Prenatal and pubertal exposure to 17α-ethinylestradiol disrupts folliculogenesis and promotes morphophysiological changes in ovaries of old gerbils (Meriones unguiculatus).

17α-Ethinylestradiol is an endocrine-disrupting chemical that make up most contraceptive pills and can be found in the environment. Exposure to ethinylestradiol in different development periods may promote changes in morphophysiological parameters of reproductive and endocrine organs. Considering that the effects of low doses (15 µg/kg/day) of ethinylestradiol in ovaries from 12-month-old female gerbils (Meriones unguiculatus) were investigated. Four experimental groups used were control (without treatment), EE/PRE (treated from the 18th to the 22nd gestational day), EE/PUB (treated from the 42nd to the 49th day of life), and EE/PRE-PUB (treated in the both periods). The animals were euthanized at 12 months. Testosterone and 17ß-estradiol levels were measured. The ovaries were stained with Hematoxylin and Eosin, Periodic Acid Schiff, and Gomori's Trichome. The follicles, corpus luteum, interstitial gland, lipofuscin, ovarian epithelium, and tunica albuginea were analyzed. Estradiol was higher in EE/PRE and EE/PUB groups, while testosterone was higher only in EE/PUB group. The main changes in follicle count occurred in EE/PUB and EE/PRE-PUB groups, with higher primordial follicle count and lower maturation of follicles. The corpus luteum was more evident in EE/PRE group. No differences were found in atretic follicles count. A higher area occupied by interstitial gland cells and lipofuscin deposit in these cells was noted in EE/PUB and EE/PRE-PUB groups. Higher epithelium height and thicker tunic albuginea were showed in treated groups. These results suggest that exposure to doses of EE2 in prenatal and pubertal periods of the development leads to morphological changes in senile ovaries.

An estrogen (17α-ethinyl estradiol-3-sulfate) reduces mortality in a swine model of multiple injuries and hemorrhagic shock.

BACKGROUND: Although 17α-ethinyl estradiol-3-sulfate (EES) reduces mortality in animal models of controlled hemorrhage, its role in a clinically relevant injury model is unknown. We assessed the impact of EES in a swine model of multiple injuries and hemorrhage. METHODS: The study was performed under Good Laboratory Practice, with 30 male uncastrated swine (25-50 kg) subjected to tibial fracture, pulmonary contusion, and 30% controlled hemorrhage for an hour. Animals were randomized to one of five EES doses: 0 (control), 0.3, 1, 3, and 5 mg/kg, administered postinjury. Subjects received no resuscitation and were observed for 6 hours or until death. Survival data were analyzed using Cox-proportional hazard regression. Left ventricular pressure-volume loops were used to derive preload recruitable stroke work as a measure of cardiac inotropy. Immediate postinjury preload recruitable stroke work values were compared with values at 1 hour post-drug administration. RESULTS: Six-hour survival for the 0, 0.3, 1, 3, and 5 mg/kg groups was 0%, 50%, 33.3%, 16.7%, and 0%, respectively. Following Cox regression, the hazard (95% confidence interval) of death was significantly reduced in the 0.3 (0.22 [0.05-0.93]) and 1 (0.24 [0.06-0.89]) mg/kg groups but not the 3 (0.49 [0.15-1.64]) and 5 (0.46 [0.14-1.47]) mg/kg groups. Mean survival time was significantly extended in the 1 mg/kg group (246 minutes) versus the 0 mg/kg group (96 minutes) (p = 0.04, t test). At 1 hour post-drug administration, inotropy was significantly higher than postinjury values in the 0.3 and 1 mg/kg groups (p = 0.003 and p < 0.001, respectively). Inotropy was unchanged in the 3 and 5 mg/kg groups but significantly depressed in the control (p = 0.022). CONCLUSION: Administration of EES even in the absence of fluid resuscitation reduces mortality and improves cardiac inotropy in a clinically relevant swine model of multiple injuries and hemorrhage. These findings support the need for a clinical trial in human trauma patients.

Less Carcinogenic Chlorinated Estrogens Applicable to Hormone Replacement Therapy.

