POSTER SESSION - uu-pharmfaculty50years.seuu-pharmfaculty50years.se/wp-content/uploads/2018/10/Poster-session... · i FACULTY OF PHARMACY 50 YEARS POSTER SESSION 26 October 2018 Full

POSTER SESSION

POSTER SESSION

BOOK OF ABSTRACTS

Faculty of Pharmacy 50 Years

26 October 2018

POSTER SESSION

POSTER SESSION

BOOK OF ABSTRACTS

Faculty of Pharmacy 50 Years

26 October 2018

Current research at the Faculty of Pharmacy is in focus during the poster session.

The 58 posters on display demonstrate the multidisciplinary aspects of pharmaceutical sciences.

Posters are presented by the new generation of scientists.

the Anniversary Organisation Committee and the Dean of The Faculty of Pharmacy

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FACULTY OF PHARMACY 50 YEARS POSTER SESSION

26 October 2018

Full abstracts on QR:

1. THE ADDUCT FORMATION IN HILIC-ESI-MS IS STRONGLY AFFECTED BY THE INORGANIC ION CONCENTRATION OF THE SAMPLES. I. Erngren, J. Haglf, M. Engskog, T. Arvidsson, C. Pettersson. ida.erngren@ilk.uu.se 2. IN VITRO LIPOLYSIS-PERMEATION ASSAY REFLECTS FENOFIBRATE ABSORPTION FROM LIPID-BASED FORMULATIONS IN PIGS. Janneke Keemink, Elin Mrtensson, Christel A.S. Bergstrm. janneke.keemink@farmaci.uu.se 3. SYNTHESIS OF SULFONIMIDAMIDE (SIA) BASED AMINO ACID BUILDING BLOCKS. Praveen Kumar Chinthakindi, Andrea Benediktsdottir, Edouard Zamaratski, Per I Arvidsson, Yantao Chen, and Anja Sandstrm. praveen.chinthakindi@ilk.uu.se 4. INTEGRATION OF CLINICAL PHARMACOKINETICS AND PHARMACODYNAMICS OF CLOFAZIMINE AND PYRAZINAMIDE TO A MULTISTATE TUBERCULOSIS PHARMACOMETRIC MODEL: EXPLAINS AN UNEXPECTED OUTCOME IN PHASE IIA TRIAL. Alan Faraj, Robin J Svensson, Ulrika SH Simonsson. alan.faraj@farmbio.uu.se 5. ASYMMETRIC HINDLIMB MOTOR RESPONSE TO FOCAL TRAUMATIC BRAIN INJURY IS CONTROLLED BY SIDE-SPECIFIC OPIOID MECHANISM. H. Watanabe, M. Zhang (b,c), D. Sarkisyan, O. Kononenko, F. Clausen, T. Iakovleva, N. Marklund and G. Bakalkin. olga.kononenko@farmbio.uu.se 6. ADVANCED BIOMARKER DISCOVERY THROUGH INVESTIGATION OF GUT MICROBIOTA AND HUMAN HOST CO-METABOLISM - LINKING METABOLOMICS WITH CHEMICAL BIOLOGY METHODOLOGIES. Mrio S. P. Correia, Caroline Ballet, Louis P. Conway, Neeraj Garg, Daniel Globisch (a, b). mario.correia@ilk.uu.se 7. DEEP-SEA, DEEP SEQUENCING: WGS OF THE DEMOSPONGE GEODIA BARRETTI. Karin Steffen and Paco Crdenas. karin.steffen@ilk.uu.se 8. BORONIC ESTER MACROCYCLES AS NEW E.COLI TYPE I SIGNAL PEPTIDASE INHIBITORS. Natalia Szaaj, Lu Lu, Andrea Benediktsdottir, Edouard Zamaratski, Sha Cao c, Gustav Olanders, Charles Hedgecock, Anders Karln, Mt Erdlyi, Diarmaid Hughes, Sherry L. Mowbray, Peter Brandt. andrea.benediktsdottir@ilk.uu.se 9. INVESTIGATION OF PULMONARY DISSOLUTION OF LOW SOLUBILITY DRUG USING AN EX VIVO LUNG MODEL AND COMPUTATIONAL ANALYSIS. J. Eriksson, H. Thrn, E. Sjgren, L. Holmstn, K. Rubin, H. Lennerns. johanna.eriksson@farmaci.uu.se 10. LANSOPRAZOLE PRE-TREATMENT INCREASES CYTOTOXICITY OF NANOFORMULATION (DOXIL) IN 2D CELL MODEL. F. Kullenberg (a,), O. Degerstedt, N. Pavlovic, F. Heindryckx, and H. Lennerns. fredrik.kullenberg@farmaci.uu.se 11. A MICROSERVICE-BASED INFRASTRUCTURE FOR LIGAND-BASED MODELING. Jonathan Alvarsson, B Ernst Ahlberg Helgee, A. Arvid Berg, A. Ola Spjuth. jonathan.alvarsson@farmbio.uu.se 12. A HIGH-THROUGHPUT METHOD TO PREDICT THE IN VIVO EXPOSURE OF DIFFERENT DRUG DELIVERY SYSTEMS. C. Alvebratt, J. Keemink, K. Edueng, O. Cheung, M. Strmme, C. A.S. Bergstrm. caroline.alvebratt@farmaci.uu.se 13. SYNERGY CONFORMAL PREDICTION. Niharika Gauraha, Ola Spjuth. niharika.gauraha@farmbio.uu.se 14. ISOLATION AND IDENTIFICATION OF THE MAIN STEROL FROM THE SPONGE HALICLONA SP. FROM TAIWAN. Merran Dunford (a,b), Sanjeevan Ranjendran, Patric Jonsson, Ping-Jyun Sung, Fang-Rong Chang, Achyut Adhikari, Sunithi Gunasekera, Paco Crdenas. paco.cardenas@ilk.uu.se

15. A DIGESTION/PERMEATION MODEL BASED ON ARTIFICIAL MEMBRANES FOR STUDIES OF PERFORMANCE OF LIPID-BASED FORMULATIONS. O. J. Hedge, C. A. S. Bergstrm. oliver.hedge@farmaci.uu.se 16. MAPPING THE DISTRIBUTION OF NEUROPEPTIDES THROUGH MASS SPECTROMETRY IMAGING. Heather E Hulme a, Halla Gunnarsdttir a, Elva Fridjonsdottir a, Anna Nilsson a, Mohammadreza Shariatgorji a, Erwan Bezard b, Per E. Andrn a. heather.hulme@farmbio.uu.se 17. DOCKING OF MACROCYCLES: COMPARING RIGID AND FLEXIBLE DOCKING IN GLIDE. Hiba Alogheli, Gustav Olanders, Wesley Schaal, Peter Brandt, Anders Karln. gustav.olanders@ilk.uu.se 18. HOME VS. SELF-INITIATED ART REFILL: CLINICAL, IMMUNOLOGICAL, AND VIROLOGIC OUTCOMES. Rory F. Leisegang, Jane Ball, Mark Cotton, David Dowdy, Keri Calkins, Susan Cleary, Gary Maartens, Jean B. Nachega. rory.leisegang@farmbio.uu.se 19. CHEMOSELECTIVE PROBES FOR THE DETECTION OF MICROBIAL BIOMARKERS OF DISEASE - COMBINING METABOLOMICS WITH CHEMICAL BIOLOGY METHODOLOGIES. Neeraj Garg, Louis P. Conway, Caroline Ballet, Mrio S. P. Correia, Daniel Globisch.. louis.conway@ilk.uu.se 20. LCMS-BASED UNTARGETED METABOLOMICS USING GUINEA PIG PERILYMPH: HIGHLIGHTING DIFFERENCES IN METABOLOME WITH AND WITHOUT A POSTERIORI HYDROGEN GAS ADMINISTRATION FOLLOWING LOUD NOISE EXPOSURE. K. Pirttil, A. E. Fransson, J. Haglf, M. Engskog, P. V. Pierre, G. Laurell, T. Arvidsson (a, d), C. Pettersson. kristian.pirttila@ilk.uu.se 21. POPULATION MODELING OF UNI-AND THREE-DIMENSIONAL AND DENSITY-BASED TUMOR MEASUREMENTS IN GASTRO-INTESTINAL STROMAL TUMOR PATIENTS TREATED WITH IMATINIB. Sreenath M. Krishnan, Emilie Schindler, Gaia Schiavon (b,c), Ron Mathijssen, Lena E. Friberg. sreenath.krishnan@farmbio.uu.se 22. POPULATION PHARMACOKINETIC ANALYSIS OF FACTOR VIII ACTIVITY FOLLOWING TREATMENT WITH MOROCTOCOG ALFA IN MODERATE TO SEVERE HAEMOPHILIA A SUBJECTS. Joo A. Abrantes, Elisabet I. Nielsen, Joan Korth-Bradley, Lutz Harnisch, Siv Jnsson. joao.abrantes@farmbio.uu.se 23. PROBABILISTIC PREDICTIONS OF METABOLISM USING VENN-PREDICTORS. Staffan Arvidsson Mc Shane, Lars Carlsson, Paolo Toccaceli, Ola Spjuth. staffan.arvidsson@farmbio.uu.se 24. CONTRALESIONAL HINDLIMB MOTOR RESPONSE INDUCED BY UNILATERAL BRAIN INJURY: EVIDENCE FOR EXTRA- SPINAL MECHANISM. N. Lukoyanov, L. Carvalho, H. Watanabe, M. Zhang (c, d), D. Sarkisyan, O. Kononenko, I. Bazov, T. Iakovleva, J. Schouenborg and G. Bakalkin. olga.kononenko@farmbio.uu.se 25. EXPLORING THE USEFULNESS OF MORPHOLOGICAL PROFILING OF CELLS TO STUDY TOXICITY MECHANISMS. P. Georgieva, Tanya Aggarwal, W. Schaal, O. Spjuth. polina.georgiev@farmbio.uu.se 26. EXPLORING THE USEFULNESS OF MORPHOLOGICAL PROFILING OF CELLS TO STUDY TOXICITY MECHANISMS. P. Georgieva, T. Aggarwal, W. Schaal, O. Spjuth. tanya.aggarwal@farmbio.uu.se 27. DISCREPANCY IN STABLY EXPRESSED PROTEINS WITHIN AND ACROSS HUMAN TISSUE TYPES. C. Wegler (a,b), M. lander, P. Artursson. christine.wegler@farmaci.uu.se 28. EARLY-LIFE EXPOSURE TO TO AN ALGAL NEUROTOXIN INDUCES LONG-TERM CEREBELLAR NEURODEGENERATION AND LYSOSOMAL CHANGES. J Swan, L Ersson, A-L Berg, O Karlsson(a,c), E Brittebo. julia.swan@farmbio.uu.se 29. MASS SPECTROMETRY LIPIDOMICS: A PLATFORM TO UNRAVEL LIPID BIOLOGY. David Balgoma, Alfhild Grnbladh, Sofia Zelleroth, Mathias Hallberg, Curt Petterson, Mikael Hedeland. david.balgoma@ilk.uu.se 30. DISCOVERY AND CHARACTERIZATION OF SMALL PEPTIDE TOXINS FROM THE EPIDERMAL MUCUS OF LINEUS LONGISSIMUS, THE LONGEST ANIMAL ON EARTH. Erik Jacobsson, Hkan S. Andersson, Malin Strand, Steve Peigneur, Eline K. M. Lebbe, K. Johan Rosengren, Jan Tytgat , Ulf Gransson. erik.jakobsson@ilk.uu.se 31. CLINICAL METABOLOMICS: THE STETHOSCOPE FOR THE 21ST CENTURY? Stephanie Hermana (a,b), Payam Emami Khoonsari, Valter Niemel, Torbjrn kerfeldta, Ola

