Substrate Report for: Capecitabine

Capecitabine and irinotecan (CPT-11) combination regimen (XELIRI) is used for colorectal cancer treatment. Capecitabine is metabolized to 5-fluorouracil (5-FU) by three enzymes, including carboxylesterase (CES). CES can also convert CPT-11 to 7-ethyl-10-hydroxycamptotecin (SN-38). CES is involved in the metabolic activation of both capecitabine and CPT-11, and it is possible that drug-drug interactions occur in XELIRI. Here, a physiologically based pharmacokinetic (PBPK) model was developed to evaluate drug-drug interactions. Capecitabine (180 mg/kg) and CPT-11 (180 mg/m(2)) were administered to rats, and blood (250 microL) was collected from the jugular vein nine times after administration. Metabolic enzyme activities and K(i) values were calculated through in vitro experiments. The plasma concentration of 5-FU in XELIRI was significantly decreased compared to capecitabine monotherapy, and metabolism of capecitabine by CES was inhibited by CPT-11. A PBPK model was developed based on the in vivo and in vitro results. Furthermore, a PBPK model-based simulation was performed with the capecitabin dose ranging from 0 to 1000mol/kg in XELIRI, and it was found that an approximately 1.7-fold dosage of capecitabine was required in XELIRI for comparable 5-FU exposure with capecitabine monotherapy. PBPK model-based simulation will contribute to the optimization of colorectal cancer chemotherapy using XELIRI.

Capecitabine, an orally available prodrug of 5-FU, requires activation by carboxylesterase (CES) enzymes present in the liver to generate 5'-deoxy-5-flurocytidine ribose (5'-DFCR). The deamination of the latter by cytidine deaminase gives 5'-deoxy-5-fluorouridine ribose (5'-DFUR). Finally, the conversion of 5'-DFUR to the cytotoxic drug 5-FU, occurs primarily in the tumour and is catalyzed by thymidine phosphorylase (TP). Accordingly, it was surmised that events associated with an increase of TP levels should enhance the potency of capecitabine and its metabolites. EGFR inhibition was found to be one such event. The observed synergy between gefitinib and 5'-DFUR has inspired the design of single molecules capable of acting as prodrugs of both an EGFR inhibitor and 5-FU. Here, we report on the synthesis and characterization of one such molecule, ZRX1, that consists of an acetylated 5'-DFCR moiety linked to a quinazoline inhibitor of EGFR through an alkyl dicarbamate spacer that requires CES activation to generate the two active metabolites. Our results showed that ZRX1 was ineffective as an intact molecule. However, when CES was present, ZRX1 induced an increase in EGFR inhibition, TP expression, DNA damage and apoptosis. ZRX1 was, at least, 3-fold more potent than capecitabine and 5'-DFUR and recapitulated the effects of the combination treatments. LC-MS analysis showed that in the presence of CES, ZRX1 is metabolized into a mixture of bioactive quinazoline derivatives and 5'-DFCR derived metabolites. Our results in toto, suggest that capecitabine-based EGFR targeting combi-molecules of the same type than ZRX1, have the potential to induce stronger growth inhibitory potency than capecitabine, 5'-DFUR or single EGFR inhibitors and equivalent potency when compared with combinations of EGFR inhibitors + 5'-DFUR.

Our understanding of the detailed recognition and processing of clinically useful therapeutic agents has grown rapidly in recent years, and we are now able to begin to apply this knowledge to the rational treatment of disease. Mammalian carboxylesterases (CEs) are enzymes with broad substrate specificities that have key roles in the metabolism of a wide variety of clinical drugs, illicit narcotics and chemical nerve agents. Here, the functions, mechanism of action and structures of human CEs are reviewed, with the goal of understanding how these proteins are able to act in such a non-specific fashion, yet catalyze a remarkably specific chemical reaction. Current approaches to harness these enzymes as protein-based therapeutics for drug and chemical toxin clearance are described, as well as their uses for targeted chemotherapeutic prodrug activation. Also included is an outline of how selective CE inhibitors could be used as co-drugs to improve the efficacy of clinically approved agents.

