CH5137291 and fulvestrant were synthesized in our laboratories. Bicalutamide was purchased as Casodex tablets (AstraZeneca, London, UK) and purified in our laboratories. Synthetic androgen R1881 was purchased from NEN Life Science Products (Boston, MA, USA). Progesterone, mifepristone, dexamethasone, and β-estradiol were purchased from Sigma-Aldrich (St. Louis, MO, USA). Aldosterone was purchased from Acros Organics (Geel, Belgium). Abiraterone acetate was synthesized in our laboratories.

LNCaP, VCaP, PC3, HeLa, and COS-7 cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). LNCaP-BC2andLNCaP-CS10cellswere previously established in our laboratory (28,29). LuCaP35V cells that progressed in castrated male mice and expressed wild-type AR were a gift from Professor Robert L. Vessella (University of Washington, Seattle, WA, USA) (38). LNCaP cells were maintained in RPMI-1640 medium (Invitrogen, Carlsbad, CA, USA) supplemented with 10% FBS. VCaP cells were maintained in DMEM (high glucose) (Invitrogen) supplemented with 10% FBS. LNCaP-BC2 cells were maintained in phenol red-free RPMI-1640 supplemented with 10% FBS and 2 μM bicalutamide. LNCaP-CS10 cells were maintained in phenol red-free RPMI-1640 supplemented with 10% dextran-coated charcoal-treated FBS (DCC-FBS; Hyclone Laboratories, Logan, UT, USA) and 10 μM bicalutamide. PC3 cells were maintained in F-12 Kaighn’s medium (Invitrogen) supplemented with 10% FBS. HeLa cells were maintained in EMEM medium (Sigma-Aldrich) supplemented with 10% FBS, 1 mM sodium pyruvate (Invitrogen), 0.1 mM non-essential amino acids (Invitrogen), and 1.5 g/l sodium bicarbonate (Sigma-Aldrich). COS-7 cells were maintained in DMEM (high glucose) supplemented with 10% FBS, 1.5 mM L-glutamine (Sigma-Aldrich), 4.5 g/l D-glucose (Sigma-Aldrich), and 1.5 g/l sodium bicarbonate. The characteristics in terms of hormone sensitivity, AR status, and AR mutation of these cell lines are summarized in Table I.

Five-week-old male C.B-17/Icr-scid Jcl severe combined immune-deficient (SCID) mice were purchased from CLEA Japan (Tokyo, Japan). Four- to 6-year-old cynomolgus monkeys (Macaca fascicularis) were bred in the Chugai Research Institute for Medical Science (Kanagawa, Japan). All animals were housed in a pathogen-free environment under controlled conditions (temperature 20–26°C, humidity 35–75%, light/dark cycle 12/12 h). Chlorinated water and irradiated food were provided ad libitum. The health of the animals was monitored by daily observation. The protocols of the animal studies were reviewed and approved by the Institutional Animal Care and Use Committee of Chugai Pharmaceutical Co., Ltd., and all animal experiments were performed in accordance with the Guidelines for the Accommodation and Care of Laboratory Animals promulgated in Chugai Pharmaceutical Co., Ltd.

LNCaP cells in 10% DCC-FBS medium were plated 24 h before transfection. To analyze intracellular translocation of AR, we used HaloTag system. The cells were transfected with HaloTag pHT2-hAR-WT, HaloTag pHT2-W741C, or HaloTag pHT2-T877A using FuGENE HD transfection reagent (Roche, Basel, Switzerland). Forty-eight hours after the transfection, HaloTag TMR ligand (Promega, Fitchburg, WI, USA) was added, and the cells were incubated for 0.5 h. CH5137291 or bicalutamide (10 μM) was added in the presence or absence of R1881 (0.5 nM). After 2-h incubation, fluorescence imaging of the living cells was performed with a BioZero fluorescence microscope (Keyence, Osaka, Japan).

