Methods and compositions for increasing NAD+ metabolome in a healthy middle-aged population

Abstract

This invention provides a composition for increasing NAD levels in human subjects using effective amounts of D-ribose and nicotinamide. [Solution] A composition administered to a human subject to increase the NAD metabolome in the human subject, comprising an effective amount of D-ribose and nicotinamide, wherein the effective amount is administered to the subject morning and evening for 8 days, and by the 5th day, the serum total glutathione level increases by at least 10% compared to baseline.

Description

Technical Field

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63137720, filed on January 14, 2021, which is hereby incorporated by reference in its entirety.

Background Art

[0002] The nicotinamide adenine dinucleotide (NAD+) metabolome, including NAD+, NADH, NADP+, and NADPH, is extremely important for human health. Since NAD+ is associated with energy production and oxidative stress, its decline is correlated with aging and diseases. NAD+, NADH, NADP+, and NADPH are recyclable coenzymes for various oxidation-reduction (redox) reactions and for energy homeostasis, which decomposes nutrients and converts them into energy in the form of adenosine triphosphate (ATP). NAD+ and NADP+ are the consumable substrates of enzyme reactions that regulate important biological processes, including gene expression, energy homeostasis, DNA repair, apoptotic cell death and lifespan, calcium signaling, glucose homeostasis, and circadian rhythm. [1-5] As a coenzyme, the NAD+ metabolome is involved in more than 60% of the reactions in cell metabolism, and their homeostasis is a determining factor for maintaining the redox balance and metabolism. [1,2] As a consumable substrate, the NAD+ concentration is directly related to NAD-consuming enzymes, including aging, premature aging [6] and fat composition [7], poly(ADP-ribose) polymerase (PARP), sirtuin (SIRT1-7), and cADP-ribose synthase (CD38), and has a wide range of effects on the health and diseases, especially aging and age-related chronic degenerative diseases.

[0003] As shown below, there are four NAD+ biosynthetic pathways that act in mammals [9], including the de novo pathway starting from the amino acid tryptophan and the following three alternative pathways of pyridine salvage. Pathway #1: The de novo pathway from the amino acid tryptophan, which is represented as follows. Trp →NAD Pathway #2: The salvage pathway for nicotinamide (Nam), which can be described as follows: NAM+PRPP→NMN+ATP→NAD Pathway #3: The salvage pathway for nicotinic acid (Na), which can be expressed as follows: NA+PRPP→NAMN+ATP→NAAD→NAD Pathway #4: The salvage pathway for nicotinamide riboside (NR), which can be described as follows: NR+ATP→NAD During the ceremony, ATP = Adenosine triphosphate NA = Nicotinic acid NAAd = Nicotinate adenine dinucleotide NAD = Nicotinamide adenine dinucleotide NAM = Nicotinamide NAMN = Nicotinic acid mononucleotide NMN = Nicotinamide mononucleotide, NR = Nicotinamide riboside PRPP = Ribose Pirophosphate Trp = Tryptophan

[0004] PRPP and / or ATP are required in each of the three salvage pathways (pathways #2 to #4). Both PRPP and ATP are known to be expanded products of D-ribose (i.e., D-ribose + ATP PRPP). Pyridine, NA, NAM, and NR are collectively referred to as niacin or vitamin B3

[10] which can be produced from dietary intake and / or intracellular NAD+ catabolism. In the nascent pathway (pathway #1), the starting material for tryptophan is from dietary protein sources such as eggs, meat, and cheese. Nascent NAD synthesis is generally considered insufficient to maintain normal NAD homeostasis

[11] . Vitamin B3, commonly found in fortified foods and beverages, is limited in quantity because high intake of nicotinic acid (NA) causes flushing.

[12] Most NAD+ in mammals is synthesized from nicotinamide (NAM) via the amidation salvage pathway. NAM salvage is catalyzed by nicotinamide phosphoribosyltransferase (NAMPT)

[13] , which is under the regulation of circadian rhythms [4]. Some researchers believe that age-related decline in NAD+ is due to a decrease in NAMPT with aging.

[14] However, further evidence has been reported that NAD+ uptake can be increased by NAD+ uptake enzymes such as PARP, sirtuins, and CD38

[15] . More importantly, age-related NAM salvage capacity in human skeletal muscle can be reserved by exercise.

[16] Therefore, it is possible to increase NAD+ by regularly supplementing with NAM.

[0005] In the last decade, there has been considerable research into enhancing NAD+ by supplementing with NR.

[17] NR is a more advanced precursor in the NAD+ biosynthesis salvage pathway. NR is thought to be converted to NMN by NR kinase (NRK1 / 2) using ATP as a cosubstrate.

[18] Supplementing with NR has been shown to increase NAD levels, enhance oxidative metabolism, and reduce steatosis and hepatic steatohepatia. [17,18] However, its benefits are controversial. Firstly, several experiments have demonstrated that NR is very rapidly broken down into nicotinamide (NAM) and ribose, especially when taken orally. [19,20] Therefore, the reported benefits of NR are likely due to circulating nicotinamide or nicotinamide and ribose. Secondly, supplementing with NR in healthy subjects can reduce their exercise capacity, [21,22] and its use may be limited in healthy activity populations. To address these issues and overcome the shortcomings of these NRs, the applicant initiated a research study using different combinations of nicotinamide and D-ribose.

