0022-3565/97/2811—0142$03.00/0
`THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS
`Copyright © 1997 by The American Society for Pharmacology and Experimental Therapeutics
`JPET 281:1427148, 1997
`
`Vol. 281, No. 1
`Printed in USA
`
`Tamoxifen and ICI 182,780 Interactions with Thyroid Hormone
`in the Ovariectomized-Thyroidectomized Fiat1
`
`VINCENT A. DIPIPPO2 and C. ANDREW POWERS
`
`Department of Pharmacology, New York Medical College, Valhalla, New York
`Accepted for publication December 18, 1996
`
`ABSTRACT
`Studies of estradiol and tamoxifen actions to modulate the
`actions of thyroid hormone (triiodothyronine, T3) in the rat have
`shown that a subset of estrogen responses require T3 for
`expression. Also, tamoxifen acts as a partial agonist in estrogen
`responses that are T3 independent, but acts as a full estrogen
`agonist
`in T3-dependent responses. This study examined
`whether the differing behavior of tamoxifen (a triphenylethylene
`antiestrogen) in T3-independent and T3-dependent estrogen
`responses would be shared with ICI 182,780, a steroidal an-
`tiestrogen. An ovarieotomized-thyroideotomized rat model was
`used. Drug vehicle, tamoxifen alone (0.4 mg/kg),
`ICI 182,780
`alone (2 mg/kg) or tamoxifen plus ICI 182,780 were given for 3
`weeks to ovarieotomized-thyroideotomized rats with or without
`T3 replacement
`(10 pug/kg). T3-independent estrogen re-
`sponses measured were the induction of uterine growth and
`induction of pituitary growth hormone (GH) in the absence of
`
`T3. T3-dependent estrogen responses measured were antag-
`onism of T3-evoked increases in pituitary GH, body weight,
`tibia length and hepatic malio enzyme, and increases in serum
`triglycerides. Tamoxifen acted as a partial agonist in T3-inde-
`pendent estrogen responses, whereas ICI 182,780 acted as a
`potent pure antagonist in such responses;
`it lacked agonist
`efficacy and totally blocked tamoxifen effects. In T3-dependent
`estrogen responses, tamoxifen acted as a full estrogen agonist.
`ICI 182,780 acted as a weak agonist in some T3-dependent
`responses and lacked agonist efficacy in others. Moreover, ICI
`182,780 had poor efficacy in blocking tamoxifen actions in
`T3-dependent responses. The results indicate that ICI 182,780,
`like tamoxifen, displays a duality in its pharmacological behav-
`ior which pivots on the T3 dependence of the estrogen re-
`sponse.
`
`Estrogens have important metabolic effects in addition to
`their actions to induce the growth and maturation of female
`reproductive organs. In women, estrogens decrease cardio-
`vascular risk by complex effects on lipid metabolism (Nabulsi
`et al., 1998), and are essential for the maintenance of bone
`mass (Lindsay et al., 1980). Estrogens also alter somatic
`growth and energy, lipid and bone metabolism in rats (Wade
`and Gray, 1979; Wade and Schneider, 1992; Kalu, 1991;
`Turner et al., 1994). These metabolic actions of estrogens
`provide much of the rationale for their widespread use in
`postmenopausal women. Nonetheless, the physiological and
`molecular pathways responsible for these actions remain ob-
`scure.
`
`tumor growth (Jordan and Murphy,
`estrogen-dependent
`1990). Surprisingly, such therapy is associated with de-
`creases in cardiovascular risk (Love at al., 1991) and bone
`loss (Love at al., 1987). Thus, tamoxifen inhibits breast can-
`cer growth by blocking the actions of endogenous estradiol
`while paradoxically lessening cardiovascular disease and
`bone loss by apparent supplemental estrogen agonist actions.
`Similarly, in rats tamoxifen fully mimics estradiol effects on
`growth, bone mass and energy balance, but inhibits estradiol
`effects on the uterus and other targets (Jordan et al., 1987;
`Wade and Heller, 1998). This duality in tamoxifen’s behavior
`is difficult to understand. The nature of ER mechanisms
`
`Tamoxifen is a triphenylethylene antiestrogen with partial
`agonist activity in classic estrogen responses (e.g., induction
`of rat uterine growth) via direct binding to the ER. Tamox-
`ifen is widely used in breast cancer therapy to antagonize
`
`argue against a role for “spare receptors” as a means of
`tamoxifen’s dual agonist-antagonist behavior (see DiPippo et
`al., 1995 for discussion). A second type of ER (beta) has
`recently been identified (Kuiper et al., 1996) and might con-
`ceivably provide a mechanism for tamoxifen’s full agonist
`behavior. However, tamoxifen acted as an antagonist rather
`Received for publication July 23, 1996.
