Recapitulation of human pathophysiology and identification of forensic biomarkers in a translational model of chlorine inhalation injury.

Chlorine gas (Cl2) has been repeatedly used as a chemical weapon, first in World War I and most recently in Syria. Life-threatening Cl2 exposures frequently occur in domestic and occupational environments, and in transportation accidents. Modeling the human etiology of Cl2-induced acute lung injury (ALI), forensic biomarkers, and targeted countermeasures development have been hampered by inadequate large animal models. The objective of this study was to develop a translational model of Cl2-induced ALI in swine to understand toxico-pathophysiology and evaluate whether it is suitable for screening potential medical countermeasures and to identify biomarkers useful for forensic analysis. Specific pathogen-free Yorkshire swine (30-40 kg) of either sex were exposed to Cl2 (≤240 ppm for 1 h) or filtered air under anesthesia and controlled mechanical ventilation. Exposure to Cl2 resulted in severe hypoxia and hypoxemia, increased airway resistance and peak inspiratory pressure, and decreased dynamic lung compliance. Cl2 exposure resulted in increased total leucocyte and neutrophil counts in bronchoalveolar lavage fluid, vascular leakage, and pulmonary edema compared with the air-exposed group. The model recapitulated all three key histopathological features of human ALI, such as neutrophilic alveolitis, deposition of hyaline membranes, and formation of microthrombi. Free and lipid-bound 2-chlorofatty acids and chlorotyrosine-modified proteins (3-chloro-l-tyrosine and 3,5-dichloro-l-tyrosine) were detected in plasma and lung tissue after Cl2 exposure. In this study, we developed a translational swine model that recapitulates key features of human Cl2 inhalation injury and is suitable for testing medical countermeasures, and validated chlorinated fatty acids and protein adducts as biomarkers of Cl2 inhalation.NEW & NOTEWORTHY We established a swine model of chlorine gas-induced acute lung injury that exhibits several features of human acute lung injury and is suitable for screening potential medical countermeasures. We validated chlorinated fatty acids and protein adducts in plasma and lung samples as forensic biomarkers of chlorine inhalation.

Formation of flavorant-propylene Glycol Adducts With Novel Toxicological Properties in Chemically Unstable E-Cigarette Liquids.

INTRODUCTION: "Vaping" electronic cigarettes (e-cigarettes) is increasingly popular with youth, driven by the wide range of available flavors, often created using flavor aldehydes. The objective of this study was to examine whether flavor aldehydes remain stable in e-cigarette liquids or whether they undergo chemical reactions, forming novel chemical species that may cause harm to the user. METHODS: Gas chromatography was used to determine concentrations of flavor aldehydes and reaction products in e-liquids and vapor generated from a commercial e-cigarette. Stability of the detected reaction products in aqueous media was monitored by ultraviolet spectroscopy and nuclear magnetic resonance spectroscopy, and their effects on irritant receptors determined by fluorescent calcium imaging in HEK-293T cells. RESULTS: Flavor aldehydes including benzaldehyde, cinnamaldehyde, citral, ethylvanillin, and vanillin rapidly reacted with the e-liquid solvent propylene glycol (PG) after mixing, and upward of 40% of flavor aldehyde content was converted to flavor aldehyde PG acetals, which were also detected in commercial e-liquids. Vaping experiments showed carryover rates of 50%-80% of acetals to e-cigarette vapor. Acetals remained stable in physiological aqueous solution, with half-lives above 36 hours, suggesting they persist when inhaled by the user. Acetals activated aldehyde-sensitive TRPA1 irritant receptors and aldehyde-insensitive TRPV1 irritant receptors. CONCLUSIONS: E-liquids are potentially reactive chemical systems in which new compounds can form after mixing of constituents and during storage, as demonstrated here for flavor aldehyde PG acetals, with unexpected toxicological effects. For regulatory purposes, a rigorous process is advised to monitor the potentially changing composition of e-liquids and e-vapors over time, to identify possible health hazards. IMPLICATIONS: This study demonstrates that e-cigarette liquids can be chemically unstable, with reactions occurring between flavorant and solvent components immediately after mixing at room temperature. The resulting compounds have toxicological properties that differ from either the flavorants or solvent components. These findings suggest that the reporting of manufacturing ingredients of e-liquids is insufficient for a safety assessment. The establishment of an analytical workflow to detect newly formed compounds in e-liquids and their potential toxicological effects is imperative for regulatory risk analysis.

