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IDENTIFICATION OF THE CYTOSOLIC CARBOXYLESTERASE CATALYZING THE 5'-DEOXY-5-FLUOROCYTIDINE FORMATION FROM CAPECITABINE IN HUMAN LIVER

Drug Metabolism and Toxicology, Division of Pharmaceutical Sciences, Graduate School of Medical Science, Kanazawa University, Kanazawa, Japan (T.T., M.K., M.N., T.Y.); and Department of Legal Medicine, Dokkyo University School of Medicine, Tochigi, Japan (S.T.)

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 K_m and V_max 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.

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