- Estrogen receptor α (ERα): binds with four- to five-fold preference over ERβ; affinity is four-fold lower compared to ethinylestradiol (EE) and E2. [1]
- Estrogen receptor β (ERβ). [1]
- Does not bind sex hormone-binding globulin (SHBG). [2]

- In human umbilical vein endothelial cells (HUVEC), E4 (10⁻¹⁰ to 10⁻⁸ M, 48 h) significantly induced nitric oxide (NO) production and eNOS enzymatic activity. However, E4 was significantly less effective than equimolar E2. The concentration-effect curve was bell-shaped: higher concentrations (10⁻⁸ M) resulted in lower stimulation than lower concentrations (10⁻⁹ M). E4 also increased eNOS protein expression and Ser1177 phosphorylation. These effects were reduced by the pure estrogen receptor antagonist ICI 182,780. [2]
- In HUVEC co-treated with pregnancy-like E2 (10⁻⁸ M) and E4 (10⁻¹⁰ to 10⁻⁸ M, 48 h), E4 significantly reduced E2-induced NO synthesis, eNOS expression, and eNOS activity. [2]
- In HUVEC treated with postmenopausal-like E2 (10⁻¹⁰ M) plus E4, E4 did not significantly reduce E2-induced NO synthesis or eNOS expression. [2]
- Rapid treatment (30 min) of HUVEC with E4 (10⁻¹⁰ to 10⁻⁸ M) significantly increased NO release and eNOS enzymatic activity, and induced rapid phosphorylation of eNOS on Ser1177 without increasing eNOS expression. Increasing E4 concentrations beyond 10⁻⁹ M did not further increase NO synthesis. ICI 182,780 reduced these rapid effects. [2]
- E4 (10⁻¹⁰ M, 30 min) increased Akt phosphorylation (Thr308) in HUVEC; higher concentrations (10⁻⁹ to 10⁻⁸ M) resulted in progressive decrease. ICI 182,780 reduced Akt phosphorylation induced by low-dose E4. [2]
- In HUVEC, E4 decreased in a concentration-dependent manner eNOS and Akt phosphorylation induced by pregnancy-like E2 (10⁻⁸ M), but did not affect these phosphorylations when co-administered with postmenopausal-like E2 (10⁻¹⁰ M). [2]
- E4 treatment (3 weeks) in ovariectomized mice did not significantly affect platelet aggregation in washed platelets stimulated by thrombin, U46619 (thromboxane A2 analog), or collagen, as assessed by Light Transmission Aggregometry (LTA). [1]
- Ex vivo flow-based adhesion assay: Whole blood from E4-treated mice (6 mg/kg/day for 3 weeks) perfused over a collagen matrix at arterial shear rate (60 dynes/cm²) formed smaller thrombi compared to blood from untreated mice. At high pathological shear rate (160 dynes/cm²), thrombi from E4-treated mice grew at a slower rate. Platelet interaction with fibrinogen was not affected. [1]

- In ovariectomized (OVX) mice treated with E4 (6 mg/kg/day via subcutaneous osmotic minipump for 3 weeks), tail-bleeding time after 3-mm tail-tip transection was significantly prolonged compared to vehicle-treated OVX mice. [1]
- E4 treatment protected mice from acute systemic thromboembolism induced by intravenous injection of collagen (0.4 mg/kg) and epinephrine (60 mg/kg): 50% of E4-treated mice survived, while most vehicle-treated mice died within 10 min. [1]
- In a ferric chloride (FeCl₃)-induced carotid artery thrombosis model, 70% of E4-treated mice were protected against occlusive thrombosis. [1]
- In inferior vena cava (IVC) stasis (24 h) and stenosis (48 h) models, E4 treatment significantly reduced thrombus weight compared to OVX controls (p < 0.001). [1]
- In bone marrow chimeric mice: E4 treatment prolonged tail-bleeding time in mice engrafted with wild-type bone marrow (ERα-AF2⁺/⁺) but not significantly different in mice engrafted with mutant bone marrow lacking nuclear ERα activity (ERα-AF2⁰). However, the protective effect of E4 against thromboembolism was lost in mice reconstituted with ERα-AF2⁰ bone marrow (16 of 17 died within 10 min), demonstrating that hematopoietic nuclear ERα activation contributes to E4's protective action. [1]

