阅读说明：本技术 用于治疗/预防骨骼肌疾病的还原型烟酰胺核糖核苷 (Reduced nicotinamide riboside for the treatment/prevention of skeletal muscle diseases ) 是由 C·坎托阿尔瓦雷斯 S·克里斯滕 M·P·吉内 J·吉鲁-格贝坦特 S·莫科 于 2020-06-03 设计创作，主要内容包括：本发明提供包含还原型烟酰胺核糖核苷的化合物和组合物,其用于预防和/或治疗骨骼肌疾病和/或病症的方法中。在本发明的一个实施方案中,本发明的所述化合物和组合物通过以下方式来改善骨骼肌：维持或改善肌肉功能；维持或增加肌肉质量；维持或改善肌肉力量；以及改善损伤或手术后的肌肉恢复和再生。在本发明的另一个实施方案中,本发明的化合物和组合物可用于预防和/或治疗诸如以下的骨骼肌疾病和/或病症的方法中：恶病质或恶病质前期；肌少症、肌病、营养不良和/或剧烈运动、肌肉损伤或手术后的恢复。(The present invention provides compounds and compositions comprising reduced nicotinamide riboside for use in methods of preventing and/or treating skeletal muscle diseases and/or disorders. In one embodiment of the invention, the compounds and compositions of the invention improve skeletal muscle by: maintenance or improvement of muscle function; maintaining or increasing muscle mass; maintaining or improving muscle strength; and to improve muscle recovery and regeneration following injury or surgery. In another embodiment of the invention, the compounds and compositions of the invention are useful in methods of preventing and/or treating skeletal muscle diseases and/or disorders such as: cachexia or pre-cachexia; sarcopenia, myopathy, malnutrition and/or recovery after strenuous exercise, muscle damage or surgery.)

1. Reduced nicotinamide riboside for use in a method of increasing intracellular NAD + in a subject, the method comprising delivering reduced nicotinamide to a subject in need thereof in an effective unit dosage form to prevent and/or treat a skeletal muscle disease or disorder.

2. Reduced nicotinamide riboside for use according to claim 1, wherein the reduced nicotinamide riboside is selected from the group consisting of:

(i)1, 4-dihydro-1- β -D-ribofuranosyl-3-pyridinecarboxamide;

(ii)1, 2-dihydro-1-beta-D-ribofuranosyl-3-pyridinecarboxamide or

(iii)1, 6-dihydro-1-beta-D-ribofuranosyl-3-pyridinecarboxamide.

3. Reduced nicotinamide riboside for use according to claim 1 or 2, wherein the reduced nicotinamide riboside is 1, 4-dihydro-1- β -D-ribofuranosyl-3-pyridinecarboxamide.

4. A composition comprising reduced nicotinamide riboside according to any one of claims 1 to 3, wherein the composition is for use in the prevention and/or treatment of a skeletal muscle disease or disorder.

5. The composition of claim 4, wherein the composition consists essentially of reduced nicotinamide riboside, free of other NAD + precursors, for use in the prevention and/or treatment of a skeletal muscle disease or disorder.

6. The composition of any one of claims 4 to 5, comprising reduced nicotinamide riboside for use in maintaining or increasing skeletal muscle function in a subject.

7. The composition according to claim 6, wherein increased muscle function is measured by an increase in the number of muscle stem cells and/or myoblasts and/or myotubes.

8. The composition of any one of claims 4 to 5, comprising reduced nicotinamide riboside for use in maintaining or increasing skeletal muscle mass in a subject.

9. The composition according to any one of claims 4 to 5, comprising reduced nicotinamide riboside for use in preventing or reducing skeletal muscle atrophy in a subject.

10. A composition according to any one of claims 4 to 5 comprising reduced nicotinamide riboside for use in enhancing skeletal muscle recovery following strenuous exercise.

11. The composition according to any one of claims 4 to 5, comprising reduced nicotinamide riboside for use in enhancing recovery of skeletal muscle following injury.

12. The composition according to any one of claims 4 to 5, comprising reduced nicotinamide riboside for use in enhancing skeletal muscle recovery following trauma or surgery.

13. The composition according to any one of claims 4 to 12, wherein the composition is a nutritional composition selected from the group consisting of: a food or beverage product comprising a food additive, food ingredient, functional food, dietary supplement, medical food, nutraceutical, Oral Nutritional Supplement (ONS), or food supplement.

14. The reduced nicotinamide riboside or composition thereof of any one of claims 1-13, wherein the skeletal muscle disease and/or disorder is selected from the group consisting of: cachexia or pre-cachexia; sarcopenia, myopathy, malnutrition and/or recovery after strenuous exercise, muscle damage or surgery.

15. The reduced nicotinamide or composition thereof of any of claims 1-14, wherein cachexia is associated with a disease selected from the group consisting of: cancer, chronic heart failure, renal failure, chronic obstructive pulmonary disease, AIDS, autoimmune disorders, chronic inflammatory disorders, cirrhosis of the liver, anorexia, chronic pancreatitis, metabolic acidosis, and/or neurodegenerative diseases.

16. A method for increasing intracellular NADH in a mammal in a subject, said method comprising delivering to said mammal in need of such treatment an effective amount of reduced nicotinamide riboside of any one of claims 1 to 15 in a unit dosage form effective to prevent and/or treat a skeletal muscle disease or disorder.

17. The method of claim 16, wherein the skeletal muscle disease or disorder is selected from the group consisting of: cachexia or pre-cachexia; sarcopenia, myopathy, malnutrition and/or recovery after strenuous exercise, muscle damage or surgery.

18. The method according to any one of claims 16 to 17 for use in the prevention and/or treatment of a skeletal muscle disease or disorder in a subject in need thereof, the method comprising the steps of:

i) providing to said subject a composition consisting essentially of reduced nicotinamide riboside, and

ii) administering the composition to the subject.

19. The method of claim 18, wherein the subject is selected from the group consisting of: human, dog, cat, cow, horse, pig or sheep.

