title: A sensitive new method for clinically monitoring cytarabine concentrations at the DNA level in leukemic cells.
abstract: Cytarabine (ara-C), a major antileukemic agent, is phosphorylated in the cell to cytarabine triphosphate (ara-CTP), which is then partly incorporated into DNA. The drug incorporation into DNA poisons the extending primer against further incorporation of deoxyribonucleotides including dCTP, ultimately inhibiting DNA synthesis. While intracellular ara-CTP concentration has been found to predict clinical outcome, cytotoxicity in vitro is determined primarily by the extent of drug incorporation into DNA. However, clinically appropriate quantitation methods for ara-C at the DNA level have not been available. We developed a sensitive new method for monitoring ara-C incorporated into DNA in vivo. After DNA from leukemic cells was fractionated using the Schmidt-Thannhauser-Schneider method, it was degraded to constituent nucleosides to release ara-C, which was isolated from the nucleosides using HPLC and then measured by radioimmunoassay. Recovery for DNA fractionation, ara-C release by degradation, and ara-C isolation were 92.0+/-6.4%, 90.7+/-9.4%, and 98.5+/-1.4%, respectively. The method was found to determine ara-C incorporation into DNA of ara-C-treated HL 60 cells in vitro with minimal interassay variation. The values determined were compatible with those determined by scintillation counting in parallel experiments using tritiated ara-C. Our method could be used to monitor DNA-incorporated ara-C concentrations during ara-C therapy, together with plasma ara-C and intracellular ara-CTP concentrations. ara-C incorporation into DNA appeared to be associated with intracellular retention of ara-CTP or persistence of plasma ara-C. Thus, the present method is sensitive, accurate, precise, and may permit therapeutic drug monitoring at the DNA level for better individualization of antileukemic regimens.
authors.affiliation: First Department of Internal Medicine, University of Fukui, 23 Shimoaizuki, Matsuoka, Fukui 910-1193, Japan. tyamauch@fmsrsa.fukui-med.ac.jp <tyamauch@fmsrsa.fukui-med.ac.jp>
authors.forename: Takahiro
authors.initials: T
authors.lastname: Yamauchi
authors.forename: Takanori
authors.initials: T
authors.lastname: Ueda
mesh_terms: D000368:Aged; D000964:Antimetabolites, Antineoplastic / Q000032:analysis / Q000378:metabolism; D003561:Cytarabine / Q000032:analysis* / Q000378:metabolism; D004273:DNA, Neoplasm / Q000032:analysis* / Q000378:metabolism; D016903:Drug Monitoring / Q000379:methods; D005260:Female; D018922:HL-60 Cells; D006801:Humans; D007938:Leukemia / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D012680:Sensitivity and Specificity; D014407:Tumor Cells, Cultured
chemical_list: D000964:Antimetabolites, Antineoplastic; D004273:DNA, Neoplasm; D003561:Cytarabine