Study Design
This was a randomised, controlled, crossover clinical study carried out at a single clinical site in Uppsala, Sweden. The study was registered in the ISRCTN registry (ISRCTN17828518). Approval was given by an independent ethics committee (Central Swedish Ethics Committee, Uppsala, Sweden; reference number 2019-06294) prior to study commencement. The study was conducted in compliance with the study protocol, the Declaration of Helsinki, the International Council for Harmonisation (ICH) Guideline E6 for Good Clinical Practice (GCP), the Food and Drug Administration (FDA) GCP Code of Federal Regulations Title 21 (part 56) and the European regulation EU 536/2014, including regulations relating to the protection of subjects’ personal data. Written informed consent was obtained from all subjects prior to participation in the study and before undergoing any study procedures, including screening assessments.
Participants
Among potential recruits attending a screening visit, 35 healthy male or female subjects who met the inclusion criteria were enrolled in the study. Participants were aged 19–55 years with no clinically relevant abnormal findings on physical examination, vital signs assessment, electrocardiogram, clinical laboratory evaluations, oral mucosa examination or in their medical history. All participants were current daily snus users who had regularly used snus products (pouch weight ≥ 0.8 g, containing ≥ 8 mg nicotine) under their upper lip for at least 6 months. They were also current smokers of ≥ 5 cigarettes per week who had been smoking for at least a year prior to the study. Women of childbearing age were required to use two accepted forms of contraception for approximately 1 month before and after the study.
The main exclusion criteria were pregnancy or breastfeeding (women); self-reported non-inhalation of cigarettes; presence of dental work (including missing molars) that might affect the conduct of the study; presence or history of significant oral and/or pharyngeal inflammation, oral lesions and/or gum disease or temporomandibular joint dysfunction; history of significant hypersensitivity to any excipients of the study product formulations or severe hypersensitivity to any drugs; and the donation of ≥ 400 mL of blood within 90 days, plasma within 7 days, or platelets within 6 weeks before the study. In addition, individuals who reported the postponement of a decision to quit using tobacco- or nicotine-containing products in order to participate in the study or who had attempted to quit using tobacco- or nicotine-containing products within the 28 days before the study were also excluded.
All subjects were informed that they were free to quit smoking and withdraw from the study or to withdraw their consent to participate at any time. All subjects were able to ask for advice to stop using tobacco/nicotine products and were to be provided with a smoking cessation helpline number and/or web address for appropriate smoking cessation services if required.
Investigational Products
The following products were examined in this study: (1) a combustible cigarette (Pall Mall Red, BAT; 0.66 mg ISO nicotine yield); (2) Lyft mint NP (BAT; 10 mg nicotine/pouch); (3) Zyn (Wet) spearmint NP (Swedish Match; 10 mg nicotine/pouch); (4) Nordic Spirit mint NP (Japan Tobacco International; 9 mg nicotine/pouch); (5) Skruf Super White Fresh Stark mint NP (Imperial Brands; 8 mg nicotine/pouch); and (6) On! mint NP (Altria; 6 mg nicotine/pouch). The five NPs were commercially available or a commercial specification product (Lyft) at the time of the study.
Study Procedures
At screening, subjects underwent testing to ensure that they met all inclusion and no exclusion criteria, and they completed a smoking/smokeless product use history questionnaire. They were admitted to the clinic site in the afternoon of the day before the first study product use session (Day −1) and were confined to the site until the last assessments at the end of the study (i.e. for a period of ~8 days). On admission to the clinic site, subjects underwent an eligibility criteria review, vital signs assessments, a symptom-oriented physical examination if indicated, and an oral mucosa examination. At the end of the study, they underwent clinical laboratory testing, an oral mucosa examination, and a physical examination if indicated. After using at least four of the study products, subjects were given the option to go home for 1 day, returning to the clinic the following day to complete their product use sessions.
