A single mid-follicular dose of CDB-2914, a new antiprogestin, inhibits folliculogenesis and endometrial differentiation in normally cycling women

Pamela Stratton1,6, Beth Hartog2, Negin Hajizadeh1, Johann Piquion1, Dorett Sutherland3, Maria Merino4, Young Jack Lee5 and Lynnette K. Nieman1

1 Pedriatric and Reproductive Developmental Endocrinology Branch, National Institute of Child Health and Human Development, Building 10 Room 9D42, 10 Center Dr. MSC 1583, Bethesda, MD 20892–1583, 2 Division of Reproductive Endocrinology and Infertility, Department of Obstetrics and Gynecology, George Washington University Medical Center, Washington DC, 3 Department of Nursing, Warren Grant Magnusen Clinical Center, 4 Surgical Pathology, National Cancer Institute, National Institutes of Health, and 5 Division of Epidemiology, Statistics, and Prevention, National Institute of Child Health and Human Development, Bethesda, MD 20892–1583, USA


    Abstract
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Previous studies in women have shown that the antiprogestin mifepristone delays or inhibits folliculogenesis. The purpose of this study was to explore whether a new analogue, CDB-2914, has similar effects on folliculogenesis, ovulation, or on subsequent luteal phase endometrial maturation. Forty-four normally cycling, healthy women recorded urine LH and vaginal bleeding during pre-treatment, treatment, and post-treatment cycles. At a lead follicle diameter of 14–16 mm, a single oral dose (10, 50, 100 mg) of CDB-2914 or placebo was given, and daily ultrasound, oestradiol and progesterone were obtained until follicular collapse; an endometrial biopsy was obtained 5–7 days later. Single doses of CDB-2914 were well tolerated. Mid-follicular CDB-2914 suppressed lead follicle growth, causing a dose-dependent delay in folliculogenesis and suppression of plasma oestradiol. At higher doses, a new lead follicle was often recruited. Although luteinized unruptured follicles were observed at the 100 mg dose, all women had follicular collapse. There was a significant delay in endometrial maturation after CDB-2914 at all doses. The treatment cycle was lengthened by 1–2 weeks in 30% at 100, 27% at 50 and 9% at 10 mg. CDB-2914 altered ovarian and endometrial physiology without major effects on menstrual cyclicity and may have therapeutic utility.

Key words: antiprogestin/CDB-2914/endometrium/follicle


    Introduction
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
The antiprogestin mifepristone (RU 486; Roussel-Uclaf, Romaineville, France) inhibits endometrial maturation or induces early menses when progesterone concentrations are high (Nieman et al., 1987Go; Gemzell-Danielsson et al., 1994Go), suggesting that these agents compete for the endometrial progesterone receptor to antagonize progesterone action. Surprisingly, mifepristone also has significant effects when circulating progesterone concentrations are low, causing a delay or inhibition of folliculogenesis, steroidogenesis, and ovulation (Liu et al., 1987Go; Shoupe et al., 1987Go; Batista et al., 1992bGo, 1994Go). These effects in the follicular phase may represent inhibition of oestradiol action by the mifepristone–progesterone receptor complex, independent of progesterone per se, as has been demonstrated in vitro (McDonnell et al., 1994Go). Alternatively, mifepristone binding to the progesterone receptor in the absence of progesterone might alter availability of transcription factors available to the oestrogen receptor–ligand complex (Gronemeyer et al., 1992Go). Although the mechanism of action is not understood, the clinical results (suppression of steroidogenesis and endometrial maturation) have been the rationale for exploring mifepristone efficacy for emergency contraception (Glasier et al., 1992Go; Bygdeman et al., 1997Go; WHO, 1999), and for the treatment of endometriosis and fibroids (Kettel et al., 1994Go; Murphy and Castellano, 1994Go).

Follicular phase administration of mifepristone blocks or delays ovulation in a dose-dependent fashion. At doses of 1–10 mg, ovulation is delayed but not abolished (Spitz et al., 1993Go). At higher doses, 200–600 mg, a new follicle is often recruited (Liu et al., 1987Go; Shoupe et al., 1987Go), so that a single 600 mg dose is more likely to cause a 1 week delay in menses (36%) than 10 mg (18%) (World Health Organization, 1999Go).

