Workshop: Applying Exposure Science to Increase the Utility of Non-Animal Data in Efficacy and Safety Testing, 16 Feb. 2017
Presentation Outline Should we account for kinetics in vitro and in vivo for quantitative in vitro-in vivo extrapolation (QIVIVE) purposes? 0 QIVIVE 0 Toxicokinetis 0 Free concentration 0 Repeated Dosing
% affected people In Vitro Assays in Risk Assessment 0 Toxicity Testing in 21 st Century 0 National Research Council (2007), Toxicity Testing: A Vision and a Strategy, DOI: 10.17226/11970 0 From hazard identification to hazard characterization 0 Quantitative In Vitro-In Vivo Extrapolation (QIVIVE) 0 Estimating chemical exposures producing target tissue exposures in humans equivalent to those associated with effects in in vitro toxicity tests? ADI? Dose (mg/kg bw)
Louisse et al. (2010) Tox. Sci. 118: 470 QIVIVE In vitro toxicodynamics Mouse embryonic stem cell test Input: BMC values metabolite alkoxyacetic acid Blood Blood Fat Fat In vitro toxicokinetics Biotransformation kinetics in rat hepatocytes Rapidly perfused tissue Slowly perfused tissue Liver Rapidly perfused tissue Slowly perfused tissue Liver Evaluation of IVIVE model with in vivo embryotoxic doses alkoxyacetic acid formation GI tract Output: predicted oral embryotoxic dose parent glycol ethers
% of living cells In Vitro Assays 0 Toxicodynamics EC 50 0 Toxicokinetics concentration
Toxicokinetics Input Parameters Passive diffusion down C gradient J = P app *SA*C e.g. Caco-2, PAMPA Diffusion into tissue Non-saturable protein binding, fu Well-stirred tissue Whole-body D perfusion-limited e.g. RED, SPME Bessems et al. (2014) Reg. Toxicol. Pharmacol. 68, 119 Peyret and Krishnan (2011) SAR QSAR Environ. Res. 22, 129 Liao et al. (2007) Risk Anal. 27, 1223 Wetmore (2015) Toxicol. 332, 94 e.g. microsomes, cryopreserved hepatocytes e.g. RPTEC/TERT1
Dose Metrics in QIVIVE oral dose C plasma C tissue Hermens et al (2007) JTEH-A 70: 727 Kretschmann et al (2012) Env. Tox. Chem 31: 2014
Distribution of a chemical in vitro Differences between in vitro assays influencing the fraction of test chemical in cells: evaporation PLASTIC (well) protein binding plastic binding free in medium MEDIUM cell binding CELL metabolism target free free in in cell cell 0Serum concentration 0Cell concentration 0Well plate dimensions 0Open vs closed systems 0Exposure time 0pH 0Temperature 0Dosing regimen 03D matrix 0Metabolic capacity 0Transporter presence 0Monolayer vs 3D structure 0Transwell vs well plate Groothuis et al., 2015 Toxicol. 332: 30
Distribution of a chemical in vitro that determine free concentrations in culture medium Properties affecting affinity for in vitro constituents, including: K d /LogP/LogD7.4/ K OW H pk a Properties affecting reactivity, degradation, metabolism Groothuis et al. (2015) Toxicol. 332: 30
Serum Protein Binding 0 Surfactants 0 Relationships with standard phys. chem. properties do not apply 0 Benzalkonium chlorides (C6-C18) 0 Measured K MW using IAM column, TRANSIL 0 Measured PPB using Resolvosil BSA-7 column 0 RTgill-W1 basal cytotoxicity assay (Kramer et al., 2012, Chem. Res. Toxicol. 25:436-45) 0 10% FBS, 4 g/l BSA in medium Groothuis et al., manuscript in preparation
Serum Protein Binding 0 Hypothesis 0 CMB Narcosis ~100 mmol/kg lipid 0 EC50 basal cytotoxicity (mm) x K MW (L/kg lipid) = CMB (mmol/kg lipid) 0 PPB increases with increasing alkyl chain length 0 Discrepancy hypothesized & measured EC50 increases with increasing alkyl chain length 0 Hypothesized EC50 = measured nominal EC50 for C6, C8 BAC 0 Measured unbound EC50 = hypothesized EC50 for BAC-C6 to C14 Groothuis et al., manuscript in preparation
