ER/Estrogen Receptor; Endogenous Metabolite

In vitro activity: Ethinyl Estradiol increases respiratory chain activity in both cultured rat hepatocytesand HepG2 cells. Ethinyl estradiol is a strong promoter of hepatocarcinogenesis. Ethinyl Estradiol enhances the transcript levels of nuclear genome- and mitochondrial genome-encoded genes and respiratory chain activity in female rat liver, and also inhibits transforming growth factor beta (TGFbeta)-induced apoptosis in cultured liver slices and hepatocytes from female rats. Ethinyl Estradiol increases the transcript levels of the mitochondrial genome-encoded genes cytochrome oxidase subunits I, II, and III in cultured female rathepatocytes. Ethinyl Estradiol significantly increases both the levels of glutathione (reduced [GSH] and oxidized [GSSG] forms) per mg protein in mitochondria and nuclei, while the percentage of total glutathione in the oxidized form is not affected.

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Ethinyl estradiol (EE) is a strong promoter of hepatocarcinogenesis. Treatment of rats with EE and other hepatic promoters induces a mitosuppressed state characterized by decreased hepatocyte turnover and reduced growth responsiveness. Previously, we identified several nuclear and mitochondrial genome-encoded mitochondrial genes whose transcripts were increased during EE-induced hepatic mitosuppression in rats and in EE-treated HepG2 cells (Chen et al. Carcinogenesis, 17, 2783-2786, 1996 and Carcinogenesis, 19, 101-107, 1998). In both cultured rat hepatocytes and HepG2 cells, EE increased respiratory chain activity (reflected by increased mitochondrial superoxide production detected as increased lucigenin-derived chemiluminescence (LDCL). In this paper, we provide additional characterizations of these effects. Increased LDCL was detected in mitochondria isolated from EE-treated rats, documenting that these estrogen effects on mitochondrial function are not confined to cells in culture. EE and estradiol (E2) increased LDCL in cultured rat hepatocytes and HepG2 cells in a dose- (beginning at 0.25 microM levels) and time-dependent response. Inhibition of P450-mediated estrogen metabolism inhibited, while direct exposure to E2 catechol metabolites enhanced LDCL. Co-treatment with glutathione ester or with the specific antiestrogen, ICI 182708 inhibited LDCL. In contrast, estrogen-induced LDCL was enhanced by glutathione depletion, and by inhibition of catechol-o-methyltransferase. These results support a working hypothesis that in liver cells, increased respiratory chain activity induced by estrogen treatment requires both metabolism to catechols and an estrogen receptor-mediated signal transduction pathway. [1]

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Ethinyl estradiol (EE) is a strong promoter and weak hepatocarcinogen in rats. Previously, we demonstrated that EE enhanced the transcript levels of nuclear genome- and mitochondrial genome-encoded genes and respiratory chain activity in female rat liver, and also inhibited transforming growth factor beta (TGFbeta)-induced apoptosis in cultured liver slices and hepatocytes from female rats. In this study, using cultured female rat hepatocytes, we observed that EE, within 24 h, increased the transcript levels of the mitochondrial genome-encoded genes cytochrome oxidase subunits I, II, and III. This effect was accompanied by increased mitochondrial respiratory chain activity, as reflected by increased mitochondrial superoxide generation, and detected by lucigenin-derived chemiluminescence and cellular ATP levels. EE also enhanced the levels of Bcl-2 protein. Biochemical analyses indicated that EE significantly increased both the levels of glutathione (reduced [GSH] and oxidized [GSSG] forms) per mg protein in mitochondria and nuclei, while the percentage of total glutathione in the oxidized form was not affected. This finding was supported by confocal microscopy. These effects caused by EE may contribute, at least in part, to the EE-mediated inhibition of hepatic apoptosis [2].

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The proton-coupled amino acid transporter, PAT1, is known to be responsible for intestinal absorption drug substances such as gaboxadol and vigabatrin. The aim of the present study was to investigate, if 17-α-ethinyl-estradiol (E-E2) and 17-β-estradiol (E) inhibit PAT1-mediated intestinal absorption of proline and taurine in vitro in Caco-2 cells and in vivo using Sprague-Dawley rats to assess the potential for taurine-drug interactions. E and E-E2 inhibited the PAT1-mediated uptake of proline and taurine in Caco-2 cells with IC50 values of 10.0-50.0 μM without major effect on other solute carriers such as the taurine transporter (TauT), di/tri-peptide transporter (PEPT1), and serotonin transporter (SERT1). In PAT1-expressing oocytes E and E-E2 were non-translocated inhibitors. In Caco-2 cells, E and E-E2 lowered the maximal uptake capacity of PAT1 in a non-competitive manner. Likewise, the transepithelial permeability of proline and taurine was reduced in presence of E and E-E2 [4].

Ethinyl Estradiol (50 mg/kg/day) increases anogenital distance and reduces pup body weight at postnatal day 2, accelerates the age at vaginal opening, reduces F1 fertility and F2 litter sizes, and induces malformations of the external genitalia (5 mg/kg) in the female Long-Evans rat. Ethinyl Estradiol increases the number of low density lipoprotein (LDL) receptors in livers of rats, thereby producing a profound fall in plasma cholesterol levels. Ethinyl Estradiol exerts the same effect in livers of male and female rabbits and that the increase in receptor number is correlated with a 6- to 8-fold increase in the levels of receptor mRNA.