On admission on Day –1, subjects were given two of the study NPs to try in order to familiarise themselves with NPs and ensure their tolerance of these study products. A minimum familiarisation period of 30 min per product was stipulated, with a gap of 1–2 h between the use of each product.
Before each study day, subjects were required to abstain from using any nicotine product for a period of at least 12 h before their product use session the following day. On each study day, subjects consumed a light breakfast at least 1 h before product use and ate a standardised lunch after the 6-h blood sample was drawn. Product use sessions were conducted in the morning of each study day.
On each study day, subjects used a single study product in accordance with a randomisation schedule. For the NPs, subjects were instructed to place the pouch between their top lip and their gum for 60 min. At the end of the 60-min period, they removed the pouch from their mouth. For the combustible cigarette, subjects were asked to smoke a single cigarette ad libitum (i.e. they were not instructed how to puff on the cigarette) for a 5-min period. If the cigarette was still lit at the end of the 5-min period, no further puffing was allowed. If the subject reached the end of the cigarette before the 5 min had elapsed, no further cigarettes were allowed to be smoked. The number of puffs taken was counted by clinic staff. Subjects were allowed to use their own brand snus or NPs after completion of the product use and blood sampling session until 12 h prior to the next product use session. During this period, the subjects were only allowed to use their own products, which were dispensed by the clinic staff on request.
Blood Sampling for Nicotine Pharmacokinetics
Venous blood samples were collected from an indwelling cannula inserted into a forearm vein at the start of each study day. Blood samples (4 mL) were taken at –5, 3, 5, 7, 10, 20, 45, 60, 65, 75, 120, 240 and 360 min relative to the start of each product use session. Blood was collected into a K2-EDTA vacutainer tube. After collection, blood samples were mixed by inverting the tube 10 times to ensure anticoagulation. Samples were then held at 4 ± 4 °C until centrifugation at 1900 ± 38 g at 4 ± 4 °C for 10 min. The time between blood collection and placement in the centrifuge did not exceed 60 min. After centrifugation, the blood plasma fraction was separated into two aliquots of approximately 0.7 mL each. After separation, plasma was stored at –20 ± 5 °C at the clinical site until shipment to the bioanalytical laboratory. The time between the end of centrifugation and aliquot storage did not exceed 180 min. Plasma nicotine analysis was performed by Anapharm Bioanalytics, Barcelona, Spain. Nicotine was extracted from each sample (study sample, calibration standard or quality control) using a protein precipitation procedure. Briefly, an internal standard (Nicotine-D4) was added to tubes containing plasma, followed by extraction with acetonitrile. Processed samples were stored at 4 °C until analysis. Samples were analysed using an ACQUITY UPLC® I-Class system (Waters) equipped with an ACUITY UPLC® Binary Solvent Manager, Sample Manager with Flow-Through Needle, Sample Organizer and BEH C18 column (Waters), which was coupled to a QTRAP 6500 mass spectrometer (Sciex).
The mobile phase was an isocratic water/acetonitrile (10/90) mixture with 2.5 mM ammonium acetate at pH 5. The flow rate was 0.50 mL/min and the injection volume was 10 µL. The calibration range was 0.40–39.92 ng/mL, with a lower limit of quantitation (LLOQ) of 0.4 ng/mL.
Subjective Effects Assessments
At the end of the product usage period, subjects completed a product liking questionnaire and an intent-to-use-again questionnaire to evaluate the subjective effects of study product use. Answers were given on a scale of 0 (strongly dislike/not at all) to 10 (strongly like/very much).
