and / or increase their bioavailability
`(7—1 1) have been synthesized and shown to possess pure and
`potent antiestrogenic activity in the most rigorous in vitro
`and in viva systems (3, 11), including human breast cancer
`cells in vitro (3, 7, 9, 10) and in vivo in nude mice (4, 8, 12, 13).
`These 7a-alkyl and 11 B-amidoalkoxyphenyl derivatives of
`estradiol, however, have low oral bioavailability, thus lim-
`iting their acceptability for the treatment of breast cancer. We
`have thus developed a series of even more potent estrogen
`antagonists that possess high oral bioavailability in the
`
`mouse, rat, monkey, and human. The present study com-
`pares the characteristics of the new antiestrogen EM-800
`(14 —17) on well characterized estrogen-sensitive parameters
`in the mouse with the effects of lCl 182 780 (4, 12) and
`toremifene, an analog of tamoxifen (18—22).
`
`Materials and Methods
`
`Animals
`
`Female BALB/c mice (BALB/cAnNCrlBR), approximately 50 days
`old and weighing 18—20 g, were obtained from Charles—River (St. Con—
`stant, Canada) and housed 4—5 / cage in a temperature (23 i 1 C)— and
`light (12 h light / day, lights on at 0715 h)—controlled environment. The
`mice were fed rat chow and tap water ad libitum. The animals were
`ovariectomized (OVX) under general anesthesia (Avertin) Via bilateral
`flank incisions and randomly assigned to groups of 9—10 animals. Ten
`mice were kept intact as controls.
`
`Chemicals
`
`EM—800 [( + )—7—pivaloyloxy—3—(4 ’—pivaloyloxyphenyl)—4—methyl-2—(4"—
`( ’”—piperidinoethoxy)phenyl)—2H—benzopyran]
`(14) and lCl 182 780
`were synthesized in the medicinal chemistry division of our laboratory.
`Both compounds analyzed under Good Laboratory Practice (GLP) con—
`ditions were more than 99% pure. Toremifene citrate was provided by
`Orion Corp. (Farmos, Finland). The structures of these antiestrogens are
`illustrated in Fig. 1.
`
`Treatments
`
`Received June 27, 1997.
`Address all correspondence and requests for reprints to: Prof. Fer—
`nand Labrie, Laboratory of Molecular Endocrinology, CHUL Research
`Center, 2705 Laurier Boulevard, Quebec, Canada G1V 4G2.
`
`For the experiment described in Figs. 2—5, EM—800 and lCl 182 780
`were injected sc once daily at different doses (0.1, 0.3, 1.0, 3.0, or 10
`ng/injection), whereas in Figs. 6—9, EM—800, lCl 182 780 and toremifene
`were administered orally by gavage once daily at a dose of 0.3, 1.0, 3.0,
`
`2486
`
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`
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`
`

`

`COMPARATIVE ANTIESTROGENIC ACTIVITY OF EM-SOO
`
`2487
`
`
`
`
`
`HO
`
`”0" (CH219SO(CH2)3CQF5
`ICI 182780
`
`O
`/ O
`
`'
`/—/N—
`O
`
`G O
`
`Toremjfene
`
`FIG. 1. Chemical structures of antiestrogens.
`
`EO)
`
`s 140
`1:
`,g
`120
`
`.EM-800
`.ICI182780
`
`ER assays
`
`Binding assay. Preparation of cytosol: Vaginal and uterine tissues were
`minced and homogenized at 4 C with two 10—sec bursts of a Polytron PT
`10—ST (Brinkmann Instruments, Westbury, NY) in 20 vol buffer A (25 mM
`Tris—HCl, 1.5 mM EDTA disodium salt, 10 mM a—monothioglycerol, 10%
`glycerol, and 10 mM sodium molybdate, pH 7.4). For each group, three
`different homogenates were used, and each homogenate was prepared
`from a pool of two or three uteri or vaginas. The homogenates were then
`centrifuged at 105,000 X g for 60 min at 4 C. The steroid binding assay
`was performed with freshly prepared cytosol. The protein concentration
`of cytosol was determined using the method of Bradford (23) with BSA
`as standard.
`
`Estrogen binding assay: [2,4,6,7—3H]Estradiol (SA, 87 Ci / mmol) was
`purchased from New England Nuclear (Boston, MA), whereas dieth—
`ylstilbestrol was obtained from Sigma Chemical Co. (St. Louis, MO).
