Bioreductive prodrug PR-104 improves the tumour distribution and titre of the nitroreductase-armed oncolytic adenovirus ONYX-411NTR leading to therapeutic benefit.

Advances in the field of cancer immunotherapy have stimulated renewed interest in adenoviruses as oncolytic agents. Clinical experience has shown that oncolytic adenoviruses are safe and well tolerated but possess modest single-agent activity. One approach to improve the potency of oncolytic viruses is to utilise their tumour selectivity to deliver genes encoding prodrug-activating enzymes. These enzymes can convert prodrugs into cytotoxic species within the tumour; however, these cytotoxins can interfere with viral replication and limit utility. In this work, we evaluated the activity of a nitroreductase (NTR)-armed oncolytic adenovirus ONYX-411NTR in combination with the clinically tested bioreductive prodrug PR-104. Both NTR-expressing cells in vitro and xenografts containing a minor population of NTR-expressing cells were highly sensitive to PR-104. Pharmacologically relevant prodrug exposures did not interfere with ONYX-411NTR replication in vitro. In vivo, prodrug administration increased virus titre and improved virus distribution within tumour xenografts. Colonisation of tumours with high ONYX-411NTR titre resulted in NTR expression and prodrug activation. The combination of ONYX-411NTR with PR-104 was efficacious against HCT116 xenografts, whilst neither prodrug nor virus were active as single agents. This work highlights the potential for future clinical development of NTR-armed oncolytic viruses in combination with bioreductive prodrugs.

Design, Synthesis and In-Vitro Biological Evaluation of Antofine and Tylophorine Prodrugs as Hypoxia-Targeted Anticancer Agents.

Phenanthroindolizidines, such as antofine and tylophorine, are a family of natural alkaloids isolated from different species of Asclepiadaceas. They are characterized by interesting biological activities, such as pronounced cytotoxicity against different human cancerous cell lines, including multidrug-resistant examples. Nonetheless, these derivatives are associated with severe neurotoxicity and loss of in vivo activity due to the highly lipophilic nature of the alkaloids. Here, we describe the development of highly polar prodrugs of antofine and tylophorine as hypoxia-targeted prodrugs. The developed quaternary ammonium salts of phenanthroindolizidines showed high chemical and metabolic stability and are predicted to have no penetration through the blood-brain barrier. The designed prodrugs displayed decreased cytotoxicity when tested under normoxic conditions. However, their cytotoxic activity considerably increased when tested under hypoxic conditions.

Subcellular Location of Tirapazamine Reduction Dramatically Affects Aerobic but Not Anoxic Cytotoxicity.

Hypoxia is an adverse prognostic feature of solid cancers that may be overcome with hypoxia-activated prodrugs (HAPs). Tirapazamine (TPZ) is a HAP which has undergone extensive clinical evaluation in this context and stimulated development of optimized analogues. However the subcellular localization of the oxidoreductases responsible for mediating TPZ-dependent DNA damage remains unclear. Some studies conclude only nuclear-localized oxidoreductases can give rise to radical-mediated DNA damage and thus cytotoxicity, whereas others identify a broader role for endoplasmic reticulum and cytosolic oxidoreductases, indicating the subcellular location of TPZ radical formation is not a critical requirement for DNA damage. To explore this question in intact cells we engineered MDA-231 breast cancer cells to express the TPZ reductase human NADPH: cytochrome P450 oxidoreductase (POR) harboring various subcellular localization sequences to guide this flavoenzyme to the nucleus, endoplasmic reticulum, cytosol or inner surface of the plasma membrane. We show that all POR variants are functional, with differences in rates of metabolism reflecting enzyme expression levels rather than intracellular TPZ concentration gradients. Under anoxic conditions, POR expression in all subcellular compartments increased the sensitivity of the cells to TPZ, but with a fall in cytotoxicity per unit of metabolism (termed 'metabolic efficiency') when POR is expressed further from the nucleus. However, under aerobic conditions a much larger increase in cytotoxicity was observed when POR was directed to the nucleus, indicating very high metabolic efficiency. Consequently, nuclear metabolism results in collapse of hypoxic selectivity of TPZ, which was further magnified to the point of reversing O2 dependence (oxic > hypoxic sensitivity) by employing a DNA-affinic TPZ analogue. This aerobic hypersensitivity phenotype was partially rescued by cellular copper depletion, suggesting the possible involvement of Fenton-like chemistry in generating short-range effects mediated by the hydroxyl radical. In addition, the data suggest that under aerobic conditions reoxidation strictly limits the TPZ radical diffusion range resulting in site-specific cytotoxicity. Collectively these novel findings challenge the purported role of intra-nuclear reductases in orchestrating the hypoxia selectivity of TPZ.

Identification of one-electron reductases that activate both the hypoxia prodrug SN30000 and diagnostic probe EF5.

