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  • 1.
    Coulthard, Sally
    et al.
    Newcastle University, UK.
    Redfern, Christopher
    Newcastle University, UK.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Lindqvist Appell, Malin
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Skoglund, Karin
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Jakobsen Falk, Ingrid
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Hall, Andrew
    Newcastle University, UK.
    Taylor, Gordon
    Newcastle University, UK.
    Hogarth, Linda
    Newcastle University, UK.
    Increased sensitivity to thiopurines in methylthioadenosine phosphorylase-deleted cancers in PURINERGIC SIGNALLING, vol 6, issue , pp 33-332010In: PURINERGIC SIGNALLING, Springer Science Business Media , 2010, Vol. 6, p. 33-33Conference paper (Refereed)
    Abstract [en]

    The thiopurines, 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are used in the treatment of leukaemia. Incorporation of deoxythioguanosine nucleotides (dGs) into the DNA of thiopurine-treated cells causes cell death but there is also evidence that thiopurine metabolites, particularly the 6-MP metabolite methylthioinosine monophosphate (MeTIMP), inhibit de novo purine synthesis (DNPS). The toxicity of DNPS inhibitors is influenced by methylthioadenosine phosphorylase (MTAP), a gene frequently deleted in cancers. Since the growth of MTAP-deleted tumour cells is dependent on DNPS or hypoxanthine salvage, we would predict such cells to show differential sensitivity to 6-MP and 6-TG. To test this hypothesis, sensitivity to 6-MP and 6-TG was compared in relation to MTAP status using cytotoxicity assays in two MTAP-deficient cell lines transfected to express MTAP: the T-cell acute lymphoblastic leukaemic cell line, Jurkat, transfected with MTAP cDNA under the control of a tetracycline-inducible promoter, and a lung cancer cell line (A549-MTAP-ve) transfected to express MTAP constitutively (A549-MTAP+ve). Sensitivity to 6-MP or methyl mercaptopurine riboside, which is converted intra-cellularly to MeTIMP, was markedly higher in both cell lines under MTAP-ve conditions. Measurement of thiopurine metabolites support the hypothesis that DNPS inhibition is a major cause of cell death with 6-MP, whereas dGs incorporation is the main cause of cytotoxicity with 6-TG. These data suggest that thiopurines, particularly 6-MP, may be more effective in patients with deleted MTAP.

  • 2.
    Folkesson, Maggie
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Sadowska, Natalia
    Linköping University, Department of Medical and Health Sciences, Division of Cardiovascular Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Karlsson, Matts
    Linköping University, Department of Management and Engineering, Applied Thermodynamics and Fluid Mechanics. Linköping University, Faculty of Science & Engineering. Linköping University, Center for Medical Image Science and Visualization (CMIV).
    Carlhäll, Carl-Johan
    Linköping University, Department of Medical and Health Sciences, Division of Cardiovascular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Heart and Medicine Center, Department of Clinical Physiology in Linköping. Linköping University, Center for Medical Image Science and Visualization (CMIV).
    Länne, Toste
    Linköping University, Department of Medical and Health Sciences, Division of Cardiovascular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Heart and Medicine Center, Department of Thoracic and Vascular Surgery.
    Wågsäter, Dick
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Jensen, Lasse
    Linköping University, Department of Medical and Health Sciences, Division of Cardiovascular Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pharmacology.
    Differences in cardiovascular toxicities associated with cigarette smoking and snuff use revealed using novel zebrafish models2016In: Biology Open, ISSN 2046-6390, Vol. 5, no 7, p. 970-978Article in journal (Refereed)
    Abstract [en]

    Tobacco use is strongly associated with cardiovascular disease and the only avoidable risk factor associated with development of aortic aneurysm. While smoking is the most common form of tobacco use, snuff and other oral tobacco products are gaining popularity, but research on potentially toxic effects of oral tobacco use has not kept pace with the increase in its use. Here, we demonstrate that cigarette smoke and snuff extracts are highly toxic to developing zebrafish embryos. Exposure to such extracts led to a palette of toxic effects including early embryonic mortality, developmental delay, cerebral hemorrhages, defects in lymphatics development and ventricular function, and aneurysm development. Both cigarette smoke and snuff were more toxic than pure nicotine, indicating that other compounds in these products are also associated with toxicity. While some toxicities were found following exposure to both types of tobacco product, other toxicities, including developmental delay and aneurysm development, were specifically observed in the snuff extract group, whereas cerebral hemorrhages were only found in the group exposed to cigarette smoke extract. These findings deepen our understanding of the pathogenic effects of cigarette smoking and snuff use on the cardiovascular system and illustrate the benefits of using zebrafish to study mechanisms involved in aneurysm development.

  • 3.
    Gad, Helge
    et al.
    Karolinska Institute, Sweden .
    Koolmeister, Tobias
    Karolinska Institute, Sweden .
    Jemth, Ann-Sofie
    Karolinska Institute, Sweden .
    Eshtad, Saeed
    Karolinska Institute, Sweden .
    Jacques, Sylvain A.
    Karolinska Institute, Sweden .
    Ström, Cecilia E.
    Karolinska Institute, Sweden .
    Svensson, Linda M.
    Stockholm University, Sweden .
    Schultz, Niklas
    Karolinska Institute, Sweden .
    Lundbäck, Thomas
    Karolinska Institute, Sweden .
    Osk Einarsdottir, Berglind
    University of Gothenburg, Sweden; Sahlgrenska University Hospital, Sweden .
    Saleh, Aljona
    Stockholm University, Sweden .
    Göktürk, Camilla
    Karolinska Institute, Sweden .
    Baranczewski, Pawel
    Karolinska Institute, Sweden Uppsala University, Sweden .
    Svensson, Richard
    Karolinska Institute, Sweden; Uppsala University, Sweden .
    Berntsson, Ronnie P. -A.
    Stockholm University, Sweden .
    Gustafsson, Robert
    Stockholm University, Sweden .
    Strömberg, Kia
    Karolinska Institute, Sweden .
    Sanjiv, Kumar
    Karolinska Institute, Sweden .
    Jacques-Cordonnier, Marie-Caroline
    Karolinska Institute, Sweden .
    Desroses, Matthieu
    Karolinska Institute, Sweden .
    Gustavsson, Anna-Lena
    Karolinska Institute, Sweden .
    Olofsson, Roger
    University of Gothenburg, Sweden; Sahlgrenska University Hospital, Sweden .
    Johansson, Fredrik
    Stockholm University, Sweden .
    Homan, Evert J.
    Karolinska Institute, Sweden .
    Loseva, Olga
    Karolinska Institute, Sweden .
    Bräutigam, Lars
    Karolinska Institute, Sweden .
    Johansson, Lars
    Karolinska Institute, Sweden .
    Höglund, Andreas
    Karolinska Institute, Sweden .
    Hagenkort, Anna
    Karolinska Institute, Sweden .
    Pham, Therese
    Karolinska Institute, Sweden .
    Altun, Mikael
    Karolinska Institute, Sweden .
    Gaugaz, Fabienne Z.
    Karolinska Institute, Sweden; Uppsala University, Sweden .
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Evers, Bastiaan
    Karolinska Institute, Sweden .
    Henriksson, Martin
    Karolinska Institute, Sweden .
    Vallin, Karl S. A.
    Karolinska Institute, Sweden .
    Wallner, Olov A.
    Karolinska Institute, Sweden .
    Hammarström, Lars G. J.
    Karolinska Institute, Sweden .
    Wiita, Elisee
    Karolinska Institute, Sweden .
    Almlöf, Ingrid
    Karolinska Institute, Sweden .
    Kalderén, Christina
    Karolinska Institute, Sweden .
    Axelsson, Hanna
    Karolinska Institute, Sweden .
    Djureinovic, Tatjana
    Stockholm University, Sweden .
    Carreras Puigvert, Jordi
    Karolinska Institute, Sweden .
    Häggblad, Maria
    Stockholm University, Sweden .
    Jeppsson, Fredrik
    Karolinska Institute, Sweden .
    Martens, Ulf
    Stockholm University, Sweden .
    Lundin, Cecilia
    Karolinska Institute, Sweden .
    Lundgren, Bo
    Stockholm University, Sweden .
    Granelli, Ingrid
    Stockholm University, Sweden .
    Jenmalm Jensen, Annika
    Karolinska Institute, Sweden .
    Artursson, Per
    Karolinska Institute, Sweden; Uppsala University, Sweden .
    Nilsson, Jonas A.
    University of Gothenburg, Sweden, Sahlgrens University Hospital, Sweden .
    Stenmark, Pål
    Stockholm University, Sweden .
    Scobie, Martin
    Karolinska Institute, Sweden .
    Warpman Berglund, Ulrika
    Karolinska Institute, Sweden .
    Helleday, Thomas
    Karolinska Institute, Sweden .
    MTH1 inhibition eradicates cancer by preventing sanitation of the dNTP pool2014In: Nature, ISSN 0028-0836, E-ISSN 1476-4687, Vol. 508, no 7495, p. 215-221Article in journal (Refereed)
    Abstract [en]

    Cancers have dysfunctional redox regulation resulting in reactive oxygen species production, damaging both DNA and free dNTPs. The MTH1 protein sanitizes oxidized dNTP pools to prevent incorporation of damaged bases during DNA replication. Although MTH1 is non-essential in normal cells, we show that cancer cells require MTH1 activity to avoid incorporation of oxidized dNTPs, resulting in DNA damage and cell death. We validate MTH1 as an anticancer target in vivo and describe small molecules TH287 and TH588 as first-in-class nudix hydrolase family inhibitors that potently and selectively engage and inhibit the MTH1 protein in cells. Protein co-crystal structures demonstrate that the inhibitors bindin the active site of MTH1. The inhibitors cause incorporation of oxidized dNTPs in cancer cells, leading to DNA damage, cytotoxicity and therapeutic responses in patient-derived mouse xenografts. This study exemplifies the non-oncogene addiction concept for anticancer treatment and validates MTH1 as being cancer phenotypic lethal.

  • 4.
    Green, Henrik
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Division of Gene Technology, Royal Institute of Technology, Solna, Sweden/ Royal Institute Technology, Sweden; National Board Forens Med, Department Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Hasmats, Johanna
    Royal Institute Technology, Sweden.
    Kupershmidt, Ilya
    Royal Institute Technology, Sweden; NextBio, CA USA.
    Edsgard, Daniel
    Royal Institute Technology, Sweden.
    de Petris, Luigi
    Karolinska Institute, Sweden; Karolinska University Hospital, Sweden.
    Lewensohn, Rolf
    Karolinska Institute, Sweden; Karolinska University Hospital, Sweden.
    Blackhall, Fiona
    Christie Hospital, England; University of Manchester, England.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Besse, Benjamin
    University of Paris 11, France.
    Lindgren, Andrea
    Linköping University, Department of Medical and Health Sciences. Region Östergötland, Heart and Medicine Center, Department of Clinical Physiology in Linköping. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Respiratory Medicine. Linköping University, Faculty of Medicine and Health Sciences.
    Branden, Eva
    Karolinska Institute, Sweden; Karolinska University Hospital, Sweden.
    Koyi, Hirsh
    Karolinska Institute, Sweden; Karolinska University Hospital, Sweden.
    Peterson, Curt
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology.
    Lundeberg, Joakim
    Royal Institute Technology, Sweden.
    Using Whole-Exome Sequencing to Identify Genetic Markers for Carboplatin and Gemcitabine-Induced Toxicities2016In: Clinical Cancer Research, ISSN 1078-0432, E-ISSN 1557-3265, Vol. 22, no 2, p. 366-373Article in journal (Refereed)
    Abstract [en]

    Purpose: Chemotherapies are associated with significant interindividual variability in therapeutic effect and adverse drug reactions. In lung cancer, the use of gemcitabine and carboplatin induces grade 3 or 4 myelosuppression in about a quarter of the patients, while an equal fraction of patients is basically unaffected in terms of myelosuppressive side effects. We therefore set out to identify genetic markers for gemcitabine/carboplatin-induced myelosuppression. Experimental Design: We exome sequenced 32 patients that suffered extremely high neutropenia and thrombocytopenia (grade 3 or 4 after first chemotherapy cycle) or were virtually unaffected (grade 0 or 1). The genetic differences/polymorphism between the groups were compared using six different bioinformatics strategies: (i) whole-exome nonsynonymous single-nucleotide variants association analysis, (ii) deviation from Hardy-Weinberg equilibrium, (iii) analysis of genes selected by a priori biologic knowledge, (iv) analysis of genes selected from gene expression meta-analysis of toxicity datasets, (v) Ingenuity Pathway Analysis, and (vi) FunCoup network enrichment analysis. Results: A total of 53 genetic variants that differed among these groups were validated in an additional 291 patients and were correlated to the patients myelosuppression. In the validation, we identified rs1453542 in OR4D6 (P = 0.0008; OR, 5.2; 95% CI, 1.8-18) as a marker for gemcitabine/carboplatin-induced neutropenia and rs5925720 in DDX53 (P = 0.0015; OR, 0.36; 95% CI, 0.17-0.71) as a marker for thrombocytopenia. Patients homozygous for the minor allele of rs1453542 had a higher risk of neutropenia, and for rs5925720 the minor allele was associated with a lower risk for thrombocytopenia. Conclusions: We have identified two new genetic markers with the potential to predict myelosuppression induced by gemcitabine/ carboplatin chemotherapy. (C)2015 AACR.