Cells were plated onto poly-D-lysine coated plates (Becton-Dickinson, Heidelberg, Germany) in 10% DCC-FBS medium. After 3-day incubation, 10 μM of CH5137291 or bicalutamide was added and the plates were incubated for another 24 h. Then, nuclear and cytoplasmic fractions were prepared by using NE-PER Nuclear and Cytoplasmic Extraction Reagents (Pierce Biotechnology, Rockford, IL, USA), and a total fraction was prepared using cell lysis buffer (Cell Signaling Technology, Danvers, MA, USA) supplemented with Complete Protease Inhibitor Cocktail Tablets (Roche). AR-specific antibody N-20, IκBα-specific antibody C-15, and c-Jun-specific antibody H-79 (Santa Cruz Biotechnology, Santa Cruz, CA, USA) were used for immunoblotting.

VCaP, LNCaP, LNCaP-BC2, and LNCaP-CS10 cells were pre-incubated in phenol red-free RPMI-1640 medium supplemented with 10% DCC-FBS for 2–4 days and were then plated onto poly-D-lysine-coated plates (Becton-Dickinson) (VCaP, 1×10⁴ cells/well; LNCaP, LNCaP-BC2, and LNCaP-CS10, 5×10³ cells/well) in 10% DCC-FBS medium. After overnight incubation, CH5137291 or bicalutamide at concentrations of 4.6–30 μM was added in the presence of R1881 (VCaP, 0.1 nM; LNCaP, 0.1 nM; LNCaP-BC2, 0.01 nM) or absence of R1881 (LNCaP-CS10). After an additional 10-day incubation, the number of remaining cells were estimated using a DNA quantity assay (FluoReporter Blue Fluorometric dsDNA Quantitation kit; Invitrogen).

The following plasmids were used: GMLUC, an MMTV-luciferase reporter plasmid created by replacing the reporter of GMCAT (ATCC) with luciferase; phRL-CMV (Promega), a Renilla luciferase plasmid; and human AR expression plasmids corresponding to mutations (pcDNA3.1-hAR-WT, pcDNA3.1-hAR-W741C and pcDNA3.1-hAR-T877A).

Twenty-four hours before transfection, the PC3 cells in 10% DCC-FBS medium were plated onto 96-well plates at 1×10⁴ cells/well. The cells were co-transfected with GMLUC (50 ng/well), phRL-CMV (0.5 ng/well), and human AR expression plasmid (10 ng/well) using a TransIT-Prostate Transfection kit (Mirus, Madison, WI, USA). Six hours after the transfection, various doses of CH5137291 or bicalutamide were added in the presence of R1881 (WT, 0.05 nM; W741C, 0.5 nM; T877A, 0.5 nM) or absence of R1881. Cell lysates were collected 48 h after the treatment with the compounds, and the luciferase activity of each sample was measured using a Dual-Luciferase Reporter Assay system (Promega).

Twenty-four hours before transfection, HeLa cells in 10% DCC-FBS medium were plated onto 96-well plates at 1×10⁴ cells/well for the assays of PR and ERα. COS-7 cells were plated at 0.75×10³ cells/well for the assays of GR and MR. The cells were co-transfected with GMLUC (50 ng/well), phRL-CMV (0.5 ng/well), and expression plasmids of the nuclear receptors (10 ng/well of pSG5-hPR, pRShGR, or pRShMR) using FuGENE 6 transfection reagent (Roche). For ERα, the cells were co-transfected with ERE-reporter vector (50 ng/well), phRL-CMV (0.5 ng/well), and ERα expression vector HEG0 (1 ng/well) using FuGENE 6 transfection reagent. Six hours after transfection, CH5137291 or bicalutamide (10 μM) were added in the presence or absence of the corresponding agonist (10 nM progesterone for PR, 10 nM dexamethasone for GR, 1 nM aldosterone for MR, and 1 nM estradiol for ERα). Cell lysates were collected 48 h after treatment with the compounds. Mifepristone (1 nM), mifepristone (10 nM), progesterone (10 nM), and fulvestrant (100 nM) were used as positive antagonists for PR, GR, MR and ERα, respectively.

Models of CH5137291 docking with wild-type and W741C-mutant ARs were built based on the X-ray crystal structure of the W741L-mutant AR in complex with bicalutamide (Protein Data Bank accession code: 1z95). The three-dimensional structure of CH5137291 was modeled using Sybyl software (Tripos Inc., St. Louis, USA) with a Tripos force field.