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Spjuth, Joachim Burman, Kim Kultima. stephanie.herman@medsci.uu.se 32. SINGLE BEAD INVESTIGATION OF A CLINICAL DRUG DELIVERY SYSTEM. E. Ahnfelt, J. Gernandt, Y. Al-Tikriti, E. Sjgren, H. Lennerns, and P. Hansson. emelie.ahnfelt@farmaci.uu.se 33. CLASSIFICATION OF DRUG INDUCED TOXICOLOGY IN TISSUE SAMPLES USING MALDI MASS SPECTROMETRY IMAGING. A. Nilsson, R.J. Goodwin, B.Forngren, S. Iverson, J. Lindberg, P.E. Andrn. anna.nilsson@farmbio.uu.se 34. SCILIFELAB DRUG DISCOVERY AND DEVELOPMENT PLATFORM- MEDICINAL CHEMISTRY. U. Yngve and Y. Gravenfors. ulrika.yngve@scilifelab.uu.se 35. ELEVATED BRAIN LEVELS OF L-DOPA AND STRUCTURE SPECIFIC CHANGES IN DOPAMINE METABOLISM IN L-DOPA INDUCED DYSKINESIA REVEALED BY MASS SPECTROMETRY IMAGING. Elva Frijnsdttir, Mohammadreza Shariatorji, Anna Nilsson, Theodosia Vallianatou, Per Svenningsson, Erwan Bezard, Per E. Andrn. elva.fridjonsdottir@farmbio.uu.se 36. A MASS SPECTROMETRY IMAGING APPROACH FOR INVESTIGATING HOW DRUG-DRUG INTERACTIONS INFLUENCE DRUG BLOOD-BRAIN BARRIER PERMEABILITY. Theodosia Vallianatou, Nicole Strittmatter, Anna Nilsson, Mohammadreza Shariatgorji, Gregory Hamm, Marcela Pereira, Patrik Kllback, Per Svenningsson, Maria Karlgren, Richard J.A. Goodwin, A. Per E. Andrn. theodosia.vallianatou@farmbio.uu.se 37. ANABOLIC ANDROGENIC STEROIDS INDUCE TOXIC EFFECTS IN RAT PRIMARY CORTICAL CELL CULTURES. Sofia Zelleroth, Erik Nylander, Fred Nyberg, Alfhild Grnbladh, Mathias Hallberg. sofia.zelleroth@farmbio.uu.se 38. CAUSAL FACTORS FOR POST-CRYOPRESERVATION VARIABILITY IN ATTACHMENT AND CULTURE MORPHOLOGY OF PRIMARY HUMAN HEPATOCYTES. M. lander, J.R. Wisniewski, I. Flrkemeier, A. Norn, and P. Artursson. magnus.olander@farmaci.uu.se 39. COMPLETE KNOCKOUT OF ENDOGENOUS MDR1 (ABCB1) IN MDCK WILDTYPE AND MDCK-HMDR1 CELLS BY CRISPR-CAS9. M. Karlgren (a, b), I. Simoff, M. Backlund (a, b), C. Wegler (a, c), M. Keiser, N. Handin, J. Mller, P. Lundquist, A-C. Jareborg, S. Oswald, P. Artursson (a, b). maria.karlgren@farmaci.uu.se 40. TARGETED BRAIN DELIVERY OF METHOTREXATE BY GLUTATHIONE PEGYLATED LIPOSOMES: HOW CAN THE FORMULATION MAKE A DIFFERENCE? Yang Hu, Pieter J. Gaillard, Elizabeth C.M. de Lange, and Margareta Hammarlund-Udenaes. yang.hu@farmbio.uu.se 41. MAPPING THE BRAIN NEUROTRANSMITTER NETWORK WITH MALDI MASS SPECTROMETRY IMAGING. M. Shariatgorji, A. Nilsson, L. Odell, E. Fridjonsdottir, T. Vallianatou, L. Katan, J. Svmarker, E. Bezard, P. Svenningsson, P. E. Andren. reza.shariatgorji@farmbio.uu.se 42. THE INNATE/ADAPTIVE IMMUNE RESPONSE TRIGGERED UPON LOCAL IMMUNOTHERAPY OF ORTHOTOPICALLY GROWING BLADDER CANCER TUMORS. Iliana K. Kerzeli, Andrea Kurtinovic, Aikaterini Nasi, Ivan Stepnek, Martin Lord, Sara M. Mangsbo. iliana.kerzeli@farmbio.uu.se 43. THE UPPSALA UNIVERSITY DRUG OPTIMIZATION AND PHARMACEUTICAL PROFILING FACILITY (UDOPP) EMERGING NATIONAL INFRASTRUCTURE FOR ADME INVESTIGATIONS IN SWEDEN. Pawel Baranczewski, Maria Backlund, Maria Karlgren, Shibu Krishnan, Annika Lindqvist, Maria Mastej, Aljona Saleh, Ivailo Simoff, Richard Svensson and Per Artursson. pawel.baranczewski@farmaci.uu.se 44. METABOLIC CONTROL OF DENDRITIC CELL IMMUNOGENICITY. Aikaterini Nasi (a.b), Vishnu Priya Bollampalli, Meng Sun, Yang Chen, Sylvie Amu, Susanne Nyln, Liv Eidsmo, Antonio Gigliotti Rothfuchs, Bence Rthi. aikaterini.nasi@farmbio.uu.se 45. STABILIZED CYCLIC PEPTIDES AS SCAVENGERS OF AUTOANTIBODIES: NEUTRALIZATION OF ANTI-CITRULLINATED PROTEIN/PEPTIDE ANTIBODIES IN RHEUMATOID ARTHRITIS. Sunithi Gunasekera, Ctia Fernandes-Cerqueira, Stefan Wennmalm, Heidi Whmaa, Yngve Sommarin, Anca I. Catrina, Per-Johan Jakobsson, Ulf Gransson. sunithi.gunasekera@ilk.uu.se 46. PHOSPHOLIPID BUT NOT NEUTRAL LIPID CONTENT IS A MAJOR DETERMINANT OF INTRACELLULAR BINDING OF DRUGS. Andrea Treyer,

Andr Mateus, Hinnerk Boriss, Pr Matsson, Per Artursson. andrea.treyer@farmaci.uu.se 47. THE PROTEOME OF PRIMARY HUMAN HEPATOCYTE SPHEROIDS CULTURED AND USED IN HIGH- THROUGHPUT SCREENING FORMAT. Niklas HANDIN, Wojciech SENKOWSKI, Magnus LANDER, Frida NYBERG, Mrten FRYKNS, Per ARTURSSON. Niklas.Handin@farmaci.uu.se 48. TRANSLATING LONG-READ SEQUENCING TO THE CLINIC: A CASE STUDY USING BCR-ABL1 MUTATION SCREENING IN CHRONIC MYELOID LEUKEMIA. Wesley Schaal, Adam Ameur (b,c,) Ulla Olsson-Strmberg, Lucia Cavelier, Ola Spjuth (a,c). wesley.schaal@farmbio.uu.se 49. PALLADIUM(0)-CATALYZED CARBONYLATIVE SYNTHESIS OF N-ACYLSULFONAMIDES USING MOLYBDENUM HEXACARBONYL AS A SOLID CARBON MONOXIDE SOURCE. Luke S. Schembri, Luke R. Odell. luke.schembri@ilk.uu.se 50. TRANSFORMATION OF KR-12 DERIVED CROSS-LINKED CYCLIC DIMERS INTO STABLE AND POTENT ANTIMICROBIAL DRUG LEADS. Taj Muhammad, Adam A. Strmstedt,, Sunithi Gunasekera, Ulf Gransson. taj.muhammad@fkog.uu.se 51. TIME-DEPENDENT EFFECTS ON SMALL INTESTINAL TRANSPORT BY ABSORPTION-MODIFYING EXCIPIENTS. Dahlgren, David; Roos, Carl; Tannergren, Christer4; Lundqvist, Anders; Sjblom, Markus; Sjgren, Erik; Lennerns, Hans. david.dahlgren@farmaci.uu.se 52. EFFECT OF ABSORPTION-MODIFYING EXCIPIENTS, HYPOTONICITY, AND ENTERIC NEURAL ACTIVITY IN AN IN VIVO MODEL FOR SMALL INTESTINAL TRANSPORT. Dahlgren, David1; Roos, Carl; Tannergren, Christer; Lundqvist, Anders; Sjblom, Markus; Sjgren, Erik; Lennerns, Hans. david.dahlgren@farmaci.uu.se 53. THE EFFECTS OF THREE ABSORPTION-MODIFYING CRITICAL EXCIPIENTS ON THE IN VIVO INTESTINAL ABSORPTION OF SIX MODEL COMPOUNDS IN RATS AND DOGS. Dahlgren, David; Roos, Carl; Johansson, Pernilla; Tannergren, Christer; Lundqvist, Anders; Langguth, Peter; Sjblom, Markus; Sjgren, Erik; Lennerns, Hans. david.dahlgren@farmaci.uu.se 54. PRECLINICAL EFFECT OF ABSORPTION-MODIFYING EXCIPIENTS ON RAT INTESTINAL TRANSPORT OF MODEL COMPOUNDS AND THE MUCOSAL BARRIER MARKER 51CR-EDTA. Dahlgren, David; Roos, Carl; Tannergren, Christer; Lundqvist, Anders; Langguth, Peter; Sjblom, Markus; Sjgren, Erik; Lennerns, Hans. david.dahlgren@farmaci.uu.se 55. TRANSFER LEARNING WITH DEEP CONVOLUTIONAL NEURAL NETWORKS FOR CLASSIFYING CELLULAR MORPHOLOGICAL CHANGES. Alexander Kensert, Philip J Harrison, Ola Spjuth. ola.spjuth@farmbio.uu.se 56. SUCCESSFUL METHOD VALIDATION AND ABSOLUTE QUANTITATION BY MALDI MASS SPECTROMETRY IMAGING. Patrik Kllback, Theodosia Vallianatou, Anna Nilsson, Mohammadreza Shariatgorji, Nicoletta Schintu, Marcela Pereira, Florian Barr, Henrik Wadensten, Per Svenningsson and Per E. Andrn. patrik.kallback@farmbio.uu.se 57. JEJUNAL ABSORPTION OF APREPITANT FROM SUSPENSIONSIN RAT: ROLE OF PARTICLE SIZE, PRANDIAL STATE AND MUCUS LAYER. Carl Roos, David Dahlgren, Erik Sjgren, Markus Sjblom, Mikael Hedeland, Hans Lennerns. carl.roos@farmaci.uu.se 58. MECHANISTIC MODELLING OF INTESTINAL DRUG ABSORPTION THE IN VIVO EFFECTS OF NANOPARTICLES, HYDRODYNAMICS, AND COLLOIDAL STRUCTURES. Carl Roos, Jan Westergren, David Dahlgren, Hans Lennerns, Erik Sjgren. carl.roos@farmaci.uu.se 59. DEVELOPMENT OF SCAFFOLD-BASED PEPTIDE SCAVENGERS FOR INHIBITION OF AUTOANTIBODIES IN RHEUMATOID ARTHRITIS. Camilla Eriksson, Sunithi Gunasekera, Ctia Fernandes-Cerqueira, Per-Johan Jakobsson, Ulf Gransson. Camilla.Eriksson@fkog.uu.se HTTP://UU-PHARMFACULTY50YEARS.SE/WP-CONTENT/UPLOADS/2018/10/POSTER-SESSION-BOOK-OF-ABSTRACT.PDF

