Luteal phase dose–response relationships of the antiprogestin CDB-2914 in normally cycling women*

Maureen D. Passaro1,5, Johann Piquion1,6, Nancy Mullen2,7, Dorette Sutherland2, Suoping Zhai3, William D. Figg3, Richard Blye4 and Lynnette K. Nieman1

1 Pediatric and Reproductive Endocrinology Branch, National Institute of Child Health and Human Development, 2 Department of Nursing, Warren Grant Magnuson Clinical Center, 3 Molecular Pharmacology Section, National Cancer Institute, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892 and 4 Contraception & Reproductive Health Branch, National Institute of Child Health and Human Development, Executive Bldg, Rm 8B07, 6100 Executive Blvd MSC 7510, Bethesda, MD 20892-7510, USA

5 Present address: 650 Pennsylvania Ave SE, Suite 50, Washington, DC 20003, USA.

6 Current address: 12 Wexford Glenn, Pittsford, NY 14534, USA.

7 Present address: 3 Chevy Chase Circle, Chevy Chase, MD 20815, USA

8 To whom correspondence should be addressed. e-mail: NiemanL{at}nih.gov


    Abstract
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
BACKGROUND: Progesterone receptor modulators have potential therapeutic use in progesterone-dependent conditions such as endometriosis, fibroids and induction of labour. The synthetic steroid CDB-2914 binds to the progesterone and glucocorticoid receptors. In animals it has antiprogestational activity at doses 50-fold less than those required for antiglucocorticoid effects. METHODS AND RESULTS: We evaluated the biological activity, blood levels and safety of CDB-2914 at escalating single doses, in 36 normally cycling women at mid-luteal phase. CDB-2914 at doses of 1–100 mg did not change luteal phase length, but after 200 mg, all women had early endometrial bleeding. Four women with early menses had concurrent functional luteolysis (one at 10, 50, 100 and 200 mg). There were no biochemical or clinical signs of toxicity, and no effect on urinary cortisol or circulating thyroxine, prolactin, adrenocorticotrophic hormone or renin levels. Higher serum equivalents of CDB-2914 were observed by radioimmunoassay than by high performance liquid chromatography detection, indicating a considerable contribution of metabolites. CONCLUSIONS: Mid-luteal administration of CDB-2914 antagonizes progesterone action on the endometrium, in a dose-dependent fashion, without apparent antiglucocorticoid effects. Further study of CDB-2914 is needed to determine its clinical role.

Key words: CDB-2914/menses/normally cycling women/progesterone receptor modulator


    Introduction
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
The progesterone receptor modulator mifepristone (Figure 1) has activity as an abortifacient and postcoital contraceptive in women (Webb et al., 1992Go; Ulmann and Silvestre, 1994Go). When given chronically it also ameliorates the pain of endometriosis and decreases the size of endometriomas and uterine leiomyomas (Murphy et al., 1993Go; Kettel et al., 1996Go), presumably by a direct effect on uterine tissue and by inhibition of normal folliculogenesis (Spitz et al., 1994Go), with a resultant decrease in ovarian estrogen production.



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Figure 1. The chemical structure of progesterone, mifepristone (RU486) and CDB-2914.

 
Mifepristone also exhibits significant antiglucocorticoid activity. After single doses of the agent, or at daily doses of up to 200 mg for 8 days, individuals with an intact hypothalamic–pituitary–adrenal axis usually overcome glucocorticoid blockade, as shown by increased urinary cortisol excretion and the lack of symptoms that are suggestive of glucocorticoid insufficiency (Gaillard et al., 1984Go; Bertagna, 1994Go). Because of this compensation, and the greater potency of the agent as an antiprogestin (Nieman and Loriaux, 1988Go), the antiglucocorticoid properties of mifepristone do not limit its clinical application for single dose use for antiprogestin effects. However, at higher doses and when given chronically, mifepristone may have adverse effects related to glucocorticoid blockade. At a daily dose of 200–400 mg, anorexia, nausea, fatigue and loss of well-being occurred in 2/11, 4/10 and 2/5 subjects receiving the agent in trials for breast cancer, meningioma and Alzheimer’s disease (Klijn et al., 1989Go; Lamberts et al., 1991Go; 1992; Pomara et al., 2002Go). These symptoms could be ameliorated with prednisone 7.5 mg daily, confirming their antiglucocorticoid aetiology (Lamberts et al., 1991Go; 1992). At a higher dose (10 mg/kg) in normal men, 8/11 individuals developed an exanthem and one had symptoms of adrenal insufficiency (Laue et al., 1990Go). Additionally, the increase in adrenocorticotrophic hormone (ACTH) during chronic administration of mifepristone stimulates adrenal secretion of androstenedione as well as cortisol, resulting in peripheral conversion to estrogens, an undesirable effect in women with breast cancer (Heikinheimo et al., 1997Go; 2000; Klijn et al., 2000Go). Increased circulating estrogen concentrations also may underlie the massive endometrial hyperplasia reported in a woman receiving the agent chronically (Newfield et al., 2001Go).

Despite the therapeutic promise of mifepristone, its undesirable side-effects related to glucocorticoid antagonism have led to efforts to develop other compounds with more pure antiprogestational action. Other structurally similar compounds with an 11{beta}-aromatic substitution and antiprogestational activity (Teutsch and Philibert, 1994Go) have been synthesized, but few have entered clinical trials and none are commercially available. Thus, further development of such agents is desirable.

