Synthesis, bioanalysis and pharmacology of nucleoside and nucleotide analogs

Abstract

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.