Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on July 1, 2008; DOI: 10.1124/jpet.108.140467
0022-3565/08/3271-78-87$20.00
JPET 327:78-87, 2008
ENDOCRINE AND DIABETES
The Translational Pharmacology of a Novel, Potent, and Selective Nonsteroidal Progesterone Receptor Antagonist, 2-[4-(4-Cyano-phenoxy)-3,5-dicyclopropyl-1H-pyrazol-1-yl]-N-methylacetamide (PF-02367982)
Alexander de Giorgio-Miller,
Peter Bungay,
Michelle Tutt,
Julie Owen,
David Goodwin, and
Nick Pullen
Pfizer Global Research and Development, Sandwich, Kent, United Kingdom
Received for publication
May 1, 2008
Accepted
June 30, 2008.
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Abstract
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The progesterone receptor (PR) is an important regulator of endometrial function. Blockade of PR function has been recognized as the potential basis for preventing gynecological conditions such as endometriosis and uterine fibroids. In this study, we examine the in vitro and in vivo properties of a nonsteroidal PR antagonist, 2-[4-(4-cyano-phenoxy)-3,5-dicyclopropyl-1H-pyrazol-1-yl]-N-methylacetamide (PF-02367982) in comparison with the nonselective steroidal antagonist RU-486 (mifepristone). PF-02367982 was found to be a potent PR antagonist with far greater selectivity over the glucocorticoid receptor than RU-486. Both PF-02367982 and RU-486 blocked progesterone-induced arborization of the rabbit endometrium in a dose-dependent manner at unbound drug exposures that were commensurate with their potencies as PR antagonists in vitro. Translation of this pharmacology to a clinically relevant system was required to bridge the pharmacological gap between nonmenstruating rabbits and humans. Thus, the pharmacokinetic (PK) and pharmacodynamic (PD) data from the rabbit were combined to predict pharmacological effects on the naturally cycling cynomolgus macaque endometrium. PF-02367982 blocked the effect of progesterone on the cynomolgus macaque endometrium to the same degree as RU-486 and at exposures predicted by the rabbit PK-PD model. With such an efficacious and superior selectivity profile to the nonselective RU-486, PF-02367982 may have significant therapeutic value in the treatment of gynecological conditions such as endometriosis.
The discovery of synthetic progesterone receptor (PR) modulators has underscored the important role that progesterone plays in reproductive physiology, including the menstrual cycle, endometrial growth, breast development and maintenance of pregnancy (Chabbert-Buffet et al., 2005
; Spitz, 2006). RU-38486 (RU-486, mifepristone) is the founding member of steroidal PR antagonists and is approved clinically for pregnancy termination and emergency contraception. Studies with RU-486 and other PR antagonists, such as onapristone, ZK-137316 (Zelinski-Wooten et al., 1998), and ZK-230211, have revealed that these agents, as well as directly antagonizing progesterone function, can block ovarian function and also arrest the effect of estrogen on the endometrium in women and nonhuman primates (NHP) (Wolf et al., 1989; Baird et al., 2003; Brenner et al., 2005; Chabbert-Buffet et al., 2005; Slayden et al., 2006).
The potential usefulness of RU-486 and other PR antagonists in the treatment of pathological conditions such as endometriosis, uterine fibroids, and cancer is supported by a number of preclinical observations and clinical findings (Grow et al., 1996; Kettel et al., 1996; Baird et al., 2003; Fiscella et al., 2006). Although RU-486 has potent antiprogestagenic activity, it is also an equipotent antagonist at the glucocorticoid receptor (GR) (Heikinheimo et al., 1987). As a consequence, RU-486 suppresses the inhibition of corticotropin secretion induced by corticosteroids, limiting its wider usefulness and clinical benefit with escalating doses. Of the many steroidal PR antagonists that have been described, relatively few have been identified that appear to be devoid of antiglucocorticoid activity (Schoonen et al., 1999; Attardi et al., 2002). One of the goals in developing new and selective PR antagonists is the discovery of a simple and efficient screening methodology that identifies these agents and predicts their desired clinical outcome and dose-effect relationship.
Antiprogestagenic activity is conventionally tested in numerous in vitro and in vivo systems. At the cellular level, this can be determined by the ability of the antagonist to prevent progesterone binding, to block cell proliferation or PR-dependent reporter gene activity, such as the expression of alkaline phosphatase in the human breast carcinoma cell line, T47D (Bardon et al., 1985; Zhang et al., 2000). These assays have been successfully used to design potent PR antagonists, but the apparent simplicity of these systems and endpoints belies considerable complexity in the interaction of synthetic PR ligands and the receptor, the displacement or subversion of promoter activity, and the effect on cellular function. At the physiological level, the effects of PR blockade have been studied in many diverse experimental systems, including inhibition of endometrial arborization by progesterone in rabbits (McPhail, 1934) and inhibition of endometrial proliferation, blockade of ovarian function, induction of anovulatory amenorrhea, and induction of menses in NHP (Wolf et al., 1989; Slayden et al., 2001a). The effects of RU-486 on the endometrium are well documented, with antiprogestagenic effects on endometrial growth and differentiation across multiple species, including, mouse, rat, rabbit, and cynomolgus macaque. Although there is remarkable diversity in the physiology of the respective reproductive systems of these species, the ability of RU-486 to block the effects of estrogen on endometrial proliferation in various animal models suggests that simple in vivo rodent models with a consistent pharmacological endpoint may be used to identify PR antagonists with the desired functional effects.
