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Characteristics of the Formation and Membrane Transport of 7-Hydroxymethotrexate in Freshly Isolated Rabbit Hepatocytes¹

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

The cellular pharmacology of methotrexate was evaluated in freshly isolated rabbit hepatocytes in suspension with an analysis of drug metabolism by high-performance liquid chromatography. After exposure of hepatocytes at a cytocrit of 5% to 5 µM [³H]methotrexate, intracellular 7-hydroxymethotrexate appears rapidly within the cell; within 15 sec, the level of 7-hydroxymethotrexate exceeds the level of intracellular methotrexate, although the latter has not achieved the dihydrofolate reductase binding capacity. Within 20 min, virtually all methotrexate is hydroxylated. There is minimal formation of methotrexate polyglutamyl derivatives even after exposure of cells to very high levels of methotrexate, and 7-hydroxymethotrexate polyglutamates do not accumulate in the cell at all after incubation with [³H]-7-hydroxymethotrexate. Because of the rapidity of the hydroxylation of methotrexate, transport of this agent could not be characterized. However, some aspects of the transport properties of 7-hydroxymethotrexate could be studied since the catabolite is neither bound nor metabolized in this system. Net 7-hydroxymethotrexate transport was reduced by the addition of 5-formyltetrahydrofolate. As observed for 4-aminoantifolate transport in other cell systems, net 7-hydroxymethotrexate transport was markedly stimulated by sodium azide, an inhibitor of energy metabolism.

The data suggest that hydroxylation of methotrexate proceeds at a rate at least comparable to the rate of association of the drug with dihydrofolate reductase and that transport of methotrexate into rabbit hepatocytes is slow relative to the rate of catabolism to the 7-hydroxy derivative. Rabbit hepatocytes may be a useful model for exploring methotrexate catabolism at the cellular level and may provide insights into the interaction between methotrexate and/or other 4-aminoantifolates and the human liver.

¹ This investigation was supported by USPHS Grants CA-16906 and AM-18976 awarded by NIH.

² Exchange scientist under the Clinical Cancer Research Program Area of the United States-France (National Cancer Institute-Institut National de la Santé et de la Recherche Médicale) Cancer Program (G50111). Present address: INSERM SC16, 27 Boulevard Jean Moulin, 13385 Marseille, Cedex 5, France.

³ Present address: INSERM SC16, 27 Boulevard Jean Moulin, 13385 Marseille, Cedex 5, France.

⁴ To whom requests for reprints should be addressed.

Received 5/24/84. Accepted 11/19/84.