Enhanced NAD+ Compositions and Methods of Making and Using 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 NAD+ deficiency, as well as methods for preparing and using the same. The present disclosure further relates to methods for producing NAD+ drug products that are stable, safer to use, and have a longer shelf life. The present disclosure also provides novel methods for increasing intracellular NAD and methods for treating central nervous system diseases and disorders, including drug addiction and substance abuse.

Description

[Technical Field]

[0001] Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 63 / 380,186, filed October 19, 2022, which is incorporated herein by reference in its entirety.

[0002] Field of Disclosure The present disclosure relates to the field of chemical compound compositions. In particular, the present disclosure relates to chemical compound compositions and pharmaceutical formulations for treating conditions, diseases and disorders. [Background technology]

[0003] Background of the Disclosure Nicotinic acid and nicotinamide, collectively referred to as "niacin," are vitamin forms of nicotinamide adenine dinucleotide (NAD+), which are found in all living cells. NAD+ plays an important 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 decomposition, and chemical synthesis. Therefore, NAD+ is an important nutrient for animal health. Eukaryotes synthesize NAD+ de novo from tryptophan via the kynurenine pathway (Krehl, et al. (1945) Science 101:489-490; Schutz and Feigelson (1972) J. Biol. Chem. 247:5327-5332). Nicotinic acid is phosphoribosylated to form 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 (1958) J. Biol. Chem. 233:488-492; Preiss and Handler (1958b) J. Biol. Chem. 233:493-50).

[0004] Depleted levels of NAD+ are known to result in several conditions and disorders in humans. For example, drug and alcohol abuse depletes NAD+ levels, which has been shown to make recovery from addiction a physically and psychologically demanding process. NAD+ supplementation is applied in many diseases and conditions. For example, niacin supplementation prevents pellagra, which can occur in populations that consume a diet poor in tryptophan. O'Holleran, U.S. Pat. No. 3,412,190, teaches the use of NADPH to treat lipids in the bloodstream. NADPH has also been used to treat alcoholism (see, e.g., Canadian Patent No. 670,909). However, currently available commercially produced NAD+ is chemically and biologically unstable. It has a limited shelf life and requires special storage conditions (e.g., refrigerated or dry storage) for manufacturing and production. Furthermore, NAD+ is rapidly metabolized in the body, therefore, higher dosages are required for effectiveness. Furthermore, until now, NAD+ supplementation has been via IV, which has been reported to be painful, and the dosage required to be therapeutic was high due to instability. Risks associated with IV NAD+ administration include fluid overload in patients with renal, hepatic, and cardiac dysfunction, and the side effect profile included pain in the back of the neck, paresthesia at the base of the tongue, dizziness, and nausea (Pritsos et al. (1999) U.S. Patent No. 5,888,532). Oral formulations of NAD+ and several phosphate derivatives of NAD+ with slightly different biological activities and resistance to metabolic deactivation have been developed (U.S. Pat. No. 5,888,532). However, NAD+ is not completely absorbed by the gastric acid intestinal mucosa, and therefore, this oral form also requires higher dosages for effectiveness. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 3,412,190 [Patent Document 2] Canadian Patent No. 670,909 [Patent Document 3] U.S. Patent No. 5,888,532 [Non-patent literature]

[0006] [Non-Patent Document 1] Krehl, et al. (1945) Science 101:489-490 [Non-patent document 2] Schutz and Feigelson (1972) J. Biol. Chem. 247:5327-5332 [Non-patent document 3] Preiss and Handler (1958) J. Biol. Chem. 233:488-492 [Non-patent document 4] Preiss and Handler (1958b) J. Biol. Chem. 233:493-50 Summary of the Invention [Means for solving the problem]

[0007] Thus, there is an unmet need for improved NAD+ compositions and pharmaceuticals with enhanced stability and bioavailability.

[0008] Summary of the Disclosure It has been discovered that NAD+, when prepared by vacuum filtration, may have enhanced stability and higher bioavailability compared to commercially available NAD+ supplements. This discovery has been utilized to provide the present disclosure, which provides enhanced NAD+ compositions, pharmaceutical formulations, and methods of producing and using the same.

[0009] In one aspect, the present disclosure provides enhanced NAD+ compositions having increased shelf life, stability, and bioavailability compared to NAD+ (e.g., NAD+ monohydrate), e.g., commercially available and other known forms of NAD. In some embodiments, the enhanced compositions comprise vacuumed NAD+ and phosphorus. In certain embodiments, the compositions consist essentially of vacuumed NAD+ and phosphorus. In some embodiments, the enhanced compositions comprise vacuumed NAD+ and polyethylene glycol. In some embodiments, the enhanced compositions comprise vacuumed NAD+.

[0010] In some embodiments, the unit dose of NAD+ in the compositions described herein is about 5 mg to about 4000 mg. In some embodiments, the unit dose of NAD+ is about 500 mg.

[0011] 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 4000 mg.

[0012] In some embodiments, the composition comprises about 5 mg to about 4000 mg of NAD+. In certain embodiments, the composition comprises about 100 mg, 200 mg, 300 mg, 400 mg, 500 mg, 600 mg, 700 mg, 800 mg, 900 mg, 1000 mg, 1100 mg, 1200 mg, 1300 mg, 1400 mg, 1500 mg, 1600 mg, 1700 mg, 1800 mg, 1900 mg, 2000 mg, or 4000 mg of NAD+.

[0013] One aspect of the present 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%.

[0014] In some embodiments, the concentration of NAD+ in the compositions described herein is about 200 mg / ml to about 1000 mg / ml, hi some embodiments, the concentration of NAD+ is about 500 mg / ml.

[0015] In some embodiments, the enhanced NAD+ composition is in powder form. In certain embodiments, the composition has a longer shelf life relative to commercially available NAD+, such as NAD+ monohydrate.

[0016] In another aspect, the present disclosure provides a pharmaceutical formulation comprising the enhanced NAD+ composition described above and at least one component that facilitates administration to a subject and / or can further treat a disease or condition in a subject.

[0017] In some embodiments, the pharmaceutical formulation comprises polyethylene glycol (PEG) of any molecular weight, such as, but not limited to, PEG300, PEG400, PEG monomethyl ether (MME)550, PEG600, PEG1000, PEGMME2000, PEG3350 and / or PEG4000, and / or combinations thereof.

[0018] In some embodiments, the composition is a powder.

[0019] Another aspect of the present disclosure relates to a composition comprising liothyronine, polyethylene glycol, microporous glucose and a pharmaceutically acceptable excipient.

[0020] Another aspect of the present disclosure relates to a composition comprising LevaDopa and polyethylene glycol and a pharmaceutically acceptable excipient.

[0021] In some embodiments, the PEG is PEG300, PEG400, PEG monomethyl ether (MME)550, PEG600, PEG1000, PEGMME2000, PEG3350, and / or PEG4000. In some embodiments, the PEG is PEG3350. In some embodiments, the PEG has a molecular mass greater than 4000 g / mol. In some embodiments, the PEG is a higher molecular weight PEG than commercially available. An exemplary method for PEG synthesis is described in Khanal, A., Fang, S. Chemistry. 2017 Oct. 26; 23(60):15133-15142, which is incorporated herein by reference in its entirety.

[0022] In some embodiments, the composition further comprises a trace amount 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.

[0023] Another aspect of the present disclosure relates to a process for making an NAD+ composition (e.g., an enhanced NAD+ composition). The process includes blending NAD+ and PEG and evacuating the blended mixture of NAD+ and PEG under vacuum and inert gas conditions. In some embodiments, evacuating includes placing the mixture of NAD+ and PEG under reduced pressure at a pressure between about 24 inches of Hg and about 30.25 inches of Hg. Units of measure for pressure can be readily interconverted by one of ordinary skill in the art. In another embodiment, evacuating further includes replacing the vacuum with an inert gas into the blended mixture to achieve a pressure of about 1 atm in the blender. In some embodiments, the process further includes pretreating the NAD+ by passing it through a 0.25 mm sieve. In some embodiments, the process further includes pretreating the NAD+ to produce particles that are 0.152 mm in size. Without wishing to be bound by theory, co-formulating NAD+ with PEG increases product stability. For example, such pretreatment can be performed before mixing NAD+ and PEG. In some embodiments, such pretreatment is performed after mixing NAD+ and PEG (e.g., pre-forming the NAD+ / PEG mixture). In some embodiments, a measured portion of PEG is pre-evacuated before adding NAD+ to the composition.

[0024] 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 greater than 4000 g / mol.

[0025] In some embodiments, blending NAD+ (e.g., pre-formulated NAD+) and PEG (e.g., pre-formulated PEG) is performed in a blender.

[0026] In some embodiments, applying a vacuum comprises placing the mixture of NAD+ and PEG under a stable vacuum.

[0027] In some embodiments, applying a vacuum further comprises pumping an inert gas into the blended mixture under vacuum to restore the pressure in the blender 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.

[0028] In some embodiments, the applying of vacuum is performed at least two times.

[0029] A further aspect of the present disclosure relates to NAD+ compositions produced by the processes described herein. In some embodiments, the process includes blending NAD+ and PEG and evacuating the blended mixture of NAD+ and PEG under vacuum and inert gas conditions.

[0030] In some embodiments, the NAD+ compositions described herein contain water in an amount less than about 0.2%.

[0031] In some embodiments, the dose of the NAD+ compositions described herein is between about 5 mg and 4000 mg. In some embodiments, the dose of the NAD+ composition is about 500 mg per gram of composition.

[0032] 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.

[0033] In some embodiments, the composition further comprises a trace amount 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.

[0034] 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).

[0035] In some embodiments, the ratio of NAD+ to PEG in the compositions of the present disclosure is 2:1 (wt / wt). In some embodiments, the ratio of NAD+ to PEG in the compositions of the present disclosure is 3:1 (wt / wt). In some embodiments, the ratio of NAD+ to PEG in the compositions of the present disclosure is 4:1 (wt / wt). In some embodiments, the ratio of NAD+ to PEG in the compositions of the present disclosure is 5:1 (wt / wt). In some embodiments, the ratio of NAD+ to PEG in the compositions of the present disclosure is 6:1 (wt / wt). In some embodiments, the ratio of NAD+ to PEG in the compositions of the present disclosure is 7:1 (wt / wt). In some embodiments, the ratio of NAD+ to PEG in the compositions of the present disclosure is 8:1 (wt / wt). In some embodiments, the ratio of NAD+ to PEG in the compositions of the present disclosure is 9:1 (wt / wt). In some embodiments, the ratio of NAD+ to PEG in the compositions of the present disclosure is 10:1 (wt / wt). In some embodiments, the ratio of NAD+ to PEG in the compositions of the present disclosure is 20:1 (wt / wt).

[0036] In certain embodiments, the composition comprises about 3.0% to about 11.0% (wt / wt) phosphorus. In some embodiments, the ratio of NAD+ to PEG is about 1:1 (wt / wt). In some embodiments, the ratio of NAD+ to PEG3350 is about 1:1 (wt / wt). The enhanced NAD+ composition, in some embodiments, is in powder form.

[0037] In certain embodiments, pharmaceutical preparations are suitable for oral administration to subjects.In certain embodiments, pharmaceutical preparations are in the form of powder, liquid, aqueous, tablet, suppository or capsule.In some embodiments, preparations are suitable for subcutaneous implantation, and may be formulated as nasal spray, cream, toothpaste or shampoo.

[0038] In some embodiments, the enhanced NAD+ pharmaceutical formulation, when administered to a subject, increases NAD levels in the subject by at least about 10% relative to the baseline level of intracellular NAD (icNAD) in the subject. In some embodiments, the pharmaceutical agent 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 the baseline level of intracellular NAD in the subject.

[0039] In certain embodiments, the pharmaceutical formulations are capable of increasing intracellular levels of NAD in a subject, and in certain embodiments, the formulations are capable of increasing intravascular levels of NAD in a subject. In some embodiments, the formulations do not significantly alter extracellular NAD levels in a subject when administered to the subject.

[0040] In some embodiments, the subject to whom the enhanced NAD+ pharmaceutical formulation is administered has or is at risk of having a disease or disorder correlated with reduced 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 nonalcoholic 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.

[0041] In some embodiments, the pharmaceutical formulation, when administered to a subject, is capable of improving liver and / or kidney function in the subject by, as needed, reducing serum levels of gamma-glutamyltransferase (GGT), bilirubin, alkaline phosphatase (ALP) and / or albumin, and, as needed, reducing oxidative stress in the subject by, as needed, decreasing serum GGT, increasing plasma gamma-glutamylglutamine (L-Glu-L-Gln), decreasing circulating glutamate, decreasing at least one histidine metabolite, upregulating glutamate utilization, increasing the 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), and, as needed, upregulating mitochondrial CA5A; and and / or plasma urea and / or aspartate, and / or improve ammonia clearance, and optionally may not significantly increase inflammation in the subject as measured by circulating inflammatory biomarkers, e.g., cardiac CRP, hsCRP, TNF, IL-6, and / or white blood cell count, and optionally may improve lipid transport and / or metabolism by lowering the LDL / HDL ratio, increasing the plasma abundance of FABP1 and / or RBP2, reducing circulating medium-chain and / or long-chain PUFAs, lowering plasma cholesterol levels, and / or increasing oxidized cholesterol metabolites, and may not significantly increase fasting serum glucose levels in the subject, and / or may increase the plasma abundance of at least one NAD+ metabolite, e.g., 1-methyl-nicotinamide (MeNAM), N1-methyl-2-pyridone-5-carboxamide (2PY), indole lactate, and / or urate.

[0042] In certain embodiments, when the pharmaceutical preparation is administered to a subject, it can modulate the expression of at least one biomarker in the subject. In certain embodiments, the at least one biomarker is a protein or metabolite indicating endothelial, liver, and / or overall mitochondrial function and / or integrity, one of upregulated fatty acid binding proteins, or one of downregulated proteins associated with endothelial, liver, and / or pancreatic injury. In some embodiments, the at least one biomarker is a marker for opiates in the subject's blood. In certain embodiments, the biomarker is an increase in circulating serotonin receptor 1A (5-HT1AR), dopamine release in the nucleus accumbens, and / or at least one liver enzyme level. In some embodiments, the presence of at least one biomarker is associated with substance use, for example, addiction to opiates and other drugs, including, but not limited to, an increase in circulating serotonin receptor 1A (5-HT1AR), dopamine release in the nucleus accumbens, and / or at least one liver enzyme level. The presence of a biomarker may also or alternatively be associated with neurodegenerative and / or neuropsychiatric disorders, including, for example, 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 a biomarker may also or alternatively be associated with cardiovascular disease, including, for example, improved LDL / HDL ratios, vascular and / or endothelial health, injury resistance, and / or oxidative stress. In some embodiments, the presence of a biomarker may also or alternatively be associated with exocrine pancreatic and / or digestive function, including, for example, reduced circulating levels of pancreatic enzymes. The presence of a biomarker may also or alternatively be associated with inflammation and / or immune function, including, for example, improved adaptive or innate immune function or inflammation.In some embodiments, the presence of the biomarker may also or instead be associated with preproduction function, including, for example, INSL3; with metabolic disease and / or obesity, including, for example, GALNT2, FABP1 and / or RBP2; and / or with skin and / or bone health, including, for example, collagen synthesis, skin inflammation in psoriasis and / or atopic dermatitis and / or bone development, including, for example, PPIB, WFDC12, INSL4 and / or MATN3.

[0043] In some embodiments, the pharmaceutical formulations provided herein do not increase any anxiety, stress, depression, fatigue, or any combination thereof in a subject.

[0044] In some embodiments, the pharmaceutical formulations provided herein do not induce unfavorable change(s) in vital signs, do not induce unfavorable change(s) in body composition, do not induce unfavorable change(s) in clinical test results, or any combination thereof.

[0045] In certain embodiments, the present disclosure provides enhanced NAD+ pharmaceutical formulations for use in methods of alleviating drug addiction withdrawal symptoms in a subject in need thereof, alleviating 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 with decreased levels of nicotinamide adenine dinucleotide (NAD+) in a subject in need thereof.

[0046] In yet another aspect, the present disclosure provides kits comprising the enhanced NAD+ compositions or enhanced NAD+ pharmaceutical formulations provided herein.

[0047] In yet another aspect, the present disclosure provides a method for making the enhanced NAD+ composition described herein. The method includes evacuating 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, evacuating the NAD+ includes placing the NAD+ under vacuum or a reduced pressure of between about 24 inches Hg and about 30.25 inches Hg and replacing the vacuum with an inert gas to increase the pressure in the container to about 1 atmosphere (atm). In certain embodiments, evacuating is performed at least twice. In some embodiments, the inert gas that replaces the vacuum includes argon, helium, nitrogen, and / or xenon. In certain cases, the NAD+ is evacuated more than once, more than twice, or more than three times.

[0048] In some embodiments, NAD is pre-treated before being vacuumed.In certain embodiments, pre-treatment comprises processing NAD+ to obtain a pre-selected average NAD+ particle size.In certain embodiments, processing comprises passing NAD+ through a sieve and / or grinding, milling or jet-milling NAD+.

[0049] The present disclosure also provides enhanced NAD+ compositions made by the methods described herein.

[0050] In yet another aspect, the present disclosure provides methods of making an enhanced NAD+ pharmaceutical formulation described herein, comprising mixing an enhanced NAD+ composition of the present disclosure, e.g., as described above, with a compound, component, and / or ingredient to form a blended mixture. The secondary compound, component, or ingredient facilitates administration to a subject and / or also has therapeutic efficacy. In some embodiments, the ingredient comprises PEG of any molecular weight or a mixture of PEGs with various molecular weights.

[0051] In some embodiments, NAD+ is pretreated as described above to provide NAD+ particles of a preselected particle size for homogeneous mixing with a secondary compound, component, or ingredient. In some embodiments, NAD+ is pretreated before being mixed with PEG in a blender. Pretreatment may additionally or alternatively include evacuating either NAD+ or PEG before they are mixed. The blended mixture of NAD+ and PEG is then evacuated in the blender under vacuum, or in some embodiments, under reduced pressure between about 24 inches of Hg and about 30.25 inches of Hg. The vacuum is then replaced with an inert gas or CO2 to increase the pressure in the blender to about 1 atmosphere (atm). In certain embodiments, evacuating before or after mixing is performed at least twice. In some embodiments, the inert gas used to replace the vacuum includes argon, helium, nitrogen, and / or xenon.

[0052] In yet another aspect, the present disclosure provides a method for alleviating drug addiction withdrawal symptoms in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of the enhanced NAD+ pharmaceutical formulation, the amount effective to alleviate at least one drug addiction withdrawal symptom in the subject.

[0053] In some embodiments, administration of the formulation increases NAD levels in a subject by at least 5%. In other embodiments, administration of the formulation increases NAD levels in a 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 increase in NAD amount is intracellular, and in certain embodiments, the intracellular level of NAD is increased to greater than about 35 μM. In certain embodiments, the intracellular level of NAD is increased to greater than about 35 μM, greater than about 36 μM, greater than about 37 μM, greater than about 38 μM, greater than about 39 μM, greater than about 40 μM, greater than about 41 μM, greater than about 42 μM, greater than about 43 μM, greater than about 44 μM, or greater than about 45 μM. In certain embodiments, the method is effective to increase the plasma level of N-methyl-nicotinamide (MeNAM) or N-methyl-2-pyridone-5-carboxamide (2PY) in a subject by at least 5%. In some embodiments, the extracellular level of NAD is instead or additionally increased. In some embodiments, the administered amount is effective to increase the level of icNAD to a level within a selected physiological range. In certain embodiments, the minimum value of the selected physiological range is at least 5% to at least 50% higher than the 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 the baseline level of intracellular NAD in the subject. In certain embodiments, administration of the pharmaceutical formulation results in at least a 5% increase in the subject's urinary excretion of nicotinamide or its metabolites.

[0054] In some embodiments, the drug addiction withdrawal symptoms suffered by the subject prior to administration of the formulation are associated with detoxification from tobacco, heroin, opium, morphine, dihydromorphine, meperidine, codeine, cocaine, amphetamines, barbiturates, alcohol, tranquilizers, and / or opioids.

[0055] In some embodiments, the subject to whom the pharmaceutical formulation is administered scored at least about 3 or higher on the Drug Abuse Screening Test (DAST) prior to administration. In other embodiments, the subject to whom the pharmaceutical formulation is administered scored about 6 or higher on the Drug Abuse Screening Test-10 (DAST-10) prior to administration. In some embodiments, the amount of the pharmaceutical formulation administered is effective to alleviate 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 the formulation administered is effective to alleviate 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 detoxification.

[0056] In some embodiments, pharmaceutical preparations are administered to subjects as hydrated, dissolved or suspended powder.In certain embodiments, preparations are administered orally, buccal, intramuscularly, systemically, anally, vaginally or sublingually.In certain examples, preparations are administered by the protocol of multiple sips, rinsing the oral cavity and swallowing.

[0057] In certain embodiments, the amount of NAD+ in the pharmaceutical formulation administered to a subject is about 5 mg to about 4000 mg. In certain embodiments, the amount of enhanced NAD+ composition in the formulation is 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 1200 mg, about 1400 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 850 mg, about 900 mg, about 1000 mg, about 1200 mg, about 1400 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, 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+.

[0058] In some embodiments, the amount of the pharmaceutical formulation administered comprises a daily dose of about 1 mg / kg to about 100 mg / kg of the subject's body weight. In certain embodiments, the amount of the pharmaceutical formulation administered comprises about 1 mg / kg, about 2 mg / kg, about 3 mg / kg, about 4 mg / kg, about 5 mg / kg, about 6 mg / kg, about 7 mg / kg, about 8 mg / kg, about 9 mg / kg, about 10 mg / kg, about 11 mg / kg, about 12 mg / kg, about 13 mg / kg, about 14 mg / kg, about 15 mg / kg, about 16 mg / kg, about 17 mg / kg, about 18 mg / kg, about 19 mg / kg, about 20 mg / kg, about 22 mg / kg, about 24 mg / kg, about 26 mg / kg, about 27 mg / kg, or about 30 mg / kg of the subject's body weight. Including maximum daily doses of 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.

[0059] In some embodiments, the method further comprises administering to the subject a pharmaceutically effective amount of an analgesic or a nonsteroidal anti-inflammatory drug (NSAID). In certain embodiments, the pharmaceutical formulation further comprises a pharmaceutically effective amount of an analgesic or a nonsteroidal anti-inflammatory drug (NSAID). In certain 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 comprises administering a pharmaceutically effective amount of an antidiarrheal agent, such as, but not limited to, loperamide; an antiemetic agent, such as, but not limited to, ondansetron; an antispasmodic agent, such as, but not limited to, tizanidine; and / or a sedative, such as, but not limited to, promethazine.

[0060] In some embodiments, the method further comprises monitoring multiple vital signs of the subject after administration of the formulation. In certain embodiments, the method further comprises measuring intracellular NAD or extracellular NAD concentrations in the subject before and / or after administration of the formulation, and the dosage of the pharmaceutical formulation to be administered can be selected based on the measured intracellular NAD or extracellular NAD concentrations in the subject.

[0061] In some embodiments, the pharmaceutical formulation is administered over a period of at least two days, and may be administered over a period of about two, three, four, or five or more days. In certain embodiments, the pharmaceutical formulation is administered in multiple doses within a period of about 10 to about 16 hours. In some embodiments, about one to about four doses are administered within a period of 10 to 16 hours. In certain embodiments, the formulation 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 is administered immediately before the subject goes to bed. In some embodiments, the first daily dose of the formulation is administered after the subject has fasted for about 12 hours.

[0062] The method may further include monitoring at least one vital sign of the subject before and after administration of the composition and / or measuring intracellular NAD or extracellular concentrations of NAD in the subject before and after administration of the pharmaceutical formulation, and / or selecting a dosage of the composition to be administered to the subject based on the measured baseline intracellular NAD or extracellular concentrations of NAD in the subject.

[0063] The present disclosure also provides a method for increasing NAD levels in a subject, the method comprising administering to the subject an amount of an enhanced NAD+ pharmaceutical formulation provided herein that is effective to increase NAD levels in the subject. In some embodiments, the subject is a healthy human subject. In other embodiments, the subject is suffering from or at risk of a disease or disorder, and administration of the formulation reduces the risk of and / or alleviates at least one symptom of the disease or disorder. In certain embodiments, the method may further comprise monitoring at least one vital sign of the subject before and after administration of the pharmaceutical formulation, and / or measuring the intracellular concentration of NAD or the extracellular concentration of NAD in the subject before and after administration of the pharmaceutical formulation, and / or selecting a dosage of the composition to be administered to the subject based on the measured baseline intracellular concentration of NAD or the extracellular concentration of NAD in the subject.

