Publications by authors named "Margret Thorsteinsdottir"

31 Publications

Design of experiments for development and optimization of a liquid chromatography coupled to tandem mass spectrometry bioanalytical assay.

J Mass Spectrom 2021 Apr 2:e4727. Epub 2021 Apr 2.

Faculty of Pharmaceutical Sciences, University of Iceland, Reykjavik, Iceland.

Design of experiments (DoE) is a valuable tool for optimization of a quantitative bioanalytical method utilizing liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Liquid chromatography mass spectrometry (LC-MS) is composed of several processes, including, liquid introduction and analyte ionization. The goal is to transfer analytes from atmospheric pressure to vacuum and maintain conditions that are compatible for both the LC and the MS. These processes involve many experimental factors which need to be simultaneously optimized to obtain maximum sensitivity and resolution at minimum retention time. In this tutorial the basic concepts of DoE will be explained with focus on practical use of DoE, and then three case studies about utilization of DoE for optimization of liquid chromatography tandem mass spectrometry (LC-MS/MS) quantitative assays will be presented.
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http://dx.doi.org/10.1002/jms.4727DOI Listing
April 2021

Genetic variability in the absorption of dietary sterols affects the risk of coronary artery disease.

Eur Heart J 2020 07;41(28):2618-2628

Laekning, Medical Clinics, Lágmúli 5, 108 Reykjavik, Iceland.

Aims: To explore whether variability in dietary cholesterol and phytosterol absorption impacts the risk of coronary artery disease (CAD) using as instruments sequence variants in the ABCG5/8 genes, key regulators of intestinal absorption of dietary sterols.

Methods And Results: We examined the effects of ABCG5/8 variants on non-high-density lipoprotein (non-HDL) cholesterol (N up to 610 532) and phytosterol levels (N = 3039) and the risk of CAD in Iceland, Denmark, and the UK Biobank (105 490 cases and 844 025 controls). We used genetic scores for non-HDL cholesterol to determine whether ABCG5/8 variants confer greater risk of CAD than predicted by their effect on non-HDL cholesterol. We identified nine rare ABCG5/8 coding variants with substantial impact on non-HDL cholesterol. Carriers have elevated phytosterol levels and are at increased risk of CAD. Consistent with impact on ABCG5/8 transporter function in hepatocytes, eight rare ABCG5/8 variants associate with gallstones. A genetic score of ABCG5/8 variants predicting 1 mmol/L increase in non-HDL cholesterol associates with two-fold increase in CAD risk [odds ratio (OR) = 2.01, 95% confidence interval (CI) 1.75-2.31, P = 9.8 × 10-23] compared with a 54% increase in CAD risk (OR = 1.54, 95% CI 1.49-1.59, P = 1.1 × 10-154) associated with a score of other non-HDL cholesterol variants predicting the same increase in non-HDL cholesterol (P for difference in effects = 2.4 × 10-4).

Conclusions: Genetic variation in cholesterol absorption affects levels of circulating non-HDL cholesterol and risk of CAD. Our results indicate that both dietary cholesterol and phytosterols contribute directly to atherogenesis.
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http://dx.doi.org/10.1093/eurheartj/ehaa531DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7377579PMC
July 2020

Lipidomic study of cell lines reveals differences between breast cancer subtypes.

PLoS One 2020 14;15(4):e0231289. Epub 2020 Apr 14.

Faculty of Pharmaceutical Sciences, University of Iceland, Reykjavík, Iceland.

Breast cancer (BC) is the most prevalent type of cancer in women in western countries. BC mortality has not declined despite early detection by screening, indicating the need for better informed treatment decisions. Therefore, a novel noninvasive diagnostic tool for BC would give the opportunity of subtype-specific treatment and improved prospects for the patients. Heterogeneity of BC tumor subtypes is reflected in the expression levels of enzymes in lipid metabolism. The aim of the study was to investigate whether the subtype defined by the transcriptome is reflected in the lipidome of BC cell lines. A liquid chromatography mass spectrometry (LC-MS) platform was applied to analyze the lipidome of six cell lines derived from human BC cell lines representing different BC subtypes. We identified an increased abundance of triacylglycerols (TG) ≥ C-48 with moderate or multiple unsaturation in fatty acyl chains and down-regulated ether-phosphatidylethanolamines (PE) (C-34 to C-38) in cell lines representing estrogen receptor and progesterone receptor positive tumor subtypes. In a cell line representing HER2-overexpressing tumor subtype an elevated expression of TG (≤ C-46), phosphatidylcholines (PC) and PE containing short-chained (≤ C-16) saturated or monounsaturated fatty acids were observed. Increased abundance of PC ≥ C-40 was found in cell lines of triple negative BC subtype. In addition, differences were detected in lipidomes within these previously defined subtypes. We conclude that subtypes defined by the transcriptome are indeed reflected in differences in the lipidome and, furthermore, potentially biologically relevant differences may exist within these defined subtypes.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0231289PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7156077PMC
July 2020

Cellular and Molecular Mechanisms of Kidney Injury in 2,8-Dihydroxyadenine Nephropathy.

J Am Soc Nephrol 2020 04 21;31(4):799-816. Epub 2020 Feb 21.

Institute of Pathology,

Background: Hereditary deficiency of adenine phosphoribosyltransferase causes 2,8-dihydroxyadenine (2,8-DHA) nephropathy, a rare condition characterized by formation of 2,8-DHA crystals within renal tubules. Clinical relevance of rodent models of 2,8-DHA crystal nephropathy induced by excessive adenine intake is unknown.

Methods: Using animal models and patient kidney biopsies, we assessed the pathogenic sequelae of 2,8-DHA crystal-induced kidney damage. We also used knockout mice to investigate the role of TNF receptors 1 and 2 (TNFR1 and TNFR2), CD44, or alpha2-HS glycoprotein (AHSG), all of which are involved in the pathogenesis of other types of crystal-induced nephropathies.

