| 1. |
- Karlgren, Maria, et al.
(författare)
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Classification of Inhibitors of Hepatic Organic Anion Transporting Polypeptides (OATPs) : Influence of Protein Expression on Drug - Drug Interactions
- 2012
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Ingår i: Journal of Medicinal Chemistry. - : American Chemical Society (ACS). - 0022-2623 .- 1520-4804. ; 55:10, s. 4740-4763
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Tidskriftsartikel (refereegranskat)abstract
- The hepatic organic anion transporting polypeptides (OATPs) influence the pharmacokinetics of several drug classes and are involved in many clinical drug-drug interactions. Predicting potential interactions with OATPs is, therefore, of value. Here, we developed in vitro and in silico models for identification and prediction of specific and general inhibitors of OATP1B1, OATP1B3, and OATP2B1, The maximal transport activity (MTA) of each OATP in human liver was predicted from transport kinetics and protein quantification. We then used MTA to predict the effects of a subset of inhibitors on atorvastatin uptake in vivo. Using a data set of 225 drug-like compounds, 91 OATP inhibitors were identified. In silico models indicated that lipophilicity and polar surface area are key molecular features of OATP inhibition. MTA predictions identified OATP1B1 and OATP1B3 as major determinants of atorvastatin uptake in vivo. The relative contributions to overall hepatic uptake varied with isoform specificities of the inhibitors.
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| 2. |
- Kundu, Snehangshu, et al.
(författare)
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Linking FOXO3, NCOA3, and TCF7L2 to Ras pathway phenotypes through a genome-wide forward genetic screen in human colorectal cancer cells
- 2018
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Ingår i: Genome Medicine. - : Springer Science and Business Media LLC. - 1756-994X. ; 10
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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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. |
- Prasad, Bhagwat, et al.
(författare)
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Toward a Consensus on Applying Quantitative Liquid Chromatography-Tandem Mass Spectrometry Proteomics in Translational Pharmacology Research : A White Paper
- 2019
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Ingår i: Clinical Pharmacology and Therapeutics. - : WILEY. - 0009-9236 .- 1532-6535. ; 106:3, s. 525-543
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Tidskriftsartikel (refereegranskat)abstract
- Quantitative translation of information on drug absorption, disposition, receptor engagement, and drug-drug interactions from bench to bedside requires models informed by physiological parameters that link in vitro studies to in vivo outcomes. To predict in vivo outcomes, biochemical data from experimental systems are routinely scaled using protein quantity in these systems and relevant tissues. Although several laboratories have generated useful quantitative proteomic data using state-of-the-art mass spectrometry, no harmonized guidelines exit for sample analysis and data integration to in vivo translation practices. To address this gap, a workshop was held on September 27 and 28, 2018, in Cambridge, MA, with 100 experts attending from academia, the pharmaceutical industry, and regulators. Various aspects of quantitative proteomics and its applications in translational pharmacology were debated. A summary of discussions and best practices identified by this expert panel are presented in this "White Paper" alongside unresolved issues that were outlined for future debates.
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| 6. |
- Treyer, Andrea, et al.
(författare)
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Intracellular Drug Bioavailability : Effect of Neutral Lipids and Phospholipids
- 2018
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Ingår i: Molecular Pharmaceutics. - : AMER CHEMICAL SOC. - 1543-8384 .- 1543-8392. ; 15:6, s. 2224-2233
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Tidskriftsartikel (refereegranskat)abstract
- 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.
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| 7. |
- Urdzik, Jozef, et al.
(författare)
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Global proteome changes in liver tissue 6 weeks after FOLFOX treatment of colorectal cancer liver metastases
- 2016
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Ingår i: PROTEOMES. - : MDPI AG. - 2227-7382. ; 4:4
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Tidskriftsartikel (refereegranskat)abstract
- (1) Oxaliplatin-based chemotherapy for colorectal cancer liver metastasis is associated with sinusoidal injury of liver parenchyma. The effects of oxaliplatin-induced liver injury on the protein level remain unknown. (2) Protein expression in liver tissue was analyzed-from eight patients treated with FOLFOX (combination of fluorouracil, leucovorin, and oxaliplatin) and seven controls-by label-free liquid chromatography mass spectrometry. Recursive feature elimination-support vector machine and Welch t-test were used to identify classifying and relevantly changed proteins, respectively. Resulting proteins were analyzed for associations with gene ontology categories and pathways. (3) A total of 5891 proteins were detected. A set of 184 (3.1%) proteins classified the groups with a 20% error rate, but relevant change was observed only in 55 (0.9%) proteins. The classifying proteins were associated with changes in DNA replication (p < 0.05) through upregulation of the minichromosome maintenance complex and with the innate immune response (p < 0.05). The importance of DNA replication changes was supported by the results of Welch t-test (p < 0.05). (4) Six weeks after FOLFOX treatment, less than 1% of identified proteins showed changes in expression associated with DNA replication, cell cycle entry, and innate immune response. We hypothesize that the changes remain after recovery from FOLFOX treatment injury.
