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Intracellular Drug Bioavailability: Effect of Neutral Lipids and Phospholipids
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci.ORCID-id: 0000-0002-4533-7761
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci.ORCID-id: 0000-0001-6870-0677
Max Planck Inst Biochem, Dept Prote & Signal Transduct, Biochem Prote Grp, D-82152 Martinsried, Germany.
Sovicell GmbH, D-04103 Leipzig, Germany.
Vise andre og tillknytning
2018 (engelsk)Inngår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, nr 6, s. 2224-2233Artikkel i tidsskrift (Fagfellevurdert) 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.

sted, utgiver, år, opplag, sider
AMER CHEMICAL SOC , 2018. Vol. 15, nr 6, s. 2224-2233
Emneord [en]
intracellular drug bioavailability, lipid, phospholipid, drug binding membrane partitioning, proteomics, 3T3-L1, unbound concentration
HSV kategori
Identifikatorer
URN: urn:nbn:se:uu:diva-358082DOI: 10.1021/acs.molpharmaceut.8b00064ISI: 000434491800015PubMedID: 29709195OAI: oai:DiVA.org:uu-358082DiVA, id: diva2:1243094
Forskningsfinansiär
EU, FP7, Seventh Framework Programme, 60751Swedish Research Council, 2822Swedish Research Council, 2017-01951Åke Wiberg FoundationTilgjengelig fra: 2018-08-30 Laget: 2018-08-30 Sist oppdatert: 2018-12-18bibliografisk kontrollert
Inngår i avhandling
1. Characterization of parameters influencing intracellular bioavailability and prediction of intracellular drug exposure
Åpne denne publikasjonen i ny fane eller vindu >>Characterization of parameters influencing intracellular bioavailability and prediction of intracellular drug exposure
2019 (engelsk)Doktoravhandling, med artikler (Annet vitenskapelig)
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.

sted, utgiver, år, opplag, sider
Uppsala: Acta Universitatis Upsaliensis, 2019. s. 59
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 266
Emneord
intracellular drug bioavailability, unbound drug concentration, drug disposition, ADME, drug transport, drug metabolism membrane partitioning, phospholipid, drug-drug interaction, antisense oligonucleotide, cell-penetrating peptide
HSV kategori
Forskningsprogram
Farmaceutisk vetenskap
Identifikatorer
urn:nbn:se:uu:diva-369705 (URN)978-91-513-0542-4 (ISBN)
Disputas
2019-02-15, Room B41, BMC, Husargatan 3, Uppsala, 09:15 (engelsk)
Opponent
Veileder
Tilgjengelig fra: 2019-01-23 Laget: 2018-12-18 Sist oppdatert: 2019-02-18

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