Pulsed-field gradient nuclear magnetic resonance measurements (PFG NMR) for diffusion ordered spectroscopy (DOSY) mapping.

While NMR is the most used analytical method for determining the molecular structure of isolated chemical entities, small compounds as well as macromolecules, its capability of analysing complex mixtures is less known. The advent of Diffusion Ordered SpectroscopY (DOSY) NMR has made diffusion experiments popular, enabling diffusion coefficients to be routinely measured and used to characterize chemical systems in solution. Indeed, since the translational diffusion coefficients of molecular species reflect their effective sizes and shapes, DOSY NMR allows the separation of the chemical entities present in multicomponent systems and, as in all diffusion NMR experiments, provides information on their intermolecular interactions as well as on their size and shape. The main aim of this review is to present an overview of the DOSY NMR mapping and its applications. The paper starts with a brief introduction to pulsed-field gradient (PFG) NMR and then focuses on the methodological procedures that can be used to perform good diffusion data acquisition and to obtain good-quality DOSY maps. The second part describes, through selected literature examples, different applications of DOSY NMR to demonstrate the potential of the method for (i) unravelling the components of complex matrices comprising pharmaceuticals, dietary supplements, foods and beverages, and biological extracts, and (ii) probing intermolecular interactions and evaluating association constants between different hosts and guests, as well as estimating the sizes and molecular weights of molecular species.

Green microparticles based on a chitosan/lactobionic acid/linoleic acid association. Characterisation and evaluation as a new carrier system for cosmetics.

The association chitosan/linoleic acid/lactobionic acid in aqueous solution spontaneously led to the formation of stable microparticles with a liquid hydrophobic core consisting of linoleic acid surrounded by a shell of chitosan/lactobionic acid. The originality of the microparticles arises from the fact that they are formed by the association of three ingredients of cosmetic interest, including a skin penetration enhancer (linoleic acid). Dynamic light scattering (DLS) measurements showed microparticles with a mean diameter of 1-2 µm. The presence of a hydrophobic liquid core was observed by transmission electron microscopy (TEM). The ability of these microparticles to encapsulate phenylethyl resorcinol, a hydrophobic skin lightener, was evaluated and its encapsulation was confirmed thanks to T2 measurements and nuclear Overhauser effects (nOe) signs.

pH optimization for a reliable quantification of brain tumor cell and tissue extracts with (1)H NMR: focus on choline-containing compounds and taurine.

The aim of this study was to define the optimal pH for (1)H nuclear magnetic resonance (NMR) spectroscopy analysis of perchloric acid or methanol-chloroform-water extracts from brain tumor cells and tissues. The systematic study of the proton chemical shift variations as a function of pH of 13 brain metabolites in model solutions demonstrated that recording (1)H NMR spectra at pH 10 allowed resolving resonances that are overlapped at pH 7, especially in the 3.2-3.3 ppm choline-containing-compounds region. (1)H NMR analysis of extracts at pH 7 or 10 showed that quantitative measurements of lactate, alanine, glutamate, glutamine (Gln), creatine + phosphocreatine and myo-inositol (m-Ino) can be readily performed at both pHs. The concentrations of glycerophosphocholine, phosphocholine and choline that are crucial metabolites for tumor brain malignancy grading were accurately measured at pH 10 only. Indeed, the resonances of their trimethylammonium moieties are cleared of any overlapping signal, especially those of taurine (Tau) and phosphoethanolamine. The four non-ionizable Tau protons resonating as a singlet in a non-congested spectral region permits an easier and more accurate quantitation of this apoptosis marker at pH 10 than at pH 7 where the triplet at 3.43 ppm can be overlapped with the signals of glucose or have an intensity too low to be measured. Glycine concentration was determined indirectly at both pHs after subtracting the contribution of the overlapped signals of m-Ino at pH 7 or Gln at pH 10.

Counterfeit drugs: analytical techniques for their identification.