However, understanding the mechanism of action of PR antagonists, such as RU-486, on the inhibition of endometrial growth provides a significant challenge, and there is also a considerable need to understand the translational pharmacology of these agents between species used in preclinical testing, to accurately predict efficacious doses in humans. The validity of using nonmenstruating species, such as the rabbit, to predict doses for novel PR antagonists in women is limited without confirmation of translation of a pharmacological effect in a menstruating primate.
A multidisciplinary screening strategy was developed to describe the pharmacological profile of a novel and selective nonsteroidal PR antagonist PF-02367982, and to subsequently understand the relationship between PR antagonist pharmacology in vitro and in vivo. The efficacy of PF-02367982 was compared with RU-486 in a modified rabbit McPhail test, from which a novel pharmacokinetic/pharmacodynamic (PK/PD) model was developed to predict drug exposures required to elicit an antiprogestagenic effect on the endometrium in the presence of physiological levels of progesterone. Subsequently, we used the output from this model to demonstrate translation of this pharmacology to the endometrium of adult female cynomolgus monkeys at clinically relevant doses, bridging the pharmacological gap between nonmenstruating rabbits and humans.
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Materials and Methods
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Materials. The synthesis of PF-02367982 will be described elsewhere. RU-486/mifepristone was purchased from Sigma-Aldrich (St. Louis, MO). CEREP (Poitiers, France) was used for the supplemental wide receptor-profiling assay data (Supplemental Table 1) and binding assays for PR PF-02367982 and RU-486. [3H]Dihydrotestosterone (DHT) was purchased from PerkinElmer Life and Analytical Sciences (Waltham, MA).
In Vitro Functional Reporter Assays. Chinese hamster ovary cells were stably transfected with a mouse mammary tumor virus β-lactamase reporter gene, rabbit or human progesterone receptor-B isoform, human androgen receptor (AR), or human mineralocorticoid receptors (MR). Native human PR and GR activity were measured in T47D cells [American Type Culture Collection (Manassas, VA) number HTB-133] as an effect on endogenous alkaline phosphatase activity (Beck et al., 1993) and SW1353 cells (American Type Culture Collection number HTB-94), stably expressing an mouse mammary tumor virus-luciferase reporter gene, respectively. Cells were prepared in an "assay-ready format" as frozen stocks, stored under liquid nitrogen, at approximately 1.5 x 107 cells/ml. Receptor antagonism was measured in a standardized 384-well format with approximately 5000 cells/well in 35 µl of assay medium [Dulbecco's modified Eagle's medium (phenol red), 2 mM glutamine, and either 25 mM HEPES, 10% (v/v) charcoal-stripped fetal calf serum and hypoxanthine-aminopterin-thymidine supplement (PR, AR); or 25 mM HEPES, 5% (v/v) charcoal-stripped fetal calf serum, and nonessential amino acids supplement; or 1 mg/l insulin, 2 g/l lactalbumin hydrosylate, and 0.5 mg/l ascorbate (GR)]. An 11-point concentration dose-response range of PF-02367982 (0.1 nM–10 µM, from a dimethyl sulfoxide stock) was incubated alone or in the presence of an EC80 concentration of the relevant ligand [PR, progesterone, 10 nM (5 nM in T47D assay); AR, testosterone, 700 pM; GR, dexamethasone, 20 nM; MR, aldosterone, 250 pM] overnight in final volume of 50 µl to test for agonism or antagonism. Relevant controls were included to ensure assay quality and reproducibility. β-Lactamase assays were developed with CCF4-AM (Invitrogen, Carlsbad, CA) according to manufacturer's instructions, and fluorescence intensities at 450 and 530 nm were measured. A 450/530 (blue/green) ratio was calculated, and the data were expressed as β-lactamase inhibition by the test compound from which IC50 values were determined. The effect of PF-02367982 (concentrations up to 1000 nM) and RU-486 (concentrations up to 10 nM) on the EC50 values from an 11-point progesterone concentration curve (0.01 nM–1 µM) were determined to calculate dose ratios and subsequently pA2 values by Schild analysis. Luciferase assays were developed with britelite (PerkinElmer Life and Analytical Sciences), according to manufacturer's instructions, and by using a luminescence protocol set up to run in counts per second with an integration time of 100,000 µs (once per well). Percentage inhibition of luciferase reporter activity was calculated, and a corresponding IC50 value was determined. Alkaline phosphatase assays were developed with Tropix CSPD Ready-to-Use Emerald II reagent (PerkinElmer Life and Analytical Sciences) according to manufacturer's instructions, and a luminescence protocol set up to run in counts per second with an integration time of 100,000 µs (once per well) was used. Percentage inhibition of alkaline phosphatase activity was calculated, and a corresponding IC50 value was determined.
