Priority Report
`
`Cancer
`Research
`
`Interactions of Abiraterone, Eplerenone, and Prednisolone
`with Wild-type and Mutant Androgen Receptor: A Rationale
`for Increasing Abiraterone Exposure or Combining
`with MDV3100
`
`Juliet Richards1, Ai Chiin Lim1, Colin W. Hay3, Angela E. Taylor4, Anna Wingate1, Karolina Nowakowska1,
`Carmel Pezaro1,2, Suzanne Carreira1, Jane Goodall1, Wiebke Arlt4, Iain J. McEwan3,
`Johann S. de Bono1,2, and Gerhardt Attard1,2
`
`Abstract
`
`Prostate cancer progression can be associated with androgen receptor (AR) mutations acquired following
`treatment with castration and/or an antiandrogen. Abiraterone, a rationally designed inhibitor of CYP17A1
`recently approved for the treatment of docetaxel-treated castration-resistant prostate cancer (CRPC), is often
`effective, but requires coadministration with glucocorticoids to curtail side effects. Here, we hypothesized that
`progressive disease on abiraterone may occur secondary to glucocorticoid-induced activation of mutated AR. We
`found that prednisolone plasma levels in patients with CRPC were sufficiently high to activate mutant AR.
`Mineralocorticoid receptor antagonists, such as spironolactone and eplerenone that are used to treat side effects
`related to mineralocorticoid excess, can also bind to and activate signaling through wild-type or mutant AR.
`Abiraterone inhibited in vitro proliferation and AR-regulated gene expression of AR-positive prostate cancer cells,
`which could be explained by AR antagonism in addition to inhibition of steroidogenesis. In fact, activation of
`mutant AR by eplerenone was inhibited by MDV3100, bicalutamide, or greater concentrations of abiraterone.
`Therefore, an increase in abiraterone exposure could reverse resistance secondary to activation of AR by residual
`ligands or coadministered drugs. Together, our findings provide a strong rationale for clinical evaluation of
`combined CYP17A1 inhibition and AR antagonism. Cancer Res; 72(9); 2176–82. Ó2012 AACR.
`
`Introduction
`
`The small-molecule CYP17A1 inhibitor, abiraterone acetate
`(Zytiga, Janssen), was recently approved for the treatment of
`men with castration-resistant prostate cancer (CRPC) progres-
`sing after docetaxel chemotherapy. Despite a significant sur-
`vival advantage with 1,000 mg abiraterone daily and objective
`tumor responses in up to 60% of patients with CRPC, progres-
`sive disease on treatment invariably develops (1, 2). MDV3100
`is a novel antiandrogen (3, 4) that has also recently been
`
`Authors' Affiliations: 1Section of Medicine, The Institute of Cancer
`Research; 2The Royal Marsden NHS Foundation Trust, Sutton, Surrey;
`3School of Medical Sciences, University of Aberdeen, Foresterhill, Aberd-
`een; and 4Centre for Endocrinology, Diabetes and Metabolism, School of
`Clinical and Experimental Medicine, University of Birmingham, Birming-
`ham, United Kingdom
`
`Note: Supplementary data for this article are available at Cancer Research
`Online (http://cancerres.aacrjournals.org/).
`
`J.S. de Bono and G. Attard are joint senior authors.
`
`Corresponding Author: Gerhardt Attard, Section of Medicine, The Insti-
`tute of Cancer Research and the Royal Marsden NHS Foundation Trust,
`Downs Road, Sutton, Surrey SM2 5PT, United Kingdom. Phone: 0044-
`7793077493; Fax: 0044-2086427979; E-mail: Gerhardt.attard@icr.ac.uk
`
`doi: 10.1158/0008-5472.CAN-11-3980
`
`Ó2012 American Association for Cancer Research.
`
`reported to confer a survival advantage in patients with CRPC
`progressing after docetaxel (5). As prostate-specific antigen
`(PSA) level often increases at progression on both these agents,
`we have hypothesized that resistance occurs secondary to
`reactivation of androgen receptor (AR) signaling. Inhibition
`of CYP17A1 results in significant suppression of androgens and
`estrogens but also of cortisol that is associated with a com-
`pensatory increase in adrenocorticotropic hormone level (2).
