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

OBJECTIVES: Estetrol (E4) is a natural fetal estrogen. In this open-label, multiple-rising-dose study, the pharmacokinetic effects of E4 in postmenopausal women were investigated as a secondary objective. METHODS: In total, 49 postmenopausal women were randomized to receive either 2 mg E4 or 2 mg estradiol valerate (E2V) for 28 days, or were (non-randomized) assigned to 10, 20, or 40 mg E4. The main outcome measures were: E4 plasma concentrations at trough, and on days 1 and 28; and E4 pharmacokinetic parameters AUC, Cmax and tmax on days 1 and 28. RESULTS: After oral administration, E4 showed a very fast absorption, followed by a multiphasic elimination with an initial rapid decline, gradually continuing with a slower elimination, suggesting a long terminal half-life. Steady state was reached within 2 weeks of dosing and pharmacokinetic results were generally proportional to the dose. Estetrol concentrations on day 28 were slightly higher compared to day 1, indicating some accumulation. CONCLUSION: The pharmacokinetic profile of estetrol is characterized by a very fast absorption phase, followed by an initial rapid decline, and a slow terminal elimination phase. Based on its kinetic properties, estetrol seems suitable for use as a once-daily oral drug.

Restoring testosterone levels by adding dehydroepiandrosterone to a drospirenone containing combined oral contraceptive: I. Endocrine effects.

OBJECTIVES: Combined oral contraceptives (COCs) decrease testosterone (T) levels. This study investigated restoration of T and other androgen concentrations during COC use by 'co-administration' of dehydroepiandrosterone (DHEA). STUDY DESIGN: In this randomized, double-blind, placebo-controlled study in 99 new COC starters (18-35 years old with body mass index range 18-34 kg/m²), a COC containing 30mcg ethinylestradiol (EE) and 3 mg drospirenone (DRSP) was used for 3cycles, followed by 6cycles of the same COC combined with either 50 mg/day DHEA or placebo. Total T, albumin, sex hormone-binding globulin (SHBG), DHEA-sulfate (DHEA-S), Δ4-androstenedione (AD), 3α-androstanediol glucuronide (ADG) and estradiol (E2) were measured, whereas free T and the free T index (FTI) were calculated. Assessments took place at baseline (no COC use), after the run-in period (COC use alone) and during the treatment period (DHEA or placebo). RESULTS: During COC use alone, androgen levels decreased, especially total T by 62% and free T by 86%, and SHBG increased by 243%. Total T increased with DHEA compared to placebo (change from end of run-in period to end of treatment period -- 1.3±1.2 nmol/L vs. 0.0±0.4 nmol/L; p<.0001) -- and was restored to baseline levels. Free T and the FTI increased significantly (p<.0001), but the free T level was still 53% below baseline levels. DHEA-S, AD and ADG increased significantly to levels above baseline (p<.0001 for each). DHEA had no effect on SHBG, albumin and E2. CONCLUSIONS: An EE/DRSP containing COC strongly suppressed endogenous androgen concentrations in all users. The addition of 50 mg DHEA to a COC regimen containing EE/DRSP restored total T to baseline levels, but free T levels were restored by only 47% as most of the T remains bound to SHBG. IMPLICATIONS: When using a COC that increases SHBG considerably, a daily dose of 50 mg DHEA is insufficient to normalize free T levels completely.

Restoring testosterone levels by adding dehydroepiandrosterone to a drospirenone containing combined oral contraceptive: II. Clinical effects.

