Sublingual NMN raises early blood levels of breakdown products 2PY and 4PY more than oral NMN in healthy men

No adverse events considered causally related to the consumption of the test substance occurred in this study.

This study was approved by the Ueno-Asagao Clinic Ethics Review Board on 02/11/2022 (approval number: 2022-38). The board is composed of independent third-party members not involved in the trial. All procedures were conducted in accordance with the ethical principles for medical research involving human subjects as stipulated in the Declaration of Helsinki. The study was registered in the University Hospital Medical Information Network (UMIN) clinical registry system prior to participant enrollment (trial ID: UMIN000049530, registration date: 17/11/2022). The trial was conducted by TES Holdings at the Ueno Asagao Clinic in Tokyo, Japan, and the study period, from screening to the final visit, took place between 11/2022 and 12/2022.

As this was an exploratory study, the sample size was determined as follows. The sample size was established based on a statistical power analysis with the following parameters: a significance level (α) of 0.05 for a two-sided test, a statistical power (1-β) of 0.80, and an effect size (Cohen’s d) of 0.8, which represents a large effect. The choice of effect size was informed by the lack of previous studies directly comparing sublingual and oral NMN administration. However, sublingual absorption is theoretically expected to result in substantially higher systemic delivery by avoiding the hepatic first-pass effect, a significant barrier for oral administration. Based on this rationale, we anticipated a large difference between the two routes and thus adopted a Cohen’s d of 0.8. The calculation, performed using a statistical analysis software package (BellCurve for Excel, Social Survey Research Information Co., Ltd. Tokyo, Japan), indicated that a minimum of 12 participants would be necessary to achieve the desired power. To accommodate for potential attrition or data loss during the study, the target sample size was set at 14 participants .

An allocation manager, independent of all other study personnel, used block randomization to assign the 14 eligible participants to one of two treatment sequences (n = 7 per sequence). Participants in Group A received oral administration in the first period, followed by sublingual administration in the second period. Conversely, participants in Group B began with sublingual administration, followed by oral administration.

This study was conducted as a randomized, two-period, two-group crossover trial with an 8-day washout period between interventions to minimize carryover effects. On the day before each test, participants consumed a standardized dinner (806 kcal; 131 g of carbohydrate, 29 g of protein, and 18 g of fat) and were instructed to finish the meal by 21:00. Thereafter, they were required to fast, with only the provided water permitted until bedtime; alcohol consumption was prohibited. On the morning of each test day, participants arrived at the facility in a fasted state, with water intake (up to one cup, approx. 200 mL) permitted until two hours prior to arrival. Upon arrival, a compliance check and a physical examination—including measurements of body weight, body fat percentage, BMI, blood pressure, and pulse rate, as well as a medical interview by a physician—were performed prior to the administration of the test substance. Participants were administered one tablet via two different methods. For oral administration, the tablet was swallowed with 100 mL of water after the mouth was rinsed. For sublingual administration, after rinsing the mouth, the tablet was placed under the tongue and held for 3 min to dissolve in saliva, without swallowing. After the 3-min period, the accumulated saliva and any tablet residue were swallowed without water. Blood samples were collected from the median cubital vein at baseline (pre-administration) and at 5, 15, 30, and 60 min post-administration. For both oral and sublingual administration, participants consumed an additional 100 mL of water immediately after the 5-min blood draw.

Throughout the study period, spanning from the initial screening to the conclusion of the second observation period, participants were instructed to adhere to a set of guidelines to ensure the integrity of the data. They were required to maintain their usual lifestyle concerning diet, exercise, and sleep, and to avoid irregularities such as sleep deprivation or overeating. Strenuous exercise and lack of sleep were specifically prohibited on the day preceding each testing session. Participants were also directed to refrain from excessive alcohol consumption and to avoid abrupt changes in their drinking habits. The consumption of any health foods, including Foods for Specified Health Uses (FOSHU) and Foods with Function Claims (FFC), as well as smoking, was strictly prohibited. In the event of unavoidable use of medications (including topical agents), quasi-drugs, or Kampo medicines, participants were required to notify the study coordinator and record the product name, manufacturer, and reason for use in a daily diary. This diary was to be completed daily and submitted on scheduled dates. Furthermore, participants were forbidden from enrolling in any other clinical studies or engaging in any other activities that could potentially interfere with the study outcomes.

Tablets containing 250 mg of β-NMN per tablet, together with 245 mg of excipient (SWELWiCK®, manufactured by Daicel Corporation) and 5 mg of lubricant (calcium stearate), were prepared. Crystalline NMN (lot no. HNH04-JP210401↗; purity, 99.9%) was purchased from Japan Bulk Co., Ltd (Osaka, Japan). The purity of NMN was determined using HPLC.