`Abiraterone acetate has therefore been developed in combi-
`nation with exogenous glucocorticoids. However, up to 40% of
`patients on prednisone/prednisolone alone and 55% of
`patients on abiraterone acetate and prednisone/prednisolone
`develop a syndrome of secondary mineralocorticoid excess
`characterized by hypokalemia, hypertension, and fluid over-
`load that can be controlled by increasing the dose of predni-
`sone or adding a mineralocorticoid receptor antagonist (MRA)
`such as eplerenone (1). Eplerenone is currently recommended
`in preference to spironolactone as previous studies showed
`that eplerenone did not bind and activate wild-type (WT)-AR
`(2, 6). However, as eplerenone is not invariably available,
`spironolactone is also being used.
`Point mutations of the AR, which appear to cluster in the
`ligand-binding domain, are rare in therapy naive patients but
`occur in 15% to 45% of castration-resistant disease and can
`increase AR affinity for a wide range of steroids (7, 8). Over 100
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`mutations have been described and many have been shown to
`give a functional advantage to maintain AR signaling. We
`hypothesized that progressive disease on abiraterone acetate
`could occur secondary to activation of mutated "promiscuous"
`AR by steroidal agents administered to patients to prevent or
`treat side effects of mineralocorticoid excess.
`
`Materials and Methods
`
`Materials
`FBS and charcoal-stripped serum (CSS) were purchased
`from Gibco. Bicalutamide, dexamethasone, prednisone, and
`titrated [3H]-
`dihydrotestosterone (DHT; Sigma-Aldrich),
`R1881 (Perkin-Elmer), R1881 (Steraloids), eplerenone and spir-
`onolactone (Tocris-Bioscience) were obtained from commer-
`cial sources. Abiraterone and MDV3100 were synthesized using
`the publicly available chemical structures and checked by mass
`spectrometry. Drugs were dissolved in dimethyl sulfoxide
`(DMSO) and then diluted to a maximum DMSO concentration
`of 0.2%. LNCaP, VCaP, PC-3, DU145, and COS-7 cells were
`obtained from American Type Culture Collection (ATCC; LGC
`Standards), grown according to ATCC recommendations, used
`less than 6 months from receipt and freeze down and con-
`firmed mycoplasma free.
`
`Luciferase reporter assays
`We constructed a PSA-ARE3-luc luciferase reporter plasmid
`that was cotransfected with a human AR expression plasmid,
`F527-AR [wild-type or mutant as stated; mutations confirmed
`by sequencing (Beckman Coulter Genomics)] into PC-3 cells.
`These were seeded in white opaque 384-well plates and grown
`in 10% CSS-supplemented phenol red–free RPMI-1640 for 30
`hours. Cells were then treated with the indicated concentra-
`tion of compound and R1881 for 16 hours. Luciferase activity
`was determined by adding ONE Glo (Promega) and measuring
`luminescence on a TopCount plate reader (Perkin-Elmer).
`Transfection efficiency and protein expression are shown in
`Supplementary Fig. S1.
`
`Cell viability
`LNCaP and VCaP cells were seeded in 96-well plates and
`grown in CSS-supplemented phenol red–free or FBS-supple-
`mented media for 7 days. Cells were treated with compound at
`24 and 96 hours after plating and cell viability was determined
`on day 7 by adding CellTiter Glo (Promega) and measuring
`luminescence.