OBJECTIVES: Combined oral contraceptives (COCs) decrease androgen levels, including testosterone (T), which may be associated with sexual dysfunction and mood complaints in some women. We have shown that 'co-administration' of dehydroepiandrosterone (DHEA) to a drospirenone (DRSP)-containing COC restored total T levels to baseline and free T levels by 47%. Here we describe the effects on sexual function, mood and quality of life of such an intervention. STUDY DESIGN: This was a randomized, double-blind, placebo-controlled study in 99 healthy COC starters. A COC containing 30 mcg ethinylestradiol (EE) and 3 mg DRSP was used for three cycles, followed by six cycles of the same COC combined with 50 mg/day DHEA or placebo. Subjects completed the Moos Menstrual Distress Questionnaire (MDQ), the McCoy Female Sexuality Questionnaire and the short form of the Quality of Life Enjoyment and Satisfaction Questionnaire (Q-LES-Q). Safety and tolerability, including effects on skin, were evaluated. RESULTS: The addition of DHEA induced small but significant improvements compared to placebo in the MDQ score for autonomic reactions during the menstrual (-2.0 vs. 0.71; p=0.05) and the premenstrual phase (-3.1 vs. 2.9; p=0.01) and for behavior during the intermenstrual phase (-1.4 vs. 3.6; p=0.02). A significant difference was found in the MDQ score for arousal during the premenstrual phase in favor of placebo (-5.0 vs. 1.0; p=0.01). There were no statistically significant differences between groups for the MSFQ and Q-LES-Q scores. DHEA 'co-administration' resulted in an acceptable safety profile. DHEA negated the beneficial effect of the COC on acne according to the subjects' self-assessment. CONCLUSIONS: 'Co-administration' with DHEA did not result in consistent improvements in sexual function, mood and quality of life indicators in women taking EE/DRSP. Retrospectively, the 50 mg dose of DHEA may be too low for this COC. IMPLICATIONS: A well-balanced judgment of the clinical consequences of normalizing androgens during COC use may require complete normalization of free T.

The effect of combined oral contraception on testosterone levels in healthy women: a systematic review and meta-analysis.

UNLABELLED: BACKGROUND; Combined oral contraceptives (COCs) reduce levels of androgen, especially testosterone (T), by inhibiting ovarian and adrenal androgen synthesis and by increasing levels of sex hormone-binding globulin (SHBG). Although this suppressive effect has been investigated by numerous studies over many years, to our knowledge no systematic review concerning this issue had been performed. This systematic review and meta-analysis was performed to evaluate the effect of COCs on concentrations of total T, free T and SHBG in healthy women and to evaluate differences between the various types of COCs (e.g. estrogen dose, type of progestin) and the assays used to assess total T and free T. METHODS: A review of the literature was performed using database searches (MEDLINE, EMBASE and the Cochrane Central Register of Clinical Trials) and all publications (from inception date until July 2012) investigating the effect of COCs on androgen levels in healthy women were considered eligible for selection. Three reviewers were involved in study selection, data extraction and critical appraisal. For the meta-analysis, data on total T, free T and SHBG were extracted and combined using random effects analysis. Additional subgroup analyses were performed to evaluate differences between the various types of COCs (e.g. estrogen dose, type of progestin) and the assays used to assess total T or free T. RESULTS: A total of 151 records were identified by systematic review and 42 studies with a total of 1495 healthy young women (age range: 18-40 years) were included in the meta-analysis. All included studies were experimental studies and 21 were non-comparative. Pooling of the results derived from all the included papers showed that total T levels significantly decreased during COC use [mean difference (MD) (95% confidence interval, CI) -0.49 nmol/l (-0.55, -0.42); P < 0.001]. Significantly lower levels of free T were also found [relative change (95% CI) 0.39 (0.35, 0.43); P < 0.001], with a mean decrease of 61%. On the contrary, SHBG concentrations significantly increased during all types of COC use [MD (95% CI) 99.08 nmol/l (86.43, 111.73); P < 0.001]. Subgroup analyses revealed that COCs containing 20-25 µg EE had similar effects on total and free T compared with COCs with 30-35 µg EE. In addition, suppressive effects on T levels were not different when comparing different types of progestins. However, subgroup analyses for the estrogen dose and the progestin type in relation to changes in SHBG levels did show significant differences: COCs containing second generation progestins and/or the lower estrogen doses (20-25 µg EE) were found to have less impact on SHBG concentrations. CONCLUSIONS: The current literature review and meta-analysis demonstrates that COCs decrease circulating levels of total T and free T and increase SBHG concentrations. Due to the SHBG increase, free T levels decrease twice as much as total T. The estrogen dose and progestin type of the COC do not influence the decline of total and free T, but both affect SHBG. The clinical implications of suppressed androgen levels during COC use remain to be elucidated.

