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  • 1.
    Hammer, H. S.
    et al.
    Univ Tubingen, Nat Wissensch & Med Inst, Reutlingen, Germany..
    Poetz, O.
    Univ Tubingen, Nat Wissensch & Med Inst, Reutlingen, Germany..
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Wegler, Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Development of MS- based immunoassays for Cytochrome P450 and transporter quantification2016In: Naunyn-Schmiedeberg's Archives of Pharmacology, ISSN 0028-1298, E-ISSN 1432-1912, Vol. 389, no 1, p. S47-S47Article in journal (Other academic)
  • 2.
    Karlgren, Maria
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University Drug Optimization and Pharmaceutical Profiling Platform, Science for Life Laboratory.
    Simoff, Ivailo
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Backlund, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University Drug Optimization and Pharmaceutical Profiling Platform, Science for Life Laboratory.
    Wegler, Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. AstraZeneca.
    Keiser, Markus
    Department of Clinical Pharmacology, Center of Drug Absorption and Transport, University Medicine of Greifswald, Greifswald, Germany..
    Handin, Niklas
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Müller, Janett
    Department of Clinical Pharmacology, Center of Drug Absorption and Transport, University Medicine of Greifswald, Greifswald, Germany..
    Lundquist, Patrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Jareborg, Anne-Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Immunology, Genetics and Pathology. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Oswald, Stefan
    Department of Clinical Pharmacology, Center of Drug Absorption and Transport, University Medicine of Greifswald, Greifswald, Germany..
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University Drug Optimization and Pharmaceutical Profiling Platform, Science for Life Laboratory.
    A CRISPR-Cas9 Generated MDCK Cell Line Expressing Human MDR1 Without Endogenous Canine MDR1 (cABCB1): An Improved Tool for Drug Efflux Studies.2017In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 106, no 9, p. 2909-2913Article in journal (Refereed)
    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.

  • 3.
    Mateus, André
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Treyer, Andrea
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Wegler, Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. AstraZeneca R&D, Cardiovasc & Metab Dis Innovat Med, DMPK, SE-43183 Molndal, Sweden..
    Karlgren, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Matsson, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Intracellular drug bioavailability: a new predictor of system dependent drug disposition2017In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, p. 1-12, article id 43047Article in journal (Refereed)
    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).

  • 4.
    Wegler, Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Proteomics-informed analysis of drug disposition in the human liver and small intestine2019Doctoral 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.

    List of papers
    1. Variability in Mass Spectrometry-based Quantification of Clinically Relevant Drug Transporters and Drug Metabolizing Enzymes
    Open this publication in new window or tab >>Variability in Mass Spectrometry-based Quantification of Clinically Relevant Drug Transporters and Drug Metabolizing Enzymes
    Show others...
    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
    2. Subcellular fractionation of human liver reveals limits in global proteomic quantification from isolated fractions
    Open this publication in new window or tab >>Subcellular fractionation of human liver reveals limits in global proteomic quantification from isolated fractions
    2016 (English)In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 509, p. 82-88Article in journal (Refereed) Published
    Abstract [en]

    The liver plays an important role in metabolism and elimination of xenobiotics, including drugs. Determination of concentrations of proteins involved in uptake, distribution, metabolism, and excretion of xenobiotics is required to understand and predict elimination mechanisms in this tissue. In this work, we have fractionated homogenates of snap -frozen human liver by differential centrifugation and performed quantitative mass spectrometry -based proteomic analysis of each fraction. Concentrations of proteins were calculated by the "total protein approach". A total of 4586 proteins were identified by at least five peptides and were quantified in all fractions. We found that the xenobiotics transporters of the canalicular and basolateral membranes were differentially enriched in the subcellular fractions and that phase I and II metabolizing enzymes, the cytochrome P450s and the UDP glucuronyl transferases, have complex subcellular distributions. These findings show that there is no simple way to scale the data from measurements in arbitrarily selected membrane fractions using a single scaling factor for all the proteins of interest. This study also provides the first absolute quantitative subcellular catalog of human liver proteins obtained from frozen tissue specimens. Our data provide quantitative insights into the sub cellular distribution of proteins and can be used as a guide for development of fractionation procedures.

