Development and validation of a sensitive solid-phase-extraction and high-performance liquid chromatography assay for the bioreductive agent tirapazamine and its major metabolites in mouse and human plasma for pharmacokinetically guided dose escalation

Hernani Robin, Suresh Senan, Paul Workman, Martin A. Graham*

*Corresponding author for this work

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A sensitive solid-phase-extraction and high-performance liquid chromatography (HPLC) method has been developed to investigate the pharmacokinetics and metabolism of the hypoxic-cell cytotoxic agent tirapazamine (1,2,4-benzotriazine-3-amine 1,4-di-N-oxide; WIN 59075, SR 4233), currently in phase I/II studies in the United Kingdom and United States. A sample extraction and concentration process was devised using strong cationexchange Bond Elut cartridges. Tirapazamine, the mono and zero-N-oxide metabolites (WIN 64012, WIN 60109) were isocratically resolved using a μBondapak phenyl HPLC column and measured using photodiode-array detection. The minimal quantifiable level (MQL) of tirapazamine was 40 ng/ml in mouse plasma and 20 ng/ml in human plasma. Recovery was consistently greater than 80% for all compounds over the concentration range of 20 ng/ml to 20 μg/ml. No significant decomposition was observed following up to three freeze/thaw cycles and storage at -70°C for 52 days. The assay was accurate and reproducible, with measured values lying within the limits of defined acceptance criteria. Additional studies to investigate the degree of plasma protein binding showed that tirapazamine did not bind extensively to plasma proteins (binding, 9.7%±0.1% and 18.7%±1.3% in mouse and human plasma, respectively). These small species differences in protein binding are unlikely to have any major impact on the extrapolation of pharmacokinetic data from mice to humans. The assay has now been successfully applied to investigate the pharmacokinetics and metabolism of tirapazamine in mice and patients as part of a pharmacokinetically guided dose-escalation strategy for phase I clinical trials.

Original languageEnglish
Pages (from-to)266-270
Number of pages5
JournalCancer Chemotherapy and Pharmacology
Issue number3
Publication statusPublished - 1 May 1995

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