Enhanced NAD + compositions and methods of making and using the same

Abstract

The present disclosure provides enhanced nicotinamide adenine dinucleotide (NAD+) compositions and pharmaceutical formulations, useful in the treatment of disorders and diseases regulated by deficiencies in NAD+ including neurodegenerative diseases and disorders and / or age-related diseases and disorder, and methods of preparation and use of the same.

Description

ENHANCED NAD+COMPOSITIONS AND METHODS OF MAKING AND USING THE SAMERELATED APPLICATION

[0001] This application claims priority to U.S. Provisional Patent Application Serial Nos. 63 / 665,794, filed June 28, 2024, and 63 / 688,293, filed on August 28, 2024, which are incorporated herein by reference in their entireties for all purposes.FIELD OF DISCLOSURE

[0002] The present disclosure relates to the field of therapeutic methods. In particular, the disclosure relates to chemical compound compositions and pharmaceutical formulations or medical food for treating conditions, diseases, and disorders.BACKGROUND OF THE DISCLOSURE

[0003] Neurodegenerative diseases such as Parkinson’s disease (PD), Multiple Sclerosis (MS), and Amyotrophic Lateral Sclerosis (ALS) and Alzheimer’s disease (AD) are diseases of the central nervous system with some possible overlapping disease processes and points of therapeutic intervention. They share many similar symptoms, and at times can often be mistaken for one another, especially in their earlier stages. However, there are key differences in their symptoms and prognoses that make each one a unique and challenging condition. These diseases affect thousands of new patients each year in the US. There is no curative treatment for these diseases.

[0004] Nicotinic acid and nicotinamide, collectively “niacins,” are the vitamin forms of nicotinamide adenine dinucleotide (NAD+) found in all living cells. NAD+plays an essential role in the synthesis of adenosine triphosphate (ATP), an organic compound that provides energy for many processes in living cells, such as muscle contraction, nerve impulse propagation, condensate dissolution, and chemical synthesis, and as such NAD+is a crucial nutrient for animal and human health.Eukaryotes synthesize NAD+de novo from tryptophan via the kynurenine pathway (Krehl, et al. Science 101:489-490 (1945); Schutz and Feigelson J. Biol. Chem. 247:5327-5332 (1972)). NAD+can also be resynthesized from nicotinamide (NAM) via the salvage pathway; and from nicotinic acid (NA) via the Preiss-Handler pathway where NA is phosphoribosylated to nicotinic acid mononucleotide (NMN), which is then adenylated to form nicotinic acid adenine dinucleotide (NAAD), which in turn is amidated to form NAD+(Preiss and Handler J. Biol. Chem. 233:488-492 (1958); Preiss and Handler Biol. Chem. 233:493-50 (1958)).

[0005] Depleted levels of NAD+are known to lead to a number of conditions and disorders in humans. For example, drug and alcohol abuse has been shown to deplete NAD+levels, causing recovery from addiction to be a physically and psychologically challenging process.

[0006] NAD+is a key metabolite in aging, and depleted levels of NAD+are known to lead to anumber of conditions and disorders in humans. For example, levels of NAD+have been shown to decrease with age and are depleted in neurodegenerative diseases. In addition, low intracellular NAD+increases reactive oxygen species (ROS) production, mitochondrial dysfunction, cellular senescence. Further, animal models demonstrate benefits of NAD+repletion in modulating aging pathways.

[0007] Supplementation of NAD+has application in numerous diseases and conditions. For example, niacin supplementation prevents the pellagra that can occur in populations with a tryptophan-poor diet. U.S. Pat. No. 3,412,190 to O'Holleran describes the use of NADPH for the lipids in the blood stream. NADPH has also been used to treat alcoholism (see, e.g., Canadian Patent 670,909). However, currently available commercially produced NAD+is chemically and biologically unstable. It has a limited shelflife, and requires special storage condition requirements (such as, for example, cooling or dry storage) to manufacture and produce it. In addition, NAD is metabolized in the body rapidly, and thus a high dosage is required for efficacy. In addition, up to this point, NAD+supplementation has been by IV which has been reported to be difficult, and the dosage required to be therapeutic has been high due to instability. The risks associated with the administration of IV NAD+include fluid overload in patients with compromised renal, hepatic or cardiac function, and the side effect profile has included pain in the back of the neck, paresthesias at the base of the tongue, dizziness, flushing, and nausea (U.S. Patent No. 5,888,532).

[0008] An oral formulation of NAD+and some phosphate derivatives of NAD+that have a slightly different bioactivity ad resistance to metabolic deactivation has been developed (U.S. Patent No. 5,888,532). However, NAD+bioavailability is debated. For instance, endogenous NAD+is predicted to not be orally bioavailable due to (i) its large size (663 g / mol) and charge, and rapid degradation on the shelf and in the GI tract. NAD+is not completely absorbed by the gastric acid intestinal mucosa, and thus this oral form also requires a higher dosage for efficacy. Additionally, the widespread expression of NAD+hydrolases which causes enzymatic degradation, coupled with acidic pH, and metabolism by microbiome.

[0009] Current therapies, primarily NAD precursors, show limited benefit in diseases of aging: variable conversion rates and delayed onset constrain therapeutic potential. Accordingly, there is an unmet need for improved NAD+compositions and pharmaceuticals with enhanced stability and bioavailability for the treatment of disease, including age-related diseases and disorders and neurodegenerative diseases.SUMMARY OF THE DISCLOSURE

[0010] It has been discovered that NAD+can change gene expression, and accordingly, can change the resulting plasma level of certain proteins encoded by certain genes in subjects having a neurodegenerative disease or disorder. This discovery has been exploited to provide the instantdisclosure, which provides methods of using enhanced NAD+compositions.

[0011] The present disclosure relates to enhanced NAD+pharmaceutical compositions and methods for their use in treating neurodegenerative diseases / disorders and / or age-related diseases / disorders, such as Alzheimer’s Disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS). In addition to pharmaceutical applications, the enhanced NAD+compositions can be used in precision nutrition medical food interventions.

[0012] In one aspect, the disclosure provides a method of preventing or treating a neurodegenerative disease in a subject, the method comprising administering a therapeutically effective amount of an enhanced NAD+pharmaceutical formulation or medical food to the subject. In some examples, the neurodegenerative disease is Parkinson’s disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis or Alzheimer’s disease. In some examples, the subject is human.

[0013] In another aspect, the present disclosure provides a method of administering a precision nutritional composition comprising enhanced NAD+to a human subject diagnosed with a condition associated with impaired NAD+metabolism deficiency. In some embodiments, the method comprises orally administering a therapeutically effective amount of enhanced NAD+medical food formulated to bypass degradation in the gastrointestinal tract. The medical food can be administered to the human subject under physician supervision to meet the distinct nutritional requirement for NAD+in the human subject and restore intracellular NAD+homeostasis.

[0014] In some examples, the administration changes the level of gene expression of a protein involved in neurodegenerative disease. In certain examples, the gene encodes a protein selected from the group of SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Receptor 1A (HTR1A), Insulin Receptor (INSR), Superoxide Dismutase 2 (SOD2), Artemin (ARTN), Fas Cell Surface Death Receptor (FAS), Interleukin 2 Receptor Subunit Beta (IL2Rb), Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), CD163 Molecule, Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), and Neurofilament Light Chain (NfL).

[0015] In certain examples, administration of the enhanced NAD+formulation increases the level of at least one of a protein involved in neurodegenerative disease, as compared to the level of the at least one protein in an unadministered subject. In some examples, the plasma level of the at least one protein is increased in the administered subject. In certain examples, the level of at least one of SIRT1 (Sirtuin 1), 5 -Hydroxytryptamine Receptor 1A (HTR1A), Artemin (ARTN), Interleukin 2 Receptor Subunit Beta (IL2Rb), CD 163 Molecule, and / or Platelet-Derived Growth Factor Receptor Alpha (PDGFRA) is increased as compared to the level of the at least one protein in an unadministered subject.

[0016] In other examples, the administration decreases a level of at least one protein involved in neurodegenerative disease, as compared to the level of the at least one protein in an unadministeredsubject. In certain examples, the level of the at least one protein is decreased in the plasma of the administered subject as compared to the level of the at least one protein an unadministered subject. In specific examples, the level of at least one of C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Insulin Receptor (INSR), Superoxide Dismutase 2 (SOD2), Fas Cell Surface Death Receptor (FAS), Platelet and / or Endothelial Cell Adhesion Molecule 1 (PECAM1) is decreased as compared to the level of the at least one protein in an unadministered subject.

[0017] In some examples, the neurodegenerative disease is Parkinson’s disease, and the administration changes the level of at least one protein in the subject, the protein being selected from the group consisting of SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5- Hydroxytryptamine Receptor 1A (HTR1A), and Insulin Receptor (INSR). In certain examples, the plasma level of the at least one protein is changed.

[0018] In other examples, the neurodegenerative disease is Multiple Sclerosis, and the administration changes the level of at least one protein selected from the group consisting of SIRT1 (Sirtuin 1), C-X- C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Fas Cell Surface Death Receptor (FAS), Interleukin 2 Receptor Subunit Beta (IL2Rb), Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), CD163 Molecule, and Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), Matrix Metallopeptidase 9 (MMP9), and Neurofdament Light Chain (NfL). In certain examples, the plasma level of the at least one protein is changed.

[0019] In yet other example, the neurodegenerative disease is Amyotrophic Lateral Sclerosis, and the administration changes the level of at least one protein selected from the group consisting of SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), Superoxide Dismutase 2 (SOD2), Artemin (ARTN), and Neurofdament Light Chain (NfL). In certain examples, the plasma level of the at least one protein is changed.

[0020] In another aspect, the disclosure provides a method of changing the level of at least one protein involved in a neurodegenerative disease in a subject, the method comprising administering to the subject an amount of an enhanced NAD+formulation effective to change the level of the protein in the subject. In some examples, the level of the protein in the plasma of the administered subject is changed. In specific examples, the protein involve in a neurodegenerative disease is selected from the group consisting of SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Receptor 1A (HTR1A), Insulin Receptor (INSR), Superoxide Dismutase 2 (SOD2), Artemin (ARTN), Fas Cell Surface Death Receptor (FAS), Interleukin 2 Receptor Subunit Beta (IL2Rb), Platelet and Endothelial Cell Adhesion Molecule 1(PECAM1), CD163 Molecule, Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), and Neurofilament Light Chain (NfL).

[0021] In some examples, the level of the protein involved in neurodegenerative disease is increased, as compared to the level of the protein in an unadministered subject. In certain examples, the level of at least one of SIRT1 (Sirtuin 1), 5 -Hydroxytryptamine Receptor 1A (HTR1A), Artemin (ARTN), Interleukin 2 Receptor Subunit Beta (IL2Rb), CD 163 Molecule, and / or Platelet-Derived Growth Factor Receptor Alpha (PDGFRA) is increased as compared to the level of the protein in an unadministered subject

[0022] In other examples, the level of the protein involved in neurodegenerative disease is decreased, as compared to the level of the at least one protein in an unadministered subject. In specific examples, the level of at least one of C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Insulin Receptor (INSR), Superoxide Dismutase 2 (SOD2), Fas Cell Surface Death Receptor (FAS), Platelet, Endothelial Cell Adhesion Molecule 1 (PECAM1), and / or Neurofilament Light Chain (NfL) is decreased as compared to the level of the at least one protein in an unadministered subject.

[0023] In some examples, subject suffers from a neurodegenerative disease. In certain examples, the neurodegenerative disease is Parkinson’s disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, or Alzheimer’s disease. In some embodiments, a subject having the neurodegenerative disease, such as Parkinson’s disease, Multiple Sclerosis, Amyotrophic Lateral Sclerosis, or Alzheimer’s disease, has an elevated level of circulating Neurofilament Light Chain (NfL). In some embodiments, the enhanced NAD+ formulation and / or medical food reduces the level of circulating Neurofilament Light Chain (NfL).

[0024] In yet another aspect, the disclosure provides an enhanced NAD+composition with increased shelf life, stability, and bioavailability relative to NAD+(e.g., NAD+monohydrate), such as commercially obtainable and other known forms of NAD. In some embodiments, the enhanced composition comprises vacuumized NAD+and phosphorus. In particular embodiments, the composition consists essentially of vacuumized NAD+, and phosphorus. In some embodiments, the enhanced composition comprises vacuumized NAD+and polyethylene glycol. In some embodiments, the enhanced composition comprises vacuumized NAD+.

[0025] In some embodiments, the unit dose of NAD+in the composition described herein is about 5 mg to about 4000 mg. In some embodiments, the unit dose of NAD+is about 500 mg.

[0026] In some embodiments, the unit dose of NAD+is about 5 mg, about 25 mg, about 50 mg, about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1500 mg, about 1700 mg, about 2000 mg, about 2200 mg, about 2400 mg, about 2500 mg, about 2600 mg, about 2800 mg, about 3000 mg, about 3200 mg, about 3400 mg, about 3600 mg, about 3800 mg, or about 4000mg.

[0027] In some embodiments, the composition comprises from about 5 mg to about 4000 mg NAD+. In certain embodiments, the composition comprises about 100 mg, about 200 mg, about 300 mg, about 400 mg, about 500 mg, about 600 mg, about 700 mg, about 800 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, or about 4000 mg NAD+.

[0028] Another aspect of the instant disclosure relates to a composition comprising nicotinamide adenine dinucleotide (NAD+), polyethylene glycol (PEG), and a phosphorus content of about 3.0% to about 11.0%. In some embodiments, the composition contains a phosphorus content of about 3.0% to about 9.5%. In some embodiments, the composition contains a phosphorus content of about 3.0% to about 9.33%.

[0029] In some embodiments, the concentration of NAD+in the composition described herein is about 200 mg / ml to about 1000 mg / ml. In some embodiments, the concentration of NAD+is about 500 mg / ml.

[0030] In some embodiments, the enhanced NAD+composition is in powder form. In certain embodiments, the composition has a longer shelflife relative to a commercially obtained NAD+, e.g., NAD+monohydrate.

[0031] In an additional aspect, the disclosure provides a pharmaceutical formulation or medical food comprising the enhanced NAD+composition described supra, and at least one ingredient which facilitates administration to a subject and / or which can further treat the subject’s disease or condition.

[0032] In some embodiments, the pharmaceutical formulation comprises polyethylene glycol (PEG ) of any molecular weight and / or combinations thereof, such as, but not limited to, PEG 300, PEG 400, PEG monomethyl ether (MME) 550, PEG 600, PEG 1000, PEG MME 2000, PEG 3350, and / or PEG 4000.

[0033] In some embodiments, the composition is a powder.

[0034] In some embodiments, the PEG is PEG 300, PEG 400, PEG monomethyl ether (MME) 550, PEG 600, PEG 1000, PEG MME 2000, PEG 3350, and / or PEG 4000. In some embodiments, the PEG is PEG 3350. In some embodiments, the PEG has a molecular mass over 4000 g / mol. In some embodiments, the PEG is a higher molecular weight PEG than is commercially available. Illustrative methods of PEG synthesis are described in Khanal and Fang, Chemistry 23(60): 15133-15142 (2017), which is incorporated herein by reference in its entirety.

[0035] In some embodiments, the composition further comprises traces of an inert gas. In some embodiments, the inert gas comprises Nitrogen, Helium, Xenon, or Argon. In some embodiments, the inert gas comprises Nitrogen. In some embodiments, the inert gas comprises Helium. In some embodiments, the inert gas comprises Xenon. In some embodiments, the inert gas comprises Argon.

[0036] An additional aspect of the disclosure relates to a process of making a NAD+composition (such as an enhanced NAD+composition). The process comprises blending NAD+and PEG andvacuumizing the blended mixture of NAD+and PEG under vacuum and inert gas conditions. In some embodiments, vacuumizing comprises placing the mixture of NAD+and PEG under reduced pressure under vacuum at a pressure between about 24 inches of Hg and about 30.25 inches of Hg. Units of measurements of pressure may be readily interconverted by one skilled in the art. In another embodiment, vacuumizing further comprises replacing the vacuum with an inert gas into the blended mixture to bring the pressure in the blender to about 1 atm. In some embodiments, the process further comprises pre-treating the NAD+by passing the NAD+through a 0.25 mm sieve. In some embodiments, the process further comprises pre-treating the NAD+to produce a particle that is 0. 152 mm in size. Without wishing to be bound by theory, the co -formulation of the NAD+with PEG provides increased product stability. For example, such pre-treating may be performed prior to mixing NAD+and PEG. In some embodiments, such pre-treating is performed after mixing NAD+and PEG (e.g., pre -formulating the NADVPEG mixture). In some embodiments, a measured portion of PEG is pre -vacuumized before adding NAD+to the composition.

[0037] In some embodiments, the PEG is PEG 300, PEG 400, PEG monomethyl ether (MME) 550, PEG 600, PEG 1000, PEG MME 2000, PEG 3350, and / or PEG 4000. In some embodiments, the PEG is PEG 3350. In some embodiments, the PEG has a molecular mass over 4000 g / mol.

[0038] In some embodiments, the blending of NAD+(e.g., the pre-formulated NAD+) and PEG (e.g., the pre-formulated PEG) is performed in a blender.

[0039] In some embodiments, the vacuumizing comprises placing the mixture of NAD+and PEG under a stable vacuum.

[0040] In some embodiments, the vacuumizing further comprises pumping an inert gas into the blended mixture under vacuum to bring the pressure in the blender back to about 1 atmosphere (atm). In some embodiments, the inert gas comprises Helium, Nitrogen, Xenon, and / or Argon. In some embodiments, the inert gas comprises Nitrogen. In some embodiments, the inert gas comprises Helium. In some embodiments, the inert gas comprises Xenon. In some embodiments, the inert gas comprises Argon.

[0041] In some embodiments, the vacuumizing is performed at least twice.

[0042] A further aspect of the instant disclosure relates to NAD+compositions made by the process described herein. In some embodiments, the process comprises blending NAD+and PEG and vacuumizing the blended mixture of NAD+and PEG under vacuum and inert gas conditions.

[0043] In some embodiments, a NAD+composition described herein comprises water in an amount less than about 0.2%.

[0044] In some embodiments, the dose of the NAD+composition described herein is about 5 mg to 4000 mg. In some embodiments, the dose of the NAD+composition is about 500 mg per gram of composition.

[0045] In some embodiments, the PEG is PEG 300, PEG 400, PEG monomethyl ether (MME) 550, PEG 600, PEG 1000, PEG MME 2000, PEG 3350, and / or PEG 4000. In some embodiments, thePEG is PEG 3350.

[0046] In some embodiments, the composition further comprises traces of an inert gas. In some embodiments, the inert gas comprises Nitrogen, Helium, Xenon, or Argon. In some embodiments, the inert gas comprises Nitrogen. In some embodiments, the inert gas comprises Helium. In some embodiments, the inert gas comprises Xenon. In some embodiments, the inert gas comprises Argon.

[0047] In some embodiments, the ratio of NAD+to PEG in the composition is about 1: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 2: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 3: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 4: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 5: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 6: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 7: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 8: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 9: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 10: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 1:2 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 1:3 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 1:4 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 1:5 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 1:6 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 1:7 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 1:8 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 1:9 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 1: 10 (wt / wt). In some embodiments, the ratio of NAD+to PEG in the composition is about 1:20 (wt / wt).

[0048] In some embodiments, the ratio of NAD+to PEG in a composition of the present disclosure is 2: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in a composition of the present disclosure is 3 : 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in a composition of the present disclosure is 4: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in a composition of the present disclosure is 5: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in a composition of the present disclosure is 6: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in a composition of the present disclosure is 7: 1 (wt / wt). In some embodiments, the ratio of NAD+toPEG in a composition of the present disclosure is 8: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in a composition of the present disclosure is 9: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in a composition of the present disclosure is 10: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG in a composition of the present disclosure is 20: 1 (wt / wt).

[0049] In certain embodiments, the composition comprises phosphorus at about 3.0% to about 11.0%(wt / wt). In some embodiments, the ratio of NAD+to PEG is about 1: 1 (wt / wt). In some embodiments, the ratio of NAD+to PEG 3350 is about 1: 1 (wt / wt). The enhanced NAD+composition is in powder form in some embodiments.

[0050] In certain embodiments, the pharmaceutical formulation or medical food is suitable for oral administration to a subject. In particular embodiments, the pharmaceutical formulation is in powder, liquid, aqueous, tablet, suppository, or capsule form. In some embodiments, the formulation is suitable for subcutaneous implant and may be formulated as a nasal spray, cream, toothpaste, or shampoo.

[0051] In some embodiments, the enhanced NAD+pharmaceutical formulation is medical food comprising enhanced NAD+ of the present disclosure.

[0052] In some embodiments, the enhanced NAD+pharmaceutical formulation or medical food, when administered to a subject, increases NAD levels in the subject by at least about 10% relative to a baseline level of intracellular NAD (icNAD) in the subject. In some embodiments, the pharmaceutical is capable of increasing NAD+levels in the subject by at least about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, about 50%, about 52%, about 55%, about 60%, about 65%, about 70%, about 75%, 80%, about 85%, about 90%, about 95%, or about 100%, relative to a baseline level of intracellular NAD in the subject.

[0053] In particular embodiments, the pharmaceutical formulation or medical food is capable of increasing a subject’s intracellular levels of NAD, and in certain embodiments the formulation is capable of increasing intravascular levels of NAD in the subject. In some embodiments, the formulation does not significantly change extracellular NAD levels in the subject when administered to the subject.

[0054] In some embodiments, the subject to which the present enhanced NAD+pharmaceutical formulation or medical food is administered, has, or has a risk of having, a disease or disorder correlated with decreased NAD levels. In some embodiments, the subject has a cardiovascular disorder, a neurodegenerative disorder, a neuropsychiatric disorder, a liver disorder [e.g., nonalcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH)], a kidney disorder, a disorder of oxidative stress, a disorder of lipid, carbohydrate, or amino acid metabolism, an addiction to a substance use, a digestive disorder, a pancreatic disorder, an endocrine disorder, an immune system disorder, an autoimmune disorder, a reproductive system disorder, a metabolic system disorder, obesity, a skin disorder, a collagen disorder, and / or a bone disorder.

[0055] In particular embodiments relevant to ALS treatment, the enhanced NAD+pharmaceutical formulation or medical food demonstrates a unique pharmacokinetic profde optimized for central nervous system (CNS) delivery. Following administration, the formulation provided herein can further achieve:1. Rapid systemic bioavailability, with increases in blood NAD+and NADP+levels within approximately 15 minutes of oral administration;2. Central nervous system penetration, with measurable NAD+elevation in spinal cord tissue within approximately 2 hours post-administration;3. Sustained elevation of NAD+levels in neural tissue for at least 12 hours beyond the initial peak, maintaining therapeutic concentrations; and4. Dose-dependent increases in both systemic and tissue-specific NAD+levels.This profile of rapid CNS penetration and sustained elevation is particularly advantageous for ALS treatment, where therapeutic agents may need to reach and maintain effective concentrations in spinal cord motor neurons, and where oxidative stress can be a hallmark and central driver of pathophysiological changes.

[0056] In some embodiments, the present pharmaceutical formulation or medical food, which, when administered to a subject, is capable of: improving liver and / or kidney function in the subject, optionally by decreasing serum levels of gamma-glutamyl transferase (GGT), bilirubin, alkaline phosphatase (ALP), and / or albumin; decreasing oxidative stress in the subject, optionally by decreasing serum GGT, increasing plasma gamma-glutamyl glutamine (L-Glu-L-Gln), decreasing circulating glutamate, decreasing at least one of histidine metabolites, upregulating glutamate utilization, increasing plasma abundance of glutathione S-transferase alpha-1 (GSTA1), increasing mitochondrial carbonic anhydrase 5 (CA5A), decreasing plasma mitochondrial superoxide dismutase 2 (SOD2), and / or increasing plasma sirtuin 1 (SIRT1) and / or alcohol dehydrogenase 4 (ADH4); decreasing nitrosative stress and / or improves ammonia clearance, optionally by upregulating CA5A and / or decreasing plasma urea and / or aspartate; not significantly increasing inflammation in the subject, optionally measurable by circulating inflammatory biomarkers such as cardiac CRP, hsCRP, TNF, IL-6, and / or white blood cell counts; improving lipid transport and / or metabolism, optionally by decreasing LDL / HDL ratio, increasing plasma abundance of FABP 1 and / or RBP2, decreasing circulating medium- and / or long-chain PUFAs, decreasing plasma cholesterol levels, and / or increasing oxidated cholesterol metabolites; not significantly increasing fasting serum glucose levels in the subject; and / or increasing plasma abundance of at least one of NAD+metabolites, such as 1- methyl-nicotinamide (MeNAM), N1 -methyl -2 -pyri done-5 -carboxamide (2PY), indolelactate and / or urate.

[0057] The pharmaceutical formulation or medical food’s effects on oxidative stress are particularly relevant in ALS pathology. In some embodiments involving experimental ALS model systems, the enhanced NAD+formulation or medical food can modulate glutathione homeostasis in affected spinal cord tissue. Specifically, treatment can result in rapid increases in GSH levels in spinal cord tissue within approximately 15 to 30 minutes. The formulation can also improve the GSH / GSSG ratio, indicating a reduced oxidative burden, with these effects sustained for up to 48 hours. Additionally, treatment can enhance GST activity, thereby improving the tissue’s capacity to manage oxidative stress. These combined effects on the glutathione system represent a mechanism by which the enhanced NAD+formulation can slow ALS progression, as oxidative stress can be a primary driver ofmotor neuron death in this disease.

[0058] In particular embodiments, the pharmaceutical formulation or medical food is capable of modulating expression of at least one biomarker in a subject when administered to the subject. In certain embodiments, the at least one biomarker is a protein or metabolite that indexes endothelial, liver, and / or overall mitochondrial functioning and / or integrity, one of upregulated fatty acid-binding proteins, or one of downregulated proteins associated with endothelial, liver, and / or pancreatic injuries. In some embodiments, at least one biomarker is a marker for an opiate in the blood of a subject. In certain embodiments, the biomarker marker is an increased level of circulating serotonin receptor 1A (5-HT1AR), circulating cortisol or cortisone, and / or at least one liver enzyme. In some embodiments, the presence of the at least one biomarker is related to an addiction to a substance use, such as opiates and other drugs, including, but not limited to, e.g., increased circulating levels of serotonin receptor 1A (5-HT1AR), increased circulating cortisol or cortisone, and / or increased levels of at least one liver enzyme. The presence of the biomarker may also or alternatively be related to a neurodegenerative and or neuropsychiatric disease, including, e.g., increased levels of proteins with neurological effects, improved levels associated with neurodegenerative diseases, depression, anxiety, or chronic pain, oxidative stress, and / or nitrosative stress. The presence of the biomarker may also or alternatively be related to a cardiovascular disease, including, e.g., improved LDL / HDL ratio, vascular and / or endothelial health, injury resistance, and / or oxidative stress. In some embodiments, the presence of the biomarker may also or alternatively be related to an exocrine pancreas and / or digestion function, including, e.g., decreased circulate levels of pancreatic enzymes. The presence of the biomarker may also or alternatively be related to an inflammation and / or immune function, including, e.g., improved adaptive or innate immune function or inflammation. In some embodiments, the presence of the biomarker may also or alternatively be related to: a preproduction function, including, e.g., INSL3; a metabolic disease and / or obesity, including, e.g., GALNT2, FABP1, and / or RBP2; and / or skin and / or bone health, such as collagen synthesis, skin inflammation in psoriasis and / or atopic dermatitis, and / or bone development, including, e.g., PPIB, WFDC12, INSL4, and / or MATN3.

[0059] In embodiments relating to ALS treatment, the enhanced NAD+pharmaceutical formulation or medical food modulates specific biomarkers indicative of therapeutic response. For pharmacokinetic markers, the pharmaceutical formulation or medical food can result in blood NAD+elevation within approximately 15 minutes and spinal cord NAD+elevation within approximately 2 hours. For oxidative stress markers, the formulation can increase GSH levels and improve the GSH / GSSG ratio in spinal cord tissue, as well as reduce markers of lipid peroxidation. For functional markers, treatment can preserve compound muscle action potential (CMAP) amplitude and maintain motor unit number estimation (MUNE) stability. For disease progression markers, the formulation can affect circulating neurofilament light chain (NfL) levels and SOD 1 aggregation in neuronal and muscle tissues, particularly in relevant genetic subtypes. These biomarkers can support both doseoptimization and treatment response monitoring in ALS patients.

[0060] In some embodiments, neurofilament light chain (NfL) can serve as a clinically relevant biomarker for monitoring neuroaxonal damage and therapeutic response in subjects with neurodegenerative diseases. NfL is a cytoskeletal protein highly expressed in neuronal axons, and its presence in cerebrospinal fluid or blood can reflect the extent of axonal injury. In some embodiments, elevated NfL levels can be associated with disease activity or progression in conditions such as amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Parkinson’s disease (PD), and Alzheimer’s disease (AD). In certain embodiments, administration of an enhanced NAD+formulation or medical food can reduce circulating NfL concentrations, thereby indicating a potential protective effect on neuronal integrity and axonal structure.

[0061] In some embodiments relating to ALS-specific applications, the pharmaceutical formulation or medical food of the present disclosure demonstrates measurable functional benefits in preserving neuromuscular integrity. In validated ALS disease models (SOD1-G93A), treatment with the enhanced NAD+formulation over a 30-day period can show that, at certain tested doses, the pharmaceutical formulation or medical food can exhibit an overall trend (P=0.061) toward preservation of compound muscle action potential (CMAP) amplitude. In one treatment group, this can correspond to approximately an 18% reduction in muscle and motor neuron deterioration compared to controls. These functional outcomes, while preliminary, suggest that the enhanced NAD+formulation may not only address biochemical markers of disease but also translate to preservation of clinically relevant neuromuscular function.

[0062] In some embodiments, the pharmaceutical formulation or medical food provided herein does not increase any anxiety, stress, depression, fatigue, or any combination thereof in a subject.

[0063] In some embodiments, the pharmaceutical formulation or medical food provided herein does not elicit unfavorable change(s) of vital signs, does not elicit unfavorable change(s) to body compositions, does not elicit unfavorable change(s) to a clinical laboratory test result, or combination thereof.

[0064] In certain embodiments, the disclosure provides an enhanced NAD+pharmaceutical formulation or medical food for use in a method of: mitigating chemical addiction withdrawal symptoms in a subject in need thereof; mitigating opioid withdrawal symptoms in a subject in need thereof; increasing nicotinamide adenine dinucleotide (NAD+) levels in a subject; and / or preventing or treating a disease or disorder correlated to decreased levels of nicotinamide adenine dinucleotide (NAD+) in a subject in need thereof. In a further embodiment, the chemical addiction is related to a neurological or neurodegenerative disease or disorder.

[0065] In yet another aspect, the disclosure provides a kit comprising the enhanced NAD+composition or the enhanced NAD+pharmaceutical formulation or medical food provided herein.

[0066] In still another aspect, the disclosure provides a method of making the enhanced NAD+composition as described herein. The method comprises vacuumizing NAD+(e.g., NAD+monohydrate) in a container under vacuum; and replacing the vacuum in the container with an inert gas or CO2. In some embodiments, vacuumizing the NAD+comprises: placing the NAD+under a vacuum or under a reduced pressure of between about 24 inches of Hg and about 30.25 inches of Hg.; and replacing the vacuum with an inert gas to increase the pressure in the container to about 1 atmosphere (atm). In certain embodiments, vacuumization is performed at least twice. In some embodiments, the inert gas which replaces the vacuum comprises Helium, Nitrogen, Xenon, and / or Argon. In certain cases, the NAD+is vacuumized more than once, more than twice, or more than three times.