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Many chemicals released into the environment display estrogenic activity including the oral contraceptive Ethinyl estradiol (EE2) and the plastic monomer bisphenol A (BPA). Ethinyl estradiol (EE2) is present in some aquatic systems at concentrations sufficient to alter reproductive function of fishes. Many concerns have been raised about the potential effects of BPA. The National Toxicology Program rated the potential effects of low doses of BPA on behavior and central nervous system (CNS) as an area of "some concern," whereas most effects were rated as of "negligible" or "minimal" concern. However, the number of robust studies in this area was limited. The current study was designed to determine if maternal exposure to relatively low oral doses of EE2 or BPA in utero and during lactation would alter the expression of well-characterized sexually dimorphic behaviors or alter the age of puberty or reproductive function in the female Long-Evans rat offspring. Pregnant rats were gavaged with vehicle, EE2 (0.05-50 microg/kg/day), or BPA (2, 20, and 200 microg/kg/day) from day 7 of gestation to postnatal day (PND) 18, and the female offspring were studied. EE2 (50 microg/kg/day) increased anogenital distance and reduced pup body weight at PND2, accelerated the age at vaginal opening, reduced F1 fertility and F2 litter sizes, and induced malformations of the external genitalia (5 microg/kg). F1 females exposed to EE2 also displayed a reduced (male-like) saccharin preference (5 microg/kg) and absence of lordosis behavior (15 microg/kg), indications of defeminization of the CNS. BPA had no effect on any of the aforementioned measures. These results demonstrate that developmental exposure to pharmacologically relevant dosage levels of EE2 can permanently disrupt the reproductive morphology and function of the female rat [3].

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In male Sprague Dawley rats pre-dosed with Ethinyl estradiol (EE2) a decreased maximal plasma concentration (Cmax) of taurine and increased the time (tmax) to reach this was indicated, suggesting the possibility for an in vivo effect on the absorption of PAT1 substrates. In conclusion, 17-α-ethinyl-estradiol and 17-β-estradiol were identified as non-translocated and non-competitive inhibitors of PAT1 [4].

Estradiol and Ethinyl estradiol (EE2) Inhibit PAT1-Mediated Uptake of Taurine and Proline [4]
The uptake of solute carrier substrates such as taurine, proline, glycyl-sarcosine (Gly-Sar) and 5-hydroxytryptamine was investigated in Caco-2 cells in the presence of 100 μM E2 and E-E2 (Fig. 1). The uptake of both taurine and proline was higher under slightly acidic condition compared to neutral pH in the uptake buffer (Fig. 1 A and B), consistent with proton-coupled transport via PAT1. 100 μM E2 and E-E2 inhibited the uptake of proline and taurine at pH 6.0, but not at pH 7.4. Likewise, the ...

Dosage Levels and Administration of Chemicals [3]
Pregnant rat dams were randomly assigned to treatment groups on GD7 within each cohort prior to dosing. Laboratory-grade corn oil, Ethinyl estradiol (EE2), and BPA were used. Dams were dosed via oral gavage from GD7 through PND18 with 0 (corn oil, vehicle control), Ethinyl estradiol (EE2) at 0.05, 0.5, 1.5, 5, 15, or 50 μg/kg/day, or BPA at 2, 20, or 200 μg/kg/day. The doses were delivered in 0.5-ml corn oil/kg body weight; thus, all dams in the study received the same amount of vehicle per body weight. The dams were weighed daily during the dosing period to adjust the administered dose for body weight changes during pregnancy and lactation and to monitor their health.
This study was performed in two blocks. The first block involved 169 dams with 13–29 dams per treatment group: oil vehicle, Ethinyl estradiol (EE2) (0.05, 0.5, 5, or 50 μg/kg/day), or BPA (2, 20, or 200 μg/kg/day). The second block (block 2) was performed to expand the range of EE2 doses tested and involved 82 dams with 6–14 dams per treatment group: oil vehicle, EE2 (0.05, 0.15, 0.5, 1.5, 5, 15, or 50 μg/kg/day), or BPA (20 or 200 μg/kg/day).
The effects of Ethinyl estradiol (EE2) and BPA on the dams and the F1 male offspring were previously published by Howdeshell et al. (2008). Maternal body weight gain during pregnancy was calculated from inception of dosing (GD7) to GD20, and the analysis of these data included only those dams that were pregnant and survived through GD20. Maternal body weight gain during lactation was calculated from PND2 through the end of dosing (PND18) and included only those dams with pups surviving to weaning.

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Pilot study with control male and female LE rats. [3]
Prior to the study of animals treated with Ethinyl estradiol (EE2) or BPA, a study of saccharin preference was conducted with untreated LE rats to confirm that the behavioral methods we were planning to use to assess the effects of in utero EE2 and BPA displayed the expected sexual dimorphism. This experiment also was designed to determine if female rats displayed a greater preference for either 0.50% wt/vol (5 g of saccharin was added to a liter of deionized, distilled water) or 0.25% wt/vol (2.5 g of saccharin was added to a liter of water) saccharin solutions. Adult male (n = 3–5 per dose group) and adult female intact (n = 4 per dose group) LE rats were purchased from Charles River Laboratories. Animals were housed individually upon receipt and allowed 2 weeks to acclimate before testing.

