Early ADMET profiling of anti-inflammatory alkaloids using validated LC-MS/MS methods Inauguraldissertation zur Erlangung der Würde eines Doktors der Philosophie vorgelegt der Philosophisch-Naturwissenschaftlichen Fakultät der Universität Basel von Evelyn Andrea Jähne aus Höri, Zürich Basel, 2016 Original document stored on the publication server of the University of Basel edoc.unibas.ch This work is licenced under the agreement „Attribution Non-Commercial No Derivatives – 3.0 Switzerland“ (CC BY-NC-ND 3.0 CH) The complete text may be reviewed here: creativecommons.org/licenses/by-nc-nd/3.0/ch/deed.en Genehmigt von der Philosophisch-Naturwissenschaftlichen Fakultät auf Antrag von Prof. Dr. Matthias Hamburger Prof. Dr. Jürgen Drewe Basel, den 21.06.2016 Prof. Dr. Jörg Schibler Dekan Attribution-NonCommercial-NoDerivatives 3.0 Switzerland (CC BY-NC-ND 3.0 CH) You are free: to Share — to copy, distribute and transmit the work Under the following conditions: Attribution — You must attribute the work in the manner specified by the author or licensor (but not in any way that suggests that they endorse you or your use of the work). 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Quelle: creativecommons.org/licenses/by-nc-nd/3.0/ch/deed.en Datum: 12.11.2013 Für meine Familie und Freunde Table of contents List of abbreviations ...............................................................................................................................9 Summary ...............................................................................................................................................11 Zusammenfassung ................................................................................................................................13 1 Aim of work .....................................................................................................................................15 2 Introduction .....................................................................................................................................19 2.1 Natural product based lead discovery .....................................................................................20 2.2 Alkaloids from Isatis tinctoria L. as potential leads for anti-inflammatory drugs ...............23 2.2.1 Woad (Isatis tinctoria L.) ......................................................................................................24 2.2.2 Tryptanthrin ...........................................................................................................................26 2.2.3 Indirubin ................................................................................................................................28 2.2.4 Indolinone derivative .............................................................................................................31 2.3 Pharmacokinetics and early ADMET profiling in drug discovery .......................................38 2.3.1 Drug discovery and development process .............................................................................39 2.3.2 Pharmacokinetics in drug discovery......................................................................................40 2.3.3 The gastrointestinal tract (GIT) and intestinal epithelium ....................................................43 2.3.3.1 Rule-based approaches and in silico models ................................................................44 2.3.3.2 In vitro permeability models .........................................................................................45 2.3.3.3 In situ models ................................................................................................................47 2.3.4 The blood-brain barrier (BBB) ..............................................................................................48 2.3.4.1 Structure BBB permeation relationships and in silico models .....................................49 2.3.4.2 In vitro BBB models .....................................................................................................49 2.3.4.3 In vivo BBB models ......................................................................................................50 2.3.5 The hERG channel ................................................................................................................51 2.3.5.1 Structure activity based relationships and in silico approaches ....................................52 2.3.5.2 In vitro hERG methods .................................................................................................53 2.3.5.3 In vivo hERG methods ..................................................................................................54 7 2.4 Bioanalysis ......................................................................................................................................59 2.4.1 Definition and current techniques .........................................................................................59 2.4.2 LC coupled to MS/MS and HR-MS ......................................................................................60 2.4.3 Sample preparation ................................................................................................................62 2.4.4 Bioanalytical quantification using validated LC-MS/MS methods .......................................63 2.4.5 Method development .............................................................................................................64 2.4.6 Method validation .................................................................................................................65 2.4.7 Sample analysis .....................................................................................................................67 