Materials and methods

This was an open-label, two-armed, randomized, longitudinal study designed to assess the effects on various oral variables in daily tobacco-based snus users who completely substituted their snus with either the dry or moist non-tobacco-based nicotine pouch formulations. The primary endpoint was the change in lesion severity from baseline to day 28. The investigational products (IPs) consisted of unflavored (Smooth) or flavored (Cool Mint and Citrus) NPs with different nicotine strengths (dry 6 mg and moist 9 mg). The difference in nicotine content was compensated for by the higher nicotine extraction from the dry formulation compared with the moist formulation, resulting in an overall comparable nicotine delivery profile. According to previously published chemical analyses [3], the dry nicotine pouch formulation contained fillers, a stabilizer, pH adjusters, nicotine salt, flavorings, and a sweetener. The moist formulation additionally contained water, glycerol, and sodium chloride.

The study was monitored by Clinical Trial Consultants AB (CTC), which systematically and independently reviewed all study-related activities and documentation to verify that they were conducted, and that data was recorded, analyzed, and reported, in accordance with the study protocol and other applicable regulatory requirements.

Risk and benefit assessment

All participants were daily snus users for at least one year, familiar with the effects of nicotine, and at no risk of developing new dependence. Potential adverse effects were expected to be minor based on prior clinical trials [9–12], and individuals belonging to any risk groups, as defined by the exclusion criteria, were excluded. Nicotine exposure did not exceed participants’ usual intake, minimizing acute risks. No serious adverse events (AEs) have been reported in previous studies, aside from typical nicotine-related effects such as salivation, nausea, and dyspepsia.

As in a standard Phase I study, there was no direct benefit for participants, aside from an oral examination. Participant safety and well-being were prioritized.

Ethical conduct of the study

The study and the clinical study protocol were approved by the Swedish Ethical Review Authority (approval number 2022-03919-01).

The study was conducted in accordance with ethical principles that have their origin in the Declaration of Helsinki and in compliance with the International Conference of Harmonization (ICH) of Technical Requirements for Pharmaceuticals for Human Use E6(R2) guideline for Good Clinical Practice guidance, the EU Clinical Trials Directive, and applicable local regulatory requirements. The study was registered in the International Standard Randomized Controlled Trial Number (ISRCTN) registry (ISRCTN13243849).

Recruitment

Participants were recruited from CTC’s database of healthy volunteers and through strategic marketing campaigns. Advertisements on social media and other media channels (e.g., newspapers, internet, radio), and local distribution of flyers were used to reach the target audience. All advertising texts were approved by the independent ethics committee.

Participant information and consent

All participants were given adequate verbal and written information before any study-specific assessments were performed. Participants were given the opportunity to ask questions about the study and sufficient time to consider participation before signing the informed consent form (ICF). A copy of the participant information, including the signed ICF, was provided to each participant.

Method of assigning subjects to product test groups

At Visit 3, participants were randomized to one of two treatment arms: a dry nicotine pouch formulation (ZYN® Dry) or a moist nicotine pouch formulation (ZYN® Moist). Within each arm, participants could select and change flavor as desired. Randomization was computer-generated using SAS Proc Plan (Version 9.4; SAS Institute Inc., Cary, NC, USA), and the randomization list, including subject IDs and treatment allocations, was prepared by CTC.

Blinding

This was an open-label study; therefore, no formal blinding procedures were implemented.

Number of participants

The study included 45 randomized participants, with the aim of obtaining 40 fully evaluable participants. In total, 59 subjects were screened, 45 were randomized, and all 45 completed the study, i.e., they used the IPs ad libitum for 4 weeks and attended all study visits. Of the randomized participants, 16 were female (36%) and 29 were male (64%). Specifically, 23 participants (13 male and 10 female) were randomized to the dry formulation, and 22 participants (16 male and 6 female) were randomized to the moist formulation (Table 1). The study was conducted between 22 November 2022 and 2 May 2023. Participant flow throughout the study is presented in Supplementary Figure S1.

In total, 14 participants were withdrawn prior to randomization at Visit 3. There were four screening failures, three participants withdrew consent for personal reasons, and five participants were designated as reserves. Two participants were lost to follow-up prior to randomization after failing to attend consecutive scheduled visits and could not be reached despite multiple contact attempts.

 | Total (n = 59)
Screened subjects | 59
Withdrawn prior to dose | 14
Reason for withdrawal prior to dose | 
Screening Failure | 4
Withdrawal Of Consent | 3
Reserve | 5
Other | 2
Subjects included in study | 45
Randomized to arm | 
ZYN Dry | 23
ZYN Moist | 22
Withdrawn subjects | 0
Completed subjects | 45
Included in Full analysis set | 45
Subjects at each visit | 
Visit 1 (screening) | 59
Visit 2 | 46
Visit 3 | 45
Visit 4 | 45
Visit 5 | 45

Inclusion criteria

To be included in the study, participants had to meet the following criteria: be healthy males or females aged 21–55 years and be willing and able to provide written informed consent. Participants were required to have used tobacco-based snus for ≥ 1 year, with a minimum weekly consumption of three cans, and to obtain ≥ 90% of their total nicotine intake from tobacco-based snus. They also had to be non-smokers (defined as having smoked no more than five packs in total ever and none during the past year). In addition, participants were required to have normal stimulated salivary secretion rate (> 0.7 mL/min), at least 24 natural teeth, and overall good oral health, as assessed by the investigator.

