The infected bone marrow cells were then injected into the sub-lethally irradiated (600 cGy) recipient NOD/SCID mice at 1106 cells/mouse

The infected bone marrow cells were then injected into the sub-lethally irradiated (600 cGy) recipient NOD/SCID mice at 1106 cells/mouse. To evaluate therapeutic efficacy in the C3H leukemia model, mice were treated beginning the second day after cell inoculation with (1) imatinib (150 mg/kg, toxicity analysis of targeted TG101209 C3H mice were treated with targeted TG101209 micelles (20 mg/kg or 40 mg/kg, analysis, sample sizes were chosen to ensure adequate power to detect a pre-specified effect size. and its downstream JAK2 and STAT5. The effective Nadolol dosage to overcome therapy resistance identified in an setting serves as a guidance to develop the proper drug formulation for efficacy. A targeted formulation was developed to achieve sustained bone marrow TG101209 concentration at or above 17.5 M for effective killing of CML cells correlation, bone marrow targeting, therapy-resistance, chronic myeloid leukemia Introduction Philadelphia chromosome-positive chronic myeloid leukemia (CML) is caused by constitutive activation of the oncogenic p210BCR-ABL tyrosine kinase as result of a reciprocal translocation between chromosomes 9 and 22 (1). Therefore, CML treatment in clinic has been focused on blocking kinase activity of the fusion protein with tyrosine kinase inhibitors (TKIs) such as imatinib, dasatinib, nilotinib, and recently, ponatinib (2C5). However, patients develop resistance to these targeted therapy drugs (6, 7). Among the potential mechanisms for therapy resistance include development of new mutations in the fusion gene such as the T315I mutation (7), unfavorable pharmacokinetics and biodistribution of TKIs (8, 9) and a protective bone marrow microenvironment (10, 11). Strategies to address these critical issues will be effective in treating CML. To identify a dose range for effective cell killing, CML cells have traditionally been treated with TKIs for 24~72 hours in cell culture (12C14). However, it is impossible to maintain the therapeutic magnitude and duration of the drugs due to their rapid drug metabolism and clearance, as the plasma half-lives for dasatinib and nilotinib are 2 hours and 1 hour in mice, respectively (15, 16). Although the IC50 value for nilotinib on inhibition of Ba/F3 cells overexpressing the BCR-ABL fusion protein in cell Nadolol culture is less than 10 nM (14), a daily dosage of 75~100 mg/kg is needed to treat animals in order to achieve a desirable therapeutic efficacy in murine CML models (17, 18). Since the peak plasma drug concentration could already reach 14 M at a 25 mg/kg treatment Nadolol dosage (16), which is over 1,000 folds of the IC50 value in cell culture, the peak plasma concentration at these therapeutic dosages will be even higher. Thus, the cell growth inhibition Nadolol study provided little guidance on the design of efficacy studies. Therefore, more reliable approaches are needed to predict therapeutic outcome based on the cell killing data. In the current study, we applied TG101209 to treat CML cells that are resistant to BCR-ABL targeted therapy in order to 1) establish an – correlation on treatment dosage, and 2) develop an effective treatment for therapy-resistant CML. The TG compounds (TG101209 and TG101348) were originally developed as inhibitors of the JAK2/STAT5 signaling (19, 20). STAT5 is one of the critical mediators for CML initiation, maintenance and TKI resistance (21). Upon BCR-ABL inhibition, CML progenitor cells depend on high levels of cytokine-mediated JAK2/STAT5 activation for continued viability inside the bone marrow (22). So targeting the JAK2/STAT5 signaling with inhibitors such as TG101209 is an ideal approach to prevent CML cell escape from BCR-ABL-targeted therapy. Interestingly, a recent study indicated that TG101209 could also inhibit the p210BCR-ABL tyrosine kinase activity (21). Thus, TG101209 might serve as a multi-kinase inhibitor to block p210BCR-ABL tyrosine kinase-dependent and independent pathways. Since CML cells carrying a T315I mutation in the BCR-ABL gene (p210T315I) are resistant to imatinib and dasatinib (6), and clinical cases of resistance to ponatinib have also been identified RICTOR (7), we applied cells with overexpressed p210T315I to test drug efficacy in this study. We performed transient treatments of murine myeloid 32D cells overexpressing p210T315I with TG101209 in cell culture, and identified the concentration range where CML cells were sensitive to TG101209 treatment. We then developed a bone-targeted formulation to achieve bone marrow TG101209 concentration at or above the effective concentration range in a sustained manner so as to effectively kill leukemia cells. Subsequently, we applied two murine leukemia models to demonstrate therapeutic efficacy. Materials and Methods Cell culture 32D cells overexpressing wide-type BCR-ABL (32Dp210WT) or T315I.

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