Decomposition of 3'-azido-2',3'-dideoxythymidine 5'-monophosphate (AZTMP) prodrugs in biological media studied by on-line solid-phase extraction coupled to liquid chromatography mass spectrometry.

Using a column-switching HPLC method previously described, we studied the behavior of some mononucleotide prodrugs (pronucleotides) of 3'-azido-2',3'-dideoxythymidine in various biological media. From UV data, this method allowed quantification of transient metabolites resulting from prodrug bioconversion. The kinetic data related to the successive steps were calculated according to pseudo-first-order kinetic models and optimized using mono- or poly-exponential regressions. Various metabolites were identified by co-injection with authentic samples and/or ESI-MS coupling. The results led us to propose, for each considered pronucleotide, a global decomposition pathway ending in the selective delivery of the corresponding mononucleotide. Associated to the determination of other parameters (lipophilicity, aqueous solubility), the present study contributes to the search of suitable pharmacological properties for further in vivo evaluations.

Pharmacology and pharmacokinetics of the antiviral agent beta-D-2',3'-dideoxy-3'-oxa-5-fluorocytidine in cells and rhesus monkeys.

Beta-D-2',3'-dideoxy-3'-oxa-5-fluorocytidine (D-FDOC) is an effective inhibitor of human immunodeficiency virus 1 (HIV-1) and HIV-2, simian immunodeficiency virus, and hepatitis B virus (HBV) in vitro. The purpose of this study was to evaluate the intracellular metabolism of d-FDOC in human hepatoma (HepG2), human T-cell lymphoma (CEM), and primary human peripheral blood mononuclear (PBM) cells by using tritiated compound. By 24 h, the levels of D-FDOC-triphosphate (D-FDOC-TP) were 2.8 +/- 0.4, 6.7 +/- 2.3, and 2.0 +/- 0.1 pmol/10(6) cells in HepG2, CEM, and primary human PBM cells, respectively. Intracellular D-FDOC-TP concentrations remained greater than the 50% inhibitory concentration for HIV-1 reverse transcriptase for up to 24 h after removal of the drug from cell cultures. In addition to d-FDOC-monophosphate (D-FDOC-MP), -diphosphate (D-FDOC-DP), and -TP, D-FDOC-DP-ethanolamine and d-FDOC-DP-choline were detected in all cell extracts as major intracellular metabolites. D-FDOC was not a substrate for Escherichia coli thymidine phosphorylase. No toxicity was observed in mice given D-FDOC intraperitoneally for 6 days up to a dose of 100 mg/kg per day. Pharmacokinetic studies in rhesus monkeys indicated that D-FDOC has a t(1/2) of 2.1 h in plasma and an oral bioavailability of 38%. The nucleoside was excreted unchanged primary in the urine, and no metabolites were detected in plasma or urine. These results suggest that further safety and pharmacological studies are warranted to assess the potential of this nucleoside for the treatment of HIV- and HBV-infected individuals.

Metabolism of the anti-hepatitis C virus nucleoside beta-D-N4-hydroxycytidine in different liver cells.

Beta-D-N4-hydroxycytidine (NHC) was found to have selective anti-hepatitis C virus (HCV) activity in the HCV replicon system (clone A). The intracellular metabolism of tritiated NHC was investigated in the HCV replicon system, Huh-7 cells, HepG2 cells, and primary human hepatocytes. Incubation of cells with 10 microM radiolabeled NHC demonstrated extensive and rapid phosphorylation in all liver cells. Besides the 5'-mono, -di-, and -triphosphate metabolites of NHC, other metabolites were characterized. These included cytidine and uridine mono-, di-, and triphosphates. UTP was the predominant early metabolite in Huh-7 cells and primary human hepatocytes, suggesting deamination of NHC as the primary catabolic pathway. The intracellular half-lives of radiolabeled NHC-triphosphate and of CTP and UTP derived from NHC incubation in Huh-7 cells were calculated to be 3.0 +/- 1.3, 10.4 +/- 3.3, and 13.2 +/- 3.5 h (means +/- standard deviations), respectively. Studies using monkey and human whole blood demonstrated more-rapid deamination and oxidation in monkey cells than in human cells, suggesting that NHC may not persist long enough in plasma to be delivered to liver cells.

Nucleoside inhibitors of human immunodeficiency virus type 1 reverse transcriptase.

The development of novel compounds that can effectively inhibit both wild type and the most consensus resistant strains of human immunodeficiency virus type 1 (HIV-1) is the primary focus in HIV disease management. Combination therapy, comprising at least three classes of drugs, has become the standard of care for acquired immunodeficiency syndrome (AIDS) or HIV-infected individuals. The drug cocktail can comprise all three classes of HIV inhibitors, including nucleoside reverse transcriptase inhibitors (NRTI), non-nucleoside reverse transcriptase inhibitors (NNRTI) and protease inhibitors (PI). Due to their competitive mode of inhibition and requirement for metabolic activation, almost all NRTI drugs lack the virological potency of NNRTI or PI drugs. However, data from clinical trials indicate that sustained viral suppression could not be achieved with NRTI, NNRTI or PIs alone. Therefore, the NRTIs will remain essential components of highly active antiretroviral therapy (HAART) for the foreseeable future, because they enhance the virological potency of the regimen, they do not bind excessively to protein and most regimens are small pills/tablets given once a day. It has become apparent in recent years that the prolonged use of certain NRTIs exhibits adverse events as a class, limiting the length of time for which they can be safely used. Of major clinical concern is their association with the potentially fatal lactic acidaemia and hepatic steatosis. These class events, as well as individual drug effects, such as peripheral neuropathy, are linked to delayed mitochondrial destruction. In addition to toxicity, the development of resistance-conferring mutations against exposure to nucleoside analogs currently in use influences long-term therapeutic benefits. Of critical importance for the evaluation of new NRTIs are recent studies showing that the efficiency of discrimination or excision by pyrophosphorolysis in the presence of nucleotides of a given NRTI is a key determinant in the emergence of one or the other resistance pathway.