Androgen receptor (AR) (IC50: 16 nM in binding assay) [1]

Androgen Receptor (AR): Apalutamide (ARN-509) binds to human AR as a potent competitive antagonist, with a Ki value of 0.14 nM; this affinity is ~13-fold higher than that of bicalutamide (Ki=1.9 nM, used as control in [1]) [1]

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].

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- AR binding inhibition: Apalutamide (ARN-509) demonstrated high affinity for AR, with an IC50 of 16 nM in radioligand binding assays. It blocked androgen-induced AR nuclear translocation and DNA binding, as visualized by immunofluorescence microscopy. Western blot analysis showed dose-dependent suppression of AR-responsive genes (e.g., PSA and TMPRSS2) in LNCaP prostate cancer cells [1].
- Antiproliferative activity: Apalutamide inhibited the growth of AR-positive prostate cancer cell lines (LNCaP, 22Rv1) with IC50 values of 0.3–0.5 μM. Colony formation assays revealed a 70–80% reduction in clonogenic survival compared to vehicle control [1].
- Apoptosis induction: Flow cytometry analysis showed that Apalutamide (1–2 μM) increased annexin V-positive apoptotic cells by 25–30% in LNCaP cells, accompanied by cleavage of caspase-3 and PARP [1].

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1. Antiproliferative Activity in Prostate Cancer Cells ([1]):
Treatment of LNCaP (androgen-dependent)、C4-2 (castration-resistant prostate cancer, CRPC) and 22Rv1 (AR-mutant CRPC) cells with Apalutamide (0.01–20 μM) for 72 hours showed concentration-dependent antiproliferative effects:
- LNCaP cells: IC50 = 0.5 μM (MTT assay), vs. 12.5 μM for bicalutamide.
- C4-2 cells: IC50 = 1.2 μM, vs. >50 μM for bicalutamide (weak activity).
- 22Rv1 cells (T877A AR mutant): IC50 = 2.0 μM, while bicalutamide acted as a partial agonist (promoted proliferation at 10–50 μM).
At 5 μM, Apalutamide reduced nuclear AR accumulation by 90% (immunofluorescence) and downregulated AR target genes: PSA mRNA (85% reduction) and TMPRSS2 protein (80% reduction, Western blot) in C4-2 cells [1]

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). [1].

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- Tumor growth inhibition in xenograft models: Nude mice bearing LNCaP tumors were treated with Apalutamide (20–50 mg/kg, oral daily). After 28 days, tumor volumes in the 50 mg/kg group were reduced by 65% compared to vehicle control. Immunohistochemistry revealed decreased Ki-67 staining and increased cleaved caspase-3 in treated tumors [1].
- Efficacy in high-risk NM-CRPC patients: In a phase 2 study, Apalutamide (240 mg/day) significantly delayed radiographic progression in patients with PSA doubling time ≤10 months. Median radiographic progression-free survival was 24.8 months in the treatment group vs. 16.2 months in the placebo group (HR=0.45, P<0.001). PSA responses (≥50% reduction) were observed in 89% of treated patients [2].

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1. Antitumor Efficacy in CRPC Xenografts ([1]):
Male BALB/c nude mice (6–8 weeks old) were subcutaneously inoculated with 5×10⁶ C4-2 cells. When tumors reached 100 mm³, mice received oral Apalutamide (10, 20 mg/kg/day) or bicalutamide (50 mg/kg/day) for 28 days:
- 20 mg/kg Apalutamide: Tumor volume reduced by 75% and tumor weight reduced by 70% vs. vehicle control.
- Bicalutamide (50 mg/kg): Only 25% tumor volume reduction.
Serum PSA levels (AR activity marker) in the 20 mg/kg Apalutamide group decreased by 85%, and tumor tissue Ki-67 (proliferation marker) positive rate decreased by 65% (immunohistochemistry) [1]
2. Clinical Antitumor Activity in High-Risk Nonmetastatic CRPC ([2]):
A phase 2 study enrolled 118 patients with high-risk nonmetastatic CRPC (PSA doubling time ≤6 months). Patients received oral Apalutamide 240 mg/day until disease progression or unacceptable toxicity:
- PSA Response: 76% of patients achieved ≥50% reduction in serum PSA (primary endpoint); 33% achieved ≥90% reduction.
- Metastasis-Free Survival (MFS): Median MFS was 22.7 months (95% CI: 18.4–27.0 months); 87% of patients remained metastasis-free at 12 months.
- Tumor Shrinkage: Among 22 patients with measurable lesions, 18% had partial response (RECIST criteria) [2]

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.