SN30000 is a second-generation benzotriazine-N-oxide hypoxia-activated prodrug scheduled for clinical trial. Previously we showed that covalent binding of the hypoxia probe EF5 predicts metabolic activation of SN30000 in a panel of cancer cell lines under anoxia, suggesting that they are activated by the same reductases. However the identity of these reductases is unknown. Here, we test whether forced expression of nine oxidoreductases with known or suspected roles in bioreductive prodrug metabolism (AKR1C3, CYB5R3, FDXR, MTRR, NDOR1, NOS2A, NQO1, NQO2 and POR) enhances oxic or anoxic reduction of SN30000 and EF5 by HCT116 cells. Covalent binding of (14)C-EF5 and reduction of SN30000 to its 1-oxide and nor-oxide metabolites was highly selective for anoxia in all lines, with significantly elevated anoxic metabolism of both compounds in lines over-expressing POR, MTRR, NOS2A or NDOR1. There was a strong correlation between EF5 binding and SN30000 metabolism under anoxia across the cell lines (R(2)=0.84, p=0.0001). Antiproliferative potency of SN30000 under anoxia was increased most strongly by overexpression of MTRR and POR. Transcript abundance in human tumours, evaluated using public domain mRNA expression data, was highest for MTRR, followed by POR, NOS2A and NDOR1, with little variation between tumour types. Immunostaining of tissue microarrays demonstrated variable MTRR protein expression across 517 human cancers with most displaying low expression. In conclusion, we have identified four diflavin reductases (POR, MTRR, NOS2A and NDOR1) capable of reducing both SN30000 and EF5, further supporting use of 2-nitroimidazole probes to predict the ability of hypoxic cells to activate SN30000.

The Role of Bystander Effects in the Antitumor Activity of the Hypoxia-Activated Prodrug PR-104.

Activation of prodrugs in tumors (e.g., by bioreduction in hypoxic zones) has the potential to generate active metabolites that can diffuse within the tumor microenvironment. Such "bystander effects" may offset spatial heterogeneity in prodrug activation but the relative importance of this effect is not understood. Here, we quantify the contribution of bystander effects to antitumor activity for the first time, by developing a spatially resolved pharmacokinetic/pharmacodynamic (SR-PK/PD) model for PR-104, a phosphate ester pre-prodrug that is converted systemically to the hypoxia-activated prodrug PR-104A. Using Green's function methods we calculated concentrations of oxygen, PR-104A and its active metabolites, and resultant cell killing, at each point of a mapped three-dimensional tumor microregion. Model parameters were determined in vitro, using single cell suspensions to determine relationships between PR-104A metabolism and clonogenic cell killing, and multicellular layer (MCL) cultures to measure tissue diffusion coefficients. LC-MS/MS detection of active metabolites in the extracellular medium following exposure of anoxic single cell suspensions and MCLs to PR-104A confirmed that metabolites can diffuse out of cells and through a tissue-like environment. The SR-PK/PD model estimated that bystander effects contribute 30 and 50% of PR-104 activity in SiHa and HCT116 tumors, respectively. Testing the model by modulating PR-104A-activating reductases and hypoxia in tumor xenografts showed overall clonogenic killing broadly consistent with model predictions. Overall, our data suggest that bystander effects are important in PR-104 antitumor activity, although their reach may be limited by macroregional heterogeneity in hypoxia and reductase expression in tumors. The reported computational and experimental techniques are broadly applicable to all targeted anticancer prodrugs and could be used to identify strategies for rational prodrug optimization.

Identification of human reductases that activate the dinitrobenzamide mustard prodrug PR-104A: a role for NADPH:cytochrome P450 oxidoreductase under hypoxia.

Hypoxia is a common trait found in many solid tumours and thus represents a therapeutic target with considerable potential. PR-104, a hypoxia-activated prodrug currently in clinical trial, is a water-soluble phosphate ester which is converted in vivo to the corresponding alcohol, PR-104A. This 3,5-dinitrobenzamide-2-nitrogen mustard is activated by reduction to the corresponding 5-hydroxylamine (PR-104H) and 5-amine (PR-104M) in hypoxic cells. The clinical effectiveness of PR-104 will depend in part on the expression of reductases within tumours that can effect this reduction. Here, we evaluate the roles of NADPH:cytochrome P450 oxidoreductase (CYPOR; E.C.1.6.2.4) and NAD(P)H:quinone oxidoreductase (NQO1; E.C.1.6.99.2) as candidate PR-104A reductases. A weak correlation was observed between NQO1 activity and aerobic cytotoxicity in a panel of eight tumour cell lines. However, overexpression of human NQO1 did not increase cytotoxicity of PR-104A or the formation of PR-104H/M, showing that PR-104A is not a substrate for NQO1. Overexpression of human CYPOR did, however, increase the hypoxic cytotoxicity of PR-104A, and its metabolism to PR-104H and PR-104M, demonstrating it to be a PR-104A reductase. To assess the contribution of CYPOR to overall activation of PR-104A in hypoxic SiHa cells, a combination of siRNA transfection and antisense expression were used to suppress CYPOR protein by 91% (+/-3%), a phenotype which conferred 45% (+/-7%) decrease in cytotoxic potency of PR-104A. Regression analysis of all CYPOR depletion data was found to correlate with cytoprotection and metabolism (p<0.001). Residual PR-104A reductase activity could be inhibited by the flavoprotein inhibitor diphenyliodonium. We conclude that CYPOR is an important PR-104A reductase, but that other flavoenzymes also contribute to its activation in hypoxic SiHa cells.