A tumor cell specific Zona Pellucida glycoprotein 3 RNA transcript encodes an intracellular cancer antigen.

Background: Expression of Zona Pellucida glycoprotein 3 (ZP3) in healthy tissue is restricted to the extracellular Zona Pellucida layer surrounding oocytes of ovarian follicles and to specific cells of the spermatogenic lineage. Ectopic expression of ZP3 has been observed in various types of cancer, rendering it a possible therapeutic target. Methods: To support its validity as therapeutic target, we extended the cancer related data by investigating ZP3 expression using immunohistochemistry (IHC) of tumor biopsies. We performed a ZP3 transcript specific analysis of publicly available RNA-sequencing (RNA-seq) data of cancer cell lines (CCLs) and tumor and normal tissues, and validated expression data by independent computational analysis and real-time quantitative PCR (qPCR). A correlation between the ZP3 expression level and pathological and clinical parameters was also investigated. Results: IHC data for several cancer types showed abundant ZP3 protein staining, which was confined to the cytoplasm, contradicting the extracellular protein localization in oocytes. We noticed that an alternative ZP3 RNA transcript, which we term 'ZP3-Cancer', was annotated in gene databases that lacks the genetic information encoding the N-terminal signal peptide that governs entry into the secretory pathway. This explains the intracellular localization of ZP3 in tumor cells. Analysis of publicly available RNA-seq data of 1339 cancer cell lines (CCLs), 10386 tumor tissues (The Cancer Genome Atlas) and 7481 healthy tissues (Genotype-Tissue Expression) indicated that ZP3-Cancer is the dominant ZP3 RNA transcript in tumor cells and is highly enriched in many cancer types, particularly in rectal, ovarian, colorectal, prostate, lung and breast cancer. Expression of ZP3-Cancer in tumor cells was confirmed by qPCR. Higher levels of the ZP3-Cancer transcript were associated with more aggressive tumors and worse survival of patients with various types of cancer. Conclusion: The cancer-restricted expression of ZP3-Cancer renders it an attractive tumor antigen for the development of a therapeutic cancer vaccine, particularly using mRNA expression technologies.

Estetrol Prevents Hot Flushes and Improves Quality of Life in Patients with Advanced Prostate Cancer Treated with Androgen Deprivation Therapy: The PCombi Study.

Background: Androgen deprivation therapy (ADT) for prostate cancer (PCa) is accompanied by side effects affecting health-related quality of life (HRQL). Objective: To assess the effects of the fetal estrogen estetrol (E4) on symptoms related to estrogen and androgen deficiency, and on HRQL measured using the validated Functional Assessment of Cancer Therapy-Prostate (FACT-P) questionnaire. Design setting and participants: This was a phase 2, double-blind, randomized, placebo-controlled study in patients with advanced PCa. Intervention: Patients receiving ADT were randomly assigned at a 2:1 ratio to daily treatment with a high dose of E4 (HDE4; n = 41) or placebo (n = 21) for 24 wk. Outcome measurements and statistical analysis: The primary outcome was the effect of HDE4 cotreatment on hot flushes (HFs). Secondary outcomes were the Q-Man questionnaire for evaluation of the effect on estrogen and androgen deficiency symptoms, and the FACT-P questionnaire for evaluating HRQL. Results and limitations: At 24 wk, the number of patients experiencing HFs was significantly lower in the HDE4 group than in the placebo group (14.3% vs 60.0%; p < 0.001). HDE4 treatment was associated with lower incidence of night sweats, arthralgia, and fatigue, but more nipple tenderness and gynecomastia. At 24 wk, the mean HRQL score favored HDE4 over placebo for the FACT-P total score (122.2 ± 12.3 vs 118.7 ± 19.7) and for several other FACT subscales. Conclusions: Daily HDE4 coadministration almost completely prevented HFs in patients with advanced PCa treated with ADT. HDE4 also had positive effects on HRQL and counteracted other estrogen deficiency symptoms caused by ADT. These data support the dual efficacy concept of ADT and HDE4 to improve HRQL and increase the antitumor effect of ADT. Patient summary: For patients on androgen deprivation therapy for advanced prostate cancer, cotreatment with a high dose of estetrol almost completely prevents the occurrence of hot flushes and improves quality of life and well-being, but nipple sensitivity and an increase in breast size may occur.

Maintaining bone health by estrogen therapy in patients with advanced prostate cancer: a narrative review.

