Cancer Chemotherapy and Pharmacology

, Volume 72, Issue 3, pp 597–606 | Cite as

The membrane transport and polyglutamation of pralatrexate: a new-generation dihydrofolate reductase inhibitor

  • Michele Visentin
  • Ersin Selcuk Unal
  • Rongbao Zhao
  • I. David Goldman
Original Article



To characterize, directly and for the first time, the membrane transport and metabolism of pralatrexate, a new-generation dihydrofolate reductase inhibitor approved for the treatment for peripheral T-cell lymphoma.

Experimental design

[3H]pralatrexate transport was studied in unique HeLa cell lines that express either the reduced folate carrier (RFC) or the proton-coupled folate transporter (PCFT). Metabolism to active polyglutamate derivatives was assessed by liquid chromatography. These properties were compared to those of methotrexate (MTX).


The pralatrexate influx K t, mediated by RFC, the major route of folate/antifolate transport at systemic pH, was 0.52 μΜ, 1/10th the MTX influx K i. The electrochemical potential of pralatrexate within HeLa cells far exceeded the extracellular level and was greater than for MTX. In contrast, MTX transport mediated by PCFT, the mechanism of folate/antifolate absorption in the small intestine, exceeded that for pralatrexate. After a 6 h exposure of HeLa cells to 0.5 μM pralatrexate, 80 % of intracellular drug was its active polyglutamate forms, predominantly the tetraglutamate, and was suppressed when cells were loaded with natural folates. There was negligible formation of MTX polyglutamates. The difference in pralatrexate and MTX growth inhibition was far greater after transient exposures (375-fold) than continuous exposure (25-fold) to the drugs.


Pralatrexate’s enhanced activity relative to MTX is due to its much more rapid rate of transport and polyglutamation, the former less important when the carrier is saturated. The low affinity of pralatrexate for PCFT predicts a lower level of enterohepatic circulation and increased fecal excretion of the drug relative to MTX.


Methotrexate PCFT Polyglutamation Pralatrexate RFC 




AICAR transformylase

Phosphoribosylaminoimidazole carboxamide formyltransferase


Dihydrofolate reductase


Folylpolyglutamate synthetase




Proton-coupled folate transporter


Reduced folate carrier



This study was supported by Spectrum Pharmaceutical (Irvine, CA) and a Grant from the National Institutes of Health National Cancer Institute [CA82621].

Conflict of interest

No conflict to disclose.


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Copyright information

© Springer-Verlag Berlin Heidelberg 2013

Authors and Affiliations

  • Michele Visentin
    • 1
  • Ersin Selcuk Unal
    • 2
  • Rongbao Zhao
    • 1
    • 3
  • I. David Goldman
    • 1
    • 3
  1. 1.Department of Molecular PharmacologyAlbert Einstein College of MedicineBronxUSA
  2. 2.Department of Medicine, Jacobi Medical CenterAlbert Einstein College of MedicineBronxUSA
  3. 3.Department of MedicineAlbert Einstein College of MedicineBronxUSA

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