Abstract 5491: Marked augmentation of the polyglutamate derivatives as the basis for the enhanced activity of pralatrexate relative to methotrexate.
Authors: Visentin Michele , Unal Ersin Selcuk , Zhao Rongbao , Goldman I David 
Published: August 2014

Abstract
Pralatrexate (PDX) is a new-generation dihydrofolate reductase (DHFR) inhibitor with high affinity for the reduced folate carrier (RFC) and for folypolyglutamyl synthetase (FPGS), the enzyme that mediates the addition of glutamate moieties at the γ-carboxyl of folates and antifolates (Sirotnak et al., Cancer Chemother Pharmacol. 42:313, 1998). The polyglutamate derivatives are better accumulated and retained into the cells. Recently, PDX received FDA approval for the treatment of relapsed or refractory peripheral T-cell lymphoma. This report represents the first analysis of PDX cellular pharmacology using the tritiated derivative, comparing its properties with methotrexate (MTX) in HeLa cells. The influx Kt was determined at ∼0.5 μM in comparison to a MTX influx Ki of ∼3 μM. Consistent with RFC-targeting, [³H]PDX was a lesser substrate for the proton-coupled folate transporter (PCFT) than MTX. While the net uptake of [³H]MTX reached steady-state conditions within 30 min, net uptake of [³H]PDX continued to increase, exceeding the [³H]MTX level by a factor of 15, by 6 hrs. This difference in net uptake could be attributed to a marked increase in PDX polyglutamates. At 24 hrs, the PDX mono-, di- and triglutamate levels in the cells were 14%, 12%, and 70% of the total antifolate, respectively. When polyglutamation was suppressed, the subsequent intracellular [³H]PDX level was markedly decreased now exceeded the [³H]MTX level by a factor of only 3. The importance of PDX polyglutamation was reflected in the marked difference in growth inhibition relative to MTX when exposure to the drugs was transient. When cells were exposed continuously to PDX or MTX for six days the IC50 for the former was ∼ 1/25th the latter (7 nM vs 180 nM, respectively). However, when the exposure was limited to 6 hrs followed by growth in drug-free medium, the pharmacological advantage of PDX over MTX was markedly increased with an IC50 of 70 nM and 30 μM,respectively, now ∼ 1/450th that of MTX. Hence, in the first direct measurement of the transport and polyglutamation of PDX, the data confirm a much higher affinity for RFC relative to MTX along with a markedly greater rate of polyglutamation in cells. It is this difference in the extent of accumulation of these active polyglutamate derivatives that is the basis for the marked difference in the antitumor activities between these antifolates. The apparent low affinity of PDX for PCFT suggests that changes at the nitrogen-10 position are one determinant of the affinity of 4-amino antifolates for this transporter. Further, since PCFT is the mechanism by which folates and antifolates are absorbed across the proximal small intestine, the data suggests that the low affinity of PDX for this carrier would result in a low rate of re-absorption of this antifolate during its enterohepatic circulation, with a consequent higher level of fecal excretion, than for MTX.
Citation Format: Michele Visentin, Ersin Selcuk Unal, Rongbao Zhao, I David Goldman. Marked augmentation of the polyglutamate derivatives as the basis for the enhanced activity of pralatrexate relative to methotrexate. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 5491. doi:10.1158/1538-7445.AM2013-5491