`
`Ligand-binding assay
`PC-3 cells transfected with wild-type or T877A mutant AR or
`LNCaP cells were seeded in 24-well plates and grown in CSS-
`supplemented phenol red–free media for 24 hours. To deter-
`mine the kinetics of [3H]-R1881 binding to the wild-type and
`T877A AR, cells were treated with 0.25 to 25 nmol/L [3H]-R1881
`for 2 hours, then washed, lysed, and radioactivity was measured
`(1900CA analyzer, Perkin-Elmer). The Kd and Bmax were deter-
`mined by nonlinear regression using GraphPad Prism soft-
`ware. When the concentration of [3H]-R1881 required to
`almost saturate AR in both wild-type and T877A AR mutant
`
`Interaction of AR with Abiraterone and Eplerenone
`
`transfections was established (5 nmol/l), displacement of [3H]-
`R1881 by test compound was determined. The concentration
`at which 50% of [3H]-R1881 was displaced (EC50) was estab-
`lished using nonlinear regression (GraphPad Prism).
`
`Quantitative real time-PCR
`LNCaP and VCaP cells were seeded in 6-well plates and
`grown in CSS-supplemented phenol red–free media for 24
`hours and then treated for 5 hours as indicated. Following
`RNA extraction and cDNA synthesis, quantitative PCR
`(qPCR) was carried out on the Mx3000P QPCR System
`(Agilent) using the RT2 SYBR Green ROX qPCR Mastermix
`(SABiosciences). Every sample was run in duplicate and each
`reaction contained 50 ng of cDNA in a total volume of 20 mL.
`DCt for each gene was determined after normalization to
`actin
`and
`glyceraldehyde-3-phosphate
`dehydrogenase
`(GAPDH), and DDCt was calculated relative to the designat-
`ed reference sample. Gene expression values were set equal
`to 2DDCt (Applied Biosystems). Primers were purchased
`from SABiosciences.
`
`Measurement of plasma prednisolone
`Plasma was collected from patients with CRPC after 48 days
`of continuous daily abiraterone acetate and prednisolone. All
`patients provided written, informed consent to blood with-
`drawal for research purposes, and this study was approved by
`the Royal Marsden Hospital ethics review committees. Pred-
`nisolone was quantified by comparison to a calibration series
`ranging from 5 to 500 ng/mL prepared in 50:50 methanol:water.
`A Waters Xevo mass spectrometer with Acquity uPLC system
`was used, fitted with a HSS T3, 1.8 mm, 1.2  50 mm2 column
`(Waters). The column temperature was maintained at 60C
`and the settings used were an electrospray source in positive
`ionization mode; capillary voltage 4.0 kV; source temperature,
`150C; and desolvation temperature, 500C.
`
`Results
`
`The selective mineralocorticoid receptor antagonist,
`eplerenone, activates mutant AR
`We first cotransfected PC-3 AR-negative prostate cancer
`cells with PSA-ARE2-luc and either wild-type (WT)-AR or 3
`mutations previously described in CRPC (T877A-AR, D879G-
`AR, and W741C-AR). The T877A mutation has been identified
`in several studies in patients treated with flutamide (8, 9) and
`has been extensively studied as it is found in the LNCaP
`prostate cancer cell line (Supplementary Table S1). D879G
`and W741C mutations have been identified in patients previ-
`ously treated with bicalutamide (8, 9). We then compared
`activation of wild-type or mutant AR by synthetic androgen
`(R1881) to activation by the MRAs, eplerenone, and spirono-
`lactone. In keeping with previous reports, spironolactone
`activates WT-AR (7) and also T877A-AR, D879G-AR, and
`W741C-AR only 2-log less potently than R1881 does (Fig. 1A
`and B and Supplementary Fig. S2). Eplerenone does not
`activate WT-AR, D879G-AR, or W741C-AR but importantly
`can activate T877A-AR with a dose-proportional response
`and an EC50 value of 5.2 mmol/L [95% confidence interval (CI),
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`Figure 1. Eplerenone activates T877A-AR and spironolactone activates both T877A-AR and wild-type (WT)-AR. Sigmoidal dose–response curves show
`activation of WT-AR by R1881 and spironolactone (A) and T877A-AR by R1881, spironolactone, eplerenone, prednisolone, dexamethasone. (B) Fold
`change from the DMSO control was plotted and EC50 values calculated using nonlinear regression (GraphPad). EC50 values and 95% CIs are given. C, LNCaP
`and VCaP prostate cancer cells in CSS were treated with eplerenone or spironolactone alone or in combination with 0.1, 1, or 5 mmol/L abiraterone, 10 mmol/L
`bicalutamide, or 10 mmol/L MDV3100 for 7 days and then analyzed for cell viability. Fold change from the DMSO control was then calculated and plotted.
