The first of the five steps of our systems toxicology strategy for Reduced-Risk Product (RRP) assessment is focused on the production of high-quality, systems-wide experimental data, which we generate through a combination of in vitro, in vivo and clinical systems. We use whole smoke / aerosol exposure techniques for our in vitro models.

In Vitro Systems

In 2007, the US National Research Council published a new strategic plan for toxicological assessment in order to update and advance our knowledge of the toxicity and mode of action of environmental agents¹. To generate better data on the potential risks to humans, the strategy recommends the use of computational toxicology and systems biology in combination with human-relevant in vitro models that allow multiple dose testing.

For details and references, please read below.

In-Vitro

In Vivo Systems

In vivo models (usually rodents) are used to assess the systemic toxicological impact of compounds and products. We are applying the in vivo testing guidelines defined by the Organization for Economic Co-operation and Development (OECD) and are committed to efforts to “Replace, Reduce, Refine” in vivo testing¹, as evidenced by our significant investment into the advancement in vitro methods. In the assessment of Reduced-Risk Products (RRPs), we use in vivo disease models that respond to cigarette smoke^2,3,4,5,6.

For details and references, please read below.

In Vivo

Clinical Systems

The aim of clinical systems biology at PMI is to conduct observational, non-interventional clinical research studies to generate human systems biology data for our systems biology assessment of Reduced-Risk Products (RRPs). Samples are collected as part of our clinical assessment work, allowing us to conduct systems analysis as exploratory endpoints.

For details and references, please read below.

Clinical Assessment

Whole Smoke/Aerosol Exposure

There are a variety of whole smoke exposure systems available for the generation, dilution and delivery of cigarette smoke or Reduced-Risk Product (RRP) aerosols in vitro, all of which aim to ensure that there are limited changes between generation and exposure⁷.

For details and references, please read below.

Whole smoke aerosol exposure

In Vitro Systems

Human 2D models and High Content Screening

Human 3D models

In accordance with the well-accepted 3R strategy (the effort to “replace, reduce, refine” the use of animal models in research²), we have devised a series of human in vitro models which will avoid species translatability issues. These models are composed of human primary cells, cultured in two- or three-dimensions, which can be exposed to individual smoke constituents and fractions (eg, 2D cultures such as normal human bronchial epithelial cells or human endothelial cells) or to whole aerosols from Reduced-Risk Products (RRPs) or cigarette smoke (eg, 3D cultures).

By using systems toxicological risk assessment approaches, combining computable biological network models and gene expression changes, the molecular alterations triggered after different exposure conditions are analyzed and integrated with other biological endpoints. The measured biological alterations are also quantified, allowing the direct comparison of the impact triggered by exposure to cigarette smoke or RRP aerosols, thus providing evidence on the potential risk reduction.

Human 2D models and High Content Screening

High Content Screening (HCS) is an automated technology used to carry out functional / biological assays in a rapid, robust and cost-effective manner. The technology is based on the integration of three elements:

A state-of the art fluorescence microscope that allows the visualisation of cells and cellular structures.

A high-throughput image acquisition system that allows the automatic acquisition of hundreds of microscope images in a rapid and reproducible manner.

A software tool that enables the analysis and quantification of the biological signals in each one of the acquired images.

Using a combination of antibodies (capable of identifying specific components in the cell) and fluorescent dyes, the HCS technology can detect changes in target intensity, localisation and cellular morphology in live or fixed cell³.

At PMI, we use a large battery of HCS assays to investigate how specific substances alter the phenotype of a cell in a particular manner. Currently, we have established more than 10 different assays, allowing us to measure more than 15 different cellular parameters in cellular types representing the lung (normal human bronchial epithelial cells) and the cardiovascular system (human coronary artery endothelial cells)⁴.

Human 3D models

While 2D models have their uses, traditional 2D cell cultures in plastic dishes or flasks are limited in that the culture medium that covers them does not allow for direct exposure to complex aerosols. In addition, 2D cell cultures do not satisfactorily recapitulate the physiological cell-cell interaction conditions that cells experience in vivo.

