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Intracellular unbound drug concentrations: Methodology and application for understanding cellular drug exposure
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Pharmacy, Department of Pharmacy. Faculty of Pharmacy, University of Lisbon.ORCID iD: 0000-0001-6870-0677
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 [en]
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: urn:nbn:se:uu:diva-276095ISBN: 978-91-554-9496-4 (print)OAI: oai:DiVA.org:uu-276095DiVA: diva2:908586
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
List of papers
1. Rapid Measurement of Intracellular Unbound Drug Concentrations
Open this publication in new window or tab >>Rapid Measurement of Intracellular Unbound Drug Concentrations
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.

Keyword
intracellular unbound concentrations, drug binding, drug transport, drug accumulation, membrane partitioning
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-204292 (URN)10.1021/mp4000822 (DOI)000320015600037 ()
Available from: 2013-07-29 Created: 2013-07-29 Last updated: 2017-12-06
2. A High-Throughput Cell-Based Method to Predict the Unbound Drug Fraction in the Brain
Open this publication in new window or tab >>A High-Throughput Cell-Based Method to Predict the Unbound Drug Fraction in the Brain
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.

National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-224730 (URN)10.1021/jm401963n (DOI)000334572000017 ()
Available from: 2014-05-22 Created: 2014-05-19 Last updated: 2017-12-05
3. Impact of drug transporters on intracellular drug concentrations
Open this publication in new window or tab >>Impact of drug transporters on intracellular drug concentrations
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(English)Manuscript (preprint) (Other academic)
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-276093 (URN)
Available from: 2016-02-09 Created: 2016-02-09 Last updated: 2016-04-04
4. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil
Open this publication in new window or tab >>CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil
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2016 (English)In: Nature Communications, ISSN 2041-1723, E-ISSN 2041-1723, Vol. 7, 11040Article in journal (Refereed) Published
Abstract [en]

Target engagement is a critical factor for therapeutic efficacy. Assessment of compound binding to native target proteins in live cells is therefore highly desirable in all stages of drug discovery. We report here the first compound library screen based on biophysical measurements of intracellular target binding, exemplified by human thymidylate synthase (TS). The screen selected accurately for all the tested known drugs acting on TS. We also identified TS inhibitors with novel chemistry and marketed drugs that were not previously known to target TS, including the DNA methyltransferase inhibitor decitabine. By following the cellular uptake and enzymatic conversion of known drugs we correlated the appearance of active metabolites over time with intracellular target engagement. These data distinguished a much slower activation of 5-fluorouracil when compared with nucleoside-based drugs. The approach establishes efficient means to associate drug uptake and activation with target binding during drug discovery.

National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-276077 (URN)10.1038/ncomms11040 (DOI)000372887500001 ()27010513 (PubMedID)
Funder
The Karolinska Institutet's Research FoundationSwedish Research CouncilSwedish Cancer SocietyKnut and Alice Wallenberg Foundation
Note

Artursson, P., Martinez-Molina, D och Nordlund, P. delar sistaförfattarskapet.

Available from: 2016-02-09 Created: 2016-02-09 Last updated: 2017-11-30Bibliographically approved
5. Impact of intracellular drug bioavailability on cellular drug response
Open this publication in new window or tab >>Impact of intracellular drug bioavailability on cellular drug response
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(English)Manuscript (preprint) (Other academic)
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-276089 (URN)
Available from: 2016-02-09 Created: 2016-02-09 Last updated: 2016-04-04

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