Background. Phase 1 clinical studies have shown that an orally bioavailable syk kinase inhibitor, R788/406, is very well tolerated in human volunteers for up to 7 days (and in primates for up to 28 days) at doses achieving concentrations in excess of 5 micromolar (ED50 for in vivo biomarker of syk-inhibition in humans is 1 micromolar). In biochemical kinase assays, R788/406 inhibits syk and FLT3 at less than 10 nM, and in cell-based assays at less than 100 nM. The demonstration of biological activity and excellent tolerability in humans, and the equipotent inhibition of FLT3 and syk kinases in biochemical assays led us to explore the potential for use of R788/406 in a xenograft of a human AML FLT-3ITD mutant cell line.

Objective: To evaluate the in vitro and in vivo activity of R788/406 in a human AML FLT3-ITD mutant cell line, MV411, a model system for examination of FLT3 mutant AML.

Methods: MV411 cells were treated with R788/406 and evaluated for cell viability and markers of apoptosis (Annexin-V/PI and caspase) by FACS. Cell cycle analysis was performed on cells stained with PI. 5 X 106 MV411 cells harvested in logarithmic phase growth were injected with Matrigel SC in NOD/SCID mice. Treatment with R788/406 began when tumors reached a predetermined size (mean volume of 100 mm3) and continued for 26 days. At sacrifice, tumor xenografts were lysed, immunoprecipitated with anti-FLT-3, and probed with anti-phosphotyrosine 4G10 or anti-FLT-3.

Results: R788/406 potently and selectively induced dose-dependent cytotoxicity of MV-411 AML cells in vitro with an ED50 of 20nM. Pretreatment of cells with R788/406 promoted dephosphorylation of constitutively active pFLT3, as well as a reduction of pStat5 and pErk1/2 in the FLT3 signaling cascade. Moreover, R788/406 induced cell cycle arrest in the G1 phase and subsequent apoptosis in MV411 cells in a dose-dependent manner. Twice daily administration of R788/406 to NOD/SCID mice bearing SC MV411 tumors reduced tumor growth significantly in a dose dependent manner. When compared to vehicle controls, daily doses of 40 and 80mg/kg R788/406 resulted in 45% and 82% inhibition of mean tumor volumes, respectively. At study termination mean tumor volumes were 686.90 ± 115.56 and 224.45 ± 49.80 for 40 and 80 mg/kg R788/406-treated animals compared to 1255.48 ± 182.94 for vehicle controls with a final %T/C of -0.4 (range of %T/C throughout study was −7.9 to −0.4). During the study, no significant body weight loss was observed in any of the animals in this study. Ex vivo analyses of subcutaneous tumors from MV411 tumor-bearing mice showed that R788/406 completely inhibited constitutive FLT3 activation and downstream signaling events.

Conclusions: R788/406 is well tolerated in humans (and primates) at concentrations well in excess of those that inhibit syk in vivo. Given the equipotent inhibition of syk and FLT3, the in vivo activity against human syk, and the xenograft data reported here, R788/406 may be a promising agent for FLT-3 AML.

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