In the docking model of CH5137291 for W741C-mutant AR, CH5137291 was manually docked to the W741C-mutant AR such that: i) the crystallographic structure of the W741C-mutant AR was fixed, ii) helix 12 of the AR was removed from the AR model because of the intensive collision between CH5137291 and helix 12, iii) the crystallographic structure of the trifluoromethyl benzonitrile moiety of bicalutamide and its binding mode were used for CH5137291 and kept fixed during the docking, iv) the binding mode of the portion other than the trifluoromethyl benzonitrile moiety of CH5137291 was manually determined so as to avoid a steric collision between CH5137291 and the AR without helix 12, and v) energy minimization of the ‘compound/AR without helix 12’ complex was performed using a molecular mechanics method with the Tripos force field, on condition that the coordinates of AR without helix 12 were fixed. After the energy minimization, the crystallographic coordinates of helix 12 were re-built into the AR structure.

The docking models of CH5137291 or bicalutamide for wild-type AR were built based on the docking model of CH5137291 or bicalutamide for W741C-mutant AR, respectively. C741 was changed to W741 such that the conformation of W741 of the model would be identical to that of the crystallographic structure of W741 of wild-type AR (PDB ID: 1e3g).

Xenograft models were prepared and plasma PSA was quantified as described previously (29). Briefly, LNCaP (2×10⁶ cells), LNCaP-BC2 (2×10⁶ cells), LNCaP-CS10 (2×10⁶ cells), or LuCaP35V (blocks of xenografted tumor) was subcutaneously inoculated into non-castrated or castrated 6- to 8-week-old male SCID mice. When the tumor size reached 90–400 mm³, the animals were randomized into control and treatment groups. The agents or vehicle (5% gum arabic; Sigma-Aldrich) were orally administered at 10 or 100 mg/kg once a day for 2–17 cycles of 5 days on/2 days off starting from the day of randomization. The antitumor efficacy was evaluated by tumor volume (TV) and the percentage of tumor growth inhibition (TGI%). TV was estimated by using the equation TV = ab²/2, where a and b are the length and width of the tumor, respectively. TGI% was calculated as follows: TGI% = [1 − (mean change in TV in each group treated with antitumor drugs/mean change in TV in control group)] × 100. The plasma PSA levels were measured by ELISA (Eiken Chemical, Tokyo, Japan). Relative PSA concentration (%) = (PSA concentration/PSA concentration on day 0 of administration) × 100. Serum testosterone concentrations were measured using LC/MS/MS (ASKA Pharma Medical, Tokyo, Japan) at the end of the experiment.

To evaluate the efficacy of CH5137291 on tumors that had become resistant to bicalutamide, LNCaP-xenografted non-castrated mice were orally administered bicalutamide (100 mg/kg) until resistant tumors appeared. The mice bearing resistant tumors were selected, re-randomized into two groups, and moved on to secondary treatment. Bicalutamide (100 mg/kg) was administered in one group and CH5137291 (100 mg/kg) in the other group. The tumors with acquired resistance were defined as those that fulfilled both of the following two criteria: i) (PSA concentration on day 10 of administration/PSA concentration on day 0 of administration) × 100 < 85%, and ii) the tumor volume on the day of re-randomization/minimum tumor volume during primary treatment > 1.4.

For the exposure assay, the monkeys received a single oral administration of CH5137291 at doses of 0.1, 1, 10 or 100 mg/kg. The vehicle was 1% hydroxypropylcellulose (Nippon Soda, Tokyo, Japan). The serum concentration of CH5137291 was measured by using LC/MS/MS (API3200; Applied Biosystems, Foster City, CA, USA). To assess the effect of CH5137291 on serum PSA, we selected 4- to 6-year-old monkeys with initial serum PSA concentrations of >0.25 ng/ml, as measured by using chemiluminescent immunoassay at BML, Inc. (Tokyo, Japan). The animals were randomized, and CH5137291 was orally administered daily for 7 days at doses of 0.1, 1, 10 or 100 mg/kg. Twenty-four hours after the last administration, serum was harvested and PSA concentration was measured.

