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IDENTIFICATION OF POLYMORPHISMS IN THE NUCLEAR
RECEPTORS (PXR, CAR AND HNF4α) GENES IN THE LOCAL
POPULATION
HOR SOK YING
NATIONAL UNIVERSITY OF SINGAPORE
2007
ACKNOWLEDGEMENTS
I would like to thank my supervisor, Dr Theresa Tan, for her invaluable ideas,
suggestions and contributions for this project. Not to mention the countless amendments
for both my manuscript and thesis. Thanks for being so understanding and patient with
me all these years.
I would like to thank HaoSheng, Li Yang, Weiqi, Bai Jing, Yang Fei, Jasmine, and
Thomas for all the helps, suggestions and countless ideas for my project. For that and
much, much more I am extremely grateful. Thanks for being such great friends.
Most importantly, I would like to thank my husband, Dewayne, for being so supportive
and encouraging throughout my course of study. A big “Thank You” for my parents and
parents-in-law for all the help they have rendered to me.
Great thanks also go to: Dr Goh Boon Cher and Dr Lee Soo Chin for their precious
samples and suggestions; Lai San, for helping me with all the statistical analysis; Huiling
and Jiayi, for helping me with BigDye sequencing; and Rex, for helping me with
Pyrosequencing.
I am also grateful to the followings for permission to reproduce copyright material:
Elsevier
Lippincott & Williams Wilkins
The American Association for the Advancement of Science
And lastly, I would like to thank Biomedical Research Council of Singapore (BMRC
01/1/26/18/060) for their generosity in supporting this work.
i
TABLE OF CONTENTS
Acknowledgements…………………………………………………………………….. i
Summary……………………………………………………………………………….. iv
List of Tables…………………………………………………………………………... vi
List of Figures………………………………………………………………………….. viii
List of Abbreviations…………………………………………………………………... x
1.
Introduction……………………………………………………………………. 1
1.1 Drug Metabolism and Disposition…………………………………………1
1.2 Cytochrome P450………………………………………………………….4
1.2.1
CYP3A…………………………………………………………….. 8
1.2.2
CYP3A4……………………………………………………………10
1.2.3
CYP3A5……………………………………………………………12
1.2.4
CYP3A7……………………………………………………………12
1.2.5
CYP3A43…………………………………………………………..13
1.3 Nuclear Receptor………………………………………………………….14
1.3.1
Pregnane X Receptor………………………………………………17
1.3.2
Constitutive Androstane Receptor…………………………………18
1.3.3
Hepatocyte Nuclear Receptor 4-alpha……………………………..19
1.4 Regulation of CYP3A4 Expression by PXR, CAR and HNF4α………….23
1.5 Docetaxel………………………………………………………………….26
1.5.1
2.
Docetaxel Metabolism and Elimination Pathway…………………28
Objectives and Overview of the Study………………………………………...31
ii
3.
Materials and Method…………………………………………………………..35
3.1 Materials…………………………………………………………………...35
3.2 Methods……………………………………………………………………36
4.
3.2.1
Study Population………………………………………………….. 36
3.2.2
Genotyping…………………………………………………………38
3.2.3
Alignment of Sequences……………………………………………46
3.2.4
Statistical Analysis………………………………………………… 47
Results…………………………………………………………………………..49
4.1 Screening of PXR, CAR and HNF4α Genes in Local Healthy Population...49
4.1.1
Amplification of Exons and Sequencing…………………………...49
4.1.2
Variants in the PXR, CAR and HNF4α Genes……………………...53
4.2 Screening of PXR, CAR and HNF4α Genes in the Breast Cancer
Population………………………………………………………………….60
4.3 Comparing the Allele Frequencies between Local Healthy and
Breast Cancer Population………………………………………………….63
4.4 Pharmacokinetics Correlations……………………………………….……64
5.
Discussion……………………………………………………………………... 75
5.1 Exonic Variants in PXR, CAR and HNF4α genes…………………………76
5.2 PXR, CAR and HNF4α Genotypes and Docetaxel Pharmacokinetics…….79
6.
Conclusion …………………………………………………………………….81
7.
Publications…………………………………………………………………….82
8.
References……………………………………………………………………...83
iii
SUMMARY
The nuclear receptor (NR) superfamily is a large class of pharmacologically important
receptors that play vital roles in the defence mechanisms in the human body. It is
responsible for protecting the body from a diverse array of harmful endogenous and
exogenous toxins by modulating the expression of the genes involved in drug metabolism
and disposition. The detoxifying and elimination of these toxins is mainly mediated by
cytochrome P450 (CYP) enzymes, along with phase I and phase II drug metabolising
enzymes, as well as drug transporters.
Three closely related nuclear receptors, namely the pregnane X receptor (PXR),
constitutive androstane receptor (CAR) and hepatocyte nuclear factor 4-alpha (HNF4α)
have recently been identified as the master transcriptional regulators of CYPs expression.
The human CYP3A sub-family collectively comprises the largest portion of CYP
proteins expressed in the liver and they are involved in the metabolism of more than 60%
of all currently prescribed drugs. CYP3A4, the most abundantly expressed CYP3A
isoform, is considered as the main oxidase for these drugs in the liver. In recent years,
much work had been carried out to identify single nucleotide polymorphisms (SNPs) in
the three receptor genes (PXR, CAR and HNF4α) and to examine the significance of
these SNPs in relation to CYPs expression in terms of drug disposition or responsiveness.
It is hypothesized that genetic variation in these nuclear receptors may contribute to
human inter-individual variation in drug metabolism and also drug-drug interactions.
iv
The first part of this study aims to identify SNPs in the exons of the PXR, CAR and
HNF4α genes in the local healthy population. We identified a 5’ UTR variant for the
PXR gene (- 24381 A > C), one variant for the CAR gene (Pro180Pro) and two coding
variants for the HNF4α gene (Met49Val and Thr130Ile). The second part of this study
was conducted to screen for SNPs in breast cancer patients administered with docetaxel
in their chemotherapy treatment. The objective of the second study was to address the
clinical significance of the SNPs identified in the receptor genes in relation to docetaxel
kinetics. Docetaxel is an anti-cancer agent that is metabolised by CYP3A4. Thus, any
SNPs in these receptor genes could possibly affect docetaxel clearance in the breast
cancer patients. From our data, the same four variants were again identified in the breast
cancer cohort. No additional SNP was observed. Statistically, no significant correlation
was noted for the docetaxel clearance, body-surface-area normalised docetaxel clearance,
area under curve and half-life for PXR, CAR and HNF4α genes. In conclusion, the SNPs
identified in the PXR, CAR and HNF4α genes in this study appear not to have any
significant contribution to the variability in docetaxel clearance among the breast cancer
patients.
v
LIST OF TABLES
Table 1
Summary of the major drug metabolising cytochrome P450 enzymes,
their main tissue localisation and the anti-cancer agents which
they metabolise……………………………………………………………. 6
Table 2
Summary of the tissue distribution and type of reactions catalyzed by
some human cytochrome P450 enzymes involved in the maintenance
of cellular homeostasis……………………………………………………. 7
Table 3
List of reagents needed for this study and the suppliers………………….. 35
Table 4
A set of PCR forward and reverse primers that were used to amplify
each individual exonic region of the PXR gene…………………………. 40
Table 5
A set of PCR forward and reverse primers that were used to amplify
each individual exonic region of the CAR gene……………………….… 41
Table 6
A set of PCR forward and reverse primers that were used to amplify
each individual exonic region of the HNF4α gene……………………… 42
Table 7
Forward and reverse primers for pyrosequencing………………………. 45
Table 8
Polymorphisms identified in the PXR, CAR and HNF4α gene in the
healthy control population (n = 287)…………………………………… 56
Table 9
Genotypic distribution and allele frequencies of PXR, CAR
and HNF4α variants in healthy control………………………………... 57
Table 10 Comparison of SNP frequencies of PXR exon 1 variant……………… 58
vi
Table 11 Comparison of SNP frequencies of CAR exon 5 variant……………… 58
Table 12 Comparison of SNP frequencies of HNF4α exon 1C variant…………. 59
Table 13 Comparison of SNP frequencies of HNF4α exon 4 variant……………59
Table 14 Genotypic distribution and allele frequencies of PXR, CAR
and HNF4α variants in breast cancer patients (n = 101)……………….61
Table 15 Genotypic distribution and allele frequencies of PXR, CAR
and HNF4α for the different ethnic groups in the breast cancer
population (n = 101)………………………………………………….. 62
vii
LIST OF FIGURES
Figure 1
Pie chart illustrations of phase I and phase II drug metabolising
enzymes……………………………………………………………….. 3
Figure 2
The schematic organization of the human CYP3A locus………………9
Figure 3
Transcription factor binding sites within the regulatory regions
of human CYP3A4 gene……………………………………………….11
Figure 4
Structure of a typical nuclear receptor……………………………….. 15
Figure 5
The structure of HNF4α gene and its spliced isoforms……………… 22
Figure 6
An illustration of the effects of docetaxel in tumour cell……………. 27
Figure 7
Proposed metabolic pathways of docetaxel by CYP3A enzymes…... 30
Figure 8
A summary of the functions of PXR, CAR and HNF4α in drug
detoxification and elimination………………………………………. 33
Figure 9
Flow chart showing the study approach to identify PXR, CAR
and HNF4α SNPs in healthy subjects and breast cancer patients….. 34
Figure 10 The principle of Pyrosequencing……………………………………..44
Figure 11 PCR amplification of all nine PXR exons from patient genomic
DNA……………………………………………………………….... 50
Figure 12 PCR amplification of all eight CAR exons from patient genomic
DNA………………………………………………………………… 50
viii
Figure 13 PCR amplification of all twelve HNF4α exons from patient
genomic DNA…………………………………………….……….... 51
Figure 14 PCR amplification of PXR exon 1, CAR exon 5, HNF4α
exon 1C and HNF4α exon 4 from patient genomic DNA using
Pyrosequencing primers……….…………………………….……... 51
Figure 15 Electropherograms of PXR, CAR and HNF4α SNPs……….….….. 52
Figure 16 Docetaxel clearance (L/h/m2) against PXR exon 1, CAR
exon 5, HNF4α exon 1C and HNF4α exon 4 genotypes...………... 66
Figure 17 BSA normalised docetaxel clearance (L/h/m2) against PXR
exon 1, CAR exon 5, HNF4α exon 1C and HNF4α exon 4
genotypes…………………………………………………………... 68
Figure 18 Maximum concentration of docetaxel, Cmax, (mg/L) against
PXR exon 1, CAR exon 5, HNF4α exon 1C and HNF4α exon 4
genotypes……………………………………………………………70
Figure 19 Area under curve, AUC, (mg/L*h) against PXR exon 1, CAR
exon 5, HNF4α exon 1C and HNF4α exon 4 genotypes………..… 72
Figure 20 Half life, t1/2, (hours) against PXR exon 1, CAR exon 5, HNF4α
exon 1C and HNF4α exon 4 genotypes…………………………… 74
ix
LIST OF ABBREVIATIONS
ADH
Alcohol dehydrogenase
AF-1
Activation function 1
AF-2
Activation function 2
ALDH
Aldehyde dehydrogenase
APS
Adenosine 5’ phosphosulfate
AUC
Area under the concentration-time curve
Bp
Base pair
CAR
Constitutive androstane receptor
CCD
Charged coupled device
CL
Clearance
CLEM
Constitutive liver enhancer module
Cmax
Maximum concentration
COMT
Catechol O-methyl-transferase
CRE
cAMP response element
CYP
Cytochrome P450
CYPOR
cytochrome P450 oxidoreduactase
DBD
DNA binding domain
DME
Drug metabolizing enzyme
dNTP
Deoxyribonucleotide triphosphate
DPD
Dihydropyrimidine dehydrogenase
DR
Direct repeat
ER
Everted repeat
EST
Expressed-Sequence Tag
GRE
Glucocorticoid responsive element
GST
Glutathione S-transferase
HMT
Histamine methyl-transferase
HNF4α
Hepatocyte nuclear factor 4- alpha
HSP
Heat shock protein
Ile
Isoleucine
x
IR
Inverted repeat
k
Elimination rate constant
LBD
Ligand binding domain
Met
Methionine
MDR
Multidrug resistance
MODY
Maturity-onset diabetes in the young
MRP
Multidrug resistance associated protein
NADPH
Nicotinamide adenine dinucleotide
NAT
N-acetyl-transferase
NHR
Nuclear hormone receptor
NQO1
NADH:quinone oxidoreductase or DT diaphorase
NR
Nuclear receptor
NUMI
National University Medical Institute
OATP
Organic anion transporter
PAR
Pregnane activated receptor
PCN
Pregnenolone 16α-carbonitrile
PCR
Polymerase chain reaction
P-gp
P-glycoprotein
PPi
pyrophosphate
Pro
Proline
PXR
Pregnane X-receptor
RE
Response element
RORα
Retinoic acid receptor alpha
RXR
Retinoid X receptor
SAP
Shrimp Alkaline Phosphatase
SNP
Single nucleotide polymorphism
ST
Sulfotransferase
SULT
Sulfotransferase
SXR
Steroid and xenobiotic receptor
t1/2
Half life
TAE
Tris-Acetate-EDTA
xi
Thr
Threonine
TPMT
Thiopurine methyl-transferase
UGT
Uridine 5’-triphosphate glucuronosyltransferases.