- eNOS activity assay: HUVEC lysates were incubated with ^3H-arginine, and conversion to ^3H-citrulline was measured using an acidic ion-exchange resin. Results were expressed as picomoles of converted citrulline per milligram of protein extract. Extracts incubated with the eNOS inhibitor (N-nitro-L-arginine methyl ester) were used as blanks. [2]

- NO production (nitrite assay): HUVEC were treated with E4 and/or E2 for 48 h or 30 min. The fluorescent product derived from reaction of nitrite with 2,3-diaminonaphthalene was measured (excitation/emission 365/450 nm). Standard curves were constructed with sodium nitrite. Non-specific fluorescence was determined in the presence of NG-monomethyl-L-arginine. [2]
- Western blotting: HUVEC lysates were separated on 10% SDS-PAGE, transferred to PVDF membranes, and probed with antibodies against eNOS, Ser1177-p-eNOS, Akt, Thr308-p-Akt, ERα, and ERβ. Blots were blocked in 5% BSA, incubated with primary antibody at 4°C overnight, then with HRP-conjugated secondary antibody for 2 h at room temperature. Chemiluminescence was recorded, and densitometric analysis was performed using ImageJ. Anti-actin antibody was used as loading control. [2]
- Light Transmission Aggregometry (LTA): Washed platelets from OVX mice treated or not with E4 were stimulated with thrombin, U46619, or collagen. Aggregation was assessed with a dual-channel aggregometer with stirring at 900 rpm at 37°C for 5 min. [1]
- Ex vivo flow-based thrombus formation assay: Heparinized whole blood from E4-treated or control mice was perfused through a collagen-coated microcapillary at arterial shear rate (60 dynes/cm², 1500 s⁻¹) for 2 min, or at low shear (20 dynes/cm², 500 s⁻¹) followed by high shear (160 dynes/cm², 4000 s⁻¹). Thrombus growth was monitored by video microscopy, and surface coverage (%) was analyzed using ImageJ software. [1]

- Ovariectomy: Female C57BL/6J mice (4 weeks old) were anesthetized with ketamine (25 mg/kg) and xylazine (10 mg/kg) intraperitoneally, then ovariectomized. Treatments started approximately 2 weeks after ovariectomy. E4 was dissolved in 60% ethanol and 40% PBS. OVX mice were implanted subcutaneously with osmotic minipumps releasing either vehicle or E4 at 6 mg/kg/day. Mice were euthanized after 3 weeks of treatment. [1]
- Tail-bleeding time: After anesthesia, a 3-mm tail-tip transection was made. Blood drops were removed every 15 seconds with a paper filter. Bleeding was considered stopped if it did not recur within 30 seconds of cessation. Experiments were terminated after 30 minutes if no cessation occurred. [1]
- Thromboembolism model: Collagen (0.4 mg/kg) and epinephrine (60 mg/kg) mixture was injected into the right jugular vein of anesthetized mice. Mice were euthanized 10 min after injection, lungs were excised, formalin-fixed, paraffin-sectioned (5 μm thick), stained with hematoxylin-eosin, and analyzed histologically for thrombi. [1]
- Inferior vena cava (IVC) stasis model: IVC was ligated below the renal veins with an 8.0 polypropylene suture to obtain complete blood stasis. Mice were euthanized after 24 h, thrombi were dissected, weighed, and processed for histology. [1]
- IVC stenosis model: IVC ligation was performed over a 30-gauge needle placed outside the vessel to decrease vascular lumen by about 90% without endothelial injury. Needle was then removed. Mice were euthanized after 48 h, thrombi were weighed. [1]
- Carotid artery thrombosis: FeCl₃ solution (7%) saturated on a 1×4-mm paper strip was applied to the adventitial surface of the left carotid artery for 2 min then removed. Blood flow was monitored continuously with flow probes connected to a flow meter. [1]
- Bone marrow transplantation: Recipient OVX mice were lethally irradiated (9.2 Gy, γ source) then intravenously reconstituted with bone marrow cells from ERα-AF2⁰ mice or wild-type littermates (ERα-AF2⁺/⁺). Three weeks later, transplanted mice were implanted or not with E4 osmotic minipumps (6 mg/kg/day). Enrofloxacin was added to drinking water for 3 weeks after transplantation. Efficiency was confirmed by PCR. [1]

