Quantitative evaluation of intracellular uptake of daunorubicin in acute myeloid leukemia: a method analysis

Abstract

Critical technical parameters to establish a reliable method for quantifying the intracellular content of anthracyclines were evaluated in patients with acute myelocytic leukemia (AML); two methods were used for the isolation of leukocytes from the peripheral blood and two methods, for the extraction of daunorubicin (DNR), daunorubicinol (DOL), and doxorubicin (DOX) from these cells, followed by drug analysis using high-performance liquid chromatography (HPLC). At 0-4 degrees C the recovery of leukocytes after methylcellulose separation was low (64%). Cold hypotonic lysis gave better recovery (100%) when performed at the same temperature. After low-volume (2 ml extraction mixture) drug extraction from isolated leukocytes, the recoveries of DNR, DOL, and DOX from the cells were low, and they were inversely related to the cellularity of the sample, irrespective of the amount of drug in the cells. With high-volume extraction (5 ml extraction mixture) the recoveries were better (up to 95%), but they remained dependent on the cellularity. A correction factor accounting for these cellularity-related recoveries was applied to calculate the DNR and DOL contents of the leukocytes. Finally, using this information, plasma and cellular DNR and DOL levels were measured in seven patients with AML during their first course of remission induction therapy. The cellular DNR levels appeared to vary over a broad range and did not correlate with plasma pharmacokinetics.