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Filppula, A. M., Parvizi, R., Mateus, A., Baranczewski, P. & Artursson, P. (2019). Improved predictions of time-dependent drug-drug interactions by determination of cytosolic drug concentrations. Scientific Reports, 9, Article ID 5850.
Open this publication in new window or tab >>Improved predictions of time-dependent drug-drug interactions by determination of cytosolic drug concentrations
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2019 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 9, article id 5850Article in journal (Refereed) Published
Abstract [en]

The clinical impact of drug-drug interactions based on time-dependent inhibition of cytochrome P450 (CYP) 3A4 has often been overpredicted, likely due to use of improper inhibitor concentration estimates at the enzyme. Here, we investigated if use of cytosolic unbound inhibitor concentrations could improve predictions of time-dependent drug-drug interactions. First, we assessed the inhibitory effects of ten time-dependent CYP3A inhibitors on midazolam 1′-hydroxylation in human liver microsomes. Then, using a novel method, we determined the cytosolic bioavailability of the inhibitors in human hepatocytes, and used the obtained values to calculate their concentrations at the active site of the enzyme, i.e. the cytosolic unbound concentrations. Finally, we combined the data in mechanistic static predictions, by considering different combinations of inhibitor concentrations in intestine and liver, including hepatic concentrations corrected for cytosolic bioavailability. The results were then compared to clinical data. Compared to no correction, correction for cytosolic bioavailability resulted in higher accuracy and precision, generally in line with those obtained by more demanding modelling. The best predictions were obtained when the inhibition of hepatic CYP3A was based on unbound maximal inhibitor concentrations corrected for cytosolic bioavailability. Our findings suggest that cytosolic unbound inhibitor concentrations improves predictions of time-dependent drug-drug interactions for CYP3A.

National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-382453 (URN)10.1038/s41598-019-42051-x (DOI)000463984600008 ()
Funder
Swedish Research Council, 01951
Available from: 2019-04-25 Created: 2019-04-25 Last updated: 2019-05-03Bibliographically approved
Treyer, A., Mateus, A., Wisniewski, J. R., Boriss, H., Matsson, P. & Artursson, P. (2018). Intracellular Drug Bioavailability: Effect of Neutral Lipids and Phospholipids. Molecular Pharmaceutics, 15(6), 2224-2233
Open this publication in new window or tab >>Intracellular Drug Bioavailability: Effect of Neutral Lipids and Phospholipids
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2018 (English)In: Molecular Pharmaceutics, ISSN 1543-8384, E-ISSN 1543-8392, Vol. 15, no 6, p. 2224-2233Article in journal (Refereed) 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.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2018
Keywords
intracellular drug bioavailability, lipid, phospholipid, drug binding membrane partitioning, proteomics, 3T3-L1, unbound concentration
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-358082 (URN)10.1021/acs.molpharmaceut.8b00064 (DOI)000434491800015 ()29709195 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 60751Swedish Research Council, 2822Swedish Research Council, 2017-01951Åke Wiberg Foundation
Available from: 2018-08-30 Created: 2018-08-30 Last updated: 2018-12-18Bibliographically approved
Llona-Minguez, S., Höglund, A., Wiita, E., Almlof, I., Mateus, A., Calderon-Montano, J. M., . . . Helledayt, T. (2017). Identification of Triazolothiadiazoles as Potent Inhibitors of the dCTP Pyrophosphatase 1. Journal of Medicinal Chemistry, 60(5), 2148-2154
Open this publication in new window or tab >>Identification of Triazolothiadiazoles as Potent Inhibitors of the dCTP Pyrophosphatase 1
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2017 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 60, no 5, p. 2148-2154Article in journal (Refereed) Published
Abstract [en]

The dCTP pyrophosphatase 1 (dCTPase) is involved in the regulation of the cellular dNTP pool and has been linked to cancer progression. Here we report on the discovery of a series of 3,6-disubstituted triazolothiadiazoles as potent dCTPase inhibitors. Compounds 16 and 18 display good correlation between enzymatic inhibition and target engagement, together with efficacy in a cellular synergy model, deeming them as a promising starting point for hit -to-lead development.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2017
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-319535 (URN)10.1021/acsjmedchem.6b01786 (DOI)000396296100037 ()28145708 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research CouncilEU, European Research CouncilGöran Gustafsson Foundation for promotion of scientific research at Uppala University and Royal Institute of TechnologySwedish Cancer SocietySwedish Childhood Cancer FoundationTorsten Söderbergs stiftelseRagnar Söderbergs stiftelse
Available from: 2017-04-06 Created: 2017-04-06 Last updated: 2018-01-13Bibliographically approved
Mateus, A., Treyer, A., Wegler, C., Karlgren, M., Matsson, P. & Artursson, P. (2017). Intracellular drug bioavailability: a new predictor of system dependent drug disposition. Scientific Reports, 7, 1-12, Article ID 43047.
Open this publication in new window or tab >>Intracellular drug bioavailability: a new predictor of system dependent drug disposition
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2017 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 7, p. 1-12, article id 43047Article in journal (Refereed) Published
Abstract [en]