USF-1
Upstream stimulatory factor 1
UTR
Untranslated region
Val
Valine
XREM
Xenobiotic-responsive enhancer module
xii
1.
INTRODUCTION
1.1
Drug Metabolism and Disposition
The body’s first line of defence against the accumulation of potential toxic
endogenous and exogenous lipophilic compounds is the liver. This is the site
where drugs and toxic xenobiotics are being transformed to less toxic water
soluble metabolites that subsequently can be excreted out of the body. In multicellular organisms, two different defence mechanisms have evolved for this
purpose, biotransformation and transport. Biotransformation and transport
processes comprise three phases; phase I (functional reaction) and phase II
(conjugative reaction) form the biotransformation or drug metabolism pathway
while phase III forms the drug transportation and disposition pathway (Gibson
and Skett, 2001).
The phase I enzymes are responsible for primary modification of lipophilic
compounds into more polar forms. Phase I reactions generally include oxidation,
reduction, hydrolysis, hydration, dethioacetylation and isomerisation. On the other
hand, Phase II reactions include glucuronidation, glycosidation, methylation, Nacetylation, sulfation, amino acid and glutathione conjugation. (Gibson and Skett,
2001; Handschin and Meyer, 2003) Phase II comes into play by acting on phase I
metabolites or on the parent compounds to further convert or detoxify to inactive
derivatives, which accounts for bulk of the excreted products. Thus, phase II
1
reactions are considered the true “detoxification” pathway. However, there are
instances where phase II reactions lead to reactive metabolites. Phase III
comprises the transport and elimination steps where the parent drug and its
metabolites are exported out of the cell and eventually removed from the body
through the bile or urine. Figure 1 illustrates the contribution of phase I and phase
II enzymes to the metabolism of drugs.
2
Figure 1: Pie chart illustrations of phase I and phase II drug metabolising
enzymes. The relative size of each section on the charts show the relative
contribution of each phase I and phase II enzymes to drug metabolism. Phase I
enzymes are responsible for modification of functional groups and phase II is
involved in conjugation with endogenous substituents. ADH, alcohol
dehydrogenase; ALDH, aldehyde dehydrogenase; CYP, cytochrome P450; DPD,
dihydropyrimidine dehydrogenase; NQO1, NADH:quinone oxidoreductase or
DT diaphorase; COMT, catechol O-methyl-transferase; GST, glutathione Stransferase; HMT, histamine methyl-transferase; NAT, N-acetyl-transferase;
STs, sulfotransferase; TPMT, thiopurine methyl-transferase; UGTs, uridine 5’triphosphate glucuronosyltransferases. Adapted from Evans and Relling, 1999,
with permission from The American Association for the Advancement of
Science.
3
1.2
Cytochrome P450
Cytochrome P450 (CYPs) enzymes were first discovered in 1958 by Martin
Klingenberg while studying the spectrophotometric properties of rat liver
microsomal pigments. The name P450 was derived from the property of these
pigments which has a maximum absorbance reading at 450nm (Hasler et al.,
1999). CYPs constitute a superfamily of heme-thiolate containing proteins that
belong to a group of enzymes involved in hepatic detoxification of endogenous
and exogenous compounds (phase I enzymes). CYP, together with its reducing
counterpart nicotinamide adenine dinucleotide (NADPH) - cytochrome P450
oxidoreductase (CYPOR), is able to catalyze mono-oxygenase reactions with
lipophilic compounds by allowing the attachment of a hydroxyl group as a
reactive group that can later be modified by phase II enzymes (Handschin and
Meyer, 2003).
CYPs play an important role in the maintenance of the human cellular
homeostasis. Predominately express in the human liver, CYPs metabolize a wide
spectrum of endogenous steroid hormones, bile acids, fatty acids and xenobiotic
substrates such as drugs, carcinogens, food additives, pollutants and
environmental chemicals.
In human beings, there are 18 known CYP gene families and 43 subfamilies
(Kretschmer and Baldwin, 2005; Nelson DR, 2003). Only three of these, CYP1,
4
CYP2 and CYP3, are actively involved in drugs and xenobiotics metabolism.
Members of CYP1A, CYP2B, CYP2C and CYP3A gene subfamilies are highly
inducible by a diverse array of xenobiotics (Handschin and Meyer; 2003). Besides
being involved in drug metabolism, these CYPs also play an important role in
cholesterol biosynthesis, vitamin D metabolism, bile acid metabolism,
biosynthesis and catabolism of steroids (Pascussi et al., 2003; Nelson DR, 1999).
In the CYP family, the major isoforms responsible for drug metabolism are
CYP2C9, CYP2C19, CYP2D6 and CYP3A4 (Ingelman-Sundberg, 2004). Table 1
shows the main tissue localisation of these CYPs and their anti-cancer substrates
(Ingelman-Sundberg, 2004; Van Schaik, 2005).
Different forms of CYP are found to be expressed in intestine, lung and kidneys
but the liver is the major site for CYP-mediated oxidative metabolism, with
CYP3A family as the dominant class. In this study, the focus will be on the
CYP3A sub-family members. This is because of their dominant role in drug
metabolism in the liver, and their regulation by nuclear receptors. Table 2 shows
the reactions catalysed by CYPs in humans and their tissue localization.
5
Cytochrome P450
Main Tissue
Enzyme
Localisation
CYP2C9
CYP2C19
CYP2D6
Liver
Liver
Liver
Anti-cancer Agent
Cyclophosphamide
Tamoxifen
Ifosfamide
Tegafur
Thalidomide
Ifosfamide
Cyclophosphamide
Tamoxifen
Tamoxifen
Gefitinib / Iressa
CYP3A4
Liver
Intestine
Cyclophosphamide
Imatinib / Gleevec
Ifosfamide
Irinotecan
Docetaxel
Paclitaxel / Taxol
Etoposide
Teniposide
Flutamide
Tamoxifen
Gefitinib / Iressa
Vinca-alkaloids
Table 1: Summary of the major drug metabolising cytochrome P450 enzymes,
their main tissue localisation and the anti-cancer agents which they metabolise.
Adapted from Ingelman-Sundberg, 2004; Van Schaik, 2005.
6
Tissue
Liver and Intestine
Function
(i)
Bile acid formation
(ii)
Polyunsaturated fatty acid epoxidation
(iii) Xenobiotic metabolism
► N- & O-dealkylations
► Alcohol oxidation
► Alkane & Arene oxygenation
► Aromatic hydroxylation
Kidney
(i)
Omega hydroxylation of fatty acids
Adrenal
(i)
21-OH of Progesterone
Placenta
(i)
17α-OH of Pregnenolone
Ovary
(i)
Aromatase
Table 2: Summary of the tissue distribution and type of reactions catalyzed by
some human cytochrome P450 enzymes involved in the maintenance of cellular
homeostasis. Reactions include (a) synthesis and degradation of prostaglandins
and other unsaturated fatty acids, (b) metabolism of cholesterol to bile acids and
(c) metabolism of endogenous and exogenous compounds (Hasler et al., 1999)
7
1.2.1
CYP3A
The human CYP3A sub-family is relatively small, comprising only four members;
CYP3A4, CYP3A5, CYP3A7 and CYP3A43 which are mapped on human
chromosomal position 7q21-q22.1 (Figure 2) (Gellner et al., 2001; Plant, 2007).
CYP3As can be induced by a large array of compounds. These include naturally
occurring and synthetic glucocorticoids, pregnane compounds such as
pregnenolone 16α-carbonitrile (PCN) and macrolide antibiotics like rifampicin
(Quattrochi and Guzelian, 2001). Inter-individual variability in induction of
CYP3A activity by these compounds could be due to the genetic variation of
CYP3A sub-family members or its transcription regulators. To date, the number of
variants for CYP3A4, 3A5, 3A7 and 3A43 are 40, 24, 7 and 5 respectively. This
information is published on the official allele nomenclature committee website
(http:www.imm.ki.se/CYPalleles) (Plant, 2007).
As the most abundantly expressed CYP3A isoform in the human liver and
intestine, CYP3A4 is one of the best studied member of the CYP3A gene subfamily. CYP3A4 plays a crucial role in the metabolic elimination of a broad range
of structurally diverse substrates and thus contributes critically to the first-pass
and systemic metabolism in the human body.
8
CYP3A43
CYP3A4
CYP3A7
CYP3A5
Figure 2: The schematic organization of the human CYP3A locus. The assembled 231kb
sequence contains the four CYP3A sub-family members, CYP3A4, CYP3A5, CYP3A7 and
CYP3A43. This cluster is localised on chromosome 7q21-7q22.1 (Burk and Wojnowski,
2004; Finta and Zaphiropoulos, 2000). Both CYP3A4 and CYP3A5 genes contain 502
amino acids each and they have molecular weights of 57299 Dalton and 57109 Dalton
respectively. CYP3A7 and CYP3A43 genes each contain 503 amino acids with molecular
weights of 57526 Dalton and 57670 Dalton respectively. This information is published on
http://www.genecards.org.
9
1.2.2
CYP3A4
CYP3A4 is most abundantly expressed in the liver and small intestine. Accounting
for 30-40% of the total CYPs in the liver, CYP3A4 is considered as the main
oxidase for xenobiotics in this organ. It is catalytically effective on cyclosporine,
macrolide antibiotics, anti-cancer agents such as taxol and is responsible for the
metabolism of more than 60% of the prescribed drugs marketed today (Hasler et
al., 1999; Kretschmer and Baldwin, 2005; Plant, 2007). CYP3A4 is also expressed
weakly in stomach, colon, lung and adrenal (Guengerich, 2005).
Other than its own genetic variations that could possibly contribute to intervariation in drug metabolism, genetic variation in its regulatory transcriptional
partners could also affect how efficiently the gene is transcribed and expressed.
This would eventually have an impact on drug clearance processes, which is an
important determinant of drugs efficacy and toxicity. In recent studies, pregnane
X-receptor (PXR), constitutive androstane receptor (CAR) and hepatocyte nuclear
factor 4- alpha (HNF4α) have been identified to serve key roles in regulating
CYP3A4 transcription (Quattrochi and Guzelian, 2001; Burk and Wojnowski,
2004). This seems reasonable as PXR, CAR and HNF4α binding sites have been
revealed in the CYP3A4 gene (Figure 3) and would be further discussed in Section
1.4 Thus, any variability in these transcriptional controls could also either upregulate or down-regulate CYP3A4 activity.
10
-11400
HNF 4
HNF 1
HNF 4
E-Box
(USF1)
E-Box
(USF1)
CRE
(AP-1)
E-Box
(USF1)
-10900
CLEM4 Region
-7840
-7270
P/GRE
HNF 4
RORα
CAAT
PXRE
PXRE
P/GRE
XREM Region
Figure 3: Transcription factor binding sites within the regulatory regions of human
CYP3A4 gene. The two regulatory regions shown are CLEM4 and XREM that lies
upstream of CYP3A4 promoter (Plant, 2007). HNF4α binding sites have been identified
in both CLEM4 and XREM regions of CYP3A4 gene. PXR and CAR bind to the same
binding site, PXRE, in the XREM region.
11
1.2.3
CYP3A5
CYP3A5 or H1p3 has approximately 85% sequence identity to CYP3A4. CYP3A5
is found to be expressed in liver, small intestine, kidney, lung, prostate and
adrenal gland. CYP3A5 accounts for approximately 20% of total hepatic CYPs.
Unlike CYP3A4, CYP3A5 is polymorphically expressed in fetal liver. The
regulation and catalytic selectivity of CYP3A5 has also been documented.
Comparison of the metabolic capabilities of the CYP3A isoforms for a series of
CYP3A substrates (including midazolam, alprazolam, triazolam, clarithromycin,
tamoxifen, testosterone, estradiol, diltiazem, nidefipine and 7-benzyloxy-4trifluoromethylcoumarin) showed that CYP3A5 generally has lower affinities for
these substrates than CYP3A4 (Williams et al., 2002). In addition, the clearance
values were also lower for most of the substances except for the clearance for 1’hydroxy midazolam.
1.2.4
CYP3A7
CYP3A7 was initially named as HFLa. It is the main CYP present in human fetal
liver, when CYP3A4 is not expressed. CYP3A7 was believed to be significantly
down regulated after birth, even though low levels of approximately less than 2%
of the total CYPs in adult liver has been detected in some individuals (De Wildt et
al., 1999; Guengerich, 2005). Other known expression sites include kidney, lung
12
and adrenal. Compared to other CYP3A members, less work has been done on
CYP3A7 in relation to drug metabolism. However, it has been shown that
CYP3A7 has a significantly weaker metabolic capacity (in terms of affinity and
clearance) when compared to CYP3A4 (Williams et al., 2002).
1.2.5
CYP3A43
CYP3A43 was first characterized in 2001 by three groups and is found to be
expressed in liver, kidney, pancreas and prostate (Domanski et al., 2001; Gellner
et al., 2001; Westlind et al., 2001). The level of expression of CYP3A43 was
considerably low in human liver, accounting for only approximately 0.1% of
CYP3A isoforms (Guengerich, 2005). Since, the main site of drug metabolism in
the human system is the liver, CYP3A43 is deemed to make little contribution to
this aspect due to its low expression.
