Structural determination of a glucuronide conjugate of flucytosine in humans.

The structure of a glucuronide metabolite of flucytosine (FC; 5-fluorocytosine), found in the urine of all patients treated with this antifungal drug, was determined. This compound is the O2-beta-glucuronide of FC. Its structure was established after isolation from urine and by comparing its spectroscopic characteristics with those of three FC glucuronides previously synthesized. This study is the first report of the identification of a glucuronide of a fluoropyrimidine drug in humans.

[Cardiotoxicity of 5-fluorouracil: a question of formulation]. / Cardiotoxicité du 5-fluorouracile: une question de formulation.

The cardiotoxicity of 5-fluorouracil (FU) was attributed to degradation compounds present in the injected vials, fluoroacetaldehyde (Facet) and fluoromalonaldehydic acid (FMald). These compounds are formed with time in the basic medium necessary to solubilize FU. FU-NaOH vials were much less cardiotoxic than FU-Tris vials on the isolated perfused rabbit heart model since, in FU-Tris vials, Facet and FMald are stored in stable "depot" forms, which are adducts with Tris, whereas, in FU-NaOH vials, they are extensively chemically transformed. Cardiotoxic fluoroacetate (FAC), arising from Facet metabolization, was found in urine of patients, with a ratio FAC/FU catabolites 10-30 fold lower in patients treated with FU-NaOH than in those treated with FU-Tris.

Chemical and biological evaluation of hydrolysis products of cyclophosphamide.

31P NMR spectroscopy was used to study the products of the decomposition of cyclophosphamide (1) in buffered solutions at pH's ranging between 1.2 and 8.6 at 20 degrees C and at pH 7.4 at 37 degrees C. At pH 1.2, 1 undergoes a rapid breakdown (t1/2 = 1.4 days) of the two P-N bonds, giving compounds 2 [HN(CH2CH2Cl)2] and 3 [H2N(CH2)3OP(O)(OH)2] as hydrochlorides. No intermediates were detected. At pH's between 5.4 and 8.6, hydrolysis of 1 during 17 days leads to the sole and previously unknown nine-membered ring compound 13. 13 results from the intramolecular alkylation of 1 giving the bicyclic compound 7 followed by the exothermal hydrolytic breakdown of the P-N bond of its six-membered ring. At pH 2.2 and 3.4, the two hydrolytic pathways coexist since, beside compounds 2 and 3, the hydrochloride of compound 9 [Cl(CH2)2NH(CH2)2NH(CH2)3OP(O)(OH)2] is formed, resulting from the acid-catalyzed breakdown of the P-N bond in the nine-membered ring compound 13. At pH 2.2, the presence of chloride ion affected neither the stability of 1 nor the contribution of the two competing hydrolytic pathways. At pH's ranging from 3.4 to 8.6, there is little degradation of 1 since more than 95% of initial 1 was still present after 7 days at 20 degrees C. Under physiological conditions (pH 7.4, 37 degrees C) after 6 days, 45% of 1 is hydrolyzed (t1/2 = 6.6 days), leading essentially (30% of initial 1) to the nine-membered ring compound 13. The rate of hydrolysis of 13 and the nature of its hydrolysis products were found to depend on pH over the range 0-8.6. After a single ip injection to mice, compounds 3, 9, and 13 were less toxic than 1. They did not exhibit any direct cytotoxic efficacy on the colony-forming capacity of L1210 cells in vitro, and they had no antitumor activity in vivo against P388 leukemia.

Determination of the urinary excretion of ifosfamide and its phosphorated metabolites by phosphorus-31 nuclear magnetic resonance spectroscopy.

