Monitoring of Intracellular l‐βD‐Arabinofuranosylcytosine 5′‐Triphosphate in 1‐β‐D‐Arabinofuranosylcytosine Therapy at Low and Conventional Doses

Abstract

1‐β‐D‐Arabinofuranosylcytosine (ara‐C) is used empirically at a low, conventional, or high dose. Ara‐C therapy may be optimal if it is directed by the clinical pharmacokinetics of the intracellular active metabolite of ara‐C, 1‐β‐D‐arabinofuranosylcytosine 5′‐triphosphate (ara‐CTP). However, ara‐CTP has seldom been monitored during low‐ and conventional‐dose ara‐C therapies because detection methods were insufficiently sensitive. Here, with the use of our newly established method (Cancer Res., 56, 1800‐1804 (1996), ara‐CTP was monitored in leukemic cells from acute myelog‐enous leukemia patients receiving low‐ or conventional‐dose ara‐C [subcutaneous ara‐C administration (10 mg/m2) (3 patients), continuous ara‐C infusion (20 or 70 mg/m2/24 h) (7 patients), 2‐h ara‐C infusion (70 mg/m2) (4 patients), and 2‐h infusion of N4‐behenoyl‐l‐β‐D‐arabinofuranosylcy‐tosine, a deaminase‐resistant ara‐C derivative (70 mg/m2) (6 patients)]. Ara‐CTP could be determined at levels under 1μM. There was a close correlation between the elimination half‐life values of the plasma ara‐C and the intracellular ara‐CTP. The presence of ara‐C in the plasma was important to maintain ara‐CTP. The continuous ara‐C and the 2‐h N4‐behenoyl‐l‐β‐D‐arabinofura‐nosylcytosine infusions maintained ara‐CTP and the plasma ara‐C longer than the subcutaneous ara‐C or the 2‐h ara‐C infusion. They also afforded relatively higher ara‐CTP concentrations, and consequently produced ara‐CTP more efficiently than the 2‐h ara‐C infusion. Different administration methods produced different quantities of ara‐CTP even at the same dose.

Characterization of cytarabine-resistant leukemic cell lines established from five different blood cell lineages using gene expression and proteomic analyses

“…2-4). The fact that four of the five cell lines had alterations in ara-CTP-related factors is in agreement with other studies that intracellular activation of ara-C to ara-CTP is the most crucial factor for determination of cellular sensitivity to ara-C (11)(12)(13)(14)18). Our study did not detect an increase in total cellular glutathione, which we previously identified in leukemic cells that were resistant to both ara-C and daunorubicin (36), suggesting that the mechanism of resistance to ara-C of the clones established here might be ara-C-specific.…”

“…Since ara-C cytotoxicity depends on ara-CTP incorporation into DNA and therefore depends on ara-CTP concentration, ara-CTP levels thus represent an index of ara-C cytotoxicity. Intracellular ara-CTP levels also have clinical value since a correlation between the intracellular pharmacokinetics of ara-CTP and response to ara-C therapy has been established (11)(12)(13)(14).…”

“…Because the production of ara-CTP depends on the cellular activation of ara-C, cellular factors that can regulate ara-C activation play a role in ara-C resistance (11)(12)(13)(14)(15)(16). Ara-C catabolism results from rapid deamination by cytidine deaminase to the non-toxic metabolite ara-U (2), while cytosolic 5'-nucleotidase II (cN-II) dephosphorylates ara-CMP (7), Characterization of cytarabine-resistant leukemic cell lines established from five different blood cell lineages using gene expression and proteomic analyses…”

Simple and sensitive method for quantification of fludarabine triphosphate intracellular concentration in leukemic cells using isocratic liquid chromatography

“…The mixture was vortexed for 10 s, and allowed to stand for 15 min at 4 • C. The acidic supernatant was isolated by centrifugation of the sample (15,600×g, 20 s, 4 • C), and then mixed with 100 l of 0.5N potassium hydroxide for neutralization. After another centrifugation (15,600 × g, 20 s, 4 • C), the neutralized supernatant was obtained as an acid soluble fraction (ASF), a nucleotide pool [12,15]. The sample loop for the HPLC had a 500-l space, and the dead volume for the aspiration of the sample by the autosampler was 170 l. By adding 500 and 170 l and some room, the volume of each ASF sample was adjusted to 700 l by the addition of water, and the 500-l aliquot was applied to the chromatographic analysis.…”

“…In clinic, no correlation has been found between the therapeutic outcome and F-ara-ATP in treating chronic lymphocytic leukemia [10]. However, as demonstrated in the case of cytarabine, the parameter for predicting the clinical efficacy of a nucleoside analogue is not the plasma drug concentration but its triphosphate form in leukemic cells [11,12]. Therefore, taken together with the understanding of the mechanism of action, the pharmacokinetic evaluation of F-ara-ATP may provide crucial information on scheduling and dosing fludarabine.…”