`[3H]Estradiol binding was measured using the dextran—coated charcoal
`absorption technique, essentially as previously described (24 —27). In
`brief, 0.2—ml aliquots of the cytosol preparation were incubated with 0.1
`ml [3H]estradiol (final concentration, 3—5 nM) in the presence or absence
`of a 100fold excess of diethylstilbestrol for 3 h at room temperature.
`Unbound steroids were separated by incubation for 15 min at 4 C with
`0.3 ml of 0.5% Norit-A and 0.05% dextran T—70 in buffer B (1.5 mM EDTA
`disodium salt, 10 mM a—monothioglycerol, and 10 mM Tris—HCl, pH 7.4)
`and centrifugation at 3000 X g for 15 min. Aliquots of the supernatant
`(0.3 ml) were then taken for radioactivity measurement after the addition
`of 10 ml liquid scintillation cocktail.
`
`Enzyme immunoassay (ER-ETA). Preparation of cytosol: Cytosols were pre—
`pared as described for the binding assay, except that vaginal tissue was
`homogenized in 10 vol of a different buffer (10 mM Tris—HCl, 1.5 mM
`EDTA disodium salt, 1 mM a—monothioglycerol, and 5 mM sodium
`molybdate, pH 7.4). For each group, only one homogenate prepared
`from a pool of three vaginas was used.
`ER—EIA assay: The ER—ElA monoclonal kit from Abbott Laborato—
`ries (Diagnostics Division, North Chicago, IL) used for this assay is
`an enzyme immunoassay based on the sandwich principle, using
`monoclonal rat antibodies. A detailed description of the method used
`is given in the instruction manual supplied with the kit. In brief,
`cytosols (100 pil) were incubated at 4 C for 18 h with antibody (rat
`anti—ER; D547)—coated polystyrene beads that bind ER proteins. A
`second antibody (rat anti—ER; H222) conjugated to horseradish per—
`oxidase was then incubated with the beads at 37 C for 1 h to label the
`
`bound receptor. A further incubation (30 min at room temperature)
`of the beads with the enzyme substrate hydrogen peroxide and the
`color reagent / electron donor o—phenylenediamine-ZHCI produced a
`phenazine polymer chromophore that was measured at 492 nm. Each
`incubation step was completed by washing the beads with distilled
`water, and the enzyme reaction was stopped by the addition of 1 N
`sulfuric acid. A standard curve was obtained by plotting the ER
`concentration of ER standards (0 and dilutions of 500 meI/ ml ER
`stock, supplied in the Abbott kit) vs. their absorbance.
`
`Statistical analysis
`
`The effects of drugs and doses were analyzed using a two—factor
`nested ANOVA model. When main effects were significant (P < 0.05),
`a posteriori pairwise comparison between the control and treated groups
`was performed with Fisher’s least significant difference test
`(LS—
`MEANS), requiring P < 0.01 to declare significance. Original scale val—
`ues were used to analyze the variables with normally distributed re-
`siduals and respecting the hypothesis of homoscedasticity. If not, a log
`or a rank transformation was applied to the measurements before anal—
`ysis. The two routes of administration (sc and oral) were analyzed
`separately using a common intact group. All data are presented as the
`mean : SEM.
`
`Results
`
`To assess the relative efficacy of EM-800 and the steroidal
`antiestrogen ICI 182 780 under optimal conditions of bio-
`availability, the two compounds were first injected sc daily
`for 9 days, starting 2 days after ovariectomy and 3 days
`
`E, 100
`v so
`5’
`m
`E
`g
`a
`5
`a
`g
`
`60
`40
`—
`20—
`D -
`
`..
`
`lNTACT
`
`u
`H
`CONT
`
`
`
`
`
`g
`a
`7
`g
`7
`/
`7
`/
`
`com
`
`0.1
`
`
`ANTIESTROGEN (ug, s.c., ID)
`
`ESTRONE (0.0m s.c., BID)
`
`OVARIECTOMIZED
`
`FIG. 2. Effect on uterine weight of increasing concentrations of EM-
`800 and ICI 182 780 injected sc for 9 days to ovariectomized mice
`simultaneously treated With estrone. **, P < 0.0001 vs. estrone-
`treated control.
