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Women’s Reproductive Health Research Center, Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine (K.L.B.-T., E.E., K.G.O.), Nashville, Tennessee 37232; and Division of Endocrinology and Reproductive Disorders, Women’s Health Research Institute, Wyeth Research (Z.Z., R.C.W.), Collegeville, Pennsylvania 19426
Address all correspondence and requests for reprints to: Dr. Kevin G. Osteen, Women’s Reproductive Health Research Center, Department of Obstetrics and Gynecology, Vanderbilt University School of Medicine, 1161 21st Avenue S, MCN B-1100, Nashville, Tennessee 37232. E-mail: kevin.osteen{at}vanderbilt.edu.
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Abstract |
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Objective: Reduced progesterone responsiveness results in an elevated endometrial expression of matrix metalloproteinases (MMPs) during the secretory phase of the menstrual cycle in women with endometriosis. Although cyclic MMP expression is critical for endometrial growth and remodeling, the failure of progesterone to down-regulate MMPs may impair nidation and promote the invasive establishment of endometriosis. In the current study we examined the ability of a newly developed progesterone receptor (PR) agonist, tanaproget (TNPR), to down-regulate endometrial MMP expression in vitro and regress experimental endometriosis in vivo.
Setting: This study was performed at a university-based medical center.
Participants: Asymptomatic volunteers and patients with endometriosis were studied.
Main Outcome Measures: We examined the ability of TNPR to down-regulate endometrial MMP expression in vitro compared with that of natural progesterone and two currently marketed synthetic steroidal progestins. Using a human/mouse model of endometriosis, we also tested the in vivo ability of TNPR to regress ectopic lesions established by tissues with reduced progesterone sensitivity.
Results: TNPR effectively down-regulated MMP expression in vitro and induced significant reduction of lesions in mice with disease established by tissues from endometriosis patients.
Conclusion: Given the positive preclinical pharmacological profile of TNPR that has recently been reported, additional development of this compound for the treatment of endometriosis is warranted.
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Introduction |
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Emerging research from a number of laboratories suggests that alterations in the basic biology of the eutopic endometrium of endometriosis patients may be intimately linked to the pathophysiology of the disease (2). For example, reduced progesterone (P) responsiveness in the endometrium of these patients affects the regulation of multiple genes, including genes associated with the expression and regulation of matrix metalloproteinases (MMPs) (3, 4). Although matrix degradation is a normal component of cyclic remodeling in the eutopic endometrium, altered expression of MMPs is associated with disruption of endometrial function, potentially affecting the establishment of pregnancy as well as the development of diseases, including endometriosis (5). Endometrial preparation for pregnancy is complex; however, the ability of ovarian P to directly affect the function of specialized stromal fibroblasts at the maternal-fetal interface appears to play a key role in reducing MMP expression by maternal cells during the invasive process of implantation (6). For example, during the secretory phase of the menstrual cycle, we have shown that P acts not only to down-regulate the cell-specific expression of pro-MMP-3 by endometrial stromal cells, but also to prevent locally produced proinflammatory cytokines from stimulating the expression of this enzyme (7). Importantly, we have also shown that the ability of P to inhibit pro-MMP-7 expression by epithelial cells requires stromal-derived paracrine signals (8).
In the absence of pregnancy, the loss of P support leads to increased levels of MMP expression by multiple cell types during menstruation, providing the enzymatic potential for the invasive establishment of ectopic sites of endometrial growth. Using a chimeric human/mouse model system, we have shown that biological agents that impact the expression and action of endometrial MMPs dramatically affect both tissue invasion (9) and the establishment of a vascular supply (10). Interestingly, in vitro treatment of endometrial tissues acquired from women with endometriosis with P fails to fully suppress either pro-MMP-3 or pro-MMP-7 secretion and fails to prevent the ability of these tissues to establish experimental disease in mice (3). We have recently identified a loss of P receptor-B (PR-B) expression compared with the expression of PR-A in the endometrium of endometriosis patients (11), perhaps explaining the reduced P responsiveness that we and others have noted in these women. Taken together, research findings using endometrial tissue obtained from endometriosis patients suggest that effective, progestin-based therapies in these women must take into consideration their reduced endometrial responsiveness to endogenous P.
