Plasma Nicotine Pharmacokinetics of Oral Nicotine Pouches Across Varying Flavours and Nicotine Content *

The present randomised, controlled, crossover clinical study was conducted at a single site in Kansas City, KS, USA. The study was registered on the U.S. Clinical-Trials.gov registry (NCT04846088). Approval was given by an Institutional Review Board (IRB; Ethics (WIRB Copernicus Group, Puyallup, WA, USA; study reference number 1305801) before study commencement. The study was conducted in accordance with the U.S. Code of Federal Regulations (CFR) governing Protection of Human Subjects (21 CFR Part 50), Financial Disclosure by Clinical Investigators (21 CFR Part 54), and IRBs (21 CFR Part 56). It was also carried out in accordance with the protocol and under the principles of the International Council for Harmonisation (ICH) of Technical Requirements for Pharmaceuticals for Human Use Guideline for Good Clinical Practice (GCP) E6 (R2). All participants provided written informed consent before enrollment and before undergoing any study procedures, including screening. At any time, they were free to quit smoking, withdraw their consent, or withdraw from the study.

Forty-two healthy male or female participants who met the inclusion criteria were enrolled in the study within 45 days of their screening visit to ensure that a minimum of 36 participants completed the study. An attempt was made to recruit a balance of sexes with no less than 40% of either sex represented and to recruit at least 15–20% black/African American participants to reflect the reported percentage of U.S. smokers in this group. Participants were aged 22–65 years inclusive and generally healthy as determined by clinical laboratory evaluations (including haematology, clinical chemistry, urinalysis, serology, urine drug and urine/breath alcohol screen), medical history, physical examination including oral examination, vital signs assessment and 12 lead electrocardiograms. All participants were current smokers with a minimum 1-year smoking history and who were smoking at least 10 per day of filtered non-menthol or menthol cigarettes between 83 and 100 mm in length as their primary source of tobacco use. Exhaled carbon monoxide and urine cotinine were assessed to confirm cigarette smoking status. Participants also self-reported use of smokeless tobacco products (e.g., moist snuff or snus) at least once or twice in their lifetime before screening. Female participants underwent a serum pregnancy test at screening and a urine pregnancy test at checkin to the clinical site. Women of childbearing potential were required to use an accepted form of contraception for 30 days before and after the study.

The main exclusion criteria were:

pregnancy or breastfeeding (women only);

The study products were nine Velo portioned oral NPs including a 4-mg/pouch of nicotine with one flavour (Velo Wintergreen) and 7-mg/pouch of nicotine with eight flavours (Velo Max Wintergreen, Spearmint, Peppermint, Citrus Burst, Black Cherry, Coffee, Dragon Fruit, and Cinnamon). The contents of each Velo or Velo Max pouch include a powdered cellulosic substrate, pharmaceutical grade tobacco-derived nicotine, pH adjusters, sweeteners, and other ingredients specific to each flavour. The powdered mixture is pouched in a porous wrapping material referred to as “fleece”.

All participants were screened against the inclusion and exclusion criteria and completed a questionnaire on their tobacco product use. Within 45 days of screening, they were admitted to the clinic on Day 1 of the study and were confined to the site for approximately 10 days until the last assessments had been made. On admission, participants’ eligibility was reconfirmed and they underwent vital signs assessments, physical and oral examinations, and a urine pregnancy test (females only). At the end of the study, they underwent clinical laboratory testing, physical and oral examinations, vital signs assessments, and a urine pregnancy test (females only).

On Day 1, all participants took part in a product familiarisation session lasting at least 45 min in which they tried the Velo Max 7-mg nicotine and peppermint flavour study NP. Throughout the study, participants were required to abstain from using any nicotine product for at least 12 h before the product use session the following morning. In each product use session, participants used a single study product as specified by predetermined randomisation sequences. Within 30 min of the start of the session, participants were asked to rinse their mouth with approximately 50 mL water and were then instructed to place a single Velo pouch in their mouth anywhere between their gum and their upper lip and allow the pouch to absorb saliva and moisten. Participants moved the pouch from one side of the mouth to the other approximately every 10 min, and the pouch remained in the mouth for a period of 45 min.

After product use and blood sampling was completed, participants were allowed to smoke their own brand of combustible cigarettes ad libitum until the 12 h abstinence period prior to the next product use session.

Venous blood samples were collected by direct venepuncture or through an indwelling cannula. Blood samples (4 mL) were taken at –5, 2, 5, 10, 15, 30, 45, 60, 90 and 180 min relative to product administration into K₂-EDTA vacutainer tubes. To ensure anticoagulation, the tubes were inverted 10 times and centrifuged within 60 min (1,500 g, 4 °C, 10 min). The plasma was stored in two aliquots at –20 °C. The time from blood sample collection to plasma storage did not exceed 90 min.

