DR. MARIANNA D GACA Manager, Pre-Clinical Assessment R&D, British American Tobacco

Transcription

1 National Academy of Sciences, Engineering and Medicine Washington 21st February 2017 Pre-clinical and clinical testing of ENDS: Results from in vitro assays DR. MARIANNA D GACA Manager, Pre-Clinical Assessment R&D, British American Tobacco

2 CONFLICT OF INTEREST STATEMENT I declare that this work was fully funded by British American Tobacco (Investments) Ltd and that myself and my co-workers were full time employees of British American Tobacco (Investments) Ltd for the duration of the research.

3 OVERVIEW Types of aerosols and dosimetry The in vitro response: cigarette versus e-cigarette o High content screening o Classical toxicology o In vitro biology o Systems biology o Adverse Outcome Pathways (AOPs) Summary

4 GENERATING THE TEST ARTICLE e-liquid ENDS 3R4F E-Liquid Submerged exposure directly to e-liquid Particulate Matter (PM) Submerged exposure to filter trapped particles eluted in solvent Aqueous Extract (AqE) Submerged exposure to aerosol bubbled media or buffer Aerosols Whole aerosol or vapour phase only exposure at the air-liquid/agar interface

5 IN VITRO DOSIMETRY Tools and technologies to assess test articles Chemical composition at source, through generation and delivery systems and at exposure interface e-liquids PM [1] AqE [2] Aerosol Quantitation/ dosimetry Nicotine Tar Nicotine nm CO Nicotine Carbonyls Mass-(QCMs) [3] Nicotine [4] Carbonyls [5] 1. ISO 4387: Taylor et al.toxicol. Mech. Methods , Adamson et al. Chemistry Central, : Adamson et al. Applied In Vitro Toxicology 2017 In press 5. Majeed et al. Chem Cent J, :62 NICOTINE Cross-category marker In situ of exposure QCM surface (CFP), PBS, cell culture insert Conversion of dilution to delivered nicotine QCM Real time data generation In situ of exposure

6 PRODUCTS Product Description Aerosol Generation Standardised Method for Aerosol Generation US blended reference cigarette from the University of Kentucky (3R4F) Combustion and pyrolysis of tobacco Health Canada Intense regime (HCI): 55ml puff volume; 2 sec puff duration; 30 sec interval between puffs [6] A closed system vaping device (Vype epen, 18mg/ml Tobacco Blend) Heating of a tobacco-free nicotine solution CORESTA regime (CR81): 55ml puff volume; 3 sec puff duration; 30 sec interval between puff [7] 6. Health Canada Official Method T-115. Determination of Tar, Nicotine and Carbon Monoxide in Mainstream Tobacco Smoke. Prepared by the Department of Health, December 31, CORESTA Recommended Method No 81, Routine analytical machine for e-cigarette aerosol generation and collection Definitions and Standard Conditions. CRM No 81, 2015.

7 IN VITRO ENDPOINTS ASSESSED WITH E-CIGARETTES High Content Screening Classical Toxicology Cytotoxicity In Vitro Disease Endpoints Many endpoints: Cell cycle: necrosis/ apoptosis, DNA damage, oxidative stress Ames assay (DNA mutations) Neutral red uptake assay ApoLive-Glo assay (Caspase 3/7 activation) Oxidative stress Cancer CVD COPD DCF assay (free radical production) GSH/GSSG ratio (antioxidant depletion) Gamma-H2AX assay (DNA double strand breaks) Bhas 42 assay (transformed foci formation) Wound healing assay (cell migration) 3D organotypic respiratory tissue systems Cytotoxicity ARE reporter assay (responsiveness of ARE reporter) Systems Biology Gene expression changes: gene ontology enrichment: identification of network perturbations

8 HIGH CONTENT SCREENING FOR E-LIQUIDS Undifferentiated human bronchial epithelial cells 4 hour treatment e-liquid A e-liquid B Cell count * * - * Nuclear size - * - * DNA structure Mitochondrial mass Mitochondrial membrane potential Oxidative stress Glutathione content * * * * Cellular ATP * * - * 24 hour treatment e-liquid A e-liquid B Cell count * * - * Nuclear size - * - * DNA structure * * - * Mitochondrial mass Mitochondrial membrane potential * Oxidative stress * Glutathione content * * * * Cellular ATP * * * * - = No biological response * = Significant change against control >1 fold

9 CLASSICAL REGULATORY TOXICOLOGY OECD* TG 471: Bacterial Reverse Mutation Test [8] S. typhimurium TA98 E-cigarettes gave no response even at 900 puffs Exposure to reference cigarette smoke caused mutations in a dose dependent manner; e-cigarettes gave no response 9. Thorne et al. (2016) Mutation Research 812: Thorne et al (in draft) *Organisation for Economic and Cooperative Development

