ANTICANCER RESEARCH 26: 3983-3988 (2006)

Correlation between the Urinary Dihydrouracil- Ratio and the 5-FU Plasma Concentration in Patients Treated with Oral 5-FU Analogs

YOSHIFUMI NAKAYAMA, KENTARO MATSUMOTO, YUZURU INOUE, TAKEFUMI KATSUKI, KOJI KADOWAKI, KAZUNORI SHIBAO, YOSUKE TSURUDOME, KEIJI HIRATA, TATSUHIKO SAKO, NAOKI NAGATA and HIDEAKI ITOH

Department of Surgery 1, University of Occupational and Environmental Health, Kita-kyushu, Japan

Abstract. Background: The determination of dihydropyrimidine UH2/Ura (treated/no treated) can predict the plasma 5-FU dehydrogenase (DPD) deficiency is important in avoiding severe concentration levels or DPD deficiencies. 5-fluorouracil (FU) toxicity. The dihydrouracil (UH2)-uracil (Ura) ratio (UH2/Ura) in plasma might be an important 5-fluorouracil (5-FU) and its analogs remain widely used in indicator of the risk of 5-FU catabolic deficiency. In order to the treatment of gastrointestinal carcinomas and other clarify this possibility, the pyrimidine metabolites and the carcinomas. Recently, S-1, a novel oral formulation of tegafur, UH2/Ura were measured in urine and the plasma level of 5-FU oxonic acid and 5-chloro-2, 4-dihydroxypyridine (CDHP) at a was evaluated in patients with gastric and colorectal cancer. molar ratio of 1:0.4:1, is frequently used for patients with Patients and Methods: Patients with primary gastric (n=14) gastric or colorectal cancer in Japan. This drug displayed and colorectal (n=8) cancer who had undergone surgery were powerful effects with a reported response rate of 53.6% for recruited in this study. These patients were divided into the S-1 recurrent gastric cancer in an early phase II study (1). treatment group, which drug is a novel oral formulation of However, as CDHP is a dihydropyrimidine dehydrogenase tegafur, oxonic acid and 5-chloro-2, 4-dihydroxypyridine (DPD) inhibitor, the rate of the adverse bone marrow (CDHP) (n=14) and a group receiving other drugs which suppression after S-1 administration increased in comparison include UFT (Uracil/Tegafur) or oral doxifluridine (n=8). The with that after the administration of other oral 5-FU analogs urinary levels of UH2 and Ura were measured by high- (1). Moreover, some powerful combination regimens of performance liquid chromatography (HPLC) using column chemotherapy for colorectal cancer, such as oxaliplatin/5- swiching. The plasma level of 5-FU was assessed by gas FU/leucovolin (LV) combination and irinotecan/bolus 5- chromatography-mass spectrometry (GC-MS). Results: The FU/LV combination, are now used in Japan. Several UH2/Ura or UH2/Ura (treated/no treated) in the S-1 group pharmacological population studies indicated a higher 5-FU significantly decreased in comparison to that in the other-drug concentration with these new drugs or regimens in group and the plasma 5-FU concentration in the S-1 group comparison to those under previous regimens (2-4). significantly increased compared to that in the group treated Therefore, accurately predicting the adverse side-effects of 5- with other drugs. The plasma 5-FU concentration levels FU is considered clinically important. significantly indicated a positive correlation with urinary Ura. DPD is the key enzyme of pyrimidine and fluoropyrimidine Moreover, UH2/Ura treated with 5-FU analogs or UH2/Ura catabolism. In pyrimidine metabolism, uracil (Ura) and (treated/no treated) significantly showed a negative correlation thymidine are first converted to dihydrouracil (UH2) and with the plasma 5-FU concentration levels. Conclusion: Our dihydrothymidine by DPD and are then metabolized to findings indicate that either urinary Ura, the UH2/Ura or ‚-ureidopropionic acid and ‚-ureidoisobutyric acid, respectively, by . These metabolites are converted subsequently into ‚-alanine and ‚-aminoisobutyric acid, respectively (5). Approximately 80% of an administered Correspondence to: Y. Nakayama, Department of Surgery 1, dose of 5-FU is degraded by this pathway. The remnant of 5- University of Occupational and Environmental Health, 1-1 Iseigaoka, Yahata-nishi-ku, Kita-kyushu 807-8555, Japan. Tel: (+81) 93 691 FU is then metabolized and exerts its cytotoxic effect. DPD is 7441, Fax: (+81) 93 603 2361, e-mail: [email protected] mainly expressed in the liver, but it is also present in such tissues as peripheral blood mononuclear cells (PBMCs), bone Key Words: 5-FU, cancer, dihydrouracil/uracil, S-1. marrow, intestinal mucosa and the spleen (6). The

