Interactions between 7-hydroxymethotrexate and methotrexate at the cellular level in the Ehrlich ascites tumor in vitro.
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 Km of 9 microM in comparison to the MTX Km of 5 microM. 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 the 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 microM. 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.
D000818:Animals; D001386:Azides / Q000494:pharmacology; D001667:Binding, Competitive; D001692:Biological Transport / Q000187:drug effects; D002286:Carcinoma, Ehrlich Tumor / Q000378:metabolism*; D005493:Folic Acid Antagonists / Q000378:metabolism*; D005947:Glucose / Q000494:pharmacology; D007700:Kinetics; D008727:Methotrexate / Q000031:analogs & derivatives* / Q000378:metabolism*; D051379:Mice; D019810:Sodium Azide; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism
D001386:Azides; D005493:Folic Acid Antagonists; D019810:Sodium Azide; D013762:Tetrahydrofolate Dehydrogenase; D005947:Glucose; C011864:7-hydroxymethotrexate; D008727:Methotrexate