The ability of Pilaralisib (XL147) to inhibit this endogenous phosphorylation of AKT, p70S6K, and S6 is examined following a single oral dose of 10, 30, 100, or 300 mg/kg. The tumors are harvested 4, 24, or 48 hours postdose and homogenized in lysis buffer. Tumor lysates from each animal (n=4) are then pooled for each group and analyzed for levels of total and phosphorylated AKT, p70S6K, and S6 by Western immunoblotting. Administration of Pilaralisib (XL147) causes a dose-dependent decrease in phosphorylation of AKT, p70S6K, and S6 in the tumors, reaching a maximum of 81% inhibition of AKT phosphorylation at 300 mg/kg at 4 hours. The dose-response relationships derived from the 4-hour time point predict 50% inhibition of AKT, p70S6K, and S6 phosphorylation at doses of approximately 100 mg/kg (pAKTT308), 54 mg/kg (pAKTS473), 71 mg/kg (p-p70S6K), and 103 mg/kg (pS6). The inhibition of AKT, p70S6K, and S6 phosphorylation in MCF7 tumors following a 100 mg/kg dose of Pilaralisib (XL147) is maximal at 4 hours, reaching 55% to 75%; however, the level of inhibition decreased to 8% to 45% by 24 hours, and only minimal or no inhibition was evident by 48 hours. Following a 300 mg/kg dose of Pilaralisib (XL147), inhibition is also maximal at 4 hours (65%-81%). However, in contrast with the 100 mg/kg dose, inhibition at 24 hours (51%-78%) is almost comparable with that seen at 4 hours, and partial inhibition (25%-51%) persisted through 48 hours[1].