Abstract 2272: Comparison of bioreductive metabolism of tirapazamine, its second generation analogue SN 30000 and hypoxia imaging agent EF5 in human tumor cell lines

Abstract

Abstract We have developed a novel bioreductive prodrug, SN 30000, an analogue of tirapazamine (TPZ), which has improved tissue penetration and higher hypoxic selectivity compared to TPZ. The activation of SN 30000 depends on one electron reduction that is also exploited by the hypoxia probe EF5. 18F-EF5 is currently in clinical development for PET imaging of tumour hypoxia. In this study we investigate whether activation of SN 30000, TPZ and EF5 depends on the same enzymes in hypoxic human tumor cells, and therefore whether EF5 imaging might profile both hypoxia and the reductases required for SN 30000 activation. We developed 96-well plate based microassays for reduction of SN 30000 (and TPZ) to their corresponding 1-oxide and nor-oxide metabolites using sensitive and rapid electrospray ionisation tandem mass spectrometry methods with a run time of 6 min on an Agilent 6460 UPLC/MS/MS (ultrahigh pressure liquid chromatography/tandem mass spectrometry). Octa-deuterated internal standards of SN 30000 and its 1-oxide and nor-oxide metabolites were used as internal standards. We used these methods to compare reductive metabolism of SN 30000 and TPZ in parental HCT116 cells (HCT116) and HCT116 cells overexpressing cytochrome P450 oxidoreductase (CYPOR; HCT116/CYPOR) and also for a SiHa parental and CYPOR-overexpressing pair (SiHa and SiHa/CYPOR). This assay showed excellent sensitivity, with a lower limit of quantitation of < 0.01 µM for all analytes. CYPOR over-expression increased SN 30000 1-oxide formation by 2.8- and 2.4-fold in hypoxic HCT116 and SiHa cells, with a 5.4- and 4.0-fold increase in nor-oxide production, respectively. Similarly, we developed a microplate assay for quantifying covalent binding of 14C-EF5. The assay demonstrated enhancement of EF5 binding by 3.0- and 3.8-fold in hypoxic HCT116/CYPOR and SiHa/CYPOR cells, respectively, relative to parental lines. This demonstrates that SN 30000 and EF5 are substrates for CYPOR, as previously shown for TPZ. Additionally, our initial data for 12 human cancer cell lines shows a significant correlation between SN 30000 metabolism and covalent binding of 14C-EF5 (r2 =0.44, p = 0.018). Excluding one apparent outlier (Panc-01) improved the correlation (r2 = 0.67, p = 0.002). TPZ metabolism showed a similar relationship with EF5 binding. In conclusion, our data imply SN 30000, TPZ and EF5 share similar reductases in human tumor cells for their hypoxic reduction. Therefore, we propose that EF5 might be used not only to evaluate hypoxia in tumours, but also as a non-invasive probe for enzymatic activity of the 1-e oxidoreductases that activate TPZ and SN 30000. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 2272.