[0064] In yet another aspect, the present disclosure provides a method of preventing or treating a disease or disorder correlated with reduced levels of NAD in a subject in need thereof, the method comprising administering an amount of an enhanced NAD+ pharmaceutical formulation 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., nonalcoholic fatty liver disease (NAFLD) and nonalcoholic 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 reducing serum levels of gamma-glutamyltransferase (GGT), bilirubin, alkaline phosphatase (ALP) and / or albumin, optionally by decreasing serum GGT, increasing plasma gamma-glutamylglutamine (L-Glu-L-Gkn), decreasing circulating glutamate, decreasing at least one of histidine metabolites, upregulating glutamate utilization, increasing the 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), optionally by increasing mitochondrial CA5A. and / or improve ammonia clearance, and optionally do not significantly increase inflammation in the subject as measurable by circulating inflammatory biomarkers, e.g., cardiac CRP, hsCRP, TNF, IL-6, and / or white blood cell count, and optionally improve lipid transport and / or metabolism, and optionally do not significantly increase fasting serum glucose levels in the subject, and / or improve the plasma abundance of at least one NAD+ metabolite, e.g., 1-methyl-nicotinamide (MeNAM), N1-methyl-2-pyridone-5-carboxamide (2PY), indole lactate, and / or urate, by lowering the LDL / HDL ratio, increasing the plasma abundance of FABP1 and / or RBP2, decreasing circulating medium-chain and / or long-chain PUFAs, decreasing plasma cholesterol levels, and / or increasing oxidized cholesterol metabolites.

[0065] In some embodiments, by administering enhanced NAD+ preparations to a subject, the expression of at least one biomarker in the subject is modulated.In certain embodiments, at least one biomarker is a protein or metabolite associated with endothelial, liver and / or overall mitochondrial function and / or integrity, or is an upregulated fatty acid binding protein or a downregulated protein associated with endothelial, liver and / or pancreatic injury.In certain embodiments, at least one biomarker is associated with substance use, for example, opiates and other drugs, including, for example, an increase in the circulating level of serotonin receptor 1A (5-HT1AR), an increase in dopamine release in the nucleus accumbens and / or an increase in the level of at least one liver enzyme; associated with neurodegenerative disease and / or neuropsychiatric disease, including, for example, an increase in the level of a protein with neurological effects, an improvement in the level associated with neurodegenerative disease, depression, anxiety or chronic pain, oxidative stress and / or nitrosative stress.

[0066] In certain embodiments, the at least one biomarker is associated with cardiovascular disease, including, for example, improved LDL / HDL ratio, vascular and / or endothelial health, injury resistance and / or oxidative stress; associated with exocrine pancreatic and / or digestive function, including, for example, reduced circulating levels of pancreatic enzymes; associated with inflammation and / or immune function, including, for example, improved adaptive or innate immune function or inflammation; associated with preproduction function, including, for example, INSL3; associated with metabolic disease and / or obesity, including, for example, GALNT2, FABP1 and / or RBP2; and / or associated with skin and / or bone health, including, for example, collagen synthesis, dermatitis in psoriasis and / or atopic dermatitis and / or bone development, including, for example, PPIB, WFDC12, INSL4 and / or MATN3.

[0067] In some embodiments, administration of the enhanced NAD+ pharmaceutical formulation does not result in a significant increase in at least one adverse symptom, such as, but not limited to, anxiety, stress, depression, fatigue, and / or significant unfavorable changes in vital signs, body composition, and / or clinical test results.

[0068] In some embodiments, the amount of pharmaceutical formulation 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 the baseline level of intracellular NAD in the subject.

[0069] The method may further include monitoring at least one vital sign of the subject before and after administration of the pharmaceutical formulation, and / or measuring the intracellular concentration of NAD or the extracellular concentration of NAD in the subject before and after administration of the pharmaceutical formulation, and / or selecting a dosage of the composition to be administered to the subject based on the measured baseline intracellular concentration of NAD or the extracellular concentration of NAD in the subject.

[0070] 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 NAD+ composition disclosed herein effective to increase intracellular NAD levels.

[0071] Another aspect of the present disclosure relates to a method of treating a subject suffering from or preventing an age-related disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an NAD+ composition disclosed herein.

[0072] The present disclosure also provides NAD+ compositions for use in the manufacture of a medicament or supplement for treating age-related diseases.

[0073] In another aspect, the present disclosure relates to a method for treating or preventing a central nervous system disorder or disease, comprising administering a therapeutically effective amount of an NAD+ composition described herein to a subject in need thereof.

[0074] Another aspect of the present disclosure relates to a method for treating (e.g., reducing) a subject for drug addiction or substance abuse, the method comprising administering to the subject a therapeutically effective amount of an NAD+ composition described herein.

[0075] 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 NAD+ composition described herein effective to increase SIRT1 in the subject.

[0076] 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 described herein effective to increase PLB1 in the subject.

[0077] Another 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 NAD+ composition described herein effective to increase MME in the subject.

[0078] Another aspect of the present disclosure relates to a method for treating a subject suffering from a disease or disorder modulated by SIRT1, the method comprising administering to the subject a therapeutically effective amount of an NAD+ composition described herein.

[0079] 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 NAD+ composition described herein.

[0080] Another aspect of the present disclosure relates to a method for treating a subject suffering from a disease or disorder modulated by a membrane metalloendopeptidase (MME), comprising administering to the subject a therapeutically effective amount of an NAD+ composition described herein.

[0081] Another aspect of the present disclosure relates to a method for increasing the plasma proteome in a subject in need thereof, comprising administering to the subject an amount of an NAD+ composition described herein effective to increase the plasma proteome in the subject.

[0082] In another aspect, the NAD+ composition is provided as a component in a liquid formulation for use in treating a subject suffering from an age-related disorder and / or for use in treating a subject suffering from a CNS disease or disorder. In some embodiments, the CNS disorder is drug addiction. In another embodiment, the CNS disorder is substance abuse. In another aspect, the NAD+ composition is provided as a component in a liquid formulation for use in enhancing the energy of autophagy, the process by which cellular debris is removed. In another aspect, the NAD+ composition is provided as a component in a liquid formulation for use in extending lifespan and preventing disease in humans, animals, and / or plants.

[0083] Another aspect of the present disclosure relates to a method of promoting cellular NAD+ metabolism or NAD+ cell homeostasis in a subject, comprising administering to the subject an amount of a composition described herein effective to promote NAD+ metabolism or NAD+ cell homeostasis.

[0084] 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 a composition described herein, wherein the at least one condition is elevated blood pressure, hyperglycemia, excess body fat around the waist, abnormal cholesterol or triglyceride levels, or any combination thereof.

[0085] Another aspect of the present disclosure relates to a method of increasing the amount of at least one functional polypeptide 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 a composition described herein effective to increase the amount of at least one functional polypeptide of SIRT1, PLB1, NPL, ENPP5, GLSTA, GALNT3, FABP1, NRCAM, NLGN2, ARTN, UPB1, MME, CTSB, CTSL, or HRAS polypeptide.

[0086] Certain APIs, such as thyroid hormones, contain bound water, which can be removed by azeotroping the solvent to provide product stability. In some embodiments, the thyroid hormone is dissolved in a solvent and combined with pharmaceutically acceptable excipients to form a mixture, and the solvent is removed under vacuum, resulting in a powder suitable for further processing.

[0087] The foregoing and other objects of the present disclosure, its various features, as well as the disclosure itself, may be more fully understood from the following description when read in conjunction with the accompanying drawings, in which: [Brief explanation of the drawings]

[0088] [Figure 1] FIG. 1 is a graph demonstrating the intracellular increase in NAD following treatment with an illustrative NAD+ composition (“A”) compared to placebo treatment (“B”).

[0089] [Figure 2] FIG. 2 is a graph depicting the effect of treatment with an illustrative NAD+ composition (“A”) compared to placebo treatment (“B”) on total bilirubin (μmol / L) over time.

[0090] [Figure 3] FIG. 3 is a graph depicting the effect of treatment with an illustrative NAD+ composition (“A”) compared to placebo treatment (“B”) on total bilirubin (μmol / L) over time.

[0091] [Figure 4] FIG. 4 is a graph depicting the effect of treatment with an illustrative NAD+ composition (“A”) compared to placebo treatment (“B”) on total bilirubin (μmol / L) over a series of time points (days).

[0092] [Figure 5] FIG. 5 is a graph depicting the effect of treatment with an exemplary NAD+ composition (“A”) on alkaline phosphatase as measured by enzyme activity in liver and units / liter compared to placebo treatment (“B”) over time.

[0093] [Figure 6] FIG. 6 is a graph depicting the effect of treatment with an exemplary NAD+ composition (“A”) on alkaline phosphatase as measured by enzyme activity in liver and units / liter compared to placebo treatment (“B”) over time.

[0094] [Figure 7] FIG. 7 is a graph depicting the effect of treatment with an illustrative NAD+ composition (“A”) on alkaline phosphatase as measured by enzyme activity in liver and units / liter compared to placebo treatment (“B”) over a series of time points.

[0095] [Figure 8]FIG. 8 is a graph depicting the effect of treatment with an illustrative NAD+ composition (“A”) compared to placebo treatment (“B”) on gamma-glutamyltransferase (GGT) over time.

[0096] [Figure 9] FIG. 9 is a graph depicting the effect of treatment with an illustrative NAD+ composition (“A”) compared to placebo treatment (“B”) on gamma-glutamyltransferase (GGT) over time.

[0097] [Figure 10] FIG. 10 is a graph depicting the effect of treatment with an illustrative NAD+ composition (“A”) compared to placebo treatment (“B”) on gamma-glutamyltransferase (GGT) over a series of time points.

[0098] [Figure 11] FIG. 11 is a graph depicting the effect of treatment with an illustrative NAD+ composition (“A”) compared to placebo treatment (“B”) on the LDL / HDL ratio over time.

[0099] [Figure 12] FIG. 12 is a graph depicting the effect of treatment with an illustrative NAD+ composition (“A”) compared to placebo treatment (“B”) on the LDL / HDL ratio over time.

[0100] [Figure 13] FIG. 13 is a graph depicting the effect of treatment with an illustrative NAD+ composition (“A”) compared to placebo treatment (“B”) on the LDL / HDL ratio over a range of time points for treatment.

[0101] [Figure 14] FIG. 14 is a schematic diagram of NAD+ metabolism.

[0102] [Figure 15]Figure 15A is a graph depicting plasma concentrations of the 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 = 7.59 x 10-11.

[0103] Figure 15B is a graph depicting plasma concentrations of the NAD metabolite, N1-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 = 1.03 x 10 -13 .

[0104] [Figure 16] FIG. 16 is a panel of graphs depicting plasma concentrations 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).

[0105] [Figure 17] Figure 17 is a graph depicting the set of the top 25 enriched metabolites 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.

[0106] [Figure 18] FIG. 18 is a collection of graphs demonstrating the intracellular increase in NAD following treatment with illustrative NAD+ compositions compared to placebo treatment and the change relative to baseline (“BL”; pre-treatment) for individual participants within each treatment group.

[0107] [Figure 19] FIG. 19 is a collection of graphs depicting plasma concentrations of the 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.

[0108] [Figure 20] FIG. 20 is a collection of graphs showing plasma concentrations of the NAD metabolite, N1-methyl-2-pyridone-5-carboxamide, measured in NAD- or placebo-treated participants at three time points during the study described in Examples 5 and 6.

[0109] [Figure 21] FIG. 21 is a collection of graphs depicting the plasma concentrations of SIRT1 measured in NAD+-treated 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.

[0110] [Figure 22] FIG. 22 is a collection of graphs showing plasma concentrations of methionine synthase (MTR) measured in NAD+-treated or placebo-treated participants at three time points during the study described in Examples 5 and 6.

[0111] [Figure 23] FIG. 23 is a panel of graphs showing plasma concentrations of glutathione S-transferase alpha-1 (GSTA1) measured in NAD+-treated or placebo-treated participants at three time points during the study described in Examples 5 and 6.

[0112] [Figure 24] FIG. 24 is a panel of graphs representing plasma concentrations of mitochondrial superoxide dismutase 2 measured in NAD+-treated or placebo-treated participants at three time points during the study described in Examples 5 and 6.

[0113] [Figure 25] FIG. 25 is a panel of graphs representing plasma concentrations of hypoxanthine measured in NAD+-treated or placebo-treated participants at three time points during the study described in Examples 5 and 6.

[0114] [Figure 26] FIG. 26 is a panel of graphs representing plasma concentrations of gamma-glutamyltransferase (GGT) measured in NAD+-treated or placebo-treated participants at three time points during the study described in Examples 5 and 6.

[0115] [Figure 27] FIG. 27 is a panel of graphs representing plasma concentrations of total bilirubin (TBILI) measured in NAD+-treated or placebo-treated participants at three time points during the study described in Examples 5 and 6.

[0116] [Figure 28] FIG. 28 is a table summarizing changes in several proteins that modulate neuronal signaling pathways or neuronal growth following treatment with NAD+ during the studies described in Examples 5 and 6.

[0117] [Figure 29] FIG. 29 is a table summarizing the changes in several immune and inflammation-related proteins following treatment with NAD+ during the study described in Examples 5 and 6.

[0118] [Figure 30] FIG. 30 is a panel of graphs representing plasma concentrations 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.

[0119] [Figure 31]FIG. 31 is a panel of graphs representing plasma concentrations 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.

[0120] [Figure 32-1] Figure 32A is a graph of the TGA thermogram for Sample 6, Replicate 1 (Sample 6-1). Figure 32B is a graph of the TGA thermogram for Sample 6, Replicate 2 (Sample 6-2). Figure 32C is an overlay of the TGA thermograms for Sample 6 Replicates (Samples 6-1 and 6-2). [Figure 32-2] Same as above. [Figure 32-3] Same as above.

[0121] [Figure 33-1] Figure 33A is a graph of the TGA thermogram for Sample 7, Replicate 1 (Sample 7-1). Figure 33B is a graph of the TGA thermogram for Sample 7, Replicate 2 (Sample 7-2). Figure 33C is an overlay of the TGA thermograms for Sample 7 replicates (Samples 7-1 and 7-2). [Figure 33-2] Same as above. [Figure 33-3] Same as above.

[0122] [Figure 34-1] Figure 34A is a graph of the TGA thermogram for Sample 8, Replicate 1 (Sample 8-1). Figure 34B is a graph of the TGA thermogram for Sample 8, Replicate 2 (Sample 8-2). Figure 34C is an overlay of the TGA thermograms for Sample 8 Replicates (Samples 8-1, 8-2). [Figure 34-2] Same as above. [Figure 34-3] Same as above.

[0123] [Figure 35-1]Figure 35A is a graph of the TGA thermogram for Sample 9, Replicate 1 (Sample 9-1). Figure 35B is a graph of the TGA thermogram for Sample 9, Replicate 2 (Sample 9-2). Figure 35C is an overlay of the TGA thermograms for Sample 9 Replicates (Samples 9-1, 9-2). [Figure 35-2] Same as above. [Figure 35-3] Same as above.

[0124] [Figure 36] FIG. 36 is an overlay of the TGA thermograms of samples 6, 7, 8 and 9. DETAILED DESCRIPTION OF THE INVENTION

[0125] Detailed Description The present disclosure relates to enhanced NAD+ compositions useful in treating diseases and disorders regulated by intracellular levels of NAD. The present disclosure further relates to methods for producing NAD+ drug products that are stable, safer to use, and have a longer shelf life. The present disclosure also provides novel methods for increasing intracellular NAD and methods for treating central nervous system diseases and disorders, including drug addiction and substance abuse.

[0126] The present disclosure provides enhanced delivery of NAD+ to target cells for treating substance abuse, including but not limited to, drug or alcohol addiction.

[0127] NAD+ is known to be chemically and biologically unstable. The present disclosure provides herein NAD+ compositions with enhanced stability compared to traditional NAD+ compositions. For example, exemplary 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 less stringent storage requirements (e.g., refrigerated or dry storage) compared to control NAD+ compositions (e.g., NAD+ compositions from commercial sources) that do not have the properties described herein or are not produced by the conditions and processes described herein.

[0128] NAD+ is an important nutrient for animal health. The present disclosure provides NAD+ compositions with enhanced properties, which can be administered to individuals (e.g., humans) as nutrients and / or pharmaceuticals to prevent, improve, and / or treat diseases or disorders associated with insufficient icNAD in the body. In some embodiments, the enhanced NAD+ compositions described herein increase icNAD concentrations and / or NAD+ degradation in an individual. In some embodiments, the individual is consuming a tryptophan-deficient diet. In some embodiments, the individual has a genetic mutation and / or other mechanisms (e.g., living or environmental conditions, dietary or drug effects, etc.) that result in reduced or inhibited NAD production, reduced NAD stability, and / or increased 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 back to NAD. The individual may have another genetic mutation that affects the production, transport, storage, and / or utilization of NAD in the individual. The enhanced NAD+ compositions described herein can enhance NAD-related biochemical and biological reactions in individuals in need thereof. For example, because NAD is a cofactor for oxidoreductases, the enhanced NAD+ compositions described herein can enhance the biological function of oxidoreductases in individuals, e.g., enhancing synthetic enzymes such as poly(ADP-ribose) polymerase and cADP-ribose synthase. Thus, if an individual has a disease or disorder associated with insufficient poly(ADP-ribose) polymerase and cADP-ribose synthase or other genes or gene products downstream of or affected by NAD, the individual can be administered the enhanced NAD+ compositions described herein to prevent, ameliorate, and / or treat the disease or disorder. Furthermore, the enhanced NAD+ compositions described herein, due to their enhanced stability, can be a substitute for current commercially available NAD+ compositions or other NAD+ compositions that do not have the same properties or are not produced by current conditions or processes.For example, because NAD+ is unstable even under non-extreme conditions, such as in aqueous solution, the enhanced NAD+ compositions described herein can be used as supplements and / or pharmaceuticals with improved bioavailability for oral administration (or by other routes of administration) to individuals. Compared to traditional NAD+ compositions, the enhanced NAD+ compositions described herein can be more stable in direct interaction with water, radiation, oxygen, and / or UV light, and can be resistant to hydrolysis in the small intestine by brush border cells.

[0129] 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 in the art as of the date of the disclosure set forth and claimed herein. In the event of any conflict between the patents, patent applications, and publications and the present disclosure, the present disclosure will control.

[0130] 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. Unless otherwise stated, the first definition provided for a group or term herein applies to that group or term throughout this specification, either individually or as part of another group.

[0131] In the remainder of the disclosure, repeated use of the phrase "in one embodiment" does not necessarily refer to the same embodiment.

[0132] As used herein, the articles "a" and "an" refer to one or to more than one (i.e., to 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.

[0133] As used herein, the term "about" is understood by those skilled in the art and varies to some extent depending on the context in which it is used. As used herein, when referring to a measurable value such as an amount, a time period, etc., the term "about" means to encompass a variation of ±20% or ±10%, including ±5%, ±1%, and ±0.1% from the specified value, and therefore, the variation is appropriate for carrying out the disclosed method.

[0134] As used herein, the term "NAD" refers to "nicotinamide adenine dinucleotide" and encompasses NAD+ and / or NADH. As used in this disclosure, the term "NAD+" describes a composition consisting of NAD+.

[0135] The terms "composition," "enhanced NAD+ composition," "formulation," and "enhanced NAD+ pharmaceutical formulation," as used in this disclosure, include compositions and pharmaceutical formulations containing NAD+ prepared according to this disclosure.

[0136] "Vacuumizing" or "vacuumization" is a procedure for purifying active principles, components (i.e., excipients), intermediates, secondary compounds, ingredients, or components, and final products (e.g., but not limited to, powders and tablets). Vacuuming consists of subjecting a composition or component mixture to reduced pressure or vacuum, followed by replacing the vacuum with an inert gas or CO2. In some embodiments, one vacuum cycle involves subjecting a composition or component mixture to reduced pressure or vacuum, followed by replacing the vacuum with an inert gas or CO2. The cycle begins with a vacuum under a specific pressure, followed by subsequent and sequential ones that replace the air with an inert gas to compensate or resaturate the pressure created by the vacuum at a similar pressure. These vacuum cycles azeotrope volatile contaminants from the active pharmaceutical ingredient (API) and associated excipients. In the non-limiting examples described herein, NAD+ and combinations of NAD+ and PEG are vacuumized to provide the compositions and pharmaceutical formulations described herein.

[0137] "NAD+ monohydrate" or "β-NAD [β-nicotinamide adenine dinucleotide, oxidized free acid hydrate]" is a compound that is commercially available and may contain phosphorus.

[0138] The term "blended" refers to mixed components or ingredients, e.g., NAD+ is mixed with secondary components, e.g., various weights of one or more excipients (e.g., but not limited to, PEG, including preformulated PEG), to achieve a homogenous distribution of the active ingredient in the final drug product. The mixing and blending described herein prevents small amounts of the active agent (NAD+) from unintentionally distributing into or becoming concentrated in small compartments or portions of the formulation device surface due to irregularities in particle size distribution and / or small dimensions and depressions on the surface of the blending device.

[0139] The term "substrate" or "excipient substrate" refers to a therapeutically suitable excipient that can be used to bind or immobilize an active agent or drug to its surface through non-covalent intermolecular forces or interactions according to the methods of the present disclosure. The optimal selection of the substrate can vary depending on the solvent and the active agent or drug, so that the active agent or drug can interact with the substrate and become immobilized thereon. The substrate can also be selected depending on the final dosage form of the product. Any classic therapeutically acceptable excipient(s), such as microcrystalline cellulose, can be used as a substrate according to the present disclosure because they are (i) known to be non-toxic and therefore compatible with GRAS compounds, (ii) compatible with a wide range of solvents, and (iii) are believed to exhibit a surface topology (e.g., but not limited to, porosity) and surface chemistry suitable for drug immobilization without covalent chemical interactions. The compound or drug immobilized on microcrystalline cellulose is, for example, compatible with other excipients and can be blended with other excipients to create a formulation, which can be formed into a tablet by direct compression. Powder streams of microcrystalline cellulose with immobilized active agents or drugs may also be used to fill capsules.

[0140] The term "decompose," as used herein, means to chemically break down. Decomposition differs from melting in that it involves breaking chemical bonds or forming new chemical bonds. Methods of decomposition can include hydrolysis, solventolysis, pyrolysis, or oxidation.

[0141] A "subject" or "patient" can be a mammal, such as, 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.

[0142] "Effective amount," when used in connection with a pharmaceutical preparation, encompasses an amount that produces a particular result, such as reducing or eliminating at least one symptom or negative effect of a disorder or disease, or prevents the onset of a disease or disorder in a subject described herein. An effective amount also refers to an amount that can produce a predetermined or preselected result, change, or activity.

[0143] The term "carrier," as used in this disclosure, encompasses carriers, excipients, and diluents, and refers to a material, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, solvent, or encapsulating material, that is involved in carrying or transporting a pharmaceutical agent from one organ or part of the body to another organ or part of the body of a subject. A "carrier" can be used to manipulate the release rate of an API (e.g., NAD+) to include rapid release or sustained release.

[0144] As used herein, the terms "treat" and "treatment" are synonymous with the term "prevent" and are meant to refer to postponing the onset of a disease, preventing the onset of a disease, and / or reducing the severity of symptoms that have occurred or are predicted to occur. Thus, these terms include alleviating existing disease symptoms, preventing additional symptoms, alleviating or preventing the underlying cause of symptoms, inhibiting a disorder or disease, for example, stopping the onset of a disorder or disease, relieving a disorder or disease, causing regression of a disorder or disease, alleviating the condition caused by a disease or disorder, or stopping or alleviating the symptoms of a disease or disorder.

[0145] The term "disorder" is used in this disclosure to mean, and is used interchangeably with, the term disease, condition, or illness, unless otherwise specified. The terms "administer," "administering," or "administration," as used in this disclosure, refer to any direct administration of a pharmaceutical formulation to a subject. The term "container" encompasses any vessel, dish, or other structure that can hold NAD+ or a mixture of NAD+ and any other chemical components and that can withstand reduced pressure or vacuum. A container can also be a blender or vessel that allows for mixing of the components.

[0146] The terms "administer," "administering," or "administration," as used in this disclosure, refer to administering to a subject directly a disclosed salt or composition, or to administering to a subject a prodrug derivative or analog of the salt or composition that is capable of forming an equivalent amount of the active salt in the subject's body.

[0147] The present disclosure relates to enhanced NAD+ compositions and pharmaceutical formulations containing the same, which are useful in treating diseases, disorders, and conditions associated with reduced intracellular NAD levels. The disclosure further relates to methods for producing stable, therapeutic, and bioavailable enhanced NAD+ compositions and pharmaceutical formulations or "drug products." The disclosure also provides novel methods for increasing intracellular levels of NAD and treating various disorders associated with reduced intracellular NAD. For example, the disclosure provides enhanced delivery of NAD+ to target cells for treating substance abuse, including, but not limited to, drug or alcohol addiction. Also disclosed are methods for detecting biomarkers associated with reduced intracellular levels of NAD.