Poster session Faculty of Pharmacy 50 Year Anniversary

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THE ADDUCT FORMATION IN HILIC-ESI-MS IS STRONGLY AFFECTED BY THE INORGANIC ION CONCENTRATION OF THE SAMPLES

I. Erngren (a), J. Haglf (a), M. Engskog (a), T. Arvidsson (a,b), C. Pettersson (a)

ida.erngren@ilk.uu.se

(a) Department of Medicinal Chemistry, Uppsala University, (b) Medical Product Agency, Uppsala Introduction. In metabolomics electrospray-mass spectrometry (ESI-MS) hyphenated with liquid chromatography (LC) is one of the most commonly used techniques. However, in ESI-MS adduct formation is very common and often seen as a major pitfall which lead to complicated data sets and sensitivity loss where metabolites are represented by several different adducts. Adding to this complexity, inorganic ions such as sodium and potassium show retention similar to many metabolites in hydrophilic interaction liquid chromatography (HILIC) leading to co-elution of metabolites and inorganic ions which could affect the adduct formation of these metabolites. The correlation between metabolite adduct formation and co-elution with sodium and potassium was studied by looking at trends between adduct formation and retention of metabolites in cell-samples analysed by HILIC-ESI-QTOF-MS. Methods. Head and neck cancer cells were cultivated and harvested, the intracellular metabolome was extracted using chloroform:methanol:water 4:4:2.85. The aqueous phase was isolated and analysed using an Acquity UPLC I-class system from Waters (Manchester, UK) coupled to a G2S Synapt Q-TOF equipped with an electro spray ionization (ESI) source (Waters). The separation was performed on an Acquity UPLC BEH Amide Column (1.7 M, 2.1x50 mm) from Waters and the detection was performed in positive ionization mode using data independent acquisition, MSE. The adduct formation of forty annotated metabolites was investigated, correlation between the types of adducts, retention time and co-elution with sodium and potassium formate clusters was studied. Preliminary data. It was found that sodium and potassium adducts were formed in much higher abundance for those metabolites that co-eluted with sodium and potassium formate clusters. In the retention time periods of sodium cluster (RT=8-9 min) and potassium cluster (RT= 7-8 min) almost all metabolites were detected as sodium or potassium adducts respectively. To test if the adduct formation was a result of the different metabolites structures; since both retention and adduct formation is dependent upon molecule structure or if it in fact was the co-elution of metabolites and alkali metal ions that affected the adduct formation, post-column infusion experiments were carried out. Ten metabolites were infused post-column while standard solutions with varying concentrations of sodium and potassium formate (1 - 100mM) was injected. The adduct formation of the infused metabolites was tracked during the elution of sodium and potassium. It was found that the adduct formation of the infused metabolites were strongly affected by the elution of sodium and potassium. The enhancement of the sodium and potassium adducts were as high as 8106% as compared to a solvent blank injection for some metabolites such as phenylalanine and guanosine. While the suppression of the [M+H] adduct was as high as 97% as compared to baseline in the same samples. The enhancements of the sodium and potassium adducts were not increased linearly with increasing sodium and potassium concentration. In an analytical point of view this renders several issues that need to be addressed, in relative quantitation where no internal standards are used, there is an obvious risk to use the adduct signals for quantitation since the differences might be a secondary effect of different levels of alkali metal ions in the samples. The adduct formation also yields numerous signals from each metabolite making data interpretation much more complicated. Novel aspect. The outcome of relative quantitations in MS could be strongly influenced by the sample alkali metal ion content.


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IN VITRO LIPOLYSIS-PERMEATION ASSAY REFLECTS FENOFIBRATE ABSORPTION FROM LIPID-BASED FORMULATIONS IN PIGS

Janneke Keemink, Elin Mrtensson, Christel A.S. Bergstrm

janneke.keemink@farmaci.uu.se

Department of Pharmacy, Uppsala University, Uppsala Biomedical Center, P.O. Box 580, SE-751 23 Uppsala, Sweden

Purpose. Lipid-based formulations (LBFs) are a delivery strategy to enhance intestinal absorption of poorly soluble compounds by keeping them solubilized during their transit in the gastrointestinal tract [1]. Unfortunately, in vitro lipolysis studies, typically used to evaluate performance of LBFs, do not accurately predict the in vivo behavior of LBFs [2]. One of the main reasons for this poor correlation is the absence of an absorption compartment in the in vitro lipolysis setup [1]. To better predict in vivo drug absorption from LBFs, we developed an in vitro method that simultaneously evaluates digestion and absorption. Methods. In vitro lipolysis was carried out in a lipolysis-permeation setup consisting of two chambers, representing a digestion and an absorption chamber, separated by a Caco-2 monolayer. Digestions of three fenofibrate-loaded-LBFs were performed with immobilized lipase. Samples were collected from both chambers to determine (i) fenofibrate solubility in the aqueous phase of the digestion medium and (ii) fenofibrate permeation across the Caco-2 monolayer. In vivo data from the literature were used to perform an in vitro-in vivo correlation [2]. Results and Discussion. Fenofibrate concentrations in the aqueous phase of the digestion medium predicted significantly higher exposure upon administration of type IIIA formulations than of the type IV formulation. However, mass transfer of fenofibrate was similar for the three formulations, thereby indicating that in vivo exposure of fenofibrate would also be similar. Conclusions. Drug transfer across the Caco-2 membrane accurately reflected in vivo drug exposure upon administration of three different LBFs loaded with fenofibrate. Acknowledgements. This work has received support from the European Research Council Grant 638965 and the Swedish Research Council Grant 2014-3309. References [1] O.M. Feeney et al. Adv. Drug Deliv. Rev. 101 (2016), [2] B.T. Griffin et al. Eur. J. Pharm. Biopharm. EV. 86 (2014) 427437.


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SYNTHESIS OF SULFONIMIDAMIDE (SIA) BASED AMINO ACID BUILDING BLOCKS

Praveen Kumar Chinthakindi (a), Andrea Benediktsdottir (a), Edouard Zamaratski (a), Per I Arvidsson

(c), Yantao Chen (b), and Anja Sandstrm (a) praveen.chinthakindi@ilk.uu.se

(a) Department of Medicinal Chemistry, Drug Design and Discovery, BMC, Uppsala University, Sweden. (b) Cardiovascular and Metabolic Diseases, IMED, AstraZeneca R&D, Gothenburg, Sweden(c) Medical

Biochemistry and Biophysics, Karolinska Institutet-Scilife Lab, Stockholm, Sweden Amide bond isosteres and unnatural amino acids are important building blocks in the development of pseudopeptides and peptidomimetics.[1] They can modulate a peptide physiochemical propertiesas well as selectivity against biological targets. Expanding on the recent tactical application of bioisosteres[2] i.e., sulfur-aza class of analogues,[3] Arvidsson group recently highlighted an emerging interest in sulfonimidamides (SIA) in contemporary drug design.[4] SIA is a chiral isostere of sulfonamide that offers number of advantages in drug design, for instance building highquality compound libraries.[5] SIAs have an extra "N" atom/handle in comparison with sulfonamide and imine N-substituents of sulfonimidamide are known to tune the physicochemical and biological properties.[6,7] Furthermore, in some cases single atom modifications are known to increase increase the activity of the parent molecule.[8] Therefore, the full potential of sulfonimidamides in regard to stability as well as biological activity should be further exploited in biologically relevant molecules. Acknowledgements. Anja Sandstrm gratefully acknowledges Kjell and Mrta Beijer Foundation for financial support. P. K. Chinthakindi acknowledges the Department of Medicinal Chemistry, Uppsala University, Sweden, for a postdoc fellowship. The authors wish to thank master students Ayah Ibrahim and Ataa Wared for their contributions. References: 1) E. Valeur, S. M. Guret, H. Adihou, R. Gopalakrishnan, M. Lemurell, H. Waldmann, T. N. Grossmann, A. T. Plowright, Angew. Chemie Int. Ed. 2017, 56, 1029410323. 2) N. A. Meanwell, J. Med. Chem. 2011, 54, 25292591. 3) U. Lcking, Angew. Chemie Int. Ed. 2013, 52, 93999408. 4) P. K. Chinthakindi, T. Naicker, N. Thota, T. Govender, H. G. Kruger, P. I. Arvidsson, Angew. Chemie Int. Ed. 2017, 56, 41004109. 5) F. W. Goldberg, J. G. Kettle, T. Kogej, M. W. D. Perry, N. P. Tomkinson, Drug Discov. Today 2015, 20, 1117. 6) L. D. Pennington, D. T. Moustakas, J. Med. Chem. 2017, 60, 35523579. 7) S. R. Borhade, R. Svensson, P. Brandt, P. Artursson, P. I. Arvidsson, A. Sandstrm, ChemMedChem 2015, 10, 455460. 8) D. L. Boger, J. Org. Chem. 2017, 82, 1196111980.


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INTEGRATION OF CLINICAL PHARMACOKINETICS AND PHARMACODYNAMICS OF CLOFAZIMINE AND PYRAZINAMIDE TO A MULTISTATE TUBERCULOSIS

PHARMACOMETRIC MODEL: EXPLAINS AN UNEXPECTED OUTCOME IN PHASE IIA TRIAL

Alan Faraj, Robin J Svensson, Ulrika SH Simonsson

alan.faraj@farmbio.uu.se

Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden Tuberculosis (TB) is the main cause of death, within infectious diseases. In a Phase IIa clinical trial aiming to scrutinize the early bactericidal activity (EBA) of clofazimine (CFZ) and pyrazinamide (PZA) monotherapy, no significant drug effects was observed. Despite being an important part of the sterilizing multi-drug standard treatment of tuberculosis, a paradoxical numeric increase of bacterial load was observed, post CFZ treatment. Using non-linear mixed effects modeling, we characterized statistically significant exposure-response relationships of both drugs, and discovered a quantitative model-based explanation for the paradoxical increase in colony forming units (CFU) after CFZ treatment. Simulations based on the final model suggested that a linearly concentration-dependent effect on dormant tubercular bacilli, explains the increase in utilized bacterial biomarker. Sensitive analysis tools are desired to reduce impact of noisy data, and to accurately explore drug effects to advance clinical trials. We propose that this quantitative semi-mechanistic approach to study drug effects, provides a rational framework for analysing Phase IIa EBA studies, and can accelerate anti-TB drug development. Acknowledgements. We express gratitude towards all patients that participated in the clinical trial, as well as the principal investigators and staff that executed the trial. We thank the authors of the original publication for sharing data in accordance with GDPR, and enabling this interdisciplinary work. Contributions: A.Faraj developed the PK/PD model and initiated the writing of the manuscript. AH Diacon was the principal investigator of the clinical trial and provided clinical expertise regarding scientific plausibility of modeling results. USH Simonsson was the principal expert in the modeling and simulation analysis work, providing quality assurance in the data analysis. RJ Svensson provided hands on support and supervised A. Faraj during the master thesis work that resulted in this work. 1. A. H. Diacon, R. Dawson, F. von Groote-Bidlingmaier, G. Symons, A. Venter, P. R. Donald, C. van Niekerk, D. Everitt, J. Hutchings, D. A. Burger, R. Schall, C. M. Mendel, Bactericidal activity of pyrazinamide and clofazimine alone and in combinations with pretomanid and bedaquiline, Am. J. Respir. Crit. Care Med. 191, 943953 (2015). 2. R. J. Svensson, S. H. Gillespie, U. S. H. Simonsson, Improved power for TB Phase IIa trials using a model-based pharmacokinetic-pharmacodynamic approach compared with commonly used analysis methods, J. Antimicrob. Chemother. 72, 23112319 (2017). 3. R. Svensson, U. Simonsson, Application of the Multistate Tuberculosis Pharmacometric Model in Patients With RifampicinTreated Pulmonary Tuberculosis, CPT Pharmacomet. Syst. Pharmacol. 5, 264273 (2016). 4. O. Clewe, L. Aulin, Y. Hu, A. R. M. Coates, U. S. H. Simonsson, A multistate tuberculosis pharmacometric model: a framework for studying anti-tubercular drug effects in vitro, J. Antimicrob. Chemother. 71, 964974 (2016).


