Estriol (Oestriol) targets:
Estrogen receptor alpha (ERα) (agonist)
Estrogen receptor beta (ERβ) (agonist)
G protein-coupled receptor 30 (GPR30) (antagonist)
No IC50, Ki, EC50, or DC50 values are reported in the literature for these targets. [1]

MTT assays using G-1 shows that, in SkBr3 cells, the proliferative effect induced by 100 nM G-1 is abolished in the presence of 1 μM estriol which acts as an antagonist of GPR30-dependent pathway. A cell-free transcription assay demonstrates that the antiestrogenic activity exhibited by estriol is because of interferring with estradiol-induced positive cooperative binding and receptor dimerization, binding of hER complexes to ERE, as well as reducing estradiol-dependent transcription in a dose-dependent manner. A recent study shows that estrogen (estrone, estradiol, and estriol) inhibits Alzheimers disease-associated low-order Aβ oligomer formation, and among them, estriol shows the strongest in vitro activity.

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Cell Assay: For the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay, cells are cultured in plastic 96-well plates under 200 μL of growth medium and at an initial density of 10,000 cells per well. Cells are washed once they have attached and further incubated in medium containing 2.5% charcoal-stripped FBS with the indicated treatment. The medium is changed every 2 days (with treatment). Where applicable, 200 ng of the indicated plasmids are transfected every 2 days before treatments using Fugene6 Reagent, as recommended by the manufacturer. Following 6 days of incubation, the assay mixture (10µL per well) containing 1mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) is added to each well and incubated at 37 ◦C for 4 h in a 5% CO2 atmosphere. The yellow tetrazolium MTT, reduced by metabolically active cells, results in intracellular purple formazan, which is released after overnight incubation with 200 µL 1% sodium dodecyl sulfate in 0.01N HCL and quantified spectrophotometrically by reading absorbance at 570nm using an enzyme-linked immunosorbent assay plate reader.

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Estriol (Oestriol) (1 μM) did not trigger ERK1/2 phosphorylation but prevented ERK activation induced by 1 μM G-1 (selective GPR30 agonist) and 5 μM 4-hydroxytamoxifen (OHT) in ER-negative SkBr3 breast cancer cells after 10 min exposure. [1]
Estriol (Oestriol) (1 μM) abolished c-fos and connective tissue growth factor (CTGF) expression induced by 1 μM G-1 and 5 μM OHT at both mRNA (2 h exposure) and protein (2 h exposure) levels in SkBr3 cells. [1]
Estriol (Oestriol) (1 μM) inhibited the proliferative effects induced by 100 nM G-1 in SkBr3 cells as assessed by MTT assay after 6 days of incubation (medium changed every 2 days with treatments). [1]
In ER-positive MCF7 cells, Estriol (Oestriol) (1 mM) induced up-regulation of ERα target genes pS2, progesterone receptor (PR), cathepsin D, and cyclin D1 at mRNA level after 24 h treatment, mimicking E2. [1]
Estriol (Oestriol) (1 mM) stimulated proliferation of MCF7 cells, which was inhibited by 1 μM ICI 182,780 (ER antagonist). [1]
In SkBr3 cells co-transfected with ERα or ERβ expression plasmids, Estriol (Oestriol) (100 nM) transactivated both ER isoforms in luciferase reporter assays. [1]

In mPTEN+/- mice, estriol treatments resulted in a 187.54% gain in the relative ratio of uterine wet weight to body weight; estriol also increases the ratio to 176.88% in wild-type mice. Estriol treatment (20 mg/kg ip), in vivo, sensitizes Kupffer cells to LPS via mechanisms dependent on an increase in CD14 by elevated portal blood endotoxin caused by increased gut permeability in rats; while one-half of the rats given estriol intraperitoneally 24 hours before an injection of a sublethal dose of LPS (5 mg/kg) died within 24 hours.

Competitive binding assay: SkBr3 cells were grown in 10-cm dishes, washed twice, and incubated with 1 nM [2,4,6,7-3H]E2 (89 Ci/mmol) in the presence or absence of increasing concentrations of non-labeled competitors (E2, E3, G-1, and OHT). Cells were incubated for 2 h at 37°C, washed three times with ice-cold PBS, and the radioactivity was collected by 100% ethanol extraction and measured by liquid scintillation counting. Competitor binding was expressed as a percentage of maximal specific binding. [1]
Molecular modeling and docking simulations: All ligand structures (E2, G-1, E3, OHT) were built and energy minimized. A homology model of GPR30 was built using bovine rhodopsin (PDB code 1U19, 40% similarity) as template with MODELLER. The model was energy minimized using ESFF force field and stereochemistry optimized with REFMAC5. Receptor and ligands were prepared using ADT (AutoDock tool). Polar hydrogens were added, Kollman charges assigned, and atomic solvation parameters added. AutoDock 3.05 was used for blind docking with a grid encompassing the whole protein surface. Lamarckian Genetic Algorithm with populations of 256 individuals, mutation rate 0.02, evolved for 100 generations. Cluster analysis based on RMSD (0.5 Å) was performed. The lowest energy conformation of the more populated cluster was considered as the most promising bioactive candidate. [1]

