0013-7227/98/$03.00/0
`Endocrinology
`Copyright © 1998 by The Endocrine Society
`
`Vol. 139, No. 9
`Printed in USA.
`
`Effect of the High-Affinity Estrogen Receptor Ligand ICI
`182,780 on the Rat Tibia"<
`
`JEAN D. SIBONGA, HARALD DOBNIG, RYAN M. HARDEN, AND
`RUSSELL T. TURNER
`
`Department of Orthopedic Research (J.D.S., HD., R.MH, RTT.) and Biochemistry and Molecular
`Biology (R.T. T.), Mayo Clinic, Rochester, Minnessota 55905
`
`ABSTRACT
`We examined the specificity of the steroidal antiestrogen ICI
`182,780 (ICI) on bone and reproductive tissues in adult and growing
`female rats. Using a 1.5-mg/kg dose (sc), we evaluated the effects of
`ICI on the bone, body weight, uterine weight, serum cholesterol, and
`serum estradiol in either adult and/or growing rats. ICI increased
`serum estradiol cholesterol in ovary-intact rats, had no effect on
`uterine weight in ovariectomized rats, and resulted in uterine atrophy
`in ovary-intact animals comparable with ovariectomy. In contrast,
`ICI had no effect on body weight. In bone, ICI significantly increased
`the rate of periosteal bone formation in long bones of growing and
`mature female rats. In contrast, ICI had no effect on longitudinal bone
`growth in rapidly growing rats. When ICI was administeredto mature
`rats with or without ovaries, two-factor ANOVA revealed significant
`interaction (P S 0.05) between ovariectomy and ICI treatment for
`cancellous bone area and labeled bone perimeter. ICI increased skel-
`
`
`etal indices ,of bone turnover in the cancellous bone of ovary-intact rats
`but reduced these indices of bone turnover in the cancellous bone of
`ovariectomized rats. The increase in bone turnover was associated
`with a reduction in cancellous bone area in the ovary-intact rats. A
`reduction in bone turnover was similarly associated with an increase
`in bone area in the ICI-treated ovariectomized rats. In summary, ICI
`exhibited complete estrogen antagonism in cortical and cancellous
`bone, partial agonism in cancellous bone, and no activity on tibial
`longitudinal growth rate of growing ovary-intact rats. The effects in
`adult rats were influenced by circulating levels of estradiol. ICI had
`no activity on body weight and complete antagonism on uterine
`weight. These results demonstrate that a ligand with high binding
`affinity to the estrogen receptor(s) can elicit an array of estrogen-
`mediated regulation of bone metabolism. (Endocrinology 139: 3736—
`3742, 1998)
`
`NTlESTROGENS is the term originally applied to com-
`pounds which block the physiological response of
`reproductive tissues to estrogen. It was initially assumed that
`these agents would antagonize estrogen in all target cells.
`Estrogen analogs can act as antiestrogens by competing with
`estrogen for the receptor to produce an inactive ligand-re-
`ceptor complex, but the analog-receptor complex can also
`display variable degrees of estrogen agonism (1). One class
`of estrogen analogs is the pharmaceutically-developed C7-
`alkyl substituted steroid analogs of estrogen (2—4). In the
`rodent, these antiestrogens possess both high affinity for the
`estrogen receptor and complete absence of uterotrophic ac-
`tivity. Although the lCl compound 164,384 has been reported
`to disable the ligand-receptor complex from binding to DNA
`(5), there is another report that the formed complex facilitates
`binding to DNA (6). It is the absence of estrogen agonism in
`reproductive tissues to which the terminology, pure anties-
`trogens, was initially referenced (2—5).
`Because of their high binding affinity and their lack of
`estrogen agonism, these estrogen antagonists have great po-
`tential as clinical chemoagents for breast cancer. The ability
`of lCl 182,780 (lCl) to inhibit cell growth, in fact, has been
`reported in MCF-7, as well as Br10 human breast cancer cells
`
`Received November 6, 1997.
`Address all correspondence and requests for reprints to: Russell T.
`Turner, Ph.D., Mayo Clinic, Department of Orthopedics, Medical Sci—
`ence Building 3—69, 200 First Street SW, Rochester, Minnesota 55905.
`E—mail: rolbiecki.lori@mayo.edu.
`* These studies were supported by NIH Grant AR—41418 and the
`Mayo Foundation.
