Society of Toxicology In Vitro & Alternative Methods Section Webinar, May 18 th 2017 Cardiotoxicity Adverse Outcome Pathways C-AOP STAR Center Diversity in a dish: A human population-based organotypic in vitro model for cardiotoxicity testing Fabian Grimm Department of Veterinary Integrative Biosciences Texas A&M University 1 E-mail: fgrimm@cvm.tamu.edu
Acknowledgements Society of Toxicology – In Vitro and Alternative Methods Specialty Section Molecular Devices LLC Texas A&M University Ivan Rusyn, MD PhD Oksana Sirenko, PhD Weihsueh Chiu, PhD David Threadgill, PhD National Toxicology Program/ NIEHS William Klaren, PhD Yasuhiro Iwata, DVM Raymond Tice, PhD Sarah Burnett Kristen Ryan, PhD Alec Wright Mamta Behl, PhD Frederick Parham, PhD North Carolina State University Funding Fred Wright, PhD David Reif, PhD U.S. Environmental Protection Agency: John House, PhD STAR RD83516602 / RD83580201 Cellular Dynamics International Society of Toxicology: Blake Anson, PhD 2015-2016 Colgate-Palmolive Fellowship 2017-2018 Syngenta Fellowship
Introduction: Bridging Emerging Technologies And Chemical Safety Assessments Advances in stem cell technologies and organotypic culture methods have the potential to overcome major limitations in contemporary risk assessment: Limited interpretability of animal model-derived data Low-throughput asssociated with in vivo testing (“Chemical Data Gap”) Standardized, rather than chemical-specific population-level adjustment factors The implementation of organotypic culture models in human health safety assessments Adverse Outcome is impeded by the lack of multidimensional high-throughput testing strategies that are: Pathway Functionally and physiologically-relevant 1,2,3 Medium- to high-throughput applicable format 1,2,3 Amenable for in vitro -to- in vivo extrapolation 3 Capable of estimating inter-individual susceptibilities to adverse chemical effects Goal: Demonstrate the potential of organotypic culture systems to fill crucial needs in chemical risk assessment using a population-based in vitro cardiotoxicity model 1. Grimm FA, Iwata Y, Sirenko O, Bittner M, Rusyn I. Assay Drug Dev Technol . (2015) 13: 529-46 2. Grimm FA, Iwata Y, Sirenko O, Chappell GA, Wright FA, Reif DM, Braisted J, Gerhold DL, Yeakley JM, Shepard P, Seligmann B, Roy T, Boogaard PJ, Ketelslegers HB, Rohde AM, Rusyn I. Green Chem . (2016) 18: 4407-19 3 3. Sirenko O, Grimm FA, Ryan KR, Iwata Y, Behl M, Wignall JA, Parham F, Anson B, Cromwell EF, Rusyn I, Tice RR. Toxicol Appl Pharmacol (2017) 98: 120-128
Advances in Induced Pluripotent Stem Cell (iPSC) Technologies Enable Population-wide studies Inter-Species Variability Intra-Species Variability (10-fold) (10-fold) Uncertainty in extrapolating from animals to humans: Dichloromethane IRIS: “ The use of PBPK models to extrapolate internal doses from rats to humans reduces toxicokinetic uncertainty in extrapolating from the rat liver lesion data but does not account for the possibility that humans may be more sensitive than rats to dichloromethane due to toxicodynamic differences .” Uncertainty in human variation (from average to sensitive): Dichloromethane IRIS: “ The probabilistic human PBPK model used in this assessment incorporates the best available information about variability in toxicokinetic disposition of dichloromethane in humans but does not account for humans who may be sensitive due to toxicodynamic factors .” 4
Cardiotoxicity Adverse Outcome Pathways – A Three-Tiered Approach 1: Assay Multiplexing and Quality Assessment Functional Cytotoxicity Mechanistic [Ca 2+ -Flux] [HC-Imaging] [Transcriptomics] 0.1 µM 1 µM 10 µM 100 µM 2: Bioactivity Profiling in iCell Cardiomyocytes 3: Population Variability Assessment Cardiotox Screening QC Dose-Response Profiling 100 “healthy” donors [no known cardiovascular disease] Variability in chemical Inherent Biological Variability treated cardiomyocytes In untreated cardiomyocytes “Point -of- Departure” Bioactivity Profiling Population Variability Assessment Functional, Cytotox & In a Single Individual [phenotypic effects modeling and IVIVE] Mechanistic Panels 5
