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In solid tumors like head and neck cancer (HNC), chronic and acute hypoxia have serious adverse clinical consequences including poorer overall patient prognosis, enhanced metastasis, increased genomic instability, and resistance to radiation-, chemo-, or immuno-therapies. However, cells in the two-dimensional monolayer cultures typically used for cancer drug discovery experience 20%-21% O2 levels (normoxic) which are 4-fold higher than O2 levels in normal tissues and ≥10-fold higher than in the hypoxic regions of solid tumors. The oxygen electrodes, exogenous bio-reductive markers, and increased expression of endogenous hypoxia-regulated proteins like HIF-1α generally used to mark hypoxic regions in solid tumors are impractical in large sample numbers and longitudinal studies. We used a novel homogeneous live-cell permeant HypoxiTRAK™ (HPTK) molecular probe compatible with high content imaging detection, analysis, and throughput to identify and quantify hypoxia levels in live HNC multicellular tumor spheroid (MCTS) cultures over time. Accumulation of fluorescence HPTK metabolite in live normoxic HNC MCTS cultures correlated with hypoxia detection by both pimonidazole and HIF-1α staining. In HNC MCTSs, hypoxic cytotoxicity ratios for the hypoxia activated prodrugs (HAP) evofosfamide and tirapazamine were much smaller than have been reported for uniformly hypoxic 2D monolayers in gas chambers, and many viable cells remained after HAP exposure. Cells in solid tumors and MCTSs experience three distinct O2 microenvironments dictated by their distances from blood vessels or MCTS surfaces, respectively; oxic, hypoxic, or intermediate levels of hypoxia. These studies support the application of more physiologically relevant in vitro 3D models that recapitulate the heterogeneous microenvironments of solid tumors for preclinical cancer drug discovery.

Tumor hypoxia contributes to resistance to anticancer therapies. Hypoxia-activated prodrugs (HAPs) selectively target hypoxic cells and their activity can extend to well-oxygenated areas of tumors via diffusion of active metabolites. This type of bystander effect has been suggested to be responsible for the single agent activity of the clinical-stage HAP evofosfamide (TH-302) but direct evidence is lacking. To dissect the contribution of bystander effects to TH-302 activity, we implemented a Green's function pharmacokinetic (PK) model to simulate the spatial distribution of O2, TH-302 and its cytotoxic metabolites, bromo-isophosphoramide mustard (Br-IPM) and its dichloro derivative isophosphoramide mustard (IPM), in two digitized tumor microvascular networks. The model was parameterized from literature and experimentally, including measurement of diffusion coefficients of TH-302 and its metabolites in multicellular layer cultures. The latter studies demonstrate that Br-IPM and IPM cannot diffuse significantly from the cells in which they are generated, although evidence was obtained for diffusion of the hydroxylamine metabolite of TH-302. The spatially resolved PK model was linked to a pharmacodynamic (PD) model that describes cell killing probability at each point in the tumor microregion as a function of Br-IPM and IPM exposure. The resulting PK/PD model accurately predicted previously reported monotherapy activity of TH-302 in H460 tumors, without invoking a bystander effect, demonstrating that the notable single agent activity of TH-302 in tumors can be accounted for by significant bioreductive activation of TH-302 even in oxic regions, driven by the high plasma concentrations achievable with this well-tolerated prodrug.

Hypoxic cells pose a problem in anticancer chemotherapy, in which often drugs require oxygen as an electron acceptor to bring about the death of actively cycling cells. Bioreductive anticancer drugs, which are selectively activated in the hypoxic regions of tumours through enzymatic one-electron reduction, are being developed for combination with chemotherapy-, radiotherapy- and immunotherapy-containing regimens to kill treatment-resistant hypoxic cells. The most clinically-advanced bioreductive drug, evofosfamide (TH-302), which acts by releasing a DNA-crosslinking mustard, failed to extend overall survival in combination with doxorubicin, a topoisomerase II inhibitor, for advanced soft tissue sarcoma in a pivotal clinical trial. However, the reasons for the lack of additive efficacy with this combination are unknown. Here, we show that the radical anion of evofosfamide undergoes electron transfer to doxorubicin in kinetic competition to fragmentation of the radical anion, thus suppressing the release the cytotoxic mustard. This electron transfer process may account, at least in part, for the lack of overall survival improvement in the recent clinical trial. This study underlines the need to consider both redox and electron transfer chemistry when combining bioreductive prodrugs with other redox-active drugs in cancer treatment.