[0067] In some embodiments, the NAD is pretreated before being vacuumized. In particular embodiments, pretreatment comprises processing the NAD+to obtain a preselected average NAD+particle size. In certain embodiments, processing comprises passing the NAD+through a sieve, and / or grinding, milling, or jet-milling the NAD+.

[0068] The present disclosure also provides an enhanced NAD+composition made by a method as described herein.

[0069] In still another aspect, the disclosure provides a method of making an enhanced NAD+pharmaceutical formulation as described herein, comprising mixing the enhanced NAD+composition of the disclosure, such as that described above, with a compound, component, and / or ingredient which to form aa blended mixture. The secondary compound, component or ingredient is one that facilitates administration to a subject and / or which also has therapeutic efficacy. In some embodiments, the ingredient comprises PEG of any molecular weight or mixtures of PEG having different molecular weights.

[0070] In some embodiments, the NAD+is pretreated as described above to provide NAD+particles of a preselected particle size for homogeneous mixing with the secondary compound, component, or ingredient. In some embodiments, the NAD+is pretreated before being mixed with PEG in a blender. Pretreatment may also or alternatively comprise vacuumization of either the NAD+or PEG before they are mixed. The blended mixture of NAD+and PEG is then vacuumized in the blender under vacuum or under a reduced pressure, in some embodiments, of between about 24 inches of Hg and about 30.25 inches of Hg. The vacuum is then replaced with an inert gas or CO? to increase the pressure in the blender to about 1 atmosphere (atm). In certain embodiments, vacuumization before or after mixing is performed at least twice. In some embodiments, the inert gas which replaces the vacuum comprises Helium, Nitrogen, Xenon and / or Argon.

[0071] In yet another aspect, the disclosure provides a method of mitigating chemical addiction withdrawal symptoms in a subject in need thereof. The method comprises: administering a therapeutically effective amount of the present enhanced NAD+pharmaceutical formulation or medical food to the subject, the amount being effective to mitigate at least one chemical addiction withdrawal symptom in the subject. In some embodiment, the chemical addiction is related to a neurological or neurodegenerative disease or disorder.

[0072] In some embodiments, administration of the formulation or medical food increases the level of NAD in the subject by at least 5%. In other embodiments, administration of the formulation or medical food increases the level of NAD in the subject by at least 5% to 10%, at least about 6%, at least about 7%, at least about 8%, at least about 9% or at least about 10%. In certain embodiments, the increased NAD amount is an intracellular level, and in particular embodiments, an intracellular level of NAD is increased to greater than about 35 pM. In certain embodiments, intracellular level of NAD is increased to greater than about 35 pM, greater than about 36 pM, greater than about 37 pM, greater than about 38 pM, greater than about 39 pM, greater than about 40 pM, greater than about 41 pM, greater than about 42 pM, greater than about 43 pM, greater than about 44 pM, or greater than about 45 pM. In certain embodiments, the method is effective to increase a plasma level of N- methyl-nicotinamide (MeNAM) or N-methyl-2-pyridone-5-carboxamide (2PY) by at least 5% in the subject. In some embodiments extracellular level of NAD alternatively or also is increased. In some embodiments, the amount administered is effective to increase a level of icNAD to a level within a selected physiological range. In specific embodiments, a minimum value of the selected physiological range is from at least 5% to at least 50% higher than a baseline level of intracellular NAD in the subject, or at least 5%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, or at least 50% higher than a baseline level of intracellular NAD in the subject. In specific embodiments, administration of the pharmaceutical formulation or medical food results in at least a 5% increase in the subject’s urinary excretion of nicotinamide or its metabolites.

[0073] In some embodiments, the chemical addiction withdrawal symptoms suffered by the subject before administration of the formulation or medical food are related to detoxification from tobacco, heroin, opium, morphine, dihydromorphine, meperidine, codeine, cocaine, amphetamines, barbiturates, alcohol, tranquilizers, and / or opioids.

[0074] In some embodiments, the subject to whom the pharmaceutical formulation or medical food is administered had scored at least about 3 or greater on the Drug Abuse Screening Test (DAST) before administration. In other embodiments, the subject to whom the pharmaceutical formulation or medical food is administered had scored about 6 or greater on the Drug Abuse Screening Test - 10 (DAST- 10) before administration. In some embodiments, the amount of the pharmaceutical formulation or medical food administered is effective to mitigate opioid withdrawal symptoms such that the subject has a Clinical Opioid Withdrawal Scale (COWS) score of 12, 11, 10, 9, 8, 7, or 6 or lower. In some embodiments, the amount of formulation or medical food administered is effective to mitigate opioid withdrawal symptoms such that the client maintains a Clinical Opioid Withdrawal Scale (COWS) score of 12 or lower over the first about four days of acute detox.

[0075] In some embodiments, the pharmaceutical formulation or medical food is administered to the subject as a hydrated, dissolved, or suspended powder. In specific embodiments, the formulation is administered via an oral, buccal, intramuscular, systemic, anal, vaginal, or sublingual route. In aparticular example, the formulation is administered via a multi-sip oral swish and swallow protocol.

[0076] In certain embodiments, the amount of NAD+in the pharmaceutical formulation or medical food administered to the subject is from about 5 mg to about 4000 mg. In particular embodiments, the amount of the enhanced NAD+composition in the formulation comprises about 5 mg, about 10 mg, about 15 mg, about 20 mg, about 30 mg, about 40 mg, about 50 mg, about 60 mg, about 70 mg, about 80 mg, about 90 mg, about 100 mg, about 120 mg, about 140 mg, about 160 mg, about 180 mg, about 200 mg, about 220 mg, about 240 mg, about 260 mg, about 280 mg, about 300 mg, about 350 mg, about 400 mg, about 450 mg, about 500 mg, about 550 mg, about 600 mg, about 650 mg, about 700 mg, about 750 mg, about 800 mg, about 850 mg, about 900 mg, about 1000 mg, about 1100 mg, about 1200 mg, about 1300 mg, about 1400 mg, about 1500 mg, about 1600 mg, about 1700 mg, about 1800 mg, about 1900 mg, about 2000 mg, about 2200 mg, about 2400 mg, about 2600 mg, about 2800 mg, about 3000 mg, about 3200 mg, about 3400 mg, about 3600 mg, about 3800 mg, or about 4000 mg of NAD+.

[0077] In some embodiments, the amount of the pharmaceutical formulation or medical food administered comprises a daily dose of about 1 mg per kg to about to about 100 mg / kg body weight of the subject. In some specific embodiments, the amount of the pharmaceutical formulation or medical food administered comprises a maximum daily dose of about 1 mg per kg, about 2 mg per kg, about 3 mg per kg, about 4 mg per kg, about 5 mg per kg, about 6 mg per kg, about 7 mg per kg, about 8 mg per kg, about 9 mg per kg, about 10 mg per kg, about 11 mg per kg, about 12 mg per kg, about 13 mg per kg, about 14 mg per kg, about 15 mg per kg, about 16 mg per kg, about 17 mg per kg, about 18 mg per kg, about 19 mg per kg, about 20 mg per kg, about 22 mg per kg, about 24 mg per kg, about 26 mg per kg, about 27 mg per kg, about 28 mg per kg, about 30 mg per kg, about 32 mg per kg, about 34 mg per kg, about 36 mg per kg, about 38 mg per kg, about 40 mg per kg, about 42 mg per kg, about 44 mg per kg, about 46 mg per kg, about 48 mg per kg, about 50 mg per kg, about 52 mg per kg, about 54 mg per kg, about 56 mg per kg, about 58 mg per kg, about 60 mg per kg, about 65 mg per kg, about 70 mg per kg, about 75 mg per kg, about 80 mg per kg, about 85 mg per kg, about 90 mg per kg, about 95 mg per kg, or about 100 mg per kg of the subject’s weight.

[0078] In some embodiments, the method further comprises administering to the subject a pharmaceutically effective amount of an analgesic or a non-steroidal anti-inflammatory drug (NSAID). In specific embodiments, the pharmaceutical formulation further comprises a pharmaceutically effective amount of an analgesic or a non-steroidal anti-inflammatory drug (NSAID). In specific embodiments, the analgesic or NSAID is ibuprofen, acetaminophen, naproxen, diclofenac, celecoxib, mefenamic acid, etoricoxib, indomethacin, aspirin, etodolac, nabumetone, oxaprozin, codeine, fentanyl, hydrocodone, meperidine, methadone, naloxone, naltrexone, morphine, tramadol, gabapentin, or oxycodone. In other embodiments, the method further comprising administering a pharmaceutically effective amount of: an anti-diarrhea medication, such as, but not limited to, loperamide; an anti-nausea medication such as, but not limited to, ondansetron; anantispasmodic medication such as, but not limited to, tizanidine; and / or a sedative such as, but not limited to, promethazine.

[0079] In some embodiments, the method further comprises monitoring a plurality of vital signs of the subject after administration of the formulation or medical food. In certain embodiments, the method further comprises measuring an intracellular NAD or extracellular concentration of NAD in the subject before and / or after administration of the formulation or medical food, and the dosage of the pharmaceutical formulation or medical food to be administered may be selected based on the measured intracellular NAD or extracellular concentration of NAD in the subject.

[0080] In some embodiments, the pharmaceutical formulation or medical food is administered over a period of at least two days, and may be administered over about two, three, four, or five or more days. In certain embodiments, the pharmaceutical formulation or medical food is administered multiple doses within an about a 10 hour to about 16 hour period. In some embodiments, about one to about four doses are administered within a 10-16 hour period. In particular embodiments, the formulation or medical food is administered in about one to about five daily doses of about 500 mg each. In certain embodiments, the final daily dose of the formulation or medical food is administered immediately before the subject’s bedtime. In some embodiments, a first daily dose of the formulation or medical food is administered following about 12 hours of fasting by the subject.

[0081] The method may further comprise: monitoring at least one vital signs of the subject before and after administration of the composition: and / or measuring an intracellular NAD or extracellular concentration of NAD in the subject before administration and after administration of the pharmaceutical formulation or medical food; and / or selecting a dosage of the composition to be administered to the subject based on the measured baseline intracellular NAD or extracellular concentration of NAD in the subject.

[0082] In another aspect, the disclosure also provides a method of increasing NAD levels in a subject, the method comprising administering to the subject an amount of the enhanced NAD+pharmaceutical formulation or medical food as provided herein, which is effective to increase NAD levels in the subject. In some embodiments, the subject is a healthy human subject. In other embodiments, the subject suffers from or is at risk for a disease or disorder, and administration of the formulation or medical food decreases a risk of and / or ameliorates at least one symptom of the disease or disorder. In certain embodiments, the method may further comprise: monitoring at least one vital signs of the subject before and after administration of the pharmaceutical formulation or medical food: and / or measuring an intracellular concentration of NAD or extracellular concentration of NAD in the subject before administration and after administration of the pharmaceutical formulation or medical food; and / or selecting a dosage of the composition to be administered to the subject based on the measured baseline intracellular concentration of NAD or extracellular concentration of NAD in the subject.

[0083] In yet another aspect, the disclosure provides a method of preventing or treating a disease ordisorder correlated with a decreased level of NAD in a subject in need thereof, the method comprising: administering an amount of the enhanced NAD+pharmaceutical formulation or medical food as described herein effective to prevent or treat at least one symptom of the disease or disorder. In certain embodiments, the disease or disorder is a cardiovascular disorder, a neurodegenerative disorder, a neuropsychiatric disorder, a liver disorder [e.g., non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH)], a kidney disorder, a disorder of oxidative stress, a disorder of lipid metabolism, an addition to a substance use, a digestive disorder, a pancreatic disorder, an endocrine disorder, an immune system disorder, an autoimmune disorder, a reproductive system disorder, a metabolic system disorder, obesity, a skin disorder, and / or a bone disorder. In some embodiments, administering the formulation: improves liver and / or kidney function in the subject, optionally by decreasing serum levels of gamma-glutamyltransferase (GGT), bilirubin, alkaline phosphatase (ALP), and / or albumin; decreases oxidative stress in the subject, optionally by decreasing serum GGT, increasing plasma gamma-glutamyl glutamine (L-Glu-L-Gkn), decreasing circulating glutamate, decreasing at least one of histidine metabolites, upregulating glutamate utilization, increasing plasma abundance of glutathione S-transferase alpha-1 (GSTA1), increasing mitochondrial carbonic anhydrase 5 (CA5A), decreasing plasma mitochondrial superoxide dismutase 2 (SOD2), and / or increasing plasma sirtuin 1 (SIRT1) and / or alcohol dehydrogenase 4 (ADH4); decreases nitrosative stress and / or improves ammonia clearance, optionally by upregulating CA5A and / or decreasing plasma urea and / or aspartate; does not significantly increase inflammation in the subject, optionally measurable by circulating inflammatory biomarkers such as cardiac CRP, hsCRP, TNF, IL-6, and / or white blood cell counts; improves lipid transport and / or metabolism, optionally by decreasing LDL / HDL ratio, increasing plasma abundance of FABP1 and / or RBP2, decreasing circulating medium- and / or long-chain PUFAs, decreasing plasma cholesterol levels, and / or increasing oxidated cholesterol metabolites; does not significantly increase fasting serum glucose levels in the subject; and / or increases plasma abundance of at least one of NAD+metabolites, such as 1 -methyl -nicotinamide (MeNAM), N1 -methyl -2 -pyridone-5 -carboxamide (2PY), indolelactate and / or urate.

[0084] In some embodiments, administering the enhanced NAD+formulation or medical food to a subject modulates expression of at least one biomarker in the subject. In certain embodiments, the at least one biomarker is a protein or metabolite associated with endothelial, liver, and / or overall mitochondrial functioning and / or integrity or is an upregulated fatty acid-binding protein or a downregulated protein associated with endothelial, liver, and / or pancreatic injuries. In particular embodiments, the at least one biomarker is related to: a substance use, such as opiates and other drugs, including, e.g., increased circulating levels of serotonin receptor 1A (5-HT1AR), increased circulating cortisol or cortisone s, and / or increased levels of at least one liver enzyme; a neurodegenerative and or neuropsychiatric disease, including, e.g., increased levels of proteins with neurological effects, improved levels associated with neurodegenerative diseases, depression, anxiety,or chronic pain, oxidative stress, and / or nitrosative stress.

[0085] In particular embodiments, the at least one biomarker is related to: a cardiovascular disease, including, e.g., improved LDL / HDL ratio, vascular and / or endothelial health, injury resistance, and / or oxidative stress; an exocrine pancreas and / or digestion function, including, e.g., decreased circulate levels of pancreatic enzymes; an inflammation and / or immune function, including, e.g., improved adaptive or innate immune function or inflammation; a preproduction function, including, e.g., INSL3; a metabolic disease and / or obesity, including, e.g., GALNT2, FABP1, and / or RBP2; and / or skin and / or bone health, such as collagen synthesis, skin inflammation in psoriasis and / or atopic dermatitis, and / or bone development, including, e.g., PPIB, WFDC12, INSL4, and / or MATN3.

[0086] In some embodiments, administering the enhanced NAD+pharmaceutical formulation or medical food does not significantly increase at least one adverse symptom, such as, but not limited to, anxiety, stress, depression, fatigue, and / or a significant unfavorable change of vital signs, body compositions, and / or a clinical laboratory test result.

[0087] In some embodiments, the amount of pharmaceutical formulation or medical food administered is effective to increase the level of icNAD in the subject by at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 52%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, or 100% relative to a baseline level of intracellular NAD in the subject.

[0088] The method may further comprise: monitoring at least one vital signs of the subject before and after administration of the pharmaceutical formulation or medical food: and / or measuring an intracellular or extracellular concentration of NAD in the subject before administration and after administration of the pharmaceutical formulation or medical food; and / or selecting a dosage of the composition to be administered to the subject based on the measured baseline intracellular or extracellular concentration of NAD in the subject.

[0089] Another aspect of the present disclosure relates to a method of increasing intracellular NAD levels in a subject in need thereof, comprising administering to the subject an amount of an enhanced NAD+composition or medical food disclosed herein effective to increase the intracellular NAD level.

[0090] Another aspect of the present disclosure relates to a method of treating a subject suffering from, or preventing in a subject, an age-related disease comprising administering to the subject, a therapeutically effective amount of an enhanced NAD+composition or medical food disclosed herein.

[0091] The disclosure also provides NAD+compositions or medical food for use in the manufacture of a medicament, supplement, or formulation for treating age related diseases.

[0092] In another aspect, the present disclosure relates to a method for treating or preventing central nervous system disorders or diseases, comprising administering to a subject in need thereof a therapeutically effective amount of an enhanced NAD+composition or medical food described herein.

[0093] Yet another aspect of the present disclosure relates to a method for treating a subject for (e.g., reducing) chemical addiction or substance abuse, comprising administering to the subject atherapeutically effective amount of an enhanced NAD+composition or medical food described herein. In a further embodiment, the disclosure pertains to the treatment of the chemical addition resulting from a neurological or neurodegenerative disorder or disease.

[0094] Still another aspect of the present disclosure relates to a method for increasing SIRT1 in a subject in need thereof, comprising administering to the subject an amount of an enhanced NAD+composition or medical food described herein effective to increase SIRT1 in the subject.

[0095] Another aspect of the present disclosure relates to a method for increasing cryptococcal phospholipase (PLB1) in a subject in need thereof, comprising administering to the subject an amount of an NAD+composition or medical food described herein effective to increase PLB1 in the subject.

[0096] An additional aspect of the present disclosure relates to a method for increasing membrane metalloendopeptidase (MME) in a subject in need thereof, comprising administering to the subject an amount of an enhanced NAD+composition or medical food described herein effective to increase MME in the subject.

[0097] Another aspect of the present disclosure relates to a method for treating a subject suffering from a disease or disorder modulated by SIRT1, comprising administering to the subject a therapeutically effective amount of an enhanced NAD+composition or medical food described herein.

[0098] Yet another aspect of the present disclosure relates to a method for treating a subject suffering from a disease or disorder modulated by cryptococcal phospholipase (PLB1), comprising administering to the subject a therapeutically effective amount of an enhanced NAD+composition or medical food described herein.

[0099] Still another aspect of the present disclosure relates to a method for treating a subject suffering from a disease or disorder modulated by membrane metalloendopeptidase (MME), comprising administering to the subject a therapeutically effective amount of an enhanced NAD+composition or medical food described herein.

[0100] An Additional aspect of the present disclosure relates to a method for increasing a plasma level of a protein in a subject in need thereof, comprising administering to the subject an amount of an enhanced NAD+composition or medical food described herein effective to increase the plasma proteome in the subject.

[0101] In another aspect, enhanced NAD+compositions or medical food are provided as a component in solution for use in the treatment of a subject suffering from an age-related disorder, and / or for use in the treatment of a subject suffering from a CNS disease or disorder. In some embodiments, the CNS disorder is chemical addiction. In another embodiment, the CNS disorder is substance abuse. In another aspect, NAD+compositions or medical food are provided as a component in solution for use in the enhancement of energy for autophagy, a process in which cellular debris is removed. In another aspect, NAD+compositions or medical food are provided as a component in solution for use in increasing longevity and prevention of disease in humans, animals, and / or plants.

[0102] Yet another aspect of the present disclosure relates to a method of promoting cellular NAD+metabolism or NAD+cellular homeostasis in a subject, comprising administering to the subject an amount of the composition or medical food described herein effective to promote NAD+metabolism or NAD+cellular homeostasis.

[0103] Still another aspect of the present disclosure relates to a method of treating a patient suffering from at least one condition of metabolic syndrome, comprising administering to the subject a therapeutically effective amount of the composition or medical food described herein, wherein the at least one condition is increased blood pressure, high blood sugar, excess body fat around the waist, abnormal cholesterol or triglyceride levels, or any combination thereof.

[0104] Another aspect of the present disclosure relates to a method of increasing the amount of functional polypeptide of at least one of SIRT1, PLB1, NPL, ENPP5, GLSTA, GALNT3, FABP1, NRCAM, NLGN2, ARTN, UPB1, MME, CTSB, CTSL, or HRAS polypeptide in a subject in need thereof, comprising administering to the subject an amount of the composition or medical food described herein effective to increase the amount of functional polypeptide of at least one of SIRT1, PLB1, NPL, ENPP5, GLSTA, GALNT3, FABP1, NRCAM, NLGN2, ARTN, UPB1, MME, CTSB, CTSL, or HRAS polypeptide.

[0105] Certain APIs like thyroid hormones contain bound water that can be removed by azeotroping solvents to provide product stability. In some embodiments, the thyroid hormone is dissolved in a solvent and combined with a pharmaceutically acceptable excipient in a mixture and the solvent is removed under vacuum to result in a powder that is suitable for further processing.BRIEF DESCRIPTION OF THE FIGURES

[0106] The foregoing and other objects of the present disclosure, the various features thereof, as well as the disclosure itself may be more fully understood from the following description, when read together with the accompanying drawings in which:

[0107] FIG. 1 is a graph demonstrating the intracellular increase of NAD following treatment with an illustrative NAD+composition (“A”) as compared to placebo treatment (“B”).

[0108] FIG. 2 is a graph depicting the impact of treatment with an illustrative NAD+composition (“A”) on total bilirubin (pmol / L) as compared to placebo treatment (“B”) over time.

[0109] FIG. 3 is a graph depicting the impact of treatment with an illustrative NAD+composition (“A”) on total bilirubin (pmol / L) as compared to placebo treatment (“B”) over time.

[0110] FIG. 4 is a graph depicting the impact of treatment with an illustrative NAD+composition (“A”) on total bilirubin (pmol / L) as compared to placebo treatment (“B”) over a series of time points (days).

[0111] FIG. 5 is a graph depicting the impact of treatment with an illustrative NAD+composition (“A”) on liver and alkaline phosphatase measured by enzyme activity in Units / Liter as compared to placebo treatment (“B”) over time.

[0112] FIG. 6 is a graph depicting the impact of treatment with an illustrative NAD+composition (“A”) on liver and alkaline phosphatase measured by enzyme activity in Units / Liter as compared to placebo treatment (“B”) over time.

[0113] FIG. 7 is a graph depicting the impact of treatment with an illustrative NAD+composition (“A”) on liver and alkaline phosphatase measured by enzyme activity in Units / Liter as compared to placebo treatment (“B”) over a series of time points.

[0114] FIG. 8 is a graph depicting the impact of treatment with an illustrative NAD+composition (“A”) on gamma-glutamyl transferase (GGT) as compared to placebo treatment (“B”) over time.

[0115] FIG. 9 is a graph depicting the impact of treatment with an illustrative NAD+composition (“A”) on gamma-glutamyl transferase (GGT) as compared to placebo treatment (“B”) over time.

[0116] FIG. 10 is a graph depicting the impact of treatment with an illustrative NAD+composition (“A”) on gamma-glutamyl transferase (GGT) as compared to placebo treatment (“B”) over a series of time points.

[0117] FIG. 11 is a graph depicting the impact of treatment with an illustrative NAD+composition (“A”) on LDL / HDL ratio as compared to placebo treatment (“B”) over time.

[0118] FIG. 12 is a graph depicting the impact of treatment with an illustrative NAD+composition (“A”) on LDL / HDL ratio as compared to placebo treatment (“B”) over time.

[0119] FIG. 13 is a graph depicting the impact of treatment with an illustrative NAD+composition (“A”) on LDL / HDL ratio as compared to placebo treatment (“B”) over a series of time points relative to treatment;

[0120] FIG. 14 is a schematic of NAD+metabolism.

[0121] FIG. 15A is a graph depicting the plasma concentration of NAD metabolite 1- methylnicotinamide (MeNAM) measured in treatment (A) and placebo (B) participants at three time points during the Study described in Example 6. P=7.59x 10-11.

[0122] FIG. 15B is a graph depicting the plasma concentration of NAD metabolite N 1 -methyl -2- pyridone-5 -carboxamide (2PY ) measured in treatment (A) and placebo (B) participants at three time points during the Study described in Example 6. P=1.03x IO-13.

[0123] FIG. 16 is a panel of graphs depicting the plasma concentration of hypoxanthine, 4- hydroxyphenylpyruvate, docosahexaenoylcholine, cortisone, gamma-glutamylglutamine, urate, indolelacetate, glycohyocholate, tetrahydrocortisone glucuronide, and glutamate measured in treatment (A) and placebo (B) participants at three time points during the Study described in Example 6. (P<0.05 for all graphs).

[0124] FIG. 17 is a graph depicting the top 25 enriched metabolite sets observed in participants treated with NAD+. Metabolite enrichment analysis (MetaboAnalyst 5.0) identified NAD+, histidine, aspartate / glutamate metabolism, and arginine biosynthesis as the most affected (Kyoto Encyclopedia of Genes and Genomes) KEGG metabolic pathways.

[0125] FIG. 18 is a set of graphs demonstrating the intracellular increase of NAD followingtreatment with an illustrative NAD+composition as compared to placebo treatment, and the changes relative to baseline (“BL”; prior to treatment) for individual participants within each treatment group .

[0126] FIG. 19 is a set of graphs depicting the plasma concentration of NAD metabolite 1- methylnicotinamide (MeNAM) measured in NAD- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group.

[0127] FIG. 20 is a set of graphs depicting the plasma concentration of NAD metabolite N 1 -methyl - 2-pyri done-5 -carboxamide measured in NAD- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group.

[0128] FIG. 21 is a set of graphs depicting the plasma concentration of SIRT1 measured in NAD+- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group .

[0129] FIG. 22 is a set of graphs depicting the plasma concentration of methionine synthase (MTR) measured in NAD+- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group.

[0130] FIG. 23 is a set of graphs depicting the plasma concentration of glutathione S -transferase alpha- 1 (GSTA1) measured in NAD+- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group.

[0131] FIG. 24 is a set of graphs depicting the plasma concentration of mitochondrial superoxide dismutase 2 measured in NAD+- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group.

[0132] FIG. 25 is a set of graphs depicting the plasma concentration of hypoxanthine measured in NAD+- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group.

[0133] FIG. 26 is a set of graphs depicting the plasma concentration of gamma-glutamyl transferase (GGT) measured in NAD+- or placebo -treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group.

[0134] FIG. 27 is a set of graphs depicting the plasma concentration of total bilirubin (TBILI) measured in NAD+- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group.

[0135] FIG. 28 is a table summarizing changes in a number of proteins that modulate neuronalsignaling pathways or neuron growth after treatment with NAD+during the Study described inExamples 5 and 6.

[0136] FIG. 29 is a table summarizing changes in a number of immune and inflammatory related proteins after treatment with NAD+during the Study described in Examples 5 and 6.

[0137] FIG. 30 is a set of graphs depicting the plasma concentration of fatty -acid binding protein 1 (FABP1) measured in NAD- or placebo -treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group.

[0138] FIG. 31 is a set of graphs depicting the plasma concentration of retinol binding protein 2 (RBP2) measured in NAD- or placebo-treated participants at three time points during the Study described in Examples 5 and 6, and the change relative to baseline for individual participants within each treatment group.

[0139] FIG. 32A is a graph of the TGA thermogram for Sample 6, replicate 1 (Sample 6-1).

[0140] FIG. 32B is a graph of the TGA thermogram for Sample 6, replicate 2 (Sample 6-2).

[0141] FIG. 32C is an overlay of the TGA thermograms for Sample 6 replicates (Samples 6-1, 6-2).

[0142] FIG. 33A is a graph of the TGA thermogram for Sample 7, replicate 1 (Sample 7-1).

[0143] FIG. 33B is a graph of the TGA thermogram for Sample 7, replicate 2 (Sample 7-2).

[0144] FIG. 33C is an overlay of the TGA thermograms for Sample 7 replicates (Samples 7-1, 7-2).

[0145] FIG. 34A is a graph of the TGA thermogram for Sample 8, replicate 1 (Sample 8-1).

[0146] FIG. 34B is a graph of the TGA thermogram for Sample 8, replicate 2 (Sample 8-2).

[0147] FIG. 34C is an overlay of the TGA thermograms for Sample 8 replicates (Samples 8-1, 8-2).

[0148] FIG. 35A is a graph of the TGA thermogram for Sample 9, replicate 1 (Sample 9-1).

[0149] FIG. 35B is a graph of the TGA thermogram for Sample 9, replicate 2 (Sample 9-2).

[0150] FIG. 35C is an overlay of the TGA thermograms for Sample 9 replicates (Samples 9-1, 9-2).

[0151] FIG. 36 is an overlay of the TGA thermograms for Samples 6, 7, 8 and 9.

[0152] FIG. 37 shows an experimental design for the studies described in Example 12.

[0153] FIG. 38 is a graph depicting the impact of treatment with an illustrative NAD+composition (LNAD+) on intracellular NAD (“icNAD”) concentration as compared to placebo treatment over time.

[0154] FIG. 39 is a graph depicting the plasma concentration of NAD metabolites 1- methylnicotinamide (MeNAM) (left) and N1 -methyl -2 -pyridone-5 -carboxamide (2PY) (right) measured in treatment (LNAD+) and placebo participants at three time points during the Study described in Example 12.

[0155] FIG. 40 provides a schematic flow diagram showing various pathways through which NAD+is synthesized.

[0156] FIG. 41 shows a set of graphs depicting the plasma concentration of fatty-acid binding protein 1 (FABP1), Superoxide Dismutase 2 (SOD2), SIRT1 (Sirtuin 1), Granulin Precursor (GRN), Matrix Metallopeptidase 9 (MMP9), and C-X-C Motif Chemokine Ligand 10 (CXCL10), measured inLNAD+or placebo-treated participants at three time points during the Study described in Example 12.

[0157] FIG. 42 is a panel of graphs depicting the plasma concentration of hypoxanthine, urate, glutamate, gamma-glutamylglutamine, l-pentadecanoyl-2-arachidonoyl-GPC, and cortisone measured in treatment (LNAD+) and placebo participants at three time points during the Study described in Example 12.

[0158] FIG. 43 is a graph showing the overlap between certain proteins modulated by an enhanced NAD+pharmaceutical formulation or medical food according to one embodiment of the present invention and the top genes prioritized by PandaOmics for Alzheimer’s Disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and multiple sclerosis (MS).

[0159] FIG. 44A is a graph illustrating elevation of blood NAD+level within 15 minutes following oral administration of the enhanced NAD+formulation at a human equivalent dose (HED) of 300 mg.

[0160] FIG. 44B is a graph illustrating elevation of blood NADP+level within 15 minutes following oral administration of the enhanced NAD+formulation at a human equivalent dose (HED) of 300 mg.

[0161] FIG. 45A is a graph illustrating elevation of NAD+level in spinal cord tissue postadministration.

[0162] FIG. 45B is a graph illustrating elevation of NADP+level in spinal cord tissue postadministration, which were sustained for more than 24 hours.

[0163] FIG. 46A is a graph illustrating elevated GSH level in spinal cord tissue following oral administration of the enhanced NAD+formulation.

[0164] FIG. 46B is a graph illustrating elevated GSSG level in spinal cord tissue following oral administration of the enhanced NAD+formulation.

[0165] FIG. 46C is a graph illustrating elevated GSH / GSSG ratio in spinal cord tissue following oral administration of the enhanced NAD+formulation.

[0166] FIG. 47A is a graph illustrating CMAP amplitude over time by treatment group.

[0167] FIG. 47B is a graph illustrating CMAP response at different enhanced NAD+doses.DETAILED DESCRIPTION

[0168] The present disclosure provides for enhanced delivery of NAD+to target cells to treat neurodegenerative diseases and disorders, age-related diseases and disorders, and / or substance abuse including, but not limited to, drug or alcohol addiction.

