Indotecan was administered intravenously through a central line over 1 h for the daily schedule (days 1–5, followed by 23 days without drug in 28-day cycles) and over 3 h for the weekly schedule (days 1, 8, and 15 in 28-day cycles). An MTD of 60 mg/m2/day was established for the daily regimen, compared to 90 mg/m2 for the weekly regimen.

Human recombinant Top1 was purified from Baculovirus. 117-bp DNA oligonucleotide (Integrated DNA Technologies) encompassing the previous identified Top1 cleavage sites identified in the 161-bp fragment from pBluescript SK(−) phagemid DNA was employed. This 117-bp oligonucleotide contains a single 5′-cytosine overhang, which was 3′-end labeled by fill-in reaction with [•-32P]-dGTP in React 2 buffer (50 mM Tris-HCl, pH 8.0, 100 mM MgCl2, 50 mM NaCl) with 0.5 units of DNA polymerase I (Klenow fragment, New England BioLabs). Unincorporated 32P-dGTP was removed using mini Quick Spin DNA columns (Roche, Indianapolis, IN), and the eluate containing the 3′-end-labeled DNA substrate was collected. Approximately 2 nM of radiolabeled DNA substrate was incubated with recombinant Top1 in 20 μL of reaction buffer [10 mM Tris-HCl (pH 7.5), 50 mM KCl, 5 mM MgCl2, 0.1 mM EDTA, and 15 μg/mL BSA] at 25 °C for 20 min in the presence of various concentrations of compound 5 (Indotecan). The reactions were terminated by adding SDS (0.5% final concentration) followed by the addition of two volumes of loading dye (80% formamide, 10 mM sodium hydroxide, 1 mM sodium EDTA, 0.1% xylene cyanol, and 0.1% bromophenol blue).