Title

LC-MS based bio-analytical methods for DMPK studies, toxicity screening and identification of DNA/protein adducts in the presence of bioreactors

Date of Completion

January 2009

Keywords

Health Sciences, Pharmacology|Chemistry, Analytical|Chemistry, Biochemistry

Degree

Ph.D.

Abstract

Toxicity is one of main issues involving chemicals to which we are exposed. For example, in drug discovery and development, toxicity is often not identified sufficiently early for new pharmaceutical candidates. Rapidly and accurately predicting in vivo metabolism, pharmacokinetics and toxicity of drug candidates early in the development process is a major toxicity-related challenge in drug discovery. Thus, there is an urgent need for new, faster, lower cost molecular methodologies for in vitro metabolism studies that simplify sample workup while providing high quality kinetic and structural information. In this dissertation, new methods utilizing enzyme-coated silica nanoparticles for the first time in combination with LC-MS/MS for drug metabolism pharmacokinetic studies (DMPK) and toxicity screening assays have been developed. The first part of the approach addresses the study of drug metabolism in the presence of assembled liver metabolic enzymes on silica nanoparticles (bioreactors). These bioreactors are designed for metabolite profiling, identification of minor metabolites, pharmacokinetic and drug-drug interaction studies. The second part features the assembly of enzymes and DNA or peptides on silica nanoparticles for rapid genotoxicity and hepatotoxicity screening. It is designed to identify reactive metabolites and their sites of reaction with DNA/peptide as well as to measure DNA/peptide adduct formation rates. This approach has been integrated into a 96 well plate high throughput semi-automated method facilitating rapid sample preparation in a single step, followed by simultaneous filtration and transfer to an autosampler for LC-MS/MS analysis. The use of microsomes as sources of metabolic enzymes on the nanoparticles offers distinct advantages including significant numbers of metabolic enzymes, good stability, longer storage time, and rapid separation of reaction products from microsomal enzymes.^ The applicability and versatility of these methodologies are illustrated by concurrently studying in vitro oxidation and conjugation reactions, enzyme specific metabolism and drug-drug interactions, identifying reactive metabolites and elucidating metabolic pathways, and screening for DNA and protein adducts. These approaches are designed to be combined with current bioanalytical and microbiological assays to provide a more comprehensive assessment of drug metabolism.^