`
`or 10.0 Mg / animal. Treatment with the antiestrogens was initiated 2 days
`after ovariectomy, whereas treatment with estrone (0.06 Mg, sc, twice
`daily) was started 3 days later (5 days postovariectomy). Thereafter,
`estrone and antiestrogens were administered in combination for a 6—day
`period. Compounds were dissolved in a 50:50 (vol/vol) mixture of
`polyethylene glycol 600 and ethanol and administered in a 1% (wt /vol)
`gelatin-0.9% NaCl solution (final concentration of polyethylene glycol
`600/ ethanol was 8%). Mice in the intact and OVX control groups re—
`ceived the vehicle alone during the 9—day period. The animals were
`killed by cervical dislocation on the 11th morning after ovariectomy. The
`uteri and vagina were rapidly dissected and weighed, and then frozen
`in liquid nitrogen and stored at —80 C.
`
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`
`AstraZeneca Exhibit 2159 p. 2
`
`

`

`2488
`
`COMPARATIVE ANTIESTROGENIC ACTIVITY OF EM-800
`
`Endo I 1998
`Vol 139 - No 5
`
`estradiol receptors from 426 i 31 to 128 i 14 fmol/mg
`protein (P < 0.0001) was observed, whereas further inhibi-
`tion was observed at the 3.0- and 10-ug doses, thus decreas-
`ing uterine ER levels to 46 i 3 fmol/ mg protein (89% re-
`duction; P < 0.0001) and 9 i 3 fmol/mg protein (98%
`reduction; P < 0.0001), respectively. ICI 182 780, on the other
`hand, had no significant effect on the same parameter at the
`0.3-, 1.0-, and 3.0-pLg daily doses, whereas a 65% stimulation
`of uterine ER levels (705 i 105 fmol/mg protein; P < 0.001)
`was observed at the highest dose used (10 Mg). It can also be
`seen in Fig. 4 that ovariectomy increased uterine ERs from
`418 i 6 fmol/mg protein in intact animals to 1235 i 163
`fmol/ mg protein (P < 0.0001 US. intact) in ovariectomized
`rats, an increase that was completely reversed by the ad-
`ministration of estrone to ovariectomized animals.
`
`before treatment with estrone. As illustrated in Figs. 2 and 3,
`EM-800 was approximately 10-fold more potent than ICI 182
`780 in blocking the stimulatory effect of estrone (0.06 prg, sc,
`twice daily) on uterine (Fig. 2) and vaginal (Fig. 3) weight,
`respectively. The P values associated with the treatment and
`dose (treatment) nested effects are reported in Table 1 for
`both oral and sc routes. In fact, at the lowest daily dose used
`(0.1 ug or ~0.005 mg/ kg), EM-800 caused a 33% decrease
`(P < 0.0001) in uterine weight, whereas doses of 0.3, 1.0, 3.0,
`and 10 pug caused respective 72%, 84%, 91%, and 87% de-
`creases (P < 0.0001 for all doses 05. control) of estrone-
`stimulated uterine weight. ICI 182 780, on the other hand,
`had no significant effect at the two lowest doses used and
`caused 38%, 75%, and 90% decreases in uterine weight at
`doses of 1.0, 3.0, and 10 Mg, respectively (P < 0.0001 at all
`doses 275. control).
`A similar 10-fold higher activity of EM-800 compared with
`ICl 182 780 was observed on estrone-stimulated vaginal
`weight (Fig. 3). The daily 0.1-pLg dose of EM-800 caused a 37%
`(P < 0.0001) inhibition of estrone-stimulated vaginal weight,
`whereas doses of 0.3, 1.0, 3.0, and 10 pug caused 66%, 91%,
`98%, and 96% (P < 0.0001 for all groups vs. control) inhibi-
`tions of vaginal weight, respectively. ICI 182 780, on the other
`hand, had no detectable effect at
`the two lowest doses,
`whereas the 1.0-, 3.0-, and 10-pLg daily sc doses caused re-
`spective 32%, 53%, and 90% inhibitions of estrone-stimulated
`vaginal weight (P < 0.0001 for all of these groups vs. control).
`As illustrated in Fig. 4, treatment with EM-800 caused an
`almost complete loss of ER measured by binding assay in the
`uterus of mice supplemented with estrone. The daily 0.3-pLg
`dose of EM-800 caused a 30% (P < 0.01) loss of uterine ER
`levels. With the daily 1.0-pLg dose, a 70% loss of uterine
`
`
`
`
`
`
`
`Comparable effects were observed on vaginal ER levels
`measured by binding assay (Fig. 5). Thus, sc injection of 0.3,
`1.0, 3.0, and 10 ug EM-800 caused 40% (P < 0.01), 82% (P <
`0.001), 97% (P < 0.0001), and 99% (P < 0.0001) inhibitions of
`vaginal ER levels, respectively. ICI 182 780, on the other
`hand, had no significant effect on this parameter under the
`assay conditions used.
