Application of Animal Models for Human Glucuronidation Shujuan Chen - PowerPoint PPT Presentation

Application of Animal Models for Human Glucuronidation Shujuan Chen Tukey Laboratory Departments of Chemistry & Biochemistry and Pharmacology, UCSD 2012 SCDMDG

Human UGTs: 19 UGT1A5 Tertiary Amines isoforms UGT1A3 Heterocyclic Aromatics UGT1A4 Bilirubin, irinotecan UGT1A1 UGT1A6 Drugs, 66% 1 Pollutants, UGT1A7 C 18 Steroids UGT1A8 UGT1A10 UGT1A9 41% UGT2B10 Bioactive lipids UGT2B28 UGT2B11 UGT2B7 Bile acids UGT2B4 B UGT2B15 Androgens 59% 2 UGT2B17 UGT2A1 A Odorant molecules UGT2A2 UGT2A3

Bilirubin Clearance Heme UGT1A1 HOO COO C H Bilirubin Bilirubin Glucuronides N N H H N N H H O O Bilirubin

Neonatal Hyperbilirubinemia Jaundice Light Therapy Kernicterus

Tg-UGT1 Mice Kidney Liver S.I. Colon Chen S, et al. JBC, 2005

Ugt1 -/- Mice +/- -/- WT BglII digestion 6.0 kb (mut) 4.8 kb (wt) [genomic probe] Ad -/- +/- WT mRNA UGT1A UGT1A UGT1A1 Protein Actin Nguyen N, et al. JBC, 2008

Longevity of Ugt1 -/- Mice Ugt1 +/- Ugt1 -/- 40 35 Number of Mice 30 25 20 15 10 5 0 1 2 3 4 5 6 7 8 9 10 11 12 Life Span of Ugt1 -/- mice (days)

Generation of Humanized UGT1 Mice Ugt1 +/- Tg-UGT1 Ugt1 +/- UGT1/Ugt1 +/- UGT1/Ugt1 -/- ( h UGT1 )

Humanized UGT1 Mice as a Model for Hyperbilirubinemia and Brain Toxicity Normal Kernicterus A B 7-day 7-day Ugt1 -/- Ugt1 +/- C D 15-day 15-day hUGT1 hUGT1

TSB Levels in Ugt1-null and Humanized UGT1 Mice 30 Seizures 25 Bilirubin (mg/dL) Ugt1 -/- 20 15 10 Neonatal Hyperbilirubinemia 5 0 17 10 12 13 14 15 16 18 19 20 21 22 1 2 3 4 5 6 7 8 9 11 Days after birth Fujiwara R, et al. PNAS, 2010

NRs Agonists v.s. Bilirubin Levels 15 Fat tissues Bilirubin (mg/dL) Ctrl Ctrl PCN 10 5 0

Generation of hUGT1 Mice with NR-null background Car -/- /Ugt1 +/+ Pxr -/- /Ugt1 +/+ TgUGT1/Ugt1 -/- TgUGT1/Ugt1 -/- ( Car-null ) ( hUGT1 ) ( hUGT1 ) ( Pxr-null ) TgUGT1/Pxr +/- /Ugt1 +/- TgUGT1/Car +/- /Ugt1 +/- ⊗ ⊗ TgUGT1/Pxr -/- /Ugt1 -/- TgUGT1/Car -/- /Ugt1 -/- ( hUGT1/Car -/- ) ( hUGT1/Pxr -/- ) TgUGT1/Pxr -/- / Car -/- /Ugt1 -/- ( hUGT1/Pxr -/- /Car -/- )

hUGT1 Mice with Pxr-null and/or Car-null *** 20 *** n.s Bilirubin (mg/dL) 15 Fold of Induction UGT1A1 18 10 12 5 6 0 0 I I I I hUGT1/Pxr +/- hUGT1/Pxr -/- Chen S, et al. Hepatology, 2012

Neonatal hUGT1 Hepatocyte Treated with siPXR PXR UGT1A1 ** 1.2 6 Fold Gene Expression Fold Gene Expression 0.9 4 ** ** 0.6 ** 2 0.3 0 0 Control siPXR_bio siPXR_sc Control siPXR_bio siPXR_sc

ChIP Assay PXR IgG Input

Hypothesis of PXR Repression & De-repression HDM ② NCoR/SMRT ① OFF P R Ac X X 1 2 3 4 5 R R HDM ON P X 1 2 3 4 5 R Ac Repressive Histone Methylation Marks Active Histone Methylation Marks

UGT1A1 Gene Expression v.s. siRNA Fold Gene Expression UGT1A1 8 ** 6 ** Fold Gene Expression (SiRNA gene targets) 1.5 4 1.0 2 ** ** 0.5 0 0 Control siSMRT siNCoR SiCtrl siSMRT siNCoR Fold Gene Expression UGT1A1 4 ** 3 ** 2 Fold Gene Expression (SiRNA gene targets) 1.5 1.0 1 ** ** ** 0.5 0 0 siHDAC2 SiCtrl siHDAC1 siHDAC2 siHDAC3 SiCtrl siHDAC1 siHDAC3

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.