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Saccharin preference in experimental females. [3]
Following the pilot study, saccharin preference was measured in female rats exposed during gestation and lactation to varying doses of Ethinyl estradiol (EE2) or BPA in the first block of the study (sample sizes are shown in Fig. 5). Saccharin preference for 0.25% wt/vol saccharin solution versus deionized, distilled water was determined over a 5-day period, and the average saccharin preference over the 5-day period was then analyzed using litter means on PROC GLM.
Subsequently, females were ovariectomized and the activity levels measured a second time to determine if the removal of the ovaries and endogenous estrogens reduced the behavior to male levels. Females were then tested a third time after oral exposure to Ethinyl estradiol (EE2) at 0 (corn oil at 0.5 μl/g body weight), 175, or 275 μg/kg/day EE2 daily for 14 days. Based on the results of this experiment, we administered 275 μg EE2/kg daily for 14 days to restore activity levels in females developmentally exposed to EE2 and BPA.

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Assessment of Figure-8 maze activity in Ethinyl estradiol (EE2)- or BPA-treated females. [3]
Figure-8 maze activity was measured in ovariectomized females from the first block of the study. After recovering from surgery, Figure-8 maze activity levels were measured (trial 1), as above. Subsequently, females were treated orally by gavage for 14 days with 275 μg/kg Ethinyl estradiol (EE2) and retested in the Figure-8 mazes (trial 2; sample sizes are shown in Fig. 6).
Preliminary study of lordosis behavior and uterine weight in SD and LE rats after oral Ethinyl estradiol (EE2) treatment: dose-response to EE2.Lordosis behavior was studied using untreated adult ovariectomized females given Ethinyl estradiol (EE2) followed by progesterone to activate the behavior. Female rats that have been defeminized by neonatal estrogen exposures display low levels of lordosis behavior (Gorski, 1986).
Adult, ovariectomized SD and LE rats as well as adult, intact stimulus SD males were used. Female rats were housed two per cage in a room on a reversed light cycle (lights on from 9:00 P.M. to 11:00 A.M.) and given at least 2 weeks to recover from surgery and to acclimate to the altered light cycle prior used for behavioral testing. In this experiment, ovariectomized adult female LE rats were given varying doses of Ethinyl estradiol (EE2) by oral gavage in oil (0, 2.5, 5, 10, 12.5, 19, 25, 37.5, 50, 65, 80, 95, 110, 125, and 250 μg/kg) for 2 days, followed by 0.5 mg progesterone (sc) in oil on the third day in order to determine an optimal oral dose of EE2 that induces this behavior reliably in control females. Females were retested at different dosage levels two to three times. This experiment (Fig. 7) included both LE and SD female rats to determine if there was a strain difference in the dose-response to EE2.
A similar experiment compared the dose-related effects of Ethinyl estradiol (EE2) on uterine weight in LE and SD rats to determine which estrogen-dependent end point was more sensitive to low doses of Ethinyl estradiol (EE2), increased uterine weight, or induction of lordosis behavior. Ovariectomized female LE and SD rats were treated for 2 days by gavage with EE2 at 0, 0.5, 1, 2.5, 5, 10, 25, 50, or 250 μg/kg and necropsied on the third day, and the wet weight of the uterus was measured.
We used Ethinyl estradiol (EE2) to activate estrogen-dependent behaviors and uterine weight (Fig. 7) because our initial studies demonstrated that it can be used to induce female-like maze activity as well as lordosis behavior, whereas based upon unpublished studies, we suspected that a single daily dose of estradiol would not induce activity as effectively.