CDB-2914, 17{alpha}-acetoxy-11{beta}-(4-N,N-dimethylaminophenyl)-19-norpregna-4,9-diene-3,20-dione (Figure 1), is a 19-nor steroid that is structurally similar to mifepristone. In vitro, CDB-2914 binds competitively to the progesterone, glucocorticoid and androgen receptors, but has minimal affinity for the estrogen or mineralocorticoid receptors (D.Blithe, unpublished data, IND 49,381; Cook et al., 1994Go; Wagner et al., 1999Go; Attardi et al., 2002Go). The compound has antiprogestational activity in rats, rabbits and monkeys, with additional antiglucocorticoid and antiandrogen activity at doses ~50 times higher than those needed for antiprogestational activity (Tarantal et al., 1996Go; Reel et al., 1998Go; Hild et al., 2000Go). In women, a single mid-follicular phase dose of CDB-2914 (compared with placebo, 10, 50 or 100 mg) caused dose-dependent inhibition of folliculogenesis and steroidogenesis (Stratton et al., 2000Go). However, its effects in the luteal phase have not been examined. This trial was designed to evaluate the safety, adverse effects, pharmacokinetics and biological activity of single escalating doses of CDB-2914 in the luteal phase of normally cycling women not at risk for pregnancy.


    Subjects and methods
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Subjects
Thirty-seven women met the following general eligibility criteria. They were: (i) between 18 and 42 years of age, (ii) in good health, as determined by a medical examination, including normal pelvic and breast examinations and routine laboratory screening, (iii) free of any chronic disease, (iv) on no current medications, and had not used any form of glucocorticoids in the previous year nor taken hormonal contraception in the last 3 months, (v) within 20% of ideal body weight, (vi) not lactating, and (vii) had normal menstrual cycle length between 26 and 34 days and agreed to use mechanical or sterilization methods of contraception throughout the study. Each also met criteria for an ovulatory baseline cycle as described below.

Study design
The study protocol (95-CH-0168) was approved by the Institutional Review Board of the National Institute of Child Health and Human Development (NICHD). After giving informed written consent, all participants underwent a history, physical examination and routine laboratory analysis. Women used an in-home test (OvuquickTM; Quidel, USA) to detect the urinary LH surge and documented this date as well as basal body temperature, vaginal bleeding or spotting, and any constitutional symptoms on a menstrual calendar.

During the first (pre-treatment) cycle, participants reported to the clinic 6–8 days after detection of urinary LH, for measurement of serum progesterone. Women lacking an LH surge or a luteal phase progesterone level were excluded.

During the second (treatment) cycle, participants were admitted to the Clinical Center for administration of either placebo or CDB-2914 at one of five doses (1, 10, 50, 100 or 200 mg) on luteal day 6–8. After an overnight fast, the test compound was given between 08:00 and 10:00 h, and women remained at bed rest for 24 h. Blood samples were drawn at 15 and 1 min before and 15, 30, 60, 90 min, 2, 3, 4, 6, 8, 12, 16, 24 and 36 h after administration of the test compound for cortisol, ACTH and CDB-2914 measurements. Blood was drawn at 0, 4, 8, 12 and 24 h for FSH, LH, estradiol, progesterone, prolactin and renin measurements. After discharge at 36 h, women returned daily until vaginal bleeding commenced for measurement of vital signs and serum estradiol and progesterone levels. Women collected urine for three consecutive days following CDB-2914 administration. Urinalysis, haemogram (CBC), blood chemistry, kidney and liver function tests were obtained at baseline and on days 1, 2 and 7 to monitor for signs of toxicity. Serum thyroxine was measured at baseline and 7 days after treatment.

The initial CDB-2914 dose of 1 mg was increased incrementally following the absence of adverse findings at each dose level. The six placebo doses were interspersed randomly by the pharmacy within the active compound groups. Within each dosage group, neither participants nor investigators were aware of the identity of the agent administered.

In the third (post-treatment) cycle, women returned to the clinic on luteal day 6–8 for measurement of serum progesterone to document normal luteal function.

CDB-2914 and other steroids
Pure crystalline CDB-2914 and its putative metabolites, and the 3-carboxymethyloxime–bovine serum albumin (BSA) and 3-carboxymethyloxime–histamine conjugates of CDB-2914 were synthesized by the Southwest Foundation for Biomedical Research (USA) under contract N01-HD-1-3137 to the Contraception and Reproductive Health Branch, NICHD, and were a gift from that branch (Rao et al., 1999Go). The Clinical Center Pharmaceutical Development Service sieved the powder with a 125 mm mesh sieve and formulated gelatin capsules containing either 1, 10, 50, 100 or 200 mg of CDB-2914, or inert powder (AvicelTM microcrystalline cellulose; FMC Corp., USA). Mifepristone was purchased from Sigma–Aldrich Company (USA).

Hormone and CDB-2914 assays
All hormone measurements except serum thyroid-stimulating hormone (TSH) were performed at Covance Laboratories (USA), using established assays. Plasma FSH and LH (Odell et al., 1967Go), prolactin, renin and cortisol (Chrousos et al., 1984Go) were measured using direct radioimmunoassay, and ACTH (Chrousos et al., 1984Go), estradiol (Jiang et al., 1969Go; Abraham et al., 1972Go) and progesterone (DeVilla et al., 1972Go) were measured by radioimmunoassay after extraction. Urinary free cortisol (UFC) was measured by radioimmunoassay (Nichols Institute Diagnostics, USA). Serum TSH was measured at the Clinical Center using a standard second generation assay (Diagnostic Products Co., USA).