Nicotine analysis was done by Altasciences (Laval, Quebec, Canada) using liquid chromatography with tandem mass spectrometry detection as previously described (28). In brief, nicotine was extracted from 0.15 mL plasma by protein precipitation and analysed using a Waters XBridge C18 column on an AB Sciex API 5000 quadrupole mass spectrometer in positive ion mode for the detection of nicotine.

Analyst® software version 1.6.3, was used to acquire and review chromatograms. The internal standard was nicotine-D₄, and nicotine was quantified over a theoretical concentration range of 0.2–100.0 ng/mL. For the analysis only non-smokers prepared the spiking solutions, calibrant and quality control samples.

Furthermore, blank samples were injected before the pre-test to check for the presence of nicotine in the system. In addition to blank and zero standards, all runs had a set of 11 non-zero standards and 4 levels of Quality Control samples prepared with analyte-free human plasma. Incurred Sample Reanalysis evaluation was assessed concurrently to the sample analysis with at least 10% of the first 1000 analysable study samples and 5% of the remaining samples reassayed and compared to their original values. The sample analysis was conducted in accordance with U.S. Food and Drug Administration Guidance for Industry, (29) and European Medicines Agency Guideline on Bioanalytical Method Validation (30).

At the end of the pharmacokinetic session (180 min relative to the start of product use), participants completed a single product-liking questionnaire to evaluate the subjective effect of study product use. Answers were given as a numeric rating score from 0 to 10, with 0 corresponding to “strong disliking”, 5 corresponding to “neither like nor dislike” and 10 corresponding to “strong liking”.

Adverse events (AEs) were defined as any untoward medical occurrence or condition experienced by a participant after signing the informed consent form until completion of the study, irrespective of whether it was considered to be related to the use of study products. An AE could be any unfavourable and unintended sign (e.g., abnormal laboratory finding), symptom or disease, without any judgment about causality.

All AEs, whether volunteered, elicited or noted on the physical examination/oral examination at the end of the study, were recorded throughout the study. The start and stop date and time of all AEs was captured. Participants who presented with unresolved or new AEs at study conclusion or early termination were followed up until the AE had resolved or stabilised. A product-emergent adverse event (PEAE) was defined as an AE that was not present prior to study product use or an AE that was present but worsened in intensity or frequency after study product use.

A serious adverse event (SAE) was defined as any medical occurrence that resulted in death or was life-threatening, required inpatient hospitalisation or prolongation of an existing hospitalisation, resulted in persistent or significant incapacity or substantial disruption of a person’s ability to conduct normal life functions, was a congenital anomaly or birth defect or was a severe medical event that required medical or surgical intervention to prevent one of the above outcomes.

Using data from prior studies, it was estimated that 36 participants would be needed to have at least an 80% chance of obtaining a 95% confidence interval with a half-width of up to 20% of the means for the primary endpoints. The target number of participants to be recruited into this study was 42 participants, which allowed for approximately a 14% dropout rate with a goal of 36 participants completing the study.

Raw nicotine concentrations and derived baseline-adjusted concentrations were determined by compartmental methods using Phoenix® WinNonlin® version 8.0 (Certara, Princeton, NJ, USA). The primary endpoint nicotine pharmacokinetic parameters maximum plasma nicotine concentration (C_max) and area under the nicotine concentration-versus-time curve from time zero to 180 min after the start of study product use (AUC_{nic 0–180}) were adjusted for baseline plasma nicotine concentration under the assumption that nicotine elimination follows first-order kinetics. Negative concentrations resulting from baseline adjustment were forced to zero. The secondary pharmacokinetic endpoint time to maximum plasma nicotine concentration (T_max) was also calculated based on the baseline-adjusted plasma nicotine concentrations. Observed plasma nicotine concentration values below the lower limit of quantification were set at half the lower limit of quantification, and missing data were considered as missing at random and not imputed. Demographic and pharmacokinetic parameters presented here are for the pharmacokinetic population, which included 41 participants who used at least one study product and it had sufficient data to derive at least one pharmacokinetic parameter.

The pharmacokinetic parameters C_max and AUC_{nic 0–180} were summarised using descriptive statistics for each study product. These statistics included the number of nonmissing data points, mean, standard deviation (SD), coefficient of variation, minimum, median, and maximum. Additionally, geometric means, geometric SD, and geometric coefficient of variation calculated for C_max and AUC_{nic 0–180} are reported. For T_max only non-missing data points, minimum, median, and maximum values are reported.