`Significance is shown for stimulation by eplerenone or spironolactone compared with DMSO control ( , horizontal) and for inhibition by bicalutamide,
`MDV3100, or abiraterone when compared with stimulated levels ( , vertical). D, LNCaP and VCaP cells were treated with 0.1 nmol/L R1881 or 0.1 to 10 mmol/L
`eplerenone for 5 hours. RNA was extracted and cDNA synthesized for analysis by qPCR to determine relative levels of PSA and TMPRSS2 mRNA expression.
`Significance compared with DMSO controls is shown. Data shown for all experiments are the mean (error bars, SEM) of 3 independent experiments of 16
`replicates (A and B) or in duplicate (C and D). , P < 0.05; , P < 0.01; , P < 0.001, one-way ANOVA with the Bonferroni correction.
`
`2.89–9.37 mmol/L; Fig. 1A and B and Supplementary Fig. S2).
`Pharmacokinetic studies with eplerenone report a Cmax of 1.72
` 0.28 mg/mL (equivalent to 4.2  0.7 mmol/L) and a half-life of
`3 hours with 100 mg eplerenone (6); doses of eplerenone
`between 50 and 200 mg are used to treat toxicities secondary
`to mineralocorticoid excess from abiraterone in patients with
`CRPC (Supplementary Table S2). We proceeded to confirm
`that both spironolactone and eplerenone (1 and 10 mmol/L)
`increased proliferation of hormone-stripped LNCaP (T877A-
`AR) but only spironolactone increased the proliferation of
`VCaP (WT-AR; Fig. 1C). The increase in proliferation was
`inhibited by AR antagonism, suggesting this effect was sec-
`ondary to binding to and activation of the AR (Fig. 1C).
`Similarly, eplerenone significantly increased expression of the
`androgen-regulated and clinically important genes PSA and
`TMPRSS2 in LNCaP but not in VCaP (Fig. 1D).
`
`Exogenous glucocorticoids can activate mutant AR at
`clinically relevant doses observed in CRPC patients
`treated with abiraterone acetate
`Prednisolone or its precursor prednisone are commonly
`administered in combination with abiraterone acetate
`although 2 phase II studies combined abiraterone acetate with
`dexamethasone (2, 10). Prednisone and dexamethasone do not
`activate WT-AR but activate T877A-AR with EC50 values of 25.1
`mmol/L (95% CI, 12.64–36.83 mmol/L) and 21.6 mmol/L (95% CI,
`12.53–50.26 mmol/L), respectively (Fig. 1A and B). Previous
`reports have shown that other AR mutations such as T877A in
`combination with L701H are highly sensitive to glucocorticoids
`with activation by concentrations as low as 10 nmol/L (11). We
`therefore proceeded to measure plasma levels of prednisolone
`in 15 patients with CRPC on continuous daily treatment with
`1,000 mg abiraterone acetate and 10 mg prednisolone.
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`Interaction of AR with Abiraterone and Eplerenone
`
`to confirm downregulation by qPCR of PSA and TMPRSS2 in
`LNCaP cells treated with abiraterone (Fig. 3D).
