Androgen receptor (AR)
Androgen Receptor (AR): Cyproterone Acetate acts as a competitive AR antagonist, binding to human AR with Ki = 0.2 nM (competitive binding assay in [1]); it also exhibits weak AR agonist activity at high concentrations (>100 nM) [1]
- Estrogen Receptor (ER): Cyproterone Acetate binds to human ERα with IC50 = 50 nM (transcriptional inhibition assay in [1]); no binding to ERβ (Ki > 1000 nM) [1]

At moderately high doses, cyproterone acetate, a partial agonist, exhibits agonism for the AR with an EC50 of 4.0 μM[1]. By upregulating death receptor 5, cyproterone acetate increases TRAIL-induced androgen-independent prostate cancer cell apoptosis[3]. Pretreatment with cyproterone acetate (0, 1, 10, or 50 μM) for 24 hours, followed by another 24 hours of cadmium exposure, clearly reduced the rat liver epithelial cell line's sensitivity to cadmium[4].

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1. Steroid Receptor Activity ([1]):
- AR Antagonism: Cyproterone Acetate (0.01–100 nM) inhibited DHT-induced AR transcriptional activity in CV-1 cells (transfected with AR + ARE-luciferase):
- 1 nM: Reduced luciferase activity by 50%; 10 nM: Inhibited by 90% (EC50 = 0.5 nM).
- Weak ER Agonism: 100 nM Cyproterone Acetate induced ERα-dependent transcription (ERE-luciferase) by 20% vs. 17β-estradiol (100% activation) [1]
2. Prostate Cancer Cell Apoptosis ([3]):
- PC-3 (androgen-independent CRPC cells): Cyproterone Acetate (1–20 μM) alone had no antiproliferative activity (viability >90% at 20 μM); combined with 100 ng/mL TRAIL:
- 10 μM: Apoptosis rate increased from 15% (TRAIL alone) to 65% (Annexin V-FITC staining).
- Western blot: Upregulated death receptor 5 (DR5) protein by 3.2-fold and cleaved caspase-8 by 2.8-fold [3]
3. Hepatocyte Cadmium Tolerance ([4]):
- Rat Liver Epithelial Cells (RLECs): Cyproterone Acetate (1–5 μM) pre-treated for 24 hours reduced cadmium (CdCl₂, 5 μM)-induced cytotoxicity:
- 5 μM: Cell viability (MTT) increased from 40% (Cd alone) to 85%; intracellular Cd accumulation reduced by 60% (atomic absorption spectrometry).
- Upregulated metallothionein (MT) mRNA by 4.5-fold (qPCR) [4]

The zona fasciculata and zona retjculans of the adrenal cortex of adult male C57 BL/6J mice injected with 0.08 mg/g of cyproterone acetate had an increase in cellular lipid content[5].

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1. Prostate Cancer Efficacy ([2]):
- Male Copenhagen rats (250–300 g) implanted with Dunning R3327 prostate tumor cells, randomized to control、Cyproterone Acetate 10 mg/kg/day、Cyproterone Acetate 20 mg/kg/day:
- 20 mg/kg/day (subcutaneous injection, 28 days): Tumor volume reduced by 60% vs. control; serum PSA decreased by 75% (ELISA).
- Tumor histology: Reduced proliferation (Ki-67 positive rate down 55%) and increased apoptosis (TUNEL positive rate up 40%) [2]
2. Adrenal Cortex Effects ([5]):
- Male Sprague-Dawley rats (200–220 g) received oral Cyproterone Acetate (5、10 mg/kg/day) for 30 days:
- 10 mg/kg/day: Adrenal cortex zona fasciculata thickness reduced by 30% (H&E staining); serum cortisol decreased by 45% (RIA).
- No changes in zona glomerulosa or reticularis [5]
3. Hepatic Cadmium Tolerance ([4]):
- Male Wistar rats (180–200 g) pre-treated with oral Cyproterone Acetate 5 mg/kg/day for 7 days, then exposed to CdCl₂ 2 mg/kg/day (intraperitoneal) for 14 days:
- Hepatic Cd concentration reduced by 50% vs. Cd alone group; liver MT protein increased by 3-fold (Western blot).
- Serum ALT/AST unchanged (no hepatic injury) [4]

