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剂和新的抗病毒药物的结构和纯度，（2）确定这些化合物及其代谢产物的物理、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）-嘧啶核苷（zebularine）及其类似物是目前感兴趣的主要化合物。一系列的生物分析方法已经和正在设计用于测量生物样品中的zebularine及其细胞内代谢产物。一个合作的努力，导致了快速和灵敏的HPLC方法的开发和验证，用于测量药物介质和生物样品中的这种代理。Zebularine在酸性和中性pH下具有令人印象深刻的水解稳定性，并且可以在饮用水中长时间口服给药于啮齿动物。测量血浆中zebularine所需的小样本量允许在单个啮齿动物中测定药代动力学。后一种方法既适用于当前的临床前药理学研究，也适用于未来的毒理学和临床研究。已经进行了协作药代动力学研究，以确定个体大鼠中zebularine的血浆动力学和口服生物利用度。这些初步研究表明，静脉推注10 - 100 mg/kg zebularine后血浆清除迅速。等效剂量的zebularine口服给药表明可变的生物利用度，范围从低（1%）到中等（31%）。计划进行进一步评估zebularine生物利用度和处置的其他研究，以便可以改进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和代谢活化。 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，在所有细胞系中观察到含有完整zebularine碱的主要磷酸化缀合物。基于色谱、酶和光谱学证据，我们初步鉴定了这种新的代谢产物为3 '，5'-环状二磷酸乙醇胺加合物，并假设它是由zebularine-5 '-triphosphate与乙醇胺偶联而产生的。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。人们认为，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。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。 使用毛细管电泳测量细胞内核苷酸池和代谢物的方法的发展继续强调zebularine的细胞内代谢活化的快速，非放射性评价。CE已被用于表征假定的zebularine的二磷酸乙醇胺加合物，并表明它是zebularine-5 '-单磷酸的潜在贮库来源。我们以前的工作已经证明，一个100至160倍的信号增强，可以获得的合成核苷酸的混合物的CE分析，但一个显着的峰宽加宽和分辨率的损失是在生物样品的样品堆积。这种分离度的恶化部分与样品离子强度有关。样品和/或运行缓冲液离子强度已在个体基础上进行控制，以增强各种合成核苷酸混合物的次要组分的测定，并表征细胞基质（如培养的MOLT-4细胞）中的核苷酸药物代谢物。克服这种影响的样品制备方法和分析策略仍在研究中。CE与样品堆叠也显着增加的速度和灵敏度的测定中产生的寡核苷酸产物的回文寡核苷酸定向酶法测定正在开发的细胞内脱氧和双脱氧核苷酸的测量，以更全面地表征各种抗逆转录病毒疗法。目前正在进行的研究是针对应用CE测定细胞内核苷类药物代谢和连接CE与质谱进行结构分析。

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 and new antiviral drugs, (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 and are being devised for the measurement of zebularine and its intracellular metabolites in biological 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 rodent. This latter method is suitable for both current preclinical pharmacology studies and 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. These preliminary studies showed rapid plasma clearance after intravenous bolus doses of 10 - 100 mg/kg zebularine. Oral administration of zebularine at equivalent doses indicated variable bioavailablity, ranging from low (1%) to moderate (31%). Additional studies to further assess zebularine bioavailability and disposition are planned so that a previously developed species-scalable physiological pharmacokinetic model for nucleoside-based prodrugs can be refined and extended. 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, 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. Based on chromatographic, enzymatic and spectrospcopic evidence, we have tentatively identified this new metabolite as a 3',5'-cyclic diphosphoethanolamine adduct and postulate that it arises from coupling of zebularine-5'-triphosphate with ethanolamine. 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 with emphasis on the rapid, nonradiometric evaluation of the intracellular metabolic activation of zebularine. CE has been used to characterize the postulated diphosphoethanolamine 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 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 to interfacing CE with mass spectrometry for structural analysis.