Identification of the Novel Capecitabine Metabolites in Capecitabine

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Cite This: Chem. Res. Toxicol. 2018, 31, 1069−1079

Identification of the Novel Capecitabine Metabolites in Capecitabine-Treated Patients with Hand-Foot Syndrome Yan Lou,† Qian Wang,† Jinqi Zheng,‡ Xi Wang,§ Weiqin Jiang,† Yi Zheng,† Qingwei Zhao,† Yunqing Qiu,*,† and Su Zeng∥

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†

State Key Laboratory for Diagnosis and Treatment of Infectious Disease, Collaborative Innovation Center for Diagnosis and Treatment of Infectious Diseases, Zhejiang Provincial Key Laboratory for Drug Clinical Research and Evaluation, The First Affiliated Hospital, Zhejiang University, 79 QingChun Road, Hangzhou, Zhejiang 310000, People’s Republic of China ‡ Zhejiang Institute for Food and Drug Control, Hangzhou, Zhejiang 310004, People’s Republic of China § Department of Oncology, The 117th Hospital of PLA, 14 Lingyin Road, Hangzhou, Zhejiang 310013, People’s Republic of China ∥ Laboratory of Pharmaceutical Analysis and Drug Metabolism, Zhejiang Province Key Laboratory of Anti-Cancer Drug Research, College of Pharmaceutical Sciences, Zhejiang University, Hangzhou, Zhejiang 310058, People’s Republic of China S Supporting Information *

ABSTRACT: Hand-foot syndrome (HFS), the most common side effect of capecitabine, is a dose-limiting cutaneous toxicity with only rare therapeutic options. The causative mechanisms of HFS are still unclear. Many studies suggested that capecitabine or its metabolites caused the toxicity. This study is attempting to determine if there are any new metabolites that may be present and be linked to toxicity. For this purpose, 25 patients who ingested capecitabine orally were enrolled and divided into HFS positive and negative groups. Urine and plasma samples were collected before administration and five cycles after administration. Eleven phase I and phase II metabolites of capecitabine were detected and identified by ultraperformance liquid chromatography coupled with quadrupole time-of-flight tandem mass spectrometry with a metabolomic approach and MetaboLynxXS. Nine novel metabolites of capecitabine were identified herein, which were not observed in the HFS negative group. Their structures were confirmed by chemical synthesis and nuclear magnetic resonance spectroscopy. The cytotoxities of capecitabine and its metabolites on HaCaT cells were measured. Among them, M9/10 exhibited significant inhibitory activity, and they were produced via acetylation mainly by N-acetyltransferase 2. Our study comprehensively described the metabolism of capecitabine in patients with HFS and detected the novel pathways of capecitabine, which was a positive significance for the mechanism of HFS.

1. INTRODUCTION Hand-foot syndrome (HFS), also called palmar plantar erythrodysestesia, which is the common dose-dependent clinical adverse event occurring more frequently with some chemotherapeutical agents, such as capecitabine, 5-FU, pegylated liposomal doxorubicin (PLD), cytarabine, and docetaxel. Newer targeted multikinase inhibitors such as sorafenib also induced a toxicity referred to the hands and feet.1−4 The clinical manifestation of this reaction was different somewhat and was only involved in these agents, which has been named ‘“hand-foot skin reaction”’ (HFSR) and its incidence was associated with tumor type.4 The occurrence of HFS varies widely ranging from 6% to 60.5%. It was reported that capecitabine had the highest incidence of HFS at 45−68%.4 The risk factors of HFS include dosage, female sex, © 2018 American Chemical Society

and genetic polymorphisms affecting drug metabolism. Once HFS develops, the quality of life is significantly impaired, causing the cessation of therapy or dose reduction.5 Moreover, infectious complications are uncommon but may be fatal in some rare cases.6 At present, there are no effective preventive and therapeutic means for HFS since the mechanism remains unknown. According to the previous scientific evidence, each type of agent may present a unique mechanism for the development of HFS.3 Capecitabine is a prodrug of 5-FU, which is a new generation of oral fluorouracil broad-spectrum antitumor drugs. With the advantages of tumor selectivity and high Received: June 5, 2018 Published: September 19, 2018 1069

