The objective of this project is the research and development 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 are the major analaytical tools that are employed. The orally active DNA methyltransferase inhibitor 2(1H)-pyrimidinone riboside (zebularine) and its analogues are currently the major compounds of interest. A range of bioanalytical methods have been devised and applied for the measurement of zebularine and its intracellular metabolites in biological and pharmaceutical samples. A collaborative effort has resulted in the development and validation of rapid and sensitive HPLC methods for the measurement of this agent in pharmaceutical media and biological samples. Zebularine exhibits impressive hydrolytic stability at acid and neutral pH and can be administered orally to rodents for extended periods in drinking water. The small sample size required for the measurement of zebularine in plasma allows pharmacokinetics to be determined in an individual animal. This method has been applied to exploratory preclinical pharmacology studies and is adaptable for future toxicology and clinical studies. Collaborative pharmacokinetic studies have been carried out to define the plasma kinetics and oral bioavailability of zebularine in individual rats. Oral doses of 10 - 100 mg/kg zebularine result in variable bioavailablity, ranging from low (1%) to moderate (31%). Because of the low bioavailability observed in other species (monkeys), additional in vitro and in vivo studies are planned or are ongoing to further assess zebularine disposition and possible first-pass metabolism. Zebularine is a very poor substrate for both bacterial and human pyrimidine phosphorylase, and a collaborative study of its possible catabolism by aldehyde oxidase in human liver is in progress. This data 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 metabolic activation. 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 major phosphorylated conjugate containing the intact zebularine base is observed in all cell lines. This new metabolite has been identified as zebularine-5'-diphosphocholine and is postulated to arise from coupling of zebularine-5'-triphosphate with choline. It possesses a longer intracellular elimination half-life than the other phosphorylated metabolites and is a potential depot source of the 5'-monophosphate. Zebularine is incorporated into both DNA and RNA with RNA incorporation predominating by 7- to 30-fold depending on the cell line. It is thought 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. 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. The development of methods using capillary electrophoresis to measure intracellular nucleotide pools and metabolites continues. CE has been used to characterize the diphosphocholine adduct of zebularine and show that it is a potential depot source of zebularine-5'-monophosphate. Our previous work has demonstrated that a 100- to 160-fold signal enhancement can be obtained for the CE analysis of mixtures of synthetic nucleotides, but that a marked peak width broadening and loss of resolution is noted during sample stacking of biological samples. This deterioration in resolution is partially related to sample ionic strength. Sample and/or run buffer ionic strength has been controlled on an individual basis to enhance the determination of minor components of various synthetic nucleotide mixtures and to characterize nucleotide drug metabolites in cellular matrices such as cultured Molt-4 and Hela cells. Sample preparation methods and analysis strategies to overcome this effect remain under investigation. CE with sample stacking also dramatically increases the speed and sensitivity of the determination of the oligonucleotide products generated in a palindromic oligonucleotide-directed enzymatic assay being developed for the measurement of intracellular deoxy- and dideoxynucleotides in order to more fully characterize various antiretroviral therapies. Ongoing research is currently directed toward the application of CE for the determination of intracellular nucleoside drug metabolism and toward off-line interfacing of CE with mass spectrometry for structural analysis.
