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Characterization of parameters influencing intracellular bioavailability and prediction of intracellular drug exposure
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.ORCID iD: 0000-0002-4533-7761
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
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 266
Keywords [en]
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
Identifiers
URN: urn:nbn:se:uu:diva-369705ISBN: 978-91-513-0542-4 (print)OAI: oai:DiVA.org:uu-369705DiVA, id: diva2:1271759
Public defence
2019-02-15, Room B41, BMC, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2019-01-23 Created: 2018-12-18 Last updated: 2019-02-18
List of papers
1. 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
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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

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