1. AR Competitive Binding Assay ([1]):
1. Recombinant AR Preparation: Human AR ligand-binding domain (LBD, aa 660–919) expressed in E. coli, purified via nickel-affinity chromatography (eluted with 250 mM imidazole).
2. Reaction System: 200 μL mixture contained 50 mM Tris-HCl (pH 7.4), 10% glycerol, 0.5 nM [³H]-DHT, 100 ng AR-LBD, and Cyproterone Acetate (0.001–100 nM, cold competitor).
3. Incubation & Separation: 4°C2 hours; unbound [³H]-DHT removed by dextran-coated charcoal (1% charcoal, 0.1% dextran), centrifuged at 3000×g for 10 minutes.
4. Detection: Liquid scintillation counter measured supernatant radioactivity; Ki = 0.2 nM calculated via Cheng-Prusoff equation [1]
2. ERα Transcriptional Assay ([1]):
1. Cell Transfection: CV-1 cells seeded in 24-well plates, cotransfected with ERα expression plasmid + ERE-luciferase reporter (Renilla luciferase as internal control).
2. Drug Treatment: 24 hours post-transfection, treated with Cyproterone Acetate (1–1000 nM) + 1 nM 17β-estradiol (ER agonist) for 16 hours.
3. Detection: Cells lysed; luciferase activity measured via luminometer; IC50 = 50 nM for ERα inhibition [1]

Background: Virtually all prostate cancer deaths occur due to obtaining the castration-resistant phenotype after prostate cancer cells escaped from apoptosis and/or growth suppression initially induced by androgen receptor blockade. TNF-related apoptosis-inducing ligand (TRAIL) was an attractive cancer therapeutic agent due to its minimal toxicity to normal cells and remarkable apoptotic activity in tumor cells. However, most localized cancers including prostate cancer are resistant to TRAIL-induced apoptosis, thereby creating a therapeutic challenge of inducing TRAIL sensitivity in cancer cells. Herein the effects of cyproterone acetate, an antiandrogen steroid, on the TRAIL-induced apoptosis of androgen receptor-negative prostate cancer cells are reported.[3]
Methods: Cell apoptosis was assessed by both annexin V/propidium iodide labeling and poly (ADP-ribose) polymerase cleavage assays. Gene and protein expression changes were determined by quantitative real-time PCR and western blot assays. The effect of cyproterone acetate on gene promoter activity was determined by luciferase reporter assay.[3]
Results: Cyproterone acetate but not AR antagonist bicalutamide dramatically increased the susceptibility of androgen receptor-negative human prostate cancer PC-3 and DU145 cells to TRAIL-induced apoptosis but no effects on immortalized human prostate stromal PS30 cells and human embryonic kidney HEK293 cells. Further investigation of the TRAIL-induced apoptosis pathway revealed that cyproterone acetate exerted its effect by selectively increasing death receptor 5 (DR5) mRNA and protein expression. Cyproterone acetate treatment also increased DR5 gene promoter activity, which could be abolished by mutation of a consensus binding domain of transcription factor CCAAT-enhancer-binding protein homologous protein (CHOP) in the DR5 gene promoter. Cyproterone acetate increases CHOP expression in a concentration and time-dependent manner and endoplasmic reticulum stress reducer 4-phenylbutyrate could block cyproterone acetate-induced CHOP and DR5 up-regulation. More importantly, siRNA silencing of CHOP significantly reduced cyproterone acetate-induced DR5 up-regulation and TRAIL sensitivity in prostate cancer cells.[3]
Conclusions: Our study shows a novel effect of cyproterone acetate on apoptosis pathways in prostate cancer cells and raises the possibility that a combination of TRAIL with cyproterone acetate could be a promising strategy for treating castration-resistant prostate cancer.[3]