[0006] Nicotinamide (NAM) is a preferred treatment for pellagra.

[23] NAM is also used for acne and non-melanoma skin cancer.

[24] More recently, NAM has been considered a potential candidate for increasing the NAD+ metabolome for anti-aging applications.

[25] High doses of NAM did indeed enhance NAD+ levels and improve disease in obese rat models.

[26] Given its long-term application, the applicants identified several limiting factors. The recommended daily dietary intake to prevent vitamin B3 deficiency is only about 15 mg for adults.

[27] Doses exceeding 3 g per day may cause side effects, including hepatotoxicity.

[28] Accordingly, the tolerable upper dietary intake of NAM per day is set at 900 mg in the EU ("Opinion of the Scientific Committee on Food on the Tolerable Upper Intake Levels of Nicotinic Acid and Nicotinamide," published by the Scientific Committee on Food), 500 mg in Canada, and 5 mg / kg in Japan ("Overview of Dietary Reference Intakes for Japanese," published by the Ministry of Health, Labour and Welfare). Therefore, it is only practical to use NAM in a relatively low dose range, especially 100-500 mg per day. It is also important to note that NAM does not have the effect of increasing NAD+ at doses of less than 90 mg per day.

[26] Above 900 mg, adjustment challenges arise. Therefore, it is preferable to maximize its ability to increase NAD+ in the low dose range.

[0007] As disclosed in International Patent Application No. PCT / US2019 / 031889 (Publication No. WO2019 / 217935), data from the applicant's prior preclinical animal studies to determine the pharmacodynamics and tissue distribution of NAD+ metabolites have been constructed, which are incorporated herein by reference in their entirety. The applicant has developed a novel combination of NAM and D-ribose that amplifies NAM's ability to enhance NAD and reduces its potential aspects. This novel combination of NAM and D-ribose is available from Bioenergy Life Science, Inc., 13840 Johnson Street NE, Ham Lake, MN USA 55304, under the trademark RiaGev®.

[0008] As disclosed herein, the applicant conducted a clinical trial using the RiaGev product in a randomized, triple-blind, controlled, crossover pilot study investigating the efficacy and safety of RiaGev through the assessment of NAD+ metabolome and various health-related parameters in healthy adults aged 35–65 years. The clinical trial confirmed that supplementation with the RiaGev product increased the NAD+ metabolome in subjects and was safe and effective in preventing oxidation-reduction imbalances caused by intensive aerobic exercise in healthy, active middle-aged human subjects. [Brief explanation of the drawing]

[0009] [Figure 1] This is a diagram from a clinical trial. [Figure 2] This chart summarizes the number of participants screened, the number of participants who failed screening and the reasons for those failures, the number of participants who were randomized, and how the participants were grouped. [Figure 3] This table shows the mean and median clinical assessment values ​​for participants in the control-IP group and the IP-control group. [Figure 4A] This graph shows the primary NAD+ outcome for the control group and the RiaGev group. [Figure 4B] This graph shows the primary NADP+ outcome for the control group and the RiaGev group. [Figure 4C] Graph of the changes in NAD+ and NADP+ levels over time in the comparison control group and the RiaGev group. [Figure 5] Graph of the total serum ATP and ADP in the comparison control group and the RiaGev group. [Figure 6] Graph of the total serum glutathione in the comparison control group and the RiaGev group. [Figure 7] Graph of the salivary cortisol in the comparison control group and the RiaGev group. [Figure 8A] Graph of the OGTT blood glucose on day 8 vs. day 1 in the RiaGev group. [Figure 8B] Graph of the OGTT insulin on day 8 vs. day 1 in the RiaGev group. [Figure 8C] Graph of the OGTT blood glucose on day 8 vs. day 1 in the comparison control group. [Figure 8D] Graph of the OGTT insulin on day 8 vs. day 1 in the comparison control group. [Figure 9A] Graph of the change in NADPH / NADP+ in the comparison control group and the RiaGev group before and after exercise on day 1 vs. day 8. [Figure 9B] Graph of the change in GSH / GSSG in the comparison control group and the RiaGev group before and after exercise on day 1 vs. day 8. [Figure 10A] Graph of the CIS physical fatigue in the comparison control group and the RiaGev group from day 1 to day 8. [Figure 10B] Graph of the CIS concentration in the comparison control group and the RiaGev group from day 1 to day 8. [Figure 10C] Graph of the CIS motivation in the comparison control group and the RiaGev group from day 1 to day 8. [Figure 10D] Graph of the CIS total score in the comparison control group and the RiaGev group from day 1 to day 8.

[0010] Description In healthy adults aged 35 - 65 years, the efficacy and safety of nicotinamide (NAM) and D - ribose composition (sold under the trademark RiaGev®) were investigated in a randomized triple - blind comparative - control crossover study through the evaluation of the NAD+ metabolome and health - related parameters. This trial was approved by IntegReview, Internal Review Board (protocol code 19RNHB(1918)), and the study is registered on ClinicalTrials.gov with identifier NCT04483011.

