Comparative study of calcium sulphate and cellulose based nicotine pouch on pharmacokinetics, pulse rate and nicotine extraction

Comparative study of calcium sulphate and cellulose based nicotine pouch on pharmacokinetics, pulse rate and nicotine extraction | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Clinical trial Comparative study of calcium sulphate and cellulose based nicotine pouch on pharmacokinetics, pulse rate and nicotine extraction Anna Franzen, Jesper Loof, Ulrik Birgersson, Glebs Kiselovs, Maria Pokosta, and 1 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-5953198/v1 This work is licensed under a CC BY 4.0 License Status: Under Review Version 1 posted 10 You are reading this latest preprint version Abstract This study presents a comparative analysis of nicotine pouches using calcium sulphate and cellulose as carrier materials. A randomized, controlled, cross-over nicotine pharmacokinetic study of 12 healthy volunteer subjects with the primary objective to evaluate the pharmacokinetics, pulse rate, and nicotine extraction efficiency of three different formulations: SERATEK 3mg, SERATEK 6mg, and dry ZYN 6mg was conducted. SERATEK formulations utilized calcium sulphate, while ZYN contained cellulose as nicotine carrier. The study aims to address the need for rapid nicotine uptake, akin to facilitate replacement of smoking or vaping, by developing an innovative nicotine pouch with enhanced bioavailability. Results indicate that SERATEK formulations provide a faster initial release and higher nicotine uptake compared to ZYN, particularly within the first 5 minutes of use. This rapid uptake is attributed to higher pH for nicotine loaded into calcium sulphate granules in the SERATEK formulations. The findings suggest that calcium sulphate-based nicotine pouches could offer a more effective alternative to traditional nicotine delivery methods, potentially driving a shift from smoking and vaping to nicotine pouches. The study has been registered at clinicaltrials.gov with ID: NCT06170138 at 2023-11-24 Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Nicotine pouches have emerged as a significant alternative to traditional nicotine delivery methods such as smoking and snus, particularly focusing on dry and wet formulations based on cellulose as carrier/bulk material. These products offer a discreet and smokeless mode of nicotine consumption, catering to a market segment that prioritizes reduced risk and convenience. The current technology behind nicotine pouches emphasizes delivering nicotine effectively while minimizing the harmful byproducts associated with combustion. The release and uptake kinetics of nicotine from the pouches are controlled by the release rate of nicotine and the pH of the product [ 1 ]. Despite the advancements, there remains a critical need for nicotine pouches that can provide a more rapid nicotine uptake, akin to the swift delivery experienced in smoking or vaping. Achieving this could significantly drive the transition from traditional cigarettes and electronic nicotine delivery systems to nicotine pouches, aligning with public health initiatives aimed at reducing smoking prevalence. Development poised to address these gaps by developing an innovative nicotine pouch featuring a novel carrier and loading method. This technology aims to enhance the bioavailability and rapidity of nicotine delivery, potentially outperforming existing products and encouraging a shift from traditional smoking and vaping to pouches. This approach could significantly impact public health by offering a more effective and appealing alternative to conventional nicotine consumption methods [ 2 ]. The future of nicotine pouches lies in continued research and development, focusing on optimizing the delivery systems to meet user preferences and health standards. By integrating advanced materials science, pharmacokinetics, and user behavior analysis, the next generation of nicotine pouches could represent a paradigm shift in harm reduction and nicotine replacement therapy [ 3 ]. Efforts to develop a new innovative formulation of nicotine pouches, based on calcium sulphate bioceramic technology. Calcium sulphate has several uses in the food industry and is on the list of permitted food additives [ 4 ]. Calcium sulphate is generally recognized as safe (GRAS) by the United States Food and Drug Administration (FDA) [ 5 ]. This report presents a comparative analysis of the nicotine exposures of three different formulations: SERATEK 3 mg, SERATEK 6 mg, and dry ZYN 6mg. Where SERATEK 3 mg and SERATEK 6 mg are based on loading nicotine into a calcium sulphate bioceramic formulation and ZYN in a cellulose formulation. The study aims to evaluate the pharmacokinetic (PK) parameters, pulse rate and nicotine extraction following single oral use of these products. 2. Materials Nicotine was incorporated into calcium sulphate dihydrate granules by dissolving nicotine salt (nicotine bitartrate) into the hydration liquid used during the setting process. This method ensures homogeneous distribution of nicotine throughout the calcium sulphate matrix as it transitions from the hemihydrate to the dihydrate form. To prepare the nicotine-loaded calcium sulphate granules, the specific amount of nicotine was first dissolved in the required volume of hydration liquid (water). The nicotine solution was then added to calcium sulphate hemihydrate powder and thereby initiating the hydration and setting process. As the material begins to set, the calcium sulphate hemihydrate reacts with the water to form calcium sulphate dihydrate, trapping the dissolved nicotine within the pore system. This ensures that the nicotine is well-integrated into the granules. The nicotine loaded granules were mixed with mint flavor, pH regulator (sodium carbonate) and pouched. Two study groups based on calcium sulphate as carrier material were prepared containing 3 mg and 6 mg nicotine (SERATEK). ZYN cool mini dry mint, 6 mg was used as control product. SERATEK contains in descending order of weight; calcium sulphate (E516), aromas, pharmaceutical grade nicotine, pH adjuster (E500), flavor enhancer (table salt) and sweetener (E950). 3. PK-study Outline A randomized, controlled, cross-over nicotine pharmacokinetic study of 12 healthy volunteer subjects was conducted. The primary objective of the study was to assess the pharmacokinetics (PK) of nicotine following the administration of nicotine pouches (3 mg and 6 mg) to current, daily nicotine users. Secondary objectives included nicotine extraction from the pouches during use and pulse rate increase of the subjects. Non-adjusted and baseline-adjusted PK parameters for nicotine were measured as primary objective, including: Area under the curve (AUC) from time 0 to infinity (AUC 0-∞ ) and from time 0 to 30 minutes (AUC 0-30min ) Maximum plasma concentration (Cmax). Time to maximum concentration (Tmax). Terminal elimination half-life (T₁/₂). The Secondary Objectives include Nicotine Extraction Assessment: To determine the amount of nicotine extracted from the pouches during use by daily nicotine users. In vivo extracted amount of nicotine (mg/unit). Extracted fraction (%) of nicotine for each individual product (IP). Pulse Rate Assessment: To assess the impact of nicotine pouches on pulse rate. Highest recorded increase (Emax) in pulse rate from baseline. Change from baseline in pulse rate following administration. Plasma nicotine concentration, time to maximum concentration, and pulse rate data were analyzed using Shapiro-Wilk normality tests to determine the distribution characteristics of each dataset. Subsequently, both paired parametric tests (paired t-tests) and paired non-parametric tests (paired Wilcoxon signed-rank tests) were utilized where appropriate. These evaluations specifically examined differences in pharmacokinetic parameters, time to maximum concentration, and pulse rate effects between ZYN 6mg and SERATEK 3mg, and also between ZYN 6mg and SERATEK 6mg products. All p-values are reported as two-sided with the significance level set at p < 0.05. Descriptive statistics such as mean, standard deviation, median, minimum, maximum, and sample size for continuous data, along with count and percentage for categorical data, were provided where appropriate. The clinical study protocol, the subject information and the informed consent form was approved by the Swedish ethical review authority. The study was conducted in the accordance with the ethical principles from the Declaration of Helsinki, International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Good Clinical Practice (GCP) E6 (R2) guidance, applicable parts of the European Union (EU) Clinical Trials Directive 2001/20/EC, and applicable local regulatory requirements. 