`
`(3). The effectiveness of the C7 alkyl-substituted compounds
`in chemotherapy contrasts with tamoxifen, a pharmaceuti-
`cally-developed antiestrogen of a triphenylethylene struc-
`ture. Tamoxifen is the chemoagent of choice for defense in
`breast cancer (7—10). Tamoxifen though possesses partial es-
`trogen agonism in reproductive tissue. It is this degree of
`agonism that is thought to contribute to the acquired toler-
`ance of breast tumor cells after extended tamoxifen therapy
`(4, 11). Antiestrogens with complete absence of estrogen
`agonism, on the other hand, may be more rapid, more potent
`tumorstatic agents with longer lasting results (4, 12).
`In addition, the variable display of estrogen agonism by
`antiestrogens seems to be tissue-specific. A potential mech-
`anism for the tissue-selectivity of antiestrogens involves dif-
`ferential transactivational gene transcription by the estrogen
`receptor (13). The model proposes that capacity of the ligand-
`bound estrogen receptor to activate gene transcription is
`mediated by two distinct regions within the receptor mole-
`cule. Analogs of different antiestrogen classes could confer
`conformational changes to the estrogen receptor, thereby
`affecting the interaction, either positively or negatively, be-
`tween the ligand-bound estrogen receptor and DNA. Alter-
`natively, a second type of estrogen receptor, the estrogen
`receptor-[3, could account for tissue differences based on
`variable tissue distributions of receptors (14). Because of the
`tissue-selectivity and the variable degree of agonism in an-
`tiestrogens, it is important to investigate the tumorstatic es-
`trogen analogs, in terms of their side effects in nontumor,
`estrogen-responsive tissues. There are reports that these
`
`8786
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`

`EFFECTS OF ICI 182,780 ON THE RAT TIBIA
`
`8737
`
`compounds may have detrimental effects on the skeletal
`system (15).
`Estrogen has similar effects on the rat and human skele-
`tons, and the response of the laboratory animal model to
`estrogen antagonists has successfully predicted the outcome
`in women (16). Thus, we investigated the effects of the ICI on
`skeletal tissue of female rats that were growing or sexually
`mature and either with or without ovaries.
`
`Materials and Methods
`
`Animal procedures
`
`Procedures used in all animal experiments were approved by the
`institutional animal care and use committee at the Mayo Clinic in ac—
`cordance with the NIH Guide for the Care and Use of Laboratory
`Animals. All rats were fed laboratory chow ad libitum.
`
`Exp 1. Forty 10week—old female Sprague—Dawley rats (Harlan, India—
`napolis, IN) were used to evaluate the effects of ICI on skeletal growth
`and modeling. Thirty of the rats were divided into three groups of
`ovary—intact rats (n = 10): one group was injected with ICI, the second
`group was injected with the sesame oil vehicle, and the third served as
`the baseline control group. The baseline group was killed at the start of
`the treatment. A fourth group (n = 10) was ovariectomized 1 week
`before the start of the study (OVX group). ICI powder (Zeneca Phar—
`maceuticals, Macclesfield, Cheshire, UK) was resuspended in 95% eth—
`anol to a 100 mg / ml Stock Solution. Each day, an aliquot of the stock
`solution was resuspended in sesame oil and sonicated for the delivery
`of a 1.5—mg /kg BW dose of ICI, based on an averaged weekly weight of
`rats for the 3—week study. A vol of 0.1 ml was injected sc at the back of
`the neck. Ovariectomized rats were injected with vehicle. The fluoro—
`chrome tetracycline (20 mg/ kg; Sigma Chemicals, St. Louis, MO) was
`injected at the base of the tail on the first day of treatment (day 1) and
`2 days before death (day 19). Another fluorochrome calcein (20 mg/kg;
`Sigma Chemicals, St. Louis, MO) was similarly injected 9 days before
`death (day 13). Rats were anesthetized by C02 inhalation and killed by
`guillotine decapitation. Wet weights of uteri were recorded. Tibiae were
`removed and fixed by immersion in 70% ethanol.