[33]

[0011] The middle - aged period was selected as the study population because many health problems that directly affect healthy aging occur during this period. It is also the period of the greatest stress burden in life. Oxidative stress is a known factor for many chronic diseases and is harmful to healthy aging. [29,30] Two of the most common chronic diseases associated with aging are obesity and diabetes. In 2016, the World Health Organization (WHO) reported that approximately 1.6 million deaths were attributable to diabetes. Half of these individuals had high blood glucose by the age of 70 (3). Therefore, it is important to actively control blood glucose and oxidative stress during middle age. Therefore, stress parameters and blood glucose are secondary outcomes following the primary outcomes of the NAD+ metabolome in the clinical trial.

[0012] Study Population This study included healthy, active men and women aged 35–65 years. The primary selection criteria for participants included: having a body mass index (BMI) of 18.5–29.9 kg / m2; female participants were non-childbearing; participants were healthy based on laboratory results, medical history, physical examination, and electrocardiogram; participants agreed to avoid supplementation with tryptophan, vitamin B3, or its derivatives (niacin, nicotinic acid, niacinamide) one week prior to randomization and during the study; participants were able to complete maximal and submaximal exercise tests; participants agreed to maintain their current diet, activity level, and sleep cycle during the study; and participants agreed to follow all research procedures with voluntary, written, informed consent. Participants with disease or inflammatory conditions were excluded from this study. Detailed selection and exclusion criteria are listed under identifier NCT04483011 on ClinicalTrials.gov.

[33]

[0013] Comparison with the investigational drug The investigational drug (IP), RiaGev®, contained 1280 mg of D-ribose, 240 mg of nicotinamide, and 480 mg of palm oil in three capsules. D-ribose and nicotinamide were the active ingredients, and palm oil was an excipient. The batch number for RiaGev was S0776313. The control group contained 1280 mg of dextrose and 480 mg of palm oil, and was filled into three capsules of the same size and color as the IP. Dextrose was used to match the calorie content of D-ribose in the IP. The batch number of the control group was SI126314. Both the IP and the control group were provided by Bioenergy Life Science, Inc., 13840 Johnson Street NE, Ham Lake, MN, USA 55304.

[0014] Screening and assignment of participants to groups Referring to Figures 1 and 2, a total of 50 healthy men and women aged 35–65 years were screened as potential participants for the clinical trial. All potential participants were identified by initials and date of birth, and each was assigned a participant number at their initial screening visit (visit 1). Of the 50 individuals screened, 18 were determined to meet all selection criteria and not meet any exclusion criteria. These 18 eligible individuals were recruited for the study. At visit 2 (baseline), each of the 18 individuals (hereinafter referred to as participants) was assigned a randomized number by a blinded researcher. The randomized participant numbers were generated via a randomized list generator. Subsequently, the 18 participants were randomly assigned to two matching groups of nine participants each, referred to as the "IP to comparison group" and the "comparison group to IP group," based on demographic and physical information such as age, sex, weight, and height. The BMI, heart rate, and hemoglobin A1c of each group were also statistically similar.

[0015] Administration All participants were instructed to take two capsules daily, once in the morning and once in the evening. Each dose consisted of three capsules, administered immediately before breakfast and immediately before dinner. During both supplementation periods, only the evening dose was administered on day 1, and only the morning dose was administered on day 8. Participants were instructed to retain all unused and opened capsule packages and return them for compliance assessment. If a dose was missed, participants were instructed to take the missed dose at any time during the same day, except at bedtime. Participants were advised not to exceed the two daily doses.

[0016] During each supplementation period, one group received a dose of IP capsules twice daily, while the other group received a control dose of capsules twice daily. After the replenishment period, all participants were washed for 7 days, after which they switched to taking capsules of the other product. For clarity, participants in the IP to control group were initially administered IP capsules during the first replenishment period of the study (i.e., the first set of days 1-8). After the 7-day washing period, the IP to control group were then administered the control capsules during the second replenishment period of the study (i.e., the second set of days 1-8). Conversely, participants in the control to IP group were initially administered the control capsules during the first replenishment period of the study (i.e., the first set of days 1-8). After the 7-day washing period, the control to IP group were administered IP capsules during the second replenishment period of the study (i.e., the second set of days 1-8).

[0017] Blind The clinical trial was a triple-blind study conducted by Prism Clinical Research, Minneapolis, MN. The IP and control subjects were sealed in identical packages, each labeled according to the requirements of ICH-GCP guidelines and applicable local regulatory guidelines. Labeling of the IP and control packages was performed by an open-label staff member at Prism Clinical Research who was not involved in any trial assessment. A randomization schedule was created and provided to the Prism Clinical Research investigators, indicating the order of randomization. All Prism Clinical Research investigators, including the principal investigator, other on-site personnel, and participants, were blinded to the IP and control subjects.

[0018] Clinical assessment, blood sampling, and analysis Prism Clinical Research measured participants' height, weight, blood pressure, and heart rate using standard procedures. For participants of childbearing potential, Prism Clinical Research performed urine pregnancy tests (Henry Schein One Step+) at visits 1 and 2. Figure 3 shows tables of mean and median measured parameters and clinical assessments for each of the IP-control group and the control-IP group.