CDB-2914 was measured in serum by radioimmunoassay using iodinated CDB-2914 tracer and rabbit antisera against a 3-carboxymethyloxime–BSA conjugate of CDB-2914, as previously described (Larner et al., 2000Go). BIOQUAL, Inc. (USA) performed the assays under NICHD contract N01-HD-6-3259. The N-mono and N-didemethylated putative metabolites of CDB-2914 showed 76 and 59% cross-reactivity with antiserum 67192 respectively (Larner et al., 2000Go). Thus, results from this assay should be considered to represent the parent compound and possible metabolites of unknown biological potency in people. Progesterone, mifepristone, estradiol, estrone, cortisol, testosterone, and the putative 17{alpha}-hydroxy metabolite of CDB-2914 exhibited cross-reactivities with antiserum 67192 of <1%.

The usable range of the CDB-2914 standard curve was 1 to 400 pg/tube with a mid-point of 30 ± 2 pg/tube (mean ± SE: n = 10) and a mean slope of –0.91 ± 0.03 (n = 10). The average limit of detection was 1 ± 0.1 pg/tube. The recovery of CDB-2914 from low, mid- and high concentration quality control human serum pools run with each assay averaged 100 ± 8% (n = 3). Selected serum samples from three of the study subjects were assayed for CDB-2914 and immunoreactive metabolites over a series of dilutions. Using a four-parameter logistic plot of percentage bound versus log10 concentration, the diluted serum samples behaved in a linear and parallel fashion as compared with the CDB-2914 standard curve. The mean slope of the curves for the diluted serum samples (n = 3) was –0.83 (95% Cl, –0.72 to –0.94).

The same preparation of [125I]CDB-2914 was used in all assays. CDB-2914 and its immmunoreactive metabolites were extracted with methanol from serum samples and assayed as described using a 1:510 000 final antiserum dilution. The inter- and intra-assay coefficients of variation were 7.7 and 7.7% (n = 10) respectively, as calculated from CDB-2914 spiked human serum quality control samples run for each assay.

High performance liquid chromatography (HPLC) assay
CDB-2914 serum concentrations were determined at the 100 and 200 mg doses by HPLC, and were evaluated at these doses in five and four subjects respectively. In brief, steroids were extracted from 0.25 ml serum by vortexing it with 1 ml acetonitrile for 30 s. The samples were centrifuged at 10 000 rpm for 5 min at 4°C and the supernatant was transferred to a glass tube and evaporated to dryness. The sample was reconstituted with 200 µl mobile phase and vortexed. A total of 150 µl was injected into a Water Nova-Pak C18 column and eluted at 1 ml/min with a gradient mobile phase containing 0.02 mmol/l ammonium acetate (solvent A) and acetonitrile (solvent B). A gradient profile was started with 50% A and 50% B. At 8 min, A/B ratio was gradually changed so that by 11 min into the run, the concentration of solvent B was 70%. This ratio was maintained until 14 min into the run where solvent concentration was returned to 50%. The total run time was 21 min. CDB-2914 had an eluting time of 12 min and was detected from UV absorbance at 303 nm. The assay had a lower limit of quantification of 25 ng/ml. The three putative metabolites of CDB-2914 (the 17{alpha}-hydroxy, monodemethylated and didemethylated analogues) were added to sera, and were detected at different eluting times (3.2, 6.1, 6.7 min) so that there was no interference with the peak of the parent compound (Figure 2). We did not assess the presence of these metabolites in subjects’ sera. The mean extraction recovery of CDB-2914 was 95%. The intra- and inter-assay coefficients of variation were 1.4 and 3.6%.



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Figure 2. High performance liquid chromatography (HPLC) chromatogram of human plasma spiked with CDB-2914, and its three putative metabolites: M1, CDB-3963 the 17{alpha}-hydroxy analogue; M2, CDB-3877A the monodemethylated analogue; M3, CDB-3236 the didemethylated analogue.

 
Binding of CDB-2914 to serum proteins
We investigated the interaction of [N-C3H]CDB-2914 (specific activity 80 Ci/mmol), [N-C3H]mifepristone (specific activity 80 Ci/mmol, Contraception and Reproductive Health Branch, NICHD, Bethesda, MD, USA) and 1,2-[3H]progesterone (specific activity, 50 Ci/mmol) with purified human serum proteins (all compounds) and diluted whole serum ([3H]CDB-2914 and [3H]mifepristone) using equilibrium dialysis. The serum specimen was a basal sample from one of the normal volunteers.

A physiological concentration of each purified protein, 12.2 µmol/l human {alpha}1-acid glycoprotein (hAGP; orosomucoid), 606 µmol/l human serum albumin (HSA) or 8.9 µmol/l purified human gamma-globulin, in phosphate-buffered saline (PBS, pH 7.4) containing 5% ethanol was placed in one chamber of the equilibrium dialysis unit and a 5 nmol/l solution of [3H]progesterone, [3H]CDB-2914 or [3H]mifepristone in PBS/5% ethanol was placed in the unit’s second chamber. Incubations were carried out as previously described (Larner et al., 2000Go).

Analysis of data
Menstrual cycle length was defined as days from the first day of bleeding until and including the day before the next menses/endometrial bleeding. Follicular phase length was defined as the days from the first day of bleeding until and including the first day of detectable urinary LH. The luteal phase length was defined as days after the urinary LH surge until the day before the next menses/endometrial bleeding.

Early endometrial bleeding was defined as bleeding per vagina that occurred earlier than that seen in the pre-treatment cycles, all of which had a luteal phase >12 days. Functional luteolysis was defined as a decreasing estradiol value to <180 pmol/l over >=3 days, and either a concurrent decrease of progesterone to <16 nmol/l, or decreasing progesterone values over >=3 days, reaching a nadir >=50% of the baseline.

