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Women’s Health Research Institute (H.A.H., Y.P.K., J.M., B.S.K., R.C.W., D.E.F., R.A.H., Y.Z.) and Chemistry and Screening Sciences (R.E.M., C.P.M.), Wyeth Research, Collegeville, Pennsylvania 19426; Cardiovascular and Metabolic Diseases, Wyeth Research (L.M.A., Y.L., J.C.K.), Cambridge, Massachusetts 02140; and Chemistry and Screening Sciences, Wyeth Research (M.S.M.), Monmouth Junction, New Jersey 08852
Address all correspondence and requests for reprints to: Heather A. Harris, Ph.D., Women’s Health Research Institute, RN 3256, 500 Arcola Road, Collegeville, Pennsylvania 19426. E-mail: harrish{at}wyeth.com.
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Introduction |
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, was cloned in 1986 (2), and a knockout mouse (KO) was created in 1993 (3). The phenotype of the ERKO mouse was consistent with many of the known functions of estrogens and the ER at that time (4, 5, 6). For example, there were profound effects in the uterine and mammary phenotypes, and negative feedback of gonadotropin secretion was lost. Some estrogenic responses were retained in the ERKO mouse, but these responses could perhaps be explained by the presence of alternate functional ER transcripts in these mice (7). In fact, at least one of these estrogenic responses is lost in a more recent, complete ERKO mouse (8). Early studies of ERß focused on its tissue distribution and ligand specificity (9). Because ERß is widely expressed, but is not the dominant ER in the uterus, there was much optimism about ERß as a drug target (10). A logical hypothesis, given the expression of ERß in bone, colon, endothelial cells, bladder, and areas of the brain important for cognition, was that an ERß-selective ligand would be the third generation selective ER modulator. Such a compound would be expected to retain a number of features of traditional hormone therapy [e.g. alleviation of vasomotor instability (hot flushes) and prevention of osteoporosis], but lack the stimulatory effect on the uterus and possibly the breast. In addition, its high expression in ovarian granulosa cells suggested that an ERß ligand might be a contraceptive agent.
Because of the scientific as well as commercial interest in elucidating ERß biology, it has been an intense area of investigation by both academic as well as industrial scientists, and more than 1500 papers/abstracts have been published to date. Broadly, these studies fall into three categories: tissue/cell line distribution, in vitro activity, and characterization of KO mice (ERßKO, as well as revisiting residual estrogenic responses in ERKO). Although each of these approaches has merit, none gives a complete picture. For example, the activity seen on promoter constructs in cell lines overexpressing ER and/or ERß may not reflect in vivo activity. As no conditional ERKO mouse has been described, the observed phenotype may in part result from loss of function during development. Furthermore, a consensus on the phenotype of the ERßKO mouse has not been reached (11) (Harris, H. A., et al., unpublished observations).
One approach to elucidating ERß function that has not yet been fully exploited is the use of selective ligands. Exploitation of these tools awaits the design/discovery of truly functionally selective ligands, as naturally occurring compounds (e.g. genistein) are only modestly selective and show evidence of ER activity in vivo (9, 12, 13). Recently, an ER-selective ligand [propyl pyrazole triol (PPT)] has been characterized in several clinically relevant models (14). These studies showed that selective stimulation of ER is sufficient to elicit many of the biological responses attributed to estrogen action. For example, PPT fully stimulates uterine wet weight increase, prevents bone resorption, ameliorates vasomotor instability, and prevents ovariectomy-induced weight gain. However, as ERß is coexpressed in a number of these target tissues (e.g. bone, brain, and adipose tissue), the possibility exists that, except for the uterus, an ERß-selective ligand would have a similar profile. In addition, defining the capabilities of ER only indirectly helps define the function of ERß.
Current published medicinal chemistry efforts have yielded only modestly selective ligands (15, 16, 17). These compounds have been characterized primarily in radioligand binding assays and cotransfection assays, with no in vivo data being reported. We describe here the biological profile of a highly selective, orally active ERß agonist that is representative of a family of compounds we have studied. We characterized this compound in numerous animal models and report that its biological profile is radically different from that of nonselective or ER-selective ligands. Specifically, our data suggest that an ERß-selective ligand will not be useful for hormone therapy or as a contraceptive agent. However, this compound has a dramatic beneficial effect in two models of inflammation involving different organ systems: the HLA-B27 transgenic rat model of inflammatory bowel disease and adjuvant-induced arthritis in the Lewis rat. These data, coupled with a lack of activity on uterus and mammary tissue, suggest that ERß-selective ligands may be novel antiinflammatory agents with significant therapeutic value for treating intestinal and joint inflammation.
