Fluorine nuclear magnetic resonance spectroscopy of human biofluids in the field of metabolic studies of anticancer and antifungal fluoropyrimidine drugs

Clinica Chimica Acta 366 (2006) 61 – 73 www.elsevier.com/locate/clinchim

Review

Fluorine nuclear magnetic resonance spectroscopy of human biofluids in the field of metabolic studies of anticancer and antifungal fluoropyrimidine drugs Myriam Malet-Martino, Ve´ronique Gilard, Franck Desmoulin, Robert Martino * Groupe de RMN Biome´dicale, Laboratoire SPCMIB (UMR CNRS 5068), Universite´ Paul Sabatier, Toulouse, France Received 5 August 2005; received in revised form 13 October 2005; accepted 14 October 2005 Available online 6 December 2005

Abstract Fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy provides a highly specific tool for the detection, identification and quantification of fluorine-containing drugs and their metabolites in biofluids. The value and difficulties encountered in investigations on drug metabolism are first discussed. Then the metabolism of three fluoropyrimidines in clinical use, 5-fluorouracil, 5-fluorocytosine and capecitabine are reported. Besides the parent drug and the already known fluorinated metabolites, 12 new metabolites were identified for the first time with 19F NMR in human biofluids. Nine of them can only be observed with this technique: fluoride ion, N-carboxy-a-fluoro-halanine, a-fluoro-h-alanine conjugate with deoxycholic acid, 2-fluoro-3-hydroxypropanoic acid, fluoroacetic acid, O2-h-glucuronide of fluorocytosine, fluoroacetaldehyde hydrate and its adduct with urea, fluoromalonic acid semi-aldehyde adducts with urea. This emphasizes the high analytical potential of 19F NMR for the furtherance in the understanding of fluoropyrimidine catabolic pathways. 19F NMR should also play a role in the therapeutic monitoring of FU and its prodrugs in specific groups of patients, e.g. hemodialyzed patients or patients with deficiency in FU catabolic enzymes. D 2005 Elsevier B.V. All rights reserved. Keywords:

19

F NMR; Biological fluids; Metabolism; Fluorouracil; Fluorocytosine; Capecitabine

Contents 1. 2. 3.

Introduction. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Advantages and limitations of NMR biofluids analysis for drug metabolism studies Fluorouracil (FU) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1. FU catabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2. FU degradative pathway and FU cardiotoxicity . . . . . . . . . . . . . . . . 4. Fluorocytosine (FC) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5. Capecitabine (CAP) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6. Conclusion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

. . . . . . . . .

61 62 63 63 66 66 69 71 71

1. Introduction * Corresponding author. Groupe de RMN Biome´dicale, Laboratoire SPCMIB, Universite´ Paul Sabatier, 31062 Toulouse cedex 9, France. Tel.: +33 5 61 55 62 71; fax: +33 5 61 55 76 25. E-mail address: [email protected] (R. Martino). 0009-8981/$ - see front matter D 2005 Elsevier B.V. All rights reserved. doi:10.1016/j.cca.2005.10.013

Nuclear Magnetic Resonance spectroscopy (NMR) is the study of molecules by recording the interaction of radiofrequency electromagnetic radiation with the magnetically

62

M. Malet-Martino et al. / Clinica Chimica Acta 366 (2006) 61 – 73

active nuclei of molecules placed in a strong magnetic field. Modern NMR is the preeminent method for determining the structure of organic compounds including the three-dimensional structures of proteins and other biological macromolecules in solution. NMR is also widely used to determine the structures and characterize the solution chemistry of inorganic and organometallic compounds. But NMR has also found many applications in biomedical field since it allows the direct study at the molecular level of biological samples, from biofluids, cell or tissue extracts, excised tissues, cell pellets (in vitro studies) to isolated perfused biological systems (cells or organs) (ex vivo studies) and finally intact biological systems (bacteria, plants, animals and humans) (in vivo studies). Consequently, NMR is particularly well suited to analysis of the metabolism of both endogenous and xenobiotic compounds such as drugs. Several active nuclei can be routinely used in drug metabolism and disposition such as proton, carbon-13, fluorine-19, phosphorus-31, lithium-7. Nevertheless, the majority of studies in the literature concerns fluorine-19 NMR (19F NMR). Indeed, this nucleus presents favorable NMR characteristics, including a nuclear spin of 1/2, relatively narrow lines, 100% natural abundance, high sensitivity (83% that of proton), large chemical shift range (about 500 ppm), and short longitudinal relaxation times (T1) which permit rapid pulsing with a corresponding improvement in the signal-to-noise ratio per unit time. As a number of fluorinated drugs are currently in clinical use, 19F NMR offers a powerful method of monitoring their pharmacokinetics and metabolism either in vitro or in vivo. This article will focus on the application of 19F NMR to metabolic studies of fluoropyrimidine (FP) drugs from human biofluid analysis, emphasizing the usefulness of the technique for the quantitative detection of novel and unexpected metabolites. The advantages and limitations of NMR, especially 19F NMR, for drug metabolism studies in biofluids are first discussed. Then, we present the 19F NMR studies dealing with three FP in clinical use: the anticancer drug 5fluorouracil (FU), the mainstay of antimetabolite treatment for solid tumors, the antifungal agent 5-fluorocytosine (FC) and the recent oral prodrug of FU, capecitabine (CAP), which is a fluorocytidine derivative.

2. Advantages and limitations of NMR biofluids analysis for drug metabolism studies NMR has several significant advantages over conventional chromatographic and electrophoretic methods. It enables the direct study of any intact biofluid without prior treatment, avoiding the problems stemming from chemical derivatization and from the pH-sensitivity of many metabolites. The method is non-selective and requires no prior knowledge of the structures of metabolites since all the

molecules bearing the nucleus under investigation are simultaneous detected in a single analysis. A NMR spectrum contains numerous structural information, and the observed chemical shifts, spin-coupling patterns, integrals, as well as the two-dimensional experiments leading to identify resonances that are connected by through-bond scalar coupling (COSY, TOCSY, HMQC, etc.) or linked by through-space dipole –dipole interactions (NOESY, etc.) or chemical exchange (EXSY), provide valuable clues on the molecular structure of metabolites, novel or not, present in a mixture. Nevertheless, the unequivocal determination of the structures of the new metabolites requires their extraction and purification. Under appropriate recording and processing conditions [checking that excitation is uniform over the whole frequency range when a large spectral width is observed which is often the case in 19F NMR, avoidance of perturbation of the relative intensities of the resonance peaks due to their different T1 values and that of differential nuclear Overhauser enhancements when proton decoupling is applied, adequate digitization of the signals, optimization of data processing such as zero-filling, exponential multiplication to improve signal-to-noise ratio, careful correction of phase and baseline distortions of the spectrum, integration], the area of each NMR peak is directly proportional to the number of corresponding nuclei. Thus, at variance with other techniques, the response factor is not dependent on the molecular structure. Absolute concentration determinations can be assessed with calibrating on a standard that gives resonance signal(s) in the excited frequency range observed. One can use an internal or external standard. Since the external standard is contained in a coaxial capillary put into the larger NMR tube, its apparent concentration must be previously calibrated against solutions of adequate compounds at known concentrations. Moreover, it must be taken into account that accuracy and precision of the measurements are greatly dependent on the signal-to-noise ratio (S/N) of the resonance peak considered. A S/N of at least 150 is required for an uncertainty of 1%, but an acceptable level of precision is obtained for a S/N å 10 (with S/N å 30 or 10, the precision is å 3% or å 8%, respectively) [1]. NMR is thus a suitable technique for the quantitative detection of drugs and metabolites in biofluids. Nevertheless, the binding of drugs and/or metabolites to macromolecules in plasma or the presence of micellar substructures in bile can induce significant signal broadening that leads to reduced signal-to-noise ratio or even to NMR-invisibility of the signal. Some sample pre-treatment may be required in such cases. All the details of 19F NMR spectra recording conditions for quantitative analysis of bulk solutions (biofluids, cell or tissue extracts, perfusates) are reported in several papers [2 –10]. An important advantage, specific to 19F NMR, is that the negligible level of endogenous mobile fluorinated