Absorption, Distribution and Excretion
Estetrol is rapidly absorbed from the gastrointestinal tract. According to a pharmacokinetic study, the peak plasma concentration (Cmax) of Estetrol is 18 ng/mL, and the AUC is 36.4 ng•h/mL. When Estetrol and drospirenone are administered as a single formulation, a maximum serum concentration of approximately 48.7 ng/mL can be reached within 1–3 hours. The bioavailability of this combination is between 76% and 85%. According to a clinical study, the time to peak concentration (Tmax) is 0.5 to 2 hours, and the time to steady-state plasma concentration is approximately 4 days. Estrogens are typically excreted as sulfate and glucuronidated derivatives. Approximately 69% of the Estetrol dose is excreted in the urine, and approximately 22% is excreted unchanged in the feces. Limited distribution of Estetrol in erythrocytes has been demonstrated. Metabolism/Metabolites Estetrol is rapidly metabolized after oral administration. Phase II metabolism of estrogen forms glucuronide and sulfate conjugates, the latter of which have negligible estrogenic activity in vitro. In vitro metabolic studies show that UGT2B7 catalyzes the formation of E4-16-glucuronide. Estetrol is used in combination with drospirenone in formulations. Hepatic cytochrome P450 enzyme CYP3A4 metabolizes drospirenone into two major metabolites: one is the drospirenone acid form, generated through ring-opening of the lactone ring; the other is 4,5-dihydrodrospirenone, generated by reduction followed by sulfation. Neither of these metabolites has pharmacological activity.
Biological Half-Life
The elimination half-life of Estetrol is approximately 27 hours. The half-life range may be between 19 and 40 hours.

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- Average half-life in humans: 28 hours, approximately two-fold longer than E2. [2]
- E4 is minimally metabolized and completely excreted in urine unaltered. [2]
- E4 does not inhibit cytochrome P450 liver enzymes. [2]

Protein Binding
Estetrol binds to plasma proteins at a rate of 46-50%. It does not bind to sex hormone-binding globulin (SHBG). One study showed that Estetrol binds moderately to human plasma proteins (45.5%-50.4%) and human serum albumin (58.6%), but binds less strongly to human α1-glycoprotein (11.2%).

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- In a phase II clinical trial as an oral contraceptive (daily dose 20 mg E4 combined with a progestin), E4 had little or no effect on sex hormone-binding globulin, angiotensinogen, or coagulation factors, suggesting a “liver friendly” profile and not increasing the risk of thromboembolic events based on current knowledge. [1]

[1]. Effect of estetrol, a selective nuclear estrogen receptor modulator, in mouse models of arterial and venous thrombosis. Mol Cell Endocrinol. 2018 Dec 5;477:132-139.

[2]. Estetrol Modulates Endothelial Nitric Oxide Synthesis in Human Endothelial Cells. Front Endocrinol (Lausanne). 2015 Jul 22;6:111.

Estetrol (E4) is a 3-hydroxy steroid, formed by replacing the 15α and 16α positions of 17β-Estetrol with two additional hydroxyl groups. It is a natural estrogen produced only by the fetal liver during pregnancy. It has multiple functions, including as an estrogen, estrogen receptor agonist, human metabolite, exogenous substance metabolite, and oral contraceptive. It is a 3-hydroxy steroid, 17β-hydroxy steroid, 16α-hydroxy steroid, 15α-hydroxy steroid, and steroid hormone. It is derived from the hydrogenation of Estetrol. Natural or synthetic steroid estrogens have a wide range of medicinal uses, ranging from hormonal contraception to treating menopausal symptoms. Estetrol (E4) is a natural estrogen produced naturally during pregnancy, but it can also be synthesized from plants and used for contraception. It is more effective and safer than ethinylEstetrol (EE2), the synthetic estrogen found in 97% of oral contraceptives, and reduces the accumulation of harmful endocrine disruptors (EDCs) in the environment, which often lead to harmful epigenetic effects. On April 15, 2021, Estelle/Nextstellis, an oral contraceptive from Mayne Pharma Group Limited and Mithra Pharmaceuticals, received FDA approval. This drug is a combination of drospirenone and Estetrol. Estetrol is the first novel estrogen introduced to the United States in over 50 years and the first Estetrol product approved globally. The combination of drospirenone and Estetrol provides women seeking contraceptive treatment with a safer new option. In Canada, Nextstellis was approved in March 2021; it was developed by Mithra and marketed by Searchlight Pharma. Anhydrous Estetrol is an estrogen. Its mechanism of action is as an estrogen receptor agonist. Therapeutic Estetrol is a synthetic steroid similar to or identical to endogenous Estetrol. It is a short-acting estrogen with dual estrogen receptor agonist and antagonist activities. After oral administration, therapeutic Estetrol binds to estrogen receptors, acting as a selective estrogen receptor modulator (SERM). It exhibits estrogen agonist activity in some tissues and estrogen antagonist activity in others. Estrogenol exhibits weak estrogenic activity in the uterus but acts as an estrogen antagonist in breast tissue. Endogenous estriol is produced solely by the human fetal liver and is the main metabolite of estrogen biosynthesis in the human fetal liver. Estriol is a metabolite of estriol with a 15-α-hydroxyl group. Estriol can be converted from estriol sulfate or dehydroepiandrosterone sulfate in the placental-fetal unit. Drug Indications: Estriol, in combination with drospirenone, is used for pregnancy prevention.

