The expression and localisation of membrane transporters and P450 enzymes along the ...

323 Pages·2015·11.23 MB·English

The expression and localisation of
membrane transporters and P450 enzymes
along the longitudinal and crypt-villus axes
of the rat intestine and their response to
oral imatinib

Megan Jo Webster MRes. B.Sc. (Hons)

Submitted for the degree of Doctor of
Philosophy

Epithelial Research Group
Institute for Cell and Molecular Biosciences
The Medical School
University of Newcastle upon Tyne
September 2014
Abstract
A reduction in oral bioavailability of a wide range of drug compounds may occur following
efflux of drug substrates into the intestinal lumen mediated by ATP dependent ABC
transporters MDR1, MRP2 and BCRP and/or by enteric metabolism of drug compounds by
CYP450 enzymes. Furthermore, inter-individual variability in the expression and function of key
drug disposition proteins, which may occur following a regime of drug treatment, has the
potential to cause altered pharmacokinetic profiles leading to failure of therapy.
This thesis aimed to establish mRNA and protein expression of key drug transporter proteins
and CYP450 enzymes both along the length of the intestine and across the crypt-villus axis
using NanoString mRNA technology and immunohistochemistry. Key findings include; a)
evidence for cytoprotection of the crypt cells distinct from enterocytes, b) a lack of correlation
between Mrp2 mRNA and protein expression in the rat small intestine, with a paucity of brush-
border protein in all regions, c) using the M70 antibody, Bcrp protein was immunolocalised to
the lateral cell membranes of cryptal epithelial in the duodenum, jejunum and ileum with no
apparent protein expression above the crypt-villus junction.
Oral administration of the tyrosine kinase inhibitor imatinib mesylate was found to cause
significant induction of Cyp1a1 mRNA expression with maximal induction in ileum samples
showing an average 169-fold increase in expression. Immunohistochemistry indicates an
associated increase in Cyp1a1 protein levels. In addition intestinal exposure to imatinib was
found to cause significant changes in mRNA expression levels of genes including Mdr1a and
Bcrp.
This thesis presents key differences with regards to protein localisation of ABC transporters
proposed to play an important role in conferring intestinal drug resistance between rat and
human tissues and highlights the high levels of complexity associated with oral drug delivery
and intestinal absorption.
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Acknowledgements
Firstly I would like to thank my supervisor Prof. Nicholas Simmons for his support, commitment
and motivation throughout my PhD studies. I wish to thank Dr Gary Wilkinson not only for his
input and supervision throughout my placement at AstraZeneca but also for his continued
support throughout my studies whilst based at Newcastle University.
I would like to acknowledge several employees of AstraZeneca, without whom I would have
been unable to utilise novel technologies and equipment whilst based at the Alderley Edge
site. I wish to thank Margaret Veldmann-Jones, for her expertise and advice on design and
implementation of NanoString mRNA analysis technology, Janet Kelsall for her advice
surrounding molecular biology techniques and Kevin Randall for his support with
immunohistochemical work. Thanks must also go to Peter Jones, Patricia Pimlott and Kathryn
Pickup for their help in design and implementation of the in vivo work carried out during this
project. I would also like to thank Constanze Hilgendorf for her help in obtaining human
intestinal samples.
Finally, I would like to thank my friends and family for their motivational speeches in my hour
of need and for helping to make this experience enjoyable.