Variability in Constituents of E-Cigarette Products Containing Nicotine Analogues.

This study characterizes and quantifies constituents in 2 e-cigarette products to assess product consistency and inform future risk assessments.

Synthetic Cooling Agent and Other Flavor Additives in "Non-Menthol" Cigarettes Marketed in California and Massachusetts After Menthol Cigarette Bans.

This study uses a bioassay and chemical analysis to determine the proportion of newly introduced "non-menthol" cigarette brands with sensory cooling effects, cooling agents added, and any other flavor additives after menthol cigarette bans.

TRPA1 controls inflammation and pruritogen responses in allergic contact dermatitis.

Allergic contact dermatitis is a common skin disease associated with inflammation and persistent pruritus. Transient receptor potential (TRP) ion channels in skin-innervating sensory neurons mediate acute inflammatory and pruritic responses following exogenous stimulation and may contribute to allergic responses. Genetic ablation or pharmacological inhibition of TRPA1, but not TRPV1, inhibited skin edema, keratinocyte hyperplasia, nerve growth, leukocyte infiltration, and antihistamine-resistant scratching behavior in mice exposed to the haptens, oxazolone and urushiol, the contact allergen of poison ivy. Hapten-challenged skin of TRPA1-deficient mice contained diminished levels of inflammatory cytokines, nerve growth factor, and endogenous pruritogens, such as substance P (SP) and serotonin. TRPA1-deficient sensory neurons were defective in SP signaling, and SP-induced scratching behavior was abolished in Trpa1(-/-) mice. SP receptor antagonists, such as aprepitant inhibited both hapten-induced cutaneous inflammation and scratching behavior. These findings support a central role for TRPA1 and SP in the integration of immune and neuronal mechanisms leading to chronic inflammatory responses and pruritus associated with contact dermatitis.

Introduction of Nicotine Analogue-Containing Oral Pouch Products in the United States.

Background: In 2023, 6-methyl nicotine (6MN), a synthetic nicotine analogue, was introduced in US-marketed electronic cigarette products advertised as exempt from regulation. It is unknown whether the use of 6MN has spread to the oral nicotine pouch product category that has become increasingly popular. Methods: Industry reports and the USPTO database were searched for informationon 6-methyl nicotine products. The search terms "Metatine", "Nixotine", "Imotine" and "pouches" were shortlisted and used to identify US-marketed pouch products. Ingredient contents were compared to popular products, and safety warnings and regulatory statements assessed in context with US state and federal regulations. Results: Two US-based brands, "MG" and "Hippotine" pouches, were identified in August 2024, advertised to contain "Imotine"-trademarked 6MN. MG Pouches are marketed in four youth-appealing flavors. "Hippotine"-branded pouches are marketed in two flavors. 6MN contents ranged between 8mg - 25mg with almost identical ingredient lists otherwise. Products list extensive addiction and health warnings, including warnings not to operate vehicles. Vendors state that these are not a tobacco product, implicating that federal and state tobacco regulations do not apply. Conclusions: The spread of nicotine analogues to additional product categories such as oral pouches is concerning, especially given the high 6MN contents that exceed nicotine contents in popular US-marketed oral nicotine pouch products. Legislators and regulators need to provide certainty about the regulatory status of nicotine analogues to prevent further erosion of tobacco flavor bans and other regulations.

Marketing of nicotinamide as nicotine replacement in electronic cigarettes and smokeless tobacco.

In the United States, the Food and Drug Administration (FDA) requires tobacco product manufacturers to submit Premarket Tobacco Product Applications (PMTA) for new products, granting marketing approval only if deemed appropriate for the protection of public health. Historically, the tobacco industry has exploited loopholes in the Tobacco Control Act (TCA), especially related to the definitions of nicotine, tobacco product and characterizing flavors, to circumvent the PMTA requirement. In 2023, the industry introduced several 'PMTA-exempt' e-cigarette and smokeless products, including products containing 6-methyl nicotine, a synthetic nicotine analog that is pharmacologically more potent than nicotine. In late 2023 and early 2024, the major US e-cigarette suppliers Nicotine River and ECBlend introduced 'PMTA-exempt' products with the brand names 'Nixamide' or 'Nixodine' or 'Nixotine', with nicotinamide as the main active ingredient. Nicotinamide is a form of vitamin B3 with no known pharmacological activity at nicotinic receptors. Here, we report that the marketing claims for these products, suggesting them and be nicotine substitute products designed to target nicotinic receptors and provide the same experience as nicotine, is deceptive and misleading to consumers. We also inform that these products have evolved further to contain a combination of nicotinamide and 6-methyl nicotine. The regulatory implications of these newly introduced products are discussed.