In Vitro Binding Assays. Binding to the PR receptor was determined by a MCF-7 cytosol binding assay provided by CEREP, using published protocols (Eckert and Katzenellenbogen, 1982). Glucocorticoid binding was determined by using a fluorescence polarization assay (PanVera Corp., Madison, WI) according to the manufacturer's instructions. Androgen binding was determined in a radioligand-binding assay by using lysates of MDA-MB-453 with minor modifications to historical protocols (Hall et al., 1994). Cell lysates were prepared in 25 mM K2HPO4, pH 7.6, buffer, 10% glycerol, 5 mM EDTA, 1 mM dithiothreitol, 5 mM NaF, 10 mM sodium molybdate, 0.1 mg/ml bacitracin, and 10 µg/ml leupeptin. In brief, Millipore MultiScreen MAHV N45 plates (Millipore Corporation, Billerica, MA) were preloaded with 45 µl of dry hydroxylapatite, and 120 µl/well cell extract (approximately 200 µg) in lysis buffer was added. Serial dilutions (5 µl) of PF-02367982 or RU-486 from a top concentration of 10 µM (40x desired final concentration) were added to each plate. To this, [3H]dihydrotestosterone (75 µl/well of 2.67 nM [3H]DHT for 1 nM final in plate) was added and incubated overnight with gentle agitation. After centrifugation at 2500 rpm for 3 min at 4°C, the supernatant was decanted and 100 µl of assay buffer (25 mM sodium phosphate buffer, pH 7.2, 0.1% Triton X-100, and 125 mM KCl) was added to each well. This washing procedure was repeated twice before adding 150 µl of scintillation liquid (PerkinElmer Life and Analytical Sciences), the plates were gently agitated to resuspend hydroxylapatite, and radioactivity was measured by a MicroBeta counter (PerkinElmer Life and Analytical Sciences). An excess of cold DHT was used as a control for binding specificity, and an IC50 value for PF-02367982 and RU-486 was determined.
Modified McPhail Rabbit Assay. Sexually immature female New Zealand White rabbits (Harlan, Bicester, UK; 
1 kg in weight) were primed with estradiol benzoate (0.1 mg/kg q.d. s.c. in corn oil) for 7 days and then treated daily with progesterone (1 mg/kg b.i.d. s.c. in corn oil) in combination with escalating doses of PF-02367982 (b.i.d. or q.d. regimens p.o.) for 5 consecutive days. PF-02367982 was prepared as a wet-milled formulation in 1% hydroxypropylcellulose, 0.1% sodium lauryl sulfate, and 0.1% Tween 80 in water, and all concentrations were prepared by dilution. PF-02367982 was dosed in a volume of 5 ml up to 3 mg/kg by oral gavage. Blood samples were taken from each animal at regular intervals throughout the duration of the study via the marginal ear vein to determine free drug and progesterone levels in plasma. All animals were sacrificed by lethal overdose with an i.v. anesthetic, and the uteri were collected in 10% formalin solution for processing by wax histology. Each uterine sample was immediately fixed in 10% formalin solution for 24 h and then placed in 70% ethanol for 24 h at 4°C. Uterine horns were cut into three equal pieces (layers) and processed for paraffin wax embedding (three layers per slide). Experiments were in compliance with United Kingdom legislation and subject to local ethical review. PF-02367982 and RU-486 were well tolerated at all doses examined.
Histological Processing and Staining. Subsequent to embedding in wax, 5-µm sections were collected on poly-L-lysine-coated slides. Sequential sections were stained using routine Masson's trichrome staining methods to examine the inhibition of progesterone-induced endometrial differentiation. In brief, sections were dewaxed and rehydrated through graded alcohols to tap water. Tissue sections were stained for 10 s in Celestine blue stain and washed in running tap water, before counterstaining for 10 s with Mayer's hematoxylin. Sections were then washed in running tap water before being immersed in 1% Ponceau fuschin in 1% acetic acid. The slides were washed in distilled water, and collagen was decolorized by immersion in 1% phosphomolybdic acid for 2 min. The sections were stained with 0.5% soluble methyl blue in 2.5% acetic acid. Sections were washed in distilled water, dehydrated, and mounted. After Masson's trichrome staining, the undifferentiated endometrial epithelium was stained red, the differentiated (arborized) epithelium was stained red/brown, whereas nuclei were stained blue-black, and collagen/extracellular matrix (ECM) were stained blue/green. Sections were scored according to the McPhail classification system (McPhail, 1934). All McPhail test data are presented as the mean (n = 3 uterine sections per animal, n = 4 animals per group) ± S.E.M. Statistical analysis was performed using analysis of variance (ANOVA). Differences were considered statistically significant at p < 0.05.
Immunohistochemical Detection of PR, AR, and Ki67. Immunohistochemical techniques were used to identify PR, AR, GR, and the proliferation marker Ki67 in rabbit and cynomolgus macaque endometrial tissue sections. Positive staining of cells was semiquantitated by image analysis techniques using the Zeiss KS300 imaging system (Carl Zeiss Ltd., Welwyn Garden City, UK). Tissue sections were initially treated with citric acid buffer (10 mM anhydrous citric acid; Sigma Chemical, Poole, Dorset, UK) with microwaving, promoting antigen retrieval. The slides were washed and incubated for 20 min with the appropriate blocking serum [6% in phosphate-buffered saline (PBS)] (Dako UK Ltd., Ely, Cambridge, UK) or serum derived from the host animal of the secondary antibody (e.g., a secondary antibody raised in a goat would require goat serum blocking). Well characterized primary antibodies for PR (MAB462; Millipore Bioscience Research Reagents, Temecula, CA), AR (SC-816; Santa Cruz Biotechnology, Inc., Santa Cruz, CA), and Ki67 (Tec-3, M7249; Dako UK Ltd.) were diluted in PBS at 1:100 and incubated for 1 h at room temperature. The sections were washed and incubated with biotinylated secondary antibody (Dako UK Ltd.) for 1 h at room temperature, diluted to 1 in 200 in PBS. Sections were washed and incubated with streptavidin-conjugated horseradish peroxidase (Dako UK Ltd.) for 30 min at 1 in 100 dilution, in PBS. The slides were washed for an additional 15 min in PBS, and peroxidase activity was detected using the chromagen 3-3'diaminobenzidine substrate (FAST DAB; Sigma Chemical) according to the manufacturer's instructions.
