Diana Busch

Absolute protein quantification of clinically relevant cytochrome P450 enzymes and UDP-glucuronosyltransferases by mass spectrometry-based targeted proteomics

Cytochrome P450 (CYP) enzymes and UDP-glucuronosyltransferases (UGT) are major determinants in the pharmacokinetics of most drugs on the market. To investigate their impact on intestinal and hepatic drug metabolism, we developed and validated quantification methods for nine CYP (CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, CYP2E1, CYP3A4 and CYP3A5) and four UGT enzymes (UGT1A1, UGT1A3, UGT2B7 and UGT2B15) that have been shown to be of clinical relevance in human drug metabolism. Protein quantification was performed by targeted proteomics using liquid chromatography with tandem mass spectrometry (LC-MS/MS)-based determination of enzyme specific peptides after tryptic digestion using in each case stable isotope labelled peptides as internal standard. The chromatography of the respective peptides was performed with gradient elution using a reversed phase (C18) column (Ascentis(®) Express Peptide ES-C18, 100mm×2.1mm, 2.7μm) and 0.1% formic acid (FA) as well as acetonitrile with 0.1% FA as mobile phases at a flow rate of 300μl/min. The MS/MS detection of all peptides was done simultaneously with a scheduled multiple reaction monitoring (MRM) method in the positive mode by monitoring in each case three mass transitions per proteospecific peptide and the internal standard. The assays were validated according to current bioanalytical guidelines with respect to specificity, linearity (0.25-50nM), within-day and between-day accuracy and precision, digestion efficiency as well as stability. Finally, the developed method was successfully applied to determine the CYP and UGT protein amount in human liver and intestinal microsomes. The method was shown to possess sufficient specificity, sensitivity, accuracy, precision and stability to quantify clinically relevant human CYP and UGT enzymes.

Variability in Mass Spectrometry-based Quantification of Clinically Relevant Drug Transporters and Drug Metabolizing Enzymes

Many different methods are used for mass-spectrometry-based protein quantification in pharmacokinetics and systems pharmacology. It has not been established to what extent the results from these various methods are comparable. Here, we compared six different mass spectrometry-based proteomics methods by measuring the expression of clinically relevant drug transporters and metabolizing enzymes in human liver. Mean protein concentrations were in general quantified to similar levels by methods using whole tissue lysates. Methods using subcellular membrane fractionation gave incomplete enrichment of the proteins. When the enriched proteins were adjusted to levels in whole tissue lysates, they were on average 4-fold lower than those quantified directly in whole tissue lysates. The differences in protein levels were propagated into differences in predictions of hepatic clearance. In conclusion, caution is needed when comparing and applying quantitative proteomics data obtained with different methods, especially since membrane fractionation is common practice for protein quantification used in drug clearance predictions.

Caco‐2 cells – expression, regulation and function of drug transporters compared with human jejunal tissue

BACKGROUND Induction or inhibition of drug transporting proteins by concomitantly administered drugs can cause serious drug-drug interactions (DDIs). However, in vitro assays currently available are mostly for studying the inhibitory potential of drugs on intestinal transporter proteins, rather than induction. Therefore, this study investigated the suitability of the frequently used intestinal Caco-2 cell line to predict transporter-mediated DDIs as caused by induction via activation of nuclear receptors. METHODS TaqMan® low density arrays and LC-MS/MS based targeted proteomics were used to evaluate transporter expression in Caco-2 cells in comparison with jejunal tissue, in culture-time dependence studies and after incubation with different known inducers of drug metabolism and transport. Additionally, studies on ABCB1 function were performed using Transwell® assays with [3 H]-digoxin and [3 H]-talinolol as substrates after incubation with the prototypical inducers rifampicin, St Johns wort, carbamazepine and efavirenz. RESULTS The gene and protein expression pattern of drug transporters in Caco-2 cells and jejunal tissue differed considerably. For some transporters culture-time dependent differences in mRNA expression and/or protein abundance could be determined. Finally, none of the studied prototypical inducers showed an effect either on mRNA expression and protein abundance or on the function of ABCB1. CONCLUSION Differences in transporter expression in Caco-2 cells compared with jejunal tissue, as well as expression dependence on culture time must be considered in in vitro studies to avoid under- or overestimation of certain transporters. The Caco-2 cell model is not suitable for the evaluation of DDIs caused by transporter induction. Copyright