Intracellular drug exposure is influenced by cell-and tissue-dependent expression of drug-transporting proteins and metabolizing enzymes. Here, we introduce the concept of intracellular bioavailability (F-ic) as the fraction of extracellular drug available to bind intracellular targets, and we assess how Fic is affected by cellular drug disposition processes. We first investigated the impact of two essential drug transporters separately, one influx transporter (OATP1B1; SLCO1B1) and one efflux transporter (P-gp; ABCB1), in cells overexpressing these proteins. We showed that OATP1B1 increased Fic of its substrates, while P-gp decreased Fic. We then investigated the impact of the concerted action of multiple transporters and metabolizing enzymes in freshly-isolated human hepatocytes in culture configurations with different levels of expression and activity of these proteins. We observed that Fic was up to 35-fold lower in the configuration with high expression of drug-eliminating transporters and enzymes. We conclude that Fic provides a measurement of the net impact of all cellular drug disposition processes on intracellular bioavailable drug levels. Importantly, no prior knowledge of the involved drug distribution pathways is required, allowing for high-throughput determination of drug access to intracellular targets in highly defined cell systems (e.g., single-transporter transfectants) or in complex ones (including primary human cells).

National Category
Medical Biotechnology
Identifiers
urn:nbn:se:uu:diva-317940 (URN)10.1038/srep43047 (DOI)000394530900001 ()28225057 (PubMedID)
Available from: 2017-04-01 Created: 2017-04-01 Last updated: 2019-07-23Bibliographically approved
Llona-Minguez, S., Höglund, A., Ghassemian, A., Desroses, M., Calderon-Montano, J. M., Moron, E. B., . . . Helleday, T. (2017). Piperazin-1-ylpyridazine Derivatives Are a Novel Class of Human dCTP Pyrophosphatase 1 Inhibitors. Journal of Medicinal Chemistry, 60(10), 4279-4292
Open this publication in new window or tab >>Piperazin-1-ylpyridazine Derivatives Are a Novel Class of Human dCTP Pyrophosphatase 1 Inhibitors
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2017 (English)In: Journal of Medicinal Chemistry, ISSN 0022-2623, E-ISSN 1520-4804, Vol. 60, no 10, p. 4279-4292Article in journal (Refereed) Published
Abstract [en]

The dCTP pyrophosphatase 1 (dCTPase) is a nucleotide pool "housekeeping" enzyme responsible for the catabolism of canonical and noncanonical nucleoside triphosphates (dNTPs) and has been associated with cancer progression and cancer cell sternness. We have identified a series of piperazin-1-ylpyridazines as a new class of potent dCTPase inhibitors. Lead compounds increase dCTPase thermal and protease stability, display outstanding selectivity over related enzymes and synergize with a cytidine analogue against leukemic cells. This new class of dCTPase inhibitors lays the first stone toward the development of drug-like probes for the dCTPase enzyme.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2017
National Category
Medicinal Chemistry
Identifiers
urn:nbn:se:uu:diva-326233 (URN)10.1021/acs.jmedchem.7b00182 (DOI)000402498200013 ()28508636 (PubMedID)
Funder
Knut and Alice Wallenberg FoundationSwedish Research CouncilEU, European Research CouncilGöran Gustafsson Foundation for Research in Natural Sciences and MedicineSwedish Cancer SocietySwedish Childhood Cancer FoundationTorsten Söderbergs stiftelseRagnar Söderbergs stiftelse
Available from: 2017-08-10 Created: 2017-08-10 Last updated: 2018-01-13Bibliographically approved
Mateus, A., Gordon, L. J., Wayne, G. J., Almqvist, H., Axelsson, H., Seashore-Ludlow, B., . . . Artursson, P. (2017). Prediction of intracellular exposure bridges the gap between target- and cell-based drug discovery. Proceedings of the National Academy of Sciences of the United States of America, 114(30), E6231-E6239
Open this publication in new window or tab >>Prediction of intracellular exposure bridges the gap between target- and cell-based drug discovery
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2017 (English)In: Proceedings of the National Academy of Sciences of the United States of America, ISSN 0027-8424, E-ISSN 1091-6490, Vol. 114, no 30, p. E6231-E6239Article in journal (Refereed) Published
Abstract [en]

Inadequate target exposure is a major cause of high attrition in drug discovery. Here, we show that a label-free method for quantifying the intracellular bioavailability (F-ic) of drug molecules predicts drug access to intracellular targets and hence, pharmacological effect. We determined F-ic in multiple cellular assays and cell types representing different targets from a number of therapeutic areas, including cancer, inflammation, and dementia. Both cytosolic targets and targets localized in subcellular compartments were investigated. F-ic gives insights on membrane-permeable compounds in terms of cellular potency and intracellular target engagement, compared with biochemical potency measurements alone. Knowledge of the amount of drug that is locally available to bind intracellular targets provides a powerful tool for compound selection in early drug discovery.

