A novel phenotypic method to determine fludarabine triphosphate accumulation in T-lymphocytes from hematopoietic cell transplantation patients

Abstract

Purpose: Fludarabine is an integral anticancer agent for patients with chronic lymphocytic leukemia (CLL) and those receiving conditioning regimens prior to allogeneic hematopoietic cell transplantation (HCT). An individual's response to fludarabine may be influenced by the amount of CD4(+) and CD8(+) T-lymphocyte suppression. Fludarabine undergoes cellular uptake and activation to form the cytotoxic metabolite, fludarabine triphosphate (F-ara-ATP).

Methods: We have previously developed a highly sensitive LC-MS method to quantitate intracellular F-ara-ATP concentrations in a leukemic cell line. However, quantitation of F-ara-ATP concentrations within CD4(+) and CD8(+) T-lymphocytes from pharmacokinetic blood samples obtained from patients receiving fludarabine therapy is not feasible because of the limited number of T-lymphocytes that can be isolated from each blood sample. Thus, we sought to determine F-ara-ATP accumulation after ex vivo exposure of freshly isolated human CD4(+) or CD8(+) T-lymphocytes to fludarabine. The method was optimized in T-lymphocytes obtained from healthy volunteers, and proved to be a feasible method to determine F-ara-ATP accumulation in patients undergoing HCT.

Results: Considerable variability was observed in F-ara-ATP accumulation in HCT patients (10.5- and 12.5-fold in CD4(+) and CD8(+) cells, respectively), compared to healthy volunteers (1.6- and 1.9-fold in CD4(+) and CD8(+) cells, respectively). Larger variability was also observed in gene expression of transporters and enzymes involved in F-ara-ATP accumulation in HCT patients; however, F-ara-ATP accumulation was not correlated with gene expression, which is in agreement with previous studies.

Conclusions: The quantitation of F-ara-ATP accumulation in T-lymphocytes provides a novel tool to evaluate patient sensitivity to fludarabine. This tool can be used in future studies to evaluate whether intracellular F-ara-ATP accumulation is associated with efficacy and/or toxicity in patients receiving fludarabine.

Figure 1.. Intracellular disposition of fludarabine.

The schematic displays the intracellular uptake and metabolism of fludarabine to the active metabolite fludarabine triphosphate (F-ara-ATP). Abbreviations: ecto-5’-nucleotidase (CD73), human equilibrative nucleoside transporter 1 (hENT1), human equilibrative nucleoside transporter 2 (hENT2), human concentrative nucleoside transporter 3 (hCNT3), deoxycytidine kinase (dCK), dephosphorylation by 5’-nucleotidase (CN-II), deoxynucleotidase-1 (dNT-1), adenylate kinase (AK), and nucleoside diphosphate kinase (NDK).

Figure 2.. Variability in F-ara-ATP accumulation in CD4⁺ and CD8⁺ T-lymphocytes.

Range of F-ara-ATP accumulation in CD4⁺ (●) and CD8⁺ (◯) T-lymphocytes isolated from healthy volunteers and patients awaiting HCT. Solid lines indicate the mean values (pmol/1×10⁶ cells/4 hours) in each group.

Figure 3.. F-ara-ATP accumulation in CD4⁺ and CD8⁺ T-lymphocytes from HCT patients.

Correlation between F-ara-ATP accumulation (pmol/1×10⁶ cells/4 hours) in CD4⁺ and CD8⁺ T-lymphocytes in HCT patients (n = 34).

Figure 4.. Fludarabine-induced apoptosis in CD4⁺ and CD8⁺ T-lymphocytes from healthy volunteers.

Concentration-dependent (panel A) and time-dependent (panel B) fludarabine-induced apoptosis was evaluated by determining percentage of viable T-lymphocytes (CD4⁺: solid line; CD8⁺: dotted line) from 3 healthy volunteers (designated ●, ∎, and ▴) at variable fludarabine concentrations over a range of incubation times. Concentration dependence was evaluated with a 24 hour exposure to fludarabine. Time dependence was evaluated in T-lymphocytes exposed to 5μM fludarabine. Viable cells were those that did not bind either Annexin V or 7AAD dyes.