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Artursson, Per
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Publications (10 of 110) Show all publications
Ölander, M., Wiśniewski, J. R., Flörkemeier, I., Handin, N., Urdzik, J. & Artursson, P. (2019). A simple approach for restoration of differentiation and function in cryopreserved human hepatocytes. Archives of Toxicology, 93(3), 819-829
Open this publication in new window or tab >>A simple approach for restoration of differentiation and function in cryopreserved human hepatocytes
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2019 (English)In: Archives of Toxicology, ISSN 0340-5761, E-ISSN 1432-0738, Vol. 93, no 3, p. 819-829Article in journal (Refereed) Published
Abstract [en]

Primary human hepatocytes are used in all facets of liver research, from in vitro studies of xenobiotic disposition and toxicity to the clinical management of liver failure. Unfortunately, cellular stress during isolation and cryopreservation causes a highly unpredictable loss of the ability to attach and form cell-matrix and cell-cell interactions. Reasoning that this problem could be mitigated at the post-thawing stage, we applied label-free quantitative global proteomics to analyze differences between attached and non-attached fractions of cryopreserved human hepatocyte batches. Hepatocytes that were unable to attach to a collagen matrix showed many signs of cellular stress, including a glycolytic phenotype and activation of the heat shock response, ultimately leading to apoptosis activation. Further analysis of the activated stress pathways revealed an increase in early apoptosis immediately post-thawing, which suggested the possibility of stress reversal. Therefore, we transiently treated the cells with compounds aimed at decreasing cellular stress via different mechanisms. Brief exposure to the pan-caspase apoptosis inhibitor Z-VAD-FMK restored attachment ability and promoted a differentiated morphology, as well as formation of 3D spheroids. Further, Z-VAD-FMK treatment restored metabolic and transport functions, with maintained sensitivity to hepatotoxic insults. Altogether, this study shows that differentiation and function of suboptimal human hepatocytes can be restored after cryopreservation, thus markedly increasing the availability of these precious cells.

Keywords
Apoptosis, Cellular stress, Cryopreservation, Hepatotoxicity, Human hepatocytes, Proteomics
National Category
Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-372723 (URN)10.1007/s00204-018-2375-9 (DOI)000463730100019 ()30560367 (PubMedID)
Funder
Swedish Research Council, 2822Swedish Research Council, 01951
Available from: 2019-01-08 Created: 2019-01-08 Last updated: 2019-04-30Bibliographically approved
Nies, A. T., Handin, N., Schaeffeler, E., Haag, M., Hofmann, U., Winter, S., . . . Schwab, M. (2019). Comprehensive drug uptake transporter proteomics and metabolomic profiling of human liver. Paper presented at 85th Annual Meeting of the German-Society-for-Experimental-and-Clinical-Pharmacology-and-Toxicology (DGPT) / 21st Annual Meeting of the Association-of-the-Clinical-Pharmacology-Germany (VKliPha), FEB 25-28, 2019, Stuttgart, GERMANY. Naunyn-Schmiedeberg's Archives of Pharmacology, 392(Suppl. 1), S8-S8, Article ID 24.
Open this publication in new window or tab >>Comprehensive drug uptake transporter proteomics and metabolomic profiling of human liver
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2019 (English)In: Naunyn-Schmiedeberg's Archives of Pharmacology, ISSN 0028-1298, E-ISSN 1432-1912, Vol. 392, no Suppl. 1, p. S8-S8, article id 24Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
SPRINGER, 2019
National Category
Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-377795 (URN)10.1007/s00210-019-01621-6 (DOI)000458266900019 ()
Conference
85th Annual Meeting of the German-Society-for-Experimental-and-Clinical-Pharmacology-and-Toxicology (DGPT) / 21st Annual Meeting of the Association-of-the-Clinical-Pharmacology-Germany (VKliPha), FEB 25-28, 2019, Stuttgart, GERMANY
Available from: 2019-02-28 Created: 2019-02-28 Last updated: 2019-02-28Bibliographically approved
Artursson, P. (2019). Experimental approaches to understand intracellular based drug bioavailability. Drug Metabolism and Pharmacokinetics, 34(1), S3-S3
Open this publication in new window or tab >>Experimental approaches to understand intracellular based drug bioavailability
2019 (English)In: Drug Metabolism and Pharmacokinetics, ISSN 1347-4367, E-ISSN 1880-0920, Vol. 34, no 1, p. S3-S3Article in journal, Meeting abstract (Other academic) Published
Place, publisher, year, edition, pages
JAPANESE SOC STUDY XENOBIOTICS, 2019
National Category
Pharmaceutical Sciences Pharmacology and Toxicology
Identifiers
urn:nbn:se:uu:diva-378238 (URN)10.1016/j.dmpk.2018.09.014 (DOI)000458519400011 ()
Available from: 2019-03-15 Created: 2019-03-15 Last updated: 2019-03-15Bibliographically approved
Ölander, M., Handin, N. & Artursson, P. (2019). Image-based quantification of cell debris as a measure of apoptosis. Analytical Chemistry, 91(9), 5548-5552
Open this publication in new window or tab >>Image-based quantification of cell debris as a measure of apoptosis
2019 (English)In: Analytical Chemistry, ISSN 0003-2700, E-ISSN 1520-6882, Vol. 91, no 9, p. 5548-5552Article in journal (Refereed) Published
Abstract [en]