M. Malet-Martino et al. / Clinica Chimica Acta 366 (2006) 61 – 73

metabolites of low molecular mass (the only ones that can be detected) eliminates interfering backgrounds signals and dynamic range problems as encountered in proton NMR due to the strong signal of water protons that must be suppressed. The volume of biological fluid needed for a NMR analysis is generally not a hindrance as the total volume required ranges between 0.3 and 0.7 or 2.0 and 3.0 mL with 5 or 10 mm tubes, respectively, depending if a coaxial capillary is or not inserted in the tube. However, compared with most chromatographic and other spectroscopic techniques, 19F NMR is relatively insensitive, which represents (in addition to the high cost of the spectrometers) the principal drawback of the technique and limits its use to the observation of those drugs that are present at relatively high concentrations. The sensitivity limit is the low micromolar range for one fluorine atom. Indeed, the detection thresholds reported in the literature range between 1 and 3 AM depending on the proton resonance frequency of the spectrometer (300 to 500 MHz), the recording time (10 to 24 h), and the NMR diameter tube (5 or 10 mm) [6,9 – 12]. The detection sensitivity may be increased by recording the spectra with continuous 1H decoupling which induces the nuclear Overhauser enhancement of the signal intensities. A detection threshold of å 0.3 AM is thus reached but, under these conditions, the signal integrals will not be strictly proportional to concentration and large quantitation errors ( 50%) are expected [10]. The accuracy and precision of the NMR concentration determinations are generally on the order of T5–10% for concentrations superior to 50 AM, but are considerably lowered for concentrations near the limit of detection: T 1 AM [6].

3. Fluorouracil (FU) Since its introduction in clinical use more than 45 years ago, FU has become a component of the standard therapy for a variety of malignancies including gastrointestinal tract, head and neck, and breast cancers [13]. FU is a prodrug that requires intracellular complex metabolic conversion to fluoronucleosides (FNUCs) and then to cytotoxic fluoronucleotides (FNUCt). Besides this biochemical activation pathway, called anabolism, there is a degradative pathway called catabolism that leads to the drug elimination from the body. FU and all its metabolites can diffuse back out of the cell, except FNUCt that are trapped within the cells due to the presence of the charged phosphate group. Biological fluid analyses are thus useful in studies of FU metabolism, especially catabolism that is the major route of elimination. Consequently, only the catabolic pathway is presented in this article and illustrated in Fig. 1 (for reviews devoted to the biochemistry, mechanism of action and pharmacology of FU, see Refs. [13 – 16]).

63

3.1. FU catabolism As for natural pyrimidine bases, uracil and thymine, FU is degraded through enzymatic catabolism (Fig. 1). The major fraction of the administered FU dose is converted to 5,6-dihydro-5-fluorouracil (FUH2) by a trans-addition of two hydrogen atoms to the C5 – C6 double bond of the pyrimidine ring. This reversible first step of FU catabolism requires the high-energy cosubstrate nicotinamide adenine dinucleotide phosphate in its reduced form (NADPH), and is catalyzed by the enzyme dihydropyrimidine dehydrogenase (DPD; E.C. 1.3.1.2). FU catabolism further proceeds through reversible hydrolytic pyrimidine ring opening between C3 and C4 atoms catalyzed by dihydropyrimidinase (E.C. 3.5.2.2), leading to a-fluoro-h-ureidopropionic acid (FUPA). The major part of FUPA is converted via irreversible decarboxylation and deamination reactions into a-fluoro-h-alanine (FBAL) by h-alanine synthase or hureidopropionase (E.C. 3.5.1.6). From CO2 and NH4+, urea is formed by the urea cycle. FBAL, the most important product of FU catabolism is optically active and exhibits a R configuration indicating the stereospecificity of the metabolic hydrogenation step of the C5 – C6 double bond of FU [17,18]. FBAL can react non-enzymatically with bicarbonate ion (HCO3 ) to give N-carboxy-a-fluoro-h-alanine (CFBAL). This is in accordance with the well-known equilibrium between compounds with an amino group and their corresponding carbamate (N-carboxy) derivatives in weakly alkaline aqueous carbonate solution. Due to its acid lability, CFBAL is only detected in urine at pH  6.8 – 7.3 depending on the bicarbonate ion concentration, and in plasma [4,6,19]. FBAL can also serve as a substrate for further enzymatic reactions with the release of fluoride ion (F ) and the formation of 2-fluoro-3-hydroxypropanoic acid (FHPA) and fluoroacetic acid (FAC) as well as that of FBAL conjugates with bile acids. Twenty years ago, the 19F NMR detection of elevated levels of F in acidic urine of rats treated with FBAL coupled to the fact that FBAL is defluorinated chemically in basic medium only led to the conclusion that F most likely results from the metabolic cleavage of the C – F bond of FBAL [20]. More recently, Porter et al. demonstrated that the release of F from racemic (S,R)-FBAL is catalyzed by the enzyme l-alanine-glyoxylate aminotransferase II (E.C. 2.6.1.44), the metabolic (R)-FBAL being the preferred enantiomer for the defluorinating activity in rat liver homogenates [21]. The 19F NMR detection of low amounts of two fluorinated analogs of h-alanine metabolites (FHPA and FAC) in perfusates of isolated perfused rat liver (IPRL) injected with pure FU or FBAL and in urine of rats treated with pure FU showed that the metabolism of h-alanine and its fluorinated analog are similar [22]. The first step in halanine catabolism is a transamination reaction to form malonic acid semi-aldehyde (MASAld) catalyzed by hepatic

64

M. Malet-Martino et al. / Clinica Chimica Acta 366 (2006) 61 – 73 O H

N

O

F

4 3 5 2 16 N

H

H

FU NADPH

NADPH

Dihydropyrimidine dehydrogenase (1.3.1.2)

NADP +

NADP + O H

H N

O

F H N

H

H

FUH 2 Dihydropyrimidinase (3.5.2.2)

H 2O

H2O

HOOC H 2N O

N

H F H H

H

FUPA β-ureidopropionase (3.5.1.6) HOOC

H

H 2O

H2O

HCO3-

HOOC

F H - OOC-HN

H

H F H

H2O

HCO3 -

CFBAL Alanine-glyoxylate

H 2N

FBAL

+ CO2 + NH 3

H H+ H 2 NCONH 2

Urea

aminotransferase II (2.6.1.44)

CholylCoA-synthetase (6.2.1.7) Bile acid-CoA:amino acid N-acyltransferase (2.3.1.65)

Fβ-alanine-pyruvate aminotransferase (?) (2.6.1.18)

NADH

NAD +

HO C CH 2 O F

FAC

+ NADH NAD

CO2 H C CH2

NADH NAD+

Conjugates of FBAL with bile acids

O F

Facet

H C CH C OH O F

O

FMASAld

HO CH 2 CH C OH NADH NAD +

F

O

FHPA

Fig. 1. Catabolic pathway of 5-fluorouracil. All the compounds (except CFBAL) are represented in neutral form. FU, 5-fluorouracil; FUH2, 5,6-dihydro-5fluorouracil; FUPA, a-fluoro-h-ureidopropionic acid; FBAL, a-fluoro-h-alanine; CFBAL, N-carboxy-a-fluoro-h-alanine; F , fluoride ion; FMASAld, fluoromalonic acid semi-aldehyde; FHPA, 2-fluoro-3-hydroxypropanoic acid; Facet, fluoroacetaldehyde; FAC, fluoroacetic acid. The ellipses denote fluorinated metabolites identified for the first time with 19F NMR. Non-detected fluorinated intermediates are represented in brackets.