Results: Adenine-enriched diet in mice induced 2,8-DHA nephropathy, leading to progressive kidney disease, characterized by crystal deposits, tubular injury, inflammation, and fibrosis. Kidney injury depended on crystal size. The smallest crystals were endocytosed by tubular epithelial cells. Crystals of variable size were excreted in urine. Large crystals obstructed whole tubules. Medium-sized crystals induced a particular reparative process that we term . In this process, tubular cells, in coordination with macrophages, overgrew and translocated crystals into the interstitium, restoring the tubular luminal patency; this was followed by degradation of interstitial crystals by granulomatous inflammation. Patients with adenine phosphoribosyltransferase deficiency showed similar histopathological findings regarding crystal morphology, crystal clearance, and renal injury. In mice, deletion of significantly reduced tubular CD44 and annexin two expression, as well as inflammation, thereby ameliorating the disease course. In contrast, genetic deletion of , , or had no effect on the manifestations of 2,8-DHA nephropathy.

Conclusions: Rodent models of the cellular and molecular mechanisms of 2,8-DHA nephropathy and crystal clearance have clinical relevance and offer insight into potential future targets for therapeutic interventions.
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http://dx.doi.org/10.1681/ASN.2019080827DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC7191925PMC
April 2020

Valuable Fatty Acids in Bryophytes-Production, Biosynthesis, Analysis and Applications.

Plants (Basel) 2019 Nov 19;8(11). Epub 2019 Nov 19.

Department of Biotechnology and Biomedicine, Technical University of Denmark, Søltofts Plads 223, 2800 Kongens Lyngby, Denmark.

Bryophytes (mosses, liverworts and hornworts) often produce high amounts of very long-chain polyunsaturated fatty acids (vl-PUFAs) including arachidonic acid (AA, 20:4 △5,8,11,14) and eicosapentaenoic acid (EPA, 20:5 △5,8,11,14,17). The presence of vl-PUFAs is common for marine organisms such as algae, but rarely found in higher plants. This could indicate that bryophytes did not lose their marine origin completely when they landed into the non-aqueous environment. Vl-PUFA, especially the omega-3 fatty acid EPA, is essential in human diet for its benefits on healthy brain development and inflammation modulation. Recent studies are committed to finding new sources of vl-PUFAs instead of fish and algae oil. In this review, we summarize the fatty acid compositions and contents in the previous studies, as well as the approaches for qualification and quantification. We also conclude different approaches to enhance AA and EPA productions including biotic and abiotic stresses.
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http://dx.doi.org/10.3390/plants8110524DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6918284PMC
November 2019

Urinary 2,8-dihydroxyadenine excretion in patients with adenine phosphoribosyltransferase deficiency, carriers and healthy control subjects.

Mol Genet Metab 2019 Sep - Oct;128(1-2):144-150. Epub 2019 May 28.

Faculty of Medicine, School of Health Sciences, University of Iceland, Reykjavik, Iceland; Children's Medical Center, Landspitali-The National University Hospital of Iceland, Reykjavik, Iceland. Electronic address:

Background: Adenine phosphoribosyltransferase (APRT) deficiency is a rare autosomal recessive disorder of adenine metabolism that results in excessive urinary excretion of the poorly soluble 2,8-dihydroxyadenine (DHA), leading to kidney stones and chronic kidney disease. The purpose of this study was to assess urinary DHA excretion in patients with APRT deficiency, heterozygotes and healthy controls, using a recently developed ultra-performance liquid chromatography - tandem mass spectrometry (UPLC-MS/MS) assay.

Methods: Patients enrolled in the APRT Deficiency Registry and Biobank of the Rare Kidney Stone Consortium (http://www.rarekidneystones.org/) who had provided 24-h and first-morning void urine samples for DHA measurement were eligible for the study. Heterozygotes and healthy individuals served as controls. Wilcoxon-Mann-Whitney test was used to compare 24-h urinary DHA excretion between groups. Associations were examined using Spearman's correlation coefficient (r).

Results: The median (range) 24-h urinary DHA excretion was 138 (64-292) mg/24 h and the DHA-to-creatinine (DHA/Cr) ratio in the first-morning void samples was 13 (4-37) mg/mmol in APRT deficiency patients who were not receiving xanthine oxidoreductase inhibitor therapy. The 24-h DHA excretion was highly correlated with the DHA/Cr ratio in first-morning void urine samples (r = 0.84, p < .001). DHA was detected in all urine samples from untreated patients but not in any specimens from heterozygotes and healthy controls.

Conclusions: High urinary DHA excretion was observed in patients with APRT deficiency, while urine DHA was undetectable in heterozygotes and healthy controls. Our results suggest that the UPLC-MS/MS assay can be used for diagnosis of APRT deficiency.
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http://dx.doi.org/10.1016/j.ymgme.2019.05.015DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6864267PMC
April 2020

New Aromatic Bisabolane Derivatives with Lipid-Reducing Activity from the Marine Sponge sp.

Mar Drugs 2019 Jun 22;17(6). Epub 2019 Jun 22.

Faculty of Pharmaceutical Sciences, University of Iceland, Hofsvallagata 53, 107 Reykjavik, Iceland.

The previously reported 1-(2,4-dihydroxy-5-methylphenyl)ethan-1-one (), (1'Z)-2-(1',5'-dimethylhexa-1',4'-dieny1)-5-methylbenzene-1,4-diol (), and 1,8-epoxy-1(6),2,4,7,10-bisaborapentaen-4-ol () together with four new structures of aromatic bisabolane-related compounds (, , , ) were isolated from the marine sponge sp. Compounds , , and were identified based on spectral data available in the literature. The structures of the four new compounds were experimentally established by 1D and 2D-NMR and (-)-HRESIMS spectral analysis. Cytotoxic and lipid-reducing activities of the isolated compounds were evaluated. None of the isolated compounds were active against the tested cancer cell lines; however, lipid-reducing activity was found for compounds - and in the zebrafish Nile red fat metabolism assay. This class of compounds should be further explored for their suitability as possible agents for the treatment of lipid metabolic disorders and obesity.
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http://dx.doi.org/10.3390/md17060375DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6627430PMC
June 2019

Mononuclear phagocytes orchestrate prolyl hydroxylase inhibition-mediated renoprotection in chronic tubulointerstitial nephritis.

Kidney Int 2019 08 5;96(2):378-396. Epub 2019 Mar 5.