`
`Exp 2. Rats which were either ovariectomized or ovary—intact were
`treated with either ICI or vehicle. Forty female Sprague—Dawley rats
`(Harlan, Indianapolis, IN) at 6 months of age, were divided into 4 groups
`of approximately 10 (:2) rats each. Two groups were ovariectomized 1
`week before the start of treatment, and the other groups remained
`ovary—intact. One ovariectomized group and one ovary—intact group
`were treated groups and injected with ICI; the other ovariectomized and
`ovary—intact group were treatment controls and injected with vehicle. A
`small group of ovary—intact rats (n = 6) was killed on the first day of
`treatment. The bone fluorochrome tetracycline (20 mg/kg; Sigma Chem—
`icals, St. Louis, MO) was injected at the base of the tail on the first day
`(day 1) of the 8—week study and 2 days before death (day 54). The bone
`fluorochrome calcein (20 mg/ kg; Sigma Chemicals, St. Louis, MO) was
`similarly injected 12 days before death (day 44). The averaged body
`weight of the rats was recorded weekly. An aliquot of a 5% wt / vol stock
`solution of ICI in sesame oil (Zeneca Pharmaceutical, Macclesfield,
`Cheshire, UK) was diluted further in sesame oil for delivery of a 1.5—
`mg/ kg-day dose, based on the weekly averaged body weight. As in Exp
`1, a 0.1—ml vol was injected sc at the back of the neck daily throughout
`the 8—week study. Rats were anesthetized by C02 inhalation and killed
`by guillotine decapitation. Blood was drained from the carcass, clotted,
`and centrifuged for serum isolation. Serum aliquots were stored at —80
`C before assay. Wet weights of uteri were recorded. Tibiae were re—
`moved and fixed by immersion in 70% ethanol.
`
`Exp 3. Skeletal effects were evaluated in 8—month old ovary—intact female
`Sprague—Dawley rats (Harlan, Indianapolis, IN), treated for 52 days with
`ICI (n = 7). Rats were injected sc with ICI (Zeneca Pharmaceutical,
`Macclesfield, Cheshire, UK) or sesame oil vehicle (1.5 mg/ kg BW ICI).
`Rats were anesthetized by Ethrane inhalation and killed by exsangui—
`nation. Blood was obtained from the abdominal aorta. Tibiae were
`
`harvested and fixed, as in previous experiments, for cancellous bone
`histomorphometry.
`
`Bone histomorphometry
`
`Measurements for Exp 1 and 2 were performed with an SMI—Micro—
`comp semiautomatic image analysis system (Southern Micro Instru—
`ments, Inc., Atlanta, GA), which consists of a Compaq computer with
`microcomp software interfaced with a microscope and image analysis
`software. Measurements for Exp 3 were executed as for Exps 1 and 2,
`except for the image analysis software (OsteoMetrics, Inc., Atlanta, GA).
`Skeletal indices are measured by registering the movement of a digi—
`tizing pen across a graphics tablet as a tracing is superimposed on an
`image of the section displayed on the video screen. As regions of interest
`are traced in the bone specimen, the computer software records the
`lengths of tracings and calculates the enclosed areas.
`
`Cortical bone measurements. All corticalbone measurements of Exp 1 were
`made on unstained cross—sections obtained from the tibia—fibula synos—
`tosis using a low-speed saw equipped with a diamond wafer blade
`(Isomet, Buehler, Lake Bluff, IL). Cross—sections (150 um) were ground
`to 15—20 am on a roughened glass plate and permanently mounted.
`Fluorochrome labeling was visualized under reflected UV light. Cortical
`bone histomorphometry included cross—sectional area, medullary area,
`cortical area, periosteal bone formation rate, and periosteal mineral
`apposition rate and were performed as previously described (17), except
`for a bone growth period of 21 days.
`
`Cancelloas bone measurements. Proximal tibial metaphyses were dehy—
`drated in 95% ethanol for 1 day, followed by 6 days in 100% ethanol
`before embedding, without demineralization, in a mixture of methyl—
`methacrylate—2—hydroxyethyl—methacrylate (12.521). Parasagittal
`sec—
`tions were cut from the middle of the proximal tibia (5 pm thick) with
`a Reichert Jung microtome.
`All measurements, except for longitudinal growth rate, were con—
`ducted in a standard sampling site located in the secondary spongiosa
`of the metaphyseal region of the proximal tibia. This sampling site was
`located 1 mm from the most distal point of the epiphyseal growth plate,
`extended bilaterally, but excluded endocortical bone and encompassed
`a 2.88—mm2 tissue area (TAr). Mean longitudinal growth rate was de—
`termined as the distance from the calcein label to the metaphyseal
`growth plate cartilage at five equidistant sites across the growth plate.
`The mean distance was divided by the growth period of 9 days.
