Abstract A028: In vitro and in vivo efficacy of the androgen receptor antagonist darolutamide in prostate cancer models
Authors: Sugawara Tatsuo , Baumgart Simon J. , Reichert Kristin , Lejeune Pascale , Haendler Bernard 
Published: February 2019

Abstract
Darolutamide is a novel, high-affinity androgen receptor (AR) antagonist currently in clinical phase 3 studies for nonmetastatic castration-resistant prostate cancer and metastatic hormone-sensitive prostate cancer. Its efficacy and mode of action were analyzed in detail using two different prostate cancer models.
In vitro efficacy was determined by measuring cell proliferation with alamarBlue® and spheroid formation with the ImageJ program, following treatment of single-cell suspensions in a 3D culture model. Gene expression was determined with RT2 profiler arrays and by real-time PCR, AR protein levels by ELISA and AR or RNA polymerase II binding to DNA by ChIP-qPCR. In vivo efficacy studies were performed following subcutaneous injection of LAPC-4 cells into noncastrated, testosterone-supplemented SCID male mice. Treatment started on day 26 post tumor inoculation at a mean tumor range of 137-150 mm3. Tumor size was measured with calipers and converted in volume using the formula: (length x width2)/2. For gene expression analysis, RNA was extracted from tumors using the RNeasy® Plus Mini Kit.
Treatment of androgen-stimulated VCaP or LAPC-4 prostate cancer cells with darolutamide, diastereomers (S,R)- and (S,S)-darolutamide and the main circulating metabolite keto-darolutamide led to a superior inhibition of proliferation, demonstrating a continuous high pressure on AR. A strong reduction of spheroid formation and dose-dependent reduction of androgen-stabilized AR protein levels were observed in both cell lines. There was marked downregulation in expression of androgen target genes such as KLK3/PSA, TMPRSS2, and FKBP5 with reduced binding of the AR to the KLK3/PSA upstream regulatory region. Furthermore, we observed a reduction of RNA polymerase II recruitment. In vivo efficacy studies revealed a significant reduction of tumor growth when darolutamide was dosed orally, daily or twice daily, compared to castrated, untreated mice. At day 56, the ΔT/C was 25% for the 100 mg/kg bi-daily dose and 23% for the 200 mg/kg once-daily dose. All the treatment schedules were well tolerated, as indicated by the limited body weight loss observed (-3% maximal mean body weight loss). Gene expression analysis of the tumors at the end of treatment showed a 50% reduction of FKBP5 transcript levels for the highest treatment doses.
In conclusion, darolutamide shows a consistent high pressure on AR. Significant antitumor efficacy was observed in vitro and in vivo in prostate cancer models. RNA-seq and ChIP-seq studies are currently ongoing for the determination of the complete transcription profiles and AR binding pattern of treated tumor models. Altogether, these data further support the evaluation of darolutamide in clinical studies for prostate cancer.
Conflict of Interest: All authors are employees and/or shareholders of Bayer. Darolutamide is jointly developed by Bayer and Orion.
Citation Format: Tatsuo Sugawara, Simon J. Baumgart, Kristin Reichert, Pascale Lejeune, Bernard Haendler. In vitro and in vivo efficacy of the androgen receptor antagonist darolutamide in prostate cancer models [abstract]. In: Proceedings of the AACR Special Conference: Prostate Cancer: Advances in Basic, Translational, and Clinical Research; 2017 Dec 2-5; Orlando, Florida. Philadelphia (PA): AACR; Cancer Res 2018;78(16 Suppl):Abstract nr A028.