Background Sunitinib (SUN) is approved for the treatment of advanced progressive pNETs. Hypoxia induced by SUN could foster the activation of the prodrug evofosfamide (EVO), designed to release the DNA alkylator bromo-isophosphoramide mustard under hypoxic conditions. The SUNEVO trial seeks to exploit the potential synergy of SUN + EVO in advanced pNETs. Methods Phase-II, single-arm, multicenter trial that included 17 patients (pts) with metastatic G1/G2 pNETS naive for systemic treatment (tx) other than somatostatin analogues (SSA). Tx consisted on EVO 340 mg/m2 on days 8, 15 and 22 every 4 weeks (q4w), and sunitinib 37.5 mg/day continuously. Objective response rate (ORR) was selected as the primary endpoint and evaluated q8w by RECIST 1.1. Results Median age was 62.4 years, 41.2% had prior SSA, 47% had a Ki-67 >10%, 70.6% had liver involvement. Patients received a median of 5 (1-21) and 4 (0-21) cycles of SUN and EVO, respectively. After a median follow-up time of 15.7 months (m), 23.5% achieved a partial (N = 3) or complete response (N = 1), 11 pts had stable disease (64.7%) and 1 pt (5.3%) had progressive disease as best response. Median time to response was 1.9m (1.4-8.8) and duration of response was 18.5 m (4.2-38.3). Median PFS was 10.3 m (2.6-18.0). Treatment-related adverse events of >G3 or were observed in 52.9% pts, being the most frequent being neutropenia (18.8%), hypertension (12.5%), ALT increase (12.5%), thrombopenia (6.3%), and fatigue (6.3%). 4 pts (26.7%) discontinued at least one drug due toxicity. Conclusions SUN + EVO is active in pts with advanced pNETs, but the toxicity profile of the combination negatively affects tolerability. Biomarker analyses to select patients most likely to benefit from this therapy are ongoing. Clinical trial identification EUDRACT Number: 2014-004072-30 Approval date for the trial: 22-January-2015. Editorial acknowledgement Mfar Clinical Research S.L. Legal entity responsible for the study Grupo Espanol de Tumores Neuroendocrinos y Endocrinos (GETNE). Funding Molecular Templates Inc. and Pfizer Inc. Disclosure E. Grande: Speaker Bureau / Expert testimony, Public speaking and expert: Pfizer, Ipsen, BMS, Eisai, Roche, MSD, Sanofi-Genzyme, Adacap, Novartis, Eusa Pharma, Pierre Fabre, Lexicon, Celgene; Leadership role, Principal Investigator: AstraZeneca, Pfizer, Ipsen, Mtem/Threshold Lexicon; Honoraria (institution), Medical Education Grant: MSD, Roche; Non-remunerated activity/ies, Advasory Board Member: Enets; Non-remunerated activity/ies, Directory Board Member: Getne; Non-remunerated activity/ies, Directory Board Member: Gethi. C. Lopez: Honoraria (self): Pfizer, Ipsen, Roche; Advisory / Consultancy: Pfizer, Ipsen, Roche; Research grant / Funding (self): Pfizer, Ipsen, Roche; Travel / Accommodation / Expenses: Pfizer, Ipsen, Roche; Honoraria (self): Novartis. T. Alonso Gordoa: Research grant / Funding (self): Roche; Travel / Accommodation / Expenses: Sanofi, Pfizer. J. Capdevila: Advisory / Consultancy: Eisai, Bayer, Exelixis, Novartis, Pfizer, Adacap, Merck Serono; Speaker Bureau / Expert testimony: Eisai, Bayer, Exelis, Ipsen, Novartis, Pfizer, Adacap, Merck Serono; Research grant / Funding (self): Eisai, Exelixis, Adacap, AstraZeneca, Novartis; Travel / Accommodation / Expenses: Ipsen, Pfizer, Eisai. A. Teule: Advisory / Consultancy: Pfizer, Ipsen, Novartis. I. Sevilla: Advisory / Consultancy: Ipsen, Novartis, Pfeizer, Lilly; Travel / Accommodation / Expenses: Ipsen. P. Gajate: Advisory / Consultancy: IPSSEN; Speaker Bureau / Expert testimony: Ipsen; Travel / Accommodation / Expenses: Ipsen. J. Molina-Cerrillo: Speaker Bureau / Expert testimony: Ipsen, Janssen. J. Hernando Cubero: Speaker Bureau / Expert testimony: Eisai, Ipsen, Roche, Angelini Pharma; Travel / Accommodation / Expenses: Ipsen, Novartis, Advanced Accekeratir Applications, Roche, AstraZeneca, Eisai. R. Garcia-Carbonero: Honoraria (self): Ipsen, Roche, Sanofi, BMS, Servier, Novartis, Pfizer, Merck, PharmaMar, Adacap; Honoraria (self), An intermediate Family Member: Merck, PharmaMar, Roche, BMS, AZ, Lilly, Boerhinger, Gilead Sc, Servier, Sysmex; Advisory / Consultancy: Ipsen, Novartis, Pfizer, AAA; Speaker Bureau / Expert testimony: Ipsen, Pfizer; Research grant / Funding (institution): Pfizer, BMS, MSD, AstraZeneca; Travel / Accommodation / Expenses: Roche, Merck. All other authors have declared no conflicts of interest.