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  • 1.
    Treyer, Andrea
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Characterization of parameters influencing intracellular bioavailability and prediction of intracellular drug exposure2019Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    This thesis work investigates factors influencing intracellular drug disposition. An experimental method for measurement of intracellular bioavailability (Fic), was used throughout. Fic is defined as the ratio between the unbound drug concentration inside the cell and the compound concentration in the cell exterior.

    First, the impact of transporter proteins—such as the uptake transporter OATP-1B1 and the efflux transporter P-gp—on Fic was assessed in isolation in singly transfected, well-characterized cell models. The net impact of ADME proteins on Fic, including drug transporter proteins and metabolic enzymes, was assessed in primary human hepatocytes. The results indicated that the Fic measurement accurately reflected system-dependent functionality of these proteins.

    Second, the impact of cellular lipids on Fic was studied, in particular phospholipids (a major constituent of cellular membranes) and neutral lipids (in the form of neutral lipid droplets in adipocytes). Drug partitioning to phospholipids was found to be the major determinant of intracellular fraction of unbound drug (fu,cell), while neutral lipid droplets and cellular proteins played a relatively smaller role. Therefore, the importance of phospholipids, and their major four subspecies—phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI)—was investigated in a cell-free approach with purified phospholipids.

    Finally, Fic was applied in two ways to drug discovery settings. First, Fic successfully harmonized system-dependent CYP450 enzyme inhibition values (IC50) obtained in human hepatocytes and human liver microsomes. Fic measured in suspended human hepatocytes also reflected hepatic enrichment factors of CYP450 inhibitors used in physiologically-based pharmacokinetic modelling. Second, Fic was used as a complementary tool to study the effect of cell-penetrating peptides on intracellular disposition of targeted antisense oligonucleotide conjugates.

    Overall, the thesis contributes to the mechanistic understanding of Fic and demonstrates its use for drug compound profiling at an early stage in drug discovery settings.

    List of papers
    1. Intracellular Drug Bioavailability: Effect of Neutral Lipids and Phospholipids
    Open this publication in new window or tab >>Intracellular Drug Bioavailability: Effect of Neutral Lipids and Phospholipids
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    2018 (English)In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, no 6, p. 2224-2233Article in journal (Refereed) Published
    Abstract [en]

    Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by multiple processes, including the extent of drug binding to intracellular structures. The aim of this study was to evaluate the effect of neutral lipid (NL) and phospholipid (PL) levels on intracellular drug disposition. The NL and/or PL content of 3T3-L1 cells were enhanced, resulting in phenotypes (in terms of morphology and proteome) reminiscent of adipocytes (high NL and PL) or mild phospholipidosis (only high PL). Intracellular bioavailability (F-ic) was then determined for 23 drugs in these cellular models and in untreated wild-type cells. A higher PL content led to higher intracellular drug binding and a lower F-ic. The induction of NL did not further increase drug binding but led to altered F-ic due to increased lysosomal pH. Further, there was a good correlation between binding to beads coated with pure PL and intracellular drug binding. In conclusion, our results suggest that PL content is a major determinant of drug binding in cells and that PL beads may constitute a simple alternative to estimating this parameter. Further, the presence of massive amounts of intracellular NLs did not influence drug binding significantly.

    Place, publisher, year, edition, pages
    AMER CHEMICAL SOC, 2018
    Keywords
    intracellular drug bioavailability, lipid, phospholipid, drug binding membrane partitioning, proteomics, 3T3-L1, unbound concentration
    National Category
    Pharmaceutical Sciences
    Identifiers
    urn:nbn:se:uu:diva-358082 (URN)10.1021/acs.molpharmaceut.8b00064 (DOI)000434491800015 ()29709195 (PubMedID)
    Funder
    EU, FP7, Seventh Framework Programme, 60751Swedish Research Council, 2822Swedish Research Council, 2017-01951Åke Wiberg Foundation
    Available from: 2018-08-30 Created: 2018-08-30 Last updated: 2018-12-18Bibliographically approved
    2. Impact of intracellular bioavailability on metabolic drug-drug interactions
    Open this publication in new window or tab >>Impact of intracellular bioavailability on metabolic drug-drug interactions
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Pharmaceutical Sciences
    Identifiers
    urn:nbn:se:uu:diva-369686 (URN)
    Available from: 2018-12-16 Created: 2018-12-16 Last updated: 2018-12-18
    3. Effect of cell penetrating peptides on GLP-1R-mediated intracellular delivery of antisense oligonucleotides
    Open this publication in new window or tab >>Effect of cell penetrating peptides on GLP-1R-mediated intracellular delivery of antisense oligonucleotides
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    (English)Manuscript (preprint) (Other academic)
    National Category
    Pharmaceutical Sciences
    Identifiers
    urn:nbn:se:uu:diva-369687 (URN)
    Available from: 2018-12-16 Created: 2018-12-16 Last updated: 2018-12-18
    4. A cell free approach for determination of cell specific drug binding based on phospholipid speciation
    Open this publication in new window or tab >>A cell free approach for determination of cell specific drug binding based on phospholipid speciation
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    (English)Manuscript (preprint) (Other academic)
    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-369684 (URN)
    Available from: 2018-12-16 Created: 2018-12-16 Last updated: 2019-01-04
    5. Intracellular drug bioavailability: a new predictor of system dependent drug disposition
    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
  • 2.
    Treyer, Andrea
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Mateus, André
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Wisniewski, Jacek R.
    Max Planck Inst Biochem, Dept Prote & Signal Transduct, Biochem Prote Grp, D-82152 Martinsried, Germany.
    Boriss, Hinnerk
    Sovicell GmbH, D-04103 Leipzig, Germany.
    Matsson, Pär
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala Univ, Dept Pharm, S-75123 Uppsala, Sweden.
    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: Effect of Neutral Lipids and Phospholipids2018In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, no 6, p. 2224-2233Article in journal (Refereed)
    Abstract [en]