[0148] NAD+ is known to be chemically and biologically unstable. The present disclosure provides NAD+ compositions and formulations herein that have enhanced stability compared to traditional NAD+ compositions. For example, exemplary 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 less stringent storage requirements (e.g., refrigerated or dry storage) compared to control NAD+ compositions (e.g., NAD+ compositions from commercial sources) that do not have the properties described herein or are not produced by the conditions and processes described herein. Enhanced NAD+ Composition

[0149] The present disclosure provides NAD+ compositions with enhanced stability and bioavailability compared to NAD+ (e.g., NAD+ monohydrate)-containing compositions. The 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 less stringent storage condition requirements (e.g., but not limited to, refrigerated or dry storage) compared to control NAD+ not produced by the conditions and processes described herein.

[0150] The chemical name and structure of NAD+ are shown below: [ka]

[0151] In some embodiments, 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-1-ium-1-yl)-3,4-dihydroxyoxolan-2-yl]methyl hydrogen phosphate. In some embodiments, the chemical formula of Formula I is 21 H 28 N7O 14 P2 + In some embodiments, the molecular weight of protonated NAD+ (positively charged, anhydrous) is 664.44 g / mol. In some embodiments, the CASRN of Formula I is 53-84-9 and the source is PubChem (CID 5893). In some embodiments, the molecular weight of NAD+ (zwitterion, anhydrous) is 663.43 g / mol.

[0152] The enhanced NAD+ compositions described herein, due to their enhanced stability, may be a substitute for current commercially available NAD+ compositions or other NAD+ compositions that do not have the properties or are not produced by current conditions or processes. For example, because NAD+ is unstable even under non-extreme conditions, such as in aqueous solution, the enhanced NAD+ compositions described herein may be used as supplements and / or pharmaceuticals with improved bioavailability for oral administration (or by other routes of administration) to individuals. Compared to commercially available NAD+ compositions, the enhanced NAD+ compositions described herein may be more stable in direct interaction with water, radiation, oxygen, and / or UV light.

[0153] This compound is the active ingredient in various enhanced compositions and pharmaceutical formulations of the present disclosure (described below) and is present in an amount of 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 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 amount 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 about 200 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.

[0154] Enhanced NAD+ compositions according to the present disclosure may also contain phosphorus as a minor component originally derived from commercially available NAD+ (e.g., NAD+ monohydrate) and remaining after the vacuumization process (described below) or which may be added. In some embodiments of the present disclosure, the phosphorus content is about 0.5% to about 9.33%. For example, enhanced NAD+ compositions may contain about 0.5% (wt / wt) to about 5.5% (wt / wt). In some embodiments, the phosphorus content is about 1.0% to about 5.0%. In some embodiments, the phosphorus content is about 4.5% to about 5.0%. In some embodiments, the phosphorus content is about 3.5% to about 6.0%. In some embodiments, the phosphorus content is about 3.0% to about 5.5%. In some embodiments, the phosphorus content is about 4.0% to about 5.5%. In some embodiments, the phosphorus content is about 3.5% to about 5.5%. In certain embodiments, the phosphorus content is about 4.5% to about 4.7%. In other embodiments, the phosphorus content is about 4.5% to about 5.5%. In some embodiments, the phosphorus content is about 3.0%, about 3.5%, about 4.5%, about 4.58%, 4.6%, about 4.67%, about 4.7%, about 4.8%, or about 4.9%. In some embodiments, the phosphorus content 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%.

[0155] In a specific, non-limiting embodiment, the NAD+ composition comprises 3.0% (wt / wt) NAD+ and about to 9.33% (wt / wt) phosphorus.

[0156] In some embodiments, the enhanced NAD+ composition further contains trace amounts of an inert gas. For example, the NAD+ composition may contain trace amounts of argon, nitrogen, xenon, or helium, and may also contain anhydrous CO2.

[0157] In some embodiments, the enhanced compositions of the present disclosure contain an amount of water, moisture and / or solvent that is less than about 0.2% (w / w) as measured by standard methods (e.g., Karl Fischer titration).

[0158] In some embodiments, the enhanced NAD+ composition of the present disclosure has a concentration of about 0.51 g / cm 3 In some embodiments, the NAD+ compositions of the present disclosure have a density of about 0.4 g / cm 3 In some embodiments, the enhanced NAD+ compositions of the present disclosure have a density of about 0.27 g / cm 3 ~Approx. 0.51g / cm 3 In some embodiments, the enhanced NAD+ compositions of the present disclosure have a density of about 0.52 g / cm 3 It has a density of In some embodiments, the enhanced NAD+ composition of the present disclosure has a concentration of about 0.53 g / cm 3 In some embodiments, the enhanced NAD+ compositions of the present disclosure have a density of about 0.55 g / cm 3 In some embodiments, the enhanced NAD+ compositions of the present disclosure have a density of about 0.56 g / cm 3 In some embodiments, the enhanced NAD+ compositions of the present disclosure have a density of about 0.57 g / cm 3 In some embodiments, the enhanced NAD+ compositions of the present disclosure have a density of about 0.58 g / cm 3 In some embodiments, the enhanced NAD+ compositions of the present disclosure have a density of about 0.59 g / cm 3 In some embodiments, the enhanced NAD+ compositions of the present disclosure have a density of about 0.6 g / cm 3 In some embodiments, the enhanced NAD+ compositions of the present disclosure have a density of about 0.51 g / cm 3 ~Approx. 0.75g / cm 3 It has a density of

[0159] The enhanced NAD+ compositions described herein have enhanced stability relative to control NAD+. "Control NAD+" refers to NAD+ (e.g., NAD+ monohydrate) that can be synthesized or commercially available. For example, the 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+, and other exemplary enhanced NAD+ compositions according to the present 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 control NAD+. Such stability may be expressed in terms of degradation rate, half-life, shelf life, or other measures known to those of skill in the art.

[0160] The enhanced NAD+ composition of the present disclosure is also stable under physiological conditions. In some embodiments, the NAD+ composition has higher bioavailability in cells and organs compared to commercially available NAD+ supplements. In some embodiments, the NAD+ composition has higher bioavailability in target cells (e.g., liver, kidney, immune cells) compared to commercially available NAD+ supplements.

[0161] In some embodiments, the NAD+ compositions described herein exhibit slow degradation. For example, the enhanced NAD+ compositions may be stored 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), for up to 36 months without signs of degradation. In some embodiments, the enhanced NAD+ compositions have enhanced solid-state stability. In some embodiments, the enhanced NAD+ compositions of the present disclosure exhibit greater stability than commercially available NAD+ supplements.

[0162] In some embodiments, the enhanced NAD+ compositions also exhibit increased stability in aqueous solution. In some embodiments, significant degradation of the NAD+ compositions in aqueous solution does not occur for up to 72 hours. In other embodiments, degradation of the enhanced NAD+ compositions is less than 5% after 4 days in aqueous solution. The compositions have 10% or less degradation yet remain stable in aqueous solution after 5 days. Thus, in some embodiments, the enhanced NAD+ compositions have enhanced aqueous stability.

[0163] In some embodiments, the enhanced NAD+ composition of the present disclosure has a concentration of about 0.51 g / cm 3 In some embodiments, the compositions of the present disclosure have a density of about 0.4 g / cm 3 In some embodiments, the compositions of the present disclosure have a density of about 0.27 g / cm 3 ~Approx. 0.51g / cm 3 In some embodiments, the compositions of the present disclosure have a density of about 0.52 g / cm 3 In some embodiments, the compositions of the present disclosure have a density of about 0.53 g / cm 3 In some embodiments, the compositions of the present disclosure have a density of about 0.55 g / cm 3 In some embodiments, the compositions of the present disclosure have a density of about 0.56 g / cm 3 In some embodiments, the compositions of the present disclosure have a density of about 0.57 g / cm 3 In some embodiments, the compositions of the present disclosure have a density of about 0.58 g / cm 3 In some embodiments, the compositions of the present disclosure have a density of about 0.59 g / cm 3 In some embodiments, the compositions of the present disclosure have a density of about 0.6 g / cm 3 In some embodiments, the compositions of the present disclosure have a density of about 0.51 g / cm 3 ~Approx. 0.75g / cm 3 It has a density of

[0164] In some embodiments, the enhanced NAD+ composition of the present disclosure comprises a crystalline component comprising polyethylene glycol and an amorphous component comprising amorphous NAD+. In some embodiments, the enhanced NAD+ composition further comprises 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 between about 300 and about 4000. In some embodiments, the polyethylene glycol is PEG3350.

[0165] In some embodiments of the enhanced NAD+ compositions of the present disclosure, which include a crystalline component that includes polyethylene glycol and an amorphous component that includes amorphous NAD+, the composition is characterized by having a first onset temperature between 179.39°C (±0.10°C) and 182.61°C (±0.10°C) by thermogravimetric analysis (TGA) thermogram.

[0166] In some embodiments of the enhanced NAD+ compositions of the present disclosure, which include a crystalline component comprising polyethylene glycol and an amorphous component comprising amorphous NAD+, the compositions are characterized by having a weight loss of 14.39% to 16.98% by weight over the range of about 170°C to about 300°C according to a thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 300°C under nitrogen gas at a ramp rate of 10°C / min.

[0167] In some embodiments of the enhanced NAD+ compositions of the present disclosure, which include a crystalline component comprising polyethylene glycol and an amorphous component comprising amorphous NAD+, the compositions are characterized by having a weight loss of 57.83% to 58.55% by weight over the range of about 300°C to about 800°C according to a thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 800°C under nitrogen gas at a ramp rate of 10°C / min.

[0168] In some embodiments of 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 Figures 33A, 33B, 34A, 34B, 35A, or 35B. In some embodiments, the TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with Figure 33A or 33B. In some embodiments, the TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with Figure 34A or 34B. In some embodiments, the TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with Figure 35A or 35B.

[0169] In some embodiments of the enhanced NAD+ compositions of the present disclosure that include a crystalline component that includes polyethylene glycol and an amorphous component that includes amorphous NAD+, the composition comprises: (1) a first weight loss of 2.42 wt % to 3.25 wt % in the range of ambient temperature to about 170°C, as measured by a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to at least 170°C; and / or (2) a second weight loss of 13.66 wt % to 14.61 wt % in the range of about 170° C. to about 300° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to at least 300° C.; and / or (3) a third weight loss of 58.73 wt % to 60.37 wt % in the range of about 300°C to about 800°C according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to at least 800°C; and / or (4) a fourth weight loss of 20.14 wt % to 21.46 wt % in the range of about 800° C. to about 900° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to about 800° C. and under air at a ramp rate of 10° C. / min from about 800° C. to about 900° C.; and / or (5) a first starting temperature of 179.39°C to 180.15°C, and / or (6) Second starting temperature: 369.03℃~370.82℃ The thermogravimetric analysis (TGA) thermogram has the following formula: In some embodiments, the composition is characterized by one feature selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by two features selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by three features selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by four features selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by five features selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by all six features selected from (1), (2), (3), (4), (5), and (6).

[0170] In some embodiments of the enhanced NAD+ compositions of the present disclosure that include a crystalline component that includes polyethylene glycol and an amorphous component that includes amorphous NAD+, the composition comprises: (1) a first weight loss of 2.32 wt % to 3.55 wt % in the range of ambient temperature to about 170°C, as measured by a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to at least 170°C; and / or (2) a second weight loss of 14.39 wt % to 16.22 wt % in the range of about 170° C. to about 300° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to at least 300° C.; and / or (3) a third weight loss of 57.83 wt % to 58.55 wt % in the range of about 300° C. to about 800° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to at least 800° C.; and / or (4) a fourth weight loss of 22.62 wt % to 22.76 wt % in the range of about 800° C. to about 900° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to about 800° C. and under air at a ramp rate of 10° C. / min from about 800° C. to about 900° C.; and / or (5) a first starting temperature of 178.62°C to 180.11°C, and / or (6) Second starting temperature: 365.73℃~370.47℃ The thermogravimetric analysis (TGA) thermogram has the following formula: In some embodiments, the composition is characterized by one feature selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by two features selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by three features selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by four features selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by five features selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by all six features selected from (1), (2), (3), (4), (5), and (6).

[0171] In some embodiments of the enhanced NAD+ compositions of the present disclosure that include a crystalline component that includes polyethylene glycol and an amorphous component that includes amorphous NAD+, the composition comprises: (1) a first weight loss of 3.62 wt % to 3.96 wt % in the range of ambient temperature to about 170°C, as measured by a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to at least 170°C; and / or (2) a second weight loss of 15.17 wt % to 16.98 wt % in the range of about 170° C. to about 300° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to at least 300° C.; and / or (3) a third weight loss of 58.05 wt % to 59.66 wt % in the range of about 300° C. to about 800° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to at least 800° C.; and / or (4) a fourth weight loss of 22.85 wt % to 25.87 wt % in the range of about 800° C. to about 900° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to about 800° C. and under air at a ramp rate of 10° C. / min from about 800° C. to about 900° C.; and / or (5) a first starting temperature of 179.83°C to 182.61°C, and / or (6) Second starting temperature: 363.24℃~366.16℃ The thermogravimetric analysis (TGA) thermogram has the following formula:

[0172] In some embodiments, the composition is characterized by one feature selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by two features selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by three features selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by four features selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by five features selected from (1), (2), (3), (4), (5), and (6). In some embodiments, the composition is characterized by all six features selected from (1), (2), (3), (4), (5), and (6). Pharmaceutical preparations

[0173] The present disclosure also provides pharmaceutical formulations comprising NAD compositions with enhanced properties, which can be administered to individuals (e.g., humans) as nutrients and / or pharmaceuticals to prevent, ameliorate, and / or treat diseases or disorders associated with insufficient NAD in the body. In some embodiments, the enhanced NAD+ pharmaceutical formulations described herein increase NAD concentrations and / or NAD degradation in an individual. In some embodiments, the individual is consuming a tryptophan-deficient 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.) that result in reduced or inhibited NAD production, reduced NAD stability, and / or increased NAD degradation or turnover in the individual. For example, an individual may have a genetic mutation in the kynurenine pathway, thus reducing or inhibiting the nicotinic acid to NMN to NAD to NAD production chain. An individual may have another genetic mutation that affects the production, transport, storage, and / or utilization of NAD+ in the individual.

[0174] The enhanced NAD+ compositions described herein can also enhance NAD-related biochemical and biological reactions in individuals in need thereof. For example, because NAD is a cofactor for oxidoreductases, the enhanced NAD+ pharmaceutical formulations described herein can enhance the biological function of oxidoreductases in individuals, e.g., enhancing synthetic enzymes such as poly(ADP-ribose) polymerase and cADP-ribose synthase. Thus, if an individual has a disease or disorder associated with insufficient poly(ADP-ribose) polymerase and cADP-ribose synthase or other genes or gene products downstream or affected by NAD, the individual can be administered the enhanced NAD+ pharmaceutical formulations described herein to prevent, ameliorate, and / or treat the disease or disorder.

[0175] Furthermore, the enhanced NAD+ pharmaceutical formulations described herein may be alternatives to current commercially available NAD+ compositions or other NAD+ compositions that do not have the properties or are not produced by current conditions or processes for enhanced stability. For example, because NAD+ is unstable even under non-extreme conditions, such as in aqueous solution, the enhanced NAD+ compositions described herein may be used as supplements and / or pharmaceuticals with improved bioavailability for oral administration to individuals (or by other routes of administration). Compared to commercially available NAD+ compositions, the enhanced NAD+ compositions described herein may be more stable in direct interaction with water, radiation, oxygen, and / or UV light, and, when administered as part of a pharmaceutical formulation, are resistant to hydrolysis in the small intestine by brush border cells.

[0176] The enhanced NAD+ pharmaceutical formulations of the present disclosure include the enhanced NAD+ composition described above and at least one additional secondary component, compound, or ingredient that can assist in the administration and / or treatment of a subject.

[0177] In some embodiments, this component is PEG (a polymer of ethylene glycol), which is "Generally Regarded as Safe" (GRAS) under the U.S. Federal Drug Administration (FDA). PEGs come in a variety of sizes, depending on their molecular weight, which is indicated by the term "PEG." In some embodiments, enhanced NAD+ compositions include PEG300, PEG400, PEGMME (polyethylene glycol monomethyl ether) 550, PEG600, PEG1000, PEGMME2000, PEG3350, and / or PEG4000. The disclosed compositions include PEG of any molecular weight at a concentration of about 10% to about 75%. In some embodiments, the compositions include 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%.

[0178] In some embodiments, the ratio of any molecular weight of NAD+ to PEG in the NAD+ compositions of the present disclosure is 1:1 (wt / wt), 1:2 (wt / wt), 1:3 (wt / wt), 1:4 (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), 1:13 (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).

[0179] In some embodiments, the ratio of any molecular weight of NAD+ to PEG 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).

[0180] The enhanced NAD+ pharmaceutical formulations of the present disclosure are also stable under physiological conditions. In some embodiments, these formulations have higher bioavailability in cells and organs 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 increase intracellular NAD (Matasic et al., 2018). In some embodiments, the present NAD+ formulations have higher bioavailability in target cells (e.g., liver, kidney, immune cells) compared to commercially available NAD+ supplements.

[0181] These formulations can be in solid as well as liquid dosage forms, such as tablets, capsules, powders, troches, suppositories, syrups, elixirs, sterile solutions, suspensions or emulsions, pastes, ointments, jellies, waxes, oils, lipids, encapsulation in lipid (cationic or anionic)-containing vesicles (e.g., liposomes, microparticles, microcapsules or LIPOFECTIN™), DNA conjugates, anhydrous absorbent pastes, oil-in-water and water-in-oil emulsions, carbowaxes, semi-solid gels and semi-solid mixtures containing carbowax.

[0182] The amount of pharmaceutical formulation to be administered will vary depending 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, etc., as would be known to a practitioner treating a subject.

[0183] These pharmaceutical formulations may contain carriers, excipients, flavoring agents, dyes and / or other agents that provide for suitable delivery, tolerance, etc. Such useful and suitable agents can be found in the formulary known to all pharmacists: Remington's Pharmaceutical Sciences, Mack Publishing Company, Easton, PA.

[0184] In some embodiments, the pharmaceutical formulation comprises about 0.5% (wt / wt) magnesium stearate, hi 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.

[0185] In still other embodiments, the tablet form may further comprise glucose, e.g., about 10% to about 75% glucose. Some non-limiting embodiments of NAD+ formulations include glucose at concentrations 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%.

[0186] 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%.

[0187] Useful dyes include, but are not limited to, Allura Red AC (Red 40) or Blue No. 1. For example, the composition can include Allura Red AC at a concentration of about 0.1% to about 3%. In some non-limiting embodiments, the composition includes 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%.

[0188] In some embodiments, the enhanced NAD+ pharmaceutical formulations of the present disclosure further comprise a flavoring agent, such as, but not limited to, methyl salicylate. For example, the methyl salicylate may be present in a concentration of about 0.1 ml / kg to about 2.0 ml / kg. In some non-limiting embodiments, the methyl salicylate may 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.

[0189] The enhanced NAD+ pharmaceutical formulation may further include other therapeutic compounds or drugs, such as, but not limited to, painkillers. For example, a powdered formulation may include acetylsalicylic acid at a concentration of about 0.1% to about 0.7%. In some non-limiting embodiments, the NAD+ formulation includes 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%.

[0190] Pharmaceutical compositions may include, but are not limited to, for example, magnesium stearate, glucose, polyethylene glycol, Red 40, methyl salicylate, Blue No. 1, or any combination thereof.

[0191] Various delivery systems are known and can be used to administer the pharmaceutical formulations described herein depending on the delivery route. For example, the pharmaceutical formulations of the present disclosure can be administered orally, anally, systemically, intramuscularly, ocularly, vaginally, bucally, subcutaneously, etc. Routes of administration and delivery systems are known to those skilled in the art. For example, NAD+ formulations in liquid form can be administered by any conventional 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, intratracheal, epidural, and oral routes, and can be administered together with other biologically active substances.

[0192] In one embodiment of the present disclosure, the compositions described herein are in powder form. Preparation Process

[0193] NAD+ was placed in a container under vacuum pressure or a vacuum between about 24 inches Hg and about 30.25 inches Hg, and the vacuum was replaced with an inert gas to increase the pressure in the container to about 1 atmosphere (atm). In some embodiments, the inert gas replacing the vacuum includes argon, helium, nitrogen, and / or xenon. The vacuum may be applied more than once, at least twice. The vacuum may be applied more than once, for example, once, twice, or at least twice.

[0194] In another embodiment, the inert gas used for evacuating includes argon. In another embodiment, the inert gas used for evacuating includes xenon. In another embodiment, the inert gas used for evacuating includes anhydrous, high purity nitrogen. In another embodiment, the inert gas used for evacuating includes helium.

[0195] NAD+ can be pre-treated before being subjected to vacuum. For example, NAD+ can be treated to obtain a pre-selected average NAD+ particle size. Treatment can include, but is not limited to, passing NAD+ through a sieve and / or grinding, milling, or jet-milling NAD+ to obtain a pre-selected particle size. In some non-limiting embodiments, NAD+ is ground or milled into particles of about 0.05 mm to about 0.4 mm in size. In some embodiments, the NAD+ particles are 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.

[0196] The enhanced NAD+ pharmaceutical preparation according to the present disclosure can be prepared by mixing NAD+ with any other components to obtain a blended mixture, which is then evacuated under vacuum and inert gas conditions.Therefore, the evacuated mixture may contain trace amounts of inert gas.For example, the obtained evacuated mixture contains trace amounts of argon, nitrogen, xenon or helium. Preparation of enhanced pharmaceutical formulations

[0197] An API such as that according to the present disclosure can be initiated by combining vacuumized or non-vacuumized NAD+ with other components in a container, such as a blender, to form a blended mixture. The term "blended mixture" refers to the components of a mixed composition, such as varying amounts of one or more components and another component.

[0198] In one embodiment, NAD+ is mixed with PEG of any molecular weight, which may be commercially obtained and optionally pre-treated as described herein to achieve a homogenous distribution of NAD+ and PEG in the final pharmaceutical formulation (or "drug product") to obtain a pre-selected particle size. In some embodiments, commercially available PEG of any particular molecular weight or weight, as described above, is GRAS under the FDA and is used in the preparation.

[0199] Blending as described herein prevents small amounts of NAD+ from unintentionally distributing into or becoming concentrated in small sections or portions of the formulation device surface due to irregularities in particle size distribution and / or small dimensions and depressions on the surface of the blending device.

[0200] In some embodiments of the present disclosure, the preparation process involves pre-forming or pre-treating commercially available NAD+ and / or PEG. In some embodiments, the pre-treated NAD+ and / or PEG are vacuumed before mixing. Alternatively, the NAD+ or PEG are pre-treated and vacuumed before mixing. Alternatively, both the NAD+ and PEG are vacuumed separately before mixing, before or after sizing, and / or before, and / or before mixing.

[0201] The processing or pre-processing of NAD+ is carried out in a manner that allows for homogeneous distribution with excipients in the NAD+ formulations of the present disclosure.

[0202] In some non-limiting embodiments, the NAD+ is ground or milled into particles of about 0.05 mm to about 0.4 mm in size.

[0203] Another aspect of the present disclosure relates to a process for making an enhanced NAD+ composition, said process comprising blending NAD+ and polyethylene glycol and evacuating the blended mixture of NAD+ and PEG under vacuum and / or inert gas conditions.

[0204] In one embodiment of the present disclosure, the preparation process includes pre-forming the NAD+. In certain embodiments, pre-forming the NAD+ includes grinding or milling the NAD+. In certain 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 NAD+ formulations of the present disclosure.

[0205] In yet another embodiment of the present disclosure, the process of pre-forming NAD+ involves grinding or milling the NAD+ and then vacuuming the ground or milled NAD+ product itself before blending.

[0206] In some embodiments of the present disclosure, the preparation process further comprises pre-forming commercially available PEG. In some embodiments, pre-forming the PEG comprises grinding or milling the commercially available PEG. In certain embodiments, grinding and / or milling the commercially available PEG is performed to reduce the commercially available PEG to ultrafine-sized particles to allow for homogeneous distribution with the excipients and active ingredient (NAD+) in the NAD+ formulations of the present disclosure.

[0207] In yet another embodiment of the process of preparation, pre-forming the PEG involves vacuuming commercially available PEG and then grinding or milling the vacuumed PEG product itself before blending.

[0208] In some embodiments, the pre-treated NAD+ and / or PEG are vacuumed before mixing. Alternatively, the NAD+ or PEG are pre-treated and vacuumed before mixing. Alternatively, both the NAD+ and PEG are vacuumed separately before mixing, before or after sizing, and / or before, and / or before mixing.