Norethindrone acetate (NETA) and medroxyprogesterone acetate (MPA) are synthetic steroidal progestins often used in the treatment of women with endometriosis (12). Although these compounds have been used with some success, they also exhibit unwanted side effects due to activation of cell signaling pathways after interactions with other closely related steroid receptors. Recently, the molecular and pharmacological properties of tanaproget (TNPR), a novel, nonsteroidal PR agonist developed by Wyeth Research, have been described (13, 14). These studies demonstrated that TNPR is highly selective for PR, binding with high affinity and demonstrating full efficacy and enhanced potency in the rat ovulation inhibition assay compared with MPA (14). However, unlike PR agonists currently available for medical therapy, TNPR exhibits only limited interaction with other steroid receptors, potentially limiting the side effects generally associated with currently marketed progestins. In the present study we examined the potential efficacy of TNPR as a human therapeutic agent using in vitro cell and organ culture systems and an in vivo model of experimental endometriosis. We found that endometrial tissues obtained from normal women respond equally well to natural P, MPA, and TNPR. However, TNPR and MPA were more effective than natural P in down-regulating MMP secretion in tissues from women with endometriosis. Compared with P, TNPR was significantly better at reducing the ectopic growth of human tissue in our experimental model of endometriosis.
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Materials and Methods |
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Normal endometrial tissues (n = 15) were acquired by Pipelle (Unimar, Inc., Wilton, CT) biopsy during the proliferative phase (d 9–12) of the menstrual cycle from a donor population (age, 18–45 yr) exhibiting normal menstrual cycles and no history of endometriosis. Endometrial tissues from women with surgically confirmed endometriosis (n = 9) were also obtained by biopsy during the proliferative phase. An endometrial thickness of 9 mm or more (confirmed by vaginal ultrasound) and a serum P level of 1.5 ng/ml or less were required for inclusion in this study. Individuals with a recent (
3 months) history of hormone therapy (i.e. oral contraceptives) were excluded. Biopsies were washed in prewarmed, phenol-red free DMEM/Ham’s F-12 medium (Sigma-Aldrich Corp., St. Louis, MO) to remove residual blood and mucous before culture. Informed consent was obtained before biopsy, and the use of human tissues was approved by Vanderbilt University’s institutional review board and committee for the protection of human subjects.
Organ cultures of human tissues
Endometrial biopsies were dissected into small cubes (
1 x 1 mm3), and eight to 10 pieces of tissue per treatment group were suspended in tissue culture inserts (Millipore Corp., Bedford, MA) as previously described (3, 9). Organ cultures were maintained under serum-free conditions in DMEM/F-12 supplemented with 1% insulin-transferrin-selenium (ITS+, Collaborative Biomedical, Bedford, MA) and 0.1% Excyte (Miles Scientific, Kankakee, IL) and incubated at 37 C in a humidified chamber with 5% CO2. Treatments included 17ß-estradiol [E; 272.4 pg/ml (1 nM)], E plus P [1 nM and 157.25 ng/ml (500 nM), respectively], or 1 nM E plus synthetic progestins at various concentrations. Specifically, doses of NETA included 340.46 pg/ml (1 nM), 34.05 pg/ml (100 pM), 3.405 pg/ml (10 pM), and 0.3405 pg/ml (1 pM). MPA was administered at 386.54 pg/ml (1 nM), 38.65 pg/ml (100 pM), 3.865 pg/ml (10 pM), and 0.3865 pg/ml (1 pM). TNPR was used at 297.38 pg/ml (1 nM), 29.74 pg/ml (100 pM), 2.974 pg/ml (10 pM), and 0.2974 pg/ml (1 pM). Cultures were maintained for 24 h before injection into mice or 48–72 h before analysis of protein expression (pro-MMP-3 and pro-MMP-7). E, P, NETA, and MPA were purchased from Sigma-Aldrich Corp.; TNPR was prepared by the Medicinal Chemistry Department at Wyeth Research. The chemical and crystal structures of TNPR have recently been reported (13).
