Use of a rapid human primary cell-based disease screening model, to compare next generation products to combustible cigarettes

Abstract

A growing number of public health bodies, regulators and governments around the world consider electronic vapor products a lower risk alternative to conventional cigarettes. Of critical importance are rapid new approach methodologies to enable the screening of next generation products (NGPs) also known as next generation tobacco and nicotine products. In this study, the activity of conventional cigarette (3R4F) smoke and a range of NGP aerosols (heated tobacco product, hybrid product and electronic vapor product) captured in phosphate buffered saline, were screened by exposing a panel of human cell-based model systems using Biologically Multiplexed Activity Profiling (BioMAP® Diversity PLUS® Panel, Eurofins Discovery). Following exposure, the biological activity for a wide range of biomarkers in the BioMAP panel were compared to determine the presence of toxicity signatures that are associated with specific clinical findings. NGP aerosols were found to be weakly active in the BioMAP Diversity PLUS Panel (≤3/148 biomarkers) whereas significant activity was observed for 3R4F (22/148 biomarkers). Toxicity associated biomarker signatures for 3R4F included immunosuppression, skin irritation and thrombosis, with no toxicity signatures seen for the NGPs. BioMAP profiling could effectively be used to differentiate between complex mixtures of cigarette smoke or NGP aerosol extracts in a panel of human primary cell-based assays. Clinical validation of these results will be critical for confirming the utility of BioMAP for screening NGPs for potential adverse human effects.

Keywords: ACM, aerosol collected mass; AhR, Aryl hydrocarbon receptor; Alternative methods; COPD, Chronic obstructive pulmonary disease; EGFR, epidermal growth factor receptor; ELISA, enzyme-linked immunosorbent assay; EVP, Electronic vapor product; HDFn, Human neonatal dermal fibroblasts; HTP, Heated Tobacco Product; HUVEC, Human umbilical vein endothelial cells; HYB, Hybrid product containing e-liquid drawn through a tobacco plug; IL, interleukin; ISO, International Organization for Standardization; In vitro assays; MOA, Mechanism of action; M−CSF, Macrophage colony-stimulating factor; NGP, Next generation product; NRC, National Research Council; NRF2, Nuclear factor erythroid 2-related factor 2; Next generation products; PBMC, Peripheral blood mononuclear cells; PBS, Phosphate buffered saline; Panel; Phenotypic screening; SRB, Sulforhodamine B; TCR, T cell receptor; TF, Tissue factor; TLR, toll-like receptor; TNFα, tumor necrosis factor alpha; TPM, Total particulate matter; Toxicity signature; bPBS, Bubbled phosphate buffered saline; mTOR, mechanistic target of rapamycin.

Graphical abstract

Fig. 1

BioMAP profiles of cigarette and NGPs in the Diversity PLUS Panel: A. 3R4F, B. HTP, C. HYB, D. EVP. The X-axis lists the measured protein-based biomarker readouts in each system. The Y-axis represents a log₁₀-transformed ratio of the biomarker readouts for the test article-treated sample (n = 1) over vehicle controls (n ≥ 6). The grey region around the Y-axis represents the 95% significance envelope generated from historical vehicle controls. Antiproliferative effects are indicated on the profile plot by thick grey arrows above the X-axis. Where the biomarker changes at 2 or more concentrations and follow the same directional trend they are annotated on the chart with grey arrows. Line colors indicate different test concentrations.

Fig. 2

Toxicity Signature Analysis for the Diversity PLUS panel. Evaluation of the presence of Toxicity Signatures within the BioMAP profile of the tested article and concentrations detected. Toxicity Signatures are made up of 2–5 biomarker activities that have been correlated to an increased risk of certain toxicity effects in vivo. Concentrations are listed if the signature for the toxicity was detected. Not detected (ND) indicates the signature was not detected at any of the concentrations tested.