Safety Assessments
Adverse events (AEs)—defined as any untoward medical occurrences in the subjects, including a clinically significant abnormal clinical laboratory finding, symptom or disease temporally associated with the use of a study product (whether or not related to the use of the product)—were self-reported by subjects during the study, either spontaneously or when asked through open-ended questioning by study staff. A product use-emergent AE (PEAE) was defined as an AE not present prior to the use of a study product or an AE already present that worsened in intensity or frequency following the use of a study product. All AEs reported following the use of a study product were considered PEAEs. Each PEAE was assigned to the last product used by the subject, with the assignment performed irrespective of any determination of the relationship between the PEAE and product use. A serious adverse event (SAE) was defined as any untoward medical occurrence that results in death, is life-threatening, requires inpatient hospitalisation or prolongation of existing hospitalisation, results in persistent or significant disability or incapacity (defined as a substantial disruption of a person’s ability to conduct normal life functions), is a congenital anomaly or birth defect, or is an important medical event (including development of drug dependence or drug abuse) that may jeopardise the subject or may require intervention to prevent one of the other outcomes listed.
Sample Size and Statistical Methods
Based on two-sided pairwise comparisons, and using an eight-product and eight-sequence Williams design, we calculated that a minimum of 32 subjects (4 subjects per sequence) would be necessary to achieve a balanced design and a power of 0.825 with an α-level of 0.05. This would enable us to detect a difference of 20% in the log scale with an intrasubject coefficient of variation (CV) of 0.3.
Individual raw nicotine concentration data and derived concentrations were calculated using Phoenix® WinNonlin® version 8.1 (Certara, Princeton, NJ, USA). The pharmacokinetic parameters maximum plasma concentration (Cmax), area under the plasma concentration-time curve between 0 and 6 h (AUC0–6h) and time to reach Cmax (Tmax) were calculated using non-compartmental procedures. Reported plasma nicotine concentrations and pharmacokinetic parameters were not corrected for baseline plasma nicotine concentrations; a sensitivity analysis comparing uncorrected and corrected concentrations showed no differences in statistical outcomes. Samples with nicotine concentration values that were below the LLOQ that preceded the first quantifiable concentration were assigned a value of zero for linear plots and for all calculations and summary statistics, but were excluded from semi-logarithmic plots. All other nicotine concentrations below the LLOQ were treated as missing for all analyses. For samples above the upper limit of quantitation (ULOQ), a fourfold dilution factor was applied, and the samples were reanalysed, along with low dilution quality controls.
Descriptive statistics were calculated for nicotine concentrations at each individual time point and for all pharmacokinetic parameters. Individual concentrations, actual sampling times and pharmacokinetic parameters were summarised per study product by the following descriptive statistics: number of observations (N), minimum, arithmetic mean, geometric mean, median, maximum, standard deviation (SD) and CV. The geometric mean nicotine Cmax and AUC0–6h were compared among the study products using a mixed-effects analysis of variance (ANOVA) model with treatments, periods and sequences as fixed effects and subjects nested within sequences as random effects. Product comparisons were conducted by using the fitted model with an adjusted significance level of 0.025. Data were analysed on a natural log (ln)-transformed scale.
Statistical analysis was performed with SAS® version 9.4 for Windows (SAS Institute Inc., Cary, NC, USA). The reported data correspond to the pharmacokinetic subject set, which was a subset of the full analysis set and excluded subjects who had relevant protocol violations (or circumstances) that affected the evaluation of pharmacokinetic parameters. One protocol deviation occurred due to problems inserting the cannula into a study subject on a single study day when using an oral NP. Consequently, all blood samples after 3 min were missing. A further subject had three missing consecutive blood draws due to an issue with early blood sampling during their combustible cigarette smoking period.
Subjective measures data were summarised by product with frequencies (n, %) using the original rating (0–10) and a three-category rating (below neutral, neutral, and above neutral, where an original rating of 5 was regarded as neutral). Post-hoc statistical analysis was performed on the original ratings using ANOVA with post-hoc Tukey adjustments.
The randomisation code was not available to the personnel of the bioanalytical facility until the bioanalytical tables had been finalised and audited by the quality assurance department. The statistician and pharmacokineticist were also blind to the study product used on each study day until the final analyses had been performed.