Apoptosis is a controlled form of cell death that can be induced by various diseases and exogenous toxicants. Common apoptosis detection methods rely on fluorescent markers, which necessitates the use of costly reagents and time-consuming labeling procedures. Label-free methods avoid these problems, but often require specialized instruments instead. Here, we utilize apoptotic cell disintegration to develop a novel label-free detection method based on the quantification of subcellular debris particles in bright-field microscopy images. Debris counts show strong correlations with fluorescence-based annexin V staining, and can be used to study concentration-dependent and temporal apoptosis activation. The method is rapid, low-cost, and easy to apply, as the only experimental step comprises bright-field imaging of culture media samples, followed by automated image processing. The late-stage nature of the debris measurement means that the method can complement other, established apoptosis assays, and its accessibility will allow a wider community of researchers to study apoptotic cell death.

National Category
Pharmaceutical Sciences Analytical Chemistry
Identifiers
urn:nbn:se:uu:diva-382403 (URN)10.1021/acs.analchem.9b01243 (DOI)000467642100015 ()31001971 (PubMedID)
Funder
Swedish Research Council, 2822Swedish Research Council, 01951
Available from: 2019-04-24 Created: 2019-04-24 Last updated: 2019-06-19Bibliographically approved
Treyer, A., Ullah, M., Parrott, N., Molitor, B., Fowler, S. & Artursson, P. (2019). Impact of Intracellular Concentrations on Metabolic Drug-Drug Interaction Studies. AAPS Journal, 21(5), Article ID 77.
Open this publication in new window or tab >>Impact of Intracellular Concentrations on Metabolic Drug-Drug Interaction Studies
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2019 (English)In: AAPS Journal, ISSN 1550-7416, E-ISSN 1550-7416, Vol. 21, no 5, article id 77Article in journal (Refereed) Published
Abstract [en]

Accurate prediction of drug-drug interactions (DDI) is a challenging task in drug discovery and development. It requires determination of enzyme inhibition in vitro which is highly system-dependent for many compounds. The aim of this study was to investigate whether the determination of intracellular unbound concentrations in primary human hepatocytes can be used to bridge discrepancies between results obtained using human liver microsomes and hepatocytes. Specifically, we investigated if Kp(uu) could reconcile differences in CYP enzyme inhibition values (K-i or IC50). Firstly, our methodology for determination of Kp(uu) was optimized for human hepatocytes, using four well-studied reference compounds. Secondly, the methodology was applied to a series of structurally related CYP2C9 inhibitors from a Roche discovery project. Lastly, the Kp(uu) values of three commonly used CYP3A4 inhibitorsketoconazole, itraconazole, and posaconazolewere determined and compared to compound-specific hepatic enrichment factors obtained from physiologically based modeling of clinical DDI studies with these three compounds. Kp(uu) obtained in suspended human hepatocytes gave good predictions of system-dependent differences in vitro. The Kp(uu) was also in fair agreement with the compound-specific hepatic enrichment factors in DDI models and can therefore be used to improve estimations of enrichment factors in physiologically based pharmacokinetic modeling.