transaminases, namely h-alanine-pyruvate aminotransferase (E.C. 2.6.1.18), h-alanine-oxoglutarate aminotransferase (E.C. 2.6.1.19) and d-3-aminoisobutyrate aminotransferase (E.C. 2.6.1.40) [23,24]. MASAld is converted either by spontaneous decarboxylation to acetaldehyde, which is further oxidized into acetate through an aldehyde deshydrogenase catalysis requiring nicotinamide adenine dinucleotide (NAD) in its oxidized form (NAD + ), or enzymatically to h-hydroxypropanoic acid (HOCH2-CH2COOH) by a propionate deshydrogenase, which needs NAD in its reduced form (NADH) ([23] and references quoted in). By analogy with the metabolism of h-alanine,

we proposed that FBAL leads to the formation of FHPA and FAC according to the metabolic pathway depicted in Fig. 1. Since FU is a competitive inhibitor of h-alanine for E.C. 2.6.1.19 and E.C. 2.6.1.40, and FBAL inactivates E.C. 2.6.1.40 [25], FBAL transamination to give fluoromalonic acid semi-aldehyde (FMASAld) is probably catalyzed by E.C. 2.6.1.18 when FBAL results from FU metabolism. The FMASAld metabolic pathways are very likely catalyzed by the same enzymes than those involved in MASAld metabolism. The aldehydes being very reactive, FMASAld and its decarboxylated analog, fluoroacetaldehyde (Facet), are not detected in our experiments. Similarly, during the

M. Malet-Martino et al. / Clinica Chimica Acta 366 (2006) 61 – 73

metabolization of fluorinated ethanes into FAC in rats, intermediate Facet was undetected in urine and kidney extracts [26]. FHPA and FAC are detected in urine of patients treated with FU using 19F NMR [7,27], FHPA was also detected by Hull et al. in urine of patients treated with FU but not identified [6]. As FU commercial formulations are not pure (see below), FAC and FHPA can arise from the metabolism of impurities or that of FU or both. Nevertheless, Lemaire et al. demonstrated that FHPA detected in urine of patients treated with FU comes, at least partly, from FU metabolism [27]. Biliary excretion of fluoropyrimidine drugs in humans is low: 2 –3% for radiolabeled FU as measured by scintillation counting [28] and 0.8% for the FU prodrug, 5V-deoxy-5fluorouridine (5VdFUR), as determined by 19F NMR [29]. In the last case, F and FBAL represented å 10% of the excreted metabolites and unknown compounds accounted for å 90% [29]. These metabolites were identified as conjugates of FBAL with cholic acid (choloFBAL) and

OH

H 3C CH 3

NH

H

CH 3

O

R COOH

H

F

H

HO

OH H

choloFBAL H 3C CH3

NH

H

CH 3

H

O

COOH

F

H

HO

R

OH H

chenoFBAL OH

H 3C CH 3

NH

H

CH 3

H

O

H

R

COOH

F

HO H

dcholoFBAL Fig. 2. Structures of conjugates of a-fluoro-h-alanine (FBAL) with cholic acid (choloFBAL), chenodeoxycholic acid (chenoFBAL) and deoxycholic acid (dcholoFBAL).

65

chenodeoxycholic acid (chenoFBAL) in a 74/26% ratio in bile of patients with external bile drainage (Fig. 2). This result is in agreement with the fact that only the ‘‘primary’’ bile acids (cholic and chenodeoxycholic acids in a ratio of å 3/1) are present in case of bile derivation. The ‘‘secondary’’ bile acids (mainly deoxycholic acid) are no longer synthesized as the enterohepatic circulation is suppressed. In a bile sample obtained at surgery from a patient treated with intrahepatic FU, three conjugates of FBAL were detected, choloFBAL (54%), chenoFBAL (17%) and the conjugate with the third major bile acid in human bile, deoxycholic acid, deoxycholic acid (dcholoFBAL) (29%) (Fig. 2) [29]. Independently and at the same time, Heggie et al. [28] found, from an HPLC analysis of biliary excretion of radiolabeled FU administered to patients with external bile drainage, that 80 – 90% of the FU biliary metabolites were previously unrecognized. These metabolites were identified later using mass spectrometry and enzymatic degradation after isolation as conjugates of FBAL with cholic acid and chenodeoxycholic acid [30,31]. Comparing the 19F NMR spectra of FBAL conjugates with the bile acids in human bile to those synthesized from racemic FBAL showed that only one of the two diastereoisomers of each FBAL conjugate forms in vivo, in accordance with the fact that only the (R) enantiomer of FBAL is metabolically produced from FU [18]. Two steps are involved in the formation of bile acid – amino acid conjugates, the first being the conversion of the bile acid (RCOOH) to an acyl-CoA thioester (RCO-S-CoA) by the cholyl-CoA synthetase (E.C. 6.2.1.7). The bile acid-CoA:amino acid N-acetyltransferase (E.C. 2.3.1.65) from human liver catalyzes the transfer of the bile acid moiety from the acyl-CoA thioester to the amino group (RVNH2) of taurine, glycine or FBAL (but not h-alanine) to form a N-acyl bile conjugate (RCO-NHRV) [32] (Fig. 2). In summary, 19F NMR has helped to improve the knowledge of FU catabolic pathway, the present understanding of which is summarized in Figs. 1 and 2. All these fluorinated metabolites were detected and quantified in a single-run analysis, the ellipses circling those identified for the first time by this method: F , CFBAL, conjugates of FBAL with cholic, chenodeoxycholic and deoxycholic acids, FHPA and FAC, all of them being only observed with 19F NMR except FBAL conjugates with cholic and chenodeoxycholic acids. Moreover, 19F NMR confirms the importance of the urinary excretion of FU and metabolites demonstrated in the sixties by Mukherjee and Heidelberger [33]. They reported that 90% of 6-[14C]-FU administered dose was excreted in patients’ urine within 24 h mainly as FBAL. Using 19F NMR as the analytical method, a daily total urinary excretion profile of FU and metabolites following a daily intravenous (i.v.) bolus administration during a 6-day chemotherapy shows that (i) the daily excretion is nearly constant and reaches 95 T 10% of the administered dose (a.d.), (ii) FBAL is by far the major metabolite accounting