`
`Binding of abiraterone or eplerenone to the AR is
`confirmed by competitive displacement of [3H]-R1881
`To confirm that AR antagonism by abiraterone and agon-
`ism by eplerenone (both previously undescribed) occurred
`secondary to binding to the AR ligand–binding domain, we
`used a competitive radiolabeled assay to show displacement
`of R1881 from PC-3 cells transfected with either WT-AR or
`T877A-AR. The EC50 value of eplerenone for WT-AR was
`6-fold higher than T877A-AR (EC50, 2.4 mmol/l; 95% CI, 2.0–
`2.9 mmol/L; Fig. 4A and B). In keeping with the inhibitory
`activity of abiraterone observed in our reporter luciferase
`studies, abiraterone displaced ligand from both WT-AR
`(EC50, 13.4 mmol/L; 95% CI, 10.3–17.4 mmol/L) and T877A
`(EC50, 7.9 mmol/L; 95% CI, 6.7–9.3 mmol/L; Fig. 4A and B). We
`also confirmed displacement of radiolabeled R1881 from
`LNCaP with abiraterone (EC50, 2.6 mmol/L; 95% CI, 1.0–6.8
`mmol/L) and eplerenone (EC50, 4.3 mmol/L; 95% CI, 2.4–7.8
`mmol/L; Supplementary Fig. S5).
`
`Mutant AR activation by eplerenone can be inhibited by
`abiraterone or bicalutamide but most effectively by
`MDV3100
`We observed dose-proportional growth inhibition with
`abiraterone of LNCaP cells stimulated by eplerenone and of
`LNCaP and VCaP cells stimulated by spironolactone (Fig. 1C).
`Similar levels of inhibition were observed with bicalutamide,
`with more profound inhibition by MDV3100 (Fig. 1C). Abir-
`aterone, MDV3100, and bicalutamide achieved similar levels
`of
`inhibition of upregulation of PSA by eplerenone but
`MDV3100 inhibited induction of TMPRSS2 expression more
`significantly than bicalutamide or abiraterone (Fig. 3D). Sim-
`ilarly, MDV3100 showed more significant inhibition of spir-
`onolactone-stimulated PSA and TMPRSS2 expressions than
`abiraterone or bicalutamide (Supplementary Fig. S6). Also,
`abiraterone (5 mmol/L) significantly inhibited activation of
`T877A-AR (in transfected PC-3) by 1 mmol/L eplerenone but
`not by 10 mmol/L eplerenone; stimulation by 10 mmol/L
`eplerenone was significantly inhibited by both bicalutamide
`and MDV3100 (Fig. 4C).
`
`Increased hormone levels reduce AR inhibition by
`MDV3100
`Recent studies have suggested that intratumoral testoster-
`one levels increase in patients treated with MDV3100 (12). We
`found that 1mmol/L and 10 mmol/L MDV3100 significantly
`inhibited WT-AR luciferase activity stimulated by 0.1 nmol/L
`R1881 or 1 nmol/L DHT, respectively, but 50mmol/L
`MDV3100 was required to significantly inhibit AR stimulated
`by 1 nmol/L R1881 (Fig. 4D) or 10 nmol/L DHT (Supplementary
`Fig. S7).
`
`Discussion
`
`Abiraterone was developed as a specific CYP17A1 inhib-
`itor (13). Previous studies have failed to identify binding of
`
`Figure 2. Plasma concentrations (nmol/L) of prednisolone in 15 patients
`with CRPC treated with abiraterone acetate measured with liquid
`chromatography/tandem mass spectrometry. The median concentration
`of 152 nmol/L (SD, 100 nmol/L) is marked by the solid line. The 10 nmol/L
`limit above which activation of T877A-L701H-AR has been previously
`reported to occur is shown by the dashed line.
`
`Prednisolone levels were less than 4 nmol/L in 2 patients but
`more than 30 nmol/L in the other 13 patients. The median
`concentration was 153 nmol/L (range, <4–305 nmol/L; Fig. 2
`and Supplementary Table S2).