To address these issues, in recent years researchers have looked for ways to culture cells in a more physiologically relevant manner. It has been demonstrated that by culturing normal human bronchial epithelial cells at the air-liquid interface, they are able to differentiate into a bronchial epithelium-like tissue with all the morphological properties that define the bronchial epithelium in vivo⁵. A 3D airway cellular model has also been built where cells express a comparable gene expression profile to tracheal and bronchial epithelial cells that have been collected in the clinical setting by brushing human airways⁶.

Cell culture in 3D has been touted as ‘biology's new dimension’⁷ and the National Institutes of Health, the US Food and Drug Administration, the Defense Advanced Research Projects Agency and the Defense Threat Reduction Agency are sponsoring a variety of projects focused on developing 3D organ models⁸.

At PMI we are using human 3D cultures that represent the different airway epithelia (nasal, oral, gingival and bronchial) that are directly exposed to cigarette smoke when inhaled by a smoker. These models are cultured with an air-liquid interface, allowing us to test whole cigarette smoke and RRP aerosols using the VITROCELL® system. We recently demonstrated that nasal, bronchial and oral in vitro systems are reliable models to assess RRPs by comparing their response to cigarette smoke exposure against in vivo tissue counterparts^9,10,11,12.

[1] National Research Council Committee on Toxicity Testing Assessment of Environmental Agents. Toxicity testing in the 21st century: a vision and a strategy. 2007: National Academies Press.

[2] Russell, WMS, RL Burch, and CW Hume. The principles of humane experimental technique. 1959.

[3] Thermo Scientific. CellInsight NXT High Content Screening Platform Brochure. 2012.

[4] Gonzalez-Suarez, I, et al. Systems biology approach for evaluating the biological impact of environmental toxicants in vitro. Chem Res Toxicol, 2014. 27(3): p. 367-76.

[5] Karp, PH, et al. An in vitro model of differentiated human airway epithelia. Methods for establishing primary cultures. Methods Mol Biol, 2002. 188: p. 115-37.

[6] Pezzulo, AA, et al. The air-liquid interface and use of primary cell cultures are important to recapitulate the transcriptional profile of in vivo airway epithelia. Am J Physiol Lung Cell Mol Physiol, 2011. 300(1): p. L25-31.

[7] Abbott, A. Cell culture: Biology's new dimension. 2014.

[8] Baker, M. Tissue models: a living system on a chip. Nature, 2011. 471(7340): p. 661-5.

[9] Mathis, C, et al Human bronchial epithelial cells exposed in vitro to cigarette smoke at the air-liquid interface resemble bronchial epithelium from human smokers. Am J Physiol Lung Cell Mol Physiol, 2013. 304(7): p. L489-503.

[10] Iskandar, AR, et al. Systems approaches evaluating the perturbation of xenobiotic metabolism in response to cigarette smoke exposure in nasal and bronchial tissues. Biomed Res Int, 2013. 2013: p. 512086.

[11] Talikka, M, et al. The response of human nasal and bronchial organotypic tissue cultures to repeated whole cigarette smoke exposure. Int J Toxicol, 2014.

[12] Schlage, WK, et al. In vitro systems toxicology approach to investigate the effects of repeated cigarette smoke exposure on human buccal and gingival organotypic epithelial tissue cultures. Toxicol Mech Methods, 2014. 24(7): p. 470-87.

In Vivo Systems

To maximise the value of our studies and gain mechanistic insights into the different effects of cigarette smoke and RRP aerosol exposure, we have developed the concept of OECD+ studies in which we combine standard toxicological assessment methods with systems toxicology (an approach we have tested with a prototypic RRP⁷).

In Vivo Disease Models

Cardiovascular disease

Chronic obstructive pulmonary disease (COPD)

Lung cancer

OECD+

Testing a prototypic RRP in an in vivo model of COPD

In Vivo Disease Models

In the assessment of our Reduced-Risk Products (RRPs) the key diseases of interest are cardiovascular disease, chronic obstructive pulmonary disease (COPD) and lung cancer, each of which has well established epidemiological links between their development and cigarette smoking.

