More than Smoke and Patches: The Quest for Pharmacotherapies to Treat Tobacco Use Disorder.

Tobacco use is a persistent public health issue. It kills up to half its users and is the cause of nearly 90% of all lung cancers. The main psychoactive component of tobacco is nicotine, primarily responsible for its abuse-related effects. Accordingly, most pharmacotherapies for smoking cessation target nicotinic acetylcholine receptors (nAChRs), nicotine's major site of action in the brain. The goal of the current review is twofold: first, to provide a brief overview of the most commonly used behavioral procedures for evaluating smoking cessation pharmacotherapies and an introduction to pharmacokinetic and pharmacodynamic properties of nicotine important for consideration in the development of new pharmacotherapies; and second, to discuss current and potential future pharmacological interventions aimed at decreasing tobacco use. Attention will focus on the potential for allosteric modulators of nAChRs to offer an improvement over currently approved pharmacotherapies. Additionally, given increasing public concern for the potential health consequences of using electronic nicotine delivery systems, which allow users to inhale aerosolized solutions as an alternative to smoking tobacco, an effort will be made throughout this review to address the implications of this relatively new form of nicotine delivery, specifically as it relates to smoking cessation. SIGNIFICANCE STATEMENT: Despite decades of research that have vastly improved our understanding of nicotine and its effects on the body, only a handful of pharmacotherapies have been successfully developed for use in smoking cessation. Thus, investigation of alternative pharmacological strategies for treating tobacco use disorder remains active; allosteric modulators of nicotinic acetylcholine receptors represent one class of compounds currently under development for this purpose.

An Open-Label, Randomized, Controlled, Crossover Study to Assess Nicotine Pharmacokinetics and Subjective Effects of the JUUL System with Three Nicotine Concentrations Relative to Combustible Cigarettes in Adult Smokers.

INTRODUCTION: This randomized, open-label, crossover clinical study evaluated nicotine pharmacokinetics (PK) and subjective effects of the JUUL System (JS; Juul Labs, Inc.) with three nicotine concentrations compared to the usual brand (UB) cigarettes in 24 adult smokers. METHODS: At five study visits, subjects used either the JS in 59 mg/mL, JS 18 mg/mL (two visits), and JS 9 mg/mL (all tobacco-flavored) or smoked their UB cigarette first during a controlled puffing sequence (CPS) and then ad libitum (5 min) use sessions. Blood samples were taken at specified timepoints for 60 min in each session. The modified Product Evaluation Scale assessed subjective effects 30-min post-use in the CPS session. RESULTS: Maximum plasma nicotine concentration (Cmax-BL), total nicotine exposure (AUC0-60-BL), and rate of plasma nicotine rise were significantly lower for all JS products compared to subjects' UB cigarette in CPS and ad libitum use sessions. In both use sessions these PK parameters were significantly higher for JS 59 mg/mL compared to 18 and 9 mg/mL. Subjective measures of cigarette craving relief and "Enough Nicotine" for JS 59 mg/mL did not differ significantly from UB cigarettes, but JS 18 and 9 mg/mL were rated significantly lower than JS 59 mg/mL and UB cigarettes. CONCLUSIONS: Nicotine exposure and subjective relief were directly related to JS nicotine concentration: higher nicotine concentrations gave rise to significantly greater plasma nicotine levels and relief from craving. Heavier and more dependent smokers may require the greater nicotine delivery of JS 59 mg/mL to successfully transition away from cigarettes. IMPLICATIONS: It has been suggested that electronic nicotine delivery systems (ENDS) and other alternative nicotine delivery products that more closely mimic the nicotine pharmacokinetics (PK) of cigarettes may facilitate smokers transitioning away from cigarettes. We examined nicotine PK and subjective effects of JUUL System (JS) ENDS with three nicotine concentrations (59, 18 and 9 mg/mL) compared to combustible cigarettes. Nicotine delivery from JS ENDS was nicotine concentration dependent, with higher nicotine concentrations giving rise to higher nicotine exposure. These findings suggest that heavier and more dependent smokers may require ENDS with nicotine concentrations greater than 20 mg/mL to successfully transition away from cigarettes.

Mechanisms of Herb-Drug Interactions Involving Cinnamon and CYP2A6: Focus on Time-Dependent Inhibition by Cinnamaldehyde and 2-Methoxycinnamaldehyde.

