A new high-performance liquid chromatography method determines low production of 9-beta-D-arabinofuranosylguanine triphosphate, an active metabolite of nelarabine, in adult T-cell leukemia cells.

The 9-beta-D-arabinofuranosylguanine (ara-G), an active compound of nelarabine, demonstrates potent cytotoxicity specifically on T-cell malignancies. In cells, ara-G is phosphorylated to ara-G triphosphate (ara-GTP), which is subsequently incorporated into DNA, thereby inhibiting DNA synthesis. Because ara-GTP is crucial to ara-G's cytotoxicity, the determination of ara-GTP production in cancer cells is informative for optimizing nelarabine administration. Here, we developed a new, sensitive isocratic-elution HPLC method for quantifying ara-GTP. Samples were eluted isocratically by using phosphate buffer at a constant flow rate. Ara-GTP was clearly separated from other nucleotides by using an anion-exchange column and it was quantitated by its peak area at 254 nm. The standard curve was linear with low variability and a sensitive detection limit (10 pmol). Furthermore, due to ara-G's specificity to T-cells we hypothesized that nelarabine might be effective against adult T-cell leukemia (ATL). The ara-GTP production was compared between T-lymphoblastic leukemia CCRF-CEM and ATL cell lines in vitro. When CEM cells were incubated with ara-G, the ara-GTP production increased in a concentration- and time-dependent manner. In contrast, 5 ATL cell lines accumulated lower ara-GTP in the same condition. While ara-G inhibited the growth of CEM cells with a 50% growth inhibition concentration of 2 microM, the inhibitory-concentration values were >1 mM in 8 of the 12 ATL cell lines. This ineffectiveness appeared to correspond with the low ara-GTP production. The present study is the first to evaluate the potential of ara-G against ATL cells; our results suggest that nelarabine would not be effective against ATL.

Evaluation of the combination of nelarabine and fludarabine in leukemias: clinical response, pharmacokinetics, and pharmacodynamics in leukemia cells.

PURPOSE: A pilot protocol was designed to evaluate the efficacy of fludarabine with nelarabine (the prodrug of arabinosylguanine [ara-G]) in patients with hematologic malignancies. The cellular pharmacokinetics was investigated to seek a relationship between response and accumulation of ara-G triphosphate (ara-GTP) in circulating leukemia cells and to evaluate biochemical modulation of cellular ara-GTP metabolism by fludarabine triphosphate. PATIENTS AND METHODS: Nine of the 13 total patients had indolent leukemias, including six whose disease failed prior fludarabine therapy. Two patients had T-acute lymphoblastic leukemia, one had chronic myelogenous leukemia, and one had mycosis fungoides. Nelarabine (1.2 g/m(2)) was infused on days 1, 3, and 5. On days 3 and 5, fludarabine (30 mg/m(2)) was administered 4 hours before the nelarabine infusion. Plasma and cellular pharmacokinetic measurements were conducted during the first 5 days. RESULTS: Seven patients had a partial or complete response, six of whom had indolent leukemias. The disease in four responders had failed prior fludarabine therapy. The median peak intracellular concentrations of ara-GTP were significantly different (P =.001) in responders (890 micromol/L, n = 6) and nonresponders (30 micromol/L, n = 6). Also, there was a direct relationship between the peak fludarabine triphosphate and ara-GTP in each patient (r = 0.85). The cellular elimination of ara-GTP was slow (median, 35 hours; range, 18 to > 48 hours). The ratio of ara-GTP to its normal counterpart, deoxyguanosine triphosphate, was higher in each patient (median, 42; range, 14 to 1,092) than that of fludarabine triphosphate to its normal counterpart, deoxyadenosine triphosphate (median, 2.2; range, 0.2 to 27). CONCLUSION: Fludarabine plus nelarabine is an effective, well-tolerated regimen against leukemias. Clinical responses suggest the need for further exploration of nelarabine against fludarabine-refractory diseases. Determination of ara-GTP levels in the target tumor population may provide a prognostic test for the activity of nelarabine.

High-performance liquid chromatography method for the determination and quantitation of arabinosylguanine triphosphate and fludarabine triphosphate in human cells.

A gradient anion-exchange high-performance liquid chromatographic assay was developed for the simultaneous determination and quantitation of the cytotoxic triphosphates of arabinosylguanine (ara-GTP) and fludarabine (F-ara-ATP). The method was validated with respect to selectivity, recovery, linearity, precision, and accuracy using authentic standards. To test this assay in a more complex biological matrix, perchloric acid extracts of circulating human leukemia cells spiked with known concentrations of ara-GTP and F-ara-ATP were examined. Finally, to assess the clinical utility of our method, perchloric acid extracts of circulating human leukemia cells isolated from patients treated with fludarabine and nelarabine were analyzed. The range of quantitation was 0.0125-10 nmol for the ara- and native NTPs in cellular extracts. This assay should be helpful in establishing the mechanistic rationales for drug scheduling and combinations of nelarabine and fludarabine, and for correlating the therapeutic efficacy and levels of the cytotoxic triphosphates in target cells.

Highly sensitive high-performance liquid chromatographic assay for 1-beta-D-arabinofuranosylcytosine-5'-stearyl phosphate (cytarabine-ocfosfate).

