AR/androgen recepto

With endogenous AR gene amplification, the human prostate cancer cell line VCaP is treated with RD162 (1–10 μM) for four days to suppress growth and induce apoptosis[1]. In an in vitro fluorescence polarization assay, RD162 exhibits little to no binding to the progesterone, estrogen, or glucocorticoid receptors[1].

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RD162 binding to AR was specific, as there was little to no binding to the progesterone, estrogen or glucocorticoid receptors in an in vitro fluorescence polarization assay (table S1). We next compared the effects of RD162 and MDV3100 versus bicalutamide on androgen-dependent gene expression in LNCaP/AR cells. Expression of the AR target genes PSA and transmembrane serine protease 2 (TMPRSS2) was induced by bicalutamide but not by RD162 or MDV3100 (Fig. 1C), indicating that RD162 and MDV3100 do not have agonist activity in a castration-resistant setting. Both RD162 and MDV3100 antagonized induction of PSA and TMPRSS2 by the synthetic androgen R1881 in parental LNCaP cells (fig. S1). In the human prostate cancer cell line VCaP which has endogenous AR gene amplification, RD162 and MDV3100 suppressed growth and induced apoptosis whereas bicalutamide did not (Fig. 1D, E). This growth suppression was reversed by co-treatment with the synthetic androgen R1881 which competes for AR binding (fig S2A) and was not observed in the AR-negative DU145 human prostate cancer cells (fig S2B). In addition, RD162 and MDV3100 inhibited the transcriptional activity of a mutant AR protein (W741C) isolated from a patient with acquired resistance to bicalutamide (fig. S3). The W741C substitution in the AR LBD causes bicalutamide to act as a pure agonist[1].

All tumors regress when RD162 (10 mg/kg; oral gavage; daily; 28 days) is administered[1]. In human LNCaP/AR xenografts produced in castrated male mice, RD162 (0.1, 1, 10 mg/kg; oral gavage; daily; for 5 days) reliably lowers luciferase activity, but lower doses of 0.1 and 1.0 mg/kg/day have negligible effect. After five days, RD162 significantly lowers cellular proliferation[1]. Following oral administration, RD162 (20 mg/kg; gavage) has a serum half-life of roughly 30 hours and is approximately 50% bioavailable[1].

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To evaluate the activity of RD162 in vivo we first determined its pharmacokinetic properties in mice. RD162 was ∼50 percent bioavailable after oral delivery with a serum half-life of about 30 hours (Fig. 2A, table S2A). Trough levels observed 24 hours after a single 20 mg/kg oral dose (∼23 μM) exceeded concentrations expected to block AR activity based on the in vitro studies (∼1-10 μM). We evaluated the pharmacodynamic effects of RD162 on AR function in vivo by measuring luciferase activity of human LNCaP/AR xenografts grown in castrated male mice. These tumors coexpressed exogenous AR and the AR-dependent reporter construct ARR2-Pb-Luc. Luciferase activity was consistently reduced relative to vehicle control in mice treated for 5 days with 10 mg/kg RD162 daily by oral gavage (Fig. 2B), whereas lower doses of 0.1 and 1.0 mg/kg/day had minimal effect (fig. S4A). Commensurate with reduced AR transcriptional function, cellular proliferation of LNCaP/AR xenografts as measured by Ki-67 staining was substantially reduced after 5 days of RD162 treatment (fig. S4B).[1]

Ligand Binding Studies [1]
The relative binding affinity in cells of RD162 and MDV3100 to AR, relative to dihydrotestosterone (DHT) and bicalutamide, was determined using a competition assay in which increasing concentrations of cold competitor are added to cells pre-incubated with 18F-FDHT. LNCaP/AR (codon-switch) cells were propagated in Iscove’s or RPMI media supplemented with 10% FBS. Cells were trypsinized, washed in PBS, and triplicate cell samples were mixed with 20,000 cpm 18F-FDHT and increasing amounts of cold competitor (1 pM to 1μM). The solutions were shaken on an orbital shaker at ambient temperature, and after 1 hour the cells were isolated and washed with ice cold Tris-buffered saline using a Brandel cell harvester. All the isolated cell samples were counted using a scintillation counter, with appropriate standards of total activity and blank controls, and the specific uptake of 18F-FDHT determined. These data were plotted against the concentration of the cold competitor to give sigmoidal displacement curves. The IC50 values were determined using a one site model and a least squares curve fitting routine with the R2 of the curve fit being >0.99.

