Restorin^® NR is a Highly Efficient NAD⁺ Precursor

Abstract

Introduction

Nicotinamide adenine dinucleotide (NAD+) is vital for maintaining normal cellular function. Aging-associated NAD+ decline is linked to the onset and progression of age-related diseases. Consequently, oral supplementation of NAD+ precursors has been shown to improve disease-relevant phenotypes and physiological outcomes in model organisms. Among NAD+ precursors, nicotinamide riboside (NR) has attracted interest owing to its potent in vivo NAD+ biosynthetic activity. However, NR exhibits poor stability in the circulatory system. Thus, there is considerable interest in discovering effective NAD+ precursor forms that can address these limitations. Here, we report Restorin NR, a proprietary crystalline form of NR fumarate, as a novel NAD+ enhancer.

Methods

The current study was performed to compare the effectiveness of three different NAD+ precursors, including nicotinamide mononucleotide (NMN), NR chloride, and Restorin NR in boosting NAD+ levels in an aged murine model.

Results

The results showed that Restorin NR significantly increased NAD+ levels above equivalent doses of NMN or NR chloride. Notably, Restorin NR at the low dose of 0.33 mmol/kg/day induced blood NAD+ elevation statistically equivalent to that of a higher dose of NR chloride or NMN (1 mmol/kg/day, n=11). At the matched dose of 1 mmol/kg/day, Restorin NR yielded significantly higher circulating NAD+ levels than either comparison treatment. These results confirm Restorin NR’s superiority to the tested precursors for NAD+ concentrations in circulation.

Discussion

Overall, our research identifies a new crystalline salt form of NR and classifies Restorin NR as a promising new therapeutic agent to enhance NAD+ levels and combat aging.

Introduction

Nicotinamide adenine dinucleotide (NAD⁺) serves as a vital element for sustaining normal cellular function, with its roles covering energy metabolism, the regulation of redox balance, and the control of gene expression.1,2 During the aging process, NAD⁺ levels tend to decrease, and this decline has been associated with the occurrence and progression of age-related illnesses.3,4 Studies have further expanded on the link between NAD⁺ deficiency and diseases, such as compromised hematopoiesis,5 liver tumorigenesis,6 kidney injury,7 and diastolic heart failure.8 Considering these observations, NAD⁺ supplementation therapy exhibits significant potential in combating aging and aging-related functional decline. Supplying NAD⁺ can boost anti-inflammatory M2 macrophages, reduce neurodegeneration, trigger autophagy and mitophagy, and preserve genome stability, which may offer health benefits such as reduced inflammation, diminished atrophy, improved brain function, and enhanced insulin sensitivity.9

Fundamentally, the NAD⁺ de novo synthesis pathway begins with the use of the amino acid tryptophan (TRYP) as its starting material (Figure 1). Tryptophan is metabolized stepwise by seven enzymes, with some enzymes being selectively inactivated in various human organs.10 The NAD⁺ produced by the liver is thought to be delivered to peripheral cells through the bloodstream’s circulation. Recent studies have reported that the membrane-bound enzyme cluster of differentiation (CD) 73 degrades extracellular NAD⁺ into nicotinamide (NAM), and NAM is then used as a raw material for intracellular NAD⁺ generation. In general, NAD⁺ synthesis from NAM—known as the primary salvage pathway—represents the primary mechanism for producing NAD⁺ in peripheral cells.11 Beyond the primary salvage pathway, the Preiss–Handler pathway also plays a key role in cellular NAD⁺ metabolic processes (Figure 1). This pathway initiates with nicotinic acid (NA), which undergoes metabolism via nicotinic acid phosphoribosyltransferase 1 (NAPRT1) to form nicotinic acid mononucleotide (NAMN)—a product that then feeds into the de novo pathway. The NAD⁺ generated through this route is utilized by a range of proteins involved in diverse biological processes, such as sirtuins, CD38, and poly(ADP-ribose) polymerases (PARPs), with this utilization ultimately leading to the release of nicotinamide (NAM).1,11

Figure 1.

The scheme of the NAD⁺ metabolic pathway. Arrows illustrate the synthesis route of NAD⁺.

Abbreviations: NA, nicotinic acid; NAD⁺, nicotinamide adenine dinucleotide; NAM, nicotinamide; NAMN, nicotinic acid mononucleotide; NAMPT, nicotinamide phosphoribosyltransferase; NAPRT, nicotinic acid phosphoribosyltransferase; NMN, nicotinamide mononucleotide; NMNAT, nicotinamide mononucleotide adenylyl transferase; NR, nicotinamide riboside; NRK, nicotinamide riboside kinase; QPRT, quinolinate phosphoribosyltransferase; TRYP, tryptophan.