[0169] NAD+is known to be chemically and biologically unstable. The present disclosure provides herein NAD+compositions or medical food having an enhanced stability compared to traditional NAD+compositions. For example, the illustrative enhanced NAD+compositions or medical food may have a slower degradation rate and / or a longer half-life (in vitro and / or in vivo), a longer shelflife, and / or a less stringent storage condition requirement (e.g., cooling or dry storage), compared to acontrol NAD+composition without the property or not produced by the conditions and processes described herein (e.g., aNAD+composition from a commercial source).

[0170] NAD+is a crucial nutrient for animal health. The present disclosure provides herein NAD+compositions or medical food with enhanced properties, which may be administered to an individual (e.g. , a human) as a nutrient and / or a pharmaceutical agent to prevent, ameliorate, and / or treat a disease or disorder related to insufficient icNAD in the body. In some embodiments, the enhanced NAD+compositions or medical food described herein increases the icNAD concentration and / or NAD+degradation in the individual. In some embodiments, the individual is having tryptophan-poor diet. In some embodiments, the individual has a genetic mutation and / or other mechanisms (e.g., living or environmental conditions, food or drug effects, etc.) resulting in a reduction or inhibition of NAD production, a reduction of NAD stability, and / or an increase of NAD degradation or turnover in the individual. For example, the individual may have a genetic mutation in the kynurenine pathway, thus reducing or inhibiting the production chain from nicotinic acid to NMN to NAD and then to NAD. The individual may have another genetic mutation affecting the production, transportation, storage, and / or utilization of NAD in the individual. The enhanced NAD+compositions or medical food described herein may also enhance NAD-related biochemical and biological reactions in an individual in need thereof. For example, since NAD is a co-factor of oxidoreductases, the enhanced NAD+compositions or medical food described herein may enhance the bio-functions of oxidoreductases in the individual, e.g., enhancing synthesizing enzymes such as poly(ADPribose) polymerases and cADPribose synthases. Thus, if the individual has a disease or disorder related to insufficient poly(ADPribose) polymerases and cADPribose synthases, or other genes or gene products downstream or affected by NAD, the enhanced NAD+compositions or medical food described herein may be administered to the individual to prevent, ameliorate, and / or treat the disease or disorder. In addition, the enhanced NAD+compositions or medical food described herein may be a substitute of current commercially available NAD+compositions, or other NAD+compositions without the property or not produced by the current conditions or process, for its enhanced stability. For example, since NAD+is unstable even under conditions that are not drastic such as in aqueous solution, the enhanced NAD+compositions or medical food described herein may be used as a supplement and / or a pharmaceutical agent with improved bioavailability for oral administration (or through other administration routes) to an individual. Compared to traditional NAD+compositions, the enhanced NAD+compositions or medical food described herein may be more stable in the direct interaction with water, radiation, oxygen, and / or UV light, and may be resistant to hydrolysis in the small intestine by brush border cells.

[0171] The present disclosure relates to enhanced NAD+compositions, pharmaceutical formulations, and medical food containing the same useful in the treatment of diseases, disorders, and conditions related to decreased levels of intracellular NAD. The present disclosure further relates to methods of manufacturing enhanced NAD+compositions, pharmaceutical formulations, medical food, or “drugproducts” that are stable, therapeutic, and bioavailable. The present disclosure also provides new methods of increasing intracellular levels of NAD and of treating various disorders associated with a decrease in intracellular NAD. For example, the present disclosure provides for enhanced delivery of NAD+to target cells to treat neurodegenerative diseases and disorders, age-related diseases and disorders, and / or substance abuse including, but not limited to, drug or alcohol addiction. Also disclosed are methods of detecting biomarkers associated with decreased intracellular levels of NAD.

[0172] NAD+is known to be chemically and biologically unstable. The present disclosure provides herein NAD+compositions, formulations, and medical food having an enhanced stability compared to traditional NAD+compositions. For example, the illustrative enhanced NAD+compositions or medical food may have a slower degradation rate and / or a longer half-life (in vitro and / or in vivo), a longer shelf-life, and / or a less stringent storage condition requirement (e.g., cooling or dry storage), compared to a control NAD+composition without the property or not produced by the conditions and processes described herein (e.g., a NAD+composition from a commercial source).

[0173] The present disclosure provides herein NAD+compositions or medical food having an enhanced stability compared to traditional NAD+compositions. For example, the illustrative enhanced NAD+compositions or medical food may have a slower degradation rate and / or a longer half-life (in vitro and / or in vivo), a longer shelf-life, and / or a less stringent storage condition requirement (e.g., cooling or dry storage), compared to a control NAD+composition without the property or not produced by the conditions and processes described herein (e.g. , a NAD+composition from a commercial source). “Enhanced NAD+” or any derivatives or precursors thereof can comprise any of the compositions disclosed in or produced according to the methods and processed disclosed in U.S. Provisional Application No. 63 / 687,107, filed on August 26, 2024, and PCT / US23 / 35557. In embodiments, “enhanced NAD+” refers to a stable, oral formulation of NAD+.

[0174] Aspects of the invention are drawn towards therapeutic preparations. As used herein, the term “therapeutic preparation” can refer to any compound or composition that can be used or administered for therapeutic effects. As used herein, the term “therapeutic effects” can refer to effects sufficient to result in amelioration of symptoms, e.g., treatment, healing, prevention or amelioration of the relevant medical condition, or an increase in rate of treatment, healing, prevention or amelioration of such conditions. “Therapeutic effects” can refer to increasing intracellular NAD+, increasing circulating NAD+, or a combination thereof.

[0175] Embodiments as described herein can be administered to a subject in the form of a pharmaceutical composition or therapeutic preparation prepared for the intended route of administration. Such compositions and preparations can comprise, for example, the active ingredient(s) and a pharmaceutically acceptable carrier. Such compositions and preparations can be in a form adapted to oral, subcutaneous, parenteral (such as, intravenous, intraperitoneal), intramuscular, rectal, epidural, intratracheal, intranasal, dermal, vaginal, buccal, ocularly, or pulmonary administration, such as in a form adapted for administration by a peripheral route or issuitable for oral administration or suitable for parenteral administration. Other routes of administration are subcutaneous, intraperitoneal and intravenous, and such compositions can be prepared in any manner known to the person skilled in the art. The compositions and preparations can appear in conventional forms, for example, solutions and suspensions for injection, capsules and tablets, in the form of enteric formulations, e.g. as disclosed in U.S. Pat. No. 5,350,741, and for oral administration.

[0176] Solutions or suspensions used for parenteral, intradermal, or subcutaneous application can include the following components: a sterile diluent such as water for injection, saline solution, fixed oils, polyethylene glycols, glycerin, propylene glycol or other synthetic solvents; antibacterial agents such as benzyl alcohol or methyl parabens; antioxidants such as ascorbic acid or sodium bisulfite; chelating agents such as ethylenediaminetetraacetic acid; buffers such as acetates, citrates or phosphates and agents for the adjustment of tonicity such as sodium chloride or dextrose. pH can be adjusted with acids or bases, such as hydrochloric acid or sodium hydroxide. The parenteral preparation can be enclosed in ampoules, disposable syringes or multiple dose vials made of glass or plastic.

[0177] Pharmaceutical compositions suitable for injectable use include sterile aqueous solutions (where water soluble) or dispersions and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include physiological saline, bacteriostatic water, Cremophor EM™ (BASF, Parsippany, N.J.) or phosphate buffered saline (PBS). In all cases, the composition must be sterile and should be fluid to the extent that easy syringeability exists. It must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms such as bacteria and fungi. The carrier can be a solvent or dispersion medium containing, for example, water, ethanol, a pharmaceutically acceptable polyol like glycerol, propylene glycol, liquid polyethylene glycol, and suitable mixtures thereof. The proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersion and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, and thimerosal. In many cases, it can be useful to include isotonic agents, for example, sugars, polyalcohols such as mannitol, sorbitol, and sodium chloride in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate and gelatin.

[0178] Sterile injectable solutions can be prepared by incorporating the compound in the required amount in an appropriate solvent with one or a combination of ingredients enumerated herein, as required, followed by filtered sterilization. Generally, dispersions are prepared by incorporating the active compound into a sterile vehicle which contains a basic dispersion medium and the required other ingredients from those enumerated herein. In the case of sterile powders for the preparation ofsterile injectable solutions, examples of useful preparation methods are vacuum drying and freeze- drying which yields a powder of the active ingredient plus any additional desired ingredient from a previously sterile-filtered solution thereof.

[0179] Oral compositions can include an inert diluent or an edible carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the active compound can be incorporated with excipients and used in the form of tablets, troches, or capsules. Oral compositions can also be prepared using a fluid carrier for use as a mouthwash, wherein the compound in the fluid carrier is applied orally and swished and expectorated or swallowed. Oral formula of the drug can be administered once a day, twice a day, three times a day, or four times a day, for example, depending on the half-life of the drug.

[0180] Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition administered to a subject. The tablets, pills, capsules, troches and the like can contain any of the following ingredients, or compounds of a similar nature: a binder such as microcrystalline cellulose, gum tragacanth or gelatin; an excipient such as starch or lactose, a disintegrating agent such as alginic acid, Primogel, or com starch; a lubricant such as magnesium stearate or sterotes; a glidant such as colloidal silicon dioxide; a sweetening agent such as sucrose or saccharin; or a flavoring agent such as peppermint, methyl salicylate, or orange flavoring.

[0181] Systemic administration can also be by transmucosal or transdermal means. For transmucosal or transdermal administration, penetrants appropriate to the barrier to be permeated are used in the formulation. Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents, bile salts, and fusidic acid derivatives. Transmucosal administration can be accomplished through the use of nasal sprays or suppositories. For transdermal administration, the active compounds are formulated into ointments, salves, gels, or creams as generally known in the art.

[0182] In embodiments, administering can comprise the placement of a pharmaceutical composition into a subject by a method or route which results in at least partial localization of the composition at a desired site such that desired effect is produced.

[0183] For example, the pharmaceutical composition can be administered by bolus injection or by infusion. A bolus injection can refer to a route of administration in which a syringe is connected to the IV access device and the medication is injected directly into the subject. The term “infusion” can refer to an intravascular injection.

[0184] Embodiments as described herein can be administered to a subject one time (e.g., as a single injection, bolus, or deposition). Alternatively, administration can be once or twice daily to a subject for a period of time, such as from about 2 weeks to about 28 days. Administration can continue for up to one year. In embodiments, administration can continue for the life of the subject. It can also be administered once or twice daily to a subject for period of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 times per year, or a combination thereof.

[0185] In embodiments, compositions as described herein can be administered to a subject chronically. “Chronic administration” can refer to administration in a continuous manner, such as to maintain the therapeutic effect (activity) over a prolonged period of time.

[0186] The pharmaceutical or therapeutic carrier or diluent employed can be a conventional solid or liquid carrier. Examples of solid carriers are lactose, terra alba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia, magnesium stearate, stearic acid or lower alkyl ethers of cellulose. Examples of liquid carriers are syrup, peanut oil, olive oil, phospholipids, fatty acids, fatty acid amines, polyoxyethylene and water. Similarly, the carrier or diluent can include any sustained release material known in the art, such as glyceryl monostearate or glyceryl distearate, alone or mixed with a wax.

[0187] When a solid carrier is used for oral administration, the preparation can be tableted, placed in a hard gelatin capsule in powder or pellet form or it can be in the form of a troche or lozenge . The amount of solid carrier will vary widely but will usually be from about 25 mg to about 1 g.

[0188] When a liquid carrier is used, the preparation can be in the form of a syrup, emulsion, soft gelatin capsule or sterile injectable liquid such as an aqueous or non-aqueous liquid suspension or solution.

[0189] The composition and / or preparation can also be in a form suited for local or systemic injection or infusion and can, as such, be formulated with sterile water or an isotonic saline or glucose solution. The compositions can be in a form adapted for peripheral administration only, with the exception of centrally administrable forms. The compositions and / or preparations can be in a form adapted for central administration.

[0190] The compositions and / or preparations can be sterilized by conventional sterilization techniques which are well known in the art. The resulting aqueous solutions can be packaged for use or filtered under aseptic conditions and lyophilized, the lyophilized preparation being combined with the sterile aqueous solution prior to administration. The compositions and / or preparations can contain pharmaceutically and / or therapeutically acceptable auxiliary substances as required to approximate physiological conditions, such as buffering agents, tonicity adjusting agents and the like, for instance sodium acetate, sodium lactate, sodium chloride, potassium chloride, calcium chloride, etc.

[0191] Embodiments are also drawn towards methods of treating a disease, disorder, or condition such as an age-related disease or disorder. An “age-related disease or disorder” can refer to a disease or disorder associated with normal or diseased aging. In certain embodiments, the phrase “age-related disease or disorder” refers to any condition, ailment, or process of normal aging associated with reduced intracellular NAD+, reduced circulating NAD+, or a combination thereof. An “age related disease or disorder” can refer to a disease or disorder that is more prevalent in populations of adult humans who are over the age of 55 years. Non-limiting examples of age-related diseases or disorders include Alzheimer’s (AD) Parkinson’s (PD), Amyotrophic, Lateral Sclerosis (ALS), and Multiple Sclerosis (MS).

[0192] In various embodiments, the enhanced NAD+pharmaceutical formulations or medical food of the present disclosure can be used in the treatment of a variety of conditions, diseases and disorders. For example, they can be used in preventing or ameliorating normal aging processes, in treating or preventing age-related disorders, treating CNS disorders, in increasing intracellular NAD levels, or any combination thereof.

[0193] The present disclosure relates to enhanced NAD+compositions or medical food useful in the treatment of diseases and disorders regulated by intracellular levels of NAD. The present disclosure further relates to methods of manufacturing of NAD+drug products that are stable, safer for use and that have a longer shelf life. The present disclosure also provides new methods of increasing intracellular NAD and methods of treatment of central nervous system diseases and disorders including chemical addiction and substance abuse.

[0194] NAD+is known to be chemically and biologically unstable. The present disclosure provides herein NAD+compositions or medical food having an enhanced stability compared to traditional NAD+compositions. For example, the illustrative enhanced NAD+compositions may have a slower degradation rate and / or a longer half-life (in vitro and / or in vivo), a longer shelf-life, and / or a less stringent storage condition requirement (e.g., cooling or dry storage), compared to a control NAD+composition without the property or not produced by the conditions and processes described herein (e.g., a NAD+composition from a commercial source).

[0195] NAD+is a crucial nutrient for animal health. The present disclosure provides herein NAD+compositions or medical food with enhanced properties, which may be administered to an individual (e.g. , a human) as a nutrient and / or a pharmaceutical agent to prevent, ameliorate, and / or treat a disease or disorder related to insufficient icNAD in the body. In some embodiments, the enhanced NAD+compositions or medical food described herein increases the icNAD concentration and / or NAD+degradation in the individual. In some embodiments, the individual is having tryptophan-poor diet. In some embodiments, the individual has a genetic mutation and / or other mechanisms (e.g., living or environmental conditions, food or drug effects, etc.) resulting in a reduction or inhibition of NAD production, a reduction of NAD stability, and / or an increase of NAD degradation or turnover in the individual. For example, the individual may have a genetic mutation in the kynurenine pathway, thus reducing or inhibiting the production chain from nicotinic acid to NMN to NAD and then to NAD. The individual may have another genetic mutation affecting the production, transportation, storage, and / or utilization of NAD in the individual. The enhanced NAD+compositions or medical food described herein may also enhance NAD-related biochemical and biological reactions in an individual in need thereof. For example, since NAD is a co-factor of oxidoreductases, the enhanced NAD+compositions or medical food described herein may enhance the bio-functions of oxidoreductases in the individual, e.g., enhancing synthesizing enzymes such as poly(ADPribose) polymerases and cADPribose synthases. Thus, if the individual has a disease or disorder related to insufficient poly(ADPribose) polymerases and cADPribose synthases, or other genes or gene productsdownstream or affected by NAD, the enhanced NAD+compositions or medical food described herein may be administered to the individual to prevent, ameliorate, and / or treat the disease or disorder. In addition, the enhanced NAD+compositions or medical food described herein may be a substitute of current commercially available NAD+compositions, or other NAD+compositions without the property or not produced by the current conditions or process, for its enhanced stability. For example, since NAD+is unstable even under conditions that are not drastic such as in aqueous solution, the enhanced NAD+compositions described herein may be used as a supplement and / or a pharmaceutical agent with improved bioavailability for oral administration (or through other administration routes) to an individual. Compared to traditional NAD+compositions, the enhanced NAD+compositions described herein may be more stable in the direct interaction with water, radiation, oxygen, and / or UV light, and may be resistant to hydrolysis in the small intestine by brush border cells.

[0196] The present disclosure relates to enhanced NAD+compositions, pharmaceutical formulations, and medical food containing the same useful in the treatment of diseases, disorders, and conditions related to decreased levels of intracellular NAD. The present disclosure further relates to methods of manufacturing enhanced NAD+compositions, pharmaceutical formulations, medical food or “drug products” that are stable, therapeutic, and bioavailable. The present disclosure also provides new methods of increasing intracellular levels of NAD and of treating various disorders associated with a decrease in intracellular NAD. For example, the present disclosure provides for enhanced delivery of NAD+to target cells to treat substance abuse including, but not limited to, drug or alcohol addiction. Also disclosed are methods of detecting biomarkers associated with decreased intracellular levels of NAD.

[0197] NAD+is known to be chemically and biologically unstable. The present disclosure provides herein NAD+compositions, formulations, and medical food having an enhanced stability compared to traditional NAD+compositions. For example, the illustrative enhanced NAD+compositions may have a slower degradation rate and / or a longer half-life (in vitro and / or in vivo), a longer shelf-life, and / or a less stringent storage condition requirement (e.g., cooling or dry storage), compared to a control NAD+composition without the property or not produced by the conditions and processes described herein (e.g., a NAD+composition from a commercial source).

[0198] The disclosures of the patents, patent applications, and publications disclosed herein are hereby incorporated by reference in their entireties into this application in order to more fully describe the state of the art as known to those skilled therein as of the date of the disclosure described and claimed herein. The instant disclosure will govern in the instance that there is any inconsistency between the patents, patent applications, and publications and this disclosure.Definitions

[0199] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs.The initial definition provided for a group or term herein applies to that group or term throughout the present specification individually or as part of another group, unless otherwise indicated.

[0200] In the following description of the disclosure, repeated usage of the phrase “in one embodiment” does not necessarily refer to the same embodiment.

[0201] As used herein, the articles “a” and “an” refer to one or to more than one (z. e. , at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0202] As used herein, the term “about” will be understood by persons of ordinary skill in the art and will vary to some extent on the context in which it is used. As used herein when referring to a measurable value such as an amount, a temporal duration, and the like, the term “about” is meant to encompass variations of ±20% or ±10%, including ±5%, ±1%, and ±0. 1% from the specified value, as such variations are appropriate to perform the disclosed methods.

[0203] As used herein, the term “NAD” refers to “nicotinamide adenine dinucleotide” and encompasses NAD+and / or NADH. The term “NAD+” as used in this disclosure describes compositions consisting of NAD+.

[0204] The terms “composition,” “enhanced NAD+composition”, “formulation,” and “enhanced NAD+pharmaceutical formulation”, as used in this disclosure encompass compositions and pharmaceutical formulations comprising NAD+that has been prepared according to the present disclosure.

[0205] The term “medical food” refers to a category of nutritional products specifically formulated to manage distinct nutritional requirements arising from certain diseases or medical conditions. As defined under the U.S. Orphan Drug Act and FDA regulations (21 U.S.C. § 360ee(b)(3)), medical foods are intended for the dietary management of a disease or condition that has distinctive nutritional needs, which cannot be met by normal diet alone. Medical food must be used under physician supervision and is typically administered as part of a clinical treatment plan. Unlike conventional foods or dietary supplements, medical foods address well-established, scientifically recognized nutritional deficiencies associated with specific pathological states. In the context of the present disclosure, the enhanced NAD+composition can be administered as a medical food to manage NAD+deficiencies or impairments in subjects with neurodegenerative or metabolic disorders, providing targeted nutritional support to restore intracellular NAD+homeostasis.

[0206] ‘ ‘vacuumizing” or “vacuumization” is a procedure that purifies active primary ingredients, components (z'.e., excipients), intermediates, secondary compounds, ingredients, or components, and final products (such as, but not limited to, powder and tablets). Vacuumizing consists of subjecting a composition or mixture of components to decreased pressure or vacuum, followed by the replacement of the vacuum with an inert gas or CO2. In some embodiments, one vacuumization cycle comprises subjecting a composition or mixture of components to decreased pressure or vacuum, followed by thereplacement of the vacuum with an inert gas or CO2. The cycles are subsequent and sequential starting with vacuum under specific pressure and followed with replacement of the air with an inert gas to compensate or re-saturate the pressure created by the vacuum at similar pressure. These cycles of vacuumization azeotrope the volatile contaminants from the active pharmaceutical ingredient (API) and associated excipients. In a non-limiting example described herein, NAD+and combinations of NAD+and PEG are vacuumized to provide the compositions and pharmaceutical formulations described herein.

[0207] ‘ ‘NAD+monohydrate” or “[3-NAD [ -Nicotinamide Adenine Dinucleotide, Oxidized free acid hydrate]” is a compound which is commercially available, and which may include phosphorus.

[0208] The term “blended” refers to mixed components or ingredients, for example, NAD+is mixed with a secondary component such as one or more excipients of various weight (e.g., PEG, including but not limited to, pre -formulated PEG) to achieve a homogeneous distribution of the active ingredient in the final drug product. Mixing and blending as described herein prevents small amounts of the active agent (NAD+) from becoming inadvertently partitioned to, or concentrated within, small compartments or portions of the formulation equipment surfaces due to particle size distribution and / or small dimensional irregularities and cavities on the surfaces of the blending equipment.

[0209] The term “substrate” or “excipient substrate” refers to a therapeutically suitable excipient that may be used to bind or immobilize an active agent or drug to its surface through non-covalent intermolecular forces or interactions according to methods of the present disclosure. The optimal choice for a substrate may depend on the solvent and the active agent or drug, such that the active agent or drug may interact with and become immobilized to the substrate . The substrate may also be chosen depending on the final dosage form of the product. Any classic therapeutically acceptable excipient(s), such as micro-crystalline cellulose, etc., may be used as a substrate according to the present disclosure because they are considered (i) GRAS compound because they have been shown to be non-toxic, (ii) compatible with a wide range of solvents, and (iii) present a surface topology (e.g., but not limited to, porosity) and surface chemistry suitable for drug immobilization without covalent chemical interactions. Compounds or drugs immobilized to micro -crystalline cellulose, for example, are compatible with other excipients and may be blended with other excipients to make formulations that may be formed into tablets by direct compression. A micro-crystalline cellulose powder flow having the active agent or drug immobilized to it may also be used for fdling capsules.

[0210] The term “degrade,” as used herein, means to chemically decompose. Degradation is distinct from melting in that it involves the breaking of chemical bonds or the formation of new chemical bonds. Methods of degradation can include hydrolysis, solventolysis, thermolysis, or oxidation.

[0211] Embodiments as described herein can involve isolating, collecting, or obtaining a biological sample from a subject. As used herein, the term “collecting a sample” or “isolating a sample”, for example, can refer to any process for directly or indirectly acquiring a biological sample from a subject. For example, a biological sample may be obtained (e.g. , at a point-of-care facility, e.g. , aphysician's office, a hospital, laboratory facility) by procuring a tissue sample (such as a skin biopsy) from a subject. Alternatively, a biological sample can be obtained by receiving the biological sample (e.g. , at a laboratory facility) from one or more persons who procured the sample directly from the subject. The biological sample may be, for example, a tissue (e.g., biopsy), fluid (e.g., cerebrospinal fluid, plasma, blood, serum) or cell (e.g., skin fibroblast cells, peripheral blood cells) of a subject.

[0212] The term “sample” can refer to a biological sample obtained or derived from a source of interest, as described herein. In some embodiments, a source of interest comprises an organism, such as an animal or human. In some embodiments, a biological sample comprises biological tissue or fluid. In some embodiments, a biological sample is or comprises bone marrow; spleen, blood; blood cells; blood mononuclear cells; serum; plasma; ascites; tissue or fine needle biopsy samples; cellcontaining body fluids; free floating nucleic acids; sputum; saliva; urine; cerebrospinal fluid, peritoneal fluid; pleural fluid; feces; lymph; gynecological fluids; skin swabs; vaginal swabs; oral swabs; nasal swabs; washings or lavages such as a ductal lavages or bronchoalveolar lavages; aspirates; scrapings; bone marrow specimens; tissue biopsy specimens; surgical specimens; feces, other body fluids, secretions, and / or excretions; and / or cells therefrom, etc. In some embodiments, a biological sample is or comprises cells obtained from an individual. In some embodiments, a sample is a “primary sample” obtained directly from a source of interest by any appropriate means. For example, in some embodiments, a primary biological sample is obtained by methods selected from the group consisting of biopsy {e.g., fine needle aspiration or tissue biopsy), surgery, collection of body fluid {e.g., blood, lymph, feces etc.), etc. In some embodiments, the term “sample” refers to a preparation that is obtained by processing (e.g., by removing one or more components of and / or by adding one or more agents to) a primary sample. For example, filtering using a semi-permeable membrane. Such a “processed sample” may comprise, for example nucleic acids or proteins extracted from a sample or obtained by subjecting a primary sample to techniques such as amplification or reverse transcription of mRNA, isolation and / or purification of certain components, etc.

[0213] A “subject” or “patient” can be a mammal, e.g., but not limited to, a human, mouse, rat, guinea pig, dog, cat, horse, cow, pig, chordate, or non-human primate, such as, but not limited to, a monkey, chimpanzee, or baboon. The term “subject” or “patient” can refer to any organism to which aspects of the invention can be performed, e.g., for experimental, diagnostic, prophylactic, and / or therapeutic purposes. Subjects to which methods as described herein are performed comprise mammals, such as primates, for example humans. For veterinary applications, a wide variety of subjects are suitable, e.g., livestock such as cattle, sheep, goats, cows, swine, and the like; poultry such as chickens, ducks, geese, turkeys, and the like; and domesticated animals and pets such as dogs and cats. For diagnostic or research applications, a wide variety of mammals are suitable subjects, including rodents (e.g., mice, rats, hamsters), rabbits, primates, and swine such as inbred pigs and the like. The term “living subject” can refer to a subject noted herein or another organism that is alive. The term “living subject” can refer to the entire subject or organism and not just a part excised (e.g., aliver or other organ) from the living subject. The term “normal subject” can refer to a subject that is not afflicted with a disease or condition, such as a subject that is not afflicted with an age-related disorder.

[0214] As used herein, the phrase “therapeutic agent” can refer to any agent that elicits a desired pharmacological effect when administered to a subject. In some embodiments, an agent is considered to be a therapeutic agent if it demonstrates a statistically significant effect across an appropriate population. In some embodiments, the appropriate population may be a population of model organisms. In some embodiments, an appropriate population may be defined by various criteria, such as a certain age group, gender, genetic background, preexisting clinical conditions, etc. In some embodiments, a therapeutic agent is any substance that can be used to alleviate, ameliorate, relieve, inhibit, delay onset of, reduce severity of, and / or reduce incidence of one or more symptoms or features of a disease, disorder, and / or condition.

[0215] An “effective amount” when used in connection with a pharmaceutical formulation or medical food encompasses an amount that causes a particular result, such as reducing or eliminating at least one symptom or negative effect of ae disorder or disease, or which prevents the manifestation of the disease or disorder in a subject as described herein. An effective amount also refers to that amount which can cause a predetermined or preselected result, change, or activity.

[0216] The term “therapeutically effective amount”, as used herein, can refer to an amount of a therapeutic agent whose administration, when viewed in a relevant population, correlates with or is reasonably expected to correlate with achievement of a particular therapeutic effect. The therapeutic effect may be objective (z.e., measurable by some test or marker) or subjective (z.e., subject gives an indication of or feels an effect). In some embodiments, a therapeutically effective amount of a substance is an amount that is sufficient, when administered to a subject suffering from or susceptible to a disease, disorder, and / or condition, to treat, diagnose, prevent, and / or delay and / or alleviate one or more symptoms of the disease, disorder, and / or condition. Disease progression can be monitored by clinical observations, laboratory and imaging investigations apparent to a person skilled in the art. A therapeutically effective amount is administered in a dosing regimen that can comprise multiple unit doses. For a therapeutic agent, a therapeutically effective amount (and / or an appropriate unit dose within an effective dosing regimen) can vary, for example, depending on route of administration, on combination with other pharmaceutical agents. Also, the specific therapeutically effective amount (and / or unit dose) for a patient can depend upon a variety of factors including the disorder being treated and the severity of the disorder; the activity of the specific pharmaceutical agent employed; the specific composition employed; the age, body weight, general health, sex and diet of the patient; the time of administration, route of administration, and / or rate of excretion or metabolism of the specific fusion protein employed; the duration of the treatment; and like factors as is well known in the medical arts. Furthermore, an effective amount may be administered via a single dose or via multiple doses within a treatment regimen. In some embodiments, individual doses or compositions areconsidered to contain a “therapeutically effective amount” when they contain an amount effective as a dose in the context of a treatment regimen. Those of ordinary skill in the art will appreciate that a dose or amount may be considered to be effective if it is or has been demonstrated to show statistically significant effectiveness when administered to a population of patients; a particular result need not be achieved in a particular individual patient in order for an amount to be considered to be therapeutically effective as described herein.

[0217] The term “carrier”, as used in this disclosure, encompasses carriers, excipients, and diluents and means a material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, involved in carrying or transporting a pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body of a subject. The “carrier” can be used to manipulate the release rate of the API (e.g., NAD+) to include rapid or sustained release.

[0218] As used herein, the term “unadministered” subject refers to a subject that has not been administered the enhanced NAD+formulation according to the disclosure and may be also called an “untreated” or control subject.

[0219] As used herein, the terms “treat” and “treatment” are synonymous with the term “prevent” and are meant to indicate a postponement of development of diseases, preventing the development of diseases, and / or reducing severity of such symptoms that will or are expected to develop. Thus, these terms include ameliorating existing disease symptoms, preventing additional symptoms, ameliorating, or preventing the underlying causes of symptoms, inhibiting the disorder or disease, e.g., arresting the development of the disorder or disease, relieving the disorder or disease, causing regression of the disorder or disease, relieving a condition caused by the disease or disorder, or stopping or alleviating the symptoms of the disease or disorder. The term “treating” can mean the medical management of a subject, e.g. an animal or human, with the intent that a prevention, cure, stabilization, or amelioration of the symptoms or condition will result. This term includes active treatment, that is, treatment directed specifically toward improvement of the disorder; palliative treatment, that is, treatment designed for the relief of symptoms rather than the curing of the disorder; preventive treatment, that is, treatment directed to prevention of disorder; and supportive treatment, that is, treatment employed to supplement another specific therapy directed toward the improvement of the disorder. The term “treatment” also includes symptomatic treatment, that is, treatment directed toward constitutional symptoms of the disorder. “Treating” a condition with the compounds of the invention involves administering such a compound, alone or in combination and by any appropriate means, to a patient. For example, “treating” an age-related disease can refer to (or be indicated by) raising intracellular NAD+levels in a patient or subject. Treatment can be administered to a subject who does not exhibit signs of a disease, disorder, and / or condition (e.g., prior to an identifiable disease, disorder, and / or condition), and / or to a subject who exhibits only early signs of a disease, disorder, and / or condition for the purpose of decreasing the risk of developing pathology associated with the disease, disorder,and / or condition. In embodiments, treatment is administered to healthy aging (or aged) adult humans.

[0220] The term “disorder” is used in this disclosure to mean, and is used interchangeably with, the terms disease, condition, or illness, unless otherwise indicated. The term “administer”, “administering”, or “administration” as used in this disclosure refers to either directly administering a pharmaceutical formulation or medical food to a subject. The term “container” encompasses any vessel, dish, or other structure into which NAD+or a mixture of NAD+and any other chemical component is placed and which can withstand decreased pressure or vacuum. A container may also be a blender or a container that enables mixing of components.

