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Characterization of Trimetrexate Transport in Human Lymphoblastoid Cells and Development of Impaired Influx as a Mechanism of Resistance to Lipophilic Antifolates

Parke-Davis Pharmaceutical Research Division, Warner-Lambert Company, Ann Arbor, Michigan 48105

The purpose of this study was to characterize the transport properties of trimetrexate in WI-L2 human lymphoblastoid cells and determine the mode of resistance that had developed in a subline, WI-L2/TMQ, that was grown in increasing concentrations of trimetrexate. WI-L2/TMQ cells were 62-fold resistant to trimetrexate and 68- and 96-fold cross-resistant to the other lipophilic antifolates metoprine and piritrexim (BW 301U). No cross-resistance was observed with vincristine or doxorubicin, and sensitivity was not increased with 5 µg/ml of verapamil, indicating that it was not a typical multidrug resistance phenotype. WI-L2/TMQ exhibited a 2-fold increase in dihydrofolate reductase; however, this did not contribute significantly to the observed resistance, since these cells retained full sensitivity to methotrexate. Nor were there any kinetic alterations in dihydrofolate reductase toward trimetrexate or differences in the levels of thymidylate synthase. The major difference between the sensitive and resistant cell line was a 50% decrease in the influx rate of WI-L2/TMQ cells which produced a corresponding decrease in cellular trimetrexate at the steady state. No difference in efflux rates was detected nor were there any differences in intracellular water or metabolism of trimetrexate. Additional characterization of trimetrexate transport in WI-L2 showed that influx was nonsaturable up to 5 mM extracellular trimetrexate, relatively insensitive to sodium azide, and exhibited a Q₁₀ of 2.7. Influx was, however, inhibited in a dose-dependent manner by concentrations of p-chloromercuribenzylsulfonate above 10 µM. Efflux studies revealed a large nonexchangeable fraction of trimetrexate that was well above the dihydrofolate reductase binding capacity and varied depending on the initial level of cell-associated drug. The intracellular exchangeable trimetrexate concentration at the steady state was always several-fold higher than the extracellular concentration. Retention of trimetrexate appeared to be coupled to some component of energy metabolism, since the presence of sodium azide stimulated this process by 2- to 3-fold. The data suggest that trimetrexate enters cells by passive diffusion but then is distributed and concentrated within the cell through more complex mechanisms which may involve energy coupling, compartmentation, or binding to macromolecules or organelles, although some type of carrier-mediated process cannot be ruled out. Furthermore, this study indicates that resistance to lipophilic antifolates can occur by mechanisms other than alterations in the cellular level or kinetics of dihydrofolate reductase or thymidylate synthase and that an impaired influx can develop for these compounds that may contribute to resistance.

Received 4/ 7/88. Revised 8/ 4/88. Accepted 9/ 2/88.