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

Abstract

Purpose: 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: [(3)H]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).

Results: 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.

Conclusions: 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.

Fig. 1. Initial rates and kinetic analysis of [³H]pralatrexate and [³H[MTX uptake

The time-course of drug uptake over 15 min at an extracellular concentration of 0.05 M (Panel a) or over 80 sec at an extracellular concentration of 0.5 M (Panel b) at pH 7.4. The data are the mean ± SEM from three independent experiments. Panel c: [³H]pralatrexate influx as a function of substrate concentration assessed over 1 min at pH 7.4 in presence or absence of 5 M non-labeled MTX. The lines are best-fit to the Michaelis-Menten equation (V = Vmax[S]/(K_t + [S])). Panel d: A double-reciprocal plot of the data (Lineweaver-Burk). Results are the mean ± SEM from four independent experiments.

Fig. 2. Antifolate transport mediated by PCFT

Influx of tritiated pralatrexate, MTX and pemetrexed at an extracellular concentration of 0.5 M in HeLa R1-PCFT cells that express only PCFT or HeLa R1-11 cells that lack both RFC and PCFT. The transport rates were assessed either at pH 6 (Panel A) or pH 7.4 (Panel B). Results are the mean ± SEM from three independent experiments.

Fig. 3. An analysis of pralatrexate accumulation and metabolism in HeLa cells

HeLa cells were grown in RPMI with the usual folic acid concentration of 2.0 μM (Panel A) or with 100 M folic acid (Panel B) for three days. Following this, the time-course of uptake of 0.5μM [³H]MTX, [³H]pralatrexate and [³H]pemetrexed was monitored in RPMI (2.0 μM folic acid) . The data are the mean ± SEM from three independent experiments. Panel C: HPLC analysis of intracellular tritiated constituents after exposure of cells grown in RPMI medium to [³H]pralatrexate with the usual folic acid concentration of 2 μM or, Panel D, with 100 μM folic acid. The extracellular [³H]pralatrexate concentration was 0.5 M; incubation time was 6h. The data is representative of three independent experiments.

Fig. 4. A comparison of pralatrexate and methotrexate growth inhibition in HeLa cells

HeLa cells were grown for 6 days continuously exposed to drug (Panel A) or pulsed for 6h with drug (Panel B) at the indicated concentrations. Growth in the absence of drug is indicated as 100%. Data are the mean ± SEM from three independent experiments. The vertical line intercepts the x axis at the concentration at which growth inhibition is 50% of the level of growth in the absence of drug (IC₅₀).

Fig. 5. Impact of RFC expression on pralatrexate influx and growth inhibition

HeLa cells, HeLa-RFC7, that overexpress RFC, and HeLa-R1-PCFT4 that express constitutive levels of PCFT but do not express RFC, were exposed to pralatrexate for 6h at the indicated concentrations then grown for an additional 6 days in the absence of drug. Growth in the absence of pralatrexate is indicated as 100%. The vertical line intercepts the x axis at the concentration at which growth inhibition is 50% of the level of growth in the absence of drug (IC₅₀). Data are the mean ± S.E.M from three independent experiments.