[0221] The term “administer”, “administering”, or “administration” as used in this disclosure refers to either directly administering a disclosed salt or a composition to a subject, or administering a prodrug derivative or analog of the salt or composition to the subject, which can form an equivalent amount of active salt within the subject's body.

[0222] The present disclosure relates to enhanced NAD+compositions useful in the treatment of diseases and disorders regulated by intracellular levels of NAD. The present disclosure further relates to methods of manufacturing of NAD+drug products that are stable, safer for use and that have a longer shelf life. The present disclosure also provides new methods of increasing intracellular NAD and methods of treatment of central nervous system diseases and disorders including neurodegenerative diseases and disorders and / or age-related diseases and disorders. In some embodiments, chemical addiction and substance abuse can result from neurodegenerative diseases and disorder.Enhanced NAD+Compositions

[0223] The present disclosure provides NAD+compositions or medical food having an enhanced stability and bioavailability compared to NAD+(e.g., NAD+monohydratej-containing compositions. The present enhanced NAD+compositions or medical foods have a slower degradation rate and / or a longer half-life (in vitro and / or in vivo), a longer shelf-life, and / or a less stringent storage condition requirement (e.g. , but not limited to, cooling or dry storage), compared to a control NAD+not produced by the conditions and processes described herein.

[0224] The chemical name and structure of NAD+are shown below:Formula I

[0225] In some embodiment the chemical name of Formula I is: [[(2R,3S,4R,5R)-5-(6-aminopurin-9- yl)-3,4-dihydroxyoxolan-2-yl]methoxy-hydroxyphosphoryl] [(2R,3S,4R,5R)-5-(3-carbamoylpyridin- l-ium-l-yl)-3,4-dihydroxyoxolan-2-yl]methyl hydrogen phosphate. In some embodiment the chemical formula of Formula I is C2iH28N?Oi4P2+. In some embodiment the molecular weight of protonated NAD+(positively charged, anhydrate) is 664.44 g / mol. In some embodiment the CASRN of Formula I is 53-84-9 and the source is PubChem (CID 5893). In some embodiment the molecular weight of NAD+(zwitterion, anhydrate) is 663.43 g / mol.

[0226] The enhanced NAD+compositions or medical foods described herein may be a substitute of current commercially available NAD+compositions, or other NAD+compositions without the property or not produced by the current conditions or process, for its enhanced stability. For example, since NAD+is unstable even under conditions that are not drastic such as in aqueous solution, the enhanced NAD+compositions described herein may be used as a supplement and / or a pharmaceutical agent with improved bioavailability for oral administration (or through other administration routes) to an individual. Compared to commercially available NAD+compositions, the enhanced NAD+compositions described herein may be more stable in the direct interaction with water, radiation, oxygen, and / or UV light,

[0227] This compound is an active component of various enhanced compositions and pharmaceutical formulations or medical foods of the present disclosure (described below), where it is present from about 5 mg to about 4000 mg. In some embodiments of the present disclosure, the amount of NAD+in the enhanced composition or pharmaceutical formulation or medical food is about 100 mg to about 3500 mg. In some embodiments of the present disclosure, the amount of NAD+is about 20 mg to about 2500 mg. In some embodiments of the present disclosure, the dose of NAD+is about 50 mg to about 2000 mg. In some embodiments of the present disclosure, the amount of NAD+is about 100 mg to about 1500 mg. In some embodiments of the present disclosure, the amount of NAD+is about200 mg to about 1000 mg. In further embodiments, the amount of NAD+is about 300 mg to about 900 mg. In further embodiments, the amount of NAD+is about 400 mg to about 800 mg. In further embodiments, the amount of NAD+is about 500 mg to about 700 mg. In further embodiments, the amount of NAD+is about 550 mg to about 650 mg. In some embodiments, the amount of NAD+is about 500 mg. In some embodiments, the amount of NAD+is about 600 mg. In some embodiments, the amount of NAD+is about 700 mg. In some embodiments, the amount of NAD+is about 800 mg. In some embodiments, the amount of NAD+is about 900 mg. In some embodiments, the amount of NAD+is about 1000 mg. In some embodiments, the amount of NAD+is about 400 mg. In some embodiments, the amount of NAD+is about 300 mg. In some embodiments, the amount of NAD+is about 200 mg.

[0228] Enhanced NAD+compositions according to the disclosure may also include phosphorus as a minor component originally from the commercially obtained NAD+(e.g., NAD+monohydrate) which remains after the vacuumization process (described below), or which may be added. In some embodiments of the present disclosure, the content of phosphorus is from about 0.5% to about 9.33%. For example, an enhanced NAD+composition may comprise from about 0.5% (wt / wt) to about 5.5% (wt / wt). In some embodiments, the content of phosphorus is about 1.0% to about 5.0%. In some embodiments, the content of phosphorus is about 4.5% to about 5.0%. In some embodiments, the content of phosphorus is about 3.5% to about 6.0%. In some embodiments, the content of phosphorus is about 3.0% to about 5.5%. In some embodiments, the content of phosphorus is about 4.0% to about 5.5%. In some embodiments, the content of phosphorus is about 3.5% to about 5.5%. In certain embodiments, the content of phosphorus is about 4.5% to about 4.7%. In other embodiments, the content of phosphorus is about 4.5% to about 5.5%. In some embodiments, the content of phosphorus is about 3.0%, about 3.5%, about 4.5%, about 4.58%, 4.6%, about 4.67%, about 4.7%, about 4.8%, about 4.9%. In some embodiments, the content of phosphorus is about 5.0%, about 5.1%, about 5.2%, about 5.3%, about 5.4%, about 5.5%, about 5.6%, about 5.6%, about 5.8%, about 5.9%, about 6.0%, about 6.5%, about 7.0%, about 7.5%, about 8.0%, about 8.5%, about 9.0%, or about 9.33%.

[0229] In particular nonlimiting embodiments, the NAD+composition comprises 3.0% (wt / wt) NAD+and about to 9.33% (wt / wt) phosphorus.

[0230] In some embodiments, the enhanced NAD+composition further contains traces of an inert gas. For example, the NAD+composition may include s traces of Argon, Nitrogen, Xenon, or Helium, and may also include anhydrous CO2.

[0231] In some embodiments, the enhanced composition of the present disclosure comprises an amount of water, moisture, and / or solvent that is less than about 0.2% (weight / weight) measured by standard methods (e.g., Karl Fischer titration).

[0232] In some embodiments, the enhanced NAD+compositions of the present disclosure have a density of about 0.51 g / cm3. In some embodiments, the NAD+compositions of the present disclosure have a density of about 0.4 g / cm3. In some embodiments, the enhanced NAD+compositions of thepresent disclosure have a density from about 0.27 g / cm3to about 0.51 g / cm3. In some embodiments, the enhanced NAD+compositions of the present disclosure have a density of about 0.52 g / cm3. In some embodiments, the enhanced NAD+compositions of the present disclosure have a density of about 0.53 g / cm3. In some embodiments, the enhanced NAD+compositions of the present disclosure have a density of about 0.55 g / cm3. In some embodiments, the enhanced NAD+compositions of the present disclosure have a density of about 0.5 6 g / cm3. In some embodiments, the enhanced NAD+compositions of the present disclosure have a density of about 0.57 g / cm3. In some embodiments, the enhanced NAD+compositions of the present disclosure have a density of about 0.58 g / cm3. In some embodiments, the enhanced NAD+compositions of the present disclosure have a density of about 0.59 g / cm3. In some embodiments, the enhanced NAD+compositions of the present disclosure have a density of about 0.6 g / cm3. In some embodiments, the enhanced NAD+compositions of the present disclosure have a density from about 0.51 g / cm3to about 0.75 g / cm3.

[0233] The enhanced NAD+compositions or medical food described herein have enhanced stability relative to control NAD+. “Control NAD+” refers to NAD+(e.g., NAD+monohydrate) which may be synthesized or which is commercially available. For example, enhanced NAD+compositions described herein are at least 10%, at least 20%, at least 30%, at least 40%, 50%, at least 60%, at least 70%, 80%, at least 90%, at least 100%, at least 150%, at least 200%, at least 250%, at least 300%, at least 350%, at least 400%, at least 500%, at least 600%, at least 700%, at least 800%, or at least 900% more stable than control NAD+, Other exemplary enhanced NAD+compositions or medical food according to the disclosure are 10-fold, 15-fold, 20-fold, 30-fold, 40-fold, 50-fold, 60-fold, 70-fold, 80-fold, 90-fold, 100-fold, or more, stable than a control NAD+. Such stability may be represented by the degradation rate, half-life, shelf-life, or other measurements known to those with skill in the art.

[0234] The enhanced NAD+compositions or medical food of the instant disclosure are also stable under physiological conditions. In some embodiments, the instant NAD+compositions or medical food have a higher bioavailability in cells and organs as compared to commercially available NAD+supplements. In some embodiments, the instant NAD+compositions or medical food have a higher bioavailability in target cells (e.g., liver, kidney, immune cells) as compared to commercially available NAD+supplements.

[0235] In some embodiments, the NAD+compositions or medical food described herein display a low rate of degradation. For example, the instant enhanced NAD+compositions or medical food can be stored without signs of degradation for 36 months at room temperature (RT) (20 °C to 22 °C) and / or under suitable storage conditions, including, but not limited to, refrigeration (0 °C to 4 °C) or freezing (0 °C to -20 °C). In some embodiments, the enhanced NAD+composition or medical food has enhanced solid form stability. In some embodiments, the enhanced NAD+composition or medical food of the instant disclosure shows greater stability than commercially available NAD+supplements.

[0236] In some embodiments, the instant enhanced NAD+compositions or medical food also show increased stability in aqueous solution. In some embodiments, no significant degradation of theinstant NAD+compositions or medical food in aqueous solution phase occurs for up to 72 hours. In other embodiments, degradation of the enhanced NAD+composition or medical food is less than 5% after 4 days in an aqueous solution. The compositions or medical food remain stable in aqueous solution after 5 days with 10% degradation or less. Thus, in some embodiments, the enhanced NAD+composition or medical food has enhanced aqueous stability.

[0237] In some embodiments, the enhanced NAD+compositions of the present disclosure have a density of about 0.51 g / cm3. In some embodiments, the compositions of the present disclosure have a density of about 0.4 g / cm3. In some embodiments, the compositions of the present disclosure have a density from about 0.27 g / cm3to about 0.51 g / cm3. In some embodiments, the compositions of the present disclosure have a density of about 0.52 g / cm3. In some embodiments, the compositions of the present disclosure have a density of about 0.53 g / cm3. In some embodiments, the compositions of the present disclosure have a density of about 0.55 g / cm3. In some embodiments, the compositions of the present disclosure have a density of about 0.56 g / cm3. In some embodiments, the compositions of the present disclosure have a density of about 0.57 g / cm3. In some embodiments, the compositions of the present disclosure have a density of about 0.58 g / cm3. In some embodiments, the compositions of the present disclosure have a density of about 0.59 g / cm3. In some embodiments, the compositions of the present disclosure have a density of about 0.6 g / cm3. In some embodiments, the compositions of the present disclosure have a density from about 0.51 g / cm3to about 0.75 g / cm3.

[0238] In some embodiments, the enhanced NAD+compositions of the present disclosure comprise a crystalline component comprising polyethylene glycol, and an amorphous component comprising amorphous NAD+. In some embodiments, the enhanced NAD+composition further comprises an amorphous polyethylene glycol. In some embodiments, the ratio of NAD+to polyethylene glycol in the composition is about 1: 1 (wt / wt). In some embodiments, the ratio of NAD+to polyethylene glycol in the composition is 1: 1 (wt / wt). In some embodiments, the polyethylene glycol has an average molecular weight of between about 300 and about 4000. In some embodiments, the polyethylene glycol is PEG3350.

[0239] In some embodiments of the enhanced NAD+compositions of the present disclosure comprising a crystalline component comprising polyethylene glycol and an amorphous component comprising amorphous NAD+, the composition is characterized by having a first onset temperature of between 179.39 °C (± 0.10 °C) and 182.61 °C (± 0.10 °C) by thermogravimetric analysis (TGA) thermogram.

[0240] In some embodiments of the enhanced NAD+compositions of the present disclosure comprising a crystalline component comprising polyethylene glycol and an amorphous component comprising amorphous NAD+, the composition is characterized by having a weight loss from 14.39 wt% to 16.98 wt% in the range from about 170 °C to about 300 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 300 °C under nitrogen gas at a ramp rate of 10 °C / min.

[0241] In some embodiments of the enhanced NAD+compositions of the present disclosure comprising a crystalline component comprising polyethylene glycol and an amorphous component comprising amorphous NAD+, the composition is characterized by having a weight loss from 57.83 wt% to 58.55 wt% in the range from about 300 °C to about 800 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 800 °C under nitrogen gas at a ramp rate of 10 °C / min.

[0242] In some embodiments of the enhanced NAD+compositions of the present disclosure comprising a crystalline component comprising polyethylene glycol and an amorphous component comprising amorphous NAD+, the composition is characterized by having a TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with any one of FIG. 33A, FIG. 33B, FIG. 34A, FIG. 34B, FIG. 35A, or FIG. 35B. In some embodiments, the TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with FIG. 33A or FIG. 33B. In some embodiments, the TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with FIG. 34A or FIG. 34B. In some embodiments, the TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with FIG. 35A or FIG. 35B.

[0243] In some embodiments of the enhanced NAD+compositions of the present disclosure comprising a crystalline component comprising polyethylene glycol and an amorphous component comprising amorphous NAD’ the composition is characterized by having a TGA thermogravimetric analysis (TGA) thermogram having:(1) a first weight loss from 2.42 wt% to 3.25 wt% in the range from ambient temperature to about 170 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 170 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or(2) a second weight loss from 13.66 wt% to 14.61 wt% in the range from about 170 °C to about 300 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 300 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or(3) a third weight loss from 58.73 wt% to 60.37 wt% in the range from about 300 °C to about 800 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 800 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or(4) a fourth weight loss from 20. 14 wt% to 21.46 wt% in the range from about 800 °C to about 900 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at about 800 °C under nitrogen gas at a ramp rate of 10 °C / min and from about 800 °C to about 900 °C under air at a ramp rate of 10 °C / min, and / or(5) a first onset temperature from 179.39 °C to 180.15 °C, and / or(6) a second onset temperature from 369.03 °C to 370.82 °C.

[0244] In some embodiments, the composition is characterized by having one characteristic selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having two characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, thecomposition is characterized by having three characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having four characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having five characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having all six characteristics selected from (1), (2), (3), (4), (5), and (6).

[0245] In some embodiments of the enhanced NAD+compositions of the present disclosure comprising a crystalline component comprising polyethylene glycol and an amorphous component comprising amorphous NAD+, the composition is characterized by having a TGA thermogravimetric analysis (TGA) thermogram having:(1) a first weight loss from 2.32 wt% to 3.55 wt% in the range from ambient temperature to about 170 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 170 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or(2) a second weight loss from 14.39 wt% to 16.22 wt% in the range from about 170 °C to about 300 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 300 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or(3) a third weight loss from 57.83 wt% to 58.55 wt% in the range from about 300 °C to about 800 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 800 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or(4) a fourth weight loss from 22.62 wt% to 22.76 wt% in the range from about 800 °C to about 900 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at about 800 °C under nitrogen gas at a ramp rate of 10 °C / min and from about 800 °C to about 900 °C under air at a ramp rate of 10 °C / min, and / or(5) a first onset temperature from 178.62 °C to 180.11 °C, and / or(6) a second onset temperature from 365.73 °C to 370.47 °C.

[0246] In some embodiments, the composition is characterized by having one characteristic selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having two characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having three characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having four characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having five characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having all six characteristics selected from (1), (2), (3), (4), (5), and (6).

[0247] In some embodiments of the enhanced NAD+compositions of the present disclosure comprising a crystalline component comprising polyethylene glycol and an amorphous component comprising amorphous NAD+, the composition is characterized by having a TGA thermogravimetricanalysis (TGA) thermogram having:(1) a first weight loss from 3.62 wt% to 3.96 wt% in the range from ambient temperature to about 170 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 170 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or(2) a second weight loss from 15.17 wt% to 16.98 wt% in the range from about 170 °C to about 300 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 300 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or(3) a third weight loss from 58.05 wt% to 59.66 wt% in the range from about 300 °C to about 800 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 800 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or(4) a fourth weight loss from 22.85 wt% to 25.87 wt% in the range from about 800 °C to about 900 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at about 800 °C under nitrogen gas at a ramp rate of 10 °C / min and from about 800 °C to about 900 °C under air at a ramp rate of 10 °C / min, and / or(5) a first onset temperature from 179.83 °C to 182.61 °C, and / or(6) a second onset temperature from 363.24 °C to 366.16 °C.

[0248] In some embodiments, the composition is characterized by having one characteristic selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having two characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having three characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having four characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having five characteristics selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by having all six characteristics selected from (1), (2), (3), (4), (5), and (6).Pharmaceutical Formulations or Medical Foods

[0249] The present disclosure also provides pharmaceutical formulations or medical foods comprising NAD compositions with enhanced properties, which may be administered to an individual (e.g. , a human) as a nutrient, a pharmaceutical agent, and / or medical food to prevent, ameliorate, and / or treat a disease or disorder related to insufficient NAD in the body. In some embodiments, the enhanced NAD+pharmaceutical formulations or medical foods described herein increases the NAD concentration and / or NAD degradation in the individual. In some embodiments, the individual is having tryptophan-poor diet. In other embodiments, the individual has a genetic mutation and / or other mechanisms (e.g., living or environmental conditions, food or drug effects, etc.) resulting in a reduction or inhibition of NAD production, a reduction of NAD stability, and / or an increase of NAD degradation or turnover in the individual. For example, the individual may have a genetic mutation inthe kynurenine pathway, thus reducing or inhibiting the production chain from nicotinic acid to NMN to NAD and then to NAD. The individual may have another genetic mutation affecting the production, transportation, storage, and / or utilization of NAD+in the individual.

[0250] The enhanced NAD+compositions or medical foods described herein may also enhance NAD-related biochemical and biological reactions in an individual in need thereof. For example, since NAD is a co-factor of oxidoreductases, the enhanced NAD+pharmaceutical formulations or medical foods described herein may enhance the bio-functions of oxidoreductases in the individual, e.g., enhancing synthesizing enzymes such as poly(ADPribose) polymerases and cADPribose synthases. Thus, if the individual has a disease or disorder related to insufficient poly(ADPribose) polymerases and cADPribose synthases, or other genes or gene products downstream or affected by NAD, the enhanced NAD+pharmaceutical formulations or medical food described herein may be administered to the individual to prevent, ameliorate, and / or treat the disease or disorder.

[0251] In addition, the enhanced NAD+pharmaceutical formulations or medical foods described herein may be a substitute of current commercially available NAD+compositions, or other NAD+compositions without the property or not produced by the current conditions or process, for its enhanced stability. For example, since NAD+is unstable even under conditions that are not drastic such as in aqueous solution, the enhanced NAD+compositions described herein may be used as a supplement, a pharmaceutical agent, or medical food with improved bioavailability for oral administration (or through other administration routes) to an individual. Compared to commercially available NAD+compositions, the enhanced NAD+compositions or medical foods described herein may be more stable in the direct interaction with water, radiation, oxygen, and / or UV light, and if administered as part of a pharmaceutical formulation, is resistant to hydrolysis in the small intestine by brush border cells.

[0252] The enhanced NAD+pharmaceutical formulations of the present disclosure comprise the enhanced NAD+composition as described above, as well as at least one additional secondary component, compound, or ingredient that can aid in administration and / or treatment of a subject.

[0253] In some embodiments, this ingredient is PEG, a polymer of ethylene glycol) which is “generally recognized as safe” (GRAS) under the US Federal Drug Administration (FDA). PEG has various sizes depending on its molecular weight which follows the term “PEG.” In some embodiments, the enhanced NAD+composition comprises PEG300, PEG400, PEGMME (Polyethylene glycol monomethyl ether) 550, PEG600, PEG 1000, PEGMME 2000, PEG3350, and / or PEG4000. The composition of the present disclosure comprises PEG of any molecular weight at a concentration of about 10% to about 75%. In some embodiments, the composition comprises polyethylene glycol at a concentration of about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 60%, about 70%, or about 75%.

[0254] In some embodiments, the ratio of NAD+to PEG of any molecular weight in an NAD+composition of the present disclosure is l: l (wt / wt), 1:2 (wt / wt), 1:3 (wt / wt),l:4 (wt / wt), 1:5 (wt / wt),1:6 (wt / wt), 1:7 (wt / wt), 1:8 (wt / wt), 1:9 (wt / wt), 1: 10 (wt / wt), 1: 11 (wt / wt), 1: 12 (wt / wt), l: 13(wt / wt), 1: 14 (wt / wt), 1: 15 (wt / wt), 1: 16 (wt / wt), 1: 17 (wt / wt), 1: 18 (wt / wt), 1: 19 (wt / wt), or 1:20 (wt / wt).

[0255] In some embodiments, the ratio of NAD+to PEG of any molecular weight in a composition of the present disclosure is 2: 1 (wt / wt), 3: 1 (wt / wt), 4: 1 (wt / wt), 5: 1 (wt / wt), 6: 1 (wt / wt), 7: 1 (wt / wt), 8: 1 (wt / wt), 9: 1 (wt / wt), 10: 1 (wt / wt), 11: 1 (wt / wt), 12: 1 (wt / wt), 13: 1 (wt / wt), 14: 1 (wt / wt), 15: 1 (wt / wt), 16: 1 (wt / wt), 17: 1 (wt / wt), 18: 1 (wt / wt), 19: 1 (wt / wt), or 20: 1 (wt / wt).

[0256] The enhanced NAD+pharmaceutical formulations or medical foods of the instant disclosure are also stable under physiological conditions. In some embodiments, these formulations have a higher bioavailability in cells and organs as compared to commercially available NAD+supplements. Without wishing to be bound by theory, due to low bioavailability, preclinical studies have used nicotinamide mononucleotide (NMN), nicotinamide riboside (NR), or nicotinamide (NAM) to elevate intracellular NAD (Matasic et al., Am J Physiol Heart Circ Physiol. 314(4):H839-H852 (2018)). In some embodiments, the instant enhanced NAD+formulations have a higher bioavailability in target cells (e.g., liver, kidney, immune cells) as compared to commercially available NAD+supplements.

[0257] These formulations can be in solid as well as liquid dosage forms, such as tablets, capsules, powders, lozenges, suppositories, syrups, elixirs, sterile solutions, suspensions or emulsions, pastes, ointments, jellies, waxes, oils, lipids, encapsulation in lipid (cationic or anionic) containing vesicles (such as liposomes, microparticles, microcapsules, or LIPOFECTIN™, DNA conjugates, anhydrous absorption pastes, oil-in-water and water-in-oil emulsions, carbowax, semi-solid gels, and semi-solid mixtures containing carbowax.

[0258] The amount of pharmaceutical formulation or medical food to be administered depends on the concentration or amount of NAD+in the formulation, the mode of administration, the disorder to be treated, the severity of the disorder being treated, the weight of the patient, and the like, as known by the professional treating a subject.

[0259] These pharmaceutical formulations may include carriers, excipients, flavorings, dyes, and / or other agents that provide suitable delivery, tolerance, and the like to the subject. Such useful and appropriate agents can be found in the formulary known to all pharmaceutical chemists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.

[0260] In some embodiments, the pharmaceutical formulation comprises about 0.5% (wt / wt) magnesium stearate. In some embodiments, the pharmaceutical formulation comprises about 0.1% (wt / wt), about 0.2% (wt / wt), about 0.3% (wt / wt), about 0.4% (wt / wt), about 0.5% (wt / wt), about 0.6% (wt / wt), about 0.7% (wt / wt), about 0.8% (wt / wt), about 0.9% (wt / wt), or about 1% (wt / wt) magnesium stearate.

[0261] In yet other embodiments, the tablet form may further comprise glucose, e.g., at from about 10% to about 75% glucose. Some non-limiting embodiments of the enhanced NAD+formulations comprise glucose at a concentration of about 10%, about 20%, about 30%, about 40%, about 41%,about 41.42%, about 45%, about 50%, about 60%, about 70%, or about 75%.

[0262] In yet another embodiment, the composition of the present disclosure further comprises polyethylene glycol at a concentration of about 50%. In some embodiments, the composition comprises polyethylene glycol at a concentration of about 10%, about 20%, about 30%, about 40%, about 45%, about 50%, about 60%, about 70%, or about 75%.

[0263] Useful dyes include, but are not limited to, Allura Red AC (Red 40) or Blue #1. For example, the compositions may comprise Allura Red AC at a concentration of from about 0. 1% to about 3%. In some nonlimiting embodiments, the composition comprises Allura Red AC at a concentration of about 0.1%, about 0.5%, about 1%, about 1.25%, about 1.5%, about 1.75%, about 2%, about 2.5%, or about 3%.

[0264] In some embodiments, the enhanced NAD+pharmaceutical formulation of the present disclosure further comprises a flavoring such as, but not limited to, methyl salicylate. For example, methyl salicylate can be present at a concentration of about 0.1 ml / kg to about 2.0 ml / kg. In some nonlimiting embodiments, methyl salicylate can be present in the composition at a concentration of about 0.1 ml / kg, about 0.2 ml / kg, about 0.3 ml / kg, about 0.4 ml / kg, about 0.5 ml / kg, about 0.6 ml / kg, about 0.7 ml / kg, about 0.8 ml / kg, about 0.9 ml / kg, about 1 ml / kg, about 1.5 ml / kg, about 1.7 ml / kg, or about 2 ml / kg.

[0265] The enhanced NAD+pharmaceutical formulations may additionally include other therapeutic compounds or drugs, such as, but not limited to analgesic agents. For example, a powder form of the formulation may include acetylsalicylic acid at a concentration of about 0. 1% to about 0.7%. In some non-limiting embodiments, the enhanced NAD+formulations comprises acetylsalicylic acid at a concentration of about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, or about 0.7%.

[0266] In some embodiments, the enhanced NAD+pharmaceutical formulation is medical food comprising enhanced NAD+ of the present disclosure.

[0267] The pharmaceutical composition may include, but is not limited to, for example, magnesium stearate, glucose, polyethylene glycol, Red 40, salicylate methyl, Blue #1, or any combination thereof.

[0268] Various delivery systems are known and can be used to administer the pharmaceutical formulations described herein, depending on the delivery route. For example, pharmaceutical formulations of the present disclosure may be administered orally, anally, systemically, intramuscularly, optically, vaginally, buccally, subcutaneously, etc. The route and delivery system for administration are known by those with medical skill in the art. For example, liquid forms of the enhanced NAD+formulation may be administered by any convenient route, for example by infusion or bolus injection, by absorption through epithelial or mucocutaneous linings (e.g., oral mucosa, rectal and intestinal mucosa, etc.), for example, by intradermal, intramuscular, intraperitoneal, intravenous, subcutaneous, intranasal, intra-tracheal, epidural, and oral routes, and may be administered together with other biologically active.

[0269] In an embodiment of the present disclosure, the composition described herein is in a powder form.Process of Preparation

[0270] The NAD+is placed in a container under reduced pressure of vacuum or under a reduced pressure of between about 24 inches of Hg and about 30.25 inches of Hg; and replacing the vacuum with an inert gas to increase the pressure in the container to about 1 atmosphere (atm). In some embodiments, the inert gas which replaces the vacuum comprises Helium, Nitrogen, Argon, and / or Xenon. Vacuumization can be performed more than once and at least twice. Vacuumization can be performed more than once, e.g., once, twice, or at least twice.

[0271] In another embodiment, the inert gas used for vacuumization comprises Xenon. In another embodiment, the inert gas used for vacuumization comprises Nitrogen. In another embodiment, the inert gas used for vacuumization comprises anhydrous, high purity Argon. In another embodiment, the inert gas used for vacuumization comprises Helium.

[0272] The NAD+may be pretreated before being vacuumized. For example, the NAD+can be processing to obtain a preselected average NAD+particle size. Processing can include, but not limited to, passing the NAD+through a sieve, and / or grinding, milling, or jet-milling the NAD+to obtain the preselected particle size. In some non-limiting embodiments, the NAD+is ground or milled into a particle about 0.05 mm to about 0.4 mm in size in size. In some embodiments, the NAD+particle is about 0.05 mm, about 0.07 mm, about 0.09 mm, about 0.1 mm, about 0.110 mm, about 0.120 mm, about 0.140 mm, about 0.150 mm, about 0.153 mm, about 0.155 mm, about 0.160 mm, about 0.180 mm, about 0.2 mm, about 0.220 mm, about 0.250 mm, about 0.270 mm, about 0.300 mm, about 0.320 mm, about 0.340 mm, about 0.350 mm, about 0.370 mm, or about 0.4 mm in size.

[0273] The enhanced NAD+pharmaceutical formulations according to the present disclosure can be prepared by mixing NAD+with any other components, to obtain a blended mixture, which is then vacuumized under vacuum and inert gas conditions. Accordingly, the vacuumized mixture may contain traces of an inert gas. For example, the resulting vacuumized mixture contains traces of Xenon, Nitrogen, Argon, or Helium.Preparation of the Enhanced Pharmaceutical Formulations

[0274] APIs such as those according to the disclosure may be initiated by mixing vacuumized or unvacuumized NAD+with other components in a container, e.g. , in a blender, to form a blended mixture. The term “blended mixture” refers to mixed components of the composition, e.g., various amounts of one or more ingredient with another or others.

[0275] In one embodiment, NAD+is mixed with PEG of any molecular weight, which may be commercially-obtained, and which may be pretreated as described herein to obtain a preselected particle size, to achieve a homogeneous distribution of NAD+and PEG in the final pharmaceuticalformulation (or “drug product”). In some embodiments, the commercially obtained PEG of any particular molecular weight or weights, as described above, is used in the preparation is GRAS under the FDA.

[0276] Mixing as described herein prevents small amounts of NAD+from becoming inadvertently partitioned to, or concentrated within, small compartments or portions of the formulation equipment surfaces due to particle size distribution and / or small dimensional irregularities and cavities on the surfaces of the blending equipment.

[0277] In some embodiments of the disclosure, the process of preparation comprises pre -formulating or pretreating commercially obtained NAD+and / or PEG. In some embodiments, the pretreated NAD+and / or PEG is vacuumized prior to mixing. Alternatively, the NAD+or PEG is vacuumized prior to being pretreated and mixed. Alternatively, both NAD+and PEG are separately vacuumized before mixing, before or after being sized, and / or before and / or before mixing.

[0278] Processing or pretreatment of NAD+is performed to allow for homogeneous distribution with excipients in the enhanced NAD+formulation of the present disclosure.

[0279] In some non-limiting embodiments, the NAD+is ground or milled into a particle about 0.05 mm to about 0.4 mm in size.

[0280] Another aspect of the disclosure relates to a process of making an enhanced NAD+composition, said process comprising blending NAD+and polyethylene glycol and vacuumizing the blended mixture of NAD+and PEG under vacuum and / or inert gas conditions.

[0281] In one embodiment of the disclosure, the process of preparation comprises pre -formulating NAD+. In a certain embodiment, pre -formulating the NAD+involves grinding or milling the NAD+. In particular embodiments, grinding and / or milling the NAD+is performed to produce NAD+particles approximately 0.152 mm in size to allow for homogeneous distribution with excipients in the enhanced NAD+formulation of the present disclosure.