`The following indices were obtained or calculated from measure—
`ments performed in the metaphyseal sampling site, as previously de—
`scribed (17), and according to Parfitt et al. (18): cancellous bone area,
`cancellous bone perimeter, labeled bone perimeter, mineral apposition
`rate, bone formation rate, and osteoclast-covered perimeter. Indices of
`cancellous bone architecture, such as trabecular number, trabecular
`thickness and trabecular separation, were estimated according to stan—
`dard formulas (19).
`
`Serum measurements
`
`Measurements of 17B—estradiol in Exp 3 were made using a double—
`antibody RIA that has a minimum detectable limit of 1.4 pg /ml (Diag—
`nostic Products Corporation, Los Angeles, CA). Cholesterol measure—
`ments in Exp 2 were measured by the Immtmochemical Core Facility at
`the Mayo Clinic using an automated procedure (Roche Diagnostic Sys—
`tem, Los Angeles, CA).
`
`Statistical analysis
`
`Comparisons between multiple pairs of groups were accomplished
`by application of Fisher’s protected least-significant difference (PLSD)
`post hoc test, after determination of significance by one—way ANOVA
`(Exp 1). Two—way ANOVA was performed to determine whether there
`is a significant effect of either ovarian status or ICI treatment or whether
`there is a significant interaction between the two factors (Exp 2). Sub—
`sequently, one-way ANOVA, followed by multiple—group comparisons
`with Fisher’s PLSD, were conducted to determine the significance of ICI
`treatment in the ovary—intact and in the ovariectomized rats, Student’s
`t test was performed for comparisons between ovary—intact control and
`ICI—treated rats (Exp 3). Statistical significance was considered at P
`values S 0.05.
`
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`

`3738
`
`EFFECTS OF ICI 182,780 ON THE RAT TIBIA
`
`Endo - 1998
`Vol 139 I No 9
`
`Results
`
`Body growth, uterine weight, serum estradiol, and
`cholesterol
`
`For Exps 1—3, ICI had no negative effects on the husbandry
`or overall health status of the female rats. ICI had no direct
`
`effect on the final body weights of the treated animals, when
`compared with the vehicle controls, regardless of ovarian
`status (Table 1).
`
`Exp 1. Ovariectomy increased final body weight. Ovariec-
`tomy and ICI reduced uterine weight (Table 1).
`
`Exp 2. Ovariectomy increased final body weight, but there
`was no interaction of ovariectomy and ICI, by two-way
`ANOVA, on final body weight (Table 1). Two-way ANOVA
`revealed no effect of ovariectomy, a significant effect of ICI,
`and a significant interaction between ovariectomy and ICI on
`serum cholesterol levels. ICI significantly increased serum
`cholesterol in ovary-intact (25%) but not in ovariectomized
`rats (2%) (see Table 3).
`
`Exp 3. ICI increased circulating levels of 17B-estradiol almost
`10-fold in the ovary-intact rats (18.8 i 3.5 pg/ml ICI US. 1.8 i
`0.8 pg/ml VEH, P 5 0.0001). Serum 17B-estradiol was not
`detectable in ovariectomized rats.
`
`Skeletal data
`
`Exp 1. ICI had no effect on cross-sectional area, medullary
`area, and cortical area measured at
`the tibia diaphysis,
`whereas ovariectomy and ICI similarly increased rates of
`mineral apposition and bone formation measured at the
`same site (Table 2). Ovariectomy increased longitudinal
`growth at the proximal tibial metaphysis in the young rats,
`whereas ICI treatment of intact rats had no effect on longi-
`tudinal growth rate (Fig. 1).
`
`Exp 2. Ovariectomy increased dynamic indices of bone for-
`mati on (labeled bone perimeter, mineral apposition rate, and
`bone formation rate) in cancellous bone (P 5 001-0005),
`increased the percent of osteoclast—covered perimeter (Fig. 2),
`and decreased indices of cancellous bone volume (bone area,
`trabecular number), had no effect on trabecular thickness,
`and increased trabecular separation (Table 3). ICI had no
`effect on labeled bone perimeter and on bone formation rate
`but decreased mineral apposition rate. However, there was
`an interaction between ovarian status and ICI on the labeled
`
`bone perimeter, such that ICI increased labeled perimeter in
`ovary-intact rats and decreased this measurement in ovari-
`ectomized animals. Neither of these effects was significant in
`group comparisons after one-way ANOVA. As presented in
`Table 3 and Fig. 2, significant interaction between ovarian
`status and ICI was evident for cancellous bone area (BAr/
`TAr), bone-forming perimeters (LPm/TPm), and osteoclast-
`covered perimeter (Och), and for the architectural indices
`of trabecular number (TbN) and separation (TbSp), Le. the
`direction of ICI’s actions was dependent on ovarian status
`(Table 3 and Fig. 2). ICI decreased measurements related to
`bone volume and bone-forming surfaces in ovary-intact rats
`but increased these values in the ovariectomized rats.