Place, publisher, year, edition, pages
NATL ACAD SCIENCES, 2017
Keywords
intracellular drug bioavailability, drug exposure, target engagement, published kinase inhibitor set, MAPK14
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-332843 (URN)10.1073/pnas.1701848114 (DOI)000406189900026 ()28701380 (PubMedID)
Funder
Swedish Research Council, 2822Carl Tryggers foundation Magnus Bergvall FoundationÅke Wiberg FoundationScience for Life Laboratory - a national resource center for high-throughput molecular bioscienceEU, FP7, Seventh Framework Programme, 607517
Available from: 2017-11-09 Created: 2017-11-09 Last updated: 2018-07-30Bibliographically approved
Almqvist, H., Axelsson, H., Jafari, R., Dan, C., Mateus, A., Haraldsson, M., . . . Nordlund, P. (2016). CETSA screening identifies known and novel thymidylate synthase inhibitors and slow intracellular activation of 5-fluorouracil. Nature Communications, 7, Article ID 11040.
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, article id 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: 2018-01-10Bibliographically approved
Gordon, L. J., Allen, M., Artursson, P., Hann, M. M., Leavens, B. J., Mateus, A., . . . West, A. (2016). Direct Measurement of Intracellular Compound Concentration by RapidFire Mass Spectrometry Offers Insights into Cell Permeability. Journal of Biomolecular Screening, 21(2), 156-164
Open this publication in new window or tab >>Direct Measurement of Intracellular Compound Concentration by RapidFire Mass Spectrometry Offers Insights into Cell Permeability
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2016 (English)In: Journal of Biomolecular Screening, ISSN 1087-0571, E-ISSN 1552-454X, Vol. 21, no 2, p. 156-164Article in journal (Refereed) Published
Abstract [en]

One of the key challenges facing early stage drug discovery is understanding the commonly observed difference between the activity of compounds in biochemical assays and cellular assays. Traditionally, indirect or estimated cell permeability measurements such as estimations from logP or artificial membrane permeability are used to explain the differences. The missing link is a direct measurement of intracellular compound concentration in whole cells. This can, in some circumstances, be estimated from the cellular activity, but this may also be problematic if cellular activity is weak or absent. Advances in sensitivity and throughput of analytical techniques have enabled us to develop a high-throughput assay for the measurement of intracellular compound concentration for routine use to support lead optimization. The assay uses a RapidFire-MS based readout of compound concentration in HeLa cells following incubation of cells with test compound. The initial assay validation was performed by ultra-high performance liquid chromatography tandem mass spectrometry, and the assay was subsequently transferred to RapidFire tandem mass spectrometry. Further miniaturization and optimization were performed to streamline the process, increase sample throughput, and reduce cycle time. This optimization has delivered a semi-automated platform with the potential of production scale compound profiling up to 100 compounds per day.

Keywords
intracellular drug concentrations, RapidFire-MS, cell penetration, drug accumulation
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-273527 (URN)10.1177/1087057115604141 (DOI)000368325000007 ()26336900 (PubMedID)
Funder
Swedish Research Council, 2822
Available from: 2016-01-15 Created: 2016-01-15 Last updated: 2018-01-10Bibliographically approved
Mateus, A. (2016). Intracellular unbound drug concentrations: Methodology and application for understanding cellular drug exposure. (Doctoral dissertation). Uppsala: Acta Universitatis Upsaliensis
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. p. 69
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Pharmacy, ISSN 1651-6192 ; 211
Keywords
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: 2018-01-10
Vildhede, A., Mateus, A., Khan, E. K., Lai, Y., Karlgren, M., Artursson, P. & Kjellsson, M. C. (2016). Mechanistic modeling of hepatic pitavastatin disposition: a proteomics-informed bottom-up approach. Paper presented at 20th North American Meeting of the International-Society-for-the-Study-of-Xenobiotics (ISSX), OCT 18-22, 2015, Orlando, FL. Drug metabolism reviews (Softcover ed.), 48, 56-57
Open this publication in new window or tab >>Mechanistic modeling of hepatic pitavastatin disposition: a proteomics-informed bottom-up approach
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2016 (English)In: Drug metabolism reviews (Softcover ed.), ISSN 0360-2532, E-ISSN 1097-9883, Vol. 48, p. 56-57Article in journal, Meeting abstract (Other academic) Published
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-303429 (URN)000380744900118 ()
Conference
20th North American Meeting of the International-Society-for-the-Study-of-Xenobiotics (ISSX), OCT 18-22, 2015, Orlando, FL
Available from: 2016-11-21 Created: 2016-09-19 Last updated: 2018-01-13Bibliographically approved
Organisations
Identifiers
ORCID iD: ORCID iD iconorcid.org/0000-0001-6870-0677

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