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 (KM) 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 Po/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,