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50 mg/kg

Rats

Absorption, Distribution and Excretion
Following oral administration of 30 µg ethinylestradiol, the peak plasma concentration (Cmax) was 74.1 ± 35.6 pg/mL, the time to peak concentration (Tmax) was 1.5 ± 0.5 h, and the area under the curve (AUC) was 487.4 ± 166.6 pgh/mL. Following administration of 1.2 mg via a patch, the peak plasma concentration (Cmax) was 28.8 ± 10.3 pg/mL, the time to peak concentration (Tmax) was 86 ± 31 h, and the area under the curve (AUC) was 3895 ± 1423 pgh/mL. 59.2% of ethinylestradiol is excreted in urine and bile, and 2-3% is excreted in feces. Over 90% of ethinylestradiol is excreted unchanged.
The apparent volume of distribution (VOD) of 30 µg orally was 625.3 ± 228.7 L, and that of 1.2 mg topically was 11745.3 ± 15934.8 L.
The intravenous clearance of ethinylestradiol was 16.47 L/h, and the estimated renal clearance was approximately 2.1 L/h. The clearance of 30 µg orally was 58.0 ± 19.8 L/h, and that of 1.2 mg topically was 303.5 ± 100.5 L/h.
Ethinylestradiol is rapidly and almost completely absorbed. When comparing the lowest and highest strength tablets (0.100 mg desogestrel/0.025 mg ethinylestradiol and 0.150 mg desogestrel/0.025 mg ethinylestradiol) with solutions, the relative bioavailability of ethinylestradiol was 92% and 98%, respectively.
The distribution of exogenous estrogens is similar to that of endogenous estrogens. Estrogens are widely distributed throughout the body, typically reaching higher concentrations in sex hormone target organs. Ethinyl estradiol in the blood is primarily bound to albumin. Although ethinyl estradiol does not bind to sex hormone-binding globulin (SHBG), it can induce SHBG synthesis. Estradiol, estrone, and estriol are excreted in urine along with glucuronide and sulfate conjugates. Twenty-five healthy women of reproductive age who had never previously used oral contraceptives each took one tablet containing 50, 80, or 100 micrograms of ethinyl estradiol. Blood samples were collected before administration and at 1, 2, 4, and 24 hours after administration. An anti-ethinyl estradiol antibody with an initial dilution of 1:100,000 was used. The techniques used are described in detail. Peak plasma ethinyl estradiol concentrations were obtained from blood samples collected 1 hour after administration. After 24 hours, the drug was undetectable in the plasma of 4 out of 5 subjects (who received 50 mcg) and 1 out of 5 subjects (who received 80 mcg). Drug clearance curves fluctuated considerably with ethinylestradiol administration, with peaks typically occurring at 2 hours, but occasionally at 4 hours. Measurable serum ethinylestradiol levels were detected after 24 hours at all three dose levels. These levels were reached slowly and were lower than those at the time of ethinylestradiol administration. Unlike the natural estrogen ethinylestradiol, estrogens bind primarily to plasma proteins through nonspecific binding, thus they are less likely to affect ethinylestradiol metabolism compared to endogenous steroids. Furthermore, large doses of ethinylestradiol were administered. Unlike the natural estrogen ethinylestradiol, estrogens bind in vitro in a diynylated form. The pharmacokinetics of ethinylestradiol differ from those of natural estrogens. This makes measurements of estrone and estradiol in plasma or urine difficult to interpret. For more complete data on the absorption, distribution, and excretion of ethinylestradiol (7 metabolites), please visit the HSDB record page.

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Metabolism/Metabolites
Ethinylestradiol can be glucuroninated by UGT1A1, UGT1A3, UGT1A4, UGT1A9, and UGT2B7. It can also be sulfated by SULT1A1, SULT1A3, and SULT1E1. Ethinylestradiol can also be hydroxylated at positions 2, 4, 6, 7, and 16 by CYP3A4, CYP3A5, CYP2C8, CYP2C9, and CYP1A2. These hydroxylated metabolites can be methylated by catechol-O-methyltransferases. Methoxylated metabolites can be further sulfated or glucuroninated.
Exogenous estrogens are metabolized in the same way as endogenous estrogens. Circulating estrogens are in a dynamic equilibrium of metabolic interconversions. These conversions mainly occur in the liver. Estradiol is reversibly converted to estrone, and both can be converted to estriol, which is the main urinary metabolite. Estrogens also undergo enterohepatic circulation via sulfate and glucuronide conjugation in the liver. These conjugates are secreted into the intestine via bile and are hydrolyzed and reabsorbed in the intestine. In postmenopausal women, a significant portion of circulating estrogen exists as sulfate conjugates, especially estrone sulfate, which serves as a circulating reservoir for the synthesis of more potent estrogens. Ethinyl estradiol metabolism is extensive, including oxidation and conjugation with sulfate and glucuronide. Sulfate is the main circulating conjugate of ethinyl estradiol, while glucuronide is mainly found in urine. The main oxidative metabolite is 2-hydroxyethinyl estradiol, produced by the CYP3A4 isoenzyme of cytochrome P450. The first-pass metabolism of ethinyl estradiol is thought to occur in the gastrointestinal mucosa. Ethinyl estradiol may undergo enterohepatic circulation. Due to reduced hepatic metabolism, ethinyl estradiol clearance is much slower. Studies on ethinyl estradiol metabolism have been conducted in rats, rabbits, guinea pigs, dogs, and monkeys. Ethinyl estradiol is rapidly and efficiently absorbed by the rat intestine; no significant metabolic transformation has been reported during absorption. The main metabolic pathway of ethinyl estradiol in rats is 2-hydroxylation of the aromatic ring; hydroxylation of the B ring (C-6/C-7) is negligible. The main metabolites in the rat liver are 2-hydroxyethinyl estradiol and its methyl ethers, namely 2-methoxyethinyl estradiol and 2-hydroxyethinyl estradiol-3-methyl ether. This metabolic pathway is also important in humans. Almost all metabolites of ethinyl estradiol in rats are excreted in feces. For more complete data on the metabolism/metabolites of ethinyl estradiol (10 metabolites in total), please visit the HSDB record page.
Known human metabolites of ethinylestradiol include 17-ethynyl-13-methyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthrene-3,4,17-triol and 17-ethynyl-13-methyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthrene-2,3,17-triol.
Ethinylestradiol is a known human metabolite of mestriol.
It is metabolized in the liver. Quantitative analysis shows that the main metabolic pathway of ethinylestradiol in rats and humans is the same as that of natural estrogens, namely aromatic hydroxylation.
Half-life: 36 ± 13 hours

