A Randomized Crossover Clinical Study to Assess the Effect of Oral Nicotine Pouches Used for Different Durations on Plasma Nicotine Pharmacokinetics in Healthy Oral Pouch Consumers

Abstract

Oral nicotine pouches (NPs), although addictive and not risk‐free, have potential application for a tobacco harm reduction approach. This study aims to further characterize their ability to deliver nicotine effectively. This randomized seven‐way crossover PK study in healthy oral pouch consumers evaluated 11 and 20 mg NPs used for 10, 20, and 30 min, and for 30 min with expulsion of saliva. Used pouches were analyzed for extracted nicotine, flavor components, and sweeteners. The C_max and AUC_0–4 h increased with nicotine strength and usage time (18.9–21.6 and 26.7–33.6 ng/mL; 24.6–43.0 and 36.4 to 65.6 h ng/mL for the 11 and 20 mg NPs, respectively). Extracted nicotine was greater for the 11‐mg than for the 20‐mg NP, (10 min, 28.0% vs. 22.4%; 20 min, 35.8% vs. 29.6%; 30 min, 43.7% vs. 36.9%, respectively). Only 1.8% of the nicotine measured in the reference NPs was detected in saliva collected over the 30 min use period. Use of 20‐mg NP for 30 min resulted in the extraction of 30.1% flavor components and 17.3% sweeteners, but only 0.16% and 3.4% of the amount measured in the reference NPs were detected in saliva, respectively. Thus, very little nicotine, flavor components, and sweeteners were swallowed during NP use. This study shows NPs can deliver nicotine effectively to satisfy smokers’ nicotine desire. Nicotine delivery to the consumer increases with the duration of use. Our findings suggest that smokers who switch completely to an NP can obtain their accustomed amount of nicotine during normal product use.

International Standard Registered Clinical Trial number: ISRCTN12265853.

Introduction

Smoking is a cause of lung cancer, other cancers, respiratory disease, and cardiovascular disease, owing to the inhalation of cigarette smoke containing multiple known toxicants and carcinogens. ¹ To reduce the projected public health burden from cigarette smoking, a policy of tobacco harm reduction (THR), in which smokers are encouraged to switch from conventional cigarettes to less harmful alternative products, has been recommended by the US National Academy of Medicine (formerly Institute of Medicine). ² Currently, several public health bodies support a policy of encouraging smokers who will not otherwise quit to switch to potentially reduced‐risk nicotine products such as e‐cigarettes. ³ , ⁴

In this regard, a model continuum of risk for nicotine and tobacco products has been proposed, ⁵ with combusted tobacco products such as cigarettes at the most risky end of the continuum, and nicotine replacement therapies at the least risky end. ⁶ , ⁷ , ⁸ , ⁹ Tobacco and nicotine products with no tobacco combustion, such as Swedish snus, e‐cigarettes, and tobacco heating products, are positioned toward the less risky end of the spectrum. ⁹ , ¹⁰ , ¹¹ In particular, long‐term epidemiological data indicate that Swedish snus (a product comprised of a small pouch of specially processed tobacco placed next to the gum), has considerably lower health risks as compared with conventional cigarettes, ¹² , ¹³ and the U.S. Food and Drug Administration has recently authorized some snus brands to be marketed as a modified risk products with certain claims. ¹⁴

Since the mid‐2010s, a new category of potentially reduced‐risk product, nicotine pouches (NPs), has become available in the United States and some European countries. These oral NPs are similar to portioned Swedish snus, which is a form of smokeless tobacco, in appearance and usage. However, NP do not contain tobacco leaf; they contain pharmaceutical‐grade nicotine along with food‐grade ingredients, including flavor components and sweeteners, which are added to satisfy adult consumer preferences. These NP are also different from American‐style smokeless tobacco, which can be used in a dip format, often placed between the gum and the cheek of the lower lip. These products induce excessive saliva production, and the excess saliva is expectorated. ¹⁵ Unlike smokeless tobacco products, NP have a simpler composition ⁹ and elicit reduced biological responses in vitro ¹⁶ as compared with Swedish snus, and thus have potential application in a THR approach. For example, Shrestha et al. ¹⁷ have suggested that NPs might contribute to THR in countries such as India where use of particularly hazardous types of smokeless tobacco (e.g., gutkha, zarda and paan‐tobacco) is prevalent and the incidence of oral cancer is high. Thus, there are calls for further studies of these relatively new products, including pharmacokinetic (PK) and abuse liability studies for NPs of varying nicotine strengths, ¹⁸ and for more research on what role NPs might play in reducing smoking prevalence. ¹⁹

The aims of the present seven‐way crossover clinical study were: to assess and compare the use of two NPs (11 and 20 mg nicotine) for three different durations (10, 20, and 30 min) in order to evaluate nicotine delivery by these products; to test the hypothesis that nicotine extraction and absorption will increase with both NP nicotine strength and duration of use; and, to explore whether nicotine absorption occurs solely through the buccal mucosa or if a significant proportion is swallowed. For the last study aim, participants used one NP and expelled saliva that was collected and analyzed for nicotine, sweeteners, and selected flavor components to allow an estimation of how much of the content of NPs may be swallowed during use. Our hypothesis was that the NP ingredients present in saliva during use—a surrogate for the amount swallowed—will be small and that the nicotine swallowed during NP use will have no impact on total nicotine absorption.

Methods

Prior to study commencement, ethical approval was obtained from the Swedish Ethical Review Authority (reference number 2022‐00703‐01, approval date February 28, 2022) and the study was performed in accordance with the Declaration of Helsinki ²⁰ and with ICH/GCP, E6 (R2), EU Clinical Trials Directive, and local regulatory requirements. All participants provided written informed consent prior to undergoing any study procedures, including screening. Because the study was conducted while there was still a risk of COVID‐19 infection, specific protocols to control for infection were adhered to throughout. The study was registered on the ISRCTN registry (ISRCTN 12265853).

Study Design

The present single‐use open‐label randomized seven‐way crossover PK study was carried out among healthy adult regular consumers of snus or NPs in Uppsala, Sweden, by CTC Clinical Trial Consultants AB. The study involved two clinic visits and one follow‐up telephone call approximately 1 week after the end of product use (Table S1) and was performed between April and September 2022.

Study Products

Two variants of commercially available NPs were used in the study: Velo Freeze, 11 mg of nicotine (BAT, “Product 1”); and Velo Freeze, 20 mg of nicotine (BAT, “Product 2”). These NPs are designed to be placed between the gum and upper lip for short durations, during which nicotine and flavor components are released and nicotine is absorbed through the oral mucosa. Product 1 was used in three PK sessions for durations of 10 min (Product 1a), 20 min (Product 1b), and 30 min (Product 1c). Product 2 was used in three PK sessions for durations of 10 min (Product 2a), 20 min (Product 2b), and 30 min (Product 2c), as well as for 30 min with saliva expelled (Product 2d) (Table 1). All study products were stored at −20°C and allowed to acclimatize to room temperature (15–25°C) 24–48 h prior to use. They were allocated to participants according to the randomization list and labelled on the outer box with the participant number, product use session, and date.

Table 1.