DOI: 10.1021/acs.chemrestox.8b00150 Chem. Res. Toxicol. 2018, 31, 1069−1079

Article

Chemical Research in Toxicology

phosphate, entacapone, potassium phosphate, potassium dihydrogen phosphate, dithiothreitol, odium bicarbonate, and sodium chloride were purchased from Sinopharm Chemical Reagent (Beijing, China). All HLMs and S9 fractions were frozen and stored at −80 °C until use. Solvents were mass spectrometry grade and obtained by Mallinckrodt Baker (Phillipsburg, NJ). All other reagents were supplied by Sigma-Aldrich (St. Louis, MO). 2.2. Subjects and Study Design. This study included 25 patients treated at the 117 Hospital of PLA or the First Affiliated Hospital of Zhejiang University between June 2013 and July 2015 (13 females, 12 males, mean age 56 ± 5 years). Institutional review board approval of our hospital was obtained for this study (2015178). All patients involved in this study gave their informed consent, which was carried out according to the Helsinki Declaration. Inclusion criteria were defined as follows: (1) only patients who prospectively received capecitabine monotherapy; (2) capecitabine was administered according to the standard protocol: oral capecitabine 1250 mg/m2 twice daily on days 1−14, repeated every 3 weeks; (3) other fluorouracil agents were not allowed; (4) maintain the same chemotherapy regimen during observation; (5) tumor types included colon cancer, other gastrointestinal tumors, and breast cancer, (6) patients with kidney or liver damage were excluded from the study, and (7) patients at least more than 18 years old, ambulatory (Karnofsky performance status ≥70%), and the survival period is at least three months. 2.3. Sample Collection and Preparation. Doctors assessed the HFS at each patient visit and graded them according to NCI CTC criteria. For the purpose of this study, the enrolled patients underwent the maximum HFS grade during the treatment, which was used as the end point of the study. The grade 0−2 of HFS was considered nontoxic or low toxic, while the grade 3 HFS was highly toxic and defined as impaired skin function accompanied by pain. The participants were divided into two groups (10 HFS-positive patients and 15 HFS-negative patients) according to the development of HFS and were asked to fast at least 6 h before the sampling. For plasma and urine samples, predose and five cycles of therapy after administration were collected. Blood was collected in a vacuum tube containing the chelating agent ethylenediaminetetraacetic acid (EDTA) and subjected to centrifugal separation at 4 °C at 16,000 rpm for 10 min. Plasma samples were pretreated with the mixture of acetonitrile and methanol (50:50, v/v, 300 μL), followed by vortex mixing for 10 s and centrifuging at 23, 100 g for 10 min to precipitate proteins. Urine samples were precipitated with triple volumes of acetonitrile. After centrifugation, plasma and urine samples were analyzed by UPLC-QTOF MS. 2.4. In Vitro Investigations. 2.4.1. Cell Culture and Treatments. HaCaT cells were cultured in Dulbecco’s modified Eagle medium (DMEM) base medium which contained 10% fetal bovine serum (FBS) and 1% penicillin−streptomycin saturated with 5% CO2 at 37 °C. After the cells reached confluence, they were treated for 5 min with a 0.25% trypsin-0.02% EDTA solution (w/v) at 37 °C. The cells were diluted further with 10% FBS DMEM culture medium at a final concentration of 2 × 105 cells/mL and plated onto 96-well plates for cell viability assay. 2.4.2. Cell Viability Assay. Cell viability was determined by MTT assay. In brief, cells were inoculated in 96-well plates and treated with various concentrations of capecitabine and its metabolites (0.001, 0.01, 0.1, 1, 10, 100, and 200 μM). After incubation for 48 h, the medium was discarded, MTT solution (5 mg/mL) was added, and cells were maintained for 4 h at 37 °C. Solubilization solution (DMSO, 150 μL) was added to each well, dissolving the formazan. The absorbance was recorded by spectrophotometer (Bekman Corporation, Fullerton, CA, USA) at 490 nm. All reactions were repeated four times. In order to evaluate the toxicity of compounds, the IC50 values (inhibition of cell survival rate to 50% of the control group) were measured. IC50 values were determined by nonlinear regression fits using Graph Pad Prism (Version 5, GraphPad Software, Inc., LaJolla, USA).

bioavailability, capecitabine has been widely used in advanced or metastatic colorectal cancer and taxane-refractory breast cancer.7,8 After taking capecitabine orally, it was widely absorbed by the gastrointestinal tract and transformed into 5FU through a three-step enzyme cascade reaction and two intermediary metabolites, 5-deoxy-5-fluorocytidine (5′-DFCR) and 5-deoxy-5-fluorouridine (5′-DFUR). 5′-DFUR is then transported out of hepatocytes and entered into the circulation system to arrive at the tumor tissue to form fluorouracil (5FU). 5-FU is further converted to form 5-fluorouridine 5′triphosphate (FUTP), 5-fluoro-2′-deoxyuridine 5′-triphosphate (FdUTP), and 5-fluoro-2′-deoxyuridine 5′-monophosphate (FdUMP) via ribosylation.9 Most clinicians believed that HFS resulted from inflammation in the hands and feet. This toxicity may be caused by capecitabine or a byproduct of capecitabine.5 For example, the recent research showed that capecitabine-related HFS may be formed by reducing the number of keratinocytes through activating caspase-dependent apoptotic pathways to stimulate mitochondrial dysfunction in cells.10 In an in vitro experiment, capecitabine was highly cytotoxic to breast cancer and lung cancer cells, but not to normal gastric cells.11 The toxic metabolite of capecitabine related-HFS is still unclear. Previous studies have demonstrated that drug metabolism, particulary reactive metabolites, played a key role in the HFS of capecitabine. So far, information about capecitabine metabolism was mainly derived from monitoring these known metabolitesin in vitro experiments, animal studies, and in the liver of patients treated with capecitabine.9,12−16 Few studies have been published on the metabolites of capecitabine in HFS patients. In the present work, we investigate the metabolites in capecitabine-treated patients, in human S9 fraction, and recombinant enzymes. Moreover, data obtained from urine or plasma of patients with HFS were compared to those without this toxicity. Novel metabolites of capecitabine which might be associated with the HFS in patients were reported in this study using UPLC-Q-TOF MS as the analytical method. The full structural characterization of these metabolites was applied by nuclear magnetic resonance (NMR) spectroscopy. We found that M9/M10 possessed significant toxicity toward HaCaT cells. Furthermore, NAT1 and NAT2 contributed to the formation of acetylation metabolites M9 and M10. NAT2 was the main enzyme for the capecitabine O-acetylation. These findings can contribute to understanding capecitabine-related HFS from the perspective of metabolic activation. As far as we know, our study is the first case to describe the capecitabine metabolism in HFS patients.

2. MATERIALS AND METHODS 2.1. Chemicals and Reagents. Capecitabine (purity ≥98%) was supplied by National Institutes for Food and Drug Control (Beijing, China). Pooled and individual human liver microsomes (HLMs), pooled human liver S9 fractions (S9), and recombinant enzymes (NAT1 and NAT2) were obtained from the Research Institute for Liver Diseases (Shanghai, China). Human epidermal keratinocytes (HaCaT cells) were generously provided by Dr. Xingguang Liang (Zhejiang University, China). DMEM and FBS were obtained from GIBCO Ltd. (Invitrogen Life Technologies, USA). Trisodium isocitric acid, isocitric dehydrogenase, β-NADP, its reduced form (β-NADPH), and acetyl CoA were purchased from Sigma-Aldrich (St. Louis, MO, USA). 3-[4,5-Dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide (MTT) was purchased from Sangon Biotech Co., Ltd. (Shanghai, China). Magnesium chloride, dipotassium hydrogen 1070