13
1.3
Nuclear Receptors
Nuclear hormone receptors (NHRs) constitute a superfamily of ligand-dependent
and ligand-independent transcription factors that govern important physiological
processes such as development, homeostasis and disease. To date, more than 50
nuclear receptors have been identified in various species. They generally have two
transcription activation function domains (AF-1 and AF-2) located at the amino
and carboxyl termini respectively, a zinc finger DNA binding domain and a
ligand binding domain as illustrated in Figure 4. NHRs are able to induce or
regulate drug metabolism by binding to small lipophilic ligands. Following ligand
binding and dimerization, they then bind to DNA element repeats of the
nucleotide hexamers in different arrangements like the ones found in drugresponsive enhancers of CYPs. The hexamers can be arranged either as direct
repeats (DR), everted repeats (ER) or inverted repeats (IR). Upon binding to a
specific ligand, the receptor may undergo a conformational change that either
facilitates the binding of co-activator proteins or interaction to fellow transcription
factors which eventually regulates the transcriptional activity of the target gene.
14
N
AF-1
DBD
Hinge
LBD
AF-2
Figure 4: Structure of a typical nuclear receptor. Nuclear receptors share a
common modular structure which consists of activation function 1 (AF-1) domain
located at the amino-terminal and AF-2 domain at the carboxy-terminal. The
DNA binding domain (DBD) is connected to the ligand binding domain (LBD) by
a flexible hinge. Upon ligand binding to the LBD, AF-2 will undergo a
conformational change that disrupts interaction with transcriptional co-repressors
and allows the interaction with transcriptional co-activators. The activated nuclear
receptor will then bind to the response elements on the regulatory regions of
target genes and initiates transcription.
15
C
NHRs are categorized into three main classes. Class I receptors bind to steroid
hormones and in absence of ligand, these receptors are associated with molecular
chaperones like heat shock proteins (HSPs). Class II receptors bind to thyroid
hormone, vitamin D3, 9-cis-retinoic acid and trans-retinoic acid. Upon binding of
ligands, class II receptors dimerizes with retinoid X receptor (RXR). In both
cases, upon binding ligand binding, the receptors may undergo conformational
changes that eventually cause the dissociation of co-repressors and binding of coactivators. Class III belongs to a group of receptors whose physiological ligands
have not yet been identified (Handschin and Meyer, 2003; Pascussi et al., 2003)
Over the past decades, some members of the NHR superfamily were termed
‘orphan’ receptors because at the time of their cloning, nothing was known about
their physiological ligands or co-activators. Till now, the term remains for these
receptors even though their ligands are now known. The first ‘orphan’ receptor
was identified in 1988 and since then, the number of orphan receptors has
increased tremendously (Wang and LeCluyse, 2003; Kliewer, 2005). Scientists
are not only interested in researching on the novel physiological ligands of these
orphan receptors but also the biological functions and signaling cascades that
these receptors elicit. The next part of the introduction will focus on three
members of the NHR superfamily (PXR, CAR and HNF4α) and their relationship
with the CYP family.
16
1.3.1
Pregnane X Receptor
The pregnane X receptor (PXR, NR1I2), also known as pregnane activated
receptor (PAR) or the steroid and xenobiotic receptor (SXR), is a member of the
NHR superfamily. The PXR gene is located on chromosome 3q12-q13.3 and
consists of nine exons. The size of this gene is approximately 38kb. The length of
the unprocessed precursor protein is 434 amino acid long and it has a molecular
weight of 49762 Dalton (http://www.expasy.org/uniprot/O75469). PXR was first
discovered in 1997 from a search performed on the Washington University Mouse
Expressed-Sequence Tag (EST) database (Kliewer et al., 1998). It derived its
name based on its activation by 21-carbon steroids (pregnanes), namely
pregnenolone 16α-carbonitrile (PCN) (Willson and Kliewer, 2002; Kliewer,
2005)
PXR coordinates the induction and regulation of phase I and II DMEs, and phase
III drug transporters that accelerate systemic clearance upon drug exposure. Phase
I enzymes that are regulated by PXR include CYP3As, CYP2Bs and CYP2Cs
(Bertilsson et al., 1998; Blumberg et al., 1998; Kliewer et al.,1998; Lehmann et
al., 1998). Phase II enzymes include uridine diphospho-glucuronosyltransferases
1A1 (UGT1A1), glutathione-S-transferase 1 (GST1) and sulfotransferase 2A1
(SULT 2A1), and phase III enzymes include drug efflux transporters such as
multidrug resistance 1 (MDR1), multidrug resistance associated proteins 2, 3 and
4 (MRP2, MRP3 and MRP4) as well as uptake transporter like Na+-independent
17
organic anion transporter 2 (Oatp2) (Kretschmer and Baldwin, 2005; Lamba et
al., 2005).
PXR resides in the cytoplasm. Upon activation by ligand binding, PXR
heterodimerised with RXR and subsequently binds to PXR response elements in
the promoter region of target genes, such as the CYP3A genes. PXR, like its
primary target CYP3A4, is expressed predominantly in the liver and to a lesser
extent in the colon and small intestine (Blumberg et al., 1998; Kliewer et al.,
1998; Lehmann et al., 1998; Lamba et al., 2005)
1.3.2
Constitutive Androstane Receptor
The constitutive androstane receptor (CAR, NR1I3) was initially known as
MB67. CAR regulates the drug metabolism and disposition pathway and the gene
encoding for CAR is located on chromosome 1q23.3 and consists of 9 exons. The
size of this gene is approximately 8.5kb. The length of the unprocessed precursor
protein is 352 amino acid long and it has a molecular weight of 39942 Dalton
(http://www.expasy.org/uniprot/Q14994). CAR was first identified in 1994
through screening of a cDNA library using a nuclear receptor DNA binding
domain (DBD)-based oligonucleotide probe. It was originally known as
constitutive activated receptor as it could transactivate target gene as a
18
heterodimerised complex with RXR in the absence of ligands (Wang and
LeCluyse, 2003; Lamba et al., 2005)
CAR, like PXR, is predominately expressed in liver and intestine, and regulates a
variety of drug detoxifying genes (Pascussi et al., 2003). These include the
CYP3As, CYP2Bs, CYP2Cs, UGT1A1, GST, SULT, MDR1, MRP2, MRP3 and
MRP4 (Handschin and Meyer, 2003; Lamba et al., 2005). CAR resides in the
cytoplasm and translocates into the nucleus upon binding to its ligand (Kawamoto
et al., 1999; Savkur et al., 2003; Tirona and Kim, 2005). Interestingly, there are
evidences that suggest ligand binding is not always necessary for CAR
translocation (Goodwin and Moore, 2004). Thus, it appears that there might be
some unidentified cellular signaling cascade regulations, crosstalk or feedback
mechanisms that could trigger the translocation and activation of CAR.
1.3.3
Hepatocyte Nuclear Receptor 4-alpha
Hepatocyte nuclear receptor 4-alpha (HNF4α, NR2A1) is a member of the NHR
that was first identified in 1989 in crude rat liver nuclear extracts (Costa et al.,
1989; Sladek et al., 1990). It plays important roles in metabolic processes in the
liver and is involved in glucose homeostasis and insulin secretion in the pancreas.
HNF4α also plays a critical role in development and cell differentiation (Odom et
19
al., 2004). HNF4α is highly expressed in liver, kidney and intestine and to a
lesser extent in pancreas and stomach (Sladek and Seidel, 2001)
This gene is localized on chromosome 20q12-q13.1 and consists of 10 exons. The
size of this gene is approximately 75.6kb. The length of the unprocessed precursor
protein is 474 amino acid long and it has a molecular weight of 52785 Dalton
(http://www.expasy.org/uniprot/p41235). At least nine possible HNF4α isoforms
can be generated through alternative promoters (P1 and P2) usage and splicing
(Figure 5), although to date, only four have been detected in vivo. The P2
promoter lies at approximate 45.5kb upstream of P1 promoter. The different
isoforms expression varies with development stages, differentiation and tissue
origin. In adult liver and kidney, the expression of HNF4α is initiated mainly at
the P1 promoter (Nakhei et al., 1998; Sladek and Seidel, 2001). It was also
demonstrated that the HNF4α P1 promoter transcription site exhibits stronger
transcriptional activity and recruits co-activators more efficiently as compared to
the P2 promoter. This could be explained by the presence of the activation
function domain AF-1 which is encoded by exon 1A proximal to P1 promoter
initiation site. AF-1 plays a crucial role in HNF4α transcriptional potential and
interaction with its co-activators (Green et al., 1998; Kistanova et al., 2001;
Eeckhoute et al., 2001; Eeckhoute et al., 2003).
HNF4α was originally known as an ‘orphan’ nuclear receptor as its physiological
ligands were not identified. However, fatty acyl-CoA thioesters were recently
20
shown to be the natural putative ligand of HNF4α (Dhe-Paganon et al., 2002).
Like PXR and CAR, HNF4α also plays a significant role in the regulation of
CYPs. But unlike them, HNF4α is not associated with RXR and functions as
homodimers that subsequently bind to specific DNA response elements.
21
Figure 5: The structure of HNF4α gene and its spliced isoforms. Adapted from Sladek and
Seidel, 2001, with permission from Elsevier
22
1.4
Regulation of CYP3A Expression by PXR, CAR and HNF4α
PXR and CAR are considered to be the master regulators of drug clearance in the
body because of their close relationship with the key DMEs, CYP3A4 and
CYP2B6. This was further supported by studies which show that PXR-RXRα and
CAR-RXRα heterodimers are capable of binding to the proximal promoter region
of the CYP3A4 gene and mediates PXR or CAR transactivation of the CYP3A4
promoter respectively (Bertilsson et al., 1998; Goodwin et al., 2002; Akiyama
and Gonzalez, 2003). The activation of CYP3A4 promoter by the nuclear
receptors heterodimer is dependent upon ligand binding leading to the binding of
the nuclear receptors to the response elements (REs) in the 5’ flanking region of
CYP3A4. Transactivation of CYP3A4 by PXR and CAR upon ligand activation is
mediated by the “proximal ER6” element located at -153bp to -170bp, and by the
DR3 motif at the distal xenobiotic-responsive enhancer module (XREM)
(Goodwin et al., 2002) (Figure 3). It has also been shown that regulation by PXR
and CAR extended well beyond the CYP3A4 gene. Together, PXR and CAR coregulate members of CYP2B, CYP2C, GST, SULT, MRP2 and UGT families
(Kliewer et al., 2002).
In 2004, a second enhancer module that confers constitutive activation of the
CYP3A4 gene was identified by Matsumura’s group. It is known as the
constitutive liver enhancer module (CLEM4) and is located between -11400bp
and -10900bp upstream of CYP3A4 promoter (Figure 3). Because of the poor
23
sequence conservation of CLEM4 between the CYP3A gene members, CLEM4
appears to be specific for regulation of CYP3A4 expression (Matsumura et al.,
2004; Plant, 2007).
Beside PXR and CAR, HNF4α also emerges as one of the widely acting
transcription factor in the liver. Not only does HNF4α regulates DMEs such as
CYP3A4, CYP3A5, CYP2A6, CYP2B6, CYP2C9 and CYP2D6, it is also
involved in transcriptional regulation of glucose, cholesterol, fatty acid, urea and
bile acid metabolism (Akiyama and Gonzalez, 2003). In addition, mutations in
human HNF4α gene is linked to maturity-onset diabetes in the young (MODY1)
and is characterized by defective secretion of insulin by the pancreatic-β cells
(Ryffel, 2001; Gupta et al., 2005). Most importantly, an HNF4α binding site was
identified in both the PXR and CYP3A4 promoter region (Kamiya et al., 2003;
Burk and Wojnowski, 2004). In addition, two other binding sites have been
identified in the CYP3A4 gene, one within the CLEM4 region and the other within
the XREM region (see Figure 3). The HNF4 binding site identified within the
CLEM4 has been assessed and was found to be necessary for maximal enhancer
activity (Plant, 2007). The HNF4α binding site (DR1) identified in the CYP3A4
XREM region lies between -7783bp and -7771bp, adjacent to the DR3 motif. In
the studies done by Tirona et al (2003), it was demonstrated that the binding of
HNF4α to DR1 can confers maximal PXR-mediated transcriptional activation.
Similar to that observed for PXR, HNF4α was also able to augment the activation
of the CYP3A4 promoter by CAR.
24
Other sources of evidence that support the importance of PXR, CAR and HNF4α
as the regulators of CYP3A induction came from work in animal models. PXR and
CAR null mice failed to induce CYP3A activity upon induction by respective
agonists. HNF4α deficient fetal mice, too, demonstrated absence of CYP3A and
PXR mRNAs. Reduced level CYP3A mRNA was also observed in adult mice
with deleted hepatic Hnf4a gene (Honkakoski et al., 2003). Thus, it is now
evident that HNF4α not only influences the basal activity of the CYP3A promoter,
it is also necessary for maximal PXR- or CAR- mediated transcriptional activation.
Hence, any sequence variations in the three nuclear receptors, PXR, CAR and
HNF4α would likely contribute to altered induction of target DME genes such as
CYP3A and this would eventually affects the systemic clearance of xenobiotics,
homeostasis, development and disease.