The threshold minimal dose of antiprogestin that alters endometrial maturation is lower than that for ovarian effects (Danielsson et al., 1997Go). Batista et al. demonstrated a delay in luteal phase maturation with mifepristone 1 mg daily, but minimal effects on ovulation (Batista et al., 1992aGo). Interestingly, although endometrial maturation has been extensively investigated after luteal phase administration (Greene et al., 1992Go; Gemzell-Danielsson et al., 1994Go; Cameron et al., 1997Go) it has not been examined after follicular phase use.

CDB-2914, a new agent with antiprogesterone and antiglucocorticoid activity, was developed by the National Institute of Child Health and Human Development (NICHD). As shown in Figure 1Go, CDB-2914 is a synthetic steroid analogue with structural similarity to progesterone and mifepristone. In a phase I safety and toxicity study of a single mid-luteal dose (placebo, 1, 10, 50, 100 and 200 mg) in normally cycling women, no adverse events or antiglucocorticoid activity were noted (Passaro et al., 1997Go). The compound and its metabolites, as detected by radioimmunoassay, have a terminal elimination half-life of 12–136 h (L.Nieman, personal communication). Early menses consistently occurred at the highest dose, demonstrating antiprogestational activity.



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Figure 1. The chemical structures of progesterone, mifepristone and CDB-2914.

 
The present study was designed to characterize the action of CDB-2914 when given in the mid-follicular phase to normally cycling women. The effect of a single dose of CDB-2914, 10–100 mg, on folliculogenesis, ovulation, and subsequent luteal phase endometrial maturation was studied. The aim of the study was to determine a dose of CDB-2914 that would inhibit endometrial development and steroidogenesis, with minimal effects on menstrual cyclicity. It was postulated that CDB-2914 effects on these parameters might well differ from the effects of mifepristone, just as the selective oestrogen receptor modifiers have different actions in various tissues.


    Materials and methods
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Subjects
Healthy women, aged 18–43 years, with regular ovulatory menstrual cycles every 24–35 days were studied. All were within 20% of ideal body weight, were not currently using an intrauterine device, and had not used reproductive hormonal medications or glucocorticoids for at least 3 months before the study. Women agreed to prevent pregnancy by abstinence or use of barrier contraception, or they were protected by tubal ligation or vasectomy. The study was approved by the Food and Drug Administration, and the NICHD institutional review board. Written informed consent was obtained from each subject.

Preparation of CDB-2914
Pure crystalline CDB-2914 was synthesized by Southwest Foundation for Biomedical Research (San Antonio, TX, USA). The Clinical Center Pharmaceutical Development Service sieved CDB-2914 and formulated 10, 50 and 100 mg doses of active agent, or inert material (placebo) in gelatin capsules. Initially, only the placebo, 10, and 50 mg dose were studied and randomization was done in three blocks of six and one block of three for a total sample size of 21. A 100 mg dose was added, at the time when data from the phase I study were available. Randomization for the next 24 subjects occurred in one block, assigning most to the 100 mg dose and some to each of the other three doses to achieve a total sample size of 45 and to ensure that at least 10 women in each dose group completed the study. The final dose groups each included 10–12 women (placebo or 10 mg, n = 12; 50 mg, n = 11; and 100 mg, n = 10). Investigators and subjects were not aware of the dose or agent administered.

Study design
Women participated in the study for three consecutive menstrual cycles (Figure 2Go). The first and third were intended to document menstrual cycle length and ovulation, while the second was the treatment cycle. At study entry, each woman was instructed in the use of an in home urinary LH kit, menstrual calendar, coital log and basal body temperature chart to be completed for all three cycles. Urinary LH was measured using a self-test or one-step test (OvuQUICKTM; Quidel, San Diego, CA, USA); each assay had a threshold of 40 mIU/ml and identified the LH surge on the same day as radioimmunoassay 98% of the time (Quidel, unpublished data) and on the same day as enzyme immunoassay 96% of the time (Elkind-Hirsch et al., 1986Go). In the first cycle, a plasma progesterone concentration was measured 1 week after the reported urine LH surge. Women progressed to the treatment cycle if the first cycle was ovulatory, with normal follicular maturation, and had normal length. Menstrual cycle length was considered normal if it was in the range 24–35 days with a luteal phase at least 12 days in length. A menstrual cycle was defined as ovulatory if the mid-luteal plasma progesterone measurement was >4 ng/ml.



