Gene Expression and Protein Abundance of Nuclear Receptors in Human Intestine and Liver: A New Application for Mass Spectrometry-Based Targeted Proteomics.

BACKGROUND: Unwanted drug-drug interactions (DDIs), as caused by the upregulation of clinically relevant drug metabolizing enzymes and transporter proteins in intestine and liver, have the potential to threaten the therapeutic efficacy and safety of drugs. The molecular mechanism of this undesired but frequently occurring scenario of polypharmacy is based on the activation of nuclear receptors such as the pregnane X receptor (PXR) or the constitutive androstane receptor (CAR) by perpetrator agents such as rifampin, phenytoin or St. John's wort. However, the expression pattern of nuclear receptors in human intestine and liver remains uncertain, which makes it difficult to predict the extent of potential DDIs. Thus, it was the aim of this study to characterize the gene expression and protein abundance of clinically relevant nuclear receptors, i.e., the aryl hydrocarbon receptor (AhR), CAR, farnesoid X receptor (FXR), glucocorticoid receptor (GR), hepatocyte nuclear factor 4 alpha (HNF4α), PXR and small heterodimer partner (SHP), in the aforementioned organs. METHODS: Gene expression analysis was performed by quantitative real-time PCR of jejunal, ileal, colonic and liver samples from eight human subjects. In parallel, a targeted proteomic method was developed and validated in order to determine the respective protein amounts of nuclear receptors in human intestinal and liver samples. The LC-MS/MS method was validated according to the current bioanalytical guidelines and met the criteria regarding linearity (0.1-50 nmol/L), within-day and between-day accuracy and precision, as well as the stability criteria. RESULTS: The developed method was successfully validated and applied to determine the abundance of nuclear receptors in human intestinal and liver samples. Gene expression and protein abundance data demonstrated marked differences in human intestine and liver. On the protein level, only AhR and HNF4α could be detected in gut and liver, which corresponds to their highest gene expression. In transfected cell lines, PXR and CAR could be quantified. CONCLUSIONS: The substantially different expression pattern of nuclear receptors in human intestinal and liver tissue may explain the different extent of unwanted DDIs in the dependence on the administration route of drugs.

Transcriptional and Post-Transcriptional Regulation of Duodenal P-Glycoprotein and MRP2 in Healthy Human Subjects after Chronic Treatment with Rifampin and Carbamazepine.

To predict the outcome of intestinal drug transporter induction on pharmacokinetics, signaling of the DNA message along with messenger RNA (mRNA) transcription and protein translation leading to transporter function must be understood. We quantified the gene expression of PXR and CAR, gene expression and protein abundance of P-glycoprotein (P-gp), multidrug-resistance-associated protein 2 (MRP2) and breast-cancer-resistance protein, the content of 754 microRNAs in human duodenal biopsy specimens, and pharmacokinetics of talinolol and ezetimibe before and after the treatment with rifampin and carbamazepine. Rifampin significantly induced the transcription of ABCB1 and ABCC2 and protein abundance of P-gp but not of MRP2. The abundance of P-gp was significantly correlated to the plasma exposure of ezetimibe and its glucuronide. Carbamazepine induced the mRNA expressions of CAR, ABCB1, and ABCC2 but did not elevate protein abundance. Using in silico prediction tools and luciferase reporter assays, microRNAs were identified that can contribute to ligand-specific regulation of intestinal drug transporters and different changes in drug disposition after induction with rifampin and carbamazepine.

Membrane Transporters in Human Parotid Gland-Targeted Proteomics Approach.

Salivary glands provide secretory functions, including secretion of xenobiotics and among them drugs. However, there is no published information about protein abundance of drug transporters measured using reliable protein quantification methods. Therefore, mRNA expression and absolute protein content of clinically relevant ABC (n = 6) and SLC (n = 15) family member transporters in the human parotid gland, using the qRT-PCR and liquid chromatography‒tandem mass spectrometry (LC-MS/MS) method, were studied. The abundance of nearly all measured proteins ranged between 0.04 and 0.45 pmol/mg (OCT3 > MRP1 > PEPT2 > MRP4 > MATE1 > BCRP). mRNAs of ABCB1, ABCC2, ABCC3, SLC10A1, SLC10A2, SLC22A1, SLC22A5, SLC22A6, SLC22A7, SLC22A8, SLCO1A2, SLCO1B1, SLCO1B3 and SLCO2B1 were not detected. The present study provides, for the first time, information about the protein abundance of membrane transporters in the human parotid gland, which could further be used to define salivary bidirectional transport (absorption and secretion) mechanisms of endogenous compounds and xenobiotics.

