FLT3/ITD is an in vivo target of TTT-3002. BALB/c mice (female, age 6 to 8 weeks) received 2 × 106 Ba/F3-ITD Luc+ cells by tail vein injection on day 0. Engraftment was confirmed on day 7 by luminescence imaging, and mice were treated with TTT-3002 (6 mg/kg twice per day) or vehicle control (1 mM HCl twice per day) for 28 days. (A) Tumor cell abundance on days 7 and 17 by luminescence imaging; representative mice (n = 10) from each treatment cohort shown. (B) Overall survival of vehicle-treated (n = 18; median survival, 17.5 days) and TTT-3002–treated (n = 18; median survival, >100 days) mice at day 100 (P < .0001). (C) Histopathology of PB and BM preparations (Wright-Giemsa stain) of representative mice from each cohort euthanized at day 18 (vehicle) or day 35 (TTT-3002). Red arrows represent examples of leukemic blasts, yellow arrow represents a normal lymphocyte, and green arrow indicates a normal neutrophil. Slides were imaged on an Olympus BX46 microscope with an Olympus DP72 camera at ×20 and ×100 magnifications with 0.5 and 1.3 apertures, respectively; Olympus cellSens Standard 1.5 image acquisition software was used. (D) BALB/C mice (female, age 6 to 8 weeks) received 2 × 106 Ba/F3-ITD Luc+ cells by tail vein injection, and mice were administered a single dose of TTT-3002 (6 mg/kg) anywhere from day 17 to day 20 as they were succumbing to disease when they had high tumor burden. Spleen and BM cells and plasma samples were harvested from these mice at the indicated time points. (E) Total plasma concentrations of TTT-3002 as measured by liquid chromatography/tandem mass spectrometry; n = 3 mice per time point, represented as average ± SD. (F) pFLT3 and pSTAT5 are inhibited in vivo by the earliest time point (4 hours postdosing) and maintained for 12 to 24 hours. Fraction of pFLT3/FLT3 and pSTAT5/STAT5 relative to time 0 hours is indicated below each blot. Western blot is representative of 3 independent experiments.