0250-7005/2006 $2.00+.40 3983 ANTICANCER RESEARCH 26: 3983-3988 (2006) pharmacokinetic data on 5-FU clearance showed that 60-90% Table I. Characteristics of the patients. of the administered 5-FU is metabolized by DPD in the liver No. of patients 22 (7, 8), thus, suggesting that DPD in the liver is critically Gender (M/F) 13/9 important in determining 5-FU toxicity. However, collecting Age (yr) (mean±S.D.) 64.4±10.6 hepatic samples in order to measure the DPD levels remains difficult to perform. Site of carcinoma Patients with genetic fluorouracil catabolic deficiencies stomach 14 colorectum 8 are at high risk for severe toxicity of pyrimidine chemotherapy agents, such as 5-FU or 5-FU analogs. Drug Previous population studies indicated that a complete DPD S-1 14 deficiency is estimated to occur in about 0.1% of patients others 8 and the relative DPD deficiency is projected to have an incidence of 2.7% to 5.8% in cancer patients (8-10). Therefore, a prediction of such DPD deficiency is important in avoiding severe 5-FU toxicity. Cre was measured using an autoanalyzer (13-15). The HPLC To predict 5-FU catabolic deficiencies and toxic side- system consists of a reversed-phase column as the first column, a effects, there are several methods available for measuring cation-exchange column as the secound column, four sets of DPD levels. In general, the DPD activity is measured in ultraviolet absorbance detectors, a microcomputer and other conventional equipments. The detailed procedures of HPLC with PBMCs by ELISA. However, this method is both time- column swiching have been previously described (13). consuming and expensive. Moreover, the DPD levels in PBMCs are not significantly correlated to either the 5-FU Measurement of 5-FU plasma levels. The plasma level of 5-FU was systemic clearance or liver DPD levels (11). On the other assessed by gas chromatography-mass spectrometry (GC-MS) (16). hand, Gamelin et al. indicated the importance of the GC-MS was carried out using Trace GC and Trace MS (Thermo UH2/Ura ratio in plasma level as an indicator of the risk of Electron K.K., Yokohama, Japan) with Xcalibur (Ver. 1.2) 5-FU catabolic deficiency (12). They measured the plasma (Thermo Electron K.K., Yokohama, Japan) as a control system. The detailed procedures of GC-MS were previously described (16). pyrimidine and dehydrogenated metabolite levels using high performance liquid chromatography (HPLC). The urinary Evaluation of the parameters. UH2 and Ura levels and the UH2/Ura pyrimidine metabolites were measured and the importance ratio were compared between the S-1 group and the other-drug of the UH2/Ura ratio level as an indicator of either a 5-FU group. The UH2/Ura (treated/no treated) ratio indicates the catabolic or a DPD deficiency was evaluated in this study. UH2/Ura ratio in treated state with drugs-the UH2/Ura ratio in no treated state ratio for each patient. The relationships between these Patients and Methods parameters and the plasma 5-FU concentration were evaluated.