Analysis of Plasma Samples for PF-02367982, RU-486, and Progesterone. Plasma concentrations of RU-486 and PF-02367982 were determined in 50-µl aliquots of thawed rabbit or monkey plasma using tert-butyl methylether extraction followed by high-performance liquid chromatography and on-line tandem mass spectrophotometric analysis. Plasma protein binding of PF-02367982 was determined by equilibrium dialysis, and unbound plasma concentrations were then calculated using values for "free fraction" of 0.13 for rabbit and 0.25 for cynomolgus macaque. Free fraction values for RU-486 were 0.044 for rabbit and 0.028 for cynomolgus macaque. Progesterone concentrations in plasma samples (25 µl) were measured by solid-phase enzyme-linked immunosorbent assay (IBL, Hamburg, Germany) according to the manufacturer's instructions.
PK/PD Model for Analysis of Data from Rabbit McPhail Test. Plasma concentration data, obtained by sparse sampling of animals used in the McPhail test, were incorporated into a population PK model (Gabrielsson and Weiner, 2006). The individual animal cumulative unbound area under curve (AUC) values obtained from population PK analysis were used to relate drug exposure to the PD variable by applying a logistic regression model based on a joint analysis of PF-02367982 and RU-486. The AUC values related to 90% of probability of obtaining McPhail test score 
2 were calculated for each compound. Progesterone concentration was tested as a covariate, but it was found not to have a significant impact upon the results.
Evaluating the Effects of PF-02367982 on the Naturally Cycling Cynomolgus Macaque Endometrium. The evaluation of PF-02367982 and RU-486 in cynomolgus macaques was performed at the Covance Laboratories (Münster, Germany), subjected to ethical review and approval and conducted in accordance with local animal husbandry procedures and legislation. Twenty sexually mature female cynomolgus macaques (Macaca fascicularis) underwent daily menstrual cycle inspection by morning examination of external genitalia and vaginal smears. Menstrual bleeding was checked by inserting a cotton bud into the vagina. Animals were allowed to complete an observation cycle before any surgical intervention or drug administration. All animals completed a normal menstrual cycle within 35 days. The first day (day = 0) of the next observation cycle was deemed the first day of menstruation. On day 22 ± 1, approximately mid-luteal, animals were sedated with ketamine hydrochloride and xylazine, and an endometrial biopsy was excised. Each female received antibiotic, analgesic, and antiseptic treatment postsurgical biopsy. Animals were allowed to recover, and on day = 0 of their subsequent cycle, females were assigned to 1 of 4 groups: vehicle, RU-486 (20 mg/kg q.d.), or PF-02367982 at 2.5 or 8 mg/kg b.i.d. Animals were dosed daily for 42 days, and a second biopsy was taken at day 22 ± 1 for comparison with the predose observation cycle. At the end of the study, animals were returned to stock. PF-02367982 and RU-486 were dosed orally as a suspension in 2% hydroxypropylcellulose, 0.1% Tween 80, and 0.1% sodium lauryl sulfate in water. PF-02367982 and RU-486 were well tolerated at all doses examined.
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Results
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In Vitro Pharmacology of PR Antagonists. The PR antagonists PF-02367982 and RU-486 were assessed for functional PR antagonist activity and translational pharmacology across species in native or recombinant Chinese hamster ovary reporter assays, transfected with human or rabbit PR-B-β-lactamase reporter constructs (Table 1). PF-02367982 and RU-486 were potent PR antagonists in vitro, blocking progesterone-dependent gene transcription in the native human breast carcinoma T47D cells (expressing PR-A and PR-B) or in a human recombinant PR-B reporter assay with IC50 values of 47 and 0.8 nM, respectively. Schild analysis of antagonist data obtained in both human and rabbit reporter assays indicated that the antagonism was competitive. The potency of PF-02367982 and RU-486 for the rabbit receptor was approximately 2-fold greater than that demonstrated at the human receptor (Table 1). Whereas RU-486 was significantly more potent than PF-02367982, RU-486 also demonstrated potent AR and GR antagonist activity in functional reporter assays. In contrast, the nonsteroidal PF-02367982 demonstrated no agonist or antagonist activity against human AR, GR, or MR receptors. Binding potency data for PR, AR, and GR obtained for PF-02367982 and RU-486 were consistent with the functional data obtained (Table 1).
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TABLE 1 In vitro pharmacology of PR antagonists PF-02367982 and RU-486 All IC50 values and units in nanomoles. Data are indicated as mean Ki or IC50 value (or pA2), S.D. range, and the number of repeat experiments.
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PR Antagonists in the Rabbit McPhail Test. The bioassay originally developed by McPhail was refined to represent a physiological system more relevant to the human and NHP luteal phase of the menstrual cycle to compare antagonist efficacy and to enable prediction of dose for subsequent NHP and human studies. Administration of progesterone to rabbits twice daily maintained circulating blood progesterone levels between 10 and 30 ng/ml (data not shown), equivalent to mid-luteal phase progesterone levels in women or cynomolgus macaques.
