uu.seUppsala University Publications
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf
Proteomics-informed analysis of drug disposition in the human liver and small intestine
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.ORCID iD: 0000-0002-2810-7518
2019 (English)Doctoral thesis, comprehensive summary (Other academic)
Description
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 [en]
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: urn:nbn:se:uu:diva-389741ISBN: 978-91-513-0694-0 (print)OAI: oai:DiVA.org:uu-389741DiVA, id: diva2:1339161
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
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

Open Access in DiVA

fulltext(1661 kB)54 downloads
File information
File name FULLTEXT01.pdfFile size 1661 kBChecksum SHA-512
75aa144ec20cfe51e21bc38c2d345843fb357503546440c2f5ec43fd60559a14d7d63796d87fa3350c6be3dc9f4c21b5b6c2746c8afc3083aa2a91d7d2fbe47e
Type fulltextMimetype application/pdf

Authority records BETA

Wegler, Christine

Search in DiVA

By author/editor
Wegler, Christine
By organisation
Department of Pharmacy
Pharmaceutical Sciences

Search outside of DiVA

GoogleGoogle Scholar
Total: 54 downloads
The number of downloads is the sum of all downloads of full texts. It may include eg previous versions that are now no longer available

isbn
urn-nbn

Altmetric score

isbn
urn-nbn
Total: 240 hits
CiteExportLink to record
Permanent link

Direct link
Cite
Citation style
  • apa
  • ieee
  • modern-language-association
  • vancouver
  • Other style
More styles
Language
  • de-DE
  • en-GB
  • en-US
  • fi-FI
  • nn-NO
  • nn-NB
  • sv-SE
  • Other locale
More languages
Output format
  • html
  • text
  • asciidoc
  • rtf