Hypoxia is a characteristic of solid tumours and a potentially important therapeutic target. PR-104, a prodrug designed to target hypoxic cells, is currently in phase II clinical trial. PR- 104 is converted systemically to the alcohol PR-104A which is reduced selectively under hypoxia to its hydroxylamine and amine metabolites. This thesis aims to determine the mechanism(s) of cytotoxicity of PR-104 in preclinical tumour models. Specific objectives were to identify biomarkers of response to PR-104, and to ascertain which features of tumours determine their sensitivity to PR-104 monotherapy. PR-104A demonstrated hypoxia-selective cytotoxicity for all nine human tumour cell lines investigated in culture, but with widely differing hypoxic/oxic differentials because of large differences in aerobic cytotoxicity. Hypoxic cytotoxicity correlated with DNA interstrand crosslink (ICL) frequency (measured using the alkaline comet assay), suggesting ICL are invariably responsible for hypoxic cytotoxicity. There was a similar relationship between ICL and aerobic cytotoxicity, except for four lines with very low rates of aerobic PR-104A reduction. Nonetheless, monotherapy activity of PR-104 in tumour xenografts of all nine cell lines showed a strong correlation with ICL at 24 hours (r2 = 0.733, p<0.001) indicating that DNA crosslinking is the dominant mechanism of cytotoxicity in the pharmacologically relevant dose range. A hypoxia-selective increase in the DNA double strand break marker γH2AX was also observed in all cell lines after PR-104A in vitro, with kinetics and cell cycle distribution consistent with DNA replication arrest at ICL. This marker also correlated with cytotoxicity in tumours (r2 = 0.465, p<0.001). The wide variation in γH2AX levels between cell lines at equivalent cytotoxicity suggested γH2AX is less useful than ICL as an absolute biomarker, but has possible utility for comparing response of aerobic and hypoxic cells within the same tumour. Reduced PR-104A metabolites in tumours also correlated with cytotoxicity (r2 = 0.585, p<0.05), but less well than for ICL. Unexpectedly, the results suggest that rates of intratumour activation of PR-104A are not primarily determined by hypoxia (measured using pimonidazole binding) or expression of the major hypoxic reductase cytochrome P450 oxidoreductase, and that aerobic nitroreduction is a major contributor to its monotherapy antitumour activity. / Whole document restricted, but available by request, use the feedback form to request access.

Hypoxia is a characteristic of solid tumours and a potentially important therapeutic target. PR-104, a prodrug designed to target hypoxic cells, is currently in phase II clinical trial. PR- 104 is converted systemically to the alcohol PR-104A which is reduced selectively under hypoxia to its hydroxylamine and amine metabolites. This thesis aims to determine the mechanism(s) of cytotoxicity of PR-104 in preclinical tumour models. Specific objectives were to identify biomarkers of response to PR-104, and to ascertain which features of tumours determine their sensitivity to PR-104 monotherapy. PR-104A demonstrated hypoxia-selective cytotoxicity for all nine human tumour cell lines investigated in culture, but with widely differing hypoxic/oxic differentials because of large differences in aerobic cytotoxicity. Hypoxic cytotoxicity correlated with DNA interstrand crosslink (ICL) frequency (measured using the alkaline comet assay), suggesting ICL are invariably responsible for hypoxic cytotoxicity. There was a similar relationship between ICL and aerobic cytotoxicity, except for four lines with very low rates of aerobic PR-104A reduction. Nonetheless, monotherapy activity of PR-104 in tumour xenografts of all nine cell lines showed a strong correlation with ICL at 24 hours (r2 = 0.733, p<0.001) indicating that DNA crosslinking is the dominant mechanism of cytotoxicity in the pharmacologically relevant dose range. A hypoxia-selective increase in the DNA double strand break marker γH2AX was also observed in all cell lines after PR-104A in vitro, with kinetics and cell cycle distribution consistent with DNA replication arrest at ICL. This marker also correlated with cytotoxicity in tumours (r2 = 0.465, p<0.001). The wide variation in γH2AX levels between cell lines at equivalent cytotoxicity suggested γH2AX is less useful than ICL as an absolute biomarker, but has possible utility for comparing response of aerobic and hypoxic cells within the same tumour. Reduced PR-104A metabolites in tumours also correlated with cytotoxicity (r2 = 0.585, p<0.05), but less well than for ICL. Unexpectedly, the results suggest that rates of intratumour activation of PR-104A are not primarily determined by hypoxia (measured using pimonidazole binding) or expression of the major hypoxic reductase cytochrome P450 oxidoreductase, and that aerobic nitroreduction is a major contributor to its monotherapy antitumour activity. / Whole document restricted, but available by request, use the feedback form to request access.