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Materials and Methods |
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. Briefly, the ligand-binding domain of human, rat, or mouse ER or ERß (expressed in Escherichia coli) was incubated with 2 nM [3H]17ß-estradiol (NEN/PerkinElmer, Boston, MA) and various concentrations of test compound for 18 h at room temperature in high binding microtiter plates. The IC50 was defined as the concentration of radioinert compound that reduced maximal binding by 50%.
IGF binding protein-4 (IGFBP4) assay
This assay was performed as previously described (14). Briefly, SAOS-2 cells were engineered to express either ER or ERß using adenovirus vectors (18). Cells were then treated with compound for 24 h, and IGFBP4 mRNA levels were measured by real-time quantitative RT-PCR. Fold induction was calculated by comparing the compound-treated groups’ IGFBP4 mRNA values to those of the vehicle-treated group.
Uterotrophic assay
The effect of compounds on increasing the wet weight of the sexually immature rat uterus was evaluated as previously described (14). Briefly, rats (19 d of age) were fed a casein-based diet (5K96C, Purina Mills, Richmond, IN) and treated sc for 3 d with test compound in a vehicle of 50% dimethylsulfoxide (DMSO; JT Baker, Phillipsburg, NJ)/50% 1x Dulbecco’s PBS (Life Technologies, Inc./Invitrogen, Grand Island, NY). Approximately 24 h after the last dose, rats were euthanized, and their uteri were excised and weighed after expressing any luminal fluid. A one-way ANOVA (Dunnett’s test with Huber weighting) was used to assess differences between groups, with the vehicle group set as the comparator. These and all other animal studies were conducted under protocols approved by our institutional animal care and use committees.
Mammotrophic assay
Twenty-eight-day-old Sprague Dawley rats (Taconic Farms, Germantown, NY) were ovariectomized and rested for 9 d. Animals were housed under a 12-h light, 12-h dark cycle, fed a casein-based Purina Laboratory Rodent Diet 5K96 (Purina Mills, Richmond, IN), and allowed free access to water. Rats were then dosed sc for 6 d with vehicle (50% DMSO/50% 1x Dulbecco’s PBS), 17ß-estradiol (0.1 mg/kg; Sigma-Aldrich Corp., St. Louis, MO), or ERB-041 (20 mg/kg). For the final 3 d, rats were also dosed sc with progesterone (30 mg/kg; Wyeth compound library). On the seventh day, rats were euthanized, and a mammary fat pad was excised. This fat pad was analyzed for casein kinase II mRNA as a marker of end-bud proliferation. Casein kinase II mRNA was analyzed by real-time RT-PCR. Briefly, RNA was isolated after TRIzol (Life Technologies/Invitrogen) according to the manufacturer’s directions. Samples were treated with deoxyribonuclease I using a DNA-free kit (Ambion, Austin, TX), and casein kinase II mRNA levels were measured by real-time RT-PCR using the TaqMan Gold procedure (PerkinElmer, Boston, MA). A total of 50 ng RNA was analyzed in triplicate using a casein kinase II-specific primer pair (5' primer, CACACGGATGGCGCATACT; 3' primer, CTCGGGATGCACCATGAAG) and a customized probe (TAMRA-CGGCACTGGTTTCCCTCACATGCT-FAM). Casein kinase II mRNA levels were normalized to the 18S ribosomal RNA contained within each sample reaction using primers and probe supplied by PE Applied Biosystems (Foster City, CA). A one-way ANOVA (Dunnett’s test with Huber weighting) was used to assess differences between groups, with the vehicle group set as the comparator.
Tissues used for morphological studies were placed in fixative (30% methanol, 64% acetone, and 0.26% paraformaldehyde). The tissues remained in the fixative for a minimum of 4 h. They were then placed in a 20% sucrose solution in 0.1 M sodium phosphate (pH 7.0) and incubated at 4 C overnight. Tissues were embedded in OCT medium (Miles, Elkhart, IN), and frozen sections were cut at 10 µm at -35 C. Slides were stored at -80 C until stained with hematoxylin and eosin for morphological evaluation.
