PIA results compared with standard curve for KW-2449. (A) Plasma was collected from patients receiving KW-2449 on the clinical trial 2, 8, and 12 hours after ingesting drug. The plasma samples underwent conventional pharmacokinetic analysis for concentrations of KW-2449 and the metabolite, M1. In parallel, plasma from the same time points was used in PIA assays for FLT3. On the x axis, the concentration of active drug is plotted for individual time point by the following formula: [KW-2449] + [M1] / 3.6. On the y axis, the degree of FLT3 inhibition is plotted as percent control. The pretreatment plasma sample for each patient was used as the control for each individual PIA assay. Thus, each circle on the graph represents a single time point for which the concentration of KW-2449 plus metabolite in a plasma sample is plotted against how well that plasma inhibited FLT3 phosphorylation in Molm14 cells (the PIA assay). (B) This graph is the standard curve of KW-2449 inhibiting FLT3 in plasma shown in Figure 2. (C) This graph shows the graph from panel A overlying the standard curve from panel B, demonstrating how the results of the PIA assay, using the concentrations of KW-2449 and M1 divided by 3.6, correspond to the standard curve. (D) The same experiment as in panel C, but using the PIA assay for STAT5 rather than FLT3.