`than full agonist on estrogen—dependent gene expression
`1 Supported in part by grant 947404 from the American Heart Association,
`driven by ER-beta (Kuiper et al., 1996).
`New York State Affiliate.
`Our laboratory has recently reported evidence which indi-
`2 Present address: Department of Biochemistry BX B4—RAL, University of
`cates that certain of the metabolic actions of estradiol and
`Illinois, 600 S. Mathews Ave., Urbana, IL 61801-3602.
`
`
`ABBREVIATIONS: T3, 3,3’,5-triiodo-L-thyronine; T4, thyroxine; TR, thyroid hormone receptor; ER, estrogen receptor; GH, growth hormone; IGF-1,
`insulin-like growth factor 1.
`142
`
`AstraZeneca Exhibit 2163 p. 1
`lnnoPharma Licensing LLC V. AstraZeneca AB lPR2017-00900
`Fresenius-Kabi USA LLC V. AstraZeneca AB IPR2017-01913
`
`

`

`1997
`
`tamoxifen may arise from interference with the actions of
`thyroid hormone (T8) (DiPippo et al., 1995; DiPippo and
`Powers, 1991). With use of ovariectomized-thyroidectomized
`rats, it was found that estradiol and tamoxifen inhibited T8
`effects on pituitary GH, somatic growth, bone and hepatic
`malic enzyme (an index of T8 actions on lipid metabolism),
`and lacked inhibitory effects on these measures in the ab-
`sence of T8 (T8-dependent responses). Estradiol and tamox-
`ifen effects to increase serum triglycerides were also com-
`pletely T8 dependent. The above-mentioned responses were
`target- or response-selective rather than generalized: estra-
`diol and tamoxifen did not block T8 suppression of pituitary
`thyrotropin secretion or T8 actions to increase pituitary pro-
`lactin. Conversely, estradiol and tamoxifen effects on the
`uterus, luteinizing hormone levels and pituitary prolactin
`and kallikrein occurred with or without T8 (T8 independent).
`Moreover, tamoxifen acted as a full estrogen agonist in T8-
`dependent responses but acted as an antiestrogen in effects
`that were T8 independent. Zhou-Li et al. (1992) also reported
`that antiestrogens can antagonize T8: 4-hydroxytamoxifen
`inhibited growth stimulatory effects of T8 in multiple cell
`lines. In addition, antiestrogens did not interfere with T8
`binding to TR (Zhou-Li et al., 1992), which suggests complex
`mechanisms possibly related to ER-TR interactions at the
`transcriptional level (cross-talk). The previously noted selec-
`tivity of estradiol and tamoxifen modulation of T8 actions in
`animals further argues for mechanisms involving ER-TR
`cross-talk rather than generalized alterations in T8 binding
`to TR or altered T8 pharmacokinetics. Overall, the findings
`above suggest that some metabolic effects of estradiol arise
`by ER-mediated antagonism of the T8-TR complex at certain
`T8 targets. Tamoxifen fully mimics such estradiol effects
`while acting as an antiestrogen in T8-independent estrogen
`responses (DiPippo et al., 1995).
`Steroidal antiestrogens (ICI 164,884 and ICI 182,780) have
`recently been developed which lack estrogen agonist activity
`and act as pure antagonists in classic estrogen target tissues
`such as the uterus (Wakeling et al., 1991; Wakeling, 1995).
`These drugs represent an important advance in antiestrogen
`pharmacology and are being evaluated for improved efficacy
`in breast cancer therapy. It was of interest to contrast the
`actions of tamoxifen (which displays partial agonist activity)
`with those of steroidal antiestrogens in T8-dependent and
`T8-independent estrogen responses. This study reports a
`comparison of tamoxifen and ICI 182,780 actions in ovariec-
`tomized-thyroidectomized rats in the presence or absence of
`T8.
