Androgen receptor (IC50 = 16 nM)

In radioligand binding experiments, aparalutamide (ARN-509) demonstrates a low micromolar affinity for GABAA receptors (IC50 3 μM), suggesting that it may potentially antagonize GABAA at levels that are inhibitory [1]. Strongly inhibiting the AR ligand-binding domain, apelutamide blocks the transcription of AR gene targets, DNA binding, and nuclear translocation of the androgen receptor (AR) [2].

Apalutamide (ARN-509) supports oral treatment once day in mice and dogs due to its long plasma half-life, excellent oral bioavailability, and low systemic clearance. The steady-state plasma levels of apalutamide rose in repeated dosing experiments, which is consistent with its extended terminal half-life. This led to high levels of C24 hours and a low peak-to-trough ratio (ratio: 2.5). Apalutamide at doses of 1, 10, or 30 mg/kg/day was administered to castrated male mice with LNCaP/AR xenograft tumors. On day 28, >50% tumor volume reduction was seen in 13 out of 20 animals treated with Apalutamide (30 mg/kg/day), compared to 19 out of 19 mice treated with MDV3100 (30 mg/kg/day). just 3[1].

Ligand binding studies [1]
Whole cell LNCaP/AR: Whole-cell competitive binding assays were performed in LNCaP/AR(codon-switch) (LNCaP/AR(cs)) (harbors a mixture of exogenous wild-type AR and endogenous mutant AR (T877A)) and cells propagated in Iscove’s or RPMI media supplemented with 10% fetal bovine serum (FBS), or during the assay with 10% charcoal-stripped, dextran-treated fetal bovine serum (CSS). Cells were pre-incubated with 18F-FDHT, increasing concentrations (1pM to 1μM) of cold competitor were added, and the assay was performed according to published procedures to measure specific uptake of 18F-FDHT (4). IC50 values were determined using a one site binding model with least squares curve fitting and R2 > 0.99.

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Whole-cell extract MDA-MB-453 cells: MDA-MB-453 cells (endogenous wild-type AR;ATCC: HTB131) were cultured in RPMI 1640 containing 20 mM HEPES, 4 mM l-glutamine, 10 μg/mL human insulin, 10% FBS and 20 μg/mL gentamicin. After reaching 90% confluence, cells were harvested, resuspended in TEGM (10 mM Tris-HCl pH 7.2, 1 mM EDTA, 10% glycerol, 1 mM -mercaptoethanol, 10 mM sodium molybdate), and frozen in liquid nitrogen in 10 mL aliquots containing 4x107 cells/mL. Binding reactions (60uL) were carried out in 96-well plates in TEGM, and typically contained 24 μL cell lysate, 1.2 nM 3H-R1881 (Perkin Elmer), and 10-10-10-4 M of the respective competitive ligand. Reactions were incubated at 4 °C overnight. Bound and unbound ligands were separated by ultrafiltration using a Unifilter-96 GF/C filter plate (Perkin Elmer). Bound 3H-R1881 was eluted in 30 uL/well Microscint-20 and quantified using a Top Count. Ki was calculated according to Cheng-Prusoff (5) as Ki = IC50/(1 + ([3H-R1881]/Kd)).

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In vitro: Competitor assay kits (green) were used according to published procedures (4) to determine relative in vitro binding affinities of ARN-509 for the rat AR ligand binding domain (LBD), human progesterone receptor (PR) LBD, and full-length human estrogen receptor-alpha (ER) and human glucocorticoid receptor (GR). Each hormone dose was performed in triplicate, relative error was calculated from the standard error of the mean (SEM), and binding curves were fit using a single binding site competition model (Prism statistical analysis software package) with R2 > 0.8. Experiments were conducted multiple times with SEM < 0.3 log units from the average logIC50 value. Ki values were calculated as averages across experiments with SEM, and binding affinities were reported as a percentage relative to the tight-binding ligand control for that receptor.

Proliferation assays[1]
Trypsinized VCaP cells were adjusted to a concentration of 100,000 cells per mL in phenol-red-free RPMI 1640 (with 5% CSS), and dispensed in 16 µL aliquots into CellBIND 384 well plates. Cells were incubated for 48 hours, after which ligand was added in a 16 µL volume to the RPMI culture medium. For the antagonist mode assay, the ligands were diluted in culture medium also containing 30 pM R1881 (final [R1881] = 15 pM). After 7 days’ incubation, 16 µL of CellTiter-Glo Luminescent Cell Viability Assay was added and Relative Luminescence Units (RLUs) measured.
In the agonist mode assay, percent viability of the samples was calculated as: % viability=[RLU sample-RLU medium without cells]/[RLU DMSO treated cells-RLU medium without cells]. In the antagonist mode assay, the percent viability of the samples was calculated as: % viability=[RLU sample-RLU VCaP without R1881]/[RLU R1881-treated cells - RLU VCaP without R1881].

