Metabolism of Capecitabine, an Oral Fluorouracil Prodrug: F Nmr Studies in Animal Models and Human Urine

Capecitabine (Xeloda; CAP) is a recently developed oral antineoplastic prodrug of 5-fluorouracil (5-FU) with enhanced tumor selectivity. Previous studies have shown that CAP activation follows a pathway with three enzymatic steps and two intermediary metabolites, 5 -deoxy-5-fluorocytidine (5 -DFCR) and 5 -deoxy-5-fluorouridine (5 -DFUR), to form 5-FU preferentially in tumor tissues. In the present work, we investigated all fluorinated compounds present in liver, bile, and perfusate medium of isolated perfused rat liver (IPRL) and in liver, plasma, kidneys, bile, and urine of healthy rats. Moreover, data obtained from rat urine were compared with those from mice and human urine. According to a low cytidine deaminase (3.5.4.5) activity in rats, 5 -DFCR was by far the main product in perfusate medium from IPRL and plasma and urine from rats. Liver and circulating 5 -DFCR in perfusate and plasma equilibrated at the same concentration value in the range 25 to 400 M, which supports the involvement of es-type nucleoside transporter in the liver. 5 -DFUR and -fluoro-ureidopropionic acid (FUPA) -fluoro-alanine (FBAL) were the main products in urine of mice, making up 23 to 30% of the administered dose versus 3 to 4% in rat. In human urine, FUPA FBAL represented 50% of the administered dose, 5 -DFCR 10%, and 5 -DFUR 7%. Since fluorine-19 nuclear magnetic resonance spectroscopy gives an overview of all the fluorinated compounds present in a sample, we observed the following unreported metabolites of CAP: 1) 5-fluorocytosine and its hydroxylated metabolite, 5-fluoro-6-hydroxycytosine, 2) fluoride ion, 3) 2-fluoro-3-hydroxypropionic acid and fluoroacetate, and 4) a glucuroconjugate of 5 -DFCR. Capecitabine (CAP, N-pentyloxycarbonyl-5 -deoxy-5-fluorocytidine) is an orally available prodrug of 5 -deoxy-5-fluorouridine (5 DFUR) with limited side effects, which has recently come into clinical practice for the treatment of breast and colorectal cancers (Di Costanzo et al., 2000; Marshall, 2001; Twelves et al., 2001; Wang et al., 2001). This fluoropyrimidine carbamate was designed to pass intact through the human intestinal mucosa and take advantage of the differential enzymatic levels in tumors to achieve better targeting of 5-fluorouracil (5-FU), the active moiety of the drug (Miwa et al., 1998; Shimma et al., 2000). An activation pathway of CAP has been proposed that requires three sequential steps of enzyme reactions (Bajetta et al., 1996). CAP is first converted to 5 -deoxy-5-fluorocytidine (5 -DFCR) by carboxylesterase and then to 5 -DFUR by cytidine deaminase, mainly in the liver (Budman et al., 1998; Miwa et al., 1998). Finally, systemic 5 -DFUR is converted to 5-FU by thymidine phosphorylase, the activity of which is increased in tumor tissue (Ishikawa et al., 1998; Verweij, 1999) (Fig. 1, in part). Most of the pharmacokinetic and metabolic studies on CAP have been carried out with liquid chromatography as the main analytical tool (Reigner et al., 1998). Since F NMR spectroscopy gives an overview of all fluorinated compounds present without any prerequisite separative process, we used this method to study the metabolism of CAP. Previous work has illustrated advantages of this method for the evaluation of metabolites of antineoplastic fluoropyrimidine drugs (Martino et al., 2000; Wolf et al., 2000). The aim of the present study is to reinvestigate the metabolism of CAP using F NMR as analytical method. Regarding the high variability of localization and activities of enzymes from species to species (Camenier and Smith, 1965; Ho, 1973; Shimma et al., 2000) involved in CAP activation, various animals and experimental models have been used. Because rats have a very low activity of cytidine deaminase, isolated perfused rat livers (IPRL) and healthy rats were used as simple models to evaluate primary process of CAP activation. We investigated fluorinated compounds present in liver, bile, perfusate medium from IPRL, and in liver, kidneys, bile, plasma, and urine from healthy rats. New metab1 Abbreviations used are: CAP, N-pentyloxycarbonyl-5 -deoxy-5-fluorocytidine (capecitabine); 5 -DFUR, 5 -deoxy-5-fluorouridine; 5-FU, 5-fluorouracil; 5 DFCR, 5 -deoxy-5-fluorocytidine; IPRL, isolated perfused rat liver; 5 -DFCR-G, 2 -D-glucuronide of 5 -deoxy-5-fluorocytidine; 5-FC, 5-fluorocytosine; 5-FCOH, 5-fluoro-6-hydroxycytosine; FAC, fluoroacetate; Cr(acac)3, chromium (III) acetylacetonate; FBAL, -fluoro-alanine; 5-FUH2, 5,6-dihydro-5-fluorouracil; FUPA, -fluoro-ureidopropionic acid; FHPA, 2-fluoro-3-hydroxypropionic acid; PCA, perchloric acid; AS, acid-soluble; AI, acid-insoluble; B.W., body weight; LD, low-dose; HD, high-dose; CPT-11, Irinotecan; FBEN, sodium parafluorobenzoate; Ucap, unknown compound 0.03 ppm downfield from the CAP signal; Ufu, unknown compound close to the 5-FU signal; HPLC, high performance liquid chromatography; TFA, trifluoroacetic acid; LC-MS, liquid chromatography-mass spectometry; COSY, correlation spectroscopy; a.d., administered dose; CFBAL, N-carboxy-fluoro-alanine; , chemical shift; T1, longitudinal relaxation time; RT, retention time. Address correspondence to: Franck Desmoulin, Groupe de RMN Biomédicale, UMR Centre National de la Recherche Scientifique 5623, Université Paul Sabatier, 118 route de Narbonne, 31062 Toulouse, France. E-mail: [email protected] 0090-9556/02/3011-1221–1229$7.00 DRUG METABOLISM AND DISPOSITION Vol. 30, No. 11 Copyright © 2002 by The American Society for Pharmacology and Experimental Therapeutics 737/1018838 DMD 30:1221–1229, 2002 Printed in U.S.A. 1221 at A PE T Jornals on Jauary 7, 2018 dm d.aspurnals.org D ow nladed from