[0282] In yet another embodiment of the disclosure, the process of pre -formulation of NAD+involves grinding or milling the NAD+and then vacuumizing the ground or milled NAD+product by itself prior to blending.

[0283] In some embodiments of the disclosure, the process of preparation further comprises pre- formulating commercial PEG. In some embodiments, pre -formulating the PEG comprises grinding or milling the commercial PEG. In certain embodiments, grinding and / or milling the commercial PEG is performed to reduce the commercial PEG into particles of super fine size to allow for homogeneous distribution with excipients and the active ingredient (NAD+) in the NAD+formulation of the present disclosure.

[0284] In yet another embodiment of the process of preparation, pre -formulation of PEG comprises vacuumizing the commercial PEG and then grinding or milling the vacuumized PEG product by itself prior to blending.

[0285] In some embodiments, the pretreated NAD+and / or PEG is vacuumized prior to mixing.Alternatively, the NAD+or PEG is vacuumized prior to being pretreated and mixed. Alternatively, both NAD+and PEG are separately vacuumized before mixing, before or after being sized, and / or before and / or before mixing.

[0286] In some embodiments, the process of mixing comprises mixing the NAD+and PEG or the pretreated NAD+and / or pretreated PEG. Mixing of NAD+with PEG enables, e.g., non-covalent coating or immobilization of NAD+to PEG, results in stabilization of NAD+and prevents its degradation. Mixing can be carried out, e.g., for about 20 minutes to about an hour at room temperature (RT). For example, the mixture is blended for at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 50 minutes, or at least 1 hour, at RT.

[0287] The blended mixture of NAD+and PEG is then vacuumized by being placed under a cycle of vacuum or reduced pressure and may be followed by inert gas re -saturation.

[0288] In some embodiments of process of preparation, the blended mixture of NAD+and PEG is placed under a reduced pressure of about 22 inches of Hg to about 29 inches of Hg. In some embodiments of process of preparation, the blended mixture of NAD+and PEG is placed under a reduced pressure of about 26 inches of Hg to about 30 inches of Hg. In some embodiments, the blended mixture of NAD+and PEG is placed under a reduced pressure of about 24 inches of Hg to about 29.92 inches of Hg. In some embodiments, the blended mixture of NAD+and PEG is placed under a reduced pressure of about 24 inches of Hg to about 28 inches of Hg. In some embodiments, the blended mixture ofNAD+and PEG is placed under a reduced pressure of about 24 inches of Hg. In some embodiments, the blended mixture of NAD+and PEG is placed under a reduced pressure of about 26 inches of Hg. In some embodiments, the blended mixture of NAD+and PEG is placed under a reduced pressure of about 28 inches of Hg. In some embodiments, the blended mixture of NAD+and PEG is placed under a reduced pressure of about 30 inches of Hg. In some embodiments, the blended mixture of NAD+and PEG is placed under a reduced pressure of about 30.25 inches of Hg. While not intending to be limited to any particular explanation, the removal of solvents also results in the azeotropic and / or evaporative removal of contaminant components, such as, but not limited to, water, air, oxygen, etc., which accelerates the degradation of the NAD+product.

[0289] As described above, the process of vacuumizing may also include placing the blended mixture of NAD+and PEG under a cycle of vacuum or reduced pressure may be followed by inert gas re -saturation.

[0290] According to embodiments of the present disclosure, non-covalent coating or immobilization of an active agent (e.g., NAD+) to the selected PEG substrate stabilizes the active agent and prevents degradation. While not intending to be limited to any particular explanation, further stabilization of the product is also believed to occur by azeotropic and / or evaporative removal of contaminant components, such as, but not limited to, water and oxygen, during solvent evaporation, and the vacuum may be replaced with an inert, anhydrous gas such as, but not limited to, Nitrogen (N2), Argon (Ar), Xenon (Xe) or Helium (He). In some embodiments of the method of the disclosure, theblended mixture under vacuum is re-saturated with an inert gas to restore the pressure in the blender to 1 atm. In some embodiments, the inert gas used in the re-saturation during the vacuumization process comprises Nitrogen, Argon, Xenon, or Helium, any of which may be of high purity. Other displacement gases may also be used to achieve further advantages. For example, anhydrous carbon dioxide (CO2) may be used as an inert replacement gas that may also serve an additional purpose of acidifying the local environment of a substrate for active agents or drugs that are more stable in acidic environments.

[0291] The presence of inert gas or anhydrous CO2 in the process of vacuumization is understood to azeotrope the impurities, volatile contaminants, and / or residues present in the original NAD+and PEG. This allows for the removal of any remaining elements such as, but not limited to, water, air, oxygen, solvents.

[0292] Vacuumization of the blended mixture is performed for at least one cycle. In other embodiments, vacuumization of the blended mixture is done in at least two cycles. In other embodiments, vacuumization of the blended mixture is done in at least three, four or more cycles.

[0293] In an embodiment of the present disclosure, the commercial PEG used in the preparation is GRAS under the FDA. In some embodiments, the commercial PEG used in the preparation is PEG300. In other embodiments, the commercial PEG used in the preparation is PEG400. In yet other embodiments, the commercial PEG used in the preparation is PEGMME 550. In still other embodiments, the commercial PEG used in the preparation is PEG600. In other embodiments, the commercial PEG used in the preparation is PEG 1000. In some embodiments, the commercial PEG used in the preparation is PEGMME 2000. In yet other embodiments, the commercial PEG used in the preparation is PEG3350. In certain embodiments, the commercial PEG used in the preparation is PEG4000. In other embodiments, the commercial PEG used in the preparation is PEG300, PEG400, PEGMME 550, PEG600, PEG 1000, PEGMME 2000, PEG3350, PEG4000, or any combination thereof.

[0294] In a further embodiment of the present disclosure, the process of preparation comprises blending the pre -formulated NAD+and pre -formulated PEG.

[0295] Pharmaceutical formulations according to the disclosure can be made from the enhanced NAD+compositions according to the process described above, mixed with PEG, and may also include the addition of other components such as, but not limited to, excipients, flavorings, sugars dyes, magnesium stearate, etc. Non-limiting embodiments of such secondary ingredients include magnesium stearate, glucose, Red40, Blue# I, methyl salicylate, and water.

[0296] A further aspect of the present disclosure is to provide an enhanced NAD+composition made by the process of the instant disclosure which comprises nicotinamide adenine dinucleotide (NAD+) and polyethylene glycol (PEG) with a phosphorus content of about 1.0% (wt / wt) to about 9.33% (wt / wt). A further aspect of the present disclosure is to provide an enhanced NAD+composition made by the process of the instant disclosure which comprises nicotinamide adenine dinucleotide (NAD+)and polyethylene glycol (PEG) with a phosphorus content of about 1.0% to about 4.67%. A further aspect of the present disclosure is to provide an enhanced NAD+composition made by the process of the instant disclosure which comprises nicotinamide adenine dinucleotide (NAD+) and polyethylene glycol (PEG) with a phosphorus content of about 0.5% to about 9.33%.Methods of Treatment and Use

[0297] The enhanced NAD+pharmaceutical formulations or medical foods of the present disclosure can be used in the treatment of a variety of conditions, diseases and disorders. For example, they can be used for preventing or ameliorating anti-aging processes, in treating or preventing age-related disorders, CNS disorders, and in increasing intracellular NAD levels.

[0298] In some embodiments, the present enhanced NAD+pharmaceutical formulations or medical foods are potent and are efficacious at clinically achievable doses, are stable in a variety of potential dosage forms, possess acceptable solubility, bioavailability, acceptable pH, and have a reduced propensity to absorb and / or degrade in the presence of water, oxygen, UV light water, display ease of handling and consumption, all of which are consistent with the development, manufacture, and use of a medicament. In some embodiments, the NAD+composition comprises an amorphous component. In addition, the enhanced NAD+pharmaceutical formulations or medical foods disclosed herein offer increased biological activity toward increased cellular NAD levels, increased stability, and more physiologically acceptable pH. Additionally, the inclusion of PEG in the formulations herein provide a novel “molecular shuttle” for the delivery of NAD+, for example, but not limited to, across the buccal membranes into the blood. This formulation provides multiple advantages that are useful to assist in NAD+-dependent cellular activities.

[0299] In some embodiments, the enhanced NAD+pharmaceutical formulation is medical food comprising enhanced NAD+ of the present disclosure.

[0300] The enhanced NAD+pharmaceutical formulations or medical foods disclosure herein provide may be administered to a subject (e.g. , but not limited to, a human) as a nutrient and / or a therapeutic agent to prevent, ameliorate, and / or treat a disease or disorder related to insufficient NAD+in the body.

[0301] In some embodiments, the enhanced NAD+pharmaceutical formulations or medical foods are administered to increase the NAD concentration and / or NAD stability (i.e. , half-life) in the individual. In some embodiments, the individual is having tryptophan-poor diet, a genetic mutation and / or other characteristics (e.g., but not limited to, living or environmental conditions, food or drug effects), resulting in a reduction or inhibition of NAD production, a reduction of NAD stability, and / or an increase of NAD degradation or turnover in the individual. In one non -limiting example, the individual has a genetic mutation in the kynurenine pathway which results in the reduction or inhibition of the production chain from nicotinic acid to NMN to NAAD and then to NAD. In another nonlimiting example, the individual has another genetic mutation affecting the production,transportation, storage, and / or utilization of NAD+in the individual.

[0302] The enhanced NAD+pharmaceutical formulations or medical foods described herein may also enhance NAD+-related biochemical and biological reactions in an individual in need thereof. For example, since NAD+is a co-factor of oxidoreductases, the enhanced NAD+compositions described herein enhances the bio-functions of oxidoreductases in the individual, e.g., but not limited to, enhancing synthesizing enzymes such as poly(ADPribose) polymerases and cADPribose synthases. Thus, if the individual has a disease or disorder related to insufficient poly(ADPribose) polymerases and cADPribose synthases, or other genes or gene products downstream or affected by NAD+, an enhanced NAD+pharmaceutical formulation or medical food is administered to the individual to prevent, ameliorate, and / or treat the disease or disorder.

[0303] In one aspect, the present disclosure relates to a method of treating or preventing a neurodegenerative disease in a subject, comprising administering to the subject a therapeutically effective amount of an NAD+enhanced pharmaceutical formulation or medical food disclosed herein.

[0304] In some embodiments, the neurodegenerative disease is Parkinson’s disease (PD), Multiple Sclerosis (MS), Alzheimer’s Disease (AD), or Amyotrophic Lateral Sclerosis (ALS). In some embodiments, the administration of the NAD+enhanced pharmaceutical formulation or medical food modulates the level of expression of a gene involved in neurodegenerative disease compared to an untreated control, e.g., a subject which has not been administered the enhanced NAD+formulation (an “unadministered subject.”). In some embodiments, the administration of the NAD+enhanced pharmaceutical formulation or medical food changes the of a gene involved in neurodegenerative disease, and hence changes the level of the protein encoded by the gene, compared to the level in an untreated control. In some embodiments, the administration of the NAD+enhanced pharmaceutical formulation or medical food increases or decreases the level of expression of a gene involved in neurodegenerative disease compared to the level in an untreated control. Methods to measure gene and protein expression are well known in the art, for example DNA microarrays, RT-PCR, ChIP, Immunoassays, HPLC, and Mass Spectrometry.

[0305] In some embodiments, a subject with a neurodegenerative disease, such as Parkinson’s disease (PD), Multiple Sclerosis (MS), Alzheimer’s Disease (AD), or Amyotrophic Lateral Sclerosis (ALS), has an elevated level of neurofilament light chain (NfL). In some embodiments, neurofilament light chain (NfL) can serve as a clinically relevant biomarker for monitoring neuroaxonal damage and therapeutic response in subjects with neurodegenerative diseases. NfL is a cytoskeletal protein highly expressed in neuronal axons, and its presence in cerebrospinal fluid or blood can reflect the extent of axonal injury. In some embodiments, elevated NfL levels can be associated with disease activity or progression in conditions such as amyotrophic lateral sclerosis (ALS), multiple sclerosis (MS), Parkinson’s disease (PD), and Alzheimer’s disease (AD). In certain embodiments, administration of an enhanced NAD+formulation or medical food can reduce circulating NfL concentrations, thereby indicating a potential protective effect on neuronal integrityand axonal structure.

[0306] In some embodiments, the administration of the NAD+enhanced pharmaceutical formulation or medical food changes the plasma level of the protein involved in neurodegenerative disease compared to the plasma level in an unadministered subject.

[0307] In some embodiments, the neurodegenerative disease can be Amyotrophic Lateral Sclerosis (ALS). In particular embodiments relating to ALS, the enhanced NAD+pharmaceutical formulation or medical food can demonstrate unique pharmacokinetic properties that can enable rapid central nervous system penetration. The formulation or medical food described herein can achieve detectable increases in blood NAD+and NADP+levels in subjects with ALS-related pathology, for example, within 15 minutes of administration. Critically for neurodegenerative applications, therapeutic concentrations in spinal cord tissue can be achieved within approximately 2 hours post-administration. These elevated NAD+levels can be sustained beyond initial peak concentrations for up to 12 hours, for example, providing prolonged therapeutic benefit. Furthermore, in some embodiments, the formulation or medical food can modulate key oxidative stress markers in experimental ALS pathology - specifically, increasing glutathione (GSH) levels and the GSH / oxidized glutathione (GSH / GSSG) ratio in spinal cord tissue - thereby enhancing the tissue’s capacity to manage oxidative damage characteristic of motor neuron degeneration. The pharmaceutical formulation or medical food’s potential effects on oxidative stress can be particularly relevant in ALS pathology, where oxidative damage can be a primary driver of motor neuron death. This rapid modulation of antioxidant systems in neural tissue can represent a potential mechanism for neuroprotection in ALS, complementing the formulation’s systemic effects on NAD+metabolism.

[0308] In some embodiments relating to ALS-specific applications, administration of the enhanced NAD+pharmaceutical formulation or medical food can demonstrate functional benefits in preserving neuromuscular integrity during disease progression. Treatment can show trends toward preservation of compound muscle action potential (CMAP) amplitude, with approximately 18% reduction in muscle deterioration compared to untreated controls over a 30-day treatment period. These findings can suggest potential for slowing disease progression.

[0309] In some embodiments, at least one protein whose expression level is changed by the administration of the enhanced NAD+pharmaceutical formulation or medical food with the expression changed by the administration includes SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Receptor 1A (HTR1A), Insulin Receptor (INSR), Superoxide Dismutase 2 (SOD2), Artemin (ARTN), Fas Cell Surface Death Receptor (FAS), Interleukin 2 Receptor Subunit Beta (IL2Rb), Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), CD163 Molecule, Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), and / or Neurofilament Light Chain (NfL).

[0310] In embodiments, where the enhanced NAD+formulation increases protein expression, thelevel of at least one of SIRT1 (Sirtuin 1), 5 -Hydroxytryptamine Receptor 1A (HTR1A), Artemin (ARTN), Interleukin 2 Receptor Subunit Beta (IL2Rb), CD 163 Molecule, and / or Platelet-Derived Growth Factor Receptor Alpha (PDGFRA) is increased as compared to the level in an unadministered subject.

[0311] In embodiments, where the enhanced NAD+formulation or medical food decreases protein expression, the expression level of at least one of C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Insulin Receptor (INSR), Superoxide Dismutase 2 (SOD2), Fas Cell Surface Death Receptor (FAS), Platelet, Endothelial Cell Adhesion Molecule 1 (PECAM1), and / or Neurofilament Light Chain (NfL) is decreased as compared to the level of the at least one protein in an unadministered subject.

[0312] In some embodiments, the disease is Parkinson’s disease, and the gene encodes a protein including SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5- Hydroxytryptamine Receptor 1A (HTR1A), Insulin Receptor (INSR), and / or Neurofilament Light Chain (NfL).

[0313] In some embodiments, the disease is Multiple Sclerosis and the gene encodes a protein including SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Fas Cell Surface Death Receptor (FAS), Interleukin 2 Receptor Subunit Beta (IL2Rb), Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), CD163 Molecule, and Platelet- Derived Growth Factor Receptor Alpha (PDGFRA), Matrix Metallopeptidase 9 (MMP9), and / or Neurofilament Light Chain (NfL).

[0314] In some embodiments, the disease is Amyotrophic Lateral Sclerosis, and the gene encodes a protein including SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), Superoxide Dismutase 2 (SOD2), Artemin (ARTN), and / or Neurofilament Light Chain (NfL).

[0315] In specific embodiments where Amyotrophic Lateral Sclerosis (ALS) is treated with the enhanced NAD+pharmaceutical formulation or medical food, the method can achieve therapeutic benefit through multiple mechanisms. The formulation can enable rapid central nervous system delivery, achieving therapeutic NAD+concentrations in spinal cord tissue within approximately 2 hours of administration. It can sustain elevated NAD+levels in neural tissue beyond the initial peak concentrations. The formulation or medical food can modulate oxidative stress by increasing GSH levels and GSST activity in spinal cord tissue. It can also contribute to functional preservation by reducing neuromuscular deterioration, as measured by electrophysiological parameters including compound muscle action potential (CMAP) amplitude. Additionally, the formulation can modulate biomarkers such as NAD+ / NADH, NADP+ / NADPH, and GSH / GSSG ratios, which can serve as pharmacodynamic indicators of treatment response.

[0316] In some embodiments, the method of treating ALS can comprise monitoring treatment response through multiple modalities. These can include serial measurement of blood NAD+and NADP+levels, as well as NAD / NADH and NADP / NADPH ratios, with therapeutic response indicated by elevations occurring within approximately 15 minutes of administration. The method can also include assessment of oxidative stress markers, particularly GSH levels and the GSH / GSSG ratio. Electrophysiological monitoring using compound muscle action potential (CMAP) amplitude can serve as a functional outcome measure. Additionally, dose adjustment can be guided by pharmacokinetic and pharmacodynamic responses to optimize therapeutic efficacy.

[0317] In some embodiments, the disease is Alzheimer’s Disease and encodes a protein including SIRT1 (Sirtuin 1), Granulin Precursor (GRN), Matrix Metallopeptidase 9 (MMP9), Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), and 5 -Hydroxytryptamine Insulin Receptor (INSR).

[0318] In another aspect, the present disclosure relates to a method of modulating at least one of SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Receptor 1A (HTR1A), Insulin Receptor (INSR), Superoxide Dismutase 2 (SOD2), Artemin (ARTN), Fas Cell Surface Death Receptor (FAS), Interleukin 2 Receptor Subunit Beta (IL2Rb), Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), CD 163 Molecule, Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), and / or Neurofilament Light Chain (NfL) polypeptide expression in a subject in need thereof, comprising administering to the subject an effective amount of an enhanced NAD+pharmaceutical formulation or medical food.

[0319] In some embodiments, a method of increasing at least one of SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Receptor 1A (HTR1A), Insulin Receptor (INSR), Superoxide Dismutase 2 (SOD2), Artemin (ARTN), Fas Cell Surface Death Receptor (FAS), Interleukin 2 Receptor Subunit Beta (IL2Rb), Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), CD163 Molecule, and Platelet-Derived Growth Factor Receptor Alpha (PDGFRA) polypeptide expression in a subject in need thereof is provided, comprising administering to the subject an effective amount of an enhanced NAD+pharmaceutical formulation or medical food.

[0320] In some embodiments, a method of decreasing at least one of SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Receptor 1A (HTR1A), Insulin Receptor (INSR), Superoxide Dismutase 2 (SOD2), Artemin (ARTN), Fas Cell Surface Death Receptor (FAS), Interleukin 2 Receptor Subunit Beta (IL2Rb), Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), CD 163 Molecule, Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), and / or Neurofilament Light Chain (NfL) polypeptide expression in asubject in need thereof is provided, comprising administering to the subject an effective amount of an enhanced NAD+pharmaceutical formulation or medical food.

[0321] In some embodiments, the subject suffers from a neurodegenerative disease. In some embodiments, the neurodegenerative disease includes Parkinson’s disease, Multiple Sclerosis, Alzheimer’s Disease, or Amyotrophic Lateral Sclerosis.

[0322] In some embodiments, a method of changing the level of a protein involved in neurodegenerative disease is provided, the method comprising: administering to a subject a therapeutically effective amount of an enhanced NAD+pharmaceutical formulation or medical food.

[0323] In some embodiments, the protein includes SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Receptor 1A (HTR1A), Insulin Receptor (INSR), Superoxide Dismutase 2 (SOD2), Artemin (ARTN), Fas Cell Surface Death Receptor (FAS), Interleukin 2 Receptor Subunit Beta (IL2Rb), Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), CD163 Molecule, Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), and / or Neurofilament Light Chain (NfL).

[0324] In some embodiments, the change includes an increase or decrease in the level of the protein in the administered subject compared to that level in an unadministered subject. In some embodiments, the change comprises an increase or decrease in the plasma level of protein in the administered subject compared to that level in an unadministered subject.

[0325] Another aspect of the present disclosure relates to a method of treating or preventing an age- related disease in a subject, comprising administering to the subject, a therapeutically effective amount of an enhanced NAD+pharmaceutical formulation or medical food disclosed herein. As described above, such formulations or medical foods can be manufactured as a medicament or supplement for treating many NAD+deficiency-related diseases and disorders as described herein, such as, but not limited to, age-related diseases, central nervous system disorders, chemical addiction or substance abuse, such as, but not limited to, abuse related to tobacco, heroin, opium, morphine, dihydromorphine, meperidine, codeine, cocaine, amphetamines, barbiturates, alcohol, ketamine, or xylazine use.

[0326] In another aspect, the present disclosure relates to a method for treating a subject suffering from, or preventing in a subject, a central nervous system disorder or disease, comprising administering to the subject a therapeutically acceptable amount of a composition or medical food of enhanced NAD+described herein.

[0327] Yet another aspect of the present disclosure relates to a method for treating a subject suffering from chemical addiction or substance abuse, comprising administering to the subject a therapeutically effective amount of an enhanced NAD+composition or medical food described herein. In certain embodiments the substance abused is tobacco, heroin, opium, morphine, dihydromorphine, meperidine, codeine, cocaine, amphetamines, barbiturates, alcohol, or tranquilizers.

[0328] Other methods of treatment include methods for increasing NAD-dependent deacetylase sirtuin-1 (SIRT1), increasing cryptococcal phospholipase (PLB1), and for increasing membrane metalloendopeptidase (MME) in a subject in need thereof, comprising administering to the subject an amount of an enhanced NAD+pharmaceutical formulation or medical food to cure the deficiency.

[0329] The present disclosure also provides a method for treating a subject suffering from a disease or disorder modulated by SIRT1, comprising administering to the subject an amount of an enhanced NAD+composition or medical food described herein effective to modulate SIRT1.

[0330] Also provided is a method for treating a subject suffering from a disease or disorder modulated by cryptococcal phospholipase (PLB1), comprising administering to the subject an enhanced NAD+pharmaceutical formulation or medical food an amount effective to modulate PLB 1.

[0331] In addition, a method of treating a subject suffering from a disease or disorder modulated by membrane metalloendopeptidase (MME) is provided and comprises administering to the subject a therapeutically effective amount of an enhanced NAD+pharmaceutical formulation or medical food described herein.

[0332] The present disclosure also relates to a method for increasing a plasma proteome in a subject in need thereof, comprising administering to the subject in need thereof an amount of an enhanced NAD+pharmaceutical formulation or medical food described herein effective to increase the plasma proteome of the subject.

[0333] Also provided are methods of treating a subject suffering from an age-related disorder and for treating a subject suffering from a CNS disease or disorder, comprising administering to the subject a therapeutically effective amount of an enhanced NAD+pharmaceutical formulation or medical food described herein. In some embodiments, the CNS disorder is chemical addiction. In some embodiments, the CNS disorder is substance abuse.

[0334] In some embodiments, the method of treating is related to the treatment of a subject suffering from alcoholism and comprises administering to the subject a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food. In some embodiments, the disclosure relates to treatment of a subject suffering from alcohol -induced organ damage and comprises administering to the subject a therapeutically effective amount of the enhanced NAD+formulation or medical food.

[0335] In some embodiments, the disclosure relates to a method of treating a subject suffering from schizophrenia, comprising administering to the subject a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein. In some embodiments, the disclosure relates to a method of treating a subject suffering from ischemia, inflammation, sepsis, free radical damage following myocardial infarction, and / or oxidative stress, comprising administering to the subject a therapeutically effective amount of the enhanced NAD+composition described herein. In some embodiments, the disclosure relates to a method of treating a subject suffering from Alzheimer’s disease, comprising administering to the subject a therapeuticallyeffective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein. In some embodiments, the disclosure relates to a method of treating a subject suffering from excitotoxicity, ischemia, and / or oxidative stress, comprising administering to the subject an effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein.

[0336] In some embodiments the disclosure relates to a method of treating a subject suffering from pellagra, comprising administering to the subject a therapeutically effective amount of the enhanced NAD pharmaceutical formulation or medical food described herein.

[0337] In some embodiments, the disclosure relates to a method of treating a subject suffering from pseudohypoxia, comprising administering to the subject a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein.

[0338] In some embodiments, the disclosure provides a method of treating a subject suffering from hypertrophic cardiomyopathy (HCM), comprising administering to the subject a therapeutically effective amount of the enhanced NAD+ +pharmaceutical formulation or medical food described herein.

[0339] n some embodiments, the disclosure provides a method of treating a subject suffering from cellular senescence in brain aging, comprising administering to the subject a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein.

[0340] In some embodiments, the disclosure provides a method of treating a subject suffering from ischemic brain damage, comprising administering to the subject a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein.

[0341] In some embodiments, the disclosure provides a method of treating a subject suffering from a prion disease, comprising administering to the subject a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein.

[0342] In some embodiments, the disclosure provides a method of treating a subject suffering from bupivacaine -induced neurotoxicity, comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein.

[0343] The disclosure also provides a method of treating a subject suffering from arrhythmia, comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein.

[0344] In addition, the disclosure provides a method of treating a subject suffering from nicotinamide-induced mitophagy, comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein.

[0345] Additionally, the also disclosure provides a method of treating a subject suffering from ischemic reperfusion, comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein.

[0346] In further embodiments, the disclosure provides a method of treating a subject suffering from traumatic brain injury, comprising administering a therapeutically effective amount of the enhancedNAD+pharmaceutical formulation or medical food described herein.

[0347] In further embodiments, the disclosure provides a method of treating a subject suffering from synchrotron radiation X-ray-induced tissue injury, comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein.

[0348] In some embodiments, the disclosure provides a method of treating a subject suffering from autoimmune diseases, comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein.

[0349] In further embodiments, the disclosure provides a method of treating a subject suffering from diabetes, comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein.

[0350] In further embodiments, the disclosure provides a method of treating a subject suffering from cancer, comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein.

[0351] In further embodiments, the disclosure provides a method of treating a subject suffering from viral infection, comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein. In some embodiments, the viral infection is the coronavirus disease 2019 (COVID- 19) and variants thereof.

[0352] In some embodiments, the disclosure provides a method of promoting sobriety and / or satiety in a subject, comprising administering to the subject an amount of the enhanced NAD+pharmaceutical formulation or medical food described herein effective to promote sobriety and / or satiety.

[0353] In some embodiments, the disclosure provides a method of promoting cellular NAD+metabolism and NAD+cellular homeostasis in a subject, comprising administering to the subject an amount of the enhanced NAD+pharmaceutical formulation or medical food described herein effective to promote cellular NAD+metabolism.

[0354] In some embodiments, the disclosure provides a method of preventing or treating a subject suffering from or in a risk of having at least one of cardiovascular diseases, comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein. Such cardiovascular diseases may include, for example, diseases or disorders related to an abnormality in any one of lipid metabolism, vascular effects / hypertension, homocysteine and the folate cycle, oxidative stress, inflammation, resting heart rate, metabolite levels, or any combinations thereof.

[0355] In some embodiments, the disclosure provides a method of preventing or treating a subject suffering from or in a risk of having at least one of liver diseases, comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein. In some embodiments, the enhanced NAD+composition described herein is capableof improving liver functions, resulting in, for example, a decrease in serum GGT, bilirubin, ALP, and / or albumin.

[0356] In some embodiments, the enhanced NAD+composition described herein is capable of reducing at least one of metabolites such as 1 -palmitoyl -2 -arachidonoyl-GPC. In some embodiments, the enhanced NAD+pharmaceutical formulation or medical food described herein is capable of reducing oxidative stress.

[0357] The disclosure also provides a method of preventing or treating a subject suffering from or in a risk of having at least one of diseases or disorders related to an abnormality in exocrine pancreas function and / or digestion, comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein. In some embodiments, the enhanced NAD+pharmaceutical formulation or medical food pharmaceutical formulation described herein is capable of decreasing levels and / or functions of at least one of pancreatic enzymes [e.g., pancreatic lipase-related protein 1 (PNLIPRP1), pancreatic ribonuclease (RNASE 1), carboxypeptidase Al (CPA1), carboxypeptidase Bl (CPB1), etc.].

[0358] In some embodiments, the disclosure provides a method of preventing or treating a subject suffering from or in a risk of having a neurodegenerative disease such a Huntington’s disease or Alzheimer’s disease, and / or neuropsychiatric disease, comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein. Such neuropsychiatric diseases may include, for example, depression, anxiety, bipolar disorder, and schizophrenia. Such diseases or disorders may be correlated with abnormal expression levels and / or functions of at least one of proteins listed in the table in FIG. 28.

[0359] In some embodiments, the disclosure provides a method of preventing or treating a subject suffering from or in a risk of having at least one of diseases or disorders related to an abnormality in inflammation and / or immune modulation, the method comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein. Such diseases or disorders may include, for example, autoimmune diseases, including multiple sclerosis (MS), as described above, inflammatory bowel disease (IBD), and rheumatoid arthritis (RA). Such diseases or disorders may be correlated with abnormal expression levels and / or functions of at least one of proteins listed in the table in FIG. 29.

[0360] In some embodiments, the disclosure provides a method of preventing or treating a subject suffering from or in a risk of having at least one of diseases or disorders related to an abnormality in reproductive function and / or fertility, the method comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein.

[0361] In some embodiments, the disclosure provides a method of preventing or treating a subject suffering from or in a risk of having at least one of metabolic diseases (such as obesity and insulin resistance syndromes), the method comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein. In someembodiments, the enhanced NAD+composition described herein is capable of increasing expression levels and / or functions of at least one of N-acetyl-galactosaminyl -transferase 2 (GALNT2), fatty-acid binding protein 1 (FABP1), retinol binding protein 2 (RBP2), Glycohyocholic acid (GHCA), and Glyco-beta-muricholate. In some embodiments, the enhanced NAD+pharmaceutical formulation or medical food described herein is capable of decreasing expression levels and / or functions of at least one ofpyrraline, urea, glutamate, acisoga, nonadecanoate, and 1 -palmitoyl -2 -arachidonoyl-GPC.

[0362] In some embodiments, the disclosure provides a method of preventing or treating a subject suffering from or in a risk of having at least one of skin and / or bone diseases, the method comprising administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation or medical food described herein. In some embodiments, the enhanced NAD+composition or medical food described herein is capable of increasing expression levels and / or functions of SIRT1 and / or Matrilin-3 (MATN3). In some embodiments, the enhanced NAD+pharmaceutical formulation or medical food described herein decreases expression levels and / or functions of at least one of Peptidyl- prolyl cis-trans isomerase B (PPIB), whey-acidic protein 4-disulfide-core 12 (WFDC12), and insulinlike peptide 3 (INSL3).

