Involvement of concentrative nucleoside transporter 1 in intestinal absorption of trifluorothymidine, a novel antitumor nucleoside, in rats.

ααα-Trifluorothymidine (TFT), an anticancer nucleoside analog, is a potent thymidylate synthase inhibitor. TFT exerts its antitumor activity primarily by inducing DNA fragmentation after incorporation of the triphosphate form of TFT into the DNA. Although an oral combination of TFT and a thymidine phosphorylase inhibitor has been clinically developed, there is little information regarding TFT absorption. Therefore, we investigated TFT absorption in the rat small intestine. After oral administration of TFT in rats, more than 75% of the TFT was absorbed. To identify the uptake transport system, uptake studies were conducted by using everted sacs prepared from rat small intestines. TFT uptake was saturable, significantly reduced under Na(+)-free conditions, and strongly inhibited by the addition of an endogenous pyrimidine nucleoside. From these results, we suggested the involvement of concentrative nucleoside transporters (CNTs) in TFT absorption into rat small intestine. In rat small intestines, the mRNAs coding for rat CNT1 (rCNT1) and rCNT2, but not for rCNT3, were predominantly expressed. To investigate the roles of rCNT1 and rCNT2 in TFT uptake, we conducted uptake assays by using Xenopus laevis oocytes injected with rCNT1 complementary RNA (cRNA) and rCNT2 cRNA. TFT uptake by X. laevis oocytes injected with rCNT1 cRNA, and not rCNT2 cRNA, was significantly greater than that by water-injected oocytes. In addition, in situ single-pass perfusion experiments performed using rat jejunum regions showed that thymidine, a substrate for CNT1, strongly inhibited TFT uptake. In conclusion, TFT is absorbed via rCNT1 in the intestinal lumen in rats.

Formation pathways of gamma-butyrolactone from the furan ring of tegafur during its conversion to 5-fluorouracil.

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-beta-alanine, and gamma-butyrolactone (GBL) and its acidic form, gamma-hydroxybutyrate (GHB). GBL/GHB, which is probably generated from the furan ring of FT, inhibits tumor cell angiogenesis, contributing to the antitumor effect of FT-based 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.

TSU-16, (Z)-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone, is a potent activator of aryl hydrocarbon receptor and increases CYP1A1 and CYP1A2 expression in human hepatocytes.

(Z)-3-[(2,4-dimethylpyrrol-5-yl)methylidenyl]-2-indolinone (TSU-16), is a potent anti-angiogenic agent that inhibits the tyrosine kinase of vascular endothelial growth factor receptor-2. In clinical trials with daily or twice weekly intravenous administration of TSU-16, its increased clearance was observed. To understand the mechanism underlying this observation, we have investigated the TSU-16-mediated regulation of cytochrome P450 expression. In human hepatocytes, TSU-16 increased mRNA levels of CYP1A1 and CYP1A2, but not CYP2B6 and CYP3A4. The extent of increase and profiles of the time-dependent changes in CYP1A1 and CYP1A2 mRNA levels after TSU-16 treatment were similar to those after treatment with 3-methylcholanthrene (3MC), a well-known activator of the aryl hydrocarbon receptor (AhR). In reporter assays using a plasmid construct that contained the human CYP1A1 5'-flanking region including the region crucial for the AhR-dependent transcription of both human CYP1A1 and CYP1A2, TSU-16 treatment increased reporter activities to an extent similar to that obtained with 3MC. Treatment of HepG2 cells and human hepatocytes with AhR-targeting siRNA suppressed the increase in both mRNA levels and CYP1A activities after treatment with TSU-16 as well as after that with omeprazole or 3MC. TSU-16 also time-dependently reduced cellular AhR protein levels in HepG2 cells to a similar extent with 3MC treatment. Furthermore, we demonstrated that unlabeled TSU-16 and 3MC but not omeprazole completely inhibited the specific binding of [(3)H]-3MC to mouse Hepa1c1c7 cytosol, suggesting TSU-16 as an AhR ligand. In conclusion, our present results suggest that TSU-16 binds to and activates AhR to enhance the expression of both human CYP1A1 and CYP1A2. Because TSU-16 is metabolized mainly by CYP1A2, its increased clearance after repeated dosing may be attributed to the enhanced expression of hepatic CYP1A2.