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| 8. |
- Vessby, Johan, 1972-, et al.
(författare)
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AGPAT1 as a Novel Colonic Biomarker for Discriminating Between Ulcerative Colitis With and Without Primary Sclerosing Cholangitis
- 2022
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Ingår i: Clinical and Translational Gastroenterology. - : Ovid Technologies (Wolters Kluwer Health). - 2155-384X. ; 13:5
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Tidskriftsartikel (refereegranskat)abstract
- INTRODUCTION: Ulcerative colitis (UC) associated with primary sclerosing cholangitis (PSC-UC) is considered a unique inflammatory bowel disease (IBD) entity. PSC diagnosis in an IBD individual entails a significantly higher risk of gastrointestinal cancer; however, biomarkers for identifying patients with UC at risk for PSC are lacking. We, therefore, performed a thorough PSC-UC biomarker study, starting from archived colonic tissue.METHODS: Proteins were extracted out of formalin-fixed paraffin-embedded proximal colon samples from PSC-UC (n = 9), UC (n = 7), and healthy controls (n = 7). Patients with IBD were in clinical and histological remission, and all patients with UC had a history of pancolitis. Samples were processed by the multienzyme digestion FASP and subsequently analyzed by liquid chromatography-tandem mass spectrometry. Candidate proteins were replicated in an independent cohort (n: PSC-UC = 16 and UC = 21) and further validated by immunohistochemistry.RESULTS: In the discovery step, 7,279 unique proteins were detected. The top 5 most differentiating proteins (PSC-UC vs UC) based on linear regression analysis were selected for replication. Of these, 1-acetylglycerol-3-phosphate O-acyltransferase 1 (AGPAT1) was verified as higher in PSC-UC than UC (P = 0.009) in the replication cohort. A difference on the group level was also confirmed by immunohistochemistry, showing more intense AGPAT1 staining in patients with PSC-UC compared with UC.DISCUSSION: We present AGPAT1 as a potential colonic biomarker for differentiating PSC-UC from UC. Our findings have possible implication for future PSC-IBD diagnostics and surveillance.
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| 9. |
- Vildhede, Anna, et al.
(författare)
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Comparative Proteomic Analysis of Human Liver Tissue and Isolated Hepatocytes with a Focus on Proteins Determining Drug Exposure
- 2015
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Ingår i: Journal of Proteome Research. - : American Chemical Society (ACS). - 1535-3893 .- 1535-3907. ; 14:8, s. 3305-3314
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Tidskriftsartikel (refereegranskat)abstract
- Freshly isolated human hepatocytes are considered the gold standard for in vitro studies of liver functions, including drug transport, metabolism, and toxicity. For accurate predictions of the in vivo outcome, the isolated hepatocytes should reflect the phenotype of their in vivo counterpart, i.e., hepatocytes in human liver tissue. Here, we quantified and compared the membrane proteomes of freshly isolated hepatocytes and human liver tissue using a label-free shotgun proteomics approach. A total of 5144 unique proteins were identified, spanning over 6 orders of magnitude in abundance. There was a good global correlation in protein abundance. However, the expression of many plasma membrane proteins was lower in the isolated hepatocytes than in the liver tissue. This included transport proteins that determine hepatocyte exposure to many drugs and endogenous compounds. Pathway analysis of the differentially expressed proteins confirmed that hepatocytes are exposed to oxidative stress during isolation and suggested that plasma membrane proteins were degraded via the protein ubiquitination pathway. Finally, using pitavastatin as an example, we show how protein quantifications can improve in vitro predictions of in vivo liver clearance. We tentatively conclude that our data set will be a useful resource for improved hepatocyte predictions of the in vivo outcome.
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| 10. |
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