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Interindividual variability in human
drug glucuronidationWhat we know and what we need to know
Laboratory of Comparative and Molecular PharmacogenomicsDepartment of Pharmacology and Experimental TherapeuticsTufts University School of Medicine
Michael H Court, BVSc, PhD
Michael H. Court 2010
Why study drug glucuronidation?
Main identified metabolic clearance pathway (after CYP) for top 200 prescribed drugs in the USA.
May be more frequently encountered since selecting drug candidates for CYP stability
(Williams et al: DMD, 32:1201-1208,2004.)
Enzymes known to be involved in the metabolism of the top 200
prescribed drugs in USA
UGTs
CYPs
Michael H. Court 2010
Advantages of glucuronidation over oxidation?
Glucuronides are “stable” = non-reactive Except some acylglucuronides
Less potential for DDI Typically low affinity (high Km) enzymes Broad, overlapping substrate specificities
Less interindividual variability in activity??
Michael H. Court 2010
Objectives
Brief overview of the UGTs
Characteristics of the Tufts human liver bank
What is the extent of interindividual variability in glucuronidation? For different UGTs? Versus CYPs?
What inherent factors determine UGT variability? Gender; age; genetics; epigenetics
What external factors influence UGT variability? Smoking; alcohol; other drugs; disease
Michael H. Court 2010
UDP-glucuronosyltransferases
Substrates Drug or Phase I metabolite (also hormones, toxins, etc) -OH; -COOH; amino; (rarely -SH; -CH)
Glucuronides generally inactive except: Morphine-6-glucuronide Acyl-glucuronides (esp. NSAIDs)
10 genes expressing 19 unique UGTs Subfamilies UGT1A, 2A, 2B involved in drug metabolism [Subfamily UGT3A - bile acid UDP-N-acetylglucosaminyltransferase]
Drug + UDP-glucuronic acid Drug-glucuronide + UDP
Michael H. Court 2010
UGT gene structures
Chr. 2q37
Differential splicing of unique exon 1 to shared exons 2-5
X X X X
Chr. 4q13
Individual genes (6 exons)
Unique exon 1 – shared exons 2-6(Mackenzie et al, PGEN J 2005)
X X X X X
Michael H. Court 2010
UGT1A gene – diversity through splicing
Conserved region COO-+H3N Variable regionTransmembrane domain
ER retentionsignal
UDPGA binding domain
Substrate binding domain
Signal sequence
5a 4321A11A31A4
> 500 kb
Gene
Differential splicing of mRNA
Protein
Promoter/regulatory regionsShared exons
UGT1A1
5b
Conserved region COO-+H3N Variable region 5bInactive dominant negative inhibitor
UGT1A1-i2
(Guillemette, Bellemare et al, University of Laval, QC)
Michael H. Court 2010
Effect of UGT1A6i2 on UGT1A6 activity
0
20
40
60
80
100
120
140
160
0 ug 0.2 ug 0.5 ug 1 ug 1.5 ug 2 ug
UGT1A6_i2
** *
% c
ontro
l APA
P gl
ucur
onid
atio
n
(Court, unpublished data, 2010)
Michael H. Court 2010
UGT mRNA expression in human tissues by QPCR
Panel of 30 human tissues
QPCR using UGT isoform-specific primer sets All except UGT3A1, 3A2 Absolute quantitation using
standard curve
Summed all UGTs for each tissue and ranked top 10 Liver Kidney Adipose GI: stomach, intestines, pancreas Airway Testis Thymus
Colon (n=3)
Kidney
Liver (n=47)
Adipose
PancreasThymus
(n=9)Stomach
Trachea
Small
intestine
(n=5)
Testis (n=19)
Top 10 tissues
(Court et al, in preparation)
Michael H. Court 2010
Most UGTs expressed in adult human liver
glucuronidate drugsLiver, adult (n=47)
1A1
1A3
1A4
1A61A9
2A3
2B4
2B7
2B10
2B11
2B152B17 Significant activity
for drugs
(Court et al, in preparation)
Michael H. Court 2010
Major drug metabolizing UGTs in human liver
UGT1A1 Bilirubin, estradiol, EE Irinotecan -> SN38 “CYP2D6” of the UGTs Gilbert’s, Crigler-Najjar
UGT1A4 Quaternary N-glucuronides
Unique to primates and rabbits (?)
Antihistamines, tricyclic antidepressants, antipsychotics
UGT1A6 Small, planar aromatics Acetaminophen,valproate Serotonin (5HT) –endogenous role?