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ASYMMETRIC HINDLIMB MOTOR RESPONSE TO FOCAL TRAUMATIC BRAIN INJURY IS CONTROLLED BY SIDE-SPECIFIC OPIOID MECHANISM

H. Watanabe (a), M. Zhang (b,c), D. Sarkisyan (a), O. Kononenko (a), F. Clausen (d), T. Iakovleva (a),

N. Marklund (d) and G. Bakalkin (a) olga.kononenko@farmbio.uu.se

(a) Dept. Pharmaceutical Biosciences, Uppsala University, Sweden, (b) Neuronano Research Center, Lund University, Sweden, (c) Dept. Molecular Medicine, University of Southern Denmark, Odense,

Denmark, (d) Dept. Neuroscience, Uppsala University, Sweden

Neuropeptides exert specific, coherent effects on formation and rewiring of neural circuits and, consequently, on behavior. Most neuropeptides are expressed in tiny neuronal subpopulations that are characterized by specific distribution patterns. We examined whether these patterns are flexible and maybe changed upon transition to a new physiologic state. For this purpose, controlled cortical impact injury (CCI) and sham operation (SO) as a control were applied to perturb endogenous opioid system (EOS) brain and spinal circuits. CCI produces generalized effects that result in impairment of multiple processes, including motor and sensory functions, emotions, and cognition, all of which are regulated by the EOS. The striking feature of this system is its asymmetric coexpression patterns, which suggest side-specific regulation of selective neural circuits by opioid neurohormone. The controlled cortical impact injury-induced postural asymmetry is encoded at the spinal level. Opioid receptors mediate formation of the asymmetry and determine the flexion side. Pharmacological correction with opioid antagonists - a strategy to reverse motor deficits. Acknowledgements. Drs. DeAnna Adkins, MUSC; V. Galatenko, I. Mityakina, A. Tonevitsky, MSU, Moscow; Nikolay Lukoyanov, Universidade do Porto, Portugal. Supported by the Swedish Science Research Council


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ADVANCED BIOMARKER DISCOVERY THROUGH INVESTIGATION OF GUT MICROBIOTA AND HUMAN HOST CO-METABOLISM -

LINKING METABOLOMICS WITH CHEMICAL BIOLOGY METHODOLOGIES

Mrio S. P. Correia (a), Caroline Ballet (a), Louis P. Conway (a), Neeraj Garg (a), Daniel Globisch (a, b)

mario.correia@ilk.uu.se

(a) Uppsala University, Uppsala, Sweden, (b) Science for Life Laboratories, Uppsala, Sweden The detailed investigation of metabolites in human samples (serum, plasma, urine, saliva, feces or tissues), termed metabolomics, carries a great potential for the discovery of unknown biomarkers. This is urgently required for diseases with advanced stage prognosis such as pancreatic cancer, one of the most lethal cancers worldwide with steadily increasing patient numbers. However, this is a challenging task as biological samples are comprised of a complex mixture of biomolecules. Advanced chemical biology tools are limited for this newest omics-research field. One of the most exciting scientific developments in the past decade has been the understanding that gut microbiota profoundly impact human physiology.1,2 The complex consortium of trillions of microbes possesses a wide range of metabolic activity. This metabolic interspecies communication represents a tremendous opportunity for biomarker discovery as only limited information on this co-metabolism has been elucidated on a molecular level.3 We have developed new chemical biology techniques for an enhanced metabolomics analysis using liquid chromatography-coupled with tandem mass spectrometry (UPLC-MS/MS). Our research is based on selective biochemical conversion of metabolites for qualitative and quantitative analysis through chemical synthesis of (isotope-labeled) reference molecules. We have combined these new methods with state-of-the-art mass spectrometric and bioinformatics techniques to enhance the scope of general metabolomics studies. Our approach represents a new and unique strategy for metabolite analysis and is focused on host-microbiota cometabolism. The detailed investigation of small molecule metabolites in human samples including feces, serum/plasma and urine, represents a high potential for discovery of unknown biomarkers. Application of our new metabolite analyzing methodologies at the interface of chemistry and biology led to the identification of a series of previously unknown metabolites using mass spectrometry-based metabolomics. (1) Turnbaugh, P. J. et al. A core gut microbiome in obese and lean twins. Nature 2009, 457 (7228), 480-4. (2) Liu, R. et al. Gut microbiome and serum metabolome alterations in obesity and after weight-lossintervention. Nat. Med. 2017, 23 (7), 859-868. (3) Wikoff, W. R et al Metabolomics analysis reveals large effects of gut microflora on mammalian blood metabolites. Proc. Natl. Acad. Sci. U. S. A. 2009, 106 (10), 3698-3703.


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DEEP-SEA, DEEP SEQUENCING: WGS OF THE DEMOSPONGE GEODIA BARRETTI

Karin Steffen and Paco Crdenas

karin.steffen@ilk.uu.se

Pharmacognosy, Department of Medicinal Chemistry, BMC, Uppsala University, Uppsala, Sweden

Sponges (phylum Porifera), among the oldest extant animals, are a poorly studied organismal group. However, the sponge fauna represents today one of the most diverse macrobenthic animal groups, more diverse than corals and probably as important as corals in the ecology of shallow and deep-sea ecosystems. Sponges also have one of the greatest potential in the rapidly expanding field of marine biotechnology and more specifically in the marine natural products research. With only a few whole genomes of shallow water species published so far, genomic data is truly lacking to investigate questions on deep-sea North Atlantic sponges. The aim of our project is therefore to sequence the whole genome of the deep-sea demosponge Geodia barretti (order Tetractinellida). With this new reference genome, we wish to answer questions on the sponge chemical potential and its natural products. In addition, this is also a unique opportunity to further elucidate early animal evolution.


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BORONIC ESTER MACROCYCLES AS NEW E.COLI TYPE I SIGNAL PEPTIDASE INHIBITORS

Natalia Szaaj (a), Lu Lu (b), Andrea Benediktsdottir (a), Edouard Zamaratski (a), Sha Cao c, Gustav

Olanders (a), Charles Hedgecock (a), Anders Karln (a), Mt Erdlyi (d), Diarmaid Hughes (c), Sherry L. Mowbray (b), Peter Brandt (a)

andrea.benediktsdottir@ilk.uu.se

(a) Department of Medicinal Chemistry, Uppsala University, Box 574, SE-751 23 Uppsala, Sweden. b) Department of Cell and Molecular Biology, Uppsala University, Box 596, SE-751 24 Uppsala, Sweden. c) Department of Medical Biochemistry and Microbiology, Uppsala University, Box 582, SE-75 123 Uppsala,

Sweden. d) Department of Chemistry, BMC, Uppsala University, SE-751 23 Uppsala, Sweden.

Type I signal peptidase, with its vital role in bacterial viability, is a promising but underexploited antibacterial drug target. In the light of steadily increasing rates of antimicrobial resistance, we have developed novel macrocyclic lipopeptides, linking P2 and P1' by a boronic ester warhead, capable of inhibiting Escherichia coli type I signal peptidase (EcLepB) and exhibiting good antibacterial activity. Structural modifications of the macrocyclic ring, the peptide sequence and the lipophilic tail led us to 14 novel macrocyclic boronic esters. It could be shown that macrocyclization is well tolerated in terms of EcLepB inhibition and antibacterial activity. Among the synthesized macrocycles, potent enzyme inhibitors in the low nanomolar range (e.g. compound 42f, EcLepB IC50 = 29 nM) were identified also showing good antimicrobial activity (e.g. compound 42b, E. coli WT MIC = 16 g/mL). The unique macrocyclic boronic esters described here were based on previously published linear lipopeptidic EcLepB inhibitors in an attempt to address cytotoxicity and hemolysis. We show herein that structural changes to the macrocyclic ring influence both the cytotoxicity and hemolytic activity suggesting that the P2 to P1' linker provide means for optimizing offtarget effects. However, for the present set of compounds we were not able to separate the antibacterial activity and cytotoxic effect.


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INVESTIGATION OF PULMONARY DISSOLUTION OF LOW SOLUBILITY DRUG USING AN EX VIVO LUNG MODEL AND COMPUTATIONAL ANALYSIS

J. Eriksson (a), H. Thrn(b), E. Sjgren (a), L. Holmstn (b), K. Rubin (c), H. Lennerns (a)

johanna.eriksson@farmaci.uu.se

a) Department of Pharmacy, Uppsala University, Uppsala, 75233, Sweden, b) Pharmaceutical Technology & Development Inhalation, AstraZeneca, Pepparedsleden 1, Mlndal, 43183, Sweden, c) Respiratory,

Inflammation & Autoimmunity, Innovative Medicines and Early Development, AstraZeneca, Pepparedsleden 1, Mlndal, 431 83, Sweden

BACKGROUND: Drug dissolution in the lungs is an important process when considering pulmonary drug delivery. Among inhaled drug compounds, a large number have low solubility; consequently, dissolution is the likely rate-limiting step in the overall absorption process1. In vivo predictive models, such as the isolated perfused lung (IPL) model,2, can be used to improve the understanding of pulmonary dissolution of inhaled drug compounds. AIMS: Using the IPL model and computational analysis, the objective of this study was to improve the understanding of dissolution processes in the lungs of four selected low solubility compounds (candidate drug 1 (CD1), budesonide, fluticasone furoate and fluticasone propionate) administered as suspensions and dry powder formulations. RESULTS: The dissolution in the lungs was the rate-limiting absorption parameter for drug transport in the IPL model for all compounds except budesonide and the small particle size of CD1. This is probably due to the high solubility of budesonide and the small particle size of CD1 which generates a large surface area promoting the dissolution rate. The dry powder of the larger particle size of CD1 had a four times slower dissolution than the corresponding suspension. The estimated dissolution constant and the drug transport in the IPL model ranked accordingly to the solubility in PBS of the compounds and there was a good agreement between ex vivo and in vitro dissolution constant. CONCLUSIONS: The dissolution in the IPL model is affected by the size of the drug particles, the solubility of the compound and the formulation properties. In addition, the IPL model in combination with computational analysis has been shown to be a useful tool when investigating pulmonary dissolution and to increase the understanding of dissolution related parameters for inhaled drug compounds. REFERENCES: [1] May, S., et al. Pharm Res 31, 3211-3224(2014) [2] Eriksson, J., et al. Eur J Pharm Biopharm 124, 1-12(2018)


