Publications by authors named "Christine Wegler"

15 Publications

  • Page 1 of 1

Short- and long-term effects of body weight loss following calorie restriction and gastric bypass on CYP3A-activity - a non-randomized three-armed controlled trial.

Clin Transl Sci 2021 Aug 26. Epub 2021 Aug 26.

Section for Pharmacology and Pharmaceutical Biosciences, Department of Pharmacy, University of Oslo, Oslo, Norway.

It remains uncertain whether pharmacokinetic changes following Roux-en-Y gastric bypass (RYGB) can be attributed to surgery-induced gastrointestinal alterations per se and/or the subsequent weight loss. The aim was to compare short- and long-term effects of RYGB and calorie restriction on CYP3A-activity, and cross-sectionally compare CYP3A-activity with normal weight to overweight controls using midazolam as probe drug. This three-armed controlled trial included patients with severe obesity preparing for RYGB (n = 41) or diet-induced (n = 41) weight-loss, and controls (n = 18). Both weight-loss groups underwent a 3-week low-energy-diet (<1200 kcal/day) followed by a 6-week very-low-energy-diet or RYGB (both <800 kcal/day). Patients were followed for 2 years, with four pharmacokinetic investigations using semisimultaneous oral and intravenous dosing to determine changes in midazolam absolute bioavailability and clearance, within and between groups. The RYGB and diet groups showed similar weight-loss at week 9 (13 ± 2.4% vs. 11 ± 3.6%), but differed substantially after 2 years (-30 ± 7.0% vs. -3.1 ± 6.3%). At baseline, mean absolute bioavailability and clearance of midazolam were similar in the RYGB and diet groups, but higher compared with controls. On average, absolute bioavailability was unaltered at week 9, but decreased by 40 ± 7.5% in the RYGB group and 32 ± 6.1% in the diet group at year 2 compared with baseline, with no between-group difference. No difference in clearance was observed over time, nor between groups. In conclusion, neither RYGB per se nor weight loss impacted absolute bioavailability or clearance of midazolam short term. Long term, absolute bioavailability was similarly decreased in both groups despite different weight loss, suggesting that the recovered CYP3A-activity is not only dependent on weight-loss through RYGB.
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http://dx.doi.org/10.1111/cts.13142DOI Listing
August 2021

Influence of Proteome Profiles and Intracellular Drug Exposure on Differences in CYP Activity in Donor-Matched Human Liver Microsomes and Hepatocytes.

Mol Pharm 2021 04 19;18(4):1792-1805. Epub 2021 Mar 19.

Department of Pharmacy and Science for Life Laboratory, Uppsala University, 752 37 Uppsala, Sweden.

Human liver microsomes (HLM) and human hepatocytes (HH) are important systems for studies of intrinsic drug clearance (CL) in the liver. However, the CL values are often in disagreement for these two systems. Here, we investigated these differences in a side-by-side comparison of drug metabolism in HLM and HH prepared from 15 matched donors. Protein expression and intracellular unbound drug concentration (Kp) effects on the CL were investigated for five prototypical probe substrates (bupropion-CYP2B6, diclofenac-CYP2C9, omeprazole-CYP2C19, bufuralol-CYP2D6, and midazolam-CYP3A4). The samples were donor-matched to compensate for inter-individual variability but still showed systematic differences in CL. Global proteomics analysis outlined differences in HLM from HH and homogenates of human liver (HL), indicating variable enrichment of ER-localized cytochrome P450 (CYP) enzymes in the HLM preparation. This suggests that the HLM may not equally and accurately capture metabolic capacity for all CYPs. Scaling CL with CYP amounts and Kp could only partly explain the discordance in absolute values of CL for the five substrates. Nevertheless, scaling with CYP amounts improved the agreement in rank order for the majority of the substrates. Other factors, such as contribution of additional enzymes and variability in the proportions of active and inactive CYP enzymes in HLM and HH, may have to be considered to avoid the use of empirical scaling factors for prediction of drug metabolism.
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http://dx.doi.org/10.1021/acs.molpharmaceut.1c00053DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC8041379PMC
April 2021

Hepatocyte size fractionation allows dissection of human liver zonation.

