Submitted on June 9, 2010; resubmitted on December 23, 2010; accepted on February 9, 2011

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1 Human Reproduction, Vol.26, No.6 pp , 2011 Advanced Access publication on March 18, 2011 doi: /humrep/der058 ORIGINAL ARTICLE Fertility control Comparison of a 24-day and a 21-day pill regimen for the novel combined oral contraceptive, nomegestrol acetate and 17b-estradiol (NOMAC/E 2 ): a double-blind, randomized study S. Christin-Maitre 1,2, D. Serfaty 3, N. Chabbert-Buffet 2,4, E. Ochsenbein 5, D. Chassard 6, and J.-L. Thomas 5, * 1 Endocrine Unit, Hôpital Saint Antoine AP-HP, Paris, France 2 ER-9 UPMC Université Paris VI, Paris, France 3 Société Francophone de Contraception, 9 Rue de Villersexel, Paris, France 4 Obstetrics and Gynaecology Unit, Hôpital Tenon AP-HP, Paris, France 5 Laboratoire Théramex, 6 Avenue Albert II, BP 59, Monaco 6 SGS Aster, 3 5 Rue E. Millon, Paris, France *Correspondence address. Tel: ; mmicheletti@theramex.mc Submitted on June 9, 2010; resubmitted on December 23, 2010; accepted on February 9, 2011 background: Nomegestrol acetate/17b-estradiol (NOMAC/E 2 ) is a new monophasic oral contraceptive combining NOMAC (2.5 mg), a highly selective progesterone-derived progestogen, with E 2 (1.5 mg), which is structurally identical to endogenous estrogen. The objective of this study was to compare the effects on ovarian activity of two different NOMAC/E 2 regimens. methods: This was a double-blind, randomized study. Healthy, premenopausal women (aged years, previous menstrual cycle length days) were randomized by computer-generated code to once-daily NOMAC/E 2 for three consecutive 28-day cycles: either 24 days with a 4-day placebo interval (n ¼ 40) or 21 days with a 7-day placebo interval (n ¼ 37) per cycle. Follicular growth (primary outcome measure), plasma hormone profiles and bleeding patterns were assessed. results: There was no evidence of ovulation during treatment with either NOMAC/E 2 regimen. The largest follicle diameter was significantly smaller in the 24-day group than in the 21-day group [mean (SD) mm in cycle 2: 9.0 (3.0) versus 11.3 (5.3) (P ¼ 0.02); in cycle 3: 9.2 (3.0) versus 11.5 (6.0) (P ¼ 0.04)]. Mean FSH plasma levels were significantly lower in the 24-day versus the 21-day group on Day 24 of cycles 1 and 2. Withdrawal bleeding duration was significantly shorter in the 24-day than in the 21-day group [mean (SD) days after cycle 1: 3.5 (1.3) versus 5.0 (2.6) (P ¼ 0.002); after cycle 2: 3.9 (1.6) versus 4.8 (1.7) (P ¼ 0.03)]. conclusions: The 24-day NOMAC/E 2 regimen was associated with greater inhibition of follicular growth and shorter duration of withdrawal bleeding than the 21-day regimen, suggesting the shorter pill-free interval results in a greater margin of contraceptive efficacy and tolerability, and fewer withdrawal symptoms. Key words: estradiol / ethinylestradiol / follicle / ovulation / progestogen Introduction Combined oral contraceptives (COCs) work in a synergistic way to inhibit ovulation (Rivera et al., 1999), by inhibiting the secretion of LH and the intercycle rise in FSH. In addition to making the primary contribution to ovulation inhibition, the progestogen component of COCs also alters cervical mucus thereby providing a physical barrier to sperm entry and reduces endometrial thickness. Different COC formulations, such as pills that contain low doses of ethinyl estradiol (EE) or novel progestogens, have been developed over the years to improve tolerability and patient satisfaction but these formulations may require specific administration schedules. & The Author Published by Oxford University Press on behalf of the European Society of Human Reproduction and Embryology. All rights reserved. For Permissions, please journals.permissions@oup.com

2 NOMAC/E 2 regimen validation study 1339 For example, pills containing low doses of EE may require short pillfree periods to maintain efficacy (van Heusden et al., 1999) because follicular maturation is most likely to be initiated during a pill-free period or after a missed pill (van Heusden et al., 1999; Baerwald et al., 2004). A shorter pill-free period may lead to greater suppression of ovarian activity (Schlaff et al., 2004) and 23- or 24-day regimens have been shown to be well tolerated (Gestodene Study Group 322, 1999; Gestodene Study Group 324, 1999; Sullivan et al., 1999; Fruzzetti et al., 2001; Bachmann et al., 2004) and more efficacious than 21-day regimens (Spona et al., 1996; Sullivan et al., 1999; Nakajima et al., 2007). COCs containing estradiol (E 2 ) in place of EE have been developed over the past years. Pills containing E 2 in combination with a potent progestogen inhibit ovulation (Wenzl et al., 1993; Hirvonen et al., 