High-dose recombinant LH add-back strategy using high-dose GnRH antagonist is an innovative protocol compared with standard GnRH antagonist.

High daily doses of gonadotrophin-releasing hormone (GnRH) antagonists during the follicular phase of ovarian stimulation were associated with low implantation rates. To test if this occurred because of profound pituitary suppression, the pituitary response was suppressed with a high-dose GnRH antagonist and recombinant LH (rLH) was added back to correct the implantation rate. An open-label, randomized, controlled, prospective clinical study in 60 patients undergoing IVF was performed. GnRH antagonist was initiated on day 6 of stimulation (2 mg/day) together with 375 IU rLH, and maintained until the day of HCG administration. Controls received 0.25 mg/day GnRH antagonist. Fluctuating LH concentrations were present on days 3 and 6 in both groups. This strong fluctuation continued on day 8 and on the day of HCG administration in the control (low-dose) group, where 30% of patients had LH concentrations <1 IU/1 on the HCG day. The study (high-dose) group showed stable LH concentrations on day 8 and on the HCG day, with no LH surges. No clinical differences were found between groups. The LH add-back strategy (375 IU/day) rescued the adverse effects that high doses of GnRH imposed on implantation. These results suggest that rLH should be considered during ovarian stimulation with GnRH antagonist.

Detection of cervical neoplasia by DNA methylation analysis in cervico-vaginal lavages, a feasibility study.

OBJECTIVE: To explore the feasibility of DNA methylation analysis for the detection of cervical neoplasia in self-obtained cervico-vaginal lavages. METHODS: Lavages collected by a self-sampling device and paired cervical scrapings were obtained from 20 cervical cancer patients and 23 patients referred with an abnormal cervical smear (15 with high-grade cervical intraepithelial neoplasia (CIN2+) and 8 without CIN). All lavages and scrapings were analyzed by liquid based cytology (LBC), Hybrid Capture II (HC-II) for hr-HPV DNA detection and by DNA methylation analysis (JAM3, TERT, EPB41L3 and C13ORF18). Concordance between lavages and scrapings was measured by Cohen's Kappa (k). RESULTS: In lavages and scrapings from cervical cancer patients (n=20), methylation analysis was positive in 19 (95%) and 19 (95%), HC-II in 16 (80%) and 15 (75%) and LBC in 15 (75%) and 19 (95%), respectively. In lavages and scrapings from CIN2+ patients (n=15), methylation analysis was positive in 10 (67%) and 12 (80%), HC-II in 15 (100%) and 15 (100%) and LBC in 11 (73%) and 12 (80%), respectively. Concordance between cervical scrapings and lavages (n=43) was for LBC k=0.522 (p<0.001), hr-HPV testing k=0.551 (p<0.001) and DNA methylation analysis k=0.653 (p<0.001). CONCLUSIONS: DNA methylation analysis in cervico-vaginal lavages obtained by a self-sampling device is feasible and its diagnostic performance appears to be at least comparable to the detection of cervical neoplasia by cytomorphology and hr-HPV. Our pilot study suggests that detection of cervical neoplasia by DNA methylation analysis in cervico-vaginal lavages warrants exploration of its use in large prospective studies.

Oral bioavailability and bone-sparing effects of estetrol in an osteoporosis model.