J Cell Physiol 2021 Aug 16;236(8):5885-5894. Epub 2021 Jan 16.

Department of Pharmacy, Uppsala University, Uppsala, Sweden.

Human hepatocytes show marked differences in cell size, gene expression, and function throughout the liver lobules, an arrangement termed liver zonation. However, it is not clear if these zonal size differences, and the associated phenotypic differences, are retained in isolated human hepatocytes, the "gold standard" for in vitro studies of human liver function. Here, we therefore explored size differences among isolated human hepatocytes and investigated whether separation by size can be used to study liver zonation in vitro. We used counterflow centrifugal elutriation to separate cells into different size fractions and analyzed them with label-free quantitative proteomics, which revealed an enrichment of 151 and 758 proteins (out of 5163) in small and large hepatocytes, respectively. Further analysis showed that protein abundances in different hepatocyte size fractions recapitulated the in vivo expression patterns of previously described zonal markers and biological processes. We also found that the expression of zone-specific cytochrome P450 enzymes correlated with their metabolic activity in the different fractions. In summary, our results show that differences in hepatocyte size matches zonal expression patterns, and that our size fractionation approach can be used to study zone-specific liver functions in vitro.
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http://dx.doi.org/10.1002/jcp.30273DOI Listing
August 2021

Proteomics-Informed Identification of Luminal Targets For In Situ Diagnosis of Inflammatory Bowel Disease.

J Pharm Sci 2021 01 4;110(1):239-250. Epub 2020 Nov 4.

Department of Pharmacy, Science for Life Laboratory, Uppsala University, SE-75123 Uppsala, Sweden. Electronic address:

Inflammatory bowel disease (IBD) is a chronic condition resulting in impaired intestinal homeostasis. Current practices for diagnosis of IBD are challenged by invasive, demanding procedures. We hypothesized that proteomics analysis could provide a powerful tool for identifying clinical biomarkers for non-invasive IBD diagnosis. Here, the global intestinal proteomes from commonly used in vitro and in vivo models of IBD were analyzed to identify apical and luminal proteins that can be targeted by orally delivered diagnostic agents. Global proteomics analysis revealed upregulated plasma membrane proteins in intestinal segments of proximal- and distal colon from dextran sulfate sodium-treated mice and also in inflamed human intestinal Caco-2 cells pretreated with pro-inflammatory agents. The upregulated colon proteins in mice were compared to the proteome of the healthy ileum, to ensure targeting of diagnostic agents to the inflamed colon. Promising target proteins for future investigations of non-invasive diagnosis of IBD were found in both systems and included Tgm2/TGM2, Icam1/ICAM1, Ceacam1/CEACAM1, and Anxa1/ANXA1. Ultimately, these findings will guide the selection of appropriate antibodies for surface functionalization of imaging agents aimed to target inflammatory biomarkers in situ.
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http://dx.doi.org/10.1016/j.xphs.2020.11.001DOI Listing
January 2021

Expanding the Efflux In Vitro Assay Toolbox: A CRISPR-Cas9 Edited MDCK Cell Line with Human BCRP and Completely Lacking Canine MDR1.

J Pharm Sci 2021 01 29;110(1):388-396. Epub 2020 Sep 29.