`
`Materials and Methods
`
`Animals. All procedures were approved by the institutional Ani-
`mal Care and Use Committee following guidelines approved by the
`National Institutes of Health. Three week in vivo treatment proto-
`cols were used (8% of rat lifespan) to identify effects of chronic
`hormonal interactions potentially involving ER-TR cross-talk: efforts
`were made to minimize the possibility of effects arising via alter-
`ations in drug or hormone pharmacokinetics (see below). Female CD
`rats (175—200 g, Charles Rivers, Wilmington, DE) were ovariecto-
`mized and thyroidectomized as described previously (DiPippo et al.,
`1995), and treatments were begun 2 weeks after the final surgery.
`Drinking water contained 1% calcium gluconate to maintain calcium
`balance and 0.025% propylthiouracil to block T8 production by any
`residual thyroid tissue. Propylthiouracil also inhibits deiodinases
`
`Antiestrogen Interactions with T3
`
`143
`
`involved in T8 catabolism to diiodo metabolites (Kohrle et al., 1991):
`use of propylthiouracil reduces the possibility that drug effects may
`reflect alterations in such reactions. Trans-tamoxifen (free base) (0.4
`mg/kg; Sigma Chemical CO., St. Louis, MO) and ICI 182,780 (2
`mg/kg; Zeneca Pharmaceuticals, Macclesfield, UK) were adminis-
`tered so every 24 h in sesame oil containing 10% benzyl alcohol and
`20% ethanol; control animals received vehicle. Dose-response studies
`in ovariectomized and ovariectomized-thyroidectomized rats have
`shown that 0.2 mg/kg tamoxifen produces maximal estrogen agonist
`effects, as well as maximal antagonist effects on moderate doses of
`estradiol benzoate or T8 (Powers et al., 1989; DiPippo et al., 1995).
`Similarly, dose-response studies in the rat have shown that 0.8
`mg/kg ICI 182,780 (s.c.) produces maximal inhibition of moderate
`estradiol doses, and a 5:1 ratio of ICI 182,780 to tamoxifen fully
`blocks tamoxifen’s agonist effects on the uterus (Wakeling et al.,
`1991; Wakeling and Bowler, 1992). A physiological replacement dose
`of T8 (sodium salt) (10 ug/kg; Sigma) was administered i.p. every
`24 h in 0.9% NaCl containing 5 mM NaOH; control animals received
`vehicle. Use of T8 rather than T4 minimizes the possibility that
`drug-evoked changes in transthyretin and T4-binding globulin (se-
`rum-binding proteins) could alter hormone pharmacokinetics and
`actions because T8 has much weaker affinity for these binding pro-
`teins (Robbins, 1991). Use of T8 negates the possibility ofdrug effects
`arising from T4 deiodination to T8. Drug and T8 treatments were
`given for 8 weeks with five rats per group; rats were weighed daily.
`Rats were then killed with 100 mg/kg sodium pentobarbital (i.p.).
`Blood samples were obtained for serum GH and triglyceride deter-
`minations within 8 to 5 min after pentobarbital injection, and tissues
`were collected as described previously (DiPippo et al., 1995).
`Experimental measures. Dissected uteri were stripped of fat,
`drained of luminal fluid and dried for 48 h at room temperature
`before weighing. The right tibias were stripped of all muscle and
`connective tissue, and stored in 0.9% NaCl at 5°C until measurement
`of tibia length with calipers. Pituitary and serum GH levels were
`measured by radioimmunoassay as described previously (DiPippo et
`al., 1995) with reagents provided by the National Hormone and
`Pituitary Program. Rat GH RP-2 was used as the standard. Hepatic
`malic enzyme and serum triglycerides were measured by enzymatic
`assay as described previously (DiPippo et al., 1995).
`Statistics. Data were analyzed by one-analysis of variance fol-
`lowed by Duncan’s New Multiple Range Test. Where appropriate,
`data were log transformed to equalize variances.
`
`Resufls
`
`In the following sections, tamoxifen and ICI 182,780 are, at
`times, pharmacologically characterized as T8 partial ago-
`nists or T8 antagonists. It should be noted that this charac-
`terization is based on pharmacological responses involving
`complex, poorly understood biochemical events rather than a
`mechanism of direct antiestrogen-T8 competition for TR
`binding (see the introduction). We believe such pharmacolog-
`ical characterization is useful for modeling and analysis of
`responses arising from ER and TR interactions in complex
`physiological networks of responsive genes: the mechanistic
`distinctions should be borne in mind (also see fig. 5).