Cell culture and in vitro treatment
Isolated endometrial stromal cells were obtained by enzymatic digestion and filter separation (15). Two hundred thousand stromal cells were seeded onto 1.9-cm2 wells coated with type I collagen. Isolated cells were maintained in phenol-red-free DMEM/F-12 supplemented with 5% charcoal-stripped calf serum plus 1 nM E and 1x antibiotic-antimycotic solution at 37 C. Subconfluent cells were removed to serum-free medium with 1 nM E in the presence or absence of 500 nM P or synthetic progestin as noted. Some cultures additionally received 200 pg/ml (11 pM) IL-1
for the last 24 h of culture. After 3–5 d in culture, medium was removed and analyzed for the expression of pro-MMP-3.
Western analysis of protein expression
Medium was collected from cell and organ cultures, and secreted proteins were quantified using the Coomassie Plus Protein Assay (Pierce Chemical Co., Rockford, IL) and 20 µg total protein subjected to 10% SDS-PAGE. Proteins were transferred to a polyvinylidene difluoride membrane (Amersham Biosciences, Arlington Heights, IL) and blocked in PBS with 10% nonfat milk and 0.05% Tween 20. Blots were incubated overnight at 4 C in PBS/milk/Tween with a primary antibody, washed, and incubated with secondary antibody for 1 h. Proteins were visualized by chemiluminescence (Amersham Biosciences) and autoradiography. As a negative control, identical blots were incubated without a primary antibody. Antibodies directed against human pro-MMP-3 (mouse monoclonal; Oncogene Research Products, Cambridge, MA), pro-MMP-7 (rabbit polyclonal; Chemicon International, Temecula, CA), and the secondary antibodies (horseradish peroxidase-conjugated; Amersham Biosciences) were commercially obtained as described.
Experimental model of endometriosis
The model of endometriosis was performed as previously described (3, 9, 16). Briefly, 5-wk-old athymic (ncr/nude), ovariectomized mice (Harlan Sprague Dawley, Indianapolis, IN) were anesthetized with isoflurane (Henry Schein Veterinary, Melville, NY) and sc implanted with a silastic capsule containing 8 µg E. Twenty-four hours later, mice were injected sc with a PBS suspension of eight to 10 human endometrial tissue fragments per mouse at the ventral midline just below the umbilicus. For 24 h immediately preceding injection, tissue fragments were established as organ cultures and treated with 1 nM E. Ten to 12 d after tissue injection, the presence of sc lesions was assessed via external visual examination (Fig. 1
), and all positive animals were randomly segregated and assigned as appropriate to receive placebo control (2% Tween 80/0.5% methylcellulose) daily by gavage or a single, slow-release silastic capsule containing 25 µg P or 300 µg/kg TNPR (in 2% Tween 80/0.5% methylcellulose) daily by gavage. Fourteen to 16 d after initiation of the treatment regimen, mice were again anesthetized and killed by cervical dislocation for direct examination of lesion size and number. The experiments described in this report were approved by Vanderbilt University institutional animal care and use committee in accordance with the Animal Welfare Act.
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Each mouse was assigned a lesion load score based on the number and size of the lesions. Scores were then analyzed with a Mann-Whitney U test using PRISM software (GraphPad, Inc., San Diego, CA) (17). Significance was considered to be P < 0.05. Results of both the parametric and nonparametric tests were identical.
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Results |
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Before examining the impact of each synthetic progestin on endometrial MMP regulation, we determined the lowest effective dose of natural P that suppressed the in vitro secretion of pro-MMP-3 in isolated human stromal cells maintained in the presence of a constant level of E. Additionally, because the ability of P exposure to block the stimulatory impact of proinflammatory cytokines is important to blocking MMP expression in vivo (7), we challenged similarly cultured cells with IL-1.