Cell culture: Human breast cancer MCF7 and SkBr3 cells were maintained in DMEM with phenol red and RPMI1640 without phenol red respectively, supplemented with 10% FBS. For treatments, cells were switched to medium without serum and phenol red 24 h before treatments. [1]
Transfection and luciferase assays: MCF7 cells were transfected with ERE-luciferase reporter (XETL) and pRL-CMV using Fugene6 in DMEM with 1% charcoal-stripped FBS. After 5-6 h, medium was replaced with serum-free DMEM lacking phenol red plus 1% CS-FBS, treatments added, incubated 16-18 h. SkBr3 cells were transfected similarly with reporter, pRL-CMV, and ERα or ERβ expression vectors where applicable. Luciferase activity measured with Dual Luciferase Kit. [1]
Reverse transcription and real-time PCR: Total RNA extracted using Trizol. cDNA synthesized using murine leukemia virus reverse transcriptase. Real-time PCR performed using Step One detection system. Gene-specific primers used for pS2, PR, cathepsin D, cyclin D1, c-fos, CTGF, and 18S (control). Assays performed in triplicate, relative standard curve method used. [1]
Gene silencing: SkBr3 cells plated onto 10-cm dishes, maintained in serum-free medium for 24 h, then transfected for additional 24 h or 48 h before treatments using Fugene6. Short hairpin construct against human GPR30 (shGPR30) and a GPR30 rescue vector containing silent mutations in the shRNA targeted sequence were used. [1]
Immunoblotting: Cells exposed to ligands, then lysed in lysis buffer (50 mM HEPES pH 7.5, 150 mM NaCl, 1.5 mM MgCl2, 1 mM EGTA, 10% glycerol, 1% Triton X-100, protease inhibitors). Protein concentrations determined by Bradford method. Lysates (10-50 μg protein) electrophoresed through reducing SDS/10% polyacrylamide gel, electroblotted onto nitrocellulose membrane. Antibodies against ERα, ERβ, c-fos, CTGF, β-actin, phosphorylated ERK1/2, ERK2, GPR30, and others were used. Proteins detected with HRP-linked secondary antibodies and ECL system. [1]
MTT assay: Cells cultured in 96-well plates at initial density of 10,000 cells per well in 200 μl growth medium. After attachment, cells incubated in medium containing 2.5% charcoal-stripped FBS with indicated treatments; medium changed every 2 days (with treatments). Where applicable, 200 μg of plasmids were transfected every 2 days before treatments. After 6 days, 10 μl MTT (1 mg/mL) added per well, incubated 4 h at 37°C in 5% CO2. Formazan released with 200 μl 1% sodium dodecyl sulfate in 0.01 N HCl overnight, absorbance measured at 570 nm. [1]

Dissolved in ethanol and corn oil; 4 μg/g/day; s.c. administration mPTEN+/- and wild-type mice

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Absorption, Distribution and Excretion
Estriol is readily absorbed after vaginal administration. Peak serum estriol concentrations are typically observed within 2 hours of vaginal administration and remain elevated for up to 6 hours. Systemic bioavailability after vaginal administration is superior to oral administration. In women with senile vaginal epithelial atrophy, intravaginal administration of 1 mg estriol achieves serum levels similar to those of oral administration of 10 mg estriol. Following intravaginal application of Gynest cream, plasma estriol levels increase approximately 50-fold from <90 pmol/L (26 pg/mL) within several hours. Eight to 10 hours after administration, estriol levels remain above 90 pmol/L (26 pg/mL) in 50% of women. Estriol circulates in the bloodstream, with approximately 14% existing in free form, 8% bound to sex hormone-binding globulin (SHBG), and the remainder bound to albumin. Over 95% of estriol is excreted in the urine, primarily as glucuronide.

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Metabolism/Metabolites
The main metabolites of estriol include 16-α-glucuronide, 3-glucuronide, 3-sulfate, and 3-sulfate 16-α-glucuronide.
Estrogen metabolic pathways include oxidative metabolism (primarily hydroxylation) and conjugation metabolism, the latter including glucuronidation, sulfonation, and/or O-methylation. Estradiol is converted to estrone by 17β-hydroxysteroid dehydrogenase; the resulting estrone is further metabolized to 16α-hydroxyestradiol, which is then converted to estriol.
Estriol is a common metabolite of estrone and estradiol-17β in animals and humans. In the human body, estriol is 2-hydroxylated and excreted in the form of conjugated and unconjugated 2-hydroxyestriol.