No / PURPOSE: AQ4N is a novel bioreductive prodrug under clinical investigation. Preclinical evidence shows that AQ4N penetrates deeply within tumors and undergoes selective activation to form AQ4, a potent topoisomerase II inhibitor, in hypoxic regions of solid tumors. This proof-of-principle, phase I study evaluated the activation, hypoxic selectivity, and safety of AQ4N in patients with advanced solid tumors. EXPERIMENTAL DESIGN: Thirty-two patients with cancer (8 glioblastoma, 9 bladder, 8 head and neck, 6 breast, and 1 cervix) received a single 200 mg/m(2) dose of AQ4N before elective surgery. AQ4 and AQ4N levels in 95 tissues (tumor, healthy tissue) were assessed by liquid chromatography-tandem mass spectrometry. Tissue sections were also analyzed for AQ4 fluorescence using confocal microscopy, and for expression of the hypoxia-regulated glucose transporter, Glut-1. RESULTS: Activated AQ4 was detected in all tumor samples with highest levels present in glioblastoma (mean 1.2 microg/g) and head and neck (mean 0.65 microg/g) tumors; 22 of 32 patients had tumor AQ4 concentrations > or = 0.2 microg/g, levels previously shown to be active in preclinical studies. In 24 of 30 tumor samples, AQ4 was detected at higher concentrations than in adjacent normal tissue (tumor to normal ratio range 1.1-63.6); distant skin samples contained very low concentrations of AQ4 (mean 0.037 microg/g). Microscopic evaluation of tumor sections revealed that AQ4 colocalized within regions of Glut-1+ hypoxic cells. CONCLUSIONS: AQ4N was activated selectively in hypoxic regions in human solid tumors. Intratumoral concentrations of AQ4 exceeded those required for activity in animal models and support the evaluation of AQ4N as a novel tumor-targeting agent in future clinical studies.

No / AQ4N (banoxatrone) (1,4-bis-{[2-(dimethylamino-N-oxide)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione) is an example of a bioreductive prodrug in clinical development. In hypoxic cells AQ4N is reduced to the topoisomerase II inhibitor AQ4 (1,4-bis- {[2-(dimethylamino)ethyl]amino}-5,8-dihydroxyanthracene-9,10-dione). By inhibition of topoisomerase II within these hypoxic areas, AQ4N has been shown to sensitise tumours to existing chemo- and radiotherapy treatments. In this study the distribution of AQ4N and AQ4 in treated H460 human tumour xenografts has been examined by imaging matrix-assisted laser desorption/ionisation mass spectrometry. Images of the distribution of AQ4N and AQ4 have been produced that show little overlap. The distribution of ATP in the tumour xenografts was also studied as an endogenous marker of regions of hypoxia since concentrations of ATP are known to be decreased in these regions. The distribution of ATP was similar to that of AQ4N, i.e. in regions of abundant ATP there was no evidence of conversion of AQ4N into AQ4. This indicates that the cytotoxic metabolite AQ4 is confined to hypoxic regions of the tumour as intended.

Hypoxia (low physiological O₂ levels) is a characteristic of solid tumours. It not only alters the chemical microenvironment of a tumour but initiates a number of mechanisms which enable cells to cope and thrive under these conditions, resulting in therapy-resistant and aggressive tumours. The replication stress induced by severe hypoxia activates a DNA damage response which involves the kinases ATR and Chk1. Moreover, periods of hypoxia are often followed by reoxygenation, which induces DNA damage. Chk1 inhibitors have been used to potentiate chemotherapy with cytotoxic agents and have recently been proposed as single agents in tumours with high levels of replication stress. However, inhibition of Chk1 also affects normal DNA replication, cell cycle progression and DNA repair. The herein presented study chose known inhibitors of Chk1 and, with methods of synthetic organic chemistry, modified them into agents to selectively target hypoxic cells. Three different Chk1 inhibitors were selected and bioreductive analogues synthesised which were evaluated in chemical, biochemical and cellular assays. We found a convenient route to access a precursor of the bioreductive 2-nitroimidazole group and established a three-step protocol for the testing of bioreductive drugs. This protocol allows us to determine whether a bioreductive drug undergoes reduction and prodrug activation. In addition, bioreductive Chk1 inhibitors were shown to induce DNA damage and cellular toxicity in a hypoxia-selective fashion. While reduction of the prodrugs occurred in all three cases, fragmentation was always the rate-limiting step. We propose that the use of bioreductive Chk1 inhibitors is a promising strategy to target the most therapy-resistant tumour fraction while sparing normal tissue.