Data are given as mean ± SE except where noted. To analyse the data statistically, we used the computer program StatView (version 4.51; Abacus Concepts Inc., USA) on a Power Computing computer to perform analysis of covariance for repeated measurements of the same variable, or to compare variables across groups. P < 0.05 was considered significant.

Radioimmunoassay data were analysed by the RiaSmartTM four parameter sigmoidal computer program (RiaSmartTM Immunoassay Data Reduction program; Packard Instrument Co., USA).

The pharmacokinetic data were analysed by both non-compartmental and compartmental analysis. Pharmacokinetic parameters for CDB-2914 were calculated using non-compartmental analysis. The terminal elimination rate constant (Ke) of CDB-2914 was determined from linear regression analysis for the HPLC data only because of the apparent measurement of metabolites and parent compounds by the radioimmunoassay. Apparent volume of distribution (Vd) was determined as: Vd = CL/Ke. The serum elimination half-life (t1/2) was calculated as: t1/2 = 0.693/Ke. The area under the serum concentration–time curve (AUC) from HPLC data was calculated using the linear trapezoidal method from time zero to the last concentration time-point obtained and extrapolated to infinity by dividing the last concentration by the terminal elimination rate constant. For data from radioimmunoassay, AUC was calculated using the linear trapezoidal method from time zero to 72 h. CDB-2914 oral clearance was determined as CL = dose/AUC. For compartmental analysis, ADAPT II version 4 (Biomedical Stimulation Resource, University of Southern California, USA) was used (D’Argenio and Schumitzky, 1979Go). Model selection was determined using Akaike’s Information Criterion, Schwartz criterion and visual examination of the fitted versus observed concentrations (Yamaoka et al., 1978Go).


    Results
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 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Thirty-seven women began the trial. No data are reported for one woman who was withdrawn prior to receiving CDB-2914 due to protocol violations. Another woman in the 200 mg group became pregnant during the post-treatment cycle, so her post-treatment cycle length was excluded from the analysis. The length of the baseline cycle did not differ among dose groups, but the 50 mg dose group was significantly younger than the 10 mg and 200 mg groups, and the 100 mg dose group was significantly younger than the 10 mg group (Table I).


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Table I. Demographics of participants
 
CDB-2914 consistently induced endometrial bleeding at a dose of 200 mg. All six women in this group began bleeding 2–4 days after mid-luteal administration of the agent (Table II), for a significantly shorter luteal phase length of 9.7 ± 0.3 days (range 9–11 days; P = 0.13, non-significant for 200 mg versus 50 mg; P < 0.02 versus all other groups) (Figure 3 and Table II). Early endometrial bleeding occurred less frequently in women receiving CDB-2914 at 1 (1/6), 10 (1/6), 50 (3/6) or 100 (2/7) mg. The total cycle length decreased in women receiving the 200 mg dose compared with those receiving 10 and 100 mg (Table II).


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Table II. Effects of CDB-2914 on bleeding pattern and luteal phase length
 


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Figure 3. Length of luteal phase (bars) and lowest serum progesterone (filled circles) within 1 day of endometrial bleeding in women receiving CDB-2914 or placebo. Open bars indicate normally timed bleeding; lightly hatched bars indicate early bleeding (<11 day luteal phase) without functional luteolysis; darker hatched bars indicate early bleeding with concurrent functional luteolysis.

 
Five of the 13 women with early endometrial bleeding had hormonal evidence of functional luteolysis at that time (Figure 3). Three other women had evidence for transient luteolysis at the time of early bleeding, with a decrease in progesterone and estradiol, but a later ‘rebound’ in progesterone to luteal phase values. The remaining eight women with early endometrial shedding did not meet criteria for functional luteolysis. These women subsequently had decreased progesterone values >=3 days after the beginning of vaginal bleeding, on day 5–9 after receiving CDB-2914. This time of luteolysis was similar to that observed in women without early bleeding, which occurred 4–8 days after CDB-2914 or placebo.

Each woman had only one episode of endometrial bleeding after CDB-2914 or placebo. Women with concomitant functional luteolysis and bleeding characterized the duration (4.9 ± 0.2 days, range 3–8) and quality of bleeding as ‘normal’. However, women with early endometrial bleeding without concomitant functional luteolysis had a significantly longer duration of bleeding (10.3 ± 2.1 days, range 5–20; P < 0.001 versus women with concomitant luteolysis), characterized by the subjects as having more spotting at the beginning and ending than the usual menses.

The follicular phase length in the post-treatment cycle, when defined by the onset of endometrial bleeding, was significantly longer in women with early bleeding and no functional luteolysis compared with women who had concurrent luteolysis and bleeding (17.3 ± 1.1 versus 14.3 ± 0.5 days, P < 0.01), presumably because of the dissociation between bleeding and functional luteolysis. Post-treatment cycles were ovulatory in all women.

CDB-2914 had no apparent effect on the hypothalamic–pituitary–adrenal axis or other endocrine function: there was no statistically significant change in urinary free cortisol, plasma renin activity, or plasma prolactin, thyroxine, LH or FSH levels following CDB-2914 administration, as compared with baseline. The diurnal variation of cortisol (0800 h: 330 ± 20 nmol/l versus 2000 h: 85 ± 8 nmol/l, P < 0.0001; no dose effect) remained intact during the 24 h immediately following CDB-2914 administration. There was no difference in urinary cortisol excretion between groups, either 0–12, 12–24 or 24–48 h after test agent administration, and no difference between the groups in plasma ACTH or cortisol at any time-point; there were no clinical signs of adrenal insufficiency.