Nicotinamide riboside (NR) serves as an extra precursor in the salvage pathway for NAD⁺, and it undergoes either a two-step12 or three-step13 metabolic process to synthesize NAD⁺ (Figure 1). This specific metabolic route is known as the NRK pathway.14 In the two-step NRK pathway, NR is first phosphorylated to nicotinamide mononucleotide (NMN) by two isoforms of nicotinamide riboside kinases—NMRK1 (nicotinamide riboside kinase 1) and NMRK2 (nicotinamide riboside kinase 2). This step bypasses the rate-limiting enzyme nicotinamide phosphoribosyltransferase (NAMPT) in the classical NAD⁺ salvage pathway, avoiding the energetic cost of phosphoribosyl pyrophosphate (PRPP) and feedback inhibition by NAD⁺ itself. Subsequently, NMN is converted to NAD⁺ by NMN/NaMN adenylyltransferases (NMNATs).15 The alternative three-step process involves initial degradation of NR to nicotinamide (NAM) by purine nucleoside phosphorylase (NP); NAM is then converted to NMN via NAMPT, followed by NMNAT-mediated conversion to NAD⁺, thereby integrating with the classical NAMPT-dependent salvage pathway. Additionally, NR via the NRK pathway elevates NAD⁺ in multiple tissues (liver, skeletal muscle, brown adipose tissue), and activates NAD⁺-dependent sirtuins (SIRT1, SIRT3) to regulate metabolism/mitochondrial function.16

As a single agent, NAM has no therapeutic efficacy for combating metabolic complications. This lack of effectiveness may be partly due to the fact that it acts as an end-product inhibitor of NAD⁺-consuming enzymes.17 This has attracted significant attention to the application of NR and NMN as dietary supplements. To date, oral supplementation of NMN and NR has been widely studied, and both are commercially accessible for health maintenance.1 Both compounds provided protection against age-associated physiological decline in mice.3,18 NR, especially, has been demonstrated to extend the lifespan of all species that have been tested to date, including mice.19,20 However, NMN and NR supplements also have certain limitations of their own. At commonly studied human-relevant oral doses, these precursors generally produce modest increases in circulating NAD⁺, often approximately 1.5- to 2.5-fold.21 The other limitations include the fact that NR has poor stability in blood circulation in vivo and is prone to degradation.22

It is worth noting that emergent evidence has suggested different pharmacokinetics and functions of these NAD⁺ precursors.23 More specifically, previous studies have demonstrated that NR and NMN differ in the extent to which they increase NAD⁺ concentration in several mammalian cell lines.24 The chloride salt of NR has been generally recognized as safe (GRAS) by the FDA and is commercially available as a dietary supplement.25 However, the most potent precursor for boosting NAD⁺ levels and its crystalline salt form have yet to be fully clarified. In this study, we aimed to explore whether other crystalline salt forms of NAD⁺ precursors could be suitable for promoting NAD⁺. Through this premise, we identified Restorin NR, a crystalline salt form of NR, as a new NAD⁺ oral supplement. Restorin NR showed an unprecedented ability to increase NAD⁺ in mice, as it was more potent than NR chloride and NMN. Overall, we establish Restorin NR as a new potential oral supplement for NAD⁺-based therapies.

Materials and Methods

Animal Experiments

All animal experiments were conducted in California, USA. A total of 53 C57BL/6 mice (27 males and 26 pup-naive females) were purchased from Charles River Laboratories at the age of 64 weeks. Another 11 C57BL/6 mice (6 males and 5 pup-naive females) were purchased at the age of 10 weeks as a young control. Mice were housed in groups of up to four per cage under standard conditions, including a 12-hour light/dark cycle (lights on at 7:00 AM), ambient temperature of 22±2°C, and relative humidity of 50±10%. Prior to dosing, all mice underwent a 14-day acclimatization period. The aged mice were randomly assigned to five treatment groups and dosed daily by oral gavage for 7 days: Restorin NR at 0.33 mmol/kg/day (n = 11; 6 females, 5 males) or 1.0 mmol/kg/day (n = 11; 5 females, 6 males); NR chloride at 0.33 mmol/kg/day (n = 9; 5 females, 4 males) or 1.0 mmol/kg/day (n = 11; 5 females, 6 males); and NMN at 1.0 mmol/kg/day (n = 11; 5 females, 6 males). NMN was included as a reference comparator at 1.0 mmol/kg/day, a dose slightly higher than the commonly reported effective dose of 100mg/kg/day in murine studies.26 Because the aged mice were 64 weeks old at purchase and underwent a 14-day acclimatization followed by 7 days of treatment, animals were approximately 67 weeks of age at the day-7 sample collection. Blood samples were collected via cheek bleeding on day 0 (baseline) and day 7 (post-treatment). Young mice received daily oral gavage of saline, and their blood was collected on day 0 (baseline) as a positive control group. All animal studies were conducted by a licensed contract research organization (HD Biosciences, San Diego, CA) operating under institutional animal care and use committee (IACUC) oversight (animal approval ethics number: HDB23-008). All procedures were performed in accordance with applicable national and institutional guidelines for the care and use of laboratory animals, including the Guide for the Care and Use of Laboratory Animals. The animals in this study were C57BL/6 mice and did not require a license/permit for third-party research use. No animals were euthanized as part of this study.