Human estrogens prescribed for hormone replacement therapy (HRT) are known to be potent carcinogens. To find safer estrogens, several chlorinated estrogens were synthesized and their carcinogenic potential were determined. A pellet containing either 2-chloro-17ß-estradiol (2-ClE2) or 4-chloro-17ß-estradiol (4-ClE2) was implanted subcutaneously for 52 weeks into August Copenhagen Irish (ACI) rats, a preferred animal model for human breast cancer. 17ß-Estradiol (E2) frequently induced mammary tumors while both 2-ClE2 and 4-ClE2 did not. Their 17α-ethinyl forms, thought to be orally active estrogens, were also synthesized. Neither 2-chloro-17α-ethinylestradiol (2-ClEE2) nor 4-chloro-17α-ethinylestradiol (4-ClEE2) induced tumors. The less carcinogenic effects were supported by histological examination of mammary glands of ACI rats treated with the chlorinated estrogens. A chlorine atom positioned at the 2- or 4-position of E2 may prevent the metabolic activation, resulting in reducing the carcinogenicity. 2-ClE2 and 4-ClE2 administered subcutaneously and 2-ClEE2 and 4-ClEE2 given orally to ovariectomized rats all showed uterotrophic potency, albeit slightly weaker than that of E2. Our results indicate that less carcinogenic chlorinated estrogens retaining estrogenic potential could be safer alternatives to the carcinogenic estrogens now in use for HRT.

Ethinyl estradiol sulfate acts without fluid resuscitation through estrogen receptors to rapidly protect the cardiovascular system from severe hemorrhage.

BACKGROUND: Our in vivo rodent and pig model evidenced that estrogen and its derivative, ethinyl estradiol sulfate (EES), promote survival following hemorrhagic shock. To determine its mechanism, we first confirmed EES binding to estrogen receptor (ER) and improving/restoring cellular signaling, countering the assumption that EES, an ethinyl estradiol metabolite, is inactive. In addition, we examined if EES acts rapidly, consistent with nongenomic signaling. We selected the biomarkers of cardiovascular performance, reduction of apoptosis and proinflammatory responses, and elaboration of nitric oxide (NO) to validate efficacy. METHODS: A rat trauma-hemorrhage model, consisting of a midline laparotomy and controlled bleeding (60% blood loss) without fluid resuscitation, was used. At 30 minutes after hemorrhage, heart performance was monitored, and Western blots were used to quantify biochemical analytes. The specificity of EES for ER was profiled with ER antagonists. Binding studies by Sekisui XenoTech (Kansas City, KS) determined an LD50 value for EES binding the rat ER. RESULTS: The EES IC50 value was 1.52 × 10-8 Mol/L, consistent with pharmacologic efficacy. Ethinyl estradiol sulfate raised mean arterial pressure and ±derivative of pressure over time (dP/dT) significantly (but did not fully restore) within a 30-minute window. Levels of apoptosis and activation of NF-κB were dramatically reduced, as was elaboration of nitric oxide (NO) by inducible nitric oxide synthase. Phospho-endothelial nitric oxide synthase (eNOS) was restored to physiological levels. The restoration of cellular signaling occurs before restoration of cardiac contractility. CONCLUSION: Ethinyl estradiol sulfate is a potent drug for improving heart performance, which also dramatically reduces damage by apoptosis, proinflammatory activity, and NO production, validating that EES can blunt multiple harmful outcomes arising from hypoxia and hypovolemia. The actions are dependent on receptor engagement, where specificity is confirmed by ER antagonists. The constraint of a 30-minute sampling window affirms that the responses are nongenomic and very likely restricted to cell-surface receptor engagement. The rapidity of these responses makes EES promising for intervention in the "golden hour."

Comparison of change in body weight between contraception containing 30-µg ethinylestradiol/2-mg chlormadinone acetate or 30-µg ethinylestradiol/3-mg drospirenone: a randomised controlled trial.

