In Vitro Disease Model Development Current Challenges

Transcription

1 Advances in In Vitro Cell Culture, May 2012, Utrecht, The Netherlands In Vitro Disease Model Development Current Challenges Damien Breheny British American Tobacco, Group Research and Development, Southampton, SO15 8TL, UK

2 Presentation outline Objective Strategy Challenges Summary

3 In vitro model development - objective To develop a number of models with endpoints which reflect pathological processes in smoking-related diseases : - Cardiovascular disease (CVD) - Chronic obstructive pulmonary disease (COPD) - Cancer - Inflammation and oxidative stress To gain acceptance of the models by the external scientific community

4 Current strategy Identify and develop in vitro models with endpoints reflective of disease processes in physiologically relevant cell types and culture systems to: - gain a better understanding of smoke toxicity and fundamentals of disease mechanisms - assist in biomarker discovery and qualification - generate biological data to inform the design of future products - apply to product testing Technically validate in vitro models

5 Challenges Bioassay design - appropriate exposure method - target tissue/cell - relevant endpoints Assessment of metabolic competency Development of chronic exposure models Technical validation Endorsement and acceptance by external scientific and industrial community and regulatory bodies

6 Exposure Method and Cell Type

7 Generating smoke for in vitro testing Particulate Phase Whole smoke aerosol Phase Gas + Particulate To Exhaust From cigarette smoke generator Whole Smoke Aqueous extracts (CSE) Total particulate matter (TPM) Whole smoke Cells Growth media Media CSE TPM in DMSO Media/CSE/TPM Cells Cell culture at the air-liquid interface Submerged cell culture

8 Increased physiological relevance Cell types Tumour derived cell lines (e.g. H292) - Easy to culture and consistent results Immortalised cell lines (e.g. BEAS-2B) - Genetically more normal than tumour-derived cells - Useful in cancer studies Primary cells (e.g. HBEC, HUVEC) - Retain metabolic capability and physiological characteristics - Donor variation - Limited lifespan in culture Ex vivo tissue cultures (e.g. lung slices) - Cells retain spatial orientation and intercellular interactions - Short lifespan after slice preparation

9 In Vitro Disease Model Endpoints

10 Oxidative stress Caused by - an increase in oxidant generation - a decrease in antioxidant protection, e.g. glutathione - a failure to repair oxidative damage Antioxidant/oxidant imbalance in favour of oxidants occurs Oxidative stress associated with numerous pathologies including cancer, cardiovascular disease and chronic obstructive pulmonary disease (COPD)

11 Oxidative stress and inflammation endpoints Intracellular free radical levels Inflammation/ Oxidative Stress Intracellular antioxidant status Gene and protein expression

12 % increase in fluorescence Intracellular free radical production DCF fluorescence in H292 cells exposed to cigarette smoke extracts Control 25µg/ml PM 50µg/ml PM 100µg/ml PM Medium DMSO (1%) PM (mg/ml) TPM treatment (µg/ml)

13 GSH GSSG Ratio Intracellular glutathione status GSH:GSSG ratio * * * * PM (µg/ml)

14 CVD endpoints Monocyte adhesion Gene and protein expression Angiogenesis CVD Vascular smooth muscle cell (VSMC) chemotaxis Platelet function and activity Endothelial cell migration

15 Simulation of early events in atherosclerosis: Monocyte adhesion & VSMC chemotaxis Monocytes and VSMC are able to sense whether endothelial cells are in the pro-inflammatory state

16 Morphological changes under flow Static culture Anti-atherogenic smooth high laminar flow Pro-atherogenic disturbed/oscillatory flow Changes in cell morphology = changes in gene expression

17 Monocyte adhesion Adherent THP-1 cells Media Control CSEaq treatment Cell Counting

18 COPD endpoints Gene and protein expression Squamous cell metaplasia COPD Alveolar damage Goblet cell hyperplasia

19 H292 Cell viability (% air control) MMP-1 secretion (pg/ml) WS cytotoxicity and inflammatory mediator secretion Cytotoxicity MMP1 secretion Increasing conc. of smoke Whole 3R4F WS Smoke n = 29 EC 50 = 1: Vapour 3R4F VP Phase n = 29 EC 50 =1:46 = 1: Increasing conc. of smoke RM20S Smoke Smoke dilution Dilution (1:x) WS 1:100 WS 1:75 WS 1:60 WS 1:50 VP 1:100 VP 1:75 VP 1:60 Exposure Smoke & smoke dilution dilution VP 1:50 Air control

