ARN-509: a novel antiandrogen for prostate cancer treatment

Abstract

Continued reliance on the androgen receptor (AR) is now understood as a core mechanism in castration-resistant prostate cancer (CRPC), the most advanced form of this disease. While established and novel AR pathway-targeting agents display clinical efficacy in metastatic CRPC, dose-limiting side effects remain problematic for all current agents. In this study, we report the discovery and development of ARN-509, a competitive AR inhibitor that is fully antagonistic to AR overexpression, a common and important feature of CRPC. ARN-509 was optimized for inhibition of AR transcriptional activity and prostate cancer cell proliferation, pharmacokinetics, and in vivo efficacy. In contrast to bicalutamide, ARN-509 lacked significant agonist activity in preclinical models of CRPC. Moreover, ARN-509 lacked inducing activity for AR nuclear localization or DNA binding. In a clinically valid murine xenograft model of human CRPC, ARN-509 showed greater efficacy than MDV3100. Maximal therapeutic response in this model was achieved at 30 mg/kg/d of ARN-509, whereas the same response required 100 mg/kg/d of MDV3100 and higher steady-state plasma concentrations. Thus, ARN-509 exhibits characteristics predicting a higher therapeutic index with a greater potential to reach maximally efficacious doses in man than current AR antagonists. Our findings offer preclinical proof of principle for ARN-509 as a promising therapeutic in both castration-sensitive and castration-resistant forms of prostate cancer.

Conflict of interest statement

Potential conflicts of interest: Patent applications: JW, CT, SO, MJ, CLS are co-inventors of ARN-509 and MDV3100; AD, OO, HZ, GY are co-inventors of an ARN-509 synthetic method. CLS, MEJ are consultants to Aragon and own Aragon stock. JJ, KG, EDB, AA, GS, JQ, JS, MRH, CB, BD, NDS, PJR, RAH, JHH are Aragon employees and own Aragon stock. NJC owns Aragon stock.

Figures

Figure 1. ARN-509 activity in vitro in human prostate-cancer cells

(A) ARN-509 structure. (B) Representative competitive binding-assay vs 18F-FDHT (LNCaP/AR(cs)). IC50-values: 11.5.nM (FDHT), 16nM (ARN-509), 21.4 nM (MDV3100), 160nM (Bic (bicalutamide)) (error-bars:SD, n=3). Inset: IC50-values (mean ± SEM) from five replicate experiments. (C) qRT-PCR analysis of AR-regulated genes (normalized to GAPDH). LNCaP/AR(cs) cultured (5% CSS) 3d with/without 1nM R1881 and DMSO (Veh) or drug (1,3,10μM). (D) VCaP cultured (5%CSS) for 2d, then treated for 7d. Left-panel:agonist-mode. Right-panel:antagonist-mode with 30pM R1881. Viable cells (CellTiter-GLO) plotted as %vehicle-control (DMSO) (n=3, mean±SEM).

Figure 2. ARN-509 impairs AR nuclear-localization and inhibits DNA-binding

(A) Representative confocal microscopic images (scale-bars:10μm) of LNCaP with AR-EYFP cultured (5% CSS) and treated with DMSO (Veh) or drug. Nuclear:cytoplasmic fluorescence-intensity of individual cells was quantified (n=3, mean±SEM). (B) ChIP of AR in LNCaP/AR(cs) cultured (5%CSS) and treated 1h with/without 1nM R1881 and either DMSO (Veh) or 10μM anti-androgen. RT-PCR: PSA, TMPRSS2 enhancers (mean±SD, n=2). (C) Activation of an androgen-regulated 4XARE-Luc reporter gene by wtAR and VP16-AR in LNCaP/AR-luc or Hep-G2 cells treated for 40–48h with/without 100 pM R1881 and either DMSO (Veh), or anti-androgen. Luciferase assay conducted on cell lysates: light-units relative to vehicle-control (n=3, mean±SEM).

