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Artursson, Per
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Publications (10 of 100) Show all publications
Ölander, M., Wiśniewski, J. R., Flörkemeier, I., Handin, N., Urdzik, J. & Artursson, P. (2018). A simple approach for restoration of differentiation and function in cryopreserved human hepatocytes. Archives of Toxicology
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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2018 (English)In: Archives of Toxicology, ISSN 0340-5761, E-ISSN 1432-0738Article in journal (Refereed) Epub ahead of print
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)30560367 (PubMedID)
Funder
Swedish Research Council, 2822, 01951
Available from: 2019-01-08 Created: 2019-01-08 Last updated: 2019-01-16Bibliographically 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: 2018-06-11Bibliographically 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
Kundu, S., Ali, M. A., Handin, N., Padhan, N., Larsson, J., Karoutsou, M., . . . Sjöblom, T. (2018). Linking FOXO3, NCOA3, and TCF7L2 to Ras pathway phenotypes through a genome-wide forward genetic screen in human colorectal cancer cells. Genome Medicine, 10, Article ID 2.
Open this publication in new window or tab >>Linking FOXO3, NCOA3, and TCF7L2 to Ras pathway phenotypes through a genome-wide forward genetic screen in human colorectal cancer cells
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2018 (English)In: Genome Medicine, ISSN 1756-994X, E-ISSN 1756-994X, Vol. 10, article id 2Article in journal (Refereed) Published
Abstract [en]

Background:

The Ras pathway genes KRAS, BRAF, or ERBBs have somatic mutations in similar to 60% of human colorectal carcinomas. At present, it is unknown whether the remaining cases lack mutations activating the Ras pathway or whether they have acquired mutations in genes hitherto unknown to belong to the pathway.

Methods:

To address the second possibility and extend the compendium of Ras pathway genes, we used genome-wide transposon mutagenesis of two human colorectal cancer cell systems deprived of their activating KRAS or BRAF allele to identify genes enabling growth in low glucose, a Ras pathway phenotype, when targeted.

Results:

Of the 163 recurrently targeted genes in the two different genetic backgrounds, one-third were known cancer genes and one-fifth had links to the EGFR/Ras/MAPK pathway. When compared to cancer genome sequencing datasets, nine genes also mutated in human colorectal cancers were identified. Among these, stable knockdown of FOXO3, NCOA3, and TCF7L2 restored growth in low glucose but reduced MEK/MAPK phosphorylation, reduced anchorage-independent growth, and modulated expressions of GLUT1 and Ras pathway related proteins. Knockdown of NCOA3 and FOXO3 significantly decreased the sensitivity to cetuximab of KRAS mutant but not wild-type cells.

Conclusions:

This work establishes a proof-of-concept that human cell-based genome-wide forward genetic screens can assign genes to pathways with clinical importance in human colorectal cancer.

Keywords
Forward genetics, piggyBac transposon, Colorectal cancer, Ras pathway
National Category
Medical Genetics
Identifiers
urn:nbn:se:uu:diva-341500 (URN)10.1186/s13073-017-0511-4 (DOI)000419613600002 ()29301589 (PubMedID)
Available from: 2018-02-14 Created: 2018-02-14 Last updated: 2018-02-14Bibliographically approved
Visnes, T., Cazares-Korner, A., Hao, W., Wallner, O., Masuyer, G., Loseva, O., . . . Helleday, T. (2018). Small-molecule inhibitor of OGG1 suppresses proinflammatory gene expression and inflammation. Science, 362(6416), 834-839
Open this publication in new window or tab >>Small-molecule inhibitor of OGG1 suppresses proinflammatory gene expression and inflammation
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2018 (English)In: Science, ISSN 0036-8075, E-ISSN 1095-9203, Vol. 362, no 6416, p. 834-839Article in journal (Refereed) Published
Abstract [en]

The onset of inflammation is associated with reactive oxygen species and oxidative damage to macromolecules like 7,8-dihydro-8-oxoguanine (8-oxoG) in DNA. Because 8-oxoguanine DNA glycosylase 1 (OGG1) binds 8-oxoG and because Ogg1-deficient mice are resistant to acute and systemic inflammation, we hypothesized that OGG1 inhibition may represent a strategy for the prevention and treatment of inflammation. We developed TH5487, a selective active-site inhibitor of OGG1, which hampers OGG1 binding to and repair of 8-oxoG and which is well tolerated by mice. TH5487 prevents tumor necrosis factor-alpha-induced OGG1-DNA interactions at guanine-rich promoters of proinflammatory genes. This, in turn, decreases DNA occupancy of nuclear factor kappa B and proinflammatory gene expression, resulting in decreased immune cell recruitment to mouse lungs. Thus, we present a proof of concept that targeting oxidative DNA repair can alleviate inflammatory conditions in vivo.