Summary of the Seven Study Products

Product ID	Nicotine Pouch	Description	Usage Time
1a	Velo Freeze 11 mg	Nicotine oral pouch, 0.7 g; nicotine content 11 mg (15.7 mg/g)	10 min
1b	Velo Freeze 11 mg	Nicotine oral pouch, 0.7 g; nicotine content 11 mg (15.7 mg/g)	20 min
1c	Velo Freeze 11 mg	Nicotine oral pouch, 0.7 g; nicotine content 11 mg (15.7 mg/g)	30 min
2a	Velo Freeze 20 mg	Nicotine oral pouch, 1.3 g; nicotine content 20 mg (15.4 mg/g)	10 min
2b	Velo Freeze 20 mg	Nicotine oral pouch, 1.3 g; nicotine content 20 mg (15.4 mg/g)	20 min
2c	Velo Freeze 20 mg	Nicotine oral pouch, 1.3 g; nicotine content 20 mg (15.4 mg/g)	30 min
2d	Velo Freeze 20 mg	Nicotine oral pouch, 1.3 g; nicotine content 20 mg (15.4 mg/g)	30 min + saliva expelled

Study Participants

Regular adult consumers (age 19–55 years) of snus or NPs were recruited from a database of healthy volunteers held at the clinic and via advertisements placed in appropriate social media, newspapers, and radio. To assess study eligibility, potential participants were invited to attend a screening session where they underwent physical examination (oral examination, vital signs assessment, and 12‐lead electrocardiogram [ECG]) and clinical laboratory evaluations (hematology, clinical chemistry, urine cotinine analysis, and drug/alcohol screen). Information on the history of nicotine oral pouch/snus use and on medical/surgical history was also obtained by interview.

The main inclusion criteria were: (1) daily use (≥5 pouches/day) of snus and/or NPs (≥0.6 g pouch weight) for at least the past 12 months, and willingness to use NPs with a nicotine content of ≥15 mg/g nicotine. (2) Aged 19–55 years and in good general health. (3) Body mass index between 18.5 and 30 kg/m². (4) Clinically normal medical history, physical findings, vital signs, ECG, and laboratory values at screening. (5) Willing to abstain from nicotine and tobacco products for 12 h before each product use session until the end of PK blood sampling. (6) Positive urine cotinine test (≥200 ng/mL) at screening and on Day −1. (7) For women of childbearing potential, willing to use a sufficient contraceptive method for the duration of the study.

The main exclusion criteria were: (1) history of a clinically significant disorder that might put the participant at risk or malignancy within the past 5 years. (2) Clinically significant illness or medical/surgical procedure in the 2 weeks before the study. (3) Resting systolic blood pressure of <90 or >140 mmHG, or diastolic blood pressure of <50 or >90 mmHg, or pulse of <40 or >90 bpm. (4) Prolonged QTcF (>450 ms), cardiac arrhythmia, or clinically significant abnormalities in the resting ECG at screening; or implanted defibrillator or pacemaker (5) Presence or history of significant oral and/or pharyngeal inflammation, oral lesions, gum disease, or temporomandibular joint dysfunction. (6) Pregnancy or lactation (females). (7) Positive screening for drugs of abuse or alcohol. There were no restrictions over concomitant medications or therapies for participants who were on a stable course of medication for the duration of the study. Refer to Table S2 for a full list of the inclusion and exclusion criteria.

Those who met the inclusion criteria and agreed to comply with general restrictions regarding nicotine use and diet (Supplementary Methods in Supporting Information) were enrolled and asked to return to the clinic for the confinement study within 28 days of screening. All participants were free to withdraw from the study at any time and for any reason. They could also be withdrawn from the study in the case of severe adverse events (SAEs) or non‐compliance with the study protocol.

Study Objectives

The primary objectives were: (1) to determine the kinetics of plasma nicotine absorption, including maximum plasma concentration of nicotine (C_max), time to reach C_max (T_max), and total plasma nicotine/area under the curve (AUC_0–4 h), among participants using 11 and 20 mg NPs for three different durations; (2) to compare nicotine delivery between the two products at each usage time specified above: and (3) to compare nicotine delivery among the usage times for each product.

The secondary objectives were (1) to compare the kinetics of nicotine absorption (C_max, T_max, AUC_0–4 h) between Product 2 used normally (Product 2c) and Product 2 used with expulsion of saliva (Product 2d); (2) to evaluate subjective reports of overall product liking (OPL) and intent to use again via a visual analogue scale (VAS; 0–100 mm); (3) to determine the amount of nicotine extracted in vivo (as mg/unit and % fraction) with increasing usage time (Products 1a–1c, 2a–2c); (4) to determine the amount of residual nicotine, flavor components and sweeteners in the pouches after use (Products 2c and 2d only); (5) to estimate the amount of nicotine, flavor components and sweeteners swallowed (Product 2d) and to evaluate the safety and tolerability of each study product by reporting adverse events (AEs) and clinically significant changes in ECG, vital signs and laboratory values.

Study Protocol

The study protocol is outlined in Figure 1. On the first visit (Day −28 to −2), potential participants were screened for eligibility and completed a familiarization session, where they used a 20‐mg NP for 30 min to ensure product acceptability and tolerance. Participants were admitted to the clinic on Day −1 of the study for a confinement period of 8 days. On the morning of Day 1, after eligibility was reconfirmed, they were randomized to one of five sequences (Supplementary Methods in Supporting Information) stating the order of product use; the randomization list contained participant number, randomization sequence, day, and product, and was computer‐generated in Proc Plan, SAS version 9.4 (SAS Institute, Inc., Cary, NC).

Figure 1.

Overview of the study protocol.

On Days 1–7, the study product designated by the randomization sequence was weighed and administered to the participant, who placed it under their top lip next to the gum for the specified time (10, 20, or 30 min). After use, all study products were reweighed and stored at −20°C until the analysis of residual analyte content.

At designated time points from baseline (1–5 min prior to the start of product use) to 4 h after the start of product use, blood samples were taken for PK measurements, vital signs, and 12‐lead ECGs were recorded, and questionnaires on the product were completed. During the use of Product 2d only, participants expelled saliva into a container whenever they felt the need to swallow their saliva. This sample represented the saliva that would have been swallowed during use and was used to estimate the amount of nicotine, sweeteners, and specific flavor components swallowed during a typical use period. A baseline saliva sample (≥5 mL) was also collected 5–10 min before use, and participants were allowed to rinse their mouth with water after providing this sample. The volume and weight of the baseline and “during use” saliva samples were recorded. At the end of the product use session on Day 7 prior to discharge, participants underwent a final health check, including physical and oral examinations, vital signs assessments, and clinical laboratory testing.

On Days 1–7, participants were not allowed to eat, drink, chew gum, or brush their teeth from 30 min before until 30 min after product use. On Days −1 to 6, they were required to refrain from using nicotine products for at least 12 h before the product use session the following morning. They were allowed to use their own nicotine products (except combustible products) between the completion of the last PK sample (4 h after use) and the start of the 12 h abstinence period. Participants brought a sufficient supply of their usual products for the 8‐day confinement period.

Study Assessments

Nicotine PK Measurements

Venous blood samples for nicotine PK measurements (≈4 mL each) were collected via an in‐dwelling catheter at baseline, 5, 7, 10, 15, 20, 25, 30, 35, 40, 60, 120, and 240 min, and centrifuged for 10 min at 1900 × g and 4°C within 60 min of collection. The plasma obtained was divided into aliquots and stored at −20°C until analysis for nicotine by Anapharm Bioanalytics, Barcelona, Spain, as previously described, ⁸ with a lower limit of quantitation (LLOQ) of 0.4 ng/mL. PK parameters (C_max, T_max, and AUC_0–4 h) were calculated by Non‐Compartmental Analysis using the software Phoenix WinNonlin® version 8.1 or later (Certara, Radnor, PA). Nicotine baseline adjustment occurred under the assumption that nicotine elimination followed first‐order kinetics. AUC_0–4 h was determined by integration of the plasma concentration versus time curve using linear interpolation for increasing plasma levels and logarithmic interpolation for decreasing plasma levels (Linear Up‐Log Down method).