Effect of dimethylnitrosamine-induced liver dysfunction on the pharmacokinetics of 5-fluorouracil after administration of S-1, an antitumour drug, to rats.

OBJECTIVES: The anti-tumour agent S-1 comprises tegafur (a prodrug of 5-fluorouracil; 5-FU), gimeracil (2-chloro-2,4-dihydroxypyridine (CDHP); a competitive inhibitor of 5-FU metabolism) and oteracil potassium. The effect of hepatic dysfunction induced by dimethylnitrosamine (DMN) on the pharmacokinetics of 5-FU after administration of S-1 to rats was investigated. METHODS: S-1 (5 mg/kg) was administered intravenously and orally to rats with DMN-induced liver dysfunction. Plasma concentrations of S-1 components and 5-FU were measured by HPLC and LC/MS-MS. Blood tests and in-vitro enzymatic investigations were also conducted. KEY FINDINGS: DMN treatment induced hepatic dysfunction and decreased the conversion of tegafur to 5-FU in the liver without altering renal function or dihydropyrimidine dehydrogenase activity. Following intravenous administration of S-1, the blood concentration-time profiles of CDHP were similar between control rats and rats with hepatic dysfunction, but the half-life of tegafur was significantly prolonged. The maximum plasma concentration (C(max)) of 5-FU was significantly reduced and the area under the blood concentration-time curve (AUC) was reduced by 22%. Following oral administration, the C(max) of tegafur, 5-FU and CDHP were significantly decreased and half-lives significantly increased. Hepatic dysfunction had a less pronounced effect on the AUC of 5-FU (13.6% reduction). CONCLUSIONS: The pharmacokinetic profiles of tegafur, 5-FU and CDHP were altered by changes in the elimination rate of tegafur induced by a decrease in the conversion of tegafur to 5-FU. However, hepatic dysfunction had less of an effect on the AUC of 5-FU, which correlates with anti-tumour effect, after the oral administration of S-1.

Thymidylate synthase, dihydropyrimidine dehydrogenase, orotate phosphoribosyltransferase mRNA and protein expression levels in solid tumors in large scale population analysis.

It has been reported that the expression of thymidylate synthase (TS), dihydropyrimidine dehydrogenase (DPD) and orotate phosphoribosyltransferase (OPRT) may predict the clinical efficacy of 5-fluorouracil (5-FU)-based therapy in cancer patients. We investigated the differences in the mRNA and protein expression of these enzymes in various tumor tissues. A total of 17,613 specimens of head and neck, gastric, colorectal, breast, lung and pancreatic cancer were collected from multiple facilities in Japan, and the mRNA and protein expression levels of the above enzymes were examined in 4,830 and 12,783 of these specimens, respectively. The mRNA levels were analyzed using RT-PCR in laser-captured microdissected formalin-fixed paraffin-embedded specimens, while the protein levels were analyzed by enzyme-linked immunosorbent assays. The median values of the relative TS, DPD and OPRT mRNA levels were 2.06, 0.803 and 1.17, respectively, while the median protein levels were 22.1, 134.8 and 3.81 ng enzyme/mg protein, respectively. The carcinomas were classified into two sets of four groups each using the overall median levels of TS and DPD or TS and OPRT as cutoff values. Approximately 60% of the gastric cancers exhibited elevated mRNA and protein expression levels of DPD, while >65% of the colorectal cancers showed low levels of DPD expression. Overall, 75% of the head and neck cancers exhibited high expression levels of DPD. Among the lung and pancreatic cancers, 50-74% showed low TS/high DPD expression. In conclusion, the mRNA expression and protein levels of TS, DPD and OPRT differed according to the type of cancer. The results of this large-scale population analysis are expected to be useful as reference data for predicting the relationship between the respective enzyme levels and the efficacy of 5-FU-based chemotherapy.

Identification of human liver cytochrome P450 isoforms involved in autoinduced metabolism of the antiangiogenic agent (Z)-5-[(1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-propanoic acid (TSU-68).

