Biochemical modulation of cytarabine triphosphate by clofarabine

Purpose: Clofarabine has proven to be effective in the treatment of adult and pediatric acute myelogenous leukemia (AML). To investigate if clofarabine could be used with success in biochemical modulation strategies, we investigated the biochemical modulation of cytarabine triphosphate (ara-CTP) by clofarabine in a myeloid leukemia cell line and the effect of this combination on cytotoxicity. Experimental design: K562 cells were incubated with clofarabine and ara-C either sequentially or simultaneously to evaluate the combination effect on their phosphorylated metabolites. Clonogenic assays were used to determine the cytotoxicity of each agent alone and in combination. Deoxynucleotide analysis was performed to assess the effect of clofarabine on dNTPs. Results: Clofarabine added either simultaneously or in sequence increased ara-CTP accumulation. The maximal modulation of ara-CTP accumulation occurred with 1 mu M clofarabine. This level was achieved at the maximum tolerated dose for adult and pediatric patients with AML. With 10 mu M ara-C alone, 86 mu M ara-CTP had accumulated after 3 h. The optimal sequence for the drug combination, i.e., clofarabine followed 4 h later by ara-C, resulted in 248 mu M ara-CTP at 3 h. Clofarabine accumulated maximally in the monophosphate form. Preincubation with ara-C did not affect the triphosphate form, but it lowered clofarabine monophosphate. Clofarabine resulted in the intracellular decrease of dATP and dGTP levels. Clonogenic assays revealed that the combination of clofarabine and ara-C produced synergistic killing of myeloid leukemia cells. Conclusions: These findings demonstrate that combination of clofarabine followed by ara-C results in a biochemical modulation of ara-CTP and synergistic cell kill. These studies provide a compelling rationale for clinical trials using this combination regimen for adult and pediatric patients with AML.