3.2. Study Population A total of 12 subjects were enrolled in the study as planned. One subject withdrew consent after visit 3 and did not complete the study. However, all remaining subjects successfully completed the study, and all were included in both the pharmacokinetic (PK) and pharmacodynamic (PD) analysis sets. The participating subjects had the following descriptive information: Sex (Female/Male): 7 (58%) / 5 (42%) Age Mean (SD): 31.9 (8.7), Median: 29.0, Range: [25.0, 55.0] Height Mean (SD): 172.7 (6.7), Median: 174.5, Range: [163.0, 187.0] Weight Mean (SD): 72.0 (11.2), Median: 68.7, Range: [52.5, 90.5] BMI Mean (SD): 24.1 (3.4), Median: 23.7, Range: [19.8, 30.4] All subjects had a history of oral tobacco/nicotine use for at least one year, with a minimum daily consumption of 5 or more nicotine pouches containing 3–9 mg nicotine/pouch. Subjects were excluded if they had any history of clinically significant disease or disorder that, in the investigator’s opinion, could pose a risk to the subject due to study participation, or could potentially affect the study’s results or the subject’s ability to participate. 3.3. Methodology Subjects participated in a total of four visits, including a screening visit (Visit 1) followed by three study visits (Visits 2–4) on separate days. At least one day separated each study visit, during which subjects were allowed to use their regular nicotine products ad libitum. Screening (Visit 1) Screening occurred within four weeks before the first use visit. During screening, eligibility was assessed based on: - A review of smoking and oral tobacco/nicotine use. - A physical examination, including the measurement of vital signs (pulse rate and blood pressure), electrocardiogram (ECG), height, weight, and BMI. - Collection of medical history. Study Visits (Visits 2–4) Participants were required to abstain from all nicotine and tobacco products (including cigarettes and e-cigarettes) for at least 12 hours prior to each study visit. All nicotine pouch administrations took place in the morning (08:00 to 12:00) to facilitate compliance with the abstinence period. During each study visit, a randomized, single pouch was administered. The nicotine pouches were placed between the subject's upper lip and gum for a 30-minute duration. Subjects were instructed to: - Gently run their tongue over the pouch at 10 and 20 minutes, but to avoid further manipulation with their tongue or lips. - Refrain from eating, drinking, chewing gum, or brushing their teeth for 30 minutes before, during, and after the use of the pouch. 3.3. Nicotine Extraction and Blood sampling After the 30-minute use period, the used pouches were collected and immediately frozen at temperatures below − 20°C for later analysis of residual nicotine content. Unused pouches from the same batch were similarly stored and served as reference samples. Blood samples for the measurement of nicotine plasma levels were collected at pre-determined timepoints, starting from pre-administration and extending up to 8 hours after each pouch use. These samples were used to calculate various PK parameters. The PD effects were evaluated by pulse rate measurements. Throughout the study, adverse events (AEs) were monitored and recorded systematically. This methodology allows for the detailed evaluation of nicotine pharmacokinetics, extraction efficiency, and physiological responses following the use of nicotine pouches by daily nicotine users. To analyze the nicotine content in the blood samples, a liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was utilized, employing the Xevo TQ-S analytical instrument, manufactured by Waters Corporation. Plasma nicotine concentration, time to maximum concentration, and pulse rate data were analyzed using Shapiro-Wilk normality tests to determine the distribution characteristics of each dataset. Subsequently, both paired parametric tests (paired t-tests) and paired non-parametric tests (paired Wilcoxon signed-rank tests) were utilized where appropriate. These evaluations specifically examined differences in pharmacokinetic parameters, time to maximum concentration, and pulse rate effects between ZYN 6mg and SERATEK 3mg, and also between ZYN 6mg and SERATEK 6mg products. All p-values are reported as two-sided with the significance level set at p < 0.05. Descriptive statistics such as mean, standard deviation, median, minimum, maximum, and sample size for continuous data, along with count and percentage for categorical data, were provided where appropriate. 4. Results 4.1 PK-data Nicotine exposures, both in terms of Cmax and AUCs, increased with the increased nicotine content of the SERATEK 6 mg and ZYN products as compared to SERATEK 3 mg, see Table 1 . The largest nicotine exposures were seen following use of SERATEK 6 mg and the lowest following use of SERATEK 3 mg, see Fig. 1 . The Cmax was the highest after use of SERATEK 6 mg and was reached after 30 minutes for both SERATEK products, as compared to 40 minutes for ZYN, see Fig. 2 . Consistent results were obtained based on non-baseline and baseline adjusted nicotine plasma concentrations. Both the 3mg and 6mg doses of SERATEK exhibit a faster initial release (0-5min) compared to the 6mg ZYN, see Fig. 3 . Out of the 12 subjects, 10 subjects in the ZYN group show lower plasma nicotine concentration at 5 min compared to SERATEK 3mg and 6mg. There were no serious adverse events (SAEs), or AEs leading to withdrawal from the study. Table 1 Baseline adjusted PK parameters. Assessment (unit) SERATEK 3mg (N = 12) SERATEK 6mg (N = 11) ZYN 6mg (N = 12) T max (min), Median (min, max) 30.5 (15, 40) 30 (15, 50) 40 (30, 50) C max (ng/mL), Mean (SD) 6.1 (1.5) 11.2 (2.6) 9.9 (3.9) AUC 0 − 30min (h*ng/mL), Mean (SD) 2,1 (0.7) 3.6 (0.9) 2.8 (1.2) AUC 0−∞ (h*ng/mL), Mean (SD) 16.7 (4.2) 34.7 (8.4) 28.9 (11.7) T1/2 (h), Mean (SD) 2.4 (0.8) 2.4 (0.6) 2.3 (0.5) When comparing SERATEK 6mg and ZYN 6mg, the AUC 0-8h was non-significant with higher values for SERATEK 6mg (p = 0.083), while the AUC 0-30min was significantly higher for SERATEK 6mg (p = 0.034), see Table 1 . The Cmax 0-8h was non-significant but higher for SERATEK 6mg (p = 0.14) as was Cmax ₀-₃₀min (p = 0.16). However, Cmax0-5min was significantly higher for SERATEK 6mg (p = 0.039). The Tmax 0-8h was significantly faster for SERATEK 6mg (p = 0.0028). Comparing the pharmacokinetic parameters between SERATEK 3mg and ZYN 6mg, the following results were observed: The area under the curve (AUC 0 − 8h ) was significantly higher for ZYN (p < 0.001), while the AUC 0 − 30min was non-significant but still higher for ZYN (p = 0.077). The maximum plasma concentration (Cmax 0 − 8h ) was significantly higher for ZYN (p = 0.0021), as was the Cmax 0 − 30min (p = 0.0058). However, the Cmax 0 − 5min was non-significant with higher values for SERATEK 3mg (p = 0.25). The time to maximum concentration (Tmax₀-₈H) was significantly faster for SERATEK 3 mg (p = 0.0012). 