Abstract

A TCL-1 transgenic mouse model of CLL was recently described that develops a slowly proliferating CD19+/CD5+/IgM+ clonal leukemia at 10–13 months of age, characterized by lymphocytosis, splenomegaly, and lymphadenopathy. To determine if this model has therapeutic properties similar to human CLL, we performed in vitro cytotoxicity assays using splenic B-cells isolated from leukemic TCL-1 mice. Results indicated that fludarabine, a common treatment for CLL, as well as experimental agents, 17-AAG, valproic acid, flavopiridol, and a novel kinase inhibitor OSU03012, each have activity against the mouse TCL-1-mediated leukemia at concentrations similar to that observed in human CLL cells in vitro. Subsequent in vivo studies in TCL-1 transgenic mice with evidence of leukemia were performed with low dose fludarabine (34 mg/kg IP, day 1–5, every month) versus saline control (n=10 per group) and were treated for three cycles. Mice receiving fludarabine had a notable decline in blood lymphocyte count. Furthermore, the median survival time for the fludarabine treated mice was 61 days, as compared to 31 days for the control mice (p=0.046). However, as seen in human CLL, treated mice became resistant to fludarabine over time as determined by lymphocyte counts. Thus, our preliminary data suggest the TCL-1 transgenic mouse model of CLL has a similar therapeutic response pattern to human CLL. We next assessed the ability of a novel PDK1/AKT inhibitor OSU03012 to mediate in vivo activity in this model. This agent previously has been shown to mediate activity against both CLL cells and TCL-1 leukemia cells in vitro through a caspase and bcl-2 independent pathway. Surprisingly, OSU03012 demonstrated no early or late in vivo activity at a dose (200 mg/kg) previously demonstrated to inhibit prostate xenograft tumor growth in vivo. The lack of efficacy with this compound in the TCL-1 transgenic mouse prompted us to examine in vivo properties such as protein binding, non-linear absorption of drug that could prevent attaining the concentration of free drug necessary to mediate apoptosis, or stromal cell contact that could promote cellular resistance to OSU03012. As measured using a liquid chromatography/tandem mass spectrometry assay, plasma levels of OSU03012 reached peak levels of 7.5–10μM in the treated mice. Interestingly, very high protein binding of OSU03012 to murine plasma (98–99%) was detected. This finding was further corroborated by parallel in vitro cytotoxicity studies in fetal bovine serum and mouse serum, in which the IC50 concentrations were 3.558μM and 14.15μM, respectively. These studies indicated that plasma levels of OSU03012 sufficient to mediate cytotoxicity against TCL-1 leukemia cells were not achieved in vivo. A second therapeutic study using higher doses of OSU03012 is underway to attain these higher concentrations to confirm in vivo activity of OSU03012. Preclinical studies of other highly effective agents (e.g., Flavopiridol) have recently demonstrated the importance of defining differences in plasma protein binding and schedule of administration of novel agents. It is important to consider the impact of plasma protein binding in designing early phase I studies in patients. Hopefully, this murine model will facilitate future preclinical studies to optimize the schedule and dose of drug administration to confirm the in vivo activity of promising new agents, including OSU03012.

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