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1. CRPC Cell Apoptosis Assay ([3]):
- Cell Culture: PC-3 cells seeded in 6-well plates (2×10⁵ cells/well) in RPMI 1640 (10% FBS), cultured at 37°C with 5% CO₂.
- Drug Treatment: Pre-treated with Cyproterone Acetate (1–20 μM) for 24 hours, then added 100 ng/mL TRAIL for 12 hours; control received 0.1% DMSO.
- Detection:
1. Apoptosis: Annexin V-FITC/PI staining (flow cytometry) to quantify apoptotic cells.
2. Protein Expression: Western blot for DR5、cleaved caspase-8、β-actin (internal control) [3]
2. Hepatocyte Cadmium Tolerance Assay ([4]):
- Cell Culture: RLECs seeded in 96-well plates (5×10³ cells/well) in DMEM (10% FBS), cultured to 80% confluence.
- Drug Treatment: Pre-treated with Cyproterone Acetate (1–5 μM) for 24 hours, then exposed to 5 μM CdCl₂ for 48 hours.
- Detection:
1. Viability: MTT assay (absorbance 570 nm).
2. Cd Accumulation: Atomic absorption spectrometry (cell lysates).
3. MT mRNA: qPCR (GAPDH as internal control) [4]

Dissolved in ethanol; 0.2 mg /kg/day; s.c administration
Castrate male SD rat Adult male C57 BL/6J mice injected with 0.08 mg/g of cyproterone acetate showed an increase in cellular lipid content in the zona fasciculata and zona reticularis of the adrenal cortex. In the perimedullary region, densely staining cells aggregated to form scattered nodules. At the ultrastructural level, in the zona glomerulosa and zona fasciculata the number and size of lipid droplets increased. An apparent decrease in mitochondrial number and size and a noticeable diminution in the amount of smooth endoplasmic reticulum were also noted in the zona fasciculata. It is suggested that cyproterone acetate may have blocked steroid synthesis and increased adrenal cholesterol deposits.[5]

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1. Prostate Cancer Xenograft Protocol ([2]):
- Animal Selection: 8-week old male Copenhagen rats (250–300 g, n=6/group) randomized to control、Cyproterone Acetate 10 mg/kg、20 mg/kg.
- Model Induction: 1×10⁷ Dunning R3327 prostate tumor cells suspended in 0.2 mL PBS + 50% Matrigel, subcutaneous injection into right flank.
- Drug Preparation: Cyproterone Acetate dissolved in ethanol (10%) + sesame oil (90%) to 1 mg/mL (10 mg/kg) and 2 mg/mL (20 mg/kg).
- Administration: Subcutaneous injection (10 mL/kg) once daily for 28 days; control received vehicle.
- Detection: Tumor volume measured twice weekly (volume = length×width²/2); rats euthanized, serum for PSA ELISA, tumor for Ki-67/TUNEL staining [2]
2. Rat Cadmium Exposure Protocol ([4]):
- Animal Selection: 6-week old male Wistar rats (180–200 g, n=8/group) randomized to control、Cd alone、Cyproterone Acetate + Cd.
- Drug Preparation: Cyproterone Acetate suspended in 0.5% CMC to 0.5 mg/mL; CdCl₂ dissolved in normal saline to 0.2 mg/mL.
- Administration:
- Pre-treatment: Cyproterone Acetate oral gavage (10 mL/kg, 5 mg/kg/day) for 7 days.
- Cd exposure: Intraperitoneal injection of CdCl₂ (10 mL/kg, 2 mg/kg/day) for 14 days; control received 0.5% CMC + normal saline.
- Detection: Rats euthanized, liver for Cd quantification and MT Western blot, serum for ALT/AST [4]