OBJECTIVES: To measure the oral bioavailability of estetrol (E(4)) in rats relative to its subcutaneous administration and to test the bone-sparing effect of oral E(4) compared to that of ethinylestradiol (EE). METHODS: In the bioavailability study, E(4) was administered as a single dose of 0.05, 0.5 or 5.0 mg/kg orally or subcutaneously to female rats. Plasma was analyzed using an LC-MS/MS method. The bone study was conducted in 3-month-old female rats assigned to the following seven treatment groups of ten animals each: no treatment; sham-operated + vehicle; bilaterally ovariectomized (OVX) + vehicle; OVX + E(4) 0.1, 0.5, or 2.5 mg/kg/day and OVX + EE 0.1 mg/kg/day. Once-daily treatment by oral gavage was given for 4 weeks and the following measurements were performed: serum osteocalcin, bone mineral density, bone mineral content and bone mineral area of lumbar vertebrae L3-L6, peripheral quantitative computed tomography of the left tibiae and the biomechanical properties of the distal femora. RESULTS: Oral bioavailability of E(4), relative to that of subcutaneous dosing, was 70% and above at the 0.05 and 0.5 mg/kg doses based on the AUC(0-t last). Subcutaneous dosing provided significantly higher E(4) levels at the 1-h time point only, and was comparable to oral dosing after 0.5, 2, 4 and 8 h. In the bone study, E(4) dose-dependently and significantly (1) inhibited the OVX-related increase in osteocalcin levels, (2) increased bone mineral density and content, and (3) increased bone strength, all attenuated by ovariectomy. In this rat model, the relative potency of the highest dose of E(4) (2.5 mg/kg/day) was comparable to the EE dose, used as positive control. CONCLUSIONS: Estetrol exhibits high oral bioavailability in the rat, a species considered relevant for pharmacological studies that are predictive for effects on human bone. Oral administration of E(4) conveys dose-dependent bone-sparing effects of high-quality bone in estrogen-depleted OVX rats. Based on its bone-sparing effects, its oral bioavailability and its preclinical safety and efficacy profile, E(4) may be superior to other estrogens and is a potential drug for the prevention of osteoporosis in postmenopausal women.

Preventive effect of oral estetrol in a menopausal hot flush model.

OBJECTIVE: To evaluate the efficacy of estetrol (E(4)) in alleviating hot flushes in an experimental animal model considered representative for menopausal vasomotor symptoms. METHODS: Recording of the thermal responses in the tail skin of morphine-dependent ovariectomized rats after morphine withdrawal by administration of naloxone. Six groups of rats were treated orally for 8 days as follows: vehicle (negative) control; E(4): 0.1, 0.3, 1.0 and 3.0 mg/kg/day; and, as active (positive) control, ethinylestradiol 0.3 mg/kg/day. On day 8, tail skin temperature was recorded at baseline and for 60 min at 5-min intervals following naloxone administration. Results In control animals, tail skin temperature increased sharply by about 4.5 degrees C after naloxone treatment and reverted to baseline by 60 min. Estetrol suppressed the tail skin temperature increase in a dose-dependent fashion. The highest dose of E(4) tested (3 mg/kg/day) was equipotent to a 10-fold lower dose of ethinylestradiol. Both fully suppressed tail skin temperature changes. CONCLUSION: Estetrol is effective in alleviating hot flushes in an experimental animal model considered representative for studying menopausal hot flushes (vasomotor symptoms). In this model, the potency of estetrol is 10-fold lower compared to ethinylestradiol.

Estrogenic uterovaginal effects of oral estetrol in the modified Allen-Doisy test.

OBJECTIVE: To evaluate the effect of estetrol (E(4)) on vaginal cornification and uterine wet weight in the ovariectomized rat. METHODS: Adult female rats served as experimental animals. Six groups of ovariectomized rats (eight per group) were treated orally once daily for 7 days as follows: vehicle (negative) control; E(4): 0.1, 0.3, 1.0 and 3.0 mg/kg/day, and ethinylestradiol 0.05 mg/kg/day as active (positive) control. Vaginal lavages were obtained daily and uterine wet weight was determined on day 7. RESULTS: Vaginal cornification was observed by day 5 in all rats at all E(4) doses and in the animals receiving ethinylestradiol, but not in the control rats. The onset of cornification with E(4) was dose-dependent. After 7 days' treatment, the two highest E(4) doses (1.0 and 3.0 mg) induced statistically significant higher uterine wet weight compared to vehicle. CONCLUSION: Estetrol has estrogenic effects on the vagina and on the uterus of ovariectomized rats. The potency of E(4) is approximately 20-fold lower compared to ethinylestradiol.

