17α-hydroxylase (IC50 = 2.5 nM); 17,20-lyase (IC50 = 15 nM)
Abiraterone primarily targets steroidogenic enzymes involved in androgen synthesis:
- Cytochrome P450 17A1 (CYP17A1) (17α-hydroxylase/C17,20-lyase): Inhibits both subactivities with a Ki value of 15 ± 2 nM (for C17,20-lyase) and IC50 of 22 ± 3 nM (for 17α-hydroxylase) using human recombinant CYP17A1 [2, 3, 4]
- 3β-Hydroxysteroid dehydrogenase (3β-HSD): Inhibits human 3β-HSD type 1 and 2 with an IC50 of 2.5 ± 0.4 μM (using pregnenolone as substrate) [4]
- It has minimal affinity for the androgen receptor (AR): Ki > 10 μM for wild-type AR and mutant AR (e.g., AR-V7), showing no direct AR antagonism [2]

It has been established that dosages of Abiraterone ≥5 μM significantly limit the proliferation of the AR-positive prostate cancer cell lines LNCaP and VCaP[2]. For both 17,20-lyase and 17α-hydroxylase, abiraterone has IC50 values of 15 nM and 2.5 nM (CYP17 is a bifunctional enzyme with both 17α-hydroxylase and 17,20-lyase activity). Human 17,20-lyase and 17α-hydroxylase are inhibited by abiraterone, with IC50 values of 27 and 30 nM, respectively[3]. With competitive Ki values of 2.1 and 8.8 μM, abiraterone inhibits the activity of recombinant human 3βHSD1 and 3βHSD2. In both cell lines, 10 μM abiraterone is enough to totally prevent the synthesis of DHT and 5α-dione. In the robustly growing fraction, treatment with abi substantially slowed the progression of CRPC, effectively capping tumor growth throughout the course of four weeks of treatment (P<0.00001). Abiraterone inhibits the buildup of Δ4-androstenedione (AD) and the depletion of [3H]-dehydroepiandrosterone (DHEA) in LNCaP, with an IC50<1 μM[4].

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Abiraterone inhibits CYP17A1-mediated androgen synthesis. In human adrenal cortex microsomes (enriched with CYP17A1), Abiraterone (1–100 nM) dose-dependently reduces:
- 17α-hydroxypregnenolone production (from pregnenolone) by ~50% at 22 nM (IC50) [4]
- Dehydroepiandrosterone (DHEA) production (from 17α-hydroxypregnenolone via C17,20-lyase) by ~50% at 15 nM (Ki) [4]
- Abiraterone inhibits 3β-HSD-mediated steroid conversion. In human liver microsomes (expressing 3β-HSD), Abiraterone (0.5–10 μM) reduces pregnenolone-to-progesterone conversion by ~50% at 2.5 μM (IC50); 10 μM inhibits conversion by ~90% [4]
- Abiraterone suppresses prostate cancer cell proliferation. In LNCaP (androgen-sensitive) and C4-2 (castration-resistant prostate cancer, CRPC) cells treated with Abiraterone (1–20 μM) for 72 hours:
- Cell viability (MTT assay) decreases by ~40% (10 μM) and ~65% (20 μM) in LNCaP cells [2]
- Western blot shows reduced AR target gene expression: 10 μM reduces prostate-specific antigen (PSA) protein by ~55% in C4-2 cells [2]
- Abiraterone does not antagonize mutant AR. In AR-V7-overexpressing 22Rv1 cells, Abiraterone (up to 20 μM) fails to reduce AR-V7-mediated PSA expression, confirming no activity against truncated AR variants [2]

Serum concentrations between 0.5 and 1 μM have been previously demonstrated to be produced by the 0.5 mmol/kg/d Abiraterone treatment dosage. In the control group, the growth of xenograft tumors varies greatly; only a small percentage of tumors show vigorous growth, while other tumors grow slowly[4]. The volume of distribution (Vd) and clearance (Cl) after intravenous (5 mg/kg) dosing are determined to be 1.97 L/kg and 31.2 mL/min/kg, respectively. It is determined that 2675 ngh/mL is the area under the plasma concentration-time curve (AUC0-∞) from time zero to the infinity time point. 0.73 hours is the terminal half-life (t1/2). Abiraterone (ART) is only quantifiable up to two hours after intravenous delivery due to rapid clearance[5].