Information is scarce regarding pharmacokinetic-based herb-drug interactions (HDI) with trans-cinnamaldehyde (CA) and 2-methoxycinnamaldehyde (MCA), components of cinnamon. Given the presence of cinnamon in food and herbal treatments for various diseases, HDIs involving the CYP2A6 substrates nicotine and letrozole with MCA (KS = 1.58 µM; Hill slope = 1.16) and CA were investigated. The time-dependent inhibition (TDI) by MCA and CA of CYP2A6-mediated nicotine metabolism is a complex process involving multiple mechanisms. Molecular dynamic simulations showed that CYP2A6's active site accommodates two dynamic ligands. The preferred binding orientations for MCA and CA were consistent with the observed metabolism: epoxidation, O-demethylation, and aromatic hydroxylation of MCA and cinnamic acid formation from CA. The percent remaining activity plots for TDI by MCA and CA were curved, and they were analyzed with a numerical method using models of varying complexity. The best-fit models support multiple inactivator binding, inhibitor depletion, and partial inactivation. Deconvoluted mass spectra indicated that MCA and CA modified CYP2A6 apoprotein with mass additions of 156.79 (142.54-171.04) and 132.67 (123.37-141.98), respectively, and it was unaffected by glutathione. Heme degradation was observed in the presence of MCA (48.5% ± 13.4% loss; detected by liquid chromatography-tandem mass spectrometry). In the absence of clinical data, HDI predictions were made for nicotine and letrozole using inhibition parameters from the best-fit TDI models and parameters scaled from rats. Predicted area under the concentration-time curve fold changes were 4.29 (CA-nicotine), 4.92 (CA-letrozole), 4.35 (MCA-nicotine), and 5.00 (MCA-letrozole). These findings suggest that extensive exposure to cinnamon (corresponding to ≈ 275 mg CA) would lead to noteworthy interactions. SIGNIFICANCE STATEMENT: Human exposure to cinnamon is common because of its presence in food and cinnamon-based herbal treatments. Little is known about the risk for cinnamaldehyde and methoxycinnamaldehyde, two components of cinnamon, to interact with drugs that are eliminated by CYP2A6-mediated metabolism. The interactions with CYP2A6 are complex, involving multiple-ligand binding, time-dependent inhibition of nicotine metabolism, heme degradation, and apoprotein modification. An herb-drug interaction prediction suggests that extensive exposure to cinnamon would lead to noteworthy interactions with nicotine.

Use of a physiologically-based pharmacokinetic model to explore the potential disparity in nicotine disposition between adult and adolescent nonhuman primates.

The widespread use and high abuse liability of tobacco products has received considerable public health attention, in particular for youth, who are vulnerable to nicotine addiction. In this study, adult and adolescent squirrel monkeys were used to evaluate age-related metabolism and pharmacokinetics of nicotine after intravenous administration. A physiologically-based pharmacokinetic (PBPK) model was created to characterize the pharmacokinetic behaviors of nicotine and its metabolites, cotinine, trans-3'-hydroxycotinine (3'-OH cotinine), and trans-3'-hydroxycotinine glucuronide (3'-OH cotinine glucuronide) for both adult and adolescent squirrel monkeys. The PBPK nicotine model was first calibrated for adult squirrel monkeys utilizing in vitro nicotine metabolic data, plasma concentration-time profiles and cumulative urinary excretion data for nicotine and metabolites. Further model refinement was conducted when the calibrated adult model was scaled to the adolescents, because adolescents appeared to clear nicotine and cotinine more rapidly relative to adults. More specifically, the resultant model parameters representing systemic clearance of nicotine and cotinine for adolescent monkeys were approximately two- to three-fold of the adult values on a per body weight basis. The nonhuman primate PBPK model in general captured experimental observations that were used for both model calibration and evaluation, with acceptable performance metrics for precision and bias. The model also identified differences in nicotine pharmacokinetics between adolescent and adult nonhuman primates which might also be present in humans.

Electrospun α-Lactalbumin Nanofibers for Site-Specific and Fast-Onset Delivery of Nicotine in the Oral Cavity: An In Vitro, Ex Vivo, and Tissue Spatial Distribution Study.