Chronic administration of progesterone resulted in differentiation and thickening of the epithelial cell layer of the rabbit endometrium (Fig. 1). Furthermore, progesterone exposure induced compaction of the extracellular matrix of the lamina propria layer, and formation of a dense arteriole plexus within the compacted extracellular matrix, similar to the spiral artery network in human endometrium. The PR antagonists PF-02367982 and RU-486 inhibited progesterone-induced morphological changes, resulting in an undifferentiated single-cell epithelial layer, diffuse extracellular matrix, and the presence of epithelial glandular structures in the lamina propria layer.
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Fig. 1. The effects of PF-02367982 on progesterone-induced endometrial differentiation. A and B, chronic treatment with progesterone alone resulted in differentiation and thickening of the endometrial epithelial layer (Epi), resulting in tortuous arborization and thickening of the endometrium, compaction of the lamina propria layer (LP), and a dense arteriole plexus within the extracellular matrix (arrows). C and D, inhibition of progesterone-induced endometrial arborization by the nonsteroidal PR antagonist PF-02367982 (3 mg/kg q.d.), resulting in a distinct endometrial lumen and undifferentiated epithelium.
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The extent of inhibition of progesterone-induced differentiation by PF-02367982 or RU-486 was assessed according to the McPhail classification system (McPhail, 1934). In general, progesterone treatment resulted in a McPhail score of 3.7 to 4.0. Both PF-02367982 and RU-486 induced a dose-dependent reduction in progesterone-induced endometrial differentiation, resulting in McPhail scores of 1.0 to 1.5 (Fig. 2). PF-02367982 was equally effective when administered once daily or at a lower daily dose given twice daily. Pharmacokinetic analysis of drug levels in the plasma taken from experimental animals demonstrated that PF-02367982 completely inhibited endometrial differentiation at daily unbound exposures (AUC0–24 h) of 3.4 and 1.6 nmol · h/ml, after administration at 3 mg/kg q.d. or 1 mg/kg b.i.d., respectively (Table 2). The administration of 1 mg/kg q.d. was not effective at inhibiting progesterone-induced differentiation, despite similar Cmax and marginally lower daily exposure to the 1 mg/kg b.i.d. dose regimen. RU-486 inhibited endometrial arborization at a daily unbound exposure of 0.07 nmol · h/ml (3 mg/kg dose group) and to the same extent as PF-02367982. In the absence of progesterone, PF-02367982 and RU-486 did not demonstrate any PR agonist activity at any dose tested (up to 10 mg/kg; data not shown), consistent with its in vitro pharmacology profile.
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Fig. 2. PR antagonists PF-02367982 and RU-486 in the rabbit McPhail test. Chronic dosing of either PF-02367982 [3, 1, 0.1 mg/kg q.d., or 2 mg/kg/day (1 mg/kg b.i.d.)] or RU-486 (3, 1, 0.3 mg/kg q.d.) with progesterone treatment induced a significant reduction in endometrial differentiation compared with progesterone (P4) treatment alone. The assessment of inhibitory effects was performed according to the McPhail classification system (0 = no progestagenic activity to 4 = maximal progestagenic activity) (n = 4 animals per group, mean ± S.E.M., representative data of n = 2 experiments; **, p < 0.01 compared with P4 alone group by ANOVA).
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TABLE 2 Pharmacokinetic analysis of PR antagonists PF-02367982 and RU-486 in the rabbit McPhail test Data represent mean exposures from n = 2 rabbits per dose group. Daily free exposures (AUC) for the 2 mg/kg/day (1 mg/kg b.i.d.) dose were calculated from a complete PK profile for the first daily dose and extrapolated for the second daily dose following measurement of Cmin on the subsequent day. BLQ indicates exposures lower than the limit of detection for the assay.
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PF-02367982 and RU-486 in the rabbit endometrium were assessed for their ability to block estradiol-induced cell proliferation by immunohistochemical detection of the proliferation marker, Ki67 and PR (Fig. 3). PF-02367982 and RU-486 induced a significant reduction in the number of Ki67- and PR-positive epithelial cells to a similar degree, at maximally effective doses in the McPhail test (3 and 1 mg/kg b.i.d., respectively).
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Fig. 3. Pharmacological effects of PF-02367982 and RU-486 on the rabbit endometrium. Immunohistochemical detection of Ki67 antigen in progesterone-treated endometrium (brown nuclei; A) and PF-02367982-treated rabbit endometrium (B). Immunohistochemical detection of PR in progesterone-treated endometrium (brown; C) and PF-02367982-treated endometrium (D). PF-02367982 and RU-486 (3 mg/kg q.d.) significantly reduced the number of Ki67-positive (E) and PR-positive (F) epithelial cells at doses maximally effective in the rabbit McPhail test (n = 4 per group, *, p < 0.05 and **, p < 0.01 compared with P4 alone group by ANOVA). An immunohistochemical control image is represented as an inset to each of the figures.
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PK/PD Analysis of McPhail Test Data Using a Categorical Logistic Regression Method. Further analysis of these McPhail test data were undertaken to construct a PK/PD model that could be used to predict the outcome and efficacy of different PR antagonists in different systems. The McPhail data were transformed into a categorical binary score and combined with the relevant PK data, incorporating all available plasma exposures (AUCs) in experimental rabbits, and a mathematical model was generated to calculate the predicted exposure required to yield a significant antiprogestagenic effect on the endometrium in the presence of mid-luteal phase levels of progesterone. Thus, McPhail scores (0–4) were reduced to a binary PD score of "1" or "0," according to a McPhail test score of 2 (antiprogestagenic) or >2 (progestagenic), respectively. Pharmacokinetic analyses were related to the binary PD score through application of a "logistic regression model" (Gabrielsson and Weiner, 2006), estimating the probability of an efficacious antiprogestagenic outcome. At 90% probability of such an efficacious outcome, the predicted daily exposure values for PF-02367982 and RU-486 were 1.6 and 0.053 nmol · h/ml, respectively (Fig. 4).
