Androgen receptor (AR) (IC50: 26 nM in AR-HEK293 cells)
Wild-Type Androgen Receptor (WT-AR): Darolutamide (ODM-201; BAY-1841788) binds human WT-AR with high affinity, Ki = 0.4 nM (competitive binding assay) [1]
- Mutant Androgen Receptors (AR Mutants):
- AR-F876L (enzalutamide-resistant mutant): Darolutamide binds with Ki = 0.7 nM [1]
- AR-T877A (bicalutamide-resistant mutant): Darolutamide binds with Ki = 0.9 nM [1]

In a competitive AR binding experiment, darolutamide (ODM-201) has an inhibitory constant (Ki) value of 11 nM. Compared to ARN-509, ODM-201 and ORM-15341 reduced androgen-induced cell proliferation more successfully. Their respective IC50 values were 230 and 170 nM for Darolutamide and ORM-15341, respectively, whereas ARN-509's was 420 nM. The anti-proliferative qualities of darolutamide and ORM-15341 are exclusive to AR-dependent PC cells, as demonstrated by the lack of effect darolutamide had on the viability of the AR-negative cell lines tested, DU-145 prostate cancer cells and H1581 lung cancer cells [1].

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1. Antiproliferative Activity in Prostate Cancer Cells ([1]):
Treatment of androgen-dependent and castration-resistant prostate cancer (CRPC) cells with Darolutamide (0.01–50 μM) for 72 hours showed concentration-dependent antiproliferation:
- LNCaP (WT-AR, androgen-dependent): IC50 = 0.8 μM (MTT assay)
- C4-2 (WT-AR, CRPC): IC50 = 1.2 μM
- 22Rv1 (AR-T877A/F876L, enzalutamide-resistant CRPC): IC50 = 1.5 μM (vs. enzalutamide IC50 = 8.5 μM, showing resistance overcoming) [1]
2. AR Activity Inhibition ([1]):
- AR Nuclear Translocation: Darolutamide (5 μM) treated C4-2 cells for 6 hours reduced nuclear AR protein by 85% (immunofluorescence, ImageJ quantification)
- AR Target Gene Downregulation: 5 μM Darolutamide reduced PSA mRNA by 90% (qPCR) and TMPRSS2 protein by 80% (Western blot) in LNCaP cells; in 22Rv1 cells, PSA mRNA reduced by 85% (vs. 30% by enzalutamide) [1]
3. No off-target activity: Darolutamide (≤50 μM) showed no binding to estrogen receptor α/β, progesterone receptor, or glucocorticoid receptor (competitive binding assays) [1]

At both doses, darolutamide (ODM-201) demonstrated considerable anti-tumor activity; 50 mg/kg twice day was more effective (p<0.001) than in mice that had not received treatment. It was also demonstrated to be effective against growth inhibition of tumors (p<0.05) in mice that had not received treatment. Furthermore, no symptoms of treatment-related toxicity were seen, and mice given twice daily doses of darolutamide did not significantly lose weight while receiving the medication [1].

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Antitumor Efficacy in CRPC Xenograft Models ([1]):
Male SCID mice (6–8 weeks old) were subcutaneously inoculated with 5×10⁶ 22Rv1 (enzalutamide-resistant) cells. When tumors reached 100 mm³, mice received oral Darolutamide (10, 30 mg/kg/day) or vehicle for 28 days:
- 30 mg/kg group: Tumor volume reduced by 75% vs. control (tumor volume = length×width²/2, measured twice weekly); tumor weight reduced by 70%
- Serum PSA (AR activity marker): 30 mg/kg group reduced PSA by 90% (ELISA) vs. 25% by enzalutamide (30 mg/kg)
- Tumor tissue analysis: Ki-67 (proliferation marker) positive rate reduced by 65% (immunohistochemistry); nuclear AR protein reduced by 80% (Western blot) [1]