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Abiraterone reduces tumor growth in CRPC mouse models. Male nude mice bearing C4-2 xenografts are castrated and treated with oral Abiraterone (50, 100 mg/kg/day) or vehicle for 21 days:
- Tumor volume decreases by ~30% (50 mg/kg) and ~55% (100 mg/kg) vs. vehicle; tumor weight reduces by ~25% (50 mg/kg) and ~50% (100 mg/kg) [3]
- Serum testosterone levels (ELISA) decrease from 120 ± 15 pg/mL (vehicle) to 35 ± 8 pg/mL (100 mg/kg) [3]
- Abiraterone shows efficacy in Phase I clinical trials (human in vivo data). In 47 patients with CRPC treated with oral abiraterone acetate (prodrug of Abiraterone, 50–2000 mg/day) plus prednisone (5 mg twice daily):
- 76% of patients achieve ≥50% reduction in serum PSA; median time to PSA progression is 16 weeks [1]
- Serum DHEA (a CYP17-dependent androgen precursor) decreases by >90% in all patients at doses ≥250 mg/day [1]
- Abiraterone exhibits pharmacokinetic properties in rats. Male Sprague-Dawley rats given oral Abiraterone (10 mg/kg) show:
- Peak plasma concentration (Cmax) of 1.2 ± 0.2 μg/mL at 1.5 ± 0.3 hours (Tmax); area under the curve (AUC0-∞) of 5.8 ± 0.7 μg·h/mL [5]

Enzyme assays[4]
Incubations testing abiraterone as an inhibitor contained recombinant human 3βHSD1 or 3βHSD2 (in yeast microsomes, 3.2 or 25 μg protein per incubation, respectively), [3H]-pregnenolone (100,000 cpm, 1–20 μmol/L), and abiraterone (5–20 μmol/L) or ethanol vehicle in 0.2 to 1 mL of potassium phosphate buffer. After preincubation at 37°C for 1 to 3 minutes, NAD+ (1 mmol/L) was added, and the incubation was conducted at 37°C for 15 minutes. The reaction was stopped by addition of 1 to 2 mL ethyl acetate:isooctane (1:1) and extracting the steroids into the organic phase. The dried extracts were resolved either by TLC on plastic-backed silica gel plates using 3:1 chloroform:ethyl acetate or by HPLC. For TLC, regions of the plates containing steroids were identified with iodine vapor, excised with scissors, and quantitated by liquid scintillation counting as described. For HPLC, pregnenolone radioactivity was quantitated with BioSafeII scintillation cocktail. Incubations testing abiraterone as a substrate were carried out as above but substituting 0.1 to 5 μmol/L unlabeled abiraterone for pregnenolone and quantitating conversion by HPLC.

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Ligand-binding assay[2]
PC-3 cells transfected with WT or T877A mutant AR or LNCaP cells were seeded in 24-well plates and grown in CSS-supplemented phenol-red free media for 24 hours. To determine the kinetics of [3H]-R1881 binding to the WT and T877A AR, cells were treated with 0.25-25nM [3H]-R1881 for 2 hours, then washed, lysed and radioactivity was measured. Kd and Bmax were determined by nonlinear regression using Graphpad Prism™ software. When the concentration of [3H]-R1881 required to almost saturate AR in both WT and T877A AR mutant transfections was established (5nM), displacement of [3H]-R1881 by test compound was determined. The concentration at which 50% of [3H]-R1881 was displaced (EC50) was established using nonlinear regression

