A B S T R A C T Human subjects and rhesus monkeys receiving the antitumor agent methotrexate at the high dose levels recently introduced into clinical use (> 50 mg/kg) excrete significant amounts of the metabolite 7-hydroxymethotrexate. The metabolite is not detected in these species after methotrexate therapy at conventional dose levels. The evidence indicates that in primates, the in vivo conversion of methotrexate to 7-hydroxymethotrexate is a dose-dependent phenomenon, with the enzyme system (s) catalyzing the reaction having a low affinity for the drug. INTRODUCTIONThe folate antagonist methotrexate (MTX; 4-amino-4-deoxy-N10-methylpteroylglutamate) 1 is in general clinical use as a single agent or in combination with other antineoplastic agents in the treatment of choriocarcinoma, Burkitt's lymphoma, acute leukemia of childhood, epidermoid carcinoma of the head and neck, and breast cancer (1, 2). Recently, "high-dose" MTX therapy (> 50 mg MTX/kg) with leucovorin rescue has been shown to be of value in the treatment of osteogenic sarcoma (3, 4). The toxicity of this regimen is significant, with renal failure having been observed, in addition to the gastrointestinal ulceration and bone marrow depression seen with conventional MTX therapy. Earlier studies of the clinical pharmacology of MTX administered at conventional dose levels indicate that in man and other primates, the compound is not metabo-

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In a number of mammalian species, including man, the 4-amino analogs of pteroylglutamate persist in liver and kidney for many months after administration (1-4). In the rat (3) and mouse (4), the binding site appears to be the enzyme dihydrofolate reductase [also termed folate reductase (5) and tetrahydrofolate dehydrogenase.1] Studies with partially purified dihydrofolate reductase obtained from several sources (6-8) have shown the affinity of this enzyme for the 4-amino analogs to be extremely high.Recent work with tritium-labeled pteroylglutamate in vivo has shown that, in man, the labeled compound can be displaced from cells several days after its administration, both by unlabeled pteroylglutamate (9, 10) and by other compounds that possess an affinity for the enzyme dihydrofolate reductase (11). If, as animal experiments suggest, the 4-amino analogs bind to the same enzyme, a similar procedure should also bring about the displacement of these compounds. The studies reported here show that, in man, tritiated methotrexate (amethopterin; 4-amino-AN10-methylpteroylglutamic acid) can be readily displaced