Female participants of childbearing potential were required to use an adequate contraceptive method for the duration of the study. Sexual abstinence was permitted if it was the participant´s usual and preferred lifestyle.

Exclusion criteria

Participants were excluded if they had a history of diagnosed hypertension or any cardiovascular disease. Any surgical or medical condition, including abnormal salivation (also pharmaceutically induced), or a history of such conditions that, in the investigator´s judgment, could interfere with the absorption, distribution, metabolism, or excretion of the investigated product (IP), pose a risk to the participant, influence the study results, or impair participation also resulted in exclusion. Participants who were pregnant, breastfeeding, or intending to become pregnant during the study were excluded. Additional exclusion criteria included a history of severe allergy/hypersensitivity, or ongoing manifestations thereof, to aroma compounds (including fragrances and/or flavorings), as assessed by the investigator. Participants with severe oral conditions such as open caries lesions, severe periodontal disease, soft-tissue lesions (other than gingival hyperplasia related to snus use), or extensive prosthetic work (e.g., multiple implants, partial dentures, or dental veneers) were also excluded. Further exclusion criteria included current or past alcohol abuse and/or the use of anabolic steroids or drugs of abuse, as judged by the investigator. Use of antibiotics within 4 weeks prior to screening was prohibited. Participants who intended to change their nicotine consumption habit, including plans to stop using nicotine products, within 4 months of the screening visit were also excluded, as judged by the investigator. Finally, participants undergoing other dental treatments during the study period were excluded.

Study design

Participants attended a screening visit followed by four treatment visits on separate days. All tests and screenings were carried out at the research laboratory at the Department of Cariology, Institute of Odontology, University of Gothenburg. The screening visit (Visit 1) took place within 5 weeks prior to the first treatment visit and included an eligibility assessment, including evaluations of smoking and oral tobacco/nicotine use, a brief oral examination, urine pregnancy testing (for women of child-bearing potential only), and collection of medical history. Salivary secretion rate and buffer capacity were also assessed. At the end of the screening, participants were asked about their flavor preferences for the IPs.

During the first treatment visit (Visit 2, Day − 28), participants attended the clinic for a routine dental examination, including assessment of oral mucosal lesions. They electronically recorded the number of tobacco-based snus pouches used per day, the timepoints of the first and last pouch of the day, and the estimated duration of use per pouch over a 4-week period (up to Visit 3, day 0).

At the second treatment visit (Visit 3, day 0), a routine dental examination, including assessment of oral mucosal lesions, was performed, and saliva was collected for cytokine analysis. Visit 3 was selected as the baseline assessment because it represented the final evaluation immediately prior to substitution of tobacco-based snus with nicotine pouches and served as the predefined reference point for all subsequent treatment comparisons. Participants continued using their usual tobacco-based snus products between Visit 2 and Visit 3, and lesion severity appeared similar at both assessments. Prior to the assessment of oral mucosal lesions, clinical photographs were taken. Biopsies were obtained from the upper vestibular mucosa corresponding to the participant’s habitual pouch placement site. At Visit 5, efforts were made to collect the biopsy from a comparable anatomical area based on the clinical appearance and location of the lesion. However, minor variations in biopsy location between visits could not be completely excluded. Participants were then randomized to substitute their tobacco-based snus with either the dry formulation or the moist formulation for 4 weeks. They were provided with the IPs and instructed to use them ad libitum, according to their usual pattern of use. Participants were also instructed to electronically record the number of nicotine pouches used per day, the timepoints of the first and last pouch of the day, and the estimated duration of use per pouch for 4 weeks (until Visit 5, day 28).

At the third treatment visit (Visit 4, day 14), a routine dental examination was performed, and clinical photographs of the oral lesions were taken. Additional IPs were provided if needed, and participants could change flavor upon request.

During the fourth treatment visit (Visit 5, day 28), a routine dental examination (including assessment of oral mucosal lesions) was performed, and saliva was collected for cytokine analysis. A biopsy of the oral mucosa was also obtained from the representative area.

AEs were collected through participant interviews from the start of substitution of tobacco-based snus with nicotine pouches (Visit 3) until the final treatment visit (Visit 5).