Product Questionnaires

Four hours after the start of product use, participants rated their overall liking of the product by using a visual analogue scale (VAS 0–100 mm) to answer the question: “How much do you like the product, overall?” They also rated their intent to use the product again by using VAS to answer the question: “If given the opportunity, would you use this product again?”

Analysis of Nicotine, Sweetener, and Major Flavor Components in Used Pouches and Saliva

All NPs used in the study, including those from the familiarization session, were retained for analysis of residual nicotine (Products 1a, 1b, 1c, 2a, 2b, and 2c) or sweetener (Product 2d) and major flavor components (familiarization product). All used pouches were stored at −20°C until analysis at Labstat, Kitchener, Canada. Baseline saliva samples and saliva expelled during the use of Product 2d were stored at −20°C until analysis for residual nicotine, sweetener, and major flavor components at Labstat, Kitchener, Canada, as described in reference ²¹ with a nicotine LLOQ of 5 µg/mL.

Adverse Events

All AEs and SAEs were recorded from the start of study product use on Day 1 until the end‐of‐study follow‐up. AEs were documented on the eCRF, including signs and symptoms; start and stop dates and times; intensity; causal relationship to study product; action taken; and outcome.

An AE was defined as any untoward medical occurrence (e.g., unfavorable sign or symptom) in a participant using a study product, whether or not considered related to the product. AEs were coded on the basis of the Medical Dictionary of Regulatory Activities. The intensity of any AEs was graded by the researcher as mild (transient; needing minimal treatment or therapeutic intervention), moderate (alleviated with additional specific therapeutic intervention; causing discomfort but no significant of permanent risk or harm), or severe (interrupting activities of daily living; significantly affecting clinical status; or requiring intensive therapeutic intervention).

An SAE was defined as any AE that resulted in death, was life‐threatening at the time, required in‐patient hospitalization, resulted in significant disability/incapacity, or was an important medical event (i.e., not immediately life‐threatening or resulting in death/hospitalization, but potentially putting the participant at risk or requiring intervention to prevent the outcomes defined above).

Data Management

Clinical data were entered directly from the source documents or the bedside onto an electronic case report form (eCRF) with internal quality checks for inconsistent, incomplete, or inaccurate data. Each eCRF was completed as soon as possible during or after the participant's visit. To avoid interobserver variability, the same researcher who made the initial baseline determinations completed all corresponding follow‐up evaluations as far as possible. The eCRFs were evaluated for completeness and consistency.

Data Analysis

Sample Size Calculation

No formal sample size calculation was performed; instead, the sample size was based on previous nicotine PK studies, which typically ranged from 10 to 34 participants, ⁸ , ²² , ²³ , ²⁴ and were considered sufficient to provide adequate information for the study objectives.

Statistical Analysis

Continuous data were presented as arithmetic mean ± standard deviation (SD) or median (min–max). For the parameters, AUC_0–4 h and C_max, geometric mean, and percentage coefficient of variation (CV%) were additionally presented. Categorical data were presented as numbers (percentages). Where appropriate, summary data were presented by product and assessment time. Differences in C_max and AUC_0–4 h were compared between study products by LS means of fixed effects, where treatment, period, and sequence were included as fixed effects, and subject within sequence was included as a random effect in the model. A Holm–Bonferroni correction was applied for multiple comparisons at an overall significance level of α = 0.05 across m = 10 hypotheses by sequentially comparing the ordered p‐values to adjusted thresholds of α / (m − i + 1), where i is the rank of the p‐value defined by AUC_0–4 h (see Table S4 [Comparison of unadjusted PK parameters] and Table S5 [Comparison of baseline‐adjusted C_max and AUC_0–4 h: Final model results (PK analysis set)]). Hypotheses were rejected until a p‐value exceeded its corresponding threshold. T_max was analyzed for the same pairwise product comparisons as for C_max and AUC_0–4 h, but using the Wilcoxon signed rank test. All descriptive summaries and statistical analyses were performed using SAS Version 9.4 or later.

Results

Study Participants

During the study period, 69 potential participants were screened. Among these, 20 failed the screen, 13 were excluded for other reasons, and 1 individual met the eligibility criteria but was not required (Figure S1). Thus, the target sample size of 35 was achieved. Using a Latin square design, participants were randomized to one of five sequences for the order of product use. Allocation was balanced with seven participants in each sequence group (Table 2).

Table 2.

Baseline Characteristics and Demographics of the Study Participants by Randomization Sequence ^a

Characteristic	Sequence 1 (N = 7)	Sequence 2 (N = 7)	Sequence 3 (N = 7)	Sequence 4 (N = 7)	Sequence 5 (N = 7)	Overall Average (N = 35)
Age (years)
Mean ± SD	27.6 ± 6.9	28.9 ± 5.6	23.0 ± 3.3	29.9 ± 10.5	25.4 ± 3.3	26.9 ± 6.6
Median (min–max)	27.0 (20–40)	31.0 (21–35)	22.0 (20–30)	27.0 (22–53)	26.0 (19–29)	27.0 (19–53)
Height (cm)
Mean ± SD	174.0 ± 8.1	176.1 ± 9.9	175.3 ± 9.0	175.9 ± 9.8	170.0 ± 10.2	174.3 ± 9.2
Median (min–max)	178.0 (160–181)	175.0 (160–192)	174.0 (165–193)	180.0 (162–186)	166.0 (157–189)	175.0 (157–193)
Weight (kg)
Mean ± SD	76.6 ± 11.2	72.3 ± 10.5	72.7 ± 16.0	72.3 ± 14.0	67.0 ± 15.3	72.2 ± 13.1
Median (min–max)	78.0 (59–90)	69.0 (58–89)	69.0 (54–101)	69.0 (56–97)	68.0 (50–94)	69.0 (50–101)
BMI (kg/m2)
Mean ± SD	25.1 ± 2.1	23.2 ± 1.5	23.4 ± 3.2	23.2 ± 2.6	22.9 ± 2.8	23.6 ± 2.5
Median (min–max)	24.6 (22.8–28.1)	23.6 (20.7–25.3)	22.3 (19.8–28.7)	22.2 (21.1–28.3)	22.5 (18.7–26.3)	22.8 (18.7–28.7)
Sex
Female	2 (28.6%)	3 (42.9%)	2 (28.6%)	2 (28.6%)	5 (71.4%)	14 (40.0%)
Male	5 (71.4%)	4 (57.1%)	5 (71.4%)	5 (71.4%)	2 (28.6%)	21 (60.0%)
Race
Black/African American	1 (14.3%)	0	1 (14.3%)	0	0	2 (5.71%)
White	6 (85.7%)	7 (100%)	6 (85.7%)	7 (100%)	7 (100%)	33 (94.3%)
Ethnicity
Hispanic/Latino	1 (14.3%)	0	0	0	0	1 (2.86%)
Not Hispanic/Latino	6 (85.7%)	6 (85.7%)	7 (100%)	7 (100%)	7 (100%)	33 (94.3%)
Unreported	0	1 (14.3%)	0	0	0	1 (2.86%)

Abbreviations: BMI, body mass index; SD, standard deviation.