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Figure 1. Metabolomic analysis of HFS positive and negative groups in urine or plasma samples. Separation of HFS positive and negative group in urine (A) or in plasma (B) in OPLS-DA score plot. The t[1] and to[1] values represent the score of each sample in principal components 1 and 2, respectively. Loading S-plot generated by OPLS-DA analysis of urine samples (C) or plasma samples (D). The axes that are plotted in the S-plot from the predictive component are w1 vs p(corr)1, representing the magnitude (modeled covariation) and reliability (modeled correlation), respectively. was 40 V, source temperature was 100 °C, desolvation temperature was 500 °C, cone gas flow rate was 50 L/h, and desolvation gas (N2) flow rate was 800 L/h. In order to ensure accuracy and reproducibility, the analyses were performed using the lockspray. The [M + H]+ ion at 556.2771 Da from leucine-enkephalin (5 ng/ mL) was used as mass reference. Data acquisition was achieved using MSE, which has two separate scan functions: Function 1 (low energy): mass-scan range was 50−1200, scan time was 0.2 s, interscan time was 0.015 s, and the collision energy was 6 eV. Function 2 (high energy): mass-scan range was 50−1200, scan time was 0.2 s, interscan time was 0.015 s, and collision energy ramp was 20−30 eV. Obtaining data in this way enabled us to gather the information on intact precursor ions and fragment ions. 2.6. Synthesis of M2−M10. M2−M10 were obtained by chemical synthesis. M9 and M10 were obtained together as a colorless oil, others as monomers. The detailed synthesis methods of M2−10 are shown in the Supporting Information. 2.7. Nuclear Magnetic Resonance (NMR) Analysis. NMR data which included 1D NMR (1H and 13C NMR) and the 2D NMR spectra (heteronuclear single quantum coherence (HSQC) and heteronuclear multiple-bond connectivity (HMBC)) were recorded on a AV-400 spectrometer. Chemical shifts in ppm were measured relative to the residual solvent signal as an internal standard (δ 2.50 and 40.3 ppm in the 1H and 13C NMR, respectively). Proton coupling patterns were expressed as singlet (s), doublet (d), triplet (t), quartet (q), multiplet (m), double doublet (dd), and broad singlet and broad (brs). All coupling constants (J) were described in Hertz (Hz). All assignments were based on extensive NMR data which is depicted in Tables S1−S8. 2.8. Data Processing. A multivariate data matrix containing information on sample identity, ion identity, and ion abundance was provided by progenesis QI Version 2.0 software (Waters Corp., Milford, MA, USA), which was further conducted by SIMCA-13.0 software (Umetrics, Kinnelon, NJ). Orthogonal projection to latent structures-discriminant analysis (OPLS-DA) was performed on Pareto-scaled data. Potential metabolites were identified by MetaboLynxXS program (Waters Corp., Milford, MA, USA), using a list of expected metabolites based on potential biotransformation reactions.17 The software automatically detects and identifies metabolites, which is based on the comparison of sample and control to eliminate endogenous interfering ions in complex matrices. The detailed steps for MetabolynxXS program are described in ref 17.

2.4.3. Metabolism of Capecitabine in Human Liver S9 Fraction or Human Liver Microsomes. The reaction was conducted by incubating human liver microsomes (1.0 mg/mL) or human liver S9 fraction protein (1.0 mg/mL) in a medium containing Tris-HCl (0.1 mol/L, pH 7.4), D,L-isocitrate trisodium (12 mmol/L), MgCl2 (15 mmol/L), EGTA (1.0 mmol/L), isocitrate dehydrogenase (0.08 unit), capecitabine (100 μmol/L), and phosphate buffer (150 μL, 5 mmol/L, pH 7.8). The reaction mixture was pre-incubated for 3 min at 37 °C, followed by adding NADP/NADPH solution (2 μL) to initiate the reaction. Phosphate buffer (5 mmol/L, pH 7.8, 1.5 μL) containing 25 mM S-adenosylmethionine was added to the reaction when it was incubated for 30 min at 37 °C. After incubation for 90 min at 37 °C, ice-cold methanol (150 μL) was added to terminate the reaction. After centrifugation, the supernatant was analyzed by UPLCQ-TOF MS. Control samples containing inactivated enzymes were included. The experiments were performed in triplicate. 2.4.4. Incubations with NAT1 and NAT2. The incubation was performed as follows: 0.025 mg/mL NAT1/2 cytosol was incubated with the reaction mixture which contained 100 mM potassium phosphate buffer (pH 7.4), 200 μM capecitabine, 1 mM acetyl CoA, 100 μM dithiothreitol, and 100 μM EDTA for 60 min. Ice-cold acetonitrile (300 μL) was added to terminate the reaction. After mixing (30 s) and centrifuging (10 min at 13,000 rpm), the supernatant was used for UPLC-MS analysis. Control samples containing no capecitabine were included. The experiments were performed in triplicate. 2.5. LC-MSn Method for Capecitabine Metabolites Identification. 2.5.1. Instrumentation and Conditions. The UPLC-QTOF MS spectrometer (Waters, Manchester, UK) coupled to the Waters ACQUITY UPLC H-Class system through an electrospray ionization (ESI) interface. Chromatography was performed on an Waters HSS T3 C18 column (100 mm × 2.1 mm, 1.8 μm particle size), using the UPLC system equipped with a conditioned autosampler at 4 °C. The mobile phase consisted of water with 0.1% formic acid for solvent A and acetonitrile for solvent B in a gradient elution manner. The gradient program was as following: 0− 0.5 min, 3% B, 0.5−8 min, 3−80% B, 8−8.1 min, 80−95% B, 8.1−11 min, 95% B, 11−11.1 min, 95−3% B, followed by re-equilibration with starting conditions for 3.9 min. The flow rate was set to 0.3 mL/ min. The injection volume was 1 μL. The ESI source was operated in the positive ionization mode. Typical interface conditions were optimized for maximum intensity of the precursor ions as follows: Capillary voltage was 3 kV, sample cone 1071


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Figure 2. Mass spectrum of capecitabine obtained on Q-TOF mass spectrometry at high collision energy (A) and tentative structures of the most informative fragment ions for capecitabine (B).