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LANSOPRAZOLE PRE-TREATMENT INCREASES CYTOTOXICITY OF NANOFORMULATION (DOXIL) IN 2D CELL MODEL

F. Kullenberg (a,), O. Degerstedt (a), N. Pavlovic (b), F. Heindryckx (b), and H. Lennerns (a)

fredrik.kullenberg@farmaci.uu.se

(a) Department of Pharmacy, Uppsala University, Box 580, 751 23 Uppsala, Sweden (b) Department of Medical Cell Biology, Uppsala University, Box 571, Uppsala, Sweden

In recent years, countless nanoformulations of the cytostatic drug doxorubicin (DOX) have been developed with the aim of treating various recent cancers.[1] Doxil (named Caelyx in Europe), a liposomal formulation of DOX, was the first nanoformulation to receive regulatory approval in 1995 and is still one of the best example of successful clinical nanomedicines.[2] Much research has investigated the synergistic effects observed when giving a proton pump inhibitor (PPI) as a concomitant medication to conventional DOX treatment. Several clinical studies are ongoing and encouraging results have already been seen both in-vitro and in-vivo. [3] PPIs appear to increase the amount of DOX located in the cell nucleus, by increasing extracellular pH and thereby reducing endosomal sequestration of the drug, resulting in higher cytotoxicity. [4] However, very little is known about how PPI pre-treatment will affect uptake and distribution of nanoformulations with DOX in tumor cells. The primary objective of this study was to compare the effect of pre-treating cancer cells with different concentrations of a common proton pump inhibitor (PPI) before exposure to Doxil or DOX in solution. Though the objective was to compare the results in four different cell lines, this study was delayed due to contamination issues. As such, only the HCC cell line Huh7's results being shown in this poster. It is clear that the pre-treatment with 500 M PPI improves the effect of this DOX nanoformulation by more than one order of magnitude (p


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A MICROSERVICE-BASED INFRASTRUCTURE FOR LIGAND-BASED MODELING

Jonathan Alvarsson (a), B Ernst Ahlberg Helgee (b), A. Arvid Berg (a), A. Ola Spjuth (a) jonathan.alvarsson@farmbio.uu.se

(a) Department of Pharmaceutical Biosciences, Uppsala University, (b) Predictive Compound ADME &

Safety, Drug Safety & Metabolism, AstraZeneca IMED Biotech Unit

New data is continously being generated and prediction models should be rebuilt to keep up. We are building a microservice-based system for producing and publishing ligand-based prediction models. Models are built in a workflow that can be re-run when data is updated. The model is then packaged and published as a microservice in a Docker container in Kubernetes / OpenShift. The model can be accessed in a web based user interface or used in scripts via a REST based API published according to the Swagger standard.


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A HIGH-THROUGHPUT METHOD TO PREDICT THE IN VIVO EXPOSURE OF DIFFERENT DRUG DELIVERY SYSTEMS

C. Alvebratt (a), J. Keemink (a), K. Edueng (a), O. Cheung (b), M. Strmme (b), C. A.S. Bergstrm (a)

caroline.alvebratt@farmaci.uu.se

(a) Department of Pharmacy, Uppsala University, Sweden, (b) Department of Engineering Sciences, Uppsala University, Sweden

Supersaturating drug delivery systems (SDDS) have been suggested as a promising strategy to overcome poor aqueous solubility and hence increase in vivo exposure of drug compounds. Commonly used SDDS include amorphous solid dispersions (ASD), carrier-based delivery systems and lipid-based formulations (LBF). Methods used to determine release and absorption from these delivery systems are unfortunately significantly different, which limits the possibilities to perform head-to-head comparisons of formulations. In this work, we present a method that allows real time monitoring of release and absorption of felodipine from several different SDDS. The Diss ProfilerTM was combined with a slightly modified FLUXTM system, which herein was based on Caco-2 cell monolayers as the absorptive membrane. To reduce particle interference with analytics, which in this system is based on in situ UV-probes, a protective filter composed on nylon was placed on the UV-probe in the donor chamber [1]. The release and absorption were determined from crystalline drug, three types of LBFs, one ASD and one drug-loaded mesoporous carrier. A modified lipolysis buffer was used to ensure that the pH would remain constant (pH 6.5) during digestion of the LBFs [2]. One of the LBFs showed extensive absorption at the same wavelength as felodipine, and therefore HPLC-UV was utilized to determine the donor concentrations of this LBF. Significant differences with regard to degree of supersaturation reached in the donor compartments were observed between the SDDSs. The absorption rate was despite that similar for all formulations; the supersaturation rank order was not directly related to the flux across the membrane. To conclude, a method that enables monitoring and comparison of the release and permeation behavior of felodipine from various SDDs has been developed. The method is a valuable addition to currently available dissolution and absorption models, and is applicable to a wide range of drug delivery systems. References: [1] Alvebratt et al., 2018, AAPS PharmSciTech. 1-7 [2] Mosgaard et al., 2015, Eur J Pharm Biopharm, 94:493-500.


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SYNERGY CONFORMAL PREDICTION

Niharika Gauraha, Ola Spjuth niharika.gauraha@farmbio.uu.se

Department of Pharmaceutical Biosciences, Uppsala University

Conformal Prediction is a machine learning methodology that produces valid prediction regions under mild conditions. Ensembles of conformal predictors have been proposed to improve the informational efficiency of inductive conformal predictors by combining p-values, however, the validity of such methods has been an open problem. We introduce Synergy Conformal Prediction, which is an ensemble method that combines monotonic conformity scores, and is capable of producing valid prediction intervals. We study the applicability in two scenarios; where data is partitioned in order to reduce the total model training time, and where an ensemble of different machine learning methods is used to improve the overall efficiency of predictions. We evaluate the method on 10 data sets and show that the synergy conformal predictor produces valid predictions and improves informational efficiency as compared to inductive conformal prediction and existing ensemble methods. The results indicate that synergy conformal prediction has advantageous properties compared to contemporary approaches, and we also envision that it will have an impact in Big Data and federated environments.


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ISOLATION AND IDENTIFICATION OF THE MAIN STEROL FROM THE SPONGE HALICLONA SP. FROM TAIWAN

Merran Dunford (a,b), Sanjeevan Ranjendran (a), Patric Jonsson (a), Ping-Jyun Sung (c), Fang-Rong

Chang (d), Achyut Adhikari (e), Sunithi Gunasekera (a), Paco Crdenas (a) paco.cardenas@ilk.uu.se

(a) Pharmacognosy, Department of Medicinal Chemistry, BMC, Uppsala University, Uppsala, Sweden, b)

Department of Pharmacy and Pharmacology, University of Bath, Bath BA2 7AY, UK, c) National Museum of Marine Biology and Aquarium, Pingtung 944, Taiwan, d) Graduate Institute of Natural

Products, College of Pharmacy, Kaohsiung Medical University, Kaohsiung 80708, Taiwan, e) International Centre for Chemical and Biological Sciences, H. E. J. Research Institute of Chemistry, University of

Karachi, Pakistan

Sponges are known to harbor a wide spectrum of bioactive compounds of potential pharmaceutical value. In the current study, sterols were isolated from the shallow water Haliclona sp. from South Taiwan, and their antimicrobial activity was investigated. A normal phase, open-column chromatography procedure was adopted to isolate sterols from the sponge extract. From the initial NMR experiments, the most abundant compound in the sponge extract was suggested to be -sitosterol present in a mixture with several other sterols. Subsequently a preparative TLC was utilized to further separate the sterol mixture by using -sitosterol as a reference. The NMR analysis of the isolated peak having similar Rf value as -sitosterol from the preparative TLC procedure, gave evidence of a partially purified sterol mixture with the presence of a sterol with an additional double bond to -sitosterol. The partially purified sterol mixture was determined to have no activity on Escherichia coli and Staphylococcus aureus at a concentration of less than 0.5 mg/mL. Financial support from the EU Erasmus programme, the EU Horizon 2020 programme SponGES under grant agreement No. 679849 and an International Collaborative Research Grant (Swedish Research Council).


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A DIGESTION/PERMEATION MODEL BASED ON ARTIFICIAL MEMBRANES FOR STUDIES OF PERFORMANCE OF LIPID-BASED FORMULATIONS

O. J. Hedge, C. A. S. Bergstrm

oliver.hedge@farmaci.uu.se

Department of Pharmacy, Uppsala University, Uppsala Biomedical Center, P.O. Box 580, SE-751 23 Uppsala, Sweden

Lipid-based formulations (LBFs) may be a viable strategy for oral delivery of poorly water-soluble drug compounds, since they may increase bioavailability and reduce food effects of many contemporary drug molecules. There is, however, a lack of in vitro performance assessment assays that correlate well to in vivo data. One reason could be focus on precipitation during dispersion and digestion in simulated intestinal fluid (SIF), without taking absorption into account (1). Attempts to incorporate simultaneous absorption into the lipolysis assay have been made using cell monolayers (2). While these are promising, the cells are sensitive to the enzymes used and to date, digestion in the presence of cells is based on immobilized recombinant lipase that is not reflecting the diversity of the pancreatic enzymes. We therefore identified an artificial membrane that withstands the harsh environment of LBF digestion. Polymer filter supports were treated with oil/lipid solutions to form artificial membranes. LBFs were dispersed in fasted state SIF, spiked with the impermeable marker Lucifer Yellow (LY), and sampled during dispersion and digestion. Digestion was initiated after 10 minutes of dispersion, through addition of porcine pancreatic extract. Formulations of three different LBF classes loaded with 80 mg/g fenofibrate (FEN) were assayed using the novel ENA system with sampling at defined time points from donor and receiver compartments. Donor samples were filtered (0.1 m) to separate precipitates from the matrix. Samples were quantified for FEN using LC-MS and LY using fluorescence detection. Of the physical membranes explored, LIDO applied to PVDF membranes gave the lowest flux of LY between the ENA compartments. The model produced significant (p < 0.05) differences in flux of FEN between the formulations. The rank order of the flux between the formulations was comparable to that of the fraction FEN solubilized in the donor compartment. While the system could withstand the digestion process of several types of LBFs, the in vitro in vivo correlation (IVIVC) was not improved as compared to the previously explored methods (3). However, previous studies in the ENA system utilizing Caco-2 monolayers have shown rank order IVIVC for the formulations tested here (4). As FEN is not a known transporter substrate, this could indicate other mechanisms at play, e.g. uptake of lipids and bile salts. Further studies comparing the structural evolution of the donor matrix over time in a cell-based model could therefore be required to elucidate the mechanics of LBF mediated drug absorption. References: (1) O. M. Feeney et al., Adv. Drug Deliv. Rev. 101, 167194 (2016). (2) J. Keemink, C. A. S. Bergstrm, Pharm. Res. 35 (2018), doi:10.1007/s11095-017-2327-8. (3) B. T. Griffin et al., Eur. J. Pharm. Biopharm. 86, 427437 (2014). (4) J. Keemink et al., Submitted manuscript (2018).