[0019] Prism Clinical Research collected blood samples from each participant at visits 1 through 9 and analyzed them using the following procedure: 1) Whole blood was collected using a heparinized plasma tube (BD vacutainer, sodium heparin 95 USP Units, REF 367878); 2) After blood collection, the tube was gently mixed by inverting it 5-6 times, and a 400 μL aliquot of (accurately measured) blood was quickly prepared using a 2.0 mL pre-cooled Eppendorf tube (see catalog number below) at 4°C on ice; 3) The aliquot was immediately frozen using a dry ice bucket, then transferred to a -80°C freezer; 4) The frozen aliquot was transported on dry ice to the Northwest Metabolomics Research Center, University of Washington, Seattle, Washington, USA, for coenzyme analysis. Sufficient dry ice was placed in the transport box to ensure that the samples remained frozen until they were received.

[0020] Safety endpoints for whole blood count (differentiated WBC count, RBC count, hemoglobin, hematocrit, platelet count, RBC index (MCV, MCH, MCHC, RDW)), liver function (AST, ALT, bilirubin), and renal function blood tests (creatinine, eGFR, electrolytes) were analyzed using standardized procedures from blood samples collected at visits 1 (screening), 5, 6, and 9 by HCMC Pathology Lab, USA. Glucose and insulin were also analyzed by HCMC Pathology Lab at visits 2, 5, 6, and 9 using standardized procedures.

[0021] At visits 2, 5, 6, and 9, the Northwest Metabolomics Research Center analyzed glutathione (GSH), glutathione disulfide (GSSG), adenosine triphosphate (ATP), adenosine diphosphate (ADP), and adenosine monophosphate (AMP) using established NMR methodologies

[31] . At visits 2 through 9, NAD+, NADP+, and NADPH were also analyzed by the Northwest Metabolomics Research Center using established NMR methodologies

[31] .

[0022] CIS Questionnaire This study used the standard Checklist Individual Strength (CIS) questionnaire. The CIS questionnaire consists of 20 questions scored on a 7-point scale to measure subjective fatigue experiences, decreased concentration, decreased motivation, and decreased physical activity levels.

[32] Questions and scoring methods are as described in the references. The CIS questionnaire was administered to participants by Prism Clinical Research during visits 2 through 8.

[0023] Saliva collection Participants collected salivary cortisol samples using a Salivette collection device. Except for the screening visit (visit 1), saliva samples were collected within 15 minutes of waking up and before meals on all visits, in the morning. Participants received instructions from Prism Clinical Research to ensure proper collection.

[0024] Treadmill exercise data collection To determine the maximum heart rate of participants, Prism Clinical Research conducted an incline treadmill test following the ramp-type Bruce protocol during each participant's initial screening (Visit 1). Participants continued through the stages until they voluntarily fatigued. Throughout the test, participants' heart rates were monitored and recorded using a chest strap heart rate monitor. A good competition of the test was achieving 85% or more of the age-predicted maximum heart rate (220 - age).

[0025] Each participant performed additional treadmill exercises on visits 2 (period 1, day 1), 5 (period 1, day 8), 6 (period 2, day 1), and 9 (period 2, day 8). On the day of the treadmill exercise, participants were instructed to warm up on the treadmill for 5 minutes at a slow walking pace. Once participants were ready, they were instructed to increase the speed to 60% of their maximum heart rate (HR), set the incline to 5%, and walk on the treadmill for 30 minutes, or until fatigued.

[0026] Food records Participants were asked to record their food intake during the study. Participants' food records were analyzed using Nutritics software (Nutritics, 2019) to calculate daily calorie, macronutrient, and micronutrient intake throughout the study. Food records were reviewed by trained staff from Prism Clinical Research at each participant visit. Participants received counseling from Prism Clinical Research staff and dietary suggestions as needed. All participants were provided with instructions on how to complete their food records.

[0027] compliance Each participant's compliance with the study procedures was recorded by Prism Clinical Research staff in the relevant section of the compliance report at each visit. Each participant's compliance with the administration of IP and control was assessed by counting the unused IP and control capsules returned at each visit. Compliance was calculated by dividing the number of units taken by the number of units that were expected to have been taken multiplied by 100.

[0028] Recording of adverse events During the study, each participant recorded adverse events (AEs) in a diary. At each visit, participants were asked: "Have you experienced any difficulties or problems since our last meeting?" AEs reported by participants were recorded in the study log and categorized according to their description, duration, severity, frequency, and outcome. Principal investigators at Prism Clinical Research assessed the AEs and determined causal relationships.

[0029] statistical analysis For this study, the following analytical populations were defined: the Intention to Treat (ITT) population and the Per Protocol (PP) population. The ITT population consisted of all participants who received either product and for whom efficacy information after randomization was available. The ITT population was used to present all efficacy information according to the treatment to which subjects were randomized. The PP population consisted of all participants who received at least 80% of the IP and control doses, had no significant protocol violations, and completed all study visits and procedures related to the measurement of key variables.

[0030] For categorical variables, both counts and percentages were presented. Unless otherwise specified, the denominator for each percentage was the number of subjects within the research group. Potential differences between groups were assessed, where necessary, using two-tailed chi-squared tests or Fisher's exact tests.

[0031] For interval variables, the arithmetic mean, standard deviation, median, and minimum / maximum range were presented to two decimal places. These were accompanied by the number of participants included in the analysis at that time. Potential differences between groups at screening / baseline visits were assessed using ANOVA. For each group, changes in each outcome between study time points were assessed using a paired Student's t-test for normally distributed outcomes, or a Wilcoxon signed-rank test for other outcomes.

