The Analytical Chemistry of Anti-AIDS Agents

本项目的目标是研究、开发和应用适当的生物分析方法，以便：（1）确定潜在的抗艾滋病剂、新的抗病毒药物和选定的抗肿瘤剂的结构和纯度（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，和（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血浆和细胞内药代动力学。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。口服活性的DNA甲基转移酶抑制剂2（1H）-嘧啶酮核苷（zebularine）及其类似物和前药是目前感兴趣的主要化合物。以前开发的生物分析方法测量zebularine及其细胞内代谢物的生物和药物样品已被完善，并应用于建立这些化合物的酶和水解稳定性，并确定磷酸化zebularine代谢物的细胞内形成和降解的动力学。Zebularine本身是细菌和人嘧啶磷酸化酶的非常差的底物，尽管在人肝脏中通过醛氧化酶从其广泛催化产生的尿苷被快速降解为尿嘧啶。这种首过代谢为我们在大鼠中观察到的低和可变的口服生物利用度（1-31%）和其他人在猴中观察到的口服生物利用度（<1%）提供了可能的解释。这些结果将用于改进和扩展先前开发的基于核苷的前药的物种可扩展的生理药代动力学模型。该模型被用于研究各种生理和生化过程对药物处置和活化的影响，重点是胃肠道吸收，血脑屏障渗透到CNS和细胞内代谢活化。该模型将用于指导zebularine前药的选择和评价。 在选定的人和鼠细胞系中进行了关于zebularine代谢活化的合作研究。在T-24膀胱癌细胞以及Molt-4成淋巴细胞和鼠MC-38结肠癌细胞中，zebularine容易以剂量和时间依赖性方式经历细胞内磷酸化以形成相应的5 '-单磷酸、二磷酸和三磷酸。除了这些预期的代谢物外，一种新的代谢物（推测是由zebularine-5 '-triphosphate与胆碱偶联产生的）已被暂时鉴定为zebularine-5'-diphosphocholine。这种磷酸化缀合物是所有三种细胞系中的主要代谢产物，并且由于其具有比其他磷酸化代谢物长得多的细胞内消除半衰期而持续存在。Zebularine掺入DNA和RNA中，其中RNA掺入占主导地位，取决于细胞系为7至30倍。据推测，在药物通过与甲基转移酶形成紧密复合物而发挥甲基转移酶抑制剂的作用之前，需要将zebularine掺入DNA中。由于DNA掺入需要活化的2 '-脱氧核苷酸，DNA掺入的代谢瓶颈似乎是通过核糖核苷酸还原酶将zebularine-5'-二磷酸转化为2 '-脱氧zebularine-5'-二磷酸。我们观察到的非常有限的DNA掺入表明，这是相对于其他DNA甲基化抑制剂具有相同活性但效力较低的原因。这些观察结果还表明，2 '-脱氧泽布拉林-5'-单磷酸的前药可以避免这种代谢效率低下的活化并增加药物效力。 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。

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.