Inhibitory Effect of 5-Fluorouracil on Cytochrome P450 2C9. Activity in Cancer Patients. Arzu Gunes1, Ugur Coskun2, Cem Boruban3, Nazan Gunel2, Melih O.
C Basic & Clinical Pharmacology & Toxicology 2006, 98, 197–200. Printed in Denmark . All rights reserved Copyright C ISSN 1742-7835
Inhibitory Effect of 5-Fluorouracil on Cytochrome P450 2C9 Activity in Cancer Patients Arzu Gunes1, Ugur Coskun2, Cem Boruban3, Nazan Gunel2, Melih O. Babaoglu4, Orhan Sencan3, Atila Bozkurt4, Anders Rane5, Moustapha Hassan6, Hakan Zengil1 and Umit Yasar4 Department of Pharmacology and 2Department of Oncology, Gazi University, Medical Faculty, Ankara, 3 Department of Oncology, SSK Ankara Hospital, 4Department of Pharmacology, Hacettepe University, Medical Faculty, Ankara, 5Department of Clinical Pharmacology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, and 6Laboratory of Haematology, Division of Haematology, Karolinska Institutet, Karolinska University Hospital Huddinge, Stockholm, Sweden 1
(Received August 3, 2005; Accepted September 19, 2005) Abstract: Drug interactions have been reported between 5-fluorouracil and cytochrome P450 2C9 (CYP2C9) substrates, S-warfarin and phenytoin. This study was performed to determine the influence of 5-fluorouracil on cytochrome P450 2C9 (CYP2C9) activity in colorectal cancer patients (nΩ17) receiving 5-fluorouracil. Losartan was used as a marker to assess CYP2C9 activity. Losartan and its CYP2C9 dependent metabolite, E-3174, were determined in urine. The ratios of urinary losartan/E-3174 before and after the 5-fluorouracil treatment were compared for each patient. Genotyping was performed to detect the CYP2C9*2 and CYP2C9*3. At the end of the first cycle of 5-fluorouracil, losartan/E-3174 ratio was increased by 28.0% compared to the pre-treatment values (PΩ0.15). In five patients recruited for phenotyping after three 5-fluorouracil cycles, the metabolic ratio was increased significantly by 5.3 times (PΩ0.03). The results suggest that in most patients 5-fluorouracil inhibited CYP2C9 activity. This inhibition was more pronounced when the total administered dose increased. This finding may help explain the mechanism of interaction between 5-fluorouracil and CYP2C9 substrates.
5-Fluorouracil is a fluorinated pyrimidine analogue used in the treatment of a variety of solid tumours including gastrointestinal, breast, head and neck cancer (Rich et al. 2004). The cytotoxicity of 5-fluorouracil is mainly due to the formation of fluoro-deoxyuridine-monophosphate metabolite, which inhibits thymidylate synthetase and incorporates into ribonucleic acid (Rich et al. 2004). 5-Fluorouracil has a rather short elimination half-life in plasma and is mostly eliminated by dihydropyrimidine dehydrogenase (Gonzalez & Fernandez-Salguero 1995). Due to many complications during chemotherapy, many other drugs are co-administrated during treatment with 5-fluorouracil. Increasing number of drug-drug interactions between warfarin and 5fluorouracil-based chemotherapies have been reported. These interactions result in bleeding complications due to the prolongation of prothrombin time (Brown 1997 & 1999; Kolesar et al. 1999). 5-Fluorouracil has also been reported to interact with phenytoin, resulting in elevated phenytoin plasma concentrations and neurotoxic side effects (Gilbar & Brodribb 2001; Konishi et al. 2002; Rosemergy & Findlay 2002; Brickell et al. 2003). Both S-warfarin and phenytoin are established CYP2C9 substrates (Lee et al. 2002). The mechanism of these interactions is unclear. Available evidence from case reports and in vitro studies in liver microAuthor for correspondence: Umit Yasar, Hacettepe University, Faculty of Medicine, Department of Pharmacology, 06100 Sihhiye, Ankara, Turkey (fax π90 312 3105312, e-mail uyasar/hacettepe.edu. tr).
somes suggests that the interaction is not due to a competitive inhibition of CYP2C9 (Brown 1999; Park & Kim 2003). The effect of 5-fluorouracil on in vivo CYP2C9 activity in man has not been studied. Prospective clinical studies are needed to clarify the mechanism of the interaction between the substrates of CYP2C9 and 5-fluorouracil. CYP2C9 is a polymorphic enzyme that catalyses the metabolism of a large number of clinically used drugs. Losartan is a selective angiotensin II receptor antagonist that is converted to its active metabolite, E-3174 by CYP2C9 (Yasar et al. 2001). Urinary losartan/E-3174 ratio after a single oral dose of losartan (25 mg) has been suggested as a useful measure of CYP2C9 activity in healthy volunteers (Yasar et al. 2002; Babaoglu et al. 2004). Therefore, this study was conducted to investigate the acute and chronic effects of 5fluorouracil on losartan oxidation in cancer patients.