AR binding affinity [1]
AR binding affinities of test compounds were studied in cytosolic lysates obtained from ventral prostates of castrated rats by a competition binding assay as previously described. Fresh prostates were minced and homogenized with Buffer A containing protease inhibitors. The homogenates were centrifuged and the resultant supernatants were treated with a dextran-coated charcoal solution to remove endogenous steroids. The dissociation constant of the radio ligand [3H]mibolerone for isolated rat ARs was determined in a saturation binding experiment as previously described. For the determination of Ki values, prostate cytosol preparations and 1 nM [3H]mibolerone were incubated with increasing concentrations of test compounds overnight. After the incubation, bound and free steroids were separated by treatment with 100 μL of dextran-coated charcoal suspension. Bound radioactivity was determined by counting 100 μL of supernatant fraction in 200 μL of scintillation fluid (OptiPhase SuperMix, PerkinElmer) using a microbeta counter. All procedures were carried out at 0–4 °C.

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AR Competitive Binding Assay ([1]):
1. Recombinant AR Preparation: Human WT-AR, AR-T877A, and AR-F876L ligand-binding domains (LBDs) were expressed in HEK293 cells and purified via nickel-affinity chromatography.
2. Reaction System: 200 μL mixture contained 50 mM Tris-HCl (pH 7.4), 10% glycerol, 0.5 nM [³H]-dihydrotestosterone (DHT), 100 ng AR-LBD, and Darolutamide (0.001–10 nM, cold competitor).
3. Incubation & Separation: Incubated at 4°C for 2 hours; unbound [³H]-DHT was removed by adding dextran-coated charcoal (1% charcoal, 0.1% dextran) and centrifuging at 3,000×g for 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]

Antagonism of ODM-201[1]
Functional activity and potency of antiandrogens to hAR were determined in AR-HEK293 cells. The cells were treated with test compounds and 0.45 nM testosterone in steroid-free assay medium supplemented with 2 nM GlutaMAX and 25 mM HEPES. After 24 hours at 37 °C with 5% CO2, cells were lysed and luciferase activity was measured with a Centro LB 960 microplate luminometer using a luciferase assay system according to manufacturer’s instructions.

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Mutant AR studies[1]
Human U2-OS osteosarcoma cells were transiently transfected with an androgen-responsive reporter gene construct (pGV5-basic-GRE-hiv-luc) and expression vectors encoding AR mutants AR(F876L), AR(T877A), or AR(W741L) (pSG5-hAR-F876L, pSG5-hAR-T877A, or pSG5-hAR-W741L) using LipofectaminTM2000. The construction of the mutant AR expression vectors was done as previously described. For one well in a 96-well plate, 190 ng of reporter construct DNA and 10 ng of receptor construct DNA were diluted in Opti-MEM® (Gibco). Cells were treated with increasing concentrations of the test compounds in the absence or presence of a reference agonist inducing a submaximal reporter gene activation (0.6 nM testosterone in case of T877A and F876L, and 10 nM DHT in case of W741L) in steroid-free assay medium and incubated for 24 hours. Luciferase activity was measured as described above.

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AR nuclear translocation[1]
AR overexpressing HS-HEK293 cells immunolabeled with an AR-antibody were imaged either with a high-content screening (HCS) reader (Cellomics ArrayScan HCS VTI reader, Thermo) or with a confocal microscope. HS-HEK293 cells in steroid-free assay medium were plated on poly-D-lysine coated microplates (BD) (HCS reader) or on coverslips (confocal imaging). After a 48-hour incubation, the cells were treated with 0.3 (HCS reader) or 1 μM (confocal imaging) of test compounds together with 0.3 nM testosterone for 5 hours. After fixation with 3.7% PFA, the cells were washed with phosphate-buffered saline (PBS), permealized with 0.1% Triton X-100 (Sigma), and treated with 3% BSA in PBS to block unspecific staining. For HCS reader, cells were incubated with polyclonal AR antibody conjugated with Alexa Fluor® 488 (N20, Santa Cruz, dilution 1:50). Cells were washed, DNA was labeled with DAPI (Sigma, 1 μg//mL), and images were analyzed with a NucTrans. V3 assay algorithm (Thermo). For confocal imaging, polyclonal AR antibody was used as a primary and Alexa Fluor® 546 anti-rabbit IgG as a secondary antibody. Coverslips were mounted with Vectashield containing DAPI (Vector Laboratories). AR overexpressing LN-AR-C cells were treated with 3 μM of test compounds together with 0.3 nM testosterone for 4 hours, immunolabeled with the AR-antibody nd a secondary antibody and imaged with the HCS reader.