Protein Abundance of Clinically Relevant Drug‐Metabolizing Enzymes in the Human Liver and Intestine: A Comparative Analysis in Paired Tissue Specimens

This work revises and complements existing findings on the distribution of drug‐metabolizing enzymes in the first‐pass effect organs. We explored gene expression (quantitative polymerase chain reaction) and protein abundance (liquid chromatography/ tandem mass spectrometry) of CYP1A2, CYP2B6, CYP2C8/9/19, CYP2D6, CYP2E1, CYP3A4/5, UGT1A1/3, UGT2B7/15 in the liver, duodenum, jejunum, ileum, and colon in paired tissues from nine organ donors. All proteins were detected in the liver, but in the intestine CYP2C9/19, CYP2D6, CYP3A4/5, UGT1A1/3, and UGT2B7 were found. CYP3A4 showed comparable abundance in the liver and jejunum, whereas other enzymes were markedly higher in the hepatic tissue. Nearly all detected enzymes showed their highest abundance in the jejunum. Significant correlations between mRNA and protein levels in liver or intestine were found for most enzymes. CYP3A4 and CYP3A5 protein abundance, but not other enzymes, were significantly correlated in the liver and the small intestine. Our data may contribute to an improved understanding of hepatic and intestinal drug metabolism.

LC-MS/MS method for the simultaneous quantification of intestinal CYP and UGT activity

HIGHLIGHTSDevelopment of an analytical method for the simultaneous quantification ofprobe substrates for human intestinal enzymesSimultaneous quantification of CYP and UGT metabolites (so far almost exclusively CYP substrates).Comprehensive method validation according to current bioanalytical guidelines.Similar/superior sensitivity compared to previously published methods.Application of the method to characterize the metabolic activity of intestinalCYP2C9 / 2C19 / 2D6 / CYP3A and UGT1A1 / 2B7 ABSTRACT Many orally administered drugs are subject to first‐pass metabolism by cytochrome P450 (CYP) enzymes and uridine 5′‐diphospho‐glucuronosyltransferases (UGT). While their hepatic activity is well characterized, respective information about the intestine are very scare due to limited availability of tissue, very low microsomal protein content and the heterogeneity of the individual segments. As a consequence, determination of enzyme kinetic parameters is challenging. It was therefore the aim of this study to develop a sensitive liquid chromatography tandem mass spectrometry method for the simultaneous quantification of CYP and UGT metabolites formed by clinically relevant intestinal biotransformation enzymes: 4‐hydroxydiclofenac (CYP2C9), 5‐hydroxyomeprazole (CYP2C19), dextrorphan (CYP2D6), 1‐hydroxymidazolam (CYP3A), ezetimibe glucuronide (UGT1A) and naloxone glucuronide (UGT2B7). After precipitation of microsomal protein with acetonitrile, analytes were chromatographically separated on a C18 column with gradient elution using acetonitrile and water, both containing 0.1% formic acid and detected with a tandem mass spectrometer operating in positive mode with electron spray ionization. The assay was validated according to current bioanalytical guidelines regarding linearity, accuracy, precision, stability, recovery and matrix effects spanning an analytical range from 1 to 200 nmol/L for each analyte. The developed method was successfully applied to a proof of concept experiment using pooled human jejunal microsomes (50 &mgr;g protein/mL) in order to determine enzyme kinetic parameters. Formation of all monitored metabolites followed Michaelis‐Menten kinetics and allowed calculation of KM and Vmax values. The developed method may be useful for characterization of enzymatic activity in the human intestine which may allow more precise insights into the intestinal contribution to first pass metabolism of drugs.

