The objective of this project is the research and development of suitable bioanalytical methods to: (1) establish the structure and purity of potential anti-AIDS agents and new antiviral drugs, (2) determine the physical, chemical and biochemical properties, including octanol-water partition coefficients, of these compounds and their metabolites, and (3) measure these drugs and their metabolites in biological samples to elucidate pharmacology and to determine pharmacokinetics. High-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and mass spectrometry are the emphasized techniques. The Phase II drug 2'-b -fluoro-2',3'-dideoxyadeonsine (F-ddA, lodenosine) continues as a compound of interest because of its high oral bioavailability and its rapid metabolism by adenosine deaminase (ADA) to the anti-HIV-active metabolite 2'-b-fluoro-2',3'-dideoxyinosine (F-ddI). Our physiological pharmacokinetic model, that was constructed using F-ddA as a model compound to study the disposition of selected ADA-activated F-ddI prodrugs, has been extended to include oral dosing. This model is being used to investigate the effects of various physiological and biochemical process with emphasis on gastrointestinal absorption, blood-brain-barrier penetration into the CNS, and metabolic activation. Concentration data in plasma and brain following intravenous and oral administration of F-ddA in rats, monkeys and humans are being used for model validation and interspecies scaling. The toxicity, metabolism and biochemical pharmacology of 2'-fluoro-2'-deoxyadenosine (2'-F-dA) have been investigated to determine the role of this trace constituent in the toxicity profile of lodenosine. Studies using MOLT-4 human lymphocytes have shown that 2'-F-dA is more cytotoxic than lodenosine and that even trace amounts (1%) of 2'-F-dA are rapidly phosphorylated and incorporated into DNA. Measured concentrations of 2'-F-dA in available samples of bulk lodenosine did not exceed 0.15%. Evaluation of this trace constituent of lodenosine continues. Direct fluorogenic derivatization of cellular extracts in conjunction with paired-ion HPLC has been employed for the nonradiochemical determination of sub- and low picomole amounts of intracellular F-ddATP, the active metabolite of both F-ddA and F-ddI. F-ddATP can be measured in peripheral blood mononuclear cells from patients treated with F-ddA, but sufficient data is not available to correlate with observed anti-HIV activity. The development of methods using capillary electrophoresis to measure intracellular nucleotide pools and metabolites has been initiated. Large-volume sample stacking has been quantitatively evaluated in terms of linearity, reproducibility and chromatographic resolution for the CE analysis of nucleotides. Signal enhancements of up to 160-fold could be achieved for the analysis of nucleotides using this technique although there was some loss in electrophoretic resolution. Sample stacking has been applied to characterize the minor components of synthetic nucleotide mixtures and to profile endogenous intracellular nucleotides in cultured MOLT-4 lymphocytes. Ongoing research is currently directed toward the application of CE for the determination of intracellular nucleoside drug metabolism and to interfacing CE with mass spectrometry for structural analysis. AIDS Title: The Analytical Chemistry of Anti-AIDS Agents