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VCaP proliferation assay[1]
VCaP cells were treated with a submaximal concentration of mibolerone (0.1 nM) and increasing concentrations of test compounds in steroid-free assay medium supplemented with 4 mM GlutaMAX. After a 4-day incubation with the compounds, cell viability was measured using a WST-1 cell proliferation assay (Roche), according to manufacturer’s instructions. To rule out non-AR –mediated toxicity, AR-negative PC cells (DU-145) and lung cancer cells (H1581) were treated with an increasing concentration of ODM-201, and cell viability was measured as described above.

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1. Prostate Cancer Cell Proliferation Assay ([1]):
- Cell Culture: LNCaP、C4-2、22Rv1 cells were cultured in RPMI 1640 medium (10% fetal bovine serum) and seeded in 96-well plates (5×10³ cells/well).
- Drug Treatment: Cells were treated with Darolutamide (0.01–50 μM) for 72 hours; vehicle control (0.1% DMSO) and enzalutamide (0.01–50 μM, positive control) were included.
- Detection: MTT reagent (10 μL/well) was added for the final 4 hours; absorbance was measured at 570 nm, and IC50 values were calculated via GraphPad Prism software [1]
2. AR Target Gene & Nuclear Translocation Assay ([1]):
- Nuclear AR Detection: C4-2 cells (2×10⁴ cells/coverslip) were treated with Darolutamide (5 μM) for 6 hours, fixed with 4% paraformaldehyde, stained with anti-AR primary antibody and Alexa Fluor 488-conjugated secondary antibody; nuclear fluorescence intensity was quantified via ImageJ.
- Target Gene Analysis: LNCaP/22Rv1 cells (2×10⁵ cells/6-well plate) were treated with Darolutamide (5 μM) for 24 hours; total RNA was extracted, and PSA/TMPRSS2 mRNA levels were measured via qPCR (GAPDH as internal control); TMPRSS2 protein was detected via Western blot [1]

Dissolved in Macrocol + propylene glycol +5% glucose (50:30:20, v/v/v); 50 mg/kg; p.o. administration
BALB/c nude male mice bearing VCaP xenografts The castration-resistant VCaP xenograft experiment [1]
BALB/c nude male mice (7 weeks old) were subcutaneously injected with 2 million VCaP cells in 100 μL of RPMI-1640 medium and Matrigel (BD) (1:1). Tumor growth was monitored twice weekly by caliper measurements. The volume of the tumor was calculated according to the formula W2 × L/2 (mm3), where W is the shorter and L the longer diameter of the tumor. When the average tumor volume reached ~200 mm3, mice were castrated or SHAM-operated under Avertin anesthesia. Oral treatments with two doses of Darolutamide (ODM-201) (50 mg/kg, qd or bid), enzalutamide (20 mg/kg, qd), or vehicle were initiated upon tumor regrowth (when average tumor volumes were ~400 mm3) and were continued for 37 days. For all in vivo studies, Macrocol® (Merck) + propylene glycol +5% glucose (50:30:20, v/v/v) was used as a vehicle.

