ReviewFluorine nuclear magnetic resonance spectroscopy of human biofluids in the field of metabolic studies of anticancer and antifungal fluoropyrimidine drugs
Introduction
Nuclear Magnetic Resonance spectroscopy (NMR) is the study of molecules by recording the interaction of radiofrequency electromagnetic radiation with the magnetically 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 5-fluorouracil (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.
Section snippets
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
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
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,
Capecitabine (CAP)
N4-Pentyloxycarbonyl-5′-deoxy-5-fluorocytidine, more commonly called capecitabine (CAP) or Xeloda®, is a recent prodrug of 5′dFUR, another FU prodrug, that is administered orally to circumvent the unacceptable toxicity of 5′dFUR without compromising its antitumor efficacy [47]. Since the main limitation of 5′dFUR 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
Conclusion
Despite its limited sensitivity in the low micromolar range, 19F 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:
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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
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