3. RESULTS 3.1. Profiling Capecitabine Metabolism in HFS Positive Group and HFS Negative Group Using a Metabolomic Approach. As shown in the OPLS-DA, urine samples (Figure 1A) and plasma samples (Figure 1B) yielded distinct separation of the HFS positive group from the negative group. The S-plots (Figure 1C,D), provided in OPLSDA, showed ion contribution to separation between the two groups in urine or plasma. The top ranking ions appearing in the upper-right quadrant of S-plots were identified as capecitabine metabolites (Figure 1C,D) which were further analyzed by Metabolynxxs software. Capecitabine and metabolites were detected in urine and plasma, but mainly in urine. 3.2. Identification of Capecitabine Metabolites in Patients with HFS by Metabolynxxs Software. 3.2.1. MS Behaviors of Capecitabine. MS analysis of the parent compound capecitabine in positive ionization mode was investigated in order to facilitate the identification of metabolites. The fragment ions of the parent drug were m/z 266, 244, 174, 156, 152, 130, 113, 112, 87, and 85 (Figure 2), which were derived from a loss of C3H9O3, C5H8O3, C 1 0 H 1 8 O 3 , C 1 0 H 2 0 O 4 , C 1 0 H 1 5 N 2 O 3 F, C 9 H 1 9 NO 3 F,

C11H21NO5, C11H20O6, C12H18N2O4F, and C12H21NO6, respectively. 3.2.2. Capecitabine Metabolites in Patients. Urine and plasma samples were collected in patients to detect of the parent compound and metabolites. The structural elucidation depended on the high mass accuracy with the selected narrowmass window (2 mDa), which provided only one feasible formula for each metabolite. The detected metabolites were confirmed by the product ion scan, and the results are shown in Table 1. The putative structure of each metabolite was established by comparing these results with the CID behavior of capecitabine. Six phase I and five phase II metabolites were identified, nine of which have not been reported previously. Metabolites M2−M10 were detected in the HFS positive group, but not detected in the HFS negative group. The metabolites in plasma were less abundant and showed a large variation between the HFS positive and negative group, and the major peaks were hydroxylated metabolites (M5−M7). Unlike in plasma, the metabolites M1−M11 were detected in urine of patients with HFS, and unchanged capecitabine still stayed predominant in urine. The peaks were identified based on the LC-MSn data. 1072


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Chemical Research in Toxicology Table 1. Identification of Capecitabine Metabolites using UPLC-Q-TOF Mass Spectrometry metabolites

description

retention time (min)

formula

measured mass [M + H]+

calcd mass [M + H]+

M0

parent

7.33

C15H22FN3O6

360.1571

360.1554

M1

5′-DFCR

2.46

C9H12FN3O4

246.0890

246.0872

M2

5.51

C10H14 FN3O4

260.1024

260.1047

4.21

C10H14 FN3O4

260.1025

260.1047

5.13

C10H14 FN3O4

260.1032

260.1047

5.13

C15H22FN3O7

376.1508

376.1520

5.51

C15H22FN3O7

376.1485

376.1520

6.65

C15H22FN3O7

376.1513

376.1520

M8

methylated 5′DFCR methylated 5′DFCR methylated 5′DFCR hydroxylated metabolite hydroxylated metabolite hydroxylated metabolite degradation

6.73

C10H14FN3O3

244.1078

244.1097

M9

acetylation

7.81

C17H24FN3O7

402.1673

402.1677

M10

acetylation

7.88

C17H24FN3O7

402.1673

402.1677

M11

5′-DFUR

6.05

C9H11FN2O5

247.0730

247.073

M3 M4 M5 M6 M7

fragment ions 85, 112, 113, 130, 156, 174, 244, 266 52, 130, 113, 112, 85, 73 168, 156, 152, 130, 112 85 240, 226, 215, 210, 206, 198, 130 222, 167, 159, 152, 130,113 174, 156, 130, 113, 112 174, 156, 130, 113, 112 174, 156, 130, 113, 112 176, 156, 130, 116, 113, 112 342, 288, 266, 159, 152, 130, 113, 112 342, 288, 266, 159, 152, 130, 113, 112 131, 117

found in plasma

found in urine

found in S9

found in HLM

no

yes

yes

no

no

yes

yes

no

no

yes

yes

no

no

yes

no

no

no

yes

no

no

yes

yes

yes

no

yes

yes

no

no

yes

yes

no

no

no

yes

yes

no

no

yes

no

no

no

yes

no

no

no

yes

no

no

Figure 3. Cytotoxic effects of capecitabine and its metabolites on HaCaT cells. (A) 5-FU, M9/M10 and M11; (B) CAP, M3, and M7; (C) M1, M5, and M6; (D) M2, M4, M8, FBAL, UREA, 5-FUH2.

3.3. Cytotoxities of Capecitabine and Its Metabolites on HaCaT Cells. The cytotoxities of capecitabine and metabolites on HaCaT cells are shown in Figure 3. The cell growth was significantly inhibited in a dose-dependent manner by 5-FU and M9/10, and their IC50 values were 0.21 and 0.76 μM, respectively. Moreover, M11, M5, M1, and M6 exhibited higher toxicity (IC50, 9.8, 10.7, 24.1, 36.3 μM, respectively) than capecitabine (IC50, 48.72 μM) against HaCaT cells. In addition, M3 and M7 showed lesser toxicity than capecitabine. Other metabolites (M2, M4, M8, UREA, FBAL, 5-FUH2)

showed no cytotoxic effect on HaCaT cells. The calculated IC50 values are shown in Table 2. 3.4. Metabolism of Capecitabine in Human Liver S9 Fraction or Human Liver Microsomes. Multiple metabolites, including M1−2, M5, and M8, have been observed after incubation of capecitabine in human liver S9 (Figure 4H), but no metabolite was detected in liver microsomal fractions (Figure 4G). We confirmed the identity of the metabolite peaks produced in vitro in hepatic S9 by comparing characteristic fragmentation ions and LC retention times with those for synthetic reference compounds. These 1073


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Chemical Research in Toxicology Table 2. Concentration of Capecitabine and Its Metabolites Causing the Death or Growth Inhibition of 50% of Cells (IC50) in HaCaT Cell Lines compounds

IC50 (μM)

5-FU M9/M10 M11 M1 M5 M6 capecitabine M7 M3 M4 M2 M8 UREA FBAL 5-FUH2

0.2072 0.7618 9.840 10.70 24.14 36.29 48.72 74.63 90.69 >200.0 >200.0 >200.0 >200.0 >200.0 >200.0

Figure 5. Metabolites M9 and M10 formed by recombinant NAT1 and NAT2 isoenzymes.