Patients. Between December 2001 and April 2003, patients with Statistical analysis. Data are expressed as the mean±SD and were primary gastric (n=14) and colorectal (n=8) cancer who had statistically analyzed using the Student’s t-test and the regression undergone surgery at the Department of Surgery 1, University theory, as appropriate. The relationships between parameters were Hospital of Occupational and Environmental Health, Japan, were assessed statistically using Pearson’s correlation coefficient test recruited into this study. The clinical data for these patients are using Stat View-J. Statistical significance was established at the summarized in Table I. The patients were divided into the S-1 p<0.05 levels. treatment group (n=14), and the group treated with other drugs which include UFT (Uracil/Tegafur) or oral doxifluridine (n=8). Results Informed consent was obtained from all patients prior to starting the study. The urinary concentration of Ura, UH2 and the UH2/Ura Urine and blood samples. To evaluate the 5-FU plasma levels, ratio in no treated state with 5-FU analogs were not venous blood samples were collected using a tube containing different in the two groups (Table II). On the other hand, EDTA-Na. The samples were centrifuged for 10 min at in the S-1 group, the concentration of Ura was increased approximately 1000 xg. Plasma was then collected 4 h after and the concentration of UH2 significantly decreased administration of 5-FU analogs and samples were stored at compared to that in the other-drug group which is treated <–20ÆC until analysis. To evaluate the urinary levels of Ura and with UFT or oral doxifluridine. The UH2/Ura ratio treated UH2, urine samples were also collected 4 h after administration of with drugs in the S-1 group was significantly decreased in 5-FU analogs and were stored at <–20ÆC until analysis. comparison to that in the group treated with other drugs Measurement of urinary levels of Ura and DH2. The urinary levels and the plasma 5-FU concentration treated with drugs in of Ura and UH2 were measured by high-performance liquid S-1 group significantly increased compared to that in other- chromatography (HPLC) with column swiching (13), while urinary drug group. Moreover, the UH2/Ura (treated/no treated)

3984 Nakayama et al: Correlation between Urinary UH2/Ura and 5-FU Blood Concentration

Table II. The concentrations of uracil and dihydrouracil, values of the Table III. The concentrations of uracil and dihydrouracil and values of dihydrouracil/uracil ratio and 5-FU concentrations, treated or no treated the dihydrouracil/uracil ratio, treated with 5-FU analogs. with 5-FU analogs. No drug Other drugs S-1 Group Ura (Ìmol/g.Cre.) 33.3±20.5 230.7±279.6 428.6±211.6a S-1 group Other-drug group p-value UH2 (Ìmol/g.Cre.) 16.4±14.9 56.5±45.3b 11.0±4.9 UH2/Ura (Ìmol/g.Cre.) 0.553±0.361 0.395±0.257 0.029±0.014c Not treated Ura (Ìmol/g.Cre.) 30.2±16.0 38.7±27.1 0.3586 All values are shown as the mean±standard deviation (Student’s t-test). UH2 (Ìmol/g.Cre.) 17.8±18.0 14.1±7.0 0.5903 Ura; uracil. UH2; dihydrouracil. Cre.; creatinin. UH2/Ura 0.586±0.352 0.496±0.396 0.5890 asignificantly increased compared to no drug (p<0.0001) or other drugs Treated (p=0.0021). Ura (Ìmol/g.Cre.) 428.6±211.6 230.7±279.6 0.0749 bsignificantly increased in comparison to no drug (p=0.0009) or S-1 UH2 (Ìmol/g.Cre.) 11.0±4.9 56.5±45.3 0.0011 (p=0.0011). UH2/Ura 0.029±0.014 0.395±0.257 <0.0001 csignificantly increased in comparison to no drug (p<0.0001) or other 5FU (ng/ml) 82.4±44.0 18.5±10.7 0.0007 drugs (p<0.0001). The patients in other-drug group were treated with UH2/Ura (treated/ 0.070±0.056 0.846±0.312 <0.0001 UFT (Uracil/Tegafur) or oral doxifluridine. not treated)