(Z)-5-[(1,2-Dihydro-2-oxo-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-propanoic acid (TSU-68) is a new anticancer drug that inhibits angiogenic receptor tyrosine kinases, which play a crucial role in tumor-induced vascularization. TSU-68 undergoes hepatic oxidation and glucuronidation. Incubation of TSU-68 with human liver microsomes in the presence of NADPH resulted in the formation of three major metabolites: 5-, 6-, and 7-hydroxyindolinone derivatives. The 5-, 6-, and 7-hydroxylation followed simple Michaelis-Menten kinetics with V(max)/K(m) values (an indicator of intrinsic clearance) of 13, 25, and 6 microl/min/mg, respectively. Of the 10 cDNA-expressed human cytochrome P450 isoforms examined, only CYP1A1 and CYP1A2 exhibited appreciable TSU-68 hydroxylation activity. Inhibition studies with alpha-naphthoflavone (a selective CYP1A2 inhibitor) and anti-CYP1A2 antibody also indicated the almost exclusive role of CYP1A2 in microsomal TSU-68 hydroxylation. Treatment of human hepatocytes with 10 microM TSU-68 resulted in a 28- to 140-fold increase in CYP1A1/2-mediated ethoxyresorufin O-deethylase activity. The protein levels of CYP1A2 were increased in TSU-68-treated hepatocytes, and those of CYP1A1, which were undetectable in control hepatocytes, were also increased to detectable levels in the TSU-68-treated hepatocytes. Thus, TSU-68 was shown to induce CYP1A1/2 expression, which was responsible for its hydroxylation. The observation that TSU-68 treatment resulted in a 10- to 45-fold increase in 5-, 6-, and 7-hydroxylation directly demonstrated the autoinduced hydroxylation of TSU-68. In conclusion, TSU-68 has the potential to cause induction of its own CYP1A1/2-mediated oxidative metabolism in humans. This autoinductive effect provides a clear explanation for the clinically observed decrease in TSU-68 plasma concentrations during repeated administration of the drug.

Time-dependent induction of rat hepatic CYP1A1 and CYP1A2 expression after single-dose administration of the anti-angiogenic agent TSU-68.

The anti-angiogenic agent TSU-68 is known to rapidly induce cytochrome P450 activity responsible for its own hydroxylation in rats. In this study, we identified CYP1A1 and CYP1A2 as the TSU-68-induced P450 and temporally characterized the rapid induction of these isoforms. Protein and mRNA levels of CYP1A1 and CYP1A2 along with CYP1A activities were examined in rat liver after a single oral administration of 500 mg/kg TSU-68. CYP1A-mediated ethoxyresorufin O-deethylation and TSU-68 hydroxylation activities reached the maximum at 12 hr. The activities were maintained up to 24 hr and then slowly decreased down to control levels. Protein levels of both CYP1A1 and CYP1A2 were also rapidly induced with temporal profiles similar to the profile of CYP1A activity. In contrast, unlike CYP1A2 mRNA levels, which peaked at 12 hr and almost returned to control levels by 48 hr, CYP1A1 mRNA levels peaked as early as 3 hr and returned to control levels by 24 hr. Thus, CYP1A1 showed more rapid elevation and turnover of its mRNA than CYP1A2. In conclusion, TSU-68 administered to rats rapidly induced mRNA and protein of CYP1A1 and CYP1A2 as well as CYP1A activity. Furthermore, the data showed a difference in the time-dependent induction between CYP1A1 and CYP1A2 mRNAs.

Decrease in plasma concentrations of antiangiogenic agent TSU-68 ((Z)-5-[(1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-propanoic acid) during oral administration twice a day to rats.