  • 5.
    Haglund, Sofie
    et al.
    Regional Jonköping County, Sweden; Karolinska Institute, Sweden.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Almer, Sven
    Karolinska Institute, Sweden; Karolinska University Hospital, Sweden.
    Söderman, Jan
    Linköping University, Department of Clinical and Experimental Medicine, Division of Cell Biology. Linköping University, Faculty of Medicine and Health Sciences. Regional Jonköping County, Sweden.
    Combination treatment with 6-mercaptopurine and allopurinol in HepG2 and HEK293 cells - Effects on gene expression levels and thiopurine metabolism2017In: PLoS ONE, ISSN 1932-6203, E-ISSN 1932-6203, Vol. 12, no 3, article id e0173825Article in journal (Refereed)
    Abstract [en]

    Combination treatment with low-dose thiopurine and allopurinol (AP) has successfully been used in patients with inflammatory bowel disease with a so called skewed thiopurine metabolite profile. In red blood cells in vivo, it reduces the concentration of methylated metabolites and increases the concentration of the phosphorylated ones, which is associated with improved therapeutic efficacy. This study aimed to investigate the largely unknown mechanism of AP on thiopurine metabolism in cells with an active thiopurine metabolic pathway using HepG2 and HEK293 cells. Cells were treated with 6-mercaptopurine (6MP) and AP or its metabolite oxypurinol. The expression of genes known to be associated with thiopurine metabolism, and the concentration of thiopurine metabolites were analyzed. Gene expression levels were only affected by AP in the presence of 6MP. The addition of AP to 6MP affected the expression of in total 19 genes in the two cell lines. In both cell lines the expression of the transporter SLC29A2 was reduced by the combined treatment. Six regulated genes in HepG2 cells and 8 regulated genes in HEK293 cells were connected to networks with 18 and 35 genes, respectively, present at known susceptibility loci for inflammatory bowel disease, when analyzed using a protein-protein interaction database. The genes identified as regulated as well as the disease associated interacting genes represent new candidates for further investigation in the context of combination therapy with thiopurines and AP. However, no differences in absolute metabolite concentrations were observed between 6MP+AP or 6MP +oxypurinol vs. 6MP alone in either of the two cell lines. In conclusion; the effect of AP on=gene expression levels requires the presence of 6MP, at least in vitro. Previously described AP-effects on metabolite concentrations observed in red blood cells in vivo could not be reproduced in our cell lines in vitro. APs effects in relation to thiopurine metabolism are complex. The network-identified susceptibility genes represented biological processes mainly associated with purine nucleotide biosynthetic processes, lymphocyte proliferation, NF-KB activation, JAK-STAT signaling, and apoptotic signaling at oxidative stress.

  • 6.
    Haglund, Sofie
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Söderman, Jan
    Division of Medical Diagnostics, Laboratory Medicine, Ryhov Hospital, Jönköping;.
    Hindorf, Ulf
    Department of Gastroenterology, Lund University Hospital, Lund.
    Grännö, Christer
    Department of Medicine, Ryhov Hospital, Jönköping.
    Danelius, Margareta
    Department of Internal Medicine, Ersta Hospital, Stockholm, Sweden.
    Coulthard, Sally
    Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom.
    Peterson, Curt
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology.
    Almer, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Center, Department of Endocrinology and Gastroenterology UHL.
    The Role of Inosine-5'-Monophosphate Dehydrogenase in Thiopurine Metabolism in Patients With Inflammatory Bowel Disease2011In: Therapeutic Drug Monitoring, ISSN 0163-4356, E-ISSN 1536-3694, Vol. 33, no 2, p. 200-208Article in journal (Refereed)
    Abstract [en]

    BACKGROUND:: There is a large interindividual variability in thiopurine metabolism. High concentrations of methylthioinosine-5'-monophosphate (meTIMP) and low concentrations of 6-thioguanine nucleotides (6-TGNs) have been associated with a lower response rate and an increased risk of adverse events. In this study, the role of inosine-5'-monophosphate dehydrogenase (IMPDH) for differences in metabolite patterns of thiopurines was investigated.

    METHODS:: IMPDH activity and thiopurine metabolite concentrations were determined in patients with inflammatory bowel disease and a normal thiopurine methyltransferase (TPMT) phenotype and meTIMP/6-TGN concentration ratio > 20 (n = 26), in patients with a metabolite ratio ≤20 (n = 21), in a subgroup with a metabolite ratio <4 (n = 6), and in 10 patients with reduced TPMT activity. In vitro studies were conducted on human embryonic kidney cells (HEK293) with genetically engineered IMPDH and TPMT activities.

    RESULTS:: Patients with metabolite ratios >20 had lower IMPDH activity than those with ratios ≤20 (P < 0.001). Metabolic ratios >20 were only observed in patients with normal TPMT activity. Downregulation of IMPDH activity in HEK293 cells was associated with an increase in the concentration of meTIMP (fold change: 17 up to 93, P < 0.001) but, unexpectedly, also of 6-thioguanosine monophosphate (fold change: 2.6 up to 5.0, P < 0.001).

    CONCLUSIONS:: These data question the general view of IMPDH as the rate-limiting enzyme in the phosphorylation of thiopurines. Investigations of other mechanisms are needed to more fully explain the various metabolite patterns and outcomes in patients under treatment.

  • 7.
    Haglund, Sofie
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences.
    Zimdahl Kahlin, Anna
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Vikingsson, Svante
    Linköping University, Faculty of Health Sciences. Linköping University, Department of Medical and Health Sciences, Division of Drug Research.
    Almér, Stefan
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences.
    Söderman, Jan
    Division of Medical Diagnostics, Ryhov County Hospital, Jönköping, Sweden.
    P658 Effects of allopurinol on thiopurine metabolism and gene expression levels in HepG2 cells2014Conference paper (Other academic)
  • 8.
    Lee, Mi-Young
    et al.
    Karolinska Institute, Sweden.
    Apellaniz-Ruiz, Maria
    Spanish National Cancer Research Centre CNIO, Spain.
    Johansson, Inger
    Karolinska Institute, Sweden.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Bergmann, Troels K.
    University of Southern Denmark, Denmark.
    Brosen, Kim
    University of Southern Denmark, Denmark.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. National Board Forens Med, Department Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Rodriguez-Antona, Cristina
    Spanish National Cancer Research Centre CNIO, Spain; ISCIII Centre Biomed Research Rare Disease CIBERER, Spain.
    Ingelman-Sundberg, Magnus
    Karolinska Institute, Sweden.
    Role of cytochrome P450 2C8*3 (CYP2C8*3) in paclitaxel metabolism and paclitaxel-induced neurotoxicity2015In: Pharmacogenomics (London), ISSN 1462-2416, E-ISSN 1744-8042, Vol. 16, no 9, p. 929-937Article in journal (Refereed)
    Abstract [en]

    Aim: The CYP2C8*3 allele has been suggested as a risk factor for paclitaxel-induced neuropathy but the data hitherto published are conflicting. Materials and methods: In total 435 patients were investigated with respect to maximum neuropathy grade and accumulated paclitaxel dose. The enzymatic properties of CYP2C8.3 variant were analyzed using heterologous mammalian HEK293 cell expression system. Results: No significant association between CYP2C8*3 allele and neuropathy was found, although a trend was observed. The paclitaxel and amodiaquine metabolism by CYP2C8.3 were found similar to CYP2C8.1, whereas CYP2C8.3 was more efficient in the metabolism of rosiglitazone. Conclusion: These results indicate a difference in substrate specificity between CYP2C8.1 and CYP2C8.3; however, the CYP2C8*3 allele has no major impact on paclitaxel metabolism in vitro or of paclitaxel-induced neuropathy in vivo. Original submitted on 6 February 2015; revision submitted on 9 April 2015

  • 9.
    Lentini, Antonio
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Lagerwall, Cathrine
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Mjoseng, Heidi K.
    Univ Edinburgh, Scotland.
    Douvlataniotis, Dimitrios Karolos
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    Vogt, Hartmut
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center of Paediatrics and Gynaecology and Obstetrics, H.K.H. Kronprinsessan Victorias barn- och ungdomssjukhus.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Meehan, Richard R.
    Univ Edinburgh, Scotland.
    Benson, Mikael
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Heart and Medicine Center, Allergy Center.
    Nestor, Colm
    Linköping University, Department of Clinical and Experimental Medicine, Division of Children's and Women's health. Linköping University, Faculty of Medicine and Health Sciences.
    A reassessment of DNA-immunoprecipitation-based genomic profiling2018In: Nature Methods, ISSN 1548-7091, E-ISSN 1548-7105, Vol. 15, no 7, p. 499-+Article in journal (Refereed)
    Abstract [en]

    DNA immunoprecipitation followed by sequencing (DIP-seq) is a common enrichment method for profiling DNA modifications in mammalian genomes. However, the results of independent DIP-seq studies often show considerable variation between profiles of the same genome and between profiles obtained by alternative methods. Here we show that these differences are primarily due to the intrinsic affinity of IgG for short unmodified DNA repeats. This pervasive experimental error accounts for 50-99% of regions identified as enriched for DNA modifications in DIP-seq data. Correction of this error profoundly altered DNA-modification profiles for numerous cell types, including mouse embryonic stem cells, and subsequently revealed novel associations among DNA modifications, chromatin modifications and biological processes. We conclude that both matched input and IgG controls are essential in order for the results of DIP-based assays to be interpreted correctly, and that complementary, non-antibody-based techniques should be used to validate DIP-based findings to avoid further misinterpretation of genome-wide profiling data.

  • 10.
    Lopez-Contreras, Andres J.
    et al.
    Spanish National Cancer Research Centre CNIO, Spain.
    Specks, Julia
    Spanish National Cancer Research Centre CNIO, Spain.
    Barlow, Jacqueline H.
    NCI, MD 20892 USA.
    Ambrogio, Chiara
    Spanish National Cancer Research Centre CNIO, Spain.
    Desler, Claus
    University of Copenhagen, Denmark.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Rodrigo-Perez, Sara
    Spanish National Cancer Research Centre CNIO, Spain.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. National Board Forens Med, Department Forens Genet and Forens Toxicol, SE-58185 Linkoping, Sweden.
    Juel Rasmussen, Lene
    University of Copenhagen, Denmark.
    Murga, Matilde
    Spanish National Cancer Research Centre CNIO, Spain.
    Nussenzweig, Andre
    NCI, MD 20892 USA.
    Fernandez-Capetillo, Oscar
    Spanish National Cancer Research Centre CNIO, Spain.
    Increased Rrm2 gene dosage reduces fragile site breakage and prolongs survival of ATR mutant mice2015In: Genes & Development, ISSN 0890-9369, E-ISSN 1549-5477, Vol. 29, no 7, p. 690-695Article in journal (Refereed)
    Abstract [en]

    In Saccharomyces cerevisiae, absence of the checkpoint kinase Mec1 (ATR) is viable upon mutations that increase the activity of the ribonucleotide reductase (RNR) complex. Whether this pathway is conserved in mammals remains unknown. Here we show that cells from mice carrying extra alleles of the RNR regulatory subunit RRM2 (Rrm2(TG)) present supraphysiological RNR activity and reduced chromosomal breakage at fragile sites. Moreover, increased Rrm2 gene dosage significantly extends the life span of ATR mutant mice. Our study reveals the first genetic condition in mammals that reduces fragile site expression and alleviates the severity of a progeroid disease by increasing RNR activity. Supplemental material is available for this article.

