mERα (IC50 = 2.3 nM); hERα (IC50 =28 nM); HBx

Estradiol benzoate is an asynthetic ester of the natural estrogen estradiol, more precisely its 3-benzoyl ester. The development of the female genotype during embryogenesis and puberty is regulated by estrogens. The primary estrogen that the premenopausal ovary secretes is estradiol. In order to induce estrus in domestic livestock, estradiol benzoate, an estradiol analog with a benzyl ester at the C-3 position, is frequently used in conjunction with a progestin. With IC50 values between 22 and 28 nM, this compound binds to the estrogen receptor α (ERα) in humans, mice, and chickens. When compared to estradiol, this indicates a binding affinity reduction of 6–10 fold.[1]

In the Ovx female model, estradiol benzoate (20–100 μg/kg, subcutaneous injection, once daily for 4-5 weeks) enhances learning and memory behavior [1]. Estradiol benzoate (subcutaneous injection, single dose, 0.015–15000 μg/kg)

HBx plays a significant role in the cccDNA epigenetic modification regulating the hepatitis B virus (HBV) life cycle and in hepatocyte proliferation and carcinogenesis. By using the sleeping-beauty transposon system, we constructed a tetracycline-induced HBx-expressing stable cell line, SBHX21. HBx with a HiBiT tag can be quickly detected utilizing a NanoLuc-based HiBiT detection system. By screening a drug library using SBHX21 cells, we identified estradiol benzoate as a novel anti-HBx agent. Estradiol benzoate also markedly reduced the production of HBeAg, HBsAg, HBV pgRNA, and HBV DNA in a dose-dependent manner, suggesting that estradiol benzoate could be an anti-HBV agent. Docking model results revealed that estradiol benzoate binds to HBx at TRP87 and TRP107. Collectively, our results suggest that estradiol benzoate inhibits the HBx protein and HBV transcription and replication, which may serve as a novel anti-HBV molecular compound for investigating new treatment strategies for HBV infection.[3]

Cytotoxicity Assay[3]
The cytotoxic effects of estradiol benzoate and 17β-estradiol were assessed by the Cell Counting kit-8 (CCK8) assay. The HepG2 cells were cultured in 96-well plates at 1 × 104 cells per well with three replicates per group in a concentration gradient of 1 µM, 50 µM, 100 µM, 150 µM, 200 µM, 250 µM, 300 µM, 350 µM, 400 µM, 450 µM, 500 µM, 550 µM, 600 µM, 650 µM, and 700 µM. The DMSO was normalized to 1% in all treatment groups. After 24 h, the medium was replaced with 10% CCK-8 diluted in fresh DMEM. CCK-8 solution and culture media (no cells) were added to the blank control. The absorbance (OD value) was recorded. Cell survival rate curves and IC50s were calculated using GraphPad Prism.

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Western Blot[3]
To analyze the expression of HiBiT-HBx, the SBHX21 cells were induced with Dox (1 µg/mL) for 48 h in a 12-well plate. Then, the cells were treated with different concentrations of estradiol benzoate and 17β-estradiol for 24 h without Dox. We used 0.5% DMSO DMEM as the control group. After treatment, the cells were lysed with 100 µL of RIPA buffer. The cell extracts were separated on sodium dodecyl-sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and blotted onto a nitrocellulose (NC) blotting membrane. The protein expression was analyzed using mouse monoclonal to Hepatitis B Virus X antigen and was visualized with an Odyssey CLX System.

Animal/Disease Models: Ovx mouse [1]
Doses: 20, 100 or 200 μg/kg
Route of Administration: subcutaneous injection
Experimental Results: Significant It reverses Ovx-induced decrease in uterine weight. There was no significant effect on the locomotor activity of Ovx mice. Effectively reverses Ovx-induced changes in synaptic cleft enlargement and PSD thickness reduction. Replacement with EB (100 μg/kg) for 4 weeks increased the number and density of synaptic vesicles.

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There is increasing evidence that estrogen is involved in CNS activity, particularly memory. Several studies have suggested that estrogen improves memory by altering neuronal plasticity, including increased hippocampus CA1 dendritic spine density and enhanced long-term potentiation (LTP). In the present study, we investigated the effects of estrogen on the ultrastructural modifications in cerebral frontal cortex and hippocampus of female ovariectomized mice. One week after ovariectomy (Ovx), ICR female mice received daily injection of estradiol benzoate (EB, 20, 100, 200 microg/kg, s.c.) for 4-5 weeks. Spatial memory was then tested in the water maze, and the overall locomotor activity was monitored in open field. Synaptic morphologic parameters were examined using a graph analyzer. The results from open field did not show any alterations in locomotor activity following Ovx and EB replacement. Both the latency to find the platform and the distance to reach the platform were significantly reduced in Ovx mice by EB at 20 or 100 microg/kg when compared to vehicle treated Ovx mice. The results from synaptic ultrastructural measurement and analysis did not show any differences in hemispheric or hippocampal volumes, the numeric synaptic density, the length of active zones, or the curvature of synaptic interface among Sham, Ovx, and Ovx plus EB replacement mice. However, EB replacement effectively normalized the changes induced by Ovx, reducing the width of the synaptic cleft, enlarging the thickness of postsynaptic density (PSD), and increasing the number of synaptic vesicles in the presynapse in both cerebral cortex Fr1 and hippocampus CA1 areas. These results suggest that the beneficial effects of EB on improving memory behavior of Ovx female mice are associated with the changes of some subtle structural parameters of synapses, including the width of PSD and synaptic cleft rather than some basic and permanent structure in frontal cortex and hippocampus regions.[1]

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The current project was designed to determine the dose-response relationship of the prostate gland to estradiol exposure during the developmentally critical neonatal period in the rat. Male Sprague-Dawley (SD) rats were treated on Days 1, 3, and 5 of life by s.c. injections of a 7-log range of doses (0.015 microg/kg to 15.0 mg/kg) of beta-estradiol-3-benzoate (EB) in 25 microl of peanut oil (Arachis) as vehicle. In a separate block, neonatal Fisher 344 (F344) rats received 0.15, 15.0, or 1500.0 microg EB/kg. Rats were killed on Postnatal Day (PND) 35 or 90, and the prostates were microdissected, weighed, and frozen for immunohistochemistry. Preputial separation and hepatic testosterone hydroxlase activities were monitored and measured to determine the onset of puberty. On PND 35, there was an increase in prostate weights of SD rats treated with low doses of EB and a decrease in prostate weights of SD rats treated with high doses. The low-dose effect was entirely abolished by PND 90, and only high-dose suppression of organ sizes was found. The transient nature of the effect in low-dose animals suggests an advancement of puberty as the cause for increased reproductive organ weights on PND 35. F344 rats were more sensitive than SD rats to the suppressive effects of high doses of neonatal EB on PND 90. Despite this heightened responsiveness in the F344 rats, a low-dose estrogenic effect on adult prostate weights was not observed. Thus, in the rat model a sustained effect at low doses of natural estrogens is not present in the prostate glands.[2]

Metabolism / Metabolites
Exogenous estrogens are metabolized using the same mechanism as endogenous estrogens. Estrogens are partially metabolized by cytochrome P450.

Protein Binding
Estrogens circulate in the blood largely (>95%) bound to sex hormone binding globulin (SHBG) and to albumin.

[1]. Effects of estradiol benzoate on learning-memory behavior and synaptic structure in ovariectomized mice . Life sciences, 2006, 79(16): 1553-1560.

[2]. Neonatal low-and high-dose exposure to estradiol benzoate in the male rat: I. Effects on the prostate gland . Biology of reproduction, 2001, 65(5): 1496-1505.

[3]. Identification of Estradiol Benzoate as an Inhibitor of HBx Using Inducible Stably Transfected HepG2 Cells Expressing HiBiT Tagged HBx . Molecules, 2022, 27(15): 5000.

[4]. Investigation of Antiparasitic Activity of Two Marine Natural Products, Estradiol Benzoate, and Octyl Gallate, on Toxoplasma gondii In Vitro . Frontiers in Pharmacology, 2022, 13: 841941.

[5]. Effect of estradiol benzoate on reproductive organs and fertility in the male rat . European Journal of Obstetrics & Gynecology and Reproductive Biology, 1983, 15(3): 189-198.

[6]. Macromolecular prodrugs XI. Synthesis and characterization of polymer-estradiol conjugate. Int J Pharm. 2004 Nov 5;285(1-2):35-41.

[7]. Role of sex steroid hormones in bacterial-host interactions. Biomed Res Int. 2013;2013:928290.

[8]. Estradiol benzoate.

17beta-estradiol 3-benzoate is a benzoate ester resulting from the formal condensation of benzoic acid with the phenolic hydroxy group of 17beta-estradiol. It has a role as a xenoestrogen and an estrogen receptor agonist. It is a benzoate ester and a 17beta-hydroxy steroid. It is functionally related to a 17beta-estradiol.
Estradiol Benzoate is a pro-drug ester of [DB00783], a naturally occurring hormone that circulates endogenously within the human body. Estradiol is the most potent form of all mammalian estrogenic steroids and acts as the major female sex hormone. As a pro-drug of estradiol, estradiol benzoate therefore has the same downstream effects within the body through binding to the Estrogen Receptor (ER) including ERα and ERβ subtypes, which are located in various tissues including in the breasts, uterus, ovaries, skin, prostate, bone, fat, and brain. [DB00783] is commonly produced with an ester side-chain as endogenous estradiol has very low oral bioavailability on its own (2-10%). First-pass metabolism by the gut and the liver quickly degrades the estradiol molecule before it gets a chance to enter systemic circulation and exert its estrogenic effects. Esterification of estradiol aims to improves absorption and bioavailability after oral administration (such as with Estradiol valerate) or to sustain release from depot intramuscular injections (such as with Estradiol Cypionate) through improved lipophilicity. Following absorption, the esters are cleaved, resulting in the release of endogenous estradiol, or 17β-estradiol. Ester pro-drugs of estradiol are therefore considered to be bioidentical forms of estrogen. Estradiol benzoate is not currently available in Canada or the US.
Estradiol Benzoate is the synthetic benzoate ester of estradiol, a steroid sex hormone vital to the maintenance of fertility and secondary sexual characteristics in females. As the primary, most potent estrogen hormone produced by the ovaries, estradiol binds to and activates specific nuclear receptors. This agent exhibits mild anabolic and metabolic properties, and increases blood coagulability. (NCI04)
See also: Estradiol (has active moiety); Estradiol Benzoate; Progesterone (component of); Estradiol Benzoate; Testosterone Propionate (component of) ... View More ...

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Drug Indication
Estradiol benzoate is not currently available in any FDA or Health Canada approved products.

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Mechanism of Action
Estradiol enters target cells freely (e.g., female organs, breasts, hypothalamus, pituitary) and interacts with a target cell receptor. When the estrogen receptor has bound its ligand it can enter the nucleus of the target cell, and regulate gene transcription which leads to formation of messenger RNA. The mRNA interacts with ribosomes to produce specific proteins that express the effect of estradiol upon the target cell. Estrogens increase the hepatic synthesis of sex hormone binding globulin (SHBG), thyroid-binding globulin (TBG), and other serum proteins and suppress follicle-stimulating hormone (FSH) from the anterior pituitary.

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Pharmacodynamics
Estradiol, the principal intracellular human estrogen, is substantially more active than its metabolites, estrone and estriol, at the cellular level.

C25H28O3

376.49

376.203

C, 79.76; H, 7.50; O, 12.75

50-50-0

Alpha-Estradiol;57-91-0;Estradiol (Standard);50-28-2;Estradiol-d3;79037-37-9;Estradiol-d4;66789-03-5;Estradiol-d5;221093-45-4;Estradiol-13C2;82938-05-4;Estradiol (cypionate);313-06-4;Estradiol benzoate;50-50-0;Estradiol enanthate;4956-37-0;Estradiol hemihydrate;35380-71-3;Estradiol-d2;53866-33-4;Estradiol-13C6;Estradiol-d2-1;3188-46-3;rel-Estradiol-13C6; 979-32-8 (valerate); 113-38-2 (dipropionate); 57-63-6 (ethinyl); 172377-52-5 (sulfamate); 3571-53-7 (undecylate)

222757

White to off-white solid powder

1.2±0.1 g/cm3

531.2±50.0 °C at 760 mmHg

191-198 °C(lit.)

212.0±22.9 °C

0.0±1.5 mmHg at 25°C

1.604

6.24

1

3

3

28

582

5

O([H])[C@@]1([H])C([H])([H])C([H])([H])[C@@]2([H])[C@]3([H])C([H])([H])C([H])([H])C4C([H])=C(C([H])=C([H])C=4[C@@]3([H])C([H])([H])C([H])([H])[C@@]21C([H])([H])[H])OC(C1C([H])=C([H])C([H])=C([H])C=1[H])=O

UYIFTLBWAOGQBI-BZDYCCQFSA-N

InChI=1S/C25H28O3/c1-25-14-13-20-19-10-8-18(28-24(27)16-5-3-2-4-6-16)15-17(19)7-9-21(20)22(25)11-12-23(25)26/h2-6,8,10,15,20-23,26H,7,9,11-14H2,1H3/t20-,21-,22+,23+,25+/m1/s1

[(8R,9S,13S,14S,17S)-17-hydroxy-13-methyl-6,7,8,9,11,12,14,15,16,17-decahydrocyclopenta[a]phenanthren-3-yl] benzoate

Estradiol monobenzoate; Benzo-Gynoestryl; Ovasterol-B; Oestradiol benzoate; Oestradiol benzoate; Benovocylin; Estradiol 3-benzoate; Estradiol monobenzoate; Benzo-Gynoestryl; Estradiol Benzoate

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: 19~100 mg/mL (265.61~50.5 mM)
H2O: ~0.1 mg/mL (~0.3 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (6.64 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.)