1995; Csemiczky et al., 1996). E 2 -containing pills may have different effects on hormone levels and metabolic variables than EE-containing pills (Mashchak et al., 1982). However, early E 2 -containing COCs were originally associated with poor cycle control and a tendency towards prolonged and frequent bleeding (Astedt et al., 1979; Serup et al., 1981; Wenzl et al., 1993; Hirvonen et al., 1995; Csemiczky et al., 1996). More recently, E 2 has been used successfully as E 2 valerate (E 2 V), administered in a four-phasic, 26-day pill regimen (Endrikat et al., 2008). This E 2 V-containing pill is effective and has a bleeding profile comparable to that of an EE-containing OC (Ahrendt et al., 2009), but a complex administration regimen. A new monophasic oral contraceptive pill, combining 17b-E 2 with nomegestrol acetate (NOMAC), is currently in development. Oral E 2 is structurally identical to endogenous estrogen. NOMAC is a highly selective, antigonadotropic, 19-norprogesterone derivative that is structurally similar to endogenous progesterone (Couzinet et al., 1996). NOMAC is well tolerated (Basdevant et al., 1991; Dorangeon et al., 1993) and is able to suppress ovulation when given alone at doses of mg/day (Bazin et al., 1987; Couzinet et al., 1999). The combination of 2.5 mg NOMAC and 1.5 mg E 2 inhibits ovulation in women of childbearing age when administered orally for 21 out of 28 days, and may have a synergistic antigonadotropic action (Chabbert- Buffet et al., 2011). The objective of this Phase 2 study was to examine whether suppression of follicular growth by the combination of NOMAC/E 2 (2.5 mg/1.5 mg) is increased by a shorter pill-free interval, by comparing the effects of two dose regimens on ovarian activity. Materials and Methods This Phase 2, double-blind, randomized, parallel-group study (02-ESC/ NOM-1-RD) was conducted between November 2002 and October 2003 at a single centre in France. The study duration was sufficient to assess five ovulatory cycles: one pretreatment cycle, three consecutive treatment cycles and one post-treatment cycle. The study was conducted in accordance with Good Clinical Practice, the Declaration of Helsinki, Directive 2001/83/CE (The Rules Governing Medicinal Products in the European Community) and French law. The protocol and amendment was approved by an Independent Ethics Committee. All subjects provided written, informed consent before participating in the study. Participants Healthy, non-pregnant, premenopausal women volunteers (aged years) with a previous menstrual cycle length of days were eligible for study inclusion. Use of oral contraception, intrauterine device or contraception implants was stopped at least 2 months before the start of the study. Interventions Subjects were randomized in blocks of four to one of two treatment groups, using a computer-generated randomization code, to receive either NOMAC/E2 (2.5 mg/1.5 mg) for 24 days with a 4-day placebo interval per cycle or NOMAC/E 2 (2.5 mg/1.5 mg) for 21 days with a 7-day placebo interval per cycle. Dose selection was based on a dose-ranging study (Chabbert-Buffet et al., 2011). The first day of menstrual bleeding is referred to as Day 1 of each cycle. Treatment was started on Day 1, 2 or 3 of menstrual bleeding and continued for three consecutive 28-day cycles. Treatment was taken by subjects once daily at bedtime and blinding was maintained by administering capsules that were identical in appearance. All clinical and statistical personnel involved in the clinical trial, including the ultrasonographers, were blinded to treatment allocation. The blind was broken only after the database was locked and the blind review performed. Subjects attended six clinic visits: pretreatment cycle (Day 13 of the cycle; screening); cycle 1 (Days 1 and 24); cycle 2 (Day 13); cycle 3 (Day 13); post-treatment cycle (Day 13). The following time points were used for ultrasounds and blood samples: pretreatment cycle (Day 20); cycle 1 (Days 1, 21, 24 and 27); cycle 2 (Days 2, 5, 8, 11, 13, 16, 21, 24 and 27); cycle 3 (Days 2, 5, 8, 11, 13, 16 and 21); post-treatment cycle (Day 20). Endovaginal ultrasounds were performed in the same clinic by the same two operators, using the same ultrasonograph. Blood samples were taken in the morning, 12-h post dose. Outcome measures Primary efficacy outcome: ovarian activity Ovarian activity was assessed by measuring follicular growth using endovaginal ultrasound. Data were collected on: mean diameter of the largest follicle during each cycle; mean diameter of the largest follicle at each time point; number of women with at least one follicle.10 mm diameter; number of women with at least one follicle.13 mm diameter. Secondary efficacy outcomes Mean hormone concentrations in blood