Isolated endometrial stromal cells
The results demonstrated that normal endometrial stromal cells cultured in the presence 1 nM E exhibited a rapid decline in pro-MMP-3 secretion in response to a broad range of P doses (Fig. 2A, upper gel). Only at the 1-nM P dose did this steroid fail to completely suppress the secretion of pro-MMP-3. Similarly cultured stromal cells, maintained with 1 nM E and exposed to P, also secreted little pro-MMP-3 after a challenge with IL-1 (Fig. 2A, lower gel), although MMP-3 secretion was detected in the presence of both 10 and 1 nM P.
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Endometrial stromal cells were isolated from women with endometriosis and cultured with 1 nM E and the same doses of P as those reported above. In cells from endometriosis patients, only the highest dose of natural P (500 nM) was found to suppress pro-MMP-3 expression in vitro (Fig. 2B
, upper gel). After IL-1 challenge, previous P exposure failed to prevent IL-1-stimulated expression of pro-MMP-3 at any dose tested (Fig. 2B, lower gel).
Regulation of MMP expression in normal endometrium: isolated stromal cells
In the next series of studies, a dose-response analysis was conducted to examine the ability of NETA, MPA, and TNPR to suppress MMPs compared with natural P. As shown in Fig. 3A, at the 1-nM dose level, each synthetic progestin was able to suppress pro-MMP-3 secretion more effectively compared with cells treated at the same dose of natural P (Fig. 2). Interestingly, MPA and TNPR were more effective in suppressing pro-MMP-3 secretion in vitro than NETA at all doses tested. As shown in Fig. 3B, P, MPA, and TNPR were each effective at preventing IL-1-mediated stimulation of pro-MMP-3 secretion compared with stromal cells treated with E and IL-1 alone. Only at the highest doses tested (1 and 100 pM), was NETA as effective as 500 nM P in preventing the stimulatory impact of IL-1 on pro-MMP-3 secretion.
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Normal endometrium.
After the dose-response studies using isolated stromal cells, we examined the impact of P and synthetic progestins on the secretion of pro-MMP-3 as well as pro-MMP-7, an epithelial-specific member of the MMP family, using endometrial organ cultures established from women with no history of endometriosis. As shown in Fig. 4A, 500 nM P readily suppressed MMPs, as did 1 nM MPA and TNPR. At the 100-pM dose, TNPR was most effective. NETA was ineffective at either dose tested.
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). NETA failed to suppress MMP secretion in any organ culture examined. Regression of experimental endometriosis
Normal endometrium.
We previously demonstrated that our experimental endometriosis model using human lesions growing on the parietal peritoneum of mice can be used to assess the efficacy of potential therapeutic agents (10, 18). In the current study we examined the ability of TNPR compared with natural P to regress previously established human lesions in nude mice. Our results, outlined in Table 1, indicate that 100% of mice receiving human tissues and placebo demonstrated continued growth of ectopic lesions at the end of the treatment period. In contrast, treatment with natural P significantly regressed lesions, resulting in a complete absence of lesions in the majority of animals (67%). In animals receiving TNPR, complete regression of human lesions was noted in more than half the treated animals (58%). Importantly, in animals receiving therapeutic treatment with either TNPR or natural P, the lesions observed at the end of therapy were smaller and fewer in number than the lesions in placebo-treated mice.
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, although all animals in either the placebo group or the natural P treatment group exhibited persistent lesion growth, 33% of mice receiving TNPR therapy were completely free of detectable human lesions. Additionally, all animals treated with TNPR demonstrated a reduction in lesion size and number compared with animals treated with P or placebo (Fig. 5).
