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，以确定新的抗癌药物的物理、化学和生化性质，(2)测量新药物、其代谢物和生物样品中潜在的生物调节剂，(3)体外和体内药理学和药代动力学研究，以及(4)确定独特的结构特征，以阐明分子治疗靶点和促进发现新的药物先导。高效液相色谱(HPLC)、毛细管电泳法(CE)、荧光法和紫外可见光谱是主要的分析手段。丙型肝炎病毒(丙型肝炎病毒)，通常与肝硬变和肝细胞癌有关，利用计算机模拟来识别其病毒蛋白酶的潜在抑制物，正被作为开发部位特异性抗病毒药物的目标。毛细管电泳法是监测多肽裂解产物的理想方法，目前正在发展成为筛选这些选定的蛋白酶抑制剂的高通量方法。纳升样品需求和较短的分析时间使该方法对我们的常规分析非常有吸引力，因为分析物有限。腺苷脱氨酶(ADA)是一种普遍存在的人类酶，也是腺嘌呤类化疗药物的生物调节剂，它的催化结合部位正在使用灵活但构象偏差的合成腺嘌呤核苷获得新的见解。酶动力学、核磁共振和分子模拟研究表明，“Northern”构型的呋喃核糖类似物具有优先催化作用，并确定了产生催化关键空间位阻的取代基位。利用这一知识，我们理想的人工底物显示出比腺苷更好的催化速率常数，腺苷是ADA的天然底物。这为设计作为嘌呤和嘧啶代谢调节剂的新型化疗药物提供了一条新的合理途径。应用荧光化学衍生法成功地测定了外周血淋巴细胞中腺嘌呤核苷酸的三磷酸水平，结果表明，接受LMCH抗艾滋病新药洛地诺辛治疗的患者淋巴细胞中的三磷酸浓度处于低分子水平。虽然这种方法最初是为了测量一种新的抗艾滋病毒药物的细胞浓度，但在生物样本有限且无法使用放射性同位素的情况下，这种方法将有助于测量其他抗癌和抗病毒的腺嘌呤核苷酸。在一项针对恶性黑色素瘤的临床基因治疗试验中，正在确定更昔洛韦递增剂量的人体药代动力学。该试验使用表达单纯疱疹病毒胸苷激酶的腺病毒进行药物激活。

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.