20 Goblet cell hyperplasia Normal mucociliary differentiation Undifferentiated cells Goblet cell hyperplasia Haswell L.E. et al. (2010), Toxicology In Vitro

21 Cancer endpoints Cell cycle changes Morphological transformation Anchorageindependent growth Cancer Oxidative DNA damage Epithelialmesenchymal transition DNA doublestrand breaks

22 DNA double-strand breaks gh2ax assay - early DNA damage recognition - measures the direct DNA damage and not only the fixed DNA damage - rapid and highly sensitive - complements conventional genotoxicity screening DMSO Etoposide

23 Intensity Units Cell Viability (%) gh2ax assay Etoposide hr RCC hr RCC Vehicle Concentration Log µm

24 Metabolic Competency

25 Metabolism - a key challenge In vitro xenobiotic metabolism is often overlooked - Incorrect classification of toxic compounds False ve - Underestimation of detoxification potential False +ve Metabolism-driven bioactivation is critical to generate meaningful in vitro toxicology data (e.g. NNK)

26 Metabolic characterisation of in vitro models Testing NHTBEs (plus H292, A549) metabolic competency CYP1A1/1B1 and CYP2A6/2A13 activity is conserved in cultures of differentiated primary human tracheobronchial epithelial cells Toxicology in Vitro, Volume 25, Issue 4, June 2011, Pages Nik Newland, Andrew Baxter, Katherine Hewitt, Emmanuel Minet Activity of CYP2A6/2A13? Bioactivation of NNK Activity of CYP1A1/1B1? Bioactivation of PAHs 7 28 days cultures

27 CYP activity in NHTBEs 7 days after differentiation RA essential for activity Donor to donor variation A549 always negative CYP1A1/1B1

28 RLU/mg/min pm/mg/min BEAS-2B metabolic competency P450s activity in BEAS-2B was compared to a metabolically active cell line (HepG2) and an inactive cell line (A549). CYP1A1/1B1 CYP2E BEAS-2B HEP-G2 A BEAS-2B HEP-G2 A Probe Probe + Inhibitor +TCDD Probe Probe + Inhibitor No TCDD 0 Probe (pm) Probe + Inhibitor (pm)

29 Metabolic competency - summary Many cell lines do not have metabolic competency representative of parent tissue - Addressed by using exogenous metabolic activation systems (e.g. Aroclor induced rat S9) - Only suitable for short-term treatment - Enzymatic activity not always representative of humans Novel approaches to increase and sustain metabolic competency in culture are available - E.g. Kirkstall s Quasi-Vivo system, co-cultures etc.

30 Chronic Exposure

31 Chronic exposure in vitro Our current models are based on acute treatments - Does not truly represent the long term exposure of a lifetime smoker Advanced cell culture techniques allow primary cells to be cultured for longer in a fully differentiated state - E.g. MucilAir TM Future model development will focus on longer term smoke exposure using physiologically relevant cell systems - Primary culture - Repeated vs continuous treatment - Dose??

32 Technical Validation

33 Technical validation Process of demonstrating and documenting that the performance characteristics of a procedure and its underlying method meet the requirements for the intended application and that the assay is thereby suitable for its intended use Bioassay validation should be performed when development has been completed

34 Bioassays The Modular Approach: 7 Information Modules Berg, N. et al. (2011). Toxicology in the 21 st century working our way towards a visionary reality. Toxicology in Vitro 25,

35 Summary

36 Summary In vitro models - integral part of future product assessment strategy - physiologically relevant - metabolically competent cells - model specific aspects of disease processes - quantitative endpoints - technically validated - should be used in combination (integrated approach)

37 Further Information

38 British American Tobacco s Research & Development website About science By scientists For scientists

39 Informal forum of European companies with an active interest in in vitro testing to be used in regulatory/safety testing or to be used in the compound discovery and development process. We are also supportive of applying, where possible, the principle of the 3Rs:replacing, reducing and refining animal testing. Our members represent companies in the chemical, cosmetics, consumer products and pharmaceutical sector.

40 Acknowledgements CVD Frazer Lowe Karina McQuillan Natalia Cockcroft COPD David Azzopardi Gary Phillips Katherine Hewitt Linsey Haswell Nathan Asquith Sarah Corke Cancer Anisha Banerjee Carolina Garcia-Canton Christine Roach John McMullan Tobi Oke Inflammation/Ox Stress Ieisha Pentland Mark Taylor Tony Carr

41 Any Questions?