3.5. Mass Spectrometric Results for Capecitabine Metabolites. The accurate mass data and structural information on capecitabine and its metabolites in human plasma and urine samples are summarized in Table 1. The fragment ions and the MS2 spectra of the proposed metabolites are illustrated in Figure 6. The proposed metabolic pathway in human was shown in Figure 7. The rationale for the structural characterization is described below. 3.5.1. M1:5′-DFCR. The metabolite M1 was eluted at a retention time of 2.46 min and had a protonated ion at m/z 246 which was 115 Da (C6H10O2F) lower than the protonated molecule of capecitabine, implying M1 was a degradation product. The major fragment ions of M1 were m/z 152, 130, 113, 112, 85, and 73, three of which (m/z 152, 130, and 85) were the same as those of the parent compound. Moreover, the fragment ion at m/z 112 was formed by the loss of C5H9O4 (134 Da) from the precursor ion, and its subsequent loss of C2HN (39 Da) generated the fragment ion at m/z 73. M1 had the same retention time and characteristic fragment ions as the

metabolic pathways were also the major biotransformation observed in vivo. The metabolites found in the hepatic S9 incubations showed the same chromatographic and mass spectrometric behaviors as those formed in vivo studies. However, we failed to detect M3−4, M6−7, and M9−10 in hepatic S9 samples. Because M9 and M10 significantly inhibited the growth of HaCaT cells in a dose-dependent manner, the possible metabolic pathways were investigated. When capecitabine was incubated with recombinant human NAT1 and NAT2, M9 and M10 were formed both by NAT1 and NAT2. NAT2 was found to take the major responsibility for capecitabine acetylation and NAT1 with a minor contribution (Figure 5).

Figure 4. UPLC/MS chromatograms of capecitabine and its metabolites. (A) Blank sample, (B) standard sample, (C) standard urine sample in positive group, (D) standard plasma sample in HFS positive group, (E) standard urine sample in HFS negative group, (F) standard plasma sample in HFS negative group, (G) HLM sample, and (H) S9 sample. 1074


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Figure 6. UPLC−MS/MS spectra. (A) M2 (m/z 260), (B) M3 (m/z 260), (C) M4 (m/z 260), (D) M5−7(m/z 376), (E) M8 (m/z 244), (F) M9−10 (m/z 402).

(38 Da) to generate the fragment ion at m/z 222, which further lost CON and CH2 (55 Da) to give the ion at m/z 167. Then the protonated molecular ion lost C3H2N2OF (101 Da) to form the fragment ion at m/z 159. In view of this analysis, M4 was thus identified as methylated product, and the methylation occurred on the hydroxyl group. 3.5.3. M5−M7: Hydroxylated Metabolite. In our study, three hydroxylation metabolites (M5−M7) were observed. Metabolites M5, M6, and M7 were eluted at 5.13, 5.51, 6.65 min, respectively. All of them had the protonated molecular ion at m/z 376. The elemental composition was C15H22FN3O7, suggesting they are the hydroxylated form of M0. Moreover, the characteristic fragment ions of capecitabine (m/z 174, 156, 130, 113, and 112) were observed. M6 and M7 had same fragment products as M5 at m/z 174, 156, 130, 113, and 112. Therefore, M5−M7 were isomers and identified as hydroxylated capecitabine. Importantly, the modification of M5 occurred in the methyl, and others were occurred on the amyl ether. 3.5.4. M8: Degradation Metabolite. Metabolite M8 was eluted at 6.73 min and exhibited the protonated ion at m/z 244, which was 116 Da (C5H8O3) lower than that of capecitabine, revealing that the metabolite was a degradation product. In addition, the major fragment products at m/z 174, 156, 130, 113, and 112 were identical to those of M0, which indicated that the aldopentose was lost. Accordingly, M8 was tentatively identified as degradation of M0.

commercially available corresponding standards. Therefore, M1 was identified as 5′-DFCR. 3.5.2. M2−M4: Methylated 5′-DFCR. Metabolites M2, M3, and M4 were eluted at retention times of 5.51, 4.21, and 5.13 min, respectively. Their protonated ions were 14 Da (CH2) higher than that of M1, suggesting the addition of a methyl group. Moreover, the fragment ions were observed at m/z 168, 156, 152, 130, 112, and 85. The fragment ions at 152, 130, 112, and 85 were identical to those of M1. The loss of C4H14NO (199 Da) from the precursor ion (m/z 367) formed the fragment ion at m/z 168. Further, the fragment ion at m/z 156 was the same as M0, indicating that the methylation occurred on the secondary amino group. The fragment products of M3 were at m/z 240, 226, 215, 210, 206, 198, and 130. The major fragment ion at m/z 130 was identical to that of M2. The protonated ion lost a HF (30 Da) to generate m/z 240, followed by loss of H2O2 (34 Da) and C2H (25 Da) to yield the fragment ions at m/z 215 and 206, respectively. The fragment ion at m/z 215 further lost OH (17 Da) to form the ion at m/z 198. The fragment ion at m/z 226 was due to a loss of NH2 and OH (34 Da) from the protonated ion. Additional loss of O (16 Da) to generate the fragment ion at m/z 210, indicating the methylation occurred on the secondary amino group. The major fragment products of M4 at m/z 152, 130, and 113 were identical to those of M1, implying that M4 was another methylation product. The protonated ion lost H3OF 1075


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Figure 7. Proposed metabolic pathways of capecitabine.

3.5.5. M9−M10: Acetylation Metabolite. Metabolites M9 and M10 were detected with the retention times of 7.81 and 7.88 min, respectively. The two metabolites showed the same protonated molecular ion at m/z 402 which was 42 Da (C2H3O) higher than the protonated molecule of capecitabine (m/z 360). This implied they were acetylation metabolites of capecitabine. The fragment ions of M9 were m/z 342, 288, 266, 159, 152, 130, 113, and 112. The fragment ions at m/z 342 and 288 were generated by the loss of C4H2 (50 Da) and C6H12NO (120 Da) from the protonated ion (m/z 402), respectively. The other fragment products (266, 159, 152, 130, 113, and 112) were the same as those of the parent drug. Accordingly, the acetylation occurred on the hydroxyl group. Moreover, the characteristic fragment ion at m/z 159 further confirmed the acetylation occurred on the aldopentose. The fragment ions of M10 were identical to M9, implying the two metabolites were isomers of each other and M10 was another acetylation metabolite of capecitabine. 3.5.6. M11:5′-DFUR. M11 was detected at a retention time of 6.05 min and displayed a protonated molecule at m/z 245. The major fragment products of M11 at m/z 131 and 117 were observed, which were generated by the loss of C5H8O3 (116 Da) and 129 Da (C4H2FN2O2) from the precursor ion, respectively. Comparing its retention time and characteristic fragment ions to reference standard, M11 was identified as 5′DFUR. 3.6. NMR Analysis of M2−M10. To further characterize the structures of these isomers, NMR analysis was performed.

