Liquid chromatography/tandem mass spectrometry method for simultaneous quantification of eight endogenous nucleotides and the intracellular gemcitabine metabolite dFdCTP in human peripheral blood mononuclear cells
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Herein, we describe a one‐pot synthesis, in a mixture of water/acetonitrile, of nucleoside 5′‐polyphosphates and some derivatives starting from their corresponding nucleoside 5′‐monophosphates. Phosphorimidazolide intermediates are formed in the presence of imidazole and 2‐chloro‐1,3‐dimethylimidazolinium hexafluorophosphate. Under these mild conditions, nucleoside 5′‐di‐ and 5′‐triphosphates, dinucleoside 5′,5′‐polyphosphates, as well as some nucleotide analogues modified either on the nucleoside or on the phosphate moieties are obtained in moderate to high yields.
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Background: Modified nucleoside and nucleotide analogs are now the cornerstone of antiviral and anticancer chemotherapies. However, these compounds are not active on their own and need, after entering the cell, to be metabolized to their active 5'-triphosphate form. Methods: Limitations of these metabolic processes led to development of nucleoside/nucleotide prodrugs in which nucleosides are masked with different groups that can be intracellularly cleaved either chemically or enzymatically. Results: Several prodrug approaches have been successfully developed in order to increase the efficacy, bioavailability, penetration in target organ, and selectivity of nucleoside/nucleotide analogs. Conclusion: The concept of nucleoside/nucleotide prodrug is now a well-established approach that led to the approval of numerous drugs for the treatment of HIV, HBV, HCV, HSV and cancer. Keywords: Antiviral agents, prodrug, nucleoside, enzyme, monophosphate, triphosphate.
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The paper deals with phosphorylation in possible prebiotic nonaqueous solvents. To this end, phosphorylation of nucleosides using inorganic phosphates in amide solutions is studied at room and elevated temperatures. Reaction proceeds most readily in formamide and N-methylformamide. Products obtained at elevated temperature are nucleotides, nucleoside 2',3'-cyclic phosphates, and when the phosphate concentration is high, nucleoside diphosphates. At room temperature, adenosine afforded a mixture of nucleotides, but none of the cyclic nucleotide. Conditions leading to the highest relative percentage of cyclic nucleotide involve the use of low concentrations of phosphate and an excess of nucleoside.
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The kidneys of mice and other small mammals contain relatively high phosphomonoesterase levels. When 5′-nucleotides of 9-β-d-arabinofuranosyladenine and 9-(β-d-arabinofuranosyl)-6-mercaptopurine were administered to mice, they were rapidly dephosphorylated and excreted in the same form and at the same rate as if the nucleosides were given. However, human kidney samples had much lower phosphomonoesterase levels, and when 5′-nucleotides of the 2 nucleoside analogs were administered to patients they were relatively slowly converted to nucleosides and provided sustained blood plasma levels of the nucleosides. This appears to have potential advantages both for providing sustained dosage and for a better dosage formulation, the latter because of the greater solubility of the nucleotides.
