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The Proteome of Filter-Grown Caco-2 Cells With a Focus on Proteins Involved in Drug Disposition
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
Max Planck Inst Biochem, Biochem Prote Grp, Dept Prote & Signal Transduct, D-82152 Martinsried, Germany.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.ORCID iD: 0000-0002-9094-2581
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
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2016 (English)In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 105, no 2, p. 817-827Article in journal (Refereed) Published
Abstract [en]

Caco-2 cells are widely used in studies of intestinal cell physiology and drug transport. Here, the global proteome of filter-grown Caco-2 cells was quantified using the total protein approach and compared with the human colon and jejunum proteomes. In total, 8096 proteins were identified. In-depth analysis of proteins defining enterocyte differentiation—including brush-border hydrolases, integrins, and adherens and tight junctions—gave near-complete coverage of the expected proteins. Three hundred twenty-seven absorption, distribution, metabolism and excretion proteins were identified, including 112 solute carriers and 20 ATP-binding cassette transporters. OATP2B1 levels were 16-fold higher in Caco-2 cells than in jejunum. To investigate the impact of this difference on in vitro-in vivo extrapolations, we studied the uptake kinetics of the OATP2B1 substrate pitavastatin in Caco-2 monolayers, and found that the contribution of OATP2B1 was 60%-70% at clinically relevant intestinal concentrations. Pitavastatin kinetics was combined with transporter concentrations to model the contribution of active transport and membrane permeation in the jejunum. The lower OATP2B1 expression in jejunum led to a considerably lower transporter contribution (<5%), suggesting that transmembrane diffusion dominates pitavastatin absorption in vivo. In conclusion, we present the first in-depth quantification of the filter-grown Caco-2 proteome. We also demonstrate the crucial importance of considering transporter expression levels for correct interpretation of drug transport routes across the human intestine.

Place, publisher, year, edition, pages
2016. Vol. 105, no 2, p. 817-827
National Category
Pharmaceutical Sciences
Identifiers
URN: urn:nbn:se:uu:diva-277281DOI: 10.1016/j.xphs.2015.10.030ISI: 000381768500048PubMedID: 26869432OAI: oai:DiVA.org:uu-277281DiVA, id: diva2:904408
Funder
Swedish Research Council, 2822Carl Tryggers foundation Available from: 2016-02-18 Created: 2016-02-18 Last updated: 2019-04-24Bibliographically approved
In thesis
1. Proteomic and Functional Analysis of In Vitro Systems for Studies of Drug Disposition in the Human Small Intestine and Liver
Open this publication in new window or tab >>Proteomic and Functional Analysis of In Vitro Systems for Studies of Drug Disposition in the Human Small Intestine and Liver
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

To reach the bloodstream, an orally administered drug must be absorbed through the small intestine and avoid extensive clearance in the liver. Estimating these parameters in vitro is therefore important in drug discovery and development. This can be achieved with cellular models that simulate human organ function, such as Caco-2 cells and primary hepatocytes. No model fits every scenario, however, and this thesis aimed at using proteomic and functional analysis to better understand and increase the applicability of in vitro models based on Caco-2 cells and human hepatocytes.

First, the proteome of filter-grown Caco-2 cells was analyzed. This included near-complete coverage of enterocyte-related proteins, and over 300 ADME proteins. Further, by scaling uptake transport kinetics from Caco-2 cells to human jejunum, the importance of considering in vitro­-in vivo expression differences to correctly interpret in vitro transport studies was demonstrated.

Focus was then turned to hepatocytes, where proteomics was used as a basis for the successful development of an apoptosis inhibition protocol for restoration of attachment properties and functionality in suboptimal batches of cryopreserved human hepatocytes. As a spin-off project, image-based quantification of cell debris was developed into a novel apoptosis detection method.

Next, the in vivo heterogeneity of human hepatocytes was explored in an in vitro setting, where it was observed that human hepatocyte batches contain a wide range of cell sizes. By separating the cells into different size fractions, it was found that hepatocyte size corresponds to the microarchitectural zone of origin in the liver. Size separation can thus be used to study zonated liver functions in vitro.

Finally, the proteomes of the major types of non-parenchymal liver cells were analyzed, i.e. liver sinusoidal endothelial cells, Kupffer cells, and hepatic stellate cells. The different cell types all had distinctly different proteomes, and the expression of certain important ADME proteins indicated that non-parenchymal cells participate in drug disposition.

In conclusion, this thesis has improved the phenotypic understanding and extended the applicability of Caco-2 cells and primary human hepatocytes, two of the most important in vitro models for studies of small intestinal and hepatic drug disposition.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 59
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 272
Keywords
proteomics, drug disposition, ADMET, drug transport, drug metabolism, hepatotoxicity, small intestine, liver, caco-2, human hepatocytes, cryopreservation, apoptosis, liver zonation, non-parenchymal cells
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-382406 (URN)978-91-513-0668-1 (ISBN)
Public defence
2019-06-14, Room B41, Biomedical center, Husargatan 3, Uppsala, 09:15 (English)
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Available from: 2019-05-21 Created: 2019-04-24 Last updated: 2019-06-18

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