[0363] In some embodiments, the disclosure provides a method of preventing or treating a subject suffering from post-viral long-term symptoms such as Long Covid. In other embodiments, the disclosure provides a method of preventing post-viral long-term symptoms. In some embodiments, the method comprises identifying a subject suffering from, or having a risk of suffering from, post- viral long-term symptoms such as Long Covid; and administering, e.g., orally, to the subject an amount of an enhanced NAD+pharmaceutical formulation or medical food to the subject, wherein the amount is effective to increase a level of icNAD by at least 5% in the subject, thereby mitigating or preventing post-viral long-term symptoms in the subject. In some embodiments, the composition or medical food is administered to the subject after a period of acute illness due to a viral disease such as Covid-19. In some embodiments, the subject is diagnosed based on a positive test result for SARS- CoV-2 and persistence of one or more Long Covid symptoms over a period of months. Non-limiting embodiments of Long Covid symptoms include fatigue, post-exertional malaise, fever, difficulty breathing, shortness of breath, coughing, chest pain, heart palpitations, difficulty concentrating, headache, sleep disorder, dizziness, lightheadedness, pins-and-needles feeling, change in smell or taste, depression, anxiety, diarrhea, stomach pain, joint or muscle pain, rash, and changes in menstrual cycle.Process of Preparation

[0364] In a further embodiment, the process of blending comprises mixing the pre-formulated NAD+and PEG for at least 20 minutes at room temperature. In another embodiment, the process of blending involves mixing the pre -formulated NAD+and PEG for at least 30 minutes at room temperature. In yet another embodiment, the process of blending involves mixing the pre -formulated NAD+and PEGfor at least 40 minutes at room temperature. In still another embodiment, the process of blending comprises mixing the pre -formulated NAD+and PEG for at least 50 minutes at room temperature. In another embodiment, the process of blending comprises mixing the pre -formulated NAD+and PEG for at least 1 hour at room temperature.

[0365] In a further embodiment of the disclosure, the process of preparation further comprises pre formulating commercial PEG. In one embodiment, pre-formulating the PEG comprises grinding, sieving, or milling the commercial PEG. In certain embodiments, grinding and / or milling the commercial PEG is performed to reduce the commercial PEG into particles of super fine size to allow for homogeneous distribution with excipients and the active ingredient. In certain embodiments, the mixture is processed through a sieve of 2.5 mm or other size as best to allow for homogeneous distribution and compatibility with excipients.

[0366] In yet another embodiment of the disclosure, the process of pre -formulation of PEG comprises grinding, sieving, or milling the commercial PEG and then vacuumizing the ground, sieved, or milled PEG product by itself prior to blending.

[0367] In yet another embodiment of the process of preparation, pre -formulation of PEG comprises vacuumizing the commercial PEG and then grinding, sieving, or milling the vacuumized PEG product by itself prior to blending.

[0368] In an embodiment of the present disclosure, the commercial PEG used in the preparation is GRAS under the FDA. In some embodiments, the commercial PEG used in the preparation is PEG300. In other embodiments, the commercial PEG used in the preparation is PEG400. In yet other embodiments, the commercial PEG used in the preparation is PEGMME 550. In still other embodiments, the commercial PEG used in the preparation is PEG600. In other embodiments, the commercial PEG used in the preparation is PEG 1000. In some embodiments, the commercial PEG used in the preparation is PEGMME 2000. In yet other embodiments, the commercial PEG used in the preparation is PEG3350. In certain embodiments, the commercial PEG used in the preparation is PEG4000. In other embodiments, the commercial PEG used in the preparation is PEG300, PEG400, PEGMME 550, PEG600, PEG 1000, PEGMME 2000, PEG3350, PEG4000, or any combination thereof.Medical Food Preparation

[0369] In some embodiments, the enhanced NAD+medical food can be a specialized nutritional composition formulated to meet the distinctive metabolic requirements of individuals suffering from neurodegenerative diseases / disorders and / or age-related diseases / disorders, or other conditions associated with NAD+deficiency. The medical food formulation can be designed for the dietary management of patients who require therapeutic levels of NAD+that cannot be achieved by modification of a normal diet alone. The enhanced NAD+medical food can support cellular energy metabolism, redox balance, and neuroprotection through controlled and sustained delivery ofNAD+or its precursors.

[0370] In certain embodiments, preparation of the enhanced NAD+medical food can involve vacuumizing NAD+in combination with polyethylene glycol (PEG) and optionally phosphorus, under inert gas conditions, to stabilize the NAD+content and improve its shelf life and bioavailability. The formulation can be provided in powder or liquid form, suitable for oral or enteral administration, and can be used under the supervision of a healthcare provider as part of a clinical nutrition protocol for managing specific medical conditions.

[0371] In some embodiments, the enhanced NAD+medical food can be manufactured in compliance with current Good Manufacturing Practices (cGMP), using ingredients that are Generally Recognized As Safe (GRAS). The final composition can be optimized for stability, solubility, and tolerability, and can be administered as a single or divided dose depending on the subject’s clinical needs.Labeling and instructions for use can reflect its status as a medical food, indicating its intended use under medical supervision for the dietary management of conditions with altered NAD+metabolism.Enumerated Embodiments

[0372] Embodiment 1. An enhanced nicotinamide adenine dinucleotide (NAD+) composition, comprising a crystalline component comprising crystalline polyethylene glycol, and an amorphous component comprising amorphous NAD+, wherein the composition is characterized by having a first onset temperature of between 179.39 °C (± 0.10 °C) and 182.61 °C (± 0.10 °C) by thermogravimetric analysis (TGA) thermogram.

[0373] Embodiment 2. An enhanced nicotinamide adenine dinucleotide (NAD+) composition, comprising a crystalline component comprising crystalline polyethylene glycol, and an amorphous component comprising amorphous NAD+, wherein the composition is characterized by having a weight loss from 14.39 wt% to 16.98 wt% in the range from about 170 °C to about 300 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 300 °C under nitrogen gas at a ramp rate of 10 °C / min.

[0374] Embodiment 3. An enhanced nicotinamide adenine dinucleotide (NAD+) composition, comprising a crystalline component comprising crystalline polyethylene glycol, and an amorphous component comprising amorphous NAD+, wherein the composition is characterized by having a weight loss from 57.83 wt% to 58.55 wt% in the range from about 300 °C to about 800 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 800 °C under nitrogen gas at a ramp rate of 10 °C / min.

[0375] Embodiment 4. The composition of any one of the preceding embodiments, further comprising an amorphous polyethylene glycol.

[0376] Embodiment 5. An enhanced nicotinamide adenine dinucleotide (NAD+) composition, comprising a crystalline component comprising crystalline polyethylene glycol, and an amorphous component comprising amorphous NAD+, wherein the composition is characterized by having a TGAthermogravimetric analysis (TGA) thermogram substantially in accordance with any one of FIG.33A, FIG. 33B, FIG. 34A, FIG. 34B, FIG. 35A, or FIG. 35B

[0377] Embodiment 6. The composition of embodiment 5, which is characterized by having a TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with FIG. 33 A or FIG. 33B

[0378] Embodiment 7. The composition of embodiment 5, which is characterized by having a TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with FIG. 34 A or FIG. 34B

[0379] Embodiment 8. The composition of embodiment 5, which is characterized by having a TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with FIG. 35 A or FIG. 35B

[0380] Embodiment 9. An enhanced nicotinamide adenine dinucleotide (NAD+) composition, comprising a crystalline component comprising crystalline polyethylene glycol, and an amorphous component comprising amorphous NAD+, wherein the composition is characterized by having a TGA thermogravimetric analysis (TGA) thermogram having: a first weight loss from 2.42 wt% to 3.25 wt% in the range from ambient temperature to about 170 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 170 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or a second weight loss from 13.66 wt% to 14.61 wt% in the range from about 170 °C to about 300 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 300 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or a third weight loss from 58.73 wt% to 60.37 wt% in the range from about 300 °C to about 800 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 800 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or a fourth weight loss from 20.14 wt% to 21.46 wt% in the range from about 800 °C to about 900 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at about 800 °C under nitrogen gas at a ramp rate of 10 °C / min and from about 800 °C to about 900 °C under air at a ramp rate of 10 °C / min, and / or a first onset temperature from 179.39 °C to 180.15 °C, and / or a second onset temperature from 369.03 °C to 370.82 °C.

[0381] Embodiment 10. An enhanced nicotinamide adenine dinucleotide (NAD+) composition, comprising a crystalline component comprising crystalline polyethylene glycol, and an amorphous component comprising amorphous NAD+, wherein the composition is characterized by having a TGA thermogravimetric analysis (TGA) thermogram having: a first weight loss from 2.32 wt% to 3.55 wt% in the range from ambient temperature to about 170 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 170 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or a second weight loss from 14.39 wt% to 16.22 wt% in the range from about 170 °C to about 300 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 300 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or a third weightloss from 57.83 wt% to 58.55 wt% in the range from about 300 °C to about 800 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 800 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or a fourth weight loss from 22.62 wt% to 22.76 wt% in the range from about 800 °C to about 900 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at about 800 °C under nitrogen gas at a ramp rate of 10 °C / min and from about 800 °C to about 900 °C under air at a ramp rate of 10 °C / min, and / or a first onset temperature from 178.62 °C to 180.11 °C, and / or a second onset temperature from 365.73 °C to 370.47 °C.

[0382] Embodiment 11. An enhanced nicotinamide adenine dinucleotide (NAD+) composition, comprising a crystalline component comprising crystalline polyethylene glycol, and an amorphous component comprising amorphous NAD+, wherein the composition is characterized by having a TGA thermogravimetric analysis (TGA) thermogram having: a first weight loss from 3.62 wt% to 3.96 wt% in the range from ambient temperature to about 170 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 170 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or a second weight loss from 15.17 wt% to 16.98 wt% in the range from about 170 °C to about 300 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 300 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or a third weight loss from 58.05 wt% to 59.66 wt% in the range from about 300 °C to about 800 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 800 °C under nitrogen gas at a ramp rate of 10 °C / min, and / or a fourth weight loss from 22.85 wt% to 25.87 wt% in the range from about 800 °C to about 900 °C by thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at about 800 °C under nitrogen gas at a ramp rate of 10 °C / min and from about 800 °C to about 900 °C under air at a ramp rate of 10 °C / min, and / or a first onset temperature from 179.83 °C to 182.61 °C, and / or a second onset temperature from 363.24 °C to 366.16 °C.

[0383] Embodiment 12. The composition of any of the preceding embodiments, wherein the ratio of NAD+to polyethylene glycol in the composition is 1: 1 (wt / wt).

[0384] Embodiment 13. The composition of any of the preceding embodiments, wherein the polyethylene glycol has an average molecular weight of between about 300 and about 4000.

[0385] Embodiment 14. The composition of any of the preceding embodiments, wherein the polyethylene glycol is PEG3350.

[0386] Embodiment 15. The composition of any of the preceding embodiments, which is in powder form.

[0387] Embodiment 16. The composition of any of the preceding embodiments, which is suitable for oral administration to a subject.

[0388] Embodiment 17. The composition of any of the preceding embodiments, wherein the composition has a shelflife of at least 6 months at room temperature.

[0389] Embodiment 18. The composition of any of the preceding embodiments, wherein the composition has a shelflife of at least 1 year at room temperature.

[0390] Embodiment 19. A pharmaceutical formulation comprising a composition of any one of embodiments 1-18.

[0391] Embodiment 20. The formulation of embodiment 19, which, when administered to a subject, increases NAD levels in the subject by at least about 10% relative to a baseline level of intracellular NAD in the subject.

[0392] Embodiment 21. The formulation of embodiment 19 or 20, which, when administered to a subject, increases intracellular levels of NAD in the subject.

[0393] Embodiment 22. The formulation of any one of embodiments 19-21, which, when administered to a subject, modulates expression of at least one biomarker in the subject.

[0394] Embodiment 23. The formulation of any one of embodiments 19-22, for use in a method of mitigating chemical addiction withdrawal symptoms in a subject in need thereof; mitigating opioid withdrawal symptoms in a subject in need thereof; increasing nicotinamide adenine dinucleotide (NAD) levels in a subject; and / or preventing or treating a disease or disorder correlated to decreased levels of NAD in a subject in need thereof.

[0395] Embodiment 24. A kit comprising the composition of any one of embodiments 1-18.

[0396] Embodiment 25. A method of preparing the enhanced NAD+composition of any one of embodiments 1-18, comprising (1) subjecting NAD+to at least one vacuumization cycle to form vacuumized NAD+and (2) blending vacuumized NAD+with polyethylene glycol to form a blended mixture.

[0397] Embodiment 26. The method of embodiment 25, wherein the inert gas comprises argon or nitrogen.

[0398] Embodiment 27. The method of embodiment 25 or 26 wherein step (1) comprises subjecting NAD+to at least three vacuumization cycles.

[0399] Embodiment 28. The method of any one of embodiments 25-27, wherein the polyethylene glycol has an average molecular weight of between about 300 and about 4000.

[0400] Embodiment 29. The method of any one of embodiments 25-28, wherein the polyethylene glycol is PEG3350.

[0401] Embodiment 30. The method of any one of embodiments 25-29, further comprising subjecting the blended mixture to at least one additional vacuumization cycle.

[0402] Embodiment 31. The method of any one of embodiments 25-29, further comprising subjecting the blended mixture to at least three additional vacuumization cycles.

[0403] Embodiment 32. The method of any one of embodiments 25-29, further comprising subjecting the blended mixture to at least six additional vacuumization cycles.

[0404] Embodiment 33. The method of any one of embodiments 25-32, further comprising pretreating the NAD+prior to step (1).

[0405] Embodiment 34. The method of embodiment 33, wherein pretreating the NAD+comprises passing the NAD+through a sieve, and / or grinding, milling, or jet-milling the NAD+.

[0406] Embodiment 35. An enhanced NAD+composition prepared by the method of any one of embodiments 25-34.

[0407] Embodiment 36. A method of making an enhanced NAD+pharmaceutical formulation, comprising mixing the enhanced NAD+composition of any of embodiments 1-18 with and at least one ingredient or excipient that facilitates administration to a subject and / or which can further treat the subject’s disease or condition.

[0408] Embodiment 37. A method of mitigating chemical addiction withdrawal symptoms in a subject in need thereof, the method comprising: administering a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation of any one of embodiments 19-23 to the subject, the amount being effective to mitigate chemical addiction withdrawal symptoms in the subject.

[0409] Embodiment 38. The method of embodiment 36 or 37, wherein the therapeutically effective amount increases the level of NAD in the subject by at least 5%.

[0410] Embodiment 39. The method of any one of embodiments 36-38, wherein the amount of the pharmaceutical formulation administered is effective to mitigate opioid withdrawal symptoms such that the client maintains a Clinical Opioid Withdrawal Scale (COWS) score of 12 or lower.

[0411] Embodiment 40. The method of any one of embodiments 37-39, wherein the level of NAD increased in the subject is an intracellular level of NAD.

[0412] Embodiment 41. The method of any one of embodiments 37-40, wherein an intracellular level of NAD is increased to greater than about 35 pM.

[0413] Embodiment 42. The method of any one of embodiments 37-41, wherein the level of NAD increased in the subject is an extracellular level of NAD.

[0414] Embodiment 43. The method of any one of embodiments 37-42, wherein the administration of the enhanced NAD+pharmaceutical formulation is effective to increase a plasma level of N- methyl-nicotinamide (MeNAM) or N-methyl-2-pyridone-5-carboxamide (2PY) by at least 5% in the subject.

[0415] Embodiment 44. The method of any one of embodiments 37-43, wherein the amount of NAD+in the pharmaceutical formulation administered to the subject is from about 5 mg to about 4000 mg.

[0416] Embodiment 45. The method of any one of embodiments 37-44, wherein the amount of the pharmaceutical formulation administered comprises a maximum daily dose of about 1 mg per kg to about to about 100 mg / kg body weight of the subject.

[0417] Embodiment 46. The method of any one of embodiments 37-45, further comprising administering to the subject a pharmaceutically effective amount of an analgesic or a non-steroidal anti-inflammatory drug (NSAID).

[0418] Embodiment 47. The method of any one of embodiments 37-45, wherein the pharmaceuticalformulation further comprises a pharmaceutically effective amount of an analgesic or a non-steroidal anti-inflammatory drug (NSAID).

[0419] Embodiment 48. The method of any one of embodiments 37-47 further comprising monitoring at least one vital sign of the subject before and after administration of the pharmaceutical formulation.

[0420] Embodiment 49. The method of any one of embodiments 37-48, further comprising measuring an intracellular NAD or extracellular concentration of NAD in the subject before administration and after administration of the pharmaceutical formulation.

[0421] Embodiment 50. The method of embodiment 49, further comprising selecting a dosage of the composition to be administered to the subject based on the measured baseline intracellular concentration of NAD or extracellular concentration of NAD in the subject.

[0422] Embodiment 51. A method of increasing nicotinamide adenine dinucleotide (NAD) levels in a subject, the method comprising: administering an amount of the enhanced NAD+pharmaceutical formulation of any one of embodiments 19-23 or medical food effective to increase NAD levels in the subject.

[0423] Embodiment 52. The method of embodiment 51, wherein the subject is a healthy adult.

[0424] Embodiment 53. A method of preventing or treating a disease or disorder correlated to decreased levels of NAD in a subject in need thereof, the method comprising: administering an amount of the enhanced NAD+pharmaceutical formulation of any one of embodiments 19-23 effective to prevent or treat the disease or disorder in the subject.

[0425] Embodiment 54. A method of treating a subject suffering from a chemical addiction, comprising administering to the subject a pharmaceutically effective amount of the enhanced NAD+pharmaceutical formulation of any one of embodiments 19-23.

[0426] Embodiment 55. The method of embodiment 54, wherein the chemical is tobacco, heroin, opium, morphine, dihydromorphine, meperidine, codeine, cocaine, amphetamines, barbiturates, alcohol, tranquilizers, or opioids.

[0427] Embodiment 56. A method of promoting cellular NAD metabolism or NAD cellular homeostasis in a subject, comprising administering to the subject an amount of the enhanced NAD+pharmaceutical formulation of any one of embodiments 19-23 effective to promote NAD metabolism or NAD cellular homeostasis.

[0428] Embodiment 57. A method of treating a patient suffering from at least one condition of metabolic syndrome, comprising administering to the subject a therapeutically effective amount of the enhanced NAD+pharmaceutical formulation of any one of embodiments 19-23, wherein the at least one condition is increased blood pressure, high blood sugar, excess body fat around the waist, abnormal cholesterol or triglyceride levels, or any combination thereof.

[0429] Embodiment 58. A method of increasing at least one of SIRT1, PLB 1, NPL, ENPP5, GLSTA, GALNT3, FABP1, NRCAM, NLGN2, ARTN, UPB1, MME, CTSB, CTSL, or HRASpolypeptide in a subject in need thereof, comprising administering to the subject an amount of the enhanced NAD+pharmaceutical formulation of any one of embodiments 19-23 effective to increase SIRT1, PLB1, NPL, ENPP5, GLSTA, GALNT3, FABP1, NRCAM, NLGN2, ARTN, UPB1, MME, CTSB, CTSL, or HRAS polypeptide.

[0430] Embodiment 59. A method of preparing the enhanced NAD+composition of any one of embodiments 1-18, comprising (1) blending NAD+with polyethylene glycol to form a blended mixture, and (2) subjecting the blended mixture to at least one vacuumization cycle, wherein each vacuumization cycle comprises placement in a container under vacuum and back-fdling the container with an inert gas or CO2.

[0431] Embodiment 60. A method of preparing an enhanced NAD+composition comprising a crystalline component comprising crystalline polyethylene glycol, and an amorphous component comprising amorphous NAD+, the method comprising (1) blending NAD+with polyethylene glycol to form a blended mixture, and (2) subjecting the blended mixture to at least one vacuumization cycle, wherein each vacuumization cycle comprises placement in a container under vacuum and back-fdling the container with an inert gas or CO2.

[0432] Embodiment 61. The method of embodiment 59 or 60, wherein the inert gas comprises argon or nitrogen.

[0433] Embodiment 62. The method of one of embodiments 59-61 wherein step (2) comprises at least three vacuumization cycles.

[0434] Embodiment 63. The method of any one of embodiments 59-61, wherein step (2) comprises at least six vacuumization cycles.

[0435] Embodiment 64. The method of any one of embodiments 59-63, wherein the polyethylene glycol has an average molecular weight of between about 300 and about 4000.

[0436] Embodiment 65. The method of any one of embodiments 59-64, wherein the polyethylene glycol is PEG3350.

[0437] Embodiment 66. The method of any one of embodiments 59-65, further comprising pretreating the NAD+prior to step (1).

[0438] Embodiment 67. The method of embodiment 66, wherein pretreating the NAD+comprises passing the NAD+through a sieve, and / or grinding, milling, or jet-milling the NAD+.

[0439] Embodiment 68. The method of any one of embodiments 59-67, wherein the NAD+is subjected to at least one vacuumization cycle prior to step (1).

[0440] Embodiment 69. An enhanced NAD+composition prepared by the method of any one of embodiments 59-68.

[0441] Embodiment 70. A method of preventing or treating a neurodegenerative disease in a subject, the method comprising administering a therapeutically effective amount of an enhanced NAD+pharmaceutical formulation or medical food to the subject.

[0442] Embodiment 71. The method of embodiment 70, wherein the neurodegenerative disease isParkinson’s disease, Multiple Sclerosis, Alzheimer’s Disease, or Amyotrophic Lateral Sclerosis.

[0443] Embodiment 72. The method of embodiment 70 or embodiment 71, wherein the administration changes a level of a protein involved in neurodegenerative disease.

[0444] Embodiment 73. The method of embodiment 72, wherein the change comprises an increase in the level of a protein involved in neurodegenerative disease compared to the level in an unadministered subject.

[0445] Embodiment 74. The method of embodiment 72, wherein the change comprises a decrease in the level of a protein involved in neurodegenerative disease compared to the level in an unadministered subject.

[0446] Embodiment 75. The method of any one of embodiments 72 to 74, wherein the administration comprises a change in the plasma level of the protein involved in the neurodegenerative disorder, as compared to the plasma level in an unadministered subject.

[0447] Embodiment 76. The method of any one of embodiments 70 to 74, wherein the protein is selected from the group consisting of SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Receptor 1A (HTR1A), Insulin Receptor (INSR), Superoxide Dismutase 2 (SOD2), Artemin (ARTN), Fas Cell Surface Death Receptor (FAS), Interleukin 2 Receptor Subunit Beta (IL2Rb), Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), CD163 Molecule, Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), and / or Neurofilament Light Chain (NfL).

[0448] Embodiment 77. The method of any one of embodiments 70 to 76, wherein the disease is Parkinson’s disease and the protein is selected from the group consisting of SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), C- X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Receptor 1A (HTR1A), Insulin Receptor (INSR), and Neurofilament Light Chain (NfL).

[0449] Embodiment 78. The method of any one of embodiments 70 to 76, wherein the disease is Multiple Sclerosis and the protein is selected from the group consisting of SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Fas Cell Surface Death Receptor (FAS), Interleukin 2 Receptor Subunit Beta (IL2Rb), Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), CD163 Molecule, and Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), Matrix Metallopeptidase 9 (MMP9), and Neurofdament Light Chain (NfL).

[0450] Embodiment 79. The method of any one of embodiments 70 to 76, wherein the disease is Amyotrophic Lateral Sclerosis and the protein is selected from the group consisting of SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), Superoxide Dismutase 2 (SOD2), Artemin (ARTN), and Neurofdament Light Chain (NfL).

[0451] Embodiment 80. The method of any one of embodiments 70 to 79, wherein the subject is ahuman.

[0452] Embodiment 81. A method of changing the level of at least one of SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5- Hydroxytryptamine Receptor 1A (HTR1A), Insulin Receptor (INSR), Superoxide Dismutase 2 (SOD2), Artemin (ARTN), Pas Cell Surface Death Receptor (FAS), Interleukin 2 Receptor Subunit Beta (IL2Rb), Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), CD163 Molecule, Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), and / or Neurofilament Light Chain (NfL) in a subject in need thereof, comprising administering to the subject an effective amount of an enhanced NAD+pharmaceutical formulation or medical food.

[0453] Embodiment 82. The method of embodiment 81, wherein the subject suffers a neurodegenerative disease.

[0454] Embodiment 83. The method of embodiment 82, wherein the neurodegenerative disease is Parkinson’s disease, Multiple Sclerosis, Alzheimer’s Disease, or Amyotrophic Lateral Sclerosis.

[0455] Embodiment 84. The method of any one of embodiments 81 to 83, wherein the change comprises a change in the plasma level of the protein as compared to the plasma level of the protein in an unadministered subject.

[0456] Embodiment 85. The method of any one of embodiments 81 to 83, wherein the change comprises an increase in the level of the protein, as compared to the level of the protein in an unadministered subject.

[0457] Embodiment 86. The method of any one of embodiments 81 to 83, wherein the change comprises a decrease in the level of the protein, as compared to the level of the protein in an unadministered subject.

[0458] Embodiment 87. The method of any one of embodiments 70 to 86, wherein the subject is a human.

[0459] Embodiment 88. A method of treating Amyotrophic Lateral Sclerosis (ALS) comprising administering the enhanced NAD+pharmaceutical formulation or medical food of any one of the preceding embodiments, wherein the formulation or medical food achieves therapeutic NAD+concentrations in spinal cord tissue within 2 hours of administration.

[0460] Embodiment 89. The method of Embodiment 88, wherein the administering modulates oxidative stress markers in spinal cord tissue.

[0461] Embodiment 90. The method of Embodiment 89, wherein the modulating is increasing GSH levels and GSH / GSSG ratio and / or GSST activity.

[0462] Embodiment 91. The method of any one of Embodiments 88 to 90, wherein the administration results in functional preservation of neuromuscular integrity as measured by electrophysiological parameters.

[0463] Embodiment 92. The method of any of Embodiments 88 to 91, wherein the administrationresults in approximately 18% reduction in CMAP amplitude deterioration over a 30-day treatment period.

[0464] Reference will now be made to specific examples illustrating the disclosure. It is to be understood that the examples are provided to illustrate exemplary embodiments and that no limitation to the scope of the disclosure is intended thereby.EXAMPLESExample 1: Process for the Preparation of Enhanced NAD+Composition

[0465] Pre-treated (ground, milled or jet-milled) NAD+and PEG including particle sizing and “vacuumizing” were weighed and combined into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids. The Blender was tightly sealed. The mixture was blended for a minimum of 20 minutes. The blended NAD+was then vacuumized for 3 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+under vacuum and replacing the vacuum with Xenon, Nitrogen, Argon, or Helium. The blended, enhanced vacuumized product was then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which was size-matched to the product volume. The liner bag was then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.Example 1A: Process for the Preparation of Enhanced NAD+Composition

[0466] Pre-treated (ground, milled or jet-milled) NAD+and PEG including particle sizing and “vacuumizing” were weighed and combined into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids. The Blender was tightly sealed. The mixture was blended for a minimum of 20 minutes. The blended NAD+was then vacuumized for 3 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+under vacuum and replacing the vacuum with Nitrogen. The blended, enhanced vacuumized product was then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which was size-matched to the product volume. The liner bag was then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.Example IB: Process for the Preparation of Enhanced NAD+Composition

[0467] Pre-treated (ground, milled or jet-milled) NAD+and PEG including particle sizing and “vacuumizing” are weighed and combined into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids. The Blender is tightly sealed. The mixture is blended for a minimum of 20 minutes. The blended NAD+is then vacuumized for 2 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+under vacuum and replacing the vacuum with Xenon, Nitrogen, Argon, or Helium. The blended, enhanced vacuumized product is then dispensed into a bag by the dispensing port to the bottom while simultaneously nestingthe bag into a container which is size-matched to the product volume. The liner bag is then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.Example 2: Process for Preparation of Vacuumized NAD+

[0468] Commercial NAD+was placed into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids. The Blender was tightly sealed. The NAD+was blended for a minimum of 20 minutes. The blended NAD+was then vacuumized for 3 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+under vacuum and replacing the vacuum with Xenon, Nitrogen, Argon, or Helium. The blended, enhanced vacuumized NAD+product was then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which was size-matched to the NAD+product volume. The liner bag was then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.Example 2A: Process for Preparation of Vacuumized NAD+

[0469] Commercial NAD+was placed into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids. The Blender was tightly sealed. The NAD+was blended for a minimum of 20 minutes. The blended NAD+was then vacuumized for 3 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+under vacuum and replacing the vacuum with Nitrogen. The blended, enhanced vacuumized NAD+product was then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which was size-matched to the NAD+product volume. The liner bag was then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.Example 2B: Process for Preparation of Vacuumized NAD+

[0470] Commercial NAD+is placed into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids. The Blender is tightly sealed. The NAD+is blended for a minimum of 20 minutes. The blended NAD+is then vacuumized for 2 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+under vacuum and replacing the vacuum with Xenon, Nitrogen, Argon, or Helium. The blended, enhanced vacuumized NAD+product is then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which is size-matched to the NAD+product volume. The liner bag is then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.Example 3: Process for Preparation of Vacuumized PEG

[0471] Pre-qualified commercial PEG was placed into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids. The Blender was tightly sealed. The preparation was blended for a minimum of 20 minutes. The blended NAD+was then vacuumized for3 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+under vacuum and replacing the vacuum with Xenon, Nitrogen, Argon, or Helium. The blended, enhanced vacuumized product was then dispensed into the bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which was size-matched to the product volume. The liner bag was then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.Example 3B: Process for Preparation of Vacuumized PEG

[0472] Pre-qualified commercial PEG was placed into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids. The Blender was tightly sealed. The preparation was blended for a minimum of 20 minutes. The blended NAD+was then vacuumized for 3 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+under vacuum and replacing the vacuum with Nitrogen. The blended, enhanced vacuumized product was then dispensed into the bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which was size-matched to the product volume. The liner bag was then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.Example 3B: Process for Preparation of Vacuumized PEG

[0473] Pre-qualified commercial PEG is placed into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids. The Blender is tightly sealed. The preparation is blended for a minimum of 20 minutes. The blended NAD+is then vacuumized for 2 cycles, which each cycle lasting at least 20 minutes and comprising placing the blended NAD+under vacuum and replacing the vacuum with Xenon, Nitrogen, Argon, or Helium. The blended, enhanced vacuumized product was then dispensed into the bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which is size-matched to the product volume. The liner bag is then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.Example 4: Process for the Preparation of Enhanced NAD+Composition

[0474] Pre-treated (ground, milled or jet-milled) NAD+at 50% NAD+G / 100 g and PEG including particle sizing and “vacuumizing” were weighed and combined into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids. The Blender was tightly sealed. The mixture was blended for a minimum of 20 minutes. The blended mixture was placed under vacuum and the vacuum was replaced with Xenon, Nitrogen, Argon, or Helium, and the mixture was then vacuumized for 3 cycles lasting at least 20 minutes each. The blended, enhanced vacuumized product was then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which was size-matched to the product volume. The liner bag was then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.Example 4A: Process for the Preparation of Enhanced NAD+Composition

[0475] Pre-treated (ground, milled or jet-milled) NAD+at 50% NAD+G / 100 g and PEG including particle sizing and “vacuumizing” were weighed and combined into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids. The Blender was tightly sealed. The mixture was blended for a minimum of 20 minutes. The blended mixture was placed under vacuum and the vacuum was replaced with Nitrogen and the mixture was then vacuumized for 3 cycles lasting at least 20 minutes each. The blended, enhanced vacuumized product was then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which was size-matched to the product volume. The liner bag was then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.Example 4B: Process for the Preparation of Enhanced NAD+Composition

[0476] Pre-treated (ground, milled or jet-milled) NAD+at 50% NAD+G / 100 g and PEG including particle sizing and “vacuumizing” are weighed and combined into a clean Cone Blender through a sieve to disperse clumps and remove any metallic or other solids. The Blender is tightly sealed. The mixture is blended for a minimum of 20 minutes. The blended mixture is placed under vacuum and the vacuum is replaced with Xenon, Nitrogen, Argon, or Helium, and the mixture is then vacuumized for 2 cycles lasting at least 20 minutes each. The blended, enhanced vacuumized product is then dispensed into a bag by the dispensing port to the bottom while simultaneously nesting the bag into a container which is size-matched to the product volume. The liner bag is then zip-tied prior to sealing the container with a secure, airtight lid for storage or shipping.Example 5: Testing of Biological Activity of an Enhanced NAD+ / PEG Composition

[0477] A colorimetric assay that measures NAD+and NADH present in biological samples was performed. To measure NAD+, a cell lysate sample was added to a microcentrifuge tube, combined with 0.1N HC1, and mixed thoroughly. The tube was incubated at 80 °C and protected from light. Assay Buffer was added to the tube to shift the pH of the sample back to neutral. Sample pH was adjusted to between 6.0 and 8.0, accordingly with acid or base.