20. The method of claim 19, wherein the subject is a human.

Technical Field

The present invention provides compounds and compositions comprising reduced nicotinamide riboside for use in methods of preventing and/or treating skeletal muscle diseases and/or disorders. In one embodiment of the invention, the compounds and compositions of the invention improve skeletal muscle by: maintenance or improvement of muscle function; maintaining or increasing muscle mass; maintaining or improving muscle strength; and to improve muscle recovery and regeneration following injury or surgery. In another embodiment of the invention, the compounds and compositions of the invention are useful in methods of preventing and/or treating skeletal muscle diseases and/or disorders such as: cachexia or pre-cachexia; sarcopenia, myopathy, malnutrition and/or recovery after intense muscle movement, injury or surgery.

Background

Skeletal muscle regeneration is an important mechanism for the repair and maintenance of muscle mass and function throughout life. NAD + plays an important role in skeletal muscle development, regeneration, aging and disease. Lower NAD + levels are known to be detrimental to muscle health, while higher NAD + levels are known to enhance muscle health.

At the cellular level, NAD + affects mitochondrial biogenesis, transcription of extracellular matrix components and tissue (Goody, m.f.2018). In skeletal muscle, NAD + localization in mitochondria is important for muscle function, with 95% of NADH localization in mitochondria in skeletal muscle.

Thus, there is an urgent, unmet need to address skeletal muscle diseases and/or disorders with novel compounds, compositions, and methods of prevention and/or treatment that affect NAD +.

Disclosure of Invention

The present invention provides compounds and compositions for use in methods of preventing and/or treating skeletal muscle disorders and diseases.

In one embodiment, the composition is selected from the group consisting of: food or beverage products, food supplements, Oral Nutritional Supplements (ONS), medical foods, and combinations thereof.

In another embodiment, the invention provides a method for increasing intracellular Nicotinamide Adenine Dinucleotide (NAD) in a subject⁺) The method comprising administering a compound or composition of the invention effective to increase NAD⁺The biosynthetic amount administers to the subject a reduced nicotinamide riboside composition.

In another embodiment, reduced nicotinamide riboside, as a precursor for NAD + biosynthesis, can increase NAD + biosynthesis and provide one or more beneficial effects on skeletal muscle function.

In another embodiment, the invention provides a unit dosage form of a composition consisting of reduced nicotinamide riboside comprising an effective amount of reduced nicotinamide riboside to increase NAD + biosynthesis.

In one embodiment of the invention, a composition comprising reduced nicotinamide riboside is provided to maintain or increase skeletal muscle function in a subject.

In another embodiment of the invention, a composition comprising reduced nicotinamide riboside is provided to maintain or increase skeletal muscle mass in a subject.

In yet another embodiment of the invention, a composition comprising reduced nicotinamide riboside is provided to prevent or reduce skeletal muscle atrophy in a subject.

In another embodiment of the invention, a composition comprising reduced nicotinamide riboside is provided to enhance recovery of skeletal muscle following strenuous exercise.

In yet another embodiment of the invention, a composition comprising reduced nicotinamide riboside is provided to enhance recovery of skeletal muscle following injury.

In another embodiment of the invention, a composition comprising reduced nicotinamide riboside is provided to enhance recovery of skeletal muscle following trauma or surgery.

In another embodiment of the invention, the composition is a nutritional composition selected from the group consisting of: a food or beverage product comprising a food additive, food ingredient, functional food, dietary supplement, medical food, nutraceutical, Oral Nutritional Supplement (ONS), or food supplement.

In another embodiment of the invention, the composition is a nutritional composition comprising reduced nicotinamide riboside, wherein the increase in muscle function of the muscle is measured by an increase in the number of muscle stem cells and/or myoblasts and/or myotubes.

In another embodiment of the invention, there is provided a composition comprising reduced nicotinamide riboside for the prevention or treatment of cachexia or pre-cachexia; sarcopenia, myopathy, malnutrition and/or recovery after strenuous exercise, muscle damage or surgery.

In another embodiment of the invention, a composition of the invention comprising reduced nicotinamide riboside is provided for use in the prevention or treatment of cachexia, wherein cachexia is associated with a disease selected from the group consisting of: cancer, chronic heart failure, renal failure, chronic obstructive pulmonary disease, AIDS, autoimmune disorders, chronic inflammatory disorders, cirrhosis of the liver, anorexia, chronic pancreatitis, metabolic acidosis, and/or neurodegenerative diseases.

In a preferred embodiment of the present invention, the nutritional composition of the present invention is provided for use in the prevention or treatment of cancer-related cachexia or pre-cachexia.

In another preferred embodiment of the present invention, the nutritional composition of the present invention is provided for use in the treatment of cachexia associated with a cancer selected from pancreatic cancer, esophageal cancer, gastric cancer, intestinal cancer, lung cancer and/or liver cancer.

Drawings

FIG. 1: chemical structure of oxidized (NR) and reduced (NRH) forms of nicotinamide riboside

1: 1-b-D-ribofuranosyl-3-pyridinecarboxamide salts

2: 1, 4-dihydro-1-b-D-ribofuranosyl-3-pyridinecarboxamide

3: 1, 2-dihydro-1-b-D-ribofuranosyl-3-pyridinecarboxamide

4: 1, 6-dihydro-1-b-D-ribofuranosyl-3-pyridinecarboxamide

X^-: anions (e.g. triflate)

FIG. 2: dose response experiments show that NRH can significantly increase NAD + better than NR

Starting at a level of 10 μ M, NRH achieves increases in intracellular NAD + levels similar to those achieved with a 50-fold higher concentration of NR. NRH achieves the greatest effect on NAD + synthesis in the about millimolar range, achieving an increase in intracellular NAD + levels by more than 10-fold.

FIG. 3: NHR works rapidly after 5 minutes of treatment.

NRH action is also extremely rapid, as a significant increase in NAD + levels was observed within 5 minutes after NRH treatment. Peak levels of NAD + were achieved between 45 minutes and 1 hour after treatment.

FIG. 4: NRH leads to NAD + biosynthesis via an adenosine kinase dependent pathway.

AML12 cells were treated with an adenosine kinase inhibitor (5-IT; 10mM) for 1 hour, followed by NRH treatment at the indicated doses. Then, after 1 hour, an acidic extract was obtained to measure NAD⁺And (4) horizontal. All values in the figure are expressed as mean +/-SEM of 3 independent experiments. Indications at P relative to the corresponding vehicle treatment groups<Statistical differences at 0.05.