TSU-68 ((Z)-5-[(1,2-dihydro-2-oxo-3H-indol-3-ylidene)methyl]-2,4-dimethyl-1H-pyrrole-3-propanoic acid) is a new drug under investigation that inhibits receptor tyrosine kinases involved in tumor angiogenesis. In clinical pharmacokinetic studies, lower plasma concentrations of orally administered TSU-68 are observed after the second dose given within 12 h after the first dose. We examined the cause of this observation through in vivo and ex vivo approaches using rats in which a rapid decrease in the exposure was shown as in humans. In rats, the area under the concentration-time curve after the second dose was decreased to 26% of that after the first dose during administration of TSU-68 (200 mg/kg) twice a day. Plasma clearance of TSU-68 intravenously administered 12 h after oral administration was 1.5-fold higher and the half-life was 2-fold shorter compared with those after the single intravenous administration. The amount of absorbed TSU-68, as indicated by the radioactivity totally excreted in the bile and urine following oral administration of [(14)C]TSU-68, was unchanged by the prior oral administration. These results demonstrate that administered TSU-68 causes an increase in its elimination but not a decrease in its absorption after the subsequent administration. Furthermore, rat liver taken 12 h after administration of TSU-68 exhibited 6-fold higher activity of its microsomal oxidase than untreated liver. This result suggests that TSU-68 induced its own oxidative metabolism (i.e., autoinduction). In conclusion, the decrease in plasma concentrations of TSU-68 during the administration twice a day to rats was due to the rapid autoinduction. The same mechanism is probably at work in the clinical setting.

Distribution of (7alpha)-21-[4-[(diethylamino) methyl]-2-methoxyphenoxy]-7-methyl-19-norpregna-1,3,5(10)-trien-3-ol-20-[14C]2-hydroxy-1,2,3-propanetricarboxylate ([14C]TAS-108) and its metabolites after single oral administration to rats bearing 7,12-dimethylbenz(alpha)anthracene-induced mammary tumor.

(7Alpha)-21-[4-[(diethylamino)methyl]-2-methoxyphenoxy]-7-methyl-19-norpregna-1,3,5(10)-trien-3-ol 2-hydroxy-1,2,3-propanetricarboxylate (TAS-108) is a novel steroidal antiestrogen, modulating the differential recruitment of transcriptional cofactors by liganded estrogen receptors and representing a promising agent for the treatment of breast cancer. To understand better the relationships between the drug exposure and the efficacy or toxicity of TAS-108, we investigated the metabolism and distribution of TAS-108 after oral administration of [14C]TAS-108 to rats bearing a 7,12-dimethylbenz(alpha)anthracene-induced mammary carcinoma. The metabolites (7alpha)-21-[4-[(ethylamino)methyl]-2-methoxyphenoxy]-7-methyl-19-norpregna-1,3,5(10)-trien-3-ol (deEt-TAS-108), (7alpha)-21-[4-[(diethylamino)methyl]-2-methoxyphenoxy]-7-methyl-19-norpregna-1,3,5(10)-trien-3-ol-N-oxide (TAS-108-N-oxide), and 3-methoxy-4-[(7alpha)-7-methyl-19-norpregna-1,3,5(10)-trien-3-ol-21-yl]oxybenzoic acid (TAS-108-COOH) were identified as the major metabolites in the plasma, and in addition, (7alpha)-21-[4-[(ethylamino)methyl]-2-methoxyphenoxy]-3-methoxy-7-methyl-19-norpregna-1,3,5(10)-triene (O-Me-deEt-TAS-108) was identified as a novel metabolite in this study. The time-concentration profiles of TAS-108 and its metabolites in the plasma were compared with those in the tumor and uterus of the rats. Radioactivity was found at a high level in various organs including lung, liver, spleen, ovary, and many glands at 12 h and was relatively higher in tumor tissue than in plasma. On the other hand, the levels of radioactivity in the brain and eyeball were very low or not detectable. TAS-108, deEt-TAS-108, and O-Me-deEt-TAS-108 were extensively distributed in the rat tissues and the tumor, with corresponding tissue/plasma ratios for Cmax and area under the curve in the range of 7 to 100. In contrast, TAS-108-COOH and TAS-108-N-oxide were hardly distributed to the tissues and thus may not contribute to the efficacy or toxicity of TAS-108. Thus, TAS-108, deEt-TAS-108, and O-Me-deEt-TAS-108, being distributed highly in tumor tissue, may be more important for the efficacy and toxicity of TAS-108 in vivo than TAS-108-COOH and TAS-108-N-oxide.

Antimetastatic and anticancer activity of S-1, a new oral dihydropyrimidine-dehydrogenase-inhibiting fluoropyrimidine, alone and in combination with paclitaxel in an orthotopically implanted human breast cancer model.