[0478] 50 pL of each NAD+standard or unknown sample was added into wells of a 96-well microtiter plate. 50 pL of NAD+Cycling Reagent was added to each well. Contents of the wells were mixed thoroughly and incubated for 1-4 hours at room temperature and protected from light. This assay is continuous (not terminated) and therefore may be measured at multiple time points to follow the reaction kinetics. The assay may be stopped at a desired time point by adding 50 pL 0.5 N H2SO4. The plate was read with a spectrophotometric microplate reader at 450 nm. The concentration of NAD / NADH was calculated within samples by comparing the sample optic density (OD) to a standard curve.

[0479] Intracellular increase in NAD following NAD+treatment was compared to placebo treatment (see Table 1 in Example 6).Example 6: Multiomic Characterization of the Effects of Oral Enhanced NAD+in a Wellness Cohort

[0480] The present disclosure provides for delivery of an enhanced NAD+pharmaceutical formulation to target cells to treat substance abuse including, but not limited to, drug or alcohol addiction. As used herein “Enhanced NAD+” refers to an enhanced NAD+pharmaceutical formulation according to the instant disclosure. The NAD+is delivered as an oral NAD+pharmaceutical formulation that is chemically stable, bioavailable, and pharmacologically active.

[0481] A Research Study was conducted to investigate the multi-omic effects of oral enhanced Nicotinamide adenine dinucleotide (NAD+) in a constrained “wellness cohort” composed of healthy individuals.

[0482] The study followed a double-blind placebo-controlled design with repeated measures. Enrolled 60 participants randomized approximately equally to the enhanced NAD+or the placebo group (z.e., 30 enhanced NAD+, 30 placebo). The study sample size was determined by a preliminary analysis of statistical power carried out in G* Power 3.0 (Faul, Erdfelder, Lang, & Buchner, 2007) taking the number of blood draws and anticipated effect sizes into account, and allowing the study to attain 80% power to detect (at a nominal 5% significance level) large as well as medium-size effects.

[0483] During the study, up to five 500 mg doses were given to each participant per day, for a maximum daily dose of 2500 mg / day. The range of participant weights spans 100-300 lbs. (45.4- 136.1 kg.), this corresponds to a maximum daily dose of 18.4-55.1 mg / kg / day.Dosins Schedule

[0484] Study Days 1-7: wash-out period; Study Days 8-12: treatment dose administration; Study Day 13: Post-Study.

[0485] Following a 7-day wash-out period, participants were administered enhanced NAD+(treatment A) or placebo (treatment B) for five (5) days following a q.i.d. schedule, with four 500 mg daily doses spread approximately 3-4 hours apart within a 10-16-hour window. On Days 9 and 10 an additional dose of 500 mg was administered at night, just before bedtime. The enhanced NAD+, as well as the matching placebo, met the FDA criteria for administration in humans (including compliance with GMP regulations).

[0486] Each dose of enhanced NAD+was delivered by oral administration as a swish and swallow. The first dose on Day 8 was administered following 12h of fasting. Participants were asked to record the date and time of each administration. Participants also agreed not to significantly change their diet or to discontinue the use of supplements while participating in the study.

[0487] A designated study coordinator actively monitored dose administration and blood drawcompliance with the assistance of technology that allows the participant to receive reminders, and provide quick responses to questions pertaining to the ongoing study protocol.

[0488] Dose Administration Instructions: Doses were administered with 3-4 hours between doses. If a dose was missed, it was taken within 2 hours of the scheduled dose; another 2 hours were allowed to pass prior to resuming the dose schedule such that all the doses for that day were completed. On Days 9 and 10 there was a 5th dose to be taken at least 2 hours after the 4th dose (see Table 1 for daily dosing schedule on days 8-12).Table 1*Dose administered on day 9 and day 10 only.

[0489] Method of formulating and administering the enhanced NAD+for administration:Added dry powder contents of bottle to about 4 ounces ('A cup) of water immediately before taking. Stirred well.After stirring, enhanced NAD+was administered via small sips of the prepared solution, swished in mouth for approximately 45 seconds, then swallowed.Repeated swish and swallow until all prepared solution was swallowed (approximately 3 to 5 sips).

[0490] Inclusion Criteria:English-speaking adults in the age range between 45 and 75 years in overall self-reported good health (excluding common conditions such as cardiovascular disease, hypertension, and hyperlipidemia);Participants should be able to consent for themselves to join this research study;Able to commit to the study protocol, including a 7-day washout of potentially confounding substances via restriction of supplement intake;Able to commit to repeated venous blood draws, dose administration, and assessment schedule, tolerates wrist-worn wearable devices well (z.e., consistent use during the day as well as at night for the duration of the study).

[0491] Exclusion Criteria:Body Mass Index (BMI) > 35 kg / m2;Current chronic or acute infectious diseases (e.g., hepatitis, influenza, HIV, Lyme; COVID-19 within the past two months);Proximity to SARS-CoV-2 vaccine administration (within two weeks); current use of NAD+supplements and precursors;Use of systemic anti-inflammatory medications (excluding NSAIDs), immunosuppressants; Current cancer or hematologic conditions and / or cancer treatment (radiation, chemotherapy); Type I juvenile or Type 2 diabetes mellitus;Autoimmune disorders (multiple sclerosis, lupus, rheumatoid arthritis, psoriasis); Inflammatory bowel disease (ulcerative colitis, Crohn’s disease);Current pregnancy;Current substance abuse, including alcohol, but excluding nicotine and prescription medications (e.g., stimulants, opiates);Members of the same household.

[0492] Biospecimens: Blood draws were performed on study Days 3, 7, 10, and 13. Venous blood (up to 30 ml) was collected using venipuncture. Blood was collected twice at baseline (Day 3 or 4 of the washout period and before administration of study dose, and Day 7 of the study before administration of the treatment dose) to establish an estimate of baseline intracellular NAD levels. Upon commencement of dose administration on Day 8, blood was collected on Day 10 and on Day 13. Plasma or serum were isolated using standard centrifugation protocols, frozen, and stored until use at -80° C. Whole blood or isolated fractions were processed for further assaying using the manufacturers’ recommended practices for sample handling, randomization, and plating, where applicable.Assays

[0493] NAD Concentration Measurements: Extracellular serum NAD and intracellular NAD concentrations were measured by Jinfmiti Precision Medicine (“Jinfmiti”) per manufacturer’s protocols.

[0494] Clinical Laboratory Tests: Blood testing of Complete Blood Count (CBC) with differential, Comprehensive Metabolic Panel (CMP), and hs-CRP were performed. Vitals were collected at each blood draw, including blood pressure, heart rate, waist circumference, and weight. Height was measured at the baseline blood draw as well as collected directly from the participant as part of the intake assessment.

[0495] Targeted Proteomics: Proteomics were performed using Olink’s Proximity Extension Assays (PEA) targeted proteomics Explore 3,072 platform. PEA uses pairs of matched antibodies attached to unique DNA nucleotides to generate templates for DNA polymerase -dependent extension. It uses a next-generation sequencing read-out to provide specific estimates of protein abundance.

[0496] Untargeted Metabolomics: Metabolomics were performed using Metabolon’s ultra-high- performance liquid chromatography / tandem mass spectrometry (UHPLC / MS / MS) Global Platform, detecting approximately 1,000 small metabolites. The metabolites represent super-pathways: Lipids(449), Amino Acid (183), Xenobiotics (91), Nucleotide (35), Peptide (31), Cofactors / Vitamins (23), Carbohydrate (22), Partially Characterized (6), with >200 metabolites unassigned.

[0497] Participant-reported outcomes and wearables-derived data: Physical and mental wellbeing were assessed at Day 7, Day 10, and Day 13 using questionnaires. The administered assessments included Depression, Anxiety, and Stress Scale-21 (DASS-21; Lovibond & Lovibond, Manual for the Depression Anxiety Stress Scales (2nd. Ed.) Sydney: Psychology Foundation (1995)); Clinically Useful Anxiety Outcome Scale - Daily version (CUXOS-D; Zimmerman et al., Comprehensive Psychiatry 90:37-42 (2019)); and Daily Fatigue Impact Scale (D-FIS; Fisk & Doble QualLife Res. ll(3):263-272 (2002)).

[0498] Wearables: Activity, sleep quality, and cardiovascular fitness were monitored for one week prior to the treatment dose administration and for the duration of the study using Fitbit™ wearable devices distributed to participants. Average heart rate in BPM, heart rate variability (HRV), sleep duration (overall and by stages) and quality, physical activity metrics were collected using these devices.Statistical Analysis

[0499] Data analysis, data quality assurance and control, as well as hypothesis testing were performed using Python and R. Robust mixed ANCOVAs and mixed linear models (MLM) as the statistical approaches used to evaluate the study data.Results

[0500] Following the 5-day dose administration protocol in the treatment (A) but not the placebo (B) group, induction of intracellular NAD was observed (Table 2).Table 2

[0501] An increase in plasma abundance of NAD+metabolites was also observed (FIG. 15). Six metabolites were analyzed (2PY, 1 -methylnicotinamide, nicotinate, quinolinate, nicotinamide, N’- methylnicotinate), and two showed substantial elevation in plasma, consistent with preparation for excretion by kidneys: 1 -methylnicotinamide (MeNAM; P=7.59* 10'11) (FIG. 15A) and Nl-methyl-2- pyridone-5 -carboxamide (2PY; P=1.03x 1013) (FIG. 15B). These findings survived strict multiple comparisons corrections. The data suggest the metabolic fate of MeNAM and 2PY, two major metabolites of NAD+, following enhanced NAD+oral dose administration.

[0502] Enhanced NAD+administration induced changes in amino acid metabolism, glycolysis, TCAcycle, and fatty acid oxidation (FIG. 16) observed through metabolic evidence for enzymatic activity in liver, kidney, and colon.

[0503] Non-negligible pathway activation and enzymatic reaction enhancement was observed as evidence favoring bioavailability of administered enhanced NAD+dose. These results show that NAD+supplementation in the cohort resulted in amplification of the urea cycle (likely via the xanthine component given that hypoxanthine is an intermediate metabolite), facilitation of pyruvate «-> lactate conversion contributing to glycolysis, increased lipid oxidation (observed in lipid fractions decreasing while bile acid metabolites increase), as well as increase in glutamate «-> glutamine interconversion (FIGS. 8-10).Example 7: Multiomic Biomarker Study of the Effects of Oral Enhanced NAD+

[0504] Further study was performed following Example 6 to show that an illustrative oral enhanced NAD+pharmaceutical formulation modulates levels of various biomarkers related in several therapeutic areas, where statistically significant differences between treatment and placebo group were observed, including, but not limited to, cardiovascular diseases, liver diseases, exocrine pancreas / digestion, neurodegenerative / neuropsychiatric disease, inflammation / immune function, reproduction, metabolic disease and obesity, and skin and bone health.

[0505] Cardiovascular diseases - the enhanced NAD+composition led to improvements in markers of lipid metabolism, vascular / endothelial health and injury resistance, and oxidative stress.

[0506] Liver diseases - the enhanced NAD+composition led to improvements in several liver enzymes, suggesting improved liver functioning. The improvements in oxidative stress markers also have relevance for common liver diseases such as non-alcoholic fatty liver disease (NAFLD).

[0507] Exocrine pancreas / digestion - the enhanced NAD+composition differentially decreased circulating levels of four pancreatic enzymes.

[0508] Neurodegenerative / neuropsychiatric diseases - the enhanced NAD+composition differentially increased levels of 6 proteins with known neurological effects and decreased levels of 6 other proteins. Several of the proteins altered by the enhanced NAD+composition treatment have been previously associated with neurodegenerative diseases, depression, anxiety, or chronic pain. The observed improvements in oxidative and nitrosative stress also have relevance in neurodegenerative disease.

[0509] Inflammation / immune function - There were no differences seen between groups in clinical inflammatory markers such as hsCRP. However, there were significant increases or decreases in the oral enhanced NAD+treatment group in 15 immune proteins that play a role in regulating adaptive or innate immune function and inflammation.

[0510] Reproduction - changes were observed in one of the key protein regulators of fertility in both males and females in the oral enhanced NAD+treatment group that were not seen in the placebo group.

[0511] Metabolic disease and obesity - the enhanced NAD+composition differentially upregulated 3 proteins associated with metabolic diseases and obesity.

[0512] Skin and Bone Health - There were significant differences between groups in two proteins that are centrally involved in collagen synthesis or have been associated with skin inflammation in psoriasis and atopic dermatitis. Similar differences were also found in two proteins associated with bone development.Design and Methods

[0513] The study was performed to determine the effectiveness of the illustrative oral enhanced NAD+pharmaceutical formulation versus placebo in increasing intracellular concentration of NAD and extracellular blood concentrations of NAD in vivo in healthy adults. Intracellular levels of NAD (“icNAD”) were assessed via a colorimetric assay measuring levels of total NAD in red and white blood cells and platelets. Extracellular NAD (“eNAD”) was assessed in cell-free plasma. icNAD and eNAD levels are believed to increase by end of treatment on the formulation relative to placebo, demonstrating stability and potential clinical activity.

[0514] The study also examined the effects of the illustrative oral formulation versus placebo on a comprehensive range of clinical laboratory tests and multi -omic peripheral biomarkers associated with inflammation; lipid metabolism; oxidative stress / antioxidant capacity; liver, kidney, and mitochondrial function, as well as patient-reported outcomes, including mental well-being and daily reports of physical symptoms.

[0515] Safety endpoints for all subjects who had received at least one dose included: Individual adverse events as documented in the protocol and the self-reported daily Review of Systems (ROS) targeting 14 organ and functional systems; Vital signs collected at each blood draw visit (systolic and diastolic blood pressure, calculated MAP, heart rate) as well as data provided by the fitness tracker (Fitbit Charge 2; HRV, SpO2, heart rate, sleep duration by stage); Measures of subjective well-being: Daily anxiety and stress measured by the Clinically Useful Anxiety Outcome Scale - Daily version (CUXOS-D) and Depression, Anxiety, and Stress Scale-21 (DASS-21), and fatigue measured with the Daily Fatigue Impact Scale (D-FIS); and Biomarkers related to kidney and liver functioning and toxicity (e.g., ALT, AST, alkaline phosphatase, total bilirubin); basic metabolic health biomarkers as represented by other CMP laboratory tests.

[0516] The study examined a comprehensive array of about 4,000 circulating biomarkers utilizing blood samples from baseline, Day 11 (treatment Day 4), and Day 13 (immediate post-treatment day). Samples were biobanked and preserved in liquid nitrogen at -80 °C for batch processing and assaying of longitudinal samples from the same participant was performed simultaneously. >1,000 metabolites present on Metabolon’s Global Platform used for untargeted metabolomics of plasma samples from the study; these metabolites include amino acids, carbohydrates, lipids, nucleotides, peptides, as well as partially characterized molecules; -3,000 proteins whose abundance in plasma was assayed usingOlink’s Explore 3,072 PEA proteomics platform with NGS readout (Illumina’s HiSeq 2000); a wide range of interrogated proteins targeted a host of biological pathways, including cellular membrane maintenance, nutrient transport, oxygen species formation, lipid transport and degradation, inflammation, immune signaling, and mitochondrial functioning.

[0517] This was a randomized, double-blind, placebo-controlled, longitudinal, clinical trial with multi-omic analyses. Randomization was at enrollment following prescreening using a priori defined sex-stratified, blocked randomization scheme into treatment and placebo.

[0518] The study recruited N=76 participants; of these, N=60 successfully enrolled and were randomized into the study. N=51 received at least one dose of the illustrative oral enhanced NAD+formulation or placebo and provided at least one blood draw on treatment. Analyses were performed on the full available dataset from N=51 treated participants (25 women, 26 men) . For this analysis the distribution by randomized treatment group was enhanced NAD+treatment N=23; placebo N=28.

[0519] The illustrative enhanced NAD+pharmaceutical formulation used for treatment was an oral enhanced NAD+formulation consisting of 500 mg NAD+and 500 mg PEG 3350. The composition was taken four times daily (q.i.d.) for 5 consecutive days on Day 8 through Day 12. On each of Days 9 and 10, one additional dose was taken. For each dose, a measured amount of the composition powder was dissolved in water and participants took the dose orally using a “swish and swallow” procedure. The supplement meets the FDA criteria for administration in humans (including compliance with GMP regulations).Duration of Treatment

[0520] This was a 13 -day protocol with a 7-day run-in period involving wash-out from all dietary supplements (Days 1-7), followed by a 5-day treatment period (Days 8-12) and 1 day follow-up (Day 13). After the 7-day run-in period, participants took the oral enhanced NAD+formulation or placebo for 5 days following a q.i.d. schedule, with one additional dose administered on each of Days 9 and 10. Baseline blood draws and vital signs were obtained on Day 4 and Day 6 (prior to treatment) and these results were averaged as “baseline” for statistical analysis. Additional blood draws and vital signs were obtained on Day 11 (on -treatment) and Day 13 (post-treatment). The Day 11 blood draw corresponded to ~14 doses; the Day 13 blood draw following the last dose corresponded to ~22 doses.Results

[0521] One endpoint of the study was the change in intracellular NAD (icNAD) concentrations after treatment. The illustrative enhanced NAD+composition administration resulted in a statistically significant increase in icNAD concentrations, relative to placebo: for the interaction term Time*Group: B=3.39, SE=0.35, P=4.13e13.

[0522] Average unadjusted change in icNAD from average of two baseline draws to the predetermined timepoint corresponding to the 4th blood draw (immediate post-treatment day) (22 doses500 mg each for a cumulative dose of 11,000 mg) was estimated at 14.56 pM (95% CI from 10.85 to 18.27 pM) for the enhanced NAD+group; compared with the estimated change of -1.90 pM (95% CI from -4.04 to 0.24 pM) in the placebo group. This corresponds to an approximately 52.9% increase in icNAD relative to baseline (compared to a 4.4% decrease in the placebo group).

[0523] As shown in FIG. 18, not only was there a highly significant group difference, but every individual in the treatment group showed an increase in icNAD from baseline during the 5 -day intervention. Ten out of 23 (43%) increased their icNAD by >50%; 18 out of 23 (78%) participants increased their icNAD by >30%. The patient with the highest baseline score upon entering the study resulted in the smallest individual increase of 9.4% at the end of the study.

[0524] Oral enhanced NAD+formulation administration did not result in a statistically significant increase in eNAD (P=0.83). Low circulating eNAD concentrations are likely maintained by the activity of NAD+-cleaving enzymes. While it is possible that some NAD+is transported into cells directly, the maintenance of low eNAD concentrations is known to be mediated by ectoenzymes (e.g., cADPR synthases and pyrophosphatases) that catabolize it into NAD+precursors (NAM, NMN, NR) which are then used in the salvage pathway to replenish icNAD concentrations.NAD+Metabolites and SIRT1

[0525] Oral enhanced NAD+administration also led to a differential, statistically significant increase in plasma abundance of two key NAD+metabolites, supporting the bioavailability of the illustrative oral enhanced NAD+formulation. Specifically, increases were identified in 1 -methyl -nicotinamide (MeNAM; B=0.47, SEA).065, P=7.59* 10'11; FIG. 19) and N1 -methyl -2 -pyridone-5 -carboxamide (2PY; B=0.47, SEA).055, P=1.03* 1013; FIG. 20), compared to placebo. These changes in MeNAM and 2PY survived corrections for multiple testing using the Meff adjustment. The pattern of findings was identical when time was treated as a categorical variable in a by-time point analysis.

[0526] No changes were detected in plasma abundance of four other NAD+metabolites examined (z. e. , nicotinate, quinolinate, NAM, trigonelline; all P’s >0.05) in the continuous time-based analyses. Analyses by timepoint indicated a marginal effect of treatment on NAM by Day 4 of treatment (P=0.053).

[0527] As discussed above, SIRT1 is an NAD-dependent enzyme that typically increases in plasma when levels of NAD+are increased. SIRT1 is actively involved in the regulation of energy sensing and homeostasis, oxidative stress response, and anti-inflammatory cascades. It also relates to a number of chronic diseases, including cardiometabolic and neurodegenerative diseases.

[0528] In this study a significant differential increase was observed in SIRT1 plasma abundance in the treatment group relative to placebo (BA). 14, SEA).09, t=1.76, PA).041; FIG. 21), again suggesting the bioavailability of the illustrative oral formulation.Safety Profile Results

[0529] Analyses across 52 clinical laboratory tests did not reveal any statistically significant unfavorable changes across tested analytes (all P’s > 0.05).

[0530] Self-reported symptoms: throughout the 5 -day treatment period, a Review of Systems questionnaire targeting 14 organ and functional systems was administered daily (baseline and posttreatment data were also obtained). The study did not observe differences in incidence of adverse symptoms in the illustrative oral formulation vs. the placebo group (overall incidence for self-reported symptoms on-protocol was estimated at 2.89% in the oral formulation group vs. 2.30% in the placebo group). One participant in the active treatment group withdrew due to nausea - the symptom resolved after discontinuing the illustrative oral formulation. Another participant withdrew when diagnosed with COVID-19.

[0531] No differential changes in the enhanced NAD+vs. placebo group were detected in vital signs or body composition (SBP, DBP, MAP, Body Mass Index, Waist Circumference; all P’s >0.05) in response to NAD+administration. Analyses of wearable data revealed that enhanced NAD+administration is associated with a differential reduction in resting heart rate (P=0.033) and number of minutes it took the participants to fall asleep as estimated by the Fitbit (P=0.001), relative to placebo. No other changes were observed for levels of activity or sleep measures (all P’s>0.05).

[0532] No differences were detected in changes in measures of subjective well-being (daily anxiety and stress measured by modified daily versions of CUXOS and DASS, depression measured with DASS, and fatigue measured with FIS) in the NAD+group, relative to placebo. The study had limited resolution to detect improvements in the assessment scores given restricted clinically relevant variation in this healthy population.Exploratory Analyses

[0533] Unless noted otherwise, all indicators of statistical significance (P -values) have not been adjusted for multiple comparisons. Given the small size of the study and the number of variables measured, it would be rare for a result to remain statistically significant (P<0.05) after correction for multiple hypothesis testing. Cases where the P-value does remain significant are therefore particularly noteworthy.

[0534] The proteomic and metabolomic assays used generate results that are relative, rather than absolute (quantitative) values. This is indicated in the X-axes of the figures which are labeled either “Relative abundance” (for metabolites) or “NPX” (for proteins). NPX stands for Normalized Protein expression, which is Olink’s arbitrary unit (in Log2 scale) for proteomics reports.Cardiovascular Diseases

[0535] In the current study, several significant changes in the treatment group relative to placebo were observed that may have therapeutic implications for cardiovascular disease. Some changes are illustrated below:

[0536] Enhanced NAD+administration led to a differential decrease in LDL / HDL ratio (B=-0.03, SE = 0.01, t = -2.19, P = 0.029), compared to placebo. A lower LDL / HDL ratio has been associated with reduced atherogenic and heart disease risk. There were no differences observed in the study in levels of triglycerides, LDL- or HDL-cholesterol. The majority of study participants had lipid levels in the normal range thus the statistically significant decrease in LDL / HDL ratio was particularly noteworthy. In addition to potentially improving endogenous lipid metabolism, enhanced NAD+administration may improve dietary lipid metabolism. Analyses of the plasma lipidome as part of the untargeted metabolomic profiling revealed a coordinated decrease in abundance of 19 lipids, pointing to an enhancement of phospholipid and medium and long-chain PUPA metabolism following NAD+administration, which may have cardiovascular benefits.

[0537] Enhanced NAD+differentially impacted several proteins that have vascular effects and are known to play a role in hypertension or stroke. In this study, the orally administered enhanced NAD+pharmaceutical formulation significantly increased the levels of FGF12, EDIL3, FLT1 (VEGFR1), and FLRT2.

[0538] Fibroblast growth factor 12 (FGF12) is a potent inhibitor of the vascular Smooth Muscle Cell (SMC) phenotype switch, resulting in healthier blood vessels. FGF12 strongly induces a healthier phenotype of human aortic SMCs and results in a more favorable response to arterial injury in rats. Conversely, lower FGF12 levels have been implicated in pulmonary arterial hypertension and associated mechanistically with vascular damage in this disease in animal models.

[0539] EGF-like repeat and discoidin I-like domain-containing protein 3 (EDIL3) is an extra-cellular matrix protein that acts as a pro-angiogenic factor, mediator of the immune and anti-inflammatory response, and a regulator of endothelial cell adhesion and migration. Overall, EDIL3 plays an important role in mediating angiogenesis and may be important in vessel wall remodeling and development. The effect of the modest increase in circulating levels in healthy patients is unknown.

[0540] Vascular endothelial growth factor receptor 1 (VEGFR1) protein binds to VEGFR-A, VEGFR-B and placental growth factor and plays an important role in angiogenesis and vasculogenesis. Expression of this receptor is found in vascular endothelial cells, placental trophoblast cells and peripheral blood monocytes. It is involved in the regulation of angiogenesis by regulating endothelial cell proliferation and senescence. In mice, VEGFR1 levels are increased in ischemic muscle tissue compared to non-ischemic muscle and VEGFR1 appears to play a role in vascular recovery from ischemia.

[0541] Fibronectin Leucine-rich Repeat Transmembrane protein 2 (FLRT2) is a member of a family of cell adhesion molecules that regulate early embryonic vascular and neural development. In particular, FLRT2 is essential for development of the epicardium and loss of this protein impairs expansion of the ventricular myocardium and reduces endocardial volume . Its role in cardiac function in adults is unclear.

[0542] In addition, the illustrative enhanced NAD+pharmaceutical formulation differentiallydecreased circulating levels of the protein SERPINI1 (neuroserpin) . Neuroserpin inhibits the activity of an enzyme called tissue plasminogen activator (tPA), which plays a role in cell migration, blood clotting, and inflammation. In mice, administration of neuroserpin following aortic allograft transplant exerted an anti-inflammatory effect, reducing plaque growth and CD3+T cell invasion. Following ischemic stroke, neuroserpin levels in the brain increase in the region surrounding the lesion, providing a neuroprotective action.Homocysteine and the Folate Cycle

[0543] Increased levels of the amino acid homocysteine are an independent risk factor for atherosclerosis, as there is a correlation between serum homocysteine and incidence of coronary, carotid and peripheral vascular disease, likely mediated by homocysteine’s adverse effects on vascular endothelium and arterial structure. As part of the folate / methylation cycle, the enzyme methionine synthase (MTR) converts homocysteine to methionine, using methylated vitamin B12 (methylcobalamin) thus decreasing levels of homocysteine.

[0544] A significant differential decrease in MTR levels in circulation was observed (FIG. 22), which may reflect optimization of the folate cycle and reduced need for generation of methionine from homocysteine (z.e., suggesting the potential that enhanced NAD+administration may have reduced homocysteine levels).

[0545] Oxidative stress is considered a major risk factor for many diseases including cardiovascular disease, with increased oxidative stress associated with arterial hypertension, arrhythmia, and atherosclerotic plaque formation. In the present study, administration of the illustrative oral enhanced NAD+formulation was associated with beneficial changes in a variety of metabolites associated with glutathione (GSH), a key cellular antioxidant. In particular, changes in metabolites that were consistent with an increase in glutathione turnover with enhanced NAD+administration included: increase in plasma gamma-glutamyl glutamine (B=0.04, SE=0.019, P=0.02) and decrease in circulating glutamate (B=-0.09, SEA).03. P=0.004).

[0546] Analyses of the plasma proteome revealed that administration of the enhanced NAD+pharmaceutical formulation was associated with an increase in plasma abundance of glutathione S- transferase alpha-1 (GSTA1; B=0.08, SE=0.037, P=0.035; FIG. 23) and a decrease in plasma abundance of mitochondrial superoxide dismutase 2 (SOD2; B=-0.10, SE=0.04, P=0.016; FIG. 24).

[0547] GSTA1 is an enzyme that utilizes GSH to bind electrophilic compounds and ROS, thus reducing oxidative stress, while SOD2 is another player in cellular response to oxidative stress that binds superoxide anions to convert them to H2O2 and O2. Importantly, SOD2 is under transcriptional regulation by NAD+-dependent sirtuins. Decreasing levels of plasma SOD2 may indicate relaxing demand for its antioxidant capacity.

[0548] In this study, no significant changes were observed for clinical inflammatory biomarkers, e.g., cardiac CRP, hsCRP, TNF or IL-6.

[0549] As noted above (Safety Profile), administration of enhanced NAD+resulted in a statistically significant decrease in resting heart rate compared to placebo. Increased resting heart rate is an independent predictor of cardiovascular disease and mortality in men and women, both with and without heart disease. Conversely, individuals with lower resting heart rate have reduced rates of cardiovascular mortality.

[0550] Metabolites related to cardiovascular health that were differentially upregulated by the illustrative oral enhanced NAD+formulation compared to placebo include homoarginine and glyco- beta-muricholate, while metabolites which were significantly decreased in the enhanced NAD+group included docosahexaenoylcholine, urea, hypoxanthine, glutamate, acisoga, and 1 -palmitoyl -2- arachidonoyl-GPC. Details on these metabolites in relation to CVD are shown below:

[0551] Homoarginine is an amino acid that appears to have a protective effect against cardiovascular disease. Homoarginine appears to act as a weak substrate for endothelial nitric oxide synthase, which improves vascular function.

[0552] Glyco-beta-muricholate is a type of bile acid. Mouse studies show muricholic acids promote resistance to hypercholesterolemia.

[0553] Docosahexaenoylcholine (choline docosahexaenoate), is a type of acyl choline, a class of molecules involved in fatty acid metabolism. Research has also shown association between this metabolite and serum lipids in Caucasian and African -American adults.

[0554] Urea cycle metabolites - Both urea and hypoxanthine are involved in the urea cycle which breaks down toxic ammonia derived from the metabolism of proteins and gut bacterial waste. Urea cycle metabolites are a statistically over-represented cluster in relation to cardiometabolic disease. Excess levels of urea are associated with oxidative stress, which plays a significant role in CVD.

[0555] Glutamate (glutamic acid) is an amino acid and an excitatory neurotransmitter. In addition to its neurological role, higher plasma glutamate has been associated with heart failure.

[0556] Acisoga is a polyamine metabolite. It has been shown to be predictive of atrial fibrillation. Recently, acisoga has also been suggested as a novel biomarker in heart failure, correlating with reduced left ventricular function.

[0557] 1 -palmitoyl -2 -arachidonoyl-GPC is a type of phosphatidylcholine, functionally related to arachidonic acid and hexadecenoic acid. This compound is predictive of ischemic stroke risk in women, but not men.Liver Diseases

[0558] Non-alcoholic fatty liver disease (NAFLD) and non-alcoholic steatohepatitis (NASH) are common, chronic liver diseases. NAFLD and NASH are strongly associated with both obesity and Type 2 diabetes, thus as those conditions have increased in prevalence globally, so has the prevalence of fatty liver diseases. In the US, it’s estimated that 1 in 4 people have NAFLD.