FIG. 5: NRH is an orally active NAD + precursor in mice.

C57Bl/6NTac mice, 8 weeks old, were orally drenched with saline (as vehicle), NR (500mg/kg) or NRH (500 mg/kg). After 1 hour, liver, skeletal muscle and kidney NAD were evaluated⁺And (4) horizontal. All values are expressed as mean +/-SEM of n-5 mice per group. Indicated P relative to saline treated mice<Statistical differences at 0.05. # indicates that mice treated with NR are in P<Statistical differences at 0.05.

FIG. 6: NRH was found to be intact in mouse tissues after administration.

8-week-old C57Bl/6NTac mice were orally drenched with saline (as vehicle) and NRH (250 mg/kg). After 2 hours, liver, skeletal muscle and kidney NRH levels were assessed. All results are expressed as mean +/-SEM of n-4 mice per group, as area under signal from LC-MS analysis, corrected for total protein content of the tissue.

Examples

Example 1: synthesis of reduced forms of Nicotinamide Riboside (NRH)

Reduced Nicotinamide Riboside (NRH) is obtained from NR (1) by reducing a pyridinium salt (e.g., triflate) to dihydropyridine (1, 2-dihydropyridine, 1, 4-dihydropyridine, and 1, 6-dihydropyridine), as shown below

1: 1-b-D-ribofuranosyl-3-pyridinecarboxamide salts

2: 1, 4-dihydro-1-beta-D-ribofuranosyl-3-pyridinecarboxamide

3: 1, 2-dihydro-1-beta-D-ribofuranosyl-3-pyridinecarboxamide

4: 1, 6-dihydro-1-beta-D-ribofuranosyl-3-pyridinecarboxamide

X^-: anions (e.g. triflate)

Sodium borohydride (NaBH)₄) And sodium dithionite (Na)₂S₂O₄) As reducing agents for N-substituted pyridinium derivatives. The regioselectivity of the reducing agent is different, resulting in only one dihydropyridine or a mixture of all 3 isomers in different ratios (2, 3, 4).

The dithionate reduction of pyridinium salts with electron withdrawing substituents in positions 3 and 5 yields almost exclusively 1, 4-dihydropyridine product. Due to the instability of the reduction product in acidic media, the reduction is carried out under mild conditions, for example in aqueous sodium bicarbonate or dipotassium hydrogen phosphate medium. To perform the reduction, the hydroxyl groups in the ribofuranose moiety are protected with a benzyl or acetyl substituent. After reduction, deprotection is then carried out by passage through a solution of sodium hydroxide in methanol under ball mill conditions.

Example 2: measurement of NRH and other NAD + related metabolites in biological samples

Methanol was used at 5:3:5 (v/v): water: a mixture of chloroform from which polar phases are recovered for hydrophilic interaction ultra performance liquid chromatography mass spectrometry (UHPLC-MS) analysis by obtaining levels of NRH and other NAD-related metabolites in a biological sample using cold liquid-liquid extraction. The UHPLC consists of a binary pump, a cooled autosampler and a cartridge (DIONEX Ultimate 3000UHPLC + Focuse, Semmer Scientific) connected to a triple electrospray ionization (H-ESI) source equipped with a heated sourceQuadrupole spectrometer (TSQ Vantage, seemer science). In each sample, 2. mu.L of the solution was injected into an analytical column (2.1 mm. times.150 mm, 5 μm pore size,HILICONfusion (P), consisting of a pre-column (2.1 mm. times.20 mm,HILICONfusion (p) protective sheathing) protection. The mobile phase (10mM ammonium acetate, pH 9, a, and acetonitrile, B) was pumped onto a linear gradient of decreasing organic solvent (0.5-16 min, 90% -25% B) at a flow rate of 0.25 mL/min, before re-equilibrating for a total run time of 30 min. MS was operated in positive mode with Multiple Reaction Monitoring (MRM) at 3500V. The software Xcalibur v4.1.31.9 (sermer technology) is used for instrument control, data acquisition and processing. Retention time and quality measurements were confirmed by authentic standards.

The structural elucidation of the NRH used for biological studies was confirmed by Nuclear Magnetic Resonance (NMR).

Example 3: NRH is a potent NAD + precursor

AML12 hepatocytes were treated with NRH, and it was observed that NRH increased intracellular NAD + over NR.

Dose response experiments showed that NRH can significantly increase NAD + levels at a concentration of 10 μ M (figure 2). Even at such relatively low doses, NRH achieves similar increases in intracellular NAD + levels as those achieved with 50-fold higher concentrations of NR. NRH achieves the greatest effect on NAD + synthesis in the about millimolar range, achieving an increase in intracellular NAD + levels by more than 10-fold.

NRH action was also extremely rapid (fig. 3) as a significant increase in NAD + levels was observed within 5 minutes after NRH treatment. Peak levels of NAD + were achieved between 45 minutes and 1 hour after treatment, as also occurred with NR.

NRH was also tested in other cell type models for its effectiveness in increasing NAD +. NRH treatment greatly increased NAD + levels in C2C12 myotubes, INS1 cells, and 3T3 fibroblasts, supporting the concept that NRH metabolism is widespread in different cell types.

Example 4: pathways for NRH-induced NAD + synthesis

A pathway where NRH will be converted to NMNH, then NADH, and this will eventually be oxidized to NAD +. Thus, NRH and NMNH could be detected intracellularly 5 minutes after NRH, but not NR treatment. Interestingly, NRH treatment also resulted in an increase in intracellular NR and NMN (greater than that triggered by NR itself), opening the possibility that NRH could synthesize NAD + by being oxidized to NR, then using the classical NRK/NMNAT pathway.

To understand the exact pathway by which NRH synthesizes NAD +, we initially assessed whether NRH can be transported into cells via the Equilibrating Nucleoside Transporter (ENT). This possibility was confirmed, with NRH largely losing its ability to act as an extracellular NAD + precursor in the presence of agents that block ENT-mediated transport such as S- (4-nitrobenzyl) -6-thioinosine (NBTI). However, the essential role of NRH was retained even after ENT blocking, suggesting that NRH may be able to enter cells through additional transporters.