Synthesis of the metabolites M2−M10 was required to ultimately confirm their structure. Nine new metabolites were obtained in the chemical synthesis which showed the same chromatographic and MS identities as those of the product generated in liver S9 incubation and in patients. We succeeded in obtaining NMR spectra of the synthetic M2− M10. For these new metabolites, the spectral data matched their corresponding authentic standards as assessed by comparison of chemical shifts and coupling patterns. These NMR data supported the proposed structures based on the initial mass spectrometric analysis of the metabolites. The 1H and 13C NMR and HMQC spectra of M2−M4 showed additional methyl group (δ3.26 (3H, s), δ29.4; δ3.46 (3H, s), δ32.0; δ3.20 (3H, s), δ28.4) in their molecule, suggesting the methyl group was added and these three metabolites were methylated metabolites. This conclusion was further firmed by the HMBC correlations, as shown in Tables S2−S4. The proton NMR spectra of M5−M7 showed an additional oxygen-bearing methylene signal or oxygen-bearing methine signal (δ3.56−3.58 (2H, m), δ65.7; δ3.66−3.69 (1H, m), δ74.6; δ3.37−3.42 (2H, m), δ61.0). Additionally, the HMBC spectrum indicated the exact position of hydroxylation as shown in Tables S5−S7. NMR spectroscopy confirmed that the identity of M8 was indeed degradation of capecitabine. Some marker signals of the aldopentose were lost, and the NMR assignments of M8 were fully assigned with 1H and 13C spectra, as shown in Table S8. Metabolites M9 and M10 were obtained together as a colorless oil. The 1H and 13C NMR and 1076


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0.76 μM, respectively. Furthermore, M9 and M10 formed via acetylation by arylamine N-acetyltransferases (NATs). NATs are metabolic enzyme of drugs and xenobiotic, catalyzing the N-acetylation of arylamines and hydrazines and the Oacetylation of N-hydroxy-arylamines. It is considered that Nacetylation is protective because the resulting arylacetamide derivatives are chemically stable. However, the O-acetylation of hydroxylamines produces acetoxy esters, which can be decomposed spontaneously into electrophilic, DNA-bound nitrogen ions.20 NATs have two isoforms, NAT1 and NAT2, which are different from each other in substrate specificity, structural stability, and tissue specific expression. NAT1 is expressed in various tissues including the liver, intestine, and bladder, whereas NAT2 is locally expressed in the liver and intestine.21 Genes encoding the NAT1 and NAT2 isoenzymes are highly polymorphic among populations, which lead to interindividual variability in N-acetylation capacity.22 In our study, capecitabine O-acetylation was primarily catalyzed by the NAT2 isozyme. We believe that NAT1 and NAT2 polymorphic variants may influence the metabolism of capecitabine. Further studies are needed for identification of which enzyme is involved in the other new pathways of capecitabine and investigation of the role of this enzyme in the disposition of capecitabine. In addition, transporters were involved in the development of capecitabine-related HFS.5 Further study is needed to elucidate the exact transporters involved in HFS. As postulated by previous studies, capecitabine-induced HFS through COX-2-mediated inflammatory in the hand and foot areas.5 Thus, further research should also focus on which COX-2-related signaling pathways were pivotal to capecitabine-induced HFS and how did capecitabine or its metabolites regulate such signaling pathways. Of note, patients who received systemic 5-FU alone suffered less from the HFS than those who were treated with capecitabine.23 Another hypothesis is that the catabolites of 5-FU cause the capecitabine-related HFS, which was supported by the evidence that 5-FU prodrugs, uracil/tegafur (UFT), containing DPD inhibitors did not induce HFS frequently.19 Capecitabine, a 5-FU prodrug not containing DPD inhibitor, facilitates the production of 5-FU catabolic metabolites, which is confirmed by the significantly higher proportions of FUH2 and FBAL in patients with capecitabine than those of 5-FU and 5′-DFUR.24,25 The toxicity of fluoropyrimidines and these novel metabolites which were detected in this study was performed by human keratinocytes. The HaCaT cells model was chosen for studying the linkage of HFS to these metabolites. It seems that the IC50 values of 5-FU or other capecitabine metabolites (M1, M3, M5−M7, M9/M10, and M11) were several orders of magnitude higher than those of 5FU decomposition products. The toxicities of 5-FUH2, UREA and FBAL were very limited in HaCaT cells, which were essentially identical with the previous results.19 These findings suggested the main 5-FU catabolites do not directly induce the development of capecitabine-related HFS. Interestingly, capecitabine metabolites 5′-DFUR, 5-FU, and FBAL had no obvious correlation with the frequency or severity of HFS.26 At present, the reactive metabolites produced by metabolism are considered as an important source of drug-induced toxicity.27 As a result, clarifying the metabolites of capecitabine is of great significance in elucidating the mechanism of HFS and its application in capecitabine-based personalized therapy. Our results provide understanding of the metabolism of

HMQC spectra of M9−M10 showed two sets of signals, the stronger one corresponded to M9 and the weaker to M10. M9 had the same molecular formula as M10, indicating that the two metabolites were isomers. The 13C and 1H NMR spectra displayed the additional methyl and carbonyl groups in the molecule compared with those of capecitabine, suggesting M9−M10 were acetylation metabolites and the modifications occurred in the aldopentose. The HMBC correlations as shown in Table S9 further confirmed this deduction.