3 Results and discussion .....................................................................................................................69 3.1 Development and validation of a LC-MS/MS method for assessment of an anti-inflammatory indolinone derivative by in vitro blood-brain barrier models. ............................................................70 3.2 Pharmacokinetics and in vitro blood-brain barrier screening of the plant-derived alkaloid tryptanthrin .........................................................................................................................................83 3.3 Development and full validation of an UPLC-MS/MS method for the quantification of the plant- derived alkaloid indirubin in rat plasma ..........................................................................................101 3.4 Caco-2 permeability studies and in vitro hERG liability assessment of tryptanthrin and indolinone ........................................................................................................................................115 4 Conclusions and outlook ...............................................................................................................135 Acknowledgments ...............................................................................................................................145 Curriculum vitae ................................................................................................................................146 8 List of abbreviations ADMET Absorption, distribution, hERG Human ether-a-go-go related gene metabolism, excretion, and HIV Human immunodeficiency virus toxicity HPLC High performance liquid AUC Area under the curve chromatography AhR Aryl hydrocarbon receptor HR-MS High resolution mass analyzer ANVISA Agência Nacional de Vigilância HTS High-throughput screening Sanitária IA Immunoassay APCI Atmospheric pressure ionization IAM Immobilized artificial membrane source IC Half maximal inhibitory BBB Blood-brain barrier 50 concentration BCRP Breast cancer resistance protein ICH International Conference on B.w. Body weight Harmonization C0 Initial concentration IgE Immunoglobulin E Caco-2 Human colon adenocarcinoma I Delayed rectifier potassium Kr cell line current Cmax Maximum Concentration IL-4 Interleukin 4 CDK Cyclin dependent kinase I.S. Internal standard CerK Ceramide kinase IUPAC International Union of Pure and CFS Cerebrospinal fluid Applied Chemistry cGMP Cyclic guanosine monophosphate I.v. Intravenous CHO Chinese hamster ovary cells iNOS Inducible nitric oxide synthase CID Collision induced dissociation JAK3 Janus kinase 3 CL Clearance Kit TK Kit ligand (stem cell factor) Cyp P450 Cytochrome P450 k Elimination rate constant e CML Chronic myelocytic leukemia 5-LOX 5-Lipoxygenase CNS Central nervous system LC-MS Liquid chromatography coupled to mass spectrometry COX-2 Cyclooxygenase-2 DSS Dextran sodium sulfate LC-MS/MS Liquid chromatography coupled to tandem mass spectrometry DYRK Dual specificity tyrosine- phosphorylation-regulated kinases LC-UV/VIS Liquid chromatography coupled to ultraviolet/visible absorbance ECG Electrocardiogram detection ELISA Enzyme-linked immunosorbent LLE Liquid-liquid extraction assay LLOQ Lower limit of quantification EMA European Medicine Agency LQTS Long QT syndrome ESI Electrospray ionization source LTB4 Leukotriene B4 FCɛRI Human high affinity receptor for IgE MDCK Madin-Darby canine kidney cell line FDA Food and Drug Administration MDR1 Multidrug resistant gene 1 GC-MS Gas chromatography coupled to MHLW Ministry of Health, Labour and mass spectrometry Welfare GIT Gastrointestinal tract MRI Magnetic resonance imaging GLP Good laboratory practices MRM Multiple reaction monitoring GP Glycogen phosphorylase MRP Multidrug resistance associated GSK-3 Glycogen synthase kinase-3β protein GST Glutathione S-transferases MS Mass spectrometry HEK 293 Human embryonic kidney cells NF- κB Nuclear factor kappa-light-chain- HEL Human erythroleukemia cell line enhancer of activated B cells 9 NSAID Nonsteroidal anti-inflammatory SRM Selected reaction monitoring drug S-SMEDD Super-saturated micro-emulsion OCT Organic cation transporter Drug delivery Systems PAMPA Parallel artificial membrane S/N Signal to noise ratio permeability assay SULT Sulfotransferase Papp Apparent permeability coefficient Syk Spleen tyrosine Kinase PET Positron emission tomography t Half-life 1/2 P-gp P-glycoprotein TCM Traditional Chinese medicine PK Pharmacokinetic TdP Torsades de pointes PP Protein precipitation TEER Transendothelial electrical PSA Polar surface area resistance OAT Organic anion transporter Th2 T helper type 2 cells QC Quality control TDM Therapeutic drug monitoring OCT Organic cation transporter t Time of maximum drug max concentration Q-TOF Quadrupole time-of-flight mass spectrometer TNF-α Tumor necrosis factor alpha RAW Mouse leukemic monocyte TQD Triple quadrupole detector macrophage cell line UGT Uridine 5'-diphospho RE Relative error glucuronosyltransferase RHB Ringer HEPES buffer UHPLC Ultra-high performance liquid RIA Radio immunoassay chromatography UPLC Ultra performance liquid RTK Receptor tyrosine kinase chromatography S.D. Standard deviation ULOQ Upper limit of quantification S.E.M. Standard error of mean V Volume of distribution SLE Supported-liquid extraction d WS Working solution SOP Standard operating procedure SPE Solid-phase extraction Trivial and systematic IUPAC names: Trivial name IUPAC name Couroupitine A indolo[1,2-h][1,7]naphthyridine-6,12-dione Indigo [2,2’-biindolinylidene]-3,3’-dione Indirubin [2,3’-biindolinylidene]-2’,3-dione Indolin-2-one (E,Z)-3-(4-hydroxy-3,5-dimethoxybenzylidene)indolin-2-one Tryptanthrin indolo[2,1-b]quinazoline-6,12-dione 10