The purpose of androgen deprivation therapy (ADT) in prostate cancer (PCa), using luteinizing hormone-releasing hormone agonists (LHRHa) or gonadotrophin-releasing hormone antagonists, is to suppress the levels of testosterone. Since testosterone is the precursor of estradiol (E2), one of the major undesired effects of ADT is the concomitant loss of E2, causing among others an increased bone turnover and bone loss and an increased risk of osteoporosis and fractures. Therefore, the guidelines for ADT indicate to combine ADT routinely with bone-sparing agents such as bisphosphonates, denosumab or selective estrogen receptor modulators. However, these compounds may have side effects and some require inconvenient parenteral administration. Co-treatment with estrogens is an alternative approach to prevent bone loss and at the same time, to avoid other side effects caused by the loss of estrogens, which is the topic explored in the present narrative review. Estrogens investigated in PCa patients include parenteral or transdermal E2, diethylstilbestrol (DES), and ethinylestradiol (EE) as monotherapy, or high-dose estetrol (HDE4) combined with ADT. Cardiovascular adverse events have been reported with parenteral E2, DES and EE. Encouraging effects on bone parameters have been obtained with transdermal E2 (tE2) and HDE4, in the tE2 development program (PATCH study), and in the LHRHa/HDE4 co-treatment study (PCombi), respectively. Confirmation of the beneficial effects of estrogen therapy with tE2 or HDE4 on bone health in patients with advanced PCa is needed, with special emphasis on bone mass and fracture rate.

Estetrol Cotreatment of Androgen Deprivation Therapy in Infiltrating or Metastatic, Castration-sensitive Prostate Cancer: A Randomized, Double-blind, Phase II Trial (PCombi).

BACKGROUND: Androgen deprivation therapy (ADT) for prostate cancer with luteinizing hormone-releasing hormone (LHRH) agonists can be improved. OBJECTIVE: To assess safety, the frequency and severity of hot flushes (HFs), bone health, and antitumor effects of high-dose estetrol (HDE4) when combined with ADT. DESIGN SETTING AND PARTICIPANTS: A phase II, double-blind, randomized, placebo-controlled study was conducted in advanced prostate cancer patients requiring ADT (the PCombi study). INTERVENTION: Patients receiving LHRH agonist treatment were randomized 2:1 to 40 mg HDE4 (n = 41) or placebo (n = 21) cotreatment for 24 wk. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS: Coprimary endpoints were frequency/severity of HFs and levels of total and free testosterone (T). Secondary endpoints included assessments of bone metabolism (osteocalcin and type I collagen telopeptide [CTX1]), prostate-specific antigen (PSA), and follicle-stimulating hormone (FSH). Efficacy analysis was based on the selected per-protocol (PP) population. RESULTS AND LIMITATIONS: Of 62 patients included in the study, 57 were suitable for a PP analysis (37 HDE4; 20 placebo). No E4-related serious cardiovascular adverse events occurred at 24 wk. Weekly HFs were reported by 13.5% of patients with HDE4 and 60.0% with placebo (p < 0.001). Daily HFs occurred in 5.9% versus 55%. Bone turnover parameters decreased significantly with HDE4 (p < 0.0001). Total and free T decreased earlier (p < 0.05), and free T was suppressed further (p < 0.05). PSA suppression was more profound and earlier (p < 0.005). FSH levels were suppressed by 98% versus 57% (p < 0.0001). Estrogenic side effects were nipple sensitivity (34%) and gynecomastia (17%). CONCLUSIONS: HDE4 cotreatment of ADT patients with advanced prostate cancer was well tolerated, and no treatment-related cardiovascular adverse events were observed at 24 wk. HFs and bone turnover were substantially reduced. Suppression of free T, PSA, and FSH was more rapid and profound, suggesting enhanced disease control by HDE4 cotreatment. Larger and longer-lasting studies are needed to confirm the results of the study reported here. PATIENT SUMMARY: Cotreatment of androgen deprivation therapy with high-dose estetrol in advanced prostate cancer patients results in fewer occurrences of hot flushes, bone protection, and other antitumor benefits. Nipple sensitivity and gynecomastia may occur as side effects.

Tumor suppression, dose-limiting toxicity and wellbeing with the fetal estrogen estetrol in patients with advanced breast cancer.

