Chemical structures of FLT3 inhibitors.

Pharmacokinetics of AC220. (A) Time course of AC220 plasma levels in NU/NU mice after a single oral administration at 10 mg/kg. The average values from 3 independent time courses are shown in black and individual values, in red. (B) Dose dependence of AC220 peak plasma levels (C_max) and exposure (AUC_{0-24 hours}). The insets show a zoomed view of the data at dosages of 10 mg/kg and below. Average values from 3 independent time courses are shown in black and individual values, in red.

Activity of AC220 in primary AML cells. (A) Inhibition of FLT3 phosphorylation. Blasts were incubated in increasing concentrations of AC220 with 10% FBS for 1 hour at 37°C. For the Western blot, cell lysates were subjected to immunoprecipitation, sodium dodecylsulfate polyacrylamide electrophoresis, and immunoblotting for phosphorylated and total FLT3. As is frequently observed in patient samples for which limited numbers of cells are available, phospho-FLT3 (pFLT3) levels are relatively low and only weakly detected by Western blot. Robust quantitation of phospho-FLT3 was achieved by a more sensitive ELISA (“Cellular assays”). The graph shows phospho-FLT3 levels, normalized for total FLT3, obtained by ELISA. The line represents a fit of the data to the Hill equation. (B) Effect of AC220 on cell viability. In parallel to the phosphorylation assay, using the same AC220 medium preparation, blasts from the same patient and the same thawing were incubated at 37°C in 5% CO₂. After 72 hours, the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium assay was performed, and the results were plotted as a fraction of untreated control. The line represents a fit of the data to the Hill equation. (C) Effect of AC220 on cell viability in primary cell samples from additional patients. The experiments were performed and the data are plotted as in panel B. The primary cells were obtained from a 65-year-old male (■; IC₅₀ = 0.8 nM), a 53-year-old male (▲; IC₅₀ = 1 nM), a 56-year-old female (●; IC₅₀ = 1 nM), and a 34-year-old male (▼; IC₅₀ = 2 nM), all with relapsed AML harboring FLT3-ITD mutations.

Small molecule kinase interaction maps for FLT3 inhibitors. Compounds were screened against a KinomeScan (http://www.kinomescan.com) panel of 402 kinase assays. Red circles indicate kinases bound, and circle size indicates binding affinity. Interactions with K_d < 3 μM are shown. The complete dataset is shown in supplemental Table 1, and is also available through an interactive website (http://www.ambitbio.com/technology/publications). The data for MLN-518, PKC-412, sunitinib, and sorafenib against a subset of 317 of the 402 assays were previously published,⁴⁴ and are reproduced here together with new data for assays not represented in the earlier panel. The kinase dendrogram was adapted and is reproduced with permission from Cell Signaling Technology Inc (http://www.cellsignal.com).

In vivo efficacy of AC220 in animal tumor models. (A) Inhibition of FLT3 activity in subcutaneous tumor xenografts. Total and phospho-FLT3 were quantitated in lysates from tumors harvested at each time point, and phospho-FLT3 levels normalized relative to the total amount of FLT3. Normalized phospho-FLT3 levels are shown as a percentage of levels in untreated animals. Each group contained 4 animals. (B) Comparison of antitumor efficacy of AC220 and sunitinib in the subcutaneous tumor model at a dose of 10 mg/kg. Day 1 of the study was the first day of treatment, when tumors had reached a size of 450 to 600 mm³. The solid bar indicates the 28-day treatment period. Tumor size was monitored for an additional 61 days after treatment was discontinued, and total study length was 89 days. Each treatment group contained 10 animals, and the time course of tumor size for each individual animal is shown in blue. The average tumor size in the vehicle control groups is shown in black (± SEM) for comparison. Animals were killed if tumor volume reached 1000 mm³. (C) Efficacy of AC220 in a bone marrow transplant model. Kaplan-Meier plots of survival. Day 1 of the study was the day after cells were implanted. The solid bar indicates the treatment period of 30 days. Each treatment group contained 5 animals. Data for the vehicle control group are shown as ■. The study was terminated on day 172.