66

M. Malet-Martino et al. / Clinica Chimica Acta 366 (2006) 61 – 73

for 75 T 10% of the injected daily dose, unchanged FU amounting to å 10%, and the other metabolites å 10% (FUH2 å 4%, FUPA å 8%, F å 2%), (iii) the excretion of FU and metabolites is rapid as it occurs for 83 T 9% within the initial 6 h [3]. Similar data were reported by Heggie et al. [28] in patients treated i.v. bolus with 6-[3H]-FU using HPLC with radioactivity quantification of each resolved peak as the analytical technique, and Hull et al. [6] in patients treated i.v. bolus with FU with or without a pretreatment with methotrexate using 19F NMR. 3.2. FU degradative pathway and FU cardiotoxicity Since a basic medium is needed for its solubilization, FU is dissolved for clinical use at a 50 mg/mL concentration in sodium hydroxide solution (FU-NaOH) at pH å 9.1 and in some cases in Tris (Trometamol) buffer (FU-Tris) at pH å 8.4. The 19F NMR analysis of commercial FU-NaOH solutions revealed the presence, besides FU, of about a hundred fluorinated signals accounting for å 1.8 mol% relative to nominal FU concentration, F being by far the major one (Fig. 3A). As FU powder is pure, all the fluorinated resonances detected in the 19F NMR spectra of FU clinical formulations are FU degradation compounds formed over time. Indeed, in basic conditions, FU is hydrolyzed to urea and FMASAld (HOOC-CHF-CHO) that is decarboxylated with time into Facet (H2FC-CHO) as well as to urea, fluoride ion and nonfluorinated aldehydes ([34,35], and unpublished results). The two fluorinated aldehydes that are under hydrate form are found at very low concentrations in vials (0.015% for FMASAld and 0.010% for Facet relative to nominal FU) [36]. Due to their high chemical reactivity, they are transformed by successive aldol condensations with each other and/or non-fluorinated aldehydes, and/or by reaction with urea into the numerous fluorinated compounds detected. On the other hand, the 19F NMR analysis of the FU-Tris formulations revealed the presence, besides FU itself, of few fluorinated compounds accounting for å 1.5 mol% relative to nominal FU concentration, with F representing less than 0.1 mol% of FU. The major ones were identified as adducts of Tris with FMASAld (1.0%) and Facet (0.3%) [7,36] (Fig. 3B). Indeed, it is known that aldehydes react with nucleophilic amino and hydroxyl groups of Tris leading to the formation of oxazolidines. The oxazolidines formed with FMASAld and Facet whose structures are presented in Fig. 3B, are stable at the pH of the FU formulation (8.4) but are in equilibrium with the starting aldehydes at physiological pH. FMASAld and Facet are thus trapped as stable depot forms in FU-Tris formulations. FMASAld and Facet (as oxazolidine adducts or under free form) are highly cardiotoxic on the isolated perfused rabbit heart (IPRH) model. The cardiotoxicity of FU

solutions on this model is correlated to the amounts of FMASAld and Facet, FU-NaOH formulations being much less cardiotoxic than FU-Tris solutions [7,36]. In the IPRH model, Facet is extensively metabolized into FAC, a highly cardiotoxic and neurotoxic poison, whereas FMASAld is converted in a very low extent to FAC and FHPA, which is also cardiotoxic on this model at high dose [7,22,36]. In the urine of patients treated with FU-Tris formulations, 19 F NMR analysis revealed the presence, besides that of FU and its classical metabolites (FUH2, FUPA, CFBAL, FBAL and F ), of FHPA and FAC as well as that of Facet and adducts of FMASAld and Facet with urea (Fig. 4). If FMASAld and Facet resulting from FU alkaline hydrolysis are certainly the causative factor of higher cardiotoxicity and neurotoxicity of FU-Tris formulations [37], the non-negligible frequency of cardiotoxic accidents observed after injection of the FU-NaOH formulations at pH 9.1 was too important to be explained by the very low levels of Facet and FMASAld found in these preparations. Consequently, the metabolism of FU itself, via the FBAL transamination reaction forming FMASAld that then gives FHPA, Facet and FAC (Fig. 1), is also involved in the cardiotoxicity of this drug.

4. Fluorocytosine (FC) FC is an antifungal agent used for the treatment of severe fungal infections, particularly when combined to amphotericin B. The antifungal activity of FC results from the intrafungal formation of FU leading to the inhibition of RNA processing and DNA synthesis via FNUCt metabolites. Susceptible fungi contain cytosine deaminase (CD; E.C. 3.5.4.1), the enzyme that converts FC to FU, whereas human cells lack this enzyme thus creating a theoretical absence of toxicity for FC in humans. However, because FC and FU toxicity profiles are quite similar, it is thought that FU may account for some FC side effects [38]. Moreover, FU and FU catabolites (FUPA, FBAL) were detected in biofluids of healthy volunteers or patients receiving FC [39 – 41]. The evidence of FC conversion into FU by viable and non-viable Escherichia coli as well as by a semi-continuous culture system mimicking human intestinal microflora was clearly demonstrated [42 – 44]. The 19F NMR analysis of biofluids (plasma and urine) from patients treated with FC provided new information concerning FC metabolism in humans (Fig. 5). More than 90% of the i.v. a.d. of FC was excreted within 24 h after the injection, unchanged FC representing å 95% of the total excreted. FU was detected in plasma only, whereas all the classical FU catabolites, FUPA, FBAL, FUH2 (only when the level of FBAL was high), and F , were identified in urine ([5], and unpublished results). Two other compounds involving a direct metabolism of FC were found. 6-

M. Malet-Martino et al. / Clinica Chimica Acta 366 (2006) 61 – 73

F-

A

-43.6

-93.2

ppm

67

FU ppm

FH 2 C-CH(OH)2

HOOC-CHF-CH(OH)2 FMASAld-hydrate -126.4 ppm

-126.0

-40

-50

-60

-70

-80

-90

-100 (ppm)

N H

CH2OH

-127.0

-110

-120

Facet-hydrate -155.1 ppm

-154.0

-130

-154.5 -155.0 (ppm)

-140

-155.5

-150

-160

O CH2OH

H HOOC-FHC

FMASAld-oxazolidine

B

FU

O CH2OH

H FH2C

N H

CH2OH

Facet-oxazolidine

F-

-43.2

-93.0

-117.2-122.3-124.8-125.9

-159.7 δ (ppm)

19

Fig. 3. F NMR spectra at 282 MHz with proton decoupling of clinical formulations of 5-fluorouracil dosed at 50 mg/mL in sodium hydroxide solutions at pH 9.1 (A) and in Tris buffer at pH 8.4 (B). The chemical shifts are expressed relative to the resonance peak of trifluoroacetic acid (TFA) (5% w/v aqueous solution) used as an external reference. F , fluoride ion; FU, 5-fluorouracil; FMASAld hydrate, fluoromalonic acid semi-aldehyde hydrate; Facet-hydrate, fluoroacetaldehyde hydrate; FMASAld-oxazolidine, two diastereoisomeric oxazolidine adducts of FMASAld with Tris; Facet-oxazolidine, oxazolidine adduct of Facet with Tris.

Hydroxy-5-fluorocytosine (OHFC) was detected in urine and plasma and represented less than 1.5% of the total excreted dose ([5], and unpublished results). A glucuroconjugate of FC (GLFC), identified as the O2-h-glucuronide of FC [45], was present in plasma, urine, and cerebrospinal fluid. It was the major FC metabolite

accounting for 1 – 4% of the total excreted dose ([5], and unpublished results). In conclusion, 19F NMR allowed to detect and identify four new FC metabolites in physiological fluids of FCtreated patients: FUH2 and F which are well-known catabolites of FU, OHFC that was already detected in

68

M. Malet-Martino et al. / Clinica Chimica Acta 366 (2006) 61 – 73

FU

FBAL

CFBAL

FHPA

$ FMASAld-urea Facet-urea

FUPA FUH 2

F-

?

FAC

$ $ ?

