Competitive Androgen Receptor Antagonist Research Articles

Antiandrogenic activity of Riboflavin 5′-phosphate (FMN) in 22Rv1 and LNCaP human prostate cancer cell lines

The androgen receptor is a hormone activated transcription factor that regulates the development and maintenance of male characteristics and represents one of the most well-established drug targets, being implicated not only in prostate cancer but also in many non-cancerous human diseases including androgenetic alopecia, acne vulgaris, and hirsutism. In this study, the antiandrogenic effects of FMN were investigated in 22Rv1 and LNCaP prostate cancer cells. FMN inhibited dihydrotestosterone (DHT)-induced protein expression of androgen receptor in 22Rv1cells. In another prostate cancer LNCaP cells, FMN decreased the protein level of DHT-induced prostate specific antigen (PSA). In addition, FMN downregulated DHT-induced mRNA expression of androgen regulated genes in both cell lines, showing less prominent inhibition in 22Rv1cells where androgen receptor had been significantly decreased by FMN. FMN was found to bind androgen receptor, demonstrating that it acted as a competitive androgen receptor antagonist. FMN increased the phosphorylation of Akt in 22Rv1 cells and this increment was abrogated by PI3K inhibitor wortmannin, resulting in a rescued androgen receptor protein level which was decreased by FMN. Additionally, FMN was found to increase the mRNA and protein level of E3 ligase mouse double minute 2. Our data suggest that the androgen receptor signaling is regulated through PI3K-Akt-MDM2 pathway in 22Rv1 cells. Together, our results indicate that FMN facilitated the degradation of androgen receptor in 22Rv1 cells and inhibited the expression of androgen regulated genes by competing the binding of DHT to androgen receptor in LNCaP cells, demonstrating its therapeutic potential as an antiandrogen.

Regulation and targeting of androgen receptor nuclear localization in castration-resistant prostate cancer.

Nuclear localization of the androgen receptor (AR) is necessary for its activation as a transcription factor. Defining the mechanisms regulating AR nuclear localization in androgen-sensitive cells and how these mechanisms are dysregulated in castration-resistant prostate cancer (CRPC) cells is fundamentally important and clinically relevant. According to the classical model of AR intracellular trafficking, androgens induce AR nuclear import and androgen withdrawal causes AR nuclear export. The present study has led to an updated model that AR could be imported in the absence of androgens, ubiquitinated, and degraded in the nucleus. Androgen withdrawal caused nuclear AR degradation, but not export. In comparison with their parental androgen-sensitive LNCaP prostate cancer cells, castration-resistant C4-2 cells exhibited reduced nuclear AR polyubiquitination and increased nuclear AR level. We previously identified 3-(4-chlorophenyl)-6,7-dihydro-5H-pyrrolo[1,2-a]imidazole (CPPI) in a high-throughput screen for its inhibition of androgen-independent AR nuclear localization in CRPC cells. The current study shows that CPPI is a competitive AR antagonist capable of enhancing AR interaction with its E3 ligase MDM2 and degradation of AR in the nuclei of CRPC cells. Also, CPPI blocked androgen-independent AR nuclear import. Overall, these findings suggest the feasibility of targeting androgen-independent AR nuclear import and stabilization, two necessary steps leading to AR nuclear localization and activation in CRPC cells, with small molecule inhibitors.

High Content Screening Using New U2OS Reporter Cell Models Identifies Harmol Hydrochloride as a Selective and Competitive Antagonist of the Androgen Receptor.

