Crenolanib inhibits FLT3 in vivo. (A) Plasma samples from patients 6 and 7 were collected as part of the phase 2 clinical trial of crenolanib. Patients received a single dose of drug on day 1, and samples were collected for PIA and pK analysis. Molm14 cells were incubated with each plasma sample for 1 hour and then lysed, immunoprecipitated for FLT3, and analyzed by immunoblot for phospho- and total FLT3. (B) The same time course samples from these patients were analyzed by mass spectroscopy for serum drug concentrations of crenolanib. (C) Blasts from patient 4 (relapsed D835V) were used for a PIA assay with steady-state plasma samples from 5 different patients enrolled in the crenolanib trial. Blasts were incubated in plasma for 1 hour and then lysed, immunoprecipitated for FLT3, and analyzed by immunoblot for phospho- and total FLT3. Densitometry was performed on this blot and serum concentrations of crenolanib from the corresponding time points were determined by pK analysis. In the figure, the densitometry result (expressed as a percentage of control) and the crenolanib concentration for a given time point are listed directly below each sample on the blot.