Topoisomerase I (Top1) is an established molecular target for anti‐cancer therapeutics. Top1 inhibitors function by trapping the catalytic intermediate of Top1 covalently attached to the DNA substrate, termed the Top1 covalent complex (Top1cc). We have developed and validated an immunoassay to measure Top1cc directly, in support of Phase 0 clinical trials for experimental Top1 inhibiting drugs. A simple Top1cc isolation step which separates Top1cc from free Top1 is followed by a quantitative infrared immunoblotting detection technique. Recombinant Top1 is used as the standard for quantification. The assay was used to measure Top1cc in cell lines that exhibit varied growth inhibitory responsiveness to topotecan (A375, HCT‐116, HT‐29 and SKMEL28). Cells were treated with single doses of Top1 inhibitors, topotecan and indenoisoquinoline and Top1cc levels were found to be dose dependent with a maximal response at/or before 30 min to 1h of exposure. To establish fitness‐for‐purpose for clinical applications the assay was tested on samples of human tumor xenografts from mice treated with topotecan and indenoisoquinolines. These results demonstrate the potential to quantify Top1cc levels as a pharmacodynamic maker for Top1 inhibitors in solid tumor extracts. Funded by NCI Contract No. HHSN261200800001E.

Citation Information: Mol Cancer Ther 2009;8(12 Suppl):C224.