Prostate cancer is the most commonly diagnosed malignancy in men. Its growth mainly relies on the activity of the androgen receptor (AR), justifying the use of androgen deprivation therapy as a gold standard treatment for the metastatic disease. Inhibition of the androgen axis using second generation antagonists has improved patients’ survival, but is systematically confronted to resistance mechanisms, leading to a median survival that does not exceed 5 years. Counteracting this resistance has been the object of a large number of investigations, with a particular emphasis towards the identification of new AR inhibitors, whether they antagonize the receptor by a competitive or a non-competitive binding. To this end, many high content screens have been performed, to identify new non-steroidal AR antagonists, using a variety of approaches, but reported somewhat controversial results, depending on the approach and on the cell model that was used for screening. In our study, we used the U2OS osteosarcoma cells stably transfected with AR or ARv7 and a luciferase reporter as a previously validated model to screen the Prestwick Phytochemical library. The results of our screen identified ellipticine, harmol, and harmine hydrochloride as confirmed hits. Surprisingly, we could demonstrate that harmol hydrochloride, previously identified as a non-competitive inhibitor of AR or a weak inhibitor of androgen signaling, was actually a competitive antagonist of AR, which inhibits the growth of VCaP prostate cancer line, at concentrations for which it did not affect the growth of the AR negative DU145 and PC3 cells. Interestingly, we also report for the first time that harmol hydrochloride was selective for AR, as it could not alter the activity of other nuclear receptors, such as the glucocorticoid receptor (GR), the progesterone receptor (PR), or the mineralocorticoid receptor (MR). Additionally, we demonstrate that, conversely to enzalutamide, harmol hydrochloride did not show any agonistic activity towards the pregnane X receptor (PXR), a master regulator of drug metabolism. Together, our results shed light on the importance of the cellular context for the screening of new AR antagonists. They further indicate that some of the potential hits that were previously identified may have been overlooked.

Intermittent bicalutamide in recurrent non-metastatic castrate sensitive prostate cancer: A single institution experience.

307 Background: Despite radical treatment, a proportion of men with prostate cancer will develop localized recurrence. Intermittent LHRH analogue (LHRHa) therapy has shown equivalence to long-term hormone manipulation with reduced toxicity. The non-steroidal competitive androgen receptor antagonist, bicalutamide, offers disease control with a further reduced toxicity profile. We report our institution’s experience of intermittent bicalutamide therapy (IBT) in recurrent non-metastatic castrate sensitive disease. Methods: Patients with biochemical recurrence and non-metastatic status on conventional imaging treated with IBT April 2016-November 2017 are reported. Previous LHRHa in the radical setting was allowed if testosterone recovery had occurred. For inclusion, one full course of IBT therapy had to be completed and treatment break commenced. Dose was 150mg with all other treatment decisions were at clinician discretion. Results: 103 men were identified with 4 were excluded due to insufficient data or failure to start treatment break. Mean age at initial diagnosis was 64.5yrs and, at commencement of IBT, 71.7. Mean overall time on IBT was 47months (20 and 27 months on and off treatment respectively). The longest recorded time on IBT was 153 months. 1 patient completed 8cycles of IBT. Mean number of cycles was 2.8 (range 1-8). Mean length of treatment break reduced with successive cycles of therapy measuring less than time on treatment after cycle 4. Of 39 patients with follow up therapies recorded, 77% received continuous bicalutamide as second line therapy. Conclusions: IBT offers a valid alternative to intermittent LHRHa therapy in recurrent non-metastatic castrate sensitive prostate cancer with the advantage of an improved toxicity profile benefiting patients. Further work to assess long-term outcomes in this patient population is warranted.[Table: see text]

Abstract P2-08-04: Phase 1/2 study of oral seviteronel (VT-464), a dual CYP17-lyase inhibitor and androgen receptor (AR) antagonist, in patients with advanced AR positive triple negative (TNBC) or estrogen receptor (ER) positive breast cancer (BC)