Department of Internal Medicine 4-Nephrology and Hypertension, Friedrich-Alexander-University Erlangen-Nürnberg (FAU) and University Hospital Erlangen, Erlangen, Germany. Electronic address:

Prolyl hydroxylase domain enzyme inhibitors (PHDIs) stabilize hypoxia-inducible factors (HIFs), and are protective in models of acute ischemic and inflammatory kidney disease. Whether PHDIs also confer protection in chronic inflammatory kidney disease models remains unknown. Here we investigated long-term effects of PHDI treatment in adenine-induced nephropathy as a model for chronic tubulointerstitial nephritis. After three weeks, renal dysfunction and tubulointerstitial damage, including proximal and distal tubular injury, tubular dilation and renal crystal deposition were significantly attenuated in PHDI-treated (the isoquinoline derivative ICA and Roxadustat) compared to vehicle-treated mice with adenine-induced nephropathy. Crystal-induced renal fibrosis was only partially diminished by treatment with ICA. Renoprotective effects of ICA treatment could not be attributed to changes in adenine metabolism or urinary excretion of the metabolite 2,8-dihydroxyadenine. ICA treatment reduced inflammatory infiltrates of F4/80+ mononuclear phagocytes in the kidneys and supported a regulatory, anti-inflammatory immune response. Furthermore, interstitial deposition of complement C1q was decreased in ICA-treated mice fed an adenine-enriched diet. Tubular cell-specific HIF-1α and myeloid cell-specific HIF-1α and HIF-2α expression were not required for the renoprotective effects of ICA. In contrast, depletion of mononuclear phagocytes with clodronate largely abolished the nephroprotective effects of PHD inhibition. Thus, our findings indicate novel and potent systemic anti-inflammatory properties of PHDIs that confer preservation of kidney function and structure in chronic tubulointerstitial inflammation and might counteract kidney disease progression.
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http://dx.doi.org/10.1016/j.kint.2019.02.016DOI Listing
August 2019

Identification of Cyanobacterial Strains with Potential for the Treatment of Obesity-Related Co-Morbidities by Bioactivity, Toxicity Evaluation and Metabolite Profiling.

Mar Drugs 2019 May 10;17(5). Epub 2019 May 10.

Interdisciplinary Centre of Marine and Environmental Research (CIIMAR/CIMAR), University of Porto, Avenida General Norton de Matos, s/n, 4450-208 Matosinhos, Portugal.

Obesity is a complex disease resulting in several metabolic co-morbidities and is increasing at epidemic rates. The marine environment is an interesting resource of novel compounds and in particular cyanobacteria are well known for their capacity to produce novel secondary metabolites. In this work, we explored the potential of cyanobacteria for the production of compounds with relevant activities towards metabolic diseases using a blend of target-based, phenotypic and zebrafish assays as whole small animal models. A total of 46 cyanobacterial strains were grown and biomass fractionated, yielding in total 263 fractions. Bioactivities related to metabolic function were tested in different and models. Studying adipogenic and thermogenic gene expression in brown adipocytes, lipid metabolism and glucose uptake in hepatocytes, as well as lipid metabolism in zebrafish larvae, we identified 66 (25%) active fractions. This together with metabolite profiling and the evaluation of toxicity allowed the identification of 18 (7%) fractions with promising bioactivity towards different aspects of metabolic disease. Among those, we identified several known compounds, such as eryloside T, leptosin F, pheophorbide A, phaeophytin A, chlorophyll A, present as minor peaks. Those compounds were previously not described to have bioactivities in metabolic regulation, and both known or unknown compounds could be responsible for such effects. In summary, we find that cyanobacteria hold a huge repertoire of molecules with specific bioactivities towards metabolic diseases, which needs to be explored in the future.
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http://dx.doi.org/10.3390/md17050280DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6562398PMC
May 2019

Determining gradient conditions for peptide purification in RPLC with machine-learning-based retention time predictions.

J Chromatogr A 2019 Aug 29;1598:92-100. Epub 2019 Mar 29.

Department of Engineering and Chemical Sciences, Karlstad University, SE-651 88 Karlstad, Sweden. Electronic address:

A strategy for determining a suitable solvent gradient in silico in preparative peptide separations is presented. The strategy utilizes a machine-learning-based method, called ELUDE, for peptide retention time predictions based on the amino acid sequences of the peptides. A suitable gradient is calculated according to linear solvent strength theory by predicting the retention times of the peptides being purified at three different gradient slopes. The advantage of this strategy is that fewer experiments are needed to develop a purification method, making it useful for labs conducting many separations but with limited resources for method development. The preparative separation of met-enkephalin and leu-enkephalin was used as model solutes on two stationary phases: XBridge C18 and CSH C18. The ELUDE algorithm contains a support vector regression and is pre-trained, meaning that only 10-50 peptides are needed to calibrate a model for a certain stationary phase and gradient. The calibration is done once and the model can then be used for new peptides similar in size to those in the calibration set. We found that the accuracy of the retention time predictions is good enough to usefully estimate a suitable gradient and that it was possible to compare the selectivity on different stationary phases in silico. The absolute relative errors in retention time for the predicted gradients were 4.2% and 3.7% for met-enkephalin and leu-enkephalin, respectively, on the XBridge C18 column and 2.0% and 2.8% on the CSH C18 column. The predicted retention times were also used as initial values for adsorption isotherm parameter determination, facilitating the numerical calculation of overloaded elution profiles. Changing the trifluoroacetic acid (TFA) concentration from 0.05% to 0.15% in the eluent did not seriously affect the error in the retention time predictions for the XBridge C18 column, an increase of 1.0 min (in retention factor, 1.3). For the CSH C18 column the error was, on average, 2.6 times larger. This indicates that the model needs to be recalibrated when changing the TFA concentration for the CSH column. Studying possible scale-up complications from UHPLC to HPLC such as pressure, viscous heating (i.e., temperature gradients), and stationary-phase properties (e.g., packing heterogeneity and surface chemistry) revealed that all these factors were minor to negligible. The pressure effect had the largest effect on the retention, but increased retention by only 3%. In the presented case, method development can therefore proceed using UHPLC and then be robustly transferred to HPLC.
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http://dx.doi.org/10.1016/j.chroma.2019.03.043DOI Listing
August 2019

Two new spongian diterpene analogues isolated from the marine sponge sp.

Nat Prod Res 2020 Apr 25;34(8):1053-1060. Epub 2018 Dec 25.

Faculty of Pharmaceutical Sciences, University of Iceland, Reykjavik, Iceland.