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Discussion |
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Our laboratory has focused on developing therapeutic strategies for the treatment of endometriosis based on the ability of P to directly and indirectly regulate the expression of the endometrial MMP system as well as the activity of these enzymes within the peritoneal microenvironment. Endometriosis is believed to most often result from the ectopic attachment of refluxed menstrual tissue; however, most cells within the menses are terminally differentiated and would be unlikely to retain the capacity for ectopic invasion and estrogen-mediated growth. Although certain endometrial cells within the menses clearly retain growth potential, the complexity of identifying and separating these cells from other components of the menstruum precludes the use of human menses as a workable experimental model. Therefore, to avoid these complexities, we have used biopsies of human endometrial tissue acquired during the proliferative phase to establish an experimental model of endometriosis in nude mice (9). Because any biological agent potentially involved in the regulation of the endometrial MMP system can be directly targeted in vitro or in vivo in this system, our experimental endometriosis model has proven useful for identifying factors with therapeutic potential for the treatment of endometriosis (10, 18). For example, nude mice receiving normal human endometrial tissue treated in vitro with natural P exhibit reduced expression of MMPs and thus have a reduced capacity to establish ectopic lesions in these animals compared with tissues treated with E alone (9). In contrast, endometrial biopsies acquired from endometriosis patients and treated in a similar fashion in vitro readily establish endometrial lesions in vivo regardless of P treatment (3). At this juncture, the role that reduced P-mediated regulation of endometrial MMP gene or protein expression plays in a woman’s overall risk for developing endometriosis is unknown. Nevertheless, the failure of endogenous P to down-regulate the expression of MMPs in the endometrium of women with endometriosis is clearly associated with an increased capacity for ectopic invasion in our nude mouse model (3, 9). Importantly, the reduced ability of P to regulate the expression of numerous genes in the eutopic endometrium of endometriosis patients (3, 4, 5) probably explains the variable effectiveness of natural P as a therapeutic agent in some women with this disease.
As noted previously, although most treatments for endometriosis are based on the concept that this is an estrogen-driven disease, it is becoming increasingly clear that endometriosis is also a disease of reduced P responsiveness (5). During normal endometrial maturation, the ability of P to down-regulate the expression of the MMP system is intimately linked to the ability of this steroid to block the action of locally produced, proinflammatory cytokines. For example, we have shown that 24–48 h of exposure to P either in vivo or in vitro reduces the subsequent stimulatory impact of IL-1 on pro-MMP-3 expression by specialized stromal fibroblasts (7). Equally important, agents that reduce the ability of P to protect stromal cells from the stimulatory impact of proinflammatory cytokines lead to significant increases in pro-MMP-3 secretion (5, 11). The critical antiinflammatory role of P during the process of stromal maturation suggests that examining the in vitro responsiveness of these cells to proinflammatory cytokines may be useful in determining the efficacy of progestin-based therapeutics. In the current study we examined the therapeutic potential of TNPR, a novel nonsteroidal PR agonist, determining the ability of this agent to both down-regulate MMP secretion levels in vitro and reduce the ectopic growth of human endometrial tissue in nude mice. Initially, we established cultures of isolated endometrial stromal cells to determine the ability and effective doses of P, NETA, MPA, and TNPR to down-regulate pro-MMP-3 secretion in the presence and absence of a short IL-1 challenge. We subsequently used endometrial organ cultures to examine the ability of these compounds to suppress both pro-MMP-3 and pro-MMP-7 expression in endometrial tissues obtained from normal tissue donors and women with endometriosis.