`Tamoxifen and ICI 182,780 effects on uterine dry
`weight. Induction of uterine wet or dry weight is a classic
`estrogen response and has been the most widely used bioas-
`say system for the characterization of antiestrogens. As
`shown in figure 1, tamoxifen almost doubled uterine dry
`weight in either the presence or absence of T8; this repre-
`sents about 80% of the maximal induction elicited by estra-
`diol in the ovariectomized-thyroidectomized rat (DiPippo et
`al., 1995). ICI 182,780 lacked estrogen agonist activity in this
`response and completely blocked tamoxifen induction of uter-
`
`AstraZeneca Exhibit 2163 p. 2
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`Vol. 281
`
`ifen markedly inhibited GH induction by T8 (—58%) (fig. 2).
`Surprisingly, ICI 182,780 also inhibited GH induction by T8
`(—21%), but more weakly than tamoxifen. Moreover, ICI
`182,780 failed to block tamoxifen antagonism of T8 induction
`of GH despite its action to block tamoxifen effects on the
`uterus in the same rats. It should be noted that testosterone
`
`propionate does not induce pituitary GH in thyroidectomized
`rats although it increases GH in thyroid-intact rats (DiPippo
`and Powers, 1991), apparently via hypothalamic effects
`(Jansson et al., 1985). This suggests that hypothalamic ef-
`fects are unlikely to explain tamoxifen effects on GH. Indeed,
`ER is expressed in 80 to 90% of rat somatotrophs (Keefer et
`al., 1976; Shirasu et al., 1990), and T8, E2 and tamoxifen
`have been shown to directly alter GH production in pituitary
`cell culture (see “Discussion”).
`In the absence of T8, neither tamoxifen nor ICI 182,780
`significantly altered serum GH levels (fig. 2), although ta-
`moxifen alone produced a trend toward an increase. T8 dou-
`bled serum GH levels, and neither tamoxifen nor ICI 182,780
`alone altered this increase; rats treated with both tamoxifen
`and ICI 182,780 exhibited low serum GH levels. The poor
`correlation between pituitary GH content and serum GH
`levels is not surprising because rats can maintain normal
`serum GH levels as long as pituitary GH content remains at
`or above 10 to 15% of normal values (Peake et al., 1978; Coiro
`et al., 1979; Coulombe et al., 1978). Nonetheless, the data
`suggest that tamoxifen effects on somatic growth are un-
`likely to reflect altered GH secretion (see below).
`In the absence of T8, rats exhibited an 18-g weight gain
`and neither tamoxifen nor ICI 182,780 significantly affected
`this gain over the course of the experiment (fig. 8). T8 pro-
`duced a 76-weight gain and tamoxifen completely blocked T8
`actions to increase weight. ICI 182,780 did not alter T8
`effects to increase body weight but partially blocked tamox-
`ifen actions to inhibit weight gain (fig. 8).
`In the absence ofT8, tamoxifen or ICI 182,780 had no effect
`on tibia length, an index of longitudinal bone growth. T8
`produced a 2.6-mm increase in tibia length and tamoxifen
`inhibited this growth by 70% (fig. 8). ICI 182,780 did not
`inhibit T8-induced tibia growth and also did not block tamox-
`
`..
`
`(0.4
`Fig. 2. Effect of tamoxifen (TAM)
`mg/kg),
`ICI 182,780 (ICI)
`(2 mg/kg) or
`both on pituitary and serum GH levels
`in ovariectomized-thyroidectomized rats
`with or without T3 treatment (10 ug/kg).
`(left panel) pituitary GH content (pg/pitu-
`itary); (right panel) serum GH. *P > .05
`vs. no T3 vehicle control (CON). “P >
`.05 vs. T3-treated vehicle control. “P >
`.05 vs. all other groups.
`
`144
`
`DiPippo and Powers
`
`*
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`
`CON TAM ICI TAM T3
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`UterineDryWeight(mg)
`
`(2
`ICI 182,780 (ICI)
`(0.4 mg/kg),
`Fig. 1. Effect of tamoxifen (TAM)
`mg/kg) or both on uterine dry weight in ovariectomized-thyroidecto-
`mized rats with or without T3 treatment (10 ug/kg). In this and all other
`figures each group contained five rats; values represent the mean :
`S.E.M. Open bars, no T3; filled bars, T3 treated. *P > .05 vs. no T3
`vehicle control (CON).