Objectives: The aim of this study was to compare changes in body weight in women using a combined oral contraceptive (COC) consisting of 30-µg ethinylestradiol (EE) and 2-mg chlormadinone acetate (CMA) or a COC consisting of 30-µg EE and 3-mg drospirenone (DRSP).Methods: This randomised double-blind controlled trial (ClinicalTrials.gov NCT01608698) was conducted at a university hospital-based clinic in Thailand between June 2012 and September 2015. A total of 102 women were enrolled in the study, 99 of whom were randomised to EE/CMA (n = 45) or EE/DRSP (n = 54). Each participant was treated for six cycles. Body weight and other parameters as well as side effects were recorded at baseline and at the end of the third and sixth cycles of treatment.Results: A significant difference was observed in mean body weight change between the EE/CMA and EE/DRSP groups from both baseline to third cycle (0.51 ± 1.36 kg vs -0.43 ± 1.56 kg; p = .003) and baseline to sixth cycle (1.00 ± 1.84 kg vs -0.20 ± 2.23 kg; p = .013). The mean difference in body mass index and waist circumference had a similar trend to that of the mean difference in body weight. There was no significant difference in side effects between groups.Conclusion: A COC containing 30-µg EE/3-mg DRSP tended to confer a significantly more favourable change in body weight over a 6-month period compared with a COC containing 30-µg EE/2-mg CMA, which was associated with an increase in body weight.

Last-Step Pd-Mediated [11C]CO Labeling of a Moxestrol-Conjugated o-Iodobenzyl Alcohol: From Model Experiments to in Vivo Positron Emission Tomography Studies.

The fast, efficient, and functional group tolerant last-step radiolabeling of bioconjugates is crucial for positron emission tomography (PET) applications. In this context, o-iodobenzyl alcohol based structures were identified as ideal tags for an easy Pd-catalyzed carbonylation after bioconjugation, and a moxestrol-conjugated precursor was chosen as the model compound for the further studies. Despite scale and time constraints, conditions developed with [12C]CO and [13C]CO were easily transferred to the 11C isotope, and the desired radioactive product was obtained in amounts up to 740 MBq with radiochemical purities higher than 99%. Radio-high-performance liquid chromatography analyses of rat blood samples demonstrated excellent in vivo stability within the time of the acquisition. MicroPET-magnetic resonance imaging showed excretion pathways similar to moxestrol, and molecular modeling was also performed to evaluate the potential ability of this conjugate to bind estrogen receptors α. Thus, being both synthetically and biologically suitable, this strategy clears the path to potential novel biotracers for preclinical PET imaging.

Treatment of traumatic brain injury with 17α-ethinylestradiol-3-sulfate in a rat model.

OBJECTIVE 17α-ethynylestradiol-3-sulfate (EE-3-SO4) is a highly water-soluble synthetic estrogen that has an extended half-life (∼ 10 hours) over that of naturally occurring estrogen (∼ 10 minutes). In this study, EE-3-SO4 was evaluated in a lateral fluid percussion-induced traumatic brain injury (TBI) model in rats. METHODS A total of 9 groups of Sprague-Dawley rats underwent craniectomy. Twenty-four hours later, lateral fluid percussion was applied to 6 groups of animals to induce TBI; the remaining 3 groups served as sham control groups. EE-3-SO4 (1 mg/kg body weight in 0.4 ml/kg body weight) or saline (vehicle control) was injected intravenously 1 hour after TBI; saline was injected in all sham animals. One day after EE-3-SO4/saline injection, intracranial pressure (ICP), cerebral perfusion pressure (CPP), and partial brain oxygen pressure (PbtO2) were measured in Groups 1-3 (2 TBI groups and 1 sham group), and brain edema, diffusion axonal injury, and cerebral glycolysis were assessed in Groups 4-6 using MRI T2 mapping, diffusion tensor imaging (DTI), and FDG-PET imaging, respectively. Four days after dosing, the open-field anxiety of animals was assessed in Groups 7-9 by measuring the duration that each animal spent in the center area of an open chamber during 4 minutes of monitoring. RESULTS EE-3-SO4 significantly lowered ICP while raising CPP and PbtO2, compared with vehicle treatment in TBI-induced animals (p < 0.05). The mean size of cerebral edema of TBI animals treated with EE-3-SO4 was 25 ± 3 mm3 (mean ± SE), which was significantly smaller than that of vehicle-treated animals (67 ± 6 mm3, p < 0.001). Also, EE-3-SO4 treatment significantly increased the fractional anisotropy of the white matter in the ipsilateral side (p = 0.003) and cerebral glycolysis (p = 0.014). The mean duration that EE-3-SO4-treated animals spent in the center area was 12 ± 2 seconds, which was significantly longer than that of vehicle-treated animals (4 ± 1 seconds; p = 0.008) but not different from that of sham animals (11 ± 3 seconds; p > 0.05). CONCLUSIONS These data support the clinical use of EE-3-SO4 for early TBI treatment.

