Author(s): TAKAHIRO YAMAUCHI, RIE NISHI, TAKANORI UEDA
Background/Aim: An active metabolite of the anti-leukemia agent clofarabine (Cl-F-ara-A) is an intracellular triphosphate form, Cl-F-ara-ATP. Monitoring this active form could provide crucial information for optimizing treatment regimens based on Cl-F-ara-A. A simple, isocratic HPLC method was developed. Materials and Methods: Samples (500 μl) from leukemic cells were loaded onto an anion-exchange column and eluted with a phosphate-acetonitrile buffer (flow rate: 0.7 ml/min) at ambient temperature. The Cl-F-ara-ATP concentration was determined by measuring absorbance at 254 nm. Results: The standard curve was linear, with minimal within-day and inter-day variability. Recovery was excellent; low and high quantitation limits were 10 pmol and 5,000 pmol, respectively. Cl-F-ara-ATP eluted independently of ATP, GTP, UTP, and CTP. Production of Cl-F-ara-ATP was successfully measured in cultured leukemia HL-60 cells treated in vitro with Cl-F-ara-A. Conclusion: This method is simple, sensitive and applicable for determination of the ClF-ara-ATP content of biological materials. Clofarabine, 2-chloro-2’-arabinofluoro-2’-deoxyadenosine (Cl-F-ara-A), is a second-generation purine nucleoside analog, notable for having halogenated purine and ribose rings (1). In 2004, Cl-F-ara-A received accelerated approval from the US Food and Drug Administration for the treatment of pediatric patients with refractory acute lymphoblastic leukemia (2-4). The agent is currently undegoing several clinical trials targeting other malignancies, including acute myeloid leukemia.