Abstract Background: Seviteronel (Sevi), a CYP17-lyase (L) inhibitor (reduces testosterone (T) and estradiol (E2) biosynthesis) and a competitive AR antagonist, has activity in castration resistant prostate cancer at a dose of 600mg nightly. Sevi potently inhibits the growth of ER(+)/AR(+) MCF7, tamoxifen-resistant (TAMR) MCF7, and ER(-)/AR(+) MDA-MB-453 cells. In a TAMR xenograft BC model, Sevi decreases tumor growth greater than enzalutamide (Enza), an AR antagonist (Ellison et al, SABCS 2015). Nearly all subtypes of BC, including AR(+) TNBC, are potential targets for Sevi based on its mechanism of action (MOA). Phase (Ph) 1 of this study established the recommended Ph 2 dose (RP2D) of Sevi in women with BC as 450mg once nightly, based upon preliminary tolerability and pharmacokinetics (PK) (Bardia et al, ASCO 2016). The primary objective of Ph 2 is to estimate the activity of Sevi, as measured by clinical benefit rate (CBR) at 16 and 24 weeks (wks) for AR(+) TNBC and ER(+) BC, respectively. The secondary objectives include an estimation of Sevi tolerability and pharmacodynamics (PD) (NCT02580448). Methods: Women with advanced AR(+) TNBC (stratified by prior Enza use) or ER(+) BC were enrolled using 3 parallel Simon's 2-stage designs powered to evaluate CBR. ER(+) BC patients must have had ≥1 prior line of endocrine therapy; no limit to prior treatment for TNBC. AR(+) status was confirmed using central IHC analysis in all patients, with a ≥10% tumor cell nuclear staining cutoff for evaluable TNBC patients. Sevi was administered once nightly with dinner at 450mg (28d cycle). Tumor and blood samples were collected for PK and PD analysis (circulating tumor cells, ctDNA, sex steroids). Response was assessed every 8 wks for 52 wks, then every 12 wks thereafter. Current tolerability and PD results are presented herein for this ongoing Ph 2 study. Results: As of June 7, 2016, 17 patients received Sevi at 450mg nightly between Ph1 and Ph2 with 10 in screening. 14 patients are currently on study in Cycles 1-6. The most common adverse events (AEs > 10% regardless of causality or grade) were tremor (24%), pain (18%), fatigue (18%) and dyspnea (18%), nausea (12%), AST increase (12%), ALT increase (12%) and abdominal pain (12%), all of which were Grade 1 or 2 except for Grade 3 dyspnea (n=1; unrelated). No dose reductions were reported and there were no drug-related discontinuations. Nine patients underwent central AR testing (4 AR(+) of 6 TNBC; 3 AR(+) of 3 ER(+) BC). Median AR tumor cell nuclear staining was 90% (15-100%). Preliminary sex steroid analyses from 6 Ph 1 patients receiving Sevi at 450, 600, or 750mg nightly (n=2 at each dose) for 1 cycle showed a median decline in E2 concentration of 52% (-29 to -87%) to 12.4pmol/L (4 to 33pmol/L) from baseline. There was a similar magnitude of decline for T. Conclusions: Sevi was well-tolerated at 450mg nightly with exposures similar to the RP2D in men (600mg nightly). The CYP17-L inhibition activity of Sevi was demonstrated with an early and potent reduction in E2 and T. Sevi's unique CYP17-L and AR antagonist MOA may provide a new novel treatment option for AR(+) TNBC or ER(+) BC. Citation Format: Gucalp A, Bardia A, Gabrail N, DaCosta N, Danso M, Elias AD, Ali H, Lemon SJ, Riley EC, Eisner JR, Fleming RA, Kurman MR, Moore WR, Traina TA. Phase 1/2 study of oral seviteronel (VT-464), a dual CYP17-lyase inhibitor and androgen receptor (AR) antagonist, in patients with advanced AR positive triple negative (TNBC) or estrogen receptor (ER) positive breast cancer (BC) [abstract]. In: Proceedings of the 2016 San Antonio Breast Cancer Symposium; 2016 Dec 6-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2017;77(4 Suppl):Abstract nr P2-08-04.

Design and synthesis of indoline thiohydantoin derivatives based on enzalutamide as antiproliferative agents against prostate cancer

A novel scaffold of indoline thiohydantoin was discovered as potent androgen receptor (AR) antagonist through rational drug designation. Several compounds showed good biological profiles in AR binding and higher selective toxicity than enzalutamide toward LNCaP cells (AR-rich) versus DU145 cells (AR-deficient). In addition, the docking studies supported the rationalization of the biological evaluation. Among these compounds, the representative compound 48c exhibited the strongest inhibitory effect on LNCaP growth and also acted as a competitive AR antagonist. Further preliminary mechanism study confirmed that 48c exerted its AR antagonistic activity through impairing AR nuclear translocation. All these results indicated that the novel scaffold compounds demonstrated AR antagonistic behavior and promising candidates for future development were identified.

