Competitive Protein-binding Assay for Trimetrexate

A competitive protein-binding assay has been developed for trimetrexate (TMTX) based on the tight binding of this drug to human dihydrofolate reductase (tetrahydrofolate dehydrogenase). In this assay, TMTX competes with 3H-methotrexate for binding to the enzyme. Free drug is separated from that bound to reductase by adsorption with dextran-albumin-coated charcoal. TMTX is measurable over a range from 2 X 10(-9) to 5 X 10(-8) M in plasma, with a coefficient of variation of less than 10%. Measurements of TMTX in plasma, cerebrospinal fluid, and urine agreed closely with parallel determinations in aqueous solutions.