Department of Pharmacy, Drug Delivery Research Group, Uppsala University, Uppsala, Sweden; Department of Pharmacy, Uppsala University Drug Optimization and Pharmaceutical Profiling Platform (UDOPP), Uppsala, Sweden. Electronic address:

The Breast Cancer Resistance Protein (BCRP) is a key transporter in drug efflux and drug-drug interactions. However, endogenous expression of Multidrug Resistance Protein 1 (MDR1) confounds the interpretation of BCRP-mediated transport in in vitro models. Here we used a CRISPR-Cas9 edited Madin-Darby canine kidney (MDCK) II cell line (MDCK) for stable expression of human BCRP (hBCRP) with no endogenous canine MDR1 (cMDR1) expression (MDCK-hBCRP). Targeted quantitative proteomics verified expression of hBCRP, and global analysis of the entire proteome corroborated no or very low background expression of other drug transport proteins or metabolizing enzymes. This new cell line, had similar proteome like MDCK and a previously established, corresponding cell line overexpressing human MDR1 (hMDR1), MDCK-hMDR1. Functional studies with MDCK-hBCRP confirmed high hBCRP activity. The MDCK-hBCRP cell line together with the MDCK-hMDR1 easily and accurately identified shared or specific substrates of the hBCRP and the hMDR1 transporters. These cell lines offer new, improved in vitro tools for the assessment of drug efflux and drug-drug interactions in drug development.
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http://dx.doi.org/10.1016/j.xphs.2020.09.039DOI Listing
January 2021

Proteomics-Informed Prediction of Rosuvastatin Plasma Profiles in Patients With a Wide Range of Body Weight.

Clin Pharmacol Ther 2021 03 18;109(3):762-771. Epub 2020 Oct 18.

Department of Pharmacy and Science for Life Laboratory, Uppsala University, Uppsala, Sweden.

Rosuvastatin is a frequently used probe to study transporter-mediated hepatic uptake. Pharmacokinetic models have therefore been developed to predict transporter impact on rosuvastatin disposition in vivo. However, the interindividual differences in transporter concentrations were not considered in these models, and the predicted transporter impact was compared with historical in vivo data. In this study, we investigated the influence of interindividual transporter concentrations on the hepatic uptake clearance of rosuvastatin in 54 patients covering a wide range of body weight. The 54 patients were given an oral dose of rosuvastatin the day before undergoing gastric bypass or cholecystectomy, and pharmacokinetic (PK) parameters were established from each patient's individual time-concentration profiles. Liver biopsies were sampled from each patient and their individual hepatic transporter concentrations were quantified. We combined the transporter concentrations with in vitro uptake kinetics determined in HEK293-transfected cells, and developed a semimechanistic model with a bottom-up approach to predict the plasma concentration profiles of the single dose of rosuvastatin in each patient. The predicted PK parameters were evaluated against the measured in vivo plasma PKs from the same 54 patients. The developed model predicted the rosuvastatin PKs within two-fold error for rosuvastatin area under the plasma concentration versus time curve (AUC; 78% of the patients; average fold error (AFE): 0.96), peak plasma concentration (C ; 76%; AFE: 1.05), and terminal half-life (t ; 98%; AFE: 0.89), and captured differences in the rosuvastatin PKs in patients with the OATP1B1 521T
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http://dx.doi.org/10.1002/cpt.2056DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7984432PMC
March 2021

A Comparative Analysis of Cytochrome P450 Activities in Paired Liver and Small Intestinal Samples from Patients with Obesity.

Drug Metab Dispos 2020 01 4;48(1):8-17. Epub 2019 Nov 4.

Section for Pharmacology and Pharmaceutical Biosciences, Department of Pharmacy, University of Oslo, Oslo, Norway (V.K., I.R., A.Å., H.C.); Department of Transplantation Medicine, Oslo University Hospital, Rikshospitalet, Oslo, Norway (V.K., A.Å.); Research and Early Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca Gothenburg, Gothenburg, Sweden (A.P., C.W., S.A., T.B.A.); Center for Psychopharmacology, Diakonhjemmet Hospital, Oslo, Norway (M.K.K.); Department of Health Sciences, OsloMet-Oslo Metropolitan University, Oslo, Norway (M.K.K.); Department of Pharmacy, Uppsala University, Uppsala, Sweden (C.W., P.A.); The Morbid Obesity Centre, Vestfold Hospital Trust, Tønsberg, Norway (P.C.A., J.H.); Department of Surgery, Vestfold Hospital Trust, Tønsberg, Norway (P.C.A.); Department of Endocrinology, Morbid Obesity and Preventive Medicine, Institute of Clinical Medicine, University of Oslo, Oslo, Norway (J.H.); Late-Stage Development, Cardiovascular, Renal and Metabolism, BioPharmaceuticals R&D, AstraZeneca Gothenburg, Gothenburg, Sweden (C.K.); Department of Molecular and Clinical Medicine, Institute of Medicine, Sahlgrenska Academy, University of Gothenburg, Gothenburg, Sweden (C.K.); and Department of Physiology and Pharmacology, Section of Pharmacogenetics, Karolinska Institutet, Stockholm, Sweden (T.B.A.).

