Tuesday, April 24, 2012 Workshop: Genetic Toxicology: Opportunities to Integrate New Approaches Hepatotoxicity Test by Stem Cell derived Hepatocyte Seiichi Ishida National Institute of Health Sciences Division of Pharmacology
Critical Path to New Medical Products lead discovery lead optimization candidate selection pre-clinical development clinical development approval Drug development 1) costs more than $ 1,000,000,000-. 2) takes 10 to 15 years. 3) gets the one hit out of 22,000 candidates.
Critical Path to New Medical Products lead discovery lead optimization candidate selection pre-clinical development clinical development approval Drug development First in Human Patients 1) costs more than $ 1,000,000,000-. 2) takes 10 to 15 years. 3) gets the one hit out of 22,000 candidates.
Toxicities Leading to Drug Withdrawal from the US Market Nat Rev Drug Discov. 2007, 6, 904
Regulatory Actions due to DILI (1995-2006) http://www.fda.gov/medwatch/safety.htm Withdrawals bromfenac troglitazone pemoline Second Line felbamate tolcapone Trovafloxacin Warnings acetaminophen leflunomide nefazodone nevirapine pyrazinamide/rifampin terbinafine valproic acid zifirlukast atomoxetine interferon 1b 1b and 1a saquinavir infliximab bosentan telithromycin (kava, lipokinex)
What Dose the Liver Do? Liver converts excess glucose to starch for storage. regulates blood clotting. metabolizes proteins and cholesterol. excretes bile for fat digestion. excretes wastes via bile. clears drugs, chemicals etc. in the blood. and more hepatic lobule sinusoid central vein hepatocyte portal vein Bile duct Nature Reviews Immunology 2006, 6, 244
Drug Uptake, Metabolism, Excretion in the Hepatocyte hepatocyte Discov Med. 2012 Jan;13(68):19-34 modified % drugs metabolized by CYP enzymes CYP 2C9 14% CYP 2C19 11% CYP2D6 23% CYP 1A2 14% CYP 3A4-5 33% CYP2E1 5%
Model of Acetaminophen Hepatotoxicity reactive metabolite metabolism of paracetamol (acetaminophen) showing proposed metabolic activation and its involvement in the toxicity
Drug Development & ADME Evaluation lead discovery lead optimization candidate selection pre-clinical development clinical development approval ADME evaluation using model systems Absorption Distribution Metabolism Excretion animal models difficulties of extrapolation to humans human tissue models primary hepatocyte fresh cryo-preserved liver microsome immortalized hepatocyte ( HepG2 etc.) liver resemblance easiness reproducibility to access /
Aim of Our Research is to establish the culture system which has 1) easy availability 2) human liver-like functions 3) good reproducibility.
Schematic Presentation of Fetal Liver Development hepatocyte fertilized egg endoderm hepatoblast stem cells cholangiocyte STEM CELLS 2009, 27, 577
Development of a Novel Drug Toxicity Testing System Using Human ips Cells ips cell fibroblast from human skin establishment of ips cell induction of differentiation regenerative medicine new drug development toxicity testing efficacy testing disease cause study application for human prerequisite of safety assurance doable with current technologies application for in vitro study
Hepatic Differentiation of Human Stem Cells Transduced with Three Factors stem cells endoderm heoatocyte progenitor hepatocyte Ad-SOX17 Ad-HEX Ad-HNF4a Mol Ther. 2012 Jan;20(1):127-37 without transduction with transduction primary hepatocyte binucleiar cell
relative gene expression Exprssions of Drug Metabolism relating Genes phase I enzyme phase II enzyme transporter marker PH : primary hepatocyte 48 hr after plating Mol Ther. 2012 Jan;20(1):127-37
relative gene expression Toxicity Testing with Metabolic Activation phase I enzyme phase II enzyme transporter marker PH : primary hepatocyte 48 hr after plating Mol Ther. 2012 Jan;20(1):127-37
Strengths and Weaknesses of Stem Cell derived Hepatocyte Are the ips cells useful tool to establish the culture system which has 1) easy availability? 2) human liver-like functions? 3) good reproducibility?