[0032] The change in the continuous endpoint from screening / baseline was calculated as follows: Change to Ti = Value at Ti - T screening / Value at baseline

[0033] Changes in the primary outcome and each secondary outcome were compared between groups using repeated measures mixed-model analysis of covariance (ANCOVA) for normally distributed or log-normal distributions. Each model included study group* time (study visit) as a fixed effect, baseline value of the dependent variable as a covariate, and subject as a random effect. P-values ​​between groups were obtained from the final model.

[0034] Descriptive analyses were provided for pre- and post-event adverse events (AEs) reported in this study. Furthermore, the relationship between outcomes and the study product was reported for each AE classified as potentially related to the study product. The number of participants with at least one AE was compared between study treatment groups using Fisher's exact test. Vital signs, hematological, and clinical chemistry parameters were summarized as mean, standard deviation, median, and minimum / maximum range. Changes from screening / baseline were assessed using paired t-tests.

[0035] All hypothesis tests were performed at a 5% (two-sided) significance level unless otherwise specified. P-values ​​were rounded to three decimal places. P-values ​​less than 0.001 were reported as <0.001, and P-values ​​of 0.05 or less were considered statistically significant. All analyses were performed using R Statistical Package version 3.6.3 for Microsoft Windows (R Core Team, 2020). Results - Primary Outcome - NAD + Metabolome

[0036] The primary outcome of this study was the NAD+ metabolome, particularly NAD+ levels, after supplementing the IP ("RiaGev group") compared to supplementing the control group ("control group"). As shown in Figure 4A, NAD+ levels in the RiaGev group steadily increased over baseline (day 1) after supplementation. On day 5, NAD+ concentrations in the RiaGev group were significantly higher than baseline, with a 10.4% increase (p=0.034), which was also significantly higher than the control group (p=0.044). On day 8, NAD+ levels also showed a significant increase of 6.4% from baseline (p=0.07). In comparison, NAD+ levels in the control group did not change significantly during the period.

[0037] We noticed an unexpectedly significant increase in NADP+ levels compared to a mild increase in NAD+ levels (Figure 4B). Significant intragroup increases of 19.1%, 27.6%, and 19.6% compared to baseline were recorded in the RiaGev group on days 3, 5, and 8, respectively (p ≤ 0.008). NADP+ concentrations in the RiaGev group were also significantly higher than in the control group (p ≤ 0.040).

[0038] Referring to Figure 4C, when NAD+ and NADP+ concentrations were combined on days 3, 5, and 8, the RiaGev group showed significantly higher levels than the control group on each day (p ≤ 0.029). Furthermore, the RiaGev group reported significant intragroup concentration increases of 9.4%, 14.8%, and 9.7% on days 3, 5, and 8, respectively (p ≤ 0.032).

[0039] In contrast to NAD+ and NADP+, NADPH concentrations did not change significantly throughout the study period, except for a significant decrease in NADPH concentration within the group after exercise using a control group on day 1 (p=0.039). NADH levels were not measured in this study because preservatives used during shipping disrupted the NMR signal. Whole blood 1-methylnicotinamide (MeNAM) and nicotinic acid adenine dinucleotide phosphate (NAAD(P)) were below the detection limit in blood samples.

[0040] In summary, compared to baseline (day 1) and the control group, the RiaGev group had higher blood NAD levels on day 5. + The concentration was 10.4% higher than the baseline (p=0.034) and the control group (p=0.044). NADP in the RiaGev group on day 5. + The concentration was 27.6% higher than baseline (p=0.007) and higher than the control group (p=0.033). NAD levels in the RiaGev group on day 5. + and NADP +The concentration combinations were 15% compared to the baseline (0.004) and 15% compared to the control group (p=0.014).

[0041] Results - Secondary Outcomes ATP is a universal energy carrier. Figure 5 shows the total serum ATP and ADP levels from day 1 to day 8 for the control group and the RiaGev group. Total ATP and ADP levels are reported because their sum is more accurately measured by NMR analysis than ATP alone. The total high-energy phosphate (ATP and ADP) was significantly higher in the RiaGev group than in the control group on day 5 (7.3% higher, p=0.029).

[0042] Glutathione is a circulating antioxidant produced in the body. Both reduced (GSH) and oxidized (GSSG) glutathione were measured in the blood. Total serum glutathione (GSH + GSSG) concentrations in the blood are shown in Figure 6. A significant increase in total glutathione was observed after RiaGev supplementation. Total glutathione concentrations in the RiaGev group on days 3 and 5 were 10.2% and 11.6%, respectively, higher than on day 1 (p ≤ 0.016). There was no significant change in total glutathione levels in the control group during the same period. In summary, total serum glutathione in the RiaGev group on day 5 was 11% higher than baseline (day 1), exceeding that of the control group (p = 0.003).

[0043] Awakened salivary cortisol levels are shown in Figure 7. In the RiaGev group, cortisol levels steadily decreased from day 1 onward with continued supplementation, while in the control group, cortisol levels fluctuated throughout the study period. There were significant intergroup differences in awakened salivary cortisol levels on days 5 and 8, with the RiaGev group showing significantly lower levels than the control group (p<0.044).