Artificial Sweeteners in US-Marketed Oral Nicotine Pouch Products: Correlation with Nicotine Contents and Effects on Product Preference.

Introduction: Artificial sweeteners are listed as ingredients of oral nicotine pouches (ONPs), a new product category with rapidly growing market share. The exact sweetener contents of ONPs remain unknown. Artificial sweeteners in ONPs may facilitate initiation and encourage consumption behavior. Aims and Methods: Artificial sweetener contents in major US-marketed ONP brands (Zyn, on!, Velo) were determined by Liquid Chromatography-Mass Spectrometry (LC-MS). Sweetener effects during the initiation of ONP consumption were modeled in single- and two-bottle tests, offering mice ONP extracts calibrated to contain nicotine levels similar to saliva of people who use smokeless tobacco. To examine the contribution of sweet taste perception, consumption behavior was compared between wild-type mice and mice deficient in the sweet taste receptor (Tas1r2-/-). Results: Acesulfame-K was detected in on!, Zyn and Velo ONPs (~0.3-0.9 mg/pouch), including products marketed as "Unflavored" or "Flavor ban approved". In Velo ONPs, sweetened with sucralose (0.6-1.2 mg/pouch), higher nicotine strength products contained higher sucralose levels. Tas1r2-/- mice consumed less ONP extracts than wild-type mice in both sexes. ONP extracts with both higher nicotine and sweetener strengths were tolerated by wild-type mice, but produced stronger aversion in Tas1r2-/- mice. Conclusions: ONPs contain significant amounts of artificial sweeteners, with some brands adding more sweetener to ONPs with higher nicotine strengths. Artificial sweeteners, at levels present in ONPs, increase nicotine consumption. Increasing sweetener contents facilitates consumption of ONPs with higher nicotine strengths. Sweetness is a key determinant of ONP use initiation, likely reducing the aversive sensory effects of nicotine and other ONP constituents. Implications: Artificial sweeteners such as acesulfame-K or sucralose reduce aversion and facilitate initiation and continued consumption of ONPs. The marketing of some artificially sweetened ONPs as "Unflavored" of "Flavor ban-approved" suggests that the tobacco industry rejects sweet taste as a determinant for the presence of a characterizing flavor. Sweetness as imparted by artificial sweeteners in tobacco products needs to be addressed by regulators as a component of a characterizing flavor, with the aim to reduce product appeal and initiation by never users, and especially youth attracted to sweet flavors.

High Variability in Nicotine Analog Contents, Misleading Labeling, and Artificial Sweetener in New E-Cigarette Products Marketed as "FDA-Exempt".

The recent introduction of electronic cigarette products containing a synthetic nicotine analog, 6-methyl nicotine (6MN), challenges FDA's tobacco regulatory authority. A similar strategy is pursued by vendors of recently introduced e-cigarette liquids containing nicotinamide (NA), marketed as 'Nixotine' or 'Nixamide'. Compared to nicotine, 6MN is pharmacologically more potent at nicotinic receptors, and more toxic, raising concerns about increased addictiveness and adverse effects. Here, combinations of gas chromatography, high performance liquid chromatography and mass spectrometry were used to determine nicotine analogs, flavor and sweetener contents of e-cigarette liquids of the brands "SpreeBar" and ECBlend "Nixotine" products. All SpreeBar products, labelled as containing 5% 6-methyl nicotine, contained only 0.61-0.64% 6-methylnicotine, while "Nixotine" samples contained 7-46% less of the declared nicotinamide contents. Although "Nixotine" product labels did not list 6MN as an ingredient, small amounts of 6-methyl nicotine were detected. All 'SpreeBar' samples contained the artificial sweetener neotame (0.20-0.86µg/mg). Results identified significant discrepancies between declared and measured constituents of e-cigarette products containing nicotine alternatives. The discrepancy is misleading for consumers and raises concerns about production errors. 'SpreeBar' products also contained neotame, a high-intensity sweetener with high heat stability, likely increasing appeal to young and first-time users. Novel e-cigarette products with misleading labels containing nicotine analogs instead of nicotine on the US market is concerning and should be urgently addressed by lawmakers and regulators.