[0209] In some embodiments, the mixing process includes mixing NAD+ and PEG or pre-treated NAD+ and / or pre-treated PEG. Mixing NAD+ with PEG allows, for example, non-covalent coating or immobilization of NAD+ to PEG, stabilizing NAD+ and preventing its degradation. Mixing can be performed, for example, at room temperature (RT) for about 20 minutes to about 1 hour. 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.

[0210] The blended mixture of NAD+ and PEG can then be evacuated by placing it under a vacuum or reduced pressure cycle, followed by inert gas resaturation.

[0211] In some embodiments of the process of preparation, the blended mixture of NAD+ and PEG is placed under a vacuum of about 22 inches Hg to about 29 inches Hg. In some embodiments of the process of preparation, the blended mixture of NAD+ and PEG is placed under a vacuum of about 26 inches Hg to about 30 inches Hg. In some embodiments, the blended mixture of NAD+ and PEG is placed under a vacuum of about 24 inches Hg to about 29.92 inches Hg. In some embodiments, the blended mixture of NAD+ and PEG is placed under a vacuum of about 24 inches Hg to about 28 inches Hg. In some embodiments, the blended mixture of NAD+ and PEG is placed under a vacuum of about 24 inches Hg. In some embodiments, the blended mixture of NAD+ and PEG is placed under a vacuum of about 26 inches Hg. In some embodiments, the blended mixture of NAD+ and PEG is placed under a vacuum of about 28 inches Hg. In some embodiments, the blended mixture of NAD+ and PEG is placed under a vacuum of about 30 inches of Hg. In some embodiments, the blended mixture of NAD+ and PEG is placed under a vacuum of about 30.25 inches of Hg. Without intending to be limited to any particular explanation, removal of the solvent also results in azeotropic and / or evaporative removal of contaminating components such as, but not limited to, water, air, oxygen, etc., that accelerate decomposition of the NAD+ product.

[0212] As noted above, the process of evacuation may also include subjecting the blended mixture of NAD+ and PEG to cycles of vacuum or reduced pressure, followed by inert gas resaturation.

[0213] According to embodiments of the present disclosure, non-covalent coating or immobilization of an active agent (e.g., NAD+) on a selected PEG substrate stabilizes the active agent and prevents decomposition. While not intending to be limited to any particular explanation, further stabilization of the product is believed to occur through azeotropic and / or evaporative removal of contaminating components, such as, but not limited to, water and oxygen, during solvent evaporation, and the vacuum can be replaced with an inert, anhydrous gas, such as, but not limited to, nitrogen (N2), argon (Ar), or xenon (Xe). In some embodiments of the disclosed method, the blended mixture under vacuum is resaturated with an inert gas to restore the pressure in the blender to 1 atm. In some embodiments, the inert gas used in resaturation during the evacuation process includes nitrogen, argon, xenon, or helium, any of which can be highly pure. Other replacement gases can also be used to achieve additional benefits. For example, anhydrous carbon dioxide (CO2) can be used as an inert displacement gas which can serve the additional purpose of acidifying the local environment of an active agent or drug substrate that is more stable in an acidic environment.

[0214] It is understood that the presence of inert gas or anhydrous CO2 in the vacuum process azeotropes impurities and volatile contaminants and / or residues present in the original NAD+ and PEG, thereby allowing for the removal of any remaining elements, such as, but not limited to, water, air, oxygen, and solvents.

[0215] The blended mixture is evacuated for at least one cycle. In other embodiments, the blended mixture is evacuated for at least two cycles. In other embodiments, the blended mixture is evacuated for at least three, four, or more cycles.

[0216] In one embodiment of the present disclosure, the commercially available PEG used in the preparation is GRAS under the FDA. In some embodiments, the commercially available PEG used in the preparation is PEG300. In other embodiments, the commercially available PEG used in the preparation is PEG400. In still other embodiments, the commercially available PEG used in the preparation is PEGMME550. In still other embodiments, the commercially available PEG used in the preparation is PEG600. In other embodiments, the commercially available PEG used in the preparation is PEG1000. In some embodiments, the commercially available PEG used in the preparation is PEGMME2000. In still other embodiments, the commercially available PEG used in the preparation is PEG3350. In certain embodiments, the commercially available PEG used in the preparation is PEG4000. In other embodiments, the commercially available PEG used in the preparation is PEG300, PEG400, PEGMME550, PEG600, PEG1000, PEGMME2000, PEG3350, PEG4000, or any combination thereof.

[0217] In a further embodiment of the present disclosure, the process of preparation comprises blending pre-formulated NAD+ and pre-formulated PEG.

[0218] Pharmaceutical formulations according to the present disclosure can be made from the enhanced NAD+ composition 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 examples of such secondary components include magnesium stearate, glucose, Red 40, Blue No. 1, methyl salicylate, and water.

[0219] A further aspect of the present disclosure provides an enhanced NAD+ composition produced by the process of the present disclosure, comprising nicotinamide adenine dinucleotide (NAD+) and polyethylene glycol (PEG) and having a phosphorus content of about 1.0% (wt / wt) to about 9.33% (wt / wt). A further aspect of the present disclosure provides an enhanced NAD+ composition produced by the process of the present disclosure, comprising nicotinamide adenine dinucleotide (NAD+) and polyethylene glycol (PEG) and having a phosphorus content of about 1.0% to about 4.67%. A further aspect of the present disclosure provides an enhanced NAD+ composition produced by the process of the present disclosure, comprising nicotinamide adenine dinucleotide (NAD+) and polyethylene glycol (PEG) and having a phosphorus content of about 0.5% to about 9.33%. Methods of Treatment and Use

[0220] The enhanced NAD+ pharmaceutical formulations of the present disclosure can be used in the treatment of various conditions, diseases, and disorders, for example, to prevent or ameliorate anti-aging processes, in the treatment or prevention of age-related disorders, CNS disorders, and in increasing intracellular NAD levels.

[0221] In some embodiments, the enhanced NAD+ pharmaceutical formulations are potent and effective at clinically achievable doses, stable in a variety of potential dosage forms, have acceptable solubility, bioavailability, and an acceptable pH, exhibit reduced tendency to absorb and / or degrade in the presence of water, oxygen, or UV light, and exhibit ease of handling and consumption, all of which are compatible with pharmaceutical development, manufacture, and use. In some embodiments, the NAD+ composition comprises an amorphous component. Furthermore, the enhanced NAD+ pharmaceutical formulations disclosed herein provide increased cellular NAD levels, increased stability, and increased biological activity toward a more physiologically acceptable pH. Furthermore, the inclusion of PEG in the formulations herein provides a novel "molecular shuttle" for delivery of NAD+, for example, but not limited to, across the buccal membrane and into the blood. This formulation offers several advantages useful for assisting in NAD+-dependent cellular activities.

[0222] The enhanced NAD+ pharmaceutical formulations provided herein by the present disclosure can be administered to a subject (e.g., but not limited to, a human) as a nutrient and / or therapeutic agent to prevent, ameliorate, and / or treat a disease or disorder associated with insufficient NAD+ in the body.

[0223] In some embodiments, the enhanced NAD+ pharmaceutical formulation is administered to increase NAD concentration and / or NAD stability (i.e., half-life) in an individual. In some embodiments, the individual is on a tryptophan-deficient diet, has a genetic mutation and / or other characteristics (e.g., but not limited to, living or environmental conditions, food or drug effects) that result in reduced or inhibited NAD production, reduced NAD stability, and / or increased 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 reduced or inhibited production chain from nicotinic acid to NMN to NAAD to NAD. In another non-limiting example, the individual has another genetic mutation that affects the production, transport, storage, and / or utilization of NAD+ in the individual.

[0224] The enhanced NAD+ pharmaceutical formulations described herein can also enhance NAD+-related biochemistry and biological reactions in individuals in need thereof. For example, because NAD+ is a cofactor for oxidoreductases, the enhanced NAD+ compositions described herein enhance the biological function of oxidoreductases in individuals, including, but not limited to, synthetic enzymes such as poly(ADP-ribose) polymerase and cADP-ribose synthase. Thus, if an individual has a disease or disorder associated with insufficient poly(ADP-ribose) polymerase and cADP-ribose synthase or other genes or gene products downstream of or affected by NAD+, the enhanced NAD+ pharmaceutical formulation is administered to the individual to prevent, ameliorate, and / or treat the disease or disorder. How to use

[0225] Another aspect of the present disclosure relates to a method for treating or preventing age-related diseases in a subject, comprising administering to the subject a therapeutically effective amount of an NAD+-enhancing pharmaceutical formulation disclosed herein. Such formulations can be manufactured as medicines or supplements to treat numerous NAD+ deficiency-related diseases and disorders described herein, including, but not limited to, age-related diseases, central nervous system disorders, drug addiction or substance abuse, including, but not limited to, abuse associated with tobacco, heroin, opium, morphine, dihydromorphine, meperidine, codeine, cocaine, amphetamine, barbiturate, alcohol, or tranquilizer use.

[0226] In another aspect, the present disclosure relates to a method for treating a subject suffering from a central nervous system disorder or disease or for preventing a central nervous system disorder or disease in a subject, comprising administering to the subject a therapeutically acceptable amount of an NAD+ composition described herein. Yet another aspect of the present disclosure relates to a method for treating a subject suffering from drug addiction or substance abuse, comprising administering to the subject a therapeutically effective amount of an NAD+ composition described herein. In certain embodiments, the abused substance is tobacco, heroin, opium, morphine, dihydromorphine, meperidine, codeine, cocaine, amphetamine, barbiturate, alcohol, or a tranquilizer.

[0227] Other methods of treatment include methods for increasing NAD-dependent deacetylase sirtuin-1 (SIRT1), increasing cryptococcal phospholipase (PLB1), and increasing membrane metalloendopeptidase (MME) in a subject in need thereof, comprising administering to the subject an amount of an enhanced NAD+ pharmaceutical formulation the deficiency.

[0228] The present disclosure also provides a method for treating a subject suffering from a disease or disorder modulated by SIRT1, the method comprising administering to the subject an amount of an NAD+ composition described herein effective to modulate SIRT1.

[0229] 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 amount of an enhanced NAD+ pharmaceutical formulation effective to modulate PLB1.

[0230] Further provided are methods of 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+ pharmaceutical formulation described herein.

[0231] The present disclosure also relates to a method for increasing the plasma proteome in a subject in need thereof, comprising administering to a subject in need thereof an amount of an enhanced NAD+ pharmaceutical formulation described herein effective to increase the plasma proteome of the subject.

[0232] Also provided are methods for treating a subject suffering from age-related disorders and methods 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 described herein. In some embodiments, the CNS disorder is drug addiction. In some embodiments, the CNS disorder is substance abuse.

[0233] In some embodiments, the method of treatment relates to treating a subject suffering from alcoholism and comprises administering to the subject a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation. In some embodiments, the present disclosure relates to treating a subject suffering from alcohol-induced organ damage and comprises administering to the subject a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation.

[0234] In some embodiments, the present disclosure relates to a method of treating a subject suffering from schizophrenia, the method comprising administering to the subject a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. In some embodiments, the present disclosure relates to a method of treating a subject suffering from ischemia, inflammation, sepsis, free radical damage and / or oxidative stress following myocardial infarction, the method comprising administering to the subject a therapeutically effective amount of an enhanced NAD+ composition described herein. In some embodiments, the present disclosure relates to a method of treating a subject suffering from Alzheimer's disease, the method comprising administering to the subject a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. In some embodiments, the present disclosure relates to a method of treating a subject suffering from excitotoxicity, ischemia and / or oxidative stress, the method comprising administering to the subject an effective amount of an enhanced NAD+ pharmaceutical formulation described herein.

[0235] In some embodiments, the present disclosure provides a method of treating a subject suffering from pellagra, comprising administering to the subject a therapeutically effective amount of an enhanced NAD pharmaceutical formulation described herein. In some embodiments, the present disclosure provides a method of treating a subject suffering from pseudohypoxia, comprising administering to the subject a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. In some embodiments, the present disclosure provides a method of treating a subject suffering from hypertrophic cardiomyopathy (HCM), comprising administering to the subject a therapeutically effective amount of an enhanced NAD++ pharmaceutical formulation described herein. In some embodiments, the present 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 an enhanced NAD+ pharmaceutical formulation described herein. In some embodiments, the present disclosure provides a method of treating a subject suffering from ischemic brain injury, comprising administering to the subject a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. In some embodiments, the present disclosure provides a method of treating a subject suffering from Parkinson's disease, the method comprising administering to the subject a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. In some embodiments, the present disclosure provides a method of treating a subject suffering from a prion disease, the method comprising administering to the subject a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein.

[0236] The present disclosure also provides a method of treating a subject suffering from Parkinson's disease, comprising administering an enhanced NAD+ pharmaceutical formulation described herein. In some embodiments, the present disclosure provides a method of treating a subject suffering from prion disease, comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. In some embodiments, the present disclosure provides a method of treating a subject suffering from bupivacaine-induced neurotoxicity, comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. The present disclosure also provides a method of treating a subject suffering from arrhythmia, comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. Furthermore, the present disclosure provides a method of treating a subject suffering from nicotinamide-induced mitophagy, comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. The present disclosure also provides a method of treating a subject suffering from ischemic reperfusion, comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein.

[0237] In a further embodiment, the present disclosure provides a method of treating a subject suffering from a traumatic brain injury, the method comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. In a further embodiment, the present disclosure provides a method of treating a subject suffering from synchrotron radiation X-ray induced tissue injury, the method comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein.

[0238] In some embodiments, the present disclosure provides a method of treating a subject suffering from an autoimmune disease, comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. In a further embodiment, the present disclosure provides a method of treating a subject suffering from diabetes, comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. In a further embodiment, the present disclosure provides a method of treating a subject suffering from cancer, comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. In a further embodiment, the present disclosure provides a method of treating a subject suffering from a viral infection, comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. In some embodiments, the viral infection is coronavirus disease 2019 (COVID-19) and variants thereof.

[0239] In some embodiments, the present disclosure provides methods of promoting sobriety and / or satiety in a subject, the methods comprising administering to the subject an amount of an enhanced NAD+ pharmaceutical formulation described herein effective to promote sobriety and / or satiety. In some embodiments, the present disclosure provides methods of promoting cellular NAD+ metabolism and NAD+ cellular homeostasis in a subject, the methods comprising administering to the subject an amount of an enhanced NAD+ pharmaceutical formulation described herein effective to promote cellular NAD+ metabolism.

[0240] In some embodiments, the present disclosure provides a method for preventing or treating a subject suffering from or at risk of having at least one cardiovascular disease, comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. Such cardiovascular diseases can include, for example, diseases or disorders associated with abnormalities in any one of lipid metabolism, vasoactivity / hypertension, homocysteine ​​and folate cycles, oxidative stress, inflammation, resting heart rate, metabolite levels, or any combination thereof.

[0241] In some embodiments, the present disclosure provides a method for preventing or treating a subject suffering from or at risk of having at least one liver disease, comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. In some embodiments, the enhanced NAD+ compositions described herein can improve liver function, for example, resulting in a decrease in serum GGT, bilirubin, ALP, and / or albumin. In some embodiments, the enhanced NAD+ compositions described herein can reduce at least one metabolite, for example, 1-palmitoyl-2-arachidonoyl-GPC. In some embodiments, the enhanced NAD+ pharmaceutical formulations described herein can reduce oxidative stress.

[0242] The present disclosure also provides methods for preventing or treating a subject suffering from or at risk of having at least one disease or disorder associated with abnormalities in exocrine pancreatic function and / or digestion, comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. In some embodiments, the enhanced NAD+ pharmaceutical formulation described herein is capable of reducing the level and / or function of at least one pancreatic enzyme (e.g., pancreatic lipase-related protein 1 (PNLIPRP1), pancreatic ribonuclease (RNase 1), carboxypeptidase A1 (CPA1), carboxypeptidase B1 (CPB1), etc.).

[0243] In some embodiments, the present disclosure provides a method for preventing or treating a subject suffering from or at risk of having at least one neurodegenerative and / or neuropsychiatric disorder, comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. Such neurodegenerative diseases may include, for example, Alzheimer's disease (AD), Parkinson's disease, amyotrophic lateral sclerosis (ALS), and Huntington's disease. 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 function of at least one of the proteins listed in the table in Figure 28.

[0244] In some embodiments, the present disclosure provides a method for preventing or treating a subject suffering from or at risk of having at least one disease or disorder associated with abnormalities in inflammation and / or immune modulation, comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. Such diseases or disorders may include, for example, autoimmune diseases, including multiple sclerosis (MS), inflammatory bowel disease (IBD), and rheumatoid arthritis (RA). Such diseases or disorders may be correlated with abnormal expression levels and / or function of at least one of the proteins listed in the table in Figure 29.

[0245] In some embodiments, the present disclosure provides methods of preventing or treating a subject suffering from or at risk of having at least one disease or disorder associated with abnormalities in reproductive function and / or fertility, the method comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein.

[0246] In some embodiments, the present disclosure provides a method for preventing or treating a subject suffering from or at risk of having at least one metabolic disease (e.g., obesity and insulin resistance syndrome), comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. In some embodiments, the enhanced NAD+ compositions described herein are capable of increasing the expression level and / or function of at least one of N-acetyl-galactosaminyltransferase 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 formulations described herein are capable of decreasing the expression level and / or function of at least one of pyrraline, urea, glutamate, acisoga, nonadecanoate, and 1-palmitoyl-2-arachidonoyl-GPC.

[0247] In some embodiments, the present disclosure provides methods for preventing or treating a subject suffering from or at risk of having at least one skin and / or bone disease, the methods comprising administering a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation described herein. In some embodiments, the enhanced NAD+ compositions described herein are capable of increasing the expression level and / or function of SIRT1 and / or matrilin-3 (MATN3). In some embodiments, the enhanced NAD+ pharmaceutical formulations described herein decrease the expression level and / or function of at least one of peptidyl-prolyl cis-trans isomerase B (PPIB), whey acidic protein 4-disulfide-core 12 (WFDC12), and insulin-like peptide 3 (INSL3).

[0248] In some embodiments, the present disclosure provides a method for preventing or treating a subject suffering from a long-term viral infection symptom, such as Long Covid. In other embodiments, the disclosure provides a method for preventing a long-term viral infection symptom. In some embodiments, the method includes identifying a subject suffering from or at risk of suffering from a long-term viral infection symptom, such as Long Covid, and orally administering to the subject, for example, an amount of an enhanced NAD+ pharmaceutical formulation, the amount increasing the level of icNAD in the subject by at least 5%, thereby effective to reduce or prevent the long-term viral infection symptom in the subject. In some embodiments, the composition 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 the persistence of one or more Long Covid symptoms over several months. Non-limiting embodiments of Long Covid symptoms include fatigue, post-exertional malaise, fever, difficulty breathing, shortness of breath, cough, chest pain, heart palpitations, difficulty concentrating, headache, trouble sleeping, dizziness, lightheadedness, tingling sensations, changes in smell or taste, depression, anxiety, diarrhea, stomach pain, joint or muscle pain, rash, and changes in menstrual cycle.

[0249] Another aspect of the present disclosure relates to a composition comprising liothyronine, polyethylene glycol, microporous glucose and a pharmaceutically acceptable excipient.

[0250] Another aspect of the present disclosure relates to a composition comprising levadopa and polyethylene glycol and a pharmaceutically acceptable excipient. Preparation process

[0251] In a further embodiment, the blending process comprises mixing the pre-formulated NAD+ and PEG at room temperature for at least 20 minutes. In another embodiment, the blending process comprises mixing the pre-formulated NAD+ and PEG at room temperature for at least 30 minutes. In yet another embodiment, the blending process comprises mixing the pre-formulated NAD+ and PEG at room temperature for at least 40 minutes. In yet another embodiment, the blending process comprises mixing the pre-formulated NAD+ and PEG at room temperature for at least 50 minutes. In another embodiment, the blending process comprises mixing the pre-formulated NAD+ and PEG at room temperature for at least 1 hour.

[0252] In a further embodiment of the present disclosure, the preparation process further comprises pre-forming commercially available PEG. In one embodiment, pre-forming the PEG comprises grinding, sieving, or milling the commercially available PEG. In certain embodiments, grinding and / or milling the commercially available PEG is carried out to reduce the commercially available PEG to ultrafine particles to allow for homogeneous distribution with excipients and active ingredients. In certain embodiments, the mixture is processed through a 2.5 mm or other size sieve to maximize homogeneous distribution and compatibility with excipients.

[0253] In yet another embodiment of the present disclosure, the process of pre-forming PEG involves grinding, sieving, or milling commercially available PEG, and then vacuuming the ground, sieved, or milled PEG product itself before blending.

[0254] In yet another embodiment of the process of preparation, pre-forming the PEG involves vacuuming commercially available PEG and then grinding, sieving, or milling the vacuumed PEG product itself before blending.