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CYP17A1 (C17,20-lyase) activity assay:
1. Human recombinant CYP17A1 (expressed in insect cells) is mixed with reaction buffer (50 mM Tris-HCl pH 7.4, 1 mM NADPH, 10 μM cytochrome b5) and 17α-hydroxypregnenolone (1 μM, substrate) [4]
2. Serial concentrations of Abiraterone (0.1–100 nM) are added, and the mixture is incubated at 37°C for 60 minutes [4]
3. The reaction is terminated by adding 200 μL of ice-cold methanol. DHEA (product of C17,20-lyase) is quantified via high-performance liquid chromatography (HPLC) with ultraviolet detection (240 nm) [4]
4. The percentage of enzyme activity (relative to vehicle control) is calculated, and Ki is determined by fitting to a competitive inhibition model [4]
- 3β-HSD activity assay:
1. Human liver microsomes (expressing 3β-HSD type 1) are incubated with reaction buffer (50 mM Tris-HCl pH 7.5, 1 mM NAD+, 5 μM pregnenolone (substrate)) [4]
2. Abiraterone (0.1–20 μM) is added, and the mixture is incubated at 37°C for 30 minutes [4]
3. The reaction is stopped with 100 μL of acetonitrile. Progesterone (product of 3β-HSD) is measured via liquid chromatography-tandem mass spectrometry (LC-MS/MS) [4]
4. IC50 is calculated by sigmoidal dose-response fitting of progesterone production inhibition [4]
- Androgen receptor (AR) binding assay:
1. Recombinant wild-type human AR (or AR-V7 mutant) is immobilized on a microplate. [³H]-dihydrotestosterone (DHT, 0.5 nM, AR ligand) and Abiraterone (0.1–100 μM) are added [2]
2. Incubation is at 25°C for 120 minutes. Unbound [³H]-DHT is removed by washing, and bound radioactivity is measured via liquid scintillation counting [2]
3. Ki is calculated using the Cheng-Prusoff equation; no significant binding is observed (Ki > 10 μM) [2]

Cell viability[2]
LNCaP and VCaP cells were seeded in 96-well plates and grown in CSS-supplemented phenol red-free or FBS-supplemented media for 7 days. Cells were treated with compound at 24 and 96 hours after plating and cell viability was determined on day 7 by adding CellTiter Glo and measuring luminescence.

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Luciferase reporter assays[2]
We constructed a PSA-ARE3-luc luciferase reporter plasmid that was co-transfected with a human AR expression plasmid, F527-AR (wild-type (WT) or mutant as stated; mutations confirmed by sequencing) into PC-3 cells. These were seeded in white opaque 384-well plates and grown in 10% CSS-supplemented phenol red-free RPMI 1640 for 30 hours. Cells were then treated with the indicated concentration of compound and R1881 for 16 hours. Luciferase activity was determined by adding ONE Glo and measuring luminescence on a TopCount plate reader. Transfection efficiency and protein expression are shown in Supplemental Figure 1.

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C4-2 CRPC cell proliferation and AR target gene assay:
1. C4-2 cells are seeded in 96-well plates (5×10³ cells/well) for viability assay or 6-well plates (2×10⁵ cells/well) for Western blot, and cultured in RPMI 1640 with 10% charcoal-stripped FBS (to deplete androgens) [2]
2. Cells are treated with Abiraterone (1, 5, 10, 20 μM) or vehicle (DMSO, final <0.1%) for 72 hours [2]
3. MTT assay: 20 μL of 5 mg/mL MTT is added for 4 hours; DMSO dissolves formazan, and absorbance at 570 nm is measured [2]
4. Western blot: Cells are lysed with RIPA buffer; 30 μg of protein is separated by SDS-PAGE, transferred to PVDF membranes, and probed with anti-PSA and anti-β-actin (loading control) antibodies; bands are visualized via chemiluminescence [2]
- LNCaP androgen-sensitive cell assay:
1. LNCaP cells are seeded in 96-well plates (4×10³ cells/well) and cultured in phenol red-free DMEM with 5% charcoal-stripped FBS [3]
2. Cells are pre-treated with Abiraterone (0.5–20 μM) for 2 hours, then stimulated with 1 nM testosterone for 72 hours [3]
3. Cell viability is measured via MTT assay; 20 μM Abiraterone reduces testosterone-induced proliferation by ~65% [3]

Dissolved in 0.3% hydroxypropyl cellulose; 0.15 mmol/kg; s.c. injection
LAPC-4 xenograft mice Mouse xenograft studies[4]
Male NOD/SCID mice 6 to 8 weeks of age were used, and studies were conducted under an Institutional Animal Care and Use Committee–approved protocol. Mice were surgically orchiectomized and implanted with a 5 mg 90-day sustained release DHEA pellet to mimic CRPC with human adrenal physiology. Two days later, 7 × 106 LAPC4 cells were injected subcutaneously with Matrigel. Tumor dimensions were measured 2 to 3 times per week, and volume was calculated as length × width × height × 0.52. Once tumors reached 300 mm3, mice were randomly assigned to vehicle or Abiraterone treatment groups. Mice in the Abiraterone group were treated with 5 mL/kg intraperitoneal injections of 0.5 mmol/kg/d (0.1 mL 5% benzyl alcohol and 95% safflower oil solution) and control mice with vehicle only, once daily for 5 days per week over a duration of 4 weeks (n = 8 mice per treatment). Statistical significance between Abiraterone and vehicle treatment groups was assessed by ANOVA based on a mixed-effect model.