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MAPPING THE DISTRIBUTION OF NEUROPEPTIDES THROUGH MASS SPECTROMETRY IMAGING

Heather E Hulme (a), Halla Gunnarsdttir (a), Elva Fridjonsdottir (a), Anna Nilsson (a),

Mohammadreza Shariatgorji (a), Erwan Bezard (b), Per E. Andrn (a) heather.hulme@farmbio.uu.se

(a) Medical Mass Spectrometry Imaging, National Resource for Mass Spectrometry Imaging, Science for

Life Laboratory, Department of Pharmaceutical Biosciences, Uppsala University, Box 591 BMC, 75124 Uppsala, (b) Universit de Bordeaux, Institut des Maladies Neurodgnratives,

UMR 5293 Bordeaux, France

Neuropeptides are a group of signalling molecules, which are produced by neurons and regulate neural function. Neuropeptides are particularly interesting as changes in the levels of these molecules has been associated with various neurological disorders such as Parkinsons disease. Matrix assisted laser desorption ionisation mass spectrometry imaging (MALDI-MSI) is a beneficial technique to investigate neuropeptides as this technique can map the distribution of various molecules at once across a thin tissue sample. Therefore, this technique has the potential to image various neuropeptides at once across the brain to help further understand neuropeptides and their role in neurological disorders. We optimised a method to enhance the detection of neuropeptides by MALDI-MSI, which allowed the imaging of various neuropeptides that have not been previously visualised by MSI. This method was subsequently applied to map the distribution of neuropeptides in rodent and primate brains as well as other tissue types. MALDI-MSI was further used to compare the neuropeptide abundance and distribution in experimental models of Parkinsons disease compared to healthy controls. This study highlights the capabilities of MALDI-MSI to help further understand neuropeptides and their role in neurological disorders.


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DOCKING OF MACROCYCLES: COMPARING RIGID AND FLEXIBLE DOCKING IN GLIDE

Hiba Alogheli, Gustav Olanders, Wesley Schaal, Peter Brandt, Anders Karln

gustav.olanders@ilk.uu.se

Uppsala university

In recent years, there has been an increased interest in using macrocyclic compounds for drug discovery and development. For docking of these commonly large and flexible compounds to be addressed, a screening and a validation set were assembled from the PDB consisting of 16 and 31 macrocycle-containing protein complexes, respectively. The macrocycles were docked in Glide by rigid docking of pregenerated conformational ensembles produced by the macrocycle conformational sampling method (MCS) in Schrdinger Release 2015-3 or by direct Glide flexible docking after performing ring-templating. The two protocols were compared to rigid docking of pregenerated conformational ensembles produced by an exhaustive Monte Carlo multiple minimum (MCMM) conformational search and a shorter MCMM conformational search (MCMM-short). The docking accuracy was evaluated and expressed as the RMSD between the heavy atoms of the ligand as found in the X-ray structure after refinement and the poses obtained by the docking protocols. The median RMSD values for top-scored poses of the screening set were 0.83, 0.80, 0.88, and 0.58 for MCMM, MCMM-short, MCS, and Glide flexible docking, respectively. There was no statistically significant difference in the performance between rigid docking of pregenerated conformations produced by the MCS and direct docking using Glide flexible docking. However, the flexible docking protocol was 2-times faster in docking the screening set compared to that of the MCS protocol. In a final study, the new Prime-MCS method was evaluated in Schrdinger Release 2016-3. This method is faster compared that of to MCS; however, the conformations generated were found to be suboptimal for rigid docking. Therefore, on the basis of timing, accuracy, and ease of set up, standard Glide flexible docking with prior ring-templating is recommended over current gold standard protocols using rigid docking of pregenerated conformational ensembles.


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HOME VS. SELF-INITIATED ART REFILL: CLINICAL, IMMUNOLOGICAL, AND VIROLOGIC OUTCOMES

Rory F. Leisegang (1,2), Jane Ball (3), Mark Cotton (4), David Dowdy (5), Keri Calkins (5), Susan

Cleary (2), Gary Maartens (2), Jean B. Nachega (6) rory.leisegang@farmbio.uu.se

(1) Uppsala University, Uppsala; (2) University of Cape Town, Cape Town, South Africa, (3) AID for

AIDS, Cape Town, South Africa; (4) Stellenbosch University, Tygerberg, South Africa; (5) Johns Hopkins Bloomberg School of Public Health, Baltimore, MD, USA; (6) University of Pittsburgh, Pittsburgh, PA,

USA

Background: Antiretroviral therapy (ART) delivery by courier to the patient's home (home refill) is a novel intervention that may improve clinical outcomes and reduce indirect costs for individuals in low- and middle-income countries (LMICs). We aimed to compare clinical and virologic outcomes for patients obtaining medication refills at their local pharmacy (self refill) vs. home refill in Aid for AIDS (AFA), a large South African private sector HIV/AIDS programme. Methods: Retrospective cohort analysis of ART nave HIV-infected adults in AFA who initiated first line NNRTI based ART regimen between January 2002 and July 2010 was performed. Patients were selected to switch to home refill based on the discretion of AFA. Primary endpoint was all-cause mortality; secondary endpoints were viral suppression (VL< 400 copies/mL) and median CD4+ T-cell response (cells/l) (from baseline) at 6-month intervals. We compared the crude survival between self-refill and home refill using KaplanMeier plots and a log-rank test. We performed Cox regressions to model the individual and simultaneous effects of baseline variables and mode of ART delivery on all-cause mortality, adjusting for bias using propensity score and marginal structural models. Results: 40,939 patients, contributing 66,204 years of follow-up were recorded. The most common first line regimen was efavirenz + lamuvidine + zidovudine, followed by efavirenz + emtricitabine + tenofovir in later years. Emerging at 24 months, the home refill group had improved median CD4+ T-cell count response (451 vs. 387, respectively, p < 0.01), and the likelihood of virologic suppression (81% versus 71%, respectively, p-value


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CHEMOSELECTIVE PROBES FOR THE DETECTION OF MICROBIAL BIOMARKERS OF DISEASE - COMBINING METABOLOMICS WITH CHEMICAL BIOLOGY

METHODOLOGIES

Neeraj Garg, Louis P. Conway, Caroline Ballet, Mrio S. P. Correia, Daniel Globisch. louis.conway@ilk.uu.se

Department of Medicinal Chemistry, Uppsala University

The discovery of highly specific small-molecule biomarkers of disease is the first step in the development of rapid non-invasive diagnostics, permitting the routine screening of at-risk populations and detection of disease at an early, treatable stage. In recent years, the gut microbiota has been found to profoundly impact human health. While the co-metabolism between commensal bacteria and the human host is potentially a rich source of biomarkers of disease, this chemical environment remains relatively unexplored. LC-MS-based general metabolomics is well suited to the sensitive detection of the vast number of compounds present in a biological matrix, however, the structural identification of these metabolites has long presented a major bottleneck. Furthermore, metabolites which occur at low concentrations are often neglected entirely due to compromises in analytical methodology or the lack of appropriate chemical tools. We have therefore developed chemical biology-based probes for the highly selective modification of well-defined metabolite classes, enhancing their sensitivity under LC-MC analysis and facilitating their identification. We seek to apply these tools to the analysis of patient-derived samples, with a particular focus on disease-microbiome interactions and cancer. This study was supported by the Swedish Research Council (VR 2016-04423), the Carl Tryggers Foundation (CTS16:155),and the Science for Life Laboratory to D.G


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LCMS-BASED UNTARGETED METABOLOMICS USING GUINEA PIG PERILYMPH: HIGHLIGHTING DIFFERENCES IN METABOLOME WITH AND WITHOUT A

POSTERIORI HYDROGEN GAS ADMINISTRATION FOLLOWING LOUD NOISE EXPOSURE

K. Pirttil (a), A. E. Fransson (b), J. Haglf (a), M. Engskog (a), P. V. Pierre (c), G. Laurell (b),

T. Arvidsson (a, d), C. Pettersson (a) kristian.pirttila@ilk.uu.se

(a) Dept. Medicinal Chemistry, Uppsala University, Uppsala, Sweden. (b) Dept. of Surgical Sciences, Uppsala University, Uppsala, Sweden. (c) Div. of Audiology, Dept. Clinical science, Intervention, and Technology, Karolinska Institutet, Stockholm, Sweden. (d) Medical Product Agency, Uppsala, Sweden

Introduction. In metabolomics studies of inherently localized conditions, the use of systemic matrices such as blood plasma risks diluting metabolic differences, possibly rendering them undetectable. In such studies, the use of localized tissue, sampled in proximity to the afflicted region is preferable. In the study of conditions of the inner ear, such as noise-induced hearing loss (NIHL), the use of perilymph aspired directly from the inner ear allows a more veracious view of the current metabolic state of the studied system. In this study a protocol is presented for untargeted metabolomics of perilymph aspired from the scala tympani of guinea pigs. The protocol is further applied to study the protective effect of gaseous hydrogen on NIHL in guinea pig. Methods. Animals were exposed to noise (Noise, n=8) (156 dB, 400 impulses over 3 min) or noise followed by H2 gas administration (Noise+H2, n=9). Perilymph (1 L) was aspired from the basal turn of the noise-exposed cochlea and diluted 1:19 with water. Prior to LCMS analysis the perilymph was subjected to protein precipitation. The supernatant was injected without further treatment. UPLC-Q-TOF (Waters, Milford, USA) analysis was achieved using a BEH Amide column (50x2.1mm, 1.7m, Waters) operated in HILIC mode. Raw data was subjected to pretreatment by peak picking (XCMS), normalization by median fold change, and filtering of features with high variability in repeat injections. Multivariate data analysis was performed on pareto scaled data in the SIMCA software (Umetrics, Ume, Sweden). Preliminary data. Following normalized and filtering, a total of 689 features were retained in using positive ionization mode corresponding to 276 peak groups as determined by CAMERA. Relative standard deviation (RSD) of peak areas for 8 known stable metabolites ranged between 3.4-12.3% across all QC injections in the analytical run (n=15). Retention time RSD of the same metabolites ranged from 0.00-0.34%. Multivariate data analysis of the resulting dataset allowed a clear discrimination between the sample groups Noise and Noise+H2, whereas the Noise+H2 group appears more similar to the control groups Control (n=2, no treatment), and H2 (n=3, only hydrogen gas treatment). Principal component analysis (PCA) shows the Noise group as a distinct cluster that deviates from the other sample groups. Supervised analysis by orthogonal partial least squares discriminant analysis (OPLS-DA) on the sample groups Noise and Noise+H2 shows excellent model performance indicators (R2 0.968, Q2 809) as expected from the PCA model. The indicated features were further considered manually to filter out adducts and non-peak noise data. Univariate analysis by one-sided Wilcoxon rank-sum tests of the selected 15 metabolites showed that many of them were significantly different in the two noise exposed groups. A common pattern was also that the Noise+H2 group had similar metabolite levels as the two control sample groups, indicating that the hydrogen gas treatment has a preventive effect on the metabolic dysregulation caused by the noise exposure.