1. Assay Multiplexing in iCell Cardiomyocytes [Calcium-Flux, High-Content Cell Imaging, High-Throughput Transcriptomics] 0- 90 min Functional: Cytotoxicity: Mechanistic: Ca 2+ -Flux Live Cell Imaging Targeted Transcriptomics Hoechst MitoTracker ~ 3000 selected, “toxicologically Calcein AM relevant” transcripts ImageXpress™ Micro Confocal HiSeq 2500 24 hours 8 cardiophysiologic descriptors, 10 cytotoxicity parameters Pathway analysis for mechanistic evaluation FLIPR™ tetra All output files are quantitative Concentration-response assessment Grimm FA, Iwata Y, Sirenko O, Bittner M, Rusyn I. Assay Drug Dev Technol. (2015) 13: 529-546 Grimm FA, Iwata Y, Sirenko O, Chappell GA, Wright FA, Reif DM, Braisted J, Gerhold DL, Yeakley JM, Shepard P, Seligmann B, Roy T, Boogaard PJ, Ketelslegers HB, Rohde AM, Rusyn I. Green Chem . (2016) 18: 4407-19
1. Assay Multiplexing in iCell Cardiomyocytes [Cardiophysiologic Phenotyping and Reproducibilty Assessment] Phenotypic Resemblance of In Vivo Drug Effects Reproducibility of Baseline Cardiophysiology RFU r 2 =0.97 RFU Reproducibility of Chemical Effects in iCell Cardiomyocytes Positive Inotrope Negative Inotrope K+ channel antagonist Grimm FA, Iwata Y, Sirenko O, Bittner M, Rusyn I. Assay Drug Dev Technol. (2015) 13: 529-546 7 Grimm FA, Iwata Y, Sirenko O, Chappell GA, Wright FA, Reif DM, Braisted J, Gerhold DL, Yeakley JM, Shepard P, Seligmann B, Roy T, Boogaard PJ, Ketelslegers HB, Rohde AM, Rusyn I. Green Chem . (2016) 18: 4407-19
1. Assay Multiplexing in iCell Cardiomyocytes [Targeted Transcriptomics using the TempO-seq platform]
2. Environmental Cardiotoxicity Profiling in iCell Cardiomyocytes [69 Chemicals: Drugs, Pesticides, Flame Retardants, PAHs, Others] Quantitative Assessment Data Acquisition 9 Sirenko O, Grimm FA, Ryan KR, Iwata Y, Parham F, Wignall JA, Anson B, Cromwell EF, Behl M, Rusyn I, Tice RR. Toxicol Appl Pharmacol (2017) 98: 120-128
2. Environmental Cardiotoxicity Profiling in iCell Cardiomyocytes [In Vitro-to-In Vivo Extrapolation Using Reverse Toxicokinetics Data] Comparison between C ss and POD values Chemical margin of exposure In Vitro Pharmacokinetics Human Human donor pool donor pool Cellular assays Plasma Liver clearance protein binding - Population based IVIVE model Steady State Concentration (C ss ) Upper 95% percentile among healthy adults Reverse Dosimetry Oral Equivalent Dose (mg/Kg-day) 1. Sirenko O, Grimm FA, Ryan KR, Iwata Y, Parham F, Wignall JA, Anson B, Cromwell EF, Behl M, Rusyn I, Tice RR. Toxicol Appl Pharmacol (2017) 98: 120-128 2. Pearce RG, Setzer RW, Strope CL, Sipes NS, Wambaugh JF J. Stat. Softw. (2017) In press. 3. Wetmore BA, Wambaugh JF, Ferguson SS, Sochaski MA, Rotroff DM, Freeman K, Clewell 3rd HJ, Dix DJ, Andersen ME, Houck KA, Allen B, Judson RS, Singh R, Kavlock RJ, Richard AM, Thomas RS. Toxicol. Sci. (2012) 125, 157 – 174. 10 4. Wetmore BA, Wambaugh JF, Ferguson SS, Li L, Clewell 3rd HJ, Judson RS, Freeman K, Bao W, Sochaski MA, Chu TM, Black MB, Healy E, Allen B, Andersen ME, Wolfinger RD, Thomas RS. Toxicol. Sci. (2013) 132, 327 – 346.
3. Population Variability Assessment in iPSC Cardiomyocytes: [Study Design and Data Integration] Diversity in a Dish Concept for Cardiotoxicity Testing Experimental Design Donor Pool 100 individual, “healthy” donors iPSC reprogramming and differentiation Population Variability Screening 11
3. Population Variability Assessment in iPSC Cardiomyocytes: [Inter-Indervidual Variability in Cardiophysiology of Untreated Cardiomyocytes] 12
3. Population Variability Assessment in iPSC Cardiomyocytes: [Inter-Individual Variability in Cardiomyocytes after Chemical Treatment]
3. Population Variability Assessment in iPSC Cardiomyocytes: [Inter-Individual Variability in Cardiomyocytes after Chemical Treatment] 14
3. Population Variability Assessment in iPSC Cardiomyocytes [Population-Level Concentration-Response Assessment] 15
Summary 1. Collected Ca 2+ flux and HC-Imaging data plus cell lysates for HT-transcriptomics in concentration-response for ~140 chemicals in iPSC-derived cardiomyocytes Data acquisition is complete for first batch of cells from 27 donors Identical data sets will be generated for cells from an additional 70 donors HT-transcriptomics currently underway 2. Observable variability in baseline cardiophysiological parameters not attributable to technical variation in plating efficiencies is an important factor to be considered for evaluation of chemical effects 3. Chemical treatments qualitatively reflect the anticipated phenotypic responses Qualitative characteristics remain consistent for the vast majority of chemicals 4. Quantitative variation in responses to chemical treatments is observable Differences in chemical-associated potencies are an indicator of biol. variability 16
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