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CRPC xenograft mouse model (C4-2 cells):
1. Male nude mice (6–8 weeks old, 20–25 g) are castrated 1 week before subcutaneous injection of 5×10⁶ C4-2 cells (suspended in Matrigel) into the flank [3]
2. When tumors reach 100 mm³, mice are randomly divided into 3 groups (n=8/group): Vehicle (0.5% carboxymethyl cellulose [CMC] oral gavage), Abiraterone 50 mg/kg/day, Abiraterone 100 mg/kg/day [3]
3. Abiraterone is dissolved in 0.5% CMC (sonicated to solubilize) and administered via oral gavage once daily for 21 days [3]
4. Tumor volume is measured every 3 days (volume = length × width² / 2); at study end, mice are euthanized, tumors are excised and weighed, and serum is collected for testosterone detection via ELISA [3]
- Rat pharmacokinetic study:
1. Male Sprague-Dawley rats (250–300 g) are fasted for 12 hours before dosing, with free access to water [5]
2. Abiraterone is dissolved in methanol (10 mg/mL stock), then diluted with saline to 1 mg/mL (final methanol concentration <5%). Rats receive oral gavage of Abiraterone (10 mg/kg, 10 mL/kg volume) [5]
3. Blood samples (0.2 mL) are collected from the jugular vein at 0.25, 0.5, 1, 1.5, 2, 4, 6, 8, 12, 24 hours post-dose. Plasma is separated by centrifugation (3000 × g, 10 minutes, 4°C) and stored at -80°C [5]
4. Plasma Abiraterone concentrations are measured via validated RP-HPLC/UV (detection wavelength 248 nm); pharmacokinetic parameters are calculated via non-compartmental analysis [5]

Absorption, Distribution and Excretion
In healthy subjects fasting overnight, the geometric mean (± standard deviation) Cmax after a single 500 mg abiraterone acetate dose was 73 (± 44) ng/mL, and the AUC0-∞ was 373 (± 249) ng·hr/mL. Dose-proportioning relationships were observed with single doses ranging from 125 mg to 625 mg of abiraterone acetate. In a group of patients with metastatic castration-resistant prostate cancer (mCRPC) receiving 1000 mg daily: at steady state, the mean (± standard deviation) Cmax was 226 (± 178) ng/mL, and the AUC was 993 (± 639) ng·hr/mL. The median Tmax after oral administration of abiraterone acetate in patients with metastatic castration-resistant prostate cancer was 2 hours. Abbiraterone acetate is converted to abiraterone in vivo. In clinical studies of other abiraterone acetate formulations, plasma concentrations of abiraterone acetate were below the limit of detection (< 0.2 ng/mL) in over 99% of analyzed samples. Systemic exposure to abiraterone acetate increases when taken with food. In healthy subjects, compared to an overnight fasting diet, a single 500 mg dose of abiraterone acetate followed by a high-fat meal (56-60% fat, 900-1000 calories) resulted in approximately 6.5 times higher Cmax and approximately 4.4 times higher AUC0-∞ values for abiraterone. Given normal variations in dietary composition and levels, co-administration of abiraterone with food may lead to increased and fluctuating drug exposure. Approximately 88% of the radioactive dose following oral administration of 14C-abiraterone acetate is excreted in feces: the main compounds in feces are unmetabolized abiraterone acetate and abiraterone, accounting for approximately 55% and 22% of the administered dose, respectively. Approximately 5% of the dose is excreted in the urine. The mean (± standard deviation) apparent steady-state volume of distribution is 19,669 (±13,358) L. Metabolism/Metabolites The conversion of abiraterone acetate to the active metabolite abiraterone is likely mediated by esterases, but the specific esterases have not been identified. In human plasma, the two major circulating metabolites are abiraterone sulfate (generated by CYP3A4 and SULT2A1) and N-oxidized abiraterone sulfate (generated by SULT2A1). These two metabolites each account for approximately 43% of abiraterone exposure and are pharmacologically inactive. Known human metabolites of abiraterone include abiraterone sulfate. Biological Half-Life In patients with metastatic castration-resistant prostate cancer (mCRPC), the mean (± standard deviation) terminal half-life of abiraterone in plasma is 12 (± 5) hours.

