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Rapid Measurement of Intracellular Unbound Drug Concentrations
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.ORCID iD: 0000-0001-6870-0677
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
2013 (English)In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 10, no 6, 2467-2478 p.Article in journal (Refereed) Published
Abstract [en]

Intracellular unbound drug concentrations determine affinity to targets in the cell interior. However, due to difficulties in measuring them, they are often overlooked in pharmacology. Here we present a simple experimental technique for the determination of unbound intracellular drug concentrations in cultured cells that is based on parallel measurements of cellular drug binding and steady-state intracellular drug concentrations. Binding in HEK293 cells was highly correlated with binding in liver-derived systems, whereas binding in plasma did not compare well with cellular binding. Compound lipophilicity increased drug binding, while negative charge and aromatic functional groups decreased binding. Intracellular accumulation of unbound drug was consistent with pH dependent subcellular sequestration, as confirmed by modeling and by inhibition of subcellular pH gradients. The approach developed here can be used to measure intracellular unbound drug concentrations in more complex systems, for example, cell lines with controlled expression of transporters and enzymes or primary cells.

Place, publisher, year, edition, pages
2013. Vol. 10, no 6, 2467-2478 p.
Keyword [en]
intracellular unbound concentrations, drug binding, drug transport, drug accumulation, membrane partitioning
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-204292DOI: 10.1021/mp4000822ISI: 000320015600037OAI: oai:DiVA.org:uu-204292DiVA: diva2:638302
Available from: 2013-07-29 Created: 2013-07-29 Last updated: 2017-12-06
In thesis
1. Intracellular unbound drug concentrations: Methodology and application for understanding cellular drug exposure
Open this publication in new window or tab >>Intracellular unbound drug concentrations: Methodology and application for understanding cellular drug exposure
2016 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Most known drug targets and metabolizing enzymes are located inside cells. Interactions with these proteins are determined by intracellular unbound drug concentrations. Assessing intracellular drug exposure is technically challenging, but essential for predicting pharmacokinetic, pharmacological, and toxicological profiles of new drugs.

This thesis aims at establishing and applying a straightforward methodology to measure intracellular unbound drug concentrations. This was achieved by separately measuring cellular drug binding (fu,cell), and total intracellular drug accumulation (Kp). This allowed the calculation of intracellular drug bioavailability (Fic), which represents the fraction of the concentration added to the cells that is unbound in the cell interior.

The methodology was initially developed in HEK293 cells, where the Fic of 189 drug-like compounds was measured. Binding to HEK293 cells was governed by compound lipophilicity and was correlated with binding to more complex systems, such as hepatocytes and brain. Due to negligible expression of drug transporters, Fic in this cell line was consistent with pH-dependent subcellular sequestration of lipophilic cations in low pH compartments.

The methodology was then applied to study the effects of drug transporters on Fic. The uptake transporter OATP1B1 increased the Fic of its substrates in a concentration-dependent manner. In contrast, the Fic of P-gp substrates was decreased when P-gp was present. In human hepatocytes, the methodology allowed the determination of Fic without prior knowledge of transporter mechanisms or metabolic activity.

Finally, the methodology was applied to measure the impact of Fic on target binding and cellular drug response. Intracellular concentrations of active metabolites of pro-drugs targeting the intracellular target thymidylate synthase were in agreement with the level of binding to this target. Further, high Fic was generally required for kinase and protease inhibitors to be active in cellular assays.

In conclusion, the methodology can be used to predict if new drug candidates reach their intracellular targets in sufficient amounts. Furthermore, the methodology can improve in vitro predictions of drug clearance and drug-drug interactions, by measuring the drug available for intracellular enzymes. Finally, this work can be expanded to other xenobiotics, e.g., to predict their intracellular toxicity.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2016. 69 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 211
Keyword
intracellular unbound drug concentrations, free drug, drug binding, drug transport, drug accumulation, cellular drug response, drug target engagement
National Category
Pharmaceutical Sciences Pharmacology and Toxicology
Research subject
Pharmaceutical Science
Identifiers
urn:nbn:se:uu:diva-276095 (URN)978-91-554-9496-4 (ISBN)
Public defence
2016-04-22, room B21, Biomedical center, Husargatan 3, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2016-03-31 Created: 2016-02-09 Last updated: 2016-04-04

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Mateus, AndréMatsson, PärArtursson, Per

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