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Wisniewski, J. R., Wegler, C. & Artursson, P. (2019). Multiple-Enzyme-Digestion Strategy Improves Accuracy and Sensitivity of Label- and Standard-Free Absolute Quantification to a Level That Is Achievable by Analysis with Stable Isotope-Labeled Standard Spiking. Journal of Proteome Research, 18(1), 217-224
Open this publication in new window or tab >>Multiple-Enzyme-Digestion Strategy Improves Accuracy and Sensitivity of Label- and Standard-Free Absolute Quantification to a Level That Is Achievable by Analysis with Stable Isotope-Labeled Standard Spiking
2019 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 18, no 1, p. 217-224Article in journal (Refereed) Published
Abstract [en]

Quantification of individual proteins is an essential task in understanding biological processes. For example, determination of concentrations of proteins transporting and metabolizing xenobiotics is a prerequisite for drug disposition predictions in humans based on in vitro data. So far, this task has frequently been accomplished by targeted proteomics. This type of analyses requires preparation of stable isotope labeled standards for each protein of interest. The selection of appropriate standard peptides is usually tedious and the number of proteins that can be studied in a single experiment by these approaches is limited. In addition, incomplete digestion of proteins often affects the accuracy of the quantification. To circumvent these constrains in proteomic protein quantification, label- and standard-free approaches, such as "total protein approach" (TPA) have been proposed. Here we directly compare an approach using stable isotope labeled (SIL) standards and TPA for quantification of transporters and enzymes in human liver samples within the same LC-MS/MS runs. We show that TPA is a convenient alternative to SIL-based methods. Optimization of the sample preparation beyond commonly used single tryptic digestion, by adding consecutive cleavage steps, improves accuracy and reproducibility of the TPA method to a level, which is achievable by analysis using stable isotope-labeled standard spiking.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2019
Keywords
FASP, MED FASP, total protein approach (TPA), stable isotope labeling, quantitative analysis, drug transporter, drug metabolizing enzymes, ADME
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-375231 (URN)10.1021/acs.jproteome.8b00549 (DOI)000455285900019 ()30336047 (PubMedID)
Funder
Swedish Research Council, 1951Swedish Research Council, 5715
Available from: 2019-01-29 Created: 2019-01-29 Last updated: 2019-07-23Bibliographically approved
Wegler, C. (2019). Proteomics-informed analysis of drug disposition in the human liver and small intestine. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Proteomics-informed analysis of drug disposition in the human liver and small intestine
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Orally administered drugs are absorbed in the intestine and generally metabolized in the liver. Therefore, understanding factors determining drug distribution and elimination in these tissues is important. This thesis aimed at using mass spectrometry (MS)-based proteomics and functional studies to better understand in vitro model systems used for drug clearance predictions. Further, it aimed at understanding the changes in drug disposition caused by obesity and gastric bypass surgery (GBP).

The study was initiated by investigating factors influencing MS-based protein quantification by comparing results from different proteomics methods, and by studying protein distribution during subcellular fractionation. The largest variability in protein quantification was ascribed to insufficient enrichment from subcellular fractionation, most likely due to collection of the majority of the proteins in the initial fraction of the fractionation protocols.

Proteomics and metabolic activity analyses were then used to investigate differences in intrinsic clearance from two commonly used in vitro systems, human liver microsomes and hepatocytes. For some compounds, the faster microsomal metabolism could be explained by a higher available unbound drug concentration and CYP content in the microsomes as compared to in the hepatocytes.

Next, inter-individual protein expression variability in human liver and jejunum was explored. This showed that proteins covered a wide inter-individual variability spectrum, in which proteins with low variabilities were associated with essential cellular functions, while many proteins with high variabilities were disease-related.

Further, the effects of obesity, GBP, and weight loss on the proteomes of human liver and jejunum were analyzed. After GBP and subsequent weight loss, patients showed lower levels of jejunal proteins involved in inflammatory response and drug metabolism.