AIM OF THE STUDY: Occurrence of hand-foot syndrome (HFS) during capecitabine treatment often results in treatment interruptions (26%) or treatment discontinuation (17%), and can severely decrease quality of life. In this study, we investigated whether single nucleotide polymorphisms (SNPs) in genes involved in capecitabine metabolism - other than DPYD - are associated with an increased risk for capecitabine-induced HFS. METHODS: Patients treated with capecitabine according to standard of care were enrolled after providing written informed consent for genotyping purposes. Prospectively collected blood samples were used to extract genomic DNA, which was subsequently genotyped for SNPs in CES1, CES2 and CDA. SNPs and clinical baseline factors that were univariably associated with HFS with Ps>=s0.10, were tested in a multivariable model using logistic regression. RESULTS: Of the 446 patients eligible for analysis, 146 (32.7%) developed HFS, of whom 77 patients (17.3%) experienced HFS <=sgrade 2. In the multivariable model, CES1 1165-33 C>A (rs2244613, minor allele frequency 19%) and CDA 266s+s242 A>G (rs10916825, minor allele frequency 35%) variant allele carriers were at higher risk of HFS <=sgrade 2 (OR 1.888; 95%CI 1.075-3.315; P = 0.027 and OR 1.865; 95% CI 1.087-3.200; P = 0.024, respectively). CONCLUSIONS: We showed that CES1 1165-33 C>A and CDA 266 + 242 A>G are significantly associated with HFS grade 2 and grade 3 in patients treated with capecitabine. Prospective studies should assess whether this increased risk can be mitigated in carriers of these SNPs, when pre-emptive genotyping is being followed by dose adjustment or by alternative treatment by a fluoropyrimidine that is not substrate to CES1, such as S1.

Capecitabine and irinotecan (CPT-11) combination regimen (XELIRI) is used for colorectal cancer treatment. Capecitabine is metabolized to 5-fluorouracil (5-FU) by three enzymes, including carboxylesterase (CES). CES can also convert CPT-11 to 7-ethyl-10-hydroxycamptotecin (SN-38). CES is involved in the metabolic activation of both capecitabine and CPT-11, and it is possible that drug-drug interactions occur in XELIRI. Here, a physiologically based pharmacokinetic (PBPK) model was developed to evaluate drug-drug interactions. Capecitabine (180 mg/kg) and CPT-11 (180 mg/m(2)) were administered to rats, and blood (250 microL) was collected from the jugular vein nine times after administration. Metabolic enzyme activities and K(i) values were calculated through in vitro experiments. The plasma concentration of 5-FU in XELIRI was significantly decreased compared to capecitabine monotherapy, and metabolism of capecitabine by CES was inhibited by CPT-11. A PBPK model was developed based on the in vivo and in vitro results. Furthermore, a PBPK model-based simulation was performed with the capecitabin dose ranging from 0 to 1000mol/kg in XELIRI, and it was found that an approximately 1.7-fold dosage of capecitabine was required in XELIRI for comparable 5-FU exposure with capecitabine monotherapy. PBPK model-based simulation will contribute to the optimization of colorectal cancer chemotherapy using XELIRI.