4.3 Amount of released nicotine The mean estimated orally extracted amount of nicotine use was 1.5, 3.2 and 2.4 mg from the SERATEK 3 mg, SERATEK 6 mg and ZYN products, respectively, corresponding to extracted fractions of 56, 56 and 43%. A larger fraction of nicotine was orally extracted from the SERATEK products as compared to the ZYN product, see Fig. 4 a. For statistical significance it's essential to consider the variation among subjects. Therefore, paired statistical methods are appropriate, given that there is an inherent difference in nicotine release dependent on subject, see Fig. 3 . Out of the 12 subjects and two strengths (totaling 23, as one participant did not complete the final visit for SERATEK 6mg), 3 subjects showed a higher release from ZYN than from SERATEK formulations, see Fig. 4 b. Both SERATEK 3mg and SERATEK 6mg released a significantly higher percentage of nicotine compared to ZYN 6mg, with p-values of 0.0026 and 0.0043, respectively 4.4. Pulse The mean maximum effect (Emax) for pulse rate for the three investigational products (IPs) was between 18 and 24 beats per minute. The time to reach Emax was shorter for the SERATEK products compared to ZYN, as shown in Fig. 5. The corresponding times to reach Emax were 18 minutes for SERATEK 6mg, 10 minutes for SERATEK 3mg, and 19 minutes for ZYN. Specifically, more than half (55%) of the peak effects occurred within the first 5 minutes for SERATEK 6mg, compared to 8% for SERATEK 3mg and 25% for ZYN. In a comparison between SERATEK 3mg and ZYN 6mg, the maximum pulse rate within the first 30 minutes was found to be non-significant (p = 0.63), with ZYN showing a higher mean of 2.5 beats. However, within the first 5 minutes, the maximum pulse rate was significant (p = 0.036), with SERATEK 3mg showing a higher mean of approximately 3 beats. Additionally, the time to reach maximum concentration within the first 30 minutes was non-significant (p = 0.72), with ZYN 6mg being faster by approximately 1 minute. In a comparison between SERATEK 6mg and ZYN 6mg, the increase in maximum pulse rate within the first 30 minutes was not statistically significant (p = 0.16), despite SERATEK 6mg showing a higher average increase of 4 beats and reaching this level approximately 6 minutes faster than ZYN. However, within the first 5 minutes, the difference became statistically significant (p = 0.0032), with SERATEK 6mg showing a higher average increase of approximately 12 beats. 5. Discussion The study on the pharmacokinetics, pulse rate, and nicotine extraction in dry nicotine pouches provides valuable insights into the performance of SERATEK 3mg, SERATEK 6mg, and ZYN 6mg. Nicotine pouches have emerged as an alternative to traditional nicotine delivery methods such as smoking and snus, particularly focusing on formulations based on cellulose as carrier/bulk material. The uptake of nicotine from SERATEK at both 3 mg and 6 mg doses is significantly faster than from ZYN 6 mg. This rapid uptake is particularly evident during the initial 5 minutes. This rapid uptake reflects the mode of action of the calcium sulphate as carrier in products, with faster release from SERATEK 3mg and 6mg due to the product’s nicotine solubility characteristics (higher pH). This aligns with the introduction's emphasis on the need for nicotine pouches that can provide a more rapid nicotine uptake, akin to the swift delivery experienced in smoking or vaping. Pulse measurements, used as a measure of effect, indicate a higher initial pulse for SERATEK 3 mg and 6 mg compared to ZYN mini dry 6 mg. When comparing SERATEK 6mg and ZYN 6mg, there is no statistically significant difference in the maximum concentration (Cmax) between the products. The area under the curve (AUC) over the 8-hour observation period remains similar across products. This consistency in AUC aligns with the introduction's discussion on optimizing delivery systems to meet user preferences and health standards. The results from the study show that novel calcium sulphate carriers for the nicotine in pouches can provide more rapid uptake of nicotine. The mode-of-action for the uptake likely results from the higher pH of the calcium sulphate, in contrast to the nicotine bitartrate salt, which has a notably lower pH of 3.2, whilst calcium sulphate dihydrate maintains a pH of 7.7 [ 1 ]. At a pH of 8, approximately 57% of the nicotine is in its base form [ 6 ], which clearly contrasts with the bitartrate where 0% is in the base form. Given that the dissolution time for nicotine bitartrate is about 5–8 minutes [ 7 ], the actual difference in the early uptake and the Tmax could be mainly attributed to the pH and dissolution time difference of the nicotine in the two technologies. Additionally, the nicotine in the calcium sulphate granules is very confined in size (due to the small pores), providing a more rapid dissolution than crystalline nicotine bitartrate. 6. Conclusions The study on the pharmacokinetics, pulse, and nicotine extraction in dry nicotine pouches demonstrates that the novel calcium sulphate carriers used in SERATEK 3 mg and 6 mg pouches provide a more rapid nicotine uptake compared to the ZYN 6 mg pouches. This rapid uptake is particularly evident during the initial use. Declarations Conflicts of interest All authors are employed or consultants of Emplicure AB. AE, JL, GK, MP and HE have financial interest in Emplicure. Ethics approval The clinical study protocol, the subject information and the informed consent form was approved by the Swedish ethical review authority. The study was conducted in the accordance with the ethical principles from the Declaration of Helsinki, International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Good Clinical Practice (GCP) E6 (R2) guidance, applicable parts of the European Union (EU) Clinical Trials Directive 2001/20/EC, and applicable local regulatory requirements. Concent to participate All authors have read and approved the final manuscript. All authors consent to participate. Competing Interests All authors are employed or consultants of Emplicure AB. AE, JL, GK, MP and HE have financial interest in Emplicure. Funding The research has been funded by Emplicure AB. Author Contribution All authors contributed to the study conception and design. The manuscript preparation, data collection and analysis were performed by Hakan Engqvist, Ulrik Birgersson, Jesper Loof and Anna Franzén. Material preparation was performed by Maria Pokosta and Glebs Kiesolvs. The first draft of the manuscript was written by Hakan Engqvist and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript. Availability of data and materials On request References L. Benowitz, The central role of pH in the clinical pharmacology of nicotine: implications for abuse liability, cigarette harm reduction and FDA regulation, Clin. Pharmacol Ther. 2022 May: 111(5): 1004-1006 Travis et. Al. The potential impact of oral nicotine pouches on pulbic health: a scoping review, Nicotine and Tobacco Research, 2024, xx, pp. 1-13, https://doi.org/10.1093/ntr/ntae131 Michael McEwan, David Azzopardi, Nathan Gale, Oscar M. Camacho, George Hardie, Ian M. Fearon, James Murphy, A Randomised Study to Investigate the Nicotine Pharmacokinetics of Oral Nicotine Pouches and a Combustible Cigarette, European journal of drug metabolism and pharmacokinetics (2022) 47:211-221 LIVSFS 2013:8, Föreskrifter om ändring i Livsmedelsverkets föreskrifter [LIVSFS 2012:6] om snus och tuggtobak https://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=184.1230&SearchTerm=calcium Li, H. Zhang, et. al., Direct determination of free nicotine content in Tobacco, ACS Omega 2022, 7, 23061-23068 Chia-Yu