`We next compared the activities of EM-800, ICI 182 780,
`and toremifene administered by the oral route. Whereas
`EM-800 caused an 18% (P = NS) inhibition of estrone-stim-
`ulated uterine weight at the daily oral dose of 0.3 [.Lg (Fig. 6),
`doses of 1.0, 3.0, and 10 ug of the antiestrogen caused re-
`spective 46%, 71%, and 83% inhibitions of estrone-stimulated
`uterine weight (P < 0.0001 for all the three highest doses US.
`control). Toremifene, a close analog of tamoxifen, caused 9%
`(P = NS), 25% (P < 0.001), 48% (P < 0.0001), and 51% (P <
`0.0001) inhibitions of estrone-stimulated uterine weight at
`the 0.3-, 1.0-, 3.0-, and 10-pig doses, respectively. The only
`significant inhibitory effect of ICI 182 780, namely a 21%
`inhibition (P < 0.01), was observed at the highest dose used
`(10 Mg daily), indicating at least a 30-fold lower activity of this
`compound compared with that of EM-800.
`The daily oral administration of EM-800 led to respective
`10% (P = NS), 38%, 64%, and 88% inhibitions of vaginal
`weight (P < 0.0001 for the three highest doses used as. con-
`trol) at the 0.3-, 1.0-, 3.0-, and 10-ng doses (Fig. 7), whereas
`ICI 182 780 exerted no significant inhibitory effect on this
`parameter. Toremifene, on the other hand, caused 4% (P =
`NS), 24%, 52%, and 67% inhibitions of vaginal weight at the
`0.3-, 1.0-, 3.0-, and 10-pLg doses, respectively (P < 0.0001 for
`the three highest doses used 05. control).
`As illustrated in Fig. 8, only oral administration of the
`highest dose of EM-800 led to a significant decrease in uterine
`ER levels measured by binding assay (58% reduction; P <
`0.01), whereas ICI 182 780 and toremifene had no effect on
`this parameter at any of the doses used.
`Finally, as shown in Fig. 9, vaginal ER levels measured by
`TABLE 1. P values associated with the treatment and dose (treatment) nested effects for both the oral and sc routes of administration
` Route of administration Source Uterine wt Vaginal wt Uterine ER levels Vaginal ER levels
`
`
`
`
`
`so
`Treatment
`<0.0001
`<0.0001
`<0.0001
`<0.0001
`Dose (treatment)
`<0.0001
`<0.0001
`0.0006
`0.0029
`Treatment
`<0.0001
`<0.0001
`0.0010
`<0.0001
`Oral
`
`Dose (treatment) <0.0001 <0.0001 <0.0001 0.0249
`
`
`
`
`mouseVAGINALWEIGHT(mgI20gBodyWeight)
`
`I EM-800
`ICI 132 730
`
`140
`
`120
`
`100
`
`80
`
`60
`
`4O
`
`20
`
`
`
`
`
`ESTRONE (0.06119, s.c., BID)
`
`
`OVARIECTOMIZED
`
`
`I
`
`
`
`FIG. 3. Effect on vaginal weight of increasing concentrations of EM-
`800 and ICI 182 780 injected sc for 9 days to ovariectomized mice
`simultaneously treated with estrone. **, P < 0.0001 vs. estrone-
`treated control.
`
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`
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`
`

`

`COMPARATIVE ANTIESTROGENIC ACTIVITY OF EM-800
`
`2489
`
`I EM-800
`IC|182 780
`
`1400—
`
`1200—
`
`_A OOO
`
`
`
`UTERINE(3H)E2BINDING
`
`
`
`(fmol/mgprotein)
`
`H
`
`
`
`ANTIESTROGEN ((19, s.c., ID)
`
`ESTRONE (0.0609. s.c., BID)
`
`
`OVARIECTOMIZED
`
`
`
`
`
`
`FIG. *4. Effect on uterine ER levels of increasing concentrations of
`EM-800 and ICI 182 780 injected sc for 9 days to ovariectomized mice
`simultaneously treated with estrone. +;P < 0.01; *;P < 0.001; **,P <
`0.0001 (vs. estrone-treated control).