Kawata et al reported the inhibitory effect of CH5137291 on AR nuclear translocation by using COS-7 cells transiently transfected with human wild-type AR (37). In the present study, we examined the effect of CH5137291 on subcellular localization in prostate cancer cell lines with wild-type or mutated AR. For this purpose, we used LNCaP cells transfected with HaloTag-fused wild-type AR, W741C-mutant AR, or T877A-mutant AR. In each of these cells with three different types of AR, CH5137291 clearly inhibited AR nuclear translocation in the presence of synthetic androgen R1881. CH5137291 in the absence of R1881 did not induce AR nuclear translocation suggesting that CH5137291 does not have an agonistic effect on AR (Fig. 2A). In contrast, bicalutamide did not inhibit AR nuclear translocation in the presence of R1881 and induced AR nuclear translocation in the absence of R1881.

Next, we examined the effect of CH5137291, in comparison with bicalutamide, on the intracellular distribution of AR protein. For this purpose, LNCaP-CS10 cells were used, because AR in LNCaP-CS10 cells is androgen-independently activated. Bicalutamide reportedly acts as a full agonist on LNCaP-CS10 cells (29). We found that CH5137291 decreased the nuclear AR level and increased the cytoplasmic AR level without affecting the total AR level (Fig. 2B). On the other hand, as was expected, bicalutamide increased the nuclear AR level but did not affect the total AR level.

These results indicate that CH5137291 acts as an AR nuclear translocation inhibitor, not only in androgen-dependent prostate cancer cells with wild-type or mutant AR but also in androgen-independent prostate cancer cells having outlaw pathways.

The proliferation inhibitory activity of CH5137291, compared with bicalutamide, was examined in vitro using four prostate cancer cell lines (VCaP, LNCaP, LNCaP-BC2, and LNCaP-CS10) each with different hormone sensitivity and AR status (Table I and Fig. 3). The assays were all performed in the presence of optimal concentrations of synthetic androgen R1881 except for LNCaP-CS10, because LNCaP-CS10 does not require androgen for its proliferation (29).

In VCaP cells, growth was completely inhibited by CH5137291 at concentrations of 3–30 μM. In contrast, bicalutamide almost completely inhibited the cell growth at 3 μM, however at 30 μM, it induced cell growth. In LNCaP cells, CH5137291 inhibited cell growth more strongly than bicalutamide, and completely inhibited cell growth at concentrations of 1–30 μM. In LNCaP-BC2 cells, CH5137291 also showed stronger inhibition of cell growth than bicalutamide, and complete inhibition was observed at CH5137291 concentrations as low as 0.3 μM. Although bicalutamide showed strong inhibition of cell growth at 3 μM, cell growth was revived at higher concentrations. In LNCaP-CS10 cells, CH5137291 showed strong inhibition of cell growth, whereas bicalutamide enhanced cell growth.

The in vivo efficacy of CH5137291 on CRPCs was examined by using xenograft models of castrated SCID mice bearing LNCaP-BC2 and LNCaP-CS10. CH5137291 treatment (10 and 100 mg/kg) potently inhibited tumor growth in both xenograft models. The TGI% in each model was as follows: LNCaP-BC2, 104% and 104% on day 17 (at 10 and 100 mg/kg CH5137291, respectively); LNCaP-CS10, 88% and 88% on day 16 (at 10 and 100 mg/kg CH5137291, respectively) (Fig. 4). The plasma PSA level was also measured as a pharmacodynamic biomarker of AR antagonist activity. In both of the xenograft models tested, CH5137291 completely inhibited the increase in plasma PSA level, even at the dose of 10 mg/kg, without agonistic activity (Fig. 4). In contrast, bicalutamide treatment (10 and 100 mg/kg) showed almost no or little effect on either TV or PSA level even at the dose of 100 mg/kg.

The antagonistic effect of CH5137291 on AR transcriptional activity was examined by reporter gene assay. For this purpose, PC3 cells transiently co-transfected with AR (wild-type, W741C, or T877A) expression vectors and GMLUC reporter vectors including an MMTV promoter, a well-characterized AR-targeting promoter, were used. The W741C and T877A mutations are known to cause resistance to bicalutamide and flutamide, respectively (23,26,30).

