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LIINTOP Optimisation of Liver & Intestine In Vitro Models for Pharmacokinetics & Pharmacodynamics Studies Contract number: LSHB-CT-2006-037499 Project type: Specific Targeted Research Project (FP6) EU Contribution: € 2,933,291 Starting date: 1 January 2007 Ending date: 30 June 2010 Website: liintop.cnr.it Objectives The main aim of the project was to provide optimized protocols and experimental in vitro models for testing intestinal and liver absorption and metabolism of molecules of pharmacological interest. The scientific and technological objectives of the project can be divided in five main areas, as follows: 1. Characterisation and/or production of advanced in vitro liver and intestinal models in order to provide improved performance in screening and testing of new drugs’ absorption and metabolism: • Comparison of selected functions in the hepatic and intestinal in vitro cellular models with the corresponding normal human tissue ex vivo (i.e., primary human hepatocytes or human intestinal epithelium) • Characterisation of available intestinal cell lines and selection of the most appropriate model and of the optimal conditions to obtain good and reproducible differentiation • Optimisation of the culture conditions to make the models stable over time for specialised functions • Testing a new strategy using chromatin-remodelling agents (histone deacetylase inhibitors) to induce differentiation in primary hepatocyte-based models • Setting up new approaches to generate metabolically competent human hepatic cell lines. This includes genetic manipulation of existing cell lines (HepG2, HepaRG) transfected with key transcription factors, in order to allow an appro- 94 PROGRESS REPORTS FROM EU-FUNDED PROJECTS Progress Report 2011 & AXLR8-2 Workshop Report
priate expression of the differentiated phenotype • Optimisation of the culture conditions to differentiate adult bone marrow stem cells into functional hepatocytes • Development of more complex cell co-culture models to combine absorption and metabolism in intestine and liver. 2. Identification of drug transport and metabolism pathways in the available hepatic and intestinal in vitro models: • Modulation of their expression by culture conditions • Development of high-throughput methodologies for their study. 3. Determination of cellular and molecular targets as endpoints of drug exposure in intestine and liver with respect to: • Effects on cell proliferation (e.g., cell cycle control, apoptosis/necrosis) • Effects on differentiated functions (e.g., protein secretion, cell junctions, expression of genes involved in transport and metabolism). 4. In silico approaches to modelling the liver and the intestine: • Development of mechanismbased pharmacokinetic models • Exploration of the predictive utility of new in vitro models • Identification of areas requiring refinement for future in vitro models. 5. Determination of the transfer potential of the developed in vitro models for their utilisation within the industrial setting, that derives from the close collaboration within the project of research academic institutions and SMEs. Experimental Design & Results Achieved Partners working with cellular models have mainly dealt with objectives 1 and 2, trying to characterise and optimise the cellular models already available or under development in the areas of hepatocytes and enterocytes. The activities performed and the results achieved are described below. Hepatic Cell Models Different models and approaches were explored for the hepatic differentiation, since the major problem with hepatocytes in vitro is the rapid loss of their specialised functions. The first approach (by partners INSERM, BPI, Novomass, and Advancell) has been the extensive characterisation and optimisation of the HepaRG cell line. HepaRG, a human liver cell line of tumour origin, has been extensively characterised for the expression of bio-transformation activities such as several major CYPs, antioxidant activities and efflux transport activities (BSEP and MRP2). Differentiated HepaRG PROGRESS REPORTS FROM EU-FUNDED PROJECTS Progress Report 2011 & AXLR8-2 Workshop Report 95
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ALTERNATIVE TESTING STRATEGIES PROG
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ALTERNATIVE TESTING STRATEGIES PROG
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TABLE OF CONTENTS 1. 2. EXECUTIVE S
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Update on EPA’s ToxCast Programme
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challenges, needs, and priorities f