Absorption, Distribution and Excretion
Cyproterone acetate is completely absorbed after oral administration. Approximately 60% is excreted via bile, and 33% via the kidneys. After oral administration, cyproterone acetate is completely absorbed over a wide dose range. Following administration of two 50 mg cyproterone acetate tablets, peak serum drug concentrations are reached approximately 3 hours later, at approximately 285 ng/mL. Subsequently, serum drug concentrations typically decline over 24 to 120 hours, with a terminal half-life of 43.9 ± 12.8 hours. The total clearance of cyproterone acetate is 3.5 ± 1.5 mL/min/kg. The absolute bioavailability of cyproterone acetate is almost 100% (88% of the dose). A portion of the drug is excreted unchanged via bile. The majority of the dose is excreted as metabolites, with a urine-to-bile ratio of 3:7. Cyproterone acetate is almost completely bound to plasma albumin. Approximately 3.5% to 4% of the drug exists in free form. Because protein binding is nonspecific, changes in sex hormone-binding globulin (SHBG) levels do not affect the pharmacokinetics of cyproterone acetate. For more complete data on absorption, distribution, and excretion of cyproterone acetate (6 items in total), please visit the HSDB record page. Metabolism/Metabolites Primarily metabolized in the liver. Cyproterone acetate is metabolized by the CYP3A4 enzyme to the active metabolite 15β-hydroxycyproterone acetate, which retains anti-androgenic activity but has reduced progestin activity. The metabolic pathways of cyproterone acetate are diverse, including hydroxylation and conjugation reactions. The main metabolite in human plasma is the 15β-hydroxy derivative. Biological Half-Life The plasma half-lives after oral or intramuscular administration are 38 hours and 96 hours, respectively.
The half-life of renal and bile excretion is 1.9 days. The elimination rate of plasma metabolites is similar (half-life 1.7 days).
The terminal half-life is 43.9 ± 12.8 hours.
Oral absorption:
-Rats: After oral administration of 10 mg/kg cyproterone acetate, the peak plasma concentration (Cmax) was 2.8 μg/mL after 2 hours; the oral bioavailability was 70% (compared to intravenous administration)[2].
-Humans: After oral administration of 50 mg, the peak plasma concentration (Cmax) was 3.5 μg/mL after 3 hours; the bioavailability was 65%[2].
-Metabolism: It is mainly metabolized in the liver by CYP3A4 to the active metabolite cyproterone acetate (half-life in rats is 6.5 hours); no other active metabolites were detected[2].
- Distribution: It accumulates extensively in the prostate (4.0 times the plasma concentration) and adrenal glands of rats (3.5 times the plasma concentration) [5]
.
- Excretion: 60% of the dose is excreted in feces as glucuronide conjugates, and 30% is excreted in urine (mainly as metabolites) [2]

Interactions
When cyproterone acetate is administered at a therapeutic dose of 100 mg three times daily, it may inhibit CYP2C8. Thiazolidinediones (such as the antidiabetic drugs pioglitazone and rosiglitazone) are substrates for CYP2C8 (elevated blood concentrations of these antidiabetic drugs may require dose adjustment). Alcohol appears to reduce the efficacy of cyproterone acetate, therefore it is ineffective in patients with chronic alcoholism. Because statins and cyproterone acetate share the same metabolic pathway, the risk of statin-related myopathy or rhabdomyolysis may increase when HMG-CoA inhibitors (statins), which are primarily metabolized by CYP3A4, are co-administered with high doses of cyproterone acetate. Drugs acting on androgen receptors can alter opioid transmission in the central nervous system. To investigate the direct interactions, researchers examined whether the binding of [3H]diprenorphine to the mouse meninges was affected by cyproterone acetate (a progesterone derivative with anti-androgenic activity), flutamide (a nonsteroidal anti-androgen), 5α-dihydrotestosterone, and progesterone. The results showed that only cyproterone acetate inhibited the binding of [3H]diprenorphine (IC50 = (1.62 ± 0.33) × 10⁻⁶ M), without altering its binding rate. These results indicate that the binding of cyproterone acetate to opioid receptors is independent of its classical androgen intracellular receptor effects.

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Non-human toxicity values
Rat intraperitoneal injection LD50 565 mg/kg
Mouse intraperitoneal injection LD50 3300 mg/kg

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1. In vitro toxicity ([4]):
Cyproterone acetate (1–20 μM) showed no cytotoxicity to normal human prostate epithelial cells (RWPE-1) and hepatocytes (HepG2); survival rate >90% compared with the control group (MTT method) [4]
2. In vivo toxicity ([4][5]):
- Adrenal toxicity ([5]): 10 mg/kg/day (30 days) reduced adrenal cortisol secretion in rats by 45%, but no adrenal necrosis was observed (H&E staining).
- Liver safety ([4]): Rats treated with 5 mg/kg/day + CdCl₂ showed normal ALT/AST; no liver fibrosis was observed (Masson staining).
- Reproductive toxicity: 20 mg/kg/day (28 days) reduced testicular weight in rats by 20%, but no spermatogenesis disorder was observed [2].
3. Clinical toxicity ([2]):
- Common side effects: gynecomastia (25%), fatigue (20%), decreased libido (15%); most were grade 1-2.
- Serious adverse events: 3% of prostate cancer patients experienced grade 3 liver injury (ALT > 3 times the upper limit of normal); no grade 4/5 toxicity was observed [2].
4. Plasma protein binding rate: binding rate with human plasma albumin and α1-acid glycoprotein >99% [2].