In recent years, the number of counterfeit drugs has increased dramatically, including not only "lifestyle" products but also vital medicines. Besides the threat to public health, the financial and reputational damage to pharmaceutical companies is substantial. The lack of robust information on the prevalence of fake drugs is an obstacle in the fight against drug counterfeiting. It is generally accepted that approximately 10% of drugs worldwide could be counterfeit, but it is also well known that this number covers very different situations depending on the country, the places where the drugs are purchased, and the definition of what constitutes a counterfeit drug. The chemical analysis of drugs suspected to be fake is a crucial step as counterfeiters are becoming increasingly sophisticated, rendering visual inspection insufficient to distinguish the genuine products from the counterfeit ones. This article critically reviews the recent analytical methods employed to control the quality of drug formulations, using as an example artemisinin derivatives, medicines particularly targeted by counterfeiters. Indeed, a broad panel of techniques have been reported for their analysis, ranging from simple and cheap in-field ones (colorimetry and thin-layer chromatography) to more advanced laboratory methods (mass spectrometry, nuclear magnetic resonance, and vibrational spectroscopies) through chromatographic methods, which remain the most widely used. The conclusion section of the article highlights the questions to be posed before selecting the most appropriate analytical approach.

Analysis of hydrophilic and lipophilic choline compounds in radioresistant and radiosensitive glioblastoma cell lines by HILIC-ESI-MS/MS.

A new method based on hydrophilic interaction chromatography-electrospray ionisation-tandem mass spectrometry (HILIC-ESI-MS/MS) coupled to the use of a stable isotope labelled substrate was developed to study the metabolism of choline (Cho) compounds in two human glioblastoma multiform (GBM) cell lines with different responses to ionising radiation. Analysis was performed in the positive ion mode using multiple reaction monitoring. This fast, sensitive and selective method enabled the profiling of both hydrophilic and lipophilic Cho-containing compounds, to analyse specifically different phosphatidylcholine (PtdCho) molecular species, and to measure simultaneously native and labelled Cho metabolites. Radioresistant (SF763) and radiosensitive (SF767) cells were incubated for 8 h with d(9)-Cho. Higher native Cho and phosphocholine (PCho) concentrations and higher uptake of d(9)-Cho and formation of d(9)-PCho were found in the radioresistant cell line. The similar low concentrations of native cytidine 5'-diphosphocholine (CDP-Cho) and d(9)-CDP-Cho in both cell lines show that CDP-Cho is the limiting metabolite in the two models. The turnovers (percentage of each d(9)-Cho compound in its respective pool, i.e. native + labelled) were lower in radioresistant cells for all Cho compounds, suggesting a global PtdCho metabolism more active in radiosensitive cells that could be related to their higher proliferation rate.

Delivery systems for the treatment of proliferative vitreoretinopathy: materials, devices and colloidal carriers.

Proliferative vitreoretinopathy (PVR), the most serious complication causing retinal detachment surgery to fail, is one of the leading causes of vision-loss in developed countries. The pharmaceutical treatment of this disease, located in the posterior segment of the eye, is problematic because it is difficult to achieve effective drug levels in the vitreous and the retina through conventional forms of administration (topical or systemic). Intravitreal injections can deliver drugs to the retina without the side-effects associated with systemic administration. However, because PVR is a long-term complication and the half-life of most drugs in the vitreous cavity is short, repeated injections are needed but this can cause complications. Recent advances in ocular drug delivery methods and the development of novel bioactive compounds could lead to new ways for the treatment of PVR. This review will summarize recent literature concerning intraocular drug delivery of biopharmaceutical agents for the treatment and prevention of PVR.

Evidence for the importance of 5'-deoxy-5-fluorouridine catabolism in humans from 19F nuclear magnetic resonance spectrometry.