^a Values are given as mean ± SD, median (min–max), or number (percentage).

All participants completed the study and were included in the full analysis set (n = 35). One participant did not complete the use session for Product 2d (20 mg nicotine, 30 min duration with expulsion), and their data were excluded from saliva analysis (n = 34).

Among the 35 participants, the mean age was 26.9 ± 6.6 years (min–max, 19–53 years), and 14 (40.0%) were female (Table 2). The majority of participants were white (94.3%) and non‐Hispanic or Latino (94.3%).

Nicotine PK Analysis

In each study session, between two and five of the 35 participants had plasma nicotine levels below the limit of detection at baseline. The mean baseline values ranged from 1.45 ng/mL (Product 1a) to 1.66 ng/mL (Products 2a and 2b). All products showed a similar trend in plasma nicotine concentration over time, with a sharp rise after the start of product use (Figure 2a and Table S3). Plasma nicotine levels were highest for the 20‐mg NP used for 30 min, with and without saliva expulsion (baseline‐adjusted mean C_max, 35.9 and 33.6 ng/mL, respectively) and lowest for the 11‐mg NP used for 10 min (18.9 ng/mL) (Table 3). As hypothesized, C_max generally increased as usage time rose from 10 to 30 min (11‐mg NP, 18.9 to 21.6 ng/mL; 20‐mg NP, 26.7 to 33.6 ng/mL), although the C_max values were similar when the 11‐mg NP was used for 20 and 30 min (21.62 and 21.55 ng/mL, respectively) (Table 3). Baseline adjustment had little effect on the trend in values.

Figure 2.

Change in plasma nicotine concentration during and after NP use. (a) Geometric mean of nicotine concentrations for all NPs. (b) Effect of swallowing on nicotine absorption for the 20‐mg NP used for 30 min. Data has been adjusted for baseline nicotine levels.

Table 3.

Nicotine PK Parameters Determined for Two NPs of Different Nicotine Strength Used for Increasing Durations

Parameter a	11 mg, 10 min (N = 35)	11 mg, 20 min (N = 35)	11 mg, 30 min (N = 35)	20 mg, 10 min (N = 35)	20 mg, 20 min (N = 35)	20 mg, 30 min (N = 35)	20 mg, 30 min, with expulsion (N = 34)
Tmax (h)
Adjusted	0.17 (0.08–1.00)	0.33 (0.08–0.67)	0.50 (0.08–1.00)	0.25 (0.08–0.67) b	0.33 (0.08–0.67)	0.58 (0.17–0.72)	0.58 (0.12–0.68) b
Not adjusted	0.17 (0.08–1.00)	0.33 (0.08–0.67)	0.50 (0.08–1.00)	0.25 (0.08–0.67) b	0.33 (0.08–0.67)	0.58 (0.17–0.72)	0.54 (0.12–0.68) b
Cmax (ng/mL)
Adjusted
Mean	18.92 ± 8.68	21.62 ± 7.17	21.55 ± 6.07	26.65 ± 12.26 b	30.15 ± 9.28	33.61 ± 10.02	35.87 ± 9.75 b
Median	16.80 (6.8–43.7)	21.01 (10.8–38.2)	20.21 (10.3–39.9)	25.99 (9.3–61.1) b	29.28 (10.0–52.4)	32.60 (18.1–60.2)	32.72 (17.0–57.1) b
Geo mean	17.25 (45.0%)	20.43 (35.9%)	20.81 (26.8%)	24.00 (50.3%) b	28.72 (33.5%)	32.26 (29.6%)	34.61 (27.9%) b
Not adjusted
Mean	20.27 ± 8.91	23.06 ± 7.19	22.90 ± 6.03	28.18 ± 12.19 b	31.59 ± 9.29	34.84 ± 10.04	37.10 ± 9.88 b
Median	18.26 (7.4–44.2)	22.76 (11.7–40.8)	21.94 (11.8–41.5)	28.83 (10.1–61.9) b	31.88 (11.0–53.9)	34.27 (18.1–61.1)	35.32 (17.4–58.9) b
Geo mean	18.60 (43.3%)	21.95 (33.3%)	22.22 (25.0%)	25.67 (47.1%) b	30.21 (32.1%)	33.53 (28.5%)	35.85 (27.3%) b
AUC0–4 h (h ng/mL)
Adjusted
Mean	24.64 ± 6.20	36.52 ± 9.01	42.98 ± 8.92 b	36.44 ± 11.67 b	52.07 ± 13.82 b	65.63 ± 17.83	68.10 ± 15.64 c
Median	23.64 (13.9–39.6)	36.35 (22.2–53.7)	43.04 (27.9–71.2) b	33.71 (19.2–71.8) b	51.99 (25.9–81.3) b	62.26 (40.7–107)	67.57 (33.6–100) c
Geo mean	23.91 (25.2%)	35.45 (25.2%)	42.16 (19.8%) b	34.77 (31.7%) b	50.22 (28.4%) b	63.48 (26.3%)	66.30 (24.3%) c
Not adjusted
Mean	28.05 ± 7.72	40.07 ± 9.71	46.66 ± 9.67 b	40.14 ± 12.54 b	55.56 ± 14.32 b	68.83 ± 18.69	71.33 ± 16.39 c
Median	25.44 (15.6–46.0)	39.28 (23.3–56.4)	46.66 (32.6–74.9) b	36.80 (21.0–76.4) b	57.65 (28.9–83.7) b	65.85 (40.7–113)	68.79 (34.7–102) c
Geo mean	27.08 (27.1%)	38.92 (24.9%)	45.74 (20.3%) b	38.35 (31.3%) b	53.69 (27.5%) b	66.56 (26.5%)	69.45 (24.3%) c

Abbreviations: AUC, area under curve; C_max, maximum concentration; Geo mean, geometric mean; SD, standard deviation; T_max, time to peak concentration.

^a Values are reported as mean ± SD, median (min–max) or geometric mean (geo CV%, calculated using log‐transformed SD) and were calculated from non‐adjusted plasma nicotine levels and baseline‐adjusted plasma nicotine levels.

^b Data from 34 participants.

^c Data from 33 participants.

By LS‐means of fixed effects, a significant difference in C_max was observed when the 11‐mg NP was used for 20 or 30 min relative to 10 min (p =  .0046 and p =  .0024, respectively), and similarly when the 20‐mg NP was used for 20 or 30 min relative to 10 min (p = 0.0088 and p < .0001, respectively) (Table S4). There were also significant differences in C_max between the 11‐mg NP and 20‐mg NP used for the same duration (p < .0001 for 10min, p < .0001 for 20 min and p < .0001 for 30 min) (Table S4). However, there were no differences in C_max when the 11‐mg NP or 20‐mg NP were used for 20 and 30 min (Table S4). The differences remained when the baseline‐adjusted values were compared (Table S5).

To assess whether nicotine swallowed during use of the NPs has an impact on nicotine absorption, we compared the nicotine PK for the 20‐mg NP used for 30 min with expulsion of saliva (i.e., no swallowing) and without expulsion. Although C_max and AUC_0–4 h were slightly higher for the product with expulsion of saliva (baseline‐adjusted 35.9 vs. 33.6 ng/mL and 68.1 vs. 65.6 h ng/mL, respectively) (Table 3), the values were not significantly different (Table S4) and probably within the normal variability of the analysis.