`
`Exp 3. ICI increased the percent of double-labeled perimeter
`(62% increase) in cancellous bone of ovary-intact rats but had
`no effect on mineral apposition rate (Fig. 3). ICI increased the
`percent of double-labeled perimeter (63.8 i 11.3 mm ICI vs.
`14.6 i 4.1 mm vehicle, P 5 0.001) and the rates of mineral
`apposition (1.28 i 0.11 lam/day ICI US. 0.97 i 0.15 lam/day
`vehicle, NS) and bone formation (89.4 i 12.7 mmZ/day X
`10’3 ICI US. 36.2 i 10.2 mmZ/day X 10’3 vehicle, P 5 0.001)
`at the periosteal surface of cortical bone of ovary-intact rats.
`
`Discussion
`
`ICI binds with high affinity to both ERa and ERB (14).
`Early investigations failed to detect any transcriptional ac-
`tivity in the estrogen receptor-ICI complex, suggesting that
`the ICI-bound receptor is unproductive and implying that
`ICI functions as a pure estrogen antagonist. As a conse-
`quence, the observed skeletal response to ICI in the rat was
`unanticipated.
`ICI's effects on the uterus, however, are consistent with
`previous reports. ICI resulted in uterine atrophy comparable
`with ovariectomy in ovary-intact rats and had no uterotro-
`phic activity in the ovariectomized rat. These findings are in
`agreement with the results of Lundeen et al. (20), who re-
`ported that ICI, itself, displayed no uterotrophic activity in
`the ovariectomized rat but blocked uterine stimulation by
`17a-ethynyl estradiol when administered together. These
`effects of ICI contrast with those of triphenylethylene and
`benzothiophene antiestrogens, which, besides being com-
`petitive inhibitors of estradiol, possess partial uterotrophic
`activity (20).
`In addition, ICI contrasts with the nonsteroidal antiestro-
`
`TABLE 1. ICI has no effect on body growth but reduces uterine weight comparable with ovariectomy
`
`Exp 1
`Exp 2
`EXP 3
`Uterine
`Initial
`Uterine
`Initial
`Uterine
`Final
`Final
`Final
`Initial
`Measurement :>
`Group ‘U
`body weight body weight
`weight
`body weight body weight
`weight
`body weight body weight
`weight
`
`(g)
`(g)
`(mg)
`(g)
`(g)
`(mg)
`(g)
`(g)
`(mg)
`
`ND
`ND
`ND
`276 i 10“ 429 i 21‘“)
`270 i 10
`402 : 25‘1’1’
`238 i 2
`229 i 2
`Baseline (intact) (n = 6—10)
`595 : 64b
`284 i 6
`272 i 8
`299 i 5“
`518' : 38‘1”!)
`268 i 5
`472 i 39‘”
`258 i 3“
`227 i 3
`Intact control (n = 10—11)
`141 i 9
`284 i 5
`272 i 7
`287 i 6“
`175 i 13
`272 i 10
`170 i 11
`252 i 3“
`226 i 2
`Intact + ICI (I1 = 10—11)
`NA
`NA
`NA
`348 : 10Z7
`172 i 10
`273 i 7
`152 i 10
`299 i 317
`223 i 2
`OVX Control (11 = 11—12)
`
`OVX + ICI (n = 9—12) NA NA NA NA 268 i 4 345 : 5Z7 156 i 13 NA NA
`
`
`
`
`
`
`
`
`
`’IVVO-way ANOVA for Exp 2: the effects of ovariectomy and ICI treatment were significant, with significant interaction between the two factors
`for uterine weight (P S .0001). For final body weight, there was a significant effect of ovariectomy only (P S .0001), with no significant interaction
`between surgery and ICI treatment. One-way ANOVA; comparison to ovariectomized control rats: a P S .0001; comparison to ICI-treated intact
`rats; b P S .0001. Values are means : SE. NA, Not applicable; ND, not determined.