samples were measured using gas chromatography/mass spectrometry (E 2 ) or commercial radioimmunoassay (progesterone, FSH and LH), performed at CEPHAC (Saint-Benoit, France). The lower limits of quantification were 10 pg/ml for E 2, 0.05 ng/ ml for progesterone, 0.1 miu/ml for FSH and 0.15 miu/ml for LH. Intraand inter-assay coefficients of variation for E 2 were,10%. Individual progesterone levels.3 ng/ml were used to determine ovulation. Luteinized unruptured follicle (LUF) syndrome was determined if a follicle.13 mm in diameter was present with.3 ng/ml of progesterone and no evidence of follicle rupture on ultrasound examination. Endometrial thickness was measured during the pretreatment cycle, cycle 3 and post-treatment cycle. Cervical mucus was assessed mid-cycle using a modified Insler scoring method (Insler et al., 1972). Subjects completed a daily diary to record bleeding patterns. Parameters determined using the diary data included incidence and duration of bleeding (total, intermenstrual and withdrawal). Safety and tolerability measures All treatment-emergent adverse events (AEs), abnormal results on physical examination, abnormal results on gynecological examination, changes in vital signs and changes in laboratory parameters occurring during the study were recorded.

3 1340 Christin-Maitre et al. Treatment compliance Compliance was determined by counting unused pills, reviewing diary entries and examining NOMAC plasma levels in all blood samples collected throughout the study (except Day 27). A cycle was considered compliant if the subject missed no more than one dose (provided two doses were taken the day after the missed dose) and had NOMAC plasma levels above the lower limit of quantification (0.05 ng/ml) throughout the active treatment period. A subject was considered compliant if she met the above criteria for all treatment cycles. Statistical analyses A sample size of 60 (30 per treatment arm) was deemed sufficient to detect a between-group difference of 5 mm (SD 5.5 mm) in the mean diameter of the largest follicle in treatment cycles 2 and 3 (Sullivan et al., 1999), with a power of 90% at the 0.05 significance level. Assuming a drop-out rate of 20%, 80 subjects (40 per treatment arm) were enrolled. Efficacy was assessed in the intent-to-treat (ITT) population, defined as all subjects who took at least one pill and had at least one efficacy assessment. Supportive efficacy analyses were also performed on the perprotocol (PP) population, defined as all compliant subjects who missed no more than two ultrasound assessments. All statistical tests were twosided and a P-value,0.05 was considered significant. Student s t-tests were used to compare mean diameter of the largest follicle, mean hormone concentrations, mean duration of bleeding and endometrial thickness across treatment groups. Wilcoxon rank tests were used to compare Insler scores in cycles 2 and 3 across treatment groups. x 2 test or Fisher s exact test was used to compare number of subjects with follicle size.10 mm, number of subjects with follicle size.13 mm and number of subjects with bleeding in each cycle across treatment groups. Tolerability was assessed in all subjects who received at least one dose of study drug. Results Subject disposition A total of 80 subjects were randomized, of whom 72 (90%) completed the study (Fig. 1). No subjects discontinued due to an AE. Altogether, 77 subjects received study drug and are included in the safety population. One subject in the 24-day group who received study drug did not have any ultrasound assessments; thus, 76 subjects are included in the ITT population. A total of 65 subjects (32 in the 21-day group and 33 in the 24-day group) are included in the PP population. Baseline and pretreatment characteristics Demographic, gynecological and obstetrical characteristics at baseline were similar in both groups (Table I). During the pretreatment cycle, all 76 ITT subjects ovulated (defined by progesterone levels.3 ng/ ml). Mean plasma hormone levels measured at Day 20 of the pretreatment cycle were similar in both groups. There was no statistically significant difference in the mean diameter of the largest follicle at Day 20 of the pretreatment cycle between treatment groups (Table I). Efficacy outcomes Ovarian activity There was no ovulation or LUF syndrome with either NOMAC/E 2 regimen during the three consecutive treatment cycles. Mean diameter of the largest follicle was significantly smaller in the 24-day group than in the 21-day group (Table II, Fig. 2). In the PP population, the mean (SD) diameter of the largest follicle across all treatment Figure 1 Subject disposition. ITT, intent-to-treat population. a Sixty-five subjects screened did not meet inclusion criteria.