Effectiveness of ethinylestradiol/drospirenone for premenstrual symptoms in Japanese patients with dysmenorrhea: Open-label pilot study.

AIM: A combined oral contraceptive containing ethinylestradiol 20 µg plus drospirenone 3 mg (EE20 + DRSP) in a 24/4 regimen has been shown to alleviate the symptoms of premenstrual syndrome and premenstrual dysphoric disorder. This study was conducted to evaluate the efficacy of EE20 + DRSP in Japanese patients with premenstrual symptoms. MATERIAL AND METHODS: A multicenter, prospective, open-label, single-arm, phase IV study was performed in Japanese women with dysmenorrhea and premenstrual symptoms. They were treated with EE20 + DRSP to alleviate the symptoms of dysmenorrhea for six treatment cycles. Premenstrual symptoms were evaluated using a Premenstrual Symptoms Questionnaire at baseline and after three and six cycles of EE20 + DRSP. The degree of dysmenorrhea was also evaluated using a visual analog scale at baseline and after one, three, and six cycles of EE20 + DRSP. RESULTS: Forty-eight patients were treated with EE20 + DRSP. Most of the premenstrual symptoms were alleviated significantly by three and six cycles of EE20 + DRSP treatment. EE20 + DRSP treatment significantly improved the severity of premenstrual symptoms. We also confirmed the effectiveness of EE20 + DRSP for the treatment for dysmenorrhea. CONCLUSION: This study showed that EE20 + DRSP could be a useful treatment strategy for premenstrual symptoms in Japanese women.

17α-Ethynylestradiol-3-sulfate treatment of severe blood loss in rats.

BACKGROUND: From 2001-2011, >80% of potentially survivable United States battlefield deaths were due to severe hemorrhage. We subjected male rats to acute severe blood loss, administered a single dose of 17α-ethynylestradiol-3-sulfate (EE-3-SO4) without resuscitative fluids, and measured survival and also mean arterial pressures (MAP). METHODS: After controlled removal of 60% circulating blood volume (10-11 mL) over approximately 45 min, rats received EE-3-SO4 at 0 (vehicle controls), 0.1, 0.3, 1.0, or 3.0 mg/kg in 40 µL/100 g BW saline intravenously. MAP was recorded for 40 min after drug administration and survival was recorded for 6 h. RESULTS: The dose response curve was bell shaped with optimum survival at 1 mg/kg EE-3-SO4. Median survival times of rats receiving 1 mg/kg (360 min) were approximately 6 times that of the control group (57 min): P = 0.0001. The number of animals alive at 6 h was 16 of 20 (80%) in the 1 mg/kg group versus 0 of 20 (0%) in the control group. Early increases in MAP correlated with longer survival times. CONCLUSIONS: Administration of a single dose of 1 mg/kg EE-3-SO4 in 0.4 mL/kg of saline after controlled severe hemorrhage increased survival in rats by 6-fold. Partial recovery of blood pressure values correlated with longer survival time. These results, coupled with similar findings in a companion study in minipigs, support the further product development of EE-3-SO4 for: (1) severe hemorrhage when standard resuscitative fluids are not available, and (2) situations in which prolonged transportation periods are required for definitive treatment of the injured.

[Antitumor and antiproliferative action of the steroidal cytostatic antiestrogen cytestrol acetate on hormone-dependent tumor models].

Cytestrole acetate (CA), in the structure of which the steroidal antiestrogen component is associated with bis-ß-cloroethylamino group, exhibits a strong cytotoxic activity against hormone-dependent cancer cell lines (CaOV, HeLa, MCF-7). In doxorubicin-resistant MCF-7 cells, CA potentiates the cytotoxic effect of etoposide and doxorubicin, and the IC50 for CA in these cells is 40 times lower than that for tamoxifen (TAM). In transplantable mice breast adenocarcinoma Ca-755, the therapeutic CA dose is 25 mg/kg when administered subcutaneously in oil solution for 5 days. On the DMBA-induced mammary tumors in rats, CA injected subcutaneously led to partial regressions 4 weeks after treatment in 75% of test rats, whereas TAM produced this effect in 43% of rats. Among various drug forms of CA, the most active were oil solution of CA in gelatin capsules for oral use and liposomal emulsion for intravenous administration, since these forms exhibited the highest values of Ca-755 tumor growth inhibition index (TGI = 97 - 98%).