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1INTRODUCTION It is the aim of the
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and progress of these multidiscipli
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Table 2. Members of the AXLR8 Scien
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• OECD Joint Meeting Special Sess
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ACuteTox Optimisation & Pre-Validat
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to robotic screening platforms; and
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ased on functional parameters inclu
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Table 1. Outliers identified by com
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showed that for some compounds the
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Computer-based prediction of metabo
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three reference compounds revealed
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a calibration curve constructed. Th
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cell lines) or 48 hours (A704 cells
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multivariate CART results did not c
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probability) ~50% of the substances
Page 44 and 45: Publications 2010-11 Hoffmann S, Ki
Page 46 and 47: Per Artursson Uppsala University Up
Page 48 and 49: their genotoxic and carcinogenic po
Page 50 and 51: Table 1. Overview of carcinoGENOMIC
Page 52 and 53: D12.23 3 monthly Project M42 √ Bo
Page 54 and 55: annual carcinoGENOMICS meeting in N
Page 56 and 57: M3.5 Decision of most M40 Postponed
Page 58 and 59: M3.4 Identification of most suitabl
Page 60 and 61: WP1 with input from other partners
Page 62 and 63: Partners Project Co-ordinator Jos K
Page 64 and 65: combine knowledge of critical proce
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Page 68 and 69: WP6 is devoted to the setup of a so
Page 70 and 71: Partners Co-ordinator Bart van der
Page 72 and 73: obustness, comparing results obtain
Page 74 and 75: 6. Candéias S, Pons B, Viau M, et
Page 76 and 77: ESNATS Embryonic Stem Cell-Based No
Page 78 and 79: Table 1. List of deliverables due i
Page 80 and 81: D3.3.1 Toxicity gene expression sig
Page 82 and 83: Table 2. List of milestones due in
Page 84 and 85: Table 3. Test systems corresponding
Page 86 and 87: Publications 2010-11 1. Peters SJ,
Page 88 and 89: Marcel Leist Universität Konstanz
Page 90 and 91: The most significant achievements o
Page 92 and 93: Figure 3. The Sensor Dish Reader (S
Page 96 and 97: cells express most of the functions
Page 98 and 99: Figure 3. Scheme of lentivirus gene
Page 100 and 101: Figure 5. Co-culture set-up. Functi
Page 102 and 103: Figure 9. Phalloidin-TRITC staining
Page 104 and 105: Table 1. Values are expressed as pe
Page 106 and 107: formed with mannitol, atenolol, pro
Page 108 and 109: Figure 14. Comparison of Digoxin an
Page 110 and 111: By using the new technology of HCA
Page 112 and 113: Figure 17. (Left panel) Phalloidin-
Page 114 and 115: limited amount of data could be pro
Page 116 and 117: André Guillouzo Institut National
Page 118 and 119: Objectives The overall aim of this
Page 120 and 121: Table 2: Milestones achieved in 201
Page 122 and 123: detected iron (µg/ml) 80 70 6
Page 124 and 125: in the following order: uncoated ma
Page 126 and 127: analysis of leukocytes, expression
Page 128 and 129: 2011 the automated protocols will b
Page 130 and 131: OpenTox Promotion, Development, Acc
Page 132 and 133: Results The OpenTox Framework The O
Page 134 and 135: Figure 1. ToxPredict: OpenTox Appli
Page 136 and 137: scientifically sound manner, so as
Page 138 and 139: Figure 4. Creating a QPRF report wi
Page 140 and 141: Figure 7. MaxTox model-building app
Page 142 and 143: Figure 8. Bioclipse interaction wit
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and model predictions, which they m
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Publications 2010-11 1. Hardy B, Do
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In vitro toxicology using isolated
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(2C-Glo-CYP2C, 3A-Glo-CYP3A) while
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neuronal models 2D (primary culture
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10 and 14 days. The liver-specific
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Frédéric Bois Institut National d
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Figure 1. The Sens-it-iv sphere. 2.
