THE ANALYTICAL CHEMISTRY OF NEW ANTICANCER DRUGS

该项目的目标是开发和应用生物分析方法：(1)确定新的抗癌药物的物理，化学和生化特性，(2)测量新药，其代谢物，以及生物样品中的潜在生物调节剂，(3)阐明体外和体内药理学和药代动力学，(4)定义独特的分子特征，以促进发现新的药物先导。高效液相色谱法、荧光法和紫外可见光谱法是主要的分析工具。腺苷脱氨酶（ADA）是一种普遍存在的人类酶，也是基于腺嘌呤的化疗药物的生物调节剂，利用构象偏置的合成腺嘌呤核苷获得了对ADA催化结合位点的新见解。酶动力学、核磁共振和分子模型研究表明northern构型核呋喃基类似物具有优先催化作用。这些结果反映在结合常数的提高（K_M）和北方构型类似物的水解速率的提高，无论是在约束糖构象还是在非约束糖构象中。这为设计嘌呤代谢调节剂的新型化疗药物提供了一种新的合理途径。成功应用荧光化学衍生法测量外周血淋巴细胞中腺嘌呤核苷酸的三磷酸水平，结果表明，在接受新型抗艾滋病药物洛地甘氨酸治疗的患者淋巴细胞中，三磷酸腺苷的浓度处于低摩尔浓度。虽然最初的设计是为了测量一种新的抗艾滋病毒药物的细胞浓度，但这种方法将有助于在生物样品有限和放射性同位素不可用的情况下测量其他抗癌和抗病毒腺嘌呤核苷酸。毛细管电泳（CE）是一种筛选丙型肝炎病毒潜在抑制剂的高通量方法。纳升样品要求使得该方法在常规分析和可用于测试的药物数量有限的情况下非常有吸引力。在一项针对恶性黑色素瘤的单纯疱疹胸苷激酶表达腺病毒的临床基因治疗试验中，正在确定不断增加剂量的更昔洛韦的人体药代动力学。药物亲脂性仍然是生物系统中药物吸收的重要物理化学描述符。辛醇-水分配系数（log P<sub - >o/w</sub - >）是该参数的最直接测量，已经使用我们的新型微摇瓶技术确定了超过132种核苷类似物。这些数据的汇编导致了核苷衍生物的预测结构-log P模型，该模型直观，优于目前使用的许多计算机算法。-腺苷脱氨酶，荧光衍生化，亲脂性，毛细管电泳，丙型肝炎病毒，分配系数，

The goal of this project is the development and application of bioanalytical methods to: (1) determine physical, chemical and biochemical properties of new anticancer drugs, (2) measure novel drugs, their metabolites, and potential biomodulators in biological samples, (3) elucidate in vitro and in vivo pharmacology and pharmacokinetics and (4) define unique molecular features which facilitate discovery of new drug leads. High-performance liquid chromatography (HPLC), fluorimetry and ultraviolet-visible spectroscopy are the principle analytical tools. New insight into the catalytic binding site of adenosine deaminase (ADA), a ubiquitous human enzyme and biomodulator of adenine-based chemotherapeutic agents, is being obtained using conformationally biased synthetic adenine nucleosides. Enzyme kinetics, nuclear magnetic resonance, and molecular modeling studies show preferential catalysis of northern configured ribofuranosyl analogues. These results are reflected in improved binding constants (K_M) and enhanced rates of hydrolysis for northern configured analogues whether in constrained or unconstrained sugar conformations. This affords a new rational approach for the design of novel chemotherapeutics as modulators of purine metabolism. Successful application of fluorogenic chemical derivatization to measure triphosphate levels of an adenine nucleotide in peripheral blood lymphocytes has demonstrated concentrations at low pmole levels in lymphocytic cells of patients receiving lodenosine, a new anti-AIDS drug. Although originally designed to measure cellular concentrations of a new anti-HIV drug, this method will be conducive for the measurement of other anti-cancer and antiviral adenine nucleotides where limited biological sample is available and where utilization of radioisotopes is not feasible. Capillary electrophoresis (CE) is being explored as a high through-put method for screening potential inhibitors of hepatitis C virus. Nanoliter sample requirements make this method quite attractive for routine assay and when limited amounts of drug are available for testing. Human pharmacokinetics of escalating doses of ganciclovir are being determined in a clinical gene therapy trial targeting malignant melanoma with a herpes simplex-thymidine kinase expressing adenovirus for drug activation. Drug lipophilicity remains an important physicochemical descriptor of drug absorption in biological systems. Octanol-water partition coefficients (log P_o/w), being the most direct measure of this parameter, have been determined for more than 132 nucleoside analogues using our novel micro shake-flask technique. The compilation of this data has led to a predictive structure-log P model for nucleoside derivatives which is intuitive and superior to many computer algorithm currently in use. - adenosine deaminase, fluorogenic derivatization, lipophilicity, capillary electrophoresis, hepatitis C virus, partition coefficient,