`
`Abiraterone binds and inhibits wild-type and mutant AR
`Following the observation of activation of T877A-AR
`by eplerenone, we proceeded to evaluate the effect of abirater-
`one on wild-type and mutant AR (T877A, D879G, R629Q,
`W741C, and M749L). We did not observe an increase in
`reporter luciferase activity with doses of abiraterone up to
`25 mmol/L with WT-AR or any mutation tested (Supplemen-
`tary Fig. S3) but observed dose-proportional inhibition of
`stimulated wild-type and mutant AR activity (Fig. 3A) with
`significant inhibition observed at doses 10 mmol/L. Inhibi-
`tion was however not as potent as for same concentrations of
`MDV3100. We then proceeded to confirm our findings by
`comparing inhibition of AR activation using abiraterone or
`MDV3100 in a different model system (COS-7 cells cotrans-
`fected with AR and a GRE2-TATA-luc reporter gene and
`activated by 10 nmol/L DHT for 24 hours). Similarly we
`observed dose-proportional inhibition of WT-AR, T877A-AR,
`G142V-AR, P533S-AR, T575A-AR, and H874Y-AR by abirater-
`one (Fig. 3B). Higher concentrations of abiraterone were
`required for inhibition of R629Q-AR in this system than was
`observed in PC-3 cells transfected with an ARE3-luc assay (Fig.
`3A). We also confirmed significant inhibition of proliferation of
`the AR-positive prostate cancer cell lines LNCaP and VCaP
`with doses of abiraterone 1 mmol/L (Fig. 3C). No inhibitory
`effect was observed with the AR-negative prostate cancer cell
`lines, PC-3, and DU145 (Supplementary Fig. S4). We proceeded
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`Inhibition of wild-type and mutant stimulated AR activity by abiraterone, bicalutamide, and MDV3100. A, PC-3 cells were cotransfected with ARE3-luc
`Figure 3.
`and wild-type or mutant AR (T877A, D879G, W741C, M749L, and R629Q). Cells were treated with 0.1 to 25 mmol/L abiraterone, 10 mmol/L bicalutamide, or 10
`mmol/L MDV3100 in CSS medium containing 0.1 nmol/L R1881 for 16 hours and then analyzed for luciferase activity. Fold change from the DMSO control was
`calculated and then percentage change relative to the R1881-stimulated DMSO control was determined. Data shown are representative of 3 independent
`experiments and represent mean and SEM of 8 replicates. B, COS-7 cells were cotransfected with GRE2-TATA-luc and the wild-type or mutant human
`expression plasmid pSVARo (T877A, G142V, P533S, T575A, H874Y, R629Q). Cells were treated with 0.1 to 5 mmol/L abiraterone or MDV3100 in CSS medium
`containing 10 nmol/L DHT for 24 hours. The luciferase activities were assayed in duplicate and normalized for the amounts of expressed AR determined
`immunologically by dot blot analysis and normalized for protein concentration. The change in normalized luciferase activity relative to cells incubated
`without any compound for each AR variant was determined. Data shown represent 2 or 3 independent experiments carried out in quadruplicate.
`C, dose-proportional inhibition of proliferation of LNCaP and VCaP cells by abiraterone, MDV3100, and bicalutamide. LNCaP and VCaP prostate cancer cells
`in FBS were treated with 0.1, 1, 5 or 10 mmol/L abiraterone, 0.1 or 10 mmol/L bicalutamide, or 0.1 or 10 mmol/L MDV3100 for 7 days and then analyzed
`for cell viability. Fold change from the DMSO control was then calculated and plotted. Data shown are the mean (error bars, SEM) of 3 independent experiments
`in quadruplicate. D, LNCaP cells were treated with 0.1 nmol/L R1881 or 10 mmol/L eplerenone in combination with DMSO, 10 mmol/L bicalutamide, 10 mmol/L
`MDV3100, or 5 mmol/L abiraterone for 5 hours. RNA was extracted and cDNA synthesized for analysis by qPCR to determine relative levels of PSA and
`TMPRSS2 mRNA expression. Data shown are the mean and SEM of 3 independent experiments in duplicate. Significance is shown for , P < 0.05;
` , P < 0.01; , P < 0.001; , P < 0.0001 relative to DMSO control (one-way ANOVA with the Bonferroni correction).