CDB-2914 was well tolerated and there was no abnormality of CBC, hepatic, renal or blood chemistries after the agent. Subjectively, one woman felt warm 4 h after a 10 mg dose, and one woman developed a rash on her arm and abdomen the day after a 100 mg dose. Both reactions were mild and self-limited and considered possibly related to the agent.

Clinical pharmacology
At equilibrium, [3H]CDB-2914, [3H]mifepristone and [3H]progesterone bound to human acid glycoprotein (hAGP) (58, 92 and 9% respectively) and human serum albumin (12, 84 and 63% respectively) but not to the negative control, human gamma globulin. At equilibrium, 46% of the [3H]CDB-2914 and 90% of the [3H]mifepristone were bound to proteins in the 10-fold diluted pre-treatment serum sample. These results support the hypothesis that in-vivo binding to hAGP may influence the bioavailability and pharmacokinetics of CDB-2914.

The pharmacokinetic parameters for CDB-2914 derived from the radioimmunoassay and HPLC assays are summarized in Table III. Radioimmunoassay-measurable CDB-2914/metabolite levels were detected in all women receiving active compound. A two-compartment model best characterized the HPLC data. For the radioimmunoassay data, the maximum concentration (Cmax) and AUC of CDB-2914 increased linearly as the dose increased. At doses >100 mg, Cmax did not increase further. For the HPLC data, only samples at doses of 100 and 200 mg were analysed because of the low concentrations at other doses. The serum elimination half-life was similar at both doses (100 mg: 1.5 ± 0.26 h versus 200 mg: 2.03 ± 1.12 h). The concentration-time profiles for patients analysed by both methods showed similar temporal trends, but the concentrations were significantly lower for the HPLC data compared with those of the radioimmunoassay (Figure 4 top). Tmax was not significantly different across dose groups or methods. The AUC values obtained from the HPLC method at 0–infinity only accounted for 8.8 and 12.2% of the radioimmunoassay data at 0–72 h (100 and 200 mg dose levels respectively). The HPLC assay was specific for CDB-2914, as all three hypothesized metabolites were identified in spiked sera as discrete peaks apart from CDB-2914. These results suggest that the radioimmunoassay method detected both parent compound and metabolites.


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Table III. Pharmacokinetic parameters of CDB-2914 from radioimmunoassay and high performance liquid chromatography (HPLC)
 


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Figure 4. Serum levels of CDB-2914 and immunoreactive metabolites measured by radioimmunoassay (RIA, closed circles) and serum levels of CDB-2914 (open circles) measured by high performance liquid chromatography (HPLC) in two women receiving CDB-2914, 200 mg. Top: values to 12 h. Bottom: values to 72 h.

 
There was no threshold of peak radioimmunoassay-measurable CDB-2914 concentration that predicted early bleeding for a given woman.


    Discussion
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
In this study, mid-luteal administration of CDB-2914 had biological activity as an antiprogestin, causing dose-dependent early endometrial bleeding, with a consistent effect at a single oral dose of 200 mg. Early endometrial sloughing occurred in most women despite luteal phase progesterone levels, indicating a direct action on the endometrium, rather than an indirect action on the ovary to reduce steroid production. However, concurrent functional luteolysis was observed in a few women who had early bleeding.

The endometrial effects of CDB-2914 are similar to those of mifepristone, which induces uterine bleeding within 72 h of mid-luteal administration by acting directly on the endometrium to inhibit progesterone activity (Schaison et al., 1985Go; Shoupe et al., 1987Go; Garzo et al., 1988Go; Nieman and Loriaux, 1988Go). The data from this study suggest that CDB-2914 and mifepristone are roughly equivalent in this regard. At 200 mg (~3–4 mg/kg), CDB-2914 consistently caused early endometrial bleeding. By contrast, the minimal luteal phase dose of mifepristone needed to induce endometrial bleeding consistently has not been established. Single doses of 50 mg induced bleeding in four out of four women (Shoupe et al., 1987Go) in one study but a single dose of 5 mg/kg was not consistently effective in another (Nieman and Loriaux, 1988Go) and four daily doses of 50 mg were only 80% effective in another study (Schaison et al., 1985Go). Both agents may cause luteolysis. Luteolysis occurred in 60% of women receiving mifepristone, 100 mg for 4 days (Schaison et al., 1985Go), and in all women at a single dose of 10 mg/kg (~600 mg) (Nieman and Loriaux, 1988Go). The threshold dose for consistently achieving this effect was not reached in this study of CDB-2914 up to 200 mg.

Women who experienced early endometrial bleeding without functional luteolysis had a longer duration of bleeding after CDB-2914. Two had a dramatic increase in length of bleeding in the treatment cycle (15 and 20 days). In these women, the induced early bleeding had not stopped at the time of spontaneous luteolysis (around luteal phase day 13), so that the menstruation associated with luteolysis caused an apparent lengthening of bleeding.

The physiological effect of CDB-2914 probably depends on its metabolism, the biological activity of its metabolites, and the variable binding of the compounds to plasma proteins. The serum levels of CDB-2914 and immunoreactive metabolites detected by radioimmunoassay, both peak and AUC, were higher than those detectd by HPLC, which was specific for CDB-2914. The difference between these two methods suggests that high circulating levels of CDB-2914 metabolites are present. These metabolites presumably affect the overall antiprogestational and antiglucocorticoid activity of orally administered CDB-1914. The relative contribution of CDB-2914 metabolites to biological activity has not been determined.