It is well established that with ongoing use of cigarettes, the risk for these and other diseases increases over time. It is also known that when a smoker quits, and remains abstinent from cigarettes (smoking cessation), their risk of disease then decreases, potentially to same level of risk as someone who has never smoked. Our key aspiration with RRPs is to demonstrate that they have a risk reduction profile approaching that of cessation.

Cardiovascular disease

It has been shown that cigarette smoking aggravates and speeds up the growth of atherosclerosis plaques, leading to increased incidence of cardiovascular disease. Plaques may block the blood flow through the affected artery in organs such as the heart (where a heart attack can result), brain (which can lead to stroke) or legs (where blockage can lead to peripheral artery disease)⁸. The in vivo model we use for cardiovascular disease is the apolipoprotein E deficient (ApoE^-/-) mouse^9,2.

Chronic obstructive pulmonary disease (COPD)

Cigarette smoking is an important and significant risk factor for the development of COPD. The condition is associated with chronic inflammation, affecting mainly the lung parenchyma and the peripheral airways, and it is believed that oxidative stress caused by cigarette smoke exposure is the driving factor behind this^10,11. COPD can be considered to be a syndrome with two main pathologies, namely emphysema (lung tissue destruction) and chronic bronchitis. The in vivo models we use for COPD are the ApoE^-/- mouse² and C57BL/6 mouse^12,13. We have recently tested a prototype RRP in our in vivo model of COPD.

Lung cancer

Cigarette smoking is the largest and most significant cause of lung cancer, attributing to 80-90% of cases of the disease¹⁴. Lung cancers are classified according to their histological type, and can be broadly classified into one of two main types: non-small cell lung carcinoma (NSCLC) and small cell lung carcinoma (SCLC). There are three main subtypes of NSCLC: adenocarcinoma, squamous-cell carcinoma and large-cell carcinoma¹⁴. 40% of lung cancers due to smoking are adenocarcinomas, about 30% are squamous cell carcinomas and approximately 9% are large-cell carcinomas. The remainder of lung cancers caused by smoking are SCLCs, which are typically very aggressive⁷. The in vivo model we use for lung cancer is the AJ mouse³.

All our non-human in vivo research is restricted to those occasions where there are no alternatives available.

[1] Russell, WMS, RL Burch and CW Hume. The principles of humane experimental technique. 1959.

[2]Lietz, M, et al. Cigarette-smoke-induced atherogenic lipid profiles in plasma and vascular tissue of apolipoprotein E-deficient mice are attenuated by smoking cessation. Atherosclerosis, 2013. 229(1): p. 86-93.

[3] Stinn, W, et al. Lung inflammatory effects, tumorigenesis, and emphysema development in a long-term inhalation study with cigarette mainstream smoke in mice. Toxicol Sci, 2013. 131(2): p. 596-611.

[4] Xiang, Y, et al. Discovery of emphysema relevant molecular networks from an A/J mouse inhalation study using Reverse Engineering and Forward Simulation (REFS). Gene Regul Syst Bio, 2014. 8: p. 45-61.

[5] Phillips, BW, et al. A mechanistic study of cigarette smoke-induced COPD and cessation effects in C57BL/6 mice. International Conference on Toxicology. 2013: Seoul, South Korea.

[6] Boue, S. et al. Cigarette smoke induces molecular responses in respiratory tissues of ApoE(-/-) mice that are progressively deactivated upon cessation. Toxicology, 2013. 314(1): p. 112-24.

[7] Kogel, U, et al. A 28-day rat inhalation study with an integrated molecular toxicology endpoint demonstrates reduced exposure effects for a prototypic modified risk tobacco product compared with conventional cigarettes. Food Chem Toxicol, 2014. 68: p. 204-17.