`
`abiraterone to the AR (14). However, in this study we used
`both reporter luciferase and competitive radiolabeled assays
`to show that abiraterone binds and inhibits WT-AR. Another
`study published while our article was under review reported
`supporting evidence that abiraterone binds the AR and
`produces a dose-dependent decrease in AR levels (15). This
`study failed to identify the EC50 value with wild-type
`or mutant AR but predicted it as over 3 mmol/L. We
`also tested 8 AR mutations selected from a screen of 42
`mutations for causing a differential response to various
`hormones. We included mutations in the amino terminal
`(G142V, P533S), DNA-binding (T575A), and ligand-binding
`(W741C, M749L, T877A, D879G, and H874Y) domains and
`
`the hinge region (R629Q; Supplementary Table S1). As
`previously
`described,
`bicalutamide
`activated W741C
`(4, 16) but no agonistic activity was observed with any
`mutation and abiraterone. Similarly MDV3100 potently
`inhibited WT-AR and all mutant AR tested. However, these
`mutations were mostly identified in patients progressing on
`bicalutamide or flutamide and different, new mutations may
`develop in patients progressing on abiraterone or MDV3100.
`Abiraterone is an active treatment for CRPC due to CYP17A1
`inhibition and significant suppression of hormones (2). How-
`ever, we observed up to 32% AR inhibition with 1 mmol/L
`abiraterone, with significantly greater inhibition at 5 and 10
`mmol/L. Pharmacokinetic studies have reported maximum
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`Interaction of AR with Abiraterone and Eplerenone
`
`Figure 4. Displacement of [3H] R1881 by eplerenone and abiraterone in PC-3 cells transfected with wild-type or T877A mutant AR. PC-3 cells were transfected
`with WT-AR (A) or T877A mutant AR (B) and then treated with CSS media containing 5 nmol/L of [3H] R1881 in combination with cold R1881, DHT,
`eplerenone, abiraterone, or bicalutamide at the concentrations shown for 2 hours. Abiraterone was insoluble in cell media at concentrations greater than 25
`mmol/L. Cell-associated radioactivity was measured and the data analyzed by nonlinear regression to determine the EC50 value for each test compound
`(GraphPad Prism). Data shown are the mean and SEM of 3 independent experiments in triplicate for percentage [3H]-R1881 bound versus log10 of
`concentration (mmol/L) of cold competitor. EC50 and 95% CI values are given. C, inhibition of eplerenone-stimulated AR activation by bicalutamide, MDV3100,
`and abiraterone. PC-3 cells were cotransfected with ARE3-luc and T877A mutant AR. Cells were treated with DMSO (control) or eplerenone in combination
`with DMSO, 10 mmol/L bicalutamide, 10 mmol/L MDV3100, or 5 mmol/L abiraterone for 16 hours and then analyzed for luciferase activity. Fold change
`from the DMSO control was calculated. Data shown are from 3 independent experiments and represent mean and SEM of 13 replicates. D, increased
`hormone levels reduce AR inhibition by MDV3100. PC-3 cells were cotransfected with ARE3-luc and WT-AR. Cells were treated with R1881 in combination
`with DMSO or MDV3100 at the concentrations indicated for 16 hours and then analyzed for luciferase activity. Fold change from the DMSO control
`was calculated. Data shown are from 3 independent experiments and represent mean and SEM of 24 replicates. , P < 0.01 relative to R1881
`or DHT control with DMSO (one-way ANOVA with the Bonferroni correction).
`
`plasma levels after a single 1,000 mg dose of abiraterone acetate
`in fasting patients of 1.2 to 5 mmol/L, confirming AR antag-
`onism could occur at clinically achievable doses (Supplemen-
`tary Table S2; refs. 2, 17). Higher doses of abiraterone up to at
`least 2,000 mg daily are safely tolerated (2) and greater activity
`could be observed with increased drug exposure despite
`complete CYP17A1 inhibition at lower doses. This could be
`achieved by administration with food (2, 17). Moreover, several
`studies of abiraterone have now reported preclinical in vitro
`and in vivo antitumor activity and inhibition of AR nuclear
`localization and AR-regulated transcription that was attribut-
`ed entirely to inhibition of steroidogenesis (18, 19) but could in
`fact be partly explained by AR antagonism. Similarly, in vitro
`inhibition of LNCaP and VCaP cells in our study could also be
`explained by abiraterone's effect on steroidogenesis.