All values are shown as the mean±standard deviation (Student’s t-test). Ura;uracil. UH2; dihydrouracil. Cre.; creatinin.The patients in S-1 group were treated with S-1 (Tegafur/Oxonic acid/CDHP). The patients in other-drug group were treated with UFT (Uracil/Tegafur) or oral doxifluridine. ratio was significantly decreased in the S-1 group relative to that in the group treated with other drugs (Table II). S-1 significantly increased the urinary Ura levels and decreased the UH2/Ura ratio compared to values in the other group (Table III). On the other hand, the other drugs significantly increased the UH2 levels compared to levels in the other group (Table III). The plasma 5-FU concentration levels significantly indicated a positive correlation with urinary Ura under Figure 1. Ura levels after administration of 5-FU analogs indicated a medication with 5-FU analogs (p=0.0452, r=0.430) (Figure significantly positive correlation with plasma 5-FU concentration levels 1). However, plasma 5-FU concentration levels did not (p=0.0452, r=0.430). correlate with urinary UH2 concentration levels under medication with 5-FU analogs (data not shown). Though the urinary UH2/Ura ratio under no medication with 5-FU FU pharmacokinetics in the plasma and a close link analogs did not correlate with plasma 5-FU concentration between the toxic side-effects and individual levels (data not shown), the urinary UH2/Ura ratio treated pharmacokinetic parameters was demonstrated (12, 19). with 5-FU analogs were significantly correlated with plasma There is also a correlation between the 5-FU plasma levels, 5-FU concentration levels (p=0.0067, r=–0.552) (Figure 2). toxicity and efficacy (12). These relationships point out a Moreover, the urinary UH2/Ura (treated/no treated) ratio problem of the polymorphism of 5-FU metabolism, the significantly correlated with the plasma 5-FU concentration utility of the therapeutic range determination and of the levels (p=0.0006, r=–0.657) (Figure 3). individual 5-FU dose adaptation (12). It is therefore important to identify patients with a DPD deficiency who Discussion show unexpectedly high concentrations of 5-FU. In the present study, no patients showed any adverse drug Measuring the plasma 5-FU level is considered important reactions related to 5-FU. In a population of 150 Japanese, for the efficacy and safety of this treatment modality. The Ogura et al. reported one healthy volunteer (0.7% of the response to the treatment has been correlated with the 5- population) with a very low peripheral blood mononuclear FU dose, more precisely with the 5-FU area under the curve cell (PBMC) DPD activity due to heterozygosity for a (AUC) or the 5-FU plasma concentration at steady-state mutant allele of the DPYD gene (20). However, the (17,18). There is, however, a high individual variability of 5- frequency of heterozygotes in the normal population was

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Figure 2. The urinary UH2/Ura ratio after administration with 5-FU Figure 3. The urinary UH2/Ura (treated/no treated) ratio significantly analogs indicated a significantly negative correlation with plasma 5-FU correlated with plasma 5-FU concentration levels (p=0.0006, r=–0.657). The concentration levels (p=0.0067, r=–0.552). UH2/Ura (treated/no treated) ratio indicates the UH2/Ura ratio in treated state with drugs – the UH2/Ura ratio in no treated state ratio for each patient.