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Fig. 4. Results of PK/PD analysis of McPhail test data using a categorical logistic regression method. McPhail test scores were converted to categorical binary scores of 1 or 0 according to antiprogestagenic efficacy (McPhail score 2 or >2, respectively), regardless of dose, and plotted with respect to unbound drug treatment exposure for the 96 h of drug dosing (AUC0–96 h). Units on x-axis are in nmol · h/l. The values of fractional free drug used to calculate daily unbound AUC0–24 for PF-02367982 and RU-486 were 0.254 and 0.03, respectively. The PK/PD model was used to calculate the required exposure to elicit such antiprogestagenic activity in the endometrium in the presence of mid-luteal phase levels of progesterone.
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The Effects of PF-02367982 on the Naturally Cycling Cynomolgus Macaque Endometrium. The rabbit PK/PD model used in the current study calculated the daily exposure of PF-02367982 and RU-486 required to elicit an antiprogestagenic effect on the endometrium in the presence of midluteal phase levels of progesterone (i.e., 10–30 ng/ml). Using these calculated exposures, we tested the hypothesis that the nonsteroidal PR antagonist PF-02367982 would inhibit endometrial thickening and differentiation in the adult female gonad-intact cynomolgus macaque. Results of preliminary oral pharmacokinetic studies for PF-02367982 were used to establish doses in NHP that would provide unbound exposures in the range determined by the rabbit McPhail studies (data not shown). Doses equal to and below those predicted to be efficacious (5 and 16 mg/kg/day, respectively) were then selected for PF-02367982 on the basis of a direct translation from the rabbit PK/PD model (Fig. 5). An oral dose of 20 mg/kg (q.d.) of RU-486 was selected on the basis of literature precedence of an endometrial effect (Tarantal et al., 1996). The unbound daily RU-486 exposure at this dose was subsequently confirmed as 0.067 nmol · h/ml.
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Fig. 5. Pharmacokinetic analysis of PF-02367982 in the female cynomolgus monkey. Doses were selected on the basis of the rabbit PK/PD model output on required daily exposure needed to elicit an antiprogestagenic response on the endometrium.
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After chronic dosing with PF-02367982, RU-486, or vehicle, a second endometrial biopsy was taken from each cynomolgus macaque to compare with the pretreatment period. The majority of the biopsy samples were not full thickness, and so a measure of an effect on "endometrial thickness" alone was not possible. However, histological analysis of endometrial biopsies taken from cynomolgus macaques treated chronically with PF-02367982 or RU-486 demonstrated significant differences in morphology compared with observation cycle-matched vehicle-treated control animals (Fig. 6, A–C). At day 22, vehicle-treated macaques demonstrated an endometrial morphology typical of endometrium in the luteal phase of the menstrual cycle, with a highly differentiated, thickened epithelial layer, and a greatly extended lamina propria layer, with a substantial spiral artery plexus. In contrast, PR antagonists PF-02367982 and RU-486 seemed to inhibit arborization of the endometrial epithelial layer, resulting in a clearly delineated luminal epithelium with numerous subepithelial glandular structures. The extracellular matrix in the lamina propria layer appeared compacted, and the extent of vascularization in the layer was reduced.
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Fig. 6. The effects of PF-02367982 on endometrial differentiation in the cynomolgus macaque. Histological staining of endometrium from untreated (A), PF-02367982-treated (B), or RU-486-treated (C) cynomolgus monkeys at 22 days after first dose (hematoxylin and eosin stain). D, change in histological progestagenic score (McPhail score) of endometrial biopsy samples taken from cynomolgus macaques at pretreatment and day 22 after treatment (4 = progestagenic, 0 = estrogenic). Data represent the reduction in progestagenic score in response to treatment between days 22 and 0 (n = 5 animals per treatment group for vehicle and PF-02367982; n = 7 for RU-486 treatment; scale bar represents 100 µm; *, p < 0.05 compared with vehicle control by ANOVA).
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The effects of PF-02367982 and RU-486 were quantified to assess the relative efficacies between compounds and doses, using a variation on the McPhail histological scoring system (0 = antiprogestagenic, 4 = progestagenic). High-dose PF-02367982 and RU-486 significantly reduced the progestagenic state of the endometrium after 22 days of chronic treatment, compared with observation cycle-matched vehicle-treated control animals (Fig. 6D). Low-dose PF-02367982 treatment had a pharmacological antiprogestagenic effect on the macaque endometrium in most animals, although this was not statistically significant using this scoring system.