[0255] In one embodiment of the present disclosure, the commercially available PEG used in the preparation is GRAS under the FDA. In some embodiments, the commercially available PEG used in the preparation is PEG300. In other embodiments, the commercially available PEG used in the preparation is PEG400. In still other embodiments, the commercially available PEG used in the preparation is PEGMME550. In still other embodiments, the commercially available PEG used in the preparation is PEG600. In other embodiments, the commercially available PEG used in the preparation is PEG1000. In some embodiments, the commercially available PEG used in the preparation is PEGMME2000. In still other embodiments, the commercially available PEG used in the preparation is PEG3350. In certain embodiments, the commercially available PEG used in the preparation is PEG4000. In other embodiments, the commercially available PEG used in the preparation is PEG300, PEG400, PEGMME550, PEG600, PEG1000, PEGMME2000, PEG3350, PEG4000, or any combination thereof. Enumerated Embodiments Embodiment 1 1. An enhanced nicotinamide adenine dinucleotide (NAD+) composition comprising: a crystalline component comprising crystalline polyethylene glycol, and Amorphous components, including amorphous NAD+ Including, 1. A composition characterized by having a first onset temperature between 179.39°C (±0.10°C) and 182.61°C (±0.10°C) by thermogravimetric analysis (TGA) thermogram. Embodiment 2 1. An enhanced nicotinamide adenine dinucleotide (NAD+) composition comprising: a crystalline component comprising crystalline polyethylene glycol, and Amorphous components, including amorphous NAD+ Including, 1. A composition characterized by having a weight loss of 14.39 wt % to 16.98 wt % in the range of about 170°C to about 300°C according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to at least 300°C. Embodiment 3 1. An enhanced nicotinamide adenine dinucleotide (NAD+) composition comprising: a crystalline component comprising crystalline polyethylene glycol, and Amorphous components, including amorphous NAD+ Including, 1. A composition characterized by having a weight loss of 57.83 wt % to 58.55 wt % in the range of about 300°C to about 800°C according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to at least 800°C. Embodiment 4 The composition of any one of the previous embodiments, further comprising amorphous polyethylene glycol. Embodiment 5 1. An enhanced nicotinamide adenine dinucleotide (NAD+) composition comprising: a crystalline component comprising crystalline polyethylene glycol, and Amorphous components, including amorphous NAD+ Including, A composition characterized by having a TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with any one of Figures 33A, 33B, 34A, 34B, 35A or 35B. Embodiment 6 6. The composition of embodiment 5, having a TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with FIG. 33A or FIG. 33B. Embodiment 7 6. The composition of embodiment 5, having a TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with FIG. 34A or FIG. 34B. Embodiment 8 6. The composition of embodiment 5, having a TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with FIG. 35A or FIG. 35B. Embodiment 9 1. An enhanced nicotinamide adenine dinucleotide (NAD+) composition comprising: a crystalline component comprising crystalline polyethylene glycol, and Amorphous components, including amorphous NAD+ Including, a first weight loss of 2.42 wt % to 3.25 wt % in the range of ambient temperature to about 170°C, according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to at least 170°C; and / or a second weight loss of 13.66 wt % to 14.61 wt % in the range of about 170°C to about 300°C according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to at least 300°C; and / or a third weight loss of 58.73 wt % to 60.37 wt % in the range of about 300°C to about 800°C according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to at least 800°C; and / or a fourth weight loss of 20.14 wt % to 21.46 wt % in the range of about 800°C to about 900°C according to thermogravimetric analysis (TGA) thermograms collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to about 800°C and under air at a ramp rate of 10°C / min from about 800°C to about 900°C; and / or a first starting temperature of 179.39°C to 180.15°C, and / or Second starting temperature: 369.03℃~370.82℃ 1. A composition characterized by having a TGA thermogravimetric analysis (TGA) thermogram having: Embodiment 10 1. An enhanced nicotinamide adenine dinucleotide (NAD+) composition comprising: a crystalline component comprising crystalline polyethylene glycol, and Amorphous components, including amorphous NAD+ Including, a first weight loss of 2.32 wt % to 3.55 wt % in the range of ambient temperature to about 170°C according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to at least 170°C; and / or a second weight loss of 14.39 wt % to 16.22 wt % in the range of about 170°C to about 300°C according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to at least 300°C; and / or a third weight loss of 57.83 wt % to 58.55 wt % in the range of about 300°C to about 800°C according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to at least 800°C; and / or a fourth weight loss of 22.62 wt % to 22.76 wt % in the range of about 800°C to about 900°C according to thermogravimetric analysis (TGA) thermograms collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to about 800°C and under air at a ramp rate of 10°C / min from about 800°C to about 900°C; and / or A first starting temperature of 178.62°C to 180.11°C, and / or Second starting temperature: 365.73℃~370.47℃ 1. A composition characterized by having a TGA thermogravimetric analysis (TGA) thermogram having: Embodiment 11 1. An enhanced nicotinamide adenine dinucleotide (NAD+) composition comprising: a crystalline component comprising crystalline polyethylene glycol, and Amorphous components, including amorphous NAD+ Including, a first weight loss of 3.62 wt % to 3.96 wt % in the range of ambient temperature to about 170°C according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to at least 170°C; and / or a second weight loss of 15.17 wt % to 16.98 wt % in the range of about 170°C to about 300°C according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to at least 300°C; and / or a third weight loss of 58.05 wt % to 59.66 wt % in the range of about 300°C to about 800°C according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to at least 800°C; and / or a fourth weight loss of 22.85 wt % to 25.87 wt % in the range of about 800°C to about 900°C according to thermogravimetric analysis (TGA) thermograms collected under nitrogen gas at a ramp rate of 10°C / min from ambient temperature to about 800°C and under air at a ramp rate of 10°C / min from about 800°C to about 900°C; and / or a first starting temperature of 179.83°C to 182.61°C, and / or Second starting temperature: 363.24℃~366.16℃ 1. A composition characterized by having a TGA thermogravimetric analysis (TGA) thermogram having: Embodiment 12 10. The composition of any of the previous embodiments, wherein the ratio of NAD+ to polyethylene glycol in the composition is 1:1 (wt / wt). Embodiment 13 The composition of any of the previous embodiments, wherein the polyethylene glycol has an average molecular weight of between about 300 and about 4000. Embodiment 14 The composition of any of the previous embodiments, wherein the polyethylene glycol is PEG3350. Embodiment 15 The composition of any of the previous embodiments, which is in powder form. Embodiment 16 The composition of any of the previous embodiments, which is suitable for oral administration to a subject. Embodiment 17 The composition of any of the previous embodiments, having a shelf life of at least 6 months at room temperature. Embodiment 18 The composition of any of the previous embodiments, having a shelf life of at least 1 year at room temperature. Embodiment 19 19. A pharmaceutical formulation comprising the composition of any one of embodiments 1 to 18. Embodiment 20 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 the baseline level of intracellular NAD in the subject. Embodiment 21 21. The formulation of embodiment 19 or 20, which, when administered to a subject, increases intracellular levels of NAD in said subject. Embodiment 22 22. The formulation of any one of embodiments 19 to 21, which, when administered to a subject, modulates the expression of at least one biomarker in said subject. Embodiment 23 The formulation of any one of embodiments 19 to 22 for use in a method for alleviating drug addiction withdrawal symptoms in a subject in need thereof, alleviating 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 with decreased levels of NAD in a subject in need thereof. Embodiment 24 19. A kit comprising the composition of any one of embodiments 1 to 18. Embodiment 25 19. A method of preparing an enhanced NAD+ composition according to any one of embodiments 1 to 18, comprising: (1) subjecting NAD+ to at least one evacuation cycle to form evacuated NAD+; and (2) blending the evacuated NAD+ with polyethylene glycol to form a blended mixture; A method comprising: Embodiment 26 26. The method of embodiment 25, wherein the inert gas comprises argon or nitrogen. Embodiment 27 27. The method of embodiment 25 or 26, wherein step (1) comprises subjecting the NAD+ to at least three vacuum cycles. Embodiment 28 28. The method of any one of embodiments 25 to 27, wherein the polyethylene glycol has an average molecular weight of between about 300 and about 4000. Embodiment 29 29. The method of any one of embodiments 25 to 28, wherein the polyethylene glycol is PEG3350. Embodiment 30 30. The method of any one of embodiments 25 to 29, further comprising subjecting said blended mixture to at least one additional vacuum cycle. Embodiment 31 30. The method of any one of embodiments 25 to 29, further comprising subjecting said blended mixture to at least three additional vacuum cycles. Embodiment 32 30. The method of any one of embodiments 25 to 29, further comprising subjecting said blended mixture to at least six additional vacuum cycles. Embodiment 33 33. The method of any one of embodiments 25 to 32, further comprising pretreating the NAD+ prior to step (1). Embodiment 34 34. The method of embodiment 33, wherein pre-treating the NAD+ comprises passing the NAD+ through a sieve and / or grinding, milling, or jet-milling the NAD+. Embodiment 35 35. An enhanced NAD+ composition prepared by the method of any one of embodiments 25 to 34. Embodiment 36 A method of making an enhanced NAD+ pharmaceutical formulation, comprising mixing the enhanced NAD+ composition of any of embodiments 1 to 18 with at least one ingredient or excipient that facilitates administration to a subject and / or that can further treat a disease or condition in said subject. Embodiment 37 24. A method of alleviating drug addiction withdrawal symptoms in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation of any one of embodiments 19 to 23, wherein said amount is effective to alleviate drug addiction withdrawal symptoms in the subject. Embodiment 38 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%. Embodiment 39 The method of any one of embodiments 36 to 38, wherein the amount of the pharmaceutical formulation administered is effective to alleviate opioid withdrawal symptoms such that the client maintains a Clinical Opioid Withdrawal Scale (COWS) score of 12 or lower. Embodiment 40 40. The method of any one of embodiments 37 to 39, wherein the level of NAD that is increased in the subject is an intracellular level of NAD. Embodiment 41 41. The method of any one of embodiments 37 to 40, wherein the intracellular level of NAD is increased to greater than about 35 μM. Embodiment 42 42. The method of any one of embodiments 37 to 41, wherein the level of NAD that is increased in the subject is the extracellular level of NAD. Embodiment 43 The method of any one of embodiments 37 to 42, wherein said administering of said enhanced NAD+ pharmaceutical formulation is effective to increase plasma levels of N-methyl-nicotinamide (MeNAM) or N-methyl-2-pyridone-5-carboxamide (2PY) by at least 5% in said subject. EMBODIMENT 44 44. The method of any one of embodiments 37 to 43, wherein the amount of NAD+ in the pharmaceutical formulation administered to the subject is from about 5 mg to about 4000 mg. Embodiment 45 The method of any one of embodiments 37 to 44, wherein the amount of the pharmaceutical formulation administered comprises a maximum daily dose of about 1 mg / kg to about 100 mg / kg body weight of the subject. Embodiment 46 46. ​​The method of any one of embodiments 37 to 45, further comprising administering to the subject a pharmaceutically effective amount of an analgesic or a nonsteroidal anti-inflammatory drug (NSAID). Embodiment 47 The method of any one of embodiments 37 to 45, wherein the pharmaceutical formulation further comprises a pharmaceutically effective amount of an analgesic or a nonsteroidal anti-inflammatory drug (NSAID). Embodiment 48 48. The method of any one of embodiments 37 to 47, further comprising monitoring at least one vital sign of the subject before and after administration of the pharmaceutical formulation. Embodiment 49 49. The method of any one of embodiments 37 to 48, further comprising measuring intracellular NAD or extracellular concentrations of NAD in said subject before and after administration of said pharmaceutical formulation. Embodiment 50 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. Embodiment 51 24. A method of increasing nicotinamide adenine dinucleotide (NAD) levels in a subject, comprising administering an amount of an enhanced NAD+ pharmaceutical formulation of any one of embodiments 19 to 23 effective to increase NAD levels in the subject. Embodiment 52 The method of embodiment 51, wherein the subject is a healthy adult. Embodiment 53 24. A method of preventing or treating a disease or disorder correlated with a decreased level of NAD in a subject in need thereof, comprising administering an amount of an enhanced NAD+ pharmaceutical formulation of any one of embodiments 19 to 23 effective to prevent or treat said disease or disorder in said subject. EMBODIMENT 54 24. A method of treating a subject suffering from drug addiction, comprising administering to said subject a pharmaceutically effective amount of an enhanced NAD+ pharmaceutical formulation of any one of embodiments 19 to 23. Embodiment 55 55. The method of embodiment 54, wherein the chemical compound is tobacco, heroin, opium, morphine, dihydromorphine, meperidine, codeine, cocaine, amphetamine, barbiturate, alcohol, a tranquilizer, or an opioid. Embodiment 56 24. A method of promoting cellular NAD metabolism or NAD cell homeostasis in a subject, comprising administering to the subject an amount of an enhanced NAD+ pharmaceutical formulation of any one of embodiments 19 to 23 effective to promote NAD metabolism or NAD cell homeostasis. Embodiment 57 24. 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 an enhanced NAD+ pharmaceutical formulation of any one of embodiments 19 to 23, wherein the at least one condition is elevated blood pressure, hyperglycemia, excess body fat around the waist, abnormal cholesterol or triglyceride levels, or any combination thereof. Embodiment 58 24. A method of increasing 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 an enhanced NAD+ pharmaceutical formulation of any one of embodiments 19 to 23 effective to increase the SIRT1, PLB1, NPL, ENPP5, GLSTA, GALNT3, FABP1, NRCAM, NLGN2, ARTN, UPB1, MME, CTSB, CTSL, or HRAS polypeptide. Embodiment 59 19. A method of preparing an enhanced NAD+ composition according to any one of embodiments 1 to 18, comprising: (1) blending NAD+ with polyethylene glycol to form a blended mixture; and (2) subjecting the blended mixture to at least one vacuum cycle; Including, A method wherein each evacuation cycle includes placing a container under vacuum and backfilling said container with an inert gas or CO2. Embodiment 60 1. A method for preparing an enhanced NAD+ composition comprising a crystalline component comprising crystalline polyethylene glycol and an amorphous component comprising amorphous NAD+, (1) blending NAD+ with polyethylene glycol to form a blended mixture; and (2) subjecting the blended mixture to at least one vacuum cycle; Including, A method wherein each evacuation cycle includes placing a container under vacuum and backfilling said container with an inert gas or CO2. Embodiment 61 61. The method of embodiment 59 or 60, wherein the inert gas comprises argon or nitrogen. Embodiment 62 62. The method of any one of embodiments 59 to 61, wherein step (2) comprises at least three vacuum cycles. Embodiment 63 62. The method of any one of embodiments 59 to 61, wherein step (2) comprises at least six vacuum cycles. EMBODIMENT 64 The method of any one of embodiments 59 to 63, wherein the polyethylene glycol has an average molecular weight of between about 300 and about 4000. Embodiment 65 The method of any one of embodiments 59 to 64, wherein the polyethylene glycol is PEG3350. Embodiment 66 66. The method of any one of embodiments 59 to 65, further comprising pretreating said NAD+ prior to step (1). Embodiment 67 67. The method of embodiment 66, wherein pre-treating the NAD+ comprises passing the NAD+ through a sieve and / or grinding, milling, or jet-milling the NAD+. Embodiment 68 68. The method of any one of embodiments 59 to 67, wherein the NAD+ is subjected to at least one vacuum cycle prior to step (1). Embodiment 69 69. An enhanced NAD+ composition prepared by the method of any one of embodiments 59 to 68. [Example]

[0256] Example 1 Process for preparing enhanced NAD+ compositions Pretreated (ground, milled, or jet-milled) NAD+ and PEG, including particle sizing and "vacuuming," were weighed and mixed in a clean conical blender through a sieve to disperse agglomerates and remove any metal or other solids. The blender was tightly sealed. The mixture was blended for a minimum of 20 minutes. The blended NAD+ was then evacuated for three cycles, each lasting at least 20 minutes and involving placing the blended NAD+ under vacuum and replacing the vacuum with argon. The blended, enhanced, evacuated product was then dispensed into a bag through a bottom dispensing port, while the bag was placed in a container sized to accommodate the product volume. The liner bag was then tied off with a cable tie, and the container was then sealed with an airtight lid, safe for storage or transport. Example 1A Process for preparing enhanced NAD+ compositions

[0257] Pretreated (ground, milled, or jet-milled) NAD+ and PEG, including particle sizing and "vacuuming," were weighed and mixed in a clean conical blender through a sieve to disperse agglomerates and remove any metal or other solids. The blender was tightly sealed. The mixture was blended for a minimum of 20 minutes. The blended NAD+ was then evacuated for three cycles, each lasting at least 20 minutes and involving placing the blended NAD+ under vacuum and replacing the vacuum with nitrogen. The blended, enhanced, evacuated product was then dispensed into a bag through a bottom dispensing port, while the bag was placed in a container sized to accommodate the product volume. The liner bag was then tied off with a cable tie, and the container was then sealed with an airtight lid, safe for storage or transport. Example 1B Process for preparing enhanced NAD+ compositions

[0258] Pre-treated (ground, milled, or jet-milled) NAD+ and PEG containing particle sizing and "vacuuming" are weighed and mixed in a clean conical blender through a sieve to disperse agglomerates and remove any metal or other solids. The blender is tightly sealed. The mixture is blended for a minimum of 20 minutes. The blended NAD+ is then evacuated for two cycles, each lasting at least 20 minutes and involving placing the blended NAD+ under vacuum and replacing the vacuum with argon. The blended, enhanced, evacuated product is then dispensed into a bag through a bottom dispensing port, while the bag is placed in a container sized to accommodate the product volume. The liner bag is then tied off with a cable tie, and the container is then sealed with an airtight lid, safe for storage or transport. Example 2 The process of preparing evacuated NAD+

[0259] Commercially available NAD+ was passed through a sieve into a clean conical blender to disperse clumps and remove any metal or other solids. The blender was tightly sealed. The NAD+ was blended for a minimum of 20 minutes. The blended NAD+ was then evacuated for three cycles, each lasting at least 20 minutes and involving placing the blended NAD+ under vacuum and replacing the vacuum with argon. The blended, enhanced, evacuated NAD+ product was then dispensed into a bag through a bottom dispensing port, while the bag was placed into a container sized to accommodate the NAD+ product volume. The liner bag was then tied off with a cable tie, and the container was then sealed with an airtight lid, safe for storage or transport. Example 2A The process of preparing evacuated NAD+

[0260] Commercially available NAD+ was passed through a sieve into a clean conical blender to disperse clumps and remove any metal or other solids. The blender was tightly sealed. The NAD+ was blended for a minimum of 20 minutes. The blended NAD+ was then subjected to a vacuum for three cycles, each lasting at least 20 minutes and involving placing the blended NAD+ under vacuum and replacing the vacuum with nitrogen. The blended, enhanced, vacuum-treated NAD+ product was then dispensed into a bag via a bottom dispensing port, while the bag was placed into a container sized for the NAD+ product volume. The liner bag was then tied off with a cable tie, and the container was then sealed with an airtight lid, safe for storage or transport. Example 2B The process of preparing evacuated NAD+

[0261] Commercially available NAD+ is placed into a clean conical blender through a sieve to disperse clumps and remove any metals or other solids. The blender is tightly sealed. The NAD+ is blended for a minimum of 20 minutes. The blended NAD+ is then evacuated for two cycles, each lasting at least 20 minutes and involving placing the blended NAD+ under vacuum and replacing the vacuum with argon. The blended, enhanced, evacuated NAD+ product is then dispensed into a bag through a bottom dispensing port, while the bag is placed into a container sized to accommodate the NAD+ product volume. The liner bag is then tied off with a cable tie, and the container is then sealed with an airtight lid, safe for storage or transport. Example 3 2. Process of Preparation of Vacuumed PEG

[0262] Pre-qualified commercially available PEG was passed through a sieve into a clean conical blender to disperse clumps and remove any metal or other solids. The blender was tightly sealed. The preparation was blended for a minimum of 20 minutes. The blended NAD+ was then evacuated for three cycles, each lasting at least 20 minutes and involving placing the blended NAD+ under vacuum and replacing the vacuum with argon. The blended, enhanced vacuum product was then dispensed into a bag through a bottom dispensing port, while the bag was placed into a container sized to accommodate the product volume. The liner bag was then tied off with a cable tie, and the container was then sealed with an airtight lid, safe for storage or transport. Example 3B 2. Process of Preparation of Vacuumed PEG

[0263] Prequalified, commercially available PEG was passed through a sieve into a clean conical blender to disperse clumps and remove any metal or other solids. The blender was tightly sealed. The preparation was blended for a minimum of 20 minutes. The blended NAD+ was then subjected to a vacuum for three cycles, each lasting at least 20 minutes and involving placing the blended NAD+ under vacuum and replacing the vacuum with nitrogen. The blended, enhanced vacuum product was then dispensed into a bag via a bottom dispensing port, while the bag was placed into a container sized to accommodate the product volume. The liner bag was then tied off with a cable tie, and the container was then sealed with an airtight lid, safe for storage or transport. Example 3B 2. Process of Preparation of Vacuumed PEG

[0264] Pre-qualified, commercially available PEG is placed into a clean conical blender through a sieve to disperse clumps and remove any metal or other solids. The blender is tightly sealed. The preparation is blended for a minimum of 20 minutes. The blended NAD+ is then evacuated for two cycles, each lasting at least 20 minutes and involving placing the blended NAD+ under vacuum and replacing the vacuum with argon. The blended, enhanced, evacuated product is then dispensed into a bag through a bottom dispensing port, while the bag is placed into a container sized to accommodate the product volume. The liner bag is then tied off with a cable tie, and the container is then sealed with an airtight lid, safe for storage or transport. Example 4 Process for preparing enhanced NAD+ compositions

[0265] 50% NAD+ G / 100g of pre-processed (ground, milled, or jet-milled) NAD+ and PEG containing particle sizing and "vacuuming" were weighed and mixed in a clean conical blender, passed through a sieve to disperse agglomerates and remove any metal 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, the vacuum was replaced with argon, and the mixture was then evacuated for three cycles, each lasting at least 20 minutes. The blended, enhanced, evacuated product was then dispensed into a bag through the bottom dispensing port, and the bag was simultaneously placed into a container sized to accommodate the product volume. The liner bag was then tied off with a cable tie, and the container was then sealed with an airtight lid, safe for storage or transport. Example 4A Process for preparing enhanced NAD+ compositions

[0266] 50% NAD+ G / 100g of pre-processed (ground, milled, or jet-milled) NAD+ and PEG containing particle sizing and "vacuuming" were weighed and mixed in a clean conical blender, passed through a sieve to disperse agglomerates and remove any metal 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, the vacuum was replaced with nitrogen, and the mixture was then evacuated for three cycles, each lasting at least 20 minutes. The blended, enhanced, evacuated product was then dispensed into a bag through the bottom dispensing port, and the bag was simultaneously placed into a container sized to accommodate the product volume. The liner bag was then tied off with a cable tie, and the container was then sealed with an airtight lid, safe for storage or transport. Example 4B Process for preparing enhanced NAD+ compositions

[0267] 50% NAD+ G / 100g of pre-processed (ground, milled, or jet-milled) NAD+ and PEG containing particle sizing and "vacuuming" are weighed and mixed in a clean conical blender through a sieve to disperse agglomerates and remove any metal 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, the vacuum is replaced with argon, and the mixture is then evacuated for two cycles, each lasting at least 20 minutes. The blended, enhanced, evacuated product is then dispensed into a bag through the bottom dispensing port, and the bag is simultaneously placed into a container sized to accommodate the product volume. The liner bag is then tied off with a cable tie, after which the container is sealed with an airtight lid, safe for storage or transport. Example 5 Testing the biological activity of enhanced NAD+ / PEG compositions

[0268] A colorimetric assay was performed to measure NAD+ and NADH present in biological samples. To measure NAD+, cell lysate samples were added to microcentrifuge tubes, combined with 0.1 N HCl, and mixed thoroughly. The tubes were incubated at 80°C and protected from light. Assay buffer was added to the tubes to shift the pH of the samples back to neutral. The sample pH was adjusted accordingly to between 6.0 and 8.0 using acid or base.

[0269] 50 μL of each NAD+ standard and unknown sample was added to a well of a 96-well microtiter plate. 50 μL of NAD+ cycling reagent was added to each well. The contents of the well were mixed thoroughly and incubated at room temperature for 1-4 hours, protected from light. The assay is continuous (not terminated) and can therefore be measured at multiple time points to track reaction kinetics. The assay can be stopped at desired time points by adding 50 μL of 0.5 N H2SO4. The plate was read using a spectrophotometric microplate reader at 450 nm. The concentration of NAD+ / NADH in the sample was calculated by comparing the sample optical density (OD) to the standard curve.

[0270] The intracellular increase in NAD after NAD+ treatment was compared to placebo treatment (see Table 1 in Example 6). Example 6 Multi-OMIC Characterization of the Effects of Orally Enhanced NAD+ in a Wellness Cohort

[0271] The present disclosure provides for the delivery of enhanced NAD+ pharmaceutical formulations to target cells for treating 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 present disclosure. NAD+ is delivered as an oral NAD+ pharmaceutical formulation that is chemically stable, bioavailable, and pharmacologically active.

[0272] A research study was conducted to investigate the multi-omic effects of oral enhanced nicotinamide adenine dinucleotide (NAD+) in a restricted "wellness cohort" composed of healthy individuals.

[0273] The study followed a double-blind, placebo-controlled design with repeated measures. Sixty participants were enrolled and randomized approximately equally to either the enhanced NAD+ or placebo group (i.e., 30 enhanced NAD+, 30 placebo). The study sample size was determined based on the number of blood samples taken and the expected effect size. * A preliminary statistical power analysis performed in Power 3.0 (Faul, Erdfelder, Lang, & Buchner, 2007) determined that the study would be able to achieve 80% power to detect large and medium-sized effects (at a nominal 5% significance level).

[0274] During the study, each participant received up to five 500 mg doses per day for a maximum daily dose of 2500 mg / day. Participants' weights ranged from 100 to 300 lbs (45.4 to 136.1 kg), corresponding to a maximum daily dose of 18.4 to 55.1 mg / kg / day. Dosage schedule

[0275] Study Days 1-7: Washout period; Study Days 8-12: Treatment dose administration; Study Day 13: Post-study.

[0276] After a 7-day washout period, participants received either enhanced NAD+ (Treatment A) or placebo (Treatment B) for 5 days, with four 500 mg daily doses administered approximately 3-4 hours apart within a 10-16 hour window, on a qid schedule. On days 9 and 10, additional 500 mg doses were administered in the evening, immediately before bedtime. Enhanced NAD+ and the corresponding placebo met FDA standards for human administration, including compliance with GMP regulations.

[0277] Each dose of enhanced NAD+ was delivered orally as a mouth rinse and swallowed. The first dose on day 8 was administered after a 12-hour fast. Participants were asked to record the date and time of each dose. Participants also agreed not to significantly alter their diet or discontinue use of the supplement while participating in the study.

[0278] A designated study coordinator actively monitored dose administration and blood collection, adhering to technology assistance that allowed participants to receive reminders and provide prompt responses to questions related to the ongoing study protocol. Dosage Administration Instructions

[0279] Doses were administered with 3-4 hours between doses. If a dose was missed, it was taken within 2 hours of the scheduled dose; an additional 2 hours was allowed to elapse, after which the dosing schedule resumed so that all doses for that day were completed. On days 9 and 10, the fifth dose was administered at least 2 hours after the fourth dose (see Table 1 for the daily dosing schedule for days 8-12). Table 1 [Table 1] *Dose given on days 9 and 10 only.

[0280] Methods of formulating and administering enhanced NAD+ for administration: Immediately before use, add the dry powder contents of the bottle to approximately 4 ounces (1 / 2 cup) of water. Stir thoroughly. After stirring, the enhanced NAD+ was administered by swishing a mouthful of the prepared solution in the mouth for approximately 45 seconds and then swallowing. Rinsing and swallowing was repeated until all of the prepared solution was swallowed (approximately 3-5 sips). Inclusion criteria

[0281] English-speaking adults, ranging in age from 45 to 75 years, in good overall self-reported health (excluding common conditions such as cardiovascular disease, hypertension, and hyperlipidemia);

[0282] Participants must be able to consent to take part in this research study,

[0283] Able to follow a study protocol including a 7-day washout of potentially confounding substances with restriction of supplement intake,

[0284] Repeated venous blood sampling, dose administration and assessment schedules can be followed and the wrist-worn wearable device is well tolerated (i.e., consistent use during the day and night for the duration of the study). Exclusion criteria

[0285] Body mass index (BMI)>35kg / m 2 ,

[0286] current chronic or acute infectious disease (e.g., hepatitis, influenza, HIV, Lyme disease; COVID-19 within the past 2 months),

[0287] proximity to SARS-CoV-2 vaccine administration (within 2 weeks), current use of NAD+ supplements and precursors,

[0288] Use of systemic anti-inflammatory medications (excluding NSAIDs), immunosuppressants,

[0289] current cancer or hematological condition and / or cancer treatment (radiation, chemotherapy),

[0290] Type 1 juvenile or type 2 diabetes,

[0291] Autoimmune disorders (multiple sclerosis, lupus, rheumatoid arthritis, psoriasis),

[0292] Inflammatory bowel disease (ulcerative colitis, Crohn's disease),

[0293] Currently pregnant,

[0294] current substance abuse, including alcohol but excluding nicotine and prescription medications (e.g., stimulants, opiates);

[0295] Members of the same family. biological specimens

[0296] Blood samples were collected on days 3, 7, 10, and 13 of the study.