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Figure 2. Study scheme.

 
In the second cycle, after a negative human chorionic gonadotrophin (HCG) test excluded pregnancy, women reported to the outpatient clinic on cycle days 6–8 for ultrasound evaluation of folliculogenesis using a 3.5 MHz transvaginal probe with a real-time scanner (Ultramark 9; Advanced Technologies Laboratories, Bothell, WA, USA). All sonogram examinations and interpretations were performed by one of the investigators (BH, LN, or PS). The number of follicles in each ovary were counted and follicles of >10 mm in diameter were measured. The follicular diameter was calculated as the average of the largest diameter in the transverse and longitudinal planes. On the day of treatment, all follicles within 2 mm of the largest follicle diameter were considered lead follicles. A change in lead follicle was designated when the first lead follicle stopped growing or disappeared, and another follicle that had been at least 2 mm smaller than the lead follicle(s) on the day of dosing grew larger than the size at dosing. The day of ovulation was defined retrospectively, as the day of maximum follicular enlargement, followed the next day by a 50% reduction in size, disappearance, or echogenic filling in of the previously echolucent structure (Queenan et al., 1980Go). Luteinized unruptured follicles were defined as those that continued to grow after the LH surge and that had filling in of the previously echolucent structure.

Women received CDB-2914 or placebo when the lead follicle was 14–16 mm in diameter. Subsequently, blood for oestradiol and progesterone, and a transvaginal sonogram were obtained daily until follicular collapse. To evaluate the safety of CDB-2914, blood chemistries, a complete blood count, and hepatic panel were obtained 5–7 days after dosing.

Women reported whether the LH kit was positive at the daily visit. To confirm the urine LH kit results, plasma LH was measured retrospectively by radioimmunoassay on the days of each reported positive urinary LH test as well as the day before and after. Any plasma LH value of >30 mIU/ml was considered to indicate an LH surge.

At 5–7 days after follicular collapse by ultrasound, an endometrial biopsy was performed, transvaginal sonography was done to determine the presence of ovarian cysts, and blood was obtained for measurement of oestradiol and progesterone. On the sonographic examination, an echolucent, cystic structure measuring >15 mm in diameter was considered to be an ovarian cyst. Because a change in lead follicle had been observed in some subjects, ovarian cysts noted in the luteal phase were followed as an additional evaluation of toxicity. When an ovarian cyst was noted, a limited pelvic vaginal sonogram, and blood oestradiol and progesterone measurements were repeated in the early follicular and mid-luteal phase in each subsequent cycle until the cyst resolved. Endometrial biopsies were performed using a Pipelle endometrial suction curette (Unimar, Wilton, CT, USA) and placed in neutral buffered formalin.

The third cycle was included to document any post-treatment effects on ovulation and menstrual cycle length. Ovulation was assessed by serum progesterone measured 7 days after a reported positive urine LH test. If the menstrual cycle was of abnormal length (<24 or >35 days) or the progesterone was <4 ng/ml, the woman was observed for another menstrual cycle.

Hormone assays and analysis
Blood samples were centrifuged and plasma was stored at –20°C until assayed. A radioimmunoassay was used to quantify concentration of oestradiol (Jiang and Ryan, 1969Go; Abraham et al., 1972Go), progesterone (DeVilla et al., 1972Go), and LH (Odell et al., 1967Go). LH values are expressed in terms of the Second International Reference Preparation for human menopausal gonadotrophin (HMG). The intra-assay coefficient of variation was <=10%, and the inter-assay coefficient of variation was <=15%. The maximal plasma oestradiol concentration in the 4 days after dose and in the 4 days before follicular collapse was noted. The cumulative oestradiol concentration after dose until ovulation was calculated as the sum of the daily measurements during this time period.

Endometrial biopsies
After routine haematoxylin and eosin staining, one pathologist (M.M.), unaware of treatment, assigned a post-ovulatory date for the specimen using the evaluation criteria of Noyes (Noyes et al., 1950Go) giving a 2 day spread. Biopsies were considered to be abnormal if there were >2 days delay in the pathological date compared with the chronological day of cycle based on the next menses.