    Intracellular unbound drug concentrations are the pharmacologically relevant concentrations for targets inside cells. Intracellular drug concentrations are determined by multiple processes, including the extent of drug binding to intracellular structures. The aim of this study was to evaluate the effect of neutral lipid (NL) and phospholipid (PL) levels on intracellular drug disposition. The NL and/or PL content of 3T3-L1 cells were enhanced, resulting in phenotypes (in terms of morphology and proteome) reminiscent of adipocytes (high NL and PL) or mild phospholipidosis (only high PL). Intracellular bioavailability (F-ic) was then determined for 23 drugs in these cellular models and in untreated wild-type cells. A higher PL content led to higher intracellular drug binding and a lower F-ic. The induction of NL did not further increase drug binding but led to altered F-ic due to increased lysosomal pH. Further, there was a good correlation between binding to beads coated with pure PL and intracellular drug binding. In conclusion, our results suggest that PL content is a major determinant of drug binding in cells and that PL beads may constitute a simple alternative to estimating this parameter. Further, the presence of massive amounts of intracellular NLs did not influence drug binding significantly.

  • 3.
    Treyer, Andrea
    et al.
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
    Ullah, Mohammed
    Roche Innovat Ctr Basel, Roche Pharmaceut Res & Early Dev, Basel, Switzerland.
    Parrott, Neil
    Roche Innovat Ctr Basel, Roche Pharmaceut Res & Early Dev, Basel, Switzerland.
    Molitor, Birgit
    Roche Innovat Ctr Basel, Roche Pharmaceut Res & Early Dev, Basel, Switzerland.
    Fowler, Stephen
    Roche Innovat Ctr Basel, Roche Pharmaceut Res & Early Dev, Basel, Switzerland.
    Artursson, Per
    Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Uppsala University, Science for Life Laboratory, SciLifeLab.
    Impact of Intracellular Concentrations on Metabolic Drug-Drug Interaction Studies2019In: AAPS Journal, ISSN 1550-7416, E-ISSN 1550-7416, Vol. 21, no 5, article id 77Article in journal (Refereed)
    Abstract [en]

    Accurate prediction of drug-drug interactions (DDI) is a challenging task in drug discovery and development. It requires determination of enzyme inhibition in vitro which is highly system-dependent for many compounds. The aim of this study was to investigate whether the determination of intracellular unbound concentrations in primary human hepatocytes can be used to bridge discrepancies between results obtained using human liver microsomes and hepatocytes. Specifically, we investigated if Kp(uu) could reconcile differences in CYP enzyme inhibition values (K-i or IC50). Firstly, our methodology for determination of Kp(uu) was optimized for human hepatocytes, using four well-studied reference compounds. Secondly, the methodology was applied to a series of structurally related CYP2C9 inhibitors from a Roche discovery project. Lastly, the Kp(uu) values of three commonly used CYP3A4 inhibitorsketoconazole, itraconazole, and posaconazolewere determined and compared to compound-specific hepatic enrichment factors obtained from physiologically based modeling of clinical DDI studies with these three compounds. Kp(uu) obtained in suspended human hepatocytes gave good predictions of system-dependent differences in vitro. The Kp(uu) was also in fair agreement with the compound-specific hepatic enrichment factors in DDI models and can therefore be used to improve estimations of enrichment factors in physiologically based pharmacokinetic modeling.

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  • fi-FI
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