Facet –155.1

–154.5

–140.9

–125.9 –125.0 –124.7 –122.4

–112.6 –112.3 –110.6 –110.3 –109.8

–93.3

–4.31

δ (ppm)

Fig. 4. 19F NMR spectrum at 282 MHz with proton decoupling of a urine sample from a patient treated with a continuous i.v. infusion of FU-Tris at a dose of 650 mg/m2/day over 4 consecutive days. Urine fraction 48 – 72 h, 30-fold concentrated, pH of the sample 6.8. The chemical shifts are expressed relative to the resonance peak of TFA (5% w/v aqueous solution) used as an external reference. F , fluoride ion; FU, 5-fluorouracil; ?, unknown; FUPA, a-fluoro-hureidoproionic acid; CFBAL, N-carboxy-a-fluoro-h-alanine; FBAL, a-fluoro-h-alanine; FHPA, 2-fluoro-3-hydroxypropanoic acid; FMASAld-urea, two diastereoisomeric adducts of fluoromalonic acid semi-aldehyde with urea; FUH2, 5,6-dihydro-5-fluorouracil; FAC, fluoroacetic acid; Facet-urea, adduct of fluoroacetaldehyde with urea; Facet-hydrate, fluoroacetaldehyde hydrate. Metabolites identified for the first time with 19F NMR are represented in boxes.

unusual circumstances in a urinary gravel excreted by a FCtreated patient [41], and GLFC which was the first glucuronide of a FP drug detected in humans. Moreover, the 19F NMR study of urine of two patients treated with FC showed a direct relationship between FU catabolites and the number of intestinal enterobacillary colonies. Indeed, the percentage of FU catabolites (FUPA +

FBAL + F ) was found to be extremely low (< 0.6% of total fluorinated compounds excreted) when the number of enterobacillary colonies was low (< 103), but considerably higher (3.5% to 8.8%) when such colonies were under reconstitution or within the normal range (105 to 108) [46]. This finding demonstrated that FU-related toxicity may occur in FC-treated patients.

NH2 F

N O

N H

OH

OHFC NH 2 N O

NH 2 F N

N H

H

Cytosine deaminase (3.5.4.1)

F

O

FC

O

H

N H

cell membrane

N H

FU

FC

β-D-glucuronidase (3.2.1.3)

FUH 2 F

HN

(bacteria, fungi)

Cytosine permease

O

FUPA

FBAL

F-

(catabolic pathway)

H

FNUCs

FNUCt (anabolic pathway)

NH 2 F

N COOH O

O

N

H

OH HO OH

GLFC

Fig. 5. Metabolic pathway of 5-fluorocytosine. FC, 5-fluorocytosine; FU, 5-fluorouracil; FUH2, 5,6-dihydro-5-fluorouracil; FUPA, a-fluoro-h-ureidopropionic acid; FBAL, a-fluoro-h-alanine; F , fluoride ion; OHFC, 6-hydroxy-5-fluorocytosine; GLFC, O2-h-glucuronide of FC; FNUCs, fluoronucleosides; FNUCt, fluoronucleotides. The ellipses denote fluorinated metabolites identified for the first time in human biofluids with 19F NMR.

M. Malet-Martino et al. / Clinica Chimica Acta 366 (2006) 61 – 73

in the liver. 5VdFCR is then deaminated into 5VdFUR by cytidine deaminase (E.C. 3.5.4.5) mainly localized in liver and tumor tissues. Finally, 5VdFUR is transformed into FU under the action of TP, an enzyme with higher activity in malignant tissue than in normal tissue. Higher levels of FU are thus produced within tumors with minimal exposure of healthy tissue to FU [51]. As CAP and its two first metabolites do not show intrinsic cytotoxicity, the activation pathway is expected to combine high antitumor efficacy with improved clinical safety [51]. CAP both as a single agent and/or as a component of combination chemotherapy has proven to be effective for the treatment of a range of solid tumors. Although its approved indications remain breast and colon cancers, numerous clinical trials have been performed in other cancer types, particularly pancreatic, gallbladder and bile duct adenocarcinomas in combination with gemcitabine, the cornerstone of current chemotherapy in pancreatic and biliary cancers. The results of several phase II studies show that this combination may yield benefit in this patient population with a significant

5. Capecitabine (CAP) N 4-Pentyloxycarbonyl-5V-deoxy-5-fluorocytidine, more commonly called capecitabine (CAP) or Xeloda\, is a recent prodrug of 5VdFUR, another FU prodrug, that is administered orally to circumvent the unacceptable toxicity of 5VdFUR without compromising its antitumor efficacy [47]. Since the main limitation of 5VdFUR derives from its gastrointestinal toxicity (diarrhea) attributed to the liberation of FU in the small intestine under the action of thymidine phosphorylase (TP; E.C. 2.4.2.4) [48], CAP was designed as a prodrug of 5VdFUR that could not be metabolized by TP in the intestine. Indeed, after oral administration CAP crosses intact the gastrointestinal barrier and is rapidly and almost completely absorbed [49,50]; diarrhea should not thus occur with its use. CAP is subsequently converted into FU in a three-stage mechanism involving several enzymes (Fig. 6). In a first step, it is metabolized into 5V-deoxy-5-fluorocytidine (5VdFCR) by carboxylesterase (E.C. 3.1.1.1), almost exclusively located NH COO(CH 2 ) 4 CH3

NH 2

F

F

N O O

H3C

N

Carboxylesterase

H

N

N

H

Pyrimidine N nucleoside phosphorylase ? O

OH

OH

CAP

NH 2

NH2

O O

H3C

(3.1.1.1)

OH

69

F

F

N H

N O

H

N H

OH

OH

OHFC

FC

5' dFCR Cytidine deaminase (3.5.4.5) O O

Thymidine phosphorylase

F HN

F HN

(2.4.2.4) O

N H

H

O O

H3C

N

H

FU OH

OH

5' dFUR O H

O

H N

F H N

H

HOO C H 2N O

HOOC

H

H F H

F H

N

H2N

H

H

H

FUH 2

FUPA

FCH 2

F-

H

FBAL

C O

FAC

OH

HO CH2 CH C F

OH

O

FHPA

Fig. 6. Catabolic pathway of capecitabine from 19F NMR analysis of patients’ urine. All the compounds are represented in neutral form. CAP, capecitabine; 5VdFCR, 5V-deoxy-5-fluorocytidine; FC, 5-fluorocytosine; OHFC, 6-hydroxy-5-fluorocytosine; 5VdFUR, 5V-deoxy-5-fluorouridine; FU, 5-fluorouracil; FUH2, 5,6-dihydro-5-fluorouracil; FUPA, a-fluoro-h-ureidopropionic acid; FBAL, a-fluoro-h-alanine; F – , fluoride ion; FHPA, 2-fluoro-3-hydroxypropanoic acid; FAC, fluoroacetic acid. Metabolites identified for the first time in urine of patients with 19F NMR are represented in ellipses.

70

M. Malet-Martino et al. / Clinica Chimica Acta 366 (2006) 61 – 73

combination with taxanes. A randomized phase II study demonstrated the superiority of CAP against a standard cyclophosphamide, methotrexate and FU regimen as firstline therapy for MBC with a higher response rate and trend to improved survival time and time-to-disease progression in CAP arm [58]. A multicenter phase II study showed that the combination of CAP and paclitaxel is highly active and generally well tolerated for first-line treatment of MBC [59]. A phase III trial enrolling 511 patients indicated that the CAP/ docetaxel therapy provides clear benefits over single-agent docetaxel in anthracycline-pretreated patients with MBC. Indeed, CAP/docetaxel results in a significant superior efficacy in time-to-disease progression, overall survival and objective response rate compared with single docetaxel [60]. Clinical pharmacokinetic study has demonstrated that the excretion of the intact drug and its metabolites is both rapid and almost exclusively urinary. Indeed, the percentage of radioactive dose after oral administration of a single 2 g oral dose of 14C-radiolabeled CAP reached about 98% (95.5% in urine and 2.6% in faeces) after 6 days of cumulative collection, 84% of the urine excretion occurring in the first 12 h postdosing [49]. The mean percentage of dose excreted in urine as parent drug and its fluorinated metabolites, 5VdFCR, 5VdFUR, FU, FUH2, FUPA and FBAL up to 48 h postdosing, measured by 19F NMR, was 84.2% close to 92.3% of the radioactivity recovered at that time. This difference of less than 10% demonstrates that 19F NMR spectroscopy is a suitable technique for quantitative studies [49]. As expected, the