`
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`

`

`EFFECTS OF ICI 182,780 ON THE RAT TIBIA
`
`3739
`
`TABLE 2. Exp 1. ICI stimulates radial growth in growing, ovary-intact rats
`Periosteal mineral
`Periosteal bone
`Cross-sectional area
`Cortical area
`Medullary area
`Measurement 3;
`Groups ll
`(m1n2)
`(m1n2)
`(m1n2)
`apIEEE/lggyfiate
`fofififglfiagite
`
`Baseline (intact) (n = 10)
`0.91 i 0.06
`3.5 i 0.1
`4.4 i 0.1
`ND
`ND
`Intact -- VEH (n = 11)
`0.90 i 0.04
`3.6 i 0.1
`4.5 i 0.1
`1.78 i 0.14
`0.014 : 0.001
`
`Intact -- ICI (n = 11)
`0.85 i 0.04
`3.7 i 0.1
`4.5 i 0.1
`2.71 i 0.44”
`0.021 : 0.003”
`
`OVX + VEH (n = 11)
`0.90 i 0.03
`3.7 i 0.1
`4 6 i 0 1
`2.79 i 0.21”
`0.021 : 0.002”
`
`Means : SEM; 11, Number of measured animals per group. Cortical bone formation rate over 2 1 days. One-way ANOVA: comparison to (intact +
`VEH) group: ”P S .05.
`
`80
`
`4;
`
`
`
`
`
`LongitudinalGrowthRate(um/d)
`
`S
`
`'38
`
`O
`
`I ovx
`
`E INTACT
`
`
`
`
`
`Osteoclast-coveredPerimeter(%)
`
`U)
`
`HN
`
`O
`
`s-1
`O
`E
`O
`
`H
`U
`“
`
`.—1
`O
`EZ
`0
`
`ICI
`
`CONTROL
`
`FIG. 1. Effect of ICI on longitudinal growth rate in growing rats (Exp
`1). Estrogen deficiency with ovariectomy (dark bar) increased the
`longitudinal growth rate but ICI treatment of ovary-intact rats
`(hatched bar) had no effect on longitudinal growth rate. a, P 5 0.0001
`vs. ovary-intact group. Values are means : SE; n = 8—11.
`
`gens in its effects on body weight. Ovariectomy increases
`body weight (21, 22), and estrogen replacement therapy pre-
`vents this change (21—23), which is similarly mimicked by
`tamoxifen (17, 24) and raloxifene (22). This weight gain is
`associated with increased food consumption, exhibits a pre-
`dominant accumulation of fat, but cannot be completely pre-
`vented by matching the food intake of ovariectomized rats to
`the amount ingested by ovary-intact animals (21, 23). The
`change in body weight, therefore, suggests an alteration in
`metabolism. The exact mechanism of body weight increase
`is unclear, although it is under central regulation (25). ICI had
`no influence on the expected weight changes after ovariec-
`tomy. Our observation was similarly noted by Wakeling (3),
`which he attributed to the failure of ICI to cross the blood-
`
`brain barrier (26).
`The effects of estrogen and estrogen deficiency on serum
`cholesterol in the rat model have been recently characterized
`(20). Triphenylethylene and benzothiophene antiestrogens
`have similar levels of estrogen agonism on bone and serum
`cholesterol (20). We investigated the effects of ICI on serum
`cholesterol, but results are inconclusive. Though ovariec-
`tomy did not increase serum cholesterol in this study, ICI
`increased serum cholesterol when given in the presence of
`ovaries. An effect of ovariectomy on serum cholesterol is not
`
`FIG. 2. Effect of ICI on osteoclast-covered bone perimeter in adult
`rats (Exp 2). ICI decreases the extent of osteoclasts on bone surfaces
`of the ovariectomized rats (dark bar), P S 0.01 by Fisher PLSD; but
`tends to increase osteoclasts in the ovary-intact rats (hatched bar), NS
`by Fisher PLSD. By two-way ANOVA, effect of ovariectomy: P S
`0.001; effect ofICI: NS; interaction, P S 0.02. Values are means : SE;
`n = 5—7.
