Richard van Breemen (Presenter)
University of Illinois College of Pharmacy
Bio: Richard B. van Breemen is the Matthias C Lu Collegiate Professor of Pharmacy and Professor of Medicinal Chemistry and Pharmacognosy at the University of Illinois College of Pharmacy. He serves as Director of the UIC/NIH Center for Botanical Dietary Supplements Research and leads the Mass Spectrometry, Metabolomics and Proteomics Facility for the University of Illinois Cancer Center. Prof. van Breemen received his B.A. in chemistry from Oberlin College in 1980 and Ph.D. in pharmacology and experimental therapeutics from the Johns Hopkins University (with Catherine Fenselau) in 1985, and he carried out post-doctoral research in laser desorption mass spectrometry at Johns Hopkins with Robert Cotter. Prof. van Breemen has been on the faculty of the University of Illinois College of Pharmacy for over 20 years.
Authorship: Richard B. van Breemen, Alyssa Sprouse, Guannan Li, Dejan Nikolic
University of Illinois College of Pharmacy, and UIC/NIH Center for Botanical Dietary Supplements Research
| Short Abstract Inhibition or induction of cytochrome P450 (CYP) enzymes and transporters are responsible for most drug-botanical interactions, and MS-based assays facilitate the pre-clinical and clinical assessment of these interactions. For inhibition of CYP enzymes by botanical dietary supplements, we utilize UHPLC-MS/MS with CYP substrate cocktail inhibition assays to measure activity of multiple CYP isoforms. Preclinical models of induction of CYP enzymes and transporters are carried out using human hepatocytes supported by UHPLC-MS/MS based functional CYP assays. To confirm interactions predicted in vitro, clinical trials of drug-botanical interactions are carried out based on pharmacokinetic studies of known CYP substrates supported by UHPLC-MS/MS. |
Long Abstract
Introduction
The market for botanical dietary supplements has grown steadily over the last 20 years despite incomplete information regarding mechanism of action, standardization, efficacy, and safety (1). Just as investigations of potential drug-drug interactions are essential during drug development, studies of drug-botanical interactions are important for the safe use of botanical dietary supplements. Inhibition or induction of cytochrome P450 (CYP) enzymes and xenobiotic transporters are responsible for most drug-drug or drug-botanical interactions, and MS-based assays that facilitate the pre-clinical as well as the clinical assessment of these interactions are indispensable.
Methods and Results
To enhance the efficiency of preclinical assays of CYP enzyme inhibition, we utilize a UHPLC-MS/MS based high-throughput cocktail inhibition assay that uses 10 substrates simultaneously against 9 CYP isoforms (1A2, 2A6, 2B6, 2C8, 2C9, 2C19, 2D6, 2E1 and 3A4). This method has been validated using known inhibitors of each CYP enzyme and is being applied to the analysis of potential drug-botanical interactions involving botanical dietary supplements used by women as alternatives to hormone therapy such as licorice species (2). Preclinical models of induction of CYP enzymes and induction/inhibition of drug transporters are carried out using primary cultures of human hepatocytes as suggested by the FDA for drug-drug interaction studies. To complement the mRNA assays of gene upregulation or downregulation that are recommended by the FDA, we also carry out UHPLC-MS/MS based functional CYP enzyme assays using CYP substrates (3). Clinical trials of drug-botanical interactions are usually carried out only when indicated by unexpected consumer side effects or, preferably, by predictive in vitro studies. For example, Phase I clinical trials have confirmed clinical observations and in vitro studies that St. John’s wort (Hypericum perforatum) induces cytochrome P450 3A4/5. The design of clinical trials of drug-botanical interactions will be described as well as the quantitative UHPLC-MS/MS assays that are being used to support them.
Conclusions
The speed of UHPLC enables UHPLC-MS/MS assays to be carried out at least 5-fold faster than comparable LC-MS/MS assays. The development of cocktail assays containing multiple CYP substrates facilitates an increase of another 9-fold, and the introduction of fast SRM and fast polarity switching triple quadrupole mass spectrometers enables the substrates and/or products of multiple CYP reactions to be measured in a single analysis. Together, these experimental designed and UHPLC-MS/MS features enable investigations of drug-botanical interactions, an important consumer safety issue, that are approximately 100-fold faster than was possible previously.
References & Acknowledgements:
(1) van Breemen RB. Development of safe and effective botanical dietary supplements. J. Med. Chem. 2015; Jul 21. [Epub ahead of print]
(2) Li G, Huang K, Nikolic D, van Breemen RB. High-throughput cytochrome P450 cocktail inhibition assay for assessing drug-drug and drug-botanical interactions. Drug Metab. Dispos. 2015; 43: 1670-1678.
(3) Sprouse AA, van Breemen RB. Pharmacokinetic interactions between drugs and botanical dietary supplements. Drug Metab. Dispos. 2015; Oct 5.[Epub ahead of print]
This research was supported by NIH grants P50 AT000155, R01 AT007659 and T32 AT007533 from the Office of the Director (T32), Office of Dietary Supplements (P50) and the National Center for Complementary and Integrative Health (T32, R01 and P50). We thank Shimadzu Scientific Instruments for providing the Nexera UHPLC systems and the LCMS-8050 and LCMS-8060 triple quadrupole mass spectrometers used during these investigations.
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