In vivo pharmacodynamic studies [1]
Pharmacodynamic studies with LNCaP/AR-luc xenografts in castrate male SCID mice were performed as previously described (4). Formalin-fixed, paraffin-embedded tissue was processed and stained for hematoxylin and eosin (H&E), terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) or immunohistochemistry (IHC) for Ki67 as previously described (4). In vivo luciferase imaging of mice with LNCaP/AR-luc xenografts was performed according to published methods (4), and data analyzed using Living Image 2.30 software. As part of an Investigational New Drug (IND)-enabling toxicity and toxicokinetic study, ARN-509 was administered to male beagle dogs (aged 6 to 7 months, with body weights ranging from 9.3 to 11.2 kg), by Covance Laboratories Inc., in accordance with the United States Food and Drug Administration (FDA) Good Laboratory Practice (GLP) Regulations. ARN-509 was administered daily for 28 days at 0 mg/kg (5 dogs) or 10 mg/kg (4 dogs) by oral gavage (po). ARN-509 (3.33 mg/mL) was formulated as a suspension in labrasol (10% v/v), lactic acid (10% v/v) and soybean oil (10% v/v) and brought up to volume with 50mM phosphate buffer. The placebo oral formulation contained 0 mg/mL ARN-509.

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Mouse and dog pharmacokinetics [1]
Mouse (male CD-1) or beagle dog (Charles River) plasma samples (25 µL) were combined with 100 µL of acetonitrile:methanol:acetic acid, 1:1:0.001, v/v/v containing 500 ng/mL ARN-509-d3 as an internal standard. Precipitated proteins were removed by centrifugation at 1,500 g for 20 minutes at 5°C. Supernatant (50 µL) was diluted with 400 µL of 2:1 water:acetonitrile. ARN-509 concentrations were quantified using the LC-MS/MS method below.

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Dissolved in 15% Vitamin E-TPGS and 65% of a 0.5% w/v CMC solution in 20 mM citrate buffer (pH 4.0), and diluted in saline; 30 mg/kg/day; oral administration

Castrate male immunodeficient mice harboring LNCaP/AR-luc xenograft tumors

Absorption, Distribution and Excretion
Mean absolute oral bioavailability was approximately 100%. The median time to achieve peak plasma concentration (tmax) was 2 hours (range: 1 to 5 hours). The major active metabolite N-desmethyl apalutamide Cmax was 5.9 mcg/mL (1.0) and AUC was 124 mcg·h/mL (23) at steady-state after the recommended dosage. Administration of apalutamide to healthy subjects under fasting conditions and with a high-fat meal (approximately 500 to 600 fat calories, 250 carbohydrate calories, and 150 protein calories) resulted in no clinically relevant changes in Cmax and AUC. The median time to reach tmax was delayed approximately 2 hours with food. Following administration of the recommended dosage, apalutamide steady-state was achieved after 4 weeks and the mean accumulation ratio was approximately 5-fold. Apalutamide Cmax was 6.0 mcg/mL (1.7) and AUC was 100 mcg·h/mL (32) at steady-state. Daily fluctuations in apalutamide plasma concentrations were low, with the mean peak-to-trough ratio of 1.63. Oral administration of four 60 mg apalutamide tablets dispersed in applesauce resulted in no clinically relevant changes in Cmax and AUC compared to the administration of four intact 60 mg tablets under fasting conditions.
Apalutamide and its main active metabolite are subject to both renal and focal elimination. Up to 70 days following a single oral administration of radiolabeled apalutamide, 65% of the dose was recovered in urine (1.2% of dose as unchanged apalutamide and 2.7% as N-desmethyl apalutamide) and 24% was recovered in feces (1.5% of dose as unchanged apalutamide and 2% as N-desmethyl apalutamide).
The mean apparent volume of distribution at steady state of apalutamide was approximately 276 L.
The CL/F of apalutamide was 1.3 L/h after single dosing and increased to 2.0 L/h at steady-state after once-daily dosing likely due to CYP3A4 auto-induction. The auto-induction effect likely reached its maximum at the recommended dosage because exposure to apalutamide across the dose range of 30 to 480 mg is dose-proportional.

