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Endocrinology, doi:10.1210/en.2008-0532
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Endocrinology Vol. 149, No. 10 5219-5226
Copyright © 2008 by The Endocrine Society

ICI 182,780 Penetrates Brain and Hypothalamic Tissue and Has Functional Effects in the Brain after Systemic Dosing

Peter D. Alfinito, Xiaohong Chen, James Atherton, Scott Cosmi and Darlene C. Deecher

Women’s Health and Musculoskeletal Biology (P.D.A., X.C., S.C., D.C.D.), Drug Safety and Metabolism (J.A.), Wyeth Research, Collegeville, Pennsylvania 19426

Address all correspondence and requests for reprints to: Darlene C. Deecher, Ph.D., Wyeth Research, RN 3164, 500 Arcola Road, Collegeville, Pennsylvania 19426. E-mail: deeched{at}wyeth.com.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Previous reports suggest the antiestrogen ICI 182,780 (ICI) does not cross the blood-brain barrier (BBB). However, this hypothesis has never been directly tested. In the present study, we tested whether ICI crosses the BBB, penetrates into brain and hypothalamic tissues, and affects known neuroendocrine functions in ovariectomized rats. Using HPLC with mass spectrometry, ICI (1.0 mg/kg·d, 3 d) was detected in plasma and brain and hypothalamic tissues for up to 24 h with maximum concentrations of 43.1 ng/ml, and 31.6 and 38.8 ng/g, respectively. To evaluate antiestrogenic effects of ICI in the brain after systemic dosing, we tested its ability to block the effect of 17 {alpha}-ethinyl estradiol (EE) (0.3 mg/kg, 8 d) on tail-skin temperature abatement in the morphine-dependent model of hot flush and on body weight change. In the morphine-dependent model, EE abated 64% of the naloxone-induced tail-skin temperature increase. ICI pretreatment (1.0, 3.0 mg/kg·d) dose dependently inhibited this effect. ICI (3.0 mg/kg·d) alone showed estrogenic-like actions, abating 30% the naloxone-induced flush. In body weight studies, EE-treated rats weighed 58.5 g less than vehicle-treated rats after 8 d dosing. This effect was partially blocked by ICI (3.0 mg/kg·d) pretreatment. Similar to EE treatment, rats receiving 1.0 or 3.0 mg/kg·d ICI alone showed little weight gain compared with vehicle-treated controls. Thus, ICI crosses the BBB, penetrates into brain and hypothalamic tissues, and has both antiestrogenic and estrogenic-like actions on neuroendocrine-related functions.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
THE ANTIESTROGEN ICI 182,780 (ICI) is a steroidal compound that has been widely used in both preclinical and clinical research studies. Reportedly, the compound does not cross the blood-brain barrier (BBB) (1, 2, 3), and has been used in preclinical studies to understand the biological roles of estrogen receptor (ER) signaling and distinguish peripheral vs. central effects of estrogens (1, 2, 3, 4, 5). Results from clinical studies suggest that ICI (fulvestrant) may be useful in treating advanced, hormone receptor-positive breast cancer in postmenopausal women (6, 7). In contradiction to its reported inability to cross the BBB, clinical studies in premenopausal women report that ICI treatment causes hot flushes (8). Because the hypothalamus is thought to play a major role in temperature regulation and thermoregulatory dysfunction (9, 10), this raises the question as to whether ICI can cross the BBB and exert functional effects in the brain, and, more specifically, the hypothalamus, a key site for modulation of neuroendocrine functions.

Results from several initial studies with ICI suggested that the compound did not cross the BBB. ICI failed to block the uptake of [3H]estradiol into nuclei of hypothalamic cells after parenteral administration in ovariectomized (OVX) rats or hamsters but did block uptake in uterine, pituitary, and adipose tissues (2, 3). In addition, systemic treatment with ICI in intact cycling rats did not mimic the effects of OVX on body weight gain or changes in plasma levels of FSH or LH (1). Conclusions from these studies about brain penetrability of ICI have been contradicted by data showing antiestrogenic effects of ICI on some central nervous system (CNS)-mediated endpoints. ICI treatment reportedly reduced both receptive (lordosis) and proceptive sexual behaviors (ear wiggling/darting/ hopping) in OVX estradiol benzoate (EB)-treated rats and hamsters (2, 3, 4); behaviors that appear to be regulated through estrogenic actions in the hypothalamus and not in the periphery (11, 12, 13). ICI treatment is also associated with an increased incidence of hot flushes in premenopausal women (8). This effect most likely occurs through blockade of ER signaling in the brain because hot flushes are thought to be an estrogen-dependent thermoregulatory dysfunction of hypothalamic origin (9, 10). Finally, ICI treatment in OVX rats or hamsters has blocked the effect of tamoxifen or EB, respectively, on body weight gain (2, 3), a physiological endpoint that can be regulated by ER signaling in the hypothalamus (14, 15, 16). Based on these findings, we hypothesize that ICI is capable of crossing the BBB and exerting functional effects in the CNS, and in particular the hypothalamus, after systemic dosing.