  • 11.
    Sivik, Tove
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oncology. Linköping University, Faculty of Health Sciences.
    Vikingsson, Svante
    Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Greén, Henrik
    Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Jansson, Agneta
    Linköping University, Department of Clinical and Experimental Medicine, Oncology. Linköping University, Faculty of Health Sciences.
    A validated and rapid high-performance liquidchromatography method for the quantification ofconversion of radio-labelled sex steroids2010In: Hormone Molecular Biology and Clinical Investigation, ISSN 1868-1891, Vol. 3, no 1, p. 375-381Article in journal (Refereed)
    Abstract [en]

    The 17b -hydroxysteroid dehydrogenase enzymes modify the availability of potent sex steroids and have thus attracted interest in the study of several steroid-dependent pathologies including breast, endometrial and prostate cancers. An increased awareness of the importance of steroidogenic enzymes has brought forth a demand for efficient assays to study the effects of individual enzymes on steroid levels. Methods used for assessing steroid conversion are often laborious and frequently involve hazardous sample preparation steps. We developed and validated an optimised simple method for sample preparation of sex steroids using protein precipitation by the addition of zinc sulphate/sodium hydroxide. The interconversion of radio-labelled oestrogens and androgens was quantified using high-performance liquid chromatography separation of oestrone, oestradiol, androstenedione and testosterone followed by online radiometric flow scintillation analysis. The method, which can be applied for assessing, e.g., the efficacy of inhibitors of steroidogenic enzymes, was successfully used for evaluating oestrogenic interconversion in breast cancer cell lines MCF7 and T-47D.

  • 12.
    Sivik, Tove
    et al.
    Linköping University, Department of Clinical and Experimental Medicine, Oncology. Linköping University, Faculty of Health Sciences.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Gréen, Henrik
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Jansson, Agneta
    Linköping University, Department of Clinical and Experimental Medicine, Oncology. Linköping University, Faculty of Health Sciences.
    Expression Patterns of 17β-Hydroxysteroid Dehydrogenase 14 in Human Tissues2012In: Hormone and Metabolic Research, ISSN 0018-5043, E-ISSN 1439-4286, Vol. 44, no 13, p. 949-956Article in journal (Refereed)
    Abstract [en]

    17βHSD enzymes catalyze the stereospecific oxidation/reduction at carbon 17β of androgens and estrogens, and are important players in intracrine sex hormone synthesis. The biological relevance of 17βHSD14, first named retSDR3, is largely unknown. We generated and validated an antibody targeting the 17βHSD14 antigen and used this for immunohistochemical evaluation of expression patterns in 33 healthy human tissues. Furthermore, sex steroid conversional activity in HSD17B14 overexpressing HEK293 and MCF10A cells was investigated by assessing interconversion products of estrone, estradiol, androstenedione, testosterone, and dehydroepiandrosterone. Immunohistochemical staining patterns of 17βHSD14 with the enzyme being primarily expressed in glandular epithelial tissue reveal an enzyme with possible implications in the secretion or conversion of externally derived compounds. A role for 17βHSD14 in sex steroid metabolism is supported by the finding that 17HSD14 oxidizes both estradiol and testosterone into less bioactive steroid metabolites estrone and androstenedione, respectively.

  • 13.
    Skoglund, Karin
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Boiso, Samuel
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Jönsson, Jan-Ingvar
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Carlsson, Björn
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Pharmacology.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Single-nucleotide polymorphisms of ABCG2 increase the efficacy of tyrosine kinase inhibitors in the K562 chronic myeloid leukemia cell line2014In: Pharmacogenetics & Genomics, ISSN 1744-6872, E-ISSN 1744-6880, Vol. 24, no 1, p. 52-61Article in journal (Refereed)
    Abstract [en]

    ObjectiveThe tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukemia are substrates for the efflux transport protein ATP-binding cassette subfamily G member 2 (ABCG2). Variations in ABCG2 activity might influence pharmacokinetics and therapeutic outcome of TKIs. The role of ABCG2 single-nucleotide polymorphisms (SNPs) in TKI treatment is not clear and functional in-vitro studies are lacking. The aim of this study was to investigate the consequences of ABCG2 SNPs for transport and efficacy of TKIs [imatinib, N-desmethyl imatinib (CGP74588), dasatinib, nilotinib, and bosutinib].Materials and methodsABCG2 SNPs 34Ggreater thanA, 421Cgreater thanA, 623Tgreater thanC, 886Ggreater thanC, 1574Tgreater thanG, and 1582Ggreater thanA were constructed from ABCG2 wild-type cDNA and transduced to K562 cells by retroviral gene transfer. Variant ABCG2 expression in cell membranes was evaluated and the effects of ABCG2 SNPs on transport and efficacy of TKIs were measured as the ability of ABCG2 variants to protect against TKI cytotoxicity.ResultsWild-type ABCG2 had a protective effect against the cytotoxicity of all investigated compounds except bosutinib. It was found that ABCG2 expression provided better protection against CGP74588 than its parent compound, imatinib. ABCG2 421Cgreater thanA, 623Tgreater thanC, 886Ggreater thanC, and 1574Tgreater thanG reduced cell membrane expression of ABCG2 and the protective effect of ABCG2 against imatinib, CGP74588, dasatinib, and nilotinib cytotoxicity.ConclusionThese findings show that the ABCG2 SNPs 421Cgreater thanA, 623Tgreater thanC, 886Ggreater thanC, and 1574Tgreater thanG increase the efficacy of investigated TKIs, indicating a reduced transport function that might influence TKI pharmacokinetics in vivo. Furthermore, the active imatinib metabolite CGP74588 is influenced by ABCG2 expression to a greater extent than the parent compound.

  • 14.
    Skoglund, Karin
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Moreno, Samuel Boiso
    Linköping University, Department of Medical and Health Sciences, Division of Radiological Sciences. Linköping University, Faculty of Health Sciences. Linköping University, Center for Medical Image Science and Visualization (CMIV).
    Jönsson, Jan-Ingvar
    Linköping University, Department of Clinical and Experimental Medicine, Division of Microbiology and Molecular Medicine. Linköping University, Faculty of Health Sciences.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Carlsson, Björn
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Pharmacology.
    Gréen, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Influence of variant ABCG2 on tyrosine kinase inhibitor transport and efficacy in the K562 chronic myeloid leukemia cell lineManuscript (preprint) (Other academic)
    Abstract [en]

    Objective: The tyrosine kinase inhibitors (TKIs) used in the treatment of chronic myeloid leukemia are substrates for the efflux transport protein ABCG2. Variations in ABCG2 activity might influence pharmacokinetics and therapeutic outcome of TKIs. The role of ABCG2 single nucleotide polymorphisms (SNPs) in TKI treatment is not clear and functional in vitro studies are lacking. The aim of this study was to investigate the consequences of ABCG2 SNPs for transport and efficacy of TKIs (imatinib, N-desmethyl imatinib (CGP74588), dasatinib, nilotinib and bosutinib). Methods: ABCG2 SNPs 34G>A, 421C>A, 623T>C, 886G>C, 1574T>G and 1582G>A were constructed from ABCG2 wild type cDNA and transduced to K562 cells by retroviral gene transfer. The ability of variant cells to express ABCG2 in the cell membrane and protect against TKI cytotoxicity was investigated. Results: Wild type ABCG2 had a protective effect against the cytotoxicity of all investigated compounds except bosutinib. It was found that ABCG2 expression provided a better protection against CGP74588 than its parent compound, imatinib. ABCG2 421C>A, 623T>C, 886G>C and 1574T>G reduced cell membrane expression of ABCG2 and the protective effect of ABCG2 against imatinib, CGP74588, dasatinib and nilotinib cytotoxicity. The most prominent effect was found for the 623T>C SNP which resulted in undetectable ABCG2 expression and low protection against TKI cytotoxicity. Conclusion: These findings show that the ABCG2 SNPs 421C>A, 623T>C, 886G>C and 1574T>G impair ABCG2 transport function and might influence TKI pharmacokinetics in vivo. Furthermore, the active imatinib metabolite CGP74588 is to a greater extent than the parent compound transported by ABCG2.

  • 15.
    Skoglund, Karin
    et al.
    Linköping University, Department of Medical and Health Sciences. Linköping University, Faculty of Health Sciences.
    Richter, J.
    Department of Hematology and Coagulation, Skåne University Hospital, Lund, Sweden.
    Olsson-Strömberg, U.
    Department of Medical Sciences, Uppsala University and Department of Hematology, University Hospital, Uppsala, Sweden.
    Bergquist, J.
    Analytical Chemistry, Department of Chemistry – Biomedical Center and Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
    Aluthgedara, W.
    Analytical Chemistry, Department of Chemistry – Biomedical Center and Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
    Ubhayasekera, K.
    Analytical Chemistry, Department of Chemistry – Biomedical Center and Science for Life Laboratory, Uppsala University, Uppsala, Sweden.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Svedberg, A.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Söderlund, S.
    Department of Medical Sciences, Uppsala University and Department of Hematology, University Hospital, Uppsala, Sweden.
    Sandstedt, Anna
    Linköping University, Department of Social and Welfare Studies. Linköping University, Faculty of Health Sciences.
    Johnsson, A.
    Department of Internal Medicine, Motala Hospital, Motala, Sweden.
    Aagesen, J.
    Department of Medicine, Ryhov County Hospital, Jönköping, Sweden.
    Alsenhed, J.
    Department of Internal Medicine, Västervik Hospital, Västervik, Sweden.
    Hägg, Staffan
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Peterson, Curt
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology.
    Lotfi, Kourosh
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    In vivo CYP3A activity and pharmacokinetics of imatinib in relation to therapeutic outcome in chronic myeloid leukemia patientsManuscript (preprint) (Other academic)
    Abstract [en]

    Introduction: The hepatic enzymes CYP3A4 and CYP3A5 metabolize the tyrosine kinase inhibitor imatinib into a large number of metabolites including the pharmacologically active N-desmethyl imatinib (CGP74588). Because the metabolic activity of CYP3A varies considerably between individuals and a previous pilot study suggested an inverse association between in vivo CYP3A metabolic activity and therapeutic outcome of imatinib, the primary aim of this study was to investigate the influence of CYP3A metabolic activity on the outcome of imatinib therapy in chronic myeloid leukemia patients.

    Methods: Fifty-five patients were included and CYP3A activity was estimated in vivo using quinine as a probe drug. Imatinib and CGP74588 trough concentrations in the plasma were determined at steady state in 34 patients. Cytogenetic and molecular responses after 12 months of first-line imatinib were retrospectively collected from patients’ medical records.

    Results: Patients with optimal response to imatinib (complete cytogenetic response (CCgR) or molecular response of BCR-ABL <1%) did not have different levels of CYP3A activity compared to non-optimal responders. Similar results were found when analyzing the molecular response and CCgR separately. Neither the imatinib trough concentration nor the CGP74588/imatinib ratio were significantly associated with CYP3A activity.

    Conclusion: CYP3A enzyme activity, as measured by quinine metabolic ratio, does not correlate with the plasma concentrations of imatinib or CGP74588 and is not predictive of imatinib therapeutic outcome. These results indicate that even though imatinib is metabolized by CYP3A enzymes, this activity is not the   ratelimiting step in imatinib metabolism and excretion. Future studies should focus on other pharmacokinetic processes such as plasma protein binding or transport protein activity to look for the major contributor to patient variability in imatinib plasma concentration.

