Darolutamide (ODM-201;BAY-1841788)

Androgen receptor (AR) (IC50: 26 nM in AR-HEK293 cells)

In a competitive AR binding experiment, darolutamide (ODM-201) has an inhibitory constant (Ki) value of 11 nM. Compared to ARN-509, ODM-201 and ORM-15341 reduced androgen-induced cell proliferation more successfully. Their respective IC50 values were 230 and 170 nM for Darolutamide and ORM-15341, respectively, whereas ARN-509's was 420 nM. The anti-proliferative qualities of darolutamide and ORM-15341 are exclusive to AR-dependent PC cells, as demonstrated by the lack of effect darolutamide had on the viability of the AR-negative cell lines tested, DU-145 prostate cancer cells and H1581 lung cancer cells [1].

At both doses, darolutamide (ODM-201) demonstrated considerable anti-tumor activity; 50 mg/kg twice day was more effective (p<0.001) than in mice that had not received treatment. It was also demonstrated to be effective against growth inhibition of tumors (p<0.05) in mice that had not received treatment. Furthermore, no symptoms of treatment-related toxicity were seen, and mice given twice daily doses of darolutamide did not significantly lose weight while receiving the medication [1].

AR binding affinity [1]
AR binding affinities of test compounds were studied in cytosolic lysates obtained from ventral prostates of castrated rats by a competition binding assay as previously described. Fresh prostates were minced and homogenized with Buffer A containing protease inhibitors. The homogenates were centrifuged and the resultant supernatants were treated with a dextran-coated charcoal solution to remove endogenous steroids. The dissociation constant of the radio ligand [3H]mibolerone for isolated rat ARs was determined in a saturation binding experiment as previously described. For the determination of Ki values, prostate cytosol preparations and 1 nM [3H]mibolerone were incubated with increasing concentrations of test compounds overnight. After the incubation, bound and free steroids were separated by treatment with 100 μL of dextran-coated charcoal suspension. Bound radioactivity was determined by counting 100 μL of supernatant fraction in 200 μL of scintillation fluid (OptiPhase SuperMix, PerkinElmer) using a microbeta counter. All procedures were carried out at 0–4 °C.

Antagonism of ODM-201[1]
Functional activity and potency of antiandrogens to hAR were determined in AR-HEK293 cells. The cells were treated with test compounds and 0.45 nM testosterone in steroid-free assay medium supplemented with 2 nM GlutaMAX and 25 mM HEPES. After 24 hours at 37 °C with 5% CO2, cells were lysed and luciferase activity was measured with a Centro LB 960 microplate luminometer using a luciferase assay system according to manufacturer’s instructions.

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Mutant AR studies[1]
Human U2-OS osteosarcoma cells were transiently transfected with an androgen-responsive reporter gene construct (pGV5-basic-GRE-hiv-luc) and expression vectors encoding AR mutants AR(F876L), AR(T877A), or AR(W741L) (pSG5-hAR-F876L, pSG5-hAR-T877A, or pSG5-hAR-W741L) using LipofectaminTM2000. The construction of the mutant AR expression vectors was done as previously described. For one well in a 96-well plate, 190 ng of reporter construct DNA and 10 ng of receptor construct DNA were diluted in Opti-MEM® (Gibco). Cells were treated with increasing concentrations of the test compounds in the absence or presence of a reference agonist inducing a submaximal reporter gene activation (0.6 nM testosterone in case of T877A and F876L, and 10 nM DHT in case of W741L) in steroid-free assay medium and incubated for 24 hours. Luciferase activity was measured as described above.

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AR nuclear translocation[1]
AR overexpressing HS-HEK293 cells immunolabeled with an AR-antibody were imaged either with a high-content screening (HCS) reader (Cellomics ArrayScan HCS VTI reader, Thermo) or with a confocal microscope. HS-HEK293 cells in steroid-free assay medium were plated on poly-D-lysine coated microplates (BD) (HCS reader) or on coverslips (confocal imaging). After a 48-hour incubation, the cells were treated with 0.3 (HCS reader) or 1 μM (confocal imaging) of test compounds together with 0.3 nM testosterone for 5 hours. After fixation with 3.7% PFA, the cells were washed with phosphate-buffered saline (PBS), permealized with 0.1% Triton X-100 (Sigma), and treated with 3% BSA in PBS to block unspecific staining. For HCS reader, cells were incubated with polyclonal AR antibody conjugated with Alexa Fluor® 488 (N20, Santa Cruz, dilution 1:50). Cells were washed, DNA was labeled with DAPI (Sigma, 1 μg//mL), and images were analyzed with a NucTrans. V3 assay algorithm (Thermo). For confocal imaging, polyclonal AR antibody was used as a primary and Alexa Fluor® 546 anti-rabbit IgG as a secondary antibody. Coverslips were mounted with Vectashield containing DAPI (Vector Laboratories). AR overexpressing LN-AR-C cells were treated with 3 μM of test compounds together with 0.3 nM testosterone for 4 hours, immunolabeled with the AR-antibody nd a secondary antibody and imaged with the HCS reader.