    Keywords
    Human liver, Subcellular fractionation, Absolute quantitative proteomics, Total protein approach, Drug metabolism, Drug transport
    National Category
    Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
    Identifiers
    urn:nbn:se:uu:diva-303255 (URN)10.1016/j.ab.2016.06.006 (DOI)000380866800013 ()27311553 (PubMedID)
    Funder
    Swedish Research Council, 2822
    Available from: 2016-09-16 Created: 2016-09-15 Last updated: 2019-07-26Bibliographically approved
    3. 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
    Show others...
    (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
    4. Global expression variability of proteins across and within human tissues
    Open this publication in new window or tab >>Global expression variability of proteins across and within human tissues
    Show others...
    (English)In: Article in journal (Other academic) Submitted
    Place, publisher, year, edition, pages
    Uppsala:
    Keywords
    Expression variability, Human liver, Human jejunum, Proteomics, Transcriptomics, Reference genes
    National Category
    Cell and Molecular Biology
    Research subject
    Pharmaceutical Science
    Identifiers
    urn:nbn:se:uu:diva-389738 (URN)
    Available from: 2019-07-23 Created: 2019-07-23 Last updated: 2019-07-26
    5. 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
    Show others...
    (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
    6. Proteomics-informed prediction of rosuvastatin clearance in donors with and without obesity
    Open this publication in new window or tab >>Proteomics-informed prediction of rosuvastatin clearance in donors with and without obesity
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Keywords
    Proteomics, Plasma drug distribution, Pharmacokinetics, Uptake clearance, Prediction model
    National Category
    Pharmaceutical Sciences
    Research subject
    Pharmaceutical Science
    Identifiers
    urn:nbn:se:uu:diva-389740 (URN)
    Available from: 2019-07-23 Created: 2019-07-23 Last updated: 2019-07-26
  • 5.
    Wegler, Christine
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. AstraZeneca R&D, Innovat Med & Early Dev Biotech Unit, Cardiovasc & Metab Dis, S-43150 Molndal, Sweden..
    Gaugaz, Fabienne Z.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Andersson, Tommy B.
    AstraZeneca R&D, Innovat Med & Early Dev Biotech Unit, Cardiovasc & Metab Dis, S-43150 Molndal, Sweden..
    Wisniewsk, Jacek R.
    Max Planck Inst Biochem, Dept Prote & Signal Transduct, Biochem Prote Grp, D-82152 Martinsried, Germany..
    Busch, Diana
    Univ Med Greifswald, Dept Clin Pharmacol, Ctr Drug Absorpt & Transport, D-17489 Greifswald, Germany..
    Gröer, Christian
    Univ Med Greifswald, Dept Clin Pharmacol, Ctr Drug Absorpt & Transport, D-17489 Greifswald, Germany..
    Oswald, Stefan
    Univ Med Greifswald, Dept Clin Pharmacol, Ctr Drug Absorpt & Transport, D-17489 Greifswald, Germany..
    Norén, Agneta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Upper Abdominal Surgery.
    Weiss, Frederik
    Univ Tubingen, NMI Nat & Med Sci Inst, D-72770 Reutlingen, Germany..
    Hammer, Helen S.
    Univ Tubingen, NMI Nat & Med Sci Inst, D-72770 Reutlingen, Germany..
    Joos, Thomas O.
    Univ Tubingen, NMI Nat & Med Sci Inst, D-72770 Reutlingen, Germany..
    Poetz, Oliver
    Univ Tubingen, NMI Nat & Med Sci Inst, D-72770 Reutlingen, Germany..
    Achour, Brahim
    Univ Manchester, Ctr Appl Pharmacokinet Res, Manchester M13 9PL, Lancs, England..
    Rostami-Hodjegan, Amin
    Univ Manchester, Ctr Appl Pharmacokinet Res, Manchester M13 9PL, Lancs, England..
    van de Steeg, Evita
    TNO, Netherlands Org Appl Sci Res, NL-3700 AJ Zeist, Netherlands..
    Wortelboer, Heleen M.
    TNO, Netherlands Org Appl Sci Res, NL-3700 AJ Zeist, Netherlands..
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Variability in Mass Spectrometry-based Quantification of Clinically Relevant Drug Transporters and Drug Metabolizing Enzymes2017In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 14, no 9, p. 3142-3151Article in journal (Refereed)
    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.