An important concern with the anticancer drug capecitabine (Cp), an oral prodrug of 5-fluorouracil, are dose-limiting adverse effects, in particular hand-foot syndrome (HFS) and diarrhea. Here we evaluated the association of genetic variability in all enzymes of the Cp-activation pathway to 5-fluorouracil with Cp-related early-onset toxicity in 144 patients receiving Cp. We identified a haplotype encompassing five variants in the carboxylesterase 1 (CES1) gene region including an expression quantitative trait locus associated with early-onset Cp-toxicity (Haplotype A3: ORadditive = 2.2, 95% CI 1.2-4.0, Padjusted = 0.012; ORrecessive = 10.3, 95% CI 2.1-49.4, Padjusted = 0.0038). Furthermore, the association of two linked cytidine deaminase (CDA) promoter variants (c.1-451C>T: ORdominant = 4.3, 95% CI 1.3-14.2, Padjusted = 0.017; and c.1-92A>G: ORdominant = 4.4, 95% CI 1.3-14.5, Padjusted = 0.015) with Cp-related diarrhea was replicated. This first study identifying an association of genetic variation in CES1 with Cp-related toxicity provides further evidence for the existence of a functional noncoding CES1-variant with a possible regulatory impact.

Before being able to develop a pharmacodynamic effect, a number of drugs have to be activated by enzymes, which are known to be potentially influenced by manifold factors, leading to a possible alteration of their activity behaviour. Based on capecitabine, we report a simple and rapid method for the estimation and comparison of the so-called 'apparent enzyme activity' (R), not only intra- (different dose levels) but also inter-schedule, to contribute to therapeutic success. Dividing the area under the curve (AUC) of the product by the AUC of the precursor generates a factor which indicates the apparent activity of the enzyme involved in the biotransformation of a compound. Our own data as well as data from the literature was used to calculate those R levels revealing that the formation of 5'-DFUR - the immediate precursor of 5-fluorouracil - was not affected by concomitant medication within the dosing range investigated. Calculated hypothetical means of R for carboxylesterase (1.49 +/- 0.66) and for cytidine deaminase (1.17 +/- 0.65) were obtained. Additionally, it is important to note that the method described in this report is of general use and not limited to chemotherapeutic agents, as soon as enzymes are involved in drug activation. (c) 2015 S. Karger AG, Basel.

BACKGROUND: The approved capecitabine regimen as monotherapy in metastatic breast cancer (MBC) is 1,250 mg/m(2) twice daily for 2 weeks on and 1 week off (Cint). Dose modifications are often required because of severe hand-foot syndrome (HFS). We tested a continuous regimen with a lower daily dose but a similar cumulative dose in an attempt to reduce the severity of adverse events (AEs) while maintaining efficacy.
METHODS: We randomized 195 patients with HER-2/neu-negative MBC to capecitabine 800 mg/m(2) twice daily throughout the 21-day cycle (Ccont) or to Cint to assess noninferiority in the percentage of patients free of progression at 1 year. Secondary endpoints included efficacy and safety. Associations between polymorphisms in capecitabine metabolism-related genes and drug response were assessed.
RESULTS: The percentage of patients free of progression at 1 year was 27.3% with Cint versus 25.3% with Ccont (difference of -2.0%; 95% confidence interval: -15.5% to 11.5%, exceeding the 15% deemed noninferior). Differences regarding other efficacy variables were also not found. Grade 3-4 HFS was the most frequent AE (41.1% in Cint vs. 42.3% in Ccont). Grade 3-4 neutropenia, thrombocytopenia, diarrhea, and stomatitis were more frequent with Cint. A 5' untranslated region polymorphism in the carboxylesterase 2 gene was associated with HFS. One polymorphism in cytidine deaminase and two in thymidine phosphorylase were associated with survival. CONCLUSION: Our study was unable to show noninferiority with the continuous capecitabine regimen (Ccont) compared with the approved intermittent regimen (Cint). Further investigation is required to improve HFS. Polymorphisms in several genes might contribute to interindividual differences in response to capecitabine.