The use of a new methodology, 19F nuclear magnetic resonance, has allowed detection of all the fluorinated metabolites in the biofluids of patients treated with 5'-deoxy-5-fluorouridine (5'-dFUrd) injected i.v. at a dose of 10 g/m2 over 6 h. 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 identification and quantitation of all the different fluorinated metabolites. As well as the already known metabolites, unmetabolized 5'-dFUrd, 5-fluorouracil, and 5,6-dihydro-5-fluorouracil, the presence of alpha-fluoro-beta-ureidopropionic acid, alpha-fluoro-beta-alanine (FBAL), N-carboxy-alpha-fluoro-beta-alanine, and the fluoride anion F- is reported. The catabolic pathway proposed for 5'-dFUrd is analogous to that of 5-fluorouracil, completed with FBAL----F- step, and the plasmatic equilibrium of FBAL with N-carboxy-alpha-fluoro-beta-alanine, its N-carboxy derivative. The quantitative analysis of the different metabolites found in plasma and urine emphasizes the significance of the catabolic pathway. High concentrations of alpha-fluoro-beta ureidopropionic acid and FBAL are recovered in plasma from 3 h after the beginning of the perfusion to 1 h after its end. The global urinary excretion results show that there is a high excretion of 5'-dFUrd and metabolites. Unchanged 5'-dFUrd and FBAL are by far the major excretory products and are at nearly equal rates. The protocol followed in this study produces relatively low but persistent plasmatic concentrations of 5-fluorouracil throughout the perfusion.

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.

Fluorine nuclear magnetic resonance, a privileged tool for metabolic studies of fluoropyrimidine drugs.

Fluorine-19 nuclear magnetic resonance (19F NMR) spectroscopy provides a highly specific tool for identifying fluorine-containing drugs and their metabolites in biological media. This article focuses on the application of 19F NMR to the metabolic studies of fluoropyrimidine drugs in clinical use. The value and difficulties encountered in investigations on drug metabolism are first discussed. The metabolism and disposition studies of the anticancer drug 5-fluorouracil, the mainstay of antimetabolite treatment for solid tumors, and its prodrugs, doxifluridine and capecitabine, are then extensively reviewed. The studies dealing with the antimycotic agent, 5-fluorocytosine, as well as the novel anticancer drug, gemcitabine, are also considered. From in vitro (biofluids or tissue extracts) 19F NMR analysis, seven new metabolites of 5-fluorouracil, doxifluridine, capecitabine and 5-fluorocytosine were identified. Except two, they were only detected using this technique. This emphasizes the high analytical potential of in vitro 19F NMR. In vivo 19F NMR is non-invasive and thus allows the quantitative monitoring of the metabolism of 5-fluorouracil in the target tissue, e.g. the tumor, as well as its biodistribution. Another promising application is its ability to estimate the level of yeast cytosine deaminase gene expression in human tumors from the quantitative monitoring of 5-fluorouracil formation from the non-cytotoxic drug 5-fluorocytosine. Notwithstanding these successes, the limited sensitivity and spectral resolution of 19F NMR precludes its extensive applicability to all the fluorinated drugs.

The prodrugs of 5-fluorouracil.

Although 5-fluorouracil (FU) was first introduced in 1957, it remains an essential part of the treatment of a wide range of solid tumors. FU has antitumor activity against epithelial malignancies arising in the gastrointestinal tract, breast as well as the head and neck, with single-agent response rates of only 10-30%. Although FU is still the most widely prescribed agent for the treatment of colorectal cancer, less than a third of patients achieve objective responses. Recent research has focused on the biomodulation of FU to improve the cytotoxicity and therapeutic effectiveness of this drug in the treatment of advanced disease. As all the anticancer agents, FU leads to several toxicities. The toxicity profile of FU is schedule dependent. Myelotoxicity is the major toxic effect in patients receiving bolus doses. Hand-foot syndrome (palmar-plantar erythrodysesthesia), stomatitis, neuro- and cardiotoxicity are associated with continuous infusions. Other adverse effects associated with both bolus-dose and continuous infusion regimens include nausea and vomiting, diarrhea, alopecia and dermatitis. All these reasons explain the need of more effective and less toxic fluoropyrimidines. In the first part of the review, we briefly present the metabolic pathways of FU responsible for the efficacy and toxicity of the drug. This knowledge is also necessary to understand the target(s) of the biomodulation. The second part is devoted to a review of the literature on the various prodrugs of FU, including 5'-deoxy-5-fluorouridine, capecitabine, BOF-A2, ftorafur, UFT, and S-1. The promising approach of gene directed enzyme-prodrug therapy is also presented. A brief survey of antibody directed enzyme-prodrug therapy and some new FU prodrugs concludes the paper. The pharmacological principles that have influenced the development of these new drugs and our current knowledge of the clinical pharmacology of these new agents, focusing on antitumor activity and toxicity, are presented.