Finally, proteomics data from patients with and without obesity was combined with parameters from in vitro transport kinetics, and a mechanistic model to predict drug disposition was developed. The model successfully predicted rosuvastatin plasma concentrations in the patients.

In conclusion, this thesis has provided insights into factors influencing protein quantification and function in vitro. Furthermore, this thesis demonstrates how proteomics contributes to improved understanding of inter-individual and physiological differences, and how it can be used for in vitro-in vivo scaling of drug clearance.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 79
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 273
Keywords
proteomics, protein concentration, drug disposition, drug transport, drug metabolism, human small intestine, human liver, human hepatocytes, human liver microsomes, inter-individual variability, drug clearance, obesity, prediction model
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-389741 (URN)978-91-513-0694-0 (ISBN)
Public defence
2019-09-13, B42, Biomedical center, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-08-22 Created: 2019-07-26 Last updated: 2019-08-22
Weiss, F., Hammer, H. S., Klein, K., Planatscher, H., Zanger, U. M., Norén, A., . . . Poetz, O. (2018). Direct Quantification of Cytochromes P450 and Drug Transporters-A Rapid, Targeted Mass Spectrometry-Based Immunoassay Panel for Tissues and Cell Culture Lysates. Drug Metabolism And Disposition, 46(4), 387-396
Open this publication in new window or tab >>Direct Quantification of Cytochromes P450 and Drug Transporters-A Rapid, Targeted Mass Spectrometry-Based Immunoassay Panel for Tissues and Cell Culture Lysates
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2018 (English)In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 46, no 4, p. 387-396Article in journal (Refereed) Published
Abstract [en]

The quantification of drug metabolizing enzymes and transporters has recently been revolutionized on the basis of targeted proteomic approaches. Isotope-labeled peptides are used as standards for the quantification of the corresponding proteins in enzymatically fragmented samples. However, hurdles in these approaches are low throughput and tedious sample prefractionation steps prior to mass spectrometry (MS) readout. We have developed an assay platform using sensitive and selective immunoprecipitation coupled with mass spectrometric readout allowing the quantification of proteins directly from whole cell lysates using less than 20,000 cells per analysis. Peptide group-specific antibodies (triple X proteomics antibodies) enable the enrichment of proteotypic peptides sharing a common terminus. These antibodies were employed to establish a MS-based immunoassay panel for the quantification of 14 cytochrome P450 (P450) enzymes and nine transporters. We analyzed the P450 enzyme and transporter levels in genotyped liver tissue homogenates and microsomes, and in samples from a time course induction experiment in human hepatocytes addressing different induction pathways. For the analysis of P450 enzymes and transporters only a minute amount of sample is required and no prefractionation is necessary, thus the assay platform bears the potential to bridge cell culture model experiments and results from whole organ tissue studies.

Place, publisher, year, edition, pages
AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-353108 (URN)10.1124/dmd.117.078626 (DOI)000426966600007 ()29343608 (PubMedID)
Funder
Swedish Research Council, 2822Swedish Research Council, 5715
Available from: 2018-06-11 Created: 2018-06-11 Last updated: 2019-07-23Bibliographically approved
Karlgren, M., Simoff, I., Backlund, M., Wegler, C., Keiser, M., Handin, N., . . . Artursson, P. (2017). A CRISPR-Cas9 Generated MDCK Cell Line Expressing Human MDR1 Without Endogenous Canine MDR1 (cABCB1): An Improved Tool for Drug Efflux Studies.. Journal of Pharmaceutical Sciences, 106(9), 2909-2913
Open this publication in new window or tab >>A CRISPR-Cas9 Generated MDCK Cell Line Expressing Human MDR1 Without Endogenous Canine MDR1 (cABCB1): An Improved Tool for Drug Efflux Studies.
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2017 (English)In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 106, no 9, p. 2909-2913Article in journal (Refereed) Published
Abstract [en]

