Table Of ContentEarly 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
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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
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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
Description:such as publicity or privacy rights. •. Notice — For any 2.2 Alkaloids from Isatis tinctoria L. as potential leads for anti-inflammatory drugs 23.