The effect of NRH was also NAMPT independent based on experiments with the NAMPT inhibitor FK 866. If the formation of NRH via NMNH results in NAD + synthesis, this hypothetical pathway would require phosphorylation of NRH to NMNH. In view of the fundamental and rate-limiting role of NRK1 in NR phosphorylation, we wondered whether NRH's ability to increase NAD + levels is NRK1 dependent. To answer this question, we evaluated the NRH effect in primary hepatocytes from control or NRK1 knock-out (NRK1KO) mice. Although NR failed to increase NAD + levels in NRK1 KO-derived primary hepatocytes after 1 hour of treatment, NRH effects were not affected by NRK1 deficiency. These results indicate that NRH action is NRK1 independent. Furthermore, they exclude the possibility that NRH-induced NAD + transport is driven by the oxidation of NRH to NR.

Given the molecular structure of NRH, we conclude that alternative nucleoside kinases may be responsible for the phosphorylation of NRH. IT was confirmed that the Adenosine Kinase (AK) inhibitor 5-iodotubercidin (5-IT) completely ablated the effect of NRH. A structurally different second AK inhibitor, ABT-702, was used to confirm the role of AK in NRH-mediated NAD + synthesis. Metabolomic analysis further confirmed that upon inhibition of AK, the production of NMNH, NADH and NAD + was completely blunted even though NRH efficiently entered the cells. Interestingly, the 5-IT treatment also prevented the formation of NR and NMN after NRH treatment.

This indicates that the appearance of NR after NRH treatment cannot be simply attributed to direct intracellular oxidation of NRH to NR. As a whole, these experiments describe adenosine kinase as an enzymatic activity that catalyzes the conversion of NRH to NMNH, thereby triggering the conversion to NAD + in this way.

As a subsequent step, NMNAT enzyme can catalyze the conversion from NMNH to NADH. Thus, the use of gallotannin as NMNAT inhibitor after NRH treatment greatly compromises NAD + synthesis. However, when NRH is used at the maximum dose, a partial effect of NRH is retained after gallotannin treatment. However, NRH action was completely blocked by gallotannin at sub-maximal doses, suggesting that the residual effect at 0.5mM may be attributable to incomplete inhibition of NMNAT activity by gallotannin. Taken together, these results indicate a pathway by adenosine kinase and NMNAT vertebrate NRH leading to NAD + synthesis via NADH.

Example 5: NRH is detectable in the loop after IP injection

The degradation of NR to NAM has been proposed as a limitation of its pharmacological efficacy. To assess whether NRH is also susceptible to degradation to NAM, we added NRH or NR to isolated mouse plasma. After 2 hours incubation, NR levels in plasma decayed, parallel to the increase in NAM. In contrast, NAM was not generated by NRH, as its levels remained stable during the 2 hour test. We also tested the stability of NRH in other matrices. Given our previous experiments in cultured cells, we demonstrated that NRH does not degrade to NAM in FBS supplemented media, as occurs with NR. Finally, we also demonstrated NRH stability in water (pH 7, room temperature) for 48 hours.

The above results suggest that we tested whether NRH can act as a potent NAD + precursor in vivo. To this end, we first injected mice Intraperitoneally (IP) with NR or NRH (500 mg/kg). After 1 hour, both compounds increased NAD + levels in liver (fig. 5), muscle and kidney. As expected, NAM levels greatly increased in the circulation upon NR administration, while only a very mild increase was observed under NRH. Importantly, NRH can be detected in the loop after IP injection.

Surprisingly, NR was detectable in the circulation after NRH treatment at a level much higher than that detected after NR injection itself. Since NRH incubation in isolated plasma does not result in NR production, the appearance of NR is likely the result of intracellular production and release into the circulation. Similarly, the residual appearance of NAM after NRH treatment can be explained by degradation of the released NR or by the release of intracellular NAM as a product of NAD + degradation, since NRH does not significantly alter NAM levels when incubated in isolated plasma.

Example 6: NRH is detectable after oral administration as an oral bioavailable to overcome direct degradation in plasma NAD + precursors for use

Oral administration of NRH resulted in very similar results to those observed after IP administration. First, NRH has a stronger effect on liver NAD + levels than NR. NRH was detectable in plasma 1 hour after oral administration. In contrast, no NR levels could be detected at 1 hour after NR administration. As expected, the NR treatment resulted in a large increase in circulating NAM, which was about 4 times higher than those observed after NRH treatment. Quantitative measurements showed that after oral drenching, NRH concentrations in plasma reached 11.16 ± 1.74 micromolar, which was sufficient to drive NAD + synthesis efficiently. These results show that NRH is a potent orally bioavailable NAD + precursor that overcomes direct degradation to NAM in plasma.

Example 7: NRH was found to be intact in liver, kidney and muscle following oral administration

NRH was not only present in the circulation, but was also found to be intact at high levels in the liver, kidney and muscle of mice 2 hours after drenching (fig. 6). This indicates that oral administration of NRH allows for effective biodistribution in the target tissue.

Detailed Description

Definition of

All percentages expressed herein are by weight of the total weight of the composition, unless otherwise indicated. As used herein, "about" and "substantially" are understood to mean a number within a range of values, for example in the range of-10% to + 10% of the number referred to, preferably-5% to + 5% of the number referred to, more preferably-1% to + 1% of the number referred to, most preferably-0.1% to + 0.1% of the number referred to.

All numerical ranges herein should be understood to include all integers or fractions within the range. Additionally, these numerical ranges should be understood to provide support for claims directed to any number or subset of numbers within the range. For example, a disclosure of 1 to 10 should be understood to support a range of 1 to 8, 3 to 7, 1 to 9, 3.6 to 4.6, 3.5 to 9.9, and so forth.

As used in the present invention and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a component" or "the component" includes two or more components.