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.

Defining Human Pathways of Drug Metabolism In Vivo through the Development of a Multiple Humanized Mouse Model.

Variability in drug pharmacokinetics is a major factor in defining drug efficacy and side effects. There remains an urgent need, particularly with the growing use of polypharmacy, to obtain more informative experimental data predicting clinical outcomes. Major species differences in multiplicity, substrate specificity, and regulation of enzymes from the cytochrome P450-dependent mono-oxygenase system play a critical role in drug metabolism. To develop an in vivo model for predicting human responses to drugs, we generated a mouse, where 31 P450 genes from the Cyp2c, Cyp2d, and Cyp3a gene families were exchanged for their relevant human counterparts. The model has been improved through additional humanization for the nuclear receptors constitutive androgen receptor and pregnane X receptor that control the expression of key drug metabolizing enzymes and transporters. In this most complex humanized mouse model reported to date, the cytochromes P450 function as predicted and we illustrate how these mice can be applied to predict drug-drug interactions in humans.

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.

Protein Abundance of Clinically Relevant Drug Transporters in the Human Liver and Intestine: A Comparative Analysis in Paired Tissue Specimens.

Bioavailability of orally administered drugs is partly determined by function of drug transporters in the liver and intestine. Therefore, we explored adenosine triphosphate-binding cassette (ABC) and solute carriers family transporters expression (quantitative polymerase chain reaction) and protein abundance (liquid chromatography tandem mass spectrometry (LC-MS/MS)) in human liver and duodenum, jejunum, ileum, and colon in paired tissue specimens from nine organ donors. The transporter proteins were detected in the liver (permeability-glycoprotein (P-gp), multidrug resistance protein (MRP)2, MRP3, breast cancer resistance protein (BCRP), organic anion-transporting polypeptide (OATP)1B1, OATP1B3, OATP2B1, organic cation transporter (OCT)1, OCT3, organic anion transporter 2, Na+-taurocholate cotransporting polypeptide, monocarboxylate transporter (MCT)1, and multidrug and toxin extrusion 1) and the intestine (P-gp, multidrug-resistance protein (MRP)2, MRP3, MRP4, BCRP, OATP2B1, OCT1, apical sodium-bile acid transporter (only ileum), MCT1, and peptide transporter (PEPT1)). Significantly higher hepatic gene expression and protein abundance of ABCC2/MRP2, SLC22A1/OCT1, and SLCO2B1/OATP2B1 were found, as compared to all intestinal segments. No correlations between hepatic and small intestinal protein levels were observed. These observations provide a description of drug transporters distribution without the impact of interindividual variability bias and may help in construction of superior physiologically based pharmacokinetic and humanized animal models.

Dysregulation of Mucosal Membrane Transporters and Drug-Metabolizing Enzymes in Ulcerative Colitis.

Intestinal transporters and metabolizing enzymes are the important factors of the intestinal absorption barrier. Because there is evidence that their expression and function may be affected during inflammatory conditions, we investigated gene expression, protein abundance, and regulation of relevant intestinal transporters and metabolizing enzymes in the intestinal mucosa of patients with ulcerative colitis (UC). Specimens from inflamed and noninflamed tissues of 10 patients with UC as well as colonic control tissues of 10 patients without inflammation were subjected to gene (9 enzymes, 15 transporters, 9 cytokines) and microRNA (N = 54) expression analysis. Protein abundance was quantified by liquid chromatography-tandem mass spectrometry-based targeted proteomics. Gene expression of several metabolizing enzymes (e.g., CYP2C9, UGT1A1) and transporters such as ABCB1 (ABCB1), ABCG2 (ABCG2), and monocarboxylate transporter 1 (MCT1, SLC16A1) were significantly decreased during inflammation and negatively correlated to microRNAs. On contrary, multidrug resistance-protein 4 (MRP4, ABCC4), organic anion-transporting polypeptide 2B1 (OATP2B1, SLCO2B1), and organic cation transporter-like 2 (ORCTL2, SLC22A18) were significantly elevated in inflamed tissue. However, at protein level, these findings could only be confirmed for MCT1. UC is associated with complex changes in the intestinal expression of enzymes, transporters, cytokines, and microRNAs, which may affect efficacy of anti-inflammatory drug therapy or the disease state itself.

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

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 µg 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.

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.

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

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 ±â€¯SD, 1.7 ±â€¯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 ±â€¯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.