  • 6.
    Wegler, Christine
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Gaugaz, Fabienne Z.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Andersson, Tommy B.
    AstraZeneca R&D, Cardiovasc & Metab Dis iMed DMPK, Molndal, Sweden..
    Wisniewski, Jacek R.
    Max Planck Inst Biochem, Dept Prote & Signal Transduct, Martinsried, Germany..
    Busch, Diana
    Univ Med Greifswald, Ctr Drug Absorpt & Transport C DAT, Greifswald, Germany..
    Oswald, Stefan
    Univ Med Greifswald, Ctr Drug Absorpt & Transport C DAT, Dept Pharmacol & Clin Pharmacol, Greifswald, Germany..
    Weiss, Frederik
    Univ Tubingen, NMI Nat & Med Sci Inst, Tubingen, Germany..
    Hammer, Helen
    Univ Tubingen, NMI Nat & Med Sci Inst, Tubingen, Germany..
    Joos, Thomas O.
    Univ Tubingen, NMI Nat & Med Sci Inst, Tubingen, Germany..
    Poetz, Oliver
    Univ Tubingen, NMI Nat & Med Sci Inst, Tubingen, Germany..
    Wortelboer, Heleen M.
    TNO, Zeist, Netherlands..
    van de Steeg, Evita
    TNO, Zeist, Netherlands..
    Achour, Brahim
    Univ Manchester, Manchester Pharm Sch, Ctr Appl Pharmacokinet Res, Manchester, Lancs, England..
    Rostami, Amin
    Univ Manchester, Manchester Pharm Sch, Ctr Appl Pharmacokinet Res, Manchester, Lancs, England..
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Protein quantification of human hepatic drug transporters and metabolizing enzymes: an inter-laboratory and methodological comparison2016In: Drug metabolism reviews (Softcover ed.), ISSN 0360-2532, E-ISSN 1097-9883, Vol. 48, p. 98-98Article in journal (Other academic)
  • 7.
    Wegler, Christine
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University.
    Matsson, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Krogstad, Veronica
    Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo.
    Urdzik, Jozef
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Upper Abdominal Surgery.
    Christensen, Hege
    Department of Pharmaceutical Biosciences, School of Pharmacy, University of Oslo.
    Andersson, Tommy B
    DMPK, Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Bridging differences in CYP activity between donor-matched human liver microsomes and hepatocytesManuscript (preprint) (Other academic)
  • 8.
    Wegler, Christine
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Prieto Garcia, Luna
    DMPK, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
    Klinting, Signe
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Robertsen, Ida
    Department of Pharmacy, University of Oslo, Oslo, Norway.
    Wisniewski, Jacek R
    Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
    Hjelmesæth, Jøran
    Morbid Obesity Centre, Department of Medicine, Vestfold Hospital Trust, Tønsberg, Norway.
    Åsberg, Anders
    Department of Pharmacy, University of Oslo, Oslo, Norway.
    Jansson-Löfmark, Rasmus
    DMPK, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
    Andersson, Tommy B
    DMPK, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Proteomics-informed prediction of rosuvastatin clearance in donors with and without obesityManuscript (preprint) (Other academic)
  • 9.
    Wegler, Christine
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Robertsen, Ida
    Department of Pharmacy, University of Oslo, Oslo, Norway.
    Wisniewski, Jacek R
    Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
    Hjelmesæth, Jøran
    Morbid Obesity Centre, Department of Medicine, Vestfold Hospital Trust, Tønsberg, Norway.
    Åsberg, Anders
    Department of Pharmacy, University of Oslo, Oslo, Norway.
    Andersson, Tommy B
    DMPK, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Effects of obesity and weight loss on global protein expression in human liver and jejunumManuscript (preprint) (Other academic)
  • 10.
    Wegler, Christine
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Wikvall, Kjell
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Norlin, Maria
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmaceutical Biosciences.
    Effects of osteoporosis-inducing drugs on vitamin D-related gene transcription and mineralization in MG-63 and Saos-2 cells2016In: Basic & Clinical Pharmacology & Toxicology, ISSN 1742-7835, E-ISSN 1742-7843, Vol. 119, no 5, article id BCPT12612Article in journal (Refereed)
    Abstract [en]