In the present study, we directly measured plasma, brain, and hypothalamus levels of ICI over a 24-h period after repeated systemic administration in OVX rats. In addition, we tested the ability of ICI to block the effect of 17 {alpha}-ethinyl estradiol (EE) treatment on tail-skin temperature (TST) abatement in the morphine-dependent (MD) model of hot flush (17) and on body weight change, two physiological endpoints shown to be regulated by estrogenic actions in the brain (15, 16, 18). Our results demonstrate that ICI is capable of crossing the BBB and exerting functional (antiestrogenic and estrogenic like) effects on CNS-mediated endpoints after systemic dosing.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Animals
Pharmacokinetic studies and the MD model of hot flush were conducted using female OVX Sprague Dawley rats (3–4 months, 250–375 g) from Taconic Farms (Germantown, NY). The animals used for body and uterine weight studies were 2 months old with a mean initial weight of 214.8 ± 1.6 g (mean standard ± SEM). A total of 131 animals were used for these studies. Animals were housed two per cage and maintained at 25 C on a 12-h light, 12-h dark cycle (lights on at 0600 h) with food and water available ad libitum. For the MD model, experimental procedures were performed in a room maintained at 21 C as described previously (17). Data were collected between 2 and 5 wk after OVX. All animal work was in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and the local Animal Care and Use Committee approved all procedures.

Compounds and dosing
The ER agonist EE (Sigma-Aldrich Corp., St. Louis, MO) and the antiestrogen ICI (Discovery Medicinal Chemistry group of Wyeth Research) were dissolved in 10% ethanol in sesame oil and delivered sc in a 0.5 ml final volume. We chose to use EE over other estrogens because we have previous experience with this compound in our MD model of hot flush (19, 20). For brain penetration studies, ICI was administered at 0900 h each day for 3 d. For all other studies, ICI and EE were administered at 0800 and 0900 h, respectively, for 8 d.

Collection of blood and brain samples
In pharmacokinetic studies, plasma, brain, and hypothalamus samples were collected 0.5, 1.0, 4.0, 8.0, and 24.0 h after administration of the last ICI dose (Fig. 1AGo) as described previously (21). In the MD model, plasma, brain, and hypothalamus samples were collected 2.5 h after ICI administration. For sample collection, rats were anesthetized with 3% isoflurane in 100% oxygen, and the chest cavity was opened. One milliliter of blood was drawn by cardiac puncture from the right atrium, collected in Microtainer tubes containing EDTA (BD, Franklin Lakes, NJ), and centrifuged at 4 C at 3700 x g for 4 min. Rats were perfused with 40 ml cold PBS through the left ventricle. After decapitation the brain was removed and the hypothalamus dissected with curved forceps. Pituitary tissue was excluded due to the reduced stringency of the BBB in this region of the brain (22). Brain and hypothalamic tissues were weighed, placed in 5 or 0.25 ml, respectively, cold deionized water, and maintained on ice until homogenization. The brain was homogenized with a Tempest Virtishear tissue homogenizer (VirTis Co., Inc., Gardiner, NY) for approximately 45 sec, followed by pulsed sonication (0.5 sec on, 0.1 sec off) with an ultrasonic sonicator (Heat Systems, Farmingdale, NY) for 10 sec. Hypothalamic tissue was homogenized by sonication only, as described previously. Power settings were five for brain and 4.5 for hypothalamus.


Figure 1
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  		FIG. 1. Detection of ICI in the brain, hypothalamus, and plasma of OVX Sprague Dawley rats over time. A, Schematic representation of ICI administration (1.0 mg/kg·d, sc) for 3 d. Plasma and brain and hypothalamic tissue were collected at 0.5, 1.0, 4.0, 8.0, and 24 h (hr) after administration. B, ICI detection in plasma and brain and hypothalamic tissues over time. Data are reported as ng/ml for plasma, and ng/g for brain and hypothalamic tissues (mean ± SEM). n = 4 rats per time point.