Nicotine replacement therapy (NRT) formulations for oromucosal administration induce a delayed rise in nicotine blood levels as opposed to the immediate nicotine increase obtained from cigarette smoking, this being a shortcoming of the therapy. Here, we demonstrate that α-lactalbumin/polyethylene oxide (ALA/PEO) electrospun nanofibers constitute an efficient oromucosal delivery system for fast-onset nicotine delivery of high relevance for acute dosing NRT applications. In vitro, nicotine-loaded nanofibers showed fast disintegration in water, with a weight loss up to 40% within minutes, and a faster nicotine release (26.1 ± 4.6% after 1 min of incubation) of the loaded nicotine compared to two relevant marketed NRT formulations with a comparable nicotine dose (i.e., 7.9 ± 5.1 and 2.2 ± 0.3% nicotine was released from a lozenge and a sublingual tablet, respectively). Model-fitting of the release data indicated that the release mechanism of nicotine from the hydrophilic nanofibers was possibly governed by more than one type of release phenomena. Remarkably, ex vivo studies using porcine buccal mucosa demonstrated a more efficient permeation of the nicotine released from the nanofibers [flux of 1.06 ± 0.22 nmol/(cm2·min)] compared to when dosing even a ten-fold concentrated nicotine solution [flux of 0.17 ± 0.14 nmol/(cm2·min)]. Moreover, matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MS) imaging of ex vivo porcine buccal mucosa exposed to nicotine-loaded nanofibers clearly revealed higher amounts of nicotine throughout the epithelium, as well as in the lamina propria and submucosa of the tissue. Our findings suggest that nicotine-loaded ALA/PEO nanofibers have potential as a mucosal, fast-releasing, and biocompatible delivery system for nicotine, which can overcome the limitations of the currently marketed NRTs.

Pharmacokinetic Comparison of a Novel Non-tobacco-Based Nicotine Pouch (ZYN) With Conventional, Tobacco-Based Swedish Snus and American Moist Snuff.

INTRODUCTION: The single-dose pharmacokinetics (PK) of a novel, non-tobacco-based nicotine pouch, ZYN, 3 and 6 mg, were compared with 8 mg General snus (part 1) and ZYN 8 mg was compared with 18 mg Longhorn moist snuff (part 2). The present study demonstrates the characteristics of three strengths of a novel tobacco-free oral snus, ZYN, viz. the extraction of nicotine from the oral cavity and its uptake into the systemic blood circulation. Comparison is made to Swedish General snus and American Longhorn moist snuff and from literature 4 mg Nicorette gum and mean of 13 brands of e-cigarettes. AIMS AND METHODS: A single-dose randomized crossover design was used. In vivo extraction and PK parameters were determined. RESULTS: Part 1. The AUCinf of ZYN 3 mg was 27% smaller than that of 8 mg General and the AUCinf of ZYN 6 mg was 34% larger than that of 8 mg General. Less nicotine was extracted from ZYN 3 mg (1.5 mg) and more from ZYN 6 mg (3.5 mg) than from 8 mg General (2.4 mg). The extracted fractions of nicotine for both ZYN products (56% and 59%) were significantly larger than for 8 mg General (32%). RESULTS: Part 2. Close to identical plasma nicotine curves, AUCinf and Cmax were found for ZYN 8 mg and Longhorn Natural 18 mg moist snuff. The extracted amount of nicotine from ZYN 8 mg (3.8 mg) was larger than the amount extracted from Longhorn Natural 18 mg (3.0 mg), but smaller than the extracted amount of nicotine from General 2 × 8 mg snus pouches (5.0 mg). The extracted fraction of nicotine for ZYN 8 mg (50%) was larger than for Longhorn Natural (19%) and General 2 × 8 mg snus pouches (33%). CONCLUSIONS: The two higher doses of ZYN (6 and 8 mg) deliver nicotine as quickly and to a similar extent as existing smokeless products, with no significant adverse effects.

Fourth generation e-cigarette vaping induces transient lung inflammation and gas exchange disturbances: results from two randomized clinical trials.