  • 16.
    Skoglund, Karin
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Richter, Johan
    Skåne University Hospital, Sweden.
    Olsson-Stromberg, Ulla
    Uppsala University, Sweden.
    Bergquist, Jonas
    Uppsala University, Sweden.
    Aluthgedara, Warunika
    Uppsala University, Sweden.
    Ubhayasekera, S. J. Kumari A.
    Uppsala University, Sweden.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Karolinska Institute, Sweden.
    Svedberg, Anna
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Söderlund, Stina
    Uppsala University, Sweden.
    Sandstedt, Anna
    Linköping University, Department of Social and Welfare Studies. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Haematology.
    Johnsson, Anders
    Region Östergötland, Local Health Care Services in West Östergötland, Department of Medical Specialist in Motala.
    Aagesen, Jesper
    Ryhov County Hospital, Sweden.
    Alsenhed, Jonas
    Vastervik Hosp, Dept Internal Med, Västervik, Sweden.
    Hägg, Staffan
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Diagnostics, Department of Clinical Pharmacology.
    Peterson, Curt
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology.
    Lotfi, Kourosh
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Haematology.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. National Board Forens Med, Department Forens Genet and Forens Toxicol, Linkoping, Sweden.
    In Vivo Cytochrome P450 3A Isoenzyme Activity and Pharmacokinetics of Imatinib in Relation to Therapeutic Outcome in Patients With Chronic Myeloid Leukemia2016In: Therapeutic Drug Monitoring, ISSN 0163-4356, E-ISSN 1536-3694, Vol. 38, no 2, p. 230-238Article in journal (Refereed)
    Abstract [en]

    Background: Cytochrome P450 3A (CYP3A) isoenzyme metabolic activity varies between individuals and is therefore a possible candidate of influence on the therapeutic outcome of the tyrosine kinase inhibitor imatinib in patients with chronic myeloid leukemia (CML). The aim of this study was to investigate the influence of CYP3A metabolic activity on the plasma concentration and outcome of imatinib in patients with CML. Methods: Forty-three patients with CML were phenotyped for CYP3A activity using quinine as a probe drug and evaluated for clinical response parameters. Plasma concentrations of imatinib and its main metabolite, CGP74588, were determined using liquid chromatography-mass spectrometry. Results: Patients with optimal response to imatinib after 12 months of therapy did not differ in CYP3A activity compared to nonoptimal responders (quinine metabolic ratio of 14.69 and 14.70, respectively; P = 0.966). Neither the imatinib plasma concentration nor the CGP74588/imatinib ratio was significantly associated with CYP3A activity. Conclusions: The CYP3A activity does not influence imatinib plasma concentrations or the therapeutic outcome. These results indicate that although imatinib is metabolized by CYP3A enzymes, this activity is not the rate-limiting step in imatinib metabolism and excretion. Future studies should focus on other pharmacokinetic processes so as to identify the major contributor to patient variability in imatinib plasma concentrations.

  • 17.
    Svedberg, Anna
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. National Board Forens Med, Department Forens Genet and Forens Toxicol, SE-58758 Linkoping, Sweden; KTH Royal Institute Technology, Sweden.
    Vikström, Anders
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Respiratory Medicine.
    Lundeberg, Joakim
    KTH Royal Institute Technology, Sweden.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    A validated liquid chromatography tandem mass spectrometry method for quantification of erlotinib, OSI-420 and didesmethyl erlotinib and semi-quantification of erlotinib metabolites in human plasma2015In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 107, p. 186-195Article in journal (Refereed)
    Abstract [en]

    A liquid chromatography tandem mass spectrometry method was developed and validated for quantification of erlotinib and its metabolites in human plasma. The method is suitable for therapeutic drug monitoring and pharmacokinetic studies. The substances were extracted using protein precipitation, separated on a BEH XBridge C18 column (100 x 2.1 mm, 1.7 mu m) by gradient elution at 0.7 mL/min of acetonitrile and 5 mM ammonium acetate. The concentration was determined using a Waters Xevo triple quadrupole mass spectrometer in a multi reaction monitoring mode. The total run time was 7 min. Deuterated erlotinib and OSI-597 were used as internal standard for erlotinib and its metabolites, respectively. Erlotinib, OSI-420 and didesmethyl erlotinib were quantified in the concentration range 25-5000 ng/mL, 0.5-500 ng/mL and 0.15-10 ng/mL, respectively. Precision and accuracy was less than14% except for OSI-420 at LLOQ (17%). Extraction recovery was above 89%, 99% and 89% for erlotinib, OSI-420 and didesmethyl erlotinib, respectively. The human liver microsomes generated 14 metabolites, three of them not previously reported. Twelve metabolites were measured semi-quantitatively and validated with respect to selectivity, precision and stability. (C) 2014 Elsevier B.V. All rights reserved.

  • 18.
    Svedberg, Anna
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Vikström, Anders
    Linköping University, Department of Clinical and Experimental Medicine. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Respiratory Medicine.
    Hornstra, Niels
    Kalmar Cty Hosp, Sweden.
    Kentson, Magnus
    Linköping University, Department of Medical and Health Sciences. Linköping University, Faculty of Medicine and Health Sciences. Ryhov Hosp, Sweden.
    Branden, Eva
    Gavle Cent Hosp, Sweden; Uppsala Univ Reg Gavleborg, Sweden.
    Koyi, Hirsh
    Gavle Cent Hosp, Sweden; Uppsala Univ Reg Gavleborg, Sweden.
    Bergman, Bengt
    Univ Gothenburg, Sweden.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Erlotinib treatment induces cytochrome P450 3A activity in non-small cell lung cancer patients2019In: British Journal of Clinical Pharmacology, ISSN 0306-5251, E-ISSN 1365-2125, Vol. 85, no 8, p. 1704-1709Article in journal (Refereed)
    Abstract [en]

    Aims Erlotinib is a tyrosine kinase inhibitor used in the treatment of non-small cell lung cancer highly metabolized by the cytochrome P450 (CYP) 3A. Hence, CYP3A4 activity might be a useful predictor of erlotinib pharmacokinetics in personalized medicine. The effect of erlotinib on CYP3A activity was therefore studied in non-small cell lung cancer patients. Methods The study included 32 patients scheduled for erlotinib monotherapy. CYP3A activity was assessed using quinine as a probe before and during erlotinib treatment. Plasma from blood samples drawn 16 hours post quinine administration were analysed using HPLC with fluorescence detection to determine the quinine/3-OH-quinine ratio. Results Matched samples, available from 13 patients, showed an induction of CYP3A activity (P = 0.003, Wilcoxons signed rank test) after 2 months of treatment. The quinine/3-OH-quinine ratio decreased from 20.2 (+/- 13.4) at baseline to 11.0 (+/- 4.34). Single-point samples, available from 19 patients, supported the decrease in ratio (P = 0.007, Mann-Whitney U-test). Generally, females had a higher CYP3A activity both at baseline and after two months of treatment. Statistical analysis by gender also showed significant increase in CYP3A activity (males, n = 10, P = 0.001, and females, n = 22, P = 0.001). Conclusions An induction of CYP3A activity was observed after 2 months of erlotinib treatment which was also seen when subdividing based on gender. It could be important to take this into consideration for patients co-administering other CYP3A-metabolizing drugs during erlotinib treatment and also makes it difficult to use baseline CYP3A activity to predict erlotinib pharmacokinetics.

    The full text will be freely available from 2020-04-03 11:24
  • 19.
    Truver, Michael T.
    et al.
    Sam Houston State Univ, TX 77340 USA.
    Watanabe, Shimpei
    Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Åstrand, Anna
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Swortwood, Madeleine J.
    Sam Houston State Univ, TX 77340 USA.
    Kronstrand, Robert
    Not Found:Linkoping Univ, Dept Med and Hlth Sci, Div Drug Res, Linkoping, Sweden; Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Linkoping, Sweden.
    5F-MDMB-PICA metabolite identification and cannabinoid receptor activityIn: Drug Testing and Analysis, ISSN 1942-7603, E-ISSN 1942-7611Article in journal (Refereed)
    Abstract [en]

    According to the European Monitoring Center for Drugs and Drug Addiction (EMCDDA), there were 179 different synthetic cannabinoids reported as of 2017. In the USA, 5F-MDMB-PINACA, or 5F-ADB, accounted for 28% of cannabinoid seizures 2016-2018. The synthetic cannabinoid, 5F-MDMB-PICA, is structurally similar to 5F-MDMB-PINACA with an indole group replacing the indazole. Limited data exist from in vivo or in vitro metabolic studies of these synthetic cannabinoids, so potential metabolites to identify use may be missed. The goals of this study were to (a) investigate 5F-MDMB-PICA and 5F-MDMB-PINACA in vitro metabolism utilizing human hepatocytes; (b) to verify in vitro metabolites by analyzing authentic case specimens; and (c) to identify the potency and efficacy of 5F-MDMB-PICA and 5F-MDMB-PINACA by examining activity at the CB1 receptor. Biotransformations found in this study included phase I transformations and phase II transformations. A total of 22 5F-MDMB-PICA metabolites (A1 to A22) were identified. From hepatocyte incubations and urine samples, 21 metabolites (B1 to B21) were identified with 3 compounds unique to urine specimens for 5F-MDMB-PINACA. Phase II glucuronides were identified in 5F-MDMB-PICA (n = 3) and 5F-MDMB-PINACA (n = 5). For both compounds, ester hydrolysis and ester hydrolysis in combination with oxidative defluorination were the most prevalent metabolites produced in vitro. Additionally, the conversion of ester hydrolysis with oxidative defluorination to pentanoic acid for the first time was identified for 5F-MDMB-PICA. Therefore, these metabolites would be potentially good biomarkers for screening urine of suspected intoxication of 5F-MDMB-PICA or 5F-MDMB-PINACA. Both 5F-MDMB-PICA and 5F-MDMB-PINACA were acting as full agonists at the CB1 receptor with higher efficacy and similar potency as JWH-018.

  • 20.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Development of new methodology for therapeutic drug monitoring of thiopurine treatment2012Doctoral thesis, comprehensive summary (Other academic)
    Abstract [en]

    The three thiopurine drugs azathioprine (AZA), 6-mercaptopurine (6-MP) and 6-thioguanine (6-TG) are used to treat several diseases, including inflammatory bowel disease (IBD). They are pro-drugs and are believed to act through the formation of thioguanine nucleotides (TGNs). Other important metabolites are the methylthioinosine nucleotides (meTINs). These metabolites are active in the white blood cells (WBCs).Most patients respond well to the thiopurine drugs but up to a third have to modify or discontinue their treatment due to adverse events or a lack of therapeutic effects. This could be caused by inter-patient variability in the metabolism of the drugs. Therapeutic drug monitoring (TDM) of thiopurine nucleotides in red blood cells (RBCs) is used to guide treatment. Current routine assays measure the nucleotides after hydrolysation to nucleic bases and are therefore unable to distinguish between mono-, di-, and triphosphates. Recently it was shown that these assays failed to predict the clinical outcome in about 40% of the patients. It has been suggested that measuring thioguanosine triphosphate (TGTP) (believed to be the most active of the TGNs) separately might increase the clinical value.An assay suitable for measuring thioguanosine mono- (TGMP) and diphosphate (TGDP) and TGTP, as well as methylthioinosine mono- (meTIMP), di- (meTIDP) and triphosphate (meTITP) separately in RBCs in clinical samples has been developed. In clinical studies of 82 IBD patients, we found no correlation between the thiopurine dose and metabolite levels in RBCs, thus illustrating the importance of metabolite measurements in the TDM of thiopurines.The TGN peak measured by the routine assay during TDM of patients treated with thiopurines consisted of TGTP and TGDP with a small contribution from TGMP. The meTIN also consisted of mono-, di- and triphosphates, but in different proportions, indicating differences in the formation. The inter-individual differences in nucleotide distribution were very small and a strong correlation between the different nucleotides and their respective sums was observed. As a consequence, measuring the mono-, di- and triphosphates separately was not beneficial in predicting remission, which was confirmed by the results from the clinical study.Further research into the metabolism and mode of action of thiopurine drugs is needed to understand the inter-patient variability in response and metabolite formation. An assay suitable for such studies, measuring TGNs and meTINs in cultured cells, has also been developed.