`Significant activation of both wild-type and mutant AR is
`observed with spironolactone that should be avoided in all
`patients with CRPC. We also show activation of T877A-AR by
`
`eplerenone that was developed as a novel, non–AR-binding
`MRA (6). This could underlie clinical resistance in a proportion
`of patients. Similarly, exogenous glucocorticoids that are
`currently administered in combination with abiraterone
`reach levels in patients that have been previously shown to
`activate mutant L701H T877A AR (11). Activation of "promis-
`cuous" AR by coadministered drugs or residual hormones (as
`we reported recently ref. 20) could be inhibited by increasing
`the dose of abiraterone or possibly more effectively, combining
`with a potent antiandrogen such as MDV3100. Because of
`toxicity the dose of MDV3100 selected for phase III develop-
`ment was 160 mg daily that achieves median plasma concen-
`trations up to approximately 35 mmol/L (3, 4). AR inhibition at
`these concentrations could be overcome by an increase in
`hormones that would be prevented by combination with
`abiraterone acetate. Overall these observations provide a
`strong rationale for clinical evaluation of combined CYP17A1
`inhibition and AR antagonism.
`
`www.aacrjournals.org
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`Cancer Res; 72(9) May 1, 2012
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`Richards et al.
`
`Disclosure of Potential Conflicts of Interest
`Abiraterone acetate was developed at The Institute of Cancer Research, which
`therefore has a commercial interest in the development of this agent. J.S. de Bono
`has received consulting fees from Ortho Biotech Oncology Research and Devel-
`opment (a unit of Cougar Biotechnology), consulting fees and travel support
`from Amgen, Astellas, AstraZeneca, Boehringer Ingelheim, Bristol-Myers Squibb,
`Dendreon, Enzon, Exelixis, Genentech, GlaxoSmithKline, Medivation, Merck,
`Novartis, Pfizer, Roche, Sanofi-Aventis, Supergen, and Takeda, and grant support
`from AstraZeneca. G. Attard has received consulting fees from Janssen-Cilag,
`Veridex and Millennium Pharmaceuticals, lecture fees from Janssen-Cilag, Ipsen
`and Sanofi-Aventis, and grant support from AstraZeneca. G. Attard is on The ICR
`rewards to inventors list of abiraterone acetate. No potential conflicts of interest
`were disclosed by the other authors.
`
`Acknowledgments
`The authors thank Elaine Barrie in the Centre for Cancer Therapeutics, The
`Institute of Cancer Research, Sutton, Surrey for helpful discussions on exper-
`imental design; Elizabeth Folkerd and Mitch Dowsett in the Royal Marsden
`Hospital Academic Biochemistry Department, Fulham Road, London for assis-
`tance with radiolabeled ligand-binding studies; and Penny Flohr, Cancer Bio-
`
`markers, The Institute of Cancer Research, Sutton, Surrey for assistance with
`processing clinical samples.
`
`Grant Support
`The Institute of Cancer Research authors are employees of the Section of
`Medicine that is supported by a Cancer Research UK programme grant and
`an Experimental Cancer Medical Centre (ECMC) grant from Cancer Research
`UK and the Department of Health (ref: C51/A7401). G. Attard is also
`supported by an NIHR clinical lectureship, a Welcome Trust Starter Grant
`for Clinical Lecturers and a young investigator award from the Prostate
`Cancer Foundation, Santa Monica, CA. J. Richards and A. Wingate were
`supported by Prostate Action, London, UK. W. Arlt is in receipt of a Medical
`Research Council (MRC) UK Strategic Biomarker Grant (G0801473). C.W. Hay
`was supported by the Chief Scientist's Office, Scottish Government. The
`authors also acknowledge NHS funding to the RMH NIHR Biomedical
`Research Centre.
`
`Received December 7, 2011; revised January 30, 2012; accepted February 22,
`2012; published OnlineFirst March 12, 2012.
`
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