17α-hydroxylase (IC50 = 2.5 nM); 17,20-lyase (IC50 = 15 nM)

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].

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 ng*h/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].

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

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.

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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Dissolved in 0.3% hydroxypropyl cellulose; 0.15 mmol/kg; s.c. injection

LAPC-4 xenograft mice

Absorption, Distribution and Excretion
Geometric mean (± SD) Cmax was 73 (± 44) ng/mL and AUC0-∞ was 373 (± 249) ng x hr/mL following a single dose of 500 mg abiraterone acetate in overnight-fasted healthy subjects. Dose proportionality was observed in single doses of abiraterone acetate ranging from 125 mg to 625 mg. A group of patients with mCRPC received a daily dose of 1,000 mg: at steady-state, the mean (± SD) Cmax was 226 (± 178) ng/mL and AUC was 993 (± 639) ng x hr/mL. Following oral administration of abiraterone acetate to patients with metastatic castration-resistant prostate cancer, the median Tmax was two hours. _In vivo_, abiraterone acetate is converted to abiraterone. In clinical studies of other abiraterone acetate formulations, abiraterone acetate plasma concentrations were below detectable levels (< 0.2 ng/mL) in > 99% of the analyzed samples. Systemic exposure to abiraterone is increased when abiraterone acetate is administered with food. Abiraterone Cmax was approximately 6.5-fold higher, and AUC0-∞ was 4.4-fold higher when a single dose of abiraterone acetate 500 mg was administered with a high-fat meal (56-60% fat, 900-1,000 calories) compared to overnight fasting in healthy subjects. Given the normal variation in the content and composition of meals, taking abiraterone with meals has the potential to result in increased and highly variable exposures.
Following oral administration of ¹⁴C-abiraterone acetate, approximately 88% of the radioactive dose is recovered in feces: the major compounds present in feces are unchanged abiraterone acetate and abiraterone, accounting for approximately 55% and 22% of the administered dose, respectively. Approximately 5% of the dose is recovered in urine.
The mean (± SD) apparent steady-state volume of distribution is 19,669 (± 13,358) L.

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Metabolism / Metabolites
The conversion of abiraterone acetate to abiraterone, the active metabolite, is likely to be mediated by esterases, although specific esterases have not been identified. In human plasma, the two main circulating metabolites are abiraterone sulfate, which is formed by CYP3A4 and SULT2A1, and N-oxide abiraterone sulfate, which is formed by SULT2A1. These metabolites each account for about 43% of abiraterone exposure and are pharmacologically inactive.
Abiraterone has known human metabolites that include abiraterone sulfate.

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Biological Half-Life
In patients with mCRPC, the mean (± SD) terminal half-life of abiraterone in plasma is 12 (± 5) hours.

Hepatotoxicity
Serum aminotransferase elevations occur in up to 13% of patients treated with abiraterone compared with 1% to 8% receiving placebo or a comparator drug, but the abnormalities are generally mild, transient and not associated with symptoms or jaundice. ALT elevations above 5 times the upper limit of normal (ULN) occur in 6% of abiraterone treated vs
Likelihood score: C (probable rare cause of clinically apparent liver injury).

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Protein Binding
Abiraterone is highly bound (>99%) to the human plasma proteins, albumin and alpha-1 acid glycoprotein.

[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 mediates its pharmacological actions. Abiraterone decreases serum testosterone and other androgens. A change in serum prostate-specific antigen (PSA) levels may be observed.

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.)