An electronic cigarette pod system delivering 6-methyl nicotine, a synthetic nicotine analog, marketed in the United States as "PMTA exempt".

As of April 14, 2022, the United States Food and Drug Administration (FDA) has been authorized to regulate tobacco products containing nicotine from any source, including synthetic, requiring manufacturers to submit a premarket tobacco product application (PMTA). A recent report by the World Health Organization (WHO) warned that non-nicotine tobacco alkaloids or other synthetic nicotine analogs could be used by manufacturers to bypass regulatory schemes focusing on nicotine alone. From October 2023 on, vape stores in the United States started selling a new electronic cigarette pod system, named Spree Bar, advertised as "PMTA exempt", with youth-appealing flavors and advertising. The products are marketed as containing "Metatine", a trademarked name for 6-methyl nicotine, a synthetic nicotine analog patented by a Chinese electronic cigarette manufacturer. Here we used liquid chromatography-mass spectrometry (LCMS) to confirm the presence of a chemical species with the molecular weight of 6-methyl nicotine in Spree Bar e-liquids. The FDA needs to determine whether, in its view, 6-methyl nicotine is a form of "nicotine" within the meaning of the Tobacco Control Act, or whether 6-methyl nicotine can be regulated as a drug under the Federal Food, Drug, and Cosmetic Act (FDCA).

Synthetic Cooling Agent and Candy Flavors in California-marketed "non-Menthol" Cigarettes.

RATIONALE: The ban of menthol cigarettes is one of the key strategies to promote smoking cessation in the United States. Menthol cigarettes are preferred by young beginning smokers for smoking initiation. Almost 90% of African American smokers use menthol cigarettes, a result of decades-long targeted industry marketing. Several states and municipalities already banned menthol cigarettes, most recently California, effective on December 21, 2022. In the weeks before California's ban took effect, the tobacco industry introduced several "non-menthol" cigarette products in California, replacing previously mentholated brands. Here, we hypothesize that tobacco companies replaced menthol with synthetic cooling agents to create a cooling effect without using menthol. Similar to menthol, these agents activate the TRPM8 cold-menthol receptor in sensory neurons innervating the upper and lower airways. METHODS: Calcium microfluorimetry in HEK293t cells expressing the TRPM8 cold/menthol receptors was used to determine sensory cooling activity of extracts prepared from these "non-menthol" cigarette brands, and compared to standard menthol cigarette extracts of the same brands. Specificity of receptor activity was validated using TRPM8-selective inhibitor, AMTB. Gas chromatography mass spectrometry (GCMS) was used to determine presence and amounts of any flavoring chemicals, including synthetic cooling agents, in the tobacco rods, wrapping paper, filters and crushable capsule (if present) of these "non-menthol" cigarettes. RESULTS: Compared to equivalent menthol cigarette extracts, several California-marketed "non-menthol" cigarette extracts activated cold/menthol receptor TRPM8 at higher dilutions and with stronger efficacies, indicating substantial pharmacological activity to elicit robust cooling sensations. Synthetic cooling agent, WS-3, was detected in tobacco rods of several of these "non-menthol" cigarette brands. Crushable capsules added to certain "non-menthol" crush varieties contained neither WS-3 nor menthol but included several "sweet" flavorant chemicals, including vanillin, ethyl vanillin and anethole. CONCLUSION: Tobacco companies have replaced menthol with the synthetic cooling agent, WS-3, in California-marketed "non-menthol" cigarettes. WS-3 creates a cooling sensation similar to menthol, but lacks menthol's characteristic "minty" odor. The measured WS-3 content is sufficient to elicit cooling sensations in smokers, similar to menthol, that facilitate smoking initiation and act as a reinforcing cue. Regulators need to act quickly to prevent the tobacco industry from bypassing menthol bans by substituting menthol with synthetic cooling agents, and thereby thwarting smoking cessation efforts.

Synthetic Cooling Agent in Oral Nicotine Pouch Products Marketed as "Flavor-Ban Approved".

