Protein Abundance of Clinically Relevant Multidrug Transporters

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Protein Abundance of Clinically Relevant Multidrug Transporters along the Entire Length of the Human Intestine Marek Drozdzik,† Christian Gröer,‡ Jette Penski,‡ Joanna Lapczuk,† Marek Ostrowski,§ Yurong Lai,∥ Bhagwat Prasad,⊥ Jashvant D. Unadkat,⊥ Werner Siegmund,‡ and Stefan Oswald*,‡ †

Department of Experimental and Clinical Pharmacology, Pomeranian Medical University, Szczecin, Poland Department of Clinical Pharmacology, University Medicine Greifswald, D-17487 Greifswald, Germany § Department of General and Transplantation Surgery, Pomeranian Medical University, Szczecin, Poland ∥ Department of Pharmacokinetics, Dynamics and Metabolism, Pfizer Global Research and Development, Groton, Connecticut 06340, United States ⊥ Department of Pharmaceutics, University of Washington, Seattle, Washington 98195-7610, United States ‡

S Supporting Information *

ABSTRACT: 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 ( ABCB1 (0.29−1.06 pmol/mg) > ABCC3 (0.51−0.70 pmol/mg) > ABCG2 (0.19−0.41 pmol/mg). A further quarter of the total transporter protein abundance was ascribed to the uptake carriers OCT1 (0.57−0.84 pmol/mg) > OATP2B1 (0.43−0.56 pmol/mg) > ASBT (0.01−1.58 pmol/mg) > OCT3 (0.05− 0.06 pmol/mg) (Figures 1−3). In the colon, the major transporter was the basolateral efflux carrier ABCC3 (1.53−2.11 pmol/mg), which comprised more than one-third of the total transporter protein abundance. The efflux carriers ABCC2 (0.95−1.77 pmol/mg) > ABCB1 (0.15− 0.37 pmol/mg) > ABCG2 (0.04−0.16 pmol/mg) and the uptake transporters OCT1 (0.47−0.73 pmol/mg) = OATP2B1 (0.48−0.73 pmol/mg) > PEPT1 (0.19−0.31 pmol/mg) > OCT3 (0.11−0.14 pmol/mg) > ASBT (traces) accounted for the other two-thirds (Figures 1−3). The mRNA expression and protein content of ABCB1, ABCG2, ASBT, and PEPT1 were found to be significantly higher in the small intestine than in colonic tissue (each p < 0.001; Figures 2 and 3). In the case of ABCC2, a significantly lower level of mRNA expression but not protein content was found in the colon compared to the segments of the small intestine. By contrast, significantly higher protein levels in the colon than in the small intestine were observed for ABCC3 and OCT3, whereas mRNA expression did not differ. For OCT1 and OATP2B1, neither mRNA expression nor protein amounts differed between the small and large intestine (Figure 3). The protein content of ABCB1 and ASBT significantly increased along the small intestine (duodenum < jejunum < ileum) (Figure 4). Significant positive correlations between gene expression in the small intestine and protein content were observed for ABCG2 (r = 0.494; p = 0.006), ASBT (r = 0.498; p = 0.006), OCT3 (r = 0.595; p < 0.001), and PEPT1 (r = 0.578; p < 0.001).

