The Analytical Chemistry of Anti-AIDS Agents

该项目的目的是研究、开发和应用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)在生物样品中测定这些药物及其代谢物，以阐明药理学和测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（MS）是主要的分析工具。具有口服活性的DNA甲基转移酶抑制剂2(1H)-嘧啶核苷（zebularine）及其类似物和前药是目前研究的主要化合物。先前开发的用于测量生物和药物样品中斑马碱及其细胞内代谢物的生物分析方法已经得到改进，并应用于建立这些化合物的酶促和水解稳定性，并确定磷酸化斑马碱代谢物的细胞内形成和降解动力学。对于细菌和人的嘧啶磷酸化酶来说，西莨菪碱本身都是一个很差的底物，尽管它在人肝脏中被醛氧化酶广泛分解代谢产生的尿苷会迅速降解为尿嘧啶。这种首过代谢可能解释了我们在大鼠和猴子中观察到的低且多变的口服生物利用度（1-31%）（<1%）。这些结果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细胞内代谢激活。该模型将用于指导西布拉林前药的选择和评价。

The objective of this project is the research, development and application 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 (MS) are the major analaytical tools that are employed. The orally active DNA methyltransferase inhibitor 2(1H)-pyrimidinone riboside (zebularine) and its analogues and prodrugs are currently the major compounds of interest. Previously developed bioanalytical methods for the measurement of zebularine and its intracellular metabolites in biological and pharmaceutical samples have been refined and applied to establish the enzymatic and hydrolytic stability of these compounds and to determine the kinetics of intracellular formation and degradation of phosphorylated zebularine metabolites. Zebularine itself is a very poor substrate for both bacterial and human pyrimidine phosphorylase, although the uridine produced from its extensive catabolism by aldehyde oxidase in human liver is rapidly degraded to uracil. This first-pass metabolism offers a possible explanation for the low and variable oral bioavailability (1-31%) that we have observed in rats and others have observed in monkeys (<1%). These results 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 intracellular metabolic activation. This model will be used to guide zebularine prodrug selection and evaluation. 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 new metabolite, which presumably arises from the coupling of zebularine-5'-triphosphate with choline, has been provisionally identified as zebularine-5'-diphosphocholine. This phosphorylated conjugate is a major metabolic product in all three cell lines and persists since it possesses a much longer intracellular elimination half-life than the other phosphorylated metabolites. Zebularine is incorporated into both DNA and RNA with RNA incorporation predominating by 7- to 30-fold depending on the cell line. It is postulated 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. Since DNA incorporation requires the activated 2'-deoxynucleotide, the metabolic bottleneck for DNA incorporation appears to be the conversion of zebularine-5'-diphosphate to 2'-deoxyzebularine-5'-diphosphate by ribonucleotide reductase. 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. These observations also suggest that prodrugs of 2'-deoxzebularine-5'-monophosphate may circumvent this metabolically inefficient activation and increase drug potency. The development of methods using capillary electrophoresis to measure intracellular nucleotide pools and metabolites continues.