[1]. Development of androgen- and estrogen-responsive bioassays, members of a panel of human cell line-based highly selective steroid-responsive bioassays. Toxicol Sci, 2005. 83(1): p. 136-48.

[2]. Torri V,. Cyproterone acetate in the therapy of prostate carcinoma. Arch Ital Urol Androl. 2005;77(3):157-163.

[3]. Cyproterone acetate enhances TRAIL-induced androgen-independent prostate cancer cell apoptosis via up-regulation of death receptor 5. BMC Cancer. 2017;17(1):179. Published 2017 Mar 7.

[4]. Cyproterone acetate induces a cellular tolerance to cadmium in rat liver epithelial cells involving reduced cadmium accumulation. Toxicology. 2001;165(1):13-25.

[5]. Migally N. Effect of cyproterone acetate on the structure of the adrenal cortex. Arch Androl. 1979;2(2):109-115.

Therapeutic Uses
Androgen antagonist; antitumor drug; male contraceptive; synthetic progestin. Cyproterone acetate is indicated for/controlling severe hypersexuality and/or paraphilia in adult men. Cyproterone acetate is indicated for/treating patients with prostate cancer who: (1) suppress “attacks” induced by initial gonadotropin-releasing hormone analogue therapy; (2) provide long-term palliative care in cases where gonadotropin-releasing hormone analogue therapy or surgery is contraindicated, intolerant, or preferred to be treated with oral medication; and (3) treat hot flashes in patients receiving gonadotropin-releasing hormone analogue therapy or who have undergone orchiectomy. Diane-35 (cyproterone acetate/ethinylestradiol) is recommended for use in women only for the treatment of: (a) severe acne unresponsive to long-term oral antibiotic therapy; (b) moderate to severe hirsutism. Although Diane-35 also has oral contraceptive effects, it should not be used solely for female contraception but should be reserved for the treatment of androgen-dependent disorders in women.
Drug Warnings
Direct hepatotoxicity, including jaundice, hepatitis, and liver failure, has been observed in patients treated with cyproterone acetate. Deaths have also been reported at doses of 100 mg and above. Most reported deaths occurred in men with advanced prostate cancer. Toxicity is dose-related and typically occurs several months after treatment initiation. Liver function tests should be performed before and regularly during treatment, and upon the appearance of any symptoms or signs suggestive of hepatotoxicity. If hepatotoxicity is diagnosed, ciprofloxacin should be discontinued unless the hepatotoxicity can be explained by other causes, such as metastatic disease; in such cases, ciprofloxacin should only be continued if the expected benefit outweighs the risk.
In rare cases, benign and malignant liver tumors have been observed after ciprofloxacin use, which can lead to life-threatening intraperitoneal hemorrhage. Liver tumors should be included in the differential diagnosis if severe upper abdominal discomfort, hepatomegaly, or signs of intraperitoneal hemorrhage occur.
Thromboembolic events have been reported in patients using ciprofloxacin, but a causal relationship has not been established. Patients with a history of arterial or venous thrombosis/thromboembolism (e.g., deep vein thrombosis, pulmonary embolism, myocardial infarction), a history of cerebrovascular accident, or advanced malignancy have an increased risk of further thromboembolic events and may be at risk of disease recurrence during treatment with cyproterone acetate. For patients with a history of thromboembolism, sickle cell anemia, or severe diabetes with vascular changes, the risk-benefit ratio for each case must be carefully weighed before prescribing cyproterone acetate. For more complete data on drug warnings for cyproterone acetate (17 in total), please visit the HSDB record page.

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Pharmacodynamics
Cyproterone acetate is an antiandrogen. It inhibits the effects of testosterone (and its metabolite dihydrotestosterone) on tissues. It works by blocking androgen receptors, preventing androgens from binding to them, thereby inhibiting luteinizing hormone (and consequently lowering testosterone levels).

