Disposition of leucovorin and its metabolites in the plasma, intestinal epithelium, and intraperitoneal L1210 cells of methotrexate-pretreated mice

Abstract

Leucovorin (LV or 5-CHOFH4) has had long-standing clinical use as a rescue agent from the systemic toxic effects of methotrexate (MTX). Because the mouse has been the animal model most used to investigate MTX therapy, direct tissue assessment of LV and its reduced-folate metabolites was undertaken in the plasma, intestinal epithelium, and intraperitoneal L1210 cells of MTX-pretreated mice using a ternary-complex-based assay method. The results show that total folate accumulation and depletion in tissues is closely related to plasma levels, with somewhat greater persistence occurring in tissues, presumably due to polyglutamylation. Examination of individual folates in plasma showed that the combined 5,10-methylenetetrahydrofolate (CH2FH4) plus tetrahydrofolate (FH4) pool was the most extensively elevated pool other than that of the parent compound [S]-5-formyltetrahydrofolate ([S]-5-CHOFH4). The dihydrofolate (FH2) also became elevated, whereas the 5-methyltetrahydrofolate (5-CH3FH4) remained unchanged. Individual folates that were elevated in tissues were generally the same as those elevated in plasma, the exception being a significant accumulation of 10-formyltetrahydrofolate (10-CHOFH4) in both intestinal epithelial and L1210 cells. The elevation of FH2 in L1210 cells was greater and persisted longer than that in intestinal epithelium, whereas the opposite was true for CH2FH4 + FH4. This differential effect in tumor versus epithelial tissue is consistent with the selective rescue of normal tissue by LV.

A Phase I clinical study of the antipurine antifolate lometrexol (DDATHF) given with oral folic acid

“…Interestingly, 10-formyltetrahydrofolate, which competes with lometrexol for GARFT, was found to constitute only 15 and 32% of total intracellular folates in human breast and colon cell lines, respectively, and a rapid efflux of a large portion of intracellular folates was observed 2 hours after removal of 5-formyltetrahydrofolate. Similarly, in vivo in murine intestinal epithelial cells, leucovorin led to only a transient elevation of 10-formyltetrahydrofolate levels, in comparison to the increases seen in other reduced folate cofactor pools [29]. These results raise the possibility that the limited efficacy of leucovorin in rescuing lometrexol toxicities in the current study may have been due to a failure to form or maintain adequate intracellular 10-formyltetrahydrofolate concentrations.…”

A Structurally Altered Human Reduced Folate Carrier with Increased Folic Acid Transport Mediates a Novel Mechanism of Antifolate Resistance

“…3 H]FdUMP (20 Ci/mmol) (55,56). Frozen cell pellets were thawed (25 ϫ 10 6 cells/ml in an extraction buffer containing 50 mM Tris-HCl, 50 mM sodium ascorbate, and 1 mM EDTA, pH 7.4.…”

The Structure and Competitive Substrate Inhibition of Dihydrofolate Reductase from Enterococcus faecalis Reveal Restrictions to Cofactor Docking

“…17 However, peritoneal spaces have a demonstrated capacity to serve as reservoirs for folate metabolites, so this result is not surprising. 45 Our investigations were able to only marginally reproduce this effect for E. faecalis , limited to an increase in the MIC of one dilution or less for TMP or for the anti-folate series under investigation. Interestingly, the most striking increase in MIC was for the synergistic combination of TMP with SMZ.…”

“…Continuing studies sought to address the impact of those observations within an animal model of peritoneal infection and documented an inferior outcome with TMP-SMX treatment . However, peritoneal spaces have a demonstrated capacity to serve as reservoirs for folate metabolites, so this result is not surprising . Our investigations were able to only marginally reproduce this effect for E. faecalis , limited to an increase in the MIC of one dilution or less for TMP or for the anti-folate series under investigation.…”