COLO205 cells (3 × 106 to 5 × 106 cells per mouse) were subcutaneously xenografted into BALB/c nude mice (7 weeks old, female). TAK-931 suspended in methylcellulose aqueous solution was orally administered to xenografted mice with tumor volumes of 150 to 400 mm3 at the indicated doses. After a single administration, tumors and plasma were collected at the indicated time points for the measurement of drug concentrations by liquid chromatography–tandem MS (LC-MS/MS). The pMCM2 levels in the tumors at the indicated time points were detected by immunoblotting or immunohistochemistry.

For efficacy studies, the mice bearing tumors (100 to 250 mm3) were selected and randomly categorized into vehicle and TAK-931 groups (five mice per group). The xenografted mice were orally administered TAK-931 at the indicated regimens.

T/C (%) (or %T/C) was calculated using the following formulaT/C (%)=[(TAK-931 tumor volumeTAK931 tumor volume on day 0)/(vehicle tumor volumevehicle tumor volume on day 0)]×100

All in vivo procedures were performed in accordance with the protocols approved by the Takeda Experimental Animal Care and Use Committee (experimental protocol number: 00004407).

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