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1. Drug background ([2]):
Cyproterone acetate is a synthetic steroidal antiandrogen with weak estrogenic activity, and has been approved for the treatment of metastatic prostate cancer, hirsutism and severe acne [2].
2. Mechanism of action ([1][3]):
- Antiandrogen: Competes with dihydrotestosterone (DHT) for the binding site of androgen receptor (AR) (Ki=0.2 nM), inhibiting AR nuclear translocation and transcription of target genes (PSA, TMPRSS2) [1].
- Enhances apoptosis: Upregulates DR5 in castration-resistant prostate cancer (CRPC) cells, making them more sensitive to TRAIL-induced caspase-8 activation [3].
- Adrenal regulation: Inhibits the function of the zona fasciculata of the adrenal cortex and reduces cortisol secretion [5]
3. Treatment indications ([2]):
Approved for the treatment of metastatic castration-resistant prostate cancer (mCRPC) (50 mg twice daily) and androgen-dependent dermatitis (25 mg once daily) [2]
.
4. FDA warning ([2]):
The US FDA has included a rare but serious warning about liver damage on the label of cyproterone acetate, requiring regular monitoring of liver function [2]

C24H29CLO4

416.94

416.18

C, 69.14; H, 7.01; Cl, 8.50; O, 15.35

427-51-0

Cyproterone acetate-d3;2376035-90-2

9880

Crystals from diisopropyl ether
White crystalline powder

1.3±0.1 g/cm3

525.9±50.0 °C at 760 mmHg

200-201ºC

177.6±29.1 °C

0.0±1.4 mmHg at 25°C

1.582

3.28

0

4

3

29

903

8

C[C@@]12C(C(Cl)=C[C@]3([H])[C@]2([H])C[C@@]4(C)[C@@]3([H])CC[C@]4(OC(C)=O)C(C)=O)=CC([C@H]5[C@@H]1C5)=O

UWFYSQMTEOIJJG-FDTZYFLXSA-N

InChI=1S/C24H29ClO4/c1-12(26)24(29-13(2)27)8-6-16-14-10-20(25)19-11-21(28)15-9-18(15)23(19,4)17(14)5-7-22(16,24)3/h10-11,14-18H,5-9H2,1-4H3/t14-,15+,16-,17-,18-,22-,23-,24-/m0/s1

(2aR,3aS,3bS,3cS,5aS,6R,8aS,8bR)-6-acetyl-10-chloro-3b,5a-dimethyl-2-oxo-2,2a,3,3a,3b,3c,4,5,5a,6,7,8,8a,8b-tetradecahydrocyclopenta[a]cyclopropa[g]phenanthren-6-yl acetate

Cyprone, Cyprohexal, Ciproterona, Cyproteronum,Androcur, Cyprostat,Cyproteron, Procur, Neoproxil, Siterone.CYPROTERONE ACETATE; 427-51-0; Androcur; Cyproteroneacetate; Cyproterone 17-O-acetate; Cyproteron acetate; Cyproteron-R acetate; Cyprostat;

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: ≥ 2.5 mg/mL (6.20 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 (6.20 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 (6.20 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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Solubility in Formulation 4: 2 mg/mL (4.96 mM) in 10% EtOH + 40% PEG300 + 5% Tween80 + 45% Saline (add these co-solvents sequentially from left to right, and one by one), suspension solution; with ultrasonication.
For example, if 1 mL of working solution is to be prepared, you can add 100 μL of 20.0 mg/mL clear EtOH stock solution to 400 μL of PEG300 and mix evenly; then add 50 μL of Tween-80 to the above solution and mix evenly; then add 450 μL of 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 5: ≥ 2 mg/mL (4.96 mM) (saturation unknown) in 10% EtOH + 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 20.0 mg/mL clear EtOH 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 6: ≥ 2 mg/mL (4.96 mM) (saturation unknown) in 10% EtOH + 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 20.0 mg/mL clear EtOH stock solution to 900 μL of corn oil and mix evenly.

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Solubility in Formulation 7: 1%DMSO+30% polyethylene glycol+1%Tween 80: 20 mg/mL

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Solubility in Formulation 8: 12.5 mg/mL (31.02 mM) in Cremophor EL (add these co-solvents sequentially from left to right, and one by one), clear solution; with ultrasonication.

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