To evaluate the antitumor and antimetastatic efficacy of oral fluoropyrimidines, alone and combined with taxane on human breast cancer xenografts model, we developed a breast cancer model that spontaneously metastasizes to the lung by orthotopic implantation of MDA-MB-435S-HM tumors into the mammary fat pad (mfp) of SCID mice. The activity of the 5-fluorouracil (5-FU)-degrading enzyme dihydropyrimidine dehydrogenase (DPD) was significantly higher in the metastatic tumors than in the primary tumors. Based on this enzymatic characteristic of pulmonary metastases of breast cancer in regard to 5-FU metabolism, we investigated the antitumor activity of two types of oral 5-FU prodrugs, with and without paclitaxel, on both orthotopically implanted breast tumors and metastatic lung tumors in mice. The drugs and doses used were: S-1, a new oral DPD-inhibiting fluoropyrimidine (DIF) 8.3 mg/kg/day, capecitabine 360 mg/kg/day as a non-DIF, and paclitaxel 50 mg/kg, all of which display minimal toxicity in mice. In the primary tumors, paclitaxel and S-1 displayed a significant antitumor activity, with 57 and 41%, respectively inhibition of tumor growth (p < 0.01), but capecitabine had no effect. When S-1 and paclitaxel were combined, they synergistically caused tumor regression (tumor growth inhibition ratio 94%, p < 0.01) in mice compared to capecitabine plus paclitaxel, without any toxicity. In the pulmonary metastasis model, paclitaxel, and both S-1 alone and combined with paclitaxel, but not capecitabine alone or combined with paclitaxel, diaplayed almost complete antimetastatic activity. These results strongly suggest that combination of S-1, as a DIF with taxanes will show a potent high antitumor and antimetastatic effect on refractory human breast cancers, especially those expressing strong DPD activity.

Pharmacokinetic modeling of species-dependent enhanced bioavailability of trifluorothymidine by thymidine phosphorylase inhibitor.

TAS-102, a new oral drug, is composed of an antitumor drug, alpha,alpha,alpha-trifluorothymidine (FTD), and its metabolic inhibitor, 5-chloro-6-(2-iminopyrrolidine-1-yl)methyl-2,4(1H,3H)-pyrimidinedione hydrochloride (TPI). It has been reported that the oral administration of TAS-102 increases the AUC of FTD in rodents and monkeys in different manners. In this study, a pharmacokinetic model was developed, in an attempt to evaluate the bioavailability of FTD in these animals after the co-administration of TPI. Since TPI inhibits FTD metabolism competitively, a time-dependent as well as concentration-dependent model for the hepatic intrinsic clearance of FTD was developed including the time courses of both FTD and TPI. Based on this modeling, we were able to quantitatively explain the TPI dose-dependent enhancement of AUC of FTD in monkeys, while little increase was observed in rats. These results are consistent to observations that thymidine phosphorylase (TPase) is predominantly expressed in monkeys; while uridine phosphorylase (UPase) is superior to TPase in rats. Since TPase is also predominantly expressed in humans, the pharmacokinetic model developed in this study can be used to explain the bioavailability of TAS-102 in humans.

Functional characterization of single nucleotide polymorphisms with amino acid substitution in CYP1A2, CYP2A6, and CYP2B6 found in the Japanese population.

As a part of the studies conducted by the Pharma SNPs Consortium (PSC), the enzyme activities of CYP1A2, CYP2A6 and CYP2B6 variants with altered amino acids as a result of single nucleotide polymorphisms (SNPs) found among the Japanese population were analyzed under a unified protocol using the same lots of reagents by the laboratories participating in the PSC. Mutations in CYP1A2, CYP2A6 and CYP2B6 were introduced by site-directed mutagenesis and the wild type and mutated CYP molecules were expressed in Escherichia coli. The expressed cytochrome P450s were purified and the enzyme activities were measured in reconstitution systems. CYP1A2 and CYP1A2Gln478His did not show any differences in 7-ethoxyresorufin O-deethylase activity. CYP2A6 and CYP2A6Glu419Asp metabolized coumarin to form 7-hydroxycoumarin in a similar manner, whereas CYP2A6Ile471Thr showed low activity compared to the wild-type CYP2A6. CYP2B6, CYP2B6Pro167Ala and CYP2B6Arg487Cys showed the same activity for 7-ethoxy-4-triflouromethyl-coumarin O-deethylation. However, CYP2B6Gln172His was roughly twice as active as CYP2B6 and the other CYP2B6 variants for 7-ethoxy-4-triflouromethylcoumarin O-deethylation activity. Although higher inter- and intra-laboratory variations were observed for the calculated Km and V(max) values because the studies were conducted in several different laboratories, the degree of variations was reduced by the increased number of analyses and the adoption of a simple analysis system.

