Part Two: Pharmacokinetic Evaluation of E-Liquid Flavors of Vuse Solo Electronic Nicotine Delivery System, An Unblinded, Parallel, Randomized Study To Assess Nicotine Uptake In Smokers

Kyung Soo Hong (  hongk@rjrt.com ) RAI Services Company, 401 N. Main Street, Winston-Salem Patricia DeLuca RAI Services Company, 401 N. Main Street, Winston-Salem Tao Jin RAI Services Company, 401 N. Main Street, Winston-Salem Bobbette A. Jones Retired employee of RAI Services company Paul Nelson Retired employee of RAI Services company Eckhardt Schmidt Reynolds American (United States) Elaine Round RAI Services Company, 401 N. Main Street, Winston-Salem


Introduction
Cigarette smoking is a leading cause of preventable premature death, and signi cantly increases the risk of developing lung cancer, heart disease, chronic bronchitis, chronic obstructive pulmonary disease and other serious diseases and adverse health conditions 1 . Whereas smoking conventional cigarettes requires combustion of tobacco, use of electronic nicotine delivery systems (ENDS) does not. ENDS were developed as potential reduced-harm alternative products for cigarette smokers. ENDS heat a nicotinecontaining solution (e-liquid), which results in the generation of an aerosol containing fewer and lower levels of toxicants than are found in cigarette smoke 2,3 , and reduced toxicant exposure to consumers who switch from cigarettes to ENDS [4][5][6][7][8][9] . Several public health authorities, such as Public Health England 10 , Royal College of Physicians 11 , and National Academies of Sciences, Engineering, and Medicine 12 , have recognized the potential public health bene t of current smokers switching to ENDS. A review by the National Academies of Sciences, Engineering and Medicine (NAS) concluded that "[t]he evidences about harm reduction suggests that across a range of studies and outcomes, e-cigarettes pose less risk to an individual than combustible cigarettes" 12 .
This publication is the second part of a three-part series describing the clinical assessment of Vuse Solo.
The clinical studies included in this publication series describe the nicotine pharmacokinetics (PK) of Vuse Solo across four e-liquid avors; an assessment of the abuse liability of Vuse Solo as compared to CC and a nicotine replacement therapy product 13 ; and a study to assess whether use of Vuse Solo results in a reduction in exposure to harmful and potentially harmful constituents (HPHCs) after smokers are switched to the product for ve days 14 .
This report presents ndings from a clinical study that assessed nicotine PK parameters of Vuse Solo during and following short-term, ad libitum use of one of four avor variants of Vuse Solo products by smokers and dual smokers/ENDS users. The e-liquid avors included Original (tobacco avor), Mint, Tropical and Fusion. The study was executed following the parallel-group study design in which subjects were randomized to a single product, and nicotine PK was evaluated during and after a 10 min ad libitum product use following a week of ambulatory product acclimation (at-home).

Study Population
A total of 148 subjects were enrolled and randomized; All subjects were randomized to one of four Vuse Solo investigational products (IPs) and 122 (82.4%) subjects completed all scheduled PK assessments. A total of 26 subjects (17.6%) were randomized but terminated early from the study for reasons unrelated to study products. Of these 122 study completers, four subjects were excluded from the nal PK analysis due to C max less than 1.0 ng/mL, indicating the IP was used, but subjects did not use it appropriately during the product use period. Thus, 118 (96.7% of study completers) subjects completed the study with evaluable PK data. The number of subjects who completed the study (and the associate percentage of completers with evaluable PK data) in each IP group are as follows: Original (n=30, 93.3%), Mint (n=34, 100%), Tropical (n=29, 96.5%), and Fusion (n=29, 96.5%).
The demographic and baseline characteristics of subjects are summarized in Supplementary Table S1.
Subjects were generally male (55.4%), white (56.8%), and predominantly non-Hispanic (74.3%). The mean age was 33.7 years. Subjects enrolled in the study included 135 smokers (91.2%) and 13 dual smokers and ENDS users (8.7%). Subjects reported a mean smoking duration of 18 years with a mean of 16 cigarettes per day.
Product use ENDS product use, determined by volume of e-liquid used, was assessed over two IP use periods as a surrogate for product use and to evaluate how much e-liquid was used. The use periods included oneweek ambulatory (at-home) trial period and four 10-minute use periods per PK assessment. The differences between initial and nal cartridge weights were calculated for both periods and results are shown in Table 1.