Cycle phase lengths
A menstrual cycle was the number of days from the first day of menses to the beginning of the next menses. The follicular phase included the first day of menses to the day of the positive urine LH test. The luteal phase was defined as the day after the reported urinary LH surge until the next menses. In the treatment cycle, the number of days from the urinary LH surge until follicular collapse was calculated. The time interval from the day of treatment to follicular collapse was also determined.

Statistical analyses
A sample size of 50 women was sought to have at least 40 completing the study. The hypothesis tested was that CDB-2914 would delay ovulation so that 90% of women in the placebo group would ovulate within 6 days of dosing, compared with 10% at the highest dose. The other two dose groups would have a probability of a collapsed follicle within 6 days of 10–90%. A sample size of 40 would give the study 90% power to detect an absolute difference between 90% in the placebo group and 10% at 100 mg at the 0.05 level with a two-sided test. The frequency of adverse events, ovarian cysts, and laboratory abnormalities was determined to evaluate the safety of the drug.

Baseline characteristics among the dose groups were compared using analysis of variance (ANOVA). The treatment menstrual cycle parameters were compared with baseline and post-treatment cycles and ANOVA. Dose–response trends among treatment groups were assessed by ANOVA, Jonckeheere–Terpstra, or Kruskal–Wallis for continuous variables, e.g. oestradiol concentration, follicular diameter, and the relationship between oestradiol concentration and follicular diameter, and by {chi}2 or Cochran–Armitage for discrete variables such as altered endometrial maturation and presence of luteinized unruptured follicles. Values are expressed as mean ± SE. P < 0.05 in a two-sided test was considered to be statistically significant.


    Results
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 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
Demographics
A total of 51 healthy non-pregnant women volunteered for the study. Six were excluded because of abnormal follicular growth in the treatment cycle defined as no lead follicle measuring 14–16 mm by cycle day 20 (n = 4) or ovarian cyst noted at first study ultrasound (n = 2). Of the remaining 45 women with ovulatory baseline cycles, five had cycle lengths that were abnormal according to the study parameters. One had a 37 day cycle and four others had luteal phase lengths of <12 days, 9 (n = 2) and 10 days (n = 2). The progesterone concentrations for these five women documented ovulatory cycles and they were allowed to continue in the study. Of the 45 women who received study compound, one at 10 mg was excluded from analysis because of a protocol violation related to delayed dosing.

The mean age of the 44 women included in the analysis was 33 years (range 21–42). Twenty-two were Caucasian, 18 were Black, two were Hispanic and two were Asian. Of the 28 who had been pregnant, 25 had children and three had therapeutic abortions. The mean body mass index was 24.2 kg/m2; range 17.4–28.5 kg/m2. There was no significant difference in race, age, follicle number at baseline, body mass index, gravidity, and parity among any of the treatment groups.

Follicle number and growth
Women aged <30 years had more follicles on the day of dosing than those aged >40 years (16 ± 1.1 versus 9 ± 2.0 follicles; P = 0.0006). Between two and six women in each treatment group had more than one follicle measuring >10 mm on the day of dose (P = 0.3).

CDB-2914 suppressed growth of the lead follicle during the 4 days after treatment (P < 0.001) (Figure 3Go). Sometimes the stunted follicle would resume growth after a few days; at other times, the initial lead follicle stopped growing and was replaced by a new lead follicle. Five women, one each at placebo, 10 mg and 50 mg, and two at 100 mg, had at least two lead follicles on the day of dosing, one of which remained the lead follicle. CDB-2914 caused a dose-dependent change to a new lead follicle for 11 women: one at placebo, two at 10 mg, three at 50 mg, and five at 100 mg had a change in lead follicle (P < 0.03 for trend) (Table IGo). One woman, in the 50 mg dose group, developed two lead follicles that grew to >20 mm. After dosing, the first lead follicle had little growth for a week, but then grew to 45 mm diameter. The second lead follicle emerged 1 week after dosing, grew to 22.8 mm and then collapsed 18 days after dosing.