Figure 3. ARN-509 is active in models of castration-resistant prostate cancer

(A) Luciferase-imaging of castrate male mice harboring LNCaP/AR-luc tumors pre-treatment (d0) and post-treatment (d17), normalized to tumor volume. (B) Castrate male mice bearing LNCaP/AR(cs) tumors (mean tumor-volume 200mm3) treated by daily-gavage with vehicle, or drug. %-change in individual tumor volume (9–10 tumors/treatment-group) after 28d. Unpaired t-test:Vehicle vs ARN-509: p=0.0001; Vehicle vs Bic: p=0.004; ARN-509 vs Bic: p=0.002. Plasma-concentrations measured 24h post-dose on d28 (mean±SEM: Bic 30.74±3.68; ARN-509 9.69±1.58). (C) Castrate male mice bearing LNCaP/AR tumors >100mm3 treated 5d by daily-gavage with vehicle or 10 mg/kg/day anti-androgen. Ki67 IHC performed on tumor-tissue resected 2h after final dose. # positively-stained cells calculated out of 300 cells counted in each of 5 random views (n=3 tumors/treatment group), and expressed relative to vehicle-controls (error:SD; Veh vs ARN-509 and Bic vs ARN-509: p>0.05 (1-way ANOVA with Bonferroni’s post-test)). (D) Castrate male mice bearing LNCaP/AR tumors treated as in (C) for 25d. TUNEL-staining performed on tumor-tissue resected after final dose. # positively-stained cells calculated, out of 300 cells counted in each of 5 random views (n=3 tumors/treatment-group), expressed relative to vehicle-controls (error:SD).

Figure 4. ARN-509 achieves similar efficacy with lower steady-state plasma-levels than MDV3100 in LNCaP/AR xenograft models of castration-resistant prostate cancer

A–C, Castrate male mice bearing LNCaP/AR(cs) tumors treated by oral daily-gavage with vehicle, ARN-509 or MDV3100. Unpaired t-test used for statistical comparisons. (A) 1,10 mg/kg dose: %change in individual tumor volume (n=7–8/treatment-group) after 28d-treatment. p<0.05: drug vs vehicle. (B) 10, 30 mg/kg dose: %change in individual tumor volume (n=8/treatment-group) after 42d-treatment. p<0.05: drug vs vehicle. (C) 30, 100 mg/kg dose: %change in individual tumor volume (n=19–20 tumors/treatment-group) after 28d-treatment. * p<0.05 drug vs vehicle or drug vs drug.

Figure 5. ARN-509 induces castrate-like changes in dog prostate and epididymis

A–C, Intact male beagle dogs treated by oral-gavage for 28d with vehicle (n=5) or ARN-509 10 mg/kg/day (n=4). Organs resected 24h after final-dose. Mean plasma-concentration of ARN-509 measured at sacrifice, 24h after final dose, was 17.5 μg/mL. (A) Prostate and epididymis weights (mean±SEM). p<0.05:Veh vs ARN-509 in prostate (2-way ANOVA, with Bonferroni post-test). Comparison for epididymis is insignificant. (B) H&E staining of transverse prostate-sections (upper-panels) and longitudinal epididymis sections (lower-panels) at low-power (100x, scale bar:200μm) and high-power (600x, scale-bar:20μm). Upper-panels: Fully-mature, active acini in vehicle-treated prostate containing intraluminal secretions (arrowhead), lined with folds of a single-layer of columnar epithelial cells with abundant cytoplasm. ARN-509-treated prostates: bilayer of cuboidal epithelial cells (arrow), small lumens with rare eosinophilic secretory material (arrowhead). Lower-panels: Vehicle-treated epididymis: tall columnar pseudo-stratified epithelium with lumens containing abundant spermatozoa (arrowhead). ARN-509-treated epididymis: cuboidal epithelium, lumens with little-to-no spermatozoa, containing multi-nucleated round cells suggestive of sloughed degenerate germ cells (arrowhead). (C) Dog prostates stained with antibody to Ki67 (200x, scale-bar:100μm) or TUNEL (400x, scale-bar:50μm). Ki67-staining (arrows) and TUNEL (arrowheads). No TUNEL-staining is evident in ARN-509-treated prostates. Results quantified as mean percentage (± SEM) of positive cells from representative areas from each prostate (n=5 for vehicle, n=4 for ARN-509). p<0.05 for TUNEL staining of Veh vs ARN-509 (1-way ANOVA with Bonferroni’s multiple comparison post-test). (D) Intact male mice bearing LNCaP/AR(cs) tumors, treated for 28d by oral daily-gavage with vehicle, ARN-509 or MDV3100 (10, 30 mg/kg). %change in individual tumor volume (n=5–8/treatment-group) after 28d-treatment.. * p<0.05 for each treatment relative to vehicle (unpaired t-test).