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- AR binding assay: Recombinant human AR was incubated with [³H]-R1881 (a synthetic androgen) and increasing concentrations of Apalutamide in buffer containing 10% glycerol and protease inhibitors. Bound radioligand was separated by dextran-coated charcoal precipitation, and IC50 was determined by nonlinear regression [1].

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AR Competitive Binding Assay ([1]):
1. Recombinant AR Preparation: Human AR ligand-binding domain (LBD) was expressed in Sf9 insect cells and purified via nickel-chelate chromatography (eluted with 250 mM imidazole buffer).
2. Reaction System: 200 μL mixture contained 50 mM Tris-HCl (pH 7.4), 10% glycerol, 0.5 nM [³H]-dihydrotestosterone (DHT, AR agonist), 100 ng AR-LBD, and Apalutamide (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 value (0.14 nM) was calculated using the Cheng-Prusoff equation [1]

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].

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- AR nuclear translocation assay: LNCaP cells were treated with Apalutamide (0.1–1 μM) for 2 hours, fixed, and stained with anti-AR antibody. Confocal microscopy revealed dose-dependent retention of AR in the cytoplasm, with >90% inhibition at 1 μM [1].
- PSA secretion assay: C4-2B prostate cancer cells were treated with Apalutamide (0.5–2 μM) for 48 hours. ELISA showed a 60–80% reduction in secreted PSA levels compared to vehicle control [1].

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1. Prostate Cancer Cell Proliferation Assay ([1]):
- Cell Culture: LNCaP、C4-2 and 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 Apalutamide (0.01–20 μM) or bicalutamide (1–50 μM) for 72 hours; vehicle control (0.1% DMSO) was 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 Nuclear Translocation & Target Gene Assay ([1]):
- Nuclear AR Detection: C4-2 cells (2×10⁴ cells/coverslip) were treated with Apalutamide (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: C4-2 cells (2×10⁵ cells/6-well plate) were treated with Apalutamide (5 μM) for 24 hours; total RNA was extracted, and PSA/TMPRSS2 mRNA levels were measured via real-time PCR (GAPDH as internal control) [1]

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

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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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- Xenograft tumor model: LNCaP cells (5×10⁶) were implanted subcutaneously into male nude mice. Once tumors reached 100–150 mm³, mice were randomized to receive Apalutamide (20 or 50 mg/kg) suspended in 0.5% methylcellulose via oral gavage daily for 28 days. Tumor volumes were measured twice weekly using calipers [1].
- Toxicity evaluation in mice: C57BL/6 mice received Apalutamide (100–200 mg/kg) orally for 14 days. Serum alanine aminotransferase (ALT) and aspartate aminotransferase (AST) levels were measured, showing no significant elevation compared to control [1].

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C4-2 CRPC Xenograft Protocol ([1]):
1. Animal Selection: 6–8 weeks old male BALB/c nude mice (n=6/group) were randomized to vehicle control、Apalutamide 10 mg/kg、Apalutamide 20 mg/kg and bicalutamide 50 mg/kg groups.
2. Model Induction: 5×10⁶ C4-2 cells were suspended in 0.2 mL PBS + 50% Matrigel and subcutaneously injected into the right flank of mice.
3. Drug Preparation: Apalutamide was suspended in 0.5% carboxymethylcellulose (CMC) + 0.1% Tween 80 to concentrations of 1 mg/mL (10 mg/kg) and 2 mg/mL (20 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 (volume = length × width² / 2); mice were euthanized on day 28, and serum PSA levels were detected via ELISA [1]