Chen, K. D. Papadopoulos, Temperature and salting out effects on nicotine dissolution kinetics in saline solutions, ACS Omega, 2020, 5, 7738-7744 Additional Declarations Competing interest reported. All authors are employed or consultants of Emplicure AB. AE, JL, GK, MP and HE have financial interest in Emplicure. Cite Share Download PDF Status: Under Review Version 1 posted Editorial decision: Revision requested 18 Apr, 2025 Reviewers agreed at journal 17 Apr, 2025 Reviews received at journal 15 Apr, 2025 Reviewers agreed at journal 03 Apr, 2025 Reviews received at journal 11 Mar, 2025 Reviewers agreed at journal 07 Mar, 2025 Reviewers invited by journal 06 Mar, 2025 Editor assigned by journal 06 Mar, 2025 Submission checks completed at journal 04 Mar, 2025 First submitted to journal 03 Feb, 2025 You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-5953198","acceptedTermsAndConditions":true,"allowDirectSubmit":false,"archivedVersions":[],"articleType":"Clinical trial","associatedPublications":[],"authors":[{"id":437872529,"identity":"7674b046-f2a7-4323-a6dd-028ea20e1577","order_by":0,"name":"Anna Franzen","email":"","orcid":"","institution":"Emplicure AB","correspondingAuthor":false,"prefix":"","firstName":"Anna","middleName":"","lastName":"Franzen","suffix":""},{"id":437872530,"identity":"876c241f-4ec8-49d5-bccc-7fa7bddb9b1d","order_by":1,"name":"Jesper Loof","email":"","orcid":"","institution":"Emplicure AB","correspondingAuthor":false,"prefix":"","firstName":"Jesper","middleName":"","lastName":"Loof","suffix":""},{"id":437872531,"identity":"665fe1cd-0239-4fbf-8a2a-4535588e7ebd","order_by":2,"name":"Ulrik Birgersson","email":"","orcid":"","institution":"Emplicure AB","correspondingAuthor":false,"prefix":"","firstName":"Ulrik","middleName":"","lastName":"Birgersson","suffix":""},{"id":437872532,"identity":"a976bdb5-61ff-44ec-9465-5056f9dbadde","order_by":3,"name":"Glebs Kiselovs","email":"","orcid":"","institution":"Emplicure AB","correspondingAuthor":false,"prefix":"","firstName":"Glebs","middleName":"","lastName":"Kiselovs","suffix":""},{"id":437872533,"identity":"70029e30-9df4-47a4-a4f0-703d14307581","order_by":4,"name":"Maria Pokosta","email":"","orcid":"","institution":"Emplicure AB","correspondingAuthor":false,"prefix":"","firstName":"Maria","middleName":"","lastName":"Pokosta","suffix":""},{"id":437872534,"identity":"d3617ab9-3e51-4b6a-9a0c-1e799209dd96","order_by":5,"name":"Hakan Engqvist","email":"data:image/png;base64,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","orcid":"","institution":"Emplicure AB","correspondingAuthor":true,"prefix":"","firstName":"Hakan","middleName":"","lastName":"Engqvist","suffix":""}],"badges":[],"createdAt":"2025-02-03 18:38:05","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-5953198/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-5953198/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":79915657,"identity":"26a34ca7-3877-4765-a9a5-7ae57ba3cccc","added_by":"auto","created_at":"2025-04-04 12:34:56","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":613733,"visible":true,"origin":"","legend":"\u003cp\u003eNicotine plasma concentration over time, each point represents the mean plasma concentration for all subjects. (a) represents the full 8h study time and (b) the 30 minutes the subjects used the individual products.\u003c/p\u003e","description":"","filename":"image1.png","url":"https://assets-eu.researchsquare.com/files/rs-5953198/v1/1cd94bd166793a0a7a654681.png"},{"id":79915662,"identity":"a08f21a7-2cb6-4222-9109-d1b61e273e8c","added_by":"auto","created_at":"2025-04-04 12:34:56","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":497276,"visible":true,"origin":"","legend":"\u003cp\u003eDistribution of the subjects C\u003csub\u003emax\u003c/sub\u003e and T\u003csub\u003emax\u003c/sub\u003e for the three investigational products.\u003c/p\u003e","description":"","filename":"image2.png","url":"https://assets-eu.researchsquare.com/files/rs-5953198/v1/86fe9aec79d957632639f7ff.png"},{"id":79915658,"identity":"ab018db2-c7d7-48c3-bff3-cd9d62d7d6b8","added_by":"auto","created_at":"2025-04-04 12:34:56","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":699047,"visible":true,"origin":"","legend":"\u003cp\u003eComparison between (a) SERATEK 3mg and (b) 6mg and ZYN 6mg. The subject´s nicotine plasma concentrations have been compared towards ZYN, i.e. SERATEK 3mg – ZYN 6mg and SERATEK 6mg – ZYN 6mg, using box-and-dot plots and mean lines.\u003c/p\u003e","description":"","filename":"image3.png","url":"https://assets-eu.researchsquare.com/files/rs-5953198/v1/86e80ed260aa52daff469325.png"},{"id":79917977,"identity":"f1dc5444-9b20-4679-a9c5-91da8f700789","added_by":"auto","created_at":"2025-04-04 13:06:56","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1403811,"visible":true,"origin":"","legend":"\u003cp\u003e(a) Nicotine release comparison between SERATEK 3mg and SERATEK 6mg to ZYN 6mg. (b) Individual subject-level comparison of nicotine release between SERATEK 3mg and SERATEK 6mg versus ZYN 6mg .\u003c/p\u003e","description":"","filename":"image4.png","url":"https://assets-eu.researchsquare.com/files/rs-5953198/v1/ed4a8fa2b336c134765e52c1.png"},{"id":79915660,"identity":"b3590cb7-5e6e-4c5c-80a0-3532e2a21dc1","added_by":"auto","created_at":"2025-04-04 12:34:56","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":624825,"visible":true,"origin":"","legend":"\u003cp\u003ePulse comparison between pulse difference between (a) SERATEK 3mg and (b) SERATEK 6mg to ZYN, using box-and-dot plots and mean lines.\u003c/p\u003e","description":"","filename":"image5.png","url":"https://assets-eu.researchsquare.com/files/rs-5953198/v1/2e3632daad1f0a696dfaeda2.png"},{"id":79917978,"identity":"cc00e2f1-3ce5-4331-998e-56b7dee3f73c","added_by":"auto","created_at":"2025-04-04 13:07:02","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4362970,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-5953198/v1/e9b322d6-31a9-4684-9d53-450332dd04a1.pdf"}],"financialInterests":"Competing interest reported. All authors are employed or consultants of Emplicure AB. AE, JL, GK, MP and HE have financial interest in Emplicure.","formattedTitle":"Comparative study of calcium sulphate and cellulose based nicotine pouch on pharmacokinetics, pulse rate and nicotine extraction","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eNicotine pouches have emerged as a significant alternative to traditional nicotine delivery methods such as smoking and snus, particularly focusing on dry and wet formulations based on cellulose as carrier/bulk material. These products offer a discreet and smokeless mode of nicotine consumption, catering to a market segment that prioritizes reduced risk and convenience. The current technology behind nicotine pouches emphasizes delivering nicotine effectively while minimizing the harmful byproducts associated with combustion. The release and uptake kinetics of nicotine from the pouches are controlled by the release rate of nicotine and the pH of the product [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eDespite the advancements, there remains a critical need for nicotine pouches that can provide a more rapid nicotine uptake, akin to the swift delivery experienced in smoking or vaping. Achieving this could significantly drive the transition from traditional cigarettes and electronic nicotine delivery systems to nicotine pouches, aligning with public health initiatives aimed at reducing smoking prevalence.