PURPOSE: The aim of this study (the ABCE4 study) was to assess dose-limiting toxicity (DLT), safety, tolerability and preliminary efficacy of high doses of the fetal estrogen estetrol (E4) in postmenopausal patients with heavily pretreated, locally advanced and/or metastatic ER+/HER2-breast cancer, resistant to anti-estrogens. METHODS: This was a multicenter, open-label, phase IB/IIA, dose-escalation study with a 3 + 3 cohort design, whereby successive cohorts of three patients received 20 mg, 40 mg or 60 mg E4 per day for 12 weeks by oral administration. DLTs, safety and wellbeing were evaluated after 4, 8 and 12 weeks of treatment. Anti-tumor effects were investigated by computer tomography scanning and evaluated according to RECIST criteria before and after 12 weeks of treatment. Wellbeing was judged weekly by the investigator and by quality-of-life questionnaires by the patients. In view of the small number of patients, no statistical testing was performed. RESULTS: All 12 patients enrolled had progressive, heavily pre-treated advanced breast cancer. No treatment-related serious adverse events or DLTs occurred during the first 4 weeks of E4 treatment allowing the investigation of all three doses. Five of nine patients completing 12 weeks of E4 treatment showed objective anti-tumor effects and six of nine patients reported improved wellbeing. CONCLUSION: High doses of estetrol seem to be safe and are well tolerated during 12 weeks of treatment without dose-limiting toxicity and with anti-tumor effects in five of nine heavily treated patients with progressive, anti-estrogen resistant, advanced breast cancer.

A Dose-Escalating Study With the Fetal Estrogen Estetrol in Healthy Men.

Context: Luteinizing hormone-releasing hormone (LHRH) agonists have replaced estrogens for endocrine treatment of advanced prostate cancer (PC) because of cardiovascular side effects. The fetal estrogen estetrol (E4) may be safer for PC treatment and is expected to decrease testosterone (T) and prevent estrogen deficiency. Objective: To investigate the safety and T-suppressive effect of E4 in healthy men. Design: Double-blind, randomized, placebo-controlled, dose-escalating study. Setting: The study was conducted at a phase I clinical unit (QPS, Netherlands). Participants: Healthy male volunteers aged 40 to 70 years. Intervention(s): Three treatment cohorts of 15 volunteers with placebo (n = 5) and E4 (n = 10). Estetrol doses tested were 20, 40, and 60 mg/d. Subjects were treated for 4 weeks. Main Outcome Measures: Subjective side effects, pharmacodynamic effects on hemostatic variables, lipids, glucose, bone parameters, and endocrine parameters related to T metabolism. Results: Total and free T decreased dose-dependently and significantly. Nipple tenderness occurred in 40% and decrease of libido occurred in 30% of E4-treated men. The unwanted estrogenic effects on hemostasis were small, dose dependent, and in some cases significant. Lipid and bone parameters showed a favorable trend. Conclusion: The effect of E4 on testosterone levels is insufficient for standalone PC treatment. Taking all clinical and pharmacodynamic variables into consideration, a daily dose of 40 mg E4 seems safe for further evaluation of endocrine PC treatment in combination with LHRH analogs.

Maintaining physiologic testosterone levels during combined oral contraceptives by adding dehydroepiandrosterone: II. Effects on sexual function. A phase II randomized, double-blind, placebo-controlled study.