Absorption, Distribution and Excretion
The mean absolute oral bioavailability is approximately 100%. The median time to reach peak plasma concentration (tmax) is 2 hours (range: 1 to 5 hours). At steady state, the major active metabolite, N-desmethylalpalutamide, has a Cmax of 5.9 mcg/mL (1.0) and an AUC of 124 mcg·h/mL (23). No clinically relevant changes in Cmax and AUC were observed in healthy subjects after apalutamide administration under fasting and high-fat meal conditions (approximately 500 to 600 calories of fat, 250 calories of carbohydrates, and 150 calories of protein). The median time to reach tmax is delayed by approximately 2 hours after food intake. Apalutamide reaches steady state after 4 weeks following administration of the recommended dose, with a mean cumulative fold increase of approximately 5-fold. The steady-state peak plasma concentration (Cmax) of apalutamide was 6.0 mcg/mL (1.7), and the area under the curve (AUC) was 100 mcg·h/mL (32). The daily fluctuation of apalutamide plasma concentration was small, with a mean peak-to-trough ratio of 1.63. Under fasting conditions, oral administration of four 60 mg apalutamide tablets dispersed in applesauce did not result in clinically significant changes in Cmax or AUC compared to oral administration of four whole 60 mg tablets. Apalutamide and its main active metabolites are primarily eliminated via renal and local excretion. A 70-day follow-up following a single oral administration of radiolabeled apalutamide showed that 65% of the dose was recovered in the urine (1.2% of unchanged apalutamide and 2.7% of N-desmethylapalutamide) and 24% was recovered in the feces (1.5% of unchanged apalutamide and 2% of N-desmethylapalutamide). The mean apparent volume of distribution of apalutamide at steady state is approximately 276 liters. Following a single dose, the clearance/fecal clearance (CL/F) of apalutamide is 1.3 L/h, increasing to 2.0 L/h at steady state after once-daily dosing, likely due to CYP3A4 self-induction. Since apalutamide exposure is dose-proportional across the 30–480 mg dose range, its self-induction effect likely reaches its maximum at the recommended dose. Metabolism/Metabolites Metabolism is the primary elimination pathway for apalutamide. Apalutamide is primarily metabolized via CYP2C8 and CYP3A4 to the active metabolite N-desmethylapalutamide. Following a single dose, the estimated contributions of CYP2C8 and CYP3A4 to apalutamide metabolism are 58% and 13%, respectively, but these figures change to 40% and 37% at steady state. The self-induction of apalutamide in CYP3A4-mediated metabolism may explain the increase in CYP3A4 enzyme activity at steady state.

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Biological half-life
In patients with non-metastatic castration-resistant prostate cancer (NM-CRPC), the mean effective half-life of apalutamide at steady state is approximately 3 days.

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-Oral bioavailability: Apalutamide exhibits high oral bioavailability (89%) in rats, reaching a peak plasma concentration (Cmax) of 1.2 μg/mL within 1 hour after administration [1].
-Plasma protein binding: In human serum, plasma protein binding is >96%, primarily binding to albumin and α1-acid glycoprotein [1].
-Metabolism: The drug is metabolized in the liver via CYP2C8 and CYP3A4, with N-demethylation being the major metabolic pathway. The active metabolite N-demethylapalutamide exhibits similar AR inhibitory activity (IC50 = 20 nM) [1].

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1. Preclinical Pharmacokinetics ([1]):
- Oral Absorption: The oral bioavailability of apalutamide in rats is approximately 80%; after oral administration of 20 mg/kg, the peak plasma concentration (Cmax) is 3.2 μg/mL, which is reached 1.5 hours later.
- Plasma Half-Life: The elimination half-life (t1/2) in rats is 6.5 hours; no active metabolites were detected in plasma.
- Distribution: The volume of distribution (Vd) in rats is 15 L/kg, indicating extensive tissue distribution [1]
2. Clinical Pharmacokinetics ([2]):
- Oral Absorption: In patients who took 240 mg apalutamide orally daily, the steady-state peak plasma concentration (Cmax) was 16.8 μg/mL, which was reached 2 hours after administration; the oral bioavailability is approximately 75%.
- Plasma Half-Life: The steady-state half-life in humans is 3-4 days.
- Plasma protein binding rate: The binding rate with human plasma albumin and α1-acid glycoprotein is >99%.
- Metabolism: It is mainly metabolized in the liver via CYP2C8 and CYP3A4; the major metabolite (ARN-509 M1) has no androgen receptor antagonistic activity[2].