 
Determination of ICI concentrations in plasma, brain, and hypothalamus
The ICI compound was extracted from plasma, brain, and hypothalamus by protein precipitation using acetonitrile. Experimental and calibration samples (tissue from untreated animals) were prepared by adding a 100 µl aliquot of plasma or 200 µl brain or hypothalamus into the appropriate well of a 96-well disposable plate. ICI was not detected in plasma and brain and hypothalamic tissue in the untreated animals. Therefore, calibration samples were further prepared by adding 20 (plasma) or 40 (brain or hypothalamus) µl of the appropriate concentration of ICI compound prepared in water: acetonitrile (50:50) to generate a standard curve with the following points: 1, 2, 5, 10, 20, 50, 100, 200, 500, 1000, or 2000 ng/ml. A 10-µl volume of internal standard (IS) [Wyeth Ayerst (WAY)-207800; molecular weight = 351.8) was added to both calibration and experimental samples, followed by 400 µl acetonitrile. Samples were vortexed then centrifuged at 2000 x g for 5 min. The supernatants were removed and evaporated to dryness under nitrogen atmosphere at 40 C. The resulting material was reconstituted in 100 µl acetonitrile to deionized water (1:1). Forty microliters of the reconstitute were injected onto a Phenomenex Gemini C18 (50 x 2.0 mm, 3 µm) reversed-phase column (Phenomenex, Torrance CA). For analytical separation, a gradient using 5 mM ammonium acetate buffer (pH 4.5) (mobile phase A) and acetonitrile (mobile phase B) at a flow rate of 0.4 ml/min was used (held at 90% A for 1 min followed by a linear gradient to 95% B in 1.5 min). All analyses were performed using the Agilent 1100 LC system (Agilent Technologies, Inc., Palo Alto, CA) interfaced with a Micromass Quattro Ultima mass spectrometer (Waters Corp., Milford, MA). The liquid chromatograph eluate was introduced into the mass spectrometer equipped with an electrospray ionization source (Electro Scientific Industries, Inc., Portland, OR) operating in the negative ion mode. A capillary voltage of 2.5 kV, cone voltage of 100 V, and desolvation temperature of 350 C were used. The deprotonated molecular ions of ICI [mass/charge (m/z) 605.4] and IS (m/z 350.2) were selected by quadrupole (Q) 1 set at unit resolution. Collision-activated dissociation was performed in Q2 with an argon setting of six. The collision energy and declustering potential for ICI and IS were optimized empirically. Fragment ions of ICI (m/z 427.24) and IS (m/z 290.0) were mass analyzed by Q3 at unit resolution. Data acquisition and reduction were performed with Masslynx version 4.0 (Waters Corp.). ICI was quantified against a standard curve using the peak area ratio of ICI to IS and Watson software version 7.0.1 (InnaPhase Corp., Philadelphia, PA). The lower limit of quantification was 1.0 ng/ml using a 100-µl aliquot of plasma or 200 µl aliquot of brain or hypothalamus homogenate. Area under the curve calculations were made for determining exposure levels using Prism GraphPad (version 4.0; GraphPad Software Inc., San Diego, CA).

MD model of hot flush
ICI was evaluated for its ability to block estrogenic abatement of a naloxone-induced rise in TST in MD OVX rats. This model has been detailed previously (17, 23). Briefly, rats received either ICI (1 or 3 mg/kg, sc) or vehicle 1 h before receiving either EE (0.3 mg/kg, sc) or vehicle for 8 d (Fig. 2AGo). The 0.3-mg/kg dose of EE was chosen because it is approximated to be the ED50 value for EE in this model. Morphine dependence was induced by implantation of two slow-release morphine pellets sc (75 mg/pellet; Murty Pharmaceuticals, Lexington, KY) in the dorsal scapular region on d 2 dosing (Fig. 2AGo). Six days after morphine pellet implantation, withdrawal was induced with the general opioid antagonist naloxone (1.0 mg/kg, sc; Research Biochemicals Intl., St. Louis, MO). The ICI compound was administered 3 h before naloxone (Fig. 2BGo). Ethinyl estradiol was administered 2 h before naloxone. Rats received ketamine (40 mg/kg, im, Ketaject; Phoenix Pharmaceuticals, Belmont, CA) 1 h before naloxone. The mild sedation induced by ketamine was necessary to reduce stress associated with thermistor probe attachment and mild restriction of movement, which can cause variability in the baseline temperatures. After ketamine administration a thermistor probe connected to a MacLab data acquisition system was taped to the base of the rat’s tail (CB Sciences, Dover, NH). The first three readings (35, 30, and 25 min before naloxone administration) were averaged and used as each rat’s own baseline TST value. Naloxone administration was designated as time zero, and data were analyzed at 5-min intervals. Data from each time point were reported as a change ({Delta}) in TST from baseline.


Figure 2
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  		FIG. 2. Schematic representation of compound administration and experimental design for the MD OVX rat model of hot flush. A, Rats were administered vehicle or ICI (solid arrow) sc 1 h before either EE or vehicle (dotted arrow). B, Experimental design for the MD model on test d 8. Statistical differences between treatment groups were evaluated 15 min after naloxone administration.