in duplicate using custom, predesigned TaqMan low-density array cards, and real-time RT-PCR analysis was performed using the 7900HT Sequence Detection System and the SDSRQ Manager 1.2 software for data analysis (Applied Biosystems). Mean Ct (cycles of threshold) values were used for analysis using 18S rRNA as the reference gene and the 2−ΔCt values for relative quantitative comparisons. Protein Quantification by LC−MC/MS. Sample preparation and protein quantification of ABCB1, ABCC2, ABCC3, ABCG2, OATP1A2, OATP2B1, OCT1, OCT3, PEPT1, and ASBT were accomplished by mass spectrometry-based targeted proteomics using validated LC−MS/MS methods as recently described.24 Accuracy (error) and precision (CV) during sample analysis were both below 20%. Final protein expression data (picomoles per milligram) were calculated by normalization to the total protein content of the isolated membrane fraction as determined by the bicinchoninic acid assay (Pierce, Rockford, IL). All samples were digested and measured in duplicate. To prove the functionality of our assay, wild-type HEK-293 cells and OATP1A2-transfected HEK-293 cells generated in our department and described elsewhere25 as well as healthy brain tissue that was kindly provided by S. Bien (Department of Neurosurgery, University Medicine Greifswald) were analyzed in the same manner as the intestinal samples. Statistical Analysis. Intestinal target gene expression was calculated as expression relative to the endogenous control 18S rRNA (2−ΔCt values). All mRNA and protein expression data are presented as means ± the standard deviation (median as well as minimum and maximum values are given in Table 1 of the Supporting Information). Sample differences were evaluated using the nonparametric Mann−Whitney-U test and statistical trends using the Jonckheere−Terpstra test. Correlations between samples were assessed using the Spearman rank test. p values of jejunum > ileum) of PEPT1 along the human small intestine may be due to the mode of action of this transporter. The PEPT1-mediated transport is an electrogenic H+-coupled cotransport driven by the presence of an inward H+ gradient.43 Considering, furthermore, that the luminal pH increases (i.e., H+ concentration decreases) from the proximal (pH 6.0) to distal small intestine (∼7.5),44 it becomes obvious that the intestinal pH gradient together with the transporter expression pattern creates the aforementioned proximal absorption window for PEPT1 substrates. A second consideration is that the transporter with the highest relative protein abundance in the colon is efflux carrier ABCC3, which is also expressed in all segments of the small intestine, although with a protein abundance lower than those of ABCC2 and ABCB1. ABCC3 serves as a major driver for the enterohepatic circulation of bile acids in coordinate interaction with ABCC2, ASBT, and OSTα/OSTβ.30 Despite its rather high level of expression along the entire intestine, its role in drug absorption remains uncertain and should be considered in future studies. ABCC3 shares many substrates with ABCC2, 3552

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have to be taken into account to explain the aforementioned drug interactions. An example is the recently published interaction of green tea β-blocker nadolol, which was shown to be an in vitro substrate of OATP1A2.56 While green tea and grapefruit juice were shown to be potent OATP1A2 inhibitors in vitro, only green tea, not grapefruit juice, caused a significant drug interaction with nadolol in healthy subjects.50,52,56 Because of the descriptive character of our expression analysis, we must acknowledge that our observed protein expression data may not necessarily translate directly to transporter function, considering the different transporter kinetics (e.g., high affinity and high capacity for ABCB1 but low affinity and high capacity for PEPT1), their different driving forces (e.g., ATP for ABC transporters; protons for PEPT1), and their distinct localization and activity. These aspects should be addressed in future studies using appropriate ex vivo systems (e.g., permeability studies with the Ussing chamber). Finally, although our study is, to the best of our knowledge, the first of its kind that has analyzed the intrasubject transporter expression along the entire length of the human intestine, we have included samples from only six body donors. Consequently, our expression data show high variability and should be replicated in a larger sample set. Data for intestinal transporter protein expression and function do not solely explain the complexity of oral drug absorption but are an important additional aspect for gaining deeper insights into intestinal drug absorption. Thus, intestinal transporter data should always be seen in context with other factors such as liberation characteristics of the particular drug dosage form, intestinal transit time, absorptive surface area, the real (free) intestinal drug concentration, the intestinal solubility of the drug with its dependence on the luminal pH and the availability and kinetics of free intestinal water, and, finally, the expression of metabolizing enzymes. Conclusions. Our study provides, for the first time, a comprehensive overview of the gene and protein expression patterns of clinically relevant drug transporters along the entire length of the human intestine. Along with in vitro affinity data, our results may represent further pieces of the puzzle of physiological data required by physiologically based pharmacokinetic modeling approaches such as SimCyp and GastroPlus to predict the extent and rate of intestinal drug absorption or intestinal drug interactions.21 This work thereby may improve our understanding of intestinal drug absorption.