10 CYTOTOXICITY TESTING NCI-H292 human bronchial epithelial cells NEUTRAL RED UPTAKE- ICCVAM [11]* (WHOLE AEROSOL) APO-LIVE GLO (AQUEOUS EXTRACT) E-cigarette vapour induced significantly less cytotoxicity than a reference cigarette 12. Azzopardi et al (2016) Toxicol. Mech. Methods 26: Taylor et al. (2016) Toxicol. Mech. Methods 26: * Interagency Coordinating Committee on the Validation of Alternative Methods

11 OXIDATIVE STRESS NCI-H292 human bronchial epithelial cells INTRACELLULAR ANTIOXIDANT LEVELS (AQUEOUS EXTRACT) REACTIVE OXYGEN SPECIES GENERATION (AQUEOUS EXTRACT) ANTIOXIDANT RESPONSE ELEMENT (ARE) (AQUEOUS EXTRACT) E-cigarettes induced no oxidative stress in human lung H292 epithelial cells compared to reference cigarettes 2. Taylor et al. (2016) Toxicol. Mech. Methods 26:

12 DNA DAMAGE AND CELL TRANSFORMATION BEAS2B human lung epithelial cells BHAS 42 cells γ-h2ax ASSAY (WHOLE AEROSOL) E-cigarettes were non-genotoxic; cigarette smoke induced positive genotoxic responses 13. Thorne D et al (2016) Toxicol. Letters 265: BHAS 42 CELL TRANSFORMATION ASSAY* (TPM) Cigarette smoke induced promotion activity; e-cigarette was negative at all concentrations tested 15. Breheny D et al submitted *14. OECD (2013) Draft test guideline

13 ENDOTHELIAL CELL MIGRATION Comparison of human endothelial cell (HUVEC) migration rates over a 20 hour exposure period Cigarette smoke induced a concentration-dependant inhibition of HUVEC migration, e-cigarettes demonstrated no significant inhibition of migration MIGRATION (AQUEOUS EXTRACT) 16. Taylor et al Submitted

14 IRRITANCY ASSESSMENT OF AEROSOLS Comparison of cytotoxicity after cigarette and e-cigarette exposure using EpiAirway TM IRRITANCY (WHOLE AEROSOL) No cytotoxicity with e-cigarette exposed EpiAirway TM Schematic representation of the VITROCELL VC 01 Smoking Robot, mammalian 12/6 CF stainless-steel exposure module, and EpiAirway TM tissue model. (A) VC 01 single port smoking robot, enclosed in a ventilation hood with a piston/syringe that draws and delivers smoke or aerosol to the dilution bar. (B) Dilution bar, where smoke or aerosol is diluted, mixed, and delivered to the exposure module. Diluted smoke/aerosol within the dilution bar transits to exhaust. (C) 12/6 CF stainless-steel exposure module, where EpiAirway TM inserts are housed during exposure. (D.I) Culture insert on which EpiAirway TM tissue culture is supported at the air liquid interface with smoke/aerosol distributing trumpet sitting 2 mm above the surface of the tissue. (D.II) EpiAirwayTM human airway epithelium. (D.III) Fresh culture media (AIR-100 maintenance media) basally feeding human airway epithelium. Transmission electron micrograph (magnification 20,000) showing (E.I) cilia and (E.II) tight junctions. Haematoxylin and eosin stained cross-sections (magnification 360) of (E.III) pseudostratified mucocilary morphology of EpiAirway TM tissue and (F) excised human bronchial epithelium for comparison. 17. Neilson et al 2016 Toxicol. In Vitro; 29(7):

15 FUNCTIONAL ANALYSIS- CILIARY BEAT FREQUENCY (CBF) Comparison of cigarette smoke and e-cigarette aerosol using MucilAir TM Cigarette smoke reduces cilia beat frequency however with e-cigarettes the cilia continue to beat Microscopy with digital high speed image recording and analysis software to measure CBF

16 COMPARING TRANSCRIPTIONAL PERTURBATIONS IN MUCILAIR 48, 854 genes/ RNA features screened 3R4F 8197 significant genes/rna features Vype epen 49 significant genes/rna features Vype epen* 113 significant genes/rna features RNA-seq data mapped onto 131 pathway-focussed gene sets with specific biological function and disease proceses 3R4F cigarette smoke 1/30 epen aerosol 1/7 epen aerosol 1/3 3R4F e-cig e-cig* 3R4F e-cig* Toxicogenomics RNA-seq differential gene expression e-cig * X2 nicotine dose 18. Haswell et al, in submission Gene enrichment analysis: heatmap indicating fold change for RNAs significant at pfdr<0.05

17 ADVERSE OUTCOME PATHWAYS (AOPs) Describes a sequential chain of causally linked events at different levels of biological organisation, that lead to an adverse effect 19. Ankley et al (2010) Environ.Toxicol.Chem 29(3): Collaboration to Support AOP Build Two AOPs (BAT/PMI/SELVENTA) Oxidative Stress Leading to Hypertension 20.Lowe et al. (2017) Applied In Vitro Toxicology (in press) EGFR Activation Leading to Decreased Lung Function Luettich et al.(2017) Applied In Vitro Toxicology (in press)

