Establishing Metronidazole as a Novel Biomarker of CYP2A6 Activity
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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
Ph.D. (Doctor of Philosophy)