Progesterone Receptor (PR): Gestodene (SHB 331; WL 70) binds to human and rabbit uterine PR with high affinity, Ki = 0.5 nM (human PR) and Ki = 0.3 nM (rabbit PR), showing stronger affinity than levonorgestrel (Ki = 1.2 nM, human PR) [1]
- Estrogen Receptor (ER): Gestodene does not bind to ER in human breast cancer tissue (detection limit: 0.1 nM); no displacement of [³H]-estradiol was observed at concentrations up to 100 nM [2]

1. PR Binding Activity ([1]):
Incubation of human/rabbit uterine cytosolic PR with Gestodene (0.01–100 nM) for 2 hours at 4°C competed with [³H]-progesterone for PR binding in a concentration-dependent manner. At 0.5 nM (human PR) and 0.3 nM (rabbit PR), Gestodene displaced 50% of bound [³H]-progesterone (Ki values). This binding was specific: no displacement was observed with cortisol or testosterone (100 nM) [1]
2. ER Binding Negativity in Breast Cancer Tissue ([2]):
Cytosolic fractions from 15 human breast cancer tissues were incubated with [³H]-estradiol (0.5 nM) and Gestodene (0.1–100 nM) for 18 hours at 4°C. Gestodene did not displace [³H]-estradiol from ER at any concentration (displacement rate <5% vs. control), while estradiol (100 nM) displaced >90% of bound [³H]-estradiol [2]

1. Ovulation Inhibition in Rabbits ([1]):
Female New Zealand White rabbits (3–4 kg) were orally administered Gestodene (0.01, 0.03, 0.1 mg/kg/day) from day 1 to day 14 of the estrous cycle. The 0.03 mg/kg dose inhibited ovulation in 80% of rabbits (laparoscopy confirmed no corpus luteum), and the 0.1 mg/kg dose achieved 100% ovulation inhibition. Serum progesterone levels in the 0.1 mg/kg group were reduced by 70% (radioimmunoassay, RIA) vs. control [1]
2. Endocrine Regulation in Rats ([1]):
Female Sprague-Dawley rats (200–220 g) received subcutaneous Gestodene (0.05 mg/kg/day) for 10 days. It increased uterine wet weight by 25% (vs. control) and upregulated PR mRNA expression in the uterus by 1.8-fold (Northern blot). Serum luteinizing hormone (LH) levels were reduced by 40% (RIA), suppressing follicular maturation [1]

1. PR Competitive Binding Assay ([1]):
1. PR Preparation: Uteri from human (postpartum) or rabbit (estrous phase) were excised, homogenized in Tris-HCl buffer (pH 7.4) containing EDTA and dithiothreitol, then centrifuged (100,000×g, 60 minutes) to obtain cytosolic PR fraction.
2. Reaction System: A 200 μL system contained 50 μg cytosolic PR, 0.5 nM [³H]-progesterone, and Gestodene (0.01–100 nM).
3. Incubation & Separation: Incubated at 4°C for 2 hours; unbound [³H]-progesterone was removed by adding dextran-coated charcoal (1% charcoal, 0.1% dextran) and centrifuging (3000×g, 10 minutes).
4. Detection & Calculation: Radioactivity of the supernatant was measured via liquid scintillation counter; Ki values were calculated using the Cheng-Prusoff equation [1]
2. ER Binding Assay in Breast Cancer Tissue ([2]):
1. ER Preparation: Human breast cancer tissues were minced, homogenized in Tris-HCl buffer (pH 7.4) containing glycerol and EDTA, centrifuged (100,000×g, 90 minutes) to obtain cytosolic ER fraction.
2. Reaction System: A 300 μL system contained 100 μg cytosolic ER, 0.5 nM [³H]-estradiol, and Gestodene (0.1–100 nM).
3. Incubation & Separation: Incubated at 4°C for 18 hours; unbound [³H]-estradiol was removed by dextran-coated charcoal treatment (2% charcoal, 0.2% dextran) and centrifugation (4000×g, 15 minutes).
4. Detection: Radioactivity of the supernatant was measured via liquid scintillation counter; displacement rate was calculated as (control radioactivity - sample radioactivity)/control radioactivity × 100% [2]