OBJECTIVE: The objective was to evaluate the effect of combined oral contraceptives (OCs) on sexual function, either alone or together with dehydroepiandrosterone (DHEA). STUDY DESIGN: An exploratory randomized, double-blind, placebo-controlled, comparative, crossover study was conducted in 81 OC users. Subjects discontinued their OC for one cycle before being randomized for 10cycles to a 30-mcg ethinyl estradiol (EE)/levonorgestrel (LNG) OC or a 30-mcg EE/drospirenone (DRSP) OC, along with daily use of 50mg dehydroepiandrosterone (DHEA) or placebo during five OC cycles before crossing over from DHEA to placebo or the reverse for another fivecycles. First, the effect on sexual function of five OC cycles + placebo was compared to baseline. Then, the effect of five OC cycles + DHEA was compared to the OC+placebo. Results regarding endocrine changes have been published separately. Primary efficacy outcomes of the current study were genital response (measured by vaginal pulse amplitude [VPA]) and sexual feelings (measured by the subjective self-assessment questionnaire [SSAQ]) to self-induced erotic fantasy and visual sexual stimuli in a laboratory setting and measures of desire and arousability using a sexual function diary (SFD). Secondary efficacy outcomes were the Female Sexual Function Index (FSFI) and the Female Sexual Distress Scale Revised. RESULTS: Eighty-one women were enrolled, and 74 women completed the study. Five cycles of OC+placebo resulted in a significant decline compared to baseline of four out of six SFD self-ratings of sexual desire and arousability with both OCs. The LNG OC also resulted in significant declines in the FSFI scores (baseline vs. LNG OC+placebo: total score, 28.7±3.7 vs. 25.6±7.4; arousal, 5.0±0.7 vs. 4.5±1.4; lubrication, 5.2±0.9 vs. 4.6±1.7; pain, 4.9±0.9 vs. 4.5±1.4), but no changes were observed using the DRSP OC. In the laboratory setting, five cycles of OC+DHEA showed no significant differences with placebo except for a significant increase in genital sensations (SSAQ) during erotic fantasy (OC+placebo vs. OC+DHEA: 3.3±1.4 vs. 3.6±1.5; p<.05). No significant changes were observed for genital response (VPA) and the other two variables of the SSAQ assessed after visual erotic stimulus exposure. Using the SFD, 5 out of 10 variables showed a significant improvement with DHEA. Partner's initiative was rejected less often with OC+DHEA compared to placebo (OC+placebo vs. OC+DHEA: 1.1±1.5 vs. 0.8±1.0; p<.05). Women with free testosterone levels in the upper quartile during DHEA co-administration showed significantly better effects on sexual arousal and desire compared to the three lower quartiles (lower vs. upper quartiles: sexual arousability: 25.0±19.8 vs. 41.2±29.0; sexual desire: 5.6±3.7 vs. 9.6±8.0; desire for sex with partner: 4.9±3.1 vs. 8.6±7.4; number of sex fantasies: 3.0±3.2 vs. 5.5±4.4; all p<.05). CONCLUSIONS: In this exploratory study, OC use was associated with decreases in some measures of sexual functioning, whereas others remained unchanged. Maintaining or restoring physiological testosterone concentrations by the co-administration of DHEA to the OC may prevent these effects on sexuality, particularly in women with relatively high but physiologic levels of free testosterone during DHEA co-administration. IMPLICATIONS: The results of this exploratory study warrant further testing of the hypothesis that restoration and/or preservation of physiologic testosterone levels during OC use by co-administration of DHEA has favorable effects on those aspects of sexual function compromised by OCs.

Estetrol combined with drospirenone: an oral contraceptive with high acceptability, user satisfaction, well-being and favourable body weight control.

OBJECTIVES: This study evaluated acceptability, user satisfaction, body weight control and general well-being of estetrol (E4) combined with either drospirenone (DRSP) or levonorgestrel (LNG). METHODS: In this open-label, multi-centre, dose-finding, 6-cycle study, 396 healthy women of reproductive age were randomised into five treatment groups in a 24/4-day regimen: 15 mg or 20 mg E4 combined with either 3 mg DRSP or 150 µg LNG, and as reference estradiol valerate (E2V) combined with dienogest (DNG). Data on acceptability, user well-being, satisfaction and body weight were collected. RESULTS: The number of completers was the highest in the 15 mg E4/DRSP group (91.1%), and the lowest for 20 mg E4/LNG (70.1%). The largest proportion of treatment satisfaction was reported for 15 mg E4/DRSP (73.1%), and the lowest for 15 mg E4/LNG (50.6%). The number of women willing to continue with the assigned study treatment was the highest in the 15 mg E4/DRSP group (82.1%) and the lowest for 20 mg E4/LNG (58.3%). Well-being with E4/DRSP combinations was statistically significantly better than with E4/LNG combinations: OR (95% CI) 2.00 (1.13; 3.53) and 1.93 (1.06; 3.56) for 15 and 20 mg E4, respectively, and comparable to E2V/DNG. Proportion of women with a 2 kg or more weight loss after 3 and 6 cycles was the highest in the 15 mg E4/DRSP group (30.7 and 36.7%, respectively). CONCLUSIONS: The present study shows that 15 mg estetrol combined with 3 mg DRSP is associated with a high-user acceptability and satisfaction, and with a favourable body weight control.

Mood disturbances during combined oral contraceptive use and the effect of androgen supplementation. Results of a double-blind, placebo-controlled, single-case alternation design pilot study.

