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、新的抗病毒药物和选定的抗肿瘤药物的结构和纯度；(2)确定这些化合物及其代谢物的物理、化学和生化特性；(3)在生物样品中测量这些药物及其代谢物，以阐明药理学并确定血浆和细胞内的药代动力学。高效液相色谱（HPLC）、毛细管电泳（CE）和质谱是主要的分析工具。口服活性DNA甲基转移酶抑制剂2(1H)-嘧啶核苷（zebullarine）及其类似物是目前研究的主要化合物。一系列的生物分析方法已经被设计和应用于测量生物和药物样品中的斑马碱及其细胞内代谢物。通过合作的努力，开发并验证了快速、灵敏的高效液相色谱方法，用于在制药介质和生物样品中测量该试剂。斑马碱在酸性和中性pH值下表现出令人印象深刻的水解稳定性，可以在饮用水中长期口服啮齿动物。测量血浆中西布拉林所需的小样本量允许在单个动物中确定药代动力学。该方法已应用于探索性临床前药理学研究，并适用于未来的毒理学和临床研究。协作药代动力学研究已经开展，以确定血浆动力学和口服生物利用度在个体大鼠。10 - 100mg /kg的口服剂量导致不同的生物利用度，范围从低（1%）到中等（31%）。由于在其他物种（猴子）中观察到的生物利用度较低，因此正在计划或正在进行更多的体外和体内研究，以进一步评估斑马碱的处置和可能的首过代谢。对于细菌和人的嘧啶磷酸化酶来说，西莨菪碱都是一种很差的底物，关于它在人肝脏中被醛氧化酶分解代谢的可能性的合作研究正在进行中。这些数据将用于完善和扩展先前开发的基于核苷的前药的物种可扩展生理药代动力学模型。该模型被用于研究各种生理生化过程对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。

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, new antiviral drugs and selected antitumor agents (2) determine the physical, chemical and biochemical properties of these compounds and their metabolites, and (3) measure these drugs and their metabolites in biological samples to elucidate pharmacology and to determine plasma and intracellular pharmacokinetics. High-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and mass spectrometry are the major analaytical tools that are employed. The orally active DNA methyltransferase inhibitor 2(1H)-pyrimidinone riboside (zebularine) and its analogues are currently the major compounds of interest. A range of bioanalytical methods have been devised and applied for the measurement of zebularine and its intracellular metabolites in biological and pharmaceutical samples. A collaborative effort has resulted in the development and validation of rapid and sensitive HPLC methods for the measurement of this agent in pharmaceutical media and biological samples. Zebularine exhibits impressive hydrolytic stability at acid and neutral pH and can be administered orally to rodents for extended periods in drinking water. The small sample size required for the measurement of zebularine in plasma allows pharmacokinetics to be determined in an individual animal. This method has been applied to exploratory preclinical pharmacology studies and is adaptable for future toxicology and clinical studies. Collaborative pharmacokinetic studies have been carried out to define the plasma kinetics and oral bioavailability of zebularine in individual rats. Oral doses of 10 - 100 mg/kg zebularine result in variable bioavailablity, ranging from low (1%) to moderate (31%). Because of the low bioavailability observed in other species (monkeys), additional in vitro and in vivo studies are planned or are ongoing to further assess zebularine disposition and possible first-pass metabolism. Zebularine is a very poor substrate for both bacterial and human pyrimidine phosphorylase, and a collaborative study of its possible catabolism by aldehyde oxidase in human liver is in progress. This data will be used to refine and extend a previously developed 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 drug disposition and activation, with emphasis on gastrointestinal absorption, blood-brain-barrier penetration into the CNS, and metabolic activation. Collaborative studies on the metabolic activation of zebularine have been conducted in selected human and murine cell lines. In T-24 bladder carcinoma cells as well as in Molt-4 lymphoblasts and murine MC-38 colon cancer cells, zebularine readily undergoes intracellular phosphorylation to form the corresponding 5'-mono-, di- and triphosphates in a dose- and time-dependent manner. In addition to these expected metabolites, a major phosphorylated conjugate containing the intact zebularine base is observed in all cell lines. This new metabolite has been identified as zebularine-5'-diphosphocholine and is postulated to arise from coupling of zebularine-5'-triphosphate with choline. It possesses a longer intracellular elimination half-life than the other phosphorylated metabolites and is a potential depot source of the 5'-monophosphate. Zebularine is incorporated into both DNA and RNA with RNA incorporation predominating by 7- to 30-fold depending on the cell line. It is thought that incorporation of zebularine into DNA is required before the drug can function as an inhibitor of the methyltranferase by formation of a tight complex between it and the enzyme. The very limited DNA incorporation that we have observed suggests that this is the reason for the equivalent activity but lesser potency relative to other inhibitors of DNA methylation. The development of methods using capillary electrophoresis to measure intracellular nucleotide pools and metabolites continues. CE has been used to characterize the diphosphocholine adduct of zebularine and show that it is a potential depot source of zebularine-5'-monophosphate. Our previous work has demonstrated that a 100- to 160-fold signal enhancement can be obtained for the CE analysis of mixtures of synthetic nucleotides, but that a marked peak width broadening and loss of resolution is noted during sample stacking of biological samples. This deterioration in resolution is partially related to sample ionic strength. Sample and/or run buffer ionic strength has been controlled on an individual basis to enhance the determination of minor components of various synthetic nucleotide mixtures and to characterize nucleotide drug metabolites in cellular matrices such as cultured Molt-4 and Hela cells. Sample preparation methods and analysis strategies to overcome this effect remain under investigation. 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 toward off-line interfacing of CE with mass spectrometry for structural analysis.