Phosphorus-31 nuclear magnetic resonance spectroscopy was used to analyze urine samples obtained from patients treated with ifosfamide (IF). This technique allows the individual assay of all phosphorated metabolites of IF in a single analysis without the need for prior extraction. In addition to the classic IF metabolites 2-dechloroethylifosfamide (2DEC1IF), 3-dechloroethylifosfamide (3DEC1IF), carboxyifosfamide (CARBOXYIF), and isophosphoramide mustard (IPM), several signals corresponding to unknown phosphorated compounds were observed. Four of them were identified: one is alcoifosfamide (ALCOIF), two come from the degradation of 2,3-didechloroethylifosfamide (2,3-DEC1IF), and one results from the decomposition of 2DEC1IF. The total cumulative drug excretion as measured over 24 h in nine patients was 51% of the injected IF dose; 18% of the dose was recovered as unchanged IF. The major urinary metabolites were the dechloroethylated compounds, with 3DEC1IF excretion (11% of the injected dose) always being superior to 2DEC1IF elimination (4% of the injected dose). Degradation compounds of 2DEC1IF and 2,3DEC1IF represented 0.4% of the injected dose. The metabolites of the dechloroethylation pathway always predominated over those of the activation pathway (CARBOXYIF, ALCOIF, and IPM, representing 3%, 0.8%, and 0.2% of the injected dose, respectively). In all, 14% of the injected dose was excreted as unknown phosphorated compounds. The interpatient variation in levels of IF metabolites was obvious and involved all of the metabolites. Renal excretion was not complete at 24 h, since 11% of the injected dose was recovered in the 24- to 48-h urine samples.

Cardiotoxicity of high-dose continuous infusion fluorouracil: a prospective clinical study.

PURPOSE: A prospective clinical study was performed to determine the incidence of high-dose continuous intravenous infusion fluorouracil (5FU-CIV) cardiotoxicity. PATIENTS AND METHODS: Three hundred sixty-seven patients who were given first-cycle high-dose 5FU-CIV were monitored for cardiac function by clinical examination, ECG, and laboratory tests. 5FU-CIV was administered during a 96- or 120-hour period at doses that ranged from 600 to 1,000 mg/m2/d. Associated drugs included cisplatin (56%), mitomycin (12.5%), folinic acid (leucovorin) (7%), and others (14%). Thirty-nine patients (10.5%) received 5FU as a single agent. RESULTS: 5FU-induced cardiac events occurred in 28 patients (7.6%; 95% confidence interval, 4.9% to 10.3%). Nine of them had a history of cardiac disease. Primary tumors included head and neck (n = 13), gastrointestinal (n = 6), breast (n = 3), and others (n = 6). The mean onset time of cardiac symptoms was 3 days (range, 2 to 5). Inaugural symptoms included angina pectoris (n = 18), hypotension (n = 6), hypertension (n = 5), malaise (n = 4), dyspnea (n = 2), arrhythmia (n = 1), or sudden death (n = 1). At 5FU discontinuation, six patients' cardiac symptoms returned to baseline, but 21 patients experienced unstable angina (n = 8), hypotension/cardiovascular collapse (n = 11), pulmonary edema (n = 1), or sudden death (n = 4). The lethality rate was 2.2% (five sudden deaths plus three irreversible collapses). ECG showed repolarization changes (ST segment deviation; T-wave inversion) in 65% and/or diffuse microvoltage in 22% of the patients who presented with cardiac events. Echocardiography showed partial or global hypokinesia in nine of the 16 patients who were examined, and one case of prolonged akinesia. Cardiac enzymes rarely showed an increase (n = 2). In severe but reversible cases, clinical, ECG, and echographic parameters returned to baseline status within 48 hours after the drug discontinuation. A fluorine 19 nuclear magnetic resonance (19F NMR) analysis of urine was performed on 14 patients; six had cardiac symptoms and eight did not. Fluoroacetate (FAC), a known cardiotoxic compound, was detected in all cases. CONCLUSION: In our study, the incidence of high-dose 5FU-CVI cardiotoxicity was 7.6%. The hypothesis of a toxic cardiomyopathic process requires further confirmation.

Magnetic resonance spectroscopy: a powerful tool for drug metabolism studies.

Studies on the metabolism and disposition of drugs using nuclear magnetic resonance spectroscopy (MRS) as the analytical technique are reviewed. An overview of the main studies classed in terms of the observed magnetic nucleus (1H, 2H, 7Li, 13C, 19F, 31P, 77Se) is followed by some typical examples of the way in which 19F and 31P MRS can be profitably employed to gain more understanding about the metabolism and disposition of the anticancer fluoropyrimidines (5-fluorouracil (FU) and its prodrugs) and ifosfamide (IF). The results of three recent studies carried out in our laboratory are developed. They concern the direct quantitative monitoring of the hepatic metabolism of FU in the isolated perfused mouse liver, the elucidation of the origin of the cardiotoxicity of FU and the metabolism of IF from an analysis of biofluids of patients. Finally, the advantages and limitations of MRS for investigations on drug metabolism are discussed.