Materials and Methods The present study was performed in 17 patients with colorectal cancer from Turkey (12 male/5 female) with median age of 60 (range 34–76) and median body weight of 65 kg (range 45–80). All patients had histologically or cytologically confirmed malignant diseases. The study did not interfere with the standard treatment protocol of the patients who received more than one cycles of 5-fluorouracil chemotherapy with 21 days interval. Each treatment cycle consisted of 5-fluorouracil (425 mg/m2) plus folinic acid (20 mg/m2) as a 1 hr infusion for at least three consecutive days. A single oral dose of 25 mg losartan (CozaarA, Merck Sharp & Dohme) was given to the patients at least two days prior to the first chemotherapy cycle and urine was collected during the following 8 hr.
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An aliquot of 10 ml was stored at ª20 æ until analysis. On the last day of the first chemotherapy cycle, the single dose of losartan administration was repeated immediately after the 5-fluorouracil infusion. Only in five patients (2 females/3 males) this procedure could be performed at the end of the third chemotherapy cycle due to the difficulty in following patients for a relatively long period and also the difficulty in finding patients without any concomitant therapy. Losartan was given to the patients after a 3-hr fasting period. The patients did not receive any other drug mainly known to be metabolised by CYP2C9 or any drug that may affect CYP2C9 activity, during the study period. All the patients were out-patients. Patients with hepatic or renal dysfunction and debilitated individuals were excluded from the study. Written informed consent was obtained from the patients. The study was performed in accordance with the Declaration of Helsinki and approved by the local Ethics Committee at Gazi University Hospital, Ankara, Turkey. HPLC analysis of losartan and its carboxy metabolite. Analysis of losartan and its carboxy metabolite, E-3174, in urine was performed by high-pressure liquid chromatography system (HPLC) with a fluorescence detection as described previously (Babaoglu et al. 2004). In brief, urine samples were mixed with 0.5 M citrate buffer and isopropanol (4/1/1: ml/ml/ml, respectively) to yield a pH of 4.3. Twenty ml of this mixture was injected to the HPLC instrument
through a Zorbax SB-Phenyl column (4.6 mm ID¿25 cm). The mobile phase (pH: 2.3) consisted of 10 mM sodium di-hydrogen phosphate – acetonitrile (64/36:v/v, respectively) with a flow rate of 1.5 ml/min. (Babaoglu et al. 2004). All samples were analysed in duplicates. The limits of detection for losartan and E-3174 were 20 nM and 10 nM, respectively. The coefficients of variation were less than 10% for losartan and the metabolite (nΩ10). Urinary losartan/ E-3174 ratio was calculated from the recoveries of losartan and E3174 in the 8-hr urine sample. CYP2C9 genotyping. Genomic DNA was isolated from peripheral blood cells using QIAamp DNA blood kit (QIAGEN, Hilden, Germany). The patients were genotyped with respect to the CYP2C9*1, CYP2C9*2 and *3 alleles with a previously described PCR-RFLP method (Babaoglu et al. 2004). PCR amplification was performed using the following forward and reverse primers for CYP2C9*2: 5øTAC AAA TAC AAT GAA AAT ATC ATG-3ø and 5ø-CTA ACA ACC AGA CTC ATA ATG-3ø, respectively. For CYP2C9*3, the forward and reverse primers were 5ø-TGC ACG AGG TCC AGA GATGC-3ø and 5ø-GAT ACT ATG AAT TTG GGA CTTC-3ø, respectively (Yasar et al. 1999). The PCR products were digested by the endonucleases AvaII (5U) and NsiI (5U) for the determination of CYP2C9*2 and CYP2C9*3 alleles, respectively (Babaoglu et al. 2004).