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POPULATION MODELING OF UNI-AND THREE-DIMENSIONAL AND DENSITY-BASED TUMOR MEASUREMENTS IN GASTRO-INTESTINAL STROMAL TUMOR

PATIENTS TREATED WITH IMATINIB

Sreenath M. Krishnan (a), Emilie Schindler (a), Gaia Schiavon (b,c), Ron Mathijssen (b), Lena E. Friberg (a)

sreenath.krishnan@farmbio.uu.se

(a) Department of Pharmaceutical Biosciences, Uppsala University, Sweden. (b) Department of Medical Oncology, Erasmus University Medical Center, Rotterdam, The Netherlands.

(c) Translational Science, Oncology iMed, AstraZeneca, Cambridge, UK. Objectives: Three-dimensional (3D) and density-based tumor metrics have been suggested to better discriminate tumor response to treatment than the traditional unidimensional (1D) metrics for gastro-intestinal stromal tumor (GIST) which often exhibit non-uniform size changes. This study aims to characterize the longitudinal 1D, 3D and density responses of liver metastases in imatinib-treated GIST patients and quantify the inter-individual (IIV) and inter-lesion variability (ILV) and investigate model-predicted tumor metrics as predictors of overall survival (OS) and time to treatment failure (TTF). Methods: Data were obtained from a retrospective, non-interventional study involving 77 GIST patients treated with oral imatinib at a starting dose of 400 mg/800 mg q.d. (74/3) [1]. Maximum trans-axial diameter (MTD), software-calculated segmented volume (Vs), calculated ellipsoidal volume (Ve) and density data were collected from 137 lesions (1-2 per patient) at baseline and at least one post-baseline visit with the median follow-up time of 360 days. Tumor growth inhibition (TGI) models with constant, exponential or Gompertz growth were explored to describe MTD, Vs and Ve data. Indirect response models (IDR) with inhibition of the production or stimulation of the loss of response were investigated for tumor density. IIV and ILV were tested in all parameters. All models were combined into a joint model to explore correlations. Parametric time-to-event (TTE) models were developed for exploring potential predictors for OS and TTF. Results: Tumor modeling: TGI models with an exponential growth and a linear dose-driven drug effect that washes out over time [2] best characterized the MTD, Vs and Ve data. A mixture model described the bimodal distributions of the baseline data and accounted for ILV. The model-predicted doubling time was typically lower for Ve (1.3 years) and Vs (1.5 years) than for MTD (8.7 years). The growth rate constants and drug effect parameters were associated with large IIV but no ILV. An IDR model with stimulation of the loss of response adequately described tumor density data. Large IIV (120 %CV) and ILV (53 %CV) were identified in the drug effect. High correlations (>99%) were estimated between MTD, Vs and Ve model parameters. Correlations between the density model parameters and MTD, Vs and Ve model parameters were low (


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POPULATION PHARMACOKINETIC ANALYSIS OF FACTOR VIII ACTIVITY FOLLOWING TREATMENT WITH MOROCTOCOG ALFA IN MODERATE TO

SEVERE HAEMOPHILIA A SUBJECTS

Joo A. Abrantes (a), Elisabet I. Nielsen (a), Joan Korth-Bradley (b), Lutz Harnisch (c), Siv Jnsson (a) joao.abrantes@farmbio.uu.se

(a) Department of Pharmaceutical Biosciences, Uppsala University, Uppsala, Sweden, (b) Pfizer Inc,

Collegeville, Pennsylvania, USA, (c) Global Clinical Pharmacology, Pfizer, Sandwich, UK

Objectives: Moroctocog alfa is a B-domain deleted recombinant factor VIII (FVIII) indicated for the treatment and prophylaxis of bleeding in adults and children with haemophilia A. The aim of this study is to develop a population pharmacokinetic model of FVIII activity following moroctocog alfa treatment in moderate to severe haemophilia A patients using all available clinical study data. Methods: A population pharmacokinetic model (NONMEM 7.3) was developed with data from 13 trials involving IV administration of moroctocog alfa products (Refacto, Refacto AF, Xyntha Wyeth Pharmaceuticals Inc. [Pfizer], Philadelphia, USA). The studies were conducted from 1993 to 2013 in 25 countries, and included rich sampling (10 samples post-dose, n=4), sparse sampling (2-3 samples per occasion, n=5) or both (n=4). The influence of age, measures of body size, race, ethnicity, inhibitor status and titre, assay, year of study, study and country, were investigated. Data below the lower limit of quantification were handled using the M5 method [1] and correlated residual errors for repeated analyses were accounted for by using the L2 data item [2]. Qualification of the models included pcVPCs and parameter uncertainty was obtained with SIR [3]. Results: A total of 259 children and adolescents and 497 adults with moderate to severe haemophilia A (FVIII 5 IU/dL) were included in the analysis. Age ranged from 1 day to 73 years and weight from 3 to 134 kg. A two-compartment model with allometrically scaled body weight on disposition parameters was found to adequately describe the combined data. Pre-dose activity observations were described by estimating two endogenous FVIII activity levels at 0.474 IU/dL and 1.59 IU/dL together with an additive component accounting for residual activity from previous unknown doses (2.84 IU/dL at time 0, declining over time in line with FVIII disposition). For a 70-kg, 20-year-old patient with moderate or severe haemophilia A, FVIII activity clearance was estimated at 2.76 dL/h, central volume of distribution at 24.5 dL, peripheral volume of distribution at 9.23 dL, and inter-compartmental clearance at 25.1 dL/h. Conclusions: The model is capable of describing and predicting reasonably well the data and when used with patient observations in a therapeutic drug monitoring context may be an aid in dose individualization, with the ultimate goal of a safer and more effective treatment with moroctocog alfa. References: [1] Ahn JE, Karlsson MO, Dunne A, Ludden TM. 2008. Likelihood based approaches to handling data below the quantification limit using NONMEM VI. J Pharmacokinet Pharmacodyn 35(4):401-21 [2] Karlsson MO, Beal SL, Sheiner LB. 1995. Three new residual error models for population PK/PD analyses. J Pharmacokinet Biopharm 23:651672 [3] Dosne AG, Bergstrand M, Karlsson, MO. Application of Sampling Importance Resampling to estimate parameter uncertainty distributions. PAGE 22 (2013) Abstr 2907 [www.page-meeting.org/?abstract=2907]


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PROBABILISTIC PREDICTIONS OF METABOLISM USING VENN-PREDICTORS

Staffan Arvidsson Mc Shane (a), Lars Carlsson (b), Paolo Toccaceli (c), Ola Spjuth (a) staffan.arvidsson@farmbio.uu.se

(a) Department of Pharmaceutical Biosciences, Uppsala University, (b) Quantitative Biology, Discovery

Sciences, Innovative Medicines & Early Development, AstraZeneca, (c) Department of Computer Science, Royal Holloway

Prediction of drug metabolism is an important topic in the drug discovery process, and we here present a study using probabilistic predictions applying Cross Venn-ABERS Predictors (CVAPs) on data for site-of-metabolism. We used a dataset of 73599 biotransformations, applied SMIRKS to define biotransformations of interest and constructed five datasets where chemical structures were represented using signatures descriptors. The results show that CVAP produces well-calibrated predictions for all datasets with good predictive capability, making CVAP an interesting method for further exploration in drug discovery applications.


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CONTRALESIONAL HINDLIMB MOTOR RESPONSE INDUCED BY UNILATERAL BRAIN INJURY: EVIDENCE FOR EXTRA- SPINAL MECHANISM

N. Lukoyanov (a), L. Carvalho (a), H. Watanabe (b), M. Zhang (c, d), D. Sarkisyan (b), O. Kononenko

(b), I. Bazov (b), T. Iakovleva (b), J. Schouenborg (c) and G. Bakalkin (b) olga.kononenko@farmbio.uu.se

(a) Instituto de Investigao e Inovao em Sade, Instituto de Biologia Molecular e Celular,

Departamento de Biomedicina da Faculdade de Medicina da Universidade do Porto, Portugal; (b) Dept. Pharm. Biosciences, Uppsala University, Sweden; (c) Neuro-nano Research Center, Lund University,

Sweden; and (d) Dept. Mol. Medicine, University of Southern Denmark, Odense, Denmark

INTRODUCTION. The neurological dogma states that motor deficits secondary to traumatic brain injury and stroke arise due to the aberrant activity of neural pathways descending from injured brain to the spinal cord. OBJECTIVE. We tested this dogma by analyzing hindlimb motor response to unilateral brain injury in the rats with completely transected spinal cord. METHODS. The spinal cord was transected at T2/T3 levels first, and then the cortical hindlimb representation area was unilaterally ablated. Motor outputs were analyzed under pentobarbital anesthesia during 3-4 hours. Formation of hindlimb postural asymmetry was assessed as differences between the contra- and ipsilesional legs in a) their position; b) hip, knee and ankle joint angles, and (c) stretch forces. The nociceptive withdrawal reflexes, quantified as EMG responses evoked by electrical stimulation of digits and heel, were compared between the ipsi- and contralesional hindlimb muscles. Expression of plastic genes was analyzed in the lumbar spinal cord, i.e. below the transection level by qRT-PCR. RESULTS. Behavioral, electrophysiological and molecular evidence demonstrated the development of the contralesional-side specific response to the unilateral brain injury. The spinal rats, which then received cortical ablation but not sham operation, developed asymmetric hindlimb posture with flexion of the contralesional leg. The hindlimb withdrawal reflexes of the flexor muscles (the extensor digitorum longus and semitendinosus) were enhanced on the contralesional side while extensor muscle (the interosseous) on the ipsilesional side. Unilateral cortical ablation in spinal rats produced robust changes in the expression of neuroplastic genes in the lumbar spinal cord. mRNA levels were elevated on either the ipsilesional (c-Fos) or contralesional (TGF-beta) side resulting in the asymmetric expression patterns, or bilaterally (Gap43). Unilateral brain injury failed to induce postural asymmetry in the hypophysectomized spinal rats. CONCLUSIONS. These results demonstrate that, in parallel with neural pathways, signals elicited by injured brain are transmitted to the lumbar spinal cord by alternative, likely endocrine mechanism. Spinal plastic and hindlimb motor responses induced by these signals are side specific.


