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期核苷逆转录酶抑制剂2 '-b-氟-2'，3 '-双脱氧腺苷（F-ddA，碘腺苷）仍然是感兴趣的化合物。我们已经产生的广泛的临床前和临床药理学和药代动力学数据正在用于改进基于核苷的前药的物种可扩展的生理药代动力学模型。该模型用于研究各种生理和生化过程对前药处置和活化的影响，重点是胃肠道吸收、血脑屏障渗透到CNS和代谢活化。该模型也被扩展到包括核苷类药物，而不是前药，如口服活性DNA甲基转移酶抑制剂2（1H）-嘧啶酮核苷（zebularine）。已经启动了协同的生化药理学和大鼠药代动力学研究，以确定zebularine的细胞内代谢、处置和口服生物利用度。初步结果表明，即使zebularine是口服活性，其生物利用度非常低。 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。使用毛细管电泳测量细胞内核苷酸池和代谢物的方法的开发仍在继续。大体积的样品堆积已定量评价的线性，重现性和色谱分离度的CE分析monoclastide混合物。使用这种技术可以实现高达160倍的信号增强的核苷酸的分析，虽然灵敏度的增强是在生物基质，因为在电泳分辨率的损失，由于离子强度的影响。目前正在研究克服后一种影响的样品制备方法和分析策略。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。CE与样品堆叠也显着增加的速度和灵敏度的测定中产生的寡核苷酸产物的回文寡核苷酸定向酶法测定正在开发的细胞内脱氧和双脱氧核苷酸的测量，以更全面地表征各种抗逆转录病毒疗法。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 nucleoside reverse transcriptase inhibitor 2'-b-fluoro-2',3'-dideoxyadeonsine (F-ddA, lodenosine) continues to be a compound of interest. The extensive preclinical and clinical pharmacological and pharmacokinetic data that we have generated is being used to refine a species-scalable physiological pharmacokinetic model for nucleoside-based prodrugs. This model is being used to investigate the effects of various physiological and biochemical processes on prodrug disposition and activation, with emphasis on gastrointestinal absorption, blood-brain-barrier penetration into the CNS, and metabolic activation. This model is also being extended to include nucleoside-based agents that are not prodrugs such as the orally active DNA methyl transferase inhibitor 2(1H)-pyrimidinone riboside (zebularine). Collaborative biochemical pharmacology and rat pharmacokinetic studies have been initiated to define the intracellular metabolism, disposition and oral bioavailability of zebularine. Preliminary results show that even though zebularine is orally active, its bioavailability is very low. 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 continues. Large-volume sample stacking has been quantitatively evaluated in terms of linearity, reproducibility and chromatographic resolution for the CE analysis of mononucleotide mixtures. Signal enhancements of up to 160-fold could be achieved for the analysis of nucleotides using this technique, although the sensitivity enhancement is less in biological matrices because of loss in electrophoretic resolution due to ionic strength effects. Sample preparation methods and analysis strategies to overcome this later effect are under investigation. 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. CE with sample stacking also dramatically increases the speed and sensitivity of the determination of the oligonucleotide products generated in a palindromic oligonucleotide-directed enzymatic assay being developed for the measurement of intracellular deoxy- and dideoxynucleotides in order to more fully characterize various antiretroviral therapies. 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