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)测量新的药物、它们的代谢物和生物样品中的潜在生物调节剂；(3)研究体外和体内药理学和药代动力学；(4)定义独特的结构特征，阐明分子治疗靶点，促进发现新的药物先导。高效液相色谱法（HPLC）、毛细管电泳法（CE）、荧光法和紫外可见光谱法是主要的分析工具。丙型肝炎病毒（HCV）通常与肝硬化和肝细胞癌相关，目前正在利用计算机建模来鉴定其病毒蛋白酶的潜在抑制剂，以开发位点特异性抗病毒药物。毛细管电泳，理想的监测肽裂解产物，正在开发作为筛选这些选定的蛋白酶抑制剂的高通量方法。纳升样品要求和较短的分析时间使这种方法对我们的常规分析非常有吸引力，在有限的分析物可用。腺苷脱氨酶（ADA）是一种普遍存在的人类酶和基于腺嘌呤的化疗药物的生物调节剂，目前正在使用柔性但构象偏倚的合成腺嘌呤核苷获得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，“北方”构型核呋喃基类似物具有优先催化作用，并确定了对催化产生临界位阻的取代基位点。利用这些知识，我们理想配置的人工底物显示出催化速率常数优于ADA的天然底物腺嘌呤。这为设计嘌呤和嘧啶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，在接受低分子多苷（一种新的低分子多苷抗艾滋病药物）治疗的患者淋巴细胞中，三磷酸腺苷的浓度处于低摩尔水平。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。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。

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) study in vitro and in vivo pharmacology and pharmacokinetics and (4) define unique structural features which elucidate molecular therapeutic targets and facilitate discovery of new drug leads. High-performance liquid chromatography (HPLC), capillary electrophoresis (CE), fluorimetry and ultraviolet-visible spectroscopy are the principle analytical tools. Hepatitis C virus (HCV), often associated with cirrhosis and hepatocellular carcinoma is being targeted for development of site-specific anti-viral agents using computer modeling for identification of potential inhibitors of its viral protease. Capillary electrophoresis, ideal for monitoring peptide cleavage products, is being developed as a high throughput method for screening these selected protease inhibitors. Nanoliter sample requirements and short analysis time make this method quite attractive for our routine assay where limited analyte is available. New insight into the catalytic binding site of adenosine deaminase (ADA), a ubiquitous human enzyme and biomodulator of adenine-based chemotherapeutics, is being obtained using flexible but conformationally biased synthetic adenine nucleosides. Enzyme kinetics, nuclear magnetic resonance, and molecular modeling studies show preferential catalysis of "northern" configured ribofuranosyl analogues and identify substituent sites producing critical steric hindrance to catalysis. Harnessing this knowledge, our ideally configured artificial substrates show catalytic rate constants superior to those of adensoine, the natural substrate of ADA. This affords a new rational approach for the design of novel chemotherapeutics as modulators of purine and pyrmidine 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 LMCH 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. 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.