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Racemization
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Nucleoside analogs are an important class of drugs in anticancer and antiviral therapy. The compounds are, however, only active after intracellular conversion to their mono-, di- and triphosphate nucleotide form. In this thesis the development of sensitive liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) assays to quantitate nucleoside and nucleotide analogs in cells is described. These assays were then applied to preclinical and clinical studies. Synthesis of nucleotide analogs In chapter 1 the synthesis of small amounts of nucleotide analogs from nucleoside analogs is described. These nucleotide analogs were required as reference and internal standards in quantitative analytical assays. Bioanalysis of intracellular nucleoside and nucleotide analogs First, a literature overview of liquid chromatography - mass spectrometry (LC-MS) methods for the quantitative determination of nucleotide analogs in cells is given (chapter 2.1). The development and validation of an assay for cladribine nucleotides is described in chapter 2.2. The nucleotides were quantified in cells and growth medium. In chapter 2.3, a similar assay was developed and validated for the 6 nucleotide forms of emtricitabine and tenofovir. The method was applied to samples from a patient. The development of a novel chromatographic system for the separation of nucleosides and nucleotides using porous graphitic carbon without ion-pairing agents is outlined in chapter 2.4. In chapter 2.5 this method was applied to biological matrices. We were able to separate the nucleoside and nucleotide forms of the anticancer agent gemcitabine and of its deaminated metabolite (2’,2’-difluorodeoxyuridine; dFdU). The method was validated for the simultaneous quantification of these 8 analytes in white blood cells. Chapter 2.6 compares two common methods for the quantification of the number of white blood cells in a sample. A DNA-based quantitation method was found to be superior over a protein-based method because it was unaffected by red blood cell contamination. This DNA-base method was finally validated. Pharmacology of nucleoside analogs In chapter 3.1 the role of the drug-efflux pump breast cancer related protein (BCRP) in resistance against anti-cancer nucleoside analogs was investigated. It was found that cells that overexpressed this drug-efflux pump were less sensitive to nucleoside analogs. We concluded that BCRP extruded both cladribine and its monophospate from cells. Chapter 3.2 describes a clinical study in which low doses of gemcitabine were orally administered to patients. The exposure to gemcitabine and its triphosphate was very low due extensive first-pass metabolism. A lethal hepatic toxicity observed was possibly related to accumulation of the triphosphate of deaminated gemcitabine, a previously unrecognized metabolite. Chapter 3.3 provides a literature overview on the formation and pharmacological activity of deoxyuridine analog nucleotides during deoxycytidine analog therapy. It is concluded that many deoxycytidine analogs are converted to deoxyuridine nucleotides in cells and that these nucleotides possess pharmacological activity. In chapter 3.4 the nucleotides of decitabine were determined in white blood cells from 3 patients with myelodysplastic syndrome. The decitabine triphosphate levels corresponded to the clinical effect. Accumulation of decitabine triphospate during treatment indicated that a less intensive dosing scheme without hospitalization could be as effective as the current dosing scheme.
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Three general methods for the synthesis of acyl nucleotides (mono-, di-, and triphosphates) have been developed and applied to different HIV inhibitors. These new types of compounds, where a fatty acid moiety is linked to the nucleotide phosphate chain by an acyl phosphate bond, were designed as lipophilic prodrugs of HIV inhibitors metabolites. Acyl nucleoside monophosphates 1a,b were prepared by acylation of the corresponding nucleoside monophosphates. Acyl nucleoside diphosphates 2a−c and 3a,b were synthesized directly from the free nucleosides using DCC activation of acyl pyrophosphates. Acyl nucleoside triphosphates 4a−c and 5a were obtained using phosphoromorpholidate chemistry and acyl pyrophosphates as nucleophiles. Hydrolysis of acyl nucleotides liberated the corresponding nucleotides by selective cleavage of the acyl phosphate bond, with half lives ranging from 51 to 185 h at 37 °C in triethylammonium acetate buffer pH 7.0. Their antiretroviral activity, measured by the inhibition of cytopathogenicity and reverse transcriptase activity in the cultures supernatants, did not reveal any differences between an acyl nucleotide and its corresponding nucleotide. These results are explained in term of rapid aminolysis of the acyl phosphate bond in culture media.
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The cyclic nucleotide forms of 6-mercaptopurine and 6-methylmercaptopurine have been found to be cytotoxic to rat hepatoma cells. Studies with an inhibitor of phosphodiesterase suggest that the cytotoxicity of both cyclic nucleotides results principally from conversion to the 5'-nucleotide. A comparison of the two thiopurine cyclic nucleotides with their nucleoside counterparts has suggested that (a) the thio derivatives act by a common mechanism which is different from that exerted by the methylthio derivatives, and (b) the methylthio cyclic nucleotide acts, at least in part, by a mechanism which differs from that exerted by the methylthio nucleoside.
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Thiopurine methyltransferase
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