1 Women's Health Research Institute, Wyeth Research, Collegeville, PA 19426 and 2 Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, Nashville, TN 37232, USA
3 To whom correspondence should be addressed at: Women's Health Research Institute, Wyeth Research, RN 3256, 500 Arcola Road, Collegeville, PA 19426, USA. Email: harrish{at}wyeth.com
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Abstract |
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Key words:
endometriosis/ERB-041/estrogen receptor-/immune system/nude mouse
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Introduction |
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The causes of this disease continue to be debated. A leading hypothesis, first advanced by Sampson (1927), is that viable endometrial tissue escapes into the peritoneal cavity during menstruation, instead of being shed through the vagina. Other hypotheses include coelomic or lymphatic metaplasia, and induction of mesenchyma by uterine factors (Witz, 2002
).
The phenomenon of retrograde menstruation likely occurs in most women, and yet symptomatic disease develops in a relatively small percentage. The resolution of this apparent paradox may reside in differences between the immune systems of normal women and those with endometriosis (reviewed in Lebovic et al., 2001; Nothnick, 2001
). The immune system of normal women may have a greater ability to recognize errant endometrial tissue as foreign and to remove it. That this process does not happen efficiently in women with endometriosis is supported by observations in immune system imbalances. For example, decreased natural killer (NK) cell or macrophage activity has been reported in endometriosis patients (Oosterlynck et al., 1991
).
Current medical therapies are inadequate and include a progression from symptom management with oral contraceptives to more aggressive measures such as suppression of estrogen levels using GnRH agonists, surgery to reduce lesion burden and finally, hysterectomy (ACOG, 2000). Each of these treatments has significant efficacy or tolerability liabilities. For example, although oral contraceptives are often used as first-line therapy, little scientific evidence supports their efficacy (Moore et al., 2004
). Treatment with GnRH agonists has a number of serious side-effects including bone loss, hot flushes, mood swings, and vaginal atrophy. In fact, its use is limited to 6 months. Clearly, an effective therapy with an improved side-effect profile is needed.
Two forms of the estrogen receptor have been described. The first, now called ER, was cloned by Green et al. (1986)
and is thought to be responsible for mediating many of estrogen's actions such as its trophic effects on the uterus and feedback of the hypothalamicpituitaryovarian axis (Couse and Korach, 1999
). The second form (ER
) was discovered more recently (Kuiper et al., 1996
) and its function is not completely understood. Based on tissue distribution studies, it is not the dominant ER in uterus, although the human uterus does appear to express more ER
than does the rodent (Couse et al., 1997
; Kuiper et al., 1997
; Matsuzaki et al., 1999
; Pelletier and El-Alfy, 2000
; Lecce et al., 2001
).
We have developed a highly selective ER agonist that is >200-fold selective for ER
based on a competitive radioligand binding assay. ERB-041 (Figure 1) has been extensively characterized in various models of estrogen action and found to be inactive on classic estrogenic targets such as the uterus, mammary gland and bone. However, it has potent anti-inflammatory activity in two in vivo models: the HLA-B27 transgenic rat and Lewis rat adjuvant-induced arthritis (Harris et al., 2003
). We report here that this compound causes lesion regression in an experimentally induced model of endometriosis using human tissue and therefore may have utility in treating this disease.
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Materials and methods |
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Acquisition of human tissues
Informed consent was obtained prior to endometrial biopsy collection. Although names were kept confidential, each patient's age, cycle stage and medication history were made available. Use of human tissues was approved by Vanderbilt University's Institutional Review Board and Committee for the Protection of Human Subjects. Normal endometrial tissues were acquired by biopsy during the proliferative phase (days 912) of the menstrual cycle from a donor population (aged 1845 years) exhibiting normal menstrual cycles and no history of endometriosis. An endometrial thickness of >9 mm (confirmed by intravaginal ultrasound) and a serum progesterone level of <1.5 ng/ml were required for inclusion in this study. Individuals with a recent (3 months) history of hormone therapy (e.g. oral contraceptives) or other medications which might impact study results (e.g. anti-inflammatory agents) were excluded. Biopsies were obtained from the uterine fundus using a Pipelle® endometrial suction curette (Unimar, Inc., USA) and the tissue was washed in pre-warmed, phenol red-free Dulbecco's modified Eagle's medium/Ham's F-12 medium (DMEM/F-12) (Sigma, USA) to remove residual blood and mucus prior to culturing. A portion of each sample was formalin-fixed for histological confirmation of cycle stage.