most notably glucuronides and sulfates of lipophilic compounds (e.g., bilirubin, estrogens, and dehydroepiandrosterone) or large anionic substances (e.g., leukotrienes, gadoxetate, and rosuvastatin).45,46 Because ABCC3 is localized to the basolateral membrane of enterocytes, it may be involved in effluxing drugs or drug conjugates formed by intestinal UDPglucuronosyltransferases or sulfotransferases into portal blood, an alternative route to the luminally directed efflux via ABCC2. There is also some evidence of interplay of ABCC3 with apical uptake carriers (PEPT1 and OATP2B1) allowing vectorial apical-to-basal transfer from the gut lumen toward portal venous blood, as hypothesized for cefadroxil (PEPT1), talinolol, and fexofenadine (OATP2B1).47−49 Finally, we could not detect OATP1A2 in the intestine, although this protein was previously detected by immunohistochemistry in human intestinal samples.50 Our findings are in line with the lack of SLCO1A2 (encoding OATP1A2) gene expression that was also found in other previous studies.9,10 To confirm the functionality of our proteomic assay for OATP1A2, we applied our method to OATP1A2-transfected HEK293 cells and human brain tissue as a known site of OATP1A2 expression. As expected, OATP1A2 could not be detected in parental HEK cells, whereas the OATP1A2 protein content was 6.92 ± 1.05 pmol/mg in transfected cells and 0.25 ± 0.07 pmol/mg in human brain tissue, which are comparable to the protein amounts that have been observed by Uchida et al. (Figure 6).51

Figure 6. Protein levels of OATP1A2 as observed in HEK293 cells (left), OATP1A2-trasfected HEK293 cells (middle), and human brain tissue (right). All data were generated from three individual samples. Protein amounts (picomoles per milligram) are presented as means ± the standard deviation (the asterisk denotes a value below the limit of quantification).

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ASSOCIATED CONTENT

S Supporting Information *

Detailed mRNA expression data for the additionally measured transporters and nuclear receptors involved in the intestinal absorption of drugs, nutrients, and endogenous compounds as observed in intestines from six donors (Table 1) and correlations between intestinal mRNA expression of ABC transporters and nuclear receptors in intestinal tissues from six donors (Table 2). This material is available free of charge via the Internet at http://pubs.acs.org.

According to the current understanding, OATP1A2 is assumed to be an intestinal uptake carrier for several drugs such as talinolol, fexofenadine, and aliskiren, as concluded from in vitro studies using OATP1A2-transfected cells and drug interaction studies with inhibitors of OATP1A2.52−54 However, considering our findings, this conclusion remains questionable. By contrast, we observed moderate and homogeneous OATP2B1 protein abundance throughout all segments of the human gut. In our opinion, it appears, therefore, reasonable to conclude that most of the hypothesized OATP1A2-mediated intestinal uptake processes can be attributed to OATP2B1 because most OATP1A2 substrates also have affinity for OATP2B1, and experimental inhibitors for OATP1A2 (e.g., ingredients of grapefruits, oranges, apples, and other herbs) also modulate intestinal OATP2B1 function.53,55 However, in some cases, other transporters or even completely other mechanisms

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AUTHOR INFORMATION

Corresponding Author

*Department of Clinical Pharmacology, Center of Drug Absorption and Transport, University Medicine Greifswald, Felix-Hausdorff-Str. 3, D-17487 Greifswald, Germany. Phone: +49-3834-865643. Fax: +49-3834-865631. E-mail: stefan. [email protected]. 3553

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Author Contributions

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M.D. and C.G. acquired data, analyzed and interpreted data, and contributed to a draft of the manuscript. J.P., J.L., M.O., B.P., and Y.L. acquired and analyzed data. J.D.U. and W.S. developed the study concept and revised the manuscript. S.O. acquired data, developed the study concept, analyzed data, contributed to a draft of the manuscript, revised the manuscript, and supervised the study. Notes

The authors declare the following competing financial interest(s): Y.L. was an employee of Pfizer Inc. while this study was being conducted. All other authors declare no conflict of interest.

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ACKNOWLEDGMENTS This study was supported by the German Federal Ministry for Education and Research (Grant 03IPT612X, InnoProfileTransfer).

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dx.doi.org/10.1021/mp500330y | Mol. Pharmaceutics 2014, 11, 3547−3555