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Figure 3. The lead follicle diameter (mean in mm ± SE) compared across doses of CDB-2914 (placebo, 10, 50 and 100 mg) in the treatment cycle before and on the 4 days after dosing (P < 0.001).

 

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Table I. CDB-2914 increased the time to follicular collapse. Values are given as means with ranges in parentheses
 
All women ovulated in the treatment cycle, as assessed by a urine LH surge, growth and collapse or filling in of a lead follicle, and a progesterone concentration >4 ng/ml within 1 week of ovulation. CDB-2914 caused a dose-dependent delay in the time interval from treatment to follicular collapse (P < 0.0001), even for those without a change in lead follicle (P < 0.0001) (Table IGo). All but three women receiving placebo or 10 mg ovulated within one week of treatment, while all those receiving 50 or 100 mg ovulated >1 week after treatment. At all doses of CDB-2914, women with a change in lead follicle ovulated significantly later than those who did not have a change in lead follicle (P < 0.05).

The rate of lead follicle growth during the 4 days before follicular collapse was similar in all groups, although the maximal lead follicular diameter was greater at 100 mg of CDB-2914 (Figure 4Go). Three women who had a sonographic pattern consistent with luteinized unruptured follicles had the largest observed follicular diameters and accounted for the significant difference (P < 0.01) in maximal diameter observed between women receiving 100 mg and those receiving other doses. The finding of luteinized unruptured follicles in three of the 10 women who received 100 mg of CDB-2914, was significantly different from the absence of luteinized unruptured follicles in all other groups (P < 0.01; {chi}2 test). Progesterone concentrations of those with luteinized unruptured follicles rose to luteal phase concentrations after the LH surge, before the follicle was completely filled in, suggesting early luteinization of the follicle.



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Figure 4. The lead follicle diameter (mean in mm ± SE) compared across doses of CDB-2914 (placebo, 10, 50 and 100 mg) in the treatment cycle in the 4 days before ovulation (not significant).

 
Oestradiol secretion
During the first 4 days after dosing, CDB-2914 suppressed plasma oestradiol concentrations in a dose-dependent fashion (P < 0.001) (Figure 5Go). Peak oestradiol concentrations in the 4 days after dosing were also significantly suppressed (placebo: 343 ± 21 pg/ml, 10 mg: 267 ± 36 pg/ml, 50 mg: 181 ± 25 pg/ml, and 100 mg: 121 ± 13 pg/ml, P < 0.001). Oestradiol concentrations showed a strong linear relationship with the follicular diameter in the placebo and 10 mg groups, with increasing diameter associated with increasing oestradiol concentrations (Figure 6Go). In contrast, minimal follicular growth was associated with minimal changes in oestradiol concentration at 50 and 100 mg, representing a significant difference between placebo and 10 mg compared with 50 and 100 mg (P < 0.001).



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Figure 5. The plasma oestradiol concentration (pg/ml, mean ± SE) compared across doses of CDB-2914 (placebo, 10, 50 and 100 mg) in the treatment cycle before and on the 4 days after dosing (P < 0.001).

 


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Figure 6. The plasma oestradiol concentration (pg/ml, mean ± SE) plotted against the lead follicular diameter (mean in mm ± SE) for each dose of CDB-2914 (placebo, 10, 50 and 100 mg) in the treatment cycle on day 4 after dosing. There was a significant difference (P < 0.001) between the placebo and the 10 mg dose on the one hand, compared with the 50 and 100 mg doses, on the other.

 
During the 4 days before follicular collapse, CDB-2914 also suppressed daily oestradiol production (P < 0.005) (Figure 7Go) and the peak oestradiol concentration in a dose-dependent fashion (placebo: 343 ± 21 pg/ml, 10 mg: 286 ± 31 pg/ml, 50 mg: 263 ± 22 pg/ml, 100 mg: 224 ± 25 pg/ml, P < 0.001). However, the cumulative (summed) oestradiol concentration after treatment until follicular collapse (Figure 7Go, inset) was similar in all groups (placebo: 1290 ± 124 pg/ml, 10 mg: 1151 ± 125 pg/ml, 50 mg: 1425 ± 157 pg/ml and 100 mg: 1607 ± 255 pg/ml, P = 0.3), as was the maximal follicular diameter (Figure 4Go, P = 0.12). Daily increases in follicular diameter were related to increasing oestradiol concentrations at placebo only, but not with any dose of CDB-2914 (P < 0.002) (Figure 8Go).



