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

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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.

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Abbreviations

AK:

Adenylate kinase

AUC:

Area under the plasma concentration–time curve

CD73:

Ecto-5′-nucleotidase

CLL:

Chronic lymphocytic leukemia

CN-II:

5′-nucleotidase

dCK:

Deoxycytidine kinase

dNT-1:

Deoxynucleotidase-1

F-ara-ADP:

Fludarabine diphosphate

F-ara-AMP:

Fludarabine monophosphate

F-ara-ATP:

Fludarabine triphosphate

GUS:

β-Glucuronidase

hCNT3:

Human concentrative nucleoside transporter 3

HCT:

Hematopoietic cell transplantation

hENT1:

Human equilibrative nucleoside transporter 1

hENT2:

Human equilibrative nucleoside transporter 2

LC–MS:

Liquid chromatography–mass spectrometry

NBMPR:

Nitrobenzylthioinosine

NDK:

Nucleoside diphosphate kinase

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Acknowledgments

The authors would like to thank Dr. John T. Slattery for his thoughtful discussions about the manuscript, Dr. Aaron G. Ren for his methodical experiments to show linearity over fludarabine concentration incubation, and Thomas Kalhorn for his contribution to the initial development of the LC–MS method. Supported by grants from the National Institutes of Health (CA18029, CA15704, CA78902, DK56465, HL36444, and HL91744). ELW is supported by the Elmer M. and Joy B. Plein Fellowship for Excellence in Pharmacy Education, School of Pharmacy, Seattle, WA, USA.

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  1. Erica L. Woodahl

    Present address: Department of Biomedical and Pharmaceutical Sciences, University of Montana, Missoula, MT, USA

Authors and Affiliations

  1. Clinical Research Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA

    Erica L. Woodahl, Joanne Wang, Shelly Heimfeld, Brenda M. Sandmaier, Paul V. O’Donnell, Brian Phillips, Linda Risler, David K. Blough & Jeannine S. McCune

  2. School of Pharmacy, University of Washington, Seattle, WA, USA

    Erica L. Woodahl, Joanne Wang, David K. Blough & Jeannine S. McCune

  3. School of Medicine, University of Washington, Seattle, WA, USA

    Brenda M. Sandmaier & Paul V. O’Donnell

  4. Department of Pharmacy, University of Washington, Box 357630, Seattle, WA, 98195, USA

    Jeannine S. McCune

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Correspondence to Jeannine S. McCune.

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Woodahl, E.L., Wang, J., Heimfeld, S. et al. A novel phenotypic method to determine fludarabine triphosphate accumulation in T-lymphocytes from hematopoietic cell transplantation patients. Cancer Chemother Pharmacol 63, 391–401 (2009). https://doi.org/10.1007/s00280-008-0748-0

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