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Interactions between 7-Hydroxymethotrexate and Methotrexate at the Cellular Level in the Ehrlich Ascites Tumor in Vitro¹

Departments of Medicine and Pharmacology, Medical College of Virginia, Richmond, Virginia 23298

Studies were undertaken to characterize the cellular pharmacology of 7-hydroxymethotrexate (7-OH-MTX) in Ehrlich ascites tumor cells, compare it to that of methotrexate (MTX), and define the interactions between the parent compound and its catabolite. Transport of 7-OH-MTX is mediated by the MTX-tetrahydrofolate cofactor carrier, with a K_m of 9 µM in comparison to the MTX K_m of 5 µM. Both compounds mutually inhibit their influx and steady-state levels of free drug accumulated. While influx of 7-OH-MTX is slower than influx of MTX, 7-OH-MTX efflux is likewise slower, so that the steady-state level of 7-OH-MTX achieved is comparable to that of MTX. Influx of 7-OH-MTX is inhibited by extracellular 5-formyltetrahydrofolate and trans-stimulated in cells preloaded with this tetrahydrofolate cofactor. The energetics of 7-OH-MTX transport is similar to that of MTX in that influx and net transport are stimulated by sodium azide, while net transport is reduced by glucose. As observed for MTX, 7-OH-MTX transport is sensitive to the anionic composition of the extracellular compartment and was shown to be inhibited by organic and inorganic phosphates.

7-OH-MTX does not, alone, inhibit [³H]deoxyuridine incorporation into DNA at concentrations of up to 50 µM. However, the catabolite reduces MTX inhibition of deoxyuridine metabolism, presumably due to the reduction in the free level of intracellular MTX achieved. These findings support the possibility that when 7-OH-MTX accumulates to high levels relative to MTX in clinical regimens, it may modulate the pharmacological effects of MTX.

¹ This investigation was supported by USPHS Grant CA-16906 awarded by the National Cancer Institute and NIH.

² Exchange Scientist under the Clinical Cancer Research Program Area of the U. S. France (NCI-INSERM) Cancer Program (G5.0111). Present address: INSERM SC 16, Laboratoire de Pharmacocinetique et Toxicocinetique, Faculte de Pharmacie, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France.

³ Supported by USPHS Training Grant CA-09340 from the National Cancer Institute.

⁴ Present address: INSERM SC 16, Laboratoire de Pharmacocinetique et Toxicocinetique, Faculte de Pharmacie, 27 Boulevard Jean Moulin, 13385 Marseille Cedex 5, France.

⁵ To whom requests for reprints should be addressed.

Received 7/ 8/83. Accepted 11/23/83.