 
Collection of uterine tissue
Rats were euthanized by decapitation on d 8 dosing. Both uterine horns were removed and weighed after trimming associated fat and removal of luminal fluid.

Statistical analysis
Statistically significant changes in TST after naloxone administrations in the MD model of hot flush were analyzed with a two-way repeated measure ANOVA for "treatment" and "time" using SAS software version 8.2 (SAS Institute Inc., Cary, NC) (17). Multiple comparisons (least significant difference P values) between treatment groups at each time point were used for post hoc analysis. Statistically significant differences between treatment groups were assessed 15 min after naloxone treatment when the maximal change in TST is typically observed. Statistically significant differences in body weight changes and uterine wet weights between treatment groups were determined using one-way ANOVA with Bonferroni’s post hoc analysis.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
ICI crosses the BBB and penetrates into brain and hypothalamic tissues
An earlier report suggested that ICI did not cross the BBB of OVX rats because it failed to block nuclear uptake of [3H]estradiol in hypothalamic tissue after once daily dosing at 1.0 mg/kg sc for 3 d (2). However, the ability of ICI to cross the BBB and penetrate brain tissues was not directly tested in these experiments. In the present study, the same dosing paradigm was followed as reported by Wade et al. (2) (Fig. 1AGo), and ICI levels were measured in plasma and brain (total brain minus hypothalamus and pituitary) and hypothalamic tissues over time. The ICI compound was detected in all samples and at all time points tested (Fig. 1BGo). The concentrations and pharmacokinetic profiles of ICI in plasma, brain, and hypothalamus were found to be similar (Fig. 1BGo and Table 1Go). ICI levels were stable in plasma and brain and hypothalamic tissues over the entire 24-h testing period, and the concentrations of ICI in plasma and hypothalamic tissue were similar at the 24-h time point.


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  		TABLE 1. Pharmacokinetic profile of ICI (1.0 mg/kg, sc, 3 d) in plasma and brain and hypothalamic tissues of OVX Sprague Dawley rats over time

 
The ratio of brain to plasma exposure is an indication of a compound’s ability to cross the BBB. Based on 24-h exposure values, brain and hypothalamus to plasma ratios for ICI were 0.33 and 0.66, respectively (Table 1Go). These results demonstrate that ICI crosses the BBB, is present in brain and hypothalamic tissues, and persists at a constant level in plasma and brain and hypothalamic tissues for up to 24 h after systemic dosing.

ICI blocks estrogenic actions in the MD model of hot flush but showed estrogenic-like effects when administered alone
The MD model of hot flush is based on measuring naloxone-induced increases in TST in MD OVX rats (17, 23). Previous work indicates that estrogen’s ability to abate TST elevations in the MD model of hot flush occurs through its actions in the brain (18). To determine whether ICI can exert functional effects on a CNS-mediated endpoint after systemic dosing, we tested its ability to block the effects of EE in the MD model of hot flush. OVX rats were administered either vehicle (sc), EE (0.3 mg/kg·d, sc), EE in combination with ICI (1.0, 3.0 mg/kg·d, sc), or ICI alone (1.0, 3.0 mg/kg·d) for 8 consecutive days (Fig. 2AGo). In these studies, ICI was administered 1 h before EE treatment. Rats receiving either EE or ICI treatment alone were also administered vehicle at the appropriate time points. Statistically significant changes in TST were observed among treatment groups [F(5,45) = 13.43; P < 0.0001] (Fig. 3AGo). At 15 min after naloxone administration, TST in vehicle-treated rats increased 5.7 ± 0.7 C, as expected (23). In rats treated with EE alone, naloxone-induced TST increases were abated 64% compared with vehicle-treated rats. The effect of EE was dose dependently inhibited by pretreatment with 1.0 and 3.0 mg/kg·d ICI. Rats treated with EE and 1 mg/kg ICI showed 36% abatement of the naloxone-induced flush, whereas the effect of EE was completely blocked in rats treated with 3 mg/kg ICI at 15 min after naloxone treatment.


Figure 3
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  		FIG. 3. ICI has antiestrogenic and weak estrogenic-like effects in the MD OVX rat model of hot flush. A, Administration of vehicle, EE alone (0.3 mg/kg·d), or EE in combination with 1.0 or 3.0 mg/kg·d ICI (1 h before EE) once daily for 8 d. Naloxone-induced TST elevations 15 min after administration were abated by 64% in EE-treated rats, and dose dependently inhibited by pretreatment with ICI at 1.0 and 3.0 mg/kg·d. B, Treatment with 1.0 mg/kg·d ICI alone for 8 d had no effect on naloxone-induced TST increases, whereas treatment with 3.0 mg/kg·d ICI alone abated 30% of the increase in TST. Data are reported as mean ± SEM. *, P < 0.001 compared with vehicle treatment; +, P < 0.005 compared with EE treatment, two-way ANOVA for repeated measures (n = 6–10 rats per group). Arrow denotes naloxone administration.