Irinotecan Metabolism and Toxicities Blood Liver GI Epithelium GI Lumen Neutropenia Blood CE CPT-11 SN-38 UGT1A1 SN-38G SN-38G SN-38G (Bile) Glucuronidase UGT1A1 SN-38 SN-38 Table1. SN-38 glucuronic activity studies in mouse liver microsomes (MLM) and small intestine microsomes (MSIM). Late Diarrhea Michaelis-Menten MLM MSIM Km (µM) 10.9 ± 0.3 8.22 ± 1.22 Vmax 69.1 ± 1.0 217 ± 13 (pmol/min/mg)

Target Constructs

Conditional Deletion of the Ugt1 Locus Hepatocytes deletion (Alb-Cre) F/F Δ HEP 3 Bilirubin (mg/dl) F/F Δ HEP 1 2 3 4 5 6 2 mUGT1A1 1 mUGT1A1 Loading Δ mUGT1A1 0 Ugt1 F/F Ugt1 Δ HEP Intestinal Deletion (Vil-Cre) Δ GI 3 Bilirubin (mg/dl) F/ Δ GI F/F Δ GI F/F 2 mUGT1A1 mUGT1A1 Δ mUGT1A1 Loading 1 0 Ugt1 F/F Ugt1 Δ GI

Δ Hep v.s. Δ GI ___Survival CPT-11_100mg/kg CPT-11_75mg/kg 100 100 F/F F/F Δ Hep Δ Hep Survival (%) Survival (%) 50 50 n.s n.s 0 0 0 0 7 14 21 7 14 21 Time (days) Time (days) CPT-11_50mg/kg CPT-11_25mg/kg 100 100 F/F Δ GI Survival (%) Survival (%) F/F Δ GI 50 50 *** p<0.01 *** p<0.001 0 0 0 0 7 14 21 7 14 21 Time (days) Time (days)

Δ Hep v.s. Δ GI ___Weight Loss CPT-11_75 mg/kg CPT-11_50 mg/kg 120 120 Remaining Weight (%) Remaining Weight (%) *** *** 100 100 80 80 60 60 40 40 20 20 0 0 1 2 3 4 5 8 5 1 2 3 4 5 Time (Days ) Time (Days ) F/F F/F Δ Hep Δ GI *** P<0.001

Irinotecan-induced Intestinal Toxicity Jejunum Colon Ki67 TUNEL Control CPT-11

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.

Acknowledgements Dr. Robert Tukey Collaborations: Dr. Ronald Evans Dr. Mei-Fei Yueh Salk Institute, San Diego Dr. Ryoichi Fujiwara Camille Konopnicki Dr. Masahiko Negishi Nghia Nguyen NIEHS, North Carolina Deirdre Beaton-La Placa Autumn Bonner Dr. Michael Karin University of California, San Diego Dr. Dora Brites University of Lisbon, Portugal Pfizer Dr. Jeffrey Stevens Dr. Hongliang Cai Dr. Michael Zientek Ying Jiang

Bilirubin Transport and Metabolism in Hepatocytes OAT2 OATP1B1 OATP2B1 OATP1B3 NTCP OCT1 OAT7 Bilirubin-Gluc Bilirubin UGT1A1 Bilirubin-Gluc Bilirubin MRP3 P-gp MRP4 MRP2 BCRP MRP6 Bile BSEP MATE1

Ugt1 gene locus and targeting constructs A. FRT FRT 5’ Homology arm 3’ Homology arm TGA ATG 5 2 Neo pA SII S 1 3 4 PGK 47200 48000 48800 49600 50400 51200 52000 52800 53600 54400 55200 56000 56800 57600 58400 59200 60000 60800 61600 62400 FLP B. FRT loxP loxP 5’ Homology arm 3’ Homology arm TGA ATG 5 2 1 3 4 47200 48000 48800 49600 50400 51200 52000 52800 53600 54400 55200 56000 56800 57600 58400 59200 60000 60800 CRE C. loxP 5’ Homology arm 3’ Homology arm ATG 5 TGA 1 2 47200 48000 48800 49600 50400 51200 52000 52800 54400 55200 56000 56800 57600 58400 59200 60000 60800 Deletion of exon 3 & 4