`
`always seen in our laboratory (27) or in others (28). In one
`instance, we observed an increase in serum cholesterol in
`ovariectomized rats (22), whereas in another study, we saw
`no change (27). Estrogen's regulation of circulating choles-
`terol levels in the rat involves a complex array of processes,
`many of which remain to be defined (20). We and others,
`however, have seen a consistent reduction of circulating cho-
`lesterol in ovariectomized rats treated with estrogen, triph-
`enylethylene, and benzothiophene antiestrogens (20, 22, 27,
`28). In this study, ICI had no effect on circulating cholesterol
`in the ovariectomized rat when analyzed by one-way
`ANOVA.
`
`The skeletal effects of ovariectomy and estrogen replace-
`ment have been well characterized in the rat (21, 23, 29—31).
`The ovariectomized reference groups in this study consis-
`tently displayed the skeletal effects of estrogen deficiency
`(21, 23, 29—32) at the examined sites in the tibia. The antic-
`ipated increases in radial and longitudinal bone growth and
`cancellous osteopenia were all manifested. The cancellous
`bone of the adult ovariectomized rat exhibited increased
`
`indicies of bone turnover (30, 31, 33).
`Young ovary-intact rats were selected specifically to in-
`
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`AstraZeneca Exhibit 2164 p. 4
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`

`

`TABLE 3. Exp 2. The effects of ICI treatment on cancellous bone histomorphometry and serum cholesterol are influenced by ovarian status
`
`
`Measurement é
`Cancellous
`Labeled
`Mineral
`Bone
`Trabecular
`Trabecular
`Trabecular
`Serum Cholesterol
`
`Group U’
`bone area
`bone pe’rlmeter
`app0s1t10n rate
`formatlon rate
`thlckness
`number
`separation
`(mg/d1)
`(% BAr/TAr)
`(.% LPm/TPIn)
`(Mm/day)
`(mm2/day)
`(Mm)
`(mm 1)
`(Mm)
`
`0171.8
`
`Intact
`Control
`ICI
`Ovariectomy
`Control
`ICI
`2-way ANOVA
`Ovariectomy P S
`ICI Treatment P S
`Interaction P S
`Group comparisons post
`1-way ANOVA
`.0001
`NS
`.005
`NS
`NS
`NS
`.05
`Intact : ICI P S
`
`Ovariectomy : ICI P S
`NS
`NS (.06)
`.05
`NS
`NS
`NS
`NS
`
`0.67 i 0.07
`0.48 i 0.04
`
`0.73 1‘ 0.06
`0.69 i 0.03
`
`0.050 : 0.009
`0.058 : 0.013
`
`0.157 : 0.031
`0.103 : 0.016
`
`45.7 i 2.7
`44.3 i 2.6
`
`43.0 i 6.2
`44.4 i 3.0
`
`NS
`
`.0005
`NS
`NS
`
`NS
`NS
`NS
`
`3.1 i .2
`2.0 i .3
`
`1.4 i .2
`1.9 i .2
`
`.001
`NS
`.005
`
`346 i 34
`553 i 72
`
`833 i 123
`618 i 95
`
`.01
`NS
`.05
`
`108.6 : 5.2
`135.5 : 4.5
`
`122.1 : 4.4
`124.7 : 2.9
`
`NS
`.001
`.01
`
`14.4 i 1.8
`8.8 i 1.5
`
`5.9 i 1.2
`9.0 i 1.2
`
`.01
`NS
`.01
`
`7.2 i 0 9
`11.5 i 2 5
`
`21.3 i 4.1
`14.6 i 2.0
`
`.01
`NS
`.05
`
`Values are means : SEM; n = 6 —12 animals per group. ’IWo-way ANOVA was performed to evaluate significant effects of surgery, ICI treatment or significant interaction between
`the two factors. One-way ANOVA, followed by Fisher PLSD, was performed to evaluate significant effects of ICI treatment in rats per ovarian status. Intact, OvaIy-intact rats;
`TPm, Total bone perimeter.
`
`
`
`VIHLLiLVHHHLNOOSL‘ZBIIOI.E[OSiLOELflcIIH
`
`
`
`
`
`Bone Formation Rate (x 10-3 umzld)
`
`
`
`ICI
`
`Mineral Apposition Rate (um/d)
`
`Double-labeled Perimeter (%)
`
`
`
`001
`
`0'9[
`
`OOZ
`
`
`
`|II||II|IIIIIIIIIi
`
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`
`AstraZeneca Exhibit 2164 p. 5
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`

`EFFECTS OF ICI 182,780 ON THE RAT TIBIA
`
`8741
`
`vestigate the effects of ICI on bone growth. Radial and lon-
`gitudinal bone growth normally change with age in parallel
`and both are inhibited by estrogen (23, 34). ICI increased the
`periosteal bone formation and mineral apposition rates in
`growing ovary-intact rats to a magnitude nearly identical to
`ovariectomy, indicating that the drug is a potent estrogen
`antagonist on the periosteum.
