SLIDE 1
Application of Animal Models for Human Glucuronidation
Shujuan Chen
Tukey Laboratory Departments of Chemistry & Biochemistry and Pharmacology, UCSD 2012 SCDMDG
SLIDE 2 Human UGTs: 19 isoforms
UGT1A5 UGT1A3 UGT1A4 UGT1A1 UGT1A6 UGT1A7 UGT1A8 UGT1A10 UGT1A9 UGT2B28 UGT2B11 UGT2B7 UGT2B4 UGT2B15 UGT2B17
59% 41% 66%
1 2 B A
UGT2A1 UGT2B10 UGT2A2
Drugs, Pollutants, C18 Steroids Odorant molecules Bioactive lipids Androgens Bile acids Bilirubin, irinotecan Tertiary Amines Heterocyclic Aromatics
UGT2A3
SLIDE 3
Bilirubin Clearance
Bilirubin Glucuronides Bilirubin
UGT1A1
Bilirubin Heme
HOO C N H N H O COO H N H N H O
SLIDE 4
Neonatal Hyperbilirubinemia
Jaundice Light Therapy Kernicterus
SLIDE 5 Tg-UGT1 Mice
Liver Kidney Colon S.I.
Chen S, et al. JBC, 2005
SLIDE 6 Ugt1-/- Mice
Nguyen N, et al. JBC, 2008
4.8 kb (wt) 6.0 kb (mut) BglII digestion [genomic probe]
+/-
WT UGT1A UGT1A1 UGT1A Actin
Ad
mRNA Protein +/-
WT
SLIDE 7 Number of Mice Life Span of Ugt1-/- mice (days)
5 10 15 20 25 30 35 40
1 2 3 4 5 6 7 8 9 10 11 12
Ugt1+/- Ugt1-/-
Longevity of Ugt1-/- Mice
SLIDE 8
Generation of Humanized UGT1 Mice
(hUGT1)
Ugt1+/- Tg-UGT1 UGT1/Ugt1+/- UGT1/Ugt1 -/- Ugt1+/-
SLIDE 9
Humanized UGT1 Mice as a Model for Hyperbilirubinemia and Brain Toxicity
7-day Ugt1-/- 15-day hUGT1 7-day Ugt1+/- 15-day hUGT1
Normal Kernicterus
A B C D
SLIDE 10 Bilirubin (mg/dL) Days after birth
5 10 15 20 25 30 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22
TSB Levels in Ugt1-null and Humanized UGT1 Mice
Ugt1-/- Seizures Neonatal Hyperbilirubinemia
Fujiwara R, et al. PNAS, 2010
SLIDE 11 NRs Agonists v.s. Bilirubin Levels
Bilirubin (mg/dL) 5 10 15
Ctrl Ctrl PCN
Fat tissues
SLIDE 12
Generation of hUGT1 Mice with NR-null background
Car-/-/Ugt1+/+ TgUGT1/Ugt1-/- (Car-null) (hUGT1) TgUGT1/Car+/-/Ugt1+/- TgUGT1/Car-/-/Ugt1-/-
(hUGT1/Car-/-)
Pxr-/-/Ugt1+/+ (Pxr-null)
⊗
TgUGT1/Pxr+/-/Ugt1+/- TgUGT1/Pxr-/-/Ugt1-/-
(hUGT1/Pxr-/-)
⊗
TgUGT1/Pxr-/-/ Car-/-/Ugt1-/-
(hUGT1/Pxr-/-/Car-/-)
TgUGT1/Ugt1-/- (hUGT1)
SLIDE 13 hUGT1 Mice with Pxr-null and/or Car-null
Chen S, et al. Hepatology, 2012
I I I I
Bilirubin (mg/dL) 5 10 15 20
*** *** n.s hUGT1/Pxr-/- hUGT1/Pxr+/- 18 12 6
Fold of Induction UGT1A1
SLIDE 14
Control siPXR_bio siPXR_sc
0.9 2 0.6 4 6
Fold Gene Expression Control siPXR_bio siPXR_sc
1.2 0.3
Fold Gene Expression ** ** ** ** UGT1A1 PXR
Neonatal hUGT1 Hepatocyte Treated with siPXR
SLIDE 15
ChIP Assay
PXR IgG Input
SLIDE 16 Hypothesis of PXR Repression & De-repression
1 2 3 4 5
P X R
NCoR/SMRT
P X R
R X R
OFF
HDM
Ac
① ② 1 2 3 4 5 ON
HDM
Ac
Repressive Histone Methylation Marks Active Histone Methylation Marks
SLIDE 17 2 4 6 8 SiCtrl siSMRT siNCoR Fold Gene Expression ** **
0.5 1.0 1.5 Control siSMRT siNCoR Fold Gene Expression (SiRNA gene targets) ** **
UGT1A1 Gene Expression v.s. siRNA
SiCtrl siHDAC1 siHDAC3 siHDAC2
0.5 1.0 1.5 Fold Gene Expression (SiRNA gene targets)
SiCtrl siHDAC1 siHDAC3 siHDAC2 1 2 3 4 Fold Gene Expression
UGT1A1 UGT1A1
** **
** ** **
SLIDE 18
Conclusions
1. Humanized UGT1 mice develop neonatal hyperbilirubinemia and serve as a model for brain toxicity. 2. Humanized UGT1 mice are subject to regulation by nuclear receptor agonists. 3. Deletion of the Pxr gene, but not the Car gene, lowers serum bilirubin in hUGT1 mice. 4. PXR binds to the UGT1A1 promoter, and the genetic deletion of Pxr leads to the upregulation of UGT1A1 in hUGT1/Pxr-/- mice.
