Formation Pathways of γ-Butyrolactone from the Furan Ring of Tegafur during Its Conversion to 5-Fluorouracil

Abstract

ABSTRACT:Tegafur (FT) is a 5-fluorouracil (5-FU) prodrug that has been clinically used for various cancer chemotherapies. The following metabolites of FT were identified in patients: 5-FU, fluoro-␤-alanine, and ␥-butyrolactone (GBL) and its acidic form, ␥-hydroxybutyrate (GHB). GBL/ GHB, which is probably generated from the furan ring of FT, inhibits tumor cell angiogenesis, contributing to the antitumor effect of FTbased therapies. In the present study, we identified the metabolites formed from the furan ring of FT by CYP2A6 and thymidine phosphorylase (TPase) using 2,4-dinitrophenylhydrazine derivatization procedures and clarified the metabolic pathway of FT to GBL/GHB. Succinaldehyde (SA) and 4-hydroxybutanal (4-OH-BTL) were produced as the metabolites because of the cleavage of the furan ring of FT during its conversion to 5-FU in cDNA-expressed CYP2A6 and purified TPase, respectively; however, GBL/GHB was hardly detected in cDNA-expressed CYP2A6 and purified TPase. GBL/GHB was formed after human hepatic microsomes or cDNA-expressed CYP2A6 mixed with cytosol were incubated with FT. Furthermore, 4-OH-BTL was converted to GBL/GHB in the microsomes and cytosol. These results suggest that GBL/GHB is generated from FT through the formation of SA and 4-OH-BTL but not directly from FT. Furthermore, the amount of 5-FU and GBL/GHB formed in the hepatic S9 was markedly decreased in the presence of a CYP2A6 inhibitor, suggesting that GBL/ GHB may be mainly generated through the CYP2A6-mediated formation of SA.

Enantioselectivity in the cytochrome P450‐dependent conversion of tegafur to 5‐fluorouracil in human liver microsomes

“…; Yamamiya et al. ). Incubation mixture for hepatic microsomal metabolism contained FT, microsomes (1 mg protein/mL), 0.1 mmol/L CDHP, and an NADPH‐generating system consisting of 1.3 mmol/L β‐NADP + , 3.3 mmol/L glucose‐6‐phosphate, 3.3 mmol/L magnesium chloride, and 0.4 units glucose‐6‐phosphate dehydrogenase in 100 mmol/L Tris (pH 7.4).…”

“…) (Yamamiya et al. ). FT is a chiral molecule and contains an asymmetric carbon at 2′‐position of its tetrahydrofuran moiety as shown in Figure .…”

Species variation in the enantioselective metabolism of tegafur to 5-fluorouracil among rats, dogs and monkeys

“…It has been reported that cytochrome P450 (CYP) and thymidine phosphorylase (TPase) in the liver are main contributors to the conversion of FT to 5‐FU [1–3]. As illustrated in Figure , our previous study clarified that CYP and TPase catalyze the 5′‐hydroxylation and 2′‐hydolysis of the tetrahydrofuran ring of FT, respectively, followed by sequential decomposition of the metabolites to 5‐FU [4]…”

Effect of CYP2A6 Genetic Polymorphism on the Metabolic Conversion of Tegafur to 5-Fluorouracil and Its Enantioselectivity

“…As 5-FU formed from FT was subjected to extensive metabolism by dihydropyrimidine dehydrogenase contamination in enzyme preparations, a potent dihydropyrimidine dehydrogenase inhibitor, CDHP, was always added to prevent the unintended loss of 5-FU (Ikeda et al, 2000;Yamamiya et al, 2010Yamamiya et al, , 2013. The reaction mixture (0.1 ml) in the assay of insect microsomal metabolism contained FT, 50-100 pmol/ml CYP2A6, 0.1 mM CDHP, and an NADPH-generating system consisting of 1.3 mM b-NADP + , 3.3 mM glucose 6-phosphate, 3.3 mM magnesium chloride, and 0.4 units of glucose-6-phosphate dehydrogenase in 100 mM Tris-HCl (pH 7.4).…”

“…1, FT is a chiral molecule and contains an asymmetric carbon at the 29-position of its tetrahydrofuran ring. Our previous study clarified that cytochrome P450 (P450) and thymidine phosphorylase catalyze 59-hydroxylation and 29-hydrolysis, respectively, of the tetrahydrofuran ring of FT, followed by sequential decomposition of FT to 5-FU (Yamamiya et al, 2010). Recently we found that CYP2A6 was responsible for the enantioselective metabolism of FT. CYP2A6 catalyzed the conversion of both FT enantiomers to 5-FU most efficiently among P450 isoforms, and the intrinsic clearance of R-FT was significantly higher than that of S-FT in human liver microsomes (HLMs) (Damle et al, 2001;Yamamiya et al, 2013).…”