`
`
`
`
`
`
`
`MOUSEUTERINEWEIGHT(mgl209BodyWeight)
`
`FIG. 6. Effect on uterine weight of increasing concentrations of EM-
`800; ICI 182 780; and toremifene administered orally for 9 days to
`ovariectomized mice simultaneously treated with estrone. +, P <
`0.01; *, P < 0.001; **, P < 0.0001 (113. estrone-treated control).
`
`140
`
`120
`
`100
`80
`
`60
`
`40
`
`20
`
`EM-BOO
`ICI 182 780
`
`El TOREMIFENE
`
`+
`
`OVARIECTOMIZED
`
`INTACT
`
`CONT
`
`CONT
`
`0.3
`
`1.0
`
`3.0
`
`10
`
`ANTIESTROGEN (pg, per os, ID)
`ESTRONE (0.0609, s.c., BID)
`
`1 4o
`
`120
`100
`
`80
`
`60
`
`40
`
`20
`
`EM-800
`ICI 182 780
`IE TOREMIFENE
`
`OVARIECTOMIZED
`
`INTACT
`
`CONT
`
`CONT
`
`0.3
`
`1.0
`
`3.0
`
`10
`
`ANTIESTROGEN (pg, per 05, ID)
`ESTRONE (0.0m, s.c., BID)
`
`FIG. 7. Effect on vaginal weight of increasing concentrations of EM-
`800; ICI 182 780; and toremifene administered orally for 9 days to
`ovariectomized mice simultaneously treated with estrone. **; P <
`0.0001 (vs. estrone-treated control).
`
`toremifene (administered orally) on vaginal ER protein levels
`measured by ER-EIA. As shown in Table 2, treatment with
`increasing doses of EM-800 inhibited vaginal ER protein
`levels by 26%, 40%, and 71% at daily doses of 1.0, 3.0, and 10
`ug, respectively. As measurements of ER protein levels by
`EIA were performed on a pooled tissue homogenate, no
`statistical analysis could be performed. In animals treated
`with ICI 182 780, only a small 10 —11% decrease in vaginal ER
`protein levels was observed at doses ranging from 1—10 Mg,
`whereas treatment with toremifene caused a 27% inhibition
`
`of this parameter only at the highest dose used (10 pug).
`
`Discussion
`
`The present data show that EM-800 administered under
`conditions of maximal bioavailability (sc) is approximately
`10 times more potent than ICI 182 780, the most potent of the
`known steroidal antiestrogens (4, 5, 12, 28, 29) previously
`studied in phase 11 clinical trials (4, 12, 30). In fact, a similar
`difference in the estimated potencies were obtained when the
`effects of the two antiestrogens were measured on estrone-
`
`300—
`
`+ I EM-800
`ICI182780
`
`|
`
`I
`
`NOO
`
`
`
`(fmol/mgproteIn) 8D
`
`
`
`VAGINAL(3H)E2BINDING
`
`
`
`MOUSEVAGINALWEIGHT(mg/209BodyWeight)
`
`
`
`
`
`
` INT
`
`CONT CONT
`
`
`
`
`
`ANTIESTROGEN (ug, s.c., ID)
`ESTRONE(0.05I19,S.C., BID)
`OVARIECTOMIZED
`
`|_ l
`FIG. 5. Effect on vaginal ER levels of increasing concentrations of
`EM-800 and ICI 182 780 injected sc for 9 days to ovariectomized mice
`simultaneously treated with estrone. +;P < 0.01; *;P < 0.001; **;P <
`0.0001 (vs. estrone-treated control).
`
`binding assay decreased from 157 i 12 fmol/ mg protein in
`estrone-treated rats to 123 i 5 fmol/ mg protein (P = NS) in
`animals treated with the 0.3-ug daily dose of EM-800,
`whereas daily treatment with the 1.0-, 3.0-, and 10-)ug doses
`reduced vaginal ER levels to 89 i 0.1 fmol / mg protein (43%
`reduction; P < 0.0001), 87 i 10 fmol/mg protein (45% re-
`duction; P < 0.0001), and 30 i 4 fmol/mg protein (81%
`reduction; P < 0.0001), respectively. Toremifene, on the other
`hand, caused 36% (P < 0.001), 21% (NS), 47% (P < 0.0001),
`and 45% (P < 0.0001) inhibitions of vaginal ER levels at daily
`doses of 0.3, 1.0, 3.0, and 10 ug, respectively. ICI 182 780 had
`no significant effect on vaginal estradiol receptor levels un-
`der the experimental conditions used.