In the three transfectants, CH5137291 inhibited transcriptional activity of AR in a dose-dependent manner in the presence of R1881 (Fig. 5). Bicalutamide showed different behaviors in the three transfectants. With wild-type AR, bicalutamide showed dose-dependent inhibition in transcriptional activity ≤3 μM; however, resurgence was observed at higher bicalutamide concentrations (Fig. 5A). With W741C-mutant AR, bicalutamide had no inhibitory effect throughout the concentrations used (Fig. 5B). With T877A-mutant AR, bicalutamide showed a weaker inhibitory effect compared with CH5137291. In the absence of R1881, CH5137291 showed no agonistic effect on the transcriptional activity of AR in any of the three transfectants (Fig. 5). In contrast, bicalutamide strongly increased the transcriptional activity of W741C-mutant AR in a dose-dependent manner (Fig. 5B). With wild-type and T877A-mutant AR, bicalutamide increased the transcriptional activity at relatively high concentrations (Fig. 5A and C).

The folding of helix 12, present in the ligand-binding domain of AR, is reported to be necessary for a ligand-induced agonist effect on AR transcriptional activity (39,40). The crystal structure of the bicalutamide/W741C-mutant AR complex revealed that bicalutamide does not collide with the C741 of the AR as it does with W741 in the wild-type AR; therefore, helix 12 of the bicalutamide/W741C-mutant AR complex is folded in the same manner as when a ligand, such as R1881, binds to it (Fig. 6) (41). Reportedly, this is a reason for bicalutamide exhibiting an agonist effect in W741C-mutant AR. To investigate the differences in the mechanisms of CH5137291 and bicalutamide with respect to agonist/antagonist effects against wild-type or W741C-mutant ARs, we examined a docking model of CH5137291 against wild-type and W741C-mutant ARs. The docking model indicated that the terminal sulfonamide group of CH5137291 intensively collided with the M895 residue of helix 12 in both the wild-type AR and the W741C-mutant AR. The collision would cause a complete inhibition of helix 12 folding and would result in a pure antagonist effect in both wild-type and W741C-mutant ARs. The varying interactions between the compound and AR may lead to the differing agonist and antagonist characteristics of CH5137291 and bicalutamide, respectively, in wild-type and W741C-mutant ARs.

Because of the highly similar structures within the nuclear receptor superfamily, we used a reporter gene assay to profile the effect of CH5137291 on the transcriptional activity of other nuclear receptors, including PR, GR, MR and ERα, in the presence or absence of ligands. CH5137291 exhibited weaker antagonistic effects on the progesterone receptor as compared to bicalutamide. Moreover, it exhibited neither agonistic nor antagonistic effects on glucocorticoid receptor, mineralocorticoid receptor, or estrogen receptor α (Fig. 7).

The effects of CH5137291 was examined against LuCaP35V xenografted tumors that progressed in castrated mice and LNCaP xenografted tumors that had failed to respond to initial bicalutamide treatment. In the LuCaP35V xenograft model, CH5137291 treatment (10 and 100 mg/kg) potently inhibited the tumor growth and plasma PSA level. The TGI% was 82% and 85% on day 48 (at 10 and 100 mg/kg, respectively) (Fig. 8A). In contrast, bicalutamide treatment (10 and 100 mg/kg) showed almost no effect on TV or PSA level. In the LNCaP xenograft model, 50 days after initiation of bicalutamide treatment, animals with tumors that showed resistance to bicalutamide were selected and randomized into two groups. One group continued receiving bicalutamide and the other group was switched to CH5137291 treatment. Only in the group switched to CH5137291 treatment did the tumor growth become static and the plasma PSA level decrease (Fig. 8B).

Long-term tumor growth inhibition against hormone-sensitive prostate cancer was examined by using an LNCaP xenograft in non-castrated SCID mice (Fig. 8C). The average time taken for the tumor volume to exceed double the initial volume was 113 days in the CH5137291 group and 58 days in the bicalutamide group. In addition, CH5137291 suppressed the plasma PSA concentration and the reduction in body weight during the observation period. These results indicate that CH5137291 can control tumor growth of hormone-sensitive prostate cancer for nearly twice as long as the period of control with bicalutamide.

Finally, to estimate the clinical therapeutic potential of CH5137291, cynomolgus monkeys were treated with CH5137291. Serum concentration of the drug and PSA as a pharmacodynamic biomarker was measured. The CH5137291 concentration dose-dependently increased. PSA concentration decreased in inverse proportion to the dosage of CH5137291 with a maximum 80% inhibition at 100 mg/kg (Fig. 9).