4 NOMAC/E 2 regimen validation study 1341 Table I Demographic, gynecological and obstetrical characteristics at baseline (ITT population). 21-day NOMAC/E 2 (n 5 37) 24-day NOMAC/E 2 (n 5 39) Total (n 5 76)... Age, mean (SD) [range] years 26.3 (4.9) [19 38] 28.5 (4.8) [20 37] 27.4 (4.9) [19 38] BMI, mean (SD) [range] kg/m (2.7) [17 29] 22.7 (3.1) [18 30] 22.6 (2.9) [17 30] Race, n (%) White or Caucasian 23 (62) 30 (77) 53 (70) Black or African American 14 (38) 8 (20) 22 (29) Asian 0 1 (3) 1 (1) Current smoker (,10 cigarettes/day), n (%) 18 (49) 14 (36) 32 (42) Age at first menstruation, mean (SD) [range] years 12.7 (1.5) [9 16] 12.7 (1.4) [10 16] 12.7 (1.4) [9 16] Duration of previous menstrual cycle, mean (SD) [range] days 28.7 (1.6) [25 32] 28.4 (1.3) [25 32] 28.6 (1.4) [25 32] Duration of previous menstruation, mean (SD) [range] days 4.1 (1.8) [0 10] 4.6 (3.2) [1 18] 4.4 (2.6) [0 18] Number of pregnancies, n (%) 0 22 (60) 21 (54) 43 (57) 1 10 (27) 11 (28) 21 (28) 2 5 (14) 7 (18) 12 (16) Number of childbirths, n (%) 0 30 (81) 30 (77) 60 (79) 1 5 (14) 5 (13) 10 (13) 2 2 (5) 4 (10) 6 (8) Insler score, n (%) (22) 7 (18) 15 (20) (38) 7 (18) 21 (28) (14) 8 (21) 13 (17) 9 10 (27) 17 (44) 27 (36) Pretreatment cycle (Day 20) Diameter of largest follicle, mean (SD) [range] mm a 9.8 (6.0) [0 31] 8.0 (4.1) [0 18] 8.8 (5.1) [0 31] Follicle size.10 mm, n per group (%) 15 (41) 10 (26) 25 (33) Follicle size.13 mm, n per group (%) 10 (27) 5 (13) 15 (20) There were no significant between-group differences at baseline. a Follicle diameter was assessed by ultrasound on Day 20 of the pretreatment cycle. A follicle could not be detected in five women (three in the 21-day group and two in the 24-day group). For these women, mean follicle diameter was recorded as 0 mm. Table II Follicular maturation (ITT population). 21-day NOMAC/E 2 24-day NOMAC/E 2 Treatment group difference P-value (n 5 37) (n 5 39) (95% confidence interval)... Diameter of largest follicle, mean (SD) mm Cycle (5.8) 6.9 (2.3) 1.7 ( 0.3, 3.7) 0.08 Cycle (5.3) 9.0 (3.0) 2.3 (0.3, 4.3) 0.02 Cycle (6.0) 9.2 (3.0) 2.3 (0.1, 4.5) 0.04 All cycles 13.0 (7.5) 9.9 (3.4) 3.1 ( 0.8, 7.0) 0.02 Follicle size.10 mm during treatment, 19.0 (51.4) 13.0 (33.3) 6.0 ( 3.9, 39.9) 0.11 n per group (%) Follicle size.13 mm during treatment, 12.0 (32.4) 6.0 (15.4) 6.0 ( 1.8, 35.9) 0.08 n per group (%) Follicle diameter was assessed by ultrasound on: Days 1, 21, 24 and 27 of cycle 1; Days 2, 5, 8, 11, 13, 16, 21, 24 and 27 of cycle 2; Days 2, 5, 8, 11, 13, 16 and 21 of cycle 3.