UGT1A9 Bulky phenols Propofol, flavopiridol, salicylates,
mycophenolic acid
UGT2B7 “CYP3A4” of the UGTs Retinoids, fatty acids, steroids AZT, morphine, opioids, NSAIDS
UGT2B15 Oxazepam, lorazepam, 4OH-
tamoxifen, 5OH-rofecoxib Androgens, bisphenol A
Michael H. Court 2010
Identification of isoform-selective probes for
measurement of glucuronidation by major UGTs
Vec
tor
1A1*
*
1A3
1A4*
*
1A6*
*
1A7
1A8
1A9*
*
1A10
2B4
2B7*
*
2B15
**
2B17
Estradiol-3-UGT*
Trifluoperazine-UGT*
Serotonin-UGT
Propofol-UGT
AZT-UGT
S-oxazepam-UGT
0102030405060708090
100
UGT isoforms
% of
highest
activity
*Patten, Dehal, et al (2001)
(Court: Methods Enzymol. 400:104-16, 2005)
Michael H. Court 2010
Tufts human liver bank (n=55)LTCDS (Liver Tissue Cell Distribution Service at U. Minnesota)
50 transplant quality livers Failed to match or leftover from pediatric transplant Head trauma/gunshot/stroke
NDRI (National Disease Research Interchange) 5 adjacent healthy tissue in surgical patients Liver cancer/metastases
1 gram to 500 gramsMicrosomes on all; RNA and DNA on most
49 White non-Hispanic; 4 African-Americans; 2 White Hispanic38 males; 17 females2 – 80 years old; median 40 yearsSmoking/alcohol/prescription drug use
Michael H. Court 2010
UGT activity trends for individual liversActivities normalized by Z-score (SD units; 0 = mean)
-3
-2
-1
0
1
2
3
4
5
1 3 5 7 9 11 13 15 17 19 21 23 25 27 29 31 33 35 37 39 41 43 45 47 49 51 53 55
Liver ID #
Z-s
co
re
E-3-UGT TFP-UGT 5HT-UGT Propofol-UGT AZT-UGT S-oxazepam-UGT Mean Z-score
Excluded from
published studies
(mean Z-score < -1.0)
(Court: Drug Metabolism Reviews, 2010)
Michael H. Court 2010
UGT1A1 shows highest UGT variabilityRivals CYP3A variability
(Court: Drug Metabolism Reviews, 2010)
0.01
0.1
1
10
Estradiol-3-UGT
Trifluoperazine-UGT
Serotonin-UGT
Propofol-UGT
Zidovudine-UGT
S-oxazepam-UGT
Midazolam-1'-OH
Chlorzoxazone 6-OH
Acti
vit
y r
ela
tive t
o m
ean
of
all
liv
ers
1A1 1A4 1A6 1A9 2B7 2B15 3A 2E1CV% 92 53 76 55 45 72 111 35
UGT CYP450
Human liver microsome bank (n=54)
Michael H. Court 2010
UGT1A1 and CYP3A show highest variability in vivo
Variability in clearance values for probe drugs
0
5
10
15
20
25
30
35
40
45
50
UGT1A1
UGT1A4
UGT1A6
UGT1A9
UGT2B7
UGT2B15
CYP3A
CYP2E1
%C
V
(Dorne, Walton, and Renwick, FoodChemTox, 2001, 2003, 2005 and FDA drug insert information)
Michael H. Court 2010
UGT1A1*28 is a major determinant of
UGT1A1 variability
(Court et al, unpublished)
UGT1A1*28: 6/6 6/7 7/7
Bilirubin glucuronidation
(UGT1A1)
0
0.5
1
1.5
2
2.5
nm
ole
s / m
in / m
g p
rote
in p =0.043 (ANOVA)
Bilir
ubin
glu
curo
nida
tion
Michael H. Court 2010
UGT1A1*28 predicts adverse effects of irinotecan
SN-38
Irinotecan (prodrug)
SN-38 glucuronide
CES1 (activate)
UGT1A1 (inactivate)
(Marcuello et al. British J Cancer, 2004)
01020304050607080
*1/*1 *1/*28 *28/*28UGT1A1 genotype
% p
ati
en
ts
Neutropenia
Severe diarrhea
Adverse effects of Camptosar®
in 95 patients with metastatic colon cancer
Michael H. Court 2010
UGT1A9 -275t>a is associated with
increased glucuronidation
UGT1A9 -275t>a: t/t t/a a/a
Propofol glucuronidation
(UGT1A9)
0
0.5
1
1.5
2
2.5
3
nm
ole
s /
min
/ m
g p
rote
in
p =0.047 (t-test)
(Girard et al: PGENJ, 2006)
Michael H. Court 2010
UGT1A9 -275t>a is associated with lower MPA exposure
and increased risk of renal transplant rejection
(Kuypers et al: CPT, 2005)
Lower mycophenolic acid levels
(van Schaik et al: CPT, 2009)
Higher transplant rejection risk
Michael H. Court 2010
Effect of sex on UGTs
Higher UGT2B15 activity in males
No differences for all other isoforms
0
0.5
1
1.5
2
2.5
Est
radio
l-3-U
GT
Triflu
operaz
ine-U
GT
Ser
otonin
-UG
T
Pro
pofol-U
GT
Zidovu
dine-
UG
T
S-o
xazepam
-UG
T
Female
Male*A
ctiv
ity re
lativ
e to
mea
n of
all
liver
s
Michael H. Court 2010
Oxazepam clearance is ~30% higher
in males versus females
(Greenblatt et al: JPET, 1980)
Oxazepam clearance
by human subjects
0
5
10
15
20
25
30
35
Males (n = 18) Females (n = 20)
mL
/ m
in /
kg
P < 0.05
Michael H. Court 2010
UGT2B15 is regulated by sex steroids in cell linesBut effect is opposite to expected
(Hu and Mackenzie: MolPharm, 2009)
MCF 7 breast cancer - by estradiol?