 
In the same study, treatment with 1.0 mg/kg·d ICI alone had no effect on naloxone-induced TST increases (Fig. 3BGo). In contrast, rats receiving 3.0 mg/kg·d ICI showed a 30% abatement of the naloxone-induced increase in TST. Two replicate studies with 3 mg/kg·d ICI for 8 d showed 22.1 and 43.5% abatement (data not shown). These findings suggest that when given alone, ICI may have weak estrogenic-like actions in the brain.

To verify that ICI was present in brain and hypothalamic tissues of the rats used in these MD model experiments, five rats from each ICI-treated group were euthanized after completing data collection (2.5 h after ICI administration). ICI was detected in plasma and brain and hypothalamic tissues of all rats, and ICI levels were found to increase with ascending dose (Table 2Go). In addition, ICI levels in plasma and brain and hypothalamic tissues were unaffected by EE treatment. Importantly, ICI concentrations in the 1.0 mg/kg·d group were in good agreement with results reported in Fig. 1BGo.


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  		TABLE 2. Levels of ICI in plasma, brain, and hypothalamus 2.5 h after administration in the MD OVX rat model compared with functional activity

 
ICI blocks estrogenic actions on body weight but showed estrogenic-like effects when administered alone
The hypothalamus plays a prominent role in estrogenic regulation of body weight (15, 16). To confirm that ICI can exert functional effects on CNS-mediated endpoints and, more specifically, a hypothalamic function, we tested its ability to block the effect of EE on body weight change in OVX rats. Treatment groups were the same as described previously for the MD model (Fig. 2AGo). Average body weights for each treatment group were not different on d 1 dosing and ranged from 212–218 g. Data were reported as average body weight change between d 8 and baseline (d 1) calculated using each rat as its own baseline (Table 3Go). After 8 d, vehicle-treated rats gained 45.5 g. In rats receiving EE (0.3 mg/kg·d for 8 d), body weight decreased 13 g. The effect of EE on body weight was not inhibited by pretreatment with 1.0 mg/kg·d ICI but was partially blocked by pretreatment with 3.0 mg/kg·d ICI. These data, together with results generated in the MD model, support the idea that ICI can block estrogenic signaling in the brain and, specifically, a hypothalamic-mediated function, after systemic dosing.


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  		TABLE 3. Effect of EE and ICI on body weight in OVX rats

 
In OVX rats treated with either 1.0 or 3.0 mg/kg·d ICI alone for 8 d, body weight increased 11.8 and 7.8 g, respectively (Table 3Go). Thus, similar to EE treatment, rats receiving ICI alone showed little weight gain compared with vehicle-treated controls. These data confirm the idea that when given alone, ICI may have weak estrogenic-like activity in the brain (24, 25).

ICI does not have estrogenic-like effects in the uterus at 1.0 or 3.0 mg/kg·d for 8 d
To determine whether ICI had estrogenic-like effects on a peripheral reproductive tissue, uteri from rats used in the aforementioned body weight study were dissected and weighed on the last day of dosing. In rats receiving EE alone (0.3 mg/kg·d) or the combination of EE with 1.0 or 3.0 mg/kg·d ICI, uteri weighed 403, 406, and 410 g, respectively (Fig. 4Go). Uteri from rats treated with vehicle or ICI (1.0 or 3.0 mg/kg·d) alone weighed 97.4, 114, or 123 g, respectively. Thus, in agreement with a previous report (26), ICI does not have estrogenic-like effects on uterine tissue at the doses tested. ICI has already been shown to have antiuterotrophic effects in OVX estradiol-treated rats (1). The inability of ICI to block the effect of EE on uterine growth in this study is expected because the uterus is highly sensitive to estrogens, and the 0.3 mg/kg dose of EE used in these studies is 500-fold greater than the ED50 value for EE on uterine growth (27).