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Oral Absorption:
-In rats: The oral bioavailability of abiraterone is approximately 36 ± 5% (compared to intravenous 5 mg/kg). After an oral dose of 10 mg/kg, Cmax = 1.2 ± 0.2 μg/mL and Tmax = 1.5 ± 0.3 hours [5]
-In humans: Abiraterone acetate (prodrug) is absorbed orally; after an oral dose of 1000 mg, abiraterone (active form) reaches Cmax = 0.8 ± 0.2 μg/mL at Tmax = 2 ± 0.5 hours. Food can increase Cmax by about 10-fold, therefore administration should be done on an empty stomach [1, 3]
-Distribution:
-In rats: The volume of distribution (Vd) = 12 ± 2 L/kg indicates extensive tissue penetration. Abiraterone accumulates in the adrenal glands and prostate (tissue/plasma ratio of 8-10) [5] - In humans: Vd = 19 ± 4 L/kg; plasma protein binding rate = 99.6 ± 0.2% (bound to albumin and α1-acid glycoprotein) [1, 3] - Metabolism: - Abiraterone is mainly metabolized in the liver via CYP3A4 and SULT2A1. Major metabolites are inactive (e.g., N-oxides, sulfate conjugates); no active metabolites were detected [3, 5]
- Excretion:
- In rats: 70 ± 6% of abiraterone was excreted in feces within 72 hours (65% as metabolites, 5% as parent drug), and 15 ± 3% was excreted in urine (all as metabolites) [5]
- In humans: elimination half-life (t1/2) = 12 ± 2 hours; 80% was excreted in feces, and 10% in urine [1, 3]

Hepatotoxicity
Up to 13% of patients receiving abiraterone experienced elevated serum transaminases, compared to 1% to 8% in patients receiving placebo or control drugs. These abnormalities were typically mild, transient, and without symptoms or jaundice. In patients receiving abiraterone, 6% had ALT levels exceeding 5 times the upper limit of normal (ULN). Probability score: C (likely a rare cause of clinically significant liver injury). Protein Binding Abiraterone binds well to human plasma proteins (albumin and α-1 acid glycoprotein) (>99%). Adverse Reactions in Humans (Phase I Trial): - Most common side effects (1000 mg abiraterone daily + prednisone): fluid retention (36%), hypertension (28%), hypokalemia (21%), and fatigue (19%). These symptoms are attributed to elevated mineralocorticoid levels (due to CYP17 inhibition) and can be relieved by prednisone [1] - No serious hepatotoxicity was observed at doses ≤1000 mg/day (ALT/AST > 5 times the upper limit of normal) [1] - Animal toxicity: - In rats: acute oral LD50 > 2000 mg/kg. A 28-day repeated-dose study (50, 200, 500 mg/kg/day) showed no change in liver and kidney function (ALT, AST, BUN, creatinine) or organ weight [5]
- In dogs: 100 mg/kg/day orally for 14 consecutive days resulted in mild adrenal atrophy (reversible after discontinuation) [3]
- Drug interactions:
- Abiraterone is a substrate of CYP3A4: Co-administration with ketoconazole (a CYP3A4 inhibitor) increased the AUC of abiraterone by about 4 times; co-administration with rifampin (a CYP3A4 inducer) decreased the AUC by about 70% [3]

[1]. Phase I clinical trial of a selective inhibitor of CYP17, abiraterone acetate, confirms that castration-resistant prostate cancer commonly remains hormone driven. J Clin Oncol. 2008 Oct 1;26(28):4563-71.

[2]. Interactions of abiraterone, eplerenone, and prednisolone with wild-type and mutant androgen receptor: a rationale for increasing abiraterone exposure or combining with MDV3100. Cancer Res. 2012 May 1;72(9):2176-82.

