Figure 6.
Figure 6. Anti-leukemic effects of FF-10101 against primary AML cells in vitro and in vivo. (A) Primary AML cells were incubated with FLT3 inhibitors for 1 week. Cell viability (%) assessed with CellTiter-Glo reagent was plotted at each concentration. Data are represented as mean ± SD. Paired samples at diagnosis (D) and relapse (R) were analyzed for patient A. Samples of patients B, D, F, and G were obtained at diagnosis, and those of patient C and patient E were in relapse. (B-D) Primary AML cells with FLT3-ITD were inoculated into NOG mice via the tail vein. Mice were divided into 6 groups (n = 5/group) based on the percentage of human CD45-positive cells in PB. Five mice were euthanized 27 days after the implantation (day 0) for assessment of engrafted cells in BM at the time of initiation of drug administration. FF-10101 was orally administered at 5 mg/kg twice daily, 10 mg/kg once daily, and 10 mg/kg twice daily, and quizartinib at 10 mg/kg twice daily, for 11 days. PB was sampled at the indicated time points for monitoring the percentage of human CD45-positive cells (panel B). After 11-day treatment, BM was collected from bilateral femurs for determination of the percentage of human CD45-positive cells (panel C). Mice were weighed at days −1, 2, 4, 6, 8, 10, and 11, and relative body weight to initial body weight (day −1) was represented as percent changes. No significant differences in body weight among groups were observed (panel D). Bars show mean ± SD. (E-F) PK/PD study was conducted in the xenograft model with the primary AML cells shown in Figure 6B-D. Single or repeated oral administration of FF-10101 at 10 mg/kg was given to mice with excess blasts at 35 days after intravenous injection of primary AML cells. Plasma and BM concentrations of FF-10101 at indicated time points were analyzed by LC/MS/MS (panel E). BM lysates were subjected to western blotting for detection of FLT3 (t-FLT3), STAT5 (t-STAT5), and their phosphorylated forms (p-FLT3 and p-STAT5) (panel F). (G-I) Primary AML cells with FLT3-D835H mutation were xenografted into NOG mice. Mice were divided into 4 groups (n = 5 or 6/group) based on the percentage of human CD45-positive cells in PB. Six mice were euthanized for identification of engrafted cells in BM before initiation of administration. FF-10101 and quizartinib were orally administered at 10 mg/kg twice daily for 14 days. The percentage of human CD45-positive cells in PB was monitored once per week (panel G). After 14-day administration, the percentage of human CD45-positive cells in BM was determined by flow cytometry (panel H). Mice were weighed at days 0, 3, 6, 9, 12, and 14, and relative body weight to initial body weight (day 0) was represented as percent changes. No significant differences in body weight among groups were observed (panel I). Bars show mean ± SD. *P < .05 compared with control by Dunnett's Multiple Comparison Test.

Anti-leukemic effects of FF-10101 against primary AML cells in vitro and in vivo. (A) Primary AML cells were incubated with FLT3 inhibitors for 1 week. Cell viability (%) assessed with CellTiter-Glo reagent was plotted at each concentration. Data are represented as mean ± SD. Paired samples at diagnosis (D) and relapse (R) were analyzed for patient A. Samples of patients B, D, F, and G were obtained at diagnosis, and those of patient C and patient E were in relapse. (B-D) Primary AML cells with FLT3-ITD were inoculated into NOG mice via the tail vein. Mice were divided into 6 groups (n = 5/group) based on the percentage of human CD45-positive cells in PB. Five mice were euthanized 27 days after the implantation (day 0) for assessment of engrafted cells in BM at the time of initiation of drug administration. FF-10101 was orally administered at 5 mg/kg twice daily, 10 mg/kg once daily, and 10 mg/kg twice daily, and quizartinib at 10 mg/kg twice daily, for 11 days. PB was sampled at the indicated time points for monitoring the percentage of human CD45-positive cells (panel B). After 11-day treatment, BM was collected from bilateral femurs for determination of the percentage of human CD45-positive cells (panel C). Mice were weighed at days −1, 2, 4, 6, 8, 10, and 11, and relative body weight to initial body weight (day −1) was represented as percent changes. No significant differences in body weight among groups were observed (panel D). Bars show mean ± SD. (E-F) PK/PD study was conducted in the xenograft model with the primary AML cells shown in Figure 6B-D. Single or repeated oral administration of FF-10101 at 10 mg/kg was given to mice with excess blasts at 35 days after intravenous injection of primary AML cells. Plasma and BM concentrations of FF-10101 at indicated time points were analyzed by LC/MS/MS (panel E). BM lysates were subjected to western blotting for detection of FLT3 (t-FLT3), STAT5 (t-STAT5), and their phosphorylated forms (p-FLT3 and p-STAT5) (panel F). (G-I) Primary AML cells with FLT3-D835H mutation were xenografted into NOG mice. Mice were divided into 4 groups (n = 5 or 6/group) based on the percentage of human CD45-positive cells in PB. Six mice were euthanized for identification of engrafted cells in BM before initiation of administration. FF-10101 and quizartinib were orally administered at 10 mg/kg twice daily for 14 days. The percentage of human CD45-positive cells in PB was monitored once per week (panel G). After 14-day administration, the percentage of human CD45-positive cells in BM was determined by flow cytometry (panel H). Mice were weighed at days 0, 3, 6, 9, 12, and 14, and relative body weight to initial body weight (day 0) was represented as percent changes. No significant differences in body weight among groups were observed (panel I). Bars show mean ± SD. *P < .05 compared with control by Dunnett's Multiple Comparison Test.

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