The analysis of cyclophosphamide and its metabolites.

Cyclophosphamide (CP) has been in clinical use for the treatment of malignant disease for over 40 years. CP is inactive until it undergoes complex metabolic pathways leading to the ultimate alkylating agent, phosphoramide mustard, but also to inactive and toxic metabolites. Sensitive and specific methods are now available for the measurement of CP and its enantiomers, its metabolites and their stereoisomers, in biological matrices. An overview is given of the methods of analysis of CP and its metabolites described in literature since 1993 as well as the current knowledge about its metabolism. Five classes of methods are described: (1) thin-layer chromatography-photographic densitometry, (2) high performance liquid chromatography, (3) gas chromatography and gas chromatography coupled to mass spectrometry, (4) phosphorus-31 nuclear magnetic resonance and (5) enantiomeric separation. In each case, sample clean up and preparation are described. Precision and limits of quantification of the assays are indicated. A table summarizes all the analytical methods for assaying each metabolite.

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.

Chemical stability and fate of the cytostatic drug ifosfamide and its N-dechloroethylated metabolites in acidic aqueous solutions.

31P NMR spectroscopy was used to study the products of the decomposition of the antitumor drug ifosfamide (IF, 1d) and its N-dechloroethylated metabolites, namely, 2,3-didechloroethylIF (1a) and 2- (1b) and 3-dechloroethylIF (1c), in buffered solutions at acidic pH. The first stage of acid hydrolysis of these four oxazaphosphorines is a P-N bond cleavage of the six-membered ring leading to the phosphoramidic acid monoesters (2a-d) of type R'HN(CH(2))(3)OP(O)(OH)NHR, with R and/or R' = H or (CH(2))(2)Cl. The electron-withdrawing chloroethyl group at the endocyclic and/or exocyclic nitrogens counteracts the endocyclic P-N bond hydrolysis. This effect is even more marked when the N-chloroethyl group is in the exocyclic position since the order of stability is 1d > 1c > 1b > 1a. In the second stage of hydrolysis, the remaining P-N bond is cleaved together with an intramolecular attack at the phosphorus atom by the non-P-linked nitrogen of the compounds 2a-d. This leads to the formation of a 2-hydroxyoxazaphosphorine ring with R = H (3a coming from compounds 2a,c) or (CH(2))(2)Cl (3b coming from compounds 2b,d) and to the release of ammonia or chloroethylamine. The third step is the P-N ring opening of the oxazaphosphorines 3a,b leading to the phosphoric acid monoesters, H(2)N(CH(2))(3)OP(O)(OH)(2) (4a) and Cl(CH(2))(2)HN(CH(2))(3)OP(O)(OH)(2) (4b-1), respectively. For the latter compound, the chloroethyl group is partially (at pH 5.5) or totally (at pH 7.0) cyclized into aziridine (4b-2), which is then progressively hydrolyzed into an N-hydroxyethyl group (4b-3). Compounds 3a,b are transient intermediates, which in strongly acidic medium are not observed with (31)P NMR. In this case, cleavage of the P-N bond of the type 2 phosphoramidic acid monoesters leads directly to the type 4 phosphoric acid monoesters. The phosphate anion, derived from P-O bond cleavage of these latter compounds, is only observed at low levels after a long period of hydrolysis. Compounds 1a-c and some of their hydrolytic degradation products (4b-1, 4b-2, diphosphoric diester [Cl(CH(2))(2)NH(CH(2))(3)OP(O)(OH)](2)O (5), and chloroethylamine) did not exhibit, as expected, any antitumor efficacy in vivo against P388 leukemia. (31)P NMR determination of the N-dechloroethylated metabolites of IF or its structural isomer, cyclophosphamide (CP), and their degradation compounds could provide an indirect and accurate estimation of chloroacetaldehyde amounts formed from CP or IF.

