Competitive protein binding assay for methotrexate.

Abstract

(1) and osteogenic sarcoma (2). Both systemic and intrathecal regimens have been hampered by the occasional development of serious toxicities, such as neurologic sequelae (3), renal failure (4), and myelosuppression. While the etiology of these complications is not well understood, they appear in some cases to be associated with delayed elimination of drug from cerebrospinal fluid (5) or the systemic circulation (6), and pharmacokinetic monitoring may be helpful in their prevention and management (6).Heretofore, routine monitoring of methotrexate pharmacokinetics has not been possible in most institutions due to the complexity of existing assay methods. In the present report, we describe the development of a highly specific, rapid, and sensitive assay based on the high affinity binding of methotrexate to dihydrofolate reductase. In addition to its advantages for routine clinical use, the assay has broad potential application to antifolate research in that it utilizes techniques which allow direct measurement of the association constant of antifolates and dihydrofolate reductase under optimum binding conditions. MATERIALS AND METHODSPartially purified dihydrofolate reductase from dichloromethotrexate-resistant Lactobacillus casei (New England Enzyme Center, Boston, Mass.) was used throughout this study. This enzyme preparation formed 4.6 Aumol of tetrahydrofolate/min per mg protein at pH 7.5 and 370 and was able to bind 1.9 nmol of methotrexate per mg of protein at pH 6.2 and 23'.Unlabeled methotrexate (0.15 M potassium phosphate buffer, pH 6.2, and 23°) (Drug Research and Development Branch, National Cancer Institute) and 4-amino-4-deoxy-10-methylpteroic acid (a gift of Dr. David Johns, National Cancer Institute) were purified by chromatography on DEAE-cellulose with a HC03-elution gradient as previously described (7,8). [3',5',9(n)-3H]methotrexate, 9.5 Ci/ mmol (Amersham-Searle) was diluted in 0.1 M potassium phosphate, pH 8.0, to a final concentration of 2 mCi/ml, and stored at -70°. This methotrexate preparation proved unstable and decomposed, forming radioactive decomposition products which were not adsorbable by charcoal, thus increasing the background. For this reason, the labeled methotrexate was also repurified by chromatography on DEAE-cellulose as described above whenever the background exceeded 150 cpm.Plasma samples containing 100 units of heparin per ml were obtained from patients receiving high dose methotrexate infusion and cerebrospinal fluid was obtained from patients receiving intrathecal methotrexate. All samples were stored at -20°. Prior to analysis samples were acidified to Immediately following addition of the enzyme-NADPH solution, the tubes were mixed by Vortex agitation and 25 Al of a charcoal slurry was added (Norit A, 10 g/100 ml; bovine serum albumin, Fraction V, 2.5 g/100 ml; and high-molecular-weight dextran, 0.1 g/100 ml, all from Sigma Chemical Co.) and pH was adjusted to 6.2 with 1 N HCL. Samples were again mixed by Vortex agitation and centrifuged at 700 X g for 30 min. A 200 ...

Synthesis, Retention, and Biological Activity of Methotrexate Polyglutamates in Cultured Human Breast Cancer Cells

“…The chosen conditions were those associated with little or maximal MTXPG synthesis (Table IV). In all instances, drug incubations associated with significant MTXPG formation led to marked inhibition of [3H]dU incorporation at the end of incubation with drug, and this inhibition persisted even after 24 (7)(8)(9) and rat hepatoma (13) formed predominantly the shorter chain length derivatives (1-3 glutamyl residues), although difference in drug concentrations, duration of exposure, and different methods of analysis (ion exchange or gel filtration) could account for these differences in comparison with the present work. The profile of MTXPG formed appears to have an important effect on the ability of cells to retain drug after removal of MTX from the cell culture medium.…”

“…The rates of disappearance from cells decreased with increasing glutamyl chain length. All of the 4-NH2-10-CH3-PteGlu5 and 47 and 38% of the 4-NH2-10-CH3-PteGlu4 remained in the MCF-7 and ZR-75-B cells, respectively, and could be identified in the cytosol after 24 h in drug-free medium. The retention of MTX polyglutamates in these two cell lines in excess of dihydrofolate reductase binding capacity led to prolonged inhibition of thymidylate synthesis and loss of cell viability after removal of extracellular MTX.…”

“…MTX and MTXPG were separated using a recently developed HPLC assay (16) 24 h as described previously. After the incubation, the cells were washed once with ice-cold PBS and placed in drug-and folate-free IMEM for 24 h. The medium was changed after 1 and 6 h of efflux.…”

“…In contrast, MDA-231 cells showed lesser accumulation of MTX, and only 32% (4.06 nmol/g) of the intracellular drug was in the form of polyglutamates, a difference that could only partially be explained by decreased ability of these cells to take up free drug from the medium. When and ZR-75-B cells containing polyglutamates were transferred to drug-free medium for 24 h, 22 and 51% of the total intracellular drug were, respectively, retained in each cell line. The loss of intracellular drug was primarily accounted for by disappearance of parent compound and polyglutamates containing [1][2][3] additional glutamyl residues.…”

Blast cell methotrexate-polyglutamate accumulation in vivo differs by lineage, ploidy, and methotrexate dose in acute lymphoblastic leukemia.

“…The column eluent was collected in fractions determined by the elution times of each polyglutamated metabolite (MTX-glu2 to MTXglu7, obtained from Schircks Laboratories, Jona, Switzerland). Each fraction was dried to completion and assayed using a radio-ligand binding assay (The Enzyme Center, Inc., Malden, MA) (36). Separate calibration curves were used for quantitation of MTX and each polyglutamated metabolite.…”