[0044] A postprandial oral glucose tolerance test (OGTT) was performed to assess blood glucose and insulin responses to standardized meals before and after 7 days of RiaGev supplementation. Figures 8A and 8B show postprandial blood glucose and insulin levels on day 1 (before supplementation) and day 8 (after supplementation on day 7), respectively, for the RiaGev group. In the RiaGev group, whole blood glucose (area under the rising curve, iAUC) on day 8 was significantly lower than on day 1 (61% decrease, p=0.013). However, overall insulin (iAUC) in the OGTT on day 8 was not significantly different from that on day 1 (p=0.793). On the other hand, the insulin peak on day 8 was higher than on day 1 (74 vs. 67 mcU / mL, respectively), but the glucose peak remained the same (116 vs. 114 mg / dL at 15 minutes postprandial on day 8 vs. day 1, respectively). As a result, glucose concentration on day 8 decreased more sharply than on day 1. Figures 8C and 8D show the postprandial blood glucose and insulin levels of the control group on day 1 (before supplementation) and day 8 (after supplementation on day 7), respectively. The blood glucose and insulin profiles of the control group did not differ significantly between day 8 and day 1.

[0045] Figures 9A and 9B show NADPH / NADP+ and GSH / GSSG for the control group and the RiaGev group before and after exercise from day 1 to day 8, respectively. Treadmill exercise was performed with each participant on day 1 (before supplementation) and day 8 (after supplementation). The treadmill incline and speed were gradually increased until the subject reached a maximum of 60% of VO2 maxima, and the subject continued at that pace until fatigue. Blood was collected immediately before and after exercise, and the redox balance, including NADPH / NADP+ and GSH / GSSG, as well as the energy charge ATP / AMP, were analyzed. Change is defined as the difference between the pre-exercise measurement and the post-exercise measurement. The NADPH / NADP+ and GSH / GSSG ratios decreased significantly on day 1 (p=0.003 and p=0.022), particularly in the control group, indicating redox impairment due to the submaximal exercise regimen. This redox perturbation was prevented by supplementing with RiaGev, and the redox ratio was maintained even during exercise on day 8. The control group did not exhibit the significant redox increase expected from the functionality of glucose within the control group.

[0046] As shown in Figure 9A, the NADPH / NADP+ ratio significantly decreased after exercise on day 1 in the control group (p=0.004), indicating that submaximal exercise significantly disrupts the target redox balance. This oxidative damage was prevented by 7 days of RiaGev supplementation, as shown on day 8, with the NADPH / NADP+ ratio remaining relatively unchanged before and after exercise. It is interesting to note that supplementation in the control group resulted in a slight increase in the NADPH / NADP+ and GSH / GSSG ratios. This is consistent with the function of the glucose component (dextrose) in the control group.

[0047] The Checklist Individual Strength (CIS) questionnaire includes a standard set of 20 questions with subscales for physical fatigue, mental focus, motivation, and physical activity.

[31] The total CIS score (Figure 10D) represents physical and mental fatigue with four subscales reflecting physical fatigue (Figure 10A), focus (Figure 10B), motivation (Figure 10C), and physical activity (not shown). Both the RiaGev group and the control group showed improvement in quality of life scores during the study. However, the improvement in the RiaGev group was consistently greater than that of the control group across all subscales. Specifically, on days 3, 5, and 8, the total CIS score improved by 21.5% (p=0.04) versus 10.4% (p=0.07), 18.3% (p=0.014) versus 6.2% (p=0.049), and 12.7% (p=0.15) versus 4.1% (p=0.361), respectively, in the RiaGev group versus the control group. However, the difference between the two groups did not reach a significant level (p=0.224).

[0048] Regarding the subscales, physical fatigue (Figure 10A) showed the greatest improvement relative to baseline and the greatest difference between groups. On days 3, 5, and 8, physical fatigue was 24.3% (p=0.003) vs. 13.6% (p=0.041), 21.2% (p=0.009) vs. 11.6% (p=0.08), and 15.1% between the RiaGev group and the control group, respectively. The improvement was 7.4% (p=0.17) compared to (p=0.132). Referring to Figure 10B, the concentration improved significantly in the RiaGev group on days 3, 5, and 8, at 22.9% (p=0.014), 19.8% (p=0.012), and 14.3% (p=0.118), respectively, while the control group showed no significant improvement on any of these days. A similar trend was observed for motivation (Figure 10C), with the RiaGev group improving by 20.4% (p=0.13), 22.2% (p=0.015), and 14% (p=0.163) on days 3, 5, and 8, respectively, compared to a statistically significant improvement in the control group. Of the subscales tested, the physical activity scale (not shown) did not improve in either the RiaGev group or the control group during the study period. This is to be expected, as a 7-day supplement period is not long enough to observe behavioral changes.

[0049] No clinically relevant changes were observed in physical measurements, vital signs, hematology, renal markers, or electrolytes in participants of this study before and after supplementation. All participants were deemed healthy by the principal investigator after both treatment periods.

[0050] In this study, a total of 12 post-occurrence adverse events (AEs) were reported by 10 participants. Of these, 9 were reported by 7 participants taking RiaGev, and 3 were reported by 2 participants taking the control group. None of the post-occurrence AEs were classified as "most likely" to be related to the product. Two AEs, weakness and loss of appetite, were classified as "possible" in the RiaGev group and as one of the joint pains in the control group. All AEs resolved by the end of the study. The principal investigator assessed that all subjects were healthy before and after the trial.

