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) and mass spectrometry are the emphasized techniques. The Phase II drug 2- b-fluoro-2,3-dideoxyadenosine (F-ddA, lodenosine) and its deaminated anti-HIV-active metabolite 2-b-fluoro-2,3-dideoxyinosine (F-ddI) remain the compounds of primary interest. Validated analytical strategies employing reversed-phase HPLC are being applied for both the routine and ultrasensitive measurement of F-ddA in HIV-infected human biological fluids. The human metabolism, distribution and pharmacokinetics of oral F-ddA, in both capsule and liquid formulation, have been determined in conjunction with a Phase I clinical trial of this agent in adult AIDS patients. Oral F-ddA given as a component of combination antiretroviral therapy is fully bioequivalent with F-ddA given as monotherapy. Direct fluorogenic derivatization of cellular extracts in conjunction with paired-ion HPLC is being employed for the nonradiochemical measurement of low picomole amounts of intracellular F-ddATP, the active metabolite of both F-ddA and F-ddI. F-ddATP levels are being measured in periperal blood mononuclear cells from patients treated with F-ddA and will be correlated with observed anti-HIV activity. Lipophilic prodrugs of F-ddI activated by adenosine deaminase (ADA) also continue under investigation as potential agents for the treatment of HIV sequestered in the central nervous system (CNS). HPLC methodology for the rapid measurement of F-ddI progrugs, such as 6- chloro-2,3-dideoxypurineriboside, and their metabolites in biological fluids and tissues is being developed and applied. A physiological pharmacokinetic model has been constructed to study the disposition of selected ADA-activated F-ddI prodrugs by using F-ddA as a model compound. This model is being used to investigate the effects of various physiological and biochemical processes with emphasis on prodrug gastrointestinal absorption, blood-brain-barrier penetration into the CNS, and metabolic activation. Concentration versus time 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.AIDS Title: The Analytical Chemistry of Anti-AIDS Agents - adenosine deaminase, AIDS, fluorogenic derivatization, liquid chromatography, pharmacokinetics, prodrugs, 2'-beta-fluoro-2',3'- dideoxyadenosine, - Human Tissues, Fluids, Cells, etc.