Osteoporosis model
The effects of test compounds on bone density after ovariectomy were evaluated as previously described (14). Briefly, rats (
250 g) were fed a casein-based diet (Purina Laboratory Rodent Diet 5K96, Purina Mills) and treated sc for 6 wk beginning 1 d after ovariectomy. Body weight, uterine weight, and bone mineral density of the proximal tibia were measured.
Ovulation model
The ability of test compounds to affect ovulation was evaluated as previously described (19). Briefly, rats were fed Purina Laboratory Rodent Diet 5K96 and treated with LHRH to synchronize their estrous cycles. For the next 8 d, rats were treated sc with test compound in a vehicle of 50% DMSO/50% 1x Dulbecco’s PBS. At the end of the study, the number of rats ovulating and the number of oocytes produced were measured.
HLA-B27 transgenic rat model of inflammatory bowel disease
Male HLA-B27 transgenic rats were obtained from Taconic Farms and provided unrestricted access to food (Purina Mills Lab diet 5001) and water. Typically, rats were used between 17–25 wk of age after they had chronic diarrhea. Rats (n = 4/group) were treated orally, once per day with test compound prepared in a vehicle of 2% Tween-80 (Fisher Scientific, Pittsburgh, PA)/0.5% methylcellulose (Sigma-Aldrich Corp.).
During treatment, stool quality was observed daily and graded according to the following scale: diarrhea = 3; soft stool = 2; and normal stool = 1. At the end of the study the rats were euthanized with CO2, and sections of colon were prepared for histological analysis. Colonic tissue was immersed in 10% neutral buffered formalin. Each specimen of colon was separated into four samples for evaluation. The formalin-fixed tissues were processed in a Tissue-Tek vacuum infiltration processor (Miles, West Haven, CT) for paraffin embedding. The samples were sectioned at 5 µm and then stained with hematoxylin and eosin for blinded histological evaluations using a scale modified from that described by Boughton-Smith (20, 21, 22, 23). After the scores were completed, the samples were unblinded, and data were tabulated and analyzed by ANOVA linear modeling with multiple mean comparisons.
Lewis rat model of adjuvant-induced arthritis
Male or female, 12-wk-old Lewis rats were obtained from Charles River Laboratories (Wilmington, MA) and housed according to standard operating procedures. They received a standard regimen of food (Purina Mills Lab diet 5001) and water ad libitum. Complete Freund’s adjuvant (lot 084H8800; Sigma-Aldrich Corp.) was used to induce arthritis; each milliliter contained 1 mg heat-killed and dried Mycobacterium tuberculosis, 0.85 ml mineral oil, and 0.15 ml mannide monooleate. Rats were injected intradermally with 0.1 ml complete Freund’s adjuvant at the base of the tail. Each day, the groups (n = 6 rats) were treated orally with either vehicle (2% Tween 80/0.5% methylcellulose) or test compound. All rats began treatment on d 8 after adjuvant injection when joints were maximally inflamed.
The degree of arthritis severity was monitored daily and scored according to the following disease indexes: hindpaw erythema, hindpaw swelling, tenderness of the joints, and movements and posture. An integer scale of 0–3 was used to quantify the level of erythema (0 = normal paw, 1 = mild erythema, 2 = moderate erythema, 3 = severe erythema) and swelling (0 = normal paw, 1 = mild swelling, 2 = moderate swelling, 3 = severe swelling of the hind paw). The maximal score per day was 12.
At the end of the study the rats were euthanized with CO2, hindlimbs were fixed in 10% buffered formalin, and the tarsal joints were decalcified and embedded in paraffin. Histological sections were stained with hematoxylin and eosin or Saffranin O-Fast Green stain. Slides were coded so that the examiner was blinded to the treatment groups. Synovial tissue from tarsal joints was evaluated based on synovial hyperplasia, inflammatory cell infiltration, and pannus formation (24). In addition, articular cartilage and bone were evaluated using Mankin’s histological grading system (25).
Statistical analysis was performed using the Super ANOVA (Abacus Concepts, Inc., Berkeley, CA). All of the parameters of interest were subjected to ANOVA with Duncan’s new multiple range post hoc testing between groups. Data are expressed as the mean ± SD, and differences were deemed significant if P < 0.05.
