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Intracellular unbound drug concentrations: Methodology and application for understanding cellular drug exposure
Uppsala universitet, Medicinska och farmaceutiska vetenskapsområdet, Farmaceutiska fakulteten, Institutionen för farmaci. Faculty of Pharmacy, University of Lisbon.ORCID-id: 0000-0001-6870-0677
2016 (Engelska)Doktorsavhandling, sammanläggning (Övrigt vetenskapligt)
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.

Ort, förlag, år, upplaga, sidor
Uppsala: Acta Universitatis Upsaliensis, 2016. , s. 69
Serie
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 211
Nyckelord [en]
intracellular unbound drug concentrations, free drug, drug binding, drug transport, drug accumulation, cellular drug response, drug target engagement
Nationell ämneskategori
Farmaceutiska vetenskaper Farmakologi och toxikologi
Forskningsämne
Farmaceutisk vetenskap
Identifikatorer
URN: urn:nbn:se:uu:diva-276095ISBN: 978-91-554-9496-4 (tryckt)OAI: oai:DiVA.org:uu-276095DiVA, id: diva2:908586
Disputation
2016-04-22, room B21, Biomedical center, Husargatan 3, Uppsala, 09:15 (Engelska)
Opponent
Handledare
Tillgänglig från: 2016-03-31 Skapad: 2016-02-09 Senast uppdaterad: 2018-01-10
Delarbeten
1. Rapid Measurement of Intracellular Unbound Drug Concentrations
Öppna denna publikation i ny flik eller fönster >>Rapid Measurement of Intracellular Unbound Drug Concentrations
2013 (Engelska)Ingår i: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 10, nr 6, s. 2467-2478Artikel i tidskrift (Refereegranskat) 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.

Nyckelord
intracellular unbound concentrations, drug binding, drug transport, drug accumulation, membrane partitioning
Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
urn:nbn:se:uu:diva-204292 (URN)10.1021/mp4000822 (DOI)000320015600037 ()
Tillgänglig från: 2013-07-29 Skapad: 2013-07-29 Senast uppdaterad: 2018-07-30
2. A High-Throughput Cell-Based Method to Predict the Unbound Drug Fraction in the Brain
Öppna denna publikation i ny flik eller fönster >>A High-Throughput Cell-Based Method to Predict the Unbound Drug Fraction in the Brain
2014 (Engelska)Ingår i: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 57, nr 7, s. 3005-3010Artikel i tidskrift (Refereegranskat) 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.

Nationell ämneskategori
Medicin och hälsovetenskap
Identifikatorer
urn:nbn:se:uu:diva-224730 (URN)10.1021/jm401963n (DOI)000334572000017 ()
Tillgänglig från: 2014-05-22 Skapad: 2014-05-19 Senast uppdaterad: 2018-07-30
3. Impact of drug transporters on intracellular drug concentrations
Öppna denna publikation i ny flik eller fönster >>Impact of drug transporters on intracellular drug concentrations
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(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Farmaceutiska vetenskaper
Identifikatorer
urn:nbn:se:uu:diva-276093 (URN)
Tillgänglig från: 2016-02-09 Skapad: 2016-02-09 Senast uppdaterad: 2018-01-10
4. CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil
Öppna denna publikation i ny flik eller fönster >>CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil
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2016 (Engelska)Ingår i: Nature Communications, E-ISSN 2041-1723, Vol. 7, artikel-id 11040Artikel i tidskrift (Refereegranskat) 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.

Nationell ämneskategori
Farmakologi och toxikologi
Identifikatorer
urn:nbn:se:uu:diva-276077 (URN)10.1038/ncomms11040 (DOI)000372887500001 ()27010513 (PubMedID)
Forskningsfinansiär
Karolinska Institutets ForskningsstiftelseVetenskapsrådetCancerfondenKnut och Alice Wallenbergs Stiftelse
Anmärkning

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

Tillgänglig från: 2016-02-09 Skapad: 2016-02-09 Senast uppdaterad: 2023-03-28Bibliografiskt granskad
5. Impact of intracellular drug bioavailability on cellular drug response
Öppna denna publikation i ny flik eller fönster >>Impact of intracellular drug bioavailability on cellular drug response
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(Engelska)Manuskript (preprint) (Övrigt vetenskapligt)
Nationell ämneskategori
Farmakologi och toxikologi
Identifikatorer
urn:nbn:se:uu:diva-276089 (URN)
Tillgänglig från: 2016-02-09 Skapad: 2016-02-09 Senast uppdaterad: 2018-01-10

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