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Figure 7. The plasma oestradiol concentration (pg/ml, mean ± SE) compared across doses of CDB-2914 (placebo, 10, 50 and 100 mg) in the treatment cycle on the 4 days before ovulation (P < 0.005). The inset shows the cumulative oestradiol values from the day of dosing to ovulation, calculated as the sum of the daily values (not significant).

 


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Figure 8. The plasma oestradiol concentration (pg/ml, mean ± SE) plotted against the lead follicular diameter (mean in mm ± SE) for each dose of CDB-2914 (placebo, 10, 50 and 100 mg) in the treatment cycle 4 days before ovulation. There was a significant difference (P < 0.002) between the placebo result and those for the three doses of CDB-2914.

 
Plasma LH concentrations
All women reported a LH surge in the pre-treatment and treatment cycles. In the treatment cycle, plasma LH concentrations confirmed a surge within 1 day of a positive urine LH result in 42 women. The remaining two women, with plasma LH concentrations of 11.5 and 19.2 IU/ml, had follicular collapse by 4 days after the urine LH surge. The peak LH concentration was similar for all groups (placebo: 71 ± 12 IU/ml, 10 mg: 97 ± 14 IU/ml, 50 mg: 64 ± 8 IU/ml, 100 mg: 73 ± 14 IU/ml). The temporal relationship between the LH surge and follicular collapse was distorted by CDB-2914. After placebo or 10 mg of CDB-2914, the lead follicle collapsed within 3 days of the LH surge. At 50 and 100 mg, the LH surge occurred >3 days before follicular collapse in four out of 11 and six out of 10 women respectively (P < 0.0001). Additionally, five women reported two LH surges that were confirmed by radioimmunoassay of plasma. All had developed a new lead follicle after CDB-2914. The first LH surge was reported at a lead follicle size of 14–20 mm; the follicle continued to grow, another LH surge was reported several days later, and then the lead follicle collapsed.

Ovarian cysts noted at endometrial biopsy
Asymptomatic luteal phase cysts were noted at the time of endometrial biopsy for two women at placebo, four at 50 mg and four at 100 mg CDB-2914 (Table IIGo). Women who developed a new lead follicle were not more likely to have luteal phase cysts. The cyst size was independent of treatment and measured 15–23 mm in five women and 29–33 mm in five others. Cysts spontaneously resolved within 2 months of treatment in nine of the 10 women. The remaining subject had a 16 mm cyst that persisted through 3 months of follow-up.


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Table II. Mid-follicular CDB-effects on luteal phase endometrium and ovary
 
Endometrial biopsy
In all, 33 endometrial biopsies were performed 5–7 days after ovulation. Eleven were obtained earlier or later due to scheduling errors or difficulty determining the day of ovulation. Compared with the placebo, there was a significant delay in maturation at all doses of CDB-2914 (P < 0.02) (Table IIGo). These results were not altered by inclusion of the 11 women with mistimed biopsies. Women with delayed endometrial maturation had a significantly lower peak follicular phase oestradiol than those with a normal biopsy (250 ± 20 pg/ml versus 321 ± 17 pg/ml; P < 0.01), but did not have a lower cumulative oestradiol from treatment to follicular collapse (1287 ± 109 pg/ml versus 1471 ± 138 pg/ml; P = 0.3). There was no discrepancy between stroma and glandular maturation.

Effects on cycle length
There was no significant difference among the treatment groups in the baseline or post-treatment menstrual cycle parameters, including the follicular or luteal phase (data not shown), or overall cycle lengths (Table IIIGo). The 50 and 100 mg dose groups had treatment cycles that were, on average, 4 days longer than the placebo or 10 mg dose groups (P < 0.01). The treatment cycle was lengthened by 1–2 weeks in 30% at 100, 27% at 50 and 9% at 10 mg. This increase was due to a 1 week delay in menses observed only in eight of the 10 CDB-2914 treated women with a change in lead follicle (one at 10 mg, three at 50 mg, and four at 100 mg) (P < 0.02 for trend).