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) for the ligand-binding domains of human, rat, and mouse ER and ERß. For comparison, the IC50 was also determined for 17ß-estradiol, a nonselective and presumably the most potent natural ligand for both ERs. As shown in Table 1, 17ß-estradiol binds to human, rat, and mouse ER and ERß with equal and high affinity (IC50 = 2–4 nM). Although ERB-041 binds to ERß with an IC50 of 3–5 nM (similar to 17ß-estradiol), it is 200-fold or more selective. Binding to the full-length ERß and ER sequences was also evaluated, and the compound was similarly selective (data not shown).
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and ERß in the human osteosarcoma cell line, SAOS-2. We compared the activities of ERB-041 and 17ß-estradiol on this end point. We found that ERB-041 behaves as a full agonist on both ERs, but is 14-fold more potent at regulating IGFBP4 mRNA via ERß than via ER (Table 2). In contrast, the nonselective ligand 17ß-estradiol was 16-fold more potent with ER than with ERß.
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Rodent uterotrophic assays
The sexually immature rodent uterus provides a standard and sensitive bioassay for estrogen action. Wet weight gain, regulation of gene expression, and histological changes are typical end points. In the rat, administration of 0.06 µg/d 17-ethynyl-17ß-estradiol causes an approximately 4-fold increase in organ weight (Table 3). However, administration of 2 mg/d ERB-041 did not increase uterine weight. In addition, ERB-041 had no antiestrogen effect, as shown by coadministration of the two compounds. Consistent with these data, no increase in organ weight was seen when ERB-041 was tested in sexually immature mice (100 mg/kg for 4 d), ovariectomized mice (10 mg/kg for 5 wk), or ovariectomized rats (10 mg/kg for 6 wk; data not shown).
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, ERB-041 was unable to substitute for 17ß-estradiol in this assay, and no increases in CK-II mRNA were seen. There were also no changes visible upon histological analysis (data not shown).
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-selective ligand (14). A second end point of this model was ovariectomy-induced weight gain, which was also prevented by these three classes of agents. However, ERB-041 had no effect on either proximal tibia bone mineral density or ovariectomy-induced weight gain (Table 5). Taken together, these data suggest that these effects of estrogens are mediated via ER.
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-ethynyl-17ß-estradiol) will interfere with the normal estrous cycle and inhibit ovulation. There are two possible targets: the hypothalamic-pituitary-gonadal axis and the ovary (e.g. granulosa cells). 17-Ethynyl-17ß-estradiol (1 µg/kg) completely blocked ovulation (data not shown). However, ERB-041 (50 mg/kg) did not affect the number of animals ovulating or the number of oocytes recovered when given to synchronized rats for 4 d (data not shown) or 8 d (Table 6).
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Oral daily administration of ERB-041 rapidly converts the chronic diarrhea these rats experience to a normal stool. Full efficacy is seen with doses as low as 1 mg/kg. Efficacy seems to diminish at doses of 0.3 and 0.1 mg/kg, as the response is slower. A composite graph of these data is shown in Fig. 2.
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. As shown in Table 7, all doses of ERB-041 were equally effective at significantly reducing total disease score.
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and ERß activity, was coadministered with ERB-041. This ER antagonist completely blocked the salutary effects of ERB-041 (Fig. 4), indicating that ERB-041 is acting via an ER in this model. As ERB-041 is selective for ERß over ER, we conclude that ERB-041 is acting via ERß.
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As shown in Fig. 5, a composite graph of three studies, joint swelling was rapidly and dramatically reduced in rats treated with ERB-041. Full efficacy was seen with a dose of 1 mg/kg, whereas a dose of 0.3 mg/kg was weakly effective. Histological analysis (Table 8) showed that each category of synovitis and Mankin scores was significantly reduced for the 20, 10, 5, and 1 mg/kg doses of ERB-041.
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Discussion
In this report we describe the biological profile of a highly selective ERß agonist, ERB-041. We show that this compound is inactive in several classic models of estrogen action, but that it has potent activity in two models of inflammation. We therefore propose that ERß-selective ligands may have utility in treating chronic inflammatory diseases.