    Vitamin D3 is important for calcium and phosphate homeostasis. To exert its effects, vitamin D3 has to be enzymatically activated into 1,25D3 (1,25-dihydroxyvitamin D3). Regulation by endogenous vitamin D metabolites of the activation and inactivation of 1,25D3 is important to maintain adequate amounts of active vitamin D3. Vitamin D deficiency and low bone mineral density have been linked to treatments with antiretroviral drugs and glucocorticoids. However, the causes of drug-induced osteoporosis remain unclear. The antiretroviral drugs efavirenz and ritonavir as well as the glucocorticoid dexamethasone were included in this study. Their effects on transcription of vitamin D-regulating enzymes in MG-63 cells were investigated. Ritonavir and dexamethasone both induced transcription of CYP27B1, the enzyme responsible for formation of 1,25D3. Efavirenz, however, suppressed CYP27B1 expression. When administered together with endogenous vitamin D metabolites, dexamethasone and efavirenz counteracted the 1,25D3-mediated up-regulation of CYP24A1, which inactivates 1,25D3. This suggests that the drugs may interfere with local regulation of the vitamin D metabolizing system in osteoblasts. Studies on mineralization were performed in MG-63 cells and Saos-2 cells by measuring calcium concentrations accumulated over time. The effects of efavirenz, ritonavir and dexamethasone and/or vitamin D metabolites were examined. 1,25D3 induced mineralization in both cell lines. Efavirenz administered alone did not affect mineralization but abrogated the inducing effects of 1,25D3 on mineralization in both MG-63 cells and Saos-2 cells. In summary, the results suggest that antiretroviral drugs and glucocorticoids may adversely affect bone by interference with the vitamin D-system in osteoblasts.