Specific protein binding of CDB-2914 was demonstrated for {alpha}1 acid glycoprotein (orosomucoid), with less binding to albumin. These results in women are similar to previous data derived from rhesus monkeys (Larner et al., 2000Go). CDB-2914 binding to orosomucoid was less than that of mifepristone, which may account for the longer half-life of mifepristone (Heikinheimo, 1997Go). Issues of pharmacokinetics and bioavailability of CDB-2914 and its metabolites must await additional studies with sufficiently sensitive and specific assays for the parent compound and putative metabolites.

None of the antiprogestins in clinical trials have had ‘pure’ antiprogestin effects. Their antiglucocorticoid activity occurs only at doses higher than those for antiprogestational activity and is normally overcome by an intact hypothalamic–pituitary–adrenal axis (Nieman and Loriaux, 1988Go; Laue et al., 1990Go; Bertagna et al., 1997Go). However, even after a single dose, mifepristone reduces the amount of REM sleep (Wiedemann et al., 1998Go), and is associated with asthenia, headache, malaise, dizziness and muscle pain (Dubois et al., 1986Go; Shoupe et al., 1987Go; Couzinet et al., 1990Go). Data from this clinical trial suggest that single doses of CDB-2914 may be tolerated at least as well as mifepristone, with minimal side-effects.

Chronic administration of mifepristone to a patient with Cushing’s syndrome at daily doses of up to 25 mg/kg was well tolerated (Nieman et al., 1985Go). However, in healthy individuals or patients without hypercortisolism, chronic administration of mifepristone at daily doses of >=200 mg has been reported to cause skin exanthem, endometrial hyperplasia and symptoms of adrenal insufficiency requiring glucocorticoid replacement (Klijn et al., 1989Go; Laue et al., 1990Go; Lamberts et al., 1991Go; Bertagna et al., 1994Go; Perrault et al., 1996Go; Newfield et al., 2001Go; Pomara et al., 2002Go). However, one study using a daily dose of 200 mg characterized the fatigue as ‘mild’ (Grunberg et al., 1991Go) and others using a daily dose of <=50 mg did not report any adverse effects (Croxatto et al., 1993Go; Kettel et al., 1996Go). Collectively, these data suggest that dose, duration and perhaps individual-specific factors influence whether side-effects occur. The presence of these undesirable side-effects has spurred a search for agents with a higher antiprogestin:antiglucocorticoid activity ratio. In this regard, CDB-2914 had no apparent clinical or biochemical antiglucocorticoid activity in this study. At the doses used it is not possible to determine whether CDB-2914 had a high antiprogestin:antiglucocorticoid activity, or whether the doses used were too low to evaluate effects on the pituitary–adrenal axis.

The information on the effects of CDB-2914 from this study supports continued clinical investigation of the agent as a promising compound with selective antiprogestin activity. Single oral doses of 1–200 mg of CDB-2914 were well tolerated and without apparent toxicity, and had a dose-dependent antiprogestational effect on the mid-luteal endometrium. Further studies are needed to determine the effects on the ovary and the endometrium during chronic administration. Such data will help determine the therapeutic usefulness of CDB-2914 in clinical medicine.


    FOOTNOTES
 
* Part of this work was previously presented and published as an abstract: Passaro, M., Piquion, J., Mullen, N., Sutherland, D., Alexander, N.J. and Nieman, L., ‘Safety and luteal phase effects of the antiprogestin CDB2914 in normally cycling women.’ Proceedings of the 79th meeting of the Endocrine Society, Minneapolis, MN, 1997, p. 227. Back


    References
 Top
 Abstract
 Introduction
 Subjects and methods
 Results
 Discussion
 References
 
Abraham, G.E., Buster, J.E., Lucas, L.A., Corrales, D.C. and Teher, R.C. (1972) Chromatographic separation of steroid hormones for use in radioimmunoassay. Analyt. Lett., 5, 509–517.[ISI]

Attardi, B.J., Burgenson, J., Hild, S.A., Reel, J.R. and Blye, R.P. (2002) CDB-4124 and its putative monodemethylated metabolite, CDB-4453, are potent antiprogestins with reduced antiglucocorticoid activity: in vitro comparison to mifepristone and CDB-2914. Mol. Cell. Endocrinol., 188, 111–123.[CrossRef][ISI][Medline]

Bertagna, X. (1997) Pituitary-adrenal response to RU 486 in man. Psychoneuroendocrinology, 22 (Suppl. 1), S51–S55.[CrossRef][ISI][Medline]

Bertagna, X., Escourolle, H., Pinquier, J.L., Coste, J., Raux-Demay, M.C., Perles, P., Silvestre, L., Luton, J.P. and Strauch, G. (1994) Administration of RU 486 for 8 days in normal volunteers: antiglucocorticoid effect with no evidence of peripheral cortisol deprivation. J. Clin. Endocrinol. Metab., 78, 375–380.[Abstract]

Chrousos, G.P., Schulte, H.M., Oldfield, E.H., Gold, P.W., Cutler, G.B., Jr and Loriaux, D.L. (1984) The corticotropin-releasing factor stimulation test. An aid in the evaluation of patients with Cushing’s syndrome. N. Engl. J. Med., 310, 622–626.[Abstract]

Cook, C.E., Lee, Y.W., Wani, M.C., Fail, P.A. and Petrow, V. (1994) Effects of D-ring substituents on antiprogestational (antagonist) and progestational (agonist) activity of 11 beta-aryl steroids. Hum. Reprod., 9 (Suppl. 1), 32–19.[Abstract]