Capecitabine, an orally available prodrug of 5-FU, requires activation by carboxylesterase (CES) enzymes present in the liver to generate 5'-deoxy-5-flurocytidine ribose (5'-DFCR). The deamination of the latter by cytidine deaminase gives 5'-deoxy-5-fluorouridine ribose (5'-DFUR). Finally, the conversion of 5'-DFUR to the cytotoxic drug 5-FU, occurs primarily in the tumour and is catalyzed by thymidine phosphorylase (TP). Accordingly, it was surmised that events associated with an increase of TP levels should enhance the potency of capecitabine and its metabolites. EGFR inhibition was found to be one such event. The observed synergy between gefitinib and 5'-DFUR has inspired the design of single molecules capable of acting as prodrugs of both an EGFR inhibitor and 5-FU. Here, we report on the synthesis and characterization of one such molecule, ZRX1, that consists of an acetylated 5'-DFCR moiety linked to a quinazoline inhibitor of EGFR through an alkyl dicarbamate spacer that requires CES activation to generate the two active metabolites. Our results showed that ZRX1 was ineffective as an intact molecule. However, when CES was present, ZRX1 induced an increase in EGFR inhibition, TP expression, DNA damage and apoptosis. ZRX1 was, at least, 3-fold more potent than capecitabine and 5'-DFUR and recapitulated the effects of the combination treatments. LC-MS analysis showed that in the presence of CES, ZRX1 is metabolized into a mixture of bioactive quinazoline derivatives and 5'-DFCR derived metabolites. Our results in toto, suggest that capecitabine-based EGFR targeting combi-molecules of the same type than ZRX1, have the potential to induce stronger growth inhibitory potency than capecitabine, 5'-DFUR or single EGFR inhibitors and equivalent potency when compared with combinations of EGFR inhibitors + 5'-DFUR.

Background:The aging process is accompanied by physiological changes including reduced glomerular filtration and hepatic function, as well as changes in gastric secretions. To investigate what effect would aging have on the disposition of capecitabine and its metabolites, the pharmacokinetics between patients >/=70 years and <60 years were compared in SWOG0030.Methods:Twenty-nine unresectable colorectal cancer patients were stratified to either >/=70 or <60 years of age, where the disposition of capecitabine and its metabolites were compared.Results:Notable increase in capecitabine area under the curve (AUC) was accompanied by reduction in capecitabine clearance in >/=70 years patients (P<0.05). No difference in 5'-deoxy-5-fluorocytidine, 5'-deoxy-5-fluorouridine (DFUR), and 5-fluorouracil (5FU) AUCs between the two age groups, suggesting that carboxylesterase and cytidine deaminase (CDA) activity was similar between the two age groups. These results suggest that metabolic enzymes involved in converting capecitabine metabolites are not altered by age. An elevation in capecitabine Cmax and reduction in clearance was seen in females, where capecitabine AUC was 40.3% higher in women. Elevation of DFUR Cmax (45%) and AUC (46%) (P<0.05) was also noted, suggesting that CDA activity may be higher in females.Conclusion:Increases in capecitabine Cmax and AUC was observed in patients >/=70 years when compared with younger patients who were >60 years.

PURPOSE: We designed this study in locally advanced rectal cancer to determine the pathological response, toxicity, and disease-free survival (DFS) with induction capecitabine plus irinotecan followed by capecitabine-based chemoradiotherapy (CRT) and analyze the gene expression of enzymes involved in the metabolism of capecitabine and irinotecan for associations with response and toxicity. METHODS: Patients with T3/T4 or node positive rectal cancer were treated with capecitabine 1,000 mg/m(2) twice daily (BID) days 1-14, and irinotecan 200 mg/m(2) on day 1 every 21 days for 2 cycles, followed by capecitabine 825 mg/m(2) BID days 1-5 per week with concurrent radiotherapy 50.4 Gy in 28 fractions. Surgical resection occurred a median of 7.4 weeks after CRT. Gene expression levels or sequencing were used to analyze carboxylesterase-converting enzymes (CES1, CES2), thymidylate synthase (TS), thymidine phosphorylase (TP), dehydropyrimidine dehydrogenase (DPD), topoisomerase I (TOPO I), and uridine-diphosphate (UDP) glucuronosyl transferase 1A1 in pre- and post-treatment tumor and normal tissue samples. RESULTS: Twenty-two patients were enrolled, and 18 completed neoadjuvant therapy and underwent R0 resection. Two patients with UGT1A1 7/7 had grade 3 and 4 neutropenic fever and sepsis. Pathological complete response (pCR) occurred in 6 of 18 patients (33 %) and 10 (56 %) had tumor and/or nodal downstaging. The 3-year DFS was 75.5 % (95 % CI, 39.7-91.8 %). Locoregional control rate was 100 %. We observed higher TP gene expression in pCR patients, but no correlations with toxicity. CONCLUSIONS: This neoadjuvant regimen was safe and demonstrated significant antitumor activity. High TP tumor gene expression was associated with obtaining pCR.