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Abbreviations
Ab Antibody
ABC ATP binding cassette
ABL Abelson tyrosine kinase gene
aCa Calcium activities

ACE Angiotensin - converting enzyme
ACTB β-actin
ADME Absorption Distribution Metabolism and Excretion
ADP Adenosine di-phosphate
Adr Adriamycin
AhR Aryl hydrocarbon receptor
Akt Protein kinase B
ALPH Alkaline phosphatase
AMP-PNP Adenylyl-imidodiphosphate
ANOVA Analysis of variance
ARG ABL related gene kinase
ARNT AhR nuclear translocator
Asn Asparagine
ATCC American Type Culture collection
ATP Adenosine tri-phosphate
AUC Area under curve
B2M β-2-microglobulin
BCA bi-cinchronic acid
BCR-ABL Breakpoint cluster- Abelson tyrosine kinase gene
BCRP Breast cancer resistance protein
Caco2 Colonic adenocarcinoma cell line
CAR Constitutive androsterone receptor
CBC cells Crypt-base columnar cells
cc1 cell conditioning treatment 1
cc2 cell conditioning treatment 2
CD147 cluster of differentiation 147 protein
cDNA complementary DNA
CFTR Cystic fibrosis transmembrane conductance regulator
CHO Chinese Hamster Ovary cell line
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c-Kit proto-oncogene c-Kit (also known as Mast/stem cell growth factor receptor)
Cmax Maximal concentration
CML chronic myeloid leukaemia
cMOAT Canalicular multispecific organic anion transporter
CNT Concentrative nucleoside transporter
Crkl Crk-like protein
cRNA Complementary RNA
CsA Cyclosporin A
Ct Cycle threshold
CYP Cytochrome
CYP450 Cytochrome P450
DAB 3, 3’-Diaminobenzidine
Dpm Disintegrations per minute
Dt Total disintegrations per minute
DTT Dithiothreitol
E.Coli Escherichia Coli
E3S Estrogen-3-sulphate
ECACC European Collection of Cell Cultures
ECL Electrochemical luminescence
EDTA Ethylenediaminetetraacetic acid
EGTA Ethylene glycol tetraacetic acid
FCS Fetal calf serum
FTC Futrimorgan C
FXR Farnesoid X Receptor
GAPDH glyceraldehyde-3-phosphate dehydrogenase
GIST gastrointestinal stromal tumours
GPR49 G-protein receptor 49
GUSB glucuronidase-β
HA- hemagglutinin
HBSS Hanks balanced salt solution
HEK Human Embryonic Kidney
HEK293 Human Embryonic Kidney293 cells
HIEC Human intestinal epithelial cells
HMBS Hydroxymethylbilane synthase
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HPLC High performance liquid chromatography
HPRT1 Hypoxanthine phosphorbosyltransferase 1
HRP Horseradish peroxidase
HS Horse Serum
HSCs Hematopoietic stem cells
HSKP Housekeeping
HSP90 Heat shock protein 90
J Flux (apical to basolateral)
(a-b)
J Flux (basolateral to apical)
(b-a)
J Net flux (J – J )
net (b-a) (a-b)
KB Kraftbrühe
KO Knock out
K0143 (3S,6S,12aS)-1,2,3,4,6,7,12,12a-Octahydro-9-methoxy-6-(2-methylpropyl)-1,4-
