Ethyl glucuronide and ethyl sulfate assays in clinical trials, interpretation, and limitations: results of a dose ranging alcohol challenge study and 2 clinical trials.

BACKGROUND: The ethanol metabolites, ethyl glucuronide (EtG) and ethyl sulfate (EtS), are biomarkers of recent alcohol consumption that provide objective measures of abstinence. Our goals are to better understand the impact of cutoff concentration on test interpretation, the need for measuring both metabolites, and how best to integrate test results with self-reports in clinical trials. METHODS: Subjects (n = 18) were administered, 1 week apart, 3 alcohol doses calibrated to achieve blood concentrations of 20, 80, and 120 mg/dl, respectively. Urinary EtG/EtS was measured at timed intervals during a 24-hour hospitalization and twice daily thereafter. In addition, participants from 2 clinical trials provided samples for EtG/EtS and drinking histories. Cutoffs for EtG/EtS of 100/50, 200/100, and 500/250 ng/ml were evaluated. RESULTS: Twelve hours following each challenge, EtG was always positive at the 100 and 200 cutoffs, but at 24 hours sensitivity was poor at all cutoffs following the low dose, and poor after 48 hours regardless of dose or cutoff. Similarly, in the clinical trials EtG sensitivity was good for detecting any drinking during the last 24 hours at the 2 lowest cutoffs, but under 40% during the last 24 to 48 hours. Sensitivity was reduced at the 500 ng/ml cutoff. Discrepancies between EtG and EtS were few. Comparison of self-reports of abstinence and EtG-confirmed abstinence indicated underreporting of drinking. CONCLUSIONS: Any drinking the night before should be detectable the following morning with EtG cutoffs of 100 or 200 ng/ml. Twenty-four hours after drinking, sensitivity is poor for light drinking, but good for heavier consumption. At 48 hours, sensitivity is low following 6 drinks or less. Increasing the cutoff to 500 ng/ml leads to substantially reduced sensitivity. Monitoring both EtG and EtS should usually be unnecessary. We recommend EtG-confirmed self-reports of abstinence for evaluation of outcomes in clinical trials.

Nicotine flux as a powerful tool for regulating nicotine delivery from e-cigarettes: Protocol of two complimentary randomized crossover clinical trials.

INTRODUCTION: Electronic cigarette (EC) use has increased rapidly in the last decade, especially among youth. Regulating nicotine delivery from ECs could help curb youth uptake and leverage EC use in harm reduction yet is complicated by varying device and liquid variables that affect nicotine delivery. Nicotine flux, the nicotine emission rate, is a parameter that incorporates these variables and focuses on the performance rather than the design of an EC. Nicotine flux therefore could be a powerful regulatory tool if it is shown empirically to predict nicotine delivery and subjective effects related to dependence. METHODS AND ANALYSIS: This project consists of two complementary clinical trials. In Trial I, we will examine the relationship between nicotine flux and the rate and dose of nicotine delivery from ECs, hence, impacting abuse liability. It will also examine the extent to which this relationship is mediated by nicotine form (i.e., freebase versus protonated). At Yale School of Medicine (YSM), study participants will puff EC devices under conditions that differ by flux and form, while arterial blood is sampled in high time resolution. In Trial II, we will assess the relationship between nicotine flux, form, and subjective effects. At the American University of Beirut (AUB), participants will use EC devices with varying nicotine fluxes and forms, while dependency measures, such as the urge to use ECs, nicotine craving, and withdrawal symptoms, will be assessed. We will also monitor puffing intensity and real-time exposure to toxicants. ETHICS AND DISSEMINATION: The protocol of Trial I and Trial II was approved by YSM and AUB IRBs, respectively. We will disseminate study results through peer-reviewed publications and conference presentations. TRIAL REGISTRATION: NCT05706701 for Trial I and NCT05430334 for Trial II.

Acute alcohol consumption alters the peripheral cytokines IL-8 and TNF-α.

BACKGROUND: Acute alcohol consumption triggers release of cytokines, which are immune signaling molecules. Dysregulated cytokine levels are associated with impaired immune function, and peripheral cytokine levels may communicate with the brain to propagate drinking-related behaviors. This exploratory study aims to characterize the peripheral cytokine response to an alcohol challenge in a well-controlled laboratory setting. METHODS: Moderate alcohol drinkers (n = 17), abstinent for >5 days, consumed alcohol calibrated to achieve blood concentrations of 120 mg/dL. Serum cytokine levels (IL-6, IL-8, IL-12, IFN-γ, TNF-α) were measured prior to drinking, 6 h after drinking, and 24 h after drinking. Linear mixed models evaluated within-subject differences in cytokine levels over time. RESULTS: The pro-inflammatory chemokine IL-8 significantly increased 6 h after alcohol [F(1,34) = 4.13, p = 0.0002, d' = 0.5]. In contrast, the pro-inflammatory cytokine TNF-α significantly decreased 6 h after alcohol [F(1,34) = -3.07, p = 0.004, d' = 0.3]. No cytokines were significantly different from baseline 24 h after alcohol. CONCLUSIONS: In our exploratory data, acute alcohol challenge (120 mg/dL) elicits dynamic changes in the pro-inflammatory molecules IL-8 and TNF-α. The findings help inform the temporal profile of cytokine response to alcohol, and identify IL-8 as a cytokine of interest for future studies of periphery-brain immune communication.

Plasma Menthol Glucuronide as a Biomarker of Acute Menthol Inhalation.