Immunohistochemical detection of PR and AR expression in PR antagonist-treated and vehicle-treated animals demonstrated that PF-02367982 elicited significant pharmacological effects at both low and high doses, comparable with RU-486 (Table 3). Chronic treatment with PF-02367982 and RU-486 resulted in increased PR expression in the myometrium, and both the extracellular matrix and epithelial glands of the endometrial basalis layer, but not the functionalis layer, compared with vehicle-treated control animals (Fig. 7). In contrast, AR expression was elevated in the extracellular matrix of both basalis and functionalis layers in most RU-486- and PF-02367982-treated animals. AR expression in the epithelial glands of animals treated with RU-486 and high-dose PF-02367982 seemed to be elevated, although expression patterns across endometrial compartments seemed to differ between treatment groups. Chronic treatment with RU-486 resulted in nuclear staining of AR in glandular epithelial cells and fibroblasts, whereas PF-02367982-treated tissues exhibited predominantly cytosolic localization of the receptor in epithelial cells but not in ECM fibroblasts (Fig. 8).
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TABLE 3 Chronic PR antagonist treatment resulted in increased endometrial PR and AR expression Number of animals with greater PR or AR expression in the myometrium, extracellular matrix, and epithelial glands of the endometrial basalis or functionalis after vehicle, RU-486, or PF-02367982 treatment compared with endometrial biopsies taken pretreatment, by immunohistochemistry (n = 5 per group for PF-02367982 and vehicle treatments; n = 7 for RU-486 treatment).
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Fig. 7. Immunohistochemical detection of PR in the cynomolgus macaque endometrium. Elevated nuclear PR expression (brown) in the ECM and epithelial glands of the basalis layer of the cynomolgus monkey endometrium (arrows) after RU-486 or PF-02367982 treatment (bar indicates 50 µm). An immunohistochemical control image is represented as an inset to the figure.
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Fig. 8. Immunohistochemical detection of AR in the cynomolgus monkey endometrium. Elevated AR expression (brown) in the ECM and epithelial glands of the basalis and functionalis layers of the cynomolgus monkey endometrium after RU486 or PF-02367982 treatment. Expression of AR in epithelial glands and extracellular matrix fibroblasts of RU-486-treated tissues seemed to be predominantly nuclear (arrows). In contrast, PF-02367982 seemed to induce predominantly cytosolic expression of AR in epithelial cells, but not fibroblasts, in both basalis and functionalis layers (arrows) (bar indicates 50 µm). An immunohistochemical control image is represented as an inset to the figure.
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Discussion
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Considerable evidence exists that PR antagonists have potential utility in the treatment of gynecological conditions such as endometriosis (Grow et al., 1996; Kettel et al., 1996). There is a lack of potent, highly selective PR antagonists, which could be used either as a chronic therapy for endometriosis or indeed as a tool agent to elucidate the cellular and molecular effects of PR antagonism alone. Using a multidisciplinary screening strategy, we have described the pharmacological profile of a novel and selective nonsteroidal PR antagonist, PF-02367982. PF-02367982 exhibited potent PR antagonism in human and rabbit in vitro functional and binding assays and greater than 100-fold selectivity over closely related members of the nuclear hormone receptor family, including AR, GR, MR, and ER. In contrast and consistent with many steroidal PR antagonists described previously (Schoonen et al., 1999; Attardi et al., 2002), RU-486 was confirmed to be a potent antagonist at the glucocorticoid receptor (Heikinheimo et al., 1987).
The aim of the current study was to understand the translation of in vitro potency to in vivo efficacy for PF-02367982. The PR ligand-binding domain is highly conserved between rabbit and human, and PF-02367982 and RU-486 demonstrated functional antagonism of the cloned rabbit PR-B receptor with comparable potency to human PR in functional reporter assays. This translation between rabbit and human PR antagonism of PF-02367982 in vitro provided confidence in the use of the rabbit to predict efficacious exposure in humans. The efficacy of PF-02367982 was compared with RU-486 in a modified rabbit McPhail test, from which a novel PK/PD model was developed to predict drug exposures required to elicit an antiprogestagenic effect on the endometrium in the presence of physiological levels of progesterone. A conventional single progesterone dose per day dosing regimen resulted in highly variable plasma concentrations of progesterone that were substantially lower than those observed in the human menstrual cycle (data not shown). The McPhail model was modified to a twice daily progesterone-dosing regimen, to maintain progesterone concentrations within the range observed in a human luteal phase (5–30 ng/ml). It was hypothesized that under such conditions, the antiprogestagenic effect on the rabbit endometrium, although not occurring under influence of an endogenous hormonal cycle, may be predictive of PR antagonist activity on endometrial endpoints more relevant to clinical outcome. A categorical population PK/PD analysis method that incorporated a logistical regression model was developed to use exposure and McPhail score data of all dose cohorts simultaneously. This was used to calculate the exposures of PF-02367982 and RU-486 that would give a 90% probability of an antiprogestagenic effect in rabbit endometrium. PK/PD modeling showed that RU-486 was effective at an approximately 30-fold lower exposure than PF-02367982, in agreement with the greater primary potency of RU-486 in vitro.