National Category
Cell and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-371530 (URN)10.1126/science.aar8048 (DOI)000450488500054 ()30442810 (PubMedID)
Funder
VINNOVAEU, European Research CouncilKnut and Alice Wallenberg FoundationSwedish Foundation for Strategic Research Swedish Research CouncilSwedish Cancer SocietySwedish Childhood Cancer FoundationRagnar Söderbergs stiftelseTorsten Söderbergs stiftelseSwedish Foundation for Strategic Research
Available from: 2019-01-07 Created: 2019-01-07 Last updated: 2019-01-07Bibliographically approved
Shrestha, N., Bouttefeux, O., Vanvarenberg, K., Lundquist, P., Cunarro, J., Tovar, S., . . . Beloqui, A. (2018). The stimulation of GLP-1 secretion and delivery of GLP-1 agonists &ITvia&IT nanostructured lipid carriers. Nanoscale, 10(2), 603-613
Open this publication in new window or tab >>The stimulation of GLP-1 secretion and delivery of GLP-1 agonists &ITvia&IT nanostructured lipid carriers
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2018 (English)In: Nanoscale, ISSN 2040-3364, E-ISSN 2040-3372, Vol. 10, no 2, p. 603-613Article in journal (Refereed) Published
Abstract [en]

Nanoparticulate based drug delivery systems have been extensively studied to efficiently encapsulate and deliver peptides orally. However, most of the existing data mainly focus on the nanoparticles as a drug carrier, but the ability of nanoparticles having a biological effect has not been exploited. Herein, we hypothesize that nanostructured lipid carriers (NLCs) could activate the endogenous glucagon-like peptide-1 (GLP-1) secretion and also act as oral delivery systems for GLP-1 analogs (exenatide and liraglutide). NLCs effectively encapsulated the peptides, the majority of which were only released under the intestinal conditions. NLCs, with and without peptide encapsulation, showed effective induction of GLP-1 secretion in vitro from the enteroendocrinal L-cells (GLUTag). NLCs also showed a 2.9-fold increase in the permeability of exenatide across the intestinal cell monolayer. The intestinal administration of the exenatide and liraglutide loaded NLCs did not demonstrate any glucose lowering effect on normal mice. Further, ex vivo studies depicted that the NLCs mainly adhered to the mucus layer. In conclusion, this study demonstrates that NLCs need further optimization to overcome the mucosal barrier in the intestine; nonetheless, this study also presents a promising strategy to use a dual-action drug delivery nanosystem which synergizes its own biological effect and that of the encapsulated drug molecule.

National Category
Chemical Sciences Physical Sciences
Identifiers
urn:nbn:se:uu:diva-341488 (URN)10.1039/c7nr07736j (DOI)000419152600012 ()29235598 (PubMedID)
Available from: 2018-02-28 Created: 2018-02-28 Last updated: 2018-02-28Bibliographically approved
Karlgren, M., Simoff, I., Backlund, M., Wegler, C., Keiser, M., Handin, N., . . . Artursson, P. (2017). A CRISPR-Cas9 Generated MDCK Cell Line Expressing Human MDR1 Without Endogenous Canine MDR1 (cABCB1): An Improved Tool for Drug Efflux Studies.. Journal of Pharmaceutical Sciences, 106(9), 2909-2913
Open this publication in new window or tab >>A CRISPR-Cas9 Generated MDCK Cell Line Expressing Human MDR1 Without Endogenous Canine MDR1 (cABCB1): An Improved Tool for Drug Efflux Studies.
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2017 (English)In: Journal of Pharmaceutical Sciences, ISSN 0022-3549, E-ISSN 1520-6017, Vol. 106, no 9, p. 2909-2913Article in journal (Refereed) Published
Abstract [en]