Relative in vitro binding affinities of RD162 for the ligand binding domains of rat AR and human progesterone receptor (PR), and full-length human estrogen receptor-alpha (ERα) and human glucocorticoid receptor (GR) were determined using competitor assay kits. Fluorescence polarization was used as a read-out, and the experiments were performed in 96-well plates (Costar® black polystyrene round-bottom assay plates #3792) and read (5 readings per well; 0.1 second integration time; entire plate read 5 to 8 times; G factor = 0.91) using the Analyst™ AD plate-reader with fluorescein excitation (485 nM) and emission (530 nM) filters. Each hormone dose was performed in triplicate and the relative error was determined by calculating the standard error of the three values from the mean. In all cases we controlled for minimal competition (vehicle alone), for no receptor, for no fluorescent ligand, and for maximal competition (10-5 M R1881, progesterone, estradiol (E2) or dexamethasone). The binding curves were fit using a single binding site competition model, with the Prism statistical analysis software package. The R2 values in all cases were greater than 0.8. Experiments were conducted multiple times to ensure reproducibility of the results, and the standard error of the mean (SEM) was less than 0.3 log units from the average logIC50 value in all cases. Ki values were calculated as averages across experiments along with SEM, and binding affinities were reported as a percentage relative to the tight-binding ligand control for that receptor. [1]

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In vitro serum protein binding [1]
Stock protein solutions of purified HSA (4.5%) and AAG (1.1%) in phosphate buffered saline (PBS) were mixed with an appropriate volume of antiandrogen stock solution (either 36X or 50X, in 40% ethanol/water) in order to obtain test samples containing protein (4% HSA + 0.1% AAG) and antiandrogen (0.4, 2.0 or 10 µM bicalutamide, RD162 or MDV3100). The test samples were thoroughly mixed and triplicate samples (250 μL to 500 μL) were transferred to glass centrifuge tubes. All ultracentrifugation tubes and pipette tips were treated with 0.1% aqueous Tween 20 prior to use to reduce non-specific binding losses of antiandrogen. 1.0 mL of acetonitrile/methanol (4/1 v/v) was added, thoroughly mixed, and kept at -20 °C or in an ice-bath for ~one hour, then centrifuged at 1000g for 10 minutes. The supernatants were transferred to a 96-well plate and analyzed for drug levels by LC-MS/MS. These samples represented the total pre-ultracentrifugation amount of antiandrogen. Approximately 0.5 mL of each spiked protein sample was then transferred in triplicate into each centrifuge tube (Amicon® Ultra, molecular weight cut off 5,000 or 10,000). The filled tubes were accurately balanced (with appropriate caps) and then placed in the centrifuge. The samples were centrifuged at ~4°C for ~1.0 hr at ~3,500g. Following ultrafiltration, triplicate samples (100 μL) of filtrates were carefully transferred from each tube to a glass centrifuge tube. 0.5 mL of acetonitrile/methanol (4/1 v/v) was added, thoroughly mixed, and kept at -20 °C or in an ice-bath for ~one hour, then centrifuged at 1000 g for 10 minutes. The supernatants were transferred to a 96-well plate and analyzed for antiandrogen levels by LC-MS/MS, using validated methods developed by the MSKCC Analytical Pharmacology Core Facility. These values represent unbound amount of antiandrogen. Results for the mean binding (n=3) of antiandrogen to serum protein were calculated, and percent of protein-bound ligand was reported. [1]

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Time resolved fluorescence resonance transfer assay [1]
Lantha Screen TR-FRET Androgen Receptor Coactivator Assay (Invitrogen) was performed using 100X drug stocks prepared by five-fold serial dilutions in DMSO. Assay samples were prepared in a 384 well plate as recommended by the manufacturer and then incubated at room temperature for 2 hours prior to data collection. For each drug treatment in each experiment, the emission values at 525 nm and 488 nm after excitation at 332 nm (with cutoff of 420 nm) were obtained by averaging values from four identically prepared wells, each read 10 times using a SpectraMax M5 plate reader. The 525/488 emission ratio for each treatment was then divided by the emission ratio obtained with vehicle to determine a fold change in emission ratio with each treatment. Data presented represents the average fold change in emission ratio of two separate experiments with standard error in the mean between experiments indicated.

Cell Viability Assay[1]
Cell Types: VCaP cells
Tested Concentrations: 1, 10 μM
Incubation Duration: 4 days
Experimental Results: Suppressed cell growth.

