The long term objectives of this application are to define the stereospecificity of leucovorin transport, metabolism and effects on 5-fluorouracil cytotoxicity in human tumor cells. The clinical formulation of leucovorin is a racemic mixture of stereoisomers that appear to differ significantly in their clinical and cellular pharmacology. A recently developed chiral HPLC method will be used to prepare pure quantities of each isomer for in vitro studies.
The specific aims of this proposal are to investigate the following in human breast and colon cancer cells: 1) the membrane transport, intracellular metabolism and biologic activity of the leucovorin stereoisomers; 2) the effect of 6(R) leucovorin on the membrane transport and intracellular metabolism of the 6(S) isomer; 3) the effects of each leucovorin stereoisomer on the cytotoxicity of 5- fluorouracil and; 4) the impact of high concentrations of 6(R) leucovorin on the ability of 6(S) leucovorin to potentiate the cytotoxicity of 5-fluorouracil. Methods will include cell culture; measurement of cell viability by cell counting and colony-forming assay; high pressure lipid chromatography of intracellular reduced folate pools; reductive cleavage of folates and HPLC chromatography of p-aminobenzoyl polyglutamates; assessment of thymidylate synthase activity by incorporation of (3H) deoxyuridine into DNA; and the use of a novel photoactive analogue of methotrexate to assess the stereospecificity of reduced folate binding to the plasma membrane carrier protein. Studies in cell-free systems have shown that reduced folates are required for formation of a stable ternary complex between 5-FdUMP and thymidylate synthase. In cell culture, the cytotoxic effects of 5-FU can be enhanced at least 3- fold by the provision of adequate leucovorin in the culture medium. Preliminary clinical trials have shown that the combination of 5- FU plus leucovorin procedures tumor regression in 40-50% of patients with colorectal cancer. In vivo, however, high concentration of 6(R) leucovorin accumulate in plasma and could potentially interfere with the biological activity of the 6(S) isomer. The experiments proposed in this application will accurately define the biological activity of leucovorin stereoisomers in human tumor cells and may enable the design of more rational 5-FU-LV treatment plans for use in patients with solid tumors.
| Lee, P P; Schilsky, R L (1990) Inhibition of thymidylate synthase by the diastereoisomers of leucovorin. Cancer Chemother Pharmacol 26:273-7 |
