Figure 1.
Chemical structure of BPR1J-340 and BPR1J-097.
BPR1J-340: N1-(3-4-[([(5-ethyl-3-isoxazolyl)amino]carbonylamino)methyl]phenyl-1H-5-pyrazolyl)-4-[(4-methylpiperazino)methyl]benzamide. BPR1J-097: N1-(3-3-[(phenylsulfonyl)amino]phenyl-1H-5-pyrazolyl)-4-(4-methylpiperazino)benzamide.
Table 1.
Specificity of kinase inhibition of BPR1J-340.
Table 2.
Anti-proliferative activity of BPR1J-340 against a panel of tumor cell lines.
Figure 2.
BPR1J-340 inhibits FLT3-dependent signaling.
(A) MV4;11 cells were treated with BPR1J-340 at the indicated concentrations for 1 hour. The phosphorylation status of FLT3 and STAT5 were evaluated by Western blot analysis. (B) HEK 293T cells were transfected with FLT3-WT-, FLT3-ITD-, or FLT3-D835Y-expressing plasmids for 24 hours and then incubated with various concentrations of BPR1J-340 for 1 hour. The FLT3 phosphorylation status in the transfected cells was evaluated by Western blot analysis.
Figure 3.
BPR1J-340 induces apoptosis in MOLM-13 and MV4;11 cells.
Western blotting revealed that BPR1J-340 was able to induce apoptosis in FLT3-ITD-driven FLT3-ITD+ AML cells. MOLM-13 (A) and MV4;11 (B) cells were treated with BPR1J-340 at the indicated concentrations for 24 hours, and the cell lysates were then subjected to Western blot analysis using antibodies against caspase-3 and PARP (poly-ADP-ribose polymerase). (full length caspase-3 (FL-caspase-3), cleavage of caspase-3 (CL-caspase-3), full length poly(ADP-ribose) polymerase (FL-PARP), cleavage of PARP (CL-PARP)).
Figure 4.
The effect of BPR1J-340 and SAHA versus drug combination on AML FLT3-ITD+ cells.
MOLM-13 or MV4;11 cells were seeded at an initial concentration of 1×105 cell mL−1 for 24 hours and then treated with BPR-1J340 either alone or in combination with SAHA in the indicated concentrations. (A) Viable cells were counted after staining with trypan blue dye at indicated time point (B) At 48-hours after treatment, cells were stained with FITC-labeled annexin V and propidium iodide and percentages of apoptotic cells were analyzed by flow cytometry. (C) The representative flow cytometry histograms of assays of annexin positive apoptotic cells at 48 hours drug treatment (D) MOLM-13 cells were treated with drugs for 20 hours, and then followed by Western blot analysis to assess changes in protein levels with the indicated antibodies. Statistical analysis was performed using Student's t-test. *Indicates significant difference compared to vehicle control (* p<0.05, * * * p<0.01). Data shown are representative of multiple independent experiments.
Figure 5.
Pharmacokinetic profile of BPR1J-340 in rats.
A single intravenous bolus dose (1.5 mg/kg) of BPR1J-340 was administered to adult male Sprague-Dawley rats (n = 3). Data illustrate mean values (n = 3) ± S.D. of plasma concentrations of BPR1J-340 at each timepoint.
Table 3.
Pharmacokinetic parameters of BPR1J-340 in rats.
Figure 6.
Antitumor activity of BPR1J-340 against FLT3-ITD-driven leukemia tumor growth in nude mice.
BPR1J-340 (i.v.) is active against human acute leukemia MOLM-13 tumors growing subcutaneously and can reduce the size of tumors even after the tumors were allowed to grow to a size of >630 mm3. (A) The in vivo antitumor effect of BPR1J-340 in the MOLM-13 xenograft nude mice model. The growth of the tumor xenograft was inhibited by BPR1J-340 [5 mg/kg, i.v.]; p<0.05 compared with vehicle treatment. (B) The subcutaneously growth of a MOLM-13 tumor of large size (>630 mm3) in nude mice could be significantly reversed by BPR1J-340 [20 mg/kg, i.v.].