Population study of expression of thymidylate synthase and dihydropyrimidine dehydrogenase in patients with solid tumors.

Thymidylate synthase (TS) and dihydropyrimidine dehydrogenase (DPD) has been suggested to be sensitivity-limiting factors of 5-fluorouracil therapy in cancer patients. We conducted a large-scale population study on the activity of TS and DPD in patients with various solid tumors. A total of 2590 clinically removed tumors, consisting of 1112 colon, 724 gastric, 520 breast, and 236 non-small cell lung cancers, were provided to measure TS and DPD activity. TS activity in the gastric, colon, and non-small cell lung cancers was significantly higher than in matched non-cancerous tissue (P<0.0002), but there was no difference in TS expression between tumor and non-cancerous tissue from breast cancer patients. Gastric, breast, and non-small cell lung cancers showed significantly higher DPD activity than their corresponding non-cancerous tissues, but colon cancers did not. There was no correlation between TS activity and DPD activity, and thus each enzyme was considered to be an independent sensitivity-limiting factor for 5-fluorouracil therapy. The median TS activity and median DPD activity in all specimens including gastric, colorectal, breast, and non-small cell lung cancers tested were 0.041 and 110.1 pmol/mg protein, respectively. We classified each of the type of carcinoma into 4 groups by using the median activity of TS and DPD as the cutoff values: a low TS/low DPD group, high TS/low DPD group, low TS/high DPD group, and high TS/high DPD group. About 50% of the gastric, 47% of the colon, 70% of the breast and 30% of the non-small cell lung cancers had high TS activity, and 60% of the gastric, 40% of the colon, 48% of the breast, and 87% of the lung cancers had high DPD activity. Moreover, breast cancer was characterized by high TS activity and lung cancer by high DPD activity as compared with gastric and colon cancers, and their high activity levels may influence to the effectiveness of 5-fluorouracil against cancers of these organs. The results for expression of TS and DPD in clinically dissected tumors would be useful to estimate the efficacy of 5-fluorouracil in the treatment of cancer patients.

A novel mutant allele of the CYP2A6 gene (CYP2A6*11 ) found in a cancer patient who showed poor metabolic phenotype towards tegafur.

In a clinical study, a newly developed anticancer drug, TS-1 capsule, which contained tegafur (FT) and 5-chloro-2,4-dihydroxypyridine, an inhibitor of dihydropyrimidine dehydrogenase, was orally administered to five gastric cancer patients (patients 1-5). The total area under the plasma FT concentration-time curve in patient 1 was four-fold higher than in other patients. Since cytochrome P450 2A6 (CYP2A6) has been reported to metabolize FT to yield 5-fluorouracil (5-FU), it was postulated that the poor metabolic phenotype of patient 1 was caused by mutations of the CYP2A6 gene. Thus, alleles for the CYP2A6 genes derived from patient 1 were completely sequenced. It was found that one allele was CYP2A6*4C, which was a whole deleted allele for the human CYP2A6 gene. The other allele was a novel mutant allele (CYP2A6*11) in which thymine at nucleotide 670 was changed to cytosine. The nucleotide change caused an amino acid change from serine at residue 224 to proline. To examine whether or not the amino acid change affected CYP2A6 activity, we expressed an intact or mutant CYP2A6 together with NADPH-P450 oxidoreductase in Escherichia coli, and compared the capacity of the wild and mutant enzymes to metabolize FT to 5-FU. The Vmax value for FT metabolism by the mutant CYP2A6 was approximately one-half of the value of the intact CYP2A6, although the Km values were nearly the same. From these results, we conclude that the poor metabolic phenotype of patient 1 was caused by the existence of the two mutant alleles, CYP2A6*4C and the new variant CYP2A6*11.