The liver and small intestine restrict oral bioavailability of drugs and constitute the main sites of pharmacokinetic drug-drug interactions. Hence, detailed data on hepatic and intestinal activities of drug metabolizing enzymes is important for modeling drug disposition and optimizing pharmacotherapy in different patient populations. The aim of this study was to determine the activities of seven cytochrome P450 (P450) enzymes in paired liver and small intestinal samples from patients with obesity. Biopsies were obtained from 20 patients who underwent Roux-en-Y gastric bypass surgery following a 3-week low-energy diet. Individual hepatic and intestinal microsomes were prepared and specific probe substrates in combined incubations were used for determination of CYP1A2, CYP2B6, CYP2C8, CYP2C9, CYP2C19, CYP2D6, and CYP3A activities. The activities of CYP2C8, CYP2C9, CYP2D6, and CYP3A were quantified in both human liver microsomes (HLM) and human intestinal microsomes (HIM), while the activities of CYP1A2, CYP2B6, and CYP2C19 were only quantifiable in HLM. Considerable interindividual variability was present in both HLM (9- to 23-fold) and HIM (5- to 55-fold). The median metabolic HLM/HIM ratios varied from 1.5 for CYP3A to 252 for CYP2C8. The activities of CYP2C9 in paired HLM and HIM were positively correlated ( = 0.74, < 0.001), while no interorgan correlations were found for activities of CYP2C8, CYP2D6, and CYP3A ( > 0.05). Small intestinal CYP3A activities were higher in females compared with males ( < 0.05). Hepatic CYP2B6 activity correlated negatively with body mass index ( = -0.72, < 0.001). These data may be useful for further in vitro-in vivo predictions of drug disposition in patients with obesity. SIGNIFICANCE STATEMENT: Hepatic and intestinal drug metabolism is the key determinant of oral drug bioavailability. In this study, paired liver and jejunum samples were obtained from 20 patients with obesity undergoing gastric bypass surgery following a 3-week low-energy diet. We determined the hepatic and small intestinal activities of clinically important P450 enzymes and provide detailed enzyme kinetic data relevant for predicting in vivo disposition of P450 substrates in this patient population.
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http://dx.doi.org/10.1124/dmd.119.087940DOI Listing
January 2020

Global variability analysis of mRNA and protein concentrations across and within human tissues.

NAR Genom Bioinform 2020 Mar 30;2(1):lqz010. Epub 2019 Oct 30.

Department of Pharmacy and Science for Life Laboratory, Uppsala University, Uppsala SE-75123, Sweden.

Genes and proteins show variable expression patterns throughout the human body. However, it is not clear whether relative differences in mRNA concentrations are retained on the protein level. Furthermore, inter-individual protein concentration variability within single tissue types has not been comprehensively explored. Here, we used the Gini index for in-depth concentration variability analysis of publicly available transcriptomics and proteomics data, and of an in-house proteomics dataset of human liver and jejunum from 38 donors. We found that the transfer of concentration variability from mRNA to protein is limited, that established 'reference genes' for data normalization vary markedly at the protein level, that protein concentrations cover a wide variability spectrum within single tissue types, and that concentration variability analysis can be a convenient starting point for identifying disease-associated proteins and novel biomarkers. Our results emphasize the importance of considering individual concentration levels, as opposed to population averages, for personalized systems biology analysis.
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http://dx.doi.org/10.1093/nargab/lqz010DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7671341PMC
March 2020

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.