Plastic Binding 0 Serum/BSA free medium 0 Measured nominal EC50 = hypothesized EC50 for BAC-C6 to C10 0 Fraction sorbed to plastic increases with increasing alkyl chain length up to BAC-C12 0 Fraction sorbed to plastic decreases with increasing concentration (saturation) 0 Measured actual conc. after 48h EC50 = hypothesized EC50 for BAC-C6 to C12 0 Homogenous dissolution issues in protein free medium for BAC-C14-C18 Groothuis et al., manuscript in preparation
Partitioning to Cells 0 Extracted BAC from cells after 48h exposure 0 LCMS/MS analysis of cell extracts 0 Nominal EC50 varied 3 orders of magnitude between BAC-C6 and C18 0 EC50 based on cell concentrations varied <1 order of magnitude between BAC-C6 and C18 0 ~1 mmol/kg cell lipid = EC50 for BAC 0 Similar mechanism of toxicity Groothuis et al., manuscript in preparation
Partitioning to Cells in a Substrate Depletion Assay 0 Substrate depletion assay 0 HepaRG in 12-well plate exposed to 3 µm pyrene 0 Medium concentrations measured at 0, 0.5, 1, 2, 4, 8, 16h 0 Biphasic depletion with distribution and elimination phase 0 k distribution (0-2h) 1.93 h -1, k elimination (2-16h) 0.14 h -1 0 Concentrations in cells (3 nmol/well) and on plastic (0.4 nmol/well) peak at 2h Groothuis et al. manuscript in preparation See also Jones and Houston (2004) Drug Metab Dispos 32:973-82.
Partitioning to Cells in Repeated Dosing Assays 0 Objective 0 27 drugs, varying mechanism of action Improve predictivity in vitro assays for adverse effects drugs after repeated dosing 0 Long-lived in vitro organotypic cell systems RPTEC/TERT1 Primary hepatocytes HepaRG 2D & 3D cocultures primary brain cells 0 Integrated omics readouts 0 Daily dosing for 14 days 0 Translate in vitro PoD to daily oral dose transcriptomics, proteomics, metabolomics Dose-response, PBPK modeling Mueller et al. (2015) Toxicol. In Vitro 30: 4
0 Objective Predict-IV WP3 Biokinetics In Vitro 0 Assess how kinetics of drugs in vitro explains variations in effects between drugs, cell types and assay setup 0 Difference in metabolic competence, transporters, dosing regimen, attachment matrix 0 Method 0 Analytically measure drugs, metabolites over time 0 Cells, labware, attachment matrices, medium Kramer et al. (2015) Toxicol. In Vitro 30: 217
Accumulation in Cells 0 Dependent on 0 Chemical 0 Lipophilicity (amiodarone vs ibuprofen) 0 Metabolism/transporter affinity (cyclosporin vs adefovir) 0 Cell type 0 Metabolic competence (hepatocytes) 0 Transporters (RPTEC/TERT1) 0 Dose 0 Time Kramer et al. (2015) Toxicol. In Vitro 30: 217
Accumulation in Cells Bellwon et al. (2015) Toxicol. In Vitro 30: 166; Bellwon et al. (2015) Toxicol. In Vitro Toxicol In Vitro. 2015 30: 62; Wilmes et al. (2013) J Proteomics 79, 180
Conclusions 0 Uptake of chemical into cells varies across in vitro assays, chemicals, dosing regimens and exposure times 0 To quantify AOPs in vitro and define in vitro PODs for QIVIVE: 0 Distinguish between kinetic and dynamic processes in vitro, esp. for repeat-dosing assays 0 Determine cell-associated concentrations over time
Aknowledgements 0 The many not mentioned here specifically 0 Floris Groothuis, Steven Droge 0 Unilever 0 Bas Blaauboer, Joop Hermens 0 EU FP7 Predict-IV 0 EU FP6 AcuteTox