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、新的抗病毒药物和选定的抗肿瘤药物的结构和纯度；(2)测定这些化合物及其代谢物的物理、化学和生化性质；(3)测定这些药物及其代谢物在生物样品中的含量，以阐明药理作用并确定血浆和细胞内的药代动力学。高效液相色谱、毛细管电泳法和质谱法是主要的分析手段。口服活性DNA甲基转移酶抑制剂2(1H)-嘧啶酮核苷(ZeBularine)及其类似物是目前研究的主要化合物。已设计并应用了一系列生物分析方法来测定生物和药物样品中的ZeBularine及其细胞内代谢物。在合作的努力下，开发和验证了快速和灵敏的高效液相色谱方法，用于测量药物介质和生物样品中的这种试剂。ZeBularine在酸性和中性pH条件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给啮齿动物。测量血浆中的泽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。已经开展了协作性药代动力学研究，以确定ZeBularine在大鼠体内的血浆动力学和口服生物利用度。口服剂量为10-100 mg/kg的ZeBularine的生物利用度从低(1%)到中等(31%)不等。由于在其他物种(猴子)中观察到的生物利用度较低，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ZeBularine的处置和可能的首过代谢。Zebuarine对于细菌和人类的嘧啶磷酸化酶来说都是一个非常差的底物，一项关于其在人体肝脏中可能通过醛氧化酶分解代谢的合作研究正在进行中。这些数据将被用来改进和扩展先前开发的核苷类前药的物种可缩放的生理药代动力学模型。这个模型被用来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。 在选定的人和小鼠细胞系中，已经对ZeBularine的代谢激活进行了合作研究。在T-24膀胱癌细胞以及Molt-4淋巴母细胞和小鼠MC-38结肠癌细胞中，ZeBularine以剂量和时间依赖的方式容易地进行细胞内磷酸化，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，在所有细胞系中都观察到了含有完整ZeBularine碱基的主要磷酸化结合物。这一新的代谢物被鉴定为ZeBularine-5‘-diphosopporine，推测是由ZeBularine-5’-三磷酸与胆碱偶联而成。它具有比其他磷酸化代谢物更长的细胞内消除半衰期，是5‘-单磷酸的潜在仓库来源。ZeBularine被结合到DNA和RNA中，其中RNA掺入占主导地位，根据细胞系的不同，RNA的掺入比例是7-30倍。人们认为，在药物能够通过在DNA和酶之间形成紧密的复合体来发挥甲基转移酶抑制剂的作用之前，需要将ZeBularine结合到DNA中。我们观察到的非常有限的DNA掺入表明，这是相对于其他DNA甲基化抑制剂具有同等活性但效力较低的原因。 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。用毛细管电泳法表征了ZeBularine的二磷胆碱加合物，表明它是一种潜在的ZeBularine-5‘-一磷酸的仓库来源。我们以前的工作已经证明，对于合成核苷酸混合物的CE分析，可以获得100到160倍的信号增强，但在生物样品的样品堆积过程中，注意到显著的峰宽展宽和分辨率损失。分辨率的下降部分与样品的离子强度有关。对样品和/或运行缓冲液离子强度进行了个别控制，以增强对各种合成核苷酸混合物的次要成分的测定，并对细胞基质(如培养的Molt-4和Hela细胞)中的核苷酸药物代谢物进行表征。克服这种影响的样品制备方法和分析策略仍在调查中。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将毛细管电泳法应用于细胞内核苷药物代谢的测定，以及将毛细管电泳法与质谱仪进行离线接口以进行结构分析。

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.