antitumor activity and a mild toxicity profile [52 – 54]. Therefore, this regimen warrants further evaluation in a prospective randomized phase III study. Two large randomized phase III trials assigning a total of 1207 patients demonstrated a superior response rate for oral CAP compared with i.v. bolus FU/leucovorine (LV), the standard chemotherapy protocol for metastatic colorectal cancer (MCRC): 22% versus 13% as assessed by an independent review committee. The median time to progression and overall survival were equivalent in the two arms, whereas the safety profile and quality of life were improved in the CAP arm. CAP is thus a suitable replacement for i.v. FU/ LV as the backbone of colorectal cancer therapy [55]. These data provide the rationale for a phase III trial to compare CAP and FU/LV administered according to the Mayo Clinic regimen as adjuvant treatment in resected stage III colon cancer. A study enrolling 1987 patients showed that disease-free survival in CAP group is at least equivalent to that in bolus FU/LV group. Moreover, CAP is better tolerated and has the added convenience of oral administration. Oral CAP is thus an effective alternative to i.v. FU/LV in the adjuvant treatment of colon cancer [56]. CAP in combination with oxaliplatin (Ox) (Xelox) is as effective as FU/LV/Ox regimen for MCRC treatment. As Xelox provides a more convenient regimen likely to be preferred by both patients and healthcare providers, it has the potential to replace FU/LV/Ox for MCRC treatment [57]. The situation is similar in metastatic breast cancer (MBC) where CAP is widely approved as monotherapy or in

FBAL

FBAL

FU

5'dFUR FHPA

FUPA -6

5'dFCR

FUH 2

?

x2

J

F-

x10

FAC

CAP

x10 FC FUPA

-40

FUH 2

FU

REF -50

-60

-70

-80

-90

x10

OHFC

-100

-110

-120

-130

-140

-150

(ppm)

Fig. 7. 19F NMR spectrum at 282 MHz with proton decoupling of a urine sample from a patient receiving oral capecitabine at a dose of 3800 mg/day administered twice daily at 12-h interval, as a second treatment 3 months after the first one. Urine fraction 0 – 12 h collected after the first dose of 1900 mg and 10-fold concentrated, pH of the sample: 5.45. The chemical shifts are expressed relative to the resonance peak of TFA (5% w/v aqueous solution) used as external reference. REF, external reference; F , fluoride ion; CAP, capecitabine; 5VdFCR, 5V-deoxy-5-fluorocytidine; 5VdFUR, 5V-deoxy-5-fluorouridine; FC, 5fluorocytosine; FU, 5-fluorouracil; ?, unknown; FUPA, a-fluoro-h-ureidopropionic acid; FBAL, a-fluoro-h-alanine; FHPA, 2-fluoro-3-hydroxypropanoic acid; OHFC, 6-hydroxy-5-fluorocytosine; FUH2, 5,6-dihydro-5-fluorouracil; FAC, fluoroacetic acid. J, 1J13C – F coupling constant.

M. Malet-Martino et al. / Clinica Chimica Acta 366 (2006) 61 – 73

major metabolite is FBAL representing å 60% of the a.d., 5VdFCR, 5VdFUR, FUPA and unmetabolized CAP being the other significant forms (å 3% to 11% of the a.d.), whereas FU and FUH2 accounted for less than 1% (range 0.3 – 0.7%) of the a.d. (Fig. 6) [49,61]. Urine of patients receiving i.v. dose of 200 to 250 mg/m2 irinotecan (CPT-11) 24 h before oral CAP treatment at a dose of 1000 to 1250 mg/m2 twice daily at 12-h interval were collected over 12 h after the first CAP dose and analyzed by 19 F NMR [9]. Pretreatment of patients with CPT-11 does not sensibly affect the pattern of CAP metabolites excreted in urine. Indeed, the total recovery of CAP and its fluorinated metabolites accounted for 71 T17% a.d. [9] close to the literature values based on the same measurement technique (84% and 86% a.d. after 48 h and 24 h urine collection, respectively) [49,61]. Moreover, CAP and its classical metabolites, 5VdFCR, 5VdFUR, FU, FUH2, FUPA and FBAL, are detected in close percentages of a.d. than in the two precited studies [49,61], except FBAL. The long half-life time of FBAL [28] and the limited 12 h period of urine collection can explain the lower amount of FBAL found in this study (46 T 4% a.d.) [9] compared to that obtained after urine recovery over 48 h (57 T 5% a.d.) and 24 h (å 61% a.d.) [49,61]. Three other FU metabolites were observed in small or very tiny amounts: F (å 0.2% a.d.), FHPA (å 0.3% a.d.) and FAC (å 0.004% a.d. only detected in 4 out of 14 samples analyzed). Since CAP formulation is pure, this demonstrates that FU can be metabolized, in humans, into FHPA and FAC via FBAL, as already shown in rats [22]. The detection of FC and FCOH in 4 out of 14 urine samples, accounting for 0.01% and 0.02% a.d., respectively, led to the identification of a novel, even minor, degradation pathway. The degradation pathway of CAP, incorporating the new fluorinated metabolites found in urine using 19F NMR, is depicted in Fig. 6, and a typical 19F NMR spectrum of urine is presented in Fig. 7.

6. Conclusion Despite its limited sensitivity in the low micromolar range, F NMR is a high potential analytical technique for metabolic studies of FP such as FU, FC or CAP. For biofluid analysis, it can be considered as concurrent to, and even in some cases, more performing than chromatographic techniques. Indeed, 19F NMR allowed to detect, identify and quantify: – 10 new FU metabolites in biofluids of patients treated with this drug: F , CFBAL, 3 FBAL conjugates with bile acids, FHPA and FAC as well as FMASAld adducts with urea, Facet and Facet adduct with urea when FU-Tris formulation was used. – 4 new FC metabolites in physiological fluids of FCtreated patients: 2 classical FU metabolites (FUH2 and F ), OHFC that was previously detected only in a urinary gravel, and O2-h-glucuronide of FC.

19

71

– 5 new CAP metabolites in urine of patients treated with this drug: 3 FU catabolites (F , FHPA and FAC), FC and its hydroxylated metabolite OHFC. Since CAP formulation is pure, the presence of FHPA and FAC in humans demonstrates unambiguously that they come from FU metabolism via transamination of FBAL. Moreover, nine of these new metabolites (F , CFBAL, FBAL conjugate with deoxycholic acid, FHPA, FAC, FCglucuronide, Facet as well as FMASAld and Facet adducts with urea) were only detected with 19F NMR. The quantitative determination of all these fluorinated metabolites demonstrates that 19F NMR spectroscopy should be used for the therapeutic monitoring of FP, more especially FU and its prodrugs. For example, the 19F NMR analysis of plasma from one patient on chronic hemodialysis treated with FU/LV over 5 consecutive days showed that pharmacokinetic parameters of FU and its first catabolite FUH2 were similar to values reported in patients without severe renal impairment. It was most likely the same for FUPA, which exhibits low renal clearance. In contrast, the final FU catabolite, FBAL, accumulated on day 5 to a concentration approximately twofold higher than that expected from the literature despite good removal by hemodialysis. The toxic metabolites FHPA and FAC were detected but remained at concentrations less than 20 and 2 Amol/L in plasma. Even if no FBAL-mediated cardiotoxicity or neurotoxicity was observed in this individual case, more intensive dialysis treatment may be warranted to reduce plasma and tissue levels of FBAL and its metabolites more effectively. The possible benefits of this treatment need further evaluation in a study enrolling a significant number of patients [10]. This is the sole 19F NMR study related to the evaluation of FP toxicity, but the technique should easily play a major role in the detection of deficiency of FU catabolic enzymes, DPD, dihydropyrimidinase and h-ureidopropionase. Indeed, enhanced plasma concentrations or higher recovery in urine of FU, FUH2 and FUPA, respectively, will thus be measured in patients treated with FU or its prodrugs. Such a study has not yet been carried out in humans. Nevertheless, the effects of ethynyluracil, a potent DPD inactivator, on FU metabolism were studied by 19F NMR in the IPRL model [8] and in mice in vitro and in vivo [62]. A tremendous increase in the level of unmetabolized FU and decrease in that of FU catabolites (by a factor of å 110 in FBAL formation in IPRL and even disappearance of FU catabolite signal in acid extracts of liver, kidney and tumor in mice bearing colon 38 tumors) were observed. References [1] Malz F, Jancke H. Validation of quantitative NMR. J Pharm Biomed Anal 2005;38:813 – 23. [2] Hull WE. Measurement of absolute metabolite concentrations in biological samples. Bruker Rep 1986;2:15 – 9.