The words "comprise/comprising" are to be interpreted as inclusive and not exclusive. Likewise, the terms "include/include" and "or" should be considered inclusive unless the context clearly prohibits such interpretation. However, the compositions disclosed herein may be free of any elements not specifically disclosed herein. Thus, disclosure of embodiments using the term "comprising" includes disclosure of embodiments "consisting essentially of and embodiments" consisting of the indicated components. Any embodiment disclosed herein may be combined with any other embodiment disclosed herein.

As used herein, the terms "example" and "such as" (especially when followed by a list of terms) are exemplary and illustrative only and should not be deemed exclusive or comprehensive. As used herein, a condition is "associated with" or "associated with" another condition means that the conditions occur simultaneously, preferably means that the conditions are caused by the same underlying condition, and most preferably means that one of the identified conditions is caused by another identified condition.

The terms "food," "food product," and "food composition" mean a product or composition intended for ingestion by an individual (such as a human being) and providing at least one nutrient to the individual. The food product typically comprises at least one of protein, lipid, carbohydrate, and optionally one or more vitamins and minerals. The term "beverage" or "beverage product" means a liquid product or liquid composition intended for oral ingestion by an individual (such as a human being) and providing at least one nutrient to the individual.

The compositions of the present disclosure (including the various embodiments described herein) may comprise, consist of, or consist essentially of: the elements disclosed herein, as well as any additional or optional ingredients, components or elements described herein or otherwise useful in the diet.

As used herein, the term "isolated" means removed from one or more other compounds or components with which the compound may otherwise be present (e.g., as found in nature). For example, "isolated" preferably means that the identified compound is separated from at least a portion of the cellular material with which it is normally found in nature. In one embodiment, the isolated compound is free of any other compounds.

"preventing" includes reducing the risk, incidence, and/or severity of a condition or disorder. The terms "treatment" and "ameliorating" include both prophylactic or preventative treatment (prevention and/or delay of progression of the targeted pathological condition or disorder) and curative, therapeutic or disease-modifying treatment, including therapeutic measures to cure, delay, alleviate the symptoms of, and/or halt the progression of a diagnosed pathological condition or disorder; and treating patients at risk of contracting a disease or suspected of contracting a disease, as well as treating patients who are ill or have been diagnosed as having a disease or medical condition. The term does not necessarily mean that the subject is treated until complete recovery. The term "treatment" also refers to the maintenance and/or promotion of health in an individual who is not suffering from a disease but who may be susceptible to developing an unhealthy condition. The terms "treat" and "ameliorating" are also intended to include the intensification or otherwise enhancement of one or more primary prophylactic or therapeutic measures. The terms "treatment" and "alleviating" are also intended to include dietary management of a disease or condition or dietary management for the prevention or prophylaxis of a disease or condition. The treatment may be patient-related or physician-related.

The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for human and animal subjects, each unit containing a predetermined quantity of a composition disclosed herein, in association with a pharmaceutically acceptable diluent, carrier or vehicle, in an amount sufficient to produce the desired effect. The specifications for the unit dosage form depend on the particular compound used, the effect to be achieved, and the pharmacodynamics associated with each compound in the host.

As used herein, an "effective amount" is an amount that prevents a defect, treats a disease or medical condition in an individual, or more generally, reduces symptoms, manages progression of its disease, or provides a nutritional, physiological, or medical benefit to an individual. The relative terms "improve", "increase", "enhance", "promoting" and the like refer to the effect of a composition disclosed herein (i.e., a composition comprising reduced nicotinamide riboside) relative to an otherwise identical composition without nicotinamide riboside. As used herein, "promoting" refers to enhancing or inducing relative to the levels prior to administration of the compositions disclosed herein.

As used herein, "reduced nicotinamide riboside" may also be referred to as protonated nicotinamide riboside, dihydronicotinamide riboside, dihydro-1- β -D-ribofuranosyl-3-pyridinecarboxamide, or 1- (β -D-ribofuranosyl) -dihydronicotinamide. A description of the synthesis of reduced nicotinamide riboside is given in example 1. The position of the protonation site may give rise to different forms of "reduced nicotinamide riboside". For example: 1, 4-dihydro-1- β -D-ribofuranosyl-3-pyridinecarboxamide; 1, 2-dihydro-1- β -D-ribofuranosyl-3-pyridinecarboxamide; and 1, 6-dihydro-1- β -D-ribofuranosyl-3-pyridinecarboxamide (Makarov and Migaud, 2019).

Skeletal muscle diseases and disorders

Cachexia and related diseases

The present invention provides compounds, compositions and methods for preventing and/or treating cachexia or skeletal muscle wasting syndrome. Cachexia is a complex metabolic syndrome associated with underlying disease and is characterized by muscle loss with or without loss of fat mass. The prominent clinical features of cachexia are adult weight loss (correction of fluid retention) or undersrowth in children (exclusion of endocrine disorders).

Cachexia is often present in patients with diseases such as cancer, chronic heart failure, renal failure, chronic obstructive pulmonary disease, AIDS, autoimmune disorders, chronic inflammatory disorders, cirrhosis, anorexia, chronic pancreatitis, and/or metabolic acidosis and neurodegenerative diseases.

There are certain types of cancer, where cachexia is particularly prevalent, such as pancreatic, esophageal, gastric, intestinal, lung, and/or liver cancer.

Internationally accepted diagnostic criteria for cachexia are based on current weight and height (body mass index [ BMI ]]<20kg/m²) Or skeletal muscle mass (measured by DXA, MRI, CT or bioimpedance), greater than 5% weight loss over a limited period of time, e.g. 6 months, or in individuals who have shown depletionGreater than 2% weight loss. Cachexia can develop gradually in various stages, i.e., cachexia develops in the early stage and then into intractable cachexia. Severity can be classified according to the extent of sustained weight loss combined with the extent of consumption of energy storage and body protein (BMI).

In particular, cancer cachexia has been defined as weight loss over the past 6 months>5% (no simple starvation); or BMI<20 and any degree of weight loss>2 percent; or limb lean body mass consistent with low muscle mass (male)<7·26kg/m²(ii) a Female with a view to preventing the formation of wrinkles<5·45kg/m²) And any degree of weight loss>2% (Fearon et al, 2011).

