Interindividual variability in human drug glucuronidation...Interindividual variability in human...

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


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??


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


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


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


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)


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)


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)


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)


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


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)


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


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)


UGT1A1 shows highest UGT variabilityRivals CYP3A variability


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)


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)


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


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


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)


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


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


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


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?


UGT2B17 mRNA is higher in male liversUGT2B15 – males?; UGT2B4 - females?

(Gallagher et al: DMD, 2010)

??

??


Effect of sex is independent of UGT2B17 deletion

UGT2B17 mRNA 17-dihydroexemestane glucuronidation

(Gallagher et al: DMD, 2010)

malesfemales malesfemales

del deldel del


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


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


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)


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)


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)


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)


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


n = 9, 18, 14, 13

UGT1A UGT2B


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

*

** *



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

** *


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)??


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

Interindividual variability in human drug glucuronidation...Interindividual variability in human...

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