We measured concentrations of NMN (primary outcome), and its metabolites (secondary outcome), NAD⁺, nicotinamide adenine dinucleotide phosphate (NADP⁺), nicotinamide (NAM), N¹-methyl-2-pyridone-5-carboxamide (2PY), and N¹-methyl-4-pyridone-3-carboxamide (4PY). As an indicator of bioavailability, the pre-ingestion or baseline (0 min) concentrations were subtracted from subsequent measures to calculate the iAUC.

Fasting first-morning urine and blood samples were collected from the median cubital vein. Hematological analysis was performed to measure the following parameters: white blood cell count, red blood cell count, hemoglobin, hematocrit, mean corpuscular volume, mean corpuscular hemoglobin, mean corpuscular hemoglobin concentration, and platelet count. Blood biochemical tests included the assessment of: total protein, creatinine, uric acid, aspartate aminotransferase, alanine aminotransferase, gamma-glutamyl transpeptidase, alkaline phosphatase, lactate dehydrogenase, C-reactive protein, total cholesterol, triglycerides, low-density lipoprotein cholesterol, high-density lipoprotein cholesterol, calculated non-HDL-cholesterol, blood glucose, and hemoglobin A1c. Urinalysis was conducted to evaluate urobilinogen, occult blood, bilirubin, ketone bodies, glucose, protein, pH, and specific gravity. All blood and urine analyses were performed by Hoken Kagaku, Inc. (Kanagawa, Japan).

Levels of NMN and related metabolites (NMN, NAM, NAD⁺, NADP⁺, 2PY and 4PY) were assessed using high-performance liquid chromatography-tandem mass spectrometry (HPLC–MS/MS, HPLC; ACQUITY H-class Bio Binary system, Waters Corporation, Milford, MA, USA, MS/MS; TQ-XS, Waters Corporation). Immediately after collecting blood samples, ice-cold 800 μL methanol was added to 200 μL whole blood. The samples were mixed thoroughly and immediately stored at − 80℃ until analysis. For analysis, mixtures were centrifuged for 10 min at 4℃, 15,000 × g. A 40 μL aliquot was mixed with 2 μL internal standard solution and evaporated using a miVac concentrator (Genevac Limited, Gothenburg, Sweden) at 30℃. The dried extract was dissolved with 420 μL citric acid (0.1 mg/mL), and the suspension was filtered through a 0.2 μm filter before HPLC–MS/MS analysis. All analyses were performed on a 2.1 × 150 mm column with a 1.8 μm particle size (ACQUITY UPLC Premier HSS T3, Waters Corporation). Mobile phases A and B were 5 mM ammonium formate and acetonitrile, respectively. The initial eluent composition was 100% A with an increase to 10% B over 3 min. The 10% B concentration was maintained for 0.5 min and then increased to 90% B over 1 min, held at 90% B for 3.5 min, and then reduced to 0% B over 3 min. The total run time was 11 min. The eluent flow was 0.3 mL/min, and the column was maintained at 45℃. Analytes were detected using electrospray ionization in the positive mode. Multiple-reaction-monitoring (MRM) was performed using characteristic fragmentation ions (NAD⁺, m/z = 664.2/428.1, ¹³C⁵ NAD⁺ _IS, m/z = 669.1/428.0; NMN, m/z = 335.2/123.0, NADP⁺, m/z = 744.0/136.0, D⁴ NMN_IS, m/z = 339.1/127.0, NAM, m/z = 123.2/79.9; 2PY, m/z = 153.1/110.0, 4PY, m/z = 153.1/136.0, ¹³C⁶ NAM_IS, m/z = 129.0/85.4). The lower limit of quantification (LLOQ) in human whole blood for this analytical method was 7.5 nM for NMN, 3.8 nM for NAM, 18.8 nM for NAD⁺, 3.8 nM for NADP⁺, 3.8 nM for 2PY, and 3.8 nM for 4PY.

Symptom severity and outcome, as well as the frequency of adverse events and side effects that occurred from the start to the end of the study phase for all study participants who had consumed the test substance at least once were recorded. Any associations of the test substance with adverse events were evaluated.

The efficacy analysis population comprised participants who completed all study assessments. The changes in blood NMN-related metabolites over time were analyzed using a two-factor repeated-measures analysis of variance followed by post hoc paired Student’s t-test. The iAUC over 0–60 min was calculated using the trapezoidal rule. Blood NMN and its metabolites were baseline-corrected using fasting values. The iAUC data for NMN and its metabolites in blood were analyzed using the paired Student’s t-test. Differences between treatments were significant at P < 0.05. Statistical analyses were performed using SPSS version 26 (IBM Corp., Armonk, NY, USA).