OBJECTIVES: To evaluate the effect of androgen supplementation in healthy combined oral contraceptive (COC) users who experience mood disturbances during COC-use only. METHODS: Six women with mood disturbances during COC-use only, received COC with co-treatment of 50 mg dehydroepiandrosterone (DHEA) during three cycles and placebo during another three cycles in an individualized random order. Daily mood rating was measured by a single item: 'In what kind of mood have you been in the past 24 h?' The results were analysed using a randomisation test for single-case experimental designs. RESULTS: The p values for the alternation design randomisation tests on the raw data of the six healthy individuals varied between 0.21 and 1, indicating that the average daily mood ratings of the active treatment DHEA are not statistically significantly larger than the average daily mood ratings of placebo. The combined p value of the subjects using a DRSP-containing pill was 0.97, and of the subjects using an LNG-containing pill was 0.65, indicating no statistically significant treatment effect for any of the pill types. CONCLUSIONS: In this single-case alternation design study, concomitant treatment with DHEA for intermittent periods of 4 weeks did not result in improvement of mood disturbances related to COC-use, but had also no side-effects.

Pharmacodynamic effects of the fetal estrogen estetrol in postmenopausal women: results from a multiple-rising-dose study.

OBJECTIVE: Estetrol (E4) is an estrogen produced exclusively by the human fetal liver during pregnancy. In this study the pharmacodynamic effects of escalating doses of E4 in postmenopausal women were investigated. METHODS: This was a partly randomized, open-label, multiple-rising-dose study in 49 postmenopausal women. Participants were randomized to receive either 2 mg E4 or 2 mg estradiol-valerate (E2 V) for 28 days. Subsequent dose-escalation groups were (non-randomized): 10, 20 and 40 mg E4. Blood samples were collected regularly for measuring endocrine and hemostasis variables, lipids and lipoproteins, fasting glucose and bone turnover markers. RESULTS: Estetrol treatment resulted in a decrease of follicle-stimulating hormone and luteinizing hormone and an increase of sex-hormone binding globulin. Changes in hemostasis variables were small. A lowering effect on low-density lipoprotein cholesterol was accompanied with an increase in high-density lipoprotein cholesterol and no or minimal changes in triglycerides. The considerable decrease in osteocalcin levels in the three highest E4 dose groups and the small decrease in C-telopeptide levels were comparable to the E2 V control group and suggest a preventive effect on bone loss. All changes observed were dose-dependent. CONCLUSIONS: In this study, estetrol treatment showed dose-dependent estrogenic effects on endocrine parameters, bone turnover markers, and lipids and lipoproteins. The effect on triglycerides was small as were the effects on hemostatic variables. These results support the further investigation of estetrol as a candidate for hormone therapy. Quantitatively, the effects of 10 mg estetrol were similar to the study comparator 2 mg estradiol valerate.

Maintaining physiological testosterone levels by adding dehydroepiandrosterone to combined oral contraceptives: I. Endocrine effects.

OBJECTIVE: To determine whether adding dehydroepiandrosterone to combined oral contraceptives (COCs) maintains physiological levels of free testosterone. STUDY DESIGN: A randomized, double-blind, placebo-controlled, two-way crossover study conducted in 81 healthy women (age range: 20-35 years; Body mass index (BMI) range: 18-35 kg/m2) using oral contraceptives. Androgens, sex hormone-binding globulin (SHBG), estradiol (E2) and estrone (E1) were measured, and free testosterone and the free testosterone index were calculated. Subjects discontinued oral contraceptive use for at least one menstrual cycle before being randomized to receive five cycles of ethinyl estradiol (EE) combined with either levonorgestrel (EE/LNG group) or drospirenone (EE/DRSP group) together with either dehydroepiandrosterone (DHEA) (50 mg/day orally) or placebo. Subsequently, all subjects crossed over to the other treatment arm for an additional five cycles. RESULTS: Both COCs decreased the levels of all androgens measured. Significant decreases (p<.05) were found with EE/LNG and EE/DRSP for total testosterone (54.5% and 11.3%, respectively) and for free testosterone (66.8% and 75.6%, respectively). Adding DHEA to the COCs significantly increased all androgens compared to placebo. Moreover, including DHEA restored free testosterone levels to baseline values in both COC groups and total testosterone levels to baseline in the EE/LNG group and above baseline in the EE/DRSP group. SHBG concentrations were significantly higher with EE/DRSP compared to EE/LNG (p<.0001). The addition of DHEA did not affect the levels of SHBG. CONCLUSIONS: Taking COCs reduces total and free testosterone levels and increases SHBG concentrations. By coadministration with DHEA, physiological levels of total and free testosterone are restored while using EE/LNG. With EE/DRSP, only the free testosterone level is normalized by DHEA coadministration. IMPLICATIONS: A daily oral dose of 50-mg DHEA maintains physiological free and total testosterone levels in women who are using an EE/LNG-containing COC.