18 SUMMARY in vitro holds promise as a part of a scientific assessment for e-cigarettes We have demonstrated significant reductions in toxicity and biological activity in vitro when exposed to e-cigarette aerosol versus tobacco smoke Extrapolation to human requires dose assessments Appropriate exposures to appropriate matrix Validation and qualification fit for purpose New tools to support screening Systems biology for global assessments; supports biomarker discovery in vitro and in the clinic: AOPs

19 THANK YOU Andrew Baxter Annette Dalrymple Anisha Banerjee Anya Terry Chris Proctor Chris Wright Clive Meredith Damien Breheny David Azzopardi David Thorne Emma Bishop Emmanuel Minet Frazer Lowe Ian Crooks Ivan Verrastro James Murphy Jason Adamson Katherine Hewitt Linsey Haswell Mark Taylor Natalia Cockcroft Oscar Camacho Sarah Corke Simone Santopietro Stela Bozhilova Tobi Oke Tomasz Jaunky Tony Carr Dr Marianna D Gaca marianna_gaca@bat.com Manager, Pre-Clinical Assessment, R&D, British American Tobacco British American Tobacco (Investments) Limited All rights reserved. No part of these materials may be reproduced in any form or by any means without the prior written consent of British American Tobacco (Investments) Limited and no responsibility or liability is accepted for any third party reliance on any data contained herein. The data and information used in these materials has been compiled from a number of sources.

20 REFERENCES 1.International Organization for Standardization Cigarettes determination of total and nicotine-free dry particulate matter using a routine analytical smoking machine. ISO 4387: E-cigarette aerosols induce lower oxidative stress in vitro when compared to tobacco smoke. Taylor et al. (2016) Toxicology Mechanisms and Methods 26; Application of dosimetry tools for the assessment of e-cigarette aerosol and cigarette smoke generated on two different in vitro exposure systems. Adamson et al. Chemistry Central, : Nicotine quantification in vitro: a consistent dosimetry marker for e-cigarette aerosols and cigarette whole smoke generation. Adamson et al. (2017) Applied In Vitro Toxicology (in press). 5. Characterization of the Vitrocell 24/48 in vitro aerosol exposure system using mainstream cigarette smoke. Majeed et al. Chem Cent J, :62 6.Health Canada Official Method T-115. Determination of Tar, Nicotine and Carbon Monoxide in Mainstream Tobacco Smoke. Prepared by the Department of Health, December 31, CORESTA Recommended Method No 81, Routine analytical machine for e-cigarette aerosol generation and collection Definitions and Standard Conditions. CRM No 81, OECD bacterial reverse mutation test, in: OECD guideline for the testing of chemicals, test guideline The mutagenic assessment of an electronic-cigarette and reference cigarette smoke using the Ames assay in strains TA98 and TA100. Thorne et al. (2016) Mutation Research/Genetic Toxicology and Environmental Mutagenesis 812: A novel testing approach for the assessment of undiluted e-cigarette aerosols in vitro using an Ames air liquid agar technique.thorne et al in draft (2017) 11. Test method protocol for BALC/C3T3 Neutral Red uptake cytotoxicity test. November Interagency Coordinating Committee on the Validation of Alternative Methods 12. Electronic cigarette aerosol induces significantly less cytotoxicity than tobacco smoke. Azzopardi et al (2016) Toxicol. Mech. Methods 26: The comparative in vitro assessment of e-cigarette and cigarette smoke aerosols using the γh2ax assay and applied dose measurements. Thorne et al. (2017) Toxicology Letters 265; Organisation for Economic and Cooperative Development. Guidance Document on the in vitro Bhas 42 cell transformation assay. Series on Testing and Assessment. No Paris The comparative tumour promotion assessment of e-cigarette and cigarettes using the in vitro Bhas 42 cell transformation assay, Breheny et al submitted 16. A comparative assessment of e-cigarette aerosols and cigarette smoke on in vitro endothelial cell migration. Taylor et al 2017 Submitted 17.Development of an in vitro cytotoxicity model for aerosol exposure using 3D reconstructed human airway tissue; application for assessment of e-cigarette aerosol. Neilson et al. (2015) Toxicology in Vitro 29(7): A new approach to decoding life: Systems Biology. Ideker et al (2001) Annual Review of Genomics and Human Genetics 2: Reduced biological effect of e-cigarette aerosol compared to cigarette smoke evaluated in vitro using normalized nicotine dose and RNA-seq-based toxicogenomics. Haswell et al in submission 20. Development of an adverse outcome pathway for the onset of hypertension by oxidative stress-mediated perturbation of endothelial nitric oxide bioavailability. Lowe et al. (2017) Applied In Vitro Toxicology (in press) 21. The adverse outcome pathway for oxidative stress-mediated EGFR activation leading to decreased lung function. Luettich et al.(2017) Applied In Vitro Toxicology (in press)