Animal/Disease Models: Castrate male mice bearing LNCaP/AR xenografts[1]
Doses: 10 mg/kg
Route of Administration: Oral gavage; daily ; for 28 days
Experimental Results: Caused all tumors regressed.

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Animal/Disease Models: Male mice[1]
Doses: 20 mg/kg (pharmacokinetic/PK Analysis)
Route of Administration: po (oral gavage) (in 0.5% hydroxy-methyl-propyl-cellulose)
Experimental Results: Had ∼50 percent bioavailable after oral delivery with a serum half-life of about 30 hrs (hours).

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Pharmacokinetic analysis [1]
RD162 was formulated as follows for pharmacokinetic studies. Oral formulation was prepared at concentrations of 2.5 mg/ml, 5 mg/ml, 7.5 mg/ml, 10 mg/ml and 15 mg/ml in 0.5% sterile hydroxymethyl-propyl-cellulose (HMPC) solution and kept at 4o C in amber glass bottles prior to use. Each RD162 suspension was used within one week of preparation. The intravenous formulation was prepared at a concentration of 2 mg/ml in 10% (w/v) sterile castor oil-PEG 35, 5% methanol solution. RD162 was administered as a single oral (p.o.) or intravenous (iv) dose of 20 mg/kg to 8 week old male B6D2F1 mice (n=3/time point) and plasma was analyzed at various time points (5 min to 24 hr) after dosing by an LC-MS/MS assay. To assess the level of the selected RD162 drug in blood, frozen plasma samples were thawed at ambient temperature and extracted with a solvent mixture of methanol and acetonitrile (1/4, v/v). Samples were incubated at – 20 ºC for approximately one hour and then centrifuged at 1000g for 10 minutes. The supernatants were transferred to a 96-well plate and analyzed by high-performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). Calibration curves were determined for RD162 to permit conversion of peak areas to the drug amounts against external reference standards. The limit of detection for RD162 was in a range of a few nanograms. The PK data were analyzed using the WinNonlin program two-compartmental model method of analysis. Parameters analyzed included the maximal plasma concentration (Cmax) and time (Tmax), area under the plasma concentration curve (AUC0-24h), area under the plasma concentration 2 curve to infinity from time zero (AUC0-inf) and the half-life (T1/2). The AUC was calculated using a two-compartmental analysis with linear interpolation to infinity. The oral bioavailability was calculated based on AUC0-inf. Oral PK profiles are displayed in fig 2a.

RD162 was ∼50 percent bioavailable after oral delivery with a serum half-life of about 30 hours

[1]. Development of a second-generation antiandrogen for treatment of advanced prostate cancer. Science. 2009 May 8;324(5928):787-90.

Metastatic prostate cancer is treated with drugs that antagonize androgen action, but most patients progress to a more aggressive form of the disease called castration-resistant prostate cancer, driven by elevated expression of the androgen receptor. Here we characterize the diarylthiohydantoins RD162 and MDV3100, two compounds optimized from a screen for nonsteroidal antiandrogens that retain activity in the setting of increased androgen receptor expression. Both compounds bind to the androgen receptor with greater relative affinity than the clinically used antiandrogen bicalutamide, reduce the efficiency of its nuclear translocation, and impair both DNA binding to androgen response elements and recruitment of coactivators. RD162 and MDV3100 are orally available and induce tumor regression in mouse models of castration-resistant human prostate cancer. Of the first 30 patients treated with MDV3100 in a Phase I/II clinical trial, 13 of 30 (43%) showed sustained declines (by >50%) in serum concentrations of prostate-specific antigen, a biomarker of prostate cancer. These compounds thus appear to be promising candidates for treatment of advanced prostate cancer.[1]

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Two lines of evidence suggest that the activity of RD162 in these mice is mediated through AR inhibition rather than through off-target effects. First, antitumor activity in the LNCaP/AR model is dose-dependent, with some slowing of tumor growth at 0.1 mg/kg RD162 and a few tumor regressions at 1 mg/kg (fig. S7), correlating closely with the effect of these same doses on AR transcriptional activity in the luciferase imaging experiment (fig. S4A). Second, neither bicalutamide nor RD162 impaired the growth of AR-negative DU145 prostate cancer xenografts (fig. S8).[1]