In these studies we found that pro-MMP-3 expression was readily down-regulated in normal endometrial stromal cells in response to P, even in the presence of IL-1. In cells obtained from the endometrium of endometriosis patients, similar in vitro P treatments failed to prevent pro-MMP-3 stimulation by this proinflammatory cytokine, regardless of the dose used. In a second series of studies, we examined the ability of NETA, MPA, and TNPR to suppress pro-MMP-3 in stromal cells isolated from normal endometrial tissue donors and endometriosis patients, again in the presence and absence of IL-1. We next examined the progestin responsiveness of organ cultures, because endometrial organ cultures contain stromal, epithelial, and immune cells and more closely resemble the cellular phenotypes within the in vivo microenvironment. In both studies we found that MPA and TNPR were each as effective as P in suppressing endometrial MMP secretion in isolated cells and organ cultures established from normal tissues. However, only the synthetic progestins were able to suppress pro-MMP-3 and pro-MMP-7 in cultures from patients with a history of endometriosis. Although NETA was as effective as P, MPA, and TNPR in normal cells in the absence of an IL-1 challenge, NETA was unable to suppress MMP expression in stromal cells challenged with IL-1 or within the organ cultures, even at doses similar to those used for P (data not shown). The failure of NETA to suppress MMPs is surprising, given its clinical effectiveness and other studies that suggest that this progestin is effective in the suppression of MMPs in vitro (22). Although the latter study examined isolated stromal cells from normal women, obtaining results similar to those presented in this report, this group did not examine cells from women with endometriosis or normal cells challenged with a proinflammatory cytokine. The current study suggests that NETA may be a weaker antiinflammatory agent than natural P and the other progestins examined, resulting in a reduced ability to down-regulate MMPs in the presence of IL-1. As discussed below, specific interactions with PR-B may also affect the antiinflammatory properties of each progestational compound used in our study. Finally, NETA is weakly estrogenic and, to a limited extent, can be converted to ethinyl estradiol in vivo (23, 24). Thus, the estrogenic action of NETA may counteract progestogenic actions.
Using the nude mouse model, we found that although natural P and TNPR were comparable in their ability to regress lesions established with normal endometrial tissues, TNPR was significantly more effective in regressing lesions established by tissues from patients with endometriosis. Additionally, lesions remaining after TNPR therapy in mice receiving tissues from patients with endometriosis were reduced in both size and number compared with those in other treatment groups. These results suggest that TNPR remains effective in endometrial tissue acquired from endometriosis patients even though this tissue exhibits a reduced expression of PR-B (11). TNPR has previously been found to be more potent than P or MPA (14); thus, this compound may have an enhanced ability to use a reduced number of receptors. Additionally, in vitro studies using human endometrial cells indicate that, compared with P, TNPR may more effectively increase the expression of some PR-B-selective genes (Yudt, Y., T. Berrodin, Z. Zhang, and R. C. Winneker, unpublished observations). Our recent observations, demonstrating that the loss of PR-B in eutopic tissues of women with endometriosis leads to a failure of natural P to down-regulate pro-MMP-3, suggest that this gene may also be regulated by PR-B. The ability of TNPR to suppress pro-MMP-3 expression in endometrial tissues despite a reduction in PR-B expression is additional evidence of its greater potency compared with the natural steroid.
TNPR significantly reduced human disease burden in nude mice bearing xenografts established with tissue from endometriosis patients; this suggests that this compound may have therapeutic value, which should be explored. For example, medical therapy with TNPR may reduce disease burden under conditions of longer-term treatments and may be valuable in delaying disease recurrence after surgery and/or in combination with other therapies targeting angiogenesis, inflammation, or estrogen action. Finally, as stated previously, because TNPR is a nonsteroidal agent that has only weak interactions with other steroid receptors, this compound may ultimately represent an appropriate choice for long-term therapeutic use. Additional studies, including a randomized controlled prospective clinical trial, will be needed to determine whether TNPR lives up to its potential.
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Acknowledgments |
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Footnotes |
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Current address of Z.Z.: Reproductive Therapeutics, Pharmaceutical Research and Development, Johnson & Johnson, 1000 Route 202, Raritan, New Jersey 08869.
K.L.B.-T. and E.E. have nothing to declare. Z.Z. was previously employed by Wyeth. R.C.W. is currenty employed by Wyeth. K.G.O. previously consulted for Wyeth.
First Published Online January 17, 2006
Abbreviations: E, 17ß-Estradiol; MMP, matrix metalloproteinase; MPA, medroxyprogesterone acetate; NETA, norethindrone acetate; P, progesterone; PR, P receptor; TNPR, tanaproget.
Received September 8, 2005.
Accepted January 6, 2006.
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References |
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