4. DISCUSSION The main objective of our study was to investigate if there are any new capecitabine metabolites that may be present and linked to HFS. The statistically significant group differences in metabolites between HFS patients with those without this toxicity by metabolomic approaches were observed. Eleven phase I and II metabolites of capecitabine were obtained in the patients with HFS by MetabolynxXS program. More surprising was the detection of the metabolites M2−M10 only in HFS positive group, and these nine human metabolites had never been reported. Eleven metabolites were observed in the urine of HFS patients. In the vitro, these metabolites were found in the human liver S9 fraction, but failed to be detected in human liver microsomes. The current investigation presented evidence for these novel metabolites of capecitabine via noncytochrome P450 pathways. Additionally, metabolites M5−M7 were also observed in plasma. Unfortunately, we failed to detect 5-FU, 5FUH2, FUPA, and FBAL in this study. Since capecitabine, 5FU, their pyrimidine metabolites, and FBAL are significantly different in polarity, the simultaneous determination of all these analytes in biological matrices with the lower limit of quantification (LLQ) of 1−10 ng/mL has been a great challenge.18 These metabolites were detected by UPLC-QTOF MS coupled with MetabolynxXS which provided unique high-throughput capabilities, excellent mass accuracy, and enhanced MSE data collection for drug metabolism research. However, certain structures of metabolites identified only based on the LC/MSn data might be incorrect, particularly in the presence of isomers. In the present study, three groups of isomers (M2, M3 and M4; M5, M6, and M7; M9 and M10) were observed, and they had identical retention time and similar mass spectrometric identities. Therefore, chemical synthesis was executed to further verify the metabolite identification work. The NMR spectroscopy (1H NMR, 13C NMR, HMQC, and HMBC) were applied to elucidate these synthesized compounds. Based on their NMR and mass spectrometry, all nine metabolites were correctly assigned. On the basis of the metabolites identified, we found several novel metabolic pathways of capecitabine such as hydroxylation, methylation, degradation, and acetylation in patients with HFS, which did not exist in patients without HFS. HFS is the most common toxicity of capecitabine. It is of critical importance to investigate the possible mechanism involving in the HFS in order to provide effective prevention and treatment. Most researchers believe the mechanism of HFS related to the generation and accumulation of capecitabine and its metabolites. The toxic substance of capecitabine-related HFS is not completely clear. One hypothesis is that the capecitabine metabolite, 5-FU, may be involved in the development of HFS.19 Our results of MTT assay displayed that 5-FU and M9/10 exhibited significant toxicity on HaCaT cells, and their IC50 values were 0.21 and 1077


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review of clinical presentation, etiology, pathogenesis, and management. J. Am. Acad. Dermatol. 71, 787−794. (5) Lou, Y., Wang, Q., Zheng, J. Q., Hu, H. H., Liu, L., Hong, D. S., and Zeng, S. (2016) Possible Pathways of Capecitabine-Induced Hand−Foot Syndrome. Chem. Res. Toxicol. 29, 1591−1601. (6) Murugan, K., Ostwal, V., Carvalho, M. D., D’souza, A., Achrekar, M. S., Govindarajan, S., and Gupta, S. (2016) Self-identification and management of hand-foot syndrome(HFS): effect of a structured teaching program on patientsreceiving capecitabine-based chemotherapy for colon cancer. Support Care Cancer 24, 2575−2581. (7) Hamzic, S., Kummer, D., Milesi, S., Mueller, D., Joerger, M., Aebi, S., Amstutz, U., and Largiader, C. R. (2017) Novel genetic variants in carboxylesterase 1 predict severe early-onset capecitabinerelated toxicity. Clin. Pharmacol. Ther. 102, 796−804. (8) Rudek, M. A., Connolly, R. M., Hoskins, J. M., Garrett-Mayer, E., Jeter, S. C., Armstrong, D. K., Fetting, J. H., Stearns, V., Wright, L. A., Zhao, M., Watkins, S. P., Jr., McLeod, H. L., Davidson, N. E., and Wolff, A. C. (2013) Fixed-dose capecitabine is feasible: results from a pharmacokinetic and pharmacogenetic study in metastatic breast cancer. Breast Cancer Res. Treat. 139, 135−143. (9) Zhang, J., Wu, J., Li, H., Chen, Q., and Lin, J. M. (2015) An in vitro liver model on microfluidic device for analysis of capecitabine metabolite using mass spectrometer as detector. Biosens. Bioelectron. 68, 322−328. (10) Chen, M., Chen, J., Peng, X. M., Xu, Z. F., Shao, J. J., Zhu, Y. R., Li, G. Q., Zhu, H., Yang, B., Luo, P. H., and He, Q. J. (2017) The contribution of keratinocytes in capecitabine-stimulated hand-footsyndrome. Environ. Toxicol. Pharmacol. 49, 81−88. (11) Li, Z., Guo, Y., Yu, Y., Xu, C., Xu, H., and Qin, J. (2016) Assessment of metabolism-dependent drug efficacy and toxicityon a multilayer organs-on-a-chip. Integr. Biol. 8, 1022−1029. (12) Satoh, T., Sugiura, S., Shin, K., Onuki-Nagasaki, R., Ishida, S., Kikuchi, K., Kakiki, M., and Kanamori, T. (2018) A multi-throughput multi-organ-on-a-chip system on a plate formatted pneumatic pressure-driven medium circulation platform. Lab Chip 18, 115−125. (13) Derissen, E. J., Jacobs, B. A., Huitema, A. D., Rosing, H., Schellens, J. H., and Beijnen, J. H. (2016) Exploring the intracellular pharmacokinetics of the 5-fluorouracil nucleotides during capecitabine treatment. Br. J. Clin. Pharmacol. 81, 949−957. (14) Guichard, S. M., Mayer, I., and Jodrell, D. I. (2005) Simultaneous determination of capecitabine and its metabolites by HPLC and mass spectrometry for preclinical and clinical studies. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 826, 232−237. (15) Siethoff, C., Orth, M., Ortling, A., Brendel, E., and WagnerRedeker, W. (2004) Simultaneous determination of capecitabine and its metabolite 5-fluorouracil by column switching and liquid chromatographic/tandem mass spectrometry. J. Mass Spectrom. 39, 884−889. (16) Van Laarhoven, H. W., Klomp, D. W., Kamm, Y. J., Punt, C. J., and Heerschap, A. (2003) In vivo monitoring of capecitabine metabolism in human liver by 19fluorine magnetic resonance spectroscopy at 1.5 and 3 T field strength. Cancer. Res. 63, 7609− 7612. (17) Lou, Y., Zheng, J., Hu, H., Lee, J., and Zeng, S. (2015) Application of ultra-performance liquid chromatography coupled with quadrupole time-of-flight mass spectrometry to identify curcumin metabolites produced by human intestinal bacteria. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 985, 38−47. (18) Licea-Perez, H., Wang, S., and Bowen, C. (2009) Development of a sensitive and selective LC-MS/MS method for the determination of alpha-fluoro-beta-alanine, 5-fluorouracil and capecitabine in human plasma. J. Chromatogr. B: Anal. Technol. Biomed. Life Sci. 877, 1040− 1046. (19) Fischel, J. L., Formento, P., Ciccolini, J., Etienne-Grimaldi, M. C., and Milano, G. (2004) Lack of contribution of dihydrofluorouracil and a-fluoro-balanineto the cytotoxicity of 50-deoxy-5-fluorouridineon human keratinocytes. Anti-Cancer Drugs 15, 969−974. (20) Wu, H., Dombrovsky, L., Tempel, W., Martin, F., Loppnau, P., Goodfellow, G. H., Grant, D. M., and Plotnikov, A. N. (2007)

capecitabine in patients with HFS. However, the present study is limited by the rather small sample size. In further research, it is nesseccary to investigate the behavior of capecitabine and the 11 metabolites in urine or plasma samples from a largescale study in order to explore biomarkers of HFS for personalized capecitabine therapy.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.chemrestox.8b00150.