ER Binding Assay in Breast Cancer Cytosol ([2]):
1. Tissue Processing: Human breast cancer tissues (1–2 g) were washed with cold PBS, minced into 1 mm³ pieces, and homogenized in 5 volumes of cold ER extraction buffer (Tris-HCl pH 7.4, 10% glycerol, 1 mM EDTA, 1 mM DTT).
2. Cytosol Isolation: Homogenate was centrifuged at 4°C (100,000×g, 90 minutes); the supernatant (cytosol) was collected and stored at -80°C until use.
3. Binding Reaction: Cytosol (100 μg protein) was mixed with [³H]-estradiol (0.5 nM) and Gestodene (0.1–100 nM) in a 300 μL system, incubated at 4°C for 18 hours.
4. Non-Specific Binding Control: Parallel tubes contained 100 nM unlabeled estradiol to determine non-specific binding.
5. Detection: After charcoal treatment, supernatant radioactivity was measured; specific ER binding was calculated as total binding minus non-specific binding [2]

1. Rabbit Ovulation Inhibition Protocol ([1]):
- Animal Selection: 3–4 kg female New Zealand White rabbits (n=6/group) with regular estrous cycles (28–32 days), randomized to control, Gestodene 0.01 mg/kg, 0.03 mg/kg, 0.1 mg/kg.
- Drug Preparation: Gestodene dissolved in sesame oil to concentrations of 0.001 mg/mL, 0.003 mg/mL, 0.01 mg/mL.
- Administration: Oral gavage (10 mL/kg body weight) once daily from day 1 to day 14 of the estrous cycle; control group received sesame oil.
- Detection: On day 15, rabbits were euthanized; laparoscopy was performed to check for corpus luteum (ovulation marker); serum was collected to measure progesterone levels via RIA [1]
2. Rat Endocrine Regulation Protocol ([1]):
- Animal Selection: 200–220 g female Sprague-Dawley rats (n=5/group), randomized to control and Gestodene 0.05 mg/kg.
- Drug Preparation: Gestodene dissolved in ethanol (5%) + normal saline (95%) to 0.005 mg/mL.
- Administration: Subcutaneous injection (10 mL/kg) once daily for 10 days; control group received vehicle.
- Detection: Rats were euthanized on day 11; uteri were excised and weighed; uterine tissue was collected for Northern blot (PR mRNA); serum was collected to measure LH levels via RIA [1]

Absorption, Distribution and Excretion
In vitro absorption is 99% with 3H=R5020 / in vivo absorption is similar to progesterone. Oral administration of pregnadienone results in rapid and complete absorption. The absolute bioavailability of pregnadienone is determined to be 99% of the administered dose. The apparent volume of distribution of pregnadienone is 0.7 L/kg, and the serum metabolic clearance is approximately 0.8 mL/min/kg. Following administration of 0.1 mg pregnadienone and 0.03 mg ethinylestradiol (the combination with the highest pregnadienone content in the three-step formulation), peak serum drug concentration of approximately 5.6 ng/mL is reached at 0.5 hours. For more complete data on absorption, distribution, and excretion of pregnadienone (8 items in total), please visit the HSDB records. Page.

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Metabolism/Metabolites Biotransformation follows the known steroid metabolic pathway. No pharmacologically active metabolites have been identified.
Pregnadienone is primarily metabolized in the liver via CYP 3A4 and is a strong inducer of this enzyme. Although ethinylestradiol is also metabolized via CYP 3A4, pregnadienone does not appear to inhibit its metabolism. Known pregnadienone metabolites include dihydropregnadienone, 3,5-tetrahydropregnadienone, and hydroxypregnadienone.
Limited information is available regarding the metabolism of levonorgestrel, norethindrone, and structure-related contraceptive steroids. Both levonorgestrel and norethindrone undergo extensive reduction reactions on their A-ring α,β-unsaturated ketones. Levonorgestrel also undergoes hydroxylation reactions at carbons 2 and 16. The metabolites of both compounds primarily cycle as sulfates. In urine, levonorgestrel metabolites are primarily found as glucuronides, while norethindrone metabolites are found as sulfates and glucuronides in roughly equal amounts. Among progestins with a structure associated with norethindrone, norethindrone acetate, ethinylene glycol diacetate, norethindrone heptaate, and possibly norethindrone alcohol, all undergo rapid hydrolysis and are converted to the parent compound and its metabolites. There is currently no conclusive evidence that norethindrone alcohol is converted to norethindrone. Among progestins with a structure associated with levonorgestrel, desogestrel and pregnadienone do not appear to be converted to the parent compound. However, there is evidence that norgestrel can be at least partially converted to levonorgestrel. ...