Strengths and Weaknesses of Stem Cell derived Hepatocyte Are the ips cells useful tool to establish the culture system which has 1) easy availability? ips cells could be a useful source for the hepatocyte differentiation. 2) human liver-like functions? ips derived hepatocytes might have ADME activities, however, their activities are not high enough right now. 3) good reproducibility? Differentiation protocol is too complicated.
Issues to Be Overcome 1 human liver-like functions accentuation of hepatic function [three dimensional (3-D) culture]
Effort to 3-D Culture System Development A) media flow B) HepG2 cells after 17 day culture B Scaffold Media flow Upper bottom side φ3 C) toluidine blue staining of thin section Side 15 φ20 radial flow bioreactor (RFB) HepG2 cells in RFB
change of expression (fold) Improvement of Hepatic Function by 3-D Culture 10 5 CYP2B6 GAD1 GSTA3 GSTP1 SRD5A2 CHST1 NAT2 SNN 2 1 1/2 phaseⅠ phaseⅡ other 1/5 transporter drug metabolism gene p-value 0.05 p-value < 0.05 (n=3) (Horiuchi et al. B.B.R.C 2009)
Issues to Be Overcome 2 human liver-like functions Reconstitution of hepatic function in vitro [co-culture system]
Liver Structure and Constituent Cells hepatic stellate cell Kupffer cell central vein sinusoid hepatocyte bile duct portal vein hepatic artery
Hepatic Stellate Cells
Development of Co-culture System mimicking Live Structure HepG2 primary hepatocyte collaboration with hepatocyte + co-culture collagen vitrigel vitrigel stellate cell LI90 stellate-shaped mesenchymal cells that exist in the space of Disse of the liver and contain many fat droplets in cytoplasm.
Issues to Be Overcome 3 good reproducibility development of simpler differentiation system [HepaRG cells]
HepaRG Cell HepaRG cells --- hepatocyte progenitor differentiation scheme collaboration with are isolated from normal part of an Edmondson grade I differentiated tumor. have the ability to differentiate towards hepatocyte-like and biliary epithelial-like cells at confluence. passage 2% DMSO Day 0 (dedifferentiation) 15 30 morphorolgy proliferation contact inhibition differentiation H day 4 day 7 day 15 day 30 B H: hepatocyte B: biliary cell
Changes of Gene Expressions during HepaRG Differentiation K-means and hierarchical clustering Functional characterization K-menas cluster number D4 D7 D15 D30(-) D30(+) Metabolism 4500 collaboration with GeneChip signals CYP2C9 CYP3A4 18000 8 15 7 Cluster 18 4000 3500 3000 2500 16000 14000 12000 10000 17 13 18 11 2000 1500 1000 500 8000 6000 4000 2000 4 19 DMSO-dependent increase 0 4 7 15 30 4 7 15 30 days days 0 6 2 20 14 3 10 Cluster 4 20000 18000 16000 14000 12000 10000 Albumin 800 700 600 500 400 CD14 9 16 12 8000 6000 4000 2000 300 200 100 5 DMSO-independent increase 0 4 7 15 30 4 7 15 30 days days 0 1 2% DMSO (day 15 to day 30) Negative control
Conclusion Are the ips cells useful tool to establish the culture system which has 1) easy availability? ips cells could be a useful source for the hepatocyte differentiation. 2) human liver-like functions? ADME activities are not high enough right now. 3D-culture & co-culture systems help to accelerate ADME activities. 3) good reproducibility? Differentiation protocol is too complicated. Alternative source might be desirable. i.e. hepatocyte progenitor cells, direct reprograming etc.
Acknowledgement This project is supported by Research on Publicly Essential Drugs and Medical Devices from Japan Health Sciences Foundation, Program for Promoting Basic Research in the Field of Health and Medical Care Health Labor Sciences Research Grant from the Ministry of Health, Labor and Welfare of Japan, and Agri-Health Translational Research Project (No.6110 ) from the Ministry of Agriculture, Forestry and Fisheries of Japan. Thank you very much for your attention!