Cardiotoxicity of commercial 5-fluorouracil vials stems from the alkaline hydrolysis of this drug.

The cardiotoxicity of 5-fluorouracil (FU) was attributed to impurities present in the injected vials. One of these impurities was identified as fluoroacetaldehyde which is metabolised by isolated perfused rabbit hearts into fluoroacetate (FAC), a highly cardiotoxic compound. FAC was also detected in the urine of patients treated with FU. These impurities were found to be degradation products of FU that are formed in the basic medium employed to dissolve this compound. To avoid chemical degradation of this antineoplastic drug, the solution of FU that will be injected should be prepared immediately before use.

A new approach to the study of ifosfamide metabolism by the analysis of human body fluids with 31P nuclear magnetic resonance spectroscopy.

[31P] nuclear magnetic resonance spectroscopy was used to analyze body fluids from patients treated with ifosfamide (IF). This technique, which requires no labeled drug, allows a direct study of the biological sample with no need for extraction or derivatization and a simultaneous detection and quantification of all the different phosphorated metabolites in a single analysis. In urine, isophosphoramide mustard was detected in addition to the already known human urinary compounds [i.e., unchanged IF, carboxyifosfamide, 2-dechloroethylifosfamide, 3-dechloroethylifosfamide, ketoifosfamide]. 2,3-Didechloroethylifosfamide itself was not found, but two of its degradation compounds were detected, thus showing a minor route of didechloroethylation of IF in humans. Several other signals corresponding to unknown metabolites or to degradation compounds of IF metabolites were observed. None of them corresponded to IF-activated metabolites (4-hydroxyifosfamide, aldoifosfamide) or to conjugates of IF or its metabolites with mesna. The urinary excretion of IF and metabolites over 24 h amounted to 39 to 50% of the injected dose. Unmetabolized IF was the major compound in 0- to 8-h and 8- to 16-h fractions. 2-Dechloroethylifosfamide and 3-dechloroethylifosfamide were the main metabolites detected in each 8-h fraction. The two unknown compounds at 19.16 ppm and 16.06 ppm represented a non-negligible fraction of the excretion, above that of carboxyifosfamide. Only unchanged IF could be detected in plasma samples. Unmetabolized IF and 3-dechloroethylifosfamide were found in a cerebrospinal fluid sample. Neither IF nor IF metabolites could be observed in the corresponding plasma sample. This indicates a long persistence of these compounds in cerebrospinal fluid.

F Nuclear Magnetic Resonance Analysis of 5-Fluorouracil Metabolism in Four Differently Pigmented Strains of Nectria haematococca.

F nuclear magnetic resonance spectroscopy was used to study the metabolism of 5-fluorouracil in four strains of Nectria haematococca which displayed similar sensitivities to growth inhibition by this compound but differed in their pigmentation. The major metabolites, 5-fluorouridine and alpha-fluoro-beta-alanine, were excreted into the medium by all four strains. The classical ribofluoronucleotides (5-fluorouridine-5'-monophosphate, -diphosphate, and -triphosphate) and alpha-fluoro-beta-alanine were identified in the acid-soluble fraction of perchloric acid extracts of mycelia. Two hydrolysis products of 5-fluorouracil incorporated into RNA were found in the acid-insoluble pool. They were unambiguously assigned to 5-fluorouridine-2'-monophosphate and 3'-monophosphate with specific hydrolysis reactions on isolated RNA. The lack of fluorodeoxyribonucleotides and the fact that the four strains incorporated similar amounts of fluororibonucleotides into their RNAs strongly suggest an RNA-directed mechanism of cytotoxicity for 5-fluorouracil. The heavily pigmented wild type differed from the three low-pigmented strains in its low uptake of 5-fluorouracil and, consequently, in its reduced biosynthesis of 5-fluorouridine and alpha-fluoro-beta-alanine. At present, it is not clear whether this change in 5-fluorouracil metabolism is a side effect of pigment production or results from another event.

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.