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Biological half-life
The half-life of 30 µg orally is 8.4 ± 4.8 hours, and the half-life of 1.2 mg topically is 27.7 ± 34.2 hours.
The pharmacokinetics of 19-nor-17α-pregn-1,3,5(10)-trien-20-ynthin-3,17-diol (ethinylestradiol, Progynon C) (EE2) have been studied in female rats, rabbits, beagle dogs, and rhesus monkeys at intravenous doses of 0.1 or 0.01 mg/kg and gavage doses of 1 mg/kg. Monkeys and baboons. After intravenous injection, the distribution of the parent drug in plasma was biphasic, with an initial half-life of 0.3 to 0.5 hours and a terminal half-life of 2.3 to 3.0 hours. Total plasma clearance was comparable to, or even higher than, total hepatic plasma flow (in rats), indicating rapid biotransformation of estrogen in the liver. Systemic bioavailability of gastric EE2 was 3% in rats, 0.3% in rabbits, 9% in dogs, 0.6% in rhesus monkeys, and 2% in baboons, significantly lower than in humans (40%). Differences in pharmacokinetics and systemic bioavailability of EE2 between laboratory animals and humans should be considered when retrospectively interpreting pharmacological and toxicological data and designing new studies. …The elimination half-life has been reported to be 13 to 27 hours.

Effects During Pregnancy and Lactation
◉ Overview of Lactation Use
This record contains specific information on the use of ethinyl estradiol alone. Users interested in oral contraceptives should consult the record titled "Combined Oral Contraceptives."
Information on the use of ethinyl estradiol alone during lactation is limited. Concentrations in breast milk appear to be very low. Based on studies of oral contraceptives containing ethinyl estradiol, immediate side effects such as breast enlargement appear to be rare. Daily doses of 30 micrograms or higher may suppress lactation. The extent of the effect on lactation may depend on the dose and the timing of postpartum initiation of medication. This is most likely to occur if estrogen is started approximately 6 weeks postpartum before milk production is fully established. A drop in estrogen levels may occur in the first few days of estrogen exposure.
◉ Effects on Breastfed Infants
As of the revision date, no published information has been found regarding the effects of ethinyl estradiol alone on breastfed infants. However, there are case reports of infants born to mothers taking combined oral contraceptives containing ethinylestradiol or its prodrug ethinylestradiol experiencing breast enlargement.
◉ Effects on Lactation and Breast Milk
As of the revision date, no published information has been found regarding the effects of ethinylestradiol on milk production. However, numerous studies on combined oral contraceptives containing ethinylestradiol or its prodrug ethinylestradiol suggest that daily doses of 30 micrograms or more may interfere with lactation. One study using a contraceptive containing 10 micrograms of ethinylestradiol found no effect on lactation.
A retrospective cohort study compared 371 women who received high-dose estrogen treatment (3 mg diethylstilbestrol or 150 micrograms ethinylestradiol daily) during puberty to reduce adult height with 409 women who did not receive estrogen treatment. There was no difference in the duration of breastfeeding between the two groups, indicating that high-dose estrogen during puberty has no effect on later breastfeeding.

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Protein Binding
Ethinyl estradiol binds to albumin in serum at a rate of 98.3-98.5%, but it also binds to sex hormone-binding globulin.