[8] American Heart Association. Atherosclerosis. 2014.

[9] Boue, S, et al. Cigarette smoke induces molecular responses in respiratory tissues of ApoE(-/-) mice that are progressively deactivated upon cessation. Toxicology, 2013. 314(1): p. 112-24.

[10] Kirkham, PA and PJ Barnes. Oxidative stress in COPD. Chest, 2013. 144(1): p. 266-73.

[11] Rahman, I. The role of oxidative stress in the pathogenesis of COPD: implications for therapy. Treat Respir Med, 2005. 4(3): p. 175-200.

[12] Stinn, W, et al. Mechanisms involved in A/J mouse lung tumorigenesis induced by inhalation of an environmental tobacco smoke surrogate. Inhal Toxicol, 2005. 17(6): p. 263-76.

[13] Phillips, BW, et al. A mechanistic study of cigarette smoke-induced COPD and cessation effects in C57BL/6 mice, in International Conference on Toxicology. 2013: Seoul, South Korea.

[14] Horn, L, W Pao and DH Johnson. Chapter 89, in Harrison's principles of internal medicine, DL Longo, et al. Editors. 2012, McGraw-Hill.

OECD+

OECD+ is the concept of combining the conventional toxicological assessment approach suggested by the Organization for Economic Co-operation and Development (OECD) with additional molecular biological endpoints. It is outlined in the image below.

Essentially, an OECD+ toxicity study is one designed for the purpose of obtaining omics endpoints. Rodents undergo the same exposure conditions at the same time as those in the OECD studies. Organs and body fluids of interest are collected and omics data are generated, computationally analysed and subsequently correlated with the endpoints described by the OECD guidelines.

OECD+ in practice

In a recent study[1], we investigated molecular perturbations accompanying histopathological changes in a 28-day rat inhalation study combining transcriptomics with classical histopathology.

Using laser-capture microdissection (PALM MicroBeam, Carl Zeiss Microscopy), we isolated areas of the lung parenchyma, the main bronchus and the first generation main branch of the bronchus, and then processed each of these areas for mRNA extraction. We also collected the nasal epithelium and compared the gene expression profiles to the lung.

In the collection of images below, the red dots show up-regulated genes and the green dots show down-regulated genes. The blue dots show genes that remained unchanged to the gene expression in tissues from sham (fresh air) exposed rats.

Using these gene expression profiles, we calculated the total change in the activation of different biological networks when compared to rats exposed to sham. As can be seen below, each of the eight biological networks we investigated were activated to different extents. Significantly, biological network activation is seen in the tissues from rats exposed to cigarette smoke at all doses. Importantly, exposure to the prototype Reduced-Risk Product (RRP) resulted in very low levels of biological network activation in all tissues investigated, even though the nicotine dose of the prototype RRP exposure was equal to the high dose of the cigarette.

Gene expression profiles Note: these data alone do not imply or represent a claim of reduced exposure or reduced risk.

This study was the first to demonstrate that within a classical OECD inhalation study, the correlative evaluation of the classical respiratory tract endpoints and gene expression analysis with advanced molecular modelling approaches (OECD+) is feasible and provides complimentary and important mechanistic information.

The qualitative and quantitative network modelling results exemplify how this approach can leverage a correlative evaluation of the histopathological changes and provide an insight into the molecular mechanisms perturbed by aerosols from different types of tobacco products.

We expect that this modelling-enhanced molecular extension of the classical OECD study design could be used not only to better understand the mechanisms leading to adaptive and inflammatory changes for cigarettes, but also to investigate the sensitivity and context specificity that is needed to investigate the risk-reduction potential of novel RRPs and/or to exclude the activation of previously unobserved toxicological pathways.

[1] Kogel, U, et al. A 28-day rat inhalation study with an integrated molecular toxicology endpoint demonstrates reduced exposure effects for a prototypic modified risk tobacco product compared with conventional cigarettes. Food Chem Toxicol, 2014. 68: p. 204-17.