A relationship between the gut flora level, particularly gram-negative enterobacilli, and the in vivo flucytosine conversion to fluorouracil has been observed in humans from the fluorine-19 magnetic resonance spectroscopy analysis of urine from two patients treated with the flucytosine- (6 to 9 g/day) amphotericin B (1 mg/kg/day) combination. Indeed the percentage of fluorouracil metabolites was extremely low (less than 0.6% of total fluorinated compounds excreted) when the number of enterobacillary colonies was low (less than 10(3] and higher (3.5 to 8.8%) when enterobacillary colonies were under reconstitution or in the normal range (10(5) to 10(8]. The intestinal microflora assessment may therefore be of high interest to predict the risk of an additive flucytosine-induced myelotoxicity suspected to be due to fluorouracil during flucytosine chronic therapy.

[Fluorine-19 nuclear magnetic resonance spectroscopy: a privileged tool for the study of the metabolism and pharmacokinetics of fluoropyrimidines]. / La spectroscopie de résonance magnétique nucléaire du fluor-19: un outil privilégié pour l'étude du métabolisme et de la pharmacocinétique des fluoropyrimidines.

This review reports on the various studies using fluorine-19 nuclear magnetic resonance spectroscopy (19F NMR) to study the metabolism of antineoplastic or antifungal fluoropyrimidines. It is divided into 2 parts: the first examines ex vivo studies, ie, of biofluids or excised tissue samples from patients. In vivo studies, ie where the biotransformation of the drug is followed by non-invasively both in animals and in humans, are described in the second part. For ex vivo studies, 19F NMR can already be considered as complementary to the classical analytical methodologies used for drug metabolism studies. In vivo 19F NMR spectroscopic studies, especially in humans, are still at an early stage of development. Several improvements, both methodological (development of volume-selective localization techniques and quantification methods) and clinical (more rigorous definition of pathologies under study and administered treatments), are a prerequisite for useful clinical application.

Study of the metabolism of flucytosine in Aspergillus species by 19F nuclear magnetic resonance spectroscopy.

The metabolism of flucytosine (5FC) in two Aspergillus species (Aspergillus fumigatus and A. niger) was investigated by 19F nuclear magnetic resonance spectroscopy. In intact mycelia, 5FC was found to be deaminated to 5-fluorouracil and then transformed into fluoronucleotides; the catabolite alpha-fluoro-beta-alanine was also detected in A. fumigatus. Neither 5-fluoroorotic acid nor 5-fluoro-2'-deoxyuridine-5'-monophosphate was detected in perchloric acid extracts after any incubation with 5FC. 5FC, 5-fluorouracil, and the classical fluoronucleotides 5-fluorouridine-5'-mono-, di-, and triphosphates were identified in the acid-soluble pool. Two hydrolysis products of 5-fluorouracil incorporated into RNA, 5-fluorouridine-2'-monophosphate and 5-fluorouridine-3'-monophosphate, were found in the acid-insoluble pool. No significant differences in the metabolic transformation of 5FC were noted in the two species of Aspergillus. The main pathway of 5FC metabolism in the two species of Aspergillus studied is thus the biotransformation into ribofluoronucleotides and the subsequent incorporation of 5-fluorouridine-5'-triphosphate into RNA.

19F nuclear magnetic resonance analysis of 5-fluorouracil metabolism in wild-type and 5-fluorouracil-resistant Nectria haematococca.

A mutant (furA3) was isolated from the S1 wild-type strain of Nectria haematococca on the basis of its resistance to 5-fluorouracil (5FU). This mutant has greatly reduced activity of uracil phosphoribosyltransferase, a pyrimidine salvage enzyme catalyzing the synthesis of UMP from uracil. The metabolism of 5FU was examined in both strains by using 19F nuclear magnetic resonance spectroscopy. In the S1 strain, 5FU appears to be metabolized by two pathways operating simultaneously: (i) conversion to fluoronucleotides and (ii) degradation into alpha-fluoro-beta-alanine. The furA3 mutant shows metabolic changes consistent with a uracil phosphoribosyltransferase lesion, since it takes up 5FU and forms a small amount of alpha-fluoro-beta-alanine but does not synthesize fluoronucleotides. Since pigment synthesis is strongly enhanced by 5FU in the S1 wild-type strain but not in the furA3 mutant, these results support the hypothesis that 5FU stimulation of secondary metabolism in N. haematococca is not mediated by the drug itself but involves a phosphorylated anabolite.

The application of nuclear magnetic resonance spectroscopy to drug metabolism studies.