PURPOSE: Hand-foot syndrome (HFS) is one of the most relevant dose-limiting adverse effects of capecitabine, an oral prodrug of 5-fluorouracil used in the standard treatment of breast and colorectal cancer. We investigated the association between grade 3 HFS and genetic variations in genes involved in capecitabine metabolism. EXPERIMENTAL DESIGN: We genotyped a total of 13 polymorphisms in the carboxylesterase 2 (CES2) gene, the cytidine deaminase (CDD) gene, the thymidine phosphorylase (TP) gene, the thymidylate synthase (TS) gene, and the dihydropyrimidine dehydrogenase (DPD) gene in 130 patients treated with capecitabine. We correlated these polymorphisms with susceptibility to HFS. RESULTS: We found an association of HFS appearance with rs532545 located in the promoter region of CDD (OR = 2.02, 95% CI = 1.02-3.99, P = 0.039). Because we found no association between the rs532545 genotype and CDD mRNA expression in Epstein-Barr virus lymphoblastoid cells, we explored additional genetic variations across the CDD promoter. We found an insertion, rs3215400, in linkage disequilibrium with rs532545 (D' = 0.92), which was more clearly associated with HFS (OR = 0.51, 95% CI = 0.27-0.95, P = 0.028) in patients and with total CDD gene expression (P = 0.004) in lymphoblastoid cells. In silico analysis suggested that this insertion might create a binding site for the transcriptional regulator E2F. Using a SNaPshot assay in lymphoblastoid cells, we observed a 5.7-fold increased allele-specific mRNA expression from the deleted allele. CONCLUSIONS: The deleted allele of rs3215400 shows an increased allele-specific expression and is significantly associated with an increased risk of capecitabine-induced HFS.

PURPOSE: To determine the maximal tolerated dose of capecitabine with oxaliplatin + radiotherapy in a phase I study of localized esophageal cancer. PATIENTS AND METHODS: Oxaliplatin (85 mg/m(2)) administered on days 1, 15, and 29. Capecitabine administered twice daily 5 days weekly; dose levels (DL) were 1, 1000; 2, 1250; and 3, 1500 mg/m(2) with 50.4 Gy radiation. RESULTS: Dose-limiting toxicity was reached at DL 3. Carboxylesterase expression in day 2 tumor specimens and induction correlated with response (p

Capecitabine is a drug that requires the consecutive action of three enzymes: carboxylesterase 2 (CES 2), cytidine deaminase (CDD), and thymidine phosphorylase (TP) for transformation into 5-fluorouracil (5FU). The metabolism of 5FU requires the activity of thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) among other enzymes. The present study prospectively examined the possible relationship between the toxicity and efficacy of capecitabine and 14 different polymorphisms in CES 2, CDD, TS and DPD. Between 2003 and 2005, a total of 136 patients with advanced breast or colorectal cancer treated with capecitabine were prospectively enrolled. The presence of two polymorphisms (CDD 943insC and CES 2 Exon3 6046 G/A) were associated with a non-statistically significant higher incidence of grade 3 hand-foot syndrome (HFS) (p=0.07) and grade 3-4 diarrhoea (p=0.09), respectively. Patients heterozygous or homozygous for the polymorphism CES 2 5'UTR 823 C/G exhibited a significantly greater response rate to capecitabine, and time to progression of disease (59%, 8.7 months) than patients with the wild type gene sequence (32%, p=0.015; 5.3 months, p=0.014). For the first time, an association between a polymorphism in the CES2 gene and the efficacy of capecitabine has been described, providing preliminary evidence of its predictive and prognostic value.