(Bao et al: The Prostate, 2008)
LNCAP prostate cancer - by DHT?
Michael H. Court 2010
UGT2B17 mRNA is higher in male liversUGT2B15 – males?; UGT2B4 - females?
(Gallagher et al: DMD, 2010)
??
??
Michael H. Court 2010
Effect of sex is independent of UGT2B17 deletion
UGT2B17 mRNA 17-dihydroexemestane glucuronidation
(Gallagher et al: DMD, 2010)
malesfemales malesfemales
del deldel del
Michael H. Court 2010
The common UGT2B15 variant (*2, D85Y) variant has
lower oxazepam glucuronosyltransferase turnover
S-oxazepam glucuronidation(corrected for UGT2B15 content)
0
1
2
3
4
0 200 400 600 800 1000
mM Oxazepam
pm
ole
s /
min
/ m
g p
rote
inUGT2B15*1
UGT2B15*2
Km = 29 mM
0
1
2
3
4
5
6
7
UGT2B15*1 UGT2B15*2
UG
T2
B p
rote
in c
on
ten
t
(rela
tive
)
UGT2B15*1 UGT2B15*2
UGT
Calnexin
(Court et al: DMD, 2002)
S-oxazepam glucuronidation(corrected for UGT2B15 content)
0
1
2
3
4
0 200 400 600 800 1000
mM Oxazepam
pm
ole
s /
min
/ m
g p
rote
inUGT2B15*1
UGT2B15*2
Km = 29 mM
0
1
2
3
4
5
6
7
UGT2B15*1 UGT2B15*2
UG
T2
B p
rote
in c
on
ten
t
(rela
tive
)
UGT2B15*1 UGT2B15*2
UGT
Calnexin
Michael H. Court 2010
Both sex and UGT2B15 D85Y genotype are major determinants
of variability in oxazepam glucuronidation
(Court et al: JPET, 2004.)
Codon 85: D/D D/Y Y/Y D/D D/Y Y/Y
Gender: Male Female
ANOVA: **p = 0.002 p = 0.95
0
2
4
6
8
10
12
14
S/R
ra
tio
S-/R-oxazepam glucuronidation ratio
0
50
100
150
200
250
300p
mo
les
/ m
in /
mg
pro
tein
S-oxazepam glucuronidation|-------*p < 0.05-------|
|-*p < 0.05-|
|--- n.s. ---|
*p = 0.02
Michael H. Court 2010
UGT2B15 D85Y genotype also predicts
oxazepam clearance in vivo
30 healthy males received 0 mg oxazepam orally
Genotyped for UGT2B15 D85Y and UGT2B17 deletion
>50% decease with 85YY
No effect of UGT2B17 deletion
UGT2B15 D85Y genotypeD/D D/Y Y/Y
Oxa
zepa
m c
lear
ance
(mL/
min
/kg)
0
1
2
3
4
5
6
7
---- --
**P=0.003*P=0.018 *P=0.034
(He et al: BrJClinPharm, 2009)
Michael H. Court 2010
Lorazepam glucuronidation is also
decreased by D85Y in vitro and in vivo
Codon 85: D/D D/Y Y/Y Codon 85: D/D D/Y Y/Y Codon 85: D/D D/Y Y/Y Codon 85: D/D D/Y Y/Y
Anova: Anova: Anova: Anova:**P < 0.001 P = 0.14 P = 0.56 P = 0.16
0
5
10
15
20
25
30
pm
ole
s /
min
/ m
g p
rote
in
S-Lorazepam
|-*p < 0.05-|
|----*p < 0.05----|
0
5
10
15
20
25
0
2
4
6
8
10
0
50
100
150
200
250
0
1
2
3
4
5
6
7
8
9
10
pm
ole
s /
min
/ m
g p
rote
in
R-Lorazepam
0
1
2
3
4
5
6
pm
ole
s /
min
/ m
g p
rote
in
E-4OH-Tamoxifen
0
100
200
300
400
500
600
700
800
pm
ole
s /
min
/ m
g p
rote
in
5OH-Rofecoxib
(Court: Methods Enzymol. 400:104-16, 2005)
Lorazepam
Lorazepam glucuronide
(Chung et al: CPT, 2005)
Time after dose (hrs)
Michael H. Court 2010
A common UGT2B7 haplotype (*1C) is associated with
higher AZT glucuronidation and clearance
0.00
0.02
0.04
0.06
0.08
0.10
0.12
0.14
nm
ole
s/m
in/m
g p
ro