Madin-Darby canine kidney (MDCK) II cells stably transfected with transport proteins are commonly used models for drug transport studies. However, endogenous expression of especially canine MDR1 (cMDR1) confounds the interpretation of such studies. Here we have established an MDCK cell line stably overexpressing the human MDR1 transporter (hMDR1; P-glycoprotein), and used CRISPR-Cas9 gene editing to knockout the endogenous cMDR1. Genomic screening revealed the generation of a clonal cell line homozygous for a 4-nucleotide deletion in the canine ABCB1 gene leading to a frameshift and a premature stop codon. Knockout of cMDR1 expression was verified by quantitative protein analysis and functional studies showing retained activity of the human MDR1 transporter. Application of this cell line allowed unbiased reclassification of drugs previously defined as both substrates and non-substrates in different studies using commonly used MDCK-MDR1 clones. Our new MDCK-hMDR1 cell line, together with a previously developed control cell line, both with identical deletions in the canine ABCB1 gene and lack of cMDR1 expression represent excellent in vitro tools for use in drug discovery.

Keywords
ABC transporters, MDCK cells, P-glycoprotein, drug transport, efflux pumps, genomics, membrane transporter, multidrug resistance transporters, permeability, proteomics
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-343598 (URN)10.1016/j.xphs.2017.04.018 (DOI)000417339900089 ()28450237 (PubMedID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2018-02-28 Created: 2018-02-28 Last updated: 2019-07-23Bibliographically approved
Mateus, A., Treyer, A., Wegler, C., Karlgren, M., Matsson, P. & Artursson, P. (2017). Intracellular drug bioavailability: a new predictor of system dependent drug disposition. Scientific Reports, 7, 1-12, Article ID 43047.
Open this publication in new window or tab >>Intracellular drug bioavailability: a new predictor of system dependent drug disposition
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, p. 1-12, article id 43047Article in journal (Refereed) Published
Abstract [en]

Intracellular drug exposure is influenced by cell-and tissue-dependent expression of drug-transporting proteins and metabolizing enzymes. Here, we introduce the concept of intracellular bioavailability (F-ic) as the fraction of extracellular drug available to bind intracellular targets, and we assess how Fic is affected by cellular drug disposition processes. We first investigated the impact of two essential drug transporters separately, one influx transporter (OATP1B1; SLCO1B1) and one efflux transporter (P-gp; ABCB1), in cells overexpressing these proteins. We showed that OATP1B1 increased Fic of its substrates, while P-gp decreased Fic. We then investigated the impact of the concerted action of multiple transporters and metabolizing enzymes in freshly-isolated human hepatocytes in culture configurations with different levels of expression and activity of these proteins. We observed that Fic was up to 35-fold lower in the configuration with high expression of drug-eliminating transporters and enzymes. We conclude that Fic provides a measurement of the net impact of all cellular drug disposition processes on intracellular bioavailable drug levels. Importantly, no prior knowledge of the involved drug distribution pathways is required, allowing for high-throughput determination of drug access to intracellular targets in highly defined cell systems (e.g., single-transporter transfectants) or in complex ones (including primary human cells).

National Category
Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-317940 (URN)10.1038/srep43047 (DOI)000394530900001 ()28225057 (PubMedID)
Available from: 2017-04-01 Created: 2017-04-01 Last updated: 2019-07-23Bibliographically approved
Wegler, C., Gaugaz, F. Z., Andersson, T. B., Wisniewsk, J. R., Busch, D., Gröer, C., . . . Artursson, P. (2017). Variability in Mass Spectrometry-based Quantification of Clinically Relevant Drug Transporters and Drug Metabolizing Enzymes. Molecular Pharmaceutics, 14(9), 3142-3151
Open this publication in new window or tab >>Variability in Mass Spectrometry-based Quantification of Clinically Relevant Drug Transporters and Drug Metabolizing Enzymes
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2017 (English)In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 14, no 9, p. 3142-3151Article in journal (Refereed) Published
Abstract [en]