25
1.5
Docetaxel
Docetaxel [4 - acetoxy - 2α - benzoyloxy - 5β, 20 – epoxy - 1, 7β - 10β trihydroxy – 9 – oxotax – 11 - ene13α - y1 - (2R, 3S) – 3 – tert – butoxycarbonyl
– amino – 2 - hydroxyphenylpropionate] or Taxotere®, RP56976, is a semisynthetic compound. It is modified from a non-toxic precursor, 10-deacetyl
baccatin III, which is extracted from needles of the European yew tree (Taxus
baccata L.). It is semi-synthetically prepared from 10-deacetyl baccatin III via a
direct acylation at the C13 position and then esterified with a synthetic side chain
(Shou et al., 1998; Clarke and Rivory, 1999). It has an anhydrous molecular
weight of 807.9 or 861.9 in the trihydrate form. The chemical formula of
docetaxel is C43H53NO14 (Clarke and Rivory, 1999). Docetaxel, a member of the
taxane family, is a highly effective broad spectrum anticancer agent used in
treatment of solid malignancies such as breast, ovarian, lung, head and neck
cancers (Baker et al., 2006).
Docetaxel acts as an anti-microtubule agent that stabilizes microtubulin assembly
and inhibits depolymerisation, thereby disrupting the microtubule dynamic
networks. This leads to a series of event including arrest in G2/M phase of the cell
cycle and apoptosis. Docetaxel also inhibits angiogenesis, the process whereby
tumors develop new capillary blood vessels (Herbst and Khuri, 2003). Figure 6
summarized the effects of docetaxel in tumour cell.
26
Docetaxel
Inactive
metabolites
EFFLUX
P-glycoprotein
CYP3A4
Docetaxel
β-tubulin
Apoptosis
G2/M
Arrest
TUMOR CELL
Figure 6: An illustration of the effects of docetaxel on tumour cell. Docetaxel
can be metabolised by CYP3A4 into inactive metabolites or export by Pglycoprotein (p-gp) as it is a substrate of this transporter. Docetaxel also acts
as an anti-microtubule agent that stabilizes microtubulin assembly and
inhibits depolymerisation. This could leads to a series of event including
arrest in G2/M phase of the cell cycle and apoptosis.
27
1.5.1
Docetaxel Metabolism and Elimination Pathway
Docetaxel is metabolized mainly by hepatic CYPs and eliminated through the
bile. The CYP3A class of enzymes dominates at this tissue site, therefore, it is
necessary to address the two important members that played a key role docetaxel
metabolism; CYP3A4 and CYP3A5 (Shou et al., 1998; Baker et al., 2006). Of
these two enzymes, CYP3A4 is considered the main enzyme in docetaxel
metabolism (Marre et al., 1996; Hirth et al., 2000). Unlike CYP3A4, CYP3A5 is
polymorphically expressed in some individuals, varying greatly by race and
ethnic group. Even though, individuals with polymorphically expressed CYP3A5
isoform is capable of contributing approximately up to 50% of total hepatic CYPs
or comparable level to CYP3A4, the latter is still the dominant contributor in
docetaxel metabolism. It is believed that low expression of CYP3A4 in some
adults is compensated by increased CYP3A5 polymorphic expression in these
individuals. This suggests that individual difference in CYP3As expression may
give rise to variability in docetaxel metabolism among individuals (Burk and
Wojnowski, 2004; Vaclavikova et al., 2004).
Docetaxel is metabolised by CYP3A4 to inactive metabolites (Figure 7). High
CYP3A4 activity would, therefore, result in poor therapeutic outcome of the drug,
while low CYP3A4 activity may cause toxicity in patients (Hirth et al., 2000; Goh
et al., 2002; Rodriguez-Antona and Ingelman-Sundberg., 2006).
28
Besides individual variability in CYP3As expression, individual variability in
nuclear receptor genes (PXR, CAR and HNF4α) that regulate CYP3As
transcription is also a factor to take note of in docetaxel metabolism. Any
sequence variation in these receptors would first affect the interaction between the
receptors and ligand or CYP3As, followed by changes in the transcription
activity. This cascade of reactions, in turn, may alter docetaxel metabolism by
these CYP3As. A combination of factors such as genetic variation of CYP3A4
regulatory genes (PXR, CAR and HNF4α) and CYP3A4 genotypes may influence
the outcome of chemotherapy treatment in cancer patients.
Docetaxel metabolism involves the initial hydroxylation of the methyl of the tertbutyl ester group on the side chain that generates a primary alcohol metabolite;
evident of CYPs metabolism. A subsequent oxidation of a putative aldehyde
yields two diastereomeric hydroxyl-oxazolidinones after cyclization (Figure 7)
(Shou et al., 1998; Baker et al., 2006). These non-cytotoxic inactive derivatives
are then disposed off in faeces and urine. Cellular efflux of the metabolites is
facilitated by intestinal efflux transporter P-glycoprotein (P-gp) (Baker et al.,
2006). It was also documented that docetaxel is also a substrate for P-gp but
rather than being transport-dependent, CYP3A metabolism is the prominent
elimination route for docetaxel. This was supported by observations whereby
mice lacking the ABCB1 gene (also known as MDR1) that encodes P-gp have
similar drug clearance as wild type mice (Baker et al., 2006). However, MDR1
29
expression could be regulated by PXR and this may also influence docetaxel
elimination.
Hydroxy - DOC
CYP3A4
CYP3A5
CYP3A4
Aldehyde - DOC
Docetaxel (DOC)
R- and S- Hydroxyoxazolidinones
Figure 7: Proposed metabolic pathways of docetaxel by CYP3A enzymes. Adapted
from Shou et al., 1998, with permission from Lippincott & Williams Wilkins.
30
2.
OBJECTIVES AND OVERVIEW OF THE STUDY
Drug metabolism and disposition is an important physiological process. With the
daily exposure to exogenous chemicals or ‘xenobiotic’, such as food additives,
pesticides, insecticides and pollutants either indigested in the diet or absorbed
through the skin or lungs, the detoxification and elimination system forms a
crucial surveillance role in the human body.
Human CYP3A4 is a major CYP enzyme that is present abundantly in human
liver and it influences the inter-individual variability of drug response and drugdrug interaction in the human system. As mentioned in the previous chapter,
nuclear receptors and transcription factors may be the important determinants of
CYP3A4 transcriptional activation and regulation (Figure 8).
In recent years, it has been acknowledged that genetic polymorphisms contribute
to great alteration in terms of drug disposition or responsiveness. A large number
of single nucleotide polymorphisms (SNPs) in the three genes (PXR, CAR and
HNF4α) have been identified and reported in many populations such as African
Americans, European and Asian like the Japanese. However, this has not been
done in the local population. Therefore, it would be of great interest to determine
the SNPs in PXR, CAR and HNF4α genes in the local population and to establish
the importance (if any) of the identified SNPs as potential determinant of altered
drug disposition profiles.
31
Singapore is a multiethnic country comprising of three major ethnic groups –
Chinese, Malay and Indian. This study was carried out firstly to determine the
polymorphisms in the coding regions of PXR, CAR and HNF4α genes in the three
ethnic groups in normal control populations. After the screening for SNPs in the
nuclear receptor genes of the ethnic groups, this study was taken further to screen
for the SNPs in breast cancer patients treated with docetaxel. In vitro studies have
demonstrated that CYP3A4 is the main enzyme involved in docetaxel metabolism
(Marre F et al., 1996; Hirth et al., 2000). As mentioned earlier, CYP3A4 is
transcriptional regulated by PXR, CAR and HNF4α (Figure 8), therefore, any
SNPs in these receptor genes could possibly contribute to variability in docetaxel
response and clearance in the cancer patients. The purpose of the second part of
this study was thus to explore the role of PXR, CAR and HNF4α polymorphisms
in the variability of docetaxel pharmacokinetics in the local population. An
illustration of the study approach is shown in Figure 9.
32
HNF4α
PXR/CAR
RXR
CYP3A4
Drug
Enzyme
CYP3A4
CLEM4 / XREM
Nucleus
Drug
OH
Drug
UGT
SULT
GST
MDR1
CONJUGATE
Cytoplasm
Drug
MRP2
Drug Elimination
/or Clearance
Figure 8: A summary of the functions of PXR, CAR and HNF4α in drug detoxification
and elimination. The nuclear receptors are activated by endogenous or exogenous drugs
and bind to the consensus sequences on the CLEM4 or XREM regions of the CYP3A4
gene. Together with the other phase I and II enzymes, the activated CYP3A4 will then
“detoxify” the drugs. The toxicants and the metabolites are then eliminated by phase III
transporters.
33
Extraction of patient genomic DNA
A
B
Normal healthy subjects
Breast cancer patients
PCR
Amplification of individual exons from
PXR, CAR and HNF4α genes
BigDye V3.1 Sequencing
Alignment of Sequences
Breast cancer population only
Pyrosequencing for identified SNPs in
PXR, CAR and HNF4α genes.
Statistical analysis
Figure 9: Flow chart showing the study approach to identify PXR, CAR and HNF4α SNPs in
healthy subjects and breast cancer patients. Screening of SNPs by BigDye V3.1 sequencing was
first conducted in normal healthy subjects. Any identified SNPs by BigDye technique was
confirmed by pyrosequencing. In the second part of the study, screening of SNPs was done for
the breast cancer population in the same manner. Statistical analysis and data correlation (drug
clearance) for the different genotype groups for the identified variants were conducted for the
breast cancer population only.
34
3.
MATERIALS AND METHOD
3.1
Materials
The reagents needed for this study and the suppliers are listed in Table 3.
Reagent
Company
Adriamycin
Aventis Pharma BV, Hoevelaken, Netherlands
Agarose
BioRad
BigDye Terminator V3.1
Applied Biosystems Inc
Deoxyribonucleotide triphosphate
New England Biolabs, Inc
(dNTP)
Docetaxel
Aventis Pharma BV, Hoevelaken, Netherlands
DyNazyme DNA polymerase
Finnzymes, Finland
Ethidium bromide
BioRad
Exonuclease I
GE Healthcare, US
Gentra DNA Purification Kit
Gentra Systems, Inc, Minneapolis, Minn
Hi-Di Formamide
Applied Biosystems Inc
Magnetic beads
Amersham Pharmacia Biotech, Uppsala, Sweden
PCR primers
Proligo Singapore Pte Ltd
Pyro sequencing primers
Proligo Singapore Pte Ltd
Shrimp Alkaline Phosphatase
GE Healthcare, US
TAE Buffer
National University Medical Institute (NUMI)
100bp DNA Ladder
BioRad
Table 3: List of reagents needed for this study and the suppliers.
35
3.2
Methods
3.2.1
Study Population
In this study, two groups of subjects were recruited. In the first group, a total of
287 healthy subjects were screened for SNPs in the PXR, CAR and HNF4α genes.
The healthy population consisted of 103 Chineses, 111 Malays and 73 Indians. In
the second group, the study population comprised of 101 female patients with
histologically or cytologically proven locally advanced or metastatic breast cancer
who were receiving primary systemic chemotherapy. The breast cancer
population consisted of 66 Chineses, 26 Malays, 7 Indians and 2 others. Subjects
were randomized to receive one of two alternating sequences of adriamycin (A)
and docetaxel (T), starting either with adriamycin 75mg/m2 as a slow bolus or
docetaxel 75mg/m2 infused over one hour, every 3 weeks for 6 cycles
(T→A→T→A→T→A or A→T→A→T→A→T). Plasma samples were obtained
from each subject when they received their first cycle of docetaxel or adriamycin
(Taxotere; Aventis Pharma BV, Hoevelaken, The Netherlands) for measurement
of drug concentrations at the following time points. Time points for docetaxel:
pre-dose, 1 hour, 2 hours, 4 hours, 7 hours, 10 hours, and 24 hours; and for
adriamycin: pre-dose, 1 hour, 2 hours, 6 hours, 8 hours, 12 hours and 24 hours,
following drug administration. Docetaxel concentrations were measured using an
isocratic liquid chromatography/tandem mass spectrometry (LC/MS/MS) method
as described previously (Guitton et al., 2005). Non-compartmental analysis for
docetaxel was performed using Kinetica 4.4 (InnaPhase Corp., Philadelphia, PA),
36
and area under the concentration-time curve (AUC) was estimated using the loglinear trapezoidal option from time 0 to infinity. The study protocol was approved
by the Institutional Ethics Committee (DSRB, National University Hospital) and
written informed consent was obtained from all patients according to institutional
and governmental guidelines.
37
3.2.2
Genotyping
DNA extraction and amplification
10ml of whole blood was collected from each subject, and genomic DNA was
extracted from peripheral mononuclear cells using the Gentra DNA Purification
Kit. The extraction was carried out according to the manufacturer’s protocol.
Each exon from the PXR, the CAR or the HNF4α gene was amplified by
polymerase chain reaction (PCR). The PCR reaction contained 100ng of genomic
DNA, 0.5U of DyNAzyme DNA polymerase, 0.2 mM dNTP and 0.4 μM each of
forward and reverse primers. The sequences of the PXR, CAR and HNF4α PCR
primers are listed in Table 4, Table 5 and Table 6 respectively.