Pharmacokinetic results
As illustrated in Figure 1, baseline-adjusted plasma nicotine concentrations increased rapidly within 15 minutes of use for each of the ENDS IPs evaluated. Mean plasma nicotine concentrations declined to less than 6 ng/mL for all IPs by 60 minutes. Baseline-adjusted nicotine PK parameters are summarized in Table 2. Nicotine uptake during the rst 15 minutes following the start of product use (AUC nic 0−15 ), C max , AUC nic0−60 and the time to reach the maximum nicotine concentration (T max ) across the 60-minute sampling period were similar for all ENDS e-liquid avor variants (Table 2).  *T max reported as median value, followed by the range for minimum and maximum.
Subjects with C max <1.0 ng/mL were excluded from this analysis.
Abbreviations: CI = con dence interval, C max = maximum nicotine concentration, AUC nic0−15 = area under the curve for nicotine exposure over 15 minutes, AUC nic0−60 = area under the curve for nicotine exposure over 60 minutes, T max = time to maximum nicotine concentration.
While this study was not designed to statistically compare avors, two of the nicotine PK parameters, C max and AUC nic0−60 , were qualitatively assessed across the ENDS avor variants to evaluate nicotine uptake across the e-liquid avors. Notched box plots of baseline-adjusted C max and AUC nic0−60 are shown in Figures 2 and 3, respectively, to visually compare the distributions and point estimates in C max or AUC nic0−60 across the avors. Overall, the nicotine uptake parameters C max and AUC nic0−60 were similar for all tested Vuse Solo avors. No evidence of statistically signi cant difference in median C max or AUC nic0−60 was found among the four avor variants, as the notches in Figures 2 or 3 overlap with each other. This suggests that avors do not appear to impact nicotine uptake in our study population.

Product Liking Results:
Overall product liking (OPL) was assessed at the 13-minute timepoint during test sessions using an 11point numeric rating scale (NRS) and results are presented in Table 3. Mean OPL scores (SD) ranged from 5.3 (2.5) for the Mint avor to 7.5 (1.9) for the Tropical avor, and were 6.4 (2.5) and 6.0 (2.5) for the Original and Fusion avors, respectively. Median values re ect a similar trend with the Mint avor with the lowest score of 5.5 and the Tropical with highest score of 8.0, and both the Original and Fusion avors with same median score of 7.0. Table 3 Summary of overall product liking scores Adverse events Nine (6%) of 148 subjects experienced nine adverse events during the study. Gastroesophageal re ux (one subject) and oropharyngeal pain (one subject) were each considered by the principal investigator (PI) to be possibly related and related to IP, respectively. All other adverse events were judged by the PI not to be related to an IP. All adverse events were of mild intensity except for moderate ocular hyperemia in one subject, which led to subject withdrawal from the study by the PI. No serious adverse events were reported.