Efficacy of 17α-ethynylestradiol-3-sulfate for severe hemorrhage in minipigs in the absence of fluid resuscitation.

BACKGROUND: Of the potentially survivable US battlefield deaths from 2001 to 2011, 80% to 91% were caused by severe hemorrhage. We subjected minipigs to acute severe blood loss, administered a single dose of 17α-ethynylestradiol-3-sulfate (EE-3-SO4) without resuscitative fluids, and determined survival as well as cardiovascular, biochemical, and physiologic response parameters. METHODS: Following controlled removal of 60% circulating blood volume over 1 hour, minipigs received EE-3-SO4 at 0, 1, 3, or 5-mg/mL saline per kilogram of body weight in Experiment 1 (n = 25) and 0-, 0.1-, 0.3-, or 1-mg/mL saline per kilogram in Experiment 2 (n = 23). Survival times and response parameters were recorded for the next 6 hours. RESULTS: Median survival times of the minipigs receiving 1 mg/kg (257 minutes and 360 minutes) were 1.8 times and 5 times those of the control group (140 minutes and 65 minutes) in Experiments 1 and 2, respectively. For both experiments combined, the log-rank p value was 0.0002, and the number of animals alive at 6 hours was 6 (50%) of 12 in the 1-mg/kg groups versus 0 (0%) of 12 in the control groups. Early increases in glucose, lactate, potassium, and phosphate as well as decreases in bicarbonate and mean arterial pressure correlated with shorter survival times. CONCLUSION: Administration of a single dose of 1-mg/kg EE-3-SO4 in 1-mL/kg of saline following severe hemorrhage increased survival in 60% acutely bled minipigs by 3.5-fold. Slightly elevated blood pressure values, more physiologic values of oxidative phosphorylation parameters, and lower elevations of possible tissue necrosis parameters correlated with longer survival time. These results support the further product development of EE-3-SO4 for the indication of severe hemorrhage when standard resuscitative fluids are not available.

Chronic exposure of mice to environmental endocrine-disrupting chemicals disturbs their energy metabolism.

We evaluated the effects of a 20-week chronic exposure of mice to a low dose of cypermethrin (CYP), atrazine (ATZ) and 17α-ethynyestradiol (EE2) on energy metabolism. Here, male mice were exposed to 50 µg/kg BW/day CYP, 100 µg/kg BW/day ATZ or 1 µg/kg BW/day EE2 supplied in their drinking water for 20 weeks. During the exposure, mice were fed a high energy diet (HD). The bodyweights were not significantly affected by chronic exposure to EDCs, while the serum-free fatty acids (FFA) levels, hepatic lipid accumulation and triacylglycerol (TG) contents increased significantly in the ATZ- and CYP-HD groups. To determine the mechanism involved, we determined the expression levels of the genes in the glucose and fat metabolism pathways in the liver and adipose tissue. The results showed that chronic exposure to ATZ and CYP increased the mRNA levels of a number of key genes involved in both the de novo FFA synthesis pathway and the transport of FFA from blood. The increased amount of FFA was partially consumed as energy through ß-oxidation in the mitochondria. Some of the FFA was used to synthesize TG in the liver by up-regulating primary genes, which resulted in increased TG levels and lipid accumulation. The results indicate that chronic exposure to EDCs has the potential to cause energy metabolic dysregulation and hepatotoxicity in mice.

Rapid relapse in depression following initialization of oral contraception with ethinyl estradiol and chlormadinone acetate.