When heated by an electronic cigarette, propylene glycol and glycerol produce a nicotine-carrying-aerosol. This hygroscopic/hyperosmolar aerosol can deposit deep within the lung. Whether these deposits trigger local inflammation and disturb pulmonary gas exchanges is not known. The aim of this study was to assess the acute effects of high-wattage electronic cigarette vaping with or without nicotine on lung inflammation biomarkers, transcutaneous gas tensions, and pulmonary function tests in young and healthy tobacco smokers. Acute effects of vaping without nicotine on arterial blood gas tensions were also assessed in heavy smokers suspected of coronary artery disease. Using a single-blind within-subjects study design, 25 young tobacco smokers underwent three experimental sessions in random order: sham-vaping and vaping with and without nicotine at 60 W. Twenty heavy smokers were also exposed to sham-vaping (n = 10) or vaping without nicotine (n = 10) in an open-label, randomized parallel study. In the young tobacco smokers, compared with sham-vaping: 1) serum club cell protein-16 increased after vaping without nicotine (mean ± SE, -0.5 ± 0.2 vs. +1.1 ± 0.3 µg/l, P = 0.013) and vaping with nicotine (+1.2 ± 0.3 µg/l, P = 0.009); 2) transcutaneous oxygen tension decreased for 60 min after vaping without nicotine (nadir, -0.3 ± 1 vs. -15.3 ± 2.3 mmHg, P < 0.001) and for 80-min after vaping with nicotine (nadir, -19.6 ± 2.8 mmHg, P < 0.001). Compared with sham vaping, vaping without nicotine decreased arterial oxygen tension for 5 min in heavy-smoking patients (+5.4 ± 3.3 vs. -5.4 ± 1.9 mmHg, P = 0.012). Acute vaping of propylene glycol/glycerol aerosol at high wattage with or without nicotine induces airway epithelial injury and sustained decrement in transcutaneous oxygen tension in young tobacco smokers. Intense vaping conditions also transiently impair arterial oxygen tension in heavy smokers.

Understanding the Fate of Environmental Chemicals Inside Living Organisms: NMR-Based 13C Isotopic Suppression Selects Only the Molecule of Interest within 13C-Enriched Organisms.

In vivo nuclear magnetic resonance (NMR) is rapidly evolving as a critical tool as it offers real-time metabolic information, which is crucial for delineating complex toxic response pathways in living systems. Organisms such as Daphnia magna (water fleas) and Hyalella azteca (freshwater shrimps) are commonly 13C-enriched to increase the signal in NMR experiments. A key goal of in vivo NMR is to monitor how molecules (nutrients, contaminants, or drugs) are metabolized. Conventionally, these studies would normally involve using a 13C-enriched probe molecule and feeding this to an organism at natural abundance, in turn allowing the fate of the probe molecule to be selectively analyzed. The drawback of such an approach is that there is a limited range of 13C-enriched probe molecules, and if available, they are extremely cost prohibitive. Uniquely, when utilizing 13C organisms, a reverse strategy of isotopic filtering becomes possible. The concept described here uses 1H detection in combination with a 13C filter on living organisms. The purpose is to suppress all 1H signals from the organism (i.e., 1H attached to 13C), leaving only the probe molecule (1H attached to 12C). Because the probe molecule can be selectively observed using this approach, it then makes it possible to follow and discern processes such as bioconversion, bioaccumulation, and excretion in vivo. As the approach uses 1H detection, it provides excellent detection limits in the nanogram range. In this article, the approach is introduced, optimized on standards, and then applied to follow nicotine biotransformation and lipid assimilation in vivo to demonstrate the concept.

Nicotine Pharmacokinetics in Rat Brain and Blood by Simultaneous Microdialysis, Stable-Isotope Labeling, and UHPLC-HRMS: Determination of Nicotine Metabolites.

