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No / The indolequinone compound EO9 has good pharmacodynamic properties in terms of bioreductive activation and selectivity for either NAD(P)H:quinone oxidoreductase-1 (NQO1)-rich aerobic or NQO1-deficient hypoxic cells. However, its pharmacokinetic properties are poor and this fact is believed to be a major reason for EO9's lack of clinical efficacy. The purpose of this study was to develop quinone-based bioreductive drugs that retained EO9's good properties, in terms of bioreductive activation, but have improved pharmacokinetic properties. Out of 11 naphthoquinone compounds evaluated, 2-aziridinyl-5-hydroxy-1,4-naphthoquinone (compound 2), 2,3-bis(aziridinyl)-5-hydroxy-1,4-naphthoquinone (compound 3), and 2-aziridinyl-6-hydroxymethyl-1,4-naphthoquinone (compound 11) were selected for further evaluation based on good substrate specificity for NQO1 and selectivity towards NQO1-rich cells in vitro. Compound 3 was of particular interest as it also demonstrated selectivity for NQO1-rich cells under hypoxic conditions. Compound 3 was not metabolised by murine whole blood in vitro (in contrast to compounds 2, 11 and EO9) and pharmacokinetic studies in non-tumour-bearing mice in vivo (at the maximum soluble dose of 60 mg kg¿1 administered intraperitoneally) demonstrated significant improvements in plasma half-life (16.2 min) and AUC values (22.5 ¿M h) compared to EO9 (T1/2 = 1.8 min, AUC = 0.184 ¿M h). Compound 3 also demonstrated significant anti-tumour activity against H460 and HCT-116 human tumour xenografts in vivo, whereas EO9 was inactive against these tumours. In conclusion, compound 3 is a promising lead compound that may target both aerobic and hypoxic fractions of NQO1-rich tumours and further studies to elucidate its mechanism of action and improve solubility are warranted.

Solid tumors are commonly subject to hypoxia. Hypoxic cancer cells have undesirable properties such as a high tendency to metastasize and resistance to chemotherapy and radiotherapy. Hypoxia-inducible factors (HIFs) respond to the changes in oxygen levels, orchestrating the transcription of many proteins that are vital for the survival of hypoxic cancer cells. With their parent drug SN-38 as an inhibitor of both topoisomerase 1 and HIF-1, hypoxia-activated SN-38s may have a dual inhibitory effect on hypoxic tumor cells due to hypoxia-targeting and HIF-1 inhibition. To develop hypoxia-activated prodrugs of SN-38; 2-, 3-, and 4-nitrobenzyl SN-38s have been synthesized with good yields (78%, 67% and 68%, respectively). Topoisomerase 1 inhibitory assay on 2- and 4-nitrobenzyl SN-38s and cell viability assay on 2-, 3- and 4-nitrobenzyl SN-38s have been performed. All three derivatives showed less toxicity on K562 cells, which meets the principle of prodrug design. Cyclic voltammetry results suggest that the reduction potentials of these three derivatives may be not high enough for these compounds to be activated. The manner of reduction of three nitrobenzyl SN-38s is quasi-reversible under the testing condition, not against the proposed mechanism of activation. Two new derivatives of SN-38 have been designed to elevate reduction potential and further reduce toxicity. They are to be synthesized and tested for future work. / October 2016

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No / Background: AQ4N (1,4-bis[[2-(dimethylamino)ethyl] amino]-5,8-dihydroxyanthracene-9, 10-dione bis-N-oxide dihydrochloride) is a prodrug which is selectively activated within hypoxic tissues to AQ4, a topoisomerase II inhibitor and DNA intercalator. Patients and methods: In the phase I study, 22 patients with oesophageal carcinoma received an i.v. infusion of AQ4N (22.5¿447 mg/m2) followed, 2 weeks later, by further infusion and radiotherapy. Pharmacokinetics and lymphocyte AQ4N and AQ4 levels were measured after the first dose. At 447 mg/m2, biopsies of tumour and normal tissue were taken after AQ4N administration. Results: Drug-related adverse events were blue discolouration of skin and urine, grade 2¿3 lymphopenia, grade 1¿3 fatigue, grade 1¿2 anaemia, leucopenia and nausea. There were no drug-related serious adverse events (SAEs). Three patients had reductions in tumour volume >50%, nine had stable disease. Pharmacokinetics indicated predictable clearance. Plasma area under the curve (AUC) at 447 mg/m2 exceeded AQ4N concentrations in mice at therapeutic doses and tumour biopsies contained concentrations of AQ4 greater than those in normal tissue. Tumour concentrations of AQ4 exceeded in vitro IC50 values for most cell lines investigated. Conclusions: No dose-limiting toxic effects were observed and a maximum tolerated dose was not established. Tumour AQ4 concentrations and plasma AUC at 447 mg/m2 exceeded active levels in preclinical models. This dose was chosen for future studies with radiotherapy.

No / The indolequinone EO9 demonstrated good preclinical activity but failed to show clinical efficacy against a range of tumours following intravenous drug administration. A significant factor in EO9's failure in the clinic has been attributed to its rapid pharmacokinetic elimination resulting in poor drug delivery to tumours. Intravesical administration of EO9 would circumvent the problem of drug delivery to tumours and the principal objective of this study is to determine whether or not bladder tumours have elevated levels of the enzyme NQO1 (NAD(P)H:quinone oxidoreductase) which plays a key role in activating EO9 under aerobic conditions. Elevated NQO1 levels in human bladder tumour tissue exist in a subset of patients as measured by both immunohistochemical and enzymatic assays. In a panel of human tumour cell lines, EO9 is selectively toxic towards NQO1 rich cell lines under aerobic conditions and potency can be enhanced by reducing extracellular pH. These studies suggest that a subset of bladder cancer patients exist whose tumours possess the appropriate biochemical machinery required to activate EO9. Administration of EO9 in an acidic vehicle could be employed to reduce possible systemic toxicity as any drug absorbed into the blood stream would become relatively inactive due to an increase in pH.

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.