D4-abiraterone (D4A ) (10 mM) almost totally prevents the conversion of D4-androstenedione (AD) into 5α-androstanedione and other 5α-reduced androgens. Abiraterone (Abi) (IC50=418 and >500 nM, respectively) has a lower affinity for the mutant and wild type androgen receptors (IC50=5.3 nM and 7.9 nM, respectively) than does D4-abiraterone (expressed in LNCaP and IC50=5.3 nM). When it comes to suppressing PSA, TMPRSS2, and FKBP5 expression in LNCAP, LAPC4, and C4-2 cell lines, D4-abiraterone performs a notably better job than Albi. The expression of AR target genes is also dose-dependently inhibited by D4-abiraterone[1].

D4-abiraterone (D4A ) (10 mM) almost totally prevents the conversion of D4-androstenedione (AD) into 5α-androstanedione and other 5α-reduced androgens. Abiraterone (Abi) (IC50=418 and >500 nM, respectively) has a lower affinity for the mutant and wild type androgen receptors (IC50=5.3 nM and 7.9 nM, respectively) than does D4-abiraterone (expressed in LNCaP and IC50=5.3 nM). When it comes to suppressing PSA, TMPRSS2, and FKBP5 expression in LNCAP, LAPC4, and C4-2 cell lines, D4-abiraterone performs a notably better job than Albi. The expression of AR target genes is also dose-dependently inhibited by D4-abiraterone[1].

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D4-abiraterone potently suppresses androgen-responsive gene expression. In LNCaP, LAPC4, and C4-2 cell lines, treatment with 1 μM D4A significantly inhibits the expression of PSA, TMPRSS2, and FKBP5 genes induced by DHT, DHEA, or the synthetic androgen R1881. This inhibition is more effective than with 1 μM abiraterone and is comparable to the potent AR antagonist enzalutamide. [1]
D4-abiraterone inhibits DHT-stimulated cell proliferation. In LNCaP cells treated with 0.5 nM DHT, co-treatment with D4A (concentration not specified in the proliferation assay results figure) significantly inhibits cell growth over 6 days, with an effect equivalent to enzalutamide and superior to abiraterone. [1]

D4-abiraterone (D4A) is ten times more effective than abiraterone (Abi) at preventing the conversion of dehydroepiandrosterone (DHEA) to D4-androstenedione (AD) by 3β-hydroxysteroid dehydrogenase (3βHSD) in LNCaP and VCaP xenografts. For the purpose of preventing AD accumulation at 48 hours in both LNCaP and VCaP xenografts, 0.1 μM D4-abiraterone is equal to 1 μM Abi. When comparing the D4-abiraterone group to the Abi acetate group, there is a significant delay in progression (P=0.011). When compared to Abi acetate, treatment with D4-abiraterone improves progression-free survival[1].

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D4-abiraterone exhibits superior antitumor activity compared to abiraterone acetate in castration-resistant prostate cancer (CRPC) xenograft models.
In VCaP xenograft models grown in orchiectomized mice supplemented with DHEA pellets, daily intraperitoneal treatment with D4-abiraterone (0.5 mmol/kg/d) significantly delayed time to tumor progression (>20% increase in volume) compared to treatment with abiraterone acetate (P=0.011). [1]
In C4-2 xenograft models under the same conditions, treatment with D4-abiraterone also significantly increased progression-free survival compared to both abiraterone acetate and enzalutamide. [1]

Enzyme assays are used to determine whether D4-abiraterone (D4A) is an inhibitor of 3βHSD. In summary, 0.5 mL of potassium phosphate buffer (pH 7.4), D4-abiraterone (5 to 20 μM), or ethanol vehicle are prepared along with recombinant human 3βHSD1 or 3βHSD2 (in yeast microsomes, 45 or 2.5 μg protein per incubation, respectively). NAD+ (1 mM) is added after a pre-incubation at 37°C for 1 to 3 min, and the incubation is carried out at 37°C for 20 min. The addition of 1 mL of ethyl acetate:isooctane (1:1) stops the reaction, after which the steroids are extracted into the organic phase and dried. In-line scintillation counting is used to quantify the steroids in the dried extracts, and HPLC is used to resolve the steroids[1].

