| 1. |
- Karlgren, Maria, et al.
(författare)
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A CRISPR-Cas9 Generated MDCK Cell Line Expressing Human MDR1 Without Endogenous Canine MDR1 (cABCB1) : An Improved Tool for Drug Efflux Studies.
- 2017
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Ingår i: Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0022-3549 .- 1520-6017. ; 106:9, s. 2909-2913
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 2. |
- Lundquist, Patrik, et al.
(författare)
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Oral absorption of peptides and nanoparticles across the human intestine : Opportunities, limitations and studies in human tissues
- 2016
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Ingår i: Advanced Drug Delivery Reviews. - : Elsevier BV. - 0169-409X .- 1872-8294. ; 106, s. 256-276
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Forskningsöversikt (refereegranskat)abstract
- In this contribution, we review the molecular and physiological barriers to oral delivery of peptides and nanoparticles. We discuss the opportunities and predictivity of various in vitro systems with special emphasis on human intestine in Ussing chambers. First, the molecular constraints to peptide absorption are discussed. Then the physiological barriers to peptide delivery are examined. These include the gastric and intestinal environment, the mucus barrier, tight junctions between epithelial cells, the enterocytes of the intestinal epithelium, and the subepithelial tissue. Recent data from human proteome studies are used to provide information about the protein expression profiles of the different physiological barriers to peptide and nanoparticle absorption. Strategies that have been employed to increase peptide absorption across each of the barriers are discussed. Special consideration is given to attempts at utilizing endogenous transcytotic pathways. To reliably translate in vitro data on peptide or nanoparticle permeability to the in vivo situation in a human subject, the in vitro experimental system needs to realistically capture the central aspects of the mentioned barriers. Therefore, characteristics of common in vitro cell culture systems are discussed and compared to those of human intestinal tissues. Attempts to use the cell and tissue models for in vitro-in vivo extrapolation are reviewed.
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| 3. |
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| 4. |
- Matsson, Pär, et al.
(författare)
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Quantifying the impact of transporters on cellular drug permeability.
- 2015
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Ingår i: TIPS - Trends in Pharmacological Sciences. - : Elsevier BV. - 0165-6147 .- 1873-3735. ; 35:5, s. 255-262
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Tidskriftsartikel (refereegranskat)abstract
- The conventional model of drug permeability has recently been challenged. An alternative model proposes that transporter-mediated flux is the sole mechanism of cellular drug permeation, instead of existing in parallel with passive transmembrane diffusion. We examined a central assumption of this alternative hypothesis; namely, that transporters can give rise to experimental observations that would typically be explained with passive transmembrane diffusion. Using systems-biology simulations based on available transporter kinetics and proteomic expression data, we found that such observations are possible in the absence of transmembrane diffusion, but only under very specific conditions that rarely or never occur for known human drug transporters.
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| 5. |
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| 6. |
- Shrestha, Neha, et al.
(författare)
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The stimulation of GLP-1 secretion and delivery of GLP-1 agonists &ITvia&IT nanostructured lipid carriers
- 2018
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Ingår i: Nanoscale. - : Royal Society of Chemistry (RSC). - 2040-3364 .- 2040-3372. ; 10:2, s. 603-613
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Ölander, Magnus, et al.
(författare)
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The Proteome of Filter-Grown Caco-2 Cells With a Focus on Proteins Involved in Drug Disposition
- 2016
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Ingår i: Journal of Pharmaceutical Sciences. - : Elsevier BV. - 0022-3549 .- 1520-6017. ; 105:2, s. 817-827
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Tidskriftsartikel (refereegranskat)abstract
- Caco-2 cells are widely used in studies of intestinal cell physiology and drug transport. Here, the global proteome of filter-grown Caco-2 cells was quantified using the total protein approach and compared with the human colon and jejunum proteomes. In total, 8096 proteins were identified. In-depth analysis of proteins defining enterocyte differentiation—including brush-border hydrolases, integrins, and adherens and tight junctions—gave near-complete coverage of the expected proteins. Three hundred twenty-seven absorption, distribution, metabolism and excretion proteins were identified, including 112 solute carriers and 20 ATP-binding cassette transporters. OATP2B1 levels were 16-fold higher in Caco-2 cells than in jejunum. To investigate the impact of this difference on in vitro-in vivo extrapolations, we studied the uptake kinetics of the OATP2B1 substrate pitavastatin in Caco-2 monolayers, and found that the contribution of OATP2B1 was 60%-70% at clinically relevant intestinal concentrations. Pitavastatin kinetics was combined with transporter concentrations to model the contribution of active transport and membrane permeation in the jejunum. The lower OATP2B1 expression in jejunum led to a considerably lower transporter contribution (<5%), suggesting that transmembrane diffusion dominates pitavastatin absorption in vivo. In conclusion, we present the first in-depth quantification of the filter-grown Caco-2 proteome. We also demonstrate the crucial importance of considering transporter expression levels for correct interpretation of drug transport routes across the human intestine.
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