OBJECTIVE: To report 2 cases of severe hypertriglyceridemia associated with the use of oral capecitabine. CASE SUMMARIES: The first patient was a 73-year-old woman with metastatic breast carcinoma who received capecitabine 2500 mg/m2/day in 2 divided doses for 2 weeks followed by a one week rest period. The baseline triglyceride level was 324 mg/dL; after 2 cycles of capecitabine, levels increased to 916 mg/dL. Although lipid-lowering treatment was initiated, triglyceride levels peaked at 1782 mg/dL by the end of the seventh cycle. Eight weeks after capecitabine treatment was stopped, triglyceride levels decreased to 118 mg/dL. The second patient was a 59-year-old man with metastatic colorectal carcinoma who was placed on capecitabine treatment at a dosage of 2500 mg/m2/day in 2 divided doses for 2 weeks followed by a one week rest period. The baseline triglyceride level was 244 mg/dL; levels peaked at 1455 mg/dL at the end of the fifth cycle. Capecitabine treatment was discontinued due to disease progression, and triglyceride levels decreased to 154 mg/dL after 11 weeks. DISCUSSION: The most frequently reported adverse effects of capecitabine are gastrointestinal and hematologic effects and palmar-plantar erythrodysesthesia. Drug-induced hyperlipidemia may appear more readily in individuals with hereditary lipoprotein lipase deficiency because decreased lipoprotein lipase activity might make these individuals more susceptible to a rise in triglyceride levels. The Naranjo probability scale indicated a probable relationship between capecitabine and severe hypertriglyceridemia.
CONCLUSIONS: Capecitabine should be prescribed with care, especially in patients with preexisting hypertriglyceridemia. The question of whether capecitabine actually causes hypertriglyceridemia needs careful consideration, and the possible mechanism by which it may cause this adverse effect requires further investigation.

Capecitabine is an oral prodrug of 5-fluorouracil that is indicated for the treatment of breast and colorectal cancers. A three-step in vivo-targeted activation process requiring carboxylesterases, cytidine deaminase, and thymidine phosphorylase converts capecitabine to 5-fluorouracil. Carboxylesterases hydrolyze capecitabine's carbamate side chain to form 5'-deoxy-5-fluorocytidine (5'-DFCR). This study examines the steady-state kinetics of recombinant human carboxylesterase isozymes carboxylesterase (CES) 1A1, CES2, and CES3 for hydrolysis of capecitabine with a liquid chromatography/mass spectroscopy assay. Additionally, a spectrophotometric screening assay was utilized to identify drugs that may inhibit carboxylesterase activation of capecitabine. CES1A1 and CES2 hydrolyze capecitabine to a similar extent, with catalytic efficiencies of 14.7 and 12.9 min(-1) mM(-1), respectively. Little catalytic activity is detected for CES3 with capecitabine. Northern blot analysis indicates that relative expression in intestinal tissue is CES2 > CES1A1 > CES3. Hence, intestinal activation of capecitabine may contribute to its efficacy in colon cancer and toxic diarrhea associated with the agent. Loperamide is a strong inhibitor of CES2, with a K(i) of 1.5 muM, but it only weakly inhibits CES1A1 (IC(50) = 0.44 mM). Inhibition of CES2 in the gastrointestinal tract by loperamide may reduce local formation of 5'-DFCR. Both CES1A1 and CES2 are responsible for the activation of capecitabine, whereas CES3 plays little role in 5'-DFCR formation.