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Synthetic methods of M2-M10. Figure S1: Structures of M2−M10. Tables S1−S8: 13C NMR (100 MHz) and 1H NMR (400 MHz) data of M2−M10 (PDF)

AUTHOR INFORMATION

Corresponding Author

*E-mail: [email protected]. ORCID

Yunqing Qiu: 0000-0003-0899-2019 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This article was supported by the National Natural Science Foundation of China (no. 81573502) and Science and Technology Department of Zhejiang Province (LGF18H310002, 2016C33066, 2016C33142).

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ABBREVIATIONS HFS, hand-foot syndrome; 5′-DFCR, 5-deoxy-5-fluorocytidine; 5′-DFUR, 5-deoxy-5-fluorouridine; 5-FU, 5-fluorouridine; 5-FUH2, 5-dihydrofluorouracil; FUPA, α-fluoro-βureidopropionate; FBAL, α-fluoro-β alanine; FUTP, 5fluorouridine-5′ triphosphate; FdUTP, 5-fluoro-2′-deoxyuridine 5′-triphosphate; FdUMP, 5-fluoro-2′-deoxyuridine 5′monophosphate; DPD, dihydropyrimidine deshydrogenase; PE, petroleum ether; EA, ethyl acetate; THF, tetrahydrofuran; DCM, dichloromethane; EDTA, ethylenediaminetetra acetic acid; BTC, benzene-1,3,5-tricarboxylic acid

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REFERENCES

(1) Bun, S., Yunokawa, M., Tamaki, Y., Shimomura, A., Shimoi, T., Kodaira, M., Shimizu, C., Yonemori, K., Fujiwara, Y., Makino, Y., Terakado, H., and Tamura, K. (2018) Symptom management: the utility of regional cooling for hand-foot syndrome induced by pegylated liposomal doxorubicin in ovarian cancer. Support Care Cancer 26, 2161−2166. (2) Shinagawa-Kobayashi, Y., Kamimura, K., Goto, R., Ogawa, K., Inoue, R., Yokoo, T., Sakai, N., Nagoya, T., Sakamaki, A., Abe, S., Sugitani, S., Yanagi, M., Fujisawa, K., Nozawa, Y., Koyama, N., Nishina, H., Furutani-Seiki, M., Sakaida, I., and Terai, S. (2018) Effect of histidine on sorafenib-induced vascular damage: Analysis using novel medaka fish model. Biochem. Biophys. Res. Commun. 496, 556− 561. (3) Macedo, L. T., Lima, J. P., dos Santos, L. V., and Sasse, A. D. (2014) Prevention strategies for chemotherapy-induced hand-foot syndrome: a systematic review and meta-analysis of prospective randomised trials. Support Care Cancer 22, 1585−1593. (4) Miller, K. K., Gorcey, L., and McLellan, B. N. (2014) Chemotherapy-induced hand-foot syndrome and nail changes: a 1078


Article

Chemical Research in Toxicology Structural basis of substrate-binding specificity of human arylamine Nacetyltransferases. J. Biol. Chem. 282, 30189−30197. (21) Stepp, M. W., Mamaliga, G., Doll, M. A., States, J. C., and Hein, D. W. (2015) Folate-Dependent Hydrolysis of Acetyl-Coenzyme A by Recombinant Human and Rodent Arylamine N-Acetyltransferases. Biochem. Biophys. Rep. 3, 45−50. (22) Hernández-González, O., Ortiz-Zamudio, J. J., Rodríguez-Pinal, C. J., Alvarado-Morales, I., Martínez-Jiménez, V. D. C., SalazarGonzález, R. A., Correa-González, L., Gómez R, C., Portales-Pérez, D. P., and Milán-Segovia, R. D. C. (2018) Genetic polymorphisms of arylamine N-acetyltransferases 1 and 2 and the likelihood of developing pediatric acute lymphoblastic leukemia. Leuk. Lymphoma 59, 1968−1975. (23) Azuma, Y., Hata, K., Sai, K., Udagawa, R., Hirakawa, A., Tohkin, M., Ryushima, Y., Makino, Y., Yokote, N., Morikawa, N., Fujiwara, Y., Saito, Y., and Yamamoto, H. (2012) Significant association between hand-foot syndrome and efficacy of capecitabine in patients with metastatic breast cancer. Biol. Pharm. Bull. 35, 717−724. (24) Nikolaou, V., Syrigos, K., and Saif, M. W. (2016) Incidence and implications of chemotherapy related hand-foot syndrome. Expert Opin. Drug Saf. 15, 1625−1633. (25) Roberto, M., Romiti, A., Botticelli, A., Mazzuca, F., Lionetto, L., Gentile, G., Paris, I., Falcone, R., Bassanelli, M., Di Pietro, F. R., Onesti, C. E., Anselmi, E., Macrini, S., Simmaco, M., and Marchetti, P. (2017) Evaluation of 5-fluorouracil degradation rate and Pharmacogenetic profiling to predict toxicity following adjuvant capecitabine. Eur. J. Clin. Pharmacol. 73, 157−164. (26) Lassere, Y., and Hoff, P. (2004) Managment of hand foot syndrome in patient treated with capecitabine (Xeloda). Eur. J. Oncol. Nurs. 8, S31−S40. (27) Guengerich, F. P., and MacDonald, J. S. (2007) Applying mechanisms of chemical toxicity to predict drug safety. Chem. Res. Toxicol. 20, 344−369.

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