Couzinet, B., LeStrat, N., Silvestre, L. and Schaison, G. (1990) Late luteal administration of the antiprogesterone RU 486 in normal women: effects on the menstrual cycle events and fertility control in a long-term study. Fertil. Steril., 54, 1039–1044.[ISI][Medline]

Croxatto, H.B., Salvatierra, A.M., Croxatto, H.D. and Fuentealba, B. (1993) Effects of continuous treatment with low dose mifepristone throughout one menstrual cycle. Hum. Reprod., 8, 201–207.[Abstract]

D’Argenio, D.Z. and Schumitzky, A. (1979) A program package for simulation and parameter estimation in pharmacokinetic systems. Comput. Programs Biomed., 9, 115–134.[CrossRef][ISI][Medline]

DeVilla, G.O., Jr, Roberts, K., Wiest, W.G., Mikhail, G. and Flickinger, G. (1972) A specific radioimmunoassay of plasma progesterone. J. Clin. Endocrinol. Metab., 35, 458–460.[ISI][Medline]

Dubois, C., Ulmann, A. and Baulieu, E. (1986) Contragestion with late luteal administration of RU 486 (Mifepristone). Fertil. Steril., 50, 595–596.

Gaillard, R.C., Riondel, A., Muller, A.F., Hermann, W. and Baulieu, E.E. (1984) RU486: A steroid with antiglucocorticoid activity that only disinhibits the human pituitary–adrenal system at a specific time of the day. Proc. Natl Acad. Sci. USA, 81, 3879–3882.[Abstract]

Garzo, V.G., Liu, J., Ulmann, A., Baulieu, E. and Yen, S.S. (1988) Effects of an antiprogesterone (RU486) on the hypothalamic–hypophyseal–ovarian–endometrial axis during the luteal phase of the menstrual cycle. J. Clin. Endocrinol. Metab., 66, 508–517.[Abstract]

Grunberg, S., Weiss, M., Spitz, I., Ahmadi, J., Sadun, A., Russell, C.A., Lucci, L. and Stevenson, L.L. (1991) Treatment of unresectable meningiomas with the antiprogesterone agent Mifepristone. J. Neurosurg., 74, 861–866.[ISI][Medline]

Heikinheimo, O. (1997) Clinical pharmacokinetics of mifepristone. Clin. Pharmacokinet., 3, 7–17.

Heikinheimo, O., Ranta, S., Grunberg, S., Lahteenmaki, P. and Spitz, I.M. (1997) Alterations in the pituitary–thyroid and pituitary–adrenal axes—consequences of long-term mifepristone treatment. Metabolism, 46, 292–296.[CrossRef][ISI][Medline]

Heikinheimo, O., Ranta, S., Grunberg, S. and Spitz, I.M. (2000) Alterations in sex steroids and gonadotropins in post-menopausal women subsequent to long-term mifepristone administration. Steroids, 65, 831–836.[CrossRef][ISI][Medline]

Hild, S.A., Reel, J.R., Hoffman, L.H. and Blye, R.P. (2000) CDB-2914: anti-progestational/anti-glucocorticoid profile and post-coital anti-fertility activity in rats and rabbits. Hum. Reprod., 15, 822–829.[Abstract/Free Full Text]

Jiang, N.S. and Ryan, R.J. (1969) Radioimmunoassay for estrogens: a preliminary communication. Mayo Clin. Proc., 44, 461–465.[ISI][Medline]

Kettel, L.M., Murphy, A.A., Morales, A., Ulmann, A., Baulieu, E.E. and Yen, S. (1996) Treatment of endometriosis with the antiprogesterone Mifepristone. Fertil. Steril., 65, 23–28.[ISI][Medline]

Klijn, J.G., de Jong, F.H., Bakker, G.H., Lamberts, S.W., Rodenburg, C.J. and Alexieva-Figusch, J. (1989) Antiprogestins, a new form of endocrine therapy for human breast cancer. Cancer Res., 49, 2851–2856.[Abstract]

Klijn, J.G., Setyono-Han, B. and Foekens, J.A. (2000) Progesterone antagonists and progesterone receptor modulators in the treatment of breast cancer. Steroids, 65, 825–830.[CrossRef][ISI][Medline]

Lamberts, S.W., Koper, J.W. and de Jong, F.H. (1991) The endocrine effects of long-term treatment with mifepristone (RU 486). J. Clin. Endocrinol. Metab., 73, 187–191.[Abstract]

Lamberts, S.W., Tanghe, H.L., Avezaat, C.J., Braakman, R., Wijngaarde, R., Koper, J.W. and de Jong, H. (1992) Mifepristone (RU 486) treatment of meningiomas. J. Neurol. Neurosurg. Psychiat., 55, 486–490.[Abstract]

Larner, J.M., Reel, J.R. and Blye, R.P. (2000) Circulating concentrations of the antiprogestins CDB-2914 and mifepristone in the female rhesus monkey following various routes of administration. Hum. Reprod., 15, 1100–1106.[Abstract/Free Full Text]

Laue, L., Lotze, M.T., Chrousos, G.P., Barnes, K., Loriaux, D.L. and Fleisher, T.A. (1990) Effect of chronic treatment with the glucocorticoid antagonist RU 486 in man: toxicity, immunological, and hormonal aspects. J. Clin. Endocrinol. Metab., 71, 1474–1480.[Abstract]

Murphy, A., Kettel, L., Morales, A., Roberts, V. and Yen, S. (1993) Regression of uterine leiomyomata in response to the antiprogesterone RU 486. J. Clin. Endocrinol. Metab., 76, 513–517.[Abstract]