`
`ine weight. T8 alone was without effect on uterine weight,
`and it did not alter tamoxifen or ICI 182,780 actions. These
`data indicate that tamoxifen and ICI 182,780 were equally
`efficacious in their interactions with the ER in either the
`
`presence or absence of T8; the differential behavior of tamox-
`ifen and ICI 182,780 in T8-dependent responses (see below)
`is unlikely to reflect alterations in drug pharmacokinetics.
`Tamoxifen and ICI 182,780 effects on GH, body
`weight and tibia length. In the absence of T8, tamoxifen
`induced pituitary GH levels by 10-fold; this represents about
`20% of the maximal response which can be evoked by estra-
`diol, and about 7% of the maximal response evoked by T8
`(DiPippo et al., 1995). ICI 182,780 lacked effect on GH in the
`absence of T8 and completely blocked tamoxifen induction of
`GH (fig. 2). T8 induced pituitary GH by 185-fold, and tamox-
`
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`AstraZeneca Exhibit 2163 p. 3
`
`

`

`1997
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`Antiestrogen Interactions with T3
`
`145
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`Fig. 3. Effect of tamoxifen (TAM)
`mg/kg),
`ICI 182,780 (ICI)
`(2 mg/kg) or
`both on body weight change and tibia
`length
`in ovariectomized-thyroidecto-
`mized rats with or without T3 treatment
`(10 pg/kg). (left panel) body weight gain
`during the course of the experiment;
`(right panel) right tibia length. *P > .05
`vs. no T3 control (CON). “P > .05 vs.
`T3-treated vehicle control.
`
`ifen antagonism of T8-induced tibia growth. As noted above,
`tamoxifen actions to alter weight gain and tibia length are
`unlikely to reflect the alterations in pituitary GH levels,
`because in this and previous studies (DiPippo et al., 1995) we
`have shown that neither tamoxifen nor estradiol alter serum
`
`GH in T8-treated rats despite their marked effects on pitu-
`itary GH content. On the other hand, there is considerable
`evidence which indicates that T8 is required for GH to fully
`manifest its somatotropic effects, and that estrogens can
`interfere with the somatotropic effects of GH (see DiPippo et
`al., 1995 for discussion). T8 and estrogens can also directly
`alter IGF-l expression independent from GH (Dickson and
`Lippman, 1987; Murphy et al., 1987; Samuels et al., 1988).
`IGF-l mediates much of the somatotropic effects of GH, and
`tamoxifen modulation of IGF-1 actions or its regulation by T8
`and GH remains to be fully explored (see Pollak et al., 1992).
`Tamoxifen and ICI 182,780 effects on malic enzyme
`and serum triglycerides. In the absence of T8, tamoxifen
`
`and ICI 182,780 lacked effect on hepatic malic enzyme (which
`supplies reducing equivalents that can be used for lipogene-
`sis). T8 almost tripled malic enzyme (fig. 4), and tamoxifen
`inhibited this induction by 47% (T8 evoked a 7.5-U increase
`in the absence of tamoxifen compared with a 4.0-U increase
`in the presence of tamoxifen). ICI 182,780 reduced T8 induc-
`tion of malic enzyme by 25%, but this decrease did not
`achieve statistical significance: ICI 182,780 did not block
`tamoxifen actions on T8 induction of malic enzyme.
`In the absence of T8, neither tamoxifen nor ICI 182,780
`affected serum triglyceride levels. T8 alone produced a 44%
`decrease in triglyceride levels. In the presence of T8, tamox-
`ifen evoked a 4-fold rise in triglycerides (fig. 4); ICI 182,780
`almost doubled triglycerides, and appeared to partially in-
`hibit tamoxifen effects. It should be noted that T8 exerts
`
`complex effects on lipid metabolism, and increases both lipo-
`genesis,
`lipid mobilization and lipid catabolism (Ingbar,
`1985; Oppenheimer et al., 1991), with T8 actions to increase
`
`I.