An ion-pair HPLC method for the determination of 1-beta-D-arabinofuranosylcytosine-5'-stearyl phosphate (cytarabine-ocfosfate I) was developed, using a phenyl-bonded column under reversed-phase conditions with a mobile phase of acetonitrile-buffered water (pH 6.8) (50:50) for isocratic elution. A reproducible sample clean-up was achieved by solid-phase extraction. In order to reach the low limit of detection of 2 ng/ml, an enrichment switching system was used. The present validation leads to a limit of quantification of 5 ng/ml with a coefficient of variation (C.V.) of 10%. The total time of measurement was shortened by a back-flush procedure to restore the conditions after each run. UV detection at 275 nm was applied. The recoveries for plasma samples ranged from 56.4 to 64.1%, regardless of drug concentrations. The intra-assay C.V. was about 4% (40 measurements at four different concentrations). The inter-assay recovery (ten measurements over ten days) at a plasma concentration of 50 ng/ml was 57% with a C.V. of 8.25%. Based on this HPLC method, the pharmacokinetics of I were measured during a clinical phase I/II study.

Detection and separation of intracellular 1-beta-D-arabinofuranosylcytosine-5-triphosphate by ion-pair high-performance liquid chromatography.

An ion-pair high-performance liquid chromatographic method, using a reversed-phase C18 column, was developed to provide an isocratic, sensitive, fast and reproducible separation of intracellular 1-beta-D-arabinofuranosylcytosine-5-triphosphate and its measurement at a low limit of 5 pmol by ultraviolet absorbance at 280 nm with a coefficient of variation lower than 10%. A rapid separation is achieved by using a backflush procedure at 16 min and the retention time is 14 min.

[Relation between plasma Ara-C and intracellular Ara-CTP levels by intermediate dose and high dose Ara-C administration in the treatment of AML].

Plasma level of cytosine arabinoside (Ara-C) and intracellular cytosine arabinoside triphosphate (Ara-CTP) in peripheral blood and bone marrow were measured in 8 patients with non treated acute myelogenous leukemia. Ara-C was administered by 1 hr infusion (3 g/m2 and 500 mg/m2) and was followed for 12 hrs. The AUC of Ara-C in plasma following 3 g/m2 infusion were greater than the 500 mg/m2 (p less than 0.05). Intracellular Ara-CTP in peripheral blood following 3 g/m2 and 500 mg/m2 infusions were on the same level, after 1 hr. But AUC of intracellular Ara-CTP following 3 g/m2 infusion was greater than 500 mg/m2. There was a correlation between AUC of Ara-C in the peripheral blood (p less than 0.01). Intracellular Ara-CTP in the bone marrow and peripheral blood showed a similar level. Intracellular Ara-CTP in bone marrow was lower than in peripheral blood, however, there was no correlation between intracellular Ara-CTP in bone marrow and in peripheral blood.

High-pressure liquid chromatographic methods for determining arabinosyladenine-5'-monophosphate, arabinosyladenine, and arabinosylhypoxanthine in plasma and urine.

New high-pressure liquid chromatographic methods for determining concentrations of arabinosyladenine (Ara-A), its 5'-monophosphate (Ara-AMP), and arabinosylhypoxanthine (Ara-H) in plasma and urine are presented. A fluorescence detector is used for Ara-A and Ara-AMP, which are first converted to highly fluorescent derivatives with chloroacetaldehyde. This increases sensitivity greatly over previous methods. The sensitivities of the methods (in micrograms per milliliter) are as follows: in plasma, Ara-AMP, 0.002; Ara-A, 0.0015; and Ara-H, 0.35; and in urine, 9 times these values, respectively. Drug concentration data are also presented, which were obtained after doses of Ara-AMP were given intramuscularly to two patients treated with this drug for severe herpes zoster. One patient was given 13 mg of Ara-AMP per kg of body weight once daily, and the other was given 6.5 mg/kg twice daily. Peak Ara-AMP and Ara-A levels in plasma occurred within 1 h after the doses, and neither exceeded 2 micrograms/ml. Ara-AMP and Ara-A concentrations in plasma fell to less than 0.01 micrograms/ml in both patients by 4 to 6 h after the doses. Peak Ara-H concentrations in plasma occurred within 1 to 2 h after doses and were 21 micrograms/ml in patient 1 and 2. The highest concentration of Ara-AMP in urine was 0.09 micrograms/ml. The highest Ara-A concentration in urine was 62 micrograms/ml, and the highest Ara-H concentration in urine was 1,080 micrograms/ml. An interfering substance of unknown nature, cochromatographing with Ara-H, was encountered sporadically in urine samples. An algorithm based on differential spectrophotometry to identify and correct for this problem is described. Estimates of the renal clearances of Ara-AMP, Ara-A, and Ara-H are also given.

Two simple high-performance liquid chromatographic methods for simultaneous determination of 2'-deoxycytidine 5'-triphosphate and cytosine arabinoside 5'-triphosphate concentrations in biological samples.

Cytosine arabinoside (ara-C) has been used in the treatment of leukemia, but its exact mechanism of cytotoxicity is not yet known. One of the proposed mechanisms for the effectiveness of this drug in treating leukemias suggests that a metabolite of ara-C, i.e., 2'-deoxycytidine 5'-triphosphate (araCTP), competes with cytosine arabinoside 5'-triphosphate (dCTP) for binding to DNA polymerase. The ratio of the drug metabolite to the endogenous nucleotide (araCTP/dCTP) may, therefore, be important in determining the effectiveness of ara-C therapy. This ratio may also play a role in drug resistance. Previously published methods have focused on either araCTP or dCTP, along with metabolites and analogues of one of these compounds. The methods presented here provide two simple, sensitive ways to measure dCTP and araCTP in the same biological sample.