Intestinal transporters are crucial determinants in the oral absorption of many drugs. We therefore studied the mRNA expression (N = 33) and absolute protein content (N = 10) of clinically relevant transporters in healthy epithelium of the duodenum, the proximal and distal jejunum and ileum, and the ascending, transversal, descending, and sigmoidal colon of six organ donors (24-54 years). In the small intestine, the abundance of nearly all studied proteins ranged between 0.2 and 1.6 pmol/mg with the exception of those of OCT3 (<0.1 pmol/mg) and PEPT1 (2.6-4.9 pmol/mg) that accounted for ∼50% of all measured transporters. OATP1A2 was not detected in any intestinal segment. ABCB1, ABCG2, PEPT1, and ASBT were significantly more abundant in jejunum and ileum than in colon. In contrast to this, the level of expression of ABCC2, ABCC3, and OCT3 was found to be highest in colon. Site-dependent differences in the levels of gene and protein expression were observed for ABCB1 and ASBT. Significant correlations between mRNA and protein levels have been found for ABCG2, ASBT, OCT3, and PEPT1 in the small intestine. Our data provide further physiological pieces of the puzzle required to predict intestinal drug absorption in humans.

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.

No abstract

No abstract

Abstract. Intestinal transporter proteins affect the oral bioavailability of many drugs in a significant manner. In order to estimate or predict their impact on oral drug absorption, data on their intestinal expression levels are needed. So far, predominantly mRNA expression data are available which are not necessarily correlated with the respective protein content. All available protein data were assessed by immunoblotting techniques such as Western blotting which both possess a number of limitations for reliable protein quantification. In contrast to this, mass spectrometry-based targeted proteomics may represent a promising alternative method to provide comprehensive protein expression data. In this review, we will summarize so far available intestinal mRNA and protein expression data for relevant human multidrug transporters. Moreover, recently observed mass spectrometry-based targeted proteomic data will be presented and discussed with respect to potential functional consequences. Associated to this, we will provide a short tutorial how to set up these methods and emphasize critical aspects in method development. Finally, potential limitations and pitfalls of this emerging technique will be discussed. From our perspective, LC-MS/MS-based targeted proteomics represents a valuable new method to comprehensively analyse the intestinal expression of transporter proteins. The resulting expression data are expected to improve our understanding about the intestinal processing of drugs.