[in Japanese]

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[in Japanese]

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In this study, the impact of inhalation of aerosol from a reference cigarette (3R4F) or a prototypic modified risk tobacco product (pMRTP), was evaluated in the lung methylome and proteome of C57Bl/6 mice, a model for cigarette smoking-induced chronic obstructive pulmonary disease (COPD). Mice were exposed to aerosol from 3R4F, pMRTP or filtered air for up to 7 months. After 2 months of exposure to 3R4F, switching and cessation groups were exposed to pMRTP or filtered air, respectively.
Differences in lung DNA methylomes analyzed with whole genome bisulfite sequencing showed that continuous exposure to 3R4F presented a larger increase in the amount of hypermethylated CpG sites over time than continuous exposure to a pMRTP, smoking cessation or switching to a pMRTP.
Changes in protein expression levels were detected with isobaric tags for absolute and relative quantification (iTRAQ®). Exposure to 3R4F induced up-regulation of biological functions such as xenobiotic metabolism. Animals exposed to pMRTP exhibited negligible changes, and smoking cessation and switching to a pMRTP resulted in a reduction of changes close to control group levels.
These data demonstrate that exposure to a pMRTP resulted in a response similar to fresh air-exposed animals, while smoking cessation and switching to a pMRTP resulted in the reversal or stabilization of parameters assessed. These results complement those obtained from other endpoints such as transcriptomics or lipidomics, and further establishes the foundation of our systems biology approach to assess the impact of conventional and modified risk tobacco products on biological systems.

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Philip Morris International is developing products with the potential to reduce the risk associated with smoking. The Tobacco Heating System (THS 2.2) operates by heating tobacco rather than burning it and results in an aerosol with substantially lower levels of harmful or potentially harmful constituents when compared to combustible cigarettes (CC) smoke. Additionally, THS 2.2 does not produce sidestream aerosol in the same manner as CC, since aerosol is only generate when puffs are taken. Thus, the impact on air quality of using THS 2.2 indoors is expected to be very different to CC. To verify this hypothesis, we built an environmentally controlled, furnished room and developed analytical methods to measure air pollutants under diverse simulated indoor environments focusing on: (i) ISO measurement standards for Environmental Tobacco Smoke and, (ii) selected carbonyls and volatile organic compounds.
A study was conducted with 3 simulate conditions (office, hospitality, residential) with conditions defined according to CEN standard EN 15251.2007. Three test items were compared: CC (Marlboro Gold 6mg), THS 2.2 and background (measured with people in the room without product use). Each study was duplicated, resulting in 18 separate session in total, each with a duration of 5 hours, with 4 hours of sample collection.
In case of statistical equivalence, no impact on air quality is reported. For CC, all analytes for the 3 conditions were above background. For THS 2.2, no difference was detected between background and THS 2.2 for 15 of the eighteen analytes investigated, irrespective of the environment conditions applied.

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The study objective was to evaluate the pharmacokinetic (PK) profile of nicotine following single use of THS 2.2 non-menthol or menthol compared to combustible cigarettes (CC) and nicotine gum (NRT).
Two open-label, randomized, two-period, four-sequence crossover studies were conducted, each in 62 healthy Japanese smokers. Each period consisted of 2 days, with 1 nicotine wash-out day, and 1 THS 2.2, CC or NRT single use day with every subject exposed to 2 of the 3 products. A total of 16 venous blood samples were collected, including 1 sample prior to product use. Urge-to-smoke was assessed using the Questionnaire of Smoking Urges-brief (QSU-b). The studies were conducted according ICH GCP and registered with ClinicalTrials.gov, number NCT01959607 and NCT01967706.
The overall shape of the nicotine concentration-time curves for THS 2.2 non-menthol and menthol were similar to CC with comparable values for AUC_(0-last) and C_max in both studies. AUC_(0-last) and C_max were higher in THS 2.2 compared to NRT in one study, with a comparable C_max and a lower AUC_(0-last) in the other study. The t_max was not different for CC and THS 2.2 and shorter for THS 2.2 compared to the NRT. The reduction of QSU-b total score observed after THS 2.2 and CC use were similar and greater than the reduction following NRT use. THS 2.2 was well tolerated.
The PK profile for both THS 2.2 was comparable to CC in both studies. A transient reduction in urge-to-smoke was observed with THS 2.2, comparable to CC and higher than NRT.

Keywords: Pharmacological effects, Modified risk tobacco product.

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The objective of this study was to assess the reduction in exposure to selected harmful and potentially harmful constituents (HPHCs) after 5 days of ad libitum use of THS 2.2 in confined conditions compared to continued smoking of combustible cigarettes (CC) and smoking abstinence (SA).
The open-label, randomized, controlled, 3-arm parallel group, confinement study was done in 160 healthy Japanese smokers aged between 23 and 65 years. Smokers smoked their CC at baseline for 2 days, and were subsequently randomized to continue to smoke CC, to switch to THS 2.2, or to stop smoking for 5 days. Biomarkers of exposure (BoExp) to 14 HPHCs were evaluated using validated analytical methods. This study was conducted in Japan according to ICH GCP and is registered in ClinicalTrials.gov, number NCT01970982.
The average daily product use between baseline and the end of exposure was similar in the THS 2.2 and CC arms. The total nicotine exposure measured as nicotine equivalents was also similar in both arms throughout the exposure period. The levels of evaluable BoExp were significantly reduced in the range of 47% to 96% in the THS 2.2 arm as compared to CC and approaching results obtained in the SA arm. The reductions in BoExp levels could be observed within 24 hours of starting THS 2.2. THS 2.2 was well tolerated.
THS 2.2 showed a significant reduction in exposure to HPHCs after 5 days of use in confinement, as compared to CC, and approaching the levels obtained after 5 days of smoking abstinence.

Keywords: Modified risk tobacco product, Reduced exposure.

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The wide applications of engineered amorphous silica nanoparticles (aSiNP) have emphasized the need for further mechanistic insight to predict the consequences of human and environmental health and safety impacts of its exposures. The present study aims to investigate the regulatory molecular mechanisms and pathways involved in aSiNP mediated toxicity with systems toxicology approach. We applied global gene expression changes with DNA microarray and non-targeted NMR based metabolome analysis, followed by individual as well as integrated pathway analysis in parallel to unravel altered cellular processes in aSiNP treated conditions. The significant gene expression changes and subsequent KEGG pathway analysis revealed that up regulation of steroid-cholesterol biosynthesis and down regulation of glutathione metabolism, xenobiotic/drug metabolism pathways. In addition, combined analysis of significantly altered genes and NMR based metabolites in IMPaLA displayed aSiNP treatment caused perturbation in glutathione conjugation and metabolism of lipids/ lipoproteins. To get detailed insight into lipid metabolism, next, targeted lipidomics was performed using GC-MS and the subsequent pathway analysis with MetaboAnalyst revealed up regulation of steroid biosynthesis pathway. Furthermore, integrated analysis of significantly altered genes and all metabolites in IMPaLA turned on cholesterol biosynthesis pathway as main mechanism of aSiNP treatment. Finally, biochemical tests for total cholesterol, total glutathione and glutathione-S-transferase enzyme activity, strongly corroborated with the hypothesis driven from bioinformatics analysis. Taken together, activation of cholesterol biosynthesis and suppression of glutathione metabolism/conjugation were found as principal mode of action of aSiNP treatment in HepG2 cells.

Key words: Amorphous Silica nanoparticles (aSiNP); Transcriptomics; Metabolomics; Integrated pathway analysis (IMPaLA); cholesterol biosynthesis; Systems Toxicology

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