For the 11‐mg NP, consistent with the usage durations, median T_max increased with usage time from 0.17 h (10 min) to 0.33 h (20 min) to 0.50 h (30 min). The same trend was observed for the 20‐mg NP, but the values were generally slightly higher (0.25, 0.33, and 0.58 h, respectively; Table 3). By the Wilcoxon signed rank test, there was a significant difference in T_max between the 11‐mg or 20‐mg NP used for 10 min and the same product used for 20 or 30 min (11‐mg NP, p = 0.0039 and p < .0001, respectively; 20‐mg NP, both p < .0001; Table S4). There were also significant differences in T_max between the 11‐mg NP or 20‐mg NP used for 20 min and the same product used for 30min (11‐mg, p = 0.0006 and 20‐mg p < .0001) (Table S4). However, there were no differences in T_max between the 11‐mg and 20‐mg NPs when used for the same durations and between the 20‐mg NP used for 30 min with expulsion of saliva and without expulsion. (Table S4). The differences between products remained when the baseline‐adjusted values were compared (Table S5).

Total nicotine exposure (AUC_0–4 h) also increased with usage time for both products. Similar to the trends in C_max, significant differences in AUC_0–4 h were observed when the 11‐mg NP was used for 20 or 30 min relative to 10 min, or the 20‐mg NP was used for 20 or 30 min relative to 10 min (all p < .0001) (Table S4). There were also significant differences in AUC_0–4 h between the 11‐mg NP or 20‐mg NP used for 20 min and the same product used for 30 min (all p < .0001) (Table S4). In addition, there were also significant differences in AUC_0–4 h between the 11‐mg NP and 20‐mg NPs used for the same duration (all p < .0001) (Table S4).

Subjective Effects

OPL and intent to use product again (IUA) were assessed by VAS (100 mm) 4 h after the start of product use (Table 4). IUA scores were similar for all products, with mean scores ranging from 54.1 to 57.6 mm, except for the 20‐mg NP used for 30 min with expulsion, which was slightly lower (49.4 mm).

Table 4.

Subjective Effects—Overall Product Liking and Intent to Use Again ^a

Assessment	11 mg, 10 min (N = 35)	11 mg, 20 min (N = 35)	11 mg, 30 min (N = 34)	20 mg, 10 min (N = 35)	20 mg, 20 min (N = 35)	20 mg, 30 min (N = 35)	20 mg, 30 min + expulsion (N = 34)	Overall (N = 243)
IUA
Mean ± SD	55.9 ± 30.9	55.7 ± 30.3	57.6 ± 27.7	54.1 ± 33.0	55.1 ± 30.7	55.3 ± 31.5	49.4 ± 29.0	54.7 ± 30.2
Median (min–max)	61.0 (0–100)	64.0 (3–100)	59.5 (2–100)	65.0 (0–100)	61.0 (0–100)	58.0 (1–100)	43.5 (0–100)	59.0 (0–100)
OPL
Mean ± SD	52.4 ± 27.5	59.5 ± 29.1	56.6 ± 24.6	54.0 ± 31.2	56.9 ± 27.5	52.4 ± 28.7	51.6 ± 28.6	54.8 ± 28.0
Median (min–max)	55.0 (0–100)	65.0 (1–100)	62.0 (2–100)	62.0 (2–100)	62.0 (8–100)	52.0 (3–100)	56.5 (0–100)	61.0 (0–100)

Abbreviations: IUA, intention to use product again; OPL, overall product liking.

^a IUA and OPL were scored by a visual analogue scale (0–100 mm). For OPL, 0 mm represented “really dislike” and 100 mm represented “really like”. For IUA, 0 mm represented “absolutely not” and 100 mm represented “absolutely”.

OPL was highest for the 11‐mg NP used for 20 min (mean 59.5 mm) and lowest for the 20‐mg NP used for 30 min with expulsion (51.6 mm).

Extraction of Nicotine From NPs During Use

To verify the nominal nicotine content of the NPs, 10 unused reference NPs for each nicotine strength were weighed accurately and analyzed for nicotine content. The mean ± SD amount of nicotine per NP was determined as 12.15 ± 0.99 mg (min–max, 10.6–13.5 mg) for 11‐mg NPs and 20.55 ± 0.77 (min–max, 19.2–21.6 mg) for 20‐mg NPs. These values, coupled with the weight of the unused study NPs, were used to estimate the pre‐use nicotine content of each study NP (Table 5).

Table 5.

Residual Nicotine and Fraction of Nicotine Extracted in Used Pouches

Assessment (unit)	11 mg, 10 min (N = 35)	11 mg, 20 min (N = 35)	11 mg, 30 min (N = 35)	20 mg, 10 min (N = 35)	20 mg, 20 min (N = 35)	20 mg, 30 min (N = 35)
Estimated nicotine in unused NP (mg/unit) a
Mean ± SD	12.53 ± 0.71	12.41 ± 0.83	12.48 ± 0.68	21.23 ± 0.90	21.19 ± 0.84	20.89 ± 0.71
Median (min–max)	12.65 (10.4–13.9)	12.65 (10.2–13.5)	12.65 (10.4–13.5)	21.14 (18.9–22.9)	21.30 (19.2–23.2)	20.66 (20.2–23.4)
Residual nicotine in used NP (mg/unit)
Mean ± SD	9.04 ± 1.36	8.00 ± 1.29	7.04 ± 1.19	16.45 ± 0.81	14.91 ± 1.93	13.18 ± 2.06
Median (min–max)	9.23 (4.0–12.4)	8.15 (5.2–11.2)	7.33 (4.0–8.9)	16.33 (14.9–18.1)	15.28 (8.4–17.7)	13.59 (4.2–15.6)
Extracted nicotine (mg/unit)
Mean ± SD	3.49 ± 1.04	4.41 ± 0.80	5.44 ± 1.03	4.78 ± 1.08	6.28 ± 1.85	7.71 ± 2.02
Median (min–max)	3.35 (1.5–8.3)	4.37 (2.1–6.2)	5.27 (3.9–7.9)	4.73 (3.0–7.7)	5.89 (3.7–13.9)	7.47 (5.3–16.5)
Extracted nicotine (%)
Mean ± SD	28.01 ± 8.73	35.79 ± 7.51	43.67 ± 8.50	22.42 ± 4.45	29.64 ± 8.48	36.92 ± 9.68
Median (min–max)	27.00 (10.4–67.4)	34.09 (15.8–49.3)	42.23 (31.1–64.8)	22.22 (14.5–34.1)	27.86 (17.5–62.2)	35.75 (25.7–79.9)

^a Estimated based on the weight of the unused NPs and the mean amount determined in weighed, unused reference NPs (11‐mg NPs, 12.15 ± 0.99 mg, N = 10; 20‐mg NP, 20.55 ± 0.77 mg, n = 10).

As expected, a greater amount of nicotine was extracted from the 11‐mg NPs as the use duration increased: 10 min, 3.5 mg; 20 min, 4.4 mg; and 30 min, 5.4 mg (Table 5). The mean nicotine extracted from the 20‐mg NPs similarly increased from 4.8 to 7.7 mg as the use duration increased from 10 to 30 min. Overall, however, the fraction of nicotine extracted was lower for the 20‐mg NP than for the 11‐mg NP for each usage time: 10 min, 22.4% versus 28.0%; 20 min, 29.6% versus 35.8%; and 30 min, 36.9% versus 43.7%, respectively (Table 5). Thus, nicotine is extracted rapidly from the NP pouch in the first 10 min of use, but the rate of extraction decreases thereafter. These observations are consistent with the observed nicotine PK curve and their statistical comparisons (Figure 2).