72

M. Malet-Martino et al. / Clinica Chimica Acta 366 (2006) 61 – 73

[3] Bernadou J, Armand JP, Lopez A, Malet-Martino MC, Martino R. Complete urinary excretion profile of 5-fluorouracil during a six-day chemotherapy schedule by 19F nuclear magnetic resonance. Clin Chem 1985;31:846 – 8. [4] Malet-Martino MC, Armand JP, Lopez A, et al. Evidence for the importance of 5V-deoxy-5-fluorouridine catabolism in humans from 19 F nuclear magnetic resonance spectrometry. Cancer Res 1986;46: 2105 – 12. [5] Vialaneix JP, Malet-Martino MC, Hoffmann JC, Pris J, Martino R. Direct detection of new fluorocytosine metabolites in human biofluids by 19F nuclear magnetic resonance. Drug Metab Dispos 1987;15: 718 – 24. [6] Hull WE, Port RE, Herrmann R, Britsch B, Kunz W. Metabolites of 5fluorouracil in plasma and urine as monitored by 19F nuclear magnetic resonance with or without methotrexate treatment. Cancer Res 1988; 48:1680 – 8. [7] Lemaire L, Malet-Martino MC, de Forni M, Martino R, Lasserre B. Cardiotoxicity of commercial 5-fluorouracil vials stems from the alkaline hydrolysis of this drug. Br J Cancer 1992;66:119 – 27. [8] Arellano M, Malet-Martino MC, Martino R, Spector T. 5-Ethynyluracil (GW 776): effects on the formation of the toxic catabolites of 5fluorouracil, fluoroacetate and fluorohydroxypropionic acid in the isolated perfused rat liver model. Br J Cancer 1997;76:1170 – 80. [9] Desmoulin F, Gilard V, Malet-Martino M, Martino R. Metabolism of capecitabine, an oral fluorouracil prodrug: 19F NMR studies in animal models and human urine. Drug Metab Dispos 2002;30:1221 – 9. [10] Rengelshausen J, Hull WE, Schwenger V, Goggelmann C, WalterSack I, Bommer J. Pharmacokinetics of 5-fluorouracil and its catabolites determined by 19F nuclear magnetic resonance spectroscopy for a patient on chronic hemodialysis. Am J Kidney Dis 2002;39: E10. [11] Kamm YJL, Rietjens IMCM, Vervoort J, et al. Effect of modulators on 5-fluorouracil metabolite patterns in murine colon carcinoma determined by in vitro 19F nuclear magnetic resonance spectroscopy. Cancer Res 1994;54:4321 – 6. [12] Kamm YJL, Heerschap A, Rosenbusch G, Rietjens IMCM, Vervoort J, Wagener DJT. 5-Fluorouracil metabolite patterns in viable and necrotic tumor areas of murine colon carcinoma determined by 19F NMR spectroscopy. Magn Reson Med 1996;36:445 – 50. [13] Peters GJ, Ackland SP. New antimetabolites in preclinical and clinical development. Expert Opin Invest Drugs 1996;5:637 – 79. [14] Diasio RB, Harris BE. Clinical pharmacology of 5-fluorouracil. Clin Pharm 1989;16:215 – 37. [15] Weckbecker G. Biochemical pharmacology and analysis of fluoropyrimidine alone and in combination with modulators. Pharm Ther 1991; 50:367 – 424. [16] Grem JL. 5-Fluoropyrimidines. In: Chabner BA, Longo DL, editors. Cancer chemotherapy and biotherapy: principles and practice. Philadelphia’ Lippincott-Raven; 1996. p. 149 – 211. [17] Duschinsky R, Walker H, Wojarowski W, Noack K, Ba¨chtold HP. (+)a-Fluoro-h-alanine (FBAL), the main metabolite of 5-fluorouracil (FU) and its enantiomer. Proc Am Assoc Cancer Res 1973;14:109. [18] Gani D, Hitchcock PB, Young DW. Stereochemistry of catabolism of the DNA base thymine and of the anti-cancer drug 5-fluorouracil. J Chem Soc Perkin Trans I 1985;1363 – 72. [19] Martino R, Malet-Martino MC, Vialaneix C, Lopez A, Bon M. 19F nuclear magnetic resonance analysis of the carbamate reaction of afluoro-h-alanine (FBAL), the major catabolite of fluoropyrimidines. Application to FBAL carbamate determination in body fluids of patients treated with 5V-deoxy-5-fluorouridine. Drug Metab Dispos 1987;15:897 – 904. [20] Martino R, Lopez A, Malet-Martino MC, Bernadou J, Armand JP. Release of fluoride ion from 5V-deoxy-5-fluorouridine, an antineoplastic fluoropyrimidine in humans. Drug Metab Dispos 1985;13:116 – 8. [21] Porter DJY, Harrington JA, Almond MR, Chestnut WG, Tanoury G, Spector T. Enzymatic elimination of fluoride from a-fluoro-h-alanine. Biochem Pharmacol 1995;50:1475 – 84.

