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A High-Throughput Cell-Based Method to Predict the Unbound Drug Fraction in the Brain
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, Science for Life Laboratory, SciLifeLab.
Uppsala University, Science for Life Laboratory, SciLifeLab. Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy.
2014 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 57, no 7, 3005-3010 p.Article in journal (Refereed) Published
Abstract [en]

Optimization of drug efficacy in the brain requires understanding of the local exposure to unbound drug at the site of action. This relies on measurements of the unbound drug fraction (f(u,brain)), which currently requires access to brain tissue. Here, we present a novel methodology using homogenates of cultured cells for rapid estimation of f(u,brain). In our setup, drug binding to human embryonic kidney cell (HEK293) homogenate was measured in a small-scale dialysis apparatus. To increase throughput, we combined drugs into cassettes for simultaneous measurement of multiple compounds. Our method estimated f(u,brain) with an average error of 1.9-fold. We propose that our simple method can be used as an inexpensive, easily available and high-throughput alternative to brain tissues excised from laboratory animals. Thereby, estimates of unbound drug exposure can now implemented at a much earlier stage of the drug discovery process, when molecular property changes are easier to make.

Place, publisher, year, edition, pages
2014. Vol. 57, no 7, 3005-3010 p.
National Category
Medical and Health Sciences
URN: urn:nbn:se:uu:diva-224730DOI: 10.1021/jm401963nISI: 000334572000017OAI: oai:DiVA.org:uu-224730DiVA: diva2:719069
Available from: 2014-05-22 Created: 2014-05-19 Last updated: 2016-04-04
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.
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 211
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
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)
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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