PCR was performed using the Eppendorf Mastercycler (Eppendorf, USA), with
an initial denaturing step at 920C to 950C for 2 minutes, followed by 30 to 35
cycles of denaturing at 92 to 950C for 90 seconds, annealing at 55.50C to 670C for
30 seconds, and extension at 720C for 1 minute. A final 10 minutes extension step
at 720C was then carried out. 8μl of the PCR product was resolved by 1%
ethidium bromide stained agarose gel electrophoresis. The size of the product was
determined by comparing it against the 100bp DNA marker. The remaining PCR
products were stored at -200C till required.
38
DNA Sequencing
1μl of amplified PCR products were treated with 2.5U of Exonuclease (Exo) I and
0.5U of Shrimp Alkaline Phosphatase (SAP) for 30 minutes at 370C to remove
unincorporated nucleotides and primers. The reaction was stopped by heating at
800C for 15 minutes. Treated PCR products were then sequenced using BigDye®
Terminator v3.1 Cycle Sequencing Kit.
The 20μl sequencing reaction mixture consisted of 4μl of Exo-SAP treated DNA
template, 1 μl of BigDye, 3.5μl of the sequencing buffer, 1μl (10μM appropriate
primer (forward or reverse primer) and 10.5 μl of water. The reaction was allowed
to proceed in the Eppendorf Mastercycler (Eppendorf, USA) for 25 cycles. Each
cycle consists of a denaturation step at 960C for 30 seconds, annealing at 500C for
5 seconds, and extension at 600C for 4 minute. 3μl of 3M sodium acetate and 50μl
of absolute ethanol were used to precipitate the extended DNA for an hour at 200C. The sample was centrifuged at 12,000 rpm for 30 minutes. The pellet was
then rinsed with 50μl of 70% ethanol and centrifuged for 10 minutes at 12,000
rpm. The air dried pellet was dissolved in 13μl of Hi-Di Formamide and run on
the ABI 3100 automated Sequence Analyzer (Applied Biosystems Inc), with the
forward PCR primer and the reverse PCR primer. Any variants identified were
confirmed by sequencing both the sense and the anti-sense strands as well as by
pyrosequencing. Pyrosequencing was done to reassure that the variants identified
were true and to re-confirm the alleles (homozygous or heterozygous) by
comparing
it
against
the
classical
Big
Dye
electropherogram.
39
40
TCTGGGATATAAATGGCTCCC
GGCAGAGATTACCGACTCC
ACTGGGGAACTGCCTAGCTT
GGAGGGTCTTCTGACACCAA
AGCCACCTGTGGATGGTAAC
GGCAAAGACAAGCTCAGTCC
CCCCTGTTTGCTTGTGTTTT
CATAGACCCCAGATGGCCTA
AACAATTCCAACCCCCATTC
ACTCCCACCTACACCCTTCC
AGGGGAGAATTGCTTGTCAC
CAAGCAGGGATGTGTGTGAC
GGTTGTGAGGGGAGAGATGA
CTGCAGTTATGGGAGGAAGG
TATGGCCTTGCTCCTCATTC
AAGCCTTGTCTCTTGGCTGA
PXR Exon 2
PXR Exon 3
PXR Exon 4
PXR Exon 5
PXR Exon 6
PXR Exon 7
PXR Exon 8
PXR Exon 9
92
92
92
92
92
92
94
92
92
Denaturing (0C)
61.8
61.8
64.2
64.2
60.5
64.2
61.8
61.8
60.5
Annealing (0C)
Table 4: A set of PCR forward and reverse primers that were used to amplify each individual exonic region of the PXR gene. The respective
forward or reverse primer was later used in BigDye V3.1 sequencing of the PCR products.
CAACATTAAGTGATTGTTTTCATGC
5’ to 3’
5’ to 3’
CCCTTTTCCTGTGTTTTTG
Reverse Sequence
Forward Sequence
PXR Exon 1
Primer
41
GTGGGAGAGCTAGATCATGAG
GAACATTAGCTAGAGGCTCTGG
GCCTGTACTTCAGAGATGGAG
GGAAGGACAAGTTGGGTGGC
GGAAGGACAAGTTGGGTGGC
GCCTCTGAGCTACTACTTTGC
CATTGCAACCACTGGGCTCC
GCTTTCTGGAGTGATCCTGTG
CAGGTTGGTGGAAGTGTATAGG
GCACCGACAGGATTTGGGTTG
CACCTGAGGTCAGTAGTTGG
GATGGTGCTAGAGCAATAGGG
CACCTCTGCCCAAATACTCAG
GAGCATGGTGAGAGAAGACAG
CAR Exon 3
CAR Exon 4
CAR Exon 5
CAR Exon 6
CAR Exon 7
CAR Exon 8
CAR Exon 9
92
94
94
94
94
94
94
94
Denaturing (0C)
67.0
62.8
62.8
66.7
62.8
62.8
62.8
66.7
Annealing (0C)
Table 5: A set of PCR forward and reverse primers that were used to amplify each individual exonic region of the CAR gene. The respective
forward or reverse primer is later used in BigDye V3.1 sequencing of the PCR products.
CAAGATGTAGGATGCCAGCC
5’ to 3’
5’ to 3’
GTAGTCCCTGCTACTCAGG
Reverse Sequence
Forward Sequence
CAR Exon 2
Primer
42
GCAGCCTCATACTGGCTGAG
CCTTGCCGTCTCTCTGAACC
CCTTGAAGGCCCTGAAGGGC
CCCTCTTCTCAGCCATTAGCC
CTCGGAGCTGAGCTGATCGC
CTGGATAGTAGCTATGTGACC
CCAGGGATAACCCTGATCGTGC
CGCCCGGCCATATTGTCTC
CACTTAGAACAGTGACTGGC
CCTCATCCAGTGGATTGCC
GGTGAAGGTGAAGGCAGTGGC
CCTGCTGTGTATATATGCAG
GGCGTGGAGGCAGGGAGAAT
GTCAGGAGGTCACTGAGTGG
CACCTTTCCAGCTCCTGGTGG
CCCACTCCTCATCAGTCACAG
CCCTCCGTTTTTACCCTGAGC
CATCAGGCACACAGAAGAGGC
GCAGTGCAGTTCCAGAATCTG
CTGCCTGTGTCTAGGAAATC
GATGGGAATGGTACACCCTAG
GCTTTATGATCTGGGACTCACAG
HNF4α Exon 1B
HNF4α Exon 1C
HNF4α Exon 2
HNF4α Exon 3
HNF4α Exon 4
HNF4α Exon 5
HNF4α Exon 6
HNF4α Exon 7
HNF4α Exon 8
HNF4α Exon 9
HNF4α Exon 10
92
92
92
92
92
94
92
92
92
94
92
94
Denaturing (0C)
63.1
58.0
58.0
58.0
63.1
66.4
58.7
63.1
65.0
66.1
55.5
66.1
Annealing (0C)
Table 6: A set of PCR forward and reverse primers that were used to amplify each individual exonic region of the HNF4α gene. The respective
forward or reverse primer is later used in BigDye V3.1 sequencing of the PCR products.
CCTTGCCGTCTCTCTGAACC
5’ to 3’
5’ to 3’
GGCGTGGAGGCAGGGAGAAT
Reverse Sequence
Forward Sequence
HNF4α Exon 1A
Primer
Pyrosequencing
The pyrosequencing PCR reactions were performed using the Eppendorf
Mastercycler (Eppendorf, US) in a total reaction volume of 30μl with 0.5U of
DyNAzyme DNA polymerase, 0.2 mM dNTP and 0.4 μM each of forward and
reverse primers listed in Table 7. For primers that required biotin labelling (PXR
exon 1 reverse primer, CAR exon 5 forward primer and HNF4α Exon 4 forward
primer), a ratio of 1:9 part (10μM) primer to universal primer stocks were made
prior to use. PCR was carried out with an initial denaturing step at 950C for 4
minutes, followed by 39 cycles of denaturing at 950C for 30 seconds, annealing at
600C for 30 seconds, and extension at 720C for 30 seconds, followed by a final 1
minute extension step at 720C.
PCR products were incubated with 3µl of streptavidin magnetic beads and 1x
binding buffer (10mM Tris–HCl, 2M NaCl, 1mM EDTA, 0.1% Tween 20) for 10
minutes at 37oC. This is followed by denaturing the product mix for 5 seconds in
0.2M NaOH solution. Washing was then carried out with annealing buffer (20mM
Tris–acetate, 2mM magnesium acetate) for 10 seconds. The single-stranded
products were then transferred to the annealing buffer containing 15pmol of the
sequencing primer (see Table 7) and incubated for 2 minutes at 80oC in a Hybaid
Maxi 14 hybridization oven (Thermo Electron, USA). Pyrosequencing was then
performed on a PSQ96MA pyrosequencer (Biotage AB, Uppsala, Sweden).
43
[A]
DNA Polymerase
(DNA)n + dNTP
[B]
(DNA)n+1 + PPi
Sulfurylase
Light
APS
+
PPi
ATP
Luciferin
Oxyluciferin
Time
Luciferase
ATP
Light
Figure 10: The principle of pyrosequencing. Pyrosequencing is sequencing by synthesis
and used for accurate and quantitative analysis of DNA sequences. [a] The first step in
Pyrosequencing involves the hybridisation of sequencing primer to a single stranded PCR
amplified DNA template in which the reaction is incubated with DNA polymerase, ATP
sulfurylase, luciferase and apyrase. The reaction mix is incubated with the substrates,
adenosine 5’ phosphosulfate (APS) and luciferin. [b] The first of the four
deoxynucleotide triphosphates (dNTP) is added to the reaction. DNA polymerase
catalyses the incorporation of the dNTP into the DNA strands. Each incorporation of
dNTP results in the release of pyrophosphate (PPi) in a quantity equimolar to the amount
of incorporated nucleotide. ATP sulfurylase quantitatively converts PPi to ATP in the
presence of APS. This ATP drives the luciferase-mediated conversion of luciferin to
oxyluciferin that generates visible light in amounts that are proportional to the amount of
ATP. This light is then detected by a charge coupled device (CCD) camera and recorded
as a peak in a program. Each light signal is proportional to the number of nucleotides
incorporated. (http://www.pyrosequencing.com)
44
Primer
Primer Sequence 5’ to 3’ end
PXR Exon 1 Forward
CGGGAAGAGGAAGCACTG
PXR Exon 1 Reverse*
GGGACACCGCTGATCGTTTACCATACCTGCTTGGTGGTAAGT
PXR Exon 1 Sequence
GCTAATACTCCTGTCCTGAA
HNF4α Exon 1C Forward*
(Biotin) ATCACAGGCATTCTGGGTGAAG
HNF4α Exon 1C Reverse
TTGCCGTCTCTCTGAACCTAG
HNF4α Exon 1C Sequence
GGGGGAGCTCACCAT
HNF4α Exon 4 Forward*
GGGACACCGCTGATCGTTTAAATGAGCGGGACCGGATC
HNF4α Exon 4 Reverse
GGCAGGATCACCCCGGTAC
HNF4α Exon 4 Sequence
TCCTCATAGCTTGACCTT
CAR Exon 5 Forward*
GGGACACCGCTGATCGTTTACACACTTCGCAGACATCAACA
CAR Exon 5 Reverse
CGGGGTGGATATACAATTTACTG
CAR Exon 5 Sequence
GGTCACTCACCGGAA
Universal Primer
(Biotin) GGGACACCGCTGATCGTTTA
Table 7: Forward and reverse primers for pyrosequencing. Primers with asterisk (*)
mean they are biotinylated primers. All except HNF4α exon 1C were not pre-tagged
with biotin during primer synthesis phase by Proligo Singapore Pte Ltd. HNF4α exon
1C and universal primer were tagged with biotin at primer synthesis phase. Universal
primer is required to be added together with the non-tagged biotin primer prior to use.
45
3.2.3
Alignment of Sequences.
The nucleotide sequences of PXR, HNF4α and CAR genes were retrieved from
GenBank. The PXR gene (GenBank accession number: AF364606), located on
chromosome 3q12-q13.3, consists of nine exons. Exon 2 to 9 contains the coding
region and exon 1 contains the 5’ untranslated region (UTR). The CAR gene
(GenBank accession number: Z30425), located on chromosome 1q23.1, consists
of 9 exons. The coding region of CAR spans from exon 2 through exon 9. The
HNF4α gene (GenBank accession number: U72959 to U72969), located on
chromosome 20q12-q13.1, consist of 10 exons with 3 sub exon 1 (1A, 1B, 1C),
all spanning the coding region (Ryffel, 2001). Sequence alignments were
performed using the Multiple Sequence Alignment program by Florence Corpet
(http://prodes.toulouse.inra.fr/multalin/multalin.html) (Corpet, 1988).
46
3.2.4
Statistical Analysis.
Descriptive statistics were used to study the population distribution of docetaxel
clearances. Analysis of genotypes with continuous pharmacokinetics parameters
such as docetaxel clearances, body surface area normalized clearance (CL),
maximum concentration (Cmax), area under the curve (AUC) and half life (t1/2)
were performed using the student’s t-test or one-way ANOVA test as appropriate.