`Paradoxically, ICI did not influence longitudinal bone
`growth in parallel to its effects on radial bone growth. Lon-
`gitudinal bone growth occurs as a result of proliferation and
`hypertrophy of cartilage cells in the growth plate (35),
`whereas radial bone growth is determined by the number
`and activity of osteoblasts at the periosteal surface (36). Our
`results suggest that estrogen either does not act to regulate
`longitudinal bone growth Via an estrogen receptor-mediated
`pathway or that ICI does not have access to estrogen target
`cells in the growth plate. The growth plate is avascular, and
`it is possible that there are differences in the ability of ICI and
`natural estrogens to diffuse through the extracellular matrix
`that separates chondrocytes. We recognize, however, that the
`effect of estrogen deficiency by ovariectomy on longitudinal
`growth rate is, itself, transient (28, 29) and subsides generally
`by 6 weeks after surgery.
`Older animals, however, were used exclusively for mea-
`surements of remodeling in cancellous bone of the tibia (37).
`The rates of radial or longitudinal bone growth measured in
`the older rats of Exps 2 and 3 were very low, which reflects
`the skeletal maturity of these animals. We only reported data
`on radial and longitudinal growth from the young animals,
`because
`formation rates
`could
`be more
`accurately
`determined.
`
`In the mature rats, we observed that the effects of ICI on
`cancellous bone mass and most indices of bone remodeling
`depended on gonadal status. The effects of ICI on cancellous
`bone area, labeled bone perimeter, trabecular number, tra-
`becular separation, and osteoclast-covered perimeter were
`consistent with a potent estrogen antagonist in the presence
`of significant levels of serum estrogen, as found in ovary-
`intact rats. In the ovariectomized rat, where serum estrogen
`is significantly less, ICI behaved as a weak partial estrogen
`agonist on the same indices of cancellous bone architecture
`and turnover. Gallagher et al. (15) also show a detrimental
`effect of ICI on cancellous bone volume in growing sham-
`operated rats, but the actions of ICI on cancellous bone vol-
`ume in ovariectomized rats were not investigated. The ob-
`servations of our study led us to the inescapable conclusion
`that, in contrast to the reproductive tissues, ICI is not a pure
`estrogen antagonist in bone, and ICI seems to be capable of
`transcriptional activation.
`This conclusion is further supported by the observed
`changes in the cancellous mineral apposition rate. The min-
`eral apposition rate generally reflects osteoblast activity,
`whereas the previously discussed labeled perimeter reflects
`osteoblast number (36). Ovariectomy results in a small in-
`crease in the mineral apposition rate, which is prevented by
`treatment with estrogen (32, 38, 39). The observed reduction
`in the mineral apposition rate of ovary-intact rats and the
`reducing trend observed in the ovariectomized rats indicate
`that the actions of ICI on osteoblast activity are of an estrogen
`agonist. These observations suggest that ICI differentially
`
`influences the estrogen-regulated pathways that mediate the
`hormone’s actions on osteoblast number and activity.
`The bone formation rate, for instance, is the product of
`osteoblast number and activity. Ovariectomy results in in-
`creases in the number and activity of osteoblasts and, as a
`consequence, leads to a large increase in the bone formation
`rate. In contrast, ICI was primarily an estrogen antagonist on
`osteoblast number and an estrogen agonist on osteoblast
`activity. As a consequence of the two opposing tendencies,
`ICI had no net effect on the calculated bone formation rate.
`
`We did not measure bone resorption, but a measurement of
`osteoclast-covered perimeter indicates that ICI was a estro-
`gen agonist on osteoclast number in the ovariectomized rats.
`And, though there is only a tendency for osteoclasts to be
`increased by ICI in the ovary-intact rat, the observed os—
`teopenia, without an accompanying reduction in bone for-
`mation, implicates an elevation in bone resorption.
`If ICI were unable to activate transcription when bound to
`the estrogen receptor in reproductive tissues, then the ob-
`served estrogen agonism of ICI on bone would be thro