5 1342 Christin-Maitre et al. Figure 2 Mean (SD) diameter of largest follicle in each group during three consecutive treatment cycles. ***P, 0.001, 21-day versus 24-day group. Data are not available for Days 24 or 27 of cycle 3. cycles was 9.7 (3.5) mm in the 24-day group compared with 11.4 (4.2) mm in the 21-day group. Although not statistically significant, the numbers of women in the ITT population with a follicle diameter.10 or.13 mm were lower in the 24-day group than in the 21-day group (Table II). In the PP population, five women in the 24-day group compared with eight women in the 21-day group had a follicle diameter.13 mm. Hormone profiles Mean progesterone levels were 0.20 ng/ml and mean LH levels were 4 miu/ml throughout treatment in both groups (Fig. 3). Mean E 2 plasma levels were significantly higher in the 24-day group than in the 21-day group on Day 24 of cycles 1 and 2 (Fig. 3). In contrast, E 2 levels were lower in the 24-day group than in the 21-day group from the last day of the pill-free period at the end of cycle 2 until Day 21 of cycle 3 (the difference reached statistical significance on Day 27 of cycle 2). Mean FSH plasma levels were significantly lower in the 24-day group than in the 21-day group on Day 24 of cycles 1 and 2 (Fig. 3). Mean LH plasma levels were significantly lower in the 24-day group than in the 21-day group on Day 27 of cycle 1 (Fig. 3). Other outcomes Mean mid-cycle cervical mucus scores decreased during treatment, with no differences between groups (mean [SD] score in cycle 2: 21-day 1.6 [1.6], 24-day 1.2 [1.2]; in cycle 3: 21-day 0.7 [1.1], 24-day 0.9 [1.5]), and then returned to pretreatment levels (mean [SD] score in post-treatment cycle: 21-day 5.2 [3.1], 24-day 5.8 [2.6]). Mean endometrial thickness decreased during treatment, with no differences between groups (mean [SD] thickness in cycle 3: 21-day 3.8 [1.4] mm, 24-day 3.6 [1.5] mm) and then returned to pretreatment levels (mean [SD] thickness in post-treatment cycle: 21-day 6.5 [2.4] mm, 24-day 6.5 [1.9] mm). Return of ovulation during the post-treatment cycle was observed in 52 women (72%), as indicated by a progesterone level of.3 ng/ml measured on or after Day 20 (a second measurement was carried out a few days later if the progesterone level was,3 ng/ml). Changes in mucus score and endometrial thickness, described above, also indicated the return to normal menstrual cycling. Bleeding profile There were no between-group differences in the incidences of intermenstrual bleeding or withdrawal bleeding during each treatment cycle. However, the mean duration of withdrawal bleeding was significantly shorter in the 24-day group than in the 21-day group (Table III). Safety and tolerability outcomes NOMAC/E 2 was generally well tolerated, with no reported serious AEs. Nineteen women had at least one treatment-emergent AE (10 in the 21-day group; 9 in the 24-day group). Thirteen women had at least one treatment-related AE (seven in the 21-day group; six in the 24-day group). The most frequently reported AEs related to treatment were headache, acne, pelvic pain and breast pain. There were no significant differences between the two regimens in the overall incidence of AEs, severity of AEs, their relationship to treatment or overall incidence of any AE. There were no clinically relevant changes in vital signs, physical findings or laboratory parameters in either treatment group. Discussion We report the first published study to compare the follicular growth effects of two different regimens of NOMAC/E 2. There was no ovulation or LUF syndrome with either NOMAC/E 2 regimen, but the 24-day NOMAC/E 2 regimen was associated with a greater inhibition of follicular growth and a shorter duration of withdrawal bleeding than the 21-day regimen. Thus, the shorter pill-free interval appears to result in greater suppression of ovarian activity and therefore a wider margin of contraceptive efficacy. The different effects of each NOMAC/E 2 regimen on suppression of follicular growth may be explained by the impact on gonadotropin levels. We observed that FSH levels were suppressed for longer at the