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The inhibitory effect of D4-abiraterone on recombinant human 3βHSD1 and 3βHSD2 was assessed. Incubations contained recombinant enzyme (in yeast microsomes), [³H]-pregnenolone substrate (1–20 μmol/L), NAD+ cofactor, and varying concentrations of D4A (5–20 μmol/L) or vehicle in phosphate buffer. After incubation at 37°C, reactions were stopped, steroids were extracted with an organic solvent, and metabolites were separated and quantified by HPLC with in-line scintillation counting. Analysis of the data using Lineweaver-Burk plots indicated that D4A acts as a mixed inhibitor for 3βHSD1 and a noncompetitive inhibitor for 3βHSD2. [1]
The inhibitory effect of D4-abiraterone on CYP17A1 activity was evaluated in intact 293 cells stably expressing human CYP17A1. Cells were treated with [³H]-pregnenolone and D4A or abiraterone (0.1–10 nM) for 3-6 hours. Medium was collected, and the conversion of pregnenolone to DHEA was analyzed by HPLC, showing D4A and abiraterone had comparable inhibitory potency. [1]
The effect of D4-abiraterone on endogenous SRD5A activity was tested in LAPC4 cells. Cells were treated with [³H]-androstenedione (AD, the preferred SRD5A substrate) and D4A, abiraterone, or enzalutamide (10-100 μM) for 24 hours. Medium was collected, treated with β-glucuronidase, extracted, and analyzed by HPLC. D4A (10 μM) nearly completely blocked the conversion of AD to 5α-reduced androgens, while abiraterone and enzalutamide had no detectable effect even at 100 μM. [1]

The indicated concentrations of D4-abiraterone (D4A) are applied to the cells for 30 minutes after they have been cultured for 48 hours in serum-free medium. The cells are lysed using RIPA buffer after being washed twice with 0.9% NaCl solution and four times with 1×PBS. A multilabel counter is used to detect the protein concentration, which is then used to normalize the intracellular radioactivity measured using a liquid scintillation counter|1].

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AR competition assays were performed in LNCaP (mutant AR) and LAPC4 (wild-type AR) cells. Serum-starved cells were treated with 0.1 nM [³H]-R1881 and increasing concentrations of D4A, abiraterone, enzalutamide, or bicalutamide for 30 minutes. Cells were washed extensively, lysed, and intracellular radioactivity was measured by scintillation counting and normalized to protein concentration, allowing calculation of IC50 values for competitor displacement of [³H]-R1881. [1]
Chromatin immunoprecipitation (ChIP) assays were conducted to assess AR occupancy on target gene promoters. LNCaP cells were serum-starved, treated with DHT and increasing concentrations of D4A or enzalutamide for 3 hours. Cells were cross-linked, chromatin was sheared, and AR-bound DNA was immunoprecipitated using an anti-AR antibody. Precipitated DNA was quantified by qPCR for PSA, TMPRSS2, and FKBP5 regulatory elements and normalized to input DNA. [1]
Quantitative PCR (qPCR) was used to measure androgen-responsive gene expression. Cells (LNCaP, LAPC4, C4-2, VCaP) were starved in phenol red-free, serum-free medium for 48 hours, then treated with androgens (DHT, DHEA, R1881) and/or drugs (D4A, abiraterone, enzalutamide) for 24 hours. RNA was extracted, reverse transcribed to cDNA, and gene expression (PSA, TMPRSS2, FKBP5) was measured by qPCR using SYBR Green, normalized to the housekeeping gene RPLP0 and vehicle-treated controls. [1]
Cell proliferation was assessed by measuring DNA content. LNCaP cells were seeded in 96-well plates and cultured in phenol red-free medium with charcoal-stripped serum, androgens (DHT), and/or drugs. Medium was changed every other day. After 2, 4, or 6 days, cells were lysed and DNA content was quantified using Hoechst stain and fluorescence measurement, serving as a proxy for cell number. [1]

In this study, male NSG mice aged 6 to 8 weeks are employed. Mice with castration-resistant prostate cancer (CRPC) are surgically orchiectomized and given a 5 mg sustained-release dehydroepiandrosterone (DHEA) pellet to be administered over a 90-day period, simulating human adrenal physiology. Two days later, matrigel is subcutaneously injected with 107 VCaP or C4-2 cells. Mice are randomly assigned, without following strict randomization, to treatment groups consisting of either vehicle (n = 9 or 10 mice for VCaP and C4-2, respectively) or D4-abiraterone (n = 10 mice for both cell lines) once tumors reach 300mm3. For up to 15 days, 5 mL per kg intraperitoneal injection of D4-abiraterone (0.5 mmol per kg per day in 0.1 mL 5% benzyl alcohol and 95% safflower oil solution) is given daily. Every day, intraperitoneal injections of 0.1 mL of a 5% benzyl alcohol and 95% safflower oil solution are given to the control groups. Every day, the volume of the tumor is measured, and the time it takes for the tumor to grow by 20% is calculated. When the tumor size doubles over the baseline or on day 15 of treatment, mice are killed[1].