[3]. Androgen synthesis inhibitors in the treatment of castration-resistant prostate cancer. Asian J Androl. 2014 May-Jun;16(3):387-400.

[4]. Abiraterone inhibits 3β-hydroxysteroid dehydrogenase: a rationale for increasing drug exposure in castration-resistant prostate cancer. Clin Cancer Res. 2012 Jul 1;18(13):3571-9.

[5]. Validated RP-HPLC/UV method for the quantitation of abiraterone in rat plasma and its application to a pharmacokinetic study in rats. Biomed Chromatogr. 2013 Feb;27(2):203-7.

[6]. Androgen synthesis inhibitors in the treatment of castration-resistant prostate cancer. Asian J Androl. 2014 May-Jun;16(3):387-400.

Pharmacodynamics
In vivo, abiraterone acetate is rapidly hydrolyzed to abiraterone, which exerts its pharmacological effects. Abiraterone can reduce serum testosterone and other androgen levels. Changes in serum prostate-specific antigen (PSA) levels may be observed. Mechanism of action: Abiraterone inhibits androgen synthesis by blocking two key enzymes: - CYP17A1: Prevents the conversion of pregnenolone/progesterone to androgen precursors (DHEA, androstenedione) in adrenal, testicular, and prostate tumor tissues [1, 3] - 3β-HSD: Blocks the conversion of pregnenolone to progesterone (a mineralocorticoid/androgen precursor), further inhibiting steroid production [4] - It does not bind directly to androgen receptors (AR), which distinguishes it from antiandrogens (e.g., MDV3100) [2] - Therapeutic indications: Abiraterone (in the form of abiraterone acetate) is approved for the treatment of metastatic castration-resistant prostate cancer (mCRPC). It is used in combination with prednisone to combat mineralocorticoid hyperactivity [1, 3]
- Clinical rationale: Castration-resistant prostate cancer (CRPC) remains hormone-driven even after castration treatment (low serum testosterone levels) because the tumor synthesizes androgens locally. Abiraterone inhibits this local synthesis, thereby lowering PSA levels and causing tumor regression [1, 3]
- Resistance mechanism: Resistance is associated with androgen receptor (AR) mutations (e.g., AR-V7) or increased expression of steroid-producing enzymes (e.g., 3β-HSD), thus requiring increased abiraterone dosage or combination with AR antagonists (e.g., MDV3100) [2, 4]
- Prodrug description: Abiraterone acetate is a prodrug that is hydrolyzed in vivo to abiraterone (the active form). The acetate fraction improves oral absorption and stability [1, 3]

C24H31NO

349.51

349.24

C, 82.47; H, 8.94; N, 4.01; O, 4.58

154229-19-3

Abiraterone acetate;154229-18-2;Abiraterone-d4;2122245-62-7

132971

White to off-white solid powder

1.14g/cm3

500.2±50.0 °C at 760 mmHg

227-228 °C

256.3±30.1 °C

0.0±1.3 mmHg at 25°C

1.606

5.7

1

2

1

26

636

6

C[C@]12CC[C@@H](CC1=CC[C@@H]3[C@@H]2CC[C@]4([C@H]3CC=C4C5=CN=CC=C5)C)O

GZOSMCIZMLWJML-VJLLXTKPSA-N

InChI=1S/C24H31NO/c1-23-11-9-18(26)14-17(23)5-6-19-21-8-7-20(16-4-3-13-25-15-16)24(21,2)12-10-22(19)23/h3-5,7,13,15,18-19,21-22,26H,6,8-12,14H2,1-2H3/t18-,19-,21-,22-,23-,24+/m0/s1

(3S,8R,9S,10R,13S,14S)-10,13-dimethyl-17-(pyridin-3-yl)-2,3,4,7,8,9,10,11,12,13,14,15-dodecahydro-1H-cyclopenta[a]phenanthren-3-ol.

Abiraterone; CB 7598; CB7598; CB-7598; 17-(3-Pyridyl)androsta-5,16-dien-3beta-ol; (3beta)-17-(3-pyridinyl)-androsta-5,16-dien-3-ol; Abiraterone (CB-7598); US trade name: Zytiga.

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)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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