Presentation to the IoM Committee

on Scientific Standards for Studies

on Modified Risk Tobacco

Products, May 9, 2011.

Standards for studies on in vitro

models of disease

Chris Proctor, Chief Scientific Officer, Group Research and Development, British American Tobacco (Investments) Ltd.

In vitro models of disease

By in vitro models of disease we mean laboratory based assays/tests, typically using human cells, that are physiologically relevant, responsive to cigarette smoke exposure, and mimicking disease processes associated with tobacco use

In our laboratories we are developing models related to

- Cancer

- Chronic obstructive pulmonary disease

- Cardiovascular diseases

- Inflammation and oxidative stress

In vitro models of disease in the context of a

modified risk tobacco product assessment

We are developing in vitro models of disease- To help us understand and investigate the effect of cigarette

smoke exposure on models of disease processes

- To evaluate disease-related biomarkers

- To assess changes in response when exposed to the smoke from potential modified risk tobacco products

The research is consistent with our approach to reduce animal experimentation

In the context of the assessment of a potential modified risk tobacco product, data from in vitromodels of disease would provide supporting information

Our proposed PREP Assessment Framework

Technology Development

Tobacco Blend/Filter & Paper

technologies/Novel

Technologies

Efficacy / Chemistry

Assessments

(Toxicology Profiling)

Product

Stewardship

Package on

Technology

Technology Available

for Use in PREP

Development

Candidate

PREP

Weight of Evidence

Bioassays

In Vitro

Models of Disease

In Vivo

Animal Models

Toxicity

Modelling

(PBPK)

Clinical

Studies

Biomarkers of Exposure and Effect

Consumer perceptions & behaviours

Dosimetry

Population

Studies

Extended

Use

Smoking Behaviour;

Mouth Level

Exposure

Data Package

for Product

Stewardship

Product Development)

Efficacy / Chemistry

Smoking Acceptability

Candidate

PREP

Clinical

Studies

Biomarkers of Exposure and Effect

Physiological Assessments

DosimetryConsumer

Acceptability TestingCandidate

PREP

Final Report &

Risk

Assessment on

PREP Status

Our current research efforts

We are developing models to include:

Cancer

Oxidative damage in lung epithelial cells (Comet assay); cell

transformation assay; anchorage-independent growth assay using

bronchial epithelial cells; epithelial-mesenchymal transition

Chronic obstructive pulmonary disease

Mediator expression in lung epithelial cells; goblet cell hyperplasia; 3-D

reconstituted human airway tissues (e.g. MucilAirTM); squamous cell

metaplasia; lung slices

Cardiovascular diseases

Endothelial migration and gene/protein expression; physiological

angiogenesis; endothelial-monocyte interactions; platelet activation;

smooth muscle chemotaxis

Inflammation and oxidative stress

Direct measurement of reactive oxygen species and intracellular

antioxidant status; transcriptional regulation; monocyte function

Internal validation during the development of

the models Demonstrate that the models are fit for purpose:

- Cell evaluation

- cell authentication

- metabolic capacity

- Relevance to disease

- cell types

- response

- Responsiveness and sensitivity

- positive and negative controls

- reference cigarette (3R4F)

- Suitability

- repeatability

- robustness

- Documentation and publication of the method

Standards for in vitro models of disease

For any model to be of value with respect to the assessment of a potential modified risk tobacco product, a variety of standards need to be applied

Generally, we are trying to take the approach advocated by the National Research Council (NRC) in “Toxicity Testing in the 21st Century – A vision and a strategy”

We are working with other groups, such as the In Vitro Testing Industrial Platform (IVTIP) to identify the practical steps that are needed to make the NRC vision a reality (Toxicology in Vitro, 25, 874-881, 2011)

Standards for in vitro models of disease

From N. Berg, B. De Wever, H. W. Fuchs, M. Gaca, C. Krul and E.L. Roggen, Toxicology in the

21st century – working our way towards a visionary reality, Toxicology in Vitro, 25, 874-881, 2011

Additional considerations for scientific

standards

While the basic pre-validation/validation framework should evaluate issues such as donor variation, the relevance of the cell cultures or co-cultures and the relevance of acute tests to chronic diseases, there are additional issues to be considered

These include:

- Standardisation of the test material generation (in the case of cigarette smoke usually by smoking machine)

- Exposure of the cells to relevant test material (depends on the disease model and biological mechanism being considered)

In addition, quality management system should be applied

Approach

Identify and characterise appropriate cigarette smoke generation and exposure systems for relevant test material

- total particulate matter (TPM)

- aqueous extracts (CSE)

- whole smoke

Standardise methodologies for generation, delivery and assessment of cigarette smoke

- smoke chemistry

- aerosol dynamics

- deposition and dosimetry

Generating Smoke for In Vitro Testing

Cell culture at the air-liquid interface

From

cigarette

smoke

generator

To

Exhaust

From

cigarette

smoke

generator

To

Exhaust

Total particulate matter (TPM)

Aqueous extracts (CSE)

Whole smoke

Cells

Growth media

Submerged cell culture

Cells

Media/CSE/TPM

Media CSE

TPM in

DMSO

Whole Smoke

Whole smoke

aerosol

Gas Phase

Particulate Phase

+Gas Particulate

Summary

We believe that in vitro models of disease hold promise as a part of a scientific assessment framework for modified risk tobacco products

A general framework for the assessment and validation of the models has been proposed by some researchers

Additional standards should cover the generation and the application of the test materials