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Toxicity Summary
Estriol levels can be used to assess the overall health of the fetus. The fetal adrenal cortex produces dehydroepiandrosterone sulfate (DHEA-S), which the placenta converts into estriol. Abnormally low levels of unbound estriol in the pregnant woman may indicate a problem with fetal development. This drug interacts with target cell receptors. When estrogen receptors bind to their ligands, they enter the target cell nucleus, regulating gene transcription and generating messenger RNA (mRNA). The mRNA interacts with ribosomes to produce specific proteins that express the effects of estriol on target cells. 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. Toxicity Data
Oral (LD50): Acute: >2000 mg/kg [Rat].

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Mol Cell Endocrinol.2010 May 14;320(1-2):162-70;Lab Invest.2006 Mar;86(3):286-96.

Therapeutic Uses
Estriol is indicated for hormone replacement therapy in postmenopausal women for atrophic vaginitis and vaginal atrophy. Estriol is indicated for the treatment of vulvar itching and dyspareunia associated with vaginal epithelial atrophy. Drug Warnings Gynest cream is not suitable for use during pregnancy. If pregnancy occurs while using Gynest cream, treatment should be discontinued immediately. Gynest cream contains peanut oil and is contraindicated in patients with known peanut allergies. Because peanut allergy may be associated with soy allergy, patients with soy allergies should also avoid using Gynest cream. A detailed personal and family medical history should be obtained before starting or restarting hormone replacement therapy. Physical examinations (including pelvic and breast examinations) should follow this guideline, along with contraindications and warnings. Regular follow-up examinations are recommended during treatment; the frequency and method of follow-up should be determined on a case-by-case basis. Women should be informed which changes in their breasts need to be reported to their doctor or nurse. Examinations, including mammograms, should be performed according to currently accepted screening methods and adjusted based on individual clinical needs. Long-term use of systemic estrogen alone increases the risk of endometrial hyperplasia and cancer. The safety of long-term or repeated use of topical vaginal estrogen on the endometrium is uncertain. Therefore, if repeated treatment is required, a follow-up examination should be conducted at least annually, with particular attention paid to any symptoms of endometrial hyperplasia or cancer. For more complete data on drug warnings regarding estriol (40 total), please visit the HSDB records page.

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Pharmacodynamics
Estriol (also known as estriol) is one of the three main estrogens produced by the human body. It is only produced in large quantities by the placenta during pregnancy. Researchers at the UCLA Geffen School of Medicine found that estriol significantly reduced symptoms in pregnant women with multiple sclerosis (MS).

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Estriol (Oestriol) has been the subject of the "Estriol Hypothesis," which proposes a protective role against breast cancer based on its ability to antagonize carcinogenic effects of E2. Clinical and epidemiological studies as well as animal model observations have supported this hypothesis, but other studies in cultured breast cancer cells and further clinical data did not confirm beneficial effects. The dual agonist/antagonist properties of E3 were previously attributed to its inhibitory action on E2-ER complex dimerization. This study provides new insights showing that E3 can act either as an ER agonist or as a GPR30 antagonist depending on the receptor expression profile in different breast cancer cell contexts. In ER-negative tumors (including triple-negative breast cancer, TNBC), which lack ER, PR, and HER-2, GPR30 signaling may play an important role, and E3 might antagonize these signals. [1]

C18H24O3

288.39

288.172

50-27-1

Estriol (Standard);50-27-1;Estriol-d3;79037-36-8;Estriol-d2;53866-32-3;Estriol-d;55727-98-5;Estriol-13C3;1255639-56-5;Estriol-d3-1;2687960-79-6

5756

White to off-white solid powder

1.3±0.1 g/cm3

469.0±45.0 °C at 760 mmHg

280-282 °C(lit.)

220.8±23.3 °C

0.0±1.2 mmHg at 25°C

1.624

2.94

3

3

0

21

411

6

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

PROQIPRRNZUXQM-ZXXIGWHRSA-N

InChI=1S/C18H24O3/c1-18-7-6-13-12-5-3-11(19)8-10(12)2-4-14(13)15(18)9-16(20)17(18)21/h3,5,8,13-17,19-21H,2,4,6-7,9H2,1H3/t13-,14-,15+,16-,17+,18+/m1/s1

(8R,9S,13S,14S,16R,17R)-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthrene-3,16,17-triol

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)

Solubility in Formulation 1: ≥ 2.08 mg/mL (7.21 mM) (saturation unknown) in 10% DMSO + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 400 μL PEG300 and mix evenly; then add 50 μL Tween-80 to the above solution and mix evenly; then add 450 μL normal saline to adjust the volume to 1 mL.
Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH₂ O to obtain a clear solution.

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Solubility in Formulation 2: ≥ 2.08 mg/mL (7.21 mM) (saturation unknown) in 10% DMSO + 90% (20% SBE-β-CD in Saline) (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of 20% SBE-β-CD physiological saline solution and mix evenly.
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.

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Solubility in Formulation 3: ≥ 2.08 mg/mL (7.21 mM) (saturation unknown) in 10% DMSO + 90% Corn Oil (add these co-solvents sequentially from left to right, and one by one), clear solution.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.8 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 4: 5% DMSO +95%Corn oil: 10 mg/mL

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