Continued 3-day hypoxia does not increase cytotoxicity of evofosfamide compared to overnight hypoxia. A panel of two NBL cell lines (CHLA-20 and SK-N-BE(2)) and two rhabdomyosarcoma cell line (RH4 and RD) were selected to assess the effects of evofosfamide on cell proliferation in vitro. Cell viability was measured by Alamar Blue assays after exposing tumor cells to increasing concentrations of evofosfamide in vitro for 72 hours under normoxia, 1% O2 for 2 hours, 1% O2 overnight, or 1% O2 for three days. Dose-response curves were fit to the data by GraphPad Prism software.
Funding
Threshold Pharmaceuticals, Merck-Serono, James Birrell Neuroblastoma Research Fund and CJ Memorial Fund/Sick Kids Foundation
ARTICLE ABSTRACT
Purpose: Tumor cells residing in tumor hypoxic zones are a major cause of drug resistance and tumor relapse. In this study, we investigated the efficacy of evofosfamide, a hypoxia-activated prodrug, and its combination with topotecan in neuroblastoma and rhabdomyosarcoma preclinical models.Experimental Design: Neuroblastoma and rhabdomyosarcoma cells were tested in vitro to assess the effect of evofosfamide on cell proliferation, both as a single agent and in combination with topotecan. In vivo antitumor activity was evaluated in different xenograft models. Animal survival was studied with the neuroblastoma metastatic tumor model.Results: All tested cell lines showed response to evofosfamide under normoxic conditions, but when exposed to hypoxia overnight, a 2- to 65-fold decrease of IC50 was observed. Adding topotecan to the evofosfamide treatment significantly increased cytotoxicity in vitro. In neuroblastoma xenograft models, single-agent evofosfamide treatment delayed tumor growth. Complete tumor regression was observed in the combined topotecan/evofosfamide treatment group after 2-week treatment. Combined treatment also improved survival in a neuroblastoma metastatic model, compared to single-agent treatments. In rhabdomyosarcoma xenograft models, combined treatment was more effective than single agents. We also showed that evofosfamide mostly targeted tumor cells within hypoxic regions while topotecan was more effective to tumor cells in normoxic regions. Combined treatment induced tumor cell apoptosis in both normoxic and hypoxic regions.Conclusions: Evofosfamide shows antitumor effects in neuroblastoma and rhabdomyosarcoma xenografts. Compared with single-agent, evofosfamide/topotecan, combined therapy improves tumor response, delays tumor relapse, and enhances animal survival in preclinical tumor models. Clin Cancer Res; 22(11); 2697–708. ©2015 AACR.