Background: US sales of oral nicotine pouches (ONPs) have rapidly increased, with cool/mint-flavored ONPs the most popular. Restrictions on sales of flavored tobacco products have either been implemented or proposed by several US states and localities. Zyn, the most popular ONP brand, is marketing Zyn-"Chill" and Zyn-"Smooth" as "Flavor-Ban Approved", probably to evade flavor bans. At present it is unclear whether these ONPs are indeed free of flavor additives that can impart pleasant sensations such as cooling. Methods: Sensory cooling and irritant activities of "Flavor-Ban Approved" ONPs, Zyn-"Chill" and "Smooth", along with "minty" varieties (Cool Mint, Peppermint, Spearmint, Menthol), were analyzed by Ca2+ microfluorimetry in HEK293 cells expressing the cold/menthol (TRPM8) or menthol/irritant receptor (TRPA1). Flavor chemical content of these ONPs was analyzed by GC/MS. Results: Zyn-"Chill" ONP extracts robustly activated TRPM8, with much higher efficacy (39-53%) than the mint-flavored ONPs. In contrast, mint-flavored ONP extracts elicited stronger TRPA1 irritant receptor responses than Zyn-"Chill" extracts. Chemical analysis demonstrated the presence of WS-3, an odorless synthetic cooling agent, in Zyn-"Chill" and several other mint-flavored Zyn-ONPs. Conclusions: Synthetic cooling agents such as WS-3 found in 'Flavor-Ban Approved' Zyn-"Chill" can provide a robust cooling sensation with reduced sensory irritancy, thereby increasing product appeal and use. The label "Flavor-Ban Approved" is misleading and may implicate health benefits. Regulators need to develop effective strategies for the control of odorless sensory additives used by the industry to bypass flavor bans.

The Concise Guide to PHARMACOLOGY 2023/24: Ion channels.

The Concise Guide to PHARMACOLOGY 2023/24 is the sixth in this series of biennial publications. The Concise Guide provides concise overviews, mostly in tabular format, of the key properties of approximately 1800 drug targets, and over 6000 interactions with about 3900 ligands. There is an emphasis on selective pharmacology (where available), plus links to the open access knowledgebase source of drug targets and their ligands (https://www.guidetopharmacology.org/), which provides more detailed views of target and ligand properties. Although the Concise Guide constitutes almost 500 pages, the material presented is substantially reduced compared to information and links presented on the website. It provides a permanent, citable, point-in-time record that will survive database updates. The full contents of this section can be found at http://onlinelibrary.wiley.com/doi/10.1111/bph.16178. Ion channels are one of the six major pharmacological targets into which the Guide is divided, with the others being: G protein-coupled receptors, nuclear hormone receptors, catalytic receptors, enzymes and transporters. These are presented with nomenclature guidance and summary information on the best available pharmacological tools, alongside key references and suggestions for further reading. The landscape format of the Concise Guide is designed to facilitate comparison of related targets from material contemporary to mid-2023, and supersedes data presented in the 2021/22, 2019/20, 2017/18, 2015/16 and 2013/14 Concise Guides and previous Guides to Receptors and Channels. It is produced in close conjunction with the Nomenclature and Standards Committee of the International Union of Basic and Clinical Pharmacology (NC-IUPHAR), therefore, providing official IUPHAR classification and nomenclature for human drug targets, where appropriate.

Menthol disrupts nicotine's psychostimulant properties in an age and sex-dependent manner in C57BL/6J mice.

Menthol is a commonly used flavorant in tobacco and e-cigarettes, and could contribute to nicotine sensitivity. To understand how menthol could contribute to nicotine intake and addiction, it is important to determine whether specific mechanisms related to sex and age could underlie behavioral changes induced by menthol-laced nicotinic products. Using a validated paradigm of nicotine-dependent locomotor stimulation, adolescent and adult C57BL/6J mice of both sexes were exposed to nicotine, or nicotine laced with menthol, as their sole source of fluid, and psychostimulant effects were evaluated by recording home cage locomotor activity for ten days. Nicotine and cotinine blood levels were measured following exposure. Results show an interaction between treatment, age, and sex on liquid consumption, indicating that mice responded differently to menthol and nicotine based on their age and sex. Adult male mice greatly increased their nicotine intake when given menthol. In female mice of both age groups, menthol did not have this effect. Despite an increase in nicotine intake promoted by menthol, adult male mice showed a significant decrease in locomotion, suggesting that menthol blunted nicotine-induced psychostimulation. This behavioral response to menthol was not detected in adolescent mice of either sex. These data confirm that menthol is more than a flavorant, and can influence both nicotine intake and its psychostimulant effects. These results suggest that age- and sex-dependent mechanisms could underlie menthol's influence on nicotine intake and that studies including adolescent and adult menthol smokers of both sexes are warranted.