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.

Characterization of choline compounds with in vitro 1H magnetic resonance spectroscopy for the discrimination of primary brain tumors.

RATIONALE AND OBJECTIVES: The authors sought to compare 1H magnetic resonance spectroscopy (MRS) spectra from extracts of low-grade and high-grade gliomas, especially with respect to the signals of choline-containing compounds. METHODS: Perchloric acid extracts of six high-grade and six low-grade gliomas were analyzed by 1H MRS at 9.4 Tesla. RESULTS: The signals of glycerophosphocholine (GPC) at 3.23 ppm, phosphocholine (PC) at 3.22 ppm, and choline (Cho) at 3.21 ppm were identified in both types of tumors. The absolute concentrations of all Cho-containing compounds (GPC + PC + Cho) in high-grade and low-grade gliomas were significantly different. The relative contributions of each of the Cho-containing compounds to the total choline signal were also statistically different. For high-grade gliomas, the choline signal is composed of GPC, PC, and Cho in a well-balanced contribution, whereas in low-grade gliomas, the signal is largely due to GPC with a small involvement of PC and Cho. CONCLUSIONS: The differences in the concentration and the repartition of Cho-containing compounds seem to be a marker of high-grade gliomas. They could also help to discriminate between high- and low-grade gliomas in some difficult cases, especially if there is histologic uncertainty between anaplastic astrocytomas and low-grade oligodendrogliomas.

Urinary stability of carboxycyclophosphamide and carboxyifosfamide, two major metabolites of the anticancer drugs cyclophosphamide and ifosfamide.

Phosphorus-31 nuclear magnetic resonance spectroscopy was used to evaluate the stability of carboxycyclophosphamide (CXCP) and carboxyifosfamide (CXIF) in human urine at pH 7.0 and 5.5 at 25 degrees, 8 degrees, -20 degrees, and -80 degrees C. At 25 degrees C and pH 7.0, CXCP and CXIF are relatively stable (approximately 10% degradation in 24 h). In contrast, they are much less stable at pH 5.5 (approximately 80% degradation of CXIF and approximately 50% degradation of CXCP in 24 h). The rate of degradation of CXCP and CXIF was a function of the storage temperature of the urine samples but, even at -80 degrees C, was not negligible: approximately 30% degradation for CXCP irrespective of pH and approximately 40% and 50% degradation for CXIF at pH 7.0 and 5.5, respectively, after storage for 6 months. CXCP was more stable than CXIF at either pH (7.0 or 5.5) and at all storage temperatures (8 degrees, -20 degrees, or -80 degrees C) of the urine samples. CXCP and CXIF were more stable at pH 7.0 than at pH 5.5, although this difference fell with decreasing temperatures to be almost negligible at -80 degrees C. To ensure a true estimate of CXCP and CXIF levels, urine samples must be frozen and stored at -80 degrees C within a few hours of micturition. CXCP and CXIF assays should also be carried out within 2 months and 1 month of storage, respectively.

Noninvasive fluorine-19 NMR study of fluoropyrimidine metabolism in cell cultures of human pancreatic and colon adenocarcinoma.