\u003c/p\u003e \u003cp\u003eDevelopment poised to address these gaps by developing an innovative nicotine pouch featuring a novel carrier and loading method. This technology aims to enhance the bioavailability and rapidity of nicotine delivery, potentially outperforming existing products and encouraging a shift from traditional smoking and vaping to pouches. This approach could significantly impact public health by offering a more effective and appealing alternative to conventional nicotine consumption methods [\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe future of nicotine pouches lies in continued research and development, focusing on optimizing the delivery systems to meet user preferences and health standards. By integrating advanced materials science, pharmacokinetics, and user behavior analysis, the next generation of nicotine pouches could represent a paradigm shift in harm reduction and nicotine replacement therapy [\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eEfforts to develop a new innovative formulation of nicotine pouches, based on calcium sulphate bioceramic technology. Calcium sulphate has several uses in the food industry and is on the list of permitted food additives [\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e]. Calcium sulphate is generally recognized as safe (GRAS) by the United States Food and Drug Administration (FDA) [\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThis report presents a comparative analysis of the nicotine exposures of three different formulations: SERATEK 3 mg, SERATEK 6 mg, and dry ZYN 6mg. Where SERATEK 3 mg and SERATEK 6 mg are based on loading nicotine into a calcium sulphate bioceramic formulation and ZYN in a cellulose formulation. The study aims to evaluate the pharmacokinetic (PK) parameters, pulse rate and nicotine extraction following single oral use of these products.\u003c/p\u003e"},{"header":"2. Materials","content":"\u003cp\u003eNicotine was incorporated into calcium sulphate dihydrate granules by dissolving nicotine salt (nicotine bitartrate) into the hydration liquid used during the setting process. This method ensures homogeneous distribution of nicotine throughout the calcium sulphate matrix as it transitions from the hemihydrate to the dihydrate form.\u003c/p\u003e \u003cp\u003eTo prepare the nicotine-loaded calcium sulphate granules, the specific amount of nicotine was first dissolved in the required volume of hydration liquid (water). The nicotine solution was then added to calcium sulphate hemihydrate powder and thereby initiating the hydration and setting process. As the material begins to set, the calcium sulphate hemihydrate reacts with the water to form calcium sulphate dihydrate, trapping the dissolved nicotine within the pore system. This ensures that the nicotine is well-integrated into the granules.\u003c/p\u003e \u003cp\u003eThe nicotine loaded granules were mixed with mint flavor, pH regulator (sodium carbonate) and pouched. Two study groups based on calcium sulphate as carrier material were prepared containing 3 mg and 6 mg nicotine (SERATEK). ZYN cool mini dry mint, 6 mg was used as control product. SERATEK contains in descending order of weight; calcium sulphate (E516), aromas, pharmaceutical grade nicotine, pH adjuster (E500), flavor enhancer (table salt) and sweetener (E950).\u003c/p\u003e"},{"header":"3. PK-study Outline","content":"\u003cp\u003eA randomized, controlled, cross-over nicotine pharmacokinetic study of 12 healthy volunteer subjects was conducted. The primary objective of the study was to assess the pharmacokinetics (PK) of nicotine following the administration of nicotine pouches (3 mg and 6 mg) to current, daily nicotine users. Secondary objectives included nicotine extraction from the pouches during use and pulse rate increase of the subjects.\u003c/p\u003e\n\u003cp\u003eNon-adjusted and baseline-adjusted PK parameters for nicotine were measured as primary objective, including:\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eArea under the curve (AUC) from time 0 to infinity (AUC\u003csub\u003e0-\u0026infin;\u003c/sub\u003e) and from time 0 to 30 minutes (AUC\u003csub\u003e0-30min\u003c/sub\u003e)\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eMaximum plasma concentration (Cmax).\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eTime to maximum concentration (Tmax).\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eTerminal elimination half-life (T₁/₂).\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003eThe Secondary Objectives include\u003c/p\u003e\n\u003cp\u003eNicotine Extraction Assessment: To determine the amount of nicotine extracted from the pouches during use by daily nicotine users.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\n \u003cp\u003e\u003cem\u003eIn vivo\u003c/em\u003e extracted amount of nicotine (mg/unit).\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eExtracted fraction (%) of nicotine for each individual product (IP).\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003ePulse Rate Assessment: To assess the impact of nicotine pouches on pulse rate.\u003c/p\u003e\n\u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eHighest recorded increase (Emax) in pulse rate from baseline.\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eChange from baseline in pulse rate following administration.\u003c/p\u003e\n \u003c/li\u003e\n\u003c/ul\u003e\n\u003cp\u003ePlasma nicotine concentration, time to maximum concentration, and pulse rate data were analyzed using Shapiro-Wilk normality tests to determine the distribution characteristics of each dataset. Subsequently, both paired parametric tests (paired t-tests) and paired non-parametric tests (paired Wilcoxon signed-rank tests) were utilized where appropriate. These evaluations specifically examined differences in pharmacokinetic parameters, time to maximum concentration, and pulse rate effects between ZYN 6mg and SERATEK 3mg, and also between ZYN 6mg and SERATEK 6mg products. All p-values are reported as two-sided with the significance level set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Descriptive statistics such as mean, standard deviation, median, minimum, maximum, and sample size for continuous data, along with count and percentage for categorical data, were provided where appropriate.\u003c/p\u003e\n\u003cp\u003eThe clinical study protocol, the subject information and the informed consent form was approved by the Swedish ethical review authority. The study was conducted in the accordance with the ethical principles from the Declaration of Helsinki, International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Good Clinical Practice (GCP) E6 (R2) guidance, applicable parts of the European Union (EU) Clinical Trials Directive 2001/20/EC, and applicable local regulatory requirements.\u003c/p\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n \u003ch2\u003e3.2. Study Population\u003c/h2\u003e\n \u003cp\u003eA total of 12 subjects were enrolled in the study as planned. One subject withdrew consent after visit 3 and did not complete the study. However, all remaining subjects successfully completed the study, and all were included in both the pharmacokinetic (PK) and pharmacodynamic (PD) analysis sets.\u003c/p\u003e\n \u003cp\u003eThe participating subjects had the following descriptive information:\u003c/p\u003e\n \u003cul\u003e\n \u003cli\u003e\n \u003cp\u003eSex (Female/Male): 7 (58%) / 5 (42%)\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eAge Mean (SD): 31.9 (8.7), Median: 29.0, Range: [25.0, 55.0]\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eHeight Mean (SD): 172.7 (6.7), Median: 174.5, Range: [163.0, 187.0]\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eWeight Mean (SD): 72.0 (11.2), Median: 68.7, Range: [52.5, 90.5]\u003c/p\u003e\n \u003c/li\u003e\n \u003cli\u003e\n \u003cp\u003eBMI Mean (SD): 24.1 (3.4), Median: 23.7, Range: [19.8, 30.4]\u003c/p\u003e\n \u003c/li\u003e\n \u003c/ul\u003e\n \u003cp\u003eAll subjects had a history of oral tobacco/nicotine use for at least one year, with a minimum daily consumption of 5 or more nicotine pouches containing 3\u0026ndash;9 mg nicotine/pouch.