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Figure 2. The updated modular struc
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Technology Module The aim of techno
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Table 1: The final compound list. R
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Table 2. The Sens-it-iv Toolbox. N
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Figure 4. A gene profile for identi
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Figure 7. In vitro T cell priming a
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sessions at congress and meetings (
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7. Gibbs S, van Montfrans C, Kroeze
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vitro assessment of allergens. Eds:
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S.F. Martin Universitätsklinikum F
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idging the gap to human volunteer s
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combinations of transcriptomics, me
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normal animals who usually exhibit
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Workshop proposals Concepts of data
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VITROCELLOMICS Reducing Animal Expe
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esulting of the project was the abi
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Figure 3. Four-compartment artifici
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embryonic stem cells differentiatin
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196 3AXLR8-2 WORKSHOP REPORT
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Workshop participants were divided
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08.45 - 09.15 ACuteTox Annette Kopp
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3.3 Workshop Presentations 202
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Table 1. A sampling of current toxi
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Table 2. EPA activities: a timeline
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Why is a Consortium Needed While th
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the development of new more relevan
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in particular for fundamental resea
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compared to controls. Most interest
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epidermis to known allergens and UV
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The Virtual Liver Spatial-Temporal
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A B C D E Figure 2. Tissue reconstr
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Figure 4. Mechanisms of how hepatoc
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in rat liver in vivo (Figure 5A). H
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The IMI eTOX Project Efforts to Dev
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deliverables, which can only be ach
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A series of publications related to
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Update on EPA’s ToxCast Programme
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Phase I of ToxCast involved the eva
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Endpoint Brief Description of Bioac
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includes approximately 150 chemical
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information. For each slice, distan
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Judson RS, Houck KA, Kavlock RJ, et
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Embryonic Stem Cell Approaches Towa
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perhaps be tested efficiently in al
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compared gene and protein expressio
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cost and time, allowing more chemic
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Toxicol Appl Pharmacol. 2011; 251,
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functions, establish relationships
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cellular behaviours are captured fr
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assay. PLoS One. 2011; 6, e18540. 7
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learning algorithm called Support V
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Compound Potency Vehicle Concentrat
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mediated inhibition of RXR function
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References Basketter DA, Evans P, F
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When considering consumer exposure,
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in the induction of skin sensitisat
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has reacted, when the peptide deple
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of presentations and discussions, t
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Contact Dermatitis. 2005; 53. 16. S
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the development of in vitro assays.
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Although the mechanisms underlying
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skin sensitisation, it will aid in
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Figure 3. Scatter plot of robust li
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7. De Smedt A, Van Den Heuvel R, Va
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The OECD Adverse Outcome Pathway Ap
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the AOP can be used in qualitative
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Table 1. Summary of the experimenta
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the significance of type 1 versus t
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plus the key events that occur acro
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Dimitrov, S.D., Low, L.K., Patlewic
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[Supporting information and databas
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Figure 1. Strategic R&D of chemical
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The Bhas 42 system can sensitively
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Table 1. Established reporter cell
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elated genes (Hand1, ADAM19, Cmyal,
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Publications Carcinogenicity Tanaka
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Case Study Approaches for Implement
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assay results together with CSBP mo
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them to support multi-day testing i
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CSBP models for both receptor-media
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Hamner Presentations in 2011 • Ch
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human health risk assessment. Risk
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There is a significant scientific c
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phototoxicity and eye irritation, f
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allergy risk assessment rather than
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‘toolbox’ of non-animal methods
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332 3.4 Breakout Group Reports
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Figure 1. An illustration of the me
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methods, and the generally poor und
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3.4.3. Break-Out Group 2: Reproduct
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- Deep-sequencing of birth defect p
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Figure 2. Specific stages of the ma
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3.4.4 Break-Out Group 3: Skin Sensi
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for further progress. Better tools
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sure doses is not straightforward.
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3.5 AXLR8 Scientific Panel Recommen
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Workshop participants underlined th
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could be co-ordinated in a focused
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focusing on scientific excellence a
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Directory of Projects & Co-ordinato
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Glossary of Terms 2D/3D 3Rs CARDAM/
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