Design and synthesis of novel androgen receptor antagonists via molecular modeling

Several androgen receptor (AR) antagonists are clinically prescribed to treat prostate cancer. Unfortunately, many patients become resistant to the existing AR antagonists. To overcome this, a novel AR antagonist candidate called DIMN was discovered by our research group in 2013. In order to develop compounds with improved potency, we designed novel DIMN derivatives based on a docking study and substituted carbons with heteroatom moieties. Encouraging in vitro results for compounds 1b, 1c, 1e, 3c, and 4c proved that the new design was successful. Among the newly synthesized compounds, 1e exhibited the strongest inhibitory effect on LNCaP cell growth (IC50=0.35μM) and also acted as a competitive AR antagonist with selectivity over the estrogen receptor (ER) and the glucocorticoid receptor (GR). A docking study of compound 1e fully supported these biological results. Compound 1e is considered to be a novel, potent and AR-specific antagonist for treating prostate cancer. Thus, our study successfully applied molecular modeling and bioisosteric replacement for hit optimization. The methods here provide a guide for future development of drug candidates through structure-based drug discovery and chemical modifications.

Sex Hormone Influence on Hepatitis in Young Male A/JCr Mice Infected withHelicobacter hepaticus

Hepatitis B virus (HBV), the leading cause of human hepatocellular carcinoma, is especially virulent in males infected at an early age. Likewise, the murine liver carcinogen Helicobacter hepaticus is most pathogenic in male mice infected before puberty. We used this model to investigate the influence of male sex hormone signaling on infectious hepatitis. Male A/JCr mice were infected with H. hepaticus or vehicle at 4 weeks and randomized into surgical and pharmacologic treatment groups. Interruption of androgen pathways was confirmed by hormone measurements, histopathology, and liver gene and Cyp4a protein expression. Castrated males and those receiving the competitive androgen receptor antagonist flutamide had significantly less severe hepatitis as determined by histologic activity index than intact controls at 4 months. Importantly, the powerful androgen receptor agonist dihydrotestosterone did not promote hepatitis. No effect on hepatitis was evident in males treated with the 5alpha-reductase inhibitor dutasteride, the peroxisome proliferator-activated receptor-alpha agonist bezafibrate, or the nonsteroidal anti-inflammatory drug flufenamic acid. Consistent with previous observations of hepatitis-associated liver-gender disruption, transcriptional alterations involved both feminine (cytochrome P450 4a14) and masculine (cytochrome P450 4a12 and trefoil factor 3) genes, as well gender-neutral (H19 fetal liver mRNA, lipocalin 2, and ubiquitin D) genes. Hepatitis was associated with increased unsaturated C(18) long-chain fatty acids (oleic acid and linoleic acid) relative to saturated stearic acid. Our results indicate that certain forms of androgen interruption can inhibit H. hepaticus-induced hepatitis in young male mice, whereas androgen receptor agonism does not worsen disease. This raises the possibility of targeted hormonal therapy in young male patients with childhood-acquired HBV.

Androgen manipulation alters oxidative DNA adduct levels in androgen-sensitive prostate cancer cells grown in vitro and in vivo

Intracellular reactive oxygen species (ROS) may cause oxidative DNA damage, resulting in the formation of adducts such as 8-oxo-7,8-dihydro-2′-deoxyguanosine (8-oxo-dG) and the cyclic pyrimidopurinone N-1, N 2 malondialdehyde-2′-deoxyguanosine (M 1dG). These adducts have been associated with carcinogenesis, genomic instability and clonal evolution. We tested two hypotheses in human prostate cancer cells grown in vitro and in a xenograft model: (1) treatment of androgen-sensitive cells with DHT increases levels of oxidative DNA adduct levels; (2) flutamide, a competitive androgen receptor antagonist, prevents DHT-induced changes. Levels of M 1dG and 8-oxo-dG adducts were determined by immunoslot blot and liquid chromatography–tandem mass spectrometry. M 1dG and 8-oxo-dG levels were significantly higher than control levels in LNCaP cells exposed to supra-physiological concentrations (25–100 nM) of DHT (both P < 0.05 by ANOVA). Flutamide pre-treatment completely prevented this increase. In the xenograft model, tumour levels of M 1dG were decreased by 46% ( P = 0.001 by Mann–Whitney Test) in flutamide-treated animals compared to controls. The changes demonstrated suggest that oxidative DNA adducts may serve as biomarkers of the efficacy of androgen manipulation in chemoprevention trials.