Madin-Darby canine kidney (MDCK) II cells stably transfected with transport proteins are commonly used models for drug transport studies. However, endogenous expression of especially canine MDR1 (cMDR1) confounds the interpretation of such studies. Here we have established an MDCK cell line stably overexpressing the human MDR1 transporter (hMDR1; P-glycoprotein), and used CRISPR-Cas9 gene editing to knockout the endogenous cMDR1. Genomic screening revealed the generation of a clonal cell line homozygous for a 4-nucleotide deletion in the canine ABCB1 gene leading to a frameshift and a premature stop codon. Knockout of cMDR1 expression was verified by quantitative protein analysis and functional studies showing retained activity of the human MDR1 transporter. Application of this cell line allowed unbiased reclassification of drugs previously defined as both substrates and non-substrates in different studies using commonly used MDCK-MDR1 clones. Our new MDCK-hMDR1 cell line, together with a previously developed control cell line, both with identical deletions in the canine ABCB1 gene and lack of cMDR1 expression represent excellent in vitro tools for use in drug discovery.

Keywords
ABC transporters, MDCK cells, P-glycoprotein, drug transport, efflux pumps, genomics, membrane transporter, multidrug resistance transporters, permeability, proteomics
National Category
Medical and Health Sciences
Identifiers
urn:nbn:se:uu:diva-343598 (URN)10.1016/j.xphs.2017.04.018 (DOI)000417339900089 ()28450237 (PubMedID)
Funder
Science for Life Laboratory - a national resource center for high-throughput molecular bioscience
Available from: 2018-02-28 Created: 2018-02-28 Last updated: 2018-03-07Bibliographically approved
Beloqui, A., Brayden, D. J., Artursson, P., Preat, V. & des Rieux, A. (2017). A human intestinal M-cell-like model for investigating particle, antigen and microorganism translocation. Nature Protocols, 12(7), 1387-1399
Open this publication in new window or tab >>A human intestinal M-cell-like model for investigating particle, antigen and microorganism translocation
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2017 (English)In: Nature Protocols, ISSN 1754-2189, E-ISSN 1750-2799, Vol. 12, no 7, p. 1387-1399Article in journal (Refereed) Published
Abstract [en]

The specialized microfold cells (M cells) in the follicle-associated epithelium (FAE) of intestinal Peyer's patches serve as antigen-sampling cells of the intestinal innate immune system. Unlike 'classical' enterocytes, they are able to translocate diverse particulates without digesting them. They act as pathways for microorganism invasion and mediate food tolerance by transcellular transport of intestinal microbiota and antigens. Their ability to transcytose intact particles can be used to develop oral drug delivery and oral immunization strategies. This protocol describes a reproducible and versatile human M-cell-like in vitro model. This model can be exploited to evaluate M-cell transport of microparticles and nanoparticles for protein, drug or vaccine delivery and to study bacterial adherence and translocation across M cells. The inverted in vitro M-cell model consists of three main steps. First, Caco-2 cells are seeded at the apical side of the inserts. Second, the inserts are inverted and B lymphocytes are seeded at the basolateral side of the inserts. Third, the conversion to M cells is assessed. Although various M-cell culture systems exist, this model provides several advantages over the rest: (i) it is based on coculture with well-established differentiated human cell lines; (ii) it is reproducible under the conditions described herein; (iii) it can be easily mastered; and (iv) it does not require the isolation of primary cells or the use of animals. The protocol requires skills in cell culture and microscopy analysis. The model is obtained after 3 weeks, and transport experiments across the differentiated model can be carried out over periods of up to 10 h.

Place, publisher, year, edition, pages
NATURE PUBLISHING GROUP, 2017
National Category
Biochemistry and Molecular Biology
Identifiers
urn:nbn:se:uu:diva-328973 (URN)10.1038/nprot.2017.041 (DOI)000403344900005 ()28617450 (PubMedID)
Funder
EU, FP7, Seventh Framework Programme, 281035
Available from: 2017-09-12 Created: 2017-09-12 Last updated: 2017-09-12Bibliographically 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
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