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Metabolism / Metabolites
Metabolism is the main route of elimination of apalutamide. Apalutamide is primarily metabolized by CYP2C8 and CYP3A4 to form active metabolite, N-desmethyl apalutamide. The contribution of CYP2C8 and CYP3A4 in the metabolism of apalutamide is estimated to be 58% and 13% following single dose but changes to 40% and 37%, respectively at steady-state. The auto-induction of CYP3A4-mediated metabolism by apalutamide may explain the increase in CYP3A4 enzymatic activity at steady-state.

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Biological Half-Life
The mean effective half-life for apalutamide in patients with NM-CRPC was approximately 3 days at steady-state.

Hepatotoxicity
In prelicensure controlled trials of apalutamide, serum aminotransferase elevations were uncommon and generally transient and mild, not requiring dose modification. Clinically apparent liver injury with jaundice attributable to apalutamide was not reported in the preregistration trials and is not mentioned as an adverse event in the product label. Since the approval and general clinical use of apalutamide, there have been no publications or descriptions of the clinical features of hepatotoxicity with jaundice associated with its use. The first and second generation androgen receptor blockers, flutamide, nilutamide, and bicalutamide, have all been linked to instances of hepatitis-like liver injury with jaundice that can be severe and even fatal. However, such cases have not been described with apalutamide and other third generation androgen receptor antagonists. Thus, clinically apparent liver injury due to apalutamide must be rare, if it occurs at all.
Likelihood score: E (unlikely cause of clinically apparent liver injury).

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Protein Binding
Apalutamide was 96% and N-desmethyl apalutamide was 95% bound to plasma proteins with no concentration dependency.

[1]. ARN-509: a novel antiandrogen for prostate cancer treatment. Cancer Res. 2012 Mar 15;72(6):1494-503.

[2]. Phase 2 Study of the Safety and Antitumor Activity of Apalutamide (ARN-509), a Potent Androgen Receptor Antagonist, in the High-risk Nonmetastatic Castration-resistant Prostate Cancer Cohort. Eur Urol. 2016 May 6. pii: S0302-2838(16)30133.

Pharmacodynamics
In androgen receptors (AR)-overexpressing LNCaP cells, apaludatamide was reported to have a 7 to 10-fold greater affinity to the AR than bicalutamide. Additionally, apalutamide still possesses total antagonistic activity in AR-overexpressing cell lines with bicalutamide-resistance mutations such as T878A and W741C. In castrate mice with LNCaP/AR(cs) tumors, apalutamide produced tumor regression (defined by >50% regression in tumor volume) in 8 mice compared to only 1 for bicalutamide. The apalutamide-treated tumors also have a 60% decrease in proliferative index and a 10-fold increase in apoptotic rate compared with vehicle. In an open-label, uncontrolled, multicenter, single-arm dedicated QT study in 45 patients with CRPC, an exposure-QT analysis suggested a concentration-dependent increase in QTcF for apalutamide and its active metabolite. Apalutamide demonstrated antitumor activity in the mouse xenograft models of prostate cancer, where it decreased tumor cell proliferation and reduced tumor volume.

C21H15F4N5O2S

477.43

477.088

C, 52.83; H, 3.17; F, 15.92; N, 14.67; O, 6.70; S, 6.72

956104-40-8

Apalutamide-d4;1638885-65-0;Apalutamide-d3;1638885-61-6;Apalutamide-13C,d3; 2376466-25-8 (acetate); 1505451-73-9 (ethanol); 1505451-74-0 (hydrate); 956104-40-8; 1505451-77-3 (DMSO)

24872560

White to off-white solid powder

1.6±0.1 g/cm3

1.659

1.3

1

9

3

33

886

0

N#CC1C(C(F)(F)F)=CC(N2C(=S)N(C3C=C(F)C(C(NC)=O)=CC=3)C3(CCC3)C2=O)=CN=1

HJBWBFZLDZWPHF-UHFFFAOYSA-N

InChI=1S/C21H15F4N5O2S/c1-27-17(31)13-4-3-11(8-15(13)22)30-19(33)29(18(32)20(30)5-2-6-20)12-7-14(21(23,24)25)16(9-26)28-10-12/h3-4,7-8,10H,2,5-6H2,1H3,(H,27,31)

4-(7-(6-cyano-5-(trifluoromethyl)pyridin-3-yl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl)-2-fluoro-N-methylbenzamide

JNJ56021927; ARN509; JNJ-56021927; ARN 509; JNJ 56021927; ARN-509; Apalutamide; Brand name: Erleada

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 (4.36 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 (4.36 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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Solubility in Formulation 3: 0.5% CMC, pH4.0:14 mg/mL

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