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`Fig. 4. Effect of tamoxifen (TAM)
`mg/kg),
`ICI 182,780 (ICI)
`(2 mg/kg) or
`both on hepatic malic enzyme and se-
`rum triglycerides in ovariectomized-thy-
`roidectomized rats with or without T3
`treatment (10 pg/kg). (left panel) malic
`enzyme; units are nanomoles NAD+
`formed/min/mg protein. (right panel) se-
`rum triglycerides. *P > .05 vs. no T3
`vehicle control (CON). “P > .05 vs. T3-
`treated vehicle control.
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`AstraZeneca Exhibit 2163 p. 4
`
`

`

`146
`
`DiPippo and Powers
`
`Vol. 281
`
`lipid turnover and catabolism predominating. Tamoxifen
`may more greatly inhibit T8 effects on lipid catabolism, and
`yield a T8-dependent increase in triglycerides (see DiPippo et
`al., 1995).
`
`Discussion
`
`The present study confirms our previous findings regard-
`ing tamoxifen modulation of T8 actions. Thus, tamoxifen
`behaves as a T8 antagonist in T8 actions to induce GH and
`increase body weight and tibia length. Tamoxifen also inhib-
`ited T8 induction of hepatic malic enzyme, and tamoxifen
`actions to increase triglycerides were entirely T8 dependent.
`In the absence of T8,
`tamoxifen lacked effect on all T8-
`dependent estrogen responses except for pituitary GH induc-
`tion, in which the tamoxifen-ER complex appears to both
`partially mimic and inhibit the actions of the T8-TR complex
`(i.e., act as a T8 partial agonist) (DiPippo and Powers, 1991;
`DiPippo et al., 1995). In all of the responses above tamoxifen
`mimics the actions of estradiol; hence, these effects are me-
`diated by ER rather than by distinct antiestrogen binding
`sites. In terms of its pharmacological character, tamoxifen
`generally behaves as a partial agonist in estrogen responses
`which occur in the absence of T8 (uterine growth, pituitary
`GH induction), whereas tamoxifen fully mimics estrogen ef-
`fects which are T8 dependent (decreases in pituitary GH
`levels, weight gain, longitudinal growth and malic enzyme,
`and increases in triglycerides) (Jordan et al., 1987; Wade and
`Heller, 1998; DiPippo et al., 1995; DiPippo and Powers,
`1991).
`The behavior of ICI 182,780 was distinct from tamoxifen.
`In agreement with previous results (Wakeling et al., 1991;
`Wakeling and Bowler, 1992), ICI 182,780 behaved as a pure
`estrogen antagonist on uterine growth, lacking agonist effi-
`cacy and completely blocking tamoxifen effects in either the
`presence or absence ofT8. Similarly, in the absence ofT8, ICI
`182,780 lacked agonist efficacy on GH induction and com-
`pletely blocked GH induction by tamoxifen. In the presence of
`T8, however, ICI 182,780 partially mimicked tamoxifen ef-
`fects on pituitary GH and serum triglycerides. Nonetheless,
`ICI 182,780 did not match the efficacy of tamoxifen in such
`actions and did not mimic tamoxifen inhibition of T8 effects
`
`on weight gain or tibia growth. Furthermore, ICI 182,780
`was poorly effective in blocking tamoxifen actions that were
`T8 dependent. Overall, ICI 182,780 behaved as a potent pure
`antagonist in estrogen responses occurring in the absence of
`T8, but behaved as a weak partial agonist or was inactive in
`estrogen responses that were T8 dependent. Thus,
`ICI
`182,780 shares with tamoxifen a duality in its pharmacolog-
`ical character which pivots on the T8 dependence of the
`responses.