Extraction of Flavor Components and Sweeteners from NPs During Use

Next, we evaluated which flavor components are present in NPs, and the fraction of flavor components extracted during use of the 20‐mg NP for 30 min. To determine the contents of NPs as sold, 10 unused reference NPs were weighed accurately and analyzed for 12 different flavor components, chosen as they are most abundant. The total mean content of the measured flavor components was 11.7 mg. Only menthol (mean 9.3–9.5 mg/pouch), and menthone (1.1 mg/pouch) were present in milligram quantities (Table 6), while pulegone, propylene glycol, isomenthone, menthofuran, menthyl acetate, neomenthol, and eucalyptol were present at the 100‐mg level and below.

Table 6.

Flavor Components ^a , Sweeteners ^b and Fraction of Extraction During NP Use

Flavor Component	Reference NPs mean ± SD (N = 10)	Study NPs Before Use mean ± SD (N = 35) c	Study NPs After Use mean ± SD (N = 35)	Amount Extracted mean ± SD (N = 35)	Fraction (%) Extracted mean ± SD (N = 35)
Flavor
A‐Pinene (µg/pouch)	1.010 ± 0.348	1.030 ± 0.105	0.900 ± 0.000	0.133 ± 0.104	12.18 ± 7.79
B‐Pinene (µg/pouch)	2.130 ± 0.467	2.164 ± 0.221	0.900 ± 0.000	1.268± 0.221	58.23 ± 3.81
Eucalyptol (µg/pouch)	113.8 ± 5.770	115.7 ± 11.84	33.89 ± 9.946	82.07 ± 13.46	70.83 ± 8.89
Isomenthone (µg/pouch)	396.8 ± 13.17	403.6 ± 41.27	219.1 ± 22.92	185.3 ± 45.05	45.46 ± 7.73
Limonene (µg/pouch)	7.670 ± 0.533	7.801 ± 0.798	1.189 ± 0.547	6.627 ± 0.748	85.07 ± 6.43
Menthofuran (µg/pouch)	9.11 ± 0.506	9.26 ± 0.947	3.07 ± 0.53	6.21 ± 0.954	66.89 ± 6.27
Menthol (mg/pouch)	9.302 ± 0.386	9.459 ± 0.967	7.088 ± 0.623	2.415 ± 1.145	24.83 ± 9.54
Menthone (mg/pouch)	1.090 ± 0.039	1.109 ± 0.113	0.571 ± 0.065	0.540 ± 0.120	48.31 ± 7.43
Menthyl acetate (µg/pouch)	214.1 ± 6.855	217.7 ± 22.27	156.9 ± 11.69	61.23 ± 23.84	27.52 ± 8.41
Neomenthol (µg/pouch)	42.0 ± 1.247	42.71 ± 4.37	31.54 ± 2.27	11.31 ± 4.80	25.81 ± 8.63
Propylene glycol (µg/pouch)	477.0 ± 26.05	484.9 ± 49.59	429.3 ± 132.0	81.79 ± 100.7	16.03 ± 18.74
Pulegone (µg/pouch)	16.3 ± 0.95	16.58 ± 1.70	8.75 ± 1.0	7.86 ± 2.05	46.86 ± 8.53
Overall mean (mg/pouch)	11.67 ± 0.417	11.87 ± 1.214	8.544 ± 0.786	3.398 ± 1.399	27.97 ± 8.99
Sweetener
Acesulfame potassium (mg/pouch)	0.824 ± 0.156	0.834 ± 0.029	0.751 ± 0.098	0.094 ± 0.079	11.19 ± 9.43
Xylitol (mg/pouch)	18.80 ± 1.01	19.09 ± 0.665	15.71 ± 2.13	3.378 ± 2.306	17.56 ± 11.75
Overall mean (mg/pouch)	9.811 ± 0.53	9.960 ± 0.347	8.230 ± 1.075	1.736 ± 1.166	14.37 ± 8.61

^a Residual flavor components were analyzed in the NPs used in the product familiarization session (20‐mg, 30 min use). The pre‐use weight of these NPs was not measured; therefore, it has been estimated as the weight measured post‐use subtracted with the mean difference in weight between used and unused NPs measured for Products 2c and 2d in the study. As a result, the sum of residual and extracted amounts (not shown) did not equal the estimated amount in the unused NPs.

^b Residual flavors were analyzed in Product 2d (20‐mg, 30 min use plus saliva expulsion).

^c For flavor analysis, estimated based on weight of unused pouch and content in reference NPs.

[Correction added on September 12, 2025, after first online publication: Table 6 has been updated in this version.]

A total of 3.4 mg of flavor components was extracted during use; the extracted flavor components mainly comprised menthol (2.4 mg), menthone (0.5 mg), isomenthone (0.2 mg), and propylene glycol (0.08 mg). Across the flavor components present in measurable quantity, the percentage extraction during use for 30 min ranged from 12.2% (α‐pinine) to 85.1% (limonene); among the main flavor components detected in reference NPs, 24.8% of menthol, 66.9% of menthofuran, 48.3% of menthone, 16.03% of propylene glycol and 45.5% of isomenthone were extracted.

We also evaluated 20‐mg NPs for the presence of two sweeteners: acesulfame‐potassium (acesulfamide‐K) and xylitol. Xylitol was found in unused NPs at 18.8–19.1 mg per pouch, and 3.4 mg (17.6%) was extracted during 30 min of use (Table 6). Acesulfamide‐K was detected in unused NPs at 0.8 mg, and a mean of 0.09 mg (11.2%) was extracted during use.

Nicotine, Flavor Components and Sweeteners Extracted in Saliva

To estimate the amounts of NP constituents that are swallowed during NP use, in the Product 2d study session, participants expelled their saliva into a container whenever they felt the need to swallow, and the total saliva sample—as a surrogate of what the participant would have swallowed during the 30 min use period—was analyzed for nicotine, sweeteners and flavor components. During the 30‐min use period of product 2d, the participants collected 9.5mL ± 5.67mL of saliva on average.

The mean amount of nicotine in saliva after use of the 20‐mg NP for 30 min was 0.36 ± 0.55 mg (adjusted for nicotine present in saliva before NP use). This indicates that, based on the amount of nicotine in unused reference NPs (20.55 mg), 1.8% of the nicotine content of NPs may be swallowed during use for 30 min. As described above, measurement of the residual nicotine content after use indicated that a mean of 7.71 mg of nicotine was extracted from 20‐mg NPs after 30‐min use (see Table 5); taking into account the extracted amount and the amount of nicotine in saliva for each of the 34 subjects included in saliva analysis, on average 4.3% of the total amount of nicotine extracted from NPs during use may be swallowed.

Among the 12 flavor components tested in saliva after use of the 20‐mg NP, only five were detected, and most of these were present at very low levels close to the analytical limit (Table S6). Regarding menthol, which was present in reference NPs at the milligram level (see Table 6), we detected mean levels of 14.4 mg in saliva after 30 min of product use (Table S6). This suggests that 0.2% of the total menthol content of the unused NPs would be swallowed during use (Table S6). Based on the residual amounts measured in the used NPs (see Table 6), 0.6% of the menthol extracted from NPs during use may be swallowed.

Overall, the average of individual flavor component content in saliva collected during 30 min use of the 20‐mg NP was 1.43 mg, corresponding to 0.16% of the total in unused reference NPs and 0.48% of the amount extracted during NP use for 30 min (Table S6). Similarly, the average sweetener content in saliva was 336.6 mg, corresponding to 3.35% of the amount in unused NPs and 9.85% of the amount extracted during NP use for 30 min (Table S6).