Quantitative characterization of UDP-glucuronosyltransferase 2B17 in human liver and intestine and its role in testosterone first-pass metabolism

Graphical abstract Figure. No Caption available. Abstract Protein abundance and activity of UGT2B17, a highly variable drug‐ and androgen‐metabolizing enzyme, were quantified in microsomes, S9 fractions, and primary cells isolated from human liver and intestine by validated LC‐MS/MS methods. UGT2B17 protein abundance showed >160‐fold variation (mean ± SD, 1.7 ± 2.7 pmol/mg microsomal protein) in adult human liver microsomes (n = 26) and significant correlation (r2 = 0.77, p < 0.001) with testosterone glucuronide (TG) formation. Primary role of UGT2B17 in TG formation compared to UGT2B15 was confirmed by performing activity assays in UGT2B17 gene deletion samples and with a selective UGT2B17 inhibitor, imatinib. Human intestinal microsomes isolated from small intestine (n = 6) showed on average significantly higher protein abundance (7.4 ± 6.6 pmol/mg microsomal protein, p = 0.016) compared to liver microsomes, with an increasing trend towards distal segments of the gastrointestinal (GI) tract. Commercially available pooled microsomes and S9 fractions confirmed greater abundance and activity of UGT2B17 in intestinal fractions compared to liver fractions. To further investigate the quantitative role of UGT2B17 in testosterone metabolism in whole cell system, a targeted metabolomics study was performed in hepatocytes (n = 5) and enterocytes (n = 16). TG was the second most abundant metabolite after androstenedione in both cell systems. Reasonable correlation between UGT2B17 abundance and activity were observed in enterocytes (r2 = 0.69, p = 0.003), but not in hepatocytes. These observational and mechanistic data will be useful in developing physiologically‐based pharmacokinetic (PBPK) models for predicting highly‐variable first‐pass metabolism of testosterone and other UGT2B17 substrates.

Expression of clinically relevant drug-metabolizing enzymes along the human intestine and their correlation to drug transporters and nuclear receptors: An intra-subject analysis

The oral bioavailability of many drugs is highly influenced not only by hepatic but also by intestinal biotransformation. To estimate the impact of intestinal phase I and II metabolism on oral drug absorption, knowledge on the expression levels of the respective enzymes is an essential prerequisite. In addition, the potential interplay of metabolism and transport contributes to drug disposition. Both mechanisms may be subjected to coordinative regulation by nuclear receptors, leading to unwanted drug‐drug interactions due to induction of intestinal metabolism and transport. Thus, it was the aim of this study to comprehensively analyse the regional expression of clinically relevant phase I and II enzymes along the entire human intestine and to correlate these data to expression data of drug transporters and nuclear receptors of pharmacokinetic relevance. Gene expression of 11 drug‐metabolizing enzymes (CYP2B6, 2C8, 2C9, 2C19, 2D6, 3A4, 3A5, SULT1A, UGT1A, UGT2B7, UGT2B15) was studied in duodenum, jejunum, ileum and colon from six organ donors by real‐time RT‐PCR. Enzyme expression was correlated with expression data of the nuclear receptors PXR, CAR and FXR as well as drug transporters observed in the same cohort. Intestinal expression of all studied metabolizing enzymes was significantly higher in the small intestine compared to colonic tissue. CYP2B6, CYP2C9, CYP2C19, CYP2D6, CYP3A4/5, SULT1A, UGT1A and UGT2B7 expression increased from the duodenum to jejunum but was markedly lower in the ileum. In the small intestine, that is, the predominant site of drug absorption, the highest expression has been observed for CYP3A4, CYP2C9, SULT1A and UGT1A. In addition, significant correlations were found between several enzymes and PXR as well as ABC transporters in the small intestine. In conclusion, the observed substantial site‐dependent intestinal expression of several enzymes may explain regional differences in intestinal drug absorption. The detected correlations between intestinal enzymes, transporters and nuclear receptors provide indirect evidence for their coordinative expression, regulation and function in the human small intestine.