6 NOMAC/E 2 regimen validation study 1343 Figure 3 Hormone levels in each group during three consecutive treatment cycles: (A) Progesterone, (B) Estradiol (E 2 ), (C) FSH, (D) LH. **P, 0.01 and ***P, 0.001, 21-day versus 24-day group. Data are not available for Days 24 or 27 of cycle 3. end of each cycle with the 24-day regimen than with the 21-day regimen. Furthermore, the FSH peak was lower and delayed, thereby providing a smaller window for follicular maturation to be initiated. LH levels were also suppressed for longer at the end of each cycle with the 24-day regimen than with the 21-day regimen. As with other studies of E 2 -containing pills (Wenzl et al., 1993; Csemiczky et al., 1996), E 2 levels measured here reflect exogenous E 2 from the administered medication on top of residual follicular activity. Thus, E 2 levels remained elevated for longer in each cycle when using the 24-day regimen rather than the 21-day regimen. On Day 24 of treatment cycles 1 and 2, due to the supply of exogenous E 2, the mean E 2 level was significantly higher with the 24-day regimen (last day of active treatment with exogenous E 2 ) than with the 21-day regimen (third day of the pill-free interval), although the antigonadotropic effect was stronger. On Days 2 21 of cycle 3 (data not collected past Day 21), E 2 levels tended to be lower in the 24-day group than the 21-day group, most likely due to a stronger follicular inhibition as a result of the shorter pill-free period in the previous two cycles. E 2 is much less potent than EE in terms of gonadotropin suppression (Mashchak et al., 1982). The results of this study confirm that in combination with a relatively weak estrogen, NOMAC exerts a suppressive effect on follicle development. In support of these findings, NOMAC is able to suppress ovulation, and thus endogenous E 2 secretion, when given alone at doses of mg/day (Bazin et al., 1987; Couzinet et al., 1999). Nevertheless, the measured levels of E 2 during NOMAC/E 2 treatment fall within the physiological range of pg/ml for premenopausal women. Several studies have compared the effects of different EE-containing pill regimens on ovarian function and cycle control (Spona et al., 1996; Sullivan et al., 1999; Nakajima et al., 2007). As in this NOMAC/E 2 study, EE/gestodene (GSD) regimen studies have shown a greater inhibition of follicular growth with a shorter pill-free interval (Spona et al., 1996; Sullivan et al., 1999). The mean follicle diameter during treatment with EE/GSD was below 10 mm in the 24-day group and around 13 mm in the 21-day group, similar to our findings with NOMAC/E 2. Furthermore, FSH levels increased during the pill-free interval but to a lesser extent with 24-day versus 21-day treatment

7 1344 Christin-Maitre et al. Figure 3 (Continued) (Sullivan et al., 1999). However, in contrast to the initial decrease in E 2 levels at the start of the pill-free period seen here, endogenous E 2 levels increased during the pill-free period after administration of EE/GSD although this increase occurred later and was lower with the 24-day versus the 21-day regimen. This difference in E 2 findings can be attributed to the exogenously administered E 2 contained within NOMAC/E 2 but not within EE/GSD. In this study, it is not appropriate to compare the diameter of the largest follicle during the pretreatment cycle with values obtained for this parameter during cycles 2 and 3, as there was only one measurement of follicle diameter in the pretreatment cycle (at Day 20, i.e. in the second part of the cycle, after ovulation). During cycles 2 and 3, ultrasound was performed several times, not only at Day 20 but also in the first part of the cycle, when follicular growth usually occurs. The diameter of the largest follicle during cycles 2 and 3 was the highest value out of all the values obtained throughout cycles 2 and 3 and therefore cannot be compared with the single pretreatment value. The mean diameter of the largest follicle at Day 20 of the pretreatment cycle was smaller in the 24-day group than in the 21-day group but the difference was not statistically significant. It should be noted that the current study was powered to detect a 5 mm difference between treatment groups in mean follicle diameter in cycles 2 and 3, whereas an actual difference of 2.3 mm was observed (P ¼ 0.02 in cycle 2 and P ¼ 0.04 in cycle 3). The sample size calculation was performed using data from a study comparing ovarian activity with 21-day and 24-day regimens of a low-dose pill containing 60 mg GSD/15 mg EE(Sullivan et al., 1999), which results in lower inhibition than NOMAC/E 2. In that study, a 3-day decrease in pill-free interval resulted in a decrease of around 33% in the mean diameter of the largest follicle (from 15 to 10 mm). In our study, the decrease ranged from 20% ( mm in cycle 3) to 26% (from 8.6 to 6.9 mm in cycle 1). When given in a 21-day regimen, NOMAC/E 2 has a stronger inhibitory effect on follicular development than the low-dose combination of GSD/EE. This was confirmed by a study showing similar inhibition with NOMAC/E 2 and drospirenone (DRSP)/EE 30 mg (Duijkers et al., 2010). It is clear that the additional inhibition obtained with a reduction of the