OBJECTIVE: Female sexual hormones (estrogens and gestagens) may affect neurocognitive functioning and mood. Thus, the use of oral hormonal contraceptives (OHC) bears the risk of psychiatric adverse drug reactions such as depression and psychosis. However, the available empiric evidence regarding this connection is conflicting, and, moreover, female sex hormones seem to feature also mood-stabilizing and antidepressive effects. Hence, individual susceptibility factors and preparation-specific pharmacologic properties might play a pivotal role in the development of mood disturbances related to OHC. Single case reports provide empiric data for further systematic approaches. METHODS: A clinical case is presented and discussed. RESULTS: A 36-year-old female patient with recurrent major depressive disorder developed rapid relapse in depression after initialization of OHC with ethinyl estradiol 30 µg/chlormadinone acetate 2 mg. This OHC combination was described to particularly feature positive effects on depressive mood. CONCLUSIONS: OHC may induce serious mood disturbances and should be administered with care, particularly in patients with affective disorders.

Molecular cloning, expression, and hormonal regulation of the chicken microsomal triglyceride transfer protein.

During an egg-laying cycle, oviparous animals transfer massive amounts of triglycerides, the major lipid component of very low density lipoprotein (VLDL), from the liver to the developing oocytes. A major stimulus for this process is the rise in estrogen associated with the onset of an egg-laying cycle. In mammals, the microsomal triglyceride transfer protein (MTP) is required for VLDL assembly and secretion. To enable studies to determine if MTP plays a role in basal and estrogen-stimulated VLDL assembly and secretion in an oviparous vertebrate, we have cloned and sequenced the chicken MTP cDNA. This cDNA encodes a protein of 893 amino acids with an N-terminal signal sequence. The primary sequence of chicken MTP is, on average, 65% identical to that of mammalian homologs, and 23% identical to the Drosophila melanogaster protein. We have obtained a clone of chicken embryo fibroblast cells that stably express the avian MTP cDNA and show that these cells display MTP activity as measured by the transfer of a fluorescently labeled neutral lipid. As in mammals, chicken MTP is localized to the endoplasmic reticulum as revealed by indirect immunofluorescence and by the fact that its N-linked oligosaccharide moiety remains sensitive to endoglycosidase H. Endogenous, enzymatically active MTP is also expressed in an estrogen receptor-expressing chicken hepatoma cell line that secretes apolipoprotein B-containing lipoproteins. In this cell line and in vivo, the expression and activity of MTP are not influenced by estrogen. Therefore, up-regulation of MTP in the liver is not required for the increased VLDL assembly during egg production in the chicken. This indicates that MTP is not rate-limiting, even for the massive estrogen-induced secretion of VLDL accompanying an egg-laying cycle.

Impact of environmental estrogens on nucleotide excision repair gene expression in embryonic zebrafish.

Estrogens and estrogen mimics are aquatic contaminants that can elicit a variety of deleterious effects in exposed fauna. One of the most potent xenoestrogens found in the aquatic environment is 17α-ethinylestradiol (EE(2)), the pharmaceutically derived semi-synthetic hormone found in oral contraceptives and hormone replacement therapies. Exposure to 100 ng/L EE(2) has previously been shown to profoundly decrease functional hepatic nucleotide excision repair (NER) processes in adult zebrafish in correlation with dramatic decreases in the abundance of hepatic XPC and XPA transcripts; however, its effects on these processes in embryos are currently unknown. Because developing organisms are known to have increased sensitivities to endocrine disrupting compounds such as EE(2), the goal of this study was to examine the impacts of estrogen exposure on mRNA expression of these two key NER genes in zebrafish embryos during the first 4 days of development. Embryos were exposed from 0 h post fertilization (hpf) to waterborne EE(2), its major metabolite, estrone (E(1)), or combinations of the two compounds and sampled at 12, 24, 48, 72 and 96 hpf. Increased abundance of vitellogenin-1 (VTG1) mRNA, a bioindicator of estrogen exposure, was evident as early as 24 hpf in embryos that were co-exposed to EE(2) and E(1) and this effect was sustained throughout 96 hpf. Embryos exposed to EE(2) alone exhibited elevated VTG1 beginning at 72 hpf. In contrast to observations from adult zebrafish exposed to EE(2), embryos did not show any change in mRNA abundance of the excision repair gene, XPC, during the first 4 days of development. However, co-exposure to EE(2) and E(1) elicited an increase in XPA mRNA abundance at 48 and 72 hpf, which was the opposite response as that observed in exposed adults where hepatic XPA mRNA abundance decreased after EE(2) exposure. These differences between embryos and adults suggest that alteration of NER gene transcription by EE(2) is operating under different stimuli during development.