`To ensure that the down-regulation of ER levels observed
`with EM-800 does not result from a failure of [3H]estradiol
`to exchange with EM-800 [or its metabolite(s)], we evaluated
`the effects of treatments with EM-800, 1C1 182 780, and
`
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`AstraZeneca Exhibit 2159 p. 4
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`

`

`2490
`
`COMPARATIVE ANTIESTROGENIC ACTIVITY OF EM-800
`
`VOThllgg : 11%):
`
`700
`600
`
`500
`
`400
`
`300
`
`200
`
`100
`
`
`
`(fmol/mgproteln)
`
`
`
`UTERINE(3H)E2BINDING
`
`I EM-BOO
`ICI 182 780
`
`E TOREMIFENE
`
`OVAHIECTOMIZED
`
`CONT CONT
`
`0.3
`
`3.0
`1.0
`ANTIESTROGEN (pg, per 05, ID)
`ESTRONE (0.06ug, s.c., BID)
`
`FIG. 8. Effect on uterine ER levels of increasing concentrations of
`EM-800, ICI 182 780, and toremifene administered orally for 9 days
`to ovariectomized mice simultaneously treated With estrone. +, P <
`0.01 (vs. estrone-treated control).
`
`300
`
`I EM-BOO
`
`Z |C|182 780
`
`
`
`
`: TOREMIFENE
`
`
`TABLE 2. Vaginal estrogen receptor levels measured by enzyme
`immunoassay (ER-ETA) after oral administration for 9 days of
`increasing doses of EM-800, ICI 182 780, and toremifene to
`ovariectomized mice supplemented With estrone
`
`Treatment
`ER levels (finol/mg protein)
`OVX (control)
`414
`
`OVX -- E1
`238
`
`EM-800 (1 pg)
`E1
`OVX
`175
`
`EM-800 (3 Mg)
`E1
`OVX
`143
`
`EM-800 (10 Mg)
`E1
`OVX
`69
`
`ICI 182 780 (1 Mg)
`E1
`OVX
`211
`
`ICI 182 780 (3 Mg)
`E1
`OVX
`212
`
`ICI 182 780 (10 Mg)
`E1
`OVX
`216
`
`toremifene (1 Mg)
`OVX El
`262
`toremifene (3 pg)
`E1
`OVX
`240
`
`OVX
`E1
`toremifene (10 pg)
`173
`
`
`
`
`Ovariectomized mice receiving the vehicle alone were used as ad-
`ditional controls.
`
`effect achieved with the daily 10-ug dose of toremifene on
`uterine weight is limited to about 51% compared with an
`inhibitory effect of 83% observed for EM-800 at the same
`dose. The maximal inhibitory effects achieved by toremifene
`and EM-800 on vaginal weight at the highest dose used (10
`Mg) were 67% and 88%, respectively (P < 0.0001 between the
`two compounds). Thus, in addition to its lower potency to
`neutralize the stimulatory effect of estrone, toremifene at
`high doses exerts a lower maximal inhibitory effect than can
`be achieved with EM-800. This situation is analogous to that
`observed with tamoxifen.
`
`Tamoxifen has long been known not to completely prevent
`binding of estrogen to the ER (31, 32). In agreement with the
`present in viva data, the pharmacology of toremifene has
`been found to be similar that of tamoxifen (33, 34). In the
`mouse uterus, toremifene has relatively potent estrogenic
`activity, whereas its estrogenic activity is weaker in the rat
`uterus (18, 33). Tamoxifen and toremifene have also shown
`cross-resistance in the clinic (35).
`It can be seen in the present study that the maximal in-
`hibitory effects of EM-800 and ICI 182 780, administered sc,
`are of similar magnitude, namely 87% and 90% inhibitions of
`estrogen-stimulated uterine weight and 96% and 90% inhi-
`bitions of estrogen-stimulated vaginal weight, respectively.
`In the rat, at the highest dose used (1 mg / kg-day), treatment
`with ICI 182 780 for 14 days led to a 92% inhibition of uterine
`weight compared with the effect of ovariectomy (5). Such
`data are in agreement with the present finding of a 90%
`inhibition at the daily 0.5 mg / kg~d ose. As mentioned above,
`the present data achieved with EM-800 and lCl 182 780 are
`very different from those observed with tamoxifen and
`toremifene, where the maximal inhibition obtained on uter-
`ine weight is about 50%, whereas it is limited to 67% for
`vaginal weight. As mentioned above, the much lower max—
`imal blockade of estrogen action on these two parameters by
`tamoxifen and toremifene can be explained by the intrinsic
`estrogenic activity of these two compounds.
