Date of Award

May 2013

Degree Type

Dissertation

Degree Name

Doctor of Philosophy

Department

Chemistry

First Advisor

Alexander (Leggy) Arnold

Committee Members

Joseph H. Aldstadt, Mark L. Dietz, Nicholas R. Silvaggi, Daniel S. Sem

Keywords

Drug Bioavailability, Drug Electrophilicity, Drug Permeability, Drug-Protein Binding, High-Throughput Screening, Pre-Clinical Drug Development

Abstract

A bioactive molecule must pass many hurdles to be designated as a "good" pharmaceutical lead or hit compound. It should have a significant activity, selectivity, bioavailability, and metabolic half-life. Many factors have been identified that influence the free drug concentration or bioavailability of orally administered drugs in the earliest development stages. In vitro pre-clinical assays have been developed to measure these parameters. The small molecule properties that are investigated here include aqueous solubility, permeability, reactivity (electrophilicity), small molecule-protein binding, and displacement of protein-bound molecules (drug-drug interactions). The development of rapid and miniaturized assays to quantify these factors is presented herein.

First, a 384-well filter plate based assay was developed to determine the aqueous compound solubility to greatly decrease the time and amount of compound necessary for analysis. Secondly, one of the most common and simple permeability assays (parallel artificial membrane permeability assay, PAMPA) was optimized using a filter membrane impregnated with a long chain alkane (hexadecane) solution as an artificial membrane. Thirdly, permeability was also determined rapidly with the use of Immobilized Artificial Membrane (IAM) and C18 stationary phases by HPLC. The solitary and sequential usage of these columns was compared.

Fourthly, a novel fluorescence-based high-throughput assay was developed to identify electrophilic molecules rapidly, in parallel, among small molecule libraries using only sub-milligram quantities. Subsequently, a filtration-based assay to estimate compound binding with plasma protein was developed for a 384-well plate format. This assay not only increases the throughput, but also addresses non-specific compound binding to the filtration apparatus, which is problematic with other ultrafiltration methods. Finally, a simple high-throughput competitive protein binding assay was developed based on the multiplexing of fluorescent small molecule probes with different spectroscopic and binding properties. The inhibition of probe-protein binding has been identified as a good indicator for plasma protein binding.

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