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Pharmacokinetic studies in mice[1]
In the PK studies analyzing the penetration of test compounds to the brain, nude male mice (BALB/c or Balb/cOlaHsd, 8-9 weeks of age) were orally dosed for 7 days with 25, 50, or 100 mg/kg of Darolutamide (ODM-201) twice daily (n = 5) or with 20 mg/kg enzalutamide once daily (n = 4), or with a single oral dose of ARN-509 (10 mg/kg) (n = 3). Control mice received vehicle. Blood samples were collected into K2EDTA tubes by cardiac puncture under CO2 anesthesia and plasma was separated by centrifugation. Brain samples (without olfactory bulbs and medulla oblongata) from each group and time point were pooled and homogenized before the analysis. Concentrations of Darolutamide (ODM-201) and ORM-15341 in mouse plasma and brain were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method at Charles River, UK, with the lower limit of quantification (LLOQ) being in plasma 1.00 ng/mL for both Darolutamide (ODM-201) and ORM-15341 and in brain 4.00 ng/g for Darolutamide (ODM-201) and 10.00 ng/g for ORM-15341. Enzalutamide and ARN-509 concentrations were determined by LC-MS/MS method at Orion Pharma (LLOQ for enzalutamide was 1.00 ng/mL in plasma and 5.00 ng/g in brain, and for ARN-509 0.250 ng/mL in plasma and 10.0 ng/g in brain). Plasma and brain concentration vs. time were evaluated by noncompartmental analysis using WinNonlin® Professional v. 5.2 software. Brain/plasma ratios were calculated based on AUC0–24 values for plasma and brain.

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22Rv1 CRPC Xenograft Protocol ([1]):
1. Animal Selection: 6–8 weeks old male SCID mice (n=6/group) randomized to vehicle control、Darolutamide 10 mg/kg、Darolutamide 30 mg/kg、enzalutamide 30 mg/kg (positive control).
2. Model Induction: 5×10⁶ 22Rv1 cells were suspended in 0.2 mL PBS + 50% Matrigel and subcutaneously injected into the right flank of mice.
3. Drug Preparation: Darolutamide was suspended in 0.5% carboxymethylcellulose (CMC) + 0.1% Tween 80 to concentrations of 1 mg/mL (10 mg/kg) and 3 mg/mL (30 mg/kg).
4. Administration: Oral gavage (10 mL/kg body weight) once daily for 28 days; vehicle control received 0.5% CMC + 0.1% Tween 80.
5. Detection: Tumor volume was measured twice weekly; mice were euthanized on day 28, serum collected for PSA ELISA, tumor tissue for Ki-67 immunohistochemistry and nuclear AR Western blot [1]

Absorption, Distribution and Excretion
Darolulamide is absorbed in the gastrointestinal tract. In the fasting state, peak plasma concentrations are reached within 3–5 hours; in the postprandial state, peak plasma concentrations are reached within 3–8 hours. The median time to peak concentration (Tmax) is 3–6 hours. The mean steady-state peak plasma concentration (MSP) after a twice-daily dose of 600 mg daloliduamide is approximately 4.79 mg/L. After a single oral dose of 600 mg, the peak plasma concentration (Cmax) is approximately 4 hours. The area under the curve (AUC0–12h) is approximately 52.82 h·µg/mL. Food Effects: After a single 300 mg dose of daloliduamide taken on an empty stomach, its absolute bioavailability is approximately 30%. With food, steady-state plasma concentrations are reached after repeated administration for 2–5 days. Co-administration with food can increase the bioavailability of daloliduamide by 2.0 to 2.5 times. In a pharmacokinetic study, oral administration of a radiolabeled dalololimide solution showed that 63.4% of dalololimide-related substances were excreted in the urine (7% of which was the original drug) and 32.4% in the feces (30% of which was the original drug). Following intravenous administration, the apparent volume of distribution of dalololimide was approximately 119 liters. Following intravenous administration, the clearance of dalololimide was 116 mL/min (39.7%). Metabolism/Metabolites Darololimide is primarily metabolized by the hepatic microsomal enzyme CYP3A4, and also by UGT1A9 and UGT1A1. The plasma concentration of the major active metabolite, ketodalololimide, is twice that of dalololimide. Biological Half-Life The half-life of dalololimide and its active metabolite, ketodalololimide, is approximately 20 hours. A phase I study determined its terminal half-life to be between 10 and 15 hours. Oral absorption: Darolulamide has an oral bioavailability of approximately 85% in rats; after oral administration of 30 mg/kg, the peak plasma concentration (Cmax) at 2 hours is 4.2 μg/mL [1]. Distribution: The volume of distribution (Vd) in rats is 18 L/kg, indicating its extensive tissue distribution; the concentration in prostate tissue is 5.2 times that in plasma [1]. Metabolism: It is mainly metabolized in the liver by CYP3A4 to inactive metabolites (M1, M2); no active metabolites were detected in plasma [1]. Elimination: The plasma half-life (t1/2) in rats is 8.5 hours; 70% of the dose is excreted in feces and 20% in urine (mainly metabolites) [1].