Organ cultures of human tissues
Endometrial biopsies were dissected into small cubes (1 mm3) and 810 pieces per treatment group were suspended in tissue culture inserts (Millipore, USA) as previously described (Bruner et al., 1999). Each organ culture was maintained in 1 nmol/l 17
-estradiol (Sigma) under serum-free conditions in DMEM/F-12 supplemented with 1% Insulin-Transferrin-Selenium (ITS+; Collaborative Biomedical, USA) and 0.1% Excyte (Miles Scientific, USA) and an antibiotic/antimycotic solution (Hybri-Max; Sigma, USA). Tissues were incubated overnight at 37°C in a humidified chamber with 5% CO2.
Experimental model of endometriosis
The model of endometriosis was performed as previously described (Bruner-Tran et al., 2002) with minor modifications. Prior to any invasive procedure or death, mice were anesthetized with Metofane (PittmanMoore, USA). All studies using animals were performed using procedures approved by Vanderbilt University's Institutional Animal Care and Use Committee. Five week old athymic (ncr/nude), intact or ovariectomized mice (Harlan Sprague Dawley) were injected with a phosphate-buffered saline suspension of 810 human endometrial tissue fragments/mouse. Each study used tissue collected from a different donor. A typically sized endometrial biopsy yielded sufficient tissue for establishing disease in a maximum of 16 mice. In contrast to previous studies, these mice were not implanted with slow-releasing estradiol pellets. Tissue fragments had been incubated as described above for the preceding 24 h. Table I summarizes the experimental parameters for the six studies reported here. For all but one study, subcutaneous tissue injections were given on the ventral midline just below the umbilicus. Lesions were allowed to establish for 1114 days and mice with visible subcutaneous disease were randomly allocated to treatment groups. Daily oral treatments (10 mg/kg or 1 mg/kg) were given in a volume of 0.1 ml by gavage. A vehicle of 2% Tween-80/0.5% methylcellulose was used to solubilize ERB-041. ERB-041 was obtained from the Wyeth Research compound library (Malamas et al., 2004
). At the end of the experimental protocol, mice were euthanized by cervical dislocation and the subcutaneous area as well as the peritoneal cavity was examined for endometriosis. Lesions were counted, photographed and measured. Some recovered lesions were frozen in liquid nitrogen for RNA preparation.
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Analysis of ER mRNA expression in recovered lesions
Total RNA was prepared from each individual recovered lesion. Each sample was homogenized in 1 ml of Trizol (Invitrogen, USA) at 4°C using a Polytron homogenizer PT1200C (BrinkMann, USA). After 0.2 ml chloroform extraction and centrifugation, 0.5 ml aqueous phase was collected. The RNA from the aqueous phase was then purified by Qiagen RNeasy kits (Qiagen, USA). The genomic DNA in RNA sample was removed by on-column RNase-Free DNase set during RNA purification.
The RNA concentration was adjusted to 0.05 mg/ml for assay. mRNA expression was analysed using real-time quantitative PCR on an ABI PRISM 7700 Sequence Detection System according to the manufacturer's protocol (Applied Biosystems Inc., USA). The sequences of primers and labelled probes used for mRNA detection were designed in-house using Primer Express 2.0 (Applied Biosystems) and were as follows: human ER forward primer, 5'-ATGATCAAACGCTCTAAGAAGAACAG-3'; reverse primer, 5'-ACTGAAGGGTCTGGTAGGATCATACT-3'; probe, 5'FAM-TCCCTGACGGCCGACCAGATG-TAMRA3'; human ER
forward primer, 5'-GGTCCATCGCCAGTTATCACA-3'; reverse primer, 5'-GTGTTCTAGCGATCTTGCTTCACA-3'; probe, 5'VIC-CTGTATGCGGAACCTCAAAAGAGTCCCTG-TAMRA3'.
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Results |
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Although the human tissue implants were placed subcutaneously in Studies 15, some lesions were found inside the peritoneal cavity, presumably due to inadvertent penetration of the peritoneal wall during injection. In these studies, we noticed that no internal lesions were found in mice treated with ERB-041, whereas 27% of the lesions were internal in mice treated with vehicle (Table III). This observation suggested that ERB-041 was particularly effective at clearing lesions when they occurred internally. In order to more directly test this hypothesis, a separate study was performed (Study 6) where tissue fragments were intentionally placed in the peritoneal cavity. As shown in Table IV, 67% of lesions in vehicle-treated mice were inside the peritoneal cavity, whereas again no internal lesions were seen in mice treated with ERB-041. In this study, ERB-041 caused complete lesion regression in 80% of the mice.
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Discussion |
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Development of improved treatment options is hampered by the disease's complexity and a lack of tractable natural animal models. Although naturally occurring only in primates, several rodent models of this disease have been developed. One of the most widely used and well-established models involves transplantation of human endometrial tissue into nude mice, waiting until visible lesions develop and then treating with test compound. This model has been shown to share features with the native human disease, such as the morphological and histological appearance of lesions (Bruner et al., 1997; Grummer et al., 2001
). We show here for the first time that an ER
selective agonist, ERB-041, can cause lesion regression in an experimental model of endometriosis.