estimated to be as high as 3% (21). In addition, the estimate of 5-FU degradation as well as of DPD levels frequency of the relative DPD deficiency detected by PBMC because 5-FU and Ura are catabolized by DPD. Therefore, DPD activity was projected to have an incidence of 2.7% of several studies measured plasma pyrimidine and their cancer patients in France (10), 2.7% of patients underwent dehydrogenated metabolite levels with HPLC (32-34). It laparotomy in France (8) and 5.8% of the breast cancer was speculated that the plasma UH2/Ura ratios might be patients in USA (9). On the other hand, several studies helpful in identifying patients with a partial or complete reported the findings of genetic analysis of the DYPD gene. DPD deficiency, thereby allowing us to identify the risk of There were several mutant DPYD alleles in patients 5-FU toxicity. Ohba et al. previously screened Japanese suffering from severe toxicity after the administration of 5- infants for abnormalities in pyrimidine metabolism and FU (22-26). The G to A mutation in the invariant GT splice detected a case of infantile dihydropyrimidinuria using a donor site [IVS14+1G>A] is the most frequent of these method that permits the analysis of UH2 and Ura in small mutations (27). The presence of IVS14+1G>A mutation volumes of urine (14). Moreover, Hayashi et al. analyzed was examined for screening individuals among various urinary UH2 and Ura levels in 1133 adults by HPLC with nationalities (23, 24, 28-30). This mutation was identified in column switching and detected a dihydropyrimidinuria (35). Dutch (23), German (24), Turkish (28), Finnish (29) and In addition, significant linear correlations with liver DPD Taiwanese (29), but not in Japanese (29, 30) or African- levels were demonstrated for the plasma and urinary Americans (29). Therefore, we speculate that little UH2/Ura ratio in a rat experimental model (36). Therefore, difference exists in the DPD activity or DYPD mutation we measured the urinary pyrimidine levels and evaluated between the Japanease and people from other countries. the importance of the UH2/Ura ratio in urinary levels as an Several strategies exist for predicting DPD deficiencies indicator of DPD deficiency or 5-FU concentration. prior to and during 5-FU treatment, including measurement In this study, some parameters were evaluated to predict of the PBMC DPD activity, a mutation analysis of the the 5-FU concentration. Pretreatment urinary Ura, UH2 and DYPD gene and the measurement and the assessment of UH2/Ura did not significantly correlate with plasma 5-FU pyrimidines in plasma or urine. According to previous concentration (data not shown). Though the number of experience, the DPD activity in PBMCs instead of the liver patients recruited in this study is small, pretreatment urinary was determined (10, 31), as PBMCs are much more pyrimidine and UH2/Ura might not be suitable for the accessible than is a liver biopsy. However, the method for prediction of the plasma 5-FU concentration. The urinary detection of PBMC DPD activity was found to be time- Ura concentration 4 h after administration of the 5-FU consuming and expensive. In addition, PBMC DPD levels analogs positively correlated with plasma 5-FU concentration were not significantly correlated to 5-FU systemic clearance (p=0.0452, r=0.430). Moreover, urinary UH2/Ura treated (11). It has become possible to predict the metabolism of with 5-FU analogs negatively correlated with the plasma FU-derived anticancer agents by DPD activity through the 5-FU concentration (p=0.0067, r=–0.552). Based on our determination of plasma or urinary pyrimidine levels. The results, a strong relationship was identified between the measurement of Ura and UH2 levels may provide an plasma 5-FU concentration levels and the UH2/Ura

3986 Nakayama et al: Correlation between Urinary UH2/Ura and 5-FU Blood Concentration

(treated/no treated) ratio (p=0.0006, r=–0.657). The reason 8 Chazal M, Etienne MC, Renee N, Bourgeon A, Tichelme H for this may be that this ratio may reflect the true individual and Milano G: Link between dihydropyrimidine dehydrogenase value of the systemic DPD level. The most suitable predictive activitiy in peripheral blood mononuclear cells and liver. Clin Cacer Res 2: 507-510, 1996. parameter for the plasma 5-FU concentration may thus be 9 Lu Z, Zhang R, Carpenter J and Diasio RB: Decreased the urinary UH2/Ura (treated/no treated) ratio. dihydropyrimidine dehydrogenase activity in a population of The results of the present study suggest that urinary patients with breast cancer: implication for 5-fluorouracil-based pyrimidine metabolites or their parameters may help us to chemotherapy. Clin Cancer Res 4: 325-329, 1998. effectively predict the plasma 5-FU concentration levels and 10 Etienne MC, Lagrange JL, Dassonville O, Fleming R, Thyss A, the urinary UH2/Ura or UH2/Ura (treated/no treated) Rwnee N, Schneider M, Demard F and Milano G: Population ratios might possibly predict DPD deficiencies. Since a study of dihydropyrimidine dehydrogenase in cancer patients. J correlation between 5-FU plasma levels and toxicity was Clin Oncol 12: 2248-2253, 1994. 11 Milano G and Etienne MC: Individualizing therapy with 5- identified (12, 19), accurate indicators of the plasma 5-FU fluorouracil related to dihydropyrimidine dehydrogenase: concentration are thus considered to be important for theory and limits. Ther Drug Monit 18: 335-340, 1996. predicting DPD deficiency. The measurement of urinary 12 Gamelin EC, Danquechin-Dorval EM, Dumesnil YF, Maillart pyrimidine metabolites is non-invasive, safe and easy to PJ, Goudier MJ, Burtin PC, Delva RG, Lortholary AH, Gesta perform. 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