    List of papers
    1. Monitoring of thiopurine metabolites in patients with inflammatory bowel disease-what is actually measured?
    Open this publication in new window or tab >>Monitoring of thiopurine metabolites in patients with inflammatory bowel disease-what is actually measured?
    2009 (English)In: Therapeutic Drug Monitoring, ISSN 0163-4356, E-ISSN 1536-3694, Vol. 31, no 3, p. 345-50Article in journal (Refereed) Published
    Abstract [en]

    Azathioprine and 6-mercaptopurine are often used in the treatment of patients with inflammatory bowel disease (IBD). They are prodrugs and undergo a complex metabolism to active and inactive metabolites. Thiopurine treatment is monitored in many laboratories by measuring metabolite concentrations in erythrocytes (red blood cells). The metabolites of interest are not measured directly but as hydrolysis products, which can be produced from several metabolites. The aim of this study was to examine which metabolites are actually measured during routine monitoring. Samples from 18 patients treated with a thiopurine were analyzed by a typical routine high-performance liquid chromatography method for therapeutic drug monitoring and by a newly developed specific method measuring thioguanosine monophosphate (TGMP), thioguanosine diphosphate (TGDP), and thioguanosine triphosphate (TGTP), as well as methylthioinosine monophosphate (meTIMP), and the results were compared. 6-Thioguanine nucleotide (TGN) values detected by the routine method were 69% (range 40%-90%) of the sum of TGMP, TGDP, and TGTP measured by the specific method. TGTP and TGDP contributed 85% (range 78%-90%) and 14% (range 10%-21%) of the TGN total, respectively. Thioguanosine was not found in any patient sample. The concentration of meTIMP obtained by the routine method was 548% of the value obtained by the specific method (range 340%-718%). The difference in TGN measurements between the routine and specific methods can be explained by low hydrolysis efficiency in the routine method, although the most likely explanation for the difference in meTIMP values is that not yet identified metabolites are codetermined in the routine high-performance liquid chromatography method. Concentrations reported as TGN during therapeutic drug monitoring of thiopurine metabolites consist of TGDP and TGTP with a minor contribution of the TGMP. Concentrations reported as meTIMP or methyl mercaptopurine consist in part of meTIMP, but other not yet identified metabolites are codetermined.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-18985 (URN)10.1097/FTD.0b013e3181a1ea58 (DOI)19363461 (PubMedID)
    Available from: 2009-06-07 Created: 2009-06-07 Last updated: 2017-12-13Bibliographically approved
    2. Monitoring of thiopurine metabolites: A high-performance liquid chromatography method for clinical use
    Open this publication in new window or tab >>Monitoring of thiopurine metabolites: A high-performance liquid chromatography method for clinical use
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    high-performance liquid chromatography method capable of measuring thiopurine mono-, di-, and triphosphates separately in red blood cells (RBCs) was developed. RBC:s were isolated from whole blood using centrifugation. Proteins were precipitated using dichloromethane and methanol. The thioguanine nucleotides (TGNs) were derivatised using potassium permanganate before analysis. Analytes were separated by ion-pairing liquid chromatography using tetrabutylammonium ions and detected using UV absorption and fluorescence. The method was designed for use in clinical trials in thiopurine therapy and proven valid by analysis of authentic patient samples.

    The method measured thioguanosine mono- (TGMP), di- (TGDP), and triphosphate (TGTP), as well as methylthioinosine mono- (meTIMP), di- (meTIDP) and triphosphate (meTITP) in RBCs collected from patients treated with thiopurine drugs (azathioprine, 6-mercaptopurine, and 6-thioguanine).

    LOQ was 0.3, 3, 2, 30, 30 and 40 pmol/8x10^8 RBC, for TGMP, TGDP, TGTP, meTIMP, meTIDP and meTITP, respectively. Between-day precision were below 14% for all analytes at all concentrations and samples were stable at 5 °C for 8 hours after sampling.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-84843 (URN)
    Available from: 2012-10-24 Created: 2012-10-24 Last updated: 2012-10-24Bibliographically approved
    3. Therapeutic drug monitoring of thiopurines in inflammatory bowel disease: Evaluating the benefit of measuring mono-, di-, and triphosphates separately
    Open this publication in new window or tab >>Therapeutic drug monitoring of thiopurines in inflammatory bowel disease: Evaluating the benefit of measuring mono-, di-, and triphosphates separately
    Show others...
    (English)Manuscript (preprint) (Other academic)
    Abstract [en]

    The thiopurines are widely used in the treatment of inflammatory bowel diseases but are limited by poor dose-effect relationship and large interindividual variability in clinical effects. Many attempts have been made to predict response by therapeutic drug monitoring of phosphorylated and methylated metabolites grouped together as thioguanine nucleotides and methylthioinosine monophosphate. We have developed a method to determine the individual metabolites, thioguanosine mono-, di-, and triphosphates, as well as methylthioinosine mono-, di-, and triphosphates, separately in red blood cells.

    This aim of this study was to assess the ability of our novel method to predict clinical outcome compared to the routine method in 82 patients with inflammatory bowel diseases.

    TPMT wild-type patients with TGN levels below the cut-off level were more likely to have an active disease when TGN was measured by both the routine method (p < 0.05), the novel method (p<0.05), and when TGTP was measured separately (p < 0.01). TGN levels and TGTP were, however, not correlated to disease activity in TPMT defective patients. Patients with meTIN levels above 1500 pmol were more likely to have an active disease (39%, 18/46 vs. 17%, 5/30; p = 0.02). We observed good correlations between the mono-, di-, and triphosphates and their respective sums (R2 > 0.88) and the TGTP ratio (TGTP/(TGDP+TGTP)) was not different in patients with active disease or in clinical remission.

    Thiopurine metabolites should still be measured by the routine method, since the novel and technically more challenging method, including determination of TGTP and TGTP ratio, does not offer a clinical advantage compared to the routine method.

    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-84844 (URN)
    Available from: 2012-10-24 Created: 2012-10-24 Last updated: 2012-10-24Bibliographically approved
    4. The Role of Inosine-5'-Monophosphate Dehydrogenase in Thiopurine Metabolism in Patients With Inflammatory Bowel Disease
    Open this publication in new window or tab >>The Role of Inosine-5'-Monophosphate Dehydrogenase in Thiopurine Metabolism in Patients With Inflammatory Bowel Disease
    Show others...
    2011 (English)In: Therapeutic Drug Monitoring, ISSN 0163-4356, E-ISSN 1536-3694, Vol. 33, no 2, p. 200-208Article in journal (Refereed) Published
    Abstract [en]

    BACKGROUND:: There is a large interindividual variability in thiopurine metabolism. High concentrations of methylthioinosine-5'-monophosphate (meTIMP) and low concentrations of 6-thioguanine nucleotides (6-TGNs) have been associated with a lower response rate and an increased risk of adverse events. In this study, the role of inosine-5'-monophosphate dehydrogenase (IMPDH) for differences in metabolite patterns of thiopurines was investigated.

    METHODS:: IMPDH activity and thiopurine metabolite concentrations were determined in patients with inflammatory bowel disease and a normal thiopurine methyltransferase (TPMT) phenotype and meTIMP/6-TGN concentration ratio > 20 (n = 26), in patients with a metabolite ratio ≤20 (n = 21), in a subgroup with a metabolite ratio <4 (n = 6), and in 10 patients with reduced TPMT activity. In vitro studies were conducted on human embryonic kidney cells (HEK293) with genetically engineered IMPDH and TPMT activities.

    RESULTS:: Patients with metabolite ratios >20 had lower IMPDH activity than those with ratios ≤20 (P < 0.001). Metabolic ratios >20 were only observed in patients with normal TPMT activity. Downregulation of IMPDH activity in HEK293 cells was associated with an increase in the concentration of meTIMP (fold change: 17 up to 93, P < 0.001) but, unexpectedly, also of 6-thioguanosine monophosphate (fold change: 2.6 up to 5.0, P < 0.001).

    CONCLUSIONS:: These data question the general view of IMPDH as the rate-limiting enzyme in the phosphorylation of thiopurines. Investigations of other mechanisms are needed to more fully explain the various metabolite patterns and outcomes in patients under treatment.

    Place, publisher, year, edition, pages
    Lippincott Williams & Wilkins, 2011
    National Category
    Medical and Health Sciences
    Identifiers
    urn:nbn:se:liu:diva-66431 (URN)10.1097/FTD.0b013e31820b42bb (DOI)000288498100010 ()21311411 (PubMedID)
    Available from: 2011-03-15 Created: 2011-03-15 Last updated: 2017-12-11Bibliographically approved
  • 21.
    Vikingsson, Svante
    et al.
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Almer, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Center, Department of Gastroentorology.
    Peterson, Curt
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology.
    Carlsson, Björn
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Pharmacology.
    Josefsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Monitoring of thiopurine metabolites - A high-performance liquid chromatography method for clinical use2013In: Journal of Pharmaceutical and Biomedical Analysis, ISSN 0731-7085, E-ISSN 1873-264X, Vol. 75, p. 145-152Article in journal (Refereed)
    Abstract [en]

    A high-performance liquid chromatography method capable of measuring thiopurine mono-, di-, and triphosphates separately in red blood cells (RBCs) was developed. RBCs were isolated from whole blood using centrifugation. Proteins were precipitated using dichloromethane and methanol. The thioguanine nucleotides (TGNs) were derivatised using potassium permanganate before analysis. Analytes were separated by ion-pairing liquid chromatography using tetrabutylammonium ions and detected using UV absorption and fluorescence. The method was designed for use in clinical trials. Ten patient samples were analysed to demonstrate clinical application and to establish pilot ranges for all analytes. less thanbrgreater than less thanbrgreater thanThe method measured thioguanosine mono-(TGMP), di-(TGDP), and triphosphate (TGTP), as well as methylthioinosine mono- (meTIMP), di- (meTIDP) and triphosphate (meTITP) in RBCs collected from patients treated with thiopurine drugs (azathioprine, 6-mercaptopurine, and 6-thioguanine). less thanbrgreater than less thanbrgreater thanLOQ was 0.3, 3, 2, 30, 30 and 40 pmol/8 x 10(8) RBC, for TGMP, TGDP, TGTP, meTIMP, meTIDP and meTITP, respectively. Between-day precision were below 14% for all analytes at all concentrations and samples were stable at 4 degrees C for 8 h after sampling.

  • 22.
    Vikingsson, Svante
    et al.
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Almer, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Endocrinology and Gastroenterology UHL.
    Peterson, Curt
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology UHL.
    Carlsson, Björn
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Diagnostics, Department of Clinical Pharmacology.
    Josefsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, The Institute of Technology.
    Monitoring of thiopurine metabolites: A high-performance liquid chromatography method for clinical useManuscript (preprint) (Other academic)
    Abstract [en]

    high-performance liquid chromatography method capable of measuring thiopurine mono-, di-, and triphosphates separately in red blood cells (RBCs) was developed. RBC:s were isolated from whole blood using centrifugation. Proteins were precipitated using dichloromethane and methanol. The thioguanine nucleotides (TGNs) were derivatised using potassium permanganate before analysis. Analytes were separated by ion-pairing liquid chromatography using tetrabutylammonium ions and detected using UV absorption and fluorescence. The method was designed for use in clinical trials in thiopurine therapy and proven valid by analysis of authentic patient samples.

    The method measured thioguanosine mono- (TGMP), di- (TGDP), and triphosphate (TGTP), as well as methylthioinosine mono- (meTIMP), di- (meTIDP) and triphosphate (meTITP) in RBCs collected from patients treated with thiopurine drugs (azathioprine, 6-mercaptopurine, and 6-thioguanine).

    LOQ was 0.3, 3, 2, 30, 30 and 40 pmol/8x10^8 RBC, for TGMP, TGDP, TGTP, meTIMP, meTIDP and meTITP, respectively. Between-day precision were below 14% for all analytes at all concentrations and samples were stable at 5 °C for 8 hours after sampling.

  • 23.
    Vikingsson, Svante
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Andersson, David
    Skåne University Hospital, Sweden; Danderyd Hospital, Sweden.
    Almer, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Division of Inflammation Medicine. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Center, Department of Gastroentorology. Karolinska Institute, Sweden; Karolinska University Hospital, Sweden.
    Peterson, Curt
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology.
    Hindorf, Ulf
    Skåne University Hospital, Sweden.
    Novel assay to improve therapeutic drug monitoring of thiopurines in inflammatory bowel disease2014In: Journal of Crohn's & Colitis, ISSN 1873-9946, E-ISSN 1876-4479, Vol. 8, no 12, p. 1702-1709Article in journal (Refereed)
    Abstract [en]

    Background and aims: The thiopurines are widely used in the treatment of inflammatory bowel disease, but are limited by poor dose effect relationship. The objective was to assess the ability of a novel assay, determining the mono-, di-, and triphosphates, of thioguanine as well as methylthioinosine as individual metabolites in erythrocytes, to predict clinical outcome compared to a routine assay, determining metabolites as sums. Methods: Samples from 79 patients with Crohns disease or ulcerative colitis treated with azathioprine or mercaptopurine were analysed by both assays. Clinical status was determined by the Harvey-Bradshaw and Walmsley indices. The genotypes of thiopurine methyltransferase (TPMT) and inosine triphosphatase were determined. Results: TPMT wild-type patients with thioguanine nucleotide (TGN) levels below the cut-off level were more likely to have active disease when TGN was measured by the novel assay (p = 0.02), and when thioguanosine triphosphate (TGTP) was measured separately (p = 0.01). When TGN was measured by the routine assay the correlation was not evident (p = 0.12). Neither TGN levels nor TGTP correlated to disease activity in TPMT deficient patients. Patients with methyl thioinosine nucleotide (meTIN) levels above 1500 pmol/8 x 10less than^greater than8 RBCs were more likely to have active disease (p = 0.07). We observed good correlations between the mono-, di-, and triphosphates and their respective sums (R-2 greater than 0.88). Conclusions: The novel TGN assay was better in predicting clinical outcome compared to the routine assay, while determination of TGTP had no clinical advantage and TGTP ratio was not correlated to disease activity. (C) 2014 European Crohns and Colitis Organisation. Published by Elsevier B.V. All rights reserved.