Chemical Reagents

The compounds tested included Restorin NR, NR chloride, and NMN. NR chloride and NMN were purchased from Effepharm. Restorin NR was obtained from Seragon Biosciences.

Quantitation of Blood NAD⁺ Levels

Blood NAD⁺ was quantified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using isotope dilution against a stable-isotope-labeled internal standard (NAD⁺-d4). The biosample was murine whole blood. For each sample, 20 µL of whole blood was added to 200 µL of cold methanol containing 1000 ng/mL NAD⁺-d4 to precipitate protein and disrupt the matrix. Samples were vortexed for 30s and centrifuged at 15,000 rcf for 20 min at 4 °C. A 100-µL aliquot of the supernatant was evaporated to dryness in a vacuum concentrator, reconstituted in 100 µL of LC-MS-grade water, sonicated on ice for 1 min, vortexed for 30s, and centrifuged again at 15,000 rcf for 20 min at 4 °C. An 85-µL aliquot of the supernatant was transferred to a vial, and 2 µL was injected onto the LC-MS/MS system. NAD⁺ and NAD⁺-d4 reference standards were obtained from MedChemExpress (MCE) and stored at −80 °C; the material supplied by MCE was used solely as a reference/internal standard, while the LC-MS/MS acquisition method was established in-house. Mobile phases were prepared from LC-MS-grade water, methanol, and ammonium acetate. Use of a co-eluting stable-isotope internal standard (NAD⁺-d4) corrected for matrix effects and supported measurement accuracy.

Statistical Analysis

All results are shown as mean ± standard error of the mean (SEM). Normality was assessed using Shapiro–Wilk tests and QQ plots, and homogeneity of variance using Levene’s test. Day-7 NAD⁺ concentrations were compared across treatment groups using analysis of covariance (ANCOVA), with biological sex and baseline (day-0) NAD⁺ concentration included as covariates. Pre-specified planned comparisons were then evaluated using Fisher’s least significant difference (LSD) test. Within-group changes from day 0 to day 7 were assessed using paired t-tests. Differences were considered statistically significant when two-sided p-values were below 0.05. Each NAD⁺ measurement corresponds to an independent animal; technical replicates were not treated as independent observations. Biological sex was a significant source of variation (p = 0.002) and was retained as a covariate in all group comparisons.

Results

As shown in Figure 2, Restorin NR increased blood NAD⁺ levels by 1.28-fold and 1.31-fold at doses of 0.33 mmol/kg/day and 1.0 mmol/kg/day, respectively. NR chloride and NMN at 1.0 mmol/kg/day also increased NAD⁺ levels by 1.18-fold (p = 0.018) and 1.19-fold (p = 0.003), respectively. Notably, NAD⁺ levels in mice treated with 0.33 mmol/kg/day Restorin NR were not significantly different from those in mice administered a threefold higher dose of NMN (1.0 mmol/kg/day, n = 11, p = 0.75) or NR chloride (1.0 mmol/kg/day, n = 11, p = 0.88), indicating an approximately threefold dose-efficiency advantage of Restorin NR for boosting blood NAD⁺ levels. Restorin NR at 0.33 mmol/kg/day produced a significantly higher NAD⁺ level than the same dose of NR chloride (p = 0.011). At the matched dose of 1.0 mmol/kg/day, Restorin NR yielded significantly higher blood NAD⁺ levels than both NR chloride (p = 0.011) and NMN (p = 0.016). Restorin NR also produced significantly higher NAD⁺ at 1.0 than at 0.33 mmol/kg/day (p = 0.008), consistent with a dose-dependent effect and underscoring its superior potency relative to the other two precursors tested.