Testing a prototypic RRP in an in vivo model of COPD

We have recently tested a prototypic heat-not-burn RRP in our in vivo model of COPD. In this study, we have used a number of molecular endpoints including transcriptomics, proteomics and lipidomics to determine the systemic impact of aersol from this prototype RRP compared to the overall impact of smoke from a 3R4F cigarette. We have also used non-molecular endpoints such and histology and lung function. The measurement of lung function in animals has recently been shown to accuratey describe the pathophysiological state of the lungs that can normally only be seen by histology. In this video you can find out more about lung function measurements, how the results can be interpreted to determine the pathological state of the lungs and how we apply this technique in order to compare the effects of cigarette smoke to the aersol from our prototypic RRPs.

All of the data that was collected in this study is currently being prepared for peer-reviewed publication. An outline of the study design and an overview of the results obtained can be viewed in the video presentation below by Dr Patrick Vanscheeuwijck.

Note: the data presented in this video alone do not imply or represent a claim of reduced exposure or reduced risk.

Clinical Systems

Biomarkers of Disease Onset

Identifying Biomarkers of Disease Onset for COPD

These studies are designed alongside the studies carried out in our in vivo models of disease. Although many more experimental manipulations can be carried out in rodent models as compared to humans, it is generally acknowledged that there is no model that represents the totality of disease as it manifests in humans, especially for smoking-related diseases. Therefore the combination of experimental manipulation of rodent models, combined with physiological and molecular measurements in human subjects, allows us to build a much more comprehensive understanding of the mechanisms underlying smoking induced incidence of diseases such as cardiovascular disease, COPD and lung cancer.

Diseases associated with the smoking of cigarettes normally take 25-30 years to develop. As such, the standard way to assess if switching to RRPs lowers the risk of disease incidence would require 25-30 years of epidemiology data, post-launch of a new product, in order to determine if there is a reduction in risk. In order to accumulate clinical scientific evidence in a significantly shorter timeframe, we use the translational systems approach to identify molecular or physiological markers which may give us an indication of a reduction in disease in a much shorter timeframe.

Biomarkers of Disease Onset

A large amount of research is being conducted by the scientific community in the area of biomarker discovery for all three diseases of interest (cardiovascular disease, COPD and lung cancer). However, much of this research is aimed at identifying biomarkers of disease stage, or at identifying biomarkers indicating the efficacy of a therapeutic intervention.

One example is in the field of COPD biomarker discovery. The severity of COPD is defined by the Global Strategy for the Diagnosis, Management and Prevention of COPD (also known as GOLD) criteria, and ranges from stage 1 (mild) to stage 4 (very severe). All of the therapeutic options for COPD aim at stopping the progression of disease from one stage to the next, thus the biomarkers of interest are those that indicate the stage of disease a patient is currently in, and those that are associated with later stages of COPD. The figure below shows a graphical representation of this:

COPD Biomarkers

In addition to these biomarkers, we are also interested in identifying biomarkers which precede the clinical onset of the disease and are associated with the exposure to cigarette smoke. We have termed these biomarkers “Biomarkers of Disease Onset”.

The ultimate challenge with Biomarkers of Disease Onset is to show that they are mechanistically linked to the development of a specific disease and are indeed biomarkers which indicate the future development of disease.

Identifying Biomarkers of Disease Onset for COPD

The first disease that we are working on for the identification of Biomarkers of Disease Onset is COPD and we have recently conducted a study in this area. The study was designed alongside our in vivo COPD studies to ensure that we gained a comprehensive understanding of the mechanisms linking cigarette smoke exposure to COPD development. An overview of the study design is available on ClinicalTrials.gov and is summarised below.