Preliminary investigation of changes in the sexually dimorphic nucleus of the rat medial preoptic area following prenatal exposure to fenitrothion

In vitro, the organophosphate insecticide fenitrothion is a potent competitive androgen receptor antagonist, whereas in vivo it affects the development of the male rat reproductive system. The purpose of this pilot study was to determine whether prenatal exposure to fenitrothion affects development of the rat sexually dimorphic nucleus of the medial preoptic area (SDN-POA). Pregnant rats (n = 5-6 litters/group) were orally dosed with corn oil (vehicle) or fenitrothion (20 or 25 mg kg(-1) day(-1)) from gestation day (GD) 12-21. Offspring were euthanized after reaching sexual maturity (females 60-65 days old and males 96-105 days old) and the SDN-POA volumes determined for two rats/sex/litter. Tremors, increased lacrimation and decreased body weight gain were observed in dams from both fenitrothion exposure groups. Reproductive effects in male offspring, including reduced anogenital distance on postnatal day (PND) 1 and increased retention of areolae (PND 13) were observed following fenitrothion exposure at these dose levels. These effects did not persist into adulthood. There was a dose-related increase in the SDN-POA volume in males and a dose-related decrease in SDN-POA volume in females exposed to fenitrothion. These SDN-POA volume changes contrast with those seen with flutamide, another potent anti-androgen, and suggest that fenitrothion may have mixed endocrine effects on the developing brain.

Spotlight on Bicalutamide 150mg in the Treatment of Locally Advanced Prostate Cancer1

Bicalutamide (Casodex) is a competitive androgen receptor antagonist that inactivates androgen-regulated prostate cell growth and function, leading to cell apoptosis and inhibition of prostate cancer growth. It is administered orally as a once-daily dose. In the EU and a number of other countries, bicalutamide 150 mg/day is approved in men with locally advanced nonmetastatic prostate cancer as immediate therapy either as an adjuvant to active treatment or as monotherapy as an alternative to surgical or medical castration. Combined analysis of the three trials that comprise the bicalutamide Early Prostate Cancer programme showed that bicalutamide administered in conjunction with standard care in men with locally advanced prostate cancer offers disease-free survival benefits over standard care alone and is generally well tolerated. Overall survival was improved to a greater extent in the subgroup of patients who received bicalutamide plus radiation therapy compared with radiation therapy alone. Men with localised prostate cancer do not benefit from the addition of bicalutamide to standard care. Combined analysis of two other studies in men with locally advanced prostate cancer show that bicalutamide monotherapy offers better tolerability and higher health-related quality-of-life scores for sexual interest and physical capacity compared with surgical or medical castration, while achieving disease-free and overall survival durations that were not significantly different. Thus, when treatment options are being evaluated, bicalutamide as adjuvant therapy or monotherapy should be considered as an alternative to other available hormonal therapies in men with locally advanced prostate cancer, especially in those who wish to maintain an active lifestyle.

Bicalutamide 150mg

Bicalutamide (Casodex) is a competitive androgen receptor antagonist that inactivates androgen-regulated prostate cell growth and function, leading to cell apoptosis and inhibition of prostate cancer growth. It is administered orally as a once-daily dose. In the EU and a number of other countries, bicalutamide 150 mg/day is approved in men with locally advanced nonmetastatic prostate cancer as immediate therapy either as an adjuvant to active treatment or as monotherapy as an alternative to surgical or medical castration. Combined analysis of the three trials that comprise the bicalutamide Early Prostate Cancer (EPC) programme showed that bicalutamide administered in conjunction with standard care in men with locally advanced prostate cancer offers disease-free survival benefits over standard care alone and is generally well tolerated. Overall survival was improved to a greater extent in the subgroup of patients who received bicalutamide plus radiation therapy compared with radiation therapy alone. Men with localised prostate cancer do not benefit from the addition of bicalutamide to standard care. Combined analysis of two other studies in men with locally advanced prostate cancer show that bicalutamide monotherapy offers better tolerability and higher health-related quality-of-life (HR-QOL) scores for sexual interest and physical capacity compared with surgical or medical castration, while achieving disease-free and overall survival durations that were not significantly different. Thus, when treatment options are being evaluated, bicalutamide as adjuvant therapy or monotherapy should be considered as an alternative to other available hormonal therapies in men with locally advanced prostate cancer, especially in those who wish to maintain an active lifestyle.