Discussion
We evaluated PK parameters of four avor variants of e-liquids used in Vuse Solo ENDS with 4.8% (57 mg/mL) nicotine content by weight following an acute exposure in predominately ENDS naïve smokers. Our data showed that subjects achieved similar overall nicotine exposure (AUC nic0−60 ), maximum plasma nicotine concentrations (C max ), and time to maximum concentrations (T max ) while using one of the four avor variants of Vuse Solo ENDS. Evaluation of PK parameters, C max and AUC nic0−60 , showed similar nicotine uptake distribution patterns across all avors variants with overlap of the 95% con dence intervals (CIs) around the medians in both C max and AUC nic0−60 (illustrated as the notches in boxplots, Figures 2 and 3). In addition to the PK assessments, product liking (OPL) for each avor was assessed at the 13-minute timepoint ( Table 3).
As stated above, the results and distribution of baseline-adjusted C max were similar across avor variants.
By comparison, baseline-adjusted maximum nicotine concentrations (C max ) of Vuse Solo avor variants in this study are higher ( The former studies utilized a crossover design (Williams design) and were conducted in an ambulatory setting. In contrast, our study utilized a parallel design in con nement. Both Stiles AL studies had approximately 7 days of at-home product acclimation as our study with instruction to use ENDS IP at least once but product use compliance was not assessed. In our study, subjects were told to use the ENDS IPs as often as they liked, and product use compliance was con rmed by weighing used cartridges at the end of the at-home trial period. Thus, differences in study design as well as level of familiarity with study products and general differences between study populations may account for variability among data reported. In addition to PK assessments, subjects were also asked to rate OPL for the Vuse Solo avor variants on an 11-point NRS. Subjects rated the Tropical avor highest and rated the Mint avor lowest with Original (tobacco) and Fusion avors in-between ( Review of our data on e-liquid consumption measured by cartridge weight before and after PK test sessions in the context of OPL scores suggest some trends but failed to show a direct linear correlation. St. Helen and colleagues examined the impact of avors (Strawberry vs. tobacco) on nicotine uptake and topography. The authors found no statistically signi cant differences in PK parameters and in pu ng behavior and noted the need for further investigation 26,27 . Of note, these differences were seen in subjects who used fruity/sweet avored e-liquids in their own ENDS products, suggesting potential subject bias towards avors that resembled their usual avors.
Similarly, in a topography study to assess the effect of avor in regular smokers, Voos et al., concluded that the avors used in their study delivered differential amounts of nicotine, potentially associated with product use topography, and differences in subjective effects are not solely a product of nicotine delivery, and recommended additional research 28 . In contrast, Cobb et. al., assessed subjective effects among young adult smokers using ENDS with three avors (cream, tropical fruit and tobacco/menthol) at nicotine concentrations ranging from 0 to 36 mg/ml nicotine and concluded that e-liquid avors did not appear to have signi cant impact on subjective effects 29 . Thus, the implications of overall product liking scores on both PK parameters and e-liquid consumption may require additional studies to further evaluate any potential relationship.
In our study, subjects were asked to familiarize themselves with ENDS products at home for one week prior to a PK test session, and the amount of e-liquid consumed was measured by differences in cartridges weights obtained before and after a week of at home use. We found that our subjects used approximately 10% of e-liquid by weight ranging from 0.16 (+/-0.17) mg to 0.21 (+/-0.25) mg across the four avors (Table 1). These results were similar to e-liquid consumption observed on the rst day of exclusive ENDS use in a biomarker of exposure study by Round and colleagues 8 and suggests that our ENDS naïve subjects were su ciently acclimated to ENDS IPs during their at-home trial period prior to the PK test sessions.
Our study had several strengths. First, we included a large sample size to reduce variability compared to previous ENDS nicotine PK studies 23,30−34 . In addition, our subjects were required to acclimate to ENDS for seven days to become familiar with ENDS products prior to con nement and PK test sessions. Subjects were also required to abstain from tobacco products for 12 hours prior to PK test sessions to ensure their blood nicotine concentrations were close to baseline prior to the start of product use.
Furthermore, we collected blood samples at intervals that allowed characterization of nicotine PK following ad libitum ENDS exposure and the nicotine concentrations of blood samples were baselineadjusted to ensure more accurate results.
Despite these strengths, the study had several limitations. Although we allowed participation of dual users, more than 98% of our subjects were exclusive cigarette smokers. As the prevalence of dual and poly use of multiple types of tobacco and nicotine products, such as ENDS or other non-combustible nicotine products, continues to increase, inclusion of a greater proportion of dual users of CC and ENDS in future studies may be useful to make study ndings more applicable to current nicotine and tobacco users. Future studies may also bene t from cross-over designs to evaluate nicotine PK with multiple avor variants to reduce inter-user variability. Lastly, for this study, subjects were allowed a 10-minute ad libitum use of the ENDS 15,16,24,28,33,34 . We chose the duration of 10-minutes of ENDS use to align with an estimated duration to smoke a single CC. However, this study design element may be modi ed in the future studies to align more closely with historical data on the time it takes to smoke a single CC, as well as current data trends around ENDS topography. Moreover, other considerations might include current reports on the time for subjects to smoke a single CC 18,35,36 or recent study designs which leverage a shorter ENDS use period 17,22,29,31,32,37−42 .
In conclusion, the primary endpoints of this study, C max and AUC nic0−60 , were similar across all four avors as evidenced by the overlap of 95% con dence intervals, suggesting that avors are not signi cant factors in nicotine exposure in an acute exposure setting. The results of this study will add to the growing body of literature regarding the effects of avors on nicotine delivery and uptake.

Methods
This study was a single-center study (ClinicalTrials.gov identi er: NCT03234010, 31/07/ 2017) designed to evaluate plasma nicotine uptake and overall product liking with use of four avor variants of the Vuse Solo ENDS (Vuse Solo Original, Mint, Tropical and Fusion) in tobacco consumers who were exclusive smokers or dual users of cigarettes and ENDS. The study was completed at a single clinical research site (DaVita Clinical Research, Lakewood, CO) and was reviewed and approved by Chesapeake Institutional Review Board (Columbia, MD).
The target number of subjects to be randomized was 35 subjects per IP group (140 subjects in total) to allow for approximately a 15% attrition rate (5 subjects per IP group) with a goal of completing 30 subjects per IP group. Attempts were made to recruit 15-20% African-Americans within each IP group in an effort to balance the study sample for the reported percentage of U.S. smokers who are African American 1 . During a screening visit, eligibility criteria were assessed to ensure that potential subjects met all criteria for inclusion and none of the exclusion criteria. Eligible subjects included male and female smokers, aged 21-60 years who self-reported smoking 10 or more cigarettes per day for at least the previous 6 months, or dual users who self-reported smoking 10 or more cigarettes per day for at least the previous 6 months, and using a nicotine-containing cig-a-like or tank-style ENDS at least weekly for at least the previous 3 months. Brief periods of abstinence longer than 30 days before screening were allowed at the discretion of the of the PI. Smoking history was con rmed at screening and reassessed on Study Day 1 with an expired carbon monoxide (ECO) assessment, and only subjects with ECO levels greater than 10 ppm were eligible to participate in the study. Informed consent was obtained from all subjects before any study procedures were performed.
The study was conducted in accordance with the ethical standards in the Declaration of Helsinki, applicable sections of the United States Code of Federal Regulations, and ICH E6 Good Clinical Practice guidelines.

