The Analytical Chemistry of Anti-AIDS Agents

本项目的目标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剂和新的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、化学和生物化学性质，包括辛醇-水分配系数，以及（3）测量生物样品中的这些药物及其代谢物以阐明药理学并确定药代动力学。高效液相色谱（HPLC）、毛细管电泳（CE）和质谱是重点研究的技术。II期药物2 ′-B -氟-2 ′，3 ′-双脱氧腺苷（F-ddA，碘腺苷）继续作为感兴趣的化合物，因为其高口服生物利用度和其通过腺苷脱氨酶（ADA）快速代谢为抗HIV活性代谢物2 ′-b-氟-2 ′，3 ′-双脱氧肌苷（F-ddI）。我们的生理药代动力学模型，这是使用F-ddA作为模型化合物，研究选定的ADA激活的F-ddI前药的处置，已扩展到包括口服给药。该模型被用于研究各种生理和生化过程的影响，重点是胃肠道吸收，血脑屏障渗透到CNS和代谢活化。在大鼠、猴和人类中静脉内和经口给予F-ddA后血浆和脑中的浓度数据正在用于模型验证和种间缩放。本文研究了2 '-氟-2'-脱氧腺苷（2 '-F-dA）的毒性、代谢和生化药理学，以确定这种微量成分在碘腺苷毒性特征中的作用。使用MOLT-4人淋巴细胞的研究表明，2 '-F-dA比碘腺苷更具细胞毒性，即使是痕量（1%）的2'-F-dA也会迅速磷酸化并掺入DNA中。在散装洛腺苷的可用样品中测得的2 '-F-dA浓度不超过0.15%。对这种微量碘腺苷成分的评价仍在继续。 细胞提取物的直接荧光衍生化结合双离子HPLC已被用于非放射化学测定亚和低皮摩尔量的细胞内F-ddATP，F-ddA和F-ddI的活性代谢产物。F-ddATP可以在接受F-ddA治疗的患者的外周血单核细胞中测量，但没有足够的数据与观察到的抗HIV活性相关。已经开始开发使用毛细管电泳测量细胞内核苷酸池和代谢物的方法。在线性、重现性和色谱分辨率方面对大体积样品堆积进行了定量评价，用于核苷酸的CE分析。信号增强高达160倍，可以实现使用这种技术的核苷酸分析，虽然有一些损失的电泳分辨率。样品堆积已被应用于表征合成核苷酸混合物的次要组分，并在培养的MOLT-4淋巴细胞中分析内源性细胞内核苷酸。目前正在进行的研究是针对应用CE测定细胞内核苷类药物代谢和连接CE与质谱进行结构分析。 抗艾滋病药物的分析化学

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