We began our studies with the hypothesis that an ERß-selective ligand would be a tissue-selective estrogen useful for hormone therapy. This idea was based primarily on its distribution, as it is expressed in a variety of tissues thought to respond positively to estrogens. Two approaches were taken to validate ERß as a drug target. First, we created an ERßKO mouse (28). The phenotype of this mouse was relatively benign, with a primary defect of subfertility, presumably due to high ERß expression in ovarian granulosa cells. Our second approach was to design selective ligands and characterize their activity in clinically relevant in vivo models. Designing highly selective ligands for ERß (over ER) was not a trivial undertaking due to the high degree of homology between the amino acid residues contacting the ligand in the binding pocket. In fact, there are only two rather conservative changes (29). However, we were able to synthesize a panel of ERß ligands with sufficient selectivity to enable us to use them to characterize their biological activity. This report focuses on ERB-041, but similar data were obtained for several other compounds in different chemical series.
Using a solid phase radioligand binding assay with a nonselective ligand as the tracer, we show that ERB-041 is 200-fold or more selective for ERß over ER in three species and has an IC50 of 3–5 nM. Binding to mouse and rat ER and ERß was assessed because although we used binding data gathered with the human receptor as our primary screen, preclinical model testing used rodents. Because it has been reported that some compounds’ selectivity changes between species (12), we needed to be certain that ERB-041 would retain its selectivity when tested in models using rats and mice. Our in vitro cell-based transcriptional assay indicated that ERB-041 was an agonist, and this prediction is consistent with the structure of ERB-041 cocrystallized with the ERß ligand-binding domain. Our medicinal chemistry efforts leading to the design of this compound will be described elsewhere, as will the cocrystal structure showing that ERB-041 places the ERß ligand-binding domain in a classic agonist conformation (Malamas, M. S., R. McDevitt, I. Gunawan, E. S. Manas, C. P. Miller, J. Bray, and H. A. Harris, manuscript in preparation).
Having established that ERB-041 was a selective ERß agonist, we sought to use it as a tool to probe and define ERß’s function. Early efforts used models relevant to characterizing a new agent for hormone therapy, with the first key model being one to assess uterine activity. The finding that ERß-selective ligands were nonuterotrophic was expected, given the relative lack of expression of this ER in the rodent uterus. Indeed, ERß is an attractive drug target for hormone therapy because it is not the dominant uterine ER. Not only is ERB-041 nonuterotrophic, we also find that it is inactive in a large panel of estrogen-responsive models. It does not prevent bone loss or weight gain after ovariectomy, it is not mammotrophic, and it does not inhibit ovulation. These results taken together with those using the ER-selective ligand, PPT (14), suggest that estrogens’ uterine, bone, and body weight effects are mediated solely via ER. PPT’s effects on ovulation or the mammary gland have not been evaluated, but we would predict a profile similar to that for 17ß-estradiol.
Clearly the biological profile of ERB-041 contradicted our initial hypothesis that an ERß-selective ligand would be a useful agent for hormone therapy, although we were correct that such a compound would be nonuterotrophic. We sought, therefore, to characterize ERB-041’s activity in other in vivo models, and the first one in which we saw activity was the HLA-B27 transgenic rat. This rat expresses HLA-B27 and human ß2-microglobulin proteins, which over time elicit a Th-1-mediated autoimmune response. Multiple organ systems are involved, with the intestinal phenotype appearing first (30, 31). These rats develop chronic diarrhea and intestinal lesions and are used to evaluate therapeutic agents for the treatment of inflammatory bowel disease (23, 32). In the course of unrelated work we discovered that 17-ethynyl-17ß-estradiol improved stool character in these rats (Harnish, D. C., L. M. Albert, Y. Leathurby, A. M. Eckert, A. Ciarletta, M. Kasaian, and J. C. Keith, Jr., manuscript submitted). This observation encouraged us to evaluate ERB-041, and we found that it had robust activity. Stool character rapidly normalized, with effects seen just 1 d after a single oral dose. Full improvement of stool character in each rat was achieved with a daily 5 mg/kg oral dose, with three of four rats fully normalized with a dose of 1 mg/kg. Histological analysis confirmed that the ERB-041 promoted healing at all doses tested, with reductions in total disease score of 50–60%. The integrity of colonic mucosa was restored, the frequency of ulceration was decreased, and the luminal barrier was intact. Numerous goblet cells were present, and protective mucus was produced. Inflammatory infiltrates in the lamina propria were reduced, and less fibrosis was seen.