[22] Arellano M, Malet-Martino MC, Martino R, Gires P. The anticancer drug 5-fluorouracil is metabolized by the isolated perfused rat liver and in rats into highly toxic fluoroacetate. Br J Cancer 1998;77:79 – 86. [23] Griffith OW. h-Amino acids: mammalian metabolism and utility as aamino acid analogues. Ann Rev Biochem 1986;55:855 – 78. [24] Tamaki N, Kaneko M, Mizota C, Kikugawa M, Fujimoto S. Purification, characterization and inhibition of D-3-aminoisobutyrate aminotransferase from the rat liver. Eur J Biochem 1990;189: 39 – 45. [25] Kaneko M, Kontani Y, Kikugawa M, Tamaki N. Inhibition of D-3aminoisobutyrate-pyruvate aminotransferase by 5-fluorouracil and afluoro-h-alanine. Biochim Biophys Acta 1992;1122:45 – 9. [26] Keller DA, Roe DC, Lieder PH. Fluoroacetate-mediated toxicity of fluorinated ethanes. Fundam Appl Toxicol 1996;30:213 – 9. [27] Lemaire L, Arellano M, Malet-Martino M, Martino R. Identification of 2-fluoro-3-hydroxypropanoic acid in urine of patients undergoing 5fluorouracil chemotherapy. Proc Intern Soc Magn Reson Med 1996;2: 1162. [28] Heggie GD, Sommadossi JP, Cross DS, Huster WJ, Diasio RB. Clinical pharmacokinetics of 5-fluorouracil and its metabolites in plasma, urine, and bile. Cancer Res 1987;47:2203 – 6. [29] Malet-Martino MC, Bernadou J, Martino R, Armand JP. 19F NMR spectrometry evidence for bile acid conjugates of a-fluoro-h-alanine as the main biliary metabolites of antineoplastic fluoropyrimidines in humans. Drug Metab Dispos 1988;16:78 – 84. [30] Sweeny DJ, Barnes S, Heggie GD, Diasio RB. Metabolism of 5fluorouracil to an N-cholyl-2-fluoro-h-alanine conjugate: previously unrecognized role for bile acid in drug conjugation. Proc Natl Acad Sci U S A 1987;84:5439 – 43. [31] Sweeny DJ, Martin M, Diasio RB. N-Chenodeoxycholyl-2-fluoro-halanine: a biliary metabolite of 5-fluorouracil in man. Drug Metab Dispos 1988;16:892 – 4. [32] Johnson MR, Barnes S, Kwakye JB, Diasio RB. Purification and characterization of bile acid-CoA:amino acid N-acyltransferase from human liver. J Biol Chem 1991;266:10227 – 33. [33] Mukherjee KL, Heidelberger C. Studies on fluorinated pyrimidines: IX. The degradation of 5-fluorouracil-6-C14. J Biol Chem 1960;235: 433 – 7. [34] Lozeron HA, Gordon MP, Gabriel T, Tautz W, Duschinsky R. The photochemistry of 5-fluorouracil. Biochemistry 1964;3:1844 – 50. [35] Rudy BC, Senkowski BZ. Fluorouracil. In: Florey K, editorR Analytical profiles of drug substances, vol. 2. Academic Press’ New York; 1977. p. 221 – 44. [36] Lemaire L, Malet-Martino MC, Martino R, de Forni M, Lasserre B. The Tris formulation of fluorouracil is more cardiotoxic than the sodium salt formulations. Oncol Rep 1994;1:173 – 4. [37] Lukaschek J, Nufer M, Maurer D, et al. Cardiotoxicity and neurotoxicity of high-dose continuous fluorouracil as a result of degradation compounds in the drug vials. J Clin Oncol 2004;22: 5022 – 5. [38] Scholer HJ. Flucytosine. In: Speller DCE, editor. Antifungal chemotherapy. New York’ Wiley; 1980. p. 35 – 106. [39] Diasio RB, Lakings DE, Bennett JE. Evidence for conversion of 5fluorocytosine to 5-fluorouracil in humans: possible factor in 5fluorocytosine clinical toxicity. Antimicrob Agents Chemother 1978; 14:903 – 8. [40] Polak A, Eschenhoff E, Fernex M, Scholer HJ. Metabolic studies with 5-fluorocytosine-6-14C in mouse, rat, rabbit, dog and man. Chemotherapy 1976;22:137 – 53. [41] Williams KM, Duffield AM, Christopher RK, Finlayson PJ. Identification of minor metabolites of 5-fluorocytosine in man by chemical ionization gas chromatography mass spectrometry. Biomed Mass Spectrom 1981;8:179 – 82. [42] Harris BE, Manning BW, Federle TW, Diasio RB. Conversion of 5fluorocytosine to 5-fluorouracil by human intestinal microflora. Antimicrob Agents Chemother 1986;29:44 – 8.

M. Malet-Martino et al. / Clinica Chimica Acta 366 (2006) 61 – 73 [43] Manning BW, Federle TW, Cerniglia CE. Use of a semicontinuous culture system as a model for determining the role of human intestinal microflora in the metabolism of xenobiotics. J Microb Methods 1987; 6:81 – 94. [44] Vermes A, Kuijper EJ, Guchelaar HJ, Dankert J. An in vitro study on the active conversion of flucytosine to fluorouracil by microorganisms in the human intestinal microflora. Chemotherapy 2003;49:17 – 23. [45] Chouini-Lalanne N, Malet-Martino MC, Gilard V, Ader JC, Martino R. Structural determination of a glucuronide conjugate of flucytosine in humans. Drug Metab Dispos 1995;23:813 – 7. [46] Malet-Martino M, Martino R, de Forni M, Andremont A, Hartmann O, Armand JP. Flucytosine conversion to fluorouracil in humans: does a correlation with gut flora status exist? A report of two cases using fluorine-19 magnetic resonance spectroscopy. Infection 1991; 19:178 – 80. [47] Lamont EB, Schilsky RL. The oral fluoropyrimdines in cancer chemotherapy. Clin Cancer Res 1999;5:2289 – 96. [48] Bajetta E, Carnaghi C, Somma L, Stampino CG. A pilot safety study of capecitabine, a new oral fluoropyrimidine, in patients with advanced neoplastic disease. Tumori 1996;82:450 – 2. [49] Judson IR, Beale PJ, Trigo JM, et al. A human capecitabine excretion balance and pharmacokinetic study after administration of a single oral dose of 14C-labelled drug. Invest New Drugs 1999;17:49 – 56. [50] Reigner B, Blesch K, Weidekamm E. Clinical pharmacokinetics of capecitabine. Clin Pharmacokinet 2001;40:85 – 104. [51] Miwa M, Ura M, Nishida M, et al. Design of a novel oral fluoropyrimidine carbamate, capecitabine, which generates 5-fluorouracil selectively in tumours by enzymes concentrated in human liver and cancer tissue. Eur J Cancer 1998;34:1274 – 81. [52] Stathopoulos GP, Syrigos K, Polyzos A, et al. Front-line treatment of inoperable or metastatic pancreatic cancer with gemcitabine and capecitabine: an intergroup, multicenter phase II study. Ann Oncol 2004;15:224 – 9. [53] Knox JJ, Hedley D, Oza A, et al. Combining gemcitabine and capecitabine in patients with advanced biliary cancer: a phase II trial. J Clin Oncol 2005;23:2332 – 8.

73

[54] Cho JY, Nam JS, Park MS, et al. A phase II study of capecitabine combined with gemcitabine in patients with advanced gallbladder carcinoma. Yonsei Med J 2005;46:526 – 31. [55] Van Cutsem E, Hoff PM, Harper P, et al. Oral capecitabine vs. intravenous 5-fluorouracil and leucovorin: integrated efficacy data and novel analyses from two large, randomised, phase III trials. Br J Cancer 2004;90:1190 – 7. [56] Twelves C, Wong A, Nowacki MP, et al. Capecitabine as adjuvant treatment for stage III colon cancer. N Engl J Med 2005;352: 2696 – 704. [57] Cassidy J, Tabernero J, Twelves C, et al. XELOX (capecitabine plus oxaliplatin): active first-line therapy for patients with metastatic colorectal cancer. J Clin Oncol 2004;22:2084 – 91. [58] O’Shaughnessy JA, Blum J, Moiseyenko V, et al. Randomized openlabel, phase II trial of oral capecitabine (Xeloda) vs. a reference arm of intravenous CMF (cyclophosphamide, methotrexate and 5-fluorouracil) as first-line therapy for advanced/metastatic breast cancer. Ann Oncol 2001;12:1247 – 54. [59] Gradishar WJ, Meza LA, Amin B, et al. Capecitabine plus paclitaxel as front-line combination therapy for metastatic breast cancer: a multicenter phase II study. J Clin Oncol 2004;22:2321 – 7. [60] O’Shaughnessy J, Miles D, Vukelja S, et al. Superior survival with capecitabine plus docetaxel combination therapy in anthracyclinepretreated patients with advanced breast cancer: phase III trial results. J Clin Oncol 2002;20:2812 – 23. [61] Cassidy J, Twelves C, Cameron D, et al. Bioequivalence of two tablet formulations of capecitabine and exploration of age, gender, body surface area, and creatinine clearance as factors influencing systemic exposure in cancer patients. Cancer Chemother Pharmacol 1999;44: 453 – 60. [62] Adams ER, Leffert JJ, Craig DJ, Spector T, Pizzorno G. In vivo effect of 5-ethynyluracil on 5-fluorouracil metabolism determined by 19 F nuclear magnetic resonance spectroscopy. Cancer Res 1999;59: 122 – 7.