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Toxicity Overview
Identification: Ethinyl estradiol is a synthetic steroid prepared from estrone. It is a white to off-white or slightly yellowish-white powder or crystal. Insoluble in water, soluble in ethanol. Indications: The most common use is as the estrogen component in combined oral contraceptives. It is also used to treat menopausal and postmenopausal symptoms, particularly vasomotor symptoms. It is used to treat female hypogonadism and as palliative care for breast and prostate cancer. It is also used to treat some cases of acne and Turner syndrome in women. Human Exposure: Major Risks and Target Organs: Acute ethinyl estradiol poisoning can cause mild, self-limiting symptoms, usually affecting the gastrointestinal tract. Chronic poisoning increases the risk of cardiovascular disease, including myocardial infarction, cerebrovascular disease, thromboembolic disease, gallbladder disease, and certain cancers in some populations. Clinical Overview: Acute ethinylestradiol poisoning symptoms are mild and resolve spontaneously. Nausea and vomiting may occur, and breakthrough vaginal bleeding is occasionally seen. Like other estrogens, chronic ethinylestradiol toxicity increases the risk of stroke, myocardial infarction, and thromboembolic diseases in certain populations. Jaundice, hypertension, nasal congestion, headache, dizziness, and fluid retention may occur. The incidence of endometrial cancer, breast cancer, and certain liver cancers may be higher than in the general population. Contraindications: The contraindications for ethinylestradiol are the same as for general estrogens, including: known or suspected breast cancer (except in certain patients receiving treatment for metastatic disease); known or suspected estrogen-dependent tumors; known or suspected pregnancy; unexplained abnormal genital bleeding; active thrombophlebitis or thromboembolic disease. A history of thrombophlebitis, thrombosis, or thromboembolic disease related to previous use of estrogen-containing compounds is also a risk factor. Absorption Route: Ethinylestradiol is rapidly and completely absorbed from the gastrointestinal tract. The alkynyl substitution at C17 inhibits first-pass metabolism. Its bioavailability is reported to be 40%. Distribution: It is widely bound to plasma proteins, primarily albumin. Due to its lipophilicity, the free molecule is widely distributed in tissues. Peak plasma concentrations occur 2 to 3 hours after oral administration. A second peak occurs at 12 hours, considered to represent extensive enterohepatic circulation. Biological half-life: The biological half-life after a single oral therapeutic dose is approximately 7.7 hours. The elimination half-life is reported to be 13 to 27 hours. Metabolism: Compared to other estrogens, its metabolism is slow. The main biotransformation pathway is through 2-hydroxylation and the formation of 2- and 3-methyl ethers. First-pass metabolism mainly occurs in the intestinal wall. Clearance via exposure: Some sulfate and glucuronide metabolites undergo enterohepatic circulation and are therefore partially excreted in feces. Kidney excretion is also possible. Mechanism of action: Toxicokinetics: Estrogen use has been reported to increase the incidence of gallbladder disease by 2 to 3 times. This is thought to be due to increased cholesterol saturation in bile and decreased bile acid secretion. Furthermore, numerous studies have explored the adverse effects of estrogens (including ethinylestradiol) on coagulation. These studies have used both the effects of estrogen alone and in combination with progesterone. However, there is currently no consensus on the final results regarding physiological or pharmacological dosages. Pharmacodynamics: Like other steroid hormones, ethinylestradiol is thought to exert its effects primarily by regulating gene expression. As a lipophilic hormone, it readily diffuses across the cell membrane and binds to estrogen receptors located in the cell nucleus. Estrogen receptors are present in the female reproductive tract, breast, pituitary gland, hypothalamus, bone, liver, and other tissues. The receptors interact with specific nucleotide sequences, leading to an increase or decrease in the transcription of hormone-regulated genes. Different tissues may respond differently to receptor activation. Its ideal therapeutic effects include action in the female reproductive tract (often used in combination with progesterone), where ethinylestradiol can stimulate the proliferation and differentiation of the fallopian tubes and enhance fallopian tube muscle activity. Ethinylestradiol can also increase the water content of cervical mucus and promote the contraction of the uterine myometrium. Estrogens, including ethinylestradiol, can inhibit bone resorption, thus having a positive effect on bone mass. It is well-established that women receiving estrogen replacement therapy, including ethinyl estradiol, have an increased risk of endometrial hyperplasia and cancer. Data from the 1970s and 1980s showed a 2 to 15-fold increased risk of endometrial cancer. The higher the dose and the longer the treatment duration, the greater the risk. However, adding progesterone to estrogen replacement therapy has a protective effect. Further research is needed to determine whether ethinyl estradiol therapy and other estrogens increase the risk of breast cancer. Conflicting research results exist. A review of data from the 1970s and 1980s indicated a moderate increase in breast cancer risk, but this increase only appeared after 5 years of treatment. Teratogenicity: Specific data on the teratogenicity of ethinyl estradiol are currently unavailable. Reports indicate that fetal exposure to estrogen has been associated with congenital abnormalities, including heart and limb defects. Other estrogens, such as diethylstilbestrol (DES), have been associated with vaginal and cervical adenocarcinoma in female offspring of mothers who took the drug in early pregnancy. DES intake during pregnancy has also been associated with several other abnormalities in male offspring, including smaller testes and urogenital abnormalities. Although no studies have been found directly linking ethinylestradiol to these findings, the pharmacological similarity of such compounds suggests caution in its use. Major adverse reactions: The major adverse reactions of ethinylestradiol at therapeutic doses are directly related to its estrogenic and metabolic effects, including sodium and water retention, which may lead to edema, weight gain, and breast tenderness. Additionally, altered libido and withdrawal vaginal bleeding have been reported. Liver dysfunction, jaundice, and gallstones may occur. Symptoms such as headache, depression, dizziness, glucose intolerance, and contact lens allergy have also been described. High doses may cause hypercalcemia when used to treat metastatic cancer. Nausea, vomiting, and diarrhea are not uncommon. Skin manifestations include melasma, rash, and urticaria. Erythema multiforme and erythema nodosum may also occur. Hypertension and thromboembolic diseases have been reported. Acute poisoning: Ingestion: Acute poisoning symptoms are mild and resolve spontaneously. Nausea, vomiting, and breakthrough vaginal bleeding have been reported after oral contraceptive overdose. Nasal congestion, visual disturbances, headache, and hypertension have also been reported after estrogen overdose. Animal/Plant Studies: Relevant Animal Data: A correlation between long-term oral contraceptive use and liver cancer was confirmed in rats. A single estradiol implantation two weeks later induced DNA breaks in hamster kidneys, but no DNA breaks were observed in livers. Animals exposed to estrogen developed tumors in the kidneys, bones, testes, uterus, and mammary glands. Mutagenicity: Estradiol induced DNA breaks in hamster kidney cells, but no DNA breaks were observed in liver cells. International Programme for Chemical Safety; Toxicological Information Monograph: Ethambutol (PIM 221) (1997). Available as of May 19, 2005: https://www.inchem.org/pages/pims.html
Estrogen diffuses to target cells and interacts with protein receptors. Target cells include the female reproductive tract, mammary glands, hypothalamus, and pituitary gland. Estrogen increases the synthesis of sex hormone-binding globulin (SHBG), thyroid-binding globulin (TBG), and other serum proteins in the liver, and inhibits the secretion of follicle-stimulating hormone (FSH) from the anterior pituitary gland. This cascade reaction begins with the binding of estrogen to its estrogen receptors. The combined use of estrogen and progesterone can suppress the hypothalamic-pituitary system, reducing the secretion of gonadotropin-releasing hormone (GnRH).