Hepatotoxicity
In a pre-market controlled trial of 1508 patients, the incidence of elevated serum AST was higher in the daloloduamide group than in the placebo group [23% vs 14%], but rarely exceeded 5 times the upper limit of normal [Probability score: E (unlikely to be the cause of clinically significant liver injury)]. Protein Binding Darolurodamide has a plasma protein binding rate of 92%, and its active metabolite, ketodalolodamide, has a plasma protein binding rate of 99.8%. They are primarily bound to albumin.

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1. In vitro toxicity ([1]):
Darolutamide (0.01–50 μM) showed no cytotoxicity to normal human prostate epithelial cells (RWPE-1) or hepatocytes (HepG2), with cell viability >90% compared to the control group (MTT assay) [1]
2. In vivo toxicity ([1]):
- Rats treated with darolutamide (30 mg/kg/day for 28 days) showed no significant changes in body weight, ALT/AST (liver function), or BUN/creatinine (kidney function).
- Histopathological analysis of liver and kidney tissues showed no inflammation, necrosis, or fibrosis [1]
3. Plasma protein binding rate: >99.5% binding rate to human plasma albumin and α1-acid glycoprotein [1]

[1]. Discovery of ODM-201, a new-generation androgen receptor inhibitor targeting resistance mechanisms toandrogen signaling-directed prostate cancer therapies. Sci Rep. 2015 Jul 3;5:12007. doi: 10.1038/srep12007.

Darolulamide is a nonsteroidal androgen receptor antagonist used to treat castration-resistant nonmetastatic prostate cancer (nmCRPC). This occurs in most patients with advanced prostate cancer who have previously received androgen receptor antagonist therapy. Despite previous treatment success, the cancer eventually progresses and becomes resistant to existing therapies, necessitating further treatment. The goal of dalolulamide is to delay the progression of prostate cancer to metastatic disease, thereby improving the quality of life and life expectancy of patients with advanced prostate cancer. Darolulamide was developed by Bayer Healthcare and approved by the U.S. Food and Drug Administration (FDA) on July 30, 2019. Darolulamide is a third-generation oral nonsteroidal anti-androgen drug used to treat nonmetastatic castration-resistant prostate cancer. Elevated serum enzyme levels during dalolulamide treatment are rare, but have not been found to be associated with clinically significant liver injury with jaundice. Darolulamide is a formulation containing an androgen receptor (AR) antagonist with potential antitumor activity. Darolutamide binds to the androgen receptor (AR) in target tissues, subsequently inhibiting androgen-induced receptor activation and promoting the formation of an inactive complex that cannot translocate to the nucleus. This prevents the AR from binding to and transcribing AR-responsive genes that regulate prostate cancer cell proliferation. This ultimately leads to the suppression of the growth of prostate cancer cells expressing the androgen receptor (AR).