[0559] Although all the participants in the study were healthy, the study provided strong evidence insupport of enhanced NAD+administration being associated with improved liver functioning.Enhanced NAD+administration was associated with a preferential decrease in serum GGT relative to placebo (B=-0.40, SE=0.19, P=0.0404; FIG. 26).

[0560] Enhanced NAD+administration was associated with a preferential decrease in serum total bilirubin (TBILI) (B=-0.025, SE=0.007, P=0.00054; FIG. 27). The finding passed the corrections for multiple hypothesis testing applied to clinical laboratory tests, with 95% CI for the interaction term from -0.0488 to -0.00007.

[0561] Enhanced NAD+administration was also found to be associated with a decrease in ALP (B=- 0.60, SE=0.28, P=0.0327) and serum albumin (B=-0.023, SE=0.011, P=0.037).

[0562] No effects were detected for the liver enzymes AST or ALT, or for BUN or Cr (all P’s >0.05).

[0563] In addition to liver enzymes, a significant differential decrease was observed in the enhanced NAD+group in the metabolite 1 -palmitoyl -2-arachidonoyl-GPC. This molecule is a type of phosphatidylcholine, functionally related to arachidonic acid and hexadecenoic acid. It was previously reported to be associated positively with liver fat in humans.

[0564] Additionally, oxidative stress has been recognized as a significant risk factor for both onset and progression of various liver diseases, including alcoholic liver disease as well as NASH.Although human evidence for use of antioxidant compounds in established liver disease is limited, there is evidence in animal models and for preventive benefits in humans. Thus, the antioxidant benefits of enhanced NAD+administration discussed above regarding cardiovascular disease are relevant for liver diseases. This is especially important given the central role of the liver in overall metabolism, including in relation to development of Type 2 diabetes.

[0565] GGT, an enzyme responsible for the breakdown and freeing of glutathione, may not only be a sensitive marker of liver damage and cholestasis but an overall sensitive marker of oxidative stress. Circulating GGT is a good biomarker of cellular redox status. Additionally, serum concentrations of GGT inversely correlate with serum antioxidants.Exocrine Pancreas Function and Digestion

[0566] Significant decreases were observed in the instant enhanced NAD+group relative to placebo in four pancreatic enzymes secreted in the gastrointestinal tract: PNLIPRP1 (pancreatic lipase -related protein 1); RNASE1 (pancreatic ribonuclease); CPA1 (carboxypeptidase Al); and CPB1 (carboxypeptidase Bl).

[0567] PNLIPRP l is a lipolytic enzyme secreted in the gastrointestinal tract. The function of this protein is unclear.

[0568] RNASE 1 belongs to the Ribonuclease A superfamily, which consists of 8 described enzymes. Pancreatic ribonuclease is a pancreatic enzyme that catalyzes the breakdown of RNA and plays a role in the digestion of RNA in vertebrate species. RNase 1 was historically considered as a digestive enzyme but is now thought to have additional functions regarding host defense similar toother members of this enzyme superfamily.

[0569] CPA1 is a member of the carboxypeptidase A family of zinc metalloproteases. This enzyme is produced in the pancreas and preferentially cleaves C-terminal branched-chain and aromatic amino acids from dietary proteins. Elevated protein levels (z.e., opposite of what was observed here) have been associated with pancreatic cancer.

[0570] CPB1 is another enzyme produced in the pancreas. Very elevated levels may be a serum marker for pancreatitis.

[0571] This observed impact of enhanced NAD+administration on pancreatic enzymes was consistent across the four different enzymes.

[0572] Neurodegenerative and Neuropsychiatric Diseases

[0573] Evidence for changes in levels of a number of proteins that modulate neuronal signaling pathways or neuron growth was obtained in the instant study as shown in FIG. 28.

[0574] Proteins that significantly decreased with enhanced NAD+administration compared to placebo included: ACOX1 (acyl-CoA oxidase 1), PLXNA4 (Plexin A-4), ASAHI (acid ceramidase), MTR (Methionine synthase), GRN (progranulin), and SERPINI1 (neuroserpin).

[0575] ACOX1 is the first enzyme of the fatty acid beta-oxidation pathway, which catalyzes the desaturation of acyl-CoAs to 2-trans-enoyl-CoAs. It donates electrons directly to molecular oxygen, thereby producing hydrogen peroxide, which has a variety of adverse effects in the body. Decreasing this protein suggests improved oxidative stress, which may be regulated by SIRT enzymes. ACOX1 seems to play a major role in neuronal function.

[0576] Plexin A4 binds to neuropilin 1 and 2 and transduces signals from Sema3A, Sema6A, and Sema6B. These Nrp-plexin and semaphorin complexes initiate cascades that regulate diverse processes in the nervous system such as axon pruning and repulsion, dendritic attraction and branching, regulation of cell migration, and vascular remodeling. Both upregulation and downregulation of Plexin A4 has been observed following neural injury suggesting a dynamic role for Plexin A4 in neural maintenance and regeneration. Plexin A-4 has been implicated in the pathology of Alzheimer’s disease.

[0577] The acid ceramidase enzyme is found in lysosomes, cell compartments that digest and recycle materials. Within lysosomes, acid ceramidase breaks down lipids called ceramides. Ceramides are typically found within the membranes that surround cells and play a role in regulating cell maturation, growth and division of cells (proliferation), and controlled cell death (apoptosis). Additionally, ceramides are a component of a fatty substance called myelin that insulates and protects nerve cells.

[0578] Methionine synthase is responsible for the regeneration of methionine from homocysteine and is the enzyme responsible for linking the SAMe cycle to one-carbon metabolism via the folate cycle.Both homocysteine and SAMe -dependent reactions have important implications for neurological health. Increased levels of homocysteine have been associated with increased levels of amyloid-beta in the brains of Alzheimer’s patients and may be an independent risk factor for neurodegenerativediseases. Methionine synthase is also an important regulator of brain anti-oxidant status and has been described as having a broad and dynamic role in coordinating metabolism in the brain during development and aging.

[0579] Progranulin is found in tissues throughout the body, but it is most active in cells that are dividing rapidly, such as skin cells (fibroblasts), immune system cells, and certain brain cells. This protein helps regulate the growth, division, and survival of these cells. Progranulin is active in several types of brain cells, however, little is known about this protein's role in the brain. It appears to be critical for the survival of neurons.

[0580] Neuroserpin inhibits the activity of an enzyme called tissue plasminogen activator (tPA), which plays a role in cell migration, blood clotting, and inflammation. As its name suggests, neuroserpin is active in the nervous system where it helps control the growth of neurons, especially axons which are required for the transmission of nerve impulses . Neuroserpin also plays a role in the development of synapses, and helps regulate synaptic plasticity, which suggests that it may be important for learning and memory. Neuroserpin has been shown to have a protective effect on the brain following stroke and has been reported to be upregulated in Alzheimer’s disease.

[0581] Proteins related to neurologic function that significantly and preferentially increased in the NAD+group compared to placebo include: NPL (N-acetylneuraminate lyase), 5-HT1AR (5-HT [serotonin] receptor 1A), FOLH1 (Glutamate carboxypeptidase 2), HRAS (GTPase Hras), CRIP2 (Cysteine-rich protein 2), and FLRT2 (Fibronectin Leucine-rich Repeat Transmembrane protein 2).

[0582] NPL is one of a family of lyase enzymes and it catalyzes the reversible cleavage and synthesis of sialic acids (monosaccharide sugars). Sialic acids have a variety of potential medical uses including anti-viral, anti-microbial and neural substrate uses. In the brain, addition (sialylation) or removal (desialylation) of sialic acid in microglia cells has been associated with neurodegenerative diseases. A specific type of sialic-acid modified molecule, polysialic acid, has also been associated with psychiatric disorders.

[0583] 5-HT1AR is involved in neuromodulation and impacts a variety of conditions. 5-HT1AR agonists (e.g., buspirone) show efficacy in anxiety and depression, and 5-HT1AR also seems to play a role in modulating chronic pain and immune function in peripheral circulation. The potential for a role of NAD+in depression is suggested by the importance of the SIRT1 pathway, which is strongly influenced by NAD, in bipolar depression and related neurological conditions. As described below, administration of enhanced NAD+significantly increased plasma SIRT1 levels.

[0584] 5 -HT1A receptor activation has also been shown to increase dopamine release in the medial prefrontal cortex, striatum, and hippocampus, and so may be useful for improving the symptoms of schizophrenia and Parkinson's disease. The potential for an impact on Parkinson’s disease is particularly interesting given that several older clinical studies found significant benefits of administering IV NADH to patients with Parkinson’s disease.

[0585] GCPII is a zinc metalloenzyme found in prostate epithelium, kidney, small intestine, and thenervous system. In the intestine, this enzyme catalyzes a reaction that results in the release of folic acid, which can then be used in the body as a vitamin. In the brain, GCPII has been shown to both indirectly and directly increase the concentration of glutamate. Potent and selective GCPII inhibitors have been shown to decrease brain glutamate and provide neuroprotection in preclinical models of stroke, amyotrophic lateral sclerosis, and neuropathic pain.

[0586] HRAS is an important regulator of signal transduction and cell cycle . The RAS signaling pathway is involved in the regulation of developmental processes, including cell growth, proliferation, and differentiation, in the central nervous system. Germline mutations in the RAS signaling pathway genes are associated with a group of neurodevelopmental disorders, collectively called RASopathies, which include neurofibromatosis type 1, Noonan syndrome, cardio-facio-cutaneous syndrome, and Costello syndrome. HRAS activation in neurons attenuates the generation of precursor cells in the hippocampus, which has the potential to impact spatial short-term memory and object recognition.

[0587] CRIP2 is found in many different tissues and is believed to play a role in the differentiation of smooth muscle tissue. CRIP2 is found in the spinal cord and may play an inhibitory role in inflammatory pain.

[0588] FLRT2 is a member of a family of cell adhesion molecules that regulate early embryonic vascular and neural development. FLRT2 is found in multiple regions of the brain and may play a role in healing after spinal cord injury.

[0589] In addition to changes in proteins, a number of significant changes were also observed in untargeted metabolomics analysis. Specifically, significant differential increases with NAD were observed in urate (uric acid), homoarginine, and indolelactate (indole lactic acid). Urate (uric acid) - while widely recognizes as being protective against aging and oxidative stress, increased levels of urate are also associated with reduced risk for Alzheimer’s and Parkinson’s disease and multiple sclerosis. Homoarginine is an amino acid may play a role in maintaining healthy brain function, with both significant excess and deficiency associated with negative impact. Indolelactate is an intermediate in the process of degrading tryptophan to indolepropionate. It may be significantly lower in individuals with multiple sclerosis and may be associated with the inflammatory processes of this disease. In cell culture, indolelactate increases neural growth.

[0590] Differential decreases in the enhanced NAD+group were also observed in glutamate (glutamic acid), hypoxanthine (FIG. 25), Cortisone, and 1,2-dipalmitoyl-GPC (16:0 / 16:0).

[0591] Glutamate is an amino acid and an excitatory neurotransmitter. Plasma glutamate levels have been found to be higher in patients with migraine and the glutamine / glutamate ratio is altered in patients with schizophrenia. Higher plasma glutamate levels have also been found in individuals who develop post-stroke depression, individuals addicted to stimulants, and individuals with neurodegenerative and metabolic disorders.

[0592] A metabolite in the urea cycle, in the brain, hypoxanthine causes mitochondrial dysfunction and neuronal death in animal models.

[0593] Cortisone is a metabolite of the steroid stress hormone cortisol. Both cortisol and cortisone are considered markers of stress, and some research suggests that plasma cortisone may be an even more sensitive marker of stress. Cortisone is increased in patients with depression. 1,2-dipalmitoyl- GPC (16:0 / 16:0) is a type of phosphatidylcholine glycerophospholipid containing two chains of palmitic acid. 1,2-dipalmitoyl-GPC may correlate with levels of multiple Alzheimer’s disease biomarkers in the cerebrospinal fluid of older adults and is one of the top 3 metabolite predictors of Alzheimer’s disease progression in a cohort with mild cognitive impairment.

[0594] Apart from the changes in specific proteins and metabolites, both oxidative stress and nitrosative stress have implications for neurodegenerative disease. Administration of enhanced NAD+had a number of beneficial effects on markers of antioxidant status. Increased oxidative stress is observed both peripherally and in the brain of patients with Alzheimer’s disease and plays an important role in the degeneration of dopamine neurons in Parkinson’s disease.

[0595] Nitrosative stress refers to the joint biochemical reactions of nitric oxide (NO) and superoxide (O2-) when an oxygen metabolism disorder occurs in the body. Thus, oxidative and nitrosative stress can exert parallel actions leading to cell damage and death and nitrosative stress has been implicated in a variety of diseases, including neurodegenerative diseases. The instant study found a coordinated signal for the enhanced NAD+’s effectiveness in enhancing protection nitrosative stress. Specifically, plasma proteome interrogation revealed a differential upregulation of CA5A (mitochondrial carbonic anhydrase 5A; B=0.09, SE=0.035, P=0.016), an enzyme that supplies liver mitochondria with HCOs in the urea / omithine cycle via conversion of carbon dioxide . At the same time, plasma metabolome analyses revealed a decrease in plasma urea (B=-0.10, SE=0.04, P=0.015) and aspartate (B=-0.096, SE=0.037, P=0.015), consistent with improved ammonia clearance.Inflammation / Immune Modulation

[0596] NAD+has also been implicated in autoimmune diseases, including multiple sclerosis (MS), inflammatory bowel disease (IBD), and rheumatoid arthritis (RA). In patients with MS, low serum NAD+levels correlate with severity and progression of the disease. SIRT1 -deficient mice developed a severe form of experimental autoimmune encephalomyelitis (considered an animal model for MS) and spontaneous autoimmunity.

[0597] In the instant study no changes were observed for circulating inflammatory biomarkers as assayed using clinical laboratory tests, nor were there any changes detected for white blood cell counts. However, several changes were observed in immune and inflammatory-related proteins in the proteomics analysis as shown below.

[0598] Plasma proteome analyses linked enhanced NAD+administration with a significant differential decrease in abundance of CXCL12 (P=0.004; see FIG. 29) and a trend in decrease for CCL8 (P=0.08). Increased levels of IL2RB, IL31, IL34 and a trend toward an increase in CXCL14 (P=0.068) were also observed. The possible implications of each of these proteins is discussed below:

[0599] The homeostatic chemokine CXCL12 is an important factor in many physiological and pathological processes because it activates and / or induces migration of hematopoietic progenitor and stem cells, endothelial cells and most leukocytes. The CXCL12-CXCL4 signaling axis is implicated in tumor biology and reductions in CXCL12 may be beneficial in this regard. CXCL12 is also thought to be a major player in interactions between the immune system and the nervous system.

[0600] CCL8 is a chemokine belonging to the CC chemokine family that acts as a macrophage chemotactant. CCL8 activates many different immune cells, including mast cells, eosinophils and basophils, which are implicated in allergic responses, and monocytes, T cells, and NK cells that are involved in the inflammatory response.

[0601] Increased CXCL14 results in attenuation of CXCL12-CXCR4 signaling. This has been suggested to be its primary function, restricting CXCL12-mediated chemotaxis required of progenitor and immune cells with a potential anti -tumor effect.

[0602] Interleukin-2 Receptor subunit beta (IL2RB) is a membrane protein and one of several subunits involved in the binding of interleukin 2. The interleukin 2 receptor is involved in T-cell- mediated immune responses.

[0603] Interleukin-31 (IL-31) is produced mainly by activated CD4+T-cells and interacts with a receptor expressed on epithelial cells and keratinocytes. IL-31 acts on a broad range of immune- and non-immune cells and therefore possesses potential pleiotropic physiological functions, including regulating hematopoiesis and immune response. IL-31 has been implicated in inflammatory bowel disease, airway hypersensitivity (asthma) and allergic dermatitis.

[0604] Interleukin-34 (IL-34) is a cytokine that promotes the differentiation and viability of monocytes and macrophages through the colony-stimulating factor- 1 receptor. The highest levels of IL-34 are found in the brain and the skin. IL-34 has been associated with several autoimmune disease, including rheumatoid arthritis and systemic lupus erythematosus.Reproductive Function and Fertility

[0605] The NAD+-dependent enzyme SIRT1 is involved in regulation of sex hormone levels which are key to reproductive function. SIRT1 knockout mice have been shown to have decreased production of gonadotropin-releasing hormone, and subsequently decreased LH and FSH levels, which contributes to the observed effect of decreased SIRT1 on male fertility. In men, SIRT1 also regulates spermatogenesis and sperm maturation.

[0606] In women, down-regulation of SIRT1 is associated with reduction of ovarian reserve and some have suggested that sirtuins are biomarkers for ovarian aging. Maternal obesity is a major cause of metabolic dysregulation in the offspring, including glucose intolerance, increased liver fat and obesity risk.

[0607] In the instant study, a significant differential effect of enhanced NAD+administration was observed on the protein INSL3 (insulin-like peptide 3). INSL3 is a protein hormone produced bygonadal tissues in both males and females. Circulating levels are higher in males than females. In males, INSL3 controls testicular descent during embryogenesis and declines with aging, presumably reflecting aging effects on the testes.

[0608] In females, INSL3 may be involved in regulating female fertility. The protein varies over the course of the menstrual cycle and decreases after menopause. (Note: Blood draws were not timed to menstrual cycle phase during the study). INSL3 may also play a role in polycystic ovarian syndrome (PCOS). The fact that INSL3 varied significantly with enhanced NAD+supplementation shows that INSL3 is a factor that is associated with NAD’s effect on fertility.Metabolic Disease and Obesity

[0609] In humans, SIRT1 levels are reduced in diabetic patients with poor glycemic control compared to those with good glycemic control.

[0610] This study demonstrated upregulation of 3 illustrative proteins in untargeted proteomics analysis that have previously been shown to play a role in obesity and metabolic diseases: GALNT2 (N -acetyl -galactosaminyl -transferase 2), FABP1 (fatty -acid binding protein 1; FIG. 30), and RBP2 (retinol binding protein 2; FIG. 31).

[0611] GALNT2 is a member of the glycosyltransferase 2 protein family. Recently, GALNT2 has attracted attention as a possible player in many clinical conditions that share the common ground of insulin resistance, including atherogenic dyslipidemia, type 2 diabetes, and obesity.

[0612] FABP 1 is critical for fatty acid uptake and intracellular transport and has an important role in regulating lipid metabolism and cellular signaling pathways. It is primarily found in the liver.FABP 1 may exert a protective effect against lipotoxicity by facilitating fatty acid oxidation or incorporation into triglycerides, binding these otherwise cytotoxic compounds. The ability to bind heme is another cytoprotective property. The role of FABP 1 in substrate availability and in protection from oxidative stress suggests that FABP1 plays a pivotal role during intracellular bacterial / viral infections by reducing inflammation. Altered expression of the protein has been linked to metabolic conditions including obesity: higher circulating levels have been reported in Chinese adults with high BMI and insulin resistance.

[0613] In addition to changes in proteins, a number of significant changes were observed in the enhanced NAD+group in untargeted metabolomics analysis. Specifically, a significant increase was observed with enhanced NAD+in bile acids or bile acid derivatives: Glycohyocholic acid (GHCA) and Glyco-beta-muricholate. Glycohyocholic acid is a glycine -conjugated form of the primary bile acid hyocholic acid. Plasma GHCA has been found to be lower in individuals with obesity and those with Type 2 diabetes. Plasma levels of GHCA increase in patients who are no longer diabetic following gastric bypass surgery and can predict diabetes remission. Muricholic acids are a type of bile acid. In obese humans, glyco-beta-muricholate has been found to correlate with insulin resistance.

[0614] In the present study, significant differential decreases in the enhanced NAD+group were found in the following metabolites: pyrraline, urea, glutamate (glutamic acid), acisoga, nonadecanoate (nonadecanoic acid), and 1 -palmitoyl -2 -arachidonoyl-GPC.

[0615] Pyrraline is a type of Advanced Glycation End product (AGE), harmful compounds formed in food and in vivo. AGEs are associated with diseases of aging and particularly Type 2 diabetes. Urinary pyrraline levels are higher in patients with Type 2 diabetes and are associated with glycemic control.

[0616] Elevated levels of urea have been found to impair insulin secretion in mouse and in vitro models. In animal models of chronic kidney disease, urea has been associated with insulin resistance, potentially mediated by its effect on oxidative stress.

[0617] Glutamate is an amino acid and an excitatory neurotransmitter. In addition to its neurological role, higher plasma glutamate has been associated with Type 2 diabetes.

[0618] Acisoga is a polyamine metabolite that is increased in children with obesity. Acisoga has also been found to be one of the metabolites significantly associated with new onset diabetes in a large longitudinal cohort in a model that controlled for other diabetes risk factors.

[0619] Nonadecanoate is a saturated fatty acid. Plasma levels of nonadecanoate were found to be significantly associated with glucose tolerance in a metabolomics study of adults with normal or impaired glucose tolerance. Nonadecanoate levels are also elevated in obese diabetic rodent models.

[0620] 1 -palmitoyl -2 -arachidonoyl-GPC is a type of phosphatidylcholine, functionally related to arachidonic acid and hexadecenoic acid. This compound has been shown to be significantly higher in patients with obesity and metabolic syndrome compared to healthy obese or non-obese individuals.Skin and Bone Health

[0621] Significant differential changes were observed in the enhanced NAD+group in two proteins that have previously been associated with skin health or skin conditions. Specifically, compared to placebo, enhanced NAD+significantly decreased PPIB and WFDC12.

[0622] PPIB (Peptidyl-prolyl cis-trans isomerase B) - PPIB localizes to the endoplasmic reticulum (ER) and participates in many biological processes, including mitochondrial metabolism, apoptosis, and inflammation. In the ER, PPIB interacts with various other proteins to facilitate protein folding, especially for type I collagen. Thus, PPIB is essential for collagen biosynthesis and post-translational modification and affects fibril assembly, matrix cross-linking, and bone mineralization.

[0623] WFDC12 (whey-acidic protein 4-disulfide-core 12) - WFDC12 belongs to a family of proteins that are involved in innate immune defense including inhibition of neutrophil serine proteases and inhibition of the inflammatory response to lipopolysaccharide (LPS). WFDC12 is produced in several tissues but primarily the lung and the skin. Elevated levels of WFDC12 have been found in the affected (inflamed) skin of patients with psoriasis and atopic dermatitis.

[0624] As noted above, PPIB plays a role in both...

Claims

CLAIMSWe claim:

1. A method of preventing or treating a neurodegenerative disease or an age-related disorder in a subject in need thereof, the method comprising administering a therapeutically effective amount of an enhanced NAD+pharmaceutical formulation to the subject.

2. The method of claim 1, wherein the enhanced NAD+pharmaceutical formulation is medical food.

3. The method of claim 1 or 2, wherein the neurodegenerative disease is Alzheimer’s Disease, Parkinson’s disease, Multiple Sclerosis, or Amyotrophic Lateral Sclerosis.

4. The method of any one of claims 1- 3, wherein the administration changes expression level of at least one protein involved in the neurodegenerative disease.

5. The method of claim 4, wherein the at least one protein is selected from the group of SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Receptor 1A (HTR1A), Insulin Receptor (INSR), Superoxide Dismutase 2 (SOD2), Artemin (ARTN), Fas Cell Surface Death Receptor (FAS), Interleukin 2 Receptor Subunit Beta (IL2Rb), Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), CD163 Molecule, Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), and Neurofilament Light Chain (NfL).

6. The method of any one of claims 1 to 5, wherein the administration increases the expression level of the at least one protein involved in the neurodegenerative disease in the administered subject, as compared to the expression level of the at least one protein in an unadministered subject.

7. The method of claim 6, wherein plasma level of the at least one protein is increased in the administered subject, as compared to the plasma level of the at least one protein in the unadministered subject.

8. The method of claim 6 or 7, wherein the least one protein with the increased expression level or the increased plasma level comprises SIRT1 (Sirtuin 1), 5 -Hydroxytryptamine Receptor 1A (HTR1A), Artemin (ARTN), Interleukin 2 Receptor Subunit Beta (IL2Rb), CD 163 Molecule, and / or Platelet-Derived Growth Factor Receptor Alpha (PDGFRA).

9. The method of any one of claims 6 to 8, wherein the at least one protein with the increasedexpression level or the increased plasma level comprises C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Insulin Receptor (INSR), Superoxide Dismutase 2 (SOD2), Fas Cell Surface Death Receptor (FAS), Platelet and / or Endothelial Cell Adhesion Molecule 1 (PECAM1).

10. The method of any one of claims 1 to 9, wherein the neurodegenerative disease is Parkinson’s disease.

11. The method of claim 10, wherein the administration changes the level of at least one protein, wherein the at least one protein is selected from the group consisting of SIRT1 (Sirtuin 1), C-X- C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Receptor 1A (HTR1A), and Insulin Receptor (INSR).

12. The method of any one of claims 1 to 9, wherein the neurodegenerative disease is Multiple Sclerosis.

13. The methods of claim 12, wherein the administration changes the level of at least one protein, wherein the at least one protein is selected from the group consisting of SIRT1 (Sirtuin 1), C-X- C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Fas Cell Surface Death Receptor (FAS), Interleukin 2 Receptor Subunit Beta (IL2Rb), Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), CD 163 Molecule, and Platelet-Derived Growth Factor Receptor Alpha (PDGFRA), and Matrix Metallopeptidase 9 (MMP9).

14. The method of any one of claims 1 to 9, wherein the neurodegenerative disease is Amyotrophic Lateral Sclerosis.

15. The method of claim 14, wherein the administration changes the level of at least one protein, wherein the at least one protein is selected from the group consisting of SIRT1 (Sirtuin 1), C-X- C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), Superoxide Dismutase 2 (SOD2), and Artemin (ARTN).

16. The method of claim 14 or 15, wherein the enhanced NAD+pharmaceutical formulation achieves detectable NAD+elevation in a spinal cord tissue approximately within 2 hours of administration.

17. The method of any one of claims 14 to 16, wherein the enhanced NAD+formulation modulates oxidative stress markers in the spinal cord tissue.

18. The method of claim 18, wherein the oxidative stress markers are glutathione and glutathione- S-transferase.

19. The method of any one of claims 14 to 18, wherein the enhanced NAD+formulation is administered orally.

20. The method of any one of claims 14 to 19, wherein the enhanced NAD+formulation achieves blood NAD+elevation approximately within 15 minutes of administration.

21. The method of any one of claims 14 to 20, wherein the administration results in preservation of neuromuscular function as measured by compound muscle action potential (CMAP) amplitude.

22. The method of claim 21, wherein the neuromuscular function is monitored with electrophysiological parameters.

23. The method of any one of claims 1 to 9, wherein the neurodegenerative disease is Alzheimer's Disease.

24. The method of claim 23, wherein the administration changes the level of at least one protein, wherein the at least one protein is selected from the group consisting of SIRT1 (Sirtuin 1), Granulin Precursor (GRN), Matrix Metallopeptidase 9 (MMP9), Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), and 5 -Hydroxytryptamine Insulin Receptor (INSR).

25. The method of any one of claims 1 to 5, wherein the administration decreases the expression level of the at least one protein involved in the neurodegenerative disease in the administered subject, as compared to the expression level of the at least one protein in an unadministered subject.

26. The method of claim 25, wherein plasma level of the at least one protein is decreased in the administered subject, as compared to the plasma level of the at least one protein in the unadministered subject.

27. The method of claim 25 or 26, wherein the at least one protein is circulating neurofilament light chain (NfL).

28. The method of any one of claims 1 to 27, wherein the subject is a human.

29. The method of any one of claims 1 to 28, wherein the subject has an elevated level of circulating NfL.

30. The method of claim 29, wherein the enhanced NAD+pharmaceutical formulation reducescirculating NfL in the subject.

31. A method of changing expression level of at least one protein involved in a neurodegenerative disease in a subject with the neurodegenerative disease, the method comprising administering to the subject a therapeutically effective amount of enhanced NAD+formulation, wherein the therapeutically effective amount is sufficient to change the expression level of the at least one protein in the subject.

32. The method of claim 31, wherein the enhanced NAD+formulation is medical food.

33. The method of claim 31 or 32, wherein plasma level of the at least one protein in the administered subject is increased, as compared to the plasma level in an unadministered subject.

34. The method of any one of claims 31-33, wherein the at least one protein is selected from the group consisting of SIRT1 (Sirtuin 1), C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), C- X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Receptor 1A (HTR1A), Insulin Receptor (INSR), Superoxide Dismutase 2 (SOD2), Artemin (ARTN), Fas Cell Surface Death Receptor (FAS), Interleukin 2 Receptor Subunit Beta (IL2Rb), Platelet and Endothelial Cell Adhesion Molecule 1 (PECAM1), CD163 Molecule, and Platelet-Derived Growth Factor Receptor Alpha (PDGFRA).

35. The method of any one of claims 31 to 34, wherein the expression level of the at least one protein involved in the neurodegenerative disease is increased in the administered subject, as compared to the expression level of the at least one protein in an unadministered subject.

36. The method of claim 35, wherein the at least one protein with the increased expression level or the increased plasma level comprises SIRT1 (Sirtuin 1), 5 -Hydroxytryptamine Receptor 1A (HTR1A), Artemin (ARTN), Interleukin 2 Receptor Subunit Beta (IL2Rb), CD 163 Molecule, and / or Platelet-Derived Growth Factor Receptor Alpha (PDGFRA).

37. The method of claim 31, wherein the expression level of the at least one protein involved in the neurodegenerative disease is decreased, as compared to the expression level of the at least one protein in an unadministered subject.

38. The method of claim 37, wherein the at least one protein with the decreased expression level comprises C-X-C Motif Chemokine Ligand 10 (CXCL10), Granulin Precursor (GRN), Glucocerebrosidase (GBA), Matrix Metallopeptidase 9 (MMP9), C-X-C Motif Chemokine Ligand 12 (CXCL12), 5 -Hydroxytryptamine Insulin Receptor (INSR), Superoxide Dismutase 2 (SOD2), Fas Cell Surface Death Receptor (FAS), Platelet Endothelial Cell Adhesion Molecule1 (PECAM1), and / or Neurofilament Light Chain (NfL).

39. The method of any one of claims 31 to 38, wherein the neurodegenerative disease is Alzheimer’s Disease, Parkinson’s disease, Multiple Sclerosis, or Amyotrophic Lateral Sclerosis.

40. The method of claim 39, wherein the neurodegenerative disease is Amyotrophic Lateral Sclerosis.

41. The method of claim 40, wherein the enhanced NAD+formulation achieves detectable NAD+elevation in a spinal cord tissue approximately within 2 hours of administration.

42. The method of claim 40 or 41, wherein the enhanced NAD+formulation modulates oxidative stress markers in the spinal cord tissue.

43. The method of claim 42, wherein the oxidative stress markers are glutathione and glutathione- S-transferase.

44. The method of any one of claims 40 to 43, wherein the enhanced NAD+formulation is administered orally.

45. The method of any one of claims 40 to 44, wherein the enhanced NAD+formulation achieves blood NAD+elevation approximately within 15 minutes of administration.

46. The method of any one of claims 40 to 45, wherein the administration results in preservation of neuromuscular function as measured by compound muscle action potential (CMAP) amplitude.

47. The method of claim 46, wherein the neuromuscular function is monitored with electrophysiological parameters.

48. The method of any one of claims 31 to 47, wherein the subject has an elevated level of circulating NfL.

49. The method of claim 48, wherein the enhanced NAD+pharmaceutical formulation reduces circulating NfL in the subject.

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