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Table III. CDB-2914: effect on menstrual cycle length
 
Six women had an abnormal menstrual cycle length in the post-treatment cycle. Of these, two women, one each at placebo and 10 mg, had long menstrual cycles (42 and 41 days respectively). Four had short cycles; one each at placebo, 10 mg and 100 mg had 23 day cycles, and one at 50 mg had a 17 day cycle. Of those with a 23 day cycle, two usually had a cycle length of 24 days and were not observed for an additional post-treatment cycle. The second post-treatment cycle was of normal length for all except one. This subject underwent surgery for a ruptured cyst and then had a 90 day cycle. Additionally, one woman became pregnant in her post-treatment cycle and had an uneventful pregnancy.

Ovulation data in the cycle following CDB-2914 administration were available for all but two women. For those with a progesterone concentration <4 ng/ml after a reported LH surge, a biphasic pattern on the basal body temperature was considered to indicate ovulation. In all, 35 out of 42 women had ovulatory cycles. Of the other seven women, three were in the placebo group, three at 50 mg and one at 100 mg, including two who also had an abnormal cycle length (cycle length 42 and 17 days). All of these women had an ovulatory cycle in the second cycle after treatment.

Safety of CDB-2914
CDB-2914 was well tolerated at all doses. All post-treatment laboratory values were normal. Two women had gastrointestinal or flu symptoms from intercurrent illness, and one, with a history of migraines, had a migraine within 2 days of dosing. Four women (three at 100 mg and one at 50 mg) reported lower abdominal pain with the collapse of the 30–51 mm follicles.


    Discussion
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
At mid-follicular doses of 10–100 mg, CDB-2914 caused a delay in ovulation that was greatest at the highest doses, but inhibited luteal phase endometrial maturation similarly at all doses. Thus, the threshold for altering endometrial morphology was lower than that for altering folliculogenesis, a finding that has not been reported previously after single dose follicular phase administration of an antiprogestin. The mechanism for this effect, noted long after the agent is given, might reflect a direct effect of the antiprogestin to prevent the change from proliferative to secretory endometrium, or may result from altered oestrogen exposure. As progesterone is not thought to be critical to the endometrium prior to ovulation, it is possible that one mechanism by which CDB-2914 might exert such an effect might be through inhibition of oestrogen action. In vitro, the antiprogestin–progesterone receptor complex acts as transdominant repressor of oestrogen action (McDonnell et al., 1994Go). This may explain the anti-oestrogen action of mifepristone on rhesus endometrial proliferation reported in the absence of progesterone (Brenner and Slayden, 1994Go). A similar action could underlie the effects on endometrial maturation seen here.

A single mid-follicular dose of CDB-2914 impeded folliculogenesis, causing a dose-dependent delay in time to ovulation and diminished oestradiol secretion. After CDB-2914, follicular growth was initially stunted and then resumed a normal linear rate of growth to reach a maximal diameter similar to placebo. Despite regaining a normal follicular growth pattern, daily steroidogenesis was diminished at all doses. Liu and colleagues reported similar reductions in oestradiol when RU-486 was given at a dose of 3 mg/kg, orally, once daily for 3 days when the dominant follicle was 1.5–2.0 cm in diameter (Liu et al., 1987Go).

The mechanism(s) by which CDB-2914 exerts these profound effects on follicular growth and maturation is unclear. One possibility is that, at higher doses, initial inhibition of lead follicle growth was irreversible. The ability of a second smaller follicle to continue growing, become the subsequent lead follicle and ultimately ovulate, suggests that the effects of CDB-2914 may be most detrimental to larger follicles. Such a putative action on developmentally vulnerable follicles is consistent with either a direct ovarian effect, or an alteration in gonadotrophin pulse patterns or concentrations. It is tempting to speculate that the CDB-2914 may inhibit the action of the progesterone receptor or oestrogen receptor ß, both of which were recently described as abundant in granulosa cells of developing follicles (Revelli et al., 1996Go; Enmark et al., 1997Go). As frequent FSH and LH concentrations were not measured in this study, we cannot distinguish between these possibilities. Other antiprogestins are known to influence LH pulsatility, however, and this may explain the demise of the gonadotrophin-dependent dominant follicle (Permezel et al., 1989Go). A change in LH pulsatility, amplitude and mean serum LH were not observed on the third day of a mid-follicular dose of mifepristone in a study by Liu (Liu et al., 1987Go).