All genetic variants were tested for departure from Hardy-Weinberg equilibrium
for each ethnic group using Chi square test with one degree of freedom. All
docetaxel kinetics data were provided by Dr Goh Boon Cher and Dr Lee Soo
Chin, Department of Haematology-Oncology, National University Hospital,
Singapore. Statistical analyses were performed using SPSS Version 11.5 (SPSS
Inc, Chicago I11), with P ≤0.05 considered to be statistically significant.
Derivation of area under curve (AUC)
AUC (0 – infinity) = Dose / CL
AUC is referred to as the area under the plasma (serum or blood) concentration
versus time curve. It is most commonly derived by the trapezoidal method. For
example, breaking up the areas in trapezoids (or segments), and making a
summation of the areas of each trapezoid which can be calculated by multiplying
the average concentration by the segment width. (http://www.boomer.org)
47
Derivation of clearance (CL)
CL = Dose / AUC
Clearance is describes how quickly drugs are eliminated, metabolised or
distributed throughout the body. Clearance can be calculated by measuring the
amount of drug eliminated during some time interval and the drug concentration.
The total amount of drug that can be eliminated is the total amount administrated,
that is the dose. (http://www.boomer.org)
Derivation of half-life (t1/2)
t1/2 = ln 2 / k
or
t1/2 = 0.693 / k
The half-life is the time taken for the plasma concentration to fall to half its
original value. k is the elimination rate constant describing removal of drug by all
elimination processes. (http://www.boomer.org)
The elimination rate constant can be calculated as follows:
Elimination rate constant (k) = 1 / time
48
4.
RESULTS
The liver is the main organ for xenobiotic metabolism and the major CYP enzyme
that is expressed in the liver is CYP3A4. The expression of CYP3A4 can be
regulated by various nuclear receptors and transcription factors. These include
PXR, CAR and HNF4α. In this study, the aim is to examine the polymorphisms in
the genes encoding these receptors. The populations used in this study include
firstly, healthy subjects (comprising of Chinese, Malays and Indians) and
secondly, a cohort of breast cancer patients.
4.1
Screening of PXR, CAR and HNF4α Genes in Local Healthy Population
4.1.1
Amplification of Exons and Sequencing
In the first part of this study, screening was carried out on all the exons of the
PXR, CAR and HNF4α genes in 287 healthy subjects comprising of 103 Chinese,
111 Malays and 73 Indians. Amplification of the exons from genomic DNA was
achieved by PCR and this was followed by sequencing (see Figure 11, 12 and 13).
Sequencing was carried out for both the sense and anti-sense strand. Sequences
from subjects that were homozygous and heterozygous were first identified via
the electropherogram obtained through Big Dye sequencing (aligned against the
wild type sequence) and then confirmed by pyrosequencing. Amplification of the
individual SNP exon for pyrosequencing reaction (see Figure 14) was achieved by
49
PCR using the respective pyrosequencing primers (see Table 7). An example of
the electropherogram showing the nucleotide changes for the three genes is shown
in Figure 15.
PXR Exon
M
1
2
3
4
5
6
7
8
9
700 bp
600 bp
500 bp
Figure 11: PCR amplification of all nine PXR exons from patient genomic DNA.
The PCR product size are (a) exon 1, 635bp; (b) exon 2, 541bp; (c) exon 3,
518bp; (d) exon 4, 721bp; (e) exon 5, 649bp; (f) exon 6, 525bp; (g) exon 7,
526bp; (h) exon 8, 504bp; and (i) exon 9, 655bp.
CAR Exon
M
2
3
4
5
6
7
8
9
800 bp
700 bp
600 bp
500 bp
400 bp
Figure 12: PCR amplification of all eight CAR exons from patient genomic DNA.
The coding region in the CAR gene begins at exon 2 and ends at exon 9. The PCR
product size are (a) exon 2, 558bp; (b) exon 3, 512bp; (c) exon 4, 582bp; (d) exon
5, 545bp; (e) exon 6, 803bp; (f) exon 7, 387bp; (g) exon 8, 525bp; and (h) exon 9,
453bp.
50
HNF4α Exon
M
1A 1B
1C
2
3
4
5
6
7
8
9
10
800 bp
700 bp
600 bp
500 bp
Figure 13: PCR amplification of all twelve HNF4α exons from patient genomic
DNA. The PCR product size are (a) exon 1A, 537bp; (b) exon 1B, 399bp; (c)
exon 1C, 537bp; (d) exon 2, 444bp; (e) exon 3, 389bp; (f) exon 4, 474bp; (g) exon
5, 612bp; (h) exon 6, 624bp; (i) exon 7, 676bp; (j) exon 8, 649bp; (k) exon 9,
551bp; and (l) exon 10, 613bp.
M
a
b
c
d
500 bp
300 bp
200 bp
100 bp
Figure 14: PCR amplification of PXR exon 1, CAR exon 5, HNF4α exon 1C and
HNF4α exon 4 from patient genomic DNA using pyrosequencing primers. The
PCR products were used for pyrosequencing reaction. The PCR products are (a)
PXR exon 1, 225bp; (b) CAR exon 5, 135bp; (c) HNF4α exon 1C, 229bp; and (d)
HNF4α exon 4, 118bp.
51
[A]
[B]
PXR exon 1 A > C (- 24381 A > C)
[C]
CAR exon 5 C > T (Pro180Pro)
[D]
HNF4α exon 1C A>G (Met49Val)
HNF4α exon 4 C>T (Thr130Ilel)
Figure 15: Electropherograms of PXR, CAR and HNF4α SNPs. Arrows indicate
the variant nucleotide positions.
52
4.1.2
Variants in the PXR, CAR and HNF4α Genes
The variants that were observed in the local population include the A>C at -24381
for the PXR gene, the C>T at the codon 180 for the CAR gene, the A>G and C>T
at the codon 49 and codon 130 for the HNF4α gene respectively (Table 8). These
had all been previously reported in other studies (Moller et al., 1997; Price et al.,
2000; Rissanen et al., 2000; Sakurai et al., 2000; Zhang et al., 2001; Ikeda et al.,
2003; Pruhova et al., 2003; Lamba et al., 2005; Ek et al., 2005; Bosch et al.,
2006). The CAR variant is a silent coding region variant (Pro180Pro). This proline
residue is located in the ligand-binding domain of the receptor (Lamba et al.,
2005). The PXR variant is a 5’UTR variant. Both the HNF4α variants are nonsynonymous coding region variants; Met49Val and Thr130Ile in exon 1C and
exon 4 respectively. The HNF4α Met49Val and Thr130Ile variants are located in
the N-terminal functional domain and the DNA binding domain of the receptor,
respectively (Hadzopoulou-Cladaras et al., 1997; Jiang and Sladek, 1997; Jakob
et al., 2005).
The genotypic distribution and allele frequencies of PXR, CAR and HNF4α
polymorphic variants in different ethnic groups shown in Table 9. The allele
frequencies for the three genes were similar for the Chinese and Malay ethnic
groups. However, for the Indian subjects, the distribution was different with the
exception of the frequencies for the HNF4α exon 4 variant, Thr130Ile. No
homozygous was observed for HNF4α Thr130Ile in all the three ethnic groups.
53
Heterozygous for HNF4α Thr130Ile reported in this study was rare with
frequency of ≤ 0.05.
A comparison of what was obtained for our subjects was also carried out with
those previously described for other population (Moller et al., 1997; Price et al.,
2000; Rissanen et al., 2000; Sakurai et al., 2000; Zhang et al., 2001; Ikeda et al.,
2003; Pruhova et al., 2003; Lamba et al., 2005; Ek et al., 2005; Bosch et al.,
2006). The results of this comparison are shown in Table 10, Table 11, Table 12
and Table 13. In general, what we had observed in our three ethnic groups were
different from these population with a few exceptions. The allele frequencies for
the Indian and Dutch population for the PXR variant were similar while the
Caucasian, the Chinese and the Malay population showed that the wild-type
occurs in about 60% of these populations. The reported value for the African
American subjects was very much lower (Table 10). However, this value may not
be accurate as the sample size was very small (n=20). It is of interest to note that
for the HNF4α exon 4 variant, the wild type predominates in more than 95% of
all populations (Table 13). In addition, no homozygous was detected for this
variant. This may be due to the fact that this amino acid residue is located in the
DNA binding domain and it may be critical for the function of this nuclear factor.
The allele frequencies for the Chinese and Malay population for the CAR exon 5
variant were similar, reporting a low wild type frequency value of 0.18 compared
to the Japanese population with almost half of the population having SNPs in this
gene (Table 11). Our study reported similar HNF4α exon 1C allele frequencies
54
between the Chinese and Malay population (wild-type frequency = 0.28) and
much lower for the Indian population (wild-type frequency = 0.12). The allele
frequencies for all the three ethnic groups in this study were very different from
those in reported population (Czech and Japanese). It is noted that the minor allele
for the HNF4α exon 1C in all three ethnic groups in the local populations is
actually the major allele in the Czech and Japanese populations (see Table 12).
These differences may be attributed to genetic selection, dietary or environmental
influences or to drug interactions. HNF4α exon 1C wild type predominates in
more than 70% in both the Czech and Japanese population (see Table 12).
The other known reported exonic or intronic SNPs beside the ones studied in this
study are listed in following web links:-
PXR gene
http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=8856
CAR gene
http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=9970
HNF4α gene
http://www.ncbi.nlm.nih.gov/SNP/snp_ref.cgi?locusId=3172
55
Gene
Location
Nucleotide position
Nucleotide
Change
Codon
Change
SNP Name
PXR
5’ UTR
-24381
A to C
-------
rs1523127
CAR
Exon 5
540
CCC to CCT
Pro180Pro
rs2307424
HNF4α
Exon 1C
145
ATG to GTG
Met49Val
rs2071197
HNF4α
Exon 4
389
ACT to ATT
Thr130Ile
rs1800961
Table 8: Polymorphisms identified in the PXR, CAR and HNF4α gene in the healthy
control population (n = 287). In the screening of the breast cancer population (n =
101), the same set of four SNPs were identified in the three genes. No additional
SNPs were found in the breast cancer population for all three genes.
56
Allele Frequency
Location and
Chinese
Subjects
(n = 103)
Malay
Subjects
(n = 111)
Indian
Subjects
(n = 73)
PXR gene
AA = 0.66
AA = 0.61
AA = 0.46
- 24381 A > C
AC = 0.31
AC = 0.36
AC = 0.44
CC = 0.03
CC = 0.03
CC = 0.10
CAR gene
CC = 0.18
CC = 0.18
CC = 0.23
Pro180Pro
CT = 0.46
CT = 0.54
CT = 0.60
TT = 0.36
TT = 0.28
TT = 0.17
HNF4α gene
AA = 0.28
AA = 0.28
AA = 0.12
Met49Val
AG = 0.42
AG = 0.44
AG = 0.44
GG = 0.30
GG = 0.28
GG = 0.44
HNF4α gene
CC = 0.95
CC = 0.98
CC = 0.95
Thr130Ile
CT = 0.05
CT = 0.02
CT = 0.05
Designation
Table 9: Genotypic distribution and allele frequencies of PXR, CAR and HNF4α
variants in healthy control. PXR gene (- 24381 A > C): AA, AC and CC are wild
type, heterozygous and homozygous respectively. CAR gene (Pro180Pro): CC,
CT and TT are wild type, heterozygous and homozygous respectively. HNF4α
gene (Met49Val): AA, AG and GG are wild type, heterozygous and homozygous
respectively. HNF4α gene (Thr130Ile): CC and CT are wild type and
heterozygous respectively.
57
Population
Number of Subjects
Allele Frequency
Reference
Singapore Chinese
103
0.66 / 0.34
-----
Singapore Malay
111
0.61 / 0.39
-----
Singapore Indian
73
0.46 / 0.54
-----
African American
20
0.27 / 0.73
Zhang et al., 2001
Caucasian
150
0.59 / 0.41
Zhang et al., 2001
Dutch
100
0.46 / 0.54
Bosch et al., 2006
Table 10: Comparison of SNP frequencies of PXR exon 1 variant. Study population
include Singaporean (comprising of Chinese, Malays and Indians), African American,
Caucasian and Dutch.
Population
Number of Subjects
Allele Frequency
Reference
Singapore Chinese
103
0.18 / 0.82
-----
Singapore Malay
111
0.18 / 0.82
-----
Singapore Indian
73
0.23 / 0.77
-----
Japanese
253
0.48 / 0.52
Ikeda et al., 2003
Table 11: Comparison of SNP frequencies of CAR exon 5 variant. Study population
include Singaporean (comprising of Chinese, Malays and Indians) and Japanese.
58
Population
Number of Subjects
Allele Frequency
Reference
Singapore Chinese
103
0.28 / 0.72
-----
Singapore Malay
111
0.28 / 0.72
-----
Singapore Indian
73
0.12 / 0.88
-----
Caucasian
100
0.08/ 0.92
Price et al., 2000
Czech
61
0.73 / 0.27
Pruhova et al., 2003
Japanese
100
0.95 / 0.05
Sakurai et al., 2000
Table 12: Comparison of SNP frequencies of HNF4α exon 1C variant. Study population
include Singaporean (comprising of Chinese, Malays and Indians), Caucasian, Czech and
Japanese.