Place, publisher, year, edition, pages
SPRINGER, 2019
Keywords
drug-drug interaction, intracellular bioavailability, physiologically based pharmacokinetic modeling, scaling factor, unbound drug concentrations
National Category
Pharmacology and Toxicology Pharmaceutical Sciences
Identifiers
urn:nbn:se:uu:diva-390072 (URN)10.1208/s12248-019-0344-8 (DOI)000472114600002 ()31214810 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 60751Swedish Research Council, 2822Swedish Research Council, 2017-01951
Available from: 2019-08-07 Created: 2019-08-07 Last updated: 2019-08-07Bibliographically approved
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
Wisniewski, J. R., Wegler, C. & Artursson, P. (2019). Multiple-Enzyme-Digestion Strategy Improves Accuracy and Sensitivity of Label- and Standard-Free Absolute Quantification to a Level That Is Achievable by Analysis with Stable Isotope-Labeled Standard Spiking. Journal of Proteome Research, 18(1), 217-224
Open this publication in new window or tab >>Multiple-Enzyme-Digestion Strategy Improves Accuracy and Sensitivity of Label- and Standard-Free Absolute Quantification to a Level That Is Achievable by Analysis with Stable Isotope-Labeled Standard Spiking
2019 (English)In: Journal of Proteome Research, ISSN 1535-3893, E-ISSN 1535-3907, Vol. 18, no 1, p. 217-224Article in journal (Refereed) Published
Abstract [en]

Quantification of individual proteins is an essential task in understanding biological processes. For example, determination of concentrations of proteins transporting and metabolizing xenobiotics is a prerequisite for drug disposition predictions in humans based on in vitro data. So far, this task has frequently been accomplished by targeted proteomics. This type of analyses requires preparation of stable isotope labeled standards for each protein of interest. The selection of appropriate standard peptides is usually tedious and the number of proteins that can be studied in a single experiment by these approaches is limited. In addition, incomplete digestion of proteins often affects the accuracy of the quantification. To circumvent these constrains in proteomic protein quantification, label- and standard-free approaches, such as "total protein approach" (TPA) have been proposed. Here we directly compare an approach using stable isotope labeled (SIL) standards and TPA for quantification of transporters and enzymes in human liver samples within the same LC-MS/MS runs. We show that TPA is a convenient alternative to SIL-based methods. Optimization of the sample preparation beyond commonly used single tryptic digestion, by adding consecutive cleavage steps, improves accuracy and reproducibility of the TPA method to a level, which is achievable by analysis using stable isotope-labeled standard spiking.

Place, publisher, year, edition, pages
AMER CHEMICAL SOC, 2019
Keywords
FASP, MED FASP, total protein approach (TPA), stable isotope labeling, quantitative analysis, drug transporter, drug metabolizing enzymes, ADME
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-375231 (URN)10.1021/acs.jproteome.8b00549 (DOI)000455285900019 ()30336047 (PubMedID)
Funder
Swedish Research Council, 1951Swedish Research Council, 5715
Available from: 2019-01-29 Created: 2019-01-29 Last updated: 2019-07-23Bibliographically approved
Islam, M. K. K., Strand, M., Saleeb, M., Svensson, R., Baranczewski, P., Artursson, P., . . . Evander, M. (2018). Anti-Rift Valley fever virus activity in vitro, pre-clinical pharmacokinetics and oral bioavailability of benzavir-2, a broad-acting antiviral compound. Scientific Reports, 8, Article ID 1925.
Open this publication in new window or tab >>Anti-Rift Valley fever virus activity in vitro, pre-clinical pharmacokinetics and oral bioavailability of benzavir-2, a broad-acting antiviral compound
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2018 (English)In: Scientific Reports, ISSN 2045-2322, E-ISSN 2045-2322, Vol. 8, article id 1925Article in journal (Refereed) Published
Abstract [en]