Discussion

The present study has evaluated the effect of the studied NPs' nicotine strength and use duration, as well as the effect of swallowing, on nicotine absorption based on usage times that are likely to be representative of those recently reported among European NP consumers. ²⁴ The findings add to earlier oral nicotine product PK studies by addressing gaps in knowledge regarding higher nicotine strength products and shorter usage times. As hypothesized, maximum blood levels of nicotine (C_max) and total nicotine in the blood after use (AUC_0–4 h) generally increased with both nicotine strength and usage time, while the time to maximum plasma concentration (T_max) of NPs was associated with the usage time (i.e., it occurred at or soon after pouch removal). Also, as hypothesized, the amount of nicotine measured in saliva was negligible, indicating minimal ingestion by swallowing of nicotine occurs during NP use with little to no impact on the level and rate of nicotine absorption into the blood.

Notably, all NPs tested over all durations showed a similar and rapid increase in plasma nicotine concentrations during the first 10 min of use (Figure 2a). Thereafter, C_max and AUC continued to rise for products used for 20 or 30 min, but at a much slower rate. Thus, the greatest delivery of nicotine was in the first 10 min of product use, regardless of product strength, indicating the efficient delivery of nicotine by NPs. This was also reflected in the amount of nicotine extracted from used NPs, which showed that the percentage extracted was greater for 11‐mg NPs than for 20‐mg NPs used for the same duration (Table 5).

Prior studies have characterized the nicotine PK of NPs, with initial findings indicating that NPs may deliver nicotine as efficiently as snus. ²⁴ Few existing data indicate precisely how consumers are using various types of these relatively new products. Reported use duration is as high as 60 min per pouch, and pouch nicotine content has varied widely (1.5–10.1 mg) in the available PK studies. ⁸ , ²⁴ , ²⁶ , ²⁷ , ²⁸ , ²⁹ A recent survey among NP consumers in Europe indicates that the most frequently reported single pouch use duration is in the range of 10‒20 min in some countries, while a few respondents report using NPs for 1–10 min per pouch. ²⁶ A mean pouch use duration of 19 min has been observed among Danish consumers, although Swedish consumers, on average, reportedly use single NPs for much longer (47 min), possibly because of the historical use of snus pouches for much longer durations in Sweden. ²⁹ In addition, the European survey found that most consumers reported using a nicotine strength of 6–15 mg, and only a small proportion (16.5%) used NPs containing less than 6 mg or more than 20‐mg (8.7%) of nicotine. ²⁵ Although the sample sizes were relatively small, these data inform PK studies to estimate nicotine delivery among NP consumers.

In addition, previous studies of PK parameters for the absorption of nicotine from NP use have variously reported C_max values from 3.5 to 18.4 ng/mL for NPs with nicotine strengths of 1.5–10 mg and use durations of up to 60 min. ⁸ , ²³ , ²⁵ , ²⁶ , ²⁷ , ²⁸ In the current study, the baseline‐adjusted C_max values for the 11‐mg NP used for 10–30 min were 18.9–21.6 ng/mL, respectively, which are consistent with the higher end values previously reported for 10 mg NPs. While the C_max for the 20‐mg nicotine NP (26.7–33.6 ng/mL, for 10–30 min use, respectively) was higher than any previously reported values, PK analysis of a 20 mg nicotine NP has not been reported before. However, these C_max values are not unprecedented for oral tobacco and nicotine products; for example, use of Swedish snus (8 mg) for 30 min was reported to result in a C_max value of 29.0 ng/mL. ³¹ This was discussed in a recent scoping review ³¹ the authors looked at data from 7 different PK studies and concluded that the data suggested a lower plasma nicotine concentration for lower‐strength NPs (<4 mg) compared to cigarettes and snus, while higher‐strength NPs (≥6 mg) may deliver comparable or higher nicotine than conventional snus products and cigarettes. ³² It is important to stress that these individual values should not be viewed in isolation when calculating total nicotine consumption from use of a particular product; in addition to the nicotine strength of a product, the daily consumption must be taken into account together with use duration.

Regarding total nicotine exposure from 10–30 min use, AUC_0–4 h ranged from 24.6 to 43.0 h ng/mL for the 11‐mg NP, and from 36.4 to 65.6 h ng/mL for the 20‐mg NP. Because other studies used different strength NPs and use durations, it is difficult to compare these values; however, they are in the same order as previous studies; for example, Liu et al. ²⁹ reported an AUC_0–3 h value of 24 h ng/mL for an 8 mg NP with 30 min use, while McEwan et al. reported an AUC_0–6 h values of 35.8–53.7 h ng/mL for 10 mg NPs used for 60 min. ⁸ As mentioned earlier in the discussion, the longer use durations applied in previous studies may not be representative of the use‐behavior of regular NP consumers. ²⁵ , ³⁰ Among regular consumers in Denmark and Sweden, overall use duration was found to decrease as nicotine strength increased, while the mean use duration was 19 min for NPs up to 11 mg in strength in Denmark ³⁰ ; therefore, consumers who choose 20‐mg NPs may use them for much shorter durations than 30 min.

In the present study, T_max was closely linked to use duration, which is consistent with previous studies: for example, in studies with 60 min use time, T_max ranged from 60–65 min, ⁸ , ²⁶ while in studies with 30‐ and 20‐min use times, T_max ranged 30–35 min ²⁷ , ²⁹ and 22–26 min, respectively. ²⁸

Collectively, the current data indicate the efficient delivery of nicotine by NPs and thus provide additional support for their potential use in a THR approach. ¹⁷ For both the 11 and 20‐mg NPs, the rapid rise in plasma nicotine levels after the start of product use (Figure 2a) is similar to the effects of smoking a combustible cigarette, ⁸ , ²⁹ suggesting that NPs may provide effective nicotine delivery to satisfy smokers who switch to an NP. The C_max of nicotine absorption from NP use in this study was higher than values reported for cigarettes (11.6–16.3 ng/mL ⁸ , ²⁷ , ²⁸ , ²⁹ ). The T_max value reported for these studies is longer in the experimental conditions than is typically found in real‐world data and unlikely represents consumers’ T_max in ad libitum use, owing to the mouth hold times being highly variable among consumers. The reported AUC of nicotine absorption for cigarettes varies from 13.4 to 25.2 h ng/mL, ⁸ , ²⁸ , ²⁹ which is at the lower end of the AUC_0–4 h values measured for the NPs in the present study (24.6–65.6 h ng/mL). However, cigarette C_max falls within the overall range of values reported for NPs from 4‐mg nicotine products used for 60 min (8.5 ng/mL ²⁶ ) to 11 mg products used for 30 min (21.6 ng/mL; this study) and 20 mg nicotine products used for 10 min (26.7 ng/mL; this study), suggesting that smokers who switch completely to an NP can obtain their accustomed amount of nicotine during normal product use. Moreover, while these single‐use data provide insight into the efficacy of nicotine delivery, total product usage that is, average daily consumption, must be considered. When evaluated in the context of average daily consumption, which may vary widely from individual to individual and has been reported as ≈1–5 pouches (Germany and Switzerland) and ≈8–10 pouches (Denmark and Sweden) for daily NP consumers, ²⁵ , ³⁰ and 10–15 cigarettes for daily smokers (e.g., references ³⁰ , ³³ ), the total nicotine exposure over the course of a day is likely to be similar for NP consumers.