Newfield, R.S., Spitz, I.M., Isacson, C. and New, M.I. (2001) Long-term mifepristone (RU486) therapy resulting in massive benign endometrial hyperplasia. Clin. Endocrinol. (Oxf.), 54, 399–404.[CrossRef][ISI][Medline]

Nieman, L.K. and Loriaux, D.L. (1988) The use of anti-progesterones as a medical IUD. Baillière’s Clin. Obstet. Gynecol., 2, 609–616.[ISI][Medline]

Nieman, L.K., Chrousos, G.P., Kellner, C., Spitz, I.M., Nisula, B.C., Cutler, G.B., Merriam, G.R., Bardin, C.W. and Loriaux, D.L. (1985) Successful treatment of Cushing’s syndrome with the glucocorticoid antagonist RU 486. J. Clin. Endocrinol. Metab., 61, 536–540.[Abstract]

Odell, W.D., Rayford, P.L. and Ross, G.T. (1967) Simplified, partially automated method for radioimmunoassay of human thyroid-stimulating, growth, luteinizing, and follicle stimulating hormones. J. Lab. Clin. Med., 70, 973–980.[ISI][Medline]

Perrault, D., Eisenhauer, E.A., Pritchard, K.I., Panasci, L., Norris, B., Vandenberg, T. and Fisher, B. (1996) Phase II study of the progesterone antagonist mifepristone in patients with untreated metastatic breast carcinoma: a National Cancer Institute of Canada Clinical Trials Group study. J. Clin. Oncol., 14, 2709–2712.[Abstract]

Pomara, N., Doraiswamy, P.M., Tun, H. and Ferris, S. (2002) Mifepristone (RU 486) for Alzheimer’s disease. Neurology, 58, 1436.

Rao, P.N., Acosta, C.K., Cessac, J.W., Bahr, M.L. and Kim, H.K. (1999) Synthesis of N-desmethyl derivatives of 17alpha-acetoxy-11beta-(4-N,N-dimethylaminophenyl)-19-norpregna-4,9-diene-3,20-dione and mifepristone. 1: Substrates for the synthesis of radioligands. Steroids, 64, 205–212. [CrossRef][ISI][Medline]

Reel, J.R., Hild-Petito, S. and Blye, R.P. (1998) Antiovulatory and postcoital antifertility activity of the antiprogestin CDB-2914 when administered as single, multiple, or continuous doses to rats. Contraception, 58, 129–136.[CrossRef][ISI][Medline]

Schaison, G., George, M., Lestrat, N., Reinberg, A. and Baulieu, E.E. (1985) Effects of the anti-progesterone RU486 during mid-luteal phase in normal women. J. Clin. Endocrinol. Metab., 61, 484–489.[Abstract]

Shoupe, D., Mishell, D., Lahteenmaki, P., Heikinheimo, O., Birgerson, L., Madkour, H. and Spitz, I.M. (1987) Effects of the antiprogesterone RU 486 in normal women. Am. J. Obstet. Gynecol., 157, 1415–1420.[Medline]

Spitz, I.M., Croxatto, H.B., Lahteenmaki, P., Heikinheimo, O. and Bardin, C.W. (1994) Effect of mifepristone on inhibition of ovulation and induction of luteolysis. Hum. Reprod., 9 (Suppl. 1), 69–76.[ISI][Medline]

Stratton, P., Hartog, B., Hajizadeh, N., Piquion, J., Sutherland, D., Merino, M., Lee, Y.J. and Nieman, L.K. (2000) A single mid-follicular dose of CDB-2914, a new antiprogestin, inhibits folliculogenesis and endometrial differentiation in normally cycling women. Hum. Reprod., 15, 1092–1099.[Abstract/Free Full Text]

Tarantal, A.F., Hendrickx, A.G., Matlin, S.A., Lasley, B.L., Gu, Q.Q., Thomas, C.A., Vince, P.M. and Van Look, P.F. (1996) Effects of two antiprogestins on early pregnancy in the long-tailed macaque (Macaca fascicularis). Contraception, 54, 107–115.[CrossRef][ISI][Medline]

Teutsch, G. and Philibert, D. (1994) History and perspectives of antiprogestins from the chemist’s pont of view. Hum. Reprod., 9 (Suppl. 1), 12–31.[ISI][Medline]

Ulmann, A. and Silvestre, L. (1994) RU486: the French experience. Hum. Reprod., 9 (Suppl. 1), 126–130.[ISI][Medline]

Wagner, B.L., Pollio, G., Giangrande, P. et al. (1999) The novel progesterone receptor antagonists RTI 3021-012 and RTI 3021-022 exhibit complex glucocorticoid receptor antagonist activities: implications for the development of dissociated antiprogestins. Endocrinology, 140, 1449–1458.[Abstract/Free Full Text]

Webb, A.M., Russell, J. and Elstein, M. (1992) Comparison of Yuzpe regimen, danazol and Mifepristone (RU486) in oral postcoital contraception. Br. Med. J., 305, 927–931.[ISI][Medline]

Wiedemann, K., Lauer, C.J., Hirschmann, M., Knaudt, K. and Holsboer, F. (1998) Sleep–endocrine effects of mifepristone and megestrol acetate in healthy men. Am. J. Physiol., 274, E139–145.[ISI][Medline]

Yamaoka, K., Nakagawa, J. and Uno, T. (1978) Application of Akaike’s information criterion (AIC) in the evaluation of linear pharmacokinetic equations. J. Pharmacokinet. Biopharm., 2, 165–175.

Submitted on October 21, 2002; resubmitted on April 11, 2003; accepted on May 9, 2003.





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