The Natural and Unnatural Diastereomers of Leucovorin: Aspects of their Cellular Pharmacology
A mixture of the natural ((6S)-LV) and unnatural ((6R)-LV) diastereomers of leucovorin (LV, (6RS)-LV) is administered clinically in combination with 5-fluorouracil (FUra) to overcome drug resistance due to insufficient intracellular folate concentrations (1). Initially described as a growth factor for Pediococcus cerevesiae, LV has a S chirality at the 6 carbon of the tetrahydropterine ring (2). Following intravenous administration of a mixture of the (6S) and (6R) isomers of leucovorin to patients, plasmatic (6S)-LV disappears rapidly with a T0.5 of 30 min. while (6R)-LV has a slower clearance (To.5 of 7.5 hours) (1,3). The natural isomer is readily transported into cells where it has no known folate cofactor activity. Greenberg was the first to show that LV was converted to another folate involved in purine biosynthesis (4). The first enzymatic step involved was later found to be methenyltetrahy-drofolate synthetase (5-formyltetrahy-drofolate cyclodehydrase, EC 220.127.116.11) which catalyzes the irreversible and stereospecific ATP and Mg2+-dependent transformation of (6S)-LV to N5–10-methenyltetrahy-drofolate. Sheep liver enzyme activity was first partially purified (5) and the enzyme was more recently highly purified and characterized from Lactobacillus casei (6) and rabbit liver (7). We have recently obtained and characterized highly purified human liver methenyltetrahydrofolate synthetase (8) and will describe in the following paper its purification and characteristics. We next took advantage of the enzyme characteristics to prepare stereochemically pure (6R)-LV (9) and will present its transport characteristics and biological activity in human leukemic CCRF-CEM cells.
KeywordsLactobacillus Casei Magnesium Acetate Chiral HPLC Anionic Buffer Sodium Hydrosulfite
Unable to display preview. Download preview PDF.
- (1).MACHOVER D, GOLDSCHMIDT E, CHOLLET P, METZGER G, ZITTOUN J,MARQUET J, VANDENBULCKE JM, MISSET JL, SCHWARZENBERG L,FOURTILLAN JB, G.GET H, MATHE G., Treatment of advanced colorectal and gastric adenocarcinomas with 5-fluorouracil and high-dose folinic acid, J. Clin. Oncol., 4: 685–696, 1986.PubMedGoogle Scholar
- (9).BERTRAND R, JOLIVET J., Synthesis, transport characteristics and biological activity of the unnatural diastereomer of leucovorin, in preparation.Google Scholar
- (10).MORAN RG, COLMAN PD. A simple procedure for the synthesis of high specific activity tritiated (6S)-5-formyltetrahydrofolate, Anal. Biochem., 122: 70–78, 1982.Google Scholar
- (12).RABINOWITZ JC. Preparation and properties of 5,10-methenyltetrahydrofolic acid and 10-formyltetrahydrofolic acid, Meth.in Enzymol., 814–815, 1963.Google Scholar
- (14).SIROTNAK FM, GOUTAS LJ, JACOBSEN DM, MINES LS,BARRUECO JR, GAUMONT Y, KISLIUK RL., Carrier-mediated transport of folate compound in L1210 cells, Biochem. Pharmacol., 36, 10: 1659–1667, 1987.Google Scholar
- (16).RABINOWITZ JC., in The Enzymes (Boyer P.D., Lardy H. and Myrback K., eds.), Vol 2: 185–252, Academic Press, New-York, 1960.Google Scholar
- (17).CHOI KE, SCHILSKY RL., Resolution of the stereoisomers of leucovorin and 5-methyltetrahydrofolate by chiral HPLC, Anal. Biochem., 168: 398–404, 1988.Google Scholar
- (18).BLAIR JA, SAUNDERS KJ., A convenient method for the preparation of 5-methyltetrahydrofolic acid, Anal. Biochem., 34: 376, 1970.Google Scholar
- (21).McKENZIE RE.,Biogenesis and interconversion of substituted tetrahydrofolates, in Folates and Pterins, R.L. Blakley and S.J. Benkovik eds., New-York, 293–296, 1984.Google Scholar
- (22).McGUIRE JJ, BERTINO JR., Enzymatic synthesis and function of folylpolyglutamates, Mol. Cell. Biochem., 39: 19–48, 1981.Google Scholar