SLIDE 19 Irinotecan Metabolism and Toxicities
Blood Liver GI Epithelium GI Lumen
CPT-11 SN-38 Blood Neutropenia SN-38G
SN-38
SN-38G (Bile) Late Diarrhea SN-38 UGT1A1 Glucuronidase CE SN-38G UGT1A1
- Table1. SN-38 glucuronic activity studies in mouse liver microsomes
(MLM) and small intestine microsomes (MSIM).
Michaelis-Menten MLM MSIM Km (µM) 10.9±0.3 8.22±1.22 Vmax (pmol/min/mg) 69.1±1.0 217±13
SLIDE 20
Target Constructs
SLIDE 21
mUGT1A1 ΔmUGT1A1 mUGT1A1 Loading 1 2 3 4 5 6 F/F ΔHEP 3 2 1 Bilirubin (mg/dl) Ugt1F/F Ugt1ΔHEP mUGT1A1 ΔmUGT1A1 mUGT1A1 Loading ΔGI 3 2 1 Bilirubin (mg/dl) Ugt1F/F Ugt1ΔGI
Conditional Deletion of the Ugt1 Locus
Hepatocytes deletion (Alb-Cre) Intestinal Deletion (Vil-Cre)
F/F ΔHEP F/F ΔGI F/F F/ΔGI
SLIDE 22 ΔHep v.s. Δ GI___Survival
n.s
F/F ΔHep 100 50 7 14 21
CPT-11_100mg/kg
Survival (%)
n.s
F/F ΔHep 100 50 7 14 21
CPT-11_75mg/kg
Survival (%) Time (days) Time (days) F/F ΔGI 100 50 7 14 21
CPT-11_50mg/kg
Survival (%) F/F ΔGI 100 50 7 14 21
CPT-11_25mg/kg
Survival (%)
*** p<0.001 *** p<0.01
Time (days) Time (days)
SLIDE 23 ΔHep v.s. Δ GI___Weight Loss
Time (Days ) Remaining Weight (%) 20 40 60 80 100 120 1 2 3 4 5 20 40 60 80 100 120 1 2 3 4 5 Time (Days ) Remaining Weight (%) F/F ΔHep F/F ΔGI
CPT-11_75 mg/kg CPT-11_50 mg/kg
*** *** *** P<0.001
5 8
SLIDE 24
Irinotecan-induced Intestinal Toxicity
Ki67 TUNEL Control CPT-11 Jejunum Colon
SLIDE 25
Summary
1. Tissue specific Ugt1 deletion were generated by adopting Cre/loxP recombination system. 2. Extrahepatic clearance of bilirubin occurs in the absence of functional hepatic Ugt1a1. 3. Intestinal Ugt1a1 plays an important role in preventing CPT-11 induced toxicity.
SLIDE 26 Acknowledgements
- Dr. Robert Tukey
- Dr. Mei-Fei Yueh
- Dr. Ryoichi Fujiwara
Camille Konopnicki Nghia Nguyen Deirdre Beaton-La Placa Autumn Bonner
Collaborations:
Salk Institute, San Diego
NIEHS, North Carolina
University of California, San Diego
University of Lisbon, Portugal
Pfizer
- Dr. Jeffrey Stevens
- Dr. Hongliang Cai
- Dr. Michael Zientek
Ying Jiang
SLIDE 27
SLIDE 28
OCT1 OAT2 OAT7 OATP1B1 OATP1B3 OATP2B1 NTCP MRP3 MRP4 MRP6 BCRP P-gp MRP2 BSEP MATE1
Bilirubin Bilirubin-Gluc
Bilirubin Bilirubin-Gluc Bile UGT1A1
Bilirubin Transport and Metabolism in Hepatocytes
SLIDE 29 Ugt1 gene locus and targeting constructs
FRT ATG TGA FRT
47200 48000 48800 49600 50400 51200 52000 52800 53600 55200 56000 56800 57600 58400 59200 60000 60800
1 2 5 5’ Homology arm 3 4 3’ Homology arm
A. B.
47200 48000 48800 50400 51200 52000 52800 54400 55200 56000 57600 58400 59200 60000 60800
1 2 5 5’ Homology arm 3’ Homology arm loxP
C.
47200 48000 48800 49600 50400 51200 52000 52800 53600 54400 55200 56000 56800 57600 58400 59200 60000 60800 61600 62400
loxP 1 2 5
PGK Neo pA SII S
5’ Homology arm 3 4 3’ Homology arm loxP FRT
54400
ATG TGA ATG TGA
Deletion of exon 3 & 4 FLP CRE
49600 56800