Population
Number of Subjects
Allele Frequency
Reference
Singapore Chinese
103
0.95 / 0.05
-----
Singapore Malay
111
0.98 / 0.02
-----
Singapore Indian
73
0.95 /0.05
-----
Caucasian
100
0.99 / 0.01
Price et al., 2000
Chinese
52
0.96 / 0.04
Rissanen et al., 2000
Czech
61
0.98 / 0.02
Pruhova et al., 2003
Danish
239
0.98 / 0.02
Moller et al., 1997
Danish
369
0.95 / 0.05
Moller et al., 1997
Danish
4726
0.97 / 0.03
Ek et al., 2005
Japanese
100
1.0 / 0
Sakurai et al., 2000
Swedish
666
0.95 / 0.05
Moller et al., 1997
Table 13: Comparison of SNP frequencies of HNF4α exon 4 variant. Study population
include Singaporean (comprising of Chinese, Malays and Indians), Caucasian, Chinese,
Czech, Danish, Japanese and Swedish.
59
4.2
Screening of PXR, CAR and HNF4α Genes in the Breast Cancer Population
PXR, CAR and HNF4α are known to be master transcriptional regulators of
CYP3A4 gene, which played an eminent role in drug metabolism. We postulated
that any SNPs in these receptor genes could affect CYP3A4 gene transcription
which may lead to possible variation in drug clearance in the patients.
In the second part of this study, 111 female breast cancer patients were recruited
and the exons of the PXR, CAR and HNF4α genes were analysed. This cohort
included 66 Chinese, 26 Malays, 7 Indians and 2 of other ethnic origins. The
mean age of the study population was 45.6 ± 12 years. The variants that were
observed in the three genes studied were identical to those observed for the
healthy local population (see Table 8 and Table 14). The genotypic distribution
and frequency of PXR, CAR and HNF4α polymorphic variants for all 101 breast
cancer patients are summarised in Table 14. Allele frequency of the four variants
in the different ethnic groups is shown in Table 15.
60
Location and Designation
Allele Frequency
PXR gene
AA = 0.60
-24381 A > C
AC = 0.31
CC = 0.09
CAR gene
CC = 0.26
Pro180Pro
CT = 0.41
TT = 0.33
HNF4α gene
AA = 0.24
Met49Val
AG = 0.41
GG = 0.35
HNF4α gene
CC = 0.98
Thr130Ile
CT = 0.02
TT = 0
Table 14: Genotypic distribution and allele frequencies of PXR, CAR and HNF4α
variants in breast cancer patients (n = 101). PXR gene (- 24381 A > C): AA, AC
and CC are wild type, heterozygous and homozygous respectively. CAR gene
(Pro180Pro): CC, CT and TT are wild type, heterozygous and homozygous
respectively. HNF4α gene (Met49Val): AA, AG and GG are wild type,
heterozygous and homozygous respectively. HNF4α gene (Thr130Ile): CC, CT
and TT are wild type, homozygous and heterozygous respectively.
61
Allele Frequency
Variant
Chinese
(n = 66)
Malay
(n = 26)
Indian
(n = 7)
Others
(n = 2)
PXR gene
AA = 0.56
AA = 0.77
AA = 0.29
AA = 1.00
- 24381 A > C
AC = 0.36
AC = 0.19
AC = 0.29
AC = 0
CC = 0.08
CC = 0.04
CC = 0.42
CC = 0
CAR gene
CC = 0.24
CC = 0.30
CC = 0.29
CC = 0
Pro180Pro
CT = 0.48
CT = 0.35
CT = 0.14
CT = 0
TT = 0.28
TT = 0.35
TT = 0.57
TT = 1.00
HNF4α gene
AA = 0.23
AA = 0.27
AA = 0
AA = 1.00
Met49Val
AG = 0.38
AG = 0.46
AG = 0.71
AG = 0
GG = 0.39
GG = 0.27
GG = 0.29
GG = 0
HNF4α gene
CC = 0.97
CC = 1.00
CC = 1.00
CC = 1.00
Thr130Ile
CT = 0.03
CT = 0
CT = 0
CT = 0
TT = 0
TT = 0
TT = 0
TT = 0
Table 15: Genotypic distribution and allele frequencies of PXR, CAR and HNF4α
for the different ethnic groups in the breast cancer population (n = 101). In the
cohort of 101 patients, there are 66 Chinese, 26 Malays, 7 Indians and 2 of other
ethnic origins. PXR gene (- 24381 A > C): AA, AC and CC are wild type,
heterozygous and homozygous respectively. CAR gene (Pro180Pro): CC, CT and
TT are wild type, heterozygous and homozygous respectively. HNF4α gene
(Met49Val): AA, AG and GG are wild type, heterozygous and homozygous
respectively. HNF4α gene (Thr130Ile): CC, CT and TT are wild type,
homozygous and heterozygous respectively.
62
4.3
Comparing the Allele Frequencies between Local Healthy and Breast Cancer
Population
It was not appropriate to compare the allele frequencies between different ethnic
groups of both healthy and breast cancer population as the sample size differs
greatly. The sample size for Chinese (n = 66), Malay (n = 26) and Indian (n = 7)
in the breast cancer cohort were too small to conduct a fair comparison against the
healthy patient cohort. Therefore, we compared the allele frequency observed in
the three ethnic groups for the local healthy population against the breast cancer
population as a whole. The allele frequencies for the Chinese, Malay and breast
cancer patients for the PXR variant were similar, showing that the wild-type
occurs in about 60%. CAR exon 5 variant (Pro180Pro) and HNF4α exon 1C
variant (Met49Val), too, showed similar allele frequencies distribution for the
Chinese, Malay and breast cancer population. The only variant that displayed
similar allele frequency across all the ethnic groups and the breast cancer
population is HNF4α exon 4 variant (Thr130Ile). As discussed in previous
section, HNF4α exon 4 wild-type predominates in more than 95% of all
populations. This may be because this amino acid residue plays a crucial
functional role for this nuclear factor.
63
4.4
Pharmacokinetics Correlations
Following the sequencing and genotyping for the breast cancer population,
statistical analysis test was carried out to determine if there were any correlations
between drug clearance and individual genotype groups. Analysis was also done
to determine whether the SNPs in the PXR, CAR and HNF4α genes affected the
metabolism of docetaxel in these breast cancer patients. The correlation was done
for only 97 subjects as 4 subjects dropped out in the midst of study. No significant
correlations were observed between the genotypes and the docetaxel clearance;
body surface area (BSA) normalized docetaxel clearance, Cmax, AUC or half-life
(t1/2).
The docetaxel clearances by PXR, CAR and HNF4α genotype are shown in Figure
16. BSA normalised docetaxel clearance, Cmax, AUC and half life by PXR, CAR
and HNF4α genotype are also shown in Figure 17, Figure 18, Figure 19 and
Figure 20, respectively.
64
120
2
Docetaxel Clearance (L/h/m )
A
100
80
60
40
20
0
AA
AC
CC
PXR Exon 1 Genotype
120
2
Docetaxel Clearance (L/h/m )
B
100
80
60
40
20
0
CC
CT
TT
CAR Exon 5 Genotype
65
2
Docetaxel Clearance (L/h/m )
C
120
100
80
60
40
20
0
AA
AG
GG
120
2
D
Docetaxel Clearance (L/h/m )
HNF4α Exon 1C Genotype
100
80
60
40
20
0
CC
CT
HNF4α Exon 4 Genotype
Figure 16: [A] Docetaxel clearance (L/h/m2) against PXR exon 1 genotype. AA, AC, and CC
represent wild type, heterozygous and homozygous respectively. [B] Docetaxel clearance
(L/h/m2) against CAR exon 5 genotype. CC, CT, and TT represent wild type, heterozygous
and homozygous respectively. [C] Docetaxel clearance (L/h/m2) against HNF4α exon 1C
genotype. AA, AG, and GG represent wild type, heterozygous and homozygous respectively.
[D] Docetaxel clearance (L/h/m2) against HNF4α exon 4 genotype. CC and CT represent
wild type and heterozygous respectively. The bar indicates the mean docetaxel clearance
value. Statistical analysis result in p-values > 0.05.
66
B
BSA Normalised Docetaxel Clearance
2
(L/h/m )
BSA Normalised Docetaxel Clearance
(L/h/m2)
A
70
60
50
40
30
20
10
0
AA
CC
AC
CT
CC
PXR Exon 1 Genotype
70
60
50
40
30
20
10
0
TT
CAR Exon 5 Genotype
67
BSA Normalised Docetaxel Clearance
(L/h/m2)
C
70
60
50
40
30
20
10
0
AA
AG
GG
D
BSA Normalised Docetaxel Clearance
2
(L/h/m )
HNF4α Exon 1C Genotype
70
60
50
40
30
20
10
0
CC
CT
HNF4α Exon 4 Genotype
Figure 17: [A] BSA normalised docetaxel clearance (L/h/m2) against PXR exon 1 genotype. AA,
AC, and CC represent wild type, heterozygous and homozygous respectively. [B] BSA
normalised docetaxel clearance (L/h/m2) against CAR exon 5 genotype. CC, CT, and TT represent
wild type, heterozygous and homozygous respectively. [C] BSA normalised docetaxel clearance
(L/h/m2) against HNF4α exon 1C genotype. AA, AG, and GG represent wild type, heterozygous
and homozygous respectively. [D] BSA normalised docetaxel clearance (L/h/m2) against HNF4α
exon 4 genotype. CC and CT represent wild type and heterozygous respectively. The bar
indicates the mean BSA normalised docetaxel clearance value. Statistical analysis result in pvalues > 0.05.
68
A
35
Cmax (mg/L)
30
25
20
15
10
5
0
AA
AC
CC
PXR Exon 1 Genotype
B
35
Cmax (mg/L)
30
25
20
15
10
5
0
CC
CT
TT
CAR Exon 5 Genotype
69
C
35
Cmax (mg/L)
30
25
20
15
10
5
0
AA
AG
GG
HNF4α Exon 1C Genotype
D
35
Cmax (mg/L)
30
25
20
15
10
5
0
CC
CT
HNF4α Exon 4 Genotype
Figure 18: [A] Maximum concentration of docetaxel, Cmax, (mg/L) against PXR Exon 1
genotype. AA, AC, and CC represent wild type, heterozygous and homozygous respectively.
[B] Maximum concentration of docetaxel, Cmax, (mg/L) against CAR Exon 5 genotype. CC,
CT, and TT represent wild type, heterozygous and homozygous respectively. [C] Maximum
concentration of docetaxel, Cmax, (mg/L) against HNF4α exon 1C genotype. AA, AG, and
GG represent wild type, heterozygous and homozygous respectively. [D] Maximum
concentration of docetaxel, Cmax, (mg/L) against HNF4α exon 4 genotype. CC and CT
represent wild type and heterozygous respectively. The bar indicates the mean Cmax value.
Statistical analysis result in p-values > 0.05.
70
A
25
AUC (mg/L*h)
20
15
10
5
0
AA
AC
CC
PXR Exon 1 Genotype
B
25
AUC (mg/L*h)
20
15
10
5
0
CC
CT
TT
CAR Exon 5 Genotype
71
C
25
AUC (mg/L*h)
20
15
10
5
0
AA
AG
GG
HNF4α Exon 1C Genotype
D
25
AUC (mg/L*h)
20
15
10
5
0
CC
CT
HNF4α Exon 4 Genotype
Figure 19: [A] Area under curve, AUC, (mg/L*h) against PXR Exon 1 genotype. AA, AC,
and CC represent wild type, heterozygous and homozygous respectively. [B] Area under
curve, AUC, (mg/L*h) against CAR Exon 5 genotype. CC, CT, and TT represent wild type,
heterozygous and homozygous respectively. [C] Area under curve, AUC, (mg/L*h) against
HNF4α exon 1C genotype. AA, AG, and GG represent wild type, heterozygous and
homozygous respectively. [D] Area under curve, (mg/L*h), against HNF4α exon 4 genotype.
CC and CT represent wild type and heterozygous respectively. The bar indicates the mean
AUC value. Statistical analysis result in p-values > 0.05.
72
A
Half Life (Hours)
25
20
15
10
5
0
AA
AC
CC
PXR Exon 1 Genotype
B
25
Half Life (Hours)
20
15
10
5
0
CC
CT
TT
CAR Exon 5 Genotype
73
C
25
Half Life (Hours)
20
15
10
5
0
AA
AG
GG
HNF4α Exon 1C Genotype
D
25
Half Life (Hours)
20
15
10
5
0
CC
CT
HNF4α Exon 4 Genotype
Figure 20: [A] Half life, t1/2, (hours) against PXR Exon 1 genotype. AA, AC, and CC represent
wild type, heterozygous and homozygous respectively. [B] Half life, t1/2, (hours) against CAR
Exon 5 genotype. CC, CT, and TT represent wild type, heterozygous and homozygous
respectively. [C] Half life, t1/2, (hours) against HNF4α exon 1C genotype. AA, AG, and GG
represent wild type, heterozygous and homozygous respectively. [D] Half life, t1/2, (hours) against
HNF4α exon 4 genotype. CC and CT represent wild type and heterozygous respectively. The bar
indicates the mean t1/2 value. Statistical analysis result in p-values > 0.05.