tein
P = 0.021
Noncarrier Carrier
UGT2B7*1C
AZT
glu
curo
nida
tion
Human liver bank
0
20
40
60
80
100
120
140
No
rm
alized
CL
/F (
mL
/min
/kg
)
P = 0.007
Noncarrier Carrier
UGT2B7*1C
AZT
cle
aran
ce
PK study
(Kwara et al: JClinPharm, 2009)
Michael H. Court 2010
Fetal liver
(n=63)
8% of adult
Adult liver
(n=47)
All UGTs
Effect of age on UGT expressionFetal livers do not express any UGT1As
Liver, adult (n=47)
1A1
1A3
1A4
1A61A9
2A3
2B4
2B7
2B10
2B11
2B152B17
Liver, fetal (n=63)
2B4
2B7
2B10
2B11 2A3
2B15
(Court et al, unpublished)
Michael H. Court 2010
Effect of age on UGT activities
Lower UGT1A activity in children/teens (<21 years)
No effect of old age (>60 years)
0
0.5
1
1.5
2
2.5
Est
radio
l-3-U
GT
Triflu
operaz
ine-U
GT
Ser
otonin
-UG
T
Pro
pofol-U
GT
Zidovu
dine-
UG
T
S-o
xazepam
-UG
T
2-20 yrs
21-40 yrs
41-60 yrs
61+ yrs
* * *
Act
ivity
rela
tive
to m
ean
of a
ll liv
ers
(Court: Drug Metabolism Reviews, 2010)
n = 9, 18, 14, 13
UGT1A UGT2B
Michael H. Court 2010
Smoking/alcohol history and UGT activities
0
0.5
1
1.5
2
2.5
3
Est
radio
l-3-U
GT
Trifl
uop
eraz
ine-
UGT
Ser
otoni
n-UGT
Pro
pofo
l-UGT
Zidov
udin
e-UGT
S-o
xaze
pam
-UGT
Non-smoker
Smoker
Act
ivity
rel
ativ
e to
mea
n of
all
liver
s
*
0
0.5
1
1.5
2
2.5
3
3.5
Est
radio
l-3-U
GT
Trifl
uop
eraz
ine-
UGT
Ser
otoni
n-UGT
Pro
pofo
l-UGT
Zidov
udin
e-UGT
S-o
xaze
pam
-UGT
2 or less drinks/day
>2 drinks/day
Act
ivity
rel
ativ
e to
mea
n of
all
liver
s
*
** *
(Court: Drug Metabolism Reviews, 2010)
Michael H. Court 2010
Alcohol history and UGT activitiesSubjects 21 years and over
0
0.5
1
1.5
2
2.5
3
3.5
Est
radio
l-3-U
GT
Triflu
operaz
ine-U
GT
Ser
otonin
-UG
T
Pro
pofol-U
GT
Zidovu
dine-
UG
T
S-o
xazepam
-UG
T
2 or less drinks/day
>2 drinks/day
Act
ivity
rela
tive
to m
ean
of a
ll liv
ers
** *
Michael H. Court 2010
Conclusions
The human liver bank is a useful tool for characterizing interindividual variability in drug glucuronidation
Interindividual variability in drug glucuronidation is comparable to CYP mediated drug metabolism BUT variability is dependent on UGT isoform UGT1A1 and UGT2B15 have highest variability
Genetics, sex and age affect drug glucuronidation UGT1A1 and UGT2B15 - genetic polymorphism Male>female for UGT2B15/17 – sex steroids? Lower UGT1A glucuronidation in infants/children – epigenetics? Alcohol effect on UGT1A – transcription factor?? Role for regulation via protein-protein interaction (UGT-i2)??
Michael H. Court 2010
Acknowledgements
NIH grant R01-GM61834 (2000 – 2011)
Pfizer Global Research and Development
BD Gentest
David Greenblatt and Lisa von Moltke (Tufts Pharmacology)
Chantal Guillemette (Laval University, Quebec)
Court lab - past and present personnel Soundar Krishnaswamy Qin Hao Su Duan Su Hazarika
Michael H. Court 2010
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