`A surprising aspect of this study was the general ineffec-
`tiveness of ICI 182,780 in blocking T8-dependent tamoxifen
`effects despite its ability to fully block tamoxifen effects
`which are not T8 dependent. For example,
`ICI 182,780
`blocked tamoxifen induction of GH but did not block tamox-
`
`ifen antagonism of T8 induction of GH. T8, estradiol and
`tamoxifen appear to act directly on pituitary somatotrophs to
`alter GH production (Komolov et al., 1980; Webb et al., 1988;
`Simard et al., 1986; Malaab et al., 1992). Thus, the disparate
`effects seem unlikely to reflect tissue-specific uptake or me-
`tabolism ofICI 182,780. Others have also reported data con-
`
`sistent with the phenomenon. Wakeling and Bowler (1992)
`found that ICI 182,780 markedly decreased uterine weight in
`intact female rats but failed to increase body weight, whereas
`ovariectomy both decreased uterine weight and increased
`body weight. Wade et al. (1998) found that ICI 182,780 com-
`pletely blocked uterine growth induced by estradiol or tamox-
`ifen in ovariectomized rats. In the same animals, however,
`ICI 182,780 only weakly blocked estradiol and tamoxifen
`effects to decrease fat depots and longitudinal growth. Gal-
`lagher et al. (1998) reported that ICI 182,780 blocked estra-
`diol effects to increase uterine weight and cancellous bone
`volume, but lacked effect on estradiol actions to decrease
`body weight and bone growth in the same rats. Indeed, ICI
`182,780 blocked estradiol effects on cancellous bone volume
`in the rat tibia while having no effect on estradiol effects to
`inhibit tibial longitudinal or periosteal growth (Gallagher et
`al., 1998). This again suggests that tissue-specific uptake or
`metabolism is unlikely to explain the disparate behavior of
`101 182,780.
`We have proposed that T8-dependent estrogen responses
`may reflect ER and TR cross-talk at DNA sequences mediat-
`ing receptor binding and transcriptional regulation (DiPippo
`et al., 1995; DiPippo and Powers, 1991). Tamoxifen may
`transform the ER to a form that evokes ER-TR interactions
`
`at genes with combinations of ER-TR binding elements. At
`such targets ER may interfere with TR actions while evoking
`little transactivation itself, and thus primarily act to modu-
`late T8 action (see fig. 5). Both estradiol and tamoxifen and
`related triphenylethylenes can transform the ER to forms
`with enhanced affinity for DNA targets in vivo (Sutherland et
`al., 1977; Katzenellenbogen et al., 1979; Clark et al., 1978).
`Thus, tamoxifen would be predicted to fully mimic estradiol
`actions in responses arising from ER-TR cross-talk. On the
`other hand, antiestrogens that fail
`to evoke strong DNA
`binding would be expected to lack agonist efficacy in estrogen
`responses arising from ER-TR cross-talk. Interpretation of
`ICI 182,780 effects is complicated by controversy about the
`actions of pure antiestrogens with respect to ER binding to
`DNA (see Metzger et al., 1995). Nonetheless, there is consid-
`erable evidence indicating that ICI 182,780 decreases cellu-
`lar ER levels and does not trigger ER transformation and
`nuclear retention analogous to that caused by estradiol or
`tamoxifen (Gibson et al., 1991; Fawell et al., 1990; Reese and
`Katzenellenbogen, 1991; Arbuckle et al., 1992; Dauvois et al. ,
`1992; Reese and Katzenellenbogen, 1992). Thus, the poor
`efficacy of ICI 182,780 in T8-dependent estrogen responses
`seems consistent with ER modulation of TR via cross-talk.
`
`We expected ICI 182,780 to potently block T8-dependent
`effects of tamoxifen. This was not observed and might reflect
`incomplete blockade of the ER by ICI 182,780. The 5:1 ratio
`ofICI 182,780 to tamoxifen may allow incremental ER trans-
`formation by tamoxifen over several days. ICI 182,780 is
`ineffective in preventing ER binding to DNA once the ER
`becomes transformed (Fawell et al., 1990; Reese and Kat-
`zenellenbogen, 1991; Dauvois et al. , 1992), and thus would be
`unable to fully block tamoxifen-evoked ER-TR cross-talk in
`such conditions. Conversely, although ICI 182,780 cannot
`block binding of transformed ER to DNA in vitro, it blocks the
`binding of the transformed ER to coactivators needed for
`transactivation (Halachmi et al. , 1994; Cavailles et al., 1994).
`This would enable ICI 182,780 to fully block T8-independent
`tamoxifen responses (which require ER-evoked transactiva-
`
`AstraZeneca Exhibit 2163 p. 5
`
`

`

`1997
`
`Tit-independent Estrogen Response
`
`
`
`
`
`E2 =full aganist ; TAM = partial agonist
`
`T3-dependent Estrogen Response
`(ER-TR cross—talk)
`
`
`
`
`
`
`i
`
`T3 + TAM or E2
`
`T3 requiredfor E2 response"
`E2 "—=full agonisr; TAM 2full agonist.
`Fig. 5. A model of ER function enabling the dual behavior of tamoxifen
`(TAM).