[0051] observation RiaGev® has been found to primarily enhance NADP+ rather than NAD+ (27% vs. 11% increase in studies), which is unprecedented among precursors that enhance NAD+. NADP+ is a more advanced product than NAD+, and its production requires additional high-energy phosphate. Therefore, a higher yield of NADP+ means a higher energy state in the body. This is consistent with an increase in high-energy phosphates (ATP and ADP) and glutathione in the circulating blood. It is also consistent with reduced fatigue, improved concentration, and increased motivation reported by subjects in the CIS questionnaire.

[0052] RiaGev supplementation was observed to alleviate physiological signs of stress compared to the control group, via salivary cortisol levels. This finding was supported by significant improvements of up to 24% or more in subjective fatigue, concentration, motivation, and total CIS score with RiaGev.

[0053] RiaGev was found to be safe and well-tolerated in healthy adults aged 35–65 years. Only two minor adverse events (weakness and appetite) were observed. Notably, there were no adverse events related to skin flushing, a common side effect of NAD precursor supplements. Furthermore, no changes related to clinical chemistry or hematology were observed.

[0054] It is noteworthy that MeNAM and NAAD(P) were not detected in the blood samples supplemented with RiaGev in this study. MeNAM and NAAD(P) are very common byproducts of NR and NAM supplements. [12,34] The combination of D-ribose and NAM in RiaGev clearly reduced the formation of MeNAM and NAAD(P). This is consistent with the enhanced production of the NAD+ metabolome and the potentially reduced side effects demonstrated in this study. Therefore, the combination of D-ribose and NAM provides a way to safely and effectively adopt NAM at higher doses.

[0055] Human clinical trials focused on a healthy, active middle-aged population. Previous studies had focused on either obese or older populations where oxidative stress had not been considered a contributing factor.[19,34,35] Oxidative stress is well-established as a common factor leading to disease and premature aging.[29,30,36] Some animal studies have shown that other NAD+ fortifiers, including NR, do not protect subjects from exercise-induced oxidative stress. On the contrary, NAD+ fortifiers, including NR, contribute to greater oxidative stress by depleting NADPH and glutathione.[21,22] Greater oxidative stress is potentially serious, as it is an integral part of daily life, especially for all active people in middle age. This study demonstrated that RiaGev enhances energy and glutathione levels and protects subjects from oxidative damage. This is clearly demonstrated in the preservation of redox homeostasis during intensive aerobic exercise, and in the preservation of stable, low salivary cortisol levels in the RiaGev group compared to the higher, more variable salivary cortisol levels in the control group.

[0056] Oxidative stress can be induced not only by everyday activities but also by some foods and beverages we consume daily, primarily from the intake of high-glycemic index foods, which leads to glucose intolerance and insulin resistance. RiaGev has not been found to acutely lower postprandial blood glucose peaks like D-ribose alone.

[37] Rather, RiaGev enhances glucose clearance from the bloodstream, causing glucose peaks to decrease more quickly. More importantly, it achieves an overall reduction in blood glucose without increasing insulin secretion. This suggests that RiaGev improves insulin sensitivity and glucose intolerance. This result is particularly important because the subjects in the RiaGev group have, firstly, high hemoglobin (HbA1c), which reflects the average daily glucose level in the blood. The mean HbA1c in the RiaGev group was 5.5%, which is typical for people around 50 years old in the United States and close to the upper limit for the healthy population (=5.7%). The significant overall reduction in blood glucose levels due to RiaGev supplementation in this population is particularly important, both scientifically and practically.

[0057] Despite its success, the clinical trial has its limitations. One obvious limitation was its relatively short duration. As this was RiaGev's first clinical trial, it was designed based on the applicant's preclinical animal trials, which are typically shorter in duration. Due to the short duration, the final clinical visit, day 8, saw high activity levels, resulting in sampling in the afternoon, significantly later than the sampling times on days 1, 3, and 5. This difference in sampling time is likely the main reason why the measurements on day 8 did not follow the trend of the measurements on days 3 and 5, and why the NAD+ metabolome and measurements from the CIS questionnaire were lower than expected. Previous studies have shown that the NAD+ metabolome is highly regulated by circadian rhythms, with lower NAD+ metabolome levels typically observed in the afternoon.

[0058] Both RiaGev and the control group showed improvement in CIS scores from day 1 onwards during the study period. This may be due to the fact that participants were required to keep food and sleep diaries during the study period, leading to more regular eating and sleeping habits, and consequently, improvements in blood glucose and CIS scores for all participants.

[0059] Blood glucose and insulin levels in the RiaGev group were consistently higher than those in the control group, particularly at baseline on day 1. This reflects the fact that the RiaGev-control and control-RiaGev sequence groups were not sufficiently identical in this aspect. Glycosylation The initial hemoglobin (HbA1c) levels in the RiaGev-comparison group were 5.50% (p=0.108) compared to 5.25% (p=0.108) in the comparison-RiaGev group. This means that the mean blood glucose level in the RiaGev group was initially more than 7 mg / dL higher than in the comparison group. After 7 days of supplementation, the overall blood glucose level in the RiaGev group decreased significantly (61%, p=0.013), and on day 8, the overall blood glucose levels were essentially the same between the two groups. This strongly suggests that RiaGev significantly improves blood glucose levels.