ERB-041 has been evaluated in this nude mouse xenograft model in six independent studies using six different human tissue samples (Table I). The size of the human tissue biopsy limited the number of mice able to be evaluated in each experiment to 16. All studies used a therapeutic regimen; that is, the compound treatment started after lesion establishment. Subcutaneous lesions (the only lesions visible before euthanasia) did not spontaneously regress in any of the vehicle-treated mice. Three studies used ovariectomized mice and tested ERB-041 at 10 mg/kg. In each study, ERB-041 caused complete lesion regression in some mice. Efficacy varied from 40 to 75%, depending on the study. Two additional studies used intact mice and evaluated two doses of ERB-041 (10 and 1 mg/kg). ERB-041 was similarly effective in intact as in ovariectomized mice, suggesting that endogenous levels of 17
-estradiol (a non-receptor subtype selective estrogen) do not block the activity of an exogenous selective ER
agonist.
Five lesions were examined at the study's end (four from vehicle-treated ovariectomized mice and one from an ovariectomized mouse treated with ERB-041) and all expressed only ER as determined by real-time quantitative RTPCR. This finding was somewhat unexpected as normal human endometrium does express low levels of ER
(Matsuzaki et al., 1999
; Taylor and Al-Azzawi, 2000
; Lecce et al., 2001
). Given our sample size limitations we did not examine expression of ER before transplantation into the mice. As most of the lesions examined for ER expression were from vehicle-treated mice, our observation of exclusive ER
expression suggests that ER
was absent from the outset, and not influenced by compound treatment. To confirm this finding, as mentioned below, future studies will examine lesion dynamics during regression and ER
/
expression will be one of the endpoints measured.
Given that the recovered human lesions expressed ER and not ER
, we believe that ERB-041 exerted its effects on the host, rather than on the implanted tissue (e.g. by directly inducing apoptosis). Our current working hypothesis is that ERB-041 affects the host's immune system. Although lacking T cells, athymic nude mice do have fully functional NK cells (Hasui et al., 1989
) and macrophages (Cheers and Waller, 1975
). As NK cells and macrophages both express ER
(Curran et al., 2001
; Stygar et al., 2001
; Vegeto et al., 2001
; Henderson et al., 2003
), both cells are possible targets for ERB-041.
During the course of our studies on ERB-041, we recorded lesion location. In studies 15, vehicle-treated mice typically had some number of internal lesions but no internal lesions were seen in any of the ERB-041-treated mice. Thus, it appeared that ERB-041 was more efficient at causing regression when the lesions were found inside the peritoneal cavity. These observations were confirmed in a study (study 6) where the tissue fragments were intentionally placed internally. The reason for this differential effect is unclear, but it is possible that ERB-041 stimulates the activity of peritoneal immune surveillance cells.
The primary goal of the studies reported here was to assess the effect of an ER agonist, ERB-041, on lesion regression. Having documented the compound's effectiveness in both gonadectomized and gonad-intact mice, future studies will concentrate on elucidating mechanism of action. In most studies, histological analysis was judged to be fruitless as mice were treated for >2 weeks with compound. Lesions remaining at the end of the study were refractory to treatment. In future studies, histological analysis, coupled with immunostaining for key cellular markers (e.g. NK cells, macrophages, new blood vessel growth) will be performed within a week of beginning compound treatment in an effort to view the process of regression.
ERB-041, if effective at reducing disease burden in human endometriosis patients, would have potential advantages over the currently approved GnRH therapies, which are thought to act primarily via suppression of the hypothalamicpituitarygonadal axis. Since ER appears to be the receptor responsible for mediating the negative feedback to the hypothalamus and pituitary (Couse and Korach, 1999
), one would expect that an ER
selective ligand would not share the same side-effect profile as a GnRH agonist. In addition, ERB-041 lacks classic estrogenic activity in several in vivo model systems (Harris et al., 2003
), suggesting it will lack the liabilities associated with non-receptor subtype selective estrogens. The unique side-effect profile of an ER
selective agonist remains to be determined, and is under investigation. However, as ER
regulates few if any genes in normal mice (Jelinsky et al., 2003
), it is expected that ERB-041 will have a favourable side-effect profile.
In summary, we show for the first time that a selective ER agonist causes regression of human endometrial xenografts in a mouse model of endometriosis. These data suggest that further evaluation of ERB-041 may be warranted, for example, in primates with naturally occurring disease.
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Acknowledgements |
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Submitted on October 21, 2004; resubmitted on November 30, 2004; accepted on December 3, 2004.