[0297] Venous blood (up to 30 ml) was collected using venipuncture. Blood was collected twice at baseline (on days 3 or 4 prior to the washout period and study dose administration, and on study day 7 prior to treatment dose administration) to establish an estimate of baseline intracellular NAD levels. Dose administration began on day 8, and blood was collected on days 10 and 13.

[0298] Plasma or serum was isolated using standard centrifugation protocols, frozen, and stored at −80°C until use. Whole blood or isolated fractions were processed for further assays using manufacturer-recommended methods for sample handling, randomization, and plating, where applicable. Assay NAD concentration measurement

[0299] Extracellular serum NAD and intracellular NAD concentrations were measured by Jinfiniti Precision Medicine (“Jinfiniti”) according to the manufacturer's protocol. Laboratory tests

[0300] Blood tests included a complete blood count (CBC) with differential, a comprehensive metabolic panel (CMP), and hs-CRP. Vitals, including blood pressure, heart rate, waist circumference, and weight, were collected at each blood draw. Height was measured at baseline blood draw and collected directly from participants as part of the intake assessment. Targeted Proteomics

[0301] Proteomics was performed using Olink's Proximity Extension Assays (PEA) targeted proteomics platform, which uses pairs of matched antibodies attached to unique DNA nucleotides to generate templates for DNA polymerase-dependent extension, thereby providing specific estimates of protein abundance. Untargeted Metabolomics

[0302] Metabolomics was performed using Metabolon's ultra-high performance liquid chromatography / tandem mass spectrometry (UHPLC / MS / MS) global platform, detecting approximately 1,000 small metabolites representing superpathways: lipids (449), amino acids (183), xenobiotics (91), nucleotides (35), peptides (31), cofactors / vitamins (23), carbohydrates (22), partially characterized (6), and >200 unassigned metabolites. Participant-reported outcomes and wearable-derived data

[0303] Questionnaires were used to assess physical and mental health on days 7, 10, and 13. Administered assessments included the Depression, Anxiety, and Stress Scale-21 (DASS-21; Lovibond & Lovibond, 1995); the Clinically Usable Anxiety Outcome Scale-Everyday Version (CUXOS-D; Zimmerman et al., 2019); and the Daily Fatigue Impact Scale (D-FIS; Fisk & Doble, 2002). Wearables

[0304] Participants were provided with Fitbit™ wearable devices to monitor activity, sleep quality, and cardiovascular fitness for one week prior to treatment dose administration and throughout the study period. These devices were used to collect average heart rate in BPM, heart rate variability (HRV), sleep duration (total and stage-specific) and quality, and physical activity metrics. statistical analysis

[0305] Data analysis, data quality assurance and management, and hypothesis testing were performed using Python and R. Robust mixed ANCOVA and mixed linear models (MLM) were the statistical approaches used to evaluate the study data. result

[0306] After a 5-day dosing protocol, induction of intracellular NAD was observed in the treatment (A) but not the placebo (B) group (Table 2). Table 2 [Table 2]

[0307] Increased plasma abundance of NAD+ metabolites was also observed (Figure 15). Six metabolites were analyzed (2PY, 1-methylnicotinamide, nicotinate, quinolinate, nicotinamide, and N'-methylnicotinate), and two showed substantial elevations in plasma, consistent with preparation for excretion by the kidney: 1-methylnicotinamide (MeNAM; P = 7.59 x 10 -11 ) (Figure 15A) and N1-methyl-2-pyridone-5-carboxamide (2PY; P = 1.03 × 10 -13 ) (FIG. 15B). These findings remained after rigorous multiple comparison correction. The data suggest metabolic fates of MeNAM and 2PY, two major metabolites of NAD+, following oral administration of enhanced NAD+ doses.

[0308] Enhanced NAD+ administration induced changes in amino acid metabolism, glycolysis, the TCA cycle, and fatty acid oxidation (Figure 16), observed through metabolic evidence of enzyme activity in the liver, kidney, and colon.

[0309] The observed pathway activation and enhanced enzymatic reactions were significant evidence in favor of the bioavailability of the administered enhanced NAD dose. These results indicate that NAD supplementation in the cohort resulted in an amplification of the urea cycle (possibly via xanthine components, considering that hypoxanthine is an intermediate metabolite), an enhancement of pyruvate-to-lactate conversion contributing to glycolysis, an increase in lipid oxidation (observed in the decrease in lipid fractions while increasing bile acid metabolites), and an increase in glutamate-to-glutamine interconversion (Figures 8-10). Example 7 Multi-omic biomarker study of the effects of oral enhanced NAD+

[0310] Further studies were conducted according to Example 6 to show that the illustrative oral enhanced NAD+ pharmaceutical formulations modulate the levels of various biomarkers associated with several therapeutic areas, including, but not limited to, cardiovascular disease, liver disease, exocrine pancreas / digestion, neurodegenerative / neuropsychiatric diseases, inflammation / immune function, reproduction, metabolic disease and obesity, and skin and bone health, where statistically significant differences were observed between treatment and placebo groups.

[0311] Cardiovascular disease-enhancing NAD+ compositions led to improvements in lipid metabolism, vascular / endothelial health and injury resistance, and markers of oxidative stress.

[0312] Liver Disease - The NAD+-boosting composition led to improvements in several liver enzymes, suggesting improved liver function. Improvements in oxidative stress markers also have relevance to common liver diseases such as non-alcoholic fatty liver disease (NAFLD).

[0313] The exocrine pancreas / digestion-enhancing NAD+ composition differentially reduced circulating levels of four pancreatic enzymes.

[0314] Neurodegenerative / Neuropsychiatric Disorders—The enhanced NAD+ composition differentially increased the levels of six proteins with known neurological effects and decreased the levels of six other proteins. Some of the proteins altered by enhanced NAD+ composition treatment have previously been associated with neurodegenerative disease, depression, anxiety, or chronic pain. The observed improvements in oxidative and nitrosative stress also have relevance to neurodegenerative diseases.

[0315] Inflammation / Immune Function - Clinical inflammatory markers such as hsCRP did not differ between groups, but there were significant increases or decreases in 15 immune proteins with roles in regulating adaptive or innate immune function and inflammation in the orally enhanced NAD+ treatment group.

[0316] Reproduction - Changes in one of the key protein regulators of fertility were observed in both men and women in the oral enhanced NAD+ treatment group, which were not seen in the placebo group.

[0317] Metabolic Disease and Obesity - The enhanced NAD+ composition differentially upregulated three proteins associated with metabolic disease and obesity.

[0318] Skin and bone health—There were significant differences between groups in two proteins that are centrally involved in collagen synthesis or have been linked to skin inflammation in psoriasis and atopic dermatitis. Similar differences were also found in two proteins associated with bone development. Design and Methods

[0319] A study was conducted to determine the efficacy of an exemplary oral enhanced NAD+ pharmaceutical formulation versus placebo in increasing intracellular concentrations of NAD and extracellular blood concentrations of NAD in vivo in healthy adults. Intracellular levels of NAD ("icNAD") were assessed by a colorimetric assay measuring total NAD levels in red and white blood cells and platelets. Extracellular NAD ("eNAD") was assessed in cell-free plasma. icNAD and eNAD levels were expected to increase by the end of treatment with the formulation relative to placebo, demonstrating stability and potential clinical activity.

[0320] The study also examined the effect of the illustrative oral formulation versus placebo on a comprehensive range of clinical tests and multi-omic peripheral biomarkers associated with patient-reported outcomes, including inflammation; lipid metabolism; oxidative stress / antioxidant capacity; liver, kidney, and mitochondrial function, as well as daily reports of mental well-being and physical symptoms.

[0321] Safety endpoints for all subjects receiving at least one dose included: individual adverse events as described in the protocol and self-reported daily review of systems (ROS) targeting 14 organs and functional systems; vital signs collected at each blood collection visit (systolic and diastolic blood pressure, calculated MAP, heart rate) and data provided by fitness trackers (Fitbit Charge 2; HRV, SpO2, heart rate, staged sleep duration); measures of subjective well-being: daily anxiety and stress measured by the Clinically Useful Anxiety Outcome Scale-Daily Version (CUXOS-D) and the Depression, Anxiety and Stress Scale-21 (DASS-21), and fatigue measured using the Daily Fatigue Impact Scale (D-FIS); and biomarkers associated with kidney and liver function and toxicity (e.g., ALT, AST, alkaline phosphatase, total bilirubin); and basal metabolic health biomarkers as represented by other CMP laboratory tests.

[0322] The study examined a comprehensive array of approximately 4,000 circulating biomarkers using blood samples from baseline, day 11 (day 4 of treatment), and day 13 (the day immediately following treatment). Samples were stored in a biobank and in liquid nitrogen at -80°C for batch processing, allowing assays of longitudinal samples from the same participants to be performed simultaneously. Metabolon's global platform, used for untargeted metabolomics of plasma samples from the study, features >1,000 metabolites; these metabolites include amino acids, carbohydrates, lipids, nucleotides, peptides, and partially characterized molecules. Approximately 3,000 proteins were assayed for plasma abundance using Olink's Explore 3,072 PEA proteomics platform with next-generation sequencing (NGS) readouts (Illumina HiSeq 2000). The broad range of proteins examined targeted numerous biological pathways, including cell membrane maintenance, nutrient transport, oxygen species formation, lipid transport and degradation, inflammation, immune signaling, and mitochondrial function.

[0323] This was a randomized, double-blind, placebo-controlled, longitudinal clinical trial with multi-omic analysis. Randomization to treatment and placebo was at enrollment after prescreening using a prospectively defined, sex-stratified, block randomization scheme.

[0324] The study recruited N=76 participants, of which N=60 were successfully enrolled and randomized into the study. N=51 received at least one dose of an exemplary oral enhanced NAD+ formulation or placebo and provided at least one blood draw during treatment. Analysis was performed on the complete available dataset from N=51 treated participants (25 females, 26 males). For this analysis, the distribution by randomized treatment group was N=23 enhanced NAD+ treatment, N=28 placebo.

[0325] The exemplary enhanced NAD+ pharmaceutical formulation used for treatment was an oral NAD+ formulation consisting of 500 mg of NAD+ and 500 mg of PEG3350. The composition was taken four times daily (qid) for five consecutive days from days 8 through 12. One additional dose was taken on each of days 9 and 10. For each dose, a measured amount of composition powder was dissolved in water, and participants orally administered the dose using a "rinse and swallow" procedure. The supplement meets FDA standards for human administration, including compliance with GMP regulations. Duration of treatment:

[0326] This was a 13-day protocol that included a 7-day run-in period (Days 1–7), including washout from all dietary supplements, followed by a 5-day treatment period (Days 8–12) and a 1-day follow-up (Day 13). After the 7-day run-in period, participants received either an oral NAD+-enhanced formulation or a placebo on a qid schedule for 5 days, with one additional dose administered on Days 9 and 10. Baseline blood draws and vital signs were obtained on Days 4 and 6 (before treatment), and these results were averaged as "baseline" for statistical analysis. Additional blood draws and vital signs were obtained on Days 11 (during treatment) and 13 (after treatment). The Day 11 blood draw represented approximately 14 doses, and the Day 13 blood draw, taken after the last dose, represented approximately 22 doses. result:

[0327] One endpoint of the study was the change in intracellular NAD (icNAD) concentration after treatment. Administering the exemplary Enhanced NAD+ composition resulted in a statistically significant increase in icNAD concentration relative to placebo for the interaction term, time*group: B = 3.39, SE = 0.35, P = 4.13e -13 .

[0328] The mean unadjusted change in icNAD from the mean of the two baseline blood draws to the fourth blood draw (the day immediately after treatment) (corresponding to 22 doses of 500 mg each for a cumulative dose of 11,000 mg) was estimated to be 14.56 μM (95% CI, 10.85 to 18.27 μM) for the enhanced NAD+ group, compared with an estimated change of −1.90 μM (95% CI, −4.04 to 0.24 μM) in the placebo group. This corresponds to an approximately 52.9% increase in icNAD over baseline (compared to a 4.4% decrease in the placebo group).

[0329] As shown in Figure 18, not only were there highly significant group differences, but every individual in the treatment group showed an increase in icNAD from baseline over the 5-day intervention. 10 of 23 (43%) participants increased icNAD by >50%, and 18 of 23 (78%) participants increased icNAD by >30%. The patient with the highest baseline score at study entry produced the smallest individual increase of 9.4% at the end of the study.

[0330] Oral administration of enhanced NAD+ preparations did not result in a statistically significant increase in eNAD (P=0.83). Low circulating eNAD concentrations may be maintained by the activity of NAD+ cleavage enzymes. Although some NAD+ may be directly transported into cells, maintenance of low eNAD concentrations is known to be mediated by ectoenzymes (e.g., cADPR synthase and pyrophosphatase) that catabolize it into NAD+ precursors (NAM, NMN, NR), which are then used in a salvage pathway to replenish icNAD concentrations. NAD+ metabolites and SIRT1

[0331] Oral enhanced NAD administration also led to differential statistically significant increases in the plasma abundance of two important NAD metabolites, supporting the bioavailability of the illustrative oral NAD formulations. Specifically, 1-methyl-nicotinamide (MeNAM; B = 0.47, SE = 0.065, P = 7.59 × 10 -1119) and N1-methyl-2-pyridone-5-carboxamide (2PY; B = 0.47, SE = 0.055, P = 1.03 × 10 -13 Increases were identified in MeNAM and 2PY compared to placebo (Figure 20). These changes in MeNAM and 2PY remained after correction for multiple testing using the Meff adjustment. The pattern of findings was identical when time was treated as a categorical variable in the timepoint analysis.

[0332] No changes were detected in the plasma abundance of four other NAD metabolites examined in the continuous time-based analysis (i.e., nicotinate, quinolinate, NAM, trigonelline; all P > 0.05). Time-point analysis showed a marginal effect of treatment on NAM by the fourth day of treatment (P = 0.053).

[0333] As discussed above, SIRT1 is an NAD-dependent enzyme that is normally elevated in plasma when NAD+ levels are increased. SIRT1 is actively involved in regulating energy sensing and homeostasis, oxidative stress response, and anti-inflammatory cascades. It is also associated with several chronic diseases, including cardiometabolic and neurodegenerative disorders.

[0334] In this study, a significant differential increase in SIRT1 plasma abundance was observed in the treatment group versus placebo (B=0.14, SE=0.09, t=1.76, P=0.041; Figure 21), again suggesting bioavailability of the exemplary oral formulation. Safety profile results

[0335] Analysis across 52 clinical tests revealed no statistically significant adverse changes across the analytes tested (all P>0.05).

[0336] Self-reported symptoms: A systems review questionnaire targeting 14 organs and functional systems was administered daily throughout the 5-day treatment period (baseline and post-treatment data were also obtained). The study observed no difference in the incidence of adverse symptoms between the exemplifying oral formulation and placebo groups (the overall incidence of self-reported symptoms was estimated per protocol to be 2.89% in the oral formulation group vs. 2.30% in the placebo group). One participant in the active treatment group discontinued due to nausea; symptoms resolved after interrupting the exemplifying oral formulation. Another participant discontinued upon being diagnosed with COVID-19.

[0337] No differential changes in vital signs or body composition (SBP, DBP, MAP, body mass index, waist circumference; all P > 0.05) were detected in the enhanced NAD+ versus placebo groups in response to NAD+ administration. Analysis of wearable data revealed that enhanced NAD+ administration was associated with differential reductions in resting heart rate (P = 0.033) and the number of minutes it took participants to fall asleep as estimated by Fitbit (P = 0.001) versus placebo. No other changes were observed in activity levels or sleep measures (all P > 0.05).

[0338] No differences were detected in changes in measures of subjective well-being (daily anxiety and stress measured by the modified daily version of the CUXOS and DASS, depression measured using the DASS, and fatigue measured using the FIS) in the NAD+ group versus placebo. Given the limited clinically relevant variability in this healthy population, the study had limited resolution to detect improvements in assessment scores. Exploratory analysis

[0339] Unless otherwise stated, all indicators of statistical significance (P values) have not been adjusted for multiple comparisons. Given the small size of the studies and the number of variables measured, it will be rare for results to remain statistically significant (P < 0.05) after correction for multiple hypothesis testing. Therefore, cases where P values ​​remain significant are particularly noteworthy.

[0340] The proteomic and metabolomic assays used generate results that are relative rather than absolute (quantitative) values. This is indicated on the X-axis of the figures, which are displayed as either "relative abundance" (for metabolites) or "NPX" (for proteins). NPX represents normalized protein expression, which is Olink's arbitrary unit (on a Log2 scale) for proteomic reports. cardiovascular disease

[0341] In this study, several significant changes were observed in the treatment group versus placebo, which may have relevance for the treatment of cardiovascular disease. Some changes are illustrated below:

[0342] Enhanced NAD+ administration led to a differential reduction in the LDL / HDL ratio compared with placebo (B = -0.03, SE = 0.01, t = -2.19, P = 0.029). Lower LDL / HDL ratios are associated with reduced risk of atherogenic and cardiac disease. No differences were observed in the study of triglyceride, LDL-cholesterol, or HDL-cholesterol levels. The majority of study participants had lipid levels within the normal range, so the statistically significant reduction in the LDL / HDL ratio is particularly noteworthy. Enhanced NAD+ administration may improve dietary lipid metabolism in addition to potentially improving endogenous lipid metabolism. Analysis of the plasma lipidome as part of untargeted metabolomic profiling revealed a coordinated decrease in the abundance of 19 lipids, indicating enhanced phospholipid and medium- and long-chain PUFA metabolism after NAD+ administration, which may have cardiovascular benefits.

[0343] Enhanced NAD+ differentially affected several proteins known to have vasoactive effects and to play a role in hypertension or stroke. In this study, an orally administered enhanced NAD+ pharmaceutical formulation significantly increased the levels of FGF12, EDIL3, FLT1 (VEGFR1), and FLRT2.

[0344] Fibroblast growth factor 12 (FGF12) is a potent inhibitor of the vascular smooth muscle cell (SMC) phenotypic switch, resulting in healthier blood vessels. FGF12 potently induces a healthier phenotype in human aortic SMCs, resulting in a more favorable response to arterial injury in rats. Conversely, lower FGF12 levels are associated with pulmonary arterial hypertension and are mechanistically linked to vascular injury in animal models of this disease.

[0345] EGF-like repeat and discoidin I-like domain-containing protein 3 (EDIL3) is an extracellular matrix protein that acts as a pro-angiogenic factor, a mediator of immune and anti-inflammatory responses, and a regulator of endothelial cell adhesion and migration. Overall, EDIL3 plays a key role in mediating angiogenesis and may be important in vascular wall remodeling and development. The effects of moderate increases in circulating levels in healthy patients are unknown.

[0346] The vascular endothelial growth factor receptor 1 (VEGFR1) protein binds to VEGFR-A, VEGFR-B, and placental growth factor receptors, and plays an important role in angiogenesis and vasculogenesis. Expression of this receptor is found in vascular endothelial cells, placental trophoblasts, and peripheral blood monocytes. It is involved in regulating angiogenesis by regulating endothelial cell proliferation and senescence. In mice, VEGFR1 levels are increased in ischemic muscle tissue compared with non-ischemic muscle, suggesting that VEGFR1 plays a role in vascular recovery from ischemia.

[0347] 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 epicardial development, and loss of this protein impairs ventricular myocardial expansion and reduces intracardiac volume. Its role in adult cardiac function is unknown.

[0348] Furthermore, exemplary NAD+-enhancing pharmaceutical preparations differentially reduced 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 coagulation, and inflammation. In mice, administration of neuroserpin after transplantation with aortic allografts exerted anti-inflammatory effects and reduced plaque growth and CD3+ T cell infiltration. After ischemic stroke, neuroserpin levels in the brain increased in areas surrounding the lesion, providing neuroprotection. Homocysteine ​​and the folate cycle

[0349] Increased levels of the amino acid homocysteine ​​are an independent risk factor for atherosclerosis, as there is a correlation between serum homocysteine ​​and the incidence of coronary, carotid, and peripheral vascular disease, likely mediated by the deleterious effects of homocysteine ​​on the vascular endothelium and arterial structure. As part of the folate / methylation cycle, the enzyme methionine synthase (MTR) uses methylated vitamin B12 (methylcobalamin) to convert homocysteine ​​to methionine, thus reducing homocysteine ​​levels.

[0350] A significant differential reduction in circulating MTR levels was observed (Figure 22), which may reflect optimization of the folate cycle and a reduced need for the production of methionine from homocysteine ​​(i.e., suggesting the potential that enhanced NAD+ administration may have reduced homocysteine ​​levels).

[0351] Oxidative stress is considered a major risk factor for numerous diseases, including cardiovascular disease, and increased oxidative stress is associated with arterial hypertension, cardiac arrhythmias, and atherosclerotic plaque formation. In this study, administration of an exemplary oral NAD+ preparation was associated with beneficial changes in various metabolites related to glutathione (GSH), an important cellular antioxidant. Notably, metabolite changes consistent with increased glutathione turnover with enhanced NAD+ administration included: an increase in plasma gamma-glutamylglutamine (B = 0.04, SE = 0.019, P = 0.02) and a decrease in circulating glutamate (B = -0.09, SE = 0.03, P = 0.004).

[0352] Analysis of the plasma proteome revealed that administration of the enhanced NAD+ pharmaceutical formulation was associated with an increase in the plasma abundance of glutathione S-transferase alpha-1 (GSTA1; B=0.08, SE=0.037, P=0.035; Figure 23) and a decrease in the plasma abundance of mitochondrial superoxide dismutase 2 (SOD2; B=-0.10, SE=0.04, P=0.016; Figure 24).

[0353] GSTA1 is an enzyme that utilizes GSH to bind electrophilic compounds and ROS, thus reducing oxidative stress, while SOD2 is another player in the cellular response to oxidative stress, binding superoxide anions and converting them to H2O2 and O2. Importantly, SOD2 is under transcriptional regulation by NAD+-dependent sirtuins. A decrease in plasma SOD2 levels may indicate a relaxation of the demand for its antioxidant capacity.

[0354] In this study, no significant changes in clinical inflammatory biomarkers, such as cardiac CRP, hsCRP, TNF, or IL-6, were observed.

[0355] As shown 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 both men and women with and without heart disease. Conversely, individuals with a lower resting heart rate have a reduced risk of cardiovascular mortality.

[0356] Metabolites associated with cardiovascular health that were differentially upregulated by the exemplary oral enhanced NAD+ formulation compared to placebo included homoarginine and glyco-beta-muricholate, while metabolites significantly decreased in the enhanced NAD+ group included docosahexaenoylcholine, urea, hypoxanthine, glutamate, asisoga, and 1-palmitoyl-2-arachidonoyl-GPC. More details regarding these metabolites in relation to CVD are provided below: - Homoarginine is an amino acid that appears to have protective effects against cardiovascular disease. Homoarginine appears to act as a weak substrate for endothelial nitric oxide synthase, improving vascular function. Glyco-beta-muricholate is a bile acid. Studies in mice have shown that muricholate promotes resistance to hypercholesterolemia. Docosahexaenoylcholine (choline docosahexaenoate) is a member of the acylcholine family, a class of molecules involved in fatty acid metabolism. Studies have also shown an association between this metabolite and serum lipids in Caucasian and African-American adults. - Urea cycle metabolites - Both urea and hypoxanthine are involved in the urea cycle, which breaks down toxic ammonia derived from protein metabolism and gut bacterial waste products. Urea cycle metabolites are a statistically overrepresented cluster associated with cardiometabolic disease. Excessive levels of urea are associated with oxidative stress, which plays a significant role in CVD. - Glutamate (glutamic acid) is an amino acid and an excitatory neurotransmitter. In addition to its neurological role, higher plasma glutamate is associated with heart failure. - Asisoga is a polyamine metabolite. It has been shown to predict atrial fibrillation. Recently, Asisoga has also been suggested as a novel biomarker in heart failure, correlating with reduced left ventricular function. 1-Palmitoyl-2-arachidonoyl-GPC, a phosphatidylcholine functionally related to arachidonic acid and hexadecenoic acid, predicts risk of ischemic stroke in women but not in men. Liver disease

[0357] Nonalcoholic fatty liver disease (NAFLD) and nonalcoholic steatohepatitis (NASH) are common chronic liver diseases. NAFLD and NASH are strongly associated with both obesity and type 2 diabetes, and therefore, as the prevalence of these conditions increases worldwide, the prevalence of fatty liver disease has also increased. In the United States, it is estimated that one in four people has NAFLD.