The disposition and metabolism of nicotine in the brain is an important determinant of its exposure. We have developed a novel method for the dynamic determination of nicotine and its metabolites in rat brain and blood by simultaneous microdialysis sampling, stable-isotope labeling, and ultra high-performance liquid chromatography-high-resolution mass spectrometry (UHPLC-HRMS) assaying. Microdialysis probes were inserted into both the right striatum and jugular vein of Sprague-Dawley rats. The collections of dialystes after nicotine intraperitoneal injection were analyzed by the optimized UHPLC-HRMS. Nicotine-pyridyl- d4 was used as a metabolic tracer, and several stably labeled isotopes were applied to calibrate the in vivo recoveries of retrodialysis. The quadrupole-Orbitrap HRMS provided reliable characterization of the nicotine derivatives with less than 3.5 ppm mass measurement accuracy. Good precision and accuracy were obtained for different analytes within the predefined limits of acceptability and the range of the standard curve. Nicotine and its 11 metabolites were identified in most microdialysis samples from the blood and brain tissue samples. Besides cotinine as the main metabolic product of nicotine, trans-3'-hydroxy-cotinine, nicotine- N-oxide, and norcotinine were proven to be the second most abundant metabolites. The other seven nicotine products, including 4-oxo-4-(3-pyridyl)-butanoic acid, 4-hydroxy-4-(3-pyridyl)-butanoic acid, cotinine- N-oxide, nicotine- N-glucuronide, cotinine- N-glucuronide, and trans-3'- hydroxy-cotinine- O-glucuronide, which have not been determined previously in animal brain, were present in minor amounts. The pharmacokinetic profile of nicotine metabolites indicated that the metabolic characteristic of nicotine in the brain was relatively different from that in the blood. The present work would provide comprehensive evidence for clarifying the differences between nicotine metabolism in the brain and peripheral system.

Mitochondria as a possible target for nicotine action.

Mitochondria are multifunctional and dynamic organelles deeply integrated into cellular physiology and metabolism. Disturbances in mitochondrial function are involved in several disorders such as neurodegeneration, cardiovascular diseases, metabolic diseases, and also in the aging process. Nicotine is a natural alkaloid present in the tobacco plant which has been well studied as a constituent of cigarette smoke. It has also been reported to influence mitochondrial function both in vitro and in vivo. This review presents a comprehensive overview of the present knowledge of nicotine action on mitochondrial function. Observed effects of nicotine exposure on the mitochondrial respiratory chain, oxidative stress, calcium homeostasis, mitochondrial dynamics, biogenesis, and mitophagy are discussed, considering the context of the experimental design. The potential action of nicotine on cellular adaptation and cell survival is also examined through its interaction with mitochondria. Although a large number of studies have demonstrated the impact of nicotine on various mitochondrial activities, elucidating its mechanism of action requires further investigation.

Evaluating metronidazole as a novel, safe CYP2A6 phenotyping probe in healthy adults.

AIMS: CYP2A6 is a genetically polymorphic enzyme resulting in differential substrate metabolism and health behaviours. Current phenotyping probes for CYP2A6 exhibit limitations related to procurement (deuterated cotinine), toxicity (coumarin), specificity (caffeine) and age-appropriate administration (nicotine, NIC). In vitro, CYP2A6 selectively forms 2-hydroxymetronidazole (2HM) from metronidazole (MTZ). The purpose of this study was to evaluate MTZ as a CYP2A6 phenotyping probe drug in healthy adults against the well-established method of measuring trans-3-hydroxycotinine (3HC)/cotinine (COT). METHODS: A randomized, cross-over, pharmacokinetic study was completed in 16 healthy, nonsmoking adults. Separated by a washout period of at least 2 weeks, MTZ 500 mg and NIC gum 2 mg were administered and plasma was sampled over 48 hours and 8 hours, respectively. Correlations of plasma metabolite/parent ratios (2HM/MTZ; 3HC/COT) were assessed by Pearson coefficient. CYP2A6 genotyping was conducted and incorporated as a variable of plasma ratio response. RESULTS: Correlations between the plasma ratio 2HM/MTZ and 3HC/COT were ≥ 0.9 at multiple time points (P < 0.001), demonstrating a wide window during which 2HM/MTZ can be queried post-MTZ dose. CYP2A6 genotype had significant impacts on both MTZ and NIC phenotyping ratios with decreased activity predicted phenotypes demonstrating 2HM/MTZ ratios ≤58% and 3HC/COT ratios ≤56% compared with extensive activity predicted phenotypes at all time points examined in the study (P < 0.05). No adverse events were reported in the MTZ arm while 38% (n = 6) of participants reported mild adverse events in the NIC arm. CONCLUSIONS: Metronidazole via 2HM/MTZ performed well as a novel, safe phenotyping probe for CYP2A6 in healthy adults.