Pre-cachexia can be defined as weight loss ≦ 5% along with anorexia and metabolic changes. Currently, there are no robust biomarkers to identify those pre-cachectic patients who are likely to progress further or the rate at which they will progress further. Refractory cachexia is defined essentially based on the clinical characteristics and condition of the patient.

It is to be understood that the compounds, compositions and methods of the present invention may be beneficial in the prevention and/or treatment of pre-cachexia as well as cachectic conditions, in particular in the maintenance or improvement of skeletal muscle mass and/or muscle function.

In one embodiment of the invention, the invention provides a method of treating cachexia or pre-cachexia comprising administering to a human or animal subject an effective amount of a compound of the invention.

In another embodiment of the invention, the invention provides a method of treating cachexia or pre-cachexia comprising administering to a human or animal subject an effective amount of a compound of the invention, wherein cachexia or pre-cachexia is associated with a disease selected from the group consisting of: cancer, chronic heart failure, renal failure, chronic obstructive pulmonary disease, AIDS, autoimmune disorders, chronic inflammatory disorders, cirrhosis of the liver, anorexia, chronic pancreatitis, metabolic acidosis, and/or neurodegenerative diseases.

In a preferred embodiment of the present invention, the present invention provides a method of treating cancer cachexia associated with a cancer selected from the group consisting of: pancreatic cancer, esophageal cancer, gastric cancer, intestinal cancer, lung cancer and/or liver cancer.

In yet another embodiment of the invention, the invention provides a method of treatment wherein the treatment of cancer cachexia is measured by reducing weight loss, preventing weight loss, maintaining weight, or increasing weight.

In another embodiment of the invention, the compounds or compositions of the invention may be used in a method of treatment, wherein cancer cachexia is the result of treatment of the cancer with a chemotherapeutic agent.

In another embodiment of the invention, the compounds or compositions of the invention may be used in a method of preventing or treating cachexia in combination with dietary intervention of high calorie, high protein, high carbohydrate, vitamin B3, vitamin B12 and/or vitamin D supplements, antioxidants, omega fatty acids and/or polyphenols.

Sarcopenia and related disorders

Sarcopenia may be characterized by one or more of low muscle mass, low muscle strength, and low physical fitness.

The sarcopenia of an individual may be diagnosed based on the definition of AWGSOP (senior sarcopenia asian working group), e.g., as described by Chen et al 2014. Low muscle mass can generally be based on low extremity lean mass (ALM index) normalized to height squared, in particular ALM index less than 7.00kg/m2 for men and less than 5.40kg/m2 for women. Low physical performance may generally be based on walking speed, in particular walking speed less than 0.8 m/sec. Low muscle strength may generally be based on low grip strength, in particular less than 26kg for men and less than 18kg for women.

The sarcopenia of an individual may be diagnosed based on the definition of EWGSOP (geriatric sarcopenia european working group), e.g. as described by Crutz-Jentoft et al 2010. Low muscle mass can generally be based on low limb lean body mass (ALM index) normalized to height squared, in particular ALM index less than 7.23kg/m2 for men and less than 5.67kg/m2 for women. Low physical performance may generally be based on walking speed, in particular walking speed less than 0.8 m/sec. Low muscle strength may generally be based on low grip strength, in particular less than 30kg for men and less than 20kg for women.

The individual may be diagnosed for sarcopenia based on the definition of the national institute of health Foundation (FNIH), e.g., as described by Studenski et al, 2014. Low muscle mass can generally be based on low extremity lean body mass (ALM) normalized to body mass index (BMI; Kg/m2), specifically male ALM to BMI less than 0.789, and female ALM to BMI less than 0.512. Low physical performance may generally be based on walking speed, in particular walking speed less than 0.8 m/sec. Low muscle strength may generally be based on low grip strength, in particular less than 26kg for men and less than 16kg for women. Low muscle strength can also be generally based on low grip: body mass index, in particular the male grip: body mass index less than 1.00, and female grip: the body mass index is less than 0.56.

D3-creatine dilution is another method for measuring muscle mass. This approach is becoming more widely accepted as a robust standard and is expected to replace DXA in the future. The D3-creatine dilution method has been previously described in Clark et al, (1985) and Stimpson et al, (2013).

It is to be understood that the compounds, compositions, and methods of the present invention may be beneficial in the prevention and/or treatment of sarcopenia and/or related conditions, in particular to maintain or improve skeletal muscle mass and/or muscle function.

Myopathy and related disorders

Myopathy is a neuromuscular disorder, the primary symptom being muscle weakness due to dysfunction of muscle fibers. Other symptoms of myopathy may include muscle spasms, stiffness, and convulsions. Myopathies may be inherited (such as muscular dystrophy) or acquired (such as common muscle spasms).

Myopathies were grouped as follows: (i) congenital myopathy: it is characterized by a developmental delay in motor skills; skeletal and facial abnormalities are occasionally evident at birth; (ii) muscular dystrophy: it is characterized by gradual muscle weakness at will; sometimes evident at birth; (iii) mitochondrial myopathy: caused by genetic abnormalities of mitochondria (cellular structures that control energy); including Kearns-Sayre syndrome, MELAS and MERRF muscle glycogen storage disease: caused by genetic mutations in enzymes that control the metabolism of glycogen and glucose (blood glucose); including Pompe, Andersen, and Cori diseases; (iv) myoglobinuria: caused by a disturbance in the metabolism of the fuel (myoglobin) necessary for the muscle to work; including McArdle's disease, Tarui's disease, and DiMauro's disease; (v) dermatomyositis: inflammatory myopathies of the skin and muscle; (vi) myositis ossificans: characterized in that bone grows in muscle tissue; (vii) familial periodic paralysis: it is characterized by the onset of weakness in the arms and legs; (viii) polymyositis, inclusion body myositis and related myopathies: skeletal myositis myopathy; (ix) neuromuscular rigidity: it is characterized by alternating episodes of twitching and stiffness; and stiff person syndrome: it is characterized by the onset of stiffness and reflex spasms (common muscle spasms and stiffness), and (x) tetany: it is characterized by prolonged twitching of the arms and legs. (reference: https:// www.ninds.nih.gov/disorders/all-disorders/myopathy-information-page).