dioxopyrazino[1',2':1,6]pyrido[3,4-b]indole-3-propanoic acid 1,1-dimethylethyl
ester
LAP Leucine amino peptidase
LAP3 Leucine amino peptidase 3
LGR5 Leucine-rich repeat-containing G-protein coupled receptor 5
LogP Partition coefficient
mBcrp Mouse breast cancer resistance protein
MCT1 Monocarboxylate transporter 1
MDCK Madin Darby Canine Kidney cell
MDR1 Multidrug resistance transporter 1
MDR1A Multidrug resistance transporter isoform a
MDR1B Multidrug resistance transporter isoform b
MEM Modified eagles medium
MeOH Methanol
MOPS 3-(N-morpholino)propanesulfonic acid
Mr Molecular weight
mRNA messenger ribonucleic acid
MRP Multidrug resistance protein
MSD Membrane spanning domain
MTS 3-(4, 5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-2H-tetrazolium
MXR Mitoxantrone resistance transporter
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NADPH Nicotinamide adenine dinucleotide phosphate
NBD Nucleotide binding domain
NBF Neutral buffered formalin
NEAA Non-essential amino acids
Nkcc1 Na-K-Cl cotransporter 1
NR Nuclear-receptor
OAT Organic anion transporter
OATP Organic anion-transporting polypeptide
OCT/OCTN Organic cation transporter
P.D. Potential difference
Papp Apparent permeability
PBS Phosphate buffered saline solution
PCNA Proliferating cell nuclear antigen
PCR Polymerase chain reaction
PDGFR Platelet derived growth factor receptor
PEPT1 Peptide transporter 1
PFA Paraformaldehyde
PFO Perfluorooctanesulfonate
P Inorganic phosphate
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PI3K Phosphatidylinositol-4, 5-bisphosphate 3-kinase
PK Pharmacokinetic
PMEA Para-methoxyl-N-ethylamphetamine
PNGase F Peptide -N-Glycosidase F
pNPP 4-nitrophenyl phosphate
PPAR Peroxisome proliferator-activated receptor
PXR Pregnane-X-receptor
qPCR Quantitative polymerase chain reaction
RAR Retinoic acid receptor
RIN RNA integrity value
RNA Ribonucleic acid
Rpm Revolutions per minute
RT-PCR Real time – polymerase chain reaction
RXR Retinoid-X-receptor
SEM Standard error of the mean
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SEYFP Super enhanced yellow fluorescent protein
SGLT1 Sodium glucose co-transporter 1
Ship SH2-containing Inositol 5′-Phosphatase
SLC Multi-specific solute carrier
SLCO Solute Carrier Organic Anion
SP Side population
Stat5 Signal Transducer and Activator of Transcription 5
SUR1 Sulfonylurea receptor 1
SUR2 Sulfonylurea receptor 2
TBS Tris-buffered saline
TCDD 2, 3, 7, 8-Tetrachlorodibenzodioxin
TEER Transepithelial electrical resistance
TF Transcription factor
TEM Transmission electron microscopy
TK Tyrosine kinase
Tm melting temperature
TNFα Tumour necrosis factor - α
TR- Transport deficient
TUBA1 Tubulin alpha 1
VDR Vitamin D receptor
XRE Xenobiotic response element