Figure 4
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  		FIG. 4. ICI alone showed no estrogenic-like actions on uterine tissue in OVX rats. Administration of vehicle, EE alone, EE in combination with 1.0 or 3.0 mg/kg·d ICI, or 1.0 or 3.0 mg/kg·d ICI alone once daily for 8 d. Uterine wet weight measurements were obtained on d 8. Uterine weights increased significantly in groups receiving EE treatment compared with vehicle-treated rats, and pretreatment with either 1.0 or 3.0 mg/kg·d ICI did not reduce the EE-induced increase. Treatment with ICI alone for 8 d had no effect on uterine wet weights. Data are reported as mean ± SEM. *, P < 0.001 compared with vehicle treatment, one-way ANOVA with Bonferroni’s post hoc analysis (n = 10 rats per group).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
In the present study, we tested whether ICI crosses the BBB, penetrates into the hypothalamus, and affects known neuroendocrine functions in OVX rats. When administered systemically administered at 1 mg/kg·d for 3 d, ICI was detected in plasma, brain, and hypothalamic tissues 30 min after administration, and was detected for up to 24 h. In addition, ICI had good exposure in both brain and hypothalamic tissues, relative to plasma levels. Thus, in contrast to previous conclusions (1, 2, 3), these results demonstrate that ICI rapidly crosses the BBB and persists for an extended period of time in plasma and brain and hypothalamic tissues. Consistent with its ability to penetrate brain tissues, repeated systemic administration of ICI blocked the effects of EE on naloxone-induced TST elevations in the MD model of hot flush and on body weight change. Interestingly, ICI administration alone (1.0 or 3.0 mg/kg·d) demonstrated weak estrogenic-like activity in these models. We conclude that ICI is a brain-penetrable compound that can exert functional (antiestrogenic and estrogenic) effects in the CNS, and specifically the hypothalamus, after systemic dosing.

Previous studies have concluded that ICI does not cross the BBB because at up to 1.0 mg/kg·d, it failed to block uptake of [3H]estradiol into nuclei of hypothalamic cells in OVX rats (2) and failed to mimic the effects of OVX on body weight gain and plasma gonadotropin levels in intact female rats (1). However, it is possible that these previous studies did not use a high enough dose to observe inhibitory effects on these endpoints. For example, despite the presence of ICI in brain and hypothalamic tissue after systemic administration of 1.0 mg/kg·d, we found that this dose of ICI did not inhibit the effect of EE on all functional endpoints. The 1.0 mg/kg·d dose of ICI did partially inhibit the effect of EE on TST increases in the MD model but did not block EE’s effect on body weight change. This functional selectivity may be explained by the fact that ICI’s inhibitory effect on different estrogen-mediated brain functions can vary depending on the endpoint being studied. For example, Steyn et al. (28) have shown that intracerebroventricular administration of ICI inhibited estrogen-induced GnRH pulse frequency but did not block estrogenic effects on progesterone receptor expression in the hypothalamus or on antepartum prolactin surges. The authors concluded that there might be a wide range of sensitivities to ICI in the brain that could cause variable results across different functional endpoints. Thus, it is possible that inhibition of [3H]estradiol uptake into nuclei of hypothalamic cells or blockade of estrogen’s effect on body weight change may require higher levels of ICI than other functional endpoints. This idea is supported by our results showing that ICI treatment at 3.0 mg/kg·d for 8 d did partially block the effect of EE on body weight change. Because some previous studies (1, 2) only tested ICI at up to 1.0 mg/kg·d, it is unknown if higher doses would have inhibited [3H]estradiol uptake into nuclei of hypothalamic cells in OVX rats, or induced body weight gain or increased plasma gonadotropin levels in intact female rats.

Several lines of evidence suggest that estrogens regulate body weight primarily through central mechanisms that reduce meal size (15, 16, 29, 30, 31, 32). Lesions of the ventromedial nucleus of the hypothalamus blocked the effect of systemically administered EB on body weight change and food intake in OVX rats (29), infusion of estradiol directly into the paraventricular nucleus or medial preoptic nucleus of the hypothalamus reduced body weight and/or food intake in OVX rats (30, 31), and direct administration of EB to the hindbrain just above the nucleus tractus solitarius reduced food intake in OVX rats (32). However, these results have not been reproduced in all laboratories (33), and several peripheral feedback mechanisms have been hypothesized. Therefore, it remains possible that the ability of ICI to block the effect of EE on body weight change in the present study could occur through peripheral not central mechanisms. However, direct involvement of peripheral mechanisms in mediating estrogens effect on body weight is lacking. It has been shown previously that estradiol treatment does not inhibit feeding by modulating orosensory stimuli (15, 16). Estradiol treatment can inhibit ghrelin-induced feeding, but this effect does not occur through reduction in meal size as is well established for estrogens (15). Estradiol does increase the satiating potency of cholecystokinin, but this effect likely occurs through an estradiol-induced increase in neuronal activity within the brainstem, not through regulation of signaling in the periphery (15, 16). Finally, leptin signaling does not appear to directly mediate estrogen’s effect on body weight because estradiol has reduced body weight and food intake in both leptin-deficient and leptin receptor-deficient mice (34). Thus, based on current evidence, estrogenic regulation of body weight appears to be mediated through central mechanisms, and is an appropriate endpoint for predicting whether ICI crosses the BBB and exerts functional effects in the CNS.

