The objective of this project is the research, development and application of suitable bioanalytical methods to: (1) establish the structure and purity of potential anti-AIDS agents, new antiviral drugs and selected antitumor agents (2) determine the physical, chemical and biochemical properties of these compounds and their metabolites, and (3) measure these drugs and their metabolites in biological samples to elucidate pharmacology and to determine plasma and intracellular pharmacokinetics. High-performance liquid chromatography (HPLC), capillary electrophoresis (CE) and mass spectrometry (MS) are the major analaytical tools that are employed. The orally active DNA methyltransferase inhibitor 2(1H)-pyrimidinone riboside (zebularine) and its analogues and prodrugs are currently the major compounds of interest. Previously developed bioanalytical methods for the measurement of zebularine and its intracellular metabolites in biological and pharmaceutical samples have been refined and applied to establish the enzymatic and hydrolytic stability of these compounds and to determine the kinetics of intracellular formation and degradation of phosphorylated zebularine metabolites. Zebularine itself is a very poor substrate for both bacterial and human pyrimidine phosphorylase, although the uridine produced from its extensive catabolism by aldehyde oxidase in human liver is rapidly degraded to uracil. This first-pass metabolism offers a possible explanation for the low and variable oral bioavailability (1-31%) that we have observed in rats and others have observed in monkeys (<1%). These results will be used to refine and extend a previously developed species-scalable physiological pharmacokinetic model for nucleoside-based prodrugs. This model is being used to investigate the effects of various physiological and biochemical processes on drug disposition and activation, with emphasis on gastrointestinal absorption, blood-brain-barrier penetration into the CNS, and intracellular metabolic activation. This model will be used to guide zebularine prodrug selection and evaluation.? ? Collaborative studies on the metabolic activation of zebularine have been conducted in selected human and murine cell lines. In T-24 bladder carcinoma cells as well as in Molt-4 lymphoblasts and murine MC-38 colon cancer cells, zebularine readily undergoes intracellular phosphorylation to form the corresponding 5'-mono-, di- and triphosphates in a dose- and time-dependent manner. In addition to these expected metabolites, a new metabolite, which presumably arises from the coupling of zebularine-5'-triphosphate with choline, has been provisionally identified as zebularine-5'-diphosphocholine. This phosphorylated conjugate is a major metabolic product in all three cell lines and persists since it possesses a much longer intracellular elimination half-life than the other phosphorylated metabolites. Zebularine is incorporated into both DNA and RNA with RNA incorporation predominating by 7- to 30-fold depending on the cell line. It is postulated that incorporation of zebularine into DNA is required before the drug can function as an inhibitor of the methyltranferase by formation of a tight complex between it and the enzyme. Since DNA incorporation requires the activated 2'-deoxynucleotide, the metabolic bottleneck for DNA incorporation appears to be the conversion of zebularine-5'-diphosphate to 2'-deoxyzebularine-5'-diphosphate by ribonucleotide reductase. The very limited DNA incorporation that we have observed suggests that this is the reason for the equivalent activity but lesser potency relative to other inhibitors of DNA methylation. These observations also suggest that prodrugs of 2'-deoxzebularine-5'-monophosphate may circumvent this metabolically inefficient activation and increase drug potency.? ? The development of methods using capillary electrophoresis to measure intracellular nucleotide pools and metabolites continues. ?
