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药物和新的抗病毒药物的结构和纯度；(2)测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)测量这些药物及其代谢物在生物样品中的含量，以阐明药理和确定药代动力学。重点介绍了高效液相色谱(HPLC)、毛细管电泳法(CE)和质谱法。第二相核苷逆转录酶抑制剂2‘-b-氟-2’，3‘-二脱氧脱氧脱氧核苷(F-Dda，lodenosine)仍然是一个令人感兴趣的化合物。我们生成的广泛的临床前和临床药理学和药代动力学数据正被用于完善核苷类前药的物种可扩展的生理药代动力学模型。该模型正被用来研究各种生理生化过程对前体药物处置和激活的影响，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。这一模式还扩展到包括非前体药物的核苷类药物，如口服活性DNA甲基转移酶抑制剂2(1H)-嘧啶酮核苷(ZeBularine)。协同生化药理学和大鼠药代动力学研究已经开始，以确定Zebuarine的细胞内代谢、处置和口服生物利用度。初步结果表明，尽管ZeBularine具有口服活性，但其生物利用度很低。 细胞提取物的直接荧光衍生化结合成对离子高效液相色谱法已被用于非放射化学测定细胞内亚皮摩尔和低皮摩尔含量的F-ddATP，F-ddA和F-ddi的活性代谢物。可以在接受F-Dda治疗的患者的外周血单核细胞中检测到F-ddATP，但没有足够的数据与观察到的抗HIV活性相关联。使用毛细管电泳法测量细胞内核苷酸池和代谢物的方法的发展仍在继续。从线性、重现性和层析分辨率等方面对大容量样品堆积进行了定量评估，用于单核苷酸混合物的CE分析。使用该技术对核苷酸的分析可以实现高达160倍的信号增强，尽管由于离子强度效应导致的电泳分辨率损失，在生物基质中的灵敏度增强较小。克服后一种影响的样品制备方法和分析策略正在研究中。样品堆积已被应用于表征人工合成的核苷酸混合物的次要成分，并分析培养的MOLT-4淋巴细胞中的内源性核苷酸。采用样品堆积的毛细管电泳法还大大提高了回文寡核苷酸导向酶分析产生的寡核苷酸产物的速度和灵敏度，该方法正在开发中，用于测量细胞内脱氧和双脱氧核苷酸，以便更全面地描述各种抗逆转录病毒治疗的特征。目前正在进行的研究方向是应用毛细管电泳法测定细胞内核苷药物的代谢，并将毛细管电泳法与质谱仪相结合进行结构分析。 艾滋病题目：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