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For antitumor efficacy studies in xenograft models, male NSG mice were surgically orchiectomized and implanted subcutaneously with a 5 mg, 90-day sustained-release DHEA pellet to mimic adrenal androgen production. Two days later, 10^7 VCaP or C4-2 prostate cancer cells mixed with Matrigel were injected subcutaneously. When tumors reached approximately 300 mm³, mice were assigned to treatment groups.
D4-abiraterone was dissolved in a vehicle of 5% benzyl alcohol and 95% safflower oil. It was administered via intraperitoneal injection at a dose of 0.5 mmol/kg body weight per day. Control groups received the vehicle alone, and comparison groups received abiraterone acetate prepared in the same vehicle and via the same route and dose. Treatment continued daily for up to 15 days. Tumor volume was measured daily, and the endpoint was time to progression, defined as a >20% increase in tumor volume from baseline. [1]

D4-Abiraterone was identified as a metabolite of abiraterone. The conversion of abiraterone to D4A is catalyzed by 3β-hydroxysteroid dehydrogenase (3βHSD). [1] D4-Abiraterone was detected in the serum of mice that received intraperitoneal injection of abiraterone acetate. [1] D4-Abiraterone was also detected in the serum of patients with castration-resistant prostate cancer (CRPC) who received oral abiraterone acetate (1000 mg daily). Blood samples were collected 2 to 14 hours after administration. Serum concentrations appeared to vary among patients. [1]

Mice treated with D4-abiraterone for an extended period did not show a significant increase in serum deoxycorticosterone (DOC) levels compared to the carrier control group. This contrasts sharply with abiraterone treatment, which increases DOC levels and may lead to hypertension and hyperkalemia. [1]

[1]. Conversion of abiraterone to D4A drives anti-tumour activity in prostate cancer. Nature. 2015 Jul 16;523(7560):347-51.

D4-Abiraterone (D4A) is a Δ4,3-keto metabolite derived from abiraterone by the conversion of abiraterone to the more potent metabolite D4-Abiraterone. This conversion has been observed in mice and patients with prostate cancer. [1] The mechanism of action of D4-Abiraterone is thought to be multi-target inhibition: it effectively inhibits key steroid-producing enzymes (CYP17A1, 3βHSD, and SRD5A) required for the synthesis of dihydrotestosterone (DHT) in castration-resistant prostate cancer (CRPC), and is also a potent competitive antagonist of the androgen receptor (AR). This combined effect may explain its superior antitumor effect compared to abiraterone, which primarily targets CYP17A1. [1] This study suggests that the clinical efficacy of abiraterone acetate may be partly attributed to its conversion to the more potent metabolite D4-Abiraterone. The authors suggest that direct use of D4A to treat castration-resistant prostate cancer (CRPC) may be more effective than abiraterone. [1]

C24H29NO

347.49316

347.225

C, 82.95; H, 8.41; N, 4.03; O, 4.60

154229-21-7

154229-21-7

196941

White to off-white solid powder

5.606

0

2

1

26

676

5

C[C@@]12C(C3=CC=CN=C3)=CC[C@@]1([H])[C@]4([H])CCC5=CC(CC[C@]5(C)[C@@]4([H])CC2)=O

GYJZZAJJENTSTP-NHFPKVKZSA-N

InChI=1S/C24H29NO/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/h3-4,7,13-15,19,21-22H,5-6,8-12H2,1-2H3/t19-,21-,22-,23-,24+/m0/s1

(8R,9S,10R,13S,14S)-10,13-dimethyl-17-pyridin-3-yl-1,2,6,7,8,9,11,12,14,15-decahydrocyclopenta[a]phenanthren-3-one

CB 7627; D4A; Δ4-Abiraterone

2934.99.9001

Powder      -20°C    3 years

                     4°C     2 years

In solvent   -80°C    6 months

                  -20°C    1 month

Note: Please store this product in a sealed and protected environment (e.g. under nitrogen), avoid exposure to moisture.

Room temperature (This product is stable at ambient temperature for a few days during ordinary shipping and time spent in Customs)

DMSO : ~50 mg/mL (~143.89 mM)

Solubility in Formulation 1: ≥ 2.5 mg/mL (7.19 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 2: ≥ 2.5 mg/mL (7.19 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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 (Please use freshly prepared in vivo formulations for optimal results.)