74
5.
DISCUSSION
In recent years, drug therapy has placed its focus exclusively on factors
determining systemic drug disposition (distribution and clearance). The
application of pharmacogenetic approaches to optimize drug dosage and treatment
regimes is critically important as it provides therapeutic control in the
pharmacotherapy in individual patients (Brouwer and Pollack, 2002).
The nuclear hormone receptors PXR, CAR and HNF4α play important roles as
xenobiotic-sensing receptors, transcription enhancers and regulators that serve to
regulate the expression of drug metabolizing enzymes and drug transporters.
SNPs, insertions, deletions and microsatellite polymorphism in these three nuclear
receptors may contribute to clinically significant variations in drug metabolizing
enzyme activities, which may in turn have major effects on drug clearance,
efficacy, and toxicity.
As discussed earlier, the main drug metabolising CYP isoform is CYP3A4. There
is considerable inter-individual variation in CYP3A4 activity and expression.
Approximately 90% of these CYP3A4 variations were thought to be caused by
genetic factors (Ingelman-Sundberg, 2004). From previous studies, the CYP3A4
variants identified are either rare among populations or occur with low allele
frequencies (Dai et al., 2001; Hsieh et al., 2001; Lamba et al., 2002; Bosch et al.,
2006; Du et al., 2006). This makes it difficult to relate CYP3A4 genotype to the
pharmacokinetics and pharmacodynamics of CYP3A4 substrates (Bosch et al.,
75
2006). Thus, it is necessary to examine the genes that regulate CYP3A4
transcription. Looking into the genetic variations of the regulatory genes such as
PXR, CAR and HNF4α may help to explain any possible genetic basis for the
inter-individual variability in the expression of CYP3A4 (Ingelman-Sundberg,
2004; Bosch et al., 2006).
5.1
Exonic Variants in PXR, CAR and HNF4α Genes
We sequenced the human PXR, CAR and HNF4α genes and identified a total of
four SNPs and determined their allelic frequencies in both the healthy control and
breast cancer population. The four variants that were observed were: PXR 5’-UTR
variant (-24381A>C), CAR (Pro180Pro), HNF4α (Met49Val) and HNF4α
(Thr130Ile).
For the PXR gene, the SNP that was observed was in the 5’UTR. Comparison of
the three Singaporean ethnic groups with other populations (Table 10) showed
that the allele frequencies for this variant were similar for the Chinese and Malay
population. This is also similar to that reported in a study with Caucasian subject
(Zhang et al., 2001). However, the local Indian population have a lower
occurrence of the wild-type allele and shows similar occurrence as that of a Dutch
study (Bosch et al., 2006). It is unclear what functional consequence this A>C
change may have. It may be possible that this change which is not within the
76
open-reading frame of PXR may influence transcription initiation. Alternatively, it
may also impact on translation of the PXR transcription by influencing ribosomal
binding.
The only CAR variant found in this study is a synonymous SNP in exon 5
(Pro180Pro). This proline residue is located within the ligand-binding domain. In
all the three local ethnic groups, more than 45% of the population are
heterozygous and the allele frequency is more than 0.75. In contrast, the
frequency reported in for a Japanese study was 0.52 (Ikeda et al., 2003). What
functional consequence this variant has is currently unclear. It may be that there is
no functional consequence since this is a synonymous SNP and most variations in
CAR function are associated with splice variants rather than coding region
polymorphisms (Okey et al., 2005)
Two non-synonymous SNPs in exon 1C (Met49Val) and exon 4 (Thr130Ile) were
detected for the HNF4α gene, In the Singaporean population, the allele frequency
for Met49Val is very different from that of Czech and Japanese population (Table
12). The variant predominates at a much higher frequency than that previously
reported. The Met49Val variant is located in the N-terminal functional domain
AF-1, also referred to as the A/B domain. The AF-1 plays a crucial role in
HNF4α-mediated transcription regulation and recruitment of co-activators. It has
been demonstrated that AF-1 is indispensable for HNF4α function. Absence of
AF-1 led to decrease in both the transcriptional potential and interaction with co-
77
activators (Green et al., 1998; Eeckhoute et al., 2001; Kistanova et al., 2001;
Eeckhoute et al., 2003). However, to date, there has been no study carried out to
examine the effect of mutating Met49 to Val. Thus, it is not possible to determine
as yet if such a change has any functional consequence although in our breast
cancer cohort, this variant is not associated with any of the examined parameters
of docetaxel pharmacokinetics.
The other non-synonymous SNP for the HNF4α gene was Thr130Ile. This variant
has been shown to occur in very low frequencies in most populations examined
(this study and that of in the Caucasian, Chinese, Czech, Danish, Japanese and
Swedish studies). Thr130Ile variant is located in the D domain, which is involved
in DNA binding. In vitro transactivation studies have been carried out for this
missense polymorphism. When compared to the wild-type, a significant decrease
in transactivation activity was observed for the Thr130Ile variant (Ek et al.,
2005). This together with the observation that no heterozygous was observed
certainly indicates that Thr130Ile is critical to the function of HNF4α.
78
5.2
PXR, CAR and HNF4α Genotypes and Docetaxel Pharmacokinetics
CYP3A4 is the main enzyme involved in the metabolism of docetaxel. The
expression of CYP3A4 is transcriptionally regulated by various nuclear receptors
including PXR, CAR and HNF4α. Hence, in this study, we also sequenced the
exons of the human PXR, CAR and HNF4α genes of a cohort of breast cancer
patients undergoing docetaxel treatment. Similar to that observed for our healthy
population, four variants, namely PXR 5’ UTR variant (-24381 A>C), CAR
(Pro180Pro), HNF4α (Met49Val) and HNF4α (Thr130Ile) were observed in the
breast cancer patients. The allele frequencies observed in the breast cancer
population were similar to that observed for the Chinese and Malays.
Pharmacokinetics analysis was performed for the breast cancer population and no
clear correlation was found between the different genotypes and docetaxel
pharmacokinetics. What was noteworthy was the lack of significant correlation
for the HNF4α (Met49Val). There was no difference in all pharmacokinetic
parameters between patients who are wild-type, heterozygous or homozygous
despite the fact that this SNP occurs in the AF-1 domain which is important for
interaction and recruitment of co-activators.
One possible explanation to why no clear correlation was found could be due
unequally distribution or numbers of patients in each individual ethnic group. By
combining the three ethnic groups data for pharmacokinetics analysis could have
79
skewed the result of genotypes correlation to docetaxel pharmacokinetics data.
Table 15 illustrated that different ethnic groups have different allele frequencies
and not at all similar across the board. As known, the three ethnic groups have
different dietary preferences, different racial origin and possible different gene
selection.
To improve the accuracy of analysis, a larger sample size for each race should be
recruited. In addition, a more robust index should be set for the measurement of
plasma concentration or docetaxel pharmacokinetics, although all these would be
logistically demanding.
80
6.
CONCLUSION
In conclusion, our study identified four exonic variants in the PXR, CAR and
HNF4α genes. All variants were observed in each of the three major ethnic
groups examined but the frequencies were different between that of the Indians
when compared to the Chinese and Malays. The allele frequencies observed for
the local population was also generally different from that of other studies carried
out with mainly Caucasian and Japanese subjects.
Given the role of PXR, CAR and HNF4α in regulating the expression of CYP3A4,
we had hypothesized that genetic polymorphisms of PXR, CAR and HNF4α
would affect CYP3A4 expression and this would in turn alter docetaxel clearance
and kinetics. However, we did not observe any correlation. Therefore, to
completely rule out the lack of correlation, various addition factors need to be
examined. A detail examination for polymorphisms in the PXR, CAR and HNF4α
genes has to be carried out to include polymorphisms in the promoter regions of
the genes as well as polymorphisms in the intron / splice sites. In addition, a
larger cohort of patients with sufficient representation of the various ethnic groups
needs to be recruited.
Given the major role of CYP genes and its associated transcriptional regulators
such as PXR, CAR and HNF4α in drug elimination, the variability of SNPs
examined in both ethnically diverse healthy and breast cancer populations might
81
be helpful in providing a clearer interpretation of inter-individual variability in
drug elimination capacity.
7.
PUBLICATIONS
Hor SY, Lee SC, Wong CI, Lim YW, Lim RC, Wang LZ, Fan L, Guo JY, Lee
HS, Goh BC, Tan T. PXR, CAR and HNF4alpha genotypes and their association
with pharmacokinetics and pharmacodynamics of docetaxel and doxorubicin in
Asian patients. Pharmacogenomics J. 2007 Sep 18; ahead of print.
Tham LS, Holford NH, Hor SY, Tan T, Wang L, Lim RC, Lee HS, Lee SC, Goh
BC. Lack of Association of Single-Nucleotide Polymorphisms in Pregnane X
Receptor, Hepatic Nuclear Factor 4{alpha}, and Constitutive Androstane
Receptor with Docetaxel Pharmacokinetics. Clin Cancer Res 2007; 13(23):71267132.
82
8.
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[...]... PXR-RXRα and CAR- RXRα heterodimers are capable of binding to the proximal promoter region of the CYP3A4 gene and mediates PXR or CAR transactivation of the CYP3A4 promoter respectively (Bertilsson et al., 1998; Goodwin et al., 2002; Akiyama and Gonzalez, 2003) The activation of CYP3A4 promoter by the nuclear receptors heterodimer is dependent upon ligand binding leading to the binding of the nuclear receptors. .. number of orphan receptors has increased tremendously (Wang and LeCluyse, 2003; Kliewer, 2005) Scientists are not only interested in researching on the novel physiological ligands of these orphan receptors but also the biological functions and signaling cascades that these receptors elicit The next part of the introduction will focus on three members of the NHR superfamily (PXR, CAR and HNF4α) and their... structure which consists of activation function 1 (AF-1) domain located at the amino-terminal and AF-2 domain at the carboxy-terminal The DNA binding domain (DBD) is connected to the ligand binding domain (LBD) by a flexible hinge Upon ligand binding to the LBD, AF-2 will undergo a conformational change that disrupts interaction with transcriptional co-repressors and allows the interaction with transcriptional... liver and intestine, and regulates a variety of drug detoxifying genes (Pascussi et al., 2003) These include the CYP3As, CYP2Bs, CYP2Cs, UGT1A1, GST, SULT, MDR1, MRP2, MRP3 and MRP4 (Handschin and Meyer, 2003; Lamba et al., 2005) CAR resides in the cytoplasm and translocates into the nucleus upon binding to its ligand (Kawamoto et al., 1999; Savkur et al., 2003; Tirona and Kim, 2005) Interestingly, there... 9-cis-retinoic acid and trans-retinoic acid Upon binding of ligands, class II receptors dimerizes with retinoid X receptor (RXR) In both cases, upon binding ligand binding, the receptors may undergo conformational changes that eventually cause the dissociation of co-repressors and binding of coactivators Class III belongs to a group of receptors whose physiological ligands have not yet been identified (Handschin... (Handschin and Meyer, 2003; Pascussi et al., 2003) Over the past decades, some members of the NHR superfamily were termed ‘orphan’ receptors because at the time of their cloning, nothing was known about their physiological ligands or co-activators Till now, the term remains for these receptors even though their ligands are now known The first ‘orphan’ receptor was identified in 1988 and since then, the. .. co-activators The activated nuclear receptor will then bind to the response elements on the regulatory regions of target genes and initiates transcription 15 C NHRs are categorized into three main classes Class I receptors bind to steroid hormones and in absence of ligand, these receptors are associated with molecular chaperones like heat shock proteins (HSPs) Class II receptors bind to thyroid hormone, vitamin... binding domain and a ligand binding domain as illustrated in Figure 4 NHRs are able to induce or regulate drug metabolism by binding to small lipophilic ligands Following ligand binding and dimerization, they then bind to DNA element repeats of the nucleotide hexamers in different arrangements like the ones found in drugresponsive enhancers of CYPs The hexamers can be arranged either as direct repeats (DR),... comprises the transport and elimination steps where the parent drug and its metabolites are exported out of the cell and eventually removed from the body through the bile or urine Figure 1 illustrates the contribution of phase I and phase II enzymes to the metabolism of drugs 2 Figure 1: Pie chart illustrations of phase I and phase II drug metabolising enzymes The relative size of each section on the charts... CYP2D6 and CYP3A4 (Ingelman-Sundberg, 2004) Table 1 shows the main tissue localisation of these CYPs and their anti-cancer substrates (Ingelman-Sundberg, 2004; Van Schaik, 2005) Different forms of CYP are found to be expressed in intestine, lung and kidneys but the liver is the major site for CYP-mediated oxidative metabolism, with CYP3A family as the dominant class In this study, the focus will be on the ... the amino-terminal and AF-2 domain at the carboxy-terminal The DNA binding domain (DBD) is connected to the ligand binding domain (LBD) by a flexible hinge Upon ligand binding to the LBD, AF-2... European and Asian like the Japanese However, this has not been done in the local population Therefore, it would be of great interest to determine the SNPs in PXR, CAR and HNF4α genes in the local population. .. Goodwin et al., 2002; Akiyama and Gonzalez, 2003) The activation of CYP3A4 promoter by the nuclear receptors heterodimer is dependent upon ligand binding leading to the binding of the nuclear receptors