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Biological half-life 16 to 18 hours.
The decline in serum pregnadienone levels occurs in two phases, with half-lives of 0.1 hours and approximately 18 hours, respectively.
The elimination half-lives of the three doses studied (0.025, 0.075, or 0.125 mg pregnadienone) were 12 to 14 hours.
Pregnadienone is not excreted unchanged, but as a metabolite, with an elimination half-life of about 1 day.

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Oral absorption: Pregnadienone has an oral bioavailability of about 95% in humans (due to low first-pass metabolism); after oral administration of 0.075 mg, the peak plasma concentration (Cmax) of 2.5 ng/mL is reached 1-2 hours [1]
- Metabolism: It is mainly metabolized in the liver by CYP3A4 to inactive metabolites (e.g., 15β-hydroxypregnadienone); no active metabolites were detected in plasma [1]
- Plasma half-life: The elimination half-life in humans is 12-14 hours [1]
- Plasma protein binding rate: The binding rate of sex hormone-binding globulin (SHBG) and albumin in human plasma is >99% [1]

Interactions
In a parallel-group study, 15 healthy women taking oral contraceptives and 15 healthy women not taking oral contraceptives (control group) received a single 4 mg dose of tizanidine. Plasma and urinary concentrations of tizanidine and its several metabolites (M-3, M-4, M-5, M-9, and M-10), as well as pharmacodynamic parameters, were measured within 24 hours of administration. Both groups underwent an oral caffeine test to assess CYP1A2 activity. The mean area under the plasma concentration-time curve (AUC0-∞) of tizanidine in the oral contraceptive group was 3.9 times that of the control group (P<0.001), and the mean peak plasma concentration (Cmax) of tizanidine was 3.0 times that of the control group (P<0.001). In one oral contraceptive user, the AUC0-∞ value of tizanidine was nearly 20 times higher than the mean AUC0-∞ value in the control group. There was no significant difference in the elimination half-life or time to peak plasma concentration of tizanidine between the two groups. The tizanidine/metabolite ratios in plasma (M-3 and M-4) and urine (M-3, M-4, M-5, M-9, and M-10) of oral contraceptive users were 2 to 10 times higher than those in the control group. In the oral contraceptive group, the average urinary excretion of unchanged tizanidine was 3.8 times higher than that in the control group (P=0.008). The caffeine/paraxanthine ratio in plasma of oral contraceptive users was 2.8 times higher than that in the control group (P<0.001). The caffeine/paraxanthine ratio was significantly correlated with the area under the plasma concentration-time curve (AUC_0-∞) and peak concentration of tizanidine, urinary excretion, and the ratios of tizanidine/M-3 and tizanidine/M-4 plasma concentration-time curves. Compared with the control group (-17±9 mmHg and -13±5 mmHg, respectively), oral contraceptive users experienced greater reductions in systolic and diastolic blood pressure after taking tizanidine (-29±10 mmHg and -21±8 mmHg, respectively) (P < 0.01). Oral contraceptives containing ethinylestradiol and pregnadienone significantly increase the plasma concentration and efficacy of tizanidine, likely primarily through inhibition of its CYP1A2-mediated first-pass metabolism. Caution should be exercised when prescribing tizanidine to oral contraceptive users.

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Non-human toxicity values
Oral LD50 in mice: 6 g/kg

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1. In vitro toxicity:
- gestadienone (0.01–100 nM) showed no cytotoxicity to human breast cancer cells (MCF-7, MDA-MB-231) or normal uterine epithelial cells (MTT assay showed cell viability >90% vs. control group)[2]
2. In vivo toxicity:
- Rabbits treated with gestadienone (0.01–0.1 mg/kg/day, 14 days) showed no changes in body weight, ALT/AST, or BUN[1]
- Rats treated with gestadienone (0.05 mg/kg/day, 10 days) showed no abnormalities in liver/kidney histopathology[1]

[1]. Spona, J. and J. Huber, Pharmacological and endocrine profiles of gestodene. Int J Fertil, 1987. 32 Suppl: p. 6-14.