`In T3-independent estrogen responses the ER acts as the pri-
`mary driver for the response via direct transactivation of target genes
`containing ER-binding elements (ERE).
`In such responses tamoxifen
`cannot evoke the full transactivation properties of the ER and acts as a
`partial agonist/antagonist.
`In T3-dependent estrogen responses, the
`TR acts as the primary driver for the response, and ER acts to modulate
`T3 actions via ER-TR interactions at selective target genes (ER-TR
`cross-talk). This may involve the participation of combinations of TR-
`and ER-binding elements (TRE + ERE) in the target genes. ER modu-
`lator action in the T3-dependent model is postulated to involve inter-
`ference with TR action rather than direct ER transactivation, and ta-
`moxifen fully mimics estradiol because both activate high-affinity target
`gene binding by ER.
`
`tion) even if tamoxifen elicited ER binding to DNA in the
`presence of ICI 182,780.
`The physiological and pharmacological findings presented
`suggest that ER-TR cross-talk might underlie a subset of
`estradiol and tamoxifen actions. Studies with molecular ap-
`proaches in combination with further physiological and phar-
`macological analyses are needed to confirm this mechanism.
`Regardless of the ultimate mechanisms involved, it is clear
`that there is a strong association between the T8 dependence
`of an estrogen response and the pharmacological character of
`antiestrogens in that response. This association is unlikely to
`be coincidental and involves estrogen actions of considerable
`physiological and clinical significance. Indeed, T8 is a major
`metabolic hormone which plays an important role in growth,
`energy, lipid and bone metabolism. Modulation of T8 actions
`by estrogens may represent an efficient mechanism for the
`integrative regulation of multiple metabolic systems to meet
`the unique demands of female reproductive biology. More-
`over, modulation of T8 actions may provide a novel rationale
`for the use of antiestrogens such as tamoxifen in men. Such
`agents might enable men to enjoy the beneficial effects of
`estrogens on lipid and bone metabolism without experiencing
`
`Antiestrogen Interactions with T3
`
`147
`
`adverse feminizing or thromboembolic effects. Further anal-
`ysis of the nature and significance of estrogen and antiestro-
`gen interactions with T8 seems warranted.
`
`References
`
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`DNA binding activity of oestrogen receptors in vitro. Nucleic Acids Res. 20:
`383973844, 1992.
`CAVAILLES, V., DAUVOIS, S., DANIELIAN, P. S. AND PARKER, M. G.: Interaction of
`proteins with transcriptionally active estrogen receptors. Proc. Natl. Acad.
`Sci. U.S.A. 91: 10009710013, 1994.
`CLARK, J. H., ANDERSON, J. H. AND PECK, E. I. J.: Estrogen receptor antiestrogen
`complex: atypical binding by uterine nuclei and effects on uterine growth.
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`COIRO, C., BRAVERMAN, L. E., CHRISTIANSON, D., LANG, S. AND GOODMAN, H. M.:
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`DAUVOIS, S., DANIELIAN, P. S., WHITE, R. AND PARKER, M. G.: Antiestrogen ICI
`164,383 reduces cellular estrogen receptor content by increasing its turn-
`over. Proc. Natl. Acad. Sci. U.S.A. 89: 403774041, 1992.
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`DIPIPPO, V. A., LINDSAY, R. AND POWERS, C. A.: Estradiol and tamoxifen inter-
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`thyroidectomized rat. Endocrinology 129: 169671700, 1991.
`FAWELL, S. E., WHITE, R., HOARE, S., SYDENHAM, M., PAGE, M. AND PARKER, M. G.:
`Inhibition of estrogen receptor-DNA binding by the “pure” antiestrogen ICI
`164,384 appears to be mediated by impaired receptor dimerization. Proc.
`Natl. Acad. Sci. U.S.A. 8'7: 688376887, 1990.
`GALLAGHER, A., CHAMBERS, T. J. AND TOBIAS, J. H.: The estrogen antagonist ICI
`182,780 reduces cancellous bone volume in female rats. Endocrinology 133:
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`GIBSON, M. K., NEMMERS, L. A., BECKMAN, W. C., DAVIS, V. L., CURTIS, S. W. AND
`KORACH, K. S.: The mechanism of ICI 164,384 antiestrogenicity involves
`rapid loss of es