OBJECTIVES: Menthol is often added to cigarettes and e-cigarette solutions for its cooling and anti-irritant effects, and may contribute to development of nicotine dependence, particularly in vulnerable populations such as adolescents, and among African Americans. Menthol is rapidly metabolized to menthol glucuronide (MG) with little or no unconjugated menthol measurable in venous blood. Human challenge studies of the effects of inhaled menthol, and of its interactions with nicotine, would benefit from a quantitative measure of acute menthol exposure. Our objective was to determine whether plasma MG concentrations might be a suitable quantitative biomarker of acute menthol exposure following its inhalation. METHODS: We performed a secondary analysis of plasma MG concentrations obtained during a study of the effects of inhaled menthol on behavioral responses to intravenous nicotine. MG concentrations were followed over time in venous plasma from 48 participants following inhalation of aerosols from e-cigarettes employing solutions containing either of 2 menthol concentrations or placebo. RESULTS: Whereas plasma MG concentrations were variable, they showed a dose-dependent increase following menthol inhalation. CONCLUSIONS: Measurement of plasma MG may be useful to assess inter-individual differences in acute menthol exposure in human challenge studies involving menthol inhalation.

The effects of alcohol-containing e-cigarettes on young adult smokers.

BACKGROUND: The liquids (e-liquids) used in an electronic cigarette (e-cigarette) contain myriad chemicals without adequate human inhalation safety data. Furthermore, the absence of e-liquid labeling requirements poses a formidable challenge to understanding how e-liquid constituents may promote nicotine addiction and/or have independent or synergistic biological effects when combined with nicotine. Ethyl alcohol is such a constituent, but has received little scientific interest in this context. METHODS: Using a randomized, double blind, crossover design, acute changes in subjective drug effects, motor performance and biochemical measures of alcohol and nicotine intake were evaluated after directed and ad lib puffing from two commercially available e-liquids containing nicotine (8 mg/ml), vanilla flavor and either 23.5% (high) or 0.4% (trace) alcohol. RESULTS: While no differences in subjective drug effects were observed between alcohol conditions, performance on the Purdue Pegboard Dexterity Test (PPDT) improved under the trace, but not under the 23.5% alcohol condition. Although plasma alcohol levels remained undetectable during testing, urine ethyl glucuronide (EtG), an alcohol metabolite, became measurable in three participants after puffing from the 23.5% alcohol e-cigarette. CONCLUSIONS: Brief use of a widely available type of e-cigarette containing an e-liquid purchased from an internet vendor can negatively impact psychomotor performance and in some instances, produce detectable levels of a urine alcohol metabolite. Given the widespread and unregulated use of e-cigarettes, especially by youth and other vulnerable populations, further studies are needed to evaluate both the acute safety and long-term health risks of using alcohol-containing e-cigarettes.

Rapid nicotine clearance is associated with greater reward and heart rate increases from intravenous nicotine.

The ratio of nicotine metabolites (trans-3'-hydroxycotinine (3HC) to cotinine) correlates with nicotine clearance. In previous studies, high nicotine metabolite ratio (NMR) predicted poor outcomes for smoking cessation treatment with nicotine patch. The underlying mechanisms that associate NMR with treatment outcomes have not been fully elucidated. A total of 100 smokers were divided into quartiles based on their baseline plasma NMR. Following overnight abstinence, smokers received saline followed by escalating intravenous doses of nicotine (0.5 and 1.0 mg/70 kg) given 30 min apart. The effects of nicotine on subjective, plasma cortisol, heart rate, and systolic and diastolic blood pressure measures were obtained. Smokers in the first NMR quartile (slower metabolizers) had lower Fagerstrom Test for Nicotine Dependence (FTND) scores, suggesting lower levels of dependence. In contrast, smokers in the fourth NMR quartile (faster metabolizers) reported greater craving for cigarettes following overnight abstinence from smoking and reported greater ratings of nicotine-induced good drug effects, drug liking, and wanting more drug. Higher NMR was also associated with greater heart rate increases in response to nicotine. These results suggest that enhanced nicotine reward and cigarette craving may contribute to the poor treatment response in smokers with high NMR. These findings warrant further investigation, especially in treatment-seeking smokers undergoing cessation treatment.

Nicotine fails to attenuate ketamine-induced cognitive deficits and negative and positive symptoms in humans: implications for schizophrenia.

BACKGROUND: The uncompetitive N-methyl-D-aspartate receptor (NMDAR) antagonist, ketamine, induces a range of symptoms resembling those seen in schizophrenia. Enhancement of nicotinic acetylcholine receptor (nAChR) function may have potential as a treatment for the cognitive deficits and negative symptoms of schizophrenia. Accordingly, we examined the modulatory effects of brain nAChR systems on NMDAR antagonist-induced effects. METHODS: The interactive effects of ketamine and nicotine were evaluated in 37 healthy subjects in a randomized, placebo-controlled, double-blind, crossover counterbalanced, 2 (intravenous ketamine or placebo) × 2 (intravenous nicotine or placebo) design. Verbal and visual memory, sustained attention, working memory, response inhibition, emotion recognition, executive function, reaction time, motor function, and speed of processing were assessed once per test day, while negative and positive symptoms, perceptual alterations, and a number of feeling states were measured several times before and after administration of drugs. RESULTS: Ketamine induced cognitive deficits and negative and positive symptoms. Nicotine worsened immediate recall, auditory working memory, response inhibition, and executive function and serial processing. Nicotine decreased (improved) reaction time on the sustained attention and choice reaction time tasks. Nicotine did not reduce ketamine-induced cognitive deficits or negative and positive symptoms. CONCLUSIONS: At blood levels comparable with tobacco smoking, nicotine infusion does not appear to alleviate the ketamine-induced transient cognitive and behavioral effects in healthy subjects that resemble those seen in schizophrenia. The lack of an effect of nicotine on a spectrum of ketamine effects suggests that the consequences of NMDAR antagonism are not likely under the direct influence of nAChR.