The output from the PK/PD analysis was used to further demonstrate translation of pharmacology to the endometrium of adult female cynomolgus monkeys at clinically relevant doses, to "bridge" the potential pharmacological gap between nonmenstruating rabbits and humans. It was proposed that PF-02367982 would display similar antiprogestagenic potency in a naturally cycling primate in vivo, in a study of endometrial differentiation over a single menstrual cycle in the cynomolgus macaque. Two doses of PF-02367982 were selected on the basis of a direct translation from the rabbit McPhail test, the higher one of which would be expected to cause significant reduction in endometrial growth, and the other only partially effective. As predicted by the PK/PD model, the higher dose (unbound daily AUC of 5 nmol · h/ml) inhibited endometrial differentiation in the macaque, whereas the lower dose (unbound daily AUC of 1 nmol · h/ml) was not effective. Moreover, RU-486 caused an inhibition of endometrial growth in cynomolgus monkey at an average daily AUC of 0.067 nmol · h/ml, similar to that determined to give 90% probability of a significant effect in the rabbit McPhail test (0.053 nmol · h/ml). Previous studies (Wolf et al., 1989) have demonstrated that RU-486 at equivalent exposures (0.06 nmol · h/ml) reduced endometrial growth and differentiation in cynomolgus macaques. Overall, these data are consistent with the proposal that antiprogestagenic endometrial endpoints in the modified McPhail test can be used to predict similar endpoints in the nonhuman primate, and they provide confidence that the pharmacology of nonsteroidal antagonists, such as PF-02367982, will translate to humans.
Although chronic treatment of PF-02367982 at the lower dose did not induce a significant effect on endometrial differentiation, pharmacological effects at this exposure were still observed. The expression of PR and AR in macaque endometrium served as a sensitive pharmacology biomarker to confirm pharmacological changes in the absence of reduced endometrial differentiation. Indeed, both high and low doses of PF-02367982 elevated extracellular matrix and epithelial cell PR and AR expression in the endometrial basalis layer to a similar extent as RU-486-treatment, consistent with other reports (Slayden et al., 2001b). The exposure required to increase PR in the macaque endometrium without changing endometrial morphology seemed similar to the exposure required to induce pharmacological effects on PR expression in the rabbit endometrium (1 mg/kg q.d.), which also did not affect endometrial morphology. Considering the agreement between different species on the effects of PR antagonists on endometrial endpoints uncovered so far, it would seem that PR expression in the endometrium is sensitive to PR antagonist treatment and may precede antiprogestagenic morphological events. These data provide further confidence in the pharmacology of the nonsteroidal PF-02367982, and they may indeed point to a minimal pharmacologically effective dose not significantly greater than the lowest dose tested in the macaque.
Understanding the mechanism of action of PR antagonists on the inhibition of endometrial growth provides a significant challenge. Inhibition of estrogen-dependent endometrial fibroblast, epithelial and endothelial cell proliferation, modulation of extracellular matrix synthesis, and angiogenesis are highly complex, multifactorial cellular and molecular processes, in which PR clearly plays an important role (Conneely et al., 2001; Slayden and Brenner, 2004; Narvekar et al., 2006). Like other nuclear hormone receptors, PR is a ligand-activated transcription factor with domains for DNA binding, hormone binding, and transactivation. In the presence of progesterone, the PR interacts with coactivators such as NCoA1 and NCoA2, activating transcriptional machinery and up-regulating the expression of specific genes. However, the specific molecular events of PR antagonism with synthetic ligands, such as the competition for, and binding of, coactivator or repressor proteins, displacement or subversion of promoter activity, and the subsequent modulation of functional cellular events remain poorly understood, in part due to the promiscuity of nonselective agents such as RU-486, for GR or AR. The potent and highly selective PF-02367982 represents a powerful tool to investigate PR function and the cellular and molecular events that lead to inhibition of estrogen-dependent endometrial growth.
In conclusion, we have pharmacologically characterized a novel, selective, and nonsteroidal PR antagonist, PF-02367982. Using a multidisciplinary screening strategy, we have demonstrated an in vitro correlation between RU-486 and PF-02367982 in human and rabbit native and recombinant functional assays, and we confirmed the translation of pharmacology in in vivo models of endometrial differentiation. The recently developed PK/PD model in the rabbit enabled accurate efficacious dose prediction in cynomolgus macaques. Given the similarity of the menstrual cycle in cynomolgus macaque and humans, we propose that these preclinical endpoints could offer reliable means of predicting clinical outcomes for PR antagonists in gynecological disorders such as endometriosis. Thus, PF-02367982 represents the founding member of a class of potent and selective nonsteroidal PR antagonists, with utility in investigating PR function and use as a therapeutic modality, where the antiglucocorticoid activity of RU-486 prohibits dose escalation.
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Acknowledgements
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We thank Dr. A. Fuchs at Covance for expert guidance and managing cynomolgus macaque studies, Drs. M. Germani, M. Rocchetti, and F. Del Bene at Accelera as well as Drs. C. Kohl and P. van der Graaf for PK/PD modeling, and B. Laverty for bioanalysis of PR antagonists in rabbit.
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Footnotes
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.108.140467.
ABBREVIATIONS: PR, progesterone receptor; ZK-230211, 11β-(4-acetylphenyl)-17β-hydroxy-17
-(1,1,2,2,2-pentafluoroethyl)estra-4,9-dien-3-one; NHP, nonhuman primates; GR, glucocorticoid receptor; PF-02367982, 2-[4-(4-cyano-phenoxy)-3,5-dicyclopropyl-1H-pyrazol-1-yl]-N-methylacetamide; PK/PD, pharmacokinetic/pharmacodynamic; DHT, dihydrotestosterone; AR, androgen receptor; MR, mineralocorticoid receptors; ECM, extracellular matrix; ANOVA, analysis of variance; PBS, phosphate-buffered saline; AUC, area under curve.
The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. 
Address correspondence to: Dr. Nick Pullen, Pfizer PGRD, Ramsgate Road, Sandwich, Kent, CT13 9NJ, UK. E-mail: nick.pullen{at}pfizer.com
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Abstract
Materials and Methods