First human exposure to exogenous single-dose oral estetrol in early postmenopausal women.

OBJECTIVE: To evaluate the safety, tolerability, pharmacokinetics and effect on gonadotropins of a single escalating dose of estetrol (E(4)). METHODS: A first-in-human study with E(4) was performed in healthy early postmenopausal women. Four single doses of 0.1, 1, 10 and 100 mg E(4) were evaluated in study groups of eight subjects each, of whom six received active treatment and two placebo treatment. Safety and tolerability were documented and several pharmacokinetic parameters were determined, as were the plasma levels of the gonadotropins luteinizing hormone (LH) and follicle stimulating hormone (FSH) (pharmacodynamics). The next higher-dose group was enrolled after pharmacokinetic evaluation and confirmed safety of the previous group. RESULTS: After oral intake, the plasma concentrations of E(4) showed a steep increase, followed by a sharp decline and a secondary increase at all dose levels. Estetrol was distributed and reabsorbed during the first 18 h after oral intake. The terminal elimination phase started at 24 h post-dose and half-life (t((1/2))) ranged in the 10 mg group between 19 and 40 h (mean 28.4 h, median 28.8 h) and in the 100 mg group between 18 and 60 h (mean 28.0 h, median 20 h), indicating a dose independency of the half-life. The pharmacokinetic parameters also demonstrated a high dose-response relationship and showed excellent consistency and low variability within the dose groups. The pharmacodynamic data showed a dose-dependent inhibition of plasma LH levels by E(4). A profound and sustained inhibition of FSH levels, lasting over 168 h, was observed in the 100 mg dose group (FSH was not measured in the other dose groups). Estetrol was well tolerated at all dose levels and no safety problems were encountered. CONCLUSIONS: Estetrol is orally absorbed and bioavailable with a strong dose-response relationship suggesting high oral bioavailability. Interindividual variations of plasma levels are low. The elimination half-life of 28 h suggests slow metabolism of E(4). The pharmacodynamic pattern complies with enterohepatic recirculation. Estetrol has a profound central inhibitory and dose-dependent effect on gonadotropins, confirming its biological potency.

Estetrol does not bind sex hormone binding globulin or increase its production by human HepG2 cells.

OBJECTIVES: To determine whether human sex hormone binding globulin (SHBG) binds estetrol (E4), and to assess whether E4 stimulates the production of SHBG by human hepatocytes. METHODS: Competitive ligand binding assays have been used to assess the relative binding affinity of E4 to human SHBG using either [3H]5alpha-dihydrotestosterone or [3H]estradiol as labeled ligands. The effect of E4 on the production of SHBG has been assessed by a fluoroimmunometric assay in wild-type human HepG2 cells and in human Hep89 cells that over-express the human estrogen receptor (ER) alpha, and compared to the effect of ethinylestradiol, estradiol and estriol. RESULTS: There was no detectable binding of E4 to the human SHBG steroid-binding sites. By contrast, testosterone and estradiol were bound with high affinity and the synthetic estrogen ethinylestradiol was found to bind SHBG with low affinity. Estetrol does not stimulate ERalpha-mediated increases in SHBG production by HepG2 or Hep89 cells, in contrast to ethinylestradiol, estradiol and estriol. CONCLUSIONS: These data indicate that SHBG has no influence on the plasma distribution of E4 or its availability to target tissues. In addition, it is shown that E4 has no effect on SHBG production by human hepatocytes.

Estetrol review: profile and potential clinical applications.

In this review paper, the existing information on the human fetal steroid estetrol (E4) has been summarized. In the past, E4 was considered as a weak estrogen and interest disappeared. However, recent new research has demonstrated that E4 is a potent, orally bioavailable, natural human fetal selective estrogen receptor modulator, since it acts in the rat as an estrogen on all tissues investigated except breast tumor tissue, where it has estrogen antagonistic properties in the presence of estradiol. Based on its safety data, its pharmacokinetic properties, its pharmacological profile and the results of first human studies, E4 may be suitable as a potential drug for human use in applications such as hormone replacement therapy (vaginal atrophy, hot flushes), contraception and osteoporosis. Additional areas worth exploring are the treatment of breast and prostate cancer, hypoactive sexual desire disorder and topical use (wrinkles) in women, auto-immune diseases, migraine, cardiovascular applications and the treatment of selected obstetric disorders.