  • 24.
    Vikingsson, Svante
    et al.
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Andersson, David
    Department of Gastroenterology, Skåne University Hospital, Lund, Sweden.
    Almer, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Centre, Department of Endocrinology and Gastroenterology UHL.
    Peterson, Curt
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology UHL.
    Hindorf, Ulf
    Department of Gastroenterology, Skåne University Hospital, Lund, Sweden.
    Therapeutic drug monitoring of thiopurines in inflammatory bowel disease: Evaluating the benefit of measuring mono-, di-, and triphosphates separatelyManuscript (preprint) (Other academic)
    Abstract [en]

    The thiopurines are widely used in the treatment of inflammatory bowel diseases but are limited by poor dose-effect relationship and large interindividual variability in clinical effects. Many attempts have been made to predict response by therapeutic drug monitoring of phosphorylated and methylated metabolites grouped together as thioguanine nucleotides and methylthioinosine monophosphate. We have developed a method to determine the individual metabolites, thioguanosine mono-, di-, and triphosphates, as well as methylthioinosine mono-, di-, and triphosphates, separately in red blood cells.

    This aim of this study was to assess the ability of our novel method to predict clinical outcome compared to the routine method in 82 patients with inflammatory bowel diseases.

    TPMT wild-type patients with TGN levels below the cut-off level were more likely to have an active disease when TGN was measured by both the routine method (p < 0.05), the novel method (p<0.05), and when TGTP was measured separately (p < 0.01). TGN levels and TGTP were, however, not correlated to disease activity in TPMT defective patients. Patients with meTIN levels above 1500 pmol were more likely to have an active disease (39%, 18/46 vs. 17%, 5/30; p = 0.02). We observed good correlations between the mono-, di-, and triphosphates and their respective sums (R2 > 0.88) and the TGTP ratio (TGTP/(TGDP+TGTP)) was not different in patients with active disease or in clinical remission.

    Thiopurine metabolites should still be measured by the routine method, since the novel and technically more challenging method, including determination of TGTP and TGTP ratio, does not offer a clinical advantage compared to the routine method.

  • 25.
    Vikingsson, Svante
    et al.
    Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Carlsson, Björn
    Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Almer, Sven H C
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Peterson, Curt
    Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Oncology UHL.
    Monitoring of thiopurine metabolites in patients with inflammatory bowel disease-what is actually measured?2009In: Therapeutic Drug Monitoring, ISSN 0163-4356, E-ISSN 1536-3694, Vol. 31, no 3, p. 345-50Article in journal (Refereed)
    Abstract [en]

    Azathioprine and 6-mercaptopurine are often used in the treatment of patients with inflammatory bowel disease (IBD). They are prodrugs and undergo a complex metabolism to active and inactive metabolites. Thiopurine treatment is monitored in many laboratories by measuring metabolite concentrations in erythrocytes (red blood cells). The metabolites of interest are not measured directly but as hydrolysis products, which can be produced from several metabolites. The aim of this study was to examine which metabolites are actually measured during routine monitoring. Samples from 18 patients treated with a thiopurine were analyzed by a typical routine high-performance liquid chromatography method for therapeutic drug monitoring and by a newly developed specific method measuring thioguanosine monophosphate (TGMP), thioguanosine diphosphate (TGDP), and thioguanosine triphosphate (TGTP), as well as methylthioinosine monophosphate (meTIMP), and the results were compared. 6-Thioguanine nucleotide (TGN) values detected by the routine method were 69% (range 40%-90%) of the sum of TGMP, TGDP, and TGTP measured by the specific method. TGTP and TGDP contributed 85% (range 78%-90%) and 14% (range 10%-21%) of the TGN total, respectively. Thioguanosine was not found in any patient sample. The concentration of meTIMP obtained by the routine method was 548% of the value obtained by the specific method (range 340%-718%). The difference in TGN measurements between the routine and specific methods can be explained by low hydrolysis efficiency in the routine method, although the most likely explanation for the difference in meTIMP values is that not yet identified metabolites are codetermined in the routine high-performance liquid chromatography method. Concentrations reported as TGN during therapeutic drug monitoring of thiopurine metabolites consist of TGDP and TGTP with a minor contribution of the TGMP. Concentrations reported as meTIMP or methyl mercaptopurine consist in part of meTIMP, but other not yet identified metabolites are codetermined.

  • 26.
    Vikingsson, Svante
    et al.
    Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Carlsson, Björn
    Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Laboratory Medicine, Department of Clinical Pharmacology.
    Almer, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre for Medicine, Department of Endocrinology and Gastroenterology UHL.
    Peterson, Curt
    Linköping University, Department of Medicine and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Centre of Surgery and Oncology, Department of Oncology UHL.
    How Should Thiopurine Treatment be Monitored? Methodological Aspects2010In: Nucleosides, Nucleotides & Nucleic Acids, ISSN 1525-7770, E-ISSN 1532-2335, Vol. 29, no 04-Jun, p. 278-283Article in journal (Refereed)
    Abstract [en]

    Monitoring of thiopurine metabolites is important due to a complex metabolism with large interindividual variation, but the suitability of currently used methods has been questioned. The drawbacks include poor reproducibility, the inability to differentiate between the different analytes, as well as the use of a nontarget matrix. Further research should be directed toward measuring thiopurine metabolites in mononuclear cells, measuring the different nucleotides specifically, as well as measuring the incorporation of thioguanine into DNA. The studies should not be limited to thioguanosine nucleotides but include methylthioinosine nucleotides as well.

  • 27.
    Vikingsson, Svante
    et al.
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Carlsson, Björn
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Diagnostics, Department of Clinical Pharmacology.
    Coulthard, S
    Newcastle University, Northern Institute for Cancer Research.
    Josefsson, Martin
    National Board of Forensic Medicine, Department of Forensic Genetics and Forensic Toxicology, Linkoping.
    Almer, Sven
    Linköping University, Department of Clinical and Experimental Medicine, Gastroenterology and Hepatology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Heart and Medicine Center, Department of Endocrinology and Gastroenterology UHL.
    Peterson, Curt
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences. Östergötlands Läns Landsting, Center for Surgery, Orthopaedics and Cancer Treatment, Department of Oncology.
    Rapid Method to Measure Thioguanine Incorporation Into DNA2011In: European Journal of Cancer, ISSN 0959-8049, E-ISSN 1879-0852, Vol. 47, no Supplement 1, p. S650-S650Article in journal (Other academic)
    Abstract [en]

    Background: The thiopurine drugs, 6-mercaptopurine, azathioprine, and thioguanine, are used in the treatment of acute lymphoblastic leukaemia (ALL). During treatment the thioguanine nucleotides formed are incorporated into the DNA, causing apoptosis due to the cells inability to repair the resulting damage. This mechanism is believed to be important for the effects of thiopurine drugs. We have developed a novel method for the determination of thioguanine incorporation into DNA which is both faster and cheaper than earlier methods.

    Monitoring the effects of thiopurine treatment by measuring thiopurine metabolites in erythrocytes has proven to be elusive due to the lack of good correlation between measured concentrations and thiopurine effects. If the incorporation is the main mechanism of thiopurine action, a reliable method capable of measuring the incorporation in an ordinary blood sample, such as the method we have developed, should provide a significantly better correlation with treatment effect.

    Material and Methods: Briefly, DNA extracted from buffy coat is degraded using nuclease P1 and alkaline phosphatase to produce free nucleosides which are purified by filtration. Thioguanosine and thymidine are separated and detected using an LC-MS/MS system and the ratio between the bases provides a measurement of the extent of thioguanine incorporation in DNA. The method has been successfully applied to cell culture samples as well as samples from patients treated orally with thiopurines.

    Results: In 8 inflammatory bowel disease patients treated with azathioprine the measured incorporation ranged from 2.2 to 8.4 thioguanine bases for every 10 000 thymidine bases (median 5.2). This is in agreement with earlier reports on incorporation in childhood leukemia patients.

    Conclusions: With the presented method it is possible to determine the incorporation of thioguanine into DNA during thiopurine treatment in a cost effective manner, but further research is needed to determine if there is a place for this type of methods in the monitoring of thiopurine treatment.

    An ongoing study aims to compare the incorporation to treatment effects as well as conventional measurements of erythrocyte metabolite levels. By this study we hope to determine if incorporation is a more reliable measurement to predict treatment effect and if the erythrocyte metabolite levels correlate with the incorporation.

  • 28.
    Vikingsson, Svante
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Dahlberg, Jan-Olof
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Hansson, Johan
    Karolinska University Hospital Solna, Sweden.
    Hoiom, Veronica
    Karolinska University Hospital Solna, Sweden.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. National Board Forens Med, Department Forens Genet and Forens Toxicol, Artillerigatan 12, S-58758 Linkoping, Sweden.
    Simple and cost-effective liquid chromatography-mass spectrometry method to measure dabrafenib quantitatively and six metabolites semi-quantitatively in human plasma2017In: Analytical and Bioanalytical Chemistry, ISSN 1618-2642, E-ISSN 1618-2650, Vol. 409, no 15, p. 3749-3756Article in journal (Refereed)
    Abstract [en]

    Dabrafenib is an inhibitor of BRAF V600E used for treating metastatic melanoma but a majority of patients experience adverse effects. Methods to measure the levels of dabrafenib and major metabolites during treatment are needed to allow development of individualized dosing strategies to reduce the burden of such adverse events. In this study, an LC-MS/MS method capable of measuring dabrafenib quantitatively and six metabolites semi-quantitatively is presented. The method is fully validated with regard to dabrafenib in human plasma in the range 5-5000 ng/mL. The analytes were separated on a C18 column after protein precipitation and detected in positive electrospray ionization mode using a Xevo TQ triple quadrupole mass spectrometer. As no commercial reference standards are available, the calibration curve of dabrafenib was used for semi-quantification of dabrafenib metabolites. Compared to earlier methods the presented method represents a simpler and more cost-effective approach suitable for clinical studies.

  • 29.
    Vikingsson, Svante
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. National Board Forens Med, Department Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Editorial Material: Putting Designer Drugs Back in Pandoras Box: Analytical Challenges and Metabolite Identification in CLINICAL CHEMISTRY, vol 62, issue 1, pp2016In: Clinical Chemistry, ISSN 0009-9147, E-ISSN 1530-8561, Vol. 62, no 1Article in journal (Other academic)
    Abstract [en]

    n/a

  • 30.
    Vikingsson, Svante
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Gréen, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping Sweden.
    Brinkhagen, Linda
    Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping Sweden.
    Mukhtar, Shahzabe
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Josefsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping Sweden.
    Identification of AB-FUBINACA metabolites in authentic urine samples suitable as urinary markers of drug intake using liquid chromatography quadrupole tandem time of flight mass spectrometry.2016In: Drug Testing and Analysis, ISSN 1942-7603, E-ISSN 1942-7611, Vol. 8, no 9, p. 950-956Article in journal (Refereed)
    Abstract [en]

    Synthetic cannabinoids are a group of psychoactive drugs presently widespread among drug users in Europe. Analytical methods to measure these compounds in urine are in demand as urine is a preferred matrix for drug testing. For most synthetic cannabinoids, the parent compounds are rarely detected in urine. Therefore urinary metabolites are needed as markers of drug intake. AB-FUBINACA was one of the top three synthetic cannabinoids most frequently found in seizures and toxicological drug screening in Sweden (2013-2014). Drug abuse is also reported from several other countries such as the USA and Japan. In this study, 28 authentic case samples were used to identify urinary markers of AB-FUBINACA intake using liquid chromatography quadrupole tandem time of flight mass spectrometry and human liver microsomes. Three metabolites suitable as markers of drug intake were identified and at least two of them were detected in all but one case. In total, 15 urinary metabolites of AB-FUBINACA were reported, including hydrolxylations on the indazole ring and the amino-oxobutane moiety, dealkylations and hydrolysis of the primary amide. No modifications on the fluorobenzyl side-chain were observed. The parent compound was detected in 54% of the case samples. Also, after three hours of incubation with human liver microsomes, 77% of the signal from the parent compound remained. Copyright © 2015 John Wiley & Sons, Ltd.