For successful use in a THR approach, an alternative nicotine product should also be appealing to smokers who would not otherwise quit smoking. Apart from Product 2d, which included saliva expulsion during use, the mean OPL scores ranged from 52 (20‐mg NP, 30 min use) to 60 (11‐mg NP, 20 min use) and suggest that the NPs were reasonably well liked. Intent to use again scores were also similar. Notably, the 20‐mg NP with 30 min use had the lowest OPL and IUA scores, consistent with the use of higher strength NPs for shorter durations among regular NP consumers in other studies. ²⁵ , ³⁰

In this study, we also measured the nicotine remaining in NPs to assess how much nicotine is extracted during use. As expected, both the amount and fraction of nicotine extracted increased with use duration; however, a larger fraction of nicotine was extracted from the 11‐mg than from the 20‐mg NP for the same use durations (10 min, 28.0% vs. 22.4%; 20 min, 35.8% vs. 29.6%; 30 min, 43.7% vs. 36.9%). The PK curve showed that absorption of nicotine is rapid during the first 10 min of use and slows thereafter until the NP is removed, when plasma nicotine levels start to fall (Figure 2). This decreasing rate of nicotine absorption mirrors the residual nicotine analysis; for example, 22.4% of nicotine was extracted from the 20‐mg NP after 10 min, but only a further 7.2% was extracted between 10 and 20 min (29.6% extracted), and a further 7.3% between 20 and 30 min (36.9% extracted; Table 5).

Because NPs are oral products, it is important to assess the effect of saliva swallowing on nicotine absorption and the potential ingestion of sweeteners and flavor components during use. We therefore assessed the use of the 20‐mg NP for 30 min with and without expulsion of saliva. The plasma nicotine‐time curves were similar for the two groups (Figure 2b), there were no statistically significant differences in C_max or AUC (Table 3), and also no noticeable secondary peak after 2–3 h following normal use that is typically observed when relatively large quantities of nicotine are swallowed, ⁸ , ³⁴ suggesting that swallowed nicotine during use of the study NPs does not have a notable impact on absorption. In support of this conclusion, only 1.8% of the nicotine content of the NP was detected in the collected saliva sample, equating to 4.3% of the total amount of nicotine extracted during use of the NP. In short, less than 5% of the total nicotine extracted from the NP was swallowed during 30 min of use, consistent with the nicotine PK curves. Similarly, on average, only 0.38% of each extracted flavor component and 9.85% of each extracted sweetener were detected in saliva, again suggesting that relatively small amounts of flavor components and sweeteners are swallowed during NP use. Furthermore, the absolute amounts of nicotine, flavor components and sweeteners that are swallowed by consumers are likely to be much lower because, as mentioned above, the typical usage duration among NP consumers (e.g., 19 min in Denmark) is considerably lower than the 30‐min evaluation period used in the present study and, with regard to nicotine, a majority of consumers consume NPs with a nicotine strength much lower than 20 mg.

During the 30 min use period of Product 2d, participants collected 9.5 mL of saliva on average. Typical unstimulated saliva production volumes have been quoted as 0.2–0.6 mL/min or 6–18 mL over a 30‐min period. ³⁵ , ³⁶ , ³⁷ , ³⁸ Normal stimulated salivary flow rate averages 1–3 mL/min or 30–90 mL over a 30‐min period. ³⁵ , ³⁶ , ³⁹ , ⁴⁰ The volume of saliva generated and collected by the participants during the study lies within reported average unstimulated saliva production ranges and is considerably lower than reported average volumes of saliva produced when stimulated. This is supported by Ferguson (1999), who reported a flow rate of 5 µL/cm²/min from the labial salivary glands. ⁴¹ These data suggest that saliva production during use of NPs remains within the normal unstimulated range and supports the earlier findings that low amounts of nicotine are swallowed during NP use. ⁸ The study has several strengths. The crossover design facilitates a more efficient comparison of products than a parallel study design (i.e., fewer participants are required because each serves as their own control, minimizing the risk of confounding effects). A period of at least 24 h between the start of each product use session was incorporated, as well as a minimum 12 h abstinence period from any nicotine products, to ensure that measured blood nicotine levels reflected study product use only. Last, randomization was used to minimize bias in the assignment of participants to study product use sequence and to increase the likelihood that known and unknown subject attributes (e.g., demographic and baseline characteristics) were evenly balanced across sequence groups.

The study also has some limitations. Owing to the unique historical background of snus use and the requirement to enroll regular consumers of higher strength NPs, the study was conducted only among Swedes in Sweden. It would be useful to extend the study to wider populations who may have different typical use patterns. In addition, the single‐use design of the study generated one set of nicotine PK data per NP/use duration combination. In applying them to estimations of the nicotine exposure of actual consumers, these data should be viewed in the wider context of average daily consumption and actual use durations. An additional limitation of this study is the potential impact of metabolic differences on nicotine PK. In particular, CYP2A6, a phase I metabolizing enzyme, is known to have functional polymorphisms that impact nicotine metabolism, and these polymorphisms differ substantially by ancestry. ⁴² Additionally, it has been reported that nicotine metabolism is faster in women than in men and is faster in women taking oral contraceptives compared with those who are not. ⁴³ This study employed a crossover design where subjects served as their own controls, so CYPA26 polymorphisms and differences in CYP2A6‐mediated metabolism should not impact any comparison of the products within the study. However, the demographics show that 94.3% of the study population are from one racial group; therefore, conclusions may not be reflective of other racial groups.‘’

Conclusions

The present study has shown that NPs can deliver nicotine quickly and sufficiently to satisfy smokers’ desires for nicotine. As expected, the amount of nicotine delivered to the consumer increases with the duration of use; however, the nicotine extraction rate falls off after the first 10 min of use, supporting the short use durations recently reported in surveys of NP consumers. Coupled with nicotine PK data for a 4‐mg NP used for 60 min, ²⁶ our findings suggest that smokers who switch completely to an NP can obtain their accustomed amount of nicotine during normal product use. Only a small fraction of nicotine and flavor components are swallowed during use, and the nicotine that is swallowed has little effect on nicotine concentrations in the blood. Collectively, these data support the use of NPs in a potential THR approach.

Author Contributions

Study design: David Azzopardi, Elaine Brown, George Hardie, and Michael McEwan; Methodology: David Azzopardi, Elaine Brown, Filimon Meichanetzidis, George Hardie, Stacy Fiebelkorn, and Michael McEwan; Formal analysis: Filimon Meichanetzidis and Stacy Fiebelkorn; Writing—review and editing: David Azzopardi, Elaine Brown, Filimon Meichanetzidis, Stacy Fiebelkorn, Linsey E. Haswell, and Michael McEwan; Data visualization: David Azzopardi, Filimon Meichanetzidis, Stacy Fiebelkorn, Linsey E. Haswell, and Michael McEwan.

Conflicts of Interest

At the time the study was conducted, all authors were employees of BAT, which was the sponsor and funding source of this study. All authors have read and agreed to the published version of the manuscript.

Funding

This study was funded by BAT (Investments) Ltd.

Supporting information

Supporting Information

Data Availability Statement

BAT is committed to the responsible sharing of data with the wider research community. Data access is administered for this study through an internal Data Sharing Committee on reasonable request following completion of a data sharing request form and if applicable, a Data Access Agreement. Requests for data sharing in the first instance should be emailed to the corresponding author.