American Society of Hematology

Figure 7

$Figure 7. Intermittent inhibition of FLT3 in vivo. (A) Parallel flasks of Molm14 cells were incubated in culture medium containing escalating doses of KW-2449. For one set (4 hours, twice daily; dashed line), cells were exposed to KW-2449 for 4 hours, then washed in warm drug-free medium and resuspended in DMSO control medium. This was repeated twice daily for a total of 52 hours (5 total “doses”). Control cells (continuous; solid line) were treated in continuous fashion. The continuously treated cells were likewise washed twice daily, but then resuspended in KW-2449. One set of cells had drug incubated on the cells for 4-hour periods twice daily and the other set had continuous exposure to KW-2449. At the 52-hour time point each flask was analyzed by Trypan blue, MTT, and annexin V (data not shown for Trypan blue or annexin V). The results display reduced cytotoxicity with intermittent exposure to KW-2449. (B) Peripheral blood blasts were isolated by Ficoll gradient centrifugation (“Methods”) from a 60-year-old woman with relapsed/refractory AML harboring an FLT3/ITD mutation and incubated in culture medium containing increasing concentrations of KW-2449. After 48 hours, the MTT assay was performed. Blasts were isolated at the beginning of cycle 1 and at the beginning of cycle 2. (C) PIA results for cycle 1 for this patient, showing phosphorylated FLT3 and STAT5 on days 1 and 14 for different time points. Vertical lines have been inserted to indicate a repositioned gel lane. (D) Graph of WBCs over time. The horizontal black lines denote the time period during which the patient received treatment with KW-2449. The trial dictated a 5- to 7-day break from therapy between cycles. The patient was taken off study for progressive disease after completing the second cycle. (E) FLT3 phosphorylation in circulating blasts. These immunoblots show P-FLT3 immediately prior to, and 2 hours after, dosing with KW-2449 during the first and second cycles. Densitometry was performed and the density of the 2-hour time point was expressed as a percent of the pretreatment sample.$

Intermittent inhibition of FLT3 in vivo. (A) Parallel flasks of Molm14 cells were incubated in culture medium containing escalating doses of KW-2449. For one set (4 hours, twice daily; dashed line), cells were exposed to KW-2449 for 4 hours, then washed in warm drug-free medium and resuspended in DMSO control medium. This was repeated twice daily for a total of 52 hours (5 total “doses”). Control cells (continuous; solid line) were treated in continuous fashion. The continuously treated cells were likewise washed twice daily, but then resuspended in KW-2449. One set of cells had drug incubated on the cells for 4-hour periods twice daily and the other set had continuous exposure to KW-2449. At the 52-hour time point each flask was analyzed by Trypan blue, MTT, and annexin V (data not shown for Trypan blue or annexin V). The results display reduced cytotoxicity with intermittent exposure to KW-2449. (B) Peripheral blood blasts were isolated by Ficoll gradient centrifugation (“Methods”) from a 60-year-old woman with relapsed/refractory AML harboring an FLT3/ITD mutation and incubated in culture medium containing increasing concentrations of KW-2449. After 48 hours, the MTT assay was performed. Blasts were isolated at the beginning of cycle 1 and at the beginning of cycle 2. (C) PIA results for cycle 1 for this patient, showing phosphorylated FLT3 and STAT5 on days 1 and 14 for different time points. Vertical lines have been inserted to indicate a repositioned gel lane. (D) Graph of WBCs over time. The horizontal black lines denote the time period during which the patient received treatment with KW-2449. The trial dictated a 5- to 7-day break from therapy between cycles. The patient was taken off study for progressive disease after completing the second cycle. (E) FLT3 phosphorylation in circulating blasts. These immunoblots show P-FLT3 immediately prior to, and 2 hours after, dosing with KW-2449 during the first and second cycles. Densitometry was performed and the density of the 2-hour time point was expressed as a percent of the pretreatment sample.

This Feature Is Available To Subscribers Only