Respirable aerosol exposures of nicotine dry powder formulations to in vitro, ex vivo, and in vivo pre-clinical models demonstrate consistency of pharmacokinetic profiles.

Background: Nicotine, because of its volatility, has a complex dosimetry following inhalation as a vapor/aerosol mix. To better control the dosimetry, nicotine could be formulated with a suitable dry powder excipient for use in a clinical inhaler. Aim and Methods: The aim of this study was to investigate the pharmacokinetic PK profile of two dry powder formulations containing 2.5% or 5% nicotine using three experimental models associated to the PreciseInhale™ aerosolization system: the in vitro DissolvIt dissolution system; the ex vivo isolated, ventilated, and perfused lung (IPL) of the rat; and the in vivo intratracheally intubated rat. Results and Discussion: Following exposure, both nicotine formulations had very rapid and similar dissolution and absorption kinetics in both the DissolvIt and IPL exposure models, with an initial half-time of absorption to the single-pass perfusate of 34 and 72 seconds, respectively. In the intratracheally intubated rat, following a rapid initial equilibration between the lungs and systemic compartments, nicotine had a systemic elimination half-time of 2.3-2.4 hours for both formulations. The rapid pulmonary PK of nicotine was likely close to the theoretical equilibration of a low-binding substance with a tissue-blood partition coefficient close to 1. Conclusions: The data generated with the three experimental models provided a comprehensive picture of the inhalation PK of the two nicotine formulations. In particular, the results showed a very rapid dissolution and absorption of the two nicotine formulations and these results could be highly useful for improving the design and calibration of physiologically based PK models to produce more robust predictions. Abbreviations: AED: animal equivalent dose; BW: body weight; HPLC: high-performance liquid chromatography; IPL: isolated, ventilated, and perfused lung; PK: pharmacokinetics; SEM: scanning electron microscopy; USP: United States Pharmacopeia.

Assessment of priority tobacco additives per the requirements of the EU Tobacco Products Directive (2014/40/EU): Part 3, Smoking behavior and plasma nicotine pharmacokinetics.

This publication is part of a series of 3 publications and describes the clinical assessment performed to fulfill the regulatory requirement per Art. 6 (2) of the EU Tobacco Products Directive 2014/40/EU under which Member States require manufacturers and importers of cigarettes and Roll Your Own tobacco containing an additive that is included in the priority list established by Commission Implementing Decision (EU) 2016/787 to carry out comprehensive studies (European Union, 2016). In our clinical study, two distinct end points were investigated, namely measuring plasma nicotine pharmacokinetics as a measure of nicotine uptake, and analyses of changes in smoker puffing behavior as a measure of cigarette smoke inhalation. This clinical study indicated that the inclusion of none of the priority additives either as single additive or as part of a chemical mixture, facilitated nicotine uptake. Furthermore, the data did not suggest that differences in the inhalation pattern of cigarette smoke of any of the Priority Additives tested occurred when compared to the additive-free reference cigarette. Finally, it is concluded that neither the scientific literature nor our study gave circumstantial indications of increased addictiveness for cigarettes containing these priority additives.

e-Cigarettes for smoking cessation: Do they deliver?

WHAT IS KNOWN AND OBJECTIVE: Electronic nicotine delivery systems (ENDS) are battery-powered devices that allow nicotine and/or other substances to be inhaled in aerosolized form. e-Cigarettes (electronic cigarettes), the most commonly used ENDS, have been proposed to be smoking cessation aids. However, despite the rapid surge in their popularity, little is known about long-term health consequences of e-cigarette usage. We assess published data to see if they deliver what they promise. COMMENT: e-Cigarettes may contain uncertain quantities of various ingredients, and evidence of adulteration has been identified. Flavouring agents can alter the pharmacokinetics of nicotine and have uncertain impact on the nature of e-cigarette use (eg ab initio use vs smoking cessation). WHAT IS NEW AND CONCLUSION: Although e-cigarettes have been proposed to be a safe approach to encouraging smoking cessation, there are inconsistencies in available data. And further data are needed regarding long-term implications of primary and secondary exposure to e-cigarette products.

Simultaneous determination of plasma nicotine and cotinine by UHPLC-MS/MS in C57BL/6 mice and its application in a pharmacokinetic study.