Reduced hemostatic effects with drospirenone-based oral contraceptives containing estetrol vs. ethinyl estradiol.

OBJECTIVE: The effects of estetrol (E4), a natural fetal estrogen, combined with drospirenone (DRSP) were evaluated on plasma levels of sex hormone-binding globulin (SHBG), angiotensinogen and 12 hemostasis markers. STUDY DESIGN: Combinations of 3 mg DRSP with 5 or 10 mg E4 were compared with YAZ® (20 mcg ethinyl estradiol and 3 mg DRSP; EE/DRSP) in parallel groups of 15-18 healthy young women. Main outcome was the relative change from pretreatment to the end (day 24±1) of the third treatment cycle. RESULTS: All E4 combinations showed low estrogen impact compared to EE/DRSP. Effects on SHBG and angiotensinogen of 10 mg E4 combined with DRSP were 15%-20% that of EE/DRSP. Both E4/DRSP combinations reduced D-dimer level and the 5 mg E4/DRSP combination also decreased fragment 1+2. CONCLUSIONS: The reduction in coagulation markers suggests an anticoagulant effect from DRSP. The indications of a low thrombosis risk for E4 preparations should be validated in larger studies. IMPLICATION STATEMENT.

Clinical effects of the fetal estrogen estetrol in a multiple-rising-dose study in postmenopausal women.

OBJECTIVE: Estetrol (E4) is a natural fetal estrogen. The safety of increasing doses of E4 and its preliminary effects on the vagina, endometrium and menopausal vasomotor symptoms were investigated. STUDY DESIGN: This was a partly randomized, open-label, multiple-rising-dose study in 49 postmenopausal women. Subjects with an intact uterus were randomized to receive either 2mg E4 or 2mg estradiol-valerate (E2V) for 28days. Subsequent dose-escalation groups (non-randomized) were: 10mg E4 (intact uterus and ≥35 hot flushes/week); and 20mg and 40mg E4 (hysterectomized subjects). MAIN OUTCOME MEASURE: Adverse events (AEs) and vaginal cytology were evaluated in all treatment groups; hot flushes/sweating and endometrial proliferation were analyzed with 2 and 10mg E4 and 2mg E2V. RESULTS: Estetrol appeared to be safe, without serious drug-related AEs. In all the groups there was a clear shift from parabasal to superficial vaginal cells, indicating an estrogenic effect and a potential for the treatment of vulvovaginal atrophy. The endometrial thickness remained stable in the 2mg E4 group and increased with E2V and 10mg E4. A decrease in the mean number of hot flushes and sweating was seen with 2 and 10mg E4 and 2mg E2V. CONCLUSIONS: Estetrol in a dose range of 2-40mg per day improved vaginal cytology and vasomotor symptoms in postmenopausal women. Endometrial proliferation occurred with the 10mg dose. Estetrol seems a safe and suitable candidate to develop further for hormone therapy.

Bleeding pattern and cycle control with estetrol-containing combined oral contraceptives: results from a phase II, randomised, dose-finding study (FIESTA).

OBJECTIVES: This study aims to assess vaginal bleeding patterns and cycle control of oral contraceptives containing estetrol (E4) combined with either drospirenone (DRSP) or levonorgestrel (LNG). STUDY DESIGN: An open-label, multicentre, randomised, dose-finding study lasting six cycles in healthy women aged 18-35 years was used. Four treatments (15 mg or 20 mg E4, combined with either 3 mg DRSP or 150 mcg LNG) were administered in a 24/4-day regimen. A marketed dosing regimen of estradiol valerate with dienogest (E2V/DNG) served as reference since it contains (like E4) a natural oestrogen. RESULTS: A total of 396 women were randomised, of whom 389 received study medication, and 316 completed the study. By cycle 6, the frequencies of unscheduled bleeding and/or spotting and absence of withdrawal bleeding were the lowest in the 15 mg E4/DRSP group (33.8% and 3.5%, respectively). In the E2V/DNG reference group, these frequencies were 47.8% and 27.1%, respectively. By cycle 6, the frequency of women with absence of withdrawal bleeding was <20% for all E4 treatment groups: 3.5-3.8% combined with DRSP and 14.0-18.5% combined with LNG. By cycle 6, unscheduled intracyclic bleeding was reported by <20% of women in the 20 mg E4/LNG group (18.9%) and in the 15 mg E4/DRSP group (16.9%). CONCLUSION: This study showed that, of the four treatment modalities investigated, the 15 mg E4/DRSP combination has the most favourable bleeding pattern and cycle control. IMPLICATIONS: Due to its favourable bleeding pattern and cycle control, the 15 mg E4/DRSP combination is the preferred combination for further phase III clinical development.