8 NOMAC/E 2 regimen validation study 1345 Table III Bleeding patterns (ITT population). 21-day NOMAC/E 2 24-day NOMAC/E 2 Treatment group difference P-value (n 5 36) (n 5 39) (95% confidence interval)... Total bleeding duration, mean (SD), days 15.5 (5.6) 12.4 (4.9) 3.1 (0.7, 5.5) 0.01 Intermenstrual bleeding incidence, n (%) Cycle 1 7 (19) 10 (26) 3 ( 12.6, 25.0) 0.53 Cycle 2 4 (11) 8 (21) 4 ( 6.6, 26.5) 0.25 Cycle 3 4 (12) 4 (11) 0 ( 13.8, 15.7) 0.9 Intermenstrual bleeding duration, mean (SD) days Cycle (2.8) 0.6 (1.1) 0.5 (0.0, 1.0) 0.32 Cycle (2.5) 0.5 (1.8) 0.2 (0.0, 1.0) 0.74 Cycle (2.8) 0.2 (0.7) 0.5 ( 0.5, 1.5) 0.34 Withdrawal bleeding incidence, n (%) Cycle 1 34 (94) 35 (90) 1 ( 7.4, 16.8) 0.68 Cycle 2 32 (91) 34 (92) 2 ( 12.3, 13.2) 1 Cycle 3 34 (100) 37 (100) 3 (0.0) Withdrawal bleeding duration a, mean (SD) days Cycle (2.6) 3.5 (1.3) 1.5 (0.5, 2.5) Cycle (1.7) 3.9 (1.6) 0.9 (0.1, 1.7) 0.03 a Withdrawal bleeding duration data were not collected after completion of cycle 3 treatment. pill-free interval is a little bit lower with a pill providing stronger inhibition when given in a 21-day regimen. The inhibition of follicular growth observed in the current study compares with that observed in the open-label, randomized, six-cycle study mentioned above comparing NOMAC/E 2 (2.5/1.5 mg in a 24/ 4-day regimen; n ¼ 32) with DRSP/EE (3 mg/30 mg in a 21/7-day regimen; n ¼ 16) (Duijkers et al., 2010). In the NOMAC/E 2 group, the mean maximum follicular diameter decreased from 19.3 mm at baseline to mm during treatment and no subjects had a follicular diameter 15 mm. In the DRSP/EE group, there was a decrease from 19.6 to mm and two subjects had a maximum follicle diameter 15 mm. Ovulation was suppressed in all women. During treatment there was a reduction in FSH levels, suppression of the LH peak and a reduction in mean E 2 concentrations from baseline in both groups; the effect on E 2 was much less pronounced with NOMAC/E 2, due to the exogenous administration of E 2. In this study, follicular development at baseline (during the control cycle) was assessed longitudinally (several measurements during the cycle) and not only on one day, as in the present study. In addition, the suppressive effects of NOMAC/E 2 were at least similar to those of the comparator, a pill containing 30 mg EE. The observed effects of NOMAC/E 2 on follicle growth appear to differ from those reported previously for other E 2 -containing pills. Previous, small-scale ultrasound studies showed complete inhibition of ovulation during 21-day monophasic treatment with E 2 -containing pills. However, some residual ovarian activity remained as illustrated by follicles 15 mm diameter in 10/29 women (Csemiczky et al., 1996) and unruptured follicles of mm diameter in 3/20 women (Wenzl et al., 1993). In a large study examining the effects of E 2 V/dienogest (DNG) on follicle growth during three cycles of 26-day treatment, ultrasound measurements showed a 3% incidence of ovulation or LUF syndrome in the group receiving the selected fourphasic dynamic dose regimen (E 2 V 1 3 mg, DNG 2 3 mg) (Endrikat et al., 2008). A lower incidence of 1% was seen with the pill containing DNG 3 4 mg, but this dose regimen was not selected for further clinical trial evaluation. Previous studies of E 2 -containing pills have reported high discontinuation rates of 10 40% due to bleeding irregularities (Astedt et al., 1979; Serup et al., 1981; Wenzl et al., 1993; Hirvonen et al., 1995), with breakthrough bleeding experienced by approximately one-third of women or in one-third of all cycles (Hirvonen et al., 1995; Csemiczky et al., 1996) and absence of withdrawal bleeding in 19% of cycles or 30% of women (Wenzl et al., 1993; Csemiczky et al., 1996). The E 2 V/DNG four-phasic dynamic dosing regimen was developed to overcome the unacceptable bleeding profiles seen with earlier E 2 pills. When compared with EE/levonorgestrel, the use of E 2 V/ DNG was associated with less bleeding overall, similar incidences of breakthrough bleeding and shorter withdrawal bleeding duration (Ahrendt et al., 2009). The bleeding profile seen here with the 24-day monophasic regimen of NOMAC/E 2 appears similar to that seen with the 26-day four-phasic dosing regimen