[1]. Increased mitochondrial superoxide production in rat liver mitochondria, rat hepatocytes, and HepG2 cells following ethinyl estradiol treatment. Toxicol Sci. 1999;51(2):224-35;
[2]. Enhanced mitochondrial gene transcript, ATP, bcl-2 protein levels, and altered glutathione distribution in ethinyl estradiol-treated cultured female rat hepatocytes. Toxicol Sci.2003;75(2):271-8. 2003;
[3]. In utero and lactational exposure to bisphenol A, in contrast to ethinyl estradiol, does not alter sexually dimorphic behavior, puberty, fertility, and anatomy of female LE rats. Toxicol Sci. 2010;114(1):133-48.
[4]. Inhibitory Effects of 17-α-Ethinyl-Estradiol and 17-β-Estradiol on Transport Via the Intestinal Proton-Coupled Amino Acid Transporter (PAT1) Investigated In Vitro and In Vivo. J Pharm Sci . 2021 Jan;110(1):354-364. .

According to an independent committee of scientific and health experts, ethinylestradiol may be carcinogenic. Ethinylestradiol is a white to off-white fine powder, belonging to the synthetic steroid class, and is often used in combination with progestins as an oral contraceptive. 17α-Ethinylestradiol is a 3-hydroxy steroid, a product of estradiol with an ethynyl group substituted at the 17-position. It is an isoestrone synthesized from estradiol and has been shown to have high estrogenic activity when taken orally. It is an isoestrone. It is a 17-hydroxy steroid, a terminal alkyne compound, and a 3-hydroxy steroid whose function is related to 17β-estradiol and estradiol. Ethinylestradiol was first synthesized in 1938 by Hans Herof Inhofe and Walter Holweg at Schering AG. The development of ethinylestradiol aimed to develop an estrogen with higher oral bioavailability. This was achieved by introducing an ethynyl group onto the 17th carbon atom of estradiol. Ethinylestradiol quickly replaced ethinylestradiol in oral contraceptives. Ethinyl estradiol was approved by the U.S. Food and Drug Administration (FDA) on June 25, 1943. Ethinyl estradiol is an estrogen. Its mechanism of action is as an estrogen receptor agonist. It has been reported in organisms with available data, including Minthostachys mollis, Elsholtzia eriostachya, and several other organisms. Ethinyl estradiol is a semi-synthetic estrogen. It binds to the estrogen receptor complex and enters the cell nucleus, activating DNA transcription of genes involved in estrogen-mediated cellular responses. The drug also inhibits 5α-reductase in epididymal tissue, thereby lowering testosterone levels and potentially delaying the progression of prostate cancer. In addition to its antitumor effects, ethinyl estradiol can prevent osteoporosis. In animal models, short-term use of the drug has been shown to provide long-term protection against breast cancer, mimicking the antitumor effects of pregnancy. (NCI04) Ethinyl estradiol is a semi-synthetic alkylated estradiol with a 17-α-ethynyl substituent. Ethinyl estradiol has high estrogenic activity after oral administration and is commonly used as the estrogen component in oral contraceptives. It is primarily marketed as a combined oral contraceptive under brand names including Alesse, Tri-Cyclen, Triphasil, and Yasmin. The FDA label includes a boxed warning stating that women over 35 who smoke should not use combined oral contraceptives due to the increased risk of serious cardiovascular side effects. It is a semi-synthetic alkylated estradiol with a 17-α-ethynyl substituent. It has high estrogenic activity after oral administration and is commonly used as the estrogen component in oral contraceptives. See also: ethinyl estradiol; norethindrone (ingredient); ethinyl estradiol; ethinylene glycol diacetate (ingredient); ethinyl estradiol; etoposide (ingredient)...see more...

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Drug Indications
Ethinyl estradiol is used in combination with other drugs for contraception, treatment of premenstrual anxiety, moderate acne, moderate to severe menopausal vasomotor symptoms, and prevention of postmenopausal osteoporosis.
FDA Label