Rift Valley fever virus (RVFV) is a mosquito-borne hemorrhagic fever virus affecting both humans and animals with severe morbidity and mortality and is classified as a potential bioterror agent due to the possible aerosol transmission. At present there is no human vaccine or antiviral therapy available. Thus, there is a great need to develop new antivirals for treatment of RVFV infections. Benzavir-2 was previously identified as potent inhibitor of human adenovirus, herpes simplex virus type 1, and type 2. Here we assess the anti-RVFV activity of benzavir-2 together with four structural analogs and determine pre-clinical pharmacokinetic parameters of benzavir-2. In vitro, benzavir-2 efficiently inhibited RVFV infection, viral RNA production and production of progeny viruses. In vitro, benzavir-2 displayed satisfactory solubility, good permeability and metabolic stability. In mice, benzavir-2 displayed oral bioavailability with adequate maximum serum concentration. Oral administration of benzavir-2 formulated in peanut butter pellets gave high systemic exposure without any observed toxicity in mice. To summarize, our data demonstrated potent anti-RVFV activity of benzavir-2 in vitro together with a promising pre-clinical pharmacokinetic profile. This data support further exploration of the antiviral activity of benzavir-2 in in vivo efficacy models that may lead to further drug development for human use.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2018
National Category
Basic Medicine
Identifiers
urn:nbn:se:uu:diva-346222 (URN)10.1038/s41598-018-20362-9 (DOI)000423663100004 ()29386590 (PubMedID)
Funder
Swedish Research Council, 2016-06251
Available from: 2018-03-19 Created: 2018-03-19 Last updated: 2018-03-19Bibliographically approved
Weiss, F., Hammer, H. S., Klein, K., Planatscher, H., Zanger, U. M., Norén, A., . . . Poetz, O. (2018). Direct Quantification of Cytochromes P450 and Drug Transporters-A Rapid, Targeted Mass Spectrometry-Based Immunoassay Panel for Tissues and Cell Culture Lysates. Drug Metabolism And Disposition, 46(4), 387-396
Open this publication in new window or tab >>Direct Quantification of Cytochromes P450 and Drug Transporters-A Rapid, Targeted Mass Spectrometry-Based Immunoassay Panel for Tissues and Cell Culture Lysates
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2018 (English)In: Drug Metabolism And Disposition, ISSN 0090-9556, E-ISSN 1521-009X, Vol. 46, no 4, p. 387-396Article in journal (Refereed) Published
Abstract [en]

The quantification of drug metabolizing enzymes and transporters has recently been revolutionized on the basis of targeted proteomic approaches. Isotope-labeled peptides are used as standards for the quantification of the corresponding proteins in enzymatically fragmented samples. However, hurdles in these approaches are low throughput and tedious sample prefractionation steps prior to mass spectrometry (MS) readout. We have developed an assay platform using sensitive and selective immunoprecipitation coupled with mass spectrometric readout allowing the quantification of proteins directly from whole cell lysates using less than 20,000 cells per analysis. Peptide group-specific antibodies (triple X proteomics antibodies) enable the enrichment of proteotypic peptides sharing a common terminus. These antibodies were employed to establish a MS-based immunoassay panel for the quantification of 14 cytochrome P450 (P450) enzymes and nine transporters. We analyzed the P450 enzyme and transporter levels in genotyped liver tissue homogenates and microsomes, and in samples from a time course induction experiment in human hepatocytes addressing different induction pathways. For the analysis of P450 enzymes and transporters only a minute amount of sample is required and no prefractionation is necessary, thus the assay platform bears the potential to bridge cell culture model experiments and results from whole organ tissue studies.

Place, publisher, year, edition, pages
AMER SOC PHARMACOLOGY EXPERIMENTAL THERAPEUTICS, 2018
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-353108 (URN)10.1124/dmd.117.078626 (DOI)000426966600007 ()29343608 (PubMedID)
Funder
Swedish Research Council, 2822Swedish Research Council, 5715
Available from: 2018-06-11 Created: 2018-06-11 Last updated: 2019-07-23Bibliographically 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
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