Quantitative Proteomics of Clinically Relevant Drug-Metabolizing Enzymes and Drug Transporters and Their Intercorrelations in the Human Small Intestine

Abstract

The levels of drug-metabolizing enzymes (DMEs) and transporter proteins in the human intestine are pertinent to determine oral drug bioavailability. Despite the paucity of reports on such measurements, it is well recognized that these values are essential for translating in vitro data on drug metabolism and transport to predict drug disposition in gut wall. In the current study, clinically relevant DMEs [cytochrome P450 (P450) and uridine 59-diphospho-glucuronosyltransferase (UGT)] and drug transporters were quantified in total mucosal protein preparations from the human jejunum (n 5 4) and ileum (n 5 12) using quantification concatemer-based targeted proteomics. In contrast to previous reports, UGT2B15 and organic anion-transporting polypeptide 1 (OATP1A2) were quantifiable in all our samples. Overall, no significant disparities in protein expression were observed between jejunum and ileum. Relative mRNA expression for drug transporters did not correlate with the abundance of their cognate protein, except for P-glycoprotein 1 (P-gp) and organic solute transporter subunit alpha (OST-a), highlighting the limitations of RNA as a surrogate for protein expression in dynamic tissues with high turnover. Intercorrelations were found within P450 [2C9-2C19 (P 5 0.002, R 2 5 0.63), 2C9-2J2 (P 5 0.004, R 2 5 0.40), 2D6-2J2 (P 5 0.002, R 2 5 0.50)] and UGT [1A1-2B7 (P 5 0.02, R 2 5 0.87)] family of enzymes. There were also correlations between P-gp and several other proteins [OST-a (P < 0.0001, R 2 5 0.77), UGT1A6 (P 5 0.009, R 2 5 0.38), and CYP3A4 (P 5 0.007, R 2 5 0.30)]. Incorporating such correlations into building virtual populations is crucial for obtaining plausible characteristics of simulated individuals. SIGNIFICANCE STATEMENTA number of drug transporters were quantified for the first time in this study. Several intercorrelations of protein abundance were reported. mRNA expression levels proved to be a poor reflection of differences between individuals regarding the level of protein expression in gut. The reported abundance of drug-metabolizing enzymes and transporters and their intercorrelations will contribute to better predictions of oral drug bioavailability and drug-drug interactions by linking in vitro observations to potential outcomes through physiologically based pharmacokinetic models.

Label-Free Quantitative Proteomics and Substrate-Based Mass Spectrometry Imaging of Xenobiotic Metabolizing Enzymes in Ex Vivo Human Skin and a Human Living Skin Equivalent Model

“…The final version may differ from this version. mass spectrometry-based quantitative proteomics studies of human liver and intestine (Couto et al, 2019(Couto et al, , 2020. A similarly high interbatch variability is seen across the two independent replicates of LabSkin.…”

“…Although several artificial human skin models have been proposed, they are limited by a lack of understanding of xenobiotic metabolism and transport in human skin. Efforts have been made to understand XME gene expression in human skin and in vitro skin models using reverse transcription polymerase chain reaction (Kazem et al, 2019); however, it is increasingly accepted that mRNA abundance is a poor proxy for protein abundance and activity (Maier et al, 2009;Couto et al, 2020). In this study, we used a combination of label-free quantitative mass spectrometry and substrate-based mass spectrometry imaging to assess xenobiotic metabolism in human skin and an in vitro artificial skin model (LabSkin) and have quantified several XMEs for the first time in these systems.…”

“…Cytochrome P450 enzymes are especially prevalent in the liver (Couto et al, 2019;El-Khateeb et al, 2019), and to a lesser extent the intestine (Couto et al, 2020). In the skin only three cytochrome P450 enzymes, namely CYP51A1 (0.98 ± 0.09 pmol mg -1 ), CYP7B1 (2.57 ± 0.45 pmol mg -1 ) and CYP8A1 (5.52 ± 4.70 pmol mg -1 ) were identified and quantified.…”

“…Several 3D skin models have been developed (Cantòn et al, 2010;Chau et al, 2013;Mathes et al, 2014), but none has been satisfactorily validated against human skin, in part because we lack understanding of xenobiotic-metabolizing enzyme (XME) pathways in human skin. The relatively few published studies on drug metabolism in human skin have mainly focussed on identifying and quantifying enzymes and pathways known to have a role in drug metabolism in other organs such as the liver and the intestine (Couto et al, 2019(Couto et al, , 2020El-Khateeb et al, 2019). Skin is a challenging tissue to analyse using traditional proteomic techniques owing to the high lipid content, and the insolubility and extensive cross-linking of proteins.…”

Effects of single green tea ingestion on pharmacokinetics of nadolol in healthy volunteers

“…8,9 Nadolol has been found to be a substrate of OATP1A2, and green tea catechins such as EGCG potently inhibit OATP1A2-mediated nadolol uptake. 6,8,9 Based on the assumption that OATP1A2 plays a role in the absorptive transport of nadolol in intestine, 14,15 it is suggested that the inhibition of OATP1A2 by catechins could lead to a reduction of intestinal absorption of nadolol. To gain insight into the effects of green tea on nadolol pharmacokinetics, the extent of green tea-nadolol interaction as indexed by % decrease in AUC of nadolol was plotted against estimated oral dose of EGCG in the different studies.…”