Our understanding of the detailed recognition and processing of clinically useful therapeutic agents has grown rapidly in recent years, and we are now able to begin to apply this knowledge to the rational treatment of disease. Mammalian carboxylesterases (CEs) are enzymes with broad substrate specificities that have key roles in the metabolism of a wide variety of clinical drugs, illicit narcotics and chemical nerve agents. Here, the functions, mechanism of action and structures of human CEs are reviewed, with the goal of understanding how these proteins are able to act in such a non-specific fashion, yet catalyze a remarkably specific chemical reaction. Current approaches to harness these enzymes as protein-based therapeutics for drug and chemical toxin clearance are described, as well as their uses for targeted chemotherapeutic prodrug activation. Also included is an outline of how selective CE inhibitors could be used as co-drugs to improve the efficacy of clinically approved agents.

Capecitabine is a novel fluoropyrimidine carbamate, orally administered and selectively activated to fluorouracil by a sequential triple-enzyme pathway in liver and tumor cells. This prospective trial aims to evaluate the therapeutic effects and systemic toxicities of capecitabine in patients with metastatic renal cell carcinoma in which immunotherapy failed. Twenty-six patients (median age, 58 years; range, 47 to 76 years) with disease in which first- or second-line immunotherapy treatment failed entered the trial. Median time of observation was 13+ months (range, 3 to 25+ months). Capecitabine was administered in the outpatient setting orally at a dose of 2,500 mg/m2/d divided into two daily doses for 14 days, followed by 7 days of rest. This schedule was repeated in 3-week intervals. Twenty-six patients are now assessable for toxicity, and 23 patients, for response. We observed a partial response to treatment in 2 patients (8.7%), minor response in 5 patients (21.7%), stable disease in 13 patients (56.5%), and continued disease progression despite treatment in only 3 patients (13.1%). Outpatient capecitabine therapy was well tolerated, and World Health Organization (WHO) grade III toxicity in these 26 patients consisted of hand-foot syndrome in 2 patients (7.7%) and anemia in 1 patient (3.8%). We did not observe WHO grade IV toxicity. Oral capecitabine appears to be a promising treatment with a favorable toxicity profile in patients with advanced renal cell carcinoma and should be evaluated in first- and second-line treatment schedules as monotherapy, as well as in combination with immunotherapy agents.

Capecitabine (N4-pentyloxycarbonyl-5'-deoxy-5-fluorocytidine) is a novel oral fluoropyrimidine carbamate, which is converted to 5-fluorouracil (5-FU) selectively in tumours through a cascade of three enzymes. The present study investigated tissue localisation of the three enzymes in humans, which was helpful for us to design the compound. Carboxylesterase was almost exclusively located in the liver and hepatoma, but not in other tumours and normal tissue adjacent to the tumours. Cytidine (Cyd) deaminase was located in high concentrations in the liver and various types of solid tumours. Finally, thymidine phosphorylase (dThdPase) was also more concentrated in various types of tumour tissues than in normal tissues. These unique tissue localisation patterns enabled us to design capecitabine. Oral capecitabine would pass intact through the intestinal tract, but would be converted first by carboxylesterase to 5'-deoxy-5-fluorocytidine (5'-dFCyd) in the liver, then by Cyd deaminase to 5'-deoxy-5-fluorouridine (5'-dFUrd) in the liver and tumour tissues and finally by dThdPase to 5-FU in tumours. In cultures of human cancer cell lines, the highest level of cytotoxicity was shown by 5-FU itself, followed by 5'-dFUrd. Capecitabine and 5'-dFCyd had weak cytotoxic activity only at high concentrations. The cytotoxicity of the intermediate metabolites 5'-dFCyd and 5'-dFCyd was suppressed by inhibitors of Cyd deaminase and dThdPase, respectively, indicating that these metabolites become effective only after their conversion to 5-FU. Capecitabine, which is finally converted to 5-FU by dThdPase in tumours, should be much safer and more effective than 5-FU, and this was indeed the case in the HCT116 human colon cancer and the MX-1 breast cancer xenograft models.