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Mechanism of Action
Ethinylestradiol is a synthetic estrogen compound. Estrogen use has a variety of effects on the body, including reduced bone density. Combined oral contraceptives suppress ovulation by inhibiting gonadotropins, thickening cervical mucus to prevent sperm motility, and preventing the changes in the endometrium necessary for implantation of a fertilized egg. Ethinylestradiol lowers luteinizing hormone levels, thereby reducing vascularization in the endometrium. It also increases sex hormone-binding globulins.
Endogenous estrogens are primarily responsible for the development and maintenance of the female reproductive system and the formation of secondary sexual characteristics. Although circulating estrogens are in a dynamic equilibrium of metabolic interconversion, estradiol is the dominant intracellular estrogen and is much more potent at receptor levels than its metabolites estrone and estriol. …After menopause, most endogenous estrogens are produced by the conversion of androstenedione secreted by the adrenal cortex into estrone in peripheral tissues. Therefore, estrone and its sulfated form—estrone sulfate—are the most abundant circulating estrogens in postmenopausal women. The pharmacological effects of ethinylestradiol are similar to those of endogenous estrogens. Estrogens exert their effects by binding to nuclear receptors in estrogen-responsive tissues. To date, two estrogen receptors have been identified. Their proportions vary in different tissues. Circulating estrogen regulates the secretion of gonadotropins, including luteinizing hormone (LH) and follicle-stimulating hormone (FSH), from the pituitary gland through a negative feedback mechanism. The role of estrogen is to reduce the elevated levels of these hormones in postmenopausal women. Estrogen plays important roles in the female reproductive, skeletal, cardiovascular, and central nervous systems, primarily through the regulation of gene expression. When estrogen binds to the ligand-binding domain of the estrogen receptor, a biological response is initiated, leading to a conformational change in the receptor, which in turn initiates gene transcription via specific estrogen response elements (EREs) of the target gene promoter; subsequently, the activation or repression of the target gene is mediated by the receptor's two distinct transcriptional activation domains (AF-1 and AF-2). Estrogen receptors also utilize different response elements (e.g., AP-1) and other signaling pathways to mediate gene transcription. Significant progress has been made in the molecular pharmacology of estrogen and its receptors in recent years, leading to the development of selective estrogen receptor modulators (e.g., clomiphene, raloxifene, tamoxifen, toremifene). These drugs can bind to and activate estrogen receptors, but their effects are tissue-specific and distinct from the mechanisms of estrogen action. The tissue-specific estrogen agonist or antagonist activity of these drugs appears to be related to structural differences in their estrogen receptor complexes (e.g., the AF-2 surface morphology of raloxifene differs from that of the estrogen (estradiol)-estrogen receptor complex). Furthermore, researchers have discovered a second estrogen receptor; the presence of at least two estrogen receptors (ER-α and ER-β) may help explain the tissue-specific activity of selective modulators. Although the role of estrogen receptors in bone, cardiovascular tissues, and the central nervous system is still under investigation, emerging evidence suggests that the mechanisms of action of estrogen receptors in these tissues differ from those in reproductive tissues. /Estrogen Overview/
Intracellular sol-binding proteins of estrogen have been identified in estrogen-responsive tissues, including female reproductive organs, breasts, pituitary gland, and hypothalamus. Estrogen-binding protein complexes (i.e., sol-binding proteins and estrogen) are distributed into the cell nucleus, where they stimulate the synthesis of DNA, RNA, and proteins. The presence of these receptor proteins is the reason why women with metastatic breast cancer respond to estrogen therapy. /Estrogen Overview/
Estrogens generally have a beneficial effect on blood cholesterol and phospholipid concentrations. Estrogens decrease low-density lipoprotein cholesterol (LDL-C) concentrations and increase high-density lipoprotein cholesterol (HDL-C) concentrations in a dose-dependent manner. The estrogen therapy-related decrease in LDL-C concentrations appears to be due to increased LDL catabolism, while the increase in triglyceride concentrations is due to increased production of large, triglyceride-rich very low-density lipoproteins (VLDL); changes in serum HDL-C concentrations appear to be primarily due to increased cholesterol and apolipoprotein A-1 content in HDL2-cholesterol and a slight increase in HDL3-cholesterol. /General Estrogen Information/
For more complete data on the mechanisms of action of ethinylestradiol (7 types), please visit the HSDB record page.

C20H24O2

296.4

296.177

C, 81.04; H, 8.16; O, 10.80

57-63-6

313-06-4 (cypionate) ; 57-63-6 (ethinyl) ; 3571-53-7 (undecylate); 50-28-2; 172377-52-5 (sulfamate); 50-50-0 (benzoate); 979-32-8 (valerate); 113-38-2 (dipropionate); 57-63-6

5991

White to off-white solid powder

1.2±0.1 g/cm3

457.2±45.0 °C at 760 mmHg

182-183 °C(lit.)

211.2±23.3 °C

0.0±1.2 mmHg at 25°C

1.624

4.52

2

2

1

22

505

5

C[C@]12CC[C@H]3[C@H]([C@@H]1CC[C@]2(C#C)O)CCC4=C3C=CC(=C4)O

BFPYWIDHMRZLRN-SLHNCBLASA-N

InChI=1S/C20H24O2/c1-3-20(22)11-9-18-17-6-4-13-12-14(21)5-7-15(13)16(17)8-10-19(18,20)2/h1,5,7,12,16-18,21-22H,4,6,8-11H2,2H3/t16-,17-,18+,19+,20+/m1/s1

(8R,9S,13S,14S,17R)-17-ethynyl-13-methyl-7,8,9,11,12,14,15,16-octahydro-6H-cyclopenta[a]phenanthrene-3,17-diol

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*Preparation of 20% SBE-β-CD in Saline (4°C，1 week): Dissolve 2 g SBE-β-CD in 10 mL saline to obtain a clear solution.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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 (Please use freshly prepared in vivo formulations for optimal results.)