[0358] Although all study participants were healthy, the study provided strong evidence supporting that enhanced NAD+ administration is associated with improved liver function. Relative to placebo, enhanced NAD+ administration was associated with a preferential decrease in serum GGT (B=-0.40, SE=0.19, P=0.0404; Figure 26).

[0359] Enhanced NAD+ administration was associated with a preferential decrease in serum total bilirubin (TBILI) (B = -0.025, SE = 0.007, P = 0.00054; Figure 27). This finding passed the correction for multiple hypothesis testing applied to clinical tests, with the 95% CI for the interaction term being -0.0488 to -0.00007.

[0360] Enhanced NAD+ administration was also found to be associated with decreases in ALP (B = -0.60, SE = 0.28, P = 0.0327) and serum albumin (B = -0.023, SE = 0.011, P = 0.037).

[0361] No effect was detected on the liver enzymes AST or ALT, or on BUN or Cr (all P>0.05).

[0362] In addition to liver enzymes, a significant differential decrease was observed in the enhanced NAD+ group in the metabolite 1-palmitoyl-2-arachidonoyl-GPC, a phosphatidylcholine functionally related to arachidonic acid and hexadecenoic acid, which has previously been reported to be positively associated with liver fat in humans.

[0363] Furthermore, oxidative stress has been recognized as a significant risk factor for both the initiation and progression of various liver diseases, including alcoholic liver disease and NASH. While human evidence for the use of antioxidant compounds in established liver disease is limited, there is evidence for preventive benefits in animal models and in humans. Thus, the antioxidant benefits of enhanced NAD+ administration discussed above regarding cardiovascular disease are relevant to liver disease. This is particularly important given the liver's central role in overall metabolism, including its association with the development of type 2 diabetes.

[0364] GGT, an enzyme involved in the breakdown and liberation of glutathione, may be a sensitive marker of not only liver injury and cholestasis but also overall oxidative stress. Circulating GGT is a good biomarker of cellular redox status. Furthermore, serum concentrations of GGT are inversely correlated with serum antioxidants. Exocrine pancreatic function and digestion

[0365] Significant decreases were observed in the enhanced NAD+ group versus placebo in four pancreatic enzymes secreted in the gastrointestinal tract: PNLIPRP1 (pancreatic lipase-related protein 1); RNase1 (pancreatic ribonuclease); CPA1 (carboxypeptidase A1); and CPB1 (carboxypeptidase B1).

[0366] PNLIPRP1 is a lipolytic enzyme secreted in the gastrointestinal tract. The function of this protein is unknown.

[0367] RNase 1 belongs to the ribonuclease A superfamily, which consists of eight described enzymes. Pancreatic ribonucleases are pancreatic enzymes that catalyze the degradation of RNA and play a role in RNA digestion in vertebrate species. While historically thought of as a digestive enzyme, RNase 1 is now believed to have additional functions related to host defense, similar to other members of this enzyme superfamily.

[0368] 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 food proteins. Elevated protein levels (i.e., the opposite of those observed here) are associated with pancreatic cancer.

[0369] CPB1 is another enzyme produced in the pancreas, and highly elevated levels may be a serum marker of pancreatitis.

[0370] This observed effect of enhanced NAD+ administration on pancreatic enzymes was consistent across four different enzymes. Neurodegenerative and neuropsychiatric disorders

[0371] Evidence for changes in the levels of several proteins that modulate neuronal signaling pathways or neuronal growth was obtained in this study, as shown in FIG.

[0372] Proteins that were significantly decreased with enhanced NAD+ treatment compared to placebo included: ACOX1 (acyl-CoA oxidase 1), PLXNA4 (plexin A-4), ASAH1 (acid ceramidase), MTR (methionine synthase), GRN (progranulin), and SERPINI1 (neuroserpin).

[0373] ACOX1 is the first enzyme in the fatty acid beta-oxidation pathway, which catalyzes the desaturation of acyl-CoA to 2-trans-enoyl-CoA. It directly donates electrons to molecular oxygen, thereby generating hydrogen peroxide, which has various harmful effects in the body. A decrease in this protein suggests an improvement in oxidative stress, which may be regulated by SIRT enzymes. ACOX1 appears to play a key role in neuronal function.

[0374] 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 cell attraction and branching, regulation of cell migration, and vascular remodeling. Both up- and down-regulation of Plexin A4 has been observed after nerve injury, suggesting a dynamic role for Plexin A4 in nerve maintenance and regeneration. Plexin A4 has also been implicated in the pathology of Alzheimer's disease.

[0375] The acid ceramidase enzyme is found in lysosomes, cellular compartments that digest and recycle materials. Within lysosomes, acid ceramidase breaks down lipids called ceramides. Ceramides are normally found in membranes surrounding cells and play a role in regulating cell maturation, growth, and cell division (proliferation) and controlled cell death (apoptosis). Additionally, ceramides are components of a fatty substance called myelin, which insulates and protects nerve cells.

[0376] Methionine synthase is an enzyme involved in the regeneration of methionine from homocysteine ​​and in 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 homocysteine ​​levels are associated with increased amyloid-beta levels in the brains of Alzheimer's patients and may be an independent risk factor for neurodegenerative diseases. Methionine synthase is also a key regulator of brain antioxidant status and has been described as having a broad, dynamic role in coordinated brain metabolism during development and aging.

[0377] Progranulin is found in tissues throughout the body, but is most active in rapidly dividing cells, 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, but little is known about this protein's role in the brain. It appears to be important for neuronal survival.

[0378] 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 regulate the growth of neurons, particularly axons, which are necessary for the transmission of nerve impulses. Neuroserpin also plays a role in synaptic development and helps regulate synaptic plasticity, suggesting that it may be important for learning and memory. Neuroserpin has been shown to have a protective effect on the brain after stroke and is reported to be upregulated in Alzheimer's disease.

[0379] Proteins associated with neurological function that were significantly and preferentially increased in the NAD+ group compared to placebo included: NPL (N-acetylneuraminic acid 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).

[0380] NPLs are a family of lyase enzymes that catalyze the reversible cleavage and synthesis of sialic acids (monosaccharide sugars). Sialic acids have a variety of potential medical uses, including antiviral, antibacterial, and neuropathological applications. In the brain, the addition (sialylation) or removal (desialylation) of sialic acids in microglial cells has been associated with neurodegenerative diseases. A specific type of sialic acid-modifying molecule, polysialic acid, has also been associated with psychiatric disorders.

[0381] 5-HT1AR is involved in neuromodulation and affects a variety of conditions. 5-HT1AR agonists (e.g., buspirone) have shown efficacy in anxiety and depression, and 5-HT1AR also appears to play a role in modulating chronic pain. The possible 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.

[0382] 5-HT1A receptor activation has also been shown to increase dopamine release in the medial prefrontal cortex, striatum, and hippocampus, and may therefore be useful for ameliorating the symptoms of schizophrenia and Parkinson's disease. The potential impact on Parkinson's disease is particularly intriguing, given that several older clinical studies have found significant benefits from administering IV NADH to patients with Parkinson's disease.

[0383] GCPII is a zinc metalloenzyme found in prostate epithelium, kidney, small intestine and nervous system.In the intestine, this enzyme catalyzes a reaction that leads to the release of folic acid, which can then be used as a vitamin in the body.In the brain, GCPII has been shown to increase glutamate levels both indirectly and directly.Potent, selective GCPII inhibitors have been shown to reduce brain glutamate and provide neuroprotection in preclinical models of stroke, amyotrophic lateral sclerosis and neuropathic pain.

[0384] HRAS is a key regulator of signal transduction and the cell cycle. The RAS signaling pathway is involved in regulating developmental processes, including cell growth, proliferation, and differentiation in the central nervous system. Germline mutations in RAS signaling pathway genes are associated with a group of neurodevelopmental disorders collectively referred to as RASopathies, including 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 may affect spatial short-term memory and object recognition.

[0385] CRIP2 is found in many different tissues and is thought 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.

[0386] 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.

[0387] In addition to protein changes, several significant changes were also observed in untargeted metabolomics analysis. Specifically, significant differential increases in urate (uric acid), homoarginine, and indole lactate (indole lactic acid) were observed when NAD was used. While urate (uric acid) is widely recognized as protective against aging and oxidative stress, increased urate levels have also been associated with reduced risk of Alzheimer's disease, Parkinson's disease, and multiple sclerosis. Homoarginine is an amino acid that may play a role in maintaining healthy brain function, and both significant excess and deficiency have been associated with negative effects. Indole lactate is an intermediate in the breakdown of tryptophan to indole propionate. It may be significantly lower in individuals with multiple sclerosis, which may be related to the inflammatory process of this disease. In cell culture, indole lactate increases neuronal growth.

[0388] In the enhanced NAD+ group, differential decreases were also observed in the following: glutamate (glutamic acid), hypoxanthine (Figure 25), cortisone, and 1,2-dipalmitoyl-GPC (16:0 / 16:0).

[0389] Glutamate is an amino acid and an excitatory neurotransmitter.Plasma glutamate levels are found to be higher in patients with migraine, and the glutamine / glutamate ratio is altered in patients with schizophrenia.Higher plasma glutamate levels are also found in individuals who develop post-stroke depression.

[0390] Hypoxanthine, a metabolite in the urea cycle in the brain, causes mitochondrial dysfunction and neuronal death in animal models.

[0391] Cortisone is a metabolite of the steroid stress hormone cortisol. Both cortisol and cortisone are considered markers of stress, and some studies suggest that plasma cortisone may be an even more sensitive marker of stress. Cortisone is elevated in patients with depression. 1,2-dipalmitoyl-GPC (16:0 / 16:0) is a phosphatidylcholine glycerophospholipid containing two chains of palmitic acid. 1,2-dipalmitoyl-GPC can correlate with the levels of multiple Alzheimer's disease biomarkers in the cerebrospinal fluid of elderly adults and is one of the top three metabolite predictors of Alzheimer's disease progression in a cohort with mild cognitive impairment.

[0392] Apart from alterations in specific proteins and metabolites, both oxidative and nitrosative stress have an impact on neurodegenerative diseases. Administration of enhanced NAD+ had several beneficial effects on markers of antioxidant status. Increased oxidative stress has been 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.

[0393] Nitrosative stress is a metabolic disorder that occurs in the body when nitric oxide (NO) and superoxide (O2 - ) refers to the cooperative biochemical reaction of oxidative and nitrosative stress. Therefore, oxidative and nitrosative stress can exert parallel actions leading to cell damage and cell death, and nitrosative stress has been implicated in various diseases, including neurodegenerative diseases. This study found a coordinated signal for the effectiveness of enhanced NAD+ in enhancing protection against nitrosative stress. Specifically, plasma proteome matching revealed that carbon dioxide is converted into HCO3 in liver mitochondria in the urea / ornithine cycle. -We found differential upregulation of mitochondrial CA5A, an enzyme that supplies ammonia (carbonic anhydrase 5A; B = 0.09, SE = 0.035, P = 0.016). Concomitantly, plasma metabolomic analysis revealed decreases 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 / Immunomodulation

[0394] 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 disease severity and progression. SIRT1-deficient mice developed experimental autoimmune encephalomyelitis (considered an animal model of MS) ​​and severe forms of spontaneous autoimmunity.

[0395] In this study, no changes were observed in circulating inflammatory biomarkers as assayed using clinical tests, and no changes were detected in white blood cell counts. However, several changes in immune- and inflammation-related proteins were observed in proteomic analyses, as shown below.

[0396] Plasma proteome analysis linked enhanced NAD+ administration to a significant differential decrease in the abundance of CXCL12 (P=0.004; see Figure 29) and a trend toward a decrease in 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 involvement of each of these proteins is discussed below:

[0397] The homeostatic chemokine CXCL12 is a key factor in numerous physiological and pathological processes, as it activates and / or induces the migration of hematopoietic progenitors and stem cells, endothelial cells, and most leukocytes. The CXCL12-CXCL4 signaling axis is involved in tumor biology, and reduction of CXCL12 may be beneficial in this regard. CXCL12 is also thought to be a key player in the interaction between the immune and nervous systems.

[0398] CCL8 is a chemokine belonging to the CC chemokine family that acts as a macrophage chemotactant and activates many different immune cells, including mast cells, eosinophils, and basophils involved in allergic responses, as well as monocytes, T cells, and NK cells involved in inflammatory responses.

[0399] Increased CXCL14 leads to attenuation of CXCL12-CXCR4 signaling, which is suggested to be its primary function and limit CXCL12-mediated chemotaxis required for potentially antitumor progenitor and immune cells.

[0400] The 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.

[0401] Interleukin-31 (IL-31) stimulates activated CD4 + IL-31 is primarily produced by T cells and interacts with receptors expressed on epithelial cells and keratinocytes. IL-31 acts on a wide range of immune and non-immune cells, and thus has the potential for pleiotropic physiological functions, including regulation of hematopoietic development and immune responses. IL-31 has been implicated in inflammatory bowel disease, airway hyperresponsiveness (asthma), and allergic dermatitis.

[0402] Interleukin-34 (IL-34) is a cytokine that promotes the differentiation and survival of monocytes and macrophages via the colony-stimulating factor-1 receptor. Highest levels of IL-34 are found in the brain and skin. IL-34 is associated with several autoimmune diseases, including rheumatoid arthritis and systemic lupus erythematosus. Reproductive Function and Fertility

[0403] The NAD+-dependent enzyme SIRT1 is involved in regulating sex hormone levels, which are key to reproductive function. SIRT1 knockout mice have been shown to have reduced production of gonadotropin-releasing hormone and subsequently reduced LH and FSH levels, which may account for the observed effects of reduced SIRT1 on male fertility. In men, SIRT1 also regulates spermatogenesis and sperm maturation.

[0404] In women, downregulation of SIRT1 is associated with reduced ovarian reserve, and some have suggested that sirtuins are biomarkers of ovarian aging. Maternal obesity is a major cause of metabolic dysregulation in offspring, including glucose intolerance, increased liver fat, and risk of obesity.

[0405] In this study, we observed a significant differential effect of enhanced NAD+ administration on the protein INSL3 (insulin-like peptide 3). INSL3 is a protein hormone produced by gonadal tissue in both males and females. Circulating levels are higher in males than in females. In males, INSL3 controls testicular descent during embryonic development and declines with age, possibly reflecting the effects of aging on the testes.

[0406] In women, INSL3 may be involved in regulating female fertility. This protein changes over the course of the menstrual cycle and declines after menopause. (Note: Blood samples were not timed to menstrual cycle phases during the study.) INSL3 may also play a role in polycystic ovary syndrome (PCOS). The fact that INSL3 changed significantly with increased NAD supplementation indicates that INSL3 is a factor associated with the effects of NAD on fertility. Metabolic Disease and Obesity

[0407] In humans, SIRT1 levels are reduced in diabetic patients with poor glycemic control compared with those with good glycemic control.

[0408] In this study, untargeted proteomic analysis demonstrated upregulation of three illustrative proteins previously shown to play a role in obesity and metabolic disease: GALNT2 (N-acetyl-galactosaminyl-transferase 2), FABP1 (fatty acid binding protein 1; Figure 30), and RBP2 (retinol binding protein 2; Figure 31).

[0409] GALNT2 is a member of the glycosyltransferase 2 protein family. Recently, GALNT2 has attracted attention as a possible player in a number of clinical conditions that share the commonality of insulin resistance, including atherogenic dyslipidemia, type 2 diabetes, and obesity.

[0410] FABP1 is important for fatty acid uptake and intracellular transport and plays a key role in regulating lipid metabolism and cell signaling pathways. It is primarily found in the liver. FABP1 may exert protective effects against lipotoxicity by promoting the oxidation of fatty acids or their incorporation into triglycerides and by otherwise binding these compounds. Its ability to bind heme is another cytoprotective property. FABP1's role in substrate availability and protection from oxidative stress suggests that it plays a central role by reducing inflammation during intracellular bacterial / viral infections. Altered protein expression has been linked to metabolic conditions, including obesity, and higher circulating levels have been reported in Chinese adults with high BMI and insulin resistance.

[0411] In addition to protein changes, several significant changes were observed in the enhanced NAD+ group in untargeted metabolomics analysis. Specifically, significant increases in bile acids or bile acid derivatives: glycohyocholic acid (GHCA) and glyco-beta-muricholate were observed with enhanced NAD+. Glycohyocholic acid is the glycine-conjugated form of the primary bile acid hyocholic acid. Plasma GHCA was found to be lower in individuals with obesity and type 2 diabetes. In patients who are no longer diabetic after gastric bypass surgery, plasma GHCA levels increase and can predict diabetes remission. Muricholate is a bile acid. In obese individuals, glyco-beta-muricholate has been found to correlate with insulin resistance.

[0412] In this study, significant differential decreases were found in the enhanced NAD+ group in the following metabolites: pyrraline, urea, glutamate (glutamic acid), asisoga, nonadecanoate (nonadecanoic acid), and 1-palmitoyl-2-arachidonoyl-GPC.

[0413] Pirarin is the end It is a type of glycation end product (AGE), a harmful compound formed in food and in vivo. AGEs are associated with diseases of aging and, in particular, type 2 diabetes. Urinary pyrraline levels are elevated in patients with type 2 diabetes and are associated with glycemic control.

[0414] Elevated levels of urea have been found to impair insulin secretion in mice and in vitro models. In animal models of chronic kidney disease, urea is associated with insulin resistance, potentially mediated by its effects on oxidative stress.

[0415] Glutamate is an amino acid and an excitatory neurotransmitter. In addition to its neurological role, higher plasma glutamate is associated with type 2 diabetes.

[0416] Ashisoga is a polyamine metabolite that is increased in children with obesity. Ashisoga has also been found to be one of the metabolites significantly associated with new-onset diabetes in a large longitudinal cohort in a model controlled for other diabetes risk factors.

[0417] Nonadecanoate is a saturated fatty acid. Plasma levels of nonadecanoate were found to be significantly associated with glucose tolerance in metabolomics studies of adults with normal or impaired glucose tolerance. Nonadecanoate levels are also elevated in obese diabetic rodent models.

[0418] 1-Palmitoyl-2-arachidonoyl-GPC is a 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

[0419] Significant differential changes were observed in the enhanced NAD+ group in two proteins previously associated with skin health or condition: specifically, enhanced NAD+ significantly reduced PPIB and WFDC12 compared to placebo.

[0420] PPIB (peptidyl-prolyl cis-trans isomerase B)—PPIB is localized in the endoplasmic reticulum (ER) and is involved in numerous biological processes, including mitochondrial metabolism, apoptosis, and inflammation. In the ER, PPIB interacts with various other proteins to promote protein folding, particularly for type I collagen. Thus, PPIB is essential for collagen biosynthesis and post-translational modification, affecting fibril assembly, matrix cross-linking, and bone mineralization.

[0421] WFDC12 (whey acidic protein 4-disulfide-core 12) - WFDC12 belongs to a family of proteins involved in innate immune defense, including the inhibition of neutrophil serine proteases and the inhibition of inflammatory responses to lipopolysaccharide (LPS). WFDC12 is produced in several tissues, primarily the lungs and skin. Elevated levels of WFDC12 are found in affected (inflamed) skin of patients with psoriasis and atopic dermatitis.

[0422] As mentioned above, PPIB plays a role in both skin and bone health, and specifically, significant changes were observed in two other proteins associated with bone - a decrease in INSL3 and an increase in MATN3.

[0423] INSL3 (insulin-like peptide 3) is a protein hormone produced in both male and female gonadal tissues. In addition to its important role in the reproductive system, INSL3 plays a role in bone and musculoskeletal function and may affect other organs. INSL3 regulates the expression of genes involved in the differentiation and maturation of primary human osteoblasts, such as ALP, COL1A1, COL6A1, and osteonectin. Treatment of primary human osteoblasts with INSL3 improved bone matrix mineralization. Furthermore, decreased INSL3 levels in patients with Klinefelter's syndrome correlated with increased serum sclerostin levels, which is involved in bone catabolism by inhibiting osteoblast differentiation and stimulating osteoclast activation.

[0424] MATN3 (Matrilin-3) - Matrilin-3 is found in the extracellular matrix surrounding the cells that make up ligaments and tendons and in nearby cartilage-forming cells (chondrocytes). Chondrocytes play an important role in bone formation. Matrilin-3 may play a role in the organization of collagen and other cartilage proteins and may be involved in osteoarthritis. Example 8 Oral administration of an NAD+-enhanced pharmaceutical formulation reduces opioid withdrawal symptoms

[0425] This example illustrates the use of an oral formulation of the enhanced NAD+ composition described herein to alleviate opioid withdrawal symptoms, thus facilitating abrupt opioid cessation in adult human subjects.

[0426] Opioid tolerance, dependence, and addiction are all expected manifestations of brain changes resulting from opioid use. Abrupt cessation of opioids in physically dependent patients results in acute withdrawal symptoms. Abnormalities in the brain's reward system resulting from opioid use necessitate continued drug use to avoid opioid withdrawal syndrome (OWS). While opioid withdrawal is not life-threatening, it acts as a barrier to entering treatment and ceasing opioid use; concerns about withdrawal prevent some patients from seeking treatment, and withdrawal symptoms contribute to relapse in patients attempting recovery. Current treatment options for abrupt withdrawal include methadone, buprenorphine, and clonidine. In clinical situations where this is possible, slow tapering with the opioid to which the patient is physically dependent is preferable to abrupt withdrawal.

[0427] The severity and duration of OWS vary as a function of patient-specific characteristics, including the opioid's half-life, the duration of opioid use, and health status. Abrupt discontinuation of short-acting opioids (e.g., fentanyl, heroin, hydrocodone, and oxycodone) is associated with severe OWS, typically beginning within 12 hours of the missed dose, peaking at 36–72 hours, and tapering gradually over the next 4–7 days. Withdrawal from the long-acting opioid buprenorphine may be less severe than withdrawal from short-acting opioids of similar duration. Withdrawal from methadone also results in milder symptoms, but they may persist for 2 weeks or longer.

[0428] The types of symptoms experienced may vary from patient to patient but are similar regardless of the type of opioid used (i.e., long-acting vs. short-acting). OWS symptoms include aches / pains, muscle spasms / twitching / tension, tremors, abdominal cramps, nausea / vomiting / diarrhea, anxiety / restlessness, irritability, and insomnia. OWS generally resolves after 5 to 14 days (depending on the half-life of the opioid), but distress can be severe during the first few days after discontinuation. Without appropriate treatment, many patients are unable to complete opioid cessation. Pain relief, relaxation, self-medication of depression and anxiety, or pleasure-seeking may be the initial reasons for opioid use, but with long-term use, avoidance of OWS is often the most powerful force driving continued use.

[0429] For example, in a study that included patients with chronic pain, 56.5% of patients who first used prescription opioids for pain relief reported that their primary reason for continuing use was to avoid OWS. In another study of opioid analgesic misuse, OWS was the most frequently cited reason for switching from opioid analgesics to heroin.

[0430] Conversely, patients also report OWS as a primary driver for seeking treatment for opioid use disorder (OUD). OUD is characterized by the chronic and persistent manifestation of several symptoms over a 12-month period, including withdrawal symptoms, tolerance development, and an uncontrollable desire to seek and use drugs despite negative consequences for the patient's daily life. The longitudinal course of this neuropsychiatric disorder is characterized by cyclical periods of exacerbated use and abstinence over several years, separated by periods of treatment and remission, during which vulnerability to relapse remains high due to persistent neuroadaptation to the brain's reward circuitry after chronic exposure to opioids. Effective treatment of OWS can stabilize patients, paving the way for them to discontinue opioid use and enter long-term treatment.

[0431] Both OUD and opioid addiction are at epidemic levels in the United States (US), with more than 3 million people having or currently suffering from OUD, and the crisis is expected to worsen. According to data from the US Centers for Disease Control and Prevention, National Center for Health Statistics, there were an estimated 100,306 drug overdose deaths in the US during the 12-month period ending in April 2021, a 28.5% increase from the 78,056 deaths during the same period the previous year.

[0432] Clinicians strive for early diagnosis and treatment of OUD to avoid progression to more severe disorders. Very early recognition of physical dependence on opioids before psychological dependence develops may prevent OUD. Patient education is helpful. Patients may not realize that opioid physical dependence can develop within weeks of starting opioids, and they may initially attribute OWS to having a cold or the flu rather than opioid withdrawal because the symptoms are very similar.