General restrictions

Participants were required to fully replace their tobacco-based snus products with nicotine pouches for 4 weeks, starting from the second treatment visit (Visit 3), and to refrain from using any other nicotine products until the end of the study (Visit 5). Participation in other clinical studies or the use of antibiotics were also prohibited during this period. Subjects were instructed to refrain from approximal tooth cleaning for 48 h and toothbrushing for 24 h prior to each treatment visit (Visit 2–5). They were also required to use their regular toothbrush (electrical or manual) and a standard fluoride-containing toothpaste (sodium fluoride; 1450 ppm F) from Visit 2 to Visit 5.

Oral mucosal lesions

Oral mucosal lesions were assessed and graded from 1 to 4 according to Axéll et al. [13]. Clinical photographs were taken at Visits 3 and 5, during which participants were instructed to lift their upper lip with their fingers. The grading of the oral mucosal lesions was categorized as follows: Degree 1 – a superficial lesion matching the color of the surrounding mucosa with slight wrinkling but no visible thickening; Degree 2 – a superficial lesion that is whitish or yellowish with wrinkling but no visible thickening; Degree 3 – a wrinkled lesion ranging from whitish-yellowish to brown, with normal-colored furrows and obvious thickening; and Degree 4 – a heavily wrinkled lesion from white-yellowish to brown with deep reddened furrows and/or significant thickening.

Histopathological evaluation

Punch biopsies (3 mm in diameter) were obtained under local anesthesia, following the routine clinical biopsy procedures at Visits 3 and 5. The procedure was not associated with any major discomfort or significant AEs.

Biopsy samples were prepared by Histocenter AB, Mölndal, Sweden. They were fixed in 4% formaldehyde, processed, mounted on glass slides, and stained with Hematoxylin and Eosin. Epithelial thickness and the presence of total inflammatory cells were analyzed using computer software (Biopix iQ 3.2.0). Epithelial thickness was measured in micrometers (µm) at the thickest and thinnest regions, excluding sample edges and papilla, and the mean thickness was calculated from these measurements. Cells were quantified based on color intensity and morphology, and results were expressed as the percentage of positively stained cells per selected tissue area. Cell counting was performed in a blinded manner by one observer after calibration with another observer.

Measurement of total protein concentration in saliva

Total protein concentration was determined using the Pierce™ Bicinchoninic Acid (BCA) Protein Assay Kit (Thermo Fisher Scientific, Gothenburg, Sweden). Reagents A and B supplied with the kit were used to prepare the working reagent. Bovine serum albumin (BSA) served as the protein standard, and serial dilutions were prepared to generate a standard curve ranging from 25 to 2000 µg/mL.

In brief, the BCA working reagent was prepared by mixing 50 parts of Reagent A with 1 part of Reagent B. For each sample and standard, 25 µL was pipetted into a 96-well microplate, followed by the addition of 200 µL of the working reagent. The plate was incubated at 37 °C for 30 min. Absorbance was measured at 562 nm using a microplate reader, and the protein concentrations were calculated by comparing sample absorbance values to the BSA standard curve.

Measurement of cytokines in saliva

Saliva (1 ml) was collected at Visits 3 and 5. To each sample, 25 µl of protease inhibitor (Sigma, Town, Country) was added, and the samples were stored at -80 °C. Before analysis, the saliva samples were centrifuged for 7 min at 1700 x g, and the supernatants were transferred to new Eppendorf tubes.

Cytokine concentrations in saliva were measured using a Multiplex immunoassay (Bio-Plex Pro Human Cytokine Screening Panel, 48-Plex; Bio-Rad Laboratories, Hemel Hempstead, United Kingdom) according to the manufacturer’s instructions. Briefly, color-coded beads coupled to antibodies specific for each target biomarker were used. Following sample addition, the antibodies on the beads bound to the corresponding biomarkers. Unbound proteins were removed through detergent washes, after which a biotinylated detection antibody was added to form a sandwich complex. A streptavidin-phycoerythrin conjugate was then introduced to bind the biotinylated antibody, completing the detection complex. Samples were analyzed on a Bio-Plex 200 instrument using BioManager analysis software (BioRad, Hercules, CA). Results were reported as PPM.

Statistics

Statistical comparisons of paired samples were performed using the Wilcoxon matched-pairs signed-rank test. A significance level of p < 0.05 was considered statistically significant. GraphPad Prism (GraphPad Software, San Diego, CA, USA) was used for data analysis and figure generation.

No formal a priori power calculation was performed due to limited prior data for reliable effect size estimation. To enhance transparency, effect sizes (r) were calculated for the primary outcome based on the Wilcoxon signed-rank test (r = Z/√N), providing an estimate of the magnitude of the observed effects.