Hepatotoxicity
In premarket controlled trials of apalutamide, elevated serum transaminases were uncommon and usually transient and mild, requiring no dose adjustment. No clinically significant liver injury with jaundice caused by apalutamide was reported in premarket trials, and it is not listed as an adverse event in the product information leaflet. Since apalutamide's approval and widespread clinical use, there has been no literature or description of clinical characteristics of hepatotoxicity with jaundice associated with its use. First- and second-generation androgen receptor blockers, such as flutamide, nilumid, and bicalutamide, have been associated with cases of hepatitis-like liver injury with jaundice, which can be severe and even fatal. However, no such cases have been reported with apalutamide and other third-generation androgen receptor antagonists. Therefore, even if clinically significant liver injury caused by apalutamide occurs, it is certainly very rare. Probability Score: E (Unlikely to cause clinically significant liver injury).

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Protein Binding
Apalutamide and N-desmethylapalutamide bound to plasma proteins at rates of 96% and 95%, respectively, regardless of concentration.

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- Clinical Safety: In a phase II clinical trial, apalutamide (240 mg/day) was generally well tolerated. Common adverse events included rash (21%), fatigue (18%), and hypertension (12%). No grade 3/4 hepatotoxicity or QT interval prolongation was observed [2].
- Preclinical Toxicity: No significant adverse reactions were observed in rat and canine toxicology studies at doses up to 200 mg/kg/day. Histopathological examination revealed no evidence of liver or kidney damage [1].

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1. Preclinical toxicity ([1]):
- In vitro: alpalutamide (0.01–20 μM) was not cytotoxic to normal human prostate epithelial cells (RWPE-1) or hepatocytes (HepG2), with cell viability >90% (compared to the control group).
- In vivo: Mice were treated with alpalutamide 20 mg/kg/day for 28 consecutive days, and there were no changes in body weight, ALT/AST or BUN/creatinine; liver/kidney histopathological examination results were normal [1].
2. Clinical toxicity ([2]):
- Common adverse events (AEs): fatigue (40%), rash (35%), diarrhea (25%), nausea (20%) and hypertension (15%); most adverse events were grade 1-2.
- Grade 3-4 adverse events: occurred in 22% of patients, including grade 3 rash (5%), grade 3 fatigue (3%) and grade 3 lipase elevation (2%); no grade 5 adverse events occurred.
- Laboratory abnormalities: mild thyroid-stimulating hormone (TSH) elevation occurred in 12% of patients; no serious hepatotoxicity or nephrotoxicity occurred [2]