Subject groups in the COPD Biomarker of Disease Onset study	*Equivalent groups in our COPD in vivo* study**
Never-smokers	Sham (fresh air) exposed mice
Ex-smokers	Cigarette smoke exposed mice switched to sham
COPD GOLD stage 1/2	Mice exposed to smoke that have early stage emphysema
Healthy smokers	No relevant group

	COPD clinical study	COPD clinical study
mRNA expression	White blood cells	White blood cells
	Nasal epithelium (scrapes)	Nasal epithelium (laser capture microdissection)
Proteomics and lipidomics	Blood	Blood
	Sputum	Bronchoalveolar lavage (BALF)
Protein inflammatory markers	In sputum and BALF GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17, MMP-9, MCP-1α, TIMP-1, TNFα	GM-CSF, IFN-γ, IL-1β, IL-2, IL-4, IL-5, IL-6, IL-10, IL-12p70, IL-17, MMP-9, MCP-1α, TIMP-1, TNFα
Cellular	Differential cell count in sputum	Differential cell count in BALF
Physiological	High Resolution Computerised Tomography (HR-CT) with radiologist scored emphysema	Pathological evaluation of lung sections with emphysema measured by destructive index
	Full lung function	Full lung function
	Blood haematoxicological screening	Blood haematoxicological screening

The endpoints measured in both studies were also designed to correlate.

The combination of the systems responses from both studies has resulted in much deeper understanding of the molecular mechanisms leading from cigarette smoke exposure to onset of COPD.

Experimental Data Production_2.2.1.3-copd-biomarker1

Experimental Data Production_2.2.1.3-copd-biomarker2_0_0

COPD Biomarker Identification Study The descriptions in this chart are for illustrative purposes only.

COPD Biomarker Identification Study + in vivo study

The descriptions in this chart are for illustrative purposes only.

By integrating the clinical and in vivo studies, we are able to fill in certain knowledge gaps that would exist if only one study was conducted. This combination and the ability to identify translatable biology provides an extremely powerful tool for the identification of new biomarkers.

Whole Smoke Exposure

Smoke/aerosol fractions and in vitro exposure

In vitro whole smoke/aerosol exposure

Whole smoke aerosol_tech_for_whole_smoke_exp

Smoke / aerosol fractions and in vitro exposure

The classical way of exposing in vitro test systems to smoke or aerosol is to separate them into their separate phases. Aerosols from RRPs and smoke from cigarettes are made up of particles (the Total Particulate Matter, or TPM) and a mixture of volatile or vapour components (the Gas-Vapour Phase, or GVP). By passing the smoke or aerosol through a Cambridge glass fibre filter pad and a solution of phosphate buffered saline (PBS), separation of the TPM and GVP can be achieved.

The diagram below shows how the smoke / aerosol fractions are collected. The TPM is captured on a Cambridge filter pad, while GVP is the fraction of the smoke / aerosol not retained by the filter pad and soluble in PBS.

Whole smoke aerosol exposure_2.2.1.4-diagram

TPM and GVP collection image

In vitro whole smoke / aerosol exposure

Although the TPM and GVP are widely used for in vitro studies, they have some important limitations². In particular, the method used for trapping them might alter the chemical composition of each fraction. Components of the collection system may leach impurities into the collected material and the solvents used might react with constituents of the smoke / aerosol fractions. Most importantly, analyses of the TPM and GVP individually may overlook aspects of the overall risk attributable to cigarette smoke.

For these reasons, researchers are increasingly using culture systems where cells are exposed to smoke / aerosols at the air-liquid interface. Culture inserts, in which cells grow at the air-liquid interface, are placed in an exposure chamber and smoke / aerosols are guided through the chamber in a highly controlled manner. Several research groups and companies have developed systems that allow whole smoke in vitro exposure^{3,4,5,6,7,8,9}.

The following diagram illustrates our use of the Vitrocell® 24/48 exposure system in one recent study². The system has a double inlet dilution / distribution system for up to seven dilution airflows and one fresh air control. It also provides an integrated, sensor-controlled heating plate as well as a climatic chamber surrounding the dilution / distribution system and cultivation base module.

Use of the Vitrocell® 24/48 exposure system

As shown here, the system was used to determine levels of nicotine and eight carbonyls in whole cigarette smoke.