levoleucovorin and 2--deoxyuridylic-acid

Combination of low doses of de novo pyrimidine biosynthesis inhibitors with 5-fluorouracil (FU) has been proposed to increase the antitumor activity of FU. Brequinar is such an inhibitor that has little clinical anti-tumor effect when used alone. We determined the clonogenic survival of MGH-U1 cells treated with FU +/- leucovorin (LV) +/- brequinar and examined the effects of these treatments on thymidylate synthase (TS). After 24 h exposure, the concentrations resulting in 50% inhibition of cell growth (IC50) for brequinar, FU, and FU+LV (100 microM) were 0.4, 20, and 10 microM, respectively. Both 24 h pretreatment and 48 h continuous treatment with the IC10 (0.1 microM) of brequinar increased the cytotoxicity of FU but did not enhance that of FU+LV. Simultaneous 24 h exposure to 0.1 microM brequinar and FU +/- LV did not increase the cytotoxicity of FU +/- LV. Intracellular cytidine triphosphate (CTP) and uridine triphosphate (UTP) pools, free TS binding sites, and levels of free fluorodeoxyuridine monophosphate (FdUMP) and deoxyuridine monophosphate (dUMP) were measured in cells pretreated with 0.1 microM brequinar for 24 h alone or followed by a 2-h exposure to FU (25 microM) +/- LV (100 microM). In brequinar-treated cells, CTP and UTP pools amounted to 68% and 46% of control values, respectively. The free TS binding sites remaining amounted to 70% of control values in cells treated with FU and 9% of control levels in those treated with FU+brequinar. Free FdUMP levels increased 5-fold in cells pretreated with brequinar as compared with those treated with FU alone. The increased formation of FdUMP was inhibited by simultaneous exposure to 100 microM hypoxanthine and 25 microM FU. Intracellular dUMP levels were not affected by brequinar. We conclude that a low dose of brequinar increases the cytotoxicity of FU but does not enhance that of FU+LV when exposure to brequinar precedes FU treatment. This potentiation appears to be mediated by the increased formation of FdUMP as a consequence of an increase in the cosubstrate phosphoribosyl pyrophosphate (PRPP).

Topics: Antineoplastic Combined Chemotherapy Protocols; Biphenyl Compounds; Cell Survival; Cytidine Triphosphate; Deoxyuracil Nucleotides; Dose-Response Relationship, Drug; Drug Interactions; Fluorodeoxyuridylate; Fluorouracil; Humans; Hypoxanthine; Hypoxanthines; Leucovorin; Phosphoribosyl Pyrophosphate; Thymidylate Synthase; Tumor Cells, Cultured; Uridine Triphosphate

The clinical formulation of leucovorin (5-CHO-FH4) is a mixture of diastereoisomers with markedly different pharmacologic properties. Comparatively little information is available concerning the cellular pharmacology of reduced folate stereoisomers, due largely to the difficulty in preparing sufficient quantities of these compounds for in vitro use. Recent improvements in HPLC technology have now facilitated this process, enabling studies of folate stereochemistry on a larger scale. Using purified (6R) and (6S) leucovorin, we examined the effects of these compounds on the enzymatic activity of Lactobacillus casei thymidylate synthase (TS) in a cell-free system. The natural (6S), unnatural (6R), and racemic (6R,S) leucovorin preparations inhibited TS activity by 50% at concentrations of 0.11, 2.1, and 0.52 mM, respectively. Dixon plots demonstrated the inhibition to be competitive, with Ki values of 85 microM, 1.59 mM, and 385 microM for (6S), (6R), and (6R,S) leucovorin, respectively. In view of the high doses of leucovorin given clinically and the slow clearance of the unnatural isomer, our observations suggest that leucovorin may have important direct inhibitory effects on folate-requiring enzymes.

Topics: Buffers; Cell-Free System; Deoxyuracil Nucleotides; Dose-Response Relationship, Drug; Formyltetrahydrofolates; Kinetics; Lacticaseibacillus casei; Leucovorin; Stereoisomerism; Tetrahydrofolates; Thymidylate Synthase

Topics: Adenocarcinoma; Animals; Bone Marrow; Colonic Neoplasms; Deoxyuracil Nucleotides; Fluorodeoxyuridylate; Fluorouracil; Humans; Intestinal Mucosa; Leucovorin; Liver; Pancreatic Neoplasms; Rats; Rats, Inbred Strains; Rectal Neoplasms; Stomach Neoplasms; Thymidylate Synthase