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：（1）确定新抗癌药物的物理、化学和生物化学性质，（2）测量生物样品中的新药物、其代谢物和潜在的生物调节剂，（3）研究体外和体内药理学和药代动力学，以及（4）定义独特的结构特征，阐明分子治疗靶点，促进新药先导物的发现。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是针对开发位点特异性抗病毒药物，使用计算机建模，以确定其病毒蛋白酶的潜在抑制剂。毛细管电泳是监测肽裂解产物的理想方法，目前正被开发为筛选这些选定蛋白酶抑制剂的高通量方法。纳升的样品要求和较短的分析时间使这种方法对于我们的常规分析非常有吸引力，其中有限的分析物可用。腺苷脱氨酶（ADA），一种普遍存在的人类酶和生物调节剂的腺嘌呤为基础的化疗药物的催化结合位点的新见解，正在获得使用灵活的，但构象偏向合成腺嘌呤核苷。酶动力学，核磁共振和分子模拟研究表明，优先催化的“北方”配置的呋喃核糖类似物，并确定取代位点产生的关键空间位阻催化。利用这一知识，我们的理想配置的人工基板显示催化速率常数优于那些腺苷，ADA的天然基板上级。这为设计新型嘌呤和嘧啶代谢调节剂提供了一种新的合理途径。成功应用荧光化学衍生法测定外周血淋巴细胞中腺嘌呤核苷酸的三磷酸水平，证明了接受新的LMCH抗AIDS药物碘腺苷的患者淋巴细胞中的浓度处于低pmole水平。虽然最初设计用于测量一种新的抗HIV药物的细胞浓度，但该方法将有助于测量生物样品有限且利用放射性同位素不可行的其他抗癌和抗病毒腺嘌呤核苷酸。在用表达单纯疱疹病毒胸苷激酶的腺病毒进行药物活化的靶向恶性黑色素瘤的临床基因治疗试验中，正在确定更昔洛韦剂量递增的人体药代动力学。

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.