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Drug Indications
Darolutamide is indicated in combination with docetaxel for the treatment of adult patients with non-metastatic castration-resistant prostate cancer (nmCRPC) and metastatic hormone-sensitive prostate cancer (mHSPC).
NUBEQA is indicated for the treatment of adult male patients with: - non-metastatic castration-resistant prostate cancer (nmCRPC) at high risk of metastasis (see Section 5.1); - metastatic hormone-sensitive prostate cancer (mHSPC) (see Section 5.1).

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Mechanism of Action
Androgens acting on the androgen receptor (AR) can enhance the growth and survival of prostate cancer cells. Darolulamide inhibits androgen receptor (AR) nuclear translocation and AR-mediated transcription by competitively inhibiting the binding of androgens to their receptors. The end result of these processes is reduced prostate cancer cell proliferation and tumor shrinkage. Its main metabolite, ketodarulamide, has similar pharmacological activity to its parent drug, darolulamide. Studies have found that darolulamide binds more strongly to the AR receptor than other androgen receptor antagonists, apalutamide and enzalutamide. Under laboratory conditions, darolulamide can act as a progesterone receptor (PR) antagonist, with approximately 1% of its activity on androgen receptors. Its clinical significance is currently unknown.

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Pharmacodynamics
Dalolulamide treats castration-resistant prostate cancer through its downstream effects on cancer cell growth. It inhibits cancer cell growth and significantly reduces prostate-specific antigen (PSA) levels through potent androgen receptor antagonism.

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1. Drug Background ([1]):
Darolutamide (ODM-201; BAY-1841788) is a third-generation oral nonsteroidal anti-androgen (NSAA) designed to overcome the resistance of castration-resistant prostate cancer (CRPC) to first/second-generation androgen receptor inhibitors (such as bicalutamide and enzalutamide) [1].
2. Mechanism of Action ([1]):
- Step 1: It binds to wild-type and mutant androgen receptors (F876L, T877A) with high affinity (Ki <1 nM), competing with endogenous androgens (DHT), thereby blocking the activation of androgen receptors.
- Step 2: It inhibits the nuclear translocation of androgen receptor (AR) (reducing the nuclear AR in CRPC cells by 80-85%) and the binding of AR to DNA androgen response elements (ARE).
- Step 3: Downregulate AR target genes (PSA, TMPRSS2) to inhibit CRPC cell proliferation and induce G1 phase cell cycle arrest [1]
3. Therapeutic potential ([1]):
Dalolutamide showed potent activity against enzalutamide-resistant CRPC cells and xenografts, supporting its clinical development for the treatment of metastatic castration-resistant prostate cancer (mCRPC) [1]

C19H19CLN6O2

398.85

398.125

C, 57.22; H, 4.80; Cl, 8.89; N, 21.07; O, 8.02

1297538-32-9

67171867

White to off-white solid powder

1.4±0.1 g/cm3

719.5±60.0 °C at 760 mmHg

388.9±32.9 °C

0.0±2.4 mmHg at 25°C

1.681

-0.04

3

5

6

28

598

1

ClC1=C(C#N)C=CC(C2=NN(C[C@H](C)NC(C3=NNC(C(O)C)=C3)=O)C=C2)=C1

ANGUXJDGJCHGOG-UHFFFAOYSA-N

InChI=1S/C25H29N5O/c1-19-8-9-22-23(27-19)6-3-7-24(22)29-16-14-28(15-17-29)12-10-20-4-2-5-21(18-20)30-13-11-26-25(30)31/h2-9,18H,10-17H2,1H3,(H,26,31)

N-((S)-1-(3-(3-chloro-4-cyanophenyl)-1H-pyrazol-1-yl)propan-2-yl)-5-(1-hydroxyethyl)-1H-pyrazole-3-carboxamide

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: ≥ 2.08 mg/mL (5.21 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 20.8 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: ≥ 2.08 mg/mL (5.21 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 20.8 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: ≥ 2.08 mg/mL (5.21 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 20.8 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.)