A shift in paradigm towards human biologybased systems for cholestatic-liver disease

Transcription

1 A shift in paradigm towards human biologybased systems for cholestatic-liver disease Fozia Noor Systems Toxicology In Vitro Metabolomics Biochemical Engineering Institute Saarland University, Germany BioMed21 A Human Pathways Approach to Disease Research Brussels, 8-9 th December 2015

2 Cholestatic-Liver Diseases - Introduction

3 Hepatobiliary System Disease of Civilization Obesity Diabetes Non-alcoholic fatty liver disease Chronic liver disease Heart disease Metabolic syndrome Other nutritional disorders Cholestatic Liver Diseases Biliary atresia Progressive familial intrahepatic cholestasis Alagille syndrome Primary biliary cirrhosis Primary sclerosing cholangitis Cholangiocarcinoma Drug induced cholestasis Functions of Bile Acids Glucose metabolism Lipid metabolism Cholesterol metabolism Energy expenditure Control of gut microbiota Xenobiotic metabolism

4 Bile acid transport system Noor, J Physiol., 2015

5 Bile acid transport system X X X Noor, J Physiol., 2015

6 Human specific cell models

7 Why human models? In-vivo animal models differ from humans in: Bile acid composition Transporters activities Milder phenotypes Effect on nuclear receptors Immune and inflammatory response CYP 450 system for metabolism and clearance Gut microbiota Mechanisms of parenchyma injury (necrosis vs. apoptosis) Common in-vitro models usually 2D Monocultures Very often rodent primary cells Cell lines Human in vivo-like models are needed! In-vivo In-vitro

8 Liver

9 In vitro 3D cultivation systems for liver Monolayer Collagen sandwich cultures of primary hepatocytes (Saskia Müller) Single cells Aggregates Alginate encapsulation etc Bioreactor 3D cultivation of primary hepatocytes (Charité, Berlin; Gerlach et al., Mueller et al.) Hepatology, 2011 Spinner flask cultures (Carrondo and Alves, IBET, Portugal) 3D organotypic cultures of primary hepatocytes (Insphero technology, Zurich) Mueller et al., Bioeng Biomed Sci 2011 Gunness et al, Tox Sci., 2013 Mueller et al., Tox in vitro, 2014 Alvetex scaffold

10 Human 3D in-vitro models Mueller et al., 2014

11 Day 35 Day 8 Human 3D in-vitro models Primary human hepatocytes Human HepaRG cell line CYP3A4 Albumin Merge MRP2 activity Albumin Fredriksson et al., 2015 CYP3A4 Gunness et al., 2013

12 HepaRG spheroids express important bile acid transporters and they can be induced BSEP MRP2 Nuclei Merge

13 Disruption of bile canaliculi network Effects of chlorpromazine on 3D structure 0 µm CPZ 100 µm CPZ Mueller et al., 2014

14 Troglitazone Viability (% of control) Chlorpromazine Tetracycline Bosentan Acetaminophen Drug induced cholestasis 14 days exposure Concentration (µm)

15 Negative Positive Drug induced cholestasis 8 days 14 days Compound IC 50 (-BA) (µm) IC 50 (+BA) (µm) Cholestatic Index (IC50 (+BA) / IC50 (-BA)) IC 50 (-BA) (µm) IC 50 (+BA) (µm) Cholestatic Index (IC50 (+BA) / IC50 (-BA)) Chlorpromazine Troglitazone Bosentan Tetracycline Acetaminophen

16 Accumulation of bile acids in HepaRG cells Upon exposure to serum concentrations in cholestatic patients Upon exposure to biliary concentrations in cholestatic patients

17 control ba 0.1x ba 0.3x ba 0.5x bos+ba 0.1x bos+ba 0.3x bos+ba 0.5x hc+ba 0.1x hc+ba 0.3x hc+ba 0.5x bos+hc+ba bos+hc+ba bos+hc+ba Mechanism of cell death: apoptosis 3 2,5 Serum bile acids ratio (A caspase3/7 /Viability) 2 1,5 1 0,5 0 [BA] 0x 1x 50x 1x 50x 1x 50x 1x 50x control BA Bos HC Bos HC ratio (A caspase3/7 /Viability) [BA] 0x 0.1x 0.3x 0.5x 0.1x 0.3x 0.5x 0.1x 0.3x 0.5x 0.1x 0.3x 0.5x Hepatic parenchyma death in rodents is via necrosis! control BA Bos HC Bos HC Biliary bile acids

18 Enabling Technologies & Human disease pathways-based approaches

19 Metabolome: the Rosetta Stone

20 In vitro Metabolomics in pathway research Application domains Screening Mode of action (MoA) Biomarker identification

21 Metabolome analysis biochemical networks

22 Leucine Glutamine Lactate Glucose Metabolome Metabolic alterations in 2D and 3D cultures upon repeated dose exposure to bosentan

23 Metabolite profiles over time

24 Adverse outcome pathway Drug induced cholestasis BSEP inhibition Bile salt accumulation + + PPARα FXR Increased insulin sensitivity HIF-1α Increased lactate secretion Cholestasis + Oxidative stress Increased energy metabolism Cell death

25 Systems approach within a disease-pathway framework

26 Opportunities and Challenges

27 Reprograming Gene Editing technology Mutant ipsc x x x x Gene editing ZFN TALENS CRISPR/Cas9 corrected ipsc x x x x Biopsy Inherited disease patient Disease model Research and screening

28 Microfluidic Systems Frey et al., Nat Comm, 2014

29 Prediction of dose and pathway related effects in humans Reverse dosimetry and metabolome analysis Control Low exposure High exposure In vitro long term data Klein et al., Tox Sci., 2015

30 Towards human in vivo computational models UGT APAPG APAPGB Drug Metabolism MRP3/4 MRP2 SULT APAPS APAPSB APAP-GS APAP APAPB CYP1A2 CYP2A6 CYP2D6 NQO1 APAP-GSB Passive Diffusion APAP CYP2E1 CYP3A4 NAPQI GSTA2 APAPGS Passive Diffusion Import NAPQIP GSR GSS ggc GCL Cys GSH Export GSSG GSH GPX GGT Synthesis Glutathione Metabolism NOS NO O2 - SOD H2O2 H2O ONOO - ROS and RNS Synthesis In vitro HepaRG One ODE system in each cell Cell to cell variability Agent-based mechanical model Blood flow Diffusion of drugs to the cells Absorption, distribution, metabolism and excretion by the rest of the body

31 Microbiome The gut microbiome the forgotten organ! Microbiome modifies the epigenome Bile acid signalling plays a role in health and disease Bile acids as therapeutic agents

32 Perspectives: towards evidence based science and personalized medicine Omics Biomarkers Human specific mechanisms Diagnosis and prognosis Advanced 3D Models PHH ipsc derived cells from patients Based on genetic background Therapy monitoring Huge clinical data available Omics Biomarkers

33 Acknowledgments NOTOX Prof. Elmar Heinzle Daniel Müller Sebastian Klein Yeda Kaminsky Prof. M. Ingelman-Sundberg Inger Johansson Lisa Fredriksson Patrina Gunness Dalilah Hendriks Ursula Müller-Viera Klaus Biemel Jens Sennhauser Christophe Chesné