Abstract 3893: Melatonin-tamoxifen drug conjugates: Mechanism against breast cancer cells, and pharmacokinetic assessment in female mice
Authors: Hasan Mahmud , Wright Thomas D. , Adhikari Saugat , Marzouk Mohamed A. , Pericoloso Alaina , Murgas Kelsey , Burgman Miranda , Miller Benton P. , Holzgrabe Ulrike , Zlotos Darius P. , Stratford Robert E. , Witt-Enderby Paula A. 
Published: August 2019

Abstract
Background: Melatonin-tamoxifen drug conjugate linked with a 5-carbon chain (C5) exhibited uterus protection in female mice, which did not occur when the same doses of unlinked melatonin and tamoxifen were co-administered. Anti-cancer actions of C5 and 4 others, with varying carbon lengths (C2, C4, C9, C15), were further tested in phenotypically diverse breast cancer (BC) cell lines—MCF-7(ER+/PR+), MMC(HER2+) and triple negatives(MDA-MB-231 and BT-549) in terms of cell viability and migration. In all BC lines, C4 and C5 exhibited superior potency and efficacy compared to C2, C9, C15 and unlinked melatonin and tamoxifen or 4-OH-tamoxifen controls. In this study, C4 and C5 were further tested for their anti-cancer effects in tamoxifen-resistant MCF-7 cells(TamR-MCF-7); their pharmacokinetic parameters were assessed in vitro and in vivo; and the mechanisms underlying C4- and C5-mediated anti-cancer actions were also determined by use of inhibitors against MEK1/2, MEK5 or PI3 kinase and western blot analysis.
Results: C4 and C5 exhibited similar potency (TamR-MCF-7 IC50 = 4.22µM and 7.21µM; MCF-7 IC50 = 3.62nM and 440mM) and efficacy (TamR-MCF-7 = 83% and 81% inhibition; MCF-7= 71% and 90% inhibition) to inhibit TamR-MCF-7 cell viability as they did in MCF-7 cells. Regarding metabolism, C4 and C5 displayed similar CYP-mediated pharmacokinetic profiles as tamoxifen in both mouse and human liver microsomes where 40% loss of tamoxifen, C4 or C5 occurred by 10 min in mouse vs. 20 min in human. In vivo, the Tmax for C5 (oral) was 24h compared to 0.5h for C4 (oral) resulting in lower Cmax (8.9 ng/mL) and AUC0-24 (1.6 hr.ng/mL) for C5 compared to C4 (Cmax =102.6 ng/mL and AUC0-24 =143.7 hr.ng/mL) and lower oral bioavailability (0.11%) for C5 compared to C4 (4.52%). Regarding mechanisms, the effect of C4- or C5-mediated protein modulation was “context-specific” dependent upon BC phenotype (i.e., ER+, HER2+ or ER-/PR-/HER2-); the number of carbons linking melatonin to tamoxifen (i.e., 4 carbons for C4 or 5 carbons for C5); and the concentration (i.e., 1nM or 10µM) of C4 or C5 tested. For MCF-7, MMC and MDA-
MB-231 cells, inhibition of MEK1/2, MEK5 and PI3K enhanced but did not block C4’s or C5’s anti-cancer actions; while for BT-549 cells, Bix02189 blocked C5’s effects. Also, in MCF-7 cells, pERK1/2, NF-κB and Runx2 were significantly modulated by C4 or C5; for MMCs, C4 and C5 modulated NF-κB and β1-integrin expression; for MDA-MB-231, MEK1/2 and MEK5 played a more central role possibly through co-modulation of NF-κB; and for BT-549, C5-mediated effects on pERK5 or PI3K-dependent regulation of pERK1/2 may underlie its anti-cancer actions.
Conclusions: Melatonin-tamoxifen drug conjugates (C4 and C5) may be superior to tamoxifen therapy alone in a variety of cancers, including TamR BC.
Citation Format: Mahmud Hasan, Thomas D. Wright, Saugat Adhikari, Mohamed A. Marzouk, Alaina Pericoloso, Kelsey Murgas, Miranda Burgman, Benton P. Miller, Ulrike Holzgrabe, Darius P. Zlotos, Robert E. Stratford, Paula A. Witt-Enderby. Melatonin-tamoxifen drug conjugates: Mechanism against breast cancer cells, and pharmacokinetic assessment in female mice [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 3893.