Plasma concentrations of nicotine and its active metabolite cotinine are highly correlated with its biological effects. A UHPLC-MS/MS method was developed, validated and applied for nicotine and cotinine analysis in mice plasma. Chromatographic separation was achieved on a BEH HILIC column using acetonitrile (0.1% formic acid) and 10 mm ammonium formate as mobile phase. The gradient elution was performed at 0.4 mL/min with a run time of 3.6 min. The quantitative ion transition was m/z 163.1 > 130.0 for nicotine, m/z 177.1 > 80.0 for cotinine and m/z 167.1 > 134.0 for nicotine-D4 (internal standard, IS). For both nicotine and cotinine, the calibration range was 5-500 ng/mL with 5 ng/mL as the lower limit of quantitation, and the intra- and inter-day bias and imprecision were -4.61-12.00% and <11.12%. The IS normalized recovery was 90.62-98.95% for nicotine and 89.18-101.53% for cotinine, and the IS normalized matrix factor was 106.00-116.44% for nicotine and 100.34-109.85% for cotinine. Both nicotine and cotinine were stable under conventional storage conditions. The validated method has been applied to a pharmacokinetic study in mice to calculate the pharmacokinetic parameters for both analytes.

Strain-specific altered nicotine metabolism in 3,3'-diindolylmethane (DIM) exposed mice.

Two indole compounds, indole-3-carbinol (I3C) and its acid condensation product, 3,3'-diindolymethane (DIM), have been shown to suppress the expression of flavin-containing monooxygenases (FMO) and to induce some hepatic cytochrome P450s (CYPs) in rats. In liver microsomes prepared from rats fed I3C or DIM, FMO-mediated nicotine N-oxygenation was decreased, whereas CYP-mediated nicotine metabolism to nicotine iminium and subsequently to cotinine was unchanged. Therefore, it was hypothesized that in mice DIM would also suppress nicotine N-oxygenation without affecting CYP-mediated nicotine metabolism. Liver microsomes were produced from male and female C57BL/6 J and CD1 mice fed 2500 parts per million (ppm) DIM for 14 days. In liver microsomes from DIM-fed mice, FMO-mediated nicotine N-oxygenation did not differ from the controls, but CYP-mediated nicotine metabolism was significantly increased, with results varying by sex and strain. To confirm the effects of DIM in vivo, control and DIM-fed CD1 male mice were injected subcutaneously with nicotine, and the plasma concentrations of nicotine, cotinine and nicotine-N-oxide were measured over 30 minutes. The DIM-fed mice showed greater cotinine concentrations compared with the controls 10 minutes following injection. It is concluded that the effects of DIM on nicotine metabolism in vitro and in vivo differ between mice and rats and between mouse strains, and that DIM is an effective inducer of CYP-mediated nicotine metabolism in commonly studied mouse strains.

Low concentration of morphine protects against cell death, oxidative stress and calcium accumulation by nicotine in PC12 cells.

OBJECTIVE: Nicotine causes cell death in many cell lines. Morphine at low concentrations has protective effects against cell death. We investigated the effects of low concentration of morphine on nicotine-induced cell death in PC12 cells. MATERIALS AND METHODS: PC12 are cells that grow in DMEM culture medium. Cell viability was detected by MTT test and cells cytotoxicity was measured by LDH test. The activity of caspase-3 was diagnosed by the caspase activity colorimetric assay kit, and detection of mitochondrial membrane potential was confirmed by rhodamine 123 and TUNEL test was performed for DNA fragmentation detection. The fura-2 AM and also rhod 2-AM was used for measurement of intracellular calcium (Ca2+) ic and mitochondrial calcium (Ca2+) m and finally, measurement of antioxidant enzyme activities was assessed. RESULTS: The low concentration of morphine increased cell viability and suppressed cell cytotoxicity, cell death and the formation of mitochondrial membrane potential compared to nicotine treated cells.  It also reduced the intracellular calcium (Ca2+) ic and mitochondrial calcium (Ca2+)m concentration, respectively. CONCLUSION: Morphine as a pain reducer drug, in low concentrations, can protect PC12 cells from nicotine-induced cell death (Fig. 7, Ref. 59).

Evaluation of (-)-[18 F]Flubatine-specific binding: Implications for reference region approaches.