  • 11.
    Wegler, Christine
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University.
    Ölander, Magnus
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Wisniewski, Jacek R
    Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
    Lundquist, Patrik
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Zettl, Katharina
    Biochemical Proteomics Group, Department of Proteomics and Signal Transduction, Max Planck Institute of Biochemistry, Martinsried, Germany.
    Åsberg, Anders
    Department of Pharmacy, University of Oslo, Oslo, Norway.
    Hjelmesæth, Jøran
    Morbid Obesity Centre, Department of Medicine, Vestfold Hospital Trust, Tønsberg, Norway.
    Andersson, Tommy B
    DMPK, Research and Early Development Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca, Gothenburg, Sweden.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Global expression variability of proteins across and within human tissuesIn: Article in journal (Other academic)
  • 12.
    Weiss, Frederik
    et al.
    Univ Tubingen, NMI Nat & Med Sci Inst, Markwiesenstr 55, D-72770 Reutlingen, Germany;SIGNATOPE GmbH, Reutlingen, Germany.
    Hammer, Helen S.
    Univ Tubingen, NMI Nat & Med Sci Inst, Markwiesenstr 55, D-72770 Reutlingen, Germany;SIGNATOPE GmbH, Reutlingen, Germany.
    Klein, Kathrin
    Dr Margarete Fischer Bosch Inst Clin Pharmacol, Stuttgart, Germany;Univ Tubingen, Dept Clin Pharmacol, Tubingen, Germany.
    Planatscher, Hannes
    Univ Tubingen, NMI Nat & Med Sci Inst, Markwiesenstr 55, D-72770 Reutlingen, Germany;SIGNATOPE GmbH, Reutlingen, Germany.
    Zanger, Ulrich M.
    Dr Margarete Fischer Bosch Inst Clin Pharmacol, Stuttgart, Germany;Univ Tubingen, Dept Clin Pharmacol, Tubingen, Germany.
    Norén, Agneta
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Surgical Sciences, Upper Abdominal Surgery.
    Wegler, Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Innovat Med & Early Dev Biotech Unit, Cardiovasc & Metab Dis, Molndal, Sweden.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Joos, Thomas O.
    Univ Tubingen, NMI Nat & Med Sci Inst, Markwiesenstr 55, D-72770 Reutlingen, Germany;SIGNATOPE GmbH, Reutlingen, Germany.
    Poetz, Oliver
    Univ Tubingen, NMI Nat & Med Sci Inst, Markwiesenstr 55, D-72770 Reutlingen, Germany;SIGNATOPE GmbH, Reutlingen, Germany.
    Direct Quantification of Cytochromes P450 and Drug Transporters-A Rapid, Targeted Mass Spectrometry-Based Immunoassay Panel for Tissues and Cell Culture Lysates2018In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 46, no 4, p. 387-396Article in journal (Refereed)
    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.

  • 13.
    Wisniewski, Jacek R.
    et al.
    Max Planck Inst Biochem, Dept Prote & Signal Transduct, Biochem Prote Grp, Klopferspitz 18, D-82152 Martinsried, Germany.
    Wegler, Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. AstraZeneca, Cardiovasc Renal & Metab, Innovat Med & Early Dev Biotech Unit, Gothenburg, Sweden.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    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 Spiking2019In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 18, no 1, p. 217-224Article in journal (Refereed)
    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.

  • 14.
    Wisniewski, Jacek R.
    et al.
    Max Planck Inst Biochem, Dept Prote & Signal Transduct, Biochem Prote Grp, D-82152 Martinsried, Germany..
    Wegler, Christine
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. AstraZeneca, Innovat Med & Early Dev Biotech Unit, Cardiovasc & Metab Dis, Pepparedsleden 1, S-43183 Molndal, Sweden..
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Subcellular fractionation of human liver reveals limits in global proteomic quantification from isolated fractions2016In: Analytical Biochemistry, ISSN 0003-2697, E-ISSN 1096-0309, Vol. 509, p. 82-88Article in journal (Refereed)
    Abstract [en]

    The liver plays an important role in metabolism and elimination of xenobiotics, including drugs. Determination of concentrations of proteins involved in uptake, distribution, metabolism, and excretion of xenobiotics is required to understand and predict elimination mechanisms in this tissue. In this work, we have fractionated homogenates of snap -frozen human liver by differential centrifugation and performed quantitative mass spectrometry -based proteomic analysis of each fraction. Concentrations of proteins were calculated by the "total protein approach". A total of 4586 proteins were identified by at least five peptides and were quantified in all fractions. We found that the xenobiotics transporters of the canalicular and basolateral membranes were differentially enriched in the subcellular fractions and that phase I and II metabolizing enzymes, the cytochrome P450s and the UDP glucuronyl transferases, have complex subcellular distributions. These findings show that there is no simple way to scale the data from measurements in arbitrarily selected membrane fractions using a single scaling factor for all the proteins of interest. This study also provides the first absolute quantitative subcellular catalog of human liver proteins obtained from frozen tissue specimens. Our data provide quantitative insights into the sub cellular distribution of proteins and can be used as a guide for development of fractionation procedures.

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