The effect of ICI on body weight has been reported previously in intact cycling female rats and OVX estrogen-treated rats (2, 24). In these studies body weight changes were unaffected by daily ICI treatment at either 1 or 1.5 mg/kg·d (higher doses were not tested), and it was concluded that ICI did not cross the BBB. Our results are consistent with these studies because the ability of ICI to block EE’s effect on body weight change was not observed at the 1.0 mg/kg·d dose. However, at the higher dose (3.0 mg/kg·d), ICI treatment did block the effect of EE on body weight change. These data suggest that in previous studies in OVX rats, ICI may not have been administered at a high enough dose to block estrogenic effects on body weight regulation.

The effect of ICI alone on body weight change in OVX rats has also been tested previously (2, 24). Results from these studies also suggest that body weight change is unaffected by ICI treatment at 1.0 or 1.5 mg/kg·d. These data are in contrast to our finding that ICI had weak estrogenic-like actions on body weight change at both 1.0 and 3.0 mg/kg·d. The discrepancy between the current work and previous studies is difficult to reconcile. There are several technical differences such as rat strain and vendor and total treatment length that might account for the discrepancy. One additional possibility is that the effect of ICI on body weight may vary depending on initial body weights. In the present work, initial body weights averaged 215 g, whereas in both other studies, initial body weights were approximately 270–276 g. It is possible that in heavier rats, ICI may have a greater volume of distribution, increased sequestration into adipose tissue, and/or increased plasma clearance. These possibilities are supported by studies showing that increased body mass can alter the pharmacokinetic properties of some drugs (35). Therefore, the net result of these effects could be to reduce ICI exposure in brain tissue and limit its ability to stimulate ER signaling. Importantly, it is unlikely that rats in our study inadvertently received EE administration because uterine weights were similar to those from vehicle-treated rats (Fig. 4Go).

Our results suggest that ICI has relatively low clearance in plasma and brain and hypothalamic tissues of OVX rats when administered at 1 mg/kg·d for 3 d, and can persist in all three compartments for at least 24 h after the last dose. In fact, the concentrations of ICI for each tissue were found to be similar at the 0.5 and 24-h time points (Fig. 1BGo). This relatively low clearance suggests that ICI could accumulate, particularly in lipid compartments such as brain and adipose tissues, after daily systemic administration. Although somewhat speculative, this type of accumulation could alter the pharmacokinetic and pharmacodynamic properties of ICI over time. Consistent with this idea, ICI treatment at 1 mg/kg·d for 2 d did not block the effect of EB on lordosis, ear wiggling, or hops and darts but reduced the effect of EB on all three parameters when administered at 1 mg/kg·d for 24 d (2). Thus, the potential functional effects of ICI on CNS-mediated endpoints may depend on both the doses tested and the time period over which dosing is conducted.

A question that occurs is what are the relative roles of ER{alpha} and β in mediating the effects of ICI on the endpoints measured in the current study (i.e. TST regulation in the MD model and body weight change). Because ICI binds to both receptors with similar affinities (36) and both receptors appear to have broad distribution in the brain, including the hypothalamus (37), the relative roles of the {alpha} and β-subtypes are not easily discerned. Regarding body weight, several studies suggest that estrogen’s effect is ER{alpha} mediated. In support of this idea, in two separate studies, the ER{alpha} agonist 4,4',4''-(4-propyl-[1H]-pyrazole-1,3,5-triyl)trisphenol decreased food intake and body weight in OVX rats, whereas the ERβ agonist 2,3-bis(4-hyroxyphenyl)-propionitrile had no effect (38, 39). In addition, EB had no effect on body weight and food intake in OVX ER{alpha} knockout mice, suggesting that the β-subtype is insufficient to mediate the effect of estrogen on these endpoints (40). Finally, other studies have shown that estrogenic inhibition of feeding occurs through ER{alpha}-expressing neurons located in the nucleus tractus solitarius (32). In contrast to these results, in a single study using oligonucleotide knockdown of ERs in the brain, only ERβ antisense probes blocked the effect of estradiol on body weight and food intake (41). However, the ability of their probes to reduce ER{alpha} expression in the brain was not reported. Thus, based on current data, it appears that the effect of ICI on body weight change observed in the current study is mediated through the ER{alpha} receptor subtype. However, this hypothesis will need to be confirmed in future studies.