1,2,3-Triazole-heme interactions in cytochrome P450: functionally competent triazole-water-heme complexes.

In comparison to imidazole (IMZ) and 1,2,4-triazole (1,2,4-TRZ), the isosteric 1,2,3-triazole (1,2,3-TRZ) is unrepresented among cytochrome P450 (CYP) inhibitors. This is surprising because 1,2,3-TRZs are easily obtained via "click" chemistry. To understand this underrepresentation of 1,2,3-TRZs among CYP inhibitors, thermodynamic and density functional theory computational studies were performed with unsubstituted IMZ, 1,2,4-TRZ, and 1,2,3-TRZ. The results indicate that the lower affinity of 1,2,3-TRZ for the heme iron includes a large unfavorable entropy term likely originating in solvent-1,2,3-TRZ interactions; the difference is not solely due to differences in the enthalpy of heme-ligand interactions. In addition, the 1,2,3-TRZ fragment was incorporated into a well-established CYP3A4 substrate and mechanism-based inactivator, 17-α-ethynylestradiol (17EE), via click chemistry. This derivative, 17-click, yielded optical spectra consistent with low-spin ferric heme iron (type II) in contrast to 17EE, which yields a high-spin complex (type I). Furthermore, the rate of CYP3A4-mediated metabolism of 17-click was comparable to that of 17EE, with a different regioselectivity. Surprisingly, continuous-wave electron paramagnetic resonance (EPR) and HYSCORE EPR spectroscopy indicate that 17-click does not displace water from the sixth axial ligand position of CYP3A4 as expected for a type II ligand. We propose a binding model in which 17-click pendant 1,2,3-TRZ hydrogen bonds with the sixth axial water ligand. The results demonstrate the potential for 1,2,3-TRZ to form metabolically labile water-bridged low-spin heme complexes, consistent with recent evidence that nitrogenous type II ligands of CYPs can be efficiently metabolized. The specific case of [CYP3A4·17-click] highlights the risk of interpreting CYP-ligand complex structure on the basis of optical spectra.

Effects of 17α-ethynylestradiol on sexual differentiation and development of the African clawed frog (Xenopus laevis).

Several studies have shown that exposure of amphibians, including the African clawed frog (Xenopus laevis), to potent estrogens at critical times during development results in feminization and/or demasculinization. However, genotyping of X. laevis has only recently become possible, so studies performed in the past were rarely able to make explicit linkages between genetic and phenotypic sex. Therefore, to further characterize this relationship, X. laevis tadpoles were exposed during development to 0.09, 0.84, or 8.81 µg/L 17α-ethynylestradiol (EE2), which is the estrogen analog commonly used in oral contraceptives. Exposure to all concentrations of EE2 tested resulted in significant delays in time to metamorphosis. Genotyping showed that genetic sex ratios were similar among treatments. However, morphological evaluation revealed that a significant number of individuals with a male genotype displayed mixed sex and abnormal phenotypes. Additionally, both genetic males and females exposed to EE2 exhibited greater presence of vitellogenin protein relative to the respective controls. Since estrogens function downstream of the initial molecular signals of sexual differentiation, it is likely that genetic male animals received mixed endogenous male and exogenous female signals that caused disordered sexual development. The production of vitellogenin was probably temporally separated and independent from primary effects on sexual differentiation, and might have contributed to delays in metamorphosis observed in individuals exposed to EE2.

The effects of 2 mg chlormadinone acetate/30 mcg ethinylestradiol, alone or combined with spironolactone, on cardiovascular risk markers in women with polycystic ovary syndrome.