Idarubicin (IDA) and mitomycin C (MC) are clinically effective quinone-containing anticancer agents used in the treatment of several human cancers. Quinone-containing anticancer drugs have the potential to undergo bioreduction by oxidoreductases to reactive species, and thereby exert their cytotoxic effects. In the present study, we investigated, for the first time, the potential of IDA, in comparison to MC, to undergo reductive activation by NADPH-cytochrome P450 reductase (P450R), NADH-cytochrome b5 reductase (b5R) and P450R-cytochrome P4502B4 (CYP2B4) system by performing both in vitro plasmid DNA damage experiments and enzyme assays. In addition, we examined the potential protective effects of some antioxidants against DNA-damaging effects of IDA and MC resulting from their reductive activation. To achieve these goals, we obtained P450R from sheep lung, beef liver and PB-treated rabbit liver microsomes, b5R from beef liver microsomes and CYP2B4 from PB-treated rabbit liver microsomes in highly purified forms. The plasmid DNA damage experiments demonstrated that P450R is capable of effectively reducing IDA to DNA-damaging species. The effective protections provided by antioxidant enzymes, SOD and catalase, as well as scavengers of hydroxyl radical, DMSO and thiourea, revealed that the mechanism of DNA damage by IDA involves the generation of ROS by redox cycling of IDA with P450R under aerobic conditions. The extent of DNA damages by both IDA and MC were found to increase with increasing concentrations of the drug or the enzyme as well as with increasing incubation time. IDA was found to have a greater ability to induce DNA damage at high drug concentrations than MC. The plasmid DNA experiments using b5R, on the other hand, showed that, unlike P450R, b5R was not able to reduce IDA to DNA-damaging reactive species. It was also found that in the presence of b5R and cofactor NADH, MC barely induced DNA strand breaks. All the purified P450Rs reduced IDA at about two-fold higher rate than that of MC as shown by the measurement of drug-induced cofactor consumption. This indicates that IDA may be a more potent cytotoxic drug than MC in terms of the generation of reactive metabolites. The results obtained from enzyme assays confirmed the finding obtained from plasmid DNA experiments that while MC is a very poor substrate for b5R, IDA is not a suitable substrate for this enzyme unlike P450R. The reconstitution experiments carried out under both aerobic and anaerobic conditions using various amounts of CYP2B4, P450R and lipid DLPC revealed that reconstituted CYP2B4 produced about 1.5-fold and 1.4-fold rate enhancements in IDA and MC reduction catalyzed by P450R alone, respectively. The present results also showed that among the tested dietary antioxidants, quercetin, rutin, naringenin, resveratrol and trolox, only quercetin was found to be highly potent in preventing DNA damage by IDA. These results may have some practical implications concerning the potential use of P450R as therapeutic agent on their own in cancer treatment strategies. Selective targeting of tumor cells with purified P450R by newly developed delivery systems such as using polymers, liposomes or antibodies may produce greater reductive activation of bioreductive drugs in tumor cells. Consequently, this strategy has a high potential to increase the efficacy and selectivity of cancer chemotherapy.

No / NAD(P)H:quinone oxidoreductase-1 (NQO1) is a potential target for therapeutic intervention but attempts to exploit NQO1 using quinone-based bioreductive prodrugs have been largely compromised by toxicity to organs that inherently express high levels of NQO1. In an attempt to circumvent this problem, this study describes the development of a tripartite quinone-based drug delivery system, the ultimate objective of which is to release a targeted therapeutic agent following the reduction of a quinone "trigger" by NQO1. Molecular modeling of drug/NQO1 interactions were conducted prior to the synthesis of N-{4-[bis-(2-chloroethyl)-amino]-phenyl}-beta,beta,2,4,5-pentamethyl-3,6-dioxo-1,4-cyclohexadiene-1-propanamide (prodrug 1). Prodrug 1 is a good substrate for purified NQO1 (V(max) and K(m) values of 11.86 +/- 3.09 micromol/min/mg and 2.70 +/- 1.14 micromol/L, respectively) and liquid chromatography-mass spectrometry analysis of the metabolites generated showed that lactone 3 and aniline mustard 4 were generated in a time- and NQO1-dependent manner. Chemosensitivity studies showed that prodrug 1 is selectively toxic to cells that overexpress NQO1 under aerobic conditions, and comet assay analysis confirmed the presence of elevated interstrand cross-links in NQO1-rich compared with NQO1-deficient cells. Hypoxic sensitization (hypoxic cytotoxicity ratio = 15.8) was observed in T47D cells that overexpress cytochrome P450 reductase. In conclusion, the results of this study provide mechanistic proof of principle that a tripartite benzoquinone drug delivery system is enzymatically reduced to release an active therapeutic agent. Further development of this concept to fine-tune substrate specificity for specific reductases and/or the inclusion of alternative therapeutic agents is warranted.