The increase in time between the LH surge and follicular collapse, and the unusual observation of two LH surges also must be integrated into a model of CDB-2914 action. In the latter case, an initial LH surge failed to initiate collapse of follicles 14–20 mm in diameter. We speculate that this represents either an inappropriately early LH surge, or an inhibition of the ovarian cascade that normally initiates follicle rupture. The observation of luteinized unruptured follicles may, in a similar way, represent a failure of the follicle to signal readiness for ovulation. The present results are similar to previous reports (Liu et al., 1987Go; Shoupe et al., 1987Go; Batista et al., 1994Go). that treatment of normal women with mifepristone during different stages of the follicular phase postpones the mid-cycle LH surge and delays ovulation. We previously reported the effects of mid-follicular administration of low dose mifepristone (1 mg daily for 5 days) in women with hypothalamic amenorrhoea undergoing ovulation induction with gonadotrophin-releasing hormone (GnRH) pulses (Batista et al., 1994Go). In that study, while follicular growth rate was not altered, an increase in the time to ovulation was associated with increased maximal follicular diameter compared with the placebo. In this study, where GnRH pulses were not held constant, lengthening of the time interval from LH surge to collapse may be similar to the blocking or attenuation of the mid-cycle LH surge observed by others after mifepristone. Shoupe et al. observed an attenuated LH surge with a lower peak LH than in the control cycles after 50 mg of mifepristone given twice a day on days 10–17 of the cycle (Shoupe et al., 1987Go). After 3 mg/kg of mifepristone was administered daily for three days in the mid-follicular phase by Liu, a delayed LH surge with a higher peak LH was observed during the treatment cycle when compared to the control cycle (Liu et al., 1987Go).

The initial inhibition of oestradiol concentrations during the 4 days after CDB-2914 is consistent with the inhibition of follicular growth; the normal positive correlation between increasing follicle diameter, granulosa cell mass, and steroidogenesis was preserved at placebo and 10 mg. We infer that the lack of follicular growth at 50 and 100 mg represents failure of granulosa proliferation and that the apparent decrease in oestradiol concentrations relative to placebo reflects this diminished granulosa cell volume. By contrast, diminished oestradiol concentration was related to the increasing follicle diameter in the 4 days before follicle collapse at any dose of CDB-2914, suggesting that the proliferative capacity was less impaired than the steroidogenic capacity. In addition, daily oestradiol concentrations were decreased, especially at the higher doses. It is unclear whether these lower oestradiol concentrations might cause any adverse effects or could be exploited for the treatment of other conditions, like endometriosis.

Those with a delay in ovulation after CDB-2914 had a longer treatment cycle. This is similar to the dose-dependent effect seen with mifepristone (World Health Organization, 1999Go). Any effect on menstrual cycle length was limited to the treatment cycle, returning to a normal length within one cycle. Post-treatment cycle abnormalities of anovulation or abnormal cycle length were uncommon and occurred at a similar rate in all groups. No women experienced breakthrough or prolonged bleeding after treatment, and the agent appeared to be safe and well tolerated when given in this way.

In conclusion, follicular phase CDB-2914 at single doses significantly delays endometrial maturation and diminishes oestradiol concentrations with minimal effects on the menstrual cycle. The good safety profile shown here allows for further exploration of the potential clinical utility of CDB-2914 for emergency contraception and the treatment of endometriosis.


    Acknowledgments
 
We would like to thank Dr Richard Blye of the Contraception and Reproductive Health Branch, Center for Population Research, NICHD for overseeing the manufacture and supply of CDB-2914. Synthesis of CDB-2914 was supported under contract NO1-HD-1–3137 of the National Institute of Child Health and Human Development and Research.


    Notes
 
6 To whom correspondence should be addressed Back


    References
 Top
 Abstract
 Introduction
 Materials and methods
 Results
 Discussion
 References
 
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Submitted on August 11, 1999; accepted on January 31, 2000.