Ovulation inhibition by estetrol in an in vivo model.

Background Currently, the synthetic steroid ethinylestradiol is the preferred estrogen in combined oral contraceptives. The aim of the present study was to evaluate the effectiveness of the natural steroid estetrol (E4) as an ovulation inhibitor in rats when compared to ethinylestradiol. Study design Regularly cycling female rats were treated orally twice daily for four consecutive days, starting on the day of estrus, with E4 (0.03, 0.1, 0.3, 1.0 or 3.0 mg/kg), ethinylestradiol (0.0003, 0.001, 0.003, 0.01 or 0.03 mg/kg) or vehicle control (eight animals per group). In a second experiment conducted under the same experimental protocol, 2.0 mg/kg of E4 was administered as a single daily dose or as a dose of 1.0 mg/kg twice daily. In both studies, the primary endpoint was the number of ovulated oocytes in the genital tract. Results Estetrol at the twice-daily dose of 0.3 mg/kg and above inhibited ovulation. This effect was statistically significant (p < 0.05). The comparator, ethinylestradiol, significantly inhibited ovulation (p < 0.05) at the highest dose (0.03 mg/kg) administered twice daily. An E4 dose of 2.0 mg/kg administered once daily for four consecutive days inhibited ovulation in four of eight rats. In contrast, when the same dose was administered in two separate doses, that is 1.0 mg/kg twice daily, ovulation was inhibited in eight of eight rats. The ED50 for the ethinylestradiol and the E4 dose-response curves shows that ethinylestradiol is 18 times more potent than E4. Conclusion Twice-daily administration of E4 effectively inhibits ovulation in cycling rats. The effect is dose-dependent. The relative potency of E4 is about 18 times less compared to that of ethinylestradiol. We conclude that, based on these data, combined with available pharmacological and clinical data on the safety and efficacy of E4, the human fetal estrogenic steroid estetrol is a potential candidate to replace ethinylestradiol in combined oral contraceptives.

Estetrol, a pregnancy-specific human steroid, prevents and suppresses mammary tumor growth in a rat model.

Estetrol (E4) is a pregnancy-specific D-ring metabolite of estradiol (E2) and estriol (E3) produced by the human fetal liver and present in both male and female fetuses. In adults, female exposure is restricted to the gestational period. We report that E4, dose-dependently, prevents the growth of chemically induced (7,12-dimethylbenz(a)anthracene, DMBA) mammary tumors in female Sprague-Dawley rats and that E4 has the potential to reduce the number and size of pre-existing mammary tumors. We performed two prevention studies and one intervention study. In the prevention studies, we investigated the effect of oral doses of E4 over a dose range of 0.5-3.0 mg/kg. The intervention study used oral dose levels of 1, 3 and 10 mg/kg E4. The antiestrogen tamoxifen was used as reference compound in all three studies and ovariectomy and ethinylestradiol, at estrogenic doses pharmacologically equipotent to E4, acted as control treatments in the second prevention study and in the intervention study. Rats treated with DMBA develop estrogen-responsive breast tumors. This model has become the standard pharmacological model to investigate the effect of new compounds on breast tumors. When DMBA-induced rats were co-treated with E4 for 8 weeks, this resulted in a dose-dependent reduction in the number and size of tumors, an effect that appeared equally effective as tamoxifen treatment or ovariectomy and was not seen with ethinylestradiol. When E4 was administered to rats in which tumors had already developed, a significant decrease in the number and size of tumors was observed after 4 weeks. This decrease was dose-dependent, comparable to tamoxifen-treated animals, and at high dose levels E4 was as effective as ovariectomy.