We aimed to characterize changes in binding of (-)-[18 F]Flubatine to α4 ß2 *-nicotinic acetylcholine receptors (α4 ß2 *-nAChRs) during a tobacco cigarette smoking challenge. Displacement of (-)-[18 F]Flubatine throughout the brain was quantified as change in (-)-[18 F]Flubatine distribution volume (VT ), with particular emphasis on regions with low VT . Three tobacco smokers were imaged with positron emission tomography (PET) during a 210 min bolus-plus-constant infusion of (-)-[18 F]Flubatine. A tobacco cigarette was smoked in the PET scanner ∼125 min after the start of (-)-[18 F]Flubatine injection. Equilibrium analysis was used to estimate VT at baseline (90-120 min) and after cigarette challenge (180-210 min), at the time of greatest receptor occupancy by nicotine. Smoking reduced VT by 21 ± 9% (average ±SD) in corpus callosum, 17 ± 9% in frontal cortex, 36 ± 11% in cerebellum, and 22 ± 10% in putamen. The finding of displaceable (-)-[18 F]Flubatine binding throughout the brain is an important consideration for reference region-based quantification approaches with this tracer. We observed displacement of (-)-[18 F]Flubatine binding to α4 ß2 *-nicotinic acetylcholine receptors in corpus callosum by a tobacco cigarette challenge. We conclude that reference region approaches utilizing corpus callosum should first perform careful characterization of displaceable (-)-[18 F]Flubatine binding and nondisplaceable kinetics in this putative reference region.

Effect of Permeation Enhancers on the Buccal Permeability of Nicotine: Ex vivo Transport Studies Complemented by MALDI MS Imaging.

PURPOSE: The purpose of this study was to assess the effect of several chemical permeation enhancers on the buccal permeability of nicotine and to image the spatial distribution of nicotine in buccal mucosa with and without buccal permeation enhancers. METHODS: The impact of sodium taurodeoxycholate (STDC), sodium dodecyl sulphate (SDS), dimethyl sulfoxide (DMSO) and Azone® on the permeability of [3H]-nicotine and [14C]-mannitol (a paracellular marker) across porcine buccal mucosa was studied ex vivo in modified Ussing chambers. The distribution of nicotine, mannitol and permeation enhancers was imaged using using matrix-assisted laser desorption ionization mass spectrometry imaging (MALDI MSI). RESULTS: Despite STDC significantly increasing permeability of [14C]-mannitol, no enhancing effect was seen on [3H]-nicotine permeability with any of the permeation enhancers. Rather, SDS and DMSO retarded nicotine permeability, likely due to nicotine being retained in the donor compartment. The permeability results were complemented by the spatial distribution of nicotine and mannitol determined with MALDI MSI. CONCLUSIONS: The buccal permeability of nicotine was affected in an enhancer specific manner, suggesting that nicotine primarily diffuses via the transcellular pathway. MALDI MSI was shown to complement ex vivo permeability studies and to be a useful qualitative tool for visualizing drug and penetration enhancer distribution in buccal mucosa.

Impact of Nicotine Transport across the Blood-Brain Barrier: Carrier-Mediated Transport of Nicotine and Interaction with Central Nervous System Drugs.

Nicotine, an addictive substance, is absorbed from the lungs following inhalation of tobacco smoke, and distributed to various tissues such as liver, brain, and retina. Recent in vivo and in vitro studies suggest the involvement of a carrier-mediated transport process in nicotine transport in the lung, liver, and inner blood-retinal barrier. In addition, in vivo studies of influx and efflux transport of nicotine across the blood-brain barrier (BBB) revealed that blood-to-brain influx transport of nicotine is more dominant than brain-to-blood efflux transport of nicotine. Uptake studies in TR-BBB13 cells, which are an in vitro model cell line of the BBB, suggest the involvement of H+/organic cation antiporter, which is distinct from typical organic cation transporters, in nicotine transport at the BBB. Moreover, inhibition studies in TR-BBB13 cells showed that nicotine uptake was significantly reduced by central nervous system (CNS) drugs, such as antidepressants, anti-Alzheimer's disease drugs, and anti-Parkinson's disease drugs, suggesting that the nicotine transport system can recognize these molecules. The cumulative evidence would be helpful to improve our understanding of smoking-CNS drug interaction for providing appropriate medication.