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EXPLORING THE USEFULNESS OF MORPHOLOGICAL PROFILING OF CELLS TO STUDY TOXICITY MECHANISMS

P. Georgieva, Tanya Aggarwal, W. Schaal, O. Spjuth

polina.georgiev@farmbio.uu.se

Uppsala University, Department of Pharmaceutical Biosciences, Uppsala, Sweden Although there have been major technological advances in how we study biological processes and systems in the last years, studying toxicity pathways still remains difficult, time-consuming and resource-intensive. High-content imaging of cells stained with multiplexed dyes, Cell Painting(1), has been successfully applied in drug screening(2) of chemical compounds based on morphological changes they induce. However its application in the field of toxicity is still rather unexplored and comprises an exciting opportunity to unravel toxicity mechanisms and pathways. We are investigating the usefulness of image-based morphology profiling toward understanding toxicology mechanisms and pathways, by studying a range of cell lines treated with different toxicants. To this end we implemented a cell painting screening assay, involving 4 - 6 fluorescent dyes, used to stain at least 6 - 8 subcellular components simultaneously, followed by subsequent imaging with an ImageXpress XLS high-content microscope. To analyse these compound-induced morphological changes we use a CellProfiler(3) analysis pipeline to compare morphological profiles and classify compounds based on mechanisms. Our initial experiments show promising results for mechanistic classification of toxicity mechanisms and we hypothesize that the method could be used for pathway enrichment. To this end we will strive towards automating the platform, and develop robust analysis pipelines to handle data/analysis to be able to study mechanisms on larger scale (1) Bray, Mark-Anthony et al. "Cell Painting, A High-Content Image-Based Assay For Morphological Profiling Using Multiplexed Fluorescent Dyes". Nature Protocols 11.9 (2016): 1757-1774. (2) Simm, Jaak et al. Repurposing High-Throughput Image Assays Enables Biological Activity Prediction for Drug Discovery. Cell Chemical Biology, 25.5 (2018), 611-618 (3) Carpenter, Anne et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biology (2006) 7:R100


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EXPLORING THE USEFULNESS OF MORPHOLOGICAL PROFILING OF CELLS TO STUDY TOXICITY MECHANISMS

P. Georgieva, T. Aggarwal, W. Schaal, O. Spjuth

tanya.aggarwal@farmbio.uu.se

Uppsala University, Department of Pharmaceutical Biosciences, Uppsala, Sweden Although there have been major technological advances in how we study biological processes and systems in the last years, studying toxicity pathways still remains difficult, time-consuming and resource-intensive. High-content imaging of cells stained with multiplexed dyes, Cell Painting(1), has been successfully applied in drug screening(2) of chemical compounds based on morphological changes they induce. However its application in the field of toxicity is still rather unexplored and comprises an exciting opportunity to unravel toxicity mechanisms and pathways. We are investigating the usefulness of image-based morphology profiling toward understanding toxicology mechanisms and pathways, by studying a range of cell lines treated with different toxicants. To this end we implemented a cell painting screening assay, involving 4 - 6 fluorescent dyes, used to stain at least 6 - 8 subcellular components simultaneously, followed by subsequent imaging with an ImageXpress XLS high-content microscope. To analyse these compound-induced morphological changes we use a CellProfiler(3) analysis pipeline to compare morphological profiles and classify compounds based on mechanisms. Our initial experiments show promising results for mechanistic classification of toxicity mechanisms and we hypothesize that the method could be used for pathway enrichment. To this end we will strive towards automating the platform, and develop robust analysis pipelines to handle data/analysis to be able to study mechanisms on larger scale (1) Bray, Mark-Anthony et al. "Cell Painting, A High-Content Image-Based Assay For Morphological Profiling Using Multiplexed Fluorescent Dyes". Nature Protocols 11.9 (2016): 1757-1774. (2) Simm, Jaak et al. Repurposing High-Throughput Image Assays Enables Biological Activity Prediction for Drug Discovery. Cell Chemical Biology, 25.5 (2018), 611-618 (3) Carpenter, Anne et al. CellProfiler: image analysis software for identifying and quantifying cell phenotypes. Genome Biology (2006) 7:R100


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DISCREPANCY IN STABLY EXPRESSED PROTEINS WITHIN AND ACROSS HUMAN TISSUE TYPES

C. Wegler (a,b), M. lander (a), P. Artursson (a)

christine.wegler@farmaci.uu.se

(a) Department of Pharmacy, Uppsala University, Uppsala, Sweden (b) Drug Metabolism and Pharmacokinetics, Cardiovascular, Renal and Metabolism, IMED Biotech Unit,

AstraZeneca Gothenburg, Gothenburg, Sweden In recent years, large-scale omics analyses have mapped human gene and protein expression to characterize and compare different cells and tissues. It has been shown that some proteins are mostly expressed in a limited number of tissues, while others are widely and uniformly distributed throughout the human body. Such uniform proteins have been proposed as references for quality control and normalization of omics data, as well as loading controls for blotting techniques. However, these studies did not specifically consider within-tissue variability across human subjects and its impact on normalization in the analysis of the same tissue type from many individuals. Here, we therefore analyze inter-sample variability in proteomic datasets from human liver. We describe the characteristics of the least and most variable proteins, and provide a set of proteins with uniform expression across tissue samples. Surprisingly, many previously described proteins with low variability across tissue types showed large within-tissue variability. We observed a wide distribution in variabilities, with highly abundant proteins showing less variable expression across individuals. The least variable proteins within tissues were generally confined to the cytosol or mitochondria, while the most variable proteins were more spread across different subcellular compartments. Further, proteins essential for cell survival were overrepresented in the low variability range, suggesting that survival requires expression of these proteins at a certain level in all individuals. Supporting this, inter-sample variability showed an inverse correlation with previously established protein turnover rates. In conclusion, the differences in variability within and across tissues highlight the difficulties in selecting single proteins for use as references in all tissue types.


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EARLY-LIFE EXPOSURE TO TO AN ALGAL NEUROTOXIN INDUCES LONG-TERM CEREBELLAR NEURODEGENERATION AND LYSOSOMAL CHANGES

J Swan (a), L Ersson(a), A-L Berg(b), O Karlsson(a,c), E Brittebo(a)

julia.swan@farmbio.uu.se

(a) Dept Pharm. Biosciences, Uppsala University, (b) Lkemedelsverket, (c) Stockholm University

Algal blooming is of increasing concern with respect to cyanobacteria (blue-green algae) since it is promoted by eutrophication of aquatic environments and global warming. Most cyanobacteria can produce the neurotoxin -N-methylamino-L-alanine (BMAA). Dietary exposure to this neurotoxin has been suggested to be involved in the etiology of sporadic neurodegenerative disease. Current research on the etiology of neurodegenerative disease is mainly focused on identifying gene mutations in the familiar forms of disease. The cause for the majority of sporadic cases (also called late onset) of neurodegenerative disease is still unknown. There is, however, considerable evidence to support a multifactorial etiology involving both environmental, life-style and genetic factors. Our previous studies revealed high transfer of BMAA to the neonatal rodent brain with a distinct localization in the hippocampus and cerebellum. Early-life exposure to the neurotoxin BMAA during the brain development period may trigger cell stress and perturbations of cell signaling and protein expression in sensitive brain regions, which eventually translates into progressive neurodegeneration at adult age. Our studies on the effects of BMAA in the hippocampus demonstrated that early-life exposure to BMAA induced behavioual changes such as deficits in learning and memory functions as well as a selective long-term neurodegeneration, calcification and intracellular fibril formation in the CA1 region. We are now studying the long-term effects of BMAA in the cerebellum following a short exposure during the neonatal period. The results demonstrated a selective long-term neurodegeneration, calcification as well as changed protein expression in the cerebellar vermis region. Studies on perturbation of protein expression in this area suggest lyzosomal changes.


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MASS SPECTROMETRY LIPIDOMICS: A PLATFORM TO UNRAVEL LIPID BIOLOGY

David Balgoma (a), Alfhild Grnbladh (b), Sofia Zelleroth (b), Mathias Hallberg (b), Curt Petterson (a), Mikael Hedeland (a)

david.balgoma@ilk.uu.se

(a) Analytical Science, Department of Medicinal Chemistry, Biomedicinskt Centrum, Uppsala universitet. (b) Biological research on drug dependence, Department of Pharmaceutical Biosciences, Uppsala

University

Lipidomics aims to study the lipid profile (lipidome) and the lipid regulation of a biological system. Lipids, which were for long time considered just a barrier of cells, play a key role in physiological and pathophysiological processes. Specifically, the polyunsaturated fatty acids are involved in the triggering and the resolution of inflammation. The state-of-the-art analytical technique in lipidomics separates the lipids by liquid chromatography hyphenated to mass spectrometry. This technique allows the detection of minor species, which is not possible in NMR, and the characterization of the structure of the lipids by fragmentation in tandem mass spectrometry. Here we present a platform to analyze the lipidome. This platform integrates: 1) sample preparation, 2) chromatographic separation, 3) mass spectrometry detection, 4) data pre-treatment, and 5) data statistical analysis. In detail: 1) We adapt the sample preparation to the specific sample type. 2) We perform the chromatographic separation by ultrahigh-performance liquid chromatography in an Acquity chromatographer (Waters) by a gradient on a BEH C18 column. 3) We detect the molecules by mass spectrometry in both negative and positive modes. We ionize the analytes by electrospray (ESI) on a Synapt G2S-Q-ToF analyzer (Waters). 4) We pre-treat the mass spectrometric data in R to identify the lipids by their mass-to-charge and their fragmentation patterns. 5) We perform in R the data analysis plan which we design at the beginning of the analytic process. Typically, we perform univariate (t-test, ANOVA) and multivariate analysis (principal component analysis, orthogonal partial least squares, elastic nets). By the application of this workflow, we could identify by multivariate analysis a general shift in the lipidome of blood plasma in rats treated with different anabolic androgenic steroids. Some lipids deviated from this general shift, which were associated to different metabolic pathways. Further studies on these shifts are warranted in order to elucidate their impact on changes in behavior and disease risk. In conclusion, we present a state-of-the-art lipidomics platform to unravel the lipid biology and generate lipid biomarkers of health and disease


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DISCOVERY AND CHARACTERIZATION OF SMALL PEPTIDE TOXINS FROM THE EPIDERMAL MUCUS OF LINEUS LONGISSIMUS, THE LONGEST ANIMAL ON

EARTH

Erik Jacobsson (a), Hkan S. Andersson (b), Malin Strand (c), Steve Peigneur (d), Eline K. M. Lebbe(d), K. Johan Rosengren (e), Jan Tytgat (d), Ulf Gransson (a)

erik.jakobsson@ilk.uu.se

(a) Pharmacognosy, Department of Medicinal Chemistry, Uppsala University, Uppsala, UPPSALA, Sweden (b) Department of Chemistry and Biomedical Sciences, Centre for Biomaterials Chemistry, Linnaeus University, Kalmar, Sweden. (c) Swedish Species Information Centre, Swedish University of Agricultural Sciences, Uppsala,

Sweden (d)Toxicology and Pharmacology, University of Leuven , Leuven, Belgium (e) School of Biomedical Sciences, The University of Queensland, Brisbane, Australia

Peptide toxins display many applications, including use in agriculture, medicine and pharmacology. Some animals have successfully been investigated for peptide toxin presence, while others have been neglected. One neglected source of small peptide toxins is the epidermal mucus secreted by nemertean worms. We have discovered and characterized a novel class of small cystine-rich neurotoxins, a-nemertides, in the mucus of the worlds longest animal: Lineus longissimus. The toxins could only be isolated in minute amounts and were sequenced using mass spectrometry in combination with transcriptomic data. The most abundant toxin, nemertide a-1, was synthetized by solid phase peptide synthesis in sufficient amounts for NMR-based structural elucidation and functional bioassays. Upon injection, nemertide a-1 induces paralysis in Carcinus maenas at a concentration of 300 fmol/kg. In an Artemia salina assay, an IC50 of less than 1 M was observed. Moreover, nemertide a-1 modulates voltage-gated sodium channels in insects at nM concentrations, while being approximately two orders of magnitude less active in vertebrates. These data indicate a potential use of a-nemertides in insecticidal applications.


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CLINICAL METABOLOMICS: THE STETHOSCOPE FOR THE 21ST CENTURY?

Stephanie Hermana (a,b), Payam Emami Khoonsari (a), Valter Niemel (c),

POSTER SESSION - uu-pharmfaculty50years.seuu-pharmfaculty50years.se/wp-content/uploads/2018/10/Poster-session... · i FACULTY OF PHARMACY 50 YEARS POSTER SESSION 26 October 2018 Full

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