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Treyer, A., Walday, S., Boriss, H., Matsson, P. & Artursson, P. (2019). A Cell-Free Approach Based on Phospholipid Characterization for Determination of the Cell Specific Unbound Drug Fraction (f(u,cell)). Pharmaceutical research, 36(12), Article ID 178.
Open this publication in new window or tab >>A Cell-Free Approach Based on Phospholipid Characterization for Determination of the Cell Specific Unbound Drug Fraction (f(u,cell))
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2019 (English)In: Pharmaceutical research, ISSN 0724-8741, E-ISSN 1573-904X, Vol. 36, no 12, article id 178Article in journal (Refereed) Published
Abstract [en]


The intracellular fraction of unbound compound (f(u,cell)) is an important parameter for accurate prediction of drug binding to intracellular targets. f(u,cell) is the result of a passive distribution process of drug molecules partitioning into cellular structures. Initial observations in our laboratory showed an up to 10-fold difference in the f(u,cell) of a given drug for different cell types. We hypothesized that these differences could be explained by the phospholipid (PL) composition of the cells, since the PL cell membrane is the major sink of unspecific drug binding. Therefore, we determined the f(u,cell) of 19 drugs in cell types of different origin.


The cells were characterized for their total PL content and we used mass spectrometric PL profiling to delineate the impact of each of the four major cellular PL subspecies: phosphatidylcholine (PC), phosphatidylethanolamine (PE), phosphatidylserine (PS) and phosphatidylinositol (PI). The cell-based experiments were compared to cell-free experiments that used beads covered by PL bilayers consisting of the most abundant PL subspecies.


PC was found to give the largest contribution to the drug binding. Improved correlations between the cell-based and cell-free assays were obtained when affinities to all four major PL subspecies were considered. Together, our data indicate that f(u,cell) is influenced by PL composition of cells.


We conclude that cellular PL composition varies between cell types and that cell-specific mixtures of PLs can replace cellular assays for determination of f(u,cell) as a rapid, small-scale assay covering a broad dynamic range.

cell-free assays, intracellular bioavailability, phospholipid membranes, unbound drug fraction
National Category
Cell and Molecular Biology
urn:nbn:se:uu:diva-397951 (URN)10.1007/s11095-019-2717-1 (DOI)000495617000001 ()31701258 (PubMedID)
Swedish Research Council, 2822Swedish Research Council, 2017-01951EU, FP7, Seventh Framework Programme, 60751
Available from: 2020-01-02 Created: 2020-01-02 Last updated: 2020-01-02Bibliographically approved
Treyer, A. (2019). Characterization of parameters influencing intracellular bioavailability and prediction of intracellular drug exposure. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
Open this publication in new window or tab >>Characterization of parameters influencing intracellular bioavailability and prediction of intracellular drug exposure
2019 (English)Doctoral 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.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2019. p. 59
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 266
intracellular drug bioavailability, unbound drug concentration, drug disposition, ADME, drug transport, drug metabolism membrane partitioning, phospholipid, drug-drug interaction, antisense oligonucleotide, cell-penetrating peptide
National Category
Pharmaceutical Sciences
Research subject
Pharmaceutical Science
urn:nbn:se:uu:diva-369705 (URN)978-91-513-0542-4 (ISBN)
Public defence
2019-02-15, Room B41, BMC, Husargatan 3, Uppsala, 09:15 (English)
Available from: 2019-01-23 Created: 2018-12-18 Last updated: 2019-02-18
Treyer, A., Ullah, M., Parrott, N., Molitor, B., Fowler, S. & Artursson, P. (2019). Impact of Intracellular Concentrations on Metabolic Drug-Drug Interaction Studies. AAPS Journal, 21(5), Article ID 77.
Open this publication in new window or tab >>Impact of Intracellular Concentrations on Metabolic Drug-Drug Interaction Studies
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2019 (English)In: AAPS Journal, ISSN 1550-7416, E-ISSN 1550-7416, Vol. 21, no 5, article id 77Article in journal (Refereed) Published
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.

Place, publisher, year, edition, pages
drug-drug interaction, intracellular bioavailability, physiologically based pharmacokinetic modeling, scaling factor, unbound drug concentrations
National Category
Pharmacology and Toxicology Pharmaceutical Sciences
urn:nbn:se:uu:diva-390072 (URN)10.1208/s12248-019-0344-8 (DOI)000472114600002 ()31214810 (PubMedID)
EU, FP7, Seventh Framework Programme, 60751Swedish Research Council, 2822Swedish Research Council, 2017-01951
Available from: 2019-08-07 Created: 2019-08-07 Last updated: 2019-08-07Bibliographically approved
Treyer, A., Mateus, A., Wisniewski, J. R., Boriss, H., Matsson, P. & Artursson, P. (2018). Intracellular Drug Bioavailability: Effect of Neutral Lipids and Phospholipids. Molecular Pharmaceutics, 15(6), 2224-2233
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
intracellular drug bioavailability, lipid, phospholipid, drug binding membrane partitioning, proteomics, 3T3-L1, unbound concentration
National Category
Pharmaceutical Sciences
urn:nbn:se:uu:diva-358082 (URN)10.1021/acs.molpharmaceut.8b00064 (DOI)000434491800015 ()29709195 (PubMedID)
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
ORCID iD: ORCID iD iconorcid.org/0000-0002-4533-7761

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