`The present study shows that a half-maximal inhibitory
`effect of estrogen action on mouse uterine weight by ICI 182
`780 administered sc is obtained at a dose of 2—3 )ULg/ day or
`0.1— 0.2 mg / kg~day. These data can be compared with a half-
`maximal inhibitory dose of 0.5 mg/ kg obtained in a recent
`
`200
`
`100
`
`
`
`(fmol/mgproteln)
`
`
`
`VAGINAL(3H)E2BINDING
`
`0
`
`CONT CONT
`
`0.3
`
`
`
`1.0
`
`3.0
`
`10
`
`
`
`ANTIESTROGEN (pg, per 05, ID)
`ESTHONE (0.06I19. s.c., BID)
`
`OVARIECTOMIZED
`FIG. 9. Effect on vaginal ER levels of increasing concentrations of
`EM-800, ICI 182 780, and toremifene administered orally for 9 days
`to ovariectomized mice simultaneously treated With estrone. *, P <
`0.001; **, P < 0.0001 (vs. estrone-treated control).
`
`stimulated uterine and vaginal weights in ovariectomized
`mice. To eliminate the potential problem of variable absorp-
`tion of the two compounds, both antiestrogens were initially
`administered by sc injection, a route of administration likely
`to lead to optimal exposure to the drugs.
`In addition, the present data confirm the low oral bio-
`availability of the steroidal antiestrogen ICI 182 780 (4, 5).
`Although much higher doses of lCl 182 780 would need to
`be used to achieve an accurate assessment of the potency of
`this antiestrogen by the oral route, the present data indicate
`that ICI 182 780 has less than 3% the activity of EM-800 after
`oral administration.
`
`On the other hand, the intrinsic estrogenic activity of
`toremifene, a close analog of tamoxifen (19, 20), is likely to
`be responsible for the limited maximal ability of this com-
`pound to reverse the stimulatory effect of estrone on mouse
`uterine and vaginal weight. In fact, the maximal inhibitory
`
`The Endocrine Society. Downloaded from press. endocrineorg by [$(individualUser.displayNameH on 18 December 2014. at 15:32For personal use only. No other uses williout permission. . All rights reserved.
`
`AstraZeneca Exhibit 2159 p. 5
`
`

`

`COMPARATIVE ANTIESTROGENIC ACTIVITY OF EM-800
`
`2491
`
`study, also performed in ovariectomized mice (5). The some-
`what higher activity of ICI 182 780 observed in the present
`study can possibly be explained at least in part by the dif-
`ferent vehicles used and the different durations of treatment.
`
`0)
`
`The lower inhibition ofvaginal ER protein levels measured
`by enzyme immunoassay compared with the protein binding
`assay indicate that EM-652, the active metabolite of the pro-
`drug EM-800, occupies part of the ER binding sites. For ICI
`182 780-treated animals, there is no apparent loss of ER-
`binding sites or ER protein. Previous studies have clearly
`shown a loss of uterine nuclear and cytosolic ER levels mea-
`sured by binding assay after administration of the steroidal
`antiestrogen ICI 164 384 (36), although ER messenger RNA
`levels were unaffected. In fact, previous data have shown
`that ICI 182 780 increases ER degradation and a loss of ER in
`the mouse uterus (36, 37). Moreover, incubation of MCF-7
`cells with ICI 182 780 caused a dramatic fall in ERa immu-
`
`noreactivity on day 2 after addition of the drug in vitm (38).
`The loss of ER has been associated with an increased turn-
`
`over of the receptor induced by the pure antiestrogen (37).
`The lack of significant inhibition of ER by ICI 182 780 in
`the present study could be related to the timing of ER assays
`performed in tissue obtained approximately 24 h after last
`administration of the antiestrogen and/ or some unknown
`mechanism related, or not, to simultaneous treatment with
`estrone. It could also be related to the different duration of
`
`treatment, namely 9 days, in the present study.