Inhibition of ovulation by administration of estetrol in combination with drospirenone or levonorgestrel: Results of a phase II dose-finding pilot study.

OBJECTIVES: The aim of the study was to evaluate the efficacy of different dosages of estetrol (E4) combined with one of two progestins in suppressing the pituitary-ovarian axis and ovulation in healthy premenopausal women. METHODS: This was an open, parallel, phase II, dose-finding, pilot study performed in healthy women aged 18 to 35 years with a documented ovulatory cycle before treatment. For three consecutive cycles in a 24/4-day regimen, participants received 5 mg or 10 mg E4/3 mg drospirenone (DRSP); 5 mg, 10 mg or 20 mg E4/150 µg levonorgestrel; or 20 µg ethinylestradiol (EE)/3 mg DRSP as comparator. Pituitary-ovarian axis activity and the occurrence of ovulation were evaluated by monitoring follicular size, serum levels of follicle-stimulating hormone, luteinising hormone, estradiol and progesterone during treatment cycles 1 and 3. Endometrial thickness was evaluated throughout the trial, and the return of ovulation was evaluated after the last intake of medication. RESULTS: A total of 109 women were included in the trial. No ovulation occurred in any treatment group. Ovarian activity inhibition seemed proportional to the E4 dosage: the highest suppression was observed in the 20 mg E4 group and was very similar to that observed with EE/DRSP. Endometrial thickness was suppressed to the same extent in all groups. Post-treatment ovulation occurred in all participants between 17 and 21 days after the last active treatment. The study combinations were well tolerated and safe. CONCLUSIONS: Combined with a progestin, E4 adequately suppresses ovarian activity, particularly when given at a dosage above 10 mg/day.

Unique effects on hepatic function, lipid metabolism, bone and growth endocrine parameters of estetrol in combined oral contraceptives.

OBJECTIVES: Estetrol (E4) is a natural estrogen produced by the human fetal liver. In combination with drospirenone (DRSP) or levonorgestrel (LNG), E4 blocks ovulation and has less effect on haemostatic biomarkers in comparison with ethinylestradiol (EE) combined with DRSP. This study evaluates the impact of several doses of E4/DRSP and E4/LNG on safety parameters such as liver function, lipid metabolism, bone markers and growth endocrine parameters. METHODS: This was a dose-finding, single-centre, controlled study performed in healthy women aged 18 to 35 years with a documented pretreatment ovulatory cycle. Participants received 5 mg or 10 mg E4/3 mg DRSP; 5 mg, 10 mg or 20 mg E4/150 µg LNG; or 20 µg EE/3 mg DRSP as a comparator for three consecutive cycles in a 24/4-day regimen. Changes from baseline to end of treatment in liver parameters, lipid metabolism, bone markers and growth endocrinology were evaluated. RESULTS: A total of 109 women were included in the study. Carrier proteins were minimally affected in the E4/DRSP and E4/LNG groups, in comparison with the EE/DRSP group, where a significant increase in sex hormone-binding globulin was observed. Similarly, minor effects on lipoproteins were observed in the E4 groups, and the effects on triglycerides elicited by the E4 groups were significantly lower than those in the EE/DRSP group. No imbalances in bone markers were observed in any groups. No alterations in insulin-like growth factor were observed in the E4 groups. CONCLUSIONS: E4-containing combinations have a limited effect on liver function, lipid metabolism, and bone and growth endocrine parameters.

Peculiar observations in measuring testosterone in women treated with oral contraceptives supplemented with dehydroepiandrosterone (DHEA).

