The Analytical Chemistry of Anti-AIDS Agents

该项目的目的是研究和开发合适的生物分析方法，以：(1)确定潜在的抗艾滋病药物和新型抗病毒药物的结构和纯度；(2)确定这些化合物及其代谢物的物理、化学和生化性质，包括辛醇-水分配系数；(3)在生物样品中测定这些药物及其代谢物，以阐明药理学和确定药代动力学。高效液相色谱（HPLC）、毛细管电泳（CE）和质谱分析是重点技术。II期药物2'-b-氟-2',3'-二脱氧腺苷（F-ddA, lodenosine）由于其高口服生物利用度和通过腺苷脱氨酶（ADA）快速代谢为抗hiv活性代谢物2'-b-氟-2',3'-二脱氧腺苷（F-ddI）而继续作为一个感兴趣的化合物。我们的生理药代动力学模型是用F-ddA作为模型化合物来研究选定的ada激活的F-ddI前药的处置，现已扩展到包括口服给药。该模型被用于研究各种生理生化过程的影响，重点是胃肠道吸收、血脑屏障进入中枢神经系统和代谢激活。在大鼠、猴子和人类中，静脉注射和口服F-ddA后血浆和大脑中的浓度数据被用于模型验证和物种间缩放。研究了2'-氟-2'-脱氧腺苷（2'-F-dA）的毒性、代谢和生化药理学，以确定该微量成分在洛地甘苷毒性谱中的作用。使用MOLT-4人淋巴细胞的研究表明，2'-F-dA比洛地甘氨酸具有更强的细胞毒性，即使微量（1%）的2'-F-dA也会迅速磷酸化并并入DNA。可获得的散装洛地甘样品中2′-F-dA的测量浓度不超过0.15%。对这种洛地甘的痕量成分的评价仍在继续。

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