  • 31.
    Vikingsson, Svante
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Josefsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. National Board Forens Med, Department Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. National Board Forens Med, Department Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Identification of AKB-48 and 5F-AKB-48 Metabolites in Authentic Human Urine Samples Using Human Liver Microsomes and Time of Flight Mass Spectrometry2015In: Journal of Analytical Toxicology, ISSN 0146-4760, E-ISSN 1945-2403, Vol. 39, no 6, p. 426-435Article in journal (Refereed)
    Abstract [en]

    The occurrence of structurally related synthetic cannabinoids makes the identification of unique markers of drug intake particularly challenging. The aim of this study was to identify unique and abundant metabolites of AKB-48 and 5F-AKB-48 for toxicological screening in urine. Investigations of authentic urine samples from forensic cases in combination with human liver microsome (HLM) experiments were used for identification of metabolites. HLM incubations of AKB-48 and 5F-AKB-48 along with 35 urine samples from authentic cases were analyzed with liquid chromatography quadrupole tandem time of flight mass spectrometry. Using HLMs 41 metabolites of AKB-48 and 37 metabolites of 5F-AKB-48 were identified, principally represented by hydroxylation but also ketone formation and dealkylation. Monohydroxylated metabolites were replaced by di- and trihydroxylated metabolites within 30 min. The metabolites from the HLM incubations accounted for on average 84% (range, 67-100) and 91% (range, 71-100) of the combined area in the case samples for AKB-48 and 5F-AKB-48, respectively. While defluorinated metabolites accounted for on average 74% of the combined area after a 5F-AKB-48 intake only a few identified metabolites were shared between AKB-48 and 5F-AKB-48, illustrating the need for a systematic approach to identify unique metabolites. HLMs in combination with case samples seem suitable for this purpose.

  • 32.
    Vikingsson, Svante
    et al.
    Linköping University, Department of Medical and Health Sciences, Clinical Pharmacology. Linköping University, Faculty of Health Sciences.
    Kronstrand, Robert
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Health Sciences.
    Josefsson, Martin
    Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Artillerigatan 12, SE-581 33 Linköping, Sweden.
    Retention of opioids and their glucuronides on a combined zwitterion and hydrophilic interaction stationary phase2008In: Journal of Chromatography A, ISSN 0021-9673, E-ISSN 1873-3778, Vol. 1187, no 01-Feb, p. 46-52Article in journal (Refereed)
    Abstract [en]

    A stationary phase combining zwitterionic ion chromatography and hydrophilic interaction chromatography (ZIC-HILIC) from SeQuant was evaluated for the chromatography of some opiates and their polar metabolites. The effects of mobile phase constitution on retention and resolution were extensively evaluated. Different aspects of mobile phase constitution such as ion strength and type of buffer, type and amount of organic modifier and pH were examined. The selectivity and retention of the opiates compared to their glucuronides could be substantially altered with small changes of the mobile phase, especially when the type of buffer, i.e., formate, or acetate and organic modifier, i.e., acetonitrile or methanol were changed. The retention on the ZIC-HILIC was dominated by hydrophilic interaction chromatography (HILIC) but considerable effects on the selectivity was observed, possibly caused by an ion exchange mechanism due to interactions with the charges on the stationary phase.

  • 33.
    Vikingsson, Svante
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Strömqvist, Malin
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden.
    Svedberg, Anna
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Hansson, Johan
    Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden.
    Höiom, Veronica
    Department of Oncology-Pathology, Karolinska Institutet, Karolinska University Hospital Solna, Stockholm, Sweden.
    Gréen, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, Linköping, Sweden.
    Novel rapid liquid chromatography tandem masspectrometry method for vemurafenib and metabolites in human plasma, including metabolite concentrations at steady-state.2016In: BMC Biomedical chromotography, ISSN 0269-3879, E-ISSN 1099-0801, Vol. 30, no 8, p. 1234-1239Article in journal (Refereed)
    Abstract [en]

    A novel, rapid and sensitive liquid chromatography tandem-mass spectrometry method for quantification of vemurafenib in human plasma, that also for the first time allows for metabolite semi-quantification, was developed and validated to support clinical trials and therapeutic drug monitoring. Vemurafenib was analysed by precipitation with methanol followed by a 1.9 min isocratic liquid chromatography tandem masspectrometry analysis using an Acquity BEH C18 column with methanol and formic acid using isotope labelled internal standards. Analytes were detected in multi reaction monitoring mode on a Xevo TQ. Semi-quantification of vemurafenib metabolites was performed using the same analytical system and sample preparation with gradient elution. The vemurafenib method was successfully validated in the range 0.5-100 µg/mL according to international guidelines. The metabolite method was partially validated due to the lack of commercially available reference materials. For the first time concentration levels at steady-state for melanoma patients treated with vemurafenib is presented. The low abundance of vemurafenib metabolites suggests that they lack clinical significance. This article is protected by copyright. All rights reserved.

  • 34.
    Vikingsson, Svante
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. National Board Forens Med, Department Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Wohlfarth, Ariane
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. National Board Forens Med, Department Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Andersson, Mikael
    National Board Forens Med, Department Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. National Board Forens Med, Department Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Roman, Markus
    National Board Forens Med, Department Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Josefsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering. National Board Forens Med, Department Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Kugelberg, Fredrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. National Board Forens Med, Department Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Kronstrand, Robert
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. National Board Forens Med, Department Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Identifying Metabolites of Meclonazepam by High-Resolution Mass Spectrometry Using Human Liver Microsomes, Hepatocytes, a Mouse Model, and Authentic Urine Samples2017In: AAPS Journal, ISSN 1550-7416, E-ISSN 1550-7416, Vol. 19, no 3, p. 736-742Article in journal (Refereed)
    Abstract [en]

    Meclonazepam is a benzodiazepine patented in 1977 to treat parasitic worms, which recently appeared as a designer benzodiazepine and drug of abuse. The aim of this study was to identify metabolites suitable as biomarkers of drug intake in urine using high-resolution mass spectrometry, authentic urine samples, and different model systems including human liver microsomes, cryopreserved hepatocytes, and a mice model. The main metabolites of meclonazepam found in human urine were amino-meclonazepam and acetamido-meclonazepam; also, minor peaks for meclonazepam were observed in three of four urine samples. These observations are consistent with meclonazepam having a metabolism similar to that of other nitro containing benzodiazepines such as clonazepam, flunitrazepam, and nitrazepam. Both metabolites were produced by the hepatocytes and in the mice model, but the human liver microsomes were only capable of producing minor amounts of the amino metabolite. However, under nitrogen, the amount of amino-meclonazepam produced increased 140 times. This study comprehensively elucidated meclonazepam metabolism and also illustrates that careful selection of in vitro model systems for drug metabolism is needed, always taking into account the expected metabolism of the tested drug.

  • 35.
    Vorkapic, Emina
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Dugic, Elma
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Roy, Joy
    Vascular Surgery, Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden.
    Mäyränpää, Mikko I.
    Department of Pathology, University of Helsinki, Helsinki, Finland / HUSLAB, Division of Pathology, Meilahti Laboratories of Pathology, Helsinki University Central Hospital, Helsinki, Finland.
    Eriksson, Per
    Karolinska University Hospital, Karolinska Institutet, Stockholm, Sweden.
    Wågsäter, Dick
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Imatinib treatment attenuates growth and inflammation of angiotensin II induced abdominal aortic aneurysm2016In: Atherosclerosis, ISSN 0021-9150, E-ISSN 1879-1484, Vol. 249, p. 101-109Article in journal (Refereed)
    Abstract [en]

    AbstractBackground Abdominal aortic aneurysm (AAA) is characterized by vascular remodeling with increased infiltration of inflammatory cells and apoptosis/modulation of vascular smooth muscle cells (SMCs). Imatinib is a selective inhibitor of several tyrosine kinases, including PDGF receptors, Abl, and c-kit. The objective of this study was to characterize the potential protective role of imatinib on AAA development and the molecular mechanisms involved. Methods Male ApoE−/− mice were infused with angiotensin (Ang) II (1000 ng/kg/min) for 4 weeks to induce AAA or saline as controls. Daily treatment with 10 mg/kg imatinib, or tap water as control, was provided via gavage for 4 weeks. Results Treatment with imatinib was found to decrease the aortic diameter and vessel wall thickness, mediated by multiple effects. Imatinib treatment in AngII infused mice resulted in a reduced cellular infiltration of CD3ε positive T lymphocytes by 86% and reduced gene expression of mast cell chymase by 50% compared with AngII infused mice lacking imatinib. Gene expression analysis of SMC marker SM22α demonstrated an increase by 48% together with a more intact medial layer after treatment with imatinib as evaluated with SM22α immunostaining. Conclusion Present findings highlight the importance of tyrosine kinase pathways in the development of AAA. Our results show, that imatinib treatment inhibits essential mast cell, T lymphocyte and SMC mediated processes in experimental AAA. Thus, our results support the idea that tyrosine kinase inhibitors may be useful in the treatment of pathological vascular inflammation and remodeling in conditions like AAA.

  • 36.
    Wallgren, Jakob
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Johansson, Anders
    Linköping University, Department of Physics, Chemistry and Biology. Linköping University, Faculty of Science & Engineering.
    Josefsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Dahlén, Johan
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Wu, Xiongyu
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Konradsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Synthesis and identification of an important metabolite of AKB-48 with a secondary hydroxyl group on the adamantyl ring2017In: Tetrahedron Letters, ISSN 0040-4039, E-ISSN 1359-8562, Vol. 58, no 15, p. 1456-1458Article in journal (Refereed)
    Abstract [en]

    Studies on the metabolism of bioactive substances containing the adamantyl moiety have shown that hydroxylation is likely to occur at a tertiary carbon of adamantane. Herein, we report the synthesis and identification of one major metabolite of AKB-48, a new illicit psychoactive substance with a hydroxyl group at a secondary carbon of the adamantyl ring. (C) 2017 Elsevier Ltd. All rights reserved.

  • 37.
    Wallgren, Jakob
    et al.
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Åstrand, Anna
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Josefsson, Martin
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Dahlén, Johan
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Wu, Xiongyu
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Konradsson, Peter
    Linköping University, Department of Physics, Chemistry and Biology, Chemistry. Linköping University, Faculty of Science & Engineering.
    Synthesis and identifications of potential metabolites as biomarkers of the synthetic cannabinoid AKB-482018In: Tetrahedron, ISSN 0040-4020, E-ISSN 1464-5416, Vol. 74, no 24, p. 2905-2913Article in journal (Refereed)
    Abstract [en]

    AKB-48 belongs to the family of synthetic cannabinoids. It has strong binding affinity to CBI receptor and is psychoactive. It is banned in many countries including USA, Japan, Germany, New Zealand, Singapore and China etc. But the difficulty in detecting the parent compound in urine samples highlights the importance of studies of its metabolites. Here we report the synthesis of 19 potential metabolites of AKB-48, among which, compounds 2, 9, 10, 30 and 31, together with the commercially available substance 5 were identified as metabolites of AKB-48 by comparison with one authentic human urine sample and human liver microsomal data. Compounds 10 and 30 could be of use as biomarkers in detecting AKB-48 in human urine samples. (C) 2018 Elsevier Ltd. All rights reserved.