[0060] Given RiaGev's favorable safety profile and its strong improvement in NAD+ metabolome and blood glucose, future studies should expand the population to include those at risk of reduced NAD+ levels, such as the elderly, those with impaired glucose tolerance, and those suffering from complex aspects of metabolic syndrome, including prediabetes and diabetes. More generally, people suffering from oxidative stress may benefit from RiaGev. Compared to NR and NMN

[38] , NAM is less expensive and has a long history of safety

[12] . The combination of D-ribose and NAM in RiaGev may improve NAM metabolism and enhance human performance

[39] , potentially offering a novel approach to enhancing the NAD+ metabolome. On the other hand, RiaGev's excellent safety profile suggests that the combination of D-ribose and nicotinamide may offer a novel approach to expanding the use and dosage of this vitamin B3 for human benefit.

[0061] The RiaGev used in clinical trials contained effective amounts of nicotinamide and D-ribose, with an optimized nicotinamide-to-D-ribose ratio of approximately 1:5, achieving the results identified above. However, it is expected that using effective amounts of nicotinamide and D-ribose in a wide range of ratios and doses of nicotinamide to D-ribose will increase NAD levels in human subjects, and glutathione levels will also increase without causing redox imbalance. For example, as disclosed in International Patent Application No. PCT / US2019 / 031889 (Publication No. WO2019 / 217935), incorporated herein by reference, an effective amount of nicotinamide and D-ribose may have a nicotinamide-to-D-ribose ratio of 0.5:10 to 10:0.5, or a nicotinamide-to-D-ribose ratio of 1:5 to 5:1, and the effective amount of nicotinamide and D-ribose may be 20 mg to 5400 mg per day, or 100 mg to 4000 mg per day.

[0062] conclusion A randomized, triple-blind, controlled, and randomized preliminary study assessed the efficacy and safety of a nicotinamide and D-ribose combination (RiaGev®) in healthy adults. RiaGev supplementation effectively increased the concentration of the circulating blood NAD+ metabolome, particularly NADP+ levels. High-energy phosphate and glutathione levels in the blood were also enhanced. The RiaGev group showed significantly improved postprandial glucose tolerance. Circulating antioxidants, including GSH and NADPH, were also enhanced with RiaGev. This aspect was more pronounced during vigorous aerobic exercise, indicating that RiaGev maintains redox homeostasis disrupted by oxidative stress. The stress hormone arousal cortisol was also consistently lower in the RiaGev group than in the control group. CIS questionnaire assessments showed that RiaGev reduced physical fatigue and improved concentration, motivation, and overall well-being of subjects. In summary, RiaGev was found to be safe and well-tolerated in healthy adults, and its favorable safety profile suggests that the combination of D-ribose and nicotinamide may help expand the use of this vitamin B3 for a wide range of applications and human benefits.

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Claims

1. A method for increasing the NAD metabolome in a human subject by administering an effective amount of D-ribose and nicotinamide to the subject.

2. The method according to claim 1, wherein NAD metabolome and glutathione levels increase in the subject.

3. The method according to claim 2, wherein the NAD metabolome and glutathione levels increase without causing redox imbalance in the subject.

4. The method according to any one of claims 1 to 3, wherein the effective amount is in a ratio of nicotinamide to D-ribose of 0.5:10 to 10:0.

5.

5. The method according to any one of claims 1 to 3, wherein the effective amount is in a ratio of nicotinamide to D-ribose of 1:5 to 5:

1.

6. The method according to any one of claims 1 to 5, wherein the effective amount is 20 mg to 5400 mg per day.

7. The method according to any one of claims 1 to 5, wherein the effective amount is 100 mg to 4000 mg per day.

8. The method according to any one of claims 1 to 7, wherein the effective amount is administered to the subject in the morning and evening.

9. The method according to claim 8, wherein the effective amount is administered to the subject immediately before the subject eats breakfast in the morning and immediately before the subject eats dinner in the evening.

10. A composition administered to human subjects that increases the NAD metabolome in human subjects, A composition comprising an effective amount of D-ribose and nicotinamide.

11. The composition according to claim 10, wherein the composition increases the NAD metabolome and glutathione levels in the subject.

12. The composition according to claim 11, wherein the composition increases the NAD metabolome and glutathione levels in the subject without causing redox imbalance in the subject.

13. The composition according to any one of claims 10 to 12, wherein the effective amount is in a ratio of nicotinamide to D-ribose of 0.5:10 to 10:0.

5.

14. The composition according to any one of claims 10 to 12, wherein the effective amount is in a ratio of nicotinamide to D-ribose of 1:5 to 5:

1.

15. The composition according to any one of claims 10 to 14, wherein the effective amount is 20 mg to 5400 mg per day.

16. The composition according to any one of claims 10 to 14, wherein the effective amount is 100 mg to 4000 mg per day.

17. The composition according to any one of claims 10 to 16, wherein the composition is administered to the subject in the morning and evening.

18. The composition according to claim 17, wherein the composition is administered to the subject immediately before the subject eats breakfast in the morning and immediately before the subject eats dinner in the evening.

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