`The present data obtained in ovariectomized mice are also in
`agreement with our recent findings obtained in intact mice, in
`which we found a 57% inhibition of uterine weight with the
`daily 10-pig oral dose of EM-800 (~05 mg/kg~day) (15) and the
`42% reduction in uterine weight observed in rats after 28 days
`of sc treatment with 0.3 mg /kg~day of ICI 182 780 (12). Com-
`parison of the data from these two studies suggested that EM-
`800 given orally was at least as potent as ICI 182 780 given by
`sc injection, although different sensitivities between the rat and
`mouse could play a role, In fact, the present data indicate that
`EM-800 administered by the oral route is 2- to 3-fold more
`potent than ICI 182 780 administered sc.
`It can be mentioned that although the elimination of es-
`trogens by ovariectomy is well known to lead to an increase
`in body weight in the rat (39, 40), treatment with ICI 182 780
`had no significant effect on body weight (5). Such findings
`suggest a lack of blockade by ICI 182 780 on the estrogenic
`mechanisms controlling body weight in analogy with the
`apparent lack of antiestrogenic activity of ICI 182 780 or ICI
`164384 on the inhibitory feedback activity of estrogens on the
`hypothalamo-pituitary-gonadotropin axis (3, 5, 13, 28).
`In summary, the present data show that the orally active
`antiestrogen EM-800 is the most potent antiestrogen known
`to date. EM-800 offers the opportunity to test the proposition
`that a pure antiestrogen should be a valuable improvement
`in the therapy of breast cancer while eliminating the sec-
`ondary effects related to the mixed agonist-antagonist es-
`trogenic action of tamoxifen and its analogs, such as
`toremifene and droloxifene.
`
`References
`
`1. McGuire WL, Carbone PP, Seard ME, Esche GC 1975 Estrogen receptors in
`human breast cancer: an overview. In: McGuire WL, Carbone PP, Vollmer EP
`
`
`
`20.
`
`21.
`
`22.
`
`23.
`
`24.
`
`25.
`
`26.
`
`10.
`
`11.
`
`12.
`
`14.
`
`
`
`(eds) Estrogen Receptors in Human Breast Cancer. Raven Press, New York, pp
`177
`. Furr BJ, Jordan VC 1984 The pharmacology and clinical uses of tamoxifen.
`Pharmacol Ther 25:1277205
`. Wakeling AE, Bowler] 1988 Biology and mode of action of pure antiestr'ogens.
`J Steroid Biochem 30:1417147
`. Wakeling AE, Dukes M, Bowler J 1991 A potent specific pure antiestrogen
`with clinical potential. Cancer Res 51:386773873
`Wakeljng AE, Bowler J 1992 ICI 182,780, a new antioestrogen with clinical
`potential. J Steroid Biochem Mol Biol 43:1737177
`. Nique F, Van de Velde P, Brémaud J, Hardy M, Philibert D, Teutsch G 1994
`11 BrAmidoalkoxyphenyl estradiols, a new series of pure antiestrogens. J Ste
`roid Biochem Mol Biol 50:21729
`Simard J, Dauvois S, Haagensen DE, Levesque C, Mérand Y, Labrie F 1990
`Regulation of pi'ogesteronerbinding breast cyst protein GCDFP724 secretion by
`estrogens and androgens in human breast cancer cells: a new marker of steroid
`action in breast cancer. Endocrinology 126:322373231
`. Dauvois S, Geng CS, Levesque C, Mérand Y, Labrie F 1991 Additive inhibr
`itory effects of an androgen and the antiestrogen EM7170 on estradiolestimr
`ulated growth of human ZR77571 breast tumors in athymic mice. Cancer Res
`51:313173135
`. de Launoit Y, Dauvois S, Dufour M, Simard J, Labrie F 1991 Inhibition of cell
`cycle kinetics and proliferation by the androgen Sardihydrotestosterone and
`antiestrogen N,
`nebutyerrmethylrl1r(16’ozrchloror3’,17Brdihydroxyrestrar
`l',3355(10')triene7’aeyl)undecanamide in human breast cancer ZR77571 cells.
`Cancer Res 51279772802
`Lévesque C, Mérand Y, Dufour JM, Labrie C, Labrie F 1991 Synthesis and
`biological activity of new halorsteroidal antiestrogens.
`J Med Chem
`34:162471630
`Labrie C, Martel C, Dufour JM, Lévesque C, Mérand Y, Labrie F 1992 Novel
`compounds inhibit estrogen formation and action. Cancer Res 5216107615
`Wakeling AE 1993 The future of new pure antiestrogens in clinical breast
`cancer. Breast Cancer Res Treat 2519
`. Gottardis MM, Jiang SY, Jeng MH, Jordan VC 1989 Inhibition of tamoxifen
`stimulate