Establishing Metronidazole as a Novel Biomarker of CYP2A6 Activity
Abstract
CYP2A6 is a polymorphically expressed enzyme with variation associated with
smoking behavior, cessation success, lung cancer risk, and differential drug exposure to
numerous medications including anti-infectious and chemotherapeutic agents. Current
CYP2A6 probes that could provide insight a priori into variability are limited due to safety,
accessibility, general applicability and/or enzyme specificity. Therefore, the purpose of this
research was to develop a novel biomarker to understand variability in CYP2A6 activity in
humans. A step-wise approach was utilized and included evaluation of the ability of
metronidazole and nicotine (current gold standard) to modulate CYP2A6 activity and
expressionin vitro, validation of a novel analytical method to determine the concentrations
of metronidazole and 2-hydroxymetronidazole in human plasma, and finally, comparison of
metronidazole to nicotine (via metabolite/parent ratio) for use as a CYP2A6 phenotyping
probe in humans. Metronidazole and nicotine had minimal effects on CYP2A6 expression or
activity in vitro at therapeutically relevant concentrations and are predicted to not lead to
meaningful clinical impact. Using a low volume of human plasma (10 µL), metronidazole
and 2-hydroxymetronidazole were simultaneously quantitated by a novel, validated UPLCMS/MS method that is among the most sensitive to date. The metronidazole probe measure
(2-hydroxymetronidazole/metronidazole ratio in plasma) proved well-tolerated, highly
specific for CYP2A6, and robust with a wide window of use. This novel probe measure was
also able to dichotomize individuals based on genotype-predicted phenotype in a way that
mirrored the nicotine probe measures. In summary, this work establishes the metronidazole
probe measure as a novel biomarker of CYP2A6 variability in humans, thus providing a tool
to understand human diversity and potentially, improve health outcomes in a diverse pool of
individuals. Future studies evaluating the use of metronidazole as a probe of CYP2A6
activity in sub-populations of humans would be useful to further investigate the performance
of this tool in special populations at risk for poor health outcomes.
Table of Contents
Introduction -- Effects of Metronidazole and Nicotine on CYP2A6 Activity and MRNA Expression in vitro -- Development of a UPLC-MS/MS Method for Quantitation of Metronidazole and 2-Hydeoxymetronidazole in Human Plasma and Its Application to a Pharmacokinetic Study -- Validation of Metronidazole as a Novel, Safe, CYP2A6 Phenotyping Probe in Humans -- Summary and Future Directions
Degree
Ph.D. (Doctor of Philosophy)