The Analytical Chemistry of Anti-AIDS Agents

抗艾滋病药物的分析化学

• 批准号: 6761655
• 负责人: james a kelley
• 金额: --
• 依托单位: DIVISION OF BASIC SCIENCES - NCI
• 依托单位国家: 美国
• 资助国家: 美国
• 起止时间: 至
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/6761655
• 关键词: 2'3' dideoxynucleoside; Primates; adenosine deaminase; analytical chemistry; antiAIDS agent; antiviral agents; capillary electrophoresis; chemical structure function; deoxyadenosines; drug design /synthesis /production; drug metabolism; fluorine; halogenation; high performance liquid chromatography; human tissue; laboratory rat; mass spectrometry; pharmacokinetics; prodrugs

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

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药代动力学。高性能液相色谱（HPLC），毛细管电泳（CE）和质谱是强调的技术。 II期核苷逆转录酶抑制剂2'-b-fluoro-2'，3'-二维扬丁酮（F-DDA，lodenosine）继续是感兴趣的化合物。我们生成的广泛的临床前和临床药理和药代动力学数据被用来完善基于核苷的前药的物种量表的生理药代动力学模型。该模型被用来研究各种生理和生化过程对前药处置和激活的影响，重点是胃肠道吸收，血脑屏障渗透到中枢神经系统中以及代谢激活。该模型也被扩展到包括基于核苷的药物，这些药物不是前药，例如口服活性DNA甲基转移酶抑制剂2（1H） - 吡啶酮核苷（Zebolarine）。已经开始了协作生物化学药理学和大鼠药代动力学研究，以定义Zebolarine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是口服活性的，它的生物利用度也很低。 细胞提取物与配对HPLC结合的直接荧光衍生化已用于非放射化学测定细胞内F-DDATP的亚和低丘疹量，这是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测量F-DDATP，但与观察到的抗HIV活性相关的数据没有足够的数据。使用毛细管电泳测量细胞内核苷酸池和代谢产物的方法的发展。大批样品堆叠已通过线性，可重复性和色谱分辨率进行了定量评估，以分析单核苷酸混合物的CE分析。使用该技术可以实现高达160倍的信号增强，尽管由于离子强度效应而导致电泳分辨率损失，因此在生物矩阵中的灵敏度增强较少。样本制备方法和分析策略以克服以后的效果。样品堆叠已用于表征合成核苷酸混合物的次要成分，并在培养的MOLT-4淋巴细胞中介绍内源性细胞内核苷酸。 CE在样品堆叠中还显着提高了确定在针对性的寡核苷酸指导的酶试验中生成的寡核苷酸产物的速度和敏感性，以测量细胞内脱氧核苷酸和二氧核苷酸的测量，以使各种抗逆转录抗逆转录剂更全面。目前，正在进行的研究针对CE的应用，以确定细胞内核苷药物代谢，并与CE与质谱法进行结构分析。 艾滋病标题：反辅助代理人的分析化学