Two new marine natural compounds, 3β-acetoxy-15-hydroxyspongia-12-en () and 3-methylspongia-3,12-dien-16-one (), were isolated from the marine sponge sp., collected in Pulau-Pulau. Compounds and represent new chemical entities of the known spongian diterpene family. Compound is a new 3-acetoxyspongia and compound presents an unreported rearranged 3-methylspongia-3-en. Their structures, including relative configurations, were fully elucidated based on 1D and 2D NMR analyses, as well as HRESTOFMS experiments. No significant bioactivities were found for these compounds. This work reports two new chemical structures, compounds and , together with the first isolation of spongian diterpenes from genus.
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http://dx.doi.org/10.1080/14786419.2018.1548448DOI Listing
April 2020

To Wash or Not to Wash? Comparison of Patient Outcome after Infusion of Cryopreserved Autologous Hematopoietic Stem Cells before and after the Replacement of Manual Washing by Bedside Thawing.

Acta Haematol 2018 9;140(3):169-175. Epub 2018 Oct 9.

Department of Hematology, Landspítali, The National University Hospital of Iceland, Reykjavik, Iceland.

Background: Prior to infusion, cryopreserved autologous peripheral blood stem cell (auto-PBSC) grafts can either be thawed at the bedside or thawed and washed at the laboratory. At our center, manual washing of grafts prior to infusion was discontinued in April 2012 and bedside thawing was implemented.

Methods: This study compares the outcomes of two patient groups who received auto-PBSC either after post-thaw washing (n = 84) or bedside thawing (n = 83).

Results: No life-threatening infusion-related side effects were reported in either group. There was no significant difference in the mean CD34+ cells/kg dose of infused auto-PBSC in the two groups (p = 0.41), nor in the number of days to neutrophils > 0.5 × 10(9)/L (p = 0.14), days to platelets > 20 × 10(9)/L (p = 0.64), or days to platelets > 50 × 10(9)/L (p = 0.62) after transplant. There was also no difference in the number of days on total parenteral nutrition (p = 0.69), days on G-CSF therapy (p = 0.48), or days with fever (p = 0.73). Finally, there was no significant difference in the number of red cell units transfused (p = 0.32), or platelet units transfused (p = 0.94) after the transplant. One-hundred-day mortality was identical in the two groups (2.4%).

Conclusion: Both thawing procedures are safe and result in acceptable engraftment and patient outcomes.
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http://dx.doi.org/10.1159/000492741DOI Listing
June 2019

Infraspecific Variation of Huperzine A and B in Icelandic Huperzia selago Complex.

Planta Med 2019 Jan 5;85(2):160-168. Epub 2018 Oct 5.

Faculty of Pharmaceutical Sciences, University of Iceland, Reykjavik, Iceland.

The alkaloids huperzine A and huperzine B were originally isolated from the Chinese club moss . They are known inhibitors of acetylcholinesterase, and especially huperzine A shows pharmaceutical potential for the treatment of Alzheimer's disease. Its supply heavily relies on natural plant sources belonging to the genus , which shows considerable interspecific huperzine A variations. Furthermore, taxonomic controversy remains in this genus, particularly in the group. With focus on Icelandic taxa, we aimed to explore the relatedness of species using multi-locus phylogenetic analysis, and to investigate correlations between huperzine A contents, morphotypes, and genotypes. Phylogenetic analysis was performed with five chloroplastic loci (the intergenic spacer between the photosystem II protein D1 gene and the tRNA-His gene, maturase K, ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit, tRNA-Leu, and the intergenic spacer region between tRNA-Leu and tRNA-Phe). Huperzine A and huperzine B contents were determined using an HPLC-UV method. The phylogenetic analysis suggests that previously proposed and should not be considered species, but rather subspecies of . Three genotypes of Icelandic were identified and presented in a haplotype networking diagram. A significantly (p < 0.05) higher amount of huperzine A was found in genotype 3 (264 - 679 µg/g) than genotype 1 (20 - 180 µg/g), where the former shows a typical green and reflexed "selago" morphotype. The huperzine A content in genotype 3 is comparable to Chinese and a good alternative huperzine A source. Genotype 2 contains multiple morphotypes with a broad huperzine A content (113 - 599 µg/g). The content of huperzine B in Icelandic taxa (6 - 13 µg/g) is much lower than that in Chinese (79 - 207 µg/g).
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http://dx.doi.org/10.1055/a-0752-0295DOI Listing
January 2019

Altered plasmalogen content and fatty acid saturation following epithelial to mesenchymal transition in breast epithelial cell lines.

Int J Biochem Cell Biol 2018 10 13;103:99-104. Epub 2018 Aug 13.

Center for Systems Biology, University of Iceland, Iceland; Biomedical Center, University of Iceland, Iceland. Electronic address:

Epithelial to mesenchymal transition (EMT) is a developmental event characterized by phenotypic switching from a polarized epithelial phenotype to an unpolarized mesenchymal phenotype. Changes to plasma membrane function accompany EMT yet the differences in lipid composition of cells that have undergone EMT are relatively unexplored. To address this the lipidome of two cell models of EMT in breast epithelial tissue, D492 and HMLE, were analyzed by untargeted LC-MS. Detected masses were identified and their abundance was compared through multivariate statistical analysis. Considerable concordance was observed in eight lipid components between epithelial and mesenchymal cells in both cell models. Specifically, an increase in phosphatidylcholine and triacylglycerol were found to accompany EMT while phosphatidylcholine- and phosphatidylethanolamine plasmalogens, as well as diacylglycerols decreased. The most abundant fatty acid lengths were C16 and C18 but mesenchymal cells had on average shorter and more unsaturated fatty acids. The results are consistent with enhanced cell mobility post EMT and reflect a consequence of oxidative stress pre- and post EMT in breast epithelial tissue.
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http://dx.doi.org/10.1016/j.biocel.2018.08.003DOI Listing
October 2018

Untargeted metabolic profiling reveals geography as the strongest predictor of metabolic phenotypes of a cosmopolitan weed.

Ecol Evol 2018 Jul 22;8(13):6812-6826. Epub 2018 Jun 22.

Natural History Museum of Denmark University of Copenhagen Copenhagen Denmark.