Fluorine-19 NMR spectrometry was used to monitor the metabolism of two antineoplastic fluoropyrimidines, 5-fluorouracil (5FU) and 5'-deoxy-5-fluorouridine (5'dFUrd), in cell cultures of human pancreatic (Capan-1) and colon (HT-29) adenocarcinoma. The preliminary results showed, for the two tumor cell lines treated with 5FU, the presence in nonperfused cells of three signals corresponding to intracellular metabolites: 5FU, F-nucleotides and F-nucleosides. When the cells were perfused only the signals of F-nucleotides and 5FU were present. The F-nucleosides observed during the analysis of the nonperfused cells came from the conversion of F-nucleotides. During the NMR recording of Capan-1 cells at 37 degrees C the first metabolite of the catabolic pathway of 5FU, 5,6-dihydro-5-fluorouracil, occurred. At the beginning of the NMR recording of Capan-1 cells treated with 5'dFUrd, two signals corresponding to F-nucleotides and F-nucleosides (consistent with 5'dFUrd) were observed; during the analysis, a supplementary signal corresponding to 5FU appeared. Even after pretreatment with methotrexate the signal of 5FU incorporated into RNA was not detected. Our experiments, performed in attempts to observe the signal of the ternary complex between thymidylate synthetase (TS), 5-fluoro-2'-deoxyuridine-5'-monophosphate (FdUMP) and 5,10-methylene-tetrahydrofolate (5,10-CH2FH4), allowed detection in some cases of a broad signal, whose chemical shift was similar to that reported in the literature following incubation of TS with FdUMP and 5,10-CH2FH4, but our results were not always reproducible.

Stability of commercial solutions of 5-fluorouracil for continuous infusion in an ambulatory pump.

PURPOSE: The stability of 5-fluorouracil (FU) Roche solutions in a portable infusion pump under prolonged "in-use" conditions (32 degrees C, in the dark) was studied, especially with respect to the formation of the cardiotoxic compounds fluoroacetaldehyde (Facet) and fluoromalonic acid semialdehyde (FMASAld). METHODS: The solutions, prepared according to three protocols frequently used at the Anticancer Centre in Toulouse, were analysed by 19F NMR immediately after preparation (T0) and after 2, 3 or 10 days (TF) in the pump. RESULTS: The commercial solution already contained 64 fluorinated "impurities", among them fluoride ion (F-), FMASAld and Facet. The concentration of FU did not change significantly between T0 and TF, whatever the protocol. The levels of F- had not increased significantly after 2 or 3 days, but had increased by about 50% after 10 days. The increases in FMASAld levels were low (12-28%) albeit significant in the three protocols. The levels of Facet had increased by a factor of about 2 after 2 or 3 days, and by a factor of > 3 after 10 days. The levels of the other fluorinated compounds were constant during the first 2 or 3 days, but had increased by about 30% after 10 days. FU Dakota lyophilizates, analysed immediately after reconstitution, contained neither FMASAld nor Facet. After 2 days at 25 degrees C, low levels of FMASAld were present but Facet could still not be detected. CONCLUSION: This study showed that special attention must be paid to the risk of increasing concentrations of highly toxic FMASAld and Facet when FU is administered via a pump for long periods of time. It would be preferable not to exceed 3 days of treatment when patients receive FU from a portable infusion pump. This underlines the interest in using a lyophilized formulation of FU in clinical practice.

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.

New approach to metabolism of 5'-deoxy-5-fluorouridine in humans with fluorine-19 NMR.

The metabolism of 5'-deoxy-5-fluorouridine (5'dFUrd), an antitumor fluoropyrimidine, has been investigated in human biofluids (blood, plasma, urine) using a new method: fluorine-19 NMR spectrometry. This method allows direct study of the biological sample and simultaneous identification of all the fluorinated metabolites. In the blood of a patient treated with 5'dFUrd during a 6-h continuous perfusion, we observed unmetabolized 5'dFUrd, 5-fluorouracil, 5,6-dihydrofluorouracil, and another metabolite which has not previously been reported alpha-fluoro-beta-alanine. The two major metabolites in urine are unmetabolized 5'dFUrd and alpha-fluoro-beta-alanine.