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Mechanism of Action
Estriol is a synthetic analog of estrogen that is naturally occurring during pregnancy. It is selective for both estrogen receptor α (ER-α) and estrogen receptor β (ER-β) and inhibits ovulation. Estriol has low to moderate affinity for human estrogen receptor α (ER-α) and estrogen receptor β, but it tends to bind more strongly to ER-α. Estrogens exhibit a unique mechanism of action through tissue-selective activity, showing estrogen receptor agonist activity in the vagina, uterus, and endometrium, while exhibiting estrogenic activity in breast tissue.

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- E4 acts as an estrogen agonist in some tissues (bone, brain, vagina, endometrium) and as an antagonist in others (breast), sharing properties with selective estrogen receptor modulators (SERMs). [1]
- Unlike E2, E4 does not elicit endothelial NO synthase activation nor accelerate endothelial healing, but rather prevents E2 actions in the endothelium. [1]
- E4 binds nuclear ERα and ERβ with four- to five-fold preference for ERα and four-fold lower affinity than E2 or EE. It modulates ER actions in a tissue-specific manner through selective nuclear (but not membrane) ERα activation. [1]
- E4 was evaluated in a phase II clinical trial as an oral contraceptive in combination with a progestin. [1]
- In mice, chronic E2 treatment decreased platelet responsiveness, increased bleeding times, and protected against thromboembolism through hematopoietic ERα, but E4 had no significant impact on platelet aggregation in suspension. [1]
- E4 is produced exclusively by the human fetal liver during pregnancy. Concentrations increase exponentially during pregnancy, peak at term, with fetal levels about 10-20 times higher than maternal levels. After delivery, E4 becomes rapidly undetectable. [2]
- In rats, E4 acts as an estrogen on bone, brain, vagina, and endometrium, but as an antagonist on breast (DMBA model), preventing new breast tumor development and stimulating regression of existing ones. [2]

C18H24O4

304.386

304.167

C, 71.03; H, 7.95; O, 21.02

15183-37-6

Estetrol-d4

27125

White to off-white solid powder

1.343 g/cm3

491.9ºC at 760 mmHg

233-236

231.7ºC

1.65

1.55

4

4

0

22

441

7

C[C@@]12CC[C@@H]3C4=CC=C(C=C4CC[C@H]3[C@@H]2[C@H]([C@H]([C@@H]1O)O)O)O

AJIPIJNNOJSSQC-NYLIRDPKSA-N

InChI=1S/C18H24O4/c1-18-7-6-12-11-5-3-10(19)8-9(11)2-4-13(12)14(18)15(20)16(21)17(18)22/h3,5,8,12-17,19-22H,2,4,6-7H2,1H3/t12-,13-,14-,15-,16-,17+,18+/m1/s1

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

15α-Hydroxyestriol; E4; ESTETROL; 15183-37-6; Estetrol anhydrous; 15alpha-hydroxyestriol; estetrolum; Estetrol

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)

DMSO : ~100 mg/mL (~328.54 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.21 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 25.0 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: ≥ 2.5 mg/mL (8.21 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 25.0 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: ≥ 2.5 mg/mL (8.21 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 25.0 mg/mL clear DMSO stock solution to 900 μL of corn oil and mix evenly.

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