Figure 2.

Restorin NR Outperforms NMN and NR Chloride. A 7-day repeated-dose study with Restorin NR in male and female C57BL/6 mice was conducted. Prior to dosing, all mice underwent a 14-day acclimatization period. The compounds tested included Restorin NR, NR chloride, and NMN. Each compound was administered daily via oral gavage at a dose of 1.0 mmol/kg/day or 0.33 mmol/kg/day for 7 days. Blood samples were collected via cheek bleeding on day 0 (baseline) and day 7 (post-treatment). Blood NAD⁺ levels were measured by LC-MS/MS and are expressed as μM. Data are presented as mean ± standard error (SE). Statistical analysis was performed using analysis of covariance (ANCOVA) with biological sex and baseline NAD⁺ as covariates, followed by pre-specified planned Fisher’s LSD comparisons. *p < 0.05 vs the indicated groups.

Abbreviations: ns, no significant difference; NR, nicotinamide riboside; NMN, nicotinamide mononucleotide; NAD⁺, nicotinamide adenine dinucleotide. Exact p-values are reported in the Results section.

Discussion and Conclusion

NMN and NR are presently the most widely utilized and prospective NAD⁺ precursors, and numerous scientific studies have shown their advantages in murine models that target age-associated physiological decline. Nevertheless, NR supplementation has so far been shown to be ineffective in increasing NAD⁺ levels in human tissue partly due to its poor stability in blood circulation in vivo. Therefore, new crystalline salt forms of NR featuring greater NAD⁺ elevation potential—ones that can surmount these limitations—are highly desirable. In our work, Restorin NR proved to be a potent NAD⁺ precursor in mice, exhibiting greater efficacy than NR chloride and NMN following oral supplementation. In fact, while NR chloride was only able to increase NAD⁺ levels by 1.18-fold at its maximum concentration (1 mmol/kg/day), Restorin NR led to a 1.28-fold NAD⁺ increase even at 0.33 mmol/kg/day. Consistent with this observation, Restorin NR at 0.33 mmol/kg/day achieved circulating NAD+ levels that were not significantly different from those attained with NR chloride at 1.0 mmol/kg/day, indicating an approximately threefold dose advantage for Restorin NR under the conditions tested.

The synergistic effect between NR and fumarate in Restorin NR may contribute to this key finding. Metabolomics analysis has implicated CD4⁺ T cell arginine and fumarate biosynthesis as mediators of NR’s immunomodulatory effect.27 More specifically, these findings support that NR-driven biosynthesis of arginine and fumarate, along with the subsequent transactivation of Nrf2 and its canonical targets, attenuated CD4⁺ T cell IL-17 production. Collectively, such findings suggest Restorin NR—by leveraging the synergistic interaction between NR and fumarate—may exert a superior effect in NAD⁺ elevation. The synergy between NR and fumarate is presented as a hypothesis, and it would be of interest to assess in upcoming experiments whether the superior ability of Restorin NR to induce NAD⁺ synthesis translates to greater therapeutic efficacy when contrasted with NAD⁺ precursors at equivalent doses.

In conclusion, oral administration of Restorin NR raised circulating NAD⁺ levels. Under the conditions tested, Restorin NR was a more effective precursor at increasing circulating NAD⁺ levels than equivalent doses of NMN and NR chloride. Future research will establish the therapeutic benefits of Restorin NR administration in animal models of disease, with a specific focus on its role in combating aging.

Limitations

These findings characterize circulating NAD⁺ responses over a 7-day treatment window. Future studies are needed to determine longer-term durability and tissue-level assessments. Because intermediate metabolites of the NAD⁺ biosynthetic pathway were not measured, the present data do not resolve the extent to which precursor utilization may approach a saturable ceiling at higher doses. Profiling these intermediates is a natural extension of this work. The underlying fumarate-related mechanism was not directly studied in this report, and future studies could determine these effects.

Disclosure

Mr Brett Weiss is an employee of Seragon Biosciences. Mr Rui Cao reports support for the manuscript from Seragon Biosciences during the conduct of the study. Mr Yongzheng Liu is an employee of Sinopharm-Seragon Biosciences. Ms Jiaqi Chen is an employee of Sinopharm-Seragon Biosciences. Dr George Marshall reports leadership or fiduciary roles from Seragon Biosciences; stock or stock options from Seragon Biosciences, outside the submitted work.