Evidence that inhibited prostatic epithelial bud formation in 2,3,7,8-tetrachlorodibenzo-p-dioxin-exposed C57BL/6J fetal mice is not due to interruption of androgen signaling in the urogenital sinus.

2,3,7,8-Tetrachlorodibenzo-p-dioxin (TCDD) inhibits the androgen-dependent processes by which the urogenital sinus (UGS) of fetal mice forms prostatic epithelial buds. This inhibition is mediated by aryl hydrocarbon receptors in UGS mesenchyme and causes prostate lobes to develop abnormally. Experiments were conducted to test the hypothesis that TCDD inhibits prostatic budding in C57BL/6J mice by inhibiting androgen signaling. In utero TCDD exposure sufficient to inhibit budding (5 microg/kg maternal dose on gestation day [GD] 13) had no effect on testicular testosterone content on GD 16 or 18. Nor did it inhibit the conversion of testosterone to 5alpha-dihydrotestosterone (DHT) by the UGS. Both hydroxyflutamide (OH-flutamide; a competitive androgen receptor antagonist) and TCDD inhibited prostatic epithelial budding by UGSs cultured in vitro with DHT. To determine if TCDD inhibits responsiveness to androgens, primary mesenchymal cells prepared from UGSs cultured for three days with DHT were transiently transfected with an androgen-responsive reporter plasmid (MMTV-luciferase). OH-flutamide prevented DHT from increasing luciferase activity in these cells but TCDD did not. The same results were obtained when the mesenchymal cells were isolated from UGSs cultured with both DHT and TCDD. The lack of effect of TCDD on androgen-dependent gene expression was not due to inability of transfected UGS mesenchymal cells to respond to TCDD, as shown by significant increases in luciferase activity after transfection with plasmids containing CYP1A1 and CYP1B1 promoters. Finally, while OH-flutamide prevented DHT from altering androgen receptor and 5alpha-reductase type II mRNA expression in UGS organ culture, TCDD had no such effects. Collectively, these results suggest that TCDD inhibits prostatic epithelial bud formation without impairing the androgen receptor signaling pathway.

Androgen receptor antagonism by the organophosphate insecticide fenitrothion.

Organophosphate insecticides represent one of the most widely used classes of pesticides with high potential for human exposure in both rural and residential environments. We investigated the interaction of the organophosphothioate pesticide fenitrothion (O,O-dimethyl O-(4-nitro-m-tolyl) phosphorothioate) with the human androgen receptor (AR). Fenitrothion blocked dihydrotestosterone-dependent AR activity in a concentration-dependent and competitive manner in HepG2 human hepatoma liver cells transiently transfected with human AR and an AR-dependent luciferase reporter gene. Schild regression analysis yielded an equilibrium dissociation constant value of 2.18 x 10(-8) M. To determine the antiandrogenic potential of fenitrothion in vivo, 7-week-old castrated Sprague-Dawley rats were dosed once a day for 7 days with testosterone propionate (50 microg/day, sc) plus gavage doses of either corn oil vehicle or fenitrothion (15 or 30 mg/kg/day). An additional group of rats was given testosterone propionate and flutamide (50 mg/kg/day). Motor activity and acetylcholinesterase activity in whole blood and brain were also assessed. Both fenitrothion and the reference antiandrogen flutamide caused significant decreases in the ventral prostate, seminal vesicle, and levator ani plus bulbocavernosus muscles tissue weights. In contrast, blood acetylcholinesterase activity, a standard biomarker of organophosphate poisoning, was only inhibited at the higher dose of fenitrothion (30 mg/kg). Our results demonstrate that fenitrothion is a competitive AR antagonist, comparable in potency to the pharmaceutical antiandrogen flutamide and more potent, based on in vitro assays, than the known environmental antiandrogens linuron and p,p'-, 2,2-bis(p-hydroxyphenyl)-1,1-dichloroethylene ( p,p'-DDE).