BACKGROUND: Polycystic ovary syndrome (PCOS) is an endocrine disorder associated with metabolic dysfunction and changes in cardiovascular risk markers, and using oral contraceptives (OCs) may exert a further negative effect on these alterations in patients with PCOS. Thus, the primary objective of this study was to assess the effects on arterial function and structure of an OC containing chlormadinone acetate (2 mg) and ethinylestradiol (30 mcg), alone or combined with spironolactone (OC+SPL), in patients with PCOS. STUDY DESIGN: This was a randomized, controlled clinical trial. Fifty women with PCOS between 18 and 35 years of age were randomized by a computer program to use OC or OC+SPL. Brachial artery flow-mediated vasodilation, carotid intima-media thickness and the carotid artery stiffness index were evaluated at baseline and after 6 and 12 months. Serum markers for cardiovascular disease were also analyzed. The intragroup data were analyzed using analysis of variance with Tukey's post hoc test. A multivariate linear regression model was used to analyze the intergroup data. RESULTS: At 12 months, the increase in mean total cholesterol levels was greater in the OC+SPL group than in the OC group (27% vs. 13%, respectively; p=.02). The increase in mean sex hormone-binding globulin levels was greater in the OC group than in the OC+SPL group (424% vs. 364%, respectively; p=.01). No statistically significant differences between the groups were found for any of the other variables. CONCLUSION: The addition of spironolactone to an OC containing chlormadinone acetate and ethinylestradiol conferred no cardiovascular risk-marker advantages in young women with PCOS.

Evaluation of efficacy, safety and effects on symptoms of androgenization of a generic oral contraceptive containing chlormadinone acetate 2 mg/ethinylestradiol 0.03 mg.

BACKGROUND: This prospective noninterventional study assessed the contraceptive efficacy, safety and the effects on signs of androgenization of the generic oral contraceptive containing 2 mg chlormadinone acetate/0.03 mg ethinylestradiol (CMA/EE) in a real-world setting. STUDY DESIGN: A total of 1440 women were investigated during a six-cycle period by 229 gynecological practices throughout Germany. RESULTS: The adjusted Pearl index was 0.136 (unadjusted: 0.271). Of 463 patients with cycle irregularities at baseline, 83.4% had regular cycles after six cycles. Likewise, 74.1% of 162 patients with spotting or breakthrough bleeding at baseline were free from these symptoms at the end of study. The percentage of patients with dysmenorrhea decreased significantly from baseline (36.5%) to visit 3 after six cycles (12.3%; p=.0001), with a significant reduction in the use of pain medication (p<.0001). Additionally, the number of patients with skin and hair problems was significantly reduced (skin: 56.3% at baseline, 19.6% after six cycles; hair: 45.7% at baseline, 13.4% after six cycles; p=.001). CMA/EE was well tolerated by the patients, and 89.44% of the gynecologists were satisfied with the treatment. CONCLUSION: Generic CMA/EE exhibits very good contraceptive efficacy, cycle control and dysmenorrhea reduction. Furthermore, treatment with generic CMA/EE led to a favorable reduction of skin and hair problems in our study.

The efficacy and safety of an oral contraceptive containing chlormadinone acetate: results of a pooled analysis of noninterventional trials in adult and adolescent women.

BACKGROUND: The study was conducted to assess the contraceptive efficacy, cycle events, dysmenorrhea symptoms and skin complaints of a combined oral contraceptive containing 2 mg chlormadinone acetate/0.03 mg ethinylestradiol (CMA/EE) (Belara®, Grünenthal GmbH, Aachen, Germany) in adolescent and adult women using a conventional- or extended-cycle regimen. STUDY DESIGN: Data were pooled from six noninterventional trials with CMA/EE intake over 4-12 cycles. RESULTS: The data pool contained 62,218 women (345,964 cycles), of whom 60,508 were analyzed (325,937.5 cycles), including 46,335 adults, 13,478 adolescents and 695 age unknown; 1710 retrospective documented patients were excluded from analysis. A total of 85 women became pregnant (including women missing pills) on the conventional-cycle regimen (21+7), giving a practical Pearl index of 0.34 [95% confidence interval (CI) 0.27-0.42]. Within this group (n=85), there were 19 pregnancies associated with regular pill intake, which represent a theoretical Pearl index of 0.08 (95% CI 0.05-0.12) per 100 women-years. Overall, cycle stability, bleeding profile, dysmenorrhea and the incidence of seborrhea/acne improved with CMA/EE, and there was no clinically relevant change in body weight or body mass index. Six venous thromboembolic events were reported, equating to an incidence of 2.4 per 10,000 women-years. CONCLUSIONS: Chlormadinone acetate/ethinylestradiol is effective and well tolerated in adolescent and adult women.