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.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2017
Keywords
drug transporters, drug metabolizing enzymes, membrane proteins, protein quantification, targeted proteomics, label-free proteomics
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-335414 (URN)10.1021/acs.molpharmaceut.7b00364 (DOI)000410005100027 ()28767254 (PubMedID)
Funder
Swedish Research Council, 2822, 5715
Available from: 2017-12-06 Created: 2017-12-06 Last updated: 2019-07-26Bibliographically approved
Hammer, H. S., Poetz, O., Artursson, P. & Wegler, C. (2016). Development of MS- based immunoassays for Cytochrome P450 and transporter quantification. Paper presented at 82nd Annual Meeting of the German-Society-for-Exerimental-and-Clinical-Pharmacology-and-Toxicology (DGPT) / 18th Annual Meeting of the Network-Clinical-Pharmacology-Germany (VKliPha), FEB 29-MAR 03, 2016, Berlin, GERMANY. Naunyn-Schmiedeberg's Archives of Pharmacology, 389(1), S47-S47
Open this publication in new window or tab >>Development of MS- based immunoassays for Cytochrome P450 and transporter quantification
2016 (English)In: Naunyn-Schmiedeberg's Archives of Pharmacology, ISSN 0028-1298, E-ISSN 1432-1912, Vol. 389, no 1, p. S47-S47Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
SPRINGER, 2016
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-347386 (URN)000398368200192 ()
Conference
82nd Annual Meeting of the German-Society-for-Exerimental-and-Clinical-Pharmacology-and-Toxicology (DGPT) / 18th Annual Meeting of the Network-Clinical-Pharmacology-Germany (VKliPha), FEB 29-MAR 03, 2016, Berlin, GERMANY
Available from: 2018-03-29 Created: 2018-03-29 Last updated: 2019-07-23Bibliographically approved
Wegler, C., Gaugaz, F. Z., Andersson, T. B., Wisniewski, J. R., Busch, D., Oswald, S., . . . Artursson, P. (2016). Protein quantification of human hepatic drug transporters and metabolizing enzymes: an inter-laboratory and methodological comparison. Paper presented at 20th North American Meeting of the International-Society-for-the-Study-of-Xenobiotics (ISSX), OCT 18-22, 2015, Orlando, FL. Drug metabolism reviews (Softcover ed.), 48, 98-98
Open this publication in new window or tab >>Protein quantification of human hepatic drug transporters and metabolizing enzymes: an inter-laboratory and methodological comparison
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2016 (English)In: Drug metabolism reviews (Softcover ed.), ISSN 0360-2532, E-ISSN 1097-9883, Vol. 48, p. 98-98Article in journal, Meeting abstract (Other academic) Published
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-303431 (URN)000380744900201 ()
Conference
20th North American Meeting of the International-Society-for-the-Study-of-Xenobiotics (ISSX), OCT 18-22, 2015, Orlando, FL
Available from: 2016-11-21 Created: 2016-09-19 Last updated: 2019-07-23Bibliographically approved
Wegler, C., Matsson, P., Krogstad, V., Urdzik, J., Christensen, H., Andersson, T. B. & Artursson, P.Bridging differences in CYP activity between donor-matched human liver microsomes and hepatocytes.
Open this publication in new window or tab >>Bridging differences in CYP activity between donor-matched human liver microsomes and hepatocytes
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(English)Manuscript (preprint) (Other academic)
Keywords
Human liver microsomes, Human hepatocytes, Proteomics, Kpuu, Clearance
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-389737 (URN)
Available from: 2019-07-23 Created: 2019-07-23 Last updated: 2019-07-26
Wegler, C., Robertsen, I., Wisniewski, J. R., Hjelmesæth, J., Åsberg, A., Andersson, T. B. & Artursson, P.Effects of obesity and weight loss on global protein expression in human liver and jejunum.
Open this publication in new window or tab >>Effects of obesity and weight loss on global protein expression in human liver and jejunum
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(English)Manuscript (preprint) (Other academic)
Keywords
Proteomics, Obesity, Gastric bypass, Human liver, Human jejunum
National Category
Cell and Molecular Biology
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-389739 (URN)
Available from: 2019-07-23 Created: 2019-07-23 Last updated: 2019-07-26
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0002-2810-7518

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