  • 38.
    Watanabe, Shimpei
    et al.
    UTS, Australia.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Roman, Markus
    National Board Forens Med, Department Forens Genet and Forens Toxicol, S-58758 Linkoping, Sweden.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. National Board Forens Med, Department Forens Genet and Forens Toxicol, S-58758 Linkoping, Sweden.
    Kronstrand, Robert
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. National Board Forens Med, Department Forens Genet and Forens Toxicol, S-58758 Linkoping, Sweden.
    Wohlfarth, Ariane
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. National Board Forens Med, Department Forens Genet and Forens Toxicol, S-58758 Linkoping, Sweden.
    In Vitro and In Vivo Metabolite Identification Studies for the New Synthetic Opioids Acetylfentanyl, Acrylfentanyl, Furanylfentanyl, and 4-Fluoro-Isobutyrylfentanyl2017In: AAPS Journal, ISSN 1550-7416, E-ISSN 1550-7416, Vol. 19, no 4, p. 1102-1122Article in journal (Refereed)
    Abstract [en]

    New fentanyl analogs have recently emerged as new psychoactive substances and have caused numerous fatalities worldwide. To determine if the new analogs follow the same metabolic pathways elucidated for fentanyl and known fentanyl analogs, we performed in vitro and in vivo metabolite identification studies for acetylfentanyl, acrylfentanyl, 4-fluoro-isobutyrylfentanyl, and furanylfentanyl. All compounds were incubated at 10 mu M with pooled human hepatocytes for up to 5 h. For each compound, four or five authentic human urine samples from autopsy cases with and without enzymatic hydrolysis were analyzed. Data acquisition was performed in data-dependent acquisition mode during liquid chromatography high-resolution mass spectrometry analyses. Data was analyzed (1) manually based on predicted biotransformations and (2) with MetaSense software using data-driven search algorithms. Acetylfentanyl, acrylfentanyl, and 4-fluoro-isobutyrylfentanyl were predominantly metabolized by N-dealkylation, cleaving off the phenethyl moiety, monohydroxylation at the ethyl linker and piperidine ring, as well as hydroxylation/methoxylation at the phenyl ring. In contrast, furanylfentanyls major metabolites were generated by amide hydrolysis and dihydrodiol formation, while the nor-metabolite was minor or not detected in case samples at all. In general, in vitro results matched the in vivo findings well, showing identical biotransformations in each system. Phase II conjugation was observed, particularly for acetylfentanyl. Based on our results, we suggest the following specific and abundant metabolites as analytical targets in urine: a hydroxymethoxy and monohydroxylated metabolite for acetylfentanyl, a monohydroxy and dihydroxy metabolite for acrylfentanyl, two monohydroxy metabolites and a hydroxymethoxy metabolite for 4-fluoro-isobutyrylfentanyl, and a dihydrodiol metabolite and the amide hydrolysis metabolite for furanylfentanyl.

  • 39.
    Wohlfarth, Ariane
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758 Linköping, Sweden.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Roman, Markus
    Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758 Linköping, Sweden.
    Andersson, Mikael
    Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758 Linköping, Sweden.
    Kugelberg, Fredrik C
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758 Linköping, Sweden.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758 Linköping, Sweden.
    Kronstrand, Robert
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Department of Forensic Genetics and Forensic Toxicology, National Board of Forensic Medicine, 58758 Linköping, Sweden.
    Looking at flubromazolam metabolism from four different angles: Metabolite profiling in human liver microsomes, human hepatocytes, mice and authentic human urine samples with liquid chromatography high-resolution mass spectrometry.2017In: Forensic Science International, ISSN 0379-0738, E-ISSN 1872-6283, Vol. 274, p. 55-63Article in journal (Refereed)
    Abstract [en]

    Flubromazolam is a triazolam benzodiazepine that recently emerged as a new psychoactive substance. Since metabolism data are scarce and good analytical targets besides the parent are unknown, we investigated flubromazolam metabolism in vitro and in vivo. 10μmol/L flubromazolam was incubated with human liver microsomes for 1h and with cryopreserved human hepatocytes for 5h. Mice were administered 0.5 or 1.0mg flubromazolam/kg body weight intraperitoneally, urine was collected for 24h. All samples, together with six authentic forensic human case specimens, were analyzed (with or without hydrolysis, in case it was urine) by UHPLC-HRMS on an Acquity HSS T3 column with an Agilent 6550 QTOF. Data mining was performed manually and with MassMetasite software (Molecular Discovery). A total of nine metabolites were found, all generated by hydroxylation and/or glucuronidation. Besides O-glucuronidation, flubromazolam formed an N(+)-glucuronide. Flubromazolam was not metabolized extensively in vitro, as only two monohydroxy metabolites were detected in low intensity in hepatocytes. In the mice samples, seven metabolites were identified, which mostly matched the metabolites in the human samples. However, less flubromazolam N(+)-glucuronide and an additional hydroxy metabolite were observed. The six human urine specimens showed different extent of metabolism: some samples had an intense flubromazolam peak next to a minute signal for a monohydroxy metabolite, others showed the whole variety of hydroxylated and glucuronidated metabolites. Overall, the most abundant metabolite was a monohydroxy metabolite, which we propose as α-hydroxyflubromazolam based on MSMS fragmentation. These metabolism data will assist in interpretation and analytical method development.

  • 40.
    Zetterlund, Eva-Lena
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Oscarsson, Anna
    Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Anaesthesiology and Intensive Care in Norrköping. Linköping University, Department of Medical and Health Sciences, Division of Drug Research.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Vrethem, Magnus
    Linköping University, Department of Clinical and Experimental Medicine, Division of Neuro and Inflammation Science. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Local Health Care Services in Central Östergötland, Department of Neurology.
    Lindholm, Maj-Lis
    Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Anaesthesiology and Intensive Care in Linköping. Kalmar Hospital, Sweden.
    Eintrei, Christina
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Region Östergötland, Anaesthetics, Operations and Specialty Surgery Center, Department of Anaesthesiology and Intensive Care in Linköping.
    Determination of loss of consciousness: a comparison of clinical assessment, bispectral index and electroencephalogram: An observational study2016In: European Journal of Anaesthesiology, ISSN 0265-0215, E-ISSN 1365-2346, Vol. 33, no 12, p. 922-928Article in journal (Refereed)
    Abstract [en]

    BACKGROUNDComputer-processed algorithms of encephalographic signals are widely used to assess the depth of anaesthesia. However, data indicate that the bispectral index (BIS), a processed electroencephalography monitoring system, may not be reliable for assessing the depth of anaesthesia.OBJECTIVEThe aim of this study was to evaluate the ability of the BIS monitoring system to assess changes in the level of unconsciousness, specifically during the transition from consciousness to unconsciousness, in patients undergoing total intravenous anaesthesia with propofol. We compared BIS with the electroencephalogram (EEG), and clinical loss of consciousness (LOC) defined as loss of verbal commands and eyelash reflex.DESIGNThis was an observational cohort study.SETTINGUniversity Hospital Linkoping, University Hospital orebro, Finspang Hospital and Kalmar Hospital, Sweden from October 2011 to April 2013.PATIENTSA total of 35 ASA I patients aged 18 to 49 years were recruited.INTERVENTIONSThe patients underwent total intravenous anaesthesia with propofol and remifentanil for elective day-case surgery. Changes in clinical levels of consciousness were assessed by BIS and compared with assessment of stage 3 neurophysiological activity using the EEG. The plasma concentrations of propofol were measured at clinical LOC and 20 and 30min after LOC.MAIN OUTCOME MEASURESThe primary outcome was measurement of BIS, EEG and clinical LOC.RESULTSThe median BIS value at clinical LOC was 38 (IQR 30 to 43), and the BIS values varied greatly between patients. There was no correlation between BIS values and EEG stages at clinical LOC (r=-0.1, P=0.064). Propofol concentration reached a steady state within 20min.CONCLUSIONThere was no statistically significant correlation between BIS and EEG at clinical LOC. BIS monitoring may not be a reliable method for determining LOC.CLINICAL TRIALS REGISTRYThis trial was not registered because registration was not mandatory at the time of the trial.

  • 41.
    Åstrand, Anna
    et al.
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences.
    Toreskog, Amanda
    Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Watanabe, Shimpei
    Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Kronstrand, Robert
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Linkoping, Sweden.
    Correlations between metabolism and structural elements of the alicyclic fentanyl analogs cyclopropyl fentanyl, cyclobutyl fentanyl, cyclopentyl fentanyl, cyclohexyl fentanyl and 2,2,3,3-tetramethylcyclopropyl fentanyl studied by human hepatocytes and LC-QTOF-MS2019In: Archives of Toxicology, ISSN 0340-5761, E-ISSN 1432-0738, Vol. 93, no 1, p. 95-106Article in journal (Refereed)
    Abstract [en]

    Recently, a number of fentanyl analogs have been implicated in overdose deaths in Europe and in the US. So far, little is known of the molecular behavior of the structurally related subgroup; the alicyclic fentanyls. In this study, reference standards of cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, and 2,2,3,3-tetramethylcyclopropyl fentanyl (TMCPF) at a final concentration of 5 mu M were incubated with cryopreserved human hepatocytes (1x10(6) cells/mL) for 0, 1, 3 and 5h. The metabolites formed were identified by liquid chromatography-quadrupole time-of-flight mass spectrometry analysis. The most abundant biotransformation found was N-dealkylation (formation of normetabolites) and oxidation of the alicyclic rings. As ring size increased, the significance of N-dealkylation decreased in favor of alicyclic ring oxidation. An example of this was cyclopropyl fentanyl, with a three-carbon ring, whose normetabolite covered 82% of the total metabolic peak area and no oxidation of the alicyclic ring was observed. In contrast, TMCPF, with a seven-carbon ring structure, rendered as much as 85% of its metabolites oxidized on the alicyclic ring. Other biotransformations found included oxidation of the piperidine ethyl moiety and/or the phenethyl substructure, glucuronidation as well as amide hydrolysis to form metabolites identical to despropionyl fentanyl. Taken together, this study provides a base for understanding the metabolism of a number of structurally related fentanyl analogs formed upon intake.

  • 42.
    Åstrand, Anna
    et al.
    Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Artillerigatan 12, S-58758 Linkoping, Sweden.
    Vikingsson, Svante
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Artillerigatan 12, S-58758 Linkoping, Sweden.
    Lindstedt, Daniel
    Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Artillerigatan 12, S-58758 Linkoping, Sweden.
    Thelander, Gunilla
    Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Artillerigatan 12, S-58758 Linkoping, Sweden.
    Green, Henrik
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Artillerigatan 12, S-58758 Linkoping, Sweden.
    Kronstrand, Robert
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Artillerigatan 12, S-58758 Linkoping, Sweden.
    Wohlfarth, Ariane
    Linköping University, Department of Medical and Health Sciences, Division of Drug Research. Linköping University, Faculty of Medicine and Health Sciences. Natl Board Forens Med, Dept Forens Genet and Forens Toxicol, Artillerigatan 12, S-58758 Linkoping, Sweden.
    Metabolism study for CUMYL-4CN-BINACA in human hepatocytes and authentic urine specimens: Free cyanide is formed during the main metabolic pathway2018In: Drug Testing and Analysis, ISSN 1942-7603, E-ISSN 1942-7611, Vol. 10, no 8, p. 1270-1279Article in journal (Refereed)
    Abstract [en]

    To further elucidate the metabolism of CUMYL-4CN-BINACA, a new synthetic cannabinoid with a cyano group, and to evaluate biomarkers, we incubated the substance in human hepatocytes and analysed 9 authentic urine specimens. We also quantified CUMYL-4CN-BINACA and cyanide in blood and provide comprehensive data on the 7 autopsy cases, 5 of them determined CUMYL-4CN-BINACA intoxications. For metabolite elucidation, CUMYL-4CN-BINACA was incubated with pooled human hepatocytes for up to 5hours, urine samples were analysed with and without enzymatic hydrolysis. Data was acquired in data-dependent mode by ultra-high performance liquid chromatography-high resolution mass spectrometry (UHPLC-HRMS) with an Agilent 6550 QTOF. For quantitative analysis of CUMYL-4CN-BINACA, blood samples were precipitated and analysed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Cyanide was determined by gas chromatography-headspace-nitrogen phosphorus detection (GC-headspace-NPD). CUMYL-4CN-BINACA was metabolised via CYP450-mediated hydroxylation at 4-butyl position generating a cyanohydrin (M12), which releases free cyanide to form an aldehyde intermediate and eventually generates 4-hydroxybutyl CUMYL-BINACA (M11) and CUMYL-BINACA butanoic acid (M10). Other minor metabolites were produced by hydroxylation, dihydroxylation, N-dealkylation, and dihydrodiol formation; glucuronidation was observed. One urine sample showed high intensities of M10 and a wide variety of metabolites; the other samples contained fewer metabolites in low abundance and 1 sample showed no metabolites. CUMYL-4CN-BINACA blood concentrations ranged from 0.1 to 8.3ng/g showing an overlap between fatal and non-fatal concentrations. One blood sample contained 0.36g/g cyanide. Release of free cyanide during metabolism is worrying as it might induce liver toxicity. As suggested earlier, CUMYL-BINACA butanoic acid is the most abundant biomarker in urine, but monitoring of additional metabolites or, even better, analysis for the parent in blood is recommended.

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