C22H16F4N4O2S

476.45

476.093

C, 55.46; H, 3.39; F, 15.95; N, 11.76; O, 6.72; S, 6.73

915087-27-3

11957756

White to off-white solid powder

1.55

4.841

1

8

3

33

883

0

JPQFGMYHKSKKGW-UHFFFAOYSA-N

InChI=1S/C22H16F4N4O2S/c1-28-18(31)15-6-5-14(10-17(15)23)30-20(33)29(19(32)21(30)7-2-8-21)13-4-3-12(11-27)16(9-13)22(24,25)26/h3-6,9-10H,2,7-8H2,1H3,(H,28,31)

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

RD162; RD 162; RD-162; 4-[7-[4-cyano-3-(trifluoromethyl)phenyl]-8-oxo-6-sulfanylidene-5,7-diazaspiro[3.4]octan-5-yl]-2-fluoro-N-methylbenzamide; N-Methyl-4-[7-(4-cyano-3-trifluoromethylphenyl)-8-oxo-6-thioxo-5,7-diazaspiro[3.4]octan-5-yl]-2-fluorobenzamide; Depyridinyl Phenyl Apalutamide; 5ZE6THH5VF;

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)

DMSO: 125 mg/mL (262.36 mM)

Note: Listed below are some common formulations that may be used to formulate products with low water solubility (e.g. < 1 mg/mL), you may test these formulations using a minute amount of products to avoid loss of samples.

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Injection Formulation 1: DMSO : Tween 80： Saline = 10 : 5 : 85 (i.e. 100 μL DMSO stock solution → 50 μL Tween 80 → 850 μL Saline)
*Preparation of saline: Dissolve 0.9 g of sodium chloride in 100 mL ddH ₂ O to obtain a clear solution.
Injection Formulation 2: DMSO : PEG300 ：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL DMSO → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)
Injection Formulation 3: DMSO : Corn oil = 10 : 90 (i.e. 100 μL DMSO → 900 μL Corn oil)
Example: Take the Injection Formulation 3 (DMSO : Corn oil = 10 : 90) as an example, if 1 mL of 2.5 mg/mL working solution is to be prepared, you can take 100 μL 25 mg/mL DMSO stock solution and add to 900 μL corn oil, mix well to obtain a clear or suspension solution (2.5 mg/mL, ready for use in animals).

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Injection Formulation 4: DMSO : 20% SBE-β-CD in saline = 10 : 90 [i.e. 100 μL DMSO → 900 μL (20% SBE-β-CD in saline)]
*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.
Injection Formulation 5: 2-Hydroxypropyl-β-cyclodextrin : Saline = 50 : 50 (i.e. 500 μL 2-Hydroxypropyl-β-cyclodextrin → 500 μL Saline)
Injection Formulation 6: DMSO : PEG300 : castor oil : Saline = 5 : 10 : 20 : 65 (i.e. 50 μL DMSO → 100 μLPEG300 → 200 μL castor oil → 650 μL Saline)
Injection Formulation 7: Ethanol : Cremophor : Saline = 10： 10 : 80 (i.e. 100 μL Ethanol → 100 μL Cremophor → 800 μL Saline)
Injection Formulation 8: Dissolve in Cremophor/Ethanol (50 : 50), then diluted by Saline
Injection Formulation 9: EtOH : Corn oil = 10 : 90 (i.e. 100 μL EtOH → 900 μL Corn oil)
Injection Formulation 10: EtOH : PEG300：Tween 80 : Saline = 10 : 40 : 5 : 45 (i.e. 100 μL EtOH → 400 μLPEG300 → 50 μL Tween 80 → 450 μL Saline)

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Oral Formulation 1: Suspend in 0.5% CMC Na (carboxymethylcellulose sodium)
Oral Formulation 2: Suspend in 0.5% Carboxymethyl cellulose
Example: Take the Oral Formulation 1 (Suspend in 0.5% CMC Na) as an example, if 100 mL of 2.5 mg/mL working solution is to be prepared, you can first prepare 0.5% CMC Na solution by measuring 0.5 g CMC Na and dissolve it in 100 mL ddH2O to obtain a clear solution; then add 250 mg of the product to 100 mL 0.5% CMC Na solution, to make the suspension solution (2.5 mg/mL, ready for use in animals).

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Oral Formulation 3: Dissolved in PEG400
Oral Formulation 4: Suspend in 0.2% Carboxymethyl cellulose
Oral Formulation 5: Dissolve in 0.25% Tween 80 and 0.5% Carboxymethyl cellulose
Oral Formulation 6: Mixing with food powders

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Note: Please be aware that the above formulations are for reference only. InvivoChem strongly recommends customers to read literature methods/protocols carefully before determining which formulation you should use for in vivo studies, as different compounds have different solubility properties and have to be formulated differently.

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