Multiple endpoint in vitro toxicity assessment of a prototype heated tobacco product indicates substantially reduced effects compared to those of combustible cigarette.

This study aimed to compare the aerosol chemistry and in vitro toxicological profiles of two prototype Heated Tobacco Product (p-HTP) variants to the 1R6F Reference Cigarette. In the neutral red uptake screen the p-HTPs were 37-39-fold less potent than 1R6F, in the micronucleus assay, responses to the p-HTPs were 8-22-fold less, and in the Ames test mutagenicity was weak or removed compared to 1R6F. The cardiovascular scratch wound assay revealed 58-fold greater wound healing impairment following exposure to 1R6F smoke extracts than the p-HTPs. Furthermore, in seven cell stress-related high content screening endpoints (cell count, cytochrome c release, mitochondrial membrane potential, GSH depletion, NFkB translocation, phosphorylation of c-jun and phosphorylation of H2AX), at 4 and 24 h, responses were substantially greater to 1R6F smoke extracts at comparable nicotine levels. The reduced in vitro effects of the p-HTPs were attributed to substantial reductions (90-97%) in selected HPHCs measured compared to in 1R6F smoke. The multiple endpoint in vitro assessment approach provides greater mechanistic insight and the first reported toxicological characterisation of these p-HTPs in the literature. Overall, the findings contribute to the growing weight of evidence that HTPs may offer a reduced harm mode of nicotine delivery to adult smokers.

A randomised, open-label, cross-over clinical study to evaluate the pharmacokinetic, pharmacodynamic and safety and tolerability profiles of tobacco-free oral nicotine pouches relative to cigarettes.

RATIONALE: Tobacco harm reduction (THR) involves encouraging adult smokers who would otherwise continue to smoke to transition to less harmful forms of nicotine delivery. These products must offer adult smokers reduced exposure to chemicals associated with tobacco combustion, satisfactory blood plasma nicotine levels and serve as an acceptable alternative. The most recent THR innovation is tobacco-free oral nicotine pouches. OBJECTIVES: This study aimed to compare pharmacokinetic, pharmacodynamic and safety and tolerability profiles of two nicotine pouch variants (ZoneX #2 (5.8 mg nicotine/pouch); ZoneX #3 (10.1 mg nicotine/pouch)) with cigarette to assess the pouches' THR potential. METHODS: This was a controlled use, randomised, open-label, cross-over clinical study with 24 healthy adult traditional tobacco users. Pharmacokinetic (plasma nicotine levels; up to 8 h post-use), pharmacodynamic (urge to smoke, product liking; up to 4 h post-use) and short-term safety and tolerability profiles were assessed. RESULTS: Distinct nicotine pouch pharmacokinetic profiles indicated nicotine absorption via the oral mucosa. Plasma nicotine levels were lower, and time to peak slower, for the nicotine pouches compared to cigarette (Cmax cigarette: 11.6 ng/ml vs. #2: 5.2 ng/ml, p < 0.0001; #3: 7.9 ng/ml, p < 0.0003) (Tmax cigarette: 8.6 min vs. #2: 26 min; #3: 22 min). All products effectively reduced subjects' urge to smoke and presented favourable product liking scores; nicotine pouches were also well tolerated following short-term use (no serious adverse events). CONCLUSIONS: Overall, the assessed ZoneX nicotine pouches may offer an acceptable alternative for adult smokers to achieve satisfactory levels of nicotine delivery and, based on the pharmacokinetic parameters and under the study conditions, likely have a lower abuse liability and addictive potential for current adult smokers compared to continued cigarette smoking. CLINICAL TRIAL IDENTIFIER: NCT04891406 (clinicaltrials.gov).

Use of Human Induced Pluripotent Stem Cell-Derived Cardiomyocytes to Predict the Cardiotoxicity Potential of Next Generation Nicotine Products.

Combustible cigarette smoking is an established risk factor for cardiovascular disease. By contrast, the cardiotoxicity potential of non-combustible next generation nicotine products (NGPs), which includes heated tobacco products (HTPs) and electronic vaping products (EVPs), and how this compares relative to combustible cigarettes is currently an area of scientific exploration. As such, there is a need for a rapid screening assay to assess this endpoint. The Cardio quickPredict is a metabolomics biomarker-based assay that uses human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM) to screen for potential structural and functional cardiac toxicants based on the changes of four metabolites, lactic acid, arachidonic acid, thymidine, and 2'-deoxycytidine. The study aims were to investigate the cardiotoxicity potential of NGPs compared to cigarettes, in addition to nicotine. To accomplish this, hiPSC-CM were exposed to smoke or aerosol bubbled PBS samples: reference cigarette (1R6F); three variants of HTP; and three EVP variants. The 1R6F bPBS was the most active, having cardiotoxic potential at 0.3-0.6% bPBS (0.4-0.9 µg/mL nicotine), followed by HTP, which displayed cardiotoxic potential at a 10 times higher concentration, 3.3% bPBS (4.1 µg/mL nicotine). Both 1R6F and HTP bPBS (at 10-fold higher concentration than 1R6F) affected all four predictive metabolites, whereas none of the EVP bPBS samples were active in the assay up to the maximal concentration tested (10% bPBS). Nicotine tested on its own was predicted to have cardiotoxic potential at concentrations greater than 80 µg/mL, which is higher than expected physiological levels associated with combustible cigarette smoking. The application of this rapid screening assay to NGP research and the associated findings adds to the weight-of-evidence indicating that NGPs have a tobacco harm reduction potential when compared to combustible cigarettes. Additionally, this technique was shown to be sensitive and robust for the assessment of different NGPs and may be considered as part of a larger overall scientific framework for NGP assessments.

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

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.