1. The applications of n.m.r. spectroscopy studies to drug metabolism, both in vitro (biofluids, isolated cells, excised tissue samples, isolated organs) and in vivo (animals, humans) are reviewed. 2. N.m.r. is a relatively insensitive technique, but it has the great advantage of being non-invasive and non-destructive, i.e. it allows a direct study of intact biological samples. 3. The majority of studies examined deal with 19F-n.m.r. spectroscopy mainly because of the favourable n.m.r. characteristics of this nucleus, and the low level of endogenous fluorine which gives no detectable 19F signal. However, the potential utility of 1H-31P- and 13C-n.m.r. is also emphasized.

Direct 19F NMR spectroscopic observation of 5-fluorouracil metabolism in the isolated perfused mouse liver model.

A direct analysis of 5-fluorouracil (FUra) metabolism in the isolated perfused mouse liver was carried out using 19F NMR spectroscopy. In livers treated with low (30 mg/kg body weight (b.w.] or high (180 mg/kg b.w.) doses of FUra, with (180 mg/kg b.w.) or without thymidine (dThd), FUra was rapidly catabolized to alpha-fluoro-beta-ureidopropionic acid (FUPA) and alpha-fluoro-beta-alanine (FBAL), the latter usually becoming by far the major catabolite. The first catabolite of FUra, 5,6-dihydro-5-fluorouracil (5FUH2), was never detected both in the liver and in perchloric acid (PCA) extracts. In the latter, it was generally split into three signals, the major one being attributed to 5-fluorouridine-5'-monophosphate. Moreover, when the liver was treated with high doses of FUra and dThd, a signal for fluoronucleoside was observed in the liver, in PCA extracts and in perfusate where it was attributed to 5-fluoro-2'-deoxyuridine (FdUrd). The injection of concurrent dThd with the high dose of FUra led to a marked reduction of catabolism due to competitive inhibition of dihydrouracil dehydrogenase. Moreover, a significant formation of FdUrd was noticed whereas 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) was not detected. dThd therefore inhibits the conversion of FdUrd to FdUMP.

Application of fluorine-19 nuclear magnetic resonance to the determination of plasma-protein binding of 5'-deoxy-5-fluorouridine, a new antineoplastic fluoropyrimidine.

Two distinct fluorine-19 nuclear magnetic resonance ((19)F NMR) signals have been observed in human serum for free and plasma-protein bound 5'-deoxy-5-fluorouridine (5'dFUrd). The binding of this drug was studied directly in serum using (19)F NMR. To evaluate the validity of this method, a parallel study was conducted with equilibrium dialysis as the reference method. Two assay methods were applied after equilibrium dialysis, UV spectrophotometry and (19)F NMR spectrometry, the UV assay being used to validate the (19)F NMR assay. A study of the binding of 5'dFUrd to human serum albumin was also reported. The reliability of (19)F NMR as a technique to measure directly the binding of the drug and as an assay after equilibrium dialysis was demonstrated. The percentage of 5'dFUrd bound to plasma proteins is low and concentration-dependent in the 0.04-3.5 mmol l(-1) range.

19F NMR spectrometry evidence for bile acid conjugates of alpha-fluoro-beta-alanine as the main biliary metabolites of antineoplastic fluoropyrimidines in humans.

The human biliary excretion of antineoplastic fluoropyrimidines was studied using 19F NMR. This method allows a direct detection of all the fluorinated metabolites of a fluorinated drug and requires no labeled compound. From a patient with an external bile derivation, treated with 5'-deoxy-5-fluorouridine (5'dFUrd), the biliary excretion of 5'dFUrd metabolites was low (0.8% of the injected dose) and made up of alpha-fluoro-beta-alanine (FBAL) and fluoride ion (F-) which represented approximately equal to 10% of the excreted metabolites and approximately equal to 90% of unknown metabolites. These unknown metabolites were conjugates of FBAL with the two "primary" bile acids only present in the bile of patients with an external bile drainage, i.e. cholic and chenodeoxycholic acids, in a 3:1 ratio. In the bile obtained at surgery from a patient treated with intrahepatic 5-fluorouracil, the major metabolites were conjugates of FBAL with the three major bile acids of human bile, i.e. cholic, deoxycholic, and chenodeoxycholic acids. Moreover, 19F NMR showed that only one of the two diastereoisomers of each conjugate of FBAL with bile acids was formed in vivo, confirming that metabolic FBAL is optically active.