Study Design
The study was designed as a randomized, open-label, parallel-cohort study to assess nicotine uptake in human subjects following ENDS use. The study examined plasma nicotine PK parameters in Vuse Solo ENDS with four avor variants: Original, Mint, Tropical and Fusion.
Each Vuse Solo cartridge consisted of a sealed unit containing 0.5 mL of e-liquid comprising 4.8% nicotine by weight [58 mg/mL], propylene glycol, glycerin, avorings, and water. The products were powered by a rechargeable power unit and included a heating element, microchips, and a sensor. Aerosol generation is activated by detection of a pressure differential within the ENDS product during pu ng.
Subjects were given a 1-week at-home trial period to become familiar with the assigned Vuse IPs. Subjects were randomized to speci c ENDS avors and were provided two cartridges with instructions for use. Product use compliance was checked by weighing ENDS cartridges three times before and after use to a sensitivity of 1 x 10 −4 grams before and after the at-home trial period.
On Study Day 1, subjects arrived at the study site and began their check-in procedures prior to con nement. Subjects returned ENDS IP e-liquid cartridges from the at-home trial period to the site. Eligibility criteria were recon rmed, and those who successfully completed all check-in procedures were con ned to the study site for approximately 24 hours. Subjects were allowed to use their assigned IP ad libitum until the start of a mandatory 12-hour abstinence from tobacco and nicotine products.
On the morning of Study Day 2, each subject was given their assigned ENDS IP for use during the PK assessment. Subjects were then allowed to use the assigned ENDS IP ad libitum for 10 minutes (± 10 seconds). Start and stop times were documented. ENDS IP cartridges were weighed three times before and after use to a sensitivity of 1 x 10 −4 grams.
During the PK assessment, blood samples were collected and processed to plasma for nicotine measurements at the following time points relative to the start of IP use: -5, -0.5, 3,5,8,10,11,12,15,20,30, and 60 minutes. The -0.5 min sample was used as the preferred baseline sample. All samples were collected, processed, and transferred to Celerion Global Bioanalytical Services (Lincoln, NE) for nicotine quantitation using a validated liquid chromatography tandem mass spectrometry method.
In addition to blood sample collection, subjects were required to provide an OPL score at 13 minutes after start of IP use on a numeric scale of 0 to 10, with 0 corresponding to "strongly dislike," 5 corresponding to "neither like nor dislike", and 10 corresponding to "strongly like." Each subject was assessed for adverse events and vital signs, and a symptom-driven physical examination was performed, if necessary, prior to discharge from the study to ensure subject's safety. Upon the PI's review of subjects' status, if there were no new adverse events or physical examination ndings to warrant further follow up, subjects were discharged from the study.

Statistical Analysis
The primary PK parameters (C max , AUC nic0−60 ) were summarized using descriptive statistics by IP group without inter-group comparisons. Standard summary statistics for quantitative and qualitative variables were calculated.
The pharmacokinetic (PK) analyses were performed by Nuventra Pharma Sciences, Inc. located in Durham, NC. PK parameters were derived from the baseline-adjusted plasma nicotine concentrations-time data by noncompartmental methods using Phoenix® WinNonlin® (Version 6.3; Certara USA Inc., Princeton, NJ).
AUCs were calculated using the linear trapezoidal rule. C max and T max were obtained directly from the baseline-adjusted plasma nicotine concentration-time data. Observed concentrations below the lower limit of quanti cation (LLOQ; 0.200 ng/mL) were set at half the LLOQ for data summarization and analysis. Baseline adjustment was done by estimating and subtracting pre-existing nicotine levels from observed levels, and all PK parameters were calculated from the adjusted concentrations. The amount of pre-existing nicotine was estimated by using a model that assumed that nicotine elimination followed rst-order kinetics 43,44 and a nicotine half-life of 120 minutes 23,35,45 . Each individual PK pro le was examined for completeness and suitability for inclusion in the analysis. Subjects with C max values less than 1.0 ng/mL were considered to be not inhaling aerosol during the 10-minute period of ad libitum IP use; therefore, sensitivity analyses excluding those subjects were performed. VuseSoloPKManuscriptSupplementaryTables.docx