Note: Consistent with HUGO Gene Nomenclature Committee guidelines human gene symbols
and related mRNA and protein expression are presented with all letters in uppercase type
(MDR1) whilst gene symbols, mRNA and protein designations of mouse, rat and canine are
presented with only the first letter in uppercase with remaining letters in lower case (e.g.
Mdr1).

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Contents
1. Introduction .......................................................................................................................... 1
1.1 The intestinal epithelium lining; an enterocytic barrier ..................................................... 1
1.1.1 Intestinal morphology; the longitudinal and crypt-villus axes ..................................... 5
1.2 The ABC superfamily of proteins ...................................................................................... 10
1.2.1 ABC transporters; structure and mechanism of action ............................................. 10
1.2.2 MDR1; The permeability glycoprotein, P-gp .............................................................. 15
1.2.3 The multidrug resistance protein family .................................................................... 16
1.2.4 Breast cancer resistance protein (BCRP) .................................................................... 21
1.3 Cytochrome P450 enzymes and their contribution to intestinal drug metabolism ......... 25
1.4 Solute carrier family of transporters – SLC proteins ......................................................... 27
1.5 Interindividual variability in response to oral drug administration and potential for
ABC/CYP450 mediated drug interactions ............................................................................... 28
1.6 Thesis aims and objectives ................................................................................................ 31
2. Materials and Methods ....................................................................................................... 32
2.1 Materials ........................................................................................................................... 32
2.1.1 Buffer Solutions .......................................................................................................... 32
2.2 Methods ............................................................................................................................ 33
2.2.1 Routine cell culture .................................................................................................... 33
2.2.2 Animal housing and sacrifice ..................................................................................... 33
2.2.3 Acquirement of duodenal, jejunal, ileal and colonic mucosal scrapes ...................... 34
2.2.4 Crypt-villus isolation and preparation validation ....................................................... 35
2.2.5 Measurement of functional MDR1 and mBcrp/BCRP transporter activity using the
Hoechst33342 dye retention assay ..................................................................................... 42
2.2.6 Determination of mRNA expression levels of key intestinal drug transporters and
cytochrome P450 enzymes in rat intestine using NanoString technology ......................... 45
2.2.7 Western blot analysis ................................................................................................. 57
2.2.8 Immunocytochemistry ............................................................................................... 60
2.2.9 Immunohistochemistry .............................................................................................. 63
2.2.10 Determination of transepithelial flux of radiolabelled MDR1 and mBcrp substrates,
digoxin and ciprofloxacin .................................................................................................... 67
2.2.11 In vivo study ............................................................................................................. 69
3. Drug transporter models and the isolation of villus tip and crypt populations.................. 71
3.1 Introduction ...................................................................................................................... 71
3.2 Methods ............................................................................................................................ 77
3.2.1 Crypt-villus isolation and preparation validation ....................................................... 77
3.2.2 Incubation of isolated fractions in KB medium in an attempt to restore metabolic
activity ................................................................................................................................. 77
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3.3 Results ............................................................................................................................... 79
3.3.1 Isolation of intestinal cells across the crypt-villus axis of the rat ileum .................... 79
3.3.2 Biochemical assessment of villus tip and crypt cell populations ............................... 84
3.3.3 Functional assessment of MDR1 and mBcrp activity in MDCKII cell lines and of Mdr1
and Bcrp in isolated rat villus tip and crypt samples .......................................................... 86
3.3.4 Assessment of crypt-villus cell viability; TEM, Na+/K+ ratios, live-DEAD cell analysis
and the MTS viability assay of crypt-villus preparations .................................................... 91
3.4 Discussion ........................................................................................................................ 101
4. Determination of mRNA expression levels of drug transporters and cytochrome P450
enzymes .................................................................................................................................... 108
4.1 Introduction .................................................................................................................... 108
4.2 Methods .......................................................................................................................... 112
4.2.1 NanoString Codeset design ...................................................................................... 112
4.2.2 Sample preparation and determination of mRNA expression levels....................... 112
4.2.3 Normalisation and statistical analysis of NanoString mRNA expression data ......... 112
4.3 Results ............................................................................................................................. 113
4.3.1 Identification and validation of housekeeping reference genes as internal controls
.......................................................................................................................................... 113
4.3.2 Experimental validation using control genes of known intestinal expression pattern
.......................................................................................................................................... 114
4.3.3. Intestinal expression of ABC drug transporter mRNA from the duodenum to colon
and across the crypt-villus axis ......................................................................................... 123
4.3.4 Expression of selected SLC transporters in the intestine ......................................... 131
4.3.5 Cytochrome P450 enzymes; intestinal expression patterns .................................... 136
4.4 Discussion ........................................................................................................................ 144
5. Intestinal localisation of Mdr1, Mrp2 and Bcrp in rat and human tissues ....................... 161
5.1 Introduction .................................................................................................................... 161
5.2 Methods .......................................................................................................................... 165
5.2.1 Western blot ............................................................................................................ 165
5.2.2 Immunocytochemistry ............................................................................................. 165
5.2.3 Immunohistochemistry ............................................................................................ 165
5.3 Results ............................................................................................................................. 166
5.3.1 Immunohistochemical detection of Mdr1 in rat intestine using the primary antibody
C19 .................................................................................................................................... 166
5.3.2 Immunohistochemical detection of Mrp2/MRP2 in rat and human intestine using
the primary antibody, M2-III-6 ......................................................................................... 186
5.3.3 Immunohistochemical detection of Bcrp in rat and human intestine using M70 and
BXP21 antibodies respectively .......................................................................................... 199
5.4 Discussion ........................................................................................................................ 221
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