\u003c/p\u003e\n \u003cp\u003eSubjects were excluded if they had any history of clinically significant disease or disorder that, in the investigator\u0026rsquo;s opinion, could pose a risk to the subject due to study participation, or could potentially affect the study\u0026rsquo;s results or the subject\u0026rsquo;s ability to participate.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Methodology\u003c/h2\u003e\n \u003cp\u003eSubjects participated in a total of four visits, including a screening visit (Visit 1) followed by three study visits (Visits 2\u0026ndash;4) on separate days. At least one day separated each study visit, during which subjects were allowed to use their regular nicotine products ad libitum.\u003c/p\u003e\n \u003cp\u003e\u003cem\u003eScreening (Visit 1)\u003c/em\u003e\u003c/p\u003eScreening occurred within four weeks before the first use visit. During screening, eligibility was assessed based on:\n\u003c/div\u003e\n\u003cdiv class=\"Section2\"\u003e- A review of smoking and oral tobacco/nicotine use.\u003c/div\u003e\n\u003cdiv class=\"Section2\"\u003e- A physical examination, including the measurement of vital signs (pulse rate and blood pressure), electrocardiogram (ECG), height, weight, and BMI.\u003c/div\u003e\n\u003cdiv class=\"Section2\"\u003e- Collection of medical history.\u003cp\u003e\u003cem\u003eStudy Visits (Visits 2\u0026ndash;4)\u003c/em\u003e\u003c/p\u003e\n \u003cp\u003eParticipants were required to abstain from all nicotine and tobacco products (including cigarettes and e-cigarettes) for at least 12 hours prior to each study visit. All nicotine pouch administrations took place in the morning (08:00 to 12:00) to facilitate compliance with the abstinence period.\u003c/p\u003e\n \u003cp\u003eDuring each study visit, a randomized, single pouch was administered. The nicotine pouches were placed between the subject\u0026apos;s upper lip and gum for a 30-minute duration. Subjects were instructed to:\u003c/p\u003e- Gently run their tongue over the pouch at 10 and 20 minutes, but to avoid further manipulation with their tongue or lips.\u003cp\u003e- Refrain from eating, drinking, chewing gum, or brushing their teeth for 30 minutes before, during, and after the use of the pouch.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n \u003ch2\u003e3.3. Nicotine Extraction and Blood sampling\u003c/h2\u003e\n \u003cp\u003eAfter the 30-minute use period, the used pouches were collected and immediately frozen at temperatures below \u0026minus;\u0026thinsp;20\u0026deg;C for later analysis of residual nicotine content. Unused pouches from the same batch were similarly stored and served as reference samples.\u003c/p\u003e\n \u003cp\u003eBlood samples for the measurement of nicotine plasma levels were collected at pre-determined timepoints, starting from pre-administration and extending up to 8 hours after each pouch use. These samples were used to calculate various PK parameters. The PD effects were evaluated by pulse rate measurements. Throughout the study, adverse events (AEs) were monitored and recorded systematically.\u003c/p\u003e\n \u003cp\u003eThis methodology allows for the detailed evaluation of nicotine pharmacokinetics, extraction efficiency, and physiological responses following the use of nicotine pouches by daily nicotine users.\u003c/p\u003e\n \u003cp\u003eTo analyze the nicotine content in the blood samples, a liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) method was utilized, employing the Xevo TQ-S analytical instrument, manufactured by Waters Corporation.\u003c/p\u003e\n \u003cp\u003ePlasma nicotine concentration, time to maximum concentration, and pulse rate data were analyzed using Shapiro-Wilk normality tests to determine the distribution characteristics of each dataset. Subsequently, both paired parametric tests (paired t-tests) and paired non-parametric tests (paired Wilcoxon signed-rank tests) were utilized where appropriate. These evaluations specifically examined differences in pharmacokinetic parameters, time to maximum concentration, and pulse rate effects between ZYN 6mg and SERATEK 3mg, and also between ZYN 6mg and SERATEK 6mg products. All p-values are reported as two-sided with the significance level set at p\u0026thinsp;\u0026lt;\u0026thinsp;0.05. Descriptive statistics such as mean, standard deviation, median, minimum, maximum, and sample size for continuous data, along with count and percentage for categorical data, were provided where appropriate.\u003c/p\u003e\n\u003c/div\u003e"},{"header":"4. Results","content":"\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e4.1 PK-data\u003c/h2\u003e \u003cp\u003eNicotine exposures, both in terms of Cmax and AUCs, increased with the increased nicotine content of the SERATEK 6 mg and ZYN products as compared to SERATEK 3 mg, see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The largest nicotine exposures were seen following use of SERATEK 6 mg and the lowest following use of SERATEK 3 mg, see Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The Cmax was the highest after use of SERATEK 6 mg and was reached after 30 minutes for both SERATEK products, as compared to 40 minutes for ZYN, see Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e. Consistent results were obtained based on non-baseline and baseline adjusted nicotine plasma concentrations. Both the 3mg and 6mg doses of SERATEK exhibit a faster initial release (0-5min) compared to the 6mg ZYN, see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Out of the 12 subjects, 10 subjects in the ZYN group show lower plasma nicotine concentration at 5 min compared to SERATEK 3mg and 6mg. There were no serious adverse events (SAEs), or AEs leading to withdrawal from the study.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eBaseline adjusted PK parameters.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAssessment (unit)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eSERATEK\u003c/p\u003e \u003cp\u003e3mg (N\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eSERATEK \u003c/p\u003e \u003cp\u003e6mg (N\u0026thinsp;=\u0026thinsp;11)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eZYN\u003c/p\u003e \u003cp\u003e6mg (N\u0026thinsp;=\u0026thinsp;12)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT\u003csub\u003emax\u003c/sub\u003e (min), Median (min, max)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e30.5 (15, 40)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e30 (15, 50)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e40 (30, 50)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eC\u003csub\u003emax\u003c/sub\u003e (ng/mL), Mean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e6.1 (1.5)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.2 (2.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e9.9 (3.9)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAUC\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;30min\u003c/sub\u003e (h*ng/mL), Mean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2,1 (0.7)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e3.6 (0.9)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.8 (1.2)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eAUC\u003csub\u003e0\u0026minus;\u0026infin;\u003c/sub\u003e (h*ng/mL), Mean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e16.7 (4.2)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e34.7 (8.4)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e28.9 (11.7)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eT1/2 (h), Mean (SD)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c2\"\u003e \u003cp\u003e2.4 (0.8)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e2.4 (0.6)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.3 (0.5)\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eWhen comparing SERATEK 6mg and ZYN 6mg, the AUC\u003csub\u003e0-8h\u003c/sub\u003e was non-significant with higher values for SERATEK 6mg (p\u0026thinsp;=\u0026thinsp;0.083), while the AUC\u003csub\u003e0-30min\u003c/sub\u003e was significantly higher for SERATEK 6mg (p\u0026thinsp;=\u0026thinsp;0.034), see Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e. The Cmax\u003csub\u003e0-8h\u003c/sub\u003e was non-significant but higher for SERATEK 6mg (p\u0026thinsp;=\u0026thinsp;0.14) as was Cmax\u003csub\u003e₀-₃₀min\u003c/sub\u003e (p\u0026thinsp;=\u0026thinsp;0.16). However, Cmax0-5min was significantly higher for SERATEK 6mg (p\u0026thinsp;=\u0026thinsp;0.039). The Tmax\u003csub\u003e0-8h\u003c/sub\u003e was significantly faster for SERATEK 6mg (p\u0026thinsp;=\u0026thinsp;0.0028).