J Proteome Res 2019 01 30;18(1):217-224. Epub 2018 Oct 30.

Department of Pharmacy , Uppsala University , S-751 23 Uppsala , Sweden.

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.
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http://dx.doi.org/10.1021/acs.jproteome.8b00549DOI Listing
January 2019

Direct Quantification of Cytochromes P450 and Drug Transporters-A Rapid, Targeted Mass Spectrometry-Based Immunoassay Panel for Tissues and Cell Culture Lysates.

Drug Metab Dispos 2018 04 17;46(4):387-396. Epub 2018 Jan 17.

NMI Natural and Medical Sciences Institute at the University of Tübingen, Reutlingen, Germany (F.W., H.S.H., H.P., T.O.J., O.P.); SIGNATOPE GmbH, Reutlingen, Germany (F.W., H.S.H., H.P., T.O.J., O.P.); Dr. Margarete Fischer-Bosch-Institute of Clinical Pharmacology, Stuttgart, Germany (K.K., U.M.Z.); Department of Clinical Pharmacology, University of Tuebingen, Tübingen, Germany (K.K., U.M.Z.); Departments of Surgical Sciences (A.N.) and Pharmacy (C.W., P.A.), Uppsala University, Uppsala, Sweden; and Cardiovascular and Metabolic Diseases, Innovative Medicines and Early Development Biotech Unit, Mölndal, Sweden (C.W.)

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.
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http://dx.doi.org/10.1124/dmd.117.078626DOI Listing
April 2018

Variability in Mass Spectrometry-based Quantification of Clinically Relevant Drug Transporters and Drug Metabolizing Enzymes.

Mol Pharm 2017 09 16;14(9):3142-3151. Epub 2017 Aug 16.

Department of Pharmacy, Uppsala University , Uppsala 75123, Sweden.

Many different methods are used for mass-spectrometry-based protein quantification in pharmacokinetics and systems pharmacology. It has not been established to what extent the results from these various methods are comparable. Here, we compared six different mass spectrometry-based proteomics methods by measuring the expression of clinically relevant drug transporters and metabolizing enzymes in human liver. Mean protein concentrations were in general quantified to similar levels by methods using whole tissue lysates. Methods using subcellular membrane fractionation gave incomplete enrichment of the proteins. When the enriched proteins were adjusted to levels in whole tissue lysates, they were on average 4-fold lower than those quantified directly in whole tissue lysates. The differences in protein levels were propagated into differences in predictions of hepatic clearance. In conclusion, caution is needed when comparing and applying quantitative proteomics data obtained with different methods, especially since membrane fractionation is common practice for protein quantification used in drug clearance predictions.
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http://dx.doi.org/10.1021/acs.molpharmaceut.7b00364DOI Listing
September 2017

A CRISPR-Cas9 Generated MDCK Cell Line Expressing Human MDR1 Without Endogenous Canine MDR1 (cABCB1): An Improved Tool for Drug Efflux Studies.

J Pharm Sci 2017 09 24;106(9):2909-2913. Epub 2017 Apr 24.

Department of Pharmacy, Uppsala University, Uppsala, Sweden; Uppsala University Drug Optimization and Pharmaceutical Profiling Platform, Science for Life Laboratory, Uppsala, Sweden.

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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http://dx.doi.org/10.1016/j.xphs.2017.04.018DOI Listing
September 2017

Intracellular drug bioavailability: a new predictor of system dependent drug disposition.

Sci Rep 2017 02 22;7:43047. Epub 2017 Feb 22.

Department of Pharmacy, Uppsala University, BMC, Box 580, Uppsala SE-751 23, Sweden.

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) as the fraction of extracellular drug available to bind intracellular targets, and we assess how F 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 F of its substrates, while P-gp decreased F. 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 F was up to 35-fold lower in the configuration with high expression of drug-eliminating transporters and enzymes. We conclude that F 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).
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http://dx.doi.org/10.1038/srep43047DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5320532PMC
February 2017

Subcellular fractionation of human liver reveals limits in global proteomic quantification from isolated fractions.

Anal Biochem 2016 09 14;509:82-88. Epub 2016 Jun 14.

Department of Pharmacy, Uppsala University, S-751 23 Uppsala, Sweden; Science for Life Laboratory, Uppsala University, S-751 23 Uppsala, Sweden.

The liver plays an important role in metabolism and elimination of xenobiotics, including drugs. Determination of concentrations of proteins involved in uptake, distribution, metabolism, and excretion of xenobiotics is required to understand and predict elimination mechanisms in this tissue. In this work, we have fractionated homogenates of snap-frozen human liver by differential centrifugation and performed quantitative mass spectrometry-based proteomic analysis of each fraction. Concentrations of proteins were calculated by the "total protein approach". A total of 4586 proteins were identified by at least five peptides and were quantified in all fractions. We found that the xenobiotics transporters of the canalicular and basolateral membranes were differentially enriched in the subcellular fractions and that phase I and II metabolizing enzymes, the cytochrome P450s and the UDP-glucuronyl transferases, have complex subcellular distributions. These findings show that there is no simple way to scale the data from measurements in arbitrarily selected membrane fractions using a single scaling factor for all the proteins of interest. This study also provides the first absolute quantitative subcellular catalog of human liver proteins obtained from frozen tissue specimens. Our data provide quantitative insights into the subcellular distribution of proteins and can be used as a guide for development of fractionation procedures.
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http://dx.doi.org/10.1016/j.ab.2016.06.006DOI Listing
September 2016

Effects of Osteoporosis-Inducing Drugs on Vitamin D-Related Gene Transcription and Mineralization in MG-63 and Saos-2 Cells.

Basic Clin Pharmacol Toxicol 2016 Nov 20;119(5):436-442. Epub 2016 May 20.

Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden.

Vitamin D is important for calcium and phosphate homeostasis. To exert its effects, vitamin D has to be enzymatically activated into 1,25D (1,25-dihydroxyvitamin D ). Regulation by endogenous vitamin D metabolites of the activation and inactivation of 1,25D is important to maintain adequate amounts of active vitamin D . Vitamin D deficiency and low bone mineral density have been linked to treatments with antiretroviral drugs and glucocorticoids. However, the causes of drug-induced osteoporosis remain unclear. The antiretroviral drugs efavirenz and ritonavir as well as the glucocorticoid dexamethasone were included in this study. Their effects on transcription of vitamin D-regulating enzymes in MG-63 cells were investigated. Ritonavir and dexamethasone both induced transcription of CYP27B1, the enzyme responsible for the formation of 1,25D . Efavirenz, however, suppressed CYP27B1 expression. When administered together with endogenous vitamin D metabolites, dexamethasone and efavirenz counteracted the 1,25D -mediated up-regulation of CYP24A1, which inactivates 1,25D . This suggests that the drugs may interfere with local regulation of the vitamin D metabolizing system in osteoblasts. Studies on mineralization were performed in MG-63 cells and Saos-2 cells by measuring calcium concentrations accumulated over time. The effects of efavirenz, ritonavir and dexamethasone and/or vitamin D metabolites were examined. 1,25D induced mineralization in both cell lines. Efavirenz administered alone did not affect mineralization but suppressed the inducing effects of 1,25D on mineralization in both MG-63 cells and Saos-2 cells. In summary, the results suggest that antiretroviral drugs and glucocorticoids may adversely affect bone by interference with the vitamin D system in osteoblasts.
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http://dx.doi.org/10.1111/bcpt.12612DOI Listing
November 2016
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