Total testosterone is considered to be decreased during the use of combined oral contraceptives. There is, however, considerable concern about the quality of testosterone assays, especially at low levels. We aimed to confirm testosterone levels measured by direct radioimmunoassay in a recent clinical trial with a state-of-the-art LC-MSMS method. Surplus specimens with known testosterone levels collected during the study (Clinical Trial Registration number ISRCTN06414473) were reanalyzed with an LC-MSMS method. This method was compared to another LC-MSMS method that had shown to concur excellently to a reference method. Follow-up experiments were designed to explain the results. In contrast to our expectation, LC-MSMS measurements did not corroborate the data obtained by radioimmunoassay. Subsequent experiments showed that this could be attributed to a strong dependency of the radioimmunoassay on SHBG. Testosterone results (n = 198) obtained by direct radioimmunoassay showed a negative correlation to SHBG levels (r = -0.676; p<0.001). By contrast, testosterone results obtained by LC-MSMS were not related to SHBG (r = 0.100; NS). In conclusion, our results indicate that total testosterone measurements during oral contraceptive use are unreliable when performed with assays sensitive to the SHBG concentration. The discrepancy with the literature can most likely be explained by the sensitivity of the immunoassay used to SHBG. Given the sharp increase in SHBG during the use of many oral contraceptives, total testosterone may not decrease, whereas its bioavailability, estimated by free testosterone levels, will be diminished. Studies aiming at restoration of testosterone homeostasis during oral contraception need to take this into account.

The pharmacokinetics and pharmacodynamics of dehydroepiandrosterone during use of an ethinylestradiol- and drospirenone-containing oral contraceptive.

BACKGROUND: Combined oral contraceptives (COCs) reduce the levels of ovarian and adrenal androgens. Co-administration of dehydroepiandrosterone (DHEA) may normalise androgen levels during COC use. OBJECTIVE: To investigate the effect of the addition of DHEA to a COC on the pharmacokinetics (PK) and pharmacodynamics (PD) of DHEA and its sulphate (DHEA-S), and on levels of total and free testosterone (T). METHODS: In a prospective, randomised, double-blind, placebo-controlled, cross-over study involving 21 female volunteers, the PK and PD of DHEA and DHEA-S were investigated during the use of one cycle of a COC containing 30 µg ethinylestradiol (EE) and 3 mg drospirenone (DRSP) with and without daily co-administration of 50 mg DHEA. RESULTS: Treatment during one cycle with a COC containing EE and DRSP reduces the exposure to DHEA and DHEA-S by at least 20%. This loss of adrenal androgens can be fully compensated by daily oral co-administration of 50 mg DHEA. With DHEA co-administration total T levels rise significantly (1.44 nmol/L with DHEA vs. 0.82 nmol/L with placebo; p < 0.001). Free T levels decrease significantly with both DHEA and placebo treatment, but significantly less during co-administration of DHEA (6.34 pmol/L with DHEA vs. 3.96 pmol/L with placebo; p < 0.001). CONCLUSION: By adding DHEA to a COC the loss of adrenal and ovarian androgens can be restored.

The bioequivalence of the contraceptive steroids ethinylestradiol and drospirenone is not affected by co-administration of dehydroepiandrosterone.

OBJECTIVES: To study the effect of co-administration of 50 mg dehydroepiandrosterone (DHEA) on the bioequivalence of ethinylestradiol (EE) and drospirenone (DRSP) in women who were using a combined oral contraceptive (COC) containing 30 µg EE and 3 mg DRSP, and to estimate whether the addition of DHEA to this COC affects the serum levels and the bioequivalence of the synthetic contraceptive steroids. METHODS: This was a randomised, double-blind, two-period crossover study. Participants received two EE/DRSP COC treatment cycles in random order, one with and one without daily 50 mg DHEA , separated by a 28-day wash-out cycle during which the subjects used an EE/levonorgestrel (LNG) COC without DHEA. Serum levels of EE and DRSP were measured according to a sampling scheme allowing pharmacokinetic evaluations. RESULTS: Addition of DHEA to an EE/DRSP COC had no effect on serum levels of EE and DRSP. The COC regimens with and without DHEA were bioequivalent. Oestradiol levels were equally suppressed during pill intake, whether with placebo or DHEA. CONCLUSION: Adding DHEA to a COC containing EE and DRSP does not affect the pharmacokinetic properties of EE and DRSP. Therefore, it will most likely not affect its contraceptive efficacy.