Plants produce a multitude of metabolites that contribute to their fitness and survival and play a role in local adaptation to environmental conditions. The effects of environmental variation are particularly well studied within the genus ; however, previous studies have largely focused on targeting specific metabolites. Studies exploring metabolome-wide changes are lacking, and the effects of natural environmental variation and herbivory on the metabolomes of plants growing remain unknown. An untargeted metabolomic approach using ultra-high-performance liquid chromatography-mass spectrometry, coupled with variation partitioning, general linear mixed modeling, and network analysis was used to detect differences in metabolic phenotypes of in fifteen natural populations across Denmark. Geographic region, distance, habitat type, phenological stage, soil parameters, light levels, and leaf area were investigated for their relative contributions to explaining differences in foliar metabolomes. Herbivory effects were further investigated by comparing metabolomes from damaged and undamaged leaves from each plant. Geographic region explained the greatest number of significant metabolic differences. Soil pH had the second largest effect, followed by habitat and leaf area, while phenological stage had no effect. No evidence of the induction of metabolic features was found between leaves damaged by herbivores compared to undamaged leaves on the same plant. Differences in metabolic phenotypes explained by geographic factors are attributed to genotypic variation and/or unmeasured environmental factors that differ at the regional level in Denmark. A small number of specialized features in the metabolome may be involved in facilitating the success of a widespread species such as into such wide range of environmental conditions, although overall resilience in the metabolome was found in response to environmental parameters tested. Untargeted metabolomic approaches have great potential to improve our understanding of how specialized plant metabolites respond to environmental change and assist in adaptation to local conditions.
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http://dx.doi.org/10.1002/ece3.4195DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC6053570PMC
July 2018

Authentication of Iceland Moss (Cetraria islandica) by UPLC-QToF-MS chemical profiling and DNA barcoding.

Food Chem 2018 Apr 21;245:989-996. Epub 2017 Nov 21.

Faculty of Pharmaceutical Sciences, University of Iceland, Hagi, Hofsvallagata 53, IS-107 Reykjavik, Iceland. Electronic address:

The lichen Cetraria islandica or Iceland Moss is commonly consumed as tea, food ingredients (e.g. in soup or bread) and herbal medicines. C. islandica, which has two chemotypes, can be difficult to distinguish from the sister species Cetraria ericetorum. They are collectively referred to as the Cetraria islandica species complex. This study aimed to use an UPLC-QToF-MS chemical profiling together with DNA barcoding to distinguish species and chemotypes of the C. islandica species complex. Our results show that the two chemotypes of C. islandica are clearly distinguishable from each other and from C. ericetorum by the chemometric approach. The RPB2 barcode was able to differentiate C. islandica from C. ericetorum with a barcode gap, but the widely used nrITS barcode failed. Neither of them could discriminate chemotypes of C. islandica. In conclusion, this integrative approach involving chemical profiling and DNA barcoding could be applied for authentication of Iceland Moss materials.
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http://dx.doi.org/10.1016/j.foodchem.2017.11.073DOI Listing
April 2018

Comparison of the effect of allopurinol and febuxostat on urinary 2,8-dihydroxyadenine excretion in patients with Adenine phosphoribosyltransferase deficiency (APRTd): A clinical trial.

Eur J Intern Med 2018 02 12;48:75-79. Epub 2017 Dec 12.

Faculty of Medicine, School of Health Sciences, University of Iceland, Reykjavik, Iceland; Division of Nephrology, Internal Medicine Services, Landspitali - The National University Hospital of Iceland, Reykjavik, Iceland. Electronic address:

Introduction: Adenine phosphoribosyltransferase (APRT) deficiency is a rare, but significant, cause of kidney stones and progressive chronic kidney disease. The optimal treatment has not been established. The purpose of this pilot study was to compare the effect of the xanthine oxidoreductase inhibitors allopurinol and febuxostat on urinary 2,8-dihydroxyadenine (DHA) excretion in APRT deficiency patients.

Materials And Methods: Patients listed in the APRT Deficiency Registry of the Rare Kidney Stone Consortium, currently receiving allopurinol therapy, were invited to participate. The trial endpoint was the 24-h urinary DHA excretion following treatment with allopurinol (400mg/day) and febuxostat (80mg/day). Urinary DHA was measured using a novel ultra-performance liquid chromatography - electrospray tandem mass spectrometry assay.

Results: Eight of the 10 patients invited completed the study. The median (range) 24-h urinary DHA excretion was 116 (75-289) mg at baseline, and 45 (13-112) mg after 14days of allopurinol therapy (P=0.036). At the end of the febuxostat treatment period, 4 patients had urinary DHA below detectable limits (<20ng/mL) compared with none of the participants following allopurinol treatment (P=0.036). The other 4 participants had a median 24-h urinary DHA excretion of 13.2 (10.0-13.4) mg at the completion of febuxostat therapy (P=0.036).

Conclusion: Urinary DHA excretion in APRT deficiency patients decreased with conventional doses of both allopurinol and febuxostat. Febuxostat was, however, significantly more efficacious than allopurinol in reducing DHA excretion in the prescribed doses. This finding, which may translate into improved outcomes of patients with APRT deficiency, should be confirmed in a larger sample.
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http://dx.doi.org/10.1016/j.ejim.2017.10.007DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5817015PMC
February 2018

DNA barcoding and LC-MS metabolite profiling of the lichen-forming genus Melanelia: Specimen identification and discrimination focusing on Icelandic taxa.

PLoS One 2017 24;12(5):e0178012. Epub 2017 May 24.

Faculty of Pharmaceutical Sciences, University of Iceland, Reykjavik, Iceland.

Taxa in the genus Melanelia (Parmeliaceae, Ascomycota) belong to a group of saxicolous lichens with brown to black foliose thalli, which have recently undergone extensive changes in circumscription. Taxa belonging to Parmeliaceae are prolific producers of bioactive compounds, which have also been traditionally used for chemotaxonomic purposes. However, the chemical diversity of the genus Melanelia and the use of chemical data for species discrimination in this genus are largely unexplored. In addition, identification based on morphological characters is challenging due to few taxonomically informative characters. Molecular identification methods, such as DNA barcoding, have rarely been applied to this genus. This study aimed to identify the Melanelia species from Iceland using DNA barcoding approach, and to explore their chemical diversity using chemical profiling. Chemometric tools were used to see if lichen metabolite profiles determined by LC-MS could be used for the identification of Icelandic Melanelia species. Barcoding using the fungal nuclear ribosomal internal transcribed spacer region (nrITS) successfully identified three Melalenlia species occurring in Iceland, together with Montanelia disjuncta (Basionym: Melanelia disjuncta). All species formed monophyletic clades in the neighbor-joining nrITS gene tree. However, high intraspecific genetic distance of M. stygia suggests the potential of unrecognized species lineages. Principal component analysis (PCA) of metabolite data gave a holistic overview showing that M. hepatizon and M. disjuncta were distinct from the rest, without the power to separate M. agnata and M. stygia due to their chemical similarity. Orthogonal partial least-squares to latent structures-discriminate analysis (OPLS-DA), however, successfully distinguished M. agnata and M. stygia by identifying statistically significant metabolites, which lead to class differentiation. This work has demonstrated the potential of DNA barcoding, chemical profiling and chemometrics in identification of Melanelia species.
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http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0178012PLOS
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5443556PMC
September 2017

New Fluvirucinins C1 and C2 Produced by a Marine Derived Actinomycete.