It is to be understood that the compounds, compositions, and methods of the present invention may be beneficial in the prevention and/or treatment of the above-mentioned diseases or disorders, in particular in the maintenance or improvement of skeletal muscle mass and/or muscle function.

Muscular dystrophy

Muscular dystrophy is a group of genetic diseases characterized by progressive weakness and degeneration of skeletal or voluntary muscles that control movement. The main types of muscular dystrophy include: duchenne muscular dystrophy, Becker muscular dystrophy, limb girdle muscular dystrophy, facioscapulohumeral muscular dystrophy, congenital muscular dystrophy, oculopharyngeal muscular dystrophy, distal muscular dystrophy, Emery-Dreifuss muscular dystrophy, and myotonic dystrophy.

(reference: https:// www.medicalnewstoday.com/articles/187618.php)

It is to be understood that the compounds, compositions, and methods of the present invention may be beneficial in the prevention and/or treatment of the above-mentioned diseases or disorders, in particular in the maintenance or improvement of skeletal muscle mass and/or muscle function.

Post-operative and muscle trauma muscle injury recovery

Muscle damage can be caused by abrasion, stretching, or tearing, causing acute or chronic soft tissue damage to the muscles, tendons, or both. Which can occur due to muscle fatigue, overuse, or misuse, for example, during strenuous exercise. It may occur after physical trauma such as a fall, break or overuse during physical activity. Muscle damage may also occur following surgical procedures such as arthroscopic joint replacement surgery.

It will be appreciated that the compounds, compositions and methods of the present invention may be beneficial in the prevention and/or treatment of the above-mentioned conditions of recovery following surgery and/or muscle trauma, in particular in the maintenance or improvement of skeletal muscle mass and/or muscle function.

Detailed description of the preferred embodiments

The present invention provides compounds and compositions comprising reduced nicotinamide riboside. Another aspect of the invention is a unit dosage form of a composition consisting of reduced nicotinamide riboside, and the unit dosage form comprises an amount effective to increase intracellular NAD in a subject in need thereof⁺Reduced nicotinamide riboside of (4).

In the prevention or treatment of skeletal muscle diseases, NAD⁺An increase in biosynthesis can provide one or more beneficial effects to an individual, such as a human (e.g., a human undergoing medical treatment), a pet or horse (e.g., a pet or horse undergoing medical treatment), or cattle or poultry (e.g., cattle or poultry used in agriculture).

For non-human mammals such as rodents, some embodiments include administering an amount of the composition that provides 1.0mg to 1.0g reduced nicotinamide riboside per kg of body weight of the non-human mammal, preferably 10mg to 500mg reduced nicotinamide riboside per kg of body weight of the non-human mammal, more preferably 25mg to 400mg reduced nicotinamide riboside per kg of body weight of the mammal, most preferably 50mg to 300mg reduced nicotinamide riboside per kg of body weight of the non-human mammal.

For humans, some embodiments include administering an amount of the composition that provides 1.0mg to 10.0g reduced nicotinamide riboside per kg body weight of the human, preferably 10mg to 5.0g reduced nicotinamide riboside per kg body weight of the human, more preferably 50mg to 2.0g reduced nicotinamide riboside per kg body weight of the human, most preferably 100mg to 1.0g reduced nicotinamide riboside per kg body weight of the human.

In some embodiments, at least a portion of the reduced nicotinamide riboside is isolated from a natural plant source. Additionally or alternatively, at least a portion of the reduced nicotinamide riboside can be chemically synthesized. For example, according to example 1 described below.

As used herein, a "composition consisting essentially of reduced nicotinamide riboside" comprises reduced nicotinamide riboside and does not include, or is substantially free of, or is completely free of any additional compounds other than "reduced nicotinamide riboside" that affect NAD + production. In a specific non-limiting embodiment, the composition consists of reduced nicotinamide riboside and an excipient or one or more excipients.

In some embodiments, the composition consisting essentially of reduced nicotinamide riboside is optionally substantially free or completely free of other NAD + precursors, such as nicotinamide riboside.

As used herein, "substantially free" means that any other compound present in the composition is no more than 1.0 wt% relative to reduced nicotinamide riboside, preferably no more than 0.1 wt% relative to reduced nicotinamide riboside, more preferably no more than 0.01 wt% relative to reduced nicotinamide riboside, and most preferably no more than 0.001 wt% relative to reduced nicotinamide riboside.

Another aspect of the invention is for increasing intracellular NAD in a mammal in need thereof⁺The method comprising increasing NAD in an effective amount⁺The biosynthetic amount administers to the mammal a composition consisting essentially of or consisting of reduced nicotinamide riboside. The method can promote cell and tissue growthNAD⁺To improve cell and tissue survival and overall cell and tissue health, for example in muscle cells and tissues, particularly skeletal muscle cells and tissues.

Nicotinamide adenine dinucleotide (NAD +) is considered a coenzyme and is an essential cofactor for energy production in cellular redox reactions. It plays a key role in energy metabolism, since oxidation of NADH to NAD + favors hydride transfer and thus ATP is generated by mitochondrial oxidative phosphorylation. It also serves as a degradation substrate for a variety of enzymes (Canto, C. et al 2015; Imai, S. et al 2000; Chambon, P. et al 1963; Lee, H.C. et al 1991).

Mammalian organisms can synthesize NAD + from four different sources. First, NAD + can be obtained from tryptophan by a 10-step de novo pathway. Second, Nicotinic Acid (NA) can also be converted to NAD + via a 3-step Preiss-Handler pathway that converges with the de novo pathway. Third, the intracellular NAD + salvage pathway from Nicotinamide (NAM) constitutes the major pathway for cells to build NAD +, and occurs through a 2-step reaction in which NAM is first converted to NAM-mononucleotide (NMN) via the catalytic activity of NAM-phosphoribosyltransferase (NAMPT), and then to NAD + via NMN adenylyltransferase (NMNAT) enzyme. Finally, Nicotinamide Riboside (NR) constitutes the fourth pathway of NAD +, which is characterized by NR kinase (NRK) initial phosphorylation of NR to NMN (Bregmaanowski, P. et al; 2004).