Statistical comparisons of C_max and AUC_{nic 0–180} between study products were performed using paired t-tests on a log-transformed scale. For T_max, comparisons were made using a sign test on the original data scale. For all pharmacokinetic parameter comparisons, Bonferroni adjustment was used during the statistical analyses. With a total of 24 comparisons (8 product comparisons × 3 parameters), a p-value less than 0.002 (0.05/24) would indicate statistically significant difference.

For the product-liking subjective effects measure, the overall product-liking numeric rating score was summarised by product using descriptive statistics (non-missing data points, mean, SD, minimum, quartile 1, quartile 3, and maximum).

A total of 42 participants met eligibility requirements and were enrolled into the study. The majority of randomised participants (95.2%) completed the study according to the protocol. All 42 participants (100%) completed Day 1, 41 participants (97.6%) completed Days 2 and 3, and 40 participants (95.2%) completed Days 4 through 9. Two participants voluntarily withdrew from the study early, for a family emergency on Day 4 (1 participant) and due to the number of blood draws on Day 1 (1 participant).

Basic participant demographic data are summarised in Table 1. The male:female ratio was 68:32; 80.5% of the participants were white and 19.5% were black/African American.

Mean plasma nicotine concentration-time curves for the Velo NPs are shown in Figure 1, while descriptive data and statistical comparisons of the pharmacokinetic parameters are summarised in Table 2 and Table 3, respectively. For each study product, the mean plasma nicotine concentration increased throughout use, reaching a peak at the end of the 45-min product use period. Mean coefficient of variation (CV) maximum nicotine concentration (C_max) with baseline-adjustment was lowest for the Velo Wintergreen 4-mg pouch (11.224 ng/mL (28.7%)) and was similar for the Velo Max 7-mg pouches regardless of flavour, ranging from 16.220 ng/mL (33.3%) for Velo Max Citrus Burst 7-mg to 17.888 ng/mL (26.2%) for Velo Max Cinnamon 7-mg (Table 2).

Figure 1.

Similarly, mean (CV) AUC_{nic 0–180} values were lowest for the Velo Wintergreen 4-mg pouch (1212.535 ng × min/mL (27.4%)) and both highest for all the Velo Max 7-mg pouches and similar between flavours, ranging from 1752.167 ng × min/mL (31.2%) for Velo Max Citrus Burst 7-mg and 1965.491 ng × min/mL (22.1%) for Velo Max Cinnamon 7-mg (Table 2).

Statistical comparison of C_max and AUC_{nic 0–180} showed that both parameters were significantly different between the Velo Wintergreen 4-mg and Velo Max Wintergreen 7-mg NPs, but not between the Velo Max Wintergreen 7-mg NP and any of the other 7-mg study products (Table 3). Median T_max values were identical at 46 min for all Velo NPs assessed, regardless of nicotine content or flavour (Table 2) and were not statistically significantly different between any of the study products (Table 3).

Data for the product-liking subjective effect assessment are presented in Table 4. Broadly speaking, mean scores for product-liking were similar for all Velo NPs assessed, regardless of flavour and nicotine content. Mean (SD) product-liking scores ranged from 5.1 (2.28) for the Velo Max Black Cherry 7-mg pouch to 6.1 (2.88) and 6.1 (2.43) for the Velo Max Coffee and Velo Max Dragon Fruit 7-mg pouches, respectively.

There were no serious or severe product-emergent adverse events (PEAEs) and no participants were withdrawn from the study for safety reasons. A total of 90 PEAEs were experienced by 28 (66.7%) of the 42 participants. Of these PEAEs, seven in total were reported by six participants (14.3%) following the use of Velo Max Peppermint during the product familiarisation session, this being the only product used during this session.

During the test sessions, the incidence of participants with PEAEs ranged from 25.0% for Velo Max Black Cherry 7-mg to 7.3% for Velo Max Wintergreen 7-mg. PEAE occurrences during the test sessions ranged from 15 follow ing use of Velo Max Peppermint 7-mg to 5 following the use of Velo Max Wintergreen 7-mg and Velo Max Spearmint 7-mg. Overall, the PEAE experienced most commonly was hiccups, reported by 5 participants (12.2%) after use of Velo Max Cinnamon 7-mg, 3 participants (7.5%) after use of Velo Max Black Cherry 7-mg, 3 participants (7.5%) after use of Velo Max Peppermint 7-mg, and 1 participant each after use of Velo Max Dragon Fruit 7-mg, Velo Max Wintergreen 7-mg, Velo Wintergreen 4-mg (2.4% each), and Velo Max Coffee 7-mg (2.5%). Of the participants who experienced at least one PEAE, most experienced PEAEs which were mild in severity (52.4%) while the remaining experienced PEAEs were moderate in severity (14.3%). A total of 21 participants (50.0%) experienced 57 product use-related PEAEs (categorised as either related or possibly related).