1. Quantitative proteomics for translational pharmacology and precision medicine research
We specialize in developing and employing robust proteomics methods to quantify drug transporters, metabolizing enzymes, and receptors in human tissues, cells, and biofluids. Unlike Western blotting, proteomics offers selective, robust, and multiplexed protein quantification, including post-translational modifications, without the need for antibodies. Using these methods, we assess the interindividual variability of these proteins across populations in various human tissues (such as liver, intestine, kidneys, placenta, lungs, heart, and brain) and biofluids. The data we collect are meticulously analyzed to explore the influence of genotype, age, sex, and disease state on the abundance of drug-related proteins. With our current techniques at WSU, we can quantify proteins using less than 1 µg of total protein and as few as 24 hepatocytes (on-column), achieving a lower limit of quantification in the attomole range.
2. Novel quantitative insights on the enterohepatic disposition of androgens and drugs
We characterized the quantitative importance of UGT2B17 in the intestinal and hepatic glucuronidation of testosterone using multi-omics approach and a series of in vitro and in vivo studies. Testosterone glucuronide formed in the intestine and the liver is secreted into intestinal lumen by multi-drug resistance associated protein 2 (MRP2), whereas MRP3 excretes testosterone glucuronide into blood, which is taken up by organic anion transporting polypeptide 1B3 (OATP1B3). Using quantitative proteomics, we developed an in vitro to in vivo scaling (IVIVE) approach for determining fractional contributions of individual drug metabolizing enzymes or transporters in drug disposition. Our recent data suggest that human gut microbiota activate testosterone glucuronide, thus allowing testosterone reabsorption. Similar approach was applied to an anticancer drug, irinotecan, where we explained its unique intestinal toxicity after intravenous dosing using differential tissue abundance data of individual enzymes and transporters. Taken together, these findings confirm that glucuronidation is not merely a drug elimination process, but in conjunction with drug transporters and gut microbiome, it plays an important role in drug distribution and increases drug half-life. In summary, a novel systems-biology approach is developed for explaining quantitative role of enterohepatic recycling of drugs and metabolites.
3. Deciphering genetic and non-genetic mechanisms of UGT2B17 variability
Uridine 5′-diphospho-glucuronosyltransferase 2B17 (UGT2B17) serves as a crucial drug and androgen metabolizing enzyme, exhibiting highly variable activity in humans. This variability leads to unpredictable pharmacokinetics (PK) of several medications with narrow therapeutic windows and can even result in complex drug-drug interactions (DDIs). UGT2B17, by influencing androgen metabolism, is also implicated in various cancers, as well as conditions such as obesity and insulin sensitivity. In order to comprehend the underlying mechanisms governing UGT2B17 variability, we conducted an investigation into the association of genetic and non-genetic factors with variations in protein abundance and in vitro activity of UGT2B17 in Human Liver Microsomes (HLMs). Understanding the individual contributions of population factors to UGT2B17 variability can aid in predicting the metabolism of androgens and other substrates of UGT2B17. Our findings reveal that, in addition to copy number variation (CNV), factors such as age, sex, and single nucleotide polymorphisms (SNPs) are correlated with UGT2B17 activity. We advocate for the inclusion of UGT2B17 in early phenotyping assays during drug discovery by pharmaceutical companies to mitigate potential late clinical failures stemming from its variability. Furthermore, our data can be utilized to stratify patients receiving drugs metabolized by UGT2B17. Additionally, we have elucidated the critical role of intestinal and hepatic UGT2B17 in the first-pass metabolism of testosterone.
4. Non-invasive metabolomics methods to predict variability in drug pharmacokinetics, pharmacodynamics, and interactions
Using endogenous metabolites as biomarkers, we have made significant contribution with respect to the discovery and validation of biomarkers of non-CYP enzymes. Particularly, we recently identified normalized testosterone as a biomarker of UGT2B17 variability, which was used for the prediction of effect of the genetic and non-genetic factors on UGT2B17 activity. Moreover, in collaboration with Drs. Kenneth Thummel and Allan Rettie (University of Washington, Seattle, WA), we developed an optimized method for quantification of biomarker of warfarin efficacy. This biomarker relies on the selective quantification of gamma-carboxylated proteoforms of prothrombin in human plasma. This method was successfully validated by comparison with international normalized ratio (INR) in clinical samples from the control and warfarin treated subjects. Such phenotypic biomarkers are broadly applicable for safe use of drugs by allowing prediction of drug disposition, toxicity, efficacy and drug interaction potential.