Fig. 1. The urinary losartan/E-3174 metabolic ratios before and after the (A) first and (B) third 5-fluorouracil (5-FU) cycles in seventeen and five patients with colorectal cancer, respectively. The patients whose metabolic ratios are presented in the part (B) were indicated in dashed line in part (A).
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INHIBITION OF CYP2C9 BY 5-FLUOROURACIL Statistics. The changes in the urinary losartan/E-3174 ratio before and after 5-fluorouracil treatment were compared with Wilcoxon matched pairs test. A P∞0.05 was accepted as statistically significant.
Results No side-effect was observed in the patients due to the administration of single oral dose of losartan. Fig. 1A shows the losartan/E-3174 ratios for each patient before and after the first chemotherapy cycle. The medians of the metabolic ratios were 0.50 (range 0.21–4.51) before 5-fluorouracil treatment and 0.64 (range 0.09–4.13) after the first chemotherapy cycle (PΩ0.15). Average increase in the metabolic ratio was 28.0% (range between ª71% and 223%; 95% CI between ª16.8% and 56.0%). The metabolic ratio was increased (i.e., losartan oxidation was inhibited) in 10 of the 17 patients and did not change in one patient. In five patients, losartan administration could be repeated after the third cycle of the 5-fluorouracil treatment (fig. 1B). The median of the metabolic ratios before the 5-fluorouracil treatment was 0.44 (range 0.30–4.51) and increased significantly 5.3 times to 2.32 (range 0.76–5.3) after the third chemotherapy cycle (PΩ0.03). In two of these five patients, the metabolic ratio was found to be decreased after the first 5-fluorouracil cycle as compared to the baseline activity. CYP2C9 genotypes of the subjects are shown in table 1. In the patients genotyped as CYP2C9*1*1, the median losartan/E-3174 ratio before treatment was 0.46 (range, 0.21–4.51, nΩ13). 5-Fluorouracil treatment increased the average enzyme activity in the *1*1 patients (table 1), but this change did not reach a statistical significance (PΩ0.14). No change in losartan metabolic ratio after 5-fluorouracil was evident in remaining cases carrying *2 or *3 variants, except one patient with *1*3 genotype (table 1). Four of the five patients who received losartan in the end of third chemotherapy (fig. 1B) were *1*1 carriers and one carried *1*3. Discussion Data obtained from this prospective investigation suggest that 5-fluorouracil treatment has an inhibitory effect on losartan oxidation, which is used as a marker for CYP2C9 activity. There was a large variation in the alteration of losartan metabolic ratios at the end of the first cycle of 5fluorouracil treatment among 17 patients in our study. In contrast to the first cycle, after the third cycle of 5-fluorouracil chemotherapy metabolic ratios of all five patients increased. Furthermore, in two of these five patients metabolic ratios were decreased after the first cycle of 5-fluorouracil. A number of case reports regarding the interactions between 5-fluorouracil and CYP2C9 substrates implied that 5-fluorouracil might have an inhibitory effect on CYP2C9 activity (Brown 1997 & 1999; Gilbar & Brodribb 2001; Konishi et al. 2002; Rosemergy & Findlay 2002; Brickell et al. 2003). However, there has been no study evaluating the nature of the interaction between 5-fluorouracil and CYP2C9 substrates in man. The interaction between warfa-
Table 1. The urinary losartan/E-3174 ratios before and after the first cycle of 5-fluorouracil treatment in the subjects with different CYP2C9 genotypes. Data represented as mean (range) in the CYP2C9*1*1 group. Genotype CYP2C9*1*1 CYP2C9*2*2 CYP2C9*2*3 CYP2C9*1*3
n 13 1 1 2
Before 5-fluorouracil After 5-fluorouracil 0.85 (0.21–4.51) 1.56 3.82 0.31 and 1.28
0.96 (0.09–3.64) 1.25 3.85 0.11 and 4.13
rin and 5-fluorouracil has caused extreme prolongation of the prothrombin time (Brown 1997 & 1999). Recently, coadministration of warfarin and capecitabine, an orally administered prodrug of 5-fluorouracil, was reported to cause pharmacokinetic interaction by increasing the AUC0–≤, decreasing the clearance and prolongation of the elimination half-life of S-warfarin after three chemotherapy cycles in patients with advanced/metastatic cancer (Camidge et al. 2005). Likewise, an interaction between phenytoin and 5fluorouracil was reported, resulting in phenytoin toxicity and increased phenytoin plasma concentrations (Rosemergy & Findlay 2002; Brickell et al. 2003). These interactions