To determine whether the effects of ERB-041 were mediated via an ER, the ER antagonist ICI-182780 was used. ICI-182780 is an antagonist for both ER and ERß and is a common reagent to assess the ER dependence of a physiological response (33, 34, 35). Rats cotreated with ERB-041 and ICI-182780 were indistinguishable from vehicle-treated rats, indicating that ERB-041’s activity is ER dependent and is not due to another unknown receptor. Because ERB-041 is selective for ERß, we infer that ERB-041 exerts its beneficial effect through this ER.
ERB-041 also has robust activity in the adjuvant-induced arthritis model. Lewis rats are particularly susceptible to inflammatory stimuli, probably due to defects in their hypothalamic-pituitary-adrenal axis, leading to hyposecretion of CRH (36, 37). Thus, a single intradermal injection of complete Freund’s adjuvant elicits a powerful immune response that is in part directed against glycoproteins in the bacterial cell walls. These antigens share epitopes with heat shock proteins, and within 8 d joints are severely inflamed. Similar to the HLA-B27 transgenic rat, a response to ERB-041 is seen the day after the first dose, with full normalization of joint scores seen approximately 10 d later. Histological analysis confirms resolution of inflammation in the joint, measured by decreases in synovitis and Mankin scores of 50–75%. The degree of synovial membrane proliferation and villus formation is significantly reduced. Inflammatory cell infiltrates are decreased, fibroplasia is reduced, and the proliferative pannus reaction is virtually eliminated. The articular cartilage is preserved, and the depletion of proteoglycans (as measured by Saffranin-O staining) is reduced. ERB-041 is similarly potent in these two inflammatory models, with maximum activity seen at 1 mg/kg (oral daily dosing).
Although we have shown that ERß mediates the effects of ERB-041, its mechanism of action beyond this receptor has yet to be determined. As ERß is expressed in both intestine and joint (38, 39, 40, 41), it is possible that ERB-041 is modulating receptor activity in these affected organs. Global gene expression analysis (GeneChip) of HLA-B27 transgenic rat whole colon tissue revealed no consistent changes (data not shown), suggesting that either the intestine is not the site of action or the target cell population was not sufficiently represented to be detected. Alternatively, ERB-041 may induce subtle changes in several genes, each one of which is below the level of detection, but which together translate into a significant physiological response.
Another possibility is that ERB-041 exerts its effects systemically, for example by modulating the immune system. Nonselective estrogens affect the immune system, and there is a wealth of literature on this subject (42, 43, 44). Nonselective estrogens also inhibit nuclear factor-
B-mediated processes (45, 46), but the suggestion has been made this phenomenon is mediated by ER, not ERß (47). We have tested ERB-041 in in vitro models of immune cell function, such as splenocyte proliferation in response to anti-CD3 antibody and lipopolysaccharides-induced cytokine release from monocytes, but have yet to see an effect (data not shown). Clearly, further work is needed to determine the molecular events preceding restitution of disease in these two models. At present our efforts are directed toward monitoring changes in serum proteins, and preliminary data suggest that acute phase proteins may be down-regulated by ERß ligands. Evaluation of ERB-041 continues in other models of inflammation to help determine a pattern of activity that will help define the mechanism and possibly suggest other therapeutic indications.
In summary, we have developed and characterized the biological profile of a highly selective ERß agonist. This compound is inactive on traditional target tissues such as the uterus and mammary gland and does not spare bone, inhibit ovulation, or prevent ovariectomy-induced weight gain in clinically predictive rat models. However, it has significant beneficial effects in two models of systemic inflammation, the HLA-B27 transgenic rat and adjuvant-induced arthritis in the Lewis rat. These results support our contention that ERB-041 and other ERß-selective ligands may be therapeutically effective in the treatment of inflammatory bowel disease and/or arthritis.
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Acknowledgments |
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Footnotes |
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Present address for D.E.F.: Pharmacia Corp., Kalamazoo, Michigan 49007.
Abbreviations: DMSO, Dimethylsulfoxide; ER, estrogen receptor; IC50, concentration of radioinert compound that reduced maximal binding by 50%; IGFBP4, IGF binding protein-4; KO, knockout; PPT, propyl pyrazole triol.
Received May 2, 2003.
Accepted for publication June 25, 2003.
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