Figure 2.

Distribution of M2698 in plasma and tumors, and targeted inhibition of p70S6K. A. Mice bearing subcutaneous MDA-MB-453 human xenograft tumors (n=3 per time point) were treated with vehicle or 20 mg/kg M2698 qd po for 4 days and plasma and tumors were harvested at 4, 8 and 24 h after the final administration. Plasma and tumor concentrations of M2698 were assessed by LC-MS/MS at each time point. B. Mice treatment and sample collection was the same as described for A. Phosphorylated and total S6 (S240/244), PRAS40, and Akt (T308) were assessed from tumor protein lysates by western blot analysis at each time point. C. Mice bearing MDA-MB-453 human breast cancer xenograft tumors (n=4 per time point) were treated with vehicle or M2698 1, 5, 10, or 20 mg/kg qd po for 7 days then euthanized at 1, 4, 8, 24, 48 and 72 h after the final administration (4 h post-treatment for vehicle-treated animals). M2698 concentrations were assessed by LC-MS/MS and the proportion of phosphorylated:total S6 (pS6/tS6) in tumor and normal brain (expressed as a percentage of control) was determined by western blot analysis at each time point. The PK/PD relationships between S6 phosphorylation in tumors and concentrations of unbound M2698 in plasma were characterized using an indirect response model (Equation 1), which generated an unbound IC₅₀ of 15 nM. h, hours; qd, once daily.