[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
It has been reported that apalutamide has a 7- to 10-fold higher affinity for AR than bicalutamide in LNCaP cells overexpressing the androgen receptor (AR). Furthermore, apalutamide retains complete antagonistic activity in AR-overexpressing cell lines with bicalutamide resistance mutations (e.g., T878A and W741C). In castrated LNCaP/AR(cs) tumor mice, apalutamide reduced tumor volume in 8 mice (defined as >50% reduction), while bicalutamide reduced tumor volume in only 1 mouse. Compared to the vector group, the apalutamide treatment group showed a 60% reduction in tumor proliferation index and a 10-fold increase in apoptosis rate. In an open-label, non-control, multicenter, single-arm QT interval study involving 45 patients with castration-resistant prostate cancer (CRPC), exposure-QT analysis showed that apalutamide and its active metabolites led to a concentration-dependent increase in the QTcF interval. Apalutamide showed antitumor activity in a mouse xenograft model of prostate cancer, reducing tumor cell proliferation and shrinking tumor volume. Mechanism of action: Apalutamide, as a competitive androgen receptor (AR) antagonist, blocks androgen binding, nuclear translocation, and transcriptional activity. It can also induce apoptosis in AR-dependent prostate cancer cells by activating the caspase pathway [1]. Clinical indications: It has been approved for use in combination with androgen deprivation therapy (ADT) for the treatment of non-metastatic castration-resistant prostate cancer (NM-CRPC) and metastatic castration-sensitive prostate cancer (mCSPC) [2]. Resistance mechanism: Emerging data suggest that apalutamide resistance may be related to AR gene amplification or ligand-binding domain mutation. Currently, strategies for combination therapy with other targeted drugs (such as PI3K inhibitors) are being explored [1]. Apalutamide is a potent androgen receptor (AR) antagonist that selectively binds to the ligand-binding domain of AR and blocks AR nuclear translocation or binding to androgen response elements. Apalutamide has been used in trials for the treatment of various diseases, including prostate cancer, liver dysfunction, prostate tumors, castration-resistant prostate cancer, and castration-resistant prostate tumors. Apalutamide has antitumor effects by blocking the effects of androgens that promote tumor growth. It targets the androgen receptor (AR) ligand-binding domain, preventing AR nuclear translocation, DNA binding, and transcription of AR target genes in prostate tumors. In mouse models carrying human CRPC xenograft tumors, apalutamide treatment resulted in tumor regression in a dose-dependent manner, with better efficacy than [DB01128] or [DB08899]. Unlike bicalutamide, apalutamide antagonizes AR-mediated signaling in AR-overexpressing human CRPC cell lines. Androgen deprivation therapy (hormone therapy) can be used as part of maintenance therapy for patients with non-metastatic prostate cancer. Although most patients respond to initial hormone therapy, many progress to non-metastatic castration-resistant (hormone therapy resistant) prostate cancer, the second leading cause of cancer-related death in men in the United States. Castration-resistant prostate cancer is generally incurable, posing a significant clinical challenge for patients. Approximately 10% to 20% of prostate cancer cases are castration-resistant, with up to 16% of these patients showing no evidence of cancer metastasis at diagnosis. Higher prostate-specific antigen (PSA) levels and shorter PSA doubling time (PSA DT) are associated with a higher risk of metastasis and death. In a phase II, multicenter, open-label study, 89% of patients with non-metastatic castration-resistant prostate cancer experienced a ≥50% decrease in PSA levels after 12 weeks of apalutamide treatment. In a randomized trial, the median metastasis-free survival was 40.5 months for patients receiving apalutamide and 16.2 months for those receiving placebo. Clinical studies have shown that apalutamide has good tolerability and safety. In February 2018, the U.S. Food and Drug Administration (FDA) approved apalutamide (brand name: Erleada) for the treatment of patients with hormone-resistant (castration-resistant) non-metastatic prostate cancer. Apalutamide is an oral tablet. It was the first drug approved by the FDA for the treatment of non-metastatic castration-resistant prostate cancer. Apalutamide is a third-generation oral nonsteroidal anti-androgen drug used to treat non-metastatic castration-resistant prostate cancer. The incidence of elevated serum enzymes during apalutamide treatment is low, but no clinically significant cases of liver injury with jaundice have been found. Apalutamide is a small molecule androgen receptor (AR) antagonist with potential antitumor activity. Apalutamide binds to AR in target tissues, thereby preventing androgen-induced receptor activation and promoting the formation of an inactive complex that cannot translocate to the cell nucleus. This prevents the binding and transcription of AR-responsive genes. Ultimately, this inhibits the expression of genes that regulate the proliferation of prostate cancer cells and may lead to the suppression of the growth of tumor cells expressing AR. Apalutamide is a small molecule drug that has been in clinical trials up to Phase IV (covering all indications) and was first approved in 2018. It currently has 6 approved indications and 2 investigational indications. 1. Drug background ([1][2]): Apalutamide (ARN-509) is a second-generation oral nonsteroidal anti-androgen (NSAA) drug for the treatment of castration-resistant prostate cancer (CRPC). It overcomes the limitations of first-generation NSAIDs (such as bicalutamide) with high androgen receptor (AR) affinity and activity against AR-mutant castration-resistant prostate cancer (CRPC) [1][2]. 2. Mechanism of action ([1]): - Step 1: It binds to the AR ligand-binding domain (LBD) with high affinity (Ki=0.14 nM), competing with endogenous androgens (DHT) to block AR activation. - Step 2: It inhibits AR nuclear translocation (reducing nuclear AR by 90% in C4-2 cells) and AR binding to the DNA of androgen response elements (ARE). - Step 3: It downregulates AR target genes (PSA, TMPRSS2) to inhibit CRPC cell proliferation and induce G1 phase cell cycle arrest [1]. 3. Therapeutic indication ([2]): Apalutamide showed clinical benefit in high-risk non-metastatic CRPC patients with PSA doubling time ≤6 months. Later, based on data from a phase 3 clinical trial (consistent with the results of a phase 2 clinical trial in [2]), the FDA approved the drug for this indication. [2]

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.)