\u003c/p\u003e \u003cp\u003eComparing the pharmacokinetic parameters between SERATEK 3mg and ZYN 6mg, the following results were observed: The area under the curve (AUC\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;8h\u003c/sub\u003e) was significantly higher for ZYN (p\u0026thinsp;\u0026lt;\u0026thinsp;0.001), while the AUC\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;30min\u003c/sub\u003e was non-significant but still higher for ZYN (p\u0026thinsp;=\u0026thinsp;0.077). The maximum plasma concentration (Cmax\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;8h\u003c/sub\u003e) was significantly higher for ZYN (p\u0026thinsp;=\u0026thinsp;0.0021), as was the Cmax\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;30min\u003c/sub\u003e (p\u0026thinsp;=\u0026thinsp;0.0058). However, the Cmax\u003csub\u003e0\u0026thinsp;\u0026minus;\u0026thinsp;5min\u003c/sub\u003e was non-significant with higher values for SERATEK 3mg (p\u0026thinsp;=\u0026thinsp;0.25). The time to maximum concentration (Tmax₀-₈H) was significantly faster for SERATEK 3 mg (p\u0026thinsp;=\u0026thinsp;0.0012).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e4.3 Amount of released nicotine\u003c/h2\u003e \u003cp\u003eThe mean estimated orally extracted amount of nicotine use was 1.5, 3.2 and 2.4 mg from the SERATEK 3 mg, SERATEK 6 mg and ZYN products, respectively, corresponding to extracted fractions of 56, 56 and 43%. A larger fraction of nicotine was orally extracted from the SERATEK products as compared to the ZYN product, see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea.\u003c/p\u003e \u003cp\u003eFor statistical significance it's essential to consider the variation among subjects. Therefore, paired statistical methods are appropriate, given that there is an inherent difference in nicotine release dependent on subject, see Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e. Out of the 12 subjects and two strengths (totaling 23, as one participant did not complete the final visit for SERATEK 6mg), 3 subjects showed a higher release from ZYN than from SERATEK formulations, see Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eb. Both SERATEK 3mg and SERATEK 6mg released a significantly higher percentage of nicotine compared to ZYN 6mg, with p-values of 0.0026 and 0.0043, respectively\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e4.4. Pulse\u003c/h2\u003e \u003cp\u003eThe mean maximum effect (Emax) for pulse rate for the three investigational products (IPs) was between 18 and 24 beats per minute. The time to reach Emax was shorter for the SERATEK products compared to ZYN, as shown in Fig.\u0026nbsp;5. The corresponding times to reach Emax were 18 minutes for SERATEK 6mg, 10 minutes for SERATEK 3mg, and 19 minutes for ZYN. Specifically, more than half (55%) of the peak effects occurred within the first 5 minutes for SERATEK 6mg, compared to 8% for SERATEK 3mg and 25% for ZYN.\u003c/p\u003e \u003cp\u003eIn a comparison between SERATEK 3mg and ZYN 6mg, the maximum pulse rate within the first 30 minutes was found to be non-significant (p\u0026thinsp;=\u0026thinsp;0.63), with ZYN showing a higher mean of 2.5 beats. However, within the first 5 minutes, the maximum pulse rate was significant (p\u0026thinsp;=\u0026thinsp;0.036), with SERATEK 3mg showing a higher mean of approximately 3 beats. Additionally, the time to reach maximum concentration within the first 30 minutes was non-significant (p\u0026thinsp;=\u0026thinsp;0.72), with ZYN 6mg being faster by approximately 1 minute.\u003c/p\u003e \u003cp\u003eIn a comparison between SERATEK 6mg and ZYN 6mg, the increase in maximum pulse rate within the first 30 minutes was not statistically significant (p\u0026thinsp;=\u0026thinsp;0.16), despite SERATEK 6mg showing a higher average increase of 4 beats and reaching this level approximately 6 minutes faster than ZYN. However, within the first 5 minutes, the difference became statistically significant (p\u0026thinsp;=\u0026thinsp;0.0032), with SERATEK 6mg showing a higher average increase of approximately 12 beats.\u003c/p\u003e "},{"header":"5. Discussion","content":"\u003cp\u003eThe study on the pharmacokinetics, pulse rate, and nicotine extraction in dry nicotine pouches provides valuable insights into the performance of SERATEK 3mg, SERATEK 6mg, and ZYN 6mg. Nicotine pouches have emerged as an alternative to traditional nicotine delivery methods such as smoking and snus, particularly focusing on formulations based on cellulose as carrier/bulk material.\u003c/p\u003e \u003cp\u003eThe uptake of nicotine from SERATEK at both 3 mg and 6 mg doses is significantly faster than from ZYN 6 mg. This rapid uptake is particularly evident during the initial 5 minutes. This rapid uptake reflects the mode of action of the calcium sulphate as carrier in products, with faster release from SERATEK 3mg and 6mg due to the product\u0026rsquo;s nicotine solubility characteristics (higher pH). This aligns with the introduction's emphasis on the need for nicotine pouches that can provide a more rapid nicotine uptake, akin to the swift delivery experienced in smoking or vaping.\u003c/p\u003e \u003cp\u003ePulse measurements, used as a measure of effect, indicate a higher initial pulse for SERATEK 3 mg and 6 mg compared to ZYN mini dry 6 mg. When comparing SERATEK 6mg and ZYN 6mg, there is no statistically significant difference in the maximum concentration (Cmax) between the products. The area under the curve (AUC) over the 8-hour observation period remains similar across products. This consistency in AUC aligns with the introduction's discussion on optimizing delivery systems to meet user preferences and health standards.\u003c/p\u003e \u003cp\u003eThe results from the study show that novel calcium sulphate carriers for the nicotine in pouches can provide more rapid uptake of nicotine. The mode-of-action for the uptake likely results from the higher pH of the calcium sulphate, in contrast to the nicotine bitartrate salt, which has a notably lower pH of 3.2, whilst calcium sulphate dihydrate maintains a pH of 7.7 [\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. At a pH of 8, approximately 57% of the nicotine is in its base form [\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], which clearly contrasts with the bitartrate where 0% is in the base form. Given that the dissolution time for nicotine bitartrate is about 5\u0026ndash;8 minutes [\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e], the actual difference in the early uptake and the Tmax could be mainly attributed to the pH and dissolution time difference of the nicotine in the two technologies. Additionally, the nicotine in the calcium sulphate granules is very confined in size (due to the small pores), providing a more rapid dissolution than crystalline nicotine bitartrate.