Five molecules are previously known to act as direct extracellular NAD + precursors: tryptophan, Nicotinic Acid (NA), Nicotinamide (NAM), nicotinic acid ribonucleoside (NaR) and Nicotinamide Ribonucleoside (NR). The present invention discloses a novel molecule, reduced Nicotinamide Riboside (NRH), which can act as an extracellular NAD + precursor. The reduction of NR molecules to NRH not only confers a much stronger ability to increase intracellular NAD + levels, but also confers different selectivity in their cellular use.

The present invention relates to NRH, a novel molecule that can act as a NAD + precursor. This reduced form of NR shows unprecedented capacity to increase NAD + and has the advantage of being more potent and faster than Nicotinamide Riboside (NR). NRH utilizes a different pathway to synthesize NAD + than NR, which is NRK independent. The present invention shows that NRH is protected from degradation in plasma and can be detected in the circulation after oral administration. These advantages of the present invention support its therapeutic efficacy.

The method comprises administering to the individual an effective amount of a composition consisting essentially of or consisting of reduced nicotinamide riboside.

In each of the compositions and methods disclosed herein, the composition is preferably a food product or beverage product, including a food additive, food ingredient, functional food, dietary supplement, medical food, nutraceutical, Oral Nutritional Supplement (ONS), or food supplement.

The composition may be administered weekly for at least one day, preferably weekly for at least two days, more preferably weekly for at least three or four days (e.g., every other day), most preferably at least five, six or seven days per week. The period of administration may be at least one week, preferably at least one month, more preferably at least two months, most preferably at least three months, for example at least four months. In some embodiments, dosing is at least daily; for example, the subject may receive one or more doses per day, in one embodiment multiple doses per day. In some embodiments, the administration is for the remaining lifespan of the individual. In other embodiments, administration occurs until no detectable symptoms of the medical condition remain. In particular embodiments, administration occurs until a detectable improvement in at least one symptom occurs, and the improvement continues to be maintained in additional instances.

The compositions disclosed herein can be administered to a subject enterally (e.g., orally) or parenterally. Non-limiting examples of parenteral administration include intravenous, intramuscular, intraperitoneal, subcutaneous, intra-articular, intrasynovial, intraocular, intrathecal, topical and inhalation. Thus, non-limiting examples of composition forms include natural foods, processed foods, natural juices, concentrates and extracts, injectable solutions, microcapsules, nanocapsules, liposomes, ointments, inhalation forms, nasal sprays, nasal drops, eye drops, sublingual tablets, and sustained release formulations.

The compositions disclosed herein can be administered therapeutically using any of a variety of formulations. More specifically, the pharmaceutical composition may comprise a suitable pharmaceutically acceptable carrier or diluent, and may be formulated into preparations in the form of solid, semisolid, liquid or gas, such as tablets, capsules, powders, granules, ointments, solutions, suppositories, injections, inhalants, gels, microspheres and aerosols. Thus, administration of the composition can be accomplished in a variety of ways, including oral, buccal, rectal, parenteral, intraperitoneal, intradermal, transdermal, and intratracheal administration. The active agent may be systemic after administration, or may be localized through the use of local administration, intramural administration, or the use of an implant that acts to maintain the active dose at the site of implantation.

In pharmaceutical dosage forms, the compounds may be administered as their pharmaceutically acceptable salts. They may also be used in appropriate combination with other pharmaceutically active compounds. The following methods and excipients are exemplary only, and not in any way limiting.

For oral formulations, the compounds can be used alone or in combination with suitable additives to prepare tablets, powders, granules or capsules, for example in combination with conventional additives, such as lactose, mannitol, corn starch or potato starch; in combination with a binder, such as crystalline cellulose, a functional derivative of cellulose, gum arabic, corn starch or gelatin; in combination with a disintegrant such as corn starch, potato starch or sodium carboxymethyl cellulose; in combination with a lubricant, such as talc or magnesium stearate; and if desired, in combination with diluents, buffers, wetting agents, preservatives and flavouring agents.

The compounds may be formulated for injection by: dissolving, suspending or emulsifying these compounds in an aqueous or non-aqueous solvent (such as vegetable oil or other similar oils, synthetic aliphatic glycerides, esters of higher aliphatic acids or propylene glycol); and these compounds are used together with conventional additives such as solubilizers, isotonic agents, suspending agents, emulsifiers, stabilizers and preservatives, if necessary.

These compounds are useful in aerosol formulations to be administered by inhalation. For example, the compounds may be formulated as pressurized acceptable propellants, such as dichlorodifluoromethane, propane, nitrogen, and the like.

In addition, these compounds can be formulated into suppositories by mixing with various bases such as emulsifying bases or water-soluble bases. The compounds may be administered rectally by means of suppositories. Suppositories may contain vehicles such as cocoa butter, carbowax (carbowax) and polyethylene glycol, which melt at body temperature but solidify at room temperature.

Unit dosage forms for oral or rectal administration may be provided, such as syrups, elixirs and suspensions, wherein each dosage unit (e.g. teaspoonful, tablespoonful, tablet or suppository) contains a predetermined amount of the composition. Similarly, unit dosage forms for injection or intravenous administration may comprise the compounds in a composition that is a solution in sterile water, physiological saline, or another pharmaceutically acceptable carrier, wherein each dosage unit, e.g., mL or L, contains a predetermined amount of the composition containing one or more of the compounds.

Compositions intended for use in non-human animals include food compositions that provide the necessary dietary needs for the animal, animal treats (e.g., biscuits), and/or dietary supplements. The composition can be a dry composition (e.g., kibble), semi-moist composition, wet composition, or any mixture thereof. In one embodiment, the composition is a dietary supplement, such as a gravy, drinking water, beverage, yogurt, powder, granule, paste, suspension, chew, treat, snack, pellet, pill, capsule, tablet, or any other suitable delivery form. Dietary supplements may contain high concentrations of UFA and NORC, as well as B vitamins and antioxidants. This allows the supplement to be administered to the animal in small amounts, or in the alternative, can be diluted prior to administration to the animal. The dietary supplement may require mixing or may be mixed with water or other diluents prior to administration to an animal.

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