Abstract
Capecitabine, a prodrug of 5-fluorouracil, is first metabolized to 5′-deoxy-5-fluorocytidine (5′-DFCR) by carboxylesterase (CES), which is mainly expressed in microsomes. Recently, we clarified that 5′-DFCR formation was catalyzed by the enzyme in cytosol as well as microsomes in human liver. In the present study, the cytosolic enzyme involved in 5′-DFCR formation from capecitabine was identified. This enzyme was purified in the cytosolic preparation by ammonium sulfate precipitation, Sephacryl S-300 gel filtration, Mono P chromatofocusing, and Superdex 200 gel filtration. The purified enzyme was identified by the amino acid sequence analysis to be CES1A1 or a CES1A1 precursor. Based on the result of the N-terminal amino acid sequence analysis, the purified enzyme has no putative signal peptide, indicating that it was CES1A1. The apparent Km and Vmax values of 5′-DFCR formation were 19.2 mM and 88.3 nmol/min/mg protein, respectively. The 5′-DFCR formation catalyzed by the purified enzyme was inhibited by both diisopropylfluorophosphate and bis(p-nitrophenyl)phosphate in a concentration-dependent manner. 7-Ethyl-10-hydroxycamptothecin (SN-38) formation from irinotecan also occurred in the purified enzyme, cytosol, and microsomes. In conclusion, the cytosolic enzyme involved in 5′-DFCR formation from capecitabine would be CES1A1. It is suggested that the cytosolic CES has significant hydrolysis activity and plays an important role as the microsomal CES in drug metabolism. It is worthy to investigate the metabolic enzyme in cytosol involved in the activation of ester-type prodrugs such as capecitabine.
Footnotes
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This study was supported by a grant-in-aid for Encouragement of Young Scientists of the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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doi:10.1124/dmd.104.000554.
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ABBREVIATIONS: 5-FU, 5-fluorouracil; 5′-DFCR, 5′-deoxy-5-fluorocytidine; CES, carboxylesterase; 5′-DFUR, 5′-deoxy-5-fluorouridine (doxifluridine); CDA, cytidine deaminase; TP, thymidine phosphorylase; CDHP, 5-chloro-2,4-dihydroxypyridine; TPI, 5-chloro-6-(2-iminopyrrolidin-1-yl)methyl-2,4-(1H,3H)-pyrimidinedione; DFP, diisopropylfluorophosphate; BNPP, bis(p-nitrophenyl)phosphate; THU, tetrahydrouridine; CPT-11, irinotecan hydrochloride trihydrate; SN-38, 7-ethyl-10-hydroxycamptothecin; PAGE, polyacrylamide gel electrophoresis; TFA, trifluoroacetic acid; HPLC, high-performance liquid chromatography.
- Received May 8, 2004.
- Accepted July 12, 2004.
- The American Society for Pharmacology and Experimental Therapeutics
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