Less is known about the respective roles of the ER{alpha} and β-receptor subtypes in temperature regulation. Both receptors have been implicated in the regulation of TST elevations in mice (42). However, research in our laboratory using rat models of temperature regulation has only been able to show efficacy with compounds that have ER{alpha} activity (unpublished data). Further research is needed to identify the ER subtype mediating estrogen’s effect on temperature regulation.

The antiestrogenic properties of ICI in the brain as well as uterine tissues have been well established (1, 28, 43, 44). However, results from our work and others suggest that ICI may not be a pure antiestrogen. Intrahippocampal infusion of ICI has mimicked the effect of EB on place learning in OVX rats (43). In addition, Sibonga et al. (24) have shown that, like 17 β-estradiol (45), ICI treatment (1.5 mg/kg·d) decreases the cancellous bone formation rate in OVX rats. Finally, in primary hippocampal neurons, both ICI and 17 β-estradiol promoted neuronal survival against excitotoxic- and β amyloid-induced cell death, induced rapid calcium influxes, increased spinophilin and Bcl-2 expression, and increased phosphorylation of ERK2 and Akt (25). Thus, ICI appears to have mixed antagonist and agonist properties, and its pharmacology now seems to be more similar to other selective ER modulators (SERMs), such as raloxifene and tamoxifen, then initially reported. The precise mechanisms supporting the mixed pharmacology of ICI are unknown, however, it may be related to the differential regulation of ER dimers in the absence or presence of an estrogen. It is well known that ER dimerization is a key step in the activation of estrogen signaling pathways. Using a yeast two-hybrid system, Wang et al. (46) found that ICI induced ER dimerization when given alone but destabilized ER dimers in the presence of an estrogen. Therefore, it is possible that ICI-induced receptor dimerization could lead to activation of estrogen responsive pathways, whereas destabilization of ER dimers in the presence of an estrogen would block signaling. In support of this idea, ICI was found to activate a subset of estrogen-responsive genes in MCF-7 cells, a breast cancer cell line, grown in hormone-depleted medium (47). Although this explanation might account for the antagonist and agonist-like effects of ICI observed in the MD model and on body weight change in the current study, it cannot be broadly applicable to all endpoints because ICI had no detectable estrogenic-like effect on uterine tissue. Currently, it is not well understood how the tissue-selective agonist/antagonist properties of SERMs, like ICI, tamoxifen, and raloxifene, manifest. It has been hypothesized that agonist/antagonist activities of SERMs result from specific ligand-induced conformational changes in ERs that alter coactivator/corepressor protein binding, and selectively influence different genomic and/or nongenomic signaling pathways (48). In addition, cell-specific promoter context could play a role in determining whether a SERM will elicit estrogenic or antiestrogenic actions.

The clinical use of ICI is not likely to be altered significantly by the results from the present work. However, our results do offer a mechanistic explanation for the occurrence of hot flushes in premenopausal women treated with fulvestrant (8). In addition, the use of fulvestrant in premenopausal women would be expected to induce other CNS-related menopause-like symptoms such as sleep disturbances, mood changes, loss of energy, weight gain, and decreased libido (49). Because most of these symptoms are not life threatening, it is unlikely that they would limit the use of fulvestrant for treating advanced breast cancer patients. Although we found that ICI has weak estrogenic-like activity on certain CNS-mediated functions, it is also unlikely that these results will affect the use of fulvestrant for treating breast cancer patients. Previous work has shown that ICI inhibits human breast cancer cell proliferation (1), and agonist-like activity would be inconsistent with the studies showing the utility of fulvestrant in treating ER-positive breast cancer (6, 7).

In summary, we have shown that, in contrast to previous conclusions, ICI is capable of crossing the BBB, penetrating brain and hypothalamic tissues, and exerting functional effects on neuroendocrine endpoints after systemic administration. We have also found that ICI is not a pure antiestrogen, and may have a mix of both agonist and antagonist activities on certain CNS-mediated functions. Therefore, future studies should consider the potential for ICI to influence estrogen-related functions in the CNS after systemic dosing.


   Acknowledgments
 
We thank Dr. Heather Harris for her assistance in dissecting uterine tissues.


   Footnotes
 
Disclosure Statement: All authors are currently employed by and have equity interest in Wyeth Pharmaceuticals.

First Published Online July 3, 2008

Abbreviations: BBB, Blood-brain barrier; CNS, central nervous system; EB, estradiol benzoate; EE, 17{alpha}-ethinyl estradiol; ER, estrogen receptor; ICI, ICI 182,780; IS, internal standard; MD, morphine-dependent; OVX, ovariectomized; Q, quadrupole; SERM, selective ER modulator; TST, tail-skin temperature.

Received April 15, 2008.

Accepted for publication June 23, 2008.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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