of E 2 V/DNG (Ahrendt et al., 2009). However, it must be noted that the E 2 V/ DNG study was conducted over seven treatment cycles in women aged up to 50 years; so, the results are not directly comparable with the findings seen here. A comparison of 24-day and 21-day regimens of EE/norethindrone acetate showed a reduction in total, withdrawal and intracyclic bleeding with the shorter pill-free period; the total bleeding duration over the initial three treatment cycles was 15 days in the 24-day group and 18 days in the 21-day group (Nakajima et al., 2007). This compares well with the bleeding duration results reported here, which indicate shorter intermenstrual and withdrawal bleeding durations with the 24-day NOMAC/E 2 regimen than with the 21-day regimen (total bleeding: 12 days in the 24-day group and 15 days in the 21-day group). In the current study, 10% of

9 1346 Christin-Maitre et al. women experienced amenorrhea (no withdrawal bleeding) in cycles 1 and 2; in cycle 3 the incidence of amenorrhea was 0%. This compares favorably with the reported incidence in trials of other E 2 -containing regimens. With E 2 /desogestrel, observed rates of amenorrhea were 19% of cycles (Csemiczky et al., 1996) and 30% of women over two cycles (Wenzl et al., 1993). For E 2 /norethisterone acetate, the observed rate was 14% in cycles 1 3 (Serup et al., 1981). With EE/ GSD, amenorrhea rates were 0% over three cycles (Spona et al., 1996), 11% in cycle 3 of a 21-day regimen and 3% in cycle 3 of a 24-day regimen (Sullivan et al., 1999), and with EE/norethisterone acetate the observed rate was 3% in cycles 1 3 (Serup et al., 1981). Effects on bleeding profile were not reported for the study comparing NOMAC/E 2 and DRSP/EE (Duijkers et al., 2010). Both NOMAC/E 2 treatment regimens altered cervical mucus and decreased endometrial thickness changes that were reversed quickly after treatment was stopped. Rapid return of ovulation, based on progesterone levels.3 ng/ml on or shortly after Day 20 of the posttreatment cycle, was observed in 72% of women. Similar outcomes for cervical mucus, endometrial thickness and return of ovulation were observed in the study comparing NOMAC/E 2 and DRSP/EE (Duijkers et al., 2010). In the current study, it is possible that the remaining women ovulated later or in the subsequent cycle, but the progesterone sampling and post-treatment follow-up did not continue long enough to confirm this hypothesis. Rapid return of ovulation (within one cycle) has been observed in almost all women taking an E 2- -containing pill for two cycles (Wenzl et al., 1993) and in 88% women taking EE/GSD for three cycles (Sullivan et al., 1999), although the time to onset of ovulation varied after the 24-day EE/GSD regimen. NOMAC/E 2 was well tolerated, with no serious AEs or withdrawals due to AEs. This compares with results reported for E 2 V/ DNG, where 2 5% of women withdrew from treatment due to AEs, and two serious AEs of ovarian cyst (one per study; each judged possibly related to treatment) occurred during three to seven cycles of treatment (Endrikat et al., 2008; Ahrendt et al., 2009). The incidences of the most common AEs reported here with NOMAC/E 2 (headache, acne, pelvic pain and breast pain) are generally similar to those reported for E 2 /cyproterone acetate (Hirvonen et al., 1995), E 2 V/DNG (Endrikat et al., 2008; Ahrendt et al., 2009) and EE-containing pills (Spona et al., 1996; Nakajima et al., 2007). Although the sample number was small, this comparative, doubleblind study used frequent ultrasound assessment and blood sampling to measure ovarian activity. Ideally, blood sampling would have been performed several times after NOMAC/E 2 treatment was stopped in cycle 3, but this would have meant taking more than 22 samples and 400 ml of blood. Nevertheless, this study showed that the 24-day NOMAC/E 2 regimen was more effective than the 21-day NOMAC/E 2 regimen at suppressing ovarian function and inhibiting follicular growth. Furthermore, the 24-day regimen was associated with a shorter bleeding duration than the 21-day regimen. The robust ovulation inhibition and good cycle control reported here should give clinicians confidence in the contraceptive efficacy and tolerability of the 24-day NOMAC/E 2 pill regimen. Authors roles J.-L.T. contributed to the study conception and design. S.C.-M., D.S. and D.C. were involved in data acquisition. N.C.-B., E.O. and J.-L.T. were involved in data analysis and interpretation. 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