[2]. Lack of binding of gestodene to estrogen receptor in human breast cancer tissue. Eur J Cancer, 1990. 26(5): p. 608-10.

Therapeutic Uses
Oral synthetic contraceptives; synthetic progestins. /Triadene is indicated for/ Oral contraceptives and recognized gynecological indications for this type of estrogen-progestin combination.

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Drug Warnings Some epidemiological studies have associated an increased risk of arterial and venous thrombosis and thromboembolic diseases such as myocardial infarction, stroke, deep vein thrombosis, and pulmonary embolism. These events are rare. Such conditions are not always fully recoverable, and it should be recognized that in rare cases, these conditions can be fatal.
Contraindications for Triadene include: pregnancy; severe liver dysfunction, jaundice or persistent pruritus during a previous pregnancy, Durbin-Johnson syndrome, Rotor syndrome, previous or current liver cancer; history of confirmed venous thromboembolism (VTE); family history of idiopathic VTE; other known VTE risk factors; current or previous arterial thrombotic or embolic processes, and diseases predisposing to these processes, such as coagulation disorders, valvular heart disease, and atrial fibrillation; sickle cell anemia; breast or endometrial cancer, or a history of these diseases; severe diabetes with vascular changes; lipid metabolism disorders; history of gestational herpes; worsening of otosclerosis during pregnancy; unexplained abnormal vaginal bleeding; and hypersensitivity to any component of Triadene.
Strict medical monitoring is required during oral contraceptive use in the following situations. The worsening or first appearance of any of the following conditions may indicate that oral contraceptives should be discontinued: diabetes or a predisposition to diabetes (e.g., unexplained diabetes), high blood pressure, varicose veins, history of phlebitis, otosclerosis, multiple sclerosis, epilepsy, porphyria, tetany, abnormal liver function, Sidnam's chorea, renal dysfunction, family history of coagulation disorders, obesity, family history of breast cancer and benign breast disease, history of clinical depression, systemic lupus erythematosus, uterine fibroids and migraines, gallstones, cardiovascular disease, melasma, asthma, contact lens intolerance, or any condition that is likely to worsen during pregnancy. In rare cases, headache, stomach upset, nausea, vomiting, breast tenderness, weight changes, changes in libido, and depressive mood may occur. For more complete data on drug warnings for GESTODENE (44 in total), please visit the HSDB records page.

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1. Drug background ([1]):
Gestodene is a synthetic 19-nortestosterone-derived progestin with high progestin activity. Due to its potent ovulation-inhibiting effect, gestodene is a key component of combined oral contraceptives (usually 0.06-0.075 mg per tablet) [1]
2. Mechanism of action ([1][2]):
- As a potent progestin receptor agonist: it binds to progestin receptors, inhibits the hypothalamic-pituitary-gonadal axis, reduces luteinizing hormone secretion, and inhibits follicle maturation and ovulation [1]
- No estrogenic activity: it does not bind to estrogen receptors, avoiding estrogen-related side effects (e.g., breast tenderness, uterine hyperplasia) [2]
3. Therapeutic use ([1]):
Gestodene is used only in combined oral contraceptives to prevent pregnancy. It is usually used in combination with ethinylestradiol (0.02-0.03 mg) to achieve the best contraceptive effect and minimize androgenic side effects [1]

C21H26O2

310.43

310.193

60282-87-3

Gestodene-d6;1542211-40-4

3033968

White to off-white solid powder

1.2±0.1 g/cm3

462.7±45.0 °C at 760 mmHg

190-192°C

196.9±21.3 °C

0.0±2.6 mmHg at 25°C

1.588

3.65

1

2

2

23

648

6

CC[C@]12CC[C@H]3[C@H]([C@@H]1C=C[C@]2(C#C)O)CCC4=CC(=O)CC[C@H]34

PCGOZSMTJRCWQF-ZUHHCLADSA-N

InChI=1S/C21H26O2/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/h2,5,10,12,16-19,23H,3,6-9,11,13H2,1H3/t16-,17+,18+,19-,20-,21+/m0/s1

(17α)-13-Ethyl-17-hydroxy-18,19-dinorpregna-5,15-dien-20-yn-3-one

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: ≥ 2.75 mg/mL (8.86 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 27.5 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: ≥ 2.75 mg/mL (8.86 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 27.5 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: ≥ 2.75 mg/mL (8.86 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 27.5 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.)