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.