[0433] Once OUD or opioid physical dependence is recognized and the patient requests or consents to treatment, a long-term treatment strategy must be established. OUD is a chronic disorder that often requires long-term, or even lifelong, treatment. Treatment goals guide OUD treatment strategies. Dependence on opioids occurs in a variety of patient types and clinical settings, leading to different patient preferences and treatment strategies.

[0434] Regardless of the ultimate treatment goal, management of acute OWS is the first step during opioid discontinuation or dose reduction.

[0435] In patients who use opioid analgesics appropriately, opioid withdrawal is usually managed by slowly tapering the analgesic (called detoxification or withdrawal management). In people who are physically dependent on opioids, tapering and withdrawal management may be supplemented with pharmaceutical interventions such as clonidine, lofexidine, naltrexone, or opioid administration such as buprenorphine or methadone.

[0436] However, treatment of OUD with methadone, buprenorphine, or naltrexone requires close medical supervision in a certified facility and is therefore not always available or desired by patients. These treatments are also long-term, may be accompanied by unwanted side effects, and, as such, have the potential for misuse or abuse (substituting one addiction for another). Naltrexone treatment can only be safely initiated after an opioid-free period of >7 days, so it is not an option for OUD patients who are unable to undergo withdrawal management first. Clonidine and lofexidine can be used to relieve many of the symptoms of opioid withdrawal, but they do not adequately treat all withdrawal symptoms, resulting in treatment discontinuation and relapse (≥65%). The time to opioid withdrawal with clonidine has been reported to be longer than with placebo, but not statistically significantly so (duration of withdrawal of 34.8 ± 3.7 days with clonidine compared with 25.5 ± 2.7 days with placebo). Lofexidine produces only a modest reduction in OWS symptoms of 12–14%, depending on the dose used. Both clonidine and lofexidine are associated with bradycardia, hypotension, orthostatic effect, somnolence, sedation, dry mouth, and serious cardiovascular effects that interfere with patient compliance and treatment success.

[0437] This study illustrates the use of an orally administered, enhanced NAD+ pharmaceutical formulation to alleviate opioid withdrawal symptoms and facilitate abrupt opioid discontinuation in adults. The formulation was designed to provide a less invasive route of administration compared to intravenously administered NAD+. Rinsing the mouth with the formulation before swallowing increases the duration of exposure to the oral mucosa, which may allow for greater bioavailability compared to orally ingested NAD+. Furthermore, compared to the only FDA-approved product for the relief of withdrawal symptoms from abrupt opioid withdrawal (Lucemyra®; NDA 209229), which carries warnings and precautions about cardiovascular and central nervous system (CNS) risks, the active ingredient, NAD+, in this formulation appears safe (and even protective) for these organ systems.

[0438] In an exemplary experiment, an oral enhanced NAD+ formulation containing 50% nicotinamide adenine dinucleotide and 50% polyethylene glycol will be used for human consumption. A Phase 1, randomized, two-part, open-label study will be conducted in healthy volunteers (e.g., 18-55 years of age, inclusive) including a bioavailability (BA) and dose-escalation study using the exemplary oral enhanced NAD+ formulation (administered via mouth rinse and swallow) compared to NAD+ IV administration. Exemplary doses include 500 mg, 1 g, 2 g, 3 g, and 4 g. Because a sufficient rise in circulating NAD+ is required for the product's therapeutic effect, a PK study in healthy adults will be conducted to assess the absolute BA (rate of systemic exposure [C]) of the proposed enhanced NAD+ oral solution product compared to NAD+ delivered via IV administration (e.g., a 750 mg infusion over 6 hours). maxThe objective of this study is to evaluate the BA (between 2000 and 2010) and extent (AUC) of NAD+. Oral rinsing with the enhanced NAD+ solution can be sustained for as long as possible (approximately 30-60 seconds) before swallowing the solution. A fasting-fed period for the highest proposed dose can be used to evaluate the effect of food on the BA of the proposed enhanced NAD+ product. In vitro drug-drug interaction (DDI) studies can be performed with NAD+ to evaluate potential metabolic and transporter-mediated drug interactions (as a substrate, inhibitor, or inducer).

[0439] In a further exemplary experiment, a randomized, placebo-controlled Phase 2 study of patients undergoing abrupt opioid discontinuation (with or without other medication use) is conducted. In Phase 1, the maximum safe dose of an exemplary enhanced NAD+ composition, such as 500 mg / day, 1 g / day, 2 g / day, 3 g / day, or 4 g / day, is determined. For the exemplary dosing regimen, the enhanced NAD+ oral solution (500 mg) is administered four times daily (every 3-4 hours) for five days (with additional doses given immediately before bedtime on days two and three). On days six and seven (tapering period), the same dose of NAD+ solution is administered twice daily. A placebo (all components of the oral solution, in the same volume, but without the API) is administered according to the same schedule. After this seven-day administration period, an additional seven-day follow-up is conducted for safety assessment. Exemplary efficacy endpoints may include: change in Clinical Opiate Withdrawal Scale (COWS) compared to placebo, assessed on days 3 and 5 of treatment, and mean COWS score over the course of treatment compared to placebo; change in Subjective Opiate Withdrawal Scale (SOWS) compared to placebo, assessed on days 3 and 5 of treatment, and mean SOWS score over the course of treatment compared to placebo; and / or other exploratory endpoints. Exemplary inclusion criteria for enrolling patients include those currently dependent on any other addictive or psychoactive substance in addition to opioids.

[0440] In a further exemplary experiment, a randomized, placebo-controlled, parallel-design Phase 3 study is conducted using a design like the illustrative Phase 2 study described above. Example 9 Relief of symptoms associated with viral infections

[0441] This example illustrates the use of an enhanced NAD+ pharmaceutical formulation to alleviate symptoms associated with viral infection, e.g., by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2).

[0442] SARS-CoV-2 has caused the global spread of coronavirus disease 2019 (COVID-19). Patients with COVID-19 typically develop severe respiratory distress, which can lead to death. According to the U.S. Centers for Disease Control and Prevention (CDC), some people infected with the SARS-CoV-2 virus that causes COVID-19 may experience prolonged effects from their infection, known as post-COVID status (PCC) or long COVID. People refer to the post-COVID state by many names, including long COVID, long-term COVID, post-acute COVID-19, post-acute sequelae of SARS-CoV-2 infection (PASC), long-term COVID effects, and chronic COVID. The post-COVID state can include a wide range of ongoing health problems; these conditions may persist for weeks, months, or years. While the post-COVID state is more common in people who have had severe COVID-19 illness, anyone infected with the virus that causes COVID-19 can experience the post-COVID state. Infected individuals who have not been vaccinated against COVID-19 may be at higher risk of developing the post-COVID state compared with previously vaccinated individuals. Most individuals with the post-COVID state have evidence of infection or COVID-19 disease, but in some cases, individuals with the post-COVID state may not have tested positive for the virus or may not know they were infected. The post-COVID state can include a wide range of new, recurring, or ongoing health problems experienced after being infected with the virus that causes COVID-19. Most people with COVID-19 begin to feel better within days to weeks of infection, so the post-COVID state can first be identified at least four weeks after infection. Anyone who is infected can experience the post-COVID state. Most people with the post-COVID state experience symptoms several days after first learning they have COVID-19, but some who later experience the post-COVID state did not know when they were infected.

[0443] People with post-COVID conditions can have a wide range of symptoms that can persist for weeks, months or even years after infection.

[0444] In an illustrative study, the enhanced NAD+ pharmaceutical formulation (oral enhanced NAD+ formulation) described herein is used to prevent or treat at least one long-term symptom of viral infection. For example, a subject is identified as suffering from or at risk of suffering from a long-term symptom of viral infection, such as long Covid. An amount of a composition containing nicotinamide adenine dinucleotide (NAD+), such as an enhanced NAD+ composition described herein, is then orally administered to the subject, the amount being effective to increase NAD levels in the subject by at least 5%, thereby alleviating or preventing the long-term symptom of viral infection in the subject. The amount of NAD+ for administration can be designed based on the results of the Phase I / II / III study in Example 7 or other disclosures in this application.

[0445] Non-limiting mechanisms of using the enhanced NAD+ pharmaceutical formulations provided herein to prevent or treat COVID-19 infection, e.g., Long Covid symptoms, may include replenishing NAD+ levels in the host that have been reduced by viral infection, may reactivate host cells, may suppress the expression of inflammatory cytokines (thus suppressing uncontrolled inflammation), etc. Example 10 Thermogravimetric analysis of unenhanced versus enhanced NAD+ powders

[0446] Five powder samples containing NAD+ and PEG (samples 6, 7, 8, 9, and 5) were prepared as described below.

[0447] Sample 6: NAD+ and PEG3350 were combined in a 1:1 weight ratio, stirred at room temperature for 20 minutes, and stored as Sample 6 (unaugmented).

[0448] Sample 7: NAD+ was subjected to three vacuum cycles (each cycle involved subjecting the solid to vacuum for approximately 20 minutes, followed by back-filling with argon). PEG3350 was then added (1:1 weight ratio with NAD+), and the solid was stirred at room temperature for 20 minutes and stored as Sample 7 (enhanced NAD+ composition).

[0449] Sample 8: A portion of Sample 7 was subjected to three additional vacuum cycles (each cycle involving subjecting the solid to vacuum for approximately 20 minutes and then backfilling with argon). The solid was stirred at room temperature for 20 minutes and stored as Sample 8 (enhanced NAD+ composition).

[0450] Sample 9: A portion of Sample 8 was subjected to three additional vacuum cycles (each cycle involving subjecting the solid to vacuum for approximately 20 minutes and then backfilling with argon). The solid was stirred at room temperature for 20 minutes and stored as Sample 9 (enhanced NAD+ composition). TGA

[0451] TGA was performed on powder samples as received. Sample analyses were performed in duplicate. Samples were heated from ambient to 800°C under nitrogen gas at a rate of 10°C / min, and ramped to 900°C under air at 10°C / min.

[0452] Sample 6 experienced four major weight loss steps. Between ambient temperature and 170°C, a small weight loss was observed, representing the loss of moisture and hydration water. Between 170°C and 800°C, there was a two-step decomposition that began at approximately 170°C and 360°C. Above 800°C, pyrolytic carbon formed during the pyrolysis of the polymer decomposed in air. After 900°C, the sample was oxidized, and anything that did not burn off was considered residue. result

[0453] For Sample 6, the thermograms are shown in Figures 32A-C. Weight loss results are shown in Table 3. Table 3. TGA results for sample 6 [Table 3]

[0454] Samples 7, 8 and 9 had a higher starting temperature than Sample 6, therefore the first dehydration process was not observed and Weight Loss 1 represented the sum of Weight Loss 1 and Weight Loss 2 in Sample 6.

[0455] For sample 7, the thermograms are shown in Figures 33A-C. Weight loss results are shown in Table 4. Table 4. TGA results for sample 7 [Table 4]

[0456] For sample 8, the thermograms are shown in Figures 34A-C. Weight loss results are shown in Table 5. Table 5. TGA results for sample 8 [Table 5-1] [Table 5-2]

[0457] For Sample 9, the thermograms are shown in Figures 35A-C. Weight loss results are shown in Table 6. Table 6. TGA results for sample 9 [Table 6]

[0458] FIG. 36 shows an overlay of the thermograms of samples 6, 7, 8 and 9. equivalent

[0459] Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents to the specific embodiments specifically described herein which equivalents are intended to be encompassed within the scope of the following claims.

Claims

1. 1. An enhanced nicotinamide adenine dinucleotide (NAD+) composition comprising: a crystalline component comprising crystalline polyethylene glycol, and Amorphous components, including amorphous NAD+ Including, 1. A composition characterized by having a first onset temperature between 179.39°C (±0.10°C) and 182.61°C (±0.10°C) by thermogravimetric analysis (TGA) thermogram.

2. 1. An enhanced nicotinamide adenine dinucleotide (NAD+) composition comprising: a crystalline component comprising crystalline polyethylene glycol, and Amorphous components, including amorphous NAD+ Including, 1. A composition characterized by having a weight loss of 14.39% to 16.98% by weight in the range of about 170°C to about 300°C according to a thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 300°C under nitrogen gas at a ramp rate of 10°C / min.

3. 1. An enhanced nicotinamide adenine dinucleotide (NAD+) composition comprising: a crystalline component comprising crystalline polyethylene glycol, and Amorphous components, including amorphous NAD+ Including, 1. A composition characterized by having a weight loss of 57.83 wt % to 58.55 wt % in the range of about 300°C to about 800°C according to a thermogravimetric analysis (TGA) thermogram collected from ambient temperature to at least 800°C under nitrogen gas at a ramp rate of 10°C / min.

4. 10. The composition of any one of the preceding claims, further comprising amorphous polyethylene glycol.

5. 1. An enhanced nicotinamide adenine dinucleotide (NAD+) composition comprising: a crystalline component comprising crystalline polyethylene glycol, and Amorphous components, including amorphous NAD+ Including, 33A, 33B, 34A, 34B, 35A, or 35B.

6. 6. The composition of claim 5, having a TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with Figure 33A or Figure 33B.

7. 6. The composition of claim 5, having a TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with Figure 34A or Figure 34B.

8. 6. The composition of claim 5, having a TGA thermogravimetric analysis (TGA) thermogram substantially in accordance with Figure 35A or Figure 35B.

9. 1. An enhanced nicotinamide adenine dinucleotide (NAD+) composition comprising: a crystalline component comprising crystalline polyethylene glycol, and Amorphous components, including amorphous NAD+ Including, a first weight loss of 2.42 wt % to 3.25 wt % in the range of ambient temperature to about 170° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to at least 170° C.; and / or a second weight loss of 13.66 wt % to 14.61 wt % in the range of about 170° C. to about 300° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to at least 300° C.; and / or a third weight loss of 58.73 wt % to 60.37 wt % in the range of about 300° C. to about 800° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to at least 800° C.; and / or a fourth weight loss of 20.14 wt % to 21.46 wt % in the range of about 800° C. to about 900° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to about 800° C. and under air at a ramp rate of 10° C. / min from about 800° C. to about 900° C.; and / or a first onset temperature of 179.39°C to 180.15°C, and / or Second onset temperature of 369.03°C to 370.82°C 1. A composition characterized by having a TGA thermogravimetric analysis (TGA) thermogram having:

10. 1. An enhanced nicotinamide adenine dinucleotide (NAD+) composition comprising: a crystalline component comprising crystalline polyethylene glycol, and Amorphous components, including amorphous NAD+ Including, a first weight loss of 2.32 wt % to 3.55 wt % in the range of ambient temperature to about 170° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to at least 170° C.; and / or a second weight loss of 14.39 wt % to 16.22 wt % in the range of about 170° C. to about 300° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to at least 300° C.; and / or a third weight loss of 57.83 wt % to 58.55 wt % in the range of about 300° C. to about 800° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to at least 800° C.; and / or a fourth weight loss of 22.62 wt % to 22.76 wt % in the range of about 800° C. to about 900° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to about 800° C. and under air at a ramp rate of 10° C. / min from about 800° C. to about 900° C.; and / or a first onset temperature of 178.62°C to 180.11°C, and / or Second starting temperature of 365.73°C to 370.47°C 1. A composition characterized by having a TGA thermogravimetric analysis (TGA) thermogram having:

11. 1. An enhanced nicotinamide adenine dinucleotide (NAD+) composition comprising: a crystalline component comprising crystalline polyethylene glycol, and Amorphous components, including amorphous NAD+ Including, a first weight loss of 3.62 wt % to 3.96 wt % in the range of ambient temperature to about 170° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to at least 170° C.; and / or a second weight loss of 15.17 wt % to 16.98 wt % in the range of about 170° C. to about 300° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to at least 300° C.; and / or a third weight loss of 58.05 wt % to 59.66 wt % in the range of about 300° C. to about 800° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to at least 800° C.; and / or a fourth weight loss of 22.85 wt % to 25.87 wt % in the range of about 800° C. to about 900° C. according to a thermogravimetric analysis (TGA) thermogram collected under nitrogen gas at a ramp rate of 10° C. / min from ambient temperature to about 800° C. and under air at a ramp rate of 10° C. / min from about 800° C. to about 900° C.; and / or a first onset temperature of 179.83°C to 182.61°C, and / or Second starting temperature of 363.24°C to 366.16°C 1. A composition characterized by having a TGA thermogravimetric analysis (TGA) thermogram having:

12. 10. The composition of any of the preceding claims, wherein the ratio of NAD+ to polyethylene glycol in the composition is 1:1 (wt / wt).

13. 10. The composition of claim 1, wherein the polyethylene glycol has an average molecular weight of between about 300 and about 4000.

14. 10. The composition of any of the preceding claims, wherein the polyethylene glycol is PEG 3350.

15. 10. The composition of any preceding claim in powder form.

16. 10. A composition according to any preceding claim suitable for oral administration to a subject.

17. 10. A composition according to any preceding claim, having a shelf life of at least six months at room temperature.

18. 10. A composition according to any preceding claim, having a shelf life of at least one year at room temperature.

19. A pharmaceutical formulation comprising a composition according to any one of claims 1 to 18.

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

21. 21. The formulation of claim 19 or 20, which, when administered to a subject, increases intracellular levels of NAD in the subject.

22. 22. The formulation of any one of claims 19 to 21, which, when administered to a subject, modulates the expression of at least one biomarker in the subject.

23. 23. The formulation of any one of claims 19 to 22 for use in a method for alleviating drug addiction withdrawal symptoms in a subject in need thereof, alleviating 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 with decreased levels of NAD in a subject in need thereof.

24. A kit comprising the composition of any one of claims 1 to 18.

25. 20. A method for preparing the enhanced NAD+ composition of any one of claims 1 to 18, comprising: (1) subjecting NAD+ to at least one evacuation cycle to form evacuated NAD+; and (2) blending the evacuated NAD+ with polyethylene glycol to form a blended mixture; A method comprising:

26. 26. The method of claim 25, wherein the inert gas comprises argon or nitrogen.

27. 27. The method of claim 25 or 26, wherein step (1) comprises subjecting the NAD+ to at least three vacuum cycles.

28. 28. The method of any one of claims 25 to 27, wherein the polyethylene glycol has an average molecular weight of between about 300 and about 4000.

29. 29. The method of any one of claims 25 to 28, wherein the polyethylene glycol is PEG 3350.

30. 30. The method of any one of claims 25 to 29, further comprising subjecting the blended mixture to at least one additional vacuum cycle.

31. 30. The method of any one of claims 25 to 29, further comprising subjecting the blended mixture to at least three additional vacuum cycles.

32. 30. The method of any one of claims 25 to 29, further comprising subjecting the blended mixture to at least six additional vacuum cycles.

33. 33. The method of any one of claims 25 to 32, further comprising pretreating the NAD+ prior to step (1).

34. 34. The method of claim 33, wherein pre-treating the NAD+ comprises passing the NAD+ through a sieve and / or grinding, milling, or jet-milling the NAD+.

35. 35. An enhanced NAD+ composition prepared by the method of any one of claims 25 to 34.

36. A method of making an enhanced NAD+ pharmaceutical formulation, comprising mixing an enhanced NAD+ composition described in any one of claims 1 to 18 with at least one ingredient or excipient that facilitates administration to a subject and / or that can further treat a disease or condition in the subject.

37. 24. A method for reducing drug addiction withdrawal symptoms in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an enhanced NAD+ pharmaceutical formulation of any one of claims 19-23, wherein the amount is effective to reduce drug addiction withdrawal symptoms in the subject.

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

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

40. 40. The method of any one of claims 37 to 39, wherein the level of NAD that is increased in the subject is an intracellular level of NAD.

41. 41. The method of any one of claims 37 to 40, wherein the intracellular level of NAD is increased to greater than about 35 μM.

42. 42. The method of any one of claims 37 to 41, wherein the level of NAD that is increased in the subject is the extracellular level of NAD.

43. 43. The method of any one of claims 37-42, wherein said administration of said enhanced NAD+ pharmaceutical formulation is effective to increase plasma levels of N-methyl-nicotinamide (MeNAM) or N-methyl-2-pyridone-5-carboxamide (2PY) by at least 5% in said subject.

44. 44. The method of any one of claims 37 to 43, wherein the amount of NAD+ in the pharmaceutical formulation administered to the subject is from about 5 mg to about 4000 mg.

45. 45. The method of any one of claims 37 to 44, wherein the amount of the pharmaceutical formulation administered comprises a maximum daily dose of about 1 mg / kg to about 100 mg / kg body weight of the subject.

46. 46. ​​The method of any one of claims 37 to 45, further comprising administering to the subject a pharmaceutically effective amount of an analgesic or a nonsteroidal anti-inflammatory drug (NSAID).

47. 46. ​​The method of any one of claims 37 to 45, wherein the pharmaceutical formulation further comprises a pharmaceutically effective amount of an analgesic or a nonsteroidal anti-inflammatory drug (NSAID).

48. 48. The method of any one of claims 37 to 47, further comprising monitoring at least one vital sign of the subject before and after administration of the pharmaceutical formulation.

49. 49. The method of any one of claims 37 to 48, further comprising measuring intracellular or extracellular concentrations of NAD in the subject before and after administration of the pharmaceutical formulation.

50. 50. The method of claim 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.

51. 24. A method of increasing nicotinamide adenine dinucleotide (NAD) levels in a subject, comprising administering an amount of an enhanced NAD+ pharmaceutical formulation of any one of claims 19 to 23 effective to increase NAD levels in the subject.

52. 52. The method of claim 51, wherein the subject is a healthy adult.

53. 24. A method for preventing or treating a disease or disorder correlated with reduced levels of NAD in a subject in need thereof, comprising administering an amount of an enhanced NAD+ pharmaceutical formulation of any one of claims 19 to 23 effective to prevent or treat said disease or disorder in said subject.

54. 24. A method of treating a subject suffering from drug addiction, comprising administering to the subject a pharmaceutically effective amount of an enhanced NAD+ pharmaceutical formulation of any one of claims 19 to 23.

55. 55. The method of claim 54, wherein the chemical compound is tobacco, heroin, opium, morphine, dihydromorphine, meperidine, codeine, cocaine, amphetamine, barbiturate, alcohol, a tranquilizer, or an opioid.

56. 24. A method for promoting cellular NAD metabolism or NAD cell homeostasis in a subject, comprising administering to the subject an amount of an enhanced NAD+ pharmaceutical formulation of any one of claims 19 to 23 effective to promote NAD metabolism or NAD cell homeostasis.

57. 24. 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 an enhanced NAD+ pharmaceutical formulation of any one of claims 19-23, wherein the at least one condition is elevated blood pressure, high blood sugar, excess body fat around the waist, abnormal cholesterol or triglyceride levels, or any combination thereof.

58. 24. A method of increasing 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 an enhanced NAD+ pharmaceutical formulation of any one of claims 19-23 effective to increase SIRT1, PLB1, NPL, ENPP5, GLSTA, GALNT3, FABP1, NRCAM, NLGN2, ARTN, UPB1, MME, CTSB, CTSL, or HRAS polypeptide.

59. 20. A method for preparing the enhanced NAD+ composition of any one of claims 1 to 18, comprising: (1) blending NAD+ with polyethylene glycol to form a blended mixture; and (2) subjecting the blended mixture to at least one vacuum cycle; Including, Each evacuation cycle consists of placing the vessel under vacuum and then filling it with an inert gas or CO ２ backfilling said container with

60. 1. A method for preparing an enhanced NAD+ composition comprising a crystalline component comprising crystalline polyethylene glycol and an amorphous component comprising amorphous NAD+, comprising: (1) blending NAD+ with polyethylene glycol to form a blended mixture; and (2) subjecting the blended mixture to at least one vacuum cycle; Including, Each evacuation cycle consists of placing the vessel under vacuum and then filling it with an inert gas or CO ２ backfilling said container with

61. 61. The method of claim 59 or 60, wherein the inert gas comprises argon or nitrogen.

62. 62. The method of any one of claims 59 to 61, wherein step (2) comprises at least three vacuum cycles.

63. 62. The method of any one of claims 59 to 61, wherein step (2) comprises at least six vacuum cycles.

64. 64. The method of any one of claims 59 to 63, wherein the polyethylene glycol has an average molecular weight of between about 300 and about 4000.

65. 65. The method of any one of claims 59 to 64, wherein the polyethylene glycol is PEG 3350.

66. 66. The method of any one of claims 59 to 65, further comprising pretreating the NAD+ prior to step (1).

67. 67. The method of claim 66, wherein pre-treating the NAD+ comprises passing the NAD+ through a sieve and / or grinding, milling, or jet-milling the NAD+.

68. 68. The method of any one of claims 59 to 67, wherein the NAD+ is subjected to at least one vacuum cycle prior to step (1).

69. 69. An enhanced NAD+ composition prepared by the method of any one of claims 59 to 68.

Images