were observed 1–6 weeks after the initiation of 5-fluorouracil treatment. Previous in vivo animal studies have shown that exposure to 5-fluorouracil can alter the expression of the hepatic CYP isozymes (Stupans et al. 1995; Afsar et al. 1996; Yoshisue et al. 2001). Afsar et al. (1996) have reported an inhibitory effect both on the content and the activity of CYP2C11 in rat seven days after injection of a single dose of 5-fluorouracil. Similar to these findings, the inhibitory effect of 5-fluorouracil was significant over a long period of time after the administration of the first dose, suggesting that 5-fluorouracil might inhibit CYP formation. In support of this view is the finding that addition of 5-fluorouracil has no direct inhibitory effect on CYP2C9 activity in human liver microsomes (Park & Kim 2003). The inhibitory effect of 5-fluorouracil does not seem to be specific to CYP2C9. After chronic doses, 5-fluorouracil decreases the protein expression of both CYP2C11 and CYP3A enzyme apoproteins with loss of the corresponding catalytic activities (Stupans et al. 1995; Afsar et al. 1996; McLeod et al. 1998). The activity of CYP1A, 2C and 3A isozymes in liver and small intestine decreased significantly after repeated administration of oral 5-fluorouracil in rat (Stupans et al. 1995; Yoshisue et al. 2001). However the pharmacokinetics of R-warfarin, the enantiomer which is metabolized mainly by CYP1A2 and CYP3A4, was reported to be unaffected by 5-fluorouracil co-administration in man (Camidge et al. 2005). 5-Fluorouracil is converted to its active metabolites, which are incorporated into cellular RNA and DNA as false pyrimidine bases (Schilsky 1998). This is probably the explanation for the divergency between the prolonged inhibitory effect of 5-fluorouracil despite the rapid systemic
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clearance with a plasma half-life of 6–13 min. (McLeod et al. 1998). Park & Kim (2003) has reported a lack of acute inhibitory effect of 5-fluorouracil on tolbutamide hydroxylation used as a marker reaction for determination of CYP2C9 activity in human liver microsomes in vitro. This report suggests a possible explanation that the inhibitory effect of 5-fluorouracil on CYP2C9 might be due to the active metabolites rather than the parent compound since microsomes do not contain the cytosolic enzyme dihydropyrimidine dehydrogenase, the enzyme catalyzing the formation of active metabolites of 5-fluorouracil (Park & Kim 2003). In the present study, we analysed only the most common allelic variants, CYP2C9*2 and CYP2C9*3, accounting for more then 95% of mutations in Caucasian patients. The CYP2C9*2 and CYP2C9*3 code for enzymes with decreased activity compared to the enzyme coded by the common variant CYP2C9*1 allele (Lee et al. 2002). Only four of the patients were identified as carriers of the *2 and/ or *3 variants in our study group. Although the degree of inhibition of losartan oxidation in one of these patients carrying the CYP2C9*1*3 genotype was the highest, no inhibition was observed in remaining patients with *2 and *3 variants. Due to the small number of patients with genetic variants, it is not plausible to conclude on either CYP2C9*2 or CYP2C9*3 variants has any contribution to the interaction between 5-fluorouracil and CYP2C9 substrates. In conclusion, the inhibitory effect of 5-fluorouracil on CYP2C9 enzyme was not evident at the end of first chemotherapy cycle in our study group. In contrary, in five patients evaluated at the end of the third chemotherapy cycle, a significant inhibition of the enzyme activity was observed. CYP2C9 is responsible for the metabolism of certain drugs with low therapeutic indices such as warfarin, acenocoumarol, phenytoin, glibenclamide and glipizide. The concomitant use of 5-fluorouracil with CYP2C9 substrates can cause elevated plasma levels and adverse effects of these drugs. Further investigations in larger populations are required to characterise the molecular mechanisms of the inhibitory effect of 5-fluorouracil and the effect of 5-fluorouracil on other drug metabolising enzymes. Acknowledgements This study was supported by grants from the Scientific and Technical Research Council of Turkey (SBAG-COST B15-2356), The Swedish Cancer Society and The Swedish Science Council (04496). U.Y. has been supported by the Turkish Academy of Science, Young Scientist Award Program (UY/TUBA-GEBIP/2005-17).
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