\u003c/p\u003e"},{"header":"6. Conclusions","content":"\u003cp\u003eThe study on the pharmacokinetics, pulse, and nicotine extraction in dry nicotine pouches demonstrates that the novel calcium sulphate carriers used in SERATEK 3 mg and 6 mg pouches provide a more rapid nicotine uptake compared to the ZYN 6 mg pouches. This rapid uptake is particularly evident during the initial use.\u003c/p\u003e"},{"header":"Declarations","content":"\u003ch2\u003eConflicts of interest\u003c/h2\u003e\n\u003cp\u003eAll authors are employed or consultants of Emplicure AB. AE, JL, GK, MP and HE have financial interest in Emplicure.\u003c/p\u003e\n\u003ch2\u003eEthics approval\u003c/h2\u003e\n\u003cp\u003eThe clinical study protocol, the subject information and the informed consent form was approved by the Swedish ethical review authority. The study was conducted in the accordance with the ethical principles from the Declaration of Helsinki, International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Good Clinical Practice (GCP) E6 (R2) guidance, applicable parts of the European Union (EU) Clinical Trials Directive 2001/20/EC, and applicable local regulatory requirements.\u003c/p\u003e\n\u003ch2\u003eConcent to participate\u003c/h2\u003e\n\u003cp\u003eAll authors have read and approved the final manuscript. All authors consent to participate.\u003c/p\u003e\n\u003ch2\u003e\u003cstrong\u003eCompeting Interests\u003c/strong\u003e\u003c/h2\u003e\n\u003cp\u003eAll authors are employed or consultants of Emplicure AB. AE, JL, GK, MP and HE have financial interest in Emplicure.\u003c/p\u003e\n\u003ch2\u003eFunding\u003c/h2\u003e\n\u003cp\u003eThe research has been funded by Emplicure AB.\u003c/p\u003e\n\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\n\u003cp\u003eAll authors contributed to the study conception and design. The manuscript preparation, data collection and analysis were performed by Hakan Engqvist, Ulrik Birgersson, Jesper Loof and Anna Franz\u0026eacute;n. Material preparation was performed by Maria Pokosta and Glebs Kiesolvs. The first draft of the manuscript was written by Hakan Engqvist and all authors commented on previous versions of the manuscript. All authors read and approved the final manuscript.\u003c/p\u003e\n\u003ch2\u003eAvailability of data and materials\u003c/h2\u003e\n\u003cp\u003eOn request\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eL. Benowitz, The central role of pH in the clinical pharmacology of nicotine: implications for abuse liability, cigarette harm reduction and FDA regulation, Clin. Pharmacol Ther. 2022 May: 111(5): 1004-1006\u003c/li\u003e\n\u003cli\u003eTravis et. Al. The potential impact of oral nicotine pouches on pulbic health: a scoping review, Nicotine and Tobacco Research, 2024, xx, pp. 1-13, https://doi.org/10.1093/ntr/ntae131\u003c/li\u003e\n\u003cli\u003eMichael McEwan, David Azzopardi, Nathan Gale, Oscar M. Camacho, George Hardie, Ian M. Fearon, James Murphy, A Randomised Study to Investigate the Nicotine Pharmacokinetics of Oral Nicotine Pouches and a Combustible Cigarette, European journal of drug metabolism and pharmacokinetics (2022) 47:211-221\u003c/li\u003e\n\u003cli\u003eLIVSFS 2013:8, F\u0026ouml;reskrifter om \u0026auml;ndring i Livsmedelsverkets f\u0026ouml;reskrifter [LIVSFS 2012:6] om snus och tuggtobak\u003c/li\u003e\n\u003cli\u003ehttps://www.accessdata.fda.gov/scripts/cdrh/cfdocs/cfcfr/CFRSearch.cfm?fr=184.1230\u0026amp;SearchTerm=calcium\u003c/li\u003e\n\u003cli\u003eLi, H. Zhang, et. al., Direct determination of free nicotine content in Tobacco, ACS Omega 2022, 7, 23061-23068\u003c/li\u003e\n\u003cli\u003eChia-Yu Chen, K. D. Papadopoulos, Temperature and salting out effects on nicotine dissolution kinetics in saline solutions, ACS Omega, 2020, 5, 7738-7744\u003c/li\u003e\n\u003c/ol\u003e\n\u003cp\u003e\u0026nbsp;\u003c/p\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":true,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"discover-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Medicine](https://link.springer.com/journal/44337)","snPcode":"44337","submissionUrl":"https://submission.springernature.com/new-submission/44337/3","title":"Discover Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-5953198/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-5953198/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eThis study presents a comparative analysis of nicotine pouches using calcium sulphate and cellulose as carrier materials. A randomized, controlled, cross-over nicotine pharmacokinetic study of 12 healthy volunteer subjects with the primary objective to evaluate the pharmacokinetics, pulse rate, and nicotine extraction efficiency of three different formulations: SERATEK 3mg, SERATEK 6mg, and dry ZYN 6mg was conducted. SERATEK formulations utilized calcium sulphate, while ZYN contained cellulose as nicotine carrier. The study aims to address the need for rapid nicotine uptake, akin to facilitate replacement of smoking or vaping, by developing an innovative nicotine pouch with enhanced bioavailability.\u003c/p\u003e \u003cp\u003eResults indicate that SERATEK formulations provide a faster initial release and higher nicotine uptake compared to ZYN, particularly within the first 5 minutes of use. This rapid uptake is attributed to higher pH for nicotine loaded into calcium sulphate granules in the SERATEK formulations. The findings suggest that calcium sulphate-based nicotine pouches could offer a more effective alternative to traditional nicotine delivery methods, potentially driving a shift from smoking and vaping to nicotine pouches.\u003c/p\u003e \u003cp\u003eThe study has been registered at clinicaltrials.gov with ID: NCT06170138 at 2023-11-24\u003c/p\u003e","manuscriptTitle":"Comparative study of calcium sulphate and cellulose based nicotine pouch on pharmacokinetics, pulse rate and nicotine extraction","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-04-04 12:34:51","doi":"10.21203/rs.3.rs-5953198/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-04-18T10:08:13+00:00","index":"","fulltext":""},{"type":"reviewerAgreed","content":"241558849601521717830214270942138860939","date":"2025-04-17T19:43:49+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-04-15T21:36:12+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"290082335170137506495183356480520450024","date":"2025-04-03T06:18:29+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-03-11T06:07:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"272699569245811185336617405042290946454","date":"2025-03-07T05:34:58+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-03-06T11:38:15+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-03-06T08:35:44+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-03-04T05:12:12+00:00","index":"","fulltext":""},{"type":"submitted","content":"Discover Medicine","date":"2025-02-03T18:22:17+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"discover-medicine","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":false,"externalIdentity":"","sideBox":"Learn more about [Discover Medicine](https://link.springer.com/journal/44337)","snPcode":"44337","submissionUrl":"https://submission.springernature.com/new-submission/44337/3","title":"Discover Medicine","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"stoa","reportingPortfolio":"Discover Series","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"f4effa81-81f3-4427-93af-f145d47fadc3","owner":[],"postedDate":"April 4th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"under-review","subjectAreas":[],"tags":[],"updatedAt":"2025-07-05T09:08:23+00:00","versionOfRecord":[],"versionCreatedAt":"2025-04-04 12:34:51","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-5953198","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-5953198","identity":"rs-5953198","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}