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VCaP proliferation assay[1]
VCaP cells were treated with a submaximal concentration of mibolerone (0.1 nM) and increasing concentrations of test compounds in steroid-free assay medium supplemented with 4 mM GlutaMAX. After a 4-day incubation with the compounds, cell viability was measured using a WST-1 cell proliferation assay (Roche), according to manufacturer’s instructions. To rule out non-AR –mediated toxicity, AR-negative PC cells (DU-145) and lung cancer cells (H1581) were treated with an increasing concentration of ODM-201, and cell viability was measured as described above.

The castration-resistant VCaP xenograft experiment
BALB/c nude male mice (7 weeks old from Charles River, Germany) were subcutaneously injected with 2 million VCaP cells in 100 μL of RPMI-1640 medium and Matrigel (BD) (1:1). Tumor growth was monitored twice weekly by caliper measurements. The volume of the tumor was calculated according to the formula W2 × L/2 (mm3), where W is the shorter and L the longer diameter of the tumor. When the average tumor volume reached ~200 mm3, mice were castrated or SHAM-operated under Avertin anesthesia. Oral treatments with two doses of ODM-201 (50 mg/kg, qd or bid), enzalutamide (20 mg/kg, qd), or vehicle were initiated upon tumor regrowth (when average tumor volumes were ~400 mm3) and were continued for 37 days. For all in vivo studies, Macrocol® (Merck) + propylene glycol +5% glucose (50:30:20, v/v/v) was used as a vehicle.

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Pharmacokinetic studies in mice
In the PK studies analyzing the penetration of test compounds to the brain, nude male mice (BALB/c or Balb/cOlaHsd from Charles River Laboratories and Harlan, Netherlands, 8-9 weeks of age) were orally dosed for 7 days with 25, 50, or 100 mg/kg of ODM-201 twice daily (n = 5) or with 20 mg/kg enzalutamide once daily (n = 4), or with a single oral dose of ARN-509 (10 mg/kg) (n = 3). Control mice received vehicle. Blood samples were collected into K2EDTA tubes by cardiac puncture under CO2 anesthesia and plasma was separated by centrifugation. Brain samples (without olfactory bulbs and medulla oblongata) from each group and time point were pooled and homogenized before the analysis. Concentrations of ODM-201 and ORM-15341 in mouse plasma and brain were determined by liquid chromatography-tandem mass spectrometry (LC-MS/MS) method at Charles River, UK, with the lower limit of quantification (LLOQ) being in plasma 1.00 ng/mL for both ODM-201 and ORM-15341 and in brain 4.00 ng/g for ODM-201 and 10.00 ng/g for ORM-15341. Enzalutamide and ARN-509 concentrations were determined by LC-MS/MS method at Orion Pharma (LLOQ for enzalutamide was 1.00 ng/mL in plasma and 5.00 ng/g in brain, and for ARN-509 0.250 ng/mL in plasma and 10.0 ng/g in brain). Plasma and brain concentration vs. time were evaluated by noncompartmental analysis using WinNonlin® Professional v. 5.2 software. Brain/plasma ratios were calculated based on AUC0–24 values for plasma and brain.

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Dissolved in Macrocol + propylene glycol +5% glucose (50:30:20, v/v/v); 50 mg/kg; p.o. administration

BALB/c nude male mice bearing VCaP xenografts

[1]. Moilanen AM, et al. Discovery of ODM-201, a new-generation androgen receptor inhibitor targeting resistance mechanisms toandrogen signaling-directed prostate cancer therapies. Sci Rep. 2015 Jul 3;5:12007. doi: 10.1038/srep12007

C19H19CLN6O2

398.85

398.13

C, 57.22; H, 4.80; Cl, 8.89; N, 21.07; O, 8.02

1297538-32-9

White to off-white solid powder

1.8

120Ų

O=C(C1=NNC(C(O)C)=C1)N[C@@H](C)CN2N=C(C3=CC=C(C#N)C(Cl)=C3)C=C2

ANGUXJDGJCHGOG-UHFFFAOYSA-N

InChI=1S/C25H29N5O/c1-19-8-9-22-23(27-19)6-3-7-24(22)29-16-14-28(15-17-29)12-10-20-4-2-5-21(18-20)30-13-11-26-25(30)31/h2-9,18H,10-17H2,1H3,(H,26,31)

N-((S)-1-(3-(3-chloro-4-cyanophenyl)-1H-pyrazol-1-yl)propan-2-yl)-5-(1-hydroxyethyl)-1H-pyrazole-3-carboxamide

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 (5.21 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 (5.21 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 20.8 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 3: ≥ 2.08 mg/mL (5.21 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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 (Please use freshly prepared in vivo formulations for optimal results.)