Nat Prod Commun 2017 May;12(5):679-682

Two new fluvirucin aglycones, named fluvirucinins C, and C2 (1-2), have been isolated from the ethyl acetate mycelial cake extract of the fermentation broth of.a marine sponge-associated actinomycete. Fluvirucinins C, (1) and C2 (2) represent a new type of 14-membered macrolactam aglycon, structurally related with the common aglycon of the known fluvirucins. Their structures were elucidated on the basis of ID and 2D NMR analyses, as well as HRESIMS experiments. The antimicrobial and cytotoxic activities of compounds 1 and 2 have been evaluated, but no significant activities found for fluvirucinins C, and C2.
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May 2017

Metabolic Profiling as a Screening Tool for Cytotoxic Compounds: Identification of 3-Alkyl Pyridine Alkaloids from Sponges Collected at a Shallow Water Hydrothermal Vent Site North of Iceland.

Mar Drugs 2017 Feb 22;15(2). Epub 2017 Feb 22.

Center for Biomedicine, European Academy of Bolzano/Bozen, Bolzano 39100, Italy.

Twenty-eight sponge specimens were collected at a shallow water hydrothermal vent site north of Iceland. Extracts were prepared and tested in vitro for cytotoxic activity, and eight of them were shown to be cytotoxic. A mass spectrometry (MS)-based metabolomics approach was used to determine the chemical composition of the extracts. This analysis highlighted clear differences in the metabolomes of three sponge specimens, and all of them were identified as (Bowerbank, 1866). Therefore, these specimens were selected for further investigation. metabolomes contained a class of potential key compounds, the 3-alkyl pyridine alkaloids (3-APA) responsible for the cytotoxic activity of the fractions. Several 3-APA compounds were tentatively identified including haliclamines, cyclostellettamines, viscosalines and viscosamines. Among these compounds, cyclostellettamine P was tentatively identified for the first time by using ion mobility MS in time-aligned parallel (TAP) fragmentation mode. In this work, we show the potential of applying metabolomics strategies and in particular the utility of coupling ion mobility with MS for the molecular characterization of sponge specimens.
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http://dx.doi.org/10.3390/md15020052DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5334632PMC
February 2017

Quantitative UPLC-MS/MS assay of urinary 2,8-dihydroxyadenine for diagnosis and management of adenine phosphoribosyltransferase deficiency.

J Chromatogr B Analyt Technol Biomed Life Sci 2016 Nov 14;1036-1037:170-177. Epub 2016 Sep 14.

University of Iceland, Reykjavik, Iceland; Division of Nephrology, Landspitali - The National University Hospital of Iceland, Reykjavik, Iceland. Electronic address:

Adenine phosphoribosyltransferase (APRT) deficiency is a hereditary disorder that leads to excessive urinary excretion of 2,8-dihydroxyadenine (DHA), causing nephrolithiasis and chronic kidney disease. Treatment with allopurinol or febuxostat reduces DHA production and attenuates the renal manifestations. Assessment of DHA crystalluria by urine microscopy is used for therapeutic monitoring, but lacks sensitivity. We report a high-throughput assay based on ultra-performance liquid chromatography coupled to tandem mass spectrometry (UPLC-MS/MS) for quantification of urinary DHA. The UPLC-MS/MS assay was optimized by a chemometric approach for absolute quantification of DHA, utilizing isotopically labeled DHA as an internal standard. Experimental screening was conducted with D-optimal design and optimization of the DHA response was performed with central composite face design and related to the peak area of DHA using partial least square regression. Acceptable precision and accuracy of the DHA concentration were obtained over a calibration range of 100 to 5000ng/mL on three different days. The intra- and inter-day accuracy and precision coefficients of variation were well within ±15% for quality control samples analyzed in replicates of six at three concentration levels. Absolute quantification of DHA in urine samples from patients with APRT deficiency was achieved wihtin 6.5min. Measurement of DHA in 24h urine samples from three patients with APRT deficiency, diluted 1:15 (v/v) with 10mM ammonium hydroxide (NHOH), yielded a concentration of 3021, 5860 and 10563ng/mL and 24h excretion of 816, 1327 and 1649mg, respectively. A rapid and robust UPLC-MS/MS assay for absolute quantification of DHA in urine was successfully developed. We believe this method will greatly facilitate diagnosis and management of patients with APRT deficiency.
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http://dx.doi.org/10.1016/j.jchromb.2016.09.018DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC5445224PMC
November 2016

Protolichesterinic Acid, Isolated from the Lichen Cetraria islandica, Reduces LRRC8A Expression and Volume-Sensitive Release of Organic Osmolytes in Human Lung Epithelial Cancer Cells.

Phytother Res 2016 Jan 9;30(1):97-104. Epub 2015 Nov 9.

Department of Biology, Section of Cell biology and Physiology, University of Copenhagen, 13 Universitetsparken, Copenhagen, DK-2100, Denmark.

We have tested the effect of protolichesterinic acid (PA) on the activity of the volume-sensitive release pathway for the organic osmolyte taurine (VSOAC) and the expression of the leucine-rich-repeat-channel 8A (LRRC8A) protein, which constitutes an essential VSOAC component. Exposing human lung cancer cells (A549) to PA (20 µg/mL, 24 h) reduces LRRC8A protein expression by 25% and taurine release following osmotic cell swelling (320 → 200 mOsm) by 60%. C75 (20 µg/mL, 24 h), a γ-lactone with a C8 carbon fatty acid chain, reduces VSOAC activity by 30%, i.e. less than PA. Stearic acid (20 µg/mL, 24 h) has no effect on VSOAC. Hence, length of PA's fatty acid chain adds to γ-lactone's inhibitory action. 5-Lipoxygenase (5-LO) activity is essential for swelling-induced activation of VSOAC. PA has no effect on cellular concentration of leukotrienes (5-HETE/LTB4 ) under hypotonic conditions, excluding that PA mediated inhibition of VSOAC involves 5-LO inhibition. A549 cells exposed to the chemotherapeutic drug cisplatin (10 μM, 24 h) reveal signs of apoptosis, i.e. 25% reduction in cell viability as well as 1.3-, 1.5- and 3.3-fold increase in the expression of LRRC8A, Bax (regulator of apoptosis) and p21 (regulator of cell cycle progression), respectively. PA reduces cell viability by 30% but has no effect on p21/Bax expression. This excludes PA as a pro-apoptotic drug in A549 cells.
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http://dx.doi.org/10.1002/ptr.5507DOI Listing
January 2016

Severe osteoarthritis of the hand associates with common variants within the ALDH1A2 gene and with rare variants at 1p31.

Nat Genet 2014 May 13;46(5):498-502. Epub 2014 Apr 13.

1] Department of Internal Medicine, Erasmus Medical Center, Rotterdam, The Netherlands. [2] Department of Epidemiology, Erasmus Medical Center, Rotterdam, The Netherlands.

Osteoarthritis is the most common form of arthritis and is a major cause of pain and disability in the elderly. To search for sequence variants that confer risk of osteoarthritis of the hand, we carried out a genome-wide association study (GWAS) in subjects with severe hand osteoarthritis, using variants identified through the whole-genome sequencing of 2,230 Icelanders. We found two significantly associated loci in the Icelandic discovery set: at 15q22 (frequency of 50.7%, odds ratio (OR) = 1.51, P = 3.99 × 10(-10)) in the ALDH1A2 gene and at 1p31 (frequency of 0.02%, OR = 50.6, P = 9.8 × 10(-10)). Among the carriers of the variant at 1p31 is a family with several members in whom the risk allele segregates with osteoarthritis. The variants within the ALDH1A2 gene were confirmed in replication sets from The Netherlands and the UK, yielding an overall association of OR = 1.46 and P = 1.1 × 10(-11) (rs3204689).
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http://dx.doi.org/10.1038/ng.2957DOI Listing
May 2014

Kinetics of γ-cyclodextrin nanoparticle suspension eye drops in tear fluid.

Acta Ophthalmol 2014 Sep 24;92(6):550-6. Epub 2013 Dec 24.

Department of Ophthalmology, Faculty of Medicine, National University Hospital, University of Iceland, Reykjavik, Iceland; Department of Clinical Science, Ophthalmology, Umeå University, Umeå, Sweden.

Purpose: We have developed nanoparticle γ-cyclodextrin dexamethasone (DexNP) and dorzolamide (DorzNP) eye drops that provide sustained high drug concentrations on the eye surface. To test these characteristics, we measured dexamethasone and dorzolamide levels in tear fluid in humans following eye drop administration.

Methods: Concentration of dexamethasone was measured by mass spectrometry. One drop of DexNP was instilled into one eye. Tear fluid was sampled with microcapillary pipettes at seven time-points after drop instillation. Control eyes received Maxidex(®) (dexamethasone). The same procedure was performed for dorzolamide with DorzNP and Trusopt(®) .

Results: Six subjects were included in each group. The peak concentration (μg/ml ± standard deviation) of dexamethasone for DexNP eye drops (636.6 ± 399.1) was up to 19-fold higher than with Maxidex(®) (39.3 ± 18.9) (p < 0.001). At 4 hr, DexNP was still 10 times higher than Maxidex(®) . In addition, DexNP resulted in about 30-fold higher concentration of dissolved dexamethasone in the tear fluid of extended time period allowing more drug to partition into the eye tissue. The overall concentration of dorzolamide was about 50% higher for DorzNP (59.5 ± 76.9) than Trusopt(®) (40.0 ± 76.7) (p < 0.05).

Conclusion: The results indicate high and extended concentration of dissolved dexamethasone with DexNP, which can explain the greater and longer lasting effect of dexamethasone in the cyclodextrin nanoparticle drug delivery platform. Dexamethasone seems to fit the cyclodextrin nanoparticle suspension drug delivery platform with longer duration and higher concentrations in tear fluid than available commercial drops, while dorzolamide is less suitable.
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http://dx.doi.org/10.1111/aos.12334DOI Listing
September 2014

Dexamethasone eye drops containing γ-cyclodextrin-based nanogels.

Int J Pharm 2013 Jan 10;441(1-2):507-15. Epub 2012 Nov 10.

Faculty of Pharmaceutical Sciences, University of Iceland, Hofsvallagata 53, IS-107 Reykjavik, Iceland.

Sustained release aqueous eye drops of dexamethasone, based on cyclodextrin (CD) nanogels, were designed and tested in vivo. γCD units were cross-linked in the form of nanogels by means of an emulsification/solvent evaporation process. The composition of the nanogels was optimized with regard to drug loading and release rate. The eye drops consisted of an aqueous solution of dexamethasone in 2-hydroxypropyl-γ-cyclodextrin (HPγCD) medium containing γCD nanogels. The nanogel eye drops (containing 25 mg dexamethasone per ml) were tested in rabbits and compared to the commercially available product Maxidex(®) (suspension with 1 mg dexamethasone per ml). One drop administration of the nanogel eye drops resulted in nearly constant dexamethasone concentration for at least 6h in the tear fluid (mean concentration±SD=295±59 μg/ml) whereas the concentration after administration of Maxidex(®) fell rapidly from 9.72±3.45 μg/ml 1 h after application to 3.76±3.26 μg/ml 3 h after application. The maximum dexamethasone concentration in the aqueous humor (2 h after application) was 136±24 mg/ml after application of the nanogel eye drops, and only 44.4±7.8 μg/ml after application of Maxidex(®). The dexamethasone nanogel eye drops were well tolerated with no macroscopic signs of irritation, redness or other toxic effects.
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http://dx.doi.org/10.1016/j.ijpharm.2012.11.002DOI Listing
January 2013

Cyclodextrin solubilization of carbonic anhydrase inhibitor drugs: formulation of dorzolamide eye drop microparticle suspension.

Eur J Pharm Biopharm 2010 Oct 15;76(2):208-14. Epub 2010 Jul 15.

Faculty of Pharmaceutical Sciences, University of Iceland, Reykjavik, Iceland.

Topically applied carbonic anhydrase inhibitors (CAIs) are commonly used to treat glaucoma. However, their short duration of action requiring multiple daily dosing can hamper patient compliance. The aim of this study was to develop novel aqueous CAI eye drop formulation containing self-assembled drug/cyclodextrin (D/CD) microparticles that enhance and prolong drug delivery to the eye. Phase-solubility of each drug tested (i.e. methazolamide, brinzolamide and dorzolamide HCl) was determined in either pure water or an aqueous eye drop medium. The pH was adjusted to maximize the fraction of unionized drug. Dorzolamide had the highest affinity for γ-cyclodextrin (γCD) and, thus, was selected for further investigation. Hydroxypropyl methylcellulose (HPMC) was the most effective polymer tested for stabilization of the dorzolamide/γCD complexes and gave the highest mucoadhesion at 0.5% w/v concentration. Thus, the dorzolamide eye drop vehicle containing γCD (18% w/v) and HPMC (0.5% w/v) was developed. The physicochemical properties of this formulation complied with the specifications of the eye drop suspension monograph of the European Pharmacopoeia. The in vivo testing of the formulation showed that the drug was delivered to the aqueous humor in rabbits for at least 24h with the maximum drug concentration at 4h. Furthermore, this formulation delivered the drug to the posterior segment of the eye after topical administration. These results indicate that this CAI eye drop formulation has the potential of being developed into a once-a-day product.
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http://dx.doi.org/10.1016/j.ejpb.2010.07.005DOI Listing
October 2010

Design of phosphodiesterase 4D (PDE4D) allosteric modulators for enhancing cognition with improved safety.

Nat Biotechnol 2010 Jan 27;28(1):63-70. Epub 2009 Dec 27.

deCODE biostructures, Bainbridge Island, Washington, USA.

Phosphodiesterase 4 (PDE4), the primary cAMP-hydrolyzing enzyme in cells, is a promising drug target for a wide range of conditions. Here we present seven co-crystal structures of PDE4 and bound inhibitors that show the regulatory domain closed across the active site, thereby revealing the structural basis of PDE4 regulation. This structural insight, together with supporting mutagenesis and kinetic studies, allowed us to design small-molecule allosteric modulators of PDE4D that do not completely inhibit enzymatic activity (I(max) approximately 80-90%). These allosteric modulators have reduced potential to cause emesis, a dose-limiting side effect of existing active site-directed PDE4 inhibitors, while maintaining biological activity in cellular and in vivo models. Our results may facilitate the design of CNS therapeutics modulating cAMP signaling for the treatment of Alzheimer's disease, Huntington's disease, schizophrenia and depression, where brain distribution is desired for therapeutic benefit.
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http://dx.doi.org/10.1038/nbt.1598DOI Listing
January 2010

Effects of 2,4-diaminoquinazoline derivatives on SMN expression and phenotype in a mouse model for spinal muscular atrophy.

Hum Mol Genet 2010 Feb 6;19(3):454-67. Epub 2009 Nov 6.

Department of Molecular and Cellular Biochemistry, College of Medicine, The Ohio State University, 1645 Neil Avenue, Columbus, OH 43210, USA.

Proximal spinal muscular atrophy (SMA), one of the most common genetic causes of infant death, results from the selective loss of motor neurons in the spinal cord. SMA is a consequence of low levels of survival motor neuron (SMN) protein. In humans, the SMN gene is duplicated; SMA results from the loss of SMN1 but SMN2 remains intact. SMA severity is related to the copy number of SMN2. Compounds which increase the expression of SMN2 could, therefore, be potential therapeutics for SMA. Ultrahigh-throughput screening recently identified substituted quinazolines as potent SMN2 inducers. A series of C5-quinazoline derivatives were tested for their ability to increase SMN expression in vivo. Oral administration of three compounds (D152344, D153249 and D156844) to neonatal mice resulted in a dose-dependent increase in Smn promoter activity in the central nervous system. We then examined the effect of these compounds on the progression of disease in SMN lacking exon 7 (SMNDelta7) SMA mice. Oral administration of D156844 significantly increased the mean lifespan of SMNDelta7 SMA mice by approximately 21-30% when given prior to motor neuron loss. In summary, the C5-quinazoline derivative D156844 increases SMN expression in neonatal mouse neural tissues, delays motor neuron loss at PND11 and ameliorates the motor phenotype of SMNDelta7 SMA mice.
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http://dx.doi.org/10.1093/hmg/ddp510DOI Listing
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC2798721PMC
February 2010

Antagonists of the EP3 receptor for prostaglandin E2 are novel antiplatelet agents that do not prolong bleeding.

ACS Chem Biol 2009 Feb;4(2):115-26

deCODE Chemistry, Woodridge, Illinois 60517, USA.

Myocardial infarction and stroke are caused by blood clots forming over a ruptured or denuded atherosclerotic plaque (atherothrombosis). Production of prostaglandin E(2) (PGE(2)) by an inflamed plaque exacerbates atherothrombosis and may limit the effectiveness of current therapeutics. Platelets express multiple G-protein coupled receptors, including receptors for ADP and PGE(2). ADP can mobilize Ca(2+) and through the P(2)Y(12) receptor can inhibit cAMP production, causing platelet activation and aggregation. Clopidogrel (Plavix), a selective P(2)Y(12) antagonist, prevents platelets from clotting but thereby increases the risk of severe or fatal bleeding. The platelet EP(3) receptor for PGE(2), like the P(2)Y(12) receptor, also inhibits cAMP synthesis. However, unlike ADP, facilitation of platelet aggregation via the PGE(2)/EP(3) pathway is dependent on co-agonists that can mobilize Ca(2+). We used a ligand-based design strategy to develop peri-substituted bicylic acylsulfonamides as potent and selective EP(3) antagonists. We show that DG-041, a selective EP(3) antagonist, inhibits PGE(2) facilitation of platelet aggregation in vitro and ex vivo. PGE(2) can resensitize platelets to agonist even when the P(2)Y(12) receptor has been blocked by clopidogrel, and this can be inhibited by DG-041. Unlike clopidogrel, DG-041 does not affect bleeding time in rats, nor is bleeding time further increased when DG-041 is co-administered with clopidogrel. This indicates that EP(3) antagonists potentially have a superior safety profile compared to P(2)Y(12) antagonists and represent a novel class of antiplatelet agents.
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http://dx.doi.org/10.1021/cb8002094DOI Listing
February 2009