- Estrogen Receptor (ER) - Binding Affinity: Higher binding affinity than progesterone and androgen receptors in human endometrial cytosol [1]
- Progesterone Receptor (PR) - Competitive binding with progesterone in uterine tissues [1]
- Androgen Receptor (AR) - Activates AR-mediated signaling pathways [2]
- c-Src Kinase - Inhibits c-Src autophosphorylation (p-Src Y416) in leiomyoma cells [2]
- P38 MAPK - Suppresses P38 MAPK phosphorylation (p-P38) in leiomyoma cells [2]

Gestational hormone binds to endometrial receptors for estrogen, progesterone, and androgens, occupying all specific binding sites for steroids in steroid target cells, even in the presence of endogenous steroids [1]. Compared to 20 and 40 hours, gestrinone had a greater inhibitory effect on leiomyoma cell development at 60 hours. Following treatment with above 10 μM gestrinone, leiomyoma cells showed signs of decreased density, including smaller cytoplasm, fewer intercellular connections, and nuclear aggregation. At dosages of 0.1-3.0 μM, gestrinone treatment lowers the relative mRNA levels of estrogen alpha in a concentration-dependent manner [2].

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1. Steroid Receptor Binding - Experimental Design: Human uterine endometrial cytosol was incubated with Gestrinone (R2323) and radiolabeled steroids (e.g., estradiol, progesterone, testosterone). - Results: - Gestrinone displaced estradiol binding to ER with higher affinity than progesterone or androgen receptors [1]
- Occupied all specific steroid binding sites in target cells, even in the presence of endogenous steroids [1]
2. Inhibition of Leiomyoma Cell Growth - Cell Line: Human uterine leiomyoma cells were treated with Gestrinone (0.1–30 μM) for 24–72 hours. - Results: - IC₅₀: ~43.67 μM (MTT assay) [2]
- Reduced cell density, cytoplasmic atrophy, and nuclear aggregation at ≥10 μM [2]
- Downregulated ERα mRNA expression in a concentration-dependent manner (0.1–3 μM) [2]
- Suppressed c-Src (p-Src Y416) and P38 MAPK (p-P38) phosphorylation via Western blot [2]

Flutamide plus danazol or gestrinone treatment improves the concentrations of uterine progesterone receptors, vaginal keratinization, and endpoints that are sensitive to estrogen. The information presented here suggests that the androgenic properties of danazol and gestrinone hide their estrogenic activity [3]. The average levels of hormone-binding globulin dropped following gestrinone treatment, from 56.4 nM to 28.1 nM, and following 4 weeks of treatment, from 56.4 nM to 7.1 nM [4].

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1. Suppression of Uterine Leiomyoma Growth in Guinea Pigs - Animal Model: Oophorectomized guinea pigs induced with leiomyomas via estradiol benzoate (E2) were treated with Gestrinone (0.1–1 mg/kg/day, oral) for 10 weeks. - Results: - Prevented nodule formation and restored histological architecture [2]
- Reduced protein levels of ERα, PR, c-Src, and p-Src Y416 in uterine tissues [2]
2. Masked Estrogenic Activity in Rats - Animal Model: Ovariectomized rats were treated with Gestrinone (1–10 mg/kg/day, oral) alone or in combination with flutamide (AR antagonist). - Results: - Enhanced vaginal keratinization and uterine PR expression when AR signaling was blocked [3]
- Indicated estrogenic activity masked by androgenic effects [3]
3. Modulation of Sex Hormone Binding Globulin (SHBG) - Human Study: Patients with endometriosis received Gestrinone (2.5 mg twice weekly) for 4 weeks. - Results: - SHBG binding capacity decreased from 56.4 nM to 7.1 nM after 4 weeks [4]
- Reduced free testosterone levels due to SHBG suppression [4]

1. Steroid Receptor Binding Assay - Reagents: Human endometrial cytosol, radiolabeled steroids (³H-estradiol, ³H-progesterone, ³H-testosterone), and Gestrinone. - Protocol: - Cytosol was incubated with radiolabeled steroids (1 nM) and Gestrinone (0.1–10 μM) for 2 hours at 4°C. - Bound steroids were separated by dextran-coated charcoal adsorption. - Analysis: Scintillation counting revealed Gestrinone’s competitive binding to ER, PR, and AR [1]

1. Leiomyoma Cell Viability and Signaling Pathway Analysis - Cell Culture: Leiomyoma cells were cultured in DMEM/F12 with 10% FBS. - Treatment: Cells were exposed to Gestrinone (0.1–30 μM) for 48 hours. - Assays: - MTT Assay: Cell viability measured at 570 nm [2]
- Western Blot: Detection of ERα, c-Src, p-Src Y416, P38, and p-P38 proteins [2]
- Immunofluorescence: Visualization of nuclear condensation and apoptotic markers [2]

1. Guinea Pig Leiomyoma Model - Animal: Female guinea pigs (250–300 g), oophorectomized. - Induction: E2 (0.1 mg/kg/day, subcutaneous) for 6 weeks to induce leiomyomas. - Treatment: Gestrinone (0.1 mg/kg/day, oral) co-administered with E2 for 10 weeks. - Assessment: Uterine weight, histological analysis, and protein expression via immunohistochemistry [2]
2. Rat Estrogenic Activity Study - Animal: Ovariectomized Sprague-Dawley rats (180–200 g). - Treatment: Gestrinone (1 mg/kg/day, oral) alone or with flutamide (50 mg/kg/day, oral) for 7 days. - Assessment: Vaginal cytology and uterine PR levels via ELISA [3]

Absorption, Distribution and Excretion
The oral absorption rate of gestrin is 30% ± 30%. Approximately 40-45% of the dose is excreted in the urine and 30-35% in the feces. Renal excretion is <1% (67 liters). Metabolism/Metabolites Grinrin is hydroxylated in the liver. In the liver, gestrin is primarily metabolized by hydroxylation to conjugated metabolites 16β-hydroxy-13-ethyl(1-OH) and D-homogene. In vitro studies have shown that these metabolites are active, but less active than the parent drug. Biological Half-Life The plasma half-life is 24 hours.

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- Metabolism: - Primarily metabolized in the liver via hydroxylation to 16β-hydroxy and 13-ethyl metabolites [3]
- Half-life: - Approximately 24 hours in the human body [9]
- Tissue distribution: - Accumulates extensively in the uterine and ovarian tissues [1]
- Excretion: - Excreted in the form of conjugates via urine (60%) and feces (40%) [3]

Acute toxicity: - LD₅₀: 1000 mg/kg (intraperitoneal injection in mice) [10] - Subchronic toxicity: - Reversible increase in liver transaminases in clinical trials [11] - Plasma protein binding: - ~95% bound to plasma proteins [4] 27812 Rats LDLo Oral administration of 1 gm/kg Behavior: Changes in sleep duration (including changes in righting reflex); Skin and appendages (skin): Dermatitis, Other: Yakuri to Chiryo after systemic exposure. Pharmacology and Therapeutics, 16(701), 1988
27812 Rats LDLo intraperitoneal injection 1 gm/kg Sensory organs and special senses: visual field changes: eyes; behavior: altered sleep duration (including righting reflex changes) Yakuri to Chiryo. Pharmacology and Therapeutics, 16(701), 1988
27812 Mice LDLo intraperitoneal injection 1 gm/kg Behavior: altered sleep duration (including righting reflex changes); behavior: seizures or effects on epileptic threshold; gastrointestinal tract: other changes Yakuri to Chiryo. Pharmacology and Therapeutics, 16(701), 1988

[1]. Gestrinone (R2323) binding to steroid receptors in human uterine endometrial cytosol. Acta Obstet Gynecol Scand. 1986;65(5):439-41.

[2]. Gestrinone inhibits growth of human uterine leiomyoma may relate to activity regulation of ERα, Src and P38 MAPK. Biomed Pharmacother. 2012 Dec;66(8):569-77.

[3]. Studies on the mechanism of action of danazol and gestrinone (R2323) in the rat: evidence for a masked estrogen component. Fertil Steril. 1989 Apr;51(4):705-10.

[4]. A comparison of the effects of danazol and gestrinone on testosterone binding to sex hormone binding globulin in vitro and in vivo. Clin Endocrinol (Oxf). 1986 May;24(5):555-63.

Gestrinone is an oxosteroid. Also known as ethylnorethindrone, gestrinone is a 19-nortestosterone-type synthetic steroid, marketed in Europe, Australia, and Latin America, but not in the United States and Canada. It is primarily used to treat endometriosis. Developed in the early 1970s, gestrinone underwent clinical trials in Europe and North America as a once-weekly oral contraceptive. Due to its lack of significant advantages compared to other oral contraceptives and its high price, gestrinone was discontinued after Phase II clinical trials. However, since 1982, it has attracted more attention due to its significant efficacy in treating endometriosis. Under different endocrine conditions, gestrinone exhibits estrogenic, progestogenic, androgenic, anti-estrogenic, and anti-progestogenic effects. It is a non-estrogenous contraceptive with weak progestogenic activity and strong anti-progestogenic properties. It is effective when taken orally once a week and can also be administered via vaginal devices. Drug Indications Endometriosis, with or without infertility. Treatment is limited to once-in-a-lifetime use for a 6-month course. Mechanism of Action Gestrinone has weak agonist activity against progesterone receptors in rabbit endometrium and progesterone antagonist activity in various other pharmacological testing systems. Additionally, it has moderate agonist activity against prostate androgen receptors in vitro. This activity has been found to be lower in some in vivo studies. Gestrinone primarily acts on the hypothalamic-pituitary axis, inhibiting the release of gonadotropins, with a weaker inhibitory effect on their synthesis. It also has anti-estrogenic activity. Suppression of the ovulatory gonadotropin surge can be observed after the first month of treatment; the decrease in ovarian hormone secretion leads to rapid endometrial atrophy. In addition to its central effects, gestrinone also has anti-progesterone activity against cellular receptors in the endometrium and extrauterine ectopic lesions. Gestrinone has no direct estrogenic and/or uterine trophic effects. One study aimed to examine the efficacy of gestrinone in emergency contraception. Data from this study suggests that the mechanism of action of gestrin, used for emergency contraception, may be through inhibiting embryo implantation by acting on the endometrium, rather than inhibiting ovulation.
Pharmacodynamics
Gestrin is a synthetic steroid hormone with androgenic, anti-estrogenic, and anti-progestin properties. Multiple studies have shown that gestrin is as effective as danazol in treating endometriosis-related infertility, and is better tolerated in terms of adverse reactions. Gestrin has moderate anti-estrogenic and anti-gonadal effects, leading to increased free testosterone levels, decreased sex hormone-binding globulin levels, inhibition of follicle-stimulating hormone (FSH) and luteinizing hormone (LH) peaks, and a reduction in mean LH levels, thereby lowering estrogen levels. Furthermore, gestrin has a direct effect on receptors in the endometrium and ectopic endometrium; its anti-progestin and anti-estrogenic effects can lead to atrophy of both the endometrium and ectopic endometrium, thus achieving a therapeutic effect. Trigestrin inhibits the release of pituitary gonadotropins, and its effect on ovarian hormone secretion leads to endometrial atrophy, thereby resolving endometriosis. Trigestrin has a structure similar to norethindrone and possesses certain androgenic and progesterone activities. However, trigestrin has an anti-progesterone effect on endometrial tissue. This study investigated the efficacy of oral gestrinone (2.5 mg every two weeks for 6 months) in 11 women diagnosed with mild to moderate endometriosis via laparoscopy. All patients experienced pain relief within 8 weeks of starting treatment. Gonadotropin, prolactin (PRL), 17β-estradiol (17β-E2), estrone (E1), progesterone (P), androstenedione (A), and dehydroepiandrosterone sulfate (DHEA-S) levels remained at physiological follicular phase levels. Total testosterone (TT) and sex hormone-binding globulin (SHBG) levels decreased, while free testosterone (FT) levels slightly increased. Metabolic studies showed a decrease in total triglyceride, very low-density lipoprotein (VLDL) triglyceride, high-density lipoprotein (HDL) cholesterol, and VLDL cholesterol levels. Studies also found that LDL cholesterol and apolipoprotein B levels increased during gestrinone treatment. This suggests that gestrinone, at therapeutic doses, exerts anti-estrogenic, anti-androgenic, and anti-progestogenic effects by acting on central and peripheral steroid receptors.

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1. Mechanism of action: - Dual antagonism: Inhibits estrogen receptor (ER) and progesterone receptor (PR) signaling pathways while activating androgen receptor (AR) [1-2]
- Oncogenic pathway inhibition: Inhibits c-Src/P38 MAPK-mediated cell proliferation [2]
2. Clinical application: - Approved for the treatment of endometriosis, taken orally once a week [8]
- Its efficacy in treating uterine fibroids and menorrhagia is under investigation [5]
3. Side effects: - Androgen-like effects (acne, hirsutism) and estrogen deficiency symptoms (hot flashes) [8]
- Reversible elevation of liver enzymes occurs in 4-8% of patients [11]

C21H24O2

308.42

308.177

C, 81.78; H, 7.84; O, 10.37

16320-04-0

27812

Off-white to light yellow solid powder

1.2±0.1 g/cm3

507.6±50.0 °C at 760 mmHg

154ºC

214.9±22.7 °C

0.0±3.0 mmHg at 25°C

1.607

3.27

1

2

2

23

713

4

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

BJJXHLWLUDYTGC-ANULTFPQSA-N

InChI=1S/C21H24O2/c1-3-20-11-9-17-16-8-6-15(22)13-14(16)5-7-18(17)19(20)10-12-21(20,23)4-2/h2,9,11,13,18-19,23H,3,5-8,10,12H2,1H3/t18-,19+,20+,21+/m1/s1

(8S,13S,14S,17R)-13-ethyl-17-ethynyl-17-hydroxy-1,2,6,7,8,14,15,16-octahydrocyclopenta[a]phenanthren-3-one

A 46 745; A-46-745; Gestrinone; 16320-04-0; Dimetriose; Ethylnorgestrienone